Chemical and Microbiological evaluation of Set yoghurt during shelf life. SalahEldein Hussein Mohammed

Chemical and Microbiological evaluation of Set yoghurt during shelf life By SalahEldein Hussein Mohammed B.Sc. (Honour) Natural Resources and Environmental Studies University of Kordofan (2005) A thesis Submitted in Partial Fullilment for the Requirements of the Degree of M.Sc in Dairy Production and Technology Supervisor Dr. Mohamed Osman Mohamed Abdalla Department of Dairy Production Faculty of Animal Production University of Khartoum November 2008

Dedication To the soul of my mother To my dear father, brothers and sisters To my friends and colleagues ftät{xäwx Ç i

ACKNOWLEDGMENTS Praise be to Allah I wish to express my sincere thanks and gratitude to Dr. Mohamed Osman Mohamed Abdalla for his helpful supervision, constructive criticism and valuable guidance throughout this work. My special thanks are due to Dr. Ibtisam Elyas Mohammed Elzubeeir, head Department of the Dairy Production for her continuous support, encouragement and kindness. My appreciation is due to Dr. Mohammed Khair Abdalla for his cooperation in choosing the suitable experimental design and analysis of the data and unlimited help freely offered to me. My thanks are due to Mr. Moawia Albirer and Best factory family for their help during collection of samples. Also my thanks are extended to my colleagues in Dairy Production and Technology, Course. Special thanks are due my friends, Mortada Mohammed, Ibrahim Homeda and Rania Abd el Gader, I wish to express special thanks to my family. ii

LIST OF CONTENTS Page DETECTION i ACKNOWLEDGMENT ii LIST OF CONTENTS iii LIST OFTABLES v ABSTRACT vi ARABIC ABSTRACT vii CHAPTER ONE: Introduction 1 CHAPTER TWO: LITRETURE REVIEW 3 2.1 Fermentation and fermented milk products 3 2.2 Starter cultures 4 2.3 Types of yoghurt 5 2.4 Manufacture of yoghurt 7 2.5 Preparation of the basic mix 9 2.6 Homogenization 9 2.7 Heat treatment of milk 9 2.8 Production of set yoghurt 10 2.9 Cooling of yoghurt 10 2.10 Packaging of yoghurt 11 2.11 Yoghurt properties 11 2.12 Fermentation and microbiological aspects 13 2.13 Shelf life of yoghurt 13 2.14 Defects of yoghurt 14 2.15 Nutritional value of yoghurt 15 CHAPTER THREEMATERIALS AND METHODS 16 3.1 Collection of samples 16 3.2 Chemical analysis of yoghurt 16 3.2.1 Determination of total solids content 16 3.2.2 Determination of titratable acidity 16 iii

3.2.3 Determination of fat content 17 3.2.4 Determination of protein content 17 3.3 Determination of wheying off 18 3.4 Microbiological examination 18 3.4.1 Sterilization of equipment 18 3.4.2 Preparation of media 18 3.4.2.1 Violet Red Bile Agar 18 3.4.2.2 Lactobacillus MRS (Man Rogosa and Sharpe) Agar 19 3.4.2.3 Acidified potato dextrose agar (P.D.A) 19 3.5 Culture Methods 19 3.6 Statistical analysis 20 CHAPTER FOUR RESULTS AND DISCUSSION 21 4.1 Chemical composition of yoghurt 21 4.2 microbiological properties of set yoghurt 22 Table (1) fat contents and protein contents of set yoghurt samples during shelf life. 23 Table (2) Total solids contents and Acidity percentage of set yoghurt samples during shelf life. 24 Table (3) Wheying off percentage separated during shelf life 25 Table (4) Coliform count of Set yoghurt samples during shelf life 27 Table (5) Yeasts and Molds count of set yoghurt samples during shelf life 28 Table (6) Lactobacillus bulgaricus set yoghurt samples during shelf life 29 CHAPTER SIX CONCLUSION AND COMMENDATIONS 30 Conclusion 30 Recommendations 30 REFERENCES 31 iv

LIST OF TABLES Table page page 1 Fat and protein contents of set yoghurt samples during shelf life. 23 2 Total solids contents and Acidity percentage of samples during shelf life. 24 3 Wheying off percentage separated during shelf life 25 4 Coliform count of Set yoghurt samples during shelf life 27 5 Yeasts and Molds count of set yoghurt samples during shelf life. 28 6 Lactobacillus bulgaricus set yoghurt samples during shelf life 29 LIST OF TABLES v

ABSTRACT A study was performed to determine the effect of shelf life on chemical and microbiological characteristics of set yoghurt manufactured in Khartoum State. Samples were collected and kept in the laboratory at refrigerator temperature. The chemical analysis was carried out to determine the contents protein, fat and total solids contents. The physiochemical analysis included titratable of acidity and wheying-off. Whereas should be analyzed and microbiological examination (coliform, lactobacillus bulgaricus and yeasts and molds counts) were performed at 1, 3, 6 and 9 day intervals. Results revealed that protein, fat, total solids and acidity and whey-off did not show any significant difference (P>0.05) effects throughout the storage period. Results of microbiological examination showed non significant variation (P>0.05) in the count of coliform bacteria, yeasts and molds and Lactobacillus bulgaricus throughout the shelf life of 9 days. vi

و أ المستخلص أجري ت ه ذه الدراس ة لتق يم الت ا ثير الكيمي اي ي والميكروب ي للزب ادي المتماس ك الخث رة خ لال فت رة ال صلاحية. جمع ت عين ات الزب ادي م ن م صنع لت صنيع الل بن ومنتجات ه بولاي ة الخرط وم و حفظ ت عل ى درج ة حرارة الثلاجة. أش تملت الدراس ة عل ى التحلي ل الكيم اي ى لمعرف ة ) ن سبة الب روتين ال دهن والجوام د الكلي ة ). و التحلي ل الفيزي اي ي لتحدي د ن سبة الحموض ه ون سبة ال شرش آم ا ت م تحلي ل العين ات لمعرف ة الت ا ثير الميكروبيول وجي بواس طة الع د الكل ى لك ل م ن الكلوروف ورم بكتيري اء لاآتوباس لس بلقاريكس والخماي ر والفطريات خلال فترة الصلاحية (عشرة أيام). أوض حت النت اي ج أن ن سبة الب روتين ال دهن الجوام د الكلي ة الحموض ة ون سبة ال شرش ل م تت ا ثر معنوي ا خ لال فت رة ال صلاحية (0.05<P) آم ا ض حت النت اي ج أن الع دد الكل ى للبكتري ا والبكتري ا المنتج ة لحم ض اللاآتي ك والخم اي ر والفطري ات ل م تت ا ثر معنوي ا خ لال فت رة ال صلاحية.(P>0.05) vii

