反复冻融对枯草芽孢杆菌St-zn-34抑制链格孢菌活性的影响

Effect of repeated freeze-thaw on inhibition of Bacillus subtilis strain St-zn-34 to Alternaria alternata

【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] The effects of repeated freeze-thaw cycles（F-Tc） on cell shape of fermented Bacillus subtilis and the inhibitory activity to Alternaria alternata, the primary infection pathogen of jujube fruit shrunken disease, were determined using an endophytic B. subtilis strain St-zn-34 isolated from winter jujube. [Methods] The fermented broth was freeze-thawed after batch fermentation of the test strain. The population of living bacteria and bacterial endospores were determined by dilution methods of plate counting, and the inhibitory activity to A. alternata was tested by filter paper disks with filtrate of fermented broth. The shape orB. subtilis with different freeze-thaw cycles was observed under environment scanning electron microscope. [Results] Changes of pH, living bacteria, bacterial endospore counts in the fermentation broth and the inhibitory activity of filtrate at different time generally increased first and decreased afterwards. The inhibitory activity at 60 h was higher than other time points. The bacterial cells fermented for 60 h were treated with F-Tc, and the living bacteria count and inhibitory activity decreased gradually when cells orB. subtilis were freeze-thawed for 3 cycles, but it had no significant difference （P〉0.05） after being freeze-thawed for more than 3 times. With the increase of freeze-thawing times, the bacteria cells became smaller than the normal cells, the surface twisted with one or more depressions, and the jelly flowed out under environment scanning electron microscope. The inhibitory activity of filtrate was determined and it showed a broad-spectrum inhibitive capability against twelve species of plant pathogens. The treatment of different temperature and proteinase to the filtrate showed that the temperature below 60℃ did not affect the inhibitive activity and had no significant difference compared with that of the control. However, the inhibition decreased with increasing of the temperature above 80 ℃ and was significantly lower than that of control. The inhibitory activity of filtrate to A. alternata decreased by treatment with proteinase K. [Conclusion] Repeated freeze-thaw cycles could affect the shape of B. subtilis and reduced the inhibitory activity of filtrate to A. alternata.