Mohammed Hassan Mohammed Haj Dr. Osman Ali Osman
ASSESSMENT OF CHEMICAL AND MICROBIOLOGICAL QUALITY OF STIRRED YOGHURT IN KHARTOUM STATE SUDAN By Mohammed Hassan Mohammed Haj B.Sc. (Honours) Animal Production University of Gezira 1999 A thesis submitted in partial fulfillment of the requirements for the Degree of Master of Dairy Production and Technology Supervisor Dr. Osman Ali Osman Department of Dairy Production Faculty of Animal Production University of Khartoum October - 2006 DEDICATION To the soul of my friend Zaki Eldein To my mother and father Hassan Badri To my brother Khalid and sisters. With love and respect Mohammed In the name of Allah, Most Gracious, Most Merciful. ACKNOWLEDGEMENT Praise be to Allah. I wish to express my sincere thanks and gratitude to Dr. Osman Ali Osman Elowni for his helpful supervision, constructive criticism, and valuable guidance throughout this work. My special thanks are due to Dr. Ibtisam Elyas Mohammed Alzubeir, the coordinator of the Dairy Production and Technology, M.Sc courses, for her continued support, encouragement and kindness. My appreciations is due to Dr. Abdelwahab, for his cooperation in choosing the suitable experimental design and analysis of the data and unlimited help freely offered to me. Iam indebted to the members of the Department of Dairy Production. Also my thanks are extended to the staff of Dairy Production laboratory, Faculty of Animal Production. My thanks are due to my colleagues in the Dairy Production and Technology, M.Sc. course Special thanks are due my friends, Nazar, Sami Elbushra, and Mohammed Abdullah. My gratitude and thanks are extended to Ibn Idris and Abdelbasit for their patience and unlimited help. My thanks extended to Mr. Zaki Eldein Ali Omer who started typing of this thesis and Mr. Abdelhamed Abdelrahim who completed the typing of this thesis, Finally, I wish to express special thanks to my family. ABSTRACT This study was carried out on stirred yoghurt samples purchased from the market. The stirred yoghurt was produced by Blue Nile Dairy Company (CAPO). Sixty samples were transported to the Faculty of Animal Production, laboratory to assess the chemical, microbiological content and shelf life of stirred yoghurt. Chemical and microbiological analysis were carried out on the first day of manufacturing and after 2, 4, 6, 8 and 10 days. Ten samples from six batches were examined. The investigations involved the analysis of fat, protein, lactose, ash, total solids, solids-non-fat and measurement of pH, acidity and viscosity. Microbiological determinations were conducted for the identification and classification of lactic acid bacteria and enumeration of total bacterial count. The chemical analysis for stirred yoghurt results showed that the fat ranges were 1.60–4.90%, protein were 1.61–5.36%, lactose were 6.75–10.92%, ash were 0.54–0.99% and total solids were 14.02– 17.74%, solids-non-fat were 11.34–14.52%, acidity were 0.80–1.50%, pH were 3.52– 4.72% and viscosity were 38.30–82.70%. The corresponding averages were 3.12%, 3.32%, 8.80%, 0.83%, 16.06%, 12.93%, 4.08%, 1.00% and 63.60%, while the microbiological isolation results showed Streptococcus thermophilus and Lactobacillus bulgaricus. The total viable bacterial count log of these organism ranged 7.00–7.78 and the average 7.33, 7.00–7.78 and the average 7.35, respectively. The log of total bacterial count (cfu) ranged from 7.00 to 8.78 and the average was 7.47. The results indicated that the storage period had significant (P< 0.001) effect on the chemical composition except on the total solids and viscosity. Also there were significant (P< 0.01) effect of the storage period on the microbiological evaluation of stirred yoghurt. ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ ﺨﻼﺼﺔ ﺍﻷﻁﺭﻭﺤﺔ أﺟﺮﻳ ﺖ ه ﺬﻩ اﻟﺪراﺳ ﺔ ﻋﻠ ﻰ اﻟﺰﺑ ﺎدى ﻣﻔﻜ ﻚ اﻟﺨﺜ ﺮة أﺣ ﺪ ﻣﻨﺘﺠ ﺎت ﺷ ﺮآﺔ اﻟﻨﻴ ﻞ اﻷزرق ﻟﻸﻟﺒﺎن )آﺎﺑﻮ( ،وﻗﺪ ﺗﻢ ﺷﺮاء اﻟﻌﻴﻨﺎت ﻣ ﻦ اﻟ ﺴﻮق وﺣﻤﻠﻬ ﺎ ﻟﻤﻌﻤ ﻞ آﻠﻴ ﺔ اﻹﻧﺘ ﺎج اﻟﺤﻴﻮاﻧﻲ ﺟﺎﻣﻌﺔ اﻟﺨﺮﻃﻮم ﻟﻠﺘﺤﻠﻴﻞ وﻟﺘﻘﻴﻴﻢ ﺟﻮدة اﻟﻤﻨﺘﺞ اﻟﻜﻴﻤﻴﺎﺋﻴﺔ واﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻴﻪ. إﺷﺘﻤﻠﺖ اﻟﺪراﺳﺔ ﻋﻠﻰ اﻟﺘﺤﻠﻴﻞ اﻟﻜﻴﻤﻴﺎﺋﻲ )ﻧﺴﺒﺔ اﻟﺪهﻦ و ﻧﺴﺒﺔ اﻟﺒﺮوﺗﻴﻦ و ﻧ ﺴﺒﺔ اﻟﻼآﺘﻮز وﻧﺴﺒﺔ اﻟﺮﻣﺎد و ﻧﺴﺒﺔ اﻟﺠﻮاﻣﺪ اﻟﻜﻠﻴﺔ وﻧﺴﺒﺔ اﻟﺠﻮاﻣﺪ ﻏﻴﺮ اﻟﺪهﻨﻴﺔ و ﺗﺤﺪﻳﺪ ﻧ ﺴﺒﺔ اﻟﺤﻤﻮﺿﺔ و ﻗﻴﺎس اﻷس اﻟﻬﻴﺪروﺟﻴﻨﻲ وﻗﻴﺎس اﻟﻠﺰوﺟﺔ(. وإﺷﺘﻤﻠﺖ اﻟﺘﺤﺎﻟﻴﻞ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻴﻪ ﻋﻠﻰ اﻟﺘﻌﺮف ﻋﻠﻰ أﻧﻮاع وﻣﺴﻤﻴﺎت اﻟﺒﻜﺘﺮﻳﺎ اﻟﻤﺴﺘﺨﺪﻣﺔ ﻓﻲ ﺻﻨﺎﻋﺔ اﻟﺰﺑﺎدي وآﺬﻟﻚ ﺣﺴﺎب اﻟﻌﺪد اﻟﻜﻠﻲ ﻟﻠﺒﻜﺘﺮﻳﺎ. أﺟ ﺮي اﻟﺘﺤﻠﻴ ﻞ اﻟﻜﻴﻤﻴ ﺎﺋﻲ و اﻟﻤﻴﻜﺮوﺑﻴﻮﻟ ﻮﺟﻲ ﻟﻠﺰﺑ ﺎدي ﻣﻔﻜ ﻚ اﻟﺨﺜ ﺮة ﻓ ﻲ اﻟﻴ ﻮم اﻷول ﻣﻦ اﻟﺘﺼﻨﻴﻊ وﺑﻌﺪ إﺛﻨﻴﻦ ،أرﺑﻌﺔ ،ﺳﺘﺔ ،ﺛﻤﺎﻧﻴﺔ وﻋﺸﺮة أﻳ ﺎم .ﻋ ﺸﺮة ﻋﻴﻨ ﺎت ﻟﻜ ﻞ ﻳﻮم ﺗﻢ ﺷﺮاﺋﻬﺎ ﻋﻠﻰ اﻟﺘﻮاﻟﻲ ﻣﻦ اﻟﺴﻮق. أﻇﻬ ﺮت اﻟﻨﺘ ﺎﺋﺞ اﻟﻜﻴﻤﻴﺎﺋﻴ ﺔ ﻟﻠﺒﺤ ﺚ أن ﻣﺤﺘ ﻮى اﻟ ﺪهﻦ ﻳﺘ ﺮاوح ﺑ ﻴﻦ %4.90 –1.60وﻧ ﺴﺒﺔ اﻟﺒ ﺮوﺗﻴﻦ %5.36 –1.61وﻧ ﺴﺒـــﺔ اﻟﻼآﺘــ ـﻮز –6.75 %10.92وﻧﺴﺒﺔ اﻟﺮﻣﺎد %0.99 – 0.54و ﻧ ﺴﺒﺔ اﻟﺠﻮاﻣ ﺪ اﻟﻜﻠﻴ ﺔ %17.74 –14.02 و ﻧ ﺴﺒﺔ اﻟﺠﻮاﻣ ﺪ ﻏﻴ ﺮ اﻟﺪهﻨﻴ ﺔ %14.52 –11.34و ﻧ ﺴﺒﺔ اﻟﺤﻤﻮﺿ ﺔ ﺗﺘ ﺮاوح ﺑ ﻴﻦ %1.50 –0.80 واﻷس اﻟﻬﻴ ﺪروﺟﻴﻨﻲ %4.72–3.52وﻗﻴ ﺎس اﻟﻠﺰوﺟ ﺔ –38.30 .%82.70و ﺑﻠﻐ ﺖ اﻟﻤﺘﻮﺳ ﻄﺎت ﻋﻠ ﻰ اﻟﺘ ﻮاﻟﻲ %3.12و %3.32و %8.80و3 %0.8و 16.06و %12.93و %4.08و %1.00و .%63.60 ﺑﻴﻨﻤ ﺎ ﺗﻮﺻ ﻠﺖ ﻧﺘ ﺎﺋﺞ اﻟﺘﺤﻠﻴ ﻞ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟ ﻮﺟﻲ ﻋﻠ ﻰ اﻟﺘﻌ ﺮف ﻋﻠ ﻰ L.bulgaricusوS.thermophilus ،وﻗ ﺪ آـــ ـﺎن اﻟـﻌـ ـﺪ اﻟﺒﻜﺘﻴـ ـﺮي ﻟﻜ ﻞ ﻣﻨﻬ ﺎ ﺑ ﺎﻟﻠﻮﻏﺮﻳﺜﻢ آ ﺎﻵﺗﻲ 7.78 –7.00 :واﻟﻤﺘﻮﺳ ﻂ 7.33و 7.78 – 7.00واﻟﻤﺘﻮﺳ ﻂ 7.35ﻋﻠﻰ اﻟﺘﻮاﻟﻲ .أﻣﺎ ﺑﺎﻟﻨ ﺴﺒﺔ ﻟﻠﻌ ﺪ اﻟﻜﻠ ﻲ ﻟﻠﺒﻜﺘﺮﻳ ﺎ ﺑ ﺎﻟﻠﻮﻏﺮﻳﺜﻢ ﻓﻘ ﺪ ﺑﻠ ﻎ 8.78 – 7.00 واﻟﻤﺘﻮﺳﻂ .7.47أﻇﻬﺮت اﻟﻨﺘﺎﺋﺞ أن ﻓﺘﺮة اﻟﺘﺨﺰﻳﻦ ﻟﻬﺎ اﺛﺮ ﻣﻌﻨﻮي ) ( P< 0.001ﻋﻠ ﻰ اﻟﺘﺮآﻴ ﺐ اﻟﻜﻴﻤﻴ ﺎﺋﻲ ﻟﻤﻨ ﺘﺞ اﻟﺰﺑ ﺎدي ﻣﻔﻜ ﻚ اﻟﺨﺜ ﺮﻩ ﻣﺎﻋ ﺪا اﻟﻤ ﻮاد اﻟﺠﺎﻣ ﺪﻩ اﻟﻜﻠﻴ ﻪ وﻗﻴ ﺎس اﻟﻠﺰوﺟﻪ. آﺬﻟﻚ هﻨﺎﻟﻚ ﺗ ﺄﺛﻴﺮ ﻣﻌﻨ ﻮي ) ( P< 0.01ﻟﻠﺘﺨ ﺰﻳﻦ ﻋﻠ ﻰ اﻟﻤﺤﺘ ﻮي اﻟﻤﻴﻜﺮوﺑ ﻲ ﻟﻤﻨﺘﺞ اﻟﺰﺑﺎدي ﻣﻔﻜﻚ اﻟﺨﺜﺮﻩ. LIST OF CONTENTS Page Dedication……………………………………………………………………………………………... i Acknowledgement …………………………………………….………………………………... ii Abstract ………………………………………………………..……………………………………... iii Arabic Abstract …………………………………………………………………………………... v List of Contents ………………………………..………………………………………………... vii List of Tables……………………………………..………………………………………………... xi List of Figures …………………………………..………………………………………………... xii CHAPTER ONE: INTRODUCTION……………………………………………... 1 CHAPTER TWO: LITERATURE REVIEW………………..……………… 3 2.1 Fermented dairy products……………..………………………….………..…………… 3 2.2 Processing of yoghurt……………..………………………………………………..…… 6 2.3 Types of yoghurt…………………………………………………………..…..…………… 9 2.4 Composition of Yoghurt…………………………………………………..…………… 12 2.5 Factors affecting quality of yoghurt………………………..………..…………… 15 2.5.1 Starter cultures……………..…………………………………….…………..…………… 15 2.5.2 Heat treatment……………..……………………………………...…………..…………… 16 2.5.3 Storage period……………..………………………………………...………..…………… 17 2.6 Additives in yoghurt……………..…………………………………………..…………… 19 2.6.1 Flavorings and sweeteners……………….…………………………..…………… 19 2.6.2 Stabilizers……………..……………………….………………………………..…………… 21 2.7 The nutritional value of yoghurt……………..…………………………..………… 21 2.8 Fermentation and microbiological aspects………………..……..…………… 22 2.9 Ratio of cocci: rods in starter culture……………………………….…………… 23 CHAPTER THREE: MATERIALS AND METHODS……..………… 27 3.1 Source of milk samples……………..…………….………………………..…………… 27 3.2 Chemical analysis……………..……………………………..………………..…………… 27 3.2.1 Fat content……………….……………………………………………….……………..…… 27 3.2.2 Total solids……….……………………………………………………………………..…… 28 3.2.3 Ash content…………………………………………….………...……………………..…… 28 3.2.4 Protein content……………….…………………..…………….……………………..…… 29 3.2.5 Lactose content……………………………………..………………………………..…… 30 3.2.6 Solid not fat (SNF) ……………………………….………………………………..…… 30 3.2.7 Titratable acidity (T.A) ………..………………….……………………………..…… 30 3.2.8 pH – values………………………….…………………….…………..………………..…… 31 3.2.9 Viscosity……..…………………………………...……….……………………………..…… 31 3.3 Microbiological examination………….………………………………………..…… 31 3.3.1 Sterilization…………………………………………………………...………………..…… 31 3.3.2 Preparation of serial dilution ………………………………………………..…… 31 3.3.3. Type of cultured media used for microbial examination………… 32 3.3.3.1 Plate count agar…………………………………………...……………………..…… 32 3.3.3.2 M – 17 medium…………………..……………………………………………..…… 32 3.3.3.3 MRS broth………………………………………………………..………………..…… 32 3.3.3.4 Nutrient agar…………………………………..…………………..………………..…… 32 3.4 Enumeration of microorganisms……………………………..………..…………… 33 3.4.1 Streptococcus thermophilus……………..…………………………..…………… 33 3.4.2 Lactobacillus bulgaricus…………….……………..……..…………….…………… 33 3.4.3 Bacterial count……………..……………………..…………………………..…………… 33 3.5 Purification of bacterial isolates……………………………………...…………… 33 3.6. Identification of organisms……………………………………………...…………… 34 3.6.1 Primary tests……………..……………………………………...……………..…………… 34 3.6.1.1 Morphological appearance……………..…………………………..…………… 34 3.6.1.2 Shape of cell……………..…………………………..…………………………..……… 35 3.6.1.3 Catalase test……………..……………………………………..…………..…………… 35 3.6.1.4 Oxidase test……………..…………….……………..…………………………..……… 35 3.6.1.5 Motility test……………..…………………………………………………..