基于细胞膜脂质体相变温度下脉冲电场致死微生物基础研究

Pulsed electric field (PEF) with high field intensity larger than 20 kV/cm, narrow pulse width and low frequency currently is an innovative non-thermal food processing technology and has been one of the research hot points in food sciences all over the world. Cytomembrane is the main part of cell membrane and is also the main sterilization acting site for PEF treatment. However, the mechanism of PEF treatment on the reversible or irreversible polarization of lipid membrane is not clear at the moment, especially the synergistic effect of PEF and critical phase transition temperature (Tm) of phospholipids is rarely reported, which limits the industrial scale application progress of this technique. Therefore, this project aims to investigate the synergetic effect of PEF and thermal treatment on the change of microstructure, fluidity and permeability of phospholipids bilayers membrane of vesicles under critical phase transition temperature in order to explore the influence of PEF on the structure, and physiochemical properties of vesicles. Escherichia coli will be used as the target bacteria. Its phospholipids composition in cell membrane will be modified through changing the culture condition thus its phase transition temperature Tm will be adjusted and controllable at lower temperature. PEF treatment will be applied around this Tm value to achieve efficient sterilization. The study can effectively reduce the pulse electric field strength required for killing microorganisms at lower temperature. Furthermore, the requirement on the PEF treatment and the energy consumption will be decreased. On the other hand, the sterilization treatment at lower temperature, such as around 20-40 ℃， can effectively retain the thermal sensitive components in food. This research is of important scientific theory value and may significantly promote the industrialization progress of non-thermal PEF technology.

高场强、窄脉宽、低频率的高强脉冲电场（PEF）开创了非热处理新途径，是国际科技前沿。脂质膜是细胞膜主体，也是PEF灭菌的主要作用位点。目前关于PEF对脂质膜的作用机制仍然不甚明了，特别对细胞膜磷脂临界相变温度的协同作用研究几乎还是空白，由此限制了其工业化应用进程。本项目采用脂质体模拟细胞膜，研究不同脂质体组成相变温度规律，PEF与温度协同对临界相变温度下脂质膜微观结构、流动性与通透性变化的影响，揭示PEF对脂质体与细胞膜结构性质及可逆极化穿孔的影响及强化调控规律。以大肠杆菌为目标菌通过外界环境和诱导因子刺激，定向修饰其细胞膜磷脂的组成从而改变其相变温度，探索相变温度附近的PEF致死规律，使得PEF杀菌技术的研究从摸索阶段转变到有效调控阶段，并以此为基础提供一种新的协同脉冲电场杀菌的定向修饰微生物细胞膜的方法，具有重大的理论意义和应用价值。

脉冲电场技术（Pulsed electric fields, PEF）作为一种新型的非热加工技术被广泛应用于液态食品灭菌过程中，是国际科技前沿。脂质膜是细胞膜主体，也是PEF灭菌的主要作用位点。目前PEF对脂质膜，特别对细胞膜磷脂临界相变温度的协同及定向修饰作用机理研究仍是空白，限制了其工业化应用进程。. 项目通过脂质体模拟细胞膜及各类细菌应用研究PEF灭菌作用机理，产生了以下结论：1）脂膜电通透性与其流动性密切相关，且脂膜的流动性越大，其电通透性越大，细胞的大小直接影响其在PEF作用下细胞膜的电穿孔效应。2）大肠杆菌对PEF抗性由细胞膜中不饱和脂肪酸含量决定，李斯特菌则通过调节细胞膜中支链脂肪酸anteiso-C15:0来改变膜特性。3）基于相变温度下的沙门氏菌，在较高脉冲电场作用下细胞膜磷脂由凝胶态转变为液晶态，膜流动性增加，提高了脉冲电场的杀灭效果。4）定向修饰协同PEF研究发现，丁香酚、肉桂醛对大肠杆菌的DNA分子有沟槽结合作用，离子稳态失衡，细胞内核酸和蛋白质大分子流出，最终导致细胞死亡。5）而相变温度附近沙门氏菌通过降低细胞膜流动性来应激PEF的压力环境，易引起细胞膜氧化损伤，且作用位点在细胞膜。6）柚皮素诱导脂肪酸生物合成相关基因（rpoS、sigB）的表达下调，导致膜蛋白构象发生显著变化，改变了苯丙氨酸，酪氨酸和色氨酸残基的微环境，从而引起细菌膜电位去极化，细胞膜破裂。7）在杀灭金黄色葡萄球菌方面，单纯香芹酚灭菌效率是0.96 log，PEF灭菌效果是1.23 log，二者协同的灭菌效果可达到2.56 log。8）百里香酚引起金黄色葡萄球菌膜脂肪酸发生变化，从而直链脂肪酸含量增加，甚至导致膜破裂。9）此外，PEF对醋酸杆菌的灭活率可达5.17 log，且相对较高浓度乙醇培养下的细胞更容易受到PEF的影响出现电穿孔现象。. 项目组共发表标注项目资助SCI论文44篇，参与著作编写5章本。申请专利25件（包括国际专利），授权6件。项目经费基本按照合同计划使用完毕，项目圆满完成计划指标。