Rennet action

The primary phase of rennet action may be monitored by measuring the formation of either product, i.e. para-tc-casein or the GMP. Para-tc-casein may be measured by SDS-polyacrylamide gel electrophoresis (PAGE),... 

The higher saturated fatty acids have been noted to inhibit rennet action, whereas the lower fatty acids enhance it. The inhibitory effect of the higher acids can be nullified by CaCl2. 

Harding and Prucha worked, on cheese ripening at Geneva, N. Y., 1900-1910, and concluded that the digestion of toe casein of cheese during ripening is due to toe collective action of native enzymes of milk, bacterial enzymes, and rennet action. 

K-Casein was first isolated by Waugh and von Hippel (1956), who showed that this protein is responsible for the stability of the casein micelles and that its micelle-stabilizing properties are lost on renneting. Only /c-casein is hydrolyzed to a significant extent during the primary phase of rennet action. The primary cleavage site is Pheios-Metioe (Delfour et al, 1965), which is many times more susceptible to hydrolysis by acid proteinases (all commercial rennets are acid proteinases) than any other peptide bond in the milk protein system. 

Thus, at the end of 24 hours, the milk coagulates less easily than an hour after the treatment. On the contrary, if the same milk is kept at ordinary temperature, it is found that at the end of ten hours it shows no notable modifications in its resistance to rennet-action. The difference in these results comes from the fact that, in the preservation at ordinary temperature, the lactic microbes have been able to develop and the activating action of the acid formed counterbalances the increasing resistance of the pure milk with age. The proof of this hypothesis is that if the lactic bacteria are prevented from developing, either by the use of formaldehyde, of mercury bichromate, or chloride, added to the milk at the temperature of the sunounding air, or quite simply by maintaining the milk at a temperature of 58 , we see that this milk acts like that kept in the refrigerator. This observation shows the precautions necessary to cany on experiments results of which shall be comparative. 

Destruction of the casein micelles in the milk with subsequent precipitation of the casein can be accomplished in a number of ways. The action of heat or the action of alcohols, acids, salts and the enzyme rennet all bring about precipitation. In commercial practise the two techniques used employ either acid coagulation or rennet coagulation mechanisms. 

Specificity. The generally claimed specific action of enzymes is not as sharply defined as is often expected. Proteases are broad in the range of amino acid bonds they hydrolyze and exhibit only a degree of specificity. Careful investigation of the range of bonds attacked, and testing for comparable action on the actual protein target, will enable an enzyme to be chosen that has suitable performance. Some proteases are extremely narrow in their action, for example the various cheese rennets. 

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. 

Although the gelation properties of whey proteins are of great importance in many foods (Mulvihill, 1992) and it is possible to form a weak gel in creams by the formation of a continuous network of fat globules, most important milk gels are those involving casein micelles which can be made to form a gel matrix either by isoelectric precipitation (acid-induced gel) or by the action of a proteolytic enzyme (rennet-induced gel). Both gel types... 

The gross proteolysis of casein is probably due solely to rennet and plasmin activity (O Keeffe et al. 1978). Bacterial proteases and peptides are responsible for subsequent breakdown of the large peptides produced by rennet and plasmin into successively smaller peptides and finally amino acids (O Keeffe et al. 1978). If the relative rate of proteinase activity by rennet, plasmin, and bacterial proteases exceeds that of the bacterial peptidase system, bitterness in the cheese could result. Bitter peptides can be produced from a,-,- or /3-casein by the action of rennet or the activity of bacterial proteinase on /3-casein (Visser et al. 1983). The proteolytic breakdown of /3-casein and the subsequent development of bitterness are strongly retarded by the presence of salt (Fox and Walley 1971 Stadhouders et al. 1983). The principal source of bitter peptides in Gouda cheese is 3-casein, and more particularly the C-terminal region, i.e., 3(193-209) and 3(193-207) (Visser et al. 1983). In model systems, bitter peptides are completely debittered by a peptidases system of S. cremoris (Visser et al. 1983). 

Penicillium caseicolum produces an extracellular aspartyl proteinase and a metalloproteinase with properties very similar to those of the extracellular enzymes produced by P roqueforti (Trieu-Cout and Gripon 1981 Trieu-Cout et al. 1982). Breakdown of casein in mold-ripened cheese results from the synergistic action of rennet and the proteases of lactic streptococci and penicillia (Desmazeaud and Gripon 1977). Peptidases of both lactic acid bacteria and penicillia contribute to formation of free amino acid and nonprotein nitrogen (Gripon et al. 1977). 

Hydrolysis of the casein micelle-stabilizing K-casein by the action of selected acid proteinases (rennets), and the resultant slow quiescent aggregation of the destabilized micelles in the presence of calcium ions ( 3 mM) at 30-36°C (e.g., for most rennet-curd cheeses such as Cheddar, Mozzarella and Gouda)... 

Renneted gels Enzymatic action followed by precipitation by Ca +... 

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