…………… 36 3.6.1.6 Oxidation fermentation test (OF) ………………………..……..…………… 36 3.6.2 Confirmatory tests……………..…………………………………………..…………… 36 3.7 Statistical analysis……………..……………………..………………………..…………… 37 CHAPTER FOUR: RESULTS AND DISCUSSION …………………… 38 4.1 Chemical composition of stirred yoghurt………………….……..…………… 38 4.1.1 Fat……………..…………………………..………………………..………………..…………… 38 4.1.2 Protein……………..…………………………..…………………………………..…………… 40 4.1.3 Lactose……………..……………………………………………………………..…………… 40 4.1.4 Ash……………..…………………………..………………………………………..…………… 41 4.1.5 Total solids (TS) ……………..……………………………….……………..…………… 42 4.1.6 Solids – non–fat (SNF) ……………………...…………………………..…………… 44 4.1.7 pH……………..………………………………………...…………………………..…………… 45 4.1.8 Acidity……………..……………………..…………..…………………………..…………… 47 4.1.9 Viscosity……………..…………………………..………………..……………..…………… 48 4.2 Microbiological results of stirred yoghurt………….……..……..…………… 50 4.2.1 Standard plate count……………..………………………………….……..…………… 50 4.2.2 Identification of isolated LAB……………..…………………………..………… 53 4.2.3 Streptococcus thermophilus……………………………………..…..…………… 53 4.2.4 Lactobacillus bulgaricus………………………………………………..…………… 56 CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS..… 61 5.1. Conclusion………………………………………………….……………………………..…… 61 5.2. Recommendations…………………………...………………………………………..…… 62 REFERENCES…………………………………………………………………………………... 63 LIST OF TABLES Table Title No. 2.1 Yoghurt and yoghurt – like products known worldwide… 5 2.2 Some typical values of major constituents of milk and yoghurt……………..………………………………………………….…..…………… 25 2.3 Selected characteristics of yoghurt starter culture…………..… 26 1 Fat and protein content of stirred yoghurt samples during storage….…..…….…..…….…..…….…..…….…..…….……....…….…..…….…..… 39 Lactose and Ash content of stirred yoghurt samples during storage….…..…….…..………….…..…….…..…….……....…….…..…… 43 Total solids (T.S) and Solids–non–fat (SNF) of stirred yoghurt samples during storage.…..………………….…..…….…..……. 46 pH and acidity development of stirred yoghurt samples during storage.…..………….…..………………………………………...…..……. 49 5 Viscosity of stirred yoghurt samples during storage….....….. 51 6 Chemical composition of stirred yoghurt using one way ANOVA analysis.…………………….…..……………………………………… 52 Total bacteria count of stirred yoghurt samples during storage.………….……………………………...……………………………………… 54 Microbiology analysis of stirred yoghurt samples during storage.………….…..……………………………..…………………………………… 58 Microbiology analysis of stirred yoghurt using one way ANOVA analysis.………….……………..……………………………………… 59 Identification of lactic acid bacteria.………….…..………………… 60 2 3 4 7 8 9 10 LIST OF FIGURES Fig. Title No. 2.1 Traditional yoghurt production in the Middle East……….… 8 2.2 Industrial production of various types of yoghurt…………..… 13 CHAPTER ONE INTRODUCTION Under unrefrigerated storage milk becomes sour in a few hours; this is due to the action of natural lactic acid bacteria on the milk sugar, turning it to lactic acid which causes the milk protein to coagulate (Lucey and Singh, 1997). Traditionally, most communities in Africa and in Sudan in particular have used this natural fermentation to produce a variety of fermented milks for home consumption (Ahmed and Ismail, 1978). Under normal dairy processing industry, selected lactic starter cultures are used to ferment milk during preparation of variety of cultured dairy products. The quality of those products vary from average or below average quality to high quality products (Tamime and Robinson, 1999). Yoghurt is a semi–solid fermented milk product which originated centuries ago in Bulgaria, it’s popularity has grown and is currently consumed in most parts of the world (Tamime and Deeth, 1980). The accepted home land of yoghurt is the Balkans peninsula and the Middle East region, and to communities living in these parts of the world this type of fermented milk product is identified and known as natural/plain unsweetened yoghurt. The per capita annual consumption is high. In Bulgaria, in particular, is 31.5 kg/head/ year (IDF, 1977). It is evident, therefore, that yoghurt plays an important role in the diets of these communities. Furthermore, it is customary for yoghurt to be consumed not only as a refreshing drink, but also as a main ingredient (Tamime and Deeth, 1980). There are three types of plain yoghurt: stirred yoghurt, set yoghurt and liquid yoghurt (with low viscosity). In the set yoghurt, the product is packaged immediately after inoculation with the starter culture and is incubated in the packages, for stirred yoghurt the milk is inoculated by culture, incubated in tank and packaged after cooling (Chandan, 1999). In many cases of lactose intolerance, cultured milk products where lactose has been partially broken down can be acceptable to sufferers of lactose intolerance (Harmann and Marth, 1984). Children suffering from infantile diarrhea recovered more rapidly when fed yoghurt than those given neomycin – kaopectate (Shahani and Chandan, 1979). The objectives of the present study are: 1- Evaluation of the chemical quality of market stirred yoghurt. 2- Evaluation of microbiological quality of market stirred yoghurt. 3- Isolation and identification of lactic acid bacteria in stirred yoghurt. Measurement of yoghurt ingredient and the microbial activity of some bacteria are taken as main parameter, to reach the objectives mentioned. CHAPTER TWO LITERATURE REVIEW 2.1 Fermented dairy products: Acidification of milk by fermentation is one of the oldest methods of preserving milk and imparting to it specific favorable organoleptic qualities. There are different methods of carrying out this fermentation in various parts of the world and these give rise to a wide range of fermented milk products, including kumiss, kefir, acidophilus milk and yoghurt (Thapa, 2000). These products vary considerably in composition, flavor and texture, according to the nature of fermenting organisms, the type of milk and the manufacturing process used (Chandan et al., 1969; Kosikowski, 1977 and Berlin, 1962). Fermentation is defined as any modification of the chemical or physical properties of milk or dairy products, resulting from the activity of microorganism or their enzymes which cause the main marked changes (Frank and Marth, 1988). Fermentation affects the carbohydrates, proteins and vitamins as well as producing flavor compounds (particularly acetaldehyde), some enzymes and of course bacterial mass (Deeth, 1984). Cultured milks therefore have a change in composition, as well as an accumulation of the products of bacterial fermentation and anti–microbial compounds emitted by culture organisms in order to inhibit the growth of other organisms (Harding 1999). Fermented milk products are widely used in the Balkan for medicinal purposes against diseases such as pneumonia, dysentery and less serious complains such as sore throat and laryngitis (Peterson, 1981). There are claims that the digestibility of milk proteins is improved by fermentation (Marshal, 1986 and Deeth and Tamime, 1981). Lactic acid fermentation products have traditionally been used by Africans for treating some human ailments and for other ends (Dirar, 1997). There are also claims that fermented milks contain some chemical factors that reduces the level of serum cholesterol in human beings (Mann and Spoery, 1974; Mann, 1977; Richardson, 1978; Grunewald, 1982 and Pulusani and Rao, 1983). Kilara and Shahani (1976) studied the lactase activity of cultured acidified dairy products including yoghurt. They concluded that the yoghurt culture released the enzyme lactase, which break down the lactose contained in the dairy product, allowing lactose intolerant people to consume these products with no subsequent problems. The word “yoghurt” is derived from the Turkish word “jugurt” and Table (2.1) shows the variety and names by which this product is known in different countries. Yoghurt is a traditional food and beverage in the Bulkans and the Middle East. However, it’s popularity has now spread to Europe and many other parts of the world (Rasic and Kurmann, 1978). Table (2.1): Yoghurt and yoghurt–like products known worldwide Traditional name Country Jugurt/Eyran Turkey Busa Turkestan Kissel Mleka Balkans Urgotnic Balkan mountains Leben/leban Lebanon and some Arab countries Zbady Egypt and Sudan Mast/Dough Iran and Afghanistan Roba Iraq Dahi/Dadhi/Dahee India Mazum/Matzoo Armenia Katyk Transcaucasia Tiaourti Greece Cieddu Italy Mezzoradu Sicily Gioddu Sardinia Tarho Hungary Fiili Finland Filmjolk / Fillbunke / Filbunk / Surmelk / Scandinavia Tettemelk / Taettemjolk Island Yoghurt/Yogurt / Yoart / Yourt / Yaourti Rest of the world “Y” is replaced by Yahourth/Yogur/Yaghourt ‘J’ in some instances After: Orla–Jensen (1931), Hammer and Babel (1957), Davidson and Passmore (1969), Pederson (1971), Nilson (1973); Kon (1972); El – Sadek et al. (1972). Although some controversy still exists regarding the exact definition of yoghurt in terms of its chemical composition and the product resulting from milk by fermentation with a mixed starter culture consisting of only Streptococcus thermophilus and Lactobacillus bulgaricus. The justification for using such an approach has been discussed (Tamime and Robinson, 1976). Similar definitions has been used in formulating existing or proposed standards for yoghurt in many countries (Dehove, 1960 and Danish standards 1968). Yoghurt is a popular fermented milk product consumed in many parts of the world. It is produced in different forms such as whole milk yoghurt, skim milk yoghurt, cream yoghurt, fruit yoghurt and liquid yoghurt (Balasubraman yam and Kulkarnis, 1991). Yoghurt is one of the most unique, yet universal dairy products. The uniqueness of yoghurt is attributable to one symbiotic fermentation involved in its manufacture. It may be defined as the solid, custard- like fermented milk product made from fortified high–solids milk using a symbiotic mixture of Streptococcus thermophilus and Lactobacillus bulgaricus as starter (Ebenezer and Vedamuth, 1991). 