CHAPTER ONE Introduction Yoghurt is a fermented milk product obtained by fermentation of lactose to lactic acid by the action of two types of lactic acid bacteria Streptococcus thermophilus (S. salivarius-ssp. thremophilus) and Lactobacillus bulgaricus (L. delbrueckii ssp. bulgaricus) and these two microorganisms must live in large numbers in the final product (Tayfour, 1994). Yoghurt is an extremely popular fermented milk food in Europe, Asia and Africa. It is known by quite different names in different parts of the world: Leben or Raib (Egypt- Saudia Arabia) Madzoon or Matzoon (Armania), Naja (Bulgaria) and Dalia (India) (Kosikowski, 1966). In many developing countries of Asia and Africa, yoghurt is more likely to be produced as naturally soured milk and consumed by the adult people more than fresh whole milk. It is generally considered a safe product and its unique flavor appeals to so many people. That consideration is incorporate in expensive nutrients make it an almost complete food in these areas.in particular, consumers in the Americans are now more aware of the fine properties of yoghurt, and its consumption is increasing particularly in large cities (Kosikowski,1966). It is produced in different forms such as whole milk yoghurt, skim milk yoghurt, cream yoghurt, fruit yoghurt and liquid yoghurt (Balasubramanyam and Kulkarnis, 1991). In 1973, Food and Agriculture Organization (FAO) and World Health Organization (WHO) of the United Nations put certain standards for types of yoghurt according to their fat content, and hence, yoghurt was classified into whole fat (above 3.0%), moderate fat (3.0-0.5%) and low fat yoghurt 1

(<0.5%), set yoghurt and liquid yoghurt (of low viscosity) (Tamime and Deeth, 1980) and these may be plain (natural) or they may contain additives like fruit or flavors, or it may be coloured (Tayfour, 1994). A major concern of yoghurt industry is the production and maintenance of a product with optimum consistency and stability. The factors known to improve consistency are increasing total solids, manipulation of processing variables and characteristics of starter culture. The objectives of study are: 1- Evaluation of chemical characteristics of set yoghurt during shelf life. 2- Evaluation of microbiological quality of set yoghurt during shelf life. 2

CHAPTER TWO LITRETURE REVIEW 2.1 Fermentation and fermented milk products Fermentation of milk was originated in the Near East and through Central and Eastern Europe. Fermentation is defined as any modification of chemical or physical properties of milk or dairy products, resulting from the activity of microorganisms or their enzymes which cause the main marked changes (Frank and Marth, 1988). Moreover, the earliest example of fermented milk was warm, raw milk from cows, sheep, goats, camels or horses of the nomads roaming the area (Elmardi, 1988). Fermentation was first used around ten thousand years ago when humans made the transition from food collectors to food producers and was a common method to extend the longevity of dairy products. Today, this process remains the same except that Streptococcus themophilus and Lactoobucillus bulgaricus can be used in the fermentation of yoghurt (Tamime and Robinson, 1991). Fermented milk products are cultured dairy products made from skim, whole or slightly concentrated milk that require specific lactic acid bacteria to develop their characteristic flavor and texture (Thapa, 2000). Fortunately, the dominant bacteria were lactic acid Streptococci and lactobacilli, which generally suppress the spoilage and pathogenic organisms very effectively (Kosikowski, 1982). Moreover, desired alternation of food microorganisms are referred to as fermentation regardless of the type of metabolism (Banwart, 1981). Similarly, fermentation is defined as anaerobic breakdown of an organic substance by enzyme in which the final hydrogen acceptor is an organic compound. These products include cultured butter milk, sour cream, yoghurt, acidophilus milk, kefir and concentrated fermented milk products 3

(Hargrove and Alford, 1972). Moreover, production of good fermented milk required a source of good flavored, low bacteria in milk, heat treatment of the milk, an active properly functioning appropriate starter, quick chilling of fermented product and high standard sanitation (Kosikowski, 1982). 2.2 Starter cultures The starter culture, Lactobacillus bulgaricus and Streptococcus thermophilus, are required for the fermentation in yoghurt production (Vedamuthu, 1991). The classical yoghurt starter culture is a mixture of Streptococcus thermophilus and Lactobacillus delbruechkii ssp. bulgaricus, with a coccirods ratio of usually 1:1 (Hasan and Frank, 2001; Hutkins, 2001). L. bulgaricus is a lacitic acid bacterium that has been found to be relatively sensitive to low temperatures. According to Vedamuthu (1991) this microorganism is usually seen as rod shaped and although typically slender, may be curved in pairs or chains. As a homofermentative thermophile, L. bulgaricus is known to be relatively tolerant to heat, with an optimum growth temperature of approximately 45 50 º C (Rasmussen, 1981; Rybka and Kailasapathy, 1995; Vedamuthu, 1991). L. bulgaricus is of strong tolerance for oxygen, therefore it is considered to be a relatively slow growing organism, due to lack of oxygen. As a result of this, until the oxygen levels are reduced during fermentation, this microorganism will not grow rapidly (Marth and Steele, 1998). The other starter microoganism involved in yoghurt production, S. thermophilus is a spherical-shaped, typically found in pairs or long chains and is noted for the ability to withstand higher temperatures contributing to the classification of homofermentative thermophile (Vedamuthu, 1991). The thermal resistance is demonstrated in the evidence of S. thermophilus survival 4

