Mycotoxin-degradation Compound Probiotics Preparation And Its Effect On Broiler Growth Performance And Gut Microbiota

The economic loss caused by mycotoxin pollution in animal husbandry is very heavy at present.Among various mycotoxins,Aflatoxin B1（AFB1）and Zearalenone（ZEA）are considered to be widely distributed and harmful,so they are the focus of this study.Three species of microbes（Bacillus subtilis K4,Lactobacillus casei and Candida utilis）with good mycotoxin-degradation activity were selected to obtain three kinds of mycotoxin-biodegradation preparations（MBDP）for degrading AFB1（MBDP 1）,ZEA（MBDP 2）or both of them（MBDP 3）by using response surface software.MBDP was added in broiler diets containing AFB1,ZEA or both to determine its effect on broiler production performance,mycotoxin residue,tissue ultrastructural changes,and intestinal microflora.This experiment was divided into three parts.The first part,compound probiotic preparation for mycotoxin biodegradation in vitro:Three species with good degrading toxins preserved in the laboratory were selected,which were designed as 3 levels at the levels of 1.0×105 CFU/mL,1.0×106 CFU/mL and 1.0×l07 CFU/mL.There were 17 test points with response surface software,the total volume was 5 mL,and incubation time was 24 h.The results showed that ZEA degradation rate was 36.42%（P<0.05）when the viable counts of Bacillus subtilis K4,Lactobacillus casei and Candida utilis were 1×107,1×105 and 1×106 CFU/mL（MBDP 1）;AFB1 degradation rate was 49.58%（P<0.05）at 1×106,1×107 and 1×107 CFU/mL（MBDP 2）;ZEA and AFB1 degradation rates were 26.29%and 38.67%（P<0.05）at 1×107,1×106 and 1×107 CFU/mL（MBDP 3）,respectively.The three species of microbes were cultured and dried,which was mixed according to the above ratios to make 3 kinds of MBDP for the following broiler feeding experiment.The second part,the effects of MBDP on production performance and detoxification of broilers.400 one-day-old Rose 308 were randomly divided into 8 groups of 5 replicates each,10 chickens per replicate.The experiment was divided into three stages,0-21d was the early stage,21-42 d was the late stage,and 43-50 d was the mycotoxin-discharge period.Group A was the control group and fed with the basal diet;Group B containing ZEA 500 μg/kg;Group C containing AFB1 50 μg/kg;Group D containing AFB1 50 μg/kg and ZEA 500 μg/K;Group E containing ZEA 500 μg/kg and MBDP 1;Group F containing AFB1 50 μg/kg and MBDP 2;Group G containing AFB1 50μg/kg,ZEA 500μg/kg and MBDP 3;Group H containing AFB1 50 μg/kg,ZEA 500 μg/kg,MBDP 3 and mycotoxin-degradation enzyme.At the end of experiment of the second stage,6 broilers（half male and female）were selected and slaughtered from each group for the relative parameter determinations.All the broilers were fed with basal diet during in the third stage.In order to determine the metabolic mechanisms of mycotoxins,3 broilers were selected and slaughtered from group A,D and G at 1 d,3 d,5 d and 7 d,respectively.The main results were listed as below.At the first stage,daily feed intake,daily weight gain and feed conversion rate in group E,F,G and H containing MBDP and mycotoxins were better than that in group B,C and D with only mycotoxins（P<0.05）.Daily weight gain in group F was 5.12%higher than group C（P<0.05）.The diarrhea rates were the highest in group C and D（P<0.05）,and mortality was the highest in group B and C.Protein metabolic rates from high to low were group B,E,H>group A,D,G>group D,F>group C（P<0.05）,which in group F was 5.65%higher than group C（P<0.05）.At the second stage,daily feed intake,daily weight gain and feed conversion rate in group E,F,G and H containing MBDP and mycotoxins were better than that in group C and D with only mycotoxins（P<0.05）.Daily weight gain in group G was 14.91%higher than group D（P<0.05）.The diarrhea rates（P<0.05）and mortality were the highest in group C and D.Protein metabolic rates from high to low were group B,E>group G,H>group A>group D,F>group C（P<0.05）.In general,MDBP could