Investigation On Liquid Fermentation Conditions And Preparation Technology Of Dry Bacterial Powder Of Bacillus Natto

Bacillus natto has very strong activity of protease,lipase and amylase, which can degradate some complex protein,fat and carbohydrates in the plant-based feedstuffs, so it can be used as micro-bioecological preparation in feed industry. However, because of bacillus natto hard to preserve in liquid culture, the application of bacillus natto was restricted in feed industry. If Bacillus natto cultured in liquid medium could be prepared as activated bacterial powder by drying, its value as an ideal strain of microbioecological preparation would be realized, the availability of cheap feed resource would be increased and would brought economic benefits for graziery.In the dissertation Bacillus natto was used as strain, good stain of high biomass was obtained by ultraviolet mutation. Based on its growing characteristic, single factor test and orthogonal design test were used to ensure the optimal culture conditions and the optimal medium of Bacillus natto in liquid state fermentation for high activity and concentration of viable bacteria and spores. The drying treatment of Bacillus natto focused on the effect of centrifugation conditions, drying vacuum for freeze and temperature under normal pressure on the residual rate of bacteria.Bacillus natto was mutagenized with ultraviolet, irradiation time was 120s. The plus-mutant N-6-20 and N-6-32 with genetic stability was obtained by screening with bacterialcolony diameter method and shaking culture method. The experiment results show that, compared with the original strain, the biomass of the mutant N-6-20 and N-6-32 increased by 10.35% and 9.58% when they were cultured for 18h.The liquid culture conditions of Bacillus natto were investigated by single factor experiments. The liquid culture conditions were found as follows: seed age of 16h, initial pH of 7.5, inoculation size of 5%, liquid media volume of 50mL/250mL, cultural temperature of 37℃and shaking speed of 200r/min. Under the optimal conditions the total number of bacterium and spores achieved 4.27×1010cell/mL and 3.93×1010spores/mL, respectively, and sporulation efficiency was about 92% when Bacillus natto was cultured for 24h.The single factor experiment and orthogonal experiment were used to optimize the liquid fermentation medium of Bacillus natto,and the optimal medium was found to be composed of glucose of 1.5%, soybean cake powder of 1.0%, CaCl——2 of 0.5%, MnSO₄ of 0.1% and NaCl of 0.5%. The total bacterium achieved 1.14×1012cell/mL and the total spores achieved 1.08×1012 spores/mL under the optimal medium.The conditions of preparing dry powder of Bacillus natto by vacuum freeze drying and normal pressure drying were investigated. The fermentation liquid was centrifuged to obtain bacteria mire before drying, therefore, the centrifugation conditions was necessary to be studied to obtain a higher viable count. The optimal centrifugation condition was found that the centrifugal speed and time were 4000r/min and 15min. The residue rate of viable bacteria was up to 83.82% under the condition, viable count achieved 1.08×1013 cell/gram. On vacuum freeze drying of Bacillus natto,de-fatted milk powder was chosen as freeze-drying-protectant and its suitable concentration was 10%, and the survival rate of freeze dried bacillus natto was up to 63.20% under the condition.About normal pressure drying, the optimum drying temperature was 60℃, and the survival rate of bacteria after drying was about 61.41%.