摘要
壳聚糖(Chitosan, CS)是甲壳素(Chitin, CH)脱乙酰的产物。研究表明,CS具有广谱的抗菌性能,可研制开发成一种安全的食品生物防腐剂,但是较高分子量(Molecular weight, Mw)的CS一般只能溶解于有机酸和部分稀的无机酸中,严重制约了其应用范围。为改善CS的溶解性,众多科技工作者致力于水溶性CS衍生物的研制开发,但就目前水溶性CS衍生物的研究与应用现状看,真正能用于产业化的制备工艺却不多,且存在或产物稳定性差、或收率不高、或反应时间过长、或工艺复杂等缺陷,因此,探究可行的CS水溶性衍生物的制备工艺,并采用合适的手段评价其抑菌性能是当前水溶性CS衍生物研究的热点。
以Mw1800kDa、脱乙酰度(Degree of deacetylation,DD)86%的CS为原料,采用醇酸降解法得到三种不同Mw(1180KDa、260KDa、94KDa)的CS。在降解过程中,随时间延长CS的Mw降低,而DD无明显变化(DD86±0.5%)。DD值及FTIR分析表明,CS在降解过程中,只有糖苷键发生了断裂,而酰胺键并未受影响,原料具有稳定的可靠性。
采用先溶胀后经界面修饰的改进CS乙酸盐(Chiotsan acetate, CA)制备工艺制得到三种不同Mw的CA(CA_a、CA_b、CA_c)。新制的CA为淡黄色粉末,易溶解于水和酸性溶液,碱性溶液中难溶。其1%(w/v)水溶液的OD420约为0.022,pH为4.91～5.01。不同Mw的CA的投入/产出比分别达到5:9 (CA_a)、5:8 (CA_b)、5:8 (CA_c)。采用自创的产品结合度测定方法,测得CA的结合度分别为: 39.6±1.2% (CA_a)、38.5±1.5% (CA_b)、39.6±1.1% (CA_c)。FTIR检测表明,CA是CS与HAc结合的离子化合物,具有–NH3+.RCOO-结构。用铝箔袋热合封装放置于室温下保存180d后,CA的各项指标均接近于新制备的CA。
以三种不同Mw(CSa:1180KDa、CSb:260KDa、CSc:94KDa)的CS为原料,采用优化的CS季铵盐(Hydroxypropyl Trimethyl ammonium chloride chitosan, HACC)制备工艺,制得三种HACC(HACCa、HACCb、HACCc)。FTIR图谱上在1490cm-1处出现了新吸收峰,表明在N上引入了EPTAC基团。HACC是CS与2,3-环氧丙基三甲基氯化铵(Epoxy propyl trimethyl ammonium chloride, EPTAC)共价结合的化合物,具有–NH2?RN+(CH3)3Cl-结构。新制HACC为淡黄色粉末,在酸性、中性和碱性溶液中均可溶,其1%(w/v)的水溶液的pH为6.60～6.71,OD420约为0.09。采用改进工艺后制备的HACC投入/产出比分别是30:59(HACCa)、15:29 (HACCb )、15:29 (HACCc);取代度分别为: 91.3±0.2% (HACCa)、91.7±0.3% (HACCb)、91.6±0.2% (HACCc)。用铝箔袋热合封装,室温保存180d。HACC各项指标均接近新制的HACC。
采用比浊法和平板涂布法分别测定CA和HACC对E.coli和S.aureus生长的抑制作用。在比浊法抑菌作用测定中:相同Mw的CA和HACC,伴随浓度升高,抑菌作用均显示出增强趋势:当CA和HACC浓度低于0.05%(w/v)时,抑菌效果不明显;当浓度达到0.1%(w/v)时,CA和HACC均显示出较强的抑菌作用。经过抑菌作用评估,在相同的浓度下,两种CS衍生物对S.aureu的抑制性均强于对E.coli的抑制性;HACC则显示出比CA更明显的抑菌作用。另外,根据本实验,不同Mw的CA和HACC对E.coli的抑菌作用与MW成正相关,而对S.aureu的抑菌作用则则与MW成负相关。
在平板涂布法抑菌作用测定中:不同浓度、不同Mw的CS制备的CA和HACC对E.coli和S.aureu的抑制作用与比浊法测定分析所得的抑菌作用有相似的特点:当衍生物的浓度达0.1%(w/v)时,两者在固体培养基上对E.coli.和S.aureus.均能产生有效抑制,镜检时视野中无可见菌落。
对比抑菌实验中,将CA、HACC与常见的化学防腐剂山梨酸钾(Potassium Sorbate, PS)进行抑制霉菌生长的对比:CA和HACC在各个浓度条件下均可达到相同浓度的PS类似的抑菌效果。其中,HACC对霉菌的抑制效果强于CA。CA和HACC的水果对比保鲜实验表明:处理相同时间时,浸泡处理的防腐效果优于喷洒,相同分子量和浓度的两种CS衍生物,HACC防腐效果优于CA。CA和HACC作为防腐剂可行性的。有望开发成一种价廉质优的食品生物防腐剂。
Chitosan (CS), as a kind of alkaline polysaccharides, is essentially deacetylated chitin. Chitosan has broad spectra of antibacterial properties with a lot of application in industrials. However, high molecular weight (MW) chitosan can only be dissolved in acid solution rather than in alkaline solution. This characteristic is limiting the application of this material to a great extent. For the aim of improving the dissolubility of chitosan, many kinds of its derivatives have been synthesized and prepared. But from the state-of-art review, there is hardly viable preparation method that can directly put into processing practice. The disadvantages of the current methods are poor stability and yield of the product, complicated process, as well as overlong reaction time. Thus, to improve the preparation method and study the antimicrobial activity of water-soluble chitosan derivative are attracting more attention.