during heating at 60 º C for 30 minutes and characterized by the used of these bacteria in various high temperature fermentations (Wilkins et al., 1986). Despite being able to survive at higher temperatures, S. thermophilus fails to grow at 10 º C, separating this microorganism from other streptococci that grow at lower temperature (Marranzini, 1987). Other apparent attributes aside from optimum, minimum and maximum growth temperature, include the inability of S. thermophilus to grow in high salt concentrations and the inability to produce ammonia from arginine (Marth and Steele, 1998). These attributes are important for identification, since S. thermophilus does not possess a necessary antigen for serological identification and therefore, physiological techniques must be utilized (Marranzini, 1987). S. thermophilus and L. bulgaricus exist in a complex cooperative relationship in yoghurt in which one bacterium produces stimulatory agents for the other (Tamime and Deeth, 1980; Wilkins et al., 1986; Vedamuthu, 1991). L. bulgaricus has been shown to produce certain amino acids such as valine, leucine and histidine, which are essential for S. thermophilus to grow. These amino acids are the result of proteolysis of casein by L. bulgaricus (Marranzini, 1989; Abu-tarboush, 1996). S. thermophilus in turn encourages the growth of L. bulgaricus by producing formic acid and carbon dioxide (Matalon and Sandine, 1986; Rajagopal and Sandine, 1990). 2.3 Types of yoghurt The various types of yoghurt differ according to their chemical composition, their method of production, their flavour and the nature of postincubation processing. The legal or proposed standards for the chemical composition of yoghurt in various countries are based on three possible types of yoghurt classified according to fat content (full, medium or low) (FAO/WHO, 1973). Such classification is used in composition standards to 5

facilitate standardization of product and protect the consumer. However, there are two main types of yoghurt, set and stirred, based on the method of production and on the physical structure of the coagulum. Set yoghurt is the product formed when fermentation/ coagulation of milk is carried out in the retail container, and the yoghurt produced is in continuous semi-solid mass, while, stirred yoghurt results when the coagulum is produced in bulk and the gel structure is broken before cooling and packaging (FAO/WHO, 1973). Fluid yoghurt may be considered as stirred yoghurt of low viscosity, since viscosity is mainly governed by the level of solids in the basic mix, fluid yoghurt can be produced by making stirred yoghurt from a mix with a low level of total solids, e.g. 11% (Rousseau, 1974). To make a good quality product, raw milk used must be low of bacterial count free from antibiotics, sanitizing chemicals, mastitis milk and colostrum and the milk also should be free from contamination by bacteriophages (Thapa, 2000). Traditionally, fluid yoghurt is manufactured by mixing equal quantities of yoghurt and water (Fig 1:1) and one of the main characteristics of such a product is the separation of the solid and the whey phases, hence, it is customary to shake the product before consumption (Tamime and Deeth, 1980). Falvouring of yoghurt is another method often used to differentiate various types of yoghurt. Flavoured yoghurts are basically divided into three categories (plain or natural, fruit and flavored yoghurt). The first type, plain or natural is the traditional yoghurt. Sometimes the sharp acidic taste of the natural product is masked by addition of sugar (Tamime and Deeth, 1980). Fruit yoghurts are made by addition of fruit, usually in the form of fruit preserves, puree or jam (Tamime and Deeth, 1980). However they added that falvoured yoghurt is prepared by adding sugar or sweetening agents and synthetic flavorings and colourings to plain or natural yoghurt. The post- incubation processing of yoghurt may lead to many different types of yoghurt. The emergence of new 6

yoghurt products has been largely due to recent developments in this field. Typical examples of these are pasteurized/ UHT yoghurt, concentrated yoghurt, frozen and dried yoghurt. These products may vary considerably in chemical composition, physical characteristics, and heat-treatment after incubation a process which leads to destruction of yoghurt starter bacteria and reduction in the level of volatile compounds associated with the flavour of yoghurt (Tamime and Deeth, 1980). Partial separation of the liquid phase from yoghurt leads to production of concentrated/condensed yoghurt of around 24% total solids (Tamime and Robison 1988; Robinson, 1977). Frozen yoghurt which can be either soft or hard, is a product whose physical state resembles ice-cream rather than yoghurt, while dried yoghurt can be produced by sun-drying, spray-drying or freeze-drying (Tamime and Robison 1988). 2.4 Manufacture of yoghurt The basic stages of production are common to all systems, and these stages are preparation of a basic process milk with 12-14% milk solids-nonfat (MSNF), heating milk to 85-95 o C for 10-20 minutes, inoculating the milk with a culture in which Lactobacillus bulgaricus and Streptococcus thermophilus are principal organisms present, incubating the inoculated milk at 42 o C until a smooth coagulum has formed together with the desired level of acidity and flavour, cooling the finished product and, unless the milk has been incubated in retail size (set yoghurt ), mixing with fruit or other ingredients and packaging the stirred yoghurt in containers prior to dispatch under chilled conditions (Robinson,1986). 7

Cool to incubation temperature Fig (1-1) Industrial production of various types of yoghurt (Tamime and Deeth, 1980) 8

2.5 Preparation of the basic mix The preparation of the basic mix involves the fortification and /or standardization of milk to achieve the desired rheological properties of the manufactured product. The most popular method of fortification is the addition of milk powder to liquid milk (Robinson, 1983). The use of ultra filtration to concentrate the solids in skim milk is being considered as a feasible alternative (Bundgaard et al., 1972). The aim of total solids is the consistency improvement imparted to the yoghurt coagulum (Robinson, 1983). To make a good quality product, raw milk used must be of low bacterial count, free from antibiotics and sanitizing chemicals, free from mastatic milk and colostrums and free from contamination by bacteriophages (Thapa, 2000). 2.6 Homogenization Is the stage that follows fortification and this treatment affects the lipid fraction and these changes tend to impact a rather smooth texture to the coagulum (Banks et al., 1981). It is also reported that homogenization can reduce the incidence of pips in yoghurt (white flacks that appear in some fruit varieties) (Davis, 1967; Robinson, 1981). 2.7 Heat treatment of milk Milk is heat treated by three different methods: vat treatment (85 C for 10-14 min), high temperature short time treatment (HTST) (98 C for 0.5 1.87 min) and ultra high temperature treatment (UHT) (140 C for 2-8 sec). The physical and sensory properties of yoghurt were substantially affected by method of heat teartment and exposure time. The most viable heating process investigated was HTST with a residence time of 1.87 min (Parnell-Clunies et al., 1986). 9

