抑制耐冷菌的乳酸菌抗菌肽筛选及其结构和抗菌机理研究

发布时间：2018-05-07 01:27

本文选题：耐冷菌 + 乳酸菌抗菌肽　；参考：《哈尔滨工业大学》2016年博士论文

【摘要】：食品腐败是指食品品质发生变化,而无法被人们所食用。引起食品腐败的原因多种多样,其中最主要的原因是由微生物引起的食品腐败。微生物污染不仅可以出现在食品原料的初期,还可能伴随着成品或半成品的加工和运输过程中。人们通常选择低温保藏或者冷冻的方法,用于控制微生物的生长和繁殖,防止微生物引起的食品腐败。但是耐冷菌的发现打破了人们对低温或冷冻可以保证食品质量的认识。乳酸菌抗菌肽作为一种天然防腐剂,在抑制耐冷菌方面具有很大潜力。本研究以乳酸菌为研究对象,旨在筛选出可以抑制假单胞菌的乳酸菌抗菌肽,并对抗菌肽的结构和抑菌机理及其理化性质进行研究,为抑制食品中耐冷菌的研究提供理论依据和基础。通过96-孔琼脂扩散法,对实验室保存的325株乳酸菌进行筛选。获得两株抑制菌耐冷菌的乳酸菌T1和Q7,通过对两株发酵液中和有机酸实验、过氧化氢排除实验、蛋白酶验证实验以及Tricine-SDS-PAGE实验,确定两株菌发酵液中的抑菌物质为抗菌肽。通过对两种抗菌肽性质研究,表明抗菌肽Enterocin T1和Plantaricin Q7可以有效抑制耐冷菌Pseudomonas flurescens AS1.1802,Pseudomonas putida AS1.1819,Pseudomonas aeruginosa CICC21636,Listeria monocytogenes ATCC19111和致病菌Escherichia coli ATCC25922,Salmonella typhimurium ATCC14028,Shigella flexneri ATCC1202 2,Shigella sonnei ATCC25931,Staphylococcus aureus。在不同pH值和温度下,抗菌肽Enterocin T1和Plantaricin Q7都具有稳定的抑菌活性。蛋白酶E、蛋白酶K、胃蛋白酶、胰蛋白酶以及木瓜蛋白酶都可以破坏两种抗菌肽的活性。两种抗菌肽对于不同的化学试剂都有很好的耐受性。通过16Sr DNA和API种属鉴定,确定产抗菌肽的二株菌种属:菌株T1为屎肠球菌(Enterococcus faecium)和菌株Q7植物乳酸杆菌(Lactobacillus plantarum)。以Enterococcus faecium T1和Lactobacillus plantarum Q7的发酵液为原料,依次采用SP-sepharose、CM-FF、Superdex Peptide 10/300 GL对乳酸菌发酵液进行分离纯化,最终获得高纯度的抗菌肽T1和抗菌肽Q7。LC-ESI/MS测定抗菌肽Enterocin T1分子量为4629 Da、抗菌肽Plantaricin Q7的分子量为4624 Da。通过指纹图谱分析两种抗菌肽序列,并未发现与两种抗菌肽含有相同的氨基酸序列。通过对两种抗菌肽的氨基酸成份和序列的分析,抗菌肽Enterocin T1含有41个氨基酸,部分氨基酸序列为:M-A-E-T-F-A-P。抗菌肽Plantaricin Q7含有40个氨基酸,部分氨基酸序列为V-H-A-K-I-K-F-D。采用荧光探针标记法研究抗菌肽Enterocin T1和Plantaricin Q7对单增李斯特菌和荧光假单胞菌的抑菌机理,结果表明抗菌肽Enterocin T1和Plantaricin Q7都是通过吸附于单增李斯特菌细胞膜表面后,插入细胞膜的内部的作用方式抑制单增李斯特菌。抑制荧光假单胞菌的作用方式是以螯合Zn2+后,破坏荧光假单胞菌的外膜,最后作用细胞膜。研究还发现抗菌肽Enterocin T1破坏细胞胞上跨膜电位和pH梯度,抗菌肽Plantaricin Q7只能破坏细胞上的pH梯度。两种抗菌肽都能引起单增李斯特菌和荧光假单胞菌细胞内K+和磷酸盐离子的泄露。选取牛奶、猪肉、带鱼作为食品模型,研究抗菌肽Enterocin T1和Plantaricin Q7在食品防腐中的应用。并以感观评价、菌落总数、TVB-N、pH值为主要指标反应抗菌肽Enterocin T1和Plantaricin Q7的保鲜效果。结果表明两种抗菌肽可以有效的抑制荧光假单胞菌,对不同类型的食品起到良好的保鲜效果,并且保鲜效果随着抗菌肽活力的增加而增强。
[Abstract]:Food corruption means that the quality of food has changed and can not be eaten by people. There are many reasons for food corruption. The main reason is the food corruption caused by microbes. Microbial pollution can not only appear in the early stage of food raw materials, but also may accompany the processing and transportation process of finished products or semi finished products. They usually choose low temperature preservation or cryosurgery to control the growth and reproduction of microorganisms to prevent food corruption caused by microorganisms. But the discovery of cold resistant bacteria breaks people's understanding of the quality of food at low temperature or freezing. In this study, lactic acid bacteria were studied in this study. The aim of this study was to screen out the antibacterial peptide of lactic acid bacteria that could inhibit Pseudomonas, and to study the structure and antibacterial mechanism and physicochemical properties of bacteriocin, and provide theoretical basis and basis for the study of the inhibition of cold resistant bacteria in food. Through the 96- pore agar diffusion method, the 325 strains of milk preserved in the laboratory were stored in the laboratory. Acid bacteria were screened. Two strains of lactic acid bacteria T1 and Q7 were obtained. Through the experiments on two strains of fermentation liquid and organic acid, hydrogen peroxide elimination experiment, protease verification experiment and Tricine-SDS-PAGE