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Rituparna Banerjee, Arun K. Verma, Mohammed Wasim Siddiqui - Natural Antioxidants - Applications in Foods of Animal Origin-Apple Academic Press (2017)

This document is an edited book about natural antioxidants and their applications in foods of animal origin. It was edited by Rituparna Banerjee, Arun K. Verma, and Mohammed Wasim Siddiqui. The book contains chapters written by various contributors and covers topics such as the mechanism of oxidation in foods of animal origin and the use of natural antioxidants to prevent or delay lipid oxidation in meat and meat products. It is intended to be a comprehensive resource on the role of natural antioxidants in improving the quality and extending the shelf life of foods derived from animal sources.

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0% found this document useful (0 votes)
199 views414 pages

Rituparna Banerjee, Arun K. Verma, Mohammed Wasim Siddiqui - Natural Antioxidants - Applications in Foods of Animal Origin-Apple Academic Press (2017)

This document is an edited book about natural antioxidants and their applications in foods of animal origin. It was edited by Rituparna Banerjee, Arun K. Verma, and Mohammed Wasim Siddiqui. The book contains chapters written by various contributors and covers topics such as the mechanism of oxidation in foods of animal origin and the use of natural antioxidants to prevent or delay lipid oxidation in meat and meat products. It is intended to be a comprehensive resource on the role of natural antioxidants in improving the quality and extending the shelf life of foods derived from animal sources.

Uploaded by

wilmerjmoreno
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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NATURAL ANTIOXIDANTS

Applications in Foods of Animal Origin


NATURAL ANTIOXIDANTS
Applications in Foods of Animal Origin

Edited by
Rituparna Banerjee, MVSc
Arun K. Verma, PhD
Mohammed Wasim Siddiqui, PhD
Apple Academic Press Inc. Apple Academic Press Inc.
3333 Mistwell Crescent 9 Spinnaker Way
Oakville, ON L6L 0A2 Canada Waretown, NJ 08758 USA
© 2017 by Apple Academic Press, Inc.
Exclusive worldwide distribution by CRC Press, a member of Taylor & Francis Group
No claim to original U.S. Government works
Printed in the United States of America on acid-free paper
International Standard Book Number-13: 978-1-77188-459-4 (Hardcover)
International Standard Book Number-13: 978-1-315-36591-6 (CRC Press/Taylor & Francis eBook)
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This book contains information obtained from authentic and highly regarded sources. Reprinted material is
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Library and Archives Canada Cataloguing in Publication

Natural antioxidants : applications in foods of animal origin / edited by Rituparna Banerjee, MVSc,
Arun K. Verma, PhD, Mohammed Wasim Siddiqui, PhD.
Includes bibliographical references and index.
Issued in print and electronic formats.
ISBN 978-1-77188-459-4 (hardcover).--ISBN 978-1-315-36591-6 (PDF)
1. Antioxidants. 2. Food of animal origin. 3. Food additives. I. Siddiqui, Mohammed Wasim, author,
editor II. Banerjee, Rituparna, author, editor III. Verma, Arun K., author, editor

TX553.A73N38 2017 613.2’86 C2017-900924-9 C2017-900925-7

Library of Congress Cataloging-in-Publication Data

Names: Banerjee, Rituparna, editor. | Verma, Arun K., editor. | Siddiqui, Mohammed Wasim, editor.
Title: Natural antioxidants : applications in foods of animal origin / editors, Rituparna Banerjee,
MVSc, Arun K. Verma, PhD, Mohammed Wasim Siddiqui, PhD.
Description: Toronto : Apple Academic Press, 2017. | Includes bibliographical references and index.
Identifiers: LCCN 2017003500 (print) | LCCN 2017004858 (ebook) (print) | LCCN 2017004858
(ebook) | ISBN 9781771884594 (hardcover : alk. paper) | ISBN 9781315365916 (ebook)
Subjects: LCSH: Antioxidants. | Food--Preservation. | Oils and fats, Edible--Deterioration. | Lipids-
-Oxidation.
Classification: LCC TX553.A73 N386 2017 (print) | LCC TX553.A73 (ebook) | DDC 613.2/86--dc23
LC record available at https://lccn.loc.gov/2017003500

Apple Academic Press also publishes its books in a variety of electronic formats. Some content that appears
in print may not be available in electronic format. For information about Apple Academic Press products,
visit our website at www.appleacademicpress.com and the CRC Press website at www.crcpress.com
ABOUT THE EDITORS

Rituparna Banerjee, MVSc


Dr. Rituparna Banerjee is presently a scientist in the discipline of livestock
products technology at the ICAR-National Research Centre on Meat,
Hyderabad, India. Dr. Banerjee is primarily involved in research pertaining
to meat science and technology. She has been awarded the prestigious Smt.
Mira Mallik Gold Medal for securing the highest marks in the BVSc and
AH course by the West Bengal University of Animal and Fishery Sciences.
She is the only candidate to qualify on an All-India basis as a Scientist in
Livestock Product Technology for Agricultural Research Service under the
Indian Council of Agricultural Research in 2015. She has published 21 peer-
reviewed research articles in international and national journals, three book
chapters, five lead/invited papers, and 24 abstracts. She is associated with
organizing training programs in microbial quality for meat food safety and
value added meat products. She was trained in the fields of microbiology,
chemistry, and technology of dairy functional foods and nutraceuticals and
food quality and safety. Dr. Banerjee is also associated with five professional
societies related to meat science and the effective dissemination of knowledge
in the field of packaging of meat products. She also participated in several
seminars and symposiums on meat science and food safety and has presented
papers on these aspects.

Arun K. Verma, PhD


Dr. Arun K. Verma is currently working as a scientist in livestock prod-
ucts technology at ICAR-Central Research Institute for Research on Goats
(ICAR), Makhdoom, Mathura, India. He is a graduate of Jawaharlal Nehru
Krishi Vishwavidyalay, Jabalpur. He obtained his MVSc and PhD degrees
from the Indian Veterinary Research Institute, Izatnagar, Bareilly, in Live-
stock Products Technology. He worked as an Assistant Professor (Livestock
Products Technology) at Maharashtra Animal and Fishery Sciences, Univer-
sity, Nagpur, and later joined the Agricultural Research Service as a scien-
tist in 2010. He has been working with functional meat products and food
safety and traceability associated with goat products. He has 25 national and
international peer-reviewed research papers, ten popular as well as review
vi About the Editors

articles, and three book chapters to his credit. He has presented more than 25
papers at various seminars, symposia, and conferences.

Mohammed Wasim Siddiqui, PhD


Dr. Mohammed Wasim Siddiqui is an Assistant Professor and Scientist
in the Department of Food Science and Post-Harvest Technology, Bihar
Agricultural University, Sabour, India, and author or co-author of 31 peer-
reviewed research articles, 26 book chapters, two manuals, and 18 confer-
ence papers. He has 11 edited and authored books to his credit, published by
Elsevier, CRC Press, Springer, and Apple Academic Press. Dr. Siddiqui has
established an international peer-reviewed journal, Journal of Postharvest
Technology. He is Editor-in-Chief of two book series (Postharvest Biology
and Technology and Innovations in Horticultural Science), published by
Apple Academic Press. Dr. Siddiqui is also a Senior Acquisitions Editor in
Apple Academic Press, New Jersey, USA, for horticultural science. He has
been serving as an editorial board member and active reviewer of several
international journals, including LWT- Food Science and Technology (Else-
vier), Food Science and Nutrition (Wiley), Acta Physiologiae Plantarum
(Springer), Journal of Food Science and Technology (Springer), Indian
Journal of Agricultural Science (ICAR), etc.
Dr. Siddiqui received the Best Young Researcher Award (2015) by
GRABS Educational Trust, Chennai, India, and the Young Scientist Award
(2015) by Venus International Foundation, Chennai, India. He was also a
recipient of the Young Achiever Award (2014) for outstanding research work
by the Society for Advancement of Human and Nature (SADHNA), Nauni,
Himachal Pradesh, India, where he is an honorary board member and life
time author. He has been an active member of the organizing committees of
several national and international seminars, conferences, and summits. He
is one of key members in establishing the World Food Preservation Center
(WFPC), LLC, USA, and is currently an active associate and supporter.
Dr. Siddiqui acquired a BSc (Agriculture) degree from Jawaharlal
Nehru Krishi Vishwa Vidyalaya, Jabalpur, India. He received his MSc
(Horticulture) and PhD (Horticulture) degrees from Bidhan Chandra Krishi
Viswavidyalaya, Mohanpur, Nadia, India, with specialization in Postharvest
Technology. He was awarded an Maulana Azad National Fellowship Award
from the University Grants Commission, New-Delhi, India. He is a member
of Core Research Group at the Bihar Agricultural University (BAU), which
provides appropriate direction and assistance in prioritizing research. He
About the Editors vii

has received several grants from various funding agencies to carry out his
research projects. Dr. Siddiqui has been associated with postharvest tech-
nology and processing aspects of horticultural crops. He is very engaged in
teaching (graduate and doctorate students) and research, and he has proved
himself as an active scientist in the area of postharvest technology.
CONTENTS

List of Contributors ....................................................................................... xi


List of Abbreviations ...................................................................................xiii
Preface ........................................................................................................ xix

1. Mechanism of Oxidation in Foods of Animal Origin...................................1


Manat Chaijan and Worawan Panpipat

2. Natural Antioxidants: Occurrence and Their Role in


Food Preservation .........................................................................................39
Ajit Singh Bhatnagar and Rewa Kulshrestha

3. Potential Applications of Natural Antioxidants in Meat and


Meat Products ...............................................................................................95
Rituparna Banerjee, Arun K Verma, Mohammed Wasim Siddiqui,
B. M. Naveena, and V. V. Kulkarni

4. Natural Antioxidants: Control of Oxidation in Fish and


Fish Products ...............................................................................................141
Ugochukwu Anyanwu and Reza Tahergorabi

5. Natural Antioxidants in Poultry Products ................................................165


A. K. Biswas, M. K. Chatli, and Gauri Jairath

6. Methods and Their Applications for Measuring and Managing


Lipid Oxidation: Meat, Poultry, and Seafood Products ..........................203
Tom Jones

7. Application of Natural Antioxidants in Dairy Foods ...............................261


Neelam Upadhyay, Veena N., Sanket Borad, Ashish Kumar Singh,
Sumit Arora, and Minaxi

8. Antioxidant Dietary Fiber: An Approach to Develop Healthy


and Stable Meat Products ..........................................................................299
Arun K. Verma, Rituparna Banerjee, and V. Rajkumar

9. Control of Lipid Oxidation in Muscle Food by Active


Packaging Technology ................................................................................343
José M. Lorenzo, Ruben Domínguez, and Javier Carballo

Index .....................................................................................................................383
LIST OF CONTRIBUTORS

Sumit Arora
Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal 132001, Haryana, India

Ugochukwu Anyanwu
North Carolina Agricultural and Technical State University, Greensboro, NC, USA

Rituparna Banerjee
ICAR-National Research Centre on Meat, Chengicherla 500092, Hyderabad, India

Ajit Singh Bhatnagar


IGNOU Regional Centre, Durg, Chhattisgarh, India

A. K. Biswas
Division of Post-Harvest Technology, ICAR-Central Avian Research Institute, Izatnagar, Bareilly
243122, Uttar Pradesh, India

Sanket Borad
Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal 132001, Haryana, India

Javier Carballo
Área de Tecnología de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, Ourense
32004, Spain

Manat Chaijan
Food Technology and Innovation Center of Excellence, Department of Agro-Industry, School of Agricul-
tural Technology, Walailak University, Thasala 80160, Nakhon Si Thammarat, Thailand

M. K Chatli
Department of Livestock Products Technology, GADVASU, Ludhiana 141004, Punjab, India

Ruben Domínguez
Centro Tecnológico de la Carne de Galicia, Rua Galicia No. 4, Parque Tecnológico de Galicia, San
Cibrao das Viñas, Ourense 32900, Spain

Gauri Jairath
Department of Livestock Products Technology, LUVAS, Hisar 125001, Haryana, India

Tom Jones
Meat Products, Global Applications and Product Development, Kalsec®, Inc., Kalamazoo, MI 49006,
USA. E-mail: [email protected]
V. V. Kulkarni
ICAR-National Research Centre on Meat, Chengicherla 500092, Hyderabad, India

Rewa Kulshrestha
Bilaspur University, Bilaspur, Chhattisgarh, India

José M. Lorenzo
Centro Tecnológico de la Carne de Galicia, Rua Galicia No. 4, Parque Tecnológico de Galicia, San
Cibrao das Viñas, Ourense 32900, Spain
xii List of Contributors

Minaxi
Agricultural Structures and Environmental Control Division, ICAR- Central Institute of Post-Harvest
Engineering and Technology, Ludhiana 141004, Punjab, India

B. M. Naveena
ICAR-National Research Centre on Meat, Chengicherla 500092, Hyderabad, India
Worawan Panpipat
Functional Food Research Unit, Department of Agro-Industry, School of Agricultural Technology,
Walailak University, Thasala, Nakhon Si Thammarat 80160, Thailand

V. Rajkumar
Goat Products Technology Laboratory, ICAR-Central Institute for Research on Goats, Makhdoom,
Farah, Mathura 281122, Uttar Pradesh, India

Mohammed Wasim Siddiqui


Department of Food Science and Postharvest Technology, Bihar Agricultural University, Sabour,
Bhagalpur 813210, Bihar, India

Ashish Kumar Singh


Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal 132001, Haryana, India

Reza Tahergorabi
Department of Family and Consumer Sciences, Food and Nutritional Sciences, North Carolina Agricul-
tural and Technical State University, Greensboro, NC, USA

Neelam Upadhyay
Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal 132001, Haryana, India

N. Veena
Dairy Chemistry Division, College of Dairy Science and Technology, Guru Angad Dev Veterinary and
Animal Sciences University, Ludhiana 141001, Punjab, India

Arun K. Verma
Goat Products Technology Laboratory, ICAR-Central Institute for Research on Goats, Makhdoom,
Farah, Mathura 281122, Uttar Pradesh, India
LIST OF ABBREVIATIONS


OH hydroxyl radical
1
O2 singlet oxygen
4-NHE 4-hydroxynonenal
ABTS 2,2ʹ-azino-bis3-ethylbenzothiazoline-6-sulphonic acid
ADF antioxidant dietary fiber
ADI acceptable daily intake
ADP adenosine diphosphate
AF antioxidant factor
AGEs advanced glycation end products
AH antioxidant
AH• L-ascorbic acid radical
AH2 L-Ascorbic acid
AI atherogenic index
ALE advanced lipid oxidation end
AOA antioxidant activity
APE allylic position equivalent
ARP anti-radical power
aw water activity
BAPE bis-allylic position equivalent
BCAAs branched chain amino acids
BHA butylated hydroxy anisole
BHT butylated hydroxy toluene
BPR bael pulp residue
C of V coefficient of variance
CA citric acid
Ca calcium
CB control biscuits
CF commercially available fat
CFR code of federal regulations
CHD coronary heart diseases
CIE International Commission on Illumination
CLA conjugated linoleic acid
CO2 carbon dioxide
CP cauliflower powder
xiv List of Abbreviations

CPFB Citrus paradisi fruit barks


CPP caseinophosphopeptides
DABCO diazabicyclooctane
DF dietary fiber
DFA desirable fatty acids
DG dodecyl gallate
DHA docosahexaenoic acid
DPP dried plum puree
DPPH 2,2-diphenyl-1-picrylhydrazyl
DW dry weight
EDTA ethylene diaminetetraacetic acid
EFSA European Food Safety Authority
EGCG epigallocatechin gallate
EKWE ethanolic kiam wood extract
EM exxenterol
EOs essential oils
EPA eicosapentaenoic acid
ESR electron spin resonance
FA ferulic acid
FDA Food and Drug Administration
Fe iron
FFA free fatty acid
FIR far infrared
FPP fresh plum puree
FRAP ferric ion reducing antioxidant power
FRH FIR treated rice hull
GADF grape antioxidant dietary fiber
GCMS gas chromatography mass spectroscopy
GL glycolipids
GOMPS good oxidation management practices
GPC grape pomace concentrate
GRAS generally regarded as safe
GSE grape seed extract
H2O2 hydrogen peroxide
Hb hemoglobin
HDA hydrogen donating antioxidants
HDL high density lipoproteins
HHE trans-4-hydroxy-2-hexanal
HO• hydroxyl radical
HOO•, hydroperoxyl radical
List of Abbreviations xv

HOX high oxygen


HP high pressure
HPLC high performance liquid chromatography
HPOs hydroperoxides
ICA iron chelating antioxidants
IDF insoluble dietary fiber
IT index of thromogenicity
IV iodine value
K potassium
Keq equilibrium constant
KRP kinnow rind powder
L*a*b* defined color space for reflectance colorimetry
LAOX linoleic acid accelerated by azo-initiators
LM liposterine
LMW low-molecular-weight
LOOH lipid hydroperoxides
LOQ limits of quantification
LOX lipoxygenase
LS lasalacid sodium salt
MAP modified atmosphere packaging
Mbg myoglobin
MDA malonaldehyde
ME mint extract
MEFS methanol extracts of fermented soybeans
MEs methanol extracts
MFGM milk fat globule membrane
MFM minced fish muscle
MHO menhaden oil
MMbg met-myoglobin
MST mechanically separated turkey
MUFA monounsaturated fatty acids
NADH nicotinamide adenine dinucleotide
NBT nitro blue tetrazolium
NDGA nordihydroguaretic acid
NL neutral lipids
NO• oxide radical
NO2•. nitrogen dioxide radical
NOMb nitrosomyoglobin
NPN non-protein nitrogen
O2•- superoxide radical
xvi List of Abbreviations

O2–• superoxide anion radical


OE oregano extract
OEO oregano essential oil
OFA undesirable fatty acids
OFB oryzanol fortified biscuits
OFF oryzanol fortified fat
OG octyl gallate
OMbg oxymyoglobin
OP olive pomace
OPE orange peel extract
ORAC oxygen radical absorbance capacity
ORP oxidation reduction potential
OS oxidative stability
Oz oryzanol
p- AV para- ansidine values
PCL polycaprolactone
PDCAAS protein digestibility corrected amino acid score
PE polyethylene
PET Polyethylene terephthalate
PG propyl gallate
PHB polydroxybutyrate
PhIP 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine
PL phospholipids
PLA poly lactic acid
PMS phenazonium methosulfate
POH phenolic compounds
POPs phytosterol oxidation products
PP pomegranate peels
PP polypropylene
PPE potato peel extract
PPO polyphenoloxidase
PRP pomegranate rind powder
PS polystyrene
PSP pomegranate seed powder
PUFA polyunsaturated fatty acids
PV peroxide value
PVC polyvinyl chloride
R radicals
RDI recommended daily intake
RE rosemary extract
List of Abbreviations xvii

RGE red ginseng extract


RNS reactive nitrogen species
RO• alkoxyl radicals
ROO• peroxyl radicals
ROS reactive oxygen species
RSA radical scavenging activity
RWGP red wine grape pomace
SAT saturated fatty acids
SDF soluble dietary fiber
Se selenium
SI selectivity indexes
SOSG singlet oxygen sensor green
TA total anthocyanin
TAG triacylglycerides
TBA thiobarbituric acid
TBARS thiobarbituric acid reactive substances
TBHQ tertiary butyl hydroquinone
TC tea catechins
TDF total dietary fiber
TEAC trolox equivalent antioxidant capacity
TF total flavonol
TM tocopherol
TOSC total oxyradical scavenging activity
TOTOX total oxidation
TPC total phenolic content
UF ultra-filtered
UMAE ultrasonic/microwave assisted extraction
UV ultraviolet
WGDF white grape antioxidant dietary fiber
WHC water holding capacity
WOF warmed-over-flavor
WWGP white wine grape pomace
PREFACE

Many food preservation strategies can be used for the control of oxidation
in foods; however, these quality problems are not yet controlled adequately.
Although synthetic antioxidant agents are approved in many countries,
their excessive use has increased pressure on food manufacturers to either
completely remove these agents or to adopt natural alternatives for the
maintenance or extension of a product´s shelf life. Therefore, the use of
natural safe and effective preservatives is a demand of food consumers and
producers.
Foods of animal origin are one of the key components of our diet supplying
several vital nutrients like protein, fats, vitamins, and minerals. Fat is one of
the most important nutrients in foods of the animal origin and is composed
of various fatty acids such as saturated, monounsaturated, and polyunsatu-
rated fatty acids. Depending upon the nature and origin of foods, propor-
tion of these fatty acids varies. As the amount of unsaturated fatty acids
increases, the animal foods become more vulnerable to oxidation. Oxidation
of fat damages the nutritional and sensory characteristics, particularly flavor
of food products, and thus affects their storage stability. Proteins in animal
foods are also susceptible to the oxidation thus affecting the quality of foods.
Moreover, during processing and storage, food products undergo changes
in their physicochemical characteristics leading to development of oxygen-
ated free radicals which initiate the oxidation of polyunsaturated fatty acids
while destruction of the endogenous antioxidant system. Various approaches
are being applied to minimize the oxidation of these foods such as use of
antioxidants and anaerobic and active packaging.
The search of new safe substances for food preservation is being
performed around the world. Many naturally occurring bioactive compounds
can be considered as good alternatives to synthetic antioxidant food addi-
tives. Natural antioxidants could be extracted from various plant and
animal sources. These are extracted using different approaches and solvents
depending upon feasibility and yield. In foods of animal origin such as
meat and meat products, dairy products, fish and fish products, and poultry
products, natural antioxidants are applied in various forms and ways. As
such, there is no compiled literature on the oxidation of animal products and
approaches for its reduction. In this book, attempts are made to address the
xx Preface

aforesaid issues and appraise the potential use of antioxidants from natural
sources to ameliorate the oxidative stress in animal products, to prevent
lipid–protein oxidation, and to improve oxidative stability considering
demand for safety and nutrition.
This book, Natural Antioxidants: Applications in Foods of Animal
Origin, will be a standard reference work describing the potential of natural
antioxidants in the animal food industries. It will also contemplate the senso-
rial and toxicological shortcomings of using natural products. This proposed
book looks forward to promoting the use of natural extracts and fulfilling
consumer demands for healthier foods.
CHAPTER 1

MECHANISM OF OXIDATION IN
FOODS OF ANIMAL ORIGIN
MANAT CHAIJAN* and WORAWAN PANPIPAT
Food Technology and Innovation Center of Excellence, Department
of Agro-Industry, School of Agricultural Technology, Walailak
University, Thasala 80160, Nakhon Si Thammarat, Thailand
Corresponding author. E-mail: [email protected]
*

CONTENTS

Abstract ........................................................................................................2
1.1 Introduction .........................................................................................2
1.2 Impact of Lipid Oxidation in Foods of Animal Origin .......................3
1.3 Basic Mechanism of Lipid Oxidation in Foods of Animal Origin .....5
1.4 Effect of Intrinsic Factors and Processing Parameters in
Lipid Oxidation in Foods of Animal Origin .....................................10
1.5 Myoglobin Oxidation in Foods of Animal Origin ............................18
1.6 Interrelationship Between Lipid Oxidation and Myoglobin
Oxidation in Foods of Animal Origin ...............................................20
1.7 Interaction Between Lipid Oxidation Products and Myoglobin .......27
1.8 Conclusion ........................................................................................28
Keywords ...................................................................................................29
References ..................................................................................................29
2 Natural Antioxidants: Applications in Foods of Animal Origin

ABSTRACT

Lipid and myoglobin oxidations significantly impair the quality of foods of


animal origin because these reactions deteriorate flavor and color, induce the
loss of nutritional value and cause technological problems during processing.
Lipid and myoglobin oxidations are coupled and such reactions can occur
via non-enzymatic and enzymatic routes. Several factors have been reported
to enhance the oxidation of lipid in muscle foods including species, muscle
type, fatty acid composition, endogenous antioxidants (AH), temperature,
metal ions, sodium chloride, muscle pH, and processing parameters. It is most
likely that the prooxidant effect of heme proteins, especially myoglobin, is
a prime factor influencing the lipid oxidation in muscle foods. On the other
hand, lipid oxidation results in a wide range of aldehyde products, which
can cause the oxidation of myoglobin. The interaction between myoglobin
and aldehydic lipid oxidation products can alter myoglobin redox stability
and finally results in muscle discoloration. As a consequence, the oxidation
of both lipid and myoglobin directly affect the quality and acceptability of
muscle foods and those reactions seems to promote each other.

1.1 INTRODUCTION

The problems associated with oxidation in foods of animal origin, partic-


ularly meat and muscle foods, have gained much interest as they relate to
flavor deterioration, discoloration, loss of nutritional value and safety, biolog-
ical damage, ageing, and functional property changes. Meat and other muscle
foods are complex foods with highly structured nutritional compositions
(Rodriguez-Estrada et al., 1997). Muscles are composed of water, proteins,
lipids, carbohydrates, vitamins, and minerals in variable amounts depending
on several factors such as breeds, muscle types, dietary, and growth perfor-
mance (Wattanachant et al., 2005). Oxidation is a major cause of quality dete-
rioration for a variety of raw and processed muscle foods during handling,
processing, and storage. Lipid, protein, pigment, and vitamin in muscle tissue
are susceptible to oxidative reactions. These changes resulted from reactions
of active oxygen species, free radicals, enzymes, and prooxidants with unsat-
urated fatty acids in lipids, amino acids in proteins, heme groups in pigments
and the chains in vitamins with conjugated double bonds. However, lipid
oxidation and the oxidation of heme proteins, particularly myoglobin, in
muscle foods are major deteriorative reactions which occur in a concurrent
manner and each process appears to enhance the other. During oxidation of
Mechanism of Oxidation in Foods of Animal Origin 3

oxymyoglobin, both superoxide anion and hydrogen peroxide are produced


and further react with iron to produce hydroxyl radical. The hydroxyl radical
has the ability to penetrate into the hydrophobic lipid region and hence
facilitates lipid oxidation. The prooxidant effect of heme proteins on lipid
oxidation is concentration-dependent. At equimolar concentrations, oxymyo-
globin shows higher prooxidative activity toward lipid than metmyoglobin.
However, the catalytic activity of metmyoglobin is promoted by hydrogen
peroxide. The reaction between hydrogen peroxide and metmyoglobin results
in the formation of two active hypervalent species, perferrylmyoglobin and
ferrylmyoglobin, which are responsible for lipid oxidation. Additionally, lipid
oxidation results in a wide range of aldehyde products, which are reported to
induce the oxidation of oxymyoglobin. Studies in muscle foods have been
focused mainly the interaction between myoglobin and aldehydic lipid oxida-
tion products. Metmyoglobin formation is generally greater in the presence of
unsaturated aldehydes than their saturated counterparts of equivalent carbon
chain length. In addition, increasing chain length of aldehydes, from hexenal
through nonenal, results in the increased metmyoglobin formation. More-
over, aldehydes alter myoglobin redox stability by increasing oxymyoglobin
oxidation, decreasing the metmyoglobin reduction via enzymatic process,
and enhance the prooxidant activity of metmyoglobin (Chaijan, 2008). There-
fore, the oxidation of both lipid and heme proteins directly affect the quality
and acceptability of muscle foods and the lowering of such a phenomenon
can enhance the shelf-life stability of those foods. To design strategies to
inhibit the progression of oxidative reactions in foods and biological systems,
it is important to understand the nature of these reactions and how they are
influenced by both intrinsic and extrinsic factors. This goal may be achieved
through a better understanding of the reaction kinetics including the rate at
which the reaction takes place, the effective factors (mainly temperature,
concentration of reactants and products, and presence of catalysts), and how
these two are related. This chapter deals with the mechanism of oxidation,
especially lipid and myoglobin, in foods of animal origin emphasizing on
meat and muscle foods. The interaction between lipid and myoglobin oxida-
tions and the effect of various food-processing applications on lipid and
myoglobin oxidations are also discussed.

1.2 IMPACT OF LIPID OXIDATION IN FOODS OF ANIMAL ORIGIN

Lipid oxidation is one of the important reactions in food and biological


systems because it has deleterious effects on polyunsaturated fatty acids
4 Natural Antioxidants: Applications in Foods of Animal Origin

(PUFA) and other lipid substrates, causing significant losses in food quality,
health, and well-being (Chaijan et al., 2006; Chaijan, 2008). Lipid oxidation
in food systems is a detrimental process. It is difficult to find a food compo-
nent that would not be capable of affecting lipid oxidation because lipids are
only a part of a food product (Kolakowska, 2002). Generally, lipid oxidation
deteriorates the sensory quality and nutritive value of a product, poses a
health hazard, and presents a number of analytical problems (Kolakowska,
2002). Lipid oxidation is also one of the main factors limiting the quality
and acceptability of meats and other muscle foods, especially following
refrigerated and frozen storages (Zamora & Hidago, 2001; Renerre, 2000;
Morrissey et al., 1998). Oxidation of lipids is accentuated in the immediate
post-slaughter period, during handling, processing, storage, and cooking.
This process leads to discoloration, drip losses, off-odor and off-flavor devel-
opment, texture defects, and the production of potentially toxic compounds
in meat products (Richards et al., 2002; Morrissey et al., 1998).
Hydroperoxide is a primary oxidation product during storage of foods
which is readily decomposed to a variety of volatile compounds including
aldehydes, ketones, and alcohols (Frankel et al., 1984). The formation of the
secondary lipid oxidation products is one of the main causes of the devel-
opment of undesirable odors in muscle foods. Human olfactory receptors
usually have remarkably low organoleptic thresholds to most of these vola-
tile compounds (Ke et al., 1975; McGill et al., 1977). The effect of lipid
oxidation and off-odor development in postmortem fish has been reported.
Lipid oxidation is mainly associated with the rejection by consumer due to
the off-odor and off-flavor. Flavor is a very complex attribute of meat palat-
ability. Rancid or fishy odor has been identified as a common off-flavor asso-
ciated with fish flesh and directly related with the formation of the secondary
lipid oxidation products (Ke et al., 1975; McGill et al., 1977; Sohn et al.,
2005; Thiansilakul et al., 2010). Varlet et al. (2006) reported that carbonyl
compounds, such as heptanal or (E,Z)-2,6-nonadienal, show a high detection
frequency and odorant intensity in salmon (Salmo salar), giving the flesh its
typical fishy odor. The fishy volatiles identified in the boiled sardine were
dimethyl sulfide, acetaldehyde, propionaldehyde, butyraldehyde, 2-ethyl-
furan, valeraldehyde, 2,3-pentanedione, hexanal, and 1-penten-3-ol (Kasa-
hara & Osawa, 1998).
Lipid oxidation usually causes a decrease in consumer acceptance.
However, in some cases, lipid oxidation leads to enhancement of product
quality such as the enzymatic production of fresh fish aromas and the cured
meat flavor derived from lipid oxidation during ripening (Ladikos & Lougo-
vois, 1990). A notable exception is observed in dry cured country hams and
Mechanism of Oxidation in Foods of Animal Origin 5

some fermented sausages and the desirable flavor of which does not occur
until hydrolysis of some of the fat and a certain degree of oxidation has taken
place during ripening (Pearson et al., 1977). On the other hand, lipid oxida-
tion during cooking may be a source of intermediate which react with other
components to give important constituents of the desirable flavor of normal
cooked meat (Enser, 1987). The types of flavor developed from the volatile
lipid oxidation compounds depend on a multitude of complex interactions,
concentration ranges, and the medium in which they are tasted (Frankel,
1984). Many of the reactions involved in the formation of volatile aroma
compounds from lipid, follow the same basic pathways for both thermal and
rancid oxidation and similar volatile products are formed. However, subtle
differences in the precise mechanisms of oxidation under storage conditions
and under thermal processing lead to mixtures of volatiles exhibiting both
qualitative and quantitative differences (Mottram, 1987).

1.3 BASIC MECHANISM OF LIPID OXIDATION IN FOODS OF


ANIMAL ORIGIN

The lipid oxidation in foods of animal origin is assumed to proceed along a


free radical route (autoxidation), photooxidation route and enzymatic route.
The oxidation mechanism is basically explained by invoking free-radical
reactions, while the photooxidation and lipoxygenase (LOX) routes differ
from it at the initiation stage only. For this reason, they can be treated as
different forms of free radical reaction initiation.

1.3.1 FREE RADICAL OXIDATION

The two major components involved in lipid oxidation are unsaturated fatty
acids and oxygen. In this process, atmospheric oxygen is added to some
fatty acids, producing unstable intermediates that finally breakdown to
form unpleasant flavor and aroma compounds (Erickson, 2003). Although
enzymatic and photogenic oxidation may play a role, the most common and
important process by which unsaturated fatty acids and oxygen interact is a
free radical mechanism (Erickson, 2003). A free radical reaction or autoxi-
dation is the main reaction involved in oxidative deterioration of food lipids,
including foods of animal origin (Hoac et al., 2006). It is a chain reaction that
consists of initiation, propagation, and termination reactions, and involves
the production of free radicals (Gunstone & Norris, 1983; Nawar, 1996;
6 Natural Antioxidants: Applications in Foods of Animal Origin

Renerre, 2000; Fig. 1.1). Oxidation is initiated by radicals present in living


organisms (e.g., hydroperoxide, hydroxide, peroxide, alcoxy, and alkyl)
or by thermal or photochemical homolytic cleavage of an R-H bond. The
oxidation activation energy and reaction rate at this stage depend on the type
of initiator and the number of unsaturated bonds in the substrate. The disso-
ciation energy of the C-H bonds in saturated fatty acid depend on the length
of the fatty acid carbon chain and is similar in fatty acid, their esters and in
triacylglycerols (Litwinienko et al., 1999). In unsaturated acids, the weakest
C-H bond is found in the bis-allylic position. The activation energies are
75, 88, and 100 kcal/mol for bis-allylic, allylic, and methylene hydrogens,
respectively (Simic et al., 1992). A three-step simplified free-radical scheme
has been postulated as follows:

1. Initiation
Initiator free radicals (R•, ROO•)

2. Propagation
R •+ O 2 ROO• ; ROO• + RH ROOH + R•

3. Termination
R• + R• R-R
R• + ROO• ROOR
ROO• + ROO• ROOR + O2
FIGURE 1.1 Mechanism of lipid oxidation via free radical route.

Initiation occurs as hydrogen is abstracted from an unsaturated fatty acid,


resulting in a lipid-free radical, which, in turn, reacts with molecular oxygen
to form a lipid peroxyl radical. Initiation is frequently attributed in most
foods, including muscle foods, to react ion of the fatty acids with active
oxygen species (Erickson, 2003).
The propagation phase of oxidation occurs by lipid-lipid interactions,
whereby the lipid peroxyl radical abstracts hydrogen from an adjacent mole-
cule, resulting in a lipid hydroperoxide and a new lipid-free radical. This
propagation continues until one of the radicals is removed by reaction with
another radical or with an antioxidant (AH) whose resulting radical (A•) is
much less reactive. Interactions of this type continue 10–100 times before
Mechanism of Oxidation in Foods of Animal Origin 7

two free radicals combine to terminate the process. The lipid radical reacts
very quickly with atmospheric oxygen making a peroxyl radical which again
may abstract hydrogen from another acyl chain, resulting in the formation
of a lipid hydroperoxide and a new radical. By themselves, lipid hydroper-
oxides are not considered harmful to food quality; however, they are further
degraded into compounds, especially aldehydes that are responsible for off-
flavors (Erickson, 2003).
To break the repeating sequence of propagating steps, two types of
termination reactions are encountered: radical–radical coupling and radical–
radical disproportionation, a process in which two stable products are
formed from free radicals by an atom or group transfer process. In both
cases, non-radical products are formed. However, the termination reactions
are not always efficient. When coupling gives rise to tertiary tetroxides, they
decompose to peroxyl radicals at temperatures above −80 °C and to alkoxyl
radicals at temperatures above −30 °C. On the other hand, secondary and
primary peroxyl radicals, terminate efficiently by a mechanism in which the
tetroxide decomposes to give molecular oxygen, an alcohol, and a carbonyl
compound (Erickson, 2003). On the other word, termination of free radical
oxidative reaction occurs when two radical species (peroxyl, alcoxyl, or
alkyl) react with each other to form a non-radical adduct. Free radical reac-
tions can also be terminated when one of the lipid radicals reacts with an
AH proper, because hydrogen abstraction by a peroxide radical from the AH
molecule produces an inert AH radical (Kolakowska, 2002).

1.3.2 PHOTOOXIDATION

Photooxidation involves the formation of hydroperoxides in a direct reac-


tion of singlet oxygen (1O2) addition to unsaturated lipids, without radical
formation. The 1O2 emerges during a reaction of sensitizers (e.g., chloro-
phyll, hemoglobin, myoglobin, and riboflavin) with atmospheric oxygen
(triplet oxygen, 3O2) (Kolakowska, 2002). Photosensitizations also can occur
in vivo (Halliwell et al., 1995). The 1O2 is 1450 times more reactive than
molecular oxygen. It is inserted at the end carbon of a double bond, which is
shifted to an allylic position in the trans configuration. The resulting hydro-
peroxides have an allylic trans double bond, which renders them different
from hydroperoxides formed during autoxidation. Hydroperoxides formed
during photooxidation are more easily cyclicized than hydroperoxy epid-
ioxides (Frankel, 1998). In addition, light, particularly ultraviolet light may
8 Natural Antioxidants: Applications in Foods of Animal Origin

be involved in initiation of the classical free radical oxidation of lipids


and catalyze other stages of the process. In the presence of light energy-
activated riboflavin, which is a sensitizer, a lipid radical can form, while
oxygen gives rise to superoxide radical anion (O2•−). During UV irradiation
of muscle lipids, the quantity of hydroperoxides and the ratio of their forma-
tion differ, depending on the origin of lipids. For instance, the photooxida-
tion ratio (slope of hydroperoxide accumulation over time of UV exposure
of lipids) of the muscle lipids of fish varied from about one to more than 20,
both within and between the species (Kolakowska, 2002). No correlations
between the photooxidation ratio and monounsaturated fatty acids (MUFA),
PUFA, eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA)
content in fish lipids were reported (Kolakowska, 2002).
Light induced oxidation is one of the main factors limiting shelf life
of milk. Exposure to visible light leads to off-flavors related to oxidation
of proteins and lipids due to excitation of photosensitizers among which
riboflavin has been recognized to play a major role (Bradley et al., 2003).
Beta-carotene absorbs light in the same region as riboflavin, and it has there-
fore been suggested to protect against photooxidation since less light then
reaches riboflavin (Airado-Rodríguez et al., 2011).
An et al. (2011) studied the effects of sensitizers and pH on the oil
oxidation of acidic O/W emulsions under light by measuring hydroperoxide
content and headspace oxygen consumption in the mixed canola and tuna
oil emulsions. The emulsions consisted of canola and tuna oil (2:1 w/w,
32%), diluted acetic acid (64%), egg yolk powder (4%), chlorophyll b or
erythrosine (5µM), and/or diazabicyclooctane (DABCO) or sodium azide
(0.5M). From the result, chlorophyll increased oil oxidation in the emul-
sion under light via 1O2 production while erythrosine did not. In contrast,
DABCO significantly decreased photooxidation of the oil containing chlo-
rophyll, suggesting 1O2 involvement. However, sodium azide increased
photooxidation of the oil containing chlorophyll possibly via azide radical
production under acidic conditions. The oil photooxidation was higher in
the emulsion containing chlorophyll at pH 6.27 than at pH 2.67 or 3.68,
primarily by 1O2 and secondarily by free radicals produced from hydroper-
oxide decomposition.

1.3.3 ENZYMATIC LIPID OXIDATION

LOX-catalyzed lipid oxidation differs from the free radical reaction by the
formation of hydroperoxides at a certain position of the chain of a free fatty
Mechanism of Oxidation in Foods of Animal Origin 9

acid. LOX use molecular oxygen to catalyze the stereo and regiospecific
oxygenation of PUFA with 1-cis, 4-cis-pentadiene moieties (Kolakowska,
2002). LOX react enzymatically with more than one methylene carbon on
the substrate molecule to yield double oxygenation sites (German et al.,
1992). The newly formed fatty acid peroxy free radical removes hydrogen
from another unsaturated fatty acid molecule to form a conjugated hydro-
peroxy diene. LOX forms a high-energy (radical) intermediate complex with
the substrate; this complex is capable of initiating the oxidation of lipids
and other compounds (e.g., carotenoids, chlorophyll, tocopherols, thiol
compounds, and protein), which can themselves interact with the enzyme
substrate complex as well (Hammer, 1993; Hultin, 1994).
Kolakowska (2002) reported that mammalian LOX are categorized
according to the positional specificity of oxygen insertion into arachi-
donic acid. Four isoform positions of arachidonate LOX have been identi-
fied: 5-LOX (E.C. 1.13.11.34), 8-LOX, 12-LOX (E.C. 1.13.11.31), and
15-LOX (E.C. 1.13.11.33). The LOX that catalyzes oxidation of linoleate
(E.C.1.13.11.12) attacks linoleic acid, both at position 9 and position 13.
In chicken meat arachidonate, 15-LOX was found to be active during
12-month storage at −20 °C (Grossman et al., 1988). In frozen-stored
fish, LOX contributes to oxidative lipid deterioration. However, LOX in
fish is also responsible for the formation of desirable fresh fish flavor, the
seaweed flavor (Lindsay, 1994). Some species show a higher activity of
12-LOX, while 15-LOX is more active in others; for this reason, the fresh
fish flavor spectrum is species dependent. The half-lives of 12- and 15-
LOX at 0 °C were less than 3 h and more than 10 h, respectively (German
et al., 1992). LOX was observed to be active in cold-stored fish after 48 h
of storage (Medina et al., 1999). The storage of herring, three weeks at
−20 °C, resulted in an increase in LOX activity. During prolonged frozen
storage of herring, a decrease in LOX activity was observed (Samson &
Stodolnik, 2001). Sae-leaw et al. (2013) reported that the development of
fishy odor in Nile tilapia skin during iced storage was mostly governed by
lipid oxidation via autoxidation or induced by LOX. Although the partici-
pation of LOX in the post mortem animal lipid oxidation is acknowledged,
the role of LOX in lipid oxidation is much more important in plant than
in animal food products. LOXs are responsible for the off flavor in frozen
vegetables (Ganthavorn et al., 1991), lipid oxidation in cereal products,
rapeseed, pea, avocado, and muscle foods, and for the beany and bitter
flavor (Frankel, 1998).
10 Natural Antioxidants: Applications in Foods of Animal Origin

1.4 EFFECT OF INTRINSIC FACTORS AND PROCESSING


PARAMETERS IN LIPID OXIDATION IN FOODS OF ANIMAL
ORIGIN

Ladikos and Lougovois (1990) reported that the nature and relative propor-
tions of the compounds formed from lipid oxidation depend at least in part on
the composition of the fat of the animal from which they are derived, which
may reflect a variety of factors including the nature of diet (Pearson et al.,
1977). Additionally, factors such as processing and storage conditions, type
of ingredients and concentration of pro- or antioxidants, are very important in
determining the rate of development and the possible deteriorative effects of
lipid oxidation. Therefore, the extent of lipid oxidation can be influenced by
both intrinsic and extrinsic factors such as the content and activity of pro- and
antioxidants, endogenous ferrous iron, myoglobin, enzymes, pH, tempera-
ture, ionic strength, irradiation, oxygen consumption reaction, surface area
in contact with oxygen, water activity (aw), and the fatty acid composition of
the meat (Andreo et al., 2003; Undeland, 2001; Harris & Tall, 1994; Renerre
& Labas, 1987; Castell et al., 1965; Nawar, 1996; Slabyj & Hultin, 1984;
Undeland et al., 2003). Erickson (2003) summarized typical responses exhib-
ited by muscle tissue during storage following various treatments. Processing
treatments like bleeding, curing, smoking, glazing, edible coating, freezing
and packaging inhibit the lipid oxidation whereas mincing, salting, rinsing
with oxidizing agent, cooking, deep fat frying, and radiation promote the
lipid oxidation. However, responses by muscle foods to some processing
treatments such as washing and skinning are varied among studies.
Slaughter of animals or fish is a necessary first step in converting the
living organism to food. Slaughter methods and the accompanying bleeding
step; however, may affect lipid oxidation through alteration in the removal
of hemoglobin catalysts. For example, bleeding was a potential means in
retarding lipid oxidation, fishy odor development, and microbial growth of
Asian seabass slices during storage in ice (Maqsood & Benjakul, 2011a,
2011b). Electrostatic interactions between hemoglobin and muscle compo-
nents may be an initial step of hemoglobin-mediated lipid oxidation
(Sannaveerappa et al., 2014).

1.4.1 SPECIES, MUSCLE TYPE, AND FATTY ACID COMPOSITION

A major cause of muscle food quality deterioration is lipid oxidation and the
changes associated with it. Lipid oxidation is a complex process whereby
Mechanism of Oxidation in Foods of Animal Origin 11

unsaturated fatty acids primarily reacting with molecular oxygen via a


free radical chain mechanism (Gray, 1978). The primary autooxidation is
followed by a series of secondary reactions which lead to the degradation of
the lipid and the development of oxidative rancidity. The main unsaturated
fatty acids comprising the lipids of animal tissues are oleic, linoleic, linolenic,
and arachidonic acids. Their autoxidation gives rise to a number of different
hydroperoxides which, in conjunction with the many different decompo-
sition pathways involved, lead to a large number of volatile compounds
(Mottram, 1987). Oxidation of lipids also occurs during postmortem storage
of muscle tissue. Meats such as fish and poultry contain a high concentration
of PUFA and are therefore more susceptible to oxidation (Pacheco-Aguilar
et al., 2000; Apgar & Hultin, 1982). Thus, fish lipids undergo more rapid
oxidation after capture, even at low temperature storage (Foegeding et al.,
1996; Pacheco-Aguilar et al., 2000). Pacheco-Aguilar et al. (2000) reported
that the shelf life of oily Monterey sardine was limited by lipid oxidation, as
shown by the increase of peroxide value (PV) during storage at 0 °C up to 15
days. Sohn et al. (2005) reported that the total lipid hydroperoxide content of
Pacific saury (Cololabis saira), Japanese Spanish mackerel (Scomberomorus
niphonius), and chub mackerel (Scomber japonicus) tended to increase in
both dark and ordinary muscle throughout four days of iced storage. Fatty
fish such as sardine and mackerel underwent rapid lipid oxidation during
iced storage due to the high content of PUFA (Chaijan et al., 2006; Chaijan
et al., 2013; Pacheco-Aguilar et al., 2000). Lipid oxidation seems to be a
distinct problem in surimi made from some dark-fleshed fish and particu-
larly surimi from mammalian and avian muscle (Lanier, 2000). Lynch et al.
(2001) demonstrated that lipid oxidation occurred progressively in stored
ground beef at 4 °C and produced a variety of aldehydes.

1.4.2 ENDOGENOUS ANTIOXIDANTS

The normal resistance of meat to the development of rancidity depends on


the balance between the presence of AH in the animal tissues and the level of
unsaturation and the concentration of the fatty acids present (Enser, 1987).
The living cells possess several protection mechanisms directed against
lipid oxidation products (Sies, 1997). Glutathione peroxidase reduces
hydroperoxides in the cellular membranes to the corresponding hydroxy-
compounds. This reaction demands supply of reduced glutathione and will
therefore cease post mortem when the cell is depleted of that substance.
The membranes also contain the phenolic compound α-tocopherol (Vitamin
12 Natural Antioxidants: Applications in Foods of Animal Origin

E) which is considered as the most important natural AH. Tocopherol can


donate a hydrogen atom to the radicals L• or LOO• functioning as the mole-
cule AH. It is generally assumed that the resulting tocopheryl radical reacts
with ascorbic acid (Vitamin C) at the lipid/water interface, regenerating
the tocopherol molecule. Muscle-based foods that contain relatively high
concentrations of α-tocopherol demonstrate greater lipid and oxymyoglobin
stability (Faustman et al., 1998). Poultry meat is composed of relatively high
levels of unsaturated fatty acids and low levels of natural tocopherols and
thus poultry products are very susceptible to the development of off-flavors
due to oxidative rancidity (Dawson & Gartner, 1983). According to Wilson
et al. (1976), turkey meat containing lower levels of natural tocopherol is
most susceptible to warmed-over-flavor (WOF) development, followed
closely by chicken, then by pork, beef, and mutton. The use of mechanically
deboned poultry meat enhances the tendency of poultry products to oxidize
(Moerck & Ball, 1974). However, the use of mechanically deboned beef
in beef meat products did not result in flavor deterioration during storage,
compared to control samples made of hand-boned beef, suggesting that lipid
oxidation is not a problem as with chicken and fish; this was attributed to
differences in the degree of unsaturation of fatty acids (Allen & Foegeding,
1981).
Other compounds, for example the carotenoids and phenols, have been
known to function as AH (Huss, 1995). Vareltzis et al. (2008) reported
that adding fish press juice to washed cod mince could inhibit hemo-
globin-mediated lipid oxidation of protein isolates obtained from cod
muscle (Gadus morhua). Press juice obtained from chicken breast muscle
also showed a potent inhibitor of hemoglobin-mediated lipid oxidation in
washed cod muscle (Li et al., 2005). The aqueous phase of chicken breast
muscle includes low-molecular-weight (LMW) components such as ascor-
bate, urate, glutathione, bilirubin, and histidine-containing dipeptides
(Chan et al., 1994). High-molecular-weight (HMW) components include
glutathione peroxidase, superoxide dismutase, catalase, transferrin, hapto-
globin, albumin, ceruloplasmin, and hemopexin (Decker, 1998). Erickson
et al. (1990) reported that both the LMW (<10 kDa) and HMW (>6–8 kDa)
cytosol fractions from flounder tissue inhibited iron-mediated lipid oxida-
tion in flounder sarcoplasmic reticulum and the effect was believed to be
due to the binding of iron. Furthermore, Slabyj and Hultin (1984) reported
that both LMW and HMW components in herring cytosol could inhibit iron-
mediated lipid oxidation in microsomes. Han and Liston (1989) reported the
ability of rainbow trout cytosol to inhibit iron-mediated lipid oxidation in
fish muscle microsomes.
Mechanism of Oxidation in Foods of Animal Origin 13

1.4.3 TEMPERATURE

Like most chemical reactions, lipid oxidation rates increase with increasing
temperature and time (Hultin, 1992). Saeed and Howell (2002) reported
that the rate of lipid oxidation in frozen Atlantic mackerel increased with
increasing storage time and storage temperature. Furthermore, freezing can
facilitate lipid oxidation, partly because of concentration effects (Foegeding
et al., 1996). The influence of long-term frozen storage, temperature (−25 and
−45 °C) and type of packaging materials (low and medium oxygen barriers)
on the lipid oxidation of frozen Atlantic herring fillets (Clupea harengus)
was studied by Tolstorebrov et al. (2014). The lowest lipid oxidation in term
of PV and thiobarbituric acid reactive substances (TBARS) was detected in
frozen Atlantic herring fillets kept at −45 °C and the packaging material with
a medium oxygen barrier. From the result, the oxygen concentration in the
package was considered to be the dominating factor for the herring’s oxida-
tion during frozen storing.
Cooked meats held in a refrigerator develop rancid odors and flavors
which usually become apparent within 48 h at 4 °C. These flavors are
particularly noticeable after reheating the meat and are referred to as WOF
(Tims & Watts, 1958). The rapid development of oxidized flavor in refrig-
erated cooked meats is in marked contrast to the slow onset of rancidity
commonly encountered in raw meats, fatty tissues, rendered fat, or lard,
which is normally not apparent until they have been stored for weeks or
months (Pearson et al., 1977).
Heating results in the release of heme-bound iron and in forming other
polymers with proteins; those polymers enhance the catalytic effect of iron.
This is also true with respect to the thermal inactivation of enzymes that
contain metals acting as prosthetic groups (e.g., LOX and peroxidases).
These enzymes, even after denaturation, are capable of catalyzing oxida-
tion. On the other hand, heating does not release iron from ferritin, but does
enhance its reduction (Kanner, 1992). The rate of oxidation in the presence
of metals is higher at lower pH than at neutral pH for Fe3+ and Fe2+ (Richards
& Hultin, 2000).
The extent of lipid oxidation in cooked meat appears to be related to the
intensity of heat treatment. Pearson et al. (1977) reported that meat heated
at 70 °C for 1 h developed rancidity rapidly. However, thiobarbituric acid
(TBA) values decreased when the cooking temperature was raised above
80 °C. According to Huang and Greene (1978), meat subjected to high
temperatures and/or long periods of heating developed lower TBA values,
than did samples subjected to lower temperature for a shorter period of time.
14 Natural Antioxidants: Applications in Foods of Animal Origin

This phenomenon was postulated to have resulted from AH substances


produced from browning reactions during the heating of meat. It has been
reported that Maillard reaction products possessed AH activity which can be
applied as natural AH in food products (Limsuwanmanee et al., 2014).
The effect of drying method on oxidative stability of microencapsulated
fish oil with ratio of 33/22, EPA: DHA which emulsified with four combina-
tions of matrices was studied by Anwar and Kunz (2011). The emulsions
were dried by spray granulation, spray drying, and freeze drying to produce
25% oil powders. A combination of 10% soybean soluble polysaccharide
and 65% octenyl succinic anhydride dried by spray granulation was the best
procedure for fish oil encapsulation due to its having a very low propanal
content. The microcapsules produced by spray granulation might be then
covered by successive layers resulting in multiple encapsulations which
provide maximum protection to the oil droplets. They also suggested that
combination of matrices, drying temperature, microcapsule morphology,
and processing time are among the most critical factors governing oxidative
stability of fish oil.

1.4.4 METAL IONS

Most biological and food studies of lipid peroxidation involve transition


metal ions (Fen+, Cun+, etc.), and it is generally accepted that iron is pivotal
in catalyzing oxidative changes in tissues (Gutteridge & Halliwell, 1990;
Kanner, 1994). It was found that iron concentration in cod muscle is very
low, with an average value of 6 ppm (Vareltzis et al., 2008). “Free” catalytic
iron in the muscle might be generated by the destruction of the heme and
release of iron. However, Undeland et al. (2003) suggested that the “free”
iron has a negligible effect on the oxidation of washed cod muscle system.
LMW iron added at a concentration of 23.2 µM did not induce oxidation of
washed minced cod lipids while hemoglobin at this concentration was very
pro-oxidative (Richards & Hultin, 2000).
Ladikos and Lougovois (1990) suggested that lipid oxidation is enhanced
by metals such as iron, cobalt, and copper which facilitate the transfer of
electrons leading to increased rates of free radical formation. The most
common way that metal ions enter food is via the water used and in some
instances via salt and spices (Taylor, 1987). The form of the metal is as
important as the amount of metal present (Taylor, 1987). Ferrous iron has
been shown to have greater pro-oxidant activity than ferric iron in cooked
uncured meats (Pearson et al., 1977). Low levels of ascorbic acid may
Mechanism of Oxidation in Foods of Animal Origin 15

increase the efficiency of iron as a catalyst for lipid oxidation, presumably


by regenerating the active ferrous form (Sato & Hegarty, 1971).
Kolakowska (2002) reported the effect of transition metals in lipid
oxidation. Hydroperoxides formed at the propagation stage of the free
radical oxidation, as well as those produced by photooxidation and enzyme-
catalyzed oxidation, can disintegrate and yield alcoxy, alkyl, and peroxyl
radicals, which reinitiate the oxidation of unsaturated fatty acid. Hydroper-
oxide decomposition may be triggered by temperature and/or light, but most
important in this respect is the activity of transition metals, mainly iron and
copper.
The Fe2+ ions are more reactive than Fe3+ ions and decompose hydrogen
peroxide over 100 times faster (Girotti, 1998). Iron occurs in human and
animal bodies, in up to 90% in a bound form in: hemoglobin, myoglobin,
cytochromes, the storage protein ferritin and hemosiderin, the iron trans-
port proteins, transferrins, and as prosthetic groups of enzymes. A small
amount of iron occurs in a “free” form, that is, primarily as LMW iron.
It complexes with organic phosphates, inorganic phosphates, amino acids
(histidine, glycine, and cysteine), and organic acids (citric acid) (Decker
& Hultin, 1992). LMW iron contributes between 2.5 and 3.8% to the total
iron content in muscle tissue of lamb, pork, and chicken. Dark muscles of
chicken, turkey, and mackerel contain twice as much LMW iron and more
ferritin than light muscles (Kanner, 1992). LMW iron acts as a catalyst.
Protein-bound Fe and Cu are minimally catalytic in oxidation. Ascorbate,
NAD(P)H, thiol compounds, reduced glutathione, cysteine, and protein thiol
groups release iron, which can catalyze the Fenton reaction. This occurs post
mortem during, for example, the storage of fish or turkey, but the amount of
reductants is then also decreased (Kanner, 1992; Hultin, 1994).

1.4.5 SODIUM CHLORIDE

Sodium chloride is able to catalyze lipid oxidation in muscle tissue


(Nambudiry, 1980; Love & Pearson, 1971). Alternatively, the Na+ may
replace iron from a cellular complex via an ion exchange reaction (Kanner
& Kinsella, 1983). The displaced iron may then participate in the initiation
of lipid oxidation. It is most likely that meat or meat products containing salt
such as surimi and cured meat are susceptible to lipid oxidation (Chaijan,
2008).
Ladikos and Lougovois (1990) reported that sodium chloride induces
rancidity in freezer-stored, cooked, cured meat, in cured pork and in raw
16 Natural Antioxidants: Applications in Foods of Animal Origin

and cooked beef, both during cooking and subsequent storage. Chaijan
(2011) reported that salting caused an increase in lipid oxidation in tilapia
muscle. The effect of sodium chloride on fat oxidation depends on the level
of free moisture in the system (Pearson et al., 1977). According to Love and
Pearson (1971), the oxidative effect of sodium chloride may be attributed to
the action of the reactive chloride ion on lipids, or to a modification of the
heme proteins catalyzing lipid oxidation.

1.4.6 MUSCLE pH

Several studies have shown that lipid oxidation in muscle foods increases
with a decrease in pH (Chen & Waimaleongora, 1981; Ogden et al., 1995;
Tichivangana & Morrissey, 1985). Acid injection and marination have been
used as a practice to improve the water-holding capacity and tenderness of
muscle foods (Ke et al., 2009). In general, the lower the pH values, the
stronger the prooxidant effect. Rapid formation of the oxidized forms of
heme proteins (methemoglobin and metmyoglobin) might contribute to the
rapid oxidation of muscle at acidic pH values (Chaijan et al., 2007). An
increase in iron solubility with decreasing pH could also increase oxida-
tion rates. Vareltzis and Hutin (2007) observed that exposing hemoglobin
and microsomes to low pH affected lipid oxidation rates. When isolated
membranes alone were exposed to low pH, they were less susceptible to
hemoglobin-promoted lipid oxidation. Exposure of cod hemoglobin to pH
3 decreased its prooxidant activity compared to untreated cod hemoglobin
(Vareltzis & Hultin, 2007). However, if cod hemoglobin and isolated cod
microsomes were exposed to low pH together, oxidation was promoted
(Vareltzis & Hultin, 2007).
Hemoglobin-mediated lipid oxidation can be accelerated by reduction in
pH and could be due to enhanced autoxidation of hemoglobin at reduced pH.
Hemoglobin from different fish is known to promote lipid oxidation in fish
muscle differently (Maqsood et al., 2012). Maqsood and Benjakul (2011a)
monitored the lipid oxidation of washed Asian seabass mince added with
hemoglobin from various fish at different pH (6, 6.5, and 7) during 10 days
of iced storage. Hemoglobins accelerated lipid oxidation more effectively
at pH 6, compared with pH 6.5 and 7 as indicated by the higher PV and
TBARS.
Mechanism of Oxidation in Foods of Animal Origin 17

1.4.7 PARTICLE SIZE REDUCTION AND TUMBLING

Ladikos and Lougovois (1990) postulated that any process causing disruption
of the muscle membrane system, such as grinding, cooking, and deboning,
results in exposure of the labile lipid components to oxygen, and thus accel-
erate development of oxidative rancidity. Destruction of the extremely well
organized structure of living animal cells will bring together lipids, oxidation
catalysts, and enzymes responsible for lipid oxidation. Pearson et al. (1977)
suggested that chopping and emulsification are at least as likely to cause
WOF as grinding or mincing of samples. Dawson and Gartner (1983) attrib-
uted the high oxidative potential of mechanically deboned poultry to the
extreme stress and aeration during the process and the compositional nature
(bone marrow, heme, and lipids) of the product; TBA values increase most
rapidly with decreasing particle sizes, as the latter are related to greater cell
disruption. On the other hand, comminuted beef has a storage life similar to
that of intact pork, despite the differences in fatty acid composition (Enser,
1987).
The mechanical force of tumbling can break the structure of the cell and
organelle membranes which could lead to an exposure of phospholipids
to cellular prooxidants (e.g., iron and heme proteins) or free radicals. The
tumbling process has also been found to promote lipid oxidation in beef
bottom round (Cheng & Ockerman, 2003). However, inhibition of lipid
oxidation by the citric acid marination could be due the removal of prooxi-
dants such as heme proteins by the marination/tumbling procedure. Alter-
nately, inhibition of lipid oxidation could be due to the presence of citric acid
since these molecules are strong metal chelators (Ke et al., 2009).

1.4.8 HIGH PRESSURE PROCESSING

High pressure (HP) processing has been shown to initiate lipid oxidation in
fresh meat, especially a threshold pressure around 500 MPa seems to exist,
which can lead to reduced quality and shelf life (Bolumar et al., 2014). Two
mechanisms have been proposed to explain the pressure-induced lipid oxida-
tion: (a) increased accessibility of iron from hemoproteins and (b) membrane
disruption. Several studies have observed that the addition of ethylenediami-
netetraacetic acid (EDTA), which can chelate metal ions like iron, can be
correlated with a reduction of the lipid oxidation in meat processed by HP,
which suggests that transition metal ion catalysis is the major factor under-
lying the increased lipid oxidation (Beltran et al., 2004; Ma et al., 2007).
18 Natural Antioxidants: Applications in Foods of Animal Origin

However, iron release was not observed after HP treatment of chicken breast
(Orlien et al., 2000). In the same study, it was also concluded that the cata-
lytic activity of metmyoglobin did not increase during HP-treatment, indi-
cating that pressure-induced changes of the metmyoglobin conformation that
facilitates the access to the catalytic heme group did not take place (Orlien
et al., 2000). So far, the role of iron in the induction of lipid oxidation of
meats treated by HP is not well established. Membrane disruption facilitates
contact between unsaturated lipids from the membrane and enzymes and
catalysts like heme, nonheme iron as well as other metal cations and, thus,
may contribute to the initiation of lipid oxidation. Recently, the formation of
free radicals during HP has been proposed as a possible mechanism behind
the induction of lipid oxidation in HP processed meats (Bolumar et al., 2011).
Radical formation in the aqueous and lipid phases from HP-treated meat was
first reported by Mariutti et al. (2008), and further studied by Bolumar et
al. (2012), who characterized the kinetics of the formation of radicals in
chicken meat during the application of different HP treatments. It was found
that there is a threshold for the formation of radicals under HP conditions
at 400 MPa at 25 °C and 500 MPa at 5 °C. The chemical mechanism which
leads to the formation of radicals in meats by HP was intensively described
by Bolumar et al. (2014) using electron spin resonance detection. The higher
level of spin adducts was observed in the beef loin than in the chicken breast
with radicals forming in the sarcoplasmic and myofibrillar fractions as well
as in the non-soluble protein fraction due to the HP treatment, indicating
that other radicals than iron-derived radicals were formed, and most likely
protein derived radicals. The addition of EDTA reduced the radical forma-
tion suggesting iron-species (protein-bound or free) catalyzes the formation
of radicals when meat systems are submitted to HP.

1.5 MYOGLOBIN OXIDATION IN FOODS OF ANIMAL ORIGIN

Myoglobin is a globular heme protein found in the muscle of meat-


producing animals (Faustman & Phillips, 2001). It has been known to be
a major contributor to the color of muscle, depending upon its redox state
and concentration. Myoglobin concentration is affected by both genetics and
environment (Giddings, 1974; Livingston & Brown, 1981; Faustman et al.,
1996). The content of myoglobin in skeletal muscle will vary depending on
the metabolic profile of the muscle, animal species, and age of the animal
(Chaijan, 2008). Chaijan et al. (2004) reported that lipid and myoglobin
contents were higher in dark muscle than in ordinary muscle of both sardine
Mechanism of Oxidation in Foods of Animal Origin 19

and mackerel, and higher contents of both constituents were found in sardine
muscle than mackerel muscle.
Myoglobin is made up of a single polypeptide chain, globin, consisting
of 153 amino acids and a prosthetic heme group, an iron (II) protoporphyrin-
IX complex (Hayashi et al., 1998; Pegg & Shahidi, 1997). This heme group
gives myoglobin and its derivatives their distinctive color (Dunn et al., 1999;
Pegg & Shahidi, 1997). The structure and chemistry of the iron atom have
an impact on the reactions and color changes that myoglobin undergoes
(Livingston & Brown, 1981). The oxidation of ferrous-oxymyoglobin (Fe2+)
to ferric-metmyoglobin (Fe3+) is responsible for discoloration of meat during
storage. Ferrous iron (Fe2+) can react with molecular oxygen to produce
superoxide anion (O2•-) with concomitant oxidation to ferric iron (Fe3+).
Hydrogen peroxide (H2O2), which may be produced by dismutation of O2•-,
can react with Fe2+ to produce hydroxyl radical (OH•) (Hultin, 1992). This
reaction termed as Fenton reaction is the principal mechanism for myoglobin
oxidation (Fig. 1.2).

Fe2+ + O2 Fe3+ + O2•-

2O2•- + 2H+ H2O2 + O2

Fe2+ + H2O2 Fe3+ + OH- + OH•


FIGURE 1.2 Reactive oxygen species generated by the Fenton reaction.

In general, fish myoglobins are more readily oxidized than the mamma-
lian counterpart (Haard, 1992). Discoloration of tuna meat during frozen
storage is associated with the formation of metmyoglobin (Haard, 1992).
This phenomenon can be influenced by many factors such as pH, tempera-
ture, ionic strength, and oxygen consumption reaction (Renerre & Labas,
1987). Metmyoglobin formation is positively correlated with lipid oxida-
tion (Chan et al., 1997a, 1997b; Lee et al., 2003a, 2003b). Benjakul and
Bauer (2001) suggested that the freeze-thaw process caused damage of cell
and heme-proteins, resulting in the release of prooxidants. Haard (1992)
also reported that fish myoglobins are at least 2.5 times more sensitive to
autoxidation than mammalian myoglobins. Autoxidation of myoglobin
becomes greater as temperature increased and pH decreased (Livingston
et al., 1981; Chaijan et al., 2007). Chaijan et al. (2007) demonstrated that
sardine myoglobin was prone to oxidation and denaturation at tempera-
ture above 40 °C and at very acidic or alkaline pHs as evidenced by the
20 Natural Antioxidants: Applications in Foods of Animal Origin

formation of metmyoglobin, the changes in tryptophan fluorescence inten-


sity as well as the disappearance of Soret absorption. Furthermore, the rate
of myoglobin autoxidation was related to oxygen concentration (Brown
& Mebine, 1969). Atmospheres enriched in carbon dioxide (CO2) are effec-
tive in delaying spoilage of meat; however, one problem is that CO2 can
promote the oxidation of oxymyoglobin to metmyoglobin, thereby causing
the discoloration (Haard, 1992). Post-harvest discoloration of fish muscle
has been reviewed by Chaijan and Panpipat (2009).
It has been suggested that myoglobin has a close relationship with
lipid oxidation (O’Grady et al., 2001; Ohshima et al., 1988). Besides the
unpleasant color, the oxidation of myoglobin is the main cause in the devel-
opment of the undesirable odor during ice storage of fish muscle. Lee et al.
(2003a) reported that surface metmyoglobin accumulation and lipid oxida-
tion of refrigerated tuna (Thunnus albacares) steaks increased during six
days of storage, leading to discoloration and lowered odor acceptability.
The total lipid hydroperoxide content and TBARS of the yellowtail (Seriola
quinqueradiata) dark muscle were higher than those of the ordinary muscle
during two days of ice storage. Those changes were accompanied with the
increasing intensity of fishy, spoiled, and rancid off-odor smells as well as
increasing metmyoglobin formation. However, no correlation was found
between the content of total lipid hydroperoxide and the odor intensities
in ordinary muscle (Sohn et al., 2005). It is believed that the formation of
metmyoglobin by the oxidation of myoglobin predominantly in dark muscle
accelerates lipid oxidation and leads to the generation of greater amounts
of hydroperoxide. Thus, the lipid oxidation associated with metmyoglobin
formation may have caused the development of the rancid off-odor and fishy
smell in dark muscle. For ordinary muscle of yellowtail which contained
a low level of metmyoglobin, the influence of myoglobin oxidation on the
development of rancid off-odor appeared to be insignificant (Sohn et al.,
2005). The suppression of myoglobin oxidation will in turn decrease lipid
oxidation and off-odor development of muscle foods.

1.6 INTERRELATIONSHIP BETWEEN LIPID OXIDATION AND


MYOGLOBIN OXIDATION IN FOODS OF ANIMAL ORIGIN

The heme proteins including hemoglobin and myoglobin are effective


promoters of lipid oxidation (Love, 1983; Han et al., 1994). Myoglobin
consists of a globin portion plus a porphyrin heme, the latter containing an
iron atom coordinated inside the heme ring (Grunwald & Richards, 2006a,
Mechanism of Oxidation in Foods of Animal Origin 21

2006b). Heme is the nomenclature used to describe the porphyrin ring


containing ferrous (Fe2+) iron, while hemin describes the porphyrin ring
containing ferric (Fe3+) iron. Ferrous myoglobin are typically either liganded
with O2 or no ligand is present (e.g., deoxymyoglobin). The problem in under-
standing the pathway by which heme proteins promote lipid oxidation is that
heme protein autoxidation, ferryl radical formation, heme dissociation, heme
destruction, and iron release can all occur in a very short time sequence and
simultaneously so that the most relevant step related to lipid oxidation is
obscured ring (Grunwald & Richards, 2006a, 2006b). Heme-initiated lipid
oxidation, especially myoglobin, has been extensively reported in meats
(Ledward, 1987; Love & Pearson, 1974; Richards & Hultin, 2002). The
interrelationship between lipid and myoglobin oxidations in muscle foods
has been reported by Chaijan (2008). Ohshima et al. (1988) proposed that the
lipid oxidation in fish muscle was promoted by autoxidation of myoglobin.
Moreover, O’Grady et al. (2001) reported a relationship between oxymyo-
globin oxidation and lipid oxidation in bovine muscle. There are numerous
potential mechanisms by which myoglobin can promote lipid oxidation in
muscle foods. The process by which ferrous myoglobin (or hemoglobin) is
converted to ferric metmyoglobin is called autoxidation. Superoxide anion
radical (O2•−) or •OOH is liberated in this process depending on whether
deoxy or oxy heme protein undergoes autoxidation (Brantley et al., 1993).
O2•− and •OOH can readily be converted to hydrogen peroxide (H2O2), which
enhances the ability of heme proteins to promote lipid oxidation. Moreover,
metmyoglobin can react with H2O2 or lipid hydroperoxides to generate ferryl
heme protein radicals, which can abstract hydrogen from PUFA and hence
initiate lipid oxidation. Alternatively, displaced hemin or released iron can
stimulate lipid oxidation. Metmyoglobin is an effective prooxidant at acidic
pH and in the presence of hydroperoxides. Morey et al. (1973) found that
H2O2 acting as an oxidizing agent caused changes in the oxidation state of the
iron in myoglobin. The reaction between H2O2 and metmyoglobin results in
the formation of red pigment, ferrylmyoglobin (MbFe(IV)=O). Under physi-
ological conditions (pH 7.4), ferrylmyoglobin is a strong prooxidant, which
is able to abstract a hydrogen atom from fatty acids with subsequent stereo-
specific addition of oxygen (Rao et al., 1994). The prooxidative activity of
ferrylmyoglobin is independent of pH and of lipid concentration (Chan et
al., 1997a, 1997b; Kanner et al., 1987).
The concentration of ferrous iron and its ability to be active in the lipid
oxidation reaction will be a key factor causing differences in lipid oxida-
tion among species. In general, dark meats tend to have more reactive iron.
Chaijan et al. (2004) reported that lipid and myoglobin contents were higher
22 Natural Antioxidants: Applications in Foods of Animal Origin

in dark muscle than in ordinary muscle of both sardine and mackerel. Satu-
ration of red color in meat was directly related to myoglobin concentration
(Faustman et al., 1992). Other constituents of meat including enzymatic and
non-enzymatic reducing systems can accelerate oxidation by converting iron
from the inactive ferric form to the active ferrous state (Foegeding et al.,
1996). Changes of PV, conjugated diene (CD) and TBARS in sardine muscle
indicated that lipid oxidation occurred throughout 15 days of iced storage.
Apart from a plenty of unsaturated fatty acids, heme protein as well as reac-
tive iron in the muscle might contribute to the accelerated oxidation (Chaijan
et al., 2006).
Under fluctuating oxygen supply and pH decrease of post mortem system,
the heme pigments like hemoglobin and myoglobin become catalytic in
lipid peroxidation by mechanisms involving both one- and two-electron
transfer processes, which are different from mechanisms for lipid oxida-
tion by the non-heme-iron LOX (Carlsen et al., 2005). Reeder and Wilson
(1998) suggested that myoglobin plays a role of photosensitizer which may
be responsible for the initial formation of lipid hydroperoxides and increase
the rate of oxygen uptake of fish oil via a photosensitized oxidation.

1.6.1 ROLE OF DEOXYMYOGLOBIN IN LIPID OXIDATION

The physiologically active myoglobin species are the purple high-spin iron
(II) myoglobin (deoxymyoglobin), which has the sixth coordination site
of the heme iron vacant, and the bright cherry-red low-spin oxy-iron (II)
myoglobin (oxymyoglobin), which bind a molecule of oxygen at the sixth
coordination of the heme iron, due to their high affinity for oxygen (Baron
& Andersen, 2002; Faustman et al., 1999; Gorelik & Kanner, 2001). Distur-
bance of the globin structure can result in binding of the unusual ligands
(e.g., the distal histidine in the heme cavity, exogenous amino acids as histi-
dine and methionine, or a hydroxyl group) at the sixth coordination of the
heme iron and induce the formation of a low-spin iron (II) species, known
as hemochromes. Hemochromes in its oxidation state II can be found either
reversible (binding to the imidazole group of the distal histidine or hydroxyl
ion) or irreversible (binding to the imidazole group of free histidine) (Baron
& Andersen, 2002).
The study regarding prooxidative activity of deoxymyoglobin in biolog-
ical system including muscle foods is scarce (Baron & Andersen, 2002).
This is mainly due to the fact that deoxymyoglobin initiated lipid oxidation
demands strictly anaerobic condition; to exclude oxymyoglobin initiated
Mechanism of Oxidation in Foods of Animal Origin 23

lipid oxidation and the subsequent propagation of lipid oxidation. However,


Richards and Dettmann (2003) postulated that perch and trout deoxyhemo-
globin could stimulate lipid oxidation in washed cod muscle during storage
at 4 °C as evidenced by the formation of lipid peroxides and TBARS. A more
rapid formation of methemoglobin from deoxygenated molecules, deoxyhe-
moglobin, likely increases the lipid oxidation (Richards et al., 2002).

1.6.2 ROLE OF OXYMYOGLOBIN IN LIPID OXIDATION

Oxymyoglobin oxidation and lipid oxidation are coupled (Yin & Faustman,
1993). A high correlation between oxymyoglobin oxidation and lipid oxida-
tion both in microsomes and liposomes was reported by Yin and Faustman
(1993, 1994) and O’Grady et al. (2001). Lipid oxidation in fresh meat is
influenced by the oxidation of oxymyoglobin since the oxymyoglobin
oxidation results in production of two species known as the prooxidants,
namely metmyoglobin and H2O2 (Chan et al., 1997a, 1997b). It has been
proposed that O2•- and H2O2 are produced during oxidation of oxymyoglobin
to metmyoglobin (Gotoh & Shikama, 1976). O2•- can further react with H2O2
and Fe3+ via the Fenton reaction to produce OH• and facilitate lipid oxida-
tion (Chan et al., 1997a, 1997b). The prooxidative effect of oxymyoglobin
toward lipid oxidation was concentration-dependent (Chan et al., 1997a,
1997b). Additionally, H2O2 can react with metmyoglobin to form a prooxi-
dative ferrylmyoglobin radical (Decker & Hultin, 1992). Kanner and Harel
(1985) reported that H2O2-activated metmyoglobin caused the rapid oxida-
tion of poultry skeletal muscle microsomes. Moreover, reactive oxygen
species including superoxide, hydroperoxyl radical (HOO•), and H2O2, orig-
inated by the autoxidation of oxymyoglobin (Kruger-Ohlsen & Skibsted,
1997), can cause damage to muscle lipids via oxidation reaction (Skulachev,
1996; Hultin & Kelleher, 2000).
Prooxidative activity of oxymyoglobin in a myoglobin-liposome system
was concentration-dependent and showed the higher activity than did
metmyoglobin. The added sperm whale myoglobin was found to promote
lipid oxidation in washed cod by which lipid oxidation occurred more rapidly
at pH 5.7 compared to pH 6.3 (Grunwald & Richards, 2006b). Prooxidative
activity of oxymyoglobin is difficult to assess because of continuous autoxi-
dation of oxymyoglobin to metmyoglobin. Chan et al. (1997a) and Yin and
Faustman (1993) reported a high correlation between oxymyoglobin oxida-
tion and lipid oxidation both in microsomes and liposomes system. The role
of oxymyoglobin oxidation in lipid oxidation was reported by Chan et al.
24 Natural Antioxidants: Applications in Foods of Animal Origin

(1997b). Hogg et al. (1994) showed that oxymyoglobin can promote oxida-
tive modification of low-density lipoprotein. Galaris et al. (1990) showed
visible absorption spectral change of oxymyoglobin upon incubation with
linoleic acid at physiological pH. This could be attributed to the formation
of the noncatalytic low-spin myoglobin derivative, hemochrome (Akhrem
et al., 1989).

1.6.3 ROLE OF METMYOGLOBIN IN LIPID OXIDATION

High-spin iron (III) myoglobin, commonly known as metmyoglobin, binds


a molecule of water at the sixth coordination site of the heme iron (Pegg
& Shahidi, 1997). Like hemochromes, the low-spin iron (III) myoglobin
species known as hemichromes can be formed by disturbance of the
globin structure. Hemichrome formation is either reversible or irreversible
depending on the type of ligand at the sixth coordination site of the iron and
the extent of globin denaturation. Hemichrome formation from iron (III)
myoglobin is the intermediate step in the heat denaturation of myoglobin
in muscle foods (Baron & Andersen, 2002). Post mortem process, espe-
cially the pH fall, continuously inactivate the reductive enzyme systems and
stimulate acid-catalyzed autoxidation of the iron (II) states to the iron (III)
state of myoglobin, resulting in the accumulation of metmyoglobin in meats
(George & Stratmann, 1954; Gotoh & Shikama, 1976).
Formation of metmyoglobin is highly correlated with lipid oxidation in
muscle foods (Andersen & Skibsted, 1991). Baron et al. (1997) found that
metmyoglobin is an effective prooxidant at acidic pH and in the presence
of hydroperoxides. In contrast, at physiological pH and in the presence of
lipids, metmyoglobin can undergo a rapid neutralization due to formation of
the noncatalytic heme pigment. However, further denaturation of the heme
proteins due to a high lipophilic environment may result in heme release
or further exposure of the heme group to the surrounding lipids, thereby
inducing lipid peroxidation (Baron & Andersen, 2002). Metmyoglobin acts
as a prooxidant in raw fish more effectively than in raw turkey, chicken,
pork, beef, and lamb (Livingston et al., 1981).
In addition, the lipid to heme protein ratio is an important factor
affecting the prooxidative activity of heme proteins (Kendrick & Watts,
1969). At lower linoleate/heme protein ratios, heme proteins become inef-
fective initiators of lipid oxidation (Nakamura & Nishida, 1971). The
mechanism responsible for the inhibition of lipid oxidation at low lino-
leate/heme protein ratios has been proposed. The fatty acid anions bind
Mechanism of Oxidation in Foods of Animal Origin 25

reversibly to metmyoglobin, resulting in a spin transition, to yield the


low-spin metmyoglobin derivative which is not prooxidative (Baron et
al., 1998). At high linoleate-to-heme ratios, metmyoglobin immediately
denatures and results in exposure or release of the heme group to the envi-
ronment that instantly initiates hematin-induced lipid peroxidation in the
system (Baron & Andersen, 2002). The result of Grunwald and Richards
(2006a) also confirmed that sperm whale metmyoglobin caused a more
rapid formation of lipid peroxides and TBARS in washed cod muscle as
compared to ferrous myoglobin during 2 °C storage.

1.6.4 ROLE OF FERRYLMYOGLOBIN IN LIPID OXIDATION

The reaction between H2O2 and metmyoglobin results in the formation of a


red pigment, ferrylmyoglobin (Baron & Andersen, 2002). During this inter-
action, the production of free radicals occurs in the globin part of the heme
protein. H2O2 activation of metmyoglobin is a necessary step in the conver-
sion of metmyoglobin to a prooxidant (Kanner & Harel, 1985). Interaction
between metmyoglobin and H2O2 is a complex mechanism, resulting in the
generation of two distinct hypervalent myoglobin species, perferrylmyo-
globin (•MbFe(IV)=O) and ferrylmyoglobin (MbFe(IV)=O) (Davies, 1990,
1991) as follows:

Metmyoglobin + H2O2 •
MbFe(IV)=O MbFe(IV)=O

Perferrylmyoglobin is a transient species with a very short half-life and


autoreduces rapidly to the more stable ferrylmyoglobin (Baron & Andersen,
2002). Ferrylmyoglobin is a relatively stable species, which is slowly
reduced back to metmyoglobin at physiological pH but with an increasing
rate at decreasing pH due to an acid-catalyzed process (Mikkelsen & Skib-
sted, 1995). Perferrylmyoglobin can effectively transfer its radical to other
proteins and subsequently induces lipid oxidation (Baron & Andersen,
2002). However, the ability of perferrylmyoglobin to initiate lipid oxida-
tion by abstracting a hydrogen atom from fatty acids (LH) was suggested by
Kanner and Harel (1985) as shown in the following reaction:

MbFe(IV)=O + LH MbFe(IV)=O + L• + H+

Ferrylmyoglobin is responsible for the oxidation of a variety of substrates


(Baron & Andersen, 2002). Under conditions similar to those found in
26 Natural Antioxidants: Applications in Foods of Animal Origin

muscle foods, ferrylmyoglobin is able to initiate lipid oxidation (Hogg et


al., 1994). However, under the conditions found in fresh meat (pH 5.5–5.8),
ferrylmyoglobin autoreduces rapidly to metmyoglobin. Nevertheless, under
physiological conditions (pH 7.4), ferrylmyoglobin is a strong prooxidant,
which is able to abstract a hydrogen atom from fatty acids with subsequent
stereospecific addition of oxygen (Rao et al., 1994). The prooxidative
activity of ferrylmyoglobin is independent of pH and of lipid concentration
(Baron & Andersen, 2002). Therefore, ferrylmyoglobin is expected to be
an effective prooxidant under the conditions found in muscle food, as well
as under physiological conditions. However, ferrylmyoglobin formation in
muscle tissues is determined by hydrogen peroxide and lipid hydroperoxide
production. Its potential to oxidize lipids depends on the concentration of
reducing agents and their compartmentalization in the muscle cells (Baron
& Andersen, 2002).
The interrelationship between myoglobin oxidation, lipid oxidation,
and discoloration in oxeye scad fish during ice storage has been reported
by Wongwichian et al. (2015). The myoglobin autoxidation rate, hydrogen
peroxide, and ferrylmyoglobin concentrations increased with increasing
storage time. The CD and PV of oxeye scad lipids tended to stabilize during
the initial phase of storage, increased in the differentiation phase and had
declined at the end of storage. However, TBARS increased markedly.
Overall, lipid and myoglobin oxidations in oxeye scad occurred in a concur-
rent manner and each process appeared to enhance the other.
Heme, hematin and hemin are normally used interchangeably to describe
the existence of non-protein bound heme-iron (or “free heme iron”). Heme
in solution is mainly found as hematin (ferriprotoporphyrin hydroxide).
Hemin is ferriprotoporphyrin chloride which readily converts to hematin
in aqueous solution and accordingly the term hematin should be used for
non-protein bound heme-iron (Carlsen et al., 2005). Grunwald and Richards
(2006b) suggested that sperm whale myoglobin having a more rapid hemin
loss rate possessed a more effective prooxidative activity than did modified
myoglobin with high hemin affinity. It was found that hemin concentrations
in mackerel light muscle increased around 3-fold during ice storage (Decker
& Hultin, 1990). Following release of hemin from the globin, hemin is
proposed to intercalate within phospholipids membranes due to hydrophobic
attractions. Also the propionate groups of hemin can bind with phospho-
lipid headgroup amines by electrostatic interactions (Cannon et al., 1984).
Hemin can react with lipid hydroperoxide to form alkoxyl radical and ferryl-
hydroxo complex (Dix & Marnett, 1985). Ferryl-hydroxo complex can react
Mechanism of Oxidation in Foods of Animal Origin 27

with another lipid hydroperoxide to form a peroxyl radical and regenerate


hemin:

hemin(3+) + LOOH LO• + hemin(4+)-OH


hemin(4+)-OH + LOOH LOO• + hemin(3+) + H2O

Alkoxyl and peroxyl radicals are capable of abstracting a hydrogen atom


from a PUFA which will stimulate the lipid oxidation processes (Grunwald
& Richards, 2006b).
Recently, a new heme protein determination method for fish muscle
overcoming such extractability problems faced by previous reported
methods was developed by Chaijan and Undeland (2015). The principle was
to homogenize and heat samples in an SDS-containing phosphate buffer to
dissolve major muscle components and convert ferrous/ferric heme proteins
to hemichromes with a unique absorption peak at 535 nm.

