\‘Jawrmw. 25:; T ‘I‘WE MICHIGAN STATE | llllllllflljfllllfllllllll 31 93 lll This is to certify that the dissertation entitled Rennet Coagulation of Salted Milk and Rheology of Soft White Cheese Prepared from It. presented by Hoda Mahmoud Elzeny has been accepted towards fulfillment of the requirements for Ph.D. (figmem Food Science & Human Nutrition / @4125“ mm Major professor o Dme February 15, 19,1 MSU is an Affirmative Anion/Equal Opportuniry Institution 0-12771 i, filling? ' “Rhine State ‘ University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity Institution c:\clrc\dateduepm3-n.[ RENNET COAGULATION OF SALTED MILK AND RHEOLOGY OF SOFI‘ WHITE CHEESE PREPARED FROM IT BY HODA MAHIVIOUD ELZENY A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1991 £552 'M/é ABSTRACT RENNET COAGULATION OF SALTED lVIILK AND THE RHEOLOGY OF SOFT WHITE CHEESE PREPARED FROM IT By Hoda Mahmoud Elzeny The texture properties and microscopic structure of white soft cheese were studied, while the following parameters of milk coagulation were varied: milk pH, renneting temperature, rennet concentration, CaCl2 concentration, and combinations of the above in the presence of NaCl. The textural measurements made on the cheese were: modulus of elasticity, hardness, elasticity, brittleness, cohesiveness, chewiness, and gumminess. The combined effect of decreasing the pH and adding NaCl to milk decreased all texture parameters (p < 0.01 and p < 0.001), and increased water retention of cheese. These parameters increased significantly (p < 0.01) with increasing rennet concentration, renneting temperature, and CaC12 concentration in milk. Scanning electron micrographs results supported the texture data. The coagulation conditions of milk which produced the maximum texture parameters were: pH 6.6, 39°C clotting temperature, 0.09% (15,000 SU) rennet concentration, and 0.02% (w/v) CaCl2 concentration. At these conditions the following texture parameters were obtained with cheese made from milk containing 10% NaCl: elasticity modulus 1.1x10 N/mz, hardness 0.9 Kg, brittleness 0.6 Kg, elasticity 30.9%, cohesiveness 59.5%, chewiness 0.4 (unitless), and gumminess 1.2 (unitless). Texture parameters of cheese made from milk containing 5% NaCl were: modulus of elasticity 1.4x10“, hardness 1.4 Kg, brittleness 0.9 Kg, elasticity 48.7%, cohesiveness 97.2%, chewiness 1.4 (unitless), and gumminess 2.9 (unitless). Addition of NaCl (0-14%) to milk significantly decreased (p < 0.001) pH from 6.64 to 6.13. Up to 6% NaCl addition induced a significant increase in soluble Ca (p < 0.001) in the milk serum. The amount of water (g) loss/g protein as a result of adding 14% NaCl was highly significant (p < 0.001). Casein micelles of salted milk had an irregular shape rather than a Spherical shape characteristic of unsalted milk. Aggregation was enhanced at high NaCl concentrations, and was dependent upon the size of the micelles and the time of exposure to salt. The degree of x-casein hydrolysis by rennet action was decreased by 43% and 61% by the addition of 5 and 10% NaCl to milk, respectively. The coagulation of salted milk increased as milk pH decreased, and rennet concentration, clotting temperature, and CaCl2 concentration in milk increased. An instrumental method was not as sensitive for detecting the clotting point of milk when compared to a visual method. DEDICATION This thesis is dedicated to very important people in my life. My husband, Dr. Abdelnabi A. Ali, his support throughout my career has enabled me to accomplished personal goals I once only dreamed of acquiring. Through his continued encouragement, nurturing, and confidence in my ability, the completion of this research has become a professional reality. For all of his love and faith in me _I will be forever grateful. My children, Ahmed, Rania, and Shereen, who have been the light of my eyes and the joy of spirit. My mother, Zeinab, who taught me the value of hard work to cope with life. For whetting my appetite for learning far and beyond non-formal education by providing me, at times, what she did not have, words are totally inadequate. Trl'i ACKNOWLEDGlVfliNTS This study was made possible through the assistance and cooperation of many people who directly and indirectly contributed to its successful completion. For this reason, I wish to express my profound gratitude to them all. First, I must express my profound gratitude to my major advisor and dissertation director, Dr. Pericles Markakis, whose resourcefulness, encouragement, and support will allows be remembered. Second, I am also indebted to Dr. Robert M. Cook, Denise Smith, and Stanly Flegler, my doctoral committee members, for their unqualified support, critical comments, and encouragement. Without their sobering and incisive criticisms, invaluable suggestions, and patient editing, it would have been difficult to complete this work. A special thanks to Dr. J. Gill for his aid in the development of the experimental design and statistical analysis for this project. I would also like to thank the following people for helping in reading and printing this manuscript; J. Harte, T. Oleas, J. Carpenter, and M. Weaver. My appreciation is extended to the people in the Cultural Bureau and Government of Egypt who provided the financial support throughout the study. Above all, my unqualified gratitude goes to the Almighty God, who alone can take the credit for facilitating the way to obtain this advanced formal education to be used in ministering to Him among my own people in Egypt. To Him be the glory, the power, and the honor forever. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES INTRODUCTION REVIEW OF LITERATURE Milk Proteins Caseins Casein micelles Physical Properties of Casein Micelles Forces Affecting the Stability of Casein Micelles Colloidal Calcium Phosphate (CCP) Casein Micelle Structure Mechanism of Milk Coagulation by Rennet Proteolysis of K-Casein (Enzymatic Phase) Aggregation (Non-Enzymatic Phase) Effect of Sodium Chloride on the Physico-chemical Properties of milk Effect of NaCl on pH and Acidity of Milk Effect of NaCl on Hydration (Solvation) of Casein Micelles Effect of NaCl on Soluble and Colloidal Calcium Effect of NaCl on Coagulation Process Effect of NaCl on Clotting Time (CT) of Milk Effect of NaCl on Casein Structure vi xi xiii xvii HOOOQQQONLIIM F—‘H A 14 Ia Texture Profile Analysis (TPA) 21 Definition of Texture Profile 21 Classification of Texture Profile 21 Texture Profile Instruments 22 Instrumental TPA Techniques 23 Texture Profile Analysis of Cheese 24 Effect of the Variables on TPA of Cheese 27 Effect of Water Content (Moisture) 28 Effect of pH 29 Effect of Sodium Chloride (NaCl) 31 Effect of Calcium Chloride (CaClz) 33 Effect of Renneting Temperature 35 Effect of Rennet Concentration 36 Effect of Protein Degradation 37 Effect of Aging, Storage Time, and Temperature of Storage 38 Effect of Mechanical Force Conditions 39 Texture of White Soft Domiati Cheese 40 Microstructure of White Soft Domiati Cheese 42 MATERIALS AND METHODS 45 Physico—chemical Properties of High Salted Milk 45 pH of Milk 45 Sodium Calcium Replacement 45 Calcium Determination 46 Stock Solution 46 Milk Samples 46 Whey Samples 46 Atomic Absorption Conditions 47 vii Casein Hydration 47 Microstructure of Casein Micelles 48 Rennet Clotting Time of Milk (RCT) 49 Apparent Viscosity Method 49 Visual Slide Testing Method 50 Measuring the Degree of K-Casein Hydrolysis by Rennet 51 Action Milk Sample Preparation 51 Glycomacropeptide (GMP) Standard Preparation 51 High Performance Liquid Chromatography (I-IPLC) 52 Analysis Texture Profile (TPA) and Physical Structure of White Soft Domiati 52 Cheese as Affected by Coagulation Process Factors Preparation of Cheese Samples 53 Effect of pH 53 Renneting Temperature 53 Rennet Concentration 55 Calcium Chloride (CaClz) Addition 55 Interaction of Milk pH, Renneting Temperature, Rennet 55 Concentration, and Added Calcium Chloride Texture Profile Evaluation 57 Cheese Microstructure 57 Chemical Analysis 58 Statistical Analysis 59 RESULTS AND DISCUSSION 61 A) Effect of NaCl on Milk pH 61 B) Replacement Ca2+ by Na+ 63 C) Effect of NaCl on Casein Hydration 65 viii D) Effect of NaCl on Microstructure of Casein Micelles E) Effect of NaCl on the Enzymatic Phase of the Rennet Coagulation Process F) Effect of Independent Variables on Rennet Coagulation Time (RCT) of Salted Milk 1) pH of Milk 2) Temperature 3) Rennet Concentration 4) CaCl2 Concentration 5) The Combined Effect of the Independent Variables G) Texture Profile of White Soft Domiati Cheese 1) Effect of Milk pH a) Cheese Composition b) Texture Profile Analysis (T PA) 2) Effect of Renneting Temperature a) Cheese Composition b) Texture Profile Analysis (TPA) 3) Effect of Rennet Concentration a) Cheese Composition b) Texture Profile Analysis (TPA) 4) Effect of Calcium Chloride a) Cheese Composition b) Texture Profile Analysis (TPA) 5) Effect of NaCl 6) Effect of Altering Coagulation Conditions on Textural Properties of Domiati Cheese H) Effect of the Independent Variables on Microstructure of White Soft Domiati Cheese 1) Effect of pH 2) Effect of Renneting Temperature 68 76 81 81 83 84 85 86 87 87 87 89 101 101 102 114 114 115 123 123 124 132 133 139 139 142 T i-tfi 3) Effect of Rennet Concentration 4) Effect of CaClz Concentration SUMMARY AND CONCLUSION APPENDICES BIBLIOGRAPHY 144 146 149 153 211 THifi LIST OF TABLES Table Page 1. Experimental design of the independent variables of coagulation 54 process of salted milk for cheese texture profile analysis 2. Experimental design of interaction among the independent variables 56 of coagulation process of salted milk for cheese texture profile analysis 3. Analysis of variance for pH of milk as affected by adding NaCl to 61 milk 4. Analysis of variance for replacement the calcium of milk by sodium 63 after adding NaCl to milk 5. Multiple regression analysis of calcium replacement by sodium 65 after adding NaCl to milk 6. Multiple regression analysis of casein micelles hydration as affected 67 by adding NaCl to milk 7. Analysis of variance for hydration of casein micelles as affected by 67 adding NaCl to milk 8. Multiple regression analysis of released glycomacropeptide after 80 action of rennet on casein of raw cows’ milk 9. Regression analysis of released glycomacropeptide after action of 80 rennet on casein during coagulation process of milk containing 5% NaCl 10. Regression analysis of released glycomacropeptide after action of 80 rennet on casein during coagulation process of milk containing 10% NaCl 11. Analysis of variance for released glycomacropeptide after action of 81 rennet on casein of raw cows’ milk 12. Analysis of variance for released glycomacropeptide after action of 81 rennet on casein during coagulation process of milk containing 5% NaCl 13. Analysis of variance for released glycomacropeptide after action of 81 rennet on casein during coagulation process of milk containing 10% NaCl 14. Effect of pH of milk on rennet coagulation time (RCT min) 82 xi 20. 21. 22. Effect of renneting temperature on rennet coagulation time (RCT min) of milk Effect of rennet concentration on the rennet clotting time (RCT min) of milk Effect of addition of CaC12 on the rennet clotting time (RCT min) of milk The combined effect of the independent variables on the rennet coagulation time (RCT min) of salted milk Effect of milk pH on the yield and composition of White Soft Domiati cheese made from milk containing 5 and 10% NaCl Effect of renneting temperature on the yield and composition of White Soft Domiati cheese made from milk containing 5 and 10% NaCl Effect of rennet concentration on the yield and composition of White Soft Domiati cheese made from milk containing 5 and 10% NaCl Effect of added CaClz to milk on the yield and composition of White Soft Domiati cheese made from milk containing 5 and 10% NaCl 84 85 86 87 88 102 114 124 LIST OF FIGURES Figure Page 1. Casein micelle model proposed by Schmdit (1980) 10 2. Schematic representation of rennet coagulation of milk 13 3. Effect of NaCl on pH of milk 62 4. Effect of NaCl on soluble and colloidal calcium of milk 64 5. Effect of NaCl on casein hydration 66 6. SEM micrographs show the effect of NaCl concentration on the 69 casein micelles of skim milk 7. SEM micrographs show the effect of time on casein micelles in 71 skim milk containing 5% NaCl 8. SEM micrographs of the casein micelles in skim milk showing the 73 effect of adding 10% NaCl to milk 9. SEM micrographs of the casein micelles in skim milk showing the 75 effect of adding 15% NaCl to milk 10. HPLC determination of glycomacropeptide (GMP) after renneting 78 times of 0, 1, 3, 10, 40, 70, and 120 min at 35°C and 0.03% calf rennet for raw unsalted milk 11. Released glycomacropeptide (GMP) after rennet action on casein 79 during coagulation process of milk 12. Texture profile curves obtained with the Instron Universal Testing 90 Machine for Domiati cheese made from milk containing 5 % NaCl at pH 6.6, 6.2, and 5.8 13. Texture profile curves obtained with the Instron Universal Testing 91 Machine for Domiati cheese made from milk containing 10% NaCl at pH 6.6, 6.2, and 5.8 14. Modulus of elasticity of Domiati cheese made from milk 93 containing 5 and 10% NaCl at pH 6.6, 6.2, and 5.8 15. Hardness of Domiati cheese made from milk containing 5 and 95 10% NaCl at pH 6.6, 6.2, and 5.8 16. Brittleness of Domiati cheese made from milk containing 5 and 95 10% NaCl at pH 6.6, 6.2, and 5.8 17. Elasticity of Domiati cheese made from milk containing 5 and 97 10% NaCl at pH 6.6, 6.2, and 5.8 xiii 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. Cohesiveness of Domiati cheese made from milk containing 5 and 10% NaCl at pH 6.6, 6.2, and 5.8 Chewiness of Domiati cheese made from milk containing 5 and 10% NaCl at pH 6.6, 6.2, and 5.8 Gumminess of Domiati cheese made from milk containing 5 and 10% NaCl at pH 6.6, 6.2, and 5.8 Texture profile curves obtained with the Instron Universal Testing Machine for Domiati cheese made from milk containing 5% NaCl at 35, 39, and 43°C renneting temperature Texture profile curves obtained with the Instron Universal Testing Machine for Domiati cheese made from milk containing 10% NaCl at 35, 39, and 43°C renneting temperature Effect of renneting temperature on modulus of elasticity of Domiati cheese made from milk containing 5 and 10% NaCl Hardness of Domiati cheese made from milk containing 5 and 10% NaCl at 35, 39, and 43°C renneting temperature Brittleness of Domiati cheese made from milk containing 5 and 10% NaCl at 35, 39, and 43°C renneting temperature Elasticity of Domiati cheese made from milk containing 5 and 10% NaCl at 35, 39, and 43°C renneting temperature Cohesiveness of Domiati cheese made from milk containing 5 and 10% NaCl at 35, 39, and 43°C renneting temperature Chewiness of Domiati cheese made from milk containing 5 and 10% NaCl at 35 , 39, and 43°C renneting temperature Gumminess of Domiati cheese made from milk containing 5 and 10% NaCl at 35, 39, and 43°C renneting temperature Effect of rennet concentration on modulus of elasticity of Domiati cheese made from milk containing 5 and 10% NaCl Hardness of Domiati cheese made from milk containing 5 and 10% NaCl with 0.03, 0.06, and 0.09% rennet concentration Brittleness of Domiati cheese made from milk containing 5 and 10% NaCl with 0.03, 0.06, and 0.09% rennet concentration Elasticity of Domiati cheese made from milk containing 5 and 10% NaCl with 0.03, 0.06, and 0.09% rennet concentration xiv 97 99 99 110 112 112 116 118 118 120 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. Cohesiveness of Domiati cheese made from milk containing 5 and 10% NaCl with 0.03, 0.06, and 0.09% rennet concentration Chewiness of Domiati cheese made from milk containing 5 and 10% NaCl with 0.03, 0.06, and 0.09% rennet concentration Gumminess of Domiati cheese made from milk containing 5 and 10% NaCl with 0.03, 0.06, and 0.09% rennet concentration Modulus of elasticity of Domiati cheese made from milk containing 5 and 10% NaCl with 0.02, 0.05, and 0.1% CaCl2 Hardness of Domiati cheese made from milk containing 5 and 10% NaCl with 0.02, 0.05, and 0.1% CaCl2 Brittleness of Domiati cheese made from milk containing 5 and 10% NaCl with 0.02, 0.05, and 0.1% CaCl2 Elasticity of Domiati cheese made from milk containing 5 and 10% NaCl with 0.02, 0.05, and 0.1% CaC12 Cohesiveness of Domiati cheese made from milk containing 5 and 10% NaCl with 0.02, 0.05, and 0.1% CaCl2 Chewiness of Domiati cheese made from milk containing 5 and 10% NaCl with 0.02, 0.05, and 0.1% CaCl2 Gumminess of Domiati cheese made from milk containing 5 and 10% NaCl with 0.02, 0.05, and 0.1% CaCl2 Combined variable effect on modulus of elasticity of Domiati cheese made from milk containing 5 and 10% NaCl. Combined variable effect on hardness and brittleness of Domiati cheese made from milk containing 5 and 10% NaCl Combined variable effect on elasticity and cohesiveness of Domiati cheese made from milk containing 5 and 10% NaCl Combined variable effect on chewiness and gumminess of Domiati cheese made from milk containing 5 and 10% NaCl Electron micrographs of fresh White Soft Domiati cheese made from milk containing 5 and 10% NaCl at pH 6.6, 6.2, and 5.8 Electron micrographs of fresh White Soft Domiati cheese made from milk containing 5 and 10% NaCl at renneting temperature of 35, 39, and 43°C XV 120 122 122 125 127 141 143 50. 51. Electron micrographs of fresh White Soft Domiati cheese made 145 from milk containing 5 and 10% NaCl with 0.03, 0.06, and 0.09% rennet concentration Electron micrographs of fresh White Soft Domiati cheese made 147 from milk containing 5 and 10% NaCl with 0.02, 0.05, and 0.1% CaCl2 LIST OF APPENDICES APPENDICES Page Appendix A. Significant mean interactions for cheese modulus of 154 elasticity Fig. A.1. Significant mean interactions among CaClz, 155 temperature, and rennet concentration (BCD) for cheese modulus of elasticity Fig. A.2. Significant mean interactions between NaCl and 156 CaCl2 (AB), NaCl and temperature (AC), and CaCl2 and temperature (BC) for cheese modulus of elasticity Fig. A.3. Significant mean interactions between CaCl2 and 157 rennet concentration (BD), and temperature and rennet concentration (CD) for cheese modulus of elasticity Appendix B. Significant mean interactions for cheese hardness 158 Fig. B.1. Significant mean interactions among NaCl, CaC12 159 and temperature (ABC) for cheese hardness Fig. B.2.~ Significant mean interactions among CaCl2 160 temperature and rennet concentration (BCD) for cheese hardness Fig. B.3. Significant mean interactions among NaCl, 161 temperature and rennet concentration (ACD) for cheese hardness Fig. B.4. Significant mean interactions among NaCl, CaCl2 162 and rennet concentration (ABD) for cheese hardness Fig. B.5. Significant mean interactions between NaCl, and 163 CaC12 (AB), NaCl and temperature (AC), and CaCl2 and temperature (BC) for cheese hardness Fig. B.6. Significant mean interactions between NaCl and 164 rennet concentration (AD), CaCl2 and rennet concentration (BD), and temperature and rennet concentration (CD) for cheese hardness Appendix C. Significant mean interactions for cheese brittleness Fig. 0.1. Significant mean interactions among NaCl, CaClz, 166 and rennet concentration (ABD) for cheese brittleness Fig. C.2. Significant mean interactions among NaCl, 167 temperature, and rennet concentration (ACD) for cheese brittleness Fig. C.3. Significant mean interactions among CaClZ, 168 temperature, and rennet concentration (BCD) for cheese brittleness Fig. C.4. Significant mean interactions between NaCl and 169 rennet concentration (AD), and CaCl, and rennet concentration (ED) for cheese brittleness Appendix D. Significant mean interactions for cheese elasticity 170 Fig. D.1. Significant mean interactions among NaCl, CaClz, 171 and temperature (ABC) for cheese elasticity Fig. D.2. Significant mean interactions among NaCl, CaClz, 172 and rennet concentration (ABD) for cheese elasticity Fig. D.3. Significant mean interactions among NaCl, 173 temperature, and rennet concentration (ACD) for cheese elasticity Fig. D.4. Significant mean interactions among CaClz, 174 temperature, and rennet concentration (BCD) for cheese elasticity Fig. D.5. Significant mean interactions between NaCl and 175 CaClz (AB), NaCl and temperature (AC) and NaCl and rennet concentration (AD) for cheese elasticity Fig. D.6. Significant mean interactions between CaCl2 and 176 temperature (BC), CaCl2 and rennet concentration (BD), and temperature and rennet concentration (CD) for cheese elasticity Appendix E. Significant mean interactions for cheese cohesiveness 177 Fig. B.1. Significant mean interactions among NaCl, 178 temperature, and rennet concentration (ACD) for cheese cohesiveness xviii Fig. G.6. Significant mean interactions between CaC12 and 193 temperature (BC), CaC12 and rennet concentration (BD), and temperature and rennet concentration (CD) for cheese gumminess Appendix H. Analysis of variance for chemical composition and 194 texture profile of cheese as affected by pH, renneting temperature, rennet concentration, and CaClZ concentration Table H.1. Analysis of variance for chemical composition of 195 White Soft Domiati cheese as affected by pH of milk Table H.2. Analysis of variance for texture parameters of 196 White Soft Domiati cheese as affected by pH of milk Table H.3. Analysis of variance for chemical composition of 197 White Soft Domiati cheese as affected by renneting temperature Table H.4. Analysis of variance for texture parameters of 198 White Soft Domiati cheese as affected by renneting temperature Table H.5. Analysis of variance for chemical composition of 199 White Soft Domiati cheese as affected by rennet concentration Table H.6. Analysis of variance for texture parameters of 200 White Soft Domiati cheese as affected by rennet concentration Table H.7. Analysis of variance for chemical composition of 201 R White Soft Domiati cheese as affected by adding CaCl2 to milk Table H.8. Analysis of variance for texture parameters of 202 White Soft Domiati cheese as affected by adding CaC12 to milk Appendix 1. Analysis of variance for texture parameters of cheese 203 as affected by the interaction of the variables of milk coagulation factors Table 1.1 Analysis of variance for cheese modulus of 204 elasticity as affected by the interaction of four variables of milk coagulation factors XX Table 1.2. Table 1.3. Table 1.4. Table 1.5. Table 1.6. Table 1.7. Analysis of variance for hardness of Domiati cheese as affected by the interaction of four variables of milk coagulation factors Analysis of variance for brittleness of Domiati cheese as affected by the interaction of four variables of milk coagulation factors Analysis of variance for elasticity of Domiati cheese as affected by the interaction of four variables of milk coagulation factors Analysis of variance for cohesiveness of Domiati cheese as affected by the interaction of four variables of milk coagulation factors Analysis of variance for chewiness of Domiati cheese as affected by the interaction of four variables of milk coagulation factors Analysis of variance for gumminess of Domiati cheese as affected by the interaction of four variables of milk coagulation factors 205 206 207 208 209 210 INTRODUCTION The addition of sodium chloride to milk is traditionally practiced in many Eastern Mediterranean countries possessing subtropical climates as a simple method to prevent microbial and enzymatic spoilage. Milk can be processed into cheeses which are characteristically ripened in salt solutions (Abd El-Salam et al., 1981, Ramet, 1985). Sodium chloride has been permitted as antimicrobial preservative for dairy products but not fluid milk. Salt is legally added to cheese milk as a means of: a) preserving milk during transportation when cooling facilities are unavailable or inadequate, and b) prevent high microbiological numbers in milk used for Domiati cheese manufacturing (Abd El-Salam et al., 1981). Therefore, Egyptian Soft White Domiati cheese is of great importance in warm climates due to the addition of NaCl to milk prior to renneting which prevents cheese deterioration prior to ripening. The effects of salting the milk includes reducing the water activity of the medium (milk or cheese) and limiting or inhibiting microbial growth, subsequent food hygienic hazards and enzymic changes (Duckworth, 1974). Many physical and chemical properties change with incorporation of NaCl into milk, resulting in poor processing characteristics. Sodium chloride has been shown to alter the following: 1) pH of milk, 2) soluble calcium 3) voluminosity and hydration of casein, 4) shape and structure of 2 casein micelles which leads to instability of the micelles and 5) May cause salting-out of milk protein. An increase in rennet activity was observed with small amounts of salt, and then, a gradual detrimental effect occurred due to increasing salt concentration (Ramet and El—Mayda, 1984; El—Mayda et al., 1985). The clotting time may be increased, the firmness of the curd decreased, its water binding capacity increased, and the rate of syneresis of the curd decreased. The effects of NaCl on the coagulation process of milk may also be attributed to several factors. The degree of K-casein hydrolysis by rennet may be inhibited. Ca—ions may be replaced by Na-ions with formation of Na—paracaseinate when rennet is added. This paracaseinate has a high water absorbing capacity. Some alteration of the milk protein including folding or aggregation may occur. Modification of the enzyme molecule due to the salting-out effect of high ionic strength has also been shown. In Egypt, a very large amount of white soft Domiati cheese is produced by developed industries (government) and small dairy factories (private) and presents a wide range of textures. One of the most important effects of NaCl on Egyptian Domiati cheese is altering the texture and body of the cheese. Texture is one of the most important factors that determines the quality of cheese. Cheese has a complicated structure due to the interaction of the milk components (water, protein, fat, salt). The cheese structure may resemble that of a three dimensional network (Masi and Addeo, 1987), it is nonhomogeneous and may vary tremendously in the same plant. Even so, each kind of cheese introduces completely different structure properties. Complete quality characterization could be achieved by increasing knowledge of both texture and 3 flavor properties that define consumer acceptability. Because of the inconsistencies of organoleptic estimation of food attributes, food scientists are continuously looking for practical and sensitive objective measurements. The textural evaluation of milk and dairy products was reported extensively in Switzerland and England during the nineteen forties (Baron and Scott Blair, 1953). Since then, automated testing systems, texture profile analysis, and a variety of instruments and methods for determining textural parameters have been developed (Szczesniak, 1966; Breane, 1975). Objective methods can quantitate physical characteristics of the food in an unbiased manner. If textural attributes can be related to one or more physical properties then these can be used for quality control through manufacturing and ripening. The demand for large amount of high quality cheese in Egypt is steadily increasing. Domiati cheese presents wide ranges of physical structure and texture properties due to its manufacture from variant sources such as bovine milk, or various mixtures of bovine and buffalo milk. Although Domiati cheese is the most popular variety of Egyptian white soft cheese, its manufacturing is not standardized. The milk may optionally be pasteurized, homogenized, hydrogen peroxide-catalase treated, phosphated, citrated, ripened with culture, partially skimmed or fortified with skim milk powder. While most of the knowledge of the physical and mechanical properties of hard cheeses and some soft cheeses have been reported (e.g. Cooper 1987; Lucisano et al., 1987), there have been no similar reports on the Egyptian Domiati cheese. A quantitative description of its physical and mechanical qualities has not been established. For this reason, cheese quality varies from one manufacturer to another, and 4 governmental agencies have not established physical standards for such cheese. This fact has delayed the improvement in Domiati cheese quality and has provided no protection for the consumer. Accordingly, the objectives of the present study were: (1) To determine the effect of NaCl concentration on the physico-chemical properties of milk such as: pH, casein hydration, soluble calcium and microstructure of casein micelles. (2) To study the effect of NaCl on the rennet coagulation process of milk by: . (a) measuring the clotting time of milk. (b) measuring the degree of x-casein hydrolysis by rennet action. (3) To evaluate the rheological properties and microstructure of Domiati cheese as affected by several independent variables of the coagulation process such as milk pH, renneting temperature, rennet concentration, CaCl2 concentration, and combinations of them. REVIEW OF LITERATURE Milk Proteins The major milk proteins can be classified into two broad categories: casein and serum (whey) proteins. Approximately 80% of the protein in bovine milk is casein. Only caseins will be discussed here. There are many publications reviewing the composition and properties of caseins (e. g. Walstra and Jenness, 1984; McMahon and Brown, 1984; Eigel et al., 1984; Ruettimann and Ladisch, 1987). Caseins The casein in cow’s milk occurs in the form of a stable colloidal dispersion which is responsible for the high turbidity of skim milk. The particles of this dispersion are spherical aggregates of casein proteins and colloidal calcium phosphate. These particles have a broad size range from 20-600 nm (Schmidt, 1982). Casein has been defined as a heterogeneous phosphoprotein that can be precipitated from raw skim milk by acidification to pH 4.6 at 20°C (Brunner, 1976). Recently, the American Dairy Science Association Committee for nomenclature of the proteins of cow’s milk (Eigel et al., 1984) recommended that caseins is identified according to their amino acid sequence. Accordingly, caseins been classified into 01,1, 01,2, '6 and x-caseins (Holt, 1986). These classes occur in a ratio of approximately 3:1:3:1 (Schmidt, 1980). Casein Micelles Many reviews have discussed the composition, structure, stability and other properties of casein micelles (Farrell and Thompson, 1974; Schmidt, 1980, 1982; Brunner, 1977; Waugh, 1971; Slattery, 1976; Walstra and Jenness, 1984; McMahon and Brown, 1984). Therefore, only some pertinent aspects of the casein micelles will be presented. Physical Properties of Casein Micelles Most casein exists in colloidal dispersion as micelles in milk. The micelles are roughly spherical with diameters ranging from 0.02 to 0.30 ,um and containing 20-150,000 casein molecules (Walstra and Jenness, 1984). Micelles contain about 93% protein. The remaining 7% comprise inorganic calcium (3%), phosphate (3%) and small amounts of Mg, Na, K, citrate and carbohydrate (Wong, 1989). These micelles range in size from 500 to 3000 A in diameter and in particle weight from 107 to 1010 daltons. The casein micelles are assembled from submicelles that are different in size and composition having diameters of 10—20 nm, and molecular weight of almost 3-6 X 105 daltons. The submicelle may contain 10—100 casein molecules (Walstra and Jenness, 1984). The variation in the size, composition, and particle weight of submicelles is determined by factors such as pH, ionic strength, concentration and temperature (Schmidt, 1982). Casein micelles hold up to 4 g H20/ g casein (McMahon and Brown, 1984; Fox and Mulvihill, 1982; Schmidt, 1982). Only a small amount of water is bound to the protein, almost 0.5 g H20/g protein; the rest is occluded within the micelle (McMahon and Brown, 1984). 7 Forces Affecting The Stability of Casein Micelles Casein micelles are in a dynamic equilibrium in milk. Walstra and Jenness (1984) summarized some of these equilibria as follows: a) equilibrium between casein molecules and submicelles, b) equilibrium between submicelles and micelles, and c) equilibrium between soluble and colloidal calcium and phosphate. There are many forces influencing the stability of the colloidal dispersion of casein micelles. These forces have been described in several reviews (McMahon and Brown, 1984; Schmidt, 1980; Bloomfield and Morr, 1973). McMahon and Brown (1984) and Schmidt (1982) proposed that the interactions between casein molecules and between micelles are electrostatic forces and hydrophobic interactions. Other forces such as hydrogen bonding, Van der Waals attraction, and steric repulsion may contribute to the stability of casein micelles (Schmidt, 1982; McMahon and Brown, 1984; Bloomfield and Morr, 1973). Colloidal Calcium Phosphate (CCP) Generally, each mole of casein may contain about 18.0 moles calcium, 9.1 moles phosphate, 2 moles K, 1.4 mole Mg, 0.9 mole Na and 0.7 mole citrate (Walstra and Jenness, 1984). The calcium and phosphate may be presented in three different forms: colloidal, soluble and ionic. Colloidal calcium may bind to carboxylic amino acids and phosphate esters of casein micelles forming a complex (McMahon and Brown, 1984). Schmidt (1980) reviewed the role of colloidal calcium phosphate (CCP) and its association in the micelles. There is a consensus on the Ca/P ratio in casein micelles (Schmidt, 1982; Walstra and Jenness, 1984). However, there is disagreement on the state of colloidal calcium phosphate (CCP) and how it is bound to casein. Lyster (1979) 8 showed that CCP more closely resembles hydroxyapatite than amorphous Ca phosphate. Many different types of possible linkages in CCP were given by Morr et a1. (1971). Schmidt (1982) suggested that it is similar to amorphous tricalcium phosphate [Cag (PO4)6]. He concluded that CCP is bound .to casein through electrostatic force and that calcium forms a bridge between CCP and the phosphoserine residue of casein. No matter what the composition or forms of CCP are, or how it is linked to casein micelles, it plays an important role in maintaining the integrity of casein micelles, since removal of CCP results in their dissociation (Schmidt, 1982; Farrell and Thompson, 1974). Casein Micelle Structure Although the exact structure of casein submicelles and micelles has not been determined, several models of the casein micelle structure have been proposed (Ruettimann and Ladisch, 1987; Schmidt, 1982; Brunner, 1981; Slattery, 1976; Farrell and Thompson, 1974). Schmidt (1982) divided the models into three categories: coat-core (Waugh, 1971; Payens, 1966), internal structure models (Rose, 1969; Gamier and Ribadeau-Dumas, 1970) and submicelle models (Schmidt, 1982; Walstra and Jenness, 1984). The submicellar structure models (Fig 1) are most widely accepted by the scientific community and to a certain extent have been confirmed through ultracentrifugation experiments, electron microscopy examination and synthetic micelle formation (Schmidt, 1982). This model was first proposed by Morr (1967), then revised by several researchers (Slattery and Evard, 1973; Schmidt, 1980; Schmidt, 1982). Finally, Walstra and Jenness (1984) adopted these models with slight modification into 9 a model which was supposed to account for most of the known physico-chemical properties of casein. According to the submicelle model, the micelle is composed of spherical submicelles which bind via CCP by electrostatic interaction. The binding takes place between the negatively charged phosphoseryl residues of the awed-and B—caseins and positively charged CCP (Ca9(PO4)6) clusters. Submicelles are formed with or without x—casein. The submicelles with x-casein are on the outside of the micelle. Submicelles without x-casein and some submicelles with x-casein are buried in the interior of the micelle. Micellar growth stops when the micelle surface is covered entirely with x-casein which acts as a "protective colloid " (protect the micelle from precipitation by Ca ions). The C-terminal region of x-casein is hydrophilic, negatively charged and contains carbohydrate moieties. Walstra and Jenness (1984) proposed that this region sticks out into the surrounding medium in the form of flexible hairs giving the micelle a hairy surface. The hairy surface is thought to help in steric repulsion and to increase voluminosity of casein micelle. Mechanism of Milk Coagulation by Rennet The coagulation of milk by rennet (chymosin) includes two separate steps: an enzymatic primary (proteolysis) phase and a secondary non-enzymatic (aggregation) phase. Numerous investigators have reviewed this phenomenon (Mackinlay and Wake, 1971; Emstrom and Wong, 1974; Dalgleish, 1982; Walstra and Jenness, 1984; McMahon and Brown, 1984). 