2.2 Processing of yoghurt: Traditionally, yoghurt has been manufactured from boiled milk which was concentrated to two thirds of the original volume (Cronshaw, 1947 and Elliker, 1949). The traditional procedure, which is illustrated in Figure 2.1, has several draw backs. First, successive inoculations of the starter cultures tend to up set the balance between the S. thermophilus and L. bulgaricus; second the low incubation temperature (blood temperature as compared with the optimum temperature of (40-45°C) results in slow acidification of milk and can promote undesirable side effects, e.g. whey syneresis, and adversely affect the quality of yoghurt, and third, such a method provides no control over the level of lactic acid produced during manufacture (Cronshaw, 1947 and Elliker, 1949). The starter, which is obtained from commercial starter manufacturers or starter banks, is propagated in rotation and inoculated into the milk under aseptic conditions, the temperature is accurately controlled and the yoghurt is cooled very quickly when the desired acidity is reached. Hence, the process can be well controlled and the quality of yoghurt is standardized (Cronshaw, 1947 and Elliker, 1949). The work on the manufacture of yoghurt has been reviewed by several authors (Humphreys and Plunkett, 1969; Tamime and Robinson, 1976; Puhan, 1976 and Bottazzi, 1977). Figure (2.1): Traditional yoghurt production in the Middle East: (Tamime TtTt and Deeth 1980) Boil milk to cause partial concentration Cool to incubation temperature, i.e. blood temperature Starter (previous day yoghurt) Inoculate with starter Incubate in bulk until firm coagulate is produced or incubate over night at room temperature Cool Dispatch Legal standards of yoghurt are mainly based on the chemical composition of the product i.e. percentage of fat, solids-non-fat (SNF) or total solids (Tamime and Robinson, 1976). The aim of using high total solids is consistency improvement the yoghurt coagulum (Robinson, 1983). Very thin liquid yoghurt caused by strong mixing, incubation (short time), low level of total solids, bad culture and low viscosity production (Tayfour, 1994). 2.3 Types of yoghurt: The industrial manufacture of different types of yoghurt is shown schematically in Fig 2.2. The various types differ according to their chemical composition, their method of production, their flavour and the nature of post – incubation 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) fat content (FAO/WHO, 1973). They added that such classification is used in composition standards to facilitate standardization of product and to 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 a 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 of low 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 2.2) 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). Flavouring 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, pure or jam (Tamime and Deeth, 1980) However they added that flavoured yoghurt are prepared by adding sugar or sweetening agents and synthetic flavorings and colorings to plain or natural yoghurt. The post – incubation processing of yoghurt may lead to many different types of yoghurt. The emergence of new yoghurt products has been largely due to recent developments in this field as they stated. Typical examples of these are pasteurized/UHT yoghurt, concentrated yoghurt, frozen and dried yoghurt. These products may vary considerably in chemical composition, physical characteristics, heat – treatment after incubation a process which leads to destruction of the yoghurt starter bacteria and reduction in the level of volatile compounds which are 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 a round 24% total solid (Tamime and Robinson 1978, 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 Robinson, 1978 and Robinson, 1977). Sun dried yoghurt is produced in many rural areas in the Middle East where the surplus of summer milk is made into yoghurt and conserved by this method for winter consumption (Tamime and Deeth, 1980). Industrial production of dried yoghurt is achieved by either spray drying or freeze – drying (Tamime and Robinson, 1976). 2.4 Composition of Yoghurt: Yoghurt is highly nutritious and easily digestible diet due to the predigested nutrients by bacterial starters, it is perishable in view of its unused lactose content which can be utilized for the growth of undesirable microorganisms responsible for spoilage (Durga et al., 1986). Musa (1997) examined yoghurt prepared from fresh cow’s milk, he reported for fat content, protein content and total solids 3.2%, 4.5% and 19.39%, respectively. In terms of over all composition, yoghurt is similar to milk. However, there are many aspects in which the composition of milk and yoghurt differs. These differences are either from deliberate addition of solids to milk or yoghurt, or from changes brought about by bacterial fermentation (Deeth and Tamime, 1981). The composition of yoghurt is approximately the same as that of whole milk; it provides an excellent protein source particularly when it is fortified with dried milk (Vieseyre, 1964). Fig. (2.2): Industrial production of various types of yoghurt (Tamime and Deeth, 1980) Preliminary treatment of milk (standardization/fortification/lactose hydrolysis) Homogenization Heat treatment Starter propagation Cool to incubation temperature Inoculation with starter Incubate Inoculate in bulk Cool Cool Concentrate Dispatch SET YOGHURT Freeze Dispatch SOFT OR DEEP FROZEN YOGHURT Mix with fruit Heat treatment Cool Pack Dispatch PASTEURIZED OR UHT YOGHURT Pack Dispatch STIRRED YUGHURT Heat treatment Pack Pack Dry Mix with equal quantity of water Pack Freeze Cool Dispatch PASTEURIZED OR UHT YOGHURT Cool Dispatch Dispatch COCENTRATED Pack YUGHURT SOFT OR Dispatch DEEP FROZEN DRIED YUGHURT YUGHURT Dispatch FLUID YUGHURT Deeth and Tamime (1981) concluded that, with all methods of fortification, the percentage of protein is increased, thus yoghurt will have an invariably high protein content than milk. Dalles and Kechagias (1984) found that the acidity of commercial types of yoghurt in Greece ranged from 1.02 to 2.15% (lactic acid). The major change resulted from fermentation is the production of lactic acid from lactose. However, several other important changes also occur. In many developing countries, “Zabadi” is produced as naturally sour milk, and makes an important contribution to the diet as a source of protein, calories and some vitamins (Ahmed and Ismail, 1978). Kim et al. (1998) reported a titratable acidity of 0.95- 0.99% for yam– yoghurt which was prepared by addition of yam to skim milk substrate and cultured with a mixed culture. Collado et al. (1994) found that the yoghurt drink and yoghurt like products had 0.56% and 0.58% total titratable acidity. Gregurek (1999) found that post acidity cation was slightly higher in yoghurt prepared with lower amount of inoculation and incubation temperature should be properly maintained, to achieve maximum bacterial viability. Djordjevic et al. (1988) found that there was a lowering of dry matter during the incubation and during storage and this lowering takes place in dry fat matter, portion dry matter and is due to the disappearance of volatile compounds formed during the process of yoghurt. Uraltas and Nazli (1998) when studied Turkish fruit yoghurt, found that dry matter content ranged from 22.2 to 23.5% and values for fat ranged from 2.2 to 2.8% and SNF values ranged from 19.4 to 23.5% for 50 samples. Agaoglu et al. (1997) examined 25 samples of cokelek (concentrated yoghurt). They found that the average dry matter content was 18.15%, fat content 1.2%, protein content 4.08% and mineral content 0.94%. 2.5 Factors affecting quality of yoghurt: Many factors affect the quality of yoghurt such as type of starter culture, heat treatment and storage condition. 