IMPROVEMENT OF BIFIDO ENRICHED CURD

USING LACTIC ACID BACTERIA

Dissertation Submitted to Bharathidasan University

in partial fulfillment of the requirement for the Degree of
Master of Science in Life Science
(Five Year Integrated)

Submitted by

N. BOOMIKA

(Reg. No. LS18308)

Under the guidance of

Dr.D. DHANASEKARAN

Associate Professor

Department of Microbiology

Bharathidasan university

Tiruchirappalli 620 024

SCHOOL OF LIFE SCIENCES

BHARATHIDASAN UNIVERSITY
TIRUCHIRAPPALLI 620 024
June 2023
 School of Life Sciences
Bharathidasan University, Tiruchirappalli – 620024
Tamil Nadu, India.

CERTIFICATE
This is to certify that this dissertation entitled “IMPROVEMENT OF BIFIDO

ENRICHED CURD USING LACTIC ACID BACTERIA” submitted by N. BOOMIKA,

Reg. No LS18308 to School of Life Sciences, Bharathidasan University, Tiruchirappalli

620024, in partial fulfillment of the requirement for the award of Master of Science in Life

Science (Five year integrated, Specialization in MICROBIOLOGY).

Internal/ Research Supervisor Program Coordinator

Submitted to M.Sc., Life Science Degree Examination, held on………………at School of Life
Sciences, Bharathidasan University, Tiruchirappalli.

External Examiner-1 External Examiner-2 Internal Examiner

 DECLARATION

I, BOOMIKA. N hereby declare that the project entitled “IMPROVEMENT OF BIFIDO

ENRICHED CURD USING LACTIC ACID BACTERIA” is based on my own work carried

out during the course of our study from January – June 2023, under the guidance and

supervision of Dr. Prakash M. Halami, Chief Scientist and Head of Department of Microbiology

and Fermentation Technology (MFT) at CSIR – Central Food Technological Research Institute,

Mysuru, India. This project work was submitted to Bharathidasan University, Tiruchirappalli,

Tamil Nadu in partial fulfilment of the requirement of award of Master of Science in Life

Sciences as a record of dissertation work.

Place: Signature of the Student

Date:
 ACKNOWLEDGEMENT

I express my deep sense of thankfulness to my honorable guide Dr. D. Dhanasekaran,

Associate professor, Department of Microbiology for his valuable guidance and time,

inspirational thoughts, suggestions and constant support for my project work. I thank Sir for his

patience and persistent help.

I also express my gratitude towards my External guide Dr. Prakash M. Halami, Chief Scientist

and Head of the Department of Microbiology and Fermentation Technology (MFT), CSIR-

CFTRI for the constant encouragement throughout my course period. He has assisted me with

the protocols, and his advice has caused a massive impact on my results. Without his dedicated

assistance, the goals of this project would not have been reached.

I would also like to thank Dr. K. Emmanuvel Rajan, Co-Ordinator, School of Life Sciences,

Bharathidasan University for his valuable support which has been a great help in this endeavor.

I also would like to acknowledge the Department - School of Life Sciences for providing me this

learning opportunity of my dissertation work.

I wish to convey my earnest thanks to my lab seniors Mrs. Aditi Goel, Mrs. Steji Raphel, Mrs. Ishrat

Jahan. P, Mr. Krishna Komatwar, Ms. Kusuma N P and Ms. Pramila Epparti for the counsel,

assistance, and support throughout my project work.

I take this opportunity to express my boundless gratitude to my mom, dad, sister and friends for their

constant encouragement and emotional support during the project tenure.

BOOMIKA N
 TABLE OF CONTENTS

CHAPTER TITLE PAGE NO.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 8

3 MATERIALS AND METHODS 32

4 RESULTS AND DISCUSSION 51

5 SUMMERY AND CONCLUSION 81

6 REFERENCES 83
 List of Figures

S.no Figure title Pg.no

1 Health benefit of probiotics 5
2 Evolution of the term probiotics 10
3 Mechanism of action of probiotics 13
4 Probiotics market size 14
5 The SCFA in human body 30
6 Oscillatory/Rotational Rheometer 45
7 Fourier transform infrared spectroscopy 46
8 Soxhlet apparatus 48
9 Protein analyser 48
10 HPLC grade Shimadzu liquid chromatograph 51
11 Gram positive B. longum and L. delbrueckii 52
12 Individual Bifido probiotic curd(a) and Improved Bifido enriched 54
probiotic curd with L. delbrueckii (b)
13 Graphical representation of Colonies in A(control), B (Oxygen
Scavengers), C (Commercial Sachet) 58
14 Control in anaerobic chamber and Control plates 59
15 Oxygen scavenger in anaerobic chamber and plate 59
16 Commercial sachet in anaerobic chamber and plate 59
17 Compatibility between B. longum(a) and L. delbrueckii (b) 60
18 Comparison of Viability of B. longum,L. delbruckeii and Complex curd 61
19 Microbial quality assessment colonies 62
20 B. longum and complex curd showing zone against Pathogens in 64
antimicrobial assay
21 Relation between viscosity (ɳ) and shear rate (per sec) (γ) 64
22 Individual Bifido curd Spectrum 65
23 Complex curd spectrum 66
24 Market curd spectrum 67
25 Day 0-15 complex curd viability plates 72
26 Graph of shelf life of Complex curd 72
27 pH of the complex curd from Day 0-15 75
28 Data about all characterization of probiotic complex curd 76
29 Freeze-dried curd powder 76
30 Enumeration of wet curd and freeze-dried curd 77
31 Plates of enumerated Bifido curd and freeze-dried curd 78
32 Chromatogram for the freeze-dried powder 79
33 Chromatogram for the Butyric acid peak 77
34 Chromatogram for the Acetic acid peak 78
35 Cell viability percentage of MTT assay in B. longum curd powder 79
36 Sensory analysis 80
 List of Tables

S.no Title Pg.no

1 Timeline of the evolution of probiotics 11

2 Diary based probiotics 16

3 Non diarybased probiotics 17

4 Bifido enriched product 21

5 Microscopic observation of bacteria 52

6 Different percentage of individual bacteria curdling 53

7 Antibiotic sensitivity and resistance for B. longum and L. delbrueckii 55

8 Resistance profile for B. longum 56

9 Resistant profile for L. delbrueckii 57

10 A (control), B (Oxygen scavenger), C (commercial sachet) 58

11 Comparison of viability of individual and complex curd 62

12 Antimicrobial activity of individual and probiotic curd 65

13 Vibrational modes of bifido probiotic curd 66

14 Vibrational modes of complex probiotic curd 67

15 Vibrational modes of market curd 68

16 BIS specification of curd and Bifidobacteria probiotic curd comparison 71

17 Studies of viability of complex curd 72

 Abbrevations

IBD - Inflammatory bowel diseases

IBS - Irritable bowel syndrome

GIT - Gastrointestinal tract

NAFLD - Non-Alcoholic Fatty Liver Disease

GI – Gastrointestinal tract

GALT – Gut-associated lymphoid tissues

LAB – Lactic acid Bacteria

FDA – Food and Drug Administration

CBER – Centre for Biologics Evaluation and Research

CGRA – Currency and gold revaluation account

FSSAI – Food Safety and Security Authority of India

NSAID - Non-Steroid Anti-Inflammatory Drugs

DMARD - Disease Modifying A Rheumatic Drug

VRBA - Violet Red Bile Agar

MRS - De Man, Rogosa and Sharpe agar

PI - Primary inoculum

SCFA - Short chain fatty acid

FTIR- Fourier transform infrared

HPLC - High-performance liquid chromatography

 ABSTRACT
Research and commercial interest in Bifidobacterium have increased in the last decade due to

their health benefits in probiotic functional foods, in dairy and non-dairy food products.

Bifidobacteria are an anaerobic bacterium which is very challenge for viability is a fermentive

bacteria for curd and it contains so much of health benefits mainly in colon cancer. The aim of

this present study is to develop the probiotic curd with the selected strain Bifidobacterium

longum (lab isolate) with the incorporation of Lactobacillus delbrueckii (lab isolate). Different

percentage of both bacteria were checked to get good settling of curd and made the complex

curd. With the standardized of probiotic curd several parameters such as Antimicrobial assay,

FTIR, Rheological for viscosity and Proximate analysis such as moisture, ash, fat, protein and

Carbohydrate, Sensory analysis were done with the comparison of Market curd. The

standardized Curd made in large quantity and undergoes a freeze-drying. The storage studies of

the finalized probiotic curd were evaluated for two weeks by keeping at 4 ⁰C with selective

enumeration of antibiotics namely Mupirocin and Clindamycin were used. Difference between

viability of wet curd enumeration and freeze-dried enumeration were determined. With the

freeze-dried powder Short chain fatty acid synthesis ability and Anti-inflammatory cytokines

were checked by MTT assay. The suitable anaerobic condition for bifidobacteria were

determined by examine Oxygen scavenger and commercial sachet. A well settled curd was

obtaining in the 10% of B. longum and 5% L. delbrueckii combination without whey separation.

In storage studies, the probiotic bacterial counts were reducing gradually from Day 0 to Day 15.

Keywords: Probiotics, Bifidobacterium longum (lab isolate), Lactobacillus delbrueckii (lab

isolate), Freeze-Drying, SCFA analysis.

INTRODUCTION

1
1. INTRODUCTION:

The gut microbiota plays an important role in human health. It provides nutrients and energy for
the host through the fermentation of non-digestible dietary components in the large intestine.
Resident bacteria within the human gut are considered to represent a crucial line of defense
against colonization by potential pathogenic micro-organisms (Servin, 2004). The
gastrointestinal tract is a natural habitat for a large and diverse microbial ecosystem. Microbial
colonization begins immediately after birth with facultative anaerobes, which include
lactobacilli, enterococci, and enterobacteria, being the first colonizers. This is followed with the
colonization by anaerobic microorganisms, such as Bifidobacterium, Bacteroides, and
Clostridium. This results in a gradual decrease of the ratio of facultative anaerobes to strict
aerobes over time (Arboleya et al, 2012). Mode of delivery (natural birth vs. caesarean section),
initial diet (breast feeding vs. formula feeding), geographical location (developed country or
developing country) and type of birth (home birth or hospitalized birth) – all contribute to the
colonization pattern (Fanaro et al, 2003).

Foods are not only intended to relieve hunger and provide important nutrients, but also to prevent
the development of nutrition-related illnesses and to promote customers' physical and emotional
well-being (Stanton et al., 2005; Arihara, 2006). So, Let's accept the adage that "Let’s food be
your medicine and medicine be your food" by words of Hippocrates which is still the foundation
of contemporary medicine. Before the concept of probiotics originated, the health benefits of
these dairy products were a matter of mythology. But, as time has gone on, a more systematic
approach to the research of fermented milks and yoghurt containing probiotics has been
developed. As a result, functional foods or food ingredients that provide benefits beyond their
nutritional value have emerged ( Gasbarrini et al., 2016) . An increasing number of people are
turning to nutraceuticals, yoga, and workout as alternatives to going towards medicine.
Individuals have become more sensitive with their health status and aware of what ingredients
are in their food Probiotic foods are highly sought-after on the market as foods that promote
health. Functional traits of probiotic bacteria include their ability to adhere to the intestinal
epithelium and tolerate conditions in the gastrointestinal tract (GIT). Two important elements of
the probiotic potential that benefits the host organism are immunomodulation and bacterial
antagonism. Probiotic bacteria must be present in concentrations of at least 6 log cfu/g of food or

2
other formulation in order to provide the health benefits. Upon ingestion, probiotic bacteria must
overcome intestinal tract bacterial competition while travelling through the stomach's extremely
acidic environment (Krasaekoopt et al., 2003) More than 70% of immune cells are found in the
gut, particularly in the small bowel, making it commonly believed that the gut microbiota plays a
significant role in both health and illness by helping the immune system develop and function at
its best. As health-promoting foods, probiotic foods are gaining popularity in the market.
Tolerance to gastrointestinal tract (GIT) conditions, adherence to the intestinal epithelium,
immune regulation, and bacterial resistance are recognized essential components of the probiotic
potential that provides health benefits to the host organism. A possible weapon against bacterial
and viral infections in humans is thought to be bacterial strains that can increase IgG levels.
Research examining the impact of probiotic bacteria on immunological function revealed that
different strains, doses, and treatment regimens had different impacts on probiotics (Dinkci et al.,
2019).

The addition of beneficial bacteria into a food substrate poses a new problem because of their
interactions with other ingredients as well as the harsh conditions frequently used during the
preparation and storage of food, which may result in significant viability rates.

1.1 PROBIOTICS

Eli Metchnikoff, the 1908 Nobel Prize winner, proposed that the long life of Bulgarian farmers
was due to their use of fermented milk products, which is when the idea of probiotics first
emerged (Williams et al., 2010). Lilly and Stillwell used the word "probiotic" in 1965 to refer to
chemicals generated by one organism that promote the development of another. They were
described as "microbial preparations or components of microbial cells that have a favourable
influence on health and well-being" by (Marteau et al., in 2002).

The feeding certain probiotics to hospitalised new-borns might considerably lower the risk of
diarrhoea and rotavirus shedding (Saavedra et al., 1994).

3
Probiotics can be utilised to control gastrointestinal tract activity as well as the immune system
and endogenous intestinal flora. Due to a more systematic approach, the evidence for various
probiotics' beneficial benefits in particular clinical settings is now established.

Probiotics have been intensively studies for a numerous health advantages they provide to the
host, as well as prospective alternative therapy options for a variety of disorders. Increased study
in the field of probiotics has resulted from a shift in consumer attitude towards healthier options,
various unique species are being found and examined for their probiotic properties. Maintenance
of physiological and gut microbial homeostasis, reduction of serum cholesterol; relief of
gastrointestinal ailments such as lactose intolerance, diarrhoea, inflammatory bowel diseases
(IBD), irritable bowel syndrome (IBS); anti-cancer, anti-diabetic, anti-allergic;
immunomodulation and neuro immunomodulation (Nagpal et al., 2012). At present, several
well-characterized strains of Lactobacilli and bifidobacteria are available for human use to
reduce the risk of gastrointestinal (GI) infections or treat such infections (Salminen et al.,
2005). Several researchers have discovered that probiotics lower fecal concentrations of enzymes
(glycosidase, B-glucuronidase, azoreductase, and nitro reductase) and secondary bile salts, as
well as the absorption of dangerous mutagens that may lead to colon cancer (Rafter, 1995).
According to Goldin and Gorbach (1980), adding L. acidophilus to the diet reduces the incidence
of chemically produced colon tumors in rats. Several studies have found that lactobacilli found in
dairy products can boost the host's immunological response. This characteristic has been
discovered in B. longum, L. acidophilus, L. casei subsp. rhamnosum, and Lactobacillus
helveticus (Isolauri, 2001). Probiotics boost intestinal microbial populations largely by
stimulating intestinal fermentation; stimulation of short-chain fatty acid (SCFA) synthesis is one
of the important reasons for probiotics' positive effects. It has been shown that probiotic
Lactobacillus causes a considerable rise in indigenous lactobacilli in the large intestine
(Tsukahara & Ushida, 2001). Ley et al., (2005) thoroughly demonstrated the function of gut flora
in the pathophysiology of insulin resistance (type 2 diabetes) and obesity. Gut flora has been
shown in animal and human studies to increase body weight gain and insulin resistance, and
these phenotypes are transmittable with gut flora during implantation studies of microbiota from
obese to normal and germ-free mice (Ley et al., 2006; Turnbaugh et al., 2006). (Fig: 1) shows the
health beneficial of probiotics such as production of SCFA, Cytokines and inhibition of
enzymes.