Hong and Goh (1979) found that yoghurt from milk heated at 85 Cwas harder than that from milk heated at 75 C or 95 C, and it received highest subjective scores for appearance, aroma and flavour. Cultured yoghurt from UHT processed whole milk (149 C for 3 sec) was lower in gel hardness and apparent viscosity but higher in spreadability and fluidity than yoghurt processed by conventional vat system (82 C for 30 min). UHT processing of milk indicated a potential method for commercial manufacture of yoghurt with consistency or low curd firmness (Labropoulos et al., 1984). Grgurek (1999) found that post acidity action was slightly higher in yoghurts prepared with lower amounts of inoculums and he concluded that the incubation temperature should be properly maintained to achieve maximum viability. 2.8 Production of set yoghurt In set yoghurt the coagulation of milk takes place in the retail containers, and hence the process involves cooling the heated milk to incubation temperature, adding the starter culture and appropriate flavoring and coloring ingredients, and filling into the retail containers. For sundae style yoghurt, the fruit is placed in the container before the milk, followed by incubation of the product to achieve the desired acidity (Robinson, 1986). 2.9 Cooling of yoghurt When yoghurt reaches the required acidity (i.e. 0.8-1.0% lactic acid) cooling of the coagulum commences, and the intention is to reduce the temperature of the coagulum to below 20ºC within an acceptable time span. Thus below 20ºC the metabolic activity of the starter organisms is sufficiently reduced to prevent yoghurt from becoming unpalatable due to excessive acidity and hence initiation of cooling depends on. The level of lactic acid required in the end product is usually between 1.2% and 1.4% lactic acid, 10

and the rate of cooling that can be achieved with the available equipment, and in amanner that does not damage the texture of yoghurt (Robinson, 1981). 2.10 Packaging of yoghurt It is an important step during production the purpose of which can be summarized as follows: protection of the product against dirt microorganisms and the environment (e.g. gases) and light, providing relevant information to the consumer (e.g. name and origin of the product ingredient instructions expiry date). The packaging material must be non-toxic, and no chemical reactions should take place between the material and the product (Robinson, 1986). 2.11 Yoghurt properties Yoghurt properties are influenced by the level of total solids in the milk. This is important for both the consistency and aroma of the manufactured yoghurt. In general, an increase in total solids will enhance these properties (Emmon and Tuckey, 1967). The level of total solids in milk varies from 9% in skim milk yoghurt to over 20% in other types of yoghurt (Tamime and Deeth, 1980). Kozhev et al. (1972) concluded that the best yoghurt is made from milk containing 15.0-16.0% total solids. Although the composition of commercial yoghurt varies considerably, most low fat yoghurt falls within the range of 14-15 % total solids. The level of total solids also affects the titratable acidity of the milk mix due to the buffering action of proteins, phosphates, citrates and other miscellaneous milk constituents (Tamime and Deeth, 1980). An increase in total solids results in an increase in the titratable acidity and reduction in the coagulation time (Humphreys and Plunkett, 1969). The viscosity of yoghurt is almost wholly dependent on protein content of milk. Hence a high protein concentration is essential for the 11

production of viscous yoghurt. However, Tamime and Deeth (1980) concluded that the addition of milk powder raised the protein level of milk. Atamer et al. (1996), showed that titratable acidity as lactic acid was higher in yoghurt samples with high total solids. If a full fat (> 3% fat) set yoghurt is made then homogenization of the mix is essential to prevent separation of cream line during incubation, but even for low fat varieties, homogenization is reported to offer certain advantages, since it reduces the average diameter of the fat globules to < 2µm, the viscosity of yoghurt is improved, the syneresis is reduced and the process ensures uniform mixing of any dry particles added to milk (Robinson, 1986). Alexiou et al. (1988) found that after homogenization of milk both consistency and organoleptic properties of yoghurt were significantly improved. The optimum conditions for heat treatment are 80-85ºC with a holding time of 30 min (Galesloot, 1969). The effect of heat treatment can be summarized as follows: there is denaturation of whey proteins (albumins and globulins) and an aggregation of casein molecules into a three dimensional network. This network traps the whey proteins and the yoghurt coagulum produced subsequently is rendered more viscous (Janness and Patton, 1959). The bacterial load in milk is reduced, and hence the starter culture has less competition from adventitious organisms (Robinson, 1983). There is reduction in the amount of oxygen in the milk (Ling, 1963) and as the normal yoghurt cultures are microaerophilic, the lowered oxygen tension encourages their growth (Robinson, 1983). Some limited damage to the milk proteins may occur during heating and the breakdown products can stimulate starter activity (Greence, 1957). The quantity of metabolic compounds produced by combined cultures was higher than that produced by single strain (Aslim, 1998). 12

After the incubation period, yoghurt is cooled in order to control the level of lactic acid in the final product. The rate of cooling can affect the structure of the coagulum. Thus, very rapid cooling can lead to whey separation due to rapid contraction of the protein filaments which, in turn, affects their hydrophilic properties (Rasic and Kurmann, 1978). 2.12 Fermentation and microbiological aspects The classification of lactic acid bacteria by Orla Jensen (1931) was still recognized as the standard method of classifying these organisms. In the 7 th edition of Bergey, s Manual (1957) all these bacteria were grouped in the family Lactobacillacaeae, which was subdivided into two tribes, Streptococcaeae (coccal shaped) and Lactobacillaceae (rod shaped). However, in the 8 th edition of Bergey, s manual (1974), lactic acid bacteria were reclassified into two families the Streptococcaceae and Lactobacillaceae. The yoghurt starter bacteria, S. thermophilus and L. bulgaricus are thermophilic, homofermentative lactic acid bacteria. Moreover S. thermophilus is distinguished from other members of the genus Streptococci by its growth at 45 o C and failure to grow at 10 o C. Unlike most Streptococci, this species lacks a recognized group antigen for serological identification and has to be identified by physiological procedures (Bergey, s Manual, 1974). Classification of S. thermophilus is well documented and clear cut, in contrast, the classification of L. bulgaricus and it is relationship to other Lactobacillus species have been the subject of some controversy (Davis, 1975). 2.13 Shelf life of yoghurt The end of shelf life of food is ultimately assessed by sensory failure. The determination in sensory quality is quantified by monitoring changes in specific attributes or re-education in over all quality acceptability using different tests that utilize different scales of measurement (Gacula, 1975). 13

