垃圾堆积点土壤中广谱拮抗菌株的分离鉴定及抑菌特性

Isolation, identification and antibacterial characteristics of broad-spectrum antagonistic strains in soils of garbage accumulation

摘要:

[目的]从积年垃圾堆积点土壤中筛选具有高效抑菌能力的广谱拮抗菌株并研究其抑菌特性，寻找乡镇垃圾堆积点污染治理的新思路。[方法]采用平板稀释法和管碟法从乡镇积年垃圾堆积点的土壤中分离拮抗真菌，并通过形态观察、生理生化特性及分子生物学鉴定分析对拮抗菌株进行鉴定。通过测定抑菌活性物质的极性，选择合适的有机试剂对拮抗真菌的发酵菌体甲醇浸提物及发酵液进行萃取，并利用TLC-Bioautography法对发酵液及发酵菌体中的抑菌活性组分进行分析。使用23种指示菌测定拮抗真菌DAZ-2的发酵菌体乙酸乙酯粗提物的抑菌广谱，测定粗提物在高温、酸碱、紫外和自然光条件下抑菌能力，以及对大肠杆菌和白色念珠球菌的最小抑菌浓度。[结果]分离得到一株传代稳定的广谱拮抗真菌DAZ-2，经过形态观察、生理生化特性测定及ITS序列分析，菌株鉴定为Aspergillus fumigatus。通过极性测定，选择乙酸乙酯对烟曲霉DAZ-2发酵菌体甲醇浸提物及发酵液进行萃取，TLC-Bioautography法分析表明两者中的抑菌活性物质不是同一组分，且存在多组分物质共同作用产生抑菌效果的可能性；菌体浸提物乙酸乙酯萃取部分（200μg/mL）对11种指示菌具有抑菌效果，对大肠杆菌和白色念珠球菌的MIC为7.50μg/mL和15μg/mL，且对高温、酸碱、紫外、自然光照都具有较强的稳定性。[结论]分离得到一株高效抑菌且传代稳定的广谱拮抗菌株烟曲霉DAZ-2，有希望为微生物药物的开发和乡镇垃圾堆积点污染治理提供科学依据。

Abstract:

[Objective] A broad-spectrum antagonistic strain with high antibacterial ability was screened from the garbage heap soil and its antibacterial properties were studied for pollution control of township garbage accumulation sites. [Methods] Antagonistic fungi were isolated from the soil of garbage accumulation sites in townships by plate dilution method and cylinder-plate method, and the antagonistic strains were identified by morphological observation, physiological and biochemical characteristics and molecular biological identification. Suitable organic reagents were selected to extract mycelia and fermentation broth of antagonistic fungi, by measuring the polarity of antibacterial active substances. The TLC-Bioautography method was used to analyze the bacteriostatic active components in the fermentation broth and mycelia. Determination of antibacterial spectrum of ethyl acetate extract of mycelium used 23 indicator bacteria. The high-temperature tolerance, acid-base tolerance, ultraviolet stability, natural light stability of the crude extract and the minimum inhibitory concentration against Escherichia coli and Candida albicans were determined. [Results] A passaging stable broad-spectrum antagonistic fungus DAZ-2 was isolated and identified as Aspergillus fumigatus by morphological observation, physiological and biochemical characteristics, and ITS sequence analysis. Selecting ethyl acetate reagent to extract methanol extract and fermentation broth of Aspergillus fumigatus DAZ-2 fermentation mycelium. The TLC-Bioautography method showed that the antibacterial active substances in the two were not the same component, and the multi-component substances cooperated to produce a bacteriostatic effect. The crude ethyl acetate extract of fermenting mycelium (200 μg/mL) has antibacterial effect on 11 indicator bacteria, and the MIC of Escherichia coli and Candida albicans is 7.50 and 15 μg/mL, and the antibacterial ability was stable, tolerated to temperature, light, ultraviolet, acid, and alkali. [Conclusion] A wide-spectrum antagonistic strain, Aspergillus fumigatus DAZ-2 with high antibacterial activity and stable passage was isolated, which is expected to provide a scientific basis for the development of microbial drugs and the pollution control of garbage accumulation sites in villages.