Research On The Mechanisms Of Stress Tolerance In Solid And Liquid Fermented Lacticaseibacillus Paracasei Zhang

Non-spore probiotics such as lactic acid bacteria have poor heat resistance and short shelf life,which are key issues to be solved in the probiotic industry.This research took Lacticaseibacillus paracasei Zhang as the research object,and analyzed the differences in stress resistance,cell structure,physiological change,metabolic process,gene expression of the cells fermented from solid-state and liquid-state fermentation.These results preliminarily reveal the resistance mechanisms of Lacticaseibacillus paracasei Zhang.The main research results are as follows:1.Solid-state fermented Lacticaseibacillus paracasei Zhang has better stress resistance than liquid-state fermented cells.The survival rate of solid-state fermented cells after being dried by cold air was 32.93%,which is significantly higher than the22.13%survival rate of liquid-state fermented cells;the survival rate of solid-state fermented cells after spray drying was 0.21%,which is significantly higher than that of liquid-state fermented cells’0.15%survival rate.2.The research determined the stress conditions under which Lacticaseibacillus paracasei Zhang had obvious changes,which were 58℃,0.6%hydrogen peroxide,30%ethanol,p H 2,all treated for 10 min.The differences in stress resistance between cells fermented from solid-state and liquid-state fermentation were compared:under the above-mentioned stress conditions,the survival rate of solid-state fermented cells were3.57 folds（heat stress）,2.6 folds（hydrogen peroxide stress）and 3.22 folds（ethanol stress）higher than those of liquid-state fermented cells;the survival rate of liquid-state fermented cells was 46.07 folds（acid stress）higher than that of solid-state fermented cells.3.After the stress treatment,the membrane structure of the solid-state fermented cells remained intact,and the intracellular p H value and the enzyme activities were stable.After heat stress and ethanol stress,the unsaturation of the cell membrane of the solid-state fermented cell membrane decreased slightly,the membrane wall structure was basically intact,and the membrane fluidity remained unchanged,while the cell membrane of the liquid-state fermented cells decreased significantly.After heat stress and ethanol stress,the intracellular p H of solid-state fermented cells does not decrease significantly,but the cells of liquid-state fermented cells decrease significantly.The ATPase activity,glutathione reductase activity and pyruvate kinase activity of solid-state fermented cells were higher than those of liquid-state fermented cells.4.Transcriptome analysis found that in solid-state fermented cells,genes related to carbohydrate transport,glycolysis and gluconeogenesis,pentose phosphate pathway,phosphoinositide metabolism pathway,pyruvate metabolism,and some genes related to environmental adaptation were up-regulated.Genes related to fatty acid synthesis were down-regulated.5.According to the results of the transcriptome,an endogenous CRISPR-Cas9gene editing method was developed and 33 related genes were knocked out.It was found that the gene glp F and glp O were related to the stress resistance and growth of Lacticaseibacillus paracasei Zhang.Compared with the wildtype strain,the survival rates of the gene glp F and glp O knockout strains after heat stress decreased by 83.67%and 78.52%,and the survival rates after oxidative stress decreased by 87.75%and98.96%,respectively.The gene glp K was only related to growth.Compared with the wildtype strain,the bacterial count of the glp K gene knockout strain decreased by24.8%.6.According to transcriptome data and molecular experiment results,it was found that adding a specific type and ratio of carbon source to the culture medium can improve the stress resistance and growth performance of Lacticaseibacillus paracasei Zhang.The comparative experiment of various carbon sources found that the heat resistance（treated at 58℃for 10 min）and oxidation（treated in 0.6%H₂O₂ solution for 10 min）resistance of the bacteria obtained by glycerol replacement culture were significantly improved compared with the bacteria cultured in the original medium.After adding 20g/L glycerol during cultivation,the heat resistance of the bacteria was increased by1304%compared with the original,and the oxidation resistance of the bacteria was increased by 809%compared with the original,but the number of bacteria was one order of magnitude lower than the original.When cellobiose,mannose,sucrose,maltose,and fructose were used to replace glucose for culture,the heat resistance of the bacteria was significantly lower than the original,but the biomass was significantly higher than that of the original group.In the combined experiment,when the ratio of mannose and glycerin was 1:3,the heat resistance of the bacteria was increased by165%,the oxidation resistance was increased by 75%,and the number of bacteria was increased by 67%.Adding specific types of sugars（mannose,cellobiose,glucose,citric acid and fructose）could significantly increase the total bacterial count of solid-state fermentation(The total bacterial counts were 3.46×10¹¹ CFU,3.6×10¹¹ CFU,3.4×10¹¹CFU,3.81×10¹¹ CFU and 3.77×10¹¹ CFU,improved by 40.08%,45.75%,37.65%,54.25%and 52.63%compared to the control).Changing the culture method can enhance the stress resistance of the bacteria.The heat resistance and oxidation resistance of the shaking culture bacteria were 1.23 times and 24.83 times than those of the static cultured bacteria,respectively.In this paper,we started to study the phenomenon that there is a significant difference in stress resistance between the solid-state and liquid-state fermentation of Lacticaseibacillus paracasei Zhang,and carried out systematic analysis from multiple levels of phenotype,structure,transcriptome and genes,and found that material absorption and transport,the intensity of energy metabolism and the formation of reducing power are all related to the stress resistance of the bacteria.Knockout of related genes and carbon source replacement experiments verified the above conclusions.The researches in this article enriched the understanding of the stress resistance mechanism of lactic acid bacteria,and provided a new method and reference for improving the stress resistance of lactic acid bacteria.