10 x-casein meteoric: "Wm" °" 0 139190416 chister Fig. 1. Casein micelles model proposed by Schmdit (1980). Schematic representation of a submicelle (A); a casein micelle composed of submicelles (B); and binding two submicelles via a Ca,,(PO.,).5 cluster (C). 11 Proteolysis of x—Casein (Enzymatic Phase) Rennet (chymosin) attacks x-casein and hydrolyzes the specific peptide bond between phenylalanine (residue 105) and methionine (residue 106), to give a highly acidic macropeptide, and a basic hydrophobic para-x-casein (Fig 2) (Mackinlay and Wake, 1971; Dalgleish, 1982; Walstra and Jenness, 1984). The macr0peptide usually referred to as caseinomacropeptide (CMP) or glycomacmpeptide (GMP) is hydrophilic, negatively charged and soluble in milk serum. Para-x—casein is hydrophobic, positively charged, and insoluble in milk serum as it remains bound to the micelle (Dalgleish, 1982; Walstra and Jenness, 1984). The susceptibility of the Phe-Met bond to hydrolysis by chymosin is attributed to the location, type and sequence of the amino acid residues in x—casein, and not to the nature of the bond itself (Emstrom and Wong, 1974; Dalgleish, 1982). Several methods have been proposed for measuring the enzymatic reaction, by estimation of the production of either GMP or para-x-casein. The liberation of GMP from x—casein may be followed by determining the increase in soluble N (NPN) in the trichloroacetic acid (TCA) filtrate of milk after rennet action. Nitschmann and Bohren (1955) and Foltmann (1959) have shown that the increase in NPN was caused by the formation of GMP. However, the sensitivity of this simple and rapid method is insufficient to determine the initial reaction rate. Determination of N -acetyl neuraminic acid (present in GMP) content of TCA filtrates (Wheelock and Knight, 1969, Marier et al., 1963; de Kong etal., 1963) is considered a specific, sensitive, but time consuming and laborious method. Measuring the turbidity of a GMP suspension formed by adding phosphotungstic acid to TCA filtrates did not give reproducible results when applied to 12 milk (Banejee and Ganguli, 1973). Also, a fluorescence technique was used by Pearce (1979) and Beeby (1980) to determine the released GMP by rennet action. This method involved the reaction of fluorescamine with a-amino groups at pH 6.0 in milk. The increase in fluorescence intensity of the reaction mixture was found to be proportional to the increase in GMP concentration. The fluorescence method was less laborious and could be applied to both milk and casein solutions. Recently, high-performance liquid chromatography (HPLC) has been used to determine GMP in milk and has been described as a very accurate and sensitive technique (Van and Olieman, 1982; Morr and Sec, 1988; Van Hooydonk et al., 1984). 13 PROTEOLYSIS N ' —‘ Para-K—casein ) PM So: Lou His Pro Pro (A) Chymosin —-—| '°° Mot Ala llo Pro Pro no . Macropeptide ) c O HYDROLYSIS O (B) O O 0 fi ’ O O Q O O C) o HYDROPHOBIC AREA O AGGREGATION Q successrm. COLLISION ' (C) 0 : Fig. 2. Schematic representation of rennet coagulation of milk (Carlson, 1985). (A) and (B) proteolysis or hydrolysis (enzymatic) phase, (C) aggregation (non-enzymatic) phase. —_I 14 Aggregation (Non Enzymatic Phase) As x-casein is hydrolyzed by chymosin, the hydrophilicity of the micelle surface is reduced (Green and Crutchfield, 1971; Pearce, 1976). The release of hydrophilic macropeptide reduces the surface charges, and micellar aggregation or coagulation occur in the presence of calcium ions (Walstra and Jenness, 1984). Consequently, there is an increase in hydr0phobic interaction which is a major force in coagulation (Payens, 1977; Dalgleish, 1983). There is a lag phase between the addition of chymosin to the milk and observable coagulation (Darling and Van Hooydonk, 1981). This lag phase represents about 60% of the clotting time (Green et al., 1978). It has been suggested that a critical degree of proteolysis of x-casein (almost 80%) must occur before coagulation takes place (Green et al., 1978; Dalgleish, 1979). Effect of Sodium Chloride on the Physico-chemical Properties of Milk Adding NaCl to milk brings about several changes in physical and chemical properties which result in poor processing characteristics. There are a few publications examining the effect of NaCl on physico-chemical properties of milk (Ramet, et al., 1983; Grufferty and Fox, 1985; Van Hooydonk et a1. , 1986). Only the physico-chemical properties of milk related to the coagulation process as affected by high concentrations of NaCl will be presented here. Effect of NaCl on pH and Acidity of Milk The pH indicates the free hydrogen ion concentration while total acidity includes lactic acid and the "apparent acidity". The apparent acidity is derived from the alkali 15 binding properties of casein, albumin, phosphates, citrates and carbon dioxide (Kosikowiski, 1982). Normal fresh milk has a pH from 6.5 to 6.7 with an average of 6.6. Acidity ranges from 0.13% and 0.22% with an average of 0.15%. Addition of up to 500 mM NaCl to milk caused a decrease in pH from 6.62 to 6.4 (Grufferty and Fox, 1985 ). Ramet et a1. (1983) have also observed that addition of NaCl decreased the pH in milk. On the other hand, the titratable acidity of milk increased on the addition of 2% NaCl. Further addition of NaCl led to a slight increase at the 4% level, beyond which the acidity titer remained constant (Fahmi et al., 1982). On adding NaCl to milk, Na+ appears to exchange with H" attached to negative groups on the casein, with a resultant decrease in pH. At less than 100 mM added NaCl, the Na+ may also neutralize negative charges on the casein, but at greater than 100 mM added NaCl, Na+ appears to exchange with Ca2+ on the casein (Van Hooydonk et al., 1986). Effect of NaCl on Hydration (Salvation) of Casein Micelles The fact that the casein micelles behave like highly hydrated spheres has been demonstrated by electron microscopy studies (Saito, 1973). The casein micelles bind a high proportion of water. The commonly quoted value is 2 g HZOI g protein while other values range from 0.5 to 4.0 g HZO/g protein (Fox and Mulvihill, 1982). An average of 3.7 g HzO/g protein was given by Payens (1979) and McMahon and Brown (1984). Part of this water must be immobilized at the surface, forming an envelope of hydration which protects and stabilizes the micelles (Payens, 1979). Darling (1982) also concluded that the micelles are stabilized by steric forces created by the formation of a hydration 16 layer. He found a positive correlation between the degree of hydration and the stability of the micelles. The hydration of casein increases progressively with the quantity of NaCl, to the point when the protein becomes completely saturated with ion pairs (Gal and Bankey, 1971). Creamer (1985) reported that acidified salted milk had a higher proportion of water absorbed into casein pellets and the quantity of unpelleted dense viscous solution of casein micelles. Grufferty and Fox (1985) found an increase in micellar hydration from 2.2 to 2.4 g HZO/g protein on the addition of 0-400 mM NaCl to milk. Greater hydration values for milk protein in late-lactation (because of increase in Na+ concentration of milk at this period) than mid-lactation were also found by Donnelly et a1. (1980). The increase in casein micelle hydration after adding NaCl to milk possibly was due to interference with the cohesive forces holding the micelle together. Sodium chloride may displace calcium or phosphate from the protein matrix with a concomitant increase in the number of ionic groups in the matrix, and a consequent increase in its volume (Creamer, 1985). Effect of NaCl on Soluble and Colloidal Calcium Calcium may be present in milk in colloidal, soluble and ionic forms (Walstra and Jenness, 1984). The distribution of calcium between the casein micelle and the soluble phase of milk depends on various factors, including temperature, pH and ionic strength (Brule and Fauquant, 1981; Pierre and Brule, 1981). Raising the ionic strength by adding NaCl to milk decreases the amount of Ca in the micelles and consequently micelle 17 size. The calcium is 'shifted from the micellar phase to the aqueous phase of milk and is accompanied by an increase in protein solubility. Van Hooydonk et al. (1986) found that centrifugation of 100 mM (NaCl) salted milk increased the concentration of Ca in the supernatant by about 1 mM. Addition of up to 5% NaCl to milk also increased the quantity of Ca in the supernatant and decreased the Ca in the pellet at pH between 6.6 and 4 (Creamer, 1985). Grufferty and Fox (1985) have also shown that the concentration of non-sedimentable (at 100,000 g x 1 hr) Ca increased from 42 mg/100 ml milk to 50 mg/ 100 ml milk on addition of up to 500 mM NaCl. Parker and Dalgleish (1981) observed that the binding of Ca to casein decreased with the amount of NaCl added. The highest amount of Ca was detected in whey when 7.5 % NaCl was added to milk. When more NaCl was added, a decrease in Ca content in whey was noted (Sharara, 1958). Fox and Morrissey (1977) and Grufferty and Fox (1985) suggested that casein bound calcium, rather than colloidal calcium phosphate, is affected by added NaCl, since centrifugation non-sedimentable Ca increased on addition of up to 500 mM NaCl. However, the same level of NaCl had no effect on the non-sedimentable inorganic phosphorus content of milk. Qvist (1979) attributed the increase in coagulation time of pasteurized milk to the addition of NaCl to disruption of colloidal calcium phosphate (CCP) by NaCl. This was indicated by a large increase in the concentration of Ca2+ in milk and serum. It appears that the increase in soluble Ca concentration following addition of NaCl to milk may be due to an exchange of Na+ for Ca“ which were attached directly to the caseins rather than the solubilization of colloidal calcium 18 phosphate (Grufferty and Fox, 1985). Effect of NaCl on Coagulation Process Addition of NaCl not only increases the ionic strength but also the concentration of the divalent cations by Na+ displacement. This will have a significant influence on the electrostatic repulsion and ionic bond formation due to the screening of charges. Accordingly, both the enzymatic and coagulation reactions may be influenced by these changes (Van Hooydonk et al., 1986). The effect of NaCl on the enzymatic activity of rennet varies: an increase in activity with small amounts of salt, and then, beyond a certain point, a gradual enzyme inhibition was observed (Hamdy and Edelsten, 1970; Ramet and El-Mayda, 1984). The N aCl inhibitory effect is probably due to the induction of a substrate transconformation, together with a modification of the enzyme molecule, owing to the salting-out effect at high ionic strength (Fox and Wailey, 1971; Ramet and El-Mayda, 1984; El-Mayda et al., 1985). The effect of addition of up to 100 mM NaCl to milk on the rate of the enzymatic reaction was studied by Van Hooydonk et a1. (1986) and Van Hooydonk and Walstra (1987). They reported that the hydrolysis of x-casein by rennet decreased approximately linearly with increasing concentrations of NaCl. Visser et al. (1980) observed a decrease in hydrolysis rate of a x-casein model substrate (residues 98—112) with increasing ionic strength (residues 98-112). They concluded that increased screening of both the positive clusters near the Phe—Met bond, and the negative charges near the active site of the enzyme, were responsible for the decreased velocity. 19 Poor curd formation may be attributed to increased content of monovalent cations (e. g. Na“) as is common in late lactation (Davies and Law, 1977). Monovalent cations do not enhance rapid aggregation of casein micelles after chymosin cleavage of the Phe (105)-Met (106) bond (Emstrom and Wong, 1974). In addition, experimental data of Dalgleish (1983) with completely rennet-converted micelles in a buffer containing calcium and various amounts of NaCl, also showed a marked decrease in the rate of aggregation with increasing amount of NaCl. Effect of NaCl on Clotting Time (CT) of Milk Clotting time (CT) of milk is defined as the time which large agglomerates of flocculated calcium caseinate become visible to the human eye (Berridge, 1955). It can also be taken as the time when the relative viscosity of milk increased to 2.6 its initial value (Christen and Virasoro, 1932, 1935) or as the time the milk forms gel (Thomasow, 1968). Adding NaCl to the milk in concentrations from 0.2% to 0.6%, depending on the rennet use (animal or microbial), yielded a minimum CT. In contrast, concentrations of NaCl higher than 0.6% with calf rennet, and 1.5% with microbial rennet, prolonged the CT (Hamdy and Edelsten, 1970). Ramet et al.(1983) recommended that salting of milk in excess of 0.4% should be avoided for cheese—making in order to prevent clotting disturbances with animal rennet. Grufferty and Fox (1985) also reported that addition of 500 mM NaCl to milk at pH 6.6 increased CT from 3 min to 18 min. They confirmed results of Berridge (1952) and O’keeffe (1984) who observed retardation of rennet coagulation of milk in the presence of NaCl. On the other hand, several publications have reported a minimum in the rennet CT of milk (Hamdy and Edelsten, 20 1970; Alais, 1972; Qvist, 1979; Ramet et al., 1983) and of pure x-casein solution (Payens and Both, 1980) with an addition of about 50 mM NaCl. They attributed the minimum CT to the optimal ionic strength conditions for the action of rennet and not to an effect of NaCl on the aggregation process. In contrast to these results, a gradual increase of rennet CT as a function of NaCl addition was observed by Grufferty and Fox (1985) and Van Hooydonk et al. (1986). The longer clotting time and softening of coagula of cheese made from salted milk might be related to significant changes, caused by Na-ions, in the original salt and colloidal balance of milk. Moreover, the enzymatic proteolysis by rennet probably induces additional destruction of proteins. Subsequently, formation of a fibrillar network in the early stages of the secondary phase and, later, after the point of gel formation, might be delayed (Ramet et al., 1983). Van Hooydonk et al. (1986) attributed the increase of clotting time (CT) of milk at higher concentration of NaCl to two reasons: a) the screening influence of the electrolyte on charge groups which impair the ionic bond formation thereby decreasing the number of effective collisions and b) an increase of steric repulsion by protruding chains of casein. Effect of NaCl on Casein Structure The technique of electron microscopy has been used by several investigators to study the shape and size distribution of the casein micelles in milk (Nitschman, 1949; Hostettler and Imhof, 1951; Knoop and Wortmann, 1960; Addachi, 1963; Sood et al., 1979; Vreeman et al., 1989). Visser et al. (1986) reported that addition of sodium citrate to milk caused a sharp increase in the number of small casein particles. Moreover, large particles still present, 21 possessed a more open structure than initially. The effect of NaCl added to milk on the structure and integrity of the casein micelles was examined by various criteria. Tessier and Rose (1964) reported that addition of almost 200 mM NaCl to some milks gave centrifugal sera significantly higher in x—casein content than the control. Grufferty and Fox (1985) found that addition of NaCl up to 200 mM did not affect the level of non-sedimentable (100,000 g x 1 h) nitrogen. N-acetylnuraminic acid was constant over the range 0 to 500 mM added NaCl. In addition, replacing Ca ions by Na ions, as a result of adding NaCl to milk, caused changes in the structure of casein micelles and in some of their functional properties (Burgess, 1982; Snoeren et al., 1984). Texture Profile Analysis (TPA) Definition of Texture Profile Many attempts have been made to define texture as well as to explain how it is actually perceived (Brandt, et al., 1963; Friedman et al., 1963; Sherman, 1969; Breene, 1975, Szczesniak, 1975; Peleg, 1983). Szczesniak (1963) stated that "texture is composed of a number of different sensations/parameters" and that the "sensory evaluation of texture is a dynamic property". Civille and Szczesniak (1973) further described texture as "the sensory manifestation of the structure or inner make-up of foods. It is perceived as "tactile" in the senses of the skin and "kinesthetic" in the muscles". More recently, Peleg (1983) defined texture as a "response of different kinds of physical and physico-chemical stimuli". Classification of Texture Profile Szczesniak (1963) classified texture characteristics into three main classes. (a) 22 mechanical characteristics related to the reaction of the food to stress, (b) geometrical characteristics related to the size, shape, and arrangement of particles within a food, and (c) others, mainly due to moisture and fat. Mechanical characteristics include 5 primary parameters and 3 secondary parameters (Civille and Szczesniak, 1973). Szczesniak (1963) pointed out that popular terms used to describe texture often denote degrees of intensity of these characteristics. The proposed classification lends itself to the use of instrumental methods (Friedman et al., 1963), and has served as a basis for the development of the quantitative rating scale of food (Szczesniak et al., 1963). Texture Profile Instruments Recently, automated texture profile analysis instruments have been developed (Szczesniak, 1966; Boume, 1968; Finny, 1969; Breene, 1975), which further refine the quantitative analysis of food texture. Denturometer (Proctor et al., 1956a, b) was the prototype for construction of a modified texture measuring unit called the Texturometer or General Food (GF) Texturometer (Friedman et al., 1963). This instrument provided several values or parameters which made up the GF Texture Profile (Szczesniak, 1975 ; Szczesniak and Hall, 1975). Drake (1965) devised a modification of the GF Texturometer called a Masticometer. Since 1968 the Instron Universal Testing Machine (IUTM) has been widely used for instrumental texture profile analysis of solid foods (Finney, 1969; Mohsenin, 1970; Bourne et al., 1966) and semi-solid foods, like cheese Breene, 1975; Emmons et al., 1980; Chen et al., 1979). Bourne (1968, 1974) was the first to apply and adapt the IUTM to perform a modified texture profile test. Henry and Katz (1969) and Henry et 23 al. (1971) used the Instron to develop a more detailed analysis of the adhesiveness portion of the texture profile curve for work with semi-solid food. The IUTM and the General Food Texturometer both have been used successfully for TPA. However, Boume (1968) and Brennan (1975) contend that the Instron is a better tool for determining TPA parameters than the Texturometer. The most recent development in TPA instruments has been to utilize computer-assisted read-out of data from the texture profile (Bourne et al., 1977) . Instrumental TPA Techniques Instrumental TPA techniques are very useful in evaluating the textural quality of foods, particularly when parameters can be correlated with sensory assessments (Breene, 1975). Certain refinements in the technique have been made to extract more information from the TPA curve (Peleg, 1976; Boume, 1978; Olkku and Rha, 1975; Law and Wigmire, 1982; Fedrick and Dulley, 1984). By analysis of the force-time curve obtained with IUTM, Bourne (1978) listed and modified the five measured and the two calculated parameters which were originally named by Szczesniak (1975) and Friedman et al., (1963): Fracturability: (once called brittleness) is defined as the force at the first 1. significant break in the first positive bite area. 2. Hardness: is defined as the peak force during the first compression cycle. 3. Cohesiveness: is defined as the ratio of the positive force area during the second compression cycle to the positive force area during the first compression cycle. 4. Adhwiveness: is defined as the negative force area for the first bite representing 24 the work required to pull the plunger away from the food sample. 5. Springiness: (originally called elasticity) is defined as the height to which the food recovers during the time that elapsed between the end of the first bite and the start to the second bite. 6. Gumminess: is defined as the product of hardness and cohesiveness. 7. Chewiness: is defined as the product of gumminess and springiness. 8.Modulus of elasticity (MOE): 5:171. AL/L where, F = Force. A = Original cross-sectional area of sample. AL = Deformation corresponding to force F. L = Initial length of the sample. Texture Profile Analysis of cheese Diverse textural characteristics were found in different cheese types (Cooper and Watts, 1981 ; Fukui et al., 1971, Cooper, 1987; Hardy and Adda, 1984; Creamer and Olson, 1982; Creamer et al., 1988; Green et al., 1981; Masi and Addeo, 1984, 1987; Parnell-Clunies et al., 1985; Chianese et al., 1986; Masi, 1987; Prentice, 1987). Texture characteristics which were evaluated in natural cheeses were: hardness, springiness, smoothness, crumbliness and firmness (Davis, 1937). Davis (1937) Portrayed some typical elastic modulus and viscosity for hard cheese and their interrelationships with some textural characteristics. Thomas and Brown (1970) added 25 stickiness, sliceability and spreadability to Davis’ textural characteristics of processed cheese. Fukui et al. (1971) concluded that the most reliable parameters for processed cheese were: hardness and cohesiveness, however, springiness, and adhesiveness should also be considered. Baron (1952) and Scott Blair (1953) have concentrated on the relationship between manufacturing parameters and the rheological quality (texture) or acceptability of the finished cheese. Firmness (hardness) has been one of the most frequently investigated textural attributes in cheese. Lee et al. (1978) evaluated the texture of 11 different cheese types and concluded that firmness was their most important textural characteristics. A number of researchers have also evaluated the firmness of different cheese types such as: cottage cheese (Emmons and Price, 1959), natural cheeses (Shanta and Sherman, 1973; Fukushima et a1. , 1964), cheddar cheese (Lindsay et al. , 1982; Boyd and Sherman, 1975; Cooper, 1987), cream cheese (Boyd and Sherman, 1975; Imoto et al., 1979), Gouda cheese (Culioli and Sherman, 1976; Vernon Carter and Sherman, 1978), Leicester cheese (Vernon Carter and Sherman, 1978), and Mozzarella cheese (Cervantes, et al., 1983). Brennan et al. (1970) measured the texture of six samples of cheese (five of cheddar of different ages and one of Edam) by three Texturometers (IUTM, GF Texturometer, and Kramer Shear Press) and taste panel’s appraisal. They concluded that instrumental hardness was the most useful measure of the firmness/hardness of cheese as judged subjectively. Brennan et al. (1975) also found a high correlation coefficient between sensory evaluation and Instron for three mechanical properties of five cheeses. Green et al. (1981) analyzed the natural cheddar cheese made from concentrated 26 milk by ultra-filtration (UP) for textural parameters by instrumental and sensory measures. The instrumental firmness, cohesiveness and compression force required to cause fracture, and the sensory firmness, crumbliness, granularity and dryness of the cheese all increased as the concentration factor of the milk increased. The adhesiveness declined and there was little change in elasticity with increase in milk concentration. Homogenization of whole milk produces Cheddar cheese that tends to retain more moisture and is less elastic and firm (Emmons et al., 1980; Peters, 1956). Emmons et al. (1980) studied the interrelationships among composition, texture and microstructure in fullefat and reduced-fat cheeses and in reduced-fat cheese made from milk receiving different degrees of homogenization. They found that reduced-fat cheese (17% fat, 44% moisture) was considerably firmerand more elastic and cohesive than full-fat cheese with the same milk solid non fat (MSNF). Lucisano et al. (1987) evaluated the texture of various Italian cheese by TPA in order to determine the optimum conditions for their characterization. They concluded that a "fingerprint" of any cheese could be achieved by carrying out TPA under at least two different sets of operating conditions (deformation at the same deformation rate) and then processing the data for the various parameters by Quantitative Descriptive Analysis. Yang and Taranto ( 1982) evaluated seven textural parameters of several analog formulations of Mozzarella cheese. Factors such as pH, fat content, salt content, that influence textural characteristics were studied. Textural properties of processed cheese which were evaluated by Park (1979) were considered economically important in retail markets. Attributes of flexibility/brittleness, stickiness, body breakdown, mealiness and 27 overall cheese matrix for processed cheese were measured at a later date. Attributes of wetness, thinness, slipperiness, cohesiveness and mealiness for Cottage cheese were tested by Cooper and Watts (1981). In recent years, cheese textural characteristics evaluation have been the subject of serious research by numerous investigators (Masi, and Addeo, 1987; Lucisano, et al. 1987; Walstra and Van Vliet, 1982; C00per, 1987; Culioli and Sherman, 1976; Weaver and Kroger, 1978; Rosenau et al., 1978; Creamer and Olson, 1982; Imoto et al., 1979; Chen et al., 1979; Sherman, 1976; Bouron, 1978; Yang et al., 1983; Green et al., 1985; Pamell-Clunies et al., 1985; Chianese et al., 1986). Effect of The Variables on TPA of Cheese Differences in cheese texture is related mainly to its structure and arrangement of the protein molecules (Lawrence et al., 1983). The relationship between the texture of cheese and its composition is complex and time dependent (Creamer and Olson, 1982). Cheese texture at any specific stage of ripening is largely dependent on its pH and the ratio of intact casein to moisture (de Jong, 1976). Several variables affect the texture of cheese such as: moisture content, fat, acidity (pH), salt content, protein degradation and temperature (Creamer and Olson, 1982; Lawrence et al. , 1984). Chen et al. (1979) measured the textural parameters of 11 different cheese types which were in close correlation with composition and pH of cheese. While textural measures were the dependent variables, protein, water content, fat, NaCl, and pH were independent variables. The textural contributions of the independent variables followed this sequence: protein > NaCl > water > pH > fat. 28 Green et al.(1981) measured the texture parameters of cheddar cheese made from UF milk as affected by the concentration factor (CF). The texture differences were related to the smaller fat content, lower level of proteolysis, stronger links in the protein matrix and reduced capacity of the fat and protein phases to move relative to each other in cheese from the more concentrated milk. Effect of Water Content (Moisture) It has been known that excessive retention of water in the curd during manufacturing produces cheese of high moisture and less firmness than optimum. Hardness of cheese can be considered a function of moisture content since the more moisture present at any pH, the softer the resultant cheese texture. The intermediate moisture cheese products manufactured at water activity (a) level of 0.81 or 0.86 were found by Kreisman and Labuza (1978) to be tough. However, cheese of higher aW (0.90—0.94) possessed a more acceptable texture. This was thought to be caused by a , corresponding reduction in the proportion of solid matter present (Prentic, 1972, Lawrence et al., 1983). On the basis of moisture content, cheese may be classified as: very hard, hard, semi-hard or semi-soft, and soft (Walstra and Hargrove, 1972; Scott, 1986). A significant negative correlation between hardness of cheese and its moisture content has been also found by Fedriek and Dulley (1984). Weak-bodied cheese is soft and is closely associated with a high moisture content (Amantea,et al., 1986). As the water content of cheese increased, the modulus of elasticity decreased and the cheese became elastic (Fukushima, et al., 1965). A high viscosity but low modulus elasticity was commensurate with a hard, elastic cheese. When both the elastic modulus 29 and viscosity are high, the cheese was hard but not elastic (Davis, 1937). Cohesiveness and springiness (elasticity), but not hardness, of processed cheese were pr0portional to the water content (F ukui et al., 1971). However, Fukushima et al. , (1965) found that elasticity and hardness were increased with low moisture cheese. The rheological properties of cheese with similar pH and sodium ions concentrations, and at a similar degree of Old-casein degradation, are regulated by their moisture contents. The firmness of Gruyere and Emmental (Eberhard, 1985) Gouda (de Jong, 1978), and Cheddar (Creamer and Olson, 1982) was reported to be influenced markedly by relatively small variations in moisture. The role of water in the texture of very high moisture cheese, however, may be more complicated than simply a variation in the proportion of solid matter since X-ray studies (Walstra and Van Vliet, 1982) show that the casein network is indeed modified by excess water. Effect of pH The influence of pH on the coagulation time and the firmness of the gel is very noticeable. The coagulation time is deeply induced and the curd’s hardening’s is strongly influenced by the pH of milk (Ramet and Weber, 1980). It has been observed that the coagulation phase is much more sensitive to a reduction in pH than the enzymatic phase (Cheryan et al., 1975). The increase in the coagulation rate produced by lowering the pH is accompanied by a perceptible increase in the rate of firming of the gel and in the maximum firmness, unless the pH is lower than 6.0. Below this pH the effects of demineralization and desegregation of the micelles are noticeable (Ramet and Weber, 1980). 