Chitosan (MW 1800 KDa, Degree of deacetylation, DD 86%) is selected as the raw material. Alkyd degradation method has been employed to prepare different kinds of low MW chitosan. The MW of the low MW product is 1180KDa, 260KDa and 94KDa, respectively. Along with the reaction time, the MW drops but the DD value remains the same in the degradation process. The DD of different products are the same, 86±0.5%. The FTIR spectra analysis and DD value determination suggested that, only the glycosidic bond breaks in the degradation process. The amido bonds remain intact after chitosan is degraded. The feature is important that can guarantee the stability of the chitosan as raw material.
The chitosan material is swelling and surface-modified. Then Chitosan acetate (CA) is prepared using the different MW CS above. The newly produced product is faint yellow powder, soluble in water and acid solution and insoluble in alkaline solution. The OD420 value of the product dissolved in water is 0.022 with a pH range from 4.91 to 5.01. In this research, the input-output ratio of different MW CA is 5:9 (CA_a), 5:8(CA_b) and 5:8 (CA_c), respectively. Using the designed substituted ratio test method, the ratio is 39.6±1.2% (CA_a), 38.5±1.5% (CA_b) and 39.6±1.1% (CA_c), respectively. FTIR spectra of CA indicate the binding site is ionic bond forming a–NH3+.RCOO- structure. After sealed in a bag for 180 d, the characteristics of CA are still similar to that of newly produced product.
Three kinds of Hydroxypropyl Trimethyl ammonium chloride chitosan (HACC) of named HACCa、HACCb、HACCc are prepared from the different MW CS using the improved method described above. Bending vibration absorption is found at 1490cm-1 in the FTIR spectra which indicates the Epoxy propyl trimethyl ammonium chloride (EPTAC) has been successfully introduced on forming covalent bonds with a–NH2?RN+(CH3)3Cl- structure. The newly produced HACC is yellowish powder, soluble in neutral, acidic and alkaline solution. The OD420 value of the product dissolved in water is 0.090 with a pH range from 6.60 to 6.71. The input-output ratio of different MW HACC is 30:59(HACCa), 15:29 (HACCb ) and 15:29 (HACCc), respectively. Substituted ratio of the product is 91.3±0.2% (HACCa), 91.7±0.3% (HACCb) and 91.6±0.2% (HACCc). After sealed in a bag for 180 d, the characteristics of HACC are still similar to that of newly produced product.
E. coli and S. aureu have been introduced in the antimicrobial activity determination. Turbidimetric analysis and spread plate method have been employed to maintain the accuracy in the test. In the Turbidimetric analysis, the antimicrobial activity rises along with the concentrations of the CA and HACC. When the concentration is lower than 0.05%(w/v), no obvious antimicrobial activity is found while it reaches 0.1%(w/v), high antimicrobial activity can be achieved. Under the same concentration, the two derivatives have better prohibitive effect on S.aureu than E.coli. Besides, the HACC performs better than CA. The high MW CS derivative is more effective to E.coli while the low MW CS derivative is more effective to S.aureu.