4
 Fig: 1 Health benefits of Probiotics adopted from (Basak et al., 2022)

1.2 FERMENTED MILK AND PROBIOTIC CURD

Probiotic curd is a form of yoghurt that contains probiotics, which are living, helpful
microorganisms. These bacteria can improve digestive health, enhance the immune system, and
reduce inflammation, among other things. Probiotic curd is prepared by fermenting milk with
probiotic bacteria strains such as Lactobacillus and Bifidobacteria. Lactose (milk sugar) is
converted into lactic acid by bacteria during the fermentation process, giving the curd its sour
flavour and thick texture. Probiotic curd can be eaten on its own or as a component in a variety
of cuisines. It is a popular meal in many cultures, and it is often used in Indian cuisine as “dahi”.
Curd is a sour, viscous mixture produced from fermented cow or buffalo milk that is most
generally known as 'Dahi' on the Indian subcontinent. The name 'Dahi' comes from the Sanskrit

5
word 'Dadhi,' which is one of the 'Panchamrita' (five elixirs of immortality), and is regarded as a
functional meal because of its high nutritional value and a variety of health-promoting properties.
Curd consumption has been shown to improve immune function and reduce susceptibility to
infections such as Candida albicans and Pseudomonas aeruginosa in HIV-AIDS patients, slowing
disease progression and infectivity. It's been related to gastrointestinal cancer prevention and
prophylaxis, as well as the treatment of liver illnesses including NAFLD (Non-Alcoholic Fatty
Liver Disease), liver cirrhosis, and hepatic encephalopathy. Curd and fermented dairy products
have also been used to treat conditions including insomnia, diabetes, hypercholesterolemia,
pancreatitis, urogenital infections, and allergies. The reaction of milk proteins, specifically casein
and whey proteins, in an acidic environment results in curdling.

1.3 BIFIDOBACTERIA

Members of the genus Bifidobacterium have attracted a lot of commercial and scientific attention
during the past 20 years. Gram-positive procaryotes known as bifidobacteria are found in the
gastrointestinal tracts of warm-blooded mammals such as humans. Bifidobacteria, which were
just recently discovered, are important commensals in human-microbe interactions and are
thought to be essential for maintaining a healthy gastrointestinal tract (Leahey et al., 2005).
Henry Tissier isolated and characterised bifidobacteria for the first time in 1899-1900. He
discovered an abundance of an irregular Y-shaped bacteria in the faeces of breast-fed infants as
compared to bottle-fed children. On average, they account for almost three-quarters of all
bacterial cells in the new-born gut, compared to just approximately 3% in the adult gut flora
(Macfarlane, 1995).

Bifidobacteria are the mostly preferred probiotic after Lactobacillus. Bifidobacteria are
important commensals in interactions between humans and microbes and are thought to be
crucial for maintaining a healthy gut (Leahey et al., 2005). Bifidobacteria can have a significant
impact on other bacteria in the colon, but these bacteria, as well as other intestinal flora, are
influenced by a variety of exogenous and endogenous causes. Bifidobacteria colonize the gut
during infancy along with other microbe from mother’s vagina and faeces. Brest milk is also
considered as a source of inoculum for bifidobacteria.

6
1.4 LACTIC ACID BACTERIA

The term Lactic Acid Bacteria (LAB) was gradually accepted in the beginning of the 20 th century
(Carol et. al., 2010). Other terms as “milk souring” and “lactic acid producing” bacteria had
previously been used for the same bacteria causing a slight confusion. Lactic Acid Bacteria are
classified based on the gram reaction and the formation of lactic acid from various fermentable
carbohydrates. Lactic Acid Bacteria are gram-positive rods and cocci that are normally
nonmotile and do not generate spores. They are unable to create ATP via creating a proton
gradient because they lack the capacity to synthesise cytochromes and porphyrins (components
of respiratory chains). The LAB can only produce ATP by fermentation, generally using
carbohydrates. Lactic acid bacteria thrive in anaerobic environments because they do not require
oxygen for energy synthesis, but they may also thrive in the presence of oxygen. Lactic acid is
the major end product of their fermentation process. Omnipresent in nature, LAB is commonly
found in fermented foods as well as the human gut (Dack,1955). They have achieved the
“generally recognized as safe” (GRAS) status, as they become part of the microbiota of human
mucosal surfaces and confer health benefits to their host (Caballero et al., 2015). Therefore, LAB
is one of the most important microorganisms used in the food industry. Some of the well-known
genera of LAB are Lactobacillus, Enterococcus, Leuconostoc, Lactococcus, Pediococcus, and
Streptococcus (Ruiz Rodríguez et al., 2019) Lactic acid bacteria are chemotrophic, which means
they get their energy from the oxidation of chemical molecules. The primary energy-producing
mechanism is the oxidation of carbohydrates. Lactic acid bacteria of the genera Lactobacillus,
Leuconostoc, and Pediococcus, which are crucial in winemaking, absorb sugars via a
homofermentative or heterofermentative route (Carol et al., 2010).

The aim of this study is to improve the Bifido enriched curd with Lactic acid bacteria and see the
various parameters and to study the shelf life of the improved product.

7
The objective of this present study is

1. To improve the Bifido enriched probiotic curd using L. delbrueckii

2. To analyse the viability, storage conditions and sensory analysis of the improved
complex curd.

REVIEW OF LITERATURE

8
2. REVIEW OF LITERATURE

2.1 GUT MICROBIOTA

Microbiota refers to the entire population of microorganisms that colonizes a particular location;
and includes not just bacteria, but also other microbes such as fungi, archaea, viruses, and
protozoans (Sekirov et al., 2010).Significant interest in the gut microbiota has emerged in the
scientific community in recent years, and the gut microbiota have been linked to a wide range of
human diseases, including luminal diseases such as inflammatory bowel disease (IBD) and
irritable bowel syndrome (IBS), metabolic diseases such as obesity and diabetes, allergic disease,
and neurodevelopmental illnesses, though the strength of evidence for many of them is lacking
(Ferreira et al., 2014) The gut microbiota is made up of roughly 100 trillion bacteria that live in
the gastrointestinal system. The microbiota has a symbiotic connection with the gut mucosa,
mediating crucial physiological processes in exchange for nutrition, room for development, and
survival, and is necessary for maintaining homeostasis (Hardy et al., 2013). Elie Metchnikoff
(1907) first put up the idea of gut microbiota regulation as a means of enhancing health and
lifespan, which laid the groundwork for the current probiotic era. After a rocky start, probiotics
are now a multimillion-dollar industry and the fastest expanding field of study (Podolsky 2012).
The probiotics industry is expected to expand to approximately US$ 65.9 billion by 2024
(Severyn and Bhatt 2018), driven by consumer demand for food items that can promote health
and prevent or treat illnesses. Gut microbes are believed to interact bidirectionally with the host,
and such dynamic interactions are essential for homeostasis maintenance (Forbes et al. 2016).
Microbes are recognised to have a critical role in the development and functioning of the host
immune system.

The individual composition of infant gut datasets revealed a large conservation of members of
the Actinobacteria with a high proportion belonging to the family Bifidobacteriaceae ranging

9
from 74% to 99.3%. Despite the fact that B. longum species dominated the overall microbiota,
the fraction of B. longum in the individual composition datasets ranged from 21.7% to 90.6%
(Podolsky 2012). Presence of bifidobacteria is also known to promote the growth of other
friendly bacteria such as lactobacilli, as well as have negative impacts on the growth of
pathogens. They involve in cross-talk with the gut, and such interactions are considered as the
reason for the bifidobacteria mediated health benefits (Hidalgo-Cantabrana et al. 2017).

A lot of bacteria species in our intestine have a metabolism that ends with SCFA production,
which carry out various functions in the intestine and systemic activity. They increase the
number of regulatory T cells in the intestine and acetylation of histone H3, a major transcription
for transcription factor with anti- inflammatory activity, reducing elaboration and macrophage
activity (MICROBIOTA, 2017).

2.2 PROBIOTICS

2.2.1 HISTORY OF PROBIOTICS

Probiotic, a word derived from Latin, means ‘for life’. It introduced in 1953 by Werner Kollath
(Fig:2) (German Scientist) for describing "active substance that are essential for the healthy
development of life"(Gasbarrini et al., 2016).

Fig:2 Evolution of the term probiotics

10
In 1907 the foundation of concept about beneficial microbes was came to the speech by Elie
Metchnikoff followed by the development of the term Probiotic by Werner Kollath it leads to
define probiotics by FAO/WHO officially (Fig: 2).

Although they were not aware of the presence of bacteria and yeasts, the early food producers
actually used them to turn milk into fermented dairy products (Nakazawa and Hosono, 1992).
FAO/WHO proposed the current definition of probiotics in a consensus meeting in 2001 as 'live
microorganisms which, when administered in adequate amounts, confer health benefit on the
host' (FAO/WHO 2002). The traditional Indian medical practice 'Ayurveda' has also highlighted
the significance of consumption of milk and fermented milk products on health (Gasbarrini et al.,
2016). However, Elie Metchnikoff (Russian Scientist) is credited for the inception of probiotics.
He has been regarded as the 'father of probiotics' for his theory of altering gut flora for a healthy
life. He correlated the regular consumption of fermented milk (sour milk) with the longevity of
the Bulgarian locals. He proposed that putrefying bacteria in the intestines can be replaced with
useful microbes found in fermented milk, which can promote health (Podolsky 2012).

The greatest influence on microbiology was made by Pasteur and his successors. As
microbiology made strides, especially at the Pasteur Institute, the notion of utilizing helpful
microbes gained popularity. Pasteur made the initial discovery of lactic acid-producing bacteria
in 1857. The isolation of these bacteria from spoiled milk was then reported by Lister in 1878.
Between 1880 and 1888, researchers at the same institution identified lactic acid bacteria from
the digestive system (Neubauer and Mollet, 2002). From 1857 to 2016 the evaluation of
probiotic microbes is discussed in Table 2.

Bifidobacterium spp. were first discovered in 1889 by Henry Tissier, a paediatrician at the
Pasteur Institute. Tissier discovered that the majority of the bacteria in breastfed infants' gut flora
are Bifidobacteria, and he gave the bacteria's scientific name, Bacillus bifidus communis.

Update of Probiotics in Human World: A Nonstop Source of Benefactions till the End of Time

Table 1. Timeline of the evolution of probiotic bacteria adopted from (Neubauer and
Mollet, 2002).

11
 S.n Year Highlights
o

01. 1857-1864 Pasteur discovers that LAB are the culprits behind spoilage of food

02. 1878 Lister isolates LAB from milk

03. 1889 Tissier describes Bifidobacterium

04. 1900 Metchnikoff finds that Bulgarian Bacillus contributes to health

05. 1907 Moro describes Bacillus acidophilus

06. 1930 Shirota commercializes a fermented milk based on Lactobacillus case

07. 1953 The term ‘probiotika’ used to refer to active compounds promoting

health

08. 1965 Lilly and Stillwell define probiotics as “microbes stimulating growth

of other microorganisms”

09. 1989 Fuller defines probiotics as “beneficial microbial supplements”

10. 2001 The WHO/FAO defines probiotics officially

11. 2003 First-ever genome sequencing of Lactobacillus plantarum

12. 2005 Relman uses high throughput 16s amplicon sequencing to systemize

gut flora

13. 2016 FDA/CBER releases guidelines for live biotherapeutic formulations

2.3 MECHANISM OF ACTION OF PROBIOTICS

The mechanism of action of probiotics requires the survival of bacteria at low pH and bile salts
in order to produce therapeutic effects (Valero-cases et al., 2020). The bacteria must
12
subsequently be able to colonise the gut and make bacteriocins, short chain fatty acids, and
reduce pH for optimal development by creating organic acids Lactic acid, acetic acid, propionic
acid, and butyric acid are among the constituents (Docarmo et al., 2018). Second, the mechanism
involves the improvement of the intestinal barrier and tight junctions between epithelial cells, in
which probiotic bacteria produce peptides and mucus cells such as defining, which have an
antimicrobial effect against pathogenic bacteria due to their ability to solubilize the cell
membrane, preventing pathogenic bacteria from adhering to the cell membrane. (Sanchez et al.,
2017) Third, the method includes immune system regulation, in which the gut associated
lymphoid tissue (GALT) is made up of immune cells that are predominantly found in the small
bowel. T-cells and cytokines are produced when immunological receptors such as Toll-like
receptors (TLRs) are activated. This activates B cells, which create antibodies, notably IgA,
which block harmful microorganisms. (Fig:3) shows Dairy and non- dairy sources adopt
probiotic bacterial survival and abilize to colonize the intestine and withstand in low pH and bile
acids in human body and production of cytokines as health beneficial.

Fig: 3 Mechanism of action of Probiotics is adopted from (Bute, 2014)

2.4 PROBIOTICS SAFETY

According to FAO/WHO (2001), several studies back up the efficacy and safety of particular
probiotic strains, but these advantages cannot be generalised to other strains without further
13
testing. According to Papadimitriou et al., (2015), a thorough inventory of the hazards has to be
evaluated in various populations, at various dosages, and utilising various delivery methods and
matrices. Probiotic strains should be tested for safety factors such as mucin breakdown,
antibiotic sensitivity, the presence of antibiotic resistance genes, haemolytic activity, hydrogen
peroxide generation, and biogenic amine synthesis. Probiotics live in the gastrointestinal system
and interact with the local commensal bacteria. By passing the resistance to intestinal pathogens,
the presence of transferable antibiotic resistance genes in probiotic strains may have detrimental
effects. It is therefore vital to evaluate the durability or transferability of antibiotic resistance of
probiotic strains due to consumer safety concerns.

