【作者】 钟建业；

【导师】 吴成业； 陈丽娇；

【作者基本信息】 福建农林大学 ， 农产品加工及贮藏工程， 2009， 硕士

【摘要】 甲壳低聚糖(Chitooligosaccharides,COS)是壳聚糖(Chitosan,CTS)的降解产物,不仅具有壳聚糖大分子的某些功能性质,而且还具有许多壳聚糖所不具备的独特的生理活性和功能性质。本文旨在通过研究不同的非专一性酶对壳聚糖的降解效果,探索酶法降解壳聚糖制备甲壳低聚糖有效方法,并对低聚糖相对分子量分布范围进行了初步研究。1、选用纤维素酶、果胶酶、木瓜蛋白酶、胃蛋白酶和枯草杆菌蛋白酶等5种酶,对壳聚糖进行降解试验。通过单因素试验,以还原糖释放量为指标,探讨以上5种酶降解壳聚糖的工艺条件。试验结果表明:在试验选取的范围内,5种酶降解壳聚糖所需时间依次为5h、3h、5h、4h和3h;果胶酶、枯草杆菌蛋白酶、纤维素酶和木瓜蛋白酶酶解壳聚糖最适温度均为50℃,胃蛋白酶为55℃;果胶酶、纤维素酶、木瓜蛋白酶和胃蛋白酶的最适加酶量均为5%,而枯草杆菌蛋白酶为7%;各酶降解壳聚糖最适底物浓度均为1%;考虑到降解产物一般在近中性的条件下使用,为方便降解产物调节适宜的pH,采用pH5.0作为各酶降解壳聚糖的酸碱条件。通过对各酶降解壳聚糖的酶活力、酶解液中还原糖释放量,以及各酶降解壳聚糖的粘度下降率3个指标的综合比较,得出各酶降解壳聚糖的能力大小为:纤维素酶>木瓜蛋白酶>胃蛋白酶>枯草杆菌蛋白酶>果胶酶。2、分别运用正交试验设计法优化纤维素酶和木瓜蛋白酶降解壳聚糖的工艺条件,得出纤维素酶降解壳聚糖最佳工艺参数为:加酶量5%,底物浓度1.25%,酶解时间6 h,酶解温度为55℃,pH5.0;木瓜蛋白酶降解壳聚糖最佳工艺参数为:加酶量7%,底物浓度1.25%,酶解时间6 h,酶解温度为55℃,pH5.0。将原料壳聚糖胶体、壳聚糖酶解液分别过Sephadex G-100凝胶色谱柱,发现原料壳聚糖相对分子量主要分布范围为3.5×10~5～1.2×10~5;纤维素酶酶解壳聚糖,产物相对分子量主要分布范围为8.4×10~3～2.2×10~3(洗脱体积为104～130 mL),小部分分布在相对分子量3.2×10~5～1.7×10~5的范围内(洗脱体积为34～46 mL);木瓜蛋白酶解降解壳聚糖,产物相对分子量分布范围分别为3.2×10~5～1.0×10~5(洗脱体积34～56 mL)、1.2×10~4～2.7×10~3(洗脱体积98～126 mL)。从分子量分布来看,纤维素酶和木瓜蛋白酶均未能对壳聚糖彻底降解。3、应用复合酶解技术,通过调整纤维素酶和木瓜蛋白酶的组合比例,探讨不同比例的复合酶对壳聚糖的降解效果。结果表明:纤维素酶与木瓜蛋白酶按1:2的比例降解壳聚糖,还原糖浓度最高。采用二次回归旋转组合试验设计方案,建立了加酶量(X1)、酶解温度(X2)、酶解时间(X3)与粘度下降率(Y1)和还原糖释放量(Y2)之间的动态数学模型。试验结果的方差分析显示,方程Y1相关系数R2为97.63 %,F值为59.5919,显著性系数为0.0001,模型的符合度为97.63%;方程Y2的相关系数R2为97.69%,F值为61.0413,显著性系数为0.0001,说明两个回归方程是极显著的,能够较为准确的预测酶解实际效果。运用响应面法对工艺条件进一步优化,得到复合酶降解壳聚糖的最佳工艺条件为:加酶量7.70%,酶解温度50℃,酶解时间5 h,壳聚糖浓度1.25%,pH5.0。在此条件下对壳聚糖糖进行降解,壳聚糖粘度下降率达到97.21%,还原糖浓度达到2.47 mmol/L,均高于纤维素酶和木瓜蛋白酶的单一酶解效果。同样将降解产物过Sephadex G-100凝胶色谱柱。洗脱曲线显示:降解产物相对分子量分布于1.0×10~4～2.7×10~3和2.3×10~5～1.1×10~5之间。与原料壳聚糖胶体的洗脱曲线相比,使用复合酶降解壳聚糖效果显著,大部分产物相对分子量低于1×10~4。更多还原

