黄曲霉拮抗菌株的分离筛选及对黄曲霉毒素的降解效果

发布时间：2018-02-13 03:32

本文关键词： 饲料霉变黄曲霉黄曲霉毒素解淀粉芽孢杆菌降解　出处：《河北农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：饲料及原料在运输和储存过程中极易受霉菌污染,霉菌污染的饲料不仅破坏其营养成分,同时霉菌及其产生的毒素影响动物的安全及生产效率,且通过食物链间接影响到人类的安全和健康。黄曲霉是引起饲料霉变的常见菌株,黄曲霉毒素是毒性最强、分布最广、污染最严重的毒素。常用的黄曲霉及毒素的防治方法有物理法、化学法及生物法,而生物法效率高且安全无污染,因此成为国内外研究的热点。本试验利用平板对峙法初筛和抑菌率法复筛,从土壤中分离得到一株高效抑制黄曲霉的菌株N-2,通过形态观察、生理生化测定和16S r DNA序列分析,确定其为解淀粉芽孢杆菌Bacillus amyloliquefaciens。通过急性毒理试验研究发现,菌株N-2安全无毒。菌株N-2通过改变黄曲霉菌丝形态及降低黄曲霉孢子萌发率抑制黄曲霉生长。菌株N-2使黄曲霉菌丝变粗而短,菌丝体内部原生质体分布不均匀,对黄曲霉孢子萌发抑制率为81.08%。通过菌株N-2抑制黄曲霉活性组分分析试验发现,菌株N-2发酵液和无N-2菌体上清液对黄曲霉的抑制率显著高于胞悬液和胞内液,而菌株N-2发酵液和无N-2菌体上清液对黄曲霉的抑制率无差异(P0.05),因此,可以推测菌株N-2抑制黄曲霉生长的活性物质为胞外分泌物。菌株N-2发酵液能降解黄曲霉毒素B1(Aflatoxin B1,AFB1),降解率为37.81%。微生物经口进入胃肠道后,必须耐受胃酸低p H及肠道中胆盐高渗环境才能发挥其作用,本试验结果表明,模拟胃液处理菌株N-2 4 h,其活菌数达96.11%。模拟胆盐处理菌株N-2 24 h,其活菌数达95.68%。菌株N-2对大肠杆菌、沙门氏菌、痢疾杆菌有抑制效果,抑菌圈直径分别为19.57 mm、18.39 mm、17.92 mm。本试验利用N-2发酵液进行饲喂肉鸡试验,通过检测肉鸡的生长性能、血液生化指标及器官的组织切片等,研究不同剂量菌株N-2的发酵液对肉鸡饲料中的黄曲霉毒素降解效果。试验结果表明,基础日粮每千克添加0.2 L菌株N-2发酵液能改善AFB1对肉鸡的毒害作用,降低了毒素对肝脏损伤,维护了肉鸡肠道菌群的平衡。本研究试验结果表明,试验分离筛选得到的解淀粉芽孢杆菌N-2对黄曲霉有显著抑制效果,其安全无毒,对胃酸和胆盐有耐受性,且能降低AFB1对动物的毒害作用,为研制黄曲霉及黄曲霉毒素生物防治菌剂提供了理论基础及试验依据。
[Abstract]:Fodder and raw materials are easily contaminated by mold in the course of transportation and storage. The fodder contaminated by mold not only destroys its nutritional composition, but also affects the safety and production efficiency of animals. Aflatoxin is the most common strain causing feed mildew, and aflatoxin is the most toxic and widely distributed. The most seriously polluted toxins. The commonly used methods of prevention and treatment of aflatoxin and toxin are physical, chemical and biological methods, while biological methods are highly efficient and safe and pollution-free, In this experiment, a strain N-2 was isolated from soil by plate confrontation method and bacteriostasis method, and a strain N-2 was isolated from the soil. The strain N-2 was obtained by morphological observation, physiological and biochemical analysis and 16s r DNA sequence analysis. It was identified as Bacillus amylolique faciens. Strain N-2 was safe and non-toxic. Strain N-2 inhibited the growth of Aspergillus flavus by changing the morphology of Aspergillus flavus filaments and reducing the germination rate of Aspergillus flavus spores. Strain N-2 made Aspergillus flavus filaments thicker and shorter, and the protoplasts of mycelium were not evenly distributed. The inhibition rate of Aspergillus flavus spore germination was 81.08%. The inhibitory rate of strain N-2 fermentation broth and non-N-2 supernatant on Aspergillus flavus was significantly higher than that of cell suspension and intracellular fluid. However, the inhibition rate of strain N-2 fermentation broth and non-N-2 supernatant on Aspergillus flavus was not different (P 0.05). It can be inferred that the active substance of strain N-2 inhibiting the growth of Aspergillus flavus is extracellular secretion. The fermentation broth of strain N-2 can degrade aflatoxin B1Aoxflatin B1 and AFB1, and the degradation rate is 37.81.After the microorganism enters the gastrointestinal tract, In order to play its role, gastric acid low pH and bile salt hyperosmotic environment must be tolerated. The number of live bacteria of simulated gastric juice treatment strain N-2 for 4 h was 96.111.The number of live bacteria of mimic bile salt treated strain N-2 for 24 h was 95.68. Strain N-2 had inhibitory effect on Escherichia coli, Salmonella and Shigella dysenteriae. The diameter of bacteriostasis circle was 19.57 mm ~ 18.39 mm ~ (-1) ~ 17.92 mm 路m ~ (-1) respectively. The broilers were fed with N-2 fermentation broth. The growth performance, blood biochemical index and organ tissue section of broiler were tested. The degradation of aflatoxin in broiler feed by fermentation broth with different doses of strain N-2 was studied. The results showed that the toxicity of AFB1 to broilers was improved by adding 0.2 L strain N-2 fermentation broth per kilogram of basal diet. The results showed that Bacillus amylolyticus N-2, which was isolated and screened by the experiment, had a significant inhibitory effect on Aspergillus flavus, and it was safe and non-toxic. It has tolerance to gastric acid and bile salt, and can reduce the toxicity of AFB1 to animals. It provides a theoretical basis and experimental basis for the development of aflatoxin biocontrol agents.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.8

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