《乳品生物化学》（英文版） 10 Chemistry and biochemistry of cheese and fermented milks.pdf_大学文库

10 Chemistry and biochemistry of cheese and fermented milks 10.1 Introduction Cheese is a very varied group of dairy products, produced mainly in Europe, North and South America. Australia and New Zealand and to a lesser extent in North Africa and the Middle East, where it originated during the gricultural Revolution, 6000-8000 years ago. Cheese production and con sumption, which vary widely between countries and regions(Appendic DA and 10B), is increasing in traditional producing countries(2-4% pa for several years)and is spreading to new areas. On a global scale, 30% of all milk is used for cheese; the proportion is about 40% in North America and about 50% in the European Union Although traditional cheeses have a rather high fat content, they are rich sources of protein and in most cases of calcium and phosphorus and have anticarigenic properties; some typical compositional data are presented in Table 10. 1. Cheese is the classical example of a convenience food: it can be d as the main course in a meal, as a dessert or snack as a sandwich filler food ingredient or condiment. There are at least 1000 named cheese varieties, most of which have very limited production. The principal families are Cheddar, Dutch, Swiss and Pasta filata(e.g. Mozzarella), which together account for about 80% of total cheese production. All varieties can be classified into three superfamilies based on the method used to coagulate the milk, i. e. rennet coagulation (representing about 75% of total production ), isoelectric(acid)coagulati and a combination of heat and acid (which represents a very minor group) Production of cheese curd is essentially a concentration process in which the milkfat and casein are concentrated about tenfold while the whey proteins, lactose and soluble salts are removed in the whey. The acid coagulated and acid heat-coagulated cheeses are normally consumed fresh but the vast majority of rennet-coagulated cheeses are ripened(matured )for a period ranging from 3 weeks to more than 2 years, during which numerous microbiological, biochemical, chemical and physical changes occur, resulting in characteristic flavour, aroma and texture. the biochemistry of cheese ripening is very complex and is not yet completely understood

10 Chemistry and biochemistry of cheese and fermented milks 10.1 Introduction Cheese is a very varied group of dairy products, produced mainly in Europe, North and South America, Australia and New Zealand and to a lesser extent in North Africa and the Middle East, where it originated during the Agricultural Revolution, 6000-8000 years ago. Cheese production and consumption, which vary widely between countries and regions (Appendices 10A and lOB), is increasing in traditional producing countries (2-4% p.a. for several years) and is spreading to new areas. On a global scale, 30% of all milk is used for cheese; the proportion is about 40% in North America and about 50% in the European Union. Although traditional cheeses have a rather high fat content, they are rich sources of protein and in most cases of calcium and phosphorus and have anticarigenic properties; some typical compositional data are presented in Table 10.1. Cheese is the classical example of a convenience food: it can be used as the main course in a meal, as a dessert or snack, as a sandwich filler, food ingredient or condiment. There are at least 1000 named cheese varieties, most of which have very limited production. The principal families are Cheddar, Dutch, Swiss and Pasta filata (e.g. Mozzarella), which together account for about 80% of total cheese production. All varieties can be classified into three superfamilies based on the method used to coagulate the milk, i.e. rennet coagulation (representing about 75% of total production), isoelectric (acid) coagulation and a combination of heat and acid (which represents a very minor group). Production of cheese curd is essentially a concentration process in which the milkfat and casein are concentrated about tenfold while the whey proteins, lactose and soluble salts are removed in the whey. The acidcoagulated and acid/heat-coagulated cheeses are normally consumed fresh but the vast majority of rennet-coagulated cheeses are ripened (matured) for a period ranging from 3 weeks to more than 2 years, during which numerous microbiological, biochemical, chemical and physical changes occur, resulting in characteristic flavour, aroma and texture. The biochemistry of cheese ripening is very complex and is not yet completely understood

