Screening High Yield Mutant From Chymosin-producing Bacillus Subtilis Irradiated By N~+Ion Beam

There are mainly three categories of chymosin in the production of cheese, including animal rennet, vegetable rennet and microbial rennet. Curd dynamic and proteolytic power are the most important indicators to evaluate advantages and disadvantages of rennet. In recent years, by increasing output of cheese, the demand of chymosin is on the increased, therefore, microbal rennet which source is extensive and its price is cheap increasingly was valued highly by people.Now, the research about mutation breeding of product rennet strains focus on mostly in the mold rarely in the bacteria. In this research a strain named XC-1with high milk-clotting activity was isolated from the merchant cheese. Using the physiology and biochemistry identification and16SrDNA sequencing, strain XC-1was identified as Bacillus subtilis. It was the first time that N+ion beam implantation was applied for mutagenesis of Bacillus subtilis. Using strain XC-1as parent strain the mutation experiment was carried out. The high yield of product rennet strain was screened. Fermentation production of industrialization of microbial rennet was laid the foundation by optimizing the fermentation technology and studying the activity of enzyme.Experiment one:A product chymosin strain was obtained by isolating and screening from Mailand cheese. The strain’s chymosin’s curd dynamic was750SU/mL, proteolytic activity was1.86U/mL, and the ratio of curd activity and proteolytic activity was403.23. Its curd effect:texture was smooth, hardness was moderate, appearance was cream yellow uniform and glossy.Experiment two:Using the physiology and biochemistry identification and16SrDNA sequencing to build a strain phylogenetic tree, the strain XC-1was identified as Bacillus subtilis.Experiment three:It was the first time that N+Ion beam implantation was applied for mutagenesis of Bacillus subtilis. Using strain XC-1as parent strain the mutation experiment was carried out and the result showed that the dose of2.08X1015ions/cm2with the power of20keV was optimal for mutagenesis of Bacillus subtilis.and a mutant strain XCYB-6was screened. The milk-clotting activity of strain XCYB-6reached2181.82SU/mL which was1.9higher than that of the parent strain. Meanwhile the proteolytic activity of strain XCYB-6was2.53U/mL and the ratio of milk-clotting activity to proteolytic activity reached up to862.38. The pass-generation (six generations)1indicated that strain XCYB-6had good genetic stability. Experiment four:Using SAS9.1software for data processing, the mathematical model of the effects of the chymosin activity from chymosin on glucose concentration, beef extract concentration, loading volume, inoculum density was built based on the single factor test of ermentation. The fit of the model was better through the variance and reliability analysis. The optimal fermentation conditions were as follows:glucose concentration was15g/L, beef extract concentration was5.0g/L, loaded liquid was50mL, inoculum dose was3.5%, and the actual chymosin activity was3055SU/mL. Verification test showed that fermentation technology model could completely imitate the fermentation production process of the chymosin.Experiment five:The enzyme activity tests showed that the chymosin had high enzyme activity on the acidic condition when pH was5.5and kept higher the curds vigor on the Weak acid to neutral and slightly alkaline conditions. Its stability of the enzyme activity was best when pH was6.0. The optimal curd temperature of chymosin was45℃Moreover, as the extension of heat preservation time, curd activity and proteolytic activity showed a trend of decline the higher the temperature, the bigger the downward trend. Enzyme is stable to light while the optimum substrate concentration was10%. Besides, ferrous ions, calcium ions and magnesium ions had a significantly activity role in curd dynamic, thereinto, the activity of calcium effect is most obvious, the curd dynamic effect of sodium, potassium and chloride was not significant, as well as the curd dynamic effect of copper ions and zinc ions had a slight inhibition.