Effects Of Mycotoxin-Biodegradation Preparation On Piglet Growth And Intestinal Microbiota And Molecular Mechanisms Of Liver Detoxification

Mycotoxins are ubiquitous and accessible in human foods and feedstuffs to cause big hazard.AFB1 and ZEA are considered to be the main mycotoxins in the contaminated feeds and feed ingredients,which not only cause several adverse effects on animals,but also cause huge economic losses and pose a threat to human security.Aflatoxin B1(AFB1)is the most fatal toxin,which causes hepatotoxic,teratogenic and carcinogenic,and was classified by the International Agency for Research on Cancer(IARC)as a human carcinogen(group 1 carcinogen).Zearalenone(ZEA)is characterized by structural similarity to estradiol,which is one-tenth of the intensity of estrogen.Zearalenone can increase the estrogenic level of female animals,and then affect the reproductivity of animals,which can induce hyperestrogenism symptoms such as changes in the morphology and function of reproductive organs,cytotoxicity and genotoxicity,hepatotoxicity,nephrotoxicity and immunosuppression in animals.Therefore,the contamination of mycotoxins in food and feed has attracted wide attention.However,the healthy assessment of mycotoxins is often limited to one or a class of mycotoxins,while the additive,synergistic and antagonistic effects of mycotoxins are neglected.Therefore,the harm of mycotoxins to human and animal health is much more serious than they were assessed.Recently,biodegradation of mycotoxin has attracted wide attention for its safety,effectiveness,specificity and operability.Therefore,the mycotoxin-biodegradation preparation(MBP)consisting of three species of probiotics and mycotoxin-degradation enzymes for degrading both AFB1 and ZEA were obtained by optimizing the combination in vitro.The protective effects of MBP on alleviating the cytotoxicity of IPEC-J2 and piglet production performance induced by AFB1 and ZEA were studied at the cellular and animal levels.16S rDNA high-throughput-sequencing technology was used to analyze the microbial flora richness and diversity in jejunum and feces of piglets.Finally,the differential protein and pathways in the liver of piglets were analyzed by quantitative proteomics,which will provide ideas for reducing the toxic effect of AFB1 and ZEA on production performance of piglets.The main results were listed below:(1)Mycotoxin-biodegradation preparation development:The present study aimed to optimize the proportion of different species of beneficial microbes by means of response surface methodology(RSM)and its combination with mycotoxin-degradation enzymes.The results indicated that AFB1 and ZEA degradation rates were 38.38%and 42.18%by individual Bacillus subtilis(P<0.05),which were increased to 45.49%and 44.90%(P<0.05)when three probiotic species such as Bacillus subtilis,Lactobacillus casein,and Candida utilis were mixed at a ratio of 1:1:1(v:v:v),corresponding with the predicted value of the RSM model.The further experiment showed that AFB1 and ZEA degradation rates were 63.95%and 73.51%(P<0.05)when the compound of three probiotic species was combined with mycotoxin-degradation enzymes from Aspergillus oryzae(A.orgzae)at a ratio of 3:2.This result indicated that the combination of probiotics with mycotoxin-degradation enzymes was a promising new approach for synchronous detoxification of AFB1 and ZEA,which provided the foundation for the further study.(2)Effect of AFB1 and ZEA on swine jejunal epithelial cell(IPEC-J2)and mouse hepatic cell(AML12)damages:The cytotoxicity of AFB1 and ZEA for IPEC-J2 cell and AML12 cell were different.The cytotoxicity was ZEA>AFB1 for IPEC-J2 cells,but AFB1>ZEA for AML12 cells.The toxicity of AFB1 and ZEA was time-dependent and dose-dependent effect on the two kinds of cells.At low concentrations,the main test results of the interaction between AFB1 and ZEA showed that there was a synergistic effect between the two toxins(PAFB1×ZEA<0.05).When the two toxins(AFB1 40 ?g/L and ZEA 500 ?g/L)acted simultaneously on IPEC-J2 cells for 24 h,the cytotoxicity showed a synergistic effect.Establishment of cell toxicity model laid a foundation for further experiments.(3)Effect of MBP on alleviating IPEC-J2 cell damage induced by AFB1 and ZEA:In order to remove AFB1 and ZEA toxicity,the combination of cell-free supernatant of compound probiotics(CFSCP)and mycotoxin-degradation enzymes(MDE)from Aspergillus oryzae was used to determine their alleviating cytotoxic effects on IPEC-J2 cell damage induced by AFB1 and ZEA.The results demonstrated that coexistence of AFB1 and ZEA had synergetic toxic actions for inhibiting cell viability(P<0.05).Cell viability was reduced with mycotoxin concentrations increasing,while it was increased with reaction time prolonging(P<0.05).The necrotic cell rates were increased when 40 ?g/L AFBi and/or 500 ?g/L ZEA were added(P<0.05),but CFSCP+MDE addition could decrease necrotic cell rates(P<0.05).Compared with the control group,viable cell rates were reduced when AFB1 and/or ZEA were added(P<0.05);however,CFSCP+MDE addition could alleviate the toxicity of AFB1 and ZEA for increasing viable cell rates(P<0.05).The relative mRNA abundances of Bcl-2,caspase 3,occluding and ZO-1 genes were up-regulated(P<0.05),while Bax,GLUT2,ASCT2 and IL6 genes were down-regulated(P<0.05)by CFSCP+MDE addition.This research provided an effective strategy to alleviate mycotoxin toxicity for keeping normal intestinal cell structure and animal health.