30 The acidity of thecurd at drainage determines the basic structure of the cheese by controlling the calcium phosphate level in the curd (Lawrence, 1983; Holmes et al., 1977). Marshall et al. (1982) found the maximum curd firming rate is around pH 5 .8. Van Hooydonk and Van Der Berg (1988) stated that "pH 6.3 gave mainly soft cheese and around pH 6.6 gave mainly hard cheese." The texture of a cheese is determined primarily by pH (Lawrence et al., 1987). For instance, a low pH is associated with less elasticity (Eberhard, 1985), firmer but crumbly (Creamer et al., 1988) and more brittle (Creamer and Olson, 1982) cheese body. As the pH of cheese curd approaches that of the isoelectric point of casein, the protein assumes an increasingly more compact conformation and the cheese becomes shorter in texture (Creamer and Olson, 1982). The compression at the yield point, one of the texture characteristics, was found to be in good correlation with the pH of cheese (Creamer et al., 1988). A cheese with a high pH was more pliant and rubbery (as shown by greater compression before fracturing) irrespective of its Ca content. However, a low pH cheese was firmer but crumbled at low deformations. Also, more acidic cheeses were more difficult to deform and fracture at lower compressions and lower applied forces (i.e. more brittle) (Creamer and Olson, 1982). Creamer et al. (1985) noted that texture of Cheddar cheese made from acidified milk was more crumbly and the force required to fracture was less. Yang et al. (1983) and Stainsby (1977) found that the highest cheese hardness was at pH 7.5 and lowering the pH resulted in a softer curd. They attributed that to the interference of the acid or base in the formation of cross-linking or weakening of the 31 cross-linking due to the changes of the protein charges. Likewise, change in pH also weakened the adhesiveness and cohesiveness of Mozzarella cheese analog made from soy protein (Yang and Taranto,1982). As the pH of the casein matrix in 5% NaCl solution decreases, the Ca and phosphate normally complexes to the matrix dissociates and the matrix tends to dissolve (Creamer, 1985). There are two conflicting effects in cheese. Firstly, a higher pH results in the protein having a greater net negative charge, which could require a greater or a lower force to fracture the cheese. Secondly, at the higher pH, a greater quantity of calcium and phosphate is transferred into the casein matrix. This could lead to a greater force being required to fracture the cheese. Walstra and Van Vliet (1982) reported that the cheese is firmer near the isoelectric point if its moisture content is not high. On the other hand, it becomes shorter and needs a smaller deformation to fracture. The effect of pH seems to depend much on the water and salt content and on protein degradation. The hardness of processed cheese reached a minimum at pH 6.2, but adhesiveness, cohesiveness and springiness exhibited a maximum at pH 6.0 (Fukui et al., 1971). Effect of Sodium Chloride (NaCl) The effect of NaCl on cheese rheology is complicated by two conflicting mechanisms: The inhibitory effect on proteolysis (Thomas and Pearce, 1981) suggests a positive correlation between salt level and hardness and a negative effect due to the exchange of Na+ ions for Ca++ ions (Lawrence et al., 1983; Green and Manning 1982) 32 and the subsequent adverse effect on the protein matrix strength. Eck (1986) found that salt affected the binding of water by protein. NaCl at low concentration increased water binding. On the other hand, at the highest concentrations, hydration was thought to be reduced. Salt tends to reduce the force between charges and leads to a fall in the curd rigidity (Cumper and Alexander, 1952). The reduction of charges due to addition of NaCl caused a tenfold reduction in adhesiveness (Yang and Taranto, 1982; Stainsby, 1977; Goodman, 1963; Huang and Rha, 1974). The modulus or the "curd tension" of rennet-induced milk gels is not affected by added NaCl up to 500 mM (Gruffery and Fox, 1985), but decreases at higher concentrations (Jen and Ashworth, 1970) and markedly so in the range from 500 to 1500 mM (Gouda, et al., 1985). Geurts (1972) reported that a higher Ca+*/Na+ratio yields a firmer and shorter cheese (more compact). The tendency of the cheese to swell decreases and its firmness increases with increasing NaCl content of cheese (around 2 % NaCl). Cervants et al. (1983) evaluated the effect of salt concentrations (0.27 to 2.4 %) on texture of Mozzarella cheese at 50 % compression and concluded that increasing salt concentration caused increases in firmness and decreases in cohesiveness. Creamer and Olson (1982) examined the effect of added salt or water in the form of 4 % (w/w) NaCl solution on the properties of cheese. It was found that up to 0.5 % (w/w) added salt did not have any effect on the force-time curves of the cheese. However, added moisture in the form of 4% (w/w) NaCl solution decreased the force at the yield point. Likewise, Fedriek and Dulley (1984) found that cheese hardness was negatively correlated with salt level in 33 cheese. Sodium chloride can affect many constituents of cheese that influence texture. Such factors as structure of casein (Fox and Walley, 1971), moisture content (Nilson, 1968), hydration of the protein network (Geurts et al., 1974), and interaction of Ca-para-caseinate complex in cheese (Geurts et al., 1972) all are influenced by salt concentration which consequently affect texture of cheese. Effect of Calcium Chloride (CaClz) Calcium chloride decreases the pH of milk and helps the second part of coagulation process (Van Hooydonk and Van der Berg, 1988; Ramet et al., 1981; Mahaia and Cheryan, 1983). It also increases the reaction rate of x-casein cleavage (Green and Marshall, 1977) and affects the inter-particle bond strength (Van Hooydonk and Van der Berg, (1988). The amount of soluble phosphate decreases after addition of CaCl2 (Tessier and Rose, 1958; Van Hooydonk et al., 1986). Calcium chloride is commonly added to milk to improve coagulation. Addition of 0.02% (about 1.8 mM) CaC12 to milk increased curd firmness by about 32%. Curd firmness may increase 81% when 10 mM CaClz is added (Jen and Ashworth, 1970). Still higher concentration of added CaClz result in decreased curd firmness (Jen and Ashworth, 1970; McMahon et al., 1984). The Federal definition and Standard of Identity allow for the addition of 0.02% anhydrous CaCl2 or equivalent to milk for cheese making (FDA, 1979). Optimum curd formation can be obtained using less rennet when CaC12 is added to cheese milk (Kelly, 1951; Lindet, 1924), and cheese yield is increased (Lindet, 1924; Ustunol and Hicks, 1990). Green and Marshall (1977) and Van Hooydonk and Van der 34 Berg (1988) reported that most of the calcium binds to the casein either by direct adsorption onto the negatively charged residues of casein or as a micellar calcium phosphate. Ramet and Weber (1980) studied the influence of adding CaCl2 and other factors on the rheological properties of curd. They concluded that adding CaCl2 induced few effects upon the rheological characteristics studied. Yang and Taranto (1982) studied the effect of CaCl2 on textural characteristics of Mozzarella cheese analog manufactured from soy-milk. They found that 2% CaCl2 strengthened the gel network by cross-linking between soy protein molecules. Calcium may contribute to a lower springiness. Satia and Raadsveld (1969) examined the effect of added calcium and calcium phosphate on the quality of Gouda cheese and found that with increased calcium content the cheese was slower to mature and harder, and had a more mealy texture. Creamer et a1. (1988) concluded that the physical properties of Colby-style cheeses were unchanged although the calcium content varied from the normal of 160-170 m mol/kg cheese down to 120 m mol/kg cheese. They also noticed that lowering the calcium content of cheddar cheese below 170 m mol/ kg compared with 190-210 In mol/kg obtained for the control cheeses did not affect the texture of cheese. Calcium could not be assigned a role in determining the texture of the cheese (Creamer et al., 1985), and it was not as important as pH (Creamer et al., 1988). Creamer et al. (1988) found that, in general, normal pH, high calcium cheese was similar to the control, whereas low pH, high calcium cheese was more brittle than the control at 6 weeks and 6 months of age. Conversely, the high pH, low calcium cheese 35 was more elastic than the control. Effect of Renneting Temperature The phenomenon of coagulation is highly temperature dependent. Below 10°C, coagulation of milk does not occur. In the range 10-20°C the coagulation rate is slow. Above 20°C it increases progressively up to 40-42°C, it then diminishes again, and above 65 °C coagulation no longer occurs, the enzyme having been inactivated (Brule and Lenoir, 1986). Coagulation temperature has a much greater influence on the rate of aggregation (secondary phase) than on the rate of the enzymatic reaction (primary phase). The reported values for the temperature coefficient (Q10) (ratio of the rate constants corresponding to a temperature interval of 10°C) for the splitting of K-casein by rennet vary between 1.3 and 2.0 (Van Hooydonk et al., 1984; Nitschman, 1955; Carlson, 1985; Mehaia and Cheryan, 1983; Tuszynski, 1971; Cheryan etal., 1975). It was considerably higher for the coagulation reaction (about 12 ) and decreased with temperature (Dalgleish, 1983; Brinkluis and Payens, 1984; Cheryan et al., 1975). At 30-40°C, it was 5.3, 11.4 and 16.7 for the enzymatic phase alone, the non-enzymatic phase and for both, respectively (Mehaia and Cheryan, 1983). Van Hooydonk and Van den Berg (1988) stated that the activation energy is 170 KJ/mol around 25°C and 70 KJ/mol around 35°C. The optimum rennet activity for clotting of milk is around 40-42°C (Scott, 1986). At higher temperature, rennet coagulation time of milk is shortened, rate of curd firming is faster and coagulum strength is stronger (McMahon et al., 1984; Bohlin et al., 1984; Marshall et al., 1982; Darling and Van-Hooydonk, 1981; Emmons and Lister, 1976). 36 However, Story and Ford (1982) found that coagulum strength decreased with an increase in temperature. Scott (1986) reported that the clotting temperature is important in determining the type of curd to be formed. At 20-27°C, the clot tends to be soft, at 30°C the clot is firmer and does not break into small particles during cutting, while at 33-36°C the clot is tough and rubbery with slow syneresis. The effect of temperature on coagulation may be attributed to an increase in temperature (McMahon et al., 1984 and Dalgleish, 1983). Changes in temperature may also affect the binding of Ca ions to casein as well as changes in calcium phosphate content of the micelles. Effect of Rennet Concentration It is known that the coagulation time (t,) is inversely proportional to the enzyme concentration, which is in accordance with Storch’s rule and expressed by Holter’s equation: t,=t,,+k/C when k = a constant, C = concentration of enzyme, and to: time elapsed between the end of the enzymatic reaction and the moment of coagulation (Collin and Grappin, 1977). When the amount of rennet and Q factor (clotting time + cutting time/clotting time) are small,the curd has little cohesion, i.e. the inter-micellar bonds are weak. On the other hand, if the amount of rennet is high, the bonds are formed very early, which produces a rapid increase in the hardness of the coagulum and renders it progressively more impermeable. Development of curd firmness vary with the amount of rennet up to an Optimal limit of the enzyme/ substrate ratio. The rheological state of the coagulum, is dependent on the amount of milk-clotting enzyme and on the Q factor (Weber, 1986). The rate of the curd firming depends on the rennet concentration (Van Hooydonk . 37 and Van der Berg, 1988; Marshall, 1982; Garnot and Olson, 1981; McMahon and Brown, 1982; Tukita et al., 1982) and a higher elastic modulus was also found with increasing rennet concentration (Van Hooydonk and Van der Berg, 1988). Most of the rennet escaped to the whey at draining, but some retained in the curd, depending upon the pH and the proportion of whey retained in the curd (Holmes et al., 1977 ; Grappin et al., 1985). However, Visser (1977) found that retention of rennet in Gouda cheese was related linearly to the proportion of rennet initially added to the cheese milk. De Jong (1978) noted that the rate of softening of relatively high moisture cheese was influenced by its rennet content. He also found that lower and higher than normal amounts of rennet gave slower and faster rates of softening, respectively and neither urn-casein degradation nor softening occurred when no active rennet was present. Effect of Protein Degradation Cheese hardness and springiness were correlated negatively with cheese proteolysis, indicating softening of the cheese as the protein matrix was broken down (Desmazeaud and Grappin 1977; Adda et al., 1982; de Jong, 1976, 1977, 1978; Fedriek and Dulley, 1984). Similarly, he more proteolysis of rind Emmental cheese, the weaker and shorter the cheese body (Eberhard, 1985). Two distinct phases in texture deve10pment take place during ripening. The first phase occurs within the first 7 to 14 days when the rubbery texture of young cheese curd is rapidly converted into a smoother, more homogeneous product (Creamer and Olson, 1982). The second phase includes a more gradual change in texture, ranging from springy through to a plastic non-cohesive, as the rest of casein is broken down (Creamer 38 and Olson, 1982) depending primarily on the pH of the curd and its calcium content. For instance, Gouda cheese becomes increasingly more cheddar-like in its texture during ripening, presumably because the submicelles are being degraded to give a range of casein aggregates that are normally associated with cheddar. Similarly, as cheddar ripens, proteolysis results in a texture that becomes increasingly less cohesive (Hall and Creamer, 1972). Effect of Aging, Storage Time and Temperature of Storage The temperature and storage time of cheese affect the cheese hardness, elasticity, and brittleness. The hardness of cheddar cheese increased, while the elasticity decreased, with the age of cheese, the greatest changes occurring during the first 30 days. The texture progressively changed by aging (Baron, 1949) due to casein proteolysis (Creamer and Olson, 1982). Fedriek and Dulley (1984) found that higher temperature storage of cheddar cheese accelerated the development of mature cheese texture. The cheese became more fracturable (brittle), and had less springiness with increasing storage time and temperature. Cheese was less firm at higher temperatures. The stress needed for a certain deformation, and that needed for fracture, both decreased at increasing temperature (Culioli and Sherman, 1976; Park, 1979). The elastic moduli of the Gouda and Cheddar cheese sharply decreased above 35°C (Taneya et al., 1979). Creamer et al. (1988) examined the changes in the rheological properties of cheddar cheese made using calf rennet and microbial coagulant. It was verified that, as 39 the cheese aged, firmness increased, the force required to fracture the cheese remained nearly constant, and the compression at fracturing decreased. Rheological characteristics of Manchego cheese, which was made from ewe’s milk, were influenced by elevating the ripening temperature (Gaya et al., 1990). The fracturability (p < 0.05), elasticity (p < 0.01), and hardness (p < 0.01) were significantly influenced by ripening temperature. Lower ripening temperature increased significantly the values of fracturability (p < 0.01), elasticity (p < 0.001) and hardness (p < 0.01). Effect of Mechanical Force Conditions Cross-head speed and compression ratio have a considerable effect on the results of instrumental TPA. Shama and Sherman (1973) mentioned that a cross-head speed greater than or equal to 200 mm per min, and a compression ratio of 38 to 62% were the best correlated with sensory hardness comparison between two cheeses varieties. However, The most commonly used speeds are in the range of 20-50 mm per min (Chen et al., 1979; Taranto et al., 1979; Lee et al., 1978; Imoto et al., 1979; Bourne and Comstock, 1981; Law and Wigmor, 1982 ; Creamer and Olson, 1982). Boyd and Sherman (1975) showed that very high crosshead speeds were impractical, due to the response time of the standard Instron recorder. The response time of the data recording system is important when the force is applied quickly (V oiscy, 1975). The behavior of cheese in compression tests is quite complex. It depends on maturity, sample dimensions, temperature, and the nature of the contact surface between the cheese and the Instron (Culioli and Sherman, 1976; Brinton and Bourne, 1972). 4O Lee et al.(1978) reported that there was a great variation in the responses of different cheese types to the experimental conditions. Cream cheese was less affected by temperature rate of loading and compression ratio than were American and Cheddar cheeses . Shama and Sherman ( 1973) described a procedure for establishing the mechanical force conditions that should be used in Instron tests, based on a correlation of sensory responses with instrumental force-compression-rate of force application data. Texture of White Soft Domiati Cheese At present time no attempt has been made to define and evaluate the Texture Profile (TP) of Domiati cheese instrumentally. Currently, describing the texture of Domiati cheese and determining its quality depend only upon sensory panel tests (Abou-Donia, 1986). Panelists proposed several scoring sheets for evaluating the texture of Domiati cheese using 100 points; giving 40 points for body and texture (El-Neshawy et al., 1982; El-Koussy et al., 1970; El-Zayat and Omer, 1987) or 35 points (El-Koussy et al., 1976; Hamdy, 1972; Abou-Donia, et al., 1985) or 30 points (El-Koussy et al., 1977, 1976; El-Gendy, 1983; El-Safty et al., 1981; Abou-Donia, 1981; Omer and Buchheim, 1983; Omer, 1987) and gave either 60 or 50 points for flavor and the rest of 15 to 20 points were divided among appearance, color, and salt. ' The texture and body of Domiati cheese as judged subjectively were first described by Fahmi and Sharara (1950). They described the cheese as having " soft body and close texture with no holes, for fresh cheese; then as ripening proceeds it 41 becomes slightly flaky and brittle, rather than elastic when broken". The texture and body of Domiati cheese were influenced by many factors: chemical composition of the cheese, flora present, composition of the pickling mixture, and temperature during storage and ripening (Fahmi and Sharara, 1950). Increasing the pressure on the curd from 20% of its weight to 40% yielded a better body and texture (El-Safty et al. , 1980). Addition of starter to cheese milk reduced the body and texture, however. The low acidity milk gave the best body and texture (El-Koussy et al., 1976). Both pasteurization (El-Koussy et al., 1975, 1976) and homogenization (Ahmed et al., 1972; El-Shibiny et al., 1972; El-Koussy et al., 1975, 1976) of milk gave a firm body and high scores for body and texture of Domiati cheese, respectively. Increasing the milk solids by adding skim milk powder (Monib et al., 1981) or mixing fresh milk with reconstituted dried milk (Mashaly et al., 1983; Hefnawy et al., 1984) produced the best organoleptic properties for Domiati cheese. However, ultrafilteration (U F) of cheese milk gave different results. Hofi (1984) found that UF had no effect on the organoleptic scores, while Omer (1987) reported that cheese made from UF milk had a uniform and closed texture, good appearance and better organoleptic properties than cheese made by conventional procedure. The effect of various additives in milk on the resultant body and texture of Domiati cheese has been researched sensorily. Some of these additives improved the body and texture such as: adding 0.05 % CaC12 to the heated cheese milk (El-Koussy et al., 1974), or milk hydrolyzates (Said and Mahran, 1984), whey protein (EL-Shibiny et al., 1973), whey protein/carboxymethyl cellulose complex (Abou-Donia, 1981) to 42 fresh milk. Also, adding B-galactosidase (El-Safty et al., 1985), hydrogen peroxide (Ghaleb, 1977a), glutamine and pectin (Ghaleb, 1977b) to cheese milk enhanced the texture of cheese. However, the other additives had an adverse effect on the body and texture of cheese. Such additives as fat (Naghmoush et al., 1978) and formalin (El—Shibiny et al., 1972). Microstructure of White Soft Domiati Cheese Domiati cheese consists of an aggregation of water, fat and protein (mainly casein) in roughly equal proportions by weight, to which salt is added. Protein primarily constitutes the structural matrix of the cheese. The protein matrix is formed after the proteolytic conversion of x-casein into para-x—casein by rennet action (Mackinlay and wake, 1971). The cheese curd (rennet gel) is formed from the para-casein micelles in the presence of Ca++ and can be formed into various types of cheese in many ways. It was shown by electron microsc0py techniques (SEM), that during the manufacturing process, casein changed progressively from spherical micelles to filaments forming large aggregates, with a granular structure, in which the fat globules are entrapped. The final cheese consisted of islands of fat entrapped in a protein matrix (Knoop and Peters, 1972); Kimber et al., 1974; Eino et al., 1979; Kalab, 1977). The structure of ripened soft white Domiati cheese changed into loose structure of fine particles (Abd El—Salam and El-Shibiny, 1973). The evidence was very clear, since the milk changed from a liquid to a gel, then to semi solid or almost solid. The properties and structure characterization of the final cheese are determined during the transformation process of para-x-casein micelles into cheese and by the condition of that 43 transformation (e. g. pH, temperature, enzyme concentration, Ca”, etc.). Recently, a few studies have examined the microstructure of Domiati cheese. The screened cheeses were made using raw milk (Abd El-Salam and El-Shibiny, 1973; Knoop et al., 1976) or both pasteurized skim milk and pasteurized high fat milk (Kerr et al., 1981). Also, cheeses made from pasteurized milk with added fi-galactosidase (Omar and El-Shibiny, 1985), from recombined milk (Omar and Buchheim, 1983), and from ultrafiltered (UF) milk (Omar, 1987) were examined. Abd El-Salam and El-Shibiny (1973) were the first to describe a technique for preparing ultrathin sections of Domiati cheese for examination using electron microscopy. They found that the internal structure of Domiati cheese was greatly changed during the ripening in highly salted solution (pickling). Thus in fresh cheese, the internal structure was found to be composed of a framework of large, spherical casein aggregates held together by bridges and enclosing fat. However, in the pickled cheese, the casein aggregates were partly dispersed into small spherical particles forming a loose structure. Knoop et al.(1976) compared the microstructure of Domiati cheese to a new type of brine cheese made from pasteurized milk, added starter culture and CaClz. They concluded that the paracasein micelles rapidly dispersed into submicelles in Domiati cheese due to the higher content of phosphatase than the experimental cheese. Scanning electron microscopy (SEM) studies, made by Kerr et al.(1981) on Domiati cheese, showed a similar matrix of globular casein micelles for both pasteurized skim milk and pasteurized high fat milk cheeses after overnight pressing. However, cheese made of pasteurized high fat milk had additional material filling the inter-micellar 44 spaces. After 8 weeks, the cheese was less distinct in structure and more compact than that made of pasteurized skim milk. Another study conducted by Omar and El-Shibiny (1985) on the microstructure of Domiati cheese made of pasteurized cow’s milk and from the same milk with added B—galactosidase (200 mg/l) showed no appreciable differences in microstructure of fresh cheese. However, after 8 weeks, the cheese containing the fi-galactosidase had considerable changes in its matrix, which became porous, possibly due to proteolysis during the ripening. Omar and Buchheim (1983) noticed differences in the microstructure of the protein matrices in ripened cheese samples made from raw milk and from recombined milk. There was a very homogeneous structure in cheese made from raw milk, compared with a slight aggregated state of the protein in cheese made from recombined milk. More recently, Omar (1987) studied the microstructure of Domiati cheese made of UF milk comparing with cheese made by the conventional method. His micrographs showed similarities in the appearance of the cheese matrix (which consists of fused casein micelles in homogeneous masses) between the two cheeses when young and during ripening. MATERIALS AND METHODS Fresh, whole cow’s milk was obtained from the herds of Michigan State University (MSU) dairy farm. Commercial calf rennet (15,000 SU), containing 90% chymosin and 10% bovine pepsin, was purchased from CHR. Hansen’s Laboratory, Inc. (Milwaukee, WI). Low heat nonfat dry milk, which was used to measure the strength of the calf rennet, and commercial food grade sodium chloride were obtained from Michigan Milk Producers Association (Novi, MI.). All chemicals and solvents were chemically pure and bought from Sigma Co. (St. Louis, MO). Physico-chemical Properties of High Salted Milk To study the effect of NaCl concentration on some physico-chemical properties of milk which relate to rennet milk coagulation process, the following experimental designs were applied: pH of Milk Whole fresh cow’s milk was divided into 15 portions of 150 milliliter in 250 m1 beakers. Sodium chloride was added to the beakers in increments of 1% from 0 to 14% followed by agitation. Samples were run in quadruplicate. Milk pH was recorded in triplicate using a Beckman pH-meter model 2500.' Sodium-Calcium Replacement Fresh cow’s milk was divided into 11 equal portions (100 ml) in a 250 ml 45 beakers. from 0 1 The be: Sampler calcium priman dropwi: comple Were ; 46 beakers. Food grade sodium chloride was added to the beakers in increments of 2 % from 0 to 14% (w/v). Milk pH was adjusted in each beaker to 6.6 after NaCl addition. The beakers were tempered at 37°C for 30 min in a thermoregulator water bath. Samples were coagulated with 0.03% calf rennet. The coagulum was removed. The calcium content of milk and whey was determined in triplicate. Calcium Determination: Stock Solution (500 ug Ca/ml) Anhydrous calcium carbonate was used to prepare the standard. To 1.219 g primary standard (CaCO3), 5-ml distilled, deionized water was added (DDW). A dropwise minimum volume of 1N HCl (approximately 10 ml) was added to effect complete solution of the CaCO3. The solution was brought to 1 liter with DDW. Milk sample To 5 milliliters of milk (pH 6.6) in a 100-ml volumetric flask, 50 ml of 24 % (w/v) trichloroacetic acid (TCA) solution were added and brought to volume with DDW. Samples were shaken at 5 min intervals for 30 min and then filtered with Whatman # 42 filter paper (Clifton, NJ). Five milliliters of the filtrate were transferred to a 50-ml volumetric flask. One milliliter of a 5% (w/v) lanthanum solution was added. The mixture was brought to volume with DDW. Whey Sampr To 5 milliliters of whey in a 100-ml volumetric flask, 50 ml of 12 % (w/v) TCA were added and brought to volume with DDW. Samples were shaken at 5-min intervals for 30 r filtrate lantham quadruj 1982). Ammk setting instrurr checks milk s: betwee (1986) in 251 throng at 10C AH-G diame imme 47 for 30 min and then filtered with Whatman # 42 filter paper. Five milliliters of the filtrate were transferred to a 50-ml Volumetric flask. One milliliter of 5% (w/v) lanthanum chloride was added. The mixture was brought to volume with DDW. Atomic Absorption Conditions Calcium in milk (total Ca) and whey (soluble Ca) sarnples was determined in quadruplicate as described by Perkin-Elmer atomic absorption manual (Norwalk, CT, 1982). Equipment and conditions utilized were as follows: Perkin-Elmer Model 2380 Atomic Absorption instrument with air-acetylene flame, wavelength at 422.7 nm and slit setting of 0.7 nm. Standards were made from a 500 rig/ml calcium stuck solution. The instrument was standardized using a 5 pug/ml standard with 1 and 2 ng/ml standard checks. A sample of 5 milliliter of DDW, which had been treated in the same way as milk samples was used as a blank. Colloidal calcium was calculated as the difference etween the total and soluble calcium as described by Polychroniadou and Vafopoulou 1986). f Casein Hydration Fresh cow’s milk was defatted and divided into 15 equal portions, 100 ml each, 250 ml beakers. Sodium chloride (0-14%) was added to the beakers followed by ough agitation. The pH was adjusted to 6.6. Samples in triplicate were centrifuged t 100,000 x g (27700 rpm) in RC 60 Sorvall Instruments Ultracentrifuge equipped with -629 rotor for 1 hr at 4°C. Casein pellets were spread over aluminum trays (5 cm iameter X 2 cm height) and weighed. Each tray was sealed with freezer type and frozen mediately at -20 F. The trays were placed in a Virtis Unitrap II Freeze-Drier, with capacity 30.5 or control of absc Torr at the va (w\v) Salts at to with fixe: C011. 0110 C0) 48 capacity of eight liters. The freeze-drier was equipped with a cylinder (43.2 cm high X 30.5 cm) and instrumentation for the control of freeze-drying variables. Automatic controls for condenser and platen temperature, vacuum adjustment and constant recording of absolute pressure was maintained with this unit. Drying was carried out at 4-6 X 102 Torr at 90 F for 48 hr. After completion of drying, nitrogen (W/P) was introduced into the vacuum chamber to establish atmospheric pressure. Samples were immediately placed in a dissector until constant weight was obtained. The difference between the weight before and after freeze-drying, expressed as a percentage of the dried casein, was taken as the water of hydration (Grufferty and Fox, 1985). Microstructure of Casein Micelles Freshly drawn cow’s milk was defatted and salted with 0, 5, 10, and 15 % NaCl (w\v). Samples preparation were done using the technique of Caroll et al. (1968). Salted milk was stored at room temperature for 0, 24, 48 and 72 hr. A 0.2 ml milk samples were fixed with 2 ml glutaraldehyde (1%) in 0.1 M phosphate buffer (pH 7.2) at room temperature for 15 min with occasional stirring. Samples were diluted (1:50) with distilled water and deposited on coverslips by dipping the coverslips in the diluted fixed milk. The coverslips with dispersed milk were mounted on stubs using double stick tape. The coverslips were mounted just slightly off-center on the stubs so that a good onductive pathway could be obtained from the top of the coverslips to the metal stubs nce they had been sputter coated. The mounted coverslips were air-dried, then ransferred to a dissector and held under vacuum for 24 hr. The dried mounted coverslips were coated with gold using EM Scope SC 500 Sputter Coater (Ashford, Kent, England] time at The fit: visual s and 10 had nc differe (35, 3' 0.05 2 const calf r the inn and A f thIl Ca 49 England) and viewed on the JEOL ISM-35 scanning electron microscope (Tokyo, Japan). Rennet Clotting Time of Milk (RCT) Two techniques were used to measure RCT of milk (the period elapsing from the time at which rennet was added to the milk to the appearance of flocculated particles). The first was the back extrusion technique of Harper et al. (197 8). The second was the visual slide method of Sharma et al. (1989). Milk was divided into three aloquots. Five and 10% NaCl was added to the first and second aliquot, respectively, while the third had no salt (control). Clotting time of salted and unsalted milk was determined under different independent variables such as pH (6.6, 6.2, and 5.8), renneting temperature (35, 39 and 43°C), rennet concentration (0.03, 0.06 and 0.09 %) and added CaCl2 (0.02, 0.05 and 0.1 %). Each variable was tested individually while the other variables were constant. The milk pH was adjusted to 6.6 after NaCl or CaCl2 addition. Commercial calf rennet (strength 1/ 15000 Soxlhet) was added (0.03 %) to milk tempered at 35 °C in a thermoregulated water bath. Apparent Viscosity Method Ten ml milk samples were transferred to 100 mm pyrex screw top tubes immediately after rennet addition. Samples were kept in a thermoregulated water bath and removed in triplicate at varying intervals (0, 3, 10, 40, 70, 100, 150, and 180 min). A flat bottomed stainless steel plunger (23 cm long with a 0.733 cm diameter) was forced through the milk gel using an Instron Universal Testing Machine (IUTM model 4202, Canton, OH) with a microcomputer (Hewlett Packard 86 B). The Instron was equipped with a 50 N (5 Kg) load cell and operated at 100 mm/ min cross head speed and 130 mm travel distar Hickson et perform th millisecond The area ur the triangle height of I Area/Lo. taken to in standard . viscosity defined calculal ViSCOSil 50 travel distance. Apparent viscosity (viscosity index) was calculated using a modified Hickson et al. (1982) procedure. A program, developed by Lever (1988), was used to perform these calculations. Distance (mm) and force (N) were read every 300 milliseconds and this data was used to calculate the back-extrusion apparent viscosity. The area under the curve formed from the distance—force curve was converted into a right the triangle containing equivalent area and travel distance (LP) to the original curve. The height of triangle was then equivalent to the maximum force (Fp) found by: Fp = 2 Area/Lp. The ratio of Fp to Lp multiplied by a constant (Hickson et al., 1982) was taken to be the apparent viscosity of the samples. Data were normalized to reduce the standard error of the measurements taken before milk coagulation. The normalized viscosity Y’ was given by: Where: N0 = initial viscosity (at 0 time) N = apparent viscosity at the intervals The viscosity index used to determine the clotting time by back extrusion was defined as 4.8 poise based on initial visual observation of the flocculated particles in milk. Microstat computer program (Ecosoft, Inc. , 1984) regression function was used to calculate the clotting time from the normalized apparent viscosity versus time data using viscosity index. Visual Slide Test Method For comparison, the clotting time of milk was also determine by dipping a glass slide in the Met Mil Fre was adjust bath. Call Inc, Mill quickly pi intervals, samples. The sam] was and et al., 1! l l coIllplet give a Whatm PhOSp 51 e in the renneted milk and observing the formation of flecks. Measuring the Degree of x-casein Hydrolysis by Rennet Action Milk Sample Preparation Fresh cow’s milk was salted with 0, 5, and 10% NaCl (w/v). The pH of milk 3 adjusted to 6.6 then equilibrated for 30 min at 35 °C in a thermoregulated water h. Calf rennet (0.03 %) with 1/ 15000 Soxhelt strength (CHR. Hansen’s Laboratory, :., Milwaukee, WI) was added followed by stirring. Two ml of renneted milk were ickly pipetted into test tubes and incubated at 35 °C. At 0, 1, 10, 40, 70, 120 min ervals, 4 ml of a 12% (w/v) TCA solution were mixed with the renneted milk nples. A blank was prepared the same way as for milk samples but without rennet. re samples were kept at 35°C for one hr, filtered with Whatman # 42 and the filtrate is analyzed using High Performance Liquid Chromatography (HPLC) (Van Hooydonk ., 1984). Glycomacropeptide (GMP) Standard Preparation Fresh cow’s skim milk was incubated at 37 °C with calf rennet (0.03 %) until plete coagulation (when a glass rod comes out of the clot clean), TCA was added to e a final concentration of 12%. The precipitate was removed by filtering through atman paper # 42 and the filtrate was dialyzed at 4°C against repeated changes of ' ed water for 14 days. The dialyzed filtrate was lyophilized using a Virtis Unitrap freeze-drier as mentioned above in casein hydration experiment. A graded series ging from 20-700 rig/ml of freeze dried GMP were weighed and dissolved in tsphate buffer at pH 6.64 The buffer consisted of a solution of potassium hydrogen phosphatt g) in 100 standard GMP rel equine flow he 710B \ Beckm "Syste. colurn phosp‘ The ll Modt chror conv the; soft 52 >sphate (1.74 g), potassium dihydrogen phosphate (12.37 g), and sodium sulfate (21.14 in 1000 ml of DDW. The eluent was filtered through a 0.45 pm millipore filter. The ndard was injected into a HPLC system. A standard curve was used to quantitate the 4P released during rennet action on x-casein. High Performance Liquid Chromatography (HPLC) Analysis The HPLC was composed of a Water’s model 600 multi-solvent delivery system ipped with a thermally regulated column compartment and two pumps each with a head capacity of 110 p1. Sample injection was done automatically using a model B Water’s Intelligent Sample Processor (WISP). The system is connected to a krnan model 166 programmable variable-wavelength UV detector on line with ’stem Gold" computer software for data manipulation. A TSK-l25 gel filtration amn (30 cm X 0.75 cm) was maintained at 35°C. The eluent composed of the same tsphate solution aforementioned. 