【摘要】：放線菌是土壤中常見的一類微生物,能產(chǎn)生多種抗生素,其中包括抗細(xì)菌、抗腫瘤、免疫抑制劑等多種次級(jí)代謝產(chǎn)物以及一些酶類。在已知的土壤放線菌中,鏈霉菌占到約50%。放線菌產(chǎn)生的生物活性物質(zhì)中大約75%是由鏈霉菌產(chǎn)生的。鏈霉菌產(chǎn)生的抗真菌物質(zhì)中很多已經(jīng)被應(yīng)用于農(nóng)業(yè)生產(chǎn),如殺稻瘟菌素、春雷霉素、有效霉素、多殺菌素、博來霉素、井岡霉素等。鏈霉菌的活菌劑也被使用于農(nóng)業(yè)生產(chǎn)中如Mycostop TM(S.griseoviridis K61),Rhizovit?(S.rimosus),其活性成分(孢子)能對(duì)多種土傳病原真菌有拮抗作用如腐霉屬、疫霉屬、鐮刀菌屬、絲核菌屬等。在鏈霉菌生長(zhǎng)和產(chǎn)生抑菌活性物質(zhì)過程中,培養(yǎng)條件的優(yōu)化顯得尤為重要,調(diào)節(jié)微生物生長(zhǎng)過程中所需的營(yíng)養(yǎng)和環(huán)境條件對(duì)其發(fā)酵工藝發(fā)展更是起到?jīng)Q定性作用。本研究是運(yùn)用單因素和正交實(shí)驗(yàn)的方法,以優(yōu)化鏈霉菌3-10的發(fā)酵培養(yǎng)基成分及其發(fā)酵條件,經(jīng)過優(yōu)化培養(yǎng)后,鏈霉菌3-10產(chǎn)生抗真菌物質(zhì)的產(chǎn)量有所提高,并且進(jìn)一步探究了鏈霉菌3-10產(chǎn)生的抗真菌物質(zhì)(antifungal substance,AFS)對(duì)油菜根腫病的防病潛力。同時(shí)也探究了鏈霉菌3-10孢子及產(chǎn)生的AFS對(duì)黃曲霉在平板上的抑制作用和在花生上的防病效果,并且測(cè)定了對(duì)黃曲霉毒素產(chǎn)量的影響。研究結(jié)果如下:1.對(duì)鏈霉菌3-10發(fā)酵培養(yǎng)基成分進(jìn)行了優(yōu)化。結(jié)果表明:相對(duì)于Mn2+和Zn2+,K+和Mg2+對(duì)鏈霉菌3-10產(chǎn)生AFS的產(chǎn)量有顯著提高,確定了最佳發(fā)酵培養(yǎng)基為: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.對(duì)鏈霉菌3-10發(fā)酵條件的優(yōu)化。將鏈霉菌3-10孢子懸浮液以5%接種于種子培養(yǎng)液搖培24 h后再將種子液以3%的量接入發(fā)酵培養(yǎng)基培養(yǎng)72 h。得到的最佳發(fā)酵條件為:28℃,150 rpm,72h,p H6.5。3.本課題主要研究鏈霉菌3-10在發(fā)酵罐中發(fā)酵過程相關(guān)參數(shù)(攪拌速度、通氣量、碳源和發(fā)酵時(shí)間)對(duì)其AFS產(chǎn)量的影響。所得結(jié)果表明,攪拌速度和通氣量對(duì)AFS產(chǎn)量的影響極為顯著。在AFS產(chǎn)生的過程中,攪拌速度和通氣量可以改變發(fā)酵過程的某些參數(shù),如溶氧量,p H,AFS產(chǎn)量等。當(dāng)發(fā)酵過程中溶氧量改變時(shí),菌絲體的長(zhǎng)勢(shì)和AFS產(chǎn)量也隨之受到一定影響。試驗(yàn)得到的優(yōu)化條件為:通氣量0.75 vvm,攪拌速度200 rpm,溫度28oC,培養(yǎng)時(shí)間72 h。根據(jù)優(yōu)化培養(yǎng)條件得到的AFS產(chǎn)量為4.02 g/L,與三角瓶搖培發(fā)酵72 h得到的AFS產(chǎn)量(4.45 g/L)十分相近。4.將鏈霉菌3-10孢子液、發(fā)酵液、乙酸乙酯提取物分別處理接種病原的油菜,以只接種病原菌P.brassicae為對(duì)照。調(diào)查發(fā)現(xiàn),通過鏈霉菌3-10孢子液、發(fā)酵液、乙酸乙酯提取物處理后的油菜的病害嚴(yán)重程度與只接種P.brassicae的處理比較分別下降了83%,77%和73%,油菜根腫病的發(fā)病率明顯降低,將鏈霉菌3-10與P.brassicae休眠孢子同時(shí)接種油菜后發(fā)現(xiàn),其病害嚴(yán)重程度顯著低于對(duì)照處理,并且在鏈霉菌3-10處理后的發(fā)病的根瘤更小且休眠孢子數(shù)量也顯著降低。以上結(jié)果表明,鏈霉菌3-10可以延遲油菜根腫病的發(fā)生。5.通過平板對(duì)峙實(shí)驗(yàn)發(fā)現(xiàn),鏈霉菌3-10可以抑制黃曲霉的生長(zhǎng),其抑菌率為41%。向PDA培養(yǎng)基中添加2.5%的鏈霉菌3-10發(fā)酵液(CF3-10,2.5%的乙酸乙酯提取物(CE3-10,2%的孢子液,對(duì)黃曲霉生長(zhǎng)的最小抑菌濃度為5μg/10μL。在發(fā)酵液(CF3-10),不同處理的抑菌率范圍從39.59%到90.26%,黃曲霉毒素產(chǎn)量的抑制率范圍從79.37%到93.38%。在花生上,鏈霉菌3-10的發(fā)酵液(CF3-10)和乙酸乙酯提取物(CE3-10)均可以抑制黃曲霉的生長(zhǎng),并且顯著降低黃曲霉毒素的產(chǎn)生。同時(shí)發(fā)現(xiàn),經(jīng)過鏈霉菌3-10發(fā)酵液處理過的黃曲霉菌絲的afl R和afl S表達(dá)量(0.09±0.03)比未處理的(0.343±0.05)有顯著降低,說明鏈霉菌3-10發(fā)酵液處理可以抑制黃曲霉毒素的產(chǎn)生。以上研究結(jié)果表明,優(yōu)化鏈霉菌3-10的培養(yǎng)基及培養(yǎng)條件可以為大規(guī)模發(fā)酵提供理論依據(jù)。鏈霉菌3-10對(duì)油菜根腫病和黃曲霉有良好的防病效果和抑制作用。本研究表明,鏈霉菌3-10具有良好的生防潛力。
[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，

本文編號(hào)：2264306