摘 要

随着生物材料领域的发展，壳聚糖及其衍生物作为功能性高分子医用材料受到了越来越广泛的关注。羟丙基壳聚糖有生物降解性，在生物体内能通过溶菌酶的作用分解。羟丙基壳聚糖能对受损的生物体诱生特殊细胞，加快创伤愈合，特别是促进愈合张力的增长。乳酸链球菌肽具有安全无毒无害的特点，在食品行业中作为国际许可的天然防腐剂使用。在医药领域，乳酸链球菌肽作为一种天然抗菌剂也逐渐得到研究。本文使用转谷氨酰胺酶催化羟丙基壳聚糖接枝乳酸链球菌肽，制备出一种水溶性良好，具有抗菌性能的壳聚糖衍生物。

本文具体研究内容和结果如下：

1. 使用转谷氨酰胺酶催化羟丙基壳聚糖接枝乳酸链球菌肽。采用单因素实验优化反应条件。实验结果表明，羟丙基壳聚糖-乳酸链球菌肽的最优制备条件是：反应温度40℃，反应时间2h，转谷氨酰胺酶的加入量为10ml(0.1g/ml)，乳酸链球菌肽与羟丙基壳聚糖质量比为1.2。最大取代度为0.448。

2. 研究羟丙基壳聚糖-乳酸链球菌肽的抗氧化能力和还原能力。结果显示，羟丙基壳聚糖-乳酸链球菌肽具有良好的自由基清除能力和还原能力，且自由基清除能力和还原能力的大小随浓度和取代度的增加而增强。抗菌性能试验表明乳酸链球菌肽接枝羟丙基壳聚糖后抗菌性能得到提高。细胞实验数据表明，老鼠胚胎成纤维细胞存活率随着羟丙基壳聚糖-乳酸链球菌肽的浓度和取代度的增加而略有降低。

关键词：羟丙基壳聚糖；乳酸链球菌肽；转谷氨酰胺酶

Abstract

With the development of the field of biological materials, chitosan and its derivatives, as functional medical polymer materials, have attracted more and more attention. Hydroxypropyl chitosan has biodegradability, broken down by lysozyme in the organism. Hydroxypropyl chitosan can induce special cells for damaged cells and accelerate wound healing, especially to promote the healing of tension. Nisin has the characteristics of safe non-toxic harmless, used as an international license in the food industry. In the field of medicine, Nisin is also research as a natural antimicrobial agent gradually. This article uses hydroxypropyl chitosan and Nisin for reaction, with transglutaminase as the catalyst, gets a chitosan derivative with good solubility in water and antibacterial properties.

The contents and results of this paper are as follows:

1. Transglutaminase as a catalyst prepare hydroxypropyl chitosan-Nisin (HPCS-Nisin). Single factor experiments optimize the reaction conditions. The optimal conditions were found to be as follows:The reaction temperature was 40 ℃, the reaction time was 2h, the dosage of the transglutaminase (0.1g/ml) is 10ml and the ratio of the quality of Nisin to HPCS is 1.2. The maximum degree of substitution is 0.448.

2. We also studied on HPCS-Nisin’s antioxidant capacity and reducing power. Antioxidant capacity reduction experiments show that HPCS-Nisin have a good free radical scavenging capacity and reducing power. The scavenging ability and reducing power are increased with the increase of the concentration and the degree of substitution. Antibacterial performance test show that streptococcus peptide after grafting hydroxypropyl chitosan antibacterial performance was improved. Cell experiments datas show that the viability of fibroblast is decreased with the HPCS-Nisin’s concentration and the degree of substitution.

Key words: Hydroxypropyl chitosan；Nisin；Transglutaminase

目 录

摘 要 I

Abstract II

目 录 III

第1章 绪论 1

1.1 壳聚糖的概述 1

1.1.1壳聚糖的来源 1

1.1.2壳聚糖的结构 1

1.1.3壳聚糖的应用 1

1.1.4壳聚糖及其衍生物的抑菌机理 2

1.2 乳酸链球菌肽 2

1.2.1 乳酸链球菌肽的性质 2

1.2.2乳酸链球菌肽的生产 2

1.2.3乳酸链球菌肽的应用 3

1.3 转谷氨酰胺酶 3

1.3.1转谷氨酰胺酶概述 3

1.3.2转谷氨酰胺酶的来源 3

1.3.3转谷氨酰胺酶的作用机理 3

第2章 羟丙基壳聚糖接枝乳酸链球菌肽的制备及表征 5

2.1引言 5

2.2实验材料 5

2.3 实验仪器 6

2.4羟丙基壳聚糖的制备 6

2.5 转谷氨酰胺酶的纯化 6

2.6羟丙基壳聚糖-乳酸链球菌肽的制备 7

2.7样品的结构表征 7

2.8羟丙基壳聚糖-乳酸链球菌肽取代度的测定 7

2.9羟丙基壳聚糖-乳酸链球菌肽抗氧化能力的测定 8

2.9.1羟丙基壳聚糖-乳酸链球菌肽清除DPPH自由基能力的测定 8

2.9.2羟丙基壳聚糖-乳酸链球菌肽清除^.OH自由基能力的测定 8

2.10羟丙基壳聚糖-乳酸链球菌肽抗菌性能测定 9

2.11羟丙基壳聚糖-乳酸链球菌肽还原能力的测定 9

第3章 结果与分析 10

3.1红外分析 10

3.2核磁分析 11

3.3羟丙基壳聚糖-乳酸链球菌肽制备条件的优化 12

3.3.1反应时间对羟丙基壳聚糖-乳酸链球菌肽制备的影响 12

3.3.2反应温度对羟丙基壳聚糖-乳酸链球菌肽制备的影响 13

3.3.3转谷氨酰胺酶用量比对羟丙基壳聚糖-乳酸链球菌肽制备的影响 14

3.3.4乳酸链球菌肽/羟丙基壳聚糖质量比对羟丙基壳聚糖-乳酸链球菌肽制备的影响 15

3.4 羟丙基壳聚糖-乳酸链球菌肽的抗氧化能力 15

3.4.1 浓度对自由基清除能力的影响 16

3.4.2 取代度对自由基清除能力的影响 16

3.5羟丙基壳聚糖-乳酸链球菌肽的还原能力 17

3.6羟丙基壳聚糖-乳酸链球菌肽抗菌性能 18

3.7羟丙基壳聚糖-乳酸链球菌肽细胞毒性实验 18

3.8本章小结 19

第4章 结论 20

参考文献 21

致 谢 23

第1章 绪论

1.1 壳聚糖的概述

1.1.1壳聚糖的来源

壳聚糖（chitosan）又称几丁聚糖、脱乙酰甲壳素、壳多糖。目前壳聚糖主要从虾、蟹壳的甲壳素中用化学方法提取^[1]。甲壳素每年再生量约百亿吨，是一种绿色可再生资源。其主要分布于软体动物的内外骨骼和微生物的细胞壁当中^[2-3]。

1.1.2壳聚糖的结构

甲壳素（chitin）又名甲壳质，几丁质，灰白色，半透明片状固体 ^[4]。结构式如图1.1所示：

图1.1 甲壳素结构式