2.5.1 Starter cultures: The culture may be of one strain microorganism which called a single – strain culture, or a number of strains and/or species called a multi – strain or mixed – strain culture (Kosikowski, 1982). Yoghurt is produced with a mixed culture of S. salivarius sub – sp. thermophilus and L. delbrueckii, sub – sp. bulgaricus in a 1:1 ratio. The coccus grows faster than the rod and it's primarily responsible for acid production, while the rod adds flavor and aroma (Kosikowski, 1982). The associative growth of the two organisms results in lactic acid production at a rate greater than that produced by either when growing alone, and more acetaldehyde (the chief volatile flavour component of yoghurt) is also produced (Jay, 1986). The symbiotic relationship between S. thermophilus and L. bulgaricus, the thermoduric, homofermentative lactic acid bacteria, has long been utilized in the manufacture of yoghurt, maintenance of the proper balance between these organisms is considered important, especially in manufacture of high quality yoghurt (Lee et al. 1974). Radke–Mitchell and Sandine (1984) assumed a symbiotic relationship between the strains. The Lactobacilli liberate amino acids from the protein, and these amino acids encourage the development of the Streptococcus that stimulates the growth of Lactobacilli by increasing the initial acidity and also by removing residual oxygen. Finally the acidity produced by Lactobacilli inhibits the growth of Streptococci (Hamill, 1968). 2.5.2 Heat treatment: Milk was processed by three different heating systems, 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 (UHT 140° C for 2-8 sec). The physical and sensory properties of yoghurt were substantially affected by type of heat processing and exposure time. The most viable heating process investigated was HTST with a residence time of 1.87 min (Parnell – clunies et al. 1986). Hong and Goh (1979) found that yoghurt from milk heated at 85° C was harder than yoghurt from milk heated at 95° C or 75° 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 spread– ability and fluidity than yoghurt processed by conventional vat system (82° C for 30 min). The UHT processing of milk indicated a potential method for commercial manufacture of yoghurt with low curd firmness (Labropoulos et al. 1984). Homogenization can reduce the incidence of pipes in yoghurt i.e. white flecks that appear in some fruit varieties (Davis, 1967; Nielsen, 1972 and Robinson, 1977). 2.5.3 Storage period: Yoghurt is not as stable as one would like, due to proteolytic activity of L. bulgaricus, yoghurt develops a bitter and a stringent taste during cold storage. Souring is also continued during storage; by these processes the quality is limited to 14 days at 7° C (Driessen, 1981). Besides this yoghurt can be spoiled (natural deterioration) after any contamination and growth of yeasts and molds. This deterioration is strongly dependent on the type of packaging material used. However, Driessen (1981) suggested several methods for prolonging the storage life of yoghurt which include aseptic manufacture, preservation by chemical agents, preservation by heat- treatment of milk and culture manipulation. Temperature control (4-5° C) is most important since higher temperatures can lead to defects such as bitterness, low temperature can induce ice–crystal formation and slow freezing is detrimental to yoghurt, but rabidly frozen yoghurt will keep for several months at 27° C (Humphreys and Plunkett, 1969). They added that cold storage and careful stock rotation play an important role in ensuring that the consumer always receives uniform first quality yoghurt. Keeping quality is essentially determined by the rate of production of flavour. As such, it is related only to the bacterial quality of milk as a bacterial strain which produces more flavours will obviously cause spoilage at a lower count (Urbach and Milne, 1987). International standards are in general agreement in that, coliform count should not exceed 10 CFU/ml in yoghurt. However, because of the different methods of controlling the air borne and ubiquitous moulds and yeasts particularly the yeast, a tolerance of 100 CFU/ml has been given to good quality yoghurt (Salji et al., 1987). Toba et al. (1983) concluded that oligosaccharides content of yoghurt progressively increased during storage. Decline in pH of yoghurt during cold storage has been reported by Tramer (1973) who related this effect to the ability of the starter culture organisms to carry out metabolic processes at storage temperatures. Most vitamins decrease during storage so the level of vitamins will largely depend on the age of yoghurt, vitamin B12 appears to be more stable during storage in yoghurt than in milk (Deeth and Tamime, 1981). Reddy et al. (1971) found that storage at 5° C caused a decrease in the level of vitamins, folic acid and vitamin B12 showed the greatest loss, while the loss of biotin, niacin and pantothenic acid was small. 2.6 Additives in yoghurt: Yoghurt may contain variable amounts of nutrient additives that are classified into flavourings, colourings, sweeteners and stabilizers. 2.6.1 Flavoruings and sweeteners: Wightwick (1970) mentioned that flavors are used for different reasons which include: 1. To provide a flavour when little or no initial flavour as in is carbonated beverages. 2. To mask an existing unpleasant flavour e.g. medicines. 3. To boost a flavour which is too weak e.g. ice cream. 4. To give a twist to a naturally occurring flavour e.g. yoghurt. The flavour used to achieve the above objectives, fall into three main categories (Wightwick, 1970). a) Natural flavours: Which are prepared by macerating vegetables, spices, herbs, fruits etc… with a suitable solvent. b) Synthetic flavours: Consist of esters, aldehydes, ketones, alcohols, lactones, etc… c) A blend of natural and synthetic flavours. The addition of fruit juice, concentrated fruit juice, fruit pieces and fruit pastes to milk depends entirely on the particular fruit used, its acidity and the level at which it is used. The flavour market covers a wide range of consumable products and extends beyond the food and beverage industry to pharmaceuticals, animal foods and tobacco of these, by far; the most important for flavours are soft drinks, dairy products and savory foods (Firth, 1990). Wightwick (1970) reported from his personal observation that, although unflavored yoghurt is in fair demand, the most popular forms appear to be “real fruit” yoghurt and flavoured yoghurt. Statistics for the consumption of fruit – flavoured milk and yoghurt showed that, in Australia the consumption of flavoured milk is approximately 9 litres per capita per year and approximately 1-2 litres per capita per year in New Zealand (Wright, 1992). 2.6.2 Stabilizers: Kosikowski (1982) claimed that properly made plain yoghurt requires no stabilizer because a stiff, smooth gel with high viscosity is attained naturally. More of a need exist for stabilizers in flavored yoghurt and generally the stabilizers used are gelatin, agar or complex mixtures consisting of guar gum, arab gum and gelatin at level of 0.40.5% (Kosikowski 1982). Different methods for adding stabilizer exist, it can be added to the mix prior to pasteurization. In other methods a concentrated stabilizer and fruit are added to incompletely cooled yoghurt and the mixture is chilled in the container (Kosikowski 1982). 2.7 The nutritional value of yoghurt: The chemical composition of food stuff provides a useful indication of it is potential nutritional value, and the data showed in Table 2.2 indicate the main components of some typical natural and fruit yoghurts (Robinson, 1977). If these figures are accepted at face value, then it is evident that yoghurt could prove an important introduction to any diet (Robinson, 1977). At the same time, it must be accepted that values reveal only part of the story, and even if the almost mystical properties ascribed to yoghurt are ignored for moment, there are some aspects of the behavior of yoghurt in the human body that not revealed by chemical analysis (Robinson, 1977). Several lactic acid organisms produce natural antibiotic for example L. bulgaricus produces Bulgarican (Reddy and Shahani, 1971). It is of some interest, therefore, to look at the constituents of yoghurt in a little more detail, and, in particular, to asses the likely nutritional importance of the materials concerned (Deeth and Tamime, 1981 and Alm, 1982). Wherever the final outcome of various controversies surrounding the influence of yoghurt on the health of human beings, there can be little doubt that it is over all nutritional value makes it a most desirable addition to any diet. If the consumer appeal of both natural and fruit yoghurts is also placed on record, together with its excellent performance in respect of public health, then it is evident why yoghurt is among the most popular fermented dairy products. (Robinson, 1977). 