(4)Effect of MBP on alleviating toxic symptoms of piglets induced by AFB1 and ZEA:MBP was added to piglet diet containing AFB1 and ZEA to feed piglets for 60 d.The results showed that the average daily gain was increased by 9.06%,diarrhea rate and mortality were decreased by 54.30%and 6.67%,and the area of vulva of female piglets was reduced by 24.97%by MBP addition,compared with the diet containing only AFB1 and ZEA(Negative control group).In addition,the digestibility of crude protein and fecal amylase activity were significantly increased by 12.23%and 29.01%(P<0.05),and the jejunal villus height and V/C ratio were also significantly increased by 116.27%and 104.84%(P<0.05)by MBP addition.MBP significantly decreased the serum enzyme activities of ALT,AST,ALP and LDH(P<0.05),compared with the negative control group.MBP had obvious alleviating effect on inflammation induced by AFB1 and ZEA from the histomorphology of liver,jejunum and uterus.The result of immunohistochemistry showed that the positive expression rate of Caspase-3 in MBP group was 29.80%lower than that in AFB1 and ZEA groups in uterus(P<0.05).The positive expression rates of NF-?B in liver and uterus in MBP group were also 49.96%and 8.71%lower than that in AFB1 and ZEA group(P<0.05).Compared with the negative control group,AFB1 residue contents in serum,liver and muscle were decreased by 41.38%,51.43%and 51.85%(P<0.05),and ZEA residue contents in liver and faeces were decreased by 46.65%and 28.83%(P<0.05)by MBP addition.In addition,serum endotoxin content was also decreased by MBP addition.MBP significantly down-regulated Bax and Caspase-3 expressions in liver,jejunum and uterus(P<0.05),up-regulated Bcl2,ZO-1 and Occludin expressions in jejunum and uterus(P<0.05),up-regulated ZO-1 and down-regulated Occludin expressions in liver(P<0.05).PepTl and GLUT2 expressions were significantly up-regulated(P<0.05),and ASCT2 and SGLT1 expressions were significantly down-regulated(P<0.05)in jejunum by MBP addition;ASCT2 and GLUT2 expressions were significantly up-regulated(P<0.05),and SGLT1 expression was significantly down-regulated(P<0.05)in liver by MBP addition;PepT1,ASCT2 and GLUT2 expressions were significantly up-regulated(P<0.05)in uterus by MBP addition,but there was no significant difference for SGLT1 expressions among three groups(P>0.05).The expressions of inflammatory cytokines such as IL10,IL8,IL1? and TNF-? in jejunum were down-regulated(P<0.05),while IL6 expression was up-regulated(P<0.05)in uterus by MBP addition;the expression levels of IL10 and IL8 were significantly up-regulated in liver by MBP addition(P<0.05);IL6 and IL8 expressions were significantly up-regulated(P<0.05),and IL10 expressions was significantly down-regulated(P<0.05)in uterus by MBP addition.In general,most of the inflammatory cytokines were negatively regulated by AFB1 and ZEA addition;however,they were regulated to the normal levels by MBP addition,indicating that the regular functions of jejunum,liver and uterus would be recovered.(5)Study on the microflora in jejunum and feces of piglets by 16S rDNA high throughput sequencing technique:The results showed that MBP significantly increased the microbial richness(Sobs index)and diversity(Shannon index)in jejunum(P<0.05),but they were not significantly different in piglet feces between the different groups(P>0.05).The microflora in jejunal contents and feces of piglets were significantly affected by AFB1 and ZEA.On the phylum level of piglet jejunal contents,MBP significantly increased the number of Bacteridetes flora by 4.99 folds(P<0.05),also increased Actinobacteria,Saccharibacteria and Verrucomicrobia by 4.64,13.00 and 6.50 folds(P<0.05).The imbalance of gut microbiota induced by AFB1 and ZEA were regulated to the normal level by MBP addition.On the phylum level of piglet feces,the number of Spirochaetae and were significantly reduced by 60.98%(P<0.05)by MBP addition,and Lentisphaerae were reduced by 45.61%.On the genus level of piglet jejunal contents,Lactobacillus was increased by 1.34 folds(P<0.05),while Clostridium_sensu stricto₁ was decreased by 38.66%by MBP addition.On the genus level of piglet feces,Lactobacillus and Prevotella₉ were significantly increased by 4.23 folds and 4.90 folds by MBP addition(P<0.05),while Rikenellaceae_RC9_gut_group was decreased by 53.41%.MBP could reduce the proportion of harmful bacteria(Clostridium_sensu_stricto₁)and increase the proportion of beneficial bacteria(Lactobacillus and Prevotella₉)to maintain the balance of intestinal microflora,ensure intestinal health and promote the growth of piglets.(6)Study on the molecular mechanism of piglet hepatic detoxification by proteomics:The protein expression profiles of piglet liver were analyzed by high throughput and label-free quantitative proteomics-DIA.208 differentially expressed proteins were screened with P<0.05 and fold change>1.5 times or<0.67 times,in which 153 proteins were up-regulated and 55 proteins were down-regulated.These proteins took part in 8 cell components,1 1 molecular functions and 18 biological processes.Among the differentially expressed proteins,10 proteins such as CYP2E1,HMGCL,ABAT,AOX1,MAOB,ACM4,ALDH5A1,ADH1-7,HK3,GSTZ1 were selected and verified by qRT-PCR.The results of qRT-PCR showed that 8 proteins were consistent with DIA in spite of HK3 and GSTZ1,indicating that quantitative proteomics were reliable.Pearson correlation analyses of ten differentially expressed proteins in liver with jejunal microflora,average daily gain of piglets,residues of AFB1 and ZEA in serum,liver and muscle of piglets were conducted.The results showed that the residues of AFB1 and ZEA in serum and liver of piglets were positively correlated with HK3 protein expression,the abundance of Clostridium_sensu_stricto₁ and Campylobacter spp.The correlation analyses revealed the molecular mechanism of mycotoxin biodegradants to alleviate the toxicity of AFB1 and ZEA.