Screen And Probiotic Mechanism Of Oxidative Stress Alleviating Lactobacillus Fermentum L8

Aging and many chronic diseases such as diabetes,cancer,cirrhosis,atherosclerosis are related to excessive free radicals which was shown to be eliminated by supplementing dietary of antioxidants.With the implementation of the "human intestinal metagenomic project",the probiotic roles of lactic acid bacteria to alleviate oxidative stress,reduce cholesterol and regulate the intestinal flora have become the hotspot of life science.Therefore,screening prominent probiotics from human intestinal microflora to relieve the stress ability of oxidation,clarifying the protective effect and mechanism of probiotics on oxidative stress injury,and developing probiotics with alleviate function of oxidative damage has certain theoretical research value,which is also the key for preparing high-quality starters and fermentation products.In this study,we isolated Lactobacillus fermentum L8 from the intestine of Rugao longevity population in Jiangsu province according to the screening model of in vitro anti-oxidative probiotics established by Jiangsu Dairy Biotechnology and Safety Control Key Laboratory.With investigating the oxidative stress injury alleviating and key indicators changing of Lfermentum L8 on the liver injury oxidative stress and high-fat diet induced oxidative stress rat models,its mechanism on oxidative stress injury was explained on the molecular,cell and gene levels.This study provided theoretical and technical support for the efficient usage of probiotics and development of related functional food.1.In vitro screening of probiotics with good antioxidant propertiesFour strains with high scavenging ability of hydroxyl radical(OH ·),DPPH free radicals was selected from 30 LAB strains obtained by the analysis of Rugao longevity population.Particularly,the ability to scavenge OH ·,the ability to scavenge DPPH and reducing power A700nm of L8 were 69.96%,85.27%and 2.853,respectively,between which there were no significant correlation,suggesting the different antioxidant substrates.The activity of SOD and GSH-Px were measured at different culture time.The SOD of L8 was 33.4U/mL and the GSH-Px was 38.2 U/mL.Further study found that all the different groups of strains(supernatant,bacteria and intracellular extracts)have antioxidant properties,but there is a certain difference.The antioxidant properties of fermentation supernatant was higher than that of biomass and intracellular extract(P<0.05).The antioxidation capacity of the fermentation broth was not equal to the sum of its antioxidant capacity.The acid and bile salt resistant and in vitro cholesterol lowering ability of the 4 LAB strains was further studied in artificial simulation gastric intestinal environment.The survival rate of strains L8 at pH 2.0 and pH 3.0 in artificial gastric fluid after 3 h was 37.01%and 68.55%and 10.28%and 23.39%after 5 h;the survival rate of strains L8 in artificial intestinal fluid after 1.5 h and 3h were 72.94%and 46.84%,respectively;the tolerance to 0.1%,0.3%and 0.5%bile salt environment were 24.11%?20.31%and 19.92%,respectively.Its cholesterol degradation rate was the highest by 63.73%at 0.1%bile salt concentration.The further study found that 0.1%bile salt concentration is the best concentration of L8 to play the role of degradation of bile salt.It was found that the supernatant of the L8 fermentation had a certain inhibitory effect on the three pathogens of Staphylococcus aureus,Escherichia coli and Salmonella.The acidity was eliminated by adjusting the pH,and the antibacterial substance was removed by heat treatment,suggesting that the antibacterial substance was polypeptide.The results showed that L8 had potential antioxidant,acid-resistant,bile-resistant and cholesterol-degrading properties.2.Identification of L.Fermentum L8 and its basic applicationThe strain L8 was identified as L.fermentum L8 by the method of physiology and biochemistry,API 80 CHL and 16S rRNA sequencing.The homology of L.fermentum L8 with L.fermentum CECT 5716 reached 100%.The growth and fermentation characteristics of L.fermentum L8 were studied.The results showed that the strain entered the logarithmic growth phase after 4 h,and reached a steady state at 10 h and decreased slightly after 34 h.The micro structure of the fermented milk was fine and the acidity of the fermented milk was small.After 5 passages,the curd time and acidity were more stable,and the post-acidification was weaker and the viscosity increased greatly,which indicated that the fermentation stability of the strain was better.L.fermentum L8 was hydrophobic and had strong adhesion to IEC-6 cells,which was similar to that of positive control