乳酸菌R8高密度培养的发酵工艺研究

Optimization of Culture Conditions and Medium of Lactic Acid Bacteria R8 Cultured in High Density Fermentation

以乳杆菌R8菌株为材料,对其高密度发酵培养工艺进行了研究。以菌体密度(OD600)及碳源(葡萄糖)的利用情况为主要参考指标,研究了中和剂、pH、接种量、初始糖含量、通气方式以及补料工艺等对菌体在发酵罐内生长的影响,并对试验菌进行250 L中试放大工艺的优化。优化菌体小试发酵工艺为:将种子液以8%(V/V)的接种量接种于装液量70%的小罐,初糖浓度40 g/L,搅拌转速100 r/min,间歇通氮气维持一定的厌氧环境,自动流加12.5%的氨水控制pH恒定在5.8,37℃恒温发酵。250 L中试发酵工艺为:将控制pH 5.8,37℃恒温发酵7 h左右的种子液以8%接种量接种于装液量为70%的250 L发酵罐,搅拌转速60 r/min,自动流加氨水控制pH 5.8,间歇通氮气不保压(每2 h以0.2 vvm的速率通氮气5 min),37℃恒温发酵10 h左右结束。250 L规模所得发酵液的活菌浓度约为8×109 CFU/mL,经真空冷冻干燥所得的菌粉活菌浓度约为1.2×1011 CFU/g。

The high density fermentation technology of Lactobacillus R8 strain was studied in this study. The main reference indexes were the density（OD600） and the utilization of carbon source（glucose）. The effects of medium and agent, pH value, inoculation amount, initial sugar content, aeration mode and feeding process on the strains growth in 7.5 L stirred fermentation tank were studied, and then the experiment bacteria were optimized by 250 L pilot-scale amplification. The optimizing small-scale fermentation process of the bacteria was： inoculating the seed liquid（8% v/v） into cannikin with medium volume of 70%,, the initial sugar concentration 40 g/L, stirring speed 100 r/min, intermittent nitrogen to maintain a certain anaerobic environment, automatic flow plus 12.5% ammonia, and the fermentation at 37 ℃ and pH 5.8. The 250 L pilot fermentation process was as follows： the pH was controlled at 5.8, the fermentation was performed at 37 ℃ for 7 h, the seed liquid was inoculated in the 250 L fermentation tank of 70% liquid at 8% inoculation amount, stirring speed was 60 r/min, 12.5% ammonia was used to maintain the pH of medium at 5.8,, intermittent nitrogen was introduced（0.2 vvm/2 h of the rate of nitrogen 5 min）, the resultant mixture was incubated for 10 h at 37 ℃. The live bacteria concentration of fermented liquid obtained from 250 L scale was about 8×109 CFU/mL, and the concentration of live bacteria was about 1.2×1011 CFU/g after vacuum freeze-drying.