Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. DEPARTMENT OF FOOD TECHNOLOGY MASSEY UNIVERSITY PALMERSTON NORTH NEW ZEALAND RENNET COAGULATION PROPERTIES OF HEATED MILKS A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQurn.EMENTS FOR THE DEGREE OF MASTER OF TECHNOLOGY IN FOOD TECHNOLOGY ALGANE WAUNGANA 1995 MASSEY UNIVERSITY LIBRARY 1 ABSTRACT The effects of heat treatment at temperatures in the range 80 - 140°C for 4 s in a spiral flow indirect UHT plant on (i) the denaturation of whey proteins, (ii) their association with the casein micelles and incorporation into rennet gels and (iii) rennet coagulation properties of skim milk were determined. The extent of denaturation of J3-lactoglobulin (J3-Lg) and a-lactalbumin (a­ La), as determined by the decrease in the amounts of native protein (Native-PAGE) in the ultracentrifugal supernatants (100,000g for 1 h) of heated milks, increased with the severity of heat treatment. At all temperatures, J3-Lg was more sensitive to thermal denaturation than a -La. The extent of association of J3-Lg and a-La with the casein micelles, as determined by the total amounts of these proteins (SDS-PAGE) remaining in the ultracentrifugal supernatants of heated milks, also increased with the severity of heat treatment. At any given temperature, association of these proteins with casein micelles was much less than the amount that denatured. The extent of incorporation of J3-Lg and a -La into rennet gels increased with temperature and could be related to the levels of denaturation of J3-Lg and its association with casein micelles . Heat treatment impaired the rennet coagulation properties of milk as indicated by an increase in gelation times and a decrease in gel strengths (both determined using the Bohlin VOR Rheometer). There was a close correlation between the extents of association of J3-Lg with the casein micelles and the changes in gelation time or gel strength. When heated milks were acidified to pH 5.5 and re-neutralised to pH 6.5 (pH cycled), the adverse effects of heat treatment on rennet coagulation were reduced, except for those milks heated at temperatures above 120°C. The rennet coagulation properties of heated milks were markedly improved by addition of low concentrations of CaC12, but no additional improvement 11 resulted when CaC12 addition was combined with pH cycling. Concentration of skim milk by ultrafiltration CUF) lengthened gelation time but increased gel strength, the effect being dependent on the volume concentration ratio (VCR). Heat treatment of milk (140°C for 4 s) before or after UF increased gelation times and lowered gel strengths with weaker gels being formed from milks heated prior to UF. pH cycling of heated milk before UF or of heated UF concentrates had an adverse effect on rennet coagulation properties of the UF concentrates. When 3X UF concentrate was heated at temperatures in the range 80 - 140°C for 4 s, gelation time did not change with temperature between 80 and 120°C but more severe heat treatments caused an increase. In contrast, the gel strength decreased gradually with increase in heating temperature. These changes in rennet coagulation properties were related to the extent of denaturation of ~-Lg and its association with the casein micelles. 111 ACKNOWLEDGEMENTS I would like to express my deepest appreciation to my supervisor, Dr. Harjinder Singh, for introducing me to this subject and for his unfailing commitment, support and expert advice in all stages of this research project. I am also grateful for the encouragement and constructive criticism received from my co-supervisor, Mr Rod Bennett, especially during compilation of this manuscript. Ms June Latham is greatly acknowledged for her help on the Bohlin Rheometer and her patience and willingness to be called up at home ( often at odd hours) to come and tend to minor difficulties with the machine. The technical assistance received from Messrs Steve Glasgow, Alistair Young, Garry Radford and Byron McKillop is greatly appreciated as is the help received from other members of staff in the Food Technology department. I would like to thank my fellow graduate students for their friendship and helpful discussions on aspects of my study particularly David Oldfield for his assistance in the techniques of gel electrophoresis . I enjoyed some great Kiwi