1.7 INTERACTION BETWEEN LIPID OXIDATION PRODUCTS AND


MYOGLOBIN

Lipid oxidation generates a wide range of secondary aldehyde products,


which are predominantly n-alkanals, trans-2-alkenals, 4-hydroxy-trans-
2-alkenals, and malondialdehyde (Lynch & Faustman, 2000). Lynch et al.
(2001) demonstrated that propional, pentenal, hexanal, and 4-hydroxynon-
enal (4-HNE) were the primary aldehydes formed during lipid oxidation
in stored ground beef at 4 °C. The aldehyde products are more stable than
free radical species and readily diffuse into the cellular media, where they
may exert toxicological effects by reacting with critical biomolecules in vivo
(Esterbauer et al., 1991). Aldehydes produced during lipid oxidation can
form adducts with proteins and this may have an impact on protein stability
and functionality as well as the color stability of meat. Aldehyde products
can alter myoglobin stability (Lynch & Faustman, 2000). Covalent modi-
fication of equine, bovine, porcine and tuna myoglobin by 4-hydroxynon-
enal (4-HNE) has been demonstrated (Faustman et al., 1999; Phillips et al.,
2001a, 2001b; Lee et al., 2003a, 2003b). Lynch and Faustman (2000) also
determined the effect of aldehydic lipid oxidation products on oxymyo-
globin oxidation, metmyoglobin reduction and the catalytic activity of
metmyoglobin as a lipid prooxidant in vitro. Metmyoglobin formation was
greater in the presence of α,β-unsaturated aldehydes than their saturated
28 Natural Antioxidants: Applications in Foods of Animal Origin

counterparts of equivalent carbon chain length (Faustman et al., 1999). The


covalent attachment of aldehydes to oxymyoglobin rendered oxymyoglobin
more susceptible to oxidation (Faustman et al., 1998). Alderton et al. (2003)
studied the effect of 4-HNE on bovine metmyoglobin formation. Increased
metmyoglobin formation was found in the presence of 4-HNE. Similar
results were observed by Faustman et al. (1999) during the incubation of
4-HNE with equine oxymyoglobin and by Lee et al. (2003a, 2003b) with
porcine and tuna oxymyoglobin. The covalent binding of α,β-unsaturated
aldehydes to oxymyoglobin at key amino acid residues may subsequently
lead to alter tertiary structure of the protein and increases susceptibility to
oxidation. This would result in a loss of physiological activity and the brown
discoloration in fresh meat (Alderton et al., 2003). Alderton et al. (2003) and
Lee et al. (2003a) demonstrated that 4-HNE covalently attached to bovine
and porcine myoglobin, respectively. The liquid chromatography-mass
spectrometry (LC-MS) spectra revealed the covalent binding of up to three
molecules of 4-HNE to bovine myoglobin and a di-adduct formed under the
reaction of 4-HNE with porcine myoglobin.

1.8 CONCLUSION

Chemical constituents in foods of animal origin including lipid, protein,


pigment, and vitamin in muscle tissue are susceptible to oxidative reactions
resulting in the loss of quality, including discoloration, development of off-
flavors, loss of nutrients, textural changes, and progression of spoilage and/
or pathogenicity. Among such compositions, lipid and heme proteins espe-
cially myoglobin are prone to oxidation. Lipid oxidation and myoglobin
oxidation in meat are coupled and both reactions appear capable of influ-
encing each other. The oxidation of oxymyoglobin results in the production
of metmyoglobin and H2O2 necessary to induce lipid oxidation. On the other
hand, aldehyde lipid oxidation products alter myoglobin redox stability,
resulting in the promoted oxidation of oxymyoglobin and the formation of
adduct with myoglobin through covalent modification. Thus, studies of the
relationship between lipid oxidation and myoglobin oxidation processes in
muscle foods are important in understanding reactions and mechanisms that
may affect the quality and acceptability and could be useful in minimizing
lipid oxidation of meats and meat products during handling, processing, and
storage.
Mechanism of Oxidation in Foods of Animal Origin 29

KEYWORDS

• mechanism
• lipid oxidation
• myoglobin
• muscle foods
• quality

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CHAPTER 2

NATURAL ANTIOXIDANTS:
OCCURRENCE AND THEIR ROLE IN
FOOD PRESERVATION
AJIT SINGH BHATNAGAR and REWA KULSHRESTHA*
Department of Food Processing and Technology, Bilaspur University,
Bilaspur, Chhattisgarh, India
*Corresponding author. E-mail: [email protected]

CONTENTS

Abstract ......................................................................................................41
2.1 Antioxidants: What, Why, and How ................................................41
2.2 Antioxidants Versus Free Radicals ..................................................43
2.3 Synthetic Antioxidants Versus Natural Antioxidants .......................44
2.4 Natural Antioxidants: Dynamics and Mechanism ...........................45
2.5 Role of Lipid Fractions in Radical Scavenging and
Antioxidant Activity ........................................................................46
2.6 Role of Fatty Acids in Oxidative Stability .......................................47
2.7 Vitamin E or Tocols (Tocopherols and Tocotrienols) ......................48
2.8 Vitamin K1 (Phylloquinone)............................................................54
2.9 Vitamin C (Ascorbic Acid) ..............................................................55
2.10 Phytosterols......................................................................................60
2.11 Phenolic Compounds .......................................................................63
2.12 Carotenoids ......................................................................................66
2.13 Phytic Acid.......................................................................................69
2.14 Sesame Lignans ...............................................................................73
40 Natural Antioxidants: Applications in Foods of Animal Origin

2.15 Oryzanol ..........................................................................................76


2.16 Applications of Natural Antioxidants/Extracts in
Food Products Preservation .............................................................80
2.17 Regulatory Status of Natural Antioxidant Extracts,
Concentrates, and Resins .................................................................83
2.18 Conclusion .......................................................................................84
Keywords ...................................................................................................85
References ..................................................................................................85
Natural Antioxidants: Occurrence and Their Role in Food Preservation 41

ABSTRACT

Antioxidants neutralize or inhibit free radicals by preventive and radical


scavenging modes. Free radicals are unstable reactive molecules; rapidly
attack the molecules in nearby cells leading to aging of body. The repair
process involves scavenging free radicals by antioxidant compounds. Anti-
oxidants have dual role: shelf-life prolongation and combating oxidative
stress. Consumers’ concerns regarding the bio-safety of synthetic antiox-
idants have pushed the food industry to seek natural alternatives such as
ascorbic acid (AA), tocols, carotenoids, phenolics, oryzanol (OZ), and so
forth. In contrast to animal foods, foods of vegetable origin usually contain
natural antioxidants, such as tocopherols, carotenoids, or flavonoids in suffi-
cient amounts due to higher degree of unsaturation. Natural antioxidants
provide preservative action in various foods, namely cereal based breakfast
foods, baked foods like bread and crackers, dried products, and processed
fruit products. This chapter provides full insight on different naturally occur-
ring antioxidants’ structure, levels, and effectiveness as food preservative.

2.1 ANTIOXIDANTS: WHAT, WHY, AND HOW

Antioxidants are compounds which neutralize free radicals or inhibit free


radicals. Free radicals are unstable molecules with an unpaired electron
and highly reactive. To become stable, they take electron from other mole-
cules/cells and in the process eventually damage the DNA and cause aging,
degenerative diseases, and may also lead to cancer. Free radicals can be
classified as reactive oxygen species (ROS) and reactive nitrogen species
(RNS). Few examples of ROS are alkoxyl radicals (RO•), peroxyl radicals
(ROO•), hydroxyl radical (HO•), and superoxide anion radical (O2−•) while
examples of RNS would be nitric oxide radical (NO•) and nitrogen dioxide
radical (NO2•). Potential sources of free radicals could be ultraviolet (UV)
or ionizing radiations, metabolic processes, inflammatory reactions, air-
pollution, and tobacco (smoking or chewing). ROS are formed in vivo both
usefully and “accidentally.” Their formation increases in all human disease,
and sometimes makes a significant contribution to disease severity (Halli-
well, 1995). Free radicals damage the cell molecules like DNA, proteins,
and lipids, potentially causing a variety of disorders, including diabetes
mellitus, hypertension, cancer, Alzheimer, and aging of body. Free radicals
like ROS and RNS are generally produced due to the oxidative stress in the
body. The oxidative stress leads to over 200 disorders including aging of
42 Natural Antioxidants: Applications in Foods of Animal Origin

body, degenerative disorders, and cancer. Oxidative stress can cause single/
multi-organ disorders/diseases including brain disorders like Alzheimer,
Parkinson, obsessive–compulsive disorder (OCD), attention deficit hyperac-
tivity disorder (ADHD), autism, migraine, stroke, trauma, and cancer; lung
disorders like asthma, chronic obstructive pulmonary disease (COPD), aller-
gies, acute respiratory distress syndrome (ARDS), and cancer; eye disor-
ders like macular or retinal degeneration and cataract; heart disorders like
coronary heart diseases (CHD), cardiac fibrosis, hypertension, ischemia,
and myocardial infarction; kidney disorders like chronic kidney diseases,
renal graft, and nephritis; bone and joint disorders like rheumatism, osteo-
arthritis, and psoriasis; blood vessel disorders like restenosis, atheroscle-
rosis, endothelial dysfunction, and hypertension; skin disorders like skin
aging, sunburn, psoriasis, dermatitis, and melanoma; multi-organ disorders
like diabetes, aging, and chronic fatigue; and immune system disorders like
chronic inflammations, auto-immune disorders, lupus, inflammatory bowel
disease (IBD), multiple sclerosis (MS), and cancer. Oxidative stress in the
body leads to the generation of free radicals like ROS and RNS which cause
the above-mentioned disorders/diseases. Now, the million-dollar question
is how to reduce the oxidative stress in the body? The answer would be by
slowing down the oxidative processes inside the body. How would it be
achieved? The answer is ANTIOXIDANTS.
As we said earlier, antioxidants are compounds which neutralize free radi-
cals or inhibit free radicals. An antioxidant can be defined as: “any substance
that, when present in low concentrations compared to that of an oxidisable
substrate, significantly delays or inhibits the oxidation of that substrate.”
This oxidisable substrate (lipid, protein, and carbohydrate) can generate
free radicals if it involves transfer of unpaired single electrons. Examples
of oxygen-centered free radicals, also known as ROS, are superoxide (O2−),
hydroxyl (HO•), peroxyl (ROO•), alkoxyl (RO•), and nitric oxide (NO•). The
hydroxyl (half-life of 10−9 s) and the alkoxyl (half-life of seconds) free radi-
cals are very reactive and rapidly attack the molecules in nearby cells, and
probably the damage caused by them is unavoidable and is dealt with by
repair processes (Gülcin, 2012). The repair process involves scavenging free
radicals by antioxidant compounds. Antioxidants have dual role: shelf-life
prolongation and combating oxidative stress. Antioxidants are often added
to foods to prevent the radical chain reactions of oxidation, and they act
by inhibiting the initiation and propagation step leading to the termination
of the reaction and delay the oxidation process (Shahidi & Wanasundara,
1992; Gülcin, 2006). Antioxidants may be broadly grouped according to
their mechanism of action into primary or chain-breaking antioxidants and
Natural Antioxidants: Occurrence and Their Role in Food Preservation 43

secondary or preventive antioxidants. The main difference with primary


antioxidants is that the secondary antioxidants do not convert free radicals
into stable molecules (Wanasundara & Shahidi, 2005). The primary anti-
oxidants consist mainly of hindered phenols and hindered aromatic amines.
They scavenge and destroy the chain propagating peroxy and alkoxy radi-
cals before they can react with the polymer. Gum guaiac was the first anti-
oxidant approved for the stabilization of animal fats, especially lard.

2.2 ANTIOXIDANTS VERSUS FREE RADICALS

The modes of action of an antioxidant can be broadly classified as preven-


tive/inhibitory and radical scavenging. Some examples of preventive/inhibi-
tory antioxidant enzymes are superoxide dismutase, catalase, and glutathione
peroxidase while radical scavenging antioxidants can be broadly classified
as hydrogen donating and iron-chelating antioxidants (ICA). Hydrogen
donating antioxidants (HDA) donate a hydrogen to peroxy radicals of lipid
or protein, which are mainly responsible for free radical chain reaction in a
biological system. Few examples of synthetic HDA are butylated hydroxy
anisole (BHA), butylated hydroxy toluene (BHT), propyl gallate (PG),
tertiary butyl hydroquinone (TBHQ), and so forth, while natural HDA are
AA (vitamin C), tocols (tocopherols and tocotrienols), carotenoids, polyphe-
nols, phenolics, phytosterols (PS), lignans, OZ, gossypol, and so forth. ICA
usually deactivate radically active trace metals by the formation of complex
ion. Examples of synthetic and natural ICA are ethylene diamine tetra acetic
acid (EDTA) and phytic acid, which form ions EDTA complex and phytate
complex, respectively. Several mechanisms by which antioxidants provide
defense mechanism against free radicals are:

1. Scavenging species that initiate peroxidation,


2. Chelating metal ions such that they are unable to generate reactive
species or decompose lipid peroxides,
3. Quenching O2− preventing formation of peroxides,
4. Breaking the autoxidative chain reaction, and/or
5. Reducing localized O2 concentrations (Nawar, 1996).

Several assays for quantifying antioxidant capacity are known and well
established. Few of them include:

1. ORAC (oxygen radical absorbance capacity).


44 Natural Antioxidants: Applications in Foods of Animal Origin

2. DPPH (2,2-diphenyl-1-picrylhydrazyl).
3. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)).
4. FRAP (ferric ion reducing antioxidant power).

It should be noted that antioxidant activity of food extracts can be deter-


mined using a variety of tests (stable free radical scavengers: galvinoxyl,
diphenylpicrylhydrazyl (DPPH); lipid oxidation: peroxide oxygen, conju-
gated dienes, Rancimat (measurements of oxygen consumption of a linoleic
acid emulsion and oxidation induction period in lard at 100 °C), oxygen
radical absorbance capacity (ORAC) values), active oxygen method, iodine
value (IV) (measure of the change in number of double bonds that bind I),
anisidine value (reaction of acetic acid p-anisidine and aldehydes to produce
a yellow color that absorbs at 350 nm), measurement of absorbance at 234 nm
(conjugated dienes) and 268 nm (conjugated trienes) to assess oxidation in
the early stages, and chromatographic methods; however, extraction proce-
dures strongly influence the composition of the extracts and, therefore, also
influence the antioxidant activity results (Halliwell, 1996; Schwarz et al.,
2001; Trojakova et al., 2001; Brewer, 2011). In addition, the effect of the
antioxidant compound in a food matrix may be significantly different than
the activity of a purified extract (Brewer, 2011).

2.3 SYNTHETIC ANTIOXIDANTS VERSUS NATURAL


ANTIOXIDANTS

The use of synthetic antioxidants (such as BHA, BHT, PG, and TBHQ)
to preserve food products for a longer shelf life with retained quality and
organoleptic attributes has become common commercially. However, the
consumers’ concerns regarding their bio-safety have motivated the food
industry to seek natural alternatives. Synthetic phenolic antioxidants BHA,
BHT, PG, and TBHQ effectively inhibit oxidation by scavenging free radi-
cals. Chelating agents, such as EDTA, can bind radically active trace metals
reducing their contribution to the process. Some natural substances like
vitamins (AA, tocols, and phylloquinone), carotenoids, polyphenols/pheno-
lics, PS, sesame lignans, OZ, and phytic acid can also perform the role of
radical scavenging and metal ion chelating effectively and as efficiently as
their synthetic counterparts. Natural antioxidants, mostly absorb light in
the UV region (100–400 nm), can effectively scavenge free radicals, and
chelate transition metals, thus stopping progressive autoxidative damage
and production of off-odors and off-tastes in food products. Consumers
Natural Antioxidants: Occurrence and Their Role in Food Preservation 45

have expressed concern about the safety of preservatives and additives in


their food (Brewer et al., 1994; Brewer & Prestat, 2002; Rojas & Brewer,
2008; Brewer, 2011). Sloan (1999) reported that one of the top 10 trends for
the food industry to watch included the sales of natural, organic, and vege-
tarian foods. There is a clear trend in consumer preference for clean labeling
(Hillmann, 2010; Brewer, 2011), for food ingredients and additives that are
organic/natural with names that are familiar, and that are perceived to be
healthy (Joppen, 2006; Brewer, 2011). In addition, the call for sustainable
sources and environment friendly production is forcing the food industry to
move in that direction (Berger, 2009; Brewer, 2011). Phenolic antioxidants
can inhibit free radical formation and/or interrupt propagation of autoxida-
tion. Fat-soluble vitamin E (α-tocopherol (α-T)) and water-soluble vitamin
C (L-ascorbic acid (AH2)) are both effective in the appropriate matrix. Plant
extracts, generally used for their flavoring characteristics, often have strong
H-donating activity thus making them extremely effective antioxidants
(Brewer, 2011).

2.4 NATURAL ANTIOXIDANTS: DYNAMICS AND MECHANISM

Chain-breaking antioxidants differ in their antioxidative effectiveness


depending on their chemical characteristics and physical location within a
food (proximity to membrane phospholipids (PL), emulsion interfaces, or in
the aqueous phase). The chemical potency of an antioxidant and solubility
in oil influence its accessibility to peroxy radicals especially in membrane,
micellar and emulsion systems, and the amphiphilic character required for
effectiveness in these systems (Wanatabe et al., 2010; Brewer, 2011). Anti-
oxidant effectiveness is related to activation energy, rate constants, oxida-
tion–reduction potential, ease with which the antioxidant is lost or destroyed
(volatility and heat susceptibility), and antioxidant solubility (Nawar, 1996;
Brewer, 2011). In addition, inhibitor and chain propagation reactions are both
exothermic. As the A:H and R:H bond dissociation energies increase, the
activation increases and the antioxidant efficiency decreases. Conversely, as
these bond energies decrease, the antioxidant efficiency increases. The most
effective antioxidants are those that interrupt the free radical chain reaction.
Usually containing aromatic or phenolic rings, these antioxidants donate H
to the free radicals formed during oxidation becoming a radical themselves.
These radical intermediates are stabilized by the resonance delocalization
of the electron within the aromatic ring and formation of quinone struc-
tures (Nawar, 1996; Brewer, 2011). In addition, many of the phenolics lack
46 Natural Antioxidants: Applications in Foods of Animal Origin

positions suitable for molecular oxygen attack. Both synthetic (BHA, BHT,
and PG) and natural antioxidants contain phenolics (flavonoids) function in
this manner. Natural extracts with antioxidant activity generally quench free
radical oxygen with phenolic compounds (POH) as well. Because bivalent
transition metal ions, Fe2+ in particular, can catalyze oxidative processes,
leading to formation of hydroxyl radicals, and can decompose hydroper-
oxides via Fenton reactions, chelating these metals can effectively reduce
oxidation (Halliwell et al., 1987; Brewer, 2011). Food materials containing
significant amounts of these transition metals (red meat) can be particularly
susceptible to metal-catalyzed reactions. Food tissues, because they are (or
were) living, are under constant oxidative stress from free radicals, ROS, and
pro-oxidants generated both exogenously (heat and light) and endogenously
(H2O2 and transition metals). For this reason, many of these tissues have
developed antioxidant systems to control free radicals, lipid oxidation cata-
lysts, oxidation intermediates, and secondary breakdown products (Naka-
tani, 2003; Agati et al., 2007; Brown & Kelly, 2007; Chen, 2008; Iacopini
et al., 2008; Brewer, 2011). These antioxidant compounds include flavo-
noids, phenolic acids, carotenoids, and tocopherols that can inhibit Fe3+/AA
induced oxidation, scavenge free radicals, and act as reductants (Khanduja,
2003; Ozsoy et al., 2009; Brewer, 2011). Spices and herbs, used in foods for
their flavor and in medicinal mixtures for their physiological effects, often
contain high concentrations of POH that have strong H-donating activity
(Lugasi et al., 1995; Muchuweti et al., 2007; Brewer, 2011).

2.5 ROLE OF LIPID FRACTIONS IN RADICAL SCAVENGING AND


ANTIOXIDANT ACTIVITY

Radical scavenging activity (RSA) tests are used to evaluate the health
impact of many bioactive compounds found in foods. RSA tests like DPPH
and galvinoxyl free radicals are generally used for seed oils and their free
radical quenching ability are measured by spectrophotometric and ESR
assays. Generally, it is accepted that the higher the degree of unsaturation of
an oil, the more susceptible it is to oxidative deterioration. RSA of seed oil
fractions like neutral lipids (NL), glycolipids (GL), and PL is also studied.
The results revealed that the PL fraction had the strongest antiradical action
followed by GL and NL, respectively (Ramadan et al., 2003). The radical
quenching property of GL was expected to be due to reducing sugars in
all GL components and the sterol moiety in steryl glucoside. Moreover,
less polar POH that have been extracted with GL may be responsible for
Natural Antioxidants: Occurrence and Their Role in Food Preservation 47

the strong antiradical action. On the other hand, four postulates have been
proposed to explain the antioxidant activity of PL:

i) synergism between PL and tocopherols;


ii) chelation of pro-oxidant metals by phosphate groups;
iii) formation of Maillard-type products between PL and oxidation prod-
ucts; and
iv) action as an oxygen barrier between oil/air interfaces (Ramadan,
2012).

2.6 ROLE OF FATTY ACIDS IN OXIDATIVE STABILITY

All edible oils and fats consist of triglycerides with a variety of fatty acids
that differ in chain-length (number of carbon atoms in molecule), degree
of saturation (number of double bond in carbon chain), position of double
bond within the carbon chain, and geometry of each double bond (cis and
trans isomers). Oleic acid is the most abundant monounsaturated fatty acid
(MUFA) in all the common edible oils (Gunstone, 2000; Abdulkarim et al.,
2007). Compared with polyunsaturated fatty acids (PUFA), oleic acid is
more stable toward oxidation both at ambient storage temperatures and at the
high temperatures that prevail during the cooking and frying of food. There-
fore, oils with high amounts of oleic acid are slower to develop oxidative
rancidity during shelf life or undergo oxidative decomposition during frying
than those oils that contain high amounts of PUFA. The various strengths
of hydrogen–carbon bond of fatty acids explain the differences of oxidation
rates of stearic, oleic, linoleic, and linoleic acids during thermal oxidation
or autoxidation. Compared with (PUFA) (ω-6 and ω-3 PUFA), oleic acid
(ω-9 MUFA) and saturated fatty acids are more stable toward oxidation both
at ambient storage temperatures and at the high temperatures that prevail
during the cooking and frying of food (Abdulkarim et al., 2007).
The oil rich in linoleic acid is more easily polymerized during deep-fat
frying than the oil rich in oleic acid. The energy required to break carbon–
hydrogen bond on the carbon 11 of linoleic acid is 50 kcal/mol (Min & Boff,
2002; Choe & Min, 2007). The double bonds at carbon 9 and carbon 12
decrease the carbon–hydrogen bond at carbon 11 by withdrawing electrons.
The carbon–hydrogen bond on carbon 8 or 11, which is α to the double
bond of oleic acid, is about 75 kcal/mol. The carbon–hydrogen bond on the
saturated carbon without any double bond next to it is ~100 kcal/mol (Min
& Boff, 2002; Choe & Min, 2007). Oxidation produces hydroperoxides and
48 Natural Antioxidants: Applications in Foods of Animal Origin

then low molecular volatile compounds such as aldehydes, ketones, carbox-


ylic acids, and short-chain alkanes and alkenes. In a study reported by Bhat-
nagar et al. (2009), it was found that the oxidative stability (OS) of oil blends
depended upon the PUFA and MUFA content of the oil blends. The higher
the PUFA and MUFA content, the lower would be the OS, while the RSA
of oil blends depended upon the total tocopherols’ content. The higher the
total tocopherols’ content the higher the DPPH scavenging activity would
be. Antioxidant decreases the frying oil oxidation, but the effectiveness of
antioxidant decreases with high frying temperature.
OS of stripped and crude seed oil was studied during 21 days under
accelerated oxidative conditions at 60 °C (Ramadan & Moersel, 2004).
Peroxide value (PV) and UV absorptivity were determined to monitor lipid
oxidation during the experiment. The crude oil had a much lower PV than
that of stripped oil over the entire storage period. PV in crude oil remained
increased at a low level over 21 days, whereas the peroxides accumulated
in the stripped oils to high levels. Absorption at 232 nm and 270 nm, due to
the formation of primary and secondary compounds of oxidation, showed a
pattern similar to that of the PV. The high content of conjugated oxidative
products is attributed to high PUFA content which is readily decomposed to
form conjugated hydroperoxides. It was concluded that the low OS of PUFA
rich oil could be partly explained by the fact that it has a high proportion of
PUFA. Aside from the fatty acid profile, factors such as oxygen concentra-
tion, metal contaminants, lipid hydroxy compounds, enzymes, and light may
also influence the OS of the oil (Ramadan, 2012).

2.7 VITAMIN E OR TOCOLS (TOCOPHEROLS AND


TOCOTRIENOLS)

Tocols (tocopherols and tocotrienols) constitute a series of benzopyranols


(or methyl tocols) that occur in plant tissues and vegetable oils and are
powerful lipid-soluble antioxidants. In the tocopherols, the C16 side chain
is saturated, and in the tocotrienols it contains three trans double bonds.
Together, these two groups are termed the tocochromanols. In essence,
the tocopherols have a 20-carbon phytyl tail (including the pyranol ring),
and the tocotrienols a 20-carbon geranyl tail with double bonds at the 3’,
7’, and 11’ positions, attached to the benzene ring. The side-chain methyl
groups have R,R,R stereochemistry. The four main constituents of the two
classes are termed—alpha (5,7,8-trimethyl), beta (5,8-dimethyl), gamma
(7,8-dimethyl), and delta (8-methyl) (Christie, 2013). The plant chloroplast
Natural Antioxidants: Occurrence and Their Role in Food Preservation 49

is site for tocol biosynthesis and the aromatic amino acid tyrosine is consid-
ered to be its precursor. The mechanism of biosynthesis of tocols involves
coupling of phytyl diphosphate with homogenestic acid (2,5-dihydroxyphen-
ylacetic acid), followed by cyclization and methylation reactions. Vitamin E
compounds include the tocopherols and tocotrienols. Tocotrienols have a
conjugated triene double bond system in the phytyl side chain, while tocoph-
erols do not. Methyl substitution affects the bioactivity of vitamin E, as
well as its in vitro antioxidant activity. Tocopherols or vitamin E have eight
known homologues, that is, α-, β-, γ-, δ- tocopherols and α-, β-, γ-, δ- toco-
trienols. Tocopherols are fat-soluble antioxidants that function as scaven-
gers of lipid peroxyl radicals. Tocopherols’ content is found to be related to
RSA and antioxidant activity of oils. Tocopherols’ content decreases during
processing of oils. There have been many reports on the protective effect of
tocopherols as food antioxidants (Dougherty, 1988). Tocopherols protect PL
and cholesterol against oxidation (Faustman et al., 1989; Li et al., 1996).
Total tocopherol content and major tocopherol homologues differ from one
oil to another (Table 2.1 and Fig. 2.1). The level of tocopherols decreases
with time of storage and heating of oils (Li et al., 1996).

R'
·a/p/7a-tocopherol -CH3 -CH3
beta-tocopherol -CH3 -H
gamma-tocopherol -H -CH3
delta-tocopherol -H -H

CH3
R"

R' tocomeools

FIGURE 2.1 Different tocopherol homologues of vegetable oils.


TABLE 2.1 Levels of Tocopherols and Tocotrienols in Some Crude Vegetable Oils (mg/kg) (Kamal-Eldin & Andersson, 1997; Bhatnagar et al., 50
2009; Codex Stan 210, 2011).
Tocopherols Coconut Groundnut Mustard oil Olive oil Palm oil Rice bran oil Safflower Sesame oil Soyabean oil Sunflower
oil oil seed oil seed oil
α-tocopherol ND-17.0 49.0–373.0 268.0–380.0 4.0–280.0 4.0–193.0 49.0–583.0 234.0–660.0 ND-3.3 9.0–352.0 403.0–935.0
β- tocopherol ND-11.0 ND-41.0 ND 1.0–10.0 ND-234.0 ND-47.0 ND-17.0 ND ND-36.0 ND-45.0
γ- tocopherol ND-14.0 88.0–389.0 426.0–550.0 1.0–10.0 ND-526.0 ND-212.0 ND-12.0 521.0–983.0 89.0–2307.0 ND-34.0
δ-tocopherol ND ND-22.0 97.0–150.0 ND ND-123.0 ND-31.0 ND 4.0–21.0 154.0–932.0 ND-7.0
α-tocotrienol ND-44.0 ND ND ND 4.0–336.0 ND-627.0 ND ND ND-69.0 ND
γ- tocotrienol ND-1.0 ND ND ND 14.0–710.0 142.0–790.0 ND-12.0 ND-20.0 ND-103.0 ND
δ-tocotrienol ND ND ND ND ND-377.0 ND-59.0 ND ND ND ND
Total ND-50.0 170.0–850.0 790.0–1050.0 5.0–300.0 150.0–1500.0 191.0–2349.0 240.0–670.0 330.0–1010.0 600.0–3370.0 440.0–1020.0
tocopherols

ND: not detected.


Natural Antioxidants: Applications in Foods of Animal Origin
Natural Antioxidants: Occurrence and Their Role in Food Preservation 51

2.7.1 ANTIOXIDATIVE MECHANISM OF TOCOPHEROLS

Vitamin E or tocopherols which are benzopyranols or methylated tocols


are natural antioxidants and integral bioactive molecules of an oil or fat.
The primary task of tocopherols is to act as antioxidants to prevent free
radical damage to unsaturated lipids or other membrane constituents of the
tissues. Tocopherols are powerful antioxidants in vitro and in vivo. They
are certainly extremely useful as antioxidants in non-biological systems,
including foods, cosmetics, pharmaceutical preparations, and so forth
(Christie, 2013). Because of their lipophillic character, tocopherols are
located in the membranes or with storage lipids where that are immedi-
ately available to interact with lipid hydroperoxides. They react rapidly in a
non-enzymic manner to scavenge lipid peroxyl radicals, that is, the chain-
carrying species that propagate lipid peroxidation. In model systems in vitro,
all the tocopherols (α > γ > β > δ) and tocotrienols are good antioxidants
(Christie, 2013). In general, the oxidation of lipids is known to proceed by a
chain process mediated by a free radical, in which the lipid peroxyl radical
serves as a chain carrier. In the initial step of chain propagation, a hydrogen
atom is abstracted from the target lipid by the peroxyl radical as shown:

LOO• + LH → LOOH + L• (2.1)

L• + O2 → LOO• (2.2)

where LH is a lipid; LOO• is the lipid peroxyl radical, and LOOH is the lipid
hydroperoxide. The main function of α-T is to scavenge the lipid peroxyl
radical before it is able to react with the lipid substrate as shown:

LOO• + TOH → LOOH + TOO• (2.3)

where TOH is tocopherol and TOO• is the tocopheroxyl radical. As shown


in eq 2.3 tocopherols thus prevent propagation of the chain reaction. The
potency of an antioxidant is determined by the relative rates of reactions eqs
2.1 and 2.2. Studies of the relative rates of chain propagation to chain inhibi-
tion by α-T in model systems have demonstrated that α-T is able to scavenge
peroxyl radicals much more rapidly than the peroxyl radical can react with
a lipid substrate (Christie, 2013). In biological systems, oxidant radicals can
spring from a number of sources, including singlet oxygen, alkoxyl radicals,
superoxide, peroxynitrite, nitrogen dioxide, and ozone. α-T is most efficient
at providing protection against peroxyl radicals in a membrane environment
52 Natural Antioxidants: Applications in Foods of Animal Origin

(Table 2.2). When a tocopheroxyl radical is formed, it is stabilized by delo-


calization of the unpaired electron about the fully substituted chromanol
ring system rendering it relatively unreactive. This also explains the high
first order rate constant for hydrogen transfer from α-T to peroxyl radicals.
Reaction of the tocopheroxyl radical with a lipid peroxyl radical yields
8α-substituted tocopherones, which are readily hydrolysed to 8α-hydroxy
tocopherones that rearrange spontaneously to form α-T quinones (Fig. 2.2).

TABLE 2.2 Approximate Biological Activity Relationships of Vitamin-E Compounds


(Akoh & Min, 2002).
Compound Activity of D-α-tocopherol (%)
D-α-tocopherol 100
L-α-tocopherol 29
DL-α-tocopherol 74
DL-α-tocopheryl acetate 68
D-β-tocopherol 8
D-γ-tocopherol 3
D-δ-tocopherol –
D-α-tocotrienol 22
D-β-tocotrienol 3
D-γ-tocotrienol –
D-δ-tocotrienol –

tocopheroxyl radical 8a -alkyldi oxytocopherone / tocopherol quinone

HO

tocopherol hydroquinone
FIGURE 2.2 Mechanism for radical quenching action of α-tocopherol.
Natural Antioxidants: Occurrence and Their Role in Food Preservation 53

2.7.2 TOCOPHEROLS IN FOOD PRESERVATION

The antioxidant function of tocopherols in various foods has been reported


by many researchers in various types of food. Instant ramen, a Japanese dried
noodle product, is fried in lard to produce the characteristic flavor. Kuwahara
et al. (1971) reported that natural vitamin E at a level of 0.03% prevented the
lard oxidation in the noodles better than the synthetic antioxidants such as
BHA and BHT (Christine, 2010). Kanematsu et al. (1972) studied the effect
of tocopherols, such as synthetic α-T, mixed natural tocopherol concentrate,
and BHA, on OS of margarines. As a control, margarine without antioxi-
dants was used. Margarines were stored at 5 or 25 °C for six months. Natural
mixed tocopherols and BHA were found to have nearly the same antioxidant
effect (Christine, 2010). King (1986) determined the effects of antioxidants
and modified atmospheres (vacuum and N2 gas) on the stability of pecan
kernels stored at 85 °C for 15 weeks. Chopped pecan kernels were treated
with various antioxidants such as α-T at 0.05%, γ-tocopherol and mixed
tocopherols at 0.02 and 0.05%, BHA, BHT, and TBHQ at 0.02% sealed in
cans with or without headspace modification by vacuum or N2 flush. α-T,
γ-tocopherol, and mixed tocopherols at 0.05% had significantly protected
the color and reduced flavor changes. γ-tocopherol and mixed tocopherols
also reduced the headspace pentane production (Christine, 2010). Ochi et
al. (1988) studied the effects of α- and δ-tocopherols on OS of cookies.
Both α- and δ-tocopherols were decreased by 20% during baking. During
storage between 25 and 60 °C, the degradation of α-T was faster than the
degradation of δ-tocopherol. But the loss of α- and δ-tocopherols decreased
with an increase in added amounts of whole milk powder and egg because
they protected the fats from oxidative degradation. Cookies with added α-T
(50 mg/100 g dough) had relatively low PV of their lipid fraction after baking
(Christine, 2010). Inagaki (1968) investigated the antioxidative components
in unshu-orange flavedo (the white part in the peel of unshu orange), which
inhibited autoxidation of limonene. The antioxidants identified by thin
layer and gas–liquid partition chromatography were: 100–160 µg/g α-T,
and 60–70 µg/g γ-tocopherol on fresh weight basis (Christine, 2010). Elez-
Martínez et al. (2007) studied the ability of α-T to extend the shelf life of
an avocado puree by determining the PV and IV of the stored puree with
and without added tocopherol (100 ppm) and sorbic acid, an antimicrobial
agent. They found that the α-T was very effective in extending the shelf
life but sorbic acid had the effect of enhancing oxidation in the avocado
puree. Therefore, the best quality product was obtained with the addition of
α-T and using low oxygen packaging (Christine, 2010). It is proposed that
54 Natural Antioxidants: Applications in Foods of Animal Origin

understanding the interfacial phenomena is a key to understand the actions


of tocopherols in heterogeneous food systems (Frankel, 1996).

2.8 VITAMIN K1 (PHYLLOQUINONE)

Vitamin K is a fat-soluble vitamin that functions as a co-enzyme and is


involved in the synthesis of a number of proteins participating in blood clot-
ting and bone metabolism (Damon et al., 2005). Vitamin K also plays a role
as a co-factor for blood coagulation and coagulation inhibitors in the liver, as
well as a variety of extra hepatic proteins such as the bone protein osteocalcin
(Shearer, 1992). The importance of vitamin K as a blood-clotting agent is
well known. Moreover, it is demonstrated that vitamin K may play a variety
of health-promoting roles. Vitamin K reduces the risk of heart disease, kills
cancer cells, and enhances skin health and have antioxidant properties (Otles
& Cagindi, 2007). A recent study concluded also that high phylloquinone
intakes are markers of a dietary and lifestyle pattern that is associated with
lower CHD risk in men (Erkkilä et al., 2007). Vitamin K1 (phylloquinone)
is a polycylic aromatic ketone which contains a functional naphthoquinone
ring and a phytyl side chain, that is, 2-methyl-1,4-naphthoquinone, with a
3-phytyl substituent (Fig. 2.3). Its molecular formula is C31H46O2 and molar
mass is 450.70 g/mol.

0 00 00 00
00
00
0
FIGURE 2.3 Structure of vitamin K1 (phylloquinone).

2.8.1 FOOD APPLICATIONS OF VITAMIN K1

The vitamin K1 (phylloquinone) level is very low in most foods


(<10 mg/100 g), and the majority of the vitamin is obtained from a few green
and leafy vegetables (e.g., spinach and broccoli). Many studies have shown
that some vegetable oils (especially soybean, cottonseed, and rapeseed oils)
Natural Antioxidants: Occurrence and Their Role in Food Preservation 55

are important dietary sources of phylloquinone (Gao & Ackman, 1995;


Piironen et al., 1997; Booth & Suttie, 1998; Koivu et al., 1999; Jakob &
Elmadfa, 2000). Niger seed oil has been characterized by extremely high
level of vitamin K1 (0.2%) (Ramadan & Moersel, 2002). Among edible oils,
the best sources of phylloquinone are niger seed oil (ca 2.0 mg/g), rape-
seed oil (ca 1.5 ug/g) and soybean oil (ca 1.3 ug/g). Sunflower oil is the
poorest source (ca 0.10 ug/g) of phylloquinone (Piironen et al., 1997). Its
levels are also moderate in olive oil (Jakob & Elmadfa, 2000; Shearer et
al., 1996; Piironen et al., 1997). A study reported that in olive oil, the mean
content of phylloquinone ranged from 12.7 to 18.9 μg/100 g while in human
plasma, phylloquinone content varied between 0.22 and 0.56 ng/mL (Otles
& Cagindi, 2007). The addition of phylloquinone-rich oils in the processing
and cooking of foods that are otherwise poor sources of vitamin K (e.g.,
peanut and corn oils) makes them potentially important dietary sources of
the vitamin. The significance of dietary vitamin K has recently increased.
Blending of niger seed oil with other vegetable oils would enrich them with
vitamin K1 (phylloquinone) (Bhatnagar & Gopala Krishna, 2015).

2.9 VITAMIN C (ASCORBIC ACID)

AA (vitamin C) is considered to be one of the most powerful, least toxic


natural antioxidants. It is a water-soluble vitamin and is found in high
concentrations in many foods or plants (Table 2.3). The varied roles of
AH2 related to its antioxidant property in foods, browning reaction, and its
anaerobic loss are discussed here. AH2 is the trivial name for L-threo-2-hex-
enono-l, 4- lactone, the molecule responsible for preventing scurvy (Fig.
2.4). AH2 or vitamin C is ubiquitous and has multiple functions in all meta-
bolically active plant and animal cells. One of the principal biochemical
reactions of AH2 is to destroy toxic free radicals (hydroxyl and perhydroxyl)
resulting from the metabolic products of oxygen. In this role, the mixture
of AH2 and its oxidation product dehydroascorbic acid (A) is thought of
as a “redox buffer” (Sapper et al., 1982; Ming-Long & Paul, 1988). When
terminating free radicals, AH2 is converted to A, which is then recycled to
AH2 by reductase enzymes and co-factors. Besides the redox functions in
cells, other physiological actions of AH2 (Loewus & Loewus, 1987; Ming
& Paul, 1988) may be related to the compound’s chelation with metals and
complexing with protein (Gorman & Clydesdale, 1983; Fleming & Bensch,
1983; Ming-Long & Paul, 1988). The uses of AH2, including those in food,
continue to increase because of the compound’s vitamin C activity, useful
56 Natural Antioxidants: Applications in Foods of Animal Origin

properties, and low toxicity. AA reacts with superoxide radical (O2), perhy-
droxyl radical (HO2), hydroxyl radical (HO•), and singlet oxygen (Fessenden
& Verma, 1978; Nanni et al., 1980; Cabelli & Bielski, 1983; Ming-Long
& Paul, 1988). Those reactions by AH2 retard lipid autoxidation. Ascorbate
radical is the initial oxidation product of two enzyme reactions that occur in
plants (Loewus & Loewus, 1987; Ming & Paul, 1988). Ascorbate oxidase
is the enzyme that oxidizes AH2 in the presence of oxygen as shown by the
overall reaction below:

2 Ascorbate + O2 → 2 Dehydroascorbic acid + 2H2O

Ascorbate peroxidase is another plant enzyme that oxidizes AH2, but it


uses hydrogen peroxide instead of oxygen as the electron acceptor.

Ascorbate + H2O2 → Dehydroascorbic acid + 2H2O

TABLE 2.3 Vitamin C in Selected Foods (Steinberg & Rucker, 2013).


Sources of vitamin C mg of ascorbic acid per 100 g of wet
weight or edible portion
Animal products
Cow’s milk 0.5–2
Human milk 3–6
Oysters (raw) 30
Beef, pork, veal 2–10
Beef, pork, Liver 20–30
Fruits
Apple 3–30
Banana 8–16
Blackberry 8–10
Cherry 15–30
Currant, red 20–50
Currant, black 150–200
Grapefruit 30–70
Kiwi fruit 80–90
Lemon, orange 40–50
Lime 30–45
Melon 9–60
Natural Antioxidants: Occurrence and Their Role in Food Preservation 57

TABLE 2.3 (Continued)

Sources of vitamin C mg of ascorbic acid per 100 g of wet


weight or edible portion
Strawberry 59–70
Pineapple 15–25
Rose hips 250–800
Vegetables
Beans, various 10–15
Broccoli 70–90
Brussels sprouts 100–120
Cabbage 30–70
Carrot 5–10
Cucumber 6–8
Cauliflower 50–70
Eggplant 15–20
Kale 70–100
Onion 10–15
Parsley 90–130
Peas 8–12
Potato 4–30
Pumpkin 15
Radish 25
Spinach 35–40
Tomato 15–20
Condiments
Chicory 30–40
Coriander (spice) 90
Garlic 15–25
Horseradish 50
Lettuce, various 10–30
Leek 15
Parsley 200–300
Pepper, various 150–200
58 Natural Antioxidants: Applications in Foods of Animal Origin

HO OH

OH
0

OH
FIGURE 2.4 Ascorbic acid.

2.9.1 ROLE OF VITAMIN C IN FOOD PRESERVATION

AA (E-300) generally regarded as safe (GRAS) substance prevents oxidative


browning in heat-processed foods, enzyme-catalyzed oxidation in frozen
fruits, rusting and rancidity in frozen fish, discolorations and rancidity in
meat products, and oxidized flavor in dairy and beverage products (Bauern-
feind, 1953). It acts synergistically with other antioxidants (known to regen-
erate α-Ts) in edible fats and also acts as flour and dough improver. It is
important to add the AA as late as possible during processing or preserva-
tion to maintain highest levels during the shelf life of the food commodity
(Wiley, 1994). The beneficial use of AA has been established for the stabili-
zation of beer (Wales, 1956) and other food applications where it can serve
to reduce the oxygen from the headspace of a closed system (Cort, 1974).
AH2 may preserve or promote the reduced oxidation state of a metal ion
in food (Hay et al., 1967). The oxidation state is an important variable in
mineral nutrition (Solomons & Viteri, 1982; Keypour et al., 1986). AA has
strong singlet oxygen and superoxide anion quenching ability and has been
shown to protect riboflavin loss in milk (Lee et al., 1998).
The acid-catalyzed degradation of AH2 is thought to be responsible for
anaerobic loss of vitamin C in foods, such as canned grapefruit and orange
juices, which have a pH of ~3–5 (Kefford et al., 1959; Smoot & Nagy, 1980;
Ming-Long & Paul, 1988). At 50 °C, the juices lose 70–95% of AH2 in 12
weeks; the degradation reaction is zero-order with respect to AH2. The anaer-
obic loss of AH2 is often one-tenth the rate of loss under aerobic conditions.
Categories of reactions AA undergoes are mentioned in Table 2.4 and the
level of permitted AA derivatives in different foods is presented in Table 2.5.
Natural Antioxidants: Occurrence and Their Role in Food Preservation 59

TABLE 2.4 Categories of Ascorbic Acid Reactions.


S. no. Type of reaction Substrate involved
1 Redox Glutathione/glutathione disulfide and ascorbic acid/
dehydroascorbic acid
2 Oxidation Ascorbic acid and dehydroascorbic acid
3 Reduction o-quinone back to phenolic compound
4 Anaerobic degradation L-ascorbic acid decarboxylate and dehydrate to give
almost quantitative yields of furfural and CO2

TABLE 2.5 Antioxidants Permitted in Foodstuffs for Infants and Young Children (Miková,
2003).
E number Name Foodstuff Maximum level
E 300 L-ascorbic acid Fruit and vegetable based 0.3 g/kg
E 301 Sodium L-ascorbate drinks, juices, and baby foods

E 302 Calcium L-ascorbate Fat-containing cereal-based 0.2 g/kg


foods including biscuits
E 304 L-ascorbyl palmitate Fat-containing cereals, *100 mg/kg individu-
E 306 Tocopherol rich extract biscuits, rusks, and baby ally or in combination
foods
E 307 α-tocopherol
E 308 γ-tocopherol
E 309 β-tocopherol
*10 mg/kg for follow-on formulae for infants in good health.

AH2 retards enzymic browning by at least two mechanisms (Golan-


Goldhirsh et al., 1984; Ming & Paul, 1988). AH2 chemically reduces benzo-
quinone intermediates to colorless o-dihydroxyphenols, and it also irrevers-
ibly denatures polyphenoloxidase (PPO). Evidence (Golan-Goldhirsh et al.,
1987; Ming-Long & Paul, 1988) suggests that PPO is denatured mainly by
Cu+2-catalyzed oxidative cleavage of imidazole groups of the histidine resi-
dues on the enzyme to aspartic acid and urea. The oxidation is mediated
through a quaternary complex thought to contain the imidazole group, cupric
ion, AH2 radical (AH•), and O2. The anti-browning effects of AA have been
widely demonstrated in several fruit fresh-cut products under a wide range
of conditions (Soliva-Fortuny et al., 2001; Senesi et al., 1999). Ascorbate
reacts with nitrite forming NO, NO2, and N2, thereby inhibits carcinogenic
nitrosamine formation.
60 Natural Antioxidants: Applications in Foods of Animal Origin

2.10 PHYTOSTEROLS

PS are steroid alcohols and resemble cholesterol in structure, the predomi-


nant sterol found in animals both in their biological function and chemical
structure. PS are fat-soluble nutrients and are biosynthetically derived from
squalene and belong to the group of triterpenes. They are made of tetracy-
clic cyclopentaphenanthrene ring and long flexible side chain at the C-17
carbon atom (Clifton, 2002; Moreau et al., 2002). The 3-hydroxyl group of
free sterols may be esterified by a fatty acid or a phenolic acid or β-linked
to a carbohydrate. Plant sterols are primarily present in plasma membrane,
the mitochondria, and endoplasmic reticulum, and to a large extent deter-
mine the properties of the membranes (Jonker et al., 1985). Plant sterols are
white powder and solid at room temperature and the melting point of sitos-
terol, campesterol, and stigmasterol are 140 °C, 157–158 °C, and 170 °C
(Nes, 1987). Plant sterols are divided into three different kinds based on
structures namely, 4-desmethyl sterols, 4-methyl sterols, and 4,4-dimethyl
sterols. The 4-desmethyl sterols family includes three different kinds of PS
which accounts for most of the total PS mass. They are β-sitosterol (include
an extra methyl group at C-24 position), campesterol (includes an additional
ethyl group at C-24 poisition), and stigmasterol (includes an additional
ethyl group at C-24 position and a double bond at C-22 position). All these
PS account for about 65, 30, and 3% of the total dietary PS intake (Weihr-
auch & Gardner, 1978; Moreau et al., 2002; Ostlund, 2002). The chemical
difference among 4-desmethyl sterols resides in number of carbon atoms in
carbon-17 branch chain and in presence or absence of a double bond at posi-
tion 22. The 4-methyl sterols and 4,4-dimethyl sterols are minor components
in plant sources. PS are structurally similar to cholesterol (steroid nucleus
and a hydroxyl group at C-3 position) and are differentiated by their degree
of saturation and side chain configuration at C-24 position (Clifton, 2002;
Moreau et al., 2002). Analysis of sterols provides a powerful tool for quality
control of vegetable oils, and for the detection of oil as well as blends not
recognized by the fatty acids’ profile (Ramadan, 2012). Structures of some
4-desmethylsterols of vegetable oils are provided in Figure 2.5. The PS
levels and composition of some vegetable oils are provided in Table 2.6.

2.10.1 FOOD APPLICATIONS OF PHYTOSTEROLS

A dark chocolate containing PS esters was developed to reduce cholesterol


in individuals. However, oxidative instability during chocolate processing
Natural Antioxidants: Occurrence and Their Role in Food Preservation 61

Cholesterol

Stigmasterol

Campesterol

HO
J3-Sitosterol
FIGURE 2.5 Different desmethylsterols of vegetable oils.
TABLE 2.6 Levels of Total Phytosterols (mg/kg) and Desmethylsterols (wt % of Total Sterols) in Some Crude Vegetable Oils (Kamal-Eldin & 62
Appelqvist, 1994; Gunstone, 2002; Codex Stan 210, 2011).
Sterols Coconut Groundnut Mustard Olive oil Palm oil Rice bran oil Safflower Sesame oil Soyabean Sunflower
oil oil oil seed oil oil seed oil
Total sterols 400–1200 900–2900 8000–8800 800–1000 300–700 10500–31000 2100–4600 4500–19000 1800–4500 2400–5000
Cholesterol ND-3.0 ND-3.8 ND-0.4 ND-0.5 2.6–6.7 ND-0.5 ND-0.7 0.1–0.3 0.2–1.4 ND-0.7
Brassicasterol ND-0.3 ND-0.2 10.0–13.2 ND-0.1 ND ND ND-0.4 0.1–0.2 ND-0.3 ND-0.2
Campesterol 6.0–11.2 12.0–19.8 30.0–34.4 3.5–4.0 18.7–27.5 11.0–35.0 9.2–13.3 10.3–20.5 15.8–24.2 6.5–13.0
Stigmasterol 11.4–15.6 5.4–13.2 ND-0.3 2.5–3.0 8.5–13.9 6.0–40.0 4.5–9.6 4.4–14.0 14.9–19.1 6.0–13.0
β-Sitosterol 32.6–50.7 47.4–69.0 40.0–47.9 93.0 50.2–62.1 25.0–67.0 40.2–50.6 57.7–61.9 47.0–60.0 50.0–70.0
δ-5-Avenasterol 20–40.7 5.0–18.8 1.0–2.1 ND-0.5 ND-2.8 ND-9.9 0.8–4.8 6.2–7.8 1.5–3.7 ND-6.9
δ-7-Stigmastenol ND-3.0 ND-5.1 0.8–1.6 ND-0.5 0.2–2.4 ND-14.1 13.7–24.6 0.5–7.6 1.4–5.2 6.5–24.0
δ-7-Avenasterol ND-3.0 ND-5.5 1.0–2.1 ND-0.5 ND-5.1 ND-4.4 2.2–6.3 1.2–5.6 1.0–4.6 3.0–7.5
Others ND-3.6 ND-1.4 ND-0.5 ND-0.5 ND ND 0.5–6.4 0.7–9.2 ND-1.8 ND-5.3
ND: not detected.
Natural Antioxidants: Applications in Foods of Animal Origin
Natural Antioxidants: Occurrence and Their Role in Food Preservation 63

and storage could reduce the PS bioactivity. Chocolate bars were prepared
containing palm oil (CONT) or 2.2 g of PS (PHYT). All samples were stored
at 20 and 30 °C during five months. A peak of hydroperoxides formation
was observed after 60 days at 20 °C and after 30 days at 30 °C. PS-enriched
samples presented higher values of hydroperoxides than control samples,
which could be attributed to the higher level of α-linolenic acid present in the
PHYT samples. All chocolate bars became lighter and softer after 90 days
of storage. However, these physical changes did not reduce their sensory
acceptability. In addition, PS bioactivity was kept during the storage, since
no significant alterations in the PS esters were observed up to five months.
However, some PS oxidation occurred in the PHYT bars, being sitostane-
triol, 6-ketositosterol, 6β-hydroxycampesterol, and 7-ketocampesterol the
major phytosterol oxidation products (POPs). The POPs/PS ratio was low
(0.001). Therefore, the dark chocolate bars developed in this study kept their
potential functionality after five months of storage at room temperature,
representing an option as a functional food (Botelho et al., 2014).

2.11 PHENOLIC COMPOUNDS

POH have aromatic hydrocarbon ring with one or more hydroxyl group.
POH contribute to the quality of edible oils by enhancing the shelf life and
flavor stability of oil (Bendini et al., 2006). Several POH have been identified
that inhibit oxidation of fats and oils by interrupting the free radical mecha-
nism of oxidation. Structural groups influencing the antioxidant activity of
POH include position and number of hydroxyl groups, polarity, solubility,
and stability of POH during processing (Soobrattee et al., 2005). Phenolics
present in fats and oils are potent antioxidants and have anticarcinogenic
properties. The antioxidant property of phenolics depends on the structure
and type of phenolics present in oil. Hydroxybenzoic acid derivatives, that
is, gallic acid, vanillic acid, and vanillin show an UV-absorption maxima
at around 280 nm while hydroxycinnamic acid derivatives, that is, caffeic,
cinnamic, p-coumaric, and ferulic acids show an UV-absorption maxima at
around 320 nm.
Virgin olive oil has a unique place among vegetable oils because of its
polyphenols and their beneficial role in human health (Visioli, 2000; Tricho-
poulou & Vasilopoulou, 2000). The polyphenols are an important class
of minor constituents linked both to the flavor of virgin olive oil and to
its keeping ability. POH present in olive oil are conventionally character-
ized as “polyphenols,” though not all of them are polyhydroxy aromatic
64 Natural Antioxidants: Applications in Foods of Animal Origin

compounds. Compounds which often appear in lists of olive oil polyphenols


are 4-acetoxy-ethyl-1,2-dihydroxybenzene, 1-acetoxy-pinoresinol, apigenin,
caffeic acid, cinnamic acid, o- and p-coumaric acids, elenolic acid, ferulic
acid, gallic acid, homovanillic acid, p-hydroxybenzoic acid, p-hydroxy-
phenylacetic acid, hydroxytyrosol, luteolin, oleuropein, pinoresinol, proto-
catechuic acid, sinapic acid, syringic acid, tyrosol, vanillic acid, and vanillin
(Morales & Tsimidou, 2000; Garcia et al., 2001; Mateos et al., 2001; Boskou,
2006). Structures of some phenolics of plant origin are provided in Figure
2.6. Phenolic acid content of some vegetable oils is provided in Table 2.7.

Good listener Good listener Good listener

Good listener Good listener Good listener

FIGURE 2.6 Some major phenolics of vegetable oils.

2.11.1 ANTIOXIDATIVE MECHANISM OF PHENOLICS

POH or “phenolics” constitute a class of chemical compounds consisting of


one or several hydroxyl group (–OH) covalently bonded to one or several
aromatic rings. It is difficult to say how many different phenolics exist on
earth. In the sole example of flavonoids, there are more than 10,000 known
formulas. Phenolics may be subdivided in various classes such as flavonoids
TABLE 2.7 Phenolic Acid Content (µg/100 g oil) of Some Vegetable Oils (Siger et al., 2008).
Vegetable oils p-hydroxybenzoic Vanillic acid Caffeic acid p-coumaric Ferulic acid Sinapic acid Total
acid acid
Soyabean 0.77–0.83 1.04–1.16 0.73–0.87 1.44–1.56 1.12–1.28 0.87–0.93 5.97–6.63
Sunflower 1.45–1.55 6.75–7.05 4.8–5.0 1.74–1.86 1.22–1.38 1.37–1.43 17.33–18.27
Rapeseed 1.55–1.65 ND 0.25–0.35 12.98–13.22 5.5–5.7 235.5–236.5 255.87–257.33
Corn 1.68–1.72 ND ND 1.82–1.98 5.7–5.9 0.57–0.63 9.77–10.23
Grape seed ND 0.75–0.85 ND ND ND 0.12–0.28 0.87–1.13
Hemp 5.94–6.06 1.90–2.10 ND 1.85–2.15 0.92–1.08 2.95–3.05 13.56–14.44
Linseed 3.03–3.17 0.85–1.15 ND ND 0.95–1.05 ND 4.83–5.37
Rice bran ND ND ND ND 0.37–0.43 ND 0.37–0.43
Pumpkin seed oil 2.95–3.25 11.3–11.5 ND 3.74–3.86 3.74–3.86 ND 21.73–22.47
ND: not detected.
Natural Antioxidants: Occurrence and Their Role in Food Preservation
65
66 Natural Antioxidants: Applications in Foods of Animal Origin

(quercetin, catechin, and condensed tannins), phenolic acids (caffeic acid)


and esters (chlorogenic acid, gallotannins), stilbenes (resveratrol), phenolic
alcohols (hydroxytyrosol), and so forth (Laguerre et al., 2014). POH are
natural antioxidants and integral bioactive molecules of an oil or fat. POH
act as free radical scavengers and metal chelators (Dai & Mumper, 2010).
POH act as free radical acceptors and chain breakers. They interfere with
the oxidation of lipids and other molecules by rapid donation of a hydrogen
atom to radicals (R) ,that is, R + POH → RH + PO•. The phenoxy radical
intermediates (PO•) are relatively stable due to resonance and therefore a
new chain reaction is not easily initiated. Moreover, the phenoxy radical
intermediates also act as terminators of propagation route by reacting with
other free radicals, that is, PO• + R• → POR. POH possess ideal structure
chemistry for free RSAs because they have, (a) phenolic hydroxyl groups
that are prone to donate a hydrogen atom or an electron to a free radical and
(b) extended conjugated aromatic system to delocalize an unpaired electron.
Several relationships between structure and reduction potential of pheno-
lics have been established. For phenolic acids and their esters, the reduction
activity depends on the number of free hydroxyl groups in the molecule.
Hydroxycinnamic acids were found to be more effective than their hydroxy-
benzoic acid counterparts, possibly due to the aryloxy-radical stabilizing
effect of the ─CH═CH─COOH linked to the phenyl ring by resonance (Dai
& Mumper, 2010).

2.12 CAROTENOIDS

Carotenoids are a class of hydrocarbons consisting of eight isoprenoid units


joined in such a manner that the arrangement of isoprenoid units is reversed
at the center of the molecule so that the two central methyl groups are in a
1,6-positional relationship and the remaining non-terminal methyl groups
are in a 1,5-positional relationship. Carotenoids are defined by their chemical
structure. The majority of carotenoids are derived from a 40-carbon polyene
chain, which could be considered the backbone of the molecule. This chain
may be terminated by cyclic end-groups (rings) and may be complemented
with oxygen-containing functional groups (Zeb & Mehmood, 2004). These
hydrocarbons are commonly known as carotenes, while oxygenated deriva-
tives of these hydrocarbons are known as xanthophylls. β-carotene, the prin-
cipal carotenoid in carrots, is a familiar carotene, while lutein, the major
yellow pigment of marigold petals, is a common xanthophyll. The struc-
ture of a carotenoid ultimately determines what potential biological function
Natural Antioxidants: Occurrence and Their Role in Food Preservation 67

that pigment may have. The characteristic pattern of alternating single and
double bonds in the polyene backbone of carotenoids allows them to absorb
excess energy from other molecules, while the nature of the specific end
groups on carotenoids may influence their polarity. The former may account
for the antioxidant properties of biological carotenoids, while the latter may
explain the differences in the ways that individual carotenoids interact with
biological membranes (Britton, 1995).
The most important carotenoids are α-carotene, β-carotene,
β-cryptoxanthin, lutein, violaxanthin, neoxanthin, and lycopene. β-carotene,
α-carotene, and β-cryptoxanthin are carotenes that are converted into vitamin
A or retinol in the body. β-carotene is the most widely studied carotenoid.
Lutein and zeaxanthin are both stored in the retina of the eye; however,
neither converts to vitamin A. Both are powerful antioxidants and may be
very important for healthy eyes. Carotenoids are singlet oxygen quenchers
and protect the oil from photo-oxidation (Psomiadou & Tsimidou, 1998). The
structure of some carotenoids of plant origin is provided in Figure 2.7. Among
the vegetable oils, palm oil is the richest source of carotenoids, especially β-
and α- carotenes. In comparison, other vegetable oils contain little amounts of
carotenoids, especially coconut, palmkernel, sesame, and groundnut oils have
very low content of carotenoids. The dark red-orange color of oil palm fruit is
due to the high concentration of carotenoids and anthocynanins. Crude palm
oil, extracted commercially by pressing, contains 400–1000 ppm of carot-
enoids, the variation being due to process conditions, species of oil palm,
and level of oxidation. Carotenoids in palm oil are α-carotene, β-carotene,
phytoene, phytofluene, cis β-carotene, cis α-carotene, δ-carotene, γ-carotene,
ζ-carotene, neurosporene, β-zeacarotene, α-zeacarotene, and lycopene (Table
2.8) (Yap et al., 1991; Jalani et al., 1997). Carotenoids are fat-soluble nutri-
ents and categorized as either xanthophylls or carotenes according to their
chemical composition. Carotenoids are singlet oxygen quenchers and protect
the oil from photo-oxidation (Psomiadou & Tsimidou, 1998).

TABLE 2.8 Composition of Carotenoids in Palm Oil, Given as % of Total Carotenoids (Yap
et al., 1991; Jalani et al., 1997).
Carotenoids Elaeis guineesis variety Elaeis oleifera variety crude
crude palm oil palm oil
Total (ppm) 500–700 4300–4600
Phytoene 1.27 1.12
Cis β-carotene 0.68 0.48
Phytofluene 0.06 Trace
68 Natural Antioxidants: Applications in Foods of Animal Origin

TABLE 2.8 (Continued)


Carotenoids Elaeis guineesis variety Elaeis oleifera variety crude
crude palm oil palm oil
β-Carotene 56.02 54.08
α-Carotene 35.06 40.38
Cis α-carotene 2.49 2.30
ζ-Carotene 0.69 0.36
γ-Carotene 0.33 0.08
δ-Carotene 0.83 0.09
Neurosporene 0.29 0.04
β-Zeacarotene 0.74 0.57
α-Zeacarotene 0.23 0.43
Lycopene 1.30 0.07

~-Carotene

a-Carotene

~-carotene

l5- carotene

FIGURE 2.7 Different carotenoids of vegetable oils.


Natural Antioxidants: Occurrence and Their Role in Food Preservation 69

2.13 PHYTIC ACID

Phytic acid is one of the bioactive compounds that are being intensively
studied to evaluate their effects on health. It has been shown to have poten-
tial as anticancer agent which only affects malignant cells and does not
affect normal cells and tissues (Vucenik & Shamsuddin, 2003). Phytic
acid is a simple ranged carbohydrate with six phosphate groups attached to
each carbon (Shamsuddin, 2002). It serves as the major phosphorus storage
compound in plant in the seed, as well as being a natural antioxidant by its
chelating properties and reduction of the catalytic activities of many divalent
transition metals (Verghese et al., 2006). The chelation ability of phytic acid
with minerals has been suggested to have beneficial effects toward lowering
serum cholesterol and triglycerides and suppression of iron-mediated oxida-
tion (Lee et al., 2005). A variety of benefits of phytic acid on human health
has also been reported including its potential as an anti-cancer property in
soft tissue, colon, prostate, metastatic, and mammary cancers. It may also act
as an inhibitor for renal stone development (Dost & Tokul, 2006). In whole
grain cereals such as corn, wheat, and rice, the ranges of phytic acid is from
1.5 to 6.4% while defatted and dehulled oilseed meals such as soy, peanut,
and sesame contain 1.5% or more of the compound (Grases et al., 2004).
Phytic acid is primarily found in the outer layers (bran) of unpolished rice.
Phytin is a white amorphous powder, odorless and tasteless, almost insol-
uble in water, soluble in dilute mineral acids and in some organic acids. One
part phytin dissolves in 10 parts of 1 N hydrochloric acid and forms a clear
solution. According to some authors, phytin contains 36% organically bound
phosphoric acid. Upon heating with dilute acids, alkali, and water, phytin hydro-
lyzes to give o-phosphoric acid and the cyclitol myo-inositol as end products.
These are obtained together with some other products of semi-degradation.
Phytic acid (myo-inositol 1,2,3,4,5,6 hexakisphosphate) is the most abun-
dant form of phosphorus in rice and is virtually indigestible by humans or
non-ruminant livestock and hampers the nutritional value of rice and its
milling by-product rice bran (Larson et al., 2000). Studies have demonstrated
that the antinutrient effect of phytic acid can be manifested only when large
quantities of phytic acid are consumed in combination with a diet poor in
oligoelements (Shamsuddin & Vucenik, 2005). IP6 (phytic acid, phytin) is a
6-phosphate ester of inositol (Saad et al., 2011). It exists in almost all plants
as its mixed calcium and magnesium salts (phytin) and, especially, seeds and
grains contain it in a lot of amount.
It is considered as the storage of organic phosphates of plants with
60~90% of entire phosphorus quantity being in the form of phytin (Saad et
70 Natural Antioxidants: Applications in Foods of Animal Origin

al., 2011). For rice bran, its concentration is particularly high and 9.5~14.5%
of rice bran is occupied by phytin (Saad et al., 2011), therefore, rice bran is
likely to be appropriate material for IP6. The most outstanding feature of
phytic acid is its strong metal chelate function (Saad et al., 2011), allowing
metal ions such as ferrum which often adversely affect the production or
storage of food in various forms to be removed or deactivated. Compared to
other chelate agents, it is distinctively effective in wider pH range (Saad et
al., 2011). Besides this function, it is known to have strong pH buffer action
(Saad et al., 2011), their derived effects of preventing the change of proper-
ties or colors and antioxidation effect (Saad et al., 2011). In recent years,
various physiologically active functions of phytic acid within living bodies
have been reported including the prevention of urinary and nephritic calculi
(Shamsuddin, 2002; Vucenik & Shamsuddin, 2003; Shamsuddin & Vucenik,
2005), prevention of colic cancer (Shamsuddin, 2002; Vucenik & Sham-
suddin, 2003; Shamsuddin & Vucenik, 2005), and suppression of bacterial
plaque formation (Shamsuddin, 2002; Vucenik & Shamsuddin, 2003; Sham-
suddin & Vucenik, 2005). In addition, other carcinostatic effects have also
been suggested (Shamsuddin, 2002; Vucenik & Shamsuddin, 2003; Sham-
suddin & Vucenik, 2005). Furthermore, as notable functions of phytic acid,
the deodorant effect of body odor, bad breath or uraroma (Shamsuddin, 2002;
Vucenik & Shamsuddin, 2003; Shamsuddin & Vucenik, 2005), prevention
of acute alcoholism (Saad et al., 2011), and enrichment of meat or fish taste
(Saad et al., 2011) are popular. These effects of phytic acid provide food
products with practical added values.

2.13.1 PHYTIC ACID IN FOOD PRESERVATION

Phytic acid (known as inositol hexakisphosphate, IP6, or phytate when in


salt form) is an organic acid extracted from rice bran (Fig. 2.8). Phytic acid is
used as an acidulant for pH adjustment. Phytic acid binds to metals strongly
because of strong chelating effect. Moreover, phytic acid shows antioxidant
action and prevention of color degradation. Phytic acid has been approved
as GRAS by the Food and Drug Administration (FDA) in the United States.
The most outstanding feature of phytic acid is its strong metal chelate func-
tion, allowing metal ions such as ferrum (Fe) which often adversely affect
the production or storage of food in various forms to be removed or deacti-
vated. Phytic acid is the best acidulant because it lowers pH level the most
at the same concentration. In conclusion, phytic acid has a mild, not a strong
or sharp acid taste relatively. Phytic acid does not affect the original taste
Natural Antioxidants: Occurrence and Their Role in Food Preservation 71

of food or beverage and only low concentration of it is required to achieve


the desired pH level. Phytic acid has the potential to prevent color degra-
dation in food or beverage including anthocyanin. Phytic acid is the most
potent natural iron chelator and has strong bacteriostatic and antioxidant
action (Graf et al., 1987; Graf & Eaton, 1990). Phytic acid is found to have
similar iron-chelating properties as desferrioxamine, a drug commonly used
to kill germs, tumor cells, or to remove undesirable minerals from the body
(Hawkins et al., 1993). The chelating stability constants of magnesium ion
and calcium ion of phytic acid are compared favorably with that of EDTA.
In fruits and vegetables, phytic acid helps to prevent oxidative browning
by inhibiting polyphenol oxidase. Phytic acid may be used as a safe preser-
vative and antioxidant in food products (Graf et al., 1987). Prevention of
browning of cut lotus root by phytic acid was investigated by immersing cut
lotus root in 0.5% phytic acid, 1.0% phytic acid, and distilled water with no
additives (control) then removed after 1 h. The cut lotus root with phytic acid
showed significant prevention of browning. The chelate action of phytic acid
compared to synthetic chelating agent, sodium metaphosphate was studied.
Sodium metaphosphate, an effective metal ion chelator has the greatest
salt forming activity among phosphates, particularly with calcium salts.
Iron chelate ability of phytic acid was superior to sodium metaphosphate
at pH 5.0. Phytic acid sequesters metal ions promoted oxidation, discol-
oration, and loss of flavor. Iron may cause discoloration in wine or fruit
juice. Hence, phytic acid can be added to chelate polyvalent iron cations to
prevent or treat these problems and make a wine more stable and commer-
cially acceptable. Phytic acid is a natural antioxidant. Phytic acid forms a
chelate with iron, thereby preventing the radical formation and oxidative
damage. It blocks the formation of hydroxyl radicals and suppresses lipid
peroxidation. In fruits and vegetables, phytic acid helps to prevent oxidative
browning by inhibiting polyphenol oxidase. Phytic acid may be used as a
safe preservative and antioxidant in food products (Graf et al., 1987). Graf
et al. (1987) reported the effects of added phytate upon iron-mediated OH
production and arachidonic acid peroxidation. Substantial amount of OH is
produced by a superoxide-generating system in the presence of iron alone.
Even greater amounts of OH are evolved if adenosine diphosphate (ADP)
is added to chelate the iron. Generation of this oxyradical, however, is
completely blocked by the addition of micromolar amounts of phytic acid. It
is important to note that the inhibition of OH generation is found over a wide
range of phytate:iron ratios from 1:4 to 20:1 (Graf et al., 1984). The effect is
due to occupation of all iron coordination sites by phytate; all iron-phytate
chelates prepared were completely soluble. Similarly, phytate prevents the
72 Natural Antioxidants: Applications in Foods of Animal Origin

peroxidation of arachidonic acid driven by AA and iron. Substantial amount


of malondialdehyde arises from arachidonic acid in the presence of free iron
or of an iron–ADP chelate. However, the addition of phytate prevents this
iron-dependent generation of malondialdehyde. The magnitude of the effect
of chelating agents on OH formation does not directly correspond to that on
lipid peroxidation, suggesting that different reactions may be involved in the
two processes and that, during lipid peroxidation, iron may catalyze several
steps, for example, OH-dependent hydrogen abstraction, OH-independent
formation of lipid peroxides, and catalysis of the formation of the final alde-
hydic cleavage products. Phytic acid is an antioxidant and chelating agent.
It suppresses oxidative reactions catalyzed by iron. In plant seeds phytic
acid helps to reduce the oxidation of its components but when ingested by
humans it may reduce the risk of colon cancer and some other IBDs. The
addition of phytic acids to foods improves its shelf life. It is also used as
an antioxidant in many industrial applications. Toxicity studies of phytic
acid revealed that single-dose test acute oral LD50 is 0.9 g/kg in the case of
mouse and is 0.41 g/kg in the case of rat. Repeat-dose studies for 12 weeks,
a non-toxic amount is 300 mg/kg/day in the case of rat. Reverse mutation
test, chromosome aberration examination test, micronucleus test, all were
found to be negative.

Patient

Patient Patient

Patient Patient

FIGURE 2.8 Structure of phytic acid.

2.13.2 FOOD APPLICATIONS OF PHYTIC ACID

Food applications of phytic acid includes, preservation of oils and fats in


tofu and deep-fried tofu; chelate action in miso, soy sauce, pickle, meat
industry products, canned foods, and soft drinks; browning prevention of
Natural Antioxidants: Occurrence and Their Role in Food Preservation 73

fruit juice; sterilization/bacteriostatic action in boiling noodles; deodorant


action in mutton meat; growth promotion action of lactic acid bacterium in
fermented foods; acidulant action in soft drinks and pickled plums; struvite
production prevention of canned foods; return prevention of bleaching; and
brightness improvement of bean jam. Recommended dosage (%) for various
food products are: soft drink 0.02~0.1; agriculture and fishery canned foods
0.02~0.2; pickle 0.02~0.1; bean jams 0.02~0.1; and boiling noodle 0.5~0.7.

2.14 SESAME LIGNANS

Crude or virgin sesame oil has these unique bioactive lignans namely
sesamin, sesamolin, sesaminol, and sesamolinol which occur with their
breakdown products like sesamol (from sesamolin) (Bhatnagar et al.,
2015). Sesame oil lignans are reported to have unique bioactive, functional,
physiological, and nutritional properties (Moazzami & Kamal-Eldin, 2006;
Smeds et al., 2007; Namiki, 2007). Sesame seeds contain 0.26–1.16% of
lignans mainly as sesamin, sesamolin, sesaminol, and sesamolinol (Moaz-
zami & Kamal-Eldin, 2006) and sesamol is a minor component of the total
lignans. Sesamin and sesamolin are usually present to an extent of 0.4 and
0.3% in sesame oil, respectively (Namiki, 2007). Sesame seeds and its oil
have unique physiological and nutritional properties, which are attributed
to the presence of oil soluble lignans such as sesamin and sesamolin and
oil insoluble lignans present as lignan glucosides namely sesaminol di- and
triglucosides, sesamolinol diglucoside, pinoresinol mono-, di-, and triglu-
cosides, and other glucosides of lariciresinol, 7-hydroxy matairesinol, and
medioresinol in minor amounts (Figs. 2.9 and 2.10) (Smeds et al., 2007);
(Namiki, 2007); (Milder et al., 2005); (Katsuzaki et al., 1992). Sesame seeds
contain 0.26–1.16% of lignans mainly as sesamin, sesamolin, sesaminol,
and sesamolinol (Moazzami & Kamal-Eldin, 2006). Lignans are a group
of natural compounds which are defined as an oxidative coupling product
of β-hydroxyphenylpropane. Sesamin has a typical lignan structure of β-β′
(8-8′) linked product of two coniferyl alcohol radicals. Sesamolin has a
unique structure involving one acetal oxygen bridge in a sesamin type struc-
ture. Both sesamin and sesamolin are characteristic lignans of sesame seed
(Namiki, 2007). Hydrolysis of sesamolin produces two breakdown prod-
ucts namely sesamol and samin (Fukuda et al., 1986a). Samin and sesamol
further combine to form sesaminol, another major sesame lignan (Nagata et
al., 1987).
74 Natural Antioxidants: Applications in Foods of Animal Origin

~ 0 0

YOH
OH

OCH,
Sesamolinol ocHl
Pinoresinol Piperitol

O':. O CH
CH,Oxi~ ,O
)C(,
I
"" '· ~ o
HO 0
HO
A
(
OH
..

~I ""I
::,... OCH,

Sesangolin Hydroxymata iresinol


OH OCH,
H

Larisiresinol

FIGURE 2.9 Oil soluble lignans of sesame seed.

Sesaminol
monoglucoside
CH,O

Ctt,OH
C H,O~I

0
OH

H
CU,OI IO 011
ott~
11 1
,: ° Pinoresinol Pinoresinol
0>0
diglucoside monoglucoside
OH 0
OH OH

Pinoresinol Pinoresi nol


triglucoside diglucoside

FIGURE 2.10 Oil insoluble lignans of sesame seed.


Natural Antioxidants: Occurrence and Their Role in Food Preservation 75

2.14.1 ANTIOXIDATIVE EFFECT OF SESAME LIGNANS

Sesaminol has sesamol as a moiety and has far stronger antioxidative activity
than sesamol because sesamol is easily dimerized and its products have lower
activity (Fukuda et al., 1986a). The markedly strong and stable antioxidative
property may be provided by the presence of a bulky samin group at the
ortho position of the phenol group in sesamol similar to the BHT molecule
(Namiki, 2007). Sesamin is the main characteristic lignan of sesame seed
with a content of about 0.4% in seed oil, but it has no free phenol group and
showed very weak or no antioxidative effect in conventional in vitro tests.
However, sesamin exhibits significant physiological activities assumed to
be due to antioxidative activity in vivo. Another important issue concerning
the stereochemical structure of sesame lignans is the fact that the artifact
episesamin, which is produced during food processing, has stronger physi-
ological activities than native sesamin (Namiki, 2007). Sesame lignans have
been found to exhibit various unique functions, such as a synergistic effect
with tocopherols on vitamin E activity and the specific inhibition of fatty
acid metabolism. These are quite different from the activities of other poly-
phenolic antioxidants, including sesamol, and they do not always appear to
be based upon their antioxidative activity. These facts suggest the existence
of some unique biochemical activity in sesame lignans due to their charac-
teristic structures in addition to their antioxidative activities (Namiki, 2007).

2.14.2 SESAME LIGNANS IN FOOD PRESERVATION

Sesame oil is highly resistant to oxidative deterioration. In ancient Egypt


it was used for making mummies, and in Japan it has been evaluated as
the best oil for deep frying tempura because of its superior stability against
deterioration by heating. There are two different kinds of sesame oil, roasted
and unroasted. The antioxidative activities of these oils were demonstrated
in experiments with other common vegetable oils that were stored at 60 °C.
Soybean oil, rapeseed oil, and others showed rapid increase after about 10
days, whereas both roasted and unroasted sesame oils were very stable.
The unroasted oil remained unchanged for 30 days, while no oxidation was
observed even after 50 days in the roasted oil (Fukuda & Namiki, 1988).
Roasted sesame seed oil has a characteristic flavor and red-brown color
probably caused by the Maillard-type reaction during roasting. The anti-
oxidative activity increases mainly in proportion to the roasting tempera-
ture along with the brown color, indicating that some products of the roast
76 Natural Antioxidants: Applications in Foods of Animal Origin

reaction contribute to the antioxidative activity. Thus, the very strong anti-
oxidative activity of the roasted oil might result from the synergistic effect
of the combination of such effective factors as sesamol produced from sesa-
molin, γ-tocopherol, sesamin, and roasted products like melanoidin (Fukuda
et al., 1986b; Koizumi et al., 1996; Fukuda et al., 1996). It has also been
shown that when foodstuff covered with wet material is fried in roasted seed
oil, as in the case of Japanese tempura, sesamol is produced by the splitting
of sesamolin, resulting in the formation of a strong antioxidative coating on
the fried food (Fukuda et al., 1986b).

2.14.3 FOOD APPLICATIONS OF SESAME LIGNANS

There are various forms and methods of using sesame seed and oil in Asian
countries, particularly in China, Korea, and Japan, and people in these coun-
tries enjoy many kinds of foods containing sesame seed and oil with superb
taste and flavor. However, the use of sesame in the Western countries is
limited in variety and sesame is utilized mostly as topping on bread and
biscuits, with low consumption. In this respect, it will be necessary to develop
various sesame foods which will suit many people’s tastes throughout the
world. For example, one recommended form of sesame may be used in salad
dressing or seasoning containing ground sesame seed and oil, which can be
used with various vegetables. This use of sesame is delicious in taste and has
good digestibility with high nutritional value in combination with sesame
lignans and various vegetable components (Namiki, 2007).

2.15 ORYZANOL

Rice has been widely cultivated as one of the major food resources and
remains as staple food. With the advancement in rice milling technology,
by-products of rice milling such as rice bran is being produced. Rice bran
contains about 10–24% of oil which can be commercially utilized to produce
rice bran oil for edible as well as cosmetic purposes. Rice bran oil is loaded
with bioactive compounds such as γ-OZ, tocopherols, tocotrienols, PS,
and so forth, which contribute to the excellent stability and functionality
of rice bran oil. γ-OZ is a lipid soluble antioxidant/nutraceutical/bioactive
compound uniquely present in rice bran and rice bran oil. It has been widely
used in foods and cosmetics around the world. It is registered as a medicine
in Japan and South Korea. γ-OZ is a naturally occurring component in rice
Natural Antioxidants: Occurrence and Their Role in Food Preservation 77

bran and rice germ which consists of a mixture of ferulic acid esters of PS
and triterpene alcohols (Fig. 2.11). There are numerous reports indicating
the benefits, efficacy, and safety of γ-OZ (Rukumini & Raghuram, 1991).

26

0 0

H,co~
I ""' o H,CO~
I ""' o
HO .&- 28 29
HO .&-

cycloartenol ferulate 24-methylenecycloartenol ferulate

0 0

H 3CO~""'
I o
H 3 C0~ 0
HO .& HO,Jl--J

campesferol ferulate ~-sitosterol ferulate

H 3 CO~O
HO~
cyclobranol ferulate

FIGURE 2.11 Different components of γ-oryzanol.

2.15.1 HEALTH BENEFITS OF ORYZANOL

Wilson et al. (2007) reported that γ-OZ reduced plasma cholesterol in hyper-
cholesterolemic hamsters. Clinically, oral intake of rice bran oil (containing
naturally occurring γ-OZ) has been shown to alleviate hypercholesterolemia
and hyperlipidemia. γ-OZ has been advocated as treatment for relieving
menopausal symptoms. Besides, Oka et al. (2010) reported that cycloartenyl
ferulate, a component of rice bran oil-derived γ-OZ, inhibits mass cells
degranulation. The anti-inflammatory effects of γ-OZ in ulcerative colitis
induced in mice have also been reported (Rukumini & Raghuram, 1991).
78 Natural Antioxidants: Applications in Foods of Animal Origin

2.15.2 SAFETY STUDIES OF ORYZANOL

Oral and intraperitoneal administration of γ-OZ (10,000 ppm) showed no


abnormality generally and upon autopsy (Rukumini & Raghuram, 1991).
Similarly, no abnormalities were observed on subcutaneous administration of
γ-OZ (500 ppm). It has been reported that no abnormal finding observed in rats
after six months of continuous oral administration of γ-OZ (30–1000 ppm)
(Rukumini & Raghuram, 1991). No fetal teratogenicity observed in mouse
with the administration of γ-OZ (6–600 ppm) during pregnancy (Rukumini
& Raghuram, 1991). Oral administration of γ-OZ (2000 ppm) was given to
mouse for 72 weeks and rat for two years, respectively. No carcinogenicity
observed at the above dosage (Rukumini & Raghuram, 1991).

2.15.3 ANTIOXIDANT EFFECT OF ORYZANOL

The antioxidant effect of γ-OZ is well documented and has been reported
to be excellent in inhibiting lipid peroxidation. Kanno et al. (1985) reported
that γ-OZ (0.5~1%) inhibited thermal oxidative polymerization of soybean
oil. The antioxidant effect of γ-OZ is contributed by ferulic acid entity, mean-
while, BHT and α-T have been revealed to be heat resistant. In addition,
Oryza Oil & Fat Chemical Co. Ltd. showed that the antioxidant effect of
γ-OZ was potentiated with rice bran/germ amino acid and showed a syner-
gistic increase in antioxidant effect of γ-OZ (Rukumini & Raghuram, 1991).
The excellent heat resistance property of γ-OZ is highly suitable for its
incorporation in heat-processed foods. Currently in Japan, γ-OZ is approved
and listed as “antioxidant” under the list of chemical composition of food
additives. In addition, γ-OZ is being incorporated as antioxidant in cosmetic
products (Rukumini & Raghuram, 1991).

