effect of some treatments on properties of some dairy

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Milk contains several nutrients that are necessary for the life of all living ...... for 15, 30 and 60 s for milk used in Danbo 45+ (semi-hard cheese with 45% fat.
Mansoura University Faculty of Agriculture Dairy Department

EFFECT OF SOME TREATMENTS ON PROPERTIES OF SOME DAIRY PRODUCTS By MOHAMED ZAKI AHMED EID B.Sc. Agric.Sci ( Dairy )1998 Fac. of Agric. Mansoura University THESIS Submitted in partial fulfillment of the requirements for the degree of Master of Science in Agricultural Sciences ( DAIRYING )

Supervision Prof .Dr ABDEL-WAHAB EL-SHAZLY KHALIL Prof. of Dairy Chem. and Tech. Faculty of Agriculture Mansoura University.

Prof .Dr EL-Tahra M. A. Ammar Prof. of Dairy Chem. and Tech. Head of Dairy Department Faculty of Agriculture, Mansoura University.

Dr. Magdy M. I. Mohamed Associate Prof. of Dairy Technology, Animal Production Research Institute, Agriculture Research Center.

Mansoura University Faculty of Agriculture Dairy Department

SUPERVISION SHEET Title of the thesis: EFFECT OF SOME TREATMENTS ON

PROPERTIES OF SOME DAIRY PRODUCTS The Researcher : MOHAMED ZAKI AHMED EID

Thesis supervised by: No

Name

Position

1

Prof .Dr Abd EL-Whab ELShazly Khalil

Prof. of Dairy Chemistry and Technology, Faculty of Agriculture, Mansoura University

2

Prof .Dr EL-Tahra M. A. Ammar

3

Dr. Magdy M. I. Mohamed

Signature

Prof. of Dairy Chemistry and Technology, Head of Dairy Department Faculty of Agriculture, Mansoura University. Associate Prof. of Dairy Technology, Animal Production Research Institute, Agriculture Research Center.

Date of discussion: 30 /12 / 2008 Head of the department Vice Dean for Post-Graduate Affairs Prof.Dr. EL-Tahra M. A. Ammar

Prof.Dr. EL-Sayed M.EL-Hdidi

Dean

Prof.Dr. Hesham.N.Abdel-Mageed

Mansoura University Faculty of Agriculture Dairy Department

APPROVAL SHEET Title of the thesis: EFFECT OF SOME TREATMENTS ON PROPERTIES OF SOME DAIRY PRODUCTS . The Researcher: MOHAMED ZAKI AHMED EID Thesis supervised by: o

Name

1

Prof .Dr Abd EL-Whab EL-Shazly Khalil

2

3

Position Prof. of Dairy Chemistry and Technology, Faculty of Agriculture, Mansoura University.

Prof .Dr EL-Tahra M. A. Ammar

Prof. of Dairy Chemistry and Technology, Head of Dairy Department Faculty of Agriculture, Mansoura University.

Dr. Magdy M. I. Mohamed

Associate Prof. of Dairy Technology, Animal Production Research Institute, Agriculture Research Center.

Signature

This thesis has been approved by: No

Name

1

Prof .Dr Abd EL-Whab EL-Shazly Khalil

2

Position

Prof .Dr EL-Tahra M. A. Ammar

Signature

Prof. of Dairy Chemistry and Technology, Faculty of Agriculture, Mansoura University. Prof. of Dairy Chemistry and Technology, Head of Dairy Department Faculty of Agriculture, Mansoura University.

3

Prof .Dr Mohamed Younis Riad Mehana

Prof. of Dairy Technology, Faculty of Agriculture, Mansoura University.

4

Prof .Dr Abdel-Hamid Aboel –Hassan Askar

Prof. of Dairying Faculty of Agriculture, Ein Shams University.

Date of discussion: 30 / 12 /2008 Head of the department Vice Dean for Post-Graduate Affairs Prof.Dr. EL-Tahra M. A. Ammar

Prof.Dr. EL-Sayed M.EL-Hdidi

Dean Prof.Dr. Hesham. N. Abdel-Mageed

ACKNOWLEDGMENTS

Thanks GODNESS by the grace and care of whom indeed, the complete of this work was possible. The author wishes to express his deep thanks and gratitude to Prof.Dr. EL-TAHRA .M A. AMMAR. Professor of Dairy chemistry and Technology, Head of Dairy Department, Faculty of Agriculture, Mansoura University, for her direct supervision, planning the research program, reviewing the manuscript, her kind and continuous invaluable help. And deep thanks and gratitude to Prof.Dr.ABD ALWHAB El-Shazly. Professor of Dairy chemistry and Technology, faculty of Agriculture, Mansoura University, for his direct supervision, planning the research program, reviewing the manuscript, his kind and continuous invaluable help. Thanks are also extended to Dr. MAGDY.M. Ismail Associate Professor of Dairy Technology, Animal Production Research Institute, for his supervision, his continuous help during the course of the experimental work and the prepration of this thesis. , and for the long hours he has offered during the writing of this manuscript. The author is also grateful to my parents and my brothers and my wife for their continuous encouragement and support, which enabled me to complete this work. The author is highly thankful to all stuff members of dairy department, Fac. of Agric., Mansoura Univ., for their contribution in the process of my education during my study period.

