Probiotic

Bacteria
This page intentionally left blank
 edited by
J. Paulo Sousa e Silva
Ana C. Freitas

Probiotic
Bacteria
Fundamentals, Therapy, and Technological Aspects
CRC Press
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 Contents

Foreword ix
Preface xi

1. Probioc Bacteria: From Science to Consumers’ Benefit 1

Manuela M. Pintado, Ana M. Gomes, and Ana C. Freitas

2. Gastrointesnal Tract: Microflora and Transit Aspects 7

Ana M. Gomes, Manuela M. Pintado, Ana C. Freitas, and
J. Paulo Sousa e Silva
2.1 Gastrointestinal Physiology 7
2.1.1 Introduction 7
2.1.2 GIT pH 9
2.1.3 Total Residence Time in the GIT 10
2.1.4 Mouth and Oesophagus 10
2.1.5 Stomach 11
2.1.6 Small Intestine 11
2.1.7 Large Intestine 12
2.2 Gut Microlora: Human ‘Virtual Organ’ 12
2.2.1 Ecological Niches 12
2.2.2 Physiological Role of Gut Microlora 15
2.2.2.1 Antagonistic mechanisms:
barrier function 16
2.2.2.2 Metabolic contribution 17
2.2.2.3 Immunomodulation 21
2.2.3 Balance between Detrimental/
Beneicial Gut Microbiota and
Intestinal Disorders 22
2.3 Selection of Target Organisms 26
2.3.1 Probiotics and Their Effects Upon Gut
Microbiota 26
2.3.2 GIT In vitro Models for Selection 28
2.4 Conclusions 33
vi Contents

3. Probiocs and Their Therapeuc Role 47

Manuela M. Pintado, Ana M. Gomes, and Ana C. Freitas
3.1 Health Potential of Probiotics: Mechanisms
of Action 47
3.2 Alleviation of Lactose Intolerance 52
3.3 Inhibition of Helicobacter pylori and
Intestinal Pathogens 55
3.3.1 Inhibition of Helicobacter pylori 55
3.3.2 Inhibition of Other Enteric Pathogenic
Bacteria and Prevention and Reduction
of Diarrhea Symptoms 60
3.3.3 Inhibition of Enteric Virus: Rotavirus 63
3.4 Prevention of Inlammatory Bowel Disease 66
3.5 Hypocholesterolemic Effect 70
3.6 Treatment and Prevention of Allergy 72
3.7 Reduction of the Risk Associated with
Mutagenicity and Carcinogenicity 75
3.8 Effect on Urogenital Infections 79
3.9 Beneits for the Healthy Function of the Liver
and Pancreas 81
3.10 Oral Health–Promoting Properties 83
3.11 Other Therapeutic Applications 84
3.12 Conclusions 85

4. Food as Vehicles of Probiocs 95

Ana C. Freitas, Dina Rodrigues, Sérgio Sousa,
Ana M. Gomes, and Manuela M. Pintado
4.1 Introduction 95
4.2 Dairy Products 96
4.2.1 Fermented Milks 96
4.2.1.1 Deinition, classiication,
market, and physiological role 97
4.2.1.2 Technological challenges for
probiotic fermented milks 101
4.2.1.3 Survival characteristics 114
4.2.2 Cheeses 115
4.2.2.1 Strains, cell probiotic
concentration, and viability 116
4.2.2.2 Cheese: Technological
aspects 128
 Contents vii

4.3 Nondairy Products 134

4.3.1 Beverages 134
4.3.2 Other Products 144
4.4 Conclusions 151

5. Immobilizaon and Microencapsulaon of Probiocs 171

Paulo J. C. Costa, Teresa Rocha-Santos, Ana M. Gomes,
Manuela M. Pintado, Sérgio Sousa, Maria H. Amaral,
J. Paulo Sousa e Silva, and Ana C. Freitas
5.1 Introduction 171
5.2 Microencapsulation 175
5.2.1 Encapsulating Materials 176
5.3 Methods of Microencapsulation 178
5.3.1 Microparticle Formation 178
5.3.1.1 Liquid matrix dispersion 178
5.3.1.2 Solid matrix techniques 194
5.3.2 Immobilization/Entrapment
Techniques 197
5.3.2.1 Solidiication 197
5.3.2.2 Coacervation 198
5.3.2.3 Gelation 201
5.3.2.4 Solvent extraction/
evaporation 203
5.3.2.5 Polymerization 205
5.3.3 Other Methods 206
5.4 Microparticle Characterization 208
5.5 Conclusions and Future Trends 209

6. Development of Probioc Dosage Forms 227

Maria H. Amaral, J. Paulo Sousa e Silva,
Paulo J. C. Costa, and Ana M. Gomes
6.1 Introduction 227
6.2 Manufacturing of Dosage Forms Containing
Probiotics 230
6.2.1 Powders 233
6.2.2 Capsules 234
6.2.3 Tablets 236
6.2.4 Vaginal Suppositories 240
6.2.5 Other Pharmaceutical Dosage Forms 243
6.2.5.1 Chewing gums and lozenges 243
viii Contents

6.2.5.2 Gels 245

6.2.5.3 Eye drops 246
6.2.5.4 Pellets 246
6.3 Dosage Forms Characterization 247
6.4 Packaging and Storage of Dosage Forms
Containing Probiotics 253
6.5 Conclusions and Future Trends 254

7. Guidelines and Regulaons 263

J. Paulo Sousa e Silva and Ana M. Gomes
7.1 Introduction 263
7.1.1 Food Standards 264
7.1.2 Drug Standards 265
7.2 Guidelines for Probiotic Selection 265
7.2.1 Safety Criteria 266
7.2.2 Functionality, Technological, and
Labeling Criteria 268
7.3 Probiotics Legal Status 272
7.3.1 Asia–Paciic 272
7.3.1.1 Australia and New Zealand 272
7.3.1.2 China 275
7.3.1.3 Japan 277
7.3.2 Europe 281
7.3.2.1 Central legislation 283
7.3.2.2 Web pages 283
7.3.3 United States of America 283
7.3.3.1 Legislation 286
7.3.3.2 Web pages 287
7.3.4 Latin America (Brazil) 287
7.3.4.1 Legislation 289
7.4 Conclusions 290

Index 295
 Contents ix

Foreword

It gives me immense pleasure to write a few words about the

upcoming book Probiotic Bacteria: Fundamentals, Therapy, and
Technological Aspects edited by Drs. J. Paulo Sousa e Silva and Ana
Cristina Freitas. Probiotic organisms have been a subject of keen
research for more than a century. Studies on probiotic organisms
have come a long way from the era of Metchnikoff in the early
1900s, and our knowledge is being enhanced with each passing
decade. Traditionally, probiotic organisms have been incorporated
in fermented products like yogurt, however, as of recent times there
are numerous probiotic products in the global market of varied
types. The literature on probiotic organisms is vast and diverse and
there is enough clinical evidence to support the health-enhancing
potential of probiotic organisms.
This book is a unique compilation of technological aspects
related to probiotic products, their beneits, and their therapeutic
and physiological implications. The information is conveniently
grouped under seven chapters. Chapter 1 details the fundamentals of
probiotic bacteria. Chapter 2 covers the gastrointestinal physiology
and its relevance to probiotic products. Chapter 3 deals with the
various therapeutic roles of probiotics beyond gut-health. Chapter 4
gives an overview on the advances in probiotic food, with judicious
insights into the technological and functional aspects. Chapter 5
provides a detailed analysis of several materials and techniques
for immobilization and microencapsulation of probiotic bacteria.
Chapter 6 is devoted to the development of probiotic dosage forms,
and Chapter 7 focuses on the guidelines and regulation pertaining to
the use of probiotic organisms.
In general, an attempt has been made to provide a comprehensive
review on the fundamentals of probiotic organisms, along with their
therapeutic and industrial aspects. The book is unique in presenting
a dedicated section on the development of several dosage forms
containing probiotic bacteria. The book provides a contemporary
update and a holistic review of the topic, and is designed to augment
related books in the market. The editorial team comprises individuals
x Foreword

with noteworthy and remarkable experience in the ield of probiotic

organisms. It is anticipated that this book should be an indispensable
resource for academicians, extension staff, and students working in
the ield of probiotic organisms and probiotic products. Also, the
book should appeal to technologists and food scientists in the related
industry.

Nagendra P. Shah
Professor of Food Science
School of Biological Sciences
The University of Hong Kong, Hong Kong
 Contents xi

Preface

Probiotic organisms, according to the Food and Agriculture

Organization (FAO) of the United Nations and the World Health
Organization (WHO), are live microorganisms that when
administered in adequate amounts could confer a health beneit on
the host. Probiotics can play a major role in human health if they can
be incorporated in food products or used as dosage forms. For this, a
detailed knowledge of the microorganisms is required, which forms
the basis of the selection and use of probiotics.
Probiotics may be useful in several functions, namely protection
against pathogenic bacteria directly via displacement of these
bacteria by competitive binding or growth inhibition, by antimicrobial
compounds or pH reduction, or indirectly by neutralization or
elimination of toxins from the intestine, improving gut barrier
integrity by ameliorating epithelial and tissue integrity through
low-dose NO synthesis, simulation of mucus production, or/and
enhancing gut epithelial cell proliferation. In addition, probiotics
have also been shown to have immunomodulation capacity, to
inhibit endogenous carcinogen production, and to provide nutrients
for enterocytes by short-chain fatty acid production. Such activities
by a speciic strain (or group) allow them to promote several
health beneits, which enables assuring of different therapeutic
applications, including alleviation of lactose intolerance, inhibition of
Helicobacter pylori or other enteric pathogenic bacteria and enteric
virus (particularly Rotavirus) with reduction of associated diarrhoea
symptoms, prevention of inlammatory bowel disease, reduction of
cholesterol level, treatment and prevention of allergy, reduction of
the risk associated with mutagenicity and carcinogenicity, reduction
and control of urogenital infections, improvement in liver and
pancreas dysfunctions, promotion of oral health.
This book, organized in seven chapters, will help to understand
what a probiotic is, how to isolate and assess the eficiency and
safety of each strain, and to elucidate about health beneits and
main mechanisms of action presenting the major current in in vitro,
animal, and human studies supporting these properties.
xii Preface

Chapter 1 introduces the theme and summarizes the steps of

launching probiotic products in the market. Chapter 2 presents an
updated overview of the human intestinal microbial ecosystem from
both endogenous and exogenous perspectives. At an endogenous
level, the chapter covers the available knowledge on the dominant
microbiota composition and stability, discusses the functional roles
bacteria play in human health and well-being, and analyzes the
consequences of homeostasis rupture among microbial balance as
far as intestinal disorders are concerned. At the exogenous level, the
chapter illustrates that target organisms are capable of modulating
gut microbiota and of promoting different physiological roles. The
chapter also gives perspectives on the use of probiotics in dietary
management and disease risk reduction.
The main goal of Chapter 3 is to describe the relevant health
potential of probiotics and current advances. The beneicial
properties assigned to probiotics and the corresponding speciic
mechanisms of action that will support each of the subsequent
therapeutic applications will be explored in this chapter.
Chapter 4 aims to provide a comprehensive overview on the
advances in probiotic food, covering the technological issues,
functionality aspects, and limitations of some foods as carriers
of probiotics. This chapter is divided in two parts: the irst part
covers dairy products, fermented milks, and cheeses that constitute
the major group of products that can carry and deliver probiotic
bacteria; the second part covers non-dairy products where
alternative functional foods with probiotics such as juices and other
food carriers are presented and discussed.
To confer health beneits to the human host, probiotics must be
kept alive until they reach their site of action. In Chapter 5 different
approaches including immobilization and/or encapsulation of
probiotics inside a protective material in order to increase the
resistance of these sensitive microorganisms against adverse
conditions have been revisited.
The probiotic strains intended to beneit health or treat illness
may be incorporated into suitable dosage forms in which they can
maintain their effectiveness. Therefore, Chapter 6 is concerned
with dosage forms, such as oral powders, capsules, oral and vaginal
tablets, vaginal suppositories, chewing gums, gels, eye drops, and
pellets, that are used to administer probiotics. The deinition of
 Preface xiii

these dosage forms, their respective processes of manufacturing,

and characterization tests are also addressed in this chapter.
In Chapter 7, which is the last chapter of the book, the global
legal framework for probiotics is addressed. Generically, probiotics
may be considered as a food, including food additives and dietary
supplements, or as a drug. The chapter underlines the safety
considerations and presents a comprehensive report on all necessary
requirements related to them.
In summary, the book intends to provide a comprehensive
overview of the fundamental concepts, mechanisms, therapeutic
actions, technological aspects, and ongoing research related with
probiotic bacteria. The book will be helpful for students and scientists
from the food science and technology, pharmacy, and nutrition
sciences ields; scientists working in the ield of gastrointestinal
disorders and other chronic diseases; companies who are designing
and marketing new functional foods or nutraceuticals; as well as
other public health professionals and clinicians. Furthermore, it
provides important information for all readers interested in the
relationship between food and health.
This scientiic work was a team effort written by a group of
scientists from the food and pharmaceutical research ields directly
involved in the development of project PROBIOCAPS (PTDC/AGR-
ALI/71051/2006; FCOMP-01-0124-FEDER-008792): Ana Gomes,
Dina Rodrigues, Helena Amaral, Manuela Pintado, Paulo Costa,
Sérgio Sousa, Teresa Rocha-Santos, and the editors of this book,
and through individual research grants (SFRH/BD/77647/2011;
SFRH/BPD/73781/2010; SFRH/BPD/65410/2009) by FCT. We
would like to thank all these scientists for their contribution, and all
others who, in different areas and skills, helped this project become
a real success. We would also like to thank the reviewers for their
professional advice and reviewing the chapters of this book.
J. Paulo Sousa e Silva
Ana C. Freitas
Winter 2013
This page intentionally left blank
Chapter 1

Probiotic Bacteria: From Science to

Consumers’ Benefit

Manuela M. Pintado,a Ana M. Gomes,a and Ana C. Freitasb,c

aCBQF, Biotechnology School of Portuguese Catholic University,

Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal

bISEIT/Viseu-Instituto Piaget, Estrada do Alto do Gaio, Galifonge,

3515-776 Lordosa, Viseu, Portugal

cCESAM & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

[email protected]

Although there is still no oficial deinition for probiotics, several

authors have tried to deine this concept throughout time. One of
the most accepted and simplest deinitions was that presented
by an expert committee organized by the Food and Agriculture
Organization (FAO) of the United Nations and the World Health
Organization (WHO), which states: “Probiotic organisms are live
microorganisms that when administered in adequate amounts
confer a health beneit on the host” (FAO/WHO, 2001). Associated
with probiotic organisms, prebiotics are deined as nondigestible
food ingredients that beneit the host by selectively stimulating the
growth and/or activity of one, or a limited number, of bacteria in

Probiotic Bacteria: Fundamentals, Therapy, and Technological Aspects

Edited by J. Paulo Sousa e Silva and Ana C. Freitas
Copyright © 2014 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4411-62-2 (Hardcover), 978-981-4411-63-9 (eBook)
www.panstanford.com
2 Probiotic Bacteria

the colon and thus improve host health (Gibson & Roberfroid, 1995).
When we combine probiotic organisms and prebiotics in a product
to obtain a presumably synergistic relationship, the term synbiotic
is used.
Although any microorganism that would produce health
beneits could be considered a probiotic, only some genera have
proven to be probiotic. The genera of bacteria and fungi that have
been employed for their probiotic properties are most commonly
species of Lactobacillus and Biidobacterium and species of the yeast
genus Saccharomyces; other bacterial genera, such as Streptococcus,
Enterococcus, and Bacillus, have also been studied. However, with
regard to the genera Enterococcus and Bacillus, particular concerns
have been raised concerning their safety properties (Hempel et al.,
2011). Some of these genera have been used as single cultures or
in mixed formulations. In a very recent revision, Bengmark (2012)
reported some probiotic starter cultures assumed with no identiiable
adverse effects in clinical studies (e.g., no effect in terms of bacteria
translocation, gastric colonization with enteric organisms, or septic
morbidity, serum C reactive protein levels or mortality). This included
isolated strains such as Lactobacillus plantarum 299v (Pro-Viva) or
L. rhamnosus GG or multiple strains, Ecologic 641 (Winclove Bio
Industries, Amsterdam, the Netherlands), a supplemented synbiotic
composition consisting of six different strains of freeze-dried, viable
bacteria: L. acidophilus, L. casei, L. salivarius, Lactococcus lactis,
Biidobacterium biidum, and B. lactis along with corn-starch and
maltodextrins, Trevis (Chr Hansen Biosystem, Denmark) with L.
acidophilus La5, B. lactis Bb-12, Streptococcus thermophilus, and L.
bulgaricus as well as VSL#3 (VSL Pharmaceuticals, Ft Lauderdale,
Florida, USA) with four strains of Lactobacillus (L. casei, L. plantarum,
L. acidophilus, and L. delbrueckii subsp. bulgaricus) along with three
strains of Biidobacterium (B. longum, B. breve, and B. infantis) and S.
salivarius subsp. thermophilus.
Several health attributes have been ascribed to probiotics, which
has increased commercial interest in exploiting different applications
leading to the rapid growth and expansion of this market sector.
From the selection of a probiotic strain to its incorporation in a inal
product, both in food matrices or pharmaceutical formulations,
several steps must be accomplished if a safe and biological active
product is to be achieved (Fig. 1.1).
 Probioc Bacteria 3

Sources: Selecon of Probioc

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-
'&
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-
+
 + 
( .

Toxicity: ,
0 
Potenal probioc

Colonizaon: 


+

Health benefits: 




+
+
 (

 

Stability



Food Pharmaceucal


 



 
applicaons applicaons





 

Guidelines and Regulaons

Immobilizaon/encapsulaon
+
-
*+-
 

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+
 
+-

Figure 1.1 Schematic representation of the sequential steps required

from probiotics selection to a inal product be launched in the
market.

Each potential probiotic strain isolated from a human, animal,

plant, or food source, apart from its accurate identiication, must be
validated regarding safety issues according to guidelines deined by
a system similar in concept and purpose at both US and European
levels, denominated as GRAS (generally recognised as safe) or QPS
(Qualiied Presumption of Safety), respectively, permitting in both
cases the identiication of what is required to make an adequate
safety assessment of a microorganism. In parallel, the probiotic
must be submitted to functional characterization, as certain
functionalities, namely, its survival through the gastrointestinal
tract (GIT) (pH conditions, enzymes, and bile salts), and instead of
help predict health beneits potential (production of antimicrobial
compounds, activity upon cholesterol, and so on). These potential
probiotics must be now tested through increasing complex systems
from in vitro studies, to animal and inally human studies requiring, in
general, double-blind, randomized, placebo-controlled human trials
or other appropriate design with sample size and primary outcomes
appropriate to determine if a strain/product is eficient. This eficacy
is assured if probiotics colonize transiently the intestine, do not
exhibit any adverse effect on the patient, and demonstrated one or
more health beneits mediated by one or more mechanisms of actions
4 Probiotic Bacteria

to be associated with one or more of the therapeutic applications:

(i) the prevention or treatment of infectious diseases, including
viral, bacterial, or antibiotic-associated diarrhoea; (ii) relief of
chronic bowel inlammatory diseases; (iii) immuno-modulation; (iv)
lowering of serum cholesterol; (v) decreased risk of colon cancer;
(vi) improvement of lactose digestion; (vii) reduction of allergies;
and (viii) effect on intestinal microbiota (Saad et al., 2013). However,
some strains are not as stable as required to guarantee the passage
throughout the GIT or to resist to the conditions and interactions
when incorporated in the food or dosage forms. This reduced
stability may be overcome by immobilization or encapsulation
of probiotic strain using different encapsulation techniques and
materials assuring the required protection for a certain environment
and pre-determined period. The safe, functional, and stable probiotic
may now be incorporated in a commercial product, either a food or a
dosage form. However, launching of these products associated with
health claims is regulated according to each country’s legislative
system and following strict guidelines. As an example, nutrition
and health claims were harmonized at the European level to better
protect consumers. EU regulation, EC No. 1924/2006, amended by
the European Parliament and the Council of 15 January 2008 (EC No.
109/2008) establishes the necessary authorization procedures to
ensure that the allegations contained on packages and in marketing
of foodstuffs are clear, precise, and based on evidence accepted by
the scientiic community.
Despite the developed research performed in the last years,
it should be emphasized that the effect of probiotics remains
ambiguous and requires more investigations in order to be
conirmed or validated. This fact may be not only due to action of
these probiotics but also due to the lack of information about the
pathogenesis of some diseases (Saad et al., 2013). Although large
investigation has been done on health beneits associated with
probiotic bacteria as far as binomials strain-therapeutic application
and strain-dose effects are concerned, experts still believe that the
studies are not yet suficient and information is still lacking. Such
expert opinions have barred the possibility of European Food Safety
Authority approval over 2012 of related health claims, limiting the
use of probiotics with recognized health beneits at food industry
level. So, in future, more studies, particularly well-designed, double-
blind, randomized, placebo-controlled trials continue to be required.
 References 5

In addition, a greater understanding of the mechanisms behind the

action of probiotics on the gastrointestinal microbiota is required in
order to better understand which probiotic is the most beneicial and
how the genetic and bacterial proiles of the patient will inluence
treatment responsiveness.

References
Bengmark, S. (2013) Gut microbiota, immune development and function.
Pharmacol. Res., 69(1), 87–113.
FAO/WHO (2001) Report on joint FAO/WHO expert consultation on
evaluation of health and nutritional properties of probiotics in food
including powder milk with live acid bacteria, Cordoba, Argentina.
Gibson, G.R. and Roberfroid, M.B. (1995) Dietary modulation of the human
colonic microbiotia: introducing the concept of prebiotics. J. Nutr.,
125, 1401–1412.
Hempel, S., Newberry, S. Ruelaz, A., Wang, Z. Miles, J.N.V., Suttorp, M.J.,
Johnsen, B., Shanman, R. Slusser, W., Fu, N., Smith, A., Roth, B., Polak,
J., Motala, A., Perry, T. and Shekelle, P.G. (2012) Safety of probiotics to
reduce risk and prevent or treat disease. Evidence Report/Technology
Assessment, 200, 1–94.
Saad, N., Delattre, C., Urdaci, M., Schmitter, J.M. and Bressollier, P. (2013) An
overview of the last advances in probiotic and prebiotic ield. LWT -
Food Sci.Technol., 50, 1–16.
This page intentionally left blank
Chapter 2

Gastrointestinal Tract: Microflora and

Transit Aspects

Ana M. Gomes,a Manuela M. Pintado,a Ana C. Freitas,b,c and

J. Paulo Sousa e Silvad
aCBQF, Biotechnology School of Portuguese Catholic University,
Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal
bCESAM & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
cISEIT/Viseu-Instituto Piaget, Estrada do Alto do Gaio, Galifonge,

3515-776 Lordosa, Viseu, Portugal

dFaculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira,

228, 4050-313 Porto, Portugal

[email protected]

2.1 Gastrointestinal Physiology

2.1.1 Introduction
Oral use is the most convenient way for administering probiotic
products not only due to the easiness of the method but also to
promote one of the main effects attributed to probiotics— improving
intestinal microbial balance. Nevertheless, the upper gastrointestinal
tract (GIT) is an adverse medium to probiotics; hence, there are

Probiotic Bacteria: Fundamentals, Therapy, and Technological Aspects

Edited by J. Paulo Sousa e Silva and Ana C. Freitas
Copyright © 2014 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4411-62-2 (Hardcover), 978-981-4411-63-9 (eBook)
www.panstanford.com
8 Gastrointestinal Tract

many advantages in understanding the physiology of GIT in order to

develop products containing probiotics. Many textbooks delve deeply
into GIT anatomy and physiology, and for detailed descriptions, one
may consult Guyton and Hall Textbook of Medical Physiology (2011).
Anatomically and physiologically, the digestive system is divided
into the tubular GIT and accessory digestive organs (Fig. 2.1). The
GIT is approximately 6 m in length and extends from the mouth to
the anus. The organs of GIT comprise oral cavity, pharynx, esophagus,
stomach, small intestine, and large intestine. The accessory digestive
organs are the teeth, tongue, and salivary glands, liver, gallbladder,
and pancreas.

Figure 2.1 Anatomic structure of the human GIT and accessory digestive
organs.

The GIT or alimentary canal wall is composed of four layers:

from outer surface to the inner surface are the serosa, muscularis,
submucosa, and mucosa. The mucosa is composed of a simple
columnar epithelium separating the highly colonized intestinal
lumen from the second, underlying layer, the lamina propria, and the
 Gastrointestinal Physiology 9

muscularis mucosa. A mucus gel layer (composed predominantly

of mucin glycoproteins) covers a large part of the GIT epithelium.
The mucosa is the absorptive layer, maximizing nutrient absorption,
but also has secretory and barrier function preventing the passage
of strange luminal components (bacteria, food components), thus
inhibiting pro-inlammatory host response (O’Hara et al., 2006).
Adjacent intestinal epithelial cells form tight junctions, which help
maintain such selective impermeable barrier function (Bron et al.,
2011). Paneth cells and goblet cells also contribute to this function
via innate immune defences. Paneth cells are responsible for the
production of several antimicrobial compounds, including defensins
and lysozyme, which prevent interaction between microorganisms
and proliferative cells in the crypts, while goblet cells produce mucins,
which confer to the epithelium its barrier effect by preventing direct
contact from luminal microorganisms (Bevins and Salzman, 2011;
Salzman, 2011). M cells or microfold cells founded in lymphoid
tissue of ileum (Peyer´s patches) are involved in the absorption of
macromolecules, particularly protein antigens. The lamina propria
is a special type of essentially sterile connective tissue that contains
lymph nodules, which are involved in the protection against disease.
The muscularis mucosa is responsible for the small folds in some
parts of GIT.

2.1.2 GIT pH
Among other factors (transit time, bacteria metabolism, and
chemical reactions) that might inluence the performance of
probiotic products, the pH is perhaps the most important due to
general sensibility of probiotics to acidic conditions. According to
Evans et al. (1988), the average pH in stomach is 1.8 and rises to 6.6
in the proximal intestine reaching 7.5 in the distal intestine, and then
it decreases to 6.3 in right colon and it rises again, until it reaches 7.1
in left colon. The pH depends on prandial condition of individuals.
Table 2.1 illustrates physiological pH in humans in the fasting and
fed states (Gray and Dressman, 1996; Dressman et al., 1998).
Apart from prandial condition, the pH in GIT is a function of
many variables, including meal volume and content, and volume
of secretions (Mudie et al., 2010). It should be noted that the
extrapolation of GIT pH data from healthy situations to pathological
conditions must be made with precaution.
10 Gastrointestinal Tract

Table 2.1 Physiological pH in the GIT in the fasted and fed states

Gastrointestinal site Fasting Fed

Stomach 1.4–2.1 3.0–7.0
Duodenum 4.9–6.4 5.1–5.2
Jejunum 4.4–6.5 5.2–6.2
Ileum 6.5–8.0 6.8–8.0

Thus, it is not surprising that GIT pH varies between and within

individuals.

2.1.3 Total Residence Time in the GIT

The residence time (transit time) of a probiotic product (e.g., dosage
form or food) in the intestine is also a determinant of the viability
of probiotics, as the exposure to the harsh conditions will depend
substantially on gastric residence time.
The oesophageal residence time is usually short (in the order
of seconds to minutes) and the small intestine residence time is
relatively constant (3–4 h), but the gastric time varies widely (5 min
to 2 h, although much longer times have been reported) according
to factors such as volume, osmolality, pH, viscosity, and nature of
ingested material or even emotional factors and age. Thus, the total
residence time is dependent upon factors such as gastric emptying
rate and low rate, and can exhibit a signiicant intraindividual and
interindividual variability (Mudie et al., 2010).

2.1.4 Mouth and Oesophagus

During mastication or chewing, the contact of food and other
products with oral mucosa is generally brief but suficient to initiate
the digestion of the starch by the saliva (salivary amylase). After
this rapid passage through the mouth, the swallowed food is driven
to the stomach by the peristaltic movements of oesophagus. The
oesophagus is a muscular tube that connects pharynx to stomach
and has a lumen pH between 5 and 6. The oesophageal transit time
for dosage forms, liquids, or boluses of solids is approximately 10–
20 s (Evans, 1993).
 Gastrointestinal Physiology 11

2.1.5 Stomach
Stomach has a J-shape with an approximate capacity of 1.5 l, but in a
fasting state, it normally contains 20–30 ml of luid (predominantly
wet mucus). Stomach acts as a reservoir for food while it is mixed
with acid, mucus, and pepsin in order to be released (as chime) into
the duodenum at a controlled rate. The gastric emptying time may
vary from few minutes to several hours, depending on the time of the
last food ingestion, anxiety, position, and level of individual activity,
among other factors.
During fasting, an interdigestive cycle of motility (migrating
myoelectric complex, MMC) composed of four phases governs
stomach activity. This cycle begins in the stomach and moves along
the small intestine into the distal ileum. The Phase III of MMC (a burst
of contractile activity of 5–15 min) opens the pylorus and clears the
stomach. The ingestion of food stops the cycle.
The peristaltic movements appear 5–10 min after the intake of
foods and remain until the gastric emptying, which lasts from 1 to
several hours depending on the meal composition (Phillips, 1993).
The hydrochloric acid secreted by parietal cells kills many bacteria
and provides the pH for pepsin to begin protein digestion (Ganong,
2005).

2.1.6 Small Intestine

The small intestine (small bowel) extending from the pyloric
sphincter of stomach to the ileocaecal valve has three distinct parts,
namely, duodenum (20–30 cm), jejunum (approx. 2 m), and ileum
(approx. 3 m). The main functions of this organ are the digestion of
foods and the absorption of nutrients and other materials.
Several intestinal adaptations, which increase the surface
area, favor absorption in small intestine. These adaptations are as
follows:
 Folds of Kerckring—submucosal folds of several millimeters
in depth
 Villi—inger-like projections of approx. 0.5 to 1.5 mm in
length
 Microvilli—brush-like structures of approx. 1 μm in length
In the lumen of the small intestine, foods and other products
are mixed with the bicarbonate (from Brunner’s glands, located
in duodenum), secretions of mucosal cells (mucus and enzymes),
12 Gastrointestinal Tract

pancreatic juice (sodium bicarbonate and enzymes), and bile (bile

acids, phospholipids, and bilirubin). The detergent property of bile
confers a potent antimicrobial activity that hampers the survival
of the microorganisms (including many probiotics) in the GIT. The
small bowel constitutes a transition zone between stomach bacterial
scarcity and highly populated colon.

2.1.7 Large Intestine

The large intestine (colon) stretches from the ileocecal valve to the
anus and has two main functions:
 the absorption of water, sodium, and chloride ions
 the storage of feces
The colon is composed of the caecum, the ascending colon, the
transverse colon, the descending colon, the sigmoid colon, and the
rectum. The slower colon motility is responsible for transit times up
to 60 h, which undoubtedly contributes to the tremendous number
of microorganisms found in the colon.
The large intestine is colonized by about 1012 bacteria per gram
of intestinal contents (about 35–50% of the volume content of the
colon), which are responsible for several metabolic reactions.
These microorganisms may be in the lumen, at the mucus gel,
or in the mucosal epithelial cells receptors (Berg, 1996; Madsen,
2001). The equilibrium of the microbial groups present in the GIT
is essential for human health. Biidobacterium and Lactobacillus
species are the main strains with identiied beneicial properties in
the indigenous GIT microlora.

2.2 Gut Microflora: Human ‘Virtual Organ’

2.2.1 Ecological Niches

The human GIT microbiota consists of about 1014 bacteria and
up to an estimated 1000–1150 different bacterial species, whose
collective genome (intestinal microbiome) consists of at least 100-
fold more genes than the human genome (Qin et al., 2010). These
microbial communities are found in a diversity of environmental
niches (digestive, urogenital, naso-bucal, and respiratory mucosal
surfaces), yet, as previously mentioned, the large intestine is the
 Gut Microflora 13

most inhabited (average of 1011 bacteria per gram of stool). Although

55 divisions of bacteria have been described, only eight bacterial
divisions have been identiied so far in the GIT. Here, recent studies,
involving culture-independent molecular microbiological techniques,
have shown that the most abundant bacterial phyla found are the
Gram-negative Bacteroidetes (include genera related to Bacteroides
corresponding to 9–42% of total bacteria on average) and the
Gram-positive, low guanine-cytosine (GC)% Firmicutes (composed
of species belonging to the genera Eubacterium, Clostridium,
Ruminococcus, Butyrivibrio); together, they may represent more
than 90% of the phylogenetic groups present in the human gut,
speciically in the distal part (Eckburg et al., 2005; Lay et al., 2005;
Aureli et al., 2011). Less abundant phyla, yet not less important
in terms of host’s health, include Actinobacteria, Fusobacteria,
Proteobacteria, and Verrucomicrobia phyla. The dominant bacterial
groups include Clostridium coccoides-Eubacterium rectale (often the
most represented between 14 to 22% of total bacteria depending on
the studies (Doré and Corthier, 2010), Clostridium leptum (including
the species Faecalibacterium prausnitzii, Ruminococcus albus, and
R. lavefaciens (at a dominance of 16–22% of total bacteria on
average) (Lay et al., 2005), Bacteroides-Prevotella, Biidobacterium
species, and Atopobium species (Diamant et al., 2011). Among the
occasionally found species are Clostridium ramosum, Eubacterium
cylindroides, Phascolarctobacterium, Verrucomicrobium, Sporomusa,
Selenomonas, or Veillonella (Doré and Corthier, 2010).
Each individual houses a unique collection of bacterial species
(including the established core of at least 57 bacterial species
considered common to all humans) that remain in a relatively
complex equilibrium (within the same log-unit equivalent in terms
of population) over time from day-to-day and even across years.
Indeed, molecular ecology tools have shown that approximately 80%
of an individual’s fecal microbiota is individual-speciic (Eckburg et
al., 2005). At the level of strains, stability is not as clear and will vary
with individual (Doré and Corthier, 2010). Furthermore, species
diversity for subdominant groups such as that of Lactobacillus is
less stable than that of dominant groups (Vanhoutte et al., 2004)
and the colon shows better stability of communities than the ileum.
Factors modulating ecological niches include transit time, pH, and
quality and quantity of exogenous substances such as prebiotics
or probiotics and endogenous mucins among others (Doré and
14 Gastrointestinal Tract

Corthier, 2010). Induction of durable alterations, especially among

dominant bacterial groups, is not easy due to capacity of bacteria
to resist change as well as resilience (capacity to recover original
make-up following application of a stress)—two determinants
responsible for maintenance of homeostasis (Doré and Corthier,
2010). Shifts in these ecological homeostatic niches (normobiosis)
may either prompt speciic disease-inducing activity (dysbiosis at
metabolic, degenerative, or immune levels) or disease-protective
activity (probiosis at similar levels of disease).
The well-established interindividual variability among microbial
communities is related with either internal (genetic factors, age,
gender, stress exposure, health status) or external factors. The
latter include type of early exposure to microorganisms of maternal
origin (faecal, vaginal, cutaneous and maternal milk (Doré and
Corthier, 2010), lifestyle factors (Dicksved et al., 2007), diet (Ley
et al., 2006), antibiotic therapy (Jernberg et al., 2010), and even
geographical inluence—differences in the microbiota composition
of healthy individuals from different locations have been reported
(Mueller et al., 2006; Fallani et al., 2010). In fact, consideration of
the highly individual gut microbial activity has been acknowledged
as an important aspect of personalized nutrition strategies and is
becoming a turning point among food industry innovation.
The diversity of the gut microbiota is relatively simple in infants
but becomes more complex with increasing age, reaching a high
degree of complexity in adults (Fanaro et al., 2003). Human gut
microbiota acquisition and development in the host may begin before
birth (bacteria have been detected in amniotic luid, umbilical cord
and meconium (DiGiulio et al., 2008; Koenig et al., 2011), although
in infants effective development occurs as a function of the irst
inoculi received from the environment, the mother’s microbiota, the
delivery mode, formula or breast-feeding, and subsequent weaning
food practices and the use of antimicrobials (Fallani et al., 2010). The
existence of a fetal microbiome has clinical implications, with greater
microbial diversity being associated with prematurity (DiGiulio et
al., 2008; Mshvildadze et al., 2010).
The indigenous microlora of infants is dominated by one or a
few genera, and among breast-fed infants, Biidobacterium strains
are dominant, and become established shortly after birth. Their
proliferation is stimulated by speciic nutrients, glycoprotein
components of κ-casein in human colostrum and, to a lesser extent,
breast-milk as well as biidogenic bacteria in breast-milk (Martín et
 Gut Microflora 15

al., 2004, 2009; Walker, 2010). Microbiota composition undergoes

signiicant shifts as solid foods are introduced into the diet (Koenig
et al., 2011). The numerical proportion of biidobacteria decreases
with increasing age of the individual and eventually becomes the
third most abundant genus (accounting for ca. 25% of the total
adult gut lora), second to the genera Bacteroides and Eubacterium
(Finegold et al., 1983).
Undoubtedly, human gut microbiota may play an important
buffer role between the host and the environment and several human
microbiome projects are in progress in order to try and explore its
relevance in health and disease. Two major projects, namely, the NIH
Human Microbiome Project (HMP: http://commonfund.nih.gov/
hmp/) in the United States and the Metagenomics of the Human
Intestine (metaHIT: http://www. metahit.eu) project in Europe, are
important contributions to this deciphering process.

2.2.2 Physiological Role of Gut Microflora

There is an increasing awareness of the role that intestinal microlora
may play in human health. Tremendous progress has been made
in this ield and results indicate that it is likely that much of this
impact is mediated through diet and the consumption of speciic
health-related foods. Growing evidence suggests that gut microbiota
inluence what the human host is able to extract from its diet,
including energy (see Fig. 2.2).

Gut
Microbiota

Functional
Foods Metabolites

Role in Host
Physiology
and Health

Figure 2.2 Possible interactions between gut microbiota-diet and

generated metabolites therefrom toward beneicial functions
in host physiology and health.
16 Gastrointestinal Tract

Considerable efforts have been made to better deine the complex

symbiotic role of the intestinal microbiota in host physiology and to
discover the underlying mechanisms (Hörmannsperger and Haller,
2010; Bron et al. 2011). These have been associated with multiple
functions, including (i) a metabolic function (including energy
homeostasis, digestion and bioavailability of nutrients, supporting
fat metabolism, fermentation of undigestible carbohydrates with
concomitant production of short-chain fatty acids, SCFAs); (ii)
a mucosal barrier function (including prevention of mucosal
infections by pathogen invasion inhibition and maintenance of an
intact intestinal barrier); and (iii) an immune modulatory function
(including enteric nerve regulation and maintenance of intestinal
epithelium homeostasis and regulation of the mucosal immune
system by acting as an important source of stimulators (Preidis and
Versalovic, 2009; Hörmannsperger and Haller, 2010; Holmes et al.,
2011).

2.2.2.1 Antagonistic mechanisms: barrier function

As previously mentioned, the intestinal epithelium constitutes an
important primary line of defence composing a physical barrier
that controls the transcellular and paracellular transit of exogenous
substances and prevents the entry of most of luminal antigens.
The commensal microbiota contribute to the “barrier effect” via
participation in paracellular permeability regulation, mucin gene
expression by goblet cells, and secretion of antimicrobial peptides
(defensins and angiogenins) by intestinal Paneth cells.
Studies in germ-free animals have demonstrated that the normal
functioning of intestinal epithelial cells is impaired in the absence
of the intestinal microbiota. Intestinal epithelium expression of
microbial recognition receptors, defensins, and antimicrobial
peptides is reduced in germ-free animals (Bouskra et al., 2008). A
good overview on the very important role of antimicrobial peptides
in the GIT and the existing dynamic interactions between the
intestinal epithelial barrier and the commensal microbiota based
on recent advances is available in the review by Muniz et al. (2012).
Further studies have shown that a modulation of the gut microbiota
through dietary supplementation with a prebiotic (i.e. oligofructose)
increases epithelial barrier integrity by increasing the expression of
tight junction proteins (i.e. zonulin ZO-1 and occludin), a mechanism
 Gut Microflora 17

that is dependent on the augmented secretion of the glucagon-like

peptide-2 intestinal hormone (Cani et al., 2009) (see Fig. 2.4).

2.2.2.2 Metabolic contribution

A growing body of evidence suggests that the gut microbiota impacts
a wide range of host metabolic pathways inluencing disease
prevention and disease risk. Gut microbiota produce an almost
limitless set of metabolites and their understanding may provide the
key to unlocking many health-promoting functions of gut microbiota,
including probiotics.
Intestinal microbiota act as a “metabolic organ” providing
additional enzymes and regulating the expression of genes involved
in the degradation of complex indigestible dietary carbohydrates and
proteins, with subsequent generation of fermentation end-products
(Falony et al., 2006), cholesterol reduction, deconjugation and
dehydroxylation of bile acids, production of vitamins (K and B group)
and isoprenoids, metabolism of amino acids, and conversion of dietary
polyphenolic compounds into their active form (van Duynhoven et al.,
2011). Indeed, the intact forms of dietary polyphenols have limited
availability presenting low levels in plasma. A major part of the
polyphenols persists in the colon, where the resident microbiota (e.g.,
Escherichia coli, Biidobacterium sp., Lactobacillus sp., Bacteroides
sp., Eubacterium sp.) produces metabolites that may suffer further
metabolism upon enteric systemic circulation. For example, dietary
lavonoids are normally present as poorly absorbed glycosides,
which need to undergo enzymatic deglycosylation in the small
intestine before being absorbed as aglycones (the nonsugar group)
(Walle, 2004). Apart from microbial deglycosylation, the microbiota
may also perform mild transformations such as dehydroxylation and
demethylation and, in addition, catabolism of polyphenols into small
fragments (see Fig. 2.3).
Apart from less active metabolites, bacterial metabolic
conversion in the gut may also lead to metabolites with increased
biological activity. For example, the pseudoestrogenic activity of
prenyllavonoids from hops (Possemiers et al., 2006), soy isolavones,
and lignans (Bowly et al., 2003) is determined by intestinal bacterial
activation followed by absorption of the microbial metabolites (van
Duynhoven et al., 2011), hence modifying host exposure to these
components and their potential health effects.
18 Gastrointestinal Tract

Other studies have shown a beneicial impact of polyphenols

on GIT microbiota proiles. Feeding wine polyphenols (57 mg/kg
body weight by gavage for 10 days) to rats resulted in predominant
fecal Bacteroides, Lactobacillus, and Biidobacterium compared with
the controls, which revealed predominant Bacteroides, Clostridium,
and Propionibacterium (Dolara et al., 2005). Parkar et al. (2008)
demonstrated how several polyphenols, including caffeic acid,
catechin, epicatehecin, coumaric acid, phloridzin, rutin, naringenin,
daidzein, genistein, and quercetin, inhibited growth and adhesion of
bacterial pathogens to human Caco-2 cells as well as enhanced the
proliferation and adhesion of Lactobacillus rhamnosus, a probiotic
strain. Further examples may be found in the recent review by
Laparra and Sanz (2010) in which reciprocal interactions between
intestinal microbiota and phytochemicals in food, and associated
consequences toward human health are duly covered.


 






   



 

 






 

 


  









  
 

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Figure 2.3 Representation of metabolic bioconversion of dietary

polyphenols in the human-microbiota ecosystem. Adapted
from van Duynhoven et al., 2011, and Possemiers et al., 2011.
 Gut Microflora 19

The speciic capacity of gut microbiota to use complex dietary

carbohydrates may contribute to harvest energy from the diet,
representing up to 10% of the daily energy supply (Falony et al.,
2006). Accordingly, comparisons between germ-free mice and mice
colonized by the conventional distal gut microbiota revealed a host’s
increased ability to extract energy from the diet brought about by the
microbiota; this energy was then stored in adipocytes, contributing
to increased body weight (Bäckhed et al., 2004; Turnbaugh et
al., 2008). Indeed, the obese microbiome was shown to extract
more energy from the same amount of kilocalories than the lean
microbiome (Turnbaugh et al., 2008).
Commensal microbiota extract energy by direct fermentation of
dietary carbohydrates, resistant to digestion, into short-chain fatty
acids (SCFAs) (e.g., acetate, propionate, and butyrate), ethanol, and
gases (e.g., carbon dioxide and hydrogen). In contrast, when proteins
are fermented, the end products include toxic compounds such as
amines and phenols (Tuohy et al., 2006; Bernalier-Donadille, 2010).
The beneicial role of SCFAs in enterocyte growth and
differentiation is well known; butyrate is preferentially metabolized
and may also promote anti-inlammatory effects. Acetate may act as
a precursor for lipid and cholesterol synthesis in the liver, whereas
propionate may impact negatively thereon; indeed, propionate is a
natural precursor for gluconeogenesis (Bernalier-Donadille, 2010).
The role of SCFAs on food intake behavior and on energy intake is
being targeted and available studies have been recently reviewed by
Cani and Delzenne (2011). Here, it was proposed that SCFAs come
directly into play following gut microbiota fermentation and may
play a role in appetite regulation; modulation of plasma SCFAs was
related to changes in gut peptides involved in appetite regulation.
Indeed, interactions between food, intestinal microbiota, and
the host are fundamental to the maintenance of homeostasis in the
ecosystem. Although many of the abovementioned roles may be
beneicial for the host colonocytes, drawbacks are associated with the
proneness to weight gain. Observations among microbiome capacity
have been generated to uphold such statements. The expression of a
monosaccharide transporter (Na+/glucose cotransporter) has been
induced in mono-colonized mice, leading to increased absorption of
dietary monosaccharides and SCFAs, which in turn lead to de novo
synthesis of lipids in the liver. Conventionalization of germ-free mice
originates increased liver expression of acetyl-CoA carboxylase and
20 Gastrointestinal Tract

fatty acid synthase, key enzymes in de novo fatty acid synthesis,

as well as increased expression of the transcriptional factors
carbohydrate response element binding protein and sterol regulatory
element binding protein-1, both involved in hepatocyte lipogenic
responses to insulin and glucose. In addition, levels of fasting-
induced adipose factor (FIAF) in the gut are decreased by microbial
colonization, which minimizes fat oxidation and promotes fat storage
(Bäckhed et al., 2004) (see Table 2.2). Additional evidence for such
interrelationships between energy homeostasis and gut microbiota
and metabolic functions are covered in a comprehensive review by
Cani and Delzenne (2011). Several mechanisms are proposed herein
on how gut microbiota control body weight and energy homeostasis
including models on how gut microbiota may contribute to the onset
of obesity and associated metabolic disorders (Fig. 2.4).

Lifestyle and gene factors:

High-fat diet and genetic obesity

Gut microbiota dysbiosis

Gut permeability – altered barrier function

(altered distribution of the tight junction
proteins ZO-1 and occludin)

Metabolic endotoxaemia

Development of low-grade inflammation and

insulin
i li resistance
it in
i the
th liver,
li muscles
l andd
adipose tissue

Figure 2.4 Possible mechanisms for describing how gut microbiota are
involved in the onset of obesity and associated metabolic
disorders. Adapted from Cani and Delzenne, 2011.

In summary, recent advances in cellular and molecular biology

have provided solid evidence on the signiicant contribution of gut
microbial metabolism to the presence of metabolites in the host
(Wikoff et al., 2009; Martin et al., 2012). Metabolomic strategies
have become an essential tool to decipher such gut microbiota–host
metabolic interaction and impact on homeostasis and host health. A
mass spectrometry based metabolomics strategy applied to germ-
free mouse model systems has demonstrated the signiicant effect of
 Gut Microflora 21

the microbiome on blood metabolites (Wikoff et al., 2009); numerous

circulating molecules were determined to arise exclusively in the
presence of gut microlora or signiicantly alter their concentration
by its presence (Wikoff et al., 2009). For example, production of
the antioxidant indole-3-propionic acid (derived from tryptophan
metabolism) was shown to be completely dependent on the
presence of gut microbiota and could be established by colonization
with the single bacterium Clostridium sporogenes. Martin et al.
(2007) underlined the different bacterial modulation of the bile acid
metabolism and enterohepatic cycle, with consequent effects on
the absorption of dietary fat and concomitant to lipid accumulation
in the livers of animals holding a nonadapted gut microbiota. With
these studies, the authors showed the importance of maintaining
a balanced and well-adapted gut microbiota to prevent onset of
microbial-related metabolic disorders. Martin et al. (2012) further
addresses nutritional approaches, in which the need for homeostatic
balance is dependent on both the host and the nutritional modulation
of the intestinal microbiota–host metabolic interactions.

2.2.2.3 Immunomodulation
The immune regulatory function of the intestinal microbiota
consists of priming the mucosal immune system and maintenance
of intestinal epithelium homeostasis. The intestinal microbiota may
inluence the content of lamina propria T cells, immunoglobulin A
producing B cells, intraepithelial T cells, and serum immunoglobulin
levels (Tlaskalová-Hogenová et al., 2004). As already stated, studies
in germ-free animals demonstrated impairment of immune function
in the absence of the intestinal microbiota. Defective development of
gut-associated lymphoid tissues, antibody production (i.e., sIgA), and
maturation of isolated lymphoid follicles have been shown in germ-
free animals, together with reduced Peyer’s patches and mesenteric
lymph node number and dimension (Bouskra et al., 2008).
In this symbiotic relationship, it is also important that immune
system adequately recognizes microbial components and establishes
a state of tolerance towards them (Aureli et al., 2011). According
to Aureli et al. (2011), such tolerance of microbiota is mediated
by complementary mechanisms, including (i) microbiota bacteria
do not express virulence factors such as antigenic structures
recognized by toll-like receptors on the surface of immune system
22 Gastrointestinal Tract

cells or secondary to chemical modiications of pathogens-associated

molecular patterns; or, (ii) several commensal bacteria are able
to suppress inlammatory processes (e.g., downregulation of the
activity of nuclear factor-kB responsible for inlammatory response
via pro-inlammatory cytokine/chemokine and AMP production).

2.2.3 Balance between Detrimental/Beneficial Gut

Microbiota and Intestinal Disorders
As previously stated, a strong ecological balance exists between
the host and its gut microbiota, in terms of impact on health, yet
consensus on the exact mechanisms by which microbes modulate
disease onsets has not yet been achieved (Holmes et al., 2011;
Collado et al., 2012).
In infants, recent indings have found associations between
microbiome composition and two diseases prevalent among babies
in neonatal intensive care units, namely, necrotizing enterocolitis
and late-onset sepsis (Alexander et al., 2011; Mai et al., 2011).
Furthermore, the implications of mode of delivery on gut colonization
have also been shown in the context of disease wherein a wide
range of immune-related diseases may be associated with cesarean
section delivery (Neu and Rushing, 2011). Other recent evidence
indicates associations between microbial ecology in children and
the onset of type 1 diabetes (Vaarala et al., 2008; Brown et al., 2011).
Collectively, these various lines of research may suggest that the
early microbiome, from fetal development through childhood, can
inluence both short-term and long-term health.
Any disturbance of the abovementioned physiological functions
may be intrinsically linked with alterations or maladaptations of
gastointestinal microbiota and can consequently impair many of these
homeostatic and physiological signals leading to varying states of
disease, including allergy, inlammatory bowel disease (IBD), obesity,
certain cancers, diabetes (Holmes et al., 2011), celiac disease (CD),
autism, cardiovascular disease as well as emergence of antibiotic-
resistant strains as far as administration of antimicrobial agents is
concerned (Jernberg et al., 2010) (see Table 2.2). Restoration of this
balance by increasing levels of Biidobacterium either by food-based
strategies (using prebiotics) or supplementation has been shown to
reduce disease intensiveness in patients and to improve well-being
 Gut Microflora 23

in healthy volunteers. Associated mechanisms that underlie these

effects will be visited in Chapter 3.

