A Report of Minor Research Project

EFFICACY OF MICROENCAPSULATION ON

SYNBIOTIC YOGURT PRODUCTION

Ms. SMITHA MATHEWS

(1939-MRP/14-15/KLMG034/UGC-SWRO)

Assistant Professor in Microbiology

Department of Zoology

Assumption Autonomous College

Changanacherry, Kottayam, Kerala, 686101

Submitted to

University Grants Commission

New Delhi

May 2017

ACKNOWLEDGEMENTS

The present study was supported by the University grants commission of India

under the grant No:1939-MRP/14-15/KLMG034/UGC-SWRO.

I would like to express my sincere gratitude and profound appreciation to

University Grants Commission (UGC), New Delhi for providing financial support for this

research.

I would like to express my sincere thanks to Dr Sr Amala SH, Principal,

Assumption College, Changanacherry for her inspiration, constant support and for

providing necessary lab facilities for carrying out this work.

I wish to thank Ms. Tessy Jose, HOD, Department of Zoology, Assumption College,

Changanacherry and my colleagues for their constant encouragement and support

throughout the period of my work.

I express my gratitude to Dr. Jickcy Isac, Department of Mathematics, Assumption

College, Changanacherry for carrying out my statistical work.

I deeply express my gratitude to my parents and family members for their constant

encouragement, valuable support and understanding.

Above all I thank God Almighty for his grace and blessings....

Smitha Mathews

CONTENTS

Abstract 1

1. Introduction 2

1.1 Probiotics and Prebiotics 2

1.2 Synbiotics 4

1.3 Viability of Probiotic organisms 4

1.4 Microencapsulation of Probiotics 5

1.5 Scope of present investigation 8

1.6 Objectives of the present investigation 9

2. Review of Literature 10

2.1 Yogurt 10

2.2 Definition of probiotics 11

2.3. Characteristics of lactobacillus 12

2.3.1. Lactobacillus acidophilus 12

2.3.2. Lactobacillus casei 13

2.4. Regulations and safety of probiotics in manufacture 13

2.5. Prebiotics 14

2.5.1. Inulin 15

2.5.2. Lactulose 16

2.6. Factors affecting the growth and survival of Lactobacillus 17

2.7. Effect of prebiotics on probiotic growth and survival. 18

2.8. Microencapsulation Technique 19

3. Materials and Methods 23

3.1 Materials 23

3.2 Methodology 24

3.2.1 Preparation of cell suspension 24

3.2.2 Production of Probiotic yogurt 24

3.2. a. Determination of pH 25

3.2. b. Determination of titratable acidity 25

3.2. c. Enumeration of L. acidophilus and L.casei in yogurt 25

3.2. d. Determination of Syneresis 25

3.3. Production of Synbiotic yogurt 26

3.3.a. Physicochemical and Microbial analysis of Synbiotic yogurt 26

3.4 Microencapsulation of Probiotic Cultures 26

3.4.1 Preparation of alginate microcapsules 27

3.4.2 Preparation of simulated gastric and intestinal juices

and inoculation of cells 28

3.4.3 In vitro release studies (GIT) 29

3.4.4 Release of entrapped bacteria 29

3.5. Co-encapsulated synbiotic yogurt Production 29

4. Results and Discussion 31

4.1 Preparation of cell suspension 31

4.2 Production of Probiotic yogurt 31

4.2.a. pH of Probiotic yogurt 31

4.2.b. Acidity (% Lactic acid) Probiotic yogurt 32

4.2.c. Bacterial counts of Probiotic yogurt 33

4.2.d. Syneresis of Probiotic yogurt 33

4.3 Synbiotic yogurt Production 34

4.3.a. pH of Synbiotic yogurt during storage 34

4.3.b. Acidity of Synbiotic yogurt during storage 35

4.3.c. Viability of Synbiotic yogurt during storage 36

4.3.d. syneresis of Synbiotic yogurt during storage 39

4.4. Microencapsulation of Probiotic yogurt 40

4.4.a. In vitro release studies of L.acidophilus and L. casei

from the microcapsules 40

4.4.b. Survival of free and microencapsulated probiotics

in simulated gastric juice 41

4.4.c Survival of free and microencapsulated probiotics

in simulated intestinal juice 43

4.5. Co-encapsulated synbiotic yogurt 44

4.5.a. pH changes during storage 45

4.5.b. Producing acidity during storage 46

4.5.c. Syneresis on storage 48

4.5.d. Viability of Lactobacillus strains grown in synbiotic yogurt 50

4.5.d.I. Survival of free and microencapsulated

synbiotic L. acidophilus in Yogurt during storage 50

4.5.d.II. Survival of free and microencapsulated

synbiotic L. casei in Yogurt during storage 51

5. Conclusion 56

6. Bibliography 58

1

ABSTRACT

Due to the perceived health benefits of probiotics, there has been an increased use

in different health based products. The viability of probiotic cells is of paramount

importance because to have their beneficial effects on the health, they must stay alive until

they reach their site of action. There are some problems pertaining to the survivability of

probiotic bacteria in dairy foods. This has encouraged developing different innovative

methods to improve the probiotic cells viability in the product incorporated. The objective

of the present study is to prepare probiotic, synbiotic and microencapsulated yogurt.

Physicochemical and microbiological properties of probiotic and synbiotic yogurt were

evaluated in the first day and thereafter every 7 days interval for 28 days of storage at 40C.

During cold storage, pH, and probiotic bacterial count decreased, while acidity and

syneresis were increased in Probiotic yogurt prepared with both organisms. Neither Inulin

nor Lactulose had significant effect on physicochemical changes (except pH) and probiotic

bacterial survival of L.acidophilus. There was a significant decrease (p<0.05) in pH of

synbiotic yogurt with inulin and lactulose. For L.casei, both prebiotics had significantly

reduced pH and increased acidity, and found to have some effect on viability of probiotic

bacteria, but inulin produced least change in syneresis.

Microencapsulation of probiotics is one of the approaches which is currently

receiving considerable attention. Here an attempt was made to determine the efficacy of

microcapsules prepared by alginate and gelatin coated alginate along with prebiotic

(lactulose or inulin) to give better protection in gastrointestinal tract and during

refrigerated storage of yogurt for 28 days. Probiotic Lactobacilli (Lactobacillus

acidophilus and Lactobacillus casei) with inulin and lactulose as prebiotics were

encapsulated in alginate and alginate –gelatin beads and to determine the effect of inulin

and Lactulose as well as microencapsulation on the physicochemical (pH, acidity, level of

syneresis) and microbiological (probiotic bacterial count) properties of synbiotic yogurt.

For both probiotics, alginate- gelatin along with prebiotic was found to give better

protection in simulated gastric juice (SGJ) and better release in gastro-intestinal

fluid(SIF). Further free cells were very susceptible to SGJ and SIF. Alginate-gelatin and

alginate microcapsules with lactulose was most effective in protecting L.acidophilus and

L.casei from simulated intestinal juice with an encapsulation efficiency of 97.38 % and

85.9 % respectively. Microencapsulated synbiotic were also used for production of yogurt.

The results showed that alginate–gelatin with inulin showed significant (p<0.05) effect on

least change in pH, acidity and syneresis and increase in bacterial count of L.acidophilus,

while alginate–gelatin with lactulose showed significant (p<0.05) effect on least change in

physicochemical and microbiological properties of L.casei. The final results showed that

L.casei had a higher viability than the level of the therapeutic minimum (>107 CFU/g)

during 28 days of storage in all the treatment while for L.acidophilus, only inulin

encapsulated in alginate–gelatin maintained the count above this limit. So co-

encapsulation of synbiotics can be used to enhance and improve the physicochemical

properties and viability of probiotic bacteria during processing and also in gastrointestinal

tract.

2

Chapter 1

INTRODUCTION

1.1 Probiotics and Prebiotics

In recent years, probiotics showed the increasing attention on its application for

improving the intestinal microbial balance of the human and animal, which was defined

as ‘‘living microorganisms” (Adhikari et al., 2000; Chandramouli et al., 2004;

FAO/WHO, 2006; Pedroso et al., 2012). Fuller (1989) has redefined a probiotic as ‘a live

microbial feed supplement which beneficially affects the host animal by improving its

intestinal microbial balance’. Lactobacillus acidophilus is frequently used in food

products, which have been reported to suppress the pathogens growth, improve lactose

utilization and stabilize the digestive system (Ouwehand et al., 1998; Kopp-Hoolihan,

2001; Kaur et al., 2002; Kim et al., 2008).

A prebiotic is a nondigestible food ingredient that beneficially affects the host by

selectively stimulating the growth and/or activity of one or a limited number of bacteria in

the colon, and thus improves host health (Gibson & Roberfroid, 1995). The majority of

simple sugars and oligosaccharides ingested and digested by humans are absorbed in the

small intestine (Bond et al., 1980). However some prebiotic such as lactulose, raffinose,

stachyose and fructooligosaccharides (such as oligofructose or inulin) are able to reach the

colon intact (Roberfroid et al., 1993). Prebiotics have been selectively manufactured to

contain several or all of the following attributes; active at low dosages, varying viscosity,

lack of side effects, varying sweetness, control of micro flora modulation, persistence

through the colon, good storage and processing stability and inhibition of pathogen

adhesion. Established and possible effects of prebiotics include nondigestibility and low

3

energy value, stool bulking effect and modulation of the gut flora, promotion of

Bifidobacteria and repression of clostridia. In the last 10 years, there has been an increasing

interest in the consumption of probiotics and functional foods in Western diets (O’sullivan,

1996). Probiotic bacteria are able to suppress potentially pathogenic microorganism in the

gastrointestinal tract and enhance the population of beneficial microorganisms (Yaeshima

et al., 1997).

The health benefits derived by the consumption of foods containing probiotic

bacteria are well documented and more than 90 probiotic products are available worldwide

(Shah, 2000). To provide health benefits, the suggested concentration for probiotic bacteria

is 106cfu/g of a product (Shah, 2000). However, studies have shown low viability of

probiotics in market preparations. A number of factors have been claimed to affect the

viability of probiotic bacteria in fermented food including acid and hydrogen peroxide

produced by bacteria, oxygen content in the product, and oxygen permeation through the

package (Shah, 2000). Viability of probiotic bacteria in fermented products declines over

time because of the acidity of the product, storage temperature, storage time, and depletion

of nutrients (Dave & Shah, 1997). Loss of viability of probiotic bacteria occurs in

fermented products, and these products have limited shelf life (Dave & Shah, 1996).

However, in order to achieve maximum viability in a product, in the gut and maximum

health benefits, there is a need to have a better understanding of this organism as an

emerging probiotic.

During use and under storage the probiotic should remain viable and stable, and be

able to survive in the intestinal ecosystem, and be able to survive in the ecosystem, and

the host animal should gain beneficially from harbouring the probiotic. It is therefore

proposed that the exogenous bacteria reach the intestine in an intact and viable form, and

establish therein and exert their advantageous properties. In order to do so, microbes must

4

overcome a number of physical and chemical barriers in the gastrointestinal tract. These

include gastric acidity and bile acid secretion. Moreover, on reaching the colon the

probiotics may be in some sort of stressed state that would probably compromise chances

of survival.

