【正文】 碩 士 學(xué) 位 論 文 廣譜高效微生物防腐劑 產(chǎn)生菌的篩選及應(yīng)用 學(xué) 科 門類 理 學(xué) 學(xué) 科 專業(yè) 微生物學(xué) 培 養(yǎng) 單位 生物工程學(xué)院 Screening of Broadspectrum Biocontrol Substance Producing Strain and Application A Dissertation Submitted for the Degree of Master Candidate： Supervisor： Prof. Associate Prof. College of Bioengineering Henan University of Technology, Zhengzhou, China 關(guān)于學(xué)位論文的獨(dú)創(chuàng)性聲明 本人鄭重聲明：所呈交的論文是本人在指導(dǎo)教師指導(dǎo)下獨(dú)立進(jìn)行研究工作所取得的成果，論文中有關(guān)資料和數(shù)據(jù)是實(shí)事求是的。盡我所知，除文中已經(jīng)加以標(biāo)注和致謝外，本論文不包含其他人已經(jīng)發(fā)表或撰寫的研究成果，也不包含本人或他人為獲得河南工業(yè)大學(xué)或其它教育機(jī)構(gòu)的學(xué)位或?qū)W歷證書而使用過(guò)的材料。與我一同工作的同志對(duì)研究所做的任何貢獻(xiàn)均已在論文中做出了明確的說(shuō)明。 若有不實(shí)之處，本人愿意承擔(dān)相關(guān)法律責(zé)任。 學(xué)位論文作者簽名： 日期： 年 月 日 學(xué)位論文使用授權(quán)書 本人完全同意河南工業(yè)大學(xué)有權(quán)使用本學(xué)位論文（包括但不限于其印刷版和電子版），使用方式包括但不限于：保留學(xué)位論文，按規(guī)定向國(guó)家有關(guān)部門（機(jī)構(gòu)）送交學(xué)位論文，以學(xué)術(shù)交流為目的贈(zèng)送和交換學(xué)位論文，允許學(xué)位論文被查閱、借閱和復(fù)印，將學(xué)位論文的全部或部分內(nèi)容編入有關(guān)數(shù)據(jù)庫(kù)進(jìn)行檢索，采用影印、縮印或其他復(fù)制手段保存學(xué)位論文。 保密學(xué)位論文在解密后的使用授權(quán)同上。 學(xué)位論文作者簽名： 日期： 年 月 日 指導(dǎo)教師簽名： 日期： 年 月 日 I 摘 要 微生物導(dǎo)致食品的腐敗變質(zhì)不僅會(huì)造成巨大經(jīng)濟(jì)損失，還會(huì)引起嚴(yán)重的食品安全問(wèn)題。而現(xiàn)有的食品防腐保鮮方法在實(shí)際生產(chǎn)應(yīng)用中具有一定的局限性，迫切需要尋求更安全有效的防腐保鮮新技術(shù)，將微生物及其代謝產(chǎn)物應(yīng)用于食品防腐保鮮的生物防治方法是現(xiàn)階段研究的熱點(diǎn)。 從輪馬熱泉、北海溫泉、大塘熱泉等騰沖溫泉附近采集土壤樣品 23 種，采用稀釋分離法分離純化得到細(xì)菌菌株 142 株。以大腸桿菌、黑曲霉、禾谷鐮刀菌等為指示菌，采用濾紙片法或平板對(duì)峙法從 142 株細(xì)菌菌株中初步篩選得到具有廣譜抑菌效果的菌株 7 株，其中菌株 LM2303 廣譜抑菌 效果最好。通過(guò)形態(tài)特征分析、生理生化特性分析和 16S rDNA 序列比對(duì)，菌株 LM2303 應(yīng)為枯草芽孢桿菌 (Bacillus subtilis)。 采用紫外線誘變、硫酸二乙酯誘變和微波誘變對(duì)菌株 LM2303 進(jìn)行選育。其中，經(jīng)硫酸二乙酯誘變處理得到突變菌株 D59，其抑菌效果較原始菌株提高了%。以抑菌物質(zhì)產(chǎn)量為指標(biāo)，從 8 種基礎(chǔ)培養(yǎng)基中篩選出菌株 D59 最佳發(fā)酵培養(yǎng)基 M2。采用單因素實(shí)驗(yàn)和響應(yīng)面分析對(duì)菌株 D59 進(jìn)行發(fā)酵條件優(yōu)化。單因素試驗(yàn)結(jié)果表明培養(yǎng)時(shí)間、溫度和轉(zhuǎn)速對(duì)菌株 D59 抑菌物質(zhì)產(chǎn)量有顯著影響(p)。利用響應(yīng)面確定菌株 D59 的最佳培養(yǎng)條件為：接種量 5%、裝液量100mL/250mL、培養(yǎng)時(shí)間 43h、溫度 33℃ 、轉(zhuǎn)速 177rpm。在上述發(fā)酵條件下培養(yǎng)， 100mL發(fā)酵液可得到抑菌物質(zhì)粗提物 g(濕重 )，是原發(fā)酵條件下的 倍，有效提高了抑菌物質(zhì)產(chǎn)量。 采用鹽酸沉淀、甲醇萃取，從拮抗菌株發(fā)酵液中得到抑菌物質(zhì)粗提物。利用薄層色譜對(duì)抑菌物質(zhì)進(jìn)行分析和初步純化，紫外及茚三酮顯色結(jié)果表明抑菌物質(zhì)含有苯環(huán)和閉合肽鍵。 紅外光譜掃描結(jié)果表明菌株 LM2303 抑菌物質(zhì)為環(huán)狀脂肽類物質(zhì)。該脂肽類物質(zhì)理化性質(zhì)穩(wěn)定，抑菌譜廣，對(duì)大腸桿菌、金黃色葡萄球菌等細(xì)菌和黑曲霉、黃曲霉、擴(kuò)展青霉、禾谷鐮刀菌等真菌均有較強(qiáng)的抑制作用。 從不同腐爛病癥的冬棗上分離得到根菌索菌、細(xì)極鏈格孢、橄欖色盾殼霉、擴(kuò)展青霉四種致腐真菌。平板對(duì)峙試驗(yàn)結(jié)果表明菌株 LM2303 可有效抑制上述四種真菌的生長(zhǎng)，菌株 LM2303 具有冬棗貯藏期致腐真菌 的生物防控應(yīng)用潛力。 ………………… 關(guān)鍵詞： 枯草芽孢桿菌；拮抗；抑菌物質(zhì)；誘變選育；發(fā)酵條件優(yōu)化 III I ABSTRACT Food spoilage caused by pathogenic microanisms not only results in great financial losses, but also serious food safety problems. And existing food antisepsis method in practical application has certain limitations, to explore an obvious safe and effective anticorrosion technology is particularly important. Microanisms and their metabolites work for food conservation is a research focus of biological control at the present stage. 