2.5 STATUS OF PROBIOTIC INDUSTRY

The global probiotics market was valued at USD 61.15 Billion in 2021 and is expected to grow
at a CAGR of 7.7% during the forecast period
(Probiotics Market Size Global Report, 2022 - 2030, n.d.)
. The growing need for consumer tendency towards healthcare that is preventive in
association with the advancement of systematic probiotic strains is primarily driving the growth
of the global probiotics market.

The data unmistakably shows that the probiotic industry will grow substantially in the years to
comes in (Fig 4). Therefore, a robust regulatory system that could manage the deceptive product
variety from the global and Indian markets is urgently needed. Currently, a small number of
major businesses worldwide control the probiotic market through the use of a select number of
well-established probiotic strains (Thakur et al., 2016).

14
Fig: 4 Probiotics market size by region 2018- 2030 adopted from
(Probiotics Market Size Global Report, 2022 - 2030, n.d.)

2.5.1 Probiotic industry in India

Pharmaceutical supplements and probiotic-containing functional foods each have a portion of the
probiotic industry. Food and Drug Administration (FDA) and Food Safety and Standards
Authority of India (FSSAI) now govern the assessment of the dependability and safety of these
items. With the support of the Food Safety and requirements Act of 2005, the FSSAI has
suggested minimal scientific requirements for the control of the production, storage, and
marketing of such items. After the creation of the ICMR-DBT recommendations, however,
attempts are being made to limit the deceptive advertising of probiotic goods through the
creation of a more precise regulatory framework that will benefit the security and wellbeing of
Indian customers (Thakur et al., 2016).

2.6 PROBIOTIC DOSAGE

The efficiency of adding probiotic bacteria is dosage dependent. They must remain viable during
the product's shelf life and survive the intestinal environment. They must establish themselves in
sufficient numbers in the gastrointestinal system to have a favourable impact on health
(Kailasapathy & Sultana, 2003). Several food organisations throughout the globe have adopted a
standard requiring a minimum of 106-107 colony forming units per gram (cfu g1) of
L. acidophilus and/or bifidobacteria in fermented milk products (IDF, 1992).

2.7 APPLICATION OF PROBIOTICS

2.7.1 Dairy based probiotics food products

15
Dairy products frequently fermented with LAB, or lactic acid bacteria, have been widely utilised
as probiotics in the market and have grown in popularity and scope in the dairy business. The
most popular dairy items are yoghurts, buttermilk, kefir, and fermented milk. Two categories of
dairy products that are sold on the market might be identified: (A) fermented, which includes
dairy products like kefir, yoghurt, and cheese. (B) Nonfermented foods like ice cream and sweet
milk. Live probiotic cultures (Lactobacillus and Bifidobacterium) have been added to ferment
milk, and various flavours of probiotic yoghurt (plain or flavoured) are offered in many nations
and have the highest market share. Table 2 discusses the many forms of dairy-based probiotic
products and microorganisms involved in their manufacturing from (Syiemlieh, & Morya 2022).

Table 2. Dairy based probiotics adopted from Syiemlieh, & Morya (2022).

S.n Dairy based Organism used Reference

o probiotics
1 Koumiss Lactobacilli, Non-lactose- fermenting Turkmen et al.,
yeasts (2019)
2 Kefir Lactobacillus kefir Kandylis et al.,
(2016)
3 Butter milk Lactococcus lactis, Lactobacillus Ranadheera et al.,
bulgaricus (2017)
4 Bifidus milk Bifidobacterium bifidum, Bifidobacterium Khorshidian et al.,
longum (2020)
5 Acido- bifidus Lactobacillus acidophilus, Bifidobacterium Hati et al., (2017)
milk sp.
6 Whey and sorghum Lactobscillus acidophilus Lactobacillus Morya et al.,
beverage casei, Lactobacillus rahmnosus (2017), Morya et
al., (2017)
7 Whey beverage Bifidobacterium lactis, Lactobacillus Shori, (2015)
acidophilus
8 Fermented skim Lactobacillus acidophilus Bifidobacterium Ranadheera et al.,

16
 milk Lactis animalis subsp. (2017)
9 Cheddar cheese Lactobacillus lactis subsp. lactis, Ulpathakumbura et
Lactobacillus helvetics, Streptococcus al., (2016)
thermophiles, Lactobacillus rhamnosus
10 Minas cheese Lactobacillus paracasei, Lactococcus Sperry et al.,
subsp. Streptococcus thermophilus (2018)

2.7.2 Limitations of Dairy based probiotic products

Lactose intolerance- It takes lactase enzymes, namely beta galactosidase, to break down this
milk sugar component, and in their absence, lactose cannot be hydrolysed or broken down into
the monosaccharide’s galactose and glucose. Those who drink milk and milk products and are
lactose intolerant have osmotic diarrhoea, gastrointestinal discomfort, and flatulence when
indigested or non-hydrolysed lactose enters the large intestines. (Khan et al., 2021; Kumar et al.,
2015).

Milk protein allergies - Cow's milk protein allergy (CMPA) is the common food allergen in
children and is defined as a reproducible allergic response to one or more cow's milk proteins
(CMP) which are usually caseins or whey beta-lacto globulin (Qamer et al., 2019).

Cholesterol content - Cow milk has a fat level of around 4-5%, whereas buffalo milk has a fat
content of about 7-8%. The quantity of fat in milk varies based on its source. High milk
consumption is anticipated to increase the necessary number of low-density lipoproteins (LDLP).
LDLP is a kind of poor cholesterol that is found in blood and has been linked to an increased risk
of heart disease, cardiovascular disease, and obesity (Natt and Katyal, 2021).

2.7.3 Non-dairy based food products

Furthermore, with an increase in vegetarianism in both developed and developing countries,

there is a high demand for plant-based probiotic products (Table 3) non-dairy probiotic
beverages are also cheaper alternative to dairy products for the delivery of probiotics in
developing countries. As a result, there is a need for the development and research of dairy

17
probiotic products that are high in nutrients and have health-promoting properties. (Kumar et al.,
2015).

Table 3. Non-Dairy based probiotics adopted from Syiemlieh, & Morya, (2022).

S.no Probiotics Probiotics microorganisms References

1 Oat flour, milk Lactobacillus plantarum, Ravindran and
Bifidobacterium lactis RadhaiSri,
(2020);
Asadzadeh et al.
(2021)
2 Wheat, millet, maize milk Lactobacillus plantarum, Tesfaye et al.,
or flour Lactobacillus acidophilus, (2019)
Lactobacillus fermentum,
Lactobacillus coprophilus,
Leuconostoc reffinolactis,
Leuconostoc mesenteroides,
Lactobacillus brevis, Saccharomyces
cerevisiae, Candida tropicalis,
Candida glabrata, Geotrichum
penicillatum, Geotrichum candidum
3 Sorghum, ragi flour Leuconostoc, Enterococcus and Salmeron et al.,
Lactobacillus brevis (2015)
4 Peanut-soy milk Pediococcus acidilactici, do Amaral Santos
Lactobacillus lactis, Lactobacillus et al., (2014)
rhamnosus, Lactobacillus acidophilus.
5 Chickpeas milk, Lactobacillus plantarum Paredes-Toledo,
Watermelon juice (2021)
6 Apple juice Lactobacillus paracasei Lactobacillus Lilio-Perez et al.,
plantarum Lactobacillus rhamnosus (2021); Aspri et
al., (2020)
7 Broccolli Lactobacillus bulgaricus, Maryati et al.,

18
 Streptococcus thermophilus, (2017)
Lactobacillus acidophilus,
Bifidobacterium bifidum
8 Mango juice Raspberry Lactobacillus casei Lilio-Perez et al.,
juice, Sugarcane (2021)
juice,Pumpkin,Aetichoke,
Cabbage juice

2.7.4 Limitation of non-dairy based food products

The viability of the beneficial bacteria during processing and storage of fruit, vegetables and
cereal foods is dependent on water activity, pH, and the type of strain. Fruit and vegetable juices
also include some necessary nutrients but, variables such as low pH, may impair probiotic
survival. Furthermore, because dairy products are often stored at temperatures close to 5°C,
probiotic cell viability is likely to be maintained throughout the product's shelf life. However, the
storage of non-dairy products is mainly at room temperature which can provide a significant
obstacle to probiotic survival (Abu-Ghanna and Rajauria, 2015).

2.7.5 Probiotics role in Non-alcoholic Fatty Liver Disease (NAFLD)

Modifications in the type and number of microorganisms that live in the intestinal lumen pose an
important role in hepatocyte function. The changes of population of the microorganisms that live
in the intestinal tract can cause serious and harmful liver dysfunctions like nonalcoholic fatty
liver disease (NAFLD). The gut microbiota contributes to inhibit NAFLD by anatomy-functional
interaction between the gut and the liver in which they block the entry of microorganisms to
blood flow and to the liver by increasing the strength of intestinal barrier (Ferolla et al., 2015).
The microorganisms that contribute to treatment of liver disease includes Lactobacillus
acidophilus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri,
Lactobacillus rhamnosus, Lactobacillus brevis, Lactococcus lactis, Bifidobacterium

19
adolescentis, Bifidobacterium longum, Bifidobacterium lactis (Meroni et al., 2019) (Syiemlieh,
I., & Morya 2022).

2.7.6 Probiotics in cancer

Many in vitro studies have revealed that probiotics have therapeutic characteristics in controlling
cancer cell growth and apoptosis. Lactobacillus rhamnosus GG strain, for example, has been
shown to reduce proliferation and cause apoptosis in mouse colon cancer HGC-27 and human
colon cancer Caco-2, DLD-1, and HT-29 cells. (Chen, 2021).

2.8 BIFIDOBACTERIAL SUPPLEMENTATION THROUGH FOOD

Food products have been increasingly used as a medium to deliver probiotic bacteria (Flach et
al., 2018). Food formulations supplemented with probiotic bacteria have been the choice of the
consumers compared to pharmaceutical formulations containing probiotics. Retaining the
viability of bacteria in an efficacious dosage is crucial for achieving the desired health benefits.
Food matrix is known to influence probiotic survival, physiology, and functionality (Sanders and
Marco 2010). In addition to the gastrointestinal stress, probiotic bacteria have to encounter
various harsh conditions during different stages of product development such as production,
down-stream processing as well as storage. Food products containing probiotic microbes are
developed in two ways: 1) Probiotic bacteria are added to the substrates where they grow,
interact, and produce metabolites resulting in final fermented product (e.g., Yoghurt); 2) Live
bacteria, lyophilised or dried powder, or encapsulated bacteria are added into the product where
there is no interaction between the bacteria and product matrix, e.g., Ice cream (Flach et al.,
2018).

In the case of fermented products, fermentation temperature, pH, oxygen, competition from
starter cultures, etc. and in case of non-fermented foods, the processing conditions have a
significant impact on the viability of bacteria. Additionally, factors such as food ingredients and
additives, temperature, pH, water activity, oxygen contents, and redox potentials, packaging

20
aspects and competing flora are known to influence the survival of bacteria in the product during
storage (Flach et al., 2018).

Bifidobacteria growth in soy milk has also been found to be strain dependent, with drastically
varied growth patterns seen between strains of the same species (Murti et al., 1993a; Scalabrini
et al., 1998). Yoghurt is the oldest fermented dairy product in the world, and it is most
commonly used as a probiotic carrier. Fruit preparations, including mango, strawberry, mixed
berry, and passion fruit, showed no influence on L. acidophilus and B. animalis subsp lactis
viability in stirred yoghurt (Godward et al., 2000; Kailasapathy et al., 2008). However, because
these bacteria are acidity sensitive, the viable count of B. bifidum in fermented milk combined
with orange and lemon fibres reduced (Sendra et al., 2008). Bifidobacteria has also been used in
the production of bread showing to be a viable alternative to Lactobacillus strains for dough
fermentation, with improved product quality and softer crumbs (Palacios et al., 2006).

2.8.1 Bifidobacteria – exclusive Dairy Products

Bifidobacteria holds a great reputation in the health aspect as they predominate the gut in breast-
fed infants. Some products are also manufactured which contain bifidobacteria exclusively and
they are enlisted as follows (Abe et al, 2009; Kurmann et al, 1992; Puniya, 2015; Yildiz, 2016).

Table 4. Bifido enriched products

Commercial Culture Product type Characteristics Viability

name count (cfu/ml)
Bifidus Milk B. bifidum, Fermented milk Should be <10⁰C 8
10 - 10
9

B. longum can be stored till

2 weeks
7
Bifighurt B. longum/ Fermented milk Acidification time 10
B. bifidum is short.
7
Bifidus Yoghurt B. longum BB536 Yoghurt Less acidic 10
8 9
Sweet Bifidus Bifidobacteria spp. Non fermented Freeze dried 10 - 10
Milk milk culture also
present

21
Bifidus Milk is the first infant product produced with bifidobacteria. It was first produced by
Mayer in 1948 in Germany. Now it is manufactured in smaller quantities in various European
countries. The final product has a pH of 4.3-4.7. It contains 108 -109 viable bifidobacteria
(Puniya, 2015).
Bifighurt is made with B. longum C KL 1969 (DSM 2054, a slime-forming variant) (Kurmann et
al, 1992). Manufacturing process is very similar to the production of Bifidus Milk. 6% inoculum
is added. The pH of the final product is 4.7. It contains more than 107 viable bifidobacteria. It
should be stored in refrigerator and it expires after 2 weeks of storage (Yildiz, 2016).
Bifidus Yoghurt is produced by the company Morinaga Milk. Co. of Japan. This utilises
Bifidobacterium BB536. This was released to the market in 1979 (Farnworth, 2008). The yoghurt
contains 107 of B. longum BB536 (Abe et al, 2009).
Sweet Bifidus Milk is produced in US, Germany and Japan. It is manufactured by inoculating
sterilised milk with freeze dried bifidobacteria. It contains 108 - 109 viable bifidobacteria
(Puniya, 2015).

2.9 VIABILITY OF BIFIDOBACTERIA

A number of bifidobacteria strains found in dairy products are unable to exert their probiotic
properties because they die due to a lack of resistance to acid production during/after
fermentation, oxygen exposure, or passage through the human stomach (Maus and Ingham,
2003). Because adequate numbers of cells cannot survive the acidity in the human stomach,
stomach acidity affects the functioning of Bifidobacterium in the large intestine. In a study of the
effect of prebiotics on bifidobacteria viability, researchers observed that B. animalis and FOS
had the highest viability (3.59-2.25-10 CFU/g), whereas B. longum had a viable number of more
than 10 CFU/g in yoghurt fortified with FOS for up to 21 days (Bruno et al., 2002) (Akalin et al.,
2004).
The use of probiotic encapsulation has been extensively investigated. In the case of
bifidobacteria, encapsulated copies of the bacteria have been introduced to mayonnaise. In a
study on bifidobacteria viability in soy milk, freeze-drying of cultures yielded more viable cells
than spray-drying, with B. longum surviving better than B. infantis.