CONTENTS CONTENT 1. NTRODUCTION 2. REVIEW OF LITERATURE 2.1. Cooling of milk. 2.1.1. Effect of cooling on the physico-chemical properties of milk. 2.1.2. Effect of cooling on the microorganisms of milk. 2.1.3. Effect of cooling of milk on the cheese and yoghurt making. 2.2. Pasteurization of milk. 2.2.1. Effect of Pasteurization on the physico-chemical properties of milk. 2.2.2. Effect of Pasteurization on the microorganisms of milk. 2.2.3. Effect of Pasteurization of milk on the cheese and yoghurt making. 3. MATERIALS AND METHODS 3.1. Materials 3.1.1. Source of milk. 3.1.2. Rennet. 3.1.3. Starter. 3.1.4. Salt. 3.1.5. Calcium chloride. 3.1.6. Chemicals. 3.2. Methods 3.2.1. Domiatii cheese manufacture. 3.2.2. Yoghurt manufacture. 3.2.3. Sampling. 3.2.4. Methods of analysis 3.2.4.1. Yield. 3.2.4.2. Total solids (TS).

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page 1 4 4 4 7 10 12 12 18 22 32 32 32 32 32 32 32 33 33 33 35 36 36 36 36

CONTENT 3.2.4.3. Fat content. 3.2.4.4. Titratable acidity (TA). 3.2.4.5. pH value. 3.2.4.6. Nitrogenous fractions. 3.2.4.6.1. Total nitrogen (TN). 3.2.4.6.2. Water Soluble nitrogen (WSN). 3.2.4.6.3. Non-protein-nitrogen (NPN). 3.2.4.7. Total volatile fatty acids (TVFA). 3.2.4.8. Ash content. 3.2.4.9. Salt content. 3.2.4.10. Analyses of amino acids composition. 3.2.4.11. Microbial analysis. 3.2.4.12. Rheological tests. 3.2.4.12.1. Rennet coagulation time (RCT). 3.2.4.12.2. Curd tension. 3.2.4.12.3. Curd syneresis. 3.2.4.13. Organoleptic properties judging. 3.2.4. 14. Statistical analysis. 4. RESULTS AND DISCUSSION part (1) 4.1.1. Effect of cold storage and mixing various lactating milk on some properties of buffaloe's milk. 4.1.1.1. Chemical composition of milk. 4.1.1.2. Rheological properties of milk. 4.1.1.3. Microbial groups of milk. 4.1.2. Effect of cold storage and mixing various lactating milk on some properties of cow's milk. 4.1.2.1. Chemical composition of milk. 4.1.2.2. Rheological properties of milk. 4.1.2.3. Microbial groups of milk. part (2) 4.2. Effect of cold storage and mixing various lactations of

II

page 37 37 37 37 37 37 37 38 38 38 38 38 39 39 39 39 40 40 41 41 42 42 45 49 52 52 55 59 63 64

CONTENT buffaloe’s milk on some properties of Domiatii cheese. 4. 2.1. Chemical composition of milk used in Domiatii cheese manufacture. 4.2.2. Yield of Domiatii cheese. 4.2.3. Chemical composition of Domiati cheese. 4.2.3.1 Acidity and pH values. 4.2.3.2. Total solids and fat contents. 4.2.3.3. Salt content. 4.2.3.4. Total nitrogen, water soluble nitrogen and nonprotein- nitrogen contents. 4.2.3.5. Total volatile fatty acids (TVFA). 4.2.3.6. Free amino acid (FAA). 4.2.3.7. Microbial profile of cheese. 4.2.3.8. Organoleptic properties. Part(3) 4.3. Effect of cold storage and mixing various lactations of cow’s milk on some properties of Domiatii cheese. 4.3.1. Chemical composition of milk used in Domiatii cheese manufacture. 4.3.2. Yield of Domiatii cheese. 4.3.3. Chemical composition of Domiati cheese. 4.3.3.1. Acidity and pH values. 4.3.3.2. Total solids and fat contents. 4.3.3.3. Salt content. 4.3.3.4. Total nitrogen, water soluble nitrogen and nonprotein- nitrogen contents. 4.3.3.5. Total volatile fatty acids (TVFA). 4.3.3.6. Free amino acid (FAA). 4.3.3.7. Microbial profile of cheese. 4.3.3.8. Organoleptic properties.

