Production of concentrated yoghurt culture using whey-based medium

CONTENTS

1. INTRODUCTION

2. REVIEW OF LITERATURE
2.1 The History of Yoghurt
2.2 Symbiotic Growth of Yoghurt Starter Bacteria
2.3 Role of Starters in Yoghurt Production
2.3.1 Lactic Acid Production
2.3.2 Proteolytic Activity
2.3.3 Aroma Production
2.3.4 Exopolysaccharide Production
2.3.5 Production of Inhibitory Compounds
2.4 Product description
2.4.1 Production of yoghurt
2.4.1.1 Raw materials
2.4.1.2 Equipments
2.4.1.3 Yoghurt preparation protocol
2.4.1.4 Traditional method of yoghurt making
2.4.1.5 Industrial method of yoghurt making
2.5 Varieties in yoghurt presentation
2.6 Therapeutic benefits of yoghurts
2.7 Factor that alter the quality of yoghurt
2.8 Preparation of direct vat starter cultures
2.8.1 Introduction
2.8.2 Direct Vat Cultures
2.8.3 Preparation of Starter Concentrates
2.8.4 Diffusion Culture
2.8.5 Batch culture
2.8.6 Preparation of frozen concentrated cultures by Batch culture
2.9 Whey as growth medium for bacteria
2.10 Freeze drying
2.10.1 The origin of freeze drying
2.10.2 Description of the Operation
2.10.3 Freeze Drying of Starter Cultures
2.10.4 Advantages of Freeze Drying Starter Cultures
2.10.5 Applications of Freeze Dried Starter Cultures

3 MATERIAL AND METHODS
3.1 Bacterial cultures
3.2 Maintenance, preservation and propagation of culture
3.2.1 Microscopic examination
3.2.2 Gram staining
3.2.3 Simple staining
3.2.4 Catalase test
3.3 Measurement of growth
3.3.1 Viable count (Pour plating
3.3.2 Growth performance in whey based medium
3.3.2.1 Direct microscopic count
3.4 Freeze drying
3.4.1 Preparation of ampoules
3.4.2 Suspending medium
3.4.3 Preparation of cultures
3.4.4 Filling of ampoules
3.4.5 Primary drying
3.4.6 Secondary drying
3.5 Preparation of concentrated yoghurt cultures for direct application
3.6 Preparation of yoghurt using preserved concentrated cultures
3.7 Analysis of yoghurt
3.7.1 Measurement of pH
3.7.2 Determination of titratable acidity (AOAC, 2007)
3.7.3 Coliforms count
3.7.4 Yeast and molds count

4
RESULTS AND DISCUSSION
4.1 Microscopic examination
4.2 Growth performance of yoghurt culture in whey media
4.2.1 Viable count
4.2.2 Direct microscopic count
4.3 Preservation of yoghurt cultures in freeze dried ampoules
4.4 Production of yoghurt culture biomass and preservation as frozen concentrated and freeze dried forms
4.5 Evaluation of preserved yoghurt cultures in preparation of yoghurt

5 SUMMARY AND CONCLUSIONS

6 REFERENCES

7 APPENDIX

ACKNOWLEDGEMENT

Area of research in life science needs a lot of patience and perseverance and I take pride in the fact that I am working in this field. This project report is the result of work whereby, I have been guided and supported by many people. It is a pleasant aspect that I now have the opportunity to express my gratitude for all of them.

At the very outset, I consider it my pleasant obligation to express my profound gratitude and extreme regard to the almighty divinity and my parents who made me so much capable to reach this height supporting me everywhere when I failed.

I am highly indebted to Dr. Surajit Mandal, Scientist, DM Division , whose gracious initiative, sublime suggestions, excellent scientific guidance, constant manuscript. It is proud privilege for me to express my profound regards and deep sense of gratitude to the entire staff of Dairy Microbiology Division for their cooperation during my work for their valuable advice, pertinent suggestions, keen supervision and ever willing help which definitely helped me in appropriate designing of experiments during my research project. My sincere gratitude is for Dr. R. P. Singh and Vinod Ji, Yogita mam who were always with me for any kind of assistance that made this endeavor possible to complete on right time.

My deepest thanks to Dr. Indrani Jadhav, Advisor, Jaipur National University, for guiding and correcting various documents of mine with attention and care. she has taken pain to go through the project and make necessary correction as and when needed.

I take immense pleasure in thanking Mr. Sandeep Bakshi, Chancellor, Prof. K.L. Sharma, Vice-Chancellor, and Prof. H.N. Verma, Pro-Vice-Chancellor for permitting me to carry out this project work and wish to convey my deep sense of gratitude to them.

I am highly indebted to Prof. C. P. Malik (Advisor Academic affairs JNU), Dr. Divya Shrivastava (Joint Director) and my faculty members without whom this project would have been a distant reality.

I appreciate my veneration to Dr. Khatri (Director), Dr. M. K. Upadhyay (Asist. Director), Dr. Indrani Jadhav (Associate Professor), School of life Sciences, Jaipur National University and my all other lecturers to make me avail buoyancy and explore hidden potential within myself.

I express my heartfelt appreciation and thanks to for their immense help, magnanimous cooperation and for endowing an excellent lab environment. I wish to express my affection for my pals Rinku Dhanker, Monika Rathi, Neha Gupta, Monika Vaishanv

My biggest strength and source of constant encouragement has been my Family and my friends. Their love, affection and blessing are greatest gift that i have received in my life.

LIST OF ABBREVIATIONS

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LIST OF TABLES

2.1 Nutritive value of whey 18

3.1 Bacterial cultures 25

4.1 Growth of culture in whey based medium 34

4.2 Evaluation of direct microscopic count 34

4.3 Growth performance of preserved yoghurt culture in freeze dried ampoules 35

4.4 Sensory characteristics of yoghurt prepared with fresh and preserved cultures during storage 36

4.5 Physicochemical characteristics of yoghurt prepared with fresh and preserved cultures during storage 36

LIST OF FIGURES

2.1 Growth curves of starter bacteria for pure cultures and mixed culture 5

2.2 Stimulatory effects of two microorganisms 6

4.1 Morphology of Streptococcus thermophillus (NCDC 74) 32

4.2 Morphology of Lactobacillus delbrueckii spp. bulgaricus (NCDC009 33

4.3 Morphology of yoghurt culture 33

1. INTRODUCTION

Interest in the role of probiotics for human health goes back at least as far as 1908 when Metchnikoff suggested that man should consume milk fermented with lactobacilli to prolong life (Hughes a Hoover, 1991; O’Sullivan, Thornton, Sullivan, a Collins, 1992).Many functional characteristics of lactic acid bacteria (LAB) are responsible for their historical and modern use in food production. Several health benefits have been claimed to be associated with the consumption of fermented milk products (Le et al., 1986; Van. Veer et al., 1989; Modler, 1990; Hughes and Hoover, 1991; Kanbe, 1992; Mital and Garg, 1992; Nakazava and Hosono, 1992; Yamamoto et al., 1994). Although yoghurt microflora (Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus) have been found to be beneficial for human health and nutrition (Deeth and Tamime, 1981; IDF, 1984).Yoghurt is a fermented dairy product obtained from the lactic acid fermentation of milk. It is one of the most popular fermented milk products in the world and produced commercially at home (Willey et al., 2008). In its commercial production, non fat or low fat milk is pasteurized cooled to 43°C and are inoculated with known cultures of microorganisms referred to as starter cultures. The starter cultures may be a pure culture of a particular species of Lactobacillus or a mixed culture of Streptococcus thermophilus and Lactobacillus bulgaricus in a 1: 1 ratio. The coccus which is the Streptococcus thermophilus grows faster than the Rod which is the Lactobacillus bulgaricus and is primarily responsible for acid production while the rod adds flavor and aroma. The growth of these Microorganisms causes the transformation of milk's sugar, lactose into lactic acid. This process gives yoghurt its texture. The associative growth of the two organisms results in acid production at a rate greater than that produced by them individually.

Specific bacteria cultures, known as starters, are used for manufacturing of fermented milk products. Traditional method, using part of a previous batch to inoculate a new batch, have been used for centuries. Such cultures lead to variable performance, however industrial production needs consistency. The method of choice is the use of bacterial strains with known physiological, biochemical and genotypic characters.

