藤椒精油对腐败解淀粉芽孢杆菌DY1a生物被膜的抑制作用

Antibiofilm effect of Zanthoxylum armatum DC. essential oil against spoilage Bacillus amyloliquefaciens DY1a

摘要:

【背景】芽孢杆菌是豆制品的重要腐败菌，在气液界面形成生物膜，对产品生产带来持续污染。【目的】探讨藤椒精油(Zanthoxylum armatum DC. essential oil，ZA-EO)对腐败解淀粉芽孢杆菌DY1a菌体及生物被膜的抑制作用与机制。【方法】采用气相色谱-质谱(gas chromatography-mass spectrometer，GC-MS)分析藤椒精油主要成分与相对含量，通过二倍稀释法测定藤椒精油对菌株的最低抑菌浓度(minimum inhibitory concentration，MIC)和最低杀菌浓度(minimum bactericidal concentration，MBC)，并分析精油对腐败菌胞外蛋白酶活性、腐败菌生物被膜形成抑制及成熟生物被膜的清除作用，采用扫描电镜结合三维光学显微镜分析生物被膜形貌结构变化，测定生物被膜胞外聚合物(extracellular polymeric substance，EPS)多糖与蛋白质含量变化；并通过细菌运动能力、细胞黏附及自聚集能力、细胞表面疏水性和Zeta电位来初步探讨藤椒精油对生物被膜的抑制机理。【结果】藤椒精油主要含有芳樟醇、D-柠檬烯和桧烯，它们的相对含量分别为52.37%、21.81%和10.94%。藤椒精油对腐败解淀粉芽孢杆菌DY1a的MIC和MBC分别为3.2 µL/mL和12.8 µL/mL。MIC浓度藤椒精油可完全抑制腐败菌胞外蛋白酶的产生及生物被膜的形成，对成熟生物被膜的清除率为25.79%。形貌分析表明，藤椒精油可减小生物被膜厚度和粗糙度，显著降低生物被膜EPS多糖和蛋白质含量，抑制细菌泳动、丛集、黏附及自聚集能力，增强细胞表面疏水性，降低细胞表面电荷。【结论】藤椒精油可抑制腐败解淀粉芽孢杆菌及其生物被膜的形成，并具有一定的生物被膜清除活性。藤椒精油可通过干扰细菌运动和自聚集能力，改变细胞表面特性，降低菌体初始黏附，干扰胞外多糖和蛋白合成而抑制或延缓生物被膜的形成。

Abstract:

[Background] Bacillus spp. are major aerobic bacteria associated with the spoilage of soy products. They form biofilms at the air-liquid interface to bring about constant microbial contamination to soy food processing line. [Objective] To examine the activity of Zanthoxylum armatum DC. essential oil (ZA-EO) against the growth and biofilm formation of a spoilage bacterium Bacillus amyloliquefaciens DY1a isolated from soy products. [Methods] Gas chromatography-mass spectrometry (GC-MS) was used for the chemical characterization of ZA-EO. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ZA-EO against DY1a were determined by the microdilution method. The inhibitory effect on extracellular protease activity and biofilm formation and the eradication of preformed biofilm by ZA-EO were evaluated. Scanning electron microscope coupled with 3D optical microscope was used to observe the surface topography and structure changes of the biofilm treated with ZA-EO. Besides, the content of exopolysaccharide and protein in the extracellular polymeric substance (EPS) were measured. The anti-biofilm mechanism of ZA-EO was explored from swimming and swarming motility, cell adhesion and auto-aggregation, cell surface hydrophobicity, and Zeta potential. [Results] ZA-EO mainly contained linalool (52.37%), D-limonene (21.81%), and sabinene (10.94%). The MIC and MBC of ZA-EO against spoilage B.amyloliquefaciens DY1a were 3.2 µL/mL and 12.8 µL/mL, respectively. Additionally, 3.2 µL/mL ZA-EO inhibited the secretion of extracellular protease and the biofilm formation, and showed the eradication efficiency of 25.79% for the preformed biofilm. Moreover, ZA-EO decreased the thickness and surface roughness of the biofilm as well as the content of exopolysaccharide and protein in EPS. Furthermore, ZA-EO treatment significantly weakened the swimming and swarming motility, inhibited cell adhension and auto-aggregation, enhanced the cell surface hydrophobicity, and reduced the surface charge of DY1a. [Conclusion] ZA-EO had activity against the growth and biofilm formation of B.amyloliquefaciens DY1a and exhibited promising eradication of preformed biofilm. The activity ofZA-EO against the biofilm formation of B.amyloliquefaciens can be attributed to the interruption against cell motility, changing of cell surface characteristics, and inhibition on cell adhension and auto-aggregation during the early stage, as well as the reduction of exopolysaccharide and protein content in EPS.