20 pl of the samples were injected automatically. = flow rate was set at 1.0 ml/ min, and the UV detector operated at 205 nm. The Data iule was programmed to allocate the base-line at the beginning and at the end of the imatogram. The response factor was calculated on the basis of the complete rersion of x-casein into para-x-casein. Peak areas were corrected by subtraction of teak area obtained with the TCA filtrate of the substrate before renneting (blank). Texture Profile (TPA) and Physical Structure of White Soft Domiati Cheese as Affected by Coagulation Process Factors The following design was used for studying the TPA and microstructure of white )omiati cheese, made from high salted milk, as affected by coagulation process factors such CaCl, Pre} Che cow’s milk 30 min in cylindrical which the batches/tr first was chemical E T by slow “Who were m at the lilCl‘mC batche 53 tors such as: pH of milk, renneting temperature, enzyme concentration, and added :12. Preparation of Cheese Samples Cheese was manufactured as described by Fahmi and Sharara (1950). Fresh v’s milk was salted with 5% and 10% NaCl (w/v). Milk was tempered at 35°C for 1min then coagulated with 0.03% diluted enzyme (1:50). The curd was ladled into rndrical aluminum moulds (11 X 7 cm). The moulds were drained for 48 hr through ich the cheese was inverted 3 times. The cheese blocks of each batch (3 :hes/ treatment) as removed from the moulds were divided to two equal portions. The t was used for TPA. The second was used for microstructure examination and mical analysis. Effect of pH The pH of 5 and 10% salted milk was adjusted to pH 6.6, 6.2 and 5.8 (Table l) zlow addition of 1N lactic acid or sodium hydroxide. The milk was then allowed to librate at 35 °C for 1 hr in a thermoregulated water bath. Three batches of cheese a manufactured as mentioned above. Renneting Temperature The 5 and 10% salted milk was adjusted to pH 6.6. Milk samples were tempered .ree coagulation temperatures; 35 , 39, and 43°C (Table 1) for 30 min in a roregulated water bath. The same manufacturing procedure was followed for three 38. Table l. Renne Rm 0210 54 3 1. Experimental design of the independent variables of coagulation process of salted milk for cheese texture profile analysis. coagulation conditions added NaCl added atment (%) C21012 (%) pH temperature rennet . (°C) conc. (%) k pH: 5 , 10 6.6 35 0.03 5 , 10 6.2 35 0.03 5 , 10 5.8 35 0.03 neting temperature: 5 , 10 0 6.6 35 0.03 5 , 10 6.6 39 0.03 5 , 10 6.6 43 0.03 net concentration 5 , 10 6.6 35 0.03 5 , 10 6.6 35 0.06 5 , 10 0 6.6 35 0.09 2 addition 5 , 10 0.02 6.6 35 0.03 5 , 10 0.05 6.6 35 0.03 5 , 10 0.10 6.6 35 0.03 Re Fi‘ at 35°C concent were m C T milk (5 min in to mal Varial com: 6ij 55 Rennet Concentration Five and 10% (w/v) salted milk was adjusted to pH 6.6. @Samples were tempered 5°C for 30 min in a thermostatically controlled water bath. Three rennet :ntrations (0.03%, 0.06%, 0.09% v/v) were used (Table 1). Three cheese batches made as aforementioned. Calcium Chloride (CaCl,) Addition Three concentrations of CaC12 (0.02, 0.05, and 0.1% w/v) were added to the salted :5 and 10% NaCl). Milk pH was adjusted to pH 6.6 and tempered at 35°C for 30 1 a thermoregulator water bath (Table 1). The rest of the procedure was followed ke three batches of cheese as mentioned above. Interaction of Milk pH, Renneting Temperature, Rennet Concentration, and Added Calcium Chloride Table 2 Ishows the experimental design for the interactions among the independent les in the coagulation processes: milk pH, renneting temperature, rennet rtration, and CaC12 added to milk. There were triplicate cheese batches for each ment. Table 2. E cc Treatmen 56 e 2. Experimental design of interaction among the independent variables of coagulation process of salted milk for cheese texture profile analysis. coagulation conditions added NaCl added atment pH temperature rennet (%) CaClz ( %) (°C) conc. (%) r 5 , 10 o 6.2 39 0.03 2 5 , 10 o 6.6 39 0.09 3 5 , 10 o 6.2 39 0.09 4 5 , 1o 0 6.2 35 0.09 5 - 5 , 10 0.02 6.6 39 0.09 6 5 , 10 0.02 6.2 39 0.09 7 5 , 10 0.02 6.2 35 0.03 8 5 , 10 0.02 6.2 35 0.09 9 5 , 10 0.02 6.2 39 0.03 propert elastici Univer used f kg) 10 with 2 cross- recon W385 bite con deri cob 57 Texture Profile Evaluation Cheese cubes (2.5 x 2.5 x 2.5 cm) were prepared from the cheese. Rheological rties of cheese such as modulus of elasticity (MOE), hardness, brittleness, ity, cohesiveness, chewiness, and gumminess were measured. The Instron rsal Testing Machine (IUTM) table model 4202 (Instron Co., Canton, OH) was ollowing of Bourne et al. (1967, 1968). The Instron was equipped with 50 N (5 1d cell in conjunction with a microcomputer (Hewlett Packard 86 B). A plunger flat plate 3.5 cm in diameter was attached to the cross-head. The speed of the read was set at 22.5 cm/sec in both upward and downward directions. The ing chart speed was 1.25 cm/min. The strain level of the plunger to the sample tat one inch (75 % deformation). One-tenth scale load range of 5 kg was used and nsecutive bites were taken. The texture evaluation was done in triplicate on each if cheese. A computer program provided by (Ralston Purina, St.Louis, MO) was » collect the data and calculate the area under the response plot. Areas 1 and 2 epresent areas under the curves formed during the first and second compression nd work done during compression. The height of the first peak during the first tresented the extent of hardness (force). While the distance of the sample under ssion during the second bite represented springiness (cm). Cohesiveness was from the ratio of Area 2/Area 1. Where gumminess was equal to hardness X eness. Chewiness was equal to gumminess and springiness. hearse Microstructure rbes (3 x 3 x 3 mm) of cheese samples were fixed in 4% glutaraldehyde in 0.1 M phosp times in osmium cheese interva1 50, 75, Balzer: agent. Specir Engla (10k) Over diffe (K0: Voll higl 58 hosphate buffer at pH 7.2 for 2 hr to fix the protein. The cubes were washed several s in 0.1 M phosphate buffer (pH 7.2) for 15 min intervals, then post fixed in 1% 'um tetroxide (OsO4) in 0.1 M phosphate buffer for 1-2 hr for fat fixation. The se samples were re-washed several times in 0.1 M' phosphate buffer for 15 min als. Then specimens were dehydrated in a series of aqueous ethanol solutions (25, 75, 95, and 100%) for 15 min each. Critical point drying was carried out using a ers instrument Model CPD010 (Balzers, Liechtenstein) and liquid C02 as the drying t. Dried samples were mounted on specimen stubs using silver conducting paint. imens were coated with gold in an EM Scope Sc 500 Sputter coater (Ashford, Kent, land) for 7 min and examined in a JEOL J SM-35 CF Scanning Electron Microscope yo, Japan) (Carroll et al., 1968). Chemical Analysis The moisture content of cheese samples was determined in duplicate by the Vacuum 1 Method (Kosikowski, 1982). The percentage moisture was calculated by the rence in weight of the original and the dried sample. The modified Babcock method ikowski, 1982), was used to determine the fat content of cheese. The modified ard test (Kosikowski, 1982) with a slight modification in sample size (2 g) for the y salted white soft Domiati cheese was used to determine the salt content of cheese. NaCl content was calculated using the following formula: 1 .lNANO - 10.1N KSCNxoooss m 0 g 3 m x100 % NaCl = weight of sample The micro-Kjeldahl procedure (AOAC. 1984. 16.274) was followed to determine total nitrc HX-20 m weighed: each sam oontainin l) and tl without heating were a! flasks w Preheat: for DD‘ acid we lilratio; 30% It autom; The fr total nitrogen. A Buchi automated nitrogen analyzer 322/342 equipped with an Epson HX-20 minicomputer was used. Approximately 0.5 g of cheese sample was accurately weighed into a 100 ml straight sided digestion tube. Samples were run in duplicate. To each sample, 5 ml of concentrated H280, and one Kjeldahl MT catalyst tablet (Fisher) containing 3.5 g KZSO4/3.5 mg Se was added. Digestion was started at low heat (setting 1) and the settings were increased at 15 min intervals until samples boiled consistently ' without danger of sample loss. At this point the temperature was turned on high and heating continued until all samples were clear (2.5-3 hr total heating time). The flasks were allowed to cool in the digestion device when digestion was completed. Cooled flasks were transferred to the automated nitrogen analyzer which had been prepared and preheated as suggested in the equipment manual. The unit controls were set at (1) 1.9 for DDW, (2) 2.1 for 30% NaOH and (3) 1.0 for distillation time. Sixty ml of 4% boric acid were added to the receiving vessel. Beginning pH was recorded and entered as the titration endpoint on the automatic titration device (Impulsomat/ 614). Water (DDW) and 30% NaOH were automatically added and distillation and titration were then completed automatically. The volume of 0.0963 N HCl added was displayed on the Dosimat/655. The following calculations to determine percent nitrogen were performed: (sample - blank) ml HCl X N HCl X 14 mg/mmole X 100 sampleweight (mg) A factor of 6.38 was used to convert the total nitrogen to percent protein. Statistical Analysis The two-way statistical analysis of variance (ANOVA), 4 factor factorial, mean separati< 010W cheese. analyze treatme compa 1968). compl combi 0n 801 W616 6O eparation and correlation required a subprogram of MSTAT microcomputer statistical rogram (ver. 4 C, 1989) to evaluate the texture parameters of white soft Domiati heese. The effect of adding different concentrations of NaCl to cheese milk were alyzed using analysis of variance and the student "t" test. The significance between eatments was determined using either the Dunnet test or Bonferroni "t" test for omparison analysis, after a significant "F" test was determined (Gill, 1978, Woolf, 968). The same program was used to analyze a factorial 4-way analysis of variance ompletely randomized design, for the interaction significant differences between the mbined factors. To examine the effect of adding different salt concentrations to milk 11 some of its physico-chemical properties, simple and multiple linear regression analysis Iere applied and "t" test was used to test means at p < 0.05 (Gill, 1978). (Tabl 6% l exch: exch: were foun acid Tab RESULTS AND DISCUSSION A) Effect of NaCl on milk pH: Addition of up to 14% NaCl to milk induced a significant (p < 0.001) decrease Table 3) in pH from 6.64 to 6.13 (Fig. 3). The rate of decline was more rapid below 5% NaCl and eventually reached a limiting value. That was probably due to an :xchange of Na+ for H+ attached to charged groups of casein. On the completion of the :xchange, no change in the pH occurred (Grufferty and Fox, 1985). These observations vere in accordance with those of Ramet et al. (1983) and Grufferty and Fox (1985) who round that the pH of milk decreased to 6.4 after adding 5% NaCl. Abd El-Hamid et .(1981) also noticed that addition of up to 5% NaCl to milk increased its titratable tcidity. Beyond 5 % NaCl the acidity of milk remained constant. ”able 3. Analysis of variance for pH of milk as affected by adding NaCl to milk. Source DF Sum Mean F value Probability of squares square Regression 1 0.4105 0.4105 151.696 1.667E-10 esidual 19 0.0524 0.0027 otal 20 0.4619 gnificance = p < 0.001 dard error of determination = 0.052 61 1mm. w. mafia» Om. zmfifl O: D: On. 31:? 62 pH 9m 9N ab 0.0 can 90 a.» a; _ r _ _ _ _ b _ L h _ a a a u a o 8 3 an a e. a Zm0_$$. 3m. w. mayo. cm 2%: o: m 94 BE? (Tat add 575 63 B) Replacement Ca2+ by N a+ Addition of 6% NaCl caused a significant increase (p < 0.001) in soluble Ca (Table 4) from 463 ppm to 575.7 ppm in the milk serum (Fig. 4). However, further addition up to 14% NaCl, caused a significant decrease (p < 0.001) in soluble Ca from 575.7 ppm to 446.3 ppm. Colloidal Ca decreased with increasing NaCl, up to 6%, from 1044.67 ppm to 932 ppm followed by an increase (1061 ppm) up to 14% NaCl. The coefficient of determination (R2) between salt concentration and soluble Ca was 0.994 (Table 5). These results were in agreement with those of Grufferty and Fox (1985), who attributed the increase in soluble Ca to an exchange of Na+ for Ca2+ bound to casein, rather than the solubilization of colloidal calcium phosphate. However, Puri and Parkash (1965) reported that the addition of NaCl to milk solubilized part of the colloidal calcium phosphate (CCP). The amount of Ca released increased with up to 4 g NaCl/ 100 ml milk and there was no noticeable change thereafter (Puri and Parkash, 1965). The maximum percentage of soluble calcium was detected in whey when 7.5% NaCl was added to milk. When more NaCl was added a decrease in the percentage of calcium in whey was noted (Sharara, 1958), due to a diminishing replacing power of NaCl. Therefore, NaCl affected the secondary (aggregation) phase, by affecting Ca2+ needed for forming the bridges between casein micelles to establish the casein network of cheese curd. Table 4. Analysis of variance for replacement the calcium of milk by sodium after adding NaCl to milk. Source DF Sum Mean F ratio Probability of squares square Regression 4 19791.976600 4947.99410 129.33 0.001 Residual 3 114.773438 38.25781 Total 7 19906.750000 1mm. 5.. Minion» O». 2N0. O: morn—in ”3Q Goa—Omn—Nu Quinn—:3 0». BEN- Calcium In mllk (ppm) amoo BooH .mooi moo- #00 t 00:059. 09 Moo —4 d _ _ — _ o M a o m .5 in .3. zmo_ $3 Em. A. mason" 04 2mg o: 832a Ea 8:93: 2:053 cm Saw. 65 Table 5. Multiple nrifrression analysis of calcium replacement by sodium after adding NaCl to ° k. Variable Regression Standard Std. Partial Std. .error of Proba- number coefficient error regrx coeffi- partial coef- bilrty crent ficient 2 4.3535E+01 7.1192E+001 0.3999E+01 6.5401E-01 0.001 4 -3.0722E+001 2.3070E+001 -O.4109E+01 3.0858E+001 0.22 5 -3.3119E-01 2.5626E-01 -0.6171E+01 4.7748E+001 0.23 6 2.3019E—02 9.0817E-02 0.5938E+01 2.3427E+001 0.03 Intercept = 463.8937 Coefficient of determination (R2) = 0.994 C) Effect of NaCl on Casein Hydration The addition of 0-400 mM NaCl to milk caused an increase in the micellar hydration from 2.2 to 2.4 g H20/ g protein (Grufferty and Fox, 1985). Addition of up to 14% NaCl decreased the water content of the casein pellet of milk from 3.2 g HZOI g protein in the control to 1.69 g HzOl g protein with 14% NaCl (Fig. 5). Casein micellar hydration did not change much when 0-2% NaCl was added to milk. Beyond that concentration, a decrease in casein micellar hydration was noticeable (p < 0.001) (Tables 6 and 7). The amount of water lost/ g protein for adding 14% NaCl was almost 50% that of the control. The coefficient of the determination (R2) was 0.998 (Table 6). These findings were in agreement with those of Eck (1986) who reported that hydration of milk protein decreased with high concentrations of NaCl in milk. NaCl bound more H20 as much as its concentration increased. Salt seems to play a major role in regulating cheese consistency (Ramanaskas, 1978). That is because sodium chloride competes with the casein micelle for the water surrounding the micelles. Consequently, adding high concentrations of NaCl disturbs the casein micelle stability and exposes its charges to the environment. ~uonmpxq uraseo no DEN J0 1091521 '$ '313 (%) IO3N - 0|» 31 71 9|» 66 Casein micellar hydration (g HZO/g protein) N 0| 0 9'8 T I r Ta 67 Table 6. Multiple regression analysis of casein micelles hydration as affected by addition of NaCl to milk. Variable Regression Standard Std. Partial Std. error of Proba- number coefficient error regr. coeffi- partial coef- bility cient ficient 1 9.0295E+01 3.9981E+01 0.7928E+001 3.5106E-01 0.05 3 -5.7651E+01 1.1316E+01 -0.8418E+01 1.6523E+001 0.001 4 5.0257E+001 1.1113E+001 0.1162E+02 2.5704E+001 0.001 5 -1.3524E—01 3.4838E-02 -0.4926E+01 1.2690E+001 0.001 Intercept = 3169.388 Coefficient of determination (R2) = 0.998 Table 7. Analysis of variance for hydration of casein micelles as affected by adding NaC to milk. Source DF Sum Mean F ratio Probability of squares square Regression 4 310610300 776525.75 458.87 0.001 Residual 4 6769.00 1692.25 Total 8 311287200 68 The amount of interacting NaCl with milk proteins increased with increasing salt concentration, decreasing the water activity (a) and moisture content (Hardy and Steinburg, 1984). An increase of NaCl bound to para-casein decreased the amount of bound moisture to para-x-casein (Ramanaskas, 1978). Geurts et al. (1974) hypothesized that the degree of NaCl interaction with para-x-casein depends on pH, NaCl and Ca-ion concentration. D) Effect of NaCl on the Microstructure of Casein Micelles Scanning electron microscopy showed spherical casein micelles of different sizes (Fig. 6, A). Salt caused changes in the casein micelle shape (Fig. 6). Addition of 5% NaCl caused some aggregation of the micelles (Fig. 6, B). When NaCl was increased to 10%, the casein micelle shape became irregular and some micelles aggregated (Fig. 6, C). The aggregation of the micelles was increased at higher NaCl concentrations (Fig. 6, D). 69 fig. 6. SEM micrographs show the effect of NaCl concentration on the casein micelles of skim milk. (A) no added NaCl; (B) 5% NaCl; (C) 10% NaCl; (D) 15% NaCl. Bar = 1.0 p. 70 Examining the milk containing 5% NaCl immediately after NaCl addition showed few casein micelles aggregates. Larger micelles showed some changes in shape (Fig. 7, A). After 24 hr, micelles in milk containing 5% NaCl aggregated (Fig. 7, B). Large micelles did not appear to aggregate (Fig. 7, B). After 48 hr, micelle aggregation increased. The large casein micelles aggregated with the small micelles (Fig. 7, C). Although there was no visual coagulation (clot) at 72 hr with 5% NaCl, the micrograph showed almost a complete network aggregation (Fig. 7, D). 71 Fig. 7. SEM micrographs show the effect of time on casein micelles in skim milk containing 5% NaCl. (A) 0 hr; (B) 24 hr; (C) 48 hr; (D) 72 hr. Bar 1.0/.1. 72 Fig. 8,A showed micellar aggregation following the addition of 10% NaCl to milk. The aggregation was increased after 24 hr of adding salt to the milk. Most of the small micelles lost their shapes and showed a bulk-like structure (Fig. 8, B). After 48 and 72 hr, the micelles were highly aggregated (Fig. 8, C and D). 73 Fig. 8. SEM micrographs of the casein micelles in skim milk showing the effect of adding 10% NaCl to milk. (A) 0 hr; (B) 24 hr; (C) 48 hr; (D) 72 hr. Bar = 1.011. 74 Micrograph 9 (A, B, C and D) illustrated the effect of adding 15% NaCl on the casein micelles microstructure. Micrograph (9, A) showed the aggregated micelles which form a network. The aggregation of the casein micelles was increased with time. Twenty four hr after adding salt to the milk, the micelles showed attached surfaces (Fig. 9, B). After 48 hr, all the casein micelles participated in the aggregated network (Fig. 9, C). A complete precipitation occurred 72 hr after salt addition (Fig. 9, D). Salt competes with the casein micelles for water surrounding the molecule. Withdrawing the surrounding water exposes the casein micelle charges to the environment. The destabilized micelle precipitates and separates from the aqueous phase. 75 Fig. 9. SEM micrographs of the casein micelles in skim milk showing the effect of adding 15 % NaCl to milk. (A) 0 hr; (B) 24 hr; (C) 48 hr; (D) 72 hr. Bar = 1.0 u 76 As a result, salting out for some casein micelles occurred at as low as 5% NaCl added to milk, depending on the casein micelle size and time after the salt addition. E) Effect of NaCl on the Enzymatic Phase of the Rennet Coagulation Process Two percentages of NaCl (5 and 10 %) were used to evaluate the effect of NaCl on the enzymatic phase of the coagulation process in comparison with unsalted milk (control). The HPLC peaks of glycomacrOpeptide (GMP), non protein nitrogen (NPN), and trichloroacetic acid (TCA) at specific incubation periods with rennet at 35 °C for unsalted milk, are shown in Fig. 10. The resolution of the two main fractions, NPN and GMP, was excellent. There was no indication that other TCA-soluble degradation products, with a molecular size differing from that of GMP, were produced during the first 10 min after rennet addition. GMP was not detected after 3 min of renneting in both 5 and 10% salted milk, but GMP was measurable after 1 min of renneting with control milk. The GMP peak was detected after 10 min in both 5 and 10% salted milks. The concentration of GMP in the unsalted milk was 5 times greater than GMP in the milk containing 5% NaCl and 10 times that of milk containing 10% NaCl after 10 min of renneting. After 40 min incubation, GMP showed an insignificant increase in control. GMP continued to increase until 120 min in both (5 and 10%) salted milks. At 120 min after renneting, there was no change in the GMP with unsalted milk. The results indicated that the addition of NaCl to milk decreased GMP by 43 % and 61% compared to the control after 120 min with 5 and 10% NaCl, respectively (Fig. 11). NaCl inhibited (p < 0.00001 and 0.00016) x-casein hydrolysis (Tables 9 and 10). The coefficients of determination (R2) were 0.926 (Table 8), 0.9534 (Table 9) and 0.987 (Table 10) for 0, 5, and 10% salted milk, respectively. Statistical analysis of variance for milk containing 0, 5, and 10% NaCl are shown in Tables 11, 12, and 13, respectively. In agreement with these results, Van Hooydonk and Walstra (1987) reported that the conversion rate of x-casein by rennet decreased approximately linearly as NaCl concentration increased when the pH of milk was not corrected and decreased even more with pH correction, due to the effect of pH on the enzymatic reaction of 77 Van Hooydonk et al., 1986). The direct interaction of NaCl with the enzyme could modify the molecule, due to a salting out effect at high ionic strength (Ramet and El—Mayda, 1984, and El-Mayda et al., 1985). Additionally, NaCl might cause some conformational changes in x-casein which renders its rennet-susceptible bonds less accessible to the enzyme. NaCl inhibited the enzymatic phase of the coagulation process either by modifying the enzyme substrate (Dalgleish, 1983) or the enzyme molecule (Ramet and El-Mayda, 1984) or both. 78 (D g,“ at... I..." a. no inject S L—t = 129 min. . fwd: - ' l -' r 0...,” no -- . O :0 t = 70 min. .. .31.: 'Wt. 00-... .0”...¢~Dcl -UOMQOOC. \ i ‘ .4 9‘ 0‘}? o \ ’o ‘b 00::32‘0:.“w.00 .0. COO...: - .P..:..f.*9....e.1.e.:.- ../ .__._..___.- . , . , RECORDER RESPONSE '0 0 ti 1: a '10 Anna. _ .,. ~w~-4;\\g C“ ‘ 7" 0 V . D t 3 a min . ./'\‘\~’—-m_-J_. I {-0 z .- lu’dfi “Mme—“mm...“ 'W ' I v g : u-w- " .' 9 o l '. .. 3 o '3“ “‘0 z t =- 1 min . , ..... . _../"v-...~-—-—~°‘:‘ “"z o" '5 .. .. wanna-coat. 0....0‘ +— ‘00 i ‘9? i ; .- 1; " ".- E ' V t . 0 min . -W' ~00... o. 0.0 0 ‘ .000‘9000v 00...... .0- o.‘-_‘ COCA-O C. 7* r 2.214 4.38 6-57 7.6 8.6 10.78 12.19 15.04 TIME,MINUTES Fig. 10. HPLC determination of 1 comacro e tide GMP after rennetin ti f l, 3, 10 40 70 and 12 n’rIin at 35 OIEllzind 0(.03% )calf rennet forgrawmtfrisgltgd milk. (A):.GMP; (13g: NPN; (C): TCA; flow rate: 1.0 m1/min; detection: UV 205 nm; injection: 2 p1. 79 Glycomacropeptide (ug/ml) moo moo r Boo moo w moo 1. no 8 mo .230 A35. 30 o: 209 mm ZmQ 3: >39 no :5 3m 2. ~58an «3603833230 833 “52. 8.52 mono: o: 88m: 955 Same—mac: @308” 2.. BE". 80 Table 8. Multiple regression analysis of released glycomacropeptide after action of rennet on casein of raw cows’ milk. Variable Regression Standard Std. Partial Std. .error of Proba- number coefficrent error regr.. coeffi- partial coef- bility crent ficrent 1 2.6335E+01 7.6971E+001 0.5788E+01 1.6918E+001 0.01 2 -4.4562E—01 1.8242E-01 -0.1145E+02 4.6869E+001 0.05 3 2.1259E-03 1.0248E—03 0.6548E+01 3.1567E+001 0.08 Intercept = 68.59305 . Coefficient of determination (R2) = 0.926 Table 9. Regression analysis of released g1 comacropeptide after rennet action on casein during coagulation process of m1 k containing 5 % NaCl. ‘ Variable Regression Standard T Probability coefficient error : 1 2.2160 0.2190 10.119 0.00016 Constant 12.5231 Coefficient of determination (R2) = 0.95 34 Table 10. Regression analysis of released glycomacropeptideafter action of rennet on casein during coagulation process of mil contamrng 10 % NaCl. Variable Regression Standard T Probability coefficient error 1 1.5680 0.0804 19.496 0.00001 Constant 4.1273 Coefficient of determination (R2) = 0.9870 81 Table 11. Analysis of variance for released glycomacropeptide after action of rennet on casein of raw cow’s milk. Source DF Sum Mean F ratio Probability of squares square Regression 3 239626.0620 79875 .3520 12.47 0.033 Residual 3 19219.3125 6406.4375 Total 6 258845 .3750 Table 12. Analysis of variance for released glycomacrope tide after action of rennet on casein nucelles during coagulation process of ml containing 5% NaCl. _ Source DF Sum Mean F ratio Probability of squares square Regression 1 61404. 8434 61404. 8434 102.389 1 .615E-04 Residual 5 29986049 599.7210 Total 6 64403.4483 Table 13. Analysis of variance for released glycomacropeptide after action of rennet on casein during coagulation process 0 mrlk containing 10 % NaCl. Source DF Sum Mean F ratio Probability of squares square Regression 1 30743.6327 30743.6327 380.096 6.552E—06 Residual 5 404.4194 80.8839 Total 6 F) Effect of Independent Variables on Rennet Coagulation Time (RCT) of Salted Mlk 1) pH of milk Table 14 shows the combined effect of pH and NaCl on the rennet clotting time (RCT) of milk. As pH decreased, the coagulation of milk containing 5 and 10% NaCl 82 was increased to a minimum clotting time beyond which the coagulation time increased with the formation of a very fine coagulum. The coagulation time was decreased at pH 6.2 with salted milks then increased at pH 5.8. According to the visual observation method, RCT was 101.3 and 153.5 min at pH 6.6 and then decreased to 67.8 and 87.2 min at pH 6.2. An increase in RCT to 194.9 and 216.3 occurred at pH 5.8 with 5 and 10% NaCl, respectively (Table 14). However, RCT decreased with decreasing the pH of unsalted milk and the minimum CT (3.8 min) was reached visually when the pH was decreased to 5 .8 while the instrumental method recorded 5.9 min. These findings were in agreement with those of Fahmi et al. (1973) who found that 7% NaCl delayed the coagulation of milk. However, as the milk acidity increased, the RCT decreased and reached a minimum at 0.26% acidity beyond which it increased until no coagulation could be observed. Table 14. Effect of pH of milk on rennet coagulation time (RCT min) NaCl (%) pH - ' 0 5 10 Visual observation method 6.6 12.4 101.3 153.5 6.2 5.1 67.8 87.2 5.8 3.8 194.9 216.3 Objective method 6.6 17.8 102.2 158.2 6.2 7.7 71.7 88.9 $8 5.9 Lower concentrations of NaCl increased milk clotting, while higher concentrations decreased the clotting. Addition of about 50 mM NaCl has been reported to minimize clotting time (Hamdy and Edelsten, 1970, Alais and Iagrange, 1972, Qvist, 1979) if the pH was not kept constant. At a constant pH, a gradual increase in the clotting time as 83 a function of NaCl addition was observed (Ramet etal., 1983, Grufferty and Fox, 1985). Addition of NaCl was reported to decrease the rate of enzymatic reaction (Van Hooydonk et al. , 1986) and also the coagulation of the renneted micelles (Dalgleish, 1983). At high ionic strength a decrease in the calcium ion activity probably occurred, which slow down the reaction (Dalgleish, 1983). While pH activates the enzyme, it neutralizes the protein charges and moves some Ca and P from the casein micelle, which is important for its stability in the aqueous phase. Sodium chloride not only interferes with the second step (gelation) of milk coagulation, but also affects enzyme activity (Van Hooydonk et al. , 1986; Van Hooydonk and Walstra, 1987) particularly at high concentrations. Sodium exchanges with Ca in the colloidal system and saturates some of the bonds that might otherwise be linked with Ca, producing dispersed noncoagulable protein (Lawrence et al. , 1983; Green and Manning, 1982). 2) Temperature Understanding the role of coagulation temperature of milk is necessary to obtain optimum conditions for cheese production. Increasing the temperature from 35 to 39°C caused a greater decrease in CT than when increased from 39 to 43°C with salted and unsalted milk (Table 15). Results agreed with those of Sharma et al. (1989) who found that the rate of x-casein hydrolysis by rennet could be increased by increasing the temperature from 28 to 32 then 37°C. Fahmi et al. (1973) noticed that the time of coagulation of milk containing 7 % NaCl was decreased by increasing the temperature from 30 to 35°C which affected both stages of clotting. At higher temperatures, rennet coagulation time was decreased and the rate of curd firming was faster (McMahon et al. , 1984; Bohlin et al., 1984; Marshall et al., 1982; Emmons and Lister, 1976). The effect of temperature on coagulation may be attributed to an increase in hydrophobic interaction of the casein micelles (McMahon et al., 1984; Dalgleish, 1983) and hastened enzyme reaction (Sharma et al., 1989). 84 Table 15. Effect of renneting temperature on rennet coagulation time (RCT min) of NaCl (%) 252213 f temper— 0 5 10 Visual observation method 35 17.1 103.7 149.6 39 ‘ 12.2 98.4 136.3 43 10.4 92.5 127.6 Objective method 35 17.8 102.2 158.2 39 17.2 96.2 145.6 43 14.9 90.9 124.2 . 3) Rennet concentration Milk coagulation time was inversely proportional to the amount of rennet used. As shown in Table 16, increasing the concentration of rennet by a factor of 2X and 3X caused a parallel decrease in RCT of milk in both methods of measurement. The same pattern of RCT was found using 0.02, 0.03, and 0.04% rennet (Sharma et al., 1989), and when three concentrations of calf rennet (1, 2, and 3 ml) were used in curdling 7% NaCl cheese milk (Fahmi et al., 1973). Increasing rennet concentration, increases the rate of hydrolysis of x—casein which, in turn, influences the aggregation of casein micelles (Bohlin et al., 1984, Dalgleish et al., 1981). 85 Table 16. Effect of rennet concentration on the rennet coagulation time (RCT min) of NaCl @) Rennet concentra- 0 5 10 tion (%) Visual observation method 0.03 16.3 98.3 150.4 0.06 9.5 50.1 82.3 0.09 6.6 33.2 60.2 Objective method 0.03 17.8 102.2 158.2 0.06 10.1 58.5 92.7 0.09 8.4 33.8 64.1 4) CaCl2 Concentration Clotting time was used to evaluate the effect of CaClz on the coagulation process of highly salted milk. Table 17 shows RCT recorded using different CaC12 concentra- tions. Increasing CaCl2 concentration in milk decreased the CT with the three salt concentrations (0, 5, and 10%). The minimum CT was recorded with using 0.1% CaCl2 with 0, 5 and 10% salted milk in both methods used. The results were in agreement with those of Abd El-Hamid et a1. (1981) and Amer et al. (1974) who reported that addition of CaC12 to different kinds of milk resulted in a gradual decrease in RCT. Addition of CaCl2 to milk decreased coagulation time and increased the rate of aggregation up to a certain concentration, above which, the rate of aggregation decreased (McMahon et al., 1984, Story and Ford, 1982, Bohlin et al., 1984). The critical concentration at which calcium ions have a maximum effect depends on the experimental conditions. 86 Table 17. Effect of addition of CaCl2 on the rennet coagulation time (RCT min) of mrlk NaClfliL CaCl2 (%) 0 5 10 Visual observation method 0 14.4 106.2 146.3 0.02 9.8 97.6 136.5 0.05 7.9 92.1 134.3 0.10 5.6 84.1 122.7 Objective method 0 17.8 102.2 158.2 0.02 12.1 101.5 1441 0.05 7.3 101.5 137.9 0.10 5.2 89.1 122.1 The effect of Ca ions on milk coagulation may involve a transfer of soluble casein (weakly bound casein) to the micelles (Bloomfield and Mead, 1975; Smietana et al., 1977), neutralization of micellar charge (Mehaia and Cheryan, 1983), formation of calcium bridges (Payens, 1977) or some other specific functions (Dalgleish, 1983). 5) The Combined Effect of the Independent Variables , As shown in Table 18 the least RCT was found in combination 6 with both salted milk (5 and 10%), followed by combination 3 in both methods. The data indicated that the rennet concentration was the most effective variable used followed by the pH of milk on decreasing the RCT of salted milk. Decreasing RCT in heavily salted milk may be due to the interaction among the independent variables of the coagulation process which lead to an increase in the enzyme reaction and, in turn, the coagulation process. Therefore, choosing the proper coagulation conditions overcomes some of the problems encountered by heavily saltin g the cheese milk. 87 Table 18. The combined effect of the independent variables on the rennet coagulation time (RCT min) of salted milk. Visual observation method Objective method Treatment 5% NaCl 10% NaCl 5% NaCl 10% NaCl 1 49.4 54.8 36.3 54.6 2 26.1 36.7 29.7 38.6 3 18.1 21.4 20.7 28.9 4 27.3 31.6 32.9 36.4 5 24.2 32.8 21.2 23.3 6 16.1 18.2 16.7 22.6 7 54.4 65.3 54.4 67.0 8 21.2 29.3 24.7 32.6 9 41.5 43.7 35.3 40.4 Treatment 1. milk pH 6.2, rennetin temperature 39°C, and rennet concentration 0.03%; Treatment 2. 6.6, 9°C, and 0.09 a; Treatment 3. 6.2, 39°C, and 0.09%; Treatment 4. 6.2, 35°C and 0.09%; Treatment 5. 6.6, 39°C, 0.09%, and 0.02% CaClz; Treatment 6. 6.2, 39°C, 0.09%, and 0.02%; Treatment 7. 6.2, 35°C, 0.03%, and 0.02%; grgggnent 8. 6.2, 35°C, 0.09%, and 0.02%; Treatment 9. 6.2, 39°C, 0.03%, and . O. - G) Texture Profile of White Soft Domiati Cheese The effect of four of the most important factors in salted milk rennet coagulation, namely: pH of milk, the renneting temperature, the amount of rennet, calcium chloride concentration, and the interaction of these factors on the texture properties of Egyptian fresh White Soft cheese (Domiati) were investigated. The results of each factor will be discussed separately. 1) Effect of Milk pH a) Cheese Composition. Table 19 shows the effect of milk pH on cheese composition and yield. Domiati cheese produced from milk containing 10% NaCl had less fat, protein, and total solids (p < 0.001), but more moisture and salt (p < 0.001) than cheese made from milk containing 5% NaCl. The greater moisture content is due to increasing water binding capacity of sodium paracaseinate. At the natural pH of milk (6.6), the moisture of the 88 cheese made from milk containing 10% NaCl was 69.7 %, but increased to 75.5% when the pH was decreased to 5.8 (p < 0.01). The moisture increased also when the pH was decreased from 6.6 to 6.2 with cheese made from milk containing 10% NaClQ) < 0.01). Moisture content increased as the salt concentration increased, but fat, and protein content decreased (p < 0.001). Also, fatzTS and proteinzTS ratio decreased by decreasing the pH and increasing the salt concentration. Statistical analysis of pH, salt, and their interaction was shown in Appendix H, Table 1. These findings were in agreement with those of Fahmi et al.