植物乳杆菌CCFM8724抑制双菌生物膜的成分初探

Identification of metabolites secreted by Lactobacillus plantarum CCFM8724 on inhibiting dual-species biofilm

摘要:

[背景] 植物乳杆菌是一种重要的益生菌，本实验室前期研究表明植物乳杆菌CCFM8724发酵液可抑制变异链球菌和白色念珠菌双菌生物膜，但植物乳杆菌发酵液中起作用的具体物质尚不清楚。[目的] 评价植物乳杆菌CCFM8724发酵液抑菌成分的特性，初步探究其物质基础。[方法] 探索温度、pH等因素对抑菌物质的影响，采用气相色谱-质谱（Gas Chromatography-Mass Spectrometry，GC-MS）联用技术分析植物乳杆菌代谢物的组成，进一步通过有机溶剂萃取、超滤等方法初步分离纯化发酵液中抑制双菌生物膜的成分，并采用液相色谱-质谱（Liquid Chromatography-Mass Spectrometry，LC-MS）联用技术进行鉴定。[结果] 通过多元统计分析，发现植物乳杆菌发酵液的主要差异标志物为有机酸（如苯乳酸、乙酸、羟基己酸和甘油酸等），经过初步提取鉴定并进行功能验证，其中有效成分主要为有机酸和环肽类化合物。[结论] 植物乳杆菌CCFM8724发酵液主要通过多种有机酸和环肽类的协同作用抑制变异链球菌和白色念珠菌生物膜，该研究为植物乳杆菌发酵液进一步的分离纯化和有效成分的生产应用提供理论依据。

关键词:植物乳杆菌;代谢组学;物质基础;有机酸;环肽类化合物

Abstract:

[Background] Lactobacillus plantarum is a kind of important probiotic. Our previous study suggested that L. plantarum CCFM8724 could inhibit the double species biofilm of Streptococcus mutans and Candida albicans. However, the specific contributing substances remain unclear. [Objective] To evaluate the characteristics of the antibacterial components and explore the material basis of L. plantarum CCFM8724. [Methods] The influence of temperature, pH and other factors on metabolites were measured. Gas chromatography-mass spectrometry (GC-MS) was performed to analyze the composition of L. plantarum metabolites. Organic solvent extraction and ultrafiltration were further applied to initially separate and purify the fermentation supernatant. The metabolites inhibiting the double species biofilms were then identified by liquid chromatography-mass spectrometry (LC-MS). [Results] Through multivariate statistical analysis, the main difference metabolites of L. plantarum fermentation supernatant were organic acids (such as phenyllactic acid, acetic acid, hydroxyhexanoic acid and glyceric acid, etc.). After preliminary extraction, identification and function verification, the active ingredients were mainly organic acids and cyclic peptide compounds. [Conclusion] The supernatant of L. plantarum CCFM8724 can inhibit the mixed-biofilm through the synergistic effect of organic acids and cyclic peptides. This study provides a theoretical basis for the further production and application of L. plantarum effective components.

Key words:Lactobacillus plantarum;metabolism;material basis;organic acids;cyclic peptides