2.15.4 ORYZANOL IN FOOD PRESERVATION

The antioxidant effect of natural OZ concentrate (15.5% OZ), purified OZ


(80% OZ), and α-T (0.1%) on oxidative and thermal stability of sunflower
oil was studied. It was concluded that sunflower oil containing a combina-
tion of 1% OZ (80% purity) and 0.1% α-T exhibited a synergistic effect in
inhibiting primary and secondary oxidation products and also showed a very
high thermal stability (Sunil et al., 2015).
Natural Antioxidants: Occurrence and Their Role in Food Preservation 79

To provide nutraceutical such as OZ through food, two instant mixes


based on the Indian traditional food cuisine Bisibele bhath and Upma
(Bhath-OZ and Upma-OZ) were developed and evaluated for shelf life. The
formulations contained cereals, pulses, and spices along with OZ enriched
oil and were packed in 200 gauge/50 gauge metallized polyester packaging
material and stored under ambient (27 °C 65% relative humidity (RH)) and
accelerated conditions (37 °C/92% RH). Samples were withdrawn periodi-
cally and PV, free fatty acid value (FFA), fatty acid composition, OZ, and
total tocopherols content were estimated. Sensory evaluation of reconsti-
tuted products was also carried out. OZ content (610 and 550 mg%) did
not change appreciably in Bhath-OZ and Upma-OZ, respectively. The PV
under ambient condition increased from 1.1 to 9.3 meq.O2/kg and 2.24
to 9.02 meq.O2/kg during the six-month storage study at 27 °C and 65%
RH, while under accelerated conditions at 37 °C and 92% RH, it increased
from 1.12 to 8.54 meq.O2/kg and 2.24 to 6.96 meq.O2/kg during two-month
storage period. Bhath-OZ and Upma-OZ packed in metallized polyester
pouches stored at 27 °C and 65% RH had a shelf life of four months without
affecting the OZ content and quality of instant mixes during the storage
period (Baby Latha et al., 2014).
Biscuit is a well-known cereal based processed food and the fortification
of OZ into the biscuits will go a long way to provide antioxidant rich, highly
stable, and acceptable functional food to the consumers. Biscuits were
prepared with commercially available fat (CF) and oryzanol fortified fat
(OFF). The control biscuits (CB) and oryzanol fortified biscuits (OFB) were
packed in 200 gauge polypropylene pouches, stored at 27 °C with different
relative humidity (RH 11, 22, 32, 44, and 56%) and analyzed for its stability
during storage of 120 days. Critical moisture content of OFB (4.8%) was
slightly less than that of CB (5.3%). The fat content of the CB (12.2%) and
OFB (12.5%) did not change during storage while free fatty acid content
(0.36 and 0.60%) and PV (0.08 and 0.17 meq.O2/100 g biscuit), respectively,
for CB and OFB were showed small but significant changes during storage.
OZ content (292 mg) and RSA (81.1%) of OFB did not change during
storage. The biscuits had a shelf life of minimum three months at 27 °C. OZ
in OFB showed good stability during baking and storage of biscuits (Pras-
anth Kumar et al., 2014).
80 Natural Antioxidants: Applications in Foods of Animal Origin

2.16 APPLICATIONS OF NATURAL ANTIOXIDANTS/EXTRACTS


IN FOOD PRODUCTS PRESERVATION

Foods of plant origin are stabilized by addition of antioxidants less


frequently than foods of animal origin, perhaps with the exception of
edible and essential oils. In contrast to animal foods, foods of vegetable
origin usually contain natural antioxidants, such as tocopherols, carot-
enoids, or flavonoids in sufficient amounts. The pro-oxidative activity of
iron and other heavy metals is less dangerous in plant materials than that
of heme derivatives in animal products, as plant materials usually also
contain metal-chelating agents. The only important oxidation catalyst in
raw materials and foods of vegetable origin is a group of lipoxygenases
and related enzymes. Synthetic antioxidants prevailed for the stabiliza-
tion of foods of plant origin in earlier applications, but in the last decade
or two, natural antioxidants have been intensively applied, following
consumers’ wishes.
Lipids in foods of vegetable origin are usually more unsaturated than
that of animal origin; therefore, the initiation rate of oxidation reactions is
higher and natural antioxidants, originally present in foods are more rapidly
consumed than in lard or tallow and other animal fats. The stabilization of
products of vegetable origin against autoxidation is thus less efficient than
the stabilization of animal products. Protection factors of natural antioxi-
dants are several times higher in lard than in edible oils.
The initial concentration of natural antioxidants in plant foods is already
near the optimum so that a further addition of antioxidants has only a small
effect, but it is useful for those cases when rapid decomposition of antioxi-
dants is expected. For instance, additional natural antioxidants can be added
to foods heated to high temperature or stored for a long time (Löliger, 1991;
Pokorný &Trojáková, 2001).
Cereal products such as dehulled rice, white flour, or grits, are not usually
stabilized. In whole grain flours, enzymes have to be inactivated to increase
shelf life. After heating, natural antioxidants from brans are sufficient for
lipid stabilization (Table 2.9). Natural antioxidants may be added to break-
fast cereals, the shelf life of which should be long. Rice bran, stabilized
by extrusion, has high natural antioxidant content, and thus it was found
suitable as a component for breakfast cereals with high stability (Saunders,
1989; Pokorný &Trojáková, 2001).
Natural Antioxidants: Occurrence and Their Role in Food Preservation 81

TABLE 2.9 Natural Antioxidants Present in Cereal Brans (Rosa et al., 1999; Hídvégi and
Lásztity, 2003).
Nutrients (values/100 g) Rice bran Corn bran Oat bran Wheat bran
Vitamin E
Tocopherols (mg) 12.0 0.4 1.0 1.5
Tocotrienols (mg) 13.6 – – –
Total carotenoids (mg) 129.3 – – –
Gamma oryzanol (mg) 300.0 – – –
Phytosterols (mg) 341.1 – – –
Phytic acid (mg) 9500 620–1170 900–1420 520–1050

Aqueous extracts of natural antioxidants from other whole grains or


brans, tea extracts and fruit extracts may be used with good results (Baublis
et al., 2000a; Pokorný & Trojáková, 2001). The catalytic effect of iron is
eliminated by phytic acid (Baublis et al., 2000b; Pokorný & Trojáková,
2001). Natural amino acids—methionine and cystine, PL and uric acid—are
as active as synthetic antioxidants in breakfast cereals (Maestro-Duran &
Borha-Padilla, 1993; Pokorný & Trojáková, 2001). The nutritional value of
breakfast cereals is increased by the addition of flavonoids and related plant
antioxidants, which extend shelf life (Shukla, 1993; Pokorný & Trojáková,
2001). Breakfast cereals, fortified with vitamin A (a very unstable compound)
were efficiently stabilized with commercial phenolic natural antioxidants
(Fritsch et al., 1975; Pokorný & Trojáková, 2001). Browning products, often
present to improve the flavor of food products, may also help in their stabi-
lization. Another kind of cereal products are extruded products, such as flat
bread. Natural antioxidants may be added with flour and other additives to the
extruder barrel. They are thus uniformly distributed in the extruded product.
Spices are useful for stabilization as they impart interesting flavor notes to
the final product. The agreeable color of extruded snack products, mainly
due to carotenoids, rapidly disappears on storage. Therefore, it needs to be
stabilized. An oil-soluble liquid rosemary extract (4942 Rosmanox) and its
mixture with tocopherols (4993 Rosmanox E) preserved the natural color-
ation for more than seven months (Marcus, 1994; Pokorný & Trojáková,
2001). Various snacks are easily stabilized with salt-containing natural anti-
oxidants (Sharma et al., 1997; Pokorný &Trojáková, 2001). Some cereal
products contain added fat, mostly hydrogenated edible oil and/or fillings
also rich in fat. Even when hydrogenated oils are rather stable against oxida-
tion, off-flavors may arise on storage. Application of natural antioxidants
82 Natural Antioxidants: Applications in Foods of Animal Origin

may be useful in such products as their shelf life is expected to be long.


Both synthetic and natural antioxidants or mixtures of both additives are
available for these specialty products. Certain spices, Maillard products
and essential oils could also be tested for this purpose. Sugar-snap cookies
usually stabilized by BHA, are being stabilized with natural antioxidants as
a replacement. Ferulic acid and sodium phytate were found to be suitable as
natural antioxidants (Hix et al., 1997; Pokorný & Trojáková, 2001). Cookies
containing phytate were sensorially fully acceptable. In sugar cookies,
BHT may be replaced by casein, whey proteins, or Maillard reaction prod-
ucts without any loss of storage stability (Ferreira et al., 1996; Pokorný &
Trojáková, 2001). Active natural antioxidants are formed during Maillard
reactions in butter cookies (Bressa et al., 1996; Pokorný & Trojáková, 2001).
Coffee bean components, such as chlorogenic acid, caffeic acid, and roasted
coffee bean powder or extract, have been added to butter cookies to good
effect. The last two natural additives are more active than tocopherol (Ochi
et al., 1994; Pokorný & Trojáková, 2001). Ascorbic and erythorbic acids,
citric acid and its isopropyl ester act as synergists of tocopherols (Ochi et al.,
1993; Pokorný & Trojáková, 2001). The addition of spices, such as extracts
from lemongrass, clove leaves, black pepper leaves, and turmeric increased
the shelf life of cakes and also contributed to their characteristic flavor (Lean
& Mohamed, 1999; Pokorný & Trojáková, 2001). The keeping quality of
crackers and cookies is of great economic importance since these products
are often stored for extended periods before they are consumed (sometimes
after opening the packaging) and they are not protected from oxidation. A
soda cracker biscuit was processed using a fine powder of marjoram, spear-
mint, peppermint, and basil, and their purified diethyl ether extracts as natural
antioxidants. Addition of ether extract from each of the above four plant
materials gave an excellent antioxidative effect compared with the effect of
BHA at concentrations of 0.01, 0.02, and 0.03%. Addition of fine powder
of all plant materials at 0.5% level gave an antioxidant effect compared to
the control sample. Addition of a 1% mixture of equal amounts of the four
plant powders caused a pro-oxidant effect (Bassiouny et al., 1990; Pokorný
& Trojáková, 2001). Carotene in bread and crackers is stabilized against
oxidative bleaching by α-T and ascorbyl palmitate (Ranhotra et al., 1995;
Pokorný & Trojáková, 2001). Large losses of coloration, otherwise observed
during baking, were thus reduced. The shelf life of fruits and vegetables
is limited by factors other than lipid oxidation, for example, antioxidants
are added to fruit and mushrooms to prevent oxidation of polyphenols,
resulting in the enzymic browning (Nisperos-Carriedo et al., 1991; Pokorný
& Trojáková, 2001). The lipid content in fruit and vegetables is about 1%
Natural Antioxidants: Occurrence and Their Role in Food Preservation 83

or less, so that the effect of their rancidification may be masked by other,


sensorily more active substances. Fruits contain natural essential oils, which
possess antioxidant activities but are also easily oxidized. They are protected
by similar antioxidants as glyceridic oils. If lipoxygenases are deactivated
by blanching, the content of natural antioxidants (mostly flavonoids) would
be sufficient to protect the lipid fraction against oxidation. Natural antioxi-
dants are applied only exceptionally, for example to protect carotenoids or
anthocyanins against oxidation. Pigmented orange juice was stabilized with
AA and phenolic acids and pasteurized (Maccarone et al., 1988; Pokorný &
Trojáková, 2001). The use of natural antioxidants is more justified for the
stabilization of dried products. The stability of dehydrated mashed potatoes
was achieved with α-T or ascorbyl palmitate or with Prolong P (a mixture
of rosemary, thyme, and marjoram) with more success than with TBHQ
(Baardseth, 1989; Pokorný & Trojáková, 2001).
The application of synthetic antioxidants will probably be reduced
further. They could be replaced by natural or nature-identical antioxidants.
We believe that only few more new natural antioxidants will be introduced
to the market in the near future in addition to the rosemary extract currently
being used. Green tea extracts (prepared from dust, old leaves, and other
tea wastes) also have a fair prospect of market success. Tocopherols and
β-carotene will probably be increasingly used. Prolongation of the shelf life
of complex foods will be achieved mainly by modifying recipes, introducing
herbs and spices with a high concentration of natural antioxidants, using
high-oleic edible oils requiring lower antioxidant levels and using protein
hydrolysates, which act as good synergists.

2.17 REGULATORY STATUS OF NATURAL ANTIOXIDANT


EXTRACTS, CONCENTRATES, AND RESINS

Synthetic antioxidants (BHA, BHT, PG, TBHQ, and EDTA) are regulated
by the FDA as direct food additives. They may be used alone or in combina-
tion not to exceed 0.02% (200 ppm) of the final product in specified food
products (21CFR172.110). These antioxidants are considered to be safe and
suitable ingredients for use in meat, poultry, and egg products, alone or in
combination, not to exceed 0.02% of the fat content (FSIS Directive 7120.1.
revision 5). Some herbs, spice extracts, and oleoresins are GRAS. Some
are considered to be indirect additives (21 CFR Vol. 3. Part 101); as such,
solvents permitted for the extraction process and solvent residues allowed are
specified. Some extracts, concentrates, and resins are regulated by the FDA
84 Natural Antioxidants: Applications in Foods of Animal Origin

“Dietary Supplement Health and Education Act of 1994” and are considered
to be one (or more) of several defined dietary ingredients a vitamin, a mineral,
an herb or other botanical, amino acid, a dietary substance for use by man to
supplement the diet by increasing the total dietary intake, or a concentrate,
metabolite, constituent, extract, or combination of any ingredient described
in clause (A), (B), (C), (D), or (E) and is excluded from regulation as a food
additive. Extracts, concentrates, and resins are also regulated under the Food
Labeling Regulation, Amendments; Food Regulation Uniform Compliance
Date; and New Dietary Ingredient Premarket Notification Final Rule (1997).
If they are added to cause flavor or color changes, they are regulated as such
and specific quantities allowable for use in various foods are set forth. Based
on the number of various classifications under which an extract, concentrate,
or resin could be covered, allowable use levels vary widely (Brewer, 2011).

2.18 CONCLUSION

Plant and animal tissues contain unsaturated fatty acids, primarily in the
PL fraction of cell membranes. These lipids are especially susceptible to
oxidation because of their electron deficient double bonds. The breakdown
products of oxidation can produce off-odors, new flavors, loss of nutrient
content, and color deterioration. To manufacture high-quality, stable food
products, the most effective solution is often the addition of antioxidants,
either synthetic or natural, which can serve as “chain breakers,” by inter-
cepting the free radicals generated during various stages of oxidation or to
chelate metals. Chain-breaking antioxidants are generally the most effec-
tive. A common feature of these compounds is that they have one or more
aromatic rings (often phenolic) with one or more −OH groups capable of
donating H· to the oxidizing lipid. Synthetic antioxidants, such as BHA,
BHT, and PG, have one aromatic ring. The natural antioxidants AA and α-T
each have one aromatic ring as well. However, many of the natural anti-
oxidants (flavonoids and anthocyanins) have more than one aromatic ring.
The effectiveness of these aromatic antioxidants is generally proportional
to the number of −OH groups present on the aromatic ring(s). Depending
on the arrangement of the −OH groups, these compounds may also chelate
pro-oxidative metals. The facts that they are natural, and have antioxidative
activity that is as good or better than the synthetic antioxidants, make them
particularly attractive for commercial food processors because of consumer
demand for natural ingredients.
Natural Antioxidants: Occurrence and Their Role in Food Preservation 85

KEYWORDS

• antioxidants
• free radicals
• preservation
• applications

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CHAPTER 3

POTENTIAL APPLICATIONS OF
NATURAL ANTIOXIDANTS IN MEAT
AND MEAT PRODUCTS
RITUPARNA BANERJEE1,*, ARUN K VERMA2,
MOHAMMED WASIM SIDDIQUI3, B. M. NAVEENA1, and
V. V. KULKARNI1
1
ICAR-National Research Centre on Meat, Chengicherla, Hyderabad
500092, Telangana, India
ICAR-Central Institute of Research on Goats, Makhdoom, Farah,
2

Mathura 281122, Uttar Pradesh, India


Department of Food Science and Postharvest Technology, Bihar
3

Agricultural University, Sabour, Bhagalpur 813210, Bihar, India


Corresponding author. E-mail: [email protected]
*

CONTENTS

Abstract ......................................................................................................96
3.1 Introduction .......................................................................................96
3.2 Antioxidants: Mechanism of Action .................................................97
3.3 Synthetic or Natural? ........................................................................99
3.4 Natural Antioxidants .........................................................................99
3.5 Natural Antioxidants in Meat System .............................................102
3.6 Market Potential ..............................................................................124
3.7 Future Prospects ..............................................................................125
3.8 Conclusion ......................................................................................126
Keywords .................................................................................................127
References ................................................................................................127
96 Natural Antioxidants: Applications in Foods of Animal Origin

ABSTRACT

In recent years, there is a lot of buzz about natural antioxidants. Scientific


advances, awareness of personal health, increasing healthcare costs, busy
lifestyles, and technical advances in the meat industry have stimulated the
“green consumerism.” Demands for the natural ingredients have forced the
researchers as well as meat industry to go for natural alternatives for synthetic
antioxidants. In this journey numerous plant materials have been screened
for their potential to prevent protein and lipid peroxidation. The extracts of
these plant materials have also been screened for their active principles and
have been attempted in different meat and meat products at various concen-
trations or levels and the quality, acceptability of the products have been
assessed. In future, we can see many more natural alternatives of synthetic
antioxidants with better potency and functionality for meat products.

3.1 INTRODUCTION

Meat as a food has a complex physical structure and chemical composition


that is very prone to oxidation (Wood et al., 2008). The oxidative stability
of meat depends upon the interaction between endogenous anti- and pro-
oxidant substances and the substrates prone to oxidation including polyun-
saturated fatty acids (PUFA), cholesterol, proteins, and pigments (Bertelsen
et al., 2000). Additionally, a variety of intrinsic properties and processing
steps can pre-dispose meat to lipid oxidation. For example, meat from non-
ruminants is more prone to lipid oxidation than that of ruminants (Tichivan-
gana & Morrissey, 1985) due to greater concentrations of unsaturated fatty
acids (Enser et al., 1996); muscle with red fibers are more susceptible than
white fibers because they contain more iron and phospholipid (Wood et al.,
2004); processing of meat will accelerate lipid oxidation as the comminution
or grinding process will incorporates oxygen and increase surface area as a
result of particle size reduction (Gray et al., 1996).
Oxidation of lipids is a complex chemical process which involves the
development of off-flavor, decreases the acceptability of meat and meat
products by deteriorating their color, texture, and nutritive value, and can
ultimately precipitate health hazards and economic losses in terms of infe-
rior product quality (Naveena et al., 2008b). The oxidation process can be
reduced or inhibited through application of antioxidants. These antioxidants
could be either from synthetic or from natural sources; the latter includes
both endogenous natural antioxidants (present in meat itself) and exogenous
Potential Applications of Natural Antioxidants in Meat and Meat Products 97

natural antioxidants (present in plant materials and other natural sources).


Several endogenous antioxidants (including ubiquinone, glutathione, lipoic
acid, spermine, carnosine, and anserine) have been studied in skeletal
muscle (Decker et al., 2000). Both carnosine and anserine are histidyl dipep-
tides and the most abundant antioxidants in meat. Carnosine is present at
around 365 mg/100 g in beef (Purchas & Busboom, 2005) and 400 mg/100 g
in lamb (Purchas et al., 2004). Anserine is especially abundant in chicken
muscle. The antioxidant activity of these dipeptides may result from their
ability to chelate transition metals (Brown, 1981) and form complexes with
copper, zinc, and cobalt. Levels of Coenzyme Q10 (ubiquinone) in meat has
been estimated to be around 2 mg/100 g in both beef and mutton (Purchas
& Busboom, 2005). Glutathione, a component of glutathione peroxidase
enzymes, has an important antioxidant function. Glutathione levels in red
meat are estimated to be 12–26 mg/100 g in beef (Jones et al., 1992). In addi-
tion, numerous Maillard reaction products formed during cooking have also
been shown to have antioxidant activities (Bailey, 1988). However, none
of these antioxidant systems, individually or combined, have been shown
to sufficiently delay oxidation in meat or meat products under common
processing conditions (Decker & Mei, 1996). Therefore, the best strategy
to combat this problem is either supplementation of exogenous antioxidants
through dietary manipulation or incorporation during processing of products.
This chapter will deal with sources of natural antioxidants such as fruits,
vegetables, herbs, and spices as well as marine macroalgae, their application
in meat and meat products and effects on various quality and acceptability.

3.2 ANTIOXIDANTS: MECHANISM OF ACTION

Antioxidants are substances that at low concentrations retard the oxidation


of easily oxidizable biomolecules, such as lipids and proteins in meat prod-
ucts, thus improving shelf life of products by protecting them against dete-
rioration caused by oxidation (Karre et al., 2013).
According to their mechanism of action, antioxidants can be classified
into different groups:

• Antioxidants that act as radical scavengers (react with free radicals):


Two basic mechanisms are involved in this free radical scavenging
activity of antioxidants: (a) A chain breaking mechanism in which the
antioxidants break the chain reaction of radicals and avoid the propa-
gation step of lipid peroxidation process by donating electrons to the
98 Natural Antioxidants: Applications in Foods of Animal Origin

free radicals present in the system (Masuda et al., 2002; Amakura et


al., 2000). (b) The second mechanism involves the removal of reac-
tive oxygen species (ROS) and reactive nitrogen species (RNS) initi-
ators by quenching the chain initiator catalyst (Hamid et al., 2010).
Examples of antioxidants which scavenge free radicals are phenolic
compounds (tocopherols, butylated hydroxytoluene (BHT), butyl-
ated hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ), propyl
gallate (PG), lignans, flavonoids, and phenolic acids, carotenoids,
and so forth.
• Antioxidants that react with transition metals to form complexes, and
thus avoid the catalytic effect of the metals in the oxidation process.
Metal chelators decrease oxidation by preventing metal redox cycling,
forming insoluble metal complexes, or providing steric hindrance
between metals and food components or their oxidation intermedi-
ates (Graf & Eaton, 1990). The most common metal chelators used
in foods contain multiple carboxylic acid (e.g., ethylene diamine tetra
acetic acid (EDTA) and citric acid) or phosphate groups (e.g., poly-
phosphates and phytate). Chelators are typically water soluble but
many also exhibit lipid solubility (e.g., citric acid), thus allowing it
to inactivate metals in the lipid phase. Lignans, polyphenols, ascorbic
acid, and amino acids such as carnosine and histidine can also chelate
metals (Decker et al., 2001). Phenolics, which possess hydroxyl and
carboxyl groups are able to bind particularly well with metals like Fe
or Cu (Jung et al., 2003).
• Antioxidants that decompose peroxides and produce stable substances
which are unable to produce radicals, such as selenium (Se) containing
glutathione peroxidase, an antioxidative enzyme, which inactivate
free radicals and other oxidants, particularly hydrogen peroxide.
• Antioxidants which inactivate the singlet form of oxygen: In the
presence of a photosensitizer, such as chlorophylls and pheophy-
tins, singlet oxygen may be formed from ordinary triplet oxygen by
the action of light. This singlet form of oxygen is very reactive; it
is extremely important to deactivate it back to the triplet form very
rapidly to prevent the photo-oxidation process. Tocopherols, carot-
enoids, curcumin, phenolics, urate, and ascorbate can quench singlet
oxygen (Das & Das, 2002; Choe & Min, 2005).
• Antioxidants which prevent the enzymatic activity required for auto-
oxidation. Examples are flavonoids, phenolic acids, and gallates,
which deactivate the lipoxygenase.
Potential Applications of Natural Antioxidants in Meat and Meat Products 99

3.3 SYNTHETIC OR NATURAL?

Though there are several different types of available antioxidants, they can
be broadly grouped into two categories: natural and synthetic. While natural
antioxidants are those that can be harvested directly from any organic source
such as herbs, fruits, vegetables, and so forth, synthetic antioxidants are
compounds produced artificially and added to processed or pre-packaged
food to prevent rancidity, browning or to preserve the flavor and texture.
Synthetic antioxidants such as BHA, BHT, TBHQ, and PG have been widely
used in meat and meat products (Biswas et al., 2004; Formanek et al., 2001;
Jayathilakan et al., 2007) by the food processors as they are cheaper than
the natural ones. But the demand for natural antioxidants, especially of
plant origin has increased in the recent years due to the growing concern
among consumers about these synthetic antioxidants because of their poten-
tial toxicological effects (Naveena et al., 2008b). However, both of these
antioxidants differ in performance level; the effectiveness can be measured
by the number of peroxides formed in lipids over time and by their ability
to provide stability under different processing conditions. Both natural and
synthetic antioxidants act by donating electron density to fat and preventing
their oxidation but synthetic antioxidants have shown to possess a higher
performance than the natural ones. They differ in their fortification values
also. The natural antioxidants are known to have higher additional health
benefits in preventing cancer and heart diseases.

3.4 NATURAL ANTIOXIDANTS

Plants are persistently the generous source to supply man with valuable
bioactive substances (Tayel & El-Tras, 2012) and thus different plant prod-
ucts are being evaluated as natural antioxidants to improve the overall
quality of meat and meat products. The focus for using natural antioxidants
for the effective preservation of meat or meat products has almost exclu-
sively been on the use of plant phenolics or phenolic-containing extracts.
Phenolic compounds are plant secondary metabolites commonly found in
herbs and fruits, vegetables, grains and cereals, tea, coffee, and red and
white wines. Phenolic acids are phenols that possess carboxylic acid func-
tionality. Phenolic compounds can be broadly divided into two categories,
flavonoids and non-flavonoid polyphenols. Among phenolic compounds
found in plants, flavonoids are the most widely studied class of polyphenols
with respect to their antioxidant and biological activities. Flavonoids may be
100 Natural Antioxidants: Applications in Foods of Animal Origin

divided into different subclasses according to the degree of oxidation of the


heterocyclic ring: anthocyanins, flavonols, flavanones, flavanol, flavones,
and isoflavones (Scalbert & Williamson, 2000). Flavonols are the most ubiq-
uitous flavonoids in foods, and the main representatives are quercetin and
kaempferol. Onions, kale, broccoli, and blueberries are the richest sources.
Flavones are much less common than flavonols in fruit and vegetables.
Celery, parsley, wheat, millet, and skin of citrus fruits are important sources
of flavones. Anthocyanins are mainly found in red wine, certain leafy and
root vegetables, and are most abundant in fruits. Flavanols exist as catechins
or proanthocyanidins. Catechin and epicatechin are the main flavanols in
fruit, whereas gallocatechin, epigallocatechin, and epigallocatechin gallate
are found in certain seeds of leguminous plants, grapes, and more impor-
tantly in tea (Arts et al., 2000). Proanthocyanidins (condensed tannins) are
responsible for the astringent character of grapes, peaches, apples, pears,
berries, tea, wine, and bear. Flavanones are common in tomatoes, mint and
to a considerable extent in citrus fruit. Soya and its processed products are
the main source of isoflavones in the human diet. The importance of all these
antioxidant constituents of plant materials and other natural sources in the
maintenance of health and protection from coronary heart disease and cancer
is raising interest among scientists, food processors, and consumers as the
future trend is moving toward functional food with specific health effects.
The extraction of bioactive compounds is the first step in utilization of
natural antioxidants as additives in meat products. Generally the plant mate-
rial is cleaned, dried, and ground into fine powder followed by extraction.
The drying process has some undesirable effects on the constituent profile
of plant material; however, freeze-drying process retains higher levels of
phenolic content in plant samples than air-drying (Abascal et al., 2005).
There are many techniques to recover antioxidants from plants, such as
Soxhlet extraction, maceration, supercritical fluid extraction, subcritical
water extraction, and ultrasound assisted extraction but the solvent extrac-
tion process is the most commonly used procedure to prepare extracts from
plant materials due to their ease of use, efficiency, and wide applicability.
Table 3.1 represents the common solvents used for extraction of antioxi-
dant compound from different plant parts. Extraction efficiency is affected
by the chemical nature of phytochemicals, the extraction method used,
sample particle size, the solvent used, as well as the presence of interfering
substances (Stalikas, 2007) whereas the yield of extraction depends on the
solvent with varying polarity, pH, temperature, extraction time, and compo-
sition of the sample (Turkmen et al., 2006). Solvents, such as methanol,
ethanol, acetone, ethyl acetate, and their combinations have been used for
Potential Applications of Natural Antioxidants in Meat and Meat Products 101

the extraction of phenolics from plant materials, with different proportions of


water. Methanol has been generally found to be more efficient in extraction
of lower molecular weight polyphenols while the higher molecular weight
flavanols are better extracted with aqueous acetone (Prior et al., 2001, Guyot
et al., 2001). Ethanol is another good solvent for polyphenol extraction (Shi
et al., 2005). Water is the safest solvent but it is less efficient than the organic
solvents in extracting all the antioxidants. On the contrary, extra precaution
should be taken to remove all the traces of organic solvent as it may not be
acceptable for consumers if residues are left in the final product. In addition,
the cost-effectiveness of extraction process should also be taken in account
in order to reduce the cost of natural antioxidants and its wider exploitation
in meat industry.

TABLE 3.1 Extraction of Antioxidant Components from Different Sources and Its Application
in Meat Products.
Source Part used Extraction solvent Reference
Fruits
Bearberry Leaf 95% ethanol and Pegg et al. (2005)
50% acetone
Citrus paradisi (grape Bark Ethyl acetate, Sayari et al. (2015)
fruit) methanol, and water
Grape Seed 80% ethanol Shan et al. (2009)
Pomace Methanol Garrido et al. (2011)
Kinnow Peel Water Devatkal et al. (2010)
Pomegranate Peel Water Devatkal et al. (2010)
70% ethanol Tayel and El-Tras (2012)
80% ethanol Shan et al. (2009)
Prunus mume Fruit Methanol Jo et al. (2006)
Herbs and spices
Clove Bud 80% ethanol Shan et al. (2009)
Cinnamon Bark 70% ethanol Tayel and El-Tras (2012)
Cinnamon stick Cortex 80% ethanol Shan et al. (2009)
Fenugreek Seed 90% ethanol Mansour and Khalil (2000)
Green tea Leaf Water Rababah et al. (2011)
Rosemary & hissop Leaf and Dimethyl sulfoxide Fernandez-Lopez et al.
secondary (2003)
branches
Rosemary Leaf Deionized water Akarpat et al. (2008)
Leaf Acetone, hexane Naveena et al. (2013)
102 Natural Antioxidants: Applications in Foods of Animal Origin

TABLE 3.1 (Continued)


Source Part used Extraction solvent Reference
Mint Leaf Water Kanatt et al. (2007, 2008)
Leaf Water, ethanol, and Biswas et al. (2012)
50% ethanol:50%
water
Vegetables
Broccoli Flowering Water Banerjee et al. (2012)
head
Cauliflower Flowering Water Banerjee et al. (2015)
head Acetone:water (1:1)
Drumstick Leaf Water Das et al. (2012); Muthu-
kumar et al. (2014)
Green leafy vegetables Leaf 70% ethanol Kim et al. (2013a)
Butterbur, chamnamul,
bok choy, Chinese chives,
crown daisy, fatsia pump-
kin, sesame stonecrop
potato Peel 90% ethanol Mansour and Khalil
(2000)

3.5 NATURAL ANTIOXIDANTS IN MEAT SYSTEM

The natural antioxidants from plants, in several forms, have been obtained
from different sources such as fruits (grapes, pomegranate, date, and
kinnow), vegetables (broccoli, potato, drumstick, and curry leaves), herbs,
and spices (tea, rosemary, oregano, cinnamon, sage, thyme, mint, ginger,
and clove) and explored to decrease the lipid oxidation (Akarpat et al., 2008;
Banerjee et al., 2012; Das et al., 2012; Devatkal et al., 2010; Huang et al.,
2011; Kanatt et al., 2007; Mansour & Khalil, 2000; McCarthy et al., 2001a,
b; Rojas & Brewer, 2007, 2008; Shan et al., 2009).

3.5.1 FRUIT-BASED ANTIOXIDANTS

Fruits have gathered interest from the public and scientific groups because of
their health-promoting properties. The benefits of fruits have been credited
to their high phenolic content, which acts as antioxidants (Zuo et al., 2002).
Numerous studies conducted on the antioxidant potential of fruits in meat
and meat products are presented below.
Potential Applications of Natural Antioxidants in Meat and Meat Products 103

3.5.1.1 BAEL

The bael fruit (Aegle marmelos L. Correa) is known in India since pre-historic
times. This fruit is native to Northern India but widely found throughout the
Indian Peninsula (Rahman & Pravin, 2014). Bael fruit pulp contains many
functional and bioactive compounds such as dietary fiber, carotenoids, pheno-
lics, alkaloids, coumarins, flavonoids, terpenoids, and other antioxidants
(Suvimol & Anprung, 2008). Major antioxidants in bael fruit are phenolics,
flavonoids, carotenoids, and vitamin C (Roy & Khurdiya, 1995). Bael fruit
is rich in carbohydrates, fibers and is also a good source of protein, vitamins,
and minerals (Ramulu & Rao, 2003). Kamalakkannan and Prince (2003a,
2003b) reported that the aqueous extract of the bael fruit pulp possesses potent
antioxidant effects. Abdullakasim et al. (2007) also reported that the bael
fruit drink was found to possess high quantities of total phenolic compounds
(83.89/37.6 mg gallic acid equivalents/100 mL) and was also a good anti-
oxidant in both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and photochemilumi-
nescence assays. Das et al. (2014) had reported the antioxidant potential of
bael pulp residue (BPR), a by-product of bael fruit pulp in goat meat nuggets.
BPR was found to be a rich source of phenolic compounds and contained
15.16 mg GAE/g dry weight (DW) total phenolics. Incorporation of BPR
(0.25, 0.5%) in goat meat nuggets improved the lightness and redness values,
whereas yellowness value remained unaffected. The lighter and redder goat
meat nuggets looked very much appealing and could be helpful in attracting
the consumers. Lower thiobarbituric acid reactive substances (TBARS) value
was recorded in BPR treated nuggets; lowest value was observed in 0.5%
treatment. Incorporation of BPR may enrich meat products with dietary fiber
and antioxidants, and can be helpful in enhancing their physiological and
functional values as well as oxidative stability.

3.5.1.2 BEARBERRY

Bearberries also known as Uva Ursi, is a member of genus Arctostaphylos


and is one of the lesser-studied source of natural antioxidants. Tradition-
ally, the astringent leaves of bearberry plant have been used in the treat-
ment of bladder infections and other afflictions of the urinary tract. The plant
contains arbutin, ursolic acid, tannic acid, gallic acid, some essential oils
(EOs), hydroquinones, phenolic glycosides, and flavonoids (Hansel et al.,
1992). O'Brien et al. (2006) investigated the antioxidant activity of several
plant extracts under oxidative stress in cells and found bearberry to be a
104 Natural Antioxidants: Applications in Foods of Animal Origin

strong antioxidant. The total polyphenol content of bearberry extract was


reported by Carpenter et al. (2007) as 57.4 g ± 1.73 GAE/100 g. Bearberry
extract reduced the lipid oxidation in raw and cooked pork patties during
storage up to 12 days at 4 °C and sensory properties were affected by its
addition. Pegg et al. (2005) had reported that bearberry leaf extract possesses
marked antioxidant activity in model and meat systems. Crude leaf extract,
and its fractions (acetone, ethanol) inhibited TBARS formation in cooked
meat systems after seven days of refrigerated storage.

3.5.1.3 CAROB FRUIT

The Carob is the fruit of an evergreen Ceratonia silique L. cultivated in the


Mediterranean area. Use of the whole carob fruit for consumption is rather
limited, due to a high level of tannins causing astringency (Avallone et al.,
1997). The two main carob pod constituents are pulp (90%) and seed (10%).
The seed coat contains antioxidants (Batista et al., 1996). Phenolic contents
of pulps and leaves from carob tree have been reported (Avallone et al.,
1997; Corsi et al., 2002; Kumazawa et al., 2002; Owen et al., 2003; Makris
& Kefalas, 2004). Flavonol glycoside, 4`-p-hydroxybenzoylisorhamnetin-
3-O-α-L-rhamnopyranoside named ceratoside, together with the known
kaempferol-3-O- α-L-rhamnopyranoside (afzelin), quercetin-3-O- α -L-arabi-
nofuranoside (auriculain), quercetin-3-O- α-L-rhamnopyranoside, β-sitosterol,
and β-sitosterol-3-O- β -D-glucoside were isolated from carob seeds (Gohar
et al., 2009). Vaya and Mahmood (2006) observed that the carob leaves are
rich in flavonoids; and more than nine compounds were identified. Researchers
had isolated and identified the major polyphenols in carob fibers (Owen et al.,
2003; Papagiannopoulos et al., 2004).
Bastida et al. (2009) evaluated the effect of adding condensed tannins
in the form of non-purified (Liposterine®) or purified (Exxenterol®) extracts
obtained from carob fruit to prevent oxidation in lipid-cooked pork meat
systems during chilling and frozen storage. The antioxidant activity of these
extracts was compared with that of α-tocopherol (TM). TBARS levels were
significantly lower in samples containing Liposterine (LM), Exxenterol
(EM), and TM than in control sample under chilled storage. TBARS forma-
tion was similar (P > 0.05) for LM and EM but significantly lower than that
for TM. Thermal oxidation compounds were lower (P < 0.05) in EM than in
LM or TM, which is also having nutritional importance as thermal oxidation
products are potentially toxic. Therefore, Carob extract has the potential to
improve the fat stability and toxicological safety of meat systems.
Potential Applications of Natural Antioxidants in Meat and Meat Products 105

3.5.1.4 CITRUS FRUITS

Citrus fruits are an important source of bioactive compounds including


antioxidants such as ascorbic acid, flavonoids, phenolic compounds, and
pectins that are important to human nutrition (Fernandez-Lopez et al., 2005;
Jayaprakasha & Patil, 2007). Flavanones, flavones, and flavonols are three
types of flavonoids which are present in citrus fruit (Calabro et al., 2004).
The main flavonoids found in citrus species are hesperidine, narirutin,
naringin, and eriocitrin (Schieber et al., 2001). Epidemiological studies
on dietary citrus flavonoids improved a reduction in risk of coronary heart
disease (Di Majo et al., 2005) and are attracting more and more attention
not only due to their antioxidant potential, but also as anti-carcinogenic and
anti-inflammatory agents because of their lipid anti-peroxidation effects
(Stavric, 1993; Elangovan et al., 1994; Martın et al., 2002). In addition,
citrus by-products represent a rich source of naturally occurring flavonoids.
The peel which constitutes almost one-half of the fruit mass, contains the
highest concentrations of flavonoids in the citrus fruit (Anagnostopoulou et
al., 2006; Manthley & Grohmann, 2001).
The antioxidant effects of orange and lemon extracts were investigated
in cooked Swedish-style meatballs by Fernandez-Lopez et al. (2005). Anti-
oxidant activities of each natural extract were expressed as stability index
(SI). The SI of orange extract (1.30) was higher (P < 0.05) than that of lemon
extract (1.19). TBARS data indicated that orange extract was superior in
reducing the lipid oxidation of cooked products in comparison to lemon
extract throughout the storage period (8 ± 1 °C, 12 days). Viuda-Martos et
al. (2009) stated that the addition of citrus waste water (5, 10%) obtained
as co-product during the extraction of dietary fiber to the bologna samples
reduced the residual nitrite levels and the degree of lipid oxidation. The
flavonoids hesperidin and narirutin were detected in all the samples. Viuda-
Martos et al. (2010) studied the effect of adding orange dietary fiber (1%),
rosemary EO (0.02%), or thyme EO (0.02%) and the storage conditions on
the quality characteristics and the shelf life of mortadella, a bologna-type
sausage. Color coordinates lightness (L*) and yellowness (b*) were affected
by the fiber content. The treatments had lower level of residual nitrite; the
extent of lipid oxidation was also reduced, and analysis of the samples
revealed the presence of the flavonoids, hesperidin, and narirutin. Anti-
oxidant and antibacterial properties of Citrus paradisi fruit barks (CPFB)
extract was evaluated in turkey sausage formulation (Sayari et al., 2015).
The CPFB water extract contained a high amount of total phenolics and
flavonoids (118 ± 4 mg GAE/g dried extract and 794 ± 8.7 mg QE/g dried
106 Natural Antioxidants: Applications in Foods of Animal Origin

extract, respectively) and showed important antioxidant and antibacterial


activities. Water extracts from CPFB showed a higher antioxidant activity
than sodium lactate.
Kinnow or Tangerine (Citrus reticulata), a citrus fruit variety is grown
in North Indian states, mainly Punjab and Rajasthan. In the process of juice
extraction, 30–34% of kinnow peel is obtained as a major by-product, which
is a very rich source of vitamin C, carotenoids, limonene, and polyphenolic
antioxidants (Anwar et al., 2008). Kinnow rind powder (KRP), pomegranate
rind powder (PRP), and pomegranate seed powder (PSP) extracts were
investigated (Devatkal et al., 2010) in goat meat patties stored at 4 ± 1 °C. It
was found that these extracts are rich in phenolic compounds and have free
radical scavenging activity. Hunter Lab L* value was significantly (P < 0.05)
lower in PRP followed by PSP and KRP treated patties. Sensory evalua-
tion indicated no significant differences among patties. Further, a significant
(P < 0.05) reduction in TBARS values during storage of goat meat patties
was observed in PRP, PSP, and KRP as compared to control patties. The
overall antioxidant effect was in the order of PRP > PSP > KRP.
Effects of salt, kinnow, and pomegranate fruit by-product powders on
color and oxidative stability of raw ground goat meat stored at 4 ± 1 °C was
evaluated by Devatkal and Naveena (2010). Five treatments were evalu-
ated including control (no treatment), meat with salt (MS) (meat + 2% salt),
KRP (meat + 2% salt + 2% KRP), PRP (meat + 2% salt + 2% PRP), and
PSP (meat + 2% salt + 2% PSP). TBARS values were higher (P < 0.05)
in MS followed by control and KRP samples compared to PRP and PSP
samples throughout storage. The PSP treated samples showed lowest
TBARS values than others. Percent reduction of TBARS values was highest
in PSP (443%) followed by PRP (227%) and KRP (123%). Salt accelerated
the TBARS formation and by-products of kinnow and pomegranate fruits
counteracted this effect. The overall antioxidant effect was in the order of
PSP > PRP > KRP > control > MS.

3.5.1.5 CRANBERRY

Cranberries are a group of evergreen dwarf shrubs or trailing vines in the


subgenus Oxycoccus of the genus Vaccinium. It is generally processed
into three basic categories: fresh (5%); sauce products, concentrate, and
various value-added applications (35%); and juice drinks (60%) (NASS,
2001). Extensive processing of cranberry for different products such as
juice yields cranberry pomace as a by-product, a cheap source of natural
Potential Applications of Natural Antioxidants in Meat and Meat Products 107

antioxidants with potential health benefits as food ingredients (Yamaguchi


et al., 1999; Koga et al., 1999). Cranberry contains various classes of poly-
phenolic compounds, including phenolic acids, flavonol glycosides, antho-
cyanins, and proanthocyanidins (Foo et al., 2000; Chen et al., 2001; Sun
et al., 2002; Zuo et al., 2002). Studies have shown that cranberry phenolic
compounds possess antioxidant activity against peroxyl (Wang & Stretch,
2001; Gunes et al., 2002; Zheng & Wang, 2003), superoxide (Wang & Jiao,
2000), hydroxyl (Wang & Jiao, 2000) and DPPH (Yan et al., 2002) radicals,
hydrogen peroxide, and singlet oxygen (Wang & Jiao, 2000). A major unde-
rutilized by-product from cranberry juice production is cranberry press cake,
containing seeds and skins. Cranberry press cake contains many phenolic
compounds (Zheng & Shetty, 2000) and could be used as a potential source
for preparing antioxidant extracts (Moure et al., 2001).
The potential of cranberry press cake and cranberry juice powder as
antioxidants in meat and poultry products has been the interest of several
researchers (Larrain et al., 2008; Raghavan & Richards, 2006, 2007). The
cranberry juice powder extract (extracted with chloroform) was superior
(P < 0.05) to cranberry press cake extract (extracted with either ethyl acetate
or ethanol) in inhibiting lipid oxidation in vacuum-packaged mechanically
separated turkey (MST) (Raghavan & Richards, 2006).
The ability of components of cranberry powder to inhibit lipid oxida-
tion processes in MST and cooked ground pork was assessed by Lee et al.
(2006). Fraction of extract enriched in flavonols showed the greatest inhibi-
tory effect on lipid oxidation of cooked ground pork with 81% inhibition, in
comparison to other fractions (phenolic acids, anthocyanin, and proanthocy-
anidin), over the entire storage period. Crude extract treated cooked ground
pork exhibited up to 51% inhibition on TBARS formation. Concentrated
cranberry juice powder (0.32%) was effective in retarding TBARS forma-
tion and rancidity development in MST during 14 days of storage at 2 °C.
Quercetin, a non-glycosylated flavonol present in cranberry powder, inhib-
ited lipid oxidation in MST at low concentrations. Ethanol was the most
effective carrier solvent of polyphenolics compared to propylene glycol and
water as carriers.

3.5.1.6 GRAPES

Grape seed extract has been reported to be one of the richest sources of natural
polyphenols, comprising flavanols, phenolic acids, catechins, proanthocy-
anidins, and anthocyanins. Among these, catechins and proanthocyanidins
108 Natural Antioxidants: Applications in Foods of Animal Origin

are the major groups representing about 77.6% of total polyphenols (Silvan
et al., 2013). The high amount of phenol groups in grape seed extract
explains their strong lipid oxidation inhibition and antimicrobial activity in
raw and cooked muscle foods (Ahn et al., 2007a; Brannan, 2008). Numerous
authors have mentioned the potent antioxidant effect of grape polyphenols
(Vitis vinifera) in pork (Carpenter et al., 2007; O’Grady et al., 2008), beef
(Rojas & Brewer, 2007, 2008), and poultries (Brannan, 2009; Mielnik et al.,
2006; Sayago-Ayerdi et al., 2009).
Kulkarni et al. (2011) compared grape seed extract (100, 300, 500 ppm)
with ascorbic acid and PG (100 ppm of fat) in lean beef sausages cooked
(70 °C), sliced and stored at −18 °C for four months and concluded that
samples prepared with the grape seed extract and PG retained their fresh-
ness, had less rancid odor and had lower TBARS values compared to
controls and ascorbic acid containing samples during the storage period. It
was also demonstrated that frankfurters prepared with addition of different
concentrations (0, 0.5, 1, 2, 3, 4, and 5%) of grape seed flour, had lower
oxidation level and enhanced protein and total dietary fiber (TDF) content
with increasing levels of grape seed flour (Ozvural & Vural, 2011). The
addition of red grape pomace extract (0.06 g/100 g) to pork burgers resulted
in color stability, lipid oxidation inhibition and yielded best overall accept-
ability after six days storage at 4 °C under aerobic conditions (Garrido et
al., 2011).
Grape seed extract (ActiVin™) and pine bark extract (Pycnogenol®)
significantly improved the oxidative stability of cooked beef at three days
of refrigerated storage. TBARS values, hexanal content, and warmed over
flavor were reduced during the storage period (Ahn et al., 2002). In another
study, grape seed extract (ActiVin™), pine bark extract (Pycnogenol), oleo-
resin rosemary (Herbalox), and BHA/BHT were used in cooked ground
beef. The control showed significantly higher TBARS and hexanal content
over storage. BHA/BHT, ActiVin™, Pycnogenol, and Herbalox retarded the
formation of TBARS by 75, 92, 94, and 92%, respectively, after nine days,
and significantly lowered the hexanal content throughout the storage period.
The color of cooked beef treated with ActiVin™ was less light (L*), more
red (a*), and less yellow (b*) than those treated with BHA/BHT, Pycno-
genols, and Herbaloxs. ActiVin™ and Pycnogenols effectively retained the
redness in cooked beef during storage (Ahn et al., 2007a). The antioxidant
effect of grape seed extract was determined in raw or cooked ground muscle
during refrigerated or frozen storage (Brannan & Mah, 2007). It was found
that grape seed extract was more effective than gallic acid in inhibiting
Potential Applications of Natural Antioxidants in Meat and Meat Products 109

oxidation. The formation of lipid hydroperoxides (LOOH) and TBARS


was inhibited by grape seed extract (0.1 and 1.0%) compared to untreated
controls. Furthermore, the results showed that grape seed extract at concen-
trations as low as 0.1% is a very effective inhibitor of primary and secondary
oxidation products in various meat systems.

3.5.1.7 GUAVA

Guava (Psidium guajava L.), being recognized as “super food” is getting


much attention in the agro-food industry due to the attractive characteris-
tics of the fruit, its health-promoting bioactive components and functional
elements. The fruit is considered as highly nutritious because it contains a
high level of ascorbic acid (50–300 mg/100 g fresh weight) and has several
carotenoids such as phytofluene, β-carotene, β-cryptoxanthin, lycopene,
rubixanthin, cryptoflavin, lutein, and neochrome (Mercadante et al., 1999).
Phenolic compounds such as myricetin and apigenin (Miean & Mohamed,
2001), ellagic acid, and anthocyanins (Misra & Seshadri, 1968) are also at
high levels in guava fruits.
Reports regarding use of guava in the meat products either as antioxi-
dant or as a source of dietary fiber are scarce. Guava powder (0.5, 1%) has
been used as a source of antioxidant dietary fiber in sheep meat nuggets
(Verma et al., 2013) and it was found that the powder was rich in dietary
fiber (43.21%), phenolics (44.04 mg GAE/g), and possessed good radical
scavenging activity as well as reducing power. Total phenolics, TDF, and
product redness values were significantly increased (P < 0.05) in nuggets
with added guava powder. Addition of powder retarded lipid peroxida-
tion of cooked sheep meat nuggets as measured by TBARS number during
refrigerated storage. Antioxidant potential of pink guava pulp (10%) was
evaluated in raw pork emulsion during refrigerated storage for nine days
under aerobic packaging (Joseph et al., 2014). The surface redness (a*
value) increased (P < 0.05) with the incorporation of pink guava pulp.
Metmyoglobin formation and lipid oxidation were lower (P < 0.05) in
guava-treated emulsions than in control. Overall, incorporation of pink
guava pulp improved the visual color and odor scores of raw pork emul-
sion. Incorporation of guava fruits having several bioactive components in
meat products would definitely enhance their physiological, functional, and
nutritional values.
110 Natural Antioxidants: Applications in Foods of Animal Origin

3.5.1.8 PLUM

Dried plums, known as prunes, have been extensively investigated for their
potential human health benefits. The phenolic compounds in dried plum prod-
ucts have been shown to inhibit low-density lipoprotein cholesterol oxida-
tion in humans (Stacewicz-Sapuntzakis et al., 2001) and have been shown to
have better antioxidant ability than vitamins C and E in vitro (Vinson et al.,
2005). Plum products exhibited antioxidant properties in a variety of meat
products under several different processing and storage conditions (Lee &
Ahn, 2005; Nuñez de Gonzalez et al., 2008a, 2008b; Yildiz-Turp & Serda-
roglu, 2010).
Leheska et al. (2006) added 5 and 10% of dried plum puree (DPP) and
dried blueberry purees in pork breakfast sausage patties, pre-cooked them
prior to sensory evaluation and total phenolic level was measured. This
research demonstrated that plum puree increased the phenolic content of the
sausage more than the blueberry puree. Yıldız-Turp and Serdaroglu (2010)
used different levels of plum puree (5, 10, and 15%) as an extender in low-
salt beef patties. Addition of plum puree slightly increased redness and
decreased yellowness and lightness both in cooked and uncooked samples.
TBARS values of treated samples were lower than control at the end of
the storage period. Lee and Ahn (2005) found that plum extract (California
Dried Plum Board, Sunsweet Growers Inc., Yuba City, CA) used at 3%
in irradiated (3 kGy) turkey breast rolls reduced (P < 0.05) lipid oxida-
tion. TBARS value for the control product was 0.95 mg MDA/kg meat
whereas the 3% plum extract sample had a reduced (P < 0.05) TBARS
value of 0.84 mg MDA/kg meat after seven days of storage at 4 °C. Nuñez
de Gonzalez et al. (2008a) added 3 and 6% each of DPP, dried plum and
apple puree, or 0.02% BHA/BHT to sausage. For the raw sausage, there
were no differences (P < 0.05) in TBARS values among treatments. The
3 and 6% DPP were about equal to the synthetic antioxidants BHA and
BHT. In the pre-cooked/refrigerated sausage TBARS values were similar to
the raw sausage, whereas the control without antioxidants exhibited higher
(P < 0.05) TBARS values which indicated lower antioxidant capacity.
Among the treatments for pre-cooked/frozen sausage only the 6% DPP
inhibited lipid oxidation such that its TBARS values (0.46 mg MDA/kg)
were similar (P < 0.05) to the raw, non-oxidized values. However, even
3% DPP was able to inhibit lipid oxidation to the same extent as BHA
and BHT. These researchers concluded that plum products could be used
as natural antioxidants and have the ability to replace BHA and BHT in
inhibiting lipid oxidation. The work was continued by Nuñez de Gonzalez
Potential Applications of Natural Antioxidants in Meat and Meat Products 111

et al. (2008b) in roast beef brine formulations. They used 2.5 and 5% each
of fresh plum puree (FPP), DPP, and spray dried plum powder (DPWD)
in addition to sodium chloride, dextrose, alkaline phosphate, potassium
lactate, and water. All treatments had reduced (P < 0.05) TBARS values
compared to the control (0.62 mg MDA/kg), which further proved that
dried plum ingredients were able to inhibit lipid oxidation.
Conversely, hams brined with the same amounts of plum products (2.5
and 5% FPP, DPP, and DPWD) and with sodium chloride, dextrose, alkaline
phosphate, potassium lactate, sodium nitrite, and sodium erythorbate did not
exhibit differences between treatments and control for TBARS values at 21
days post storage (Nuñez de Gonzalez et al., 2009). They suggested that the
hams were not susceptible to lipid oxidation due to the inclusion of sodium
nitrite and alkaline phosphates and therefore no differences were observed
among treatments.

3.5.1.9 POMEGRANATE

Pomegranates (Punica granatum) have been used extensively in the folk


medicine of many cultures (Longtin, 2003). Numerous studies on the anti-
oxidant activity have shown that pomegranate juice contains high levels
of antioxidants—higher than most other fruit juices and beverages (Gil
et al., 2000; Seeram et al., 2008). The exceptionally high antioxidative
capacity of the fruit juice might be the result of the remarkably high
content and unique composition of soluble phenolic compounds (Gil et al.,
2000; Poyrazoglu et al., 2002; Seeram et al., 2008). Phenolic concentra-
tion and composition in the pomegranate fruit are cultivar-dependent; the
most abundant components are anthocyanins, catechins, ellagic tannins,
and gallic and ellagic acids (El-Nemr et al., 1990; Gil et al., 2000; Poyr-
azoglu et al., 2002).
PRP was used at 10 mg tannic acid equivalent phenolics/100 g in fresh
chicken, and then prepared as cooked chicken patties (Naveena et al., 2008a).
Reduced TBARS values were observed (P < 0.05) in comparison to control.
Chicken patties were treated with pomegranate, cooked to an internal
temperature of 80 °C, and stored in low-density polyethylene pouches for
15 days at 4 °C. TBARS value for control was reported as 1.272 ± 0.13 mg
MDA/kg meat, and the treatment with PRP had a value of 0.203 ± 0.04 mg
MDA/kg. TBARS values also decreased 68% compared with samples
treated with BHT (100 mg BHT/100 g meat) for the same product held
under identical storage conditions. PRP and pomegranate juice powder have
112 Natural Antioxidants: Applications in Foods of Animal Origin

little effect on sensory or quality attributes when used at concentrations from


5 to 20 mg tannic acid equivalent phenolics/100 g meat (Naveena et al.,
2008a; Naveena et al., 2008b). Naveena et al. (2008b) reported a decrease
in L* values (56.71 ± 0.74) compared with the control (63.8 ± 0.73) for
cooked chicken patties with PRP at 20 mg equivalent phenolics/100 g
meat. An 8–10-member trained sensory panel found no differences for off-
odor, sweet flavor, and chicken flavor between the pomegranate samples
at any of the concentrations compared with the control; however, chicken
flavor was slightly reduced for the sample with 20 mg tannic acid equiva-
lent phenolics/100 g meat. Vaithiyanathan et al. (2011) evaluated the effect
of pomegranate fruit juice phenolics (PFJP) dipping solution on the shelf
life of chicken meat held under refrigerated storage at 4 °C. TBARS were
evaluated in two-day intervals for 28 days and it was reported that TBARS
values were lower (0.35–0.75 mg MDA/kg of meat) in samples treated with
PFJP. Sensory scores indicated that both samples treated with and without
PFPJ performed well in all sensory attributes. However, on day 4, sensory
attribute scores of samples without PFJP started to decline while scores of
samples with PFJP remained high. Additionally, the acceptability scores of
control samples decreased significantly (P < 0.05) on day 12 of storage.
These studies demonstrated the potential of pomegranate components as
antioxidants in refrigerated chicken and goat patties. Pomegranate was
effective at inhibiting lipid oxidation and does not significantly affect the
overall sensory attributes of the finished product.

3.5.2 HERBS- AND SPICES-BASED ANTIOXIDANTS

In addition to the application of herbs and spices as seasonings, meat


industry has explored their potential as a natural antioxidants and antimicro-
bials too. Spices and herbs are excellent sources of antioxidants and have a
long history of safe usage. They are rich sources of phytochemicals (Shan et
al., 2005; Srinivasan, 2014; Surh, 2002; Zheng & Wang, 2001) (Table 3.2).
Spices are derived from different parts of a plant other than the leaves while
herbs from leaves of a plant. The basil, oregano, bay leaf, and thyme come
from leaves, clove and saffron from flower or bud, clove, chilli, and black-
pepper from fruits or berries, fennel and fenugreek from seeds, cinnamon
and cassia from bark, onion and garlic from bulb, ginger and turmeric from
root, and mace from aril. Antioxidant components of herbs and spices may
be removed/concentrated as extracts, EOs, or resins.
Potential Applications of Natural Antioxidants in Meat and Meat Products 113

TABLE 3.2 Major Active Constituents and Antioxidative Capacities of Herbs/Spices


(Modified from Charles, 2013; Prior et al., 2003).
Herb/spice Active constituents Total ORAC value
(μm TE/100) g
Basil Eugenol, apigenin, limonene, ursolic acid, methyl 4805 (fresh)
cinnamate, 1,8-cineole, α-terpinene, anthocyanins,
β-sitosterol, carvacrol, citronellol, farnesol, geraniol,
kaempferol, menthol, p-coumaric acid, quercetin,
rosmarinic acid, rutin, safrole, tannin, catechin
Marjoram Sinapic acid, ferulic acid, coumarinic acid, caffeic acid, 27,297 (fresh)
syringic acid, vanillic acid, 4-hydroxybenzoic acid,
limonene, ursolic acid, α-pinene, α-terpinene, p-cymene,
rosmarinic acid, sterols, apigenin
Oregano Apigenin, rosmarinic acid, luteolin, quercetin, 13,970 (fresh)
myricetin, caffeic acid, p-coumaric acid, diosmetin,
protocatechuic acid, eriodictyol, carvacrol, thymol
Sage α-Pinene, β-pinene, geraniol, limonene, 1,8-cineole, 32,004 (fresh)
perillyl alcohol, citral, β-sitosterol, farnesol, ferulic
acid, gallic acid, β-carotene, catechin, apigenin, luteolin,
saponin, ursolic acid, rosmarinic acid, carnosic acid,
vanillic acid, caffeic acid, carnosol
Cinnamon Cinnamic aldehyde, 2-hydroxycinnamaldehyde, 131,420 (ground)
eugenol, myristicin, cinnamate, phenolics
Clove Eugenol, isoeugenol, gallic acid, flavonoids, phenolic 290,283 (ground)
acids
Ginger Zingiberone, zingiberene, ar-curcumene, gingerol, 39,041 (ground)
paradol, shogaols, zingerone, curcumin, zerumbone
Nutmeg Caffeic acid, argenteane, myristicin, lignans, catechin 69,640 (ground)
Oregano Apigenin, rosmarinic acid, luteolin, quercetin, 175,295 (dried)
myricetin, caffeic acid, p-coumaric acid, diosmetin,
protocatechuic acid, eriodictyol, carvacrol, thymol
Black Piperidine, piperine, limonene, α-pinene, β-pinene, 34,053
pepper sarmentine, guineesine, isoquercetin
Rosemary Carnosol, carnosic acid, rosmanol, ursolic acid, 165,280 (dried)
1,8-cineole, geraniol, α-pinene, limonene, β-carotene,
apigenin, naringin, luteolin, caffeic acid, rosmarinic
acid, rosmanol, vanillic acid, diosmetin
Sage α-Pinene, β-pinene, geraniol, limonene, 1,8-cineole, 119,929 (ground)
perillyl alcohol, citral, β-sitosterol, farnesol, ferulic
acid, gallic acid, β-carotene, catechin, apigenin, luteolin,
saponin, ursolic acid, rosmarinic acid, carnosic acid,
vanillic acid, caffeic acid, carnosol
114 Natural Antioxidants: Applications in Foods of Animal Origin

TABLE 3.2 (Continued)

Herb/spice Active constituents Total ORAC value


(μm TE/100) g
Thyme Thymol, carvacrol, 1,8-cineole, α-pinene, limonene, 27,426 (fresh)
apigenin, β-carotene, ursolic acid, luteolin, gallic acid,
caffeic acid, rosmarinic acid, carnosic acid, hispidulin,
cismaritin, diosmetin, naringenin, kaempferol,
quercetin, hesperidin
Turmeric Curcumin, curcuminoids, β-turmerin 127,068 (ground)
Garlic Allicin, diallyl sulfide, diallyl disulfide, diallyl trisulfide 5708 (raw)
allyl isothiocyanate, S-allylcysteine
Ginger Zingiberone, zingiberene, ar-curcumene, gingerol, 14,840 (raw)
paradol, shogaols, zingerone, curcumin, zerumbone

The herbs and spice extracts, including rosemary, oregano, clove, thyme,
and so forth have been investigated for their antioxidant potential in several
meat products. El-Alim et al. (1999) investigated the use of ground spices
and spice extracts as antioxidants in raw ground chicken and ground pork.
Ground chicken was treated with 1% of dried spices: marjoram, wild
marjoram, caraway, clove, peppermint, nutmeg, curry, cinnamon, basil,
sage, thyme, and ginger. TBARS formation was significantly inhibited in
refrigerated and frozen samples that were treated with spices. During refrig-
erated storage (4 °C for seven days), cloves showed the largest reduction
in TBARS values compared with the control. After six months of frozen
storage at −18 °C, marjoram-treated samples showed the highest inhibition
for TBARS formation. These researchers also examined use of spice extracts
of basil, sage, thyme, and ginger @ 1 ml/10g as antioxidants in ground pork.
After seven days of refrigerated storage, TBARS values for all treatments
were significantly lower than control. Sage, thyme, and basil were more
effective at inhibiting TBARS values than ginger. All treatments significantly
reduced TBARS formation after six months frozen storage compared with
the control. Efficacy of varying concentrations of dried holy basil powder
(0.07, 0.18, and 0.35%) and its ethanolic extracts (0.02, 0.05, and 0.10%)
in retarding oxidative rancidity was reported in cooked ground pork during
refrigerated storage at 5 °C for 14 days (Juntachote et al., 2007). Ethanolic
extracts of holy basil were less effective than dried holy basil powder in
controlling oxidative stability. Dried holy basil powder at a concentration of
0.35% (w/w) was the most effective in retarding lipid oxidation in cooked
ground pork during the storage period.
Potential Applications of Natural Antioxidants in Meat and Meat Products 115

Mohamed et al. (2011) reported that addition of herbal extracts of


marjoram, rosemary, and sage at concentration of 0.04% (v/w) to ground
beef prior to irradiation (2 and 4.5 kGy) significantly lowered the TBARS
values, off-odor scores, and increased color and acceptability scores. Simi-
larly, Kanatt et al. (2007) found that radiation processed lamb meat treated
with mint leaf extract (0.1 and 0.5%) showed greater antioxidant activity
and decreased lipid oxidation during four weeks chilled storage compared
with non-treated samples. Jayathilakan et al. (2007) showed that cinnamon
and cloves (250 mg/100 g meat) were significantly effective in inhibiting
TBARS formation in cooked ground beef, pork, and mutton stored at 5 °C
for six days. No difference in TBARS values was observed between samples
treated with cinnamon and samples treated with BHA, or PG at 0.02% in
ground beef and pork. Cloves exhibited higher antioxidant activity than BHA
and PG. However, TBHQ demonstrated the highest antioxidant activity of
all tested antioxidants in all three types of meat.
According to Yu et al. (2002) aqueous rosemary extracts (0, 100, 250,
and 500 ppm) improved the color stability of turkey rolls in addition to their
inhibition of lipid oxidation. Sánchez-Escalante et al. (2001) reported that
rosemary powder and rosemary along with ascorbic acid were most effec-
tive in inhibiting oxidation of both lipid and myoglobin, as revealed by the
results of TBARS and the percentage of metmyoglobin, respectively, in
beef patties stored at 2 ± 1 °C for 20 days. Both of these desirable effects
contributed in maintaining desirable sensory characteristics of fresh beef
patties in extending their shelf life. Antioxidant effectiveness of a commer-
cial rosemary extract (FORTIUMTM R20) at concentrations of 1500 and
2500 ppm in frozen and pre-cooked-frozen pork sausage, and from 500 to
3000 ppm in refrigerated, fresh pork sausage was compared with BHA/BHT
(Sebranek et al., 2005). Rosemary extract at 2500 ppm was as effective as
the maximum permitted concentrations of BHA/BHT in refrigerated, fresh
pork sausage and in cooked-frozen sausage, but was superior to BHA/BHT
in raw-frozen pork sausage patties. Three kinds of Rosmarinus officinalis
extract (powder-acetone, liquid-methanol, and liquid-acetone) were used by
Rocío Teruel et al. (2015) to examine their effects on frozen chicken nuggets
quality. The highest antioxidant activity was found for the powder-acetone
extract followed by the liquid-methanol and liquid-acetone extracts. In a
study with porcine liver pâté, Doolaege et al. (2012) found that addition
of a rosemary extract had a positive effect on retarding lipid oxidation and
maintaining higher concentrations of the antioxidants ascorbic acid, TM,
and carnosic acid. It was also noticed that the sodium nitrite concentration
in liver pâté, could be reduced from 120 to 80 ppm when rosemary extract
116 Natural Antioxidants: Applications in Foods of Animal Origin

was added, without negative effects on lipid oxidation, antioxidant level,


and color stability.
Jinap et al. (2015) reported that local spices, that is, turmeric (4 g/100 g),
lemongrass, torch ginger, and curry leaves (10 g/100 g) have effectively
reduced the level of heterocyclic amines in grilled beef, thus spices could
be exploited to avoid toxic and carcinogenic effects from these amines too.

3.5.3 ESSENTIAL OIL-BASED ANTIOXIDANTS

EOs are aromatic and volatile oily liquids that are extracted from plant mate-
rials, such as flowers, buds, roots, bark, leaves, seeds, peels, fruits, wood,
and whole plants (Hyldgaard et al., 2012; Sanchez et al., 2010; Viuda-
Martos et al., 2010) and are characterized by a strong odor (Burt, 2004).
These have been widely used for centuries for their biological and flavoring
characteristics. Some of the beneficial properties, for example, antiseptic,
antioxidant, or, anti-inflammatory, have been supported by recent scientific
investigations (Bakkali et al., 2008; Adorjan et al., 2010). The advantages
associated with use of EOs in comparison to dried spice materials include
better stability during storage, higher concentrations of flavor components,
reduced need for storage space, ease of handling, microbial safety, and stan-
dardization (Tipsrisukond et al., 1998).
The composition of an EO is influenced by the extraction method, which
ultimately influences its antioxidative properties. Among the different
extraction methods that are used to obtain EOs, steam distillation is most
commonly used on a commercial basis (Burt, 2004). Antioxidant properties
play a pivotal role in some of EOs’ biological activities. These attributes are
due to the inherent ability of some of their components, particularly phenols,
to stop or delay the aerobic oxidation of organic matter. However, there are
some phenol-free EOs that express antioxidant behavior due to the radical
chemistry of some terpenoids and other volatile constituents like sulfur-
containing components of garlic (Valgimigli, 2012).
The effectiveness of a wide range of EOs against lipid oxidation has been
demonstrated by many researchers over the years. The antioxidant activity
of pork and beef (both raw and cooked) treated with oregano and sage EOs,
during meat storage (4 °C for 12 days), was determined by Fasseas et al.
(2007). Both meat samples showed significantly lower TBARS values and
DPPH radical scavenging effects during refrigerated storage when treated
EOs. Oregano EO was found more effective as an antioxidant than sage
EO in both meat samples. Findings of Estevez et al. (2007) revealed that
Potential Applications of Natural Antioxidants in Meat and Meat Products 117

sage and rosemary EOs at the levels of 0.1% improve the oxidative stability
of lipids in liver pates during refrigerated storage for 90 days, mainly by
reducing the degradation of PUFAs and thereby preventing the formation
of residual components such as malondialdehyde and lipid-derived vola-
tiles. At 60 and 90 days, TBARS values and lipid-derived volatiles in pates
with EOs were significantly lower than in the control samples. The effect of
addition of rosemary and marjoram EOs (200 ppm) to beef patties formu-
lated with mechanically deboned poultry meat (20%) was demonstrated by
Mohamed and Mansour (2012). The findings showed that both marjoram
and rosemary EOs reduced the lipid oxidation and improved the sensory
attributes of beef patties during frozen storage for three months. The TBARS
value of beef patties prepared with marjoram and rosemary EOs remained
significantly lower compared with those of control beef patties during frozen
storage. Additionally, these natural antioxidants were superior to BHT used
in the study with regard to their role in sensory attributes besides lowering
the lipid oxidation. Moreover, the addition of EOs to the beef patties signifi-
cantly increased the flavor and overall acceptability scores of patty formulas
processed with mechanically deboned poultry meat after processing and
during the frozen storage period. Dzudie et al. (2004) prepared beef patties
by incorporating ginger and basilica EOs together with maize oil. During 18
days of storage, the TBARS values of the beef patties with EOs were stable.
Fratianni et al. (2010) investigated the effectiveness of balm and thyme EOs
as natural antioxidants on fresh chicken breast meat that had been stored
for three weeks at 4 °C. During their experiment, they found that both EOs
reduced radical formation in meat compared with untreated control. Thyme
EO, in particular, was the most effective out of the two EOs, with a DPPH
radical inhibition percentage of 25–30%. Balm oil showed 15–20% DPPH
radical inhibition and lower activity of ~10% was demonstrated in the
control.

3.5.4 VEGETABLES-BASED ANTIOXIDANTS

Vegetables account for a small part of our daily caloric intake; however, their
benefits to health surpass their caloric contribution mainly due to presence
of dietary fiber, phenolic compounds, minerals, and vitamins. Epidemiolog-
ical studies have indicated that the frequent consumption of fruits and vege-
tables significantly reduced the incidence of chronic diseases (WHO, 2003).
In order to obtain maximum health benefits, intake of sufficient amounts
of antioxidants from plant food (fruits, vegetables, etc.) is preferred. The
118 Natural Antioxidants: Applications in Foods of Animal Origin

antioxidants obtained in vegetables play the major role in maintenance of


health and prevention of diseases (Paganga et al., 1999). It has been esti-
mated that every serving increase in vegetable consumption reduces the risk
of cancer by 15%, cardiovascular disease by 30%, and mortality by 20%
(Steimez & Potter, 1996; Rimm et al., 1996), attributable to antioxidants
such as ascorbic acid, vitamin E, carotenoids, lycopenes, polyphenols, and
other phytochemicals. These antioxidants scavenge radicals and inhibit the
chain initiation or break the chain propagation (the second defense line).
Vitamin E and carotenoids also contribute to the first defense line against
oxidative stress, because they quench singlet oxygen (Krinsky, 2001; Shi et
al., 2001).

3.5.4.1 LEAFY GREEN VEGETABLES

Fresh leafy green vegetables contain important functional food components,


such as β-carotene, ascorbic acid, riboflavin, folic acid, minerals (Grusak
& DellaPenna, 1999), and a large amount of polyphenols (e.g., phenolic
acids, flavonoids, and aromatic compounds). They are also known for their
characteristic color, flavor, and therapeutic value (Gupta et al., 2005; Faller
& Fialho, 2009). Ten common vegetables were screened for their antioxi-
dant and anti-proliferative activities by Chu et al. (2002). Broccoli and
spinach had the highest amount of free phenolics, followed by yellow onion,
red sweet pepper, cabbage, carrot, potato, and lettuce. Cucumber had the
lowest free phenolics of the 10 vegetables. The total antioxidant activity
was determined by total oxyradical scavenging activity (TOSC) assay. Red
pepper, broccoli, carrot, and spinach were in the group with higher anti-
oxidant activities. The medium group comprised cabbage and yellow onion.
The remaining four vegetables in the group with lower antioxidant activities
included celery, potato, lettuce, and cucumber.
Antioxidant activities of 70% ethanolic extracts of 10 leafy green vegeta-
bles were determined and applied in raw beef patties (Kim et al., 2013a). The
extracts and BHT were separately added to patties at 0.1 and 0.5% (w/w)
concentrations and the patties were stored at 4 °C for 12 days. The addi-
tion of extracts and BHT resulted in concentration dependent decreases in
TBARS values in the beef patties and also improved meat color stability. The
fatsia (Aralia elata) extract had more effective antioxidant than the cham-
namul (Pimpinella brachycarpa). In another study, the antioxidant efficacy
of 70% ethanol and water extract of 10 leafy edible plants was evaluated
Potential Applications of Natural Antioxidants in Meat and Meat Products 119

in ground beef patties. Plant extracts (butterbur and broccoli extracts) and
BHT were separately added to the patties at 0.1 and 0.5% (w/w) concentra-
tions and stored at refrigerated conditions for 12 days. TBARS values were
significantly lower in the samples containing plant extracts or BHT than the
non-treated control. In addition, the beef patties formulated with the selected
plant extracts showed significantly better color stability than those without
antioxidants (Kim et al., 2013b).

3.5.4.2 CARROT

Carrot is one of the important widely consumed root vegetable with high
nutritional value due to its enriched healthy composition, such as phyto-
nutrients and minerals. It is a good source of natural antioxidants espe-
cially carotenoids and phenolic compounds, having the highest carotenoid
content among foods (Arabshahi-D et al., 2007; Hsieh & Ko, 2008; Soria
et al., 2009). Antioxidant activity of carrot juice (unconcentrated carrot
juice, carrot juice concentrated by 35 and 60%) in gamma irradiated (0,
3, and 4.5 kGy) beef sausage was studied by Badr and Mahmoud (2011).
Carrot juice exerted a significant antioxidant effect during the irradiation
of sausages and the formation of hydroperoxides, and TBARS significantly
decreased with increasing the concentration of the carrot juice. Formulation
of sausages with carrot juice at the different concentrations decreased the
formation of hydroperoxides by 24.68, 40.38, and 58.01%, respectively, in
sausage samples exposed to the highest irradiation dose, while decreased
the formation of TBARS in the samples by 28.86, 42.86, and 54.29%,
respectively.

3.5.4.3 POTATO

Potato (Solanum tuberosum L.) tubers possess a wide range of carotenoid


contents. Potato peel extract (PPE) was found to have the highest antioxidant
activity owing to its high content of phenolic compounds and flavonoids
(Mohdaly et al., 2010). Effectiveness of PPE in reducing lipid peroxidation
of γ-irradiated lamb meat was examined by Kanatt et al. (2005). TBARS
number and carbonyl content were reduced in irradiated meat containing
PPE in comparison to the samples without PPE. These researchers found
that the antioxidant activity of PPE was comparable to BHT and it did not
affect flavour or aroma of the radiation processed meat.
120 Natural Antioxidants: Applications in Foods of Animal Origin

3.5.4.4 CRUCIFEROUS VEGETABLES

The Brassicaceae (Cruciferae) family is composed of 350 genera and about


3500 species (Sasaki & Takahashi, 2002). Brassica is an inexpensive, though
very nutritive, source of food, providing nutrients and health-promoting
phytochemicals such as phenolic compounds, vitamins (Dekker et al., 2000;
Vallejo et al., 2002, 2003; Vallejo et al., 2004), phytic acid, fiber, soluble
sugars (Pedroche et al., 2004), glucosinolates (Fowke et al., 2003), minerals,
polyphenols (Heimler et al., 2005), fat, and carotenoids (Zakaria-Rungkat et
al., 2000). There is ever-increasing evidence that a higher consumption of
Brassica vegetables, for example, broccoli, cabbage, kale, mustard greens,
Brussels sprouts, and cauliflower, reduces the risk of several types of cancer
(Kristal & Lampe, 2002; Wang et al., 2004). The anti-carcinogenic effect
of these vegetables has been attributed to decomposition products of gluco-
sinolates, indoles, and iso-thiocyanates (Zukalova & Vasak, 2002), phyto-
alexins, and other antioxidants (Samaila et al., 2004; Hanf & Gonder, 2005).
Extracts of the different species of the Brassicaceae family show antioxidant
effects (Banerjee et al., 2012; Banerjee et al., 2015) and decrease oxida-
tive damage (Ferguson, 1999). Phenolic compounds with vitamin C are the
major antioxidants of Brassica vegetables, due to their high content and
high antioxidant activity. On the contrary, lipid-soluble antioxidants such
as carotenoids and vitamin E were responsible for up to 20% of the total
antioxidant activity of Brassica vegetables (Podsedek, 2007). The order of
the oxygen radical absorbance capacity (ORAC) values of the fresh weight
extracts reported by Cao et al. (1996) was: kale > Brussels sprouts > broc-
coli > cauliflower > cabbage. Generally, among Brassica vegetables, Brus-
sels sprouts, broccoli, and red cabbage have the highest antioxidant capacity.
Common cabbage demonstrated rather low antioxidant activity. However,
the antioxidant activities depend on the extraction method, and on the type
of the reactive species in the reaction mixture (Azuma et al., 1999; Cao et al.,
1996). Mustard leaf (Brassica juncea), a cruciferous vegetable originating
from China has attracted a lot of attention as a functional food for mainte-
nance of health and disease prevention (Kim et al., 2004). Lee et al. (2010)
demonstrated the effectiveness of mustard leaf kimchi ethanolic extract
(MK; 0.05, 0.1, and 0.2%) on microbial growth and lipid oxidation and in
extending the shelf life of raw ground pork meats during storage at 4 °C for
14 days. The TBARS values indicated that at MK @ 0.1 or 0.2% was as effec-
tive as 0.02% L-ascorbic acid, and at the level of 0.2%, it suppressed lipid
oxidation and reduced the formation of peroxides more than the ascorbic
acid treatment, indicating the high protective effect of MK against oxidation
Potential Applications of Natural Antioxidants in Meat and Meat Products 121

in ground pork. The lowest free fatty acid value was reported in 0.2% MK
treated pork. The antioxidant power of broccoli powder extract (1.0, 1.5, and
2.0%) was determined and evaluated in goat meat nuggets by Banerjee et
al. (2012). Total phenolics, radical scavenging activity, and reducing power
estimation indicated that broccoli powder has good antioxidant potential.
Among treatments, TBARS number decreased with the higher levels of
broccoli powder extract with significant effect at 2% level and its value was
similar to the product with 100 ppm BHT. The antioxidant potential of cauli-
flower (Brassica oleracea) powder (CP) was evaluated in pork meatballs by
Banerjee et al. (2015). The amount of total phenolics (mgGAE/g) was higher
in aqueous extract (29.52) of CP as compared to the extract from acetone:
water mix (24.22). Addition of CP in pork meatballs significantly increased
the amount of total phenolics and TDF. It also reduced the lipid peroxidation
and thus enhanced their oxidative stability of meatballs. Therefore, inclu-
sion of CP in meat products makes them much healthier and stable without
affecting their acceptability.

3.5.4.5 TOMATO

Tomatoes are an important dietary source of antioxidants—ascorbic acid,


lycopene and carotenoids, phenolics, and vitamin E (Frusciante et al.,
2007). Dietary intakes of tomatoes and tomato products containing lycopene
have been shown to be associated with decreased risk of chronic diseases,
such as cancer (van Breemen et al., 2011). Approximately one-third of the
total weight of tomatoes in the form of skin and seeds is discarded during
processing. However, majority of the flavonols in tomatoes are present in
the skin (Stewart et al., 2000). George et al. (2004) reported that tomato skin
had significant amounts of phenolics and ascorbic acid and on an average,
it had 2.5 times higher lycopene levels than the pulp. Hence, adding tomato,
tomato products, or lycopene to processed meat could lead to a significant
increase in the amount of all the major antioxidants in the final products.
Selgas et al. (2009) analyzed the safety and shelf life of vaccum-packed,
irradiated (2 or 4 kGy) raw hamburgers containing 30, 45, and 60 g/kg dry
tomato peel as a source of lycopene. The lycopene concentration fell to 15%
of the initial value after irradiation with 4 kGy on 17 days of storage period.
Even with this decrease, hamburgers containing 6% dry tomato peel had
a final lycopene concentration of 7.14 mg/100 g of hamburger, an amount
very close to the recommended daily intake for a healthy diet. Dry tomato
peel masked the brownish color characteristic of irradiated meat, and 6%
122 Natural Antioxidants: Applications in Foods of Animal Origin

dry tomato peel imparts a characteristic redness (a*) to the hamburger, inde-
pendent of the dose of radiation applied. The researchers also found that
the higher lycopene concentration (6 g/kg) sufficiently masked the nega-
tive effects of irradiation on sensory characteristics to ensure an acceptable
color and odor in the final product after the storage period. The nitrosomyo-
globin (NOMb) content, lycopene content, oxidation level, and the sensory
properties of frankfurters produced by both reducing the nitrite level and
adding tomato powder were analyzed by Eyiler and Oztan (2011). The pH
of the frankfurters produced with tomato powder was reduced, compared
with samples which did not contain tomato powder. The addition of 2 g
tomato powder/100 g decreased the level of oxidation; however, 4 g tomato
powder/100 g caused a slight increase compared with the samples which
did not contain tomato powder. According to sensorial evaluations, tomato
powder also improved consumer acceptability.
Sanchez-Escalante et al. (2003) analyzed the stabilization of color and
odor of beef patties using lycopene-rich tomato as a source of antioxidants,
which they found exerted a significant antioxidative effect on the beef patties,
depending on the lycopene concentration. These tomato products delayed
meat deterioration and the shelf life of treated beef patties ranged between
8 and 12 days. Mercadante et al. (2010) analyzed the oxidative stability of
sausages containing added natural pigments and stored under refrigeration.
These authors reported that the addition of lycopene (10%) produced signifi-
cant reductions in redness, although it did not exert any antioxidant effect.

3.5.5 SEAWEED-BASED ANTIOXIDANTS

Marine macroalgae or seaweeds have been used as food mainly in Asian


countries and to a lesser extent in Europe and America. Seaweeds are also
used as raw material for industrial production of some purified ingredients,
for example, agar, carrageenan, alginates, or oils. Edible seaweeds contain
good-quality protein, high concentrations of vitamins, high proportions of
essential unsaturated fatty acids, particularly long chain n-3 PUFA, bioactive
compounds with known antioxidant properties, and are an excellent source
of most minerals and dietary fiber (Kolb et al., 2004; Sánchez-Machado et
al., 2004). Among the marine organisms, marine macroalgae or seaweed
represents one of the richest sources of natural antioxidants (Cox et al.,
2010; Ruperez et al., 2002). Among the three algal groups, brown algae
generally contains higher amount of polyphenols than red and green algae
(Wang et al., 2009a). The bioactive compounds which have been isolated
Potential Applications of Natural Antioxidants in Meat and Meat Products 123

and identified from seaweeds include sulfated polysaccharides (laminarins


and fucoidans), polyphenols such as phlorotannins (Zou et al., 2008), carot-
enoid pigments such as fucoxanthin (Airanthi et al., 2011) and astaxanthin,
and sterols and mycosporine-like amino acids. The potential antioxidants
identified in seaweeds include some pigments such as fucoxanthin and astax-
anthin, polyphenols such as phlorotannins, chlorophyll related compounds,
phospholipids, flavonoids, bromophenols, and polysaccharides.
Algal phlorotannins have up to eight interconnected rings and are thus
more potent antioxidants than plant polyphenols (Wang et al., 2009a). These
phlorotannins have been reported to scavenge free radicals, superoxide
radicals (Kuda et al., 2005), peroxyl radical (Wang et al., 2009b), chelate
ferrous ions (Chew et al., 2008), and nitric oxide (Valentão et al., 2010).
Presence of polyphenols such as catechin, epicatechin, epigallocatechin
gallate, and gallic acid are reported in the green seaweed Halimada (Yoshie
et al., 2002). López et al. (2011) reported the presence of 14 polyphenols,
namely gallic acid, catechin, epicatechin, rutin, p-coumaric acid, myricetin,
quercetin and protocatechuic, vanillic, caffeic, ferulic, chlorogenic, syringic,
and gentisic acids in the solvent extracts of Stypocaulon scoparium. Onofre-
jová et al. (2010) reported the extraction of bioactive phenolic acids (proto-
catechuic, p-hydroxybenzoic, 2,93-dihydroxybenzoic, chlorogenic, caffeic,
p-coumaric, and salicylic acid), cinnamic acid, and hydroxybenzaldehydes
(p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde) from food products
of Porphyra tenera and Undaria pinnatifida. Fucoxanthin and phlorotan-
nins have been identified as active antioxidant compounds from Hijika fusi-
formis (Yan et al., 1999) and Sargassum kjellamanianum (Yan et al., 1996),
respectively.
Several researchers have investigated the antioxidant activities of the
bioactive compounds purified from seaweeds and found that DPPH, alkyl,
hydroxyl, superoxide radical scavenging, and metal chelating activities were
comparable or even higher than most of the commercial antioxidants (Athu-
korala et al., 2003; Ahn et al., 2007b; Kim et al., 2007). Since seaweeds
contain various bioactive compounds with potential health-beneficial prop-
erties, their use as functional ingredients paving the ways for their appli-
cation in food processing, including meat products (Cofrades et al., 2008;
Fleurence, 1999). However, limited attention has been paid to the use of
edible seaweeds as a source of natural antioxidants in meat products.
López-López et al. (2009) reported that the addition of edible seaweeds,
Sea Spaghetti (Himanthalia elongata), Wakame (U. pinnatifida), and Nori
(Porphyra umbilicalis) to low-salt meat emulsion model systems supplied
the meat samples with soluble polyphenolic compounds thereby enhancing
124 Natural Antioxidants: Applications in Foods of Animal Origin

the antioxidant capacity of the systems. The amount of soluble polyphenols


varied from 820 mg GAE/100 g in samples containing Wakame to values as
high as 2170 and 2570 mg GAE/100 g of sample in products with Nori and
Sea Spaghetti, respectively. The highest antioxidant activity was observed in
the samples containing Sea Spaghetti (3.69 µmol eq. Trolox/g of sample),
while products containing Nori and Wakame had similar antioxidant poten-
tials (1.18 and 1.09 µmol eq. Trolox/g, respectively). López-López et al.
(2010) also studied the effect of adding Wakame seaweed on the character-
istics of beef patties with low-salt and low-fat contents. However, no clear
effect on lipid oxidation was found with the presence of seaweed.
Sasaki et al. (2008) observed the effects of major carotenoid pigment,
fucoxanthin (from Wakame) @ 200 mg/kg on lipid peroxidation and meat
color in ground chicken breast meat upon chilled storage before and after
cooking. It was found that fucoxanthin could decrease the TBARS value
on days 1 and 6 (63.1 and 58.5%, respectively) when stored after cooking.
It also decreased the L* value and increased a* and b* values thereby
showing it to be a potent ingredient for the improvement of the appearance
and shelf life of chicken meat and its products. However, the antioxidative
activity of fucoxanthin during chilling storage after cooking was lower
than that of TM.
Thus, the use of seaweeds can play key role in the development of func-
tional foods and provide an opportunity to improve the nutritional profile of
meat products and address consumer demands. Seaweeds can also supply
meat products with valuable polyphenols to improve their oxidative stability
during processing and storage besides enriching them with dietary fiber and
minerals.