Yoghurt is generally made from a standardized mixture containing whole milk, partially defatted milk, condensed skim milk cream and non fat dry milk. Alternatively milk may be partly concentrated by removal of 15- 20% water in a vacuum pan or by heating. While the microorganisms fermenting milk confers on it certain health benefits inadequate pasteurized milk may contain microorganism of special importance to man (Boor and Murphy, 2002). In which its presence or absence in milk may reflect success or failure of good manufacturing practice (GMP) or cause infection when consumed together with food. This is of economic significance in Africa where the HIV/AIDS and cancer scourge has left the public who consume milk products immune suppressed and prone to bacterial and fungi infection. (Boor, 2001). The isolation and identification of natural starters is a need not only for the dairy industry, which still import starters abroad, but also for the preservation of natural lactic acid bacteria. With this perspective, the aim of our study was the isolation of starters from artisanal yoghurts, and their biochemical and molecular characterization.

The FAO/WHO standards No. 11(a) and 11(b) define yoghurt and flavoured yoghurt respectively as coagulated milk product obtained by lactic acid fermentation through the action of L.bulgaricus and Streptococcus thermophilus.

Whey is main liquid waste generated during manufacture of cheese and some other traditional coagulated products in dairy industry. Approximately, 55% of the total milk proteins are drained in whey. World production of whey is 168 MT with ongoing annual growth of around 2 %. The recycling of whey can deserve dual purpose, avoid environmental pollution and produce value added products (Dlamini and Peiris, 1997 ;), besides the reduction of production cost, which is an essential requirement for the food and dairy industry. Recently novel technologies are industrialized to utilize whey for production of valuable bio ingredients (Koutinas et al., 2007). Whey can be converted into many products with various process and technologies. Condensed whey, dry whey, dry modified whey, whey protein concentrate and isolates, as well as lactose (crystallized and dried) are often cited whet products. There are many others secondary and tertiary products that can be derived from whey; it may be from biocatalytic activity of microbes and some enzymes. Production of value added products such as bio-molecules and some metabolites, having industrial application. Food and agricultural wastes are rich in carbohydrates, microorganism are able to convert these cheap material in to value added products like exopolysaccharides, lactic acid, acetic acid, citric ,gluconic, vitamins, probiotics biomass and antimicrobial peptides(Kasseva et al., 2009; Katecchaki et al., 2010).

Based on above mentioned points, the project was formulated the following objectives:-

- To study the growth performance of yoghurt culture in whey based media
- Production of yoghurt culture biomass and preservation in frozen concentrated and freeze dried forms
- Evaluation of preserved cultures for preparation of yoghurt

2. REVIEW OF LTERATURE

2.1 The History of Yoghurt

For thousands of years yoghurt has been produced throughout the Middle East. Although no records are available regarding the origin, yoghurt is most likely evolved from the nomadic people living in the Middle East part of the world. The production of milk in the Middle East was seasonal, being restricted to a few months of the year. The main reason for this limited availability of milk was the lack of intensive animal production. Farming was in the hands of nomadic people who moved from one area to another. Hence they were in wilderness away from cities where they could sell their animal produce. Another factor was the climate in the Middle East. At as high as 40°C, milk sours immediately under primitive conditions. The animals were hand-milked, no cooling of milk was possible, and contamination was unavoidable. Under these conditions transportation and keeping of milk for a long time was not possible. However, the nomadic people devised a fermentation process, which as a result led them to keep milk for long times. It was heating the milk over an open fire (Tamime and Robinson, 1985).

Heating milk resulted in;

- Concentration of milk
- Modification of casein in milk
- Selection of thermophilic lactic acid bacteria resistant to high temperatures
- Destruction of pathogenic microorganisms
- Fermentation at slightly higher temperatures during cooling, and enrichment of thermophilic lactic acid bacteria.

Soured milk with thermophilic lactic acid bacteria became the preservation method of milk, and other communities learnt of this technique. As a result the product “yoghurt”, coming from the Turkish name “Yogurt”, has been widely accepted (Tamime and Robinson, 1985). Specific bacteria cultures, known as starters, are used for manufacturing of fermented milk products. Yoghurt is made from milk by the protocooperative action of well known starters, namely Lactobacillus delbrueckii spp. bulgaricus and Streptococcus thermophilus. They lead to coagulation of milk by lactic acid fermentation and other products give the characteristic properties, such as acidity, aroma, and consistency. Traditional method, using part of a previous batch to inoculate a new batch, have been used for centuries. Such cultures lead to variable performance, however industrial production needs consistency. The method of choice is the use of bacterial strains with known physiological, biochemical and genotypic characters.

2.2 Symbiotic Growth of Yoghurt Starter Bacteria

Milk fermentation for yoghurt is done by the addition of thermophilic lactic acid bacteria for thousands of years. This is about the associative growth of microorganisms in growth medium. Symbiotic growth of thermophilic lactic starter microorganisms is based on their metabolic compatibility. It was demonstrated by Robinson at 2002; inoculation of mixed cultures results in the production of lactic acid >10 g/L in 4 hours while 2 g/L and 4g/L lactic acid was obtained by the fermentation of isolated pure cultures of Lactobacillus bulgaricus and Streptococcus thermophilus. Protocooperative growth of Lactobacillus bulgaricus and Streptococcus thermophilus considerably gives better results due to cell densities, the specific growth rates, lactic acid production rates

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Figure 2.1. Growth curves of starter bacteria for pure cultures and mixed culture (Source: Tamime and Robinson 1985)

Streptococcus thermophilus grows more rapidly than Lactobacillus bulgaricus initially and begins to produce lactic acid. Lactic acid production results in a decrease in the pH of the medium. While Streptococcus thermophilus grows, it releases CO2 from the breakdown of urea and formic acid. Streptococcus thermophilus depletes the oxygen in the medium and this causes the oxidation-reduction potential more favorable for the growth of Lactobacillus bulgaricus. The increased acidity, CO2, formic acid and depletion of O2 stimulates the growth of bacilli which is more acid tolerant than Streptococcus thermophilus. Besides having a stimulatory effect on bacilli, the growth of Streptococcus thermophilus depends on the growth of Lactobacillus bulgaricus. Lactobacillus bulgaricus has higher proteolytic activity than Streptococcus thermophilus. The proteolytic enzymes of Lactobacillus bulgaricus degrade casein with the liberation of low molecular weight peptides and amino acids which have stimulatory effect on the growth of Streptococcus thermophilus (Rajagopal and Sandine, 1990). Both two microorganisms can grow at a temperature of 42-43ºC, and the optimum temperature for symbiotic growth is 42ºC. Since the optimum growth temperature for Streptococcus thermophilus is 37ºC and 45ºC for Lactobacillus bulgaricus, increasing the temperature above from 42ºC, the growth of lactobacilli will be favored while the temperatures below 42ºC results in increased growth of streptococci. Either case is resulted in a deviation in the ratio of cocci to bacilli, for the optimum yoghurt the ratio should be 1:1 (Shah, 2003).

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Figure 2.2. Stimulatory effects of two microorganisms (Source: Tekinşen, 2000)

The isolation and identification of natural starters is a need not only for the dairy industry, which still import starters abroad, but also for the preservation of natural lactic acid bacteria. With this perspective, the aim of our study was the isolation of starters from artisanal yoghurts, and their biochemical and molecular characterization

2.3 Role of Starters in Yoghurt Production

The usage of carefully selected strains as starter cultures or co-cultures in fermentation process can help to achieve in situ expression of the desired property and to maintain a product with good quality characteristics such as aroma, taste, flavour. Starter culture inoculation into the milk to during the production of yoghurt leads to production of lactic acid, aroma compounds, exopolysaccharides and inhibitor compounds which give specific characteristics to the end product.

2.3.1 Lactic Acid Production

Lactic acid can be produced by homofermentative and heterofermentative ways in D (-) and L (+) forms. Since L (+) lactic acid is produced during the early fermentation, D (-) type is began to be produced after about second hour of fermentation and increase continuously. Typical yoghurt flavour is caused by lactic acid which imparts an acidic and refreshing taste (Chaves et al., 2002). Lactic acid has an effect on regulation of hydrolysis of casein and adsorption of some amino acids, peptides, lactose and minerals (Akın, 2006).

2.3.2 Proteolytic Activity

The growth of lactic acid bacteria depends on some nutritional supplies of suitable sources of nitrogen and carbon. Free amino acids and peptides are present only to a limited degree in milk. Starter bacteria have limited biosynthetic capabilities. Therefore they require free amino acids for growth. One of the essential features of lactic acid bacteria used as starter must be that they possess an efficient proteolytic system enabling them to grow to high cell densities and that they have the ability to ferment lactose rapidly into lactic acid. Therefore a complement of proteinases and peptidases are essential for the degradation of milk proteins. By itself Streptococcus thermophilus has a lower proteolytic activity than Lactobacillus bulgaricus. Therefore, in dairy starter systems Streptococcus thermophilus is used in combination with lactobacilli which leads to impact flavour, texture and composition. The free amino acids arising from the proteolytic acitvity of Lactobacillus bulgaricus might be identified as specific growth factors for Streptococcus thermophilus (Rajagopal and Sandine 1990). Although proteolysis causes the stimulation of bacterial growth, it has some adverse effects on fermented milk products. It was demonstrated that, the production of bitter peptides have been attributed to the proteolysis by Lactobacillus bulgaricus during storage (Renz and Puhan 1975).