Table 2.2 Examples of disease models with modulation of gut

microbiota

Disease Model Host–microbiome interaction Reference

IBD Human Certain antibiotics improved the disease Barnich et
course of IBD patients, whereas several al., 2007
IBD animal models needed bacterial
colonization for inlammation
Human IBD patients revealed reduced diversity Frank et
of Firmicutes and Bacteroidetes; al., 2007
E. coli (example of mucous-invading
bacteria) may be associated with dis-
ease-speciic activity
Human Bacterial species Faecalibacterium Sokol et
prausnitzii, if present in the mucosa- al., 2008
associated microbiota of the patient’s
ileum, is protective against postopera-
tive recurrence of endoscopic signs of
inlammation 6 months after surgical
resection of the ileocecal region of the
intestine
Human IBD patients revealed intestinal dysbio- Sokol et
sis with a lower representation of the al., 2009
Clostridium leptum group among the
Firmicutes phylum
Human The differential urinary signature of Williams
IBD from Crohn’s disease and ulcera- et al., 2009
tive colitis includes alteration of hippu-
rate, 4-cresyl sulphate, and formate, all
potential metabolites of gut microbial
activity
Mouse Induction of a transforming growth Atarashi et
factor-β rich environment by indig- al., 2011
enous Clostridium species promotes T
regulatory cell accumulation in the co-
lon and resistance to ulcerative colitis

(Continued)
24 Gastrointestinal Tract

Table 2.2 (Continued)

Disease Model Host–microbiome interaction Reference

Diabetes Mouse A dysfunction in the microbial respon- Wen et al.,
sive immune system can lead to autoim- 2008
munity and diabetes; nonobese diabetic
mouse lacking the innate microbial-
recognition immune system receptor
MyD88 is resistant to type 1 diabetes
Human Sequencing results revealed a bacte- Wu et al.,
rial composition of diabetic group dif- 2010
ferent from that of the healthy group;
Bacteroides vulgatus, Biidobacterium
genus, and Clostridium leptum subgroup
have undergone changes of different
degree in diabetic group; copy number
of Biidobacterium genus is signiicantly
declining in diabetic group
Human Amount of Firmicutes signiicantly re- Larsen
duced in diabetes type 2 patients com- et al., 2010
pared with the control group. The ratio
of Bacteroidetes to Firmicutes positively
correlated with plasma glucose concen-
tration but not with body mass index.
Obesity Human Aberrant compositional development Kalliomäki
of the gut microbiota precedes over- et al., 2008
weight; biidobacterial numbers in fe-
cal samples during infancy, assessed by
luorescent in situ hybridization (FISH)
with low cytometry, were higher in
normal weight children (median: 2.19 s
109 cells/g) than in children developing
overweight (1.20 s 109 cells/g); a great-
er number of Staphylococcus aureus
were reported in overweight children;
authors propose that S. aureus consti-
tutes trigger for low-grade inlamma-
tion contributing to development of
obesity.
 Gut Microflora 25

Disease Model Host–microbiome interaction Reference

Obesity Human Signiicantly higher numbers of Collado
Bacteroides group in women with ex- et al., 2008
cessive weight gain upon pregnancy.
Positive correlation between the
number of Bacteroides, Clostridium, and
Staphylococcus on the one hand, and
the weight and BMI before pregnancy
on the other hand. The Biidobacterium
genus was present at higher numbers
in normal-weight than in overweight
women and also in women with lower
weight gain during pregnancy.
Mouse Conventionalization of germ-free mice Bäckhed
was shown to: promote body weight et al., 2004
gain and fat mass development; pro-
mote a general increase in the activity
of lipoprotein lipase (LPL), catalyzing
the release of fatty acids and triacylg-
lycerol from circulating lipoproteins
in muscle, and adipose tissue. Such in-
crease was proposed as a consequence
of suppression of the FIAF in the gut.
LPL activity is inhibited by FIAF; hence,
lack of FIAF in conventionalized germ-
free mice may lead to accumulation of
lipids in the adipose tissue.
Cancer Mouse The use of probiotics, prebiotics, or Tuohy
synbiotics was able to protect against et al., 2005
chemically induced colonic DNA dam-
age in animal models
Rats Rats inoculated with human lora and Rowland
fed a diet containing lactulose com- et al., 1996
pared with those fed a diet containing
comparable amount of sucrose revealed
colonoctyes with less DNA damage fol-
lowing oral treatment with dimethylhy-
drazine
Mouse Colitis-associated colorectal cancer can Uronis
be reversed by colonization with nor- et al., 2009
mal gut microbiota
(Continued)
26 Gastrointestinal Tract

Table 2.2 (Continued)

Disease Model Host–microbiome interaction Reference

CD Human Speciic microbial TTGE proile and a Schippa
signiicant higher biodiversity in CD et al., 2010
pediatric patients’ duodenal mucosa
was noted; Bacteroides vulgatus and E.
coli were detected more often in CD pa-
tients than in controls.
No signiicant difference was found in
the prevalence of Biidobacterium spp.
between CD patients and controls.
Autism Human Ten-fold higher numbers of Clostridium Sekirov
spp. in autistic children than in healthy et al., 2010
individuals.
Many Clostridium species may produce
neurotoxins, which could contribute to
the autism spectrum impaired GI symp-
toms

2.3 Selection of Target Organisms

2.3.1 Probiotics and Their Effects Upon Gut Microbiota

As previously mentioned in Chapter 1, probiotics are live
microorganisms, which when administered in adequate amounts,
confer a health beneit on the host. Key aspects and requirements
for probiotic strains and probiotic products have been the target
of many reviews (Gobbetti et al., 2010; Sanders and Marco, 2010;
Sanders, 2011) and issues such as strain speciicity of health
effects, mechanisms of action, required dose for probiotic products,
and safety considerations will be further detailed in the following
chapters. In this chapter, an overview of their role in the GIT will be
given.
The most common formulation of probiotics is as fresh
fermentation products or as dried bacterial supplements, and their
consumption has been associated with a variety of health beneits for
the consumer either directly (interaction with host’s mechanisms)
or via modulation of intestinal microlora. Supplementation with
probiotics is intended to either replace or reduce the number
 Selection of Target Organisms 27

of potentially pathogenic bacteria in the intestine by enhancing

the number of beneicial strains that are capable of fermenting
carbohydrates and that have little proteolytic activity. Species of the
microbial genera Lactobacillus and Biidobacterium represent the
great majority of marketed probiotics corresponding to 23 and ive
strains, respectively; other less representative strains include two
E. coli and one strain each of Bacillus, Streptococcus, Enterococcus,
and Lactococcus (Sanders, 2007; EFSA, 2010). Alternatively,
prebiotics, which are known as nondigestible food ingredients,
generally oligosaccharides, may modify the balance of the intestinal
microbiota by stimulating the activity of these health beneicial
bacteria, lactobacilli and biidobacteria.
Probiotics may play a beneicial role on human health by
interacting with the gut microbiome and impacting on host response
(Aureli et al., 2011; O’Flaherty and Klaenhammer et al., 2011; van
Baarlen et al., 2011); it is less clear whether the observed human
health beneits are actually mediated by the microbiome changes
(Sanders, 2011). The mechanisms of action whereby probiotics may
promote such health-promoting effects include competitive exclusion
of pathogenic bacteria, either directly (inhibition or competition by
probiotic strain) or indirectly (probiotic inluence on endogenous
commensal microbiota) (Corr et al., 2009); strengthening of epithelial
barrier function by modulating signaling pathways that may promote
increased mucus production (Mack et al., 2003), defensins generation
(Schlee et al., 2008) or tight junction function (Anderson et al., 2010;
Karczewski et al., 2010), and apoptosis prevention (Yan et al., 2007);
modulation of the host immune system (O’Flaherty et al., 2010).
Noticeably, probiotic microorganisms do not act exclusively in the
large intestine by affecting the intestinal lora but also mediate some
of the abovementioned mechanisms (immunological modulation or
provision of bioactive metabolites) in other organs. In particular,
some of these interactions can be inluenced by the in situ SCFA
milieu, which may affect or even mediate some of the beneicial
effects of probiotics.
Numerous studies report that probiotics can inluence the
nutritional status of mankind (Gobbetti et al., 2010; Bhat and Bhat,
2011). A plethora of beneicial compounds may be generated directly
from in situ secondary metabolism by probiotic bacteria or indirectly
from overall production in food vectors. These may include bioactive
peptides (Gobbetti et al., 2010), water-soluble vitamins, including
28 Gastrointestinal Tract

thiamine, nicotinic acid, folic acid, pyridoxine, biotin, and B12 (Lee
et al., 1999), oligosaccharides, organic acids (Bhat and Bhat, 2011),
and polyunsaturated fatty acids such as conjugated linoleic acid
(Rodrigues et al., 2011; Rodriguez-Alcalá et al., 2011). Furthermore,
iron bioavailability may be increased by L. acidophilus and bile acids
may be deconjugated by numerous lactobacilli.
An overall review of the mechanistic insights that many studies
have provided concerning probiotics and their interactions with
the composition and function of the intestinal mucosa via speciic
molecules (cell wall components such as peptidoglycan, teichoic
acids or capsular polysaccharides, and immunomodulatory proteins)
is given by Bron et al. (2012). In this review, the importance of
human individuality is discussed as previously mentioned and the
possibility of a personalized application of probiotics in the near
future is proposed (Bron et al., 2012). These mechanisms will be
further probed in Chapter 3.
Criteria for rational use of probiotics will be given in Chapter 7,
but in summary may include adequate identiication and functional
characterization of bacteria; administration of an appropriate
quantity of bacteria (which is inluenced by nature of strain
employed, whether in combination with other strains of same or
different species, and by the food/pharmaceutical matrix selected)
and their enumeration; beneits for the host and their deinition; and
safety of use.

2.3.2 GIT in vitro Models for Selection

Use of probiotics as mediators of health and well-being or as
therapeutic agents for the treatment of several GIT diseases has
been suggested as a positive management strategy; nonetheless,
their selection needs to be as target-oriented as possible as well
as the food or supplement vector better appointed. In addition,
the destiny of food compounds in the GIT, including probiotics, has
a major impact on their nutritional quality or biological stability
to reach speciic targets in GIT. As previously discussed, gut
microbiota and its metabolic capacity is among the factors that most
inluence how the host responds to nutrients/bioactive compounds.
Furthermore, different microlora proiles, as a consequence of
different pathologies, may necessarily implicate different host
responses. Hence, there is currently a big challenge to develop
 Selection of Target Organisms 29

reliable methods for providing important insights into digestion and

the gut microbiota mode of action required toward maintenance/
destruction of host health. Performance of these mechanistic studies
in vivo is sometimes dificult not only due to sampling drawbacks
but also due to ethical constraints. Due to such limitations, different
in vivo animal models have been developed; the rat (Nyman et al.,
1986) and the pig (Glitso et al., 2000) are the two main animals
used in fermentation studies. Nevertheless, in vivo experiments
are expensive and time-consuming; hence, different in vitro models
simulating conditions in the human GIT are warranted. These model
systems may potentially reduce the use of animals in testing new
strains, products, and treatments. Several such models, of varying
degrees of complexity, have been developed worldwide (Molly et al.,
1993; Nollet et al., 1997; Gmeiner et al., 2000; Spratt et al., 2005).
These in vitro models may be either upper intestinal models, which
are used for removal of digestible components and for detection of
changes in the nondigestible ones, or continuous, semi-continuous,
or batch colon models, which elucidate the role of microbiota in
the metabolism of all nondigestible parts of the diet. They provide
a baseline for studying the micro-ecology of the gut, particularly
changes induced after perturbation of lora by diet, drugs, or toxic
chemicals; metabolic capacity of GIT microbiota and their effect
on structural changes of food components (beyond that gained by
chemical analysis alone) can also be assessed as a function of time.
In addition, model systems enable assessment of a large number of
samples over a short period of time. Nonetheless, systemic effects
cannot be simulated by in vitro models, and as consequence of their
predictive nature, studies with humans are ultimately required in
order to be able to document health effects.
Most conventional methodologies have involved growing cells to
be studied, centrifugating and ressuspending the cells in an acidic
milieu, with or without washing, at pH values varying between 1.0
and 3.0 or incubating bacteria in a medium containing 0.3% oxgall
bile, porcine bile, or bile salts (Jacobsen et al., 1999). Currently, two
types of simulators are available: those that simulate the human
intestinal microbial ecosystem (in vitro colon models) and those
that simulate the gastrointestinal physiological events (in vitro
digestion models). A good example of the latter is the dynamic,
computer-controlled model termed TNO GIT Model (TIM) that was
developed by the Netherlands Organisation for Applied Scientiic
30 Gastrointestinal Tract

Research TNO (Minekus et al., 1995, 1999). It consists of four

chambers to accurately replicate the dynamic in vivo conditions in
the stomach and small intestine, such as the change of pH, bile salt
concentrations, and chime transit, when food is being digested and
absorbed. This model enables sampling at various times along the
digestion process, which allows for a real-time assessment of the
extent of release, dissolution, absorption, and bioconversion of food
and its bioactive components under various conditions in the GIT
(Gao et al., 2006).
More appropriate for probiotic selection, the in vitro colon
models maintain colonic microbiota, usually obtained from human
feces from speciic donors (according to the objectives of study),
under strictly anaerobic conditions. The methods differ from each
other in the structure of the fermentor, substrate-to-inoculum ratios,
media, operating conditions, and sampling, which make comparison
of results dificult (Aura et al., 2005a). Culture media are variable
and may be as simple as a mineral salt solution or as complex as
a medium containing vitamins, hemin, SCFA, yeast extract and
trypticase, a reducing agent, and different buffers. Cultures may be
static or several mixing methods may be used: regular swirling, a
shaking water bath, or periodic mixing (Aura et al., 2005b).
Continuous and semi-continuous colon model systems are
suitable for study of the colonic ecosystem and bacteriology (Allison
et al., 1989). Reproduction of conditions can be achieved in a multi-
stage chemostat (Allison et al., 1989). The foremost was a complex
multi-compartmental simulator, the Simulator of the entire Human
Gastrointestinal Microbial Ecosystem (SHIME; Molly et al., 1993),
which consisted of ive reactors simulating the duodenum/jejunum,
ileum, caecum and ascending colon, transverse colon, and descending
colon, respectively. A sixth reactor was later added to simulate the
stomach (Nollet et al., 1997; Gmeiner et al., 2000). This SHIME
model was recently used to study the effects of two commercially
available plant polysaccharide supplements on the structure,
composition, and metabolism of an in vitro cultured colon microbial
community (Marzorati et al., 2010). Overall, results revealed that
dietary supplements were selectively fermented along the entire
colon, promoting a positive biidogenic effect; the possibility of
enhancing species belonging to Bacteroidetes, a phylum that has
been associated with body weight management, was also discussed
(Marzorati et al., 2010).
 Selection of Target Organisms 31

At the University of Reading, an anaerobic three-stage

continuous culture system was investigated in terms of carbohydrate
metabolism and amino acid metabolism. Researchers demonstrated
that carbohydrate breakdown and SCFA production occurred mainly
in the irst reactor, whereas amino acid fermentation producing
branch-chain fatty acids occurred mainly in the second and third
reactors corresponding to reactions occurring in the ascending,
transverse, and descending colons in vivo, respectively (Macfarlane
et al., 1992; Macfarlane et al., 1998; Probert et al., 2004). Metabolic
activities of gut microbiota and associated mechanisms have also
been tested in this culture system. For example, the potential of the
lavanol monomers (–)-epicatechin and (+)-epicatechin to positively
inluence the growth of speciic bacterial groups, hence supporting
gut health via a prebiotic effect, was shown with this model (Tzounis
et al., 2008). Another study used this model to demonstrate the
impact of chain length on the selectivity of dextrans toward gut
microlora, SCFAs, and gas production (Sarbini et al., 2011).
A more robust development of a continuous in vitro model of
the colon is the validated computer-controlled TIM-2 system, which
simulates (ratio of microorganisms with composition and metabolic
activity similar to that of the colon) the dynamic processes in the
large intestine in a reproducible way (Minekus et al., 1999) under
both normal and impaired physiological conditions. The model uses
a high-density complex microbiota and includes peristaltic mixing,
water absorption, and absorption of fermentation products. The
TIM-2 model system enables continuous monitoring and control of
temperature, pH, peristaltic movements, absorption of water, and the
fermentation products, as substrate is passed from one compartment
to another through the model. The model has been validated in
terms of metabolites produced, including SCFAs, branched-chain
fatty acids, gases, ammonia, and phenolic compounds (Rehman
et al., 2012). It has been used for many applications in many
different studies, including estimation of bioavailability of iron and
phosphorous, survival of lactic acid bacteria (Minnekus et al., 1995),
bioconversion of lavonoids by the human colon microbiota (Gao
et al., 2006), prebiotic action of maize-based ibers with increased
production of health-promoting metabolites and beneicial microbes
(Maathuis et al., 2009), or the protective effect of probiotics against
antibiotic-associated disturbances (production of toxic metabolites
or destruction of endogenous microbiota) of the intestinal metabolic
32 Gastrointestinal Tract

homeostasis (Rehman et al., 2012). An important aspect with regard

to these continuous low models is their requirement for high
technical and scientiic expertise and their high costs in terms of
both hardware and running expenses.
The Enteromix tools include a small-scale, semi-continuous colon
simulator. This model consists of four sequentially attached anaerobic
vessels representing the ascending, transverse, descending, and
distal colons, respectively. All luid transfers and pH adjustments are
computer controlled and they work with small amounts of substrate,
as vessels have small working volumes (Mäkivuokko et al., 2005).
This model is considered a simple way of following adequately the
temporal production of the microbial metabolites.
A simple reproducible batch system was setup and tested
for its validity in a European interlaboratory study (Barry et al.,
1995). Fresh human feces were used as inoculum and mixed with
a carbonate phosphate buffer supplemented with trace elements
and urea. Five different carbohydrate sources were compared in ive
laboratories on three occasions in order to determine the pH, residual
non-starch polysaccharide, and SCFA production during the in vitro
fermentation. The optimal amount of carbohydrates was 10 g/l in a
fecal suspension of 167 g/l. There was also a close correspondence
between the in vitro data and an in vivo rat experiment using diets
supplemented with the same sources of carbohydrate (Barry et al.,
1995). The model has since then been applied in many fermentation
studies of nondigestible carbohydrates (Aura et al., 2005a;
Hanhineva et al., 2012) or further developed for the study of phenolic
compounds and their interaction with intestinal microbiota (Aura
et al., 2002, 2005b).
Other models include a dynamic model with two 1-l jacketed glass
beakers representing the stomach and the duodenum (Mainville
et al., 2005), which has been used to test the survival of probiotic
bacteria. Spratt et al. (2005) developed a model that realized some of
the features not captured by the other models, by designing a system
in which the low could be modeled easily and which approached
the more realistic features of dispersed plug low; the model also
intended to achieve substantial levels of mass transfer of water and
fatty acids out of the lowing system across a growing bioilm. The
system consisted of three membrane fermenters, each identical to the
single stage reported previously joined in series by three sampling
modules. Sumeri et al. (2008) designed an in vitro single bioreactor
 References 33

model that simulates food transit through the upper part of the GIT to
enable us to evaluate the probiotic potential of food products. More
recently, Mercuri et al. (2010) created the dynamic gastric model,
which was the irst dynamic in vitro model of the human stomach,
and as such, it is able to mimic the digestive processes of the gastric
compartment not only in terms of temporally varying enzymatic and
acid secretions but also in terms of simulation of the mechanical
forces exerted by the muscular tissue of the stomach, an important
model for assessment of digestion and its impact on probiotics.

2.4 Conclusions
A balanced host–intestinal microbiota relationship is essential
for intestinal homeostasis due to important roles in digestive,
immune, and metabolic functions. Gut microbiota provides an array
of functions for the host ranging from epithelial barrier function,
through protection against infectious diseases to energy recovery
from nutrients, generating SCFAs with different physiological
roles. Different studies using advanced analytical strategies have
highlighted the importance of modulation of intestinal microbiota
toward the improvement of human health.
Speciic dietary intervention programs involving food-based
strategies or supplements, such as probiotics and prebiotics, are
constantly being proposed as relevant solutions to be used in
the management of microbiota-associated disorders. The use of
probiotics in the treatment and prevention of gastrointestinal
diseases has yielded important results, and in order to select the
most adequate binomial strain-effect in vitro, gastrointestinal models
are available for a irst phase of development.

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Chapter 3

Probiotics and Their Therapeutic Role

Manuela M. Pintado,a Ana M. Gomes,a and Ana C. Freitasb,c

aCBQF, Biotechnology School of Portuguese Catholic University,
Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal
bISEIT/Viseu-Instituto Piaget, Estrada do Alto do Gaio, Galifonge,

3515-776 Lordosa, Viseu, Portugal

cCESAM & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

[email protected]

3.1 Health Potential of Probiotics:

Mechanisms of Action
Probiotics have been associated with many health-promoting effects,
primarily focused on the gastrointestinal (GI) tract beneits through
several modulating activities, but also demonstrating promising
applications at other locations of the body, namely at respiratory
and genital tract and subcutaneous tissue (Marco et al., 2006). It
is recognized that due to the numerous beneicial activities that
can be assigned to probiotics, more than one mechanism of action
is required to explain the effects of their activity that are generally
explained by direct or indirect modiication of the endogenous

Probiotic Bacteria: Fundamentals, Therapy, and Technological Aspects

Edited by J. Paulo Sousa e Silva and Ana C. Freitas
Copyright © 2014 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4411-62-2 (Hardcover), 978-981-4411-63-9 (eBook)
www.panstanford.com
48 Probiotics and Their Therapeutic Role

microbiota or the immune response. These beneicial properties are

strain dependent, and although several properties may be attributed
to a speciic strain, the multiple activities are in general achieved
by microorganism interaction either among them or with the host
(Marteau and Shanahan, 2003).
Figure 3.1 summarizes the main mechanisms of action displayed
by probiotic bacteria when in contact with gut mucosa. Concerning
the improvement of endogenous microbiota, the colonization by
probiotic bacteria involves, in irst place, the adhesion of these
bacteria to in situ cell receptors allowing for the competition for the
sites of adhesion and for nutrients, reducing number of available
receptors as well as accessible nutrients to be used by pathogenic
bacteria. This adhesion is generally considered a pre-requisite
for colonization that should not be permanent, but only transient
colonization (Ouwehand et al., 1999).
During their colonization and through their growth and
metabolism, they can release antimicrobial substances, including
bacteriocines—natural antimicrobial substances (generally of
proteinaceous nature that can have a lipid or carbohydrate moiety)
or organic acids (namely lactic and acetic acids, hydrogen peroxide,
among others) that consequently decrease the local pH. These natural
antimicrobials contribute toward in situ inhibition of pathogens.
In addition, via immunological activation, they can also promote
pathogen inhibition through induction of cytokine production or the
increase in situ IgA secretion.
Apart from that, these beneicial microorganisms can also protect
from toxins released in situ, generated from food digestion or by
microorganisms, through the entrapment of toxins and consequent
elimination from the gut. During their permanence in the gut,
they can improve epithelial and tissue integrity and functionality,
mainly through the production of low amounts of nitric oxide (NO)
synthesis, enhancement of mucus production, improvement of gut
epithelia cell proliferation, inhibition of carcinogenic substances
production or elimination through detoxiication, and generation
of nutrients, namely production of short fatty acids and vitamins
(Steidler, 2001). The mucus provides a barrier that protects the
intestinal immune cells from the high level of antigens present in
gut lumen and short-chain fatty acids (SCFAs) are the major source
of energy for colonocytes and its deiciency has been suggested to
promote colitis.
 Health Potential of Probiotics 49

Figure 3.1 Summary of the main mechanisms of action associated with

probiotic activity when in contact with gut mucosa. Probiotic
strains can displace harmless bacteria by competitive binding
(1) or killing/growth inhibition of pathogenic bacteria by
antibacterial compounds or lowering of the pH (2). Probiotics
have also proven immunomodulation ability by stimulation
of the host immune cells such as the induction of cytokine
production (3) or the increase in in situ IgA secretion (4).
Probiotic bacteria still can neutralize toxins and eliminate them
from the intestine (5). In addition, probiotics can inluence
epithelial and tissue integrity by low-dose NO synthesis (6),
stimulation of mucus production (7), enhancing gut epithelial
cell proliferation (8), inhibition of endogenous carcinogens
production (9), and providing nutrients by SCFA production
(10). This igure has been adapted from Steidler (2001).
Copyright © 2001 Elsevier Masson SAS. All rights reserved.

Some probiotic bacteria develop positive in situ effects by

strengthening the mucosal barrier by preventing and repairing
mucosal damage, whether caused by pathogens, food antigens,
or medicinal substances, and increase the pathogen-induced
transepithelial resistance of the cell monolayer to induce mucin
gene expression (Saxelin et al., 2005).
50 Probiotics and Their Therapeutic Role

In addition, the other relevant mechanism of action associated

with the probiotics include their ability to modulate the immune
system, which implies the activation of lymphoid cells of the gut-
associated lymphoid tissue present in the lamina propria and sub-
mucosa. These interactions with the lymphoid tissue may occur
through the intact microorganism and its fragments [e.g., cell wall
peptidoglycan, lipoteichoic acid (LTA) complexes, S-layer proteins,
and unmethylated cytosine–phosphate–guanine (CpG) motifs of
DNA, or metabolites produced in situ (Ouwehand et al., 1999)]. The
interaction of probiotics with host cells by adhesion might already
activate a signaling cascade leading to immune modulation, or
alternatively respond to the release of soluble factors (Oelschlaeger,
2010).
Our host’s innate immune system discriminates signals from
pathogens and from normal lora (commensals) by speciic
recognition receptors or Toll-like receptors (TLRs). Immune cells
use specialized TLRs to detect different microbial components
separately or simultaneously. The activation of different TLR
triggers diverse host responses allowing for modulation of immune
system (Marteau and Shanahan, 2003). The main immune responses
mostly associated with infection events generally involve acquired
immune system, including mainly B lymphocytes and sensitized T
lymphocytes (Th1, Th2, and Th3), and the innate immune system,
consisting mainly of macrophages and natural killer (NK) cells
(Nomot, 2005). Dendritic cells (DCs), antigen-presenting cells that
initiate in situ response in the intestinal mucosa and play a crucial
immunoregulatory role in the balance of T helper cells Th1, Th2, and
Th3 (Marteau et al., 2002).
The immunomodulation role of probiotics is also explained
(Lactobacillus rhamnosus commonly used as model) by the reduction
of the production of the proinlammatory cytokines [tumor necrosis
factor α (TNF-α) and interleukins 6 and 12 (IL-6 and IL-12)] by
immature DCs and the production of IL-12 and IL-18 by mature DCs
(Saxelin et al., 2005).
Marco et al. (2006) described the main effects of probiotics on
immune responses and intestinal barrier integrity identifying the
bacterial and host effector molecules (see Fig. 3.2).
In irst stage, DCs play a key role in initial bacterial recognition
and, consequently, in inluencing T-cell responses, favoring Th1,
 Health Potential of Probiotics 51

Th2, or Th3 immune responses. This interaction was shown to be

mediated in Lactobacillus by binding to the pattern-recognition
receptor (PRR) DC-speciic intercellular adhesion molecule
3-grabbing non-integrin (DC-SIGN). In particular, only those
Lactobacillus strains able to interact with DC-SIGN were able to
induce IL-10 producing and regulatory T-cell populations. On the
contrary, intestinal and colonic epithelial cells (IECs and CECs) also
demonstrated to be involved in bacterial recognition and immune
modulation. Interaction of Lactobacillus GG (LGG) with Caco-2
IECs leads to reducing TNF α (TNF-α)-induced IL-8 production
via the nuclear factor (NF)-kB signaling pathway. Other bacteria
(namely the probiotic mixture VSL#3 consisting of eight different
strains) showed to interact with CEC inhibiting the degradation
of the NF-kB inhibitor I-kB. In addition, these may also occur
after recognition of bacterial components (e.g., CpG DNA by TLR9
receptors). These effects positively affect the mucosa by reducing
the intestinal inlammation. Probiotic also proved to reinforce the
intestinal barrier through several mechanisms. A speciic probiotic
Escherichia coli (strain Nissle 1917) was shown to induce expression
of the antimicrobial peptide β-defensin-2 (hBD2) via the NF-kB and
activating protein 1 (AP-1) transcription factors. This activation of
hBD2 production possibly leads to the enhancement of the intestinal
mucosal barrier, and simultaneously justiies the antimicrobial
activity upon pathogens in GI tract. Inhibition of pathogen can also
be mediated by neutralizing attachment and growth by probiotic
strains possessing mannose adhesins. On the contrary, the LTAs
of probiotics have been identiied as relevant in the modulation of
speciic immune responses and D-alanine substituents seem to be
involved. The beneicial effect of probiotics at this level culminates
on reduced levels of pro-inlammatory cytokines and higher levels of
the anti-inlammatory cytokine IL-10 mediated by TLR2-mediated
signaling.
In addition, speciic heat shock proteins (Hsps) that regulate
cytoskeletal integrity were produced after interaction of VSL#3
consortiums with IECs. Similarly, interaction of LGG with CECs
also induced the production of Hsps via intracellular signaling
with several mitogen-activated protein kinases (MAPKs). The inal
effect allows preventing apoptosis by the activation of IEC signal
transduction pathway modulating the intestinal barrier function.
52 Probiotics and Their Therapeutic Role

Figure 3.2 Illustration describing the main immune responses induced by

probiotics and effects on intestinal barrier integrity. Some pro-
biotic strains are able to promote IL-10 producing, regulatory
T cells (Tregs) through DC-SIGN interaction (1). They can also
induce hyporesponsive CD4+ T-cell populations after DC inter-
action (2). LTA composition is responsible for the differential
modulation of cytokine production (3). Positive modulation of
inlammatory responses by inactivation of the NF-kB signaling
pathway is achieved through proteasome inhibition after IEC
recognition of soluble probiotic components (4) or after rec-
ognition of bacterial motifs (e.g., CpG DNA by TLR9 receptors)
(6). The induction of Hsps either via 4 or 5, stabilizing the actin
cytoskeleton, would reinforce the mucosal barrier. Pathogen
attachment and growth could be neutralized by strains pos-
sessing mannose adhesins (7) or by induction of hBD2 in IECs
(8). M cell, an epithelial cell specialized in antigen uptake and
transport. Reprinted from Marco et al. (2006), with permission
from Elsevier.

During the subsequent sections, the beneicial properties assigned

to the probiotics and the corresponding speciic mechanisms of
action that will support each of the subsequent therapy applications
will be explored.

3.2 Alleviation of Lactose Intolerance

Lactose intolerance is a syndrome associated with the decrease of
the intestinal β-galactosidase (β-gal or commonly known as lactase)
 Alleviation of Lactose Intolerance 53

activity to values lower than 10% of childhood levels. In general, the

decline of this enzyme is a natural characteristic of the maturing
intestine in the majority of the world’s population, declining from
the third year of life. However, this characteristic changes across the
world population, and among Europeans, the incidence is between 7
and 20%, compared with 65–75% in Africans and 60–90% in people
from Asia and the area around the Mediterranean (Ockeloen and
Deckers-Kocken, 2012).
This enzyme lactase transforms lactose into glucose and
galactose, which will be easily absorbed by mucosa to blood
(Fig. 3.3).


  

  




 




 

 



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Figure 3.3 Schematic description of events associated with lactose

intolerance and possible solutions to reduce lactose
malabsorption clinical symptoms.

The lack or reduction of this enzyme (Fig. 3.3) leads to a high

concentration of free lactose (not hydrolyzed) in the gut that is
utilized by the local fermentative microlora resulting in abundant
gas, short-chain organic acids, and an osmotic increase of the water
low into the lumen. These local events lead to characteristic lactose
malabsorption clinical symptoms, including bloating, latulence,
nausea, abdominal pain, and diarrhea (Vasiljevic and Shah, 2008).
Lactose intolerance treatment implies the elimination or
reduction of lactose ingestion, avoiding the consumption of food rich
54 Probiotics and Their Therapeutic Role

in lactose, in particular milk. However, fermented dairy products

contain fermentative microorganism, which eficiently use lactose
and reduce lactose contents. The marker for bacterial metabolism
of lactose in the large bowel is the production of hydrogen in breath,
and in individuals treated with fermented milk, there is lower
hydrogen content than in group treated with nonfermented milk,
indicating that lactose was metabolized (Mombelli and Gismondo,
2000). The advantage of fermented milks in lactose maldigesters is
well established, and two mechanisms have been involved: digestion
of lactose in the intestine by the lactase contained in the yoghurt
bacteria, and slower GI transit time of yoghurt than milk (Marteau,
2001).
Probiotics can be beneicial in this condition, as lactobacilli
produce lactase, which hydrolyses the lactose in dairy products as
well as in gut when it is released within the intestinal lumen once
these bacteria are lysed by bile secretions (Tuohy et al., 2003).
Different factors appear to be responsible for a better tolerance
of lactose in fermented milk, including starter culture, intracellular
β-galactosidase expressed in these cultures, and most relevant the
GI transit time. The common cultures used in milk fermentations,
namely yoghurt, such as Streptococcus thermophilus and L. bulgaricus
(but also other lactobacilli in fermented milk products), can alleviate
symptoms of lactose intolerance by using lactose as an energy
source during growth, allowing for the reduction of its content in
fermented products as well as by providing bacterial lactase to the
intestine and stomach (Singh et al., 2011). After consumption of
fermented products, the bacterial lactase may resist to GI conditions
and arrive intact to terminal ileum, in which it contributes to lactose
hydrolysis and improved lactose tolerance. Some clinical studies
have been performed in patients with lactose intolerance. Ockeloen
and Deckers–Kocken (2012) found that the abdominal pain was
improved in 94% of children after 5 months using a lactose-restricted
diet and/or probiotics and in 61% after 15 months. Nevertheless,
these results were evident for short-term use, but not for long term.
However, some studies did not found direct relation for digestion
and lactose tolerance, as even using fermented milks with different
lactase activities, no corresponding tolerance to lactose was found.
So, especially in this case, the higher viscosity of fermented product
(mainly yoghurt) can slow the gastric emptying and consequently
 Inhibition of Helicobacter pylori and Intestinal Pathogens 55

increase the time available to achieve the lactose digestion and

improve lactose tolerance (Vasiljevic and Shah, 2008).
So, as inal remark, the use of probiotic bacteria in lactose-
intolerant patients may be a successful direction to reduce
characteristic lactose malabsorption clinical symptoms, especially
when incorporated in fermented milks, as lactose is reduced in
dairy matrix by in situ fermentation and at same time in the gut by
lactase that allows the eficient hydrolysis of lactose from other food
sources.

3.3 Inhibition of Helicobacter pylori and

Intestinal Pathogens
It has been reported that probiotics might be useful in the prevention
and/or treatment of certain infections of the stomach and small and
large intestines. The main agents involved include the H. pylori in the
stomach and small intestine, and other intestinal pathogens, namely
Rotavirus, Salmonella, Campylobacter jejuni, and E. coli. The main
mechanisms reported, in general, include (Fig. 3.4) host–bacteria
interaction mechanisms that include physical bacteria–epithelium
interaction (adhesion to mucosal and epithelial cells, stimulation of
mucus secretion, production of defensive molecules, reinforcement
of gut barrier function), bacteria–immune system interaction that
comprise modulation and regulation of immune responses, and
also, bacteria–bacteria interaction, which consist of exclusion and
inhibition of pathogens by prevention of adhesion, secretion of
antimicrobial substances, competition for nutrients, and anti-toxin
effects (Salminen et al., 2010).
The role of probiotics and associated mechanism in GI tract
infectious disease caused by each type of pathogen will be explored
in the following section.

3.3.1 Inhibition of Helicobacter pylori

H. pylori was recognized for the irst time in 1982 as C. pyloridis. This
agent has been associated as a major agent with gastritis and peptic
ulcer being also considered a risk factor for gastric adenocarcinoma
or gastric mucosa associated lymphoid tissue (MALT) lymphoma.
Although this microorganism has been found in a great number
56 Probiotics and Their Therapeutic Role

 


  
 


  
 

 

   



  
 

 



 
   

 


  



 



  


  

 

 



 

   

 
   







Figure 3.4 Main mechanisms of probiotic bacteria involved in the

prevention and/or treatment of certain GI infections (based on
Salminen et al., 2010).

of population (70–90% in developing countries and 25–50% in

developed countries), disease only occurs in a few percentage, as
most of the carriers are asymptomatic (Hamilton-Miller, 2003).
The most common treatment of H. pylori infection includes
one-week triple therapy, combining acid suppression with proton
pump inhibitors and two antibiotics, among them the amoxicillin
and clarithromycin are the most common. These solutions usually
achieve high success rates, but have the disadvantage of the rapid
development of antibiotic resistance associated with relevant
side effects (Felley and Michetti, 2003), namely alterations to the
intestinal microbiota.
As the H. pylori disease is located primarily on the gastric mucosa,
a reduction of H. pylori by dietary measures might help to decrease
the risk of developing H. pylori associated diseases.
In the last decade, the use of probiotics, mainly incorporated in
food matrix, for the inhibition of H. pylori has been explored. Data
obtained in both in vitro and in vivo report an inhibitory effect of
probiotics upon H. pylori adhesion to gastric cells and reduction of
viability.
In vitro studies have proposed that they could act through the
production of organic acids and/or bacteriocins capable of inhibiting
growth and its attachment to gastric epithelial cells. The irst in vitro
 Inhibition of Helicobacter pylori and Intestinal Pathogens 57

studies reported that H. pylori growth was inhibited by the secretion

of lactic acid by the Lactobacillus tested L. acidophilus (Bhatia et al.,
1989). Later, Lorca et al. (2001) found that this strain possesses some
speciic properties, in particular, the secretion of a proteinaceous
nature compound—an autolysin, released after cell lysis possibly
involved in its anti-H. pylori activity. On the contrary, the probiotic
strain Bacillus subtilis 3 has also been shown to inhibit the growth
of H. pylori that was not related to pH or organic acid, but to two
bacteriocines, one identiied as amicoumacin A (Felley and Michetti,
2003). Two out of nine L. reuteri strains, JCM 1081 and TM 105, were
able to bind to asialo-GM1 and sulphatide and to inhibit binding of H.
pylori to both glycolipids, suggesting that selected L. reuteri strains
could help to prevent infection in an early stage of colonization of the
gastric mucosa by H. pylori (Felley and Micheti, 2003).
Several clinical trials have been performed and the most common
markers used for control of positive effects include invasive tests
such as endoscopy to observe the eradication of H. pylori in gastric
mucosa or noninvasive test such as 13C Urea breath test, in which
the presence of H. pylori is directly proportional to the 13C present
in CO2 liberated in breath resulting from hydrolysis of ingested 13C
Urea by urease produced by H. pylori. Another noninvasive method
includes the determination of serum pepsinogens I (sPGI) and II
(sPGII), serum gastrin-17 (sG-17), and IgG anti–H. pylori antibodies
(IgG-Hp), markers that relect both the morphological and functional
status of the gastric mucosa (Myllyluoma et al., 2007).
Some clinical trials produced conlicting evidence, especially
when eradication was the main expected effect and L. acidophilus
was found to be the only agent that showed this activity in published
trials (Canducci et al., 2002a). Part of these studies aimed to test the
effect of several strains of probiotic bacteria (including L. acidophilus,
L. johnsonii La1, Biidobacterium longum BB536, L. gasseri OLL) alone
on the treatment of H. pylori infections. The probiotic concentrations
used in these studies range from 109 to 1010 cfu/ml and the trials
duration range from 2 to 8 weeks of treatment. Although there is
evidence in part of these studies that probiotics bacteria cleared
H. pylori, or reduced the breath test values, indicating reduction of
H. pylori density in gastric mucosa, the number of negative cases is
much higher than the positive cases (see Table 3.1). Some studies
also showed reduction of pepsinogen (Hamilton-Miller, 2003) or
58 Probiotics and Their Therapeutic Role

gastrin-17 (Myllyluoma et al., 2007). Myllyluoma et al. (2007) also

demonstrate that the probiotic combination of ive bacteria (see
Table 3.1) exerts a beneicial effect on gastric mucosa in H. pylori
infected patients. Gotteland et al. (2008) also combine probiotics (L.
acidophilus La1) with cranberry juice assuring 23% reduction of H.
pylori against 14.9% when La1 was alone.

Table 3.1 Compilation of main regimen, number of patients, and main

results obtained in clinical trials using probiotics alone in
treatment of H. pylori infections

Number of
Study Regimen patients Main results
Hamilton- L. acidophilus 8 × 1010 for 20 Four patients
Miller, 2003 8 weeks cleared
L. acidophilus NSA in 14 Six patients cleared
“acidophilus milk” c. 5 ×
10 9 cfu/day for 8 weeks
L. johnsonii La1 20 Breath test values
supernatant, 200 ml for 2 reduced
weeks
B. longum BB536 2.5 × 68 Breath test values
1010 cfu/day for 4 weeks reduced
in pasteurized yogurt
L. gasseri OLL 2716 c. 2 × 31 Breath test and
109 cfu/day for 8 weeks in pepsinogen values
yogurt base reduced
Yogurt made with six 27 Breath tests
“lactic acid” strains 7 × 109 remained positive
cfu/day for 30 days in
26/27
Myllyluoma L. rhamnosus GG (LGG), 13 Breath test and
et al., 2007 L. rhamnosus LC705, P. gastrin-17 reduced
freudenreichii
ssp. shermanii JS, and B.
lactis Bb12, 2.5 × 109 cfu/
day for 8 weeks
Gotteland L. acidophilus La1 product 295 Breath test values
et al., 2008 (80 ml) daily with >107 reduced and
cfu/ml for 3 weeks eradication of 14.9%
alone or combined with for La1 and 22.9%
cranberry jus when combined with
cranberry juice
 Inhibition of Helicobacter pylori and Intestinal Pathogens 59

When probiotic bacteria was used as a supplement of H. pylori

triple-therapy studies with L. acidophilus (Canducci et al., 2002b)
and L. casei subsp. rhamnosus (LGG) (Armuzzi, et al., 2001), results
showed a signiicant reduction in side effects incidence, with respect
to placebo, in particular the reduction in the GI disorders in the course
of antibiotic administration, but without signiicant differences in
the eradication rate. Sabbi and Palumbo (2007) studied the effect of
probiotics in combination with traditional treatment (omeprazole,
clarithromycin, and amoxicillin) using 60 patients randomized into
three groups: the irst received LGG; the second received Bacillus
clausii, and the third received placebo. The results showed that all
patients exhibited eradication of H. pylori, and the incidence of side
effects (nausea and abdominal pain) in patients treated with LGG
and Bacillus was lower.
The main mechanisms by which the probiotics assure the
aforementioned effects are not yet completely clariied. The most
likely mechanism supporting the prevention of initiation of H. pylori
infection by probiotics is the competition for binding sites. The
adjunctive effect to antibiotic treatment has been explained as a
direct additive antibacterial effect of inhibitory metabolites, on the
antibiotic regime, complemented by a possible stimulation of the
immune system (Hamilton-Miller, 2003).
Some studies were focused in the reduction of gastric ulcer
through anti-inlammatory effect rather than other mechanisms.
These indings suggested that LGG enhanced gastric ulcer healing
via the attenuation of cell apoptosis to cell proliferation ratio and
increase in angiogenesis. Some components may regulate these
processes, namely ornithine decarboxylase (ODC) and B-cell
lymphoma 2 (Bcl-2) protein expression, vascular endothelial growth
factor (VEGF) expression and growth factor receptor (EGF receptor),
and explain the healing action of LGG on gastric ulcer (Lam et al.,
2007).
As a inal remark, it is important to point out that probiotics
might be suggested as a new tool in the management of H. pylori
colonization. However, due to the prevailing conlicting results, more
studies are required in order to improve standardization for each new
strain studied, namely, in dose, duration and conditions of regimen,
and diet of the patient. Nonetheless, on the basis of consistent
results, probiotics may have a place as adjunctive treatment in H.
60 Probiotics and Their Therapeutic Role

pylori infections and possibly in prophylaxis, but cannot, as yet, be

considered a valid alternative to conventional anti-H. pylori therapy.

3.3.2 Inhibition of Other Enteric Pathogenic Bacteria

and Prevention and Reduction of Diarrhea
Symptoms
Preservation of the intestinal ecological lora is important in pre-
venting disease by controlling overgrowth of potentially pathogenic
bacteria. Several studies have suggested that some probiotic strains
could be used to prevent colonization of the GI tract by a large va-
riety of pathogens. Although the underlying mechanism remains
unclear, it could be due to organic acid production and associated
pH reduction, depletion of nutrients, production of intestinal mucin,
and inhibition of adhesion pathogen to the epithelium and of speciic
bacterial toxins, as well as to intrinsic activities of metabolites or to
the synthesis of antimicrobial substances. In addition, adherence of
probiotics to intestinal epithelial cells ensuring temporary coloniza-
tion of the gut is probably one of most important functionalities for
their beneicial health effect upon intestinal pathogen. Although the
last mechanism is referred as one of the principal mechanism of ac-
tion, most beneicial effects are thought to be by immunomodulation
with an interaction with TLRs and intracellular pathways, activation
of macrophages and NK cells and gut lymphoid tissue, and enhance
in immunoglobulins and speciic cytokines (Forestier et al., 2001;
Salvatore et al., 2007).
Saccharomyces boulardii, a yeast used for a long time in diarrhea,
is one of the well-studied probiotics and results have proved several
mechanisms, namely neutralization of bacterial toxins, stimulation
of host immune response, inluence of inlammatory pathways
(by nuclear factor-B, microtubule-associated protein kinase),
enhancement of the maturation of brush border enzymes (lactase,
sucrase, maltase, and aminopeptidase) and increase of glucose
carriers in the enterocyte membrane via a 54-kDa protease, and the
liberation of prolamine and soluble factors (Salvatore et al., 2007).
Enteropathogenic microorganisms such as enterotoxigenic E. coli,
Shigellae, and Salmonella are the main causes of acute diarrhea in
travelers accounting for about 80% of the cases in which a pathogen
was identiied. This acute diarrhea is quite common and may occur
in 10–60% of travelers; however, depending on the capacity of
 Inhibition of Helicobacter pylori and Intestinal Pathogens 61

hydration and electrolytes reposition, usually it is a shelf-limited

disease (Sullivan and Nord, 2002).
Culture cells have been used to prove the eficacy of probiotic
bacteria inhibiting the adhesion of pathogen bacteria to gut cells.
Using intestinal Caco-2 cell line, Forestier et al. (2001) reported
the inhibitory effects of L. casei subsp. rhamnosus strain (Lcr35) on
the adherence of three pathogens, enteropathogenic E. coli (EPEC),
enterotoxigenic E. coli (ETEC), and Klebsiella pneumonia. Results
indicated that for all bacteria, there was a decrease in the number of
adhering pathogens. In addition, the supernatant obtained by LCR35
growth showed inhibition (but not bactericidal effect) upon nine
human pathogenic bacteria, namely ETEC, EPEC, K. pneumoniae,
Shigella lexneri, Salmonella typhimurium, Enterobacter cloacae,
Pseudomonas aeruginosa, Enterococcus faecalis, and Clostridium
dificile. Other probiotic strains and their combinations were also
able to signiicantly inhibit the adhesion of B. vulgatus, C. dificile,
S. aureus, and C. sakazakii (Salminen et al., 2010). L. acidophilus
and L. reuteri cell-free supernatants also prove to inhibit protozoan
parasites of the genus Cryptosporidium, a leading cause of diarrhea
in domestic livestock and humans worldwide (Glass et al., 2004).
The L. acidophilus and L. reuteri cultures reduced the infectivity of
bovine C. parvum and C. hominis in a cell culture by 21–42% and 30–
35%, respectively, as well as reduced oocyst viability 40–80% and
oocyst infectivity up to 95%. Authors hypothesized that production
of antimicrobial compounds may be a mechanism by which L.
acidophilus and L. reuteri adversely affect bovine C. parvum and C.
hominis.
It is important to highlight that this antimicrobial activity is
probiotic strain dependent and the pathogen target is also quite
variable. Tuomola et al. (1999) showed that the probiotic strains
LGG and L. rhamnosus LC-705 slightly reduced S-imbria mediated
adhesion of E. coli, while adhesion of S. typhimurium was signiicantly
inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota.
An in vitro study in a reactor showed that two probiotics, L.
plantarum 0407 and B. biidum Bb12, combined with prebiotics
(oligofructose and xylo-oligosaccharides) under batch conditions
inhibit E. coli and C. jejuni, but only C. jejuni was inhibited under
continuous conditions. Results suggested that the effect of acetate
and lactate was responsible for antimicrobial activity, rather than
the low culture pH (Fooks and Gibson, 2003).
62 Probiotics and Their Therapeutic Role

Concerning the clinical trial studies, no effect of probiotics (or

slight) to prevent traveler’s diarrhea has been demonstrated, and in
some studies, the clinical effects have been considered as minimal.
However, in some cases, a signiicant reduction was observed,
namely a reduction of clinical effects of 71% compared with 43%
in the placebo group, but in this study, the placebo group showed
a diarrhea abnormally high. The effect of drinking LGG strain was
investigated in 820 travelers who traveled to two regions in Turkey.
The effect of LGG was not signiicant in the combined results of
the two regions, but the incidence of diarrhea was signiicantly
decreased in travelers to one region (Nomoto, 2005). Others studies
did not show enough evidence, namely in a placebo-controlled study
on children, a group with invasive pathogens (Salmonella, Shigella,
Campylobacter, Yersinia, or Entameba) did not reveal beneits on
duration of diarrhea between children receiving LGG or placebo
(Sullivan and Nord, 2002).
The most frequent side effect of antimicrobial therapy is
antibiotic-associated diarrhea with relevant prevalence (5–25%).
The antimicrobial treatment alters the ecological balance of the
normal microlora, which can result in diarrhea and emergence of
pathogens such as C. dificile (Sullivan and Nord, 2002). There are
only two standard antibiotic treatments for C. dificile infection; the
vancomycin and metronidazole and resistance to metronidazole
has been increasing (McFarland, 2009). Probiotics may be used
as an adjunctive therapy (given parallel with standard antibiotics
vancomycin or metronidazole) for C. dificile infections, as several
strains produce proteases that directly degrade C. dificile toxins or
increase the immune response to C. dificile toxins A and B. Clinical
trials combining probiotics with standard antibiotics have shown,
in general, a signiicant reduction in the risk of C. dificile infections.
Wullt et al. (2003) during a clinical trial compared the use of probiotic
L. plantarum 299v (5 × 1010 cfu/day) combined with metronidazole
with metronidazole alone during 38 days in the treatment of C.
dificile infection. Results showed that the frequency of CDI relapses
in probiotic group was four of 11 cases (36%) against six of nine
cases (67%); however, due to the small size of the group (20 adults
inished the study), the data collected could not prove to be of any
statistical signiicance. On the other side, McFarland and Elmer
(1997) mentioned a study using larger size group; they observed
 Inhibition of Helicobacter pylori and Intestinal Pathogens 63

that the combination therapy of standard antibiotics (vancomycin or

metronidazole) together with S. boulardii was shown to be effective
in a placebo-controlled clinical trial of 124 with C. dificile infections.
The recurrence of disease was found to be 26% in the yeast-treated
group and signiicantly higher (45%, P < 0.05) in placebo group.
Other studies have proven eficiency or lack signiicant differences
as reported by McFarland (2009) during a complete revision,
recommended for the reason that more research is required using
eficient randomized controlled trials of suficient size to detect
signiicant differences and using different types of probiotics.
In fact, there is some evidence that different probiotics exert
speciic effects on diarrhea for speciic pathogenic bacteria, but
more studies on the mechanisms of action, particularly in clinical
situations, are also required. In addition, to reduce contradictory
results, studies must be more conscious with strain speciicity, dose,
and timing of supplementation.

3.3.3 Inhibition of Enteric Virus: Rotavirus

The major viral agents involved in human diarrhea include
rotaviruses, caliciviruses (noroviruses and sapoviruses), enteric
adenoviruses, and astroviruses. Among these, rotaviruses are one of
the most important enteric virus, as it is responsible for about one-
third of the cases of severe diarrhea in childhood in both developed
and developing countries, accounting for 600,000 deaths in children
under 5 years of age annually, almost exclusively in developing
countries where they die especially due to the lack of dehydration
(Colbére-Garapin et al., 2007). Clinically, the intestinal mucosa is
partially compromised and the GI normal microlora is disturbed,
leading to diarrhea. The irst episode is osmotic diarrhea and the
second is associated with bacterial overgrowth. The common
treatment includes oral rehydration, but several studies have
reported that combinations with probiotic microorganisms shorten
the duration of diarrhea (Sullivan and Nord, 2002).
It is well recognized that certain probiotics, such as LGG, L. casei
Shirota, L. reuteri, B. lactis Bb-12, and a number of other probiotic
strains, are effective against rotavirus by shortening the recovery
time from diarrhea approximately by 1–1.5 days, reducing the
shedding of rotaviruses or increasing the production of rotavirus-
speciic antibodies (Salminen et al., 2010).
64 Probiotics and Their Therapeutic Role

The main mechanisms against viral infection ascribed to

probiotics have been reported as follows (Colbére-Garapin et al.,
2007):
Interference with a viral cycle at several levels, by speciic
or nonspeciic mechanisms. Extracellular, they can inhibit the
adsorption of virus, involving steric hindrance, or improve the
barrier effects of mucus, glycocalyx, and intercellular junctions. If
the virus adsorbs onto probiotics, it is not available to interact with
eukaryote cell and fails to infect it.
Probiotics could also block viral attachment by competitive
inhibition if they are able to bind viral receptors at the surface of
intestinal cells. Probiotic binding to molecules adjacent to viral
receptors could also confer some protection to cells.
Clinical trial studies using probiotics to prevent or reduce
symptoms during diarrhea caused by rotavirus have demonstrated, in
general, signiicant positive results. Nomoto (2005) and Salvatore et
al. (2007) report several positive results during a complete revision.
In general, these effects are positive demonstrating a reduction of
the duration of viral diarrhea and signiicantly reduced rotavirus
shedding as well as higher level of antirotavirus immunoglobulin A
(see Table 3.2). Although some studies showed prevention of rotavirus
diarrhea, few negative results were also reported; L. paracasei strain
ST11 did not reduce the volume of fecal matter output in rotavirus
infection but improved the outcome of non-rotavirus diarrhea in
children from Bangladesh (Colbére-Garapin et al., 2007).

