A Report of Minor Research Project EFFICACY OF MICROENCAPSULATION ON SYNBIOTIC YOGURT PRODUCTION

Transcription

1 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, Submitted to University Grants Commission New Delhi May 2017

2 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

3 CONTENTS Abstract 1 1. Introduction Probiotics and Prebiotics Synbiotics Viability of Probiotic organisms Microencapsulation of Probiotics Scope of present investigation Objectives of the present investigation 9 2. Review of Literature Yogurt Definition of probiotics Characteristics of lactobacillus Lactobacillus acidophilus Lactobacillus casei Regulations and safety of probiotics in manufacture Prebiotics Inulin Lactulose Factors affecting the growth and survival of Lactobacillus Effect of prebiotics on probiotic growth and survival Microencapsulation Technique 19

4 3. Materials and Methods Materials Methodology Preparation of cell suspension Production of Probiotic yogurt a. Determination of ph b. Determination of titratable acidity c. Enumeration of L. acidophilus and L.casei in yogurt d. Determination of Syneresis Production of Synbiotic yogurt a. Physicochemical and Microbial analysis of Synbiotic yogurt Microencapsulation of Probiotic Cultures Preparation of alginate microcapsules Preparation of simulated gastric and intestinal juices and inoculation of cells In vitro release studies (GIT) Release of entrapped bacteria Co-encapsulated synbiotic yogurt Production Results and Discussion Preparation of cell suspension Production of Probiotic yogurt a. ph of Probiotic yogurt b. Acidity (% Lactic acid) Probiotic yogurt c. Bacterial counts of Probiotic yogurt d. Syneresis of Probiotic yogurt 33

5 4.3 Synbiotic yogurt Production a. ph of Synbiotic yogurt during storage b. Acidity of Synbiotic yogurt during storage c. Viability of Synbiotic yogurt during storage d. syneresis of Synbiotic yogurt during storage Microencapsulation of Probiotic yogurt a. In vitro release studies of L.acidophilus and L. casei from the microcapsules b. Survival of free and microencapsulated probiotics in simulated gastric juice c Survival of free and microencapsulated probiotics in simulated intestinal juice Co-encapsulated synbiotic yogurt a. ph changes during storage b. Producing acidity during storage c. Syneresis on storage d. Viability of Lactobacillus strains grown in synbiotic yogurt d.I. Survival of free and microencapsulated synbiotic L. acidophilus in Yogurt during storage d.II. Survival of free and microencapsulated synbiotic L. casei in Yogurt during storage Conclusion Bibliography 58

6 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 4 0 C. 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 % 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 (>10 7 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 coencapsulation of synbiotics can be used to enhance and improve the physicochemical properties and viability of probiotic bacteria during processing and also in gastrointestinal tract. 1

7 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 2

8 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 10 6 cfu/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 3

9 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 4

10 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). 5

11 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 4 0 C. 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 6

12 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 7

13 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 8

14 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. 9

15 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 10

16 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) 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. 11

17 2.3. Characteristics of Lactobacillus Lactobacilli are characterized as Gram-positive facultative bacteria, they are nonspore 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 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 C but it can tolerate temperatures as high as 45 C. The optimum ph for growth is between 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 12

18 dietary supplementation with L.acidophilus decrease the number of colon cancer cells in a does dependant manner (Rao et al., 1999) 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 10 6 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 13

19 cultures. The seal is a voluntary identification available to all manufacturers of refrigerated yogurt products contain at least 10 8 cfu per gram at the time of manufacture, and at least 10 7 cfu 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 10 6 CFU/g and ph of 4.5 at the end of manufactured of yogurt 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. 14

20 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 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 15

21 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 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. 16

22 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 10 6 cfu 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 17

23 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 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. 18

24 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 spraydrying, 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 oxygenimpermeable 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 19

25 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, 20

26 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 (10 7 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 21

27 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]. 22

28 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) 23

29 3.2 Methodology 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 CFU/gm.The cultures were then washed twice by sterile PBS and resuspended in pasteurised (63 0 C for 30 min) 10 % reconstituted skim milk (RSM) 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 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. 24

30 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 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 4 0 C 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). 25

31 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 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 a. Physicochemical and Microbial analysis of Synbiotic yogurt ph value of samples was measured using Digital ph-meter (Scientific Tech) at 25 0 C. 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 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 26

32 methods basically reported by Donthidi et al. in 2010 and Sultana et al. in 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 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 10 8 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). 27

33 Figure 3.2: Microencapsulated probiotic in (a) alginate gelatin and (b) alginate beads 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 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 37 0 C and followed by inoculation in sterile simulated intestinal juice (SIF) and incubated at 37 C for 4 hours. 28