experiment, the bacteriostat in two strains of bacteria fermentation liquid was determined as antibacterial peptide. Through the study on the properties of two kinds of antimicrobial peptides, the antibacterial peptide En was shown. Terocin T1 and Plantaricin Q7 can effectively inhibit the Pseudomonas flurescens AS1.1802, Pseudomonas putida AS1.1819, Pseudomonas aeruginosa. C25931, Staphylococcus aureus. at different pH and temperature, antibacterial peptide Enterocin T1 and Plantaricin Q7 have stable antibacterial activity. Protease E, proteinase K, pepsin, trypsin and papain can all destroy the activity of two kinds of antimicrobial peptides. Two kinds of antimicrobial peptides are well tolerated to different chemical reagents. Through the identification of 16Sr DNA and API species, the two strains of antimicrobial peptides were identified: the strain T1 was Enterococcus faecium and the strain Q7 plant lactobacillus (Lactobacillus plantarum). 300 GL was isolated and purified for lactic acid bacteria fermentation broth. Finally, high purity antibacterial peptide T1 and antibacterial peptide Q7.LC-ESI/MS were obtained for the determination of Enterocin T1 molecular weight of 4629 Da. The molecular weight of antibacterial peptide Plantaricin Q7 was 4624 Da. by fingerprint analysis, and the same amino acids were not found with the two antimicrobial peptides. By analyzing the amino acid composition and sequence of two antimicrobial peptides, the antibacterial peptide Enterocin T1 contains 41 amino acids. The sequence of partial amino acids is: M-A-E-T-F-A-P. antibacterial peptide Plantaricin Q7 contains 40 amino acids, and some amino acid sequences are V-H-A-K-I-K-F-D. using fluorescence probe labeling method to study antibacterial peptide Enterocin T1 and Plantaric The bacteriostasis mechanism of in Q7 on mono Lester and Pseudomonas fluorescens showed that the antibacterial peptide Enterocin T1 and Plantaricin Q7 were all inhibited by the insertion of the membrane on the surface of the cell membrane of the single increase of the cell membrane of Lester bacteria. The inhibition of Pseudomonas fluorescens was by chelating Zn2+, after the inhibition of Pseudomonas fluorescens. The outer membrane of the bad Pseudomonas fluorescens acted as the membrane. The study also found that the antibacterial peptide Enterocin T1 destroys the cell transmembrane potential and pH gradient, and the antibacterial peptide Plantaricin Q7 can only destroy the pH gradient on the cell. The two antimicrobial peptides can cause the leakage of K+ and phosphate ions in the cells of Lester and Pseudomonas fluorescens. Milk, pork, and fish were used as food models to study the application of antimicrobial peptide Enterocin T1 and Plantaricin Q7 in food preservation. The effect of antimicrobial peptide Enterocin T1 and Plantaricin Q7 on the fresh-keeping effect of antibacterial peptide Enterocin T1 and Plantaricin Q7 was evaluated by sensory evaluation, total colony total, TVB-N and pH value. The results showed that the two antimicrobial peptides could effectively inhibit Pseudomonas fluorescens. Different types of food had good preservation effect, and the fresh-keeping effect increased with the increase of antibacterial peptide activity.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TS201.3

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