significantly alleviate mycotoxin hazard,increase growth rate and nutrient metabolic rate,decease diarrhea rates and mortality for broilers（P<0.05）.The relative weights of liver in group E,F and G were higher than the other groups（P<0.05）,the relative weights of spleen in group E and G were higher than the other groups（P<0.05）.The relative weights of breast muscle in group B were higher than the other groups（P<0.05）,the relative weights of small and large intestines in group G were higher than most groups（P<0.05）.It was inferred that compound probiotics could increase relative organ weight of broilers（P<0.05）.The serum endotoxin contents from high to low were group B,C>group A,D,G,H>group E>group F（P<0.05）,which in group F was 21.92%lower than group C（P<0.05）,indicating that compound probiotics were able to reduce endotoxin contents for broilers.Serum ALT,TP,ALB,TG contents in group G were higher than group A and D（P<0.05）,while serum alkaline phosphatase（ALP）content in group G was 31.15%lower than group D（P<0.05）,indicating that compound probiotics could alleviate mycotoxin hazard and protect cell from damage.The residual ZEA and AFB1 in serum,liver,breast muscle,small intestine and faeces in group B,C and D containing mycotoxins were higher than the other groups（P<0.05）,which in small intestine in group G were 45.22%and 11.81%lower than group D（P<0.05）,indicating the function of MBDP for degrading mycotoxins.The contents of ZEA and AFB1 in serum,liver,breast muscle and faeces were decreased with discharge period prolonging,which got to the lowest point after 7 d discharge（P<0.05）.The contents of AFB1 in breast muscle and faeces in group G were 31.82%and 26.20%lower than group D（P<0.05）,and the contents of ZEA in liver and faeces in group G were 24.41%and 30.56%lower than group D after 7 d discharge（P<0.05）.Mycotoxins could cause jejunum and liver damages through up-regulating inflammatory factors such as Caspase-3,TNF-α and NFkB expressions by HE staining and immunohistochemical analysis;however,MBDP could down-regulated the gene expressions for alleviating mycotoxin hazards.The third part,gut microbiota analysis:The jejunal contents in group A,D and G were selected for microbiota analysis by High-throughput sequencing technology.The result showed that there were 634,707 and 671 Operational taxonomoc unit（OTUs）in group A,D and G,respectively.At phylum level of microbiota in jejunum,Firmicutes,Proteobacteria,Bacteroidetes,Actinobacteria were the dominant microbes occupying more than 90%microbiota;the abundance of Firmicutes in group D was lower than group A and G,while the abundance of Proteobacteria and Bacteroidetes were higher than group G（P<0.05）.At genus level of microbiota in jejunum,Lactobacillus,Helicobacter and Clostridium_sensu_tricto₁ were the dominant microbes.At species level of microbiota in jejunum,Lactobacillus-aviarius,Helicobacter and Clostridium_sensu_stricto₁ were the dominant microbes.Jejunal Lactobacillus-aviarius abundance in group G was 45%higher than group D（P<0.05）,inferring that Lactobacillus-aviarius might be relative to mycotoxin degradation.At species level,the correlation between the top 10 abundance and the toxin residues of the two toxins in the serum,small intestine and chest muscle,and the average daily gain of broilers were analyzed.The results showed that the toxin residues in the small intestine and chest muscles were The abundance of Lactobacillus-aviarius was significantly negatively correlated,and positively correlated with Helicobacter and Clostridium_sensu_stricto₁,revealing the molecular mechanism of mycotoxin antidote to alleviate the toxicity of AFB1 and ZEA.In conclusion,MBDP could keep gastrointestinal microbiota stable,degrade mycotoxins,decrease mycotoxin residue in organs and tissues,alleviate cell damages induced by mycotoxins,increase broiler production performance and nutrient metabolic rates,reduce diarrhea rates and mortality,and provide support for removing mycotoxin hazards.