2.8 Fermentation and microbiological aspects: The classification of lactic acid bacteria by Orla Jensen (1931) was still recognized as the standard method for classifying these organisms. In the 7th edition of Bergey’s Manual (1957) all these bacteria were grouped in the family Lactobacillaceae, which was subdivided into two tribes, the Streptococceae (Coccal shaped) and Lactobacillaceae (rod shaped). However, in the 8th edition of Bergey’s manual (1974), lactic acid bacteria are reclassified into two families the Streptococcaceae and Lactobacillaceae. The yoghurt starter bacteria, S. thermophilus and L. bulgaricus are thermoduric, homofermentative lactic acid bacteria, their general characteristics are shown in Table 2.3. Moreover S. thermophilus is distinguished from other members of the genus Streptococcus by its growth at 45° C and failure to grow at 10° C. Unlike most streptococci, this species lacks a recognizable 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 subjects of some controversy (Davis, 1975). They also reported that the organism appears to be basically the same as that isolated from Bulgarian yoghurt by Metchnikoff and Coworkers in the 1900’s 2.9 Ratio of cocci: rods in starter culture: The yoghurt starter cultures are mixed strain starters of S. thermophilus and L. bulgaricus which are normally propagated together at around 42° C (Bergey’s 1974). Successive subculturing may lead to mutation or to one organism overgrowing the other. Hence the starter strains are either monitored microscopically (Brad smear) or examined after culturing on a solid medium, whatever the method is used, the objective is to monitor the ratio of the cocci to rods (Bergey’s 1974). The ‘ratio’ as it appears in the literature, is somewhat confusing, since it might refer to the colony: colony; clump; chain: chain or cell: cell ratio of Streptococcus: Lactobacillus (Bergey’s 1974). Table (2.2): Some typical values of major constituents of milk and yoghurt: Milk Constituent units/100g Yoghurt Whole Skim Full fat Low fat Fruit Calories 67.5 36 72 64 98 Protein (g) 3.5 3.3 3.9 4.5 5.0 Fat (g) 4.25 0.13 3.4 1.6 1.25 Carbohydrate (g) 4.75 5.1 4.9 6.5 18.6 Calcium (mg) 119 121 145 150 176 Phosphorus (mg) 94 95 114 118 153 Sodium (mg) 50 52 47 51 - Potassium (mg) 152 145 186 192 254 Adapted from Deeth and Tamime (1981). Table (2.3): Selected characteristics of yoghurt starter culture: Characteristic S. thermophilus DNA G + C (%) Acid from glucose 50.3 + Gas from glucose + - Growth in media containing 2% NaCl Lactic acid in milk L. bulgaricus - - L (+) D (-) % Acid in milk 1.7 Ammonia from arginine - - Growth at 15°c - - Growth at 45°c + + Serological group × E Requirement for vitamins Thiamine - Riboflavin + Folic acid - Vitamin B12 - Carbohydrate utilization Fructose + Galactose ± ± Gelatin - Lactose + + Maltose - - Mannitol - - Sucrose + - Xylose ± - Sharpe et al. (1968); Rogosa (1974) and Deibel and Seeley (1974). + Positive reaction by 90% or more strains, - negative reaction by 90% or more strains, ± variable slow or weak reaction, X ungrouped. CHAPTER THREE MATERIAL AND METHODS 3.1 Source of milk samples: Ten stirred yoghurt samples from each of six batches processed by Blue Nile Dairy Company (CAPO) were obtained from market. The collection of samples was conducted during the period from September to December 2005. Each batch was collected separately. Samples of stirred yoghurt purchased from market are transported to the laboratory for chemical analysis and for microbiological examination. 3.2 Chemical analysis: 3.2.1 Fat content: The fat content was determined by Gerber’s methods described by AOAC (1990) as follows: In clean dry Gerber tube, 10 ml of sulphuric acid (density 1.815 gm/ml at 20° C) were poured, and then 10.94 ml of milk samples were added. Amyl alcohol (1-2 ml) was added to the mixture, followed by the addition of distilled water. The contents were thoroughly mixed till no white particles could be seen. The Gerber tubes were centrifuged at 1100 revolution per minutes (rpm) for 4-5 minutes, and the tubes were then transferred to a water bath adjusted at 65° C for three minutes. The fat percent was then read out directly. 3.2.2 Total solids: The total solids contents were determined according to the modified method of AOAC (1990). Three grams of the milk samples were weighed in dry clean flat bottomed aluminum dish and heated on a steam bath for 10-15 minutes. The dishes were placed in an oven at 100° C for three hours. Then cooled in a desicator and weighed quickly. Weighing was repeated until the difference between the two readings was < 0.1 mg. The total solids (T.S) contents were calculated as fallows: T.S% = W1/W2×100. Where: W1 = Weight of sample after drying. W2 = Weight of sample before drying. 3.2.3 Ash content: The ash content was determined according to the method described in the AOAC (1990). Five grams of the sample were weighed in a crucible and evaporated to dryness on a steam bath. The crucibles were then placed in a muffle furnace at 550-600° C until ashes were carbon free (2-3 hours), then crucibles were cooled in a desicator and weighed. The ash content was calculated using the following equation: Ash% = W1 ×100 W Where: W1 = Weight of ash. W = Weight of sample. 3.2.4 Protein content: The protein content of milk samples was determined according to Kjeldahl method as described by AOAC (1990). Ten ml of each milk samples were weighed and poured into a clean dry Kjeldahl flask. Kjeldahl tablets of CuSo4 and concentrated H2SO4 (25 ml) were added to the flasks. The flasks were heated until clean solutions were obtained (2-3 hours) and left for another 30 min. The flasks were then removed and allowed to cool. The digested milk samples were poured into volumetric flasks (100 ml) and diluted with distilled water. Then 15 ml of 40% NaOH was added to each flask and the content of the flask were distillated. The distillate was received in conical flasks (100 ml) containing ten ml of 2% boric acid plus three drops of indicator (bromocresol green + phenolphthalein red). The distillation was continued until the volume in the flasks was 75 ml then the flasks were removed from distillator. The distillate was then titrated with 0.1 HCl until the end point (red color) was obtained. The protein content was calculated as follows: N% = T × 0.1 × 0.014 × 100/W P% = N% × 6.38 Where: T = Reading of titration. W = Weight of original sample. N = Total nitrogen. P = Total protein. 3.2.5 Lactose content: The lactose content was determined by subtracting the sum of proteins %, fat % and ash % from total solids %. Lactose % = T.S% – ( proteins % + fat % + ash %). 3.2.6 Solid not fat (SNF): The solid not fat was determined by subtracting fat from total solids. SNF % = T.S% – fat %. 3.2.7 Titratable acidity (T.A): The acidity of the milk samples was determined according to the method described in the AOAC (1990). Ten ml of each sample was placed in a white porcelain dish and five drops of phenolphthalein indicator were added. Titration was carried out using N/9 NaOH until a faint pink colour appeared. The titration figure was divided by ten to get the percentage of lactic acid. (1ml of 0.1N sodium hydroxide (NaOH) =0.009 gm of lactic acid). 3.2.8 pH – values: The pH values were determined using pH – meter (HANNA – instrument, model 5A520, Rench meter). 3.2.9 Determination of viscosity: Viscosity was measured by viscometer (Visco 6–R thermo HAAKE). 3.3 Microbiological examination: 3.3.1 Sterilization: Petri dishes, test tubes, flasks and pipettes were sterilized by heating in an oven (Gallen kamb) at 180° C for one hour. 3.3.2 Preparation of serial dilution: One ml from each milk sample was taken aseptically by sterile pipette in sterile bottle. They were mixed well with 9 ml sterile ringers' solution. Serial dilutions were prepared (10-1 – 10-6) according to Richardson (1985). 3.3.3 Type of culture media used for microbial examination: 3.3.3.1 Plate count agar: (KGaA642711) This medium was prepared according to Harrigan and McCance (1976), 23.5 gms of the medium were dissolved in 1000 ml distilled water, then it was sterilized by autoclaving at 121° C for 15 minutes. The media was then distributed using the pour plate technique. 3.3.3.2 M – 17 medium: (DIFCO, L 110660-1) The medium was prepared according to Harrigan and McCance, (1976) by disolving 39.2 gms in 1000 ml distilled water. It was sterilized by autoclaving at 121° C for 15 minutes, cooled to 50° C. Then 50 ml of sterilized 10% lactose and agar (20 gm/liter) were added. 3.3.3.3 MRS broth: Oxoid, (CM 495) In 1000 ml of distilled water, 67.15 gms were disolved and sterilized by autoclaving at 121° C for 15 minutes. 