Study On The Anti-fatigue Mechanism Of Soybean And Deer Blood Transformed By Probiotics

With the improvement of living standard,more and more people are concerned about healthy lifestyle.It is well known that exercise is popular for its many health-related benefits.Physical exercise has a positive impact in reducing and managing the risk of a range of chronic diseases,mental health and life expectancy.However,intense exercise and excessive exercise can lead to the production of active free radicals,leading to oxidative damage and energy source depletion,as well as the accumulation of excessive metabolites,resulting in fatigue in the body.Nowadays,fatigue has become a serious problem,which affects human health,work efciency and quality of life.There is an urgent need to develop functional foods that can improve exercise performance and relieve fatigue without side effects.Fermentation is one of the oldest and most natural approaches to produce food,and it improves the flavor and physiological functions of food.The soybean(Glycine max)is a recognized vegetable protein with a wide range of functional components.Over the years,soybean has been successfully converted into low-cost fermented foods with high nutritional value and unique flavor.Soybean peptide has been found to have anti-fatigue effect,but its anti-fatigue mechanism has not been clarified.Deer blood is a typical animal blood,which contains a number of compounds and represents a valuable source of protein.Deer blood has a variety of physiological functions,such as anti-aging,anti-fatigue,enhancing immunity,enriching blood and calming nerves.Over the years,deer blood was only consumed as deer blood wine and deer blood powder products in Asia.However,few studies have used probiotics to ferment deer blood to enrich active components and improve its antioxidant and anti-fatigued activities.Furthermore,the mechanism by which fermented deer blood products relieve fatigue is not yet clear.At present,there is no research to compare and analyze the anti-fatigue effects and mechanisms of plant-derived and animal-derived products transformed by probiotics.The previous research had confirmed the technology of fermented soybean(FSF)and deer blood(FDB).The composition and antioxidant activity of FSF and FDB were analyzed in vitro.In this study,5-week-old male C57BL/6J mice are given treadmill exercise with or without FSF and FDB supplementation(30,150 and750mg/kg/d)for 3 weeks to explore the anti-fatigue effect and mechanism of FSF and FDB.This reasearch uses a variety of methods to analyze the serum biochemical indicators,liver function,antioxidant level,intestinal barrier function,intestinal microbiome,host and intestinal metabolome of exercise mice.These results indicate that FSF and FDB regulate the weakening of energy metabolism,the imbalance of the antioxidant system and the intestinal microecological system,etc.The main research results obtained are as follows:1.The results of common fatigue-related indicators show that FSF and FDB treatment reduce the increase in urea nitrogen,lactic acid and LDH,and reverse the changes in blood glucose and glycogen in high-intensity exercise mice.FSF and FDB can effectively improve the accumulation of metabolic waste and energy storage,thereby alleviating the fatigue of exercise mice,and the effect of FDB150 is the most obvious.The intervention of FSF and FDB increased the final body weight,daily food intake and running distance,reduced liver weight,ALT and AST levels.FSF and FDB significantly improved the loss of appetite,increased exercise ability and alleviated the liver damage in high-intensity exercise mice.2.FSF and FDB increase the activity of antioxidant enzymes(SOD and GSH-Px)and reduce MDA levels in serum and liver,restoring the balance of oxidation and antioxidant systems in the body of exercise mice,thereby alleviating fatigue.In terms of molecular mechanism,the intervention of FSF and FDB significantly reversed the decline in the expression levels of Nrf2/ARE signal pathway related factors(Nrf2,NQO1,GCLC and GCLM),and improved the body’s antioxidant capacity.Among them,FSF regulates the expression of antioxidant factors in a dose-dependent manner.The expression levels of FDB30 and FDB150 increase as the dose increases,while the effect of the FDB750(high-dose group)is not as good as the FDB30 and FDB150 group.3.The results of intestinal microecology analysis showed that the intervention of FSF and FDB increased the expression of intestinal barrier factor genes(ZO-1,Muc 2,Occludin,and Claundin 1).FDB intake significantly improved the inflammation of the ileum tissue and up-regulated Reg3γ expression.FSF and FDB increase the diversity and richness of small intestinal microbial species.FSF and FDB regulate the intestinal microecological disorder of the small intestine,by reducing the relative abundance of pathogenic bacteria Enterococcus and Enterobacter,increasing the uncultured＿f＿Muribaculaceae,uncultured＿f＿Lachnospiraceae,uncultured＿f＿Ruminococcaceae as well as Ruminococcaceae＿UCG-014 and Roseburia.High-intensity exercise has little disturbance to the intestinal microbes in the cecum contents of mice.The intervention of FSF and FDB reduced the uncultured＿f＿Muribaculaceae,uncultured＿f＿Erysipelotrichaceae,Bifidobacterium,Faecalibaculum and Candidatus＿Saccharimonas,and increased the relative abundance of probiotic Lactobacillus.At the same time,we found that FSF and FDB intake can regulate the changes of lactic acid and short-chain fatty acids in the intestine.4.The analysis of host and intestinal metabolites revealed that FSF and FDB intervention reversed the changed metabolite levels.The intervention of FSF and FDB significantly increased the level of metabolites related to the pyruvate metabolic pathway(pyruvate),and the level of metabolites related to the citric acid cycle pathway(pyruvate,2-ketoglutarate,citric acid,lactic acid,Acetic acid),which regulates the body’s energy metabolism balance.FSF and FDB intervention reduced the levels of various amino acids(isoleucine,leucine,alanine,phenylalanine,valine and methionine),acetic acid and lactic acid in the contents of the small intestine,and increased taurine,choline,β-glucose and betaine levels.Among them,FSF30,FSF150 and FDB30 showed strong regulatory effects.Metabolite analysis results show that high-intensity exercise has little effect on the metabolites of the cecal content.High-intensity exercise causes a decrease in the level of ethanol and an increase in the level of acetic acid,lysine,leucine,isoleucine and succinic acid.However,FSF and FDB treatment reduced the levels of acetate and lysine.The changed microorganisms in the contents of the small intestine and cecum are closely related to metabolites involved in pyruvate metabolism,tricarboxylic acid cycle,and amino acid metabolism pathways.5.The comparative analysis found that both FSF and FDB can relieve exercise fatigue through the following ways,including reducing the accumulation of metabolites,maintaining the intestinal barrier function,improving the imbalance of the antioxidant system,and regulating the imbalance of small intestinal microbiota and the host and intestinal metabolism.However,there are differences in the anti-fatigue effects of FSF and FDB.In terms of intestinal barrier function,compared with FSF,FDB can also significantly reduce the expression level of pro-inflammatory factors and increase the expression level of antimicrobial peptides,and the regulation effect of FDB is stronger at the same dose(low and medium dose),but it is not dose-dependent.In terms of small intestinal microbiota,FDB significantly regulates more types of small intestinal microorganism,especially microbes that are closely related to physiological indicators of mice.FDB30 intervention can significantly affect the levels of differential metabolites.In summary,this study used probiotic transformation to produce fermented soybeans and fermented deer blood that are rich in polypeptides and have antioxidant activity.Animal experiment results show that the FSF and FDB regulate the accumulation of metabolites,energy metabolism,oxidative stress,intestinal barrier function,intestinal flora structure,and host and intestinal metabolism,thereby alleviating exercise fatigue.Among them,FDB is more effective than FSF in improving the intestinal inflammation and the structure of intestinal flora,especially the significant regulation of small intestinal microorganisms related to the physiological indicators of mice.