(1973). Table 19. Effect of milk pH on theyield and com osition of White Soft Domiati cheese made from mi k containrng 5 and 10 0 NaCl. ' pH Test 6.6 6.2 5.8 6.6 6.2 5.8 5 % NaCl 10% NaCl Yield (%) 30.2 35.4 38.8 38.0 41.8 35.0* Moisture (%) 65.7 67.2 69.5 69.7 71.6 75.5 Fat (%) 13.5 12.7 11.3 11.7 10.4 8.4 Fat:TS (%) 39.4 38.6 36.9 38.4 36.5 34.2 Protein (%) 10.4 9.7 8.5 7.9 6.8 5.2 ProteinzTS (%) 30.2 29.6 27.9 26.1 23.9 21.2 NaCl (%) 3.7 3.9 4.0 7.6 7.8 8.2 NaClzTS (%) 10.8 11.8 13.0 24.9 27.3 33.7 Average of six determinations from three replicates. * Cheese curd was very soft and very hard to handle. 89 b) Texture Profile Analysis (TPA) Typical texture profile curves, obtained with Instron Universal Testing Machine (IUTM), of fresh white soft Domiati cheese made from milk containing 5 and 10% NaCl at pH’s 6.6, 6.2, and 5 .8 are shown in Fig. 12 and 13. Texture parameters of cheese made from milk containing 5% NaCl were greater than those of cheese made from milk containing 10% NaCl. These parameters decreased by decreasing the pH of milk, .Appendix H, Table 2 shows the significant interaction of pH and NaCl for each parameter measured. Force Kg) '81 , ..- ..-. .. . .. .. .. - - . .. . . . .. t . ......._.,._ ..pu-r --...._. ..... . - -.~ --.- . . ..-..-----._. -‘ _._.._._.... --..—.—o_._._-__. t..- 1-. ___._-M -.‘__,,_ ._..-.--.--...- ... - .. .ul-.. n-.. - .-.. . . .-u. .--.-.u—_ —-—--- _. , .. .-.. .. -. , -~.... ... .... o ,.-.. -- .~-- 4 -.—. .-—-_ . _. ...- .. Ih.~---.’- .. . _-._...- g-‘-- ..o— o... _.... d arntxoj, 'Zl — 'h—o— flurunnuoo ~— —~| --up. ..- _—- -‘ .-- >—— o-.- % ---._-_.. *_..._..- -. . . o - a .. .... _.___.a —. . 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V | Y— . — s .. 4‘ _I __ _..-.- ._._.._.__..__ .__..___._. ,l— t-- ' —_§u-_---—--——-—-——- ...-.._.._ .t____._.__ _ ’ - cm. ..a—' - L..- T _' __ , --.._.-..._.- , .- f _ _..__ 1 . '9 9"“ ‘29 mm: mm; spew asaoqo netwoq JO} ouruoew sunset], [esraxrun uorisul our qr ) eouei '8 __-._. 'p- ..— .. -——-.-——.. -.—.-———.. LL10 til‘ i. i; it I l 'i it i '1 ii .. ..— ....— .-.o-_--..~ -—--—.——-—. ( ‘il ‘ '— I—A-—_ O - - O-“ 'n . .1- ~. ... p....-....-..-.... , # .._.- 3..—_-~...—.--.--_..-_—-. ..__._.-- - ._..._ . . i..——-__.— ..._... a ' .—-—. - ‘ ,-_.- .. ._..- -~-—q. .. .~ -- -~o-...—o—.——o ._-...—_-_-—.-_——_—+—-_— 5..-...—-- n—. _.«.o —-—-— — c-..—_...- .- . ..-—-.- .-—--.. - ..o ,. - M..-_.--.-..., .- --..-—. w——.‘.LU—I~Q‘ ,-___- fi ..-.. ... .... . -. . . 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L'-—.g—--—-—--.d “On—o-.- .__-_._ .--.—_-—o.¢ .— -- '—- u. ,._----~'.—..-. .mo—y—u-.—— ‘.. e--.-l—- 0.. —- ’----.....—.. . . ..- -...¢---_——--.- - -~—-o. --—~-v I- F—‘ ...-..-- -.-__.....- .....-_/.._-_.- ‘. ‘___ ‘. .5" ---v- ~ .. - -~. . L__..._.__-_.. .-.. .. -.. .___ " " ' ' —-—~-.-... """'I" . .0 - -~ v~ ._.. .. . .. . .._.._._. _.,--.——o._-.-—.--_..—-.- ..- . a... ..- - __“_ , _-.,...-..—.-v.- - . . .-..... no .. . . .. .. , -. .-.-— ....-‘-. ...---.- .n-- . to- .. "'—' -.—.._-.. .. -..-._. .. -. - - . - .. . .,--.... ..«_..-. —...—.‘. -.'--.. ._......__ --.-—.--- .... -.. _ -. .. . -....--. ...... .. ..c— .,,..... ..u....¢—..—.a—.—-—.— _..-—-‘. ....... ...i— ., .-«-. uvq-n. I —.--—..--_-. —.. -- .- ...... . .. ___ _. -.-.—.-.-.‘.. -_-..-..—....-...—__ .-—...-..-.-.... ..r _ _ , »----‘-~ - ......_ . --~ _. .-. - » - . . ...- .. _.....~ .-. - . A”-.. Force (Kg) 'sr '313 or nrxo .1. ...-co...” “—9-. —. .601 3Ulut‘muoo EBJqo sauna algord i l —-—-. .... __'.---.-.. ...—.... ---.-.- _ ...... -—,—.—————--.--.-¢-._.- .— —— “WA-to.-- ——_.- --. . --. ..- . ——-. - . _...-. -..- .- .—._.—..——. —. .— ..- -. -..— _ .——. ._... -__..-_.._ _. — —. - --.. —.-.-_ _.-—_.__ --. _-.—_—-——_—— y.— -_._..—-_4_..___,._.______...-----. -...-.- .. .. o _ -... .——._._...—. _-.—_.__....._ . . . --- “...—... ... ...——. .._.._.. . .-—. , ~ - -... .. .-..---4-——...—---. - “-..—... —m...——.—........——-——.-. m-..—_ ... —..... --.-.- - .. ...—--...- -. u....-.... . «..- ..--...-_._._.. ...-.....— -...—- -. .. . ..._.__- ..-. .-- --. -.. .-.-...........--..,._.. _ .-.—...— __. . ———— _——oa— - ...—.....-m ..-—.... . ...—.... ..----L.-..._.-...... 1-- _-__.-_..-.-_.. . . -. ...—.-..y ...-_.--_ _.. ...----....._._ . .-.... .. -. -...._-.-N-_._ . ..-., .- .9002; ...—~— -— .- --.- '8'9 DU?- ‘89 ‘9'9 lid 12 tom 0 .-u m———'--_.. . n uortsul our 1mm pau :5 h IU -.- ...... . -—-—— (U10) l ‘1 I i l : v! . -l -—-.—- . -..--.. ... -. _...... .. -—.. ---... :......__. .. .. —-————. -<- vu—u— . o - — o a . ---———-——. -— . . - ~ _m_ — -- -- ~ cu - . - . . . .... -——— .... ----- -~ --.. a . ...—o ...-.— .n~—_—-—.-.--.u -__---'---——-.—_. “...—....- . .. . 1 -_ . --— ... -._»-.-.- ——.-—-—. ........ .- neuuoa JO} aurqoew 31111891, [2519A Hutu won 91"” 93°qu ° ° 7.. -. b ..‘ -.- --.-” . .- ....- . ..TI . 2.. .-...L 92 The data indicated that the modulus of elasticity decreased by decreasing the pH from 6.6 to 5.8 (Fig. 14). Decreasing the pH from 6.6 to 6.2 with cheese made from milk containing 5% NaCl decreased (p < 0.01) the modulus by 42.08%. Decreasing the pH from 6.2 to 5.8 showed no change in the modulus (p> 0.1) with cheese made from milk containing 5% NaCl. Modulus of elasticity decreased (p < 0.01) when pH was decreased from 6.6 to 6.2 and from 6.2 to 5.8 (42.7, and 28.8%, respectively) with cheese made from milk containing 10% NaCl. The cheese modulus of elasticity decreased 18.3, 19.1, and 39.9% (p < 0.001) at pH 6.6, 6.2, and 5.8, respectively when the salt was increased from 5 to 10% NaCl (Fig. 14). The correlation coefficient between pH and modulus of elasticity was 0.886 for cheese made from milk containing 5% NaCl and 0.9688 for cheese made from milk containing 10% NaCl. 93 Modulus of elasticity (X1000 N/m2) >1an Hd \‘ I 33W £8 :9. ‘1‘ y—A .e i E. t: 0! o m a N 14. 2. Q o "fi e O 3. 8. 0 it is e a. O 5’ a a g. *- O 0 ii a: :3 (N U: D. p— O 39 E Q h) F. '0 m s» 9 9 .N a 9- 9° 94 Hardness did not change (p < 0.1, and p > 0.1) when milk pH was decreased from 6.6 to 6.2 with cheese made from milk containing 5 and 10% NaCl (Fig. 15). Cheese hardness decrease by 38.4 and 28.3% (p < 0.01 and p < 0.05) when the pH decreased from 6.2 to 5 .8 with cheese made from milk containing 5 and 10% NaCl, respectively. Decreasing the pH from 6.6 to 5 .8 markedly decreased (p < 0.01) the hardness of cheese made from milk containing 5 and 10% NaCl. Cheese hardness decreased (p < 0.001 and p < 0.01) with increasing salt concentration from 5 to 10% in milk at pH 6.6 and 6.2. At pH 5 .8 no significant difference (p < 0.1) was observed in hardness. The correlation coefficients between pH and hardness were 0.969 and 0.986 for cheese made from milk containing 5 and 10% NaCl, respectively. The highest firmness was observed at pH 6.6 and the lowest was at pH 5.8. Decreasing the pH from 6.6 to 6.2 and from 6.2 to 5 .8 decreased the force required to fracture the cheese 2 and 20% with cheese made from milk containing 5% NaCl. The brittleness decreased 3.9 and 3.7% with cheese made from milk containing 10% NaCl, when the pH decreased from 6.6 to 6.2 and from 6.2 to 5 .8 respectively. A decrease in brittleness was observed (Fig. 16) when milk pH decreased from 6.6 to 5 .8 (p < 0.05) for cheese made from milk containing 5% NaCl. Cheese made from milk containing 10% NaCl showed no change in brittleness (6.7%) when pH was decreased from 6.6 to 5 .8. Decreasing the pH from 6.6 to 6.2 caused an insignificant change in brittleness with cheeses made from milk containing 5 and 10% NaCl. Furthermore, the interaction between pH and salt decreased the brittleness more than each variable individually. Brittleness decreased (p < 0.001) 43.6, 44.7, and 33.3% at pH 6.6, 6.2, and 5 .8, respectively, when salt increased from 5 to 10%. The correlation coefficients between pH and brittleness were 0.905 and 0.9992 for cheese made from milk containing 5 and,10% NaCl. - 5% NaCl t\\\\\\‘ 10% NaCl Hardness (Kg) 6.6 6.2 6.8 pH of milk Fig. 15 . Hardness of Domiati cheese made from milk containing 5 and 10% NaCl at pH 6.6, 6.2, and 5.8. 0.6 - 0.6 i 0.4 ~ - 6% NaOl 0.3- 1095 N801 //, 0.2 ‘ Brittleness (Kg) ////// // / 0.1“ W 0.0 - 6.6 6.2 5.8 pH of mllk Fig. 16. Brittleness of Domiati cheese made from milk containing 5 and 10% NaCl at pH 6.6, 6.2, and 5.8. 96 Springiness (elasticity) decreased as pH decreased (Fig. 17). Adding 10% NaCl to milk decreased the springiness of the cheese compared with cheese made from milk containing 5% NaCl. Lowest springiness was found at pH 5.8 with cheese made from milk containing 10% NaCl. The springiness markedly decreased (p < 0.01) when the pH was decreased from 6.6 to 6.2 (22.5%). Springiness decreased 25.7% when the pH was decreased from 6.6 to 5.8 in cheese made from milk containing 5% NaCl. Springiness did not change (p> 0.05) with cheese made from milk containing 10% NaCl when the pH decreased from 6.6 to 6.2 and from 6.2 to 5 .8. Springiness decreased at pH 6.6 and pH 6.2 (p < 0.001) but no change was noticed at pH 5.8 (p < 0.1) with increasing NaCl from 5 to 10%. Cheese cohesiveness was higher at pH 6.6 than at pH 6.2 and pH 5.8 (Fig. 18). At pH 5.8 the cheese had the lowest cohesiveness for cheese made from milk containing 5 and 10% NaCl. Cohesiveness decreased with cheese made from milk containing 5% NaCl (p < 0.01) and did not change with cheese made from milk containing 10% NaCl (p > 0.1) when the pH was decreased. The correlation coefficients between pH and cohesiveness were 0.979 and 0.996 with cheese made from milk containing 5 and 10% NaCl, respectively. Salt decreased cohesiveness at pH 6.6 and 6.2 (p < 0.001) while no significant differences were found at pH 5.8 (p> 0.1). 97 - 6% NaOl 1095 NaCl Elasticity (%) 5-6 6.2 5.3 pH of mllk Fig. 17. Elastici of Domiati cheese made from milk ‘ ' PH 6.6,ty6.2, and 5.8. Contammg 5 and 10% NaCl at - 5% NaCl \\\\\\\\‘ 1095 N801 Cohesiveness (96) 8 O 6.6 6.2 5.8 pH of mllk Fi . l8. Cohesiveness of Domiati cheese made from milk containin 5 g at pH 6.6, 6.2, and 5.8. g and 10% Nacr 98 Chewiness and gumminess decreased as pH decreased (Fig. 19 and 20). Chewiness was highest at pH 6.6 with cheese made from milk containing 5% NaCl and lowest at pH 5.8 with cheese made from milk containing 10% NaCl. Chewiness and gumminess of cheese made from milk containing 5% NaCl decreased (p < 0.01), but no change was found (p> 0.05 and p < 0.1) with cheese made from milk containing 10% NaCl, when milk pH decreased. The effect of salt on gumminess was less pronounced at pH 5.8 (p> 0.1) than 6.6 and 6.2 (p < 0.001). The correlation coefficients between pH and chewiness were 0.989 and 0.9608 and between pH and gumminess were 0.9987 and 0.9901 with cheese made from milk containing 5 and 10% NaCl, respectively. 92 020 0164 010“ Chewiness 005' 0.00 - 66 82 pH of milk Fig. 19. Chewiness of Domiati cheese made from milk containing 5 and 10% NaCl at pH 6.6, 6.2, and 5.8. 68 QB 006 - Q41 Gumminess 02' eel 86 6.2 pH of mllk 6.8 - 6% NaCl \\\\\\\V 1021 N801 - 5% NaCl QNN KNBNMJ Fig. 20. Gumminess of Domiati cheese made from milk containing 5 and 10% NaCl at pH 6.6, 6.2, and 5.8. 100 Results obtained were in agreement with those of Walstra and Van Vliet (1982), who reported a decrease in the brittleness of cheese by decreasing the pH of milk. The results also were in accord with those of Van Hooydonk and Van der Berg, (1988); Creamer and Olson (1982); Yang and Taranto (1982); and Fahmi et al. (1973). In general, decreasing the pH decreased all the rheological parameters: modulus of elasticity, hardness, brittleness, cohesiveness, gumminess, and chewiness. The role of pH in the texture of cheese is particularly important because changes in pH are related directly to chemical changes in the protein network of the cheese curd. As the pH of the cheese curd decreases, a concomitant loss of colloidal calcium phosphate from the casein submicelles occurred. Below pH 5.5, a progressive dissociation of the submicelles into smaller casein aggregates was found (de Jong, 197 8; Hall and Creamer, 1972; Roofs et al., 1985). These changes in the size and characteristics of the submicelles significantly increase their ability to absorb water. The swelling of the casein submicelles in renneted milk is greatly increased in the presence of sodium chloride. The role of water in the texture of high moisture cheese, however, may be more complicated than a variation in the proportion of solids, since X-ray studies (Walstra and Van Vliet, 1982) showed that the casein network is indeed modified by excess water. However, in low pH cheese, casein fractions form compact aggregates which are held together with strong ionic and hydrophobic intra-aggregate forces while the inter-aggregate forces are weaker. Most of the water in such a system is inert, interstitial, and not distributed evenly throughout the curd mass (Creamer and Olson, 1982). By contrast, at a higher pH the casein molecules are net negatively charged, and while the hydrophobic interactions persist, the ionic interactions change from attraction between the protein molecules to repulsion. Thus the tight protein aggregates absorb water partly to solvate the unneutralized ionic charges. If the pH is high enough and there is enough water present, the protein can then dissolve. In cheese, however, dissolution is prevented by the presence of calcium, which binds tightly to the casein and decreases its solubility by the limited quantity of water available (Creamer and Olson, 101 1982). Nevertheless, the tendency to absorb water into the protein matrix is strong, and there is unlikely to be interstitial water in the high pH cheese (Creamer and Olson, 1982). More moisture at any pH, results in a soft inelastic cheese. It is not surprising that various types of texture can be obtained between pH 6.6 and 5.8, since a wide range of casein aggregates are present. On the other hand, an overlap between texture types occurs since the effect of pH can be modified by other compositional factors, particularly the moisture, salt, and calcium contents (Walstra and Van Vliet, 1982; Lawrence etal., 1987). 2) Effect of Renneting Temperature a) Cheese Composition Data in Table 20 shows the effect of renneting temperatures of 35 , 39, and 43°C on yield and composition of Domiati cheese made from high salted milk (5 and 10% NaCl). Higher temperatures (39 and 43°C) led to higher enzymatic activity, producing cheese with lower moisture than cheese prepared at 35°C (p < 0.05, 0.01). Cheese prepared at 43°C had a lower yield, moisture, fatzTS ratio, and NaCl:TS ratio but greater protein:TS ratio (p < 0.01). The higher clotting temperature allowed fat globules to rise to the top of the coagulum, particularly with longer coagulation time. Fat was then significantly lost during the curd cutting and ladling (p < 0.05) at 43°C for cheese prepared from 5 and 10% salted milk. On the other hand, the solubility effect of NaCl on the casein and protein of fat membranes caused some fat and protein loss from high salted curd (10% NaCl). In general, cheese made from milk containing 10% NaCl had higher yield, more moisture, and higher ratio of NaCl to TS but lower ratio of both fat and protein to TS (p < 0.001) for the three renneting temperatures compared to that made from milk containing 5% NaCl (Appendix H, table 3). These results were in concordance with those of Fahmi et a1 (1973). 102 Table 20. Effect of renneting temperature on the yield and com osition of White Soft Domiati cheese made from milk containing 5 and 10 0 NaCl. Renneting temperaturgC) Test 35 39 43 35 39 43 5% NaCl 10% NaCl Yield (%) 29.6 27.2 24.2 35.8 33.0 31.9 Moisture (%) 68.3 67.7 66.0 71.5 70.8 69.2 Fat (%) 12.0 11.7 11.2 10.2 9.8 8.9 Fat:TS (%) 37.9 36.2 32.9 35.8 33.6 28.9 Protein (%) 10.0 10.5 11.4 7.0 7.6 8.2 ProteinzTS (%) 31.7 32.5 33.4 24.7 25.9 29.5 NaCl (%) 3.7 3.5 3.4 7.5 7.4 7.3 NaCl:TS (%) 11.5 10.8 9.9 26.4 25.2 23.6 Average of six determinations from three replicates. b) Texture Profile Analysis (TPA) . Statistical analysis of the data from the study of temperature, salt, and their interaction for each texture parameter investigated are presented in Appendix H, Table 4. Typical texture profile curves of fresh White Soft Domiati cheese made from milk containing 5 and 10% NaCl at 35, 39, and 43°C are showing in Fig. 21 and 22. it '30 runnuoo 991913214 %9 3U. 6 rmqo sa/uno sword ornixal, 'arnreradwar Sunauuar 3°97 pun ‘5g 9891, [esraxrun uorrsul our rural pcu qo pcrwoq JO} aurqoew Bu 9899 3mm war} opeur (rue) eouerstq Force K9 .- --....--|--.----.—- .._————- — ... ..-- -———_.--- __-- ....-_.. _-—.~. -.Ot-Q—or-l—n—o- . 08 ll —._—_—..—._._--_. o— —. —. —.————_—.._._- — -—-—u- ...+-..-.- I--—. 0.. p. —. .o 0....-- ._ .-..‘ -—..._-. -oo-or— .-.—— ".4 . Inn-... --.--—. - .... ~-.Q—-o‘— .- —. —. .‘*M_ ‘zz 'Sier armxa 1, gr; it: 38 0 Sum uoo run uorrsul our 1mm pourmqo siaunNo érgind I?) °omnzradurar Surrauuar 0 up ‘ ‘ 01159.]. 11291911 . 0 £7 P 69 norm 8 I 1 open: csaoqo nerruoq .10} au rut uror 3" (uro) eouersga ._ -~. 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Au.- ..._-.,—. .._—- .—..—.—-——..-...-- -.— e ... -. ... -.tc—u—g . -..- .... . _—.—.—-.... - ..--.-.——.-— o .. . 105 Results indicated that the modulus of elasticity increased with increasing renneting temperature (Fig. 23). The lowest value was found at 35°C and the highest was at 43°C for cheese made from milk containing 5% NaCl. The modulus increased 8.4 and 12.7% with cheese prepared at 39°C from milk containing 5 and 10% NaCl, respectively. The elasticity modulus increased 7.3 and 23.9% with cheese made from milk containing 5 and 10% NaCl at 43°C, respectively. The modulus decreased significantly (p < 0.001, 0.001, and 0.05) at the three temperatures investigated (35, 39, and 43°C), when 10% NaCl was added to milk. Increasing clotting temperature increased the modulus of elasticity more with cheese made from milk containing 10% NaCl than cheese made from milk containing 5% NaCl. The correlation coefficients between renneting temperature and modulus of elasticity were 0.999 and 0.979 for cheese made from milk containing 5 and 10% NaCl, respectively. 106 not '8.ng v. “M 5&269 ..H E. C) M102 mm 0 two a. 00 80 mm tw{.\ 5.1 O M 0m mm we so Em. Nu. mmmoon a». 8:33: 2mg. 395636 4.0306838 AOL m 833888 c: Boa—:5 9.. 05305 c». 622.»: 0:88 Swan ~83 BEA 8:839” u Ba 3* 107 As the clotting temperature increased cheese hardness also increased (Fig. 24). The stress needed for a certain deformation increased by increasing the temperature. Hardness increased 9.4 and 6.5% for cheese made from milk containing 5 and 10% NaCl, respectively, when the temperature was increased from 35 to 39°C. However, hardness increased 5.8 and 1.1% when the temperature was increased from 39 to 43°C with cheese made from milk containing 5 and 10% NaCl, respectively. Hardness did not change (p> 0.1) when clotting temperature was increased from 39 to 43°C with 10% salted milk. The correlation coefficients between rennetin g temperature and the hardness were 0.998 and 0.938 with cheese made from milk containing 5 and 10% NaCl, respectively. The harder the cheese, the higher the force needed to fracture it. The fracturing force was higher with cheese made from milk containing 5% NaCl than with cheese made from milk containing 10% NaCl. Increasing the coagulation temperature from 35°C to 43°C increased (p < 0.01) brittleness of the cheese made from milk containing 5 and 10% NaCl (Fig. 2 5). Increasing the concentration of NaCl to 10% decreased (p < 0.001) the brittleness to almost one-half of its values at 5% NaCl at the three temperatures investigated. The correlation coefficients between renneting temperature and brittleness were 0.981 and 0.999 with cheese made from 5 and 10% salted milk, respectively. 108 1.2 1.0 4 A 0-8 - o . é - 5% NaCl 53 0'6 ‘ 1\\\\\t 1095 NaCl 8 '9 0.4 - G3 I 0.2 "l 0.0 . ' 4a Renneting Temperature (C) Fig. 24. Hardness of Domiati cheese made from milk containing 5 and 10% NaCl at 35, 39, and 43°C renneting temperature. 1.2 1.0-4 9 v 0.8- (D 8 06 - 596 Neel 5.2 . i _ \\\\\\\i 10% NaOl g: 0.44 0.2 -l 0.0- <3Hennetlng temperature( (C)8 Fig. 25. Brittleness of Domiati cheese made from milk containing 5 and 10% NaCl at 35, 39, and 43°C renneting temperature 109 Renneting temperature had a noticeable effect on the springiness (elasticity) of the cheese (Fig. 26). As renneting temperature increased, cheese elasticity increased. The springiness increased (p < 0.01) when the temperature was increased from 35 to 39°C with cheese made from milk containing 5 and 10% NaCl. N0 increase (p < 0.1) was detected with increasing clotting temperature from 39 to 43°C with cheese made from milk containing 10% NaCl. Salt decreased the springiness at the three clotting temperatures almost 32%. So, in spite of increasing clotting temperature, salt still had a significant influence on springiness. The correlation coefficients between renneting temperature and springiness were 0.974 and 0.982 with cheese made from 5 and 10% salted milk, respectively. Cheese had the least cohesiveness when prepared at 43°C (Fig. 27). Increasing the clotting temperature decreased (p < 0.1) the cohesiveness of cheese made from milk containing 5 and 10% NaCl. Cohesiveness did not change (p > 0.1) when the temperature was increased from 35 to 39°C, but decreased (p < 0.01) when the temperature was increased from 39 to 43°C with cheese prepared from milk containing 5% NaCl. Cohesiveness decreased (p < 0.05) when the temperature increased from 35 to 39°C, but no change occurred when the temperature increased from 39 to 43°C (p> 0.1) with cheese made from milk containing 10% NaCl. Salt decreased cohesiveness (p < 0.001) at the three clotting temperature studied. The correlation coefficient between renneting temperature and cohesiveness was 0.897 for cheese made from milk containing 5% NaCl and 0.966 for cheese made from milk containing 10% NaCl. 110 600 400‘ ,8 ‘? Elasticity (%) 200- 100‘ 00- 35 - 6% NaOl 10% NaCI 39 4a Renneting Temperature (C) Fig. 26. Elasticity of Domiati cheese made from milk containing 5 and 10% NaCl at 35 , 39, and 43°C renneting temperature. 600 600- 400‘ 300- 200' Cohesiveness (%) , 100- - 6% N30! \\\\\\\\‘ 10% NaOl 00 35 39 4a Rennetlng Temperature (C) Fig. 27. Cohesiveness of Domiati cheese made from milk containing 5 and 10% NaCl at 35 , 39, and 43°C renneting temperature. 111 Likewise, increasing renneting temperature from 35 to 43°C increased chewiness. Chewiness was lower at 43°C than 39°C (p < 0.01) with cheese prepared from milk containing 5% NaCl. An increase in chewiness was observed with cheese made from milk containing 10% NaCl as temperature increased. However, increasing the salt from 5 to 10% decreased the chewiness (p < 0.001) at the three temperature investigated (Fig. 28). While increasing clotting temperature increased cheese gumminess, adding more salt to the milk decreased the gumminess (Fig. 29). Gumminess was highest (p < 0.05) at 39°C and lowest at 35°C in cheese prepared with milk containing 5% NaCl. No change (p > 0.1) in gumminess was observed with cheese made from milk containing 10% NaCl. Salt decreased the gumminess at the three temperatures (p < 0.001) studied. The correlation coefficients between renneting temperature and gumminess were 0.841 and 0.979 with cheese prepared with milk containing 5 and 10% NaCl, respectively. 112 0.4 - 0.3 ‘ a) a) g - 5% NaCi g 0.2 ‘ 10% NaCl 0.1 - 0.0 Renneting Temperature (C) Fig. 28. Chewiness of Domiati cheese made from milk containing 5 and 10% NaCl at 35, 39, and 43°C renneting temperature. 1.0 - 0.8 t (O {’3 0.6-l E - 6% NaCl E ‘ \\\\\\\V 10% N60! g 0.4 - 0.2 - 0.0 35 69 43 Rennetlng Temperature (C) Fig. 29. Gumminess of Domiati cheese made from milk containin 5 and 10? N C at 35, 39, and 43°C renneting temperature. g o a 1 113 All texture parameters increased except cohesiveness, with increasing renneting temperature. Increasing the temperature may go along with a faster rearrangement of strands and fusion of micelles, resulting in a faster increase of the moduli directly after the onset of gelation (Zoon et al., 1988). The higher parameter values may be due to two reasons. First, the optimum activity for rennet action to clot the milk is around 40-42°C (Scott, 1986). Therefore, increasing the coagulation temperature increasing the action of the rennet to break down the x-casein and increases the rate of aggregation and the curd firmness (Marshall, 1982; Walstra and Jenness, 1984). At a higher temperature, the rennet coagulation time of milk is decreased even in the presence of NaCl (Fahmi et a1. , 1973), and the rate of curd firming is faster and the coagulum is stronger (McMahon et al., 1984; Bohlin et al., 1984; Marshall et al., 1982; Darling and Van Hooydonk, 1981; Emmon and Lister, 1976). Marshall (1982) and Walstra and Jenness (1984) reported that increasing temperature increased the syneresis of the cheese curd, and consequently, increased the hardness of the finished cheese. Increasing the temperature of coagulation hampered the inhibition effect of the salt and allowed the gelation process to proceed at a faster rate. Scott (1986) noted that clotting temperature was important in determining the type of curd. At 20-27°C the unsalted clot tended to be soft, but at 30°C, the clot was firmer and did not break into small particles during cutting. At 33-36°C the clot was tough and rubbery with slow syneresis. The Q10 (temperature coefficient) values at 30-40°C for the enzymatic phase, the nonenzymatic phase and for both, were 5.31, 11.4 and 16.7, respectively (Mehaia and Cheryan, 1983). The Q10 was markedly higher for the coagulation reaction which decreased with temperature (Dalgleish, 1983; Brinkluis and Payens, 1984). The second reason for the high texture parameter values may be attributed to an increase in hydrophobic interaction at higher temperatures (McMahon et al., 1984 and Dalgleish, 1983). The results showed no change in most of the texture parameters measured when clotting temperature was increased from 39 to 43°C. Additionally, salt had no significant effect. on most of the texture parameters with cheese prepared at 39°C. So, 39°C was chosen for the combination experiments. 114 3) Effect of Rennet Concentration a) Cheese Composition Table 21 shows the results of the cheese composition and yield when the three concentrations of rennet, 0.03, 0.06, and 0.09% (15,000 SU), were used to curdle the cheese made from milk containing 5 and 10% NaCl. Increasing rennet concentration, decreased cheese yield, moisture content, and NaCl:TS ratio (p < 0.01) and increased fat:TS and protein:TS ratio (p < 0.01, 0.05). Table 21. Effect of rennet concentration on the yield and com osition of White Soft - Domiati cheese made from milk containrng 5 and 10 0 NaCl. Rennet concentration ( %) Test 0.03 0.06 0.09 0.03 0.06 0.09 5% NaCl 10% NaCl Yield (%) 29.9 28.2 27.5 36.2 33.9 31.9 Moisture (%) 68.8 67.6 66.5 70.5 69.5 68.9 Fat (%) 12.0 12.4 13.2 11.0 11.6 12.2 Fat:TS (%) 37.9 38.2 39.3 37.4 38.1 39.1 Protein (%) 10.7 11.0 11.8 7.4 7.7 8.1 ProteinzTS (%) 33.8 34.0 35.1 24.9 25.2 26.1 NaCl (%) 3.6 3.5 3.3 7.4 7.2 7.1 NaCl:TS (%) 11.3 10.8 9.7 25.0 23.7 22.9 Average of six determinations from three replicates. The acceleration of the enzymatic stage of milk coagulation produced cheese with lower moisture content (p < 0.01). Cheese made. from milk containing 10% NaCl had less fat:TS and protein:TS ratios and higher yield, moisture, and NaCl:TS ratio than cheese made from milk containing 5 % NaCl with the three concentrations of rennet used (p < 0.001). analysis of the data from the study of rennet concentration, salt, and their interaction for cheese chemical composition are presented in Appendix H, Table 5. These findings agree with those of Fahmi et al. (1973). 115 b) Texture Profile Analysis (TPA) Three concentrations of commercial calf rennet (30, 60, and 90 111/100 m1 milk with 15,000 Soxhlet Unit (SU) strength) were used for curdling milk containing 5 and 10% salt to study the effects of increasing rennet concentration on the texture of Domiati cheese (Appendix H, Table 6). Increasing rennet concentration increased the modulus of elasticity (p < 0.01). Rennet concentration had a greater effect than the clotting temperature of milk on the modulus of elasticity of the cheese. Increasing rennet concentration from 0.03 to 0.06 and from 0.03 to 0.09% increased the modulus by 15.5 and 20% with cheese made from milk containing 5% NaCl, respectively. The modulus increased 6.6 and 40.5% with cheese made from milk containing 10% NaCl when rennet concentration was increased from 0.03 to 0.06 and from 0.03 to 0.09% respectively (Fig. 30). Addition of 10% salt to milk decreased the cheese modulus (p < 0.001) compared to cheese prepared from milk containing 5% NaCl. The correlation coefficients between rennet concentration and modulus of elasticity were 0.993 and 0.921 with cheese made from milk containing 5 and 10% NaCl, respectively. 116 No.01 v. I an. 209 who \\\\\t 00. 200. .0 Na; 301 8 a m l / e N 0 0 80 00.01 0 m wX d( 0 M 0.0- 0.0 0.00 0.00 0.00 30333 0030032020: E Em. mo. mam: am 8:02 000003830: 0: 3005:... 0». 05305. cm USE»: 0:00.40 5000 :63 SEA 8:833“ u 000 8* Z» . 117 Hardness of cheese increased (p < 0.01) with increasing rennet concentration (Fig. 31). However, salt concentration had no effect on hardness of the cheese made with 0.09% rennet concentration (p < 0.1). Hardness increased 42.5, and 9.4%, with cheese made from milk containing 5% NaCl, when rennet concentration was increased from 0.03 to 0.06 and from 0.03 to 0.09%, respectively. Hardness increased 71.1 and 35.8%, with cheese made from milk containing 10% NaCl when rennet concentration was increased from 0.03 to 0.06 and from 0.03 to 0.09% , respectively. The correlation coefficients between rennet concentration and hardness were 0.956 and 0.999 with cheese made from milk containing 5 and 10% NaCl. Brittleness of cheese increased with increasing rennet concentration. Increasing rennet concentration from 0.03 to 0.06 and from 0.03 to 0.09% increased the brittleness of the cheese 24.1 and 23% with cheese made from milk containing 5% NaCl, respectively. Brittleness increased 85.2 and 4.2% with cheese made from milk containing 10% NaCl, when rennet concentration increased from 0.03 to 0.06 and from 0.03 to 0.09%, respectively (Fig. 32). The highest rennet concentration (0.09%) did not increase (p> 0.1) the brittleness of the cheese made form milk containing 10% NaCl. Salt decreased the brittleness (p < 0.001, p < 0.02, and p < 0.001) with 0.03, 0.06 and 0.09% rennet concentrations when increased from 5 to 10% in milk, respectively. The correlation coefficients between rennet concentration and brittleness were 0.909 and 0.901 with cheese made from milk containing 5 and 10 % NaCl. 118 1.0 - 0.8 « A :34" v 0.6 ~ 311 - 6x NaC! 2 t\\\\\\\‘ 109: NaCl .6 0.4 - L. ad 2 0.2 - 0.0 0.09 0.03 0.06 Rennet Concentration (%) Fig. 31. Hardness of Domiati cheese made from milk ' ° - 0-03, 0-06, and 0.09% of rennet concentratigiifltmmng 5 and 10% NaCl wrth 1.0- 0.6 - ’5 x ... 0.6- g - 6% NaCl t: \\\\\\\V 9 0.“ 10% NaCl :9 0.2 - 0.0 0.03 0.06 0.00 Rennet Concentratlon (%) 32. Brittleness of Domiati cheese made from milk containing 5 and 10% NaCl Fig. with 0.03, 0.06, and 0.09% of rennet concentration. 119 Springiness increased about 23 and 74% with cheese prepared from milk containing 5 and 10% NaCl, respectively, with 0.06% rennet. At 0.09% rennet concentration, springiness increased 26.8 and 4.1% with cheese made from milk containing 5 and 10% NaCl, respectively (Fig. 33) over cheese prepared with 0.03% rennet concentration. Increasing the salt to 10% in milk decreased cheese springiness (p < 0.001) at the three rennet concentrations. The correlation coefficients between rennet concentration and springiness were 0.995 and 0.9688 with cheese made from milk containing 5 and 10% NaCl. A 0.09% rennet concentration significantly increased cheese springiness and decreased the clotting time (p < 0.01). Clotting time was decreased from 98.3 min to 50.1 min with cheese made from milk containing 5% NaCl and from 150.4 min to 82.3 min with cheese made from milk containing 10% NaCl. An increase in cheese cohesiveness occurred when rennet concentration increased (p < 0.01). The highest cohesiveness was observed by increasing the rennet concentration, compared to the other factors such as pH and renneting temperature, as shown in Fig. 18 and 27, and 34. Cohesiveness increased 86.5 and 133.8% with cheese prepared with milk containing 5% NaCl at 0.06 and 0.09% rennet concentration, respectively. Cohesiveness increased 110.2 and 258% with cheese made from milk containing 10% NaCl, at 0.06 and 0.09% rennet concentration, respectively (Fig. 34). Salt decreased (p < 0.001) cohesiveness when increased from 5 to 10% with the three rennet concentrations. The correlation coefficients between rennet concentration and cohesiveness were 0.9523 and 0.9996 with cheese made from milk containing 5 and 10% NaCl. 120 600- 400- 38’ 30.0‘ - 6% NaCl 2? .\\\\\s 10% NaCl 3 20.0 - U) 9 LL] 100‘ 0.0 0.03 0.06 0-09 Rennet Ooncentratlon (%) Fig. 33. Elasticit560f Domiati cheese made from milk containing 5 and 10% NaCl with 0.03, 0. , and 0.09% of rennet concentration. 140.0 '- 120.0 ' 1m‘0 - 80.0- - 6% NaCl 10% N80! 60.0-l 40.0 " Cohesiveness (%) 20.0 'l 0.0 0.03 0.06 0.09 Reenet Concentration (%) Fig. 34. Cohesiveness of Domiati cheese made from milk containing 5 and 10% NaCl wrth 0.03, 0.06, and 0.09% of rennet concentration. 121 The cheese exhibited increased chewiness (p < 0.01) with increasing rennet concentration (Fig. 35). The highest chewiness was obtained with cheese prepared with 0.09% rennet from milk containing 5 and 10% salt. Chewiness was significantly lower in cheese prepared from milk containing 10% salt than cheese made from milk containing 5 % salt at all rennet concentrations. The correlation coefficients between rennet concentration and chewiness were 0.997 and 0.996 with cheese made from milk containing 5 and 10% salt. Gumminess increased (p < 0.01) with increasing rennet concentration. The highest gumminess was maintained at 0.09% rennet with cheese made from milk containing 5 and 10% salt (Fig. 36). However, gumminess decreased as NaCl in milk increased from 5 to 10% (p < 0.001). The correlation coefficients between rennet concentration and gumminess were 0.9655 and 0.9938 with cheese made from milk containing 5 and 10% salt. 122 3.0 ' 2.0 1* - 55 NaCl 1.5 ~ \\\\\\V 10% NaCI Gummlness 0.5 ”’ 0.0 0.08 0.06 0. 