本文關(guān)鍵詞： 飼料霉變 黃曲霉 黃曲霉毒素 解淀粉芽孢桿菌 降解　出處：《河北農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：飼料及原料在運(yùn)輸和儲(chǔ)存過(guò)程中極易受霉菌污染,霉菌污染的飼料不僅破壞其營(yíng)養(yǎng)成分,同時(shí)霉菌及其產(chǎn)生的毒素影響動(dòng)物的安全及生產(chǎn)效率,且通過(guò)食物鏈間接影響到人類(lèi)的安全和健康。黃曲霉是引起飼料霉變的常見(jiàn)菌株,黃曲霉毒素是毒性最強(qiáng)、分布最廣、污染最嚴(yán)重的毒素。常用的黃曲霉及毒素的防治方法有物理法、化學(xué)法及生物法,而生物法效率高且安全無(wú)污染,因此成為國(guó)內(nèi)外研究的熱點(diǎn)。本試驗(yàn)利用平板對(duì)峙法初篩和抑菌率法復(fù)篩,從土壤中分離得到一株高效抑制黃曲霉的菌株N-2,通過(guò)形態(tài)觀察、生理生化測(cè)定和16S r DNA序列分析,確定其為解淀粉芽孢桿菌Bacillus amyloliquefaciens。通過(guò)急性毒理試驗(yàn)研究發(fā)現(xiàn),菌株N-2安全無(wú)毒。菌株N-2通過(guò)改變黃曲霉菌絲形態(tài)及降低黃曲霉孢子萌發(fā)率抑制黃曲霉生長(zhǎng)。菌株N-2使黃曲霉菌絲變粗而短,菌絲體內(nèi)部原生質(zhì)體分布不均勻,對(duì)黃曲霉孢子萌發(fā)抑制率為81.08%。通過(guò)菌株N-2抑制黃曲霉活性組分分析試驗(yàn)發(fā)現(xiàn),菌株N-2發(fā)酵液和無(wú)N-2菌體上清液對(duì)黃曲霉的抑制率顯著高于胞懸液和胞內(nèi)液,而菌株N-2發(fā)酵液和無(wú)N-2菌體上清液對(duì)黃曲霉的抑制率無(wú)差異(P0.05),因此,可以推測(cè)菌株N-2抑制黃曲霉生長(zhǎng)的活性物質(zhì)為胞外分泌物。菌株N-2發(fā)酵液能降解黃曲霉毒素B1(Aflatoxin B1,AFB1),降解率為37.81%。微生物經(jīng)口進(jìn)入胃腸道后,必須耐受胃酸低p H及腸道中膽鹽高滲環(huán)境才能發(fā)揮其作用,本試驗(yàn)結(jié)果表明,模擬胃液處理菌株N-2 4 h,其活菌數(shù)達(dá)96.11%。模擬膽鹽處理菌株N-2 24 h,其活菌數(shù)達(dá)95.68%。菌株N-2對(duì)大腸桿菌、沙門(mén)氏菌、痢疾桿菌有抑制效果,抑菌圈直徑分別為19.57 mm、18.39 mm、17.92 mm。本試驗(yàn)利用N-2發(fā)酵液進(jìn)行飼喂肉雞試驗(yàn),通過(guò)檢測(cè)肉雞的生長(zhǎng)性能、血液生化指標(biāo)及器官的組織切片等,研究不同劑量菌株N-2的發(fā)酵液對(duì)肉雞飼料中的黃曲霉毒素降解效果。試驗(yàn)結(jié)果表明,基礎(chǔ)日糧每千克添加0.2 L菌株N-2發(fā)酵液能改善AFB1對(duì)肉雞的毒害作用,降低了毒素對(duì)肝臟損傷,維護(hù)了肉雞腸道菌群的平衡。本研究試驗(yàn)結(jié)果表明,試驗(yàn)分離篩選得到的解淀粉芽孢桿菌N-2對(duì)黃曲霉有顯著抑制效果,其安全無(wú)毒,對(duì)胃酸和膽鹽有耐受性,且能降低AFB1對(duì)動(dòng)物的毒害作用,為研制黃曲霉及黃曲霉毒素生物防治菌劑提供了理論基礎(chǔ)及試驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S859.8

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