Preliminary Study On Acid Resistance Mechanism Of Thermophilic Streptococcus

As a yoghurt starter Thermophilic streptococcus plays an important role in the process of acidification. This paper is aimed at a preliminary study on the acid resistance mechanisms of T.streptococcus and laid a foundation for illustrating the mechanism of acid resistance, as well as control yogurt fermentation.The growth characteristics of the T.streptococcus have been described by cultured in different lactose content of medium. Acid stimulation treatment for Ih in the early logarithmic growth of T.streptococcus was performed and proteomic analysis was used to compare the differently expressed proteins of samples before and after stimulation. The results indicated that there were665proteins expressed in4h sample while677proteins expressed in5h sample. Among them12proteins were found only expressed in5h samples. Seventy proteins that exhibited more than1.5folds upregulated in5h sample in comparison with4h sample were selected and identified with mass spectrum. According to the functions in the acid environment, these proteins could be roughly divided into the following categories:Four metal ion stress related proteins;5repair proteins in protection mechanism under stress;27proteins associated with the metabolic pathway of enzymes;11ATP synthase proteins, transcription factor related proteins;8amino acids metabolism related enzymes;10other functional proteins. Four proteins (Alpha-acetolactate decarboxylase, UreaseG, UreaseE and60KD) were chosen for further analysis using real-time quantitative PCR (RT-qPCR) to verify the expressions at transcriptome level. The results indicated that the expression changes of the genes in5h samples compered to the4h sample15.24folds,13.27folds,15.24folds and0.60folds respectively. Expression of protein Alpha-acetolactate decarboxylase, UreaseG and-UreaseE at the transcriptome level are consistent with proteomic analysis result, while the60KD protein is not.CiaH is a possible acid resistance gene. This study successfully constructed the plasmid for gene knockout, and was used for preliminary knockout experiments. Primers were synthesized according to the upstream and downstream regions of CiaH gene, and were used to amplify the641bp upstream arm and698bp downstream arm. The2fragments were inserted into the pMD18-T using enzyme digestion and ligation. Chloramphenicol resistance gene were amplified and inserted into the recombinant pMD18-T plasmid containing the flanking regions of target gene. Gene knockout experiments were performed after linearization of the recombinant plasmid. Screening knockout mutant strains on chloramphenicol plates were carried out for several rounds, but failed. This may be due to lethal mutation is formed after the gene being knocked out.At present, comprehensive research on screening the acid resistance genes of T.streptococcus has not been reported. In this study, comparative proteomics method was used to screen acid factors of T. streptococcus. This study is great helpful to elucidate mechanisms of T. streptococcus, as well as to control yogurt acidification.