【作者】 范冰；

【导师】 刘成圣；

【作者基本信息】 中国海洋大学 ， 生物化学与分子生物学， 2011， 硕士

【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。更多还原

【Abstract】 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.更多还原