De Angelis Maria, Di Cagno Raffaella, Huet Claude, Crecchio Carmine, Fox Patrick F, Gobbetti Marco
Institute of Sciences of Food Production, CNR, 70125 Bari, Italy.
Appl Environ Microbiol. 2004 Mar;70(3):1336-46. doi: 10.1128/AEM.70.3.1336-1346.2004.
Heat stress resistance and response were studied in strains of Lactobacillus plantarum. Stationary-phase cells of L. plantarum DPC2739 had decimal reduction times (D values) (D value was the time that it took to reduce the number of cells by 1 log cycle) in sterile milk of 32.9, 14.7, and 7.14 s at 60, 72, and 75 degrees C, respectively. When mid-exponential-phase cells were used, the D values decreased. The temperature increases which caused a 10-fold reduction in the D value ranged from 9 to 20 degrees C, depending on the strain. Part of the cell population treated at 72 degrees C for 90 s recovered viability during incubation at 7 degrees C in sterile milk for 20 days. When mid-exponential- or stationary-phase cells of L. plantarum DPC2739 were adapted to 42 degrees C for 1 h, the heat resistance at 72 degrees C for 90 s increased ca. 3 and 2 log cycles, respectively. Heat-adapted cells also showed increased growth at pH 5 and in the presence of 6% NaCl. Two-dimensional gel electrophoresis of proteins expressed by control and heat-adapted cells revealed changes in the levels of expression of 31 and 18 proteins in mid-exponential- and stationary-phase cells, respectively. Twelve proteins were commonly induced. Nine proteins induced in the heat-adapted mid-exponential- and/or stationary-phase cells of L. plantarum DPC2739 were subjected to N-terminal sequencing. These proteins were identified as DnaK, GroEL, trigger factor, ribosomal proteins L1, L11, L31, and S6, DNA-binding protein II HlbA, and CspC. All of these proteins have been found to play a role in the mechanisms of stress adaptation in other bacteria. Antibodies against GroES detected a protein which was induced moderately, while antibodies against DnaJ and GrpE reacted with proteins whose level of expression did not vary after heat adaptation. This study showed that the heat resistance of L. plantarum is a complex process involving proteins with various roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing, and control of ribosomal function. The physiological mechanisms of response to pasteurization in L. plantarum are fundamental for survival in cheese during manufacture.
对植物乳杆菌菌株的耐热性及热应激反应进行了研究。植物乳杆菌DPC2739的稳定期细胞在无菌牛奶中,于60、72和75℃时的十倍减少时间(D值)(D值是使细胞数量减少1个对数周期所需的时间)分别为32.9、14.7和7.14秒。当使用指数中期细胞时,D值降低。导致D值降低10倍的温度升高范围为9至20℃,具体取决于菌株。在72℃处理90秒的部分细胞群体,在7℃的无菌牛奶中孵育20天时恢复了活力。当植物乳杆菌DPC2739的指数中期或稳定期细胞在42℃适应1小时后,其在72℃处理90秒时的耐热性分别提高了约3和2个对数周期。热适应细胞在pH 5以及存在6%氯化钠的情况下也表现出生长增加。对对照细胞和热适应细胞表达的蛋白质进行二维凝胶电泳分析发现,指数中期细胞和稳定期细胞中分别有31种和18种蛋白质的表达水平发生了变化。共有12种蛋白质被共同诱导。对植物乳杆菌DPC2739热适应的指数中期和/或稳定期细胞中诱导的9种蛋白质进行了N端测序。这些蛋白质被鉴定为DnaK、GroEL、触发因子、核糖体蛋白L1、L11、L31和S6、DNA结合蛋白II HlbA以及CspC。所有这些蛋白质在其他细菌的应激适应机制中都发挥了作用。针对GroES的抗体检测到一种中等程度诱导的蛋白质,而针对DnaJ和GrpE的抗体与热适应后表达水平未发生变化的蛋白质发生反应。该研究表明,植物乳杆菌的耐热性是一个复杂的过程,涉及在细胞生理学中具有各种作用的蛋白质,包括伴侣活性、核糖体稳定性、严谨反应介导、温度传感以及核糖体功能的控制。植物乳杆菌对巴氏杀菌的生理反应机制对于其在奶酪制造过程中的存活至关重要。