Research Of A Novel Bacillus Subtilis And The Application Of Its Fermented Feed On Piglets

Soybean meal(SBM)is the most important plant protein source in animal industry.However,anti-nutritional factors in SBM restrict its application to young animals.As major anti-nutritional factors,soybean allergenic proteins are of high content and heat stable.Fermentation is an efficient method to eliminate allergenic proteins and improve the nutritive value of SBM.However,the efficacy of fermentation differs by different strains,and the study of fermentation mechanisms is also inadequate.There are 4 aims in this study:1)screen a strain which can degrade SBM allergenic proteins efficiently;2)investigate the soybean protein and fiber degradation process by the strain during fermentation;3)develop a pilot scale fermentation technique on soybean meal and corn-soybean meal mixed feed fermentation;4)form a set of feeding technique about fermented feed application on piglets to improve the growth performance of the piglets and decrease usage of antibiotics.The main results are as below:1.Screening and selection a high efficient strain for soybean meal allergic protein degradation1)A selection method based on multiple selective plates had been established in this study.3 high efficient protein degradation strains named BS1,BS11 and BS12;3 high efficient cellulose degradation ability strains named BA6,BS12 and BS17;2 efficient xylan degradation strains named BS12 and BS14 had been isolated with the selection method.Among all of the strains,BS12 showed better activities on both protein and fiber.2)After 24 h fermentation,BS12 reduced 84.77%less glycinin and 87.07%lessβ-conglycinin in SBM,better than commercial strains.2.Physical and chemical characteristics of BS12 fermented soybean meal and the degradation rule of organics during fermentation1)After 24 h solid-state fermentation by BS12,the content of cellulose and hemicellulose in soybean meal was decreased by 38.83%and 22.21%;crude protein was increased by 14.76%and the content of acid soluble protein was increased by 6 folds;contents of aspartic acid,glutamic acid,alanine,alanine,methionine,leucine,isoleucine and proline were increased(P<0.05).2)The SEM and CLSM analysis indicated soybean proteins and fibers were degraded during BS12 fermentation.The process of fiber degradation mainly occurred at 8 to 12 h and proteins were degraded during 12 to 24 h in fermentation.3)RNA-seq illustrated the main protease of BS12 during fermentation was bacillolysin and more than 14 enzymes attend in fiber degradation.Among them,endoglucanase,glucuronoxylanase and endo-1,4-beta-xylanase had higher transcription level during the fermentation process.3.Optimization of the fermentation technique of soybean meal and corn-soybean meal mixed feed in pilot scale1)The optimum condition for Bacillus subtilis BS12 in pilot scale solid fermentation of soybean meal was inoculum size,5%(v/w),initial water content,50%,environmental temperature,30 to 35℃ and incubation time,24 h.After fermentation,allergenic protein could be decreased by more than 80%.2)After fermented with Bacillus subtilis BS12 and Enterococcus faecalis EF at a two-step fermentation,the content of acid soluble protein in soybean meal was increased from 11.83%to 20.13%,and lactic acid was increased from 1.01%to 3.56%,pH was decreased from 8.01 to 4.98.3)The corn-soybean meal mixed feed was produced with the same technique.After two-step fermentation,the degradation rates of glycinin and β-conglycinin in the feed was 82.88%and 71.92%respectively;and the percentage of lactic acid was increased from 1.27%to 4.45%.4.Effect of fermented soybean meal and fermented corn-soybean meal mixed feed on piglets productionA 24-d animal experiment was designed with 324 piglets(Duroc X Large white X Yorkshire)with body weights of 9.35 ± 0.65 kg.The experiment was randomly divided into three dietary treatments:①corn-SBM based diet with 100 mg/kg olaquindox,50 mg/kg kitasamycin(control group,CON);②10%replacement of protein from SBM in the corn-SBM based diet by protein from fermented soybean meal(FSBM);③10%replacement of protein from corn soybean meal in the corn SBM based diet by protein from fermented corn-soybean meal mixed feed(FMF).1)Growth performance:Compared with the piglets in control group,piglets in 10%FSBM group had improved average daily feed intake(ADFI)at a rate of 7.73%and greater(P<0.05)average daily gain(ADG)at a rate of 12.89%;and piglets in 10%FMF group had improved ADFI at a rate of 6.39%and ADG at 7.99%.2)Intestinal morphrage and digest function:Compared with the control group,FSBM and FMF significantly increased the villus height and the villus/crypt depth in the jejunum(P<0.05).Besides,FSBM and FMF significantly increase the trypsin activity,amylase activity(P<0.05).Piglets fed with FMF diets presented higher(P<0.05)apparent nutrient digestibility of DM,protein and total P.While piglets fed with FSBM presented a greater(P<0.05)apparent total tract digestibility of total P and tendency of improvement(P = 0.08)apparent total tract digestibility of protein.3)Intestinal immune status:Compared with the control group,both FSBM and FMF decreased(P<0.05)the transcription levels of inflammatory cytokines IL-4 and IL-6.4)Colonic microbiota:There was no significant difference of α-diversity in colonic microbiota of piglets in three groups.However,compared with the control group,piglets fed with FSBM and FMF had a higher proportion of Lactobacillus.spp,at 15.48%and 42.72%,respectively.In summary,a high efficient strain named BS12 for soybean meal allergic protein degradation was isolated in this study.BS12 mainly participated in the degradation of fiber and protein in soybean meal with a secretion of various hydrolytic enzymes during fermentation.Both FSBM and FMF with BS12 and Enterococcus faecalis fermentation improved the nutritional value of feed,increased the content of lactic acid and decreased the pH of feed.The animal experiment indicated both FSBM and FMF could improve growth performance of piglets by enhancing intestinal digest function and reducing allergic inflammation.