III

page 64 65 67 69 69 71 73 74 77 78 80 83 86 87 88 90 92 92 93 94 95 97 98 100 102

CONTENT Part(4) 4.4. Effect of cold storage and mixing various lactations of buffaloe's and cow’s milk on some properties of yoghurt. 4.4.1. Chemical composition of milk used in yoghurt manufacture. 4.4.2. Chemical composition of yoghurt. 4.4.2.1 Acidity and pH values. 4.4.2.2. Total solids, fat and ash contents. 4.4.2.3. Total nitrogen, water soluble nitrogen and nonprotein- nitrogen contents. 4.4.2.4. Total volatile fatty acids (TVFA). 4.4.3. Microbial profile of yoghurt. 4.4.4. Organoleptic properties. 5- SUMMARY AND CONCLUSION 6- REFERENCES ARABIC SUMMARY

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page 105 106 107 109 109 111 112 114 114 116 119 128

LIST OF TABLES Table No

Tables

Page

1

Effect of cold storage and mixing various lactating milk on the chemical composition of buffaloe's milk.

43

2

Effect of cold storage on some rheological properties of buffaloe's milk.

46

3

Effect of cold storage on some microbial groups of buffaloe's milk.

50

4

Statistical analysis of buffaloe's milk treatments.

51

5

Effect of cold storage and mixing various lactating milk on the chemical composition of cow's milk.

53

6

Effect of cold storage on some rheological properties of cow's milk.

56

7

Effect of cold storage on some microbial groups of cow's milk.

60

8

Effect of cold storage on some microbial groups of cow's milk.

62

9

Chemical composition of buffaloe's milk used in Domiatii cheese manufacture.

66

10

Effect of cold storage and mixing various lactations of buffaloe’s milk on yield and chemical composition of Domiatii cheese.

68

11

Effect of cold storage and mixing various lactations of buffaloe’s milk on TN and some ripening indices of Domiatii cheese.

75

12

Amino acid concentrations (ug/ml) of Domiati cheese at the end of storage Period

79

13

Effect of cold storage and mixing various lactations of buffaloe’s milk on some microorganisms of Domiatii cheese.

81

14

Effect of cold storage and mixing various lactations of buffaloe’s milk on organoleptic properties of Domiatii cheese.

84

VI

Table No

Tables

15

Statistical analysis of buffaloe's milk cheese treatments.

16

Chemical composition of cow's milk used in Domiatii cheese manufacture. Effect of cold storage of cow’s milk on yield and chemical composition of Domiatii cheese. Effect of cold storage of cow’s milk on TN and some ripening indices of Domiatii cheese. Amino acid concentrations (ug/ml) of Domiati cheese at the end of storage period. Effect of cold storage of cow’s milk on some microorganisms of Domiatii cheese. Effect of cold storage of cow’s milk on organoleptic properties of Domiatti cheese.

17 18 19 20 21 22 23 24 25 26 27 28

Statistical analysis of cow's milk cheese treatments. Chemical composition of buffaloe's and cow's milk used in yoghurt Manufacture. Effect of mixing morning and evening milks and cold storage on chemical composition of yoghurt made from buffaloe's or cow's milk. Effect of mixing morning and evening milks and cold storage on TN, nitrogen fraction and TVFA of yoghurt made from buffaloe's or cow's milk Effect of mixing morning and evening milks and cold storage on some microbial groups of yoghurt made from buffaloe's or cow's milk. Effect of mixing morning and evening milk and cold storage on organoleptic properties of yoghurt made from buffaloe's or cow's milk. Statistical analysis of yoghurt treatments.

VII

Page 85 89 91 96 99 101 103 104 108 110 113 115 117 118

INTRODUCTION Milk is one of the most important products for human consumption. It is highly nutritious food, which is suitable for both children and adults as an excellent source of energy, protein, vitamins and minerals. However, due to its rich nutritional composition, it is also ideal for microbial growth. Fresh raw milk is easily deteriorated to become unsuitable for processing and human consumption. Milk’s quality relates to its chemical, microbiological, physical, and organoleptic properties, as well as to its safety. To protect milk’s quality, this food is handled under rigid sanitary conditions, resulting in low bacterial count, good flavor and appearance, satisfactory keeping quality, high nutritive value, and freedom from disease-producing organisms and foreign constituents. The distance between the farm, the dairy and the consumer became greater, as did the time lapse between milking and the drinking of milk. Milk storage on the farm, and the time taken to bridge the gap between producer and consumer gave bacteria the chance to acclimatize and grow in this nutritious liquid. It became a problem to keep milk quality at the same level as just after milking. If you lower the temperature of stored milk, chemical processes and microbiological growth will slow down, delaying the reduction in quality. This knowledge enabled farmers, transporters, and dairy organizations to provide milk to consumers after a time delay, without an unacceptable impact on quality. Refrigeration is currently recognized as the preferred milk preservation method. Refrigerating milk on the farm has two main aims:

