Abstract: In order to prepare lyophilized cells of Bifidobacterium bifidum TMC3115 with high viability, the impact of the initial pH of the medium, different carbon and nitrogen sources, and inoculum quantity on the viable cell count was studied by the one-factor-at-a-time method. Subsequently, an orthogonal array design was applied to optimize combinations of carbon and nitrogen sources based on the pH of the medium at 16 h of culture, cell density (expressed as optical density) and viable cell count. The efficacy of 18 cryoprotectants on the probiotic strain was investigated, and the optimization of cryoprotectant blends was carried out using an orthogonal array design. The results showed that when the initial medium pH was 7.2 and the inoculum amount was 4%, the viable cell count reached the highest. The optimized medium was composed of glucose 25 g/L, tryptone 20 g/L, yeast peptone 10 g/L, yeast extract FM902 15 g/L, diammonium citrate 2 g/L, anhydrous sodium acetate 5 g/L, dipotassium phosphate 2 g/L, Tween-80 1 g/L, magnesium sulfate 0.58 g/L, manganese sulfate 0.25 g/L, and L-cysteine hydrochloride 0.5 g/L. The optimal cryoprotectant was composed of skim milk 12%, trehalose 12%, glycerol 1.2%, sodium glutamate 0.6%, and L-cysteine hydrochloride 0.15%. The viable cell count of the freeze-dried culture produced under the optimized conditions reached 4.26 × 1011 CFU/g.

Key words: Bifidobacterim bifidum TMC3115, production process, inoculum quantity, high-density culture, cryoprotectant

摘要： 为制备高活性两歧双歧杆菌（Bifidobacterium bifidum）TMC3115冻干菌粉，对培养基初始pH值和初始接种量进行研究，同时对不同碳源和氮源进行初步筛选，进而以发酵16 h发酵液pH值、光密度和活菌数为评价指标，通过正交试验对碳源和氮源组合进行筛选。考察18 种冻干保护剂对两歧双歧杆菌TMC3115的冻干保护效果，并通过正交试验确定复合冻干保护剂配方。结果表明：两歧双歧杆菌TMC3115在培养基初始pH值7.2、接种量4%时所得发酵液活菌数最高；两歧双歧杆菌TMC3115发酵培养基配方为葡萄糖添加量25 g/L、胰酪蛋白胨20 g/L、酵母蛋白胨10 g/L、酵母浸粉FM902 15 g/L、柠檬酸氢二铵2 g/L、无水乙酸钠5 g/L、磷酸氢二钾2 g/L、吐温-80 1 g/L、硫酸镁0.58 g/L、硫酸锰0.25 g/L、L-半胱氨酸盐酸盐0.5 g/L；最佳冻干保护剂配方为：脱脂乳添加量12%、海藻糖12%、丙三醇1.2%、谷氨酸钠0.6%、L-半胱氨酸盐酸盐0.15%，此条件下，两歧双歧杆菌TMC3115冻干粉活菌数达到4.26×1011 CFU/g。

关键词: 两歧双歧杆菌TMC3115, 生产工艺, 接种量, 高密度培养, 冻干保护剂