3.6 MARKET POTENTIAL

From tea bags to grape seeds, the term antioxidant is used as a marketing
tool for food products especially processed foods. The increased demand for
processed meat products will undoubtedly advance the use of antioxidants
across the globe. The global market for processed meats is estimated to be
USD 362 billion in 2012 and is projected to reach USD 799 billion by 2018
with a compound annual growth rate of 14.3%. Maximum growth of this
sector is expected in China, India, Japan, and New Zealand. Similarly, the
global natural antioxidants’ market is expected to witness substantial growth
and is forecasted to reach USD 4.14 billion by 2022, particularly due to
increasing global meat consumption. Asia Pacific has experienced highest
Potential Applications of Natural Antioxidants in Meat and Meat Products 125

growth in 2014 followed by Europe and North America. Huge population and
growing health awareness in developing nations such as India and China are
influencing the growth of the antioxidants’ market in the Asia Pacific region.
Demand for vitamin C was highest accounting for over 80% of the global
demand in 2014 and this trend is likely to continue in the coming years too.
Growing demand for animal feed on account of increasing consumption of
dairy products and meat products on a global scale is expected to augment
vitamin E market growth over the forecast period. Demand-supply imbal-
ance has escalated the price of natural antioxidants. In an effort to deal with
the increasing raw material cost, food manufacturers are opting blending
of natural antioxidants, for example, TM with rosemary extract or herbal
extract with synthetic antioxidants, which produces a less expensive product
without compromising the efficacy of antioxidants.

3.7 FUTURE PROSPECTS

Strong health awareness among the modern consumers is creating a genuine


need for adopting a synthetic additive free diet, with increasing personalized
value of convenience, cost, and taste. Plant materials have drawn enough
attention as a source of natural antioxidants in meat system to maintain and
improve quality and stability. However, still there is a long way ahead to make
these herbal ingredients an appropriate substitute of synthetic antioxidants.

• Both synthetic and natural antioxidants have to face a number of


considerations for their practical usage—natural availability, extrac-
tion efficiency and purification, and economical and practical aspect
of the natural antioxidants as preservatives. The other factors consid-
ered should involve the incorporation of antioxidant into the final
meat product; effect of the incorporation on their stability, solubility
and sensory properties, and the interactions of the preservatives with
other meat ingredients. Further, there are certain important regulatory
considerations, such as generally recognized as safe (GRAS) status,
the limit of the amount that can be incorporated, and so forth.
• Plant extracts are harvested both in aqueous and organic solvents and
the extracts in organic solvents have been found to possess greater
antioxidant potential. This could be mainly due to greater solubility
and extraction of plant phenolics in organic solvents. However, for
the application in food or meat system, these organic solvents need to
be carefully removed from the extracts. It would be proper to extract
126 Natural Antioxidants: Applications in Foods of Animal Origin

plant phenolics in aqueous medium to avoid any chances of residual


organic solvents in foods. In order to enhance the yield of aqueous
plant extracts there is need to device suitable extraction process and
techniques.
• It is claimed that plant extracts have lower antioxidant potential than
synthetic counterparts. The application of microencapsulation and
nanoencapsulation should be considered to optimize delivery and
release of natural antioxidants and enhance their efficacy. Active
packaging technology can also be exploited to control the delivery
and efficacy of natural antioxidants.
• Screening of plant extracts should be done with the help of mass
spectrometry to track down any unwanted component.
• The interaction among antioxidant in different food system should
be taken in account. On one hand, the synergistic action of TM and
ascorbic acid can be utilized for preventing auto-oxidation or photo-
oxidation of lipids, on the other hand, the strong antagonistic action
between plant polyphenols and TM reported in pork fat should also
be scrutinized.

3.8 CONCLUSION

Meat and meat products because of their chemical constituents and


processing as well as post-processing conditions, become vulnerable to
oxidation which subsequently lead to deterioration in color, texture, shelf
life, and overall acceptability. Protection from such chemical and quality
changes requires various managemental, processing, and post-processing
steps. Application of natural antioxidants from plant sources in meat system
to prevent quality deterioration is one of several steps. Plant extracts are
being regularly screened for their antioxidant potential. These are applied in
the meat and meat products to evaluate their efficacy and efficiency. Several
plant extracts have been attempted in the meat system at different levels and
the quality, acceptability, and oxidative stability of the products has been
monitored through determination of protein and lipid peroxidation, color
properties, and sensory acceptability. In the coming years there are need
to carry out research work to enhance yield of plant phenolics, screen their
active principles, and control their delivery and release in the meat system.
It can be expected that more and more natural antioxidants with greater anti-
oxidant potential and tailored delivery mechanisms would be explored in
the near future to produce safer, fresh, acceptable, and stable meat products.
Potential Applications of Natural Antioxidants in Meat and Meat Products 127

KEYWORDS

• natural antioxidants
• plant material
• meat and meat products
• oxidation

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CHAPTER 4

NATURAL ANTIOXIDANTS: CONTROL


OF OXIDATION IN FISH AND FISH
PRODUCTS
UGOCHUKWU ANYANWU and REZA TAHERGORABI*
Department of Family and Consumer Sciences, Food and Nutritional
Sciences, North Carolina Agricultural and Technical State University,
Greensboro, NC, USA
*Corresponding author. E-mail: [email protected]

CONTENTS

Abstract ....................................................................................................142
4.1 Introduction .....................................................................................142
4.2 Oxidation in Fish Products .............................................................144
4.3 Oxidation Mechanism .....................................................................147
4.4 Antioxidants ....................................................................................149
4.5 Application of Natural Antioxidants in Fish Products ....................155
4.6 Market and Consumer Acceptability of Natural Antioxidants........157
4.7 Conclusion ......................................................................................158
Keywords .................................................................................................159
References ................................................................................................159
142 Natural Antioxidants: Applications in Foods of Animal Origin

ABSTRACT

Seafood is known to contain high amounts of polyunsaturated fatty acids


(PUFAs); thereby lipid oxidation is the main cause of quality loss in seafood.
Lipid oxidation may cause off-flavor as well as lowering of nutritive value,
which can be retarded by incorporation of additives having antioxidative
properties. The use of synthetic antioxidants has long been practiced in
retarding lipid oxidation. However, due to the potential health concerns of
synthetic antioxidants, natural antioxidants such as polyphenolic compounds,
essential oils, peptides, and microbial antioxidants, have been used as an
alternative natural antioxidants to retard lipid oxidation in different seafood
systems. This chapter reviews in detail the lipid oxidation in fish products,
antioxidants, and their natural sources as well as focuses on the application
of these compounds on the prevention of lipid oxidation in different seafood
systems.

4.1 INTRODUCTION

The demand for fish and fishery products in the global market has been
increasing with increase in the world population. Fish is an important part
of a healthy diet (Mozaffarian & Rimm, 2006). It is an important source
of a number of nutrients, particularly protein, vitamin D, retinol, iodine,
vitamin E, selenium, and the essential long-chain polyunsaturated fatty
acids (PUFAs), that is, eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA) (Welch et al., 2002). It is recommended that fish and seafood
products take a prominent position in the human diet due to their benefi-
cial effect on chronic degenerative diseases. The consumption of fish may
protect against cancers and cardiovascular diseases (Nestel, 2000), hence the
food industry and health authorities have a joint interest in increasing the
consumption of fish.
Oxidation as a chemical reaction that involves the transfer of electrons
from one compound to another has long been reported to have negative
effects especially in physiological context. Oxidation of molecules such
as DNA or lipids has resulted in many problems such as cancer and heart
diseases, both of which are important for proper life function. Oxidation
reactions can occur in food items mainly as a result of prolonged exposure
to the atmosphere. These reactions can cause rancidity, browning, and
development of unpleasant flavor. Oxidation is one of the main factors
Natural Antioxidants: Control of Oxidation in Fish and Fish Products 143

that determine food quality loss and shelf-life reduction. Hence, delaying
oxidation is highly relevant to food processors. Oxidative processes in
fish products can lead to the degradation of lipids and proteins which, in
turn, contribute to the deterioration in texture and color (Decker et al.,
1995).
To combat oxidation in food and physiological context, compounds
known as antioxidants are utilized. Antioxidants are generally defined as
compounds that prevent oxidation. They vary greatly in size, molecular
weight, and composition. While some are small in size others are enor-
mous and can even be macromolecules such as proteins. They provide elec-
tron density to compounds likely to undergo oxidation, hence preventing
them from losing electrons. Since there are different types and sources
of antioxidants, they can be grouped into two major categories: natural
and synthetic. Natural antioxidants are harvested directly from organic
sources or compounds found in foods consumed without much processing
whereas synthetic antioxidants are artificial compounds created in labo-
ratories and generally added to processed or pre-packaged foods acting
as preservatives. Although, synthetic antioxidants have been widely used
to inhibit lipid oxidation, the trend is to decrease their use because of the
growing concern among consumers about such synthetic materials (Chas-
tain et al., 1982). Therefore, the search for natural additives, especially
of plant origin, has increased in recent years. Compounds obtained from
natural sources such as grains, fruit, oilseeds, spices, and vegetables have
been investigated (Chen & Ho, 1997). The development and application of
natural products with antioxidant activities in fish products may be neces-
sary and useful to prolong their shelf life and potential for preventing fish
spoilage.
Natural antioxidants have shown to have significant health benefits espe-
cially in prevention of cancer and heart diseases. Due to this, many food
manufacturers have begun publicizing this fact and have achieved promi-
nence on many food labels on various food products. Although natural anti-
oxidants are known as key supplements by vitamin and health food manu-
facturers, consumers must remain aware of the sources from which these
compounds are procured as well as their concentrations. Therefore, the aim
of this chapter is to elucidate the natural antioxidants commonly applied
to fish, their mode of chemical reaction, and consumer awareness of these
compounds.
144 Natural Antioxidants: Applications in Foods of Animal Origin

4.2 OXIDATION IN FISH PRODUCTS

Oxidation in fish and fish products possess a high risk of quality loss leading
to rancid taste, off-flavor, and development of many different compounds
which have adverse effect to human health (Ames et al., 1993). Oxidation
limits storage time and this affects marketing and distribution of fish and
fish products. Oxidation is high in fish because of presence of omega-3
PUFAs susceptible to peroxidation and results in restriction to storage and
processing possibilities (Gray et al., 1996). Products of peroxidation-alde-
hydes, react with specific amino acids to form carbonyls and protein aggre-
gates which causes additional nutritional loss (Uchida & Stadtman, 1993);
for instance, in red fish such as salmon, oxidation not only deteriorates
the lipids but also affects the color, thus affecting visual consumer accept-
ability of fish products (Scaife et al., 2000). Two forms of oxidation occur
in fish products—lipid and protein oxidation and they are discussed in the
following subsections. While lipid oxidation leads to formation of unhealthy
compounds and off-flavors such as rancid, protein oxidation affects the
functional properties, including texture, and may potentially affect the taste
of fish products. Lipid and protein oxidation occur as a result of the pres-
ence of reactive oxygen species (ROS) which include oxygen radicals such
as superoxide anion (O2−), hydroxyl (HO−), peroxy (ROO−), alkoxy (RO−),
and hydroperoxy (HOO) radicals. Non-radical derivates of oxygen such as
hydrogen peroxide (H2O2), ozone (O3), and singlet oxygen (1O2−) are also
ROS (Choe & Min, 2009).
The thiobarbituric acid reactive substances (TBARS) as reported by
Botsoglou et al. (1994) are naturally present in biological specimens and
composed of lipid hydroperoxides (HPOs) and aldehydes which increase in
concentration as a response to oxidative stress. The sensitivity of measuring
TBARS has made this assay the method of choice for screening and moni-
toring lipid peroxidation which is a major indicator of oxidative stress.
TBARS assay values are usually reported in malonaldehyde (MDA) equiva-
lents, which is a compound that results from the decomposition of PUFA
lipid peroxides. This assay is well recognized and an established method for
quantifying lipid peroxides.

4.2.1 LIPID OXIDATION

Lipids are one of the important functional and structural components of


foods. They provide energy and essential nutrients such as EPA, DHA,
Natural Antioxidants: Control of Oxidation in Fish and Fish Products 145

and fat-soluble vitamins including, vitamins A, D, E, and K. Christie


(1998) defined lipids as “fatty acids, their derivatives and substances
related biosynthetically or functionally to these components.” Although
they constitute a minor component of food, contribute to the feeling of
satiety, and help in making food products palatable, they have been known
to significantly affect food quality. This effect on food quality is as a result
of constant exposure of lipids, particularly unsaturated fatty acids to air.
The susceptibility of lipids to oxidation is one of the main causes of quality
deterioration in various types of fresh food products as well as in processed
foods.
Lipid oxidation is believed to be one of the factors limiting the shelf
life of fish as well as many other complex products (Jacobsen, 1999). Lipid
oxidation is most evident in products with a high amount of unsaturated
fatty acids leading to rancidity, off-flavor, taste, color, and nutritional value
such as reductions in omega-3 fatty acids, some vitamins, and formation of
potentially toxic substances (Medina et al., 2009). Lipid oxidation to some
extent affects the safety of fish products for human consumption; hence,
a notable determinant in consumers’ preference for fish and fish products.
Within the food industry, a great deal of research and attention is spent on
the on-going oxidative processes with the aim of protecting raw materials
and products from oxidation during production process and storage. Lipid
oxidation, initiated by hemoglobin (Hb) (Christensen et al., 2011), occurs by
a reaction between free radicals and oxygen in the presence of other initia-
tors (metal, light, and heat) that results in the formation of HPOs and peroxy
radicals (Andersen et al., 2007).
The primary products undergo further reactions to form more stable
compounds such as hydroxy acids (that can contribute to bitter taste) or
epoxides (Grosch et al., 1992). The interaction of lipid HPOs and secondary
oxidation products with proteins and other components in complex food
systems, significantly impact oxidative, flavor stability and texture during
processing, cooking, and storage (Erickson, 1992). Oxidized lipids can
further react with amines, amino acids, and proteins to form brown macro-
molecular products (Frankel, 1998). According to Pan et al. (2004), color
formation is known to be primarily influenced by the degree of fatty acid
unsaturation, water activity, oxygen pressure, and the presence of phenolic
compounds. Metal, metalloproteins, and enzymes are important factors
affecting lipid oxidation in fish products. Water activity, lipid interac-
tions, proteins, and sugars are important elements affecting the quality of
processed fish.
146 Natural Antioxidants: Applications in Foods of Animal Origin

4.2.2 PROTEIN OXIDATION

Like lipid oxidation, ROS also take part in protein oxidation. Their mecha-
nisms are very similar to that of lipid oxidation and caused by metal catal-
ysis, irradiation, light exposure, and peroxidation. The negative effect of
protein oxidation is seen in the oxidation of sulfhydryl groups, reactions
with aldehydes, establishment of cross-links between proteins, formation of
oxidative adducts on amino acids, and protein fragmentations (Stadtman &
Oliver, 1991). The outcome of oxidation of the majority of amino acids leads
to the generation of several products and formation of protein carbonyls
viz., glutamic semialdehyde from alanine, hydroperoxides and carbonyl
compounds from arginine, sulfenyle chloride from cysteine, hydroperox-
ides from glutamic acids, asparatate and asparagines form histidine, alco-
hols and carbonyl compounds from isoleucine, hydroxyleucine and alcohols
carbonyls from leucine, etc. (Stadtman and Oliver, 1991).
According to Stadtman (2006), the mechanisms that can lead to protein–
protein cross-linking by reactive species are described as:

• Formation of disulfide cross-links (RSSR) as a result of oxidation of


cysteine sulfhydryl groups on the proteins.
• Interaction between active groups on different proteins such as the
reaction of an aldehyde group of a 4-Hydroxynonenal (HNE) protein
adduct and the –NH2 group of a lysine residue in two separate proteins.
• Reaction of MDA with the reactive –NH2 groups of lysine residues in
two different protein molecules.
• The formation of covalent linkages between carbon-centered radicals
in two different protein molecules.
• The interaction between a carbonyl group of a glycated protein and
an –NH2 group of lysine residue in another protein.

Oxidation of amino acids as reported by Orrenius et al. (2007) can


be used as a marker for protein oxidation and modification. In protein
oxidation, ROS abstract a hydrogen atom which leads to the creation of
a protein with a free radical (P•). The protein carbon-centered radical,
in the presence of oxygen, reacts with a peroxyl radical (POO•) and as a
result of its reactivity the peroxyl radical abstracts a hydrogen atom from
another molecule leading to the formation of alkyl peroxide (POOH•)
(Lund et al., 2011).
Protein oxidation leads to changes in protein hydrophobicity in processed
fish thereby reducing the water holding capacity (WHC) and altering the
Natural Antioxidants: Control of Oxidation in Fish and Fish Products 147

texture of the product. Metal catalyzed reactions oxidize amino acids such
as arginine, proline, and lysine into carbonyl residues (Lund et al., 2011).
Amino acids such as cysteine or methionine react in cross-linking or sulfur
derivatives. Furthermore, H2O2 reacts with metmyoglobin (Mb (Fe3+)) which
accumulates in the muscle after slaughter, leading to formation of perfer-
rylmyoglobin and ferrylmyoglobin. Lund et al. (2011) went further to state
that protein oxidation can also be catalyzed by non-heme iron and other
transition metals in the presence of H2O2. Myofibrillar proteins are oxidized
in the presence of ferric iron (Fe3+) and H2O2, resulting in formation of a
semialdehyde and ferrous iron (Fe2+). Ferrous iron in the presence of H2O2
continues the oxidative reaction degrading amino acids into hydroxyl radi-
cals. Also, reaction between H2O2 and the thio groups of amino acids, such
as cysteine leads to the formation of sulfhydryl groups. The results of the
oxidation of thio groups are sulfenic acid (RSOH), sulfinic acid (RSOOH),
and RSSR (Lund et al., 2011).
The formation of carbonyl during protein oxidation can alter the tertiary
structure of protein and lead to various degrees of irreversible and irrepa-
rable protein unfolding (Aldini et al., 2007). Protein oxidation causes the
loss of normal functions, such as enzymatic activity, channel-forming prop-
erties, and the proteins become more susceptible to proteolytic degradation.
However, with alteration in the tertiary structure and increased hydropho-
bicity by oxidation, the rate of protein degradation is reduced (Matsuishi &
Okitani, 1997).

4.3 OXIDATION MECHANISM

Fish and fish products are made up of several compounds especially lipids
that can easily undergo oxidation due to its high tendency to lose electrons.
The centerpiece of this reaction is the molecular species known as free radi-
cals. Free radicals are molecules or atoms that have unpaired electrons and
can vary greatly in their energy. Auto-oxidation of lipids triggered by expo-
sure to light, ionizing radiations, metalloprotein catalyst, or heat can have a
deteriorating effect on color, texture, flavor, quality, and safety of fish prod-
ucts (Urquiaga & Leighton, 2000). Two major components are involved in
lipid oxidation; unsaturated fatty acids and oxygen. In the oxidation process,
oxygen from the atmosphere is added to fatty acids particularly oleate, lino-
leate, and linolenate, creating unstable intermediates which break down to
form unpleasant compounds. This process involves primary auto-oxidative
reactions which are further accompanied by various secondary reactions
148 Natural Antioxidants: Applications in Foods of Animal Origin

having oxidative and non-oxidative characteristics (Scott, 2012). Fats


contained in food are chemically composed of triglycerides and oxidation
leading to the rancidity of foods occurs at the unsaturated sites of triglycer-
ides. Oxidation in fish and fish products occurs in both free fatty acids and
fatty acyl groups. The oxidation mechanism consists of three steps:
Initiation (Formation of Fatty Acid Radicals): This is the first step in
the oxidative mechanism of lipids. It involves the abstraction of hydrogen
from fatty acid to form a fatty acid radical known as the alkyl radical. Initia-
tors react by binding a hydrogen atom from an unsaturated lipid leading to
formation of a free radical. Stabilization of the free radical by delocalization
over the double bonds results in double bond shifting or formation of conju-
gated double bonds. This leads to production of cis or trans configuration
(Damodaran et al., 2007). Pro-oxidants such as ionizing radiation or light,
transition metals, and temperature are responsible for initiation of oxidation
reactions (Fennema et al., 2008).

RH → R. + H

RH → ROO. + H.

where ROO. is a lipid peroxy radical, R. is a lipid radical, and RH is an unsat-


urated lipid. The peroxides may break down to carbonyls, form polymers or
react with vitamins, proteins, pigments, and so forth. The ease of formation
of fatty acid radicals increases with an increase in unsaturation. Hydrogen
abstraction becomes easier and lipid oxidation is faster due to decrease in
carbon–hydrogen bond energy as a result of bond dissociation. For instance,
linolenic acid has been estimated to be 10–40 times more susceptible to
oxidation than oleic acid due to its rate of bond dissociation (Damodaran et
al., 2007).
Propagation (Fatty Acids Radical Reaction): The initial reaction in
this step involves the addition of an oxygen molecule which binds to the
fatty acid radical (alkyl radical) leading to the formation of peroxyl-fatty
acid radical and a weak covalent bond. As a result of the weak covalent
bonds of unsaturated fatty acids, they are susceptible to react with peroxyl
radicals. Furthermore, the high energy of peroxyl radicals allows them to
promote the abstraction of hydrogen from another molecule. This reaction
leads to formation of fatty acid hydroperoxyl and fatty acid radical. Further
hydrogen addition to the peroxyl radical results in formation of a fatty acid
HPO and a new alkyl radical on another fatty acid. Thus, the reaction moves
from one fatty acid to another.
Natural Antioxidants: Control of Oxidation in Fish and Fish Products 149

R. + O2 → ROO.

ROO. + RH → R. + ROOH

Termination: This reaction describes the combination of two fatty acid


radicals leading to the formation of non-radical products in the presence
of oxygen. The reactions between peroxyl and alkoxyl radicals take place
under atmospheric conditions while reactions between alkyl and radicals
lead to the formation of fatty acid dimers under low oxygen levels (Abidi &
Rennick, 2003).

2RO2. → O2 + RO2R

RO2. → RO2R

4.4 ANTIOXIDANTS

Antioxidants are regarded as key agents for improving oxidative stability


of fish products and act as preservatives which are added to food items at
various stages of processing (McClain & Bausch, 2003). Antioxidant effec-
tiveness is related to activation energy, oxidation–reduction potential, rate
constants, ease with which the antioxidant is lost or destroyed (volatility and
heat susceptibility), and antioxidant solubility (O’Connor & O’Brien, 2006).
They are compounds that delay auto-oxidation by inhibiting formation of
free radicals by one (or more) of several mechanisms:

1) Scavenging species that initiate peroxidation.


2) Chelating metal ions such that they are unable to decompose lipid
peroxides or generate reactive species.
3) Breaking the auto-oxidative chain reaction, and/or reducing local-
ized O2 concentrations.
4) Quenching O2− preventing formation of peroxides.

Antioxidants have been reported to play an important role in the body


by protection against oxidative damage (especially damage to the DNA),
and delay chronic health problems like cataracts (Morton et al., 2000).
Although the Food and Drug Administration (FDA) defines antioxidants
only as dietary supplements to be taken with normal food consumption,
they also serve as preservatives for packaged foods. They are added to
150 Natural Antioxidants: Applications in Foods of Animal Origin

fish products to maintain freshness, prevent rancidity and are particularly


important in food containing large amount of lipids. Therefore, addition of
antioxidants to fish products greatly extends shelf life and maintains flavor
and aroma for as long as possible. The antioxidant activity of a particular
compound, mixture of compounds, or a natural source, is generally related
to its ability to scavenge free radicals, decompose them, or quench singlet
oxygen (Shahidi, 1997).
Antioxidants can be derived naturally as well as chemically synthesized
although their performance levels differ. Both antioxidants function by
donating electron density to fats, thus preventing their breakdown. Gener-
ally, natural antioxidants are known to have higher beneficial health effects
such as their ability to prevent disease, for example, cancer and heart disease
as compared to the synthetic ones (Morton et al., 2000).

4.4.1 SYNTHETIC ANTIOXIDANTS

Synthetic antioxidants do not occur in nature hence, are chemically synthe-


sized to help prevent lipid oxidation in food. The use of synthetic antioxi-
dants dates back to the 1940s when butylated hydroxyanisole (BHA) was
found to retard oxidation and the effectiveness of several alkyl esters of
gallic acid was unraveled. Furthermore, it was also evident that the harmful
effects of transition metals such as iron and copper had to be counteracted;
hence, certain acids, such as citric acid (CA), and their derivatives, were
found to act as metal deactivators in combination with phenolic antioxidants
(Barlow & Hudson, 1990). In 1954, butylated hydroxytoluene (BHT) was
also approved for food use in the United States and tert-butylhydroquinone
(TBHQ) was commercialized in 1972. Synthetic antioxidants are divided
into primary and secondary antioxidants. Primary antioxidants which
prevent the formation of free radicals are further divided into:

• Radical terminators: These constitute the bulk of synthetic antioxi-


dants which prevent lipid oxidation by terminating the free radical
chains. Examples include: BHA, BHT, propyl gallate (PG), TBHQ,
dodecyl gallate (DG), and octyl gallate (OG).
• Oxygen scavengers: These function as reducing agents. Example:
sulphites, ascorbyl palmitate, and glucose oxidase.
• Chelating agents: These prevent lipid oxidation by binding the cata-
lysts such as heavy metals (copper, iron, etc.), either by precipitating
Natural Antioxidants: Control of Oxidation in Fish and Fish Products 151

the metal or by occupying all its coordination sites. Examples include:


ethylene diaminetetraacetic acid (EDTA) and polyphosphatases.
• Secondary antioxidants function by breaking down HPOs formed
during lipid oxidation into stable products. Examples include: dilauryl
theodipropionate and thiodipropionic acid (Naidu, 2010).

BHT and BHA are the most prevalent synthetic antioxidants in food.
Chemically, they are monohydric phenol with BHA consisting of two
isomers 3-tertiary butyl 4-hydroxyanisole and 2-tertiary butyl 4-hydroxy-
anisol in the ratio 9:1. It is available as white waxy flakes, while BHT is a
white crystalline solid and both are extremely soluble in fats but not in water
as a result of their phenolic structure with bulky hydrocarbon side chains.
Because of their carry through properties, both compounds can withstand
various processing steps such as baking and frying as well as maintaining
their functionality (Devlieghere et al., 2004). They are effective in protecting
the flavor and color of foods.
The FDA stated that the presence of synthetic antioxidants used in foods
be mentioned on food labels with an explanation of their intended usage.
Their permissible levels in food is decided on the basis of the fat content
of the food and usually limited to 0.02% total antioxidants (Naidu, 2010).
When used within recommended levels, they have shown to prevent lipid
deterioration in food thereby extending the shelf life of foods. Even though
at current levels of intake, synthetic antioxidants seem to pose no reason-
able threat to health, but long-term ingestion may aid in modifying the
acute toxicity of several carcinogenic and mutagenic chemicals and lead
to chronic side effects. Therefore, in recent time, there has been growing
concern over possible carcinogenic effects of synthetic antioxidants in
foods. BHA, TBHQ, as well as other synthetic antioxidants are no longer
allowed for food application in Japan and a number of other countries
although still in use at recommended levels in certain countries; there is
a general desire to replace synthetic antioxidants with natural ingredients
(Venkatesh, 2011).

4.4.2 NATURAL ANTIOXIDANTS

The mention of natural antioxidants brings about an association with spices


and herbs, in that they are utilized by-product developers as replacements for
synthetic antioxidants. However, other natural products such as nuts, cereals,
oilseeds, legumes, animal products, and microbial products can serve as
152 Natural Antioxidants: Applications in Foods of Animal Origin

sources of natural antioxidants. Phenols, polyphenolics, and phenolic acid


derivatives are antioxidants common to all plant sources of natural anti-
oxidants. Furthermore, modified proteins and amino acids are antioxidants
derived from animal and microbial products. The various sources of natural
antioxidants are cereals (whole wheat products, oat, rice, bran), vegetables
(leaf vegetables, potatoes), fruits (apples, bananas, berries, olives), oilseeds
(sesame seeds, hazelnuts, almonds), legumes (beans, peanuts, soybeans),
cocoa products (chocolate), beverages (tea, coffee, red wine, beer, fruit
juices), and herbs and spices (rosemary, sage, oregano, savory).

4.4.2.1 ANIMAL ORIGIN

Amino acids, peptides, and carotenoids are three animal products that could
serve as natural antioxidants. Glutathione peroxidase, superoxide dismutase,
and catalase are antioxidant enzymes present in muscle systems. Anserine,
carnosine, and ophidine are histidine-containing dipeptides reported to
chelate metals and scavenge radicals (Chan et al., 1994). L-Histidine as part
of a small peptide/protein or in the free form can scavenge hydroxyl radi-
cals and quench singlet oxygen, which can react with the double bond of
L-histidine to form a peroxyl radical (Wade & Tucker, 1998). L-Histidine
has the ability to quench singlet oxygen three-fold higher than tryptophan
and five-fold higher than methionine.
Carotenoids typically associated with the color of fruits and vegetables
are also found in many animals. Crustacea demonstrate a multitude of carot-
enoid pigments found in nature. According to Zagalsky et al. (1990), carot-
enoids of invertebrates are associated with protein in a complex defined as
carotenoprotein. The carotenoids present in the exoskeleton of crustaceans
may provide the best opportunity to develop natural antioxidants from
animal sources. Red crabs contain β-carotene and astaxanthin while blue
crabs contain 4-hydroxyechinenone, canthaxanthin, 3-hydroxycanthax-
anthin, echinenone, isocryptoxanthin, β-carotene, and astaxanthin. These
compounds are the most common and important pigments from animal
sources that serve as natural antioxidants although limited research have been
completed on the antioxidant activity of carotenoids in crustacean (Ramı́rez
et al., 2001). However, their activity would be expected to be similar to plant
carotenoids due to the structural similarities between plant and animal carot-
enoids. Development of extraction or concentration processes is required
for the production of adequate amount of natural antioxidants from animal
sources.
Natural Antioxidants: Control of Oxidation in Fish and Fish Products 153

4.4.2.2 PLANT ORIGIN

Plant-derived additives offer natural alternatives to synthetic antioxidants. In


the modern world changes in lifestyle have triggered a growing awareness
that particular ingredients in food may favorably modify diet-related prob-
lems. The interest in using naturally occurring nutritive and non-nutrient
antioxidants for food preservative purpose is due to their possible prevention
of a number of diseases, in the etiology of which oxidation mechanisms are
involved. Antioxidants can be sourced from selected herbs, spices, fruits,
nuts, and other plants (Boskou, 2006). The classes of compounds that act as
antioxidants from plant sources include: tocochromanols (lipophilic plant-
derived antioxidants) and the more polar phenolic compounds, including
phenolic acids, simple phenolics, flavonoids, anthocyanins, tannins,
hydroxytyrosol and derivatives, and constituents of essential oils (Pokorný,
2007). Frequently encountered natural antioxidants in plants are phenolic
acids and hydroxybenzoic acid (vanillic acid), hydroxycinnamic acid series
(ferulic acids, chlorogenic acid), flavonoids (quercetin, catechin, rutin),
anthocyanins (delphidin), tannins (procyanidin, ellagic acid, tannic acid),
lignans (sesamol), stibenes (resveratrol), coumarines (ortho-coumarine), and
essential oils (S-carvone).
Phenolic compounds are plant secondary metabolites and are commonly
found in herbs, vegetables, fruits, grains and cereals, coffee, red and white
wines, and green and black tea. Phenolic acids are phenols that possess
carboxylic acid functionality and they are made up of two distinguishing
constitutive carbon frameworks. The flavonoids consist of a group of low-
molecular weight polyphenolic substances. According to the degree of
oxidation of the C-ring, flavonoids can be categorized into the subclasses
flavones, isoflavones, flavanones, flavanonols (dihydroflavonols), flava-
nols (catechins), flavonols, anthocyanins, and proanthocyanidins. The anti-
oxidant activity of these compounds arises from their direct reaction with
free radicals (acting as primary antioxidants) and via their chelation of free
metals, which prevents further involvement of these metals in reactions that
finally generate radicals.
Tocochromanols are natural compounds known as tocopherols and toco-
trienols. They are found mainly in plant oils, nuts, and seeds. Experimental
data indicate that they have a radical chain-breaking activity and reducing
ability (Kamal-Eldin & Appelqvist, 1996).
154 Natural Antioxidants: Applications in Foods of Animal Origin

4.4.2.3 MICROBIAL SOURCES

Microorganisms are one of the most abundant and diverse species found on
earth and their exploitation to produce food ingredients has been going on
for the past decade. However, the isolation of microbial antioxidants became
a focus of research in the early 1980s, Forbes et al. (1958) established a
relationship between antioxidants and microorganisms and since this early
work, a vast number of compounds and microorganisms have been charac-
terized. Several studies have demonstrated the antioxidant activity of micro-
organisms. Using the thiocyanate method, the antioxidant activity of ethyl
acetate extracts of several Penicillium and Aspergillus species was evaluated
(Yen & Lee, 1996). Extracts of these species protected linoleic acid better
than the control. In a study by Yen and Chang, it was reported that sucrose or
lactose and ammonium sulphate in culture media enhanced the Aspergillus
candidus production of antioxidants (Yen & Chang, 1999). Extracts with
similar activity were produced from ethyl acetate extraction of the broth and
mycelium.
In a study conducted by Aoyama et al. (1982), 750 filamentous fungi
isolated from soil were screened. Two antioxidants were identified as
citrinin and protocatechuic. A third compound, curvulic acid, isolated from
an unidentified Penicillium was also evaluated for antioxidant activity in
linoleic acid. All three compounds were reported to have good antioxidant
activity. The curvulic acid had the largest antioxidant activity followed by
the curvulic acid methyl ester, protocatechuic acid, and citrinin.
According to Esaki et al. (1997) Aspergillus species are effective
producers of antioxidant activity compounds. In their study, 30 strains
of Aspergillus were evaluated and it was found that methanol extracts of
fermented soybeans (MEFS) prevented oxidation of methyl linoleate. The
MEFS of 28 strains had better antioxidant activity than the non-fermented
soybean while all strains were better than the control. Separation of the
MEFS revealed 2,3-dihydroxybenzoic acid as a component of the most
active fraction. Hayashi et al. (1995) also identified this compound in Peni-
cillium roquefortii IFO 5956 cultures.
In another study, Esaki et al. (1997) evaluated the antioxidant activity of
methanol extracts (MEs) of miso, natto, and tempeh and found that tempeh
was the most effective followed by miso. They further stated that fermenta-
tion by mold cultures are more active than bacterial (Bacillus natto) ones
in producing antioxidants. This was evident as a result of the antioxidant
activity of the natto ME being less than that of other fermented products
but was equivalent to that of unfermented soybeans. Hoppe et al. (1997)
Natural Antioxidants: Control of Oxidation in Fish and Fish Products 155

identified 5-(ϭ-tocopheroxy)-d-tocopherol as an antioxidant obtained from


tempeh fermented by Rhizopus oligosporus. Gallic acid has been isolated
from cultures of Penicillium and Aspergillus and is known as a phenolic acid
found in many natural sources including microbial products. Methylenebis
(5-methyl-6-tert-butyl-phenol) has been identified as an antioxidant from
Penicillium janthinellum. Eurotium species have also been found to produce
several antioxidants. Three of the seven metabolites were found to have
antioxidant activity and were identified as dihydroauroglaucin, auroglaucin,
and flavoglaucin. Furthermore, Atroventin was isolated from Penicillium
paraherquei and found to have good antioxidant activity. Demethylnaph-
terpin and Carazostatin are free radical scavengers isolated from Strepto-
myces chromofucus and Streptomyces prunicolor, respectively (Shin-Ya et
al., 1992).
Carotenoids are also group of antioxidants that can be synthesized by
microorganisms. Nelis and Leenheer reported that lycopene from Blakeslea
trispora and Streptomyces chrestomyceticus, subsp. rubescens and β-carotene
from B. trispora and Duniella salina were approved for human foods as
colorants (Johnson & Schroeder, 1996). Also, astaxanthin from microbial
sources, for example, Xanthophyllomyces dendrorhous, found to have excel-
lent singlet oxygen quenching activity has been approved for use in fish
foods. The antioxidant activity of carotenoids including lutein, β-carotene,
and astaxanthin was confirmed using a fluorometric assay (Naguib, 2000).
The use of microbial fermentation as a method for producing natural antioxi-
dants has promise; therefore, more work is needed to optimize production
conditions.

4.5 APPLICATION OF NATURAL ANTIOXIDANTS IN FISH


PRODUCTS

The necessity to stabilize food against oxidation was realized before World
War II and, surprisingly, natural antioxidants have been in use because
synthetic antioxidants for edible uses were not yet available at that time
(Musher, 1944). However, composition and efficiency of the natural prep-
arations were found extremely variable and their activity was considered
insufficient. This led to the invention of synthetic antioxidants which were
chemically pure, possessed antioxidant activity, tested for safety, then made
readily available in the market. However, natural antioxidants isolated
from herbs, tea, grapes, and seeds have gained interest as replacement for
synthetic antioxidants (Samaranayaka & Li-Chan, 2011). They are readily
156 Natural Antioxidants: Applications in Foods of Animal Origin

acceptable by consumers as they are considered safe. Natural extracts or


pure compounds have been used for supplementing food products made of
minced fish muscle or surimi. Rosemary, olive oil, ginger, vegetable extracts
especially from tea, grape seeds composed of flavonoids, terpenoids, and
so forth, have successfully inhibited the rancidity of seafood products such
as fish patties, canned fish, fermented fish, and emulsified fish (Tang et al.,
2001).
Some other natural extracts obtained from materials such as light fish
muscle have been also utilized in fish systems (Sannaveerappa et al., 2007).
Furthermore, procyanidins, catechins and their gallate esters, flavonoids,
and hydroxytyrosol have also been used in fish muscle (Pazos et al., 2008).
They have exhibited a high ability to inhibit oxidation in fish and fish
products.
Fish muscle is known to be highly susceptible to oxidation primarily
because of the high level of unsaturation found in its lipids. Antioxidant
compounds have been studied as a means of increasing the oxidative
stability of fish and fish products. Several natural antioxidants have been
used in fish oils and fish products to retard lipid oxidation. Cuppett (2001)
reported that using a surface application of a rosemary oleoresin on muscle
from fish supplemented with tocopherol enhanced the stability of rainbow
trout muscle. Dry oregano was reported to be effective in preventing oxida-
tion in mackerel oil (Tsimidou et al., 1995). Zheng and Wang (2001) reported
that phenolic antioxidants from rosemary leaves were successfully used
in sardine and cod liver oil. Another study found that green tea polyphe-
nols protected silver carp from oxidation and phenolic extracts from grape
by-products were successfully applied in fish muscle (Sánchez-Alonso et al.,
2007). Frankel et al. (1996) reported that rosemary extracts (carnosol and
carnosic acid) were effective antioxidants in fish oils tested in bulk systems.
Tea catechins were found to be more efficient than tocopherol in inhibiting
minced muscle lipid oxidation in fish patties (Tang et al., 2001). Medina et
al. (1998) reported that polyphenols extracted from virgin olive oil retarded
oxidation of canned tuna, fish oils, and horse mackerel. A combination of
polyphenols and other antioxidant have been reported to be more effective
than synthetic antioxidants in preventing lipid oxidation in marine oils and
frozen fish (Pazos et al., 2005). Medina et al. (2007) reported that common
phenolic acids, such as caffeic acid, when added at the relatively low concen-
trations of 10–50 mg/kg, are very active in inhibiting increases in TBARS
and peroxide value (PV) in horse mackerel.
Many studies have shown the antioxidative effectiveness of natural
plant polyphenolic extracts in fish model systems. In a recent study, lipid
Natural Antioxidants: Control of Oxidation in Fish and Fish Products 157

oxidation was retarded in washed seabass mince with added Hb and


menhaden oil (MHO) using ethanolic kiam wood extract (EKWE) (0.1%,
w/w). The effectiveness was demonstrated by decreased PV and TBARS
values in the samples with added EKWE (Maqsood & Benjakul, 2013).
They also found that the formation of volatile lipid oxidation compounds
in the washed seabass mince with added MHO and Hb during iced storage
could be retarded using 0.1% EKWE. Brown lead (Leucaena leucocephala)
seed extracts (100 and 200 ppm) without prior chlorophyll removal showed
a protective impact on lipid oxidation in a dose-dependent manner in minced
mackerel. Brown lead seed extract exhibited scavenging action toward reac-
tants and radicals causing lipid oxidation. The extract was also capable of
delaying lipid oxidation in emulsion and liposome model systems in a dose-
dependent way (Benjakul et al., 2013). In another study, quince extracts
(8.9 ± 0.4 mg phenolics/mL) containing procyanidin B dimer (50.8%) and
hydroxycinnamic acids (36.62%) lowered the PV and restrained the devel-
opment of TBARS in the mackerel (Scomber scombrus) fillet fat fraction
during refrigerated storage (4 °C) (Fattouch et al., 2008). The bioactive
constituents of this extract possessed antioxidant activities as evaluated by
antiradical test (Fattouch et al., 2008).
In a recent study by Anyanwu (2015), bay essential oil (BEO) which
has been reported to have high antioxidant activity was used to inhibit lipid
oxidation in surimi seafood. Alaska pollock surimi seafood was nutrified
with omega-3 PUFA-rich oils from flaxseed and salmon and stabilized with
BEO during storage time. Omega-3 oils were added at 5% by replacing ice
at 1:1 along with 0 (control), 0.5, 1% BEO, followed by cooking (90 °C
for 30 min) in hotdog casings, vacuum packed and stored at 4 °C for six
days. Whiteness of surimi gels increased significantly with the addition of
BEO between treatments and storage time. Lipid oxidation significantly
decreased over storage time for treatments with 1% BEO. Addition of BEO
and omega-3 rich oils had no detrimental effect on the texture of surimi gels.
These studies indicate and promote the efficacy of natural antioxidants in the
inhibition of lipid oxidation in fish and fish products as well as other food
products.

4.6 MARKET AND CONSUMER ACCEPTABILITY OF NATURAL


ANTIOXIDANTS

Due to ability of natural antioxidants to prevent formation of free radicals,


they have been found to be useful in preventing certain diseases such as
158 Natural Antioxidants: Applications in Foods of Animal Origin

cancer and heart diseases by decreasing the amount of plaque buildup in the
blood vessels (McClain & Bausch, 2003). Additionally, they are reported to
increase the amount of high density lipoproteins (HDL), commonly known
as “good cholesterol” in the blood, thus as preventing heart disease (Yao
et al., 2004). These beneficial properties have put natural antioxidant on
the forefront of recent advertising and public awareness concerning natural
antioxidants and their positive effect has increased greatly. Asahara (1987)
studied the antioxidant effect of natural tocopherol mixture on marinated
sardine during cold storage which was compared with the effect of BHA.
Thiobarbituric acid (TBA) value was determined on lipids of samples during
storage time (200 days). Sensory evaluation revealed no negative effect on
the organoleptic properties on the samples. Lemon balm and oregano have
been reported to have a safe history of use as herbal food ingredients, and
their natural extractives are listed as generally recognized as safe (GRAS) in
the United States. Furthermore, herbs and flower tips of Origanum vulgare
and Melissa officinalis have been allocated the status N2 by the Council
of Europe; N2 comprises admissible natural sources of flavorings (Boskou,
2006).

4.7 CONCLUSION

Antioxidants are compounds that are present either naturally or added to food
items to prevent oxidation which always leads to rancidity, browning, and
general lack of freshness. Fish and fish products contain unsaturated fatty
acids which are especially susceptible to oxidation because of their electron
deficient double bonds. The breakdown products of oxidation can produce
off-odors, loss of nutrient content, new flavors, and color deterioration. To
manufacture high-quality, stable fish products, the most effective solution
is the addition of antioxidants, especially natural, which can serve as “chain
breakers,” by intercepting generation of free radicals during various stages
of oxidation or to chelate metals. A common feature of these compounds is
that they have one or more aromatic rings (often phenolic) with one or more
–OH groups capable of donating H· to the oxidizing lipid. The facts that
they are natural, and have antioxidative activity that is as good as or even
better than the synthetic antioxidants, make them particularly attractive for
commercial food processors. It is clear that consumers are becoming increas-
ingly aware of and selective against foods that are perceived by them to be
unnatural and containing additives. This means that controlling oxidation
Natural Antioxidants: Control of Oxidation in Fish and Fish Products 159

in fish with applied antioxidants has to be carefully considered. This would


account for the trend toward investigating the use of more natural antioxi-
dants such as herb extracts. Hence, there is a need to screen for new and
perhaps more efficacious natural extracts because of consumer demand for
natural ingredients.

KEYWORDS

• seafood
• lipid oxidation
• natural antioxidants
• synthetic antioxidants

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CHAPTER 5

NATURAL ANTIOXIDANTS IN
POULTRY PRODUCTS
A. K. BISWAS1,*, M. K. CHATLI2, and GAURI JAIRATH3
1
Division of Post-Harvest Technology, ICAR-Central Avian Research
Institute, Izatnagar, Bareilly 243 122, Uttar Pradesh, India
2
Department of Livestock Products Technology, GADVASU,
Ludhiana 141 004, Punjab, India
Department of Livestock Products Technology, LUVAS,
3

Hisar 125 001, Haryana, India


Corresponding author. E-mail: [email protected]
*

CONTENTS

Abstract ....................................................................................................166
5.1 Introduction .....................................................................................166
5.2 Lipid Oxidation ...............................................................................167
5.3 Sources of Natural Antioxidants .....................................................171
5.4 Mode of Application of Antioxidants .............................................190
5.5 Synergistic Effect of Natural Antioxidants .....................................193
5.6 Measurement of Efficacy of AOA ..................................................193
5.7 Conclusions .....................................................................................195
Keywords .................................................................................................195
References ................................................................................................196
166 Natural Antioxidants: Applications in Foods of Animal Origin

ABSTRACT

Arising consumer interests in “natural” and “fresh” muscle foods with


minimum chemical preservatives have motivated researchers to explore
antioxidant efficacy of many naturally occurring compounds. Many plants
or their parts, fruits, vegetables, herbs, and spices contain diverse bioac-
tive compounds namely phenolics, flavonoids, β-carotene, α-tocopherol,
glucosides, dietary fibres, and macro-minerals especially potassium. These
bioactive compounds, especially phenolic compounds, glucosides, and
some important vitamins like vit. C and vit. E possess significant ABTS+
radical cation, 1, 1 diphenyl-2-picrylhydrazyl (DPPH) radical, and super
oxide anion scavenging activity. This antioxidant potential of leaves,
seeds, vegetables, fruits can be utilized in different form such as paste,
powder, solvent extract etc. to augment the shelf life of poultry products
and could replace the synthetic antioxidants effectively. Addition of natu-
rally derived compounds found to have substantial desirable effects on the
physico-chemical, microbiological, and sensory attributes of poultry prod-
ucts. It has been reported that poultry products are rich in diverse bioac-
tive compounds which exert chemomodulatory effects through a variety
of physiological processes. Dietary intake of fruits and vegetables rich in
phenolic/flavonoid compounds prevents excessive free radical formation in
the cell, thereby, reduces risk of coronary heart diseases including stroke
and cancers. Hence, utilization of these naturally occurring compounds in
variety of poultry products will not only increase the storage stability, but
also provide adequate health promoting effect to the consumer. In view of
these facts, large scope exists for the use of naturally occurring antioxidants
in processing of animal food products including poultry. Research studies
were carried out on the beneficial aspects of natural antioxidants in improve-
ment of poultry products quality. It is assumed that this chapter will be of
great value to the researchers, academicians, entrepreneurs, students, and
also some poultry producers/processors who are directly or indirectly linked
with the processing of various poultry products.

5.1 INTRODUCTION

Poultry products are rich source of proteins, lipids, vitamins, and minerals,
out of which lipids are considered to be the integral components of these food
products. Being an integral component, the fatty acid compositions of cell
membranes’ phospholipid fractions are especially important in determining the
Natural Antioxidants in Poultry Products 167

stability of foods and act as a site for oxidative process initiation. Further, the
fatty acids mostly exist in unsaturated state which makes them more prone to
oxidation. Lipid oxidation is influenced by the composition of phospholipids,
polyunsaturated fatty acids (PUFA), metal ions, oxygen, heme pigments,
and addition of salt and processing approaches. It is one of the major factors
affecting the quality of animal foods; however, in cured meat, due to forma-
tion of stable pink color by ferrous form of pigment, there is less oxidation as
compared to cooked uncured meat. This oxidation is initiated when PUFA react
with molecular oxygen via free radical chain mechanism form peroxides and
accomplished with discoloration and production of malodorous compounds.
These compounds not only give rancid odors but significantly affect human
health. Color and flavor are the first stimuli for the consumers to purchase
meat and meat products, and in this regard, lipid oxidation is the main limiting
factor (Gray et al., 1996). Further, the low oxidative stability of fresh meat,
precooked and restructured meat products is a problem for all those involved
in the meat production chain, including the primary producers, processors,
distributors, and retailers, and a major challenge for meat scientists. The
scientific literature pertaining to propensity of lipid oxidation in animal foods
including poultry products and other biological systems is vast and suggests
various remedies to combat this problem; however, before adopting the solu-
tion there is a need to understand the concept of lipid oxidation.

5.2 LIPID OXIDATION

Lipid oxidation is often the decisive factor in determining the useful storage
life of food products, even when their fat content is as low as 0.5–1%. Oxida-
tive rancidity is initiated by the so called “reactive oxygen species” (ROS)
(Evans & Halliwell, 2001). The ROS in-turn combines various free radicals
which not only include oxygen-centered free radicals but also non-radical
derivatives of oxygen. Free radicals contain one or more unpaired electrons
and are capable of independent existence. Lipid oxidation in muscle foods is
initiated in the highly unsaturated phospholipid fraction of subcellular bio-
membranes; unsaturated portions of fatty acid esters react with molecular
oxygen to form peroxides, hydroperoxides, and carbonyl compounds. The
lipid hydroperoxides formed during the propagation of the per-oxidation
processes are unstable and are reductively cleaved in the presence of trace
elements to give a range of new free radicals and non-radical compounds
including aldehydes, ketones, alcohols, and acids that cause the off-odor,
off-color, change in nutritive value, and safety of muscle foods. Besides
168 Natural Antioxidants: Applications in Foods of Animal Origin

all, lipid oxidation products also affect protein solubility, emulsification,


water-binding capacity, texture, and the other rheological properties through
the interactions between lipid and protein oxidation products (Hall, 1987).
Cholesterol may be oxidized to form oxysterols, which are also as toxic as
fatty acid-derived hydroperoxides (Smith & Johnson, 1989). Consumption
of oxysterol-containing foods may be potentially harmful to cellular physi-
ology (Morel & Lin, 1996). Oxidation of meat lipids is a complex process
and its dynamics depend on numerous factors including chemical composi-
tion of the meat, light, and oxygen access as well as storage temperature. The
products of lipid oxidation also interfere with the absorption of protein or
folic acid and have been found to cause pathological changes in the mucous
membrane of the digestive tract.

5.2.1 LIPID OXIDATION MECHANISM

The first step in lipid oxidation is the removal of hydrogen ion from a methy-
lene carbon of a fatty acid (RH). It becomes easier as the number of double
bonds in the fatty acid increases, which is why PUFA are particularly suscep-
tible to oxidation. The initiation step can be catalyzed by OH– or by certain
iron–oxygen complexes (e.g., ferryl or perferryl radicals).

RH + OH– R– + H2O (5.1)

The fatty acyl radical (R–) reacts rapidly with O2 to form a peroxyl radical
(ROO–):

R– + O2 ROO– (5.2)

The rate-constant (K2) for this reaction is 3 × l08 M-1 S-1. Because ROO– is
more highly oxidized than the fatty acyl radical or the fatty acid itself, it will
preferentially oxidize other unsaturated fatty acids and propagate the chain
reaction:

ROO– + RH ROOH + R– (5.3)

The rate-constant (K3) for this step is relatively low (1 × 101 M-1S-1). Lipid
hydroperoxides (ROOH) formed in the propagation reaction are both prod-
ucts of oxidation and substrates for further reaction with Fe++ and Cu+ to
yield ROO– and alkoxyl radicals (RO–). The ferrous (Fe++) reductively
cleaves ROOH (3) as follows:
Natural Antioxidants in Poultry Products 169

Fe++ + ROOH Fe+++ + RO– + OH– (5.4)

and Fe++ can be regenerated as follows:

Fe+++ + ROOH ROO– + Fe++ + H+ (5.5)

Oxygen (O2) also reduces ferric iron to ferrous and cupric copper to
cuprous in vivo, allowing a redox cycle in which the transition metal ion is
used several times:

O2 + Fe+++ Fe++ + O2

O2 + Cu++ Cu+ + O2

Other strong reductants such as ascorbic acid (AA) and parquet also
reduce Fe+++ to Fe++. Both ROO– and RO– can initiate further reactions (3)
and the following:

RO– + RH ROH + R (5.6)

The RO– can also undergo β-scission and degrade to alkyl radicals
(R– CH2-) and a range of aldehydes (R–CHO) depending on the particular
hydroperoxide present.

RO– R–CH– + R–CHO (5.7)

(R–CH2–) can initiate further chain reactions resulting in the formation


of ethane and pentane, while the aldehydes, including hexanal, malondial-
dehyde, and 4-hydroxynonenal, can react readily with e-amino groups of
proteins to yield Maillard-type complexes.

5.2.2 FACTORS PREDISPOSING LIPID OXIDATION

The propensity of meats and meat products to undergo oxidation depends


on several factors including pre-slaughter events such as stress and post-
slaughter events such as early post-mortem pH, carcass temperature, cold
shortening, and techniques such as electrical stimulation. Oxidation can
also be accelerated due to disruption of the integrity of muscle membranes
by mechanical deboning, grinding, restructuring, or cooking which alters
cellular compartmentalization and releases the catalytic metal ions.
170 Natural Antioxidants: Applications in Foods of Animal Origin

Post-slaughter changes which predispose muscle foods to oxidation are:

a) Stunning and bleeding—circulation of blood ceases.


b) Anaerobic metabolism—lactic acid accumulates and pH declines to
approximately 5.5.
c) Circulation of nutrients rapidly ceases.
d) Preventative antioxidant enzyme system namely superoxide dismutase,
catalase, glutathione peroxidase, glutathione reductase etc., unlikely
to function.
e) Acute phase proteins which scavenge Fe+ aeruloplasmin, transferrin,
haptoglobin—unlikely to be activated.
f) Sarcoplasmic reticulum loses its calcium accumulating ability.
g) Calcium dependent proteinases degrade muscle proteins.
h) Some destruction of cell compartmentalization.
i) Low molecular weight chelatable iron is released.
j) Iron-catalyzed chain reactions.
k) Membranal lipid oxidation initiated.

5.2.3 CATALYSTS OF LIPID OXIDATION

Some confusion prevails about the nature of the initiation process in lipid
oxidation, but spontaneous lipid radical formation or direct reaction of unsat-
urated fatty acids with molecular oxygen is thermodynamically unfavor-
able. Most researchers believe that the presence of transition metals, notably
iron, is pivotal in the generation of species capable of abstracting a proton
from an unsaturated fatty acid. During handling, processing, cooking, and
storage, iron is released from high molecular weight sources (e.g., hemo-
globin, myoglobin, ferritin, hemosiderin) may directly cause oxidation or
made available to low molecular weight compounds such as amino acids,
nucleotides, and phosphates with which it is believed to form chelates. These
chelates are thought to be responsible for the catalysis of lipid oxidation in
biological tissues. However, the relative contributions of the different forms
of iron have not been clearly defined. Much of the information pertaining to
lipid oxidation in meat deals with hydroperoxide dependent lipid oxidation.
Pure lipid hydroperoxides are fairly stable at physiological temperatures,
but in the presence of transition metal complexes, especially iron salts, their
decomposition is greatly accelerated.
Natural Antioxidants in Poultry Products 171

5.2.4 ANTIOXIDANTS IN PREVENTING LIPID OXIDATION

Antioxidants are organic molecules of either synthetic origin or natural


origin, which can avoid or delay the progress of oxidative rancidity. It is
mainly imparted to their phenol-derived structure. Antioxidants work by
donating hydrogen to the lipid free radical to reform the fat molecule or by
donating one hydrogen to a peroxide free radical to form a hydroperoxide
and a stable antioxidant free radical. They “sacrifice themselves” by giving
up a hydrogen atom, then rearrange to a stable conformation.

A: H + RO• → A• + ROH (5.8)

Antioxidants can be classified according to their protective properties


at different stages of the oxidation process and since they act by different
mechanisms, they are divided into two main types: primary and secondary
antioxidants.

a) Primary antioxidants can inhibit or retard oxidation by scavenging


free radicals by donation of hydrogen atoms or electrons, which
converts them to more stable products, for example vit. C and E,
phytochemicals like flavonoids.
b) Secondary antioxidants function by many mechanisms, including
binding of metal ions, scavenging oxygen, converting hydroperox-
ides to non-radical species, absorbing UV radiation, or deactivating
singlet oxygen, for example butylated hydroxyl anisole (BHA),
butylated hydroxyrotoluene (BHT), propyl gallate (PG), and metal
chelating agent (EDTA).

5.3 SOURCES OF NATURAL ANTIOXIDANTS

Natural antioxidants are presumed to be safe and are available in variable


amounts in plant and animal kingdoms. Plant phenolics, “phytochemicals,”
are multifunctional and can act as reducing agents, free radical terminators,
metal chelators, and singlet oxygen quenchers. Flavonoids and other classes
of phenolic compounds are important phytochemicals. Extracts from plants
which contribute health benefits to consumers, arising from protection from
free radical-mediated deteriorations, and which cause retardation of lipid
oxidation have stronger antioxidant activity (AOA) than that of synthetic
antioxidants (Table 5.1).
TABLE 5.1 Some Important Natural Antioxidant and Their Effective Concentration in Chicken Products. 172
Name Part Active component Effective concentration References
(products)
Plant sources
Bamboo Leaves Flavones, lactones, phenolic acids 0.5% in chicken wings (Zhang et al., 2007)
Beetroot Ethanol extract – 0.5 ml/kg in chicken meat (Packer et al., 2015)
Canola Seed extract – 500 and 1000 ppm in chicken (Wanasundara and Shahidi,
meat 2007)
Cinnamon Cinnamon powder Cinnamaldehyde, Eugenol, 2% in minced chicken meat (Yadav et al., 2002)
cinnamicaldehyde
Cocoa Leaves/extract Polyphenols 800 mg/kg in MDCM (Osman et al., 2004)
Dark green leafy Lutein Phenolic compounds 300 µg extract/100g
vegetables
Garlic Fresh garlic Allicin, diallyl sulfide and diallyl 50 g/kg in chicken sausages (Sallam et al., 2004)
trisulfide
Garlic powder – 9 g/kg in chicken sausages
Garlic oil – 0.06 g/kg in chicken sausages
Grapes Peed extract (GSE) Proanthocyaninidins 0.1% in turkey (Brannan & Mah, 2007)
GADF Flavonoids 2% in chicken hamburger (Ayerdi et al., 2009)
Pomace (dietary) – 60 parts GPC in chicken patties (Ayerdi et al., 2009)
Green tea Leaves/Extract Catechins- epicatechins,-epigallo- 200–400 mg/kg in raw poultry (Tang et al., 2001)
catechin, epigallocatechin gallate, meat
Epicatechin gallate
(Dietary) – 300 mg/kg in poultry feed (Tang et al., 2000)
Honey – – 15% in turkey slices (Antony et al., 2006)
Natural Antioxidants: Applications in Foods of Animal Origin
TABLE 5.1 (Continued)
Name Part Active component Effective concentration References
(products)
Onion Freeze-dried onion Quercetin 1.6% in cooked chicken meat (Karastogiannidou, 1999)
powder
Juices Quercetin 50% strength onion juice brine in (Tang & Cronin, 2007)
turkey breast rolls
Oregano Oil and extracts Rosmarinic acid 200 mg/kg in turkey meat (Govaris et al., 2004)
Plums Plum extract Chlorogenic acid neoclorogenic > 2% in turkey breast rolls (Lee & Ahn, 2005)
acid, cryptochlorogenic acid
Potato Peel extract Catechin, chlorogenic acid 0.04% in irradiated meat (Kanatt et al., 2005)
Pomegranate Juice rind powder Tannins, anthocyanins and 10 m.equ/100 g in chicken patties (Naveena et al., 2008)
Natural Antioxidants in Poultry Products

flavonoids
Rice Hull extract Phenolic compounds 0.1%, w/w in turkey breast (Lee & Ahn, 2005)
Sage Oil Carnosol, rosmanol, rosemadiol, 3% in chicken meat (Mariutti et al., 2008)
carnosic acid
Sesame oil Sesamol Phenolic compounds 500–2000 µg/ml
Dry soya sprouts 30 g/kg in chicken patties (Romero et al., 2008)
Animal sources
Bone protein – – 2%
hydrolysates
Milk proteins Casein phosphopeptides – 2 % in muscle foods (Sakanaka et al., 2005)
– Data not recorded.
173
174 Natural Antioxidants: Applications in Foods of Animal Origin

5.3.1 PLANT SOURCES

5.3.1.1 α-TOCOPHEROL (VIT. E)

It is a widely studied antioxidant and has the ability to function in biological


systems. Free radicals are neutralized by tocopherol before lipid oxidation
propagates among highly unsaturated fatty acids in cellular and subcel-
lular membranes. The chromanol ring of α-tocopherol is located among the
polar head groups of the phospholipids, and the phytol side chain interacts
with the unsaturated fatty acyl chains of the phospholipids through van-der-
Waals interactions in the interior of the membrane. This specific localiza-
tion of α-tocopherol in the membrane and the molecule’s lateral mobility
allow it to function very efficiently to protect highly oxidizable PUFA from
per-oxidation by ROS produced by adjacent membrane bound enzymes. A
logical hypothesis is that tocopherol quenches free radicals originating from
lipid oxidation and in-turn protects oxymyoglobin oxidation. It inhibits free
radical oxidation by reacting with peroxyl radicals to stop chain propagation
and with alkoxyl radicals to inhibit the decomposition of the hydroperoxides
and decrease the formation of aldehydes.
Various attempts have been made to reduce pigment and lipid oxidation
in meats by endogenous and exogenous vit. E treatments. Dietary supple-
mentation of vit. E at 2500 mg for 40 days resulted in a 7–10 day exten-
sion of color, shelf life of meat steaks (Taylor et al., 1994). It has also been
reported that animal fed a diet containing 75 or 1000 mg vit. E had higher
concentration of vit. E in meat and lower discoloration. High concentrations
of vit. E have been shown to exert a pro-oxidant effect (Mahoney & Graf,
1986). Post-mortem addition of vit. E was less effective in retarding the
oxidation of pigment and lipid than endogenous one. Thus, dietary vit. E
supplementation would be a safer and more effective method for retarding
pigment and lipid oxidation.
Vit. E had been reported to be effective in improving shelf life of poultry
meat and meat products through delay in oxidation. But the efficacy of oxida-
tive stability of poultry meat is related to tissue concentration of tocopherol.
As the level of vit. E in diet increases there is concomitant increase in its
concentration in tissues. High level of vit. E in poultry meat not only improves
the oxidative stability but also prevents excessive drip loss, as it is believed
that α-tocopherol reduces leakage of sarcoplasmic components from muscle
cells by maintaining the integrity of cellular membranes and thereby reduces
drip (Goni et al., 2007). The sensory quality of meat assessed from scoring
of aroma, flavor, taste, etc. is reported to be improved by adding vit. E in
Natural Antioxidants in Poultry Products 175

diets of broilers. Vit. E was reported to improve phagocytic ability of the


immune system in broilers (Konjufca et al., 2004). It has also been reported
that under heat stress condition dietary vit. E supplementation improved the
immune response of broilers. Even supplementation of vit. E decreased the
production of fishy color because of fishmeal oil in diet and warmed over
color in refrigerated cooked meat and raw frozen meat (Niu et al., 2009).
The stability of the meat has been correlated with the tissue concentration
of α-tocopherol. For poultry, levels of 7 µg/kg α-tocopherol in muscle are
recommended to prolong the keeping quality of meat. The supplemented
tissues were more stable in cold store. Feeding of vitamin supplemented
diets (10–20 ppm) showed significantly lower levels of thiobarbituric acid
reactive substances (TBARS) in the meat measured after 5 and 10 days of
storage (Lohakare et al., 2005).
The effects of vit. E in ducks are different. The effect of feeding of
α-tocopheryle acetate at different levels to day old white Pekin ducklings
revealed significantly increased concentrations of vit. E in breast, thigh,
liver, and heart tissues in dose dependant manner. Supplementation though
enhances oxidative stability, was more effective for thigh muscle than breast.
α-tocopherol, which easily gets deposited in the egg yolk, is used as
antioxidant in animal nutrition (Galobart et al., 2001). But, other existing
substances in yolks may also prohibit oxidation, as selenium or carotenoids
(Yaroshenko et al., 2004). Antioxidants are deposited in yolks according to
dietary levels. Enriching egg yolks with α-tocopherol or carotenoids can
be done in a wide range and does not affect egg quality, except that with
increasing levels of carotenoids intensity of yolk color increases. While
supplementing carotenoids to diets, it has to be considered that yellow and
red pigments should be added in ratios given by the manufacturer to avoid
off-colors (Galobart et al., 2001). The artificial carotenoids (canthaxanthin)
usually supplemented to the layer’s diet cover more than 90% of total carot-
enoids in the egg. Enriching eggs with selenium is more complicated as high
levels of selenium in the food are toxic for humans. Nevertheless, it is easy to
enrich eggs with 35 μg Se which amounts to 50% of the recommended daily
intake (RDI) for humans (Yaroshenko et al., 2004). No negative impacts of
antioxidants on any egg quality criteria are known.
Although, the eggs provide considerable amounts of vit. E and sele-
nium, but, enriching eggs with n-3 PUFA will result in a higher liability of
eggs to oxidation and to off-colors (Tserveni-Gousi et al., 2004). Off-colors
may further occur by the use of fish oil as a dietary source. Therefore, for
the production of omega-3 enriched eggs only high quality fish oil should
be used and distinct amounts of antioxidants (α- tocopherol) should be
176 Natural Antioxidants: Applications in Foods of Animal Origin

supplemented, as well (Galobart et al., 1999). Studies have shown no clear


negative impact of omega-3 enrichment on other quality criteria of eggs,
including their functional properties.

5.3.1.2 ALOE VERA

Aloe is a genus containing about four hundred species of flowering succu-


lent plants belonging to Lileaceae family (Mohammad, 2003). True aloe
vera plant is called Aloe barbadensis Miller (Shahzad et al., 2009). The
mucilaginous jelly from the parenchyma cells of the peeled, spineless leaves
of the plant is referred as aloe vera gel. The gel is a watery-thin, viscous,
colorless liquid which contains 99% water and 1–0.5% solid matter at pH
4.5 (Shahzad et al., 2009). The gel exhibits antioxidant, antibacterial, anti-
fungal, as well as antiviral activity as it contains aloin, aloe-emodin, barb-
loin, emodin, anthraquinone glycosides, glycoprotein, gamma-lanoline acid,
prostaglandins, and mucopolysaccharides (Shafi et al., 2000; Singh et al.,
2010). Ethanolic extract of fresh aloe vera juice possessed stronger radical
scavenging activity than that of aloe vera powder, and the antioxidative
effect of aloe vera extracts was correlated to its development stage (Zhang
et al., 2001).

5.3.1.3 ASCORBIC ACID

AA is a chelating agent that binds metal ions; it also scavenges free radicals
and acts as a reducing agent. At high levels (> 1000 mg/kg), AA inhibits
oxidation; however, at low levels (< 100 mg/kg) it can catalyze oxidation
and warmed over flavor (WOF) development (Ahn & Nam, 2004). In the
presence of AA, iron stimulates oxidation in muscle membranes, presum-
ably through the involvement of hydroxyl radicals. Sepe et al. (2005) found
that sodium ascorbate and sodium erythorbate more effectively maintained
red color and maintained myoglobin in the reduced state in cooked ground
meat patties than AA and ascorbyl palmitate. The solubility of ascorbate
affects its ability to prevent discoloration (Mancini et al., 2006). The lack of
effectiveness of the hydrophobic antioxidant may be a result of localization
of components responsible for bone discoloration within the aqueous phase.
AA and phosphates appear to work synergistically to inhibit lipid oxidation.
AA and tocopherol reduction of lipid oxidation in meat can be enhanced by
adding sesamol, especially as storage time increases (Ismail et al., 2008).
Natural Antioxidants in Poultry Products 177

It has been theorized that AA functions to maintain a portion of the iron


in the reduced state. The amount of AA permitted in meat products varies
depending on the route of introduction: brine, incorporation, or surface
spray. A 10% AA solution can be applied as a spray to cure carcass surfaces.
Sodium ascorbate or erythorbate is also used as cure accelerator.

5.3.1.4 BAMBOO LEAVES

Dried bamboo leaves, yellow or brown colored powder, are commonly used
as antioxidant in various food systems. The antioxidative property is mainly
due to flavones, lactones, and phenolic acids. It can either inhibit lipid auto-
oxidation chain reaction, or chelate transition metal ions, and can be used as
primary or secondary antioxidant. Moreover, bamboo leaf powder can help
in eliminating the nitrites in cured meat. It inhibits the synthesis of N-nitro-
samine, and has anti-bacterial, bacteriostatic, deodorizing, aroma enhancing,
etc. functions (Zhang et al., 2007). It is commonly used in oil-containing
food, meat products, fishery products, expanded foods, etc.

5.3.1.5 BEETROOT EXTRACTS, CLOUDBERRY, WILLOW HERB

The AOA of these plant extracts in meat system has been investigated parallel
to pure quercetin, rutin, and caffeic acid. It is found that cloudberry extract
and quercetin are the most potent; caffeic acid intermediate and pure reutin
have the lowest AOA (Reya et al., 2005). Hexanal production was inhib-
ited by the high level of beetroot, but TBARS production was not, perhaps
because the red color of beetroot extract interfered with the determination of
the pink thiobarbituric acid chromogen.

5.3.1.6 CAMELINA MEAL

The phenolic composition in camelina meal was predominated by flavo-


nols (quercetin and glycosides), hydroxycinnamic acid derivatives (sinapine
and sinapic acids), flavanols, and tocopherols inhibiting hexanal formation
(≥ 80% inhibition). The lower efficacy of camelina meal than that of rapeseed
meal is due to lower sinapine and α-tocopherol (24 μg/g) content. Camelina
meal phenolics have potential effects in inhibiting the lipids oxidation of
broiler meat when incorporated in ration (Aziza et al., 2010).
178 Natural Antioxidants: Applications in Foods of Animal Origin

5.3.1.7 CANOLA EXTRACT

The antioxidative properties of canola extract were compared with various


synthetic antioxidants. Canola extracts at 500 and 1000 ppm were more
active than BHA, BHT, and BHA/BHT/MGC8 (methyl 3,4,5-tris(n-octy-
loxy) benzoate) and less effective than tert-butylhydroquinone (TBHQ) at a
level of 200 ppm (Wanasundara & Shahidi, 1994).

5.3.1.8 CARAWAY

It is rich source of carvone and limonene. Caraway has been used as antioxi-
dant in chicken meat stored under frozen conditions (El-Alim et al., 1999).

5.3.1.9 CAROTENOIDS

Carotenoids are yellow, orange, and red lipid-soluble pigments that occur
widely in plants, fruits, and vegetables. They are 40-carbon isoprenoids
with varying structures, and can be classified as carotenes and xanthophylls.
Certain carotenoids are also referred to as pro-vitamins such as β-carotene,
α-carotene, and β-cryptoxanthin. Carotenoids are antioxidant nutrients that
act mainly as secondary antioxidants in foods by quenching singlet oxygen.
They may also prevent oxidation by trapping free radicals in the absence of
singlet oxygen. Carotenoids are a good synergist with tocopherols. Beta-
carotene, lutein, lycopene, and isozeaxanthin are typical carotenoids that
effectively retard oxidation in foods. Astaxanthin has AOA that is ten times
greater than that of β-carotene, lutein, zeaxanthin, and canthaxanthin, and
is often used in fish products. In a high-oxygen concentration, β-carotene
may exhibit a pro-oxidant, rather than an antioxidant effect in food products.
Carotenoids are natural constituents of foods and have generally recognized
as safe (GRAS) status. No permissible limits on their addition level have
been stipulated.

5.3.1.10 CHERRY

Cherry fractions contain phenolic compounds such as flavones, isoflavones,


anthocyanins, anthocyanidins, and phenolic glycosides. The effects of tart
cherry tissue added at an 11.5% level on the oxidation of lipids in raw and
Natural Antioxidants in Poultry Products 179

cooked ground meat patties and on the formation of heterocyclic aromatic


amines (HAAs) in the fried patties were investigated. TBARS values and
cholesterol oxidation for raw and cooked ground patties containing cherry
tissue were significantly lower than those for the control samples. The
formation of mutagenic/carcinogenic HAAs during frying of the patties
was inhibited by components in the cherry tissue. The concentrations of
2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP), the principal
HAA in cooked muscle foods, were reduced 93 and 87 % by cherry tissue,
respectively.

5.3.1.11 CLOVE

It has been observed that 1 and 2% clove oil have very good antioxidant and
antimicrobial effects in chicken frankfurters (Mytle et al., 2006); 0.2 and 0.5
% clove oleoresin in chicken meat marination (Carlos & Harrison, 1999);
clove powder as phyto-preservative and antimicrobial in chicken nuggets
(Kumar & Tanwar, 2011). Hao et al. (1998) applied eugenol to meat slices
or cooked chicken and it was proved that eugenol inhibited the growth of
Aeromonas hydrophila and Listeria monocytogenes.

5.3.1.12 COCOA LEAVES

These are effective antioxidant as green tea polyphenols (Osman et al.,


2004). These can be used as extract and it has lower astringency and bitter-
ness. Therefore extracts of cocoa leaves can be used in higher concentra-
tions, for higher effectiveness as antioxidant.

5.3.1.13 CURRY LEAF

Curry leaf (Murray koenigii) is native from East-Asian countries and mostly
used as a color ingredient in variety of products. The extract contains
monoterpene hydrocarbons and monoterpene-derived alcohols which have
recently been recognized for their efficacy in providing significant AOA
to the human foods (Ningappa et al., 2008). However, AOA of curry leaf
extracts may vary depending on extraction methods, purity, types, and quan-
tity of active compounds present according to climate, soil composition,
plant organ, age, and stage in the vegetative cycle. Antioxidant effects of
180 Natural Antioxidants: Applications in Foods of Animal Origin

curry leaf powder and extract have been investigated for their use in chicken
mince and patties (Biswas et al., 2006). It has been reported that use of curry
leaf powder could reduce production of malondialdehyde content in raw and
precooked chicken meat patties.

5.3.1.14 DARK GREEN LEAFY VEGETABLES

Lutein is the main active compound from extracts of dark green leafy vege-
tables. It is an oxygenated carotenoid (xanthophylls) abundantly present
and is one of the most important dietary antioxidants for eye health. Lutein
significantly reduces the risk of age-related macular degeneration, athero-
sclerosis, and UV damage (O’Connell et al., 2008). AOA of lutein is based
on its singlet oxygen quenching ability.

5.3.1.15 DRIED PLUMS

Plum derived food ingredients, due to their flavonoid and polyphenol


content, are reported to function as antioxidants, antimicrobials, fat repla-
cents, and flavorants. The principal phytochemicals in dried plums are
phenolic acid derivatives, flavonoids, and coumarins. In addition, dried
plums also contain large amounts of neochlorogenic, chlorogenic, and
cryptochlorogenic acid. Chlorogenic acid isomers have a high AOA and
inhibit low-density lipoprotein oxidation. Nunez et al. (2008) concluded
that 2.5% dried plum or fresh plum juice concentrates or spray dried plum
powder can be used in roasts without any detrimental effects and with
potential benefit of reducing lipid oxidation and warmed over flavor. Simi-
larly, it has been observed that plum extract used at > 2% in turkey breast
rolls and irradiated at 3.0 kGy was effective in retarding oxidation and
enhancing juiciness (Lee & Ahn, 2005).

5.3.1.16 DRY SOYA SPROUTS

It possesses high total phenolics and flavonoids content, and also a little
amount of vit. C. Although reduction power is not good, 1, 1 diphenyl-2-pic-
rylhydrazyl (DPPH) radical scavenging activity was comparable to BHA,
and could be used as a cheap natural antioxidant source for meat and meat
products (Romero et al., 2008).
Natural Antioxidants in Poultry Products 181

5.3.1.17 DRUMSTICK LEAF

Drumstick leaf (Moringa oleofera) is characterized by high antioxidant


potential as they are rich in phenolic compounds. Mature leaves of this plant
contain 0.68 g total phenolic per 100 g portion, high amount of β-carotene,
iron (Fe), potassium (K), calcium (Ca), and vit. C. Drumstick leaf has
the highest amount of essential amino acids and significant quantities of
minerals (Moyo et al., 2012). AOA of drumstick leaves has been reported in
meat and meat products.

5.3.1.18 EVENING PRIMROSE

Evening primrose extract contains phenolics which inhibit the formation of


conjugated dienes, hexanal, and total hydrogen peroxide (H2O2), hydroxyl
radical (•OH), and superoxide radical (O2•-) by 43.6 and 72.6% when used at
1 and 2%; w/w, respectively, in cooked comminuted meat products.

5.3.1.19 GARLIC

It is used as a flavor enhancer and known to have medicinal properties due to


wide spectrum of actions such as antibacterial, antiviral, antifungal, and anti-
protozoal. It also has beneficial effects on the cardiovascular and immune
systems (Leuschner & Ielsch, 2003). Besides its medicinal effects, garlic showed
effective AOA in-vivo and in-vitro due to organic sulfur compounds and
their precursors, allicin, diallyl sulfide, and diallyl trisulfide. Garlic is used
as fresh, powder, and oil forms in food systems. Sallam et al. (2004) graded
the AOA of the various materials in the following order fresh garlic > garlic
powder > BHA > garlic oil in raw chicken sausage during cold storage
(3 °C); however, their activity is concentration dependent. In higher concen-
tration it has very strong flavor and cannot be acceptable to the consumers.

5.3.1.20 GINGER

The AOA of ginger relies on 6-gingerol and its derivatives. Ginger extract at
2.5% has been found effective as tenderizing, antioxidant, and antimicrobial
in smoked spent hen meat (Naveena & Mendiratta, 2001) and 2, 4, and 6%
ginger paste as an antioxidant in spent hen meat balls (Rongsensusang et al.,
2005).
182 Natural Antioxidants: Applications in Foods of Animal Origin

5.3.1.21 GRAPES

Grapes are used in various forms as seed extract, dietary fiber, pomace, grape
wine, etc. Grape seed extract (GSE) is already marketed as an ingredient
to the dietary supplementation industry, the quality and price of which are
based on its phenolic content. Phenolics in GSE exist as proanthocyanidins
in the form of oligomers and polymers of polyhydroxy flavan-3-ols such as
catechin and epicatechin (Weber et al., 2007). Antioxidant effect of GSE was
evaluated on cooked turkey patties and cooled stored turkey meat (Lau &
King, 2003). Grape pomace, a concentrate of grape seeds, stems, and peel, is
a rich source of flavonoids including monomeric phenolic compounds such
as catechins, epicatechin, and epicatechin-3-O-gallate and dimeric, trimeric,
and tetrameric procyanidins. It is used as dietary supplement to increase
antioxidant capacity in breast and thigh meat of broiler chickens (Ayerdi et
al., 2009).
Grape antioxidant dietary fiber (GADF) was effectively used at 0.5, 1,
1.5, and 2% concentration in raw and cooked chicken breast hamburger.
GADF addition resulted in reduction in lightness and yellowness and an
increase in redness in raw and chicken hamburgers without affecting the
acceptability of the products (Ayerdi et al., 2009).
Grape wine has resveratrol, a strong antioxidant and a free-radical scav-
enger. It has superior AOA over quercetin, rutin, and carnosine. Antioxida-
tive effectiveness has been reported as BHA > resveratrol > PG > tripoly-
phosphate > vanillin > phenol > BHT > α-tocopherol (Bekhit et al., 2003).

5.3.1.22 GREEN TEA

Antioxidant properties are attributed to the presence of tea catechins (TC),


epicatechins, epigallocatechin, epigallocatechin gallate, and epicatechin
gallate. These compounds have high affinity for lipid bilayers of muscle
and the radical scavenging activity which prevent lipid oxidation and also
have antibacterial action. TC can also reduce the formation of peroxides
more effectively than α-tocopherol or BHA in canola oil and chicken fat and
fish muscle model system. It has been observed that TC added at a level of
300 mg/kg inhibited the pro-oxidative effect of 1% NaCl and controlled the
lipid oxidation for all the cooked patties during refrigerated storage. It (200
or 400 mg/kg) caused discoloration in cooked patties possibly by binding
with the iron component of myoglobin.
Natural Antioxidants in Poultry Products 183

5.3.1.23 HONEY

Honey alters the water activity, thereby indirectly affecting oxidation rate.
Moreover, it facilitates the Maillard reaction during the cooking process
and thus the development of an antioxidative effect. Honey (15% wt/
wt) was reported to retard lipid oxidation in turkey and chicken meat.
However, it is also reported that incorporation of honey has imparted
a slightly darker color with lower lightness values but had no effect of
redness and yellowness values (McKibben & Engeseth, 2002; Hashim et
al., 1999).

5.3.1.24 MARJORAM

Marjoram (Origanum majorana L.) essential oil inhibits formation of


initial compounds during the oxidation of unsaturated fatty acids (conju-
gated di-enes) by 50% and the generation of secondary oxidation prod-
ucts of linoleic acid by 80% in a model system (Schmidt et al., 2008).
Wild marjoram has also been shown effective in refrigerated and chilled
meat patties, however, it has been reported that addition of salt or freezing
of samples results in loss of effect. A purified component isolated from
marjoram, T3b, a phenolic substance, is a better superoxide anion radical
scavenger than BHT, BHA, α-tocopherol, AA and a variety of polyphenolic
flavonoids epigallocatechin gallate, quercetin, epicatechin. The inhibi-
tory mechanism of T3b appears to depend on the action of an endogenous
enzyme (superoxide dismutase) which destroys the superoxide anion by
converting it to H2O2.

5.3.1.25 MINT

Spearmint or garden mint (Mentha spicata L), family Lamiaceae (Labi-


atae), is often added to several meat preparations as a color enhancer. It is a
rich source of polyphenolic compounds with strong antioxidant properties
(Dorman et al., 2003) but its application in meat is yet to be explored. Kanatt
et al. (2007) investigated that mint extract (ME) had good total phenolic and
flavonoid contents with high superoxide and hydroxyl-scavenging activity
but low iron-chelating ability.
184 Natural Antioxidants: Applications in Foods of Animal Origin

5.3.1.26 OLIVE LEAF

Olive leaves extract is known to have antioxidative, antimicrobial, and anti-


inflammatory properties and to protect low-density lipoprotein from oxida-
tion, the capacity to lower blood pressure in animals and to inhibit lipid
oxidation. Carpenter et al. (2006) found that olive leaf extract (50 µg/ml)
strongly protects cells against oxidative stress.

5.3.1.27 ONION

Onion (Allium cepa L.) is much valued for its flavoring components and
has high quercetin content (284–486 mg/kg). Quercetin, a flavonoid belongs
to a group of plant phenolics, known to control rancidity in cooked ground
turkey, cooked ground lamb, and oven-cooked turkey breast (Younathan et
al., 1980; Karastogiannidou, 1999; Tang & Cronin, 2007). It may be possible
to further enhance the antioxidant role of quercetin by using a juice from a
high quercetin-yielding onion variety.

5.3.1.28 OREGANO

Oregano (Origanum vulgare L.) extracts contain high concentrations of


phenols, primarily rosmarinic acid, which can prevent color deterioration
(Hernandez et al., 2009). Phenolic carboxylic acids and glycosides are also
particularly antioxidative and effectively scavenge superoxide anion radi-
cals. It delays surface discoloration, that is, metmyoglobin formation in
ground meat. Camo et al. (2008) compared the effects of direct addition
of oregano and rosemary to the use of active films containing oregano and
rosemary on the display life of lamb. Active films containing oregano were
significantly more efficient than those with rosemary, extending fresh odor
and color from 8 to 13 days. Similarly, Grobbel et al. (2006) used oregano
in combination with AA in polyvinyl chloride (PVC) film in a high-oxygen
modified atmosphere environment.

