开菲尔粒中微生物对苯并（α）芘的非靶向代谢组学分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1074

摘要/Abstract

摘要：

本文针对新疆传统发酵剂开菲尔粒中微生物对多环芳烃类化合物苯并（α）芘的作用机制这一问题,研究50 mg/L苯并（α）芘胁迫作用下,开菲尔粒微生物在发酵乳中代谢产物发生的特征性变化。采用非靶向代谢组学技术,结合主成分分析和偏小二乘判别分析方法,确认了苯乙酸、苯酚、原儿茶酸、邻苯二甲酸二异丙酯、邻苯二甲酸等为中间代谢特征产物,在KEGG数据库共注释到56条代谢途径,其中富集代谢产物较多、显著性高、与氨基酸代谢有关的关键通路有3条,中间代谢物质在脱氢酶、双加氧酶、脱氢异构酶等一系列酶的作用下将苯环裂解,因此推断开菲尔粒中存在微生物菌株可能通过“萘途径”“邻苯二甲酸途径”“苯丙氨酸途径”3条代谢途径对苯并（α）芘进行降解。研究可以为微生物对苯并（α）芘的作用机制提供一定的理论基础,可以为降低苯丙（a）芘对人体的危害提出可能的生物防治策略,为开菲尔发酵乳制品在食品工业中的合理开发应用提供研究依据。

关键词: 开菲尔粒, 苯并（α）芘, 代谢组学, 代谢途径

Abstract:

In order to understand the mechanism of microbial action on the polycyclic aromatic hydrocarbon benzo（α）pyrene in the traditional fermenter Kefir grains from Xinjiang,and the characteristic changes in the metabolites of Kefir grains in fermented milk under the stress of 50 mg/L benzo（α）pyrene were investigated. Using non-targeted metabonomics technology,combined with principal component analysis and partial minor binary discriminant analysis,it was confirmed that phenylacetic acid,phenol,protocatechuic acid,diisopropyl phthalate and phthalic acid were intermediate metabolic characteristic products. A total of 56 metabolic pathways were annotated in the KEGG database,among which there were 3 key pathways with high enrichment of metabolites,high significance and related to amino acid metabolism. Intermediate metabolites cleaved the benzene ring under the action of a series of enzymes such as dehydrogenase,dioxygenase,and dehydroisomerase. Therefore,it is inferred that the presence of microbial strains in the Kefir grains may have degraded benzo（α）pyrene through the“naphthalene pathway”,the“phthalic acid pathway”and the“phenylalanine pathway”. Thus study may provide a certain theoretical basis for the action mechanism of microorganisms on benzo（α）pyrene,may propose possible biological control strategies for reducing the harms of benzo（α）pyrene to the human body,and may provide research basis for the rational development and application of kefir fermented dairy products in the food industry.

Key words: Kefir grains, benzo（α）pyrene, metabolomics, metabolic pathways

古丽加马力·艾萨, 邢军, 李安, 张瑞. 开菲尔粒中微生物对苯并（α）芘的非靶向代谢组学分析[J]. 生物技术通报, 2022, 38(5): 123-135.

GULJAMAL·Aisa , XING Jun, LI An, ZHANG Rui. Non-targeted Metabolomics Analysis of Benzo（α）pyrene by Microorganisms in Kefir Grains[J]. Biotechnology Bulletin, 2022, 38(5): 123-135.

图/表 11

图1 苯并（α）芘胁迫下不同发酵阶段样品主成分分析 A：绿色5个圈表示在苯并（α）芘胁迫下发酵0 h的5个开菲尔样品;B：红色5个圈表示在苯并（α）芘胁迫下发酵8 h的5个开菲尔样品;C：橙色5个圈表示在苯并（α）芘胁迫下发酵16 h的5个开菲尔样品;D：肉红色5个圈表示在苯并（α）芘胁迫下发酵24 h的5个开菲尔样品;E：黄色5个圈表示在苯并（α）芘胁迫下发酵32 h的5个开菲尔样品

Fig. 1 Principal component analysis of the samples at diff-erent fermentation stages under benzo（α）pyrene stress A：Five green circles indicate five Kefir samples fermented for 0 h under benzo（α）pyrene stress. B：Five red circles indicate five Kefir samples fermented for 8 h under benzo（α）pyrene stress. C：Five orange circles indicate five Kefir samples fermented for 16 h under benzo（α）pyrene stress. D：Five red circles indicate five Kefir samples fermented for 24 h under benzo（α）pyrene stress. E：Yellow five circles indicate five Kefir samples fermented for 32 h under benzo（α）pyrene stress

图2 苯并（α）芘胁迫下不同发酵阶段段样品PLS-DA得分图

Fig. 2 PLS-DA score map of samples at different fermen-tation stages under benzo（α）pyrene stress

图3 OPLS-DA置换检验的检验统计量（Q2）分布以及P值

Fig. 3 Distribution of the test statistic（Q2）of the OPLS-DA substitution test and the P value

图4 苯并（α）芘胁迫下不同发酵阶段样品中代谢物百分比堆积柱形图

Fig. 4 Stacked histogram of the percentages of metabolites in the samples at different fermentation stages under benzo（α）pyrene stress

图5 苯并（α）芘胁迫下不同发酵阶样品扮演生物学角色的代谢物百分比

Fig. 5 Percentage of metabolites playing a biological role in the samples of different fermentation stages under benzo（α）pyrene stress

图6 苯并（α）芘胁迫下不同发酵阶段样品代谢物差异箱式图

Fig.6 Box plot of the differences in metabolites of the samples at different fermentation stages under benzo（α）pyrene stress *P<0.05,**P<0.01,***P<0.001

图7 苯并（α）芘胁迫下不同发酵阶段样品PLS-DA代谢物重要性图

Fig.7 Importance map of PLS-DA metabolites in the samples at different fermentation stages under benzo（α）pyrene stress

图8 苯并（α）芘胁迫下不同发酵阶段样品差异代谢物显著富集的代谢通路（ORA富集分析）

Fig.8 Metabolic pathways with significant enrichment of differential metabolites in the samples at different fermentation stages under benzo（α）pyrene stress（ORA enrichment analysis）

图9 苯并（α）芘胁迫下不同发酵阶段样品差异代谢物显著富集的代谢通路拓扑分析

Fig.9 Topological analysis of metabolic pathways with significant enrichment of different metabolites in the samples at different fermentation stages under benzo（α）pyrene stress

图10 苯丙氨酸途径

Fig. 10 Phenylalanine pathway

图11 邻苯二甲酸途径

Fig. 11 Phthalic acid pathway

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