Study On Screening Of Chymosin-producing Strain From Traditional Dairy Products

Chymosin is a kind of key enzyme in production of cheese through milk-clotting. In recent years, increased cheese production has caused calf rennet supply decreased. Therefore, microbial chymosin development for substitution of calf rennet has always been the hot topic of dairy science. This work screened milk-clotting enzyme producing strains from traditional dairy products and the strains were identied. The purification of the milk-clotting enzyme was studied by ultrafiltration, glucan gel chromatography and ion exchange technology. Also, Its enzymic properties were investigated.In this study, two milk-clotting enzyme producing strains exhibited higher milk-clotting activity (MCA) and the ratio of MCA to proteolysis activity (PA) isolated from traditional dairy products. According to the characteristics of morphology,16S rDNA sequence analysis and physiology and biochemistry tests, two strains were identified separately as Bacillus amyloliquefaciens and Bacillus subtilis.Bacillus amyloliquefaciens was choosed as original strain. On the basis of single factor experiment, the best fermentation medium for the milk-clotting enzyme produced by Bacillus amyloliquefaciens were acquired by response analysis in which the MCA was the main indicator. The results showed that the suitable fermentation medium for milk-clotting enzyme produced by Bacillus amyloliquefaciens was potato extract0.5%, glucose1.13%, yeast extract powder1.76%and CaCO30.32%. Under above conditions, the MCA reached436.8Su/ml.In order to improve the yield of milk-clotting enzyme activity (MCA) produced by Bacillus amyloliquefaciens, using single factor and orthogonal test to determine optimum fermentation conditions. Culture temperature, vibration speed, initial pH and culture time was tested. The results showed that the optimum fermentation conditions were as follows: fermentation medium30mL in a100mL Erlenmeyer flask, inoculums size3%, culture temperature39℃, culture time14h, initial pH of fermentation medium6.0and vibration speed120r/min. Under the optimal conditions the MCA was923.1Su/mL. The milk-clotting enzyme from Bacillus amyloliquefaciens was concentrated by ultrafiltration. Then the concentrated enzyme was further purified by Sephadex G50and weak anionic exchange DEAE-Sephacel, which was confirmed by SDA-PAGE that showed only one band with a molecular mass of41kDa. The MCA reached1724Su/mg, which was187.4folds higher than that before purification.As for the enzymic properties:the optimum temperature for purified enzyme was65℃. When the enzyme was obtained for60min at30～45℃, the MCA was steady. The enzyme was completely inactivited by heating for30min at60℃or10min at65℃. The optimum pH for purified enzyme was5.5and the MCA was stabe in a wide range of pH4.0-10.0. The MCA increased with the decreasing of milk pH from6.5-5.8. The metal ions Ca2+, Mg2+, Ba2+, Fe3+, Fe2+, Li2+, Mn2+showed obvious stimulation on MCA especially Mn2+whereas the MCA was inhibited only slightly by K+、Na+and significantly by Cu2+.