| Lactic acid bacteria(LAB)extend their residence time in the intestine by adhesion and improving their influence on host health either by modifying the local microbial community or regulating the immune response.It must survive the adverse conditions of the oro-gastrointestinal conditions to reach the site of action alive and adhered to the intestine.At present,most studies evaluate the survival to oro-gastrointestinal stress(OGT)simulated conditions and adherence to epithelial cells independently.However,there are few results on the properties of adhesion to epithelium and mucus of microorganisms after passing through the OGT simulated conditions.The aim of the present work was to evaluate the change of adhesion ability and surface components of LAB after simulated OGT conditions using Caco-2 cell and mucins model.Finally,Sodium Dodecyl Sulfate-Polyacrylamide gel Electrohoresis(SDS-PAGE)and tandem mass spectrometry(TMT)were used to screen and identify the expression of differential proteins related to surface adhesion of LAB.The results are as follows:(1)Resistance of LAB to simulated OGT and their adhesion to intestinal.LAB viability were assessed by plating samples collected after incubation in simulated saliva(100 mg/mL lysozyme,pH 7.0)-gastric juice(3 g/L pepsin,pH 3.0)-intestinal fluid 1(0.15%bile salt,0.1%trypsin,pH 8.0)or simulated intestinal fluid 2(0.30%bile salt,0.1%trypsin,pH 8.0)in MRS agar.High adhesion LAB were then screened using intestinal mucin and intestinal epithelial Caco-2 cell models,and explored their abilities to inhibit pathogen adhesion.The result shows that after passing saliva-gastric juice-intestinal fluid 1 and intestinal fluid 2,Lactobacillus plantarum 67,S7,W198,69,Lactobacillusfermentum 57,146,Lactobacillus rhamnosus LYO,Lactobacillus paracasei m111,W125 viability stay above at 106 CFU/mL,survival rate stay above at 1%.Streptococcus thermophilus was less tolerant,showing a viability decrease of almost 4.3 log(CFU/mL).L.plantarum 67?W198?S7,L.fermentum 146 and L.paracasei W125?m111 have strong adhesion to Caco-2 cells and mucin,The adhesion rate was higher than 10%and 5%,respectively.Six strains of LAB have the ability to inhibit the intestinal adhesion of pathogenic bacteria,among them,L.plantarum S7 has the best displacement and competitive adhesion to Escherichia coli(E.coli),the adhesion inhibition rates were 86.61%and 76.83%.respectively L.fermentum 146 had the strongest inhibition on Salmonella adhesion,the inhibition rate of Salmonella exclusion,competition and displacement was over 90%.Followed by L.plantarum S7,it expressed the highest rate of exclusion and competitive adhesion to Salmonella.the adhesion inhibition rate was 72.35%and 55.60%respectively:the main adhesin of L plantarum S7?67?W198 and L.paracasei m111 were surface proteins.L.fermentum 146 and L.paracasei W125 are polysaccharides(2)Effects of OGT stress on the adhesion ability of LAB.The Caco-2 cell model was used to investigate the effect of digestive stress on the adhesion ability of lactic acid bacteria,and the effects of digestive stress on the adhesion of LAB were investigated by removing surface proteins,extracellular polysaccharides and lipophosphate from the surface of LAB by sodium periodate,bovine serum aloumin and lithium chloride,respectively.Finally,SDS-PAGE and TEM were used to explore the effects of digestive stress on the expression and morphology of LAB proteins.The result shows that the adhesion ability of L plantarum S7 and L.paracasei m111 after simulated saliva-gastric juice-intestinal fluid 1 were significantly higher than that undigested stress LAB(p<0.05),increasing 1.46 and 1.93 times respectively(p<0.05),L.plantarum 67 and L.fermentum 146 showed no significant difference(p<0.05).There was no significant difference of adhesion ability in L.plantarum S7,W198,67 after simulated saliva-gastric juice-intestinal fluid 2 compared with that undigested stress(p<0.05),the adhesion ability of L.paracasei mill increased by 1.73 times significantly(p<0.05),however,the adhesion ability of L.fermentum 146 and