2.3.3 Aroma Production

Fermented dairy production industry; flavour perception, which is a crucial characteristic of food industry as the sensory characteristic, is strongly based on the volatile components (Kalviainen, et al. 2003). Yoghurt bacteria give the desired flavour, mouthfeel and texture which is promoted by a series of biochemical pathway in which the starter culture provide the enzymes necessary. Among all flavour compounds isolated, the most prominent ones are lactic acid and a mixture of various carbonyl compounds like acetaldehyde, ethanol, acetone, diacetyl and 2-butanone. Acetaldehyde is considered as the major flavour compound for the typical yoghurt aroma reported by many researchers (Chaves, et al. 2002). The ideal yoghurt flavour is a balanced of acidity and acetaldehyde. This is achieved by culture selection, balance of rod coccus ratio, and fermentation control. The main source of acetaldeyhde is the bioconversion of threonine catalyzed by threonine aldolase of Lactobacillus bulgaricus (Frank and Hassan 1998).

2.3.4 Exopolysaccharide Production

Several types of polysaccharides can be produced by lactic acid bacteria which will then be classified according to their location in the cell (Degeest , et al. 2001). Bacterial exopolysaccharides (EPSs) are long-chain polysaccharides consisting of branched, repeating units of sugar derivates which are mainly glucose (D-glucose), galactose (D-galactose), rhamnose (L-rhamnose), mannose, N-acetylglucosamine, D-glucuronic acid in different ratios (Vaningelgem, et al. 2004). Bacterial EPSs can be classified into two groups on the basis of their composition; homopolysaccharides (HoPS) and heteropolysaccharides (HePS). Basicly HoPS can be defined as polymers composed of one type of monosaccharides while HePS are the polymers of repeating units that are composed of two or more than two types of monosaccharides. EPS is economically important because it can impart functional effects on foods and may confer beneficial health effects (Welman and Maddox 2003). Lactic acid bacteria, producing EPS, play an important role in dairy industry by improving viscosity and the texture of fermented products (Aslim, et al. 2005). There are many factors effecting EPS yield of lactic acid bacteria such as growth medium, incubation temperature, pH, oxygen tension, agitation speed and incubation time (De vuyst, et al. 2003). As a substitute for commercial stabilizers in yoghurt manufacture, EPS-producing cultures are commonly used due to reduction of synersis and improvement of product texture and viscosity. Some researchers demonstrated that EPS-producing lactic acid cultures showed higher viscosity and lower degree of synersis compared with non-EPS-producing cultures (Bouzar, et al. 1996, Folkenberg, et al. 2004).

2.3.5 Production of Inhibitory Compounds

One of valuable properties of starter cultures is their ability to inhibit growth of undesirable microorganisms. Reduction of pH and production of organic acids are the primary inhibitory actions of lactic acid bacteria. Thus, the pH of the medium is not suitable for many of other microorganisms. Lactic starter cultures also produce nonacidic microbial inhibitors. Hydrogen peroxide in small amounts, diacetyl, bacteriocins, secondary reaction products like hypothiocyanate are the inhibitory compounds produced in small amounts by lactic acid bacteria are the nonacidic inhibitory compounds produced by lactic starters. Producing inhibitory compounds is one of the important parts of maintaining food quality for long time periods as a result of preventing contamination. Although there are several advantages for producing inhibitory compounds, production of nonacidic inhibitors by lactic acid bacteria is not necessarily advantageous such as auto inhibition by nonacidic compounds.

2.4 PRODUCT DESCRIPTION

Yoghurt is a smooth, fermented milk product that evolved empirically some centuries ago through the growth of thermophilic (heat loving). Lactic acid, bacteria, Streptococcus thermophilus and Lactobacillus bulgaricus which ferment the milks lactose to produce lactic acid. It has a characteristic acidic taste possessing 0.95 -1.5% and pH ranging from 3.7-4.2 with viable and abundant fermenting microorganisms.

2.4.1 PRODUCTION OF YOGHURT

2.4.1.1 RAW MATERIALS

In yoghurt preparation the following essential materials are use in processing the product: milk or concentrated skimmed or partly concentrated skimmed milk or milk product and the starter culture Lactobacillus bulgaricus and Streptococcus thermophilus. In. the absence of pure culture one to two spoonful of commercially purchased yoghurt can be used for the inoculation. Also, there are optional ingredients like milk powder, skimmed milk powder, flavour, colours, sugar, wheat, edible casein, preservatives, stabilizers (gelatin, locust bean gum, pectin, starch) etc.

2.4.1.2 EQUIPMENTS:

They include refrigerator or cooler, boiler or heater, thermometer.

2.4.1.3 YOGHURT PREPRATION PROTOCOL

- Homogenised whole or low fat milk
- Addition of skim milk powder
- Heat treatment at 80-90̊C for 30 min
- Cooled to 40-45̊C
- Culture added (2%) at 40-45̊C
- Packaging
- Incubation at 42°C
- Storage at 4̊C

2.4.5 TRADITIONAL METHOD OF YOGHURT MAKING

The milk is evaporated to 1/3 to 1/4 water content so that it attains the required concentration. Alternatively, 4-5% non-fat dried milk (NFDM) can be added to the whole milk. It is heated to 82-93°C for 30 minutes. Then it is cooled to 42-43°C and inoculated with 2-3% starter culture (Lactobacillus bulgaricus and Streptococcus thermophilus).Then the milk is incubated at 42-43°C for 3 hours or until a titrable acidity of 0.75% lactic acid or coagulation occurs. The product is chilled to 5°C. Further acidity of 0.9% lactic acid may develop while the product is being chilled. The product can be stored satisfactorily for 1-2 weeks at 5°C.

2.4.2 INDUSTRIAL METHOD OF YOGHURT MAKING

The desired quantity of milk is weighed and heated to 80-90°C for 15-20 minutes. Then it is cooled to 45-48°C.Two to three percent of yoghurt culture is added to the milk and mixed well. The milk is kept in clean and sterilized containers for setting. The milk in the container is incubated at 45°C until the coagulation is firmer. The product is removed from the incubator and kept at 5°C until distributed to the consumers. The equipment are cleaned and kept ready for the next batch of operation.

2.5) VARIETIES IN YOGHURT PRESENTATION

Yoghurt has been described as a notoriously balanced food, containing almost the nutrients present in milk but in a more assimilable form. The can be produced from whole or skimmed milk (Ojokoh, 2006). There are large ranges of flavors enhancer available commercially that can be used in the production of yoghurt and yoghurt is typically categorized as follows:

1. SET YOGHURT: This type of yoghurt is incubated and cooled in the final package and is characterized by a firm Jelly-like texture.
2. STIRRED YOGHURT: This type of yoghurt is incubated in a tank and the final coagulum is "broken" by stirring prior to cooling and packaging. The texture of stirred yoghurt will be less firm than a yoghurt not stirred which is somewhat like a very thick cream. There is some slight reformation of the coagulum after the yoghurt has been packed, however this is slight and cannot be relied upon.
3. DRINKING YOGHURT: This type of yoghurt is very similar to stirred yoghurt, having the coagulum "broken" prior to cooling. In drinking yoghurt, the agitation used to "break" the coagulum is severe. Little care is applied if any reformation of the coagulum will reoccur after packing.
4. FROZEN YOGHURT: This is inoculated and incubated in the same manner as stirred yoghurt. However, cooling is achieved by pumping through a whipper/chiller/freezer in a fashion similar to the cream. The texture of the finished product is mainly influenced by the whipper/freezer and the size and distribution of the ice crystals produced.
5. CONCENTRATED YOGHURT: This type is inoculated and fermented in the same way as stirred yoghurt, following the "breaking" of the coagulum. The yoghurt is concentrated by boiling off some of the water. This is often done under vacuum to reduce the yoghurt often lead to protein being totally denatured and producing rough and gritty texture. This is called strained yoghurt due to the fact that the liquid that is released from the coagulum upon heating used to be "strained" off in a manner similar to making of soft cheese.
6. FLAVOURED YOGHURT: Yoghurt with various flavours and aromas has become very popular. The following are usually added at or just prior to filling into pots. Common additions are fruits or berries, usually as a pure or as whole fruit in syrup. These additives often have" as much as 50% sugar in them. However, with the trend towards healthy eating gained momentum many manufacturers offer a low sugar and low fat version of their products. Low or no sugar yoghurts are often sweetened with saccharin or more commonly aspartame. The use of a "fruit sugar" in the form of concentrated apple juice is sometimes found as a way of avoiding' "additional sugar" on the ingredients declaration. This tends to be a market ploy and has no real added benefits.