In the spread plate method test, different MW CA and HACC has similarities of the Turbidimetric test results when used at the same concentration: when the concentration reaches 0.1%(w/v), they are both effective to E. coli and S. aureu. No visible colony can be found in the microscopic examination.
For the Contrast bacteriostatic experiment, CA and HACC have been introduced in a comparison with Potassium Sorbate (PS). The result indicate that the antimicrobial activities of CS derivatives are similar to the PS in solution at given concentrations. Besides, the HACC performs better than CA as a mould inhibitor. When applied for the same period, soak processing has better effect than spray treatment. The feasibility of CS derivatives using as biopreservatives is proved by the research. These derivatives have promising and hopeful development as a new type of biopreservative.
引文
[1] Andreas B S, Margit H, Davide G. Thiolated chitosans. European Journal of Pharmaceutics and Biopharmaceuties, 2004, 57 (1): 9～17
[2] Mojca K, Marija B, Peter V, et al. Permeability of pig urinary bladder wall: the effect of chitosan and the role of calcium. European Journal of Pharmaceutical Sciences, 2005, 25(1): 113～121
[3]蒋挺大.甲壳素.北京:化学工业出版社, 2003
[4] Hong K. No, Samuel P. Meyers. Preparation and characterization of chitin and chitosan—A Review. Journal of Aquatic Food Product Technology,1995,2(4): 27～52
[5]金鑫荣,柴平海,张文清.低聚水溶性壳聚糖的制备方法及研究进展[J].化工进展,1998 , (2) 17～21
[6] Rigby G W. US Patent. 2040879, 2072771. 1936
[7]谢雅明.可溶性甲壳质的制造和用途.化学世界. 1983, 4: 118～120
[8]秦朝晖.纯天然高分子化合物中甲壳素及其衍生物.广西轻工业,1993,3: 21～31
[9] Nakamura. T.低聚壳聚糖的制造[ P ] .日本,J P 02 , 11 ,601 , 1990
[10]姚宏亮.南极磷虾虾壳制备甲壳素/壳聚糖的工艺研究.水产科学, 2004, 23(05): 34～36
[11]曾名勇.关于壳聚糖制备条件的研究.水产科学, 1992, 11(10): 9～13
[12]蒋挺大.甲壳素和壳聚糖的制备及副产物的利用.水产科学. 1997, 16(5): 31～33
[13]严锦文,姜嘉娴,姜涌明.从虾壳中提取虾蛋白.饲料研究. 1994, 3: 35～37
[14]易瑞灶.洪专,郑邦锭等.甲壳蛋白微量元素螯合物的研制与应用潜力.中国海洋药物. 1999, 2: 20～24
[15]谢长志,王井,刘俊龙.甲壳素与壳聚糖的改性及应用.材料导报, 2006, 20: 369～371
[16] Cartier N, Domard A, Chanzy H. Single crystals of chitosan. International Journal of Biological Macromolecules. 1990, 12: 289～94
[17] Kubota N, Eguchi Y. Facile preparation of water-soluble N-acetylated chitosan and molecular weight dependence of its water-solubility. Polymer Journal, 1997, 29: 123～127
[18] Aiba S. Studies on chitosan: 3. Evidence for the presence of random and block copolymer structures in partially Nacetylated chitosans. Internatinal Journal of Biological Macromolecues, 1991, 13: 40～54
[19] Rinaudo M, Domard A. Solution properties of chitosan. Chitin and chitosan.: Sources, chemistry, biochemistry, physical properties and applications. London and New York, 1989, 71～86