3.3.3.4 Nutrient agar: (S.d. Fine-Chem.Ltd 74065) Twenty eight gms of the nutrient agar were disolved in 1000 ml distilled water, sterilized by autoclaving at 121° C for 15 minutes. 3.4 Enumeration of microorganisms: 3.4.1 Streptococcus thermophilus: This was carried out using modified (M–17) medium. 0.1 ml from suitable dilutions were spread on the surface of sterile modified M–17 medium. The plates were incubated at 37° C for 48 hours. The grown colonies were considered as streptococci. 3.4.2 Lactobacillus bulgaricus: This was carried out using modified (MRS) medium. 0.1ml from suitable dilutions were spread on the surface of sterile modified (MRS) medium. The plates were incubated at 37°C for 48 hours. 3.4.3 Total bacterial count: The pour plate techniques using plate count agar medium was used for total bacterial count. The plates were incubated at 37° C for 48 hours and the colonies were counted according to Marshal (1992). 3.5 Purification of bacterial isolates: A part of typical well–isolated colony was picked by a sterile wire loop and streaked on surface of Petri dish containing a fresh solidified corresponding medium. The subculturing for each organism was repeated until a pure colony representing the organism was obtained. The process was repeated several times. The culture was checked for purification by the Gram stain. The pure cultures were streaked onto the surface of nutrient agar medium in McCartney bottles and incubated at 37° C. The bottles were then stored at 5° C till used (Barrow and Feltham, 1993). 3.6. Identification of organisms: The identification of purified colonies was carried out according to Barrow and Feltham (1993). The purified isolates were subjected to the following tests: 3.6.1 Primary tests: 3.6.1.1 Morphological appearance: The morphological appearance of each bacterium was recorded according to Barrow and Feltham (1993). Streptococci: Gram-positive cocci in pairs or chain, nonmotile. Lactobacilli: Gram-positive rods often coryneform; chain formation common; typically non- motile and not acid fast. 3.6.1.2 Shape of cell: This was done by Gram stain as described by Harrigan and McCance (1976) as follows: Crystal violet was added to smear on slides for one minute, followed by washing with distilled water. Lugol’s iodine was added for one minute, and removed by washing with distilled water. The slides were decolorized by alcohol for ten seconds and the residue was removed by distilled water. The slides were counter – stained with Bacto Gram Saffranin for 30-60 seconds and washed with distilled water. The slides were then dried with a filter paper and a drop of immersion oil was added followed by examining under microscope. Gram positive–organisms appeared purple, while Gram negative ones appeared pink. 3.6.1.3 Catalase test: The organisms to be tested were put on sterile slides. A drop of 3% hydrogen peroxide (H2O2) added to the colony and mixed. Evolution of gas immediately or after five minutes indicated a catalase positive result. 3.6.1.4 Oxidase test: Two or three drops of tetra methyl – P – phenylene diamine dihydrochloride were placed on filter paper (7 cm in diameter). Before the drops dry, the test organism that was grown on nutrient agar medium was removed with sterile glass rod and smeared a cross the impregnated paper. The development of a dark purple colour within ten seconds indicated the presence of oxidase enzyme. 3.6.1.5 Motility test: Hanging drop technique was used. The culture was transfered by loop to slide and examined under the microscope. 3.6.1.6 Oxidation fermentation test (OF): Duplicate tubes of Hugh and Lifson’s medium were inoculated by stabbing with a sterile straight wire, medium in one of the tubes was covered with a layer of soft sterile paraffin oil to a depth of about one cm, while the other tube was not covered. The tubes were then incubated at 37° C and examined daily for 14 days. Colour change to yellow in both opened and covered tubes indicated fermentative organisms, while change in the uncovered tubes only indicated oxidative organisms. 3.6.2 Confirmatory tests: Fermentation of sugars: A twenty four hours culture was inoculated into peptone broth with sugars (manitol, lactose, xylose, maltose, glucose) and incubated at 37° C for up to seven days. Change in colour to yellow indicated a positive reaction. Gas was accumulated in the Durham’ tubes when produced. 3.7 Statistical analysis: All the data of this experiment were analyzed statistically by using complete randomized design (CRD) and the least significant difference test, which was used to detect difference between means (Snedecor and Cochran, 1980). The analysis was carried out using SPSS program (Statistical Packages for Social Science), Model design: Yij = M + αi + eij. Where: M = The over all population mean. αi = The effect of the ith A class expressed as a deviation from the over all mean. eij = Random errors. CHAPTER FOUR RESULTS AND DISCUSSION 4.1 Chemical composition of stirred yoghurt: Tables (1 6) show the chemical composition of stirred yoghurt samples (fat, protein, lactose, ash, total solids, solids non fat, pH, acidity and viscosity). 4.1.1 Fat: Table (1) shows the fat content at days 0, 2, 4, 6, 8 and 10 which ranged from 4.00% – 4.90%, 3.10% – 4.10%, 1.60%– 2.40%, 2.50%–3.10%, 2.50%– 3.10% and 2.50% – 3.20%, respectively. The mean values were 4.51% ± 0.27, 3.66% ± 0.38, 2.17% ± 0.26, 2.82% ± 0.23, 2.77% ± 0.22 and 2.77% ± 0.23, respectively. Table (6) shows the statistical analysis of chemical composition of fat by using one way ANOVA. From those results it was found the fat content was significant at P < 0.001. The results observed confirmed the findings of Hofi et al. (1978). There was small variation in fat content of different samples of yoghurt because of standardization resulting in minimal compositional variation from sample to sample, similarly as milk composition vary from day to day or batch to batch. However these results are not in accordance with the findings of Athar (1986) who reported 3.6 percent fat in typical plain yoghurt. Table (1) Fat and protein content of stirred yoghurt samples during storage 0, 2, 4, 6, 8 and 10 day Fat% Days Mean ± Sd. 0 4.51 ±0.27 2 3.66 ±0.38 4 2.17 ±0.26 6 2.82 ±0.23 8 2.77 ±0.22 10 2.77 ±0.23 Protein% Minimum Maximum Mean ± Sd. Minimum Maximum d 4.00 4.90 2.6 6 ±0.70 a 1.79 3.75 c 3.10 4.10 3.16 ±0.84 a 1.61 4.11 a 1.60 2.40 3.97 ±0.56 2.86 4.65 b 2.50 3.10 3.50 ±1.01 ab 1.79 4.73 b 2.50 3.10 3.97 ±1.14 b 2.14 5.36 b 2.50 3.20 2.66 ±0.54 a 1.97 3.93 ab In this and the following tables the column being the same superscript latter are significantly (P > 0.05) not different 4.1.2 Protein: Table (1) shows the protein content at days 0, 2, 4, 6, 8 and 10 readings ranged from 1.79%–3.75%, 1.61%– 4.11%, 2.86% – 4.65%, 1.79%–4.73%, 2.14%– 5.36% and 1.97% – 3.93%, respectively. The mean values were 2.66% ± 0.70, 3.16% ± 0.84, 3.97% ± 0.56, 3.50% ± 1.01, 3.97% ± 1.14, and 2.66% ± 0.54, respectively. Table (6) shows the statistical analysis of the chemical composition of protein. The results indicates that the protein content was significant at P< 0.05. This result was in agreement with the results of Tamime and Deeth (1980), who concluded that the addition of milk powder raised the protein level of milk. These results are also in line with findings of Duitschaver and Arnott (1972), who reported that plain yoghurt was higher in protein content due to the absence of dilution effect. These results are not in line with findings of Shanley (1973) who found that the protein and ash contents of yoghurt decreased with the progress of storage period. This is due to the fact that the majority of protein and mineral contents in milk decreased with storage. 4.1.3 Lactose: Table (2) show the lactose content at days 0, 2, 4, 6, 8 and 10 were ranged from 7.03%–9.30%, 7.53%–10.29%, 6.75%–10.26%, 6.98%–10.47%, 6.79%–10.18% and 8.29%–10.92%, respectively. The mean values were 8.31%±0.85, 9.02%±0.61, 8.83%±0.90, 8.59%±0.96, 8.45% ±1.18, and 9.58% ± 0.81, respectively. Table (6) shows the statistical analysis results of lactose. These results are in line with findings of Davis and Mclachlan (1974) who found that the lactose calculated from protein was (5.05–8.74%).These results were higher than those reported by El- Sadek et al (1972) for lactose in zabady (4.5 - 4.8 %). 