2.6 THERAPEUTIC BENEFITS OF YOGHURTS

1. Yoghurt is easier to digest- Many people who cannot tolerate milk, either because of a protein allergy or lactose intolerance, can enjoy yogurt. The live active cultures create lactase, the enzyme lactose-intolerant people lack, and another enzyme contained in some yogurts (beta-galactosidase) also helps to improve lactose absorption in lactase-deficient persons. The culturing process has already broken down the milk sugar lactose into glucose and galactose, two sugars that are easily absorbed by lactose-intolerant persons.
2. Yogurt contributes to colon health - Yogurt contains lactic acid bacteria, intestine-friendly bacterial cultures that foster a healthy colon, and even lower the risk of colon cancer. Lactic acid bacteria, especially acidophilus, promote the growth of healthy bacteria in the colon and reduce the conversion of bile into carcinogenic bile acids. The more of these intestine-friendly bacteria that are present in the colon lower the chance of colon diseases. Basically, the friendly bacteria in yogurt seems to deactivate harmful substances (such as nitrates and nitrites before they are converted to nitrosamines) before they can become carcinogenic.
Yogurt is a rich source of calcium - a mineral that contributes to colon health and decreases the risk of colon cancer. Calcium discourages excess growth of the cells lining the colon, which can place a person at high risk for colon cancer. Calcium also binds cancer-producing bile acids and keeps them from irritating the colon wall.
3. Yogurt improves the bioavailability of other nutrients- Culturing of yogurt increases the absorption of calcium and B-vitamins. The lactic acid in the yoghurt aids in the digestion of the milk calcium, making it easier to absorb.
4. Yogurt can boost immunity- The bacterial cultures in yogurt have also been shown to stimulate infection-fighting white cells in the bloodstream. Some studies have shown yogurt cultures contain a factor that has anti-tumor effects in experimental animals.
5. Yogurt aids healing after intestinal infections- Some viral and allergic gastrointestinal disorders injure the lining of the intestines, especially the cells that produce lactase. This results in temporary lactose malabsorption problems. This is why children often cannot tolerate milk for a month or two after an intestinal infection. Yogurt, however, because it contains less lactose and more lactase, is usually well-tolerated by healing intestines and is a popular "healing food" for diarrhea.
A 1999 study reported in Pediatrics showed that lactobacillus organisms can reduce antibiotic-associated diarrhea.
6. Yogurt can decrease yeast infections- Yogurt that contains live and active cultures daily reduces the amount of yeast colonies and decreases the incidence of yeast infections.
7. Yogurt is a rich source of calcium- Because the live-active cultures in yogurt increase the absorption of calcium, an 8-ounce serving of yogurt gets more calcium into the body than the same volume of milk can.
8. Yogurt is an excellent source of protein- Eight ounces of yogurt that contains live and an active culture contains 20 percent more protein than the same volume of milk (10 grams versus 8 grams). Besides being a rich source of proteins, the culturing of the milk proteins during fermentation makes these proteins easier to digest. For this reason, the proteins in yogurt are often called "predigested."
9. Yogurt can lower cholesterol- There are a few studies that have shown that yogurt can reduce the blood cholesterol. This may be because the live cultures in yogurt can assimilate the cholesterol or because yogurt binds bile acids.

2.7 FACTOR THAT ALTER THE QUALITY OF YOGHURT

1. MILK QUALITY: The milk used for yoghurt manufacture should be of the highest bacterial quality available. It should also have an absence of any material that will impede or prevent the growth of the starter microorganism, (anti-biotics, preservative, disinfectants, and bacteriophages).
2. BACTERIOPHAGES: Bacteriophages are a group of virus that attacks the yoghurt starter organisms, a whole range of defects can be attributed to the action of this bacteriophage. Bacteriophage normally referred to just as "phage" are the most likely cause of long or never-ending incubations. Large manufacturers that have laboratory facilities to check incoming milk will often eliminate the possibilities of other starter inhibiting substances but "Phages" are usually found in the drains and floor gullies of a dairy producing any cultured product, poor hygiene and lack of general housekeeping increase the risk.
3. STARTER CULTURE: The starter culture is the term generally applied to organisms used to ferment a cultured product (Cheese, Yoghurt, and Kefir). The micro organisms selected for this purpose need to produce the desired effect in the product. For normal commercial yoghurt the starter must be capable of fermenting lactose and producing lactic acid, little if any carbon dioxide is required and the flavour and aroma must be clean and fresh. Traditionally when a suitable starter organism had been found on a large quantity would be grown in a suitable nutrient medium and small quantities would be used to inoculate each new batch of yoghurt. This technique with a main batch of starter culture is often referred to as using "bulk starter". The use of a bulk starter is becoming increasingly uncommon amongst commercial producers, mainly because of the risk of "Phage" attack on the bulk starter, and the subsequent lost time while a new batch of starter organisms are prepared. A technique often referred to as Direct Vat Inoculation (DVI) is becoming the industry norm. DVI involves inoculating the yoghurt mix directly with a very large number of freeze dried starter organisms. The advantage of relative immunity to “Phage” attack for outweighs the slightly longer incubation time required with this technique.
4. FAT PERCENTAGE: The percentage of fat in the final yoghurt has a significant effect on the "mouth feel", the normal range of fat content is from 0.5% to about 3.5%, however levels as low as 0% and as high as 10% are found in some specialty products.

2.8 Preparation of Direct Vat Starter Cultures

2.8.1 Introduction

Traditionally 'bulk starter' in liquid form was used to inoculate the milk for the manufacturing of cheese, yoghurt, buttermilk and other fermented products. This culture, called bulk culture, can be prepared from commercially available frozen concentrated or freeze-dried cultures, or the inoculum can be prepared at the plant. Preparing inoculum at the plant involves starting with a ‘‘mother’’ culture maintained in small amounts (approximately 100 ml) of medium. The mother culture is used to inoculate successively larger amounts of medium (using a 1% inoculum) until sufficient inoculum volume is obtained to prepare the bulk culture.

Stock culture Mother Feeder or Intermediate Bulk Preparing bulk culture inoculum at the plant is time-consuming, requiring skilled operators and carries an increased risk of phage contamination.

Over the past 10-15 years, the Direct Vat cultures have increasing being used, particularly in small plants, to replace bulk starter in cheese manufacture

2.8.2 Direct Vat Cultures

Direct vat cultures are carefully selected strains of frozen concentrated or concentrated freeze-dried cultures which can be added directly to the milk with no intermediate growth step. Direct vat cultures are also known as starter cell concentrates, Direct Vat Set (DVS) Direct Vat Inoculant (DVI). These are concentrated cell preparations containing cells in the order of 1011-1013 CFU/g. Direct vat cultures are available in two forms - Freeze dried granular form & Deep-frozen pellets (Commercial form of DVS).

2.8.3 Preparation of Starter Concentrates

Under normal conditions starter growth in milk results in a cell concentration of about 109 CFU/ml. But the growth of starters in milk is limited by a number of factors including the accumulation of lactic acid. For preparation of starter concentrates generally following methods are used-

- Removal of the lactic acid (using diffusion culture)
- Neutralisation (traditional fermentation technology, Batch culture)

2.8.4 Diffusion Culture

Diffusion culture has been described by Osborne, 1977. The culture medium, containing starter bacteria, is pumped across a suitable membrane In this method, a semi-permeable membrane is used that allows inflow of the fresh nutrients and eliminates the waste products of growth. Fresh medium is pumped across the other side of the membrane.The major factor determining the effectiveness of this method is the provision of a sufficiently large membrane area to permit adequate diffusion of the rapidly produced metabolic by products. Osborne, (1977) has indicated that 5 cm2 of membrane per ml of fermentor medium are required. Membranes are now available that can withstand heat and chemical sterilisation.This system gives total cell concentration of 1.0-1.5×1011 cfu/ml.

2.8.5 Batch Culture

This is a simple and efficient method. This technique consists of a two-stage process for producing cell biomass for starter concentrates. The two steps include:-

a) Growing the culture in broth media, and
b) Separation of cell mass or pellet by centrifugation. It offers the opportunity to change the proportion of certain type of organisms in mixed culture by altering the growth conditions.