380 DAIRY CHEMISTRY AND BIOCHEMISTRY Table 10.1 Composition of selected cheeses (per 100 g) Water Protein Fat Cholesterol Energy Cheese type (9) (g) (g) (mg) (kJ) Brie Caerphilly Camern bert Cheddar Cheshire Cottage Cream cheese Danish blue Edam Emmental Feta Fromage frais Gouda Gruyere Mozzarella Parmesan Ricotta Roquefort Stilton 48.6 41.8 50.7 36.0 40.6 79.1 45.5 45.3 43.8 35.7 56.5 77.9 40.1 35.0 49.8 18.4 72.1 41.3 38.6 19.3 23.2 20.9 25.5 24.0 13.8 3.1 20.1 26.0 28.7 15.6 6.8 24.0 21.2 25.1 39.4 9.4 19.7 22.7 26.9 31.3 23.1 34.4 31.4 3.9 47.4 29.6 25.4 29.7 20.2 7.1 31.0 33.3 21.0 32.1 11.0 32.9 35.5 100 90 15 100 90 13 95 75 80 90 70 25 100 100 65 100 50 90 105 1323 1554 1232 1708 1571 413 1807 1437 1382 1587 1037 469 1555 1695 1204 1880 599 1552 1701 10.2 Rennet-coagulated cheeses The production of rennet-coagulated cheeses can, for convenience, be divided into two phases: (1) conversion of milk to curds and (2) ripening of the curds. 10.2.1 Preparation and treatment of cheesemilk The milk for most cheese varieties is subjected to one or more pretreatments (Table 10.2). The concentrations of fat and casein and the ratio of these components are two very important parameters affecting cheese quality. While the concentrations of these components in cheese are determined and controlled by the manufacturing protocol, their ratio is regulated by adjusting the composition of the cheesemilk. This is usually done by adjusting the fat content by blending whole and skimmed milk in proportions needed to give the desired fat : casein ratio in the finished cheese, e.g. 1.0:0.7 for Cheddar or Gouda. It should be remembered that about 10% of the fat in milk is lost in the whey while only about 5% of the casein is lost (unavoidably, see section 10.2.2). With the recent commercial availability of ultrafiltration, it has become possible to increase the concentration of casein, thus levelling out seasonal variations in milk composition and consequently in gel characteristics and

CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 381 Table 10.2 Pre-treatment of cheese milk Standardization of fat: protein ratio Addition of ski Removal of some fat Addition of ultrafiltration retentate Addition of CaCI Adjustment of pH (e.g. by gluconic acid-8-lactone) ization(eg.72C×15s) Microfiltration cheese quality. The capacity of a given plant is also increased by pre concentrating milk by ultrafiltration The pH and the concentration of calcium in milk also vary, with consequential effects on the properties of renneted milk gels. The addition of CaCI, to cheesemilk (0.02%)is widely practised and adjustment and standardization of milk pH by using the acidogen, gluconic acid-d-lactone (GDL), is recommended and commercially practised on a limited scale Although raw milk is still widely used for cheese manufacture, e.g Parmigiano-Reggiano(Italy ) Emmental(Switzerland), Comte and Beaufort (France)and many less well known varieties, both on a factory and farmhouse scale, most Cheddar and Dutch-type cheeses are produced from pasteurized milk(HTST; c. 72"C x 15s). Pasteurization is used primarily to kill pathogenic and spoilage bacteria. However, desirable indigenous bac teria are also killed by pasteurization and it is generally agreed that cheese made from pasteurized milk ripens more slowly and develops a less intense flavour than raw milk cheese, apparently because certain, as yet unidentified, indigenous bacteria are absent. At present, some countries rec quire tha heese milk should be pasteurized or the cheese aged for at least 60 days (during which time pathogenic bacteria die off). a global requirement for pasteurization of cheesemilk has been recommended but would create restrictions for international trade in cheese, especially for many of those with 'Appellation d'Origine Protegee' status. Research is under way to identify the important indigenous microorganisms in raw milk cheese for use as inoculants for pasteurized milk. While recognizing that pasteurization is very important in ensuring safe cheese, pH (below about 5.2)and water activity(aw, which is controlled by addition of Nacl)are also critical safety hurdles Milk may be thermized (c. 65C x 15s)on receipt at the factory to reduce bacterial load, especially psychrotrophs, which are heat labile. Since thermization does not kill pathogens, thermized milk is usually fully pasteurized before cheesemaking

CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 381 Table 10.2 Pre-treatment of cheese milk Standardization of fat: protein ratio Addition of skim milk Removal of some fat Addition of ultrafiltration retentate Addition of CaCI, Adjustment of pH (e.g. by gluconic acid-6-lactone) Removal or killing of contaminating bacteria Thermization (e.g. 65°C x 15 s) Pasteurization (e.g. 72°C x 15 s) Bactofugation Microfiltration cheese quality. The capacity of a given plant is also increased by preconcentrating milk by ultrafiltration. The pH and the concentration of calcium in milk also vary, with consequential effects on the properties of renneted milk gels. The addition of CaCl, to cheesemilk (0.02%) is widely practised and adjustment and standardization of milk pH by using the acidogen, gluconic acid-d-lactone (GDL), is recommended and commercially practised on a limited scale. Although raw milk is still widely used for cheese manufacture, e.g. Parmigiano-Reggiano (Italy), Emmental (Switzerland), Comte and Beaufort (France) and many less well known varieties, both on a factory and farmhouse scale, most Cheddar and Dutch-type cheeses are produced from pasteurized milk (HTST; c. 72°C x 15 s). Pasteurization is used primarily to kill pathogenic and spoilage bacteria. However, desirable indigenous bacteria are also killed by pasteurization and it is generally agreed that cheese made from pasteurized milk ripens more slowly and develops a less intense flavour than raw milk cheese, apparently because certain, as yet unidentified, indigenous bacteria are absent. At present, some countries require that all cheese milk should be pasteurized or the cheese aged for at least 60days (during which time pathogenic bacteria die off). A global requirement for pasteurization of cheesemilk has been recommended but would create restrictions for international trade in cheese, especially for many of those with ‘Appellation d’Origine Protegee’ status. Research is under way to identify the important indigenous microorganisms in raw milk cheese for use as inoculants for pasteurized milk. While recognizing that pasteurization is very important in ensuring safe cheese, pH (below about 5.2) and water activity (aw, which is controlled by addition of NaCl) are also critical safety hurdles. Milk may be thermized (c. 65°C x 15s) on receipt at the factory to reduce bacterial load, especially psychrotrophs, which are heat labile. Since thermization does not kill pathogens, thermized milk is usually fully pasteurized before cheesemaking

DAIRY CHEMISTRY AND BIOCHEMISTRY Clostridium tyrobutyricum(an anaerobic spore-former) causes late gas blowing(through the production of H, and CO,)and off-flavours(butanoic acid) in many hard ripened cheeses: Cheddar-type cheeses are major exceptions. Contamination of cheese milk with clostridial spores can be avoided or kept to a very low level by good hygienic practices(soil and silage are the principal sources of clostridia) but they are usually prevented from growing through the use of sodium nitrate(naNO,)or, less frequently, lysozyme, and or removed by bactofugation(centrifugation) or microfiltra 10.2.2 Conversion of milk to cheese curd Typically, five steps, or groups of steps, are involved in the conversion of milk to cheese curd: coagulation, acidification, syneresis(expulsion of whey) moulding/ shaping and salting. These steps, which partly overlap, enable the cheesemaker to control the composition of cheese, which, in turn, has a major influence on cheese ripening and quality Enzymatic coagulation of milk. The enzymatic coagulation of milk involves modification of the casein micelles via limited proteolysis by selected proteinases, called rennets, followed by calcium-induced aggregation of the rennet-altered micelles: Rennet Casein Para-casein+ Macropept Ca2·,-30C If present, the fat globules are occluded in the gel but do not participate in the formation of a gel matrix As discussed in Chapter 4, the casein micelles are stabilized by K-casein hich represents 12-15% of the total casein and is located mainly on the rface of the micelles such that its hydrophobic N-terminal region reacts hydrophobically with the calcium-sensitive hydrophilic C-terminal region protrudes into the surrounding aqueous environment, stabilizing the micelles by a negative surface charge and steric stabilization c Following its isolation in 1956, it was found that K-casein is the only sein hydrolysed during the rennet coagulation of milk and that it was hydrolysed specifically at the Phe1os-Met1os bond, producing para-K casein(K-CN fl-105)and macropeptides(f106-169: also called glycomac ropeptides since they contain most or all of the sugar groups attached K-casein)(Figure 10.1). The hydrophilic macropeptides diffuse into the surrounding medium while the para-k-casein remains attached to the

