柠檬酸和乙酸对致腐假单胞菌的抗生物被膜研究

Antibiofilm of Citric Acid and Acetic Acid Against Spoilage Related Pseudomonas

为探究有机酸对食品中致腐假单胞菌抗生物被膜活性,本试验测定了柠檬酸(CA)和乙酸(AA)对荧光及隆德假单胞菌的最小抑菌浓度(MIC)和生物被膜抑制能力,通过结晶紫法、苯酚-硫酸法和显微镜观察等分析亚抑菌浓度有机酸处理对2种假单胞菌的生物被膜形成、胞外多糖(EPS)和被膜结构变化及菌体运动性和蛋白酶活性的影响。结果表明,CA和AA对荧光假单胞菌的MIC分别为2.1和1.0 mg·mL^(-1)。2种有机酸添加浓度为1/4 MIC、1/2 MIC时能显著降低荧光和隆德假单胞菌的生物被膜和EPS分泌量,其中,1/2 MIC CA和1/2 MIC AA处理下生物被膜形成分别减少53.00%和52.19%,EPS分泌量分别降低54.43%和57.85%。光学和共聚焦显微镜(CLSM)观察发现,假单胞菌在亚抑菌浓度有机酸处理下粘附菌量明显降低,被膜变薄,被膜内死细菌增加,其中荧光假单胞菌成熟被膜为50.0μm,而1/2 MIC AA和1/2 MIC CA处理下仅为9.8和10.2μm,且菌体群集和泳动性变得微弱,尤其是AA处理。假单胞菌的蛋白酶活性在弱有机酸作用下显著下降,其酶活性降低22.21%~34.10%。综上表明,亚抑菌浓度CA和AA具有良好的抗假单胞菌生物被膜活性,其中AA的抑制作用更强。本研究为有机酸应用于生鲜食品保鲜提供了理论依据。

To explore the inhibitory activity of organic acid on the biofilm formation by spoilage related Pseudomona from food,minimum inhibitory concentration(MIC)and antibiofilm ability of citric acid and acetic acid against Pseudomonas fluorescence and Pseudomonas lundensis were determined.The effects of the two organic acid at the sub-MIC on biofilm formation,production of exopolysaccharides(EPS),tstructure of biofilm,bacterial motility,and protease activity of Pseudomonas were measured by crystal violet staining,phenol-sulphuric acid,microscopic observation,and folin-phenol methods.The results showed that MIC of citric acid and acetic acid against two Pseudomonas was 2.1 and 1.0 mg·mL^(-1),respectively.The two organic acid at 1/4 MIC and 1/2 MIC significantly reduced biofilm formation and EPS secretion of two Pseudomonas.Additionally,citric acid and acetic acid at 1/2 MIC significantly reduced 53.00%and 52.19%of biofilm biomass,and decreased EPS by 54.43%and 57.85%in P.fluorescens and P.lundescens,respectively.Observations by optical microscopy and CLSM indicated that the adhesion and biofilm thickness of Pseudomonas treated with two organic acid at sub MIC visibly decreased on the slide,and dead bacteria in the biofilm increased.The biofilm thickness of P.fluorescence treated with 1/2 MIC citric acid and acetic acid reduced to 9.8 and 10.2μm,respectively,in contrast with 50.0μm of the control.Furthermore,bacterial swarming and swimming of two Pseudomonas treated by sub-MIC of two organic acid became weak,especially for AA treatment.The protease activity of Pseudomonas inhibited by 22.21%-34.10%.Thus,two organic acid at the sub-MIC exhibited good antibiofilm against P.fluorescens and P.lundensis,especially acetic acid,which could provide theoretical basis for the application of organic acid in raw food preservation.