顺反式肉桂醛对肉源隆德假单胞菌生物被膜和致腐性的抑制作用

Inhibitory activity of trans-cinnamaldehyde and cinnamaldehyde on biofilm formation and spoilage of Pseudomonas lundensis

摘要:

[目的] 为比较反式和顺式肉桂醛对肉源假单胞菌生物被膜和致腐性的影响。[方法] 通过平板计数测定两种肉桂醛对隆德假单胞菌的最小抑菌浓度（MIC），结晶紫法、珠涡流法、激光共聚焦显微镜观察、福林法等检测亚抑菌浓度肉桂醛处理下隆德假单胞菌生物被膜形成、运动性和胞外酶活性变化。荧光定量RT-PCR检测肉桂醛对隆德假单胞菌粘附lapA、鞭毛fliC、蛋白酶aprX和脂肪酶lip基因表达量的影响。[结果] 反式和顺式肉桂醛对隆德假单胞菌的MIC分别为200μg/mL和225 μg/mL，1/8 MIC、1/4 MIC、1/2 MIC亚抑菌浓度肉桂醛显著降低隆德假单胞菌生物被膜结晶紫和粘附性，其中1/2 MIC反式和顺式肉桂醛处理下被膜分别减少60.27%和52.05%，菌体粘附降低56.35%和61.10%。亚抑菌浓度肉桂醛显著减少被膜厚度，反式肉桂醛还能显著杀灭被膜菌。且肉桂醛能显著抑制菌体的泳动性，反式肉桂醛对生物被膜和泳动性的抑制效果更强。肉桂醛还能抑制隆德假单胞菌蛋白酶和脂肪酶活性，其中1/2 MIC反式和顺式肉桂醛处理下菌体蛋白酶分别减少61.90%和76.19%，脂肪酶降低40.17%和47.01%。且发现肉桂醛显著降低lapA、fliC、aprX和lip表达量，其中1/2 MIC反式和顺式肉桂醛分别降低4个基因表达量至对照组的0.05-0.16和0.02-0.12倍。[结论] 两种亚抑菌浓度肉桂醛异构体显著抑制隆德假单胞菌生物被膜和致腐性，其中反式肉桂醛对生物被膜抑制较强，而顺式肉桂醛更有效地降低致腐酶活性，其与肉桂醛下调相应基因表达密切相关。

Abstract:

[Objective] To investigate the inhibitory activity of trans-cinnamaldehyde (TC) and cinnamaldehyde (CD) on spoilage and biofilm formation of Pseudomonas isolated from spoiled beef. [Methods] The minimal inhibitory concentration (MIC) on Pseudomonas lundensis was measured by plate counting. Effects of sub-MIC two cinnamaldehyde on the biofilm formation and extracellular enzymes were evaluated by crystal violet staining, bead vortexing, confocal laser scanning microscope, folin method. The effects of sub-MIC TC and CD on the gene expressions of lapA, fliC, aprX and lip were detected by real-time quantitative PCR. [Results] MIC of TC and CD was 200 μg/mL and 225 μg/mL, respectively. Cinnamaldehyde at sub-inhibitory concentration of 1/8 MIC, 1/4 MIC, 1/2 MIC decreased significantly the biofilm biomass and adhesion of P. lundensis. Compared to the control, the biofilm of P. lundensis decreased by 60.27% and 52.05%, when supplemented with exogenous 1/2 MIC of TC or CD, respectively. The 1/2 MIC of TC and CD decreased the bacterial adhesion by 56.35% and 61.10%, respectively. The treatment by TC or CD resulted in the thinner biofilm thickness of P. lundensi, even partial biofilm cell was killed by TC. Two cinnamaldehyde strongly inhibited swimming motility, and the inhibitory rates were 58.08% and 42.29% by 1/2 MIC of TC and CD, respectively. Furthermore, protease of P. lundensis decreased by 61.90% and 76.19%, while lipolytic activity inhibited by 40.17% and 47.01% in the presence of 1/2 MIC of TC and CD, respectively, indicating that sub-MIC TC or CD markedly repressed the two extracellular enzymes activity. Cinnamaldehyde caused greatly down-regulated gene expressions of lapA, fliC, aprX and lip (P<0.05), except no significant difference in lapA gene treated by 1/8 MIC of CD. The expressions of lapA, fliC, aprX and lip decreased to 0.05-0.16 and 0.02-0.12 fold in P. lundensis treated with 1/2 MIC of TC and CD (P<0.05), respectively. [Conclusion] Two cinnamaldehyde isomers at sub-MIC significantly repressed biofilm formation and spoilage of P. lundensis. TC had a stronger inhibitory effect on biofilm formation, while CD was more effective in decrease of extracellular enzymes, which was closely associated with down-regulation of expression of related genes by cinnamaldehyde.