strain BB-12,which was helpful for its beneficial effect in vivo.The resistance spectrum of L.fermentum L8 was relatively narrow and had good tolerance to tobramycin,amikacin,vancomycin and kanamycin.The resistance to penicillin G and Piperacillin is more week.3.Protective effect of L.fermentum L8 on acute alcoholic liver injury in ratsA chronic alcoholic liver injury rat model was established,and L.fermentum L8 fermentation broth was used to intervene.The weight,organ index,serum biochemical parameters and liver homogenate biochemical indexes were measured.The expression of Nrf2 protein and percentage of hepatocyte apoptosis were determined by flow cytometry in each group.The results showed that the weight increased rapidly and liver index returned to the normal level after intervention with L.fermentum L8.L.fermentum L8 could significantly decrease endotoxin in serum(P<0.05),indicating that L8 in the intestinal adhesion decreased permeability of intestinal mucosa and decreased endotoxin in blood.Compared with the model group,the high concentration of L.fermentum L8 could significantly decrease the level of ALT and AST(P<0.05),increase the content of SOD(P<0.05),increase the content of SOD,GSH and GSH-Px in liver homogenate(P<0.05),and decrease the level of MDA(P<0.05).L.fermentum L8 can reduce the accumulation of lipid in the liver,scavenging free radicals,alleviating oxidative stress,effectively reducing free radical damage to the cell membrane.L.fermentum L8 significantly increased the content of ADH,Na +-K +-ATPase and Ca2 +-ATPase in the liver of rats(P<0.05).L.fermentum L8 also significantly reduced TNF-?,IL-6,VEGF TGF-KB(P<0.05).The expression of Nrf2 protein in rat liver was significantly decreased(P<0.05).The expression of Nrf2 protein was significantly increased(P<0.05)and the percentage of hepatocyte apoptosis was significantly decreased(P<0.05).The correlation analysis showed that L.fermentum L8 could increase the supply of mitochondrial membrane ATPase by increasing the speed of electron transfer chain of mitochondria and increase the efficiency of oxidative phosphorylation,and increase the level of oxidative enzymes such as GSH.L.fermentum L8 could inhibit proinflammatory cytokines,alleviate the inflammatory response of liver injury,and reduce the apoptosis of stem cells,thus played a role in alleviating oxidative stress.4.Experimental study of L.fermentum L8 on oxidative stress induced by high fat dietThe oxidative stress model induced by high fat diet was used to evaluate the ability of L.fermentum L8 to alleviate oxidative stress induced by high fat diet in vivo.The results showed that L.fermentum L8 could alleviate the oxidative stress induced by high-fat diet in the blood and liver of rats.Compared with the model group,different concentrations of intervention group,can significantly improve the rat's body coefficient(P<0.05).But there was no significant difference with the control group(P>0.05).Compared with the model group,high concentration group can significantly reduce serum total cholesterol(TC),triglyceride(TG),ALT,AST,high density lipoprotein(HDL),atherosclerosis index(AI),anti atherosclerosis index(AAI)level(P<0.05)in rats.There are significantly increase serum SOD activity(P<0.05)and liver homogenate GSH and GSH-Px levels(P<0.05).Compared with the model group,L.fermentum L8 significantly decreased cholesterol(P<0.05)and bile acid levels in the liver of mice fed with high-fat diet induced oxidative stress(P<0.05)and significantly increased the contents of T-AOC content(P<0.05).L.fermentum L8 significantly reduced the content of MDA in colon contents(P<0.05)and the percentage of hepatocyte apoptosis decreased significantly(P<0.05).Electron microscopy revealed that the damaged liver tissue and heart tissue structure were improved.The intestinal flora of the model mice induced by high-fat diet reduced the content of gut bacteria,and increased the content of the thick-walled bacteria.While under the intervention of L.fermentum L8 and its fermented milk for 4 weeks,the content of Bacteroidetes enteritidis was increased,and the content of Firmicutes was decreased.L.fermentum L8 and its fermented milk increased the content of intestinal LAB and bifidobacteria,and decreased the content of E.coli.L.fermentum L8 reversed the changes of intestinal microflora in mice fed with high fat diet.The correlation analysis showed that MDA content in colon contents was positively correlated with abundance of E.coli(P<0.05)and negatively correlated with LAB and bifidobacteria(P<0.05).The content of T-AOC in colon was significantly positively correlated with the abundance of LAB(P<0.05),but negatively correlated with the abundance of E.coli(P<0.05).