09 Rennet Concentration (56) Fig. 35 . Chewiness of Domiati cheese made from milk containin 5 and 107 N With 003, 0.06, and 0.09% of rennet concentration. g a aCl - 5% Neon \\\\\\\\‘ 10% N80! Chewiness O a l 0.0 0.08 0.06 0.09 Rennet Concentration (%) Fig. 36. Gumminess of Domiati cheese made from milk containin 5 and 109’ N with 0.03, 0.06, and 0.09% of rennet concentration. g 0 3C] 123 Increasing rennet concentration increased all texture parameters. The rate of curd firming has been reported to depend on the rennet concentration (Marshall et al., 1982; Garnot and Olson, 1981; McMahon and Brown, 1982; Tukita et al., 1982). Also the ultimate gel strength obtained after aging the renneted milk gel increased with increasing rennet concentration (Van Hooydonk and Van der Berg, 1988). Probably the main effect of increased rennet concentration was to increase the rate of para-K-casein production which, in turn, influenced casein micelle aggregation (Bohlin et al., 1984; Garnot and Olson, 1982; Dalgleish et al. , 1981; Friedenthal et al., 1981; Bingham, 1975; Castle and Wheelock, 1972) and decrease rennet coagulation time (Story and Ford, 1982; Bohlin et al., 1984; McMahon et al., 1984; Darling and Van Hooydonk, 1981). Increasing the enzyme concentration above a certain value did not affect the maximum coagulum strength, but affected the rate of aggregation and the rate of curd firming (McMahon et al., 1984; Garnot and Olson, 1982; Story and Ford, 1982; Marshall et al., 1982). This idea was supported by the fact that increasing rennet concentration caused parallel increase in the curd firming curves (Bohlin et al., 1984; Emmons and Lister, 1976). The data indicated that using 0.09 % rennet concentration gave the highest cheese texture parameters so it will be used in the combined factors experiments. Measuring the texture parameters with different rennet and salt concentrations showed that problems attributed to salt concentration could be overcome by using a suitable amount of rennet. 4) Effect of Calcium Chloride 3) Cheese Composition Adding CaCl2 to cheese milk prior to renneting had little effect on cheese moisture and protein content (p > 0.05). Table 22 shows the yield and composition of White Soft Domiati cheese made from salted milk containing 0.02, 0.05, and 0.1% CaClz. Addition of CaC12 to cheese milk increased (p < 0.01) fat incorporated into the cheese. Calcium chloride increased micelle interaction (U stunol and Hicks, 1990), and more fat globules were entrapped into the curd matrix (Table 22). Statistical analysis of the data from the study of CaClz, salt, and their interactions for cheese chemical composition are presented in Appendix H, Table 7. These findings agreed with those 124 of Ustunol and Hicks (1990) who found that addition of CaCl2 to cheese milk increased cheese yield significantly by increasing fat incorporated into the cheese. Table 22. Effect of added CaCl on the yield and composition of White Soft Domiati 0 cheese made from m' k containing 5 and 10 NaCl. CaC_12 (%) Test 0.02 0.05 0.1 0.02 0.05 0.1 5% NaCl 10% NaCl Yield (%) 26.8 26.3 27.8 32.1 31.4 33.7 Moisture (%) 66.9 66.5 65.7 69.6 68.8 68.7 Fat (%) 12.7 13.1 13.8 11.1 11.6 12.0 Fat:TS (%) 38.5 39.3 40.3 36.4 37.3 38.3 Protein (%) 11.3 11.4 11.7 9.4 9.2 9.5 ProteinzTS (%) 34.1 34.1 33.9 31.0 29.4 30.5 NaCl (%) 3.4 3.4 3.3 7.1 7.2 7.3 NaCl:TS (%) 10.1 10.2 9.8 23.5 23.1 23.4 Average of six determinations from three replicates. b) Texture Profile Analysis (TPA) Three concentrations of calcium chloride (0.02, 0.05, and 0.1%) were used to evaluate the effect of calcium chloride in fresh salted (5 and 10% NaCl) cow’s milk on the texture parameters of Egyptian White Soft cheese (Appendix H, Table 8). Addition of 0.02% CaCl2 to milk increased the modulus of elasticity 9 % and 19.1% with cheese made from milk containing 5 and 10% salt. Adding 0.05% CaCl2 caused an even greater increase in elasticity modulus of the cheese. The highest concentration of CaC12 (0.1%) increased elasticity modulus of cheese (p < 0.01) prepared from milk containing 5% salt, but no change (p> 0.1) was observed with cheese made from milk containing 10% salt. The cheese modulus was decreased (p < 0.001) when salt concentration in milk was increased from 5 to 10%, respectively (Fig. 37). The correlation coefficients between CaC12 and elasticity modulus were 0.999 and 0.951 with cheese made from milk containing 5 and 10% salt. 125 we: I a: 2.0. W. um, § 8... 2.0. M. t ) S 2 N04 ..m m e l f N at O O 0 m m a- u d m. o M at CI 0.0» 0.0m 0; duo.» $3 Em. 3. Eon—:5 cm 0.823 O Q Q 05 003mm: 0:88 33o ~83 BEA coagaam u 2i 8w... 2mg «<5. 9cm. Pom. Ba 93¢ N N. 126 As the CaCl2 concentration increased, hardness also increased in cheese prepared with milk containing 5% NaCl (Fig. 38). Hardness of cheese prepared from milk containing 10% NaCl increased as CaC12 concentration increased and reached a maximum at 0.1% CaClz. An increase in salt from 5 to 10% decreased cheese hardness at the three CaC12 concentrations. The correlation coefficients between CaClz and hardness were 0.942 and 0.997 with cheese made from milk containing 5 and 10% salt. When Jen and Ashworth (1970) added 0.02% CaC12 to cheese milk, curd firmness increased about 32%; with 50 mM CaClZ, it increased 81%. The force required to fracture the cheese increased (p < 0.01) with increasing CaCl2 concentration. The increase in brittleness was the highest at 0.1% CaCl2 concentration with cheese made from milk containing 5 and 10% salt (Fig. 39). Brittleness did not increase much (4.8%) at 0.02% CaCl2 with cheese made from milk containing 5% salt. A greater increased was found (28.7%) with cheese made from milk containing 10% salt at 0.02% CaClz. Salt decreased the brittleness (p < 0.001) at the three CaCl2 concentrations. The correlation coefficients between CaCl2 and brittleness were 0.960 and 0.980 with cheese made from milk containing 5 and 10 % salt. 127 - 515 Mac: .\\\\\\‘ m m 1.2 ‘ 0.8 — Hardness (Kg) 0.4 — ,l 0.02 0.05 0.1 CaCI2 (%) Fig. 38. Hardness of Domiati cheese made from milk containin 5 and 107 N Cl 'th 0.02, 0.05, and 0.1% CaClz. g o a W 1.5 - - 515 NaCl 10% Mac! 1.2 - ‘6 x V 0.9 - no ’2’ 2 0.6- E 0.3 - ,l 0.02 0.06 0.1 CaCI2 (%) Fig. 39. Brittleness of Domiati cheese made from milk containing 5 and 10% NaCl with 0.02, 0.05, and 0.1% CaClz. 128 Springiness was affected significantly by adding CaCl2 to milk (p < 0.01). Springiness increased with increasing CaC12 concentration but not as much as with the other factors, e. g., rennet concentration (Fig. 40). The highest percent increase in springiness was found at 0.02% CaCl2 concentration with cheese made from milk containing 5 and 10% salt. "Salt decreased the elasticity at the three CaC12 concentrations when increased to 10% NaCl (p < 0.001). The correlation coefficients between CaCl2 and springiness were 0.972 and 0.999 with cheese made from milk containing 5 and 10% salt. Cohesiveness of cheese increased by increasing CaCl2 (p < 0.01) concentra- tion. The cheese internal bonds increased 55.9, and 33.8% with cheese made from milk containing 5% salt at the three concentrations of CaClZ. Cohesiveness increased 103.9, and 54.5% with cheese made from milk containing 10% salt at the three concentrations of CaCl2 (Fig. 41). Sodium chloride decreased cohesiveness markedly (p < 0.001). The correlation coefficients between CaCl2 and cohesiveness were 0.998 and 0.937 with cheese made from milk containing 5 and 10% salt. 129 600- - 6% NaC: .\\\\\\- 40.0_ - 1095 NaCl 3 3. 30.0- .15 3’3 260- El 10.0- (10- 0.02 0.05 0.1 CaOl2 (%) Fig. 40. Elastici of Domiati cheese made from milk containing 5 and 10% NaCl with 0.02, 0. 5, and 0.1% CaClz. 180 - - on me: 150 - 10% mo: d N O l i O O 1 Cohesiveness (1.) “a" 0.02 0.05 0.1 ’ OaC|2 (%) Fig. 41. Cohesiveness of Domiati cheese made from milk containing 5 and 10% NaCl with 0.02, 0.05, and 0.1% CaClz. 130 Cheese chewiness increased with increasing CaCl2 concentration in milk (Fig. 42). The lowest chewiness was obtained at 0.02% CaC12 with cheese made from milk containing 5 and 10% salt. Chewiness showed a great increase over all the other factors with increasing the calcium concentration of milk (p < 0.01). When salt in milk was increased from 5 to 10% chewiness decreased (p .<' 0.001) at the three CaC12 concentrations examined. The correlation coefficients between CaCl2 and chewiness were 0.996 and 0.999 with cheese made from milk containing 5 and 10% salt. Gumminess increased (p < 0.001) with increasing CaCl2 concentration in milk (Fig. 43). Gumminess was lower in cheese made from milk containing 10% salt than cheese made from milk containing 5 % salt at all three CaCl2 concentrations studied. Salt decreased (p < 0.001) gumminess at 0.05 and 0.1% CaCl2 concentrations but no change was observed in gumminess at 0.02% CaCl2 (p < 0.1). The correlation coefficients between CaC12 and gumminess were 0.879 and 0.9997 with cheese made from milk containing 5 and 10% salt. 131 2.0 - 6% NaCl 10% me: E 1.0- .\\\\.{ 0.5 - § 0.02 0.06 0.1 CaCl2 (%) Fig. 42. Chewiness of Domiati cheese made from milk containing 5 and 10% NaCl thh 0.02, 0.05, and 0.1% CaClz. 2; 1. |_ ' \‘ ,l \_h§_ %— o.oz 0.05 0.1 CaCl2 (96) Fig. 43. Gumminess of Domiati cheese made from milk containing 5 and 10% NaCl wrth 0.02, 0.05, and 0.1% CaClz. 132 As reported by Dalgleish (1982), the addition of Ca2+ increased the rate of enzyme reaction probably through increasing the adsorption of chymosin to K-casein (Walstra and Jenness, 1984). Most of the added calcium binds to the casein (Green and Marshall, 1977; Van Hooydonk et al., 1986) either by direct adsorption onto the negatively charged residues of casein or as micellar calcium phosphate (Tessier and Rose, 1958; Van Hooydonk et al., 1986). The critical concentration at which Ca2+ had maximum effect depended upon the conditions used, such as pH and temperature (Marshall et al., 1982; Dalgleish, 1983). The first increment of CaClz added (0.02%) usually had the greatest effect in increasing the rate of firming (Omar, 1985). A maximum gel strength was found with 0.03% added CaCl2 (McMahon et al., 1984) and a maximum cheese yield was obtained with 0.02-0.03% CaCl2 (U stunol and Hicks, 1990). Zoon et al. (1988) noted that the positive effect of CaCl2 at constant pH must be ascribed to an increase in calcium ion activity which was probably related to shielding of negative charges on the micellar casein phosphate (MCP). An increase in Ca“+ activity may lead to a higher positive charges on the casein resulting in faster coagulation and more interactions (cross-linking) within the gel network. Holt (1986) mentioned that when Ca” concentration in milk was below 1 mM, casein dissociates. This was thought to be due to a reduction in bound Ca”. 5) Effect of NaCl Two percentages of NaCl (5 and 10% W/V) were used to evaluate the influence of NaCl on the texture parameters of Domiati cheese. All texture parameters tended to decrease (p < 0.001) when salt concentration in milk increased from 5 to 10%. When cheese was prepared with milk containing 10% salt most of the rheological parameters decreased to almost one-half of the values observed with cheese prepared from milk containing 5% salt. Calcium chloride played an important role in counteract- ing the detrimental effect of NaCl, since a higher Ca++/Na+ratio resulted in a higher cheese firmness. Probably the phosphate content played a part as well (Walstra and Vliet, 1982). Addition of salt decreased the force between charges and led to a drop in gel rigidity (Cumper and Alexander, 1952). Likewise, springiness of the milk gel was 133 reported to decrease due to the presence of salt (de Jong and Henneman, 1932). In addition, NaCl caused a shielding of the charges and, because of the net (negative) charge of casein, this may lead to less electrostatic repulsion. It was observed by Hooydonk et al. (1986) that the voluminosity of the micelles, native and renneted, increased after the addition of NaCl and decreased the elasticity modulus of the cheese. The increase in the voluminosity suggested a decrease in the interactions within the micelles or to more interparticle interactions. Increasing the voluminosity decreased the firmness of the cheese. Above 100 mM NaCl in milk, the onset of gelation was retarded and the modulus decreased. This can be partly ascribed by the decrease in the enzyme activity (Van Hooydonk et al., 1986). So, salt affected both the enzymatic and, the nonenzymatic stages, by influencing the enzymatic reaction and the Ca2+. However, if a high salt cheese were in demand, some coagulation factors should be modified to decrease the harmful effects of NaCl on texture. A study of the interaction of the most important coagulation factors was conducted to set the conditions that produce the highest texture parameters for the highly salted cheese. 6) Effect of altering coagulation conditions on textural properties of Domiati Cheese Nine combinations of milk pH, renneting temperature, rennet concentration, and calcium chloride concentration were studied, to determine their effect on texture parameters of Egyptian White Soft cheese (Fig. 44 to 47). 134 a; 18.0 3 -samacr > ”’0‘ -1095»:th fig 1‘ \ 90- . “a? . i t t l .2 \ t t t i t § § 233.60" 1 \ \ \ g 30- 004 \\ § § § § § 5% . 1 2 3 4 5 7 a 9 Treatment Fig. 44. Combined variable effect on modulus of elasticity of Domiati cheese made i from milk containing 5 and 10% NaCl. Treatment 1. milk pH 6.2, rennetin temperature 39°C, and rennet concentration 0.03 %; Treatment 2. 6.6,, 39° , and 0.09%; Treatment 3. 6.2,39°C and 009% Treatment 4. 6.2, 35 C, and 0.09%; TreatmentS. 5.5 39 c, 0.69%, and 0.62% CaClz; Treatment 5. 5.2 39°C, 0.09%, and 0.02% CaClz; Treatment 7. 5.2, 35°C 0.03%, and 0.02% CaCl ; Treatment 8. 5.2, 35°C, 0.09%, and 0.02% CaCl’z; Treatment 9. 5.2, 39°02, 0.03%, and 0.02% CaClz. 135 1:4' -5$NaCI -1os~aCt 31.21 %081 \VV : . \\ 130.6- \\\\\' \\ " ~ t\\\\,t. °-27\)))§1))§ ,. t t t t t t t , -5suacl 30.8.. , -10%NaCl go it ii 20.4- \\V\\‘\“ :5 \\\\\\\\‘ 'MMM M 123455739 Treatment Fig. 45. Combined variable effect on hardness and brittleness of Domiati cheese made from milk containing 5 and 10% NaCl. Treatment 1. milk pH 6.2, rennetin temperature 39°C, and rennet concentration 0.03%; Treatment 2. 6.66 39° , and 0.09%; Treatment 3. 6.2 °C and 009% Treatment 4. 6.2, 35 C, and 39 0.09%; Treatment 5. 6.6 395C, 0.09%, and 0.02% CaClz; Treatment 6. 6.2 39°C, 0.09%, and 0.02% CaClz; Treatment 7. 5.2, 35°C 0.03%, and 0.02% CaCl - Treatment 8. 5.2, 35°C, 0.09%, and 0.02% CaCl’z; Treatment 9. 5.2, 39°(i’0.03%, and 0.02% CaClz. 136 60 -5§NaCl 50- Wtoxnac: 2 b 35 co 1 1 1 . a 6 9 120 ... -5$NaCI 33 100” W10$Naa 0 3 am 5 3- ear 1 0 1 \‘ 1 ° 1‘ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 . 1 1 1 0 1 1 1 1 1 1 1 1 1 1 2 a 4 5 6 7 a 9 Treatment Fig. 46. Combined variable effect on elastici and cohesiveness of Domiati cheese made from milk contamin 5 and 1 % NaCl. Treatment 1. milk pH 6.2, renneting temperature 39° , and rennet concentration 0.03 %; Treatment 2. 6.6, 39 6.2, 35°C, and 009% Treatment 5. 6.6, 39° Treatment 5. 5.2, 39°d 0.09%, and 0.02% c 0.03%, and 0.02% CaClz; Treatment 8. 5.2 35°C Treatment 9. 5.2, 39°C, 0.03%, and 0.02% Caci2 C, and 0.09%; Treatment 3. 6.2, 39°C, and 0.09%; Treatment 4. C 0.09%, and 0.02% Ca 12; Treatment 7. 6.2, 35° , 0.09%, and 0.02% CaClz; Cl' Chewiness 8 a: .E E E a 0 Fig. 47. 137 - 5% mm 10$ NaCl 1.0 - 0.5 ~ 0.0 4 < 1 6 9 3.0 - -' — 2.5 _ - 6% NaC! 10% N30! 2.0 - w 1.5 - . 1.0 - 0.5 e 0.0 J 1 2 3 4 5 5 7 8 9 Treatment Combined variable effect on chewiness and gumminess of Domiati cheese made from milk containin 5 and 10% NaC. Treatment 1. milk pH 6.2, renneting temperature 39° , and rennet concentration 0.03%; Treatment 2. 6.6, 39 C, and 0.09%; Treatment 3. 6.2, 39°C, and 0.09%; Treatment 4. 6.2, 35°C, and 009% Treatment 5. 6.6, 39°C 0.09%, and 0.02% CaCl; Treatment 6. 6.2, 39°C 0.09%, and 0.02% CaClz; Treatment 7. 6.2, 35°C, 0.03%, and 0.02% Caci,; Treatment 8. 5.2 35°C 0.09%, and 0.02% CaClz; Treatment 9. 5.2, 39°C, 0.03%, and 0.02% Caci,. 138 Tables 1-7 (Appendix I) represent the Analysis of Variance (ANOVA) for interaction between salt (A), CaCl2 (B), renneting temperature (C) and rennet concentration (D). Data statistical analysis indicated that the combination of ABC, ABD, and ACD did not change the cheese modulus of elasticity (Appendix I, Table 1), while the combination of BCD influenced the elasticity modulus (Appendix A, Fig. l). The combinations of AB, AC and BC (Appendix A, Fig. 2)), BD, and CD (Appendix A, Fig. 3) affected the modulus significantly. Combining temperature and CaClz with salt did not affect the elasticity modulus (ABC interaction). CaClz, temperature and rennet concentration noticeably influenced the modulus. The interaction of ABC did not influence the cheese modulus of elasticity, it affected the hardness (Appendix B, Fig. 1). CaCl2 significantly influenced hardness (p < 0.001) at the lowest renneting temperature (35C). At both CaCl2 concentrations (0 and 0.02%), temperature markedly influenced cheese hardness (p < 0.001) in the presence of salt (Appendix B, Fig. 2). BC, and BCD affected the hardness when they interacted with salt (Appendix B, Fig. 3 and 4). Renneting temperature had the highest Significant effect on hardness (AC), followed by rennet concentration (AD), then CaCl2 (AB) when combined with NaCl (Appendix B, Fig. 5). Temperature in any interaction was the most significant factor that influenced cheese hardness (Appendix B, Fig. 6). Rennet concentration was the most important factor that influenced cheese brittleness. Interactions of ABD (Appendix C, Fig. 1), ACD (Appendix C, Fig. 2), BCD (Appendix C, Fig. 3), AD and BD (Appendix C, Fig. 4) exhibited the greatest cheese brittleness. On the contrary, ABC, AC, and AB (Appendix I, Table 1-3) interactions did not increase cheese brittleness. Cheese elasticity was affected with combinations AB, AC, AD (Appendix D, Fig. 5), ABC, ABD, ACD, BCD, BC, BD, and CD (Appendix D, Fig. 1-4 and 6). Temperature and rennet concentration had a synergetic effect on cheese elasticity. Combinations ACD, BCD, AB (Appendix E, Fig. 1~3) influenced cohesiveness of the cheese. Cheese chewiness and gumminess had the same profile with all interaction 139 treatments. A significant influence was shown with treatments ABC, ABD and ACD which gave the highest values followed by BCD (Appendix F and G, Figs.1-4) Also, combinations AB, AC, AD and CD were highly significant treatments that influenced significantly cheese chewiness and gumminess (Appendix F and G, Fig. 5). Treatments BC, BD, and CD influenced cheese gumminess (Appendix G, Fig. 6). Rennet concentration was the most effective factor on cheese chewiness and gumminess and blocked the detrimental effect of NaCl (p < 0.1). Interaction results indicated that the texture parameters of White Soft Domiati cheese made from milk containing 5 and 10 % salt were at a maximum when combining pH 6.6, 39°C renneting temperature, 0.09% rennet concentration, and 0.02% CaCl2 (treatment 5, Fig. 44 to 47). This treatment had a longer clotting time than treatment 6 which had similar texture parameters. Treatment 6 is an alternative for treatment 5 with shorter clotting time. Application of these conditions in combination interaction experiments modified the individual effect of each factor and showed the conditions that can be used for Domiati cheese manufacture to get the highest texture parameters. These parameters were modulus of elasticity (1.5 X 104 N/mz), hardness (1.4 kg), brittleness (0.9 kg), elasticity (48.7%) cohesiveness (97.2%), chewiness (1.4 unitless), and gumminess (2.9 unitless) for cheese made from milk containing 5% salt. For cheese made from milk containing 10% salt: modulus of. elasticity (1.1 X 104 N/mz), hardness (0.9 kg), brittleness (0.6 kg), elasticity (30.9%), cohesiveness (59.5%), chewiness (0.4 unitless), and gumminess (1.2 unitless) were obtained. H) Effect of the Independent Variables on Microstructure of White Soft Domiati Cheese The effect of four important variables (pH, renneting temperature, rennet concentration, and CaCl2 concentration) in Domiati cheese making on the microstructure of the cheese were studied using the scanning electron microscope. 1) Effect of pH Electron micrographs in Fig. 48 show the microstructure of Domiati cheese 140 made from milk containing 5 and 10% salt at pH’s 6.6, 6.2, and 5 .8. The micrographs showed the protein matrix of the cheese containing individual spherical particles sticking together and forming a framework. Micrographs had interspaces which either contained fat globules or whey or both. The paracasein particles were coarse, big, and more pronounced with cheese made at pH 6.6. Cheese prepared at pH 6.6 had fewer holes and protein matrix was compact. Fat globules embedded within the network, which were hard to loose in whey. However, cheese prepared at 6.2 was less compact with more smaller interspaces filled with whey than cheese made at pH 6.6. The paracaseinate particles were smaller and more coarse than the control cheese (made at pH 6.6). Cheese prepared at pH 5.8 demonstrated a loose network due to the solubilization effect of both acid and salt. Decreasing the pH to 5.8, the interspaces were smaller and more numerous. Salted milk produced more dispersed and less coagulable, Na—Ca-caseinate complex. More acid caused a greater increase in the exchanged Ca, thereby producing sodium caseinate, which was more soluble, more dispersed, and had a higher water binding capacity (Fahmi et al., 1973; Roefs, 1985). The aggregated protein materials were in finer moist particles which forms a sponge-like structure network with loose fat globules inside the matrix which escape easily. Fat globules appeared close to each other over the surface of the matrix while some of the protein particles had been adsorbed on their surfaces. The coagulum had fine and weak structure which was hard to ladle and required more caution during processing. That was due to the elimination of sufficient number of calcium bonds which renders the coagulum more permeable (Rajput et al., 1983; Zoon, 1986). ...—.....— 142 These findings agreed with the chemical composition of the cheese (Table 19) which indicated higher moisture content and salt content and less fat and protein when the pH of highly salted milk decreased. Also, the TPA cheese results fit the microstruc- ture data (Fig. 15-21). 2) Effect of Renneting Temperature Three renneting temperatures (35, 39, and 43 °C) were used for manufacturing Domiati cheese from 5 and 10% salted milk. The micrographs in Fig. 49 showed the cheese microstructure that composed of loose spherical casein particle aggregates dispersed in separated bundles in the whey phase formed a loose external surface. The fat globules were discrete in the aqueous phase concentrated at the interface. The spherical casein particle aggregates, formed a coarse, open framework with granular appearance with cheese prepared at 35 °C. The fat globules were entrapped inside the network, and there was interaction between casein micelles and fat globules. The whey was held inside pockets within the protein matrix. 1.011 Fig. 49. Electron micrographs of fresh White Soft Domiati cheese made from milk containirig 5 (left) and 10% (right) NaCl at renneting temgoerature of 35, 39, and 43° . A: fat globule, B: casein granules, C: whey. = 1.0 p. 144 The protein network tended to shrink, which increased the syneresis (Walstra and Jenness, 1984) and yielded more linkage between micelles to form a stronger network with cheese made at 39°C (Kimber et al., 1974). Cheese made at 43°C had a fibrous-like structure, where as number of casein micelles lost their spherical shape, . particularly with cheese made from 10% salted milk due to the solubilization effect of salt. The fat globules appeared on the surface and in the whey pockets from which some can be removed during the process. The effect of temperature may be due to an increase in hydrophobic interactions of casein micelles (McMahon et al., 1984; Dalgleish, 1983). McMahon et al. (1984) reported that hydrophobic bonding may be of crucial importance, as it strongly increases with temperature increasing from 0 to 40°C. The results agree with the chemical composition (Table 20) and TPA of cheese (Fig. 24-30). 3) Effect of Rennet Concentration The SEM micrographs in Fig. 50 illustrate the structure of Domiati cheese made from 5 and 10% salted milk when three rennet (15,000 SU) concentrations, 0.03, 0.06, and 0.09% were used in clotting the milk. Microstructure of cheese was less open containing fewer interspaces filled with whey. Fat globules were trapped tightly within the protein matrix which were hard to lose during processing. These findings were in agreement with the TPA (Fig. 30-36) and chemical composition (Table 21) of the cheese. 0.09% Fig. 50. Electron micrographs of fresh White Soft Domiati cheese made from milk containing 5 (left) and 10% (ngght) NaCl w1th 0.03, 0.06, and 0.09% rennet concentration. A: fat globule, : casein granules, C: whey. Bar = 1.0 [1,. 146 The higher the amount of rennet used, the harder the cheese curd. Syneresis of the curd also increased with rennet concentration (Walstra and Jenness, 1984). The main effect of enzyme concentration may be due to increasing the rate of para-casein production which, in turn, influences casein micelle aggregation (Bohlin et al., 1984; Garnot and Olson, 1982; Dalgleish et al., 1981). 4) Effect of CaCl2 Concentration The effect of three concentrations of CaCl2 (0.02, 0.05, and 0.1% w/v) on microstructure of Domiati cheese made from highly salted (5 and 10% NaCl w/v) milk was studied using SEM. The micrographs (Fig. 51) showed the spherical particles composing the framework of the cheese. The interspaces from which the fat was disappeared were visible. Fat globules were tightly bound to paracasein particles, forming a network. With further addition of CaC12 the cheese structure became more compact. Calcium increased the interaction of the casein micelles which, in turn, entrapped more of the surface fat globules into the curd matrix producing a close structure. This structure may be due to the binding of the added calcium to the casein (Green and Marshall, 1977 ; Van Hooydonk et al. , 1986) which affects the interparticle bond strength (Van Hooydonk and Van der Berg, 1988). Fig. 51. Electron micrographs of fresh. White Soft Domiati cheese made from milk containing 5 (left) and 10% (n ht) NaCl with 0.02, 0.05, and 0.1% CaClz. A: fat globule, B: casein granu es, C: whey. Bar = 1.0 #- 148 The results agree with those of El-Koussy et al. (1974) who found that addition of 0.05 % CaCl2 to heated milk gave a firm curd cheese and of Jen and Ashworth (1970) who obtained the maximum curd tension by adding 10 mM CaC12 without adjusting the pH of milk. The same results were obtained with only 3 mM CaCl2 at constant pH. Since we used high salted milk with constant pH, it would be convenient to use more CaCl2 to get a stronger network structure. Chemical composition (Table 22) and TPA (Fig. 39-45) of the cheese were in concordance with their microstructure. SUMIMARY AND CONCLUSIONS Cheese quality is usually governed by several factors including composition and texture. The arrangement of casein micelles is responsible for the structural properties of cheese. Texture characteristics of cheese generally correlate well with its microstructure, since cheese is formed by joining together of curd particles. In addition, cheese composition influences its texture properties. The rheological properties of White Soft Domiati cheese were studied under different coagulation conditions: pH of milk, renneting temperature, rennet concentra- tion, and addition of CaC12 to cheese milk as well as the combinations of these factors in the presence of two different concentrations of NaCl (5 and 10 %). The following rheological parameters of the cheese were measured: modulus of elasticity, hardness, brittleness, elasticity, cohesiveness, chewiness and gumminess using the Instron Universal Testing Machine (IUTM). The physical structure (microstructure) of cheese was correlated to its rheological properties (texture) by evaluating cheese ultrastructure under identical coagulation conditions. When the pH of milk was decreased most of the texture parameters decreased (p < 0.01, p < 0.001) with the exception of hardness and brittleness. At pH 5 .8 the cheese demonstrated a very weak texture which was difficult to measure. For the combination experiments pH 6.2 was chosen because the coagulation time was greatly decreased at that pH, while some of the important texture parameters (hardness and brittleness) were minimally affected. Increasing the coagulation temperature affected the texture parameters as well as rennet clotting time (RCT). Cheese prepared using a 39°C clotting temperature possessed higher hardness than cheese prepared at 35°C. The highest hardness was found with cheese prepared at 43°C from milk containing 5% NaCl. All of the texture parameters increased significantly (p < 0.01) when the clotting temperature was 149 150 increased. The clotting temperature of 39°C was chosen for combination experiments because no significant differences was found in hardness, and springiness when the temperature was increased from 39 to 43°C. Additionally, cheese had the lowest cohesiveness, chewiness, and gumminess when prepared at 43°C. Rennet concentration was the most important variable affecting the texture and body of the cheese. Salt had no significant effect on cheese texture parameters when prepared with 0.09% rennet concentration. So, increasing rennet concentration opposed the harmful effect of NaCl on cheese texture. All the texture parameters increased (p < 0.01) with increasing rennet concentration. A rennet concentration of 0.09% (15,000 SU) was chosen to increase the texture parameters of cheese prepared from highly salted milk. Calcium chloride was another factor which alters texture of the cheese made from salted milk. Addition of CaCl2 to milk significantly increased the cheese modulus of elasticity, hardness and springiness (p < 0.01). The force required to fracture the cheese increased (p < 0.01) with increasing CaCl2 concentration. An increase in cohesiveness, chewiness, and gumminess (p < 0.01) were noticed with increasing CaC12 concentration in milk. To find the conditions that produce Domiati cheese with maximum texture parameters, nine combinations of milk pH, clotting temperature, amount of rennet and CaC12 concentration were tested. Cheese was made from milk containing 5 and 10% NaCl. Maximum texture parameters for Domiati cheese were obtained with a milk pH 6.6, 39°C renneting temperature, 0.09% (15,000 SU) rennet concentration, and 0.02% added CaC12 (combination 5). Alternatively, combination 6 (milk pH 6.2, 39°C renneting temperature, 0.09% rennet concentration, and 0.02% CaClz) gave close texture parameters to combination 5 with shorter clotting time. The highest texture parameters obtained with combination 5 were: modulus of elasticity (1.5x10‘ N/m2 , hardness (1.4 Kg), brittleness (0.9 Kg), elasticity (48.7 %), cohesiveness (97.2 %), chewiness (1.4 unitless), and gumminess (2.9 unitless) for cheese 151 made from milk containing 5% NaCl. For cheese made from 10% NaCl: modulus of elasticity (1.1x10“ N/mz), hardness (0.9 Kg), brittleness (0.6 Kg), elasticity (30.9%), cohesiveness (59.5%), chewiness (0.4 unitless), and gumminess (1.2 unitless) were obtained. The microstructure of Domiati cheese prepared under the aforementioned conditions was also studied using scanning electron microscopy. Electron micrographs indicated that the particles were less coarse, bigger and more pronounced with cheese samples made with milk at pH 6.6, than at pH 6.2. Cheese made with milk at pH 5.8 demonstrated looser network. Also the interspaces between the protein matrix were numerous and smaller at pH 5.8 than at pH 6.6 and 6.2. Cheese structure was less open, containing fewer interspaces filled with whey and less coarse with increasing the clotting temperature, rennet concentration and CaCl2 concentration. Addition of NaCl to milk before renneting showed the following effects on the milk and the resultant Domiati cheese. The pH of milk decreased from 6.64 to 6.13 when the concentration of NaCl increased from 0 to 14%. Very little change was observed at NaCl concentration above 6%. Also, addition of up to 6% NaCl induced a significant increase in soluble Ca (p < 0.001) from 463 ppm to 575.7 ppm in the milk serum. However, adding more NaCl (up to 14%) caused a significant decrease (p < 0.001) in soluble Ca from 575.7 ppm to 446.3 ppm. The amount of Ca released increased with increasing the amount of NaCl up to 6% in milk with no measurable increase thereafter. , Casein micellar hydration decreased from 3.2 g H20/ g protein with no added NaCl to 1.69 g 1H20/ g protein with 14% NaCl addition. The casein micellar hydration did not change much between 0-2% NaCl, beyond which the decrease in hydration was noticeable (p < 0.001). The amount of water lost/ g protein by adding 14% NaCl was almost 50% of the control. Scanning electron microscopy was applied to visualize the change in shape and arrangement of the casein micelles following the addition of NaCl to milk at 0, 5, 10, and 15%. Electron micrographs were taken directly, 24, 48, and 72 hr after salt 152 addition. Casein micelles showed a spherical shape with different sizes for unsalted milk. However, addition of 5% NaCl to milk caused few aggregated micelles. When. NaCl was increased to 10% , the casein micelle shape became irregular and some micelles aggregated. The aggregation of the micelles was increased with increasing NaCl concentration. Micelles aggregation also increased with the time after salt addition at all concentrations. As a result, salting out of the protein occurred even at as low as 5% NaCl addition to milk, depending on the casein micelle size and the time of exposure to salt. Glycomacropeptide (GMP) released after the action of rennet on k-casein were followed by HPLC after renneting times of 0, l, 3, 10, 40, 70 and 120 min for unsalted (control) and highly salted milk (5 and 10% NaCl w/v). Data indicated no signs of GMP after 3 min of renneting in both 5 and 10% salted milk, while GMP was detected after 1 min of renneting with unsalted milk (control). The GMP peak started to appear after 10 min in both salted milks. After 40 min incubation, GMP ceased to increase with the control but continued to increase until 120 min in both Sand 10% salted milk. Addition of NaCl to milk decreased the released GMP by 43% with 5% salted milk and 61 % with 10% salted milk. The action of independent variables of coagulation against the deleterious effect of NaCl on rennet clotting time (RCT) was measured visually and instrumentally. As the pH of the milk decreased, the coagulation of both salted milks increased to a minimum RCT (pH 6.2) beyond which RCT increased with the formation of a very fine coagulum (pH 5.8). Also, increasing the renneting temperature from 35 to 39°C caused a decrease in RCT than from 39 to 43°C. In addition, RCT was decreased greatly by increasing the concentration of rennet and CaClz. Interaction of these independent variables significantly decreased the RCT of milk. Measuring the RCT instrumentally was less sensitive and accurate than by visual observation. The accuracy of the instrumental method can be increased by using a smaller loading cell (less than 50 N) or increase the diameter of the plunger. APPENDICES APPENDIX "A" Significant mean interactions for cheese modulus of elasticity. 