22
2.9.1 Bifidobacterium longum
B. longum has been shown in tests to reduce the incidence of gastrointestinal symptoms such as
diarrhoea and nausea after antibiotic usage and to increase the nutritional content of meals.
Lactose maldigestion: Lactose intolerance is a condition that causes lactose maldigestion. Loose
stools, stomach bloating, discomfort, gas, and nausea are all signs of this condition. Individuals
with lactose intolerance tolerate lactose in yoghurt far better than the same quantity of lactose in
raw milk. B. longum has been demonstrated to be effective in reducing lactose intolerance in
certain people. In terms of microbial infection resistance, bifidobacteria have been studied for
their antagonistic actions against various microbial pathogens. Several protective mechanisms
have been proposed, including the generation of different acids, hydrogen peroxide, or
bacteriocins, anti-toxin activity, and immune system activation. The quantity of indigenous
bifidobacteria is lower in the elderly than in children, and this drop in the digestive tract leads to
a rise in the amount of Clostridium perfringens. In animal models, B. longum also protects
against the pathogenic challenge of Salmonella typhimurium (Patil and Halami et al., 2021).

2.9.2 Anaerobic condition

This system involved the oxidation of glucose catalyzed by glucose oxidase in the presence of
water. The hydrogen peroxide formed was removed by the action of catalase. The concept was
commercialized by Scott's company, Fermco Laboratories, using the trade name "Oxyban". A
commercial enzymic oxygen scavenger in a sachet is marketed internationally by Bioka, a
Finnish company. The oxidation of hydrogen to form water may appear to be a very attractive
mechanism for oxygen scavenging. This process has been used by microbiologists for
deoxygenating anaerobic jars for cultivation of anaerobes for many years. The process was
proposed for the packaging of milk powder in cans by King (1955), and subsequently by (Abbott
et al., 1961).

2.9.3 CULTIVATION OF BIFIDOBACTERIA

Bifidobacteria are strict anaerobes that require a sufficient growth medium and an oxygen-free
environment to develop. In an adequate growing medium, the following characteristics are
advantageous for successful identification and enumeration of bifidobacteria:

23
 1) a selective culture medium that inhibits the growth of bacteria that would otherwise
overgrown bifidobacteria.
2) A distinguishing colouring agent that helps distinguish bifidobacteria colonies from other
bacteria.
3) A fresh-ingredient medium with a pH calibrated to allow bifidobacteria to live
effectively.
4) A media composition that encourages the growth of numerous species contained in the
material under consideration (Perez, 2002).

Bifidobacteria culture mediums are classified into four types: basic, elective, differential, and
selective. Although modified MRS agar (MRS + 0.05 percent L-cysteine HCI) is not
selective for bifidobacteria, it is recommended for counting them from pure cultures and has
been demonstrated in several studies. The recovery of bifidobacteria has been good and
continuous (Dave and Shah, 1996).

2.9.4 CHALLENGES OF BIFIDOBACTERIA AS A PROBIOTIC

Viability- Yoghurt, the most commonly used dairy fermented product, has recently been
fortified with certain beneficial microbes, namely probiotics, the main microbes being
Lactobacillus and Bifidobacterium. The viability of bifidobacteria in fermented dairy products,
on the other hand, rapidly degrades during storage and transit through the GIT (Sultana et al.,
2000). This is because bifidobacteria can only thrive at pH levels close to neutral; it cannot grow
at pH levels lower than 5 or higher than 8. The bifidobacterial population is rapidly declining,
falling well below the required limit of 10-10 cfu/g (Doleyres and Lacroix, 2005).

Anaerobic Condition- Most Bifidobacterium sp. are generated from human gut bacteria and are
so anaerobic in nature. Because these animals lack a complete electron transport system, they
cannot scavenge oxygen. As a result, oxygen is incompletely broken down into hydrogen
peroxide, which is harmful to the body due to the formation of reactive oxygen species (ROS).
The fatty acid profile of B. longum changed after it was exposed to oxygen in research. The

24
strains shape was changed as well, resulting in elongated cells with a rough surface and a
prolonged log phase (Ahn et al., 2001). Oxygen sensitivity in probiotics, especially in
bifidobacteria, has been observed to vary, owing to strain- dependent features (Devries and
Stouthamer 1969) Cheng and sandine discovered adequate Bifidobacterium sp. Growth in the
absence of stringent anaerobic condition (1989).

Acid and stress survival - The capacity of bifidobacteria to survive in an acidic and stressful
environment is determined by the strain chosen. Several studies on acid and bile resistance in
bifidobacteria strains and species have been undertaken. In a study, the vitality of B. lactis was
examined in the presence of L. bulgaricus and S. thermophiles. The numbers of B. lactis and
Lactobacillus acidophilus had decreased by one log cycle at the end of the storage period. Low
bifidobacteria numbers might have resulted from a drop in pH and the production of organic
acids. Bacteria are typically encased to protect them from acid damage, allowing them to survive
in a very acidic environment. B. lactis and L. acidophilus encapsulated in calcium induced
alginate-starch increased yoghurt post-acidification survival while having no effect on pre-
acidification survivability.

2.10 Dairy product fermented as a bifidobacteria and Lactis as an adjuvant

Starter culture

Studies that a murine model has been established in which the adjuvant activity of yogurt
containing L. acidophilus and Bifidobacterium spp. was demonstrated by generating a strong gut
mucosa and systemic IgA anti-CT response (Tejada-simon et al., 1999). Yogurt manufactured
with starters containing only yogurt bacteria L. bulgaricus and S. thermophilus produced
decreased IgA anti CT when compared with either the control group fed the NDM or other
groups fed different types of yogurt made with L. bulgaricus and S. thermophilus supplemented
with L. acidophilus and Bifidobacterium spp. and (Shinjin et al., 1993) a method of producing
milk-fermented food, wherein a bifidobacteria or a lactic acid bacteria or a combination of these
two bacteria are inoculated into and cultured in a culture medium composed mainly of milk, and
an isolated soybean protein or a yeast extract or a combination of these substances are added to
the culture medium or a culture obtained by cultivation of the bacteria. The bifidobacteria is one

25
or two species selected from Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium
bifidum, or Bifidobacterium infantis.

2.11 FREEZE DRYING OF PROBIOTIC PRODUCT

Lyophilization, also known as freeze drying, is a process in which water is frozen and
subsequently removed a sample, first by sublimation (primary drying) and later by desorption
(secondary drying). Freeze drying is a drying procedure in which water is evaporated from the
product after it has been frozen. Sublimation is a phenomenon that occurs when water moves
straight from the solid state (ice) to the vapour state without going through the liquid stage.
Water may sublimate at pressures and temperatures below the triple point, i.e. 4.579 mm Hg and
0.0099 degree Celsius (Avis 2018).

The substance to be dried is first frozen, then heated (through conduction, radiation, or both)
under a high vacuum such that the frozen liquid sublimes, leaving only solid, dried components
of the original liquid. The concentration differential of water vapour between the drying front
and the condenser drives water removal during lyophilization (Avis 2018). Lyophilization begins
with sample preparation and continues with freezing, primary drying, and secondary drying to
produce a final dried product with the required moisture content. The concentration differential
of water vapour between the drying front and the condenser drives water removal during
lyophilization. During the main drying process, the vapour pressure of water rises as the
temperature rises (Schwegman et al., 2009). There are four stages of freeze drying (Kunal et al.,
2019).

 Pre treatement
 Freezing
 Primary drying
 Secondary drying

2.11.1 VIABILITY OF FREEZE-DRIED PRODUCT

Freeze-drying is a common method for incorporating probiotics into dishes. However, the
viability of freeze-dried probiotic bacteria is impaired during manufacturing and storage. Cryo-

26
protectants are used to protect freeze-dried probiotic organisms, and the key challenge is to
develop protective agents that increase cellular viability during storage and application in food
(Fonseca et al, 2015). Given the storage, handling, and transit scenarios, dried concentrated
probiotic cultures are the best form for incorporation into functional meals, particularly shelf-
stable functional foods (Ramos et al, 2013). The type of probiotic inoculant used, process
conditions, reconstitution conditions, the ability of the probiotic culture to grow and retain
viability in the food environment, and the preservation of probiotic characteristics in the food
product until consumption are all challenges associated with the introduction and maintenance of
large numbers of viable probiotic cultures into foods. For optimal probiotic administration in a
food format, identifying and employing the most effective reconstitution conditions for
sustaining viability of probiotics is critical (Jofre et. al, 2014).

2.11.2 Freeze Drying of bifidobacteria

Studies have confirmed that bifidobacteria in general are very sensitive to spray-drying and
showed superior survival rates following freeze-drying in different media (Simpson et al., 2005;
Wong et al., 2010). Maintaining viability during both the drying process and storage is necessary
(Saarela, 2005). Storage temperature, exposure to oxygen, and water activity are some critical
factors that affect viability of dried probiotics.

B. longum showed good survival rates after freeze drying when trehalose was used as
cryoprotectant and at frozen temperatures for a sufficient storage period (10 to 12 months). The
combination of 0.1M sodium phosphate buffer (pH 6.8), 5% dried skim milk (DSM), and 12 4%
trehalose provided the highest recovery rate in the studies done by Celik & O’Sullivan (2013).
They also studied the effect of temperature on the viability of freeze-dried bifidobacteria during
storage. Even if there was a decrease in the recovery rate with the increase in storage
temperature, there was a 1 log reduction in the viability of freeze-dried bifidobacteria when
stored at -20⁰C. The viability remained stable for as long as 10 months at the same storage
temperature. The other conditions tested for maintaining the viability of freeze-dried
bifidobacteria were the atmosphere exposure, and water activity of the powders. Replacement of
oxygen with nitrogen while packing and keeping the water activity controlled between 0.11 and

27
0.22 were the optimal conditions for storage of freeze-dried Bifidobacteria. The study carried out
by Bruno & Shah (2003), on the other hand, showed a 2-log decrease in the viability of freeze-
dried B. longum 1941 and 1 log reduction in B. longum. The viability was stable for 12 months.

2.12 Health benefits of bifidobacteria:

Bifidobacteria exhibit some significant health benefits such as improved lactose tolerance,

lower serum cholesterol, reduction in diarrhoea, suppression of pathogens and anti – obesity

(Achi and Halami, 2017). Some of them are enlisted below:

1) Diarrhoea – Many studies have reported Bifidobacteria being used to treat

gastrointestinal disorders (Hamid et al., 2019). This is mainly by the administration of

B. breve and B. longum. They are known to prevent acute rotaviral diarrhoea (Vlasova

et al., 2016).

2) Colorectal cancer – Probiotic supplements containing B. longum have been found to

suppress the total number of colon cancer cells in rats (Gueimonde et al, 2007). Some

strains of probiotic have been used as an adjuvant in cancer treatments (Yakoob &

Pradeep, 2019).

3) Excellent antioxidant activity – B. longum showed inhibitory effects on free radicals

and reactive nitrogen species. Both intact cells and the intracellular cell-free extracts

have anti-oxidative properties.

4) Influenza – Continuous intake of B. longum BB536 decreased the incidence of

influenza and fever, probably by potentiating innate immunity – higher levels of

neutrophil phagocytic activity and NK cell activity (Namba et al., 2010).

5) Lactose intolerance – Persons suffering from lactose maldigestion can tolerate lactose

present in yoghurt better compared to the lactose in raw milk. Yoghurt bacteria have

28
shown to release lactase upon reaction with bile salts within the gastrointestinal tract.

This enzyme can then act on the ingested lactose (Leahy et al., 2005). B. longum is a

predominant probiotic that reduces lactose maldigestion (Jiang et al., 1996).

6) Immunomodulatory effects – A study done by (Arunachalam et al., 2000) showed how

a six-week administration of B. lactis to healthy elderly individuals resulted in

increased levels of interferon alpha and polymorphonuclear phagocyte capacity.

(Chiang et al., 2000) also showed similar results and in addition, there was an increase

in NK cell tumor cell-killing activity.

7) Rheumatoid arthritis - RA is one such chronic inflammatory disorder primary affecting the
synovial tissue, articular cartilage resulting in joint damage and disability. RA an autoimmune
disease characterised with joint inflammation, progressive disable systemic complications (which
includes cardiovascular, pulmonary, psychological, skeletal disorders), and mortality (McInnes
and Schett 2017). RA is known to affect 1% of the population, wis higher prevalence in women
and has a prevalence rate of 50% among the middle-aged older population (Okely et al., 2016;
McInnes and Schett 2017; Park et al., 2018). At present, RA is incurable and, the current
treatment includes Non-Steroid Anti-Inflammatory Drugs (NSAID), Disease Modifying A
Rheumatic Drug (DMARD) and cytokine inhibitors. These are known to have several sit effects,
as well as cause an economic burden on the patient (Gulácsi et al., 2015). properties of probiotic
are highly species and strain specific. (Achi and Halami, 2017) study, three different species of
probiotic bifidobacteria, B. breve NCIM 5671, B. longum NCIM 5672 and B. bifidum NCIM
5697 were evaluated for the prophylactic effect against chronic inflammatory disease,
rheumatoid arthritis in rat model.

2.13 Short Chain Fatty Acid:

Probiotics and their metabolites, such as butyrate and pyridoxine, have shown promise in
preclinical studies as anticancer agents. SCFAs provide energy to colon cells while also
maintaining the intestine's acidic environment, inhibiting the formation of high levels of

29
secondary bile acids, and promoting acidosis and apoptosis in cancer cells (Probiotics in
colorectal cancer (CRC) with emphasis on mechanisms of action and current perspectives).
Butyric acid, for example, aids in the balance of colon cell proliferation, division, and death.
Colon cell metabolism produces around 70%-90% of butyrate, and there is a significant drop in
this kind of acid in the faeces of colorectal cancer patients when compared to healthy persons.
(Macfarlane, 2003). Although SCFAs are obtained from the gut flora, the amount produced may
not be adequate to prevent the development of colorectal cancer due to individual variances. As a
result, taking probiotics can aid in increasing the daily production of SCFAs. Pathogen
proliferation can be inhibited by the presence of SCFAs. Propionic acid and butyric acid reduced
the expression of invasive genes expressed by Salmonella typhimurium in vitro, inhibiting its
assault on healthy cells. (Gantoiset al.,2006). (Fig: 5) shows that the SCFA produced by the
probiotics how they are eliminating cancer cells by the cytokine IL-17α which is produced by
Th17 cell an indicator from T cell. Also, causing Apoptosis for proliferating cells, presence of
Antibacterial peptides and inflammation.

