| In view of the serious pollution of mycotoxin and serious hazards to human and animal health, it is an unresolved issue to take effective measures for mycotoxin detoxification. The study selected some microbes for inhibiting the growth of Aspergillus flavus and degrading aflatoxin B1(AFB1) with enzymes, which was then combined with the probiotics to detoxificate AFB1, so as to provide evidence for the application of animal production.(1) In order to inhibit A. flavus growth and AFB1production, the optimal proportion of beneficial microbes such as Lactobacillus casei (L. casei), Bacillus subtilis (B. subtilis) and Pichia anomala (P. anomala) were selected, The results showed that AFB, production and mycelium weight of A. flavus was decreased by97.09%(4.69μg/L) and81.87%with the free-cell supernatants of L. casei and B. subtili (P<0.05), respectively. The optimal proportion of L. casei, B. subtilis and P. anomala was2:1:2for inhibiting A. flavus growth determined by3X3orthogonal design.(2) In order to select the microbes for degrade AFB1, the cell dry weight, optical density, pH value and content of AFB1were determined. The AFB1degradation ability of the second L. casei and4strains of B. subtilis were the most remarkable, the content of AFB1was decreased by38.38%with the forth B. subtilis. The optimal proportion of L. casei, B. subtilis and P. anomala was2:1:2for degrading AFB1, the maximum AFB1degradation was at48h incubation (82.72%, P≤0.05).(3) Orthogonal design was used to investigate effect of the combined microbes with yeast cell wall and oligosaccharide on AFB1detoxification. The result indicated the content of AFB I was decreased by88.03%with the combined microbes with yeast cell wall and oligosaccharide, and decreased by85.05%with the only microbes (P>0.05), so the compound probiotics would be used for the detoxification of AFB1in the following experiments.(4) The AFB1-degrading microbes were selected by using AFB1as the carbon source and energy, the degradation ratio of AFB1reached58.04%and77.05%. The AFB1degrading enzyme was extracted by ion-exchange chromatography and gel chromatography from solid fermentation of AFB1-degrading strain. The molecular weight of AFB1-degrading enzyme was33.7kDa by SDS-PAGE analysis, the degradation ratio of AFB1reached54.33%. The AFB1-degrading microbes were identified as B. subtilis and Aspergillus oryzae by16S rDNA and26S rDNA sequence analysis.(5) The combined microbes with AFB1-degrading enzyme was used to detoxificate AFB1. The optimal proportion of compound probiotics and AFB1-degrading enzyme was2:3for degrading AFB1, and the maximum AFB1degradation rate was78.07%(P≤0.05).(6) In order to study the effect of compound probiotics and AFB1-degrading enzyme on production performance of broilers fed with diets added with AFB1, body weight gain, feed conversion rate, mortality, enzyme activities, antioxidant indices and gene expression were determined. A3-period feeding program was adopted in feeding experiment. In the first stage (1-21d), a total of200one-day-old AA broilers were assigned to5groups,40broilers for each group consisting of5replicates. In the second stage (22-42d), a total of15022-day-old AA broilers were assigned to5groups,30broilers for each group consisting of5replicates. In the third stage (43-72d), a total of7543-day-old AA broilers were assigned to5groups,15broilers for each group consisting of5replicates. In the first and second stages, group A was given with the basal diet, group B, C, D and E was fed with the basal diet containing100μg/kg AFB1, and added with0.00%,0.05%,0.10%and0.15%compound probiotics and AFB1-degrading enzyme, respectively. In the third stage, group A was given with the basal diet; group B, C, D and E was fed with the basal diet added with400,200,400and800μg/kg AFB1, under the condition of0.15%addition of compound probiotics and AFB1-degrading enzyme except for group B.In the first stage, the final weight and average daily gain were significantly increased, and mortality rate and diarrhea rate were significantly decreased in the groups added with compound probiotics and AFB1degrading enzyme, compared with group B (P≤0.05). Compared with group B, the final weight and the average daily gain in group E were increased by11.59%and16.87%(P<0.05), mortality rate and diarrhea rate were decreased by56.58%and88.89%, and the weight of liver and bursa of Fabricius were increased. The crude fat and phosphorus digestibility in group B were