摘要
ε-聚赖氨酸(ε-PL)是天然存在的一类聚氨基酸,具有对细菌、酵母菌、霉菌的广谱抗性,且安全无毒、水溶性好、热稳定性高。作为逐渐被推广使用的食品防腐剂,其抗菌的分子机制尚未清晰阐明。
本论文通过微生物培养,原子力显微镜(AFM)和透射电子显微镜(TEM)观察,抑制消减杂交文库筛选,实时定量PCR和基因敲除等技术,并根据不同技术选取适合的研究对象,对ε-PL抑制典型致病菌(食源性、医源性)的现象和分子机制进行了深入的研究。最后采用PCR-DGGE技术,评估了口服ε-PL对小鼠肠道微生态菌群的影响。
论文分为六个章节。第一章对食品防腐剂,尤其是天然防腐剂的分布、性质、特性进行了描述,重点对天然微生物抗菌剂ε-PL的理化性质、抗菌活性及机制,应用范围和发展方向进行了系统的阐述。
在第二章中,以Escherichia coli O157:H7为模式微生物,系统探讨了ε-PL对致病菌的抑菌活性和分子机制。结果发现ε-PL拮抗E. coli0157:H7的活性随温度的升高(4℃、25℃和37℃)和处理时间的延长(0-15h)递增；在pH5-7环境中抑菌活性没有显著变化,在pH大于8时活性降低。AFM和TEM观察结果表明,-PL能破坏细胞膜的结构,形成膜穿孔,使胞质呈不均匀分布,细胞内容物泄漏。ε-PL能诱导细菌内活性氧的产生,且其浓度随着ε-PL浓度的增加而升高。RT-qPCR检测结果表明,ε-PL能对细菌产生氧胁迫,损伤细菌DNA,并抑制E.coli0157:H7的侵染毒力。
为进一步研究ε-PL的抑菌分子机理,探讨其对致病菌的基因调控影响,在第三章中以常见感染且难以抑杀的条件致病菌-白色念珠菌(Candida albicans ATCC14053)为研究对象,采用SSH技术对ε-PL抑菌前后的差异基因进行了筛选。RT-qPCR验证结果显示,在ε-PL亚致死浓度胁迫下,C. albicans有10个发生差异表达的基因,表明ε-PL能通过降低糖酵解和脂类代谢相关基因的表达,抑制C. albicans的糖代谢并导致C. albicans中脂类代谢的紊乱。蛋白质合成相关基因GCN1?精氨酸tRNA合成酶及泛素蛋白连接酶Asi3的上调表达则表明,ε-PL的抑菌活性能破坏细胞结构,并促进C. albicans蛋白的降解。
通过基因敲除造成基因缺失是进一步验证ε-PL抑菌分子机制的有效方法。肠炎沙门氏菌是污染食品的主要食源性致病菌,其双组分信号转导系统以特定的方式调控沙门氏菌的毒力,抵抗抗菌肽的抑菌作用。基于沙门氏菌的基因敲除系统有方法成熟,且操作简单的优势,在第四章中,通过敲除S. enteritidis中抵抗抗菌肽的调控系统PhoP-PhoQ和RcsFCDB中的关键基因PhoP和rcsF,比较了ε-PL对S. enteritidis野生株和基因敲除株的相关基因表达影响的差异,探讨其抑制S. enteritidis的分子机制。结果显示S. enteritidisΔphoP对ε-PL的敏感性显著增加,且PhoP-PhoQ所调控的下游基因的表达显著下降,而S. enteritidisΔrcsF在ε-PL中的生长速率较之野生株没有显著差异,表明PhoP-PhoQ调控系统在S. enteritidis抵抗ε-PL的过程中起到关键性的作用,而RcsFCDB调控系统则不能被￡-PL激活。
ε-PL作为食品防腐剂,是否会对机体肠道微生态菌群平衡造成影响,未见报道。在第五章中,采用PCR-DGGE和RT-qPCR相结合的方法,分析了灌胃ε-PL两周过程中小鼠粪便中菌群结构和数量的变化。结果显示,乳杆菌属菌群多样性有所增加。乳杆菌属、拟杆菌属和肠杆菌属的菌体数量所占比例无显著性变化,提示ε-PL小会影响小鼠肠道菌群的平衡,具有一定的食用安全性。
ε-Poly-l-lysine (ε-PL), a natural homo-poly-amino acid, is characterized as being edible, water soluble, biodegradable, stable and low toxic, and widely used as an antibacterial agent for its broad antimicrobial activity against bacteria, yeasts, molds and others. Despite of wide using in food, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated.
