Study On High Cell Density Culture And Freeze-Drying Protection Of Lactobacillus Plantarum 299

In order to improve the number of Lactobacillus plantarum 299 for providing basis in order to commercial production in the future, this study had carried on the system research on the fermentation medium, high density fermentation process, freeze-dry process, cryoprotectant and storage period,then obtained the following conclusions.By single factor experiment, PB and the response surface test analysis, optimized the best medium for L. plantarum299:peptone 2.33%, yeast extract 1.41%, glucose 2.52%, citric acid hydrogen diamine 0.40%, acetic acid sodium 0.30%, dipotassium phosphate 0.80%, tween-800.30%, and manganese sulfate 0.0025%.Then the OD600 of bacteria can reach 1.2687, it increased by 28.9% than the bacteria in the MRS medium. At the same time, the cultivation of the optimized had removed beef paste and magnesium sulfate, simplifyied the culture medium process.By determining the optimum medium and on the basis of preliminary laboratory work, this study had optimized the effect factors of high density fermentation process in the 30L semi-automatic fermentation tank and get the high density fermentation process as follows: pH5.5, the neutralizing agent of 25% ammonia water, stirring speed 100 r/min, determine the bacteria growth restriction substrate as carbon source and nitrogen source, the best ratio 3:2, at the same time in logarithmic phase two supplementary food every 4h. Under this condition, the OD600 of L. plantarum 299 can reach 1.3422, increased by 44% than the common static culture.This study had optimized the condition of vacuum freeze drying and cryoprotectant, and the best freeze-dry process is the prefreezed for-80℃,2h. best ratio of bacteria and cryoprotectant was 2:5, the optimum freeze-drying thickness of 1.2 cm, skimmed milk 16%, frceze-dried for 20 h. The number of living bacterium can reach 2.11×1011 cfu/g, freeze-dried survival rate was 89.8%.The storage conditions and storage time of L. plantarum 299 were studied. There were no difference between Aluminum Foil bag and PET-PE bag(ordinary vacuum bags) in-20℃ within a year. However, there were little difference in 4℃ and 25℃ under the preservation, and decreased as the temperature difference. Aeration using aluminum foil bag packing, found that 95% N2,5%CO2 gas when bacteria survival rate is highest.