中强度脉冲电场对大肠杆菌的灭活效果及机理分析

Inactivation Effect of Moderate-intensity Pulsed Electric Field on Escherichia coli and the Underlying Mechanism

摘要:

脉冲电场作为一种非热灭菌方法，已被广泛用于控制微生物污染。该研究探讨了中强度脉冲电场（Moderate Intensity Pulsed Electric Fields, MIPEF）在不同介质溶液（氯化钠、PBS和蛋白胨）、氯化钠质量分数（质量分数0.85%、0.10%、0.20%和0.30%氯化钠）、电场强度（0.16、0.20和0.24 kV/cm）、处理时间（20、40、60、80和100 min）和菌液密度（103、105、107和109）的条件下对大肠杆菌的灭活效果。研究考察了经MIPEF处理的大肠杆菌的形态变化、胞内大分子物质变化和胞内酶活性。在不同条件处理下发现，MIPEF对大肠杆菌的灭活效果不同。处理时间与MIPEF的灭活效果呈正相关，处理100 min时，微生物数减少了3.08 lg CFU/mL，且温差为2.10 ℃。在不同介质溶液中，MIPEF对大肠杆菌的杀菌效果不同。随着氯化钠溶液质量分数增大，MIPEF对大肠杆菌的灭活效果显著降低。菌液密度越小，MIPEF的杀菌效果越强。场发射扫描电子显微镜（Field Emission Scanning Electron Microscope, FESEM）和生物透射电镜（Transmission Electron Microscopy, TEM）表明，MIPEF处理后细胞的完整性遭到破坏。经MIPEF处理后，包括蛋白质和DNA内的大分子物质明显减少。此外，经MIPEF处理的100 min的样本相比于对照组样本中，ATP酶（Adenosine Triphosphatase, ATPase）、琥珀酸脱氢酶（Succinate Dehydrogenase，SDH）和丙酮酸激酶（Pyruvate Kinase, PK）活性显著降低（P<0.05），钠钾泵（Sodium-Potassium Pump, Na+ K+-ATPase）、钙泵（Calcium Pump, Ca2+Mg2+-ATPase）和SDH 活性分别下降了34.21%、32.26%和86.93%，PK活性降低了88.93 U/g prot。该研究可以为非热杀菌技术在食品工业中的应用及MIPEF杀菌机理的挖掘提供参考。

Abstract:

As a non-thermal sterilization method, pulsed electric field has been widely studied for its ability to control microbial contamination. The inactivation effects of moderate-intensity pulsed electric field (MIPEF) on Escherichia coli were clarified by using different media solutions (sodium chloride, phosphate-buffered saline, and peptone), mass fractions of sodium chloride (0.85%, 0.10%, 0.20%, and 0.30%), electric field intensities (0.16, 0.20, and 0.24 kV/cm), treatment times (20, 40, 60, 80, and 100 min), and bacterial densities (103, 105, 107, and 109). The morphological changes, intracellular macromolecules, and intracellular enzyme activities of E. coli treated with MIPEF were examined. The inactivation effect of MIPEF on E. coli differed under different conditions. The treatment time was positively correlated with the inactivation effect of MIPEF, and the microbial count was reduced by 3.08 lg CFU/mL after 100 min of treatment, with a temperature differential of 2.10 ℃. The bactericidal effect of MIPEF on E. coli was different in various media solutions. The inactivation effect of MIPEF on E. coli decreased significantly as the mass fraction of sodium chloride increased. MIPEF had a greater bactericidal effect when the bacterial density was lower. The results of field emission scanning electron microscopy and transmission electron microscopy demonstrated that cell integrity was compromised following MIPEF treatment. Macromolecular constituents, including proteins and DNA, were significantly reduced following MIPEF treatment. Furthermore, the activities of adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), and pyruvate kinase (PK) were significantly diminished (P<0.05) in the samples treated with MIPEF for 100 min compared with those in control samples. Furthermore, the activities of Na+K+-ATPase, Ca2+Mg2+-ATPase, and SDH were reduced by 34.21%, 32.26%, and 86.93%, respectively, whereas the activity of PK was reduced by 88.93 U/g prot. This study provides a reference for application of non-thermal sterilization technology in the food industry and clarifies the sterilization mechanism of MIPEF.