155 NO CaClZ 1.1 — S o 5 c 09 '- .9. it o *5 0.7 - 05 a r . 0.03 f 0.09 Rennet Cone. (%) 0.02% CaCl2 1.2 390 5 2" 1.1 - g 350 § 2 1 7 5 0-9 I I 1 0.03 0.09 Rennet Gone. (96) Fig. 1. Significant mean interactions among CaClz, temperature and rennet concentra- tion (BCD) for cheese modulus of elastrcrty. 155 NO CaClz 1.1 — 390 g / 8 o *5 5.7 - 0.5 , r I 0.08 . 0.09 Rennet Cone. (%) 0.02% CaCl2 1.2 390 5 g 1.1 r g ‘ 350 2 1 - . E 09 I I 1 0.03 0.09 Rennet Gone. (96) Fig. 1. Significant mean interactions among CaClz, temperature and rennet concentra- tion (BCD) for cheese modulus of elastrcrty. 156 1.4 5 01 0 ‘ 1.2 - $3 2 C .9 t . .fl _ L- 3 as» E: on r r 2 CaCl2 (961 1.3 C G 0 2 C .2 I - E E 10% N301 0 4.. E / 0'7 1 r r Temperature (C) ‘ 7‘ N d O u. C. ' p o 0.8' lnté‘ractlon Mean . , 5.029. Cam (1% 03 $0 07 Fig. 2. Significant mean int temperature (AC), and CaCl2 elasticity. . . _ . 5.9 Temperature (G) AB AC eractions between NaCl and CaCl2 (AB), NaCl and and temperature (BC) for cheese modulus o 1.2 Interaction Mean. 0.6 1.2 . interaction Mean 0.? Fig. 3. Significant mean interactions between C and temperature and rennet concentration ( 157 08' also 555 Rennet Cone. (%) 1.1 " 0.9 08' 396 /35C coo ' ' -' - 000 Rennet Cone. (%)- CD aClz and rennet concentration (131)), CD) for cheese modulus of elastrcrty. APPENDIX "B" Significant mean interactions for cheese hardness. 159 5% NaCl 2.4 . E 2 r: 2 ” .2 {no i g 1.5- 1.2 r r r e . 35 39 Rennet Gone. (95) 10% NaCl 1.6 E 2 t: .9 an E 2 1.2~ 5 1 I l i l i 35 39 Temperature (C) Fig. 1. Significant mean interactions among NaCl, CaClz, and temperature (ABC) for cheese hardness. ' 160 NO CaCl2 1.1 ~ .9 2 c 0.9 ‘ .2 8 *6 E 0.7 - 0:5 1 I l 0.08 0.09 Rennet Gone. (96). 1 2 0.02% CaCl2 890 8 o x M :- g 356 z t3 8 1 ~ .5 0.9 T i F 0.03 0.09 Rennet Gone. (96) Fig. 2. Significant mean interactions among CaClz, temperature, and rennet concentra— tion (BCD) for cheese hardness. 161 5% NaCi - A 2.e~ 390 t: . 8 2.4- 2 c . .2 8 9" It. 350 8 S 1.6' / 1.2 r , r 0.03? 0.09 Rennet Gone. (95) 10% NaCi 1.7 . ‘ - 390 g 2 ~ 35c : 3 13- 2 E 1.1 - 0.9 r r r 0.08 0.09 Rennet Cone. (%) Fig. 3. Significant mean interactions among NaCl, temperature, and rennet concentra- tion (ACD) for cheese hardness. 2.4 - Interaction Mean '0) I 1.2 0.55 I 0.59 Rennet Gone. (96) 10% NaCi -_s h 1 Interaction Mean - it; I 0.8 r r 0. 03 0.09 Rennet Gone. (96) Fig. 4. Si nificant mean interactions among NaCl, CaClz, and rennet concentration (63D) for cheese hardness. Fig. 5. 163 2.4 interaction Mean /$N.c‘ w 08 ‘ CaCi2 (is) 3 r‘ N on is) l 7‘ ‘ 1 Interaction Mean w etc“ g'i- 1 f Temperature (C) 39 d O r 1.6 1' interaction Mean ‘1 41* 0,0 0.0 86 Significant mean int ture (AC), and CaCl2 r 39 Temperature (C) and temperature (BC) for cheese hardness. AB AC BC eractions among NaCl and CaCl2 (AB), NaCl and tempera- 164 2,4 5 etc" 0 21- fi” 2 C .2 151 AD 4.. o a: I- 2 1.2r 5 as r , r 0.03 0.09 Rennet Conc. (%) g 2 g re» 8 1a- : ' ED 0 G 1.4r ‘- 2 s 1.2- .-_.- . r- . i 0.03 0.09 Rennet Conc. (%) 2.3 . sec 5 an o 2, 1.9- :: CD 3% "7' sec § 2 S 1.8- 1.1 r r O. 03 O.“ Rennet Conc. (%) Fig. 6. Significant mean interactions between NaCl and rennet concentration (AD), ' perature and rennet concentra- CaCl and rennet concentration (BD), and tem tion CD) for cheese hardness. APPENDIX "C " Significant mean interactions for cheese brittleness. 5% SALT 1.7 5 o 2 C .2 *5 1.5 . E 2 5 1.3 r I 1 0.03 0.09 Rennet Cone. (%) 10% NaCi 1.2 C d c 1 ~ 2 .5 ii 2 0.8 r 5. 0.5 . 0.03 0.09 Rennet Gone. (15) Fig. 1. Significant mean interactions among NaCl, CaClz, and rennet concentration ( D) for cheese brittleness. 167 5% N30! 1.8 1.6“ 1.5- Interaction Mean 1.3- 1.2 , T 0.08 0.09 Rennet Cone. (%) 105 NaCl 1.4 Interaction Mean Rennet Gone. (96) Fig. 2. Significant mean interactions among NaCl, temperature, and rennet concentra- tion (ACD) for cheese bnttleness. 168 NO CaCi2 1.5 5 1'4 _ 390 i C / 350 0 T6 1.2 ~ _ E .9 .5 1 a 0-8 I T l 0.03 0.09 Rennet Gone. (95) 0.02% CaCi2 1.5 390 5 o 1.4 ~ 3 I ,C as 350 a 1.2 — 2 / 5. 1 r 1 I 0.03 0.09 Rennet Gone. (95) Fig - 3. Significant mean interactions among CaClz, temperature, and rennet concentra- tion (BCD) for cheese brittleness. 169 1.7 5% NaCl 1.5 ‘ 1.3- AD , 1 _ 1011. NaCi interaction Mean 0.9 ~ I Rennet Gone. (96) 0.7 , r 0.08 0.09 1.4 1.3“ 1.2- Interaction Mean 1.1- ' 0.03 0.09 Rennet Cone. (%) . O o o e e d F1 . 4. SI mficant mean interactions between NaCl and rennet concentration (AD) an g CagClz and rennet concentration (ED) for cheese brittleness. APPENDIX "D" Significant mean interactions for cheese elasticity. 171 5% NaCl 42 - \2 g 0.02% 0‘0 g 38 r C .2 8 34 r- ; 03012 E 80 “o 26 I I I I T 55 89 Temmrature (C) 10% NaCi 27 26L Interaction Mean I\) C) I 21 I I I 39 I -1 1 9 Temperature (C) Fig. 1. Significant mean interactions among NaCl, CaClz, and temperature (ABC) for cheese elasticity. 34- 30- lnteractlon Mean 26* 22 I I 0.03 0.09 Rennet Gone. (96) 10% NaCl 28 20' v. Interaction Mean 24- I 16 Fig. 2. 3:5? I I 0.03 O. 09 Rennet Conc. (%) ficant mean interactions among NaCl, CaClz, and rennet concentration D) for cheese elast1C1ty. 173 5% NaCl 44 390 40 “ C 3 - 350 2 36 _ .5 ‘6 E 32 - 2 E 28 - 24 I I I 0.03 0.00 Rennet Gone. (96) 10% NaCl' 390 26 _ / 3 C 5C 8 2 § 22 ‘ I “ § 2 EJ 18 - 1 4 F I T 0.03 0.09 Rennet Conc. (%) Fig. 3. SignifiCant mean interactions among NaCl, temperature, and rennet concentra- tion (ACD) for cheese elasticity. 174 NO CaCi2 84 39C 5 30 — / O 356 I C 25 h .2 3 E! 22 - 2 E 18 ‘ 14 I I I 0.03 0.09 Rennet 9000. (%) 0.02% CaCl2 38 39C C , 3 a 34 ' g 35c § 8 5 80 ‘ 26 g l I ‘ 0.03 0.09 Rennet Gone. (‘5) Fig. 4. Significant mean interactions among CaClz, temperature, and rennet concentra- tion (BCD) for cheese elast1c1ty. 175 42 c \ ask .0 8 ch“ 2 3‘. i: 3 so '5 2"" 10$ NaCi 4.: .5 22» 13 , r T ' CaCi2 (%) 2 5 car 5* “30‘ 0 2 3“ c .2 30' 4.: g 20- 10$ N361 .E 22- 18 j , j r 39 36 Temperature (C) Interaction Mean 8 18 Fig. 5. Significant mean interactions between NaCl and CaCl2 (AB temperature (AC), and NaCl and rennet concentration (AD elastic1ty. 0.. can 03 . Rennet Conc. (%) AB AC AD , NaCl and 3 for cheese _ c\2 g as 0.0” 0’ 2 . : 3°” 2 ‘6 27- BC «I 3 E 24- 21 I r 77 r r 36 39 C 34 a o 2 so, .5. ED 3‘ 2a- b ‘2 22- 18 , . T 0.03 000 ‘ Rennet Cone. (%) ee . sec C as g “2” 350 CD 5; § 2 2.. 5 20 v r u one 0.0:: Rennet Conc. (%) Fig. 6. Significant mean interactions between CaC12 and temperature (BC), CaCl and rennet concentration (BD), and temperature and rennet concentration (CD for cheese elasticity. APPENDIX "E" Significant mean interactions for cheese cohesiveness. interaction Mean interaction Mean Fig. 1. 178 5% NaCi 7O .- 60- 50- 390 350 40 I I 0.03 0.59 Rennet Gone. (95) 10% NaCi 58" 48- 88- 35C 39C 28 SignifiCant mean interactions among NaCl, temperature, and rennet concentra- 0.53 I 0.59 Rennet Gone. (95) tion (ACD) for cheese cohesiveness. 179 N0 CaCi2 72 I 62 62*- interaction Mean 32 , , 0.03 0.09 Rennet Gone. (96) 0.02% CaCiZ 390 I 70 so - . "* 4 350 60F 40 interaction Mean T“ 30 i i O. 08 O. 09 Rennet Cone. (%) Fig. 2. Significant mean interactions among CaClz, temperature, and rennet concen- tration (BCD) for cheese cohesweness. --——— —-—__. m ‘ia 180 7O 62‘ I 58 54~ I 60 Interaction Mean 38 r T ’ CaCi2 (%) Fig. 3. Significant mean interactions between NaCl, CaCl,z (AB) for cheese co esiveness. APPENDIX "F " Significant mean interactions for cheese chewiness. 182 5% N30! 08 ~ 5 o 2 5 0.6 - § 2 E 0.4 r 0-2 r l r r i 35 39 Temperature (C) 10% NaCl 0.2 \‘2 03° 092* NO 0302 interaction Mean 0 a ..J I I T 0.1 00 CD 35 Temperature (C) Fig. 1. Significant mean interactions among NaCl, CaClz, and temperature (ABC) for cheese chewiness. 0.9 - 0.7 ~ 0.5 _ interaction Mean 0.1 I I I O. 03 0.09 Rennet Cone. (as) 10%“ NaCi 0.26 - 0.18~ interaction Mean 0.02 Fig. 2. Sig? I I I 0.03 O. 09 Rennet Gone. (is) fiéant mean interactions among NaCl, CaClz, and rennet concentration D) for cheese chewmess. 184 5% NaCi 1 ' 390 5 o 2 0.7 - § 350 § / 2 0.4 ~ E 01 I I i 0.03 0.09 Rennet Cone. (%) 10% NaCi 0.3 390 C °' 350 O i. 2 0.2 c _ .2 § 8 0.1 — _ 5 e O I I I 0.03 0.09 Rennet Cone. (%) Fig. 3. Significant mean interactionsoamong NaCl, temperature, and rennet concentra- tion (ACD) for cheese chewmess. 185 NO CaCiz 0.6 390 E 5 0.4 - c: 350 .2 § 2 0.2 — 5 o . r . 0.03 0.09 Rennet gone. (96) 0.02% CaCi2 07— 39C 5 é’ .5 0.5 — 360 § 3 0.3 — E 0.1 . , . 003 009 Rennet Gone. (96) Fig, 4. Significant mean interactionscamong CaClz, temperature, and rennet concentra- tion (BCD) for cheese chewmess. 186 interaction Mean Interaction Mean >w .6th. n on: >0 0.0 . For. C a 0.0 e M 8. n 0... IO- .Nq. o... r on . 20 Owen m 0903. ii m O.N r \76\ 1 m o . . . o . i .. N 0.8 0.00 Own.» 33 33:3 0020. $8 ob ob , U 0 >0 owes. . m men 06 .. 0.09} e 9.0 u M 090/9 m o; r man 0... r .I . $0 t. C m o.» . MW ow m o . W . o 98 98 a... #33388 ADV $252 0030. 33 En. m. mmmamoma 58: $83233 vegan: 2mg 85 Omar 35. 2%: Ba 833888 99. 2%: ea 3.53 0 83:2 8:09.530: 88 men 25an 883330: 39. Ba 833388 85 0:95:89 APPENDIX "G" Significant mean interactions for cheese gumminess. 188 5% NaCi 2 30‘2 r: 0.07" G G O I 1.6 — .5 0’09 3 0'8 T T i i r 35 89 Temperature (C) 10% NaCl 0.8 0.02% OaG|2 c / fl 0 I § 0.6 — “0 036‘2 h 2 E 0.4 | I l I I 35 39 Temperature (0) Fig.1. Significant mean interactions among NaCl, CaClz, and temperature (ABC) for cheese gummmess. 2.2 interaction Mean 2 63 I I _a I 0.6 . e . 0.03 g 0.09 Rennet Gone. (96) 10% NaCi 1 _ 5 o 0.8 L' 2 C o z 0.6 - § 2 S 0.4 - 0.2 i i T 0.03 0.09 Rennet Conc. (%) Fig. 2. Si nificant mean interactions among NaCl, CaClz, and rennet concentration ( D) for cheese gumminess. interaction Mean interaction Mean Fig. 3. 190 5% NaCi 25 39C 2.2 - 1.9 - 1-6 - 350 1.3 '- 1 - 0.7 r n u 0.03 0.09 Rennet Conc. (%) 10% NaCi 1 0.8 b 0.6 - O.4 . Rennet Cone. (%) Significant mean interactions among NaCl, temperature, and rennet concentra- tion (ACD) for cheese gumminess. 191 NO CaCi2 390 1.4 - 5 0 35C 5 i: 1 P .9. E 0.6 - 0.2 I I I 0.03 ' 0.09 Rennet Cone. (%) 0.02% CaCiZ 1.8 - 390 c. a i 1.4 - r: 350 .9. § 2 1 - E. 0.6 . 0.58 0.09 Rennet Conc. (%) Fig. 4. Significant mean interactions among CaClz, temperature, and rennet concen- tration (BCD) for cheese gummmess. Fig. 5. 192 LB \ 5 5'!- “‘6 g 1.4 » C .9. , 4.; o S 1015 NaCi ,2 ca — .9. 0.2 r f r 1 CaCi2 (%) 2 1.8 C ‘0‘ cu 51b“ g C .9 is? " 3 10% NaCi E oe- ;_ ' 0.2 r A f f . 35 09 Temperature (C) 5 2 I o S c 1.4 * .9 ‘6 a h 0.6 r 8 E 0.2 r r 1 0CD Significant mean interactions between NaCl and CaClz ( temperature (AC), and NaCl and rennet concentration (AD for cheese gumminess. coo Rennet Cone. (%) AB), AB AC AD NaCl and Fig. 6. 193 5 1.8- o 2 .5- 1.1- ii 5 i- 2 0.0 5 07 r 69 L6 0 2 1.2~ C .9 ‘6 2 aa- 9 .9 0.4 1 r 1 one can Rennet Cone. (%) c 1.8 390 a: O 2 ,2. sec C .9 4-0 08’ 8 i- 2 (14- .9 O ' r 0m oéo Rennet Cone. (%) CD Significant mean interactions between CaCl2 and temperature (BC), CaCl and rennet concentration (BD), and temperature and rennet concentration (CD for cheese gumminess. APPENDIX "H" Anal sis of variance for chemical composition and texture profile of cheese as affected y pH, renneting temperature, rennet concentration, and CaCl2 concentration. 195 Table 1. Anal sis of variance for chemical composition of white soft cheese as affected by p of milk Source DF Sum Mean P value Probability of squares square Yield NaCl (A) 1 32.67 32.670 142.04 0.0001 pH (B) 2 42.21 21.103 91.75 0.0001 AB 2 87.62 43.810 190.48 0.0001 Error 6 1.38 0.230 Moisture NaCl (A) 1 69.12 69.120 864.00 0.0001 pH (B) 2 47.39 23.693 296.17 0.0001 AB 2 2.24 1.120 14.00 0.005 Error 6 0.48 0.080 Fat NaCl (A) 1 16.33 16.333 204.17 0.0001 pH (B) 2 15.41 7.703 96.29 0.0001 AB ' 2 0.61 0.303 3.79 0.086 Error 6 0.48 0.080 Protein NaCl (A) 1 25.23 25.230 315.37 0.0001 pH (B) 2 10.75 5.373 67.17 0.0001 AB 2 0.32 0.160 2.00 0.216 Error 6 0.48 0.080 Salt NaCl (A) 1 48.00 48.000 600.00 0.0001 pH (B) 2 0.41 0.203 2.54 0.158 AB 2 0.06 0.030 0.37 Error 6 0.48 0.080 DF = degrees of freedom Correlation coefficient: Yield = 0.993 and 0.44 for 5 and 10 % NaCl, respectively. Moisture = 0.993 and 0.981 for 5 and 10 % NaCl, respectively. Fat = 0.988 and 0.993 for 5 and 10 % NaCl, respectively. Protein = 0.989 and 0.994 for 5 and 10 % NaCl, respectively. 196 Table 2. Analysis of variance for texture parameters of white soft Domiati cheese as affected by pH of milk. Source DF of §d113res m F value Probability Modulus of elasticity NaCl (A) 1 10655905 .33 10655905 .333 676.52(a) 0.0001 pH (B) 2 5947185 8. 17 29735929083 1887. 86(a) 0.0001 AB 2 757241.17 378620.583 24.04(b) 0.001 Error 6 94507.00 15751.167 Hardness NaCl (A) 1 0.212 0.212 42.6229(a) 0.0006 pH (B) 2 0.465 0.233 46.7925 (a) 0.0002 AB 2 0.035 0.017 3.4783(e) 0.0993 Error 6 0.030 0.005 Brittleness NaCl (A) 1 0.714 0.714 85 .4773(a) 0.0001 pH (B) 2 0.065 0.032 3.8757(d) 0.0831 AB 2 0.034 0.017 2.0317(e) 0.2119 Error 6 0.050 0.008 Elasticity NaCl (A) 1 81.797 81.797 208.4408(a) 0.00001 pH (B) 2 92.278 46.139 1 17 .5735 (a) 0.00001 AB 2 35 .408 17.704 45 . 1 147(a) 0.0002 Error 6 2. 355 0.392 Cohesiveness NaCl (A) 1 603.501 603.501 80.6730(a) 0.0001 pH (B) 2 1050.705 525.352 70.2265(a) 0.0001 AB 2 264.172 132.086 17.6566(c) 0.0031 Error 6 44.885 7.481 Chewiness NaCl (A) 1 0.013 0.013 85.5293(a) 0.0001 pH (B) 2 0.018 0.009 56.1394(a) 0.0001 AB 2 0.008 0.004 25 . 8993(b) 0.0011 Error 6 0.001 0.0002 Gumminess NaCl (A) 1 0.171 0.171 69.2983(a) 0.0002 pH (B) 2 0.263 0.132 53.2348(a) 0.0002 AB 2 0.073 0.037 14.7613(c) 0.0048 Mr 6 0.015 0.00L DF = degrees of freedom (a) = significant (p < 0.001) (b) = significant (p < 0.005) (c) = significant (p < 0.025) (d) = significant (p < 0.1) (e) = significant (p < 0.25) 197 Table 3. Analysis of variance for chemical composition of white soft cheese as affected by renneting temperature. Source DF Sum Mean F value Probability of squares square Yield NaCl (A) 1 129.36 129.3631 617.04 0.0001 Temp. (B) 2 43.45 21.723 271.54 0.0001 AB 2 2.01 1.003 12.54 0.007 Error 6 0.48 0.080 Moisture NaCl (A) l 29.87 29.875 393.28 0.0001 Temp. (B) 2 11.29 5.646 74.32 0.0001 AB 2 0.01 0.006 0.08 Error 6 0.46 0.076 Fat NaCl (A) 1 12.00 12.000 150.00 0.0001 Temp. (B) '2 2.29 1.143 14.29 0.005 AB 2 0.14 0.070 0.87 Error 6 0.48 0.080 Protein NaCl (A) 1 27.79 27.786 364.16 0.0001 Temp. (B) 2 2.33 1.667 21.84 0.001 AB 2 0.05 0.023 0.30 Error 6 0.46 0.076 Salt NaCl (A) 1 44.85 44.853 560.67 0.0001 Temp. (B) 2 0.13 0.063 0.79 AB 2 0.01 0.003 0.04 Error 6 0.48 0.080 DF = degrees of freedom Correlation coefficient: Yield = 0.998 and 0.970 for 5 and 10 % NaCl, respectively. Moisture = 0.964 and 0.975 for 5 and 10 % NaCl, respectively. A Fat = 0.99 and 0.976 for 5 and 10 % NaCl, respectively. Protein = 0.987 and 0.998 for 5 and 10 % NaCl, respectively. 198 Table 4. Analysis of variance for texture parameters of white soft Domiati cheese as affected by renneting temperature. Probability Source DF Sum Mean F value of squares square . Modulus of elasticity NaCl (A) 1 915253333 9152533333 105 .31(a) 0.0001 Temp. (B) 2 1357961150 6789805.750 78.12(a) 0.0001 AB 2 158629017 793145.083 9.13(c) 0.015 Error 6 521468.00 86911.333 Hardness NaCl (A) 1 1.506 1.506 230.0599(a) 0.00001 Temp. (B) 2 0.730 0.365 55 .78 19(a) 0.0001 AB 2 0.200 0.100 15 .2920(c) 0.0044 Error 6 0.039 0.007 Brittleness NaCl (A) 1 2.435 2.435 896.4974(a) 0.00001 Temp. (B) 2 0.909 0.455 167.3640(a) 0.00001 AB 2 0.344 0.172 63.2742(a) 0.0001 Error 6 0.016 0.003 Elasticity NaCl (A) 1 434.577 434.577 184.9265 (a) 0.00001 Temp. (B) 2 366.368 183.184 77.9265 (a) 0.0001 AB 2 16.572 8.286 Error 6 14.100 2.350 Cohesiveness NaCl (A) 1 1268.190 1268.190 241 .4882(a) 0.00001 Temp. (B) 2 545. 154 272.577 51.9040(a) 0.0002 AB 2 160.230 272.5 77 51 .9040(a) 0.0002 Error 6 31.509 5.252 ChewineL NaCl (A) 1 0.145 0.145 118.5226(a) 0.00001 Temp. (B) 2 0.019 0.010 7.7949(d) 0.0215 AB 2 0.011 0.006 4.6346(d) 0.0607 Error 6 0.007 0.001 Gumminess 0 00001 NaCl A 1 0.833 0.833 15 6.5728(a) . Temp. ((b)) 2 0.038 0.019 3.5704(e) 0.0952 AB 2 0.035 0.018 3.3230(e) 0.1068 jigor 6 0.032 0.005 DF = degrees of freedom (a) = significant (p < 0.001) (c) = significant (p < 0.025) (d) = significant (p < 0.1) (e) = significant (p < 0.25) 199 Table 5. Analysis of variance for chemical composition of white soft cheese as affected by rennet concentration. Source DF Sum Mean F value Probability of squares square Yield NaCl (A) 1 89.65 89.6531 120.67 0.0001 R.C. (B) 2 22.73 11.363 142.04 0.0001 AB 2 1.89 0.943 11.79 0.008 Error 6 0.48 0.080 Moisture NaCl (A) 1 12.00 12.000 150.00 0.0001 .R.C. (B) 2 7.65 3.823 47.779 0.0001 AB 2 0.26 0.130 1.63 0.272 Error 6 0.48 0.080 Fat NaCl (A) 1 2.61 2.613 32.67 0.001 R.C. (B) 2 2.91 1.453 18.17 0.002 AB 2 0.03 0.013 0.17 Error 6 0.48 0.080 Protein NaCl (A) 1 35.36 35.363 442.04 0.0001 R.C. (B) 2 1.68 0.840 10.50 0.010 AB 2 0.11 0.053 0.67 Error 6 0.48 0.080 ’ Salt NaCl (A) 1 42.56 42.563 532.04 0.0001 R.C. (B) 2 0.18 0.090 1.12 0.384 AB 2 0.01 0.003 0.04 Error 6 0.48 0.080 0.04 R. C. = Rennet concentration DF = degrees of freedom Correlation coefficient: _ Yield = 0.972 and 0.999 for 5 and 10 % NaCl, respectively. Moisture = 0.999 and 0.989 for 5 and 10 % NaCl, respectively. Fat = 0.982 and 999 for 5 and 10 % NaCl, respectively. Protein = 0.967 and 0.997 for 5 and 10 % NaCl, respectively. 200 Table 6. Analysis of variance for texture parameters of white soft Domiati cheese as affected by rennet concentration. Source DF of fiflr es %fé‘r’é P value Probability Modulus of elasticity NaCl (A) 1 2915330133 29153301333 249.32(a) 0.00001 R.C. (B) 2 4220650467 21103252.333 180.48(a) 0.00001 AB 2 924802.67 462401.333 3.95 ((1) 0.08001 Error 6 701575.00 116929.167 Hardness NaCl (A) 1 0.323 0.323 138.7266(a) 0.00001 R.C. (B) 2 1.908 0.954 410.0516(a) 0.00001 AB 2 0.090 0.045 19.3209(b) 0.0024 Error 6 0.014 0.002 Brittleness NaCl (A) 1 0.377 0.377 190.7634(a) 0.00001 R.C. (B) 2 1.060 0.530 268.2663(a) 0.00001 AB 2 0.078 0.039 19.7999(b) 0.0023 Error 6 0.012 0.002 Egsticitv NaCl (A) 1 110.877 110.877 816.3089(a) 0.00001 R.C. (B) . 2 558.346 279.173 2055.3470(a) 0.00001 AB 2 23.550 11.775 86.6897(a) 0.00001 Error 6 0.815 0.136 Cohesiveness NaCl (A) 1 2736.437 2736.437 12327.3637(a) 0.00001 R.C. (B) 2 10250.716 5125.358 23089.2023(a) 0.00001 AB 2 237.237 118.618 534.3637(a) 0.00001 Error 6 1.332 0.222 Chewinessf NaCl (A) 1 0.319 0.319 113.9312(a) 0.00001 R.C. (B) 2 1.198 0.599 214.0626(a) 0.00001 AB 2 0.037 0.019 6.6572(d) 0.0300 Error 6 0.017 0.003 Gum miness NaCl (A) 1 1.807 1.807 76.1546(a) 0.00001 R.C. (B) 2 6.319 3.160 133.1602(a) 0.00001 AB 2 0.168 0.084 3.5396(e) 0.0965 flor 6 0.142 0.024 R. C. = Rennet concentration DF = degrees of freedom (a) = significant (p < 0.001) (b) = significant (p < 0.005) (d) = significant (p < 0.01) (e) = significant (p < 0.025) 201 Table 7. Analysis of variance for chemical composition of white soft cheese as affected by a ding CaCl2 to milk. Source DF Sum Mean F value Probability of squares square Yield NaCl (A) 1 88.56 88.5631 107.04 0.0001 CaCl2 (B) 2 7.55 3.773 47.17 0.0001 AB 2 0.35 0.173 2.17 0.195 Error 6 0.48 0.080 Moisture NaCl (A) 1 16.33 16.333 58.33 0.0001 CaCl2 (B) 2 3.09 1.543 5.51 0.043 AB 2 1.65 0.823 2.94 0.128 Error 6 1.68 0.280 Fat NaCl (A) 1 8.00 8.003 100.04 0.0001 CaC12 (B) 2 2.01 1.003 12.54 0.007 AB 2 0.05 0.023 0.29 Error 6 0.48 0.080 Protein NaCl (A) l 13.23 13.230 165.38 0.0001 CaCl2 (B) 2 0.21 0.103 1.29 0.341 AB 2 0.06 0.030 0.38 Error 6 0.48 0.080 Salt NaC18 (A) 1 44.08 44.083 551.04 0.0001 CaCl2 (B) 2 0.01 0.003 0.04 AB 2' 0.05 0.023 0.29 Error 6 0.48 0.080 DF = degrees of freedom Correlation coefficient: Yield = 0.756 and 0.955 for 5 and 10 % NaCl, respectively. Moisture = 0.998 and 0.585 for 5 and 10 % NaCl, respectively. Fat = 0.999 and 0.832 for 5 and 10 % NaCl, respectively. Protein = 0.991 and 0.756 for 5 and 10 % NaCl, respectively. NaCl = 0.929 and 0.866 for 5 and 10 % NaCl, respectively. 202 Table 8. Analysis of variance for texture parameters of white soft Domiati cheese as affected by adding CaCl2 to milk. Source DF 0 f $3er es slqlfueaarne F value Probability Modulus of elasticity NaCl (A) 1 11620830408 116208304083 463 . 76(a) 0.00001 CaCl2 (B) 2 12041750217 60208751083 340.28(a) 0.00001 AB 2 6127396317 30636981 .583 122.27(a) 0.00001 Error 6 150346950 250578.520 Hardness NaCl (A) 1 1.964 1.964 520.7894(a) 0.00001 CaClz (B) 2 3.556 1.778 471.5174(a) 0.00001 AB 2 0.306 0.153 40.6117(a) 0.0003 Error 6 0.023 0.004 Brittleness NaCl (A) 1 0.820 0.820 675.9842(a) 0.00001 CaCl2 (B) 2 4.824 2.412 1988.6938(a) 0.00001 AB 2 0.120 0.060 49.3054(a) Error 6 0.007 0.001 Elasticity NaCl (A) 1 137.689 137.689 . 729.0180(a) 0.00001 CaC12 (B) 2 193.270 96. 635 5 1 1 .6497 (a) 0.00001 AB 2 26.796 13.398 511.6497(a) 0.00001 Error 6 1.133 0.189 Cohesiveness NaCl (A) 1 1733.945 1733.945 3956. 1371 (a) 0.00001 CaCl2 (B) 2 6699.352 3349.676 7642.5601(a) 0.00001 AB 2 270.738 135.369 308.8552(a) 0.00001 Error 6 2.630 0.438 Chewiness NaCl (A) 1 1.088 1.088 2460.7321(a) 0.00001 CaCl2 (B) 2 2.286 1.143 25 84.2863 (a) 0.00001 AB 2 0.226 0.113 256.0583(a) 0.00001 Error 6 0.003 0.0004 Gumminess NaCl (A) 1 4.409 4.409 49.5303(a) 0.0004 CaCl2 (B) 2 13.773 6.887 77.3625(a) 0.0001 AB 2 1.513 0.756 8.4958(d) 0.0178 Error 6 0.534 0.089 DF = de rees of freedom (a) = significant (p < 0.001) (d) = significant (p < 01) APPENDIX "I" Analysis of variance for texture parameters of cheese as affected by the interaction of the variables of milk coagulation factors. 204 Table 1. Analysis of variance for cheese modulus of elasticity as affected by the interaction of four variables of milk coagulation factors. Source DF Sum Mean F value Probability of squares square NaCl (A) 1 0.614 0.614 174.9235(a) 0.00001 CaCl2 (B) 1 0.278 0.278 79. 1201(a) 0.00001 AB 1 0.049 0.049 14.0788(b) 0.00001 Temp. (C) 1 0.148 0.148 42.3271(a) 0.00001 AC 1 0.013 0.013 3.8000(d) 0.0690 BC 1 0.039 0.039 11.1973(b) 0.0041 ABC 1 0.002 0.002 0.6270 Rennet (D) 1 0.228 0.228 65 .0856(a) 0.00001 AD 1 0.00004 0.00004 0.0100 BD 1 0.048 0.048 13.7315(b) 0.0019 ABD 1 0.0007 0.0007 0.0222 CD 1 0.038 0.038 10.7387(c) 0.0047 ACD 1 0.00012 0.00012 0.0341 BCD 1 0.057 0.075 16.3353(a) 0.0009 ABCD 1 0.002 0.002 0.5317 Error 16 0.056 0.0035 Std. error of the mean = 0.0105 DF = degrees of freedom (a) = significant (p < 0.001) (b) = significant (p < 0.005) (c) = significant (p < 0.025) (d) = significant (p < 0.1) Table 2. Analysis of variance for hardness of Domiati cheese as affected by the 205 interaction of four variables of milk coagulation factors. Source DF Sum Mean F value Probability of squares square NaCl (A) 1 3.897 3. 897 982.6476(a) 0.00001 CaCl2 (B) 1 0.440 0.440 110. 85 16(a) 0.00001 AB 1 0.015 0.015 3.7444(d) 0.0709 Temp. (C) 1 1.445 1.445 364.4348(a) 0.00001 AC 1 0.435 0.435 109. 8019(a) 0.00001 BC 1 0.028 0.028 7.0959(0) 0.0170 ABC 1 0.042 0.042 10.525 8(c) 0.0051 Rennet (D) 1 3.281 3.281 827 .5278(a) 0.00001 AD 1 0.116 0.116 29.3700(a) 0.0001 BD 1 0.048 0.048 12.0919(b) 0.0031 ABD 1 0.035 0.035 8. 8731(c) 0.0089 CD 1 0.282 0.282 71 .2243(a) 0.00001 ACD 1 0.119 0.119 29.9141(a) 0.0001 ABCD 1 0.020 0.020 5 . 1005 (d) 0.0382 Error 16 0.063 0.0097 Std. error of mean = 0.0112 DF = degrees of freedom (81) = significant (p < 0.001) (b) = significant (p < 0.005) (C) = significant (p < 0.025) (e) = significant (p < 0.25) Table 3. Analysis of variance for brittleness of Domiati cheese as affected by the interaction of four variables of milk coagulation factors. Source DF Sum Mean F value Probability of squares square NaCl (A) 1 2.309 2.309 854.9616(a) 0.00001 CaCl2 (B) 1 0.030 0.030 11.2306(b) 0.0041 AB 1 0.003 0.003 0.9920 Temp. (C) 1 0.178 0.178 65 .7717(a) 0.00001 AC 1 0.001 0.001 0.3352 BC 1 0.00001 0.00001 0.0035 ABC 1 0.003 0.003 0.9478 Rennet (D) 1 0.463 0.463 171.3084(a) 0.00001 AD 1 0.013 0.013 4.8316(d) 0.0430 BD 1 0.014 0.014 5 .3282(d) 0.0347 ABD 1 0.006 0.006 2.3285 (e) 0.1465 CD 1 0.003 0.003 1.1701 0.2954 ACD 1 0.023 0.023 8.5178(c) 0.0100 ABCD 1 0.024 0.024 8.8952(c) 0.0088 Error 16 0.043 0.0027 Std. error of the mean for hardness = 0.0112 Std. error of the mean for brittleness = 0.0092 DF = degrees of freedom (a) = significant (p < 0.001) (b) = significant (p < 0.005) (c) = significant (p < 0.025) (d) = significant (p < 0.1) (e) = significant (p < 0.25) 207 Table 4. Analysis of variance for elasticity of Domiati cheese as affected by the interaction of four variables of milk coagulation factors. Source DF Sum Mean F value Probability of squares square NaCl (A) 1 1222.535 1222.535 9060.9279(a) 0.00001 CaCl2 (B) 1 213.510 213.510 1582.4461(a) 0.00001 AB 1 93.424 93.424 692.42l7(a) 0.00001 Temp. (C) 1 162.983 162.983 1207 .9659(a) 0.00001 AC 1 10.881 10.881 80.6447(a) 0.00001 BC 1 7.482 7.482 55 .45 12(e) 0.1586 ABC 1 0.295 0.295 2.1872(a) 0.1586 Rennet (D) 1 345.064 345.064 (2557.4751(a) 0.00001 AD 1 9.290 9.290 68. 8552(a) 0.00001 BD 1 35.553 35 .553 263.5019(a) 0.00001 ABD 1 4.781 4.781 35.4383(a) 0.00001 CD 1 11.065 11.065 82.0109(a) 0.00001 ACD 1 0.242 0.242 1.7946(e) 0.1991 BCD 1 60.6454 60.654 449.5433(a) 0.00001 ABCD 1 9.701 9.701 71.9008(a) 0.00001 Error 16 2.159 0.1349 Std. error of the mean = 0.0649 DF = degrees of freedom (21) = significant (p < 0.001) (e) = significant (p < 0.25) Table 5. Analysis of variance for cohesiveness of Domiati cheese as affected by the 208 interaction of four variables of milk coagulation factors. Source DF Sum Mean F value Probability of squares square NaCl (A) 1 2504.918 2504.918 6.4145(0) 0.00001 CaCl2 (B) 1 2.562 2.562 0.0066 AB 1 601.693 601.693 1.5408(e) 0.2324 Temp. (C) 1 8.188 8.188 0.0210 AC 1 22.742 22.742 0.0582 BC 1 30.186 30.186 0.0773 ABC 1 16.160 16.160 0.0414 Rennet (D) 1 3882.531 3882.531 9.9422(0) 0.0062 AD 1 11.263 11.263 0.0288 BD 1 154.125 154.125 0.3947 ABD 1 25.232 25 .232 0.0646 CD 1 299.885 299.885 0.7679 ACD 1 979.707 979.707 2.5088(e) 0.1328 BCD 1 1154.140 1154.140 2.9555(e) 0.1049 ABCD 1 765 .739 765.739 1.9609(e) 0. 1805 Error 16 6248.171 390.5087 Std. error of the mean = 0.1261 DF = degrees of freedom (a) = significant (p < 0.001) (c) = significant (p < 0.025) (e) = significant (p < 0.25) Table 6. Analysis of variance for chewiness of Domiati cheese as affected by the 209 interaction of four variables of milk coagulation factors. Source DF Sum Mean P value Probability of squares square NaCl (A) 1 1.374 1.374 5481.2610(a) 0.00001 CaCl2 (B) 1 0. 167 0. 167 665.0086(a) 0.00001 AB 1 0.076 0.076 302.3428(a) 0.00001 Temp. (C) 1 0.167 0.167 664.4328(a) 0.00001 AC 1 0.101 0.101 403.3550(a) 0.00001 ABC 1 0.0001 0.0001 0.4094 ABC 1 0.001 0.001 2.3439(d) 0.14530 Rennet (D) 1 0.783 0.783 3 124.6125 (a) 0.00001 AD 1 0.203 0.203 811.0527(a) 0.00001 BD 1 0.0001 0.0001 0.7184 ABD 1 0.001 0.001 2.9920(d) 0.1029 CD 1 0.038 0.038 152.0690(a) 0.00001 ACD 1 0.032 0.032 127.0302(a) 0.00001 BCD 1 0.024 0.024 94. 1036(a) 0.00001 ABCD 1 0.005 0.005 21.4661(a) 0.0003 Error 16 0.004 0.00025 Std. error of the mean = 0.00279 DF = degrees of freedom (21) = significant (p < 0.001) (d) = significant (p < 0.1) Table 7. Analysis of variance for gumminess of Domiati cheese as affected by the 210 interaction of four variables of milk coagulation factors. Source DF Sum Mean F value Probability of squares square NaCl (A) 1 6.091 6.091 2950. 8484(a) 0.00001 CaCl2 (B) 1 0.624 0.624 302.2300(a) 0.00001 AB 1 0.083 0.083 40.3423(a) 0.00001 Temp. (C) 1 0. 663 0.663 321 .3274(a) 0.00001 AC 1 0.378 0.378 183.1555(a) 0.00001 BC 1 0.040 0.040 19.2903 (a) 0.0005 ABC 1 0.034 0.034 16.5135(a) 0.0009 Rennet (D) 1 4. 869 4.869 2358. 6686(a) 0.00001 AD 1 0.517 0.517 250.2667(a) 0.00001 BD 1 0.026 0.026 12.6141(b) 0.0027 ABD 1 0.030 0.030 14. 6706(b) 0.0015 CD 1 0.116 0.116 55.9623(a) 0.00001 ACD 1 0.209 0.109 52.9761(a) 0.00001 BCD 1 0.078 0.078 37 .9663(a) 0.00001 ABCD 1 0.00006 0.00006 _Error 16 0.033 0.00206 Std. error of the mean = 0.0080 DF = degrees of freedom (a) = significant (p < 0.001) (b) = significant (p < 0.005) BIBLIOGRAPHY BIBLIOGRAPHY Abd El-Hamid, L.B., Amer, SN, and Zedan, A.N. 1981. Rennet coagulation time of goat’s, sheep’s, Buffalo’s and cow’s milk. Egyptian J. Dairy Sci., 9:137. Abd El-Salam, M.H. and El—Shibiny S. 1973. An electron microscope study of the structure of Domiati cheese. J. Dairy Res. 40:113. Abd El-Salam, M.H., El-Shibiny, S., Moneib, A., El-Heiba, A. A., and AlKhamy, H. 1981. Pickled soft cheese-making from recombined milk with added dried buttermilk. J. Dairy Res. 48:327. Abou-Donia, SA. 1981. Pasteurization and addition of starter to milk for Domiati cheese. Indian J. Dairy Sci. 34:136. Abou-Donia, S.A., El-Banna, A., Shehata, A., and Zoueil, M. 1985. Sensory evaluation of Domiati cheese manufactured using isolated rennin substitute from the yeast Rhodotorula rubra. J. Dairy Sci. 68(Supp1.1): D118.(Abstr.) Abou-Donia, SA. 1986. Egyptian Domiati Soft White Pickled Cheese. N. Z. J. Dairy Sci. and Technol. 21:167. Adachi, S. 1963. Electron microscopic observations of alkaline earth metal—caseinate particles. J. Dairy Sci. 46:743. Adda, J ., Gripson, J .C., and Vassal, L. 1982. The chemistry of flavor and texture generation in cheese. Food Chem. 9:115. Ahmed, N.S., Abd El-Salam, M.H., and El-Shibiny, S. 1972. The effect of homogenization on the quality, yield, and chemical composition of Domiati cheese. Indian J. Dairy Sci. 25:4. Alais, C. , and Lagrange, A. 1972. Etude biochimique protease coagulante produite par Mucor miehei. 1. Activite coagulante et activite proteolytique. Le Lait. pp. 407. Amantea, G.F., Skura, B.J., and Nakai, S. 1986. Culture effect on ripening characteristics and rheological behavior of cheddar cheese. J. Food Sci. 51:912. 212 213 Amer, S.N., El-Abd, M., and Ibrahim, M...KE 1974. Factors affecting the rennet coagulation time of milk. Egyptian Dairy Sci. 2:25. AOAC. 1984. "Official Methods of Analysis" 14th ed. Association of Official Chemists. Washington, DC. Banerjee, A.K., and Ganguli, N .C. 1973. A turbidimetric method for evaluating the release of glycomacropeptide in milk by rennet. Indian J. Animal Sci. 43:314. Baron, M. 1949. Further studies of rheological properties of cheese during manufacture and ripening. Dairy Ind. 14:146. Baron, M. 1952. "The Mechanical Properties of Cheese and Butter" United Trade Press, London. Baron, M., and Scott Blair, G.W. 1953. The rheology of cheese and curd in foodstuffs, their plasticity, fluidity and consistency. North Holland, Amestrdam. Beeby, R. 1980. The use of fluorescamine at pH 6.0 to follow the action of chymosin on kappa-casein and to estimate this protein in milk. N. Z. J. Dairy Sci. and Technol. 15:94. Berridge, NJ. 1952. Some observations on the determination of the activity of rennet. Analyst. 77:57. Berridge, NJ. 1955. Purification and assay of rennet. Methods in Enzymology, Vol.II. (S.P. Colowick and W.O. Kaplan W.O., eds) Bingham, E.W. 1975. Action of rennin on x-casein. J. Dairy Sci. 58:13. Bloomfield, V.A., and Moor, CV. 1973. Structure of casein icelles: Physical methods. Neth. Milk Dairy J. 27:103. Bloomfield, V.A., and Mead, RI. 1975. Structure and stability of casein micelles. J. Dairy Sci. 58:592. Bodyflet, F.W., Tobias,J., and Trout, GM. 1988. The sensory evaluation of dairy products. AVI publ. NY. P.330. Bohlin, L., Hegg, P., and Ljusberg-Wahren, H. 1984. Viscoelastic properties of coagulating milk. J. Dairy Sci. 67:729. Bourne, M.C., Moyer, J .C., and Hand, DB. 1966. Measurement of Food Texture by a Universal Testing Machine. Food Technol. 20:522. 214 Bourne, M.C. 1967. Deformation testing of foods. 1. A precise technique for deforming the deformation test. J. Food Sci. 32:601. Bourne, M.C. 1968. Texture profile of ripening pear. J. Food Sci. 33:223. Bourne, M.C. 1974. Textural changes in ripening peaches. Can. Inst. Food Sci. Technol. J. 7:11. Bourne, M.C., Kenny, J .F., Barnard, J. 1977. Computer assisted readout of data from texture profile analysis curves. Society of rheology meeting, Madison, Wis., Oct., 21. Bourne, M.C. 1978. Texture profile analysis. Food Technol. 7:62. Bourne, M.C., and Comstock, SH. 1981. Effect of degree of compression on texture profile parameters. J. Texture Stud. 12:201. Boyd, J .V., and Sherman, P. 1975. A study of force-compression conditions associate with hardness evaluation in several foods. J. Texture Stud. 6:507. Brandt, M.A., Skinner, E.Z., and Coleman, J.A. 1963. Texture profile method. J. Food Sci. 28:404. Breene, W.M. 1975. , Application of texture profile analysis to instrumental food texture evaluations. J. Texture Stud. 6:53. Brennan, J.G., Jowitt, R., and Mughsi, O.A. 1970. Some Experiences with the General Food Texturometer. J. Texture Stud. 1:167. Brennan, J .G., Jowitt,~R., and Williams, A. 1975. An analysis of the action of the General Foods Texturometer. J. Texture Stud. 6:83. Brinkhuis, J. , and Payens, TA. 1984. The influence of temperature on the flocculation rate of renneted casein micelles. Biophy. chem. 19:75. Brinton, K.H. Jr., and Bourne, M.C. 1972. Deformation testing of foods. III. Effect of size and shape on the magnitude of Deformation. J. Texture Stud. 3:284. Brule’, G., and Fauquant, J. 1981. Mineral balance in skim milk and milk retentate: Effect of physicochemical characteristics of the aqueous phase. J. Dairy Res. 48:91. Brule’, G., and Lenior, J. 1986. The coagulation of milk. In: "Cheese-making Science and Technology.” (A. Eck, ed.), translated by Thomas,C.D. Lavoisier Inc. NY. 215 Brunner, LR. 1976. Characteristics of edible fluids of animal origin: milk. In "Principles of Food Science-1. Food Chemistry". (O.R. Fennema, ed.), P. 619. Marcel Dekker, Inc. , NY. Brunner, J .R. 1977. Milk proteins. In "Food Proteins". (J.R. Whitaker and SR. Tannenbaum, eds.) AVI Publishing Company, Inc. Brunner, J .R. 1981. Cow milk proteins: Twenty-five years of progress. J. Dairy Sci. 64:1038. Burgess, A. 1982. Ion exchange processing of skim milk for food use. J. Dairy Res. 49:749. Carlson, A. 1985. Kinetics of gel forming in enzyme coagulated milk. Biotechnol. Progr. 