Chymosin, coagulation

Throughout both acid and chymosin coagulation processes, the diffusion of a 96750 g/mol PEG was very sensitive to variations in the size of the casein particle and in the appearance of the network. Many of the structural modifications occurring in the sample at different levels could thus be revealed from PFG-NMR, and interesting conclusions on several key points of the milk coagulation processes could be drawn. In addition, it is of note that the duration of diffusion measurements can be reduced and that the use of lactic bacteria instead of GDL seems to be feasible. Both of these modifications would allow the acquisition of more diffusion data at hi pH values. 

The Daily Industiy. The first step in cheese manufacture is the coagulation of milk. Coagulation can be divided into two distinct phases, enzymatic and the non-enzymatic. In the primary enzymatic phase a proteol ic enzyme such as chymosin (rennet), or less effectively, pepsin, carries out an extremely specific and limited proteolysis, cleaving a phenylalanine-methionine bond of /c-casein, making the casein micelle metastabie. In the second, non-enzymatic phase, the... 

Microbial coagulants are now useful and are responsible for about one third of all the cheese produced worldwide, but suffer from the disadvantage of being too stable and so are threatened commercially by improved methods of produdng chymosin by recombinant DNA technology. The use of thermally destabilized microbial rennets results in residual enzyme levels in the milk product similar to or below those encountered when calf rennet is use (55). An unexpected benefit has been an increase on some occasions of the specificity of the microbial enzyme, making it virtually indistinguishable from the action of calf rennet. Also some microbial rennets help impart a flavor that is popular with consumers. 

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. 

Figure 10.3 Schematic representation of the rennet coagulation of milk, (a) Casein micelles with intact K-casein layer being attacked by chymosin (Q (b) micelles partially denuded of K-casein (c) extensively denuded micelles in the process of aggregation (d) release of macropeptides ( ) and changes in relative viscosity (0) during the course of rennet coagulation.

Coagulant. Most of the coagulant is lost in the whey but some is retained in the curd. Approximately 6% of added chymosin is normally retained in Cheddar and similar varieties, including Dutch types the amount of rennet retained increases as the pH at whey drainage is reduced. As much as 20% of added chymosin is retained in high-moisture, low-pH cheese, e.g. Camembert. Only about 3% of microbial rennet substitutes is retained in the curd and the level retained is independent of pH. 

Most current models put K-casein on the outer casein micelle surface (Heth and Swaisgood 1982 McMahon and Brown 1984A Shahani 1974). This allows the possibility that heat-induced coagulation of milk is the result of serum proteins interacting with K-casein on the micelle surface and with each other to interconnect micelles. The observation that chymosin cannot release macropeptides from K-casein in heated milk (Morrissey 1969 Shalabi and Wheelock 1976,1977) suggests that... 

Linklater (1961) reported that bovine pepsin accounted for only 0 to 6% of the milk-clotting activity of commercial rennet extracts. He used porcine pepsin as a reference standard. Bovine pepsin has increased in use as a coagulant because of the practice of extracting the stomach from older calves and adult cattle. More recently, Sellers (1982) reported that 85 to 95% of the proteolytic activity of calf rennet is due to chymosin and the remainder is from bovine pepsin. Adult bovine rennets preparations may contain 55 to 60% bovine pepsin. Mixtures of calf rennet and porcine pepsin may contain 40 to 45% chymosin, 5 to 10% bovine pepsin, and 50% porcine pepsin. Mixtures of adult bovine rennet and porcine pepsin typically contain 20 to 25% chymosin, 40 to 45% bovine pepsin, and 30 to 40% porcine pepsin activity (McMahon and Brown 1985). 

Figure 12.3 Plot of apparent absorbance (600nm) versus time after addition of chymosin to reconstitute nonfat dry milk (12g + 100 ml.OIM CaCe2). Arrows represent Formagraph coagulation time. Inset shows an expanded view of the first 3 min of coagulation. (From McMahon et at. 1984)...

