【作者】 刘慧；

【导师】 杜予民；

【作者基本信息】 武汉大学 ， 环境科学， 2004， 硕士

【摘要】 壳聚糖是由2-乙酰氨基—2-脱氧—D—吡喃葡萄糖和2-氨基-2-脱氧-D-吡喃葡萄糖通过β—(1→4)糖苷键连接的二元线性聚合物。由于它具有抗菌、抗肿瘤、增强免疫力等功能，以及其无毒、可降解和可再生等特性而倍受关注。作为抗菌剂，壳聚糖及其衍生物可以抑制多种细菌、真菌的生长。目前，对壳聚糖及其衍生物的抗菌活性、影响因素及与结构之间的关系研究还不够完整系统，而对抗菌机理也只有一些推论性的报道。本文综述了近年来国内外在壳聚糖及其衍生物的抗菌性、抗菌机理以及壳聚糖与表面活性剂在诸多方面研究应用的进展，并较系统地考察了壳聚糖及其衍生物的结构参数对抗菌活性的影响与抗菌机理。同时，还研究了壳聚糖-表面活性剂复合物的抗菌性与其表面活性，以及壳聚糖与表面活性剂分子间的相容性，探讨分子间非共价健相互作用的本质和规律，从而确立相容性—结构—抗菌性的关系，以及总结出表面活性剂对壳聚糖增强抗菌性的机理与基本规律。 主要研究内容与结论如下： 1．壳聚糖的超声波降解：对4个壳聚糖原料(Mw：154×10~4～44.7×10~4，DD：91.6～61.9％)通过超声波降解，得到28个不同分子量(Mw：98.3×10~4～9.1×10~4)与脱乙酰度(DD：92～55％)的壳聚糖降解产物，并采用GPC、UV、IR、XRD等方法测定了Mw、DD与结晶性，分析了壳聚糖原料Mw与DD对超声波降解速率、降解产物Mw、DD、结晶性的影响。结果表明：Mw大，DD低的壳聚糖降解较快；超声波降解使壳聚糖产物的Mw分布变窄，结晶度提高，且Mw愈大的原料降解后Mw分布愈窄；超声波降解会降低壳聚糖产物的DD，尤其是Mw较大(Mw：154×10~4)、DD较低(DD：72.1％)的壳聚糖原料降解后DD降到了55.1％。 2．壳聚糖及其衍生物的抗菌性：采用固体平板与液体培养方法研究了壳聚糖及其衍生物对革兰氏阳性菌金黄色葡萄球菌、表皮葡萄球菌、革兰氏阴性菌大肠杆菌、绿脓杆菌、真菌白色念珠菌等的最低抑菌浓度(MIC)、抑菌时间(IT)、抑菌率、短期杀菌效果(180min内)，并考察了分子量、脱乙酰度、降解方法、有机酸对壳聚糖抗菌性的影响，实验表明：① 0.5％壳聚糖溶液在5分钟内就杀死了1.2 109 CFU/m】的大肠杆菌，1加分钟内杀死全部的大肠杆菌与部分的金黄色葡萄球菌;它对金黄色葡萄球菌、表皮葡萄球菌、大肠杆菌、绿脓杆菌均有抑制活性，MIC值为0.025~0.05%，但对白色念珠菌无抑制效果;②酶解壳聚糖产物的抗菌性随分子t的降低而有减弱的趋势，尤其是分子量小于10000的壳低聚糖MIC值下降1倍;③超声波降解壳聚糖的产物在分子量小且脱乙酞度高时的抗菌性最强，且脱乙酞度相近的壳聚糖随分子量的降低其抗菌性增强，但分子量相近的壳聚糖的抗菌性并不一定随脱乙酞度的增加而增强，这可能与样品本身的分子结构有关:④通过分析壳聚糖衍生物如梭甲基壳聚糖、壳聚糖季胺盐、壳寡糖、壳聚糖亚硒酸盐、钠米壳聚糖与壳聚糖的季胺盐等的抗菌性认为，壳聚糖的氨基对其抗菌性是至关重要的，能增强壳聚糖分子的电荷密度，促进其与带负电荷的细菌表面的相互作用有利于抗菌性的增强，反之亦然。⑤有机梭酸如苯甲酸、水杨酸、抗坏血酸、柠檬酸与壳聚糖醋酸溶液混合后对细菌的抑菌时间增长，0.05%壳聚糖醋酸溶液+0.01%水杨酸溶液具有最强的抗菌性，pH<5 .8时混合液对细菌有较强的抑制作用。 3.壳聚糖与表面活性剂分子间的相互作用:制备了壳聚糖与两性甜菜碱型 (Betaine)、非离子烷基多昔(APG)、阴离子型十二烷基磺酸盐(sDs)与直链十二烷基苯磺酸盐(LAS)的复合物，并用IR、XRD、热分析(TG、DTG、DSC)等方法表征了其结构，并用粘度法、GPC法测定了复合溶液的GPC谱图与特性粘数(【川)，分析了壳聚糖与表面活性剂的相互作用。结果表明，壳聚糖在pH<6的酸性溶液中，C一2位上的氨基以氨基正离子的形式存在，因而它可以与带有负电荷的表面活性剂如阴离子型与两性甜菜碱型的表面活性剂以静电作用生成复合物:由于壳聚糖分子中含有大量的轻基、氨基功能团，其可以与含有轻基的表面活性剂如烷基多昔形成一OH.二OH一或司OH…NH一形式的分子间氢键，从而形成复合物。复合物的结晶性发生了变化，且易于热分解，这可能是由于复合物中壳聚糖的高级结构如聚集态、分子链构象等发生了变化。在溶液中，壳聚糖上的经基与氨基可以在分子内或分子间生成氢键，从而形成聚集体。GPC与特性粘数的测定结果初步表明，表面活性剂的加入可能使聚集态的壳聚糖趋于解聚，且分子链趋于伸展。 4.壳聚糖一表面活性剂复合物的抗菌性及其表面活性功能:用张力仪在25℃下测定了壳聚糖、APG、Betai的e与它们的复合物的表面张力，考察了壳聚糖与阴离子表面活性剂SDS与LAS、两性甜菜碱型表面活性剂、非离子型表面活性剂APG与烷基乙氧化物(A25E7)、阳离子型表面活性剂澳化十六烷基三甲基钱(CTAB)的复合物对革兰氏阳性菌金黄色葡萄球菌、表皮葡萄球菌、革兰氏阴性菌大肠杆菌、绿脓杆菌、真菌白色念珠菌等的MIC值、IT、短期杀菌效果，并研究了配比、浓度、pH值对其抗菌性的影响。结果表明:壳聚糖与表面活性剂的复合物能够抑制壳聚糖不?更多还原