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- to inhibit bacterial spoilage - to extend storage on the farm so as to decrease milk transport costs. However, it is vital to recognize that cooling is a compliment, not a substitute, for hygienic working practices, and that prevention is better than cure. Cooling is the weapon against growth so with efficient cooling and good care the battle against microorganisms can be won. The quality of all milk products rises, as does the milk quality. This leaves only one winner, human health. The rate at which milk is cooled has a major influence on the bacterial content of raw milk. Milk should be cooled to 4°C or below as soon as possible after it leaves the udder. It should be cooled to this temperature within 3½ hours of the start of milking. However, any reduction in cooling time will increase milk quality and reduce energy costs. On the other hand, pasteurization is the process of heating liquids like milk in properly designed and operated equipment at a sufficiently high temperature for a specified length of time for the purpose of destroying viruses and harmful organisms such as bacteria, protozoa, molds and yeasts. Unlike sterilization, pasteurization is not intended to kill all microorganisms in the food. Instead, pasteurization aims to achieve a "log reduction" in the number of viable organisms, reducing their number so they are unlikely to cause disease (assuming the pasteurized product is refrigerated and consumed before its expiration date). The most common time-temperatures used by processors include 63°C (145°F) for 30 minutes or 72°C (161°F) for 15 seconds. The

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pasteurized product is then cooled quickly to 3 to 4°C (38 to 40°F) to retard the growth of surviving organisms. Now, almost all over the world, governments have passed laws requiring pasteurization of milk that is produced at a dairy and intended for consumption. The new villages of Nubaria area In Egypt, one of the new reclaimed lands which owned by graduates or settlers who have only 1-2 cows, milk collection centers are not available since graduates are scattered over the big dessert area. Through extension services, the graduates gained the experience of cheesemaking, mainly Domiatii cheese. But processing of 7-14 kgs of milk daily was not neither reasonable nor economic for them. Some of the graduates cooled the milk at their refrigerators for 48 hours, while others freezed its to collect the milk production of three or four days (30-60 kgs) to process Domiatii cheese once or twice a week instead of daily processing (Abdel-Kader 1999). Therefore, our objectives in this study are to determine the effect of cooling, mixing (morning milk with evening milk) and pasteurization of buffaloe’s or cow’s milk on some chemical, microbiological and organoleptical properties of milk, Domiatii cheese and yoghurt.

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REVIEW OF LITERATURE Milk contains several nutrients that are necessary for the life of all living beings. It is also the perfect growing medium for microorganisms. Full hygiene in all aspects of milk production is essential in the production of quality milk. A critical aspect is to ensure that the growth of bacteria during the storage interval must also be curtailed. At body temperature, bacteria in milk will multiply very quickly and even milk with a low initial bacteria count will sour rapidly. Cooling is a very good method to keep the quality of milk at a high level. 2.1. Cooling of milk: 2.1.1. Effect of cooling on the physico-chemical properties of milk: Ghaleb and Rashed (1983) studied the effect of storage for 0,1, 2, 3 or 4 days at 4°C on chemical composition of cows' milk. Cold storage did not affect milk pH or fat content greatly. Storage for greater than 1 day caused increases in acidity. Volatile fatty acids and free fatty acids increased at a similar rate during storage. Milk fat globule diam. and casein micelle mol. wt. both decreased during storage. Paquet et al., (1987) found that proteose-peptone (PP)fraction was estimated by precipitation by 12% trichloroacetic acid from acid whey obtained from milk heated for 20 min at 95 degree C. In raw milk, PP represented about 3% of total N, being proportional to the non-casein protein N. On storage at 6°C, PP increased, particularly during the exponential phase of growth of

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psychrotrophic bacteria Addition of Pseudomonas fluorescens at 60 000/ml increased PP production to give 10% of total N after 4 days at 6°C vs. 4% in control raw milk. Stirring raw milk during storage increased PP production, probably by encouraging growth of psychrotrophs. PP reached a max. after about 3 days storage, followed by an increase (on electrophoresis) in low mol. wt. peptides from continuing proteolysis. Wahba et al., (1988) heated cows' (CM) and buffaloe's (BM) milk to 70, 80 or 90°C and held for 0 or 30 min, cooled to room temp. and stored at 4°C for 24 h. Clotting times were determined, using rennet, before and after these procedures. To study effects of cool-ageing on some physicochemical attributes, milk samples were also stored, raw or pasteurized (65°C for 20 min), at 4°C for 72 h. Casein N, non casein N, non protein N, titratable acidity, pH, casein micelle diam. and mol. wt., and clotting time were determined. Clotting times increased on cold storage for 24 h, more so in CM than BM. On cold storage for 72 h, acidity of milk increased, less so in pasteurized than raw samples (due to differing lactic acid bacterial counts). Casein micelle diam. and mol. wt. of raw and pasteurized CM and BM decreased on cold storage; these changes paralleled the decreases in casein N and increases in non casein and non protein N observed (probably due to dissolution of beta-casein from the micelles). Casein micelle diam. were higher in BM than in CM. Puhan (1989) reviewed chemical changes in milk during cold storage. Changes in lipids vary with type of milk product. Fat globules lose part of the membrane, especially when there is air in the milk, and free fatty acid levels