Reddy, D., Payens, T. A. and Brown, R. J. 1986. Effect of pepstatin on the chymosin-triggered coagulation of casein micelles. J. Dairy Sci. 69 (Suppl. 1), 72. 

Cheese is made by coagulating milk by the addition of rennet to produce curds. The curds are separated from the liquid whey and then processed and matured to produce a wide variety of cheeses. The active ingredient of rennet is the enzyme chymosin. Until 1990, most rennet was produced from the stomach of slaughtered newly born calves. These days, at a cost one tenth of that before 1990, chymosin is produced by genetically engineered bacteria into which the gene for this enzyme has been inserted, and is used for making cheese in the United States, Europe, and other parts of the world. 

Casein micelles are remarkably stable structures. Milk may be boiled, sometimes for several hours, without coagulating the micelles. Also, the addition of CaCU to milk does not precipitate the micelles up to concentrations greatly in excess of that required to precipitate purified whole casein. On the other hand, micelles rapidly flocculate after treatment with chymosin, at or above room temperature, and casein... 

The stomach environment is acidic as a result of HC1 secretion by the parietal cells. The acidic pH serves to denature many proteins, thus making them susceptible to proteolysis. The chief cells of the stomach produce pepsinogen, which is activated to pepsin by the HC1 (see Table 20.3). The optimum pH of peptic activity is around 2, and pepsin is inactivated at neutrality. Another stomach enzyme is rennin or chymosin, which is present in infants but not in adults. It removes a glycopeptide from milk-K-casein, disrupting the casein micelle and promoting milk protein coagulation and digestion. 

In addition to the recombinant chymosin, several coagulants for cheese-making are available from microbial origin. These are endopro-teases from Rhizomucor miehei, Rhizomucor pussilus, and incidentally the plant-derived endothiapepsin from Cryphonectria parasitica. 

Milk Coagulation. The first step in cheese manufacture is the coagulation of milk. Traditionally, this coagulation step is catalyzed by the enzyme rennet. Rennet is a saline extract of the 4th stomach of calves, usually slaughtered before they are 30 days old. The principal protease in rennet is rennin. In an attempt to avoid confusion with the hormone peptide renin, the International Enzyme Nomenclature Committee has assigned the name chymosin to the protease in calf rennet. During the growth of calves, chymosin is replaced by pepsin, the acid protease of the mature stomach. 

Cheese Ripening. Rennet plays a major role in the texture and flavor development of cheese during the ripening process. Besides the rapid cleavage of the key phenylalanine-methionine bond to coagulate milk, chymosin has been shown to hydrolyze at least 22 other bonds in the casein molecules. The favored amino acids at the point of cleavage are leucine, isoleucine and phenylalanine. 

Chymosin produces cheese free of bitter flavor and of excellent texture. The use of other acid proteases such as pepsin and many of the microbial rennets in the coagulation step often leads to bitter flavor and a soft texture after ripening. 

Castillo, M., Lucey, J.A., and Payne, F A. (2006). The effect of temperature and inoculum concentration on rheological and light scatter properties of milk coagulated by a combination of bacterial fermentation and chymosin cottage cheese-type gels. Int. Dairy J. 16, 131-146. 

Figure 19.3. Sketch of the enzymatic coagulation of casein based on the action of chymosin.

Chymosin is secreted by the abomasum in the form of an intially inactive precursor, prochymosin, which is converted autocatalytically to chymosin (see below, p. 176). Many reviews on the properties of the zymogen and the enzyme have been published. One of the most recent, by Foltmann (J), also contains references to earlier reviews. The action of chymosin on K-casein is primarily responsible for the milk-clotting process. The numerous studies of this reaction have been reviewed recently by Mackinlay and Wake (32). Because of the number of diflFer-ent milk proteins and the complexity of their interactions, especially in the casein micelle, the full details of the coagulation process are not yet understood. 

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