Some of the factors which can influence the efficiency of batch culture are growth medium, pH during growth, temperature of incubation, time of fermentation and method of harvesting.

1. Growth Medium- The composition of growth medium affects the efficiency of batch culture. The following criteria regarding selection of the medium for starter culture proliferation intended for starter concentrates have been proposed: a) Cost, b) Ability to produce high yield of active cells, c) Suitability of collecting cells by centrifugation, d) Suitability for acid production and required flavor and texture characteristics during the use of starter concentrates.
2. pH - Although maximum cell no. of lactic starters are obtained when the pH of the growth medium is maintained at 6.0-6.5(beyond these pH values the cell no. in the biomass appears to be adversely affected). However, little difference in cell biomass is observed when sodium hydroxide or ammonium hydroxide is used as neutralizer for pH adjustment.
3. Incubation Temperature - The incubation temperature for the common (mesophilic) lactic starters varies in the range of 20-30°C depending on the desired time of incubation.
4. Incubation Period - Maximum yield of bacterial concentrate is generally obtained using 1% inoculum after incubation at appropriate temperature for 14-16 hrs.
5. Method of Harvesting - A desludging type of separator is found to be superior than sharple’s centrifuge for separation of bacterial cells as the latter gives intermittent harvesting and the removal of bacterial sludge is difficult.

2.8.6 Preparation of frozen concentrated cultures by Batch culture

It involves following steps -

1. Handling of the inoculation material
2. Preparation of media
3. Propagation of cultures in fermenters under optimal conditions using pH control
4. Concentration by harvesting the cells via centrifugation or ultra filtration
5. Adding a cryoprotectant
6. Freezing rapid freezing using liquid nitrogen
7. Packaging & Storage

1. Handling of the inoculation material - Inoculation Material can be collected from following sources –

- Cultures or single strains are prepared under aseptic conditions
- Research Establishments (NCDC, ECCO, NCFB, NCIM etc.)
- Educational Colleges
- Commercial Manufacturers

It should be handled and inoculated aseptically to retain all the enzymatic and biochemical activities of the culture.

2. Preparation of media - Growth Media for the production of cultures are composed of selected milk components and supplemented with various nutrients like yeast extract, vitamins and minerals. Growth Media preferably based on milk or whey (after digesting the proteins with proteolytic enzymes) are recommended. Enzymatic Digestion serves double purpose- Making peptides for rapid growth of organism and increasing centrifugation efficiency due to clarification. The culture growth medium is heated to an ultra high temperature and cooled to either 30 or 40°C for mesophilic or thermophilic cultures respectively.

3. Propagation of cultures in fermenters - After inoculation growth is optimized by maintaining the pH at 6-6.3 for mesophilic cultures and 5.5-6.0 for thermophilic cultures. pH is maintained by addition of an alkali such as NaOH or NH4OH. Other Critical Parameters like temperature, agitation rate in the fermenters are optimized for each strain (These conditions will produce cell suspension that are 10 fold more concentrated than a normal acidified bulk starter).

4. Concentration - After fermentation, the contents are cooled and the biomass is harvested by centrifugation or membrane filtration. It is concentrated by bactofugation/microfiltration giving a further 10-20 fold concentration of the cells. During the production of starter concentrates the concentration unit should be efficient in separation of starter cells both in degree and speed, Capable of maintaining low temperature, Easily cleaned and sterilized, Capable of automatically reconstituting and discharging the cells at the required concentration without causing cell damage.

5 . Adding a cryoprotectant - The cryoprotective agents like sodium citrate, glycerol, and sodium B-glycerophosphate etc. are used. These protective solutes are hydrogen-bonding and/or ionizing groups that help to prevent cellular injury by stabilizing the cell membrane constituents during freezing and thawing.

6. Freezing - After centrifugation the concentrated bacterial cells can be filled into cans and frozen in liq.N2. Rapid freezing can also be accomplished using a dry ice–alcohol mixture. Pelletized by dipping the concentrate into an agitated bath of liquid N2.

7. Packaging and storage - After freezing, packaging is done in an inert gas atmosphere. The frozen concentrate should be stored at -196°C (liquid nitrogen) for best retention of activity, although storage at -40°C (dry ice) is also acceptable. The activity of culture is retained up to 12-24 months. Rapid thawing minimizes cell injury. This is accomplished by immersing the unopened can of cell concentrate in cool chlorinated water immediately before use.

2.9 WHEY AS GROWTH MEDIUM FOR BACTERIA

Whey or Milk Serum is the liquid remaining after milk has been curdled and strained. It is a by-product of the manufacture of cheese or casein and has several commercial uses. Sweet whey is manufactured during the making of rennet types of hard cheese like cheddar or Swiss cheese. Acid whey (also known as "sour whey") is obtained during the making of acid types of cheese such as cottage cheese.

Production

Whey is a co-product of cheese production. It is one of the components that separates from milk after curdling, when rennet or an edible acidic substance is added.

Uses

Whey is used to produce ricotta, brown cheeses, Messmör/Prim, and many other products for human consumption. It is also an additive in many processed foods, including breads, crackers, and commercial pastry, and in animal feed. Whey proteins consist primarily of α-lactalbumin and β-lactoglobulin. Depending on the method of manufacture, whey may also contain glycomacropeptides (GMP).

Dairy whey remaining from home-made cheese making has many uses. It is a flour conditioner and can be substituted for milk in most baked good recipes that require milk (bread, pancakes, muffins, etc.). Whey is also good to add protein to breakfast smoothies. If you have no use for it in the kitchen, you can pour it on acid-loving plants in your yard, such as azaleas, raspberries, rhododendrons and strawberries. The remaining whey may be made into various products by using an array of processes and technologies, or is otherwise disposed of. Whey can be condensed or concentrated, dried, fermented, delactosed, demineralized, and deproteinated. It is adaptable to ultra filtration, reverse osmosis, ion exchange, electro dialysis, and nanofiltration (Kosikowski, et al) . The main whey products are dry products: dry whey, lactose, and whey protein concentrate. These whey products are storable for later distribution over a wide area, even internationally. Condensed whey also uses a significant amount of whey, but the market is limited due to its wet form. There are many other secondary and tertiary products that can be derived from whey (Kosikowski, et al). However, the volume of whey used in these products is relatively small (Yang, et al). While whey products have found wider uses in recent years and of late have become valuable commodities, making these products was originally considered a lower-cost, last resort alternative to dumping surplus whey. Most of the components of whey can quickly deplete oxygen levels in natural water systems (Hamilton).

Whey protein (derived from whey) is often sold as a nutritional supplement. Such supplements are especially popular in the sport of bodybuilding. In Switzerland, where cheese production is an important industry, whey is used as the basis for a carbonated soft drink called Rivella. In Iceland, MS manufactures and sells liquid whey as Mysa in 1-liter cartons (energy 78 kJ or 18 kcal, calcium 121 mg, protein 0.4 g, carbohydrates 4.2 g, sodium 55 mg) (table.1.1)

Table 1.1:- Nutritive value of whey

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2.10 Freeze drying

''Freeze-drying'''(also known as lyophilization, lyophilization or cryodesiccation) is a dehydration process typically used to preserve a perishable material or make the material more convenient for transport. Freeze-drying works by freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase.

2.10.1 The origin of freeze drying

The Andean civilizations preserved potatoes using a freeze drying process. They called this foodstuff Chuño. Freeze-drying was actively developed during 2nd World war. Serum being sent to Europe for medical treatment of the wounded required refrigeration, but because of the lack of simultaneous refrigeration and transport, many serum supplies were spoiling before reaching their intended recipients. The freeze-drying process was developed as a commercial technique that enabled serum to be rendered chemically stable and viable without having to be refrigerated. Shortly thereafter, the freeze-dry process was applied to penicillin and bone, and lyophilization became recognized as an important technique for preservation of biological. Since that time, freeze-drying has been used as a preservation or processing technique for a wide variety of products. These applications include the following but are not limited to: the processing of food, pharmaceuticals, and diagnostic kits; the restoration of water damaged documents; the preparation of river-bottom sludge for hydrocarbon analysis; the manufacturing of ceramics used in the semiconductor industry; the production of synthetic skin; the restoration of historic/reclaimed boat hulls.

2.10.2 Description of the Operation

Generally, the Freeze Drying, or Lyophilization cycle is divided in three phases: An initial freezing process, carried out in such a way that: The product exhibits the desired crystalline structure. The product is frozen below its eutectic temperature.