[20]胡文玉,王姣莲.壳聚糖的性质和用途及其在农业上的应用前景.植物生理学通讯, 1994, 30(4): 294～296
[21] Hirano S, Tobetto K, Hasegawa M, et al. Permeability properties of gels and membranes derived from chitosan. Journal of Biomedial Materials Research, 1980, 14: 477～486
[22]王爱勤,李洪启,俞贤达.不同来源甲壳素和壳聚糖的吸湿性与保湿性.日用化学工业, 1999, 10(5): 22～24
[23]许晨,卢灿辉.壳聚糖季铵化衍生物的吸湿与保湿性.应用化学, 1996, 13(5): 94～95
[24]管云林,邵蕾,姚康德.交联壳聚糖/聚醚半互穿聚合物网络水凝胶中水的状态.应用化学, 1996, 13(4): 52～55
[25]吴国杰,吴炜亮,李金蔓等.聚乙烯醇-壳聚糖水凝胶制备与溶胀行为的研究.广东工业大学学报, 2006, 3(23): 16～20
[26]陈敬华,赵鹏,马莉等.光交联羟丙基壳聚糖纳米凝胶的制备及其释药性能.武汉大学学报(理学版), 2009, 55(05): 549～554
[27]梁凯,杜予民,张婷等.光交联壳聚糖膜的制备及其性能研究.分析科学学报, 2006, 22(05): 501～504
[28]蔡良良,吕国忠,陈敬华等.光交联壳聚糖水凝胶膜治疗深Ⅱ度烧伤创面的临床研究.中华损伤与修复杂志(电子版), 2010, 5(01) : 61～65
[29]吴燕,成国祥,曲波.透明温敏性羧甲基壳聚糖配合物凝胶的制备.高分子材料科学与工程, 2006, 22(3): 223～226
[30]章永望,赵会英,张娜.新型可注射温敏水凝胶的制备及其释药性能.北京化工大学学报, 2007, 34(5): 535～539
[31] Chenite A, Buschmann M, Wang D, et al. Rheological characterization of thermogelling chitosan/glycerol-phosphate solutions. Carbohydrate Polymers, 2001, 46: 39～47
[32]陈欢欢,昝佳,林莹等.壳聚糖/聚乙烯醇温敏水凝胶的制备及性质研究.清华大学学报, 2006, 36(6): 843～846
[33] Hsu S H, Whu S W, Tsai C L, et al. Chitosan as scaff old materials: effects of molecular weight and degree of deacetylation. Journal of Polymer Research, 2004, 11: 141～147
[34] Berrada M, Serreqi A, Dabbarh F, et al. A novel non-toxic camptothecin formulation for cancer chemotherapy. Biomaterials, 2005, 26(14): 2115～2120
[35] Griffon D J, Sedighi M R, Schaeffer D V, et al. Chitosan scaff olds: interconnective pore size and cartilage engineering. Acta Biomater, 2006, 2(3): 313～320
[36]井波,王志强.温敏性壳聚糖水凝胶研究进展.国际骨科学杂志, 2006, 27(2): 115～116
[37] Jia Z S, Shen D F, Xu W L. Synthesis and antibacterial activities of quaternary anlmoniuln salt of chitosan. Carbohydrate Research, 2001, 333: 1～6
[38] Hee D H, Chung K S. A chitosan hydrogel-based cancer drug delivery system exhibits synergistic antitumor effects by combining with a vaccinia viral vaccine. International Journal of Pharmaceutics, 2008, 27～34
[39] Berrada M, Serreqi A. A novel non-toxic camptothecin formulation for cancer chemotherapy. Biomaterials, 2005, 2115～2120
[40]张伟,张子德,马雯等.天然防腐剂-水溶性壳聚糖衍生物.食品工业科技, 1998, 6: 19～20
[41]王爱勤,俞贤达.烷基化壳聚糖衍生物的制备与性能研究.功能高分子学报,1998, 11(1), 83～86
[42] Karrer P, White S M. Chitin. Helvetica chimica acta, 1930, 13: 1105～1113
[43]王周玉,蒋珍菊.水溶N-酰化CTS的合成与表征.四川工业学院学报, 2004, 1: 73～75
[44]林友文,林青,郑景峰等.壳聚糖、羧甲基壳聚糖的降脂及抗氧化作用.中国海洋药物, 2003, 22(3): 16～19
[45]王敏娟,彭湘红,李琼.羧甲基壳聚糖的制备及膜性能测试.应用化工,2001, 30(2): 35～37
[46]李治,刘晓菲,管云林.O-羧甲基壳聚糖抗菌性的研究.日用化学工业,2000, 3: 10～11
[47]叶岚.甲壳质在生产上的应用.福建水产,1987, 3: 76～78
[48]杨洋,曾庆孝,阮征. CTS及其在果蔬保鲜中的应用研究进展.中国南方果树, 2002, 31(5): 71～72
[49] Knorrd D, Teutonico R A. Chitosan immobilization and permeabilization of Amaranth us tricolor cells. Journal of Agricultural and Food Chemistry, 1986, 34: 96～97
