CHYMOSINChymosin, rennet, and rennin are often used inter-changeably to refer to this enzyme. The latter, ren-nin, should not be confused with renin, which is anenzyme associated with kidneys and does not clotmilk. Chymosin is the biochemical name of theenzyme that was formerly known as rennin. It be-longs to the group of aspartic acid proteinases, EC3.4.23, that have a high content of dicarboxylic andhydroxyamino acids and a low content of basicamino acids. Its molecular mass is 40 kDa (Andren2003, Foltmann 1993).Rennet is the stomach extract that contains theenzyme chymosin in a stabilized form that is usablefor cheese making (Green 1977). While the amountof chymosin that is required for cheese making isvery small, this enzyme industry has undergone aninteresting transformation over time. It is believedthat in the early days of cheese making, milk coagu-lation occurred either by filling dried stomachs ofcalves or lambs with milk or by immersing piecesof such stomachs in milk (Kosikowski and Mistry1997). The chymosin enzyme imbedded within thestomach lining diffused into the milk and coagulatedit. This crude process of extracting coagulating en-zymes was eventually finessed into an industry thatemployed specific methods to extract and purify theenzyme and develop an extract from the fourth stom-ach of the calf or lamb. Extracts of known enzymeactivity and predictable milk clotting properties thenbecame available as liquids, concentrates, powders,and blends of various enzymes. Live calves wererequired for the manufacture of these products. Be-cause of religious and economic reasons, anotherindustry also had emerged for manufacturing alter-native milk clotting enzymes from plants, fungi, andbacteria. These products remain popular and meetthe needs of various kosher and other religious needs.Applications of genetic technology in rennet manu-facture were then realized, and in 1990 a newprocess utilizing this technology was approved in theUnited States for manufacturing rennet.RENNET MANUFACTUREFor the manufacture of traditional rennet, calves,lambs, or kids that are no more than about 2 weeks oldand fed only milk are used (Kosikowski and Mistry1997). As calves become older and begin eating otherfeeds, the proportion of bovine pepsin in relation tochymosin increases. Extracts from milk-fed calvesthat are 3 weeks old contain over 90% chymosin, andthe balance is pepsin. As the calves age and are fedother feeds such as concentrates, the ratio of chy-mosin to pepsin drops to 30:70 by 6 months of age. Ina full-grown cow there are only traces of chymosin.Milk-fed calves are slaughtered, and the unwash-ed stomachs (vells) are preserved for enzyme extrac-tion by emptying their contents, blowing them intosmall balloons and drying them. The vells may alsobe slit opened and dry salted or frozen for preserva-tion. Air-dried stomachs give lower yields of chy-mosin than frozen stomachs; 12–13 air-dried stom-achs make 1 L of rennet standardized to 1:10,000strength, but only 7–8 frozen stomachs would berequired for the same yield.Extraction of chymosin and production of rennetbegin by extracting, for several days, chopped ormacerated stomachs with a 10% sodium chloridesolution containing about 5% boric acid or glycerol.Additional salt up to a total of 16–18% is introducedfollowed by filtration and clarification. Mucine andgrass particles in suspension are removed by intro-ducing 1% of potash alum, followed by an equalamount of potassium phosphate. The suspension isadjusted to pH 5.0 to activate prochymosin (zymo-gen) to chymosin, and the enzyme strength is stan-dardized, so that one part coagulates 15,000 or10,000 parts of milk. Sodium benzoate, propyleneglycol, and salt are added as preservatives for thefinal rennet. Caramel color is also usually added.The finished rennet solutions must be kept cold andprotected from light.Powdered rennet is manufactured by saturating arennet suspension with sodium chloride or acidify-ing it with a food-grade acid. Chymosin precipitatesand secondary enzymes such as pepsin remain in theoriginal suspension. The chymosin-containing pre-cipitate is dried to rennet powder (Kosikowski andMistry 1997).A method has been developed for manufacturingrennet without slaughtering calves. A hole (fistula)is surgically bored in the side of live calves, and at
milk feeding time, excreted rennet is removed. After
the calf matures, the hole is plugged, and the animal
is returned to the herd. This method has not been
commercialized but may be of value where religious
practices do not allow calf slaughter.
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