Fig 5: The SCFA in human body adopted by (Macfarlane, 2003).

30
Short-chain fatty acids (SCFAs) are organic fatty acids of one to six carbon atoms long in the
acyl chain. They are found as the main products of anaerobic microbial breakdown of
polysaccharide, oligopolysaccharide, and proteins in the lumen and large intestine of plant-eating
animals. Acetate (C2), propionate (C3) and butyrate (C4) are the major anions of SCFAs found
in the lumen of the human large intestine. Bifidobacteria utilize various non-digestible
carbohydrates in the large intestine through their specific "bifid shunt" catabolic pathway to
subsequently produce SCFA in the human gut. SCFAs are metabolised at three main locations in
the body: colonocytes which utilise butyrate through ẞ-oxidation as the main energy source liver
cells which metabolise butyrate and propionate for glucogenesis, the third site are muscle cells
that generate energy from the oxidation of residual acetate (Roberfroid 2005).

2.14 STORAGE STUDIES

Due to their perishability and rapid spoiling, dairy products such as curd, yogurt, and cheese
have an extremely limited shelf life. As a result, any dairy product with a longer shelf life is
highly valued. After up to three days of storage at room temperature, plain dahi remained in good
shape. Plain dahi, on the other hand, was safe to eat for up to twelve days when kept in
refrigeration. It was evident that commercial dahi could only last three days at room temperature
(Tripathi et al., 2014)

2.15 BIS/FSSAI SPECIFICATIONS FOR DAHI

According to BIS (IS 9617: Dahi),

1) The curd can either be bland or flavorful.

2) To make dahi, use the following cultures:

a) Streptococcus lactis, Streptococcus diacetilactis, Streptococcus cremoris, single or in

combination with or without Leuconostoc species; and Bifodobacterium longum and breve.

31
b) Lactobacillus bulgaricus, Lactobacillus acidophilus, and Lactobacillus casei are among the
Lactobacillus species mentioned above.

3) Dahi should have a pleasant bouquet flavour that is the consequence of a combination of a
clean delicate somewhat fragrant scent and a strong but clean acid

taste.

4) Body and texture- It should be firm, solid, and consistent before shaking, with minimal whey
separation.

5) Dahi should not have any of the following flavourings:

a) Bitter taste

b) Coarse (due to over ripening)

c) Flat

d) Off odour

MATERIALS AND METHODS

32
3. MATERIALS AND METHODS

3.1. MATERIALS

3.1.1. TOOLS

 Glassware: petri plates, test tubes, beakers, conical flasks, volumetric flasks, measuring
cylinders, stoppers, L-rods, microscope slides, funnels, cuvettes, containers.

 Plasticware: pipette tips, tip boxes, Eppendorf centrifuge tubes, microcentrifuge tubes,
L-rods, rubber bands, autoclavable bags, test tube stands, centrifuge stands,
microcentrifuge stands, plastic gloves, droppers, ruler, containers, wash bottle, and spray
bottle.

 Metalware: inoculation loops, spatulas, scissors, knife, and forceps.

 Multifarious: pipettes, micropipettes, lab coat, cotton, marker pens, oven gloves,
aluminium foil, tissue paper, filter paper, butter paper, parafilm, adhesive label stickers,
transparent adhesive tape, syringes, disinfectants, and soap solution.

33
3.1.2. INSTRUMENTS

Autoclave, bacteriological incubator, compound light microscope, cooling centrifuge, freezer,

hot air oven, laminar air flow cabinet, microwave oven, pH meter, refrigerator, shaking
incubator, UV spectrophotometer, vortex mixer, water bath, weighing balance, HPLC, Muffle
furnance.

4.1.3 CHEMICALS AND REAGENTS

The chemical reagents such as Crystal violet, Gram’s iodide, Gram’s decolouriser, Safranin, 3%
H2O2, Ethanol 70%, 0.85% of Saline solution, Dried skim milk, 0.5% Sucrose, 5% L-Cysteine
Hydrochloride were procured from Himedia Laboratories Private Ltd. (Mumbai, India)

3.1.4 MEDIA AND SOLUTIONS

Composition of HiMedia Lactobacillus MRS Broth

Lactobacillus MRS Broth was supplemented with 0.05% L-Cysteine Hydrochloride, purchased
from Himedia Laboratories was used for Subculturing the organisms. The microorganisms were
grown in the culture media for 24-48hrs at 37⁰C in incubator.

Ingredients Amounts g/L

Peptone 10
Beef extract 10
Yeast extract 5
Dextrose 20
Polysorbate 80 1
Magnesium sulphate 0.1
Manganese sulphate 0.05

34
 Dipotassium hydrogen phosphate 2
Ammonium citrate 2
Sodium acetate 5
pH 6.5

Composition of HiMedia Lactobacillus MRS media

Bifidobacterium colonies were counted using Lactobacillus MRS Broth was supplemented with
0.05% L-Cysteine Hydrochloride and Agar Agar type.

Ingredients Amounts g/L

Peptone 10
Beef extract 10
Yeast extract 5
Dextrose 20
Polysorbate 80 1
Magnesium sulphate 0.1

Manganese sulphate 0.05

Dipotassium hydrogen 2
phosphate

Ammonium citrate 2

Sodium acetate 5
Agar Agar type 1 18

pH 6.5

Composition of HiMedia Plate Count Agar

Ingredients Amount g/L

35
 Tryptone 5

Yeast extract 2.50

Dextrose 1

Agar 15

pH 7

Composition of Violet Red Bile Agar

Ingredients Amount g/L

Yeast extract 3

Peptone or gelysate 7

NaCl 5

Bile salts or bile salts 1.5

Lactose 10.0

Neutral red 0.03

Crystal violet 0.03

Agar 0.002

pH 7.2

Composition of Brain heart Infusion

Ingredients Amount g/L

HM Infusion powder 12.50

36
 BHI Powder 5

Proteose peptone 10

Dextrose 2

Sodium Chloride 5

Disodium hydrogen phosphate 2.50

pH 7.4

4.1.5 Bacterial Cultures

Potential probiotic culture Bifidobacterium longum (Lab isolate), Lactobacillus delbrueckii (Lab
isolate) Which were characterise native laboratory isolates of Department of Microbiology and
Fermentation Technology at CSIR- CFTRI, Mysore was used for curd preparation. Both bacteria
were maintained in MRS medium (with L-cysteine hydrochloride for B. longum) at 37⁰C.

3.2 PREPARATION OF GLYCEROL STOCK

Glycerol stock was prepared by collecting a concentrated cell pellet and suspending in 80%
glycerol. The stocks were stocked at -80⁰C for further use.

3.3 MICROSCOPIC ANALYSIS - Gram staining

Gram-staining was invented by Danish bacteriologist Hans Christian Gram in 1884 [Gram
staining. Current protocols in Microbiology, A, 3C]. It was performed to categorize the isolated
cultures as Gram-positive or Gram negative and observe the cell morphology [Probiotic
properties analysis of isolated LAB from buffalo milk]. The procedure is based on the ability of
microorganisms to retain the colour of the stains used during the gram staining process. Standard
microbiological methods were followed.

3.3.1 Smear preparation:

37
1. Grease free dry slide was taken and cleaned with alcohol.

2. Circle was drawn on backside to the slide and showed it to the flame and used to cool.

3. Loop was heated and used to cool the heated loop resuspend the loop into MRS broth contains
Bifidobacterium longum species and Lactobacillus delbrueckii species.

4. Smear in the way circle is drawn.

3.3.2 Sample preparation:

1. Flood air-dried, heat- fixed smear of cells were washed with crystal violet (staining reagent)
and it was used to dry for 1 minute. The quality of the smear was noted (Too heavy or too light
concentration will affect the gram staining result).

2. Slide was gently washed and indirect stream of distilled water for 2 seconds.

3. Slide was flooded with the mordant gram iodine, and it was used to dry for 1 minute.

4. Slide was gently washed and indirect stream of distilled water for 2 seconds.

5. Slide was flooded with decolorizing reagent (acetone-alcohol decolourizer) and it used to dry
for 3-5 seconds.

6. Flood slide with counter strain, safranin waits for 30 seconds to 1 minute.

7. Wash slide with gentle and indirect stream of distilled water until no colour appears.

3.4 PREPARATION OF BIFIDO PROBIOTIC CURD

3.4.1 Preparation of primary inoculum

A primary inoculum of B. longum was prepared prior to subjecting the culture to the milk
substrate, culture stored in glycerol stock was first sub cultured in 5ml screw-cap glass tubes
containing Lactobacillus MRS Broth with 0.05% L- cysteine. The subsequent sub-culturing was
carried out in 15mL then 50ml Falcon Tubes by inoculating 5% of the cultures. The culture was
sub-cultured twice before the preparation of Pl. The tubes were incubated at 37°C for 24-48 hrs
and centrifuged at 8000r.p.m. for 15mins. The pellet was dispersed in 10% skimmed milk

38
 prepared by steam sterilisation and distributed into two 50ml Falcon Tubes. The volume was
made up to 50ml by adding 10% Skimmed milk and 0.5% Sucrose. The PI was mixed
thoroughly and incubated at 37°C in incubator for 4-6 hours.

Similarly, primary inoculum for L. delbrueckii was prepared prior to subjecting the culture to the
milk substrate, culture stored in glycerol stock was first sub cultured in 5ml Lactobacillus MRS
Broth in 15mL Falcon Tubes. The subsequent sub-culturing was carried out in 50mL Falcon
Tubes by inoculating 2% of the cultures. The culture was sub-cultured twice before the
preparation of Pl. The tubes were incubated at 37°C for 18-24 hrs and centrifuged at 8000r.p.m.
for 10mins. The pellet was dispersed in 10% skimmed milk prepared by steam sterilisation and
distributed into 50ml Falcon Tube. The volume was made up to 50mL by adding 10% Skimmed
milk and 0.5% Sucrose. The PI was mixed thoroughly and incubated at 37°C in incubator for 4-6
hours.

3.4.2 Optimisation of Inoculum for Curd Preparation

Primary inoculum were inoculated in freshly prepared skim milk with many different percentage
of B. longum [10%,15%,20%,25%.30%] and L.delbruckeii [2%,5%,8%,10%,12%] in different
cups. On the basis of Visual observation, colour, whey separation and pH of the different
percentage, the best percentage was selected for the combination fermentation process.

Lactobacillus MRS Broth

5% of Bifidobacterium longum supplemented 2% of Lactobacillus delbruckeii in 5 ml

with 0.05% of L-Cysteine in 5 ml

5% of Bifidobacterium longum supplemented 2% of Lactobacillus delbruckeii in 15 ml

with 0.05% of L-Cysteine in 15 ml

39
5% of Bifidobacterium longum supplemented 2% of Lactobacillus delbruckeii in 50ml
with 0.05% of L-Cysteine in 50 ml

Centrifuge at
8000rpm for 15 mins
at 4⁰C
10% of Sucrose and 0.5% of Sucrose was 10% of Sucrose and 0.5% of Sucrose was
prepared and steam sterilized and half pellet prepared and steam sterilized and the pellet of
of bacterial cell is added into it and kept in bacterial cell is added into it and kept in 37⁰C
37⁰C

The PI is obtained in 4-6 hours

The PI is inculated as per standardized

percentage into 10% of SKM in
Nandhini tone milk and kept in 37⁰C

The Final product id obtained within 7 hours

Fig: Steps involved in Curd preparation

3.5 SELECTIVE ENUMERATION OF PROBIOTIC CULTURE

Antibiotic Sensitivity assay is usually performed to check the effectiveness of culture against an
antibiotic. A comparative study of the growth and survival of the two cultures was carried out in
the presence of antibiotics. This was done to selectively enumerate the culture after fermentation.
Two antibiotics were selected to check the resistance and sensitivity against Bifidobacterium

40
longum and Lactobacillus delbrueckii. These were checked with the help of Himedia Octadisc
and Agar disc- diffusion method.

3.5.1 Antibiotic resistant profile of bacterial culture

Antibiotics which are showing quite relevant results are checked separately with Agar disc
diffusion method with different concentration with overnight grown pure cultures. Mupirocin,
Amikacin, Erythromycin, Clindamycin, Gentamycin, Vancomycin are checked from 2mcg to
512mcg.

3.5.2 Selection of optimal anaerobic condition for B. longum

To compare the growth level of anaerobic bacteria, the Oxygen Scavenger and commercial
anaerobic sachet from Himedia were used. The overnight grown pure culture was diluted in
0.85% of sterile saline and the dilution 107 and 108 was plated with Lactobacillus MRS agar
supplemented with 0.05% L-Cysteine. Same size three anaerobic chamber was labelled as A kept
as a control without keeping any anaerobic supporter and, B as Commercial sachet and C as
Oxygen Scavenger. Then the plates were kept inside anaerobic jar with appropriate supporter and
incubated in the respective chamber for 48 hrs.

3.5.3 Compatibility Studies

Compatibility test between combinations of two bacteria to make sure they are compatible each
other such as B. longum and L. delbrueckii were examined by Agar well diffusion method. The
pure culture was mixed with MRS media and pour plate was done. The wells are made then CFS
of the pure cultures were inoculates in their respective wells of other bacteria. Selected
antibiotics are also used to make a clear view in resistant and inhibition.

3.6 PRODUCT CHARACTERIZATION

The probiotic bifido enriched individual and combination curd were analysed based on different
parameters.

3.6.1 Enumeration of cultures

41
Lactobacillus MRS media was prepared and autoclaved at 121 ⁰C for 15 minutes. Dilutions of the
curd samples were done with sterile 0.85% saline. The necessary additions of antibiotics to the
media were carried out and later poured onto sterilized petri plates. The plates were kept in the
anaerobic chamber and supplemented with 0.05% L-Cysteine HCl for B. longum and incubated
at 37°C for 48hrs. The viability count was calculated in colony forming units per ml (cfu/ml).

3.6.2 Measurement of pH

pH meter (MFT Lab -3, CSIR-CFTRI, India) was used in this step. The culture was taken in a
clean and dry beaker/tube. The pH electrode was previously kept immersed in 4M KCl solution
(pH: 7). KCl is a calibration buffer and also prevents the drying of electrode. Before usage, the
pH electrode was washed with distilled water and dried with tissue. It is then immersed into the
sample. It was made sure that the glass bulb was fully covered with the sample. The pH reading
was noted down after the value on the meter became stable. After measurement, the pH electrode
was immersed in KCl solution after washing with distilled water.

