Screening And Mechanism Exploration Of Latilactobacillus Sakei With The Efficacy Of Relieving Inflammatory Bowel Disease

Latilactobacillus sakei(L.sakei)is a member of the genus Latilactobacillus.The species is widely found in foods,such as fermented meat products,fermented vegetables,fermented wine products and sourdough,as well in the human gastrointestinal tract.L.sakei can produce bacteriocins and flavor compounds,so it is often used as bio protectant and starter.In recent years,studies have shown that L.sakei has a variety of probiotic functions,which can alleviate immune diseases such as colitis and rheumatoid arthritis and metabolic diseases such as obesity.In this study,L.sakei was screened from different samples,and inflammatory bowel disease was used as the research model to explore the immune regulation ability of L.sakei from different sources and to provide a theoretical basis for further excavation of L.sakei with probiotic function.The main findings are as follows:(1)Total 72 strains of L.sakei were screened from 120 fermentation samples and fecal samples across the country,and 16 strains of L.sakei from different sources were selected to investigate the immunomodulatory ability in vitro.The Caco-2 cell was stimulated by dextran sulfate sodium(DSS)to construct an in vitro colitis model to initially screen L.sakei with immunomodulatory ability.Among 16 strains of L.sakei from different sources,L.sakei QJSSZ1L4,QJSSZ4L10,JXJ41 and QGZZYRHMT1L6 can effectively protect the activity of Caco-2 cells after DSS stimulation and immunomodulate Caco-2 cells by inhibiting the NF-κB signaling pathway.They can also up-regulate the expression of tight junction protein-related genes to protect the intestinal barrier function in Caco-2 cells.However,L.sakei QJSNT1L10 and QGZZYRHMT2L6 did not have the above regulating effects.(2)The immunomodulatory ability of the above 6 strains of L.sakei was validated in vivo using DSS-induced colitis mice as a model.The results showed that 6 strains of L.sakei showed varying degrees of alleviation on colitis in mice.Among them,L.sakei JXJ41 can effectively restore the colon length of colitis mice,alleviate colon pathological damage,reduce the content of pro-inflammatory cytokines and related inflammatory enzymes in colon tissue,while L.sakei QJSNT1L10 was less effective.Serum metabolomics showed that compared with L.sakei QJSNT1L10,L.sakei JXJ41 up regulated the content of beneficial metabolites such as phenyllactic acid in serum to effectively relieve colitis.(3)Based on comparative genomics and metabolomics,the potential mechanisms underlying the differences in the effects of differential strains to alleviate colitis were investigated.Average nucleotide identity(ANI)and phylogeny showed that L.sakei JXJ41 and L.sakei QJSNT1L10 belonged to two subspecies: L.sakei subsp.sakei and L.sakei subsp.carnosus,and the differences in their evolutionary relationships may lead to differences in their function-related genes.Clusters of orthologous groups of proteins(COGs),Kyoto encyclopedia of genes and genomes(KEGG)and carbohydrate annotation analysis showed that the differential strains had significant differences in carbohydrate utilization-related genes such as glycoside hydrolase family.Metabolomics analysis of bacterial cell-free supernatants showed that two L.sakei strains had significant differences in metabolites,among which acetylcholine and indole-3-acetic acid may be the key substances.Based on the above studies,this subject has screened a strain of L.sakei JXJ41 which can effectively relieve colitis through in vitro cell screening and in vivo animal verification.At the same time,compared with the less effective strain L.sakei QJSNT1L10,the acetylcholine and indole-3-acetic acid produced by L.sakei JXJ41 may be the key substances which were responsible for its efficacy.