These differences in the logistics of measurement, coupled with proportion of sensory quality loss perceived to be tolerated by consumers (Piga et al., 2000). Shah and Ravula (2002). found that increased sugar lead to an increase of fermentation time and decrease in yoghurt shelf life. The shelf life of yoghurt can be prolonged based on the type of storage container used in packaging (Filardo, 2005). 2.14 Defects of yoghurt The main yoghurt defects can be classified into three categories, flavour, appearance and texture defects (Tayfour, 1994) Flavour defects: bitterness caused by keeping for long time that leads to excess activity of culture, Hydrolysis of protein by proteolytic bacteria and yeasts, lack of taste and flavour caused by bad activity of culture (imbalance), incubation for short time at low temperature or low level of total solids content. Low activity level caused by activity of culture (low incubation level insufficient incubation or, at low temperature presence of inhibitors bacteriophages in milk, excess acidity caused by bad fermentation process. Long incubation period or at high temperature, low or insufficient cooling, keeping at high temperature and harsh acid flavour caused by bad culture. Appearance defects: Whey separation caused by excess acidity due to excess incubation or high temperature in storage, extra keeping period- low cooling-extra mixing (stirred yoghurt-use of extra pump-bad fruit mixingagitation of set yoghurt low level of total solids and gas production which are caused by infection by yeasts or coliforms. Texture defects: curd separation caused by agitation during transportation immediately after bad cooling in a cold room (set yoghurt), low curd 14

consistency caused by low level of incubation (short time or low temperature), agitation before complete coagulation, very liquid yoghurt caused by strong mixing, bad incubation (short- time), low level of total solids (low viscosity production - unconcentrated flavours or fruits) (Tayfour, 1994). 2.15 Nutritional value of yoghurt In Sudan it is believed that yoghurt (zabadi) is useful for the treatment of stomach disturbances, and the individuals with such complaints are advised to eat yoghurt (Dirar, 1993). Children suffering from infantile diarrhea recovered more rapidly when fed yoghurt than those given neomycin-kaopectate (Shahani and Chardan., 1979). Lactic acid is biologically active and capable of suppressing harmful microorganism's especially putrefactive ones and so has a favourable effect on human vital activities (Oberman, 1985) and lactic acid was the only antimicrobial agent active against pathogens (Deeth and Tamime, 1981). Kilara and Shahani (1976) studied lactose or lactase activity of cultured and acidified dairy products including yoghurt and concluded that yoghurt culture released the enzyme lactase which converts lactase into lactic acid, and lactose intolerant people are adviced to consume there products with no subsequent problems. 15

CHAPTER THREE MATERIALS AND METHODS 3.1 Collection of samples Seventy five samples of yoghurt were obtained from one factory in Khartoum State during period from June to July 2007, and kept in the refrigerator (8 ± 2 C) under sterile conditions for 9 days and analyzed for chemical and microbiological properties. 3.2 Chemical analysis of yoghurt 3.2.1 Determination of total solids content Total solids content was determined according to AOAC (1990). Three grams of the sample were weighed into dry clean flat bottomed aluminum dish, and heated on a steam bath for 10 min. The dish was then cooled in a desiccator weighed and heating, cooling and weighing were repeated until the difference between two readings was < 0.1 mg. The total solids content was calculated from the successive equation: W T.S = 1 100 W Where: W1 = Weight of sample after drying. W = Weight of original sample 3.2.2 Determination of titratable acidity The acidity of yoghurt was determined according to AOAC (1990). Ten grams of sample were placed in a white porcelain dish and 5 drops of phenolphthalein indicator were added. Titration was carried out using 0.1N NaOH until a faint pink colour appeared. The titration figure was divided by 10 to get the percentage of lactic acid. 16

3.2.3 Determination of fat content Fat content was determined by Gerber method according to AOAC (1990) as follows: In a clean dry Gerber tube, 10 ml of sulphuric acid (density 1.815 gm/ ml at 20 º C) were poured and then 11 gm of a well mixed yoghurt smple was gently added. One ml of amyl alcohol (density 0.815 gm/ml at 20 º C) was added to the mixture, the contents were then thoroughly mixed till no white particles could be seen. Gerber tubes were centrifuged at 1100 revolutions per minutes (rpm) for 4 minutes and the tubes were then transferred to a water bath at 65 º C for 3 minutes. The fat percent was then read out directly from the fatcolumn. 3.2.4 Determination of protein content The protein content was determined according to Kjeldahal method (AOAC, 1990). In a dry clean Kjeldahal flask, 11 gms of yoghurt were added, then 25 ml of concentrated H 2 SO 4 were added follwed by addition of two Kjeldahal tablets (CuSO 4 ). The mixture was then digested on a heater until a clean solution was obtained (3 hours) and the flasks were removed and left to cool. The digested samples were poured into a volumetric flask (100 ml) and diluted to 100 ml with distilled water. The distillate was received in a conical flask containing 25 ml of 4% boric acid plus three drops of indicator (bromocresol green plus methyl red). The distillation was continued until the volume in the flask was 75 ml. The flasks were then removed from the distillator, and the distillates were then titrated against 0.1N HCl until the end point was obtained (red color). The protein content was calculated as follows: 17

Nitrogen (%) = T 0.1 0.014 20 100 Weight of sample Protein (%) = Nitrogen X 6.38 Where: T: Titration figure (ml). 0.1: Normality of HCl. 0.014: Atomic weight of nitrogen/ 1000. 20: Dilution factor. 3.3 Determination of wheying-off Wheying-off was determined by sucking the water on the surface of the curd and pouring in a graduated cylinder. Whey collected was then measured. 3.4 Microbiological examination 3.4.1 Sterilization of equipment Glassware such as flasks, test tubes, Petri-dishes, pipettes and bottles were sterilized in a hot oven at 170 C for two hours, whereas distilled water was sterilized by autoclaving at121 C for 15 minutes (Marshall,1992). 3.4.2 Preparation of media All media were obtained in a dehydrated form stored in a hygroscopic environment in a cool dry place, away from light and prepared according to the manufactures instructions. 3.4.2.1 Violet Red Bile Agar This medium was used to determine the coliform count (Harrigan and McCance, 1976). The medium (41.53gm) was dissolved in 1L distilled water heated to boiling and then sterilized by autoclaving at 121 C for 15 minutes (15 lb pressure), cooled to 45 ± 2 C and immediately poured into sterile Petri dishes containing the dilution. 18