5.3.1.29 POMEGRANATE

Pomegranate fruit parts have bioactive compounds which are known to


possess enormous AOA. Pomegranate juice is effective in prevention of
Natural Antioxidants in Poultry Products 185

atherosclerosis, low-density lipoprotein oxidation, prostate cancer, platelet


aggregation, and various cardiovascular diseases. Ozkal and Dinc (1994)
reported the presence of tannins, anthocyanins, and flavonoids in pome-
granate rind. Pomegranate peel is a rich source of tannins and other phenolic
compounds. The utilization of pomegranate fruits for meat processing and
its potential health benefits are not well understood. The meat industry can
use these fruits or fruit byproducts as a potential source of phenolics as they
have immense nutraceutical value and can be used to produce functional
meat products of commercial interest (Naveena et al., 2008; Vaithiyanathan
et al., 2011).

5.3.1.30 POTATO PEEL EXTRACT (PPE)

The effective utilization of potato peel, a waste generated in large quantities


by the food industry, as an antioxidant in radiation processed lamb meat
was investigated by Kanatt et al. (2005). PPE has a high phenolic content
(70.82 mg, catechin equivalent/100 g), chlorogenic acid (27.56 mg/100 g
of sample) is the major component. The yield of total phenolics and chlo-
rogenic acid increased by 26 and 60%, respectively, when the extract was
prepared from γ irradiated (150 Gy) potatoes. The AOA of PPE was found
to be comparable to BHT.

5.3.1.31 RAPESEED MEAL

Rapeseed meal contains α-tocopherol (52 μg/g) and phenolic hydroxycin-


namic acid derivatives including sinapine (2400–2900 μg/g) and sinapic
acid (280 μg/g) which inhibit hexanal formation (≥85%). It is also used in
combination with commercial CO2 extract of rosemary (0.04 g/100 g meat)
and was excellent in prevention of oxidation of meat lipids (Salminen et al.,
2006).

5.3.1.32 RICE HULL EXTRACT

Rice hull can be an attractive protective source because it contains many


easily extractable antioxidant compounds. Furthermore, radiation of rice
hull with far infrared (FIR) for 2 h increased the content of phenolic
compounds in extract. FIR radiation onto rice hull is reported to liberate
186 Natural Antioxidants: Applications in Foods of Animal Origin

and activate covalently bound phenolic compounds that have antioxi-


dant activities. Therefore, rice hull extract treated by FIR can be a good
candidate to be used in irradiated meat systems as a natural antioxidant.
The antioxidant effect of FIR treated rice hull (Lee et al., 2003) extracts
(FRH) was compared with that of sesamol and rosemary oleoresin in irra-
diated turkey breast meat. FRH significantly decreased TBARS values and
volatile aldehydes (hexanal, pentanal, and propanal) and was effective in
reducing the production of dimethyl disulfide responsible for irradiation
off-odor in irradiated raw and cooked turkey meat during aerobic storage.
The AOA of FRH (0.1%, w/w) was as effective as that of rosemary oleo-
resin (0.1%). However, the addition of FRH increased red and yellow color
intensities and produced an off-odor characteristic to rice hull in raw and
cooked meat, and cannot be used in meat without further refining process
to remove off-color and off-odor compounds to increase its applicability
as an antioxidant.

5.3.1.33 ROSEMARY

The AOA of rosemary (Rosmarinus officinalis L.) extracts has been known
for the last 30 years and is due to presence of phenolic compounds, carnosol,
carnosic acid, rosmanol, isorosmanol, rosmariquinone, rosmaridiphenol,
and rosmary-diphenol. The phenolic substances react with lipid or hydroxyl
radicals and convert them into stable products. Rosemary extracts can also
chelate metal ions, such as Fe2+, resulting in a reduced rate of formation of
activated oxygen (Formanek et al., 2003). It is four times more effective than
BHA and equal to BHT as antioxidants but less than TBHQ. It also improved
the color stability of cooked turkey rolls. Rosemary extract has also been
used in the combination of various other antioxidants (McBride et al., 2007)
to have synergistic effect. However, some of the compounds in rosemary
(verbenone, borneol, and camphor) can impart an undesirable rosemary odor
to foods, even at low concentrations.
A great deal of research on the antioxidant properties of rosemary extract
in different food systems has been carried out, which clearly demonstrated
the effectiveness of its bioactives compounds with greater acceptability by
the consumers. Moreover, several authors reported that some compounds
such as phenolic di-terpenoids present in rosemary extracts have antibacte-
rial activity (Cuvelier et al., 1994).
Natural Antioxidants in Poultry Products 187

5.3.1.34 SAGE

Sage (Salvia officinalis L.) contains a variety of antioxidative substances


including carnosol, rosmanol, rosemadiol, epirosmanol, isorosmanol,
galdosol, and carnosic acid. The antioxidative activity of sage oil correlates
with the oxygenated diterpene and sesquiterpene concentration. They are
more potent in cooked meat than in raw meat (Fasseas et al., 2008). The etha-
nolic extract of sage can reduce both peroxide oxygen and TBARS values.
The polar extracts of the Salvia species exhibit better antioxidant activities
than its corresponding non-polar sub-fractions and that was comparable to
the antioxidant activities of BHT.

5.3.1.35 SEABUCKTHORN

Seabuckthorn (Hippophae rhamnoides) berry extracts are rich in antioxidant


polyphenols mainly flavonols, which are stable during short-term cooking.
Pussa et al. (2008) reported AOA of seabuckthorn in mechanically deboned
broiler meat.

5.3.1.36 SESAME OIL

Sesamol (500–2000 µg/ml) of sesame oil educed lipid oxidation in bovine


muscle model systems by an average of 97% (Joshi et al., 2005). They
further added that sesamol at a concentration of 90 µM inhibited Fe (III)-
induced oxidation by 99%. Nam and Ahn (2003) confirmed sesamol at
0.01% as superior antioxidant than gallate, tocopherol, and carnosine in pork
homogenate.

5.3.1.37 SOY PROTEIN HYDROLYSATES

These are very frequently utilized in meat products as functional ingredients.


Soy protein isolates could inhibit TBARS formation in an iron-catalyzed
liposomal system by as much as 65% depending on the proteases and hydro-
lysis conditions used. Yet, these protein hydrolysates as potential antioxi-
dants in meat products (in situ) have not been validated.
188 Natural Antioxidants: Applications in Foods of Animal Origin

5.3.1.38 THYME

A number of species of thyme (Thymus vulgaris, T caespititius, T. camphor-


ates, T. mastichina) have antioxidative activity; all contain 1, 8-cineole,
α-terpineol, linalool, carvacrol, and thymol (Lee et al., 2005). The antioxi-
dative activity of T. caespititius (250 and 500 mg/l) has been reported to be
comparable to that of vit. E and BHT. Among the compounds isolated from
thyme, the order of antioxidative activity is thyme oil > thymol > carva-
crol > gamma-terpinene > myrcene > linalool > p-cymene > limonene >
1, 8-cineole > α-pinene (Youdim et al., 2002). Thyme essential oil exhibits
very strong free radical scavenging ability and inhibits Fe2+/ascorbate and
Fe2+/H2O2 induced lipid oxidation (Bozin et al., 2006).

5.3.1.39 TOMATO

The active compound of tomatoes is lycopene. It is highly effective anti-


oxidant owing to its ability to act as free radical scavenger and has the
highest singlet oxygen quenching rate than all carotenoids tested in biolog-
ical system. It can be used as tomato paste, sun dried tomato, tomato peel,
tomato puree. Osterlie and Lerfall (2005) confirmed the antioxidant effect
of tomato paste in meat products and dry tomato peel in dry fermented
sausage. Calvo et al. (2008) reported that tomato paste can be successfully
used up to a level of 12% without any negative effect on the processing
and quality characteristics of the product throughout its storage. It is also
effective in limiting the use of nitrite in cured meat products. Moreover,
lycopene provides protection against many chronic diseases such as cancer
and cardiovascular disease.

5.3.2 ANIMAL SOURCES

5.3.2.1 BONE PROTEIN HYDROLYSATES

These can be prepared by limited alcalase hydrolysis. It possessed signifi-


cant AOA, and AOA increased with the increasing hydrolysates concentra-
tion. Bone protein hydrolysates at 2% application level are able to not only
stabilize meat color but also suppress lipid oxidation in pork patties during
storage improving sensory quality. Therefore, bone protein hydrolysates can
be used as potent natural and cheap antioxidant for meat and meat products.
Natural Antioxidants in Poultry Products 189

Further studies are needed to identify the specific compounds in hydrolysate


that are responsible for the overall antioxidative capability.

5.3.2.2 CARNOSINE

Carnosine is a naturally occurring skeletal muscle dipeptide, which consists


of alanine and histidine. Its function in muscle is not completely understood,
but it is thought to act as both a buffering agent and as an antioxidant. The
antioxidant mechanism has been postulated to be a combination of its ability
to act as a chelator, free radical scavenger, and hydrogen donor. Carnosine
is water soluble, thus permitting the inactivation of lipid oxidation cata-
lysts such as heme pigments, iron, lipoxygenase and singlet oxygen, and
free radicals in the aqueous phase of muscle. The color protecting effects of
carnosine were greater than BHT, α-tocopherol, or sodium tripolyphosphate.
Some researchers also investigated the interactions between carnosine and
the different redox states of myoglobin and concluded that it does not stabi-
lize oxymyoglobin or significantly catalyze the reduction of met-myoglobin
formation. Djenane et al. (2004) concluded that the combination of carno-
sine (50 mM) with AA (500 ppm) provided the best antioxidant protection
for meat during refrigerated storage. Surface application of carnosine or AA
combination or alone resulted in an effective delay of oxidative deterioration
of meat.

5.3.2.3 CHITOSAN

Chitosan, a linear polymer of 2-amino-2-deoxy-β-D-glucan, is a deacetylated


form of chitin, a naturally occurring cationic biopolymer. It occurs as a shell
component of crustaceans (crab and shrimp), as the skeletal substance of
invertebrates, and as the cell wall constituent of fungi and insects. Chitosan
possesses a positive ionic charge which gives its ability to bind with nega-
tively charge fat, lipid, protein, metal ions, and macromolecules. Chitosan is
GRAS by the US FDA. Chitosan retards lipid oxidation by eliminating the
pro-oxidant activity of ferrous ions. The AOA of chitosan could also be due
to its chelating ability with free iron released from myoglobin degradation.
Soultos et al. (2008) indicated that chitosan concentration of 1% in pork
sausages show 80% decrease in MDA level after 14 days while that retard
lipid oxidation upto 70% in meat products after three day storage at 4 °C.
190 Natural Antioxidants: Applications in Foods of Animal Origin

5.3.2.4 MILK PROTEINS

Milk and milk components have been frequently used in the enhancement
of nutritional and technological properties of a wide variety of foods. A
feasible application of peptides or hydrolysates as antioxidants is being
explored especially in muscle foods. The phosphorylated caseinophospho-
peptides (CPP) have the ability to scavenge free peroxyl radicals as well
as to chelate transition metals such as iron, copper, and zinc (Kim et al.,
2007). This is positively correlated with the amounts of histidine, lysine,
proline, and tyrosine. Incorporation of casein calcium peptides (2%) inhib-
ited about 70% of lipid oxidation and prevents formation of an off-color in
meat products. Rossini et al. (2009) suggested that casein peptides (20 mg/
ml) effectively inhibited lipid per-oxidation in ground meat homogenates
and mechanically deboned meat. As cooking increases the catalytic activity
of iron, the stronger chelating activity of enriched CPP may make them
more effective antioxidants in cooked muscle foods (Diaz & Decker, 2004).
Whey hydrolysates may also act as potential antioxidants in meat prod-
ucts. Pena-Ramos and Xiong (2003) evaluated the AOA of selected whey
hydrolysates in cooked pork patties. The results indicated that at an appli-
cation level of 2%, the whey protein isolates and their hydrolytic products
not only reduced the cooking loss but also suppressed lipid oxidation in
cooked pork patties during refrigerated storage. Notably, hydrolysis with
protamex improved the capability of whey protein to inhibit early-stage
lipid oxidation (formation of hydroperoxides or conjugated dienes) as well
as to retard propagation of the oxidation process. Therefore, milk proteins
can be superlatively used as nutrient, color enhancer as well as antioxidant
in processed muscle foods.

5.4 MODE OF APPLICATION OF ANTIOXIDANTS

5.4.1 DIETARY SUPPLEMENTATION

Dietary supplementation of vit. E and TC is very commonly used to improve


the meat quality. The antioxidant activities of many plants, vegetables, herbs
and their essential oils (eugenol, thymol, and carvacrol), flavonoids, cyani-
dine glycosides, etc. are also elucidated. It is considered that the AOA of
these compounds is due to their high redox properties and chemical struc-
ture, which can be responsible for neutralizing free radicals, chelating tran-
sitional metals, and quenching singlet and triplet oxygen by delocalization
Natural Antioxidants in Poultry Products 191

or decomposing peroxides. Dietary supplementation of rosemary and sage


extracts or oregano essential oil in broilers could improve the oxidative
stability of raw and precooked meat products during storage. However,
combination of oregano and rosemary essential oils had greater effect than
those fed individually or α-tocopheryl acetate on inhibiting lipid oxida-
tion. These activities could have resulted from the action of antioxidant
compounds such as the phenolic isomers, thymol, and carvacrol found in the
extracts and essential oils from the aromatic plants.
Addition of drumstick leaf extracts in feed could reflect concentrations
of AA (vit. C), α-tocopherol (vit. E), beta carotene (vit. A precursor), various
flavonoids, and other phenolic compounds of broiler meat. So, broiler
produced with this leaf revealed potent AOA, and also improve color and
flavor of meat products.
The production of lipid peroxides in the carcasses of broiler chickens
during storage could be delayed by astaxanthin. Therefore, astaxanthin
either synthetic [45 mg/kg feed) or naturally occurring (from the red yeast
Xanthophyllomyces dendrorhous, 22.5 mg/kg feed)] could be used as an
antioxidant as well as a colorant for broiler chickens (Ahn & Nam, 2004).
Xanthophyll (natural or synthetic) sources to chicks could improve color
intensity of and inhibit lipid oxidation in leg meat. The antibacterial agent,
lasalacid sodium salt (LS) added to the diet affected not only their growth
performance but also their meat yield and stress response. Withdrawal of
LS increased the plasma 2-thiobarbituric acid reactant value, an indicator of
lipid per-oxidation.

5.4.2 DIRECTION ADDITION TO THE PRODUCT

Most of the antioxidants whether in the form of extracts, powders, or any


other form are added directly. It is the most commonly used method.

5.4.3 SPRAYING

The antioxidants directly or their active principle are sprayed over the surface
of the meat. Direct addition of a natural rosemary extract on to the meat
surface by spraying 2 ml pure extract diluted in 150 ml n-pentane, according
to a ratio of 2 ml solution to 50 g meat, gave rise to a significant decrease
of color loss and lipid oxidation spraying of vit. E directly on turkey meat
resulted in a lower myoglobin oxidation.
192 Natural Antioxidants: Applications in Foods of Animal Origin

5.4.4 ACTIVE PACKAGE

It is another method that does not involve direct addition of the active agents
to the product. Antimicrobial agents in active packaging have been reported
(Appendini & Hotchkiss, 2002), but studies on antioxidant active packaging
are rarer. Nerín et al. (2006) described the promising results of a new anti-
oxidant active packaging system; a plastic film with an embodied rosemary
extract was able to inhibit both myoglobin and lipid oxidation in red meat,
leading to enhanced display life of meat. The mechanism of antioxidant
active pack is currently under investigation. Generally postulated hypoth-
esis states that mechanism involves inactivation of free radicals either by
migration of antioxidant molecules from the active film to the meat or by
scavenging of those oxidant molecules from the meat onto the active film.
Pezo et al. (2008) has demonstrated that active films react with headspace
free radicals. This allows to envisaging antioxidant active packaging with
oregano as a promising tool for increasing the display life of lamb and other
meats in retail sale. However, the legal regulatory status of active packs is so
far not clear and needs to be specifically addressed.

5.4.5 MARINATION

Various marinades according to consumers’ acceptance are being incorpo-


rated with antioxidants such as turmeric rhizomes, tamarind, lemon grass,
etc.

5.4.6 ENROBING

Enrobing is the process of making “further processed products” by applying


edible coating on the products in two distinct steps, that is, battering and
breading (Ahamed et al., 2007). Enrobing improves display attributes and
imparts crispy texture. It provides variety to the meat products with improved
juiciness, tenderness, sensory quality, nutritive value, shelf life, and reduced
total product cost, moisture, and weight loss. In addition, enrobing mate-
rial is used as a carrier for various antimicrobial and antioxidant substances
(Shelef & Liang, 1982; Giridhar & Reddy, 2001; Biswas et al., 2004). This
approach can be used to impart a strong localized functional effect without
elevating excessively the overall concentration of an additive in the food
(Guilbert et al., 1985).
Natural Antioxidants in Poultry Products 193

5.5 SYNERGISTIC EFFECT OF NATURAL ANTIOXIDANTS

Evidence based on pulse radiolysis technique and electron spin resonance


studies supports a redox mechanism, involving reduction of the tocopher-
oxyl radical intermediate by AA to regenerate tocopherol. By this syner-
gistic mechanism, α-tocopherol and AA can mutually reinforce each other
by regenerating the oxidized form of the other. Another mechanism of syner-
gism involves metal inactivating effect of AA. A mixture of α-tocopherol,
AA, or ascorbyl palmitate and phospholipids are also known for their good
synergistic effects leading to inhibition of pigment as well as lipid oxida-
tion. Ascorbate alone enhanced the lipid oxygen radical propagation but
its pro-oxidant effect was reversed when TA was added. Yin et al. (1993)
reported that 10 ppm of vit. E and 500 ppm of vit. C showed the stron-
gest synergism to inhibit pigment and lipid oxidation in meat. Adding rose-
mary to vit. E supplemented meat resulted in lower TBARS than either of
the compound alone suggesting a synergistic effect (McBride et al., 2007).
Mansour et al. (2006) reported synergism of rosemary and marjoram in
minced meat. Combination of oil seed by-products such as rapeseed meal
(0.5 g/100 g meat) or camelina meal (0.7 g/100 g) along with CO2 extract
of rosemary (0.04 g/100 g meat) showed a higher inhibition of lipid oxida-
tion than that of either alone. A very pronounced synergistic effect exists
between rosemary and citric acid. Combinations of oregano and rosemary
essential oils (150 mg/kg each) had a greater effect than those fed individu-
ally or α-tocopheryl acetate (200 mg/kg) on inhibiting lipid oxidation, and
protecting α-tocopheryl acetate concentration in refrigerated meat enriched
with n-PUFAs stored for 15 days. This combination of essential oils also
proved as effective as α-TA in retaining the sensory qualities of breast meat
after 15 days of storage, and was more effective than when these essential
oils were fed individually or at 300 mg/kg. There is a possible synergistic
effect between oregano and rosemary essential oils in preventing lipid oxida-
tion in stored meat enriched with n-3 PUFAs. However, synergistic effects
between various antioxidants need to be exploited in the meat industry.

5.6 MEASUREMENT OF EFFICACY OF AOA

Efficacy of natural antioxidants can be evaluated by various physical and


chemical methods. Physical evaluation of meat color and odor can be
measured by sensory panelists using score card. However, it is always
ensured that the sensory panelists can able to properly recognize the color
194 Natural Antioxidants: Applications in Foods of Animal Origin

and odor and able to distinguish them for different meat species. Amongst
the chemical methods, total AOA is measured by ferric reducing antioxidant
power (FRAP) assay. FRAP assay uses antioxidants as reductants in a redox-
linked colorimetric method, employing an easily reduced oxidant system
present in stoichiometric excess. At low pH, reduction of ferric tripyridyl-
triazine (Fe III–TPTZ) complex to ferrous form (which has an intense blue
color) can be monitored by measuring the change in absorption at 593 nm.
The reaction is non-specific, in that any half reaction that has lower redox
potential, under reaction conditions, than that of ferric ferrous half reac-
tion, will drive the ferrous (Fe III to Fe II) ion formation. The change in
absorbance is, therefore, directly related to the combined or “total” reducing
power of the electron donating antioxidants present in the reaction mixture.
The ability to scavenge DPPH radical by added antioxidant can be measure
at 517 nm wavelength to know efficacy of antioxidant compound. DPPH
can make stable free radicals in aqueous or ethanol solution. However,
fresh DPPH solution should be prepared before every measurement. Super-
oxide anion radical scavenging assay is based on the reduction of nitro blue
tetrazolium (NBT) in the presence of nicotinamide adenine dinucleotide
(NADH) and phenazonium methosulfate (PMS) under aerobic condition
at room temperature under the dark. Oxygen radical absorption capacity
(ORAC) assay is the measure of the oxidative degradation of the fluores-
cent molecule (either beta-phycoerythrin or fluorescein) after being mixed
with free radical generators such as azo-initiator compounds. Azo-initiators
are considered to produce the per-oxiradical by heating, which damages the
fluorescent molecule, resulting in the loss of fluorescence. Antioxidants are
considered to protect the fluorescent molecule from the oxidative degenera-
tion. The degree of protection is quantified using a fluorometer. Fluorescein
is currently used most as a fluorescent probe. Equipment that can automati-
cally measure and calculate the capacity is commercially available. ABTS
method is based on the ability of antioxidants to quench the long-lived
ABTS radical cation, a blue/green chromophore with characteristic absorp-
tion at 734 nm, in comparison to that of standard antioxidants. ABTS was
dissolved in water to a 7 mM concentration. ABTS radical cation (ABTS+)
was produced by reacting ABTS stock solution with 2.45 mM potassium
persulfate (final concentration) and allowed the mixture to stand in the dark
at room temperature for 16 h before use. As ABTS and potassium persul-
fate react sterio-chiometrically at a ratio of 1:0.5 (mol mol-1), this results
in complete oxidation of ABTS. Oxidation of ABTS commenced immedi-
ately, but the absorbance was not maximal and stable until 6 h had elapsed.
Natural Antioxidants in Poultry Products 195

The radical was stable in this form for more than two days, when stored in
the dark at room temperature. Prior to use, the stock solution was diluted
with ethanol to an absorbance of 0.70 at t0 (t = 0 min) and equilibrated at
30 °C exactly 6 min after initial mixing. The TBARS is used to determine
primary oxidation products in terms of mg malonaldehyde per kg of meat
sample while linoleic acid accelerated by azo-initiators (LAOX) is used to
determine oxidation of an aqueous dispersion of linoleic acid accelerated by
azo-initiators.

5.7 CONCLUSIONS

Natural compounds enjoy positive consumer image and have applications


in development of novel functional meat products. Antioxidants are nature’s
defense against the damaging effects of free radicals for health and to extend
shelf life of food products. Still there is various natures’ gift for the mankind
need to be explored. The appropriate combinations of natural antioxidants
can help in designing of functional foods and also tackle lipid oxidation
problems. There are various microbial peptides and plants which need to be
explored as antioxidant and scientists are developing niche based technolo-
gies, combinations, and delivery vehicles for the incorporation of effective
and economic usage of natural antioxidants.

KEYWORDS

• natural antioxidant
• lipid oxidation
• chicken meat
• turkey
• broiler
• poultry
• phenolics
• flavonoids
196 Natural Antioxidants: Applications in Foods of Animal Origin

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CHAPTER 6

METHODS AND THEIR APPLICATIONS


FOR MEASURING AND MANAGING
LIPID OXIDATION: MEAT, POULTRY,
AND SEAFOOD PRODUCTS
TOM JONES*
Meat Products, Global Applications and Product Development,
Kalsec®, Inc., Kalamazoo, MI 49006, USA
*E-mail: [email protected]

CONTENTS

Abstract ....................................................................................................204
6.1 Introduction .....................................................................................204
6.2 Analytical Methods for Quantifying Products of
Lipid Oxidation ...............................................................................208
6.3 Measuring the Consequences of Lipid Oxidation...........................250
6.4 Conclusion ......................................................................................253
6.5 Attachments ....................................................................................255
Keywords .................................................................................................256
References ................................................................................................257
204 Natural Antioxidants: Applications in Foods of Animal Origin

ABSTRACT

The most appropriate analytical method for measuring and managing lipid
oxidation in meat, poultry, and seafood products is based on the type of
information sought, the precision needed, and the intended use of the data. In
most cases, by including a sensory evaluation the significance of the analyt-
ical data is enhanced. However, in all cases the early incorporation of an
antioxidant(s) is a strict prerequisite for managing oxidation of value-added
meat products.

6.1 INTRODUCTION

In every year millions of dollars are lost because of discounting or


discarding meat products due to oxidation. For example, the appealing color
of pepperoni sausages (Photograph 6.1) can deteriorate due to lipid oxida-
tion. Understanding when and how to use available analytical methods for
measuring lipid oxidation is the first step for managing oxidation in meat,
poultry, and seafood products.

PHOTOGRAPH 6.1 Oxidative color degradation of sliced pepperoni.

This chapter reviews the principles and applications of chemical and


sensory methods for analysis of lipid oxidation and how they can be used to:
Methods and Their Applications for Measuring 205

1. Estimate the oxidative stability (shelf life) of raw meat materials and
finished products.
2. Determine the most effective, and type and usage rate for, oxidation
inhibitors.
3. Determine critical “stress” points effecting oxidative stability in the
distribution chain.
4. Measure the effect of formulation changes on oxidative stability of
value-added meat products.
5. Evaluate and improve upon good oxidation management practices
(GOMPs).

6.1.1 SEQUENCE OF CHEMICAL EVENTS, LIPID AUTOXIDATION

Lipid oxidation has also been referred to as lipid peroxidation because


peroxides are the initial products formed. Figure 6.1 shows the oxidation
of a poly-unsaturated fatty acid (PUFA). The products from lipid oxidation
are responsible for color defects and rancid flavors that diminish the value
of processed meat products. Fish, poultry, and pork are most susceptible
to oxidation (compared to lamb and beef) because higher levels of PUFAs
drive lipid oxidation.

Antioxidants (AH) intercept free


radicals and/or regenerate via other • H
antioxidants. A A

H
A
~- A

Chelators " inactivate" pro-oxidant metals:


Fe ++, Cu*

Secondary Oxidation Products:


alcohols, aldehydes, ketones

FIGURE 6.1 Overview of the sequence of chemical events associated with the autoxidation
of lipids.
206 Natural Antioxidants: Applications in Foods of Animal Origin

6.1.2 RELATIVE OXIDATIVE STABILITY OF MEAT AND FISH


LIPIDS

Given that all other processing variables are equal, the presence (not neces-
sarily the concentration) of unsaturated fatty acids drives lipid oxidation.
Figure 6.2 shows a comparison of fatty acid profiles for beef, chicken, and
salmon, as the fatty acid profile favors PUFAs the susceptibility to oxida-
tion increases and shelf-life decreases. Throughout this chapter data will be
presented showing the rate of oxidation in value-added meat products also
depends on product form, processing methods, packaging techniques, and
storage conditions.

Rate of Oxidatioin of Unsaturated Fatty Acids Relative to Stearic Acid


3000
0:
.Sl 2500
'o!
"t:l
8 2000
~ 1500
~
-~ 1000
~ 500

0
18:0, Sterle Acid 18:1 Oleic Acid 18:2, Linoleic Acid 18:3 , Linolenic Acid
Types of Fatty Acids

FIGURE 6.2 The susceptibility of a fatty acid to oxidation increases as unsaturation


increases. When all other factors are equal, unsaturation dictates the differences in expected
shelf life between value added products formulated from lamb, beef, pork, poultry or fish.
Modified from http://www.public.iastate.edu/~duahn/teaching/Lipid%20oxidation/Lipid%20
Oxidation%20An%20Overview.pdf

6.1.3 SELECTION CRITERIA AND SAMPLE PREPARATION

Various analytical methods are available for measuring the extent of lipid
oxidation. Regardless of the method selected, the more criteria, listed below,
that are met, the more valuable the information will be.

1. The method is sensitive, accurate, and precise. The method’s limit of


quantification (LOQ) distinguishes fresh from oxidized products and
less oxidized products from more oxidized products.
2. Data from the analysis are reproducible.
Methods and Their Applications for Measuring 207

3. Data from the analysis are minimally influenced by interfering


substances.
4. Analytical data vary predictably over time.
5. Data are relatable and relevant to the industry segment.
6. Results from the analysis are correlated to sensory (flavor, aroma,
and/or color) analysis.
7. Method and technique are easy to use and rapid.

For all methods, sample preparation (collecting samples, grinding,


mixing, etc.) is critically important for minimizing errors and uncertainty.
Solvent extraction of lipids from meat tissues is of particular importance
because solvent selection is critical for complete recovery of the fat for anal-
ysis (triacylgylcerides (TAG) and the unsaturated phospholipids (PL)). Lipid
oxidation is initiated in the PL fraction and then proceeds to involve lipids in
the TAG fraction. Therefore, complete extraction of TAGs and PL fractions
will provide the most accurate assessment for the extent of oxidation.
The solvent systems recommended in the procedures described in this
chapter for extracting TAG and PL lipid fractions from meat tissues have
been determined experimentally by applying the following principles. .

1. Extraction efficiency of the solvent system.


Solvent system (pairing a polar component, with a nonpolar compo-
nent) is based on the distribution coefficient, K for a given meat
matrix.

solubility in lipid phase


K= (6.1)
solubility in aqueous phase

The larger the coefficient K (greater than 1), the greater the yield of fat
containing products from lipid oxidation in the sample.

2. Amount of the solvent(s) needed.


The less solvent that is used the better, in the case of solvent extracted
lipids containing secondary oxidation products, less is more. For
example,

1
Fraction of lipid extracted = (6.2)
(1 + Vs ) / (( Vm × n K )) n
208 Natural Antioxidants: Applications in Foods of Animal Origin

where K is the distribution coefficient,


n = the number of extractions,
Vs = unit of measurement for solvent system (sp. gr. × volume),
Vm = unit of measurement for meat homogenate (g).

The equation shows that the lipid fraction (containing products of lipid
oxidation) extracted from the raw meat homogenate increases with serial
extractions, the amount of solvent(s) is minimized when the K is large.
For meat applications, serial extractions are not always practical. There-
fore, the meat sample is finely ground under controlled conditions (to mini-
mize further oxidation during preparation) to have the same efficiency as
serial extractions:

1. The secondary oxidation products extracted should not react with the
solvent system.
2. The secondary oxidation products extracted should be easily recov-
ered; low temperature roto-vaporization under vacuum is often used.
3. The solvent should have low toxicity.

6.2 ANALYTICAL METHODS FOR QUANTIFYING PRODUCTS OF


LIPID OXIDATION

6.2.1 PEROXIDES

Peroxides are the initial products formed during lipid oxidation (Fig. 6.1).
The peroxides formed are odorless and tasteless. Nonetheless they are
widely used as an indicator of the current status of lipid oxidation. As will be
discussed later in this chapter, peroxide values (PVs) can be mathematically
combined with para-anisidine values (p-AVs) for a more comprehensive
approach for determining the oxidative status of meat and meat by-prod-
ucts. Three different methods may be used to determine the PVs on solvent
extracted lipids from meat.

1. Iodometric titration (color indicators, stoichiometry).


2. Photo-spectroscopic (Beer’s law).
3. Electrochemical (Nernst equation).

The iodometric titration method is a common method for determining


PVs on solvent extracted fat from a meat sample. A sample of extracted fat
containing a starch indicator is titrated with standardized sodium thiosulfate.
Methods and Their Applications for Measuring 209

RH + O2 ROOH
(lipid) (lipid hydroperoxide)

ROOH + 2KI + CH3COOH I2 + K+ CH3 COO – + 2H2O


(purple solution)
I2 + 2Na2S2O3 Na2S4O6 + 2 NaI
(yellow translucent solution)

Because volume and normality of the standardized sodium thiosul-


fate solution are known, the PV is derived stoichiometrically (normality1
× volume1 = normality2 × volume2). This relationship between the reactants
and products is shown the Equation 6.3.

(mL Na 2S2 O3 ) × (N Na 2S2 O 4) × 0.127 meq. × 1000 Meq O 2 (from hydro peroxides)
= (6.3)
wt. (g) of sample (solvent extracted fat or oil) Kg oil

PV may also be expressed based on the weight of the sample as long as


the exact fat content is known.
Calculation for PVs is based on “equivalents,” not equivalent weight.
Higher numerical values are indicative of a more oxidized sample. PVs that
distinguish rancid foods from non-rancid foods are based on experience and
science based historical data. As a guide, specifications for acceptable PVs
should be based on the following points:

1. Stage of processing (finished or raw material).


2. Product type (pepperoni, cooked beef roast, fresh sausage, etc.).
3. Required shelf life.
4. Sensory evaluation (aroma, flavor).

The data in Figure 6.3 show that PVs can be used to assess the oxida-
tive quality of freshly rendered lard. PV data provide information about the
storage stability of the rendered lard and the effectiveness of natural oxida-
tion inhibitors.
Interpretation of PV data in Figure 6.3 can be used to improve or imple-
ment GOMPs by incorporating natural oxidation in combination with a
nitrogen (N2) sparge and/or blanket as well as decreasing the turn over time
of the stored lard.
From our experience, the level of quantification (LOQ) has been ± 2 meq
kg-1 fat or oil. The LOQ for the titrimetric measurement of PVs is based on
the precision inherent in the method, the amount of the sample collected for
fat extraction, and the efficiency of the fat extraction procedure. Advances
210 Natural Antioxidants: Applications in Foods of Animal Origin

in electrochemistry, that is the development of the understanding of the rela-


tionship between the amount of an electrical charge and the quantity of a
chemical compound, led to improvements in faster, more precise analysis
of hydroperoxides in meat samples. Electrochemical methods quantify
compounds produced from lipid oxidation potentiometrically.

Peroxide Values for Fresh Rendered Lard

120

100

(atm)
.., 80 ~Reference

ll
t!
0" 60
-•-Reference (N2
sparge/blanket)
"
E .....,._ Reference +Nat Ox Inhib
(atm)
40
- • - Reference +Nat Ox Inhib
(N2 sparge/blanket)
20

0
0 2

Weeks Storage at 130°F (54.4° C)

FIGURE 6.3 Assessing oxidative status using peroxide values (PV). Data help provide
information about the storage conditions required to achieve maximum shelf life. Data
modified from internal study.

Measuring the resistance to oxidation (e.g., oxidative stability index of


extracted fats) is an application of coulometric electrochemistry. For our
purposes, more meaningful data depend on quantifying the extent of lipid
oxidation, that is using the potentiometric methods (e.g., oxidation reduction
potentials and determination of PVs).
Ions from the meat matrix form a potential difference at the surface of
the electrode. Because the distance between the inner surfaces of the elec-
trode is small, minute potential differences can be detected. For example, the
potential difference detected by the potentiometer is given in Equation 6.4.

k× q 1mv
Potential difference = 2
= −6 = 1,000,000 mv cm –1 (6.4)
d 10 cm
Methods and Their Applications for Measuring 211

where k is a constant,
mv = millivolt,
q is magnitude of the interfacial electrical potential developed between the
meat sample and electrode,
d 2 is square of the interfacial distance between the meat and electrode.

The electrochemical amplification of the potential offers the sensi-


tivity needed when the detection of small differences is needed. This
method would be considered when the background color of the sample or
the concentration of the analyte is low (limit of detection (LOD) = 0.01 M
and LOQ = 0.16 meq. Kg–1). Potentiometric determination of hydroperox-
ides is based on Equation 6.5, an application of the Nernst principles for
electrochemistry.

E = Eo −
0.059
log10
[ reduced ] (6.5)
n [oxidized ]
Application of the Nernst equation relates the electrical potential to
aqueous concentrations of a solution under controlled conditions (Karddash-
Strochkova & Tur’ yan, 2001). The analytical instrumentation is similar
to the method for measuring PVs with potassium iodide–starch reagents.
Although both are based on titration, the potentiometric method uses a
change in an electrical signal whereas the iodometric method depends on a
visual color change to determine the concentration of the hydroperoxides in
the extracted fat (Table 6.1).

TABLE 6.1 Potentiometric Titration of Hydroperoxide with Sodium Thiosulfate in a Model


System.
Potentio metric determination of peroxide values
Titrant [Analyte] [Analyte] [Titrant] [Titrant] Eo Constant n E system
(Liters) Ox. Red. Red. Ox. V
0.0005 0.00099 0.098020 1.77 0.0592 2 0.281
0.0050 0.00909 0.081818 1.77 0.0592 2 0.312
0.0100 0.01667 0.066667 1.77 0.0592 2 0.322
0.0150 0.02308 0.053846 1.77 0.0592 2 0.329
0.0200 0.02857 0.042857 1.77 0.0592 2 0.335
0.0240 0.02703 0.035135 1.77 0.0592 2 0.337
0.0249 0.02670 0.033511 1.77 0.0592 2 0.337
0.0250 1.77 0.0592 2 0.200
212 Natural Antioxidants: Applications in Foods of Animal Origin

TABLE 6.1 (Continued)


Potentio metric determination of peroxide values
Titrant [Analyte] [Analyte] [Titrant] [Titrant] Eo Constant n E system
(Liters) Ox. Red. Red. Ox. V
0.0253 0.0336 0.003 0.08 0.0592 2 0.050
0.0260 0.0342 0.003 0.08 0.0592 2 0.090
0.0300 0.0375 0.003 0.08 0.0592 2 0.050
0.0350 0.0412 0.004 0.08 0.0592 2 0.050
0.0400 0.0444 0.004 0.08 0.0592 2 0.050

Components for potentiometric titration are:

1. Buret containing the titrant (sodium thiosulfate).


2. Known volume of the sample containing the analyte (extracted fat
or oil).
3. Reference electrode (saturated calomel or hydrogen electrode).
4. Electrode for measuring the potential, working electrode.
5. Meter to record the change during titration.

To demonstrate the application of the Nernst equation, titration of


hydrogen peroxide (H2O2) with sodium thiosulfate (Na2S2O4) in a model
system was calculated with Microsoft® Excel™ 2010 spreadsheet, Eo value
sourced from CRC Handbook of Chemistry and Physics 71st edition.

Rxn. in aqueous system 2S4O6 –2 + H2O2 – 2e– <–> 2H2O + 2e– + 2S2O3–2

RT
o
K eq = Ecell =
nF
× ln K eq ( ) (6.6a); solving for K (rxn driven strongly to the right)

E(Redox) = (Eo Red.) + (Eo Ox.) (6.6b)

Eo (analyte at T=0) = 0.281 V

ne E o + ne E o
Eq. point = = 0.200; vol. to eq. pt. = M 1 × V1 = M 2 × V2 (6.6c)
ne + ne

Keq = 1.43 × 1057


E (Redox) 1.69 V
E eq. pt. 0.200 V (estimate)
Methods and Their Applications for Measuring 213

Potentiometric titration data have been summarized in Figure 6.4.

REDOX Potentiometry
titration ofH202 by soudium thiosulfate

0.40 1:::::::::===-------------
Volts 0.30
0.20 t:=::=:::::~!!!!!!!l'~~=========
0.10
0.00

Volume (L) ofTitrant


(sodium thiosulfate)

FIGURE 6.4 Potentiometric titration, at the equivalence point (vertical grey line) the
amount of hydroperoxide is equivalent to the amount of sodium thiosulfate.

The points of underlying chemistry that make the potentiometric method


valuable for quantifying PVs are:

1. The Keq = 1.43 × 1057 for the sodium thiosulfate–hydrogen peroxide


system; therefore, the titration reaction is complete and essentially
irreversible.
2. The magnitude of the difference between Eo for sodium thiosulfate
(0.08 V in reduced format) and the Eo for hydrogen peroxide (1.77 V
in reduced format) provides a large and steep gradient for deter-
mining the equivalence point.

Meat scientists requiring a rapid, sensitive, and precise (limit of quanti-


fication = 0.16 meq kg–1 oil or fat) analytical method will prefer the electro-
chemical method useful for determining the current status of a meat product.
The relationship between the physical properties of light and the concen-
tration of chemical compound is codified in the Beer Lambert law in Equa-
tion 6.3.

Aλ = l × ∈ × C (6.7)
214 Natural Antioxidants: Applications in Foods of Animal Origin

where Aλ is absorbance at a specified wavelength,


l is path length of light (transverse axis of cuvette) in cm,
∈ is extinction coefficient,
C is concentration.

The Beers Lambert law, illustrated in Figure 6.5, states the absorbance
(A) of light at a given wavelength (λ) is related to the concentration of a
chemical compound.

Solu tion
Wavelength
selector
Detector Co lo r versio n of
figure 5.

The difference between the incident and transm itted light


indicates the absorb;mcc

Blac k and w hi te
versio n of
figure 5.

Wavelength Selector
The difference between the inc ident light and transmitted light indicates abosrbance

FIGURE 6.5 Beer’s law relates the concentration of the analyte of interest to the degree
of absorbance at a specified wavelength. The underlying principles for the components in
Equation 6.7 are presented in Table 6.2 and Figure 6.6.

TABLE 6.2 The Principle for Quantitative Spectroscopy is Based on the Relationships of
Absorbance (A), Path Length of Plane Polarized Light (l), and a Compound’s Extinction
Coefficient (ϵ). Modified from Data in Food Analysis: Theory and Practice, pp. 63–71.
2
Principle Dependent Independent R Slope Y
variable y variable x m Intercept
Strong absorbing analyte A (ϵ) Absorbance Analyte A 100% 0.25 0
Weak absorbing analyte B (ε) Absorbance Analyte B 100% 0.06 0
Path length v. absorbance (l) Absorbance Path length 100% 1.24 0
(dimension of
cuvette)
Absorbance v. concentration (C) Absorbance [analyte] 100% 0.25 0
Methods and Their Applications for Measuring 215

Figure 6.6 shows absorbance at a given wavelength and the difference in


absorbance is based on the molecular characteristics of a compound.
Practical application of Beer’s law starts with developing a standard
calibration plot of absorbance (at the specified λ) on the “y” axis and
concentration (moles L–1 or mg mL–1) on the “x” axis. The linear equation
y = mx + b can be used to determine the concentration of hydroperoxides
(or secondary oxidation products) by interpolating values from the stan-
dard curve.
The spectrophotometry is rapid, sensitive, and can be automated with
smaller systemic errors than the titrimetric technique.

Absorbance of Weakly Absorbing vs Strongly Absorbing Analyte


Variable
2.5
- Absorbance Analyte A
- Absorbance Analyte B
2.0
E
c:::
0
M
1.5
"""u
Q.l

c:::
~
..."'
0
1.0
"'
~
<(

0.5

0.0
0 2 4 6 8 10
Analyte Concentration

FIGURE 6.6 Concentration and the extinction coefficient (ϵ) of an analyte is a key principle
in quantifying the various compounds using spectrophotometric techniques. The linear
relationship between the components in Equation 6.7 is the basis of spectroscopic techniques.

Data from hydroperoxide analysis when combined with knowledge about


the meat product and process can be used to evaluate oxidation management
practices (Fig. 6.7). For example, the Figure 6.8 shows PVs collected over
several days for a meat by-product that will be used in a value-added meat
product.
The data in Figure 6.8 show a significant amount of the product produced
exceeds the specification for PVs (abbreviated USL). Product that does not
216 Natural Antioxidants: Applications in Foods of Animal Origin

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FIGURE 6.7 Flow diagram for spectrophotometric determination of hydroperoxides.
Adapted from Research and Reviews, Special Circular 183-02, Ohio State University.

Analysis of Peroxide Data for Managing Oxidation


meat by-product to be used as a meat ingredient

LSL USL

LSL
Process Data
0
~--Within
--Overall I
Target * Potential ('.\1tlin) Capabilty
USL

~"'
9.5
Cp 1.27
Sample Mean 8.7
CPL 2.32
SampleN 31
StDev(Within) 1.2481 ~ CPU
Cpk
0.21
0.21
StDev(Ov erall) 1.23774
Overall Capabiity

0.0 1.5 3.0 4.5


..J
6.0 7.5
rR
9.0 10.5
Pp
PPL
PPU
1.28
2.34
0.22
0.22
Observed Perfonna nce Exp. 'Mthin Performance Exp. Overall Performance
PPM <LSL 0.00 PPM <LSL 0.00 PPM<LSL 0.00 *
PPM >USL 258064.52 PPM >USL 260768.95 PPM>USL 259029.80
PPM Total 258064.52 PPM Total 260768.95 PPM Total 259029.80

FIGURE 6.8 Peroxide value data in the graph show significant amount of product produced
exceeds the specified limit (abbreviated USL). Data from internal spray dry data.
Methods and Their Applications for Measuring 217

meet specification limits must be “re-worked,” discounted in value, or worse


case thrown out. However, the data can be used to frame relevant ques-
tions for managing oxidation. For example, is the specification realistic or
could the process be improved in the selection of raw material quality and/
or production methods.

6.2.2 ANISIDINE VALUES (p-AV)

Secondary oxidation products of lipid oxidation may also be measured using


the spectrophotometric technique. AVs are determined spectroscopically at
absorbance of 350 nm. p-AVs are dimensionless and based on the absor-
bance of the chromagen formed through Shiff’s base mechanism during the
reaction of p-anisidine with cis-hexadecenal as shown in Figure 6.9.

) ml. di lution factor for


~ " "" "" ful " mpleo25

&
AH
3
g
~ dilute to 2Sml with
reagent grade
sprectro-pure
0.500 ~xlracled fal isooctane
]
I }
) mlofcontaining 2.0%extracted
fat+ 1 ml of 2.) g. p-ani sidine
per lite r of glac ial aceti c acid.
Dilution factor for extracted fat
sample = 1.2

¢"'
10 ml quartz cuvette

~
Para methoxJ analine NH 2
(p-anisi ine)
glycerol or glycerol
+

>==!
phosphate - OOC(CH2) :: CH2 - - H
H-

glycerol or glycero l ~
phosphate- OOC(CH2):: CH2- -
NH 2

H
H H
H H-

cis-hexadecenal fa tty acid Shi ff's Base chromagen


(nonvo lati le remnent of fatty ac id oxidation) cis- hexadecenoic amide (absorbs al 350nm)
FIGURE 6.9 The flow diagram shows sample preparation of extracted fat for
spectrophotometric analysis for secondary oxidation products with para-anisidine. Equation
6.8 is used to calculate p-AV.

25 × 1.2 × ( A1 – A2 – A3 )
p-AV = (6.8)
m
218 Natural Antioxidants: Applications in Foods of Animal Origin

where the coefficients 25 and 1.2 are dilution factors,


A1 is the absorbance of the reacted sample,
A2 is absorbance of the blank,
A0 is absorbance of unreacted sample,
m is the mass of sample.

Although the coefficient of variation (C of V) for p-AV is relatively


precise measurement, it can vary depending on the species source and
processing methods in rendering the fat. p-anisidine analysis is a valuable
measurement technique for measuring oxidation; however, correlation to
sensory evaluation has not been established.

6.2.3 TOTAL OXIDATION (TOTOX) METHOD

A single molecule of a hydroperoxide can produce a variety of different


compounds with varying chain lengths and levels of unsaturation during
oxidation. To analyze for a single product of oxidation may not enough
information. p-AVs and PVs can be combined (Equation 6.9) to provide
more information about the oxidative status of meat products.

TOTOX = 2PV + p-AV (6.9)

PVs and p-AVs (a dimensionless value that indicates the level of


non-volatile secondary oxidation products) are expressed in different
units, the formulation and the significance must be derived empirically.
Some food scientists have suggested the using thiobarbituric acid reac-
tive substance (TBARS) values to replace the p-AV values for calculating
TOTOX values (Wanasundara & Shahidi, 1995). The justification for the
formula 2PV + p-AV is based on an increase of one PV unit corresponding
to an increase of two p-AV units (Shahidi et al., 2002). Integrating PVs
and p-AVs into one equation for the purpose can result in an accumula-
tion of errors (i.e., uncertainty) by combining values derived from two
measurement techniques into a single numerical value. The accumula-
tion or propagation of errors can be estimated for TOTOX, assuming the
values for PV and p-AV are normally distributed, independent, and errors
are small. The propagation of errors (ϵ) for TOTOX can be calculated
using Equation 6.10.
Methods and Their Applications for Measuring 219

TOTOX = 2PV + p-AV

∈2 = (mean of 2 × PV)2 × (std.dev. of PV)2

+ (mean for p-AV)2 × (std.dev. of p-AV)2 (6.10)

Therefore,
∈ = √∈2

For example, the error of uncertainty of TOTOX values can be calculated


by using the data given in the Table 6.3.

TABLE 6.3 TOTOX Data Were Collected to Determine the Error of Uncertainty (the Error
Propagated when Two Measurements are Combined). Calculating the Error of Uncertainty
Assumes the Errors will be Small and the Data Are Normally Distributed. Data from Internal
Study.
TOTOX data
Variable N Mean Std. dev.
2 × PV 7 3.4 1.7
pAV 7 6.5 0.5

Solving for the error of propagation (ϵ) when two factors (PV and p-AV)
are incorporated into Equation 6.10.

∈2 = ((3.4)2 × (1.7)2) + ((6.5)2 × (0.5)2))


∈ = √(33.6 + 10.6)
∈= 6.6

The TOTOX value with the calculated error = 14 ± 6.6. If lower values
for the error or uncertainty are needed, measurement methods, processing
procedure, and/or product specifications need to be reviewed.
The application of p-AV, PV, and TOTOX data is shown in Figure 6.10.
Because lard rendering is a lengthy process involving sustained high
processing temperatures, analytical techniques that measure the more vola-
tile secondary oxidation products (2-alkenals, 2, 4 alkadienals for TBARS)
would be lost during processing. In this situation, p-AV data express the
concentration of less volatile secondary oxidation products (less saturated,
higher molecular weight fragments).
220 Natural Antioxidants: Applications in Foods of Animal Origin

Measuring Oxidative Stability of Fresh Rendered Lard During Storage

300
~ PV Reference
§ 250
.,i ---PV- Reference+ Nat. Ox.
;§ 200 Inhib.
.....
0 ...,._pAV Reference
-.:: 150
E 100 ---pAV Reference+ Nat. Ox.
~
gj Inhib.
~ 50 ---TOTOX Reference

0
.....,. TOTOX Reference+ Nat
0 (fresh) 2
Ox. Inhib.
Weeks Storage at !30°F (54.4°C) , Atmospheric Conditions

FIGURE 6.10 Data show an improvement in the storage stability of lard with natural
oxidation inhibitors (abbreviated as Nat Ox Inhib). PV or p-AV values alone would minimize
the importance of the added natural oxidation inhibitors.

TOTOX values (2PV + p-AV) provide more information because it


measured the current oxidative status (PV) and oxidation that had occurred
during processing (p-AV).

6.2.4 TBARS AND SENSORY EVALUATION

Volatile secondary oxidation products are most likely to be associated with


off aromas and off flavors in meat products. Malondialdehyde (MDA) is a
secondary oxidation product (from trienes) that at concentrations as low as
2–3 ppm produces off flavors and off aromas in value-added beef, pork, and
poultry products (Pearson & Dutson, 1985). The diagram of the reaction
shows that a prepared sample containing the secondary oxidation product
MDA (compounds in abundance in oxidized tri-ene and tetra-ene fatty acids)
reacts with thiobarbituric acid (TBA) to from a chromagen that absorbs
strongly at 532 nm (distillation method). However, some TBA reactive alde-
hydes absorb at 450 nm.
One mole of MDA reacts with two moles of TBA to form a chromagen
that absorbs strongly at 532 nm. TBA may react with other compounds
present; therefore data form this method are reported as TBARSs. The
concentration of thiobarbituric reactive substances or TBARSs (expressed
as TBARS or MDA kg–1 of sample) is determined by developing a calibra-
tion curve using appropriate concentration range (mg mL–1 or μmoles mL–1)
Methods and Their Applications for Measuring 221

of a basic solution of reagent grade MDA tetrabutylammonium added to an


acidic solution of TBA. The concentration can be determined from absor-
bance at 532 nm. Beer’s law provides a linear relationship between absor-
bance and concentration of TBARS via the linear equation y = mx + b.
From the standard curve y = mx + b
y is the absorbance of the test sample,
m is the slope from calibration with reagent grade MDA,
x is the concentration (mg mL–1) of test sample (unknown),
b is the “y” intercept from calibration with reagent grade MDA.

Based on mg mL–1 multiplied by the volume of the extract (mL), multi-


plied again by any dilution factors, divided by the weight of the meat sample,
and multiplied 1000 to express data as mg TBARS (as MDA) kg -1 of sample.
To express in µmoles per unit weight convert it by using 72 µg per µmole.
The LOQ using in this method is 1.0 µmole or 72 µgrams. This level of
sensitivity adequately discriminates between fresh and degrees of oxidiza-
tion between test samples during shelf-life studies.
Acceptable values for TBARS meat type (beef, pork, poultry, and fish),
formulation, and process specific. Table 6.4 shows the variation in accept-
able levels of TBARS for a few types of value-added meat products.

TABLE 6.4 Acceptable Values for TBARS Varies from Product to Product. Adapted from
Handbook for Meat Chemists, Edward Koniecko.
Average acceptable TBARS values for selected meat products
TBARS expressed as mg MDA 1000 g–1 of meat
Meat product Avg. TBARS
Beef bologna 0.222
Fresh Italian sausage 0.195
Smoked sausage 0.273
Beef weiner 0.197
Hard salami 0.429
Chicken roll 1.100
Boiled ham 0.105
Pepperoni 0.343
Fresh sausage patties 0.280
Bacon, sliced vacuum packaged 0.052
222 Natural Antioxidants: Applications in Foods of Animal Origin

Variation in the values for TBARS can be unduly influenced by the


method of preparation (Figs. 6.11 and 6.12).

Effect of Sample Preparation Technique on TBARS


fresh ground pork, 60% fat

0.8
~ 0.7
E 06
,.rll o:5
Jf 0.4
;3 0.3
::E 0.2
~ 0.1
0 ~---------.----------.----------.--------~
0.3 mm grind, 0.3mm grind, Sample held Sample held
single grind double grind +3°C, 72 hours +22°C, 72 hours

FIGURE 6.11 Sample preparation can cause TBARS to vary within samples from the same
treatment of between treatments. Adapted from Handbook for Meat Chemists, E.S. Koniecko,
(1979). Non-meat ingredients commonly used in formulating value-added meat products can
be a source of variation in the values for TBARS.

Effect ofNon-Meat Ingredients on TBARS


fresh ground pork, 60% fat

., 0.5
c..
§ 0.4
VJ
-,. 0.3
Jf
<( 0.2

~0.1
E

Fresh Ground Sodium Nitrie, NaCl salt, Ground Paprika,


Pork 6.25% at 0.20% 1% bywt. 1% bywt.

FIGURE 6.12 Non-meat ingredients commonly used in formulations for value added meats
can increase or decrease TBARS values. Adapted from Handbook for Meat Chemists, E.S.
Koniecko (1979).

Non-meat ingredients can increase or decrease the values for TBARS.


Ingredients that react with TBA and absorb plane polarized light at 530 nm
increase TBARS. Ingredients that react with MDA and block the reaction
with TBA (e.g., oximes) depress TBARS. In other cases co-pigmentation
Methods and Their Applications for Measuring 223

occurs (red–orange colors of carotenoid pigments) or Maillard reaction


products absorb light at or in proximity to 530 nm adding to the absorbance
of MDA–TBA adduct in the sample (Decker, 1998).
In addition to interferences from the chemical reactions, values for
TBARS are decreased by “dilution” by non-meat ingredients in the formu-
lation (water, starch, soy, etc.). Figures 6.13 and 6.14 show formula for a
chicken sausage (nitrites not added) containing 24% non-meat ingredients
and the comparison of TBARS, respectively.
The data in the Figure 6.14 show values for the TBARS for the two
treatments, one based on sample weight the other on meat weight. TBARSs
expressed on meat weight were higher than TBARSs expressed on sample
(formulation) weight.

Chicken Sausage Formula

• Chicken thigh meat +


skin emulsion
• Non-meat ingr. (water,
soy, salt, strach, etc.)

FIGURE 6.13 Formulation for a chicken sausage 76% meat ingredients and 24% non-meat
ingredients.

A study to determine the oxidative stability of frozen, minced ocean-


run pink salmon utilized the TBARS method (University of Alaska, Alaska
Fishery Development Foundation, Kalsec®, Inc.) and sensory evaluation.
The data in Figure 6.14 show the rate of change in oxidation of salmon
mince during storage. The TBARSs are expressed as μmoles MDA per
100 g of minced salmon (Fig. 6.15). The minced salmon will be frozen then
thawed as needed for processing value-added products (nuggets, patties,
sausages). Processing accelerates lipid oxidation; therefore, meat to be
224 Natural Antioxidants: Applications in Foods of Animal Origin

further processed into value-added products will need have lower values for
TBARS than that of finished products.

TBARS for Cooked Chicken Sausages


78% to 80% lean, no nitrite added
1.20
1.00

(/)
0.80
~
co
0.60 • TBARS based on
E- formula wt.
0.40
• TBARS based on
0.20 cooked meat wt.

0.00
T=O T+30
Days Stored at 40°F (4 °C) in Vacuum P ackages

FIGURE 6.14 Understanding the difference in TBARS for different types of value added
meat products requires knowledge about the formulation (high levels of non-meat ingredients
or lower level of nonmeat ingredients) to understand the significance of the data.

Oxidative Stability of Salmon Mince From Pin Bones


16.5lb . (7.5kg) blocks

100
lw 85
90
80 IUwWIIWII 1 0~
/ 1
/
"' C§ ~70
<I\
,...........
~:2-~60
f!l J! 50 r./
----.L
cJ)

,... ~ §
---
---
40 35
::>
25

---- --
30
20
..::;;..-- r- --
~
8
10
0 ~-------- -
1.8 6 10
Months Frozen at -2o•F (-29. C)

--Control Without antioxidants


- - With a blend ofna111ral oxidation inhibitors

FIGURE 6.15 TBA values for frozen salmon mince show that the maximum storage time
prior to thawing and further processing needs to be at some point in time prior to reaching the
maximum TBA value of 70. Data modified from the pink salmon study conducted by Alaska
Fishery Development Foundation, University of Alaska and Kalsec®, Inc. ca 1995.
Methods and Their Applications for Measuring 225

Two methods are used for analyzing 2-TBARSs in meat products that
are widely used in the meat industry (Fig. 6.16). The distillation method
involves partitioning secondary oxidation products in the meat sample into
the appropriate solvent then separating these compounds by distillation.
The direct method determines the concentration of secondary oxidation
products directly without partitioning the secondary oxidation products or

Methods for QuantifY ing 2- Th iobarbituri c Acid Reactive Substances in Meats


Generali ze Flow Diagram

TB ARS
I I Distillation Methods I

with
TBARS fo r Beef TBARS for Poultry
I I I

T ransfer an aliquot of lh e test TBARS for Pork


so ln. into test tube containing
I I
the recommended quant ity of
T BA reagent. I We igh sample add I :3 w/v in
trichloroacetic acid and
homogenize.
,. +
I Vacuum filter homogenate
through Watman #42 ashless
filter paper.

Cool, pipette samp le into t


cuvette, read absorbance at

t
I Add TBA reagent incubate in
530nm from calibrated boi ling fo r 40 min ..
spectrophotometer with band

!
width < 5nm . Determine cone,
us ing Beer Lambert Law Conduct "spike and
recovery" with a
small aliquot of
Pipette an aliquot o r Cool, then pipette into cuvette,
standardized soln. of
distillate from meat read absorbance at 530 nm in
MDA-bis acetaL
homogenate into test calibrated spectrophotomer,
tube w ith TBA band width < 5nm. Calculate
rea ~en t. cone. Using Beer Lambert Law

~
IPipette an aliquot of di stillate from meat
homogenate into test tube with TBA reagent.
I
~ ~
Incubate in boiling water for 35 min., cool then pi pette
sample into cuvette. Read absorbance at 530nm fro m
calibrated spectrophotometer with spectral band width
< Snm. Determine cone, use Beer Lambert Law.

FIGURE 6.16 Methods and techniques for the analysis of TBARS (thiobarbituric acid
reactive substances).
226 Natural Antioxidants: Applications in Foods of Animal Origin

distillation. The distillation method is most widely used because the assay
is relatively inexpensive, values are related to off-flavor, off-aromas, and
color degradation and the data are familiar to the meat industry. Since the
sample preparation and analysis differ for both methods, it is important to
confirm which method is used.. TBARS data in this chapter are from the
distillation method, this method is commonly used because the assay is rela-
tively easy and inexpensive. The direct method for TBARS is problematic
for samples containing carbohydrates and proteins, the distillation method
uses the distillate which avoids this problem.
Data in the Figures 6.17(a) and 6.17(b) below show the application of the
distillation method to evaluate the comparative shelf life between a natural
and synthetic antioxidant for raw frozen pork sausages. TBARS values
increase at different rates between treatments during frozen storage.

TBARS Raw Frozen 85% lean Pork Sausage Links


PVC overwrapped styrofoam trays
4.00 . . . - - - - - - - - - - - - - - -
3.50 + - - - - - - - - - - --6
-;- 3.00 +----------~­

r/.l ~ 2.50 +---------____,,___


~~ 2.00 ~ Control, w/o antiox.
E-:21.50 -l----"'"""""'~,_,...,=::!1~----- - • -Rosemary Extract
01)
E 1.00 .,.......,._ _ _ _ _ _ _ _ _--...

...
_.,_ BHAIBHT
0.50 ~~=--~~;;;;r r"''-=--~
0.00 ,._--,.---;r-----~----,
T=O 2 4 6 8
Weeks Stored in +5°F ( -l5°C) to + 10°F ( -l2°C) Freezer

FIGURE 6.17(a) Data for TBARS show differences between treatments during storage
time. Data from internal storage stability study.

TBARS values in the study correspond inversely with changes in the


color of the raw pork sausages.
Some have suggested combining PVs and TBARS as a derivative of the
previously mentioned TOTOX equation to be TOTOX (modified) = 2PV + TBA.
This derivative of the standard TOTOX equation would need further testing
to assure the data are valid.
Methods and Their Applications for Measuring 227

Raw Frozen! Thawed Pork 85% lean Sausage Colorimetry


PVC overwrapped styrofoam trays
40.00 . - - - - - - - - - - - -
Iii
....
0
.!: 35.00 --- --T-- - - - - - - - -
~
~0
~ ~
Ea::
30.00 +--___:..!'!!!!!!!!,;;;a;;......~....,..;:::--- ,
·c u ~Control, w/o antiox.
-§ ~ 25.00 +-------------.3o~
u .... - • Rosemary Extract
* ::I
u ....5l 20 .00 + - - - - - - - - - - - - - ......... BHAIBHT
..r:::
~
~ 15.00 + - - - . , - - - - , - - - - - - , - - - - - ,
T=O 2 4 6 8
Weeks Stored in +5°F (-l5°C) to +l0°F (-l2°C) Freezer

FIGURE 6.17(b) The interrelationship between lipid oxidation and raw meat pigment
color is inverse. As meat lipids oxidize, meat color also oxidizes. Data from internal storage
stability study.

Values for TBARS of raw frozen pork sausage links can be transformed
into a linear model to compare slope (rate of oxidation) and “y intercept.”
The Figure 6.17(c) shows pork sausages without an oxidation inhibitor
(R2 = 90.9%) are initially more oxidized (“y” intercept) and oxidize most
rapidly during storage, pork sausages with a natural oxidation inhibitor
(R2 = 75.7%) are initially less oxidized and the rate of oxidation is inhib-
ited with effectiveness comparative to BHA/BHT (R2 = 93.2%). Predictive
application of the linear model is reserved for interpolation, extrapolation of
the values for TBARs beyond eight weeks, in the data below, is risky. Note
linear transformation does not alter the overall results.
The TBARS method provides valuable information about the shelf-life
meat products and can be used to improve formulations, alter packaging
materials, and review raw material specifications. However, good informa-
tion can be compromised if used incorrectly. For example, using TBARS
data to measure oxidation during a storage stability study under acceler-
ated conditions can provide information in a relatively short period of time.
However, using TBARS data from an accelerated shelf-life test to predict
shelf life under different conditions carries a great deal of risk. The sche-
matic representation in Figure 6.18 shows one possible error associated with
predicting shelf life.
228 Natural Antioxidants: Applications in Foods of Animal Origin

TBARS Raw Frozen Lean Pork Sasuage Links


PVC overwrap on styrofoam trays

3.5 Variable
- - Control
a.>
c.. 3.0 - Nat. Ox. lnhib.
E - BHA/BHT
"'"'t>b 2.5

--
..:.:
<(
0
2.0

~ 1.5
100
E
Vl
co: 1.0
<(
CQ
1- 0.5
q
D
@=
0.0
0 2 3 4 5 6 7 8 9
Storage In +SF (-15C) to+ 10F (-12C) Freezer
linear model

FIGURE 6.17(c) Transforming TBARS data into a linear model can provide information
about the trend between and within the treatments. Creating a linear model has limitations and
should be used only when appropriate.

Theoretical Comparison Accelerated vs. Real Time Flavor-life

3.0
2.8
2.6
...........
/
" 2.4 /
Q.2.2
p .o maximum valn e tor I"HARS ./

..
./
r/l "' 1.8
~ 1 1.6 ~
.·.le/

-- ---
ill .(( 1.4
I- Q 1.2 -data.f~~cclerarecLshel!.JifP < Mlrl V 'IJ);Ji'
•'~ e\~
• . fron> eJ<u y·
2 1.0 . / ated sne\thf- -
~0.8 e
"0.6 ./
0.4
0.2
0.0
2 4 6 7 8 9 10 ll
"Weeks" Storage Time

FIGURE 6.18 The schematic generalized representation showing one possible type of error
in using valid data to predict shelf life by extrapolating data from accelerated shelf life studies.

As important as color is for consumer appeal of raw frozen sausages, it


is critically important for fresh, minimally processed packaged meat prod-
ucts. Consumer demand for meat products with nutritional claims demanded
analytical methods that would help to establish the oxidative stability (raw
Methods and Their Applications for Measuring 229

meat color and flavor) of these products. High oxygen (80%) modified
atmosphere packaging (HOX MAPackaging) was developed to maintain the
freshness by maintaining levels of oxygenated myoglobin pigments during
lengthy distribution and storage at retailers.

6.2.5 ANALYSIS OF OXIDATION OF RAW MEAT PIGMENTS

HOX MAPackaing provides an oxygen rich headspace to maintain high


levels of oxymyoglobin (the pigment responsible for the bright pink or red
color of ground meat and sausages). The data in the Figure 6.19 show how
headspace gases in HOX MAPackaged ground beef change during storage.
The biochemically active meat absorbs oxygen from the headspace to convert
myoblobin (a purple meat pigment) to oxymyoglobin (oxygenation) and also
promotes lipid oxidation. As oxygen is depleted in the headspace, meat color
decreases as oxymyoglobin levels decrease and lipid oxidation increases.
Photographs 6.2(a), 2(b), and 2(c) of HOX MAPackaged ground turkey
(70% ground skinless thigh meat, 30% breast trimmings) show the change in
color during storage in a cooler (Pettersen et al., 2004). From left to right the
HOX MAPackaged ground turkey becomes progressively more oxidized, raw
meat color becomes progressively less appealing (i.e., lower colorimetric a*
values). At some point, the deterioration of color would result in discounting
the price of the meat product to the consumer or discarding the product.

PHOTOGRAPHS 6.2(a), (b), and (c), left to right. For minimally processed HOX
MAPackaged products, raw meat color and lipid oxidation are inversely related. As meat
color degrades to brown, lipid oxidation (as measured by TBARS) increases. Photographs
and colorimetry data from internal study.

Photographs 6.3(a) and 3(b) of 80% lean HOX MAPackaged ground


beef show color degradation over time. Colorimetry data (colorimetric a*)
are higher in beef than that of turkey due to the increased inherent concentra-
tion of oxymyoglobin in 80% lean beef compared to that of ground turkey
with 30% breast muscle trimmings.
230 Natural Antioxidants: Applications in Foods of Animal Origin

Head space oxygen


decreases, raw red meat
color decreases

Raw meat color


Colorimetric a* = 24.4 decreases. lipid Colorimetric a* = 12.76
oxidation increases
PHOTOGRAPHS 6.3(a) and (b), left to right Raw meat color changes during storage and
is an indication of the development of oxidized flavors. By regulation (standard of identity
as stated in CFR Chpt. III, Part 319, §319.15), the types of oxidation inhibitors in HOX
MAPackaged meats is restricted. Photographs and supporting colorimetry data from internal
study. Photographs from internal study.

During the color life of HOX MAPackaged 80% lean ground beef, head-
space gases as well color were monitored. The data in Figure 6.18 show
the change in the concentration of oxygen (O2) is associated with color
degradation.

Gas Ratios in Headspace of MAPackaged 80% Lean Ground Beef


90% ~--~~----------------------

~ 80%
~ ~70%
~ !ij 60%
~~50%
~ e,_. 40% ~Oxygen (02)
~g
] ., .,I': 30% • Carbon Dioxide (C02)
:I:§ 20%
B Io%
0%
T=O T+IO days
Days Stored in 34°F (I 0 C) Cooler

FIGURE 6.19(a) At cold temperatures oxygen in the headspace gas is readily absorbed by
the raw meat. The oxygen absorbed participates in several biochemical reactions including
oxygenation of myoglobin and oxidation of lipids.
Methods and Their Applications for Measuring 231

The interrelationship between meat color and lipid oxidation (Greene,


1969) places importance on CIE (International Commission on Illumina-
tion), L*a*b* reflectance colorimetry as well as diffuse reflectance spectros-
copy is an important consideration when evaluating the oxidative stability in
value-added raw meat products.
Reflectance spectroscopy requires a light source, an object (meat
surface), and a sensor. In this case, the colorimeter describes raw meat color
in terms of L* (lightness and darkness), a* (redness), and b* (yellowness).
Fresh meat has initial high a* values (redness) (Table 6.5).

TABLE 6.5 Reflectance Spectroscopy is Used to Monitor the Raw Meat Color Stability
During Storage. Raw Meat Color and Lipid Oxidation in Raw Meats are Interrelated, as Meat
Color Changes from Red (Relatively High a*/b* in Combination with Higher L* Values)
to Brown (Relatively Low a*/b* in Combination with Low L* Values) Lipid Oxidation
Increases. Data from Internal Fresh Beef Storage Stability Study.
CIE L*a*b* reflectance colorimetry
Sample description L* a* b* a*/b* C* Hue angle a* × b*
Fresh meat color (red) 57.42 24.40 13.48 1.81 27.88 28.93 328.91
Oxidized meat color 52.94 12.76 7.51 1.70 14.81 30.49 95.83
(redish brown)

FIGURE 6.19(b) Reflectance colorimetry has three main components, a light source, an
object (i.e. meat surface) and a sensor. The sensor detects changes in redness (a*, a* ÷ b*
or a* ÷ b*), color saturation C* = chroma = 2 (a* + b* , hue angle or color (in the CIE color
sphere. For diffuse reflectance spectroscopy the intensity of light at a particular wavelength is
detected (Modified from Hunt, M.; King, A. Meat Color Guideline Measurements; American
Meat Science Association: Savoy, Illinois, 2012.)

The color of meat is based on the relative concentrations of oxymyo-


globin (red), myoglobin (purple), and metmyoglobin (brown) (Fig. 6.20).
For raw packaged meats (e.g., ground beef, pork, turkey) higher levels of
oxymyoglobin are associated with freshness and quality. Meat containing
higher levels of metmyoglobin is associated with oxidized or rancid flavors.
232 Natural Antioxidants: Applications in Foods of Animal Origin

Oxygenation
Reduced Myoglobin, Mbg Oxymyoglobin, Oxymbg
(purple) (red)
Deoxygenation

Reduction Oxidation
(electron gain) (electron loss)

FIGURE 6.20 The three main types of raw meat pigments are in equilibrium during storage.
The equilibrium shifts away from red meat pigments to brown meat pigments as the meat and
lipid oxidize. (Modified from American Meat Science Association, Meat Color Measurement
Guidelines. www.meatscience.org.)

Scientists depend on reflectance colorimetry methods for assessing


meat color because it relates well to consumer preferences. Two methods
for evaluating surface meat color frequently used are CIE L*a*b* colorim-
etry described earlier and diffuse reflectance spectroscopy. Both methods
relay on light reflectance; however, diffuse reflectance spectroscopy is more
discriminating because the method measures the intensity of light at specific
wavelengths for oxymyoglobin, myoglobin, and metmyoglobin (610, 473,
and 572nm wavelengths, respectively). Diffuse reflectance spectroscopy
(Mancini et al., 2003) describes the oxidative status of meat pigments and
indirectly the extent of lipid oxidation.
The colorimetric reflectance data in the Figures 6.21(a) and 6.21(b) show
how the distribution for the principle raw meat pigments changes during
storage.

Diffuse Reflectance Spectroscopy Colorimetry, 95% Lean Ground Beef


Minolta 3500d at T= 0 days at 36°F (2°C) -38°F (3°C)

OxyMbg, 61%

FIGURE 6.21(a) Distribution of fresh meat pigments favors the red oxymyoglobin
(OxyMbg) pigments.
Methods and Their Applications for Measuring 233

Diffuse Reflectance Spectroscopy Colorimetry, 95% Lean Ground Beef


Minolta 3500d at T+l4 days at 36°F (2°C) - 38°F (3°C)

FIGURE 6.21(b) Colorimetry data show a shift in the concentration of brown metmyoglobin
(Mbg) during storage.

Generally, for raw ground meat products, development of oxidized or


rancid flavors lags behind degradation of raw meat pigments. Pairing colo-
rimetry with methods for measuring the level of secondary oxidation prod-
ucts (e.g., TBARS or gas chromatography mass spectroscopy (GCMS)
headspace) defines whether the meat needs to be discounted or discarded.
Although visual appearance and flavor are the main drivers for consumer
acceptance of value-added meat products, nutritional value and wholesome-
ness are becoming influential considerations. The inherent nutritional value
of meat products (low caloric content and high biological value proteins
and source of essential minerals and B vitamins) is known; however, some
conventional meat products are being reformulated to respond to consumer’s
interests. For example, nutritionists and physicians are advising that calories
derived from fat be reduced to 25–35%, with less than 10% of the calories
from saturated fats (SFA), the ω-6 to ω-3 fatty acids be reduced to improve the
thrombogenicity and atherogenicity indicies (abbreviated IT and AI, respec-
tively). Based on these guidelines (American Dietetic Association, 2007), the
demand for conventional value-added meats could be marginalized unless
addressed through product reformulation and placement (Fig. 6.22).
The objectives for reformulating value-added meats to improve the
following.

1. The ω-3 to ω-6 fatty acid ratios


2. IT and AI
3. Shelf-life stability

Sodium reduction in value-added meats is of considerable interest to


consumers and health professionals. However, the role of salt (NaCl) as an
234 Natural Antioxidants: Applications in Foods of Animal Origin

essential ingredient in processed meat products and is beyond the scope of


this chapter.
As has been discussed, changes in meat formulations can significantly
alter the oxidative stability, that is shelf life of the product. Analytical
methods for measuring the oxidative status in meats have taken on renewed
interest in product development (evaluating the oxidative stability of refor-
mulated meat products).

Fatty Acid Composition for 85 Gram Portions of Raw Meat

FIGURE 6.22 Interests of nutrition conscious consumers toward meat products with
lower levels of ω-6 fatty acids and less saturated fatty acids has shifted the demand for less
conventional products. Data sourced from USDA food composition table.

As the data in the chapter show, meat with improved “health indices”
is also more susceptible to lipid oxidation and, consequently, shorter shelf
lives. Therefore, it is critical to determine and confirm the oxidative stability
of more healthful meat products. For example, HOX MAPackaged grass fed
ground beef has been in particular demand by consumers because the meat
is higher in ω-3 fatty acids, value, and convenience.
Before proceeding to the methods used the comparative shelf-life study
for HOX MAPackaged ground beef formulated from grass fed beef (high
ω-3 fatty acid content) versus grain fed beef (low ω-3 fatty acid content),
the anatomical features of α-linolenic is reviewed in Figure 6.23. Fats and
oils can be described in terms of their chemical, physical, and sensory prop-
erties. The diagram of the anatomy of a α-linolenic acid describes several
physical characteristics that determine nutritional value and oxidative
stability.
Methods and Their Applications for Measuring 235

2 4 0

5
FIGURE 6.23 Alpha linolenic (ω-3 fatty acid) fatty acid. This fatty acid is 4–5 times higher
in grass fed beef than grain fed beef, and is highly susceptible to oxidation. Modified from
ChemDraw® software CambridgeSoft®/Perkin-Elmer®.

The ω-3 fatty acids have seven important structural features.

1. The alpha carbon is associated with the carboxyl end of the molecule.
2. The approximate reactive site (allylic carbon) where peroxidation
and scission begin with the abstraction of the carbon atom.
3. The approximate reactive site (bis-allylic carbon) where peroxida-
tion and scission are most likely to occur during initial phases of
oxidation.
4. Hydrocarbon backbone (cis-Cn H2n) of the fatty acid
5. Unsaturated portion of the fatty acid (cis-Cn Hn-2)
6. ω-6 carbon
7. ω-3 carbon

To preserve the status of meat products in the consumer’s daily diet, some
meat processors offer conventional products reformulated with grass fed.
The data in Table 6.6 compare the fatty acid profiles between grass fed/grass
finished (95% lean) and grain fed (90% lean) beef forequarter and show
grass fed beef has a higher ω-3 fatty acid content. Small changes in levels
of unsaturated fatty acids significantly impact the oxidative stability (Wood
et al., 2003). Using analytical methods, oxidative stability can be monitored
when there are changes in the fatty acid composition in the meat formula.
Indices based on the fatty acid composition in raw beef, pork, poultry, and
finfish are shown. Indices that are positively correlated to nutritional value
also present shelf-life challenges.
Indices of nutritional value (bottom of Table 6.6) are IT, AI, and ratio of
desirable fatty acids (DFA) to the undesirable fatty acids (OFA) that elevates
low density lipoproteins (LDL).
236 Natural Antioxidants: Applications in Foods of Animal Origin

(C14 : 0 + C16 : 0 + C18 : 0)


IT = (6.11)
ω − 3 PUFA
(0.5 × MUFA) + (0.5 × ω − 6 PUFA) + (3 × ω − 3 PUFA) +
ω − 6 PUFA

(4 × C14 : 0) + (C16 : 0 + C18 : 0)


A= (6.12)
Σ MUFA + Σ PUFA ω − 6 + Σ PUFA ω − 3

DFA
=
desirable fatty acids
=
∑ (PUFA + C18 : 0) (6.13)
OFA undesirable fatty acids ∑ (SFA − C18 : 0)
PUFA ω − 3
(6.14)
ω − 6 PUFA

where C:14, C:16, C:18 are carbon notations for the fatty acids,
PUFA is polyunsaturated fatty acids,
MUFA is monounsaturated fatty acids,
SAT is saturated fatty acids.

The nutritional indices (Garaffo et al., 2011) were calculated and are
presented in Table 6.6. The data show grass fed beef DFA/OFA (Moawad
et al., 2013) is more favorable than grain fed beef because of the higher
levels of unsaturated fatty acids. The table also shows PUFA ω-3/PUFA ω-6

TABLE 6.6 Data Show the Fatty Acid (FA) Profile that is Nutritionally Valuable (Percent
C:183, IT, AI, DFA/OFA) are also Fatty Acids that Reduce Oxidative Stability (APE, BAPE,
IV). Data from Internal Study.
Fatty acid analysis (FAME)
Calculated derivative values for oxidative stability and nutritional value
Fatty acid carbon notation
% FA (normalized) C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 Total
Grass fed 6.3% 39.5% 23.4% 27.5% 2.00% 1.4% 100%
Grain fed 11.1% 43.2% 9.60% 33.9% 2.00% 0.30% 100%

FA distribution (millimoles)
Grass fed 0.27 1.54 0.82 0.98 0.07 0.05 3.73
Grain fed 0.49 1.68 0.34 1.20 0.07 0.05 3.83
Methods and Their Applications for Measuring 237

TABLE 6.6 (Continued)

Fatty acid analysis (FAME)


Calculated derivative values for oxidative stability and nutritional value
Fatty acid carbon notation
% FA (normalized) C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 Total
FA distribution (mole fraction)
Grass fed 7.3% 41.3% 22.0% 26.1% 1.90% 1.40% 100%
Grain fed 12.9% 44.4% 8.90% 31.7% 1.90% 0.20% 100%

Stability and Calc. IV % IV (from APE BAPE IT AI DFA/


Nutritional value C 18:3) OFA
Grass fed 31.0 0.12% 62 5 0.47 1.06 1.53
Grain fed 33.0 0.02% 72 2 1.84 2.38 0.62

ratios are more favorable for grass fed beef. Similarly, IT and AI data (not
shown) are more desirable for grass fed beef. Beef in the form of HOX
MAPacked ground fresh beef offers consumers nutritional value, quality,
and convenience they seek. However, HOX MAPackages have a finite shelf
life because, during distribution and storage, meat becomes less red and
lipid oxidation products increase. The relationship between TBARS and
sensory (raw meat color) between raw HOX (80% oxygen, 20% carbon dioxide) MAPack-
aged ground boneless forequarter meat from grain fed versus grass fed/
grass finished beef.
Color (CIE L*a*b*) and TBARS were used for both grass fed and grain
fed MAPackaged raw, lean ground beef shelf-life study. Using both methods
provided information about what consumers value most—color or flavor.
The colorimetric data in the Figure 6.24 show grass fed beef; particularly
grass fed beef with the incorporation of rosemary natural oxidation inhibitor
is more color stable than grain fed beef.
However, the TBARS data (Fig. 6.25) show the necessity of early addi-
tion of an oxidation inhibitor (TBARS value at the “y” intercept for grass
fed beef without an oxidation inhibitor at T = 0). The data also show it is the
reactivity of the fatty acids not the fat content that is responsible for driving
oxidation. Information from both methods provides the information needed
to identify and address pre-emptively shelf-life challenges during the devel-
opment of products from grass fed beef, for example, raw HOX MAPack-
aged ground beef.
238 Natural Antioxidants: Applications in Foods of Animal Origin

Co lor Stability of Grass Fed vs. Grain Fed MAPackage (HOX) Lean Ground Beef
95% Cl for the Mean
30

i'l
25
I I. l. I. I · ! I
:I .
0"' •
* 20 •
"'
u
·c:
• • I• •
t
w
15
• I• •
E

u
0 • Oxidized

I
10

FIGURE 6.24 Color stability of HOX MAPackages of ground beef from “grass fed” and
“grain fed” was studied. MAPackages of ground beef from grass fed cattle were more color
stable than MAPackages of ground beef from grain fed cattle.

Comparison of Oxidative Stability


grass fed vs. grain fed ground lean beef
8.50 ,.-----------------
8.00 +-----------------
7.50 +------------------4
+ - - - - - - - -T-- - - - - -----:*
7.00
6.50
6.00
+--------,-,,-,,;A'T~~
+------
-, ...._..__,_
, -:::,.-'"------:'Tb-- -
=- -=--=,.,..... _-_
• •,...--'-'
.•-..-:c
. .... ,c..~·-~
''- - ---"1'
.·.
5.50 +----..-:::,,......c:::...._-----;.±:-;,,....
. ....,_,.:....__ _ _ _--"'
5.00 + - -,--,, -·"'---- - -.-..,-;-...-.• 1"-- - - - - - - -
4.50 ;L~....._::---.•..,
.. ,......,c:...._---=-------- -~-Grass Fed Control
4.00 -t---- -.-.."" ··,.-'-'-'- - - - - - - - - - - ...... Grain Fed Control
3.50
3.00 +--
... .-.. :o
.....:...__•• ,. o.-'-'-- - - - - - - - - - - - -
_ _ _ _ _ _ _ _ _ _ _ __
- ~ Grass Fed w/ r'mry
2.50 "F--- - - - - - - - - - - - - - -
2.00 +----------------- ...... Grain Fed w/ r'mry
i·6g - - - - __ ,. .........-·-·-·-·-·-·-·W··
o:5o ............ .
0.00 +--------,-------------,
T=O T+ll T+l4
Days Stored in 38°F (3°C) Cooler

FIGURE 6.25 When TBARS are paired with colorimetry (Fig. 24), the susceptibility to
oxidation reflects the changes in the composition of grass fed beef, in particular α linolenic.
Methods and Their Applications for Measuring 239

6.2.6 OXIDATION REDUCTION POTENTIAL (ORP) METHOD

ORP can be an important method for measuring the general oxidative status
of meat products. The ORP method does not measure specific products of
oxidation. It can be used for evaluating oxidation inhibitors (Fig. 6.26).

Platinum REDOX electrode

Meat homogenate
(buffered)

FIGURE 6.26 Oxidation reduction potentials (ORPs) can be used to broadly measure the
oxidative status of meat products. Negative values for ORPs indicate reducing (favorable)
environment. Diagram by Tom Jones.

The ORP method is a direct measurement of the all the oxidized and
non-oxidized compounds in the meat sample (Vahabzadeh, 1986). The elec-
trical potential measured is related to the concentration of oxidized and non-
oxidized species through the Nernst equation.

0.059
E = Eo + log10 [ oxidized and nonoxidized species ] (6.15)
n
where E is the reading taken at the potentiometer in mV,
Eo is reference electrode in mV,
0.059 is a constant calculated by mathematically combining the universal
gas constant, temperature (K), Faraday’s constant,
n is the number of electrons (unknown) involved in the oxidation reduction
process, since meat systems have dilute ionic concentrations (or activities)
n can be ignored.
240 Natural Antioxidants: Applications in Foods of Animal Origin

The read-out is based on the following equation.

D Eh (ref. Pt. electrode) + Eh (meat homogenate) (6.16)

Reducing conditions will register more negative values for Eh while


oxidizing conditions will register more positive or less negative values for Eh.
The ORP method is used for measuring the effectiveness of two oxida-
tion inhibitors for stabilizing raw meat color in HOX MAPackaged ground
beef. The two oxidation inhibitors differ in their respective water/fat parti-
tion coefficients, molecular weight, and size. The data show significant
differences between the ORP values for the oxidation inhibitors (Fig. 6.27).

