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【目的】以从冬枣分离的内生枯草芽孢杆菌(Bacillus subtilis St-zn-34)为供试菌株,明确反复冻融对菌体形态及其发酵滤液抑制枣缩果病初侵染病菌(Alternaria alternata)活性的变化。【方法】对供试菌株分批发酵后进行反复冻融,采用梯度稀释计数法、滤纸片法分别测定活菌数量和芽孢含量、发酵滤液抑菌活性,对供试菌株的形态变化进行电镜观察。【结果】发酵培养中pH值、活菌量、芽孢量及抑菌活性随发酵时间的增加均呈先上升后下降的趋势,其中发酵60 h发酵滤液抑菌活性最大,对此时的发酵液反复冻融,冻融3次枯草芽孢杆菌活菌量和发酵滤液抑菌活性依次减少,以后再冻融差异不显著(P>0.05),电镜观察发现随冻融次数增加,菌体变小,表面凹陷、扭曲,胶状物流出。抑菌谱检测发现发酵滤液对12种植物病原菌具有抑菌能力。不同温度和蛋白酶处理发酵滤液表明,低于60℃以下,抑菌活性与对照相比差异不显著;80℃以上抑菌活性随温度上升而下降,与对照相比差异显著;蛋白酶K可降低抑菌活性。【结论】反复冻融影响细菌形态并降低发酵滤液的抑菌活性。
【Objective】 Bacillus subtilis St-zn-34 isolated from Dongzao jujube was used as tested strain, and the effect of morphology and fermentation filtrate of the strain Bacillus subtilis on Alternaria alternata activity. 【Method】 The strains were fermented in batches after repeated freezing and thawing. The bacterial count, spore content and ferment filtrate were determined by gradient dilution counting and filter paper respectively. The morphological changes of the tested strains were observed by electron microscopy . 【Result】 The results showed that the pH value, viable cell count, sporulation volume and antibacterial activity increased firstly and then decreased with the increase of fermentation time in the fermentation. The ferment filtrate had the highest antibacterial activity at 60 h, The number of viable cells after freeze-thaw, freeze-thaw three times and the antibacterial activity of the fermentation filtrate decreased in turn, and the difference was not significant after thawing (P> 0.05). Electron microscopy showed that the number of viable cells decreased with the increase of freeze-thaw cycles, Surface depression, distortion, jelly outflow. Antibacterial spectrum test found that fermentation filtrate on the 12 kinds of plant pathogens have antibacterial ability. The fermentation filtrates at different temperatures and protease treatments showed that the antibacterial activity was not significantly different from the control at temperatures below 60 ℃. The antibacterial activities above 80 ℃ decreased with increasing temperature, and were significantly different from the control Bacteria activity. 【Conclusion】 The freeze-thaw cycles affected the bacterial morphology and the antibacterial activity of the fermentation filtrate.