3.4.2.2 Lactobacillus MRS (Man Rogosa and Sharpe) Agar This medium was used to determine Lactobacillus bulgaricus counts. It was obtained in a dehydrated form (Hi Media Laboratories pvt.ltd). The medium (55.15 gram) was dissolved in 1 litre distilled water, heated to boilng till dissolved completely and sterilized by autoclaving at 121 C for 15 minutes, cooled to 45±1 C 3.4.2.3 Acidified potato dextrose agar (P.D.A) The medium consisted of 200 gms potatoes infusion form, 20 gms dextrose and 15 gms agar. The medium was prepared by dissolvind 39 gm of powder in one litre of distilled water, followed by boiling to dissolve the medium completely and it was sterilized by autoclaving at 121 º C for 15 minutes and cooled to 46 º C, then enough sterile 10% tartaric acid was added 3.5 Preparation of dilutions Eleven grams from a homogenous yoghurt sample were added to 99 ml of sterile distilled water in a clean sterile flask, then shaked until a homogenous dispersion was obtained to make 10-1 dilution. One ml from the above-mentioned dilution (10-1 ) was aseptically transferred to 9 ml sterile distilled water. This procedure was repeated to make serial dilutions of 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8. Cultring method from each dilution, 1 ml was transferred to duplicate Petri-dishes and the culture medium was poured aseptically into each Petri-dish, mixed gently, left to solidify and incubated in an inverted position. The typical colonies in each Petri-dish were counted using a colony counter (Houghtby et al., 1992). 19

3.6 Statistical analysis All data were analyzed using one way ANOVA by means of statistical computer software (SPSS-13). Duncan Multiple Range test was used to determine the significance at P 0.05. 20

CHAPTER FOUR RESULTS AND DISCUSSION 4.1 Chemical composition of yoghurt Table (1) presents the effect of storage period on yoghurt during shelf life. The fat content started at high value of 3.73±0.04 at the beginning of shelf life, then decreased to 3.61±0.04 at day 3 and stayed at this level throughout the shelf life of 9 days (P>0.05). The mean values of fat percentage of yoghurt samples throughout shelf life showed non significant difference between means, and this is due to the manufacturing of sample from standardized of milk to fat content of 3.5%. This is supported by Soomro et al. (2003). The protein content did non significantly change (P>0.05) throughout the storage period, although the highest content was at day 3, 3.24 ± 0.035, then gradually decreased towards the end (3.12±0.035). The results of protein content were similar to those of Tarakci and Kücükoner (2003). Total solids content gradually increased from 13.87 ± 0.14 at the beginning to a maximum at day 6, (14.59±0.14) and slightly decreased at the end, although the variation was not significant (P>0.05) Table (2). These results are in line with the findings of Younus et al. (2002), there was hardly any variation in total solids content in different samples of yoghurt, due to balanced standardization of milk and quality control measures taken to ensure consistency of end product. The acidity of yoghurt steadily increased as shelf life of yoghurt progressed (P > 0.05). The acidity was 0.94±0.011 at day one, and gradually 21

increased to 0.99 ± 0.011 at day 3, 1.13±0.011 at day 6 and 1.22±0.011 at day 9 of storage. The results of acidity in this study are in accordance with the findings of Younus et al. (2002). Table (3) presents the amount of whey collected from yoghurt during shelf life Wheying-off of yoghurt was at minimum and did not significantly increase. The value was 0.76±0.16 at day one, decreased to 0.60±0.16 at day 6, then increase to 0.69±0.16 at day 9. Shukla et al. (1988) found that wehying-off is a major defect in yoghurt therefore stabilizers and additives of milk powder are usually used to check wheying-off in yoghurt. 4.2 Microbiological properties of set yoghurt Table (4) shows the effect of storage period on the count of coliform. The count was not significantly affected (P>0.05) by storage of yoghurt although coliform bacteria started high 2.77±0.07 at day one, decreased to minimum 2.38±0.07 at day 3, then increased to 2.55±0.07 at the end of storage period. Coliform examination for yoghurt samples collected revealed no significant variation between different samples through shelf life of product. These results were higher than what reported by Al-tahir (2005) and Younus et al. (2002). In most of yoghurt samples coliform bacteria were present which might be due to improper pasteurization of pre-mix prior to its incubation and some samples of yoghurt contained less count of coliform. It might be probably due to contamination at product. Similar results have been reported by Younus et al. (2002), who reported low number of coliform in yoghurt samples. 22

Table (1) fat contents and protein contents of set yoghurt samples during Shelf life. Shelf Fat content Protein content life(days) Mean ±SE Maximum Minimum Mean ±SE Maximum Minimum 1 3.73 a ± 0.040 5.00 3.11 3.15 a ± 0.035 3.56 2.67 3 3.61 a ± 0.040 4.10 3.00 3.24 a ± 0.035 3.70 2.65 6 3.61 a ± 0.040 4.00 3.10 3.12 a ± 0.035 3.56 2.61 9 3.61 a ± 0.040 4.00 3.00 3.12 a ± 0.035 3.84 2.57 S.L N.S N.S Means in the same column followed by the same letter (s) are not significantly different at (P = 0.05) N.S = Not Significance S.L = Significance Level SE = Standard Error 23

Table (2) Total solids contents and Acidity percentage of set yoghurt samples during Shelf life. Shelf Total solids Acidity (% lactic acid) life(days) Mean ±SE Maximum Minimum Mean ±SE Maximum Minimum 1 13.87 a ± 0.144 16.24 12.63 0.94 a ± 0.011 1.47 0.87 3 14.05 a ± 0.144 16.06 12.34 0.99 a ± 0.011 1.85 0.73 6 14.59 a ± 0.144 17.00 12.90 1.13 a ± 0.011 1.37 0.91 9 14.29 a ± 0.144 16.85 12.68 1.22 a ± 0.011 1.43 0.89 S.L N.S N.S Means in the same column followed by the same letter (s) are not significantly different at (P =0.05). N.S = Not Significance S.L = Significance Level SE = Standard Error 24

Table (3) Wheying-off percentage separated during Shelf life. Shelf life(days) 1 3 6 9 S.L Mean ± SE 0.76 a ± 0.16 0.68 a ± 0.16 0.60 a ± 0.16 0.69 a ± 0.16 N.S Means in the same column followed by the same letter (s) are not significantly different at (P = 0.05). N.S = Not Significance S.L = Significance Level SE = Standard Error 25

Yeasts and molds count did not significantly change (P > 0.05) during storage period. However the count decreased to a minimum of 1.17 ± 0.14 at day 6, then increased to 1.28 ± 0.14 at the end of storage period (Table 5). The results revealed higher mean yeasts and molds count, and this result does not conform to the Sudanese Standards, which stated that yeasts and molds count should be less than Log 10 1.0 cuf/gm. Lactobacillus bulgaricus count increased from 7.24 ± 0.8 at the beginning to 7.88 ± 0.8 at day 6, then slightly decreased to 7.82 ± 0.8 towards the end of shelf life of yoghurt (Table 6). These results are in line with the findings of Mohammed et al. (2007) who found that in Japan the fermented milk and lactic beverage association has established a standard that requires the presence of >10 7 viable bacteria/ml dairy product. Moreove These results are in accordance with findings of Ministerio de La presidencia de Espana (2003) who found that the Swiss food required that such products contain 10 6 cfu/g and the Spanish Yoghurt Quality Standard requires 10 7 cfu/ml. 26