142 strains of bacteria were isolated from 23 soil samples which collected form Tengchong hot spring, such as Lunma hot spring, Beihai hot spring, Datang hot spring and so on. Filtering paper method and dual culture technique were employed to test antagonistic effect of bacteria strains, and seven of them showed efficient inhibitory, strain LM2303 has the most obvious inhibitory effect. Strain LM2303 was identified as Bacillus subtilis through the analyses of morphological and biochemical properties and 16S rDNA sequencing. Bacillus subtilis LM2303 was used as the start strain and the mutation breeding was carried out through UV light, DES and microwave radiation. Inhibition ratio of strain D59 isolated following DES mutagenesis enhance %. The production of biocontrol bubstance was taken as indexes, M2 medium was used for fermentation screened from 8 different medium. The factors and its levels influencing the production of biocontrol bubstance produced by strain D59, was determined by using singlefactor test, and the optimization of fermentation conditions was approached by RSM. The optimum fermentation conditions were as follows: temperature was ℃ , inoculation was 5%, medium volume was 100mL/250mL, rotary speed was rpm and fermentation time was h. Under the new fermentation condition, g biocontrol bubstance can be obtained from 100 mL fermentation broth and is the times of original fermentation conditions. Biocontrol bubstance were obtained with hydrochloric acid precipitate and methanol extraction method from fermentation broth, and purified by thin layer chromatography. Purified biocontrol bubstance was detected by FTIR. The results II showed that the biocontrol bubstance contained lipopeptides. Biocontrol bubstance produced by strain LM2303 has stable physical and chemical properties and wide antibacterial spectrum, exhibit strong inhibitory effect on a number of bacteria and multiple pathogenic fungi. Four putrefying fungi were isolated from the decayed tissues of Winter Jujube and identified as Rhizomorpha Fr, Alternaria tenuissima, Coniothyrium olivaceum and Penicillium expansum. Strain LM2303’s prominent inhibition on four putrefying fungi lay the foundation for putrefying fungi biocontrol of Winter Jujube. ………………… Key words: Bacillus subtilis。 antagonistic action。 biocontrol bubstance。 mutation breeding。 optimization of fermentation conditions I 目 錄 第一章 前言 ........................................................................................................... 1 研究背景 ................................................................................................... 1 生防微生物概況 ....................................................................................... 2 用于生物防治的微生物種類 ......................................................... 2 生防微生物的作用機(jī)制 ................................................................. 2 生防芽孢桿菌 ...............................................................................