Table 3.2 Positive effects (reduction of symptoms or prevention) of

probiotic bacteria upon rotavirus diarrhea (compilation of
clinical trials by Nomoto, 2005, and Salvatore et al., 2007)
observed in clinical trials

Probiotic strain Patients Main results

L. casei GG (2 × 71 children Reduced signiicantly the
1011 twice daily duration of hospitalization
for 5 days) in rotavirus diarrhea (1.4
versus 2.4 days)
L. casei GG (5 × 123 hospitalized Reduced the duration of
109 cfu/g twice children (33% with viral diarrhea (2.7 versus
daily for 5 days) rotavirus and 21% with 3.7 days) versus 75% at
bacterial diarrhea) 48 h
 Inhibition of Helicobacter pylori and Intestinal Pathogens 65

Probiotic strain Patients Main results

L. reuteri in 66 hospitalized The probiotic shortened

different doses children the duration of diarrhea
(107 and 1010 with a dose-dependent
cfu/g once a day effect (2.5 d in the placebo
for 5 days). group versus 1.9 and 1.5
d in the L. reuteri groups,
respectively)

LGG (3 × 109 — Reduction of the duration

cfu/g twice daily of diarrhea in outpatient
for a maximum of children and signiicantly
6 days) reduced rotavirus shedding

L. acidophilus 73 children with Reduction in duration (43

LB [Lacteol Fort acute diarrhea (50% versus 57 h in placebo)
(Houdan, France), rotavirus positive)
containing heat-
killed lactobacilli,
1010 cfu/ 5 doses]

Mixture of three 87 Polish children with Signiicantly shortened

L. rhamnosus infectious diarrhea the duration of rotavirus
strains (573L/1, diarrhea (76 ± 35 versus
573L/2, and 115 ± 67 h in placebo), but
573L/3) at a dose not of diarrhea of other
of 1.2 × 1010 cfu, aetiology
twice daily, for 5
days

Formula 55 hospitalized infants Decrease in the number of

supplemented the episodes of diarrhea
with B. biidum (7 versus 31% of placebo),
(1.9 × 109/g) and rotavirus shedding, and
S. thermophilus overall infections
(0.14 × 108/g)

L. reuteri SD 2222 Patients aged 6–36 Shortened the duration of

strain (1010–1011 months (75% were watery diarrhea, compared
CFU) for 5 days infected with rotavirus) with the placebo group

(Continued)
66 Probiotics and Their Therapeutic Role

Table 3.2 (Continued)

Probiotic strain Patients Main results

The test group 175 nursery school The antibody titer (anti-
was divided into children aged 6–36 rotavirus IgA - index
powdered milk months in Thailand of rotaviral infection)
group, B. Bb12- increased 4 times or more
supplemented in 30.4% of the individuals
powdered milk in the control group who
group, and B. ingested powdered milk
Bb12 and S. alone. No signiicant
thermophilus- increase in the group that
supplemented ingested B. Bb12 alone or
powdered milk with S. thermophilus.
group
Maternal milk 220 patients aged 1–18 The incidence of rotaviral
or 1010 CFU LGG months infection was signiicantly
during hospital lower in patients fed
stay compared maternal milk than in
with artiicial patients fed artiicial
milk milk, but daily preventive
administration of probiotic
did not decrease the
incidence.
L. casei GG (1 × 39 children with Signiicantly
1010 cfu twice rotavirus enteritis increased humoral
daily for 5 days) immune response—a
speciic increase in
immunoglobulins (G, A,
and M) during the acute
infection and a subsequent
higher level of antirotavirus
immunoglobulin A

Promising results from in vitro and in vivo studies have been

obtained with probiotics on rotaviral infection, but further studies
are required, in particular in the prevention of infection.

3.4 Prevention of Inflammatory Bowel Disease

Inlammatory bowel disease (IBD) is a chronic and recurrent
inlammation generally affecting the colon or the small intestine.
 Prevention of Inflammatory Bowel Disease 67

This disease includes ulcerative colitis and Crohn’s disease. The

etiology of IBD remains unclear, but there is some evidence that
the immune system reacts abnormally toward the endogenous
microlora (Sullivan, and Nord, 2002). Whereas ulcerative colitis and
Crohn’s disease are both included in the term IBD, these conditions
can be quite distinct, with different pathogenesis, inlammatory
proiles, symptoms, and treatment strategies. Crohn’s disease is
predominantly a Th1-driven immune response, characterized
initially by increased IL-12 expression, followed by interferon
(IFN)-γ and tumor necrosis factor (TNF)-α. In contrast, ulcerative
colitis has been associated with a Th2 immune response, leading to
increased production of pro-inlammatory cytokines including IL-5
(Geier et al., 2007).
Epithelial and immune cells of the intestinal mucosa recognize
speciic bacterial molecules via TLR and nucleotide-binding
oligomerization domain (NOD) proteins such as receptors (NLR), and
this interaction modulates the inlammatory response (activation
of the NF-κB pathway) (Marteau et al., 2009). Although NOD acts
in a similar fashion to TLRs, NOD is present intracellularly, in the
cytoplasm, as opposed to TLRs, which are located at the cell surface
or in vesicles.
In the healthy gut, there is a symbiotic relationship between
the host and the commensal bacteria in which contact leads to the
downregulation of inlammatory genes, inhibiting the immune
response of the gut to other pathogens. However, in the case of
IBD, genetically predisposed individuals appear to lose the normal
tolerance to commensal bacteria, leading to an elevated inlammatory
response (Geier et al., 2007).
Outstandingly, the irst genetic polymorphisms found to be
associated with a higher risk of IBD concern genes coding for NLRs
(NOD2-CARD15) and TLRs (TLR4) [2]. The genetically determined
modulation of defensin secretion is also signiicantly related with
IBD. In addition, signiicant alteration of the intestinal microbiota
has been frequently observed in patients with IBD, including
instability of prevailing microbiota, reduction in biodiversity of
Firmicutes, increased concentration of Enterobacteriaceae, and
higher concentrations of bacteria in the mucus layer at the mucosal
surface (Marteau et al., 2009)
The number of agents used for treatment of IBD has
increased, including corticosteroids, 5-aminosalicylic acid,
68 Probiotics and Their Therapeutic Role

immunosuppressives, elemental diets, antibiotics, and biological

solutions (Fiocchi, 2006). So, it is thus coherent to try treatments that
could modify intestinal microbiota in patients with IBD, including
probiotics.
There are many different preparations of probiotics of which
LGG, L. johnsonii LA1, E. coli Nissle 197, and VSL#3 (a combination
of S. thermophilus, B. breve, B. longum, B. infantis, L. acidophilus, L.
plantarum, L. casei, and L. bulgaricus), L. reuteri, and L. salivarius
have been studied in IBD either in animal studies or in clinical trials.
Several animal studies have been performed using different models
of colitis and different probiotic bacteria. Pagninia and Cominelli
(2006) extensively review these studies, which are summarized in
Table 3.3.

Table 3.3 Summary of the published studies on probiotic bacteria

utilization in animal models of IBD

Probiotic Main results Possible mechanism

L. reuteri Improvement —
LGG VSL#3 Improvement Sulhydryl compounds
L. salivarius Improvement Flora modiication
K reduction
L. plantarum Improvement and Preventing downregulation of
prevention pro-inlammatory cytokines
B. infantis and Prevention Downregulation of Th1 pro-
L. salivarius inlammatory cytokines
VSL#3 Improvement Decrease barrier permeability
VSL#3 Improvement TLR-9 mediated anti-
inlammatory effect of DNA
VSL#3 Improvement of IL-10 dependent induction of
recurrent colitis regulatory cells
LcS Improvement Downregulation of pro-
inlammatory cytokines (IL-6)
Source: Adapted from Pagninia and Cominelli (2006)

Concerning the clinical trials, several studies have been done in

different forms of IBD, including pouchitis, ulcerative colitis, and
Crohn’s disease.
 Prevention of Inflammatory Bowel Disease 69

Pouchitis is more frequent when pouch-ileoanal anastomosis has

been performed for IBD than for other forms of colonic diseases, and
is associated with an imbalance of microbiota, and the antibiotics
metronidazole and quinolone have proven eficacy (Marteau et al.,
2009). Two randomized, double-blind placebo controlled trials of
VSL#3 in adult patients with ulcerative colitis after ileal pouch-anal
anastomosis demonstrated a signiicant decrease in the incidence
of pouchitis in the VSL#3 treated groups during a 1-year follow-up
(Noble et al., 2008); however, some other studies showed reduced
evidence of positive effects on pouchitis, requiring more clinical
studies to demonstrate the real feasibility of probiotic application in
this condition.
Regarding the ulcerative colitis, three randomized, double-
blind, placebo-controlled trials have shown that E. coli Nissle 1917
(ECN), commercialized in Germany (Mutal or; Ardeypharm GmbH,
Herdecke, Germany), was as effective as low doses of mesalazine to
prevent relapse of ulcerative colitis; however, the eficacy of other
probiotics in this condition is lower (Marteau et al., 2009).
In contrast to pouchitis and ulcerative colitis, randomized
controlled trials on the use of probiotics in Crohn’s disease have
been negative. One of the few positive results in Crohn’s disease has
been obtained in a small (n = 32) randomized controlled trial with
the probiotic yeast S. boulardii versus mesalamine for preventing
relapse in inactive disease (Cabré and Gassull, 2007).
The mechanism of action that supports the probiotic positive
effects on IBD is still unclear. However, it is becoming apparent that
the different probiotic bacteria act through multiple and concurrent
pathways rather than by a single, common mechanism. The main
possible mechanisms of action of probiotic bacteria include
antagonism against pathogens, suppression of pro-inlammatory
mediators, and induction of protective factors, namely mucosal
trophism, intestinal permeability, and regulatory cytokines (Pagnini
and Cominelli, 2006). In addition, they seem to enhance epithelial
cell proliferation, inhibit apoptosis, and provide metabolic energy
for enterocytes. Some studies with live and dead LGG showed
attenuation of TNF-α-induced IL-8 production, often observed in
human IBD. In addition, the bacterial DNA from VSL#3 was able to
decrease IL-8 secretion, delay NF-κB activation, and stabilize IκB
levels (Geier et al., 2007). Fiocchi (2006) highlights the modulation
of host immune response in IBD by probiotics assuming the changes
70 Probiotics and Their Therapeutic Role

of DC phenotype and function, modulation of NF-κB and AP-1

pathways, modulation of innate immunity through TLR engagement
by CpG-DNA motifs, altered cytokine release, induction of Tregs,
modulation of apoptosis, and induction of PPR-γ.
Probiotics and prebiotics may offer a new therapeutic option
for the treatment of IBD; however, more double-blind, randomized,
placebo-controlled trials are required. In addition, a greater
understanding of the mechanisms behind their action on the GI
microbiota is required in order to determine which probiotic is the
most beneicial and how the genetic and bacterial proiles of the
patient will inluence treatment responsiveness.

3.5 Hypocholesterolemic Effect

Cholesterol-lowering effects of probiotics have been a target
extensively studied in the last years and are well documented.
Ooi and Liong (2010) recently published a review focused on
this subject taking into account the in vivo and in vitro indings.
Animal and human models have been used to evaluate the effect of
probiotics on serum lipid proiles including cholesterol (Figueroa-
González et al., 2011). Several mechanisms have been indicated
as responsible for the lowering effect by probiotics; probiotics
that modulate hypocholesterolemic effects as proposed by several
authors have been summarized in Fig. 3.5. In the review by Chen et
al. (2011), several cholesterol-lowering functional foods are listed
as well as their active ingredients and respective action mechanism;
probiotic (lactobacilli and biidobacteria) fermented dairy products
are included in which hydrolysis of conjugated bile acids is indicated
as the mechanism responsible for lowering cholesterol in serum
blood.
Although much evidence exists of hypocholesterolemic
potential effect of several probiotic strains on animals or humans
(Baroutkoub et al., 2010; Fazeli et al., 2010; Jeun et al., 2010; Ooi
et al., 2010; Shin et al., 2010) with positive effects in lowering
cholesterol with improvement of lipid proiles, this achievement is
not absent of controversy. Studies refuting the hypocholesterolemic
effect have been published by Hatakka et al. (2008b), Simmons et
al. (2006), and Lewis and Burmeister (2005) with L. rhamnosus and
Propionibacterium freudenreichii ssp shermanii (2 × 1010 cfu of each
 Hypocholesterolemic Effect 71

 


 

 

  

 

 



 



 



    


 













 
 






Figure 3.5 Proposed mechanisms by which probiotic bacteria can

modulate hypocholesterolemic effects. Adapted from Lye et al.
(2010) and Ooi and Liong (2010).

strain in two capsules daily) for 4 weeks in 38 men, L. fermentum (2

× 109 cfu/capsule, four capsules daily) for 10 weeks in 46 individuals,
and with L. acidophilus (3 × 1010 cfu in two capsules; six capsules
daily) for 6 weeks in 80 volunteers with high cholesterol. The authors
did not ind signiicant changes in the blood lipid proile. However,
according to Guo et al. (2011) who performed a meta-analysis of
13 randomized controlled trials with 485 participants with high
to normal cholesterol levels, they concluded that a diet enriched
with probiotics decreases the total and low-density lipoprotein
(LDL) cholesterol levels in plasma. Despite the strong evidences
from the literature that probiotics could be used as an alternative
way to drugs to low cholesterol in humans, more clinical evidence
is still needed to totally assure this desirable effect, as it is known
that the risk of heart attack in people with hypercholesterolemia is
much higher than those who have normal blood lipid proiles (Ooi
and Liong, 2010). More information is also needed about effective
dosage of probiotics to exert potential hypocholesterolemic effect as
well as to understand the underlying mechanisms of probiotics to
reduce cholesterol levels in blood (Figueroa-González et al., 2011).
According to Ooi and Liong (2010), the probiotic dosage to be effective
to exhibit hypocholesterolemic effects is strain speciic. In the near
future, better knowledge about dosage and associated mechanisms
will allow better formulation to combat hypercholesterolemia.
Interesting results have also been published with prebiotics (e.g.,
inulin) and symbiotic products to reduce cholesterol (Ooi et al.,
2010).
72 Probiotics and Their Therapeutic Role

3.6 Treatment and Prevention of Allergy

During the last decades, an increasing prevalence of allergies could
be observed in westernized countries. It is uncertain that genetics are
the primary causative factor due to the quickness with which allergic
disease has progressed. So, other factors are thought to be involved,
namely the loss of microbial burden resulting from the more aseptic
environment, a decrease in family size, a lack of physical exercise,
and a change in nutrition. The “hygiene hypothesis” has been used
as a base to explain the possible relation between microbial burden
and allergy, assuming that children with hygienic backgrounds
and a limited exposure to antigens from bacteria have a greater
predisposition to suffer from allergies (Chalk and Chalk, 2003).
Allergies are associated with the generation of Th2-type cy-
tokines, including IL-4, IL-5, and IL-13, which promote IgE produc-
tion and eosinophilia. Initial signals to release IL-4, and thereby IgE
and atopy, and IL-5 generated eosinophilic inlammation may origi-
nate from components of the innate immune system and structural
components of bacteria (Isolauri, 2004). The intestinal mucosa and
the mucosa-associated immune system are the primary local of aller-
gen contact and stimulation of immune responsiveness, and are de-
pendent on gut permeability. Antigens cross at the junctions between
epithelial cells and activate our immune systems, forming antigen–
antibody complexes. Antigen–antibody reactions can produce oxi-
dants that lead to inlammation at distant sites in the body. In addi-
tion, areas of intestinal mucosa with reduced integrity are frequently
brought about by local inlammation (Chalk and Chalk, 2003).
The original conception of infectious diseases providing
protection from atopic disease by causing immune deviation in
the form of TH1-driving signals that downregulate atopic TH2
responsiveness is explained by three relevant facts (Fig. 3.6).
First, the role of the indigenous intestinal microbiota might
compensate the induced stimulus caused by infections in providing
maturational signals to the infant’s immune system.
Second, the role of the innate immune system and anti-
inlammatory adaptive responses produced by TGF-β secreting TH3
cells and IL-10 secreting TR1 cells is beneicial. These intestinal
Tregs through suppressive mechanisms can control inlammation
of both TH1 and TH2 types and are thus implicated in protection
against both atopic and autoimmune disease.
Figure 3.6 Anti-inlammatory role of the immune regulation and enhancement of gut barrier by probiotics. Adapted from Rautava
Treatment and Prevention of Allergy

et al. (2004).
73
74 Probiotics and Their Therapeutic Role

Third, the development of proper and adequate immune

competence as a result of contact with microbes might be essential
not only in establishing a nonatopic immune responder phenotype
but also in protection against infectious and autoimmune disease
(Rautava et al., 2005).
In fact, speciic gut microbes may exert an immunosuppressive
function by inhibition of the transcription factor NF-kB pathway
(see Fig. 3.2). In addition, speciic probiotic strains have been shown
to counter allergy by the generation of IL-10 and TGF-β. The main
effect results in the suppression in proliferation of Th cells and
reduced secretion of pro-inlammatory cytokines, with control
of IgE responses and reduced allergic inlammation in the gut
(Isolauri, 2004). However, Viljanen et al. (2005) demonstrated that
in LGG treated infants with IgE-associated atopic eczema–dermatitis
syndrome, there was stimulation of the intestinal immune response
and induction of low-grade inlammation [concomitantly increased
IL-6 and C-reactive protein (CRP) levels] that might paradoxically
alleviate allergic symptoms.
These evidences support the use of probiotics in supporting
immunologic maturation and reducing the risk of disease (namely
atopic and autoimmune; see Section 3.5) during immunological
system development.
Most clinical studies explore the role of probiotics in the treatment
of allergic disease, namely food allergy and atopic dermatitis. Studies
in older individuals with established respiratory disease have failed
to show any improvement except for a larger study that showed
that probiotics can improve quality of life in patients with allergic
rhinitis (Prescott et al., 2007). Taylor et al. (2007) determined
whether early probiotic supplementation prevents allergic disease
in high-risk infants by using a group of infants whose mothers
had a history of allergy (n = 231) and had received L. acidophilus.
Although from the 178 infants who completed the supplementation
period, the probiotic group has shown signiicantly higher rates of
Lactobacillus colonization; they did not reduce the risk of AD in high-
risk infants. However, the presence of Lactobacillus at 6 months of
age was associated with an increased risk of subsequent cow’s milk
sensitization, which deserves to be investigated in further studies.
Recently, Ly et al. (2011) reviewed the eficacy of probiotic
application in allergy. They reported a study of 132 infants with
a family history of atopic, and the treatment with L. rhamnosus
 Reduction of the Risk Associated with Mutagenicity and Carcinogenicity 75

strain GG before and after birth split the risk of eczema [95%
conidence interval (CI) for relative risk, 0.3–0.8] but not that of
allergen sensitization. In a large and recent study of 925 mother–
infant pairs, prenatal administration of probiotics (containing four
bacterial strains) during the last month of gestation and postnatal
administration of probiotics and prebiotics from birth to age 6
months resulted in short-lived changes in the neonatal gut lora
and diminished the incidence of atopic eczema (but had no effect
on other atopic ailments or allergic sensitization) at age 2 years.
In general, these studies demonstrate an increased production of
cytokines by Treg cells (IL-10 and TGF-β) associated with a reduced
severity of atopic dermatitis in a small number of infants.
Concerning prevention of allergy, no study showed signiicant
evidences to recommend probiotics. Regardless of all of the
immunomodulatory effects described in experimental models, so
far, none of these studies has shown any clear effect on preventive
sensitization or any allergic disease other than eczema (Prescott and
Bjorkste, 2007), which alerts us to the needs of further systematic
studies. However, the study (Van Overtvelt et al., 2010) of 11 lactic
acid bacteria as an adjuvant for sublingual allergy vaccines has been
performed as a promising application, in which they compared
immunomodulatory properties of 11 strains as well as their capacity
to enhance sublingual immunotherapy eficacy in a murine asthma
model, proving that probiotics acting as a Th1/possibly Treg, but
not Th1 adjuvant, potentiate tolerance induction via the sublingual
route.

3.7 Reduction of the Risk Associated with

Mutagenicity and Carcinogenicity
Colorectal cancers (CRCs) are a signiicant cause of mortality in
Western countries. The disease development from normal epithelium
to the acquisition of the malignant phenotype is accompanied by
several biochemical and genetic alterations. It is known that a high
level of carcinogenic substances is released into the intestine through
the diet. Some carcinogens are formed in the colon, as intestinal
microbiota is capable of hydrolyzing glycosidic linkages (occurring
naturally in foods or formed in the liver and excreted via the bile)
by producing β-glucosidase, β-glucuronidase, and β-galactosidase,
76 Probiotics and Their Therapeutic Role

leading to the release of potentially carcinogenic compounds

(Hatakka et al., 2008a). So, it is evident that diet and nutrition and the
intestinal lora are key factors in modulating colon cancer onset and
progression. As described in previous subchapters, it is evident that
probiotics may modulate the activities of the intestinal microlora
via different mechanisms, and some of them may also contribute to
explain anticarcinogenic activity of probiotics. They seem to exert
different effects along the various steps of carcinogenesis, in general
summarized as antigenotoxicity, inhibition of the colic enzymes
activity, controlling the growth of potentially harmful bacteria,
interaction with colonocytes, and stimulation of the immune system
and production of physiological active metabolites (Boutron-Ruault,
2007).
Several results strengthen the hypothesis of an antiproliferative
effect of probiotics on the GI mucosa, in particular by affecting
polyamine biosynthesis. Polyamines and their metabolizing
enzymes are closely linked to neoplastic proliferation; so, any
solution that reduces these components could be an eficient way of
CRC chemoprevention and chemotherapy. This relation among cell
proliferation, polyamines, and probiotics activity could be regulated
by different elements, including (1) the speciic metabolic activities
of the probiotic strains, (2) different survival times in the lumen, (3)
the period of administration, and (4) the proliferative behavior of
different segments of GI mucosa (Linsalata and Russo, 2008).
In 2006, Capurso and collaborators analyzed existing data
from in vitro models and animal and human (epidemiological and
interventional) studies concerning the CRC. From the identiied
29 animal studies aiming to establish the effect of probiotics
administration on the incidence of CRC and/or of precursor lesions,
most studies employed lactobacilli or biidobacteria. In general,
all studies had positive results (except three studies), and when
prebiotics were also evaluated, relating the synergistic effect of such
combination. In addition, the preventive effect on the CRC seems
more important when they were administered before, and not after
the carcinogen formation.
On the basis of the very few human epidemiological studies
speciically designed to analyze the effect of probiotics on CRC
incidence, it was possible to observe several important confounding
factors, such as role of ibers, mixture with other dairy products,
and presence of vitamin D. In general, the epidemiological studies
have failed identifying signiicant effects of probiotic fermented
 Reduction of the Risk Associated with Mutagenicity and Carcinogenicity 77

milks against CRC. On the contrary, some interventional studies have

suggested reduction of biomarkers for CRC risk, but others did not
observed signiicant differences in the development of new CRC
following administration of either ibers or probiotics in patients
(Capurso et al., 2006).
A very recent study (Stein et al., 2012) demonstrated that the
combined effect of probiotics and prebiotics on CRC is in fact positive,
whereas isolated probiotics do not make enough evidence of cancer-
preventive effect. The study demonstrated that wheat aleurone
through the modulation of markers of primary chemoprevention may
possibly have a cancer-preventive potential, which could be partially
favored by the addition of the probiotics LGG and Biidobacterium
Bb12.
In general, the main mechanisms exhibited by probiotic bacteria
toward the cancer inhibition can be explained by a group of activities
that maybe dependent on the type of probiotic strain. Ouwehand et
al. (1999) earlier described the possible mechanisms of probiotics
on the inhibition of cancer (see Fig. 3.7).

Figure 3.7 Scheme of proposed mechanisms by which probiotics can

induce positive effects on colon cancer and supericial bladder
cancer; (+) upregulation or stimulation, (–) downregulation
or inhibitory activity. Reprinted from Ouwehand et al. (1999),
with permission from Elsevier.
78 Probiotics and Their Therapeutic Role

Such mechanisms include an alteration of the metabolic

activities of intestinal microlora; an alteration of physicochemical
conditions in the colon; the binding and degradation through
enzymes of potential carcinogens; quantitative and/or qualitative
modiications in the intestinal microlora implicated in producing
carcinogen(s) and promoters (e.g., bile acid metabolizing bacteria);
the production of antitumorigenic or antimutagenic compounds;
an increase of the host’s immune response; and effects on host
physiology (Rafter, 2003). Speciic strains of probiotic bacteria are
able to downregulate intestinal microbial enzyme activities, leading
to a decrease in the activity of carcinogen-activating microbial
enzymes. This activity may be exerted in the colon, the urinary
tract, and the bladder. The studies do not always ind a reduction
in the same enzymes, although indings with β-glucuronidase and
nitroreductase are most consistently positive. Hatakka et al. (2008a)
potentially demonstrated carcinogenic bacterial activity in human
colon in a randomized, double-blind, placebo-controlled study
including 38 healthy men with treatment periods of 4 weeks with
L. rhamnosus LC705 together with P. freudenreichii ssp. shermanii JS
(2 × 1010 cfu of each strain daily). The results showed an increase
in the fecal counts of lactobacilli and propionibacteria and a
decrease in the activity of β-glucosidase (10%) and urease (13%)
enzymes; however, a decrease in β-glucosidase activity was related
to the increasing counts of propionibacteria. It is also known that
colon carcinogenesis involves a cytotoxic response on the colonic
epithelium exerted by environmental compounds such as bile acids
in the aqueous phase of the stools (soluble bile acids), followed by
an increased proliferation of cells in the intestine. Administration
of probiotics resulted in a lower concentration of soluble bile acids
in feces and change of pH, which were related to tumor reduction.
The probiotics may physically bind the bile salts, using the hydrolase
active, L. reuteri, showed to control cell toxicity in the presence of
bile salts (Commane et al., 2005).
Mutagenic compounds, commonly released through the diet,
namely 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1, 2),
can be bound to the intestinal and lactic acid bacteria in vitro, and
some lactobacilli have also been shown to degrade nitrosamines.
This binding or degradation correlated well with the reduction in
mutagenicity observed after exposure to the bacterial strains (Rafter,
2003).
 Effect on Urogenital Infections 79

Simultaneously, the production of beneicial compounds such

as SCFA (e.g., butyric acid) may improve mucosal nutrition and
integrity. Another established mechanism to exert an antitumor
effect includes their antimutagenic properties and ability to
modulate immune parameters by increasing speciic and nonspeciic
mechanisms, including T-cell, NK cell, and macrophage activity,
which are important in delaying cancer development (Ouwehand et
al., 1999). On the contrary, it has been demonstrated to also induce
the production of several cytokines, resulting in an inhibition of
tumor growth. Concerning the bladder tumor, as viral infections
have been suggested to play some role in bladder cancer, the antiviral
properties of IFN-χ and TNF-α may be important.
Little evidence has been found in the application of probiotics
on other cancers. LeBlanc et al. (2005) demonstrated the immune-
regulatory capacity of milk fermented by L. helveticus R389 on the
immune response in mammary glands in presence of a local breast
tumor. Positive effects included increased IgA and CD4-positive cells,
increased IL-10, and decreased IL-6 in mammary glands.
Nackerdien (2008) described that probiotics may be employed
to reduce treatment side effects in breast cancer; probiotic adjuvant
regimens may provide natural alternatives to pharmaceutical
antiemetic therapy, currently being used in conjunction with the
chemotherapy of this cancer. In addition, as pathogenic gut microbes
have been associated with extra-intestinal tumor development
in the breast, probiotics could be also involved in the preventive
occurrence of these tumors as intestinal pathogen inhibitors. Using
L. casei Shirota in supericial bladder cancer reduced recurrence of
this tumor (Ouwehand et al., 1999).
So, an effort must thus be made to identify the strains with the
most prominent antitumoral characteristics and it will require a
greater understanding of the mechanisms involved before we can
fully justify their use as cancer prophylactics in humans. In addition,
clinical investigators should design clinical studies using well-deined
bacterial strains in predeined quantities and with appropriately
chosen characteristics to elucidate the discrepant result existing
between experimental and epidemiological evidences.

3.8 Effect on Urogenital Infections

Use of Lactobacillus-containing probiotics has been proposed to
restore commensal vaginal lora for the treatment and prophylaxis
80 Probiotics and Their Therapeutic Role

of bacterial urogenital infections. In fact, the microbiota that covers

epithelial cells in a healthy female genital tract lacks infectious
pathogens but contains high populations of lactobacilli. However,
this equilibrium can be affected by several factors, including
hormonal concentrations, sexual activity, oral contraceptive use,
glycogen content, vaginal pH, steroid therapy, immunosuppression,
and diseases. So, dysfunction of normal microbial ecosystem, in
particular decrease in lactobacilli, may lead to bacterial urogenital
infections (Hoesl and Altwein, 2005).
Several in vitro and animal studies have proven that probiotic
bacteria must accomplish at least two criteria: assure colonization
of vaginal mucosa and possess inhibitory ability against urogenital
pathogens. So, on the basis of this fact, three main Lactobacillus
strains, namely L. rhamnosus GR-1, L. fermentum RC-14, and L.
crispatus CTV-05, possess optimal properties to be effective as
probiotic therapeutics against infections in the urogenital tract (Hoesl
and Altwein, 2005). Mutalor is a possible alternative probiotic in
which the active agent is Nissle 1917 (a commensal E. coli strain that
eradicates pathogenic bacteria from the GI tract), which may have
the potential to prevent recurrent urinary tract infections (UTIs), as
it adversely affected the growth of 34 of the 43 uropathogen isolates
(Storm et al., 2011).
The eficacy of L. rhamnosus GR-1 and L. fermentum RC-14 was
evaluated in several clinical pilot studies. In 1995, a randomized,
double-blind clinical trial showed that L. rhamnosus GR-1 and L.
fermentum B-54 signiicantly reduce UTI rates (from 6 per year to
1.9 per year) when administered vaginally once weekly (Hoesl and
Altwein, 2005).
Another study of 10 women and in a randomized, placebo-
controlled trial of 64 women, daily oral intake of strains L. rhamnosus
GR-1 and L. fermentum RC-14 resulted in a restoration of a normal
vaginal lora in patients with asymptomatic bacteria vaginosis, and
a signiicant depletion of yeast and coliforms. Direct application of
lactobacilli into the vagina would be more eficient to reduce the
associated symptomatology (Reid and Burton, 2002).
A prospective clinical pilot study was performed to conirm the
safety and effectiveness of Lactobacillus vaginal suppositories against
the recurrence of bacterial UTI. The strain used was L. crispatus GAI
98322, and in this study, a signiicant reduction in the number of
recurrences was noted, without any adverse complication. The main
 Benefits for the Healthy Function of the Liver and Pancreas 81

mechanisms involved were believed to be its ability to produce

hydrogen peroxide and to bind to vaginal epithelial cells (Uehara et
al., 2006).
In general, clinical trials use Lactobacillus doses at least 1.0 × 108
cfu per solid dosage formulation (oral capsule, intravaginal capsule,
vaginal suppository, or vaginal tampon), at least 1.0 × 108 cfu/ml
milk or yogurt, and 4.0 × 1010 cfu/100 ml cranberry–lingon berry
juice administered for at least 5 days, involving at least 28 days of
follow-up (Barrons and Tassone, 2008).
Although no signiicant in vivo evidence of mechanisms of action
exists, data suggest that to be multifactorial and complex, it must
involve (1) the ability to produce antimicrobial compounds such
as lactic acid, bacteriocins, hydrogen peroxide; (2) the competitive
exclusion of genitourinary pathogens; (3) the ability to produce a
biosurfactant and collagen-binding proteins, which block pathogen
adhesion that inhibits the adhesion of uropathogens to surfaces;
and (4) the nonspeciic augmentation of the innate immune system
(Hoesl and Altwein, 2005; Uehara et al., 2006).
Most of clinical trials were insuficient to show a treatment
effect and failed to validate the dosing strategy by quantifying local
bacterial colonization. So, future randomized controlled trials of the
use of lactobacilli in bacterial genitourinary infections in women
should be adequately implemented to detect treatment effects
(Barrons and Tassone, 2008).

3.9 Benefits for the Healthy Function of the

Liver and Pancreas
The relation between the positive effects of probiotics and liver health
is not direct, but rather it seems to be more based on prevention;
probiotic microlora can avoid the production or increase the
uptake of compounds such as lipopolysacchrides (LPS) in the gut,
which could leave to reductions in low-grade inlammation (Gratz
et al., 2010). According to the review by Jonkers and Stockbrügger
(2007), the application of probiotic therapy in liver and pancreatitis
diseases is promising and its positive effects are related with
modulation of gut microbiota and the immune system being
strain speciic. Although mechanisms by which probiotics could
be related to hepatic fat metabolism remain unclear, it seems that
82 Probiotics and Their Therapeutic Role

hepatic metabolism is inluenced by the presence of probiotics in

gut microlora, modulating intestinal barrier function. According
to Gratz et al. (2010), the understanding of these mechanisms is
of utmost importance in the future, because a set of liver disorders
(low-grade inlammation, hepatic fat iniltration, hepatitis, and so
on) will probably become more prevalent due to high-fat diets and
increasing obesity; nonalcoholic fatty liver disease is one of the most
common liver disease in some countries, the incidence of which
has risen in patients suffering from obesity and type II diabetes.
Probiotic therapy (lactobacilli and biidobacteria) and/or prebiotic
therapy (which increase biidobacteria incidence in gut microlora)
modulating the gut microlora could counterpart the negative effects
of Gram-negative bacteria or pathogens, which are responsible for
hepatic oxidative stress related to ethanol production and/or LPS
(Cani and Delzenne, 2009). Similar positive effects in alcoholic liver
disease or in patients with liver cirrhosis have been reported (Cani et
al., 2007; Zhao et al., 2004). The levels of lactobacilli, biidobacteria,
and enterococci are reduced in alcoholics through probiotic therapy
that is capable of improving liver function parameters such as
alanine aminotransferase and aspartate aminotransferase (Kirpich
et al., 2008). Although more controlled trials are needed, it appears
that the microlora is an important cofactor in the aetiology of
chronic liver disease, and that probiotics might have a therapeutic
role (Gratz et al., 2010).
Bacterial translocation, due to factors such as bacterial over-
growth, disturbed intestinal motility or mucosal barrier, over-
production of proinlammatory cytokines, and so on, is one of the
major causes of infectious complications in severe acute pancrea-
titis patients, which may be prevented by probiotics (Jonkers and
Stockbrügger, 2007). However, Zhang et al. (2010), who performed
a meta-analysis of use of probiotics, prebiotics, and symbiotics in
acute pancreatitis patients, reported that neither showed a signii-
cant effect on this type of patients but reduced their length of stay
in hospital. Kumar et al. (2011) also observed a positive effect of
probiotic-fermented milk combined with chlorophyllin on gene ex-
pressions and genotoxicity during alatoxin B1 induced hepatocel-
lular carcinoma. The studies demonstrated an enhanced protective
potential of probiotic-fermented milk with LGG and L. casei strain
shirota combined with chlorophyllin (potent antimutagen) in he-
patic cells during carcinogenesis induced by alatoxin B1.
 Oral Health: Promong Properes 83

3.10 Oral Health: Promoting Properties

Oral applications have been tested in particular in the prevention
of caries. On the contrary, the effect of probiotics on improving oral
diseases such as gingivitis, periodontitis, and halitosis has been less
explored. A possible danger is that these groups of organisms are
acid producers and implicated in dental caries. Nevertheless, one
potentially useful strain of L. paracasei retains its coaggregation
activity with S. mutans when killed. There are some proofs that
probiotic organisms, such as L. reuteri, can reduce the numbers of
S. mutans in the mouth, but whether this would result in reduced
levels of caries remains to be demonstrated (Wade, 2010). Recently,
Bosch et al. (2011) summarized the clinical trials aimed to evaluate
the effect of probiotics on oral health. They reported 12 clinical
trials using probiotic strains in caries prevention, in which the
main marker was S. mutans, mainly responsible for this disease.
Most of the studies report a signiicant evident reduction of these
bacteria after probiotic application. The main carrier of probiotic
bacteria include food products, mainly dairy products—milk,
yoghurt, ice cream, and cheese—as well as tablets, capsules, rinsing
solution, chewing gums, medical device, and straw. The dose ranged
between 9 × 108 and 1.4 × 1012 cfu/day. The main probiotics used
were LGG, L. reuteri, Biidobacterium DN-173 010, B. lactis Bb-12,
and L. paracasei. Another study reported that including probiotics
reduced the prevalence of oral Candida, in particular, in the elderly.
Evidence of halitosis reduction was also reported with two bacteria:
S. salivarius K12 and Weissella cibaria. L. reuteri has been also tested
for gingivitis reduction on the basis of reduction of gingivitis and
plaque in patients, and improvement of bleeding on probing and
reduction of pro-inlammatory cytokines. L. salivarius WB 21 was
successfully tested in periodontitis as well as ProBiora (a probiotic
mouthwash) and B. subtilis.
Mechanisms of probiotic action within the oral cavity can
possibly be by direct interactions within dental plaque, including
the disruption of plaque bioilm formation through competition for
binding sites on host tissues and other bacteria, and competition
for nutrients as well as antimicrobial compounds (organic acids,
hydrogen peroxide, low-molecular-weight antimicrobial compounds,
bacteriocins, and adhesion inhibitors) that inhibit oral bacteria,
which may also have a signiicant role.
84 Probiotics and Their Therapeutic Role

Indirect probiotic actions within the oral cavity, including the

modulation of aspects of both innate and speciic immune function,
were also reported (Allakera and Douglas, 2009).

3.11 Other Therapeutic Applications

Other therapeutic applications with limited studies have been
described for probiotics. Effects of the probiotic B. infantis in the
maternal separation model of depression showed that probiotic
treatment resulted in normalization of the immune response,
reversal of behavioral deicits, and restoration of basal noradrenaline
concentrations in the brainstem. These indings point to a more
inluential role for biidobacteria in neural function and suggest that
probiotics may have broader therapeutic applications (Desbonnet et
al., 2010).
Forsyth et al. (2010) showed that LGG treatment ameliorates
alcohol-induced intestinal oxidative stress, gut leakiness, and liver
injury in a rat model of alcoholic steatohepatitis. This improvement
was associated with reduced markers of intestinal and liver oxidative
stress and inlammation and preserved gut barrier function.
Many probiotic organisms, including L. rhamnosus GR-1, have
shown a signiicant promise in supporting the immune function of
people living with HIV, as people infected with HIV often suffer from
signiicant disturbances in nutritional status. In a review, Wilson
et al. (2008) provided evidence that probiotics were supportive in
modulating aspects of gut physiology, barrier integrity, and immune
function. Probiotic use is a supportive adjunct therapy, worthy of
consideration and further research in persons infected with HIV.
Hemsworth et al. (2011) tested a micronutrient-supplemented
probiotic yogurt for people living with HIV, which had very good
acceptability and may be applied as a supportive adjunct therapy.
Another interesting application was studied by So et al. (2008)
to elucidate whether L. casei affects the induction of oral tolerance in
experimental rheumatoid arthritis. This study provides evidence that
L. casei could potentiate antigen-speciic oral tolerance and suppress
Th1-type immune responses of arthritic inlammation. Other speciic
applications have been reported, but the results did not produce
signiicant evidence, and more studies must be implemented in
order to further explore more interesting applications.
 References 85

3.12 Conclusions
The different mechanisms of action explored in this chapter may lead
to relevant health beneits, namely in infection defence, prevention
of cancer, in stabilizing or reconstituting the physiological balance
between the intestinal microbiota and its host, and inluencing
immunological processes at different locations. However, it must be
highlighted that one probiotic cannot exhibit all activities at the same
time, at least not to that extent that it could be used for the prevention
or cure of all mentioned kinds of disease. The overall effect attributed
to a certain probiotic strain depends on the metabolic properties, the
kind of surface molecules expressed, and components to be secreted
with probiotic actions (Oelschlaeger, 2010).
The studies analyzed during this chapter will increase our
knowledge concerning the functional attributes of known probiotics
and facilitate probiotic product improvements.
The capability to control and envisage probiotic-based effects
will permit both industry and the consumer to choose, based on
science, the probiotics with well-known health beneits. These
multidisciplinary efforts to develop more probiotics to be delivered
through medication or food additives will increase available solutions
to ight old and new diseases.

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Chapter 4

Food as Vehicles of Probiotics

Ana C. Freitas,a,b Dina Rodrigues,b Sérgio Sousa,c Ana M. Gomes,c

and Manuela M. Pintadoc
aISEIT/Viseu-Instituto Piaget, Estrada do Alto do Gaio, Galifonge, 3515-776 Lordosa,
Viseu, Portugal
bCESAM & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
cCBQF, Biotechnology School of Portuguese Catholic University,

Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal

[email protected], [email protected]

4.1 Introduction
The demand for functional foods has, over recent years, increased
markedly (Siro et al., 2008). These foods, when include probiotics
and prebiotics as biologically active components, produce metabolic
and physiological health beneits in addition to their nutritional
properties or at least enhance the normal homeostatic mechanisms
in the intestine. Considering that scientiic evidences indicate that
ingestion of some microbial strains provokes health beneits, which
are extensively discussed in previous chapters, no clear indications
exist about the effective dose for these strains; however, it has been
consensual that high numbers of viable bacteria are recommended
to assure probiotic foods eficiency. The viability and metabolic
activity of probiotic microorganisms are important factors that
should be controlled throughout processing operations, maturation

Probiotic Bacteria: Fundamentals, Therapy, and Technological Aspects

Edited by J. Paulo Sousa e Silva and Ana C. Freitas
Copyright © 2014 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4411-62-2 (Hardcover), 978-981-4411-63-9 (eBook)
www.panstanford.com
96 Food as Vehicles of Probiotics

period and storage of the food. Furthermore, the amount of intake

and form of the probiotic food should be adequate for dietary
purposes. The most popular functional food products on the market
are those designed for improvement of gut health, particularly
those that contain probiotics (Siró et al., 2008). Probiotic bacteria
are currently used to be incorporated in food products; however,
some technological challenges must be faced when included in food
matrices with more aggressive environments, namely high salt,
acid or oxygen concentrations, or low temperatures. Nonetheless,
in general, the beneicial effect of probiotic bacteria depends on
its viability, requiring that food matrices as carriers assure their
viability at high levels for entire shelf-life, to guarantee their passage
throughout the gastrointestinal tract (GIT) and colonization of gut
mucosa in order to exert beneicial effects.
The aim of this chapter is to provide a comprehensive overview
on the advances in probiotic food, covering the technological issues,
functionality aspects, and limitations of some foods as carriers of
probiotics.

4.2 Dairy Products

Dairy products constitute the major group of products able to carry
and deliver probiotic bacteria, and among them, fermented milks and
cheeses are the most consumed around the world. Milk and dairy
products possess nutritional characteristics that allow the growth
and the survival of probiotic bacteria, namely the high content of
lactose. In addition, some dairy products, especially fermented
milks and cheeses, due to some of their characteristics such as pH
and buffer capacity, their dense matrix, and their fat content offer
additional protection to these microorganisms in their passage
through the GIT, in particular against the acidic environment of the
stomach (Grattepanche et al., 2008; Cruz et al., 2009a; Kiliç et al.,
2009; Souza and Saad, 2009).

4.2.1 Fermented Milks

Fermented milks are a traditional food, produced by mankind for
thousands of years, which was created as a means to extend the
shelf-life of different milks (e.g., cow, goat, sheep, mare, buffalo,
camel, or yak). Historical evidence indicates the Middle East and
the Balkans as the likely origin of these products, also responsible
 Dairy Products 97

for the evolution of fermented milks through time as a consequence

of their inhabitants culinary skills (Tamime, 2002). Nowadays,
fermented milks are produced and widely consumed in many
countries worldwide combining the traditional craftsmanship of the
past with science development of the present where microbiology,
enzymology, physics engineering, chemistry, and biochemistry all
play an important role.

4.2.1.1 Definition, classification, market, and physiological

role
There are many types of fermented milks. In the late past century,
Kurmann (1984) attempted a classiication scheme on the basis of
starter culture growth requirements building a family tree for such
purpose; in order to further account for the different metabolites
produced by the prevailing microorganisms, Robinson and Tamime
(1990) proposed an additional classiication strategy (Fig. 4.1). Such
speciic cultures are responsible for the properties of fermented
milks, such as consistency, texture, taste, aroma as well as the health
beneits they may confer. Currently, existing legal standards have
been set by the Codex Alimentarius (2003) in which fermented milks
are described as belonging to one of four different groups (Fig. 4.1).
According to the Codex Alimentarius (2003), standard fermented
milks are those that are prepared from pasteurised or raw—whole,
partially, or fully skimmed—milk or milk products (concentrated
or powdered milk, whey, buttermilk powder, edible casein, or
caseinates) by the speciic action of detailed bacteria (Table 4.1)
and/or yeasts, which proceed to multiply and convert lactose into
organic acids, resulting in pH reduction with or without coagulation
(isoelectric precipitation). The removal of whey after fermentation is
not allowed in their manufacture, except for concentrated fermented
milk. An important requirement is that the starter microorganisms
added shall be abundant as well as viable and active in the product
throughout the shelf-life; nevertheless, if the product is treated
by heat after fermentation, naturally the requirement for viable
microorganisms does not apply (Codex Alimentarius, 2003). Such
speciications of live microorganisms are transversal to the probiotic
concept and in many aspects may constitute real opportunities
despite the major challenges for the food industry—the development
of functional fermented milks. Apart from the standard fermented
milks the Codex Alimentarius Standard (2003) also includes (i)
concentrated fermented milks, which correspond to fermented
98 Food as Vehicles of Probiotics

milks in which the protein has been increased to a minimum of

5.6% before to or after fermentation (labneh is a common example);
(ii) lavored fermented milks, which contain a maximum of 50%
(w/w) of nondairy ingredients (sweeteners, fruits, or vegetables
such as juices, purees, pulps, preparations, and preserves derived
therefrom, cereals, honey, chocolate, nuts, coffee, spices, and
natural lavoring compounds); and (iii) drinks based on fermented
milks, which are also composite milk products, obtained by mixing
standard fermented milk [minimum of 40% (w/w)] with water with
or without the addition of other ingredients such as whey, other
nondairy ingredients as well as lavorings.

Yoghurt
Traditional
Fermented Milks

Therapeutic and
Nature Thermophilic Intestinal Flora
strains
Gelled Miscellaneous
Viscous Dietetic
Pasty, Foamy
Frozen, Dried Fermented Milks +
Mesophilic
Concentrated Plant Material
strains Slimy Fermented
Flavoured Milks

Non-slimy

Buttermilks

Lactic and
Alcoholic Kef ir and Koumiss

Figure 4.1 The family tree of fermented milks. Adapted from Kurmann
(1984) and Robinson and Tamime (1990). All fermented
milks are lactic fermentations (largest group worldwide)
with exception of shaded boxes that may include lactic-yeast
fermentations.

Table 4.1 Commercial products containing Biidobacterium spp. and

Lactobacillus acidophilus spp.

Product Country Microorganisms

Arla A-38 Denmark Lactobacillus acidophilus,
Biidobacterium biidum, Leuconostoc
mesenteroides ssp. cremoris,
mesophilic lactococci
 Dairy Products 99

Product Country Microorganisms

Acidophilus USA L. acidophilus, Lc. mesenteroides ssp.
buttermilk cremoris, mesophilic lactococci
Progurt B. biidum, L. acidophilus, mesophilic
lactococci
Acidophilus milk Several L. acidophilus
countries
A-B Yoghurt France, B. biidum, L. acidophilus
Serbia
Cultura Denmark Ibidem
Milky Italy Ibidem
Nu-Trish A/B Milk USA Ibidem
Biomild Several Biidobacterium spp., L. acidophilus
countries
Acidophilus Several L. acidophilus, L. delbrueckii
yoghurt countries subsp. bulgaricus, Streptococcus
thermophilus
B-Active France L. acidophilus, B. biidum, L.
delbrueckii subsp. bulgaricus, S.
thermophilus
Fresh BA UK Ibidem
Kyr Italy Ibidem
Yoplus Australia Ibidem
Biogarde Germany L. acidophilus, B. biidum,
S.thermophilus
Oilus France Ibidem
Philus Norway Ibidem
Biidus milk Several B. biidum, B. longum
countries
Biighurt Germany B. biidum, S. thermophilus
Biogurt Germany L. acidophilus, S. thermophilus
Biokys Czech B. biidum, L. acidophilus, Pediococcus
Republic acidilactici
Mil-Mil Japan L. acidophilus, B. biidum, B. breve
Yakult Japan L. acidophilus, B. biidum, B. breve, L.
casei subsp. casei
(Continued)
100 Food as Vehicles of Probiotics

Table 4.1 (Continued)

Product Country Microorganisms

Biidus Milk/ Japan B. longum BB536, L. delbrueckii
Biidus yogurt subsp. bulgaricus, S. thermophilus
DanActive, USA, L casei DN114001, L. delbrueckii
Actimel Several subsp. bulgaricus, S. thermophilus
countries
BioK+ USA, L. acidophilus CL1285®, L. casei
Canada LBC80R
Activia Several B. animalis DN173 010, L. delbrueckii
countries subsp. bulgaricus, S. thermophilus
Geilus Finland L. rhamnosus GG
Source: Adapted from Gomes and Malcata (1999) and Douglas and Sanders (2008).

From a nutritional/functional perspective, functional fermented

milks provide positive health beneits to the host either directly, via
the interactions of ingested live microorganisms therewith (probiotic
effect), or indirectly via the ingestion of a wide range of important
nutrients and microbial metabolites generated as a consequence of
fermentation (biogenic effect). Microbial metabolites may include
bioactive peptides with antihypertensive activity, for example
(Papadimitriou et al., 2007; Gonzalez-Gonzalez et al., 2011) or
conjugated linoleic acid (Rodríguez-Alcalá et al., 2011). Moreover,
the production of several antimicrobial compounds (organic acids,
bacteriocins, hydrogen peroxide) during fermentation contributes
to increased protection against food contaminants or pathogens,
thus playing an important biopreservation role. Increasing scientiic
evidence conirms that the risk for many chronic diseases such as
cancer, osteoporosis, coronary heart disease, and hypertension can
be diminished by the regular consumption of fermented milks and
fermented milk supplemented with probiotics and/or prebiotics
(Sanchez et al., 2009; Panesar, 2011). The ingestion of probiotic
fermented milks, in particular, has been shown to cure antibiotic-
associated diarrhea, prevent intestinal tract infections, prevent
ulcers related to Helicobacter pylori, prevent colon cancer, control
rotavirus, promote alleviation of lactose intolerance symptoms,
reduce irritable bowel syndrome inlammatory effects, improve
immune system response, and lower cholesterol (Özer and Kirmaci,
 Dairy Products 101

2010). The health beneits of probiotics were reviewed in detail in

Chapter 3.
Several probiotic fermented milks are available on the worldwide
market, mainly incorporating members of the Lactobacillus and
Biidobacterium genera, and their existing positive image is well
positioned to capitalize the growth of healthy foods consumed for
their beneits as well as refreshing organoleptic quality. Some of
these are listed in Table 4.1. The probiotic product segment of the
functional foods market is continuously on the rise as speciied
by recent statistical data. Market research by Euromonitor (2008)
revealed that the North-American market of probiotic spoonable
yogurt market went from US$ 112 million in 2001 to US$ 294 million
in 2006. For example, the sales growth rate of fresh probiotic dairy
products in the USA in 2004 was much higher than cheese (9–10%
vs. 2%) (Granato et al., 2010; Özer and Kirmaci, 2010). Similarly in
Europe, Euromonitor (2008) reported that the consumption grew
13% and nearly 18% per annum in Western Europe and in Eastern
Europe between 2002 and 2007, respectively. The probiotic yogurt
market in Latin America expanded 32% per annum between 2005
and 2007, and accounted for 30% of total yogurt market value in 2007
(Euromonitor, 2009). The European probiotic food and beverage
market is estimated to reach $163 million by 2013 (Champagne,
2009; Özer and Kirmaci, 2010).