Oxidation Reduction Potentials 80% Lean Ground Beef


HOX MAPackaged, n=5

ll~
~ t-- -10
.5 ~
-a!
·.=c .00 -30
" ....
c;c.
c. ~ -50
§o
.g-o ·~.= -70
" c
co: .~ -90
§~
·.g 0

-g
><.c
fi -11o
Ot:,
-130

-150

T=O T+3 T+6


Days Stored in 38°F (3°C) Cooler
• Control • Anti ox # I ::: Anti ox #2

FIGURE 6.27 Oxidation reduction potentials are more negative (reducing conditions)
when an antioxidant is added.

Colorimetry was also conducted, the data in the Figure 6.28 show the
oxidation inhibitor with the most negative ORP value over time, also has a
positive effect on raw meat color stability.
Methods and Their Applications for Measuring 241

Colorimetric Data 80% Lean Ground Beef


HOX MAPackaged , n= 20

T=O T+3 T+6


Days Stored in 38°F (3°C) Cooler

• Control • Anti ox # 1 :;: Anti ox #2

FIGURE 6.28 Colorimetry data (C*, Chroma) shows the addition of an antioxidant
improves the stability of raw meat pigments during storage. Negative oxidation reduction
potentials relates to higher color stability.

The ORP method for determining the oxidative status of meat products
is precise to within 5%; however, the following factors can reduce precision
and repeatability:

1. Temperature and ionic strength


2. Concentration of myoglobin (i.e., heme-iron) in the meat sample
3. Presence of oxygen in the homogenate
4. Time for potentiometer to reach a steady state

6.2.7 HEADSPACE ANALYSIS OF VOLATILE LIPID OXIDATION


PRODUCTS (GCMS)

TBARS method for quantifying secondary oxidation products in value-


added meats has stood the test of time, the need for faster turn-around, sensi-
tivity, and precision has become a bigger. GCMS headspace advantages
include reduced sample preparation time, increased sensitivity (LOQ), and
reduced analytical time. The GCMS method is gaining wider acceptance in
research and industry. Coupling the components of GC and MS allows for
the identification and quantification of a wide range of secondary oxidation
products in a meat sample.
242 Natural Antioxidants: Applications in Foods of Animal Origin

Meat samples are prepared by first comminuting meat in a 38 °F (3 °C)


room, the appropriate solvent is added to the meat to convert mixture into a
form compatible for the analysis. Controlled heating volatilizes the extracted
compounds prior to injection via an inert gas onto the column. The column
can be thought of as consisting of a structural support (e.g., stainless steel)
lined with a stable film (stationary/liquid phase) that separates the complex
mixture of volatiles and secondary oxidation products in the meat sample.
Separation is best achieved when the polarity of the stationary phase is
similar in polarity to the compounds being analyzed.
Retention time, archived data, and experience provide the information
needed to identify “marker compounds” (aldehyde A and aldehyde B in the
Fig. 6.29).

Aldehyde B

/ L..l-n-te_rn_a_l_s_tan-dar_d_l_ll_L_,
height (h in mm)

Detector Signal
/ LI_A
_l_de_h.;..
yd_e_A_ _.

Retention
in em
Injection
Width at 0.5h mm

Chart Speed (em sec -I)

FIGURE 6.29 Concentration of the analyte is determined by using a known volume of


an internal standard. Retention time of the analytes separates from the sample for ease of
quantification and identification. Diagram by Tom Jones.

The (calibrated) detectors used for quantifying marker compounds are


selected based on their sensitivity, signal stability, and linear responsiveness
over a wide concentration range; these characteristics are critically impor-
tant for precise quantification of secondary oxidation products using GCMS
(Fig. 6.30).
Methods and Their Applications for Measuring 243

GC

MS

FIGURE 6.30 GCMS is the combination of gas chromatography and mass spectroscopy
(GCMS). When used in combination, one or several types of compounds from lipid oxidation
can be measured.

Using the read-out from the recorder, the concentration of the marker
compounds can be calculated using the following steps.

1. A known amount (usually1 µL) of an appropriate standard is injected


into the column.
2. Although peak measurement for chromatographic quantitative anal-
ysis has been computerized, the areas of peaks in the diagram have
been calculated.

Area of eluted peak (mm2) = (ht. of peak) × (peak w at 0.5 × h) (6.17)


(0.5 x h to reduce error from “tailing” at the base of the peak)

3. The peak from 1 μL of the standard is 101.5 mm; therefore, the


concentration of the eluted marker compound(s) can be calculated
(Table 6.7).
4. The separated compounds exiting the GC are bombarded by a high
energy electron to produce charged particles necessary of mass
analysis.

CH3 (CH2) 4 CHO + 1 e– CH3 (CH2) 4 CHO+ + 2 e–


(hexanal from GC) (electron beam) (+charged particle for mass analysis)
244 Natural Antioxidants: Applications in Foods of Animal Origin

TABLE 6.7 Concentration (Usually ppm) can be Calculated Based on the Known Quantity
of an Internal Standard. The Amount of Individual Compounds from Oxidation (e.g.,
Hexanal, Propanal, Nonenal, etc.) may be Calculated or Grouped Together to Calculate Total
Aldehydes).
Internal std. Aldehyde A Aldehyde B Total aldehydes
Peak h, mm 29.0 15.0 20.0
Peak w at 0.5h, mm 3.50 2.50 3.00
2
Peak area, mm 101.5 37.5 60.0 97.5
Peak area ratio 1.00 0.37 0.59 0.96
Concentration (μL) 1.00 0.37 0.59 0.96

Charged particles are accelerated and directed through the mass spec-
trometer by controlling and varying (referred to as scanning potentiometry)
the applied voltage. The electron bombardment produces singly charged
particles; therefore, the path of a charged particle will depend only on mass
or mass charge ratio (m/z). The mass analyzer segregates the charge parti-
cles based on m/z and the detector records the abundance (frequency) of
each m/z.
There is not just one single pathway for fatty acid oxidation, the types
and concentration of secondary oxidation products produced are differed
and concentrations of each can vary. In addition, the type of meat (beef,
pork, poultry, and fish) and processing conditions (cooking method, drying,
aging, fermenting, etc.) will also affect the profile of secondary oxidation
products produced from oxidation.
When samples of oxidized beef were analyzed by GCMS, hexanal,
2-hexanal, 2-nonenal, 2-heptenal, 2-octenal, nonanal, and decanal were
identified (Flores et al., 2013). These “marker compounds” increase over
time to provide information about the rates and extent of lipid oxidation
during shelf-life studies.
The mass spectrograph data in Figure 6.31 show several compounds that
were detected in oxidized ground chicken and the internal standard shown is
a part of the calibration procedure.
The GCMS method was used to monitor the oxidative stability of raw
frozen restructured beef steaks through the distribution chain. The data
from the study would provide information about the stages in distribution
(processing, storage, cooking) would have the most the greatest impact on
lipid oxidation. Restructured steaks are made by comminuting underutilized
beef cuts (chuck, round, sirloin, etc.), then formed into steak or cutlet shapes.
Methods and Their Applications for Measuring 245

Restructured steaks have the appearance and texture “whole-muscle” steaks


and cutlets (see Photograph 6.4 below) and can be engineered to meet any
portion weight, shape, lean content, or seasoning profile. Archived data were
available for a variety of value-added meat products (Fig. 6.32).

<1l
c
<1l
X
Q)
I

Relati ve
Abso rbance

-
r-i

Time(min)

FIGURE 6.31 GCMS was used to measure multiple secondary oxidation products or
“marker” compounds. These compounds were monitored during storage stability tests to
determine maximum acceptable shelf life of cooked chicken patties.

PHOTOGRAPH 6.4 Raw restructured steak.


246 Natural Antioxidants: Applications in Foods of Animal Origin

What effect on would the method for the unique method for commi-
nuting meat and the use of non-barrier packaging during storage and distri-
bution have on shelf life little restructured beef steaks and is the amount of
the natural oxidation inhibitor adequately protect the product?
The GCMS method was used to determine the oxidative stability of
restructured beef steaks during storage for the following reasons.

1. Raw restructured steaks will have low levels of secondary oxidation


products, the sensitivity of GCMS will differentiate between fresh
and low levels of lipid oxidation.
2. The low variability in the analysis (as measured by the C of V) trans-
lates to fewer samples that are needed for analysis without sacri-
ficing the quality of the information.
3. GCMS data correlate well with raw meat color and sensory.

Restructure Beef Formulation

• BeefForequarter, 85% In
:-~ Processor Grade NaC l Salt
• Sodium Polyphosphates
l.: Com Syrup Solids (20 D .E-)
• Nat. Ox. Inhib.
• Chilled, Soft Water

FIGURE 6.32 Restructured beef is formula is 10.4% nonmeat ingredients. The GCMS
method provided the sensitivity for measuring oxidative stability in value added raw meat.
In addition, the polyphosphates and corn syrup solids could interfere if TBARS method was
used. Formulation developed internally for a study.

A study was conducted to determine at what stage during distribution


in commerce affected shelf life the most. Identifying the points of greatest
oxidative stress will provide information needed to manage oxidation.

1. Stored raw, non-barrier film in a + 20 °F (–7 °C) freezer for 15 days


(processor abuse, meat is held at –15 °F (–26 °C) to –25 °F (–32 °C).
Methods and Their Applications for Measuring 247

2. Cooked to 165 °F (74 °C) at 325 °F (163 °C) and held three hours
in a135 °F (57 °C)warming oven according to hazardous analysis
critical control point (HACCP) procedures (product abuse #1).
3. Cooked to 165 °F (74 °C), stored at 35—38 °F (2 °C–3 °C) for 48
hours then reheated (product abuse #2).

The GCMS data in Figure 6.33 identify that cooking and reheating stage
in the distribution process greatly accelerates oxidation in the steaks.

Oxidative Stability of Restructured Steaks


GCMS
30

25

'§" §..15
~ "' 10 +--.~------.--..,-----
• w/o Ox. Inhib.
• w/ Ox. Inhib.
5

0
TimeO Stored in +20°F Cooked and held Cooked, held at
frzer. , 20 das. in warming oven 35°F for 48 hrs.,
reheated

FIGURE 6.33 Data show reheating precooked restructured steaks accelerates oxidation
more than freezer storage or cooking and holding in a warming oven. The data also indicate
the type and levels of the oxidation inhibitor met product requirements.

Sensory evaluation was conducted using a nine point hedonic scale


(higher scores represent more intense flavor) and was conducted by 14
trained panelists on five aroma characteristics for restructured beef steaks.
Sensory data indicate the significant differences (α = 0.05) between steaks
with a natural oxidation inhibitor and steaks without an oxidation inhibitor
occurs when the steaks were reheated. The sensory data are presented in
Figures 6.34(a), 6.34(b), and 6.34(c).
Note the flavor threshold for hexanal is 14–25 ppm and for TBARS
(MDA) is 1.5–2.5 ppm. Pairing chemical analysis (GCMS) and sensory
confirms a natural oxidation inhibitor is needed; and incidence for consumer
“abuse” would be of greatest concern.
248 Natural Antioxidants: Applications in Foods of Animal Origin

Restructured Beef Steak


T=O

Bee:ty/Brothy
9

~ Control -+-Nat. Ox. lnhib.

FIGURE 6.34(a) Sensory evaluation at time zero (t = 0) of cooked restructured steaks


immediately after cooking. Data shows little difference between steaks with or without
(control) an oxidation inhibitor. Sensory evaluation conducted internally with trained panel.

Sensory Evaluation ofRestructured Beef Steaks


cooked, stored at 35°F-38°F (1.6°C- 6°C) for 72 hrs, re-heated
before serving
Beefy/Brothy
9

~ Control - • Nat. Ox Inhib.

FIGURE 6.34(b) Sensory evaluation of restructured beef steaks shows significant


development of warm over aroma (descriptor for oxidized meat aroma) in cooked then
reheated steaks. Sensory evaluation conducted internally with trained panel.
Methods and Their Applications for Measuring 249

Restructured Beef Steak


T+l5 day at +20°F (-7°C) Storage

BeefY/Brothy
9

~ Control - • Nat. Oxlnhib.

FIGURE 6.34(c) Steaks cooked and evaluated after 15 days in +20°F (–7°C) freezer
(storage abuse during distribution). Sensory evaluation conducted internally with trained
panel.

The change in color of the steaks between time zero and after 16 days
storage in the freezer is likely due to the presence of NaCl salt in the formu-
lation. Sodium chloride in the meat has two functions—solubilizes myofibril
protein for binding the chunks of raw meat together and enhances meaty
flavors.
Based on analytical data, the critical stage in the distribution chain is
reheating of the precooked steak (e.g., reheating left overs), the incorpora-
tion of a natural oxidation inhibitor effectively inhibits oxidation at all stages
of commerce (Fig. 6.35).
Raw meat color of the restructure steaks was also evaluated during frozen
storage.
250 Natural Antioxidants: Applications in Foods of Animal Origin

Col orim etry Frozen Restru ctured Beef Steaks


95% Cl for th e Mean
26

ro
'iii
0
24
22
20
I
*ro
c 18
(;)
E 16
·g 14
I I I I I
I
0
u 12
10 I I

Minolta CR300 colorimeter C light sou rce 8mm aperature

FIGURE 6.35 Colorimetry data (a*) complement GCMS and sensory data. Raw meat color
of frozen restructured steaks in non-barrier packaging decreases over time. The data suggest
when raw meat color is a critical consumer attribute, barrier packaging may be required.

6.3 MEASURING THE CONSEQUENCES OF LIPID OXIDATION

6.3.1 WATER HOLDING CAPACITY (WHC)

Consequences of lipid oxidation is not limited to flavor, aroma, and color,


WHC is also adversely affected (Figure 6.36). As WHC decreases during
frozen storage, meat products will lose moisture during cooking resulting in
dry, “tough,” and chewy steaks.
Studies (Lund et al., 2007) conducted by meat scientists have deter-
mined that myofibrillar proteins are susceptible to chemical modification
due to oxidation. It is myofibrillar protein oxidation that has been a factor
in observed loss of WHC in raw and cooked value-added meats. Our study
was designed to determine the effect of natural antioxidants on meat protein
oxidation in raw frozen restructured beef steaks during 90 days of frozen
(–10 °F, –23 °C) storage. After 30, 60, and 90 days of frozen storage the
restructured steaks were thawed at 38 °F (6 °C) overnight and evaluated for
raw meat color (oxidation of sarcoplasmic protein) and WHC (a measure of
myofibrillar protein function). The trend in the data shows WHC decreases
Methods and Their Applications for Measuring 251

during frozen storage for the control (R2 = 100.0%), natural oxidation inhib-
itor (R2 = 100.0%) with the rate of loss (slope) for WHC was similar for
all treatments. The data suggest that inhibiting oxidation of meat proteins
will require further study, perhaps a blend of oxidation inhibitors or modi-
fied atmosphere packaging techniques or the quality of raw meat can be
considered.

Effect of Frozen Storage on th e Functiona l Properties of Meat Proteins


-ro
E 1.8 .r------------------. Variable
3: ~ WHC Nat. Ox. lnh i.
~ - WHC Control (w/o Na tOx. lnhib.l

1.7
~
-roa;
E 1.6
u
2!
~
u
>-
6 1. 5

u
"'o_ 1.4
u"'
bJ)
c
u
0 1.3
I
~ 20 30 40 50 60 70 80 90 100
s"' Days in - 1OF (-23 C) Freezer
WHC rnethod described by Hung, S.C.

FIGURE 6.36 Meat protein functionality (WHC) decreases during frozen storage. As WHC
decreases in the raw meat, the amount of moisture retained in the meat after cooking also
decreases. The lack of moisture in the cooked steak is associated with undesirable changes in
texture and palatability. Data from study conducted internally.

6.3.2 OXIDATIVE STABILITY OF SEASONINGS DURING


STORAGE (OLEORESIN PAPRIKA)

Inhibiting oxidation in value-added meat products often begins with an


assessment of the non-meat ingredients added during processing. For
example, dry soluble pepperoni seasoning contains some or all of the
following ingredients oleoresin paprika, dextrose, processors grade NaCl
salt, and an antioxidant (s). During storage and prior to use, the seasoning
can lose significant amount of color (oleoresin paprika). The data in the
Figure 6.37 show the rate of oxidative degradation of oleoresin paprika
252 Natural Antioxidants: Applications in Foods of Animal Origin

(critical component in pepperoni seasonings), during storage in a high


density polyethylene lines multi-ply bag stored at three temperatures. Infor-
mation can be used to determine the optimum storage time and temperature
to assure the color-life requirements for a pepperoni seasonings is met at
the time of use.

Oxidative Degradation Of Paprika Color in Dry Pepperoni Season in g

Variable

- Btc h #1 40F (4C)


0.15 - Btch # 1 90F (32C)
E ~ Btch #1 72F (22C)
c
~ 0.14
-<;-

0 0.13
0
u
~ 0.12
£:!
u
~ 0.11
x
LU

0.10

0 2 4 6 8 10 12
Weeks O f Storage

FIGURE 6.37 Some seasonings contain ingredients (e.g., paprika, antioxidants, seasonings)
that are susceptible to oxidation during storage and prior to use.

6.3.3 MEASUREMENT OF THE RATE OF ANTIOXIDANT


DEPLETION IN MEAT

The rate of an antioxidant which is consumed in ground beef stored under


different storage conditions is also a measure of the rate of oxidation. In this
case the rate in the disappearance of the antioxidant is measured instead of
the production of oxidation products. Data in Figure 6.38 show there are
issues in meeting the targeted usage level. The use of barrier packaging and
cold storage temperatures reduces the rate of oxidation and maximizes the
effectiveness of the antioxidant.
Methods and Their Applications for Measuring 253

Comparison of Antioxidant Consumption in Raw 80% Lean Ground Beef

• Target

~Stored in cooler, non barrier


packaging

D Stored frozen ->thawed at


35"F before analysis, barrier
(vacuum) packaging

T=O T+24 T+48 T+72


Hours Stored at 35°F (1.6°C)

FIGURE 6.38 The analysis confirms the addition of the oxidation inhibitor missed the
targeted level and frozen storage inhibits oxidation.

6.4 CONCLUSION

The methods and their application for processed meat products are described
in the chapter to emphasize there is no such thing as a routine analysis.
The information presented reviews the importance of understanding which
method provides the information appropriate for a given situation, the
importance of following good technique, the relationship between sensory
and analytical data; and the effect of ingredients have on analytical data.
As the reliance on analytical data for making good decisions increases, so
will the need for more analytical precision and reduced analytical time. For
example, advantages by modifying an existing analytical method (described
in Section 6.2.1) using electrochemical technology are illustrated in Figures
6.39(a) and 6.39(b).
Figure 6.39(b) shows an improved precision (i.e., a standard deviation
of five) by technical modification decreases the number of samples (thereby
the time for analysis) collected for analysis without sacrificing the quality of
the information.
The scope of the chapter includes description of methods that can also
be used to measure shelf life, improve processing efficiency (e.g., WHC of
meat); and, improve the quality of formulations for value-added meat prod-
ucts (e.g., color stability of paprika in Italian sausage seasoning).
254 Natural Antioxidants: Applications in Foods of Animal Origin

Required Samples to Confidently Report Analytical Information


1 sample z test
1.0
Sem~l e
Size
32
0.8 ~-- r- --- -=-- ----.---
1
Assumf>l ions

Alpha 0.05
0.6 '
-<• '
~-----~------r- StDev 10
I
(;:;
~
Alternative Not=
0
"- '
0.4 ~---
''
'
'0
,------rI
,-----T----
0.2
I 1
I
''
I

0.0 '
-6 -4 -2 0 4 6
Detection Difference For Reference Analytical Method

FIGURE 6.39(a) For the purpose of illustration, a generalized “reference” analytical


method, with a standard deviation of 10 requires that 32 samples be collected and analyzed.

Required Sarnples to Confidentll y Report Analytical Information


1 sarnple z test
1.0
I
Semple
I Size
I
I
8
0.8 ·---r- ----:------:----
1 I I
I I I
Assumptions
I I I
I I I I Alpha 0.05
-+----
I I I I
0.6 ·-<- .... -----t------·- StDev 5
(;:; 1 1 I I 1
5
I
I Alternative Not=
0 I
CL I I I
. .J ___ _
0.4 I
----L---
1
---~---
1
I I
I I
I I

0.2

-6 -4 -2 0 2 4 6
Detection Difference For Modified Analytical Method

FIGURE 6.39(b) With a standard deviation of 10 for this “modified” analytical method, 8
samples instead of 32 provides the quality of information needed.
Methods and Their Applications for Measuring 255

6.5 ATTACHMENTS

Attachment 1. Statistical basis for the inferences made for data in Figure
6.40. Making inferences about peroxide values collected to describe the
effectiveness in managing oxidation requires samples be randomly selected
and the data are normally distributed. Figures 6.40 and 6.41 show the data
collected are normally distributed.
Peroxide Values of a Meat Ingredient
12.0-r------------------------------,

~
c£\ /\
10.5 - ;\

9.0 ::\. !) I \
7.5
~ \:j ""' p
10 13 16 19 22 25 28 31
ObseiVation

!-===========================! UCL~3.235

10 13 16 19 22 25 28 31
Observation

FIGURE 6.40 PV data taken from samples collected at different times during production
show acceptable variation. This data can be used to determine if the peroxide values fit the
normal distributed curve.

Probabitity Plot of Peroxide Values


Normal - 95% CI

99~,~,~,~,~~,~,~,-,~-,~, ~,-~!-~~ ~.~~~:~, ~~ Mean 8.7


StDev 1.238

:~ rt==~=t==~=i==t=4==t=~==t==~~:~-r.- r~~t=i==t=~: N
AD
31
0.535
so ~--t--:--t--r--t--r-1--+--1--+· -:- -r--t--t--1--t--: P- Value 0.157
70 --+--:--+--t-i--t-1--+--i-- r- - ~T--t-i--t-4--+--l-
1

~ ~ lti~U~l~~:-r-rT-rT-rT_r_r_T-rT
!=:=~;:fifl~~l~t~l~ttl
rrT-r -~--·
10 11 12 13
Peroxide Values

FIGURE 6.41 The probability plot shows the data fits a normal distribution. Therefore, the
data can be used to make inferences about how well the analytical method for peroxide values
or their specification provide the degree of control for managing oxidation.
256 Natural Antioxidants: Applications in Foods of Animal Origin

KEYWORDS

• absorbance
• aldehyde
• antioxidants
• analyte
• autoxidation
• Beer’s law
• carbon dioxide
• carotenoid
• chelation or chelators
• chroma
• colorimeter
• cured meats
• decanal
• electrochemistry
• fatty acid(s) flavor threshold
• formulations
• ground beef
• ground turkey headspace
• hexanal
• heptanal
• hue
• iodometric titration
• Maillard
• meat protein
• metmyoglobin
• myoglobin
• mince
• natural oxidation
• inhibitors
• nernst equation
• nitrogen
Methods and Their Applications for Measuring 257

• nonanal
• nutritional indices
• octenal
• oxidative stability
• oxygen
• oxymyoglobin
• pepperoni
• propanal
• redox
• reflectance spectroscopy
• restructured steaks
• rendered pork fat
• sausage
• sensory
• sparge
• titrant titrimetry
• vacuum packages

REFERENCES

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of Canada: Dietary Fatty Acids; Journal of American Dietetic Association, September,
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Flores, M.; Olivares, A.; Dryahina, K.; Spanel, P. Real Time Detection of Aroma Compounds
in Meat Products by SIFT-MS and Comparison to conventional Techniques (SPME-GC-
MS). Curr. Anal. Chem. 2013, 9, 622–630.
Garaffo, A. M.; Vassallo-Agius, R.; Nengas, Y.; Lembo, E.; Rando, R.; Maisano, R.; Dugo,
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Lipids, of Raw Rose of Blue Fin Tuna (Thunnus thynnus L.) and their Salted Product
“Bottarga”. Food Nutr. Sci. 2011, 2,736–743.
Greene, B. E. Lipid Oxidation and Pigment Changes in Raw Beef. J. Food Sci. 1969,
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Hung, S. C.; Zayas, J. F. J. Food Qual. 1992, 15, 153–157.
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tion: Savoy, Illinois, 2012.
Jones, T. Science Fellow, Kalsec®, Inc., Diagrams drawn using Visio® software.
Karddash-Strochkova, E.; Tur’yan Ya, I. Redox-Potentiometric Determination of Peroxide
Values in Edible Oils without Titration. Talanta. 2001, 54, 411–416. www.elsevier.com/
locate/talents.
Koniecko, E. Handbook for Meat Chemists; Avery Publishing Group, Inc.: Wayne, NJ, 1979;
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fied Atmosphere Packaging on Protein and Lipid Oxidation in Beef Patties during Chill
Storage. Meat Sci. 2007, 76, 226–253.
Mancini, R. A.; Hunt, M. C.; Kropf, D. H. Refectance at 610nm Estimates Oxymyoglobin
Content on the Syrface of Ground Beef. Meat Sci. 2003, 64, 157–162.
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D.; Giuffrida, D. Fatty Acids Profile, Atherogenic (IA) and Thrombogenic (IT) Health Lipid
Indices, of Raw Rose of Blue Fin Tuna (Thunnus L.,) and their Salted Product “Bottarga”.
FNS. 2011, 2, 736–743.
Moawad, R. K.; Mohamed, G. F.; Ashour, M. M. S.; Enssaf, M. A.; El-Hamzy, A. J. Appl. Sci.
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183–02, Ohio State Extension Research: OH, 2001.
Pearson, A. M.; Dutson, T. R. Advances in Meat Research, Restructured Meat and Poultry
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cally Deboned Turkey Meat as Affected by Packaging Parameters and Storage Conditions.
Poultry Sci. 2004, 83, 1240–1248.
Pietrzyk, D. J.; Frank, C. W. Analytical Chemistry, an Introduction Chapters 7 and 15;
Academic Press, Inc.: New York, NY, 1979.
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2-Thiobabituric Acid Values of Pork and Beef During Storage. J. Food Sci. 1970, 35,
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Chicago, Illinois, 2006; Vol. 5.
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CHAPTER 7

APPLICATION OF NATURAL
ANTIOXIDANTS IN DAIRY FOODS
NEELAM UPADHYAY1,*, VEENA N.2, SANKET BORAD1,
ASHISH KUMAR SINGH1, SUMIT ARORA1, and MINAXI3
1
Dairy Technology Division, ICAR-National Dairy Research Institute,
Karnal 132001, Haryana, India
2
Dairy Chemistry Division, College of Dairy Science and Technology,
Guru Angad Dev Veterinary and Animal Sciences University,
Ludhiana 141001, Punjab, India
3
Agricultural Structures and Environmental Control Division, ICAR-
Central Institute of Post- Harvest Engineering and Technology,
Ludhiana 141004, Punjab, India
*
Corresponding author. E-mail: [email protected]

CONTENTS
Abstract .................................................................................................... 262
7.1 Introduction of Milk and Dairy Products ........................................ 262
7.2 Naturally Occurring Oxidants and Antioxidants in Milk and
Dairy Products ................................................................................ 264
7.3 Oxidation in Dairy Products ........................................................... 267
7.4 Quality and Safety Issues of Oxidation .......................................... 271
7.5 Methods of Measurement of Oxidation .......................................... 271
7.6 Characteristics, Types, and Mechanism of action of Antioxidants ...272
7.7 Application of Synthetic Antioxidants and Their Effects ............... 276
7.8 Need of Natural Antioxidants in Dairy Products ............................ 277
7.9 Application of Natural Antioxidants in Various Dairy Products .... 278
7.10 Conclusion ...................................................................................... 287
Keywords ................................................................................................. 288
References ................................................................................................ 288
262 Natural Antioxidants: Applications in Foods of Animal Origin

ABSTRACT

Milk, a nature’s perfect food containing high quality of almost all nutri-
ents (proteins, lipids, carbohydrates, vitamins, and minerals), is a polyphasic
secretion of the mammary glands. It is considered to be one of the indis-
pensable ingredients for the preparation of functional foods due to the pres-
ence of myriad bioactive components. Although, besides containing some
antioxidants (like lactoferrin, α-tocopherol, etc.), milk also contains few
oxidants (like polyunsaturated fatty acids (PUFA) of triglycerides, phospho-
lipids, cholesterol ester, riboflavin, ferri-porphyrin, etc.) that may lead to
development of off-flavors in milk and milk products. Some of the oxidation
products especially of lipid oxidation are toxic and reactive. Thus, several
studies have been conducted on the addition of antioxidants, both synthetic
and natural, in milk and milk products. However, natural antioxidants are
gaining wide acceptance as synthetic antioxidants are associated with poten-
tial health hazards. Also, natural antioxidants have positive health implica-
tions and enhance functionality. Several studies have been conducted on the
applications of the natural antioxidants in milk and several dairy products.
These antioxidants can be added either in the form of the essential oil or
in the form of extract (of polar and non-polar solvents) from skins, seeds,
peels, pomace, bark, or leaf of the natural source. This chapter highlights the
applications of these natural antioxidants in dairy foods.

7.1 INTRODUCTION OF MILK AND DAIRY PRODUCTS

Milk and milk products contribute a very significant proportion in our daily
diet. Milk, considered to be closest to the nature’s perfect food, is an excel-
lent source of calcium, a good source of minerals and high-quality proteins,
the only source of lactose, and a source of lipids, the most valuable compo-
nent, which also forms the basis of milk pricing (Upadhyay et al., 2014).
The milk proteins are a good source of essential amino acids and have a high
biological value. The majority of nitrogen in milk is distributed in casein
(around 80%) which is present as colloidal dispersions called as casein
micelles; and whey proteins (around 20%) which are present as true solu-
tion. However, in addition to these proteins, milk also contains two other
groups of proteins or protein-like materials, the proteose-peptone fraction
and the non-protein nitrogen (NPN) fraction (Fox & McSweeney, 1998).
As per the protein quality ranking, casein and whey proteins are reported to
have protein efficiency ratio, biological value, net protein utilization, and
Application of Natural Antioxidants in Dairy Foods 263

protein digestibility corrected amino acid score (PDCAAS) as 2.5, 3.2; 77,
104; 76, 92; and 1.00, 1.00, respectively, while these values for milk are 2.5,
91, 82, and 1.00, respectively. As can be noticed, the PDCAAS for casein,
whey protein and milk is 1.00 which is highest for a protein (Puranik & Rao,
1996; Sarwar, 1997; United States Dairy Export Council, 1999; Hoffman
& Falvo, 2004). The biological function of casein is to carry calcium and
phosphate. It forms a gel or clot in the stomach which leads to continuous
but slow release of amino acids into the blood stream (Boirie et al., 1997;
Hoffman & Falvo, 2004). Some bioactive peptides are also released from
casein on digestion that have physiological significance like, antithrombotic
peptides, antihypertensive peptides, opioid peptides, immune modulatory
peptides, antimicrobial peptides, casein phosphopeptides, glycomacropep-
tides, and so forth. Unlike casein, the plasma appearance of amino acids is
fast, high, and transient upon ingestion of whey proteins (Boirie et al., 1997;
Hoffman & Falvo, 2004). Whey proteins are rich source of branched chain
amino acids (BCAAs) that are metabolized directly into the muscle tissue
leading to replenishment of the exhausted levels followed by repairing and
rebuilding of lean muscle tissue. These are, thus important for athletes and
alike. Whey proteins are also rich and balanced source of sulfur-containing
amino acid cysteine that help boost body’s antioxidant levels as cysteine,
along with glycine and glutamic acid is a precursor of glutathione which is
the potent intracellular antioxidant and it gets oxidized to glutathione disul-
fide (GSSG) (oxidized glutathione) leading to removal of reactive oxygen
species (ROS), thus regulating the level of ROS in the cells (Haug et al.,
2007; Smithers, 2008).
The milk lipids, similar to the milk proteins, are important dietary
components for supplying nutrients as contain certain bioactive components
like short chain fatty acids, conjugated linoleic acid (CLA), branched chain
fatty acids, and so forth, and are present as emulsified droplets in globules
coated with membrane. Butyric acid (4:0) is reported to be a modulator
of gene function (German, 1999), besides inhibiting colon and mammary
tumors (Parodi, 2003). Caprylic (8:0) and capric (10:0) acids are reported
to have antiviral activities; however, caprylic acid is also reported to delay
tumor growth (Thormar et al., 1994). Sun et al. (2002) reported that lauric
acid (12:0) may have antiviral and antibacterial function, while Schuster
et al. (1980) reported that it may act as an anticaries and antiplaque agent.
An interesting observation was reported by Henry et al. (2002) that capric
and lauric acid inhibit cyclooxygenase, that is, COX-I and COX-II. Milk
fat naturally contains CLA which is shown to be anticarcinogenic and anti-
atherogenic and it has effects on body composition and fat metabolism
264 Natural Antioxidants: Applications in Foods of Animal Origin

(Ip et al., 1999; Pariza et al., 1999; Truitt et al., 1999; Benjamin & Spener,
2009). Phytanic acid, a branched chain fatty acid presents in milk fat, has
been shown to increase insulin-independent glucose uptake by cells and to
decrease liver triglyceride accumulation in some mouse models (Hein et al.,
2002; Hellgren, 2010; Palmquist, 2010). Besides fat and protein, milk is
naturally a good source of lactose and contains ~5% w/w. Lactose, present
as true solution, promotes the absorption of calcium like other sugars and
it is a ready source of energy, providing 30% of the caloric value of bovine
milk. Also, it accounts for 50% of the osmotic pressure of milk, which is
isotonic with blood and hence is essentially constant (Fox & McSweeney,
2009). The minor components present in milk include minerals (calcium,
selenium, iodine, magnesium, and zinc) and vitamins (vitamin A, vitamin
E, riboflavin, folate, and vitamin 12) whose nutritional significance is well
established.
Milk and other dairy products have been undoubtedly considered as
nature’s perfect functional foods owing to the presence of wide myriad of
bioactive components. Milk has been advocated by the food formulators for
the development of novel dairy foods because of its richness in nutrients
and bioactives and molecules that assist in development of excellent sensory
characteristics. Since time immemorial milk and milk nutrients have been
utilized for the commercial manufacture of numerous products like cream,
butter, butter oil, cheese, condensed milk, dried milks, and indigenous dairy
products that contain components only from milk. Besides this, certain other
food products are prepared from milk where milk components form the
major ingredients like kheer, khoa/mawa, rabri, khurchan, kulfi, shrikhand,
ice cream, and so forth. Probably, it is the only raw material that has been
exploited to such a great extent, not only for the manufacture of value added
food products, but also to harness the valuable ingredients for food and phar-
maceutical sector.

7.2 NATURALLY OCCURRING OXIDANTS AND ANTIOXIDANTS


IN MILK AND DAIRY PRODUCTS

The different dairy food components, namely polyunsaturated fatty acids


(PUFA), proteins, vitamins, and pigments undergo oxidative changes, which
could be both desirable and undesirable, during processing and storage.
The rate and extent of oxidation depends on the concentration and activity
of oxidizing agents, which may form the part of food naturally or used in
the food product as ingredient or may be formed during processing and
Application of Natural Antioxidants in Dairy Foods 265

subsequent storage. Thus, the knowledge of the principal oxidants and anti-
oxidants occurring in the foodstuff is indispensable for understanding the
oxidative reactions that may occur in the food components and their implica-
tion on food quality and safety. Moreover, it is must to have the information
pertaining to enzymatic and non-enzymatic catalysts of oxidation.
The various oxidants present in the dairy products include PUFA attached
to triglycerides, phospholipids, cholesterol esters, riboflavin, proteins, and
so forth. Phospholipids are considered to be pro-oxidant because of the pres-
ence of monounsaturated and PUFA group attached to diglyceride or sphin-
gosine in it (Table 7.1). However, Chen and Nawar (1991) reported that
phospholipids can act as either pro-oxidant or antioxidant in dairy products
depending on the pH and ratio of water to phospholipid species in it. Simi-
larly, the thiol groups (-SH) in milk are reported to act as pro-oxidant or anti-
oxidant depending upon the conditions. Yee and Shipe (1982) observed in a
model system, free thiol groups in the presence of copper promoted oxidation
of emulsified methyl linoleate, while it acted as antioxidant component in
the presence of haem. Milk and milk products may contain metallic compo-
nents (such as transition metal ions- cupric, ferric, and haem proteins) that
normally act as pro-oxidant by catalyzing the decomposition of preformed
hydroperoxides to initiate the new oxidation chain reactions (Labuza, 1971;
Korycka-Dahl & Richardson, 1980; Pokorny, 1987). The ferri-porphyrin
proteins, together with their juxtaposition with lipids in the milk fat globule
membrane (MFGM) have powerful pro-oxidative properties (Kendrick &
Watts, 1969; O’Connor & O’Brien, 2006). Juxtaposition of copper-protein
complex with the phospholipids of the MFGM is also an important factor
in development of oxidized flavor in liquid milk (Samuelsson, 1966). The
water-soluble vitamin, that is, riboflavin, is a potent photosensitizer and
is associated with photo-oxidation of milk. Besides riboflavin, porphyrins
and chlorophylls are also reported to be involved in the photo-oxidation
of certain milk products like cheese (Wold et al., 2005). It is reported that
concentrations of ascorbic acid above those in normal milk (~20 mg/L)
could provide antioxidant protection; however, at the normal concentra-
tions in milk, ascorbic acid acts as a pro-oxidant (O’Connor & O’Brien,
2006). Caseins are more effective as antioxidant than whey proteins. The
antioxidant activity of casein may be attributed to the hydrophobic nature
of the same (Taylor & Richardson, 1980; Eriksson, 1982) and orientation
of potential antioxidant side-chains of constituent amino acids at the lipid
interface. Among the whey proteins, lactoferrin has also been reported to
inhibit peroxidation induced by Fe2+ by chelating it (Gutteridge et al., 1981;
Allen & Wrieden, 1982).
266 Natural Antioxidants: Applications in Foods of Animal Origin

TABLE 7.1 Oxidants and Antioxidants in Milk.


Oxidants Antioxidants Both (oxidants and
antioxidants)
Polyunsaturated fatty acids of Casein Phospholipids
triglycerides, phospholipids,
cholesterol esters
Riboflavin Lactoferrin Thiol group
Proteins α- tocopherol
Transition metal ions- cupric, ferric Carotenoids (β-carotene)
Haem proteins Maltol (in heated milk)
Ferri-porphyrin
Copper-protein complex with
phospholipids
Xanthine oxidoreductase
Sulfhydryl oxidase

Milk also contains appreciable amount of antioxidants. Vitamins


including α-tocopherol, principal tocopherol of the eight vitamers of the
vitamin E, act as a free radical scavenger. The concentration of α-tocopherol
in cow’s milk ranges from 3.0 to 5.0 mg/L (Hendricks & Guo, 2006).
α-tocopherol is also reported to quench singlet oxygen (1O2) via a charge-
transfer scavenging mechanism (Yamauchi & Matsushita, 1977; Burton
& Ingold, 1981). Vitamin A and carotenoids act as antioxidant due to the
hydrophobic chain of polyene units that quench 1O2, neutralize thiyl radi-
cals and combine with peroxyl radicals and stabilize the same (Palace et
al., 1999). Beta-carotene also acts as antioxidant in milk. It is reported to
offer good protection against light-induced lipid oxidation and it inhibits
the chlorophyll-sensitized photo-oxidation of methyl linoleate (Terao et al.,
1980). In addition, it provides protection against riboflavin degradation by
competing for absorption of light. However, this protective effect disappears
outside the absorption band of carotene (i.e., <366 nm) as the protective
mechanism is not a quenching effect of radical or 1O2, but absorption (filter)
effect of the incident light (Hansen & Skibsted, 2000). Some of the indig-
enous enzymes of milk also act as antioxidants, while others may play a role
in promoting the lipid oxidation like Xanthine oxidoreductase. Sulfhydryl
oxidase is proposed to cause oxidation of thiols in ultra-high temperature
(UHT) milk, in conjunction with lactoperoxidase (to destroy the resultant
Application of Natural Antioxidants in Dairy Foods 267

H2O2) by doing away with the pro-oxidants resulting from auto-oxidation of


thiols (Swaisgood & Abraham, 1980).
The products and by-products obtained from processing of milk can also
serve as potent antioxidant. Steps associated with preparation of cream and
during pre-heating of milk for milk powder manufacture, the antioxida-
tive activity gets increased due to enhanced activation of free sulfhydryl
groups (Farkye, 2006; Keogh, 2006). It is proposed that buttermilk solids are
effective in reducing the severity of lipid oxidation during chain propaga-
tion stage; however, it is ineffective in delaying the onset of lipid oxidation.
Thus, incorporation of buttermilk solids into the food matrices can be an
approach to stabilize against lipid peroxidation (Wong & Kitts, 2003). The
antioxidant activity of buttermilk may be attributed to the presence of sulf-
hydryl content. Few of the workers (Dugan, 1980; Eichner, 1980; Eriksson,
1982) reported that carbonyl-amine reactions between lactose and milk
proteins produce intermediates having potent antioxidative activity and may
play role in stabilization of milk fat (Wyatt & Day, 1965). Maltol, an impor-
tant flavor component in the heated milk also exhibit powerful antioxidant
activity (O’Brien, 2009). However, it is also reported that browning reac-
tion products may exert anti-nutritional and toxicological effects (O’Brien
& Morrissey, 1989). Thus, a balance between pro-oxidant and antioxidant
factors is critical for the oxidative stability of milk (Stapelfeldt et al., 1999;
Morales et al., 2000).

7.3 OXIDATION IN DAIRY PRODUCTS

Oxidation of dairy products involves the addition of oxygen atom to or the


abstraction of hydrogen atom from the different chemical moieties present
in the milk, which is further associated with conversion of primary hydroxyl
groups (alcohols) to aldehyde and finally to carboxylic acid functionality by
either chemically or biochemically mediated oxidation. In few instances, the
oxidation reaction is desirable and may lead to an improvement in the product
quality, such as oxidative cross-linking of proteins to manipulate viscosity
and gelation in dairy products. However, in majority of the cases, food oxida-
tion leads to decrease in consumer acceptance, minimizes the shelf life of the
product and in few may even be associated with formation of anti-nutrients
and toxicity as well. The two principal types of oxidation that contribute to
food deterioration are autoxidation of unsaturated fatty acids and enzyme-
catalyzed reaction. The residues susceptible for oxidation are also present on
proteins and carbohydrates, besides being present on lipids. Radicals formed
268 Natural Antioxidants: Applications in Foods of Animal Origin

on oxygen or sulfur (on proteins) and oxygen excited-state species (1O2)


generated by heat, light, or metal catalyzed reactions target double bonds
and generate reactive forms of these substrates (homoradicals). Subsequent
cross-reactivity of intermediate species produces complex product matrices
including auto-oxidative molecular products (Bennett et al., 2013).
In a mixed food system, lipids are considered to be most vulnerable to
oxidation and also to initiate attack on non-lipid substances. In milk and
dairy products, lipids, besides serving as the precursor of certain flavorful
compounds like methyl ketones (Kinsella, 1969a) and lactones (Wadhwa
& Jain, 1985), also induce the formation of few undesirable compounds
that cause off-flavor defects via hydrolytic and oxidative rancidity. The
oxidative flavors are products of autoxidation of unsaturated fatty acids of
triglycerides, phospholipids, and cholesterol esters, which are essentially a
free-radical chain reaction involving initiation, propagation, and termina-
tion stages. Unsaturated fatty acids oxidize to form unstable odorless and
tasteless hydroperoxide that degrade to yield flavorful carbonyls and other
compounds (O’Connor & O’Brien, 2006). It is assumed that during autox-
idation, initially oxygen attaches itself in loose linkage to a double bond
of the unsaturated fatty acid (Gunstone & Hilditch, 1945). This results in
an activation of the adjacent (α-) methylene group, from which a hydrogen
atom or proton gets loosened leading to drawing the oxygen atom towards
it to form hydroperoxide and not peroxide, the process creates a new double
bond at a different place; which has a trans configuration as opposed to
the cis configuration of natural unsaturated fatty acids. Thus, the reaction
proceeds via free radical mechanism. It is reported that cow ghee has lesser
tendency in comparison to buffalo ghee for autoxidation because cow ghee
absorbs oxygen slowly in comparison to buffalo ghee (Rangappa & Achaya,
1975). The oxidized unsaturated fatty acids, particularly oleic, linoleic, lino-
lenic, and arachidonic acid lead to formation of n-alkanals, alk-2-enals, and
alk-2,4-dienals. The type of off-flavor perceived depends upon the quality
and quantity of carbonyls formed. Kinsella (1969b) reported that C7–C10
alkanals possess oily and tallowy odor, C7–C11 alk-2-enals exhibit oxidized
painty odors, alkanals (C5, C6, and C8) and alk-2-dienals may exhibit nutmeg
spicy odors. The potent flavor compound oct-1,3-diene produced by auto-
oxidation of linoleic acid is responsible for metallic flavor. Unsaturated
alcohol pent-1-ene-3-ol produced from linoleic acid is responsible for an
oily and grassy aroma (Forss, 1972).
Riboflavin has been traditionally considered as an active photosensi-
tizer occurring naturally in dairy products like, milk, cheese, butter and its
presence makes these products susceptible to photosensitized oxidation.
Application of Natural Antioxidants in Dairy Foods 269

Photosensitizers are the substances that absorb light and become excited to
one or more higher energy-rich state(s). They promote the photo-oxidation of
diverse substrates, when foods are exposed to visible light (Dalsgaard et al.,
2007). Photosensitizers are reported to have two excited states: singlet and
triplet. The triplet-excited state has a longer lifetime and initiates the oxida-
tion. Photo-oxidation by a photosensitizer can proceeds through two types of
reactions, that is, either type I or type II. In type I reaction, the excited sensi-
tizer (Sen*) undergoes internal reactions that ultimately results in the oxida-
tive alteration of a second molecule primarily by free radical mechanism on
the exposure of the primary substrate to UV radiation. In type I reactions,
transfer of hydrogen atoms or electrons occurs via interaction of the triplet
excited state of the sensitizer with the target, while in the type II reactions,
the excited triplet sensitizer reacts with ground state oxygen to produce 1O2
by energy transfer. 1O2 is a strong oxidant because of its higher reactivity and
has the potential to damage proteins, lipids, and so forth (Airado-Rodríguez
et al., 2011). The two reactions can occur simultaneously, in a competitive
mode (Spikes, 1988) and the same has been observed in milk (Lee & Min,
2009). However, at low oxygen concentrations, type I reactions are most
common. After photodegradation, riboflavin is reported to break down to
lumiflavin (under alkaline condition) or lumichrome (under acidic condi-
tions) (Ahmad et al., 2006) and probably formylmethylflavin. Among these,
lumichrome is reported to be a strong photosensitizer (Parks & Allen, 1977).
Riboflavin has three absorption bands. The band with maxima between 430
and 460 nm is the main band responsible for the photo-oxidation of food,
especially milk and dairy products (O’Connor & O’Brien, 2006). Wold et al.
(2006) reported the presence of five photosensitizers in butter other than ribo-
flavin: protoporphyrin, hematoporphyrin, a chlorophyll a-like molecule, and
two unidentified tetrapyrroles. Chlorophyll and porphyrin molecules absorb
light in the UV and violet region with absorption peaks of ~410 nm (the
soret band) along with the absorption of light in the red above 600 nm, and
therefore, they may be responsible for the formation of off-flavors in dairy
products when exposed to light having wavelengths longer than 500 nm
(Wold et al., 2006). Chlorophyllic compounds have also been suggested to
contribute prominently to the major part of photo-oxidation in cow’s milk
(Airado-Rodríguez et al., 2011).
Proteins are very complex molecules organized in large structures and
oxidation of proteins may have severe consequences on product quality,
their functionality and nutritional qualities like loss of essential amino acids.
Structurally, protein oxidation may lead to a number of modifications either
on its side chains or on the backbone, including amino acid changes, protein
270 Natural Antioxidants: Applications in Foods of Animal Origin

cleavage, and promotion of cross-linking. Protein oxidation can be classi-


fied as direct or indirect oxidation. Direct oxidation involves attack of the
radical species or hydrogen abstraction from protein leading to formation of
a protein radical or anion. However, in indirect oxidation, the protein inter-
acts with other components like secondary lipid oxidation products, such as
aldehydes or reducing sugars, which may lead to the formation of carbonyl
compounds on amino acid side chains or formation of protein carbonyl
groups (Baron, 2013). Oxidation of proteins at the side chain often leads to
development of protein carbonyls, alcohols, and peroxides. In dairy prod-
ucts, methionine sulfoxide, dityrosine and cysteic acid have been detected
as the oxidation products of methionine, tyrosine, and cysteine, respectively
(Toran et al., 1996; Østdal et al., 2000).
It is believed that metal-catalyzed oxidation of protein is a predominant
mechanism in foods, because of abundance of transition metals, such as iron
and copper in the food matrix. Østdal et al. (2000) showed that in milk,
the lactoperoxidase can transfer a radical to other milk proteins, such as
β- lactoglobulin, casein, and serum albumin and has been suggested to be a
key element in oxidation of milk proteins. Milk proteins are also reported
to be susceptible to photo-oxidation. Among milk proteins, casein is more
susceptible to photo-oxidation than the globular proteins, α-lactalbumin,
β-lactoglobulin, and lactoferrin (Baron, 2013).
Various dairy products are reported to show flavor defects due to the
occurrence of oxidation of different components in the products like fat
phase in ice cream besides the positive effects on the flavor, is also reported
to be associated with the flavor defect like cardboard, painty, metallic, or
oxidized that may be due to the auto-oxidation or lipolysis of fat (Marshall
et al., 2003). Auto-oxidation of fat is also reported in milk powders and the
dry milk deposits may self-ignite to cause explosion in milk powder facto-
ries (Walker & Jackson, 1978; Knipschildt, 1986). The oxidation of milk
fat and the reducing sugar-protein browning (i.e., Maillard browning) are
the most significant deteriorative changes that occur in dry milk products
that result in the defects in sensory and functional properties of the product
(Farkye, 2006). Stapefeldt et al. (1997) reported that low-heat powders are
more susceptible to severe oxidative changes than medium-heat and high-
heat powders during storage. The unsaturated fatty acids in whole milk
powder oxidize to generate saturated aldehydes (Boon et al., 1976) which
are responsible for stale, tallow, cardboard, and painty flavors, while polar
lipids oxidize to generate unsaturated aldehydes and ketones which are
responsible for oxidized flavor even at a very low level (Kinsella, 1969a;
Keen et al., 1976; Hall & Anderson, 1985).
Application of Natural Antioxidants in Dairy Foods 271

As mentioned earlier, light also influences milk flavor due to riboflavin-


sensitized effects on milk proteins via oxidation of methionine to methional
(3-methylthiopropionaldehyde) (Patton, 1954; Tada et al., 1971; Sattar et
al., 1977) and also oxidation of other amino acids in the presence of light
and riboflavin. Allen and Parks (1975) reported that exposure of milk serum
to fluorescent light chemically modified 10 amino acids in immunoglobu-
lins. Dimick (1976) reported the considerable loss (~80%) in the activity of
milk lipase due to riboflavin-photosensitized oxidation when the milk was
exposed to sunlight for 30 min. Light- and riboflavin-induced changes in
cheese have also been reported (Deger & Ashoor, 1987).

7.4 QUALITY AND SAFETY ISSUES OF OXIDATION

The secondary oxidation products of lipid oxidation especially α,


β-unsaturated aldehydes, such as 4-hydroxynonenal (4-NHE) or trans-4-
hydroxy-2-hexanal (HHE) are reported to be toxic and very reactive. These
can easily react to proteins or peptides. The interactions between sugar/
lipid oxidation/degradation products and protein often result in addition
of carbonyl groups to protein via covalent binding between the advanced
glycation end products (AGEs), carboxymethyllysine was first AGE to be
identified in milk by Tauer et al. (1999) or advanced lipid oxidation end
products (ALE). There are evidences of the role of these interactions on the
pathologies of several diseases (Baron, 2013).

7.5 METHODS OF MEASUREMENT OF OXIDATION

The methods of detection and measurement of the extent of oxidation,


pertaining to the oxidation of lipid, in the dairy foods are mainly associ-
ated with the measurement of hydroperoxides which is one of the classical
methods for quantifying the lipid oxidation. A number of methods are based
on iodometric titration, that is, hydroperoxides oxidize and liberate iodine
from potassium iodide, the test commonly referred to as peroxide value
determination or spectrophotometric methods, that is, hydroperoxides
oxidize the ferrous to ferric iron in the presence of ammonium thiocyanate
to produce ferric thiocyanate, which can be spectrophotometrically quanti-
fied at 505 nm (Loftus Hills & Thiel, 1946), also hydroperoxides of the
oxidized fat reacts with 1,5-diphenyl-carbohydrazide to yield red-colored
products (Hamm et al., 1965). The progress of auto-oxidation is measured
272 Natural Antioxidants: Applications in Foods of Animal Origin

spectrometrically at 532 nm by the reaction based on the condensation


of two molecules of thiobarbituric acid (TBA) with one of the end prod-
ucts of auto-oxidation, malondialdehyde which results in the red-colored
complex (Dunkley & Jennings, 1951). The other traditional methods
include m-phenylenediamine test, IR value, Anisidine value, Kreis test (for
aldehydes), methods based on the carbonyl content of oxidized fats, and
measurement of oxygen uptake by manometry or polarography (Henick et
al., 1954; Tappel, 1955; Mehlenbacher, 1960). Recent methods of measure-
ment include the use of instruments like electron spin resonance (ESR)
spectrometry, static and dynamic GC/MS, head space GC/MS (electronic
nose), and high performance liquid chromatography (HPLC) (Kim & Morr,
1996; Nielsen et al., 1997; Stapelfeldt et al., 1997; Kristensen & Skibsted,
1999). Certain tests are employed to determine the proneness of the fat to
autoxidize that can be measured by studying the degree of resistance that a
fat bears to autoxidation and it can be seen by iodine value, active oxygen
method, rancimat method, induction period (by measuring manometric
reading for macro work and by using Warburg apparatus for micro studies),
and so forth.
Photoreactions in milk can be measured by monitoring the formation of
1
O2 as a function of light exposure using 1O2 fluorescent probe. One of the
probes, available under the trade name singlet oxygen sensor green (SOSG)
reagent, is highly selective for 1O2, and it does not show any appreciable
response to hydroxyl radical or superoxide. SOSG is reported (Molecular
Probes, 2004; Airado-Rodríguez, 2011) to emit weak blue fluorescence
peaking at 395 and 416 nm for excitation at 372 and 393 nm. After reaction
with 1O2, it emits a green fluorescence similar to that of fluorescein (excita-
tion/emission maxima ~504/525 nm) that is measured.

7.6 CHARACTERISTICS, TYPES, AND MECHANISM OF ACTION


OF ANTIOXIDANTS

Antioxidants are the substances that inhibit, retard, or interfere with the
formation of free radicals in fat-rich foods, thus terminating the oxidative
reaction in its initial stage. From a practical standpoint, it means that when
an antioxidant system is properly selected and correctly applied to meet
the needs of a particular food item, will help to maintain the original fresh-
ness, flavor, and odor of the product for a longer period of time than would
otherwise be possible. In the food industry, a substance having the technical
function of delaying the oxidation of nutrients, such as lipids, sugars, and
Application of Natural Antioxidants in Dairy Foods 273

proteins, whose oxidation leads to an inevitable deterioration of the organo-


leptic qualities of a food due to the formation of undesirable substances like
aldehydes, ketones, and organic acids that yield off-flavors, is considered
to be an antioxidant (Saad et al. 2007; Andre´ et al. 2010). However, to
be used in foods, antioxidants must be nontoxic, inexpensive, effective at
low concentrations (0.001–0.02%), capable of surviving processing (carry-
through), stable in the finished products, and devoid of undesirable color,
flavor, and odor effects (Shahidi & Zhong, 2010). These days, there is an
extensive use of natural and synthetic antioxidants in the food and pharma-
ceutical industry.
Antioxidants can be divided into primary or chain-breaking antioxidants
and synergists or secondary antioxidants based on their mechanisms of
action. Primary antioxidants include hindered phenols and secondary aryl
amines, while secondary antioxidants include organophosphites and thioes-
ters. All the primary antioxidants commonly used in foods, have either two—
OH groups or one—OR group in the ortho or para positions (Hudson, 1990).
They are effective at extremely low concentrations of 0.01% or less and for
some of them the effectiveness decreases as concentration is increased. It
is reported that at high concentrations, they may act as pro-oxidant due to
their involvement in the initiation reactions (Cillard et al., 1980). Phenolic
(primary) antioxidants, whether naturally occurring, for example, tocoph-
erols or flavonoids or permitted synthetic compounds, such as hindered
phenolic (e.g., BHT, BHA, and TBHQ) and polyhydroxy phenolic (e.g.,
gallates), inhibit chain reactions by acting as hydrogen donors or free radical
acceptors, resulting in the formation of more stable products. They interfere
directly with the free radical propagation process and thus block the chain
reaction.
Secondary antioxidants or synergist can be accounted for metal chela-
tion (Khokhar & Owusu Apenten, 2003). They have little direct effect on
the autoxidation of lipids but are able to enhance considerably the action
of primary antioxidants. Chelating agents and sequestering agents like
citric acid and isopropyl citrate, amino acids, phosphoric acid, tartaric
acid, ascorbic acid and ascorbyl palmitate, ethylenediaminetetraacetic acid
(EDTA) chelate metallic ions (such as copper and iron) that promote lipid
oxidation through a catalytic action. The chelators are referred to as syner-
gists since they greatly enhance the action of phenolic antioxidants. Thus,
antioxidants slow down the oxidation rates of foods by a combination of
mechanisms like, scavenging free radicals; chelating pro-oxidative metals;
quenching 1O2 and photo-sensitizers, and inactivating lipoxygenase (Thorat
et al., 2013). The effectiveness of antioxidants to scavenge free radicals
274 Natural Antioxidants: Applications in Foods of Animal Origin

in foods depends on the bond dissociation energy between oxygen and a


phenolic hydrogen, reduction potential, and delocalization of the antioxi-
dant radicals (Choe & Min, 2006; Cao et al., 2007). Phenolic compounds
primarily inhibit lipid oxidation through their ability to scavenge free radi-
cals and convert the resulting phenolic radicals into a low-energy form
that does not further promote oxidation. Flavonoids are known to exhibit
a strong metal chelating activity in addition to their antioxidant properties,
with the arrangement of 4-keto and 5-OH, or 3' and 4'-OH substituents
resulting in the formation of chelating complexes between flavonoids and
divalent cations (Cheng & Breen, 2000). Carotenoids with nine or more
conjugated double bonds are good 1O2 quenchers by energy transfer. The
1
O2 quenching activity of carotenoids depends on the number of conju-
gated double bonds in the structure (Min & Boff, 2002; Foss et al., 2004)
and the substituent in the β-ionone ring (Di Mascio & Sies, 1989). Beta-
carotene and lycopene which have 11 conjugated double bonds are more
effective 1O2 quenchers than lutein which has 10 conjugated double bonds
(Viljanen et al., 2002). The reaction mechanisms of a primary antioxidant,
AH (Antunes et al., 1999) and secondary antioxidant BH, is shown in
Figure 7.1.

a) Reaction of primary antioxidant (AH) with lipid radical


AH + ROO•  ROOH + A•
RH + A•  AH + A•
AH + ROO•  [ROO•AH] Complex

b) Termination reaction
[ROO•AH]  non-radical product
A• + A•  AA
A• + R•  RA
A• + ROO•  ROOA

c) Regeneration of primary antioxidant


A• + BH  AH + B•
FIGURE 7.1 Reaction mechanism of primary antioxidant with free radical. AH, antioxidant;
ROO•, lipid peroxyl radical; ROOH, hydroperoxide; A•, antioxidant free radical; RH,
unsaturated lipid; R•, lipid radical; ROO• AH, stable compound (non-radical product); BH,
secondary hydrogen donor; B•, secondary antioxidant free radical (Antunes et al., 1999).
Application of Natural Antioxidants in Dairy Foods 275

Antioxidants are naturally present in a wide variety of raw food mate-


rials; still there is a need to add antioxidants into foods so as to provide
additional protection against oxidation. The antioxidants which are added
to food products can be natural or synthetic compounds depending on their
availability and preparations (Yanishlieva-Maslarova, 2001). Natural anti-
oxidants such as polyphenols are primarily derived from plants, while the
synthetic antioxidants are chemically produced. Antioxidants containing a
phenol group play a prominent role in biological and food system (Shui &
Leong, 2004).
The naturally occurring antioxidant substances are at times associated
with the beneficial effects of foods (Vision et al., 1999). They are avail-
able in complex forms, which include tocopherols, lycopenes, flavonoids,
nordihydroguaiaretic acid (NDGA), sesamol, gossypol, vitamins, provita-
mins and other phytochemicals, enzymes (catalase, glutathione peroxidase,
and super oxide dismutase), minerals (Zinc and Selenium), and lecithin
(Cuppett, 2001). α-tocopherol (vitamin E) is well known as one of the most
efficient naturally occurring lipid-soluble antioxidants (McCarthy et al.,
2001). The most important natural antioxidants commercially exploited are
tocopherols, ascorbic acid and more recently plant extracts, such as, from
sage (Djarmati et al., 1991), rosemary (Tena et al., 1997), green tea (Chen
et al., 2004), spinach (Aehle et al., 2004), grape (Baydar et al., 2004), and
marigold (Cetkovic et al., 2004) are also gaining acceptance. These extracts
contain mainly phenolic compounds (e.g., flavonoids and phenolic acids),
and they are well known for their antioxidant (López et al., 2001; Gülçin et
al., 2003), antimicrobial (Gülçin et al., 2003), anti-ulcer, anti-carcinogenic
(Chen et al., 2004), anti-mutagenic, and anti-inflammatory (Caillet et al.,
2006) properties, as well as for reducing the risk of cardiovascular diseases
(Cetkovic et al., 2004; Louli et al., 2004).
The antioxidant effects in milk rely primarily on endogenous compounds
(Brien & Connor, 2003). However, synthetic antioxidant compounds are
also widely used to inhibit progress of lipid oxidation. Some of the popular
synthetic antioxidants used in many countries including India are Butylated
Hydroxyanisole (BHA), t-butylhydroquinone (TBHQ), and esters of gallic
acid (Yanishlieva-Maslarova, 2001). These are mainly phenolic compounds
whose structure allows them to form low-energy radicals through stable
resonance hybrids that prevent the further propagation of the oxidation
reaction (Karovicova & Simko, 2000). Butylated hydroxytoluene (BHT) is
very effective in animal fats, low-fat food, fish products, packaging mate-
rials, paraffin, and mineral oils but is less effective in vegetable oils, and is
276 Natural Antioxidants: Applications in Foods of Animal Origin

reported to be lost during frying because of its steam volatility (Gordon &
Kourimska, 1995).

7.7 APPLICATION OF SYNTHETIC ANTIOXIDANTS AND THEIR


EFFECTS

The synthetic antioxidants are lipid soluble and terminate free-radical


chain reactions by donating hydrogen atoms or electrons to free radicals
and thus, converting them to more stable structures (Frankel, 1998). The
use of synthetic antioxidants in the prevention or retardation of autoxida-
tion in lipids and lipid containing food products has been the subject of
numerous investigations. The use of synthetic antioxidants in dairy products
is prohibited in most countries. However, certain studies on the use of these
in dairy products reveal that their effectiveness varies in different products.
While NDGA inhibits the development of oxidized flavor in liquid milk, it
promotes autoxidation in milk fat (Hammond, 1970). Tocopherols are very
effective inhibitors of spontaneous or copper-induced oxidation in liquid
milk (Dunkley et al., 1967; King, 1968) but have little effect in whole milk
powder (Abbot & Waite, 1965). Other antioxidants that have been shown to
exert protective effects are dodecyl gallate in spray-dried whole milk (Abbot
& Waite, 1962), ascorbyl palmitate in lactic butter (Koops, 1964) and propyl
gallate and quercetin in butter oil (Wyatt & Day, 1965). Anhydrous bovine
or buffalo milk fats (ghee) may be stabilized when stored in a hot climate by
combinations of phenolic antioxidants (BHA, BHT, and propyl gallate) and
ascorbic acid (Helal et al., 1976). Wade et al. (1986) reported that BHA and
BHT were effective in retarding oxidation of anhydrous milk fat but DL-α-
tocopherol acted as a pro-oxidant.
There are a number of controversies surrounding the use of synthetic
antioxidants. Since food additives are subjected to the most stringent toxi-
cological testing procedures, only a few synthetic antioxidants have been
used in foods for any length of time. Since the toxicity of some synthetic
antioxidants is not easily assessed, as a result of which, a chemical may be
considered safe by a country, tolerated in another country and forbidden in
a third one (Thorat et al., 2013). For example, TBHQ is authorized as an
antioxidant in the United States, while it is forbidden in the European Union
countries. The legal limit for the addition of BHA and BHT to most foods in
the United States is 200 mg/kg of fat. When added in combination, a total of
200 mg/kg of fat is permitted (CFR, 2001). In India, according to the Food
Safety and Standards Regulations (2011), no antioxidant other than lecithin,
Application of Natural Antioxidants in Dairy Foods 277

ascorbic acid, and tocopherol shall be added to any food. However, in ghee
and butter, BHA may be added in a concentration not exceeding 0.02%. It is
interesting to note that the addition of these artificial chemicals is restricted
by the FDA because of food safety concerns, not to mention emerging trends
for consumer preferences toward more “green” food processing applica-
tions (Yue et al., 2008). BHA has been revealed to be carcinogenic in animal
experiments. Similarly, at high doses, BHT is reported to cause internal and
external hemorrhaging, leading to death in some strains of mice and guinea
pigs (Ito et al., 1986). Natural antioxidants are, thus, generally recognized as
safe when used in accordance with food manufacturing practices and there-
fore not limited in most foods (CFR, 2001). The addition of α-tocopherol,
ascorbic acid, and ascorbyl palmitate to milk is permitted and no legal limit
exists for the use of the same. However, the presence of these must be noted
on the label and the same must not be used in higher concentrations as it may
lead to pro-oxidative effects (Frankel, 1998).

7.8 NEED OF NATURAL ANTIOXIDANTS IN DAIRY PRODUCTS

The potential health hazards of synthetic antioxidants have prompted


researchers to search for natural antioxidants from plant source (Lee & Shib-
amoto, 2002). Additionally, recent trends in the marketplace have focused
on natural and organic products that do not utilize synthetic additives which
have further spurred this research. Foods manufacturers have also been
motivated to carry out research on the use of natural antioxidants because
studies have shown that such compounds are not only beneficial to the shelf
life of food products but also as preventive medicine. Reports revealing
toxic and carcinogenic effects of BHA and BHT; and the higher manufac-
turing costs and lower efficiency of natural antioxidants such as tocopherols,
together with the increasing consciousness of consumers with regard to food
additive safety, created a need for identifying alternative natural and prob-
ably safer sources of food antioxidants (Wanasundara & Shahidi, 1998; De
Oliveira et al., 2009; Prasad et al., 2009; Gutteridge & Halliwell, 2010).
The replacement of synthetic antioxidants by natural ones may have benefits
due to health implications and functionality of the natural antioxidants such
as solubility in both oil and water that could be of interest in preparations
of emulsions in food systems. Natural antioxidants from plant products are
reported to be more effective in reducing ROS levels compared to synthetic
single dietary antioxidants due to the synergistic actions of a wide range of
biomolecules such as vitamins C and E, phenolic compounds, carotenoids,
278 Natural Antioxidants: Applications in Foods of Animal Origin

terpenoids, and phytomicronutrients (Podsędek, 2007; Serrano et al., 2007;


Pérez-Jiménez et al., 2008).
The interest for the natural antioxidants can be explained either because
of their capacity to ameliorate the quality of food and cosmetic products,
and also more recently because of their potential role in vivo against free
radicals, via feeding (health-food notion) or medication. The great diversity
of antioxidant sources observed in the plant world can be explained for the
phenolic compounds are largely expanded, and are found in every part of
plant: flowers, fruits, grains, leaves, bark, and roots. The efficacy, however,
depends not only on the amount of phenolic acids and flavonoids, but also of
tocopherol (vitamin E), ascorbic acid (vitamin C), and β-carotene content.
Sources of β-carotene include fruits, vegetables, dairy products, eggs, and
a few seafoods. Fresh fruits and green vegetables are particularly rich in
vitamin C. The crude oils, mainly that of wheat germ and nuts, are very
good sources of vitamin E. The active components of natural plant-derived
antioxidants are polyphenolic compounds. The most effective antioxidants
are those that contain two or more phenolic hydroxyl groups (Dziedzic &
Hudson, 1984; Shahidi et al., 1992). Plant phenolics compounds can either
act as reducing agents, free radical terminators, metal chelators, or 1O2
quenchers.
The antioxidants obtained from spices and herbs (oregano, thyme, dittany,
marjoram, lavender, and rosemary) were reported to have limited applica-
tions in spite of their high antioxidant activity, as they impart a characteristic
herb flavor to the food, thus deodorization steps are required (Reglero et al.,
1999). Since many plant-derived substances often have a strong, distinctive
taste of their own, plant-derived antioxidants must not only be tested for
their ability to retard oxidation but also for any sensory characteristics they
impart to the food product. Naturally occurring antioxidant substances also
need safety testing. Caution regarding an assumption of safety of natural
antioxidants has been repeatedly advised, since the fact that an antioxidant
comes from a natural source does not prove its assumed safety. Hattori et
al. (1998) summarized the requirements that antioxidants must satisfy to be
used as food additives.

7.9 APPLICATION OF NATURAL ANTIOXIDANTS IN VARIOUS


DAIRY PRODUCTS

Dairy products contain lipids rich in PUFA and their esters are easily oxidized
by molecular oxygen over time. Deleterious changes in dairy products
Application of Natural Antioxidants in Dairy Foods 279

caused by lipid oxidation include not only loss of flavor or development of


off-flavors, but also loss of color, nutrient value, and the accumulation of
compounds, which may be detrimental to the health of consumers. One of the
most effective ways of retarding lipid oxidation in dairy products is to incor-
porate antioxidants (Gad & Sayd, 2015). The growing interest in the study
of natural antioxidant compounds has been accompanied by an increase in
the market presence of functional foods or nutraceuticals or health foods.
Fortification of dairy products with bioactive components (natural antioxi-
dants compounds) enhances their antioxidant activity and anti-inflammatory
properties, which prevent the damaging effects of free radicals (Berardini et
al., 2005) and provide various health benefits.
Herbs, fruits, vegetables, spices, and other plant materials rich in phenolic
compounds are of growing interest in the food industry because they retard
oxidative degradation of lipids and thereby improve the quality and nutri-
tional value of food (Wojdyło et al., 2007). Numerous herbs have the poten-
tial to retard lipid oxidation during storage of foods which is usually medi-
ated through their intrinsic antioxidant activity. The reports on the addition
of herb and spice extracts in milk and milk products is evolving rapidly
(Pokorny & Korczak, 2001; Pawar et al., 2012) and the same is discussed as
in the next section.

7.9.1 MILK AND MILK BASED BEVERAGES

Several approaches for antioxidant incorporation in milk have been used in


an attempt to reduce lipid oxidation. Jung et al. (1998) added ascorbic acid
(from 200 to 1000 mg/kg) directly to milk and, by using dynamic headspace
analyses and gas chromatography, concluded that formation of dimethyl
disulfide decreased. The result of sensory evaluation revealed that milk
containing ascorbic acid (and therefore less dimethyl disulfide) improved in
flavor in the presence of ascorbic acid.
In a subsequent study, the effect of antioxidants, added in a single initial
dose or in weekly additions to extend shelf life of milk, was evaluated for
over six weeks of lighted storage at 4 °C (van Aardt et al., 2005). Light
induced oxidation was measured by determining pentanal, hexanal, heptanal,
and 1-octen-3-ol contents. Weekly addition of a combination of BHA and
BHT (100 mg/kg of milk fat, each) maintained heptanal content of milk at
levels comparable to light-protected milk, whereas an initial single addition
of α-tocopherol significantly decreased hexanal content over the first four
weeks of storage. Odor-active compounds associated with light-induced
280 Natural Antioxidants: Applications in Foods of Animal Origin

oxidation included 2,3-butanedione, pentanal, dimethyl disulfide, hexanal,


1-hexanol, heptanal, 1-heptanol, and nonanal. The results revealed that the
addition of BHA and BHT in a single initial addition resulted in decrease in
pentanal and hexanal odor, but not in heptanal and 1-heptanol odor, whereas
the addition of α-tocopherol and ascorbyl palmitate decreased pentanal and
heptanol odor, but not hexanal and heptanal odor.
Antioxidant capacity of blends with 13% (w/w) bilberry and black
currant extract in low-fat milk or low-fat fermented milk were assessed by
Skrede et al. (2004). Antioxidant capacity in 13% berry/fermented milk
blends packed in glass or cardboard cartridges and stored for three weeks
in the dark or under fluorescent light was determined. Anti-radical power
(ARP) and oxygen radical absorbance capacity (ORAC) values of most fruit
preparations greatly exceeded those of plain milk. Milk blends with berry
extract increased ARP values 5–13-folds and ORAC values by 40–100%.
Packaging material, illumination, and storage period had no consistent
effects on antioxidant capacity.
The use of oregano extract (OE) and oregano essential oil (OEO) as anti-
oxidants in dairy beverages enriched with 2 g/100 g linseed oil was studied
by Boroski et al. (2012). OE and OEO reduced light- and heat-induced
oxidation of omega-3 fatty acids and change in color during storage of dairy
beverages enriched with linseed oil. OE showed better antioxidant proper-
ties than OEO. Physical stability of dairy beverages was not affected by the
addition of OE or OEO. It was concluded that natural antioxidants can be
added to dairy beverages enriched with omega-3 fatty acid to effectively
inhibit oxidation during storage.
Jung et al. (2015) investigated the physicochemical and antioxidant
properties of milk supplemented with red ginseng extract (RGE) (at 0.5, 1,
1.5, and 2%) during storage at 4 °C. The antioxidant activity of milk samples
was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method,
β-carotene bleaching assay, and ferric thiocyanate assay. It was observed
that the antioxidant activity of milk samples supplemented with RGE was
higher than that of the control sample.

7.9.2 GHEE/CLARIFIED BUTTER FAT

Ghee, the most famous traditional dairy product in India, Egypt, and many
countries in Middle East, undergoes oxidative degradation during storage,
resulting in alteration of major quality parameters affecting its suitability for
consumption. Development of rancidity reduces the shelf life of the product,
Application of Natural Antioxidants in Dairy Foods 281

which ultimately affects consumer acceptability (Mehta, 2006; Mariod et al.,


2010; Pawar et al., 2012). A number of natural antioxidants have been added
during processing and reported to have elongated the shelf life and oxidative
stability of stored products.
Merai et al. (2003) reported that water insoluble fraction of Tulsi (Ocimum
sanctum L.) leaves possess good antioxygenic properties and phenolic
substances present in Tulsi leaves were the main factors in extending the
oxidative stability of ghee (Butterfat). Pankaj et al. (2013) reported that addi-
tion of ethanolic extract of Arjuna (Terminalia arjuna Wight & Arn.) bark
at 7% by the weight was highly effective in retarding the auto-oxidation of
both cow and buffalo ghee during storage. However, the ability of ethanolic
extract of Arjuna to enhance the antioxidant potential of ghee was observed
to be more pronounced in case of cow ghee than in buffalo ghee. Shelf life
of the Arjuna ghee samples was eight days at 80 ± 1 °C as compared to two
days in the control. A study was conducted by Pawar et al. (2012) for evalu-
ating the effect of Shatavari (Asparagus racemosus) on storage stability of
ghee. It was observed that the samples incorporated with ethanolic extract
of Shatavari showed a strong activity in quenching DPPH radicals than the
aqueous extract of the same herb. Gandhi et al. (2013) carried out study on
oxidative stability of ghee added with Vidarikand (Pueraria tuberosa) (both
aqueous and ethanolic) extracts and compared the same with BHA, TBHQ,
rosemary, and green tea using β-carotene bleaching assay, DPPH, and the
rancimat method. Phenolic content and antioxidative activity of ethanolic
extract of Vidarikand was more compared to its aqueous extract. Ethanolic
extract of the Vidarikand was more effective in preventing the development
of the peroxide value and conjugated diene value in ghee compared to its
aqueous extract. Aqueous and ethanolic extracts of Vidarikand were found
to be capable of retarding oxidative degradation in ghee but were less effec-
tive than natural (rosemary and green tea) and synthetic (BHA and TBHQ)
antioxidants. Similar work was carried out by Pawar et al. (2014) on anti-
oxidant activities of Vidarikand (P. tuberosa), Shatavari (A. racemosus), and
Ashwagandha (Withania somnifera) extracts (aqueous and ethanolic) which
were evaluated and compared with BHA. Antioxidant activity of the herbs
decreased in the order Vidarikand > Ashwagandha > Shatavari. Thus, the
ethanolic extract of Vidarikand was found to have the maximum antioxidant
activity among all the herbs.
A huge amount of plant biomass wastes are produced yearly as by-prod-
ucts from the agro-food industries. These wastes are attractive sources of
natural antioxidants. In one of the studies, natural antioxidants found in
peanut skins (PS), pomegranate peels (PP), and olive pomace (OP) were
282 Natural Antioxidants: Applications in Foods of Animal Origin

extracted using ethanol (80 %), ethyl acetate, and n-hexane and the oxida-
tive stability of ghee during storage under thermal oxidative conditions was
reported. Ethanol extract showed slightly better antioxidant characteristics
compared with ethyl acetate and hexane extracts. It could be due to the
reason that extracts obtained from higher-polarity solvents were more effec-
tive radical scavengers than those obtained using lower-polarity solvents.
Extracts obtained from PS exhibited strong antioxidant capacity in all assays,
followed by PP and OP extracts (El-Shourbagy & El-Zahar, 2014).
In a subsequent study, Asha et al. (2015) evaluated the antioxidant activi-
ties of BHA and orange peel powder extract in ghee stored at different storage
temperatures during the storage period of 21 days. Ghee incorporated with
orange peel extract (OPE) showed stronger activity in quenching DPPH
radicals and least development of peroxide value, free fatty acid content and
TBA than ghee incorporated with BHA and control. The study revealed that
orange peel could be a good natural source of antioxidants which could be
used in fat rich food products like ghee to retard oxidative deterioration.

7.9.3 FERMENTED MILK PRODUCTS

Yogurt is among the most common dairy products consumed around the
world (Saint-Eve et al., 2006). Yoghurt with added antioxidants from natural
sources appears to be a convenient food format to satisfy consumer interest
in terms of beneficial effects of starter cultures, and health benefits of
added antioxidants over original yoghurt nutrients. For this reason, several
attempts to produce yoghurts fortified with natural antioxidant-rich extracts
have been studied, including supplementation with polyphenol-rich wine
extract (Howard et al., 2000), Hibiscus sabdariffa extract (Lwalokun &
Shittu, 2007), pycnogenol from French marine bark extract (Ruggeri et al.,
2008), green bell pepper juice (Halah & Mehanna, 2011), quince scalding
water (Trigueros et al., 2011), apple polyphenols (Sun-Waterhouse et al.,
2012), grape and grape callus extracts (Karaaslan et al., 2011), tea infusions
(Najgebauer-Lejko et al., 2011), grape seed extracts (Chouchouli et al.,
2013), berry polyphenols (Sun-Waterhouse et al., 2013) and pomegranate
peel extracts (PPE) (El-Said et al., 2014).
Chouchouli et al. (2013) evaluated the potential of using grape seed
extracts from two Greek wine grape varieties for the production of anti-
oxidant-rich full-fat and non-fat yoghurts. Fortified yoghurts contained
more polyphenols and exhibited higher antiradical and antioxidant activity
than controls, even after 3–4 weeks of cold storage. The degradation of
Application of Natural Antioxidants in Dairy Foods 283

polyphenols and the decrement of yoghurts’ antiradical and antioxidant


activities followed first order kinetics, with full-fat yoghurts exhibiting
higher deterioration rates and lower half-lives than the non-fat ones. In one
of the studies, stirred yoghurt was prepared from reconstituted skim milk
powder fortified with 5, 10, 15, 20, 25, 30, and 35% of the aqueous extract of
whole PPE, before and after inoculation with the traditional yoghurt starter
(El-Said et al., 2014). Addition of PPE before inoculation with the starter
resulted in stirred yoghurt of higher antioxidant activities than that with PPE
added after inoculation with the starter. Also, increasing the percentage of
the added PPE increased significantly the antioxidant activities of stirred
yoghurt up to 25% and further increase in the percentage of added PPE led
no significant effect.
Najgebauer-Lejko et al. (2014) evaluated the sensory quality and anti-
oxidant capacity of yoghurts with addition of selected vegetables (carrot,
pumpkin, broccoli, and red sweet pepper) (at 10% w/w) during two weeks
of refrigerated storage. The highest ability to scavenge DPPH radicals
was stated for yoghurts with broccoli and red sweet pepper. All vegetable
yoghurts were characterized by higher ferric reducing antioxidant power
(FRAP) values (measured directly after production) than the natural yoghurt.
However, the level of this parameter significantly decreased after storage.
Authors concluded that the red sweet pepper fortified yoghurt was the most
beneficial in terms of antioxidant properties and organoleptic acceptance of
the studied yoghurts.
Singh et al. (2013) carried out a study on the effect of strawberry poly-
phenol extract (0.5 mg/mL) addition on physicochemical and antioxidant
properties of stirred dahi preparation. This fortification resulted in a 7-fold
increase in the antioxidant activity of polyphenol-enriched stirred dahi, while
pH, acidity, water-holding capacity, and viscosity remained comparable with
the control. Sensory analysis indicated that the product was acceptable up to
two weeks when stored at 7–8 °C with no significant difference (p > 0.05) in
the antioxidant activity and total phenolic content (TPC).

7.9.4 CHEESE

The essential oils obtained from aromatic plants are natural products. Many
essential oils have demonstrated antioxidant properties; specifically, the
essential oils of oregano, rosemary, laurel, and other plants have been
studied as potential natural antioxidants (Kulisic et al., 2004; Olmedo et
al., 2008; Olmedo et al., 2009; Olmedo et al., 2012; Asensio et al., 2011).
284 Natural Antioxidants: Applications in Foods of Animal Origin

Gad and Abd El-Salam (2010) monitored the effect of addition of different
concentration of rosemary extract (RE) to skim milk during processing for
production of soft cheese. The antioxidant activity of the blends of skim milk
and REs was improved by heat treatment. The addition of calcium chloride
and pasteurization further significantly increased the phenol content and the
antioxidant activity of skim milk, whereas addition of sodium chloride and
homogenization of the skim milk-REs did not affect antioxidant activity.
Skim milk with high rosemary concentration was reported to have high
antioxidant activity.
In a subsequent study, ultra-filtered (UF)-soft cheese was prepared from
UF milk retentate (1.5% fat) and supplemented with 1–5% RE and cold
stored for 30 days (El-Din et al., 2010). The TPC and antioxidant capacity
were evaluated using DPPH and FRAP methods in retentate before and after
pasteurization and salting along with the resultant cheese. Fortification of
retentate with RE increased its phenolic content and consequently, its anti-
oxidant activity. Pasteurization increased the TPC and antioxidant activity.
It was observed that addition of 3% NaCl reduced slightly the radical scav-
enging activity (RSA), TPC, and FRAP values. Moreover, it was noticed
that UF-soft cheese fortified with 1% RE retained more TPC and antioxidant
activity; also, increasing the concentration of RE to 5% had more acceptable
flavor, body and texture, and antioxidant activity until 30 days. Furthermore,
the rate of decrease in TPC, RSA (%), and FRAP values in cheese samples
containing RE after 30 days of storage were less as compared to control
cheese (without RE) (El-Din et al., 2010).
A functional cheese product containing polyphenolic compounds
was developed, and the polyphenolic retention efficiency and antioxi-
dant property of the product was evaluated by Han et al. (2011). Single
phenolic compounds, including catechin, epigallocatechin gallate (EGCG),
tannic acid, homovanillic acid, hesperetin and flavones, and natural crude
compounds, such as grape extract, green tea extract, and dehydrated cran-
berry powder, were added as functional ingredients to the prepared cheese.
Cheese curds with polyphenolic compounds at a concentration of 0.5 mg/
mL showed effective free radical-scavenging activity and showed high
retention coefficient ranging between 0.74 and 0.87. The nutritional value
of cheese product was reported to be improved by adding bioactive phenolic
compounds to the cheese curd.
Olmedo et al. (2013) evaluated the preservative effect of oregano and
rosemary essential oils on the oxidative and fermentative stabilities of
flavored cheese prepared with cream cheese base. The addition of oregano
and rosemary essential oils was reported to improve the oxidative and
Application of Natural Antioxidants in Dairy Foods 285

fermentative stability along with preventing the lipid oxidation and the
development of rancid and fermented flavors in the said cheese. As conse-
quence, these essential oils prolonged the shelf life of the product.

7.9.5 OTHER DAIRY PRODUCTS

Herbs fortified dairy products serve as a good source of antioxidant


provided the antioxidant capacity of the dairy product and herbs are not
depleted through oxidation–reduction reactions upon mixing and storage
of the products. Bandyopadhyay et al. (2007) carried out a study on the
antioxidant activities of beet (Beet vulgaris), mint (Mentha spicata L.), and
ginger (Zingiber officinale L.) alone and in combination after fortification
in sandesh (a heat desiccated product of coagulated milk protein mass). The
same was compared with the synthetic antioxidants like TBHQ, BHA, and
BHT. Among the natural sources, ginger and combination of ginger with
mint showed excellent results and value was almost equal to TBHQ (200 mg/
kg). It was suggested that the use of BHA and BHT can be substituted by
all the natural sources (beet, mint, and ginger) alone or in combined form.
Among the four forms of herbs such as paste, tray-dried powder, freeze-
dried powder, and solvent extracted form, addition of solvent extracted
form in sandesh preparation showed highest antioxidant level than any other
form. In their subsequent study, Bandyopadhyay et al. (2008) demonstrated
the antioxidant activity, peroxide value, and ultra-violet absorbance to eval-
uate the effectiveness of natural antioxidants in reducing lipid oxidation in
sandesh as compared to synthetic antioxidants. All the natural sources and
their combinations significantly improved the oxidative stability of sandesh
and their effectiveness was comparable with synthetic antioxidant TBHQ
and a combination of BHA and BHT. Among the natural sources, although
ginger had the highest antioxidant activity but mint showed better effective-
ness in the inhibition of lipid oxidation. Regarding antioxidant activity and
lipid oxidation, combination of mint or ginger with beet showed better result
as compared to beet alone.
The antioxidant activities of peels of pomegranate, lemon, and orange
extracts were studied in paneer samples. Among the three extracts pome-
granate exhibited a high percentage of antioxidant activity and phenolic
content in comparison to lemon and OPE. The ability to prevent peroxide
formation in paneer sample was in the order of pomegranate peel > lemon
peel > orange peel (Singh & Immanuel, 2014) (Table 7.2).
286 Natural Antioxidants: Applications in Foods of Animal Origin

TABLE 7.2 Application of Natural Antioxidants in Milk and Dairy Products.


