保加利亚乳杆菌的冷适应性与冷应激蛋白的研究

几乎所有单细胞生物在外界环境急剧降温的条件下都会发生应激反应,产生的一系列蛋白质被称为冷应激蛋白。冷应激蛋白在乳酸菌适应低温环境和增强抗冻能力方面发挥着重要作用。本研究从冷应激蛋白着手,研究了保加利亚乳杆菌低温适应性及冷应激蛋白的抗冻保护作用。在冷冻前进行20℃预处理可以使保加利亚乳杆菌的存活率增加100倍,而低于最低生长温度时,与对照组的存活率相差不大。通过SDS-PAGE电泳,冷处理样泳道出现了两种7ku的冷应激蛋白。利用PCR技术进一步获得保加利亚乳杆菌中冷休克蛋白的基因片断,与L.delbrueckii subsp.bulgaricus ATCC11842中CspA序列同源性达到100%。低温预处理可以增强菌体抗冻能力,且可以产生3条6.3、6.7、7.0ku的冷应激蛋白,初步推测低温诱导冷应激蛋白的表达抵御了冷冻对保加利亚乳杆菌的伤害。

Study on cold shock proteins and low-temperature response of L.delbrueckii subsp, bulgaricus

Nearly all cells respond to a decrease in temperature by inducing a set of proteins, called cold shock proteins(CSPs). These proteins are thought to play a role in the protection of cells against damage caused by freezing. In this study, low-temperature adaptation and cryoprotection were studied in L.delbrueckii subsp.bulgaricus, which actively adapted to freezing during a pretreatment at 20 ℃, resulting in an approximately 100-fold increased survival after freezing compared to mid-exponential-phase cells grown at optimal temperature of 42 ℃. No adaptation was observed when cells were exposed to a temperature(10 ℃) below the minimal growth temperature of the strain (just below 17 ℃). By SDS-PAGE 7 ku cold-induced proteins were identified. To confirm the presence of csp gene in L.delbrueckii subsp. bulgaricus, PCR strategy was used to yield products of different sizes. Sequence analysis revealed csp-like sequences were up to 100% identical to those of CspA of L.delbrueckii subsp. bulgaricus ATCC 11842. The result showed a higher freeze survival capacity of L.delbrueckii subsp. bulgaricus after exposured to a low temperature and protein synthesis was required for this adaptation, which were found in the class of 7 ku CSPs.