Research Of The Absence Of CcpA Gene And Resist Stress In Lactobacillus Delbrueckii Subsp. Bulgaricus

Lactic acid bacteria(LAB) starter cultures of high quality should remain high viability and functional activity during long-term delivery. Freeze-drying is the primary method of producing Directed Vat Set starter of LAB. However, the LAB suffer from freezing, drying and hypertonic multiple hypertonic stress during the process of lyophilization, which leads to the damage of cytomembrane, the change of cell morphology, the decrease of metabolic capability. Therefore, the cells lose their activity or even die, which results in failure of the production of starter culture. That has become the significant bottleneck in the production of Directed Vat Set starter of LAB. LAB can adjust their metabolism to enhance the resistance to a changing environment. The Ccp A protein regulate many stress response proteins and play a crucial role in the regulation of resistance.Therefore, this study analyzes the effects of Ccp A to purpose strain, analysis of strain growth metabolism, changes in resistance and discuss the possible reasons.Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was the object of this study. We obtain the mutant using the method of homologous recombination. Glucose and organic acids were determined by High Performance Liquid Chromatography; the activities of pivotal enzymes were measured by spectrophotometry and colorimetry.The results show that growth curve was lower in anaerobiosis for both strains after 12 h and was lower for the mutant strain compared to L. bulgaricus. Inactivation of Ccp A severely impaired growth. Compared with the wild strain, under the same condition, Ccp A mutation strain entered into the stable phase was 4 h slower. The densities of Ccp A mutation strain was 23.08% lower than the wild strain.Deletion of Ccp A made an influence on the rate of glucose consumption. The glucose consumption rate of wild strain was 15.30% higher than the mutation strain in aerobiosis(P < 0.05), and it was 7.98% in anaerobiosis(P < 0.05); For the same strain, the rate of glucose consumption was higher in aerobiosis than in anaerobiosis, wild strain was 5.47%(P < 0.05), mutations was 12.62%(P < 0.05), respectively. In addition, the deletion of Ccp A made an influence on the production of lactic acid and acetic acid. The lactic acid generation rate of wild strain was 47.62% higher than the mutation strain in aerobiosis(P < 0.05), and it was 21.87% in anaerobiosis(P < 0.05); For the same strain, the lactic acid generation rate was higher in anaerobiosis than in aerobiosis, wild strain was 23.94%(P < 0.05), mutations was 50.13%(P < 0.05), respectively. For acetic acid, the acetic acid generation rate of mutation strain wild strain was 47.73% higher than wild strain in aerobiosis(P < 0.05), no significant difference was found in anaerobiosis.Through analyzed of the changes of enzyme activity belong to the glycolytic pathway between wild strain and mutation strain shown that, under the aerobic condition, the activity of lactic dehydrogenase in Ccp A mutation strain was reduced by 72.22% than the wild strain(P < 0.05), the activity of fructose phosphate kinase was reduced by 60.07%(P < 0.05), the activity of pyruvate kinase was reduced by 70.58%(P < 0.05); Under anaerobic conditions, the activity of lactic dehydrogenase in Ccp A mutation strain was reduced by 36.75% than the wild strain(P < 0.05), the activity of fructose phosphate kinase was reduced by 62.87%(P < 0.05), the activity of pyruvate kinase was reduced by 29.05%(P < 0.05). Whether in aerobiosis or anaerobiosis, deletion of Ccp A made an influence on the activity of lactate dehydrogenase, fructose phosphate kinase and pyruvate kinase which were significantly lower. It shown that Ccp A protein involved in the activation of these three enzymes regulation, after the loss of Ccp A, three kinds of enzyme activity decreased.After the loss of Ccp A, heat resistance of bacteria was decreased. The densities was about 3.14% of the wild strain(P < 0.05).Under different conditions, Ccp A mutated strain tolerance to cold was different. The densities of mutant strain is 48.42% of wild strain between 0~12 d in aerobiosis. The densities of mutant strain is 8.16% of wild strain in anaerobiosis. For the same strain, the cold resistance were higher than in anaerobiosis, wild strain is 7.93% higher(P < 0.05), mutant strain is 91.52% higher(P < 0.05), respectively.To knock out the Ccp A gene of L. bulgaricus strain via homologous recombination and construct a Ccp A-deleted mutant strain of L. bulgaricus mutants. The lower activities in the mutant strain correlate with decreased gene expression. After Ccp A gene deletion, enzyme activity(LDH, PK, PFK)was significantly lower which result in weakened glycolytic pathway. Growth rate decreased with the utilization ratio of glucose reducing caused by glycolytic pathway weakened. Cold shock or heat shock proteins differentially expressed in the wild type and ccp A mutant. Ccp A regulates the expression of cold shock and heat shock response operons: in the mutant both the expression of cold shock and heat shock operons were reduced and so was the heat and cold stress tolerance.