4.2.1.2 Technological challenges for probiotic fermented

milks
Growth of the functional fermented milk sector is an opportunity
to enhance the development of probiotic fermented milks into
products with nutritional and sensory properties without resorting
to costly and complex technology (Seleet et al., 2011). Criterious
management of technological and sensory aspects of the probiotic
foods are very important aspects because only the satisfaction of
consumers demand can lead food industry to success and promote
the consumption of functional products (Mattila-Sandholm et al.,
2002). Apart from the sensory aspects, viability of probiotic bacteria
in fermented milks and the associated probiotic effect also need to
be accounted for by manufacturers. These factors have been the
objective of many studies and are well documented in the scientiic
literature (Lacroix and Yidirim 2007; Özer and Kirmaci, 2010;
Sanders and Marco, 2010).
102 Food as Vehicles of Probiotics

4.2.1.2.1 Milk selection

The delivery vehicle in itself is an important starting point to
consider, as its composition (inherently present compounds, eventual
presence of other bioactive ingredients, fermentation end-products)
inluences probiotic viability and functionality, inducing changes in
the cell composition and physiological status of the strain during
production and throughout product shelf-life (Sanders and Marco,
2010). As mentioned in this chapter’s introductory note, milk and
derived products have a distinct role in delivery of probiotic bacteria
to the human gut, as these products provide probiotic bacteria with
a suitable environment in which not only their growth, viability, and
stability are favored, but also their enzyme portfolio and consequent
metabolic potential for the production of bioactive metabolites and
lavor compounds is intensiied (Gomes and Malcata, 1999; Saarela
2009; Özer and Kirmaci, 2010). Undoubtedly, bovine milk has been
the most used carrier for fermented milk production worldwide;
nonetheless, caprine and ovine milks have gained interest, especially
in smaller traditional markets in different countries in the Middle
East and Southern Europe where small ruminants milk is commonly
produced (Gomes and Malcata, 1998; Gomes et al., 1998a; Minervini
et al., 2009; Tamime et al., 2011). From a fermented milk point of
view, ovine milk is quite adequate for its manufacture, given its high
protein and fat contents; on the contrary, caprine milk quite often
requires undergoing protein fortiication as well as adaptation of
processing conditions in order to obtain sensory attractive products
for the consumer (Hilali et al., 2011; Tamime et al., 2011). Albeit
this technological handicap as far as good consistency is concerned,
goat’s milk has been successfully employed for the production
of probiotic fermented milks with adequately selected strains.
The mixed starter including L. acidophilus La5, Biidobacterium
lactis Bb12, and Streptococcus thermophilus has been reported to
manufacture good fermented goat’s milk with an inhibitory activity
against Escherichia coli strains (Cutic et al., 2004) and the same
Lactobacillus and Biidobacterium species attained high viable cell
numbers in goat’s milk fermented in a bioreactor and maintained
good viability throughout 10 days of storage at 4°C (Kongo et al.,
2006). More recently, a selected multiple starter (S. thermophilus
CR12, L. casei LC01, L. helveticus PR4, L. plantarum 1288) was
shown to be adequate for the manufacture of a potentially functional
 Dairy Products 103

fermented goat’s milk, which maintained adequate viable cell

numbers after 45 days of storage at 4°C and contained additional
functional properties, that is, γ-aminobutyric acid and a signiicant
Angiotensin-converting-enzyme (ACE)-inhibitory activity (Minervini
et al., 2009). In addition to milk type, agro-system production mode
also seems to inluence growth behavior of B. lactis in fermented
milks. Although many defend that organic and conventional milks
are similar in nutritional quality and wholesomeness, Florence et al.
(2012) were able to show that organic milk was more suitable for
production of fermented milks by B. animalis subsp. lactis strains
Bb12 and BL04, thanks to its technological characteristics evidenced
by the improvement in probiotic counts and superior levels of
conjugated linoleic acid (CLA) isomers and α-linolenic acid (ALA) in
the fermented products.

4.2.1.2.2 Strain selection

Despite milk qualities, production of high-quality fermented milk
products containing Biidobacterium spp. and L. acidophilus remains
a major challenge to dairy industries owing to the vulnerability of
the microorganisms in these products. In particular, biidobacteria
tend to exhibit weaker growth and acid production in milk, which
implies longer fermentation periods and anaerobiosis conditions
(Gomes and Malcata, 1999), low redox potential at least in the early
phase of growth (Dave and Shah, 1997) and often the addition of
biidogenic and growth-promoting factors to the milk (Gomes et al.,
1998a; Özer and Kirmaci, 2010). Biidobacteria produce acetic and
lactic acids at the ratio 3:2 during fermentation, so excessive growth
may yield unacceptable to consumers, yet such organoleptic hurdles
have been duly overcome by tailor-made technological strategies.
As previously demonstrated in the case of caprine milk,
strains need to be carefully selected and monitored throughout
manufacture in order to guarantee desirable pH values and end-
product metabolites. The use of combined cultures of biidobacteria
and other lactic acid bacteria, viz. L. delbrueckii subsp. bulgaricus
and S. thermophilus, S. thermophilus alone, or mesophilic aromatic
cultures, has been indicated as a way to overcome many of these
problems (Tamime, 2002), reduction of fermentation time, control
of sensory and texture defects, and nutritional improvement of
probiotic fermented milks are some of the advantages that can be
achieved by the use of combined cultures. In their recent study,
104 Food as Vehicles of Probiotics

Xantophoulos et al. (2012) have been able to demonstrate the

success of such binomial: nutritive value of carrier with potential
functional properties of mixed strains. Yogurt was successfully
produced from caprine milk by using two beneicial (cholesterol
removal, resistance to bile salts, low pH, and lysozyme) lactobacilli
isolates from newborn infant feces (L. paracasei subsp. paracasei
DC412 and L. acidophilus DC602) as costarters with two strains of S.
thermophilus and two L. delbrueckii subsp. bulgaricus isolates from
traditional goat’s milk yogurt. Growth of both S. thermophilus [>109
colony-forming units (cfu) per ml] and total lactobacilli (>108 cfu/
mL) was enhanced along with improved acidiication ability in milk,
increased production of acetaldehyde, and a desirable consistency
and organoleptic evaluation (4.4/5). However, in the case of
mixed cultures, one must bear in mind the need to assess strain
interactions in order to accurately select compatible strains and
that the traceability of the probiotic strains becomes more dificult.
Nonetheless, enumeration of culturable and nonculturable methods
have evolved greatly and many validated solutions are available in
literature (Gomes et al., 2009; Rodrigues et al., 2012a)
The probiotic species most frequently employed in the
production of fermented milks are tentatively of human intestinal
origin because it is generally accepted that they are better suited
to the physiological needs of the host and can more easily colonize
its intestine than wild strains or strains from the colon of other
animals; however, it is important to note that human origin is not
a requirement for probiotic functionality (Douglas and Sanders,
2008). Common strains include Biidobacterium adolescentis, B.
biidum, B. breve, B. infantis, B. longum, L. acidophilus, L. casei, and L.
rhamnosus (Gomes and Malcata, 1999; Sanders and Marco, 2010). A
more comprehensive list is provided in Table 4.2.
General accounts of the physicochemical and technological
aspects of commercial fermented milk products containing
Biidobacterium spp. and L. acidophilus have been provided by
Tamime (2002), Sanchez et al. (2009), Ranadheera et al. (2010), and
Panesar (2011).
A number of studies have been published related to characteristics
of good probiotics and criteria for selection thereof (Sanders
and Marco, 2010) as far as milk culturing capacity is concerned,
simultaneous with the inherently required probiotic effect; these are
summarized in Fig. 4.2.
 Dairy Products 105

Table 4.2 Species currently employed in the manufacture of probiotic

products and respective sources
Species Strain Source
Biidobacterium spp.
B. infantis 35264 Procter & Gamble
B. lactis Bb12 Christian Hansen
Ceska®-star B100 CSK Food Enrichment
HN019 (DR10) Danisco
B. animalis LAFTI B94 DSM Food Specialities
DN173 010 (Biidis Danone
regularis™)
B. longum BB536 Morinaga Milk Industry Co.
Ltd.
B. breve Yakult Yakult Japan
Lactobacillus spp.
L. acidophilus Ceska®-star A900 CSK Food Enrichment
LA-1 Chr. Hansen
LAFTI L-10 DSM Food Specialities
LB Lacteol Laboratory
NCFM® Danisco
R0052 Institute Rosell-Lallemand
L. casei DN114001 Danone
(L. casei defensis™)
LAFTI L26 DSM Food Specialities
Shirota Yakult
L. fermentum VRI003 (PCC) Probiomics, Australia
L. johnsonii Lj-1 Nestle
L. paracasei CRL 431 Chr. Hansen
F 19 Medipharm
L. plantarum 299V Probi AB
L. reuteri RC-14™ Chr. Hansen
SD2112 Biogaia
ATTC 55730 Arla-Ingman
L. rhamnosus GG (LGG) Valio Dairy Ltd.
GR-1™ Urex Biotech
HN001 (DR20) Danisco
LB21 Essum AB
(Continued)
106 Food as Vehicles of Probiotics

Table 4.2 (Continued)

Species Strain Source

271 Probi AB
R0011 Institute Rosell-Lallemand
L. salivarius UCC118 University College Cork
Others
Lactococcus lactis L1A University College Cork
Propionibacterium JS (DSM 7076) Valio Dairy Ltd.
freudenreichii ssp.
shermanii
Source: Data compiled from Gomes and Malcata (1999), Douglas and Sanders (2008)
and Ozer and Kirmaci (2010).

Strain

Survival in upper
Safety Capacity for large
intestinal tract and
considerations scale production
resistance to bile salts

Milk culturing abilities

Possible addition of
Growth with 1-10% Coagulation of milk
growth-promoting Oxygen tolerance for
inoculum within 24 h within 24 h
factors for lactic growth in milk
(108-109 cfu/ml) (pH < 4.6)
acid bacteria (LAB)

Maintenance of viability

Good acidity profile Maintenance of Good rheological Good storage stability

(aciduric, pH 4.2 - 4.7) flavour and aroma properties throughout Decimal death rate > 3.5 d
Low post-acidification profiles during storage manufacture/storage Low thermoactivity (0-15ºC)

Figure 4.2 Criteria for selection of probiotic strains, in particular

Biidobacterium spp., for application as dietary adjuncts.

Important properties include tolerance to acid and bile,

fundamental to maintain high numbers of viable cell throughout
storage and passage in the digestive tract upon consumption (Gomes
and Malcata, 1999; Douglas and Sanders, 2008), and production of
antimicrobial substances and concomitant inhibition of pathogens.
The strains selected also need to produce a inal product with good
sensory properties concerning lavor and texture; the delivery
 Dairy Products 107

vehicle also plays an important role in this case, as it may enable

improved product palatability, thus promoting regular consumption
by the consumer. In order to improve the texture of fermented
milks and yoghurts, several approaches have been proposed. For
example, the application of hydrostatic or dynamic high pressure
and the use of probiotic strains able to produce exopolysaccharides
have been suggested as alternatives to the use of additives, which
although allowed by the Codex Alimentarius standard (2003), may
negatively affect the fermented milk taste, lavor, aroma, and mouth-
feel (Patrignani et al., 2006).
Not less important is that the strains selected are able to be pro-
duced in large scale (Sanders and Marco, 2010) and starter manu-
facturing technologies have largely evolved in such direction; strains
selected for Direct-Vat-Set (DVS) starters must undergo concentra-
tion of up to 1010–1011 cfu/g in order to permit the desired perform-
ance in commercial manufacture of fermented milk products. In ad-
dition, the starter must also have good stability (usually guaranteed
for 3–12 months during processing, storage, and distribution). Direct
inoculation of a dairy product with a frozen concentrated culture
(ca. 5–10 x 1010 cfu/g) is recommended at a rate of 1 kg per 10,000
l of milk (Sanders and Marco, 2010). Finally, safety assessment of
the probiotic strains is also a mandatory requirement; a tentative
strain must be nonpathogenic for technological uses. There is a large
consensus that lactic acid bacteria are benign microorganisms with
a well-established record of safety and no record of food poison-
ing outbreaks. Updated reviews on safety of probiotic bacteria have
been made available by Saarela et al. (2000), Borriello et al. (2003),
Donohue (2006), and Bernardeau et al. (2008).
From a technological perspective, it is essential that designing
of probiotic fermented milks considers ive basic factors as a
foundation for optimizing probiotic stability, namely (i) probiotic
activity, (ii) manner of addition, (iii) composition of the inished
food, (iv) processing condition and procedures, and (v) storage and
other conditions prior to consumption. A more detailed description
of each of these factors and its implications on probiotic fermented
milk manufacture is addressed in Table 4.3. The selection of probiotic
strains as well as manufacturing optimization (both formulation and
storage conditions) are of utmost importance for growth and viability
of probiotic bacteria in fermented milks (Martinez-Villaluenga et al.,
2006; Saarela et al., 2006; Özer and Kirmaci, 2010).
 108
Table 4.3 Main factors affecting probiotic viability and functionality from production to storage supported by recent studies.

Factor Description Case studies References

Improvement of stability in powders can be Corcoran et al.,
achieved by use of cryoprotectants, use of 2004; Sousa et al.,
encapsulation technology where cysteine may 2012; Borges et al.,
be added to encapsulating material, or by 2012.
application of mild sublethal stresses either
during or after fermentation.
Food as Vehicles of Probiotics

Selection of naturally occurring strains that lack Damin et al., 2008

post-acidiication capacity enables development
of fermented milks with acceptable sensory and
rheological properties.
Probiotic strain - Besides added value to food, Aerotolerance is a desired trait in probiotic Li et al., 2010
metabolic activity probiotics need to be cost strains intended for fermented milk production:
effectively produced, maximizing Li et al. have isolated and selected an
substrate-to-biomass yield and aerotolerant strain (high relative bacterial
stability during processing and growth ratio), B. animalis subsp lactis Qq08, with
shelf life. high tolerance to low pH (84% survival at pH 2)
and to bile (90% survival after 4.5 h incubation
at 0.01 g mL–1 bile.
- Probiotics need to maintain
their functionality during
storage as frozen or freeze-dried
cultures.
- Different strains will behave Study of the technological and probiotic Sanchez et al., 2010
differently under different potential of the bile-adapted strain B. animalis
manufacturing and storage subsp. lactis 4549dOx with respect to its
conditions— tolerance to harsh parental strain IPLA4549 revealed its better
conditions can be due to an ability to survive the simulated GIT conditions,
intrinsic resistance or to an and slightly higher adhesion capability to the
adaptive response. model epithelial intestinal line HT29-MTX. Both
parental and bile-resistant derivative strains
maintained viability throughout fermented milk
shelf-life.
- Different growth conditions may In vitro antimicrobial activity of probiotic strains Fayol-Messaoudi et
affect physiological activity and depended on both growth temperature and al., 2005
alter bacterial components with growth stage for cell harvesting.
claimed probiotic activity or
lavour-producing capacity.
- Certain strains are capable
of intense post-acidiication
contributing to loss of quality,
off-lavours or texture
deiciencies.

(Continued)
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109
 110

Table 4.3 (Continued)

Factor Description Case studies References

L. plantarum harvested at different growth Van Baarlen et al.,
phases or when heat treated promoted different 2009
modulation proiles of NF-kB-dependent
pathways in human mucosa (duodenal biopsies).
Food as Vehicles of Probiotics

L. johnsonii grown in fatty acid–enriched minimal Muller et al., 2011

medium (oleic, linoleic or linolenic acids)
revealed different membrane composition which
impacted acid and heat tolerance, adhesion
properties and caused a 2-fold decrease in
Salmonella enterica serovar typhimurium UK1-lux
invasion of HT-29 epithelial cells compared with
bacteria grown in minimal medium alone.
Manner of addition - Reconstitution conditions may Reconstitution conditions such as buffer, pH, Mille et al., 2004;
affect probiotic viability and duration, sugar type and content and temperature Muller et al., 2010
stability in food matrix. may lead to variations in inal viable cell numbers
as large as 1 log cycle difference.
- Addition of probiotic strains Incorporation of free and encapsulated Kailasapathy, 2006
in a controlled delivery system probiotic bacteria did not signiicantly affect the
may help regulate acidiication overall sensory characteristics of yogurts yet
capacity. microencapsulation promoted better survival of
probiotic bacteria in yogurts during storage.
Composition of - Matrix properties inevitably Tailored (addition of carbon and nitrogen Cousin et al., 2012
fermented milk affect probiotic functionality: sources) fermented milk was able to maintain
oxygen content, high viability and in vitro stress-tolerance of potential
temperature, low pH, high probiotic Propionibacterium freudenreichii
water activity, elevated solute strain during minimum 15 days at 4 °C. In vivo
concentration and the presence eficiency was demonstrated by viability and
of antimicrobial compunds and short-chain fatty acids content in the colon of
certain food additives may all piglets. When given in the form of lyophilizate
affect probiotic strain viability. the Propionibacterium strain was not able to
exert any positive effect.

- Delivery vehicle is also essential A meta-analysis has shown that delivering Sachdeva and
for potential probiotic activity— probiotic strains via fermented milk products Nagpal, 2009
metabolic activity and probiotic may be more eficient in eradication of intestinal
properties are, in some cases, H. pylori than delivering them via capsules or
only stimulated if fermented sachets.
milk is the delivery vector.

Presence of available carbon sources such as Corcoran et al.,

glucose signiicantly increase survival of L. 2005
rhamnosus GG in simulated acidic stomach
conditions.

(Continued)
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111
 112

Table 4.3 (Continued)

Factor Description Case studies References

Processing Compared to control fermentations without Oliveira et al., 2011
conditions lactulose, the addition of such a prebiotic in
and procedures skim milk increased counts of probiotics: L.
acidophilus, L. rhamnosus, L. bulgaricus and B.
lactis in co-culture with S. thermophilus, the
Food as Vehicles of Probiotics

acidiication rate and the lactic acid acidity,

and concurrently reduced the time to complete
fermentation (tpH4.5) and the pH at the end of
cold storage upon 35 days.
- Milk supplementation, culture Fermented milk quality was inluenced by both Oliveira et al.,
composition and processing the co-culture composition (probiotic added 2009a
conditions may affect to S. thermophilus) and the selected prebiotic:
acidiication, textural properties, maltodextrin, polydextrose or oligofructose.
and microbiological stability of
fermented milks.
- Manufacturing processes Among 15 different L. rhamnosus isolates, 14 Grze kowiak et al.,
and matrix may affect the of which labelled and conirmed as L. rhamno- 2011
functionality of the strains and sus GG, all revealed similar acid tolerance and
impact outcomes of clinical binding capacity to human colonic mucus; yet,
intervention studies. pathogen exclusion by inhibition and competi-
tion varied signiicantly among the different L.
rhamnosus isolates and pathogens tested.
Storage and other - Harsh environmental conditions Encapsulated probiotic bacteria were able to Krasaekoopt et al.,
conditions prior to or abuse in storage temperature survive up to 1 log cycle better in stirred yoghurt 2006
consumption or time may affect probiotic than as free cells.
functionality.

- Microencapsulation is a Viability of commercial B. lactis Bb12 and Martinez-

protective technology that may L. acidophilus La-5 in fermented milk was Villaluenga et al.,
increase stress tolerance and improved by 12% upon 21 d of refrigerated 2006;
improve viability and stability storage when rafinose was added.
during storage of fermented
milks. Viability of commercial Biidobacterium spp. was Shin et al., 2000
improved by 55% upon 4 weeks of storage at 4°C
when FOS was added.
Dairy Products
113
114 Food as Vehicles of Probiotics

4.2.1.3 Survival characteristics

Strain viability in fermented milks by the time of consumption is
mainly related to the aforementioned technological requirements.
Suggested ranges for the minimum level for probiotic bacteria in
probiotic fermented milks may vary according to the strain employed;
nevertheless, a minimum value of 107 cfu/ml or 107 cfu/g of product
is recommended (FAO/WHO, 2001; Codex Alimentarius 2003).
Maintenance of this level is a challenging objective and industry
normally applies signiicant overages during initial formulation in
order to compensate drop in viability throughout storage. There are,
however, many internal and external variables to the environment
of fermented milks that may inluence probiotic integrity, eficacy,
and shelf-life. Acidity, pH, dissolved oxygen content, redox potential,
sugar concentrations, solids content, presence of competitive
microorganisms, storage temperature, and presence of microbial
inhibitors (e.g., NaCl and H2O2) have been identiied as having an
effect on probiotic viability during the manufacture and storage of
fermented milks (Siró et al., 2008; Ranadheera et al., 2010).
Examples with loss of viability have been reported for both
Biidobacterium and L. acidophilus, which are more often during
refrigerated storage at low pH and high levels of dissolved oxygen
(Dave and Shah, 1997; Donkor et al., 2006), and relect the need
for careful strain selection. The use of microencapsulated cells to
diminish dissolved oxygen and the addition of prebiotics are some
of the most effective strategies of keeping the numbers of probiotic
bacteria high enough for therapeutic effects (Özer et al., 2005).
Electrochemical reduction of milk, deaeration, or the addition of
reducing agents such as cysteine enhanced the survival of oxygen-
sensitive biidobacteria in fermented milks (Gomes and Malcata,
1998; Gomes et al., 1998a; Bolduc et al., 2006). Probiotic bacteria, in
particular Biidobacterium strains, also need biidogenic compounds
or growth factors to be able to grow and survive in milk (Gomes and
Malcata, 1999; Østlie et al., 2003; Roy, 2005). Various substances have
been added to milk for such purpose and favorable results have been
achieved with fructooligosaccharides (Shin et al., 2000; Varga et al.,
2006; Oliveira et al., 2009b, 2011), rafinose family oligosaccharides
(Martínez-Villaluenga et al., 2006), maltodextrin and polydextrose
(Oliveira et al., 2009a), caseinomacropeptides and whey protein
concentrate (Janer et al., 2004), soy protein concentrate, soy lour
 Dairy Products 115

and lentil lour (Zare et al., 2012), lactulose (Oliveira et al., 2011),
tryptone, yeast extracts, certain amino acids, nucleotide precursors
(Gomes et al., 1998a), and a mineral source such as iron or zinc
(Seleet et al., 2011).
Other options currently pursued for improving viability include
enhancement of bacterial stress response (Corcoran et al., 2008)
and quorum-sensing interference. The release of quorum-sensing
signal molecules by probiotics may interact with human intestinal
epithelial cells and contribute to the modulation of the different
physiological functions mentioned previously in Chapter 2.
Although survival in fermented milks has been discussed herein
as a challenge for probiotic strain stability, the relevance of the
protective effect of fermented milk on passage through the GIT
(Ranadheera et al., 2010) should not be overlooked. Indeed, buffering
capacity and pH have been indicated as the major reasons for such
protection, yet how these properties regulate the metabolism and
cellular processes of the probiotics at the molecular level is still
unknown. Recent indings by Wang et al. (2012) unravel some of
these phenomena for L. casei Zhang, a strain isolated from koumiss
in Inner Mongolia, China. The authors used microarray technology
to detect the gene expression proile of the L. casei strain with
and without fermented milk (strain suspension) used as a carrier
during transit in simulated gastrointestinal juice. Interestingly, a
large proportion of genes involved in translation and cell division
were downregulated in the bacteria that were in strain suspension
during transit in simulated intestinal juice. This may hamper protein
biosynthesis and cell division and may partially explain the lower
viability of L. casei Zhang during transit in the GIT without the
fermented milk (Wang et al., 2012).

4.2.2 Cheeses
In the last decade, research efforts on the development of probiotic
cheeses containing viable cells of Lactobacillus and Biidobacterium
sp. with potential health beneits have been made to lead this type of
product into the functional foods category (Ross et al., 2002). Cheese
has been claimed as a good carrier of probiotic bacteria because it
enables their passage as viable cells throughout the GIT (Gomes
et al., 1995; Mäkeläinen et al., 2009). In addition, the organoleptic
characteristics, nutritional values, and the suitability of cheeses to
116 Food as Vehicles of Probiotics

be consumed by several age groups (Gomes and Malcata, 1999)

increase the importance of this kind of functional dairy product.

4.2.2.1 Strains, cell probiotic concentration, and viability

Nowadays, it is well known that probiotics are microorganisms,
which, when present in adequate amount in food, should be able to
resist to the passage through the GIT as well as to adhere to intestinal
cells, in order to provide beneicial effects (Gomes and Malcata,
1999; Oliveira et al., 2009c) and therefore viability of probiotics is
a key parameter in a probiotic cheese. Due to the fact that cultures
can vary signiicantly in their performance in cheese manufacture
and ripening as well as in the intestinal environment, the selection
of strains is crucial to the development of probiotic cheeses (Ross
et al., 2002). Apart from the clinical evidence that should support
the health-promoting activity of probiotic cultures, the technological
suitability of the probiotic strains is also a crucial parameter to
their use in functional foods (Ross et al., 2005). The main probiotic
strains that have been used in different types of cheeses with more
or less success are displayed in Table 4.4. The success of using these
strains into cheeses is dependent on several factors, namely strain
resistance to aggressive factors associated with the composition of
cheese, the conditions of processing and ripening, as well as tolerance
and competiveness to the lactic acid starters that must assure their
viability in the cheese matrix.
Research studies on cheeses with biidobacteria demonstrate
that not all biidobacteria species exhibit the same survivability
or have equal role on the cheese characteristics. Some of the main
constraints for the biidobacteria are related with their susceptibility
to oxygen, as they are generally anaerobic in nature (Boylston et al.,
2004). In addition, these strains differ in their ability to grow in milk,
curdled milk, and in acidiied matrices in the presence of starter
cultures. However, studies especially with Cheddar, semi-hard, and
white-brined cheeses demonstrate that some of the biidobacteria
such as B. lactis, B. longum, and B. biidum are able to survive
through relatively long periods of ripening such as 60 days at 12°C
to 6 months at 4°C (Table 4.4). According to a review of Boylston et
al. (2004) about the challenges of using biidobacteria in cheeses,
an evaluation of biidobacteria strains viability in several cheeses
such as cottage, Crescenza (Italian cheese), Canestrato Pugliese
(Italian hard cheese), Fesco cheese, white-brined Tallaga (Egyptian
Table 4.4 Diversity of potential probiotic strains that have been tested in different types of cheese.

Strain Type of cheese/ Ripening period References

Biidobacteria
B. adolescentis White brined cheese/60 days Ghoddusi and Robinson, 1996
B. breve Fresh cheese/57 days—4 or 12°C Roy et al., 1997
B. lactis B94 Semi-hard cheese/60 days—12°C Gouda semi-hard Rodrigues et al., 2011b
B. lactis BO cheese/9 weeks—13°C Gomes et al., 1995
B. longum 1941 Cheddar cheese/6 mo—4°C Ong et al., 2006, 2007
B. biidum Bb-02 White-brined cheese/90 days—4°C Cottage Yilmaztekin et al., 2004
B. infantis cheese/30 days Blanchette et al., 1996
Lactobacilli
L. acidophilus 4962 Cheddar cheese/6 mo—4°C Ong et al., 2006, 2007
L. acidophilus La-5 Curdled milk matrices/60 days—12°C Semi-soft Rodrigues et al., 2011a
L. acidophilus NCFM Goudda cheese Makelainen et al., 2009
L. acidophilus ki Goat cheese/70 days—5 and 10°C Gomes et al., 1998b
L. casei 279 Cheddar cheese/6 mo—4°C Ong et al., 2006, 2007
L. casei-01 Semi-hard cheese/60 days—12°C Rodrigues et al., 2011b
L. fermentum AB5-18 Turkish Beyaz cheese/120 days—4°C Kiliç et al., 2009
L. helveticus H100 Cheddar cheese/6 mo—4–12°C Ong and Shah, 2008
L. paracasei L26 Cheddar cheese/6 mo—4°C Ong et al., 2006, 2007
L. plantarum AB16-65 Turkish Beyaz cheese/120 days—4°C Kiliç et al., 2009
L. rhamnosus Cheddar cheese/32 weeks—9–10°C Philips et al., 2006
Enterobacteria
Enterococcus faecium PR88 Cheddar cheese/9 or 15 mo—8°C Gardiner et al., 1999a,b
Propionibacteria
P. freudenreichii ssp. sherrmanii Cheese-based dips Tharmaraj and Shah, 2004
P.freudenreichii Suisse type cheese Jan et al., 2000
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Lactococci
Lact. lactis ssp. lactis Fresh cheese/57 days—4 or 12°C Roy et al., 1997
117
118 Food as Vehicles of Probiotics

cheese), Kariesh (Egyptian cheese) indicates that B. biidum and B.

longum were among the strains with higher levels of viability during
the processing and storage, whereas B. infantis and B. adolescentis
revealed lower survivability. According to Rodrigues et al. (2011
b, c), good levels of survivability of B. lactis B94 (109–1010 cfu/g
cheese) were observed after 45–60 days of ripening, in a potential
probiotic semi-hard cheese despite the harsh conditions of low pH
values (4.1–5.1) and low moisture content (<30%, w/w).
The diversity of different strains of potential probiotic
lactobacilli that have been incorporated in the cheese matrix is
higher than those of biidobacteria (Table 4.4). The use of facultative
heterofermentative lactobacilli as mesophilic starters has a long
tradition in cheese manufacture. More recently, the concept of using
the lactobacilli strains as probiotic adjunct cultures has emerged
(Johnson and Lucey, 2006; Cogan et al., 2007). Potential probiotic
lactobacilli strains isolated from traditional foods such as cheese
have been proposed and studied in order to develop new functional
foods (Bertazzoni-Minelli et al., 2004). Lactobacilli strains possess a
long history of safe use (Maragkoudakis et al., 2006), and according
to Bernardeau et al. (2008), 32 selected species of lactobacilli possess
the Qualiied Presumption of Safety (QPS) proposed by European
Food Safety Authority (EFSA). These strains possess the added value
of being naturally resistant to cheese matrix environment through
manufacture and ripening processes being able to reach high
levels of viable cells with positive impacts on the cheese sensorial
characteristics. The potential probiotic L. casei-01 used as a starter,
without adjunct cultures, reached values of 109–1010 cfu/g in a
semi-hard cheese after 15 days of ripening, which remained almost
constant until 60 days of ripening (Rodrigues et al., 2011b,c). Several
studies about the potential probiotic action of lactobacilli of dairy
origin in GIT have been performed in the last years. For example,
Maragkoudakis et al. (2006) evaluated the probiotic potential of
29 Lactobacillus strains of dairy origin through in vitro tests: L.
acidophilus, L. casei Shirota, L. casei Imunitass, several strains of L.
paracasei, L. plantarum, and L. rhamnosus. According to the authors,
all strains revealed to be unaffected by pH = 3, pancreatin, and bile
salts, but they exhibited variable bile salt hydrolase activity; some
of the strains, namely L. casei Shirota ACA-DC 6002, L. plantarum
ACA-DC 146, and L. paracasei subsp. tolerans ACA-DC 4037, were
able to adhere to Caco-2 cells and also to inhibit the adhesion of
 Dairy Products 119

Escherichia coli and Salmonella typhimurium to Caco-2 cells. These

results, associated with the fact that these three strains also induced
the pro-inlammatory and anti-inlammatory cytokines secretion by
human peripheral blood mononuclear cells, become good evidences
of their potential probiotic action on human GIT. Probiotic strains
such as L. acidophilus NCFM and L. rhamnosus HN001 incorporated
in a semi-soft cheese survived to simulated gastrointestinal digestion
in the upper GIT model, and increased their viable cells in the colon
simulator in comparison to nonprobiotic cheese (Mäkeläinen et al.,
2009).
The use of enterococci in dairy products is controversial,
although they have been reported as part of the adventitious lora of
many raw or pasteurized milk cheeses and the general idea is that
they play an important role in the ripening process. The majority
of studies reporting the presence and inluence of enterococci in
cheese dated from the 1990s. However, some more recent works
still point the inluence of this group of microorganisms in cheeses
such as Bryndza (Jurkovič et al., 2006), Tolminc cheese (Majhenič et
al., 2005), Firode Sardo (Cosentino et al., 2004), San Simon (Garcia
et al., 2002), and Terrincho (Pimentel et al., 2007). One of the irst
strains tested and indicated as potential probiotic enterococci strain,
Enterococcus PR88, was studied as probiotic adjunct cultures in
Cheddar cheese (Table 4.4). According to Gardiner et al. (1999b),
this enterococcal strain revealed being suitable for incorporation
in cheese, as it remained viable at high levels (>108 cfu/g) after 9
months of ripening. Giraffa and coauthors, who in 1997 reviewed
the risks and potential technological use of enterococci isolated
from dairy products (Giraffa et al., 1997), published a review about
functionality of enterococci in dairy products in 2003 (Giraffa, 2003),
exposing evidences that enterococci, furnished as adjunct starters
or nonstarters, could have potential application in some fermented
dairy products, but some care in terms of safety should be provided,
as clinical research on enterococci indicates that the safety in their
use must carefully be addressed before their application.
Dairy propionibacteria in food industry as starter cultures also
have a long history of safe use. Propionibacteria have been reported
as probiotic due to their ability to modulate some enzymatic
activities and microbial lora within the gut. Mantere-Alhonen
(1995) in a review about the use of propionibacteria as probiotic
indicates the evidences that support their use as potential probiotics.
120 Food as Vehicles of Probiotics

According to Roland et al. (1998), P. freudenreichii strain, which is

normally found in the microlora of Suisse cheese type, is also able to
stimulate biidobacteria in vitro and in the human intestine; further
studies about biidogenic properties (enhancement of biidobacteria
strains) of Propionibacterium have been performed (Warminska-
Radyko et al., 2002; Kouya et al., 2007). According to Vorobjeva
et al. (2008), propionibacteria possess a set of physiological and
biochemical properties that make them promising human probiotics
with the added values that some strains are able to produce CLA
isomers; these have received increasing interest in the last years
because of their potential health effects such as antitumor, antiobese,
antiatherogenic, antidiabetic, immunomodulatory activities, and so
on.
Some studies were performed about the probiotic potential of
Lactococcus strains, although they are commonly used as starter
bacteria in the manufacture of several types of cheese. Strains of
Lact. lactis ssp. lactis are pointed as potential new probiotics by
Kimoto et al. (1999), but no intensive research on lactococci strains
was published in the last decade.
According to Douglas and Sanders (2008), although it is not
possible to accurately generalize about a minimum dose of probiotics
needed for a beneicial effect in the gut, studies showing beneicial
effects at numbers below 108 cfu per day are uncommon. According
to trends reported by Health Canada (2009), the probiotic dosage
should be referred to a food portion, that is, a serving size of product
should contain a minimum level of 1.0 × 109 cfu of one of the eligible
microorganism that is the subject of functional claim. Considering
these guidelines, probiotic cheeses have been claimed to be adequate
food products to deliver probiotic concentrations able to provide
potential health beneits throughout the shelf-life of the product,
which naturally depends on the strain as well as the technological
aspects of cheese production (Fig. 4.3).
A review on developments in cheese cultures with protective
and probiotic functionalities by Grattenpache et al. (2008) reported
the viability of several probiotic cultures in different types of cheese
from studies reported between 1994 and 2007; a range between 106
and 109 cfu/g is reported as cell concentration at the beginning of
ripening period, whereas values ranging from 104 to 109 cfu/g are
reported at the end of ripening period; the ripening period ranged
between 14 days at 4°C for the Crescenza soft cheese (Gobetti et al.,
1998) and 32 weeks at 9–10°C for Cheddar cheese (Philips et al.,
1. Strains, probiotic cell concentration and viability through process and storage

Bacteria Lactobacilli Viable cells concentration should be

Bifidobacteria technological attainable at effective cost
Enterococci
Propionibacteria
Yeasts — Viability and metabolic activity on food product
is dependent on strain and product
composition
Addition of high number of
2. Food: Cheese —Technological aspects viable cells can affect product
quality – organoleptical
Manufacture procedures Type of milk properties
Coagulant
Temperature: coagulation, syneresis, etc.
Acidification
% NaCl Principal characteristics that turn cheese as
potential food to deliver probiotics:
Ripening procedures Time - Limited acidity
Temperature - Low oxygen
Relative humidity - High lipid concentration Suitable protection carrier against
- Low storage temperatures harsh GIT tract conditions:
- Low potential redox - Acidity in the stomach;
- Enzymatic activities;
- Bile salts in the small intestine.

Figure 4.3 Probiotic cheese: parameters, keys, and limitations.

Dairy Products
121
122 Food as Vehicles of Probiotics

2006). Madureira et al. (2008) reported high levels of L. paracasei

LAFTI L26 (>108 cfu/g) in sweet whey cheeses over 21 days at 7°C.
The most recent works (2009–2012) concerning the development of
probiotic cheeses are reported in Table 4.5. In addition, Karimi et al.
(2011) in a recent review about viability of probiotic microorganisms
in cheese during production and storage described that viability of
probiotic bacteria in cheese had been satisfactory in the sense that
at the end of ripening/storage, independently of their duration, the
numbers of viable cells were above 106 cfu/g. According to the data
displayed in Table 4.5, the numbers of viable cells varied between 107
cfu/g for L. plantarum cream cheese for 90 days and 1010 cfu/g for B.
lactis semi-hard cheese for 60 days. These results allow conirming
that cheese is a good vehicle of probiotic bacteria for the regular
consumer.
Regardless of the high concentration of probiotic bacteria at the
moment of consumption, research conirming their survival and
resistance through the passage through the GIT is mandatory. Further
research should be performed to assess the complex phenomena
that probiotic cheese undergoes in each human GIT compartment.
Mäkeläinen et al. (2009) studied the viability of probiotic lactobacilli
in three models that simulated human GIT: upper GIT, human colon,
and the colonocytes in cell culture experiments. These indings
indicate that the studied lactobacilli strains in cheese matrix were
able to survive in GIT.
As the large-scale production of probiotic bacteria at high
concentrations of viable cells is sometimes dificult especially for
less resistant strains, research efforts have focused its attention in
improving the viability of probiotic strains during the production
and shelf-life of the product (Ross et al., 2005) such as cheeses.
Microencapsulation of probiotic bacteria in suitable and protecting
carriers to increase its concentration and improve its viability and
functionality is a methodology that has been studied; some of the
carriers include milk or whey proteins (Rodrigues et al., 2011d),
calcium alginate (Sousa et al., 2012), or complex carbohydrates with
prebiotic action (Chen et al., 2005). Different results, however, have
been reported; Gobetti et al. (1998) reported that immobilization
of biidobacteria strains in calcium alginate did not improved their
viability in Crescenza cheese, in comparison to those nonimmobilized.
In turn, some beneits of immobilization were reported by Dinakar
and Mistry (1994) in Cheddar cheese after 24 weeks. More recent
Table 4.5 Viability of probiotic strains used as starter or as adjunct in different types of cheese reported between 2009 and
2012.

Cheese type Probiotic strain Process remarks Ripening Viable cell counts References
parameters/ (log cfu/g)
Storage Fresh Ripened
conditions cheese cheese
Pategrás L. paracasei Probiotic strain used as adjunct; 60 days/12°C 6.0–7.8 9.0 Bergamini
cheese (semi- L. acidophillus Starter culture: Streptococcus /80 %HR 5.5–7.2 7.5–8.5 et al., 2009
hard cheese) B. lactis thermophilus 7.0–7.5 7.2–7.8
Cream cheese L. plantarum RL34 Cheese aseptically illed in plastic 90 days/4°C 8.5 7.0 Georgieva
boxes et al., 2009
Turkish Beyaz L. fermentum Mixed probiotic culture 120 days/4°C 9.4 7.9 Kiliç et al.,
cheese L. plantarum Cheeses packed in plastic bags 2009
with brine
Cremoso L. casei I90 Each probiotic strain used as 60 days/5°C – 7.4 Milesi et al.,
cheese (soft L. plantarum I91 adjunct; Starter culture: Str. 8.2 2009
cheese) L. rhamnosus I73, I77 thermophilus. 7.3–7.7
Vacuum-packed cheeses.
Pategrás L. casei I90 Each probiotic strain used as 60 days/12°C/80 – 8.7 Milesi et al.,
cheese (semi- L. plantarum I91 adjunct; Starter culture: Str. %HR 8.9 2009
hard cheese) L. rhamnosus I73, I77 thermophilus. 8.6–8.7

(Continued)
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123
 124

Table 4.5 (Continued)

Cheese type Probiotic strain Process remarks Ripening Viable cell counts References
parameters/ (log cfu/g)
Storage Fresh Ripened
conditions cheese cheese
Cheddar B. longum 1941 Mixed probiotic culture used 24 weeks/4–8°C 8.3 8.3 Ong and
cheese B. animalis subsp. as adjunct; Starter culture: 8.4 8.5 Shah, 2009
Food as Vehicles of Probiotics

lactis B94 Lactococus lactis subsp. lactis, 8.0 7.9

L. casei 279 Lact. lactis subsp. Cremoris 8.1 8.3
L. casei L26 Vacuum-packed cheeses in 8.3 8.0
L. acidophillus 4962 oxygen barrier bags 8.2 7.9
L. acidophilus L10
White-brined B. biidum Bb12 Microencapsulated probiotic 90 days/4°C 9.0–10.0 8.0–9.0 Ozer et al.,
Turkish cheese L. acidophilus La5 strains 10.0–11.0 8.5–9.5 2009
Mixed probiotic culture used as
adjunct; Starter culture: Lact.
lactis subsp. lactis, Lact. lactis
subsp. cremoris.
Cheese stored in a pasteurized
brine solution
Minas fresh L. acidophilus La5 Probiotic added solely or in 21 days/5°C 6.0–6.2 6.3–6.6 Sousa and
cheese coculture with starter culture: Saad, 2009
Str. Thermophilus
Vacuum-packed cheeses
 Cheese type Probiotic strain Process remarks Ripening Viable cell counts References
parameters/ (log cfu/g)
Storage Fresh Ripened
conditions cheese cheese
Argentinian L. paracasei A13 Mixed probiotic culture used as 60 days/12°C 6.6–6.8 8.5–8.8 Vinderola
fresh cheese B. biidum A1 adjunct. 7.3 6.7 et al., 2009
L. acidophilus A3 Starter culture: Lact. lactis A6, 6.5 7.4
Str. thermophilus
Vacuum-packed cheeses
Synbiotic L. delbrueckii UFV Probiotic strain used as adjunct; 20 days/5°C – 8.2 Araujo et
cottage cheese H2b2O Starter: Mesophilic lactic bacteria al., 2010
RA 073.
Inulin used as prebiotic. Packed
cheese.
Ras cheese L. casei Mixed probtioc culture used as 90 days/12°C – 8.3–8.4 El-Salam et
L. acidophillus adjunct; Starter: L. delbreukii al., 2011
subsp. bulgaricus and S.
thermophilus
Curdled milk L. casei 01 Each probiotic bacteria used as 60 days/12°C 7.8–8.2 8.2–8.4 Rodrigues
(similar to L. acidophillus L10 starter 8.2–8.5 8.4–8.6 et al., 2011a
fresh cheese) B. lactis B94 8.0–8.5 7.5–7.8

(Continued)
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125
 126

Table 4.5 (Continued)

Cheese type Probiotic strain Process remarks Ripening Viable cell counts References
parameters/ (log cfu/g)
Storage Fresh Ripened
conditions cheese cheese
Synbiotic L. casei 01 Each probiotic bacteria used as 60 days/12°C 7.8–8.2 8.5–8.7 Rodrigues
curdled milk L. acidophillus L10 starter. 8.2–8.5 8.0–8.2 et al., 2011a
Food as Vehicles of Probiotics

(similar to B. lactis B94 50:50 mix of 8.0–8.5 8.2–8.5

fresh cheese) fructooligosaccharides (FOS)/
Inulin used as prebiotic.
Semi-hard L. casei 01 Each probiotic bacteria used as 60 days/12°C 8.0 9.9 Rodrigues
cheese B. lactis B94 starter 8.0 10.0 et al.,
2011b, c
Synbiotic L. casei 01 Each probiotic bacteria used as 60 days/12°C 8.0 9.8–10.7 Rodrigues
semi-hard B. lactis B94 starter FOS or 50:50 mix of FOS/ 8.0 9.9 et al.,
cheese Inulin used as prebiotic 2011b, c
White soft L. johnsonii NRRL Each probiotic culture used as 30 days/7±2°C 8.39* 7.72* Effat et al.,
cheese B-2178 adjunct; Starter: L. delbreukii 8.71* 7.75* 2012
L. hhlgardi NRRL subsp. Bulgaricus and S. 8.68* 7.81*
B-1843 thermophilus.
L. curvatus NBIMC Dextrin or polydextrose
3452 (LitesseTM) used as prebiotics
(3%). Packed cheese.
 Cheese type Probiotic strain Process remarks Ripening Viable cell counts References
parameters/ (log cfu/g)
Storage Fresh Ripened
conditions cheese cheese
Domiati L. casei L. casei and E. faecium (1:1)used 60 days/25±5°C >7.0 >7.0 El-Salam et
cheese E. faecium as starter al., 2012
Cheese made with reconstituted
milk with normal and high
content of CLA
Caprine coalho L. acidophilus La5 Probiotic culture used as adjunct. 60 days/10°C 7.12–7.30 7.31–7.39 dos Santos
cheese Starter culture: Lact. Lactis et al., 2012
subsp. lactis, Lact. Lactis subsp.
cremoris.
Packed cheese.
Brazilian goat L. acidophilus Probiotic culture as well as mixed 7 days/10°C >6.0–7.0 >6.0–7.0 Garcia et al.,
semi-hard L. paracasei probiotic culture used as adjunct. 2012
cheese B. lactis Starter culture: Lact. Lactis
L. acidophilus+ L. subsp. lactis, Lact. lactis subsp.
paracasei + B. lactis cremoris.
*Lactobacillus viability obtained in white cheeses made with 3% dextrin
Dairy Products
127
128 Food as Vehicles of Probiotics

studies indicate that cheese factors could affect the integrity of

capsules, namely sodium ions, which could exchange with calcium
ions responsible for binding alginate and consequently could be
responsible for some disintegration of the capsules (Özer et al.,
2009). It is not desirable that probiotics changes the organoleptical
properties of the inal cheese, thus encapsulation could also be
regarded as an approach to modulate the effects on cheese matrix.
However, no consensual trend has been reported. For example,
in Kasar cheese, microencapsulation did not appear to affect the
composition, but higher levels of proteolysis were observed in
cheeses with encapsulated probiotic bacteria (Özer et al., 2009).
In Feta cheese, no signiicant differences were observed in textural
parameters between cheeses containing either free probiotic cells or
encapsulated cells (Kailasapathy and Masondole, 2005). According
to the facts previously discussed, it is clear that more research is
needed to ind different strategies to use probiotic strains in cheese,
and encapsulation of bacteria in food-grade matrices, which could
also protect their viability throughout gastrointestinal transit
(Settani and Moschetti, 2010), should be also a subject of further
investigation.

4.2.2.2 Cheese: Technological aspects

The viability and metabolic activity of probiotic bacteria are key
factors that must be controlled and monitored throughout processing
operations, maturation time, and storage of the food. Generally,
addition of probiotic bacteria into milk-based food systems,
including cheese, becomes a challenge in terms of maintaining
probiotic viability and functionality during manufacture and shelf-
life. The ripening period of cheeses, except for those consumed
as fresh cheeses, is characterized by periods that could range
from 20–30 days to months or even years for extremely ripened
cheeses. The survival of probiotic strains during this time, at
suficient concentration, is crucial for functionality, namely to exert
the probiotic effect in the GIT. What characteristics in cheeses are
favorable or represent a limitation for higher levels of viability and
metabolic activity of probiotic bacteria as well as a list of constraints
that probiotic bacteria need to resist from both technological and
intestinal perspectives are summarized in Table 4.6 in order to give
the reader a global and simple perspective of the main constraints.
The inluence of the main factors that could affect the viability of
Table 4.6 List of constraints that probiotic bacteria need to resist, from both technological and intestinal perspectives, and cheese
characteristics that could be favourable or unfavourable to overcome.