Product Natural Antioxidant Form Reference
Milk and Bilberry and Black currant Extract Skrede et al. (2004)
milk based
beverages
Oregano Extract and essential oil Boroski et al. (2012)
Red ginseng Extract Jung et al. (2015)
Ghee/clarified Tulsi Water insoluble fraction Merai et al. (2003)
butter fat of leaf
Arjuna (Terminalia Ethanolic extract of Pankaj et al. (2013)
arjuna Wight & Arn.) bark
Shatavari (Asparagus Ethanolic extract Pawar et al. (2012)
racemosus)
Vidarikand (Pueraria Aqueous and ethanolic Gandhi et al. (2013)
tuberosa)
Ashwagandha (Withania -Do- Pawar et al. (2014)
somnifera)
Peanut skins, Extract using ethanol, El-Shourbagy and
pomegranate peels, ethyl acetate, and El-Zahar (2014)
olive pomace n-hexane
orange peel Extract Asha et al. (2015)
Fermented Hibiscus sabdariffa -Do- Lwalokun and Shittu
milk products (2007)
Polyphenol-rich wine -Do- Howard et al. (2000)
extract
Pycnogenol from French Extract Ruggeri et al. (2008)
marine
Green bell pepper Juice Halah and Mehanna
(2011)
Quince scalding water -Do- Trigueros et al.
(2011)
Apple polyphenols -Do- Sun-Waterhouse et
al. (2012)
Grape and grape callus -Do- Karaaslan et al.
(2011)
Tea Infusions Najgebauer-Lejko et
al. (2011)
Grape seed Extracts Chouchouli et al.
(2013)
Application of Natural Antioxidants in Dairy Foods 287

TABLE 7.2 (Continued)


Product Natural Antioxidant Form Reference
Berry polyphenols -Do- Sun-Waterhouse et
al. (2013)
Pomegranate peel -Do- El-Said et al. (2014)
Strawberry polyphenol -Do- Singh et al. (2013)
Cheese Rosemary -Do- Gad and Abd El-
Salam (2010)
Paneer Pomegranate, lemon, -Do- Singh and Immanuel
and orange peels (2014)
Sandesh Beet (Beet vulgaris), mint -Do- Bandyopadhyay et al.
(Mentha spicata L.), and (2007)
ginger (Zingiber officinale
L.)

7.10 CONCLUSION

Milk and milk products are considered to be complete food due to the
presence of almost all the macro and micronutrients; and thus are also
regarded as an indispensable part of the diet. However, due to the presence
of several oxidants, they are prone to oxidation in spite of the presence
of naturally occurring antioxidants. Hence, addition of antioxidants into
the dairy products is gaining importance these days. The most widely used
synthetic antioxidants in food (BHT and BHA) are very effective in their
role as antioxidants but their use in food products has been failing off due
to their instability, as well as due to the suspected action as promoters of
carcinogenesis. Consequently, there has been considerable interest in the
use of natural antioxidants on account of safety and acceptability. Most
of the naturally occurring antioxidants not only do keep the food stable
against oxidation but can also be effective in controlling microbial growth.
However, better understanding of the role of natural antioxidants on food
stability and human health is required; and toxicological studies are mainly
carried out to establish the no-effect level for an acceptable daily intake for
humans as the origin of the antioxidant from a natural source does not prove
its assumed safety.
288 Natural Antioxidants: Applications in Foods of Animal Origin

KEYWORDS

• milk
• oxidants
• synthetic antioxidants
• natural antioxidants
• extracts
• essential oil

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CHAPTER 8

ANTIOXIDANT DIETARY FIBER: AN


APPROACH TO DEVELOP HEALTHY
AND STABLE MEAT PRODUCTS
ARUN K. VERMA1,*, RITUPARNA BANERJEE2, and V. RAJKUMAR1
1
Goat Products Technology Laboratory, ICAR-Central Institute for
Research on Goats, Makhdoom, Farah, Mathura 281122,
Uttar Pradesh, India
ICAR-National Research Centre on Meat, Chengicherla,
2

Hyderabad 500092, Telangana, India


Corresponding author. E-mail: [email protected]
*

CONTENTS

Abstract ....................................................................................................300
8.1 Introduction .....................................................................................300
8.2 Antioxidant Dietary Fiber ...............................................................301
8.3 Polyphenols and Dietary Fiber Quality ..........................................302
8.4 Dietary Fiber Processing and Quality .............................................303
8.5 Sources of Antioxidant Dietary Fiber .............................................304
8.6 Application in Meat Products .........................................................326
8.7 Conclusion ......................................................................................329
Keywords .................................................................................................329
References ................................................................................................329
300 Natural Antioxidants: Applications in Foods of Animal Origin

ABSTRACT

The findings about association between healthy diet and consumer well-
being have escalated the demands for the foods especially meat products
supplying additional healthy nutrients such as dietary fiber (DF) and natural
antioxidants. There are several reports in the literature linking the regular
intake of DF and antioxidants help in preventing various diets related non-
communicable diseases as well as degenerative problems. Researchers are
also responding very well to the consumer’s demands through the screening
of various sources of antioxidant dietary fiber (ADF) such as fruits, vegeta-
bles, seeds and their by-products as well as other miscellaneous plant mate-
rials. These ADF ingredients are being added in different meat products and
their effects on various qualities are monitored.

8.1 INTRODUCTION

Workload has considerably increased in modern society and very limited


time is spared to take care of personal health. This time scarcity has forced
us to opt for ready-to-eat and fast foods including meat products which
lack adequate level of dietary fiber (DF) required in the daily diet. Inad-
equate DF in our meal can be a cause for several diet related problems.
Several investigations have reported that one-third of all cancer cases and
one-half of cardiovascular diseases and hypertension can be attributed to
diet (Lee & Smith, 2000; Wolfe et al., 2003). The incidence of a number
of non-infectious diseases in our modern societies, such as coronary heart
disease, certain kind of cancer is partly associated to a low DF intake. Thus
DF intake may be vital in reducing colonic cancer, in lowering serum choles-
terol levels and in preventing hyperglycemias in diabetic patients (Nawirska
& Kwasniewska, 2005). The main sources of DF are plant products like
seeds, fruits, vegetables, and their products. It is widely accepted now that
increased consumption of fruits and vegetables can reduce the risk of cancer,
heart disease, and stroke (Liu, 2003). The possible beneficial health effects
of diets rich in these plant products have been attributed to their DF and
phytochemicals (Block et al., 1992; Lampe, 1999).
Meat and meat products are very much nutritious providing many key
nutrients to our body. One of the several nutrients is the fat which is a dense
source of energy and furnishes essential fatty acids. These fatty acids can
possess various degrees of unsaturation. A high degree of unsaturation accel-
erates oxidative processes leading to deterioration in meat flavor, color,
Antioxidant Dietary Fiber: An Approach to Develop Healthy 301

texture, and nutritional value (Mielnick et al., 2006). This oxidation is a highly
complex process involving numerous reactions which produce a variety of
chemical and physical changes (Sánchez-Alonso et al., 2008). The nutritional
quality of meat and meat products is often being challenged due to absence
of DF in them. However, this can be overcome by addition of DF rich ingre-
dients while processing of meat products. There are several DF ingredients
which are also gifted with many phytochemicals like polyphenols. Phenolic
compounds present in fruits have been demonstrated to possess antioxidant,
anti-inflammatory and anti-carcinogenic properties and the ability to prevent
a variety of chronic diseases (Boyer & Liu, 2004). Adding these ingredients
to meat products, which has almost negligible amount of DF, can be helpful
in enhancing their nutritional and functional values, quality, and storage
stability. Moreover, intake of natural antioxidant through these fiber rich meat
products can help fighting stress of modern day hectic lifestyle. This chapter
broadly deals with antioxidant dietary fiber (ADF), sources and effects of
their incorporation on quality of meat products.

8.2 ANTIOXIDANT DIETARY FIBER

DFs are plentiful in plant products like fruits, vegetables, and grains. They
are very well known as a beneficial component of healthy diet and their
consumption is being linked with reductions in risks associated with cardio-
vascular disease, cancer, and diabetes (Cho & Dreher, 2001). DF has hetero-
geneous chemical structures and conventionally classified according to their
solubility in water as soluble dietary fiber (SDF) and insoluble dietary fiber
(IDF). In cereal bran, the IDF is largely predominant while fruit DFs are rich
in SDF (Gorinstein et al., 2001; Grigelmo-Miguel & Martı́n-Belloso, 1998;
Prosky et al., 1988). It is important to consume the DF in a balanced propor-
tion, that is, the water-soluble fraction should represent between 30 and 50%
of the total dietary fiber (TDF) (Eastwood, 1987; Spiller, 1986).
Interests in applying fruit processing wastes as functional food ingredi-
ents is consistently increasing as they are rich source of DF, and several bene-
ficial bioactive compounds (Balasundram et al., 2006) such as polyphenols.
Such association of DF and polyphenols led to the proposal of the concept of
ADF by Saura-Calixto (1998) with the criteria that 1 g of ADF should have
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging capacity
equivalent to at least 50 mg vitamin E and DF content should be higher than
50% dry matter from the natural constituents of the material. Antioxidant DF
is thus defined as a natural product that combines the beneficial effects of
302 Natural Antioxidants: Applications in Foods of Animal Origin

DF and natural antioxidants, such as polyphenol compounds (Saura-Calixto,


1998).
The definition of DF according to the American Association of Cereals
Chemists Expert Committee (De Vries, 2003) includes “cell wall polysac-
charides, lignin and associated substances resistant to hydrolysis by the
digestive enzymes of humans.” In this definition, “associated compounds”
are included for the first time in an official definition of DF. Polyphenols
linked with DFs could be one of the associated compounds. Later the concept
of the antioxidant activity of insoluble material was introduced (Serpen et
al., 2007) as it is able to exert a marked antioxidant activity also by a solid–
liquid interaction. The insoluble materials, particularly ADFs survive in the
gastrointestinal tract for a long time and quench the soluble radicals that are
continuously formed in the intestinal tract (Babbs, 1990).
The most abundant phenolic compounds in cereals belong to the chem-
ical class of hydroxycinnamic acids (HCA). Ferulic acid (FA) is the main,
followed by diferulic acids and by sinapic acid, p-coumaric acid, and caffeic
acid; benzoic acid derivatives have also been described (Vitaglione et al.,
2008). About 95% of grain polyphenols are linked to cell wall polysac-
charides through covalent ester bonds. FA is bound to the arabinoxylans
via the acid group acetylating the primary hydroxyl at the C5 position of
α-arabinofuranosyl residues (Hatfield et al., 1999). In case of fruits and
vegetable, phenolic compounds could also be linked to pectin and to other
polysaccharide structures (Ishii, 1997; Jiménez-Escrig et al., 2001a; Saura-
Calixto & Díaz-Rubio, 2007).
The antioxidant capacity of DF linked polyphenols has been largely
underestimated because of their low water and organic solvent solubility.
In order to get actual value, it is necessary to perform multiple step extrac-
tion and an appropriate chemical hydrolysis to release phenolics and to
permit them to exert antioxidant activity in the in vitro assays. In the view
of constituents present in the ADF, it can be used on one hand as a dietary
supplement to improve gastrointestinal health and to prevent cardiovascular
diseases (Pérez-Jiménez et al., 2008), and on the other as an ingredient in
meat products to improve technological qualities and prevent lipid oxidation
(Sánchez-Alonso et al., 2007).

8.3 POLYPHENOLS AND DIETARY FIBER QUALITY

The antioxidant moiety associated with DF fundamentally determines its


structure and consequently physical properties. The presence of FA linked to
Antioxidant Dietary Fiber: An Approach to Develop Healthy 303

polysaccharide chains represents a suitable way to cross-link them through


the formation of diferulates (Bunzel et al., 2001). The principal mechanism
for crosslinking cell wall polysaccharides is ferulate dehydrodimerization
via radical coupling reactions producing a number of different diferulates
(Bunzel et al., 2004). They form bridge structures between chains of poly-
saccharides. Moreover, ferulates have a significant role in cross-linking
polysaccharides to lignin; thus they deeply influence physical parameters
of DF determining its reticulation, molecular weight, and water solubility.
The amount of diferulates found in the SDF of different cereals is more than
100-fold lower than the amount present in the corresponding IDF (Bunzel
et al., 2001). The extent of diferulates severely affects the biological signifi-
cance of DF and it is believed that the amount of DF diferulates is inversely
correlated to the fermentability by intestinal microflora (Kroon, 2000; Wang
et al., 2004). The bacterial β-glucosidases and esterases cannot attack the
highly cross-linked IDF while their action is easier when the DF is less struc-
tured such as in SDF (Kroon et al., 1997; Zhao et al., 2003). Although it is
also reported that low to moderate levels of diferulates do not interfere with
hydrolysis of non-lignified cell walls by human gut microbiota (Funk et al.,
2007). It is generally accepted that higher the SDF/IDF ratio, the higher is
DF polyphenols bioaccessibility.

8.4 DIETARY FIBER PROCESSING AND QUALITY

The by-products from cereals as well as fruits and vegetables are subjected
different processing steps and conditions to prepare the DF or ADF. The
extent and intensity of these processing steps and conditions may affect
various properties and activity of this bioactive ingredient. The treatment
like extent and intensity of blanching and drying, nature of solvent used
should be taken into consideration while processing of ADF.
DF production typically involves pre-treatment methods, such as
blanching or chemical treatments depending on the type of raw material,
prior to drying, to inactivate enzymes responsible for degradation of many
active compounds (Wolfe & Liu, 2003). However, number of sensitive
compounds may degrade during blanching, depending on the type (steam-
or water-blanching) and conditions (Zhang & Hamauzu, 2004). Loss of SDF
and solubilization of structural polymers such as protopectin may happen
while blanching of high fiber products (Maté et al., 1998). Drying can
result in oxidation, thermal degradation and other events such as collapse of
304 Natural Antioxidants: Applications in Foods of Animal Origin

microstructure that lead, directly or indirectly, to lower levels of antioxidants


or their bioaccessibility.
In a study on evolution of antioxidant compounds from lime residue
during drying Kuljarachanan et al. (2009) found that blanching decreased
both the antioxidant contents and activities of the residues due to thermal
degradation and loss with the blanched water. During drying initially
nomilin and limolin increased followed by sharp decrease due to thermal
degradation. In this case the product temperature was found to be a major
factor controlling the changes of limonoids. The amounts of vitamin C and
phenolic compounds decreased as the product temperature increased and the
moisture content decreased during drying.
Effect of air-drying temperature on physicochemical properties of
DF and antioxidant capacity of orange (Citrus aurantium v. Canoneta)
by-products (peel and pulp) was investigated (Garau et al., 2007). It was
observed that dehydration promoted important modifications affecting
both the physicochemical properties of DF and the antioxidant capacity
of orange by-products. The major modifications on the DF components
were observed when either extended drying periods, or elevated drying
temperatures were applied. Dehydration at around 50–60 °C appar-
ently promoted the minor disruption of cell wall polymers, in particular
of pectic substances. However, significant decreases in water retention
capacity, fat adsorption capacity, and solubility values were detected for
both by-products with increased drying temperature. The by-products
studied here were quite resistant to the different heat treatments applied
within the range of 40–70 °C. It was suggested that in order to preserve
the fiber quality and/or the antioxidant capacity, air drying temperature
should be controlled.

8.5 SOURCES OF ANTIOXIDANT DIETARY FIBER

Almost all the plant materials contain ample amount of DF. These mate-
rials are also enriched with various secondary metabolites acting as defense
systems which are either present in free form or associated with DF. The
plant materials as a source of ADF can be numerous; however, in the present
chapter only those sources are mentioned which have been investigated as a
source of antioxidant and DF. These sources are categorized here into four
groups (Fig. 8.1).
Antioxidant Dietary Fiber: An Approach to Develop Healthy 305

Dietary Fiber

Fruits and Vegetables and Seed and


Others

v
by-products by-products by-products

{~
Grape by-products Brassica plants Cereals Burdock root
Apple by-products Asparagus Mexican chea seed Hibiscus
Mango peel Carrot peel Cocoa husk Seaweed
Citrus by-products Amaranth and
quinoa
A~ai palm
Cactus pear
Guava
Bael pulp residue
Pineapple shell
Date by-products

FIGURE 8.1 Sources of antioxidant dietary fiber.

8.5.1 FRUITS AND BY-PRODUCTS

8.5.1.1 GRAPE BY-PRODUCTS

Grape pomace is a by-product which constitutes ~20% of the harvested


grapes (Laufenberg et al., 2003). At present, only least amounts of these
wastes are upgraded or recycled. Investigation on grape pomace is limited,
but it is undoubtedly rich in polyphenols (Amico et al., 2004; Kammerer
et al., 2004). The major constituents of grape pomace peels and seeds, has
been reported by several authors, with high polyphenols such as anthocya-
nins, catechins, flavonoids, phenolic acids as well as DF contents (Bravo
& Saura-Calixto, 1998; Valiente et al., 1995; Larrauri et al., 1999; Mazza,
1995; Mazza & Miniati, 1993). The phenolic compounds in grape pomace
include catechins, namely monomeric and oligomeric flavan-3-ols (proan-
thocyanidins) and glycosylated flavonols. Catechins, together with other
polyphenols, are potent free radical-scavengers (Ruberto et al., 2007).
306 Natural Antioxidants: Applications in Foods of Animal Origin

Several studies have highlighted the beneficial effects of grape or wine


polyphenols on human health (Ho et al., 1994; Rice-Evans et al., 1997;
Lurton, 2003; Frankel, 1999). More specifically, the antioxidative proper-
ties of many natural polyphenols may exert a chemopreventive role toward
cardiovascular and degenerative diseases (Halpern et al., 1998; Renaud &
De Lorgeril, 1992), including neurodegenerative pathologies (Esposito et
al., 2002) and cancer (Bozidar, 1995). Grape skins are rich in anthocya-
nins, a group of polyphenols well known for their beneficial properties
(Katsube et al., 2003; Wang et al., 1997; Ghiselli et al., 1998; Kong et al.,
2003; Kähkönen & Heinonen, 2003). Pérez-Jiménez et al. (2008) found that
fibers from grapes show higher reducing efficacy in lipid profile and blood
pressure with respect to oat fiber or psyllium due to combined effect of DF
and antioxidants. Wine grape pomace as ADF not only retarded human low-
density lipoprotein oxidation in vitro (Meyer et al., 1998) but also helped
to enhance the gastrointestinal health of the host by promoting a beneficial
microbiota profile (Pozuelo et al., 2012).
The general composition of two by-products of the Manto Negro red
grape (Vitis vinifera) variety, namely pomace and stem, were determined
(Llobera & Cañellas, 2007). Both by-products had high contents of TDF,
comprising three fourths of the total dry matter. Notable were the high
percentage of soluble fiber (15%) in relation to the TDF for the pomace, as
well as the high content of Klason lignin (KL) in both by-products, espe-
cially in the stem (31.6%). The free radical scavenging capacity (EC50) of
the by-products was found as 0.46 mg DM/mg DPPH (stem) and 1.41 mg
DM/mg DPPH (pomace). Thus both by-products of the vinification process,
particularly the stem, present excellent antioxidant properties as free radical-
scavengers. The methanolic extracts (MeOH) obtained from de-stemmed
grape pomace samples of five Sicilian red grape cultivars were evaluated for
their DPPH• and ABTS• radical scavenging capacity (Ruberto et al., 2007).
All the MeOH extracts showed significant antioxidant activity, with some
differences between the two methods employed. There was large variability
in the total anthocyanin (TA) and flavonol (TF) contents of the extracts,
as well as in the quantitative distribution of the single anthocyanins and
flavonols.
Grape skins, comprising on average, 82% of the wet weight of wine grape
pomace (Jiang et al., 2011), contain multiple types of polyphenols, including
39 types of anthocyanins, HCA, catechins, and flavonols (Kammerer et
al., 2004). The skins of two white wine grape pomace (WWGP) and three
red wine grape pomace (RWGP) were analyzed for their DF and phenolic
composition (Deng et al., 2011). IDF represented 95.5% of TDF, composed
Antioxidant Dietary Fiber: An Approach to Develop Healthy 307

of KL (7.9–36.1% DM), neutral sugars (4.9–14.6% DM), and uronic acid


(3.6–8.5% DM) in all five WGP varieties. WWGP was significantly lower
in DF (17.3–28.0% DM) than those of RWGP (51.1–56.3%), but extremely
higher in soluble sugar (55.8–77.5% DM vs 1.3–1.7% DM). Compared with
WWGP, RWGP had higher values in total phenolic content (TPC) (21.4–
26.7 mg GAE/g DM vs 11.6–15.8 mg GAE/g DM) and DPPH radical scav-
enging activity (32.2–40.2 mg AAE/g DM vs 20.5–25.6 mg AAE/g DM).
The total flavanol and proanthocyanidin contents were ranged from 31.0 to
61.2 mg CE/g DM and 8.0 to 24.1 mg/g DM, respectively, for the five WGP
varieties.

8.5.1.2 APPLE BY-PRODUCTS

Apples are well known and widespread fruit of the genus Malus belonging
to the family Rosaceae and play significant part in our diet. They are impor-
tant source of bioavailable polyphenolic compounds such as flavonols,
monomeric and oligomeric flavanols, dihydrochalcones, anthocyanidins,
p-hydroxycinnamic, and p-hydroxybenzoic acids (Escarpa & Gonzalez,
1998). The phenolic contents in apple are variable among different varieties,
and between the peel and the flesh; apple peels contain a higher concentra-
tion of phenolic compounds (Escarpa & Gonzalez, 1998; Vrhovsek et al.,
2004).
Apple pomace is a by-product of the apple juice processing, which is a
rich source of polyphenols, minerals, and DF (Boyer & Liu, 2004; Fernando
et al., 2005; Schieber et al., 2001; Sudha et al., 2007). Disposal of apple
pomace may present an added cost to beverage industry. Fernando et al.
(2005) evaluated some functional properties of apple pomace and reported
that fiber concentrates from apple pomace can be considered as a potential
source for fiber enrichment. According to Lu and Foo (2000), polyphenols
present in apple pomace could be a cheap and readily available source of
dietary antioxidants. The antioxidant capacity of apple pomace is related to
its phenolic profile. Procyanidins have long been recognized as the major
contributors to antioxidant activity of apples (Chinnici et al., 2004; Tsao
et al., 2005) and derivatives (Oszmianski et al., 2008), and the capacity
depends on their polymerization degree and substituents (Lotito et al., 2000).
Additionally, the antioxidant activity of hydroxycinnamic and benzoic acids
and flavonols has been ascertained (Kim et al., 2002; Tsao et al., 2005).
Apple pomace procured from fruit juice industry, contained 10.8% moisture,
0.5% ash, and 51.1% of DF. The total phenol content in apple pomace was
308 Natural Antioxidants: Applications in Foods of Animal Origin

7.16 mg/g (Sudha et al., 2007). Apple extracts have been shown to have
potent antioxidant activity and anti-proliferative activity against human
cancer cells (Boyer & Liu, 2004; Leontowicz et al., 2002).
Apple pomaces were subjected to evaluation as potential sources of anti-
oxidant phytochemicals on the basis of their total content of phenolics (from
4.22 to 8.67 mg/g), total flavonoids (from 0.45 to 1.19 mg/g) and total flavan-
3-ols (from 2.27 to 9.51 mg/g), and in vitro antiradical activities (Ćetković
et al., 2008). Some individual phenolic compounds including caffeic and
chlorogenic acids (+)-catechin, and (-)-epicatechin, rutin, quercetin glyco-
sides, and phloridzin were identified and quantified by high performance
liquid chromatography (HPLC). The antiradical activity of apple pomaces
was tested by measuring their ability to scavenge DPPH and hydroxyl
radicals. Eleven different cider apple pomaces (six single-cultivar and five
from the cider-making industry) were analyzed for low molecular phenolic
profiles and antioxidant capacity (García et al., 2009). The Folin index ranged
between 2.3 and 15.1 g gallic acid per kg of dry matter. Major phenols were
flavanols, dihydrochalcones (phloridzin and phloretin-20-xyloglucoside),
flavonols, and cinnamic acids (chlorogenic and caffeic acids). The group of
single-cultivar pomaces had higher contents of chlorogenic acid (-)-epicat-
echin, procyanidin B2, and dihydrochalcones, whereas the industrial samples
presented higher amounts of up to four unknown compounds, with absorption
maxima between 256 and 284nm. The antioxidant capacity of apple pomace,
as determined by the DPPH and ferric reducing antioxidant power (FRAP)
assays, was between 4.4 and 16.0 g ascorbic acid per kg of dry matter.
Methanolic and acetonic extracts of apple pomace were evaluated for
phenolic profiles, antioxidant properties, and antiviral effects against herpes
simplex virus type 1 (HSV-1) and 2 (HSV-2) (Suárez et al., 2010). Acetone
extraction yielded the higher amounts of phenolic compounds. The extrac-
tion method influenced the phenolic composition although antioxidant
activity correlated weakly with phenols concentration. Among the poly-
phenols analyzed, quercetin glycosides were the most important family,
followed by dihydrochalcones. It was observed that apple pomace extracts
were able to inhibit both HSV-1 and HSV-2 replication in Vero cells by more
than 50%, at non-cytotoxic concentrations. Selectivity indexes (SI) ranged
from 9.5 to 12.2.
Apple skin is rich in many health-enhancing phytonutrients including
flavonoids and phenolic acids (Boyer & Liu, 2004). Apple skin has three to
six folds more flavonoids than apple flesh and has unique flavonoids, such as
quercetin glycosides, not found in the flesh (Wolfe et al., 2003; Wolfe & Liu,
2003). Apple fruit skin is rich source of DF and phenolics. The blanched,
Antioxidant Dietary Fiber: An Approach to Develop Healthy 309

dehydrated, and ground apple skin powder contained ~41% TDF and
oxygen radical absorption capacity (ORAC) of 52 mg Trolox equivalents/g
dry weight (Rupasinghe et al., 2008).

8.5.1.3 MANGO PEEL

Mango (Mangifera indica L. Anacardiaceae) is one of the most important


tropical fruits. It is a seasonal fruit thus processed into various popular
products such as puree, nectar, leather, pickles, canned slices, and so forth
(Loelillet, 1994). Processing of mango mainly for pulp and amchur powder,
peel is a by-product which is not utilized for any commercial purpose and
discarded as a waste. This waste should be treated as a specialized residue
due to high levels of residual phenolics as well as DF (Larrauri et al., 1996a).
Peel constitutes about 20% of the whole fruit and its disposal has become a
great problem. According to Ajila et al. (2007) the polyphenol contents in
these peels ranged from 55 to 110 mg/g dry peel. DF content ranged from
45 to 78 % of peel and was found at a higher level in ripe peels. Similarly,
carotenoid content was higher in ripe fruit peels. Vitamins C and E contents
ranged from 188 to 392 and 205 to 509 µg/gm dry peels, respectively, and
these were also found at a higher level in ripe peels. The study conducted by
Ajila et al. (2008) indicated that mango peel contained 51.2% of TDF, 96 mg
GAE/g of polyphenols, and 3092 mg/g of carotenoids.

8.5.1.4 CITRUS BY-PRODUCTS

The amount of residue obtained from the citrus fruits after juice and essen-
tial oil extraction accounts for 50% of the original whole fruit mass (Cohn &
Cohn, 1997). They consist of peels (albedo and flavedo), which are almost
one-fourth of the whole fruit mass, seeds, and fruit pulp (Braddock, 1999).
Citrus peel has been reported to be a good source of pectin and DF in general,
with an equilibrated proportion of soluble and insoluble fractions (Baker,
1994; Larrauri et al., 1994). Approaches to the development of products with
increased dietary benefits from citrus peel have placed emphasis not only
on the recovery of carbohydrates and pectin (Baker, 1994) but also on the
production of potentially important secondary metabolites, such as polyphe-
nols (Manthey & Grohmann, 1996). These fiber-associated polyphenols are
known to exert important health promoting effects (Middleton & Kadas-
wami, 1994). Thus, citrus fruit by-products could be interesting not only for
310 Natural Antioxidants: Applications in Foods of Animal Origin

its important fiber content but also because of its antioxidant capacity (Kang
et al., 2006; Rehman, 2006). They have a high fiber and vitamin contents
as well as other associated bioactive compounds such as flavonoids and
terpenes possessing antioxidant properties (Lario et al., 2004). According to
Saura-Calixto (1998) DF from citrus residues has good functional properties
and low caloric content. Moreover, the residues contain more natural anti-
oxidants than do the flesh or juice (Larrauri et al., 1996b).
Limonoids, the major cause of bitterness in citrus juice, have been
reported to possess substantial antioxidant and anti-cancer activities. The
anti-carcinogenic activity of limonoids has been tested successfully in labo-
ratory animals (Lam et al., 1989; Miller et al., 1989). They appear in many
forms, including nomilin, obacunone, ichangin, and limonin. Limonin and
nomilin are the most prevalent forms of the citrus limonoids. Among the
classes of phytochemicals with anti-cancer properties designated by the
National Cancer Institute of USA, carotenoids, coumarins, flavonoids, gluca-
rates, monoterpenes, and phenolic acids are present in citrus fruits (Nagy &
Attaway, 1992). They were reported in the highest concentration in citrus
peel and were mainly composed of ferulic, sinapic, coumaric, and caffeic
acids as well as hesperidin and naringin, among others (Peleg et al., 1991;
Nagy & Attaway, 1992). Citrus flavonoids have been extensively investi-
gated because of their health-promoting properties (Middleton & Kandas-
wami, 1994). Nogata et al. (1996) reported that albedo tissue extracts from
lemon, inhibited both cyclooxygenase and lipoxygenase activities more than
those in orange, which could be related to the prevention of thrombosis,
atherosclerosis, and carcinogenesis.
The chemical components of citrus fiber (pectin, lignin, cellulose, and
hemicellulose), together with other compounds, such as flavonoids, were
analyzed in nine different industrial sources (Marín et al., 2007). Final fiber
composition was found to be more dependent on the industrial process than
on the type of citrus. The chemical changes gone by citrus fiber showed
losses of functional values; that is, SDF and ascorbic acid content decreased
when waste products were transformed into fibers. The water holding and
lipid holding capacities of analyzed citrus fibers suggested a non-linear
behavior of these properties.
High DF powders from Valencia orange and Persa lime peels were prepared
and their composition and antioxidant capacity were studied (Larrauri et al.,
1996b). Fibers from both peels had high TDF content (61–69%) with an
appreciable amount of soluble fiber (19–22%). The concentration of anti-
oxidant (AA50) required to achieve a 50% inhibition of oxidation of linoleic
acid at 40 °C was measured using the ferric-thiocyanate method. Lime peel
Antioxidant Dietary Fiber: An Approach to Develop Healthy 311

fiber (AA50) had half the value of DL-α tocopherol and 23 times lower than
orange peel fiber; the AA50 of commercial butylated hydroxyanisole (BHA)
was half the value of lime fiber. The HPLC analyses of the polyphenols
extracted from orange and lime peels fibers showed the presence of caffeic
and FAs, as well as naringin, hesperidin, and myricetin in both fruit fibers.
The different antioxidant power of these fibers could be in part explained
by the presence of ellagic acid, quercetin and kaempferol in lime peel fiber
which are strong antioxidant polyphenols.
Lemon (Citrus limon cv Fino) possesses the highest antioxidant potential
among citrus fruits and it is the most suitable fiber for dietary prevention of
cardiovascular and other diseases (Gorinstein et al., 2001). Lario et al. (2004)
used lemon juice industry by-products to obtain high DF powder. The effect
of processing variables (direct drying, and washing previous to drying) on
functional properties, fiber content and type, microbial quality and physi-
cochemical properties of the fiber were evaluated. The fiber had good func-
tional and microbial qualities as well as favorable physicochemical char-
acteristics to be used in food formulations. It was observed that processing
conditions affected fiber composition and properties. Water holding capacity
was enhanced by washing and slightly decreased by the reduction in fiber
particle size. Oil holding capacity was not affected by those factors. Acid
detergent and neutral detergent fibers were highest in powder from washed
lemon residue. Washing prevented fiber browning during drying as reflected
in color parameters. Washing water rinsed green components.
High DF powders from Persian and Mexican lime peels were prepared
and their fiber composition and antioxidant capacities were determined
(Ubando-Rivera et al., 2005). The TDF contents of both varieties were high;
70.4 and 66.7%, respectively. Both lime peel varieties had an appropriate
ratio of soluble/insoluble fractions. The water-holding capacities of fiber
concentrates were high (6.96–12.8 g/g) which was related to the SDF which
was higher in the DF concentrate of Mexican lime. Fiber concentrates of
Persian lime peel had greater polyphenol contents than those of Mexican
lime peel. The polyphenols associated with the DF in both lime peel vari-
eties showed a good antioxidant activity. It was suggested that from a nutri-
tional standpoint, DF lime concentrates may be suitable as food additives.

8.5.1.5 AÇAÍ PALM

Açaí (Euterpe oleracea), also known as cabbage palm, is a tropical species


which bears a dark purple, berry-like fruit, clustered into bunches. Recently,
312 Natural Antioxidants: Applications in Foods of Animal Origin

much attention has been paid to its antioxidant capacity and its possible role
as a functional food or food ingredient (Pozo-Insfran et al., 2006; Ribeiro et
al., 2010; Schreckinger et al., 2010). Anthocyanins, proanthocyanidins, and
other flavonoids were found to be the major phytochemicals in freeze-dried
açaí (Schauss et al., 2006) and some works have also been carried out on
antioxidant capacity of açaí pulp (de Souza et al., 2009; Rufino et al., 2009a;
Rufino et al., 2009b; Rufino et al., 2010). Rufino et al. (2011) reported the
concentrations of DF and antioxidant capacity in fruits (pulp and oil) of a
new açaí cultivar—“BRS-Pará.” The result showed that “BRS-Pará” açaí
fruit has a high content of DF (71% dry matter) and oil (20.82%) as well as
a high antioxidant capacity in both defatted matter and oil. These features
provide açaí “BRS-Pará” fruits with considerable potential for nutritional
and health applications.

8.5.1.6 CACTUS PEAR

Opuntia ficus-indica (cactus pear) is a cactus well adapted to extreme


climate and edaphic conditions. The genus Opuntia embraces about 1500
species of cactus and many of them produce edible tender stems and fruits
(Hegwood, 1994). The tender young part of the cactus stem, or cladode, is
frequently consumed as a vegetable in salads, while the cactus pear fruit is
consumed as a fresh fruit. Studies on the chemical composition of the edible
portion of cladodes and fruits from O. ficus-indica showed that these foods
have a high nutritional value, mainly due to their mineral, protein, DF, and
phytochemical contents (Bensadón, 2010). Interestingly, antioxidant activity
has also been reported (Corral-Aguayo, 2008).
The by-products are the outer coating of these plants, which is removed
before food preparation and contains spines and a large quantity of glochids
and pulp. Around 20 and 45% of the fresh weight of cladodes and fruits,
respectively, are by-products (Muñoz de Chávez & Ledesma-Solano,
2002). These by-products are rich in DF, minerals, and antioxidant bioac-
tive compounds. Bensadón et al. (2010) determined the nutritional value of
by-products obtained from cladodes and fruits from two varieties of O. ficus-
indica, examining their DF and natural antioxidant compound contents.
They found that the materials studied were rich in good quality DF and
natural antioxidants, especially Milpa Alta and Alfajayucan cultivars. It was
concluded that by-products from cladodes and fruits of Opuntia sp. could be
attractive for use as functional food ingredients.
Antioxidant Dietary Fiber: An Approach to Develop Healthy 313

8.5.1.7 GUAVA

Guava (Psidium guajava L.), now being recognized as “super food” is


getting very much attention in the agro-food business attributed to presence
of health promoting bioactive components and functional elements. The fruit
is considered as highly nutritious due to presence of high level of ascorbic
acid (50–300 mg/100 g fresh weight) and has several carotenoids such as
phytofluene, β-carotene, β-cryptoxanthin, γ-carotene, lycopene, rubixan-
thin, cryptoflavin, lutein, and neochrome (Mercadante et al., 1999). Phenolic
compounds such as myricetin and apigenin (Miean & Mohamed, 2001),
ellagic acid, and anthocyanins are also at high levels in guava fruits. Jiménez-
Escrig et al. (2001a) reported IDF, SDF, and TDF content in dried guava
as 46.72–47.65, 1.77–1.83, and 48.55–49.42%, respectively. According to
researchers, peel and pulp of P. guajava fruit has high levels of DF, indigest-
ible fraction, and phenolic compounds. Nahar et al. (1990) found a similar
relative value for IDF (91% of TDF) in the edible portion of P. guajava.
The total phenolics (on fresh mass basis) was 344.9 mg GAE/100 g in
“Allahabad Safeda” and ranged from 170.0 to 300.8 mg GAE/100 g in the
pink pulp clones (Thaipong et al., 2006). According to Corrêa et al. (2011)
total phenolics in guava varied from 158 to 447 mg GAE/100 g. Luximon-
Ramma et al. (2003) have reported that white pulp guava had higher anti-
oxidant activity and total phenolics than pink pulp guava in which the
antioxidant activity was 142.6 and 72.2 mg/100 g in white and pink pulp,
respectively, and the total phenolics was 247.3 and 126.4 mg GAE/100 g in
white and pink pulp, respectively.

8.5.1.8 BAEL PULP RESIDUE

Bael fruit pulp is endowed with many functional and bioactive compounds
such as DF, carotenoids, phenolics, alkaloids, coumarins, flavonoids, terpe-
noids, and other antioxidants (Suvimol & Pranee, 2008). Major antioxidants
in bael fruit are phenolics, flavonoids, carotenoids, and vitamin C (Morton,
1987; Roy & Khurdiya, 1995). Quantitative analyses have indicated that
the bael fruit is rich in carbohydrates and fibers and also a good source of
protein, vitamins, and minerals (Ramulu & Rao, 2003).
TPC (mg of GAE/100 g of decoction) in crude aqueous extract of bael
fruit powder was reported as 336.1 (Gheisari et al., 2011). According to
Suvimol and Pranee (2008), bael fruit pulps had TPC of 87.34 mg GAE/g
dry weight while Jain et al. (2011) reported the total polyphenols (mgGAE/g)
314 Natural Antioxidants: Applications in Foods of Animal Origin

in bael fruit extract as 95.33. Abdullakasim et al. (2007) reported that the
bael fruit drink possess high quantities of total phenolic compounds (37.6–
83.89 mg GAE/100 ml). The aqueous extract of the bael fruit pulp possesses
potent antioxidant effect (Kamalakkannan & Prince, 2003a; 2003b). The
hydro-alcoholic extract of bael pulp is also shown to possess nitric oxide
scavenging activities in vitro (Jagetia & Baliga, 2004). Suvimol and Pranee
(2008) found that bael fruit pulps had TDF, SDF, and IDF contents of 19.84,
11.22, and 8.62 g/100 g dry weight, respectively. According to these workers
the bael fruit is relatively rich in DF and it was in range of fruits which
are defined as high DF fruits. According to Parichha (2004) fresh bael pulp
without seed contains 31.8% TDF.

8.5.1.9 PINEAPPLE SHELL

Pineapple is one of the most important fruits in the world, and most of its
production is used in processing. It is consumed as canned slices, chunks,
dice, or fruit salads and in the preparation of juices, concentrates, and jams
(Salvi & Rajput, 1995). By-products obtained from industrial processing
represent 25–35% of the fruit, and the shell is the major constituent. The
shell has been used to produce alcohol, citric acid, vinegar, bromelain, wine,
sugar syrup, wax, sterols, and cattle feed (Joseph & Mahadeviah, 1988;
Salvi & Rajput, 1995). The DF content and composition of pineapple flesh
has been reported (Bartolomé & Rupérez, 1995).
Properties of a high DF powder prepared from pineapple fruit shell were
evaluated and compared to those of several commercial fruit fibers (Larrauri
et al., 1997). TDF content (70.6%) was similar to some commercial DFs
from apple and citrus fruits; however, its sensory properties were better than
those from commercial fibers. The IDF fraction accounted 99% of the TDF.
Major neutral sugars in SDF and IDF were xylose and glucose, respectively.
Total uronic acids and KL were 5.1 and 11.2%, respectively. At the concen-
tration of 0.5 g of powdered sample/100 mL in the assay mixture, pineapple
fiber showed a higher antioxidant activity (86.7%) than orange peel fiber
(34.6%). Myricetin was the major identified polyphenol in pineapple fiber.

8.5.1.10 DATE BY-PRODUCTS

Dates of date palm tree (Phoenix dactylifera L.) are popular among the
population of the Middle Eastern countries. The fruit is composed of a fleshy
Antioxidant Dietary Fiber: An Approach to Develop Healthy 315

pericarp and seed which constitutes between 10 and 15% of date fruit weight
(Hussein et al., 1998). The date seeds considered as a waste product of many
date processing plants producing pitted dates, date syrup, and date confec-
tionery. Generally, seeds are used as an animal feed; however, could be a
valuable source of DF and phenolics.
Three native sun dried date varieties from Oman (Mabseeli, Um-sellah,
and Shahal) their syrups and by-products (press cake and seed) were examined
for their proximate composition, DF, total phenolics, and total antioxidant
activity (Al-Farsi et al., 2007). Carbohydrate was the predominant compo-
nent in all date varieties, syrups, and their by-products, followed by moisture,
along with small amounts of protein, fat, and ash. The DF content in seeds and
press cakes were found to be 77.75–80.15% fresh weight and 25.39–33.81%
fresh weight, respectively. Among dates, syrups, and their by-products, seeds
had the highest contents of total phenolics (3102–4430 mg of GAE/100 g
fresh weight) and antioxidant activity (580–929 µmol of Trolox equivalents/g
fresh weight). The researcher concluded that date by-products, particularly
seeds serve as a good source of natural antioxidants and could potentially be
considered as a functional food or functional food ingredient.
Al-Farsi and Lee (2008) conducted the work to optimize extraction condi-
tions of phenolics and DF from date seeds. The effects of solvent to sample
ratio, temperature, extraction time, number of extractions, and solvent type
on phenolic extraction efficiency were observed. Two stage extractions,
each stage lasting for 1 h duration at 45 °C with a solvent to sample ratio
of 60:1, was considered optimum. Acetone (50%), and butanone were the
most efficient solvents for extraction and purification, increasing the yield
and phenolic contents of seed concentrate to 18.10 and 36.26%, respec-
tively. The TDF of seeds increased after water and acetone extractions. Nine
phenolic acids were detected in seeds with p-hydroxybenzoic, protocate-
chuic, and m-coumaric acids found to be among the highest. Protocatechuic,
caffeic, and FAs were the major phenolic acids found in the concentrates. It
was suggested that date seed concentrates could potentially be economical
source of natural DF and antioxidants.

8.5.2 VEGETABLE BY-PRODUCTS

8.5.2.1 BRASSICA PLANTS

Epidemiological studies have shown that high consumption of Brassica


vegetables, including cauliflower, cabbages, and broccoli, is associated with
316 Natural Antioxidants: Applications in Foods of Animal Origin

reduced risk of certain cancers such as lung cancer, colorectal cancer, breast
cancer, and prostate cancer (Ciska & Pathak, 2004; Higdon et al., 2007).
Brassica vegetables have been reported to contain high amount of DF and
various bioactive agents with high antioxidant activity. Phenolic compounds
and vitamin C are the major antioxidants of brassica vegetables (Podsedek,
2007). Lipid-soluble antioxidants (carotenoids and vitamin E) are respon-
sible for up to 20% of the brassica total antioxidant activity.
Cauliflower has a very high waste index (Kulkarni et al., 2001) and is
an excellent source of protein, cellulose, and hemicellulose (Wadhwa et al.,
2006). It is considered as a rich source of DF and possesses both antioxi-
dant and anticarcinogenic properties. The level of non-starch polysaccharide
(NPS) in the upper cauliflower stem remains similar to that of the floret and
both are rich in pectic polysaccharides, while the cauliflower lower stem
NPS is rich in cellulose and xylan (Femenia et al., 1998).
White cabbages (Brassica oleracea var. capitata) have been reported
to contain high amount of DF and bioactive agents with high antioxidant
activity as well as glucosinolates which are claimed to possess anticar-
cinogenic activity. About 40% of cabbage leaves, which are processed into
many products, including salads and ready-to-eat vegetables, are lost and
regarded as waste which contains high amount of DF and various phyto-
chemical compounds (Nilnakara et al., 2009). Processing of these residues
could therefore add much value to the products. The main constituents in
white cabbage are carbohydrates, comprising nearly 90% of the dry weight,
where approximately one-third is DF and two-thirds are low-molecular-
weight carbohydrates (Wennberg et al., 2004). Additionally, white cabbage
also possesses significant amounts of antioxidants such as ascorbic acid,
phenolic compounds, and tocopherols (Kim et al., 2004; Wennberg et al.,
2004; Singh et al., 2006).
The cabbage glucosinolates are the most interesting compounds. Gluco-
sinolates are a group of sulfur-containing plant secondary metabolites and
can be hydrolyzed by myrosinase to form different products, for example,
thiocyanates, isothiocyanates, epithionitrile, nitrile, and oxazolidine-thione
(Wennberg et al., 2006). The breakdown products (especially isothio-
cyanates) possess anticancer activity via modulation of phase II enzymes,
including glutathione S-transferase and quinine reductase. These enzymes
are reported to help inactivate cancer by blocking normal cells from DNA
damage (Verkerk et al., 2001). Production of DF powder from cabbage outer
leaves involves mechanical and thermal processes which may affect the
amount of glucosinolates. Several reports have indeed shown that glucosino-
lates in Brassica vegetables decreased upon blanching because of enzymatic
Antioxidant Dietary Fiber: An Approach to Develop Healthy 317

breakdown, thermal breakdown and leaching into blanching water (Verkerk


et al., 2001; Wachtel-Galor et al., 2008; Cartea & Velasco, 2008). Drying is
also reported to affect glucosinolates evolution. Mrkic et al. (2010) studied
the effect of temperature (50–100 °C) of the air that was used to dry broccoli
and reported that glucosinolates content decreased upon drying, especially
at higher drying temperatures.
Tanongkankit et al. (2012) investigated the effects of processing steps,
that is, slicing, blanching, and drying, on the changes of total glucosino-
lates in cabbage outer leaves, changes in DF composition and color. They
noted that the preparation steps did not lead to any significant changes of the
DF powder compositions. On the other hand, steam blanching was noted to
better preserve glucosinolates than water blanching. Drying methods and
conditions did not lead to any significant effect on the powder compositions;
however, vacuum drying led to better retention of glucosinolates. Color of
the DF powder was not affected by the drying methods and conditions.
Jongaroontaprangsee et al. (2007) produced high DF powder from outer
leaves of cabbage and reported that the powder contained ~41–43% TDF
(dry basis). Moreover, the powder possessed high water holding capacity and
swelling capacity. The production of DF powder associated with antioxidant
activity from cabbage outer leaves was studied by Nilnakara et al. (2009).
The effects of hot water blanching and hot air drying temperature (70–90 °C)
on the quality of DF powder produced from cabbage outer leaves were also
investigated. Parameters like proximate composition, visual color, phenolic
content, vitamin C as well as total antioxidant activity were evaluated.

8.5.2.2 ASPARAGUS

Besides their culinary quality, green asparagus spears are known for their
composition of bioactive compounds. Eastern civilizations have been using
asparagus extracts as stimulants, laxatives, antitussives, diuretics, and so
forth, for hundreds of years. Asparagus has been reported as rich in the
quality and quantity of its antioxidants (Pellegrini et al., 2003; Vinson et
al., 1998). During industrial processing, around half of the total length of
each spear is discarded, which creates significant waste for producers. It is
expected that the by-products have similar composition to the edible part
of the spears and could be a promising source of phytochemicals and fiber
(Nindo et al., 2003).
The asparagus extracts possess number of biological activities, including
anti-tumor and antioxidant activities and participate in the prevention of
318 Natural Antioxidants: Applications in Foods of Animal Origin

cardiovascular diseases (Cushine & Lamb, 2005; Nijveldt et al., 2001).


Among all the bioactive compounds present in asparagus spears, saponins,
flavonoids, and hydroxycinnamates are the main compounds responsible
for the characteristics cited above. Rutin is the most abundant flavonoid in
asparagus spears, in addition to others that have been recently described
(Fuentes-Alventosa et al., 2007, 2008).
According to Fuentes-Alventosa et al. (2009a) the method by which
asparagus by-products are treated affects the phytochemical composition
and antioxidant activity of the fiber rich powders. They studied factors such
as the treatment intensity, the solvent used, and the drying system. Among
the asparagus phytochemicals, HCA, saponins, flavonoids, sterols, and
fructans were quantified. HCA varied from 2.31 to 4.91 mg/g of fiber, the
content being affected by the drying system and, in some cases, the solvent.
Treatment intensity while isolation was found to affect the saponin content
in fibers. Saponin content ranged from 2.14 to 3.64 mg/g of fiber. Flavonoids
were most affected by processing conditions, being present (0.6–1.8 mg/g
of fiber) only in three of the samples analyzed. Sterols and fructans were
present in minor amounts, 0.63–1.03 mg/g of fiber and 0.2–1.4 mg/g of fiber,
respectively.
Fuentes-Alventosa et al. (2009b) investigated the effect of extraction
method on chemical composition and functional characteristics of high DF
powders obtained from asparagus by-products. The by-products represented
around 50% of the processed vegetable. All the fiber rich powders had
high concentrations of TDF (62–77%). The proportion of insoluble fiber to
soluble fiber decreased with the severity of treatment, in this way increasing
the physiological quality of the fiber. Functional properties, namely water
holding capacity, oil holding capacity, solubility, and glucose dialysis retar-
dation index (GDRI), varied according to the preparation procedure. These
properties make fiber rich powders from asparagus by-products a valuable
source of DF to be included in the formulation of fiber-enriched foods.

8.5.2.3 CARROT PEEL

Carrot (Daucus carota L.) is a good source of natural antioxidants, espe-


cially carotenoids and phenolic compounds (Prakash et al., 2004; Zhang &
Hamauzu, 2004). After processing, carrot residues such as peels, pomace,
are usually discarded or used as animal feed. These by-products contain high
contents of beneficial substances, especially bioactive compounds with anti-
oxidant activities (Zhang & Hamauzu, 2004). The feasibility study of using
Antioxidant Dietary Fiber: An Approach to Develop Healthy 319

carrot peels as a starting raw material to produce ADF powder was inves-
tigated (Chantaro et al., 2008). The effects of blanching and hot air drying
(60–80 °C) on the drying kinetics and physicochemical properties of DF
powder were evaluated. The results showed that blanching had a significant
effect on the fiber contents and compositions, water retention and swelling
capacities of the fiber powder. In contrast, drying temperature in the selected
range did not affect the hydration properties. As far as antioxidant activity
is concerned, thermal degradation during both blanching and drying caused
a decrease in the contents of β-carotene and phenolic compounds, hence
leading to the loss of antioxidant activity of the final product.

8.5.3 SEED AND BY-PRODUCTS

8.5.3.1 CEREALS

Cereals and legumes containing wide range of phenolics are good sources of
natural antioxidants (Krings et al., 2000). It has been reported that phenolic
compounds are concentrated in the bran portion of cereal kernels and may
contribute to the total antioxidant activities, suggesting bran a potent source
of antioxidants (Onyeneho & Hettiarachy, 1992). However, it is not clear
at which extent both free and carbohydrate-bound compound are measured
by a given assay (Zhou et al., 2004). Wheat is one of the popular cereal
grains, and its bran represents not only a good source of DFs (Alabaster
et al., 1997), but also of phenolic acids (Baublis et al., 2000). Significant
levels of antioxidant activities have been detected in wheat (Yu et al., 2003;
Zielinski & Kozlowska, 2000), and wheat-based food products (Baublis et
al., 2000), suggesting that wheat may serve as an excellent dietary source of
natural antioxidants for disease prevention and health promotion. Yu et al.
(2002) reported a significant level of TPC, free radical scavenging capac-
ities, chelating activity, and inhibitory effect on lipid peroxidation of the
three-wheat grain extracts with significant differences among the varieties.
Antioxidant activity of wheat bran and flour extracts varies with cultivar and
location (Yu et al., 2002).
Wheat bran, a by-product generated in large amounts during wheat
processing, consists of 36.5–52.4% TDF (Vitaglione et al., 2008), which
makes it a good source of DF. Additionally, wheat and wheat bran has
shown strong antioxidative activities (Li et al., 2005). Several phenolic
acids, including vanillic acid, p-coumaric acid, and, largely, FA have been
found in wheat bran extracts (Kähkönen et al., 1999). These compounds,
320 Natural Antioxidants: Applications in Foods of Animal Origin

particularly FA, are not evenly distributed in the wheat; most are found in
the bran (Baublis et al., 2002). According to Onyeneho and Hettiarachchy
(1992) extract of wheat bran, having high concentration of phenolic acids
and have stronger antioxidant activity than other fractions of wheat. Wheat
bran has been reported to be able to inhibit lipid oxidation catalyzed by either
iron or peroxyl radicals (Baublis et al., 2000). Zhou et al. (2004) reported
that wheat grain, bran, and fractions had different antioxidant activities and
TPCs. Their study also showed that FA was a major contributor to the anti-
oxidant activity.
Antioxidant activity of bran extracts from five wheat varieties indigenous
to Pakistan, that is, Punjab-96, Bhakkar-2002, Uqab-2000, SH- 2002, and
Pasban-90, was evaluated (Iqbal et al., 2007). All the bran extracts exhibited
appreciable TPC (2.12–3.37 mg GAE/g bran), total flavonoid content (epicat-
echin equivalent 262–304 mg/g bran), chelating activity (EDTA equivalent
597–716 mg/g bran), DPPH radical scavenging activity (51–79%), ABTS
radical cation scavenging activity (Trolox equivalent 27–36 mmol/g), oxygen
radical absorbance capacity (ORAC) (97–123 mmol/g), and TA content
(30–38 mg/kg bran). Tocopherol (22–26 ppm) and tocotrienol content
(59–74 ppm) were determined by reversed phase HPLC (RP-HPLC). It was
observed that all the varieties exhibited appreciable antioxidant potential
and significant differences were observed among the varieties in different
systems of antioxidant activity evaluation.
Four different types of wheat bran were extracted and analyzed for
phenolic acids using the Folin–Ciocalteu method and HPLC (Kim et al.,
2006). The extracts and their hydrolysis products were also evaluated for
antioxidant activities. The TPC of the red wheat bran was found higher
than that of the bran from white wheat. The majority of the phenolic acids
existed in a bound form in wheat bran. These phenolic acids can be released
by hydrolyzing the bran under alkaline or acidic conditions; however, the
former was more efficient. Ferulic, vanillic, and syringic acids were the
major individual phenolic acids in the studied wheat bran. The major portion
of the total FA was from alkaline hydrolysis. The alkaline hydrolysable frac-
tions had greater antioxidant activities, while the acid hydrolysable frac-
tions showed lower activities in both the red and white bran. The antioxidant
activity of bran extract was stronger than that of free phenolic acids.
The impact of thermal processing on antioxidant activity of purple
wheat bran, heat-treated purple wheat bran was evaluated to assess potential
health benefits (Li et al., 2007). TPC and ORAC values of sample extracts
were significantly affected by various extracting solvents. The conditions
selected for heat treatment did not markedly change antioxidant activity
Antioxidant Dietary Fiber: An Approach to Develop Healthy 321

of purple wheat bran. Though, there was a significant reduction in TPCs,


ORAC values, and TAs during processing of purple wheat bran. Esposito et
al. (2005) selected the fractions of durum wheat bran having different func-
tional and nutritional characteristics. Wheat bran by-products were obtained
by an industrial milling process. Beside the single fractions, two commercial
products B&B 50 and B&B 70, obtained by blending some of the durum
wheat fractions were also studied. The soluble fiber content of the durum
wheat by-product ranged between 0.9 and 4.1%; while that of insoluble fiber
between 21 and 64%. The B&B 70 had a TDF content of 61%, while B&B 50
has 42%. These workers observed that water-holding capacity of each frac-
tion is strictly related to the amount of insoluble fiber and to the granulometry
of the by-products. The antioxidant activity was found higher for the internal
bran fraction and it increases in fractions having reduced granulometry.
Wheat bran DF powders was prepared by ultrafine grinding, whose
effects were investigated on the composition, hydration, and antioxidant
properties of the wheat bran DF products (Zhu et al., 2010). The results
showed that as particle size decrease, the hydration properties (water holding
capacity, water retention capacity, and swelling capacity) of wheat bran DF
were significantly decreased and a redistribution of fiber components from
insoluble to soluble fractions was observed. Compared with DF before
and after grinding, micronized IDF showed increased chelating activity,
reducing power and total phenolic compounds yet decreased DPPH radical
scavenging activity.

8.5.3.2 MEXICAN CHIA SEED

The seeds of the species Salvia hispanica L. commonly known as “chia,”


“chia sage” and “Spanish sage,” were an important staple food, oil source
and medicine for Mesoamericans in pre-Columbian times. The curative
properties of the seeds were also appreciated, for example, for treating eye
obstructions, infections, and respiratory malaises. The seeds soaked in water
or fruit juice were and still are consumed in some regions as a refreshing
drink (Cahill, 2003). The presence of cinnamic, chlorogenic, and caffeic
acids together with the flavonoids, myricetin, quercetin, and kaempferol in
methanolic hydrolyzed extracts has also been reported (Taga et al., 1984).
Chia seeds from two different regions in the states of Jalisco and Sinaloa
were analyzed for soluble and insoluble fiber and antioxidant activity of
phenolic compounds (Reyes-Caudillo et al., 2008). The soluble and insol-
uble fiber content of the Sinaloa and Jalisco seeds was similar. The major
322 Natural Antioxidants: Applications in Foods of Animal Origin

compounds identified in hydrolyzed and crude extracts were quercetin and


kaempferol, while caffeic and chlorogenic acids were present in low concen-
trations. The crude extract of the Jalisco seed has an antioxidant activity
comparable to the commercial antioxidant Trolox used as a reference.
Different concentrations of the hydrolyzed and crude extracts of the seeds
from both regions showed antioxidant effect when tested in a model water-
in-oil food emulsion.

8.5.3.3 COCOA HUSK

Besides flavonoids, cocoa is rich in other component of significant nutri-


tional interest such as DF. Polyphenolic compounds usually accumulate in
the outer parts of plants such as shells, skins, and so forth (Bravo, 1998).
Though, information on the polyphenolic content of cocoa husks is very
limited. It has been suggested that cocoa hull may be a good source of
DF, with reported values ranging from 38 to 44% of TDF as NPSs plus
KL (Martín-Cabrejas et al., 1994; Serra-Bonvehí & Aragay-Benería, 1998).
Considering the health benefits associated to the consumption of DF and
polyphenols in the diet, the presence of both bioactive components in cocoa
bean husks could highlight the interest of this product as a potential ingre-
dient for the functional food industry.
Cocoa polyphenols have been suggested to positively influence cardio-
vascular health through inhibition of lipid peroxidation, platelet activation
or cyclo-oxygenase, and lipoxygenase activities, and enhancing levels of
the endothelial-derived relaxing factor, nitric oxide (Karim et al., 2000;
Rein et al., 2000; Schewe et al., 2002; Steinberg et al., 2003; Wan et al.,
2001; Wiswedel et al., 2004). Moreover, cocoa polyphenols have exhib-
ited antimutagenic activity (Yamagishi et al., 2000). A decreased levels of
8-hydroxy-20-deoxyguanosine, a biomarker of oxidative damage to DNA,
have been reported in rats after consumption of cocoa suggesting a potential
role in cancer (Orozco et al., 2003).
The proximate composition and DF content of a fiber-rich product
obtained from cocoa were studied (Lecumberri et al., 2007). This product
contained 60.54% (dry matter basis) of DF, composed of mainly insoluble
fiber although with appreciable amounts of SDF (10.09%). The presence of
associated polyphenolic compounds (1.32 and 4.46% of soluble polyphe-
nols and condensed tannins, respectively) provides this fiber material with
intrinsic antioxidant capacity as determined by the FRAP and trolox equiva-
lent antioxidant capacity (TEAC) methods.
Antioxidant Dietary Fiber: An Approach to Develop Healthy 323

8.5.3.4 AMARANTH AND QUINOA

Amaranth (Amaranthus hypochondriacus) is an ancient and very nutritious


food crop cultivated mainly in South America and Mexico, but grows also
very well across the world. Of late, the use of amaranth and quinoa has
broadened not only in the common diet, but also in diet of people with celiac
disease or allergies to typical cereals (Berti et al., 2005). These pseudocer-
eals seeds have high nutritional and functional values which are associated
with the quality and quantity of their proteins, fats, and antioxidant poten-
tial (Gorinstein et al., 2002; Gorinstein et al., 2007; Paśko et al., 2007).
Amaranth is a crop naturally resistant to water deficit and is a good source
of nutritious seeds. Barba de la Rosa et al. (2009) analyzed physical and
proximal-nutritional properties of amaranth seeds obtained from different
varieties (Tulyehualco, Nutrisol DGETI, and Gabriela) and characterized
their phenolic acids and flavonoids. Polyphenols as rutin (4.0–10.2 mg/g
flour) and nicotiflorin (7.2–4.8 mg/g flour) were detected.
Two varieties (Centenario and Oscar Blanco) of Andean native grain,
kiwicha (Amaranthus caudatus), were evaluated as sources of DF and of
some bioactive compounds (Repo-Carrasco-Valencia et al., 2009). The
impact of low-cost extrusion on the content of these components was studied
for technological applications. The content of TDF in Centenario was higher
(16.4%) than in Oscar Blanco (13.8%). The extrusion process decreased the
total and IDF contents in both varieties while in Centenario, the content of
SDF increased, from 2.5 to 3.1%. The content of phytic acid in raw kiwicha
was 0.3% for both varieties, and the content of total phenolic compounds was
98.7 and 112.9 mg GAE/100 g of sample, for Centenario and Oscar Blanco,
respectively. Antioxidant activity of both the varieties of raw kiwicha was
evaluated through DPPH and ABTS methods. The content of total phenolics,
phytic acid and the antioxidant activity decreased in both varieties during the
extrusion process.
The consumption of sprouts—the atypical vegetable, is becoming
a new trend which has received attention as functional foods, because of
their nutritive value including amino acid, fiber, trace elements, vitamins
as well as flavonoids, and phenolic acids (Paśko et al., 2008). Its intake has
become very popular among people interested in improving and maintaining
their health status by changing dietary habits. The sprouts of amaranth and
quinoa are new vegetables, which can be used as a source of nutrition. Total
antioxidant capacity, TPCs and anthocyanins contents were determined in
Amaranthus cruentus and Chenopodium quinoa seeds and sprouts (Paśko
et al., 2009). Sprouts activity depended on the length of their growth, and
324 Natural Antioxidants: Applications in Foods of Animal Origin

the peak values were reached on the fourth day in the case of amaranth and
on the sixth day in the case of quinoa. It was suggested that amaranth and
quinoa seeds and sprouts can be used in food, because it is a good source of
anthocyanins and total phenolics with high antioxidant activity.

8.5.4 OTHER SOURCES

8.5.4.1 BURDOCK ROOT

It is a source of inulin and popular vegetable in Japan. Burdock root has been
extensively analyzed for its components due to their antioxidant properties
(Chow et al., 1997) as well as for its extractable components having antimi-
crobial activity (Duh, 1998; Lin et al., 1996). The simultaneous ultrasonic/
microwave assisted extraction (UMAE) of inulin and production of phenols
rich DF powder from burdock root was studied (Lou et al., 2009). The DF
powder prepared from the residue of burdock root after inulin extraction
was rich in phenols (302.62 mg GAE/100 g powder). It was seen that drying
temperature in the selected range did not significantly affect the hydration
properties.

8.5.4.2 HIBISCUS

Hibiscus sabdariffa L., commonly known as roselle, red sorrel, or karkadè,


is widely grown in Africa, South East Asia, and some tropical countries
of America. Its fleshy flowers provide a soft drink consumed as a cold or
hot beverage. Pharmacological actions have been identified in H. sabdar-
iffa L. flowers, petals, and seeds (Ali-Bradeldin et al., 2005). Roselle is an
important source of vitamins, minerals, and bioactive compounds, such as
organic acids, phytosterols, and polyphenols. The phenolic content in the
plant consists mainly of anthocyanins like delphinidin-3-glucoside, sambu-
bioside, and cyanidin-3- sambubioside; other flavonoids like gossypetin,
hibiscetin, and their respective glycosides; protocatechuic acid, eugenol, and
sterols like—sitosterol and ergosterol (Ali-Bradeldin et al., 2005). The health
effects include cardioprotective action; reduction of urinary concentrations
of creatinine, uric acid, citrate, tartrate, calcium, sodium, potassium, phos-
phate; antihypertensive action; effectiveness against low-density lipoprotein
oxidation and hyperlipidemia (Herrera-Arellano et al., 2004; Jonadet et al.,
1990; Chen et al., 2003; Mojiminiyi et al., 2000; Odigie et al., 2003). In a
Antioxidant Dietary Fiber: An Approach to Develop Healthy 325

study on roselle flower and beverage prepared from it, Sáyago-Ayerdi et al.
(2007) quantified the DF, associated polyphenols, and antioxidant capacity.
It was reported that roselle flower contained DF as the largest component
(33.9%) and was rich in phenolic compounds (6.13%).

8.5.4.3 SEAWEED

Fucus vesiculosus L. is a brown-colored, perennial, dioecious edible


seaweed forming dense belts in cold rocky littoral habitats (Jiménez-Escrig
et al., 2001b; Jormalainen & Honkanen, 2004), covering large areas from a
few decimeters below the water surface to a depth of several meters (Nilsson
et al., 2004). It contains protein, minerals, iodine, vitamins, monounsatu-
rated and polyunsaturated fatty acids (Herbreteau et al., 1997; Rupérez &
Saura-Calixto, 2001; Rupérez et al., 2002; Morel et al., 2005). However,
the main components that make Fucus nutritionally significant are non-
digestible polysaccharides (DF) and polyphenols. DF from seaweeds has
proven to have a positive effect on cholesterol metabolism and blood pres-
sure (Jiménez-Escrig & Sánchez-Muñiz, 2000). DF of F. vesiculosus is
composed of fucans, alginates, laminaranes, and cellulose, with fucoidan as
the predominant polysaccharide (Rioux et al., 2007). Fucoidan as observed
in several studies in rats and humans, showing beneficial effects as an anti-
coagulant, antithrombotic, antiviral, and anti-cancer agent (Béress et al.,
1993; Aisa et al., 2005), as well as in the treatment of chronic renal failure
(Zhang et al., 2003). F. vesiculosus has shown high antioxidant capacity by
several methods (Morel et al., 2005) due to the synergic effect of phloro-
tannins, vitamin E, and certain carotenoids (Le Tutour et al., 1998; Toth &
Pavia, 2001).
The presence of the functional components DF and antioxidants in F.
vesiculosus in a higher proportion than in other edible seaweeds (Jiménez-
Escrig et al., 2001b; Rupérez & Toledano, 2003) has led to the development
of a large number of functional ingredients and dietary supplements derived
from this like fucoidan powders, F. vesiculosus capsules, or F. vesiculosus
antioxidant extracts. Díaz-Rubio et al. (2009) compared the antioxidant
capacity and polysaccharide composition of raw Fucus with those of some
common commercial nutraceuticals. All tested products contained a high
percentage of DF (45–59%), raw Fucus powder being the sample with the
highest content. Moreover, Fucus powder exhibited significantly higher
antioxidant capacity as determined by FRAP, ABTS, and ORAC assays than
the commercial fucoidans and commercial antioxidant extracts. Polyphenols
326 Natural Antioxidants: Applications in Foods of Animal Origin

(phlorotannins) seem to be the main contributors to antioxidant capacity in


both raw powder and commercial fucoidans.

8.6 APPLICATION IN MEAT PRODUCTS

In addition to promoting consumers health, application of ADF in different


meat foods can play an important role in maintaining their quality and exten-
sion of storage stability. Incorporation of ADF in meat products is a quite
new concept in the area of developing functional meat products thus the
available literatures on ADFs in meat system are limited. The positive effects
of ADF on meat and meat products are depicted in Figure 8.2. The effects of
ADFs in meat products may vary according to their source, physicochemical
characteristics, amount of phenolic compounds and TDF as well as ratio of
soluble to IDF.

Prevention of lipid
and protein
oxidation

Improvement in
Antioxidant
quality and stability

Free radical
quenching

Antioxidant Dietary Batter stability and


Fiber yield

Color

Dietary Fiber Texture

Acceptability

Health value

FIGURE 8.2 Positive effects of incorporation of ADF in meat or meat products.


Antioxidant Dietary Fiber: An Approach to Develop Healthy 327

The effect of grape antioxidant dietary fiber (GADF) addition (0, 2, and
4%) to minced fish muscle (MFM) on lipid stability during frozen storage (6
months) was studied (Sánchez-Alonso et al., 2007). GADF was character-
ized in terms of DF, total polyphenols and antioxidant capacity, and multi-
functional antioxidant assays were carried out on all the MFM samples. The
addition of red grape fiber considerably delayed lipid oxidation in minced
horse mackerel (Trachurus trachurus) muscle during the first 3 months
of frozen storage. In another study, white grape antioxidant dietary fiber
(WGDF) obtained from white grape (V. vinifera, var. Airén) pomace from
wine production was evaluated for antioxidant capacity in MFM during
frozen storage at three different levels viz., 0, 2, and 4% (Sánchez-Alonso et
al., 2008). WGDF was evaluated for DF (insoluble and soluble), total poly-
phenols and antioxidant capacity, and multifunctional antioxidant assays
were done on all the MFM samples. The addition of WGDF considerably
delayed lipid oxidation in minced horse mackerel muscle during the frozen
storage. Vacuum-packing the sample with 2% WGDF significantly enhanced
the antioxidant properties of WGDF.
Sánchez-Alonso and Borderías (2008) again investigated the effect of
adding GADF at same levels to horse mackerel minced muscle as a techno-
logical ingredient in MFM for over six months of frozen storage (−20 °C).
Protein solubility, water retention, color, mechanical properties, lipid oxida-
tion, and sensory analyses were carried out immediately after preparation
of samples, and during and after storage. Bound water after thawing and
cooking minced samples was proportional to the amount of GADF used.
Mechanical properties indicated softness and loss of cohesiveness depending
on the amount of GADF. There was inhibition of lipid oxidation during
frozen storage when GADF was added. Based on chemical, physical, and
sensory analyses it was suggested that GADF is a highly active technological
ingredient in frozen dark minced fish. Grape pomace concentrate (GPC) is
a natural source of phenolic compounds with high antioxidant capacity. The
effect of a diet containing GPC on lipid peroxidation levels via measuring
thiobarbituric reactive substances (TBARS) and antioxidant capacity
(ABTS method) of raw and cooked chicken breast meat patties stored in
chilled conditions (4 °C) for 0, 3, 6, 13, and 20 days, and frozen storage
(six months) was investigated (Sáyago-Ayerdi et al., 2009a). Chickens were
fed GPC at levels of 0, 30, and 60 mg/kg from three to six weeks of age.
Dietary GPC significantly exerted an inhibitory effect on lipid oxidation of
raw and cooked breast chicken patties compared with samples obtained from
birds fed the control diet at chilling and frozen storage. Radical scavenging
capacity was significantly increased at 20 days in cooked samples and
328 Natural Antioxidants: Applications in Foods of Animal Origin

significantly reduced at six months of storage in raw and cooked samples.


The higher concentration of dietary GPC increased the ABTS values only in
the raw samples. The researchers stated that dietary GPC could be effective
in inhibiting lipid oxidation of chilled and frozen stored chicken patties.
Efficiency of four concentrations (0.5, 1, 1.5, and 2%) of GADF on
susceptibility of raw and cooked chicken breast hamburger to lipid oxida-
tion was investigated after 0, 3, 5, and 13 days of refrigerated storage at
4 °C (Sáyago-Ayerdi et al., 2009b). A significant reduction in lightness
and yellowness and increase in redness as a result of GADF addition were
observed in raw and cooked chicken hamburgers. Addition of GADF signifi-
cantly improved the oxidative stability and the radical scavenging activity in
raw and cooked chicken hamburgers. The ability of GADF to prevent lipid
oxidation was concentration-dependent. Acceptability of chicken meat was
not affected by the addition of GADF.
Guava powder (0.5 and 1 %) was used as a source of ADF in sheep meat
nuggets and its effect was evaluated against control (Verma et al., 2013).
Guava powder was found rich in DF (43.21%), phenolics (44.04 mgGAE/g)
and possessed good radical scavenging activity as well as reducing power.
Total phenolics and TDF content significantly increased in nuggets with
added guava powder. Product redness value was significantly improved due
to guava powder. Guava powder was found to retard lipid peroxidation of
cooked sheep meat nuggets as measured by TBARS number during refriger-
ated storage. Acceptability of the product remained unchanged due to addi-
tion of guava powder.
The antioxidant potential of bael (Aegle marmelos L.) pulp residue
(BPR) and its influence at two different levels (0.25 and 0.5%) as an ADF
on the quality of goat meat nuggets was investigated (Das et al., 2015). The
antioxidant potential (total phenolics, radical-scavenging activity, and ferric
reducing antioxidant power) and DF content of BPR were evaluated. BPR
contained good amount of total phenolics (15.16 mgGAE/g dry weight) and
DF (56.91%). BPR significantly improved the emulsion stability, cooking
yield, ash, total phenolics, DF, and color characteristics of the meat products.
On another side, BPR decreased the hardness, gumminess, and chewiness.
Sensory evaluation of the products revealed significant improvement in the
appearance score and non-significant increase in the score of other attributes.
BPR decreased lipid peroxidation and microbial counts in meat products
during 21 days of refrigerated storage (4±1 °C) period. It was concluded that
BPR being a rich in bioactive components such as phenolic compounds and
DF, could be used as an ADF in muscle food products without affecting its
quality and acceptability.
Antioxidant Dietary Fiber: An Approach to Develop Healthy 329

8.7 CONCLUSION

The threats of non-communicable diseases which are associated with the


life-style and diet have forced modern consumers to look toward functional
foods especially meat products which can be the source of DF and anti-
oxidants. Almost every plant-based materials are rich in natural antioxi-
dants which may be associated with the DF leading to the concept of ADF
way back in 1998. Inclusion of these materials into meat products can help
in improving their technological and functional characteristics as well as
stability while storage. It is also supposed that regular consumption of such
functional ADF rich meat products may improve the physiological status of
consumers. The incorporation of such functional ingredients in meat prod-
ucts is a newer concept and very scant works are available in the literature.
The impacts of adding ADF on the quality, acceptability, and stability of
the meat product have been reported to be very much interesting. However,
assessment of the effects on population consuming these products is some-
what missing till now. In the near future it is hoped that a lots of ADF with
defined amount of natural antioxidants and DF would be explored. Addi-
tionally these ingredients would be attempted in the meat products and their
impact on meat products quality as well as consumer health would assessed.

KEYWORDS

• antioxidant dietary fiber


• dietary fiber
• natural antioxidants
• meat products

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CHAPTER 9

CONTROL OF LIPID OXIDATION


IN MUSCLE FOOD BY ACTIVE
PACKAGING TECHNOLOGY
JOSÉ M. LORENZO1,*, RUBEN DOMÍNGUEZ1, and
JAVIER CARBALLO2
1
Centro Tecnológico de la Carne de Galicia, Rua Galicia No. 4,
Parque Tecnológico de Galicia, San Cibrao das Viñas, Ourense
32900, Spain
Área de Tecnología de los Alimentos, Facultad de Ciencias de
2

Ourense, Universidad de Vigo, Ourense 32004, Spain


Corresponding author. E-mail: [email protected]
*

CONTENTS

Abstract ....................................................................................................344
9.1 Introduction .....................................................................................344
9.2 Lipid Oxidation ...............................................................................345
9.3 Antioxidants ....................................................................................354
9.4 Modified Atmosphere Packaging (MAP) .......................................364
9.5 Active Packaging ............................................................................367
9.6 Conclusion ......................................................................................373
Keywords .................................................................................................374
References ................................................................................................374
344 Natural Antioxidants: Applications in Foods of Animal Origin

ABSTRACT

Lipid oxidation is a major cause of deterioration in meat, and preventing this


alteration process during storage is actually a major challenge for the food
technology. The function of food packaging has evolved from a simple phys-
ical barrier to include more specific aspects of convenience for approaching
to specific concerns, and active packaging seems to be an effective tool to
prevent lipid oxidation in meat and increase shelf life.
In this chapter, after a brief reminder on the lipid oxidation in meat, the
natural and artificial antioxidants used in foods, and the modified atmo-
sphere packaging (MAP), and its effect on the oxidation processes in meat,
the active packaging as a solution against oxidative processes during meat
storage is treated. The modes of action for antioxidant packages, the criteria
of selection of the antioxidant compounds used, the methodologies for
producing antioxidant packaging systems, and the materials for food active
antioxidant packaging are addressed, and information in literature on the
use of antioxidant packaging in the preventing lipid oxidation during meat
storage are reviewed.

9.1 INTRODUCTION

Lipid oxidation is a major cause of quality deterioration in meat, which leads


to off-odors and off-flavors, which are usually described as rancid (Gray &
Pearson, 1994) as well as discoloration or texture changes of muscle foods
during refrigerated storage (Kanner, 1994; Shahidi, 2002; Gong et al., 2010).
In addition, oxidation causes loss of nutritional values, and generates and
accumulates compounds that may pose continual risks to human health
(Kanner, 1994; Min & Ahn, 2005).
Nowadays, consumers are finding less time to prepare meals. Food
industry is responding to this by increasing the availability of pre-cooked
meats. However, as in fresh meat, the major problem with precooked meats
is the development of an objectionable warmed-over flavor via lipid oxida-
tion (Ang & Lyon, 1990). Therefore, lipid oxidation needs to be controlled
during storage in order to prevent the formation of off-odors and off-flavors
in foods (Richards, 2006). The first step is know the specific changes that
these foods undergo to select appropriate packaging material and package
format options, and so minimizes quality loss (Krotcha, 2006).
One method is to reduce the concentration of oxygen in the fat by
packing the products under vacuum or nitrogen (Chu & Hwang, 2002).
Control of Lipid Oxidation in Muscle Food 345

Nevertheless the fresh meat must be packaged with an atmosphere rich


in O2 to maintain the red color of the meat. In fact, red meat is usually
packaged in modified atmosphere packaging (MAP) with 70–80% O2. The
drawback to high O2 MAP is that although it maintains redness during
storage, rancidity often develops in the meat while color is still desirable
(Jayasingh et al., 2002).
Other method is the use of synthetic antioxidants such as butylated
hydroxy anisole (BHA). However, consumers’ concerns about the use of
artificial preservatives in meat products have been increased because of their
possible toxicity to human health. Consequently, attention has focused on
the use of natural antioxidants to replace synthetic antioxidants (Min & Ahn,
2012). Different antioxidant agents, such as rosemary extract, tocopherol,
ascorbic acid, and different plant extracts may be successfully included in
bio-based films, to decrease oxidative reactions in meat products (Coma &
Kerry, 2012).
Research and development in the area of active packaging systems for
meat products has received much attention recently and will continue to
do so in the near future (Walsh & Kerry, 2002). Packaging materials with
antioxidant properties could be particularly efficient (Nabrzyski, 2002). In
fact, a number of active packaging technologies for meat-based products
have been extensively reviewed (Kerry et al., 2006; Hogan & Kerry, 2008;
O’Grady & Kerry, 2008).
Therefore, the simultaneous application of both natural antioxidant and
MAP not only meets consumer demands for replacement of synthetic preser-
vatives, but also provides stronger protective effects on lipid oxidation in
fresh meat (Min & Ahn, 2012).

9.2 LIPID OXIDATION

9.2.1 FACTORS AFFECTING THE DEVELOPMENT OF LIPID


OXIDATION IN MEAT

There are many factors that affect the development of oxidative rancidity
in meat, some of them are intrinsic, such as species, muscle type, amount
and type of fat in the diet, enzymes, differences in fat content and fatty
acid composition, endogenous antioxidants (carnosine and related dipep-
tides), and others extrinsic such as storage conditions, O2 concentration, and
processing treatments (heat, mincing, irradiation, etc.).
346 Natural Antioxidants: Applications in Foods of Animal Origin

9.2.1.1 MEAT COMPOSITION

Two factors greatly influence lipid oxidation in raw meat are fat content and
fatty acid composition. According to Min et al. (2008), the composition of
fat is more important than the amount of fat in meat, because the suscepti-
bility of muscle lipid to lipid peroxidation depends upon the degree of poly-
unsaturation in fatty acids. Unsaturated lipids are generally more susceptible
to lipid oxidation because hydrogen atoms can be more easily abstracted
from polyunsaturated fats than saturated fats (Kanner et al., 1987; Gong et
al., 2010). In fact, fats containing high proportions of linoleic or linolenic
acids are more prone to oxidation than oils high in oleic acid. Thus one nutri-
tional effect of oxidation is to reduce the essential fatty acid content of fats.
It is thought that the polyunsaturated fatty acids from polar phospholipids
rather than triglycerides are responsible for the initial development of lipid
oxidation in muscle foods (Renerre & Ladabie, 1993). During the course of
oxidation, the total unsaturated fatty acid content of lipids decreases with a
concurrent increase in the amount of primary and secondary oxidation prod-
ucts such as lipid hydroperoxides, aldehydes, ketones, hydrocarbons, and
alcohols. Therefore, rancidity in food occurs when unsaturated fatty acids
decompose into volatile compounds. Increasing levels of unsaturated fatty
acids in meat increase lipid oxidation rates (and rancidity) and thus decrease
shelf life of the muscle foods. The autoxidation rate greatly depends on the
rate of fatty acid or acylglycerol alkyl radical formation, and the radical
formation rate depends mainly on the types of fatty acid or acylglycerol.
Consequently, the susceptibility of meat to lipid peroxidation varies
among meats from different animal species and muscles from the same
animal (Min et al., 2008). There are numerous studies showing that both
the species and fat location significantly affects fatty acid composition of
the meat. Cava et al. (2003) found that muscles with higher proportions of
phospholipids also presented higher amounts of polyunsaturated fatty acids.
In the same way, Domínguez et al. (2015) also observed that Psoas major
muscle (oxidative muscle) had significantly higher amounts of polyunsatu-
rated fatty acids than Longissimus dorsi (glycolytic muscle). Differences
in fatty acid composition between oxidative and glycolytic muscles might
be due to a higher number of cellular and sub-cellular membranes, and the
difference in the ratio of mitochondria to other membranes between oxida-
tive and glycolytic muscles. Therefore, the different polyunsaturated fatty
acid amounts making lipids from oxidative muscles more susceptible to
oxidative processes than those from the glycolytic muscles.
Control of Lipid Oxidation in Muscle Food 347

Regarding to the animal species, poultry meats which contain high


levels of polyunsaturated fatty acids are most susceptible to lipid oxida-
tion, followed by pork, beef, and lamb (Cross et al., 1987). In fact, a higher
proportion of unsaturated fatty acids in the triglycerides of pork and chicken,
compared with beef or lamb, produce more unsaturated volatile aldehydes
in these meats and these compounds may be important in determining the
specific aromas of meat species (Mottram, 1991).