382 DAIRY CHEMISTRY AND BIOCHEMISTRY Clostridium tyrobutyricum (an anaerobic spore-former) causes late gas blowing (through the production of H, and CO,) and off-flavours (butanoic acid) in many hard ripened cheeses; Cheddar-type cheeses are major exceptions. Contamination of cheese milk with clostridial spores can be avoided or kept to a very low level by good hygienic practices (soil and silage are the principal sources of clostridia) but they are usually prevented from growing through the use of sodium nitrate (NaNO,) or, less frequently, lysozyme, and/or removed by bactofugation (centrifugation) or microfiltration. 10.2.2 Typically, five steps, or groups of steps, are involved in the conversion of milk to cheese curd: coagulation, acidification, syneresis (expulsion of whey), moulding/shaping and salting. These steps, which partly overlap, enable the cheesemaker to control the composition of cheese, which, in turn, has a major influence on cheese ripening and quality. Conversion of milk to cheese curd Enzymatic coagulation of milk. The enzymatic coagulation of milk involves modification of the casein micelles via limited proteolysis by selected proteinases, called rennets, followed by calcium-induced aggregation of the rennet-altered micelles: Casein Rennet ____.* Para-casein + Macropeptides Ca?', - 30°C Gel If present, the fat globules are occluded in the gel but do not participate in the formation of a gel matrix. As discussed in Chapter 4, the casein micelles are stabilized by ti-casein, which represents 12-15% of the total casein and is located mainly on the surface of the micelles such that its hydrophobic N-terminal region reacts hydrophobically with the calcium-sensitive clsl-, cts2- and 0-caseins while its hydrophilic C-terminal region protrudes into the surrounding aqueous environment, stabilizing the micelles by a negative surface charge and steric stabilization. Following its isolation in 1956, it was found that ti-casein is the only casein hydrolysed during the rennet coagulation of milk and that it was hydrolysed specifically at the Phe,,,-Met,,, bond, producing para-lccasein (K-CN fl- 105) and macropeptides (f106- 169; also called glycomacropeptides since they contain most or all of the sugar groups attached to ti-casein) (Figure 10.1). The hydrophilic macropeptides diffuse into the surrounding medium while the para-#-casein remains attached to the

CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 383 yro Glu-Glu-GIn-Asn-Gln-Glu-GIn-Pro-lle-Arg-Cys-Glu-Lys-Asp-Glu-Arg-Phe-Phe-Ser-Asp Lys-lle-Ala-Lys-Tyr-lle-Pro-lle-GIn-Tyr-Val-Leu-Ser-Arg-Tyr-Pro-Ser-Tyr-Gly-Lew Asn-Tyr-Tyr-GIn-GIn-Lys-Pro-Val-Ala-Leu-nle-Asn-Asn-GIn-Phe-Leu-Pro-Tyr-Pro-Tyr Tyr-Ala-Lys-Pro-Ala-Ala-Val-Arg-Ser-Pro-Ala-GIn-lle-Leu-GIn-Trp-GIn-Val-Leu-Ser- sn-Thr-Val-Pro-Ala-Lys-Ser-Cys-GIn-Ala-GIn-Pro-Thr-Thr-Met-Ala-Arg- His-Pro His Met-Ala-lle-Pro-Pro-Lys-Lys-Asn-GIn-Asp-Lys-Thr-Glu-lle-Pro- lle (variant B) Thr-Ile-Asn-Thr-lle-Ala-Ser-Gly-Glu-Pro-Thr- Ser-Thr-Pro-Thr--Glu-Ala-Val-Glu Thr (Variant A) Set Thr-val- Ala- Th-Leu-G u Ala serp ro) - vAle lu--.- lu- pe. sn. Asp(Variant A) Thr- Val-GIn. Val- Thr-Ser-Thr-Ala- val.OH Figure 10.1 Amino acid sequence of k-casein, showing the principal chymosin cleavage site (1) ligosaccharides are attached at some or all of the threonine residues shown micelle core(the macropeptides represent c. 30% of K-casein, i.e. 4-5% of total casein; this unavoidable loss must be considered when calculating the ield of cheese). Removal of the macropeptides from the surface of the casein micelles reduces their zeta potential from about -20 to mv and removes the steric stabilizing layer. The proteolysis of k-casein is referred to as the primary(first) phase of rennet-coagulation. When about 85% of the total K-casein in milk has been hydrolysed, the colloidal stability of the micelles is reduced to such an extent that they coagulate at temperatures greater than about 20C (c. 30 C is used in heesemaking), an event referred to as the secondary phase of rennet coagulation. Calcium ions are essential for the coagulation of rennet-altered micelles (although the binding of Ca- by casein is not affected by renneting The Phe,os-Met1o6 bond of K-casein is several orders of magnitude more sensitive to rennets than any other bond in the casein system. The reason(s) or this unique sensitivity has not been fully established but work on synthetic peptides that mimic the sequence of k-casein around this bond has provided valuable information. The Phe and Met residues themselves are not essential, e.g. both Phe1os and Met10 can be replaced or modified without drastically changing the sensitivity of the bond -in human, porcine and rodent K-caseins, Met,os is replaced by lle or Leu, and the proteinase Cryphonectria parasitica (section 10.2.2.2), hydrolyses the bond 4-Phe,os rather than Phe1os-Met106. The smallest K-casein-like pept

CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 383 1 Fyro Glu-Glu-Gln-Asn-Gln-Glu-GIn-Pro-Ile-Arg-Cys-GIu-Lys-Asp-GIu-Arg-Phe-Phe-Ser-Asp- 21 Lys-Ile-Ala-Lys-Tyr-lle-Pro-lle-GIn-Tyr-Val-Leu-Ser-Arg-Tyr-Pro-Ser-Tyr-Gly-Leu- 41 Asn-Tyr-Tyr-Gln-Gln-Lys-Pro-Val-Ala-Leu-Ile-Asn-Asn-Gln-Phe-Leu-Pro-Tyr-Pro-Tyr- 61 Tyr-AIa-Lys-Pro-Ala-Ala-Val-Arg-Ser-Pto-Ala-G1n-lle-Leu-Gln-Trp-GIn-Val-Leu-Ser- 81 Asn-Thr-Val-Pro-Ala-L ys-Ser-Cys-G1 n-Ala-Gln-Pro-Thr-Thr-Met-Ala-Arg-His-Pro-His- 101 105 106 Pro-His-Leu-Ser-Ph~et-Ala-lle-Pro-Pro-Lys-Ly~-Asn-Gln-As~-~ys-~r-Glu-IIe-Pro- 121 Ile (Variant B) Thr-He-Asn-Thr-Ile-Ala-Ser-Gly-Glu-Pro-Thr- Ser-Thr -Pro-Thr - -Glu-Ala-Val-GluThr (Variant A) 141 Ala (Variant 8) Ser-Thr -Val-Ala-Thr-Leu-Glu- -SerP - Pro-Glu-Val-lle-Glu-Ser-Pro-Pro-G1u-Ile-AsnAsp (Variant A) 161 169 Thr-Val-GIn-Val-Thr-Ser-Thr-Ala-Val.OH Figure 10.1 Amino acid sequence of K-casein, showing the principal chymosin cleavage site (I); oligosaccharides are attached at some or all of the threonine residues shown in italics. micelle core (the macropeptides represent c. 30% of Ic-casein, i.e. 4-5% of total casein; this unavoidable loss must be considered when calculating the yield of cheese). Removal of the macropeptides from the surface of the casein micelles reduces their zeta potential from about -20 to -1OmV and removes the steric stabilizing layer. The proteolysis of ic-casein is referred to as the primary (first) phase of rennet-coagulation. When about 85% of the total ic-casein in milk has been hydrolysed, the collojdal stability of the micelles is reduced to such an extent that they coagulate at temperatures greater than about 20°C (c. 30°C is used in cheesemaking), an event referred to as the secondary phase of rennet coagulation. Calcium ions are essential for the coagulation of rennet-altered micelles (although the binding of Ca2+ by casein is not affected by renneting). The Phe,,,-Met,,, bond of ic-casein is several orders of magnitude more sensitive to rennets than any other bond in the casein system. The reason(s) for this unique sensitivity has not been fully established but work on synthetic peptides that mimic the sequence of Ic-casein around this bond has provided valuable information. The Phe and Met residues themselves are not essential, e.g. both Phe,,, and Met,,, can be replaced or modified without drastically changing the sensitivity of the bond - in human, porcine and rodent Ic-caseins, Met,,, is replaced by Ile or Leu, and the proteinase from Cryphonectria parasitica (section 10.2.2.2), hydrolyses the bond Ser,,,-Phe,,, rather than Phe,,,-Met,,,. The smallest Ic-casein-like pept-