Physiological Favourable/U nfavourable

constraint Description cheese characteristic Examples of studied cases
Oxygen Probiotic biidobacteria and Cheese core can be considered as Reported values of potential redox
tolerance lactobacilli of gut origin are in anaerobic environment with low for Camembert
general anaerobic or redox potential. core cheese through ripening were
microaerophilic-strain dependent. of –330 ± –30 mV
(Abraham et al., 2007).
Acid tolerance Poor resistance under prolonged In general, cheese presents higher In 35-day ripened Camembert
acidic conditions; In general and favourable pH values than cheese pH values are 6.8 and 7.5
biidobacteria is more susceptible fermented milks or yoghurts. (Abraham et al., 2007).
than lactobacilli. The pH of hard and semi-hard
cheeses, generally above 5.0 after
production, remains relatively
constant through ripening.
Ability to grow Probiotic biidobacteria and Milk is a very nutritive product and 109–1010 cfu g of B. lactis B94 and
in milk lactobacilli of gut origin need to the majority of probiotic strains of L. casei-01 were reported in
be able to grow in milk and raise are able to ferment lactose. curdled matrices throughout 30 d
the number of viable cells through of storage at 12 °C (Rodrigues et
cheese manufacture. al., 2011a).
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(Continued)
129
 130

Table 4.6 (Continued)

Physiological Favourable/U nfavourable

constraint Description cheese characteristic Examples of studied cases
Heat tolerance Manufacturing process of some Temperatures above 40 °C affect Higher viability of biidobacteria
types of cheese involve steps of especially biidobacteria survival due to lower heating temperature
heating to temperatures above 40 in cheese. Modiication on was achieved in Canestrato
Food as Vehicles of Probiotics

°C. manufacture procedures should be Pugliese cheese (Corbo et al.,

accomplished to improve probiotic 2001) .
strains survival.
NaCl NaCl content in cheese inluences Viability of probiotic strains is Salt has been reported as the major
directly water activity, which in limited for NaCl concentrations limiting factor for the growth of
turn affects the microbial growth above 4%. probiotics in white-brined cheeses
and various enzyme activities. (Ӧzer et al., 2009).
Additionally it contributes to the
lavour of cheese.
Ripening Days to weeks at 4 to 12 °C under The refrigeration temperatures High levels of probiotic lactobacilli
high relative humidities (75–85 associated with high levels of or biidobacteria (>108 cfu g–1)
%RH) characterize the ripening humidity help to limited water have been reported in several
process of the majority of cheeses. diffusion from cheese matrix. types of cheese (Table 4.5).
 Physiological Favourable/U nfavourable
constraint Description cheese characteristic Examples of studied cases
Non starter LAB The role of NSLAB is still not totally The NSLAB presence in cheese The addition of L. casei-01 or B.
(NSLAB) with clariied. In ripened cheese, non- could be minimized through lactis as starter, without adjunct
antagonistic pathogenic adventitious bacteria incorporation of high levels of cultures, reached values of
activity (lactobacilli, etc.) can proliferate probiotic as adjuncts, starters or 109–1010 cfu g–1 in 15 to 60 days
and become the dominant both. semi-hard cheese and were the
microlora of cheese. microlora present in the cheese
throughout ripening (Rodrigues et
al., 2011b,c).
Passage through Lactobacilli are mainly acid Dairy products enhance microbial High survival for B. biidum,
gastrointestinal tolerant or aciduric; Isolates of survival in gastric juice due to a L. acidophilus and L. casei in
tract lactobacilli and biidobacteria from buffering or protecting effect that Argentinian Fresco cheese when
harsh environment of the GIT are could be related to milk proteins. subjected to a pH of 3.0 for 3 h
normally more resistant to both Cheese matrix seems to protect at 37°C (Vinderola et al., 2000).
acid and bile. probiotic bacteria in simulated GIT. Probiotic strains in cheese survived
in the simulated upper GIT and
resulted in higher numbers of
lactobacilli in the simulated human
colon (Makelainen et al., 2009).
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131
132 Food as Vehicles of Probiotics

probiotic bacteria in cheese has been the target of several published

reviews. Karimi et al. (2011) discussed the main factors affecting
viability of probiotic bacteria in cheese and categorized them into
three areas, which include formulation, processing, and packaging.
Technological aspects of probiotics were also recently reviewed
by Bhadoria and Mahapatra (2011) updating some information
reported by Cruz et al. (2009a) and Saarela et al. (2000).
It is well known that microorganisms in cheese play essential
roles in their manufacture and ripening processes leading to the de-
velopment of sensorial characteristics through their metabolism and
enzymatic activities; they also have an impact in the microbiological
safety of the product because they produce several low-molecular
weight compounds with antimicrobial activity (Grattepanche et al.,
2008). Phenomena such as glycolysis, proteolysis, and lipolysis are
the main biochemical reactions that take place during cheese rip-
ening and are responsible for the degradation of carbohydrates,
proteins, and lipids present in the curdled milk matrix. These re-
actions occur in a controlled environment as a result of the action
of different enzymes that leads to the production of a wide range
of metabolic compounds that are responsible for the organoleptic
characteristics of cheese during maturation (Azarnia et al., 2006) as
well as to the generation of hurdle factors (organic acids, pH, and so
on) that guarantee the safety of the cheeses.
In terms of gross chemical composition of cheese such as total
protein and fat, salt content, and moisture, these are normally not
inluenced by the added probiotic strains. Variation of pH as well
as the extension of glycolysis. In general cheeses with probiotic
lactobacilli and biidobacteria possess higher acetic acid content due
to heterofermentation (Gomes et al., 1998). According to Desai et
al. (2004), acetic and lactic acid are produced by biidobacteria in a
molar ratio of 2:3; in terms of lactobacilli, they also produce acetic
acid, but not so much as biidobacteria.
Lactose maldigestion can be alleviated by β-galactosidase
activities of probiotic bacteria, and according to Rodrigues et al.
(2011a), lactic acid was signiicantly produced in both L. acidophilus
and B. lactis B94 probiotic matrices and correlated well with lactose
degradation.
According to several authors, the use of probiotic cultures in
cheese does not affect necessarily the primary proteolysis, which
 Dairy Products 133

is mainly derived from activity of the coagulant agent and plasmin

and to a lesser extent from residual coagulant and starter microlora
enzymes through ripening time. Reported results from Rodrigues et
al. (2011a,b) pointed that enzymes from probiotic cultures could also
have an important role in both primary and secondary proteolysis.
Higher increases of water-soluble and nonprotein nitrogen fractions
were observed throughout ripening time than with control matrices,
which were coagulated with animal rennet but noninoculated
(Rodrigues et al., 2011a). In semi-hard probiotic cheeses, there was
a signiicant increase of nonprotein nitrogen fraction in the irst 30
days that was more pronounced for the probiotic cheeses (Rodrigues
et al., 2011b).
Inluences on the secondary proteolysis with increases in free
amino acid content are facts that have been reported in cheeses
containing probiotics-viable cells (Ong et al., 2006, 2007); higher
levels for total amino acids were recorded in probiotic cheese
inoculated with L. casei-01 than those inoculated with B. lactis B94
(Rodrigues et al., 2011b). According to Requena et al. (1993), high
activity of aminopeptidases is a characteristic of L. casei that is
probably responsible for the higher contents of free amino acids in
cheeses inoculated with this bacterium. Peptides and amino acids
inluence cheese lavor and they are potential precursors of lavor
and aroma compounds.
Lipolysis during ripening is a phenomenon that also inluences
the development of the cheese organoleptic characteristics. Several
authors agree in the fact that the addition of probiotic cultures
does not appear to affect the free fatty acid (FFA) proile of cheese,
probably due to a higher lipolytic activity of starters and some
nonstarter lactic acid bacteria (NSLAB) than probiotic cultures
(Gomes et al., 1998b). Interesting results about FFA are reported by
Rodrigues et al. (2011c) in their study about lipolysis in probiotic
cheese and in particular about CLA production. The role of bacterial
lipolytic esterases (lipases) was conirmed, as lipolytic agents in
cheese, as no increase of FFA was observed in noninoculated curdled
matrices over 60 days of ripening at 12°C (Rodrigues et al., 2011a).
According to Rodrigues et al. (2011c), increases in total FFA, three
different CLA, ALA, and γ-linolenic acids were observed during
the ripening period, especially in synbiotic cheeses containing
fructooligosaccharides (FOS) and inulin. Synbiotic food products
134 Food as Vehicles of Probiotics

result from the combination of prebiotics and probiotics; their

combination promotes health in a synergistic manner, offering more
beneicial effects over either probiotics or prebiotics alone. Prebiotics
are known as nondigestible dietary components that can reach the
colon essentially intact; in the colon, they stimulate proliferation
and activity of desirable bacteria in situ such as probiotic bacteria
(Mattila-Sandholm et al., 2002). FOS and inulin are among the most
used prebiotic compounds to formulate synbiotic foods.
The production of new functional cheeses such as synbiotic
cheese with an improved performance (with higher content of CLA)
or with selected mixed cultures of probiotic bacteria providing
several beneicial effects should be the base of the future research;
this process will require the adaptation and reformulation of
manufacturing protocols involving food technologists for the revision
of the traditional manufacture of cheese (Settani and Moschetii,
2010).

4.3 Nondairy Products

4.3.1 Beverages
Alternative functional foods with probiotics and dairy products aside
such as juices are of potential interest (Rodrigues et al., 2012b).
The growing interest in the development of nondairy beverages is
associated with the potential alternative ways to deliver probiotic
strains to lactose-intolerant people with reduced cholesterol
content, which is of interest to many regular consumers. According
to the review by Espírito Santo et al. (2011), the probiotic food with
fruits as ingredients has been indicated as consumers’ predilection.
However, fermented milks are still the preferred product to develop
probiotic beverages. Calcium and vitamin-fortiied juices as
functional drinks are consumed regularly (Bhadoria and Mahapatra,
2011), and therefore, probiotic beverages based on fruit juices are
of utmost interest, as full beneits of probiotics are increasingly
recognized by the regular consumer. Apart from allergy to dairy
products, other factors such as tradition and economic reasons
limiting the consumption of dairy products in countries such as
China or Japan reinforce the need of alternative nondairy products
to deliver probiotic agents (Granato et al., 2010).
 Nondairy Products 135

In several reports, fruit drinks have been indicated as good

probiotic carriers if some care is taken relating to organoleptic
characteristics and pH (Tuorila and Cardello, 2002; Luckow and
Delahunty, 2004; Champagne and Gardner, 2008). Fruits and
vegetables are in general considered as potential matrices because
they are rich in nutrients such as vitamins, dietary ibers, sugars,
minerals, and polyphenols known by its antioxidant properties
(Parkar et al., 2008; Soccol et al., 2010).
Champagne and Gardner (2008) studied the survival of different
lactobacilli in a commercial fruit drink based on a blend of different
fruits concentrates and yoghurt powder with a pH of 4.2, through
80 days at 4°C; strains of L. rhamnosus, L. fermentum, L. reuteri, and
L. plantarum were most resistant through the 80 days of storage,
whereas strains of L. acidophilus showed lower resistance to the
juice matrix. This fact demonstrates that acidic environment of
some fruit juices represents a technological challenge to overcome
(Sheehan et al., 2007), as probiotics should be delivered to the
consumers in adequate concentrations to provide a potential health
beneit. A strategy to increase pH in a fruit juice is blending it with
milk ingredients, as observed in the commercial juice studied
by Champagne and Gardner (2008), which alter the sensory
characteristics of the juice (Suomalainen et al., 2006) and do not
overcome the constraint of lactose and allergy to milk proteins.
The main results from studies of potential probiotic beverages
based on fermented or nonfermented juices that have been reported
between 2006 and 2011 are displayed in Table 4.7. L. paracasei is
the most pointed with potential as a functional supplement in fruit
juices, especially not only in orange, peach, or apple juices, but also
in pineapple juice. Interesting results are reported with fermented
carrot juice with L. rhamnosus and B. lactis. High values of viable cells
in fruit juices, namely between 109 and 1010 cfu/ml, are reported
after 28–50 days of storage at 4°C of carrot, peach, or orange juices.
These results are in fact good evidences that fruit juices have a
potential to deliver adequate concentration of probiotic bacteria to
consumer without the use of dairy additives.
The trends displayed in Table 4.7 agree with those reported by
a review by Espírito Santo et al. (2011) in which it is summarized
that—strains of lactobacilli such as L. casei, L. acidophilus, and
L. rhamnosus and of biiobacteria such as B. lactis are among the
 136
Table 4.7 Potential probiotic beverages based on fermented/non-fermented juices, reported between 2006 and 2011.

Other
Juice Probiotic relevant Storage Viable cell counts (log cfu/
composition strains information conditions mL)/pH Remarks References
Start End
Pomegranate L. plantarum Fermented 4°C 8.6 NA Increments of all Mousavi
L. delbrueckii through 72 h 4 weeks 8.6 NA probiotic strains et al., 2011
L. paracasei at 30 °C 8.5 NA between 24 and
Food as Vehicles of Probiotics

L. acidophilus 8.5 NA 48 h, followed by

a general decrease
until 72 h of
fermentation;
L. plantarum and L.
delbruecki survived
until two weeks of
storage with 105
cfu/mL.
Cashew L. casei Fermented 4°C 8.4/6.4 8.6/3.6–3.8 Signiicant Pereira
apple through 24 h 42 days increment of L. casei et al., 2011
at 30 °C through 24 h
of fermentation;
Cashew apple
juice as potential
probiotic juice.
 Other
Juice Probiotic relevant Storage Viable cell counts (log cfu/
composition strains information conditions mL)/pH Remarks References
Start End
Orange L. paracasei Free cells 5°C 9.2–9.5/±3.6 9.2–9.5/3.7–3.8 L. paracasei 26 Rodrigues
Peach L26 and alginate 50 days 9.5–9.6/2.9–3.4 9.5–10.5/2.9–3.4 with potential et al., 2011e
microcapsules as functional
in juices. supplement in fruit
juices as free or
encapsulated.
Model juice L. rhamnosus A model juice 4°C 8.2/3.8 NA/3.6 Higher survivability Shah et al.,
HN001 was prepared 6 weeks 8.3/3.8 NA/3.4 of all strains 2010
B. lactis with sucrose, 8.4/3.8 NA/3.3 in model juice
HN001 sodium citrate containing grape
L. paracasei and citric extract, tea extract
LPC37 acid. It was or vitamin C; after
studies model 6 weeks 106 to
juice with 107 cfu/mL was
and without observed for the
vitamins and three strains in
antioxidants. model juice with
vitamin C.
Nondairy Products

(Continued)
137
 138

Table 4.7 (Continued)

Other
Juice Probiotic relevant Storage Viable cell counts (log cfu/
composition strains information conditions mL)/pH Remarks References
Start End
Noni L. casei Fermented 4°C NA NA L. casei did not Wang et al.,
Food as Vehicles of Probiotics

L. plantarum through 48 h. 4 weeks exhibited cell 2009

B. longum viability after 3
weeks of storage;
L.plantarum and B.
longum survived
the 4 weeks.
Orange L. rhamnosus Free cells 4°C ±8.0 /2.8 Orange: Encapsulated Ding and
Apple B. longun and alginate 6 weeks 5.0–5.5/2.5–2.8 probiotic bacteria Shah, 2008
L. salivarius microcapsules Apple: survived in fruit
L. plantarum in juices. 5.0–5.5/2.2–2.9 juices throughout
L. acidophilus six weeks whereas
L. paracasei free cells lost their
B. lactis Bi- viability within ive
04/Bi-07 weeks.
 Other
Juice Probiotic relevant Storage Viable cell counts (log cfu/
composition strains information conditions mL)/pH Remarks References
Start End
Carrot B. lactis Bb12 Fermented No storage NA NA 0.7 to 1.7 log incre- Kun et al.,
B. biidum through 48 h ments after 24 h 2008
B7.1 B. at 37°C with- of fermentation
biidum out juice sup- reaching values of
B3.2 plementation. 108–109 cfu/mL.

Carrot L. rhamnosus Fermented 4°C 9.7/3.4–3.6/ 9.2–9.5/3.2–3.3 The presence Nazzaro et

L. bulgaricus through 48 h 4 weeks 9.7/3.5–3.7 9.5/3.3–3.4 of FOS or inulin al., 2008
at 37°C; Study altered neither the
of inluence viable cells of both
of FOS and lactobacilli nor
inulin. the biochemical
characteristics of
fermented juices.

(Continued)
Nondairy Products
139
 140

Table 4.7 (Continued)

Other
Juice Probiotic relevant Storage Viable cell counts (log cfu/
composition strains information conditions mL)/pH Remarks References
Start End
Orange L. salivarius Non- 4°C 7.9/±3.5 NA/±3.5 Strains of L. Sheenan et
Food as Vehicles of Probiotics

Pineaple UCC118 fermented 12 weeks salivarus were al., 2007

Cranberry L. sallvarus juices. 8.2–8.3/±3.5 NA/±3.5 detected only after
UCC500 1 week (103–107
B. lactis Bb- 7.6/±3.5 NA/±3.5 cfu/mL) whereas B.
12 lactis were detected
L. casei DN- 8.0–8.2/±3.5 6.3–7.8/±3.5 after 6 weeks (103–
114001 107 cfu/mL) in both
L. rhamnosus 7.9/±3.5 6.3–7.9/±3.5 juices.
GG L. rhamnosus,
L. paracasei 8.4±3.5 7.4/±3.5 L. casei and
NBFC43338 L. paracasei
demonstrated
good potential
as functional
supplements in
fruit juices.
 Other
Juice Probiotic relevant Storage Viable cell counts (log cfu/
composition strains information conditions mL)/pH Remarks References
Start End
Carrot and L. acidophilus Vegetables NA NA One to two log Rakin et al.,
beetroot NCDO 1748 juices increments after 8 h 2007
enriched with of fermentation.
brewers yeast
No storage.
isolate and
fermented
though 8 h ate
37°C
Orange B. animalis Different 4 and 20°C 7.5–8.4/3.7 20 °C—NA In juices sucrose- Saarela
Grape ssp. lactis forms to 6 weeks 4 °C—NA to protected cells et al., 2006
Passion fruit VTT E-012010 prepare B. ±5.0 survived better
animalis ssp. than skim milk
lactis were protected cells.
tested.
Cabbage L. plantarum Cabbage 4°C ±8.0 7.6 Fermented cabbage Young
C3 juice was 4 weeks juice with some et al., 2006
L. casei A4 fermented ±8.0 NA potential as healthy
L. delbrueckii through 48 h ±8.0 5.6 juice.
Nondairy Products

D7 at 30°C.
Note: NA - Not available
141
142 Food as Vehicles of Probiotics

most used in the formulation of new fruity probiotic products—the

viability of L. acidophilus is variable and dependent on fruit juice,
whereas L. rhamnosus has been pointed as a potential probiotic with
adequate viable counts in the most tested fruit juices.
Other attempts have been reported to increase storage stability
of Biidobacterium strains in low pH fruit juice such as the use of
ultraviolet (UV) mutagenesis combined with a stress step in low pH
to generate acid-resistant strains (Saarela et al., 2011, 2010). Such
recent attempts reveal that other strategies could be interesting to
improve probiotic viability and stability in low pH juice matrices.
The use of microencapsulation to encapsulate probiotic strains
as a way to improve viability and survivability in juices is another
approach studied. It is known that microencapsulation could provide
a favorable micro-environment for the bacteria enabling their
survival throughout processing and storage, and their release in the
appropriate location(s) in the GIT (Weinbreck et al., 2010). According
to Ding and Shah (2008) and Saarela et al. (2006), more knowledge
is still needed to ascertain the potential of encapsulation as a way to
protect probiotic bacteria to organic acids and low pH in fruit juices.
According to Rodrigues et al. (2012b) and Ding and Shah (2008),
encapsulation revealed an eficient way to protect and increase the
survival period of probiotic strains in juices over storage time (Table
4.7). Despite the good evidences that fruit juice could be used as a
potential food to deliver adequate concentration of probiotic strains
to the consumer, according to a review by Prado et al. (2008), there
are not many examples of probiotic fruit juices in the market, most
of them being commercialized in the Northern European countries.
Nowadays, an increased demand from consumers for nondairy
probiotic products has been observed (Granato et al., 2010).
The incorporation of prebiotics such as inulin and FOS has also
been attempted; carrot juice with or without inulin and FOS was
demonstrated suitable for L. rhamnosus and L. bulgaricus (Nazzaro
et al., 2008) with preservation of C-carotene content and antioxidant
activity through 1 month of cold storage. As in carrot juice with B.
lactis and B. biidum strains, a signiicant decrease of carotenoids
was observed (Kun et al., 2008) but not with L. rhamnosus and L.
bulgaricus, this suggests that the consumption of carotenoids is
genera and/or strain dependent with implications on the antioxidant
potential of the fruit juice.
 Nondairy Products 143

Fermented soy beverages, another suitable alternative as

nondairy probiotic product, have been studied and consumed.
Probiotic products based on soy extract mixed with fruit juices are
some of the new generation of foods on market (Champagne and
Gardner, 2008; Granato et al., 2010). Tang et al. (2007) studied the
fermentation of calcium-fortiied soy milk with several strains of
lactobacilli (L. acidophilus, L. casei, L. plantarum, and L. fermentum);
the viability of all strains was high after fermentation and through 14
days of storage at 4°C (>8.5 log cfu/g). According to these authors,
the fermentation of soy milk with the selected probiotic strains
can potentially enhance the calcium bioavailability and bioactive
isolavones. Champagne et al. (2009, 2010) performed studies with
probiotic bacteria in combination with S. thermophilus on fermented
soy beverages. The authors selected L. helveticus and B. longum to
assert the effect of fermentation by pure and mixed cultures on
isolavone and B-vitamin content in the fermented soy beverage
(Champagne et al., 2010); fermentation did not signiicantly improved
B1 or B6 vitamins levels, and the addition of S. thermophilus to L.
helveticus culture resulted in lower bioconversion of soy isolavones.
The authors emphasized the need for careful choice of culture for
fermentation in order to optimize the quality of the inal product.
Functionality of probiotic beverages should also be addressed
once some probiotic strains tolerance to the acidic environment
in juices is conirmed. In addition, in order to consider the fruit
juice a functional food, it will require not only sensory evaluation
toward consumer acceptance but will also need to be validated as a
suitable carrier for delivery of probiotics to consumers in adequate
amounts by performing GIT resistance studies. None of the studies
reported in Table 4.7 performed sensory analysis nor analyzed the
survival in simulated GIT conditions through storage period. Saarela
et al. (2006) and Champagne and Gardiner (2008) performed some
studies related to GIT resistance. According to these authors, the
tested strains in a commercial juice were evaluated for their survival
to the exposure to simulated GIT stresses after 35 days of storage
at 4°C. Their viability in presence of 0.3% bile salts and pancreatic
enzymes was not affected by the storage, but some susceptibility
was observed to exposition at simulated gastric stress.
Although fruit juices based functional beverages with probiotics
are at irst sight appealing to all age groups due to their sensorial
144 Food as Vehicles of Probiotics

characteristics such as freshness, sweetness, and so on and healthy

concept associated, unsuitable aromas or lavors could alter sensory
proile of the juice. Unpleasant aromas and lavors were reported by
Luckow and Delahunty (2004) in juices with L. plantarum. According
to Luckow et al. (2006), who report masking strategies for improving
the sensory quality of probiotic juice, the perceptible off-lavors
caused by probiotics causing malimpression in the consumers
could be masked with addition of tropical fruit juices. Masking some
off-lavors and reinforcing the perception in the consumer about
associated health beneits could be one strategy to lead consumer
preferring the probiotic juice to the conventional juice counterpart.

4.3.2 Other Products

Some lactic acid bacteria with probiotic properties have been used
in meat products for a very long time; however, the application of
these starter cultures in meat has been performed with the main
goal of improving meat safety rather than to produce new functional
food (Khan et al., 2011). The environment found in meat products
can have positive and negative aspects upon probiotic bacteria: on
the one hand, the fermented meat is not usually exposed to high
temperatures and it has been reported that matrix components
seem to protect the bacteria from passage through GIT (Klingberg
and Budde, 2006); on the other hand, the presence of inhibitors
such as high levels of salt, some antimicrobials from seasonings,
acidic pH, and lower water activity as a consequence of drying and/
or smoking can create adverse conditions and limit the survival of
these strains (Khan et al., 2011). So, in general, before application of
probiotic starter culture, a screening of survival should be performed
to assure that bacteria are not inhibited and can be adapted to the
speciic milieu.
Several probiotic strains have been tentatively introduced in
meat products resulting in several positive effects, in particular
on the technological and organoleptical characteristics of the inal
product as well as on the reduction of pathogen growth and related
toxin production, providing a probiotic product with additional food
safety properties (De Vuyst et al., 2008). Some of these studies are
described in Table 4.8, highlighting the probiotic strain used and
type of meat product produced.
 Nondairy Products 145

Table 4.8 Some examples of other food products containing probiotic

bacteria.

Probiotic starter Food product type Reference

culture
L. rhamnosus FERM
P-15120
Meat sausage of lean
L. paracasei ssp. Sameshima et al., 1998
pork
paracasei
FERMP-15121
L. fermentum HL57 Traditional Iberian Ruiz-Moyano et al.,
P. acidilactici dry-fermented 2011
sausages
L. reuteri Dry- fermented Muthukumarasamy
B. longum sausages and Holley 2006; 2007
L. rhamnosus E-97800,
Erkkila et al., 2001;
L. rhamnosus LC-705 Dry sausages
2003
L. plantarum ALC01
L. acidophilus Pidcock et al., 2003
LAFTI™L10
L. paracasei 5119 Hungarian salami
Lactobacillus ssp. L24
B. lactis LAFTI™B94
L. helveticus RO52 Sremska type of Radulovi et al., 2011
B. longum RO175 fermented sausages
L. casei OCK 0900 Dry-cured pork loins Stadnik and
Spanish non- Dolatowski, 2012
L. casei CECT 475 + fermented dry-cured Sayas-Barbera et al.,
orange iber sausage (Longaniza de 2012
Pascua)
Bc. subtilis var. Natto Natto–a fermented Cutting, 2011
soybean
Bc. subtilis CS90 Soybean fermented Cho et al., 2011
food cheonggukjang
L. acidophilus LA-2 Soy bar with improved Chen and Mustapha,
soy oligosaccharides 2012
digestibility

(Continued)
146 Food as Vehicles of Probiotics

Table 4.8 (Continued)

Probiotic starter Food product type Reference

culture
L. acidophilus MJLA1
B. lactis
L. rhamnosus Non-fermented frozen Heenan et al., .2004
L. paracasei ssp. vegetarian dessert
paracasei
L. casei ssp. rhamnosus Probiotics dried apple Betoret et al., 2003
L. acidophilus La-5 Probiotic strawberry Moayednia et al., 2010
L. rhamnosus GG Synbiotic fresh-cut Rößle et al., 2010
apple wedges
L. helveticus CNCM Dark and milk Possemiers et al., 2010
I-1722 chocolates
B. longum CNCMI- Probiotic and
3470 synbiotic (inulin) Aragon-Alegro et al.,
L. paracasei ssp. chocolate mousse
paracasei LBC 82 2007

L. casei Lc-01 Synbiotic (starch) ice Homayouni et al., 2008

B. lactis Bb12 cream

Sameshima et al. (1998) incorporated three selected potential

probiotics in fermented pork sausage at 107 cfu/g, but only two
of them showed to be applicable to meat fermentation to assure
product safety, namely L. rhamnosus FERM P-15120 and L. paracasei
ssp. paracasei FERM P-15121, which satisfactorily inhibited the
growth and enterotoxin production of Staphylococcus aureus. In
addition, these strains also accomplish technological requirements,
such as fermentation time and acid production, assuring their use
as a starter culture to produce new fermented meat products. Other
strains have also been used for pathogen inactivation, namely L.
reuteri ATCC 55730 and B. longum ATCC 15708, which promote the
inactivation of E. coli O157:H7 throughout sausage manufacturing
(Muthukumarasamy and Holley, 2007).
Probiotic or bioprotective L. rhamnosus strains GG, LC-705
and E-97800 as well as Pediococcus pentosaceus E-90390 and L.
plantarum E-98098 have been studied in order to know about their
ability as main fermenting organisms in the manufacturing process
of dry sausages (Erkkilä et al., 2001). The sensorial properties in
 Nondairy Products 147

probiotic sausages were similar to those produced by the commercial

meat starter culture, except for the L. rhamnosus strains GG LC-705
that reduced inal quality; L. rhamnosus E-97800, L. rhamnosus LC-
705 as well as L. plantarum ALC01 conveyed additional antilisterial
activity at an early stage of the ripening process (Erkkilä et al.,
2003). Another study developed by Pidcock et al. (2003) reported
that L. paracasei LAFTI™L26 and B. lactis LAFTI™B94 in combination
with a traditional meat starter culture did not developed negative
effects on the meat sensory properties. The same authors showed
that L. acidophilus LAFTI™L10, L. paracasei 5119, Lactobacillus spp.
L24, and B. lactis LAFTI™B94 could be used as a way to increase
Hungarian salami safety because a strong inhibition of both E. coli
O111 and Listeria monocytogenes was observed by these cultures.
Recently, Ruiz-Moyano et al. (2011) have studied the use of L.
fermentum HL57 and Pediococcus acidilactici SP979 as potential
probiotics to be used in the manufacture of traditional Iberian dry-
fermented sausages. Although survival was assured for both strains,
Iberian dry-fermented sausages with P. acidilactici SP979 guaranteed
the production of an equilibrated product from the sensory point
of view, though higher amounts of acetic acid and lipid degradation
by L. fermentum HL 57 were observed, leading to a negative impact
on the color and taste, conirming that although several probiotic
strains can survive at recommended levels (>107 cfu/g), not all can
assure the expected quality of product.
Three variants of Sremska type of fermented sausages were
produced by Radulović et al. (2011) using starter culture Bactoferm
T-SPX (Chr. Hansen): (1) control sausage variant; (2) variant with
L. helveticus RO52 (Lallemand, France); (3) variant with B. longum
RO175. The probiotic L. helveticus RO52 and B. longum RO175 were
shown to be adequate for the production of dry sausages. During
the irst ripening stage, the bacterial counts were 106 cfu/g, after
which they increased to the level of 108 cfu/g; afterward, they
remained constant until the end of ripening period. The strains did
not induce a decrease in sensory quality; nonetheless, slightly better
aroma, taste, and texture were detected in sausages produced with
L. helveticus RO52.
The combination of probiotic strains with iber as a synbiotic
product was also reported. Sayas-Barberá et al. (2012) combined
the use of L. casei CECT 475 and orange iber to produce Spanish
nonfermented dry-cured sausage (Longaniza de Pascua),
148 Food as Vehicles of Probiotics

demonstrating that probiotic strain accelerates the curing process

increasing organic acids release, establishing a possible starter
culture. The incorporation of 1% orange iber enhances the growth
and survival of lactobacilli and micrococci. This probiotic product
may explore several beneits, in particular if proper ibers capable of
reducing residual nitrite are added.
The safety of probiotic meat products has also been considered
mainly concerning the ability of biogenic amine production by these
strains. Stadnik and Dolatowski (2012) compared the contents in
4, 8, and 16-month-old samples of dry-cured pork loins inoculated
with L. casei ŁOCK 0900 and showed that histamine and spermidine
were not detected, and spermine was detected at very low levels,
while cadaverine and tryptamin were the most abundant biogenic
amines, however below the suggested toxic limits.
Microencapsulation of probiotics for incorporation in meat
products as a means of strain protection has also been tested in a
study by Muthukumarasamy and Holley (2006, 2007). They reported
that microencapsulation increased survival of L. reuteri and B.
longum, maintain sensory properties, but reduced their inhibitory
action against E. coli O157:H7.
Human studies conirming the eventual functionality of
probiotic-fermented sausages are residual up to now. De Vuyst et al.
(2008) described that the daily consumption of 50 g of probiotic (L.
paracasei LTH 2579) sausage only resulted moderately successful,
indicating a signiicant increase of L. paracasei LTH only in some of
the volunteers and exhibiting only modulation of host immunity, but
with no signiicant inluence on cholesterol and triacylglycerides
serum concentrations. Hence, more research is still required,
particularly in terms of human studies, if new probiotics and
probiotic fermented sausages are sought.
Other probiotic food products include those with dried probiotics
(e.g., breakfast cereals, infant formulas, and dry milk formulations
with dried fruit or with high-lipid contents, such as chocolate). In
some of these products, microencapsulation has been reported as
an approach to improve the viability, namely in products with harsh
environment, viz. probiotics in freeze-dried yogurt, in spray-dried
milk powder, or in spray-dried kudzu powder, a starch derived from
roots of Puerari lobata, which has traditionally been used as a food
ingredient in East Asia.
 Nondairy Products 149

Some dried fruits have been used to incorporate probiotic

bacteria. Some years ago, Betoret et al. (2003) developed a probiotic
dried apple by applying the vacuum impregnation process. They
impregnated either orange juice with Saccharomyces cerevisiae and
with whole milk or apple juice containing 107 or 108 cfu/ml of L.
casei ssp. rhamnosus. The stability of probiotic fruit was obtained by
air drying at 40°C and stored at room temperature for 60 days. The
recommended probiotic level was achieved (107 cfu/g) throughout
storage period.
More recently, Roßle et al. (2011) developed potentially synbiotic
fresh-cut apple wedges by applying probiotic L. rhamnosus GG and
prebiotics such as oligofructose and inulin. The wedges were dipped
in probiotic solution, and the prebiotic carrier used was alginate
assuring a homogeneous layer on fruit surface. Organoleptic
properties were almost not changed by applying the functional
ingredients, assuring similar quality to apple slices currently
available. All samples attained ca. 108 cfu/g over the test period,
which is suficient for a probiotic effect. The alginate coating positively
affected the stability of polyphenols and was able to preserve apple
volatiles slightly better than uncoated apple wedges. Moayednia et
al. (2010) reported on probiotic fortiication of strawberry fruit,
combining encapsulated probiotics and fruit-coating techniques,
namely dipping the fruit into sodium alginate solution (2%, w/v)
containing probiotic and after a short dripping off the residual
solution submerging them in a solution of calcium chloride. Other
vegetable materials (artichokes, cabbage, table olives, tomato, red
beet, carrot juice), mainly in fermented products, have been used
as carriers and sources of potentially probiotic lactic acid bacteria
and were extensively reviewed by Peres et al. (2012). Among plant
materials, soybean has been quite explored as matrix for probiotic
delivery. As a soybean probiotic product (fermented soybean), Natto
is normally consumed either hot or cold. In this example, it is sold
as a snack with dried soybeans coated with a ine white powder of
Bacillus subtilis var. Natto, the active ingredient required for the taste
and texture of Natto. However, the health beneits associated with
Natto imply the consumption of soybeans and bacteria, rather than
just the bacterium. Bc. subtilis var. Natto carries as many as 108 viable
spores per gram of product, and for decades, health beneits have
been associated with consumption of Natto including stimulation
of the immune system (Cutting, 2011). Another fermented soybean
150 Food as Vehicles of Probiotics

probiotic product was studied by Cho et al. (2011), in which they

attained the fermentation of soybean fermented food cheonggukjang
by a potential probiotic Bc. subtilis CS90. They reported that this
strain induced changes in 25 phytochemical contents, including
isolavones, lavonols, and phenolic acids during the fermentation,
increasing phenolic compounds and consequently antioxidant
activity. The inal probiotic cheonggukjang extract may be used as a
basis for possible commercial production of functional foods in the
future.
Chen and Mustapha (2012) introduced a functional probiotic
strain (L. acidophilus LA-2) into a soy bar product aiming a probiotic
bar with in vivo enzymatic hydrolysis of soy oligosaccharides
(mainly α-galactosides), promoting the digestibility of those
oligosaccharides. In order to maintain probiotic strain with high
viability in the product, the cells were microencapsulated and
freeze-dried using the combination of k-carrageenan and inulin
as an optimal formulation at a proportion of 1.9:0.1 (w:w) as
capsule wall materials. The probiotic soy bar showed high levels of
viable cells throughout a 14-week storage at 4°C with high level of
α-galactosidase activity, improving product digestibility.
Another soy-based product, soy low-fat frozen dessert, similar
to ice cream, was developed by Heenan et al. (2004) and proved to
be a suitable product for the delivery of bacterial probiotics with
good sensory appeal. They incorporated several probiotic strains
(L. acidophilus MJLA1, B. lactis, L. rhamnosus, and L. paracasei ssp.
paracasei), which grow well in this matrix.
Possemiers et al. (2010) proposed both dark and milk chocolates
as food vectors for oral delivery of a microencapsulated mixture
of L. helveticus CNCM I-1722 and B. longum CNCMI-3470. Both
chocolates offered higher protection than plain milk (91% and
80% survival in milk chocolate for L. helveticus and B. longum,
respectively, against 20% and 31%). The coating of the probiotics in
chocolate may constitute an excellent strategy to enable protection
of these probiotics from environmental stress conditions, opening
the opportunity toward new balanced matrices. Furthermore,
chocolate mousse, an aerated dairy dessert, has also shown a great
market potential, demonstrating high viability of the probiotic under
the storage conditions during 21 days with prebiotic ingredients
(Aragon-Alegro et al., 2007).
 Conclusion 151

Ice cream and frozen desserts based on dairy or fruit pulp or

juice are another group of food products that have demonstrated
potential as probiotic culture carrier for the consumers (Ranadheera
et al., 2010). Due to the lower storage temperatures, the ice cream is
considered favorable for viability of probiotic strains, provided that
damage by freezing and thawing, mechanical stresses of mixing and
by oxygen incorporation during ice cream manufacture is minimized,
as they can lead to decrease of probiotic viable cells at the moment
of consumption (Cruz et al., 2009b). As in general ice cream is a well
accepted and desirable food product especially for children, it has
a great potential for delivering probiotics. A review on ice cream
as a probiotic carrier was published by Cruz et al. (2009b). More
recent studies on probiotic ice cream focus on the use of prebiotic
compounds such as inulin and starch and microencapsulated cells
(Akin et al., 2007; Homayouni et al., 2008). The addition of inulin
improved viscosity and stimulated the growth of L. acidophilus and
B. lactis resulting in higher viability in ice cream with no effect on
its sensory properties (Akin et al., 2007). According to Homayouni
et al. (2008), encapsulation increased signiicantly the survival rate
of probiotic bacteria in the ice cream over storage for 180 days at
–20°C.

4.4 Conclusions
The demand for functional foods has been increased over the last
years. These foods, including probiotics and prebiotics as biologically
active components, produce metabolic and physiological health
beneits apart from their nutritional properties. The most common
probiotic strains found in functional foods belong to Lactobacillus
and Biidobacterium genera. Nowadays, a phletora of probiotic
food products are available in the market, some of them including
prebiotic compounds that demonstrate the interest and potential
of these products. The dairy-based products dominate the most
consumed probiotic products (mostly fermented milks and cheeses)
over the world, but beverages (with or without dairy components)
or other products, such as meat products, dry bars, and so on, have
been researched and some of them are already found in the market.
The most challenging aspects to overcome by using food as
vehicles of probiotics are indeed to assure their viability through
152 Food as Vehicles of Probiotics

the food processing, storage, and resistance to GIT as well as to

control their repercussions on organoleptic properties of the
inal food product. Several approaches have been attempted to
increase levels of survival and viability of probiotic strains taking
into account minimization of off-lavors. The use of prebiotic
compounds, microencapsulation techniques, and so on are some of
the ones that were discussed and have the potential to increase the
offer of different food products containing viable cells of probiotic
in adequate amounts to produce beneicial effects in the host. It is
known that viability and metabolic activity of probiotic strains on
food product are both dependent on strain itself and on product
composition. This interrelationship should be considered under the
technological point of view considering the main parameters that
inluence the inal product.

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Chapter 5

Immobilization and Microencapsulation

of Probiotics

Paulo J. C. Costa,a Teresa Rocha-Santos,b,c Ana M. Gomes,d

Manuela M. Pintado,d Sérgio Sousa,d Maria H. Amaral,a
J. Paulo Sousa e Silva,a and Ana C. Freitasb,c
aFaculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira,

228, 4050-313 Porto, Portugal

bISEIT/Viseu-Instituto Piaget, Estrada do Alto do Gaio, Galifonge, 3515-776 Lordosa,

Viseu, Portugal
cCESAM & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
dCBQF, Biotechnology School of Portuguese Catholic University,

Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal

pccosta@ff.up.pt

5.1 Introduction
The preparation of microparticles is in great development in several
industrial areas, especially in the pharmaceutical and food industries.
In recent years, the food industry has been incorporating probiotics
in microparticles in order to develop new and more diversiied
functional foods that could cause an enhancement in health state of
consumer. Probiotics have also been encapsulated for incorporation
on pharmaceutical dosage forms.

Probiotic Bacteria: Fundamentals, Therapy, and Technological Aspects

Edited by J. Paulo Sousa e Silva and Ana C. Freitas
Copyright © 2014 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4411-62-2 (Hardcover), 978-981-4411-63-9 (eBook)
www.panstanford.com
172 Immobilization and Microencapsulation of Probiotics

The terms immobilization and encapsulation are used as

synonyms in most of the scientiic literature. Although encapsulation
is the process of forming a continuous coating around an internal
matrix that is fully contained within the wall of the capsule,
immobilization refers to the capture of material within or along a
matrix (Kailasapathy 2002, Vidhyalakshmi et al. 2009). Therefore, a
small percentage of the material ixed can be exposed at the surface,
which is not the case for encapsulated material (King Alan 1995,
Kailasapathy 2002). For the sake of systematization, we will use the
term encapsulation in a more comprehensive way, also including the
concept of immobilization.
The substance that is encapsulated may be called the core
material, ill, internal phase, or payload phase and the substance
that is encapsulating may be called the coating, membrane, shell,
carrier material, wall material, or external phase (Barbosa-Cánovas
et al. 2005, Augustin and Sanguansri 2008, Zuidam and Shimoni
2010). All the raw material used for encapsulation intended for food
products or processes should be natural food components or other
ingredients that have GRAS (Generally Regarded As Safe) status
(Barbosa-Cánovas et al. 2005, Augustin and Sanguansri 2008) or be
food-grade materials (Zuidam and Shimoni 2010).
The different current techniques of encapsulation make it
possible to obtain particles with diameters from a few nanometres
to a few millimetres. Generally, it is considered that the process of
microencapsulation produces solid particles with a size varying
between 1 μm and 1000 μm (Heinzen et al. 2002, Burgess and Hickey
2006, Kinam and Yoon 2006). Other authors consider a different
range of sizes (Arshady 1989, Gibbs et al. 1999, Desai and Park 2005,
Chen and Chen 2007, Vidhyalakshmi et al. 2009).
The technology associated with the encapsulation of drugs,
colorings, lavoring and aromatic substances, bacteria, and so on is
vast. Among these technologies, the polymeric matrix systems are
widely applied in the form of microparticles. The microparticles
are subdivided according to their structure, in reservoir type
(microcapsules) and in matrix type (microspheres) and other
combinations of these main types (Arshady 1989, Gibbs et al. 1999,
Barbosa-Cánovas et al. 2005). They are called microspheres if they
are compact particles consisting of a network in which the substance
is homogeneously distributed in its solid or molecular state. They
are called microcapsules if they are particles composed of an inner
 Introduction 173

core containing the bioactive agent covered with a layer of varying

thickness (Fig. 5.1).

Figure 5.1 Some forms of microparticles. (A) Matrix type (microsphere);

(B) Regular reservoir type (plain microcapsule); (C) Regular
reservoir type (irregular microcapsule); (D) Multi-wall
reservoir type (microcapsule); (E) Multi-core reservoir type
(microcapsule). Adapted from Arshady (1989) and Gibbs et al.
(1999).

Microencapsulation can be deined as a technology of packaging

solids, liquids, or gaseous materials in miniature sealed capsules that
can release their contents at controlled rates under the inluence of
speciic conditions (Nixon 1985, Anal and Singh 2007, Champagne
and Fustier 2007, Rokka and Rantamäki 2010).
In a general way, we can classify the objectives of encapsulation
into ive categories: immobilization, protection, controlled release,
structuration and functionalization of active ingredients (Poncelet
2006).
The irst records of attempts of application of this technique dates
from the years 1930s, but the irst product with microencapsulated
material only came in 1954 (Dziezak 1988, Burgess and Hickey
2006, Kinam and Yoon 2006). The American company National Cash
Register (NCR) pioneered when it developed a carbonless copy paper
(Green 1953). This paper received a thin layer of microcapsules of a
suitable pigment. The pressure from the tip of the pencil on the paper
surface broke the microcapsules, releasing the pigment, which, by
direct contact with the acid coating applied to the front surface of the
second sheet, changed color depending on the pH, allowing to obtain
174 Immobilization and Microencapsulation of Probiotics

a copy (Green 1955). In the food industry, the irst researches took
place from the late 1950s (Gouin 2004). Lactic acid bacteria (LAB)
was irst immobilized on Berl saddles in 1975 and later Lactobacillus
lactis was encapsulated in calcium alginate beads (Gibbs et al. 1999,
Desai and Park 2005). In the pharmaceutical industry, the early
research also occurred in the same period. In this ield, there was an
important contribution because microencapsulation permitted the
development of controlled-release formulas, that is, those with the
capacity to release active agents in the organs where they shall act
only or where they will be absorbed (Suave et al. 2006).
The microparticles continue to be employed in different sectors
of industry in the present day, performing a variety of functions. The
protection afforded by the wall of polymeric microparticles increases
the lifespan of a volatile compound, extending the shelf time of many
food products and cosmetics (Leimann 2008). In the pharmaceutical
industry, the applications are very varied: concealment of lavors
or odors, converting liquids into solids, protection in relation to
atmospheric agents (light, heat and humidity, and/or oxidation),
reduction or elimination of gastric irritation or secondary effects
caused by certain drugs, reducing volatility, production of modiied-
release dosage forms, and so on (Kas and Oner 2000, Yoshizawa
2004, Burgess and Hickey 2006, Jyothi et al. 2010).
The use of microparticles in the food industry is also of great
importance, because it increases the stability of oils, lavorings,
vitamins, and so on (Leimann 2008). In this area, they have the ability
to reduce the reactivity of the core material with the environment;
decrease the rate of evaporation or the transfer of the core material
toward the outer space; facilitate the encapsulated material handling;
promote the controlled release of some substances; mask unpleasant
odor and lavor; and promote homogeneous dilution of the material
encapsulated in a food matrix (Favaro-Trindade et al. 2008).
Probiotic encapsulation was developed from the technology
involved in cell culture (immobilized cell culture technology, ICT)
(Heidebach et al. 2012). Probiotics have two limitations regarding
encapsulation, namely the size of the microorganisms (with
diameters typically between 1 and 5 μm), which prevents the use
of nanotechnology, and the fact that the cells must be kept viable
(Burgain et al. 2011). This aspect has been crucial to the choice
of encapsulation technology (Champagne and Fustier 2007). The
main purpose of the encapsulation of probiotics is to increase
 Microencapsulation 175

viability of these products during its shelf life, protect cells against
an unfavorable environment that occurs in food matrices and in the
gastrointestinal tract, and to enable their release in a viable and
metabolically active state in the gut (Sun and Grifiths 2000, Picot
and Lacroix 2004, Burgain et al. 2011, Nazzaro et al. 2012).
Different approaches have been proposed to increase the
resistance of these sensitive microorganisms against adverse
conditions (Anal and Singh 2007) such as an appropriate selection
of acid and bile-resistant strains, the use of oxygen-impermeable
containers, a two-step fermentation, a stress adaptation, an
incorporation of micronutrients such as peptides and amino acids,
protectors such as sugars and oligosaccharides, and, as previously
referred, the usage of microencapsulation.
After a successful encapsulation, the inner bioactive material
should be released to the outside, where it might implement its
beneicial action. There are four typical mechanisms by which the
core material can be released from a microparticle: mechanical
rupture of the capsule wall, dissolution of the wall, melting of the wall,
and diffusion through the wall. Less common release mechanisms
include ablation (slow erosion of the shell) and biodegradation
(Franjione and Vasishtha 1995, Barbosa-Cánovas et al. 2005).

5.2 Microencapsulation
Several procedures from many ields of application with different
goals led to a great number of encapsulation methods (Dziezak 1988,
Arshady 1989, Dulieu et al. 1999, Gibbs et al. 1999, Heinzen et al.
2002, Gouin 2004, Anal and Singh 2007). The terminology employed
varies between the different scientiic domains. Therefore, the same
technology may have different names in different ields.
The selection of the microencapsulation method depends on
the properties of the core and the coating materials, the size and
morphology of the particle, and the release mechanism desired. Many
of these processes have been adapted from the pharmaceutical and
chemical industries. The use of low-cost materials and manufacturing
processes is very important because food products generally have
lower proit margins than pharmaceutical products (Augustin and
Sanguansri 2008). Generally, microencapsulation can be divided
into three main stages (Poncelet 2006, Burgain et al. 2011) as shown
in Fig. 5.2.
176 Immobilization and Microencapsulation of Probiotics

Figure 5.2 General plan describing the three main phases of microparticles
production. Adapted from Poncelet (2006) and Burgain et al.
(2011).

The incorporation of the bioactive compound in a solid or liquid

phase is a prior step to microencapsulation itself, but is always
necessary. If this phase is liquid, this step may involve processes
of mixing or dispersion. If this phase is solid, this step may involve
processes of agglomeration, adsorption, drying, and so on.
The second step, division into smaller particles, consists
of mechanical operations: for a liquid system, it is dispersed
in air (dropping or spraying) or in another immiscible liquid
(emulsiication); for a solid system, the division can be done by
grinding and/or sieving; after that, a solution is sprayed on the
microparticles in agitation (luid bed or pan coating) until a good
and uniform coating is obtained.
The third step consists in the stabilization of the microparticles
formed in the previous phase, by chemical, physicochemical, or
physical process (Heinzen et al. 2002, Poncelet 2006).

5.2.1 Encapsulating Materials

Several materials have been used for the encapsulation of probiotic
bacteria, including alginate, gums (gellan gum and xanthan gum),
k-carrageenan, cellulose acetate phthalate, chitosan, starch, gelatine,
milk proteins, whey proteins, and many others (Table 5.1) (de Vos
et al. 2010, Rokka and Rantamäki 2010, Ain Riaz and Masud 2011,
 Microencapsulation 177

Burgain et al. 2011, Rodrigues et al. 2011, Huq et al. 2012, Sousa et
al. 2012).
The choice of the encapsulate agent depends on a number of
factors, among them, the nonreactivity with the material to be
encapsulated, the process used for the microparticle formation, and
release mechanism are the most relevant. The ideal encapsulant must
present low viscosity at high concentrations and be easy to handle
during the process; have low hygroscopicity, for ease of handling and
preventing agglomeration; have the ability to seal and hold the active
material within the structure of the capsule; provide maximum
protection to the active material against adverse conditions,
such as light, pH, oxygen, and reactive ingredients; be soluble in
solvents commonly used; have the desired release properties of
the active material; not have an unpleasant lavor in the case of oral
consumption; and be costeffective (Shahidi and Han 1993, Barbosa-
Cánovas et al. 2005, Desai and Park 2005). Biodegradable polymers
and blends of biodegradable polymers natural or synthetic are often
used in controlled release of drugs (Cerini 2001).

Table 5.1 Commonly used coating materials for food applications

Material class Example

Carbohydrates Starch and starch products, (dextrins, natural starches,
modiied starches) Sugars (fructose, glucose, maltose
sucrose, corn syrup)
Celluloses (carboximethyl cellulose,
hydroxymethylpropyl cellulose, cellulose acetate
phthalate)
Chitosan
Gums (gellan gum, xanthan gum arabic gum, alginates,
carrageenan, agar, pectin)
Cyclodextrins
Lipids Waxes (beeswax, carnauba wax)
Monoglycerides and diglycerides
Natural fats and oils
Phospholipids
Hydrogenated fats
Proteins Peptides, gluten, casein, gelatine, albumin, and other
proteins
Source: Adapted from Augustin and Sanguansri (2008)
178 Immobilization and Microencapsulation of Probiotics

5.3 Methods of Microencapsulation

The objective of this section is to provide a short review of
commonly used methods of microencapsulation and is certainly not
a complete list. More details about these processes can be found in
the references.
Microencapsulation itself can be divided into two main stages,
including the stage of division into smaller particles and the stage of
stabilization of the particles formed.

5.3.1 Microparticle Formation

The irst stage of microencapsulation is the division of probiotic-
containing phase into small particles with diameters in the
micrometres order. The formation of the microparticles depends
on the liquid or solid state of the dispersing system containing the
active substance. So, we can have liquid matrix dispersion (extrusion,
spraying, emulsiication) or several solid matrix techniques (air
suspension coating, pan coating).

5.3.1.1 Liquid matrix dispersion

The dispersion of the liquid matrix can be done in air or in another
liquid. If done in air, it can be made simply by the action of gravity,
by dripping, giving rise to a prill, or by atomization (or spraying).
Dripping is the simplest technology for production of microparticles.
Usually by dripping, the particles formed are very large (2–5 mm
diameter) and consequently nonsuitable for many biotechnological
or medical applications, and it is therefore necessary to reduce the
size of the particles using auxiliary techniques (Heinzen et al. 2002,
Burgess and Hickey 2006).
A prill is a small aggregate of a material, most often a solid sphere,
formed from a melted substance. The material to be prilled must be
a low-viscosity liquid when melted and a solid at room temperature.
It is formed by allowing the drops of the melted substance to solidify
(congeal, freeze, or coagulate).
The atomization usually forms an aerosol that is a dynamic set
of very small liquid droplets or solid particles dispersed in a gas.
If the dispersion of the liquid matrix is made in another liquid, the
technique used is the emulsiication.
 Methods of Microencapsulation 179

5.3.1.1.1 Extrusion
The extrusion can be deined as a forced passage through an opening
(or openings) giving the substance a deined format. One of the most
important features in this procedure is the size of the hole that will
require a higher or lower division of the material to extrude. The
smaller is the inner diameter of the opening (nozzle), the smaller
are the particles.
This technique has been used for many years in the food industry.
The work that originally led to the extrusion/encapsulation process
was done by Schultz et al. of the United States Department of
Agriculture Albany Laboratory (Schultz et al. 1956). Encapsulation
of lavors via extrusion was irst patented in 1957 (Swisher 1957).
The main referred advantage of the extrusion method referred is the
increased stability of lavors or other heat-labile food components,
because in this method, it is not necessary to use high temperatures
(Dziezak 1988, Gibbs et al. 1999, Madene et al. 2006). The
extrusion method is one of the most used techniques for probiotic
encapsulation for food applications (Heidebach et al. 2012). This
category of extrusion technologies presents generally no risks but
sometimes is a very complex technique for microencapsulation (de
Vos et al. 2010).
The probiotic encapsulation by extrusion thus consists in forcing
the liquid dispersion containing the cells through a nozzle using
high pressure at a low temperature. If the extrusion occurs slowly,
droplets that will be formed will fall when the action of gravity
exceeds the surface tension. If the extrusion occurs faster, a jet of
liquid will be formed. If the extrusion (droplet formation) occurs
in a controlled mode (as opposed to atomization—spraying), the
technique is known as prilling (Heinzen 2002, Kailasapathy 2002,
Burgain et al. 2011). Prilling can be a very useful technology if the
solidiication of the exterior material could occur in an instantaneous
way (Meiners 2009). If, as already mentioned, the extrusion is done
only by the action of gravity, the particles formed are large and it is
therefore necessary to reduce the size of the droplets using auxiliary
techniques.
The preparation of microparticles using the extrusion process is
well documented (Gibbs et al. 1999, Krasaekoopt et al. 2003, Gouin
2004, Madene et al. 2006, de Vos et al. 2010, Kuang et al. 2010, Rokka
and Rantamäki 2010, Burgain et al. 2011, Matalanis et al. 2011,
180 Immobilization and Microencapsulation of Probiotics

Nazzaro et al. 2012), but in general, the microparticles obtained are in

a range of few hundred micrometers. In most applications involving
immobilization of living cells, the size of microparticles needs to be
small (lower than 1 mm) and carefully controlled. The reason for
this is mostly because of diffusion limitations of nutrients within the
matrix constituting the microparticle. For example, alginate beads
made by the extrusion technique generally vary in size from 2 mm
to 4 mm (Rokka and Rantamäki 2010). In general, extrusion gives
a small range size, but it does not provide particles under 300 μm
(Burgain et al. 2011). In order to get microparticles by extrusion
below the mouth feel detection limit, referred to be 100 μm (Annan
et al. 2007), some modiications in these techniques are necessary in
order to reduce the size of the of the droplets.
Some research groups have often the incorrect assumption that
this type of procedure does not allow for the large-scale production
and is only suitable for lab-scale processes. However, huge advances
have been made in scaling of encapsulation processes using extrusion
technologies (de Vos et al. 2010) allowing large-scale microparticles
production.
One of these modiications is the use of jet-based methods. These
approaches have been demonstrated to be most economical and
versatile on the basis of the ability to handle a wide range of materials
(from micro/nano to living cellular particles) (Jayasinghe and Suter
2006). Among these methods, we can cite some very promising
ones such as the electrostatic technique, the coaxial low jetting,
the aerodynamically assisted jetting (AAJ), the nozzle resonances
technology, the spinning disk technology, and so on. (Heinzen et al.
2002, Poncelet 2006, Prüsse et al. 2008, de Vos et al. 2010, Sousa et
al. 2012).