There are four stages in the complete freeze drying process:-

1) Pretreatment
2) Freezing :- It has two steps:-
a) Primary drying
b) Secondary drying

1) Pretreatment: - Pretreatment includes any method of treating the product prior to freezing. This may include concentrating the product, formulation revision(i.e., addition of components to increase stability and /or improve processing), decreasing a high vapour pressure solvent or increasing the surface area. In many instances the decision to pretreat a product is based on theoretical knowledge of freeze –drying and its requirements, or is demanded by cycle time or product quality considerations. Methods of pretreatment include:- Freeze concentration, Solution phase concentration, Formulation to preserve product appearance, Formulation to stabilize reactive products, Formulation to increase the surface area, and decreasing high vapor pressure solvents.

2) Freezing

a) A primary drying (sublimation) phase during which:

The partial pressure of the vapor surrounding the product must be lower than the pressure of the vapor from the ice, at the same temperature. The energy supplied in the form of heat must remain lower than the product's eutectic temperature (the highest allowable product temperature during the conditions of sublimation.)

b) A secondary drying aimed at eliminating the final traces of water which remain due to absorption, and where:

The partial pressure of the vapor rising from the product will be at its lowest levels. At the completion of the process, the treated product will have retained its form, volume and original structure-as well as all its physical, chemical and biological properties. It can then be stored (provided packaging is effective to the reduction of moisture migration) for an almost indefinite period of time. As the product is porous, it can be re-dissolved by the simple addition of a proper solvent.

From this description of the process of freeze drying, three facts emerge:

- The sublimation characteristics of the product are greatly dependent of the frozen structure.
- This structure cannot be altered during the process.
- Product temperature plays an active role in all three phases, and in execution it is upon this that the choice of other parameters (vacuum, heat rate, etc.) is based.

Freeze drying steps:-

Cultures are grown in tryptone yeast extract broth/M17/MRS/Elliker’s broth at 37ºC for 24-48 h

Broth culture is centrifuged and cell pellet is separated from the supernatant fluid

Pellet/sediment is suspended in cryoprotective agent (e.g. horse serum, skim milk, whey, ascorbic acid, MSG, DMSO, PVP, corn syrup, inositol, glucose, sucrose, starch, gelatin etc.

An aliquot of half ml of resuspended culture can be freeze-dried using an Edward’s freeze drier after primary and secondary drying

Theoretical Basis of Freeze Drying

The theoretical principle of freeze drying is clearly defined in the diagram "Pressure Temperature". In order to avoid the liquid phase, it is absolutely essential to lower the partial pressure of water, below the triple point pressure. A freeze drying cycle is shown in this diagram, which has been designed to conform to a typical example (described below):

- Freezing of a product from 20° C to -20 C° at atmospheric pressure.
- Sublimation of the product at -20° C.
- Transfer of evolved vapor to the condenser at low temperature.
- Vacuum release.
- Defrost
Aspects will be highlighted which play a part in the development of a freeze drying operation:
- Freezing.
- Drying.
- Vacuum influence.
- The liquid shelf on which the product is placed.
- Essential control aspects during freeze drying.

2.10.3 Freeze Drying of Starter Cultures

Starter cultures are used to assist the fermentation process in preparation of various foods and fermented drinks. A starter culture is a microbiological culture which actually performs fermentation. Freeze drying is an effective way to preserve starter cultures.

Freeze Drying Process

The freeze drying process was developed during the Second World War for preserving medical supplies that required refrigeration. Since then freeze drying has been applied to the food industry and is now a standard process used to increase the shelf-life of many products that would otherwise spoil. Freeze drying uses a process called lyophilization to gently freeze the specimen and extract the water in the form of vapours using a high-pressure vacuum. The vapour collects on a condenser, turns back into ice and is removed. A gradual temperature rise extracts all remaining 'bound' moisture from the specimen. This process retains the physical structure of the product and preserves it for storage and/or transport. The product can easily be re-hydrated using water and in some cases can be used directly in its freeze-dried form.

2.10.3 Advantages of Freeze Drying Starter Cultures

With freeze drying, both solids and liquids can be preserved without damaging their basic chemical structure. The natural size, composition and consistency of the sample are retained. Regular drying methods have a major disadvantage as the high temperatures used can cause chemical or physical changes to the product. For biological cultures, this could render them ineffective or affect the taste or quality of the end product.

2.10.4 Applications of Freeze Dried Starter Cultures

- Cultured Milk (widely used in African countries)
- Buttermilk
- Cheese
- Sour Cream
- Yogurt
- Sourdough bread
- Probiotics

Freeze drying is also suitable for a range of other items including pharmaceuticals, flowers and documents.

Frozen starters/frozen concentrates

- Preserved in frozen form
- Produced by 2 routes:
- Deep or subzero freezing (-20 to -80°C)
- Ultra low temperature freezing (-196°C) in liquid N2
- Sterile liquid milk freshly inoculated with an active starter culture is deep frozen at -30 to -40°C to preserve the mother or feeder culture and retain activity for several months when stored at -40°C. Such cultures have now been replaced by the concentrated, frozen type for direct inoculation of bulk starter tanks. DVI of milk for the manufacture of cheese or fermented milks.

Advantage: Dispatched to a dairy in dry ice whenever required

Disadvantage:

- Freezing and prolonged storage at -40°C can lead to a deterioration in starter culture activity, and can damage certain lactobacilli

Solution: Use of media containing Skim milk, sucrose, fresh milk, NaCl2 and gelatin .

- Concentrated cells ( 1011-1012cfu/ml) frozen at -30°C in the presence of certain mixtures of cryogenic compounds Na-citrate, glycerol, Na-B-glycerophosphate, yeast extract, sucrose, cream, sterile skimmed milk, peptone. trehalose, or lactose) have been retained as active as the original cultures in the case of mesophilic organisms, Lactobacillus spp . or propionic acid bacteria (Barbour and Fonseca et al., 2000).

MATERIAL AND METHODS

3.1 BACTERAL CULTURES

The bacterial cultures used in this study were procured as freeze dried cultures ampoules from National Collection of Dairy Culture (NCDC), National Dairy Research Institute, Karnal, Haryana.

Table: 3.1. Bacterial culture

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3.2 MAINTENANCE, PRESERVATION AND PROPAGATION OF CULTURE

Freeze dried cultures were activated in skim milk at 37ºC /16 h and sub-cultured monthly. Before using for experiments ,NCDC 009, NCDC 74 cultures were activated by 2-3 transfers in MRS , M17 broth at 37ºC for 16 h.

3.2.1 Microscopic examination

3.2.1.1 Gram staining

Lb. delbrueckii ssp. bulgaricus and S. thermophilus are Gram positive bacteria and the Gram statuses of the isolated bacteria were determined by the microscopic examination of Gram-stained isolates. One loop full of overnight activated broth culture was spreaded on a glass slide and they were Gram stained after drying and fixation by exposure to a flame. The main steps in Gram Staining procedure were as follows:

- Crystal violet staining for 1 min
- Removing the excess stain by washing under tap water
- Staining with Gram’s iodine mordant for 1 min
- Washing under tap water
- Fixation with 95% alcohol for 15 s
- Counter staining with safranine for 30 s
- Washing under tap water
- Drying with cotton towels gently

Gram positive bacteria became blue-purple after Gram staining; however Gram negative bacteria became pink-red .

3.2.1.2 Simple staining

Metylene blue staining (direct staining)

One loop full of overnight activated broth culture was spreaded on a glass slide and then after drying and fixation by exposure to a flame.

- Prepare a heat fixed smear of activated broth culture
- Cover the smear with methylene blue
- Allow the dye to remain on the smear for approximately for 2 minute
- Gently wash off excess methylene blue from the slide by directing a gentle steam of water over the surface of the slide.
- Air dry the slide and then observes under microscope.
Nigrosin staining (indirect staining)
- Place a small drop of negative stain (Nigrosin stain) near the end of the slide.
- Transfer one loop full of the bacterial sample to the stain mix the two together
- Air dry the slide. Do not heat fix.
- Examine under light microscope.

Nigrosin, methylene blue and Gram’s stained smear of all cultures were examined microscopically in order to observe their morphology and staining property to check their purity.

3.2.3 Catalase test

Catalase test was performed as per slide method. Using an inoculating needle culture from a well isolated colony was placed onto a clean glass slide. A drop of 3% hydrogen peroxides solution was added to this culture and closely observed for the evolution of bubbles.

3.3 Measurement of growth

3.3.1 Viable count (Pour plating)

Protocol

The pour plate technique can be used to determine the number of microbes/mL or microbes/gram in a specimen. It has the advantage of not requiring previously prepared plates, and is often used to assay bacterial contamination of foodstuffs. The principle steps are-

1) Prepare/dilute the sample
2) Place an aliquot of the diluted sample in an empty sterile plate
3) Pour in 15 ml of melted agar which has been cooled to 45o C, swirl to mix well
4) Let plate undisturbed to solidify on a flat table top
5) Invert and incubate to develop colonies.