1.2 Synbiotics

When prebiotics are combined with probiotics, their relationship is classified as

synbiotic. A synbiotic is a combination of probiotics and prebiotics that “ beneficially

affects the host by improving the survival and the implantation of live microbial dietary

supplements in the gastro intestinal tract by selectively stimulating the growth and/or by

activating the metabolism of one or a limited number of health promoting bacteria”

(DiRienzo, 2000).This combination can improve the survival rate of the probiotics and

provide additional health benefits to the host [Collins and Gibson, 1999]. Thus together

probiotics and prebiotics have health promoting effects as well as develop of products

assortment.

1.3 Viability of Probiotic organisms

Microorganisms introduced orally have to, at least, transiently survive in the

stomach and small intestine. Although this appears to be a rather minimal requirement,

many bacteria including the yoghurt-producing bacteria L. delbrueckii subsp. bulgaricus

and S. thermophilus often do not survive to reach the lower small intestine. The reason for

this appears to be low pH of the stomach. In fasting individuals, the pH of the stomach is

between 1.0 and 2.0 and most microorganisms, including lactobacilli, can only survive

from 30 seconds to several minutes under these conditions. Therefore, in order for a

probiotic to be effective, even the selection of strains that can survive in acid at pH 3.0 for

sometime would have to be introduced in a buffered system such as milk, yoghurt or other

food. Lankaputhra and Shah (1995) showed that, among several strains of L. acidophilus

5

and Bifidobacterium sp. studied, only a few strains survived under the acidic conditions

and bile concentrations normally encountered in fermented products and in the

gastrointestinal tract, respectively. Therefore, it cannot be generalised that all probiotic

strains are acid and bile tolerant. Clark et al. (1993) and Lankaputhra and Shah (1995)

showed that Bifidobacterium longum survives better in acidic conditions and is able to

tolerate a bile concentration as high as 4%.

1.4 Microencapsulation of Probiotics

Micro encapsulation was developed as the technology to improve the stability and

viability of free cells due to their sensitivity to the acidic media and oxygen (Fung et al.,

2011; Nag et al., 2011; Pedroso et al., 2012). Moreover, the higher cell viable should be

remained because they should pass through the stomach and intestine to provide beneficial

effects (Chandramouli et al., 2004).Micro encapsulation has always been used for

providing the controlled release property for cell in the coating materials (Dembczynski et

al., 2002; Lee et al., 2004; Picot et al., 2004; Ma et al., 2014; Xing et al., 2014). As

reported by Chandramouli et al. (2004), the condition for protecting Lactic acid bacteria

was optimized. Picot et al. (2004) also found that the viable cell counts of Bifidobacterium

breve R070 and Bifidobacterium longum R023 in micro encapsulation using the whey

protein as the coating materials was increased during storage at the low temperature. As

reported by Mandal et al. (2006), the survival of coated Lactobacillus casei was better than

that for free cells at heat treatment. Moreover, according to Ding et al. (2009), during both

processing and storage, micro encapsulation could improve the survival of

microorganisms. According to the investigation of Xing et al. (2014) and Ma et al. (2014),

Lactobacillus acidophilus was also embedded with porous starch as the coating material.

Therefore, microencapsulated cell is drawing more and more interesting of many

researchers in order to improve its stability during application (Semyonov et al., 2010).

6

Micro-encapsulation and added prebiotic substances in probiotic products have been used

satisfactorily to increase the survival of probiotic organisms in high acid fermented

products such as yoghurts. Khalida et al (2000) reported a modified method involving

calcium-alginate-starch micro-encapsulation. In this study the encapsulated L. acidophilus

and Bifidobacterium spp were incorporated and set yoghurt was made and stored for 8

weeks at 40C. This study demonstrated that survival of encapsulated cultures of L.

acidophilus and Bifidobacterium showed a better survival over an 8 weeks storage period

compared to the survival of free cells.

Some authors have shown that the freezing process affects dramatically the number

of live probiotic cells (Kailasapathy and Sultana, 2003). Encapsulation has been

investigated for improving the viability of microorganisms in both dairy products and the

GI tract (Krasaekoopt et al., 2003 and Picot and Lacroix, 2004). The co-encapsulation of

probiotic with prebiotic improved the survival rate of probiotics (Chen et al., 2005).

Beneficial health properties of probiotics, prebiotics and other functional

compounds strengthened the production, the offer and requirement of functional food

products and/or pharmaceuticals worldwide. The efficacy of the administered functional

products containing probiotic bacteria largely depends on the viability of the cells and their

release in the lower intestine in sufficient number. Different techniques of

microencapsulation have been used as a potential tool to enhance the viability of probiotics

and to control release of these cells across the intestinal tract (Burgain 2011, Cook, 2012).

It is important the encapsulation method applied does not reduce viability of the cells and

does not inhibit their activity which includes resistance of the cells to gastrointestinal

environment and their ability to adhere to intestinal mucosa.

Examples of biomaterials often applied to encapsulate bacterial cells include

alginate, gelatin, chitosan, carrageenan, whey proteins, cellulose acetate phthalate, acacia

7

gum, gellan and xanthan gum and starches (Burgainand Gaiani 2011 and Cook MT,

Tzortzis 2012). Alginate beads are sensitive to the acidic environment, while cellulose

acetate phthalate and mixture of gellan-xanthan are not soluble at acidic pH and enable

high resistance towards acid conditions. Amphoteric nature of gelatin and whey protein

allows favorable interaction with anionic polysaccharides when the pH is adjusted below

the isoelectric point of the protein component thus the net charge of the protein becomes

positive (Harnsilawat T, Pongsawatmanit 2006). The behaviour of the proteins below their

isoelectric point encourages cooperation between proteins and polysaccharides in terms of

higher survival rate of probiotics during processing and after consumption. Due to certain

advantages and disadvantages of an applied method of encapsulation and coating agents,

it is important to compare different biomaterials and encapsulation methods using the same

probiotic strain.

Sodium alginate is often applied for encapsulation of probiotic cells due to its low

cost, biocompatibility and suitability to form gels easily by selective binding of divalent

ions, while properly resolve in the intestine and release entrapped cells. Alginate

microparticles improved survival of L. casei NCDC-298 in simulated GI conditions and

exposure to heat (Mandal and Puniya, 2006) and had no negative effect on adhesion of

probiotic cells to HT-29 cell line of the human epithelium. However, alginate particles are

sensitive to acid medium and they can be easily disintegrated in the presence of

monovalent ions or salts as phosphates, lactates and citrates which bond calcium ions.

Additional disadvantage of alginate particles is their porous surface (Gouin, 2004). On the

other hand, the polyelectrolyte nature of alginate enables improving its properties as

encapsulating agent by creating of electrostatic interaction with other polymers or by using

additives to cause structural modifications of alginate (Krasaekoopt and Bhandari, 2003).

Micro-encapsulation and added prebiotic substances in probiotic products have been used

8

satisfactorily to increase the survival of probiotic organisms in high acid fermented

products such as yoghurts.

1.5 Scope of present investigation

Probiotic functionality depends on the ability of a strain to confer health advantages

on the host upon oral consumption of viable cells. In recent times, there has been a growing

appreciation for the important role of commensal microbiota in human health. This has led

to attempts to manipulate or augment the microbiota through the use of probiotics (live

microorganisms that when administered in adequate amounts confer a health benefit on

the host) or prebiotics (non-digestible substances that provide a beneficial physiological

effect on the host by selectively stimulating the favourable growth or activity of a limited

number of indigenous bacteria).

In the recent past, there has been an explosion of probiotic health-based products.

Many reports indicated that there is poor survival of probiotic bacteria in these products.

Further, the survival of these bacteria in the human gastro-intestinal system is

questionable. Providing probiotic living cells with a physical barrier against adverse

environmental conditions is therefore an approach currently receiving considerable

interest. The technology of microencapsulation of probiotic bacterial cells evolved from

the immobilised cell culture technology used in the biotechnological industry.

Microencapsulation is a process by which individual particles or droplets of solid or liquid

material (the core) are surrounded or coated with a continuous film of polymeric material

(the shell) to produce capsules in the micrometer to millimeter range, known as

microcapsules. The capsule has a core surrounded by a thin membrane and the membrane

serves as a barrier to LAB release. After encapsulation technique was introduced,

microencapsulation techniques were successfully used to improve the survival of

microorganisms in dairy products (Adhikari et al., 2002). The most commonly reported

9

microencapsulation procedure is based on the calcium alginate gel capsule formation.

Kappa-carrageenan, gellan gum, gelatin and starch are also used as excipients for the

micro-encapsulation of probiotic bacteria.

1.6 Objectives of the present investigation

The objectives of the present work were the following:

Production of Probiotic and synbiotic yogurt.

Analysis of physico-Chemical and microbiological analysis of Probiotic and

synbiotic yogurt.

Microencapsulation of synbiotics.

To study the Survival of microencapsulated synbiotic at acidic pH of stomach and

bile salt of intestine.

To Study the efficacy of microencapsulation on synbiotic yogurt production.

10

Chapter 2

REVIEW OF LITERATURE

2.1. Yogurt

Yogurt is a product of the lactic acid fermentation of milk by addition of a starter

culture containing Streptococcus thermophilus and Lactobacillus delbrueckii ssp.

bulgaricus (McKinley, 2005). Although fermented milk products such as yogurts were

originally developed simply as a means of preserving the nutrients in milk, it was soon

discovered that, by fermenting with different microorganisms, an opportunity existed to

develop a wide range of products with different flavours, textures, consistencies and more

recently, health attributes. The market now offers a vast array of yogurts to suit all palates

and meal occasions. Yogurts come in a variety of textures (e.g. liquid, set and stirred curd),

fat contents (e.g. regular fat, low-fat and fat-free) and flavours (e.g. natural, fruit, cereal,

chocolate), can be consumed as a snack or part of a meal, as a sweet or savoury food. This

versatility, together with their acceptance as a healthy and nutritious food, has led to their

widespread popularity across all population subgroups (Mckinley, 2005). Yogurt was

introduced to the American diet during the 1940s. By the 1980s, it had become the product

for dieters, and the lunch of choice for young women. The use of yogurt as a calcium

source has made it one of the most rapidly growing dairy products, but presently it is more

than just a calcium source. Yogurt, Kefir, and similar fermented milk products are on the

way to becoming major nutraceuticals aimed at treating a variety of disease conditions

(Katz, 2001).

Yogurt gels are formed by the fermentation of milk with thermophilic starter

bacteria; milk is normally heated at high temperatures (e.g., 85°C for 30 min), which

causes the denaturation of whey proteins. Denatured whey proteins interact and cross-link

with κ-casein on the surface of casein micelles. There is increased casein-casein attraction

11

as the pH of milk decreases from ~6.6 to ~4.6 during yogurt fermentation, which results

in gelation as casein approach their iso-electric point. Physical properties of yogurt gels,

including whey separation play an important role in quality and consumer acceptance. An

understanding of gelation process during fermentation is critical in manipulating physical

properties of yogurt (Lee and Lucey, 2004).