Table (4) Coliform count of Set yoghurt samples during Shelf life. Shelf life (days) Mean ± SE Maximum Minimum 1 2.77 a ± 0.065 2.89 2.64 3 2.38 a ± 0.065 2.50 2.25 6 2.69 a ± 0.065 2.87 2.56 9 2.55 a ± 0.065 2.68 2.42 S.L N.S Means in the same column followed by the same letter (s) are not significantly different at (P=0.05). N.S = Not Significance S.L = Significance Level SE= Standard Error 27

Table (5) Yeasts and molds count of set yoghurt samples during Shelf life. Shelf life (days) Mean ± SE Maximum Minimum 1 1.30 a ± 0.14 2.54 0.00 3 1.36 a ± 0.14 2.15 0.00 6 1.17 a ± 0.14 2.23 0.00 9 1.28 a ± 0.14 2.20 0.00 S.L N.S Means in the same column followed by the same letter (s) are not significantly different at (P=0.05). N.S = Not Significance S.L = Significance Level SE= Standard Error 28

Table (6) Lactobacillus bulgaricus set yoghurt samples during Shelf life. Shelf life (days) Mean ± SE Maximum Minimum 1 7.24 a ±0.8 7.99 0.00 3 7.85 a ±0.8 8.38 7.46 6 7.88 a ±0.8 8.23 7.64 9 7.82 a ±0.8 8.11 7.45 S.L N.S Means in the same column followed by the same letter (s) are not significantly different at (P = 0.05). N.S = Not Significance S.L = Significance Level SE= Standard Error 29

CHAPTER SIX CONCLUSION AND RECOMMENDATIONS Conclusion The results obtained during this investigation indicated that during shelf life of yoghurt the chemical composition and microbial quality did not significant difference. Recommendations: 1. Establishment of efficient programs for assurance of high quality product. 2. Quality and safety are needed to ensure marketing of products and consumer taste. 3. Application of hygienic control using system product. 4. Adjustment of yoghurt mix should approach the standard of the yoghurt package. 30

REFERENCES Abu-Tarboush, H.M. (1996). Comparison of associative growth and proteolytic activity of yoghurt starter in whole milk from camels and cows. J. Dairy Sci. 79: 366-371. Altahir, R. (2005). A comparison on microbial conditions between traditional dairy products sold in Karak and some products produced by modern dairies. Pakistan. J. Nutri, 4(5): 345-348. Alexiou, I., Kechagias, C., Konidari, P., lambrakos. M. and garifallidis,-n. (1988). Rheological properties of stirred yoghurt from goats milk. Deltio-Ethnikis- Epitropis- Galaktos-Ellado (creece) v. 5(1) p. 20-31.And sensory characteristics of some fruit-flavored yoghurt. YYÜ Vet Fak Derg.; 14 (2): 10-14. AOAC (1990). Official of method of analysis, 15th ed.association of Official Analytical Chemists, Washington DC, USA. Aslim, B. (1998). Studying on metabolic products and antagonistic effects of combined lactobacillus bulgaricus and streptococcus thermophilus cultures. Turk- Hijen -Ve Deneysed Biyoloji- Dergisi, 55:1, 17-23. Atamer, M.M.,Caric, M.D.J Milanovic, S.D., Gavaric,-D.Dj (1996). Quality of set yoghurt manufactured from UF milk. Zbornik- Matice- Srpske- zaprirodne-nauke (Yugoslavia) on- 91p. 19-26. Balasubraman yam, B.V. and Kulkarnis, C.L. (1991). Standardization of manufacture of high fat yoghurt with natural fruit pulp. J. Food Sci. Technol., 28(6): 389-390. Banks,W., Dalgleish,D.G. and Rooks, J.A.F.(1981). In Dairy Microbiology, vol, 1, Ed Roloinson,R. K Applied Science publishers ltd. London. P (251-252). Banwart, G.J. (1981). Basic microbiological, 2 nd edition NewYork, USA. PP. 112-135. Bergey, s, (1957). Bergey, s Manual of Determinative Bacteriology, 7 th ed. Ed Breed, R. S; Murray E. G. d. and Smith, N. R., Williams and Wilkins, Co., Baltimore, USA. 31

Bergey, s, (1974). Bergey, s, Manual Determinative Bacteriology, 8 th ed Buchanan. R. E. and Gibbons. N. E., Williams and Wilkins Co, Baltimore, USA. Bundgaard, A. G., Olson, D. J. and Madsen, R. F. (1972). Dairy Ind. 37, 539. Collado, L.S., Mabesa, R.C., Sumague, M.J.V. and Mock, C.K. (1994) Yoghurt like products from rige floor and soy milk. Philippine agriculturist (July-Sept.1994).V.77 (3), P307-319.IssuedJan 1995(Philippine) Davis, J.G. (1967). Dairy ice cream industries directory, 5.Union Trade press Ltd, London. Davis, J.G. (1975). Lactic acid bacteria in beverages and food. Carr, J.G. Cutting. C. V. and Whiting, G.C. ed. Academic press Inc.(london), NWI, UK.pp n 245-263. Deeth, H.C. and Tamime, A.Y. (1981). Yoghurt: Nutritive and therapeutic aspects. J. food prot., 44(1): 78-86. Development in Japan. Food Technol.; 47: 126-135. Dirar, A. H. (1993). The Indigenous fermented foods of the Sudan: A study in African food and Nutrition.CAB International, London, U.K. El-mardi, M.A. (1988). Astudy on fermented milk Rob M.Sc. thesis, University of khartoum sudan. Emmons, D. B. Tuckey, S. L. (1967). Cottage cheese and cultured milk products, vol.3, chas pfizer and co. Inc, New York, Cited by Tamime A.V.and Deeth, H.C.(1980). FAO/WHO. (1973). Codes of principle concerning milk and milk products CX 517016 th Session, Ibid. Filardo, C.(2005).Berries: A bounce of prevention. Food processing. Itasca, 1L. Frank, F.J. and Marth, H. E. (1988). Fermentation. In: Woug, N. P., Jennes, R., keency, M. and Marth, E. H.3 rd, (Ed) Van Wostrand Reinhdel. Newyork. Gacula, M.C. (1975). The designof experiment for shelf life study. J. Food Sci; 40:399-403. 32