[50]陈思行.变废为宝水产废弃物的应用.海洋渔业,1990,5:44～46
[51]张宓,谭天伟.CTS杀虫与壳低聚糖抑菌活性研究.北京化工大学学报, 2003,30(4): 13～17
[52]马永生,邱化玉. CTS类造纸化学品的应用现状及研究进展.中国造纸, 2004, 8: 51～55
[53]马延文,张洪林.无机高分子絮凝剂的开发与应用进展.辽宁城乡环境科技, 2001, 4: 3～6
[54]刘文辉,刘小亚.CTS基生物医用材料及其应用研究进展.功能高分子学报, 2001, 14(4): 493～499
[55]郑连英,朱江峰,孙昆山.壳聚糖的抗菌性能研究.材料科学与工程,2000, 18(2): 22～24
[56]王鸿,沈月新.不同脱乙酰度壳聚糖的抑菌性.上海水产大学学报, 2001, 10(4): 380～382
[57] Tokura S K, Ueno S, Miyazaki, et al. Molecular weight dependent antimicrobial activity of chitosan. Macromolecular Symposia, 1997, 120: 1～9
[58] Issam S T, Adele M G, Adele C P, et al. Chitosan polymer as bioactive coating and film against Aspergillus niger contamination. Journal of Food Science, 2005, 70 (2): 100～104
[59] Coma V, Deschampa A, Gros A M. Bioactive packaging materi-als from edible chitosan polymer antimicrobial activity assessment on dairy related contaminants. Journal of Food Science, 2003, 68 (9): 2788～2792
[60] Liu X F, Guan Y L, Li D Z, et al. Anti-bacterial action of chitosan. Journal of Applied Polymer Science, 2001, 79: 1324～1335
[61]黎军英,李红叶.壳聚糖对桃褐腐病菌的抑菌作用.电子显微学报, 2002, 21(2): 138～140
[62]叶磊,何立千,高天洲等.壳聚糖的抑菌作用及其稳定性研究.北京联合大学学报:自然科学版,2004,18(1): 79～82
[63]孙浩,王红,孙祥煜.壳多糖及其衍生物的复合抑菌作用.大连工业大学学报,2008, 27(04): 301～303
[64]王光华,张燕婉.脱乙酞壳多精醋酸混合液对含氧包装鲜猪肉中细菌生长的影响.食品与发酵工业, 1992, 2: 1～8
[65] No H K, Park N Y, Lee S H, et al. Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. International Journal of Food Microbiology, 2002, 74: 65～72
[66]宋献周,沈月新.不同平均分子量的α-壳聚糖的抑菌作用.上海水产大学学报, 2000, 9(02): 138～141
[67] Jeon Y J, Park P J, Kim S K. Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohydrate Polymers, 2001, 44: 71～76
[68] Liu X F, Guan Y L, Li D Z, et al. Antibacterial action of chitosan. Journal of Applied Polymer Science, 2001, 79: 1324～1335
[69] Yousock S. The antibiotic effect of chitosan on bacterteria of varying cell well composition. Advances in Chitin Science, 1997, (2): 890～896
[70]孙海香,宋保强,夏牧生等.不同相对分子量的壳聚糖对大肠杆菌K88的抑制作用.中国畜牧杂志, 2005, 41(7): 30～31
[71]夏文水,吴焱楠.甲壳低聚糖功能性质.无锡轻工业大学学报,1996, 15(4): 297～302
[72]马鹏鹏,何立千,高天洲.不同脱乙酰度壳聚糖对植物病原细菌的抑制作用研究.北京联合大学学报. 2003, 17(3): 28～31
[73]吴小勇,曾庆孝,阮征等.壳聚糖的抑菌机理及抑菌特性研究进展.中国食品添加剂, 2004, 12(6): 46～49
[74]杨声,冯小强,王廷璞等.壳聚糖对大肠杆菌的抑制作用规律及抗菌机理初探.天然产物研究与开发, 2007, 19(3): 39～43
[75]钟秋平,夏文水.壳聚糖对芒果炭疽病菌、蒂腐病菌的拮抗作用.食品与机械,2005, 21(1): 25～27
[76] Sudharshan N R, Hoover D G, Knorr D. Antibacterial action of chitosan. Food Biotechnology. 1992, 6: 257～272
[77]刘艳如,余萍,郑怡.水溶性壳聚糖的抑菌作用研究.中国海洋药物,2001, 20(2): 42～44
[78]刘楠,陈西广,刘成圣等.壳聚糖抑菌性能研究进展.科学视野,2005, 10(29): 90～92
[79] Ying Y, Yuting W, Hui L. Preparation of new crosslinked chitosan with crown ether and their adsorption for silver ion for antibacterial activities. Carbohydrate polymers, 2003, 53: 425～430