Each colony represents a "colony forming unit" (CFU). For optimum accuracy of a count, the preferred range for total CFU/plate is between 30 to 300 colonies/plate.

3.3.2 Growth performance in whey based medium

Again the activated cultures were studied in whey base medium for growth performance by pour plate method.

3.3.2.1 Direct microscope count:-

The basis of a direct count is the actual counting of every organism present in a sub-sample of a population. Direct microscopic count is a determination of the number of microorganisms found within a demarcated region of a slide known to hold a certain volume of culture. This total count method of cell quantification is very rapid but has the problem of requiring high cell concentrations (e.g., 107 / ml) as well as potentially counting dead and living cells with equal probability.

Procedure

1. Clean the glass slide with 70% alcohol and let air dry.
2. Mark 1cm2 area on glass slide.
3. Prepare dilution of sub cultured tube up to 10-1.
4. Take 10 µl cultures from diluted tube and then spread this in marked area with the help of loop.
5. And first air dry it and then heat fix it.
6. Stain with Gram’s stain and then observe under microscope.
7. Count the bacterial cells in 20 fields.

3.4 Freeze drying

In the freeze drying process water is removed from the frozen sample by exposing to vacuum. Microbial cells are suspended in a suitable protective medium, frozen and exposed to a vacuum. After drying the cultures are stored in glass vials or ampoules. It has been widely used to preserve various microorganisms and is applicable to bacteria, fungi, and some viruses.

3.4.1 Preparation of ampoules

The ampoules should be of neutral glass and not alkaline. Soft glass ampoules are easy to seal and reopen. The ampoules suitable for about 0.1-0.2 ml amount are tubes approx. 6 mm internal diameter with a slight constriction at the mouth for gripping the secondary drying manifold. A numbered label is placed in each tube. The tubes are plugged with absorbent cotton wool and sterilized in the autoclave at 20 lb for 20 min (1250C). After autoclaving, they are dried in an incubator at 600C.

3.4.2 Suspending medium

The suspending media used for freeze drying are either horse serum based or skim milk based. Routinely, 12% skim milk with 5% sodium glutamate may be used.

3.4.3 Preparation of cultures

Cultures are grown aerobically or anaerobically in culture media suitable for particular culture and usually harvested by centrifugation during active growth.

3.4.4 Filling of ampoules

Tubes are filled with approximately 0.1- 0.2 ml of suspension using Pasteur pipettes with long fine capillaries. After filling each ampoule, the cotton wool plug is replaced.

3.4.5 Primary drying

The cotton plugs are removed and each ampoule is capped with a strip of sterile surgical gauze folded over the top and stapled at sides. The ampoules are loaded on the centrifugal head taking care to counterbalance the tubes by virtue of their relative positions in the centrifugal head. The centrifuge is switched on and vacuum is applied. The centrifuge is switched off after 10-15 min or attaining a vacuum of 0.1 torr. The vacuum is maintained for 3-4 hours to complete the drying.

3.4.6 Secondary drying

The drying is unloaded and the centrifuge head is removed. The tubes are plugged with non absorbent cotton wool and the plug is inserted to level of label strip. The ampoules are constricted at a point just above the upper end of cotton plug. The centrifuge head is removed and replaced with the secondary manifold. Ampoules are attached to drying manifold for drying at least for 2 hours or overnight. At a vacuum of at least 2.1 torr, the ampoules are flame sealed at the middle of the constriction. The ampoules are stored at +800C or below in the dark.

3.5 Preparation of concentrated yoghurt cultures for direct application

200 ml whey based medium was prepared and then inoculated with 2% of yoghurt mix culture and then incubated for 10 hrs at 370C and then cells were harvested by centrifugation. Pellet was dissolved in 20 ml of cryoprotective solution i.e. sodium glutamate milk. And then the culture was preserved by two methods one is freeze drying as described above and other is method is DVS preparation, in this sodium glutamate suspended culture was poured in petri plate and freezed at -200C for overnight and after this culture was dried in freeze dryer under vacuum. And then cultured was preserved in powdered form.

3.6 Preparation of yoghurt using preserved concentrated cultures

- Homogenized whole or low fat milk
- Addition of skim milk powder (4-5%)
- Heat treatment at 800C-900C for 30 min
- Cooled to 450C
- Culture added at 2% level for fresh culture, 0.2% level for concentrated liquid culture and freeze dried concentrated cultures at 400C- 450C.
- Packaging
- Incubation at 420C
- Storage at 40C

3.7 Analysis of yoghurt

3.7.1 Measurement of pH

pH was measured by electronic pH meter (Thermo scientific pH meter).

3.7.2 Determination of titratable acidity (AOAC, 2007)

Reagents

1. Alcoholic solution of phenolphthalein(0.5 g of phenolphthalein in 100ml 50% alcohol)
2. N/10 NaOH solution

Procedure

Sample (20ml) was transferred in beaker (100ml) and 3 to 5 drops of phenolphthalein was added to sample and titrated against N/10 NaOH solution with continuous stirring till faint pink color persists. The volume of NaOH solution required was measured and titratable acidity (% lactic acid) was calculated as follows:

% lactic acid= 9 NV/W grams of sample

Where-

V = volume of N/10 NaOH required (ml) and

W= volume of sample taken for analysis (20 ml)

N= normality of alkali used for neutralization

3.7.3 Coliforms count

Traditionally the agar plate count method using VRBA (violet red bile agar) and MPN (most probable number) methods are being used. The plate count method takes 24 hours to perform and can use 1 ml of 1:10 dilution and result has a sensitivity of < 10 cfu/g.

3.7.4 Yeast and molds count

The finished product of yoghurt is subjected to yeast and mold count test to ascertain quality and shelf life. Agar plate count method using PDA (potato dextrose agar) takes 3-5 days to perform and can use I ml of 1:10 dilution and result has a sensitivity of < 50-100 cfu/g.

3. RESULTS AND DISCUSSION

Yoghurt is a widely consumed traditional fermented milk beverage popular in all parts of world. Yogurt is a cultured dairy product produced by lactic acid fermentation of milk, fat content ranges from 0 to over 4% and is also a means of pre-serving the nutrients in milk (Hui, 1992; Chandan, 1989). It is generally known as cultured milk, as it is derived from the action of bacterial on all or part of the lactose to produce lactic acid, carbon dioxide, acetic acid, diacetyl, acetaldehyde and several other components that gives the product its characteristic fresh taste (Tamine and Robinson, 2004). In this investigation, attempt has been made to study of growth performance of yoghurt culture in whey based media, production of cultures biomass and preservation in concentrated form and evaluation of preserved cultures for preparation of yoghurt .

4.1 Microscopic examination

Purity of bacterial cultures was determined, through microscopic examination, by Gram staining, simple staining and cellular morphology. Lactic acid bacteria, Lactobacillus delbrueckii spp. bulgaricus was found gram positive, purple colored, long thin rod shaped bacteria, and Streptococcus thermophilus was also found gram positive, purple colored, coccus shaped in chain observed under microscope using oil immersion objective (Fig. 4.1, 4.2, 4.3).

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Fig. 4.1 Morphology of Streptococcus thermophillus (NCDC 74)

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Fig. 4.2 Morphology of Lactobacillus delbrueckii spp. bulgaricus (NCDC009)

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Fig. 4.3 Morphology of yoghurt culture

4.2 Growth performance of yoghurt culture in whey media

4.2.1 Viable count

It is a method to count total no. of viable cells in 1 ml of sample. For this each sample was serially diluted and dilutions from 10-5 to 10-8 was plated by pour plate method. The plates were incubated at 37°C for 24-48 h. Each colony obtained represents a "colony forming unit" (CFU). For optimum accuracy of a count, the preferred range for total CFU/ plate is between 30 to 300 colonies/plate and the number of colonies is multiplied by the number of times the original ml sample was diluted (the dilution factor of the plate counted). Cells of Streptococcus thermophilus colonies were small and white and Lactobacillus delbrueckii ssp. bulgaricus colonies were large and white and have a white cloudy zone (Table 4.1).

Table 4.1: Growth of culture in whey based medium

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4.2.2 Direct microscopic count

Direct microscopic count is a method to determine total cell count in a given sample. It counts both the living and the dead cells as well. In this method the cells are counted in a 1 mm2 marked area on a slide, under microscope using oil immersion objective. The no. of cells are calculated by the DMC formula (as described previously).