1:46. Carroll, R.J., Thompson, M.P., and Nutting, G.C. 1968. Glutaraldehyde fixation of casein micelles for electron microscopy. J. Dairy Sci. 51:1903. Castle, A.V. , and Wheelock, J .V. 1972. Effect of varying enzyme concentration on the action of rennin on whole milk. J. Dairy Res. 39:15. Cervantes, A., Lund, DB, and Olson, NP. 1983. Effects of salt concentration and freezing on Mozzarella cheese texture. J. Dairy Sci. 66:204. Chen, A.M., Larkin, J .W., Clark, C.J., and Irwin, W.E. 1979. Texture analysis of cheese. J. Dairy Sci. 62:901. Cheryan, M., Van Wyk, P.J., Olson, N .F., and Richardson, T. 1975. Secondary phase and mechanism of enzymic milk coagulation. J. Dairy Sci. 58:477. Chianese, L., Masi, P., Laezza, P., Petrilli, P., Frunzi,A., and Addeo, F. 1986. Proteolysis and texture changes during the ripening of a Pasta Filata cheese. Dairy Sci. Abst. 50:5845. Christen, C., and Virasoro, E. 1932. La Ioi d’action de la presure dans la coagulation du oomplexe caseinate de calcium +phosphate de calcium. Lait, 12:923. Christen, C., and Virasoro, E. 1935. Presures vegetales: extraction et proprietes. Lait, 15:354. Civille, G.V. , and Szczesniak, AS. 1973. Guidelines to training a texture profile panel. J. Texture Stud. 4:203. 216 Collin, J .C. , and Grappin, R. 1977 . Etude de La methode de measure, selon Berridge, due temperatures de coagulation due lait additionne d’une solution enzymatique. Rev. Lait Fr. 3552389. Cooper, H.R., and Watts, T.A. 1981. Evaluation of textural characteristics produced in Cottage cheese creamed with selected dressings. Can. Inst. Food Sci. and Technol. J. 14:29. Cooper, H.R. 1987. Texture in Dairy Products and its Sensory Evaluation. Chap.9. In "Food Texture Instrumental and Sensory measurements." (H.R.Moskowitz, ed.), Marcel Deckker, Inc. NY. Creamer, L., and Olson, N.F. 1982. Rheological evaluation of maturing cheddar cheese. J. Food Sci. 47:631. Creamer, L.K. 1985. Water absorption by renneted casein micelles. Milchwissensch- aft, 40:589. Creamer, L.K., Lawrence, R.C., and Gilles, J. 1985. Effect of acidification of cheese milk on the resultant cheddar cheese. N. Z. J. Dairy Sci. and Technol. 20:185. Creamer, L.K., Gilles, J ., and Lawrence, R.C. 1988. Effect of pH on the texture of Cheddar and Colby cheese. N. Z. J. Dairy Sci. and Technol. 23:23. Culioli, J., and Sherman, P. 1976. Evaluation of Gouda cheese firmness by compres- sion tests. J. Texture Stud. 7:353. Cumper, C.W.N., and Alexander, A.E. 1952. The viscosity and rigidity of gelatin in concentrated aqueous system. Rigidity Australian J. Sci. Res. A5:153. Dalgleish, D.G. 1979. Proteolysis and aggregation of casein micelles treated with immobilized or soluble chymosin. J. Dairy Res. 46:653. Dalgleish, D.G. 1981. Effect of milk concentration on the nature of curd formed during renneting. A theoretical discussion. J. Dairy Res. 48:65. Dalgleish, D.G. 1982. The enzymatic coagulation of milk. In "Development in Dairy Chemistry-1". (P.F.Fox, ed), Applied Science Publ. NY. Dalgleish, D.G. 1983. Coagulation of renneted bovine casein micelles: dependence on temperature, calcium ion concentration and ionic strength. J. Dairy Res. 50:331. 217 Darling, D.F., and Van Hooydonk, A.C.M. 1981. Derivation of a mathematical model for the mechanism of casein micelle coagulation by rennet. J. Dairy Res. 48:189. Darling, D.F. 1982. In "The effects of polymers on dispersion properties". (T.F. Tadros, ed.) Academic Press, NY, pp. 285. Davis, J .G. 1937. The rheology of cheese, butter and other milk products (the measurement of body and texture). J. Dairy Res. 8:245. Davis, D.T., and Law, A.J.R. (1977). The composition of whole casein from the milk of Ayrshire cows. J. Dairy Res. 44:447. de Jong, H.G., and Henneman, J .P. 1932. Capillary electric charge and hydration gels: Changes in volume and modulus of elasticity of agar and gelatin. Kolloid-Z. 36:123. de Jong, L. 1976. Protein breakdown in soft cheese and its relation to consistency. 1. Proteolysis and consistency of Noordhallandse Meshanger cheese. Neth. Milk Dairy J. 30:242. de Jong, L. 1977. Protein breakdown in soft cheese and its relation to consistency. 2. The influence of the rennet concentration. Neth. Milk Dairy J. 31:314. de Jong, L. 1978. The influence of the moisture content on the consistency and protein breakdown of cheese. Neth. Milk Dairy J. 32:1. de Kong, P.J., Jenness, R., and Wijnand, HP. 1963. Some aspects on the determina- tion of N-acetyl neuraminic acid, and its use for the comparison of the action of heat and rennin on casein. Neth. Milk Dairy J. 17:352. Desmazeaud, M.J., and Gripon, J .C. 1977 . General mechanism of protein breakdown during cheese ripening. Milchwissenschaft, 32:731. Donnelly, W.J., Barry, J.G., and Buchheim, W. 1980. Changes in the casein micelles in late lactation milk. Food Sci. and Technol. Res. Report, An Foras Taluntais, Dublin, pp. 14-15. Drake, B. 1965. Food crushing sounds: Comparisons of objective and subjective data. J. Food Sci. 30:556. Duckworth, R.B., ed. 1974. Water Relations of Foods. Academic Press, Paris. 218 Eberhard, P. 1985. Rheologische Eigenschaften ausgewahlter kasesorten. l. Emmentalkase. Schweiz. Milchwirtsch. Forsch. 14:3. Eck, A. 1986. Cheesemaking Science and Technology. Lavoisier Publ. Inc. NW, USA. Eigel, W.N., Butler, J.E., Ernstrom, C.A., Farrel, H.M., Harwalkar, Jr.V.R., Jenness,R. , and Whitney, Mc.L. 1984. Nomenclature of proteins of cow’s milk: Fifth Revision. J. Dairy Sci. 67:1599. Eino, M.P., Biggs, D.A., Irvin, D.M., and Stanley, D.W. 1979. Microstructural changes during ripening of cheddar cheese produced with calf rennet, bovine pepsin, and porcine pepsin. Canadian Institute of Food Sci. and Technol. J. 12:149. El-Gendy, SM. 1983. Use of salt tolerant lactic acid bacteria for manufacture of white pickled cheese (Domiati) ripened without salted whey in sealed polyethylene pouches. J. Food Protect. 46:335. El-Koussy, L.A., Tawab, E.A., Hofi, A.A., and El-Sokkary, A.M. 1970. Effect of some factors on Domiati cheese making from buffalo milk. Res. Bull. No. 688, Faculty of Agr., Ein Shams Univ., Egypt. El-Koussy, L.A., Tawab, E.A., Hofi, A.A., and El-Sokkary, A.M. 1974. Domiati cheese from pasteurized milk. I. Curd formation. Dairy Sci. Abst. 38:4037 (197$. El-Koussy, L.A., El-Kalla, M.A., and Abdel-Ghani, O. 1975. Some factors influenc- ing Domiati cheese-making. 1. Effect of milk-heating. Agri. Res. Rev. 53:119. El-Koussy, L.A., Hamdy, A.M., Nasr, M.A., and Abdel Ghani, A. O. 1976. Utilization of starter in Domiati cheese making. Milchwissenschaft, 31:428. El-Koussy, L.A., Youssef, S. , and Hamdy, A.M. 1977 . Homogenization of pasteurized milk and its effect on Domiati cheese. Milchwissenschaft, 32:604. El-Mayda, E., Paquet, D., and Ramet, JP 1985. Enzymatic proteolysis of milk proteins, in a salt environment, with a Bacillus subtilis neutral protease preparation. J. Food Sci. 50:1745. El-Neshawy, A.A., Abdel Baky, A.A., and Farahat, SM. 1982. Enhancement of Domiati cheese flavor with animal lipase preparations. Dairy Ind. Intern. 47:29. 219 El-Safty, M.S., Basily, S., Abd-El-Latif, F. 1980. A continuous method for Domiati cheese making. Egyptian J. Dairy Sci. 8:81. El-Safty, M.S., Ellen, A., and Fahmi, N. 1981. Use of salted whey to reconstitute dried milk for manufacturing white soft cheese. 1. Character of the curd. J. Food Prot. 44:652. El-Safty, M.S., El-Zayat, AL, and Ismail, A.A. 1985. The effect of partial lactose hydrolysis on the quality and ripening of Domiati cheese. Egyptian J. Dairy Sci. 12:19. El-Shibiny, s., Abd E1-Sa1am,M..,H and Ahmed, NS. 1972. The effect of formalde- hyde used as a preservative of milk on quality, yield, and chemical composition of Domiati cheese. Milchwissenschaft, 27:217. El-Shibiny, S., Abd El-Salam, M.H., and Nawal, SA. 1973. Effect of some additives on the yield, quality, and chemical composition of Domiati cheese. 11. Whey proteins. Egyptian J .Dairy Sci. 1:56. El-Zayat, A.I., and Omar, M. 1987. Kareish cheese prepared from ultrafiltered milk. J.Dairy Res. 54:545. Emmons, DB, and Price, W.V. 1959. A curd firmness test for cottage cheese. J. Dairy Sci. 42:553. Emmons, DB, and Lister, EB. 1976. Quality of protein in milk replacers for young calves. 1. Factors affecting in vitro curd formation by rennet (chymosin, rennin) from reconstituted skim milk powder. Can. J. Animal Sci. 56:317. Emmons, D.B., Kalab, M., Larmond, E., and Lowrie, RI. 1980. Milk gel structure. X. Texture and microstructure incheddar cheese made from whole milk and from homogenized low-fat milk. J. Texture Stud. 11:15. Emstrom, C.A., and Wong, N .P. 1974. Milk-clotting enzymes and their action. In "Fundamentals of Dairy Chemistry." (G.H. Webb, A.H. Johnson, and J .A. Alford, eds), Avi publ. Co., Inc. Westport, CT. Fahmi, A.H., and Sharara, H.A. 1950. Studies on Egyptian Domiati cheese. J. Dairy Res. 17:312. Fahmi, A.H., Metwally, M., Abou Dawood, A.E., and Abd El-Salam, I. 1973. The effect of the amount of rennet and renneting temperature on Domiati cheese. Egyptian J .' Dairy Sci. 1:63. 220 Fahmi, A.H., Tawab, G.A., and Abou-El-Heba, A. 1982. Effect of adding sodium chloride to milk on its keeping quality. Egyptian J. Dairy Sci. 10:23. Farrell, H.M.Jr., and Thomposon, MP. 1974. Physical equilibria: proteins. In " Fundamentals of Dairy Chemistry." 2nd ed., pp. 442-473 (B.H.Web, A.H.John- son, and J.A. Alford, eds), Westport, CTzAvi Publishing Co. Inc. Fedriek, I.A., and Dulley, J.R. 1984. The effect of elevated storage temperatures on the rheology of cheddar cheese. N. Z. J. Dairy Sci. and Technol. 19:141. Finney, E.E.Jr. 1969. Objective measurements for texture in foods. J. Texture Stud. 1:19. Foltmann, B. 1959. On the enzymatic and coagulation stages of the renneting process. XV Int. Dairy Congr., (London), 2:655. Food and Drug Administration (FDA). 1979. Code of Federal Regulations 21. US Govt. Printing Office, Washington. DC. Fox, P.F., and Walley,B.F. 1971. Influence of Sodium chloride on the proteolysis of casein by rennet and by pepsin. J. Dairy Res. 38: 165. Fox, P.F., and Morrissey, RA. 1977 . Reviews of the progress of dairy science. The heat stability of milk. J. Dairy Res. 44:627. Fox, P.F., and Mulvihill, D.M. 1982. Milk proteins: molecular, colloidal and functional properties. J. Dairy Res. 49:679. Friedman, H.H., Whitney, J .E., and Szczesniak AS 1963. The Texturometer, a new instrument for objective texture measurement. J. Food Sci. 28:390. Friedenthal, M., Siimer, E., and Kostner, A. 1981. Rennet action on skim milk. J. Dairy Sci. 64:342. Fukui, Y., Tada, M., and Milk, E. 1971. Measurement of the physical properties of processed cheese by textrumeter. Technical Bull. of F ac. of Agr., Kagawa Univ. 23:149. Cited in Dairy Sci. Abst. 35:2669 (1972). Fukushima, M., Taneya, S., and Sone, T. 1964. Viscoelasticity of cheese. J. Soc. Mater. Sci. Japan. 13:331. Fukushima, M., Sone, T., and Fudaka, E. 1965 . The effect of moisture content on the Viscoelasticity of cheese. J. Soc. Mater. Sci. Japan 14:270. 221 Gal, 8., and Bankey, D. 1971. Hydration of sodium chloride bound by casein at medium water activities. J. Food Sci. 36:800. Gamier, T., and Ribadeau-Dumas, B. 1970. Structure of the casein micelle: A proposed model. J. Dairy Res. 37:493. Garnot, P., and Olson, N.F. 1981. Study of enzymatic milk gelation with the curd-firmness tester. Neth. Milk Dairy J. 35:374. Gaya, P., Medina, M., Rodriguez-Marin, M.A., and Nunez, M. 1990. Accelerated ripening of ewes’ milk Manchego cheese: The effect of elevated ripening temperature. J. Dairy Sci. 73:26. Geurts, T.J., Walstra, P., and Mulder, H. 1972. Brine composition and the prevention of the defect "soft rind" in cheese. Neth. Milk Dairy J. 26:168. Geurts, T.J., Walstra, P., and Mudler, H. 1974. Water binding to milk protein with particular references to cheese. Neth. Milk Dairy J. 28:64. Ghaleb, H.M. 1977a. Contribution to loss in weight of Domiati cheese during pickling. 11. Cheese milk treatments. C. Use of hydrogen peroxide. Dairy Sci. Abst. 4223811 (1980). Ghaleb, H.M. 1977b. Contribution to loss in weight of Domiati cheese during pickling. II. Cheese milk treatments. B. Use of pectine, gelatine, and agar. Dairy Sci., Abst. 42:3870 (1980). Gill, J .L. 1978. Design and analysis of experiments in the animal and medical science, Vol.1. The Iowa State University Press, Ames, IA. Goodman, N. 1963. "Fiber Structure" (J .W. Hearle, and RM. Peters, eds). The Textile Institute, Butterworths, London. Gouda, A., El-Zayat, A.L., El-Shabrawy, SA. 1985. Partition of some milk salts and curd properties as affected by adding sodium chloride to the milk. Dairy Sci. Abst. 48:2877 (1986). Grappin, R., Rank, T.C., and Olson, N.F. 1985. Primary proteolysis of cheese proteins during ripening. A review. J. Dairy Sci., 68:531. Green, M.L., and Crutchfield, G. 1971. Density-gradient electrophoresis of native and rennet-treated casein micelles. J. Dairy Res. 38:151. 222 Green, M.L., and Marshall, RI. 1977. The acceleration of cationic materials on the coagulation of casein micelles by rennet. J. Dairy Res. 44:521. Green, M.L., Hobbs, D.G., Morant, S.V., and Hill, V.A. 1978. Intermicellar relationships in rennet-treated separated milk. II. Process of gel assembly. J. Dairy Res. 45:413. Green, M.L., Turvey,A., and Hobbs, D.G. 1981. Development of structure and texture in cheddar cheese. J. Dairy Res. 48:343. Green, M.L., and Manning, DJ. 1982. Development of texture and flavor in cheese and other fermented products. J. Dairy Res. 49:737. Green, M.L., Marshall, R.J., and Brooker, BE. 1985. Instrumental and sensory texture assessment and fracture mechanisms of cheddar and cheshire cheese. J. Texture Stud. 16:351. Grufferty, M.B., and Fox, RF. 1985. Effect of added NaCl on some physicochemical properties of milk. Ir. J. Food Sci. Technol. 9:1. * Hall, D.M., and Creamer, L.K. 1972. A study of the submicroscopic structure of Cheddar, Cheshire and Gouda cheese by electron microscopy. N. Z. J. Dairy Sci. and Technol. 7:95. Hamdy, A., and Edelsten, D. 1970. Some factors affecting the coagulation strength of three different microbial rennets. Milchwissenshaft, 25:450. Hamdy, A. 1972. Quality of Domiati cheese prepared by using microbial enzymes. Ind. J. Dairy Sci. 25:73. Hardy, J ., and Steinberg, MP 1984. Interaction between sodium chloride and paracasein as determined by water sorption. J. Food Sci. 49:127. Hardy, J. , and Adda, J. 1984. Les Properties Organoloptique du Fromage, Le Fromage (A. Eck, ed.) Technique et Documentation (Lavoisier), Paris, France P. 320. Harper, J .P., Suter, D.A., Dill, C.W., and Jones, ER. 1978. Effects of heat treatment and protein concentration on the rheology of bovine plasma suspensions. J. Food Sci. 43: 1204. Hefnawy, S., El-Koussy, L., Ewais, S. 1984. Manufacture of half cream pickled soft cheese. 11. Effect of supplementing cheese milk with skim milk powder. Egyptian J. Dairy Sci12:251. 223 Henry, W.F., and Katz, M.H. 1969. New dimensions relating to the textural quality of semi-solid foods and ingredient systems. Food Technol. 23:822. Henry, W.F., Katz, M.H., Pilgrim, F.G., and May, A.T. 1971. Texture of semi-solid foods: Sensory and physical correlates. J. Food Sci. 36:155. Hickson, S.W., Dill, C.W., Morgan, R.G., Sweat, V.E., Suter, D.A., and Carpenter, Z.L. 1982. Rheological properties of two heat-induced protein gels. J. Food Sci. 47:783. Hofi, M. 1984. Manufacture of Domiati cheese by ultrafiltration. Ph.D. thesis. Justus Liebig University, Giessen, FRG. Holmes, D.G., Duersch, J .W., and Ernstron, CA. 1977. Distribution of milk clotting enzymes between curd and whey and their survival during cheddar Cheesemak- ing. J. Dairy Sci. 60:862. Holt, C., Thomas, D., and Law, A.Jr. 1986. Effects of colloidal calcium phosphate content and free calcium ion concentration in the milk serum on the dissociation of bovine casein micelles. J. Dairy Res. 53:557. Hostettler, H., and Imhof, K. 1951. Elektronenoptische untersuchungen uber den Fainbau der Milch. Milchwissenschaft, 6:351. Huang, P.F., and Rha, C. 1974. Protein structure and protein fibers. a review. Polymer Eng. Sci. 14:81. Imoto, E.M., Lee, C., and Rha, C. 1979. Effect of compression ratio on the mechanical properties of cheese. J. Food Sci. 44:343. Jen, J .J ., and Ashworth, US. 1970. Factors influencing the curd tension of rennet coagulated milk. Salt balance. J. Dairy Sci. 53:1201. Kalab, M. 1977. Milk gel structure. VI. Cheese texture and microstructure. Milchwissenschaft, 32:449. Kelly, LA. 1951. Factors affecting rennet coagulation time and curd tension. Ph.D. Diss., Univ. Wisconsin, Madison. Kerr, T.J., Washam, C.J., Evans, AL. and Todd, R.L. 1981. Effects of fat content on the microstructure of Domiati-type cheese. J. Food Protec. 44:96. 224 Kimber, A., Brooker, B.E., Hobbs, D.S, and Prentice, J.H. 1974. Electron micro- scope studies on the development of structure in cheddar cheese. J. Dairy Res. 41:389. Knoop, E., and Wortmann, A. 1960. Zur Grossenverteilung der caseinteilchen in Kuhmilch, Ziegenmilch, und Frauenmilch Milchwissenschaft, 15:273. Knoop, A.M., and Peters, K.H. 1972. Dependence of the submicroscopical structure of rennet coagulum and young Camembert cheese mass on the conditions of manufacture. Milchwissenschaft, 27:153. Knoop, A—M., Omar, M.M., and Peters, K-H. 1976. Vergleichende Strukturuntersuch- ungen an agyptischem Domiati-kase und einer neuen Art vonsalzlakenkase. Milchwissenschaft, 30:745. Kosikowski, F. 1982. Cheese and fermented milk foods. 2nd. Ed., Kosikowski and Associates Publishers Brooktondale, NY. Kreisman, L.N., and Labuza, T.B. 1978. Storage stability of intermediate moisture for processed cheese food products. J. Dairy Res. 22:386. Law, B.A., and Wigmore, A. 1982. Accelerated cheese ripening with food grade proteinases. J. Dairy Res. 49:137. Lawrence, R.C., Gilles, J., and creamer, L.K. 1983. The relationship between cheese texture and flavor. N. Z. J.Dairy Sci. and Technol. 18:175. Lawrence, R.C., Heap, H.A., and Gilles, J. 1984. A controlled approach to cheese technology. J. Dairy Sci. 67:1632. Lawrence, R.C., Creamer, L.K., and Gills,J. 1987. Texture Development During Cheese Ripening. J. Dairy Sci. 70:1748. Lee, C.H., Imoto, E.M., and Rha, C. 1978. Evaluation of cheese texture. J. Food Sci. 43:1600. Lever, AC. 1988. Mathematical modling of thermal gelation of myofibrillar beef proteins and their interaction with selected hydrocolloids. Ph.D. Thesis, Michigan State Univ., East Lansing, Michigan. Lindet, L.M. 1924. Emploi due chlorine de calcium en formagerie. Lait, 4:2. 225 Lindsay, R.C., Hargett, S.M., and Buch, CS. 1982. Effect of sodium solidos potassium (1:1) chloride and low sodium chloride concentrations on quality of cheddar cheese. J. Dairy Sci. 65:360. Lucisano, M., Pompcl, C., and Casiraghi, E. 1987. Texture evaluation of some Italian cheeses by Instrumental texture profile analysis. J. Food Quality, 10:73. Lyster, R.L.J. 1979. The equilibrium of calcium and phosphate ions with the micellar calcium phosphate in cow’s milk. J. Dairy Res. 46:343. Mackinlay, A.G., and Wake, R.G. 1971. x-casein and its attack by rennin (chymosin) In: Milk proteins: Chemistry and molecular biology, Vol.2 (H.A. Mackenzie, ed.) Academic Press. NY . Marier, J.R., Tessier, H., and Rose, D. 1963. Sialic acid as an index of the K-casein content of bovine skim milk. J. Dairy Sci. 46:373. Marshall, R.J., Hatfield, D.S., and Green, M.L. 1982. Assessment of two instruments for continuous measurement of the curd-forming of rennet milk. J. Dairy Res. 49:127. Marshall, R.J._ 1982. An improved method for measurement of the syneresis of curd formed by rennet action on milk. J. Dairy Res. 49:329. Mashaly, R.I., Abou-Donia, S.A., El—Soda, M. 1983. Utilization of dried milk for Domiati cheese. Indian J. Dairy Sci. 36:93. Masi, P., and Addeo, F. 1984. Rheological characteristics of some typical Pasta Filata cheese. Dairy Sci. Abst. 4924744 (1987). Masi, P., and Addeo, F. 1987. An examination of some mechanical properties of a group of Italian cheeses and their relation to structures and conditions of manufacture. J. Food Eng. 5:217. Masi, P. 1987. The collaborative compression tests on cheese. In: "Physical Properties of Foods 2." (R.Jowitt, F.Escher, M.Kent, B.Mckenna, and M.Roques, eds.) pp 383. Elsevier Applied Sci., London. McMahon, D.J., and Brown, RI. 1982. Evaluation of Formograph for comparing rennet solution. J. Dairy Sci. 65:1639. McMahon, D.J., Brown, R.G., Richardson, G.H., and Emstron, CA. 1984. Effect of bulk culture media on coagulation properties. J. Dairy Sci. 67:930. 226 McMahon, D.J., and Brown, R]. 1984. Composition, structure, and integrity of casein micelles: A Review. J. Dairy Sci. 67:499. Mehaia, M.A., and Cheryan, M. 1983. The secondary phase of milk coagulation. Effect of calcium, pH and temperature on clotting activity. Milchwissenschaft, 38:137. Mohsenin, N .N. 1970. "Physical Properties of Plant and Animal materials." Vol. 1. Gordon and Breach, New York. Monib, A.F., Abo El-Heiba, A., Al-khamy, A.F., El-Shibiny, S., Abd El-Salam, M.H. 1981. Effect of increasing milk solids on quality, yield, and chemical composi- tion of Domiati cheese. Egyptian J. Dairy Sci. 9:37. Morr, C.V., Josephson, B., Jenness, R., and Manning, PB. 1971. Composition and properties of submicellar casein complexes in colloidal phosphate-free milk. J. Dairy Sci. 54:1555. Morr, C.V., and Seo, A. 1988. Fractionation and characterization of glycomacropep- tide from caseinate and skim milk hydrolysates. J. Food Sci. 53:80. MSTAT. 1989 Version 4C, Michigan State Univ., East Lansing, MI. Nagmoush, M.R., Abd El-Salam, M.H., Seleem, R.M., and Abd, M.M. 1978. White pickled cheese from concentrated milk. 1. Effect of fat content and addition of starter on the composition and quality of cheese. Egyptian J. Dairy Sci. 6:193. Nilson, K.M. 1968. Effect of salt concentration on the quality of Italian cheeses. Food Prod. Dev. 2:90. Nitschman, H. 1949. Elektron micrOSkopische grossen bestimmung der calcium caseinatteilchenin kuhmilch. Helv. chim. Acta. 32:1258. Nitschman, H., and Bohren, H.U. 1955. Das Lab und sein Wirkung auf das Casein der Milch X. Eine Methode zur direkten Bestimmung der GeschéLab gerinnung derMilch. Helv. Chem. Acta. 38:1953. O’keeffee, A.M. 1984. Seasonal and Lactational influences on moisture content of cheddar cheese. Ir. J. Food Sci. and Technol. 8:27. Olkku, J., and Rha, CK. 1975. Textural parameters of candy licorice. J. Food Sci. 40:1050. 227 Omar, M.M., and Buchheim, W. 1983. Composition and microstructure of soft brine cheese made from instant whole milk powder. Food Microstr. 2:43. Omar, M.M. 1985. Effect of soy protein in rennet-induced reconstituted nonfat dry milk coagulum properties. Ph.D. Thesis. Univ. of Minnesota. Omar, M.M., and El-Shibiny, S. 1985. Composition and microstructure of Domiati cheese as affected by lactase. Egyptian J. Dairy Sci. 13:33. ' Omar, M.M. 1987. Microstructure and Chemical changes in Domiati cheese made from Ultrafiltered milk. Food Chem. 25:183. Park, J .L. 1979. A study on the texture of processed cheese. Korean J. Animal Sci. 2:107. Parker, T.G., and Dalgleish, D.G. 1981. Binding of calcium ions to bovine B-casein. J. Dairy Res. 48:71. Parnella-Clunies, E.M., Irvine, D.M., Bullock, DH. 1985. Textural characteristics of Queso Blanco. J. Dairy Sci. 68:789. Payens, T.A.J. 1966. Association of caseins and their possible relation to structure of the casein micelle. J. Dairy Sci. 49:1317. Payens, T.A.J. 1977 . On enzymatic clotting processes. 11. The colloidal instability of chymosin-treated casein micelles. Biophysical Chem. 6:263. Payens, T.A.J. 1979. Casein micelles: The colloidal~chemica1 approach. J. Dairy Res. 46:291. Payens, T.A.G., and Both, P. 1980. In: Bioelectrochemistry: ions, surfaces, membranes (Adv. Chem. Ser. No 188) (M.Blank, ed.), American Chemical Society, Washington, 1980, pp. 129. Pearce, K.N. 1976. Moving boundary electrophoresis of native and rennet-treated casein micelles. J. Dairy Res. 43:27. Pearce, K.N. 1979. Use of Fluorescamine to determine the rate of release of the caseinomacropeptide in rennet-treated milk. N. Z. J. Dairy Sci. and Technol. 14:233. Peleg, G. 1976. Texture profile analysis parameters obtained by Instron Universal Testing Machine. J. Food Sci. 41:721. 228 Peleg, M. 1983. Some theoretical rheological characteristics of the mechanical signals in sensory evaluation of texture. J. Food Sci. 4821187. Perkin-Elmer Corp. 1982. Analytical methods for atomic absorption spectroscopy. Norwalk, CT. Peters, 1.1. 1956. Cheddar cheese made from pasteurized milk homogenized at various pressure. J. Dairy Res. 3921083. Pierre, M., and Brule, G. 1981. Mineral and protein equilibria between the colloidal and soluble phases of milk at law temperature. J. Dairy Res. 48:417. Polychroniadou, A., and Vafopoulou, A. 1986. Salt distribution between the colloidal and soluble phases of ewes milk. J. Dairy Res. 53:353. Prentice, J. H. 1972. Rheology and texture of dairy products. J. Texture Stud. 32415. Prentice, J.H. 1987. Cheese Rheology. In: "Cheese Chemistry, Physics and Microbiology" Vol. 1. General Aspects. (P.F. Fox, ed.) London, Elsevier Applied Science. Proctor, B.E., Davison, S., and Brody, A.L. 1956a. A recording strain-gauge denture tenderometer for foods. 11. Studies on the masticatory force and motion, and the force penetration relationship. Food Technol. 10:327. Proctor, B.E., Davison,S., and Brody, A.L. 1956b. A recording strain-gauge denture tenderometer for foods. III. Correlation with subjective tests and the denture tenderometer. Food Technol. 10:344. Puri, BR, and Parkash, S. 1965 . Exchange of colloidal calcium with other cations in milk of three species. J. Dairy Sci. 48:611. Qvist, KB. 1979. Re-establishment of the original rennetability of milk after cooling. 2. The effect of some additives. Milchwissenschaft, 34:600. Ramanauskas,R. 1978. Rheological aspects of the interaction of sodium chloride with the paracasein complex of milk. XX Int. Dairy Congr., Vol. E., (1978):265. Ramet, J .P., and Webber, F. 1980. Contribution a ‘1’etude del’ influence des facteurs de milieu sur la coagulation enzymatique due lait reconstitute. Le Lait, 60: 1. Ramet, J.P., El-Mayda, E., and Webber, F. 1981. Correction par apport de chlorue de calcium de l‘aptitude a‘la coagulation enzymatique d‘un lait reconstitute refrigere. Milchwissenschaft, 36:143. 229 Ramet, J.P., El-Mayda, E., and Webber, F. 1983. Influence of salting of reconstituted milk on curdling by rennet. J. Texture Stud. 14:11. Ramet, J .P., and E1 Mayda, E. 1984. Le salage du Lait et sa coagulation enzymatique par La pre’sure et la subtilisine. Microbial Aliment. Nutr. 2:287. Ramet, J .P. 1985. La fromagerie et les varietes de fromages du bassin mediterraneen (Etude FAO), Production et Sante Animales No 48, 1-187. Rajput, Y.S., Bhavadasan, M.K., and Ganguli, NC. 1983. Changes in the chemical status of calcium in casein micelles with the pH of milk. Milchwissenschaft, 38:211. Roefs, S.P.F.M. 1985. Structure of acid casein gels. Ph.D. Thesis. Agric. Univ., ' Wageningen, Netherlands. Rose, D. 1969. A Proposed model of micelle structure in bovine milk. Dairy Sci. Abstr. 31:171. Rosenau, J.R., Calzada, J .F, and Peleg, M. 1978. Some rheological properties of cheese-like product prepared by direct acidification. J. Food Sci. 43:948. Ruettimann, K.W. , and Ladisch, M.R. 1987. Casein Micelles: structure, properties and enzymatic coagulation. Enzyme Microb. Technol. 9:578. Said, M., and Mohran, M. 1984. Acceleration of white soft cheese ripening by using some bacterial growth factors. Egyptian J. Dairy Sci. 12:219. Saito, Z. 1973. Electron microscopic and compositional studies of casein micelles. Neth. Milk Dairy J. 27:143. Satia, H.S., and Raadsveld, C.W. 1969. The quality of Gouda cheese made from milk enriched with calcium and phosphate. Neth. Milk and Dairy J. 23:276. Schmidt, D.G., and Payens, T.A.J. 1976. In: "Surface and Colloidal Science, (E.Matijevic, ed.) Witey, New York, Vol. 9, 1976., pp. 165. Schmidt, D.G. 1980. Colloidal aspects of casein. Neth. Milk Dairy J. 34:42. Schmidt, D.G. 1982. Association of caseins and casein micelle structure. Ch. 2. In: "Developments in Dairy Chemistry-1." (P.F.Fox, ed), Appl. Sci. Publ., London. Scott Blair, G.W. 1953. "Foodstuffs, Their Plasticity, Fluidity and Consistency. " Interscience Publishers, Amsterdam, New York. 230 Scott, R. 1986. "Cheesemaking Practice, 2nd ed." Chap. 11. Elsevier Applied Science, London. Shama, F. , and Sherman, P. 1973. Evaluation of some textural pr0perties of foods with the Instron Universal Testing Machine. J. Texture Stud. 4:344. Sharma, S.K., Hill, A.R., Goff, H.D., and Yada, R. 1989. Measurement of coagulation time and curd firmness of renneted milk using a Nametre viscometer. Milchwissenschaft, 44211. Sharara, H.A. 1958. The effect of adding sodium chloride on the calcium exchange in milk. Indian J. Dairy Sci. 11:175. Sherman, P. 1969. A texture profile of foodstuffs based upon well-defined rheological properties. J. Food Sci. 34:458. Sherman, P. 1976. The textural characteristics of dairy products. In: "Rheology and Texture in Food Quality.", (De Man et al., eds.) pp. 382. AVI Publishing Co., Westport, Connecticut. Slattery, C.W. , and Evard, R. 1973. A model for the formation and structure of casein micelles from subunits of variable composition. Biochem. Biophys. Acta. 317 :529. Slattery, C.W. 1976. Review: Casein micelle structure: An examination of models. J. Dairy Sci. 5921547. Smietana, Z., Jakubowski, J ., Poznanski, S., Zuraw, J., and Hosaja, M. 1977. The influence of calcium ions and heat on size changes of casein micelles in milk. Milchwissenschaft, 32:464. Snoeren, T.H.M., Klor, H.J., Hooydonk, A.C.M., and Dammans, A.T. 1984. The voluminosity of casein micelles. Milchwissenschaft, 39:461. Sood, S.M., Sidhu, KS, and Dewan, R.K. 1979. Effect of pH on micellar solvation and heat stability of milks from buffalo and cow. Ind. J. Dairy Sci. 32:4. Stainsby, G. 1977. The gelatin gel and the sol-gel transformation. In: "The Science and Technology of Gelatin." (A.G. Ward, and A. Courts, eds.), Academic Press. New York. Story, J .E., and Ford, GD. 1982. Some factors affecting the post clotting development of coagulum strength in renneted milk. J. Dairy Res. 49:469. 231 Szczesniak, A.S. 1963. Classification of textural characteristics. J. Food Sci. 28:385. Szczesniak, A.S. 1966. Texture measurements. Food Technol. 2021292. Szczesniak, A.S. 1975. General foods texture profile revisited-ten Years perspective. J. Texture Stud. 6:5. Szczesniak, A.S. , and Hall, B.J. 1975. Application of the General Foods Texturumeter to specific food products. J. Texture Stud. 6:117. Taneya, S., Izutsu, T., and Sone, T. 1979. Dynamic Viscoelasticity of natural cheese and processed cheese. In: "Food Texture and Rheology.", (P. Sherman, ed), Academic Press, London, pp. 369. Taranto, M.V., Wan,J., Chen, S.L., and Rhee, KC. 1979. Morphological, ultrastructural and rheological characterization of cheddar and Mozzarella cheese. Scanning Electron Microscopy/l977/III,273 Tessier, H., and Rose, D. 1958. Calcium ion concentration in milk. J. Dairy Sci. 41:351. Tessier, H., and Rose, D. 1964. Influence of x-casein and B-lactoglobulin on the heat stability of skim milk. J. Dairy Sci. 4721047. Thomas, M.A., and Brown, B. 1970. Studies in processed cheese manufacture—The evaluation of physical properties. Aust. J. Dairy Technol., 25 :46. Thomas, T.D., and Pearce, K.N. 1981. Influence of salt on lactose fermentation and proteolysis in cheddar cheese. N. Z. J. Dairy Sci. and Technol. 16:253. Thomasow, J. 1968. Untersuchungen an Labgallerten van Milch mit dem Hellige-Thromb-Elastographen. Milchwissenschaft, 23:725. Tukita, M., Hikichi, K., Niki, R., and Arima, S. 1982. Dynamic viscoelastic studies on the mechanism of milk clotting process. Biorheology, 19:209. Tuszynski, W.B. 1971. A kinetic model of the clotting of Casein by rennet. J. Dairy Res. 38:115. Ustunol, Z., and Hicks, LC. 1990. Effect of calcium addition on yield of cheese manufactured with Endothia Parasitica Proteases. J. Dairy Sci. 73:17. 232 Van Hooydonk, A.C.M., and Olieman, C. 1982. A rapid and sensitive high perfor- mance liquid chromatography method of following the action of chymosin in milk. Neth. Milk Dairy J. 36:153. Van Hooydonk, A.C.M., Olieman, C., and Hagedoom, H.G. 1984. Kinetics of the chymosin-catalysed proteolysis of x-casein in milk. Neth. Milk Dairy J. 37:207. Van Hooydonk, A.C.M., Hagedoom, H.G., and Boerrigter, U. 1986. The effect of various cations on the renneting of milk. Neth. Milk Dairy J. 40:369. Van Hooydonk, A.C.M., and Walstra, P. 1987. Interpretation of the kinetics of the renneting action in milk. Neth. Milk Dairy J. 41:19. Van Hooydonk, A.C.M., and Van der Berg, 1988. Control and determination of the curd-setting during Cheesemaking. Int. Dairy Fed. Bulleten No. 225. Vernon Carter, B.J., and Sherman, P. 1978. Evaluation of the firmness of Leicester cheese by compression tests with the Instron Universal Testing Machine. J. Texture Stud. 9:311. Visser, F.M.W. 1977. Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavor development in Gouda cheese. 1. Discription of cheese and aseptic Cheesemaking techniques. Neth. Milk Dairy J. 31:120. Visser, S., van Rooijen, P.J., and Slangen, CT. 1980. Peptide substrates for chymosin (Rennin) isolation and substrate behavior of two tryptic Fregments of bovine k-casein. Eur. J. Biochem. 108:415. Visser, J., Minihan, A., Smits, P., Tjan, SB, and Heertje, I. 1986. Effects of pH and temperature on the milk salt system. Neth. Milk Dairy J. 40:351. Voisey, P. 1975. Selecting deformation rate in texture tests. J. Texture Stud. 6:253. Vreeman, H.J., Markwijk, B.W., and Both, P. 1989. The structure of casein micelles between pH 5.5 and 6.7 as determined by light scattering, electron microscopy and voluminosity experiments. J. Dairy Res. 56:463. Walstra, HE, and Hargrove, R.C. 1972. Cheese of the World, Dover, NY. Walstra, P., and Van Vliet,T. 1982. Rheology of cheese. Int. Dairy Fed. Bull. Doc. 153. Walstra, P., and Jenness, R. 1984. Dairy Chemistry and Physics. John Wiley & Sons Publishers, NY. 233 Waugh, D.F. 1971. Formation and structure of casein micelles. In: "Milk Protein: Chemistry and molecular biology", Vol. 2, pp. 3-85. (H.A. Mckenzie, ed), Academic Press, NY. Weaver, J .C., and Kroger, M. 1978. Free amino acid and rheological measurements of hydrolyzed lactase cheddar cheese during ripening. J. Food Sci. 43:579. Weber, F. 1986. Curd drainage. In: "Cheese-making Science and Technology." (A. Eck, ed), translated by Thomas, C.D. Iavoisier Inc., NY. Wheelock, J .V., and Knight, DJ. 1969. The action of rennet on whole milk. J. Dairy Res. 36: 183. Wong, D.W.S. 1989. Proteins. In " Mechanism and Theory in Food Chemistry." (D.W.S. Wong, ed.), AVI Publishing Co. Inc., Van Nostrand Reinhold, NY. Woolf, CM. 1968. "Principles of Biometry", P. 205. Van Hostran Company, Inc., Princeton, NJ. Yang, C.S.T., and Taranto, M.V. 1982. Textural properties of mozzarella cheese analogs manufactured from soybeans. J. Food Sci. 47:906. Yang, C.S.T., Taranto, M.V., and Cheryan, M. 1983. Optimization of textural and morphological properties of a soy-gelatin Mozzarella cheese analoge. J. Food Process Preser. 7:41. Zoon, P. 1988. Rheological properties of rennet-induced skim milk gels. Ph.D. Thesis. Wageningen Agric. Univ., Wageningen, Netherlands. 111111