And Fteeze-Drying Processing Lactobacillus Acidophilus NX2-6

Lactobacillus acidophilus is a kind of probiotics, it has strong resistance to acid and bile capacity and adjust the intestinal flora, alleviate the lactose intolerant, enhance immunity, antitumor and other functions.It has a high application value in industry, agriculture, food and health care and other fields of closely related to human, and has been widely used in food, medical care and livestock production. But in the practical application of Lactobacillus acidophilus, there is low effective number of living bacterium, the product of poor stability and short shelf-life and other issues, limiting its industrial production and large-scale applications.The fermentation medium, fermentation process and freeze-drying process of Lactobacillus acidophilusNX2-6 were optimized in this study. Through these expriments, an effective process for L. acidophilus NX2-6 production was obtained with high viable cell count and good stability. The main are as follows:1. Optimization of fermentation medium and culture conditions in flask of L. acidophilusNX2-6. Based on the single factor experiment, the optimal carbon source, nitrogen source and growth factor were found as lactose, yeast extract and soybean meal hydrolysate, corn steep liquor, respectively, and the optimum fermentation conditions were initial pH 6.5, inoculum size 4%, incubation temperature 37℃. PB design (Plackett-Burman Screening for Design) was used to investigate the effects of fourteen factors on the viable cell count of Lacdiophilus NX2-6. Results showed that lactose, sodium citrate and sodium acetate were the key factors affecting L.acdiophilus NX2-6 growth. On this basis, the response surface method (Response Surface Methodology, RSM) was used to study the optimal levels of the three significant factors. By solving the quadratic regression equation, the optimal conditions were as following:lactose 27.4g/L, yeast extract 6g/L, soybean meal hydrolysate 60%(v/v), sodium citrate 2g/L, sodium acetate 2.3g/L, CaCO3 6g/L, K2HPO42g/L, MgSO4-7H2O 0.4g/L, corn steep liquor 20g/L, MnSO4-H2O 0.038g/L, tween-802mL/L, pH6.5, inoculum size 4% and incubation temperature 37℃. Under these conditions, the viable cell count of L.acidophilus NX2-6 was 3.23×109CFU/mL, which was 40.38 times as high as that using conventional MRS medium (8×107CFU/mL).2. Study the fermentation tank culture conditions of L. acidophilusNX2-6. The effects of impeller speed, neutralizeing agents, pH, inoculation methods, initial sugar concentration and fed-batch fermentation on the growth of L. acidophilus NX2-6 were investigated. Based on the values of viable cell count at different culture conditions, the optimal culture conditions for L. acidophilusNX2-6 were as follows:seed was prepared using previously optimized medium, then inoculated and cultured without aeration at a impeller speed 150-200r/min; initial lactose concentration of the medium was 40g/L and pH was 6.5; the culture pH was kept constant at 5.0 with aqua ammonia (4mol/L); corn steep liquor was fed at the end of log phase and cultivation continued at 37℃ to 16h. The viable cell count of L. acidophilusNX2-6 under above culture conditions was 9.7×109CFU/mL, and was 3 times as high as that before optimized, which laid the foundations for the production and application of L. acidophilusNX2-6.3. Study the freeze-drying conditions of L. acidophilusNX2-6. Influences of centrifugation conditions on the survival rate of L. acidophilusNX2-6 were first examined. Then single-factor experiments and orthogonal experiments were carried out obtain the optimum protective agents formula according to the survival rate of L. acidophilusNX2-6 after vacuum freeze-drying. The results showed that most optimum centrifugation conditions for preparing the suspension were 8000r/min and lOmin (at 4℃). The optimal protective agent formulas for L. acidophilusNX2-6 were as following:malt extract powder 15%, sodium glutamate 3%, and inulin 7%. Using this protective agent formula, the survival rate of L. acidophilusNX2-6 after vacuum freeze-drying was up to 82.43%.