2.2. Definition of probiotics:

The explanation of probiotics has been growing over time, which used for the first

time by Lilly and Stillwell (1965) to describe compounds produced by organisms that

stimulated the growth of another. However, Parker (1974) used this term to the substances

that applied to the animals feed as supplements in purpose of health improve by

contributing to its intestinal microbial balance. This term probiotics‘ was taken by Roy

Fuller (1989) and under a continuous work he referred to these substances as life microbes

and substances supplements and give his definition as ―a live microbial feed supplement

that beneficially affects the host animal by improving its intestinal microbial balance.

Applying probiotics to the human studies show more new definitions and the essential

requirements have been moderated to suit the future researches. Food and Agriculture

Organization/World Health Organization Working Group (FAO/WHO) (2002) recognize

probiotics as ―live microorganisms which when administered in adequate amounts confer

a health benefit on the host. But the Joint International Scientific Association for Probiotics

and Prebiotics recently adopted this definition (Reid et al., 2003) ―Probiotic bacteria are

live food supplements which benefit the health of the consumer.

12

2.3. Characteristics of Lactobacillus

Lactobacilli are characterized as Gram-positive facultative bacteria, they are non-

spore forming, non-flagellated rods or coccobacilli (Hammes and Vogel, 1995). They are

strictly fermentative, so they have the ability to ferment lactose and other monosaccharides

to lactic acid predominantly with the homo-fermenters ones and to lactic acid with carbon

dioxide and ethanol for the hetero-fomenters ones. They are well use in the diet, therefore

they claim as probiotics include Lactobacillus acidophilus, L.delbrueckii subsp.

bulgaricus, L.casei, L.fermentum, L.plantarum, L.reuteri.

2.3.1. Lactobacillus acidophilus

L. acidophilus belongs to the homofermentative Group of lactobacilli. L.

acidophilus is non-motile, non-flagellated and non-sporing. It is facultative bacteria and

Gram-positive rod around 0.6 to 0.9 μm in width and 1.5 to 6.0 μm in length with rounded

ends. Cells may appear singularly or in pairs as well as in short chains. The optimum

growth occurs within 35-40°C but it can tolerate temperatures as high as 45°C. The

optimum pH for growth is between 5.5-6. Lactobacillus acidophilus offers a range of

health benefits which include: providing immune support for infections and cancer, a

healthy replacement of good bacteria in the intestinal tract following antibiotic therapy,

reducing occurrence of diarrhoea in humans (children and adults), aiding in lowering

cholesterol, improving the symptoms of lactose intolerance. Anti-tumor effect of

L.acidophilus was reported by Goldin and Gorbach (1984). Oral dietary supplements

containing viable cells of L.acidophilus decreased ß- glucuronidases, azoreductase, and

nitroreductase, bacterial enzymes, which catalyze conversion of procarcinogens to

carcinogens. Anticarcinogenic effect of L.acidophilus may be due to direct removal of

procarcinogens and activation of body’s immune system. Animal studies have shown that

13

dietary supplementation with L.acidophilus decrease the number of colon cancer cells in

a does dependant manner (Rao et al., 1999).

2.3.2. Lactobacillus casei

Lactobacillus casei is an acid sensitive, rod-shaped, facultative hetero fermentative

lactic acid bacterium that can be isolated from a variety of environments including raw

and fermented milk and meat or plant products, as well as the oral, intestinal, and

reproductive tracts of humans and animals. It is a beneficial microorganism that helps to

promote other beneficial bacteria and prevents the overgrowth of pathogenic bacteria in

the human body. It has been reported that it can improve and intensify digestion, control

diarrhoea, reduce inflammations of the gut, reduce lactose intolerance and alleviate the

symptoms of constipation, all leading to better function of the immune system (Gill Prasad,

2008)

2.4. Regulations and safety of probiotics in manufacture:

Since the early of last century beneficial effects of probiotics have been proved and

well used in the dairy Industry, which suggested of daily intake to get the beneficial effect

on the host. That directs the research and Industry organizations to suggest special

regulations for the use of probiotics in food industry to obtain the desired therapeutic

effects. Probiotics are sensitive because they die after exposure to low pH in the human

stomach. Therefore, the high-count number of probiotics recommended in the products at

a minimum count of 106 CFU/g at the expiry date (Gomes and Malcata, 1999). The more

use of probiotics in the worldwide as dairy products the more suggested the need of

principles and regulations as standard for the minimum count of viable probiotics bacteria

in the dairy and fermented milk products to get the beneficial effects. The National Yogurt

Association (NYA) of the United States suggested that any yogurt with live culture and

use for health benefit recognize as containing significant amounts of live and active

14

cultures. The seal is a voluntary identification available to all manufacturers of refrigerated

yogurt products contain at least 108cfu per gram at the time of manufacture, and at least

107cfu per gram in frozen yogurt contains at the time of manufacture. Some countries like

Australia and New Zealand are still not introduce regulations for probiotics use. However,

the Australian and New Zealand Food Standards Code (ANZFA) doesn‘t put any

minimum count of probiotics products, but its mentioned the important of viable

organisms in the manufactured of fermented milk products and that should be at least 106

CFU/g and pH of 4.5 at the end of manufactured of yogurt.

2.5. Prebiotics

The term `prebiotic’ was first coined by Gibson and Roberfroid (1995). Prebiotic

is defined as a non digestible food ingredient that beneficially affects the host by

selectively stimulating the growth and/or activity of one or a limited number of bacteria in

the colon, that can improve the host health’. The function of prebiotics is to basically

stimulate existing metabolisms in the colon (Coussement, 1996). Thus, the prebiotic

approach advocates administration of non-viable entities and therefore overcomes survival

problems in the upper gastrointestinal tract. To be an effective prebiotic an ingredient

must: Neither be hydrolysed nor absorbed in the upper part of the gastrointestinal tract;

Have a selective fermentation such that the composition of the large intestinal microbiota

is altered towards a healthier composition.

Prebiotics, as currently conceived of, are all carbohydrates of relatively short chain

length (Cummings et al., 2001), additionally carbohydrates that have escaped digestion in

the upper gastrointestinal tract form the predominant substrates for bacterial growth in the

colon (Roberfroid et al., 1993). Short-chain fatty acids are a major product of prebiotic

breakdown, but as yet, no characteristic pattern of fermentation has been identified.

15

Through stimulation of bacterial growth and fermentation, prebiotics affect bowel habit

and are mildly laxative (Cummings et al., 2001).

Prebiotics may have many advantages over probiotics. This is firstly related to

survivability problems. These include: Maintenance of viability in the product (which, for

obvious reasons, will usually be stored under conditions adverse to bacterial growth);

Gastric acidity; Bile salts; Pancreatic enzymes and proteins; Competition for colonisation

sites and nutrients with the resident gastrointestinal flora.

Gibson (2004) stated that for a dietary substrate to be classified as a prebiotic, it

has to meet at least three requirements; (1) the substrate must not be hydrolysed or

absorbed in the stomach or small intestine, (2) it must be selective for beneficial bacteria

in the colon such as the Bifidobacteria and (3) fermentation of the substrate should induce

beneficial luminal/systemic effects within the host. A range of dietary compounds has

suggested as prebiotic, most of the selected prebiotics were on their health benefits on host.

Gibson et al. (1995) presented the popularity of Inulin, Fructo-oligosaccharides (FOS) and

Galacto-oligosaccharides (GOS) as health benefit subtracts.

2.5.1. Inulin

Inulin is a blend of fructan chains found widely distributed in nature as plant

storage carbohydrates (Wang and Gibson, 1993), and is present in more than 36,000 plant

species. The majority of inulin commercially available today is extracted from chicory

roots. Chemically, inulin is a polydisperse β-(2,1) fructan. The fructose units in the mixture

of linear fructose polymers and oligomers are each linked by β-(2,1) bonds. A glucose

molecule typically resides at the end of each fructose chain and is linked by an α-(1,2)

bond, similar to sucrose. Chain lengths of these chicory fructans range from 2-60, with an

average degree of polymerisation of 10 Inulin has natural taste, colourless and minimal

16

influence on the natural characteristics of the products. The only prebiotics for which

sufficient data have been generated to allow an evaluation of their possible classification

as functional food ingredients are the inulin-type fructans, which include native inulin,

enzymatically hydrolyzed inulin or oligofructose, and synthetic fructo oligosaccharides

(Roberfroid et al., 1998).It is fermented by the intestinal flora causing increase in the

biomass, producing of short chain fatty acids and decrease in the pH, and significant

increase of Bifidobacteria in the colon and inhibits the growth of less beneficial bacteria,

(Roberfroid, 1998). So using these ingredients in food allows improving the nutrition value

of the products, by reducing the calorie content and increasing the bifidus-promoting

capacities.

2.5.2. Lactulose

Lactulose is a synthetic disaccharide in the form Gal β1-4 fructofuranose.

Lactulose has been used as a laxative as it is not hydrolysed or absorbed in the small

intestine. However, at sub-laxative doses lactulose has received attention as a bifidogenic

factor and has been administered as such (Tamura, 1983). In vitro, lactulose increased

lactobacilli and bifidobacteria and significantly decreased bacteroides in mixed continuous

faecal culture. The feeding of lactulose to rats significantly increased bifidobacteria;

however, only a limited number of bacterial groups was enumerated (Suzuki et al., 1985).

In a human trial, bifidobacteria significantly increased while clostridia,

bacteroides, streptococci and Enterobacteriaceae decreased on the feeding of 3 g/d

lactulose to eight volunteers (five male, three female) for 14 days (Terada et al., 1992).

Small decreases in bacteroides and lactobacilli during the test period were also determined.

In addition, decreases in the detrimental metabolites ammonia, indole, phenol, p-cresol

and skatole, and enzymes β-glucuronidase, nitroreductase and azoreductase supported

beneficial claims of lactulose.

17

2.6 Factors affecting the growth and survival of Lactobacillus

The consumption of probiotic bacteria within food products is the most popular

way to re-establish the gastrointestinal microflora balance. The literature had stated that

probiotic products have to present no less of 106cfu in ml of probiotic bacteria at the time

of consumption to get the beneficial health on the host (Adhikari et al., 2003).

Dairy products is one of the most common carrier have been used as probiotic food

products. Therefore, it is of interest to study some factors that affect the growth and

survival of probiotic bacteria while in transit in dairy products to human use. Many factors

have been reported to affect the growth and survival of probiotic bacteria in dairy products,

including acid and hydrogen peroxide produced by yogurt bacteria, oxygen content in the

product and oxygen permeation through the package and the storage temperature (Dave

and Shah 1997) suggested of using ascorbic acid in dairy products as scavenger to reduce

the oxygen content and redox potential of dairy products to enhance the viability of

probiotic bacteria.

The interaction among probiotic species and yogurt starter cultures is also

considered important in determining their growth and survival status in dairy products.

Various inhibitory interactions were found among these bacteria (Dave and Shah, 1997).

Growth and survival of probiotic bacteria has also found to be affected by the chemical

and microbiological composition of milk, milk solids content, and availability of nutrients

(Shah, 2000).

Viability and survival of probiotic bacteria are the most important parameters in

order to provide therapeutic functions. A number of factors have been claimed to affect

the viability of probiotic bacteria in dairy foods such as yoghurt and fermented milks,

including low pH and refrigerated storage (Shah, 2000). Micro-organisms ingested with

food begin their journey to the lower intestinal tract via the mouth and are exposed during

18

their transit through the gastrointestinal tract to successive stress factors that influence

their survival (Marteau et al., 1993). The time from entrance to release from the stomach

is about 90 min, but further digestive processes have longer residence times (Berrada et

al., 1991).

