【作者】 李军；

【导师】 崔海英；

【作者基本信息】 江苏大学 ， 食品科学与工程， 2023， 博士

【摘要】 金黄色葡萄球菌(Staphylococcus aureus,S.aureus)是一种广泛分布于自然环境中的兼性厌氧革兰氏阳性菌。作为最常见的食源性致病菌之一,金黄色葡萄球菌极易在食品的加工、运输、和储藏阶段对食品造成污染,进而引发食物中毒等多种食源性疾病。金黄色葡萄球菌主要致病机制与其产生的多种具有耐高温、耐低p H、耐盐等特性的葡萄球菌肠毒素(staphylococcal enterotoxins,SEs)密切相关,SEs通过干扰肠道神经信号,引发多种食源性疾病。传统的高温灭菌法不仅无法适用于所有食材,还会导致食品营养与风味的破坏。化学抑菌剂和抗生素的大量使用不仅对人体健康造成威胁,还导致了多重耐药菌株的产生。相比之下,毒副作用小、抑菌活性高的天然产物作为新一代食品抑菌剂具有巨大的应用潜力。其中,植物精油凭借其广谱抑菌活性、不易产生耐药性、安全可食用等特点而备受青睐。本研究选用提取自天然植物丁香的丁香精油(clove essential oil,CEO)及其主成分丁香酚作为研究对象,考察了CEO对金黄色葡萄球菌的抑菌效果,初步揭示了CEO对金黄色葡萄球菌的抑菌机制。在此基础上,本研究进一步考察了CEO对金黄色葡萄球菌生物膜的抑制和清除作用,并初步揭示了其作用机制。本研究还基于转录组学技术及磷酸化蛋白质组学技术从基因及蛋白水平上深入探讨了丁香酚对金黄色葡萄球菌及其生物膜的作用机制。同时,本研究还探讨了丁香酚对金黄色葡萄球菌辅助基因调节器(accessory gene regulator,agr)系统的影响,并进一步考察了丁香酚对金黄色葡萄球菌毒力基因表达水平和胞外毒力蛋白分泌量的抑制作用。最后,为验证CEO在食品中的可应用性,本研究以生猪肉、生牛肉和生鸡肉为食品模型,考察了CEO对生肉中的金黄色葡萄球菌及其肠毒素的抑制效果。本研究的主要内容及结论如下:(1)CEO对金黄色葡萄球菌具有显著的抑菌活性,其抑菌机制是多方面的。首先,CEO能够破坏金黄色葡萄球菌的细胞壁并改变细胞膜的通透性,导致碱性磷酸酶(alkaline phosphatase,AKP)、核酸及蛋白质等胞内大分子物质外泄,以致金黄色葡萄球菌细胞破裂死亡。其次,CEO能干扰金黄色葡萄球菌的呼吸代谢,抑制金黄色葡萄球菌糖酵解途径(embden-meyerhof-parnas,EMP)和三羧酸循环(tricarboxylic acid cycle,TCA)中能量的产生,从而抑制细菌的生长。第三,CEO的主成分丁香酚可能与金黄色葡萄球菌的DNA结合,并干扰DNA的复制、转录和修复,从而抑制细菌的生长。(2)CEO能显著抑制和清除金黄色葡萄球菌生物膜。其作用机制主要在于:在生物膜形成初期的黏附阶段,CEO能抑制葡萄球菌辅助调节子A(staphylococcal accessory regulator A,sar A)的表达水平,减少细胞壁锚定蛋白(cell wall-anchored,CWA)的表达量,从而降低菌体的黏附作用;在生物膜聚集阶段,CEO能抑制多糖细胞间黏附素(polysaccharide intercellular adhesion,PIA)的合成并降低胞外蛋白等胞外聚合物(extracellular polymeric substances,EPS)的分泌量,从而抑制金黄色葡萄球菌生物膜形成;在生物膜成熟阶段,CEO能降低金黄色葡萄球菌生物膜膜内细菌的代谢能力,从而抑制金黄色葡萄球菌生物膜的持续发展;在生物膜播散阶段,CEO能通过干扰agr系统,减少酚溶性调控蛋白(phenol-soluble modulins,PSMs)的分泌,从而抑制生物膜的播散过程。(3)CEO能够干扰金黄色葡萄球菌agr系统,显著抑制毒力基因的表达水平。CEO的主成分丁香酚能显著降低Agr A的磷酸化水平,从而干扰agr系统信号的正常转导,致使金黄色葡萄球菌毒力因子表达水平下降。丁香酚还能抑制rec A的表达,从而使得sea的表达水平显著下调。同时,通过对金黄色葡萄球菌溶血活性的检测、金黄色葡萄球菌总肠毒素的检测以及免疫印迹分析等实验,证明CEO对金黄色葡萄球菌α溶血素、肠毒素A和肠毒素B等胞外毒素的分泌具有显著的抑制作用。(4)CEO在肉制品贮藏保鲜方面具有应用潜力。在保持猪肉、牛肉和鸡肉等生肉色泽、质地的同时,CEO不仅对生肉中的金黄色葡萄球菌具有良好的抑菌效果,还能有效抑制生肉中金黄色葡萄球菌肠毒素的分泌。本研究为CEO在食品中的应用提供了实验基础和有用参考。更多还原

