一株枯草芽孢杆菌降解黄曲霉毒素B1产物分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0179

摘要/Abstract

摘要：

为丰富生物降解黄曲霉毒素B₁资源,研究菌株降解机制及产物毒性。通过液相色谱串联三重四级杆质谱仪（UPLC-MS/MS）及核磁共振氢谱（¹H NMR）检测一株枯草芽孢杆菌WTX1降解黄曲霉毒素B₁（aflatoxin B₁,AFB₁）产物,并将降解产物进行细胞毒理实验。菌株WTX1降解AFB₁机制是破坏AFB₁结构中的呋喃环二氢双键、香兰素内脂环及戊烯酮环结构,形成小分子化合物,达到脱毒效果。细胞毒理实验证实菌株WTX1降解AFB₁产物基本对细胞无毒。菌株WTX1降解机制是破坏AFB₁结构中的致毒致变异结构,产生小分子化合物。WTX1降解AFB₁的产物对细胞无致突变性,无毒性。后期研究菌株降解AFB₁起主要作用的物质,为工业化去除AFB₁毒素提供新思路。

关键词: 黄曲霉毒素B₁, 枯草芽孢杆菌, 产物分析, 细胞毒理

Abstract:

This work aims to enrich the resources of biodegradable aflatoxin B₁ and study the degradation mechanism and toxicity of the product. Triple quadrupole liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）and ¹H NMR were combined to detect the degraded products of aflatoxin B₁（AFB₁）by a strain Bacillus subtilis WTX1,and the cell toxicology tests on the degradation products were performed. The degrading mechanism of AFB₁ by the strain WTX1 was to destroy the structures of furan ring dihydrogen double bond,vanillin aliphatic ring and pentenone ring to form small molecule compounds and to achieve detoxification effect. The cell toxicology test confirmed that the degraded products by strain WTX1 was basically non-toxic to cells. The degradation mechanism of strain WTX1 is to destroy the toxicity and mutation-causing structure of AFB1 and to produce small molecule compounds. The product of degraded AFB₁ by WTX1 is not mutagenic and toxic to cells. The later study of the main role of substances from strain degrading AFB1 would provide a new idea for industrial removal of AFB₁ toxin.

Key words: aflatoxin B₁, Bacillus subtilis, product analysis, cell toxicology

唐璎, 黄佳, 邓展瑞, 杨晓楠. 一株枯草芽孢杆菌降解黄曲霉毒素B₁产物分析[J]. 生物技术通报, 2021, 37(12): 82-90.

TANG Ying, HUANG Jia, DENG Zhan-rui, YANG Xiao-nan. Product Analysis of Degrading Aflatoxin B₁ by a Strain Bacillus subtilis[J]. Biotechnology Bulletin, 2021, 37(12): 82-90.

图/表 8

图1 菌株降解液总离子流图

Fig.1 Total ion chromatogram of strain degradation fluid

图2 AFB1标准物质总离子流图（A）及质谱图（B）

Fig.2 Total ion flow chromatogram（A）and mass spectrum（B）of AFB1 standard material

图3 降解上清液提取质谱图及产物结构图 A:产物1;B:产物2;C:产物3;D:产物4;E:产物5

Fig.3 Mass spectrogram of degradation supernatant extracts and product structure A:product 1;B:product 2;C:product 3;D:product 4;E:product 5

图4 菌株降解液核磁共振氢谱谱图及主要物质结构示意图

Fig.4 1H nuclear magnetic resonance spectrum of strain degradation fluid and schematic diagram of main material structures

图5 预测菌株WTX1降解AFB1机制

Fig. 5 Predictive mechanism of strain WTX1 degrading AFB1

表1 降解产物对细胞L-02细胞生长的影响

Table 1 Effects of degraded products on the growth of L-02 cells

培养时间Incubation time	组别 Group	Viable Cell Density （×10⁶ cells/mL）±RSD	Average date for double tine±RSD/h	Average date for genration number	P
0 d	参照组	0.010±0.021	/	/	/
	试验组	0.010±0.016	/	/	/
	对照组	0.010±0.020	/	/	/
1 d	参照组	0.029±013	21.609±0.74	1.362	>0.05
	试验组	0.022±0.023	21.920±0.98	1.293	>0.05
	对照组	0.013±0.031	45.270±1.20	0.821	<0.05
2 d	参照组	0.063±0.016	22.937±0.72	2.654	>0.05
	试验组	0.053±0.022	22.603±0.46	2.437	>0.05
	对照组	0.008±0.035	57.558±1.03	0.629	<0.05
3 d	参照组	0.131±0.031	21.193±0.59	3.815	>0.05
	试验组	0.116±0.027	22.462±0.91	3.623	>0.05
	对照组	0.003±0.010	68.361±1.31	0.231	<0.05

图6 不同培养基下细胞形态变化

Fig.6 Morphological changes of cells in different media

图7 不同培养基培养细胞活性比较

Fig.7 Comparison of cell activities in different media

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一株枯草芽孢杆菌降解黄曲霉毒素B₁产物分析

Product Analysis of Degrading Aflatoxin B₁ by a Strain Bacillus subtilis

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