3.6.3 Microbiological quality assessment

The probiotic curd was tested on two different media to check contamination. Two different
media that chosen to check the contaminants and their plate count agar and VRBA Agar. Plate
count agar shows the presence of aerobic contaminants while we are VRBA was used for
detecting the presence of detecting coliform organisms in dairy products. Probiotic individual
curd and combination curve serially diluted and tested on plate count agar and VRBA Agar plate.

3.6.4 Catalase test

This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies
hydrogen peroxide by breaking it down into water and oxygen gas. When a loop full inoculum is
added to hydrogen peroxide, the quick construction of oxygen bubbles indicates the presence of
the enzyme in the bacterial sample. A lack of or insufficient bubble creation indicates a lack of
catalase.

42
From curd sample containing only Bifidobacterium was added to 3% Hydrogen peroxide, no
bubble formation [Catalase negative]. The complex curd was added to Hydrogen peroxide,
there is bubble formation because of the presence of Lactobacillus culture [Catalase positive].

3.6.5Antimicrobial Activity Assay

Subculture of Pathogens

Four pathogens were used against the curd samples – E. coli, Bacilus cereus, Micrococcus leteus
and Staphylococcus aureus. All these cultures were subculture separately in BHI broth, by
inoculating 50µl into 5ml broth. These were incubated at 30°C in shaker incubator overnight.

Preparation of Curd Samples

The curd samples were aseptically transferred to 2ml Eppendorf’s. Those were centrifuged at
8000 rpm for 10 minutes at 4°C to pellet down the curd particles. The supernatant was
transferred to another fresh set of autoclaved Eppendorf’s. The supernatants of these samples
were used in the well diffusion method.

Well Diffusion Method (Georgieva et al., 2015)

Brain heart infusion agar (BHIA) was prepared and autoclaved at 121°C for 15 minutes. The
agar was inoculated with a pathogen culture (0.1%) and mixed properly. It was then poured into
Petri plates and allowed to solidify. Wells were made on the solidified agar with the use of
microtips. 50µl of each CFS were added to separate wells. The plates were then kept in
refrigerator for an hour to allow the samples to diffuse in agar. Later they were left to dry inside
laminar air flow and incubated at 37°C for 24hrs. The zones of inhibition were observed and
measured.

3.6.6 Rheological Analysis

An understanding of the rheological properties of yoghurt is important to texture, stability, and

process design. Between all milk fermented products, yoghurt is more well-known to others and

43
has more acceptability in the world. It is easily digested, has high nutritional value and is a rich
source of carbohydrates, proteins, fat, vitamins, calcium and phosphorus.

In this study, the apparent viscosity as well as the relation between shear stress and shear rate of
the market curd and probiotic curd was determined. 50ml of each sample was prepared. Analysis
was done using Oscillatory/Rotational Rheometer (CIFS, CSIR-CFTRI) (Fig: 6). The rheometer
was equipped with plate (PP75) wherein, a gap of 1mm was set for the probe. The shear rate was
between 0-100/second. The total time of the shear rate provided to the sample was 3 minutes. A
model called Herschel-Bulkley was applied to interpret the results from rheometer.

Fig 6: Oscillatory/Rotational Rheometer

3.6.7 Fourier Transform Infrared (FTIR) spectroscopy Analysis

Recently, spectroscopy has emerged as one of the major tools for biomedical applications and
has made significant progress in the field of clinical evaluation. Research has been carried out on
a number of natural tissues using spectroscopic techniques, including FTIR spectroscopy. These
vibrational spectroscopic techniques are relatively simple, reproducible, nondestructive to the
tissue, and only small amounts of material (micrograms to nanograms) with a minimum sample
preparation are required. Fourier Transform Infrared (FTIR) spectroscopy is a rapid biochemical
fingerprinting technique (Nicolaou, Xu, & Goodacre, 2010). It can be potentially applied to
deliver results with the same accuracy and sensitivity as the reference methods in short time
(Nicolaou & Goodacre, 2008). The FTIR analysis was done in CIFS, CSIR-CFTRI (Fig: 7)

44
 Fig 7: Fourier Transform Infrared (FTIR) spectroscopy

3.6.8 Proximate Analysis

The shelf stable probiotic curd undergoes proximate analysis. The estimation of components
present in the product was performed wherein quantitate evaluation of moisture, ash, fat, protein
and carbohydrate content was carried out as described below:

Estimation of Moisture content (AOAC 20th Edition 2016 953.07)

Around 5g of sample was taken in a pre-weighed and dried borosilicate petri plate and weighed
(W1). The petri plate was transferred to the Hot air oven set at 105°C and dried for 4-5 hours.
The petri plates were then transferred to a desiccator and cooled. Once cool, the sample was
weighed once again (W2).

Moisture content%= (W2-W1)/w1*100

45
Estimation of ash content (AOAC 20th Edition 2016 923.03)

The sample weighed to around 5g (W1) in a pre-weighed crucible. The crucible containing were
no the sample was charred on low Bunsen flame or a stove till the fumes longer produced. The
samples were transferred into a Muffle furnace at 550°C for 6 hours. The crucible was weighed
(W2) when the sample turned grey, if not the sample was moved back into the furnace for further
ashing.

Total ash = W2- W1

Estimation of Fat content (AOAC 20th Edition 2016 991.36)

Around 5g of dehydrated sample was weighed (W1), wrapped in a filter paper and taken in an
extraction thimble. The sample was then transferred to a previously weighed Soxhlet and
extraction was carried out using petroleum ether in the Soxhlet apparatus (Fig: 8). The extract
obtained was then dissolvents by vacuum flash evaporation where the temperature was
controlled.

The bottle was then incubated at 80°C-90°C until the solvent is completely evaporated and the
bottle is dried. The bottle is transferred to a desiccator and cooled. Once cool, the weight of the
bottle along with the dried content was taken.

Fat % = Weight of fat/ Weight of sample × 100

Fig 8: Soxhlet apparatus

46
Estimation of Protein content

Estimation of Protein content was carried out for the samples using Protein Analyser in CIFS
lab-5 CIFS-CFTRI [Model- Flash 2000 S of Thermo Fisher Scientific] (Fig: 9) 50mg dehydrated
probiotic curd samples was weighed in the foil. Around 50mg of Chromosorb WAW 80-100
Mesh from Thermo Fisher Scientific was added in the tin foil containing sample. It was wrapped
and kept it Protein Analyser for protein content.

Fig 9: Protein analyser

Estimation of Carbohydrate content

Carbohydrate content was carried out by following NIN, ICMR, 1996. Assuming that the crude
fiber content in a dairy product would be negligible, the carbohydrate content was estimated
using the following formula:

Carbohydrate % = [100 - (Moisture % + Ash % + Fat % + Protein %)]

3.6.9 STORAGE STUDIES OF PROBIOTIC CURD

47
Probiotic Bifido curd was prepared by using the protocol developed from Section 3.8.3. with
10% primary inoculum of B. longum and 5% of L. delbrueckii. These were then stored in 4°C
refrigeration. Enumeration of cultures from the curd samples were done in 0 th day, 4th day, 7th
day, 12th day, 15th day of storage in 4°C.

Enumeration of Cultures All media – MRS were prepared and autoclaved. The necessary
additions of antibiotics to the media were carried out and later poured into sterilised petri plates.
Dilutions of the curd samples were done with sterile 0.85% saline. The plates for B. longum were
kept in the anaerobic chamber with Anerobic Sachet and supplemented with 0.05% L-Cysteine
HCl and incubated at 37°C for 48hrs. The viability count was calculated in colony forming units
per ml (cfu/ml).

Measurement of pH

Measurement of pH was done throughout the storage studies 0 th day, 4th day, 7th day, 12th day,
15th day of storage in 4°C with pH meter.

Characteristic of probiotic in storage study

The whey separation settling of curd, taste, smell is analysed from day- 0 to day-12 on basis of
BIS specification.

Freeze Drying of Probiotic Bifido Curd

Steam sterilised 10% skimmed milk powder and 0.5% sucrose were used to make 1 litre of
probiotic curd. A colony count was obtained before freeze drying by plating the serially diluted
combinations in Lactobacillus MRS medium. The curd was then freeze dries in Lyomax Freeze
dryer in Food engineering Department, CSIR-CFTRI at -80⁰C for 24 hours.

Enumeration of Freeze-dried Curd

The freeze-dried powder 1mg is mixed 1ml 0.85% of saline and diluted for plating and plates are
incubated at 37⁰C in anaerobic chamber. The contamination checking also carried out in Plate

48
count agar for aerobic organisms and VRBA for Coliform organisms. Then the plates are
incubated at 37⁰C in anaerobic chamber.

3.6.10 SHORT CHAIN FATTY ACID SYNTHESIS

The HPLC analysis was performed by Shimadzu liquid chromatograph (Fig: 10) with a HB-5ms
capillary column (30 m × 0.25 mm × 0.25 µm film thickness). The freeze-dried powder 5mg is
mixed with 1ml of milliQ water and vortex completely. The Standards were prepared in the
dilutions with milliQ of Acetic acid, Propionic acid and Butyric acid. The mobile phase was used
as 0.01ml of sulphuric acid in 500ml of milliQ water. The flow rate of helium carrier gas was set
to 1 mL/min and the column temperature was set as 40⁰C.

The data were obtained in a full-scan mode from PDA Ch1 205nm 4nm. The peaks got from the
freeze-dried sample are compared with the peaks from standards to analyze what are the SCFA
are presents.

49
 Fig 10: HPLC grade Shimadzu liquid chromatograph

3.6.11 MTT assay of freeze-dried curd using cell line studies

Inflammaging (also known as inflamm-aging or inflamm-ageing) is a chronic, sterile low-grade

inflammation that develops with advanced age, in the absence of overt infection, and may
contribute to clinical manifestations of other age-related pathologies. High levels of interleukin
(IL)-6, IL-1, tumor necrosis factor-α, and C-reactive protein are associated in the aging by avting
as pro-inflammtory cytokines. In the Bifido freeze dried curd sample the presence of anti-
inflammatory markers such as IL-10 is being analyzed.

This experiment employed three steps for analyzing the sample for their pro inflammatory
properties-

Cell viability assay (MTT assay)-

o RAW 264.7 cells were treated with various concentrations of probiotic

formulation for 24 h.

o Then MTT (stock solution 5 mg/ml) was added to a final concentration of 0.5
mg/ml, and the cells were incubated for an additional 4 h at 37 ◦C and 5% CO2.

50
 o The medium was removed and the formazan precipitate was solubilized in 100 µl
DMSO, and the absorbance was measured at 570 nm on a multifunctional
microplate reader (Flex station 3, Molecular Devices, USA).

3.6.12 Sensory analysis

A probiotic product's sensory qualities are significant in addition to its health- promoting
properties. These special characteristics play a critical role in the product's commercialisation.
Colour, texture, taste, and general acceptability are all sensory qualities of a product. Due to
differences in fat content, carbohydrate content, pre- treatment, and other factors, the sensory
qualities of the product may alter as the substrate composition changes. One such study
examined goat milk and cow milk curds fermented by Lactobacillus and found that goat milk
curd was more acceptable than cow milk curd based on sensory qualities (Ehirim and Onyeneke,
2013). Some researchers have used additives such as citrus extracts and fruit juices to improve
the overall flavour of the product (Salvadar et al. 2004).

However, in this study, 11 participants (50ml) were offered bifidobacteria probiotic curd (A) and
Complex curd (B), all of which were with market curd (C) for comparison.

51
 RESULTS AND DISCUSSION

4. RESULTS AND DISCUSSION

4.1 Reconfirmation of morphology of probiotic cultures

The aim is to reconfirm the morphology of the probiotic B. longum and L. delbrueckii with the
help of Microscope. After preparation of slide it was observed in compound microscope at 40X
magnitude.

52
 (a) (b)
Fig11: Gram positive V and Y shaped cells of Bifidobacterium longum (a) and Gram-
positive long rods of Lactobacillus delbrueckii (b) is analysed.
Table 5. Microscopic observation of probiotic cultures

S.no Sample Microscopic observation Results

Colour Shape
1 a Purple V and Y rods Gram positive
2 b Purple Long rods Gram positive
These (Fig: 11) (a) and (b) are reconfirmed as Gram-positive bacteria because they have cell
walls that contain thick layers of peptidoglycan, a substance that forms the cell walls of many
bacteria. The peptidoglycan forms about 90% of the cell wall in gram-positive bacteria and they
retain crystal violet colour. This causes them to appear purple under a Gram stain (Table-5). The
shape of the bacteria V and Y shaped rods denotes as bifidobacterial and long rods denotes
Lactobacillus culture.

4.2 IMPROVEMENT OF BIFIDO PROBIOTIC CURD

The aim was to develop the Bifido probiotic curd by standardizing all of the fermentation
settings. Many Characteristics were investigated and observed in order to get a good texture,
good taste and smell without whey formation.

4.2.2 Optimisation of Inoculum for Curd Preparation

In the optimisation for B. longum, by checking 10%, 15%, 20%, 25%, 30%. In all percentage
curdling in nice and curdling hours are less in higher percentage. 10% is chosen because the

53
 curdling duration is 8 hours and there is no whey production, and the pH is 5.03, which is
favourable for bifidobacteria. 5% L. delbruckeii was chosen since, despite requiring 8.30
minutes, the settling was satisfactory and no whey formation occurred.

Table 6. Different percentage of individual bacteria curdling

Bacterial culture Percentage Curdling time Whey separation pH

(%) (hrs)
B. longum 10 8 +++ 5.03
15 7:30 ++ 5.13
20 7 ++ 5.08
25 5 + 5.20
30 4 + 5.32
L. delbrueckii 2 >8 +++ 4.65
5 8:30 +++ 4.79
8 8 +++ 4.93
10 8 +++ 5.08
12 7 +++ 5.12
‘+’ denotes positive or curdling, ‘-’ denotes negative or no curdling, ‘++’ donotes better
curdling, + denotes curdling but not good, ‘+++’ denotes best curdling.

For the final product (Fig: 12) These were combined with the optimized percentage and made
with good settling and without whey formation.

In fig: 12(a) Bifido probiotic curd and Fig 11(b) Improved complex curd which is thick in
consistency more than bifido curd.