Cellular stress begins in the stomach, which has pH as low as 1.5 (Lankaputhra &

Shah 1995). Bile secreted in the small intestine reduces the survival of bacteria by

destroying their cell membranes, whose major components are lipids and fatty acids and

these modifications may affect not only the cell permeability and viability, but also the

interactions between the membranes and the environment (Gilliland, 1987).

One of the important characteristics of the microorganisms is their ability to

survive through the acidic conditions in the human stomach and bile concentrations in the

intestine and colonise in the gut. For this to occur, viable cells of Lactobacillus must be

able to survive the harsh condition of acidity and bile concentration commonly

encountered in the gastrointestinal tract of humans.

2.7. Effect of prebiotics on probiotic growth and survival

Synbiosis is defined as ―a mixture of probiotics and prebiotics that beneficially

affects the host by improving the survival and implantation of live microbial dietary

supplements in the gastrointestinal tract, by selectively stimulating the growth and/or by

activating the metabolism of one or a limited number of health-promoting bacteria, and

thus improving host welfare (Gibson and Roberfroid, 1995). In recent years, there has been

more focus on a combination of pre-and probiotics in a single product.

Fructooligosaccharide and Inulin are the premium prebiotics used for the purpose of

stimulating the growth and/or activity of beneficial bacteria in the large intestine.

19

Inulin and Fructooligosaccharides have their stimulating effect because of their

ability to be fermented by Bifidobacteria and Lactic acid bacteria in vivo (Gibson et al.,

1995) and in vitro. However, recent studies have determined that the ability of

Bifidobacteria to metabolize fructo-oligosaccharides and inulin is a species-dependent

feature and only to a small extent a strain-dependent one related to their enzyme content.

Prebiotic ingredients have also been used as encapsulating agents. Their

advantages over other covering materials rely on the fact that, besides being nondigestible

carbohydrates, they also have beneficial effects for the host, by selectively stimulating the

growth and/or activity of probiotic bacteria (prebiosis) within the colon (Fritzen-Freire and

others 2012). Unlikely to this, greatest stability of encapsulated GG was detected during

cold storage of prebiotic edible films supplemented with inulin (Soukoulis et al., 2014). In

the study of Fritzen-Freire et al. (2012), oligofructose-enriched inulin was found to better

protect Bifidobacterium BB-12 during storage of the microcapsules produced by spray-

drying, although oligofructose showed good protection as well. The divergence of the

reported results is difficult to be explained since clear evidences on the specific protective

action of the prebiotics such as FOS or inulin for each probiotic has not been provided yet

(FritzenFreire et al., 2012).

2.8 Microencapsulation Technique

To improve the survival of LAB, different approaches that increase the resistance

of these sensitive microorganisms against adverse conditions have been proposed,

including appropriate selection of acid- and bile-resistant strains, use of oxygen-

impermeable containers, two-step fermentation, stress adaptation, incorporation of

micronutrients, however, these methods had only a limited success. Therefore,

encapsulation of bacterial cells in alginate gels is currently gaining attention to increase

viability of probiotic bacteria in acidic products such as yoghurt and it is the commonly

20

used technique because this method is very mild and is done at room temperature in

aqueous medium by using physiologically acceptable chemicals. Encapsulation is a

process in which the cells are retained within an encapsulating membrane to reduce cell

injury or cell loss and it has been widely utilized to protect microorganisms including

probiotics during transit through the human gastro-intestinal tract (Petreska et al 2014).

The microbial cells are entrapped within their own secretions (exopolysaccharides (EPS))

that act as a protective structure or a capsule, reducing the permeability of material through

the capsule and therefore less exposed to adverse environmental factors such as gastric

acid and bile salts.

Carbohydrate polymers such as alginate have been used in various food

applications (Mladenovska and Raicki 2007). Alginate, a natural polysaccharide found in

brown algae, is a linear 1, 4 linked copolymer of -D-mannuronic acid (M) and -L-guluronic

acid (G) and has the benefits of being non-toxic to the cells being immobilized and it is an

accepted food additive. The reversibility of encapsulation, i.e. solubilizing alginate gel by

sequestering calcium ions, peptides and amino acids and the possible release of entrapped

cells in the human intestine are other advantages. However, alginate beads are not acid

resistant and it has been reported that the beads undergo shrinkage and decreased

mechanical strength during lactic fermentation (Mladenovska K, Cruaud O, 2007).

Gelatin is a protein derived from denatured collagen that contains high levels of

hydroxyproline, proline and glycine and is useful as a thermally reversible gelling agent

for encapsulation. Gelatin was selected here because of its excellent membrane-forming

ability, biocompatibility and non-toxicity. The applicability of gelatin as a hydrogel matrix

is limited because of its low network rigidity. However, its physical properties can be

improved through the addition of cross-linking agents. Because of its amphoteric nature,

21

it also is an excellent candidate for cooperation with anionic polysaccharides such as

alginate and so on (Smilkov et al 2013).

A symbiotic pastry product was previously prepared by incorporating free or

encapsulated Lactobacillus casei NCDC 298 in sodium alginate in milk chocolate together

with inulin [Mandal et al 2012]. Although cell encapsulation resulted in significant

increase of cell survival at low pH, high bile salt concentration, and during heat treatment,

the viable counts of both free and encapsulated L. casei NCDC 298 were unchanged during

the storage of milk chocolate at refrigerated conditions up to 60 days and were higher than

the recommended level by International Dairy Federation guidelines (107 cfu/g) at the end

of the product shelf-life [Mandal et al 2012]. Feeding of the symbiotic chocolate increased

the fecal lactobacilli and decreased fecal coliforms and β-glucuronidase activity in mice,

indicating that it might constitute an excellent food for delivery of probiotic lactobacilli

[Mandal et al 2012].

On the contrary, encapsulation of Lactobacillus acidophilus ATCC 4356 on

calcium alginates had no effect on cell survival compared to free cells during refrigerated

storage of yoghurts for 4 weeks [Ortakci and S. Sert 2012]. However, significantly greater

survival of encapsulated over free probiotic bacteria was observed in the in vitro assays

using artificial human gastric digestion systems [Ortakci and S. Sert 2012].

Calcium alginate microspheres can be produced by both extrusion and emulsion

techniques Extrusion is the oldest and the most common approach to make capsules with

hydrocolloids and might be achieved by simply dropping an aqueous solution of probiotics

into a gelling bath. The size and shape of the beads usually range 2–5  mm and depend on

the diameter of the needle and the distance of free fall [Krasaekoopt et al, 2003]. It offers

a small range size (smaller than emulsion), but it does not provide particles under 300  μm

[Burgain et al 2011]. Extrusion is more popular than emulsion technology due to its

22

simplicity, easy handling, low cost at least to small scale, and gentle formulation

conditions, which ensure maintenance of high cell viability (80–95%) [Krasaekoopt et al,

2003].

23

Chapter 3

MATERIALS AND METHODS

3.1 Materials

Lyophilized cultures of Probiotic bacteria, Lactobacillus acidophilus (ATCC 4356,

NCDC 014), Lactobacillus casei (NCDC 018)were obtained from National Collection of

Dairy Cultures (NCDC), Dairy Microbiology Division, NDRI, Karnal , Haryana and

commercial yogurt culture containing Streptococcus thermophilus and L. delbrueckii ssp.

bulgaricus were obtained from Kerala Agricultural university, Mannuthy.

MRS and M17 broth and agar, Skim milk powder, Prebiotics (Lactulose and

inulin), Phosphate buffer saline (PBS) - pH 7.2, Bile, Pepsin, Sodium alginate, Gelatin,

Calcium Chloride (Himedia, Mumbai).

High speed refrigerated centrifuge (KHSRC-1, Kemi), pH meter (scientific tech-

ST-2001), BOD incubator (KBOD 6S, KEMI 3),Remi Mini Rotary shaker (RS-12R),

0.25-mm needle.

Figure 3.1. Gram stained preparation of (a) L.acidophilus and (b) L. casei.

(a) (b)

24

3.2 Methodology

3.2.1 Preparation of cell suspension

Pure culture of Lactobacillus acidophilus (ATCC 4356, NCDC 014) Lactobacillus

casei (NCDC 018) and L. delbrueckii ssp. bulgaricus propagated in MRS ( de Man-

Rogasa-Sharpe) agar media and broth and Streptococcus thermophilus were in M17broth

and agar for 24 hours under aerobic conditions at 37°C.Biomasses were then harvested

by centrifuging at 5000 rpm for 10 min at 4 °C. The cell pellets were then resuspended in

10 mL phosphate buffer solution (PBS) to obtain a final cell counts of 6×108 CFU/gm.The

cultures were then washed twice by sterile PBS and resuspended in pasteurised (630C for

30 min) 10 % reconstituted skim milk (RSM).

3.2.2 Production of Probiotic yogurt

Homogenized, standardized and pasteurized milk (3.62% protein, 3.61% lactose,

1.6% fat and 9.70% total solid) was used for preparation of probiotic yogurt. All yogurt

samples were produced in hygienic conditions. Milk was heated up to 85°C for 30 min

followed by cooling down to 40°C. The yogurt starter culture was then added at a

concentration of 1:1 in all samples. Experimental preparations of yogurt including control

plain yogurt in Homogenised pasteurized milk (T1), yogurt containing 1% Lactobacillus

acidophilus (T2), yogurt containing 5% Lactobacillus acidophilus (T3), yogurt containing

10 % Lactobacillus acidophilus (T4) probiotic yogurt containing 1%, 5% and 10%

Lactobacillus casei (T5,T6,T7 respectively). The mixtures were subsequently poured into

250-ml plastic cups and incubated at 43°C and fermentation was stopped at pH 4.5 – 4.7.

Then the samples were kept at 4°C. Physicochemical characteristics (pH, titratable acidity,

syneresis) and viability of probiotic bacteria in this sample were evaluated during 21-days

of refrigerated storage.

25

3.2. a. Determination of pH

pH of the milk and yogurt samples was determined with a pH meter(scientific tech-

ST-2001) at room temperature. pH was determined in a single cup of yogurt per replication

1, 2, 3,and 4 h after inoculation, and in three cups (Lactobacillus acidophilus and

Lactobacillus casei) of yogurt per replication at every 7 days of storage.

3.2. b. Determination of titratable acidity

Titratable acidity was determined in yogurt samples at room temperature according

to the methods described in AOAC (2002). Yogurt samples (10 g) were diluted with 10

ml distilled water and titrated with 0.1 N NaOH in the presence of 0.1 % phenolphthalein.

Titratable acidity was expressed as the percent of lactic acid based on the sample weight.

Titratable acidity was determined in a single cup of yogurt after 4 hours of inoculation,

and in three cups of yogurt at every 7 days of storage at 40 C.

3.2. c. Enumeration of L. acidophilus and L.casei in yogurt

SPC, a conventional method to determine cell count, was used to quantify viable

L.acidophilus and L.casei cells. One gram of yogurt sample was diluted with 99 mL of

sterile phosphate buffer saline (PBS), pH 7.2 (Himedia, Mumbai) Subsequent 10-fold

serial dilutions were made with PBS, and 0.1 mL of the diluted Samples was spread on

MRS-bile agar. After aerobic incubation at 37 °C for 48–72h, CFU/g was calculated.

