Milk-Clotting Activity

Liquid or granulate form Anson units standardized in milk clotting activities. 

Cathepsin D (EC3.4.23.5). It has been known for more than 20 years that milk also contains an acid proteinase, (optimum pH ss 4.0) which is now known to be cathepsin D, a lysozomal enzyme. It is relatively heat labile (inactivated by 70°C x 10 min). Its activity in milk has not been studied extensively and its significance is unknown. At least some of the indigenous acid proteinase is incorporated into cheese curd its specificity on asl- and / -caseins is quite similar to that of chymosin but it has very poor milk-clotting activity (McSweeney, Fox and Olson, 1995). It may contribute to proteolysis in cheese but its activity is probably normally overshadowed by chymosin, which is present at a much higher level. 

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. 

IDF (1992) Bovine Rennets. Determination of Total Milk-clotting Activity, Provisional Standard 157, International Dairy Federation, Brussels. 

Activation of prochymosin involves the splitting of peptides from the N-terminal end of prochymosin with simultaneous reduction in molecular weight from about 36,000 to 31,000. The rate of conversion increases markedly with decreasing pH below 5.0 (Rand and Emstrom 1964). At pH 5.0, NaCl concentrations up to 2M increase the rate of activation. Milk-clotting activity plotted against activation time at pH 5.0 shows the course of activation (Fig. 12.1) to be autocatalytic. If activation is carried out in the presence of preformed chymosin, the S-shape disappears and the initial rate of the activation process increases with increasing concentration of preformed chymosin. Folt-... 

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). 

Green (1972) reported that Cheddar cheese made entirely with bovine pepsin was only slightly inferior to that made with calf rennet and Fox and Walley (1971) found no significant difference between Cheddar cheese made with bovine pepsin and rennet. Fox (1969) found that the milk-clotting activity of bovine pepsin is less pH-dependent than that... 

An ideal test for measuring milk-clotting activity has never been devised, but numerous methods have been tried. In practice, activity is determined by the speed with which the enzyme clots milk under a set of specified conditions. This differs from the usual procedure in enzyme chemistry where one measures the rate at which the products of an enzyme-catalyzed reaction appear, or conversely, the rate at which the substrate disappears. 

Antonova, T., Nachev, L., Kolev, D., Bodurska, I. and Manafova, N. 1981. Bacterial strains producing enzymes with milk-clotting activity. IX. Effect of certain factors on milk clotting. Acta Microbiol Bulg. 9, 48-53. 

Hylmar, B., Pokoma, L. and Peterkova, L. 1982. Utilization of Bacillus megaterium strains producing proteases with milk-clotting activity. Prumysl Potravin 33, 208-211. 

Kawai, M. 1973. Productivity of proteolytic enzymes and distribution of its milk clotting activity among the basidiomycetes. J. Agric. Chem. Soc. Japan 47, 467-472. 

Pang, S. H. and Ernstrom, C. A. 1986. Milk clotting activity in bovine fetal abomasa. 

Rotaru, G. 1980. The milk clotting activity characterization of an enzymatic preparation from Aspergillus niger. Bull. Univ. Galati Technol. Chimia Produselor Aliment. 3, 43-48. 

Lipase (Microbial) Activity for Medium- and Long-Chain Fatty Acids, (S3)105 Lysozyme Activity, (S3)106 Maltogenic Amylase Activity, 804 Milk-Clotting Activity, 805 Pancreatin Activity, 805 Pepsin Activity, 807 Phospholipase A2 Activity, 808 Phytase Activity, 808 Plant Proteolytic Activity, 810 Proteolytic Activity, Bacterial (PC), 811 Proteolytic Activity, Fungal (HUT), 812 Proteolytic Activity, Fungal (SAP), 813 Pullulanase Activity, 814 Trypsin Activity, 814 Enzyme Assays, 786 Enzyme-Hydrolyzed (Source) Protein,... 

In subsequent observations S7c), the authors noted that acidification of all 3 pepsinogens to pH 2 for 8 minutes, followed by incubation at various pH s, from 1.1 to 7.2, and then alkalinization at pH 7.8, leads to formation of intermediate compounds between pepsinogens and pepsins. These were designated as pepsin-pepsin-inhibitor complexes (HPPI). These complexes have no milk-clotting activity at pH 5.5 and... 

Although the general proteolytic activities of various milk clotting enzymes may vary, their milk clotting activities are apparently predicated on the same specific cleavage of the Phe-Met bond in -casein. Apparently rennin, pepsin, chymotrypsin, a microbial protease, proteases from Endo-thia parasitica, Mucor pusillus, and Mucor miehei exert the same type of activity on -casein (2, 169). Enzymes that are currently used commercially for cheesemaking in the United States include rennin, rennin-pepsin mixtures, and microbial proteases from Endothia parasitica, Mucor pusiUus, and Mucor miehei. 

It has long been known (24) that one of the peptides released on activation of pepsinogen could act as a pepsin inhibitor at pH values above 4. Peptide 1-16 from porcine pepsinogen (25) and the corresponding peptide 1-17 from bovine pepsinogen (5) can indeed inhibit the milk-clotting activity of pepsin above pH 5 (see Kumar et, Chapter 13 this volume). ... 

Since peptide Al appeared to have been derived from residues 1-16, it was of interest to see whether this peptide, generated in the course of a stopped activation, could inhibit pepsin and the homologous milk-clotting calf protein, chymosin. The results (V. Barkholt Pedersen and P. H. Ward - personal communications) show that Al had no effect on the milk-clotting activity of chymosin (confirming that all of the pepstatin must have been removed) while independently the Danish and American labs produced similar curves for the inactivation of porcine pepsin (Fig.5). Thus, it would seem that the first peptide produced on activation is the inhibitor peptide. This seems to make sense from a control point of view, i.e., the first bond being hydrolyzed on activation releases the pepsin inhibitor so it can exert its effect if required. 

Figure 5. The effect of peptide A1 on the milk-clotting activities of pepsin (— — —) and chymosin ( A pH 5.3. The peptide was premixed with the enzyme before measuring the clotting activity remaining.

To determine inhibition of the milk-clotting activity of pepsin by peptide 1-16, the turbidimetric method of McPhie (16) was used with minor modification. We found that a more consistent standard curve for pepsin was obtained when the fresh skim milk purchased locally was first centrifuged at 12,000 x g for 10 min in the cold ... 

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