增强链霉菌菌株3-10抗真菌活性最适培养基和培养条件的优化研究
发布时间：2018-10-11 13:08
【摘要】：放线菌是土壤中常见的一类微生物,能产生多种抗生素,其中包括抗细菌、抗肿瘤、免疫抑制剂等多种次级代谢产物以及一些酶类。在已知的土壤放线菌中,链霉菌占到约50%。放线菌产生的生物活性物质中大约75%是由链霉菌产生的。链霉菌产生的抗真菌物质中很多已经被应用于农业生产,如杀稻瘟菌素、春雷霉素、有效霉素、多杀菌素、博来霉素、井冈霉素等。链霉菌的活菌剂也被使用于农业生产中如Mycostop TM(S.griseoviridis K61),Rhizovit?(S.rimosus),其活性成分(孢子)能对多种土传病原真菌有拮抗作用如腐霉属、疫霉属、镰刀菌属、丝核菌属等。在链霉菌生长和产生抑菌活性物质过程中,培养条件的优化显得尤为重要,调节微生物生长过程中所需的营养和环境条件对其发酵工艺发展更是起到决定性作用。本研究是运用单因素和正交实验的方法,以优化链霉菌3-10的发酵培养基成分及其发酵条件,经过优化培养后,链霉菌3-10产生抗真菌物质的产量有所提高,并且进一步探究了链霉菌3-10产生的抗真菌物质(antifungal substance,AFS)对油菜根肿病的防病潜力。同时也探究了链霉菌3-10孢子及产生的AFS对黄曲霉在平板上的抑制作用和在花生上的防病效果,并且测定了对黄曲霉毒素产量的影响。研究结果如下:1.对链霉菌3-10发酵培养基成分进行了优化。结果表明:相对于Mn2+和Zn2+,K+和Mg2+对链霉菌3-10产生AFS的产量有显著提高,确定了最佳发酵培养基为:可溶性淀粉3%,蛋白胨0.75%,酵母提取物0.025%,大豆粉1%,K2HPO4·3H2O 0.5 g/L,KH2PO4 0.7 g/L,Mg SO4·7H2O 0.4 g/L。2.对链霉菌3-10发酵条件的优化。将链霉菌3-10孢子悬浮液以5%接种于种子培养液摇培24 h后再将种子液以3%的量接入发酵培养基培养72 h。得到的最佳发酵条件为:28℃,150 rpm,72h,p H6.5。3.本课题主要研究链霉菌3-10在发酵罐中发酵过程相关参数(搅拌速度、通气量、碳源和发酵时间)对其AFS产量的影响。所得结果表明,搅拌速度和通气量对AFS产量的影响极为显著。在AFS产生的过程中,搅拌速度和通气量可以改变发酵过程的某些参数,如溶氧量,p H,AFS产量等。当发酵过程中溶氧量改变时,菌丝体的长势和AFS产量也随之受到一定影响。试验得到的优化条件为:通气量0.75 vvm,搅拌速度200 rpm,温度28oC,培养时间72 h。根据优化培养条件得到的AFS产量为4.02 g/L,与三角瓶摇培发酵72 h得到的AFS产量(4.45 g/L)十分相近。4.将链霉菌3-10孢子液、发酵液、乙酸乙酯提取物分别处理接种病原的油菜,以只接种病原菌P.brassicae为对照。调查发现,通过链霉菌3-10孢子液、发酵液、乙酸乙酯提取物处理后的油菜的病害严重程度与只接种P.brassicae的处理比较分别下降了83%,77%和73%,油菜根肿病的发病率明显降低,将链霉菌3-10与P.brassicae休眠孢子同时接种油菜后发现,其病害严重程度显著低于对照处理,并且在链霉菌3-10处理后的发病的根瘤更小且休眠孢子数量也显著降低。以上结果表明,链霉菌3-10可以延迟油菜根肿病的发生。5.通过平板对峙实验发现,链霉菌3-10可以抑制黄曲霉的生长,其抑菌率为41%。向PDA培养基中添加2.5%的链霉菌3-10发酵液(CF3-10,2.5%的乙酸乙酯提取物(CE3-10,2%的孢子液,对黄曲霉生长的最小抑菌浓度为5μg/10μL。在发酵液(CF3-10),不同处理的抑菌率范围从39.59%到90.26%,黄曲霉毒素产量的抑制率范围从79.37%到93.38%。在花生上,链霉菌3-10的发酵液(CF3-10)和乙酸乙酯提取物(CE3-10)均可以抑制黄曲霉的生长,并且显著降低黄曲霉毒素的产生。同时发现,经过链霉菌3-10发酵液处理过的黄曲霉菌丝的afl R和afl S表达量(0.09±0.03)比未处理的(0.343±0.05)有显著降低,说明链霉菌3-10发酵液处理可以抑制黄曲霉毒素的产生。以上研究结果表明,优化链霉菌3-10的培养基及培养条件可以为大规模发酵提供理论依据。链霉菌3-10对油菜根肿病和黄曲霉有良好的防病效果和抑制作用。本研究表明,链霉菌3-10具有良好的生防潜力。
[Abstract]:Actinomyces is a kind of microorganism commonly found in soil, which can produce a variety of antibiotics, including many secondary metabolites such as anti-bacteria, anti-tumor and immunosuppressant, as well as some enzymes. In known soil actinomycetes, Streptomyces accounts for about 50%. About 75% of the bioactive substances produced by actinomycetes are produced by Streptomyces. Many antifungal substances produced by Streptomyces have been applied to agricultural production, such as kill rice blast, spring reomycin, validamycin, multi-bactericidal element, bleomycin, validamycin and the like. The living bacterial agent of Streptomyces is also used in agricultural production, such as Mycostop (TM), Rizoit? (S.rimosus), its active component (spore) can antagonize various soil-borne pathogenic fungi such as Pythium, Pythium, fusarium, Rhizoctonia, etc. The optimization of culture conditions is particularly important in the growth and production of antibacterial active substances in Streptomyces. The nutrition and environmental conditions needed to regulate the growth of microorganisms play a decisive role in the development of fermentation technology. According to the method of single factor and orthogonal experiment, the fermentation medium composition and fermentation condition of Streptomyces sp. 3-10 are optimized, and after the optimized culture, the yield of the antifungal substance generated by Streptomyces coelicolor 3-10 is improved, Furthermore, the anti-fungal agent (AFS) produced by Streptomyces coelicolor 3-10 was further studied. At the same time, the inhibition of Aspergillus flavus on the flat plate and the effect of AFS on the production of Aspergillus flavus were also studied. The results of the study are as follows: 1. The composition of 3-10 fermented culture medium was optimized. The results showed that the optimum fermentation medium was 3% soluble starch, 0. 