L.paracasei W125 decreased significantly(p<0.05)At the same time,through TEM observation,it was found that after the stress of simulated saliva-gastric juice-intestinal juice 1 or intestinal juice 2 in turn,the morphology of L plantarum S7 cells changed from spindle shape to short rod or round shape;L.paracasei m111 passed through simulated saliva-gastric juice-intestinal juice 1 or intestinal juice 2 stress,the morphology of the bacteria changes from a long spindle shape to a rounded rod shape;After the digestive stress of simulated saliva-gastric juice-intestinal juice 1 in turn.the thickness of the surface protein layer of L.plantarum S7 increased by 1.31 times.After the simulated saliva-gastric juice-intestinal juice 2 digestive stress,the surface protein layer thickness has no significant change compared with the control group(p<0.05).The digestive stress has little effect on the surface protein layer thickness of L.paracasei m111,and the surface protein layerof L.plantarum S7 before and after stress is 3-4 times thicker than that of L.paracasei m111.(3)Effects of digestive stress on the expression of LAB adhesion-related proteins.LAB undigested stress as control(group C),TMT techniques was used to screen and identify the differential protein of LAB after simulated saliva-gastric juice-simulated intestinal fluid(0.3%bile salt)(T group)digestion stress and simulated saliva-gastric juice-simulated intestinal fluid(0.3%bile salt)(S group)digestion stress.to study the effect of digestive stress on the expression of adhesion proteins on the surface of lactic acid bacteria.The results that more than 1.2 times the difference,p<0.05 is the screening criteria,a total of 1372 differential proteins were screened in the T/C group,among them,674 proteins were significantly upregulated(p<0.05),expression of 698 proteins was down-regulated after stress(p<0.05).A total of 1319 differential proteins were screened in the S/C group,601 proteins were significantly upregulated(p<0.05),718 proteins were significantly downregulated among them after stress(p<0.05).A total of 23 differential proteins were screened in the T/S group,among them,13were significantly upregulated and downregulated(p<0.05).GO and KEGG were used to annotate the functional and metabolic pathways of differential proteins,the results indicate that,digestive stress causes gene repair and recombination related proteins(single chain DNA binding proteins),ribosomal proteins(50 S ribosomal protein L10 and 50 S ribosomal protein L14),stress proteins(molecular chaperones dnak)as well as in glycolysis(3-glycerol phosphate dehydrogenase(DAPDH),enolase)and membrane transport proteins(ABC substrate-binding proteins)occurred in significant changes.At the T/C of the comparison group,digestive stress protects cells by stimulating strains to produce stress proteins(dnak)and gene repair proteins to protect cells(single-stranded DNA repair proteins),subsequently,by significantly increasing the expression of GAPDH?PGK?Eno and other adhesion related enzymes in the glycolytic metabolic pathway)to generate more ATP energy(p<0.05),Then starting the isomembrane transporter in OppA in ABC membrane transport system to make cells secrete more part-time functional proteins outside it,meanwhile,the expression AI-2 key enzymes LuxS adhesion proteins and sorting enzymes that can anchor surface proteins on the cell wall and mediate the adhesion of lactobacillus to intestinal epithelial cells was significantly up-regulated.GAPDH?PGK?Eno?LuxS?Sortase A had no significant differencein the S/C of the comparison group(p<0.05),OppA was significantly upregulatedin the S/C of the comparison group(p<0.05).Lactobacillus plantarum S7 significantly increased the expression of ABC transport substrate binding protein and glucose 6-phosphate isomerase after digestion stress with low concentration of bile salt and digestion stress with higher concentration of bile salt(p<0.05),1,3-Propylene glycol dehydrogenase and MFS transporter were significantly down-regulated(p<0.05). |
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