Glesloot, (1969) Microbiology of fermented milk. In (the microbiology) of milk products vol (2) Edited by Robinson R.K.pp. (252). Greence. (1957).Microbiology of fermented milk (the microbiology of milk products Vol.(2)Edited by Robinson R.K.PP.(252) Grgurek. L. (1999). Effect of level of starter culture on viability of probiotic in yoghurt. Mljekarstvo (croatia).49(1): 3-8. Hargrove, R.E. and Macdonough, R.E (1972). Composition of milk products, In:Fundamentals of dairy chemistry, 2nd edition. Edited by Web, H.B.; Johnson, H.A. and Alford, A.J.the AVI Publishing company Inc.West port Connecticut, USA, PP.58-86. Harrigan, W.F. and McCance, Margaret, E. (1976). Laboratory methods in food and dairy microbiology. Academic press. London- pp25-29. Hassan, A.N. and Frank, J.F. (2001). Starter cultures and their use, in Marth E.H and Steele. J.L. Applied Dairy Microbiology. Marcel Dekker, New York, 151-206. Hong, B. J. and Goh, J. S. (1979). Effect of temperature and time of pasteurization and fermentation on quality of yoghurt. Korean Dairy Sci, 1(2): 7-12. Houghtby, G.A., Maturin, L.J and Koenig, E.K. (1992). Microbiological count Methods. In: standard methods for the Examination of Dairy products Marshall, R.T. (ed.), 16 th edition, Port city press, Baltimore, Washington. Humphreys, L.C and plunkett, M (1969). Yoghurt: Are view of its manufacture. Dairy Sci. Abstr., 31(11): 607-622. Hutkins, R.W. (2001). Metabolism of Starter cultures. In Marth, E.H and Steele, J.L. Applied Dairy Microbiology, Marcel Dekker, New York, 207-241. Jenness, R. and Patton, S. (1959). Principles of Dairy chemistry. New York Johan Wiley. Cited in DAS (1969). 31(11)607-622. Kilara, A and shahani, K.M. (1976). Lactose acidity of cultured and acidified dairy products, J. Dairy. Sci., 59(12) 2031-2035. 33

Kosikowski, F. (1966). Yoghurt. In: cheese and fermented milk foods department of dairy and food science, Cornell University, Ithaca, New York. Ksoikowski, F. (1982). Cheese and fermented Food 2 nd ed. Ksoikowski and Associates. New York, USA. Kozhev, A.; penelski, I-and panova, V. (1972). Bulgaria sour milk production empolying fermentation in bulk. DSA, 34 (9): 40-57. Labropoulos, A. E., Collins, W.F. and Stone, W.K (1984). Effect of ultra high temperature and vat process on heat - induced rheological properties of yoghurt. J. Dairy. Sci, 67(2): 405-409. Ling, E. R. (1963). A Text Book of Dairy Chemistry 3 rd Edition chapman & Hall Ltd. London. Marranzini, R.M. (1987). The effect of threonine and glycine levels on threonine aldolase activity of yoghurt microorganisms grown in a modified Gainesville, FL. Marshall, R.T. (1992). Standard Methods for Examination of dairy products, 16 th Ed. Public Health Assoc., Inc., Washington. Marth, E. H. and Steele. J. L. (1998). Applied Dairy Microbiology. Marcel Dekker, New York, 151-206. Matalon, M.E. and Sandine, W.E. (1986), Lactobacillus bulgaricus, Streptococuss thermophilus and yoghurt. Areview. Cult. Dairy prod. J.; 21: 6-12. Ministerio de La presidencies de Espuma (2003). Norma de Calidad del yoghurt. RD179 /2003, BOE18 de febreo, 2003. Mohammed.H. M.Haj, Osman A. O. Elowni and Ibtisam, E. M. Elzubeir Dep. Dairy production faculty of Animal production, University of Khartoum J. Ani. and Veter. Sci. 2: 56-60, 2007. Oberman, H. (1985). Fermented milks in: Microbiology of fermented. Foods Vol - 1 Edited by wood B.J.B., Elsevier Applied science publishers, London. 34

Oral- Jensen, S. (1931). Dairy bacteriology 2nd Ed., translated by Arup, P. S. J. and A. Churchill. UK. PP. 1-53 and 109-129. Organisms. Dairy food and sanitation. 1(7): 286-289. Parnell- Clunies, M.E., Kakuda, Y., Mullen, K., Arnott, R.D., and Deman, J. (1986). Physical properties of yoghurt: A comparison of vat versus continuous heating systems of milk. J. Dairy Sci., 69 (10): 2593-2603. Piga. A.; Aquino. S.; Aaabbio, M.; Emonyi, G and Farris, G.A. (2000). In fluence of storage temperture on shelf life of minimally processced cactus pear fruit lebnensw. Wiss Technol.;33: 15-20. Rajagopal, S. N. and Sandine, W.E. (1990). Associative growth and properties of Streptococcus thermophilus and lactobacillus bulgaricuc in skim milk. J. Dairy Sci. 73, 894. Rasic. J.L. and kurmann, J.A. (1978). Yoghurt-ScientificTechnology, manufacture and preparation, Vol. (1): pp39. Technical Dairy publishing house, Copenhagen, Denmark. Rasmussen, H. L. (1981). Enumeration, identification of cultured product organisms. Dairy food and sanitation. 1(7): 286-289. Robinson, R. K. (1977). Microbiology of fermented milk. In: The microbiology of milk products. Vol. (2) edited by Robinson, R. K. pp. 248. Robinson, R.K. (1981). Dairy microbiology, volume 2 microbiology of milk products, Elsevier Applied science publishers Ltd. London and New York. Robinson, R.K. (1983). The microbiology of milk products Vol. (2) Elsilre Applied science publishers Ltd. England. pp 245-252. Robinson, R.K. (1986). Modern Dairy Technology, volume 2: Advance in milk products, Elsevier Applied science publishers. London and New York pp 21-22. Rousseau, M. J. (1974). Microbiology of fermented milk. In: the microbiology of milk products. Vol. (2). Edited by Robinson, R. K. pp 247. Rybka, S. and Kailasapathy. (1995). The survival of culture bacteria in fresh and freeze-dried AB yoghurt. The Austr. J. of Dairy techn. 50: (2)51-57. 35