Table 4.2: Evaluation of direct microscopic count

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4.3 Preservation of yoghurt cultures in freeze dried ampoules

Two flask of 100ml whey based medium was prepared, inoculum was added at 2% level and then the flasks were incubated at 370C for 10 hours of incubation. And the cells were harvested by centrifugation. Then pellet was dissolved in sodium glutamate milk. After this liquid concentrated was transferred to glass ampoules and each ampoule is capped with sterile cotton cloth cap, after this the glass ampoules were loaded on centrifugal head for primary drying. Drying is unloaded and centrifugal head is removed and then cotton plugs are replaced by non absorbent cotton wool. And then the ampoules are constricted at a point just above the upper end of cotton plug. The centrifuge head is removed and replaced with the secondary manifold. Ampoules are attached to drying manifold for drying at least for 2 hours or overnight. Culture was preserved by freeze drying and this can be used in preparation of yoghurt

Table 4.3 Growth performance of preserved yoghurt culture in freeze dried ampoules

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4.4 Production of yoghurt culture biomass and preservation as frozen concentrated and freeze dried forms

200 ml whey based medium was prepared and then inoculated with 2% of yoghurt mix culture and then incubated for 10 hrs at 370C and then cells were harvested by centrifugation. Pellet was dissolved in 20 ml of cryoprotective solution i.e. sodium glutamate milk. And then the culture was preserved by two methods one is freeze drying as described above and other is method is DVS preparation, in this sodium glutamate suspended culture was poured in petri plate and freezed at -200C for overnight and after this culture was dried in freeze dryer under vacuum. And then cultured was preserved in powdered form.

4.5 Evaluation of preserved yoghurt cultures in preparation of yoghurt

Yoghurts were prepared using fresh culture (A), concentrated liquid culture (B) and freeze dried concentrated culture (C) and evaluated for sensory, physicochemical and sensory properties in order to assess the suitability of preserved cultures in preparation of yoghurt as compared to fresh culture. Bulk cultures may be prepared separately from pure strains or frozen concentrates may be added directly to the mix. The latter eliminates the need to maintain culture transfer facilities (Kosikowski and Mistry, 1997). Yoghurt culture which is a mixture of Lactobacillus delbrueckii spp. bulgaricus and Streptococcus thermophilus used for the preparation of yoghurt for consumer acceptance and storage studies.

Table 4.5 Sensory characteristics of yoghurt prepared with fresh and preserved cultures during storage

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Table 4.6 Physicochemical characteristics of yoghurt prepared with fresh and preserved cultures during storage

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Table 4.6: Microbial analysis of fresh yoghurt samples

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Yoghurt by nature is a high-acid (low pH) product and is therefore inherently protected against defects caused by most contaminating organisms. Furthermore, the high pasteurization temperature used in processing the mix eliminates most contaminating bacteria. A weak culture that is contaminated with organisms such as psychrotrophs and coliforms will lead to unclean, and, in extreme conditions, bitter flavors. Contaminating bacteria such as coliforms and Pseudomonas spp. possess a relatively high level of diacetyl reductase which degrades diacetyl (Elliker, 1945; Seitz et al., 1963) Yoghurt stored under refrigerated conditions was analyzed for microbial counts such as coliforms, yeast and molds and lactic acid bacteria (Table 4.6)

4. SUMMARY AND CONCLUSIONS

The present study was undertaken with an objective to Study of growth performance of yoghurt culture in whey based media, production of cultures biomass and preservation in concentrated form and evaluation of preserved cultures for preparation of yoghurt. Salient findings of the study are given below:

- Lactobacillus delbrueckii spp. bulgaricus and Streptococcus thermophilus were collected from NCDC, NDRI, Karnal.
- All the cultures were activated in sterilized skim milk tubes and then examined for their purity by staining and catalase test.
- Maintained and propagated in whey based media. Each culture was transferred in whey media at 2% level incubated at 37 ̊C for 15-16 hrs, to be used for further analysis.
- Growth performance of each culture was studied individually studied in whey based medium by pour plating method and direct microscopic count.
- After that yoghurt culture was prepared by mixing both the cultures in equal proportion in whey media and incubated at 370C for 8-10 hrs. Again growth performance of yogurt culture was studied by viable count and direct microscopic count.
- Yoghurt culture was preserved by freeze drying, ready to use for preparation of yoghurt. DVS was prepared by pouring sodium glutamate suspended culture in petri plate and freezed at -200C for overnight and after this culture was dried in freeze dryer under vacuum. And then cultured was preserved in powdered form.
- Preserved culture was used in preparation of yoghurt.
- 3 samples of yoghurt A, B, and C were prepared using fresh culture, concentrated liquid culture, and freeze dried culture were used.
- For sensory characterization samples were tested by consumers for flavour, texture, body and appearance, and overall acceptability. The samples prepared from concentrated liquid culture and frozen concentrated culture show equal overall acceptability as sample prepared from fresh culture.
- For physicochemical analysis samples were studied by pour plating method for Coliform count, yeast and mold count and lactic acid bacteria count.
- Preserved cultures showed equal overall acceptability as fresh culture so these cultures are ready to use for preparation of yoghurt.

Present study has led to the production of yoghurt culture in whey based media showed enhanced growth. Yoghurt culture preserved by freeze drying can be used in preparation of yoghurt and it has similar sensory and physicochemical properties. Preserved freeze dried culture is ready to use in the preparation of yoghurt.

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APPENDIX

7. Appendix

Preparation of broth and media:-

1. Sterilized skim milk:-

12 g of skim milk powder was reconstituted in distilled water and distributed in 5 ml quantities in test tubes and 30 ml test tubes. It was then sterilized at 1210C for 20 min at 15 psi.

2. MRS broth (deMan, Rogosa and Sharpe, 1960)

Ingredients g/l

Peptone 10.0

Lab-Lemco meat extract 10.0

Yeast Extract 5.0

D(-) Glucose 20.0

Tween 80 1ml

K2HPO4 2

Sodium acetate 5.0

Triammonium citrate 2.0

MgSO4.7H2O 0.2

MnSO4.4H2O 0.05

Bromcresol purple 0.04

Deionized water 1000ml

All the ingredients were dissolved in deionized water and sterlised at 121oC for 15 min at 15 psi.

3. M17 Broth

Ingredients g/l

Peptone from soymeal 5.0

Peptone from meat 2.5

Peptone from casein 2.5

Yeast extract 2.5

Meat extract 5.0

Lactose monohydrate 5.0

Ascorbic acid 0.5

Sodium β-glycerophosphate 19.0

Magnesium sulfate 0.25

Bromcresol purple 0.04

Deionized water 1000ml

All the ingredients were dissolved in deionizeded water and sterlised at 121oC for 15 min at 15 psi.

4. M17 Agar

Ingredients g/l

Peptone from soymeal 5.0

Peptone from meat 2.5

Peptone from casein 2.5

Yeast extract 2.5

Meat extract 5.0

Lactose monohydrate 5.0

Ascorbic acid 0.5

Sodium β-glycerophosphate 19.0

Magnesium sulfate 0.25

Agar-agar 12.75

Deionized water 1000ml

All the ingredients were dissolved in deionized water and sterlised at 121oC for 15 min at 15 psi.

5. MRS Agar (deMan, Rogosa and Sharpe, 1960)

Ingredients g/l

Peptone 10.0

Lab-Lemco meat extract 10.0

Yeast Extract 5.0

D(-) Glucose 20.0

Tween 80 1ml

K2HPO4 2

Sodium acetate 5.0

Triammonium citrate 2.0

MgSO4.7H2O 0.2

MnSO4.4H2O 0.05

Agar 15.0

Deionized water 1000ml

All the ingredients were dissolved in deionized water and sterlised at 121oC for 15 min at 15 psi.

6. Violet red blie agar

Ingredients g/l

Yeast Extract 3 g

Enzymatic Digest of Gelatin 7 g

Bile Salts Mixture 1.5 g

Lactose 10 g

Sodium Chloride 5 g

Neutral red 0.03 g

Crystal Violet 0.002 g

Agar 15 g

Deionized water 1000ml

All the ingredients were dissolved in deionized water and sterlised at 121oC for 15 min at 15 psi.

7. Potato dextrose agar

Ingredients g/l

Potato Infusion from 200 g 4 g*

Dextrose 20 g

Agar 15 g

Deionized water 1000ml

*4.0 g of potato extract is equivalent to 200 g of infusion from potatoes .

All the ingredients were dissolved in deionized water and sterilized at 121oC for 15 min at 15 psi. After autoclaving media was acidified by addition of tartaric acid at 1% level.

8. Composition of whey based medium

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All the ingredients were dissolved in distilled water and sterilized at 121oC for 15 min at 15 psi.