[Tharmaraj and Shah, 2003]

3.2. d. Determination of Syneresis

To measure syneresis, at first, 25g of yoghurt weighed in centrifuge tubes, then the

tubes were centrifuged in 350 G at 10°C for 30 min. The separated liquid from the sample

that collected in the top of tube was removed and the tubes were re-weighed. Syneresis

rate was expressed as lost water per 100g of yoghurt (Gonzalez – Martinez et al., 2002).

26

3.3. Production of Synbiotic yogurt

Set yogurt was prepared using milk with 3.5 % fat that was standardized to 8.5%

solids not fat. Milk was preheated to 40°C.Inulin and Lactulose at a concentration of 1%

were added separately. Milk samples were heated at 85°C for 30 min, then cooled down

to 40°C for inoculation. The samples were inoculated with yogurt culture (1%) and

probiotic culture -Lactobacillus acidophilus and Lactobacillus casei (1%) separately. The

inoculated samples were mixed thoroughly and dispensed in 500 ml polystyrene cups with

lids then incubated at 43 °C until the pH dropped to 4.7-4.5. Control samples did not

contain any prebiotics. Duplicate bottles of each treatment were prepared. The

fermentation was stopped by transferring the cups immediately to refrigerator maintained

at 4°C.

Physicochemical characteristics (pH, titratable acidity and syneresis) and viability

of probiotic bacteria in both sample were evaluated during 28-days of refrigerated storage.

3.3. a. Physicochemical and Microbial analysis of Synbiotic yogurt

pH value of samples was measured using Digital pH-meter (Scientific Tech) at

250C. Titratable acidity was determined by AOAC method [2002]. Syneresis was

measured according to Gonzalez–Martinez et al.2002 method.

MRS – bile agar was used for the selective enumeration of probiotic bacteria in the

presence of yogurt bacteria. The plates were incubated aerobically at 37°C for at least 72

hours [Tharmaraj and Shah, 2003]. Relative survival for each strain was determined by

dividing CFU/gm on 28 day by the initial cell count and then multiplied by 100.

3.4 Microencapsulation of Probiotic Cultures

All glassware and solutions used in the protocols were sterilized at 121 °C for 15

minutes. The preparation of encapsulated microcapsules was a modified version of

27

methods basically reported by Donthidi et al. in 2010 and Sultana et al. in 2000. Briefly,

2 gm sodium alginate(Hi media, Mumbai) was added to 100 ml distilled water and boiled

until it formed a gel, then to another 2% sodium alginate, 2% gelatin (Hi media ,Mumbai)

was separately added and required concentrations of inulin (1%) and Lactulose (1%) were

added separately and stirred until they were dissolved or dispersed. Then probiotic

cultures of each bacterial species (L.acidophilus and L.casei) were transferred to the carrier

solutions with stirring under sterile conditions to ensure uniform distribution of the cells.

3.4.1 Preparation of alginate microcapsule

The conditions used in the experimental work for the probiotic cells encapsulation

were: a) 2% alginate; b) 2% alginate +1% Lactulose; c) 2% alginate + 1% inulin; d) 2%

alginate + 2% gelatin; e) 2% alginate + 2% gelatin+1% Lactulose; f) 2% alginate + 2%

gelatin+1% inulin. To form capsules, a cell suspension (equivalent of 108 CFU/g) was

mixed with a 60 ml of 20 g/L alginate or alginate-gelatin with or without inulin or lactulose

and the mixture was dripped into a solution containing 0.1 M CaCl2, with a sterile syringe.

The distance between syringe and CaCl2 solution was 10 cm. The droplets formed gel

capsule instantaneously. Microscapsules were hardened for 30 min in CaCl2, and then

rinsed with sterile NaCl (8.5 g/L).

28

Figure 3.2: Microencapsulated probiotic in (a) alginate gelatin and (b) alginate beads

3.4.2 Preparation of simulated gastric and intestinal juices and inoculation of cells

The simulated juices were prepared according to Brinques et al 2011 and Michida

et al in 2006. Simulated gastric juices were prepared by dissolving pepsin (Himedia,

Mumbai) in sterile sodium chloride solution (0.5%, w/v) to a final concentration of 3.0 g/L

and adjusting the pH to 1.5 with hydrochloric acid. Simulated intestinal juices were

prepared in sterile sodium chloride solution (0.5%, w/v), with 4.5% bile salts (Oxoid,

Basingstoke, UK) and adjusting the pH to 8.0 with sterile NaOH (0.1 M). Both solutions

were filtered for sterilization through a 0.22 μm membrane. The probiotic bacteria

L.acidophilus and L. casei were inoculated to the simulated gastro-intestinal juice

individually in six different forms, non-encapsulated, encapsulated with calcium alginate

and calcium alginate-gelatin coated with inulin or Lactulose as prebiotic. Further one gram

of freshly encapsulated bacteria samples or 1 mL of cell suspensions (free cells) were

gently mixed with 10 mL of sterile simulated gastric juice (SGJ) for 2 hours at 370 C and

followed by inoculation in sterile simulated intestinal juice (SIF) and incubated at 37 °C

for 4 hours.

29

3.4.3 In vitro release studies (GIT)

To examine the release behaviour of L.acidophilus and L.casei from microcapsules

in GIT in vitro, 1 ml of free bacteria and 1 gram encapsulated samples (probiotic bacteria

with or without prebiotic and alginate and or gelatin as encapsulating material) were added

to 10 ml SGF (pH 1.5) and incubated at physiological temperature (37 0 C) for 2 hours and

subsequently transferred into SIF (pH 8) for another 4 hours. At specific time intervals

(1 hour), 2.0 ml aliquots were removed and absorbance was measured at 600 nm in

triplicate.

3.4.4 Release of entrapped bacteria

The capsules containing probiotic bacteria were released by citrate buffer

(pH= 6.0, 1 %) reported by Mokarram et al in 2009. One gram of capsules was transferred

to 9 ml buffer. The solution was stirred on a shaker for 15 min vigorously until the bacteria

from the capsules were released completely. The counts (CFU/g) were determined by

plating on MRS agar plates and incubating for 48 hours at 37 °C. The free bacteria were

treated similarly. All samples were counted in triplicates. Encapsulation yield (EY) i.e. the

number of bacterial cells that survived the process and encapsulated inside the

microcapsules was calculated as follows:

EY = (N/N0) × 100

Where N0 is the number of viable bacteria in CFU/gm of culture and N is the number of

viable bacteria in CFU/g of microcapsules.

3.5. Co-encapsulated Synbiotic Yogurt Production

A set-type yogurt was prepared for this experiment. Homogenized whole milk (5

L) containing 3.5% fat, 8.5% solid-not-fat was heated to 45 °C. This was then heated to

85 °C for 20 min and allowed to cool. Once the standardized milk had cooled to 45 °C, a

30

commercial yogurt starter culture was inoculated (1% w/v) into it. The probiotic cultures

were added as free and co-encapsulated Probiotic cultures (Lactobacillus acidophilus and

Lactobacillus casei). The acidification profile was recorded hourly until a pH of 4.7-4.5

was reached. Fermentation was stopped by quickly cooling the yogurt. The filled yogurt

cups (200 ml) were stored at 4°C.

Physicochemical characteristics (pH, titratable acidity, syneresis) and viability of

probiotic bacteria in all sample were evaluated during 28-days of refrigerated storage.

Data analysis

The data were statistically analysed using the SPSS Software (version 17.0). In all

experiments, paired t-test was applied for sample comparison. This was done to test for

any significant differences (P<0.05) in the mean value of all the groups.

31

Chapter 4

RESULTS AND DISCUSSION

4.1. Preparation of cell suspension

Lyophilized cultures of Probiotic bacteria (Lactobacillus acidophilus,

Lactobacillus casei) and commercial yogurt culture containing L.delbrueckii ssp.

bulgaricus and Streptococcus thermophilus were propagated in MRS (de Man-Rogasa-

Sharpe) media .The cultures were harvested by centrifuging at 5000 rpm for 10 minutes at

4 °C then washed twice by sterile saline solution (0.9%) and resuspended in pasteurised

(630C for 30 min) 10 % reconstituted skim milk (RSM).

4.2 Production of Probiotic yogurt

Probiotic yogurt was prepared using Homogenized, standardized and Pasteurised

milk. Physicochemical characteristics (pH, titratable acidity, syneresis) and viability of

probiotic bacteria in this sample were evaluated during 21-days of refrigerated storage was

shown in Table 4.1 to 4.4

4.2. a. pH of Probiotic yogurt during storage at 40C

pH of yogurt prepared by Homogenised Pasteurised milk (T1-T7)vary between

4.30 to 4.55 during 4 hours of fermentation. Although no statistically significant difference

was observed in the pH of the treatment groups, the pH was found to be lower in T7(yogurt

containing 10% L.casei) after 4 hours of fermentation. The decline in pH of the batches of

yoghurt obtained during 21 days of refrigerated storage is shown in Table 4.1. The decline

in pH was similar for all the batches of yoghurt. Greater change in pH of about 0.62 was

observed for T4 (10 % L.acidophilus) and control sample containing yogurt culture

showed the least change (0.43).

32

Table 4.1: pH of various yogurt samples Homogenized, pasteurized milk (T1-T7) during

incubation period and storage up to 21 days

*Yogurt

Samples pH of yogurt for various incubation periods

4 hour 7(Day) 14(Day) 21(Day)

T1 4.55 4.35 4.15 4.12

T2 4.53 4.45 4.11 4.05

T3 4.41 4.21 3.92 3.89

T4 4.34 4.09 3.85 3.72

T5 4.53 4.33 4.15 4.06

T6 4.51 4.11 4.03 3.99

T7 4.30 4.20 3.90 3.84

*Yogurt Samples- T1- control plain yogurt in Homogenised pasteurized milk

T2-yogurt containing 1% Lactobacillus acidophilus, T3-yogurt containing 5%

L.acidophilus, T4- yogurt containing 10 % L.acidophilus,

T5,T6,T7 -Probiotic yogurt containing 1%, 5% and 10% Lactobacillus casei respectively.

4.2. b. Titratable acidity (% Lactic acid) Probiotic yogurt during storage at 40C

Titratable acidity of Probiotic yogurt prepared using Homogenised Pasteurised

milk was shown Table 4.2. There was an increase in acidity by the addition of both

probiotic cultures, but it was within limit (0.9) up to 14 days except T4 (10%

L.acidophilus). At 21 day in all samples including control, acidity goes beyond limit. So,

as the concentration of inoculum increases acidity also increases.

Table 4.2: Titratable acidity of Probiotic yogurt samples Homogenized, pasteurized milk

(T1-T7) during incubation period and storage up to 21 days

*Yogurt

samples

Titratable acidity

4 hour 7(Day) 14(Day) 21(Day)

T1 0.53 0.56 0.68 1.012

T2 0.75 0.79 0.95 1.01

T3 0.76 0.79 0.86 1.12

T4 0.77 0.79 1.23 1.35

T5 0.41 0.45 0.69 1.01

T6 0.50 0.56 0.9 1.03

T7 0.72 0.79 0.97 1.23

33

4.2. c. Bacterial counts of Probiotic yogurt during refrigerated storage at 40C

Probiotic bacterial count decreased significantly during refrigerated storage at 4 0

C (Table 4.3). After 4 hours of incubation maximum cell count was shown by yogurt

inoculated with 1% L.casei, and it also showed greater survival after 21 days of storage.

