【作者】 魏新林；

【导师】 夏文水；

【作者基本信息】 江南大学 ， 食品科学， 2004， 博士

【摘要】 本论文在研究甲壳低聚糖理化和功能性质的基础上,着重研究了甲壳低聚糖(COS)的分级分离、活性筛选、定量、结构鉴定和免疫活性及机理,对于促进甲壳素壳聚糖的研究、推动甲壳低聚糖免疫功能食品和抗癌药物的开发具有重要的学术意义和很大的应用价值。 对甲壳低聚糖(COS)的理化和功能性质进行了研究,结果表明:平均相对分子质量为1389、脱乙酰度(DD)为91%的COS在pH1～13的范围内都有优良的水溶性,当pH>5.1时,COS在水中的溶解性略有下降,COS在乙醇中的溶解性随着乙醇浓度的增加而下降;当pH<9.7时COS显示阳离子性质;当温度<80℃,COS溶液较稳定,在pH2～5.5下COS溶液的热稳定性较好,DSC分析COS热分解温度为211～219℃;COS有优良的吸湿保湿性;COS对淀粉老化具有显著的抑制作用。 用膜分离、乙醇分级沉淀和柱色谱相结合的分离手段,对COS进行分级分离,并以体外淋巴细胞增殖和体内巨噬细胞吞噬、半数溶血实验相结合的方法对分离产物跟踪筛选免疫活性组分。首先从超滤截留COS(UFR-COS)和纳滤截留COS(NFR-COS)中筛选出免疫活性组分—NFR-COS,其次从NFR-COS的75%醇析沉淀(COS-75SE)、87.5%醇析沉淀(COS-87.5SE)、87.5%醇沉上清组分(COS-87.5SU)中,筛选出平均分子量为766的免疫活性组分COS-87.5SU;然后通过离子交换树脂CM-Sephadex C-25,将COS-87.5SU采用0.01MpH4.8的缓冲液洗脱后,用0—1.2N的NaCl溶液梯度洗脱,分离得到组分COS1、COS2、COS3、COS4、COS5、COS6;同样方法筛选得到高免疫活性组分COS6和低免疫活性组分COS4和COS5;最后,HPLC分离制备高免疫活性组分COS6,得到两单体COS61和COS62,对COS62体外淋巴细胞增殖实验表明其免疫活性最高。 以氨基1～6糖混合标样作为分离对象对甲壳低聚糖HPLC分析的色谱柱选型和流动相选择进行优化,并运用该条件对甲壳低聚糖定量进行了研究。结果表明,甲壳低聚糖在相同的色谱条件下保留时间的对数与聚合度成良好的线性关系,可根据聚合度计算出氨基糖的保留时间值或根据保留时间值计算出氨基糖的聚合度;甲壳低聚糖在相同的色谱条件下聚合度与相对定量校正因子的负对数呈现极好的线性关系,可通过测得基准物单糖的定量校正因子来计算其它的低聚糖的定量校正因子。该定性定量方法经样品COS6分析验证可靠性极好。因此在没有标样或只有廉价标样-单糖的情况下可准确快速更多还原