hospitality over my two years in New Zealand from the following families to whom I extend a big thank you: Mr and Mrs W. Gartner, Mr and Mrs R. van Laar-Veth, Mr and Mrs A. Schaw and Mr and Mrs J. van Laar. Special thanks are due to Jackie Gezimati for her continued friendship and to my family for their support and encouragement throughout my studies. Finally, I would like to thank the New Zealand Ministry of Foreign Affairs and Trade for financing both my stay and study in New Zealand through the award of an ODA Scholarship. TABLE OF CONTENTS ABSTRACT ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES CHAPTER 1. INTRODUCTION CHAPTER 2. REVIEW OF LITERATURE 2.1 Milk 2.2 Milk proteins 2.2.1 Casein micelles 2.2.2 Whey proteins 2.3 Milk salts 2.4 Effect of heat on milk 2 .4 .1 Changes in milk proteins 2.4.2 Changes in milk salts 2.5 Rennet coagulation of milk 2.5.1 Mechanism of coagulation 2.5.2 Factors affecting rennet coagulation 2.6 Cheese manufacture IV l 111 iv Vlll XIV 1 3 3 3 3 6 7 8 8 11 11 12 15 22 2. 7 Incorporation of whey proteins in cheese 23 2.7.1 Use of ultra-filtration (UF) m cheese manufacture 24 2.8 The rennet coagulation properties of UF concentrates 28 2.8.1 Rennet curds formed from UF concentrates 29 V 2.9 Heat treatment of UF concentrated milks 30 2.9.1 Influence of heat treatment on rennet coagulation properties of UF concentrated milks 31 CHAPTER3.0BJECTIVES 32 CHAPTER 4. MATERIALS AND METHODS 33 4.1 Materials 33 4.1.1 Milk source 33 4.1.2 Rennet source 33 4.2 Processing methods 33 4.2.1 Preparation of skim milk 33 4.2.2 Concentration of skim milk 33 4.2 .3 Heat treatment of skim milk and UF concentrates 34 4.3 Analytical methods 34 4.3.1 Total solids 34 4.3.2 Milk protein concentration 34 4.3 .3 Polyacrylamide gel electrophoresis (PAGE) 34 4.4 Experimental procedures 40 4.4.1 Residual native whey protein and total non- sedimentable whey protein 40 4.4.2 Rennet coagulation properties 41 4.4.3 Whey protein incorporation into skim milk CHAPTER 5. rennet gels 43 INFLUENCE OF THERMAL DENATURATION AND AGGREGATION OF WHEY PROTEINS ON RENNET COAGULATION OF SKIM MILK 44 5.1 Whey protein denaturation and association with casein micelles 5 .1.1 Effect of heating temperature 5.1.2 Effect of pH at heating 5.2 Incorporation of whey proteins into rennet gels 5.2.1 Effect of heating temperature 5.2.2 Effect of pH at heating 5.3 Relationship between the state of whey proteins and Vl 45 45 49 52 52 54 their incorporation into rennet gels 54 5.4 Effect of heat treatment on rennet coagulation properties of milk 5.4.1 Effect of heating temperature 5.4.2 Effect of pH at heating 5.5 Relationship between the state of whey proteins and 57 59 63 rennet coagulation properties 66 5.5.1 Relationship between state of whey proteins and GT 67 5.5.2 Relationship between state of whey proteins and G' 5.6 Conclusions 69 72 CHAPTER 6. RENNET COAGULATION PROPERTIES OF HEATED MILKS: EFFECTS OF pH ADJUSTMENT AND CaCI2 ADDITION 74 6.1 Effect of pH cycling on the rennet coagulation properties of heated milks 7 4 6.1.1 Effect of heating temperature 6.1.2 Effect of pH at heating 6.2 Effect of CaC12 addition and pH cycling 6.3 Conclusions 74 79 81 87 vu CHAPTER 7. RENNET COAGULATION PROPERTIES OF MILKS CONCENTRATED BY ULTRAFILTRATION 88 7 .1 Concentration of skim milk by UF 89 7 .1.1 Changes in protein and total solids concentration 89 7 .1.2 Changes in rennet coagulation properties 90 7 .2 Ultra-high temperature (UHT) treatment of UF concentrates 95 7.2.1 pH cycling of UHT treated UF concentrates 99 7.3 UHT treatment of skim milk prior to concentration by UF 99 7.3.1 pH cycling of UHT treated skim milk prior to concentration by UF 103 7.4 General discussion on the effects of UHT and pH cycling prior to or after UF 103 7 .5 Heat treatment of UF concentrates (3X) at different temperatures 106 7.5.1 Rennet coagulation properties of heat treated UF (3X) concentrates 106 7 .5.2 Relationship between the state of whey proteins in the 3X UF concentrate and its rennet coagulation properties 108 7.6 Conclusions 111 CHAPTER 8. GENERAL DISCUSSION AND CONCLUSIONS 113 8.1 Mechanisms of rennet gel formation 113 8.2 Restoration of coagulation properties of heated milks 116 8.2.1 Influence of concentration by UF 118 8.3 Implications in cheese manufacture and product development REFERENCES 118 120 LIST OF FIGURES Figure Title 2.1 Structure of the casein micelle. 