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
386 DAIRY CHEMISTRY AND BIOCHEMISTRY given excellent results in cheesemaking trials on various varieties and are now widely used commercially, although they are not permitted in some countries. Significantly, they are accepted for use in vegetarian cheeses. The gene for R. miehei proteinase has been cloned in A. oryzae; the resultant product, Marzyme GM, is commercially available(Texel, Stockport, UK) and is reported to be a very effective coagulant. Coagulation of rennet-altered micelles. When c. 85% of the total k-casein has been hydrolysed, the micelles begin to aggregate progressively into a gel network. Gelation is indicated by a rapid increase in viscosity (n)( Figure 10.3). Coagulation commences at a lower degree of hydrolysis of K-casein if the temperature is increased, the ph reduced or the Ca++concentration increased 8 -OO S af visually ohserved clotting time Figure 10.3 angulation of milk. (a) casein micelles with ng attacked by C) (b) micelles partially denuded of k-casein; aggregation;(d)release of macropeptides(+ and changes in relative viscosity(O ourse of rennet coagulation
386 DAIRY CHEMISTRY AND BIOCHEMISTRY given excellent results in cheesemaking trials on various varieties and are now widely used commercially, although they are not permitted in some countries. Significantly, they are accepted for use in vegetarian cheeses. The gene for R. miehei proteinase has been cloned in A. oryzae; the resultant product, Marzyme GM, is commercially available (Texel, Stockport, UK) and is reported to be a very effective coagulant. Coagulation of rennet-altered micelles. When c. 85% of the total u-casein has been hydrolysed, the micelles begin to aggregate progressively into a gel network. Gelation is indicated by a rapid increase in viscosity (q) (Figure 10.3). Coagulation commences at a lower degree of hydrolysis of rc-casein if the temperature is increased, the pH reduced or the Ca2+ concentration increased. 0 20 40 M) RO I of visunlly ohxrvcd dolling time Figure 10.3 Schematic representation of the rennet coagulation of milk. (a) Casein micelles with intact ti-casein layer being attacked by chymosin (C); (b) rnicelles partially denuded of ti-casein; (c) extensively denuded micelles in the process of aggregation; (d) release of macropeptides (+) and changes in relative viscosity (B) during the course of rennet coagulation
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
388 DAIRY CHEMISTRY AND BIOCHEMISTRY Factor First phase Second phase Overall effect, see panel Temperature Ca Pre-heating abcde Rennet concentration 65 七 1/Rennet 96 Protein Figure 10. 4 Principal factors affecting the rennet coagulation time(RCt) of milk flocs of protein form in the film on coagulation. Several types of apparatus using this principle have been described As shown in Figure 10.3, the viscosity of milk increases sharply when milk coagulates and may be used to determine the coagulation point. Any type of viscometer may, theoretically, be used but several dedicated pieces
388 DAIRY CHEMISTRY AND BIOCHEMISTRY Factor First phase Second phase Overall effect, Temperature + ++ a +++ b +++ C PH Ca Pre-heating ++ ++++ d Rennet concentration ++++ e Protein concentration + ++++ f 20 40 60 C 0 Ca 1 /Rennet t ez 6.4 PH C 65 0 % Protein Figure 10.4 Principal factors affecting the rennet coagulation time (RCT) of milk. flocs of protein form in the film on coagulation. Several types of apparatus using this principle have been described. As shown in Figure 10.3, the viscosity of milk increases sharply when milk coagulates and may be used to determine the coagulation point. Any type of viscometer may, theoretically, be used but several dedicated pieces