【Abstract】 This research work was concerned with fractionation, and immune activity sieving of chitooligosaccharides（COS）. Their structure, quantitative analysis , immune activity ,and mechanism were studied,also their physicochemical and functional properties.The results of the physicochemical and functional properties showed that the COS with average molecular weight of 1389 and DD 91%, possessed good solubility in water from pH1 to pH13. But it showed a little decrease above pH 5.1 . The COS solubility decreased with ethanol concentration and it presented positive ion characteristic at pH<9.7 . At temperature <80℃ and pH 2⁵.5, COS solution was stable as confirmed by DSC analysis. COS had excellent water absorption , water holding capacity and can significantly resist starch aging.Fractionation and isolation of chitooligosaccharides were studied by applying membrane separation, ethanol stepwise sedimentation and column chromatography. The isolated component with higher immune activity was sieved by combination of the proliferation of splenocytes in vitro , phagocytization of PMΦ and HC₅₀ experiment in vivo. Firstly, NF retentate COS （NFR-COS） was sieved from UF retentate COS （UFR-COS） and NFR-COS . Secondly, 87.5% ethanol supernatant （COS-87.5SU）was sieved among 75% ethanol sedimentation （COS-75SE）, 87.5% ethanol sedimentation （COS-87.5SE）, 87.5% ethanol supernatant （COS-87.5SU） from NFR-COS whose molecular weight was 766 Da .Thirdly, COS-87.5SU were flowed through ion-exchange resin CM-Sephadex C-25. The elution was done by 0.01M pH4.8 buffer solution and 0—1.2N NaCl. Six components （COS1, COS2, COS3, COS4, COS5, COS6） were isolated. Component with high immune activity （COS6） and component with low immune activity （COS4 and COS5） were sieved by the above activity sieving method. Two monomers （COS61 and COS62） were got by HPLC . COS62 with highest immune activity were sieved by the proliferation of splenocytes.HPLC column and mobile phase for COS analysis were optimized using GAH/（GLcN）₂/ （GLcN）₃/（GLcN）₄/（GLcN）₅/（GLcN）₆ as mixed standards. HPLC analysis showed that 1) there was good linear relationship between logarithm of retention time（Rt） and degree of polymer（DP）, so Rt of amino-sugars could be calculated from DP and vice versa;2)there was rather good linear relationship between DP and minus logarithm of relative quantitativecorrective factor（f’） , so quantitative corrective factor（fi） of other chitooligosaccharides couldbe calculated from f_s of standard GAH （benchmark） . The qualitative and quantative analyticmethod were validated by COS6 and found that it had rather good assurance. So various kind of COS monomer could be qualitatively and quantatively determined without standards or only with cheap GAH/NAG standard.The composition and structure of the two monomers （COS61 and COS62） isolated from COS6 were identified by LC/ESI-MS. COS62 was an hexamer （ GLcN-GLcN-GLcN-GLcN -GLcN-GLcN） and represented 93.11%. COS61 was a pentamer （GLcN-GLcN-GLcN -GLcN-GLcN） and represented 6.89%.The two components （COS4 and COS5） with low immune activity were also identified by LC/ESI-MS. The component COS4 was 68.61 %of a trimer （GLcN-GLcN-GLcNAc） and 31.39% of a tetramer （GLcN-GLcN-GLcN-GLcN）. The component of COS5 was 59.84% of a tetramer （GLcN-GLcN-GLcN-GLcN ）and 40.16 % of a pentamer （GLcN-GLcN-GLcN-GLcN-GLcN）.Besides, nonspecific immune activity, cell immune and humoral immune activity mechanism, cell cycle and cell factor of COS6 with known composition and structure were studied. It showed that COS6 could promote the increase of spleen index ,thymus index, phagoiytic activity of peritoneal macrophage（PMO） and proliferation rate of splenocytes and HC50 in normal mice（NM） and immunodeficiency mice （IDM）. It also could resist decreasing immune organs’ weight, weakening phagoiytic activity of PMO, killing splenocytes by cyclophosphamide（Cy） and reducing the production of HC50 due to Cy. Thus COS6 promoted specific immune activity, nonspecific immune activity, cell immune and humoral immune activity. COS6 was mitosis source of splenocytes and it could cooperate with karyokinesis matrix. It indicated that COS6 could promote the dividing and proliferation of spleen cell of mice by cell cycle of NM using cell instrument. It could repair cell dying induced by Cy used in spleen cell cycle change experiment on IDM.Effects of （GLcN）5 and （GLcN）6 in vivo and vitro on gene transcription and translation level of IL-1, TNF-a , IL-2* IFN-y were studied, respectively by methods of relatively quantitive RT-PCR and ELISA. It showed that the mRNA expression and release of IL-K TNF-cu IFN- could be promoted more significantly by （GLcN）6 than those by （GLcN）s. Effects of （GLcN）5 and (GLcN>6 in vivo and vitro on gene expression of CR3 on the surface of PMO and splenocyte were also studied by RT-PCR. It showed that the mRNA expression of CR3 could be promoted more significantly by （GLcN）6 than those by （GLcN）5 This maybe concerned with that （GLcN）6 binded more sites of CR3 than （GLcN）s? led to change conformation of CR3 and （GLcN）6 possesed more affinity with CR3 than （GLcN）s. After （GLcN）5 and （GLcN）6 had been bound with CR3, signal transfer had been started through membrane and then leaded to stimulate gene expression of cytokines and CR3, thus immunomodulating ability for body were promoted. There was maybe one of mechanisms for elucidation of immunity of COS as well as the structure-activity relationship.更多还原