【作者】 左瑞雨；

【导师】 尹清强；

【作者基本信息】 河南农业大学 ， 动物营养与饲料科学， 2012， 博士

【摘要】 鉴于霉菌毒素B1(AFB1)的严重污染及其对人类和动物健康的严重危害,采取有效措施对霉菌毒素进行脱毒处理成为函待解决的问题。本文通过筛选得到对黄曲霉菌生长及AFB1降解效果较好的菌株,并对AFB1降解酶进行分离纯化,之后将益生菌与AFB,降解酶配合应用于肉鸡生产中，观察其对肉鸡饲料中AFB1的解毒效果,为其在动物生产中的应用提供依据。(1)通过测定菌丝重量和AFB1产量,筛选对黄曲霉菌生长和产毒抑制效果最好的乳酸菌、枯草芽孢杆菌和酵母菌,并通过正交试验筛选对黄曲霉菌生长和产毒抑制效果最好的益生菌组合。结果显示枯草芽孢杆菌上清液对黄曲霉生长的抑制效果最好,抑制率高达81.87%,乳酸菌上清液抑制产毒效果最好,上清液中AFB1含量仅为4.69μg/L,抑制率高达97.09%。乳酸菌、枯草芽孢杆菌和酵母菌单独培养后以2：1：2的比例混合,对霉菌生长和AFB1产量具有显著抑制作用(P<0.05)。(2)在微生物的液体培养基中添加高浓度的AFB1,通过测定培养液中细胞干重、吸光度值以及pH观察微生物的生长情况,初步判断乳酸菌A2和4株枯草芽孢杆菌均具有较高的AFB1降解能力,其中枯草芽孢杆菌B4的降解率最高,达到38.38%(P≤0.05)。乳酸菌、枯草芽孢杆菌和酵母菌单独培养后以2：1：2的比例混合对AFB1的降解试验中,48h时对AFB1的降解率最高,达到82.72%(P≤0.05)。(3)通过正交试验,研究益生菌组合与酵母细胞壁、寡糖配伍对AFB1的降解作用。研究表明：益生菌组合与酵母细胞壁和寡糖配伍与仅使用益生菌组合对AFB1的降解效果差异不显著(P>0.05),对AFB1的降解率分别为88.30%和85.05%。因而,在后期试验中，仅选择益生菌组合来研究其对AFB1的降解效果。(4)以AFB1作为碳源和能源进行AFB1降解菌株的筛选。筛选得到的细菌和真菌对AFB1的降解率分别为58.04%和77.05%。对降解AFB1的真菌进行固体发酵培养,提取AFB1降解酶粗酶液并通过离子交换层析和凝胶层析,对AFB1降解酶进行分离纯化。通过凝胶层析分离出蛋白分子量约为33.7KDa的AFB1降解酶,对AFB1的降解率达54.33%。经过16S rDNA和26SrDNA的鉴定结果显示,筛选得到的AFB1降解细菌为枯草芽孢杆菌,真菌为米曲霉。(5)鉴于分离纯化后AFB1降解酶的降解效率低于粗酶液,将益生菌组合与AFB1降解酶的粗酶液以不同比例混合,测定其对AFB1的降解能力。结果表明,益生菌组合与AFB1降解酶按2：3的组合比例对AFB1的降解效果最好,对AFB,的降解率达78.07%。(6)将益生菌和AFB1降解酶添加到含有AFB1的饲料中,通过测定肉鸡生产性能、血清和肝脏相关酶活性、抗氧化指标及其相关基因表达量的变化等指标,研究益生菌和AFB1降解酶对肉鸡饲料中AFB1的解毒效果。饲养试验分为前期(1-21d)、中期(22-42d)和后期(43-72d)三个阶段。前期选择1d健康AA肉仔鸡200只,分为5个处理,每个处理5个重复,每个重复8只鸡。中期在前期原处理内选择150只鸡,分为5个重复,每个重复6只鸡。后期选择75只鸡,将各组鸡只混合重新分为5组,每组5个重复,每个重复3只鸡。在前期和中期试验中,A组为基础日粮(对照组),B(负对照组)、C、D和E组分别在基础日粮添加100μg/kg的AFB1,并按照0、0.05%、0.10%和0.15%的比例分别添加益生菌和AFB1降解酶的复合物。在后期试验中，A组为基础日粮(对照组),B、C、D和E组分别在基础日粮添加AFB1量为400、200、400和800μg/kg,除B组不添加益生菌和AFB1降解酶的复合物外(负对照组),C、D和E组分别在基础日粮添加0.15%益生菌和AFB1降解酶。其主要结果如下：在试验前期,与负对照B组相比,添加益生菌和AFB1降解酶的各组肉鸡的末重、平均日增重均有不同程度的提高,死亡率和腹泻率均有显著降低(P≤0.05),其中添加量最大的E组与B组相比,末重和,平均日增重分别提高了11.59%和16.87%(P≤0.05),腹泻率和死亡率分别降低了56.58%和88.89%,且肝脏和法氏囊的重量均有显著提高(P<0.05)。