[80] Muzzarelli R A. A chitin. NEW York, Pergamon press, 1977, 134～142
[81]刘振南.壳聚糖对重金属离子吸附的研究.广西化工, 1996, 25(2): 8～11
[82]吴萼,栾兆坤.氨基葡聚糖的应用研究.环境化学, 1985, 4(2): 14～18
[83]钱和生.氨基葡萄糖盐酸盐制备及溶解度研究.中国第一届甲壳质化学学术会议论文摘要集,大连:中国化学会,1996, 10: 57
[84]杨越冬.脱乙酰甲壳质处理含铅废水.环境保护.1995(8): 9～10
[85]段新芳,孙芳利,朱玮等.壳聚糖金属配合物的防腐性能.林业科学, 2004, 40(6): 138～143
[86] Yan L, Xi G Chen, Nan L, et al. Physicochemical characterization and antibacterial property of chitosan acetates. Carbohydrate Polymers, 2007, 67 (2): 227～232
[87] Nagasawa K, Tohira Y, Inoue Y. Reaction between carbohydrates and sulfuric acid. Part I: Depolymerization and sulfation of polysaccharides, Carbohydrate Research, 1971, 18 (1): 95～102
[88] Vongchan P, Sajomsang W, Subyen D, et al. Anticoagulant activity of a sulfated chitosan. Carbohydrate Research, 2002, 337(13): 1239～1242
[89]黎碧娜,王奎兰,吴勇等.壳聚糖磺化衍生物的制备及抑菌性能研究.香精香料化妆品, 2003(1): 16～18
[90]陈凌云,杜予民,肖玲等.羧甲基壳聚糖的取代度及保湿性.应用化学, 2001, 18(1): 5～8
[91]何丽,杨文静,邓婧等.壳聚糖和羧甲基壳聚糖对口腔五种常见致病菌的抑菌活性研究.陕西医学杂志, 2008, 37(7): 809～811
[92]苏云,孙艳辉,刘克忠等.羧甲基壳聚糖的制备及抑菌性能的研究.滁州学院学报, 2009, 11(6): 116～118
[93] Long Z, Hiroshi M, Naotsugu N. Radiation synthesis and characteristic of the hydrogels based on carboxymethylated chitin derivatives. Carbohydrate Polymers, 2003, 51 (2): 169～175
[94]陈凌云,杜予民,刘义.羧甲基壳聚糖的结构与抗菌性能研究.武汉大学学报, 2000, 46(2): 191～194
[95] Muzzarelli R A, Tanfani F. The N-permethylation of chitosan and the preparation of N-trimethyl chitosan iodide. Carbohydrate Polymers, 1985, 5(4): 297～307
[96]刘振儒,赵江霞.水溶性壳聚糖季铵盐的抗菌性能.青岛科技大学学报,2006,22(4): 317～319
[97] Chang W N, Kim Y N, Ko S W. Modification of polyacrylonitrile(PAN) fiber by blending with N-(2-hydroxy) propyl-3-trimethyl-ammonium chitosan chloride. Journal of Applied Polymer Science, 1999, 74 (9): 2258～2265
[98]张艳艳,马启敏,江志华.壳聚糖季铵盐的合成及性质研究.中国海洋大学学报. 2005, 35(3): 459～462
[99]吴迪,蔡伟民.壳聚糖季铵盐的抑菌机理研究.哈尔滨工业大学学报, 2005, 37(7): 1014～1016
[100] Naoji K, Nobuhide T, Takayuki S, Kaori T. A simple preparation of half N-acetylated chitosan highly soluble in water and aqueous organic solvents. Carbohydrate Research, 2000, 324: 268～274
[101] Jeon Y J, Park P J, Kim S W. Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohydrate Polymers , 2001, 44 (2): 71～76
[102]杜兰平,徐崇泉,郭慎满等.壳聚糖的溶解性能及其溶液粘合度的研究.化学与粘合. 1989. 3(4): 213
[103] Chen X G, Zheng L, Wang Z, et al. Molecular affinity and permeability of different molecular weight chitosan membranes. Journal of Agricultural and Food Chemistry, 2002, 50 (21): 5915～5918
[104]宣寒.一点法测定壳聚糖的特性黏度.安徽建筑工业学院学报(自然科学版), 2008, 16(3): 82～84
[105] Sajomsang W, Gonil P, Tantayanon S. Antibacterial activity of quaternary ammonium chitosan containing mono or disaccharide moieties: Preparation and characterization. International Journal of Biological Macromolecules, 2009, 44 (5) 419～427
