茶多酚结合热处理对枯草杆菌芽孢细胞结构与能量代谢的影响

Effect of Tea Polyphenols Combined with Heat Treatment on the Cellular Structure and Energy Metabolism of Bacillus subtilis

使用不同温度结合0.8 g/L和1.6 g/L茶多酚处理枯草杆菌芽孢,采用平板计数法测定处理对枯草杆菌芽孢的杀灭效果;由核酸泄漏量、电导率与扫描电子显微镜研究芽孢结构的变化;通过测定三磷酸腺苷(ATP)酶、琥珀酸脱氢酶(SDH)及苹果酸脱氢酶(MDH)活力分析对枯草杆菌芽孢能量代谢的影响。结果表明:茶多酚结合热对芽孢有明显的协同杀灭作用,且随浓度和温度的增加,其杀灭效果更好。1.6 g/L茶多酚结合100℃处理芽孢后,菌落总数下降2.45 lg(CFU/mL),核酸泄露量及电导率显著增加(P<0.05),OD_(260nm)从0.05升至1.08,电导率从6.61 ms/cm增至71.46 ms/cm,说明该处理可以破坏枯草杆菌芽孢的细胞膜。扫描电子显微镜观察发现,经茶多酚结合热处理的枯草杆菌芽孢菌体形态发生皱缩、凹陷,其ATP、SDH与MDH活力均显著降低(P<0.05),具体表现为:ATP酶活力下降0.16 U/mg,SDH酶活力下降5.3 U/mg,MDH酶活力下降18.7 U/mg,说明该方法能破坏枯草杆菌芽孢的能量代谢,破坏枯草杆菌芽孢的细胞结构,同时抑制呼吸代谢途径中相关酶的活性,影响菌体正常生长,最终导致其死亡。

In this paper,Bacillus subtilis spores were treated with 0.8 g/L and 1.6 g/L tea polyphenols at different temperatures,and the inactivating effect of tea polyphenols combined with heat treatments on Bacillus subtilis spores was determined by plate counting method.The effect of tea polyphenol combined with heat on the energy metabolism of Bacillus subtilis was analyzed by measuring the activity of adenosine triphosphate(ATP)enzymes and tricarboxylic acid metabolizing enzymes(succinate dehydrogenase(SDH)and malate dehydrogenase(MDH)).The results showed that tea polyphenols combined with heat had a significant synergistic effect on Bacillus subtilis spores,and the inactivating effectwas more pronounced as the concentration and temperature increased.After 1.6 g/L tea polyphenol combined with 100°C treatment of spores,the total number of colonies decreased by 2.45 lg(CFU/mL),nucleic acid leakage and conductivity increased significantly(P<0.05),OD_(260nm) increased from 0.05 to 1.08,and conductivity increased from 6.61 ms/cm to 71.46 ms/cm,indicating that the treatment could disrupt the cell membrane of Bacillus subtilis spores.Scanning electron microscopy revealed that the morphology of Bacillus subtilis spores subjected to the tea polyphenol combined with heat treatment was wrinkled and depressed,and their ATP,SDH and MDH activities were significantly reduced(P<0.05),as shown by a decrease of 0.16 U/mg in ATPase activity,5.3 U/mg in SDH enzyme activity and 18.7 U/mg in MDH enzyme activity,indicating that the tea polyphenol combined with heat treatment could well disrupt the energy metabolism of Bacillus subtilis spores.In conclusion,tea polyphenol combined with heat treatment can disrupt the cell structure of Bacillus subtilis spores,and at the same time inhibit the activity of related enzymes in the respiratory metabolic pathway,which affects the normal growth of the bacterium and eventually leads to its death.