【Abstract】 Chitosan, a copolymer consisting of ? (1,4)- 2- acetamido - 2 - deoxy- D- glucose and 0-(1,4) -2- amino -2- deoxy -D-glucose units, has attracted considerable interests due to its biological activities such as antimicrobial, antitumor, immune enhancing effects, and its nontoxicity, degradability, renewability. As antimicrobial agent, chitosan and its derivatives can inhibit the growth of many bacteria and fungi. At present, studies about antimicrobial activity of chitosan and its derivates, factors, the tructure-activity relationship and its mechanism are not clear. The resent progresses on the antimicrobial activity of chitosan and its derivatives, its factors, mechanism and application were summarized in this paper. The effects of structure on the antimicrobial activity of chitosan and its derivatives, as well as the mechanism of antimicrobial activity, were systematically studied. And the antimicrobial and surface activity of chitosan combined with surfactants, as well as the interactions between chitosan and surfactant molecule, were also studied, so as to determine compatibility-structure-antimicrobial activity relationship, and find out the mechanism and regularity of the improved antimicrobial activity of chitosan by surfactants.The main contents and conclusions are summarized as below:1. Ultrasonic degradation of chitosan: 28 chitosan samples with different Mw (98.3 ?104-91? 104) and DD (92-55%) were prepared by ultrasonic degradation of 4 raw chitosan materials (Mw: 154 ?104-44.7 ?104, DD: 91.6-61.9%), and their Mw, DD, crytallinities were characterized by GPC, UV, IR, XRD. It showed that chitosan with higher Mw and lower DD showed faster degradation; The Mw polydispersity of treated chitosans were narrower, the higher the Mw of raw chitosan, the narrower the polydispersity of the degraded samples; The crystallinity of ultrasonic treated chitosan was higher; Ultrasonic treatment decreased the DD of chitosan, especially the DD of chitosan with higher Mw (154 ?04) and lower DD (72.1%) decreased to 55.1%.2. Antimicrobial activity of chitosan and its derivatives: The minimum inhibition concentration (MIC), inhibition time (IT), inhibition rate and biocidal effect (within 180 min) of chitosan and its derivatives, against gram-positive bacteria such as 5. aureus, S. epidermidis, gram-negative bacteria such as E. coli, P. aeruginosan, fungi C. albicans, were determined using Agar plate and liquid medium methods. ?0.5% chitosan killed 1.2 log CFU/ml E. coli in 5 min, all E. coli and some Staphylococcus aureus in 120 min , and inhibited the growth of E. coli, Pseudomonas aeruginosa, S.aureus, Staphlococcus epidermidis, but not Candida albicans. Minimum inhibition concentrations (MICs) against bacteria were in the range of 0.025~0.05%; ?the antimicrobial activity of chitosan samples gained from enzyme hydrolysis decreased with decreasing Mw, especially that with Mw lower than 10000, whose MICs could be 1 times lower; (3) the antimicrobial activity of chitosan treated by ultrasonic, with low Mw and high DD, showed strongest inhibition effect; antimicrobial activity of chitosan with similar DD increased with decreasing Mw; but the antimicrobial activity of chitosan with similar Mw was not bound to increase with increasing DD, which was related to the molecular structure of samples; ?through analyzing the antimicrobial activity of chitosan derivatives, it is considered that the amino group of chitosan is critical to its antimicrobial activity, and the modification that increases the positive charge of chitosan molecule or benefits to the interaction between chitosan and negative charged bacterial surface will improve antimicrobial activity, or the case is converse. (5) The antimicrobial activity of chitosan acetate solution combined with other organic acids such as benzole acid, salicylic acid, ascorbic acid and citric acid solutions exhibited longer inhibition time than chitosan acetate solution alone. And the mixed solution of 0.05% chitosan acetate and 0.01% salicylic acid solution showed the best antim更多还原