[106]张惟杰.糖复合物生化研究技术.杭州:浙江大学出版社. 2003
[107]郑立,陈西广,刘晨光等.几丁聚糖降解技术的研究.海洋科学,2000,24(10): 18～20
[108]卢凤琦,曹宗顺,王春香等.低分子量壳聚糖的研制.中国生化药物杂志,1997, 18(4): 178～179
[109]张立彦,曾庆孝.酶法在低壳聚糖制备上的研究及展望.湛江海洋大学学报,2000, 20(4): 72～78
[110]王伟,秦汶.脱乙酰基甲壳素的超声法降解.化学通报,1989,9(6):41～44
[111] Lim S H,Samuel M H. Synthesis and antimicrobial activity of a water-soluble chitosan derivative with a fiber-reactive group. Carbohydrate Research, 2004, 339 (2): 313～31
[112]樊木,肖玲,杜予民.壳聚糖季铵盐分子结构与吸湿保湿性研究.武汉大学学报(理学版), 2003, 49(2): 205～208
[113]蔡伟民,叶筠.壳聚糖季铵盐的合成及其絮凝性能.环境污染与防治,1999, 21(4): 1～4
[114]林友文,林青.羟丙基三甲基氯化铵壳聚糖的制备及其吸湿、保湿性能.应用化学, 2002, 19(4): 351～354
[115]许晨,卢灿辉.壳聚糖季铵盐的合成及结构表征.功能高分子学报, 1997, l0(1): 51～55
[116]张远方,彭湘红,梁燕羽.羧甲基壳聚糖的制备及应用研究.湖北化工,2002, 19(6): 34～35
[117] Sajomsang W, Tantayanon S, Tangpasuthadol V, et al.Quaternization of N-aryl chitosan derivatives: synthesis, characterization, and antibacterial activity. Carbohydrate Research. 2009, 344 (18): 2502～2511
[118] Kenawyel R, Abdel H, Shanshoury E, et al. Biologically active polymers: synthesis and antimicrobial activity of modified glycidyl methacrylate polymers having a quaternary ammonium and phosphonium groups. Controlled Release, 1998, 50 (1-3): 145～152
[119] Muzzarelli R. Tarsi R, Filippini O, et al. Antimicrobial properties of N-carboxybutyl chitosan. Antimicrob. Agents Chemother. 1990, 34(10), 2019～2023
[120] Helander I M, Nurmiabo-Lassila E L, Ahvenainen R, et al. Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. International Journal of Food Microbiol. 2001, 71(2-3), 235～244
[121] Liu H, Du Y, Wang X, et al. Chitosan kills bacteria through cell membrane damage. International Journal of Food Microbiology. 2004, 95(2): 147～155
[122] Je J Y, Kim S K, Agric J. Chitosan derivatives killed bacteria by disrupting the outer and inner membrane. Food Chemistry. 2006, 54(18): 5529～6633
[123] Kim C H, Choi K S. Synthesis and antibacterial activity of quaternized chitosan derivatives having different methylene spacers. Journal of Industrial andEngineering Chemistry. 2002, 8 (1): 71～76
[124] Kim, C H, Choi J W, Chun H J, et al. Synthesis of chitosan derivatives with quaternary ammonium salt and their antibacterial activity. Polymer Bulletin, 1997, 38: 387～393
[125] Badawy M E. Chemical modification of chitosan: Synthesis and biological activity of new heterocyclic chitosan derivatives. Polymer International, 2008, 57: 254～261
[126] Liu J, Tian S P, Meng X H, et al. Effects of chitosan on control of postharvest diseases and physiological responses of tomato fruit. Postharvest Biology and Technology, 2007, 44: 300～306