75% of protein, 0. 025% of yeast extract, 1% of soybean powder, 0.5 g/ L of K2Cr3H2O, 0. 7g/ L of KH2PO4, and the optimum fermentation medium was: 3% soluble starch, 0. 75% of protein, 0. 025% of yeast extract, 1% of soybean powder, 0.5 g/ L of K2Cr3H2O, and 0. 7 g/ L of KH2PO4. Mg SO4路7H2O 0.4 g/L.2. Optimization of fermentation conditions for Streptomyces sp. 3-10. The spore suspension of mold 3-10 was inoculated with 5% seed culture solution for 24 h, and then the seed solution was inoculated into the fermentation medium at 3% for 72 h. The optimum fermentation conditions were 28 鈩，
本文编号：2264306
[Abstract]:Actinomyces is a kind of microorganism commonly found in soil, which can produce a variety of antibiotics, including many secondary metabolites such as anti-bacteria, anti-tumor and immunosuppressant, as well as some enzymes. In known soil actinomycetes, Streptomyces accounts for about 50%. About 75% of the bioactive substances produced by actinomycetes are produced by Streptomyces. Many antifungal substances produced by Streptomyces have been applied to agricultural production, such as kill rice blast, spring reomycin, validamycin, multi-bactericidal element, bleomycin, validamycin and the like. The living bacterial agent of Streptomyces is also used in agricultural production, such as Mycostop (TM), Rizoit? (S.rimosus), its active component (spore) can antagonize various soil-borne pathogenic fungi such as Pythium, Pythium, fusarium, Rhizoctonia, etc. The optimization of culture conditions is particularly important in the growth and production of antibacterial active substances in Streptomyces. The nutrition and environmental conditions needed to regulate the growth of microorganisms play a decisive role in the development of fermentation technology. According to the method of single factor and orthogonal experiment, the fermentation medium composition and fermentation condition of Streptomyces sp. 3-10 are optimized, and after the optimized culture, the yield of the antifungal substance generated by Streptomyces coelicolor 3-10 is improved, Furthermore, the anti-fungal agent (AFS) produced by Streptomyces coelicolor 3-10 was further studied. At the same time, the inhibition of Aspergillus flavus on the flat plate and the effect of AFS on the production of Aspergillus flavus were also studied. The results of the study are as follows: 1. The composition of 3-10 fermented culture medium was optimized. The results showed that the optimum fermentation medium was 3% soluble starch, 0. 75% of protein, 0. 025% of yeast extract, 1% of soybean powder, 0.5 g/ L of K2Cr3H2O, 0. 7g/ L of KH2PO4, and the optimum fermentation medium was: 3% soluble starch, 0. 75% of protein, 0. 025% of yeast extract, 1% of soybean powder, 0.5 g/ L of K2Cr3H2O, and 0. 7 g/ L of KH2PO4. Mg SO4路7H2O 0.4 g/L.2. Optimization of fermentation conditions for Streptomyces sp. 3-10. The spore suspension of mold 3-10 was inoculated with 5% seed culture solution for 24 h, and then the seed solution was inoculated into the fermentation medium at 3% for 72 h. The optimum fermentation conditions were 28 鈩，
本文编号：2264306
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