Study On The Breeding Of Bacillus Natto With High Yield γ-PGA And Fermentation Characteristics

γ-Polyglutamic acid(γ-PGA) is the polymers produced by a variety of corynebacterium microorganism, an extracellular glutamate, connected by D-glutamic acid and L-glutamic acid γ-glutamine bond polymerization. γ-PGA can be used as bio-flocculant, wetting agents, thickeners, drug delivery agents, drug carriers, superabsorbent, biodegradable fiber, heavy metal absorber and as a food additive widely used in the food industry, the pharmaceutical industry, the cosmetics industry and sewage treatment. Compared with traditional chemical synthetic polymer, and because of its biodegradability, biocompatibility, non-petroleum resources, a number of advantages to achieve sustainable development, the development and application of microbial synthesis gradually has become a hot research in recent years and people are more and more love biopolymer. This paper study on the mutation breeding a high yield γ-PGA Bacillus natto and its fermentation characteristics.First, studied the characteristic of γ-PGA and cetyl trimethyl ammonium bromide (CTAB) solution of sodium hydroxide formed suspension by reaction, the absorbance of the reaction system to reflect its turbidity, and then established a method for the detection of y-PGA in the fermentation broth was measured by the linear relationship of the turbidity and the concentration of γ-PGA. Finding as follows:CTAB turbidimetry best detection wavelength is250nm,CTAB mass concentration of5g/L,reaction temperature is room temperature, complexing3min.The linear regression equation y=0.0335x-0.1519, R2=0.9995.The precision and recovery of this method are within2.56%and106.6%.The protoplast preparation conditions of B.natto was optimized. using UV-lithium chloride composite protoplast mutagenesis to obtain high yield γ-PGA superior strains. The results show that, under the conditions of when lysozyme concentration0.6mg/mL, hydrolysis time40min, hydrolysis temperature was35℃, the osmotic stabilizer for protoplast prepared in0.7mol/L KC1solution, the formation rate was95.46%, regeneration rate was15.83%. Complex mutagenesis with40s ultraviolet irradiation dose and6%of the concentration of lithium chloride Protoplasts obtained strain ZJ-307.y-PGA production reached15.56g/L, a36.46%increase compared to the original strain, continuously passaged cultured for10generations, genetic stability was stable.By using Plackett-Burman design and response surface analysis, with fewer test of Bacillus natto ZJ-307, the fermentation conditions were optimized. Plackett-Burman design results show that sodium glutamate, yeast extract and K2HPO4are important factors that affect the level of y-PGA fermentation.In the experimental design and analysis of the response surface, the concentration of the three factors to optimize and the obtained culture conditions optimized for:sodium glutamate39.99g/L, yeast extract25.53g/L, K2HPO42.25g/L, glucose50g/L, MgSO40.4g/L, CaCl20.3g/L, inoculum size3%, pH7.5, medium volume to80mL, incubation temperature of35℃, shaking speed200r/min, γ-PGA levels by early the beginning of culture under the conditions of15.56g/L increased to24.62g/L, the values obtained is close to the regression equation which show that the model is reliable.By using Logistic equation and Leudeking-Piret equation established B.natto ZJ-307producted γ-PGA fermentation model, three models of linear R-value all above0.99,which show that can described B.natto ZJ-307Batch fermentation process in cell growth, product formation and substrate consumption with the fermentation time change well, in a certain extent, reveals the kinetic characteristics of B.natto ZJ-307fermentation metabolic γ-PGA.Studying pH and viscosity change during Bacillus natto ZJ-307fermentation process, study the effect of temperature and pH on the fermented liquid viscosity, on this basis, separation and extraction of fermentation products. The molecular weight of the fermentation product were measured by viscosity method. Paper chromatography and infrared spectroscopy method conducted a preliminary analysis of the extraction and purification of γ-PGA samples, the results show that the fermentation product is a molecular weight of300,000units of y-PGA.