In all other samples, inoculated with probiotic bacteria, number of cells fall below 10 7

CFU/gm after refrigerated storage. For dietary cultures to be beneficial in food systems,

they are expected to be viable in the food until the time of consumption and present at

levels of at least 107 viable cells per gram or milliliter of a product (Shah and Lankaputhra,

1997; Dave and Shah, 1997).

Table 4.3: Bacterial count of Probiotic yogurt samples (T2-T7) during incubation period

and storage up to 21 days

*Yogurt

Samples Cell count(Log CFU/gm)

4 hour 7(Day) 14(Day) 21(Day)

T1 - - - -

T2 7.47 7.39 7.39 6.69

T3 7.59 7.27 6.98 6.54

T4 7.76 7.33 7.16 6.92

T5 7.82 7.76 7.60 7.40

T6 7.40 7.30 7.29 6.94

T7 7.60 7.40 7.38 6.60

The main factors for loss of viability of probiotic organisms have been attributed

to the decrease in the pH of the medium and accumulation of organic acids as a result of

growth and fermentation (Shah, 2000).

4.2. d. Syneresis of Probiotic yogurt during storage at 40C

In the first week of storage, syneresis of control sample had lower level as

compared to the other samples while syneresis percentage then increased significantly.

34

Least syneresis was shown by milk inoculated with 10 % L.acidophilus and L.casei after

4 hours of fermentation, but after 21 days they showed maximum syneresis (Table 4.4).

Table 4.4: Syneresis of Probiotic yogurt samples (T1-T7) during incubation period and

storage up to 21 days.

*Yogurt

Samples Syneresis (%)

4 hour 7(Day) 14(Day) 21(Day)

T1 7.5 7.5 7.5 10

T2 8.75 9.25 10 10

T3 7.75 8.25 9 10

T4 5 7.5 11.3 12.5

T5 7.5 7.5 9.75 10.75

T6 8 9.75 10.25 12.5

T7 5 7.5 8 10.25

As the inoculum concentration of probiotic bacteria increased there was a decrease

in pH and probiotic bacterial count below therapeutic limit in probiotic yogurt during 28

days of storage at 40C. Therefore, alternative methods like production of synbiotic yogurt

was adopted.

4.3. Synbiotic yogurt Production

Synbiotic yogurt was prepared by the incorporation of Inulin and Lactulose (1%)

separately into the probiotic yogurt containing Lactobacillus acidophilus and

Lactobacillus casei. When the pH of the sample reached in between 4.5-4.7, samples were

stored at 4°C for 28 days and during this time pH, acidity, syneresis and probiotic counts

were investigated and compared to the probiotic yoghurt (with no prebiotics).

4.3. a. pH of Synbiotic yogurt during storage at 40C

Changes in pH of Synbiotic yogurt containing Lactobacillus acidophilus and

Lactobacillus casei during refrigerated storage at 40C for 28 days were shown in figure

4.1. Due to lactic and acetic acid production by Lactobacillus strains, pH of all samples

35

were found to be decreased. For Lactobacillus acidophilus and L.casei, a significant

decrease in pH (p<0.05) was observed by inulin and Lactulose as prebiotic than with

control. However there was no significant difference between inulin and Lactulose

treatment during 28 days of storage for both the Probiotics.

Figure 4.1: pH of synbiotic yogurt during storage at 40C for 28 days - Lactobacillus

acidophilus and Lactobacillus casei.

4.3. b. Acidity of Synbiotic yogurt during storage at 40C

Acidity of Lactobacillus strains grown in synbiotic yogurt containing prebiotics

during 4 weeks of storage at 40C was shown in figure 4.2.As a result of the lactose

fermentation, the titratable acidity increased. This is slightly higher for the samples with

3.8

3.9

4

4.1

4.2

4.3

4.4

4.5

4.6

Day(0) Day 7 Day 14 Day 21 Day 28

pH

Days of storage

Lactobacillus acidophilus

Control

L.a +inulin

L.a +Lactulose

3.8

3.9

4

4.1

4.2

4.3

4.4

4.5

4.6

Day(0) Day 7 Day 14 Day 21 Day 28

pH

Days of storage

Lactobacillus casei

Control

L.c +inulin

L.c +Lactulose

36

prebiotic than that of control samples. For synbiotic yogurt containing L.acidophilus

increase in acidity with Inulin and lactulose was not significantly (p>0.05) differ from

control sample, but for Synbiotic yogurt containing L.casei both Inulin and Lactulose

showed significant (p<0.05) increase in acidity than control sample.

Figure 4.2: Acidity of Lactobacillus strains grown in synbiotic yogurt during 4 weeks of

storage at 40C

4.3. c. Viability of Synbiotic yogurt during refrigerated storage at 40C

During 28 days of refrigerated storage, count of L.acidophilus treated with

prebiotics (inulin and lactulose) was found to be increased in presence of prebiotics up to

7 days (figure 4.3 (a)) and thereafter decreased. For synbiotic yogurt containing

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

Day(0) Day 7 Day14 Day 21 Day 28

Aci

dit

y

Days of storage

Control

L.a +inulin

L.a +Lactulose

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

Day(0) Day 7 Day 14 Day 21 Day 28

Aci

dit

y

Days of storage

Lactobacillus casei

Control

L.c +inulin

L.c +Lactulose

37

L.acidophilus, greatest survival rate (table 4.5) was shown by sample treated with

Lactulose (71.4 %), but it was not significantly (p>0.05) differ from that with inulin (70.6

%) and the control. Dave et al. (1997) reported that pH was found to be the most crucial

for L.acidophilus culture, yogurt with pH <4.4 at the time of fermentation results in 3-4

log cycle reduction in L.acidophilus numbers within 20-25 days.

Figure 4.3: Viability of Lactobacillus strains grown in synbiotic yogurt during 4 weeks of

storage at 40C.

Yogurt containing Lactulose showed an initial increase of L.casei upto 7 days

(figure 4.3 (b) and thereafter decreased. However there was a significant difference in

bacterial count treated with prebiotic than control, with inulin showed maximum survival

5

6

7

8

9

10

11

Day(0) Day 7 Day14 Day 21 Day 28

Log

CFU

/ g

m

days of storage

L.acidophilus

Control

L.a +inulin

L.a +Lactulose

5

6

7

8

9

10

Day(0) Day 7 Day 14 Day 21 Day 28

Log

CFU

/ g

m

days of storage

L. casei

Control

L.c +inulin

L.c +Lactulose

38

rate of 81.3 %(Table 4.5).There was no significant difference in bacterial count between

in inulin than in Lactulose (80.9 %).The number of both probiotic bacteria found to be

decreased below 7.00 log 10 CFU/gm in 28 days of storage.

Table 4.5: Percentage viability of Lactobacillus strains grown in synbiotic yogurt during

4 weeks of storage at 40C.

4.3.d. Syneresis of Synbiotic yogurt during refrigerated storage at 40C

Syneresis of Lactobacillus strains grown in synbiotic yogurt containing prebiotics

during 4 weeks of storage at 40C was shown in figure 4.4. For L.acidophilus least syneresis

was shown by sample with inulin, but it was not significantly (p>0.05) differ from that of

control and sample with Lactulose, but synbiotic yogurt containing L.casei addition of

inulin had a significant (p<0.05) effect on decrease in syneresis compared to control and

lactulose.

Prebiotics

Strains

Reading

interval

Log CFU /gm of probiotic bacteria

Control Inulin Lactulose

Lactobacillus

acidophilus

Day 0 8.59 8.61 8.64

Day 28 5.81 6.08 6.17

Viability % 67.6 70.6 71.4

Lactobacillus casei

Day 0 8.6 8.58 8.6

Day 28 5.8 6.98 6.96

Viability % 67.4 81.3 80.9

39

Figure 4.4: Syneresis of Lactobacillus strains grown in synbiotic yogurt during 4 weeks

of storage at 40C.

The results showed that neither Inulin nor Lactulose had significant effect on

physicochemical properties (except pH) and probiotic bacterial survival of L.acidophilus.

Both Inulin and Lactulose had a significant effect on physicochemical changes and

probiotic bacterial survival of L.casei.

0

10

20

30

40

50

Day(0) Day 7 Day14 Day 21 Day 28

syn

eres

is %

days of storage

L.acidophilus

Control

L.a +inulin

L.a +Lactulose

0

5

10

15

20

25

30

35

40

45

Day(0) Day 7 Day 14 Day 21 Day 28

syn

eres

is %

days of storage

L. casei

Control

L.c +inulin

L.c +Lactulose

40

4.4. Microencapsulation of Synbiotics

L.acidophilus and L.casei along with prebiotic (Inulin and lactulose) have been

microencapsulated and exposed to simulated gastrointestinal fluid. Microencapsulated

synbiotics were also used for the production of yogurt.

4.4. a. In vitro release studies of L.acidophilus and L.casei from the microcapsules

Free cells and Microcapsule samples were treated with Simulated Gastric Juice

(SJG) and then with Simulated Intestinal Fluid (SIF) to check the continuous release

characteristics of L.acidophilus and L.casei in Gastro Intestinal Tract (GIT) and the results

are shown in Figure 4.5. In SJG (pH 1.5), the release amounts of cells were minor from

each sample of the microcapsule. Once the samples were transferred from SJG to SIF, the

larger amounts and faster release rate of L.acidophilus and L.casei cells were found,

indicated by an increase in absorbance. For L.acidophilus and L.casei faster release of cells

found to occur when they are encapsulated in alginate- gelatin with inulin as prebiotic

followed by Lactulose. For both probiotics, alginate- gelatin along with prebiotic were

found to gave better protection in gastric juice and better release in intestinal fluid. Further

free cells were found to be very susceptible to SIF.

41

Figure 4.5: Absorbance of free and microencapsulated (A) Lactobacillus acidophilus and

(B) Lactobacillus casei in simulated gastro intestinal juice.

4.4. b. Survival of free and microencapsulated probiotics in simulated gastric juice

The viability of free and encapsulated probiotic bacteria during incubation in the

simulated gastro-intestinal condition was shown in Figures 4.6 .Survival of probiotics was

lower in gastric juice and decreased further as the incubation period increased. Exposure

to simulated gastric juice for 120 minutes resulted in a considerable decrease in the total

number of free Lactobacillus acidophilus and L.casei (only 25.6 % & 17.9 % viability

respectively). However, the cell number of microencapsulated L.acidophilus and L.casei

decreased slightly after 120 minutes. Alginate – gelatin encapsulated L. acidophilus was

observed to exhibit the highest viability (94.11%) when inulin was incorporated as

prebiotic while greatest viability of 84.94 % was observed for alginate encapsulated

L.casei without any prebiotic. In the case of both L. acidophilus and L.casei, the survival

of cells in both alginate and alginate-gelatin were found to be higher when compared with

free cells. Chávarri in 2010 reported that encapsulation in chitosan-coated alginate

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

30

M

1

H

2

H

3

H

4

H

5H6H

O.D

at

60

0 n

m

Time (hours)

(A)

Free cells

L.acidophilus

L.a+lact.+ Alg.