5.3.1.1.1.1 Electrostatic generators

Amsden and Goosen (1997) developed a method on the basis of the
extrusion of a liquid through a needle with an electric ield applied.
By applying an electrostatic potential in the falling drop (Fig. 5.3),
electrical charges accumulate on its surface creating a repulsion
that opposes the surface tension (Poncelet et al. 1999, Poncelet et
al. 1999, Mattheus et al. 2005, Prüsse et al. 2008). Generation of
microparticles using this equipment is based on the formation of a
single droplet at the top of the nozzle.
 Methods of Microencapsulation 181

Figure 5.3 Principle of electrostatic generators.

A commercially available equipment is the Nisco Encapsulation

Unit—Var V1 (Nisco V1 2009–2011). This equipment mainly
consists of a high-voltage power unit 0–10 kV, a switch for tuning
of the voltage magnitude, a needle holder, and a safety cage. Other
setups have been developed by some research groups using different
applied voltage such as 6.5–7.5 kV (Manojlovic et al. 2006), 0–25 kV
(Prüsse et al. 2008), and 0–30 kV (Mattheus et al. 2005). The liquid
dispersion can be fed via an infusion pump. A shaker keeps the bath,
where solidiication occurs, in agitation to prevent the union of the
microparticles formed.
The diameter of microparticles produced is governed principally
by the nozzle diameter, voltage, and distance between needle and
solution (Amsden and Goosen 1997, Prüsse et al. 2008, Teoh et al.
2011). It is possible to obtain small sizes microparticles less than
20 μm for high voltages (10 kV). The potentials used are between
10 and 25 kV (Heinzen et al. 2002). The major disadvantage of this
technique is the very low low rate and is, nevertheless, inherently
limited to small yield, as drops are only formed one after the other.
Therefore, it is only used for the production of small batches (Senuma
et al. 2000, Heinzen et al. 2002, Poncelet 2006, Zuidam and Shimoni
2010).
The Nisco instrument is designed for research uses, that is, for
production of smaller quantities of spherical alginate beads ranging
in size from around 200 μm to 1000 μm (Nisco V1 2009–2011).
182 Immobilization and Microencapsulation of Probiotics

5.3.1.1.1.2 Coaxial flow jetting

The basic principle of this instrument (Fig. 5.4) is to use a coaxial
airlow to push the droplets that are formed in the nozzle. With
this technology, microparticles with diameters of 200 μm can be
produced (Heinzen et al. 2002).

Figure 5.4 Principle of coaxial airlow bead generator.

A commercially available equipment is the Nisco Encapsulation

Unit—Var J1 (Nisco J1 2009–2011). This instrument is designed for
the production of small quantities of spherical beads (e.g., alginic
acid) with diameters smaller than 500 μm (Nisco J1 2009–2011).
The diameter of beads produced is governed principally by the
nozzle diameter, the low rate, and the applied air low (Prüsse et
al. 2008). The major disadvantage of this technology is the very low
throughput and therefore is only suitable for small-scale production
(Heinzen et al. 2002, Zuidam and Shimoni 2010).

5.3.1.1.1.3 Aerodynamically assisted jetting

Aerodynamically assisted jetting (AAJ) is one of the main jet-
processing approaches (Arumuganathar et al. 2009). AAJ is a
phenomenon exploring a pressure differential for forming a liquid
jet, which subsequently generates a myriad of droplets (Gañán-Calvo
and Barrero 1999, Jayasinghe and Suter 2006) (Fig. 5.5).
A commercially available equipment is the Nisco Encapsulation
Unit—Var J30 (Nisco J1 2009–2011). The product enters through
a central needle, which is enclosed in a pressure chamber with an
exit through the oriice. The exit oriice, which is centrally in line
 Methods of Microencapsulation 183

with the axis of the needle, has been counter-sunk externally. The
counter sunk leads to the aerodynamical effect so that the jet has a
smaller diameter when passing the oriice than before at the needle
(Fig. 5.5). The size of the drops is determined by the product low
rate and the pressure inside the chamber. The product low rate is
controlled by a syringe pump, which is connected to the product
nozzle. With this type of equipment, it is possible to obtain very
small and homogeneous particles (up to about 10 μm) with a slight
risk of clogging (Nisco J30 2009–2011). This unique technology has
the advantage of reaching smaller particles than the needle diameter,
depending on the physical properties of the product (Fig. 5.6).

Figure 5.5 Operating principle of AAJ equipment.

Figure 5.6 Preparation methods and schema size obtained by monoaxial

extrusion technologies.
184 Immobilization and Microencapsulation of Probiotics

Four probiotic bacteria (L. paracasei L26, L. casei-01, L. acidophilus

Ki, and Biidobacterium animalis BB-12) were encapsulated using
an AAJ technique in plain alginate or alginate supplemented with
L-cysteine·HCl, and stored at different temperatures for a period
up to 6 months, and the results showed that the encapsulation was
only effective in promoting protection at freezing temperatures,
independently of the sensitivity of the strain (Sousa et al. 2012).

5.3.1.1.1.4 Nozzle resonances technology

Berkland et al. (2001) developed a method on the basis of the
extrusion of a liquid through a needle that vibrates at an ultrasonic
frequency, in order to obtain microparticles with a narrow size
distribution (Fig. 5.7). This technology is based on an old principle
described by Joseph Plateau (Plateau 1873), and later conirmed
by Lord Rayleigh (Rayleigh 1878) that demonstrates that a laminar
liquid jet when subjected to a superimposed vibration breaks into
droplets of equal sizes (McCuan 1997). This vibration has to be done
in resonance of the Plateau–Rayleigh instability and leads to very
uniform droplets size distribution (Berkland et al. 2001, Kim and
Pack 2006).

Figure 5.7 Principle of vibration technology.

During the formation of the jet from the nozzle, there is a

tendency to split into small droplets. Applying a vibration at a
particular frequency, uniform drops with a size of approximately
twice the diameter of the jet are formed. The parameters that affect
the dimensions of the particles are the frequency, the speed of the
liquid jet, and the nozzle diameter (Heinzen et al. 2002, Prüsse et
al. 2008). For large particles of about 1 mm, this method allows
 Methods of Microencapsulation 185

a high liquid low, up to several l/h. For smaller particles, low

decreases proportionally. After deining an optimal vibration, the
process becomes highly reproducible. The resonance is damped if
the viscosity of the dispersion is too high, preventing or hindering
the formation of droplets (Poncelet 2006). The process works
very well for generating droplets between 100 and 5000 μm, but
applications that can obtain either smaller or larger droplets are
known (Vidhyalakshmi et al. 2009).
If one needs uniform, monodisperse, and small particles
(diameters from 0.1 mm to 2 mm) and an up-scalable technology
(from 10 g up to several 100 kg/batch), this technology is the answer
(Heinzen et al. 2002). This technique is only suitable for lab or small-
scale production (Senuma et al. 2000, Zuidam and Shimoni 2010).

5.3.1.1.1.5 Jet cutter technology

With this technology, the liquid is pressed at high speed out of
the nozzle as a continuous jet. Directly below the nozzle, the jet
is mechanically cut in cylindrical segments by a rotating cutting
tool made of small wires attached on a stand (Prüsse et al. 1998,
Prüsse et al. 1998, Prüsse et al. 2000, Prüsse et al. 2008). Driven
by surface tension, the cylindrical segments after been cut form
spherical droplets (Fig. 5.8). One of the advantages of this method
is that the liquid low rate can be high, but it is especially suitable
for high viscosity dispersions (Poncelet 2006). The diameter of
microparticles produced is mainly governed by the nozzle diameter,
rotation frequency of the cutting tool, and number and diameter of
the wires (Prüsse et al. 2008). This technique is suitable both for
lab-scale production and industrial scale microparticles production
(Zuidam and Shimoni 2010).

Figure 5.8 Principle of jet cutter technology.

186 Immobilization and Microencapsulation of Probiotics

5.3.1.1.1.6 Spinning disk technology

Rotational suspension separation (or centrifugal suspension
separation coating or spinning disk method) is a microencapsulation
technique that was irst developed by Sparks in 1987 (Barbosa-
Cánovas et al. 2005). This process involves (1) suspending core
particles in a liqueied coating material; and (2) pouring the
suspension through a rotating disk apparatus under conditions such
that excess liquid between the core particles spreads into a ilm
thinner than the core particle diameters. The liquid jet is directed
to a rotating disk (Fig. 5.9) that uses centrifugal force to cause
the division into droplets (Dziezak 1988, Gouin 2004, Desai and
Park 2005, Augustin and Sanguansri 2008, Acosta 2009, Meiners
2009). The microparticles solidify after its departure from the disk.
This whole process can take from a few seconds to a few minutes
(Barbosa-Cánovas et al. 2005).

Figure 5.9 Principle of spinning disk technology.

The parameters that affect the size of particles are the speed
of rotation, the geometry of the disk, and the liquid jet low rate
(Meiners 2009). This method has the advantage that the liquid low
rate can be high (Poncelet 2006). In addition to the method of jet
cutting, this process is the most promising for the food industry
because the preparation conditions are mild, there is no problem of
plugging, the process is continuous, and power costs are generally
low (Teunou and Poncelet 2005).
The main objectives in the disk design are to ensure that the
liquid reaches the speed of disk and to obtain a uniform droplet size
distribution in atomized liquid. Disk diameters vary from 5 cm in
 Methods of Microencapsulation 187

small laboratory models to 40 cm for industrial size dryers. Disk

speeds range from 1000 to 50,000 rpm (Teunou and Poncelet 2005).
This is a continuous process that makes possible to produce, at a
high rate, particles of a given size with a narrow size distribution
(Oxley et al. 2000, Senuma et al. 2000, Anantachoke et al. 2006).
Particles of 30 μm up to 2 mm can be encapsulated by this process
(Barbosa-Cánovas et al. 2005, Desai and Park 2005). Bégin et
al. (1991) studied the use of a rotative lat disk atomizer for the
production of biocatalysts immobilized in alginate gel and obtained
microparticles with diameters ranging from 1 mm to 3 mm. Spinning
disk atomization has been applied to an alginate microsphere
preparation with sizes ranging from 300 to 600 μm in which yeast
was used as a model system (Senuma et al. 2000).

5.3.1.1.1.7 Centrifugal extrusion

The Southwest Research Institute (SwRI), headquartered in San
Antonio, Texas, developed the centrifugal extrusion concept in 1960s
(Barbosa-Cánovas et al. 2005).
This centrifugal extrusion system, which is similar to the spinning
disk system, is a liquid coextrusion process that uses rotational
forces to create droplets. Spinning disk and centrifugal extrusion are
both based in atomization methods (Gouin 2004).
In centrifugal extrusion, liquids are encapsulated using a rotating
extrusion head containing a modiied double-luid nozzle in which
the active ingredient is pumped through the inner part of the nozzle,
whereas the shell material is pumped through the outer part of the
nozzle (Dziezak 1988, Kailasapathy 2002, Gouin 2004, Desai and
Park 2005, Augustin and Sanguansri 2008, Vidhyalakshmi et al.
2009). In this process, a jet of core liquid is surrounded by a layer of
shell material (Fig. 5.10). As the jet moves through the air, it breaks,
owing to Rayleigh instability, into droplets of core coated with the
wall solution. Although the droplets are in light, a molten wall may
be hardened, a solvent may be evaporated from the wall solution,
or other mechanism may occur, depending on the encapsulant
material, leading to the hardening of the microparticles (Augustin
and Sanguansri 2008, Vidhyalakshmi et al. 2009). Particles produced
by this method have diameters ranging from 150 μm to 2000 μm
(Dziezak 1988).
188 Immobilization and Microencapsulation of Probiotics

Figure 5.10 Principle of centrifugal extrusion technology.

From an industrial point of view, spinning disk and centrifugal

extrusion are alternatives to conventional atomization devices such
as double-luid nozzles, pressure nozzles, and spinning wheels to
atomize an emulsion or a suspension in a coating formulation (Gouin
2004). Centrifugal extrusion is an inexpensive process for producing
particles with diameter from 400 to 2000 μm with a high production
rate, up to 22.5 kg of microparticles per hour per nozzle (Barbosa-
Cánovas et al. 2005).

5.3.1.1.1.8 Microfluidic techniques

In this kind of technique, uniform droplets are fabricated using
precise microchannels through which the encapsulant material was
extruded (Seo et al. 2005, Hyun-Jik et al. 2006, Zhang et al. 2007, Oh
et al. 2008, Matalanis et al. 2011). The formation of monodisperse
Ca-alginate microparticles using a microluidic chip and a reaction of
internal gelation was reported (Huang et al. 2007). The microluidic
chip was capable of generating relatively uniform microparticles
and has the advantages of active control of droplet diameter, simple
and low-cost process, and a high throughput. It was possible to
control the size of microparticles from 80 μm to 800 μm by altering
the relative sheath/sample low rate ratio. The great advantage of
microluidics is the production of highly reproducible droplet sizes
(Nie et al. 2005, Seo et al. 2005, Utada et al. 2005, Matalanis et al.
2011). Nevertheless, batch process for food industry using this
methodology can be dificult to scale up (Matalanis et al. 2011).

5.3.1.1.2 Spraying
Spraying (also called atomization) is one of the most used methods
in the food industry, because in addition to being economical and
lexible produces good quality products (Dziezak 1988, Kailasapathy
2002, Gharsallaoui et al. 2007, Augustin and Sanguansri 2008, de
 Methods of Microencapsulation 189

Vos et al. 2010, Zuidam and Shimoni 2010, Burgain et al. 2011,
Peighambardoust et al. 2011). Atomization followed by hot air
drying (spray drying encapsulation) has been used in the food
industry since the late 1950s (Gouin 2004, Desai and Park 2005).
The spray drying of microorganisms dates back to 1914 to the study
of Rogers on dried lactic acid cultures (Peighambardoust et al. 2011).
In 1932, the irst spray-dried lavor powders, in which the lavors
were encapsulated in a thin arabic gum ilm, were produced by an
English company A. Boake, Roberts & Co., Ltd (Barbosa-Cánovas et
al. 2005).
In the spraying process, bacteria are dispersed in a liquid phase
and this system is atomized in a stream of air (Sousa e Silva and M.
Ferreira 1998, Gharsallaoui et al. 2007, de Vos et al. 2010). In the case
of probiotics, dispersion must be made mandatory in an aqueous
phase.
A dispersion or emulsion containing the living cells of the probiotic
in a suitable liquid carrier is initially prepared, usually polymeric. This
carrier should have good emulsifying properties, have low viscosity
at high solids content, and exhibit low hygroscopicity (Barbosa-
Cánovas et al. 2005). This process is characterized by spraying the
liquid dispersion inside a chamber subjected to a controlled stream
of warm air (spray drying) or cold air (spray freeze-drying). This
dispersion is atomized into millions of small individual droplets by
a fast rotating device or a nozzle (Fig. 5.11). Through this process,
the contact surface area of the product sprayed is greatly increased,
giving rise to microparticles. A rapid vaporization of solvent takes
place from the microparticles lying inside the chamber in contact
with the stream of air. After evaporation of the solvent, the dried
microparticles are retrieved.
Atomization can also occur at low temperatures (spray-
congealing or sprayfreezing, (Dziezak 1988, Gouin 2004, Rokka and
Rantamäki 2010, Nazzaro et al. 2012). In this case, the bacterial cells
are in a solution that is atomized in a cold gas phase, which leads to
dispersion of frozen droplets. These droplets can then be dried by
freeze-drying (Wang et al. 2006, Augustin and Hemar 2009, de Vos et
al. 2010). This technique involves the consumption of large amounts
of energy, high processing times, and higher price than that of the
spray-drying technique, especially on a large scale (Gölker 1993,
Johnson and Etzel 1995, Knorr 1998, Mattila-Sandholm et al. 2002,
Burgain et al. 2011). Despite of this, many LAB cannot tolerate the
190 Immobilization and Microencapsulation of Probiotics

relatively high temperatures used during spray-drying (Porubcan

and Sellars 1979, Ananta et al. 2005), and as a consequence, freeze-
drying is a very popular method for the production of dried LAB
preparations (Mattila-Sandholm et al. 2002). However, some more
heat-resistant strains of LAB can be spray dried without a drastic
loss of viability and activity, which are comparable to that obtained
by freeze-drying (Silva et al. 2002, Ananta et al. 2005). Semyonov
et al. (2010) developed a new process using spray freeze-drying to
produce microparticles of L. paracasei with high viability.
Another alternative for atomization is the inclusion of the
bacterial cells in materials with a melting point above ambient
temperature (spraychilling or and spray-cooling). Initially, these
materials are fused and the bacterial dispersion is added. This
process is performed in an equipment similar to the one used in
spray-drying, except for the fact that the process air is not heated
(Augustin and Sanguansri 2008). After spraying, the liquid will split
it into droplets that solidify by cooling in contact with the air at room
temperature (Pedroso et al. 2012).

Figure 5.11 Schematic representation of the atomization process

(spraying).

Spraying can form microspheres or microcapsules, as the

drug is dissolved or dispersed in polymer solution. Formulation
factors inluencing the characteristics of microparticles are the
polymer concentration, surface tension and viscosity of the liquid,
temperature, and atomization low rate (Silva et al. 2003, Kinam and
Yoon 2006, Kuang et al. 2010, Zuidam and Shimoni 2010), which are
often optimized by trial and error. In general, spray drying has been
 Methods of Microencapsulation 191

found to produce microparticle sizes between 5 and 80 μm (Rokka

and Rantamäki 2010).
The advantages of spraying are the speed of preparation and
the low cost of the procedure (de Vos et al. 2010, Burgain et al.
2011). It is a simple technique that allows to obtain the inal form
without having to resort, to washes, to isolate the microparticles
or dispose of waste solvents. This technique is highly reproducible
and adaptable to industrial applications. Another advantage of the
atomization process is that it can be operated continuously. This
process also allows obtaining large amounts, even when operated
in a discontinued way. The encapsulation eficiency varies typically
between 70 and 85%, regardless of the process parameters
(Giunchedi and Conte 1995, Silva et al. 2003).
A major disadvantage of this process is that it is an immobilization
technology rather than an encapsulation technology, which implies
that some bioactive components may be exposed at the surface.
This fact is especially problematic when considering the probiotics
encapsulation, in which the bacteria may leak when some hydration
occurs (de Vos et al. 2010). Among the disadvantages of atomization
are the cost of the equipment and the dificulty in obtaining spherical
particles (Giunchedi and Conte 1995). Another disadvantage of this
process is that the high temperatures used in the drying phase may
not be suited for the encapsulation of bacterial cells either because
they can affect the properties of polymers or the survival of bacteria
(Aaftabrouchad and Doelker 1992, Gardiner et al. 2000, O’Riordan
et al. 2001, de Vos et al. 2010, Burgain et al. 2011, Nazzaro et al.
2012). The turmoil caused during atomization can lead to a wide
variation of particle size formed. However, a proper control of inlet
and outlet temperature in the chamber of atomization can allow
getting probiotics encapsulated with the desired dimensions.
In order to improve probiotic survival, protectants can be added
to the media prior to drying. Rodrigues et al. (2011) studied the
survival rates of L. acidophilus Ki, L. paracasei L26, and B. animalis
BB-12® after whey protein microencapsulation via spray-drying,
with or without L-cysteine-HCl and showed that the effect of
Lcysteine-HCl was dependent on the probiotic strain. Trehalose has
been shown to enhance the survival rate of L. salivarius subjected to
spray freeze-drying (Zayed and Roos 2004). Berner and Viernstein
(2006) tested the effect of several different protectants on the
viability of Lactococcus lactis subjected to freeze-drying and found
the protectants based on skim milk or MRS-broth (all including
192 Immobilization and Microencapsulation of Probiotics

sugars) to be the most effective. Sucrose, trehalose, and sorbitol

were tested as cryoprotectants in the freeze-drying process of
L. plantarum and L. rhamnosus GG, with sucrose presenting the
best results between the three (Siaterlis et al. 2009). The addition
of polydextrose to reconstituted skim milk (RSM) improved L.
rhamnosus E800 survival to the spray-drying process, when
compared with RSM alone (Corcoran et al. 2004). Trehalose and
monosodium glutamate were tested, presenting positive results, as
a way to preserve viability of two L. rhamnosus strains, during spray-
drying (Sunny-Roberts and Knorr 2009). Monosodium glutamate,
sucrose, and fructooligossacharides in combination with RSM were
found to have a protective effect on the viability of two probiotic L.
keir strains during spray drying (Golowczyc et al. 2011).

5.3.1.1.3 Emulsification
The microparticles can also be produced by emulsiication. An
emulsion is a mixture of two immiscible liquids in which one of
them is in small droplets (the dispersed phase, which may contain
probiotics) within another liquid (the dispersing phase) to form a
stable mixture. Emulsions are thermodynamically unstable and
therefore do not form spontaneously, and it is necessary to provide
energy to obtain them through agitation. Surfactant agents are
mandatory substances added to emulsions to increase its stability.
The simplest method to produce emulsions consists in a reactor
with a turbine for agitation (Fig. 5.12). Emulsions can also be obtained
using continuous systems with static mixers. These systems consist
of tubes wherein elements are inserted in such a way as to cause the
ine division of the liquids. These systems allow the production of
emulsions in a fraction of seconds at a high liquid low rate (Poncelet
2006).

Figure 5.12 Microparticle preparation scheme by simple emulsion

technique (hydrophilic active ingredients).
 Methods of Microencapsulation 193

The emulsion technique as a general method for immobilization

of sensitive living cells (microbial, algal, plant, and animal cells) was
irst developed by Nilsson et al. (1983).
The smaller the internal phase particle size of the emulsion, the
smaller the inal microparticles will be. The size of the microparticles
is controlled essentially by the agitation rate, and can vary between
25 μm and 2 mm (Krasaekoopt et al. 2003). Emulsiication allows
the production of a wide particle size range from 0.2 to 5000 μm
(Burgain et al. 2011). Although the obtained microparticles are
of small diameter, the main disadvantage of this method is that
it provides large size range and shape. For example, alginate
microparticles made by the emulsion technique generally vary in
size from 20 μm to 2 mm (Rokka and Rantamäki 2010). Using the
emulsion technique for the encapsulation of L. acidophilus LA1, the
diameter of microparticles formed ranged between 100 and 300 μm
(Sabikhi et al. 2010).
The emulsion technique is easy to scale-up and gives a high
survival rate of probiotic bacteria (Chen and Chen 2007, Burgain et
al. 2011). However, it might be more expensive if vegetable oil has to
be removed, and the microparticles have to be washed to eliminate
the residual oil on its surface (Zuidam and Shimoni 2010). The
emulsion method is one of the techniques most used for probiotic
encapsulation aimed at food applications (Heidebach et al. 2012).
After the emulsion is formed, it is necessary to stabilize the
microparticles obtained by any process. There are various techniques
for the stabilization of microparticles, as for example, evaporation of
the solvent, solvent extraction, cooling, thermal and chemical cross-
linking, and ionic interaction (Kinam and Yoon 2006).
Özer et al. (2009) utilized an emulsion technique to encapsulate
B. biidum BB-12 and L. acidophilus LA-5 in white-brined cheese,
verifying a more limited decrease in viability when bacteria were
microencapsulated, and no differences in sensory properties were
observed. Ding and Shah (2009) tested ive different encapsulation
materials on the stability of 10 probiotic bacteria, using the emulsion
technique. Their results showed that in general, maicroparticles made
of alginate, xanthan gum, and carrageenan gum greatly improved
the survival of probiotic bacteria when exposed to acidic conditions
and bile salts. Shima et al. (2007) studied the incorporation of L.
acidophilus into the inner phase of waterinoilinwater (W/O/W)
194 Immobilization and Microencapsulation of Probiotics

emulsion, and found that it improved the bacterial survival rate in a

model gastric juice.

5.3.1.2 Solid matrix techniques

The division of solid particles can be done by grinding and/or sieving.
After that, a solution is sprayed on the microparticles under agitation
(air suspension coating or pan coating) until a good and uniform
coating is obtained. During these operations, the microparticles can
agglomerate, due to same problems. However, if well controlled, the
agglomeration may allow the formation of larger particles from ine
powders (Poncelet 2006).

5.3.1.2.1 Air suspension coating

Air-suspension particle coating (or fluid bed coating) is a process
wherein thin coatings are applied to powder particles. This method
is different to encapsulation by spray drying, because this last one
produces particles consisting of a homogeneously blended matrix
of the polymer entrapping the particle, in contrast to the previous
method in which a deined ilm coating is produced over an existing
solid core (Werner et al. 2007).
Originally developed as a technique for coating pharmaceutical
dosage forms, air suspension coating has been increasingly applied
in the food industry (Dewettinck and Huyghebaert 1999, Werner
et al. 2007, Werner et al. 2007, Kuang et al. 2010). This coating
technique was originally developed by Dale Wurster, professor of
Pharmacy at the University of Wisconsin, in the 1950s (Wurster
1959) for coating pharmaceutical tablets. The instrument thus
created (Bottom Spray or Wurster coating) consists, in general, lines
(Kleinbach and Riede 1995, Prista et al. 2007, Werner et al. 2007) by
a vertical tube, a compressor system, a heating system, an adjustable
plate system, and one or several atomizers (Fig. 5.13). In the vertical
tube, the particles to be coated are introduced; the compressor
system launches an air current lift, heated by the heating system,
which prevents the deposition of the particulate matter by the action
of gravity; the adjustable plate system regulates the speed of the air;
the atomizers release the coating liquid onto the particles (Dziezak
1988).
So, this type of coating occurs when an upward low of air is
driven through a bed of particles reaching suficient speed to suspend
 Methods of Microencapsulation 195

them, but not expel them from such air current (Dziezak 1988,
Dewettinck and Huyghebaert 1999). In conventional air suspension
coating, the basic concept of luidization depends on balancing the
gravity force experienced by the particle by an upward movement of
air low, which ensures the full luidization of those particles (Gouin
2004). In this type of encapsulation, although the core particles
are suspended in the air, the coating material is atomized into the
camera, shocking and depositing on the particle’s nucleus. As the
spraying nozzle is immersed in the airlow and sprays the coating
material concurrently into the solid particles, these droplets travel
a short distance before contacting it, resulting in a more uniform
ilm (Kinam and Yoon 2006). When the particles strike the top of
the camera, their speed becomes slow and by the action of gravity
are thrown by a descent column of air that throws them again in the
luidized bed in which they are coated once more and the cycle begins
again (Fig. 5.13). The application of a coating to a solid particle is a
very complex phenomenon. The uniform coating layer is not formed
on a single passage of the randomly oriented microparticles through
the atomization zone, but on their successive passages through the
zone, ensuring the integrity and uniformity of the coating layer.
(Dziezak 1988, Vidhyalakshmi et al. 2009).

Tangential Spray

Figure 5.13 Air suspension coating types.

Other air suspension coating systems were also created, namely

the Top Spray and Tangential Spray (Kleinbach and Riede 1995,
Desai and Park 2005, Kinam and Yoon 2006, Champagne and Fustier
196 Immobilization and Microencapsulation of Probiotics

2007, Werner et al. 2007). The three technologies illustrated in

Fig. 5.13 principally differ in the type of air luidization employed
and the site in the vessel at which the coating material is sprayed.
The probiotic bacteria are present in ine powder particles, which
are kept in motion in the vessels and are prepared by traditional
methods such as fermentation, concentration, freeze-drying, and
granulation (Champagne and Fustier 2007).
When the particles are coated by Top Spray system, the
microparticles typically have a porous surface and interstitial void
spaces; thus, the bulk density of the particles produced is generally
lower than that achieved by other methods (Kinam and Yoon 2006).
The Tangential Spray system combines centrifugal, high-
density mixing, and the eficiency of luid-bed drying leading to the
formation of a product with a higher bulk density, but it still has some
interstitial void spaces (Kinam and Yoon 2006). This method allows
to obtain less brittle and more spherical coated particles (Kinam and
Yoon 2006).

Figure 5.14 Air suspension particle coating. Adapted from Werner et al.
(2007).

The luidized bed encapsulation is one of the few technologies

that make possible that particles are coated with virtually any type
of cover material. In food applications, the coating is mostly lipid-
based, but proteins or carbohydrates can also be used (Gouin 2004,
Champagne and Fustier 2007, de Vos et al. 2010). This technique has
been extensively used in the pharmaceutical industry to coat particles
from approximately 100 μm up to the size of a few millimeters (Gouin
2004, Desai and Park 2005, Burgess and Hickey 2006, Kuang et al.
 Methods of Microencapsulation 197

2010). This technology is especially ideal for aqueous-based coating,

as it provides the high energy needed to evaporate the water (Kuang
et al. 2010).
The advantage of air suspension coating is that it is easy to scale
up (Burgain et al. 2011). As the food industry requires high volume
production with a low cost, this type of coating using the Würster
apparatus is still the irst choice, whereas the various compromises
between particle size and agglomeration can be overcome (Werner
et al. 2007). Air suspension coating, in food industry, accounts for
the second largest commercial production of encapsulated products
(Barbosa-Cánovas et al. 2005).

5.3.1.2.2 Pan coating

Relatively large particles can be encapsulated by pan coating. The
size of the solid particle must be greater than 600 μm so as to achieve
an effective coating by this method (Kinam and Yoon 2006). This
method employs a rotating pan containing the material to be coated
on which a liquid containing the coating material is released. The
rotation distributes this layer uniformly by the core particles forming
a thin layer with a high surface area that favors the evaporation of
the solvent (usually water). As the solvent evaporates, the coating
hardens and covers the nuclei (Burgess and Hickey 2006, Kinam and
Yoon 2006). Often, these pans are pierced (perforated pans) and the
coating liquid is atomized in turmoil on the nuclei to be coated. This
process is widely used in the pharmaceutical industry and is one of
the oldest processes for coating tablets (Vidhyalakshmi et al. 2009).

5.3.2 Immobilization/Entrapment Techniques

The third and inal step in the preparation of microparticles formed in
the previous phase is its stabilization, by chemical (polymerization),
physicochemical (coacervation, gelling), or physical processes
(evaporation, solidiication, coalescence).

5.3.2.1 Solidification
This method consists in the dispersion of the active substance, in
this case the living bacteria, in the molten excipient, followed by
emulsiication of this phase with a heated immiscible solvent at the
same temperature. As the formation of this phase requires heating,
198 Immobilization and Microencapsulation of Probiotics

temperatures a few degrees above the referenced value for the

excipients are being used (Kinam and Yoon 2006). The addition of a
surfactant agent helps to increase the eficiency of encapsulation, as
it increases the wettability of the active substance, which results in a
greater incorporation into the microparticles (Passerini 2003). The
mixture is then cooled below its melting point until the droplets of
internal phase solidify (Burgess and Hickey 2006, Kinam and Yoon
2006).
One of the main advantages of the use of these materials and
techniques is that it is not necessary to use organic solvents and the
raw materials are well known. One of the main disadvantages is the
use of high temperatures.

5.3.2.2 Coacervation
The earliest and one of the most widely used microencapsulation
technique involves the separation of phases by coacervation (Kinam
and Yoon 2006). This original microencapsulation technique,
described by Green (Green 1953), for the production of carbonless
copying paper was based on coacervation. It was Tiebackx (Tiebackx
1911) who irst reported the phenomenon of coacervation
without ever mentioning the word (de Kruif et al. 2004). The term
coacervation was only introduced in chemistry by Bungenberg
de Jong e Kruyt in 1929 (Bungenberg de Jong and Kruyt 1929) to
describe the phenomenon of macromolecular aggregation from a
liquid system, forming a colloidal system in which two liquid phases
separate: one rich in polymers (coacervate) and another low in
polymers (supernatant) (Dervichian 1954, Phares and Sperandio
1964, Burgess and Carless 1984, Nixon 1985, Arshady 1990, Sousa e
Silva and M. Ferreira 1998). The word derives from Latin “acervus,”
which means aggregation, and the preix “co,” which means together.
So, this word “coacervation” signiies the union of the colloidal
particles. The physical and chemical basis behind the coacervation
process is now well developed and understood (Bungenberg de Jong
and Kruyt 1929, Dervichian 1954, Bakan Joseph 1971, Burgess 1990,
Schmitt et al. 1998).
The coacervation can be deined to be simple or complex
depending on whether it has one or more polymers involved (Nixon
1985, Schmitt et al. 1998, Gibbs et al. 1999, Burgess and Hickey
2006, Kinam and Yoon 2006, Madene et al. 2006, Jyothi et al. 2010,
Zuidam and Shimoni 2010). The simple coacervation occurs when
 Methods of Microencapsulation 199

the conditions are changed, resulting in partial dehydration of

macromolecules causing a phase separation. This can be achieved
by mixing a polymer with a solvent that is incompatible or which is
poorly soluble, by changing the temperature, and so on. In solution,
these dehydrating agents promote polymer–polymer interactions
through water competition. The complex coacervation occurs when
a mixture of two or more polymers and a solvent are put together.
In the event that the polymers are highly compatible, for example,
two molecules with opposite charges, they interact and concentrate
on a poor solvent phase, whereas the solvent forms a phase poor in
polymers.
The three basic steps in coacervation are (phase 1) formation
of three immiscible phases; (phase 2) deposition of coating; and
(phase 3) hardening of coatings (Fig. 5.15). The irst step includes
the formation of three immiscible phases: the liquid vehicle, the
solid core material, and the coating material. The core material is
included in a dispersion of polymer coating. The coating material
phase, an immiscible polymer in liquid state, can be formed by (i)
change of temperature, (ii) adding a salt, (iii) adding a nonsolvent, (iv)
adding an incompatible polymer, and (v) inducing polymer–polymer
interactions. The second stage includes the deposition of these
colloidal particles on the surface of the solid core coating it. Usually,
this solid core must be compatible with the polymer and insoluble,
or slightly soluble, in the coacervation medium (Arshady 1990,
Madene et al. 2006). Finally, the micro particles can be stabilized, for
example, by cross-linking, desolvation, or heat treatment (Jyothi et
al. 2010).
This technique is complex, in operational terms, and needs
a thorough control of the experimental settings. Many factors,
including the type of polymer, pH, ionic strength, concentration,
and in the case of two or more polymers, their ratio, affect the
strength of the interaction between the polymer and the nature of
the complex formed (Augustin and Hemar 2009, de Vos et al. 2010).
The hydrodynamic conditions play an important role in determining
the properties (size) of microparticles prepared by this method
(Dobett and Pantaleo 2002).
Although the electrostatic interactions are considered responsible
for the interaction between opposite charged polymers, hydrogen
bonds and hydrophobic interactions can also contribute signiicantly
to the formation of the complex (Turgeon et al. 2007, Augustin and
Hemar 2009, de Vos et al. 2010).
200 Immobilization and Microencapsulation of Probiotics

Figure 5.15 Coacervation stages: A and B and C: phase 1; D: phase 2 and

E: phase 3; (A) polymer dispersion; (B) inclusion of solid core
into polymer dispersion; (C) coacervation; (D) coalescence of
colloidal particles at the surface of the solid core; (E) coating
hardening. (Adapted from Augustin and Hemar 2009).

A very large number of hydrocolloids systems was evaluated

for microencapsulation by coacervation (Gouin 2004), but the most
studied and best understood system is probably the arabic gum/
gelatine system (Bungenberg de Jong and Kruyt 1929, de Kruif et
al. 2004, Desai and Park 2005, Kinam and Yoon 2006, Schmitt and
Turgeon 2011). However, other coacervation systems present very
good properties such as heparin/gelatine, carrageenan, chitosan,
soy protein, gelatine/carboxymethylcellulose, β-lactoglobulin/
arabic gum, and guar/dextran (Tsung and Burgess 1997, Schmitt et
al. 2000, de Kruif et al. 2004, Gouin 2004).
The coacervation presents some advantages such as a very high
payload achievable that is one of the most important characteristic
of this method (Gouin 2004, Augustin and Sanguansri 2008). It also
presents some problems such as the price and its complexity (Gibbs
et al. 1999, Gouin 2004, Barbosa-Cánovas et al. 2005).
One of the factors that limit the use of coacervates in encapsu-
lation is their sensitivity to pH and ionic strength. To increase the
robustness of coacervates, they may be cross-linked using chemi-
cal agents, such as glutaraldehyde (an effective cross-linker) that is
toxic (Sanchez and Renard 2002, Gouin 2004, Burgess and Hickey
 Methods of Microencapsulation 201

2006, Kinam and Yoon 2006, Madene et al. 2006). Heating and add-
ing counter polyions or other cross-linking reagents are alterna-
tive cross-linking methods (Kinam and Yoon 2006, Augustin and
Sanguansri 2008). Enzymatic crosslinkers are more acceptable in
the food industry (Gouin 2004, Augustin and Hemar 2009). Both
chemical cross-linking agents and the application of heat may be
harmful to the encapsulated materials, such as live cells like probi-
otic bacteria.
Complex coacervation is mainly used for microencapsulation
of oils, but it can also be used for nutrients, vitamins, enzymes, and
probiotics (Gouin 2004, Oliveira et al. 2007, Rokka and Rantamäki
2010).

5.3.2.3 Gelation
Gelation is one of the most used entrapment techniques to obtain
microparticles. The contact of droplets of a gel-forming solution in a
gelation bath leads to the formation of these particles. The gelation
may be due to ionic bonding between polymer chains or by cooling
(Poncelet 2006).
The use of natural polymers in formulating dosage forms
containing food and drugs has received much attention because
of its excellent biocompatibility and biodegrability. Among them,
the alginic acid is a very promising natural polymer and has been
widely explored (Gouin 2004, Anal and Singh 2007, Rokka and
Rantamäki 2010, Ain Riaz and Masud 2011, Huq et al. 2012). It is
a linear heteropolysaccharide consisting of β-D-mannuronic acid
(M) and α-L-guluronic acid (G) (Anal and Singh 2007, de Vos et al.
2010, Draget and Taylor 2011), composed of blocks of homopolymer
MM or GG extracted from several species of Brown algae (Aslani and
Kennedy 1996, Tonnesen and Karlsen 2002). Figure 5.16 shows the
structure of sodium alginate that is the sodium salt of the alginic
acid. Depending on the origin, composition and sequence in M and G
vary considerably, affecting their functional properties as supporting
material (Tonnesen and Karlsen 2002, de Vos et al. 2010, Draget and
Taylor 2011, Teoh et al. 2011).
The alginic acid (or sodium alginate) can react with bivalent
cations or with polyvalent cation, as for example calcium, forming
cross-links leading to the formation of a gel (Morris et al. 1978). Gel
formation using calcium cation occurs mainly with GG and therefore
blocks (Fig. 5.17) forming a structure called “egg box” (Grant et al.
202 Immobilization and Microencapsulation of Probiotics

1973) are formed. Even if this model is commonly adopted, it has

been questioned several times and is still a subject of debate (Braccini
and Pérez 2001). A more realistic coordination pattern supported by
molecular modeling has been proposed (Mackie et al. 1983).

Figure 5.16 Sodium salt of alginic acid: blocks of (1m4)-linked

β-D-mannuronate (M) and α-L-guluronate (G) residues.

Figure 5.17 Formation of calcium alginate gel: (a) alginic acid molecules
dispersed; (b) link between the strings through the groups
of guluronic acid and calcium ions; (c) formation of the gel
network.

The gelation due to ionic bonding such as an alginate solution

dropped in calcium ions bath has been very used. The alginate gel
is relatively stable at acidic pH, but easily disintegrates in alkaline
conditions (Yoo et al. 2006).
Enhanced alginate microspheres as means of oral delivery of
bacteriophage for reducing Staphylococcus aureus intestinal carriage
 Methods of Microencapsulation 203

have been developed (Ma et al. 2012). Some other polymer systems
that can be used for ionic gelation are chitosan/triphosphate, pectin/
calcium, gelan gum/calcium, and carboxymethylcellulose/aluminum
(Kinam and Yoon 2006).
Gelation may also be obtained by spraying a thermogel (spray
chilling) or, alternatively by the extrusion method, through
emulsiication followed by cooling or pH change (Poncelet 2006,
Nag et al. 2011).
The emulsiication technique can also be used for the preparation
of calcium alginate microparticles. In this technique, an aqueous
solution of sodium alginate is dispersed in an immiscible liquid
phase and the gel droplets formed react with calcium ions, which are
added to the external phase, forming microspheres. It is possible to
get a high yield of encapsulation of hydrophilic drugs and particles
with size less than 150 μm (Wan et al. 1992). Other gelifying agents
can be used. Heidebach et al. (2009b) obtained microparticles
by transglutaminase-catalyzed gelation of casein suspensions
containing probiotic cells, namely commercial strains of L. paracasei
ssp. paracasei F19 and B. lactis Bb12. L. casei cells were successfully
entrapped into a gel matrix by a water-in-oil emulsion in which the
matrix was formed by sodium caseinate and gellan gum mixture
that was gelled by gradually decreasing pH with glucono-δ-lactone
(Nag et al. 2011). Probiotic cells L. paracasei ssp. paracasei F19 and
B. lactis Bb12 were microencapsulated in milk protein matrices by
means of an enzymatic induced gelation with rennet (Heidebach et
al. 2009a).
It is also possible to obtain microparticles of calcium alginate
by emulsiication-internal gelation (Fig. 5.18). Calcium is dispersed
in the form of an insoluble salt (e.g., calcium carbonate or calcium
citrate) in sodium alginate solution. This mixture is emulsiied in
an oily phase in order to obtain a W/O emulsion and the calcium
present in the internal phase is released by acidiication of the
external oily phase causing the gelling of alginic acid (Poncelet et al.
1992, Poncelet et al. 1995, Poncelet et al. 1999).

5.3.2.4 Solvent extraction/evaporation

Solvent extraction technique has often been employed in view
of the simplicity of the procedures involved in obtaining the
microparticles and the modulation possibilities of physical chemical
properties through the choice of components and preparation of the
204 Immobilization and Microencapsulation of Probiotics

formulation. The origin of this technique can be found in the 1960s

(Arshady 1990).

Figure 5.18 Emulsiication-internal gelation.

This technique involves the preparation of a solution of a

polymer, followed by their emulsiication originating droplets. The
two main stages of these processes are the formation of an emulsion
and the solvent removal. The active is solubilized or dispersed in the
polymer dispersion, and this mixture is emulsiied in an aqueous
solution containing a surfactant. This emulsion is then added to a
nonsolvent (liquid medium in which the polymer is insoluble) that
causes solvent extraction of the droplets. The resulting emulsion
is agitated until the volatile solvent evaporates leading to the
formation of solid microparticles (Kinam and Yoon 2006). Solvent
removal progressively increases viscosity and decreases the volume,
leading to polymer insolubilization (Arshady 1990, Sousa e Silva and
M. Ferreira 1998). This method involves the diffusion of polymer
solvent from the droplets to the outer space (continuous phase). After
this process, the removal of nonsolvent can occur by evaporation,
lyophilisation, or atomization (Arshady 1991, Aaftabrouchad and
Doelker 1992).
The solvent evaporation technique relies on the high vapor
pressure of the solvent that often leads to the use of organic solvents,
otherwise the solidiication process is slow (Kinam and Yoon 2006).
This feature makes this methodology not accessible for encapsulation
of probiotics.
 Methods of Microencapsulation 205

In the case of a nonvolatile solvent, it can be removed by extraction

into the continuous phase. This case can be done using a solvent
that has a higher solubility in the continuous phase, increasing the
concentration difference between the dispersed and continuous
phase or by the addition of a third solvent into the outer phase to
facilitate the solvent extraction (Kinam and Yoon 2006).
In solvent extraction technique, the solvent of the polymer is
chosen to be miscible with the nonsolvent (Arshady 1990, Arshady
1991). Solvent extraction leads to microparticles with better
characteristics (more regular shape, lower size, smaller variation
in diameter) than the solvent evaporation method, but the largest
particle porosity due to faster removal of the solvent increases the
surface area (greater porosity) (Giunchedi and Conte 1995, Kinam
and Yoon 2006), which leads to a faster release of its components.

5.3.2.5 Polymerization
The polymerization can be in situ or interfacial, that is, it occurs in
the continuous or in the interphase of dispersed system.
In the in situ polymerization, no reactive agents are added to
the core material, and the polymerization occurs exclusively in
the continuous phase and on the side of continuous phase, on the
interface with the core dispersed (Jyothi et al. 2010). Initially, a
prepolymer with low molecular weight will be formed, which with
the passage of time will grow in size, and will deposit on the surface
of the dispersed core material generating a solid coating (Jyothi et al.
2010). The particles obtained are very small, but, typically, have high
concentrations of associated monomer. Emulsion polymerization
in external organic phase is not so used because it requires large
amounts of organic solvents and surfactants and also due to the toxic
nature of the monomers used. It is a very fast and easily scale-up
technique (Silva et al. 2003). The particles obtained have a uniform
coating with a size between 0.2 μm and 75 μm (Vidhyalakshmi et al.
2009).
In the interfacial polymerization technique, the coating can
be formed on the surface of the particle by the polymerization of
reactive monomers (Kinam and Yoon 2006, Vidhyalakshmi et al.
2009, Jyothi et al. 2010). It is characterized by the polymerization
of a monomer in two immiscible phases (Nixon 1985, Burgain et
al. 2011). If the internal phase is a liquid, it is possible to disperse
or solubilize the monomer in this phase and emulsify the mixture
206 Immobilization and Microencapsulation of Probiotics

on the external phase in which the coreagent is added. This starts

the polymer formation on the surface of the liquid droplets. This
technique can be used to encapsulate microorganisms in order to
improve their productivity in fermentation (Yáñez-Fernández et al.
2007).
The use of the polymerization technique is limited by toxicity
associated with monomers that do not react, by the high permeability
of coating, and by the weakness of the membranes obtained (Silva et
al. 2003).

5.3.3 Other Methods

There are other microencapsulation technologies that, due to their
high price, are rarely employed, but for some speciic issues can be
employed as a solution (Desai and Park 2005, de Vos et al. 2010).
One of them is the application of liposomes, which are spherical
bilayers vesicles, which can enclose bioactive molecules (Gibbs et
al. 1999). The liposomes are formed by dispersion of polar lipids
(mostly phospholipids) in an aqueous solution and were irst
described by the British hematologist Alec D. Bangham (Bangham
and Horne 1964). Liposomes can be used as carriers for the delivery
of nutritional supplements to foods (Shoji and Nakashima 2004,
Augustin and Sanguansri 2008). They can range from 25 nm to
several micrometers in diameter (Desai and Park 2005).
Another encapsulation method is the usage of cyclodextrins,
which can envelop molecular structures by forming molecular
inclusion complexes. Cyclodextrins have a hydrophilic exterior and
a hydrophobic interior. This hydrophobic interior can be changed
by varying the number of glucose units (Del Valle 2004, Brewster
and Loftsson 2007, de Vos et al. 2010). As the encapsulation in this
technique takes place at a molecular level and the internal cavity of
cyclodextrins is very small, β-cyclodextrin cavity about 0.60–0.65
nm in diameter (Salústio et al. 2012), it cannot be used for the
encapsulation of living cells as bacteria. Cyclodextrins have long
been used to lavor encapsulation (Barbosa-Cánovas et al. 2005).
The microparticle formation technology that uses supercritical
luids has evolved in many different forms during the last years (Yeo
and Kiran 2005). A supercritical luid is any substance at a tempera-
ture and pressure above its critical point. The use of supercritical
luids can minimize the use of organic solvents and harsh manufac-
 Methods of Microencapsulation 207

turing conditions (Kinam and Yoon 2006). Supercritical luids can be

used for the production of bioactive loaded microparticles (Augustin
and Sanguansri 2008). Moolman et al. (2006) developed a method
to encapsulate B. longum in an interpolymer complex formation
between poly-(vinylpyrrolidone) (PVP) and poly-(vinylacetate-co-
crotonic acid) in supercritical carbon dioxide.
In a micromolding technique, very small molds can be used to
create microparticles with well-deined sizes and morphologies.
In this technique, polymer solution is poured into a mold with
a speciic size and shape followed by adjustment of solution or
environmental conditions to promote the gelation (Fukuda et al.
2006, Khademhosseini et al. 2006, Qiu et al. 2007, Dang et al. 2009,
Matalanis et al. 2011). This micromolding technique offers the ability
to prepare controlled shape microscale particles, even nonspherical
ones, although batch process is dificult to scale up for food industry
(Rabanel et al. 2009, Matalanis et al. 2011).
The electrostatic microencapsulation technique (electrostatic
coagulation) is a dry process in which core particles can be coated
with ine particles of different materials forming a highly porous
encapsulating shell (De et al. 2002). Due to the characteristics of
this technique, it cannot be used to encapsulate living cells such as
bacteria.
The potential use of compression coating is also an alternative
method for the encapsulation of probiotic bacteria (Chan and Zhang
2002, Calinescu et al. 2005, Vidhyalakshmi et al. 2009, Teoh et al.
2011). Previous works have demonstrated that compression coating
signiicantly improved the storage stability and protection in an
acidic medium of L. acidophilus (Chan and Zhang 2002, Chan and
Zhang 2005). In another work, succinylated β-lactoglobulin revealed
to be a suitable natural excipient to form tablets containing B. longum
and promoted their survival against gastric conditions (Poulin et
al. 2011). Carboxymethyl high-amylose starch was proposed as an
excipient for oral tablet formulation of Escherichia coli ensuring its
protection in the stomach and delivery in the intestine (Calinescu
et al. 2005). Carboxymethyl high-amylose starch and chitosan were
proposed as excipients for probiotic colon delivery of L. rhamnosus
(Calinescu and Mateescu 2008). The effects on bacterial survival
in tablets containing L. fermentum were investigated concerning
compression force , matrix-forming excipients, such as hydroxypropyl
methylcellulose phthalate (HPMCP), or other swelling agents, such
208 Immobilization and Microencapsulation of Probiotics

as sodium alginate, apple pectin, and hydroxypropyl methylcellulose

(HPMC) replacing partly HPMCP (Klayraung et al. 2009). This
strategy will be further developed in the next chapter of this book.

5.4 Microparticle Characterization

Physical properties, the release behavior, and probiotics stability
depend on the intrinsic characteristics of microparticles and the
conditions used for storage. The characterization of microparticles
is essential to ensure the encapsulation effectiveness, as well as to
predict in vivo behavior.
The properties of the microparticles that should be considered
in their characterization are general structure of the particle; ine
structure; size and granulometric distribution; composition of
the shell and the core; production yield; and release behavior and
activity.
The quality of the materials used for the encapsulation is an
important factor to obtain products with appropriate characteristics
and batch-to-batch reproducibility. In cases in which polymers
are used, these must be well characterized in terms of purity and
molecular weight. The ilm-forming characteristics of encapsulation
materials must be well known and controlled (Burgess and Hickey
2006).
The techniques used for microparticles characterization are
diversiied (Burgess and Hickey 2006, Graff et al. 2008, Alli 2011,
Doherty et al. 2011, Mamvura et al. 2011, Lee et al. 2012). For
external and internal structure, optical, electronic, and confocal
microscopy can be used. The ine structure of the microparticles can
be determined using X-ray diffraction and thermal analysis. For the
size and granulometric distribution, optical, electron microscopy,
and particle size analyzer can be used. For the composition of the
shell and the core thermal analysis, chemistry, chromatography,
and spectroscopic methods can be used. The production yield can
be determined by assay. In terms of probiotics survival and activity,
several microbiological analyses can be performed to assess the
number of viable cells and their metabolic state. The release behavior
can be veriied using release tests.
The rate of release of probiotics from the microparticles will in
many cases dictate their therapeutic action. This release is generally
 Conclusion and Future Trends 209

governed by the type of microparticles present, by the molecular

structure of the encapsulant material, the resistance of this material
to erosion or degradation, and surface area and porosity of the
microparticles (Barbosa-Cánovas et al. 2005, Burgess and Hickey
2006, Kinam and Yoon 2006).
For the characterization of the microparticles, the probiotics
release proile should be tested in vitro and whenever possible in vivo.
In the pharmaceutical industry, in vitro dissolution testing is one of
the most important tools in drug development and quality control. To
assure the survival during the gastric transit, the dissolution studies
may be performed in simulated gastric luid (SGF) or/and simulated
intestinal luid (SIF) (Lee and Heo 2000, Stadler and Viernstein
2003, Graff et al. 2008, Sabikhi et al. 2010, Alli 2011, Rodrigues et al.
2011, Heidebach et al. 2012).
The release behavior of the probiotics can also be tested in vivo
(Heidebach et al. 2012). Unfortunately, only a few in vivo studies were
experimented and they used animals such as rats or mice (Kushal et
al. 2006, Graff, Hussain et al. 2008, Heidebach et al. 2012).

