高密度发酵工艺对益生菌抗冷冻活性影响的研究

The core technologies of preparation of highly active probiotics starter cultures are high density cultivation and vacuum freeze-drying. The technology of high cell density cultivation refers to the microorganisms cultivate ways by which the population density of cells is up to ten times more than routine cells culture methods when they were cultured in liquid medium. The main purpose of high cell density cultivation of probiotics is for preparation of highly active probiotics starter cultures. Vacuum freeze-drying technology, including the techniques of freezing and drying, is one of the best methods for preparation of probiotics starter culture and preservation of strains activity. But this process can affect significantly cell viability.. A lot of research was carried out for improving cell viability, including optimization of freeze drying process and use of cryoprotective compounds, such as sucrose, lactose, trehalose, sorbitol, proteins and skim milk. Although major emphasis has been placed on the effect of the drying medium and freeze drying process, the technology of high cell density cultivation is also a critical parameter, which is likely to play a role upon survival subsequent to freeze-drying. But the latter is often ignored by probiotics starter culture producer. . In order to evaluate the relationship of the technology of high cell density cultivation and the freeze drying survival of probiotics, Lactobacillus plantarum LIP-1 was chosen as research objects in this project, which was identified as a probiotic isolate during screening of lactic acid bacteria from koumiss samples collected in Inner Mongolia, and our research showed that L. plantarum LIP-1 had cholesterol-reducing health benefits. The effect of different fermentation parameter on the freeze-drying viability will be investigated in this research, in addition, activities of sugar metabolic enzymes and changes of cell membrane of Lactobacillus plantarum LIP-1 will be determined in different growth conditions. The expression difference of proteins of the strain cultivated in different growth conditions will be detected by two-dimensional gel electrophoresis and accompanied with the analysis of mass spectrometry and bioinformatics. Base on these data, the mechanism of the effect of different fermentation parameter on the freeze-drying viability will be analyzed.. The implementation of project would provide some new ideals and methods for improving activity of probiotic starter culture, reducing the production cost and promoting the development of the industrialization of probiotic production.

在制备高活性益生菌发酵剂的过程中，为了减少菌株在真空冷冻干燥过程中的死亡率，国内外常利用合理的冷冻干燥工艺以及添加冻干保护剂来提高菌株的存活率。但有研究发现高密度培养也对益生菌发酵剂抗冷冻性有着极其重要的影响，而这经常被人们所忽视。. 本课题以益生菌植物乳杆菌LIP-1为研究对象，通过研究不同高密度发酵条件对菌株在冷冻干燥过程中存活率的影响，揭示发酵工艺与益生菌抗冻干活性的关联性；在此基础上，研究菌体糖代谢关键酶及细胞膜构成的变化，探求不同高密度发酵工艺对细胞生理特性的影响以及这些生理变化与抗冷冻干燥性能的相关性；研究菌体蛋白质表达差异，结合质谱和生物信息学技术，探讨不同高密度发酵工艺对细胞物质和能量代谢等相关蛋白的影响，进而全面解析发酵条件的改变对冻干存活率影响的机理。课题的实施将为提高益生菌发酵剂活菌数量、降低生产成本、促进我国益生菌发酵剂产业化生产提供新的思路和方法。

益生菌能够促进人体肠道菌群平衡，具有提高机体免疫力、预防心血管疾病和促进消化功能等功效，因而有着极为广阔的市场，然而在益生菌菌粉及其发酵剂的生产制备过程中，常会因为冷冻干燥处理导致活菌数量显著降低，使益生菌难以发挥应有的功效。如何改善益生菌的抗冷冻干燥性能，对于益生菌市场的发展具有重要的意义。.本课题以益生菌植物乳杆菌LIP-1为研究对象，以 MRS 培养基为对照，通过改变培养基的成分和含量（碳源、氮源、碳氮量及碳氮比，缓冲盐、微量元素及生长因子等）以及培养条件（初始pH），结合冷冻干燥后的活菌数和菌株冻干存活率，评价培养基成分和培养条件的变化对植物乳杆菌LIP-1抗冷冻干燥性能的影响。在此基础上，确定了益生菌植物乳杆菌LIP-1高密度发酵的最佳培养基和培养条件。同时选取在高密度培养后具有较高的活菌数，而菌体冻干存活率高和低的样品，测定其在不同培养基成分和培养条件下的代谢关键酶（己糖激酶、丙酮酸激、乳酸脱氢酶、ATP 酶）及细胞膜特性（细胞壁完整性、细胞膜的完整性、流动性、通透性以及脂肪酸的组成）变化，并通过电镜观察菌体形态，以培养基成分和培养条件的改变为参考，探求了不同培养工艺对细胞生理特性的影响，以及这些生理变化与抗冷冻干燥性能的相关性。此外利用原核链特异性转录组学和TMT标记蛋白质组学技术，研究所选定样品在不同培养工艺下的基因和蛋白质表达差异，探讨了高密度发酵工艺对细胞物质和能量代谢等相关基因、蛋白和代谢通路的影响，并比较分析这些差异与菌体抗冷冻干燥的相关性。.本研究确定了高密度发酵工艺对菌株冷冻干燥存活率影响的关键因素，并全面解析了不同培养基成分和培养条件的改变对冻干存活率影响的内在机理。证明在保证菌株高密度生长量的前提下，通过改变培养基成分和培养条件，可以有效提高菌株冷冻干燥抗性。