In this study, microbial cultivation, atomic force microscopy (AFM), transmission electron microscopy (TEM), real-time quantitative polymerase chain reaction (RT-qPCR), suppression subtractive hybridization (SSH) and gene knockout technology was used to investigate the antibacterial activity of ε-PL against typical foodborne and nosocomial pathogens, in order to understand the antimicrobial mechanism in molecular level. In addition, the effect of ε-PL on the fecal microbiota of mice was analyzed by using denaturing gradient gel electrophoresis (DGGE) and RT-qPCR analysis.
The whole research work is described in six chapters. In Chapter1, the type, character and antimicrobial range of food preservatives (especial from natural sources) were described. As a kind of natural food preservative, the physical and chemical characters, antimicrobial activity and mechanism, and the application range as well development tendency of ε-PL were reviewed.
In Chapter2, E. coli O157:H7(CMCC44828) was chosen as a model to investigate the antimicrobial activity and mechanism of ε-PL against pathogens. The results indicated that the antibacterial effect of ε-PL increased with rise of temperature (4℃,25℃and37℃) and prolonging of the treatment time. The inhibition effect of ε-PL was stable in the range of pH5-7, but decreased in alkaline environments (when pH up to8).
Verification of the destructive effect of ε-PL on cell structure and membrane integrity was performed by AFM and TEM. The surface of treated bacteria was rough and had an uneven shape. The cell membrane of cells exposed to ε-PL showed collapsed, lysed and rough membranes, a cavitated cell shape, and non-integral membranes. Results showed a positive correlation between ROS levels and ε-PL concentration in cells. RT-qPCR results in this work showed that ε-PL induced the up-regulation of bacterial oxidative stress response, elicited the SOS response, and reduced the transcription of virulence genes.
In order to analyze the effects of ε-PL at molecular level and examine its effect on gene expression, C. albicans, a frequent mucosal infection and hardly handling pathogen was studied as another model of pathogen in Chapter3. Suppression subtractive hybridization (SSH) was used to identify differentially expressed genes from in responses to ε-PL. A total of10subtracted clones sharing high homologies with known genes of C. albicans were isolated under the sublethal concentrations of ε-PL. RT-qPCR was used to validate these results, which suggested that ε-PL might inhibit carbohydrates metabolic and lead to lipid metabolism disorders of C. albicans through reduced expression of glycolysis and lipid metabolism associated genes. Increased expression of arginyl tRNA synthetase ARGS, translational activator GCN1and ubiquitin protein ligase Asi3suggested the cell structures of C. albicans were destroyed during ε-PL exposure, even enhancing protein degradation.
Gene absence by gene knockout technology is an effective method to verify the antibacterial mechanism of ε-PL. Salmonellae are the major foodborne pathogens in contaminated food, which uses two-component regulatory systems to regulate virulence and respond to cationic antimicrobial peptide resistance. In Chapter4, to understand the inhibition effect of ε-PL on S. enleritidis in regulatory systems(phoP and rcsF), gene of PhoP-PhoQ and RcsFCDB was knocked out. The results showed antibacterial activity of ε-PL againsted ΔphoP mutant significantly increased. The expression level of downstream genes regulated by PhoP-PhoQ increased. However, the growth rate of ΔrcsF mutant did not change when compared with the wild strain, indicating that PhoP-PhoQ but not RcsFCDB plays an important role in S. enteritidis facing the challenge of ε-PL
ε-PL is an antimicrobial peptide used in many countries as a safe and natural antibacterial food preservative. However, no studies were conducted to investigate its effect on microbial diversity in the intestine. In Chapter5, the effect of ε-PL's administration for two weeks on mice fecal microbiota was analyzed by using DGGE and RT-qPCR. Results indicated that ε-PL significantly increased the microbiota composition of Lactobacillus species. No significant variation was demonstrated in the proportion of Lactobacillus, Bacteroides, and Enterobacteriaceae when compared with the initial microbiota. The results showed that ε-PL may not significantly affect the stabilization of microbial population in the gastrointestinal tract.
引文
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