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increase. Nevertheless, the bulk of the total triglycerides remain available for lipolysis. On cold storage, caseins have an increased clotting time and form a weaker curd with increased syneresis. There is some dissociation of beta-casein from the micelles, but no change in micelle size. Changes in clotting time are correlated with pH changes. Whey nitrogen levels appear to increase, but this is due to a high proportion of cheese fines in the whey. Changes in rennet ability can be reversed by heating, by addition of CaCl2 or by pre-ripening of the milk with starters. Processing of raw cold-stored milk can lower the sensory quality of the cheese. In all cases heat treatment of milk is recommended prior to cheesemaking if milk has been stored for >2 days. Urbach (1990) stated that when raw milk was stored at refrigeration temp. (7 or 2°C), volatile carbonyls were reduced to the corresponding alcohols. Some of these carbonyls, such as acetone, were present in the fresh milk. Others were formed from the corresponding amino acids, e.g. 3-methylbutanal from leucine. The most abundant alcohols were etnanol, propan-2-ol and 3-methylbutan-l-oi. On further storage, the alcohols were partially esterified with volatile acids. Sulphur compounds, e.g. dimethyl disulpnide and 2,4-dithiapentane, were also formed. At the same bacterial cell count, off-flavour and volatiles production were much greater at 7 than at 2°C. The proportions of volatiles formed varied with the source of the milk. Headspace volatiles from cold-stored raw milk and bacterial populations increased in parallel. Celestino et al., (1997) produced whole milk powders from fresh (control) and stored (4 ± 1°C, 48 ± 2h) raw milk in three seasons (spring, summer and

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autumn), and stored for up to 8 months at 25 ± 1°C. The suitability of the milk powders for reconstitution processes was assessed on the basis of selected microbiological, physical, chemical and biochemical quality parameters. Storage of raw milk did not significantly affect total bacterial count (TPC) in the resultant powder. Free fatty acid (FFA) content in powder manufactured from stored raw milk was greater than that from the control. TPC and FFA decreased while insolubility index, FFA and peroxide value increased during storage of powder; lipase and proteinase activities did not change. All powders manufactured from control and stored raw milk, and stored for up to 8 months, were found suitable for reconstitution on the basis of TPC, moisture content and amount of scorched particles but not based on insolubility index. 2.1.2. Effect of cooling on the microorganisms of milk: Bockelmann (1974) stated that bacterial counts of (i) good and (ii) poor quality milk after storage in tanks at 2-4°C for 0, 3 and 4 days respectively were (i) 6000, 5000 and 200 000/ml and (ii) 50 000,1 million and 9 million/ml. When the milk was added to the tank in 4 equal lots at 0.5-day intervals, totalcounts were (i) 7000, 8000 and 32 000/ml and (ii) 33 000, 36 000 and 85 000. Psychrotrophic counts showed similar trends and Gram-negative non-fermenting rods predominated by day 4. Almudena et al., (1995) compared a group of 80 Pseudomonas spp. strains isolated from raw milk shortly after milking to another group of 81 obtained from the same sample after incubating it at 7°C for 3 days. Comparison of both collections of strains included growth rates at 7°C and 21°C and production of

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extracellular proteinase, lipase, and siderophores. The strains selected after cold incubation showed an average 10-fold higher growth rate at 7°C, 1,000-fold more proteolytic activity, and 280-fold more lipolytic activity than those found before the incubation. At 21°C, however, they grew half as quickly as the strains isolated before the incubation. In all but one of the 161 Pseudomonas strains tested, there was some production of siderophores, and yields were only moderately increased in the strains obtained after incubation of the milk at 7°C. These changes in spoilage-related properties took place while global Pseudomonas counts increased only 13-fold. Guinot-Thomas et al., (1995a) occurred study on raw milk with two different levels of psychrotrophic bacteria. Two treatments for raw bulk milk were compared: storage at 4°C for 48 h and storage at 8°C after inoculation with L. lactis subsp. lactis for 48 h. Analysis of the composition of milk before and after storage was conducted on the basis of microbiological and physicochemical composition: colloidal minerals and protein fractions. The results show the importance of the initial microbiological quality of raw bulk milk for its preservation and the efficiency of ripening as a storage treatment for milk with a low-bacterial level. Guinot-Thomas et al., (1995b) studied the interactions between microbial proteinases and plasmin, and their effects on milk quality during storage at 4°C. Experiments were conducted on four milk treated as follows: control milk, milk with added urokinase, milk with added urokinase and bacteriocide, and milk with added bacteriocide. This study on proteolysis in milk during storage at 4°C