L.a +inl+Alg

L.a+alg

L.a +Alg.gel

L.a +Alg.gel+ lact.

L.a +Alg.gel+inl.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

30M

1H

2H

3H

4H

5H 6H

O.D

at

60

0 n

mTime (hours)

(B)Free cellsL.acidophilus

L.c+lact.+ Alg.

L.c +inl+Alg

L.c+alg

L.c +Alg.gel

L.c +Alg.gel+ lact.

L.c +Alg.gel+inl.

42

microcapsule significantly improved the survival of Lactobacillus gasseri and

Bifidobacterium bifidum in simulated gastric juice along with pepsin. Many scientists have

also reported that the survival rate of Bifidobacteria in alginate microcapsules was higher

than that of free cells (Hansen et al 2002; Yu et al 2001; Woo et al 1999). Many studies

have shown coating the alginate matrix could increase the survival of bacteria due to

curbing the diffusion of calcium ions outside of capsules (Chávarri 2010; Mokarram 2009;

Krasaekoopt et al 2004). Mokarram et al in 2009 showed that L.acidophilus and

L.rhamnosus exposed to simulated gastric juice without pepsin had higher viability when

encapsulated in calcium alginate with double coating sodium alginate. They indicated that

the reduction of pore size and distribution of gastric juice in double layer membrane lead

to limitation of interaction between cells with the gastric juice. According to our study,

microcapsules both alginate and gelatin coated along with prebiotic (lactulose or inulin)

provided the best protection in simulated gastric juice. Furthermore, the increase in viable

counts of bacteria could be attributed to the addition of prebiotic. Alginate and prebiotics

such as inulin or oligosaccharides tend to be synergistic in gelling and as a result it may

help to maintain and improve the degree of protection to bacterial cells (Capela 2006).

43

Figure 4.6: Survival of free and microencapsulated (A) Lactobacillus acidophilus and

(B) Lactobacillus casei in simulated gastric juice.

4.4. c. Survival of free and microencapsulated bacteria in simulated intestinal juice

The effect of the simulated intestinal juice on the viability of the microencapsulated

and free probiotic bacteria after treatment with simulated gastric juice is presented in

Figure 4.7. The number of free probiotics was found to be decreased significantly as the

incubation time increased. The result indicated that alginate-gelatin microcapsules with

lactulose as prebiotic was most effective in survival of L.acidophilus in simulated

intestinal juice with an encapsulation efficiency of 97.38 % followed by inulin

encapsulated alginate beads(97.18%). This substantiate the possibility that prebiotics have

some effect on encapsulation efficiency. Similarly when L.casei after exposure to SGJ for

2 hours when introduced to SIJ, number of free cells found to be decreased drastically with

a viability of only 3.6 %. While, alginate encapsulated with lactulose showed maximum

survival (85.9 %).This is in good agreement with the results of Krasaekoopt et al in 2004

who indicated that the survival of probiotic bacteria was highly enhanced in gastro-

0

1

2

3

4

5

6

7

8

9

10

0 H 1 H 2 H

surv

iab

iliy

(lo

g C

FU/g

m)

Time(hours)

(A)

Free cells L.acidophilus

lact.+ Alg.

inl+Alg

L.A+alg

LA +Alg.gel

LA +Alg.gel+ lact.

LA +Alg.gel+inl.

0

1

2

3

4

5

6

7

8

9

10

0 H 1 H 2 H

surv

iab

iliy

(lo

g C

FU/g

m)

Time(hours)

(B)

Free cells L.casei

LC+lact.+ Alg.

LC+inl+Alg

L.c+alg

Lc +Alg.gel

Lc +Alg.gel+ lact.

Lc +Alg.gel+inl.

44

intestinal conditions when encapsulated with alginate-chitosan or poly-L-lysine. For

L.acidophilus, additional coating of sodium alginate with gelatin found to provide better

survival in simulated gastrointestinal condition, but for L.casei, gelatin was found to confer

no additional protection than alginate.

Figure 4.7: Survival of free and microencapsulated (A) Lactobacillus acidophilus and

(B) Lactobacillus casei in simulated intestinal juice.

4.5. Co-encapsulated synbiotic yogurt

Synbiotic yogurt were prepared by inoculating free, and co-encapsulated Probiotic

cultures (Lactobacillus acidophilus and Lactobacillus casei) with prebiotics (inulin and

lactulose). The acidification profile was recorded hourly until a pH of 4.7-4.5 was reached.

Fermentation was stopped by sudden cooling of the yogurt and stored at 40 C for 28 days.

Physicochemical properties (pH, titratable acidity, syneresis) and viability of probiotic

bacteria in both sample were evaluated at every 7 day interval for a period of 28-days.

0

1

2

3

4

5

6

7

8

9

3 H 4 H 5H 6H

surv

iab

iliy

(lo

g C

FU/g

m)

Time(hours)

(A)Free cellsL.acidophilus

lact.+ Alg.

inl+Alg

L.A+alg

LA +Alg.gel

LA +Alg.gel+lact.

LA +Alg.gel+inl.

0

1

2

3

4

5

6

7

8

3 H 4 H 5H 6H

surv

iab

iliy

(lo

g C

FU/g

m)

Time(hours)

(B)Free cellsL.casei

LC+lact.+ Alg.

LC+inl+Alg

L.c+alg

Lc +Alg.gel

Lc +Alg.gel+lact.

Lc+Alg.gel+inl.

45

4.5. a. pH changes during storage

The pH changes of co-encapsulated synbiotic yogurt (Lactobacillus acidophilus

and Lactobacillus casei) during storage at 4°C at every 7 day intervals for a period of 28

days are shown in Figure 4.8.

Figure 4.8: The pH changes of co-encapsulated symbiotic yogurt (A) Lactobacillus

acidophilus and (B) Lactobacillus casei during storage at 4°C for a period of 28 days.

The pH changes of co-encapsulated synbiotic yogurt during storage was found to

be lesser than the yogurt inoculated with free probiotic bacteria. The synbiotic yogurt

3.8

3.9

4

4.1

4.2

4.3

4.4

4.5

4.6

4.7

Day(0) Day 7 Day 14 Day 21 Day 28

pH

Days of storage

L. acidophilus

Control

inulin+alg

inulin+alg+gel

lactulose+alg

lactulose+alg+gel

3.8

4

4.2

4.4

4.6

4.8

Day(0) Day 7 Day 14 Day 21 Day 28

pH

Days of storage

L. casei

Control

inulin+alg

inulin+alg+gel

lactulose+alg

lactulose+alg+gel

46

containing encapsulated cells with alginate – gelatin in both of L.casei and L.acidophilus

samples showed the minimum of pH changes during storage times. Decrease in pH of

synbiotic yogurt samples containing free states of L.acidophilus was about 0.6 units and

in samples containing encapsulated bacterial cells with inulin in alginate and in alginate-

gelatin were about 0.25 and 0.23 unit respectively. But for lactulose the difference were

0.33 and 0.27 unit respectively. For L.acidophilus least change in pH was shown by inulin

encapsulated in alginate- gelatin mixture than control and alginate beads. Free cells of

L.casei inoculated in synbiotic yogurt samples was found to decreased the pH of about

0.58 units and bacterial cells with inulin encapsulated with alginate and alginate – gelatin

were decreased the pH of their environments about 0.39 and 0.4 units respectively and

with lactulose these difference were 0.29 and 0.28 units respectively. Lactulose

encapsulated in gelatin alginate was found to have significant effect on producing least

change in pH. There was significant difference (P<0.05) in pH during storage between

alginate and alginate gelatin encapsulated forms with both prebiotic for L.acidophilus and

L.casei.

4.5. b. Producing acidity during storage

Measuring the acidity at every 7- day intervals observed similar result as that of

pH for a period of 28 days of storage at 4°C. Accordingly an increase in acidity in samples

containing free L.acidophilus was approximately 0.53 %and an in increase in acidity in

samples containing encapsulated cells in alginate and alginate - gelatin with inulin were

found to be 0.34 % and 0.2 % respectively and with lactulose it was 0.31% and 0.3 %

respectively. All the treatment differ significantly from that of control with inulin in

alginate-gelatin showed least difference. For L.casei all the treatment differ significantly

from control but the microencapsulation with lactulose showed a significant difference

between alginate and alginate gelatin.

47

Figure 4.9: Percentage of acidity in microencapsulated synbiotic yogurt - Lactobacillus

acidophilus and Lactobacillus casei - storage at 4°C for a period of 28 days.

As a result, increase in acidity during storage until 28 days in sample containing

encapsulated probiotic cells were less than that of free states for both L.acidophilus and

L.casei. Therefore, microencapsulation of free cells, restricted their metabolic activity

greatly, but not completely [ISIRI number 5222 (2000)].

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

Day(0) Day 7 Day14 Day 21 Day 28

Titr

atab

le a

cid

ity(

% L

acti

c ac

id)

Days of storage

L. acidophilus

Control

inulin+alg

inulin+alg+gel

lactulose+alg

lactulose+alg+gel

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

Day(0) Day 7 Day 14 Day 21 Day 28Titr

atab

le a

cid

ity(

% L

acti

c ac

id)

Days of storage

L. casei Control

inulin+alg

inulin+alg+gel

lactulose+alg

lactulose+alg+gel

48

4.5. c. Syneresis on storage

Syneresis of microencapsulated synbiotic yogurt (Lactobacillus acidophilus and

Lactobacillus casei) during storage at 4°C at every 7-day interval for a period of 28 days

was shown in Figure 4.10. For L.acidophilus, syneresis on microencapsulation with inulin

in alginate gelatin beads showed least difference after 28 days of storage (2.7 %) compared

to inulin - alginate (3.2%) and that of control (10.6%). Microencapsulated yogurt with

lactulose in alginate and alginate - gelatin showed a difference in syneresis of 5% and 4%

respectively. So inulin in alginate –gelatin mixture showed a least difference in syneresis

(p<0.05) than other samples. For L.casei syneresis of microencapsulation with inulin in

alginate gelatin beads showed least difference during 28 days of storage (3.5 %) compared

to inulin -alginate (3.8%) and that of control (10.6%). Microencapsulated yogurt with

lactulose in alginate – gelatin and alginate showed a difference in syneresis of 3% and

3.3% respectively. So lactulose in alginate – gelatin mixture showed a least difference in

syneresis (p<0.05) than other samples.

49

Figure 4.10: Synersis of co-encapsulated synbiotic yogurt - Lactobacillus acidophilus and

Lactobacillus casei - during storage at 4°C for a period of 28 days

15

20

25

30

35

40

45

Day(0) Day 7 Day14 Day 21 Day 28

syn

eres

is %

Days of storage

L.acidophilus

Control

inulin+alg

inulin+alg+gel

lactulose+alg

lactulose+alg+gel

15

20

25

30

35

40

45

Day(0) Day 7 Day 14 Day 21 Day 28

syn

eres

is %

days of storage

L.casei

Control

inulin+alg

inulin+alg+gel

lactulose+alg

lactulose+alg+gel

50

4.5. d. Viability of Lactobacillus strains grown in microencapsulated synbiotic

yogurt

4.5. d.I Survival of free and microencapsulated synbiotic L.acidophilus in Yogurt

during storage time

The effect of encapsulation with Gelatin and alginate mixtures (2%) and that of

alginate on the viability of probiotic microorganisms in the Yogurt during 28 days of

refrigerated storage period (4°C) was investigated. Logarithmic numbers of surviving

bacteria (log cfu/gm) were measured at every seven-day intervals were shown in the

Table 4.6.