54
 (a) (b)

Fig 12: Individual Bifido probiotic curd(a) and Improved Bifido enriched probiotic curd
with L. delbrueckii (b)

Table 7. Antibiotic sensitivity and resistance for B. longum and L.

delbrueckii

Antibiotic Bifidobacterium longum Lactobacillus delbrueckii

Ciprofloxacin Resistant Intermediate

Aztreonam Sensitive Sensitive

Vancomycin Sensitive Resistant

Amikacin Sensitive Sensitive

Cefotaxime Sensitive Intermediate

Amoxycillin Sensitive Resistant

Chloramphenico Sensitive Sensitive

l

55
 Cefalexin Sensitive Resistant

Ampicilin Intermediate Resistant

Clindamycin Sensitive Intermediate

Erythromycin Sensitive Intermediate

Gentamycin Sensitive Intermediate

Tetracycline Sensitive Sensitive

Oxacillin Sensitive Resistant

Cefepime Sensitive Resistant

4.2.3 SELECTIVE ENUMERATION OF PROBIOTIC CULTURE

The aim of selective enumeration is to get the accurate data of live cells of B. longum and L.
delbrueckii individually. With pure culture Hi media octa disc was evaluated with MRS media.
Octa disc is very useful technique since it has so many antibiotics in one comb. These were
checked on two bacterial cultures to check the Antibiotic profile. Kirby-Bauer Disk Suceptibility
test method was used to interpret the results. Ciprofloxacin is only resistant to B. longum. Most
of the antibiotics are Sensitive to B. longum and Intermediate or Resistant to L. delbrueckii
(Table 7). Erythromycin, Gentamycin, Clindamycin and Vancomycin are showing positive
results. So, they are taken up for further experiments to find exact concentration.

4.2.4 Antibiotic resistant profile for Bifidobacterium longum

Antibiotics selected from section 4.2.3 are examined with different concentration to enumerate
selectively the bacteria with antibiotic and proper control was kept. From all the concentration
its shows that B. longum found as resistant for Mupirocin (Table 8) and Sensitive for L.
delbrueckii (Table 9). Other antibiotics like Amikacin, Clindamycin, Gentamycin and
Vancomycin are also resistant to B. longum but these bacteria are sensitive to L. delbrueckii. So,
these antibiotics will not work as marker for specifically B. longum. In a conclusion Mupirocin is
selected as a marker for B. longum from this antibiotic resistant profile.

Table 8. Antibiotic resistant profile of Bifidobacterium longum with different concentration

56
 Zone of inhibition (mm)
Antibiotic Mupirocin Amikacin Erythromycin Clindamycin Gentamycin Vancomycin
Concentra
tion(чg/ml)
2 - - 19 - - -

4 - - 21 - - -

8 - - 22 - - -

16 - - 24 - - -

32 - - 27 - - -

64 - - 27 - - -

128 - - 30 14 6 -

256 - - 32 29 12 -

512 - - 37 32 17 -

Zone of inhibition(mm)
Antibiotic Mupirocin Amikacin Erythromycin Clindamycin Gentamycin Vancomycin

2mcg - - 14 - - -

4mcg - - 16 - - -

8mcg 12 - 18 - - -

16mcg 15 - 19 - - -

57
 32mcg 18 - 19 - - -

64mcg 19 - 20 - 6 -

128mcg 22 - 22 - 11 -

256mcg 26 - 24 - 11 -

512mcg 29 - 27 - 13 -

4.2.5 Antibiotic resistant profile for Lactobacillus delbrueckii

L. delbrueckii also examined with different concentration to enumerate selectively the
bacteria with antibiotic. From all the concentration its shows that L. delbruckeii found as
resistant for Clindamycin (Table 9) and Sensitive for B. longum in higher concentration
(Table 8). Other antibiotics like Amikacin, Clindamycin, Gentamycin and Vancomycin
are resistant to B. longum and are sensitive to L. delbrueckii. So, these antibiotics will not
work as marker for specifically L. delbruckeii. In a conclusion Clindamycin is selected as
a marker for L. delbruckeii from this antibiotic resistant profile. Table 9: Antibiotic
sensitivity for Lactobacillus delbrueckii with different concentration

4.3 SELECTION OF OPTIMAL ANAEROBIC CONDITION FOR B. longum

Oxygen scavengers are a formulation of natural materials that react with oxygen and
eliminate it. Oxygen scavengers harness the dissolved oxygen in an inert chemical
reaction to lock up the oxygen and make it unable to damage other products within the
space. The mechanism in commercial sachet is in the presence of water, chemicals present
inside the sachet i.e. sodium bicarbonate (NaHCO3) and sodium borohydride (NaBH4) react
chemically producing hydrogen and carbon dioxide gas.

Oxygen Scavengers lock up the oxygen meanwhile Commercial sachet produces the Carbon
dioxide. In anaerobic bacteria case, (Fig 13) and (Table 9) shows that Control (Fig 14) oxygen

58
scavengers giving negative results by no growth in the plates (Fig 15), meanwhile commercial
sachets (Fig 16) are giving positive result by good number of colonies are present in the plate.

A B C
1600 1404
1400
No.of.colonies

1200
1000 812
800
600
400
200 0 0 0 0
0 Diltuions
10^7 10^8

Fig 13: Graphical representation of Colonies in A(control), B (Oxygen Scavengers),

C (Commercial Sachet)

Table 10: Colonies in A(control), B (Oxygen Scavengers), C (Commercial Sachet)

Dilution A[Control] B [Oxygen scavengers] C [Commercial Sachet]

7
10 - - 140*109
8
10 - - 812*109

Fig 14: Control in anaerobic chamber and control plates

59
 Fig 15: Oxygen Scavenger in anaerobic Chamber and plate

Fig 16: Commercial Sachet in anaerobic Chamber and plate

4.3.1 Compatibility Studies

The Compatibility of two strains is crucial to make a complex curd with two probiotic culture.
The aim is to check the compatibility between B. longum and L. delbrueckii by the compile
inoculation in one petriplate. With combination of compatibility the antibiotics which are
finalized for the two probiotic cultures are also plated to get a clear view. If the bacteria are
incompatible then one bacteria will kill the other one which leads to the zone of inhibition. If not,
then there will be no zone of inhibition which confirms the compatibility of these cultures .

60
 (a) (b)

Fig: 17 Compatibility between B. longum(a) and L. delbrueckii (b)

The compatibility between two probiotic cultures B. longum and L. delbrueckii are verified by
lack of inhibition zone in at the intersection of two colonies marked as BL and LBD in each plate
(Fig:17) resulting in the growth of two cultures. The antibiotics which are finalised Mupirocin
(16mcg) and Clindamycin (256mcg) also added in the two plates and zone of inhibition found in
Clindamycin marked as C (a) sensitive to B. longum and Mupirocin in (b) which is sensitive to
the L. delbrueckii.

From this present study its verified that both cultures are compatible each other and the
antibiotics which are resistant and sensitive also observed respective to each culture.

4.4 PRODUCT CHARACTERISATION

4.4.1 Viability of monocultures

By plating microorganisms on Lactobacillus MRS Agar plates with L-cysteine hydrochloride
and antibiotics, the viability of the microorganisms was assessed, and it was shown that
probiotics cannot provide such good advantages unless they are supplied in appropriate
proportions. The aim is to observe the enough bacterial counts in within 24 hrs to obtain the
probiotic effectiveness in our body.

It is preferable for bifidobacteria to maintain its viability in order to influence the consumer pH,
temperature, oxygen need, the presence of other species, and other host conditions are all factors

61
that affect the laws of viability Lactobacillus MRS Agar plates with inoculum derived from curd
reduced by both monoculture and complex culture by using commercially available Nandini tone
milk.

Viability of a Probiotic curd

3000
2472
2500

2000
CFU/mL

1500

1000 810
609
500

0
Bifidobacterium Lactobacillus Complex curd
longum delbrueckii

Fig 18: Graphical representation of comparison of viability of Individual Bifidobacterium

longum, Lactobacillus delbrueckii and Complex curd

The complex curd shows more colonies which is comparatively lower with L. delbrueckii and B.
longum (Fig: 18). B. longum has so less colonies when compared to other two curd samples.
Even though 609 CFU/mL is a good number of colonies for probiotic anaerobic bacteria.

4.4.2 Microbial quality assessment

Microbial quality assessment should be taken into consideration in evaluating the impact of
handling, processing and treatment procedures on maintaining the quality. The contamination
checking was done to with two different media PCA for aerobic organisms and VRBA for
coliform organisms. In PCA media bifidobacteria is expected to be no growth as it is anaerobic
organism and expected in L. delbrueckii as it is aerobic organism. For contamination checking
lower dilution was used.

Table 11: Microbial quality assessment colonies

Culture Plate count Agar Violet Red Bile Agar

62
 B. longum - -
L. delbrueckii + -
Complex curd + -
‘+’ Denotes Growth ‘-’ Denotes no growth

900
800 770
700 647
600
500
400
300
200
100
0 0 0
0
B. longum L. delbrueckii Complex Curd

PCA VRBA

Fig:19 Microbial quality assessment colonies

No colonies are observed in B. longum (Table: 11) both PCA and VRBA medium which
indicated the purity and no other organisms involved in the product. In L. delbrueckii there is
growth around 647 colonies (Fig: 19) in PCA and no colonies were observed in VRBA. In
complex curd growth was observed in PCA but not in VRBA. Around 770 colonies are observed
in complex curd this is due to L. delbrueckii. Since L. delbrueckii is a facultative aerobic
organism there is growth in PCA of individual curd and Complex curd. The coliform microbes
are absent which are proved by no colonies in VRBA.

4.4.4 Antimicrobial activity of probiotic curd

63
The antibacterial activity of the curd samples was tested against the pathogens – E. coli, Bacilus
cereus, Micrococcus leteus and Staphylococcus aureus (Table 12). Among the 3 samples – B.
longum, L.delbrueckii and complex curd sample, B. longum showed antimicrobial activity
against E.coli and Staphylococcus aureus. L. delbrueckii did not show activity against any of the
above pathogens.

Table 12. Antimicrobial activity of individual and probiotic curd

Zone of inhibition(mm)
Organisms E. coli Bacillus cereus Staphylococcus Micrococcus
aureus leteus
B. longum 13 - 12 -
L. delbrueckii - - - -
complex curd 14 - 13 -

Presence of antibacterial activity adds value to the probiotic microbes and the corresponding
product. The zone of inhibition is showing (Fig: 20 and Table: 12) that they have antimicrobial
activity against E. coli, Bacilus cereus, Micrococcus leteus and Staphylococcus aureus pathogen.

B. longum L. delbruckeii
B. longum L. delbruckeii

Complex curd
Complex curd

64
Fig 20: B. longum and Complex curd showing inhibition zone against E. coli and
Staphylococcus aureus

4.4.5 RHEOLOGICAL ANALYSIS

Rheological studies are generally carried out to study the flow properties and consistency of
various substances. The viscosity of products such as yoghurt and curd largely depend on the
total solid and fat content, enzymes and certain stabilizer agents such as k-carageenan which
enhance the structure of the products (Salvadar et al., 2004; Biliaderis et al., 2002).
ETA

20

Pa·s

16
FC BL 29.05.2023 1
14 PP75-SN16019; [d=1 mm]

 Viscosity
12
FC CS 29.05.2023 1

PP75-SN16019; [d=1 mm]

10
  Viscosity

8 FC MC 29.05.2023 1

PP75-SN16019; [d=1 mm]

6
 Viscosity

0
0 10 20 30 40 50 60 70 80 90 1/s 100
.
Shear Rate 
Fig 21: Relation between viscosity (ɳ) and shear rate (per sec) (γ) of Bifido probiotic curd
Anton Paar GmbH

(in blue), Complex curd (in red) and Market curd (in green).

Bifido probiotic curd Consistency k: 353.01 Pa·s^0.01and Flow index n: 0.01(shear-thinning)

Complex probiotic curd Flow index k: 353.01 Pa·s^0.01 Flow index n: 1.6889 (shear-thickening)
Market curd Consistency k: 211.85 Pa·s^0.01 Flow index n: 0.01(shear-thinning)

The shear thickening of complex curd is due to the low consistency. Comparing to complex curd
the individual and Market curd shows shear thinning indicates good consistency. There is
decrease in the viscosity (Fig: 21) of all three samples with prolongs shear stress which indicates
their thixotropy and confirms their Non- Newtonian nature (Shobha et al, 2013)

4.4.6 Fourier Transform Infrared (FTIR) spectroscopy Analysis

65
 FTIR is widely used in many industries and is used for the analysis of both organic and inorganic
compounds. It can confirm the composition of both solids, liquids, and gases. FTIR is mainly
used for: The identification of unknown compounds.

Fig 22: Individual Bifido curd Spectrum

Table 13: Vibrational modes of Bifido probiotic curd obtained from the FTIR spectra in
the fingerprint region from 3500 to 500cm−1 .(Fig: 22)

S.no Wavelength (cm) Vibrational mode

1 3332 Stretching vibration of bonded and non-bonded –O–H groups

2 2125 Alkanes C-H stretching

3 1636 Carbohydrates

4 1418 Aromatic compounds of C-C stretching

66
5 1155 C-N Stretching vibration of the protein

6 1119 C-4-oh typical for glucose residue of disaccharides

7 1075 Couple stretching and bending (Carbohydrates)

8 1041 Couple stretching and bending (Carbohydrates)

9 927 Aromatic compounds

10 590 C-Br stretching and bending (Carbohydrates)

Fig 23: Complex curd Spectrum

Table 14: Vibrational modes of Complex probiotic curd obtained from the FTIR spectra in
the fingerprint region from 3500 to 500cm−1 .(Fig: 23)

S.no Wavelength (cm) Vibrational mode

1 3332 O-H stretching

2 2110 Alkanes C-H stretching

3 1636 Carbohydrates

4 1156 C-N Stretching vibration of the protein

5 1120 C-4-oh typical for glucose residue of disaccharides

67
 6 1076 Couple stretching and bending (Carbohydrates)

7 1042 Couple stretching and bending (Carbohydrates)

8 927 Aromatic compounds

9 591 C-Br stretching bending (Carbohydrates)

Fig 24: Market curd Spectrum

Table 15: Vibrational modes of Market curd obtained from the FTIR spectra in the
fingerprint region from 3500 to 500cm−1 .(Fig: 24)

S.no Wavelength (cm) Vibrational mode

1 3332 O-H stretching

2 2120 Alkanes C-H stretching

3 1636 Carbohydrates

4 1417 Aromatic compounds of C-C stretching

5 1155 C-N Stretching vibration of the protein

6 1119 C-4-oh typical for glucose residue of disaccharides

68
 7 1075 Couple stretching and bending (Carbohydrates)

8 1042 Couple stretching and bending (Carbohydrates)

9 927 Aromatic compounds

10 584 C-Br stretching of alkyl halides

It was seen that the spectra at 3332 cm−1 was dominated by O-H group of amides (Fig 22,23,24).

