Study On Fermented Soybean Meal With Bacillus Amyloliquefaciens And Its Mechanism On Improving Growth Performance Of Broilers

Soybean meal（SBM）is the most commonly used plant protein source in livestock industries because of its high quality protein and relatively well-balanced amino acids.However,a variety of anti-nutritional factors（ANFs）present in SBM,such as antigenic proteins,trypsin inhibitors,and oligosaccharides,hinder the digestion and absorption of its nutrients by animals.Microbial fermentation is considered an economically viable processing technique to reduce the content of ANFs and improve the nutritional value of SBM.Therefore,the aims of this research were to screen and isolate a highly efficient antigenic-proteins-degrading Bacillus strain,to study the possible mechanism of degrading antigenic protein during solid state fermentation with Bacillus strain,and to evaluate nutrient value of fermented soybean meal（FSBM）on broilers and explore its potential mechanism on improving growth performance of broilers.This research includes seven experiments:Exp.1.The aims of this experiment were to screen and isolate a highly efficient strain that can degrade the antigenic soy proteins in SBM and assess the safety of this strain with an acute oral toxicity test.A B.amyloliquefaciens strain was successfully screened with antigenic proteins plate tests and fermentation.After solid state fermentation of SBM with B.amyloliquefaciens for 24 h,the amounts of glycinin andβ-conglycinin,two major antigenic proteins in SBM,decreased by 92.32%and 85.05%,respectively.The crude protein content in the fermented soybean meal increased by 17.54%compared with that in SBM.Notably,the trichloroacetic-acid-soluble protein（TCA-SP）content was 9.97-fold higher in FSBM than that in SBM.The contents of some essential amino acids,including valine,methionine,phenylalanine,and histidine,were significantly increased in FSBM by 10.68%,14.71%,13.31%,and 16.13%,respectively.Among the nonessential amino acids,the contents of alanine,proline,cysteine,and glutamic in FSBM increased by 10.33%,14.81%,16.67%,and 20.21%,respectively,compared with those in unfermented SBM.However,the contents of arginine and serine were significantly lower in FSBM（P<0.05）.The acute oral toxicity test suggested that the strain exerted no harmful effects on the relative organ weights,the morphological tissue structure,or the health of mice.These results indicate that the B.amyloliquefaciens strain is a safe strain for animals,and could be used to improve the nutritional quality of SBM by solid-state fermentation.Exp.2.The aims of this experiment were to explore the mechanisms of fermentation of SBM with B.amyloliquefaciens on improving its nutritional value.During the fermentation of SBM,B.amyloliquefaciens secreted 18 extracelluar hydrolases to degrade the ANFs in SBM,including non-starch polysaccharide enzymes,proteases,phytase,catalase,superoxide dismutase and chitosanase.Scanning electron microscopy revealed that the surface structure of SBM was broken down by B.amyloliquefaciens fermentation for 24h,irregluar shapes and obvious holes were mainly observed in FSBM.Fourier transform infrared spectroscopy showed that the main absorption peak in SBM presented at 1649 cm^-1（CO,CN）、1533 cm^-1（CH,CN,CC）,and 1048（CO）cm^-1,the absorption peak in FSBM were shifted from 1649 cm^-11 to 1646 cm^-11 and 1628 cm^-1,from 1533 cm^-1to 1557 cm^-1and 1537cm^-11 after fermentation with B.amyloliquefaciens,respectively.From the results above,solid state fermentation with B.amyloliquefaciens changed the structure of protein and polysaccharides in SBM.Exp.3.The aims of this experiment were to optimize the process condition of fermented SBM with mixed strains of B.amyloliquefaciens,Lactobacillus acidophilus and Saccharomyces cerevisie.The optimal ratio of mixed strains for fermentation was B.amyloliquefaciens:Lactobacillus acidophilus:Saccharomyces cerevisie=3:1:1,inoculum amount was 10%.The optimal conditions for fermentation of SBM were:feed water ratio 1:1,fermentation temperature 37℃,aerobic fermentation for 24 h and then anaerobic fermentation for 24 h.As compared with SBM,the contents of total amino acids and other 13 amino acids were significantly increased in FSBM produced by mixed strains,except for threonine,arginine,tyrosine,and serine（P<0.05）.No significant difference observed in contents of glycinin andβ-conglycinin between single strain