5.1 Loss of native ~-Lg (o) and a-La(•) from the ultracentrifugal supernatants (100,000g for 60 min) obtained from skim milk heated at temperatures in the range 80 - 140°C for 4 s (estimated using quantitative Native-PAGE). 5.2 Changes in the amounts of total ~-Lg (o) and a-La(•) (native and aggregated) remaining in the ultracentrifugal supernatants obtained from skim milk heated at temperatures in the range 80 - 140°C for 4 s (estimated using Vlll Page 5 46 quantitative SDS-PAGE). 46 5.3 Relationship between loss of native ~-Lg (from Figure 5.1) and the changes in the amounts of non-sedimentable ~-Lg (from Figure 5.2). 48 5.4 Influence of pH at heating on the amounts of total ~-Lg (o) and a-La (•) (native and aggregated) remaining in the ultracentrifugal supernatants (estimated using quantitative SDS-PAGE). Heat treatment was at 140°C for 4 s. 50 5.5 Influence of pH at heating on the formation of non­ sedimentable K-casein (estimated using quantitative SDS- PAGE) on heating skim milk at 140°C for 4 s. 50 5.6 Changes in the amounts of residual ~-Lg (o) and a-La(•) in rennet whey obtained from skim milk heated at temperatures in the range 80 - 140°C for 4 s (estimated using quantitative SDS-PAGE). 5.7 Influence of pH at heating on the amount of residual ~-Lg (o) and a-La ( ... ) in rennet whey (estimated using quantitative lX 53 SDS-PAGE). Heat treatment was at 140°C for 4 s. 53 5.8a Relationship between native ~-Lg (Figure 5.1) and residual~- Lg in rennet whey (Figure 5.6). 55 5.8b Relationship between residual ~-Lg in rennet whey (Figure 5.6) and non-sedimentable ~-Lg (Figure 5.2). 55 5.9 The changes of moduli and phase angle with time during the development of rennet gels. Complex modulus, G• (•);storage modulus, G' (o); loss modulus, G" (•); phase angle, 8 (~). Zero time indicates addition of rennet. 5.10 The changes in gel strength (G') with time during rennet coagulation of heated skim milks. Unheated control skim milk (o), skim milk heated at 80 (•), 90 (D), 100 (•), 110 (~), 58 120 ( ... ), 130 (v) or 140°C (•) for 4 s. 58 5.11 Gelation times (GT) of skim milks which have been heated for 4 sat temperatures in the range 80 - 140°C. 5 .12 Gel strengths, G', (D) and rates of curd firming K, ( •) for skim milks which have been heated for 4 s at temperatures in the 60 range 80 - 140°C. 60 5.13 Influence of pH at heating on the changes in G' with time. Skim milk samples were adjusted to pH 6.4 (•), 6.6 (~), 6.8 ( ... ), 7.0 (0) or 7.3 (*) and heated at 140°C for 4 s. The pH of all samples was readjusted to 6.5 before renneting. 5.14 Gelation times of skim milks which have been adjusted to pH values in the range 6.4 - 7 .3 and heated at 140°C for 4 s. Heated samples (•), unheated control (0). The pH of all samples was readjusted to 6.5 before renneting. 5.15 Gel strength, G', (0) and rate of curd firming, K, (•) for skim milks which have been adjusted to pH values in the range 6.4 - 7.3 and heated at 140°C for 4 s. G' for unheated control (o), K for unheated control ( • ). Renneting was carried out for all X 62 62 samples at pH 6.5. 64 5.16 The pH-dependent reversibility of the amounts of non­ sedimentable 13-Lg and K-casein. Amounts of non­ sedimentable (100,000g for 60 min) f3-Lg (o) and K-casein (•) at the heating pH. Amounts of non-sedimentable f3-Lg (•) and K-casein (~) after readjustment to pH 6.5. 5.17 Relationship between residual native f3-Lg (o) (from Figure 5.1), or non-sedimentable f3-Lg (•) (from Figure 5.2) and gelation time (from Figure 5.11). 5.18 Relationship between the amounts of residual native f3-Lg (o) (from Figure 5 .1) or non-sedimentable f3-Lg (•) (from Figure 66 67 5.2) and gel strength (G'). 70 5.19 Relationship between residual native 13-Lg (o) or non- sedimentable 13-Lg ( •) and G' or GT. 70 6.1 Experimental procedure for determining the effects of acidification and subsequent reneutralisation (i.e. pH cycling) on the rennet coagulation properties of heated milks. 6.2 Gelation times (GT) of skim milks heated at temperatures in the range 80 - 140°C for 4 s (D). Heated skim milks adjusted to pH 5.5, left for 2 hat 20°C, and readjusted to pH 6.5 (•). All heated samples were renneted at pH 6.5. 6.3 Gel strengths, G', (D) and rates of curd firming, K, (a) of skim milks heated at temperatures in the range 80 - 140°C for 4 s . G' (•) and K (.t.) of heated skim milks adjusted to pH 5.5, left for 2 hat 20°C, and readjusted to pH 6.5. 