1株黄曲霉颉颃菌的鉴定及其抑菌活性成分分析

doi:10.16431/j.cnki.1671-7236.2022.05.023

摘要/Abstract

摘要： 【目的】筛选黄曲霉颉颃菌，探究其最佳培养条件及有效抗菌成分，为实际生产中应用生物防治方法抑制黄曲霉污染提供新的理论依据。【方法】通过平板对峙法及分子生物学鉴定方法对待测菌株进行筛选鉴定。以抑菌率作为衡量标准，判断所得黄曲霉颉颃菌的最佳培养温度、培养时间、接种量、转速、pH及装液量。通过硫酸铵沉淀法从颉颃菌无菌发酵上清液中提取蛋白，观测粗提物对黄曲霉菌丝生长的影响。通过离子交换色谱和凝胶过滤色谱进一步纯化粗提蛋白，并进行质谱测序及序列对比分析，筛选鉴定颉颃菌产生的有效抑菌成分。【结果】筛选到1株解淀粉芽孢杆菌B10(菌种保藏号CCTCCNO:M2018353)。最佳培养条件为温度40 ℃，培养时间36 h，接种量3%，转速120 r/min，pH 6.0，装液量30 mL，最大抑菌率可达46.56%。其粗提蛋白能有效抑制黄曲霉生长，破坏菌丝正常形态，并从中筛选出几丁质结合蛋白、壳聚糖酶、葡聚糖酶等8种可能的有效抑菌成分。【结论】本试验分离鉴定出1株抑制黄曲霉正常生长发育的解淀粉芽孢杆菌B10，其产生的多种蛋白成分对黄曲霉有显著抑菌活性。

关键词: 黄曲霉; 解淀粉芽孢杆菌; 筛选鉴定; 抑菌蛋白; 分离纯化

Abstract: 【Objective】 This study was aimed to screen the antagonistic bacteria of Aspergillus flavus,and explore its optimal culture conditions and effective antibacterial components,so as to provide a new theoretical basis for the application of biological control methods to inhibit Aspergillus flavus pollution in practical production.【Method】 The strains to be tested were screened and identified by plate confrontation method and molecular biology identification method.Taking the inhibition rate as the measurement standard,the optimal culture temperature,culture time,inoculation amount,rotating speed,pH and liquid loading of Aspergillus flavus antagonistic bacteria were determined.In addition,the protein was extracted from the sterile fermentation supernatant of antagonistic bacteria by ammonium sulfate precipitation method,and the effect of the crude extract on the growth of Aspergillus flavus was observed.Then,the crude protein was further purified by ion exchange chromatography and gel filtration chromatography,and mass spectrometry sequencing and sequence comparative analysis were performed to screen and identify the effective antibacterial components produced by antagonistic bacteria.【Result】 A strain of Bacillus amyloliquefaciens B10 was screened (strain preservation No.CCTCCNO:M2018353).The optimum culture conditions were as follow:Temperature was 40 ℃,culture time was 36 h,inoculation amount was 3%,rotating speed was 120 r/min,pH was 6.0,liquid volume was 30 mL,and the maximum inhibition rate was 46.56%.In addition,the crude protein could effectively inhibit the growth of Aspergillus flavus and destroy the normal morphology of mycelium.Eight possible effective antibacterial components such as chitin binding protein,chitosanase and glucanase had been selected.【Conclusion】 A strain of Bacillus amyloliquefaciens B10,which inhibited the normal growth and development of Aspergillus flavus,was isolated and identified and its various protein components had significant antibacterial activity against Aspergillus flavus.

Key words: Aspergillus flavus; Bacillus amyloliquefaciens; screening and identification; bacteriostatic protein; isolation and purification

中图分类号:

S852.66

李天溪, 党萌, 龙淼. 1株黄曲霉颉颃菌的鉴定及其抑菌活性成分分析[J]. 中国畜牧兽医, 2022, 49(5): 1828-1839.

LI Tianxi, DANG Meng, LONG Miao. Identification of an Antagonistic Strain of Aspergillus flavus and Preliminary Analysis of Its Antibacterial Components[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1828-1839.

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