| In order to explore the effects produced by different initial conditional pH on the anti-freeze-drying activity of Lactobacillus plantarum LIP-1 and its intrinsic mechanism,the previous experimentally optimized medium was used as the basis.using single factor experiments,.the effects of pH(5.0,5.6,6.2,6.8,7.4)on the growth of the strain and the lyophilization survival rates were compared,Selected two groups with higher growth but significant different lyophilization survival rate,to determinate their fatty acid content,cell enzyme activity,and cell membrane integrity;and using Illumina high-throughput sequencing technology to analyze the effects and intrinsic mechanism of action of different initial conditional pH on the anti-freeze-drying activity of Lactobacillus plantarum LIP-1 at the level of transcriptomics and proteomics.The results showed that different initial pH had a certain effect on the number of live bacteria and freeze-drying survival rate.When the initial pH was 6.8(experimental group H)and 7.4(control group LB),the number of viable cells was significantly higher than that of other groups;compared with the control group,the lyophilization survival rate of the experimental group was significantly increased(p<0.05);The pH of the fermentation process was found to decrease gradually with the increase of fermentation time.At 6h,the pH of the experimental group had dropped to 5.2,while the control group decreased to 5.2 at 12h.It takes longer to be in a low acid environment.Measurement of the cell membrane fatty acid content revealed that the initial fatty acid content at pH 6.8 was significantly increased(p<0.05)compared to the initial pH 7.4.The cell membrane integrity was detected.The cell membrane damage of the strain with initial pH 6.8 was lower than that of the initial pH 7.4.The cell membrane integrity of the experimental group was significantly better than that of the control group.The activity of lactate dehydrogenase and H+-ATPase in the experimental group was significantly higher than that in the control group(p<0.05).At the transcriptional and protein levels,the initial pH 6.8 cultured Lactobacillus plantarum LIP-1 in the fatty acid biosynthesis pathway,the coding gene for citrate lyase,acetyl-CoA decarboxylase,accA,accC,compared to the initial pH 7.4 The expression levels were significantly up-regulated(p<0.05),catalyzing the formation of malonyl-CoA by acetyl-CoA,thereby promoting the production of unsaturated fatty acids.The expression levels of lactate dehydrogenase and H+-ATPase related enzymes were significantly up-regulated(p<0.05),which was consistent with the results of enzyme activity assay.In addition,compared with the control group,the expression levels of multiple cold stress proteins in the experimental group were also significantly up-regulated(p<0.05).The results showed that compared with the initial pH of 7.4,the strain cultured at initial pH 6.8 was in a low acid environment for a long time.On the one hand,acid stress promoted the increase of cyclopropane fatty acid content,prolonged the carbon chain,and improved the cell membrane fatty acid.Saturation,which in turn improves cell membrane fluidity,maintains cell membrane integrity,and reduces lyophilization of the strain.On the other hand,long-term low-acid culture significantly up-regulated the expression of genes related to fatty acid synthesis,and the cell cell enzyme activity and cold stress protein csp family expression were significantly up-regulated,(p<0.05),which improved the anti-freezing ability of the strain.The two synergistically improve the freeze-drying performance of Lactobacillus plantarum LIP-1.Accordingly,this study provides a theoretical basis for further improving the freeze-drying activity of lactic acid bacteria. |
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