6.4 Gelation times (GT) of skim milks heated at 140°C for 4 s at pH values in the range 6.4 - 7.3 (•). Skim milks heated at 140°C for 4 s at pH values in the range 6.4 - 7 .3, acidified to pH 5.5, left for 2 h at 20°C, and readjusted to pH 6.5. Raw skim milk (a), raw skim milk acidified to pH 5.5, left for 2 h at 20°C, and readjusted to pH 6.5 (.t.). All samples were X1 75 76 76 renneted at pH 6.5 . 80 6.5 Gel strengths, G', (D) and rates of curd firming, K, (o) of skim milks heated at 140°C for 4 sat pH values in the range 6.4 - 7 .3. G' (•) and K (•)of milks heated at different pH values, acidified to pH 5.5, and reneutralised to pH 6.5. G' (a) and K (•) for raw skim milk. G' (v) and K (,.) for raw milks which have been acidified to pH 5.5 and reneutralised to pH 6.5. 80 6.6 Experimental procedure for the determination of the effects of acidification and reneutralisation (pH cycling), addition of CaC12 or combined effect of pH cycling and CaC12 addition on rennet coagulation properties of heated milks. 6.7 Influence of CaC12 addition on the changes in G' with time during renneting of heated (140°C for 4 s) and unheated milks. Unheated milk with no CaC12 added (o), lmM CaC12 added ( • ), or 2mM CaC12 added (D). Heated milk with no CaCl2 added(•), lmM CaC12 added ( v), or 2mM CaCl2 added Xll 82 (•). 83 6.8 The combined effect of acidification and reneutralisation (pH cycling) and CaC12 addition on the changes of G' with time during renneting of heated milk (140°C for 4 s). No CaC12 added- raw milk (control) (o), heated milk (•); heated milk with 2m.M CaC12 added (v); heated and pH cycled milk with no CaC12 added (D); or heated and pH cycled milk with 2m.M CaC12 a~ded ( • ). 86 7.1 The changes in gel strength (G') with time during renneting at (A) natural pH of normal skim milk (o), 2X UF concentrate (D) or 3X UF concentrate (.6.) and (B) at pH 6.50 of normal skim milk(•), 2X UF concentrate(•) or 3X UF concentrate ( "). 7 .2 Experimental procedure for the effect of UHT treatment and pH cycling on the rennet coagulation properties of milk 91 concentrated by UF. 95 7 .3 The changes in G' with time during renneting at pH 6.5 of (A) unheated normal milk (o), 2X UF concentrate (D), 3X UF concentrate (6.) or (B) UHT treated normal milk ( • ), 2X UF concentrate(•), 3X UF concentrate (•)and UHT treated and pH cycled normal milk (0), 2X UF concentrate(+) or 3X UF concentrate(*). Unheated normal milk (o) is also shown in (B). 7.4: Experimental procedure for determination of the effect of concentration by UF on the rennet coagulation properties of Xlll 96 UHT treated milk. 100 7 .5 The changes in gel strength (G') with time during renneting of unheated normal milk ( o ), normal milk UHT treated before UF to VCR 2X (6.), 3X ( v), normal milk UHT treated and pH cycled(•), normal milk UHT treated and pH cycled before UF to VCR 2X (•) or 3X (' .. ). 101 7.6 The changes in gel strength (G') with time during renneting coagulation of heated 3X UF concentrates. Unheated control 3X UF concentrate (o), UF concentrate (3X) heated at 80 (•), 90 (D), 100 (•), 110 (6.), 120 (•), 130 ( '#) or 140°C (v) for 4 s. 106 7 .7 Relationship between residual native P-Lg (o), or non­ sedimentable P-Lg ( •) and gelation time. 7.8 Relationship between the amounts residual native P-Lg (o) or 109 non-sedimentable P-Lg ( •) and gel strength (G'). 109 8 .1 Schematic diagram of the attack on casein micelles by rennet. 8.2 Schematic diagram of the effect of heat treatment of milk on the attack on casein micelles by rennet. 114 116 LIST OF TABLES Table Title 2.1 Relative diameters of milk constituents. 6.1 Effect of CaC12 addition and pH cycling on the rennet coagulation properties of heated milk. 7 .1 pH, protein and total solids content of UF skim milk concentrates. 7.2 The effect of pH adjustment on the rennet coagulation properties of UF concentrated milks. 7.3 The effect of UHT treatment of UF concentrates on their rennet coagulation properties. 7.4 The effect of UHT treatment (and pH cycling) before UF on rennet coagulation properties. 7.5 Effects of heat treatment of UF concentrates (3X) on (rennet) gelation time, rate of curd firming and gel strength. 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