Protective Effect And Mechanism Of Fermented Soybean Meal On Gut Barrier In Weaned Piglets

Fermented soybean meal(FSBM)is a product of soybean meal(SBM)by microbial fermentation,which can improve the growth performance,reduce diarrhea,and enhance gut barrier function on weaned piglets.In order to analyze the protective effect and mechanism of FSBM on the gut barrier of weaned piglets,six experiments were conducted in this study.The main study contents and results were listed as follows:Exp.1 Comparative analysis of nutritional and active components between soybean meal and fermented soybean mealThe nutritional composition,anti-nutritional factors content,protein distribution,and active components of SBM and FSBM were compared and analyzed by using conventional detection methods and non-targeted metabolomics technique to identify active substances with gut protective effects in FSBM,and to preliminarily explore whether these extracts have antibacterial effects.The results showed that compared with SBM,the content of crude protein and crude fiber in FSBM was increased,and ether extract,β-conglycinin,glycinin,raffinose and stachyose were decreased(P<0.05).The content of methyl4-hydroxycinnamate,p-coumaric acid,ethyl caffeate,ethyl ferulate,cinnamic acid,guanine,cytosine,cytidine,guanosine,adenine,4-methoxyphenylacetic acid,lithosprmoside,dihydroartemisinic acid,2-pyrrolidinecarboxylic acid and atractylenolide III,which are characteristic active substances with gut protective effects in FSBM,were increased(P<0.05).FSBM extract inhibited the proliferation of ETEC K88(P<0.05).Exp.2 Effects of fermented soybean meal extract on the m RNA expression of tight junction protein,mucin and s Ig A in IPEC-J2 cellsIPEC-J2 was used as an in vitro model to study the effects of different doses of FSBM extract and SBM extract on proliferation rate and the m RNA expression of tight junction protein,mucin and s Ig A in IPEC-J2 cells,and to screen for the most suitable dose.The results showed that within the concentration range of 0.01-1 mg/m L,FSBM extract did not exhibit toxic effects on IPEC-J2 cells(P<0.05).100μg/m L FSBM extract enhanced proliferation rate of IPEC-J2,increased the m RNA expression of secretory immunoglobulin A(s Ig A)and Claudin-1(P<0.05),and improved the immune and barrier function of IPEC-J2 cells.However,SBM extract does not have an enhancing effect.Exp.3 Effects of fermented soybean meal extract on m RNA expression of tight junction protein,mucin,s Ig A and endocannabinoid receptor in IPEC-J2 cells stimulated by LPSLPS(100μg/m L,12h)was used to establish an IPEC-J2 cell damage model to study the effects of FSBM extract and SBM extract on the expression of tight junction protein,mucin,s Ig A and endocannabinoid receptor in IPEC-J2 cells stimulated by LPS.LPS treatment down-regulated the m RNA expression of Occludin,Claudin-1,(Zonula occludens-1,ZO-1),Mucin 1 and Mucin2 in IPEC-J2 cells(P<0.05),and the addition of FSBM extract alleviated the LPS-induced decrease in m RNA expression of Claudin-1,ZO-1,Mucin2 and s Ig A(P<0.05),which has the effect of improving the immune and barrier function of IPEC-J2 cells.In addition,LPS treatment down-regulated the m RNA expression of endocannabinoid receptor 2(CB2)in IPEC-J2 cells,and FSBM extract could alleviate LPS-induced decrease in CB2 m RNA expression(P<0.05).However,SBM extract has no mitigating effect.Exp.4 Effects of fermented soybean meal on growth performance,diarrhea rate,and blood immunity of ETEC K88-challenged weaned pigletsA total of 32 weaned piglets at the age of(21±2)days old,weighing 6.86±0.54 kg,were randomly divided into two groups according to the principle of similar body weight.Each group consisted of 16 piglets and were fed two different diets.One was a corn-SBM basal diet that contained 24%SBM,and the other was to replace 25%SBM in the previous diet with FSBM.After 14 days of feeding,piglets from each dietary treatment group were randomly divided into a challenge group and a non-challenge group based on an equal distribution of body weight and gender.The challenge group was fed 50 m L of ETEC K88culture medium(10~9CFU/m L),while the non-challenge group was fed an equal amount of sterilized phosphate buffer saline(PBS).The experiment was to study the effect of FSBM on growth performance,diarrhea rate,and blood immunity of ETEC K88-challenged weaned piglets.The results showed that ETEC K88 challenge leaded to decrease in F/G,plasma interleukin-10(IL-10)content and amylase activity,and increase in plasma tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)levels,and diarrhea rate of weaned piglets(P<0.05),which leaded to inflammation and growth inhibition.FSBM could alleviate the negative effects caused by ETEC K88 challenge by reducing plasma TNF-αlevel(P<0.05).Exp.5 Protective effect and mechanism of fermented soybean meal on the gut barrier of ETEC K88-challenged weaned pigletsThe experimental design was the same as Exp.4.The experiment was to study the protective effect and mechanism of fermented soybean meal on the gut barrier of ETEC K88-challenged weaned piglets.The results showed that ETEC K88 challenge caused decrease in villus height and villus height to crypt depth,tight junction protein(ZO-1,Occludin,Claudin-1)m RNA and protein expression levels,s Ig A and B-cell lymphoma-2(Bcl-2)m RNA expression,and increased in TNF-αand IL-6 m RNA expression in ileal tissue of weaned piglets(P<0.05),which damaged to intestinal morphology and gut barrier.Dietary FSBM supplementation alleviated decrease in Occludin protein expression and Bcl-2 m RNA expression and increased in TNF-αand IL-6 m RNA expression caused by ETEC K88 challenge(P<0.05),thereby improve intestinal physical and immune barrier function.ETEC K88 challenge damaged the gut microbiota barrier of weaned piglets,and increased the relative abundance of pathogenic bacteria(Escherichia-Shigella)(P<0.05),while FSBM reduced the relative abundance of pathogenic bacteria(Proteobacteria,Clostridium,and Escherichia-Shigella)(P<0.05),improving gut microbiota barrier function.In addition,ETEC K88 challenge up-regulated endocannabinoid receptor 1(CB1)m RNA expression(P<0.05)and increased gut permeability.FSBM increased the expression of CB2 m RNA(P<0.05),inhibiting gut inflammation.Exp.6 Protective effect and mechanism of fermented soybean meal on gut barrier of ETEC K88-challenged weaned piglets based on“gut microbiota-endocannabinoid system”The experimental design was the same as Exp.4.To study the effect of FSBM on the cecal microbiota and its metabolites in ETEC K88-challenged weaned piglets,and to analyze whether FSBM regulates metabolites through“gut microbiota-endocannabinoid system”pathway to protect the gut barrier.The results showed that FSBM increased the content of arachidonic acid in cecum and decreased the content of trehalose-6-phosphate of ETEC K88-challenged weaned piglets,which were metabolites in retrograde endocannabinoid signaling.Correlation analysis showed a correlation between trehalose-6-phosphate concentrations and the relative abundance Tyzzerella＿3 and Subdoligranulum,which indicated that FSBM protects gut barrier by mediating the“gut microbiota-endocannabinoid system”pathway.In conclusion,FSBM could protect the physical,chemical,immune,and microbial barriers of weaned piglets,alleviate diarrhea and improve growth performance,and its effect may be achieved by regulating the expression of endocannabinoid receptors and mediating the“gut microbiota-endocannabinoid system”pathway,and the results of this study will provide a theoretical basis for the rational utilization of FSBM.