DAIRY CHEMISTRY AND BIOCHEMISTRY (compiled from Visser et al, 1976: Visser, Langen and R Table 10.3 Kinetic parameters for hydrolysis of K-c ptides by chymosin at pH 4.7 len,1987) Peptide quence s" mM) S.F. M.A. I S.F. IP 0.114 S F M.A.I. P F M.A. I.P. PK 04-111 L.S.F. M.A 183 38.1 L.S. F.M.A.L.P. P 103-110 L.S. F M.A.L.P. P.K.K. 103-112 30 P H L.S. F M 01-10833.5 H.P. H.P.H. L.S. F.M.A. I PPK 98-111 662 2509 98-111 A-Casein 0001-0005200-2000 L.S.F. (NO2)Nle A L, OMe 12.7 pH 6.6. ide hydrolysed by chymosin is Ser. Phe. Met. Ala Ile(K-CN f104-108 ): extend- ing this peptide from its C and or n terminus increases its susceptibility to chymosin (i.e. increases kca /Km); the peptide k-CN f98-111 is as good a substrate for chymosin as whole K-casein(Table 10. 3 ). Ser,04 appears to be ssential for cleavage of the Phe1os-Met,os bond by chymosin, and the hydrophobic residues, Leu,o3, Ala107 and Ile,o8 are also important Rennets. The traditional rennets used to coagulate milk for most cheese varieties are prepared from the stomachs of young calves, lambs or kids by extraction with NaCl (c. 15%)brines. The principal proteinase in such nnets is chymosin; about 10% of the milk-clotting activity of calf rennet is due to pepsin. As the animal ages, the secretion of chymosin declines while that of pepsin increases; in addition to pepsin, cattle appear to secrete a chymosin-like enzyme throughout life Like pepsin, chymosin is an aspartyl(acid) proteinase, i.e. it has two ssential aspartyl residues in its active site which is located in a cleft in the globular molecule (molecular mass 36kDa)(Figure 10. 2 ). Its pH opti mum for general proteolysis is about 4, in comparison with about 2 for pepsins from monogastric animals. Its general proteolytic activity is low relative to its milk-clotting activity and it has moderately high specificity for bulky hydrophobic residues at the P, and Pi positions of the scissile bond. Its physiological function appears to be to coagulate milk in the stomach of the neonate, thereby increasing the efficiency of digestion, by retarding discharge into the intestine, rather than general proteolysis

384 DAIRY CHEMISTRY AND BIOCHEMISTRY Table 10.3 Kinetic parameters for hydroloysis of K-casein peptides by chymosin at pH 4.7 (compiled from Visser et al., 1976; Visser, Slangen and van Rooijen, 1987) Peptide k,,, Sequence (s- I) S.F.M.A.I. 104-108 S.F.M.A.I.P. 104-109 S.F.M.A.I.P.P. 104-1 10 S.F.M.A.I.P.P.K. 104- I 1 1 L.S.F.M.A.I. 103-108 L.S.F.M.A.I.P. 103-109 L.S.F.M.A.I.P.P. 103-110 L.S.F.M.A.I.P.P.K. 103- 11 1 L.S.F.M.A.I.P.P.K.K. 103- 112 H.L.S.F.M.A.1 102-108 P.H.L.S.F.M.A.1 101 - 108 H.P.H.P.H.L.S.F.M.A.I.P.P.K. 98- 11 1 98-111" k--Caseinb L.S.F.(NO,)Nle A.L.OMe 0.33 1.05 1.57 0.75 18.3 38.1 43.3 33.6 30.2 16.0 33.5 66.2 46.2" 2-20 12.0 8.50 9.20 6.80 3.20 0.85 0.69 0.41 0.43 0.46 0.52 0.34 0.026 0.029" 0.001-0.005 0.95 0.038 0.1 14 0.231 0.239 21.6 55.1 105.1 78.3 65.3 30.8 100.2 2509 1621" 12.7 200-2000 "pH 6.6. bpH 4.6. ide hydrolysed by chymosin is Ser.Phe.Met.Ala.Ile (K-CN fl04- 108); extending this peptide from its C and/or N terminus increases its susceptibility to chymosin (i.e. increases kcat/K,,,); the peptide K-CN f98-111 is as good a substrate for chymosin as whole K-casein (Table 10.3). Ser,,, appears to be essential for cleavage of the Phe,,,-Met,,, bond by chymosin, and the hydrophobic residues, Leu,,,, Ala,,, and Ilelo8 are also important. Rennets. The traditional rennets used to coagulate milk for most cheese varieties are prepared from the stomachs of young calves, lambs or kids by extraction with NaCl (c. 15%) brines. The principal proteinase in such rennets is chymosin; about 10% of the milk-clotting activity of calf rennet is due to pepsin. As the animal ages, the secretion of chymosin declines while that of pepsin increases; in addition to pepsin, cattle appear to secrete a chymosin-like enzyme throughout life. Like pepsin, chymosin is an aspartyl (acid) proteinase, i.e. it has two essential aspartyl residues in its active site which is located in a cleft in the globular molecule (molecular mass - 36 kDa) (Figure 10.2). Its pH optimum for general proteolysis is about 4, in comparison with about 2 for pepsins from monogastric animals. Its general proteolytic activity is low relative to its milk-clotting activity and it has moderately high specificity for bulky hydrophobic residues at the PI and Pi positions of the scissile bond. Its physiological function appears to be to coagulate milk in the stomach of the neonate, thereby increasing the efficiency of digestion, by retarding discharge into the intestine, rather than general proteolysis

CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 385 ing the cleft which contains the active site; arrows indicate B structures and cvlindeinase, 10.2 Schematic representation of the tertiary structure of an aspart helices(from Foltmann, 1987) Due to increasing world production of cheese and the declining supply of young calf stomachs(referred to as vells), the supply of calf rennet has been inadequate for many years. This has led to a search for suitable ubstitutes. Many proteinases are capable of coagulating milk but most are too proteolytic relative to their milk-clotting activity, leading to a decrease in cheese yield (due to excessive non-specific proteolysis in the cheese va and loss of peptides in the whey) and defects in the flavour and texture the ripened cheese, due to excessive or incorrect proteolysis. Only six proteinases are used commercially as rennet substitutes: porcine, bovine and chicken pepsins and the acid proteinases from Rhizomucor miehei, R pusillus nd Cryphonectria parasitica. Chicken pepsin is quite proteolytic and is used widely only in Israel (for religious reasons). Porcine pepsin enjoyed limited success about 30 years ago, usually in admixtures with calf rennet, but it is very sensitive to denaturation at ph values above 6 and may be denatured extensively during cheesemaking, leading to impaired proteolysis during ripening: it is now rarely used as a rennet substitute. Bovine pepsin is quite ffective and many commercial calf rennets contain up to 50% bovine pepsin. Rhizomucor miehei proteinase, the most widely used microbial rennet, gives generally satisfactory results. Cryphonectria parasitica pro- teinase is. in general. the least suitable of the commercial microbial rennet bstitutes and is used only in high-cooked cheeses in which extensive denaturation of the coagulant occurs, e.g. Swiss-type cheeses The gene for calf chymosin has been cloned in Kluyveromyces marxianus var. lactis, Aspergillus niger and E. coli. Microbial (cloned) chymosin have

CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 385 Figure 10.2 Schematic representation of the tertiary structure of an aspartyl proteinase, showing the cleft which contains the active site; arrows indicate p structures and cylinders the %-helices (from Foltmann, 1987). Due to increasing world production of cheese and the declining supply of young calf stomachs (referred to as vells), the supply of calf rennet has been inadequate for many years. This has led to a search for suitable substitutes. Many proteinases are capable of coagulating milk but most are too proteolytic relative to their milk-clotting activity, leading to a decrease in cheese yield (due to excessive non-specific proteolysis in the cheese vat and loss of peptides in the whey) and defects in the flavour and texture of the ripened cheese, due to excessive or incorrect proteolysis. Only six proteinases are used commercially as rennet substitutes: porcine, bovine and chicken pepsins and the acid proteinases from Rhizomucor miehei, R. pusillus and Cryphonectria parasitica. Chicken pepsin is quite proteolytic and is used widely only in Israel (for religious reasons). Porcine pepsin enjoyed limited success about 30years ago, usually in admixtures with calf rennet, but it is very sensitive to denaturation at pH values above 6 and may be denatured extensively during cheesemaking, leading to impaired proteolysis during ripening; it is now rarely used as a rennet substitute. Bovine pepsin is quite effective and many commercial calf rennets contain up to 50% bovine pepsin. Rhizomucor miehei proteinase, the most widely used microbial rennet, gives generally satisfactory results. Cryphonectria parasitica proteinase is, in general, the least suitable of the commercial microbial rennet substitutes and is used only in high-cooked cheeses in which extensive denaturation of the coagulant occurs, e.g. Swiss-type cheeses. The gene for calf chymosin has been cloned in Kluyveromyces marxianus var. lactis, Aspergillus niger and E. coli. Microbial (cloned) chymosins have

CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 387 The actual reactions leading to coagulation are not known. Caare essential but Ca-binding by caseins does not change on renneting. Colloidal calcium phosphate( CCP)is also essential: reducing the CCP concentration by more than 20% prevents coagulation. Perhaps, hydrophobic interactions, which become dominant when the surface charge and steric stabilization are reduced on hydrolysis of K-casein, are responsible for coagulation(the is soluble in urea). The adverse influence of moderately high strength on coagulation suggests that electrostatic interactions are also olved. It is claimed that ph has no effect on the secondary stage of rennet coagulation, which is perhaps surprising since micellar charge is reduced by lowering the pH and should facilitate coagulation. Coagulation is very temperature-sensitive and does not occur below about 18C, above which the temperature coefficient, QIo, is approximately 16 Factors that afect rennet coagulation. The effect of various compositional and environmental factors on the primary and secondary phases of rennet coagulation and on the overall coagulation process are summarized in Figure 10.4 o coagulation occurs below 20C, due mainly to the very high tempera ture coefficient of the secondary phase. At higher temperatures(above 65-600C, depending on pH and enzyme) the rennet is denatured. Rennet coagulation is prolonged or prevented by preheating milk at temperatures above about 70C (depending on the length of exposure). The effect is due to the interaction of B-lactoglobulin with K-casein via sulphydryl-disulphide interchange reactions; both the primary and, especially, the of coagulation are adversely affected Measurement of rennet coagulation time. A number of principles are easure the rennet coagulability of milk or the activity of rennets measure actual coagulation, i.e. combined first and second stages, but some ifically monitor the hydrolysis of k-casein. The most cor ethods are described belo The simplest method is to measure the time elapsed between the addition of a measured amount of diluted rennet to a sample of milk in a tempera- ture-controlled water-bath at, e.g. 30oC. If the coagulating activity of a rennet preparation is to be determined, a 'reference' milk, e. g low-heat milk powder reconstituted in 0.01%CaCl2, and perhaps adjusted to a certain pH e.g. 6.5, should be used A standard method has been published (IDF, 1992 and a reference milk may be obtained from Institut National de la Recherche Agronomique, Poligny, France. If the coagulability of a particu lar milk is to be determined, the ph may or may not be adjusted to a dard value. The coagulation point may be determined by placing the milk sample in a bottle or tube which is rotated in a water-bath(Figure 10.5); the fluid milk forms a film on the inside of the rotating bottle/tube but

CHEMISTRY AND BIOCHEMISTRY OF CHEESE AND FERMENTED MILKS 387 The actual reactions leading to coagulation are not known. Ca2+ are essential but Ca-binding by caseins does not change on renneting. Colloidal calcium phosphate (CCP) is also essential: reducing the CCP concentration by more than 20% prevents coagulation. Perhaps, hydrophobic interactions, which become dominant when the surface charge and steric stabilization are reduced on hydrolysis of K-casein, are responsible for coagulation (the coagulum is soluble in urea). The adverse influence of moderately high ionic strength on coagulation suggests that electrostatic interactions are also involved. It is claimed that pH has no effect on the secondary stage of rennet coagulation, which is perhaps surprising since micellar charge is reduced by lowering the pH and should facilitate coagulation. Coagulation is very temperature-sensitive and does not occur below about 18"C, above which the temperature coefficient, Qlo, is approximately 16. Factors that afect rennet coagulation. The effect of various compositional and environmental factors on the primary and secondary phases of rennet coagulation and on the overall coagulation process are summarized in Figure 10.4. No coagulation occurs below 20"C, due mainly to the very high temperature coefficient of the secondary phase. At higher temperatures (above 55-60"C, depending on pH and enzyme) the rennet is denatured. Rennet coagulation is prolonged or prevented by preheating milk at temperatures above about 70°C (depending on the length of exposure). The effect is due to the interaction of /3-lactoglobulin with K-casein via sulphydryl-disulphide interchange reactions; both the primary and, especially, the secondary phase of coagulation are adversely affected. Measurement of rennet coagulation time. A number of principles are used to measure the rennet coagulability of milk or the activity of rennets; most measure actual coagulation, i.e. combined first and second stages, but some specifically monitor the hydrolysis of K-casein. The most commonly used methods are described below. The simplest method is to measure the time elapsed between the addition of a measured amount of diluted rennet to a sample of milk in a temperature-controlled water-bath at, e.g. 30°C. If the coagulating activity of a rennet preparation is to be determined, a 'reference' milk, e.g. low-heat milk powder reconstituted in 0.01% CaCl,, and perhaps adjusted to a certain pH, e.g. 6.5, should be used. A standard method has been published (IDF, 1992) and a reference milk may be obtained from Institut National de la Recherche Agronomique, Poligny, France. If the coagulability of a particular milk is to be determined, the pH may or may not be adjusted to a standard value. The coagulation point may be determined by placing the milk sample in a bottle or tube which is rotated in a water-bath (Figure 10.5); the fluid milk forms a film on the inside of the rotating bottle/tube but