Screening And Evaluation Of Saccharomyces Cerevisiae With The Efficacy In Relieving Inflammatory Bowel Disease

Saccharomyces cerevisiae is widely used in food,wine crank and feed fields.At present,most research on it is focused on strain screening and fermentation process optimization.Besides fermentation function,it has played an important role in probiotic function,which is gradually gaining more attention.In recent years,numerous studies have demonstrated the remarkable effects of Saccharomyces cerevisiae in alleviating intestinal disorders such as diarrhea,colitis and irritable bowel.However,not all Saccharomyces cerevisiae strains have significant regulatory effects,they have differences in regulatory capacity.In addition,research on Saccharomyces cerevisiae and intestinal diseases have mainly focused on strains themselves and rarely delved into the specific material basis.Therefore,in this study,Saccharomyces cerevisiae strains from different samples were screen to alleviate intestinal inflammation in vitro.The alleviating effects of Saccharomyces cerevisiae on mice with colitis were evaluated to obtain the best strain.Further,we extracted its cell wall polysaccharide fractions to explore the anti-inflammatory effects in vitro,and we characterized the structure of the polysaccharide fractions.We provided a theoretical basis for the subsequent excavation of Saccharomyces cerevisiae strains in alleviating inflammatory bowel disease and cell wall polysaccharide fractions for application.The main findings are as follows:(1)We obtained a total of forty Saccharomyces cerevisiae strains from 208 samples nationwide.After in vitro gastrointestinal tolerance and adhesion assays to intestinal epithelial Caco-2 cells,we selected nine strains with higher survival rate and stronger adhesion ability.We used 2μg/m L lipopolysaccharide(LPS)to induce Caco-2 cells,which selected five strains with better regulatory effects using cell activity as an evaluation index.Further,we investigated the effects of above five strains on expression levels of tight junction proteins and inflammatory cytokines in Caco-2 cells,we screened for strains with protective effects against cellular inflammatory damage.The results showed that Saccharomyces cerevisiae QSXXA8L5,QSCMS5L2 and QHNLD8L1 could significantly up-regulate expression levels of tight junction protein-related genes and down-regulate expression levels of pro-inflammatory cytokine-related genes in Caco-2 cells.In contrast,Saccharomyces cerevisiae QHNSMX7L2 and QJXJA4L5 did not possess the above regulatory effects.(2)We used 2.5%dextran Sulfate Sodium(DSS)to induce colitis mice,and three strains showed good performance in vitro were evaluated in animals.The results showed that all three strains could alleviate colitis mice to different degrees.In particular,Saccharomyces cerevisiae QHNLD8L1 significantly improved physiological conditions of mice with colitis,restored colon length,alleviated histopathological colon damage,improved intestinal mucus layer,increased tight junction proteins,reduced pro-inflammatory cytokines and inflammatory enzymes in colon,and increased short-chain fatty acids in feces.The analysis of intestinal flora revealed that compared with the model group,Saccharomyces cerevisiae QHNLD8L1intervention increased diversity and improved intestinal flora structure of colitis mice,decreased the relative abundance of harmful bacteria Escherichia＿Shigella and Turicibacter,and increased the relative abundance of beneficial bacteria Faecalibaculum,Butyricimonas and Fournierella.(3)Both live and dead Saccharomyces cerevisiae QHNLD8L1 alleviated colitis,suggesting that the effect was dependent on its cell wall rather than on metabolites.We obtained MPS and DGPS as cell wall polysaccharide fractions of Saccharomyces cerevisiae QHNLD8L1by alkaline extraction method.The total sugar content of MPS was 82.57%±0.18%,that of DGPS was 75.21%±1.20%.We explored the effects of MPS and DGPS on intestinal inflammation using LPS-induced Caco-2 cells.The results showed that both MPS and DGPS increased expression levels of tight junction proteins and decreased expression levels of pro-inflammatory cytokines.The anti-inflammatory effects of MPS were stronger than that of DGPS,especially MPS at 200μg/m L.We characterized the structures of MPS and DGPS by combining molecular weight,monosaccharide composition,infrared spectroscopy and nuclear magnetic resonance.We found that MPS average molecular weight was 5.248×10~4 Da,which was composed of mannose,glucose,glucosamine and arabinose with the ratios of 97.34%,1.78%,0.82%and 0.07%.DGPS was composed of dextran,glucosamine,mannose,rhamnose and galactose with the ratios of 97.05%,1.37%,1.35%,0.16%and 0.06%.The main structure of MPS isα-D-Manp-(1→6)as the main chain and T-α-D-Manp-(1→2)and T-α-D-Manp-(1→2)-α-D-Manp-(1→2)as side chains.In summary,Saccharomyces cerevisiae QHNLD8L1 which was obtained in vitro significantly alleviated colitis in mice.Dead Saccharomyces cerevisiae QHNLD8L1 was found to be equally effective in relieving colitis.The cell wall polysaccharides of Saccharomyces cerevisiae QHNLD8L1 were extracted to obtain main components MPS and DGPS.Through inflammatory cell models in vitro,both MPS and DGPS could protect against LPS-induced inflammatory injury in Caco-2 cells,especially MPS at 200μg/m L.The main structure of MPS isα-D-Manp-(1→6)as the main chain and T-α-D-Manp-(1→2)and T-α-D-Manp-(1→2)-α-D-Manp-(1→2)as side chains.This study broadens the resource mining of probiotic Saccharomyces cerevisiae strains and their functional applications in intestinal diseases.Also,we provide new perspectives for the development of Saccharomyces cerevisiae cell wall polysaccharide fractions as bioactive substances.