Table 4.6: Logarithmic numbers of surviving bacteria (log cfu/gm) at seven-day intervals

for 28 days.

Viable count (log cfu/gm) of L.acidophilus

Day(0) Day 7 Day14 Day 21 Day 28

Control 8.59 7.3 6.98 6.61 5.81

inulin+alginate 8.37 9.28 8.12 7.67 6.84

inulin+alginate+gelatin 8.44 9.34 8.28 8.12 7.2

lactulose+alginate 8.28 8.93 8.65 7.28 6.6

lactulose+alginate+gelatin 8.4 9.34 8.98 7.92 6.81

There was a decline of about 2.78 logs over a period of 4 weeks in the cell numbers

of strains of L.acidophilus when present as free cultures, whereas there was only a 1.53

and 1.24 -log cycle decrease in cell numbers with inulin co-encapsulated in alginate and

alginate – gelatin mixture respectively. When the same organism were encapsulated with

Lactulose (1%) and alginate (2% concentration) showed a decrease of 1.68 logs, while the

encapsulated state with Lactulose and alginate – gelatin showed a decrease of 1.59 logs.

51

Therefore decrease in viable count of L.acidophilus significantly differ (p<0.05) from that

of control sample in both treatment with inulin and lactulose as prebiotic with inulin and

alginate - gelatin showed least reduction in viable cells. Mean viable count of

L.acidophilus was decreased below 7.00 log CFU/gm after treatment except inulin

encapsulated in alginate –gelatin mixture (7.2 log CFU/gm).Therefore this treatment can

be used to confer therapeutic benefit after refrigerated storage.

4.5. d.II. Survival of free and microencapsulated symbiotic L.casei in Yogurt during

storage

Viable cells of L.casei in yogurt during 28 days of storage was shown in table 4.7.

In the case of free L.casei, the cell numbers lowered substantially about 2.8 log numbers

during 28 days of storage at 4°C, however the decrease of the viable counts of

encapsulated bacteria in alginate and alginate-gelatin with inulin were 1.35 log and 1.34

log respectively and with lactulose 1.02 logs only, for both types of beads. There was a

significant difference (P<0.05) observed between mean counts of viable cells during

storage time (28 days) in free states of L.casei and both of encapsulated samples. However

there was no significant difference observed in mean viable cells during storage time

between gelatin alginate and alginate encapsulated forms. However mean viable count of

L.casei was maintained above 107 CFU /gm in all treatment to provide therapeutic benefit

in which least reduction was shown by encapsulation with lactulose. These results

indicated increase in viability and resistance of probiotic cells by encapsulation against

harsh environmental conditions at storage times.

52

Table 4.7: Logarithmic number of surviving L.casei on storage at 7 day interval for

28 days.

Viable count (log CFU/gm) of L.casei.

Day(0) Day 7 Day 14 Day 21 Day 28

Control 8.6 7.57 6.65 6.31 5.8

inulin+alginate 8.8 8.65 8.61 8.55 7.45

inulin+alginate+gelatin 8.85 8.73 8.67 8.58 7.51

lactulose+alginate 8.7 8.71 8.67 8.31 7.68

lactulose+alginate+gelatin 8.67 8.64 8.61 8.0 7.65

The presence of lactic acid combined with the low pH of yogurt might be

responsible for the low viability of free probiotic cultures in yogurt. There was a significant

increase (P < 0.05) in the viable counts of L .acidophilus in inulin coated co-encapsulated

alginate gelatin beads compared with the alginate encapsulated and free cells. Survival of

probiotics in alginate-starch beads (in the size range of 1.0 mm) was improved during

refrigerated storage in yogurt (Sultana and others 2000). It was shown that the positive

role of incorporation of prebiotic (inulin) into the alginate mix during encapsulation.

Likewise for L.casei there was a significant difference (P<0.05) between mean counts of

viable cells of both of encapsulated samples when Lactulose was used as prebiotic, but

showed no significant difference between alginate and alginate – gelatin mixture.

53

Figure 4.11: Survival of free, and co-encapsulated bacteria (L. acidophilus and L. casei)

in yogurt over a shelf life period of 4 weeks.

Sultana et al., [2000] showed that using the Hi maize in encapsulation of probiotics

can significantly improved protection of viable bacterial counts in synbiotic ice cream and

Iranian white brined cheese. Increasing cell stability of encapsulated probiotic bacteria

with calcium alginate over the refrigerated storage time has been previously reported by

Krasaekoopt, Kailasapathy and Mortazavianetal 2008, that are in agreement with the

5

5.5

6

6.5

7

7.5

8

8.5

9

9.5

10

Day(0) Day 7 Day14 Day 21 Day 28

Log

(C

FU/g

m)

Days of storage

L.acidophilus Control

inulin+alg

inulin+alg+gel

lactulose+alg

lactulose+alg+gel

5

5.5

6

6.5

7

7.5

8

8.5

9

9.5

10

Day(0) Day 7 Day 14 Day 21 Day 28

Log

(CFU

/gm

)

Days of storage

L.casei

Control

inulin+alg

inulin+alg+gel

lactulose+alg

lactulose+alg+gel

54

results obtained from present research. Also these data again confirmed advantages of

encapsulation of probiotic cells with gelatin in combination with alginate that give more

protection of viable counts of L.acidophilus compared free state caused. In addition, it was

demonstrated that, encapsulated cells required longer time to decrease one log cycle in

viable counts and encapsulated L. acidophilus with alginate gelatin and inulin as prebiotic

had the most slow rate of loss of viable cell rate. Overall the problem of sensitivity to

acidity of the probiotic culture is compounded by the fact that acidity may increase during

storage, a phenomenon known as ‘over acidification’ [Adeli et al, 2010]. Increasing rate

of viability decline of free probiotic bacteria can be attributed to the adverse effects of

accumulated organic acid by L.acidophilus and L.casei, but encapsulated bacteria

accumulating organic acid and hydrogen ions at a lower rate because of limitation

accessibility to nutrients. This fact is indicated by slower rates of pH drop and acidity

increase during refrigeration of both encapsulated types of probiotic bacteria which caused

a slower rate of viability decline compared to free probiotic bacteria cells. But the greater

loss of survival of encapsulated L.acidophilus and L.casei cells at the end of storage period

may be due to the gradual increase in the amount of organic acid molecules inside the

capsules after slow diffusion through the capsules pores. In this regard, reduction of

metabolic activity of probiotic cells in the case of coated cells with gelatin and alginate

caused a very gentle increase of acidity and so slow loss of viable cells during refrigeration

period .With comparing two strains of probiotic cells, it was found that loss of all types of

free and coated bacterial cells of L.casei from initial day to 28th day was lower than

L.acidophilus cells. In this way the survival of bacteria against unfavourable conditions

could be species dependent. This finding is in agreement with those of Haynes, 2002 and

Kailasapathy 2005.

55

According to the results obtained, microencapsulation with alginate gelatin and

inulin found to have the best efficiency to survival of L.acidophilus, while Lactulose

encapsulated in alginate/alginate-gelatin provide best protection of L.casei during

refrigerated storage, enough for therapeutic effects. Different studies have shown that

calcium alginate microcapsules are better protected in the presence of prebiotics (Sultana

et al 2000, Chen et al 2005).Adding encapsulated LAB can slower post acidification and

also reduces acid development in yoghurt during storage. Overall sensory aspects in

yoghurt do not substantially alter when encapsulated bacteria are incorporated, and

survival of probiotics during storage can be improved by microencapsulation technique

(Kailasapathy, 2006). Using calcium alginate for the microencapsulation of probiotic

bacteria in combination with prebiotics (e.g., inulin and Lactulose) in yoghurt, viability

and survivability of encapsulated cells can be improved due to reduced acid production of

LAB during storage (Sultana et al., 2000).

56

Chapter 5

CONCLUSION

The synbiotic functional food approach offers an additional health benefit by

providing probiotics and prebiotics. The growth, activity and viability of Lactobacillus

strains in RSM were dependent on prebiotics as well as strain. Physicochemical and

microbiological properties of probiotic and synbiotic yogurt were evaluated in the first day

and thereafter every 7 days interval for four weeks on refrigerated storage at 40C. During

cold storage, pH, and probiotic bacterial count decreased, while acidity and syneresis were

increased in Probiotic yogurt prepared with both organisms. Neither Inulin nor Lactulose

had significant effect on physicochemical properties (except pH) and probiotic bacterial

survival of L.acidophilus. There was a significant decrease (p<0.05) in pH of synbiotic

yogurt with inulin and lactulose. For L.casei, both prebiotics had significantly reduced pH

and increased acidity, and found to have some effect on viability of probiotic bacteria, but

inulin produced least change in syneresis.

Amongst the various approaches, microencapsulation has emerged as the best

alternative so as to overcome the problem of poor survivability of probiotic cultures in the

food matrix as well as in the gastrointestinal environment. Microencapsulation of

L.acidophilus and L.casei in calcium alginate and calcium alginate-gelatin resulted in

better in vitro release and survival of cells in simulated gastro-intestinal condition (along

with pepsin and bile salt), compared to free cells. Prebiotic addition were found to provide

better survival of both probiotic for gastro-intestinal condition. Therefore the applied

approach in this study might prove beneficial for the delivery of probiotic cultures to the

human gastro-intestinal tract. Of the six types of microcapsules with prebiotic in this

research, microencapsulated lactulose provided the best protection and survival of

57

L.acidophilus and L.casei cells in simulated gastro-intestinal condition. Survival of free

L.acidophilus and L.casei drastically decreased due to its low acid and bile resistance.

The concept of co-encapsulation offers the potential for increased efficacy of

functional foods by exploiting the synergy between prebiotic and probiotic ingredients.

According to the results of this study, microencapsulation of Probiotic bacterial cells with

alginate–gelatin with inulin showed significant (p<0.05) effect on least change in pH,

acidity, bacterial count and syneresis of L.acidophilus, while alginate–gelatin with

lactulose showed significant (p<0.05) effect on least change in physicochemical and

microbiological properties of L.casei. The final results showed that L.casei had a higher

viability than the level of the therapeutic minimum (>107 CFU/g) after 28 days of storage

in all the treatment while for L.acidophilus, only inulin encapsulated in alginate–gelatin

maintained the count above this limit. So co-encapsulation of synbiotics can be used to

enhance and improve the physicochemical properties and viability of probiotic bacteria

during processing and also in gastrointestinal tract.

Future studies need to monitor the efficacy of co-encapsulated bacteria in the gut,

using animal models. Also, the sensory evaluation of yogurt with microencapsulated

prebiotics and probiotic bacteria will reveal the consumer response to the texture and the

changes in organoleptic characteristics of the yogurt.

58

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