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for six days points to the greater importance of microbial proteinases than plasmin at this temperature. Among the physico-chemical parameters, pH and casein level of milk without bacteriocide decreased after the fourth day of storage when the bacterial count reached 106 to 107 cfu mL−1. No activation of plasminogen to plasmin by natural activators or by microbial proteinases was observed. Plasmin and plasminogen—plasmin activities decreased during the six days of storage at 4°C. Sanjuan et al., (2003) studied changes in the microbial flora of ewes' milk of medium hygienic quality (initial mean total plate count 3.3 x 105 cfu/ml) throughout refrigerated storage at 6°C.

Total plate counts after 48 h of

refrigerated storage (mean count 4.6 x 105 cfu/ml) were below current EU standards for raw ewes' milk to be used for cheesemaking after heat treatments. However, after 96 h, mean total plate count (1.6 x 107 cfu/ml) was above EU standards. Lactococci were at a higher level than Pseudomonas spp. in milk freshly drawn (54.5 and 3.5% of total plate count, respectively) and stored for 96 h at 6°C (47.9 and 33.9% of total plate count, respectively).

Lactobacilli,

enterococci, coliforms and thermodurics were at lower concn. than lactococci and Pseudomonas spp., before and after refrigerated storage (0.08% of the total plate count). Significant growth (P < 0.001) was detected for mesophiles, Pseudomonas spp. and lactococci after 96 h storage at 6°C; however, numbers of thermodurics, coliforms, lactobacilli and enterococci, did not increase (P > 0.05).

Presumptive Escherichia coli (beta-glucuronidase-

positive) counts decreased throughout storage at 6°C.

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Manzano et al., (2005) developed a rapid method for the estimation of the microbiological quality of refrigerated raw milk based on the aminopeptidase activity of Gram-negative psychrotrophic bacteria, the dominant microbial population in refrigerated foods. Two versions of the test for measuring the aminopeptidase activity are reported (direct and indirect). A good correlation (r=0.93–0.95) between bacterial counts estimated by conventional methods and the aminopeptidase activity determined in milk was obtained. The sensitivity of the indirect assay was 2×104 cfu mL−1. This sensitivity limit complies with the level of detection required to satisfy regulations in many countries. The aminopeptidase test allows the assessment of raw milk quality in approximately 2.5 h, does not require either high-cost equipment or specialised operators, and results can be interpreted both spectrophotometrically and visually. 2.1.3. Effect of cooling of milk on the cheese and yoghurt making: Ghaleb and Rashed (1983) studied the effect of storage for 0,1, 2, 3 or 4 days at 4°C of cows' milk on yoghurt properties. Time taken for yoghurt pH to reach 4.5 increased by holding milk at 4°C for 1 day, but then decreased again as storage time increased. Whey syneresis, volatile fatty acids and free fatty acids increased with storage. In yoghurt stored for 24 h at 7°C, pH was higher when milk had been stored for 1 day than if it was used fresh. However, yoghurt pH decreased when milk had been stored for longer. Yoghurt made from milk stored for 2 days had the best flavour, while sensory appraisal of acidity was best when milk stored for 3 or 4 days was used, and body and texture were most acceptable when milk had been stored for 2 or 3 days.

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Kehagias (1983) stored ewes' and cows' bulk milk for 0,1 or 2 days at 45°C; half of each sample was then pasteurized (72°C for 15 s) before making into Feta cheese using rennet alone for unpasteurized milk, and rennet + CaCl2 yoghurt culture for pasteurized milk. In most cases total bacterial count, psychrotrophic count and tyrosine value increased significantly in the milk during the 1st day of storage, Curd syneresis increased with storage time for both types of milk, increase being significant only when unpasteurized milk was used. Total N content of whey was not significantly affected by storage of milk, but soluble N content of whey from cheese made with unpasteurized milk stored for 2 days was higher than that in whey from unstored milk. Ammer (1999) studied the yield, composition and organoleptic properties of Domiati cheese made from refrigerated (4°C for 48 hrs.) or frozen (-19°C for 48 hrs.) buffalo milk, with or without the addition

of CaCl2. The rennet

coagulation time slightly decreased in cooled milk, and increased in frozen milk. The curd tension and syneresis slightly increased in cooled and frozen milk. Addition of CaCl2 remarkably minimized the effect of cold and frozen storage of milk. Domiati cheese from frozen milk had slightly less TS and fat content compared to fresh cheese, whereas, cold storage of milk had no considerable effect on cheese fat content. Also, cold and frozen storage remarkably increased cheese yield. Cold and frozen storage of milk had pronounced effect on proteolysis of cheese as indicated by increase in soluble nitrogen. Addition of CaCl2 decreased slightly cheese proteolysis. Cold storage of milk with or without CaCl2 had a noticeable effect on lipolysis by increasing TVFA, whereas