4.1.4 Ash: The ash content at days 0, 2, 4, 6, 8 and 10 were shown in table (2). The ash values ranged from 0.80%– 0.92%, 0.67%– 0.86%, 0.60%– 0.93%, 0.80%– 0.99%, 0.54%– 0.95% and 0.70%– 0.95% respectively. The mean values were 0.84% ± 0.03, 0.73% ± 0.06, 0.78% ± 0.11, 0.92% ± 0.06, 0.86% ± 0.12, and 0.86% ± 0.07, respectively. Table (6) shows the chemical composition of stirred yoghurt. The ash content was significant by different at P < 0.05, these results are in line with finding of Shanley (1973) who found that the protein and ash contents of yoghurt decreased with the progress of storage period. This is due to the fact that the majority of protein and mineral contents in milk decreased with storage. These results were supported by the findings of Hidiroglou and Proulx (1982) who reported that milk Ca, P and Mg contents were all highest during the first day of storage then decreas sharply at 2nd day. 4.1.5 Total solids (TS): Table (3) illustrates total solids content at days 0, 2, 4, 6, 8 and 10 which ranged from 15.57% – 16.90%, 16.24% – 16.92%, 14.02% – 16.69%, 15.50% – 16.42%, 15.63% – 16.28% and 14.84% – 17.74%, respectively. The mean values were 16.32% ± 0.34, 16.57% ± 0.21, 15.75% ± 0.73, 15.76% ± 0.28, 16.05% ± 0.17, and 15.88% ± 0.75, respectively. Table (6) shows that there are no variation between total solids (P > 0.05). These results are in line with finding of Hofi et al. (1978). Robinson (1983) reported that the aim of total solids is consistency improvement imparted to the yoghurt coagulum; these results are totally different from those reported by Sarkar et al. (1996). The results are in accordance with the findings of Athar (1986). There was hardly any variation in total solids of different samples of stirred yoghurt. This is most probably because of standardization of raw milk and quality control measures taken to ensure consistency of end product. But in case of milk used without subjecting to standardization thus may lead to much variation as observed in total solids content. Table (2) Lactose and Ash content of stirred yoghurt samples during storage 0, 2, 4, 6, 8 and 10 day Lactose content % Ash content % Days Mean ± Sd. Min. Max. Mean ± Sd. Min. Max. Zero day 8.31 a ±0.85 7.03 9.30 0.84 ab ±0.03 0.80 0.92 2 9.02 a ±0.61 7.53 10.29 0.73 a ±0.06 0.67 0.86 4 8.83 a ±0.90 6.75 10.26 0.78 ab ±0.11 0.60 0.93 6 8.59 a ±0.96 6.98 10.47 0.92 ab ±0.06 0.80 0.99 8 8.45 a ±1.18 6.79 10.18 0.86 b ±0.12 0.54 0.95 10 9.58 a ±0.81 8.29 10.92 0.86 b ±0.07 0.70 0.95 4.1.6 Solids – non–fat (SNF): Table (3) shows the average of solids – not – fat (SNF) content at days 0, 2, 4, 6, 8 and 10, the SNF content ranged from 11.34% – 12.39%, 12.14%–13.52%, 11.82%–14.50%, 12.40%– 13.72%, 12.93%– 13.69% and 12.34%– 14.52%, respectively. The mean values were 11.73% ± 0.31, 12.91% ± 0.49, 13.58% ± 0.79, 12.94% ± 0.40, 13.28% ± 0.29, and 13.11% ± 0.61, respectively. From the results it was found that the solids– non – fat (SNF) content were significant at P < 0.001 (Table 6). These results do not agree with the findings of Richter (1980). There was no significant variation in SNF content of different samples of plant made yoghurt because raw milk is standardized to a fixed SNF content in order to ensure consistency of end product. But in case of these results due to raw milk is used without subjecting to standardization. Hence more variation was observed in SNF content of yoghurt samples. These results were in agreement with those obtained by El – Shibiny et al. (1979a and b) who claimed that total solids content decreased proportionally during the storage period with increase in glucose plus galactose and these result are in line with findings of Tamime and Deeth (1980) who found that the production of flavour components such as acetaldehyde can arise from fat, protein or lactose and hence their results showed that any variation of content will affected the solids – non – fat content, so it is very important that to standardized the milk as well as to fix the level to acceptable standard. Tamime and Robinson (1976) found that the legal standards of yoghurt are mainly based on the chemical composition of the product i.e. percentage of fat, solids – non – fat (SNF) or total solids (TS). A minimum specification for SNF or TS is included by some countries, but the main divisions are on the basis of fat content. 4.1.7 pH: The pH values of stirred yoghurt at days 0, 2, 4, 6, 8 and 10 as shown in table (4) ranged from 3.73 – 4.72, 3.91 – 4.43, 4.03 – 4.40, 4.01 – 4.34, 3.82 – 4.14 and 3.52 – 4.04, respectively. The mean values were 4.17 ± 0.29, 4.13 ± 0.17, 4.19 ± 0.13, 4.17 ± 0.11, 4.02 ± 0.10 and 3.81 ± 0.19, respectively. Table (6) shows the result of pH were significant at P < 0.001 and the results of high acid production and decreased pH agree with findings of Kondratenko et al. (1978) and Zobkona and Plish (1980). These results are in line with the findings of Eckles et al. (1952), who reported that when acid production increased then quality of dairy product was deteriorated. As the temperature of the refrigerator was not suitable for the growth of acid producing bacteria so that acid Table (3) Total solids (T.S) and Solids–non–fat (SNF) of stirred yoghurt samples during storage 0, 2, 4, 6, 8 and 10 day Total solids (T.S) Days Solids–non–fat (SNF) Mean Mean Minimum Maximum ± Sd. Minimum Maximum ± Sd. 0 16.32 ±0.34 b 15.57 16.90 11.73 ±0.31 a 11.34 12.39 2 16.57 ±0.21 b 16.24 16.92 12.91 ±0.49 b 12.14 13.52 4 15.75 ab ±0.73 14.02 16.69 13.58 ±0.79 c 11.82 14.50 6 15.76 ab ±0.28 15.50 16.42 12.94 ±0.40 b 12.40 13.72 8 16.05 ±0.17 ab 15.63 16.28 13.28 bc ±0.29 12.93 13.69 10 15.88 ±0.75 a 14.84 17.74 13.11 bc ±0.61 12.34 14.52 Production was slow and resulting in long shelf life of yoghurt (Osborne and Pritchard 1974). The results of the study are not in line with the findings of Salji et al. (1985) and Varnam and Sutherland (1994), who found no significant variation in pH of different samples of plant made yoghurt as compared to dahi because yoghurt was incubated for specific time and temperature to attain desired pH. In case of dahi proper fermentation condition are not fully controlled. 4.1.8 Acidity The acidity percent of stirred yoghurt in table (4) shows at days 0, 2, 4, 6, 8 and 10 ranged from 0.89 – 0.97, 0.94– 1.50, 0.80 – 0.99, 0.90– 0.99, 0.97–1.00 and 0.99 – 1.20, respectively. The mean values were 0.94 ± 0.02, 1.12 ± 0.21, 0.93 ± 0.06, 0.96 ± 0.03, 0.99 ± 0.01 and 1.08 ± 0.08, respectively. From table (6) it was found that the result of acidity was significant at P < 0.001 and these results are in line with findings of Kamruzzaman et al. (2002). They found that acidity percentage of plain and dahi samples increases both at room and refrigeration temperature during storage. These results are in accordance with the findings of Davis and Mclachlan (1974), who reported less variation in acidity of different samples of plant made yoghurt as compared to dahi due to controlled incubation and post production handling and controlled storage at 4° C, so acidity remains the same through all seasons. However in case of dahi uncontrolled incubation and post production handling and uncontrolled storage lead to increase in acidity during summer and subsequent decrease during winter season. This results are in line with the findings of Kim et al. (1998) who found that titratable acidity was 0.95 – 0.99% of Yam – yoghurt which was prepared by addition of Yam to skim milk substrate and cultured with a mixed cultures. Also this findings are in line with the findings of Gregurek (1999) who found that post acidity cation showed slightly higher acidity in yoghurt prepared with lower amounts of inoculum. He concluded that the amount of inoculum and incubation temperature should be properly maintained, to achieve maximum viability. From the other side these results are not in line with the findings of Collado et al. (1994) who found that the yoghurt drink and yoghurt like products had 0.56% and 0.58% total titratable acidity. These results are not in line with the findings of Dalles and Kechagias (1984) who found that the acidity of commercial types of yoghurt in Greece ranged from 1.02 – 2.15%. 4.1.9 Viscosity: The viscosity at days 0, 2, 4, 6, 8 and 10 are shown in table (5) the viscosity results ranged from 38.30% – 82.70%, 39.40%–80.10%, 49.30% – 80.20%, 49.40%–81.10%, 55.40%– 75.30% and 56.30% – 73.00%, respectively. The mean values were 64.79% ± 13.91, Table (4) pH and acidity development of stirred yoghurt samples during storage 0, 2, 4, 6, 8 and 10 day pH Days Acidity Mean Mean Minimum Maximum ± Sd. Minimum Maximum a 0.89 0.97 b 0.94 1.50 a 0.80 0.99 a 0.90 0.99 a 0.97 1.00 b 0.99 1.20 ± Sd. b 3.73 4.72 0.94 ±0.02 b 3.91 4.43 1.12 ±0.21 b 4.03 4.40 0.93 ±0.06 b 4.01 4.34 0.96 ±0.03 b 3.82 4.14 0.99 ±0.01 a 3.52 4.04 1.08 ±0.08 0 4.17 ±0.29 2 4.13 ±0.17 4 4.19 ±0.13 6 4.17 ±0.11 8 4.02 ±0.10 10 3.81 ±0.19 61.98% ± 11.72, 62.57% ± 8.93, 62.96% ± 10.80, 64.34% ± 6.23 and 64.95% ± 6.07, respectively. Table (6) shows there was no significant P > 0.05 difference in viscosity between the samples of stirred yoghurt. 4.2 Microbiological results of stirred yoghurt: Table (7) shows the microbiological analysis of stirred yoghurt samples at days 0, 2, 4, 6, 8 and 10. 