Mechanism Of Lactiplantibacillus Plantarum P101 Alleviating Alcoholic Liver Injury In Mice And Preparation Of Probiotics

Lactic acid bacteria(LAB)mainly exist in nutritious environments,such as dairy products,fermented meat and fish,sour dough and naturally fermented fruits and vegetables.It is widely used to alleviate multi-diseases in researches,attributed to outstanding anti-inflammatory and antioxidant functions.Alcohol abuse is a significant factor in the increasing liver morbidity and mortality,most adult men drink alcohol,long-term excessive drinking will induce an increase in oxidative stress and cause liver damage.The purpose of this study was to screen one lactic acid bacteria with high antioxidant activity and probiotics from traditional fermented foods from different sources,and to explore the mechanism of the lactic acid bacteria in alleviating alcoholic liver injury in mice,and the preparation of probiotics were investigated.The chapters are described as follows:The first chapter,the function and application of lactic acid bacteria were introduced,and the research progress of the main mechanisms of relieving alcoholic liver disease by lactic acid bacteria were reviewed.The second chapter aims to screen lactic acid bacteria with high antioxidant activity from traditional fermented foods collected from different regions.Lactic acid bacteria were preliminarily screened by isolation and purification,Gram staining and catalase test.In addition,taking Lactobacillus rhamnoides GG(LGG)as a reference,1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)free radical scavenging activity,reducing L-cysteine activity,hydroxyl radical scavenging activity and anti-lipid peroxidation activity were selected to evaluate the antioxidation in vitro.The results showed that strains SC09,SLJ03,SDJA01 and P101 had higher antioxidant activity in vitro than LGG.Further 16 S r DNA sequencing and NCBI database comparison showed that SC09,SLJ03,SDJA01 and P101 were more than 99% similar to Lactiplantibacillus plantarum,they were identified as Lactiplantibacillus plantarum.Therefore,the above four strains are considered to be Lactiplantibacillus plantarum with high oxidative activity.In the third chapter aims to screen a strain with good safety and probiotics from four strains of Lactiplantibacillus plantarum with high antioxidant activity.The safety of Lactiplantibacillus plantarum were evaluated by antibiotic sensitivity,biogenic amine activity and hemolytic activity.In addition,the probiotic characteristics of lactic acid bacteria were evaluated by bacteriostasis,acid tolerance and bile salt tolerance and self-agglutination ability.The results of safety evaluation showed that the four strains of Lactiplantibacillus plantarum were sensitive to ampicillin,gentamicin,chloramphenicol and erythromycin,but not amino acid decarboxylase activity and hemolytic activity.The results of probiotic evaluation showed that the four strains of LAB had good antibacterial activity against Staphylococcus aureus,Escherichia coli,Bacillus subtilis,Pseudomonas aeruginosa and Candida albicans.In addition,the four strains of LAB all showed certain acid tolerance,bile salt tolerance and selfagglutination ability,among which Lactiplantibacillus plantarum P101 showed strong acid tolerance,bile salt tolerance and self-agglutination ability.the 2-hour survival rate was 82.97% in p H 3 medium and 88.21% in 0.3% bovine bile salt medium.Therefore,Lactiplantibacillus plantarum P101 showed better probiotics.The fourth chapter aims to clarify the mechanism of high antioxidant activity of Lactiplantibacillus plantarum P101 in alleviating alcoholic liver injury.The alcoholic liver injury model mice were established by NIAAA method,and Lactiplantibacillus plantarum P101(108 CFU/mice)and Vc(200 mg/Kg)were intervened at the same time.The results showed that the levels of serum liver function indexes(TG,ALT,AST,ALP)in the alcohol model group were significantly higher than control group.Liver histopathological analysis showed that alcohol intake could induce steatosis and focal inflammation,while the intervention of Lactiplantibacillus plantarum P101 and Vc could significantly reduce the level of liver function and alleviate liver steatosis and focal inflammation.Further determination of the level of oxidative stress in liver tissue showed that alcohol intake could induce oxidative stress and hepatocyte apoptosis,while the intervention of Lactiplantibacillus plantarum P101 could activate the expression of genes and proteins related to Nrf2/HO1 antioxidant signal pathway and hepatocyte apoptosis.Therefore,Lactiplantibacillus plantarum P101 alleviates alcohol-induced liver injury by activating Nrf2/HO1 signal pathway.In the fifth chapter aims to obtain Lactiplantibacillus plantarum P101 with high bacterial density and prepare them into preparation.The carbon source,nitrogen source,initial p H of MRS medium and inoculation amount were optimized by single factor,and orthogonal optimization was carried out on the basis of single factor optimization.In addition,Lactiplantibacillus plantarum P101 was prepared by vacuum freeze drying.The optimization results showed that 3% glucose,0.75% yeast extract,2.25% soybean peptone,initial p H=6.2 and 3% inoculum were the best fermentation conditions for Lactiplantibacillus plantarum P101,under this condition,the live bacteria pair value is10.58±0.04,which is an order of magnitude higher than before optimization.In addition,the survival rate of Lactiplantibacillus plantarum P101 was 52.24%.The above results show that the optimization of culture conditions can greatly increase the number of living bacteria and provide a better basis for the preparation of bacterial agents.To sum up,a Lactobacillus plantarum P101 with well antioxidant activity,probiotics and safety was screened from different traditional fermented foods.Lactobacillus plantarum P101 alleviates alcohol-induced liver oxidative damage by activating Nrf2/HO1 antioxidant signal pathway.In addition,in this study,Lactiplantibacillus plantarum P101 cultured at high bacterial density was made into probiotic powder,which has potential efficacy in the prevention and treatment of alcoholic liver disease.This study provides a strong theoretical basis for the development of Lactiplantibacillus plantarum P101 probiotics for alleviating alcoholic liver injury.