【Abstract】 Staphylococcus aureus(S.aureus)is a facultative anaerobic gram-positive bacterium widely distributed in natural environment.As one of the most common foodborne pathogens,S.aureus is very likely to contaminate food during the processing,transportation,and storage stages,resulting in food poisoning and other foodborne diseases.The main pathogenic mechanism of S.aureus is closely related to its production of a variety of staphylococcal enterotoxins(SEs)with high temperature resistance,low p H resistance,salt resistance and other characteristics.SEs cause a variety of foodborne diseases by interfering with intestinal nerve signals.Traditional high temperature sterilization can not be applied to all ingredients,and will lead to the destruction of food nutrition and flavor.The increasing use of chemical antibacterials and antibiotics not only poses a threat to human health,but also leads to the development of multidrug-resistant strains.In contrast,natural products with low toxicity and high antibacterial activity have great application potential as a new generation of food antibacterial agents.Among them,plant essential oils are highly favored due to their broad-spectrum antibacterial activity,low susceptibility to drug resistance,and safe edible properties.In this study,clove essential oil(CEO)extracted from natural plant clove and its main component eugenol were selected as the research object to investigate the antibacterial effect of CEO against S.aureus,and preliminarily revealed the antibacterial mechanism of CEO against S.aureus.On this basis,this study further investigated the inhibition and eradication effect of CEO on S.aureus biofilm,and preliminarily revealed its mechanism.Based on transcriptome technology and phosphorylated proteomics technology,this study further explored the mechanism of eugenol action on S.aureus and its biofilm from the level of gene and protein.In addition,the influence of eugenol on the accessory gene regulator(agr)system of S.aureus was investigated and the inhibitory effect of eugenol on the expression level of virulence gene and extracellular virulence protein secretion of S.aureus was further investigated.Finally,in order to verify the applicability of CEO in food,this study took raw pork,raw beef and raw chicken as food models to investigate the inhibitory effect of CEO on S.aureus and its enterotoxin in raw meat.The main contents and conclusions of this study are as follows:(1)CEO has significant antibacterial activity against S.aureus,and its antibacterial mechanism is multifaceted.First of all,CEO can destroy the cell wall of S.aureus and change the permeability of cell membrane,leading to the leakage of intracellular macromolecules such as alkaline phosphatase(AKP),nucleic acid and protein,resulting in the rupture and death of S.aureus cells.Secondly,CEO can interfere with the respiratory metabolism of S.aureus,inhibit the production of energy in embdenmeyerhof-parnas(EMP)and tricarboxylic acid cycle(TCA)of S.aureus,so as to inhibit the growth of bacteria.Third,eugenol,the principal component of CEO,may bind to DNA of S.aureus and interfere with DNA replication,transcription and repair,thus inhibiting bacterial growth.(2)CEO can significantly inhibit and eradicate S.aureus biofilm.The mechanism of action mainly lies in: in the adhesion stage of the initial formation of biofilm,CEO can inhibit the expression level of staphylococcal accessory regulator A(sar A),reduce the expression of cell wall-anchored(CWA),and thus reduce the cell adhesion;during the biofilm aggregation stage,the CEO can inhibit the synthesis of polysaccharide intercellular adhesion(PIA)and reduce the secretion of extracellular polymeric substances(EPS)such as extracellular proteins,thereby inhibiting the formation of S.aureus biofilm;in the mature stage of biofilm,the CEO can reduce the metabolic capacity of bacteria within the S.aureus biofilm,thereby inhibiting the sustained development of the biofilm;during the biofilm dissemination stage,CEO can inhibit the diffusion process of S.aureus biofilm by interfering with the agr system and reducing the secretion of phenol-soluble modulins(PSMs).(3)CEO can interfere with the agr system of S.aureus and significantly inhibit the expression level of virulence genes.Eugenol,the principal component of CEO,can significantly reduce the phosphorylation level of Agr A,so as to interfere with the normal signal transduction of agr system,resulting in the decreased expression level of S.aureus virulence factor.Eugenol also inhibited the expression of rec A,thus significantly downregulating the expression level of sea.Meanwhile,through the detection of the hemolytic activity of S.aureus,the detection of total enterotoxin of S.aureus and western blot analysis,it was proved that CEO had significant inhibition on the secretion of extracellular toxins such as α-hemolysin,enterotoxin A and enterotoxin B of S.aureus.(4)CEO has application potential in storage and preservation of meat products.While maintaining the color and texture of raw meat such as pork,beef and chicken,CEO not only has a good antibacterial effect on S.aureus in raw meat,but also can effectively inhibit the secretion of S.aureus enterotoxin in raw meat.This study provides experimental basis and useful reference for the application of CEO in food.更多还原