5.5 Conclusions and Future Trends

The use of microencapsulated ingredients for the release control
inside the human body is a promising alternative to solve some
problems that the food industry has been facing. Some of these
problems are related to low stability, poor absorption, poor
organoleptic characteristics, low viability, and so on.
Often, the greatest challenge is to select the appropriate
microencapsulation technique and the most appropriate
encapsulating material. Despite the wide range of encapsulated
products that have been developed, manufactured and marketed
successfully in cosmetic and pharmaceutical industries, the
microencapsulation has found a comparatively limited market in the
food industry (Gouin 2004). With regard to the different methods of
encapsulation, consumer health and safety are factors that should
be taken into account, and then only approved food materials
can be used in these techniques of encapsulation. Environmental
consciousness of the consumers also deserves special attention in
the design of future products and technology, because some of them
might not be environment-friendly.
210 Immobilization and Microencapsulation of Probiotics

For a successful implementation of probiotics microparticles in

food products, some important demands must be met. Therefore,
they should be generated by processes that do not decrease the living
cell count or induce sublethal damages, should not alter the sensory
properties of the food, and should provide protection against adverse
conditions caused by food processing and/or the environment of
the food matrix. They also should protect the probiotic cells against
stress induced by the acidic gastric conditions and should release
the cells in the intestine at a high level of activity (Heidebach and
Kulozik 2010).
The microencapsulation of probiotics raises some speciic
problems. The survival of probiotic bacteria during processing,
storage, and in gastric conditions is highly dependent on the strain
used. Stability of the strain is thus one of the main criteria in selecting
suitable probiotics (Rokka and Rantamäki 2010). The encapsulation
process must therefore contribute to an increased stability of
probiotics. Food matrix environment has to be taken into account
when selecting the materials for the encapsulation.
Probiotic encapsulation was previously developed from the
technology involved in cell culture (ICT). The majority of microparticles
produced for ICT applications in dairy systems were produced by
two main methods, the extrusion and the emulsion method, and
consequently, in most of the studies on probiotic encapsulation for
food applications, these methods were also applied. More recently,
spray drying has been also utilized to encapsulate probiotic cells
as an alternative to the encapsulation methods previously referred
based on ICT (Heidebach et al. 2012). Today, the vast majority of
probiotic microparticles are produced by these three methods: the
extrusion technique, the emulsion technique, and the spray drying
technique (Heidebach and Kulozik 2010, Rokka and Rantamäki
2010, Heidebach et al. 2012).
The development time of a microencapsulated product is quite
long and requires speciic equipment and a multidisciplinary coop-
eration. Often, the complex transition from the laboratory scale to the
large production scale has led to dificulties in the marketing of the
inal product. The low-proit margins, usually reached in food ingre-
dients, are an impediment to the development and implementation
of new technologies that could result in truly unique food products
(Gouin 2004, Desai and Park 2005). New developments in the area
of probiotics encapsulation are thus expected in upcoming years at
 References 211

the level of new encapsulation techniques, new encapsulant materi-

als, and/or new commercial applications containing these bioactive
agents.

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Chapter 6

Development of Probiotic Dosage Forms

Maria H. Amaral,a J. Paulo Sousa e Silva,a Paulo J. C. Costa,a and Ana

M. Gomesb
aFaculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira,
228, 4050-313 Porto, Portugal
bCBQF, Biotechnology School of Portuguese Catholic University,

Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal

hamral@ff.up.pt

6.1 Introduction
The use of health-beneicial microorganisms was irst introduced
in the beginning of the 20th century. As reported in the previous
chapters, probiotics represent alternative therapeutic agents. In
order to improve the clinical eficacy of probiotics, optimization will
still be achieved by the application of new approaches (Sleator and
Hill 2008). Several European Union projects have shown that with
coordinated efforts toward a scientiic approach to the selection
and application of probiotics, effective functional products can be
developed with health beneits for consumers (Saarela et al. 2000).
This chapter is concerned with formulated preparations to
administer probiotics (medicines or dietary/food supplement).

Probiotic Bacteria: Fundamentals, Therapy, and Technological Aspects

Edited by J. Paulo Sousa e Silva and Ana C. Freitas
Copyright © 2014 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4411-62-2 (Hardcover), 978-981-4411-63-9 (eBook)
www.panstanford.com
228 Development of Probiotic Dosage Forms

Dosage forms as dietary/food supplement may be capsules,

tablets, powders (in sachets), or liquids in measured doses, and as
pharmaceutical dosage forms, which also include suppositories gels
or eye drops apart from the already mentioned. Dosage forms are
products designed for the administration of bioactive agents and
should have a high potential of acceptability by the patients. For this,
special attention should be given to the steps of pre-formulation
and formulation of a probiotic product and the integration of
diverse knowledge from areas of microbiology, chemistry, and
pharmaceutical technology.
Under current FDA guidelines (FDA 2006), probiotics, as thera-
peutic agents, may it within the live biotherapeutic products (LBPs)
concept. LBPs are biological products (nonrecombinant) that:
(1) Contain live microorganisms, such as bacteria or yeast;
(2) Are applicable to the prevention, treatment, or cure of a
disease;
(3) Are not vaccines.
To confer health beneits to the host, probiotics taken orally must
survive the adverse gastrointestinal (GI) conditions and maintain
the viability when they reach their site of action. Therefore, there
is a need for formulations that protect the bacteria from the harsh
conditions of the stomach. The oral administration of probiotics, and
the technology associated with it, has been mainly studied by the food
industry. However, the stability (shelf life) required for a nutritional
product is much lower than that required for a pharmaceutical
product. The stability of an active substance within a dosage form
is a critical parameter to be taken into account in the development
of pharmaceutical products. The stability within a dosage form and
the viability after release is also applied to the administration of
probiotic bacteria.
The development of formulations containing probiotics needs
the selection and characterization of strains with proven therapeutic
properties, the selection of excipients, and the processes suitable for
large-scale production of the inal dosage form. Before the marketing
of probiotics as medicinal products, it is necessary to evaluate the
safety and eficacy of the inal products, irst with laboratory tests,
then with animal models, and inally testing in humans. After this
whole process is completed, probiotics can then be commercialized
as medicinal products. However, the dose–response relationships of
 Introduction 229

probiotic products should be further characterized, in addition to

ways of optimizing the administration of these products (Hoffman
et al. 2008).
Several studies have been conducted to develop products
containing probiotics; however, there are not many pharmaceutical
dosage forms in the market. Table 6.1 present some examples of
pharmaceutical dosage forms containing probiotics marketed in
several countries of Europe (Sweetman 2009).

Table 6.1 Pharmaceutical dosage forms containing probiotics marketed

in Europe

Probiotic product Country

Antibiophilus®
Biolorin®
Austrich
Doederlein®
Symbiolor®
Biolorin®
Belgium
Lacteol®
Antibiophilus®
Diarlac®
Lacteol® France
Lyobiidus®
UL-250®
Antibiophilus®
Hamadin®
Hylac N®
Germany
Omnisept®
Symbiolor®
Vagilor®
Lacteol®
Lactoilus®
Spain
Lactolioil®
Ultra Levura®
Bio Acidophilus®
U.K.
Biodophilus®
Lacteol®
Antibiophilus® Portugal
UL-250®
Source: Adapted from Sweetman (2009)
230 Development of Probiotic Dosage Forms

Probiotic strains must be able to grow under industrial

conditions and survive and retain their functionality during storage.
The reduction of viable cells during storage, processing, and passage
through the GI tract is a signiicant challenge in formulating dosage
forms containing probiotics. Several studies have reported that oral
doses higher than 109 colony forming units (cfu) per day are required
to restore and maintain the bacterial lora (Zárate and Nader-Macias
2006).
Because several pharmaceutical industrial processes may induce
an increase in the temperature of the handled material and may
expose it to moisture variations, the viability of probiotics must be
studied at different conditions of temperature and relative humidity
(RH). These temperatures and moisture conditions should be
selected according to the guidelines of the International Conference
Harmonization of Technical Requirements for the Registration of
Pharmaceuticals for Human Use (ICH 2002). Temperature and RH
conditions for climatic zones I—Temperate (e.g., Japan, Northern
Europe, United States, Canada, Russia) and II—Mediterranean
subtropical (United States, Japan, Southern Europe) are for real-
time test conditions 25 ± 2°C and 60 ± 5 % and for accelerated test
conditions 40 ± 2°C and 75 ± 5% (ICH, 2002, USP).
The irst probiotic products available were mostly liquid
formulations that showed low cell viability after oral administration,
mainly because bacteria did not survive in the stomach conditions.
Nowadays, the development of suitable solid dosage forms allows
obtaining higher levels of bacterial survival.

6.2 Manufacturing of Dosage Forms Containing

Probiotics
A critical aspect of product development is the implementation of a
manufacturing process that assures that a product can be produced
in a reproducible manner (Ross and Boucher 2012). Expectations
for product development of an LBP do not differ from other
regulated products that consist of live microorganisms, such as live
vaccines or microbial vectors for gene therapy. The development and
manufacturing of dietary/food supplement dosage forms is similar
to medicines; nevertheless, they do not need to follow the stringent
guidelines used in a medicinal product.
 Manufacturing of Dosage Forms Containing Probiotics 231

Safe and effective use of an LBP depends on consistent

manufacture according to valid speciications. Probiotic formulations
should include selected microorganisms with the ability to survive
during the technological process and remain viable afterward with
unaltered properties for long periods of storage (Zárate and Nader-
Macias 2006).
Probiotic products often utilize lyophilizates of microorganisms
in order to produce dosage forms. Lyophilization is a process
extensively used for preservation of biological samples. However,
during freeze-drying, the cells experience extreme environmental
conditions such as low temperature and low humidity that may
produce structural and physiological damage to the bacterial cells
resulting in the loss of viability. To reduce these undesirable effects,
protective agents are usually added to the samples before freezing
or freeze-drying (Zárate and Nader-Macias 2006).
Sutton (2008) suggested that the early development of probiotic
products should include the following steps:
1. Select and characterize a strain;
2. Document the production batches for nonclinical studies and
establish working speciications;
3. Repeat the production with few or even no modiications to
the product clinical lots;
4. Conirm or modify the speciications on the basis of clinical
results;
5. Begin stability studies.
The active ingredient of a biotherapeutic is the microorganism
that should be administered in a dosage form suitable to ensure
accurate dosing and good stability (Sutton 2008). Generally,
concentrated cultures of microorganisms are adjusted by dilution,
dry preparations are mixed with dry ingredients such as stabilizers
or diluents, and powders may be supplied with pre-packaged buffer
solutions for reconstitution. (Sutton 2008).
The reactivation of dried cultures is a critical parameter in
obtaining active and effective probiotic strains (Muller et al. 2010).
Muller et al. studied the effect of several rehydration conditions on
the viability of B. longum NCC3001 and L. Johnsonii La1. These authors
concluded that the reactivation conditions are a vital step to obtain
maximal viability counts. Furthermore, a variation can be found
between species and strains and, as a consequence, any universal
232 Development of Probiotic Dosage Forms

proceeding suitable for reactivation of bacteria cannot be designed.

The reactivation conditions should be optimized for each strain used
in order to achieve accurate numbers of viable probiotics.
Probiotic formulations in the form of solid dosage forms often
require some type of other ingredients (also termed excipients in the
pharmaceutical ield) such as diluents (to increase to the volume),
binders (to give adhesiveness), lubricants (to reduce adhesion),
glidants (to improve powder low), or even disintegrants (to facilitate
deaggregation).
The release of probiotics from dosage forms may be modiied.
In pharmaceutical ield, modiied-release dosage forms are
preparations in which the release of the drug has been deliberately
changed, regarding the rate and/or the place where it occurs,
resulting from a speciic process of formulation and/or a special
manufacturing method, and is therefore different from that achieved
with an immediately-release form (Eur. Pharmacopeia 2008).
Modiied-release dosage forms include prolonged or extended-
release (special type of modiied-release dosage form showing a
slower release of the drug than that achieved with an immediate
or conventional-release administered by the same route),
delayedrelease (special type of modiied-release dosage form
showing a release of the drug that is delayed), and pulsatile-release
(special type of modiied-release dosage form showing a sequential
release of the drug) dosage forms (Figure 6.1) (Eur. Pharmacopeia
2008).

Figure 6.1 Several types of modiied drug release.

 Manufacturing of Dosage Forms Containing Probiotics 233

Solid dosage forms may be enteric protected to ensure passage

through the adverse conditions of the stomach and the release of
probiotics in the intestine. They are called gastro-resistant (GR)
forms and can be prepared with excipients, in which the therapeutic
agents are homogeneously dispersed, preventing the entrance of
the gastric luid (enteric-matrix) or cover the dosage form (enteric-
coated). In this GR case, the release of probiotics is delayed until
the dosage form reaches the intestinal luid. The main principle
of probiotics release is to the change the gastric pH to enteric pH,
which activates its release from the solid dosage form.
With a proper selection of a solid-forming matrix, entrapped
bacteria can be more easily protected against the low pH of the
stomach. Suitable functional polymers have been studied to produce
solid dosage forms having the ability to maintain their physical
integrity in gastric luid, minimize the penetration of solvent at
acidic pH values, and increase the bacteria survival.
Several probiotic products containing speciic probiotic strains
have been developed in formulations such as powders (Ying et
al. 2010), capsules (Bruno and Shah 2003), tablets (Stadler and
Viernstein 2003; Klayraung et al. 2009), vaginal suppositories (Kale
et al. 2005), and other dosage forms (Kouimtzi et al. 1997; Çaglar et
al. 2007; Iovieno et al. 2008)

6.2.1 Powders
Oral powders are dosage forms consisting of solid, loose, dry,
inely divided particles that are intended for internal use (Eur.
Pharmacopeia 2008; USP31 2008). They may contain bioactive
compounds and/or other excipients such as coloring, lavoring, and
sweetening agents. In general, oral powders are limited to relatively
nonpotent drugs or dietary/food supplements. The administration
of oral powders could overcome the dificulty in swallowing tablets
experienced by children or even some adults. In the production of
oral powders, measures are taken to ensure an adequate particle
size, which is generally done by milling and/or sieving.
Powders containing probiotics are frequently used to disperse
in water (extemporaneous oral suspensions). Some examples of
marketed powders for oral suspension containing probiotics are
presented in Table 6.2.
234 Development of Probiotic Dosage Forms

Table 6.2 Marketed powders for oral suspensions containing probiotics

Probiotic product Composition

UL-250® Sacharomyces boulardii
Bacilor® Lactobacillus acidophilus
Antibiophilus® Lactobacillus casei
Lyobiidus® Biidobacterium biidum

In the studies of Ying et al. (2010), a commercial lyophilized

powder was encapsulated in a oil-in-water emulsion, stabilized by
whey protein and modiied resistant starch, and dried into powders
by freeze-drying or spray drying. The results of this study showed
that when the same encapsulating matrix and probiotic preparation
were used, the probiotics survival after manufacturing was similar
using freeze-drying or spray drying.

6.2.2 Capsules
Capsules are solid dosage forms with hard or soft container or shell
made from gelatin or, less often, from other suitable material (Eur.
Pharmacopeia 2008; USP31 2008). There are two types of capsules,
hard and soft (one-piece). Hard capsules are generally preferred in
order to administer probiotics.
The hard capsules, which consist of two cylindrical pieces (body
and cap) closed at one end, are typically used to contain powders.
These capsules are produced by a process based in a patent dated
from 1846 (Jones 2004). Empty standard size hard capsules are
supplied in bulk containers.
The illing of hard capsules may employ machines that form
powder plugs and eject them into the capsule bodies after having
separated the cap of the shell.
Hard capsule sizes are standard, generally ranging from n° 5
(0.13 ml), the smallest, to n° 000 (1.37 ml), although for human
administration the largest size used is n° 00 (0.95 or 0.93 ml
depending on the manufacturing). Gelatin capsules are readily
soluble in water at 37°C.
Probiotics in the form of powders are also used to ill capsules.
The powder formulations often require some type of excipients such
as illers, glidants, or even disintegrants. It is also possible to prepare
 Manufacturing of Dosage Forms Containing Probiotics 235

GR capsules that typically use GR-coated granules or in certain cases

coated shells.
As probiotic preparations include different excipients, apart
from the probiotic strains, it is technologically valuable to evaluate
the effect of these compounds on the physiology of the selected
microorganisms. For this reason, Zárate and Nader-Macias (2006)
evaluated the survival rates and probiotic properties of three human
vaginal lactobacilli lyophilized with different excipients (lactose,
skim milk, and ascorbic acid), and stored in gelatin capsules placed
in glass lasks at 5°C under darkness during 15 months. This study
demonstrated that the Lactobacillus spp. tested maintained high
viability up to 12 months in capsules containing ascorbic acid
used individually or combined with lactose, milk, or both, whereas
lyophilization and storage with only lactose or skim milk signiicantly
decreased their survival.
A study by Reid and Bruce (2006) demonstrated that daily oral
intake for 2 months of gelatin capsules containing L. rhamnosus
GR-1 and L. fermentum RC-14 resulted in a signiicant increase in
the number of vaginal lactobacillus, combined with a decrease of
coliforms and fungi.
Ya et al. (2010) studied the effectiveness of vaginal probiotic
capsules for recurrent bacterial vaginosis (BV) prevention and
concluded that the short-term probiotic prophylaxis was well
tolerated and reduced BV recurrence and G. vaginalis risk through
11 months after treatment.
Table 6.3 presents some examples of marketed capsules
containing probiotics.

Table 6.3 Marketed capsules containing probiotics

Probiotic product Composition

Bactilsubtil® Bacillus subtilis
Activecomplex Flora® Lactobacillus acidophilus and Biidobacterium
biidum
Inloran Berna® Lactobacillus acidophilus and Biidobacterium
biidum
Lacteol® Lactobacillus acidophilus
Antibiophilus® Lactobacillus casei
UL-250® Saccharomyces boulardii
236 Development of Probiotic Dosage Forms

6.2.3 Tablets
Tablets are solid dosage forms usually obtained by compressing
uniform volumes of particles (powders or particles aggregates)
(Eur. Pharmacopeia 2008). Tablets are the most widely used
pharmaceutical dosage form. The irst tablets comparable to those
used today have their roots in the invention of William Brockedon, in
the 19th century. In 1843, Brockedon obtained a patent for “Shaping
Pills, Lozenges and Black Lead by Pressure in Dies.” It is believed
that John Wyeth and his brother Frank were the irst to use the term
“compressed tablet” and register it in 1877 to protect and restrict its
use (Troy 2005).
Tablets are prepared by the application of a high pressure to
uniform volumes of powders (direct compression) or granules
(obtained by wet or dry granulation) using tablet presses (Fig. 6.2).
Tablets may be coated in order to facilitate its administration, to
protect its bioactive content, or to modify its release.

Figure 6.2 Compression of tablets.

To obtain a tablet, it is usually necessary to use some type of

excipients such as diluents, binders, disintegrants, lubricants, and
glidants. The design of tablets using functional polymers improves
the stability of probiotics, contributes to accurate dosing and ease
of administration, and provides the ability to produce dosage forms
containing probiotics at a large scale (Klayraung et al. 2009).
Generally, tablets containing probiotics are produced by direct
compression of mixtures of excipients and freeze-dried probiotics.
In the studies of Graff et al. (2008), matrix tablets containing
freeze-dried probiotics were prepared by direct compression using
hydroxypropylmethylcellulose (HPMC), which is one of the most
used hydrophilic polymers for the production of modiied release
 Manufacturing of Dosage Forms Containing Probiotics 237

systems. HPMC has the ability to form a gelling barrier limiting the
release of viable probiotics.
In the studies carried out by Klayraung et al. (2009) to evaluate
the effects of formulation and processing parameters on bacterial
viability, tablets were also prepared by direct compression. In this
study, an exactly weighed amount of powder mixture containing LAB
powder and HPMC phthalate was illed into a die and tablets were
formed under a determined force ranging from 2 to 20 kN.
Tablets can be designed to modify the release and enhance the
adhesion and colonization of the probiotic microorganisms to the
epithelial mucosa of human host by using suitable kinds of tablet
excipients (Maggi et al. 2000).
Different polymers have been studied to form the protective
matrix. For example, Rodrigues et al. (2011) studied the viability of
ive probiotic strains (L. casei 01, L. acidophilus La-5®, L. acidophilus
Ki, B. animalis BB-12®, and B. lactis Bo) immobilized on various
polymers [sodium alginate, xanthan gum, L-carrageenan, cellulose
acetate phthalate (CAP), chitosan/sodium alginate, and whey protein
concentrate] at various concentrations and concluded that alginate
and whey proteins were the most suitable polymers, except for L.
acidophilus Ki and L. casei 01 at 2% (m/v) alginate.
Some examples of polymers used in the production of GR tablets
containing probiotics are summarized in Table 6.4.

Table 6.4 Examples of polymers used in the production of GR tablets

containing probiotics

Polymers Reference
Sodium alginate in combination with Chan and Zhang, 2002, 2005
hydroxypropylcellulose
HPMC acetate succinate Stadler and Vernstein, 2003
Carboxymethyl high amylase starch Calinescu et al., 2005
Carboxymethyl high amylase starch in Calinescu and Mateescu,
combination with chitosan 2008
HPMC phthalate Klayraung et al., 2009

Chan and Zhang (2005) developed tablets with a core of L.

acidophilus ATCC 4356 cells surrounded with sodium alginate and
obtained a higher stability than the powders containing the same
bacteria after 30 days of storage at 25°C.
238 Development of Probiotic Dosage Forms

GR tablet formulations of lactic acid bacteria (LAB) were

developed by Stadler and Viernstein (2003) using HPMC acetate
succinate (HPMCAS) as well as alginates, apple pectin, and Metolose™
(methylcellulose/hydroxypropyl methylcellulose) as matrix-forming
agents. The results of this study demonstrated that a high content of
HPMCAS as well as medium or high compaction force (5 or 10 kN,
respectively) are needed to achieve gastric juice resistance. When
tablets were prepared with low amounts of HPMCAS, the addition of
sodium alginate was necessary to ensure protection against artiicial
gastric juice.
Novel excipients were tested for the protection and intestinal
delivery of probiotic bacteria. Calinescu et al. (2005) proposed three
variants of carboxymethyl high-amylase starch (CM-HAS) as new
excipients for the formulation of oral tablets containing lyophilized
E. coli. These authors also developed a new hydrophilic dosage
system on the basis of an ionic self-stabilization of CM-HAS and
chitosan for probiotic colon delivery and found that the increase
of percentage and molecular weight of chitosan caused a decrease
of bacteria release rate. The CMHAS dry-coated monolithic tablets
changed the effect of chitosan molecular weight on bacteria release
and improved the percentage of delivered bacteria in simulated
intestinal conditions.
The use of succinylated β-lactoglobulin (S β-LG) as a novel
functional tablet excipient was studied by Poulin et al. (2011). These
studies showed that tablets produced by direct compression of a
mixture of B. longum HA-135 and S β-LG enhanced bacteria survival
in gastric conditions and showed good stability after storage at 4°C
during 3 months.
Particular attention should be given to the design and
production of pharmaceutical dosage forms, intended for the vaginal
administration of living cells, as bacterial suspensions show poor
stability and are easily cleared after vaginal application. Different
types of formulations for vaginal delivery of probiotics are nowadays
commercialized in the international market. However, the largest
group of probiotic vaginal formulations consists of freeze-dried
probiotics compressed into tablets or encapsulated in a gelatin base
(Santiago et al. 2009).
A signiicant proportion of tablets containing probiotics are
designed for vaginal administration aiming the treatment of vulvo-
vaginal disorders. Throughout the production process and storage,
 Manufacturing of Dosage Forms Containing Probiotics 239

cell viability of probiotic strains should be maintained and a strict

quality control should ensure the safety and eficacy of the inal
product. Thus, a probiotic vaginal formulation should (Santiago
et al. 2009) provide a long retention time to maximize the release of
probiotics and present a suitable release, so that colonization occurs
in different parts of the vagina, does not damage the vaginal mucosa
and vaginal microlora, provides an easy form of administration, and
do not cause discomfort to the patient.
Lactobacilli are the predominant microorganisms in the healthy
human vaginal ecosystem (Lepargneur and Rousseau 2002).
Therefore, is important to know the properties of lactobacilli and
their capacity to proliferate or to maintain their viability in the
vaginal medium.
Several studies of probiotics for vaginal infections have been
reported (Falagas et al. 2006; Dover et al. 2008; Gil et al. 2010).
Most probiotic preparations for vaginal use include one or more
lactobacillus species (Elmer 2001). For example, studies using
vaginally administered L. rhamnosus GR1 in association with either
L. reuteri B54 or RC 14 have shown that vaginal colonization can be
achieved (Reid and Bruce 2006).
Mastromarino et al. (2009) determined the effectiveness of
vaginal tablets containing Lactobacillus spp. in the treatment
of BV and in the restoration of a healthy vaginal lora showing
that intravaginal administration of exogenous selected strains of
lactobacilli can restore a normal vaginal microbiota and be used in
the treatment of BV.
Fazeli et al. (2006) evaluated the viability and release of
L. acidophilus from slow-release vaginal tablets prepared using six
different retarding polymers and from two effervescent fast-release
tablets prepared using citric or adipic acid. Although fast-release
formulations produced a fast bacterial release in comparison to
polymer-based slow-release tablets, they were less stable upon
storage. All the slow-release polymers showed a high bacterial
release except carbomers with poor bacterial shedding probably
due to their high viscosity characteristics.
Maggi et al. (2000) formulated single and double-layer tablets
containing four types of lactobacilli for vaginal administration
(L. brevis, L. salivarius, L. crispatus, and L. gasseri). In this study,
double-layer tablets containing an effervescent layer for the fast
release of a percentage of bacteria and a slow-release layer to sustain
240 Development of Probiotic Dosage Forms

probiotics in vaginal environment were prepared. Three types of

polymers (HPMC-HV, HPMC-LV, and Carbopol 934 PH) were studied,
and formulations were tested regarding technological processing,
cell adhesion properties of the microorganisms, and bacterial
viability and stability. These studies showed that both fast and slow-
release tablets maintained a good stability after storage in plastic
containers at 4°C–6°C during 18 months, however, lower than the
stability showed by the freeze-dried powder.

6.2.4 Vaginal Suppositories

A suppository is a solid drug delivery system of various weights and
shapes adapted for introduction into the rectum (rectal suppository),
vagina (vaginal suppository), or urethra (urethral suppository).
Suppositories usually melt, soften, or dissolve at body temperature
(USP31 2008; Sweetman 2009).
The word suppository derives from the Latin term suppositorium
that in turn seems to come from the union of the words supositum
(low position) + torus (torus), hence its etymological roots suggest
a solid pharmaceutical form for administration into the lower body
cavities (rectum, vagina, urethra) (Prista et al. 1990). This broad
concept, although followed in North America, is not followed in
some countries. So, the name suppository in Europe designates
only the solid preparations for rectal administration, while the solid
formulations for vaginal administration are called pessaries (Eur.
Pharmacopeia 2008).
The suppositories are frequently obtained by compression or
molding (sometimes referred as fusion) techniques. The molding
technique is the most used method for the preparation of supositories
and consists in the melting of excipients, addition of probiotics at
lower temperatures, and pouring the mixture into appropriate
molds. Suppository bases usually employed are hard fat, cocoa butter,
mixtures of polyethylene glycols (PEGs), and various gelatinous
mixtures. The vaginal suppositories have certain advantages over
other dosage forms, including the fact that the uniformity of mass can
be maintained, are easy to apply into the vagina without irritation,
and does not require a large volume of dissolution medium for the
release of the active substance (Kale et al. 2005).
 Manufacturing of Dosage Forms Containing Probiotics 241

The earlier studies involving the use of vaginal probiotics date

from 1990s of the 20th century. Reid et al. (1992) examined the
recurrence of urinary tract infections (UTIs) in women who were
administered vaginal suppositories containing probiotics, and after
administration of the vaginal suppositories, only 21% of women
had recurrences of UTI, compared with 47% that were observed in
women who were given placebo. However, later studies proved that
the opposite and one of the possible causes for these results may
have been the fact that these authors used L. casei in the formulation
of vaginal suppositories, which would not be the most suitable strain
for vaginal administration (Baerheim et al. 1994).
On the contrary, more recent studies have demonstrated the
eficacy and safety of such treatments. In a study by Uehara et al.
(2006), it was shown that vaginal suppositories with L. crispatus
GAI 98332 can signiicantly reduce the recurrence of UTI, without
causing adverse complications during treatment. In this study,
vaginal suppositories containing 1 × 108 cfu were administered to
each patient, twice a day, during 1 year. The reason why this study
has proved to be so promising is related to the selection of a more
stable strain, isolated from the vagina of healthy women, known for
their ability to produce hydrogen peroxide, as well as their ability to
adhere to the vaginal epithelium.
To further evaluate the safety and eficacy of vaginal suppositories
containing L. crispatus CTV-05 for prevention of recurrent UTI,
Czaja et al. (2007) performed a randomized, double-blind study
in premenopausal women with a history of recurrent UTI and
concluded that vaginal suppositories containing this probiotic were
well tolerated and mild inlammation of the urinary tract was only
detected in some women.
Kale et al. (2005) developed formulations of vaginal supposito-
ries containing freeze-dried L. sporogenes, and then studied the in
vivo performance of the formulations developed. Three vaginal sup-
positories formulations were prepared using the molding technique,
containing cocoa butter, glycerinated gelatin, and PEG 1000, respec-
tively. Glycerinated gelatin vaginal suppositories had the most satis-
factory physical properties. Similarly, studies with different formu-
lations of vaginal tablets containing different bacterial strains have
shown to be promising in the transport of probiotics (Mastromarino
et al. 2002).
242 Development of Probiotic Dosage Forms

Kaewsrichan et al. (2007) prepared two vaginal formulations,

namely effervescent tablets and hollow-type PEG-based
suppositories, containing lactobacillus, aiming to maintain as high
as possible the live cells with proper probiotic properties. In this
study, suppositories were prepared by melting the PEG 4000 at 60°C
before the addition of PEG 400. After cooling, the mixture of PEGs
was poured into suppository molds, and a hollow centered to the
mold was made by pressing sterile stirring rod into the mixed base
prior setting. Bacterial powder was illed into the hollow followed
by capping with the mixed PEG, in order to avoid the exposure of
probiotics to wetting steps and excessive manipulation techniques.
The results of this study showed that effervescent tablets promptly
released the microorganisms and the abilities on bacteriocin and
H2O2 productions were not deteriorated by the suppositories
manipulation.
Kaewnopparat and Kaewnopparat (2009) also undertook the
development of vaginal suppositories with a mixture of PEG and
Witepsol H15, using different methods of preparation, in order to
compare the viability of L. casei. These authors prepared vaginal
suppositories by the conventional method and by the hollow-type
method. The hollow-type method (Fig. 6.3) was developed by
Watanabe and Matsumoto (1986) in order to study the effectiveness
of active substances when administered by rectal route. The vaginal
suppositories whose base excipient used was a mixture of PEGs and
whose method of preparation was the hollow-type method proved to
be the most suitable for the administration of vaginal lactobacillus,
regarding the quick release and microbiological stability. The
hollow-type method can eliminate the disadvantage of the heating
process on the survival of lactobacillus during preparation and also
the interactions between these microorganisms and the suppository
excipients.
More recently, Rodrigues (2011) developed two formulations
of vaginal suppositories with PEGs and Witepsol H12 for the
administration of vaginal L. acidophilus. The results of this study
showed that the incorporation of freeze-dried bacteria on these
excipients did not result in signiicant losses of viable bacteria,
giving rise to the idea that these vaginal suppositories possess
good properties to promote the replacement of the vaginal lora in
situations of UTI.
 Manufacturing of Dosage Forms Containing Probiotics 243

Figure 6.3 Suppositories prepared by the hollow-type method. Adapted

from Kaewnopparat et al. (2009).

Some examples of vaginal suppositories available in the

international market are presented in Table 6.5 (Nader-Macias
2008).

Table 6.5 Examples of vaginal suppositories available in the international

market (Nader-Macias 2008)

Product Probiotic composition

Vagilor® (Germany) L. acidophilus
Lactinex® (Argentina) L. acidophilus
Tropivag®(Argentina) L. rhamnosus Lcr35®
HLC Candaclear®(Canada) L. acidophilus
Intrafresh® (UK) L. acidophilus

6.2.5 Other Pharmaceutical Dosage Forms

6.2.5.1 Chewing gums and lozenges

Chewing gums are an ancient and widely popular form of confection
that recently came to be used as a drug delivery system (Sweetman
2009). Chewing gums are solid preparations with a base consisting
mainly of gum that is intended to be chewed but not swallowed (Eur.
Pharmacopeia 2008). Their active substance is released by chewing,
followed by dissolution or dispersion in saliva for local or systemic
244 Development of Probiotic Dosage Forms

treatment. Chewing gums can be made by compression or other

processes.
Lozenges are solid preparations, usually in lavored, sweetened
base, which are intended to dissolve or disintegrate slowly in the
mouth (USP31 2008; Sweetman 2009). They can be prepared
by molding (sometimes referred as pastilles) or by compression
(sometimes referred as troches) (USP31 2008).
Oral research related to the beneicial effects of probiotics has
shown that one strain with a proven intestinal effect may not be
beneicial to health in the oral environment. Recent clinical trials
have demonstrated a decrease in the prevalence of moderate or
severe gingival inlammation in adults who frequently use chewing
gums containing probiotics.
Saliva plays an important role in propagating oral bioilms.
However, sometimes, the role of saliva on bacterial survival can
be contradictory (Stamatova and Meurman 2009). Some probiotic
bacteria may be beneicial for periodontal health if they are able
to attach to the oral bioilm inhibiting the pathogen growth and
metabolism. Dental caries are formed due to interaction between
acid-producing bacteria and fermentable carbohydrates. It has been
demonstrated that probiotics in dairy products may change oral
ecology. For example, Çaglar et al. (2006) investigated the effect of
two oral delivery systems containing L. reuteri ATCC 55730 on the
levels of salivary mutans streptococci (MS) and lactobacilli (LB) and
concluded that the daily ingestion of straws or lozenges containing
lactobacilli-derived probiotics reduced the levels of salivary
bacteria.
In another study, Çaglar et al. (2007) investigated whether the
association of potential anticarie agents, such as probiotic bacteria
and xylitol, would increase the suppressive effect on dental caries
caused by bacteria in saliva. These authors evaluated the effect
of xylitol and probiotic chewing gums taken by healthy human
volunteers, three times daily after meals, on salivary MS and LB.
Chewing gums contained two strains of L. reuteri (ATCC 55730
and ATCC PTA 5289), and each pellet of the xylitol gum contained
approximately 1.0 g of xylitol as single sweetener. The results of
this study demonstrated that chewing gums containing probiotic
bacteria or xylitol may decrease signiicantly the levels of salivary
MS. However, the association of probiotic and xylitol chewing gums
did not appear to be advantageous.
 Manufacturing of Dosage Forms Containing Probiotics 245

Twetman et al. (2009) investigated the effect of short-term

use of probiotic chewing gums containing two strains of L. reuteri
(ATCC 55730 and ATCCPTA 5289) at a dose of 108 cfu/gum. The
results of this study demonstrated the signiicant dosedependent
modulating effect of a short-term intake of probiotics on the oral
immune response. In another study, Mayanagi et al. (2009) studied
the effect of the oral administration of lactobacilli on the bacterial
population in supra/subgingival plaque. Healthy volunteers without
severe periodontitis were randomized into two groups to receive
L. salivarius WB21 (2.01 × 109 cfu/day) and xylitol in tablets or
placebo with xylitol for 8 weeks. These authors concluded that the
oral administration of probiotic lactobacilli decreased the number
of ive selected periodontopathic bacteria and contributed to the
beneicial effects on periodontal conditions.
For the prevention and treatment of halitosis, the replacement of
bacteria involved in halitosis by colonization with probiotic strains
from the indigenous oral microbiota of healthy humans may have
potential application. The use of probiotics in the treatment of
halitosis has been reported in several studies. For example, it was
shown that S. salivarius K12 taken in a lozenge after a mouthwash
could reduce the oral volatile sulfur compounds levels in 85% of the
volunteers submitted to the test (Stamatova and Meurman 2009).
Iwamoto et al. (2010) also studied the inluence of probiotics
on halitosis and oral health. These authors postulated that with the
administration of L. salivarius WB21, the amount of periodontopathic
bacteria, which produce H2S and CH3SH, in the saliva of the patients
with halitosis would decrease. However, this study emphasizes the
need for better understanding of probiotic functions in the oral
cavity.

6.2.5.2 Gels
Gels are semisolid systems consisting of either suspensions made
up of small inorganic particles, forming a network of small discrete
particles (two phase gel), or large organic molecules interpenetrated
by a liquid in such a manner that no apparent boundaries exist
between them (single phase gel) (USP31 2008). In Europe, gels are
deined in a slightly different manner as “liquids gelled by means of
suitable gelling agents” (Eur. Pharmacopeia 2008).
Gels can be used to administer drugs topically, into body cavities
or orally. Few commercialized vaginal gels containing probiotics
246 Development of Probiotic Dosage Forms

are available. For example, as described before, two vaginal gels

containing L. acidophilus are marketed in France (Trophigil® and
Florgynal®).
Ahmad et al. (2008) developed a bioadhesive vaginal gel for the
treatment of vaginal infections using guar gum, xanthan gum, and
HPMC K4M as bioadhesive polymers and monosodium citrate as an
acid-buffering agent to provide a pH of 4.4. To treat vaginal infections,
clotrimazole and metronidazole were used in the formulation along
with lactobacillus spores. The results of this study showed that the
bioadhesive gel developed had better antimicrobial action than
intravaginal drug delivery systems commercially available.

6.2.5.3 Eye drops

Eye drops are sterile aqueous or oily solutions or suspensions
intended for instillation into the eye (Eur. Pharmacopeia 2008).
Iovieno et al. (2008) evaluated the eficacy of L. acidophilus eye
drops in controlling the symptoms of vernal keratoconjunctivite
(VKC). In this study, eye drops containing L. acidophilus diluted in a
saline solution (2 × 108 cfu/ml) were applied in both eyes of seven
patients with mild to moderate VKC, four times a day, during 4 weeks.
The results of this study showed that probiotic eye drops improved
symptoms of VKC. However, the authors of this study concluded that
to conirm the effects of Lactobacilli on VKC, additional double-blind
studies are needed.

6.2.5.4 Pellets
Pellets can be deined as small, free-lowing, spherical, or semi-
spherical solid units, typically from about 0.5 mm to 1.5 mm, usually
intended for oral administration and generally manufactured by
the process of extrusion-spheronization (process usually used in
the pharmaceutical industry to produce spheroids with uniform
size) (Troy 2005). The death of intestinal microlora by antibiotic
or radiation treatment can lead to overgrowth of pathogenic
microorganisms. Reintroduction of a normal lora into the
“sterilized” gut may help to resolve this problem. Pellets containing
bacteria have been prepared by extrusion-spheronization to assess
the effects of processing on bacterial survival. Gram-negative
aerobic (Escherichia coli), gram-positive aerobic (Staphylococcus
saprophyticus and B. subtilis), and gram-positive anaerobic (B.
 Dosage Forms Characterization 247

angulatum) vegetative bacteria, together with spores of B. subtilis,

were introduced separately into a formulation, which was extruded,
spheronized, and dried to produce pellets. Samples were taken at
each stage of the pellet production process and checked for bacterial
survival. The results showed that spores survived all stages of
the process. Survival levels of the gram-positive organisms after
extrusion, spheronization, and drying were signiicantly higher than
the gram-negative E. coli, with 5% B. angulatum remaining viable in
the inal dried pellets. The effects of extrusion speed, extrusion die
length-to-radius ratio, and extrusion pressure on the viability of the
more sensitive E. coli were investigated. The level of death was not
affected by extrusion speed or die length-to-radius ratio. However,
survival was inversely proportional to extrusion pressure over the
range 1–8000 kPa (Kouimtzi et al. 1997).

6.3 Dosage Forms Characterization

To orientate the development of the pharmaceutical dosage forms,
some pharmacopeial tests may be used. These tests that are fully
described in pharmacopeias were primarily developed to conirm the
quality of the medicines or probiotic dosage forms. Pharmacopeias are
oficial compendia that publish quality standards for pharmaceutical
substances and products (monographs containing directions for
their identiication, characterization, and assay). There are three
large pharmacopeias in the world, the European Pharmacopoeia
(Ph. Eur.), the United States Pharmacopeia (USP), and the Japanese
Pharmacopoeia (JP).
Uniformity of mass test (or more recently the Uniformity of
Dosage Unit test), disintegration tests, and dissolution/release
studies are some of the tests that should be performed in order to
ensure the quality of solid dosage forms. The uniformity of dosage
test can be made by two methods, namely, content uniformity
(assay of the individual content in a number of individual dosage
units) or weight variation (consists in individually weighing a
number of individual units and determining their average mass).
The disintegration test determines whether solid dosage forms
disintegrate within a prescribed time when placed in an immersion
luid at a temperature of 37 ± 0.5°C. The time for disintegration is
registered when the dosage form is completely disintegrated or
248 Development of Probiotic Dosage Forms

dissolved in the medium and the mean values are calculated from
six parallel measurements. The dissolution test is used to determine
the dissolution rate of the active ingredients of dosage forms placed
in an immersion luid at 37 ± 0.5°C. The basket (apparatus 1), the
paddle (apparatus 2), the reciprocating cylinder (apparatus 3),
or the low-through cell (apparatus 4) may be used (Fig. 6.4). At
speciied time, samples of the dissolution liquid are withdrawn,
iltered, and the amount of bioactive ingredient released is evaluated
by suitable analytical methods (USP31 2008). In the case of dosage
forms containing probiotics, the enumeration of viable cells released
should be done.

Figure 6.4 USP dissolution test apparatus.

In the pharmaceutical industry, dissolution testing is one of

the most important tools in drug development and quality control.
Dissolution studies of oral tablets may be performed in simulated
gastric luid (SGF) or/and simulated intestinal luid (SIF). In the
studies reported by Poulin et al. (2011), the dissolution of tablets
was performed in SGF and in SIF without the addition of pancreatin
because preliminary studies had demonstrated that this enzyme did
not inluence probiotics viability.
Vaginal tablets unless intended for prolonged local action can
be evaluated with the test for disintegration of suppositories with
modiications (Eur. Pharmacopeia 2008). In the case of dissolution
studies for vaginal tablets, they should be performed in simulated
vaginal luid. The understanding of the interactions of probiotic
microorganisms with the vaginal luid can help to design improved
vaginal probiotic formulations. For this purpose, in vitro conditions
 Dosage Forms Characterization 249

similar to those in vivo are needed (Tomás and Nader-Macías

2007).
In order to achieve a close in vitro/in vivo correlation for tablets
containing probiotics for oral or vaginal administration, some
parameters that must be considered when designing a dissolution
apparatus include the volume and composition of the dissolution
medium. Taking this into account, Kale et al. (2005) developed a
dissolution apparatus that could simulate the vaginal environmental
for vaginal tablets containing a probiotic agent (lactobacillus). These
tablets are a special type of dosage form in which the released
bacteria attach to the vaginal membrane in such a way that they
colonize to form a protective environment against the vaginal
pathogens. The design of the apparatus was a modiied form of a
model by Setnikar and Fantelli (1962). With this study Kale et al.
(2005) concluded that the apparatus developed presented several
advantages such as the minimization of the dissolution luid volume
and elimination of exposure of the vaginal formulation to agitation
and sampling devices, being a very promising method to be adopted
for routine quality control testing.
In the case of suppositories, it may be necessary to perform a
proper test to demonstrate that the release of the active substances
is satisfactory as, for example, the dissolution test. For hydrophilic
suppositories, the basket, paddle, or low-through cell (already
referred) can be used (Fig. 6.5). For lipophilic suppositories, a
modiied basket method, a paddle method with a wired screen
and a sinker, and a modiied low-through cell with a speciic dual
chamber suppository cell (Eur. Pharmacopeia 2008) (Fig. 6.5) have
been recommended (Siewert et al. 2003).

Figure 6.5 Dual chamber suppository cell.

250 Development of Probiotic Dosage Forms

Biologic products are not heat sterilized because active ingredi-

ents are heat labile. An important concern is related with the pre-
vention of cross-contamination with other products manufactured
in the same area and, as a consequence, product contact equipment
should be sterilized.
One of the key parameters indicating the effectiveness of dosage
forms to protect cells originating long shelf life is the stability
of probiotic cells. In quality control in addition to technological
properties, functional properties must also be taken into account.
Properties such as bile and acid stability, adhesiveness, survival
during the manufacturing process, and colonization properties must
be monitored and optimized (Forssten et al. 2011).
It is important to consider that probiotics are living organisms,
and as a consequence, their number undergoes a decrease with
time. Stability is a crucial parameter that provides the capability of a
probiotic to induce health beneices. Thus, the probiotics should be
able to withstand the processing conditions and to survive during
storage. Probiotic stability is related to factors such as gender,
species, strain biotype, and composition of other ingredients of the
dosage form. The stability is also inluenced by other factors such
as water, temperature, pH, osmotic pressure, and oxygen (Del Piano
et al. 2006).
The tests of stability are performed to guarantee that the product
continues to be safe and effective throughout the expiration date
stated on the label. Stability studies should be performed using the
same storage conditions that characterize commercial life of the
product. The dosage form should be tested in the respective container
intended for marketing and should be stored at the recommended
temperature (Sutton 2008).
Special attention is required in all steps of the formulation
development (fermentation, concentration, cell washing,
cryoprotection, freeze-drying, grinding, mixing, and packaging) in
order to guarantee probiotic cultures viability. It is important to
specify the conditions under which the bacteria are produced and
stored to minimize variability from batch to batch, improving the
eficiency of probiotic strains in the inal products (Del Piano et al.
2006).
For those inal products that are dried or lyophilized, the
moisture content is a critical parameter that may affect the viability
of microorganisms and quality of product throughout its useful life,
 Dosage Forms Characterization 251

so for this type of products, the moisture content must be controlled.

Final speciications for the inal dosage forms are established on
the basis of clinical data and manufacturing performance (Sutton
2008).
As stated above, the stability and viability of probiotics in dry
state are affected by the surrounding temperature and water activity.
Using temperatures below 30°C and water activities lower than
0.20, it is possible to maintain the probiotic viability by preserving
the integrity of microbial cell membrane. Solid dosage forms such as
powders, tablets, and capsules have a very low water activity, and as
a consequence, their storage time is longer (Forssten et al. 2011).
Klayraung et al. (2009) studied the stability of tablets containing
probiotics when stored at temperatures of 10°C and 30°C. These
authors reported that tablets stored at 10°C during 6 months showed
very good stability with almost full preservation of the number of
viable cells.
The addition of excipients that act as protecting agents of
probiotics during storage must be taken into account in the
development of a probiotic formulation with a stable shelf-life
(Zárate and Nader-Macias 2006). For example, Champagne et al.
(1996) reported the effect of the addition of gelatin, xanthan gum,
or maltodextrins on the survival during freeze-drying and stability
during storage of four LAB. The results of these studies showed
that gelatin improved the storage stability of freeze-dried L. casei, L.
rhamnosus RO11, and B. longum RO23 cultures kept at 20°C and 4°C.
In general, the additives had a detrimental effect on the stability of S.
thermophilus RO57 during storage at 20°C.
Bora et al. (2009) studied the effect of buffering agents,
encapsulating agents, diluents, disintegrants, glidants, and
lubricants on the survival of B. coagulans spores under isothermal
stress conditions at 40 ± 2°C under 75 ± 5% RH. The results of this
study demonstrated the detrimental effect of RH conditions of 75%
on the viability of the spores of B. coagulans and these probiotics
were found to be compatible with the excipients studied, with the
exception of citric acid monohydrate meglumine and sodium starch
glycolate. In another study, Brachkova (2010) studied the inluence
of the incorporation of Lactobacillus spp. into different dosage forms
keeping their viability and antibacterial activity. In order to achieve
this aim, the survival of probiotic microorganisms in freeze-dried
powders, tablets, pellets, calcium alginate beads, and ilms was
252 Development of Probiotic Dosage Forms

investigated. In this study, two strategies were used: (i) the direct
incorporation of bacterial suspensions into pellets, sodium alginate
beads, and ilms, and (ii) the mixture of freeze-dried bacteria with
tabletting excipients to produce tablets. The vegetative forms of L.
plantarum, L. rhamnosus GG, L. lactis, and L. bulgaricus and spore
form of B. subtilis were considered as model bacterial strains. With
this study, the successful production of various dosage forms for the
delivery of viable lactobacilli with an antibacterial activity against
multi-resistant bacteria was demonstrated.
As stated above, probiotics viability should also be maintained
after the manufacture of dosage forms. For example, one of the
methods described to evaluate the relationship between the applied
pressure during tablets manufacturing and bacterial viability
consisted in producing tablets using different compression forces.
After compression, the tablets are dissolved/disintegrated in an
accurate volume of SIF, and the resulting suspension is diluted and
plated in MRS (Man, Rogosa and Sharpe) agar. After incubation for
48 h at 37°C, the number of viable cells was determined. The initial
number of viable cells in the individual tablet doses of probiotics
should be also evaluated following the same procedure without
compressing the powder mixture.
In the studies of Stadler and Viernstein (2003), the degradation
of lyophilized viable LAB cells during the tablets formulation and
preparation as well as the survival in artiicial gastric juice were
evaluated. The loss of bacteria due to the compression of tablets was
evaluated by calculation from the number of viable cells in the mixed
powders before compression and in the tablets resulting from the
compression. The results of these studies showed that for achieving
the resistance of the gastric juice, high content of HPMCAS as well as
a compaction force of high or medium intensity are required.
Klayraung et al. (2009) also studied the inluence of matrix-
forming excipients and compression forces on the viability of
probiotics. The results of these studies showed that the percentage of
matrix-forming agents and the compression forces can signiicantly
inluence the tensile strength and disintegration of tablets containing
probiotics and also the survival of bacteria. These studies also
demonstrated that the tablets produced with high compression forces
exhibited a slow disintegration time and a bacterial viability greater
than 80%. Furthermore, the addition of sodium alginate resulted in
 Packaging and Storage of Dosage Forms Containing Probiotics 253

higher cell survival in simulated GI luid and a disintegration time of

approximately 5 hours.
Saccharomyces boulardii is a nonpathogenic yeast with proven
health beneits; however, the living yeast is sensitive to environmental
conditions and its viability is less than 1% in the feces after oral
administration. Thus, the studies of Graff et al. (2008) aimed to
formulate dosage forms able to protect the probiotic S. boulardii
from degradation in acidic conditions. Alginate microspheres and
tablets (50.80% of Flowlac® 100 and 13.35% of Methocel® K4M)
enabled to protect the yeast from degradation in acidic conditions
and to release viable cells at pH 6.8. However, despite similar
release proile from both dosage forms, the compression led to a
signiicant decrease in the viability of the freeze-dried yeast. These
investigators concluded that although both formulations were
eficient in protecting S. boulardii in acidic conditions, microspheres
provided higher entrapment eficiency and a faster release of the
viable probiotic in intestinal conditions than matrix tablets.
It has been reported that to improve probiotic survival, it is
necessary to induce a stresstolerance response achieved by pre-
exposing cells to sublethal stresses, such as salt, heat, bile, and low
pH. This pre-exposure can signiicantly increase probiotic survival
following subsequent exposure to lethal stress. Other strategies,
including cell immobilization technology and microencapsulation,
have also been demonstrated to improve the stress-tolerance
proiles of certain probiotic strains (Sleator and Hill 2008). Borges
et al. (2012) studied the effects of encapsulation on the viability
of probiotic strains (L. casei, L. paracasei, L. acidophilus Ki, and B.
animalis BB-12) during exposure to lethal conditions (25% NaCl,
pH 3.0 and 55–60°C). The results of this study showed that with the
exception of exposure to 25% (w/v) NaCl, L. acidophilus Ki (free and
encapsulated cells) demonstrated the highest survival rates through
exposure to lethal conditions of temperature and pH.

