Study On Separation Purification And Identification Of M-01 Bacillus Amyloliquefaciens And Its Growth Rule In Ecological Culture

The research adopts molecular biology identification technique to identify M-01 strain and study related enzyme activity change rule of M-01 which is collected, separated and purified by self from the northeast area of China. Then using the intelligent probiotic liquid fermentation device and intelligent solid ecological culture-drying equipment designed and manufacted by self to ferment and cultivate M-01 in liquid and in solid respectively. To study related indexes dynamic change rule, test running effect of solid ecological culture-drying equipment. This study can supply necessary technical reference for the factory production and application. The results are as follows:(1) Studying on morphologic features and determining physiological biochemical indexes of M-01 collected, separated and purified in own. Combining molecular biology identification technique by 16SrDNAand gyrA gene sequence phylogentic tree analysis to indentify M-01. The Identification results are:M-01 Bacillus amyloliquefaciens subsp.plantarum,and it belongs to aerobic bacterira.(2) Under the conditions of 18℃, pH 7.2, studying on amylase, protease and lipase activity change rule of M-01, the results show that:the amylase activity of M-01 reaches a higher level in 68h, is 13.02U/dL; the protease activity of M-01 tends to be stable after 56h, and achieves in 8.04U/dL in 74h; the lipase activity of M-01 reaches its peak value in 56h, is 1.159U/L.(3) Under the proper conditions of nutrition substrate and inoculation quantity, fermenting and cultivating M-01 in liquid with the intelligent probiotic liquid fermentation device through controlling reasonably and effectively fermentation temperature at 35±0.2℃, the initial pH 7.2±0.1 and gas stirring method. The results show that:after liquid for 24h, the number of viable bacteria is 146.675±4.05×108CFU/mL; the rate of spores is 91.76±1.50% in fermentation liquid; During the whole fermentation process, the pH decreases; the reducing sugar content in fermentation liquid decreases and it is 5.857±0.314mg/mL in 24h; During 0-4h of process, the ammo nitrogen content in fermentation liquid tends to increase, during 4-24h, it reduces gradually. At the same, the total microbial protein content in fermentation liquid shows a rising trend during the whole process.(4) Through L9 (33) the orthogonal experiment design, studying on the influence of culture temperature, material humidity and effect time on the M-01 spore forming rate after solid fermentation and culture for 28h. The results show that:the primary and secondary order of the factors influencing the formation of M-01 spore forming rate is:culture temperature> material humidity> culture time. So the best condition for promating M-01 spore formation rate after solid fermentation and culture for 28h is A1B3C2, which is culture temperature 45℃, material humidity 55%±5%, culture time 2h.(5) Using the intelligent solid ecological culture-drying equipment self-developed to ferment and cultivate M-01 in solid and researching on the effect solid ecological fermentation and temperature-sensitive drying through controlling the fermentation temperature and material humidity automatically, and regulating the initial pH 7.2±0.1. According to the results of orthogonal experiment design, fermentation and cultivating M-01 in stages. In the first stage, controlling the fermentation temperature at 35±1℃, material humidity at 55%±5% for solid fermentation 28h, to get higher viable bacteria number. In the second stage, controlling the fermentation temperature at 45℃ for solid fermentation 2h, to stimulate the formation of spore and get higher spore number. The results show that:the number of viable bacteria is 224.25±4.47×108CFU/g, the number of spores is 203.63±4.51×108CFU/g, the rate of spore forming is 90.8±0.73% in fermentation products in 30h. After the end of solid culture, dewatering and drying at temperature 43℃±2℃ with the intelligent solid ecological culture-drying equipment self-developed, which can keep the activity of bacteria effectively and drying effect well simultaneously. The number of spores is 210.05±4.96*108CFU/g, moisture content is 5.53±0.12%, the rate of mixed bacteria is 0.681±0.003% in fermentation products.