Studies On Screening Of Bacterial Strains Producing Chymosin And Its Enzyme Properties

The cheese is a kind of nutritional, convenient dairy with unique flavor. With theimprovement of people's living standard, ascendant trend has appeared in theproduction and consumption of the cheese. Chymosin is a kind of key enzyme inproducing cheese through milk-clotting There are many sources to get chymosin,such as from animal,plant and microbe. Because of the merits, such as wide source,short production period,low cost and simple configuration, microbe has become animportant source of chymosin. A lot of researchers pay more attention to it.This paper mainly dealt with four aspects:(1) A strain BZ-17, which could produce enzyme of high milk-clotting activityand low proteolytic activity, was isolated from cattle farm in Hunan AgricultureUniversity. The strain was identified to be Bacillus cereus without making toxinaccording to its morpha characteristics of colonies and its physiological andbiochemical properties.(2) The original strain BZ-17 was treated with Ultraviolet radiation,HNO₂ andcompound mutation technique(firstly by Ultraviolet radiation, then by HNO₂). Amutant strain (named UH-9) which could produce chymosin of higher milk-clottingactivity and low proteolytic activity was obtained. The genetic stability test of themutation strain showed that UH-9 can keep in a steady level.(3) Some researches were studied on the ingredients of culture medium such ascarbon,nitrogen,phosphate and calcium. At last, the orthogonal experiment showedthe optimum medium was(%): bran 9, whey 1, CaCl₂ 0.2, NaH₂PO₄ 0.43, Na₂CO₃0.1. The optimum culture condition test showed: age of pure culture 48h, culturetemperature 30℃, culture time 48h, the initial pH 7.0, filling liquid 40ml/300ml,vibration speed 180 r/min.(4) As for the enzymic properties: When the milk's pH was 5.5, the enzyme had the fastest reaction velocity; The milk-clotting activity kept stable in pH 6; Theoptimal reaction temperature was 45℃; Light had little influence on chymosin; It wasmore easily destructed by high temperature for milk-clotting activity compared withthe proteolytic activity; Metal ions such as Ca²⁺,Fe²⁺ and Mg²⁺ could significantlyincrease the milk-clotting activity, Al³⁺ and K⁺ had little contribution to increasingmilk-clotting activity. However, Cu²⁺,Zn²⁺ and Na⁺ could inhibit the milk-clottingactivity.