6.4 Packaging and Storage of Dosage Forms

Containing Probiotics
The packaging materials used and the storage conditions are
important for the quality and shelf-life of products containing
probiotic bacteria (Saarela et al. 2000).
254 Development of Probiotic Dosage Forms

The methods of packaging and storage must avoid moisture,

oxygen, light, microbial contamination, and high temperatures.
Generally, probiotic dosage forms contain microorganisms in the
freeze-dried form. Freeze-dried products are stored within sealed
glass ampoules, or glass vials, and for dried products, there are other
options such as high barrier plastic bags and blister packs (Morgan
et al. 2006).
Customized probiotic stick packs are also one of the best ways
to ensure that the probiotic products are suficiently protected from
the detrimental effects of humidity and oxygen. For example, several
lactobacillus strains are packaged in aluminum triple-foil, thermo-
closed packs.
There are few studies on packaging and storage, as manufacturers
are charged to establish how their products will be packaged, labeled,
and marketed. Currently, oral powders are often stored in heat-
sealable laminated sachets (individual doses) and in glass or plastic
lasks. For example, for the treatment of acute diarrhea, probiotics
are combined with oral rehydration salts. The salts and the probiotic
strain can be packed in separate sachets, or salt solution can be put
in a carton and the bacteria in a straw (i.e., suspended in oil and
dried inside the straw) (Saxelin 2008). Powdered products may be
supplied with pre-packaged buffered reconstituting solutions. Hard
capsules are generally stored in blisters (container consisting of two
layers in which one is shaped to contain the capsule) and in glass or
plastic lasks. Tablets are generally stored in blisters, strips (container
consisting of two layers, usually provided with perforations, suited
for containing one tablet), and in glass or plastic lasks. In the case of
probiotic products, the materials used in the container should have
low water-vapor transmission rate.

6.5 Conclusions and Future Trends

The use of probiotics as therapeutic agents is currently widespread.
Probiotic strains should be incorporated into suitable dosage forms
in which they can retain their viability and functionality. In addition,
probiotics should be packaged in suitable conditions that can ensure
adequate quality standards.
Highly reproducible probiotic products are required for
clinical use and, as a consequence, validation of each step of the
 Conclusions and Future Trends 255

manufacturing process is a fundamental means of controlling

product reproducibility. For these products, formulation and
packaging constitute signiicant challenges to maintain stability and
to ensure microorganisms viability.
Generally, for probiotics administration, solid dosage forms such
as powders, capsules, and tablets represent the best delivery systems,
as they provide higher stability. Tablets have several advantages
above other dosage forms and, as a consequence, these delivery
systems have been widely studied for probiotics administration.
Novel functional polymers used to improve the stability and survival
of probiotics in gastric conditions are also gaining attention.
Furthermore, the study of the best manufacturing conditions is also
important to maintain the microorganisms viability. For example,
some studies (Stadler and Viernstein 2003; Klayraung et al. 2009)
have demonstrated that probiotic tablets should be prepared with
suitable amounts of excipients and high compression forces in order
to prolong the tablet disintegration, maintaining the viability of
bacteria.
For vaginal administration of probiotics, suppositories have
also been extensively studied and developed because these dosage
forms are easy to apply into the vagina without irritation. In order
to maintain viability of probiotics, hollow-type suppositories have
been prepared. The advantage of using the hollow-type method is
that it can minimize the effect of the heating process on the survival
of bacteria during preparation. However, the industrial production
of this type of suppositories has not yet been implemented.
Although there are currently hundreds of products marketed and
labeled as containing probiotics, many of the strains they contain
have not been evaluated in scientiic trials involving humans (Saxelin
2008). It is also important to note that a higher dose does not always
mean a better effect, and health claims that are being considered
for approval should be connected to an effective daily intake and
a recommended frequency of administration of the dosage forms
containing probiotics (Hoffman 2008).
One approach that should be more investigated in the future is a
combination of probiotics and prebiotics (symbiotic action), which
can result in improved health beneicial products. However, although
the trend is to combine different types of probiotics, it is important
to know the potential antagonistic interactions between strains.
Moreover, the research and production of genetically modiied
256 Development of Probiotic Dosage Forms

therapeutic microorganisms and their incorporation in dosage

forms will allow the production of medicinal products tailored to the
individual needs of each patient.

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Chapter 7

Guidelines and Regulations

J. Paulo Sousa e Silvaa and Ana M. Gomesb

aFaculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira,

228, 4050-313 Porto, Portugal

bCBQF, Biotechnology School of Portuguese Catholic University,

Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal

paulo.silva@ff.up.pt

7.1 Introduction
To better understand the regional or country-speciic regulations,
this chapter will irst provide a broad overview of the global legal
framework concerning probiotics.
There is no universal deinition of probiotics that can be
considered generically either as a food (including food additives and
dietary supplements) or as a drug (medicines). Nevertheless, the
deinition of probiotics by a joint Food and Agriculture Organization
of the United Nations (FAO)/World Health Organization (WHO)
expert consultation in October of 2001 (already mentioned in
Chapter 1) has been widely used. These experts produced a report on
Evaluation of Health and Nutritional Properties of Probiotics in Food
Including Powder Milk with Live Lactic Acid Bacteria. This report,

Probiotic Bacteria: Fundamentals, Therapy, and Technological Aspects

Edited by J. Paulo Sousa e Silva and Ana C. Freitas
Copyright © 2014 Pan Stanford Publishing Pte. Ltd.
ISBN 978-981-4411-62-2 (Hardcover), 978-981-4411-63-9 (eBook)
www.panstanford.com
264 Guidelines and Regulations

together with another on Drafting Guidelines for the Evaluation of

Probiotics in Food developed by a joint FAO/WHO expert working
group in 2002, provides scientiic advice on the methodology for
evaluation of probiotics in food, addressing methods for strain
identiication, safety assessment and functional characterization as
well indication of what data are required to accurately substantiate
health claims. The problems associated with probiotic product
selection also become more understandable by application of
guidelines established by the International Scientiic Association
for Probiotics and Prebiotics (ISAPP, 2009). These guidelines will be
detailed in Section 7.2.

7.1.1 Food Standards

The Codex Alimentarius commission, created by FAO and WHO in
1963, is responsible for compiling the Codex Alimentarius, which
is a collection of standards, codes of practice, guidelines, and other
recommendations developed under the joint FAO/WHO Programme.
Among other objectives, this program aims to protect the health of
the consumers and ensure fair trade practices in the food market.
Independently of the type of codex documents, they are generally
designated as standards. These standards could be general or
sometimes very precise with detailed speciications related to foods,
such as practice guides concerning operation or managing operation
process. For example, Codex Standard for Fermented Milks (CODEX
STAN 243-2003) is an international standard with implications in
probiotic products.
Codex Alimentarius or food code has become a global reference
to face emerging challenges and codex standards are used in trade
disputes at the World Trade Organization (WTO) (Lähteenmäki-
Uutela 2009; WHO 2012). WTO was created after The Uruguay Round
of General Agreement on Tariffs and Trade (GATT) negotiations
(1986–1994) and begun its work in 1995. WTO aims to ensure
free (as possible) trade among nations by the establishment of
international agreements, and dispute resolutions between its 155
members (10 May 2012). Codex standards are speciically referred
to in the WTO agreements on Technical Barriers to Trade (TBT) and
on the Agreement on Sanitary and Phytosanitary Measures (SPS)
(Lähteenmäki-Uutela 2009). The TBT agreement includes technical
regulations, standards, testing, and certiication procedures. The SPS
 Guidelines for Probiotic Selection 265

agreement deines how nations can regulate food safety and apply
animal and plant health measures. The goal of these agreements is
that technical measures and health concerns do not create artiicial
obstacles to trade.
Codex Alimentarius also contributes directly to probiotics and all
related issues from a legal point of view via the Nutrition and Health
Claims standard developed and revised in the beginning of the 21st
century (Codex Alimentarius: www.codexalimentarius.net)

7.1.2 Drug Standards

Although probiotics could be regulated as a drug, based on their
physiological roles, it is more dificult and expensive to choose this
approach due to stringent legislation.
WHO supports member states in the development of medicine
policies and guidelines to implement marketing authorization
system for pharmaceutical products [Marketing Authorization
of Pharmaceutical Products with Special Reference to Multisource
(Generic) Products: A Manual for National Medicines Regulatory
Authorities (NMRAs), 2nd edition].
The International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use
(ICH) was established in 1990 with the goal of harmonizing testing
of medicines and promoting the international coordination of the
authorization procedures. ICH, which brings together the regulatory
authorities and research-based pharmaceutical industry in the
European Union, Japan, and the United States of America, meets
twice a year with the aim of reducing the cost of research by avoiding
unnecessary work duplication. It consists of six organizations:
European Union (EU), the European Federation of Pharmaceutical
Industries and Associations, Ministry of Health, Labour and Welfare
(MHLW), Japan Pharmaceutical Manufacturers Association, Food
and Drug Administration (FDA), and the Pharmaceutical Research
and Manufacturers of America. Although ICH guidelines are not
global, they are generally accepted worldwide.

7.2 Guidelines for Probiotic Selection

It is important that probiotic bacteria selected for commercial use in
foods and medicines maintain the characteristics for which they were
266 Guidelines and Regulations

originally selected and that justify their health-promoting effects.

These include growth and viability during processing and storage
as well as during transit through the gastrointestinal transit upon
consumption. Consequently, it is necessary to constantly control
strain properties, their stability during manufacture and storage,
and to ensure that they are retained in different types of foods as
well as maintained when assessed in relation to expected clinical
effects (Gueimonde, 2011). Establishment and implementation
of guidelines contributes to product safety, quality, reliability, and
level playing for all companies introducing and producing probiotic
products.

7.2.1 Safety Criteria

Regulating safety and eficacy of probiotic products is basic
“consumer protection law,” speciically if one considers that product
safety is deined as “physical safety, protecting the consumer’s body.”
Safety is a relative term implying absence of negative health effects.
In general, microbial food cultures (MFC) with a technological role
(starter cultures), be it acidiication or lavor/texture enhancement,
have a long, safe history of use in food, and have generally been
considered safe, and suitable for myriad uses. According to the U. S.
standards, these cultures hold a GRAS (generally recognized as safe)
status, which is based on either a history of presence in food prior
to 1958 or by scientiic procedures. Cultures used for traditional
fermentation with controlled metabolism and effect on food substrate
are associated with a reasonable expectation of safety. However, with
respect to probiotic strains, these not only perform technological
functionality but also promote added health beneits to consumers,
and as science and clinical relevance advances, manufacturers need
to ensure that these MFC are totally safe and reveal a safe history
of use in food. Historical data indicate that probiotic lactobacilli
and biidobacteria associated with food have been considered to
be safe, yet clinical evidences suggesting the need for high doses
(order of magnitude around 1010 colony-forming unit (cfu)) and the
development of improved fermentation and formulation techniques
have led to emerging high-dose preparations on the market that may
eventually impact negatively on the host. In this context, three areas of
safety concern have been addressed as far as probiotic authorization
is concerned: (i) potential for transmigration (accepted safety for
 Guidelines for Probiotic Selection 267

long-used probiotic isolates regarding translocation, yet rare reports

on bacteremia alert the need for continued surveillance, especially
in immunocompromised individuals); (ii) potential negative
impact of physiological and metabolic properties on the physiology,
immunology, and microbiology of the gastrointestinal tract
(deconjugated bile acid, D-lactate, need for absence of haemolytic
activity); and (iii) absence of antibiotic resistance (negative data
concerning antibiotic/gene transfer are required for each strain
under each circumstance) (Henriksson et al., 2005). An important
highlight is that special care must be taken with those individuals
presenting defective immunity, abnormal gut structure, and/or
metabolic defects with respect to safe use of probiotics (Henriksson
et al., 2005). Safety issues have been on the agenda of many food
industry committees as well as government regulatory authorities.
Sets of guidelines have been drawn up and tentatively implemented
by both probiotic manufacturers and scientiic bodies. According
to Joint FAO/WHO expert committee guidelines, strains to be used
as probiotics must be characterized with a set of tests that evaluate
antibiotic resistance patterns and horizontal transfer capacity,
certain metabolic activities, eventual side effects, and potential
virulence factors (more details are listed in the next section in Table
7.2).
The European Food Safety Authority (EFSA) has developed
a list of microbial cultures with a Qualiied Presumption of Safety
(QPS), which need no further safety determination for the purposes
of registration in the European Union (EFSA, 2010). The QPS is a
well-designed concept that lists probiotic organisms, among other
biological agents, by genus/species level and is based upon the
scientiic literature and the long history of safe use of many of the
organisms in human food. The list is annually revised and updated,
and following the most recent review, no modiications were made to
the QPS recommended biological agents list, although modiications
concerning antimicrobial susceptibility qualiications were included
(EFSA, 2010). Very recently, the bacterial species L. paracasei was
considered by EFSA to be suitable for the QPS approach (EFSA 2011).
More details on the QPS system and its principles can be found on
the EFSA Web site (http://www.efsa.europa.eu/EFSA/efsa_locale_1
178620753812_1178667590178.htm, Accessed August 2012). The
FDA also lists organisms by genus and species in its partial list of
268 Guidelines and Regulations

organisms used in food (FDA, 2001). A comparison of the assessment

schemes of the FDA and EFSA is given in Table 7.1.

Table 7.1 Comparison between assessment schemes for dietary/food

supplements in the United States (FDA GRAS system) and
proposed scheme in the EU (EFSA QPS system)

GRAS guidelines QPS guidelines

Applies to food additives, in Applies to microorganisms only
general
Determination of GRAS status by Determination of QPS status by
FDA and/or external experts EFSA
Open list Positive list
Based on common use Based on history of use and
adverse effects
Describes speciic substance or Describes taxonomic unit (e.g.,
microorganism genus, species, or strain)
Case-by-case assessment General assessment
Source: Adapted from Wassenaar and Klein (2008)
The International Food Additives Council in its position
paper considers the MFC listed in the International Dairy
Federation Inventory (http://www.effca.org/content/inventory-
microorganisms) and those having a QPS status as GRAS. It is
important to note that compared with the GRAS system used in
the United States, the QPS system seems to be more lexible and
considers additional emerging safety risks, such as acquisition of
antibiotic resistance or virulence determinants, an important aspect
that gains strength with advances in science (Wassenaar and Klein,
2008).

7.2.2 Functionality, Technological, and Labeling Criteria

The rational use of probiotics to improve physiological functions of
the host has as basis (i) functional characterization and identiication
of bacteria and viability maintenance at consumption; (ii) required
dose and enumeration techniques (inluenced by strain type, alone
or combined with other strains, food vector, and industrial form of
presentation); (iii) health beneits for the host and their deinition;
and (iv) safety of use (supported by the two FAO/WHO documents of
 Guidelines for Probiotic Selection 269

2001 and 2002 and of the concept of QPS for the safety assessment
of bacteria, recently introduced by EFSA, as previously mentioned).
These documents together with other information published in the
meantime, identify essential steps needed to select and validate
a probiotic strain (Table 7.2). Then according to each country’s

Table 7.2 Guidelines for development of a probiotic food based on

Joint Expert FAO/WHO committee guidelines (2001 and
2002) combined with the need to include the impact of the
food delivery vector—such matrix speciically contributes to
probiotic physiology, gene expression, stability, and eficacy

Factor Information required for substantiation

Thorough Genus and species determination based on
identiication of the most current genetic methods.
test product Generation of strain-speciic identiication,
with patterns from appropriate molecular
techniques.
Reproducible genetic methods such as pulsed-
ield gel electrophoresis or randomly ampliied
polymorphic DNA analysis are important tools.
Polyphasic characterization combining
phenotypic, biochemical, genotypic, and
sequencing results is suggested as a reliable
strategy to identify bacteria to the strain level.
Description of the food product vector for
probiotic. Understand how the food matrix
may affect probiotic function including, if
applicable, the impact on functionality of any
food transformation that results from probiotic
growth.
Probiotic strain must Stock cultures must be maintained under
be deposited in an appropriate conditions to ensure that the given
international culture culture maintains its beneicial properties.
collection—guarantee
historical reference
substance is available
and enables research
to be repeated
by independent
researchers
(Continued)
270 Guidelines and Regulations

Table 7.2 (Continued)

Factor Information required for substantiation

Extensive safety Description of use (dose, format, stability).
assessment Veriication that the product is manufactured
under good manufacturing practices speciic for
the product category.
Survey of literature to determine to what extent
the strain, species, or genus was involved in
adverse events.
Description of physiological and genetic
capacity for toxic activity: toxin producer,
haemolytic activity.
Description of physiological and genetic
capacity for pathogenic/opportunistic
pathogenic activity.
Genetic stability of the probiotic.
Presence of transferable antibiotic resistance
markers.
Review of the above information by regulatory
authority or a panel of experts qualiied in the
ield to evaluate the safety of the substance
for its intended use, depending on the product
category and requirements.
Eficacy assessment. Valid in vitro assessments, as dictated by
This will be speciic for speciic probiotic strain and intent of use.
the particular effects Valid animal studies using test product,
being targeted.a Only which might be useful for safety assessment,
in vitro characteristics mechanistic studies, and suggestions of
that are conirmed endpoints to conirm in human studies.
to be related to in Human eficacy studies using test product,
vivo functionality are which provide the basis for substantiation
useful. Minimum daily of health beneits and the rationale for the
amount required to dose being delivered in the product—RDBPCb
confer the purported human trials by enrolling adequate number of
health beneits is to subjects to achieve statistical signiicance. Note:
be deined explicitly dose may vary in terms of strain, food vector
on the basis of in vitro and the host (age, sex, target speciic, healthy,
and in vivo studies on and/or immunocompromised).
cell lines and animal
models as well as
human clinical trials.
 Guidelines for Probiotic Selection 271

Factor Information required for substantiation

Product labeling Genus, species, and strain designation for each
(on product label probiotic included in the product.
or in accompanying Minimum viable numbers of each probiotic
information on strain at the end of the shelf-life—important
product website) that product delivers the declared level in a
serving of stated size throughout product shelf-
life.
Expiration date.
Serving size required to deliver effective dose
for onset of labeled effects.
Health beneits description.
Identiication of proper storage conditions.
Corporate contact details for consumer
information.
Product website URL.
Post-market All stake holders need to develop some form of
surveillance system to monitor the health outcome of long-
term probiotic administration.
Potential side effects and long-term beneits to
be recorded and documented.
Proper trace back system—a pre-requisite for
surveillance of probiotic products.
Source: Adapted from Sanders and Marco (2010)
aThe traditional assertion that probiotics possess certain traits such as speciic source

of isolation (human, animal, environmental, food), acid resistance, bile resistance, and
other such traits may not be relevant for speciic applications, and therefore cannot
be considered essential.
bRandomized, double blind, placebo controlled.

speciications, an authorization dossier needs to be submitted. As

discussed later on, in the case of a dossier submitted to EFSA, this
has to include a clear identiication and characterization of the
probiotic strain, a clear statement of the claimed health beneit,
lawless evidence supporting the claimed beneit, and justiication
for why the beneit is substantiated by the evidence (Sanders and
Marco, 2010).
272 Guidelines and Regulations

7.3 Probiotics Legal Status

Regulatory standards and guidelines are crucial to prevent possibility
of products on the market that are unreliable in content and contain
strains with unvalidated scientiic eficacy. The consumer needs to be
conident that launched probiotic products are effective and fulil all
standards and recommendations through evidence-based studies.
All the leading nations have a different regulatory framework for
probiotics and products containing probiotics. The following sections
will give an overview of regulatory bodies and issues of probiotics in
different countries.

7.3.1 Asia–Pacific

7.3.1.1 Australia and New Zealand

The Australia and New Zealand food regulation system is a cooperative
arrangement between the Australian States and Territories
(including mainland—Western Australia, Northern territory, South
Australia, Queensland, Victoria, New South Wales, Australian Capital
Territory, and islands—Tasmania) and New Zealand to develop and
implement uniform food standards.
The food regulatory system is underpinned by a number of
agreements and legislative instruments, listed as follows:
• The Australia New Zealand Joint Food Standards Agreement
between Australia and New Zealand, otherwise known as The
Treaty, provides for New Zealand’s involvement in the food
regulatory system.
• The Food Regulation Agreement, initially signed by the
Council of Australian Governments in November 2000 and
amended in December 2002, is an intergovernmental agree-
ment signed by the Australian Government, States, and
Territories, and commits these signatories to a cooperative
national system of food regulation. This agreement establish-
es the Ministerial Council, which sets food policy and consid-
ers food standards for Australia and New Zealand.
• The Food Standards Australia New Zealand Act 1991 (FSANZ
Act) came into effect on 1 July 2002, and is the legislative basis
for the binational statutory authority, FSANZ. The objective
 Probiotics Legal Status 273

of the FSANZ Act is to ensure a high standard of public

health protection throughout Australia and New Zealand by
maintaining a safe food supply. All food sold in Australia and
New Zealand must be safe.
• FSANZ is the independent statutory authority in Australia
that develops (i) food standards for primary production,
processing, and food hygiene; and (ii) set residue limits for
agricultural and veterinary products. The Legislative and
Governance Forum on Food Regulation (formerly the Australia
and New Zealand Food Regulation Ministerial Council) sets
policy guidelines for the development of food standards by
FSANZ. In New Zealand, these activities are undertaken by the
New Zealand Ministry for Primary Industries.
• Once approved, the Standards are incorporated into the
Australia New Zealand Food Standards Code (the Food
Standards Code). Australian States and Territories adopt the
Food Standards Code under State and Territory Food or Health
Acts. New Zealand adopts the Food Standards Code under the
Food Act 1981.
• Enforcement of the Food Standards Code occurs through State
and Territory health departments and local councils. New
Zealand enforces the Food Standards Code through its national
enforcement body, the New Zealand Food Safety Authority.
• The Fair Trading Acts in New Zealand and the States and
Territories of Australia apply to the supply of food in trade
and commerce relating to any conduct that is considered false,
misleading, or deceptive.
With respect to probiotics, probiotic products in Australia
and New Zealand are considered either functional foods and are
regulated by FSANZ, or complementary medicine and regulated
by the Therapeutic Goods Administration (TGA). Under the Food
Standards Code, fermented milk beverages and yoghurts that claim
to be probiotic should have a minimum of 1 million live bacteria per
gram. The number of probiotic bacteria should be maintained to the
end of a product’s shelf-life to be of any health beneit.
In what concerns FSANZ and the TGA proof of eficacy for
complementary medicines (which include dietary supplements),
these are only required if the product is making high-level health
claims or is randomly audited; nevertheless, it is advisable that
274 Guidelines and Regulations

evidence for claim be held by food manufacturers for other eventual

claims required. In fact, Australia has a registration system for
dietary supplements that uses a convenient electronic application
system and a validation process. Products eligible for registration in
the Australian Register of Therapeutic Goods (ARTG) contain low-
risk ingredients in acceptable amounts that have been evaluated
and accepted by the TGA and manufactured under GMP principles,
for example, probiotics. A manufacturer must submit qualitative
formulation/ingredient information and inished good speciications.
The label may carry indications (i.e., claims about therapeutic use of
the product) only for health maintenance and health enhancement
or certain indications for nonserious, self-limiting conditions.
If high-level health claims are intended, the manufacturer must
also certify that, according to the applicable guidelines, it holds
substantiation for claims and shelf-life. The government reviews the
registration in 2–4 weeks, after which time the product can appear
on the market with an “Aust L” number that must be included on the
label. There were approximately 69.487 products on the ARTG as at
May 2012 including up to 122 probiotic products containing either
Biidobacterium (9 authorized strains) and/or Lactobacillus (26
authorized strains) probiotic strains. FSANZ is currently working
on a new Nutrition, Health and Related Claims Standard to replace
the transitional standard, in practice since 2002 that has banned the
making of most health claims. The proposed framework classiies
as health claims (i) General Level Claims that refer to the presence
of a nutrient or substance in a food and to its effect on a health
function; and (ii) High Level that implies a health claim that directly
or indirectly refers to a serious disease or a biomarker. Nutrient
content claims are not to be classiied as health claims.

7.3.1.1.1 Regulations
http://www.comlaw.gov.au/ Australian Government Law site
containing all standards included in Australian New Zealand Food
Standards Code

7.3.1.1.2 Web pages

https://www.ebs.tga.gov.au/ Australian register of therapeutic
goods
http://www.health.gov.au/ Australian Government Department of
Health and Ageing
 Probiotics Legal Status 275

http://www.tga.gov.au/index.htm Therapeutic Goods

Administration
http://www.foodstandards.gov.au/ Food Standards Australia New
Zealand
http://www.comlaw.gov.au/Details/F2009C00878 Australian
New Zealand Food Standards Code - Standard 1.1A.2 - Transitional
Standard - Health Claims

7.3.1.2 China
In China, probiotics tend to be considered a foodstuff either as
a novel food or as a health food, if health claims are invoked. The
central government authority with jurisdiction over food and health
food (as well as medicines and cosmetics) is the State Food and
Drug Administration (SFDA) but the Ministry of Health and other
entities such as the General Administration of Quality Supervision,
Inspection and Quarantine, the State Administration for Industry
and Commerce, and so on are also in charge of the regulation of
food, drug and cosmetic products. (http://www.chinafdc-law.com/
glossary.html Accessed January 2012)
The irst Food and Hygiene Law of the People’s Republic of China
was promulgated in 1995 (Kaushik and Kaushik, 2010) and the
current basic food law is Food Safety Law, which entered into force
in 1 June 2009; they both deine food as any inished product or raw
materials provided for people to eat or drink, and articles that are
traditionally food and medicine, excluding articles that are used for
the purpose of medical treatment.
Novel food has been regulated by the regulation by “Administrative
Measures on Novel Food 2007” and included four types (article 2).
Type 1 refers speciically microorganisms that are not traditionally
consumed in China. Types 2 and 3 also refer microorganisms, which
are not traditionally consumed in China or new varieties, as raw
food materials derived (type 2) or as food processing. According to
the regulation, production and import of novel food are subject to
pre-market approval by China’s Ministry of Health. Probiotic strains
have already been approved by the current regulation (Lactobacillus
paracasei GM-080 and L. paracasei GMNL-33 by GenMont in 2008)
http://www.genmont.com.tw/englishwebsite/company.html
Accessed December 2011.
276 Guidelines and Regulations

Novel food cannot bear health claims; therefore, to make this

kind of claim, the health food registration process must be followed
(Patel et al. 2008; Lähteenmäki-Uutela 2011).
Health food is deined in the interims Provisions for Health
Food Registration (SFDA Order No. 19) issued by the SFDA on 30
April 2005 as a “food claiming that it has certain health improving
functions or is able to supply vitamins and minerals. It is good for a
particular group of people and able to adjust body functions. But, it is
not used to cure certain diseases.” All health food must be approved
and registered with the SFDA, which will assess and examine the
safety, eficacy, quality, and labeling of products.
In accordance with the Provisions for Health Food Registration,
there is a particular document about Requirements for Probiotic
Microlora Health Food Application and Reviewing (interim)
formulated and issued by SFDA.
The permitted probiotics are included in a list, and to add a
new one, it is necessary to fulil an SFDA application. The following
probiotics are allowed: Biidobacterium biidum, Biidobacterium
infantis, Biidobacterium longum, Biidobacterium breve,
Biidobacterium adolescentis, Lactobacillus delbrueckii subspecies
bulgaricus, Lactobacillus acidophilus, Lactobacillus casei subspecies
casei, Lactobacillus reuteri, and Streptococcus thermophilus.
(Lähteenmäki-Uutela 2011). There is also a list of 27 categories
of health claims allowed (Patel et al. 2008), some of them can be
applied to probiotics such as enhancing immune systems, regulating
gastrointestinal lora, alleviating constipation, or facilitating
digestion. However, other new claims may be invoked if can be
proved by testing on humans and/or animals.
In the Chinese Health registration process, the applicant must
submit the product to SFDA authorized testing laboratories and
cannot apply for more than two health claims.

7.3.1.2.1 Regulations
Food Safety Law of the People’s Republic of China. June 1 2009.
(Food Safety Law)
Ministry of Health. Administrative Regulations for Health Food.
Order No. 46; 1996-06-01. (Health Food Regulation)
Ministry of Health: Technical Standards for Testing and Assessment
of Health Food. Order No. 42; 2003
 Probiotics Legal Status 277

Ministry of Health. Administrative Regulation for Novel Food. 2007.

(Novel food regulation 2007)
Food Hygiene Law of the People’s Republic of China. October 1995.
(Food Hygiene Law. Not in force)

7.3.1.2.2 Web pages

http://english.gov.cn/ Central People’s Government of the People’s
Republic of China
http://www.sfda.gov.cn SFDA
http://www.most.gov.cn Ministry of Science and Technology
http://www.fehd.gov.hk The Government of the Hong Kong Special
Administrative Region of the People’s Republic of China: Food and
Environmental Hygiene Department

7.3.1.3 Japan
Japan is a key player in probiotic-based functional foods accounting
for more than half of global probiotic foods market. These probiotic
products have different applications, including immunity, allergy,
cold-like symptoms, gastrointestinal health, blood pressure levels,
cholesterol levels, and so on (Amagase, 2008; He and Benno, 2011).
In terms of food regulation, Japan began a systematic research
on health beneits of foods in 1984. As a consequence, a deinition
for functional food was provided by the Japanese scientiic academic
community involved. In this context, functional foods were deined as
those foods with three functions: apart from nutrient provision and a
sensory function, functional foods also have additional physiological
functions contributing to health maintenance (Shimizu, 2003).
Dynamic changes began in 1991 when the Japanese Ministry of
Health, Labour and Welfare established the regulatory system “Foods
for Speciied Health Use” (FOSHU) in which foods wishing to bear
health claims on the label have to be examined and approved, prior
to use, by the Council of Pharmaceutical Affairs and Food Hygiene
(practical minimum period required for approval is approximately
one year) in terms of speciic safety and eficacy requirements
including:
(i) Proven scientiic effectiveness on the human body functions;
278 Guidelines and Regulations

(ii) Total safety (animal toxicity, identiication of effects in case of

excess intake among others);
(iii) Adequate nutrient proiles (e.g., no excessive use of sugar,
saturated fat, salt);
(iv) Product speciications must be present at time of
consumption—recommended dose of the functional food
ingredient;
(v) Quality control methods, including product speciications,
production procedure, and methods of analysis need to be
well documented.
In 2001, the regulatory range of FOSHU was broadened in order to
include pharmaceutical vehicles such as tablets and capsules, apart
from conventional foods. At that time, the MHLW established a new
regulatory system “Foods with Health Claims,” which included the
already existing FOSHU system and a second newly created “Foods
with Nutrient Function Claims” (FNFC). In this second system, the
government has set the range of nutrition and mineral content levels
and labeling standards; more precisely, 12 vitamins (vitamins A,
complex B, C, E, D) and two minerals (Ca, Fe) are standardized. Both
regulations are under the jurisdiction of Health Promotion Law,
section of Foods with Health Claims Act of 2001. These regulations
differ mainly in their target speciicity: the FOSHU system is a
product-speciic (not ingredient-speciic) approval system, whereas
the FNFC counterpart enables any product that meets the standards
to make a standardized health claim without oficial approval (He
and Benno, 2011). The FOSHU system has helped greatly in leading
consumers in their food choices; the FOSHU-approved logo and the
health claim statements clarify the relationship between food and
health.
By March 2009, there were 847 existing approved FOSHU
products with health claims classiied into eight groups, including
gastrointestinal conditions, mineral absorption, blood pressure,
blood cholesterol, bone health, dental health, and blood glucose
levels. In the FOSHU, product category targeting gastrointestinal
health probiotics are undoubtedly the most important ingredient.
Among the approved FOSHU products (with health claim), 76 were
probiotic products containing one of the 16 approved bacterial
strains (He and Benno, 2011). The approved strains include
Lactobacillus rhamnosus GG, Biidobacterium longum BB536,
 Probiotics Legal Status 279

Lactobacillus delbrueckii subsp. Bulgaricus 2038, Streptococcus

salivarius subsp. Thermophilus 1131, Lactobacillus casei Shirota,
Biidobacterium breve Yakult, Biidobacterium lactis FK120, B. lactis
LKM512, Lactobacillus acidophilus CK92, Lactobacillus herbeticus
CK60, L. casei SBR1202, Lactobacillus gaseeri SP, Biidobacterium SP,
L. casei NY1301, Lactobacillus LC1, and B. lactis Bb-12.
It is important to note that the current FOSHU approval system
includes three categories, namely, standardized FOSHU, qualiied
FOSHU, and reduction of disease FOSHU (Fig. 7.1), yet approved
probiotics only fall under standardized FOSHU (He and Benno,
2011).


  
   
         
 
 
  


    
  
  
 
  

  
           



  

  
 

      

  
   

       

 
 
 



       
 !""# $  #

  
  %


  
      
  
   

       
 
 
 
 

 
   
   &'( 

Figure 7.1 Categories of food for speciied uses for regulation purposes.

As previously mentioned, FOSHU health claims for probiotic

products are focused mainly on gastrointestinal conditions so the
scientiic evidence gathered consists mainly of data demonstrating
improvement in intestinal microlora balance, increase in local
beneicial bacteria in detriment of decrease of pathogenic species
(Clostridium perfringens), lower bacteria metabolite production, and
increased defecation frequency in human studies (Amagase, 2008;
He and Benno, 2011). On the basis of these criteria, the approved
FOSHU health claims for probiotics include:
280 Guidelines and Regulations

• reaches the intestine alive

• increases the intestinal lactobacilli/biidobacteria/beneicial
bacteria
• promotes the maintenance of a good intestinal environment
• regulates/helps maintain good gastrointestinal condition
• maintains the intestine in good health
• helps balance the intestinal lora
• reduces harmful bacteria.
It is important to note that there are many other probiotic
products marketed in Japan, which are not FOSHU. Animal and
human studies have shown promising health promoting features in
various targets, including immunity, allergy, cholesterol lowering,
and Helicobacter pylori eradication, yet related health claims are
still strictly prohibited in these cases due to lack of clinical trials and
related studies (Shimizu, 2003; He and Benno, 2011).

7.3.1.3.1 Regulations
Food for Speciied Health Uses: http://www.mhlw.go.jp/english/
topics/foodsafety/hc/02.html
Food with nutrient function claim: http://www.mhlw.go.jp/english/
topics/foodsafety/hc/01.html
Food Sanitation Law: http://www.tokio.polemb.net/iles/
Gospodarka/Handel/food-e.pdf

7.3.1.3.2 Web pages

Ministry of Health, Labour and Welfare. Available at:
http://www.mhlw.go.jp/english/topics/foodsafety/hc/index.html
Japan Health andd Nutrition Food Association. Available at:
http://www.jhnfa.org/index.html
Japan Dairy Industry Association. Available at:
http://www.jdia.or.jp/information/milk_know.html
Consumer Affairs Agency Food Labelling Division. Available at:
http://www.caa.go.jp/en/index.html
National Institute of Health Sciences. Available at:
http://www.nihs.go.jp/english/index.html
National Institute of Health and Nutrition. Available at:
http://www0.nih.go.jp/eiken/english/index.html
 Probiotics Legal Status 281

7.3.2 Europe
Probiotics are consumed by Europeans in foodstuffs (food and food
supplements) (Saxelin 2008). The legislation on foodstuffs in the 27
country states of the EU is not harmonized.
The principles concerning food safety and consumer protection
are established in the legislation of the member states. However,
EU has established general principles and requirements of food law
with the aim of protecting human life and health and to integrate a
food safety policy based on “farm to fork” approach. EU food law also
aims to harmonize member states requirements in order to allow
the free movement of food in EU. Food safety and general consumer
protection is under competence of Directorate General for Health
and Consumer (DG SANCO). For registration purposes, as already
mention in Section 7.2.1, EU has developed the QPS list for microbial
cultures, which does not need further safety determination.
Food and food supplements containing probiotics are covered by
Regulation 178/2002/EC (which is the legal general framework) and
Directive 2000/13/EC. EU Regulation 178/2002/EC deines food (or
foodstuff) as “any substance or product, whether processed, partially
processed or unprocessed, intended to be, or reasonably expected to
be ingested by humans.” The Directive 2002/46/EC relating to food
supplements [which deine as “foodstuffs the purpose of which is
to supplement the normal diet and which are concentrated sources
of nutrients (vitamins, and minerals) or other substances with a
nutritional or physiological effect, alone or in combination, marketed
in dose form, …”] establishes harmonized rules for their labeling.
Dose forms may be capsules, tablets, pastilles, pills, powders (in
sachets) or liquids in measured doses, and so on. This legislation
also aim to ensure that these products are safe and appropriately
labeled so that consumers can make informed choices.
In EU, food labels may invoke nutrition claims, if they are listed
in the annex of EC Regulation 1924/2006, and health claims, after
approval by European Commission, according to the requirements
set out in this Regulation. Any statement relating food and health
may be interpreted as a claim and should be based on generally
accepted scientiic evidence and easily understood by consumers.
Health claims are deined as any “claim that states, suggests or
implies that a relationship exists between a food category, a food
282 Guidelines and Regulations

or one of its constituents and health.” According to EC Regulation

1924/2006, health claims are the only ones that may be invoked
by probiotic products. The last mentioned regulation established
three types of health claims: (i) the reduction of disease risk; (ii)
claims referring to children’s development and health, (iii) other
health claims than those referring to the reduction of disease risk
and to children’s development and health (c1—role in the growth,
development, and functions of the body; c2—the psychological and
behavioral functions; or c3—relating to weight-control).
EFSA, established in 2002 as an independent body for risk
assessment on food chain, is responsible for evaluating the scientiic
evidence that support health claims. Food-related general function
health claims are assessed by the EFSA’s Panel on Dietetic Products,
Nutrition, and Allergies (NDA). Health claims for probiotics can be
made under article 13 or 14 of EC Regulation 1924/2006 (Rijkers et
al. 2011).
The three important areas the regulation pinpoints for assessment
in health claims for each potentially probiotic strain are the following:
(i) characterization of the strain or each of the strains in a probiotic
mix, (ii) identiication of the health relationship to beneit the
general population or a deined part of it, and (iii) demonstration
of health effects in a normal healthy target population. These three
criteria form the essential basis for the establishment of potential
human health claims for probiotic microorganisms (van Loveren et
al., 2012).
Until May 2012, none of the health claim applications for
probiotic products submitted to EFSA had been accepted (http://
ec.europa.eu/nuhclaims/ Accessed June 2012). Lack of adequate
strain characterization or only partial characterization of strains
in bacterial mixture, too general claimed health relationships
without supportive evidence of beneicial clinical outcomes, claims
oriented to subjects beyond the scope of the claims regulation,
laws in the studies designed to substantiate claims, or in the actual
measurements are among the reasons for noneligible claims; recall
that the regulation encloses claims that are designed for normal
healthy populations or populations at risk of speciic disorders (van
Loveren et al., 2012).
Alternatively, a probiotic product may encompass the legal
European deinition for medicinal product, but this will impose
 Probiotics Legal Status 283

higher and more expensive requirements to applicants or

manufactures. Nevertheless, due to special provisions or less
stringent older legislations, several approved medicinal products
that contain lactobacilli and/or biidobacteria can still be found in
many European countries.

7.3.2.1 Central Legislation

Directive 2001/83/EC of the European Parliament and of the Council
of 6 November 2001 on the Community code relating to medicinal
products for human use. (Medicinal products directive 2001)
Regulation (EC) No 178/2002 of the European Parliament and of
the Council of 28 January 2002 laying down the general principles
and requirements of food law, establishing the EFSA and laying down
procedures in matters of food safety. (General Food Law, 2002)
Directive 2000/13/EC of the European Parliament and of the
Council of 20 March 2000 on the approximation of the laws of the
Member States relating to the labeling, presentation, and advertising
of foodstuffs. (Food supplements labeling directive 2000)
Regulation (EC) No 1924/2006 of the European Parliament and
of the Council of 20 December 2006 on nutrition and health claims
made on foods. (Claim regulation 2006)
Regulation (EC) No 258/97 of the European Parliament and of
the Council of 27 January 1997 concerning novel foods and novel
food ingredients. (Novel food regulation 1997)

7.3.2.2 Web pages

http://eur-lex.europa.eu/ European Union legislation
http://www.emea.europa.eu/ema/ European Medicine Agency
http://www.efsa.europa.eu. EFSA
http://ec.europa.eu/dgs/health_consumer/index_en.htm
Directorate General for Health and Consumer (DG SANCO)

7.3.3 United States of America

In the United States, probiotics are not deined as a regulatory
product category; nevertheless, the FDA has issued draft guidance
about complementary and alternative medicine products (FDA,
2006) wherein probiotics are referred saying that they may be
284 Guidelines and Regulations

regulated as foods, dietary supplements, or drugs under the Federal

Food Drug and Cosmetic Act (Act). FDA is a regulatory, scientiic,
and public health agency that oversees food (except for meat and
poultry), drugs, cosmetics, and other related products in the United
States. Degnan (2008) stated that there are several regulatory
categories that could be applied to products containing probiotics in
the United States but four of which are fundamental: food (including
food ingredients), dietary supplement, medical food, and drug (or
biological product, FDA considers that if a probiotic is a drug, it
is also a biological product). The classiication of the products is
primarily based on their intended use (largely determined by the
claims invoked). Other factors such as type of formulation, route
of administration, target consumers, and safety should also be
considered (Hoffman et al. 2008).
Food means “articles used for food or drink for man and other
animals” (section 201(f) of Act) and food ingredient may be food
additives or GRAS substances. In a short deinition, a food additive is
a food component whose safety has to be demonstrated because it is
not GRAS or was not already used in food prior to January 1, 1958.
A dietary supplement has four main characteristics:
“supplement the diet,” “intend for ingestion,” contains or is a “dietary
ingredient,” and is not in “conventional food”(Degnan, 2008). Dietary
supplements are a special category under the general umbrella of
foods.
Medical food has a very narrow deinition “a food which is
formulated to be consumed or administered enterally under the
supervision of a physician and that is intended for the speciic
dietary management of a disease or condition for which distinctive
nutritional requirements, based on recognized scientiic principles,
are established by medical evaluation” in section 5(b) of the Orphan
Drug Act (21 U.S.C. 360ee(b)(3)).
An article intended for diagnosis, cure, mitigation, treatment, or
prevention of disease in man or other animals is a drug [the complete
deinition may be consulted in the section 201(g) (1) of the Act].
The regulatory categorization of probiotic product dictates the
authorization requirements and evidentiary burdens to establish
safety and to substantiate claims.
A probiotic product may need premarket authorization if it is a
food additive or a drug (a biological product). In case of being a food
additive, it is necessary to submit a food additive petition to Ofice
 Probiotics Legal Status 285

of Food Additive Safety (OFAS) in the Center for Food Safety and
Applied Nutrition (CFSAN) at FDA. This petition has four essential
elements for the safety assessment: identity of the additive, probable
exposure, evaluation of safety, and limitations of conditions of use.
FDA recommends that manufacturers consult the FDA about food
ingredient status (pre-petitions consultations).
If the probiotic product is considered a biologic product (a
subset of drugs), a Biologics License Application is necessary (which
includes applicant information, product/manufacturing information,
pre-clinical studies, clinical studies, labeling) and the pertinent
drug regulations, including applying for an Investigation New Drug
Application, should be fulilled.
A probiotic product categorized as a dietary supplement does not
need a premarket approval, but the act requires that to market dietary
supplements that contain “new dietary ingredients,” manufacturers
and distributors must previously notify FDA (it is necessary to wait
at least 75 days after the iling date of the notiication in order to
market the product). Dietary Supplement Health and Education Act
(DSHEA Act) of 1994, which signiicantly amends the Act, deines
“new dietary ingredients” as a dietary ingredient that was not
marketed in the United States before 15 October 1994. In a position
paper, the International Probiotics Association, the European Food
and Feed Cultures Association, and the International Food Additives
Council state that “individual strains belonging to microbial species
with a history of safe use as starter culture used for food fermentation
or as a probiotic in food are not New Dietary Ingredients under
the DSHEA Act;” on the contrary, each strain of microbial species
without a history of safe use as food (including dietary supplements
before 15 October 1994) must be evaluated individually and a
premarket notiication for a new dietary ingredient submitted to
the FDA (http://internationalprobiotics.org/news/upload/pdf/11.
pdf). There are three categories of claims that could be made for
dietary supplements and foods: health claims, structure/function
claims, and nutrient content claims. However, for food or dietary
supplements containing probiotics, only the irst and the second
claim are relevant.
Health claims describe the relationship of a food, or ingredient to
a disease or health-related condition. Legislation the 1990 Nutrition
Labeling and Education Act (NLEA), the 1997 FDA Modernization Act,
and the 2003 FDA Consumer Health Information for Better Nutrition
286 Guidelines and Regulations

Initiative] considered three “kinds” of health claims: (1) NLEA

Authorized Health Claims , (2) Heath Claims based on Authoritative
Statements, and (3) Qualiied Health Claims. In case of claims “type”1,
the manufacturer must submit a health claim petition and FDA
may authorize after an extensive review of the scientiic evidence
submitted in the petition. Claims of “type” 2, which are not applicable
to dietary supplements, may be used on labels after successful
notiication to FDA on the basis of an “authoritative statement” from
a scientiic body of the U.S. Government or the National Academy
of Sciences. If FDA found that there was not signiicant scientiic
agreement to authorize, a health claim may alternatively allow
“qualiied health claims based on less science evidence rather than
just on the standard of signiicant scientiic agreement, as long as the
claims do not mislead the consumers”(CFSAN 2006).For this “type”
of claims, the manufacturer must obtain the acquiescence of FDA,
that as a matter of its enforcement discretion, they will accept the
qualiied health claim. According to usprobiotics.org, there are no
authorized health claims for probiotics (usprobiotics.org, 2012).
Structure/function claims, which describe effects on normal
functioning of the human body, have historically appeared on the
labels of foodstuffs. These claims do not require a notiication to
FDA to be used in food, but the manufactures of dietary supplements
must submit a notiication no later than 30 days after marketing the
product (FDA 2010).
As a medical food, a probiotic product does not need to undergo
premarket review or approval by FDA, but it has to fulil its very
narrow deinition and must comply with all applicable requirements
for the manufacture of foods. Food additives regulations also apply
to the ingredients used in medical foods (CFSAN 2007).
According to usprobiotics.org in the United States, most probiotic
products are considered foods or dietary supplements and only few
are marketed as medical foods.

7.3.3.1 Legislation
Federal Food Drug and Cosmetic Act of 1938
Public Health Service Act of 1944
Nutrition Labeling and Education Act of 1990
Dietary Supplement Health and Education Act of 1994
FDA Modernization Act of 1997
 Probiotics Legal Status 287

FDA Consumer Health Information for Better Nutrition Initiative:

Task Force Final Report (2003)
FDA Food Safety Modernization Act of 2011

7.3.3.2 Web pages

http://www.fda.gov/ U.S. FDA
http://www.usprobiotics.org California Dairy Research Foundation
http://www.law.umaryland.edu/programs/health/events/
probiotics/ University Maryland Francis King Carey School of Law

7.3.4 Latin America (Brazil)

In Latin America, Brazil was the irst country to oficially recognize
functional foods in 1999 and subsequently issue speciic legislation—
“Food Functional Properties and/or Health Claims,” including
probiotics. In fact, Brazilian legislation does not deine functional
food (as Japan does), yet it does enable certain foods to claim
speciic functional health properties. In this sense, the Functional
Property Claim of a food product is deined as a claim “concerning
the metabolic or physiological role that the nutrient or non-nutrient
has in growth, development, maintenance and/or other normal
functions of the human organism” (Silveira et al., 2009). Interestingly,
one may claim a food to have “functional properties” and/or “health
properties” claims approved by the Brazilian National Sanitary
Surveillance Agency (ANVISA), yet it may not be considered as a
functional food (Stringheta et al., 2007; Silveira et al., 2009). Recall
that ANVISA in Brazil is in all similar to the FDA in the USA. Brazilian
legislation establishes the basic guidelines for risk assessment and
safety evaluation, analysis and veriication of functional, and/or
health properties claimed in labeling, as well as the conditions of
registration for such foods in the main regulations listed at the end
of this subchapter.
All these foods must also comply with general food legislation
and they may never claim medicinal or therapeutic properties
(Stringueta et al., 2012).
Speciically for probiotics, ANVISA issued, in collaboration with
the Health Ministry, at the beginning of 2002 the Executive Board
Resolution (RDC) no. 2, 7 January 2002 (Brasil, 2002), which
288 Guidelines and Regulations

approved the Technical Rules for Bioactive and Isolated Probiotic

Substances with Functional Properties or Health Claims; in this
resolution, the guidelines required for safety assessment, registration
and commercialization of bioactives, as well as probiotics claiming
functional and/or health properties are placed. Therein, probiotics
are deined as live microorganisms able to improve the microbial
balance of the bowels, resulting in beneicial effects on people’s
health; bioactive substances were deined as nonnutrients that
have speciic metabolic or physiological actions (Brazil, 2002). It is
compulsory that all probiotic products be registered and approved
by health authority ANVISA before being placed on the market, as
presented in Annex II of the RDC no. 27/2010. The registration of
food with claims and the evaluation of new claims will be made
by scientiic evidence of effectiveness of these, given the criteria
present in Resolution no. 18/99 and 19/99 (Stringueta et al., 2012).
In order to do so, the company needs to present a scientiic-technical
report containing a high number of speciic details on the product
(including description of the product, foreseen consumption,
analytical methods to assess components object of claim, chemical
composition with eventual molecular characterization, product
formulation, usage conditions, and nutritional value and scientiic
evidences) together with the basic documents demanded by basic
legislation to ANVISA for approval.
Several products, including probiotics, saw their claims being
changed in the re-evaluation process promoted by ANVISA in 2005;
such change had a better wording of claim as the main goal in
order to improve consumer’s understanding of product’s functional
properties (Brazil, 2011).
The list of approved claims in Brazil was updated in July 2008
and includes 10 probiotic strains (Lactobacillus acidophilus, L. casei
shirota, L. casei var. rhammosus, L. casei var. defensis, L. paracasei,
Lactococcus lactis, Biidobacterium biidum, B. lactis, B. longum,
Enterococcus faecium). The approved claim’s wording states: “The
(indicates the microorganism species) (probiotic) contributes to
the balance of intestinal lora. Its consumption should be associated
with a balanced diet and healthy lifestyle.” The speciic requirements
to uphold a claim associated with probiotics are listed in Table 7.3.
 Probiotics Legal Status 289

Table 7.3 Speciic requirements established by ANVISA for approved

claims concerning probiotic products

Parameter Details

Minimum In the range 1 × 108–1 × 109 cfu for daily

viable number recommendation of product ready for consumption.
Lower values may be accepted, if company proves its
effectiveness.

Documentation Report of the product analysis that prove the amount

on proof of of the minimum viable microorganism until the end of
eficacy shelf-life.
Resistance test of the culture employed in product
against the gastric acidity and bile salts.

Label/Claim The amount of viable probiotics in cfu, contained in the

daily recommendation intake in the product ready for
consumption, must be declared on the label next to the
claim.

Source: Adapted from Stringueta et al. (2012)

7.3.4.1 Legislaon
• Resolution no. 16: Procedures for registration of food and/or
new ingredients (Brazil, 1999d).
• Resolution no. 17: Risk assessment and food safety (Brazil,
1999e).
• Ministerial Order 398/1999 and Resolution no. 18: Basic
guidelines for analysis and approval of claims for functional
and/or health property mentioned on the labeling of the foods
(Brazil 1999a, 1999b).
• Resolution no. 19: Procedures for registration of foods
claiming functional and/or health properties (Brazil, 1999c).
• Resolution RDC no. 2: Technical Regulation of bioactive
substances and isolated probiotics with claim for functional
and/or health properties, annexed to that resolution (Brazil,
2002).
• Resolution RDC no. 27: Demonstrates about the categories
of food and packaging, which are excepted or compulsory to
sanitary registration, which apply to all types of food (Brazil,
2010).
290 Guidelines and Regulations

7.4 Conclusions
The global regulatory situation related with probiotics is still
undergoing development and change despite the tentative
harmonization by all countries. It is clear from the different
standpoints assumed by the different countries that probiotics are
of importance in terms of health promotion and disease prevention
among consumers. Nevertheless, there are still many challenges
to overcome until a steady, harmonized, and overall regulatory
framework is implemented worldwide to ensure the quality and
safety for proper utilization of probiotics in different countries; in
this context, regulatory bodies, food scientists, manufacturers, and
even consumers need to brace forces in order to achieve such goal.

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