静电场抑制绿针假单胞菌生长机制的研究

Electrostatic field is a novel non-thermal technology for food preservation, which is widely employed to maintain the freshness of many solid-state foods, such as meat. Data from available literature indicate electric field could be used to inhibit the growth of many microbes, including Pseudomonas. However, the inhibition mechanism has been unknown. Based on the previous works involving the screening and identification of electric field sensitive P. chlororaphis, this study would employ several modern molecular biotechnology, such as fluorescence microscope, flow cytometry, electron microscope, to investigate the mode of electrostatic field inhibiting the growth of microbe. Subsequently, the focus of the study would be concentrated on the key stress response-related proteins and stress proteomics with the aim of elucidating the effects of electrostatic field on the cellular reproduction, the biosynthesis of hot shock proteins, and the modulation of energy metabolism. Expected results would include the characterization of the reduced cell growth rate and viability, increased permeability of cell membrane, the identification of key stress response-related proteins, as well as the analysis of stress proteomics. The results of the study would provide theoretical basis and technical support for the formation of novel food preservation methods, which is based on electrostatic field.

静电场是一种非热食品保鲜技术，它广泛应用肉等固态食品的保鲜贮存过程中。目前研究表明它除了能改善肉制品的品质外，还可抑制包括假单胞菌（Pseudomonas）在内的多种微生物的生长，但尚不清楚它抑制微生物细胞生长的分子机制。基于课题组在筛选静电场敏感型绿针假单胞菌的大量前期工作，本项目将借助多种荧光染料、流式细胞仪和电子显微镜等现代生物学技术和方法，在明确微生物细胞的活力、形态及细胞膜功能变化的基础上，通过比较蛋白质组学，重点研究绿针假单胞菌在静电场中的应激反应关键蛋白（酶）和胁迫蛋白质组，从细胞分裂周期调控、热休克蛋白合成、能量代谢调控等方面对静电场抑制绿针假单胞菌生长的机制进行研究。预期结果将阐明以细胞生长速率减缓、细胞活力下降、细胞膜透性上升等为表征，以应激反应关键蛋白（酶）和胁迫蛋白质组为核心的静电场抑菌机理，为建立以静电场技术为基础的食品保鲜方法提供理论依据和技术支持。

静电场是一种非热食品保藏保鲜技术。它在保持食材原有营养、品质及风味的同时，还能抑制其中微生物的活力，甚至将其杀死。当前的研究主要集中在常温下短时间内（小于1 d）它对几种病原微生物的抑制和灭活作用上，较少研究考察冷藏条件下它对适冷腐败微生物的生存状态及蛋白质组的影响。为此，本研究从冷鲜肉中主要腐败微生物的筛选着手，重点考察静电场持续作用下固态基质上特定腐败微生物（Specific Spoilage Organisms, SSO）的生理生化、代谢活力和细胞形态等的变化情况，进而通过蛋白质学的方法揭示SSO对亚致死静电场的响应机制。通过本研究的开展获得了一株在冷藏条件下生长良好的微生物菌株，鉴定结果表明该分离株是莓实假单胞菌（Pseudomonas fragi）的一个成员，为公认的SSO。在微生物生长的初期，亚致死静电场的持续作用会减缓它的生长速率，但是这种抑制作用会随着电场处理时间的延长而逐渐减弱乃至消失。电场对微生物的逐渐减弱的抑制作用主要表现为3、4和5 d的电场处理组的生物量、胞内脱氢酶水平、胞内ATP的浓度、差异表达蛋白的数量与对照组之间的显著性逐渐消失。另外，对处理组微生物的SEM和TEM观察也表现出类似的变化趋势。但是，对所有样品的胞外β-半乳糖苷酶的活力测定结果却显示细胞膜的透性并未发生明显的变化。这些研究结果表明亚致死静电场会引起微生物胁迫应激反应，进而会导致微生物对该环境胁迫因子的适应。为了进一步提升静电场在冷鲜肉保鲜过程中的作用，还需要对电场发生设备进行改造，避免腐败微生物对亚致死电场耐受性的出现。