Effects Of Enzymatic Soybean Protein On Growth Performance And Intestinal Health Of Weaned Piglets And The Underlying Mechanism

There is a lack of research regarding the comparison of employing enzymatic soybean(ESB)to completely substitute for animal protein sources in weaning diets.Hence,the objective of the study was to assess the comparative effects of fish meal(FM),milk powder(MK)or both MK and FM replacing with ESB on growth performance,nutrients digestibility,immunological parameters,short-chain fatty acids(SCFAs),gut microbiome and plasma metabolites in weaned piglets,and the correlation analysis was used to explain the relationship among gut microbiome,plasma metabolites and piglets.And the cell experiment was performed to illustrate the underlying mechanism of ESB bioactive peptides in alleviating intestinal inflammatory injury.Collectively,the research provides innovation for the substitution of animal protein sources in the diet of weaned piglets.Experiment 1: Comparative effects of enzymatic soybean,fish meal and milk powder in diets on growth performance,immunological parameters,SCFAs production and gut microbiome of weaned pigletsThe objective of this study was to evaluate the replacement effects of MK and FM by ESB in diets on growth performance,immunological parameters,SCFAs production and gut microbiome of weaned piglets.A total of 128 piglets with initial body weight at 6.95 ± 0.46 kg,were randomly assigned into 4 dietary treatments with 8 replicates per treatment and 4piglets per replicate for a period of 14 d.Piglets were offered isonitrogenous and isoenergetic diets as follows: CON diet with MK and FM as high quality protein sources,ESB plus FM diet with ESB replacing MK,ESB plus MK diet with ESB replacing FM,and ESB diet with ESB replacing both MK and FM.No significant differences were observed in growth performance among all treatments(P > 0.05).However,piglets fed ESB plus FM or ESB diet had increased diarrhea index(P < 0.01),and lower digestibility of dry matter,gross energy or crude protein,relative to piglets fed CON diet(P < 0.01).Moreover,the inclusion of ESB in diet markedly decreased the plasma concentration of haptoglobin and fecal concentration of butyric acid(P < 0.01).The High-throughput sequencing of 16 S r RNA gene V3-V4 region of gut microbiome revealed that the inclusion of ESB in diet increased the alpha diversity,and the linear discriminant analysis effect size showed that piglets fed with ESB plus FM or ESB diet contained more gut pathogenic bacteria,such as g＿Peptococcus,g＿Veillonella and g＿Helicobacter.Non-targeted metabolomics analysis based on UHPLC-QE/MS technology was used to analyze the plasma metabolites of CON group and ESB plus FM group.Results showed that the levels of DG(18:2(9Z,12Z)/18:2(9Z,12Z)/0:0),DG(18:2(9Z,12Z)/18:2(9Z,12Z)/0:0,linoleyl carnitine,tetracosahexaenoic acid,(2-{[3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}ethyl)trimethylazanium,indoxyl and p-cresol sulfate in plasma were significantly increased,and the plasma levels of 1-methylnicotinamide,dodecanoic acid,sphingomyelin,pentadecanoic acid and heptadecanoic acid were significantly decreased,which may increase the risk of liver and intestinal disease in piglets.Correlation analysis showed that Lyso PC(18:2(9Z,12Z)),deoxycholic acid and most phosphatidylcholine were positively correlated with inflammation related pathogenic bacteria Clostridium＿sensu＿stricto＿1,Campylobacter and Fournierella,respectively,implying the relationship between intestinal health effected by ESB and microbiome structure in weaned piglets.Moreover,the blood urea nitrogen level of ESB plus FM group was higher than that of CON group(P < 0.05),suggesting that ESB replacing milk powder may induce protein metabolism disorder,and further harmful for growth and health in weaned piglets.In conclusion,the substitution of milk powder or both milk powder and fish meal with ESB in diet leaded to higher diarrhea index,which could be ascribed to the lower nutrients digestibility,more gut pathogenic bacteria and plasma metabolites.However,piglets fed diet using ESB to replace fish meal did not markedly affect grow and gut health in weaned piglets,indicating the potential for replacing fish meal with ESB in weaning diet.Experiment 2: Enzymatic soybean bioactive substances alleviate inflammatory response of porcine IPEC-J2 intestinal epithelial cellsThe results of experiment 1 showed that ESB possesses the potential for replacing fish meal without negative effect on intestinal health,and the inclusion of ESB in dietary significantly decreased the plasma level of haptoglobin in weaned piglets,indicating the bioactive functions of ESB.Researches reported that the ESB bioactive substances(ESBS)most occupy in 1000 Da.So we separated and purified ESB protein(< 1000 Da),used the enterotoxigenic Escherichia coli(ETEC)infected IPEC-J2 cell model,and further investigated the relieving effects of ESBS on inflammation and oxidative damage in IPECJ2 cells.The experiment was divided into three treatments with 6 replicates per treatment:the control group(-ETEC/-ESBS),the ETEC group(+ETEC/-ESBS)and the ESB group(+ETEC/+ESBS).After 10 MOI ETEC infected IPEC-J2 cells for 6 h,the relative expression of inflammation-related genes IL-6 and TNF-α had been markedly increased(P < 0.05),and the relative expression of antioxidant-related genes Nrf2,NQO1,GSH-px and barrier-related genes ZO-1,Claudin-1,Occludin had been markedly decreased(P < 0.05),suggesting inflammation caused by ETEC damaged the structure and function of IPEC-J2 cells.However,5 mg/m L ESBS pretreated IPEC-J2 cells for 4 h inhibited the increase of the relative expression of inflammation-related genes,alleviated the decrease of relative expression of oxidant and barrier-related genes(P < 0.05).These results indicated the critical role of ESBS on alleviating intestinal epithelial cells inflammation.In conclusion,our research showed that piglets fed diet using ESB to replace fish meal did not markedly affect grow and gut health in weaned piglets,indicating the potential for replacing fish meal with ESB in weaning diet.The higher diarrhea index caused by the substitution of milk powder or both milk powder and fish meal with ESB in diet may due to the lower nutrients digestibility,more gut pathogenic bacteria and plasma metabolites.And5 mg/m L ESBS could alleviate the inflammation of pig intestinal epithelial cells induced by ETEC.