氯化钙对食品致腐荧光假单胞菌生物被膜形成的影响

研究氯化钙(CaCl2)对食品腐败株荧光假单胞菌(Pseudomonas fluorescens)生物被膜形成特征的影响。采用结晶紫法、菌体计数、苯酚-硫酸法、共聚焦扫描显微镜和实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)检测Ca2+对荧光假单胞菌的生物被膜形成、多糖分泌、被膜结构及相关基因表达的影响。结果表明,0.1 mmol/L Ca2+刺激荧光假单胞菌生物被膜,随着浓度增加被膜形成增强,其中1 mmol/L促进最明显,而高于10 mmol/L作用减弱,并且Ca2+不影响浮游细菌生长;同时,0.1 mmol/L和1 mmol/L Ca2+对胞外多糖、薄膜和泳动性均呈现促进效果,而高浓度下呈现抑制;共聚焦扫描显微镜观察荧光假单胞菌对照组、1 mmol/L和20 mmol/L Ca2+处理组的成熟生物被膜厚度分别为20.0、40.0μm和25.0μm,其中添加1 mmol/L Ca2+显著增加PF07被膜厚度、菌体和胞外聚合物分泌量,使被膜结构更致密;实时荧光定量PCR检测显示,1 mmol/L Ca2+刺激菌体黏附素lapA、藻多糖alg、鞭毛flgA基因表达量增加3~4倍,并且Ca2+均显著刺激AHLs合成酶luxI基因的表达,提示Ca2+影响生物被膜与群体感应密切相关。可见,食品介质中CaCl2通过影响菌体黏附行为、胞外分泌物、基因表达导致荧光假单胞菌生物被膜形成特征和结构的改变,该研究为复杂的食品介质中腐败菌生物被膜形成和黏附提供依据。

Calcium Chloride Affected Biofilm Formation in Pseudomonas fluorescens as Food Spoilage Bacteria

The effect of calcium chloride on the biofilm forming-ability of Pseudomonas fluorescens was analyzed in this study. The influences of Ca2+ on biofilm formation, planktonic bacterial growth, polysaccharide secretion, biofilm structure and the expression levels of related genes in P. fluorescens were measured by crystal violet method, bacterial counting, phenol-sulfuric acid method, confocal laser scanning microscopy (CLSM) and qPCR, respectively. The results showed that Ca2+ at 0.1 mmol/L promoted biofilm formation of P. fluorescens. Biofilm biomass gradually increased with increasing concentration of Ca2+ up to 1 mmol/L, and then decreased at Ca2+ concentrations higher than 10 mmol/L. Planktonic bacterial growth was not significantly affected by various concentrations of calcium. Similarly, the production of extracellular polysaccharides, the pellicle and swimming motility were enhanced with the addition of Ca2+ at 0.1 and 1 mmol/L while an inhibitory effect was observed at higher concentrations. CLSM observations revealed that the thickness of matured biofilm in the control, 1 mmol/L Ca2+ and 20 mmol/L Ca2+ treated groups was 20.0, 40.0 and 25.0 μm, respectively and that 1 mmol/L Ca2+ resulted in significantly higher biofilm thickness and more adhered cells/extracellular secretions together with a more compact biofilm structure. qPCR revealed that the expression levels of lapA, alg and flgA genes were up-regulated about 3 to 4 folds by 1 mmol/L Ca2+. Furthermore, Ca2+ at low or high concentration positively stimulated the expression level of luxI, an acyl homoserine lactone (AHL) synthetase, suggesting that effect of Ca2+ on the biofilm may be closely related with quorum sensing. Thus, these results indicated that calcium chloride induced changes in the characteristics and structure of biofilm formation in P. fluorescens by affecting the adherence, extracellular secretions and the expression of related genes. This study provides a basis for exploring the biofilm formation and adherence characteristics of spoilage bacteria in complex food matrices.