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frozen storage had a reverse effect. Cooled and frozen milk cheese gained slightly less scores, however, all treatments were free from off-flavours. CaCl2 improved the cheese quality, as it gained relatively higher score. Tavaria et al., (2006) manufactured Serra da Estrela cheese from both refrigerated and non-refrigerated milk. Scanning electron microscopy was used to analyze the microstructure, and thus aid in understanding possible differences in their microbiological profile. The cheese were allowed to ripen under controlled conditions, and sampled at 60, 90, 120, 150 and 180 d following manufacture.

Viable

numbers

of

lactic

acid

bacteria,

staphylococci,

Enterobacteriaceae and yeasts were obtained following standard plate counting on a number of selective media. Lactococcus was the most abundant genus (above 108 cfu g−1 of cheese) up to 120 d of ripening. No significant microstructural differences were observed in cheese manufactured in different dairies over the ripening process. However, microstructural differences were apparent between cheese manufactured with refrigerated versus non-refrigerated milk. 2.2. Pasteurization of milk: 2.2.1. Effect of Pasteurization on the physico-chemical properties of milk: Yano et al., (1975) illustrated that free amino acid (FAA) levels decreased slightly in milk autoclaved at 120°C for20min, but were greatly increased by autoclaving at 120°C for 30 min. FAA content of autoclaved milk mixed with a small amount of raw milk, showed marked increases after 20 days. Salama and Youssef (1978) found that heating of cows' skim milk at 95

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and 75°C for 20 min caused marked prolongation of rennet coagulation time (RCT), and RCT was further increased on holding at 5 ±2°C for 24 h. Heat treatment also caused transfer of dissolved Ca, 4-12 mg/100 ml, to the colloidal state. Cold storage of heated milk tended to restore the soluble Ca. Micelle diam. and mol. wt. of casein decreased from 63.7 and 64.9 nm and 107 x 10-6 and 113 x 10-6 for the milk heated to 65 and 75°C resp., to 58.1 and 57.8 nm, and 81 x 10-6 and 80x10-6 for the same milk after cold storage. Dialysis of heated milk against an excess of raw fresh skim milk partially restored RCT, increasing micelle diam. to 68.3 and 62.5 nm for the 2 heat treatments. The increase in RCT of cold-stored heated milk is thought to be due to loss of colloidal calcium phosphate from the casein micelles. Warming of cold-stored raw milk at moderate temp. and times led to micellar growth and acceleration of rennet action. These effects were more pronounced when milk was warmed to 24°C for 3 h than for a 30°C, 30 min treatment. Salama et al., (1982) pasteurized cows' fresh skim milk and reconstituted dried skim milk of 7.5 and 15% SNF pasteurized at 68°C for 30 min, and then stored at 4°C for 48 h. Pasteurization increased the size of casein micelles and casein N contents of all dried milk samples, but had no significant effect on their alcohol stability. A slight increase in viscosity was observed on pasteurization, whilst it increased significantly during cold storage. Cold storage also decreased the casein N content, size of casein micelles and alcohol stability, except in the case of reconstituted skim milk of 7.5% SNF, which showed high alcohol stability. Pasteurization and cold storage increased the rennet coagulation time

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of milk, whereas the rate of whey drainage of curd decreased on pasteurization and increased upon storage. Manpal and Dutta (1986) stated that activity of xanthine oxidase increased progressively on heating milk for 5 min at up to 50°C, but decreased sharply at higher temp. and was zero at greater than or equal 75°C. Calvo and Olano (1989) studied formation of galactose, lactulose and epilactose during heating of milk. Concn. of these carbohydrates increased with pH and severity of heat treatment. The presence of proteins reduced the amount of lactulose formed, whereas it increased that of epilactose and galactose. Galactose in heated milk was derived from lactose degradation. The interaction between proteins and galactose increased the thermal stability of the monosaccharide. Prasad and Balachandran (1989) standardized buffaloe's raw milk to 6% fat and 9% SNF, preheated at various time/temp. combinations (80-95°C for 10 min, 100°C for 1 or 5 min, 110°C for 2 min, 120°C for flash preheating or for 2 min), and examined for effect on pH and heat coagulation time (HCT, determined at 120°C). pH and HCT decreased with increasing severity of heat treatment. HCT decreased in all samples compared with non-heated control raw milk (114.80 min), especially for 10 min heat treatments at temp. 85°C. The amount of whey protein complexed with micelles increased with time, reaching plateau values that, at the highest temp., were comparable with the quantity present in the original skim milk. Reaction of the whey proteins

