热休克处理调控海洋源嗜酸乳杆菌糖代谢路径的抗冻干胁迫机理研究

Previous researches indicated that the heat shock treatment significantly enhanced the survival rate and lactic acid content of Lactobacillus acidophilus SN6 which was isolated from the intestinal tract of Scomberomorus niphonius after freeze drying. However, the mechanism involved remains to be further investigated. This research tries to reveal the mechanisms of resistance to freeze drying stress of Lactobacillus acidophilum SN6 by heat shock treatment in glycometabolism pathway. The activity of rate-limiting enzymein lactic acid synthesis will be tested to determin the controlled enzyme by heat shock treatment in glycometabolism pathway.Then the qRT-PCR and western blot were used to determine the transcriptional level and protein expression level of the enzyme. After that,the gene of key control enzyme will be cloned and over-expressed in Lactobacillus acidophilus SN6 and the survival rates will be compared to validate its role of resistance to freeze drying stress. Thus the mechanisms will be explained by glycometabolism pathway. The results will provide supports for improving the viability of lactic acid bacteria and constructing strains of resistance to adversity.

前期研究表明，热休克处理可以显著提高蓝点马鲛鱼（Scomberomorus niphonius）肠道中的嗜酸乳杆菌（Lactobacillus acidophilus）SN6在冷冻干燥后的存活率和糖代谢的乳酸含量，但相关机制不明。本课题从糖代谢的角度，通过测定热休克处理的嗜酸乳杆菌SN6在热休克、预冻、冷冻干燥过程中糖代谢限速酶活性的变化，与未处理的对照组做比较，确定受热休克调控的关键酶；再分别利用western blot、实时荧光定量PCR检测该酶在热休克、预冻和冷冻干燥过程中的蛋白表达水平和转录水平的变化；进一步克隆并诱导其在嗜酸乳杆菌SN6中的过量表达，通过冻干存活率的变化，来验证其在抗冻干胁迫中的作用。研究从糖代谢路径探明热休克处理调控嗜酸乳杆菌抗冻干胁迫的机制，为提高乳酸菌抗逆能力、筛选与构建抗不良环境的菌株提供理论依据和技术支持。

热休克处理可以显著提高嗜酸乳杆菌在冷冻干燥后的存活率，但相关机制不明。本课题通过研究热休克处理对嗜酸乳杆菌冻干后糖代谢产物、酶活性、蛋白表达、mRNA表达水平的变化，揭示热休克处理调控嗜酸乳杆菌抗冻干胁迫的机理。乳酸菌培养10.5h后，与未热休克处理的对照组相比，45℃处理30 min的热休克组，冻干存活率比对照提高了18.1%，多糖产量提高38.8%，其组成成分半乳糖比例显著提高、葡萄糖比例显著降低。乳酸转化率提高。磷酸葡萄糖异构酶活性降低、磷酸葡萄糖变位酶、UDP-葡萄糖焦磷酸化酶、糖基转移酶、乳酸代谢路径乳酸脱氢酶、Na+-K+-ATPase活性显著提高。蛋白质组学结果显示差异蛋白36种下调，13种上调。糖代谢相关基因LBA0444、LBA1719、LBA1944、galT、galM和LBA0143下调，LBA0625、fbaA、pgi、tpiA、ppc、LAC30SC_08645和LBA1870上调。上述基因可能为热休克处理调控嗜酸乳杆菌糖代谢路径的关键基因。从糖代谢路径探明了热休克处理调控嗜酸乳杆菌抗冻干胁迫的机制，为提高乳酸菌抗逆能力、筛选与构建抗不良环境的菌株提供理论依据和技术支持。