植物乳杆菌冻干保护剂的优化及其保护机制

Optimization and Protective Mechanisms for Cryoprotectant of Lactobacillus plantarum CGMCC 6016

为优化以菊粉为主要基质的植物乳杆菌冻干保护剂配方,通过部分因素试验设计筛选保护剂成分,利用中心优化组合设计确定最佳保护剂配方,从菌体形态和细胞膜水平研究保护剂对植物乳杆菌的保护机制。试验结果表明,添加菊粉对植物乳杆菌的冻干存活率影响显著,谷氨酸钠和山梨醇能增强对植物乳杆菌的保护效果;优化后的植物乳杆菌冻干保护剂配方:菊粉22.4 g/100 m L,谷氨酸钠12.8 g/100 m L,山梨醇4.7 g/100 m L。在此条件下植物乳杆菌存活率达86.5%。扫描电镜观察结果表明,添加复合冻干保护剂的植物乳杆菌菌体饱满、完整,而未添加保护剂的空白对照有明显的变形、塌缩,细胞间有丝状物粘连。冻干后植物乳杆菌胞内蛋白有不同程度的泄漏,添加含菊粉的保护剂的菌体胞外蛋白量明显低于对照组。结论:以菊粉为主要基质的复合冻干保护剂,可减轻冻干对植物乳杆菌的损伤,其作用机制是通过保护细胞膜实现对菌体的保护。

The paper focuses on optimization of a protective medium with inulin as the main matrix for enhancing the cell viability of Lactobacillus plantarum CGMCC 6016（L. p） during vacuum freeze-drying. Protective agent components are screened by fractional factorial design（FFD）, then the best cryoprotectant formula of inulin was determined by central composite design（CCD） and response surface methodology（RSM）. Protection mechanisms of cryoprotectant contained inulin was checked during freeze-dried of L. p in the level of the cell membrane and cell morphology. The results showed that inulin was found to have a significantly positive effect on the survival of L. p during freeze-dring, followed by sodium glutamate and sorbitol which improved the protective effect of L.p by a 25-1FFD. After optimization, the maximum cell survival rate was achieved up to 86.5% when the concentration of inulin、glutamate and sorbitol reached 22.4 g/100 m L,12.8 g/100 m L and 4.7 g/100 m L respectively. Intact cells of L. p attached to the inulin cryoprotectant were revealed by means of scanning electron microscope（SEM） against injury during freeze-dring, whereas many damaged, flattened, and concaved characteristics were clearly observed in control. The intracellular protein of L. p was influenced during freeze-drying, inulin had a significant influence on reducing the leaking of intracellular protein compared with controls. In this study it was evident that the optimized cryoprotectant of inulin protected bacteria against injury during freeze-dried by protecting cell membrane.