Technical Study About Milk-clotting Enzyme On Liquid Fermentation

It had become a research hotspot to seek the excellent microbialchymosin as the increasing consumption of cheese and other milk products.Compared with calf rennet, chymosin synthesized from microbes have manyadvantages such as high productivity,unlimited by bio-resource, however,there are some ubiquity fault of microbial chymosin such as high proteaseactivity, high thermal stability, generate bitter peptides. Our research on liquidfermentation of chymosin from microbes to get the ideal substitute for calfrennet, has important theoretical and practical value.Research on chymosin synthesized by filamentous fungi indicates Mucormiehei ATCC16457which produced largest milk-clotting activity (21SU/mL)and grew well was the potential strain for high productivity on milk-clottingenzyme.First, mutant strain MmV-4which had high milk-clotting activity andC/P was screened by ultraviolet and HNO3mutagenesis. Throughfermentation, milk-clotting activity had arrived to40SU/mL, two times thanthat from initial strain, and C/P value to1425, increasing24%.Next, optimization of milk-clotting activity productivity on liquid fermentation by MmV-4was investigated. SCT mediun was choosen forinitial liquid fermentation medium, on which MmV-4produced two times ofmilk-clotting activity than culturing on bran solid medium. SDA medium asspores medium culturing strain as inoculums increased2times ofmilk-clotting activity than culturing on PDA medium, and the best C/N was4:1.The optimal inoculum concentration, fermentation time and culturingtemperature was3%,84h and28.After optimization of fermentationconditions, milk-clotting activity rise to60SU/mL, C/P value arrived to280.1.6%Sucrose and3%corn steep liquor as the best carbon and nitrogenfor producing milk-clotting enzyme increased highly of milk-clotting activity,C/P and specific activity. Additional feed of organic nitrogen influencedproductivity of milk-clotting enzyme greatly, of which0.5%tryptone and0.5%dusty yeast+0.3%peptone was suitable.0.2%Na2HPO4was optimal forhigher productivity of milk-clotting enzyme. Heavy metal molecule of MnCl2increased milk-clotting activity greatly, and addition of ZnSO4didn'tworked for higher productivity of milk-clotting enzyme. Finally, throughresponse surface experiment for analysis, the optimal level of factors andinteraction are obtained.399SU/mL of milk-clotting activity and1425of C/Pvalue wre obtained, which were separately20times and6.75times beforeoptimization. The specific activity of crude enzyme was390000SU/g.In addition, we preliminary study for glutinous rice mold culturefermentation process and isolation, identification of the predominant strains. We found a strain B-1-4, which milk-clotting enzyme activity is2SU/mL,expand the sources of microbial chymosin.