与其它处理组相比,B组粗脂肪和磷代谢率显著降低(P≤0.05)。日粮中添加益生菌和AFB1降解酶显著降低了十二指肠中大肠杆菌的数量(P≤0.05),显著提高了盲肠中乳酸菌的数量(P≤0.05),肌胃、十二指肠、回肠和盲肠中蛋白酶活力皆有显著提高(P≤0.05),血清中间接胆红素、低密度脂蛋白、谷酰转肽酶和总抗氧化能力显著升高(P≤0.05),各处理组基因组DNA无显著变化。总体来看,由于前期基础日粮中AFB1的含量过高,各处理组鸡只的生产性能均受到了严重抑制,但益生菌和AFB1降解酶的解毒效果得到了充分的体现。在试验中期,添加益生菌和AFB.降解酶的各组鸡只除了腹泻率显著降低外(P≤0.05),其它各项生产性能各处理组之间无显著差异(P>0.05)。与其它处理组相比,B组的粗蛋白质代谢率显著降低(P≤0.05)。D和E组全净膛率显著地高于B组(P<0.05)。添加益生菌和AFB1降解酶的各处理组鸡的十二指肠、回肠和盲肠的总细菌数均显著显著高于A和B组(P≤0.05)。与B组相比,日粮中添加益生菌和AFB1降解酶对各处理组鸡回肠淀粉酶活力和盲肠脂肪酶活力皆有显著提高(P≤0.05)。与对照组相比,添加益生菌和AFB1降解酶各组鸡血清中谷草转氨酶的活力皆有显著提高(P<0.05),谷胱甘肽过氧化物酶皆有不同程度的下降(P<0.05)；肝脏和胸肌中AFB1含量无显著差异(P>0.05),但B和D组鸡肝脏DNA有部分损伤。在试验后期,添加益生菌和AFB1降解酶显著缓解了AFB1对肉鸡生产性能的抑制。AFB1添加量为200μg/kg的C组,鸡只的末重、平均日增重、平均日采食量和料.肉比显著高于B、D和E组(P≤.05),与对照组无明显的差别(P>0.05)。而未添加益生菌和AFB1降解酶的B组和AFB1添加量达800μg/kg的E组体重则出现了负增长。同等AFB1添加量的B组和D组,因为D组添加了益生菌和AFB1降解酶,其体重、平均日增重、、平均日采食量和料肉比均显著高于B绢,平均日采食量和末重分别提高了23.39%和29.81%(P≤0.05)。与B组相比,其余各组的有机物质、粗蛋白质、钙和磷的代谢率均显著提高(P≤0.05)。随AFB1添加量的增加,显著降低了肉鸡的屠宰性能：但AFB添加量较小的C组,屠宰率、全净膛率、腹脂、脾脏和肌胃的重量与B组相比皆有显著提高(P≤0.05)。各处理组十二指肠的总菌数显著升高(P≤0.05),大肠杆菌数目显著降低(P≤0.05)；十二指肠和回肠淀粉酶活力、肝脏超氧化物歧化酶、谷胱甘肽过氧化物酶和总抗氧化能力均有显著提高(P≤0.05)。毒素添加量最大的E组,肝脏基因组DNA损伤严重。总体来看,在AFB.含量为200μ/kg的肉鸡饲料中添加0.15%的益生菌和AFB.降解酶,可以显著缓解AFB1对肉鸡生产性能的不良影响,这为AFBI的生物降解和应用奠定了基础。(7)根据生产性能指标选择43-72d时A、B和D组的公鸡各3只,将肝脏样品等比例混合后进行肝脏中相关基因表达量的测定。结果表明：对于氧化还原酶系来说,A组与B组相比,B组表达量下调的基因占表达量发生变化的基因总数的62.5%：B组与D组相比,D组的大多基因表达量均出现了上调。对于细胞生长酶系来说,B组与D组相比,D组的基因表达量均出现了下调。对于免疫过程控制相关酶系来说,B组与D组相比,D组的基因表达量发生上调和下调的各占50%。对于代谢过程控制相关酶系来说,A组与B组相比,B组的大多基因的表达量均出现了下调,B组与D组相比,D组的大多基因的表达量均出现了上调。对于调节细胞凋亡的基因来说,A组与D组相比,基因表达量均出现了下调,B组与D组相比,D组75%的基因表达量均出现了下调。由此可见,在肉鸡日粮中添加益生菌和AFB1降解酶可显著缓解因添加AFB1所导致的影响生产性能发挥的相关基因表达量发生不良改变的问题,也为今后从基因水平上研究AFB1的致病机制并采取措施缓解其危害奠定了基础。更多还原

【Abstract】 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.更多还原