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with the micelles was faster at lower pH and higher temp. Rates and extent of the reaction also changed when additional alpha-lactalbumin and betalactoglobulin isolates were added to the milk before heating. The reaction between alpha-lactalbumin and casein micelles depended to a lesser extent upon pH and temp. than interactions with beta-lactoglobulin; therefore, the more complex behavior may be attributed to the latter protein. Heat treatment of milk can result in interactions between whey proteins and casein, with effects on the functional properties of dairy products. Oldfield et al., (1998) studied

association of beta-lactoglobulin (betaLg) and alpha-

lactalbumin (alphaLa) with casein micelles in skim milk heated at 70-130°C for times ranging from 5 s to 30 min. Denaturation of the whey proteins and association with casein micelles, determined using PAGE, increased with increasing heating time and temp., with association occurring less rapidly than denaturation. betaLg was denatured more rapidly then alphaLa. The max. association of betaLg with casein micelles was approx. 55%, when most of the betaLg had been denatured. Level of association of alphaLa with the casein micelle varied with temp., the max. ranging from approx. 40% at 95-130°C to approx. 55% at 75-90°C. At 20°C; however, it could be used as an indicator of thermal abuse in stored heat-treated milk as long as initial thermal history of the products has been accurately recorded. 2.2.2. Effect of Pasteurization on the microorganisms of milk: Molska et al., (1989) indicated that thermization of milk of moderate microbiological quality, at 65 and 69°C for 15 s destroyed 54.4-92.4 and 56.996.7% of total bacteria, 76.9-96.6 and 82.9-98.7% of psychrotrophic bacteria and 52.0-93.0 and 64.5-96.7% of acid forming bacteria. Pasteurization of milk at 72°C for 15 s, reduced numbers of total, psychrotrophic and acid forming bacteria by 72.4-97.7, 87.9-99.4 and 62.9-98.1%, respectively. Psychrotrophic

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bacteria reduced to a greater extent than total or acid forming bacteria and coliform bacteria were completely eliminated by heating at all temp. After 3 days storage at 4 ± 2°C, raw milk and milk heated to 65, 69 and 72°C for 15 s showed mean total bacterial increases of 523.6, 58.8, 53.9 and 67.8%, respectively. and mean increases in psychrotrophic bacteria of 2837.8, 172.6, 151.9 and 105.0%, respectively. Acid forming bacteria multiplied relatively slowly and coliform bacteria were still absent in heat-treated samples after 3 days. It is concluded that thermization of milk at 65°C for 15 s greatly reduces contamination of the milk and extends its keeping quality to at least 3 days at 4 ± 2°C. MacDonald and Sutherland (1993) examined the possibility of the survival of Listeria spp. (L. monocytogenes strains 12/1A, SITC 404/2 and NCTC 7973 and L. innocua SITC 236/2/8) and Gram-negative psychrotrophic spoilage bacteria (which included Pseudomonas fluorescens NCDO 2085, Klebsiella ozoaenae GTE O19, Citrobacter freundii GTE O22 and Acinetobacter lwoffii GTE 024) in heat treated milk with varying fat levels. Ewes' milk, compared with cows' and goats' milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test tube. This effect was not due solely to fat content as cows' milk artificially reconstituted to 10% homologous fat was not as protective as ewes' milk. L. monocytogenes in ewes', cows' and goats' whole milk at an inoculum level of 1 x 106 cfu ml-1 was heated at 68°C for 15 s in a plate pasteurizer; survival was detected in ewes' whole milk only. Even high levels of L. monocytogenes (1 x 106 cfu ml-1) could

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not survive the current HTST plate pasteurization protocol (71.7°C/15 s) in ewes', cows' and goats' whole milk. Kwon and Choi (1998) studied effects of heat treatment (low temp.-long time (LTLT) or HTST pasteurization, UHT treatment) and storage temp. (7, 10, 13, 15°C) on bacterial counts of milk during storage. Soluble whey proteins and patterns of beta-lactoglobulin and alpha-lactalbumin were compared to assess heat treatment.

Whey proteins contents and electrophoretic patterns of beta-

lactoglobulin and alpha-lactalbumin were similar in HTST pasteurized and UHT treated milk. Standard plate count (SPC) did not increase significantly during storage at 7°C for 10 days, except in some samples of LTLT milk. LTLT milk had the highest SPC at the beginning of storage (29 000 cfu/ml in 1 sample) and reached 4.7 x 106 cfu/ml after 10 days of storage in another sample. SPC of both LTST and HTST pasteurized milk increased to >106 cfu/ml after storage for 10 days at 10, 13 and 15°C. In UHT milk, SPC increased to >106 cfu/ml during storage at 13 and 15°C, but remained at