fermented SBM and mixed strains fermented SBM.The in vitro dry matter digestibility and digestible energy of SBM increased from 62.91%to 72.52%and 76.63%and from 10.42 MJ/kg to 13.37 MJ/kg and 13.79 MJ/kg（dry matter basis）after fermentation with B.amyloliquefaciens and mixed strains,respectively.Exp.4.The objects of this experiment were to study the effects of fermented SBM by B.amyloliquefaciens in combination with non-starch polysaccharides enzymes（NSPs）on degrading antigenic protein in SBM.The optimal conditions in vitro for enzymatic hydrolysis of SBM were:temperature 50℃,pH 5.5,time 10 h,feed water ratio 1:1.2.The non-starch polysaccharide enzymes spectrum for hydrolysis of SBM were:α-galactosidase 1.2 U/g,pectinase 50U/g,cellulase 20 U/g,xylanase 10 U/g.The content of released reducing sugar was 64.97 mg/g.The contents of glycinin andβ-conglycinin in FSBM produced by B.amyloliquefaciens in combination with NSPs were higher than those of FSBM produced by B.amyloliquefaciens alone,which indicates that addition of extra non-starch polysaccharides enzymes is not necessary during B.amyloliquefaciens alone fermentation of SBM.Exp.5.The substitution method and nitrogen-free method were used to determine the Apparent Metabolizable Energy（AME）and Apparent Ileal Digestibility（AID）and Standard Ileal Digestibility（SID）of FSBM in broilers.For AME determination,A total of 72 twenty-one-day-old male Cobb 500broilers were randomly divided in to three dietary groups（six replicates per group）.The results showed that fermentation had no effect on the AME of SBM in broilers,the respective AME values were 10.29MJ/kg and 10.62 MJ/kg for SBM and FSBM.For the AID and SID determination,a total of 120 twenty-one-day-old male Cobb 500 broilers were randomly divided in to three dietary groups（six replicates per group）.The Apparent Ileal Digestibility（AID）and Standard Ileal Digestibility（SID）of some essential amino acid,including leucine,isoleucine,phenylalanine,and valine were significantly increased in FSBM.Among the nonessential amino acids,the AID and SID of cysteine,tyrosine,and Aspartic acid in FSBM were higher than those in unfermented SBM.Exp.6.The aims of this experiment were to investigate the effects of supplementation of FSBM on growth performance and serum indicators of broilers.A total of 180 one-day-old male Cobb 500 broilers were randomly divided into three dietary groups（six replicates per group）:corn-SBM diet（CC）;25%SBM replaced by FSBM（SC）;50%SBM replaced by FSBM（TC）.The feeding trial lasted for 36 days.Average daily gain（ADG）and feed/Gain（F/G）were higher in SC than CC and TC groups（P<0.05）during the growth（d 22–36）and whole（d 1–36）phases.No significant difference observed in ADG and average daily feed intake（ADFI）between CC and TC groups during any phases（P>0.05）.Dietary treatments significantly increased serum IgA,IgG,IgM,T-SOD,and mRNA gene expression of Occludin and Claudin in jejunum（P<0.05）,while significantly decreased the content of MDA and CREA（P<0.05）.Exp.7.The aims of this experiment were to study the effects of supplementation of FSBM on cecal microbiota composition of broilers.Dietary treatments increased Chao 1,observed species,and the abundance of the phylum Fimicutes but decreased the proportion of Proteobacteria（P<0.05）.Dietary treatments increased the abundance of the genera Lachnospiraceae,Lachnoclostridium,Gastranaerophilales,and Lactobacillus but decreased the abundance of Escherichia-Shigella and Clostridiales（P<0.05）.Spearman’s correlations showed that the abundance of Gastranaerophilales was positively correlated with ADG and serum immunity,whereas the genus of Anaeroplasma was strongly negatively with ADG,IgM,and IgA（P<0.05）.In addition,the abundance of genus Escherichia-Shigella was negatively correlated with FCR（P<0.05）.In conclusion,A B.amyloliquefaciens strain with highly efficient antigenic-protein-degrading was successfully screened,B.amyloliquefaciens secrets many kinds of extracelluar hydrolase that can destroy the surface structure of SBM and degrade the macromolecular antigenic protein and other anti-nutritional factors.The fermented SBM with B.amyloliquefaciens,Lactobacillus acidophilus,and Saccharomyces cerevisie improve its nutritional value,AID and SID of amino acid in broilers.FSBM supplementation in the diet improve the growth performance of broilers by modulating the cecal microbial composition and enhancing the intestinal barrier function.