9.2.1.2 PROCESSING AND STORAGE CONDITIONS

However, not only the meat composition affects lipid oxidation. Other
factors, such as processing and storage conditions have a great impact on
meat oxidation.

a) Irradiation

Irradiation is a preservation method that has been more extensively investi-


gated for preservation of poultry than red meats (Morehouse, 2002; Argyri
et al., 2012). According to Kanatt et al. (2005), who investigated the effect
of irradiation processing on the quality of chilled meat products, concluded
that irradiated samples showed significantly higher thiobarbituric reactive
substances (TBARS) values than non-irradiated. In addition, the increase
in TBARS values was dose-dependent. Katusin-Razem et al. (1992) also
reported that irradiation of pork and poultry meat accelerates lipid oxidation.
This fact is due to that when ionization radiation is absorbed by matter, ions,
and excited molecules are produced. These ions and excited molecules can
dissociate to form free radicals (Richards, 2006).
Moreover, not only the dose increases lipid oxidation, but the type of
packaging also has great importance in meat oxidation. To this regard,
Nam and Ahn (2003a, 2003b) studied the effects of combining aerobic and
anaerobic packaging and the oxidant combinations on color, lipid oxidation,
and volatile production to establish a modified packaging method to control
quality changes in irradiated raw turkey meat. These authors reported that
lipid oxidation is the major problem with aerobically packaged irradiated
turkey breast, and concluded that the combination of double packaging and
antioxidants was more effective in reducing sulfur volatiles and lipid oxida-
tion, when compared with aerobic packaging.
348 Natural Antioxidants: Applications in Foods of Animal Origin

b) Cooking

A typical treatment that greatly affects lipid oxidation is cooking. Heating


accelerates lipid peroxidation and volatile production in meat (Broncano et
al., 2009; Alfaia et al., 2010; Domínguez et al., 2014a, 2014b) by disrupting
muscle cell structure, inactivating antioxidant enzymes, and other antioxi-
dant compounds. In addition, cooking has the ability to increase iron concen-
trations in biological systems (which act as pro-oxidant) by stimulating the
release of iron from heme-proteins (Decker & Welch, 1990; Smiddy et al.,
2002; Richards, 2006). The resultant breakup of cell compartments permits
the interaction of pro-oxidants with unsaturated fatty acids and oxygen, the
generation of free radicals and propagation of the oxidative reaction (Asghar
et al., 1988). High temperature causes reduction of activation energy for
lipid peroxidation and decomposes preformed hydroperoxides to free radi-
cals, which stimulates autoxidation process and off-flavor development
further (Min et al., 2008).
In lipid oxidation, the most important parameters are the conditions
of heat treatment (temperature and time of cooking) (Byrne et al., 2002;
Domínguez et al., 2014a). The use of high temperature during cooking
causes an increase of the oxidation processes in meat (Broncano et al.,
2009). However, according to Domínguez et al. (2014a) oxidation processes
during cooking are more affected by cooking time than temperature. There-
fore, the application of heat during a long time produces higher oxidation
compared to the changes caused by the use of a higher temperature during
a shorter time.
The type of heating method used also has a great effect on the lipid
oxidation. While it is widely accepted that the microwave oven has greatly
contributed to the daily lives of modern society, the use of microwave
increase the lipid oxidation. Domínguez et al. (2014a) found that meat
cooked with microwave oven showed the highest values of TBARS. The fact
that samples cooked by microwave had high levels of oxidation compounds
suggests some interaction between microwave and meat fat which causes
oxidation of polyunsaturated fatty acids (Broncano et al., 2009).

c) High pressure

High-pressure processing is a non-thermal technology, which applies pres-


sures up to 1000 MPa for a variable time. It has been reported as a preserva-
tion method as it is able to extend the shelf life of food without modifying its
Control of Lipid Oxidation in Muscle Food 349

sensory properties or nutrient content (Cheftel & Culioli, 1997; Hendrickx


et al., 1998). However, there are several studies that relate the treatment of
meat with high pressures and increased oxidation. Regarding to this, Cheah
and Ledward (1996) showed that high pressure (800 MPa, 20 min) treated
pork mince samples revealed faster oxidation than control samples, and
that pressure treatment at greater than 300–400 MPa caused conversion of
reduced myoglobin/oxymyoglobin to the denatured ferric form. According
to Orlien and Hansen (2000), 500 MPa is a critical pressure for lipid oxida-
tion and development of rancidity in chicken breast muscle. Therefore it
appears that the iron released from metal complexes during pressure treat-
ment catalyzed lipid oxidation in meat (Cheah & Ledward, 1997) but it also
be related to membrane damage.

d) Mincing

A typical way of finding the meat is like minced meat. However, this treat-
ment has a great effect on rancidity development. It is well known that
compartmentation of cellular and extracellular reactants should be critical
in controlling rates of lipid oxidation. Therefore, mincing can cause signifi-
cant disruption of the cellular compartmentalization structure which facili-
tates the meeting of pro-oxidants with unsaturated fatty acids resulting in
the generation of free radicals and propagation of the oxidative reaction
(Buckley et al., 1995; Walsh & Kerry, 2002). According to Takama et al.
(1974), minced flesh was susceptible to rancidity due to the dispersed blood
pigments in the meat caused by the mechanical destruction of the tissue. In
addition, other study concluded that TBARS values increase most rapidly
with decreasing particle sizes, as the latter are related to greater cell disrup-
tion (Ladikos & Lougovois, 1990).

e) Light

Usually the meat is exposed in a supermarket to be attractive to consumers,


and therefore it is directly exposed to light. This fact increases the oxidation
of fatty acids. In addition, photo-oxidation is much faster than autoxida-
tion. To this regard, small amounts of O2 (for example in MAP packaging),
when combined with exposure to light, cause significant oxidative deterio-
ration of products (Jakobsen et al., 2005). This is due to ultraviolet radia-
tion decomposes existing hydroperoxides, peroxides, and carbonyl and other
350 Natural Antioxidants: Applications in Foods of Animal Origin

oxygen-containing compounds, producing radicals that initiate autoxidation


(Frankel, 1998).

9.2.1.3 PRO-OXIDANT FACTORS

a) Metals

Muscle contains notable amounts of iron, a known pro-oxidant, and trace


amounts of copper, which are potent catalysts of lipid oxidation (Richards,
2006). Iron is a part of the active site of lipoxygenase, which may participate
in lipid oxidation (Nabrzyski, 2002). These metals are believed to be pivotal
in the generation of species capable of abstracting a proton from an unsatu-
rated fatty acid (Gutteridge & Halliwell, 1990; Kanner, 1994).
The reaction between ferrous ion and oxygen produce hydrogen peroxide:

Fe2+ + O2  Fe3+ + O2−•

O2−• + 2H+  H2O2

Ferrous iron can also then react with H2O2 or preformed lipid hydroper-
oxides to produce hydroxyl or alkoxyl, and hydroxyl radicals, respectively:

Fe2+ + H2O2  Fe3+ + OH− + •OH−

Fe2+ + ROOH  Fe3+ + •OH− + RO−•

b) Heme-proteins

Hemoglobin and myoglobin are the predominant heme-proteins in muscle


foods. Therefore, it is easy to imagine that meat with higher proportion of
heme-proteins (such as pork, beef, or horse) is more susceptible to lipid
oxidation than meat with lower amounts of heme-proteins (such as chicken
or turkey). Similarly, to the above in the section of metals, heme-proteins can
react with lipid hydroperoxides to produce alkoxyl and hydroxyl radicals:

ROOH + Fe2+-complex  Fe3+-complex  RO• + OH−

ROOH + Fe3+-complex  ROO• + H+ + Fe2+-complex


Control of Lipid Oxidation in Muscle Food 351

The ability of heme pigments to accelerate the propagation step of the


free-radical chain mechanism can explain the rapid rate of oxidation in
cooked meats (O’sullivan & Kerry, 2012).

c) Enzymes

Various endogenous enzymes are of great importance in the development of


rancidity. The oxidation of fatty acids may occur either directly or indirectly
through the action of enzyme systems, of which three major groups are
involved: microsomal enzymes, peroxidases, and dioxygenases (Erickson,
2002).
Lipoxygenases is capable of the hydrogen abstraction from a polyun-
saturated fatty acid even in polar lipids bound to membrane to generate lipid
hydroperoxides. Therefore, lipoxygenase can be involved in the initiation of
lipid peroxidation of meat (Min et al., 2008).
The off-flavor is due to the volatiles that are produced from breakdown
of the lipoxygenase-derived lipid hydroperoxides (Richards, 2006). These
enzymes may also be responsible for formation of rancid odors by providing
critical amounts of lipid hydroperoxides that can be broken down by metals
or heme-proteins to produce rancid odor.

9.2.1.4 ANTIOXIDANT FACTORS

The addition of antioxidants is the most commonly used method of retarding


lipid oxidation in fat. Antioxidants increase the stability of food components,
especially polyunsaturated lipids, and maintain nutritional value and color
by preventing oxidative rancidity, degradation, and discoloration. However,
it is important to note that any compound that is antioxidative under one set
of conditions can become pro-oxidative under different conditions. As an
example of this point, ascorbate has been found to both inhibit and accel-
erate lipid oxidation depending on the concentration of linoleate hydroper-
oxides in the system (Kanner & Mendel, 1977). In addition, antioxidants are
required to be approved for the intended use. It has been suggested that an
ideal antioxidant food quality should has the following characteristics:

9 no harmful physiological effects


9 absence of undesirable effects on color, odor, or flavor
9 effective at low concentrations
352 Natural Antioxidants: Applications in Foods of Animal Origin

9 compatibility with the food and ease of application


9 survive after processing and be stable in the finished product
9 available at low cost.

The compound and its oxidation products must also be nontoxic, even at
doses much larger than those that normally would be ingested in food.
Antioxidants can be classified according to the mechanism of action into
two groups:

9.2.1.4.1 Primary Antioxidants

Primary antioxidants interfere with autoxidation by interrupting the chain


propagation mechanism. Primary antioxidants are free radical acceptors
(Wasowicz et al., 2004). The best-known and most effective primary anti-
oxidant substances are polyphenols. They react with the chain-propagating
radical species, which results in the formation of radical species incapable of
extracting hydrogen atoms from unsaturated lipids (Coma & Kerry, 2012).
Tocopherols inhibit lipid oxidation by scavenging of aqueous and
lipophilic free radicals as well as physical effects on membrane structure
(Buettner, 1993; Atkinson et al., 2008). Dietary antioxidant treatments (i.e.,
the inclusion of antioxidants in animal feed) have been shown to stabi-
lize lipids in membranes and reduce the extent of lipid oxidation in meat
during storage, but antioxidant effects in meat can differ between muscle
types (Morrissey et al., 1997; Ahn et al., 2006). Vitamin E in livestock diets
has been shown to reduce lipid oxidation in meats (Morrissey et al., 1998;
Álvarez et al., 2009). Batifoulier et al. (2002) reported that supplementation
of turkeys with α-tocopheryl acetate increased vitamin E content of micro-
somal membranes and had also a protective effect on lipid oxidation.
Some endogenous enzymes also have an antioxidant effect such as super-
oxide dismutase, catalase or glutathione peroxidase. These enzymes inhibit
lipid oxidation through the following mechanisms:

- Superoxide dismutase is present in cells and extracellular fluids


to remove −•O2 resulting in formation of oxygen and hydrogen
peroxide.
- Catalase, a heme-containing enzyme reacts with H2O2 to form water
and oxygen (Goth, 1987; Richards, 2006).
- Glutathione peroxidase reduces hydrogen peroxide and lipid
hydroperoxides to alcohols (Gong et al., 2010).
Control of Lipid Oxidation in Muscle Food 353

Therefore, the extent of these enzymes activities can be a determining


factor for the different rates of lipid oxidation in meat (Pradhan et al., 2000;
Min & Ahn, 2009).
In addition, some antioxidative peptides can be released from food
proteins. Jurewicz and Salmonowicz (1973) reported that DL-valine,
DL-methionine, OL-proline, and L-cysteine had antioxidant activity. More-
over, milk casein-derived peptides have been shown to have free radical
scavenging activity to inhibit enzymatic and non-enzymatic lipid oxidation
(Suetsuna et al., 2000; Rival et al., 2001a; Rival et al., 2001b).

9.2.1.4.2 Secondary Antioxidants

Secondary antioxidants, in opposite to the primary antioxidants, do not break


free radical chain but are able to stop the lipid oxidation through various
mechanisms (Wasowicz et al., 2004). There are some compounds who act as
secondary antioxidants:

a) Reducing agents

Reductants such as ascorbic acid, which decrease the local concentration of


oxygen, are also able to decrease the formation of peroxyl radicals (Ruiter &
Voragen, 2002). Ascorbate is believed to scavenge tocopherol free radicals
thereby regenerating tocopherol. Ascorbate can also scavenge various free
radicals such as −•O2, •OOH, and •OH. In addition, ascorbate reduces hyper-
valent to forms of heme proteins which inhibit lipid oxidation in muscle
foods (Kroger-Ohlsen & Skibsted, 1997).
Although ascorbic acid is mainly recognized as antioxidant, at low
concentrations ascorbic acid may act as a pro-oxidant, especially in the pres-
ence of metal-catalyzed oxidation. Ascorbic acid is able to reduce Fe3+ to
Fe2+. Nevertheless, the reduced Fe2+ catalyzes the breakdown of hydroper-
oxides to free radicals (as explained above) at a higher rate than Fe3+ (Ponce-
Alquicira, 2006).

b) Chelating agents

As mentioned previously, the presence of metals in fats greatly accelerates


the oxidation process. Inactivation of the catalysis effect of these metals can
be achieved by the use of a sequestering agent (Chu & Hwang, 2002).
354 Natural Antioxidants: Applications in Foods of Animal Origin

- Phosphates: Polyphosphates like sodium tri-polyphosphate are


excellent metal chelators and inhibitors against lipid oxidation.
However, when added to raw meat, they are ineffective due to rapid
hydrolysis to monophosphate by endogenous phosphatase enzyme
(Lee et al., 1998). But when this enzyme is denaturized (e.g., in
cooked meat) polyphosphates inhibited lipid oxidation (Sato &
Hegarty, 1971).
- EDTA: EDTA can inhibit lipid oxidation by forming an inactive
complex with metals.
- Citric acid: Citrate esters improve oil solubility but at least two free
carboxyl groups are needed for effective metal inactivation (Rich-
ards, 2006).
- Desferrioxamine: Desferrioxamine is often used as a metal
chelator, but this can lead to errant results since desferrioxamine
can also act as a free radical scavenger (Kanner & Harel, 1987;
Richards, 2006).
- Peptides: Both carnosine and anserine are endogenous antioxida-
tive dipeptides found in skeletal muscle at high concentrations
(Lynch & Kerry, 2000). They are known to be the most abundant
antioxidants in meats. It is capable of chelating copper, scavenging
peroxyl radicals, and forming adducts with aldehydes (Decker et al.,
2000). Histidine was found to inhibit non-enzymatic iron mediated
lipid oxidation apparently due to formation of an inactive chelate
but histidine was also found to activate enzymatic pathways of lipid
oxidation (Erickson & Hulin, 1992). In addition, carnosine, anserine,
histidine, lysine, albumin, and sulfur or amine containing compounds
have the ability to bind aldehydes and therefore decrease rancidity in
foods (Decker, 1998).

9.3 ANTIOXIDANTS

The use of molecules with antioxidant activity is the best solution for
preventing oxidative processes during storage and increasing the shelf life
of foods. Several molecules from different sources have been recognized
possessing this ability and used as antioxidants in foods, acting through
one or more of the mechanisms already described. We will comment the
most relevant compounds having this property, their characteristics, and
performances.
Control of Lipid Oxidation in Muscle Food 355

9.3.1 NATURAL ANTIOXIDANTS

Due to the damage caused by oxidations in live tissues, animals and vege-
tables accumulate antioxidant molecules as a mechanism of defense against
these undesirable changes. Most of these antioxidants are supplied by the
feed in the animals.
Several natural antioxidants are frequent in animal and vegetable tissues
as such or as precursors. We will shortly review the most representative:

9.3.1.1 PHENOLIC COMPOUNDS

Phenolic compounds are natural antioxidants widely distributed in vegetable


tissues. Its characteristic common chemical structure consist in a benzene
ring having an alcohol (hydroxyl) group bonded to a carbon atom (phenol).
Phenol itself has not antioxidant activity, but substitution of the hydrogen
atoms placed in the ortho- and para-positions with alkyl groups enhances its
reactivity toward free lipid radicals (Shahidi et al., 1992). Phenolics are clas-
sified as simple phenols or polyphenols, these having more than one phenol
unit in their molecules. Most of them are soluble and the smaller molecules
are usually volatiles.
Several polyphenols have antioxidant activity due to scavenging activity
on free radicals by donating a hydrogen atom or an electron to the free
radical and stabilizing it. They can also act as singlet oxygen quenchers and
also through the regulation of some concrete chelation reactions.
Briefly, three main groups of phenolic compound have a high-recognized
antioxidant activity in foods: tocopherols, flavonoids, and phenolic acids.
Tocopherols are a family of compounds naturally found in vegetable oils,
fish, nuts, and leafy green vegetables, which also have vitamin E activity.
Tocopherols derive from a common alcohol matrix named tocol (2-methyl-
2(4’, 8’, 12’-trimethyltridecyl)chroman-6-ol), and differ according to the
number and position of the methyl groups placed in the ring structure (chro-
manol ring), giving rise to different forms, called α, β, γ, and δ tocopherol.
The antioxidant activity of tocopherols increases from α to δ, while the
vitamin E activity and the reactivity with the peroxyl radicals decrease from
α to δ forms. Despite its low reactivity with the free radicals, the higher anti-
oxidant efficiency of the γ-tocopherol when compared to the α-tocopherol
is a consequence of the high stability of the γ-tocopherol and of the differ-
ence in the products formed in both cases during the antioxidative reactions
(Belitz et al., 2009). All the tocopherol forms have a higher rate of reaction
356 Natural Antioxidants: Applications in Foods of Animal Origin

with peroxyl radicals than BHA, due to the different nature of the radicals
formed on H-abstraction.
Tocopherols are approved as food additives with different E numbers:
E306 (tocopherol), E307 (α-tocopherol), E308 (γ-tocopherol), and E309
(δ-tocopherol).
Flavonoids are pigments widely distributed in vegetables where typically
impart a yellow color. Chemically, they have a general structure consisting
in a 15-carbon atoms skeleton integrated by two phenyl rings (named A and
B) and a heterocyclic ring (named C). Such carbon structure can be abbre-
viated C6-C3-C6 (A-C-B rings). The different classes of flavonoids differ in
the degree of oxidation and pattern of substitution in the C ring, while indi-
vidual compounds within a same class differ in the pattern of substitution
in the A and B rings (Pietta, 2000). Flavonoids, according to their chem-
ical structure are divided into five different classes: Anthoxanthins (which
include two subgroups, flavones, and flavonols), flavonones, flavanonols,
flavans (which include flavan-3-ols, flavan-4-ols, and flavan-3, 4-diols), and
anthocyanidins. The capacity of flavonoids to act as antioxidants in vitro has
been demonstrated by several studies, and important structure-antioxidant
activity relationships have been established (Pietta, 2000). Flavonoids are
generally primary antioxidants which act as free radical acceptors, breaking
the oxidation chain. Flavonols can also chelate metal ions at the 3-hydroxy-
4-keto-group, and/or the 5-hydroxy-4-keto-group (in the case in that the A
ring was hydroxylated at the fifth position).
It is generally recognized that the degree of hydroxylation and the posi-
tion of the hydroxyl groups determine the antioxidant activity of the flavo-
noids (Shahidi et al., 1992). The hydroxylation in the B ring is the major
factor for antioxidant activity. The o-dihydroxylation in the B ring actively
contributes to the antioxidant activity, and all the flavonoids with 3’-4’-dihy-
droxy configuration have antioxidant activity in more or less extent. Two
flavones, robinetin and myricetin, have an additional hydroxyl group placed
at their fifth position, which confers to these two molecules an enhanced
antioxidant activity in relation to the corresponding molecules that do not
possess such 5’-hydroxyl group (fisetin and quercetin). On the contrary, two
other flavones, naringenin, and hesperetin, have only a hydroxyl group in
the B ring, and due to this particularity they show little antioxidant activity.
Besides the hydroxylation in the B ring, other structural characteristics
affecting the A ring determine the antioxidant activity such as the presence
of a carbonyl group at the fourth position and a free hydroxyl group at third
and/or fifth positions.
Control of Lipid Oxidation in Muscle Food 357

Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one)
is the most abundant flavonoid in foods, and due to its antioxidant activity
and other beneficial properties has been object of special attention in the past
years (Alrawaiq & Abdullah, 2014). Although it has not been confirmed scien-
tifically as a specific therapeutic nor approved by any regulatory agency, it is
widely used as food supplement in the treatment of several health problems.
The Joint FAO/WHO Expert Committee on Food Additives evaluated quer-
cetin for use in food in 1977 (Harwood et al., 2007), but limited data on its
toxicity were available at the time of the evaluation which precluded the estab-
lishing an acceptable daily intake (ADI). In Japan, quercetin is permitted as
a food additive since the 1996 year (Harwood et al., 2007). Other flavonoids
such as myricetin or robinetin have a recognized high antioxidant activity. All
those compounds could be in the future efficient food antioxidant additives
after approval by the health authorities, upon proof of their harmlessness.
Phenolic acids are substances containing a phenolic ring and a carbox-
ylic function, therefore having a C6-C1 skeleton. They can be mono-, di- or
tri-hydroxybenzoic acids depending on the number of positions hydroxyl-
ated in the phenolic ring. The antioxidant activity of the phenolic acids and
their corresponding esters is determined by the number of hydroxyl groups.
Some concrete phenolic acids such as cafeic, coumaric, ferulic, gallic, and
protocatechuic acid are known as molecules possessing a not negligible
antioxidant activity. They could be in the future successfully used for this
purpose in foods after further studies on their stability and safety.

9.3.1.2 ASCORBIC ACID

Ascorbic acid (C6H8O6) ((5R)-[(1S)-1,2-Dihydroxyethyl]-3,4-dihydroxy-


furan-2(5h)-one) is a molecule with antioxidant and vitamin (vitamin C)
activities widely present in vegetables and fruits, and to a lesser extent in
animal tissues. It is oxidized with successive loss of two electrons to form
dehydroascorbic acid. It reacts with oxidants (reactive oxygen species),
such as the hydroxyl radical. Therefore, ascorbate can terminate these chain
radical reactions by electron transfer. Ascorbic acid is special because it can
transfer a single electron, due to the resonance-stabilized nature of its own
radical ion.
Ascorbic acid is an active antioxidant in aqueous media, because of its
water-soluble character, but only at high concentrations (around 10−3 mol/L).
A pro-oxidant activity is observed at lower concentrations (10−5 mol/L),
especially at high oxygen tensions and when heavy metal ions are present.
358 Natural Antioxidants: Applications in Foods of Animal Origin

This circumstance, however, seems to be irrelevant from the point of view


of the food chemistry concerns.
Ascorbic acid is, of course, approved and widely used in foods, being he
E300 additive.

9.3.1.3 CAROTENOIDS

Carotenoids are natural pigments which are synthesized by plants, being


responsible for the bright colors of several fruits and vegetables. There
are several dozen carotenoids in foods, and most of them have antioxidant
activity (Paiva & Russell, 1999). Beta-carotene and lycopene; however,
have been the best studied and more widely used ones.
Beta-carotene (C40H56) is a tetraterpene formed by eight isoprene units
having beta-rings at the two ends of the molecule. It is well known for
its provitamin A activity. With a strong lipophilic character, it acts as free
radical scavenger and therefore it has antioxidant properties widely demon-
strated in in vitro assays and in animal models. This antioxidant activity
is not lost by degradation to long chain breakdown products (Mueller &
Boehm, 2011). It shows, however, good antioxidant behavior only at partial
pressures of oxygen lower than 150 mm Hg. At higher oxygen pressure
values, β-carotene loses its antioxidant activity and, in contrary, it shows an
autocatalytic, pro-oxidant effect, particularly at relatively high concentra-
tions (higher than 5 × 10−5 mol/L). Despite of its antioxidant activity, in the
food industry is more used as colorant with the number E160a(ii).
Lycopene (C40H56) is also a tetraterpene formed by eight isoprene units,
but with a single aliphatic chain lacking of rings. As the β-carotene, lyco-
pene is highly lipophilic and it has antioxidant activity (Sies & Stahl, 1998)
due to their conjugated double bonds, but it lacks of provitamin A activity.
Contrary to the β-carotene, it is not obtained by synthesis, and purification
from natural foods, following complicated and expensive processes, is the
only source of this compound. This circumstance, together with the high
instability of the molecule notably limits its use as food additive, being used
preferably as colorant (E160d). However, tomato powder, mainly due to its
high lycopene content, was reported as an effective antioxidant in cooked
pork patties (Kim et al., 2013).
Mixtures of carotenoids or associations with other antioxidants (e.g.,
tocopherols) can increase their scavenging free radical activity.
Control of Lipid Oxidation in Muscle Food 359

9.3.1.4 ESSENTIAL OILS

Due to the doubts arose on the safety of the most common synthetic antioxi-
dants, the efforts of searching for new natural antioxidants usable in foods
have been redoubled. Together with the classical natural antioxidants already
described, other natural substances have been object of study and utilization
for this property in the recent past years.
Essential oils (EOs) are liquid mixtures of volatile compounds obtained
from plants, generally by steam distillation. Several EOs have shown a satis-
factory antioxidant capacity attributed in most cases to the presence in such
mixtures of molecules with antioxidant ability, mainly phenolic compounds
that act as antioxidants due to their high reactivity with the peroxyl radicals.
Phenolic compounds present in EOs are usually assigned to two structural
families according to their hydrocarbon skeleton: (a) terpenoids, formed
by an isoprene unit (hemiterpenoids) or by combination of two (monoter-
penoids), three (sesquiterpenoids), four (diterpenoids) or more isoprene
units, and (b) phenylpropanoids, formed by an aromatic phenyl group and
the three-carbon propene tail of cinnamic acid. Some common phenolic
compounds belonging to these two families (carvacrol or cymophenol, and
thymol, among terpenoids, and eugenol, guaiacol, syringaldehyde, umbel-
liferone, and coniferyl alcohol, among phenylpropanoids) are described as
principal components of several EOs.
EOs from Allium spp. have a chemical composition very different from
most of the other EOs. They are mainly composed of sulfur-containing vola-
tile compounds possessing antioxidant activity (Tsai et al., 2012) that has
been confirmed in different model systems (Banerjee et al., 2003; Iqbal &
Bhanger, 2007).
Being the EOs mixtures of various compounds, the antioxidant prop-
erties of a concrete EO should reflect the antioxidant activity of the most
active or the most abundant antioxidant compounds present in it. However,
it is necessary to take care with this approach, because complex interactions
depending on composition and experimental conditions take place, resulting
in synergistic or antagonistic behaviors among activities that notably affect
the whole antioxidant properties of the EOs (Kulisic et al., 2005).
Besides the botanical source, environmental factors (e.g., soil, climate,
etc.) may affect the actual composition, and therefore the antioxidant activity
of each EO.
In a recent work, Amorati et al. (2013) reviewed the antioxidant activity
of the EOs. For some selected EO, they summarized the data existing in
literature on their main components responsible for the antioxidant activity,
360 Natural Antioxidants: Applications in Foods of Animal Origin

the tests followed for activity assessment, and the antioxidant activity in rela-
tion to reference antioxidants such as butylated hydroxytoluene (BHT), or
reference EO such as those from age or bush-basil. Some EO from oregano,
thyme, or clove has good antioxidant activity, comparable to that of BHT.
In general, however, the antioxidant activity of EOs is medium or low; on
the other hand, some concrete EOs have no antioxidant or even prooxidant
activity.
EOs are promising food antioxidants when their particular aroma
compatible with the organoleptic characteristics of the foods in which they
are applied. EO from oregano seems to be the most successful one. Goulas et
al. (2007) reported that this EO in combination with modified atmospheres
and salting extend the shelf life of sea bream. Oregano EO is also able to
protect the extra virgin olive oil from oxidation during storage (Asensio et
al., 2012) and to protect minced meat from auto-oxidation (Fasseas et al.,
2008). Very recent and encouraging applications of EOs were described in
active packaging and in edible coatings as we will treat later.

9.3.1.5 PEPTIDES

Despite the fact that all the amino acids naturally present in the proteins can
react with free radicals if these have high energy, the free amino acids are in
the practice not generally effective in the prevention of oxidation processes
in foods and biological systems (Samaranayaka & Li-Chan, 2011). Some
peptides, however, possess antioxidant capacity based on their chem-
ical structure determined by the presence of some concrete amino acidic
sequences. Most of the peptides derived from food proteins having antioxi-
dant activity show molecular weights from 0.5 to 1.8 kDa and often they
have hydrophobic amino acids (as Val or Leu) in the amino-terminal posi-
tion and they include the amino acids Pro, His, Tyr, Trp, Met, and Cys in
their sequences.
The mechanism of action of the antioxidant peptides is generally based
on the free radical scavenging; the tripeptides possessing Trp or Tyr in the
carbonyl-terminal position have a strong free radical scavenging activity
(Saito et al., 2003). In other cases, the action is based in the scavenging of
oxygen-containing compounds. Some peptides act as antioxidants though
the chelation of metal ions such as Cu or Fe. The peptides chelating Cu have
the amino acid His in their sequence, being the imidazole ring of this amino
acid responsible for the union with the Cu ion. Finally, it has been proven
that antioxidant peptides can show synergistic affects with some other
Control of Lipid Oxidation in Muscle Food 361

antioxidants such as phenolic compounds (Wang & González de Mejía,


2005).
Antioxidant peptides can be obtained through digestion of both vegetable
and animal proteins by the action of exogenous or endogenous enzymes, by
microbial fermentation, during food processing, or during gastrointestinal
digestion (Samaranayaka & Li-Chan, 2011). Enzymatic hydrolysis has been
widely used in the production of antioxidant peptides from food proteins.
Some commercial enzymes or enzymatic preparations from microbial (such
as Alcalase® from Bacillus licheniformis, Flavourzyme® from Aspergillus
oryzae, and Protamex® from Bacillus spp.), vegetable (papain from Carica
papaya fruits), or animal (pepsin from stomach glands, and trypsin from
pancreas) sources have been used in the production of antioxidant peptides
(Pihlanto, 2006; Sarmadi & Ismail, 2010; Gallegos-Tintoré et al., 2011). In
foods, mainly fermented foods, the antioxidant peptides can also be produced
by the action of microorganisms or indigenous proteases (Samaranayaka &
Li-Chan, 2011).
In recent years, information on the obtaining of antioxidant peptides and
hydrolysates from vegetables (Gallegos Tintoré et al., 2013), marine foods
(Kim & Wijesekara, 2010; Di Bernardini et al., 2011), milk (Pihlanto, 2006),
eggs (Dávalos et al., 2004), and meat (muscles and by-products) (Di Bernar-
dini et al., 2011) was reviewed or directly reported, and peptidic sequences of
the most active peptides were elucidated. Regarding the vegetables, conven-
tional (proteins from soya, rice, corn, and chickpea) and non-conventional
(proteins from amaranth, buckwheat, colza, and Mexican pinon) sources
were assayed in the obtaining of antioxidant peptides. Marine species used
as sources of peptides were also diverse (jumbo squid, oyster, blue mussel,
hoki, tuna, cod, Pacific hake, capelin, scad, mackerel, Alaska pollock, conger
eel, yellowfin sole, yellow stripe trevally, silver carp, grass carp, herring,
and microalgae). Among dairy proteins, casein is the main source, although
obtaining of peptides from β-lactoglobulin was also reported. Peptides and
hydrolysates with antioxidant activity are obtained from proteins of all these
sources generally via hydrolysis with the commercial enzymes or enzymatic
preparations indicated in the previous paragraph.
Such peptides, and also less specific protein hydrolysates, can be used
as functional ingredients in foods in order to avoid or reduce undesirable
oxidation processes during storage (Samaranayaka & Li-Chan, 2011). To
date, however, few products are available in markets incorporating this
preservation system. Several reasons (problems of preparation of peptides
and/or hydrolysates at industrial scale; lack of complete and rigorous assays
confirming the activity, effectiveness, and safety; low reproducibility of the
362 Natural Antioxidants: Applications in Foods of Animal Origin

manufactured food products; modifications of the taste, color, and general


organoleptic characteristics; high production costs; etc.) are responsible for
the scarce actual exploitation of this potential solution (Samaranayaka &
Li-Chan, 2011).

9.3.2 SYNTHETIC ANTIOXIDANTS

Natural antioxidants show some disadvantages: Low antioxidant activity


(antioxidants should ideally be active at low concentrations, 0.01–0.02%),
and the fact that most of them are insoluble in water. In addition, antioxi-
dants must be stable during the food processing operations (carry through
effect) in order to show all their activity in the processed foods throughout
the storage process. Synthetic antioxidants comply with all these require-
ments and are abundantly used as food additives. We will review shortly the
synthetic molecules most used in the food industry:
BHA (tertiary-butyl-4-hydroxyanisole) (C11H16O2): It is a mix of two
isomers (2-tertiary-butyl-4-hydroxyanisole and 3-tertiary-butyl-4-hydroxy-
anisole) obtained from 4-methoxyphenol and isobutylene. The conjugated
aromatic ring from its molecule captures free radicals, sequestering them
and preventing the propagation in the oxidation processes. Approved as
food additive with the reference number E320 it is commercialized under
various trade names. Specifications have been defined in the EU legislation
in Directive 2008/128/EC and by the Joint FAO/WHO Expert Committee on
Food Additives (JECFA). The purity is specified to be not less than 98.5% of
tertiary-butyl-4-hydroxyanisole and not less than 85% of the 3-tertiary-butyl-
4-hydroxyanisole isomer.
BHT (tertiary-butyl-4-hydroxytoluene) (C15H24O): It is a non-coloring,
odorless, white solid matter. As the BHA, it is a chemical derivative of
phenol with similar sequestering capacity of the free radicals due to the
conjugated aromatic ring. It is approved as food additive with the reference
number E321.
Tert-butylhydroquinone (TBHQ) (C10H14O2): It is approved as food addi-
tive with the reference number E319. Addition to foods does not modify
color or flavor, being very effective in the enhancing of the storage life.
Ascorbyl palmitate (C22H38O7): It is an ester formed from ascorbic
acid and palmitic acid resulting in a fat-soluble form of the ascorbic acid.
Contrary to the other synthetic antioxidants, its metabolism is not suspected
to generate metabolites with a potential toxic effect. Ascorbyl palmitate is
known to be broken down (through the digestive process) into ascorbic acid
Control of Lipid Oxidation in Muscle Food 363

and palmitic acid which are absorbed into the bloodstream and metabolized
through the habitual routes for these two natural nutrients. It is an amphipa-
thic molecule, meaning one end is water-soluble and the other end is fat-
soluble. It is approved as food additive with the reference number E304.
Propyl, octyl, and dodecyl gallates: They are esters from the propanol,
octanol or dodecanol, respectively, with the gallic acid (3,4,5-trihydroxi-
benzoic acid). Propyl gallate (C10H12O5) is approved as food additive with
the reference number E310. It is obtained mainly by synthesis, although it
can be also obtained from a natural source (pods of the fruits of Caesal-
pinia spinosa). Octyl gallate (C15H22O5) and dodecyl gallate (C19H30O5) are
approved as food additives with the reference numbers E311 and E312,
respectively.
Ethoxyquin (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline): It is a
quinoline-based antioxidant molecule (C14H19NO) approved as food addi-
tive with the reference number E324. In contrast with the other synthetic
antioxidants which are widely used, ethoxyquin is only commonly used as
preservative in pet foods and in spices to prevent color loss due to oxida-
tion of the natural carotenoid pigments. It is approved as food additive in
the United States; however, it is not approved for use within the European
Union nor is it permitted for use in foods in Australia, due to speculations on
its responsibility on multiple pet health problems.
With the exception of ethoxyquin, whose particular situation was already
commented, all these synthetic antioxidants have been proved and evaluated
by both the European Food Safety Authority (EFSA) and the United States
Food and Drug Administration (FDA), which in turn fixed the upper limit of
addition for each concrete compound.

9.3.3 EFFICIENCY OF THE ANTIOXIDANTS

The efficiency of the antioxidants can be evaluated throughout comparative


tests, by using of so-called “antioxidant factor” (AF) (Belitz et al., 2009):
AF = IA/I0, were IA= oxidation induction period for a determined fat or
oil in the presence of the considered antioxidant, and I0 = oxidation induc-
tion period for the same fat or oil without the addition of the antioxidant.
The efficiency of an antioxidant increases with the increase of the AF value.
According to the results of one of such comparative assays (Belitz et
al., 2009), comparative efficiency of the most commonly used antioxidants
when added at 0.02% concentrations in refined lard is (AF values): DL-γ-
tocopherol (12), BHA (9.5), BHT (6), octyl gallate (6), D-α-tocopherol (5),
364 Natural Antioxidants: Applications in Foods of Animal Origin

ascorbyl palmitate (4). BHA and BHT have synergistic effects, and when
added together at a given total concentration they are more effective than
either antioxidant alone at the same level. In the described assay, BHA
and BHT added at a concentration of 0.01% each, show an AF value of
12, similar than that of DL-γ-tocopherol. In the same way, propyl gallate
increases the efficiency of BHA, but not that of BHT. Ascorbyl palmitate,
which alone shows a weak antioxidant efficiency, sustains the activity of the
DL-γ-tocopherol (Belitz et al., 2009).
Synergic effects can occur between antioxidants, but also between anti-
oxidants and another molecules present in foods that enhance the antioxi-
dant activity. These molecules called synergists are lecithin, amino acids,
citric, phosphoric, citraconic, and fumaric acids, and in general molecules
able to form complexes with the heavy metal ions. In this way, initiation of
heavy metal-catalyzed lipid autoxidation is prevented. A synergistic effect
of different nature is that exercised by phospholipids. The addition of dipal-
mitoylphosphatidylcholine (0.1–0.2%) to lard enhances the antioxidant
activity of α-tocopherol, BHT, BHA, and propyl gallate (Belitz et al., 2009).
Phosphatidylcholine, however, does not show this capacity.

9.4 MODIFIED ATMOSPHERE PACKAGING (MAP)

When not packaged, foods during storage are usually surrounded by air;
the main gases in dry air at sea level are N2 (78%, v/v), O2 (20.99%), Argon
(0.94%), and CO2 (0.03%) (McMillin, 2008). MAP implies the presence of a
barrier, normally a plastic film, that impedes the permeation for gases, water
vapor included, that allows the maintaining of a constant environment of the
desired gas composition during the food storage. Barrier bags are first filled
with the product, previously placed in a tray, and then sealed after flushing
with the selected gas mixture.
Package protects the products against deterioration, which usually
include discoloration, development of off-flavor and off-odor, nutrient
losses, texture changes, pathogenicity, and other measurable traits (Skibsted
et al., 1994). In the case of fresh meat the main objective of packaging is the
increase of shelf life which implies the maintaining the water content, color,
microbial quality, lipid stability, and palatability (Renerre & Labadie, 1993;
Zhao et al., 1994).
Gas composition of the modified atmosphere affects the most impor-
tant meat attributes during storage which are mainly color, flavor, and
microbiology.
Control of Lipid Oxidation in Muscle Food 365

Regarding meat color, when the choice is made consumers prefer red
color above purple color, which in turn is preferred above brown color
(Carpenter et al., 2001). Meat color depends on the quantity and form of
the myoglobin pigment. The form of the myoglobin, and its color, depends
on the state of the iron placed in the porphyrin ring in the heme group (that
can be oxidized or reduced) and on the presence or absence of O2 occupying
the sixth coordination site of the iron. Purple color is due to the deoxy-
myoglobin that is the reduced form of myoglobin (Fe2+) in the absence of
O2 (with a vacant sixth coordination site in the iron). Red bright color is
due to the oxymyoglobin that is the reduced pigment (Fe2+) in which O2
occupies the ligand position. Oxymyoglobin is usually the form in contact
with the air. Oxymyoglobin is formed by O2 binding to the ferrous (Fe2+)
ion, which occurs at high O2 tension values. The penetration of oxygen
through the meat and therefore the oxymyoglobin layer thickness depends
upon temperature, O2 partial pressure, pH, and consumption of O2 by other
respiratory processes (Mancini & Hunt, 2005). Brown or gray color is due
to the metmyoglobin form that is the oxidized state of the myoglobin (Fe3+).
Metmyoglobin is formed when pigment is exposed for extended times to
light, heat, O2, microbial growth, or freezing, all these factors determining
the oxidation of the iron to a ferric (Fe3+) state. When deoxymyoglobin is
exposed to carbon monoxide, another pigment form, the carboxymyoglobin,
is formed. Carboxymyoglobin formation, with a stable bright-red color,
occurs when CO attaches to the vacant sixth position of deoxymyoglobin,
when the environment is devoid of oxygen. In the absence of CO, the three
other states of the myoglobin may coexist in varying proportions in the same
meat piece depending on redox conditions. The desired bright red-bloomed
color is achieved by the predominance of the oxymyoglobin pigment that
is easily generated when the O2 percentages in atmosphere are higher than
5.5%, and dominates at O2 percentages higher than 13%. Deoxymyoglobin
dominates in atmosphere conditions of less than 0.2% O2, while metmyo-
globin is the main pigment form at O2 levels of 0.2–13% (Siegel, 2001).
Metmyoglobin is easily formed in fresh meat in the range of 2.6–5.3% of O2
(Sebranek & Houser, 2006).
Flavor attributes are, together with the tenderness, the most important
factors that influence the consumers’ purchase habits. Compounds deter-
mining flavor and odor are usually originated from protein and lipid compo-
nents of meat (Spanier, 1992). Undesired off-flavors from proteins are
normally generated through the action of microorganisms producing amines,
ammonia, and other odor active compounds such as sulfur compounds,
from amino acids. In this sense, MAP gas composition influences the flavor
366 Natural Antioxidants: Applications in Foods of Animal Origin

attributes through the inhibition or promotion of the growth of the different


microbial groups. MAP inhibits the habitual spoiling microorganisms, but
as will be discussed below favors the development of concrete species that
are responsible for flavor modifications in meat. Regarding lipids, they are
mainly degraded via oxidation processes. Lipid oxidation is linked to pigment
oxidation and discoloration of meat, but also causes flavor deterioration via
the formation of several volatile compounds from the hydroperoxides gener-
ated as products of the primary oxidation. As McMillin (2008) pointed out,
although initial studies did not show enhanced lipid oxidation with increased
O2 concentrations in MAP, other more recent studies (Cayuela et al., 2004;
John et al., 2005) reported higher lipid oxidation in meat packaged in high O2
concentration atmospheres, when compared with vacuum packaged or low
O2 concentration atmospheres. Other authors, however, clearly reported this
problem. Jackson et al. (1992) indicated that oxidative processes cause a real
problem in meat packaged in atmospheres with more than 21% of oxygen.
Unfortunately, this circumstance is the counterpoint to the beneficial effects
already commented of the high O2 concentrations on the meat color.
Microbiology and microbial growth during the storage are key factors
in meat quality. Anomalous unpleasant colors and odors, and surface slime
are undesirable effects of microbial growth determining the deterioration
of meat. In MAP, the extended shelf life arises a new issue because patho-
gens have extra-time for development to reach dangerous counts (Farber,
1991). Under normal O2 concentrations in air, aerobic microorganisms are
commonly present in meat surfaces, reducing the O2 tension, promoting
discoloration, and generating surface slime due to their mobile condition.
MAP notably affects the survival and growth of spoiling and pathogenic
bacteria (Blakistone, 1999). Oxygen stimulates growth of aerobic microor-
ganisms and inhibits the growth of strict anaerobic bacteria, being variable
the sensitivity to the O2 of the different anaerobic bacterial species (Church,
1994). Anoxic atmospheres favor the development of lactic acid bacteria and
another facultative anaerobic microorganisms. Nitrogen has minimum effect
on metabolic reactions occurring in meat, but as occurs with some gasses
other than oxygen, the anoxic conditions created by the use of N2 select
for anaerobic and facultative anaerobic microorganisms (Thippareddi &
Phebus, 2007). Regarding the effect of CO2 in atmospheres, gram-negative
bacteria are in general more sensitive to CO2 than the gram-positive bacteria,
because gram-positive bacteria are usually strict or facultative anaerobes
(Farber, 1991). Due to the fact that CO2 in MAP is firstly absorbed by the
components of the meat, mainly water, and lipids, until an equilibrium is
reached, an excess of CO2 should be used to obtain a desired preservative
Control of Lipid Oxidation in Muscle Food 367

effect. Concentrations of 20–60% of CO2 are necessary for complete inhibi-


tion of aerobic spoilage microorganism, but slight or no effect is observed
with CO2 values above 50–60% (Gill & Tan, 1980). In relation to CO,
the shelf life is extended by adding CO at levels above 0.5% (Clark et al.,
1976). However, the effect of CO is variable on pure microbial cultures
as reported by Gee and Brown (1980). Numerous works have studied the
effect of concrete gas mixtures on the microbiology of MAP meat and on
the growth of particular microbial groups or relevant spoiling or pathogenic
species (McMillin, 2008). Viana et al. (2005) reported that counts of lactic
acid bacteria increased during storage in pork loin packed in oxygen-free
atmospheres, while they reached the lowest counts in 100% O2 atmosphere
after 20 days of storage; the growth of Pseudomonas was limited in 100%
CO2 and 1% CO + 99% CO2 atmospheres, with the highest counts in 100%
O2. According these authors, pork treated with the 1% CO + 99% CO2 atmo-
sphere received the greatest acceptance by the consumers.
Regarding the gas mixtures commonly used, the high O2 MAP can have
25–90% O2 and 15–80% CO2 in the headspaces (Blakistone, 1999), being
80% O2+ 20% CO2 the most used gas mixture (Eilert, 2005). In relation to
color, the oxymyoglobin levels of minced meat were similar after four days
of storage in 20, 40, 60, and 80% O2 atmospheres, but in all the cases higher
than in meat stored in air, although after seven days of storage the oxymyo-
globin content decreased with the O2 decrease. At the 10th day of storage,
lipid oxidation slightly increased as the O2 percentage was higher (O’Grady
et al., 2000). Luño et al. (1998) studied the effect of a low O2 atmosphere
containing CO (24% O2 + 50% CO2 + 25% N2 + 1% CO) in comparison
with a high O2 atmosphere containing CO (70% O2 + 20% CO2 + 9% N2 +
1% CO) and with a high O2 atmosphere without CO (70% O2 + 20% CO2 +
10% N2) on the psychrotrophic counts and color stability throughout storage
of loin steaks and ground meat. Psychrotrophic counts were greatly reduced
by the low O2 atmosphere containing CO, while the bright-red color was
more stable in both atmospheres containing CO, reaching 29 days of storage
without signs of oxidation, which was confirmed using sensory analysis.

9.5 ACTIVE PACKAGING

The function of food packaging has evolved from simple preserva-


tion methods to include such aspects as convenience, point of purchase
marketing, material reduction, safety, tamper-proofing, and environmental
issues (Han, 2014). Active packaging is a novel technology which is designed
368 Natural Antioxidants: Applications in Foods of Animal Origin

to incorporate material components in the packaging that release or absorb


substances from or into the packaged food or the surrounding environment
in order to extend the shelf life and maintain or improve the condition of
packaged food. In contrast to traditional packaging, active, and intelligent
packaging may change the composition and organoleptic characteristics of
food, provided that the changes are consistent with the provisions for food.
Active packaging is receiving considerable attention as an emerging
technology that can be used to improve the quality and stability of food,
reducing the direct addition of chemicals, and the need for changes in formu-
lation. The technology provides several advantages compared to direct addi-
tion, such as lower amounts of active substances required, localization of
the activity to the surface, the migration from film to the food matrix (which
could be used to provide antioxidant effects for longer protection), and elim-
ination of additional steps within a standard process intended to introduce
the antioxidant at the industrial processing level such as mixing, immersion,
or spraying (Bolumar et al., 2011).
Active packaging is defined as a package system that deliberately incor-
porates components that release or absorb substances into or from the pack-
aged food or the environment surrounding the food to extend the shelf life
or to maintain or improve the condition of the packaged food (Regulation
(CE) No. 450/2009 (29/05/2009)). Therefore, active packaging does some-
thing more than simply providing a barrier to external detrimental factors, as
the packaging system plays an active role in food preservation and quality
during the marketing process (Lopez Rubio et al., 2004; Pereira de Abreu et
al., 2012). When designing an active package, issues that are of importance
when designing traditional food packages, such as barrier properties to gases
and moisture and the mechanical strength required for pack integrity, must
still be taken into account. So, the following aspects, which are specific to
the antimicrobial and antioxidant function of the packages, must principally
be considered (Coma & Kerry, 2012):

9 The chemical nature of the bioactive agents and their inhibition


mechanism
9 Physico-chemical characteristics of foods and the organoleptic prop-
erty of the bioactive agents
9 Packaging manufacturing processes and their influence on the effi-
ciency of bioactive additives
9 Storage environments
9 Migration mechanisms of bioactive agents into foods if needed, and
toxicity and regulatory issues
Control of Lipid Oxidation in Muscle Food 369

9 Machinability and processability of the bioactive packaging on the


packaging line materials.

A suitable selection of the antioxidant compound to be incorporated in


the packaging material is crucial. The antioxidant compound and the pack-
aging material should be compatible in order to achieve a homogeneous
distribution, and the partition coefficients of the antioxidant in the different
phases should favor its release to the food or headspace (Gómez-Estaca et
al., 2014). Once released, the solubility characteristics of the antioxidant
can determine its effectiveness, and therefore the type of antioxidant should
be selected as a function of the type of food. Non-polar antioxidants would
seem to be more suitable for foods with high lipid content and vice versa.
Lee (2014) noticed that maximum effectiveness of antioxidant packaging
systems can be achieved by fitting the antioxidant release rate with the lipid
oxidation rate. Mathematical models of diffusion probed to be a valuable
tool to predict the release profile of antioxidants into food systems (Piringer,
2000).
There are two main modes of action for antioxidant packages: The scav-
enging of undesirable compounds such as oxygen, radical oxidative species
or metal ions from the headspace or from the food and the release of anti-
oxidants to the food (Gómez-Estaca et al., 2014). Among active antioxidant
packaging materials, oxygen scavengers are the ones that are being most
widely produced by the extrusion technique. Oxygen scavengers are able
to inhibit the growth of aerobic bacteria and molds, and stop alterations of
pigments, and flavors to avoid discoloration in meat products (Vermeiren
et al., 1999). According to Day (2001), oxygen scavenging adhesive labels
are being used for a range of sliced cooked and cured meat products which
are susceptible to deleterious color changes induced by oxygen. The most
common oxygen scavengers consist of small size oxygen-permeable sachets
that contain an iron-based powder along with a catalyst. These types of scav-
engers react with water that is produced by the packaged food and generate
a hydrated metallic agent which is able to scavenge oxygen and convert it
to a stable oxide. Iron and ferrous oxide fine powders, ascorbic acid, some
nylons, photosensitive dyes and unsaturated hydrocarbons are being used
in the manufacture of extruded films with oxygen scavenging properties, as
has been reported in previous reviews (Lopez Rubio et al., 2004; Brody et
al., 2008).
Another popular group of active packaging systems are moisture
absorbers. Several companies manufacture moisture absorbers in the form
of sachets, pads, sheets, or blankets. For packaged dried food applications,
370 Natural Antioxidants: Applications in Foods of Animal Origin

desiccants such as silica gel, calcium oxide and activated clays, and minerals
are typically tear-resistant permeable plastic sachets. In addition to mois-
ture-absorber sachets for humidity control in packaged dried foods, several
companies manufacture moisture-drip absorbent pads, sheets, and blankets
for liquid water control in high activity water foods such as meats, fish, fruit,
and vegetables (Dobrucka & Cierpiszewski, 2014).
Regarding the antioxidant releasing packaging materials, one of the main
benefits, as compared to the direct addition of antioxidants to food, is that
active materials may act as a source of antioxidants that are released to the
food at controlled rates, so that a predetermined concentration of the active
compound is maintained in the food, compensating the continuous using
up of antioxidants during storage (Mastromatteo et al., 2010). There are
basically two methodologies for producing antioxidant-packaging systems
(Gómez-Estaca et al., 2014):

a) Independent devices: An independent device such as a sachet, pad


or label containing the agent separately from the food product is
added to a conventional “passive” package.
b) Antioxidant packaging materials: Antioxidant packaging mate-
rials are used in the manufacture of the package, that is, the active
agent is incorporated in the walls of the package exerting its action
by absorbing undesirable compounds from the headspace or by
releasing antioxidant compounds to the food or the headspace
surrounding it.

Many different active packaging structures can be built with active mate-
rials prepared by extrusion processes, including coextruded, and laminated
multilayers. The design is dependent on the type of agent and the type of
polymer matrix, but primarily on the packaging requirements of the food
product. Due to environmental motivation there is increasing interest in the
use of biodegradable/compostable packaging and/or edible materials. This
tendency increases when materials come from industrial waste or renew-
able resources (Gómez-Guillén et al., 2007). Four types of plastics mate-
rials could be used for food packaging (Mecking, 2004; Reddy et al., 2012;
Peelman et al., 2013; Reddy et al., 2013):

a) Non-bio-based non-biodegradable conventional plastic mate-


rials, for instance: polyethylene (PE), polypropylene (PP), poly
(ethyleneterephthalate) (PET), and polystyrene (PS).
Control of Lipid Oxidation in Muscle Food 371

b) Bio-based biodegradable plastic materials, for example: poly


lactic acid (PLA), poly hydroxybutyrate (PHB), and chitosan.
c) Bio-based non-biodegradable plastic materials, for instance: the
commonly advertised as “green” PE, PP, and PET.
d) Non-bio-based biodegradable plastic materials, for example:
aliphatic-aromatic polyesters, and polycaprolactone (PCL).

Finally, edible film and coating technology is close to active pack-


aging technology and may also be a means of reducing oxidative spoilage
in foods. The main mechanism of action is the reduction of the oxygen
transmission rate, as well as the possibility of incorporating antioxidant
compounds in the edible film or coating matrix; this vehicle has the advan-
tage of close contact between coating and food. An edible film or coating
does not act as a package itself, but it may reduce the barrier requirements
of the package.
Research on active packaging for muscle foods has focused predomi-
nantly on the use of antimicrobial agents, while the development of antioxi-
dant applications is growing. Different antioxidant agents, such as tocoph-
erol, ascorbic acid, and different plant extracts, and EOs from herbs such as
rosemary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.), and
green tea (Camellia sinensis L.) may be successfully included in bio-based
films, to decrease oxidative reactions in meat products (Table 9.1). Rose-
mary extract is one of the plant extracts that has already been incorporated
into food packaging. It is composed by flavones (apigenin, genkwanin,
hesperetin, and cirsimaritin), phenolic diterpenes (carnosic acid, carnosol,
rosmadial, epirrosmanol, rosmanol, carnosic acid o-quinone), and phenolic
acids (caffeic acid and rosmarinic acid). Carnosic acid is one of the most
important compounds responsible for antioxidant capacity (Sanches-Silva
et al., 2014). Nerin et al. (2006) observed that the active film containing
rosemary extract efficiently enhanced the stability of both myoglobin and
fresh meat against oxidation processes, thus being a promising way to
extend the shelf life of fresh meat. So, active packaging was able to extend
the shelf life of beef packaged in modified atmosphere and displayed under
conventional illumination by ~2 days, that is, 17%. In addition, Camo et al.
(2008) noticed that the use of rosemary and oregano active films resulted in
enhanced oxidative stability of lamb steaks packed in modified atmosphere.
Active films with oregano were significantly more efficient than those with
rosemary, exerting an effect similar to that of direct addition of the rose-
mary extract.
372 Natural Antioxidants: Applications in Foods of Animal Origin

TABLE 9.1 Meat and Meat Products Packed with Antioxidant Films.
Antioxidant source Meat product Reference
Oregano and pimento essential oils Fresh beef meat Oussalah et al. (2004)
Rosemary extract Fresh beef meat Nerín et al. (2006)
Rosemary and oregano EOs Fresh lamb meat Camo et al. (2008)
Thyme and oregano EOs Fresh lamb meat Karabagias et al. (2011)
Oregano Fresh beef meat Camo et al. (2011)
Rosemary extract Chicken meat patties Bolumar et al. (2011)
Thymol, carvacrol, and eugenol Fresh beef meat Park et al. (2012)
Green tea Pork sausages Siripatrawan and Noipha (2012)
Brewery and rosemary Fresh beef meat Barbosa-Pereira et al. (2014)
Oregano and green tea Fresh foal meat Lorenzo et al. (2014)
Citrus extract Cooked turkey meat Contini et al. (2014)
Carvacrol, eugenol, and thymol Fresh beef meat Tornuk et al. (2015)
Nisin, chitosan, potassium sorbate, Fresh chicken meat Soysal et al. (2015)
and zeolite

The main constituents of oregano EO are carvacrol, thymol, p-cymene, and


linalool (Karabagias et al., 2011). Oussalah et al. (2004) evaluated the ability
of milk protein-based edible film containing 1% EOs of oregano, pimento
or both applied on beef muscle slices, and showed that oregano-based films
stabilized lipid oxidation in beef muscle samples, whereas pimento-based
films presented the highest antioxidant activity. Camo et al. (2011) reported
that a concentration of at least 1% oregano extract was needed for obtaining a
significant increase of beef meat display from 14 to 23 days, while a concen-
tration of 4% in the package gave rise to an unacceptable oregano smell.
On the other hand, the use of active packaging system (packed with
active film containing a 2% of an oregano EO and 1% of a green tea extract)
showed a preservative effect in foal meat, stored under refrigeration, exerting
a protective effect against microbial spoilage, lipid and protein oxidation,
and consequently preserving color and sensorial properties (Lorenzo et al.,
2014). Green tea is a good source of polyphenolic compounds like catechin,
theaflavins, and thearubigins, which have the ability to scavenge reactive
oxygen and nitrogen species (Siripatrawan & Harte, 2010). In line with this,
the incorporation of green tea extract into chitosan film could enhance the
antioxidant and antimicrobial properties of the film and thus maintained
the qualities and prolonged the shelf life of the sausages (Siripatrawan &
Noipha, 2012).
Control of Lipid Oxidation in Muscle Food 373

According to Park et al. (2012), the antioxidant films improved the effi-
cacy of vacuum packaging and stabilized raw beef patties against oxida-
tion. The antioxidant property of prepared film containing 3% eugenol
was similar to that of the film containing 0.3% eugenol, which suggests
that eugenol is a strong antioxidant and 0.3% concentration was sufficient
to completely retard lipid oxidation in beef patties. Contini et al. (2014)
also showed that antioxidant active packaging with citrus extract is effec-
tive in reducing the lipid oxidation of cooked turkey meat during storage
and in maintaining its sensory characteristics, particularly tenderness and
overall acceptability. In line with this, lowest lipid oxidation was observed
for samples packed with chitosan-incorporated bags, which might be
helpful to limit lipid oxidation of the drumsticks during the storage period
(Soysal et al., 2015). Barbosa-Pereira et al. (2014) observed a reduction in
lipid oxidation by up to 80% in beef wrapped in films coated with natural
extracts obtained from a brewery residual waste, while Tornuk et al. (2015)
suggested that the active nanocomposite packaging had potential to extend
shelf life of fresh beef by reducing E. coli O157:H7 numbers and retarding
the meat discoloration.
Finally, active packaging can be used in combination with other
processing treatments to extend food shelf life. High pressure processing
has shown to be an effective technology to improve safety of meat and meat
products (Marcos et al., 2008; Realini et al., 2011). However, high pres-
sure processing may accelerate meat oxidation (Ma et al., 2007; McArdle
et al., 2011, 2013). In line with this, Bolumar et al. (2011) observed that
the antioxidant compounds from the antioxidant active package (rosemary
extract) suppress the oxidation in both the surface part and the inner part
of chicken meat patties and prevent formation of secondary lipid oxidation
products. So, the antioxidant active packaging was able to delay the oxida-
tion induced by high pressure processing and consequently extend the shelf
life of chicken patties.

9.6 CONCLUSION

The active antioxidant packaging is a promising emerging technology to


avoid the undesirable effects of lipid oxidation during meat storage. This
solution provides several advantages compared to direct addition of anti-
oxidants, such as lower amounts of active substances required, localization
of the antioxidant activity to the surface, progressive migration from film to
the food matrix that provides long-term antioxidant effects, and elimination
374 Natural Antioxidants: Applications in Foods of Animal Origin

of additional steps in the manufacturing process for introduction of antioxi-


dants in foods.
Positive and encouraging results were obtained in recent works. More
studies, however, are needed to find new materials (both plastic polymers
and edible coatings) with higher performances, and to select the most appro-
priate antioxidant agents for each concrete meat product in each group of
storage conditions.

KEYWORDS

• active packaging
• natural antioxidant
• lipid oxidation
• modified atmosphere packaging

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INDEX

A effectiveness, 45
Açaí (Euterpe oleracea), 311–312 efficiency of, 363–364
Active oxygen method, 44 and free radicals, 43–44
Active packaging, 367–373 future prospects, 125–126
Advanced lipid oxidation end products health hazards, 277
(ALE), 271 market potential, 124–125
Air-drying process, 100 meat and meat products packed with
Alcalase®, 361 films, 372
Aldehydes, 27 mechanism of action, 97–98
Alfajayucan cultivars, 312 natural
Algal phlorotannins, 123 ascorbic acid, 357–358
Allahabad Safeda, 313 carotenoids, 358
Alpha linolenic fatty acid, 235 essential oils, 359–360
Amaranth (Amaranthus hypochondri- peptides, 360–362
acus), 323 phenolic compounds, 355–357
Amaranthus cruentus, 323 primary, 352–353
American Association of Cereals Chem-
secondary, 353–354
ists Expert Committee, 302
from spices and herbs, 278
Andean native grain, 323
Anserine, 97 synthetic, 362–363
Anthocyanins, 99–100 Anti-radical power (ARP), 280
Antioxidant dietary fiber (ADF), 300 Apple polyphenols, 282
Arjuna (Terminalia arjuna), 281
application in meat products, 326–328
Ascorbate peroxidase, 56
beneficial component of healthy diet,
Ascorbic acid. See Vitamin C
301 Ascorbyl palmitate, 362–363
constituents present in, 302 Ashwagandha (Withania somnifera), 281
positive effects of incorporation of, 326 Asparagus, 317–318
sources of, 304–305 Atlantic herring fillets (Clupea harengus),
burdock root, 324 13
fruits and by-products, 305–315 Autoxidation, defined, 11
Hibiscus sabdariffa L., 324–325 Autoxidation of lipids
seaweed, 325–326 analytical methods
seed and by-products, 319–324 acceptable values for TBARS varies
vegetable by-products, 315–319 from product to product, 221
Antioxidants, 41–42 analysis of oxidation of raw meat
activity and total phenolic content pigments, 229–238
(TPC), 283 anisidine values, 217–218
classification of, 97–98 data for TBARS, 226
diversity of sources, 278 difference, 224
formulation for chicken sausage, 223
384 Natural Antioxidants: Applications in Foods of Animal Origin

GCMS, 241–250 Carotenoids, 43–44, 266


interrelationship between lipid oxida- chemical structure, 66
tion and raw meat pigment, 227 composition in palm oil, 67–68
methods and techniques for analysis content of, 67
of TBARS, 225
isoprenoid units in, 66
non-meat ingredients, 222
ORP method, 239–241 in vegetable oils, 68
peroxides, 208–217 Carrot (Daucus carota L.), 119, 318–319
sample preparation, 222 Caseins, 265
schematic generalized representation, Catechin, 100
228 Cauliflower (Brassica oleracea) powder
TBARS and sensory evaluation, (CP), 120–121, 316
220–229 Cereals
TBA values, 224 and legumes, 319–321
total oxidation (TOTOX) method, products, 80–81
218–220 Chain-breaking antioxidants, 45
transforming TBARS data, 228 Chamnamul (Pimpinella brachycarpa),
relative oxidative stability of meat and 118–119
fish lipids, 206 Cheese, 283–285
selection criteria and sample prepara- Chenopodium quinoa, 323
tion, 206–208 Chia seeds, 321–322
Chlorophyll and porphyrin, 269
sequence of chemical events, 205
Chocolate processing, 60, 63
B Chub mackerel (Scomber japonicus), 11
Citric acid (CA), 150
Bael (Aegle marmelos L.) pulp residue Citrus paradisi fruit barks (CPFB),
(BPR), 313–314, 328 105–106
Bay essential oil (BEO), 157 Cocoa polyphenols, 322
Bearberries (Uva Ursi), 103–104 Cod muscle (Gadus morhua), 12
Beet (Beet vulgaris), 285 Coenzyme Q10 (ubiquinone) in meat, 97
Bhakkar-2002, 320 Coffee bean components, 82
Bran extracts, 320 Color coordinates lightness and yellow-
Brassicaceae (Cruciferae) family, 120 ness, 105
Browning products, 81 Color stability of HOX MAPackages of
Brown lead (Leucaena leucocephala) ground beef, 238
seed extracts, 157 Commercial rosemary extract
BRS-Pará fruits, 312 (FORTIUMTM R20), 115
Burdock root, 324 Common edible oils, 47
Butter cookies, 82 Consequences of lipid oxidation
Butylated hydroxy anisole (BHA), 43–44 measurement of rate of antioxidant
Butylated hydroxy toluene (BHT), 43–44 depletion in meat, 252–253
oxidative stability of seasonings during
C
storage, 251–252
Cabbage, 120 WHC, 250–251
Carnosic acid, 371 Control biscuits (CB), 79
Carnosine, 97 Cooked meats, 13
Carob (Ceratonia silique L.), 104 Cooking, 347
Carotenes, 66–67, 266
Index 385

Crackers and cookies, 82 Ethanolic kiam wood extract (EKWE),


Cruciferous vegetables, 120–121 156–157
Ethoxyquin, 363
D Exxenterol®, 104
Date palm tree (Phoenix dactylifera L.),
314–315 F
Deoxymyoglobin in biological system Fatsia (Aralia elata) extract, 118–119
formation of lipid peroxides and Fat-soluble vitamin E, 45
TBARS, 23 Fatty acid (FA)
prooxidative activity of, 22–23 profile, 236–237
Desirable fatty acids (DFA), 235 role in oxidative stability, 47–48
Diazabicyclooctane (DABCO), 8 Fenton reaction, 19
Dietary fiber (DF) Fermented milk products, 282–283
absence of, 301 Ferric reducing antioxidant power
amount of, 303 (FRAP)
antioxidant capacity of, 302 assay, 193–194
definition of, 302 values, 283
ingredients, 301 Ferri-porphyrin proteins, 265
Ferriprotoporphyrin chloride, 26–27
physicochemical properties of, 304
Ferryl-hydroxo complex, 26–27
processing and quality, 303–304
Ferulic acid (FA), 82, 302
quality, polyphenols and, 302–303 Fish and fishery products, 142–143
SDF/IDF ratio, 303 antioxidants, 149
sources of, 300 natural, 151–155
Dietary Supplement Health and Educa- synthetic, 150–151
tion Act of 1994, 83–84 oxidation in
Diphenylpicrylhydrazyl (DPPH), 44, 280, lipids, 144–145
301 mechanism, 147–149
Docosahexaenoic acid (DHA), 8, 142 protein, 146–147
Drying method, 14 Fish muscle, post-harvest discoloration,
19–20
E Fish oil encapsulation, 14
Eicosapentaenoic acid (EPA), 8, 142 Flavones, 99–100
Endogenous antioxidants, 11–12 Flavonoids, 99–100, 275
Enrobing, 192 Flavonols, 99–100
Enzymatic lipid oxidation, 8–9 Flavor, 4
Epicatechin, 100 Flavourzyme®, 361
Epigallocatechin, 100 Folin–Ciocalteu method, 320
Epigallocatechin gallate (EGCG), 284 Food Labeling Regulation, Amendments,
Essential oil-based antioxidants, 359–360 84
composition of, 116 Food products preservation, applications
effectiveness, 116 of natural antioxidants/extracts in, 80
plant materials, 116 Food Regulation Uniform Compliance
TBARS values, 117 Date, 84
Ethanol, 100–101 “Free” catalytic iron in muscle, 14
Free radicals, 41–42
oxidation, 5–7
386 Natural Antioxidants: Applications in Foods of Animal Origin

Freeze-drying process, 100 Grape seed extract (ActiVin™), 108


French marine bark extract, 282 Greek wine grape varieties, 282–283
Fresh plum puree (FPP), 110–111 Green bell pepper juice, 282
Fruit-based antioxidants, 102 Green seaweed Halimada, 123
bael, 103 Green tea (Camellia sinensis L.), 371
bearberries, 103–104 extracts, 83
carob, 104 Guava (Psidium guajava L.), 109, 313
citrus fruits, 105–106 powder, 328
cranberries, 106–107
grapes, 107–109
H
guava, 109 Headspace analysis of volatile lipid
plums, 110–111 oxidation products (GCMS), 241
pomegranates, 111–112 colorimetry data, 250
Fruits and by-products concentration, 244
açaí (Euterpe oleracea), 311–312 of analyte, 242
apple by-products, 307–309 data, 247
bael pulp residue, 313–314 gas chromatography and mass spectros-
cactus pear, 312 copy, 243
citrus by-products, 309–311 mass spectrograph data, 244
date, 314–315 meat samples, 242
grape by-products, 305–307 oxidative stability, 244–245
guava, 313 oxidative stress, 246–247
mango peel, 309 raw restructured steak, 245–246
pineapple shell, 314 secondary oxidation products, 245
Fucoidan powders, 325 sensory evaluation, 247–248
Fucoxanthin, 123 steaks cooked and evaluated, 249
Fucus vesiculosus L., 325–326 steps, 243
Hematin and hemin, 26–27
G Heme, 26–27
Gallocatechin, 100 Hemichrome formation from iron (III)
Galvinoxyl, 44 myoglobin, 24
Generally regarded as safe (GRAS), 58 Hemochromes, 22
Ghee/clarified butter fat, 280–282 Hemoglobin-mediated lipid oxidation, 16
Ginger (Zingiber officinale L.), 285 Herbs- and spices-based antioxidants, 112
Glutathione, 97 active constituents and antioxidative
Glutathione disulfide (GSSG), 263 capacities of, 113–114
Glutathione peroxidase, 11 lipid oxidation, 115
Glycolipids (GL), 46 TBARS values, 115
Gossypol, 43–44 toxic and carcinogenic effects, 116
Grape Herpes simplex virus type 1 (HSV-1) and
and grape callus extracts, 282 2 (HSV-2), 308
seed extracts, 282 Hibiscus sabdariffa L., 324
Grape antioxidant dietary fiber (GADF), extract, 282
327 High density lipoproteins (HDL), 158
Grape pomace concentrate (GPC), 327
Index 387

High-molecular-weight (HMW) compo- Lipid hydroperoxides (LOOH), 108–109


nents, 12 Lipid oxidation in foods of animal origin
High-pressure processing, 17–18, impact, 3–4
348–349 basic mechanism
High-spin iron (III) myoglobin, 24 enzymatic lipid oxidation, 8–9
Hijika fusiformis, 123 free radical oxidation, 5–7
Horse mackerel (Trachurus trachurus), photooxidation, 7–8
327 effect of intrinsic factors and processing
HP-treated meat, 18 parameters in
Human olfactory receptors, 4 endogenous antioxidants, 11–12
Hydrogen donating antioxidants (HDA), high pressure processing, 17–18
43–44 metal ions, 14–15
Hydroperoxides, 4, 11, 15 muscle pH, 16
Hydroxybenzoic acid derivatives, 63 particle size reduction and tumbling,
Hydroxycinnamic acids (HCA), 302 17
4-Hydroxynonenal (4-HNE), 27–28 sodium chloride, 15–16
species, muscle type, and fatty acid
I composition, 10–11
Initiation of oxidation, 6 temperature, 13–14
Insoluble dietary fiber (IDF), 301 interaction between products and
Instant ramen, Japanese dried noodle myoglobin, 27–28
product, 53 interrelationship between, myoglobin
Iodine value (IV), 44 oxidation and, 20–21
Iron-chelating antioxidants (ICA), 43–44 role of deoxymyoglobin, 22–23
Irradiation, 347 role of ferrylmyoglobin, 25–27
role of oxymyoglobin, 23–24
J Liposterine®, 104
Japanese Spanish mackerel (Scomb- Lipoxygenase (LOX), 5, 83
eromorus niphonius), 11 LOX-catalyzed lipid oxidation, 8–9
Juxtaposition of copper-protein complex, Liquid chromatography-mass spectrom-
265 etry (LC-MS) spectra, 28
Longissimus dorsi (glycolytic muscle),
K 346
Low-molecular-weight (LMW) compo-
Kinnow or Tangerine (Citrus reticulata),
nents, 12
106
Low oxygen packaging, 53–54
Klason lignin (KL), 306
Low-salt meat emulsion model systems,
L 123–124
Lumichrome, 269
Leafy green vegetables, 118–119 Lumiflavin, 269
Lemon balm, 158 Lutein, 67
Lemon (Citrus limon cv Fino), 311 Lycopenes, 275
Light induced oxidation, 8
Lignans, 43–44 M
Limonoids, 310
Mackerel (Scomber scombrus) fillet fat
Linoleate-to-heme ratios, 24–25
fraction, 157
Linoleic acid, 47–48
Maillard products, 82
388 Natural Antioxidants: Applications in Foods of Animal Origin

Maillard reaction, 13–14 application of synthetic antioxidants


Malondialdehyde (MDA), 220 and effects, 276–277
Mango (Mangifera indica L. Anacardia- bioactive peptides, 263
ceae), 309 carbonyl-amine reactions, 267
Manto Negro red grape (Vitis vinifera), carotenoids, 266
306
commercial manufacture of, 264
Margarines, 53
components, 264
Marination, 192
Marination/tumbling procedure, 17 incorporation of buttermilk, 267
Marine macroalgae, 122 milk lipids, 263
Meat as food, 96 natural antioxidants
composition, 346–347 application of, 278–285, 286–287
extraction of antioxidant components need of, 277–278
from different sources and its applica- naturally occurring oxidants and anti-
tion, 101–102 oxidants in, 264–267
lipid oxidation non-protein nitrogen (NPN) fraction,
factors affecting development, 262
345–354 nutritional significance, 264
natural antioxidants in oxidants and antioxidants in, 266
essential oil-based antioxidants, oxidation of
116–117 auto-oxidation of fat, 270
fruit-based antioxidants, 102–112 chlorophyll and porphyrin, 267
herbs- and spices-based antioxidants, cross-reactivity of intermediate spe-
112, 114–116 cies, 267–268
seaweed-based antioxidants, 122–124 lipids, 268
vegetables-based antioxidants, methionine, 271
117–122 methods of measurement, 271–272
Melissa officinalis, 158 off-flavor defects, 268
Menhaden oil (MHO), 156–157 oxidative cross-linking of proteins,
Metal-catalyzed oxidation of protein, 270 267
Metal ions, 14–15 photosensitizers, 268–269
Methanol, 100–101 protein, 269–270
Metmyoglobin formation, 19, 24 quality and safety issues of, 271
colorimetry data, 233 riboflavin, 268–269
Mexican chia seed, 321–322 type I or type II reaction, 269
Milk and dairy products types of, 267
unsaturated fatty acids, 268
antioxidants, characteristics, types, and
phytanic acid, 264
mechanism of action of, 272
availability and preparations, 275 processing of milk, 267
chain-breaking and synergists, 273 pro-oxidant and antioxidant factors, 267
chelating and sequestering agents, proteins, 262
273 source of energy, 264
deterioration of organoleptic qualities vitamins, 266
of food, 272–273 whey proteins, 263
effects in milk, 275–276 Milk and milk based beverages, 279–280
effects of foods, 275 Milk fat globule membrane (MFGM),
reaction mechanisms, 274 265
Index 389

Milpa Alta, 312 Neutral lipids (NL), 46


Minced fish muscle (MFM), 327 New Dietary Ingredient Premarket Notifi-
Mincing, 349 cation Final Rule, 84
Mint (Mentha spicata L.), 285 Nicotinamide adenine dinucleotide
Modified atmosphere packaging (MAP), (NADH), 194
364–367 Nitro blue tetrazolium (NBT), 194
Monounsaturated fatty acids (MUFA), Nitrosomyoglobin (NOMb) content, 122
8, 47 Non-protein bound heme-iron, 26–27
Monterey sardine, 11 Nordihydroguaiaretic acid (NDGA), 275
Muscle-based foods, 12 Nori (Porphyra umbilicalis), 123–124
Muscle pH, 16
Mustard leaf (Brassica juncea), 120 O
Myoglobin autoxidation, 19–20 Oil photooxidation, 8
Myoglobin oxidation in foods of animal Oleic acid, 47
origin, 18 Oleoresin rosemary (Herbalox), 108
interrelationship between lipid oxida- Opuntia ficus-indica (cactus pear), 312
tion and, 20–21 Orange (Citrus aurantium v. Canoneta),
role of deoxymyoglobin, 22–23 304
role of ferrylmyoglobin, 25–27 Orange peel extract (OPE), 282
role of oxymyoglobin, 23–24 Oregano essential oil (OEO), 280
Oregano extract (OE), 280
N Oregano (Origanum vulgare L.), 158, 371
Natural antioxidants. See also Meat as Origanum vulgare, 158
food Oryzanol, 76
application in fish products, 155–157 antioxidant effect of, 78
aqueous extracts of, 81 components of, 77
in cereal brans, 81 in food preservation, 78–79
dynamics and mechanism, 45–46 health benefits of, 77
essential oil-based antioxidants, safety studies of, 78
116–117 Oryzanol fortified biscuits (OFB), 79
fruit-based antioxidants, 102–112 Oryzanol fortified fat (OFF), 79
herbs- and spices-based antioxidants, Oxidation of lipids, 4. See also Lipid
oxidation in foods of animal origin
112, 114–116
impact
market and consumer acceptability of, Oxidation reduction potential (ORP)
157–158 method
occurrence and their role in food preser- colorimetry data, 240–241
vation, 41–43 effectiveness, 240
quality of, 99 Nernst equation, 239
regulatory status of extracts, concen- oxidative status of meat products, 239
trates, and resins, 83–84 reducing conditions, 240
seaweed-based antioxidants, 122–124 Oxidative processes, 142–143
synthetic antioxidants and, 44–45 Oxidative rancidity, 11
use of, 83 Oxidative stability (OS)
vegetables-based antioxidants, 117–122 of oil, 48
Natural tocopherols, 12 of seasonings during storage, 251–252
390 Natural Antioxidants: Applications in Foods of Animal Origin

Oxidative stress, 41–42 Phlorotannins, 123


Oxygen radical absorbance capacity Phospholipids (PL), 45
(ORAC) antioxidant activity of, 47
assay, 194 Photooxidation, 5, 7–8
values, 44, 280 Photosensitizations, 7
Oxymyoglobin, 22 Photosensitizers, 268–269
Phylloquinone. See Vitamin K1
P Phylloquinone-rich oils, 55
Pacific saury (Cololabis saira), 11 Phytic acid
Particle size reduction and tumbling, 17 as anticancer agent, 69
Pasban-90, 320 antioxidation effect, 70
Peroxides food applications of, 72–73
assessing oxidative status, 210 in food preservation, 70–72
Beer Lambert law, 213–214 structure of, 72
calculation for PVs, 209 in unpolished rice, 69
concentration and extinction coeffi- Phytosterol oxidation products (POPs), 63
cient, 215 Phytosterols (PS), 43–44
data from hydroperoxide analysis, 215 biological function and chemical struc-
electrochemical amplification, 211 ture, 60
flow diagram for, 216 food applications of, 60–63
iodometric titration method, 208–209 in vegetable oils, 62
level of quantification (LOQ), 209–210 Pigmented orange juice, 83
Pineapple, 314
during lipid oxidation, 208
Pine bark extract (Pycnogenol®), 108
meat matrix, 210–211
Pink guava pulp, 108–109
Nernst equation, 211 Plant sterols, 60
potentiometric titration of Polyphenoloxidase (PPO), 59
hydroperoxide Polyphenols, 43–44, 63–64
with sodium thiosulfate in model Polyunsaturated fatty acids (PUFA), 3–4,
system, 211–213 47
principle for quantitative spectroscopy, Pomegranate fruit juice phenolics (PFJP),
214 112
quantifying PVs, 213 Pomegranate peel extracts (PPE), 282
solvent extracted lipids from meat, 208 Pomegranates (Punica granatum),
specification limits, 217 111–112
value data in graph, 216 Porphyra tenera, 123
Peroxide value (PV), 11, 48 Post mortem process, 24
Persa lime peels, 310–311 Potato peel extract (PPE), 119
Phenazonium methosulfate (PMS), 194 Potato (Solanum tuberosum L.), 119
Phenolic compounds Poultry meat, 12
Poultry products, 166
antioxidant property of, 63
lipid oxidation, 167
antioxidative mechanism of, 64, 66
antioxidants in preventing, 171
as free radical acceptors and chain
catalysts of, 170
breakers, 66 factors predisposing, 169–170
in vegetable oils, 64–65 mechanism, 168–169
Phenolics, 43–44
Index 391

measurement of efficacy of AOA, R


193–195 Radical–radical coupling, 7
mode of application of antioxidants Radical scavenging activity (RSA), 284
active package, 192 and antioxidant activity, role of lipid
dietary supplementation, 190–191
fractions in, 46–47
direction addition to product, 191
enrobing, 192 tests, 46
marination, 192 Rancid or fishy odor, 4
spraying, 191 Rancimat, 44
Raw meat
natural antioxidants
animal sources, 188–190 at cold temperatures oxygen, 230
and effective concentration in, color and lipid oxidation, 231
172–173 colorimetry data, 233
plant sources, 174–188 main types of, 232
sources of, 171 nutrition, 234
synergistic effect of, 193 pigments, 232
Pre-cooked/frozen sausage, 110 red oxymyoglobin pigments, 232
Press juice, 12 Red ginseng extract (RGE), 280
Primary antioxidants, 352–353 Red oxymyoglobin (OxyMbg) pigments,
Proanthocyanidins (condensed tannins), 232
100 Red wine grape pomace (RWGP),
Processing and storage conditions 306–307
cooking, 348 Reflectance colorimetry, 231
high-pressure processing, 348–349 Reflectance spectroscopy, 231
irradiation, 347 Refrigerated tuna (Thunnus albacares),
light, 349–350 20
mincing, 349 Riboflavin, 268–269
Pro-oxidant factors Rosemary (Rosmarinus officinalis L.),
enzymes, 351 371
heme-proteins, 350–351
S
metals, 350
Propagation phase of oxidation, 6–7 Salmon (Salmo salar), fishy odor, 4
Propyl gallate (PG), 43–44 Salvia hispanica L., 321–322
Protamex®, 361 Sargassum kjellamanianums, 123
Protein digestibility corrected amino acid Seafood, 142
score (PDCAAS), 262–263 Sea Spaghetti (Himanthalia elongata),
Psoas major muscle (oxidative muscle), 123–124
346 Seaweed-based antioxidants
Punjab-96, 320 antioxidant properties, 122–123
Purple wheat bran, 320–321 carotenoid pigment, 124
industrial production, 122
Q meat samples, 123–124
Quercetin, 107 research, 123
Quince scalding water, 282 use of, 124
Secondary antioxidants
chelating agents, 353–354
392 Natural Antioxidants: Applications in Foods of Animal Origin

reducing agents, 353 antioxidative mechanism of, 51–52


Seed and by-products approximate biological activity rela-
Amaranth and Quinoa, 323–324 tionships of compounds, 52
cereals and legumes, 319–321 in crude vegetable oils, 49–50
cocoa husk, 322 in food preservation, 53–54
Mexican chia seed, 321–322 mechanism for radical quenching
Sensitizers, 7–8 action, 52
Sesame oil lignans Tomatoes, 121–122
antioxidative effect of, 75 Total anthocyanin (TA), 306
contents, 73 Total dietary fiber (TDF), 301
food applications of, 76 Total oxyradical scavenging activity
in food preservation, 75–76 (TOSC) assay, 118
oil insoluble lignans of, 74 Trolox equivalent antioxidant capacity
oil soluble lignans of, 74 (TEAC) methods, 322
Sesamol, 275 Tulsi (Ocimum sanctum L.), 281
SH- 2002, 320 Tuna meat discoloration, 19
Shatavari (Asparagus racemosus), 281 Turkey meat, 12
Sliced pepperoni, oxidative color degra- Type I or type II reaction, 269
dation, 204
Sodium chloride, 15–16
U
Sodium phytate, 82 Ultra-filtered (UF)-soft cheese, 284
Soluble dietary fiber (SDF), 301 Ultra-high temperature (UHT) milk,
Soya and its processed products, 100 266–267
Spanish sage, 321–322 Ultrasonic/microwave assisted extraction
Spray granulation, 14 (UMAE), 324
Stability index (SI), 105 Undaria pinnatifida, 123
Stripped and crude seed oil, OS of, 48 Undesirable fatty acids (OFA), 235
Stypocaulon scoparium, 123 Unshu-orange flavedo, 53
Sugar-snap cookies, 82 Uqab-2000, 320
Sulfhydryl oxidase, 266 USDA food composition table, 234
Synthetic phenolic antioxidants BHA, 44
V
T Vacuum-packaged mechanically sepa-
Tea infusions, 282 rated turkey (MST), 107
Tert-butylhydroquinone (TBHQ), 150, Valencia orange, 310–311
362 Vegetable by-products
Tertiary butyl hydroquinone (TBHQ), asparagus, 317–318
43–44 brassica plants, 315–317
Thermal or photochemical homolytic carrot peel, 318–319
cleavage, 6 Vegetable oils
Thiobarbituric acid reactive substances carotenoids of, 68
(TBARS), 13 desmethylsterols of, 61
Thiobarbituric acid (TBA), 220, 272
phenolics of, 64
value, 158
tocopherol homologues of, 49–50
Tocols (tocopherols and tocotrienols),
Vegetables-based antioxidants
11–12, 43–44, 275
caloric contribution, 117
Index 393

carrot, 119 W
cruciferous vegetables, 120–121 Warmed-over-flavor (WOF) develop-
leafy green vegetables, 118–119 ment, 12
potato, 119 Water holding capacity (WHC), 250–251
prevention of diseases, 117–118 Water-soluble vitamin C, 45
role in maintenance of health, 117–118 Wheat bran, 319–321
tomatoes, 121–122 Whey proteins, 263
Vidarikand (Pueraria tuberosa), 281 White cabbages (Brassica oleracea var.
Virgin olive oil, 63–64 capitata), 316
Vitamin C White grape antioxidant dietary fiber
activity, 55–56 (WGDF), 327
antioxidants permitted in foodstuffs for White grape (V. vinifera, var. Airén), 327
infants and young children, 59 White wine grape pomace (WWGP),
306–307
categories of reactions, 59
concentrations in foods or plants, 55 X
in food preservation, 58–59
Xanthine oxidoreductase, 266
properties and low toxicity, 55–56
Xanthophyllomyces dendrorhous, 191
responsible for anaerobic loss of, 58–59
Xanthophylls, 66
in selected foods, 56–57
structure, 58 Y
Vitamin E. See Tocols (tocopherols and
tocotrienols) Yellowtail (Seriola quinqueradiata) dark
Vitamin K1 muscle, 20
Yogurt, 282
food applications of, 54–55
importance of, 54 Z
role of, 54
Zeaxanthin, 67
significance of dietary, 55
structure of, 54
Volatile secondary oxidation products,
220

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