Study On The Breeding Of Natto With Antibacterial Substances Producted And Fermentation Characteristics

Bacillus natto is a production strain of natto which is Japanese traditional fermented food, and its metabolites not only have a variety of physiological functions, but also have broad-spectrum antibacterial effect.In the food industry, the traditional food preservation methods is to add chemical preservatives, but long-term use of such food will affect the health of human. Therefore the development of natural preservatives have become a current research focus, among of it, the use of microbial production of natural preservatives is a developed field. So the development of the antibacterial of Bacillus natto have high practical value. This paper study on the breeding of Bacillus natto which is product antibacterial substances and fermentation characteristics.The main research contents are as follows:1A strain named ZX-2with high antimicrobial activity was isolated from Natto, and identification, the result was consistent with the Bergey bacterial identification, belonging to Bacillus subtilis, Natto subsppecies, refferred to as Natto. This strain has a strong antimicrobial activity to Staphylococcus aureus and Escherichia coli.2To obtain an excellent strain with a high antibacterial activity, the composite mutation of Natto protoplast was studied by UV ray and5-fluorouracil. The results showed that under the condition of0.3mg/ml enzymolysis concentration,40min enzymolysis time,35℃enzymolysis temperature and pH7.0enzymolysis, the pretoplast formation and regeneration rates are the highest. By composite mutation of UV and chemical, a strain with high antibacterial activity was obtained, and named ZX-2-4.Compared with the original strain, its antibacterial effect of Staphylococcus aureus increased by19.7%,and its antibacterial effect of E.coli increased by19.5%. The antibacterial activity kept stable after10generations of reproduction.3By using hydrolyzed soybean meal for Natto liquid fermentation to produce antibacterial substance. A Plackett-Burman design was used to evaluate the affecting factors of Natto fermented. Bran content, temperature and initial pH are significant factors; the steepest ascent experiment is used to approach the optimal conditions subsequently; finally the optimized conditions are bran content28.4g/l, temperature33℃, initial pH7.4further optimized with response surface methodology, under these conditions to ferment Natto, ΔOD value is0.416, crude extracts of bacterial inhibition rate is56.6%. 4By using of Logistic and Leudeking-Piret equation, the dymamic models of Natto growth and substrate consumption was constructed. The optimal kinetics model parameters were obtained through optimization and non-linear curve fit by Origin8.5software, the dymamic models of Natto growth and substrate consumption were x=0.083e0.222t/0.948+0.0520.222t and y0.948+0.052e0222t S(t)=2.62-0.076e0.222t/0.948+0.052e0.222t,the coefficients were0.9901and0.9908, these kinetics models can really refect the regularity of Natto growth and substrate consumption.5This paper study on the initial separation of antibacterial substances to Natto and its stability. First determine the40%-70%ammonium sulfate saturation is the best salting condition; and then the crude protein were elted through the DEAE-cellulose chromatography and three peaks were founded, peak Ⅱ and Ⅲ have not antibacterial activity, peak Ⅰ has strong antibacterial activity; finally collection peak Ⅰ to do Sephadex G-100gel filtration, get two peaks, the antibacterial experiment shows these two peaks all have antibacterial activity. And study on the stability of antibacterial crude protein, the results show that antibacterial crude protein of Natto has well stability to heat and pH, but has a certain sensitivity to the protease.