Alkanes C-H stretching have the characteristic frequency of 2110-2125. Carbohydrates have
characteristic frequency at 1636 cm−1. Frequency 1155 cm−1was dominated C-N Stretching
vibration of the protein. C-4-oh typical for glucose residue of disaccharides have characteristic
peak at 1119-1120cm−1 . The peak at 1076 cm was attributed to vibration of the Carbohydrates by
C-N stretching. The spectra 927 was attributes to aromatic compounds. The alkyl halides show
the stretch of C-Br at the frequency of 580-590 cm−1(stuart,2004) (jaya,2009).

4.4.7 PROXIMATE ANALYSIS

MOISTURE ANALYSIS

According to the Bureau of Indian Standards (1978), dahi should have a pleasant flavour and a
clean acid taste, be devoid of any unwanted flavours, have a firm, solid body and texture, and be
uniform with minimal whey separation. The proximate composition of the finished product was
examined in order to make comparisons with market curd and BIS specifications for dahi. Table
4.6 shows the results.

Table 16: BIS Specification of Curd and bifidobacteria probiotic curd comparison

Component BIS Standard (%) Bifidobacterial Market curd (%)

Probiotic curd (%)

69
 Moisture analysis 85-95 90.35 87.1

Ash content 0.75-0.79 0.71 0.68

Fat content 3-8 3.0 3.1

Protein content 3-3.4 4.7 3.1

Total carbohydrate 2-6 1.24 4.3

The proximate composition for bifidobacterial probiotic curd, Bifidobacterial probiotic curd with
lactics and Market curd were compared with that of BIS standards as shown in Table 16.

Moisture content of any product is directly related to its shelf life. In a dairy product like curd,
preventing post process contamination by spoilage microorganisms and retarding the growth of
surviving organisms remain a challenge. An intrinsic factor like moisture content has to be
within the range of BIS standards as much water in curd makes it less viscous thereby affecting
texture and mouth feel. The moisture content of Bifidobacterial probiotic curd developed in the
laboratory was 90.35%. Whereas, moisture percentage in market curd was found to be 87.1%
(BIS specification).

The ash percentage in Bifidobacterial probiotic curd developed in the laboratory was 0.71% and
which is a measure of the mineral content such as calcium, sodium and even vitamins (Panda SH
et al, 2006). The result of the ash value agreed with the specifications given by Bureau of Indian
Standards which says that the ash percentage in curd should be within the range of 0.75-0.79.

Percentage of fat is an important nutritional fact for milk and milk derivative products. Fat is
known to provide palatability to the diet as well as increase the consistency of curd along with
providing a good source of energy. According to Bureau of Indian Standards (BIS), the fat
percent in a curd product should be within the range of 3%. The fat content in Bifidobacterial
Probiotic curd developed in the laboratory was 2.52% which was not significantly different from
the standards.

70
Most of the proteins in yogurt (80%) are caseins. Alpha-casein is the most abundant. Casein
increases your absorption of minerals like calcium and phosphorus and promotes lower blood
pressure. Thus, amount of protein present in curd a vital role in overall nutritional value of the
curd. The standard for the protein content should be in the range of 3-3.4%. The protein content
in Bifidobacterial Probiotic curd developed in the laboratory was 4.7% which is significantly
higher as compared to the protein percent present in market curd which was 3.1%.

Lactose present in whey maybe the carbohydrate source in curd. In accordance with the Bureau
of Indian Standards, there should be negligible whey separation in curd, therefore it is
consequential to have low carbohydrate curd. The low carbohydrate value is attributed to the
process of fermentation which converts carbohydrate basically lactose to lactic acid. This makes
yogurt an ideal food for lactose intolerance individuals (Ehirim and Onyeneke, 2013). The
carbohydrate content in Bifidobacterial Probiotic curd developed in the laboratory was 1.24%
Market curd has a carbohydrate content of 4.3% which is not differ more when compare with the
BIS standards.

4.5 STORAGE STUDIES OF PROBIOTIC BIFIDO CURD

For Food products the shelf life is necessary especially for probiotic curd it is negligible. Storage
studies of the Probiotic Bifido curd were prepared and kept in 4 ⁰C done in Day 0 ,4,7,12,15.
Also, pH, Texture, Smell, taste is evaluated till 15th day.

4.5.1 Viability of the complex curd

The probiotic product's bacterial live count is crucial. This viability will let us know how many
days we can use the probiotic product and how long it will last on the shelf. As it is a complex

curd, both bacteria in the curd is checked by the selective enumeration till Day 15.

Total Viable count (Billion)

71
 Cultures samples Day 0 Day 4 Day 7 Day 12 Day 15

Complex curd 24 20 11 9 3
[BL+LBD]

Complex curd 5 5 3 0.3 0.1

with Mupirocin

Complex curd 5 2 1 0.6 5

with Clindamycin

Table 17: Storage studies Viability of the complex curd

Bacterial count gets by plating in Lactobacillus MRS agar supplemented with L-cysteine. The
necessary additions of antibiotics to the media were carried out. The Complex probiotic curd
shows good number of counts till Day 15 (Table 17) (Fig: 25) means the bacteria is live and the
product is good till 15 days (Fig: 26). Compare to complex probiotic curd with antibiotic counts
are less may be due to the antibiotics even though the count is considerably good.

72
 Fig 25: Day 0-12 Complex curd viability plates

SHELF LIFE OF COMBINATION CURD

Complex curd with Clindamycin

Complex curd with Mupirocin

Comlex curd

0 5 14
Day Day1012 Day 715 Day 4 20
Day 0 25 30

Fig 26: Shelf life of the Complex curd as BL+LBD with Mupirocin as BL and with
clindamycin as LBD

4.5.2 Measurement of pH of Complex curd

pH is a measure of hydrogen ion concentration in an aqueous solution. pH decreases as hydrogen

ion concentration increases, So the probiotic curd become little acidic at the end of the storage
studies and these acidic conditions can adversely affect aquatic biota. pH of complex curd is
good in Day 0 and it is getting acidifying condition as days increasing. From 5.44 to 4.50 (Fig:
27) it is reduced.

73
 6 5.44
5.03
5 4.65 4.59 4.5
4
3
pH

2
1
0
Day-0 Day-4 Day-7 Day-12 Day-15

Fig 27: pH of the Complex curd from Day-0 to Day -15

4.5.3 Product characterization

The aim of the present study is to check all characteristics of the the complex probiotic curd was
analysed under Texture, Taste, smell, colour in all characteristics. The complex curd is thick curd
during Day 7 little whey formation is founded while Day 12 and 15 (Fig: 28) it is increasing in
small quantity. The smell and taste are good throughout the storage studies and the colour still
milky white from start to end.

74
 Product Characterization
10
9
8
7
Rated from 10

6
5
4
3
2
1
0
Whey separation Taste smell Colour

Day 0 Day 4 Day 7 Day 12 Day 15

Fig 28: Data about all characteristic of probiotic complex curd till Day
15

4.6 Freeze Drying of Probiotic Bifido Curd

The presence of liquid water during the freeze-drying of food products may result in many
changes in the composition, morphology, and physical properties of foods (e.g., shrinkage). It
may also reduce the period of ensuring high quality during storage. After 24hours the Freeze-
dried curd become powder, from 1 litre of curd without any moisture around 181.65g (Fig: 29) of
powder is got from freeze drying.

Fig 29: Freeze-dried Curd powder

75
4.6.1 Enumeration of freeze-dried curd

Survival analysis of before and after freeze dried bacterial cultures

The survival of bacterial cells is depending upon the cryoprotectans and their combination.
Nowadays, food products are incorporated with these freeze-dried powders for extended viability
of foods and beverages.

218

250

200
Viability

150

100 5.3

50

Normal curd Freeze dried curd

Fig 30: Before and after freeze dried Bifido probiotic curd

Usually, the viability is significantly reduced after freeze drying (Fig:30) due to stress on the
bacterial cells during the process. The viability of the pre-freeze dried (wet) culture and the
freeze-dried culture (0th day) was checked by plating on MRS agar and incubating it at 37 ⁰C
anaerobically for 2 days. There was 21.8*107 log cfu/ml (Fig: 31(a)) reduction in the viability of
freeze-dried B. longum as 0.5* 107 log cfu/mL (Fig: 32(b)).

76
 (a) (b)

Fig 31: Survival analysis of Bifido curd(a) and Freeze-dried Powder(b)

4.6.2 SCFA OF FREEZE-DRIED POWDER

The SCFA chromatograms as illustrates in fig.32,33,34 revealed that the freeze dries curd
powder has butyric acid and acetic acid. There is peak in Butyric acid standard at 19.06 (Fig: 32)
and peak in Acetic acid standard at 6.376 (Fig: 33). The peaks of 6.079 and 19.069 confirms that
the presence of those acids in the powder (Fig: 34)

Gill et al., reported acetic acid, butyric acid as the most abundantly found SCFAs within the
human colon due to the fermentation of carbohydrates and protein by the microbiota. SCFAs
confer various health benefits to humans, as revealed by various animal model studies.
Therefore, production of SCFAs by probiotic candidates is a highly coveted bioactivity. Kim et
al., reported that acetic acid, butyric acid secreted by the gut microflora lowers the host’s blood
pressure. Canfora et al., reported that butyric acid reduces body fat accumulation and influences
insulin sensitivity. Roy et al., stated that acetic acid also helps in providing energy to the liver.
All the selected isolates produced acetic acid in common, whereas and were able to produce the
three major SCFAs of the human colon, making them the best probiotic candidates in terms of
SCFAs.

77
Fig 32: Chromatogram for the freeze-dried powder

Fig 33: Chromatogram for the Butyric acid peak

78
 Fig 34: Chromatogram for the Acetic acid peak

4.6.3 MTT assay

The MTT assay involves the conversion of the water-soluble yellow dye MTT (3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). It is a tetrazolium salt that is
commonly used to detect reductive metabolism in cells for viability, proliferation and
cytotoxicity assays. It is based on the ability of nicotinamide adenine dinucleotide phosphate
(NADPH)-dependent cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT to its
insoluble formazan, which has a purple color. Among viability assays that depend on the
conversion of substrate to chromogenic product by live cells, the MTT assay is still among one
of the most versatile and popular assays.

79
 B.longum
180

160

140

120
cell viability %

100

80

60

40

20

0
0 100 200 300 400 500 600 700 800 900 1000 1100
Concentration μg/ml

Fig 35: Cell viability percentage of MTT assay in B. longum curd powder

The cell viability percentage is increasing gradually when the concentration is more than 100
μg/ml (Fig: 35). Based on the cytokine it is evident that the cells are able to synthesize anti-
inflammatory cytokines. When increasing the concentration, the cell viability is also increasing
gradually. Requirements for correct interpretation of results are on the one hand linearity of cell
number and MTT reading and on the other hand, a sufficient number of time points over several
cell generations in order to predict growth behavior accurately.

80
4.6.4 SENSORY ANALYSIS

Participants were given Performa’s for sensory evaluation for probiotic curd in exchange for
their helpful comments in colour, texture, taste, odour and overall acceptability were assessed.

The participants were asked to give the valuable feedback by making comparison with market
curd. A method called ‘9-point Hedonic scale’ was used for scoring the parameters. Flavour
wheel was used to depict average result.

Flavour wheel for sensory Analysis

A B C
Colour
10

5
Overall acceptability Texture

odour Taste

Fig 36: Sensory analysis of Individual bifido probiotic curd(A), Complex curd(B) and
Market curd(C)

The overall acceptability of the complex probiotic product is less when compared to Bifido
probiotic curd and Market curd (Fig: 36). The colour, Odour, Texture, Taste more than 6 in an
average of ‘9-point Hedonic scale’

81
SUMMARY AND CONCLUSION

82
5. SUMMARY AND CONCLUSION

A healthy Gut microflora of 3% occupied by Bifidobacterium in human gut. The Strain

Bifidobacterium longum which is isolated from the infant intestines. Their significances as

immunomodulation, Tumour suppressor, and healthy promoters in gut is employed to humans

and other organisms also. The suitable anaerobic condition for Bifidobacteria is a Commercial

sachet because it is releasing carbon dioxide but oxygen scavengers locks up oxygen which is

not suitable for an anaerobic bacterium.

Selective enumeration is done with the help of Antibiotics namely Mupirocin and clindamycin

which are selected from Octa disc and Agar disc diffusion method. Improvement of the probiotic

curd is standardized as per the optimization of different percentage of the individual bacteria.

Curdling of complex curd nearly takes 7 hours. Antimicrobial assay against pathogens were

tested and zone of inhibition were noted. Several product characteristics such as Enumeration

with Lactobacillus MRS agar and Microbiological quality assessment with PCA and VRBA

media.

Storage studies is carried out for 2 weeks by storing in 4 ⁰C and pH, texture, colour, Taste, Smell

are also analysed through storage studies. Analysis of FTIR spectroscopy, Rheometer for

Viscosity and Quality of the product as proximate analysis were indicating the quality of the

product. Freeze drying was carried out and comparison of viability of wet curd and freeze-dried

curd are also analysed. SCFA of acetic and Butyric acids were determined by using HPLC

method. Finally, Sensory analysis are performed with participants for taste, colour, smell and

overall acceptability.

83
Further, the complex curd will be useful for consumption as a healthy diet with the clinical

studies. Also, the if the viability is increase in future research it will be preferred as a good

probiotic food.

6. REFERENCE:

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Bifidobacterium spp. in the resolution of inflammation in arthritic rats. Applied microbiology

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Arihara, K. (2014). Probiotics. Handbook of Fermented Meat and Poultry, 155-160.

Avis, K. E. (Ed.). (2018). Pharmaceutical dosage forms: parenteral medications. Routledge.

Basak, S., & Gokhale, J. (2022). Immunity boosting nutraceuticals: Current trends and

challenges. Journal of Food Biochemistry, 46(3), e13902.

Bifidobacterium longum BB536 administration on influenza infection, influenza Biotechnology,

and Biochemistry, 74(5), 939-945.

Caballero, B., Finglas, P., & Toldrá, F. (2015). Encyclopedia of food and health. Academic

Press.

Dack, G. M. (1955). symposium on methodology for salmonella in food. Bacteriological

Reviews, 19(4), 275-276.

Dinkçi, N., Akdeniz, V., & Akalin, A. S. (2019). Survival of probiotics in functional foods

during shelf life. In Food quality and shelf life (pp. 201-233). Academic Press.

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Ferreira, C. M., Vieira, A. T., Vinolo, M. A. R., Oliveira, F. A., Curi, R., & Martins, F. D. S.

(2014). The central role of the gut microbiota in chronic inflammatory diseases. Journal of

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