论文总字数：20849字

摘 要

摘要：乳酸菌是公认的食品安全生产菌株，乳酸菌抗菌肽在代谢过程中核糖体会产生一种具有抑菌活性的蛋白质或多肽，这种抗菌肽在作天然防腐剂上有着很大的市场潜力，抗菌肽的分离纯化一直受到国内外学者的广泛关注。本课题对实验室保存的乳酸菌产抗菌肽Gassericin T进行分离纯化，研究发现，采用硫酸胺沉淀法粗提细菌素后进行抑菌圈实验，发现粗细菌素中有能对指示菌产生抑菌作用的物质；粗细菌素采用Sephadex G-50凝胶层析法纯化，通过AKTA蛋白纯化系统分离之后能够获得纯细菌素，用SDS-PAGE实验结果测定抗菌肽的分子量的大小。

关键词： 乳酸菌 细菌素 新型抗菌肽Gassri T 分离纯化

Isolation, purification and identification of a novel antimicrobial peptide Gassri T

Abstract

Lactic acid bacteria are recognized as safe food production strains. During the metabolism of lactic acid bacteria antimicrobial peptides, the ribosome will produce a protein or peptide with antibacterial activity. Separation and purification has been widely concerned by scholars at home and abroad. In this project, the antibacterial peptide Gassericin T produced by the lactic acid bacteria stored in the laboratory was separated and purified. The study found that the bacteriocin was subjected to the bacteriostatic circle experiment after crude extraction of the bacteriocin by the ammonium sulfate precipitation method, and it was found that the crude bacteriocin could have an inhibitory effect on the indicator bacteria The crude bacteriocin is purified by Sephadex G-50 gel chromatography, and pure bacteriocin can be obtained after separation by AKTA protein purification system. The crude bacteriocins were purified by Sephadex g-50 gel chromatography, and purified by AKTA protein purification system. The molecular weight of antimicrobial peptides was determined by SDS-PAGE.

Key word: lactobacillus; bacteriocins; novel antimicrobial peptide ; separation and purification

目录

摘 要 I

Abstract II

第一章 文献综述 1

1.1 概述 1

1.1.1 乳酸菌抗菌肽的定义 1

1.1.2乳酸菌抗菌肽的分类 1

1.2抗菌肽的杀菌机制 1

1.3乳酸菌抗菌肽的应用 2

1.4 抗菌肽Gassericin T 2

1.5 抗菌肽的筛选方法 2

1.5.1 琼脂扩散法 2

1.5.2 PCR快速筛选 3

1.5.3生物荧光法 3

1.5.4 抗菌肽相关基因筛选法 3

1.6 抗菌肽的分离纯化研究 4

1.6.1盐析法和有机溶剂法 4

1.6.2 吸附-解析法 4

1.6.3 传统的分离方法 4

1.6.4其他分离方法 5

1.7 本课题的研究内容和研究意义 5

第二章 抗菌肽Gassri T的分离纯化及鉴定 7

2.1 实验材料以及试剂 7

2.1.1 实验菌株 7

2.1.2 实验试剂 7

2.1.3 实验设备及仪器 8

2.2 培养基与相关制剂的制备 8

2.2.1培养基 8

2.2.2 BCA蛋白定量试剂盒测定蛋白浓度 8

2.2.3 SDS-PAGE蛋白电泳相关试剂的制备 9

2.3 实验方法 11

2.3.1 发酵上清液的制备 11

2.3.2硫酸胺粗提细菌素 11

2.3.3抑菌圈实验 11

2.3.4 TCA蛋白沉淀法 12

2.3.5 Sephadex G-50柱层析系统纯化细菌素 12

2.3.6 SDS-PAGE 聚丙烯酰胺凝胶电泳 13

第三章 结果与讨论 14

3.1硫酸铵粗提抗菌肽结果 14

3.2抑菌圈实验结果 14

3.3 Sephadex G-50柱层析系统纯化细菌素 15

3.4 SDS-PAGE聚丙烯酰胺凝胶电泳结果 16

第四章 结论与展望 17

4.1 结论 17

4.2 展望 18

参考文献 19

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