4.2.1 Standard plate count: The standard plate count of bacteria at days 0, 2, 4, 6, 8 and 10 ranged from 7.00– 7.60, 7.18– 8.78, 7.18– 7.54, 7.18– 7.65, 7.30– 8.78 and 7.18– 7.70, respectively. The mean values were 7.27 ± 0.20, 7.68 ± 0.42, 7.36 ± 0.14, 7.43 ± 0.17, 7.56 ± 0.13 and 7.50 ± 0.14, respectively. Table (9) indicated that the results of plate count was significant at P < 0.01. These results are in line with findings of Riadh Al-Tahiri (2005) who found that the total bacterial count was 6 × 106 in yoghurt produced by modern dairies in Jordan. Results of this study are also in accordance with the findings of Kosikowski (1977) who found that plain yoghurt may Table (5) Viscosity of stirred yoghurt samples during storage 0, 2, 4, 6, 8 and 10 day Days Mean ± Sd. Minimum Maximum Zero day 64.79 a ±13.91 38.30 82.70 2 61.98 a ±11.72 39.40 80.10 4 62.57 a ±8.93 49.30 80.20 6 62.96 a ±10.80 49.40 81.10 8 64.34 a ±6.23 55.40 75.30 10 64.95 a ±6.07 56.30 73.00 Table (6) Chemical composition of stirred yoghurt using one way ANOVA analysis Mean square pH Acidity Fat Viscosity T.S. Protein Ash SNF Lactose Between groups 0.338 Within groups 0.059 Between groups 0.062 Within groups 0.009 Between groups 6.922 Within groups 0.074 Between groups 15.762 Within groups 100.494 Between groups 3.455 Within groups 1.932 Between groups 7.767 Within groups 2.871 Between groups 0.031 Within groups 0.015 Between groups 4.034 Within groups 0.261 Between groups 1.183 Within groups 0.937 NS: non significant (P > 0.05) *: Significant at (P < 0.05) **: Significant at (P < 0.01) ***: Significant at (P < 0.001) F Sig. 5.738 0.001*** 6.802 0.001*** 94.109 0.001*** 0.157 0.977NS 1.788 0.131NS 2.705 0.030* 2.150 0.073* 15.437 0.001*** 1.262 0.924NS Contain up to one billion live L. bulgaricus and S. thermophilus cells per ml. As yoghurt stored at 4° C becomes older, these bacteria die and the numbers will decline to few million per ml. These results are in line with findings of Masud et al. (1991) who found that total viable count as well as variation was more in dahi samples. These results are different from those reported by Davis and Mclachlan (1974). There was no significant variation in total viable count of different samples of plant made yoghurt as compared to dahi because defined starter culture is used under proper condition of fermentation for manufacture of yoghurt (Kon, 1959). 4.2.2 Identification of isolated LAB: Table (10) shows the identification of lactic acid bacteria, for S. thermophilus that is Gram positive, shape cocci, non motile, catalase positive, oxidase positive, glucose positive, lactose positive, maltose negative, sucrose positive, xylose positive and fructose positive. The L. bulgaricus is Gram positive, rod shape, non motile, catalase positive and oxidase positive, glucose positive, lactose positive, maltose negative, sucrose negative, xylose negative and fructose positive. 4.2.3 Streptococcus thermophilus: Table (8) show the growth of S. thermophilus in stirred yoghurt at days 0, 2, 4, 6, 8 and 10 the results show the count of S. thermophilus ranged from 7.00– 7.40, 7.00 – 7.60, 7.18– 7.48, 7.00 – Table (7) Total bacteria count of stirred yoghurt samples during storage 0, 2, 4, 6, 8 and 10 day Days Mean ± Sd. Minimum Maximum 0 day 7.27 a ±0.20 7.00 7.60 2 7.68 c ±0.42 7.18 8.78 4 7.36 ab ±0.14 7.18 7.54 6 7.43 ab ±0.17 7.18 7.65 8 7.56 bc ±0.13 7.30 8.78 10 7.50 bc ±0.14 7.18 7.70 7.54, 7.30 – 7.78 and 7.30 – 7.54, respectively. The mean values were 7.15 ± 0.15, 7.31 ± 0.16, 7.34 ± 0.11, 7.27 ± 0.18, 7.51 ± 0.15, and 7.41 ± 0.09, respectively. Table (10) shows that the total counts of S. thermophilus were significant at P < 0.001. These results are in accordance with the findings of Kosikowski (1977) who reported that plain yoghurt may contain up to one billion live L. bulgaricus and S. thermophilus cell per ml. As yoghurt stored at 4° C becomes older, these bacteria die and numbers will decline to few millions per ml. These results are also in line with findings of Cousin and Marth (1976) who found that the count of S. thermophilus ranged from 3.4 × 103 to 6.4 × 106 /ml. Also results were in agreement with Tamime and Deeth (1980) who reported that the quantitative standards for yoghurt bacteria differ. It is generally accepted that the yoghurt should contain 107 cfu of viable bacteria (Streptococcus thermophilus and Lactobacillus bulgaricus) per ml of yoghurt (Tamime and Deeth, 1980). Consequently there has been considerable research into ways to maintain the viability of these organisms to ensure that the live bacteria are still present in the yoghurt when it is consumed. The present results differ from the findings of Jay (1986) who found that yoghurt of the western world is cultured with Lactobacillus bulgaricus and Streptococcus thermophilus at temperature around 43° C, as freshly prepared yoghurt, has 100 million to 2 billion bacterial cells per gram. 4.2.4 Lactobacillus bulgaricus: Table (8) show the growth of L. bulgaricus in stirred yoghurt at days 0, 2, 4, 6, 8 and 10, the result shows the count of Lactobacillus bulgaricus ranged from 7.00 – 7.48, 7.18 – 7.78, 7.18 – 7.40, 7.00 – 7.48, 7.18 – 7.65 and 7.18 – 7.54, respectively. The mean values were 7.21 ± 0.15, 7.37 ± 0.16, 7.30 ± 0.10, 7.33 ± 0.15, 7.50 ± 0.14 and 7.41 ± 0.13, respectively. Table (9) show the microbiological analysis and from these results it was found that the total counts of L. bulgaricus were significant by different (P < 0.001). These results are in line with the findings of Ishibashi and Shimamura (1993) who found that in Japan the fermented milk and lactic beverages association has established a standard that requires the presence of > 107 viable bacteria/ml in dairy products. These results are in accordance with the findings of Ministerio de La Presidencia de Espana (2003) who found that the Swiss food required that such products contain ≥ 106 cfu/g, and the Spanish Yoghurt Quality Standard requires 107 cfu/ml. These results are in agreement with the findings of Harmann and Marth (1984) who found that after extensive study of survival of S. thermophilus and L. bulgaricus in commercial and experimental yoghurts recommended that yoghurt should contain at least one million viable organisms per gram at the time of sale. These results differ from those of Jay (1986) who found that yoghurt of the western world is cultured with Lactobacillus bulgaricus and Streptococcus thermophilus at temperature around 43° C and freshly prepared yoghurt has 100 million to 2 billion bacterial cell per gram. Table (8) Microbiology analysis of stirred yoghurt samples during storage 0, 2, 4, 6, 8 and 10 day Streptococcus thermophilus Days Mean Lactobacillus bulgaricus Mean Minimum Maximum ± Sd. a 0 7.15 ±0.15 2 7.31 bc 4 7.34 6 7.27 Minimum Maximum ± Sd. a 7.00 7.48 bc 7.18 7.78 ab 7.18 7.40 ±0.15 7.00 7.48 7.78 7.50 ±0.14 7.18 7.65 7.54 7.41 7.18 7.54 7.00 7.40 7.21 ±0.15 ±0.16 7.00 7.60 7.37 ±0.16 bc ±0.11 7.18 7.48 7.30 ±0.10 ab ±0.18 7.00 7.54 7.33 d 7.30 cd 7.30 8 7.51 ±0.15 10 7.41 ±0.09 ab c bc ±0.13 Table (9) Microbiology analysis of stirred yoghurt using one way ANOVA analysis M.S. Between groups Within groups 0.050 Between groups 0.154 S. thermophilus Within groups 0.021 Between groups 0.099 L. bulgaricus NS: non significant (P > 0.05) *: Significant at (P < 0.05) **: Significant at (P < 0.01) ***: Significant at (P < 0.001) Sig. 4.263 0.002** 7.420 0.001*** 5.115 0.001** 0.214 Plate count Within groups F. 0.019 Table (10) Identification of lactic acid bacteria Primary test Fermentation of sugar Gram Shape Motility Catalase Oxidase OF Glucose Lactose Maltose Sucrose S. thermophilus + Cocci - + + F + + - + + + L. bulgaricus + Rod - + + F + + - - - + Streptococcus thermophilus Lactobacillus bulgaricus +: positive -: negative F: Fermentation O: oxidation Xylose Fructose CHAPTER FIVE CONCLUSION AND RECOMMENDATIONS 5.1 Conclusion: The overall picture of stirred yoghurt quality evaluation need emphasis on quality control during processing plants. The large number of samples containing of either streptococci or lactobacilli indicates improper storage conditions of yoghurt in merchandising channels. Yoghurt is biologically active product and therefore has to be kept at about 5° C. It is likely that inadequate storage conditions and age of sample influenced the pH readings obtained. Generally the customers need to receive good value products for their money. The lack of cold storage condition during marketing responsible for variation of the product and thus influence public acceptance. 5.2 Recommendations: 1. More attention should be given to proper incubation temperature and good quality control during processing. 2. Standardization of milk for yoghurt manufacture should be observed to meet legal standards. 3. Adjustment of yoghurt mix should approach the standard of the yoghurt package label. 4. Yoghurt is biologically active product and therefore has to be kept at about 5° C until it reaches the consumers. 5. 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