decreased compared with the other groups (P≤0.05). Compared with the groups without addition of compound probiotics and AFB1-degrading enzyme, the counts of E. coli in the duodenum were reduced (P≤0.05), and the counts of lactic acid bacteria in caecum was increased; the activities of protease in gizzard, duodenum, ileum and caecum were higher (P≤0.05); the cntents of serum TB, DB, LDL, GGT and T-AOC were higher. The damage of genomic DNA of the livers in dfferent groups did not change significantly. The body weight gain of broilers was significantly inhibited because of the high content of AFB1in basal diet. In the second stage, every treatment group had no significant effect on production performances in group A, C, D and E (P>0.05), except diarrhea rate was significantly decreased, compared with group B (P≤0.05). Compared with group B, whole net carcass rate was significantly improved in the group D and E added with more compound probiotics and AFB1-degrading enzyme (P≤0.05). Meat quality was not significant difference among every treatment groups (P≤0.05). Total counts of bacteria in duodenal, ileum and appendix in the groups added with compound probiotics and AFB1-degrading enzyme were significantly higher than that in group A and B. Compared with group B, amylase activity in ileum and lipase activity in appendix were significantly increased (P≤0.05) Compared with control group, glutamic-oxalacetic transaminease activity was significantly increased and glutathione peroxidase was decreased in other groups (P≤0.05). The contents of AFB1in liver and pectorale were not significant difference among the different groups (P>0.05). The genomic DNA of liver was damaged in group B and D.In the third stage, adding with compound probiotics and AFB1degrading enzyme could significantly relieve chicken’s production performance. Compared with group B, the final weight, the average daily gain and FCR were significantly increased in group C, D and E (P≤0.05). The body weight of group B (without compound probiotics and AFB1degrading enzyme) and group E (added with800μg/kg AFB1) appeared negative growth. Because group D was added with compound probiotics and AFB1degrading enzyme, the final weight, the average daily gain, the average daily feed intake and FCR were significantly higher than that in group B, the average daily feed intake and the final weight were increased by23.39%and29.81%(P≤0.05). Compared with group B, the metabolic rates of organic matter, crude protein, calcium and phosphorus in other groups were significantly improved (P≤0.05). The adding of AFB1could significantly reduce the slaughter performance of chicken; the total viable microbial counts in duodenum in other groups were significantly increased (P≤0.05), while the counts of Escherichia coli were significantly decreased (P≤0.05). The amylase activity in duodenum and ileum, superoxide dismutase (SOD), glutathione peroxidase (GPX) and total antioxidant capability in livers in other groups were significantly increased, cmpared with group B (P≤0.05). Liver genomic DNA was damaged seriously in group E added with the largest quantity of AFB1.In general, adding0.15%probiotic bacteria and AFB1-degrading enzyme in chicken diet containing200μg/kg of AFB1could detoxificate AFB1and relieve the bad effect of AFB1on chicken’s production performance. The study could set up the foundation for detoxification of AFB1 and its application in animal feed.(7) According to the production performance of broilers in different stage, the livers of72-day-old male broilers in group A, B and D were selected to determine gene expressive quantity. The result indicated that62.5%genes of oxidoreductase in group B were down-regulated, compared with group A; and most genes in group D were up-regulated, compared with group B. All the genes for cell growth and50%genes for regulation of immune system process in group D were up-regulated, compared with group B. Most genes for regulation of metabolic process in group B were down-regulated, compared with group A and D. All the genes for cell death in group D were down-regulated, compared with group B. In general, adding0.15%probiotic bacteria and AFB1-degrading enzyme in chicken diets could significantly relieve harmful effects to the gene expression of production performance, which provided conditions for further studies on the pathogenesis of AFB1and eliminating the hazards at genetic level. |
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