【Abstract】 Chitooligosaccharides (COS), the degradation products of chitosan, not only have some features of chitosan macromolecules, but also have lots of special physiological activities and functional properties.This paper aimed at probing into the non-specific enzymatic hydrolysis technology of chitosan,and molecular weight distribution of chitooligosaccharides.1.Five kinds of enzymes including cellulase, pectinase, papain, pepsin and subtilisin were used for the degradation of chitosan,and the conditions were discussed by single-factor analysis based on the release dose of reducing sugar. The results showed that the optimum time of cellulose, papain, pepsin was 4 h, pectinase is 3 h,and subtilisin2 h to hydrolysis chitosan .The optimum temperature of pectinase, subtilisin, cellulase and papain was 50℃,and 55℃for pepsin to hydrolysis chitosan ; the optimum content of enzyme for pectinase cellulase,papain and pepsin was 5%,and 7% for subtilisin;In addition, the optimal substrate concentration for the degradation was 1%. In this paper pH5.0 was the best choice for the degradation of chitosan to facilitate the degradation products of the appropriate pH adjustment.By the comparation of enzyme activity, the release amount of reducing sugar in enzymolysis liquid and the droop rate of the viscosity of these degradation products, the degradation ability sequence was:cellulase> papain> pepsin> subtilisin> pectinase.2. The orthogonal test showed that the optimum conditions on cellulase were as follows: the optimum enzyme dosage was 5%, substrate concentration was 1.25%, enzymolysis time was 6 h, enzymolysis temperature was 55℃, pH5.0;and the the optimum conditions on papain were as follows: the optimum enzyme dosage was 7%, substrate concentration was 1.25%, enzymolysis time was 6 h, enzymolysis temperature was 55℃, pH5.0.The Sephadex G-100 was selected as the separation medium to separete raw materials of colloidal chitosan and chitosan hydrolysis liquid respectively by gel chromatography. For the raw materials, the main distribution range of chitosan molecular weight was 3.5×10~5-1.2×10~5,and the study indicated that the range was mainly between 8.4×10~3 and 2.2×10~3 after degradation with cellulase in the optimal conditions(the elution volume was 104～130 mL ),and subsection was3.2×10~5～1.7×10~5(the elution volume was 34～46 mL) ; Besides, the main ranges were 3.2×10~5～1.7×10~5 (the elution volume was 34～56 mL) and 1.2×10~4～2.7×10~3(the elution volume was 98～126 mL)after degradation with papain in the optimal conditions.3.The composite enzymatic hydrolysis technology was applicated to discuss the effects on the degradation of chitosan for different enzymes by adjusting the ratio of cellulase and papain.The result showed that the concentration of reducing sugar was highest when the ratio was 1:2.Moreover,by rotation composite design of quadratic regression, a dynamic mathematic model that showed the connection between the enzyme dosage (X1) , hydrolysis temperature (X2), hydrolysis temperature (X3)and the droop rate of viscosity (Y1), the release amount of reducing sugar (Y2).The results of variance analysis showed that the correlation coefficient R2 of equation Y1 was 97.63%, the value of F was 59.5919, and significant coefficient was 0.0001, the conformity of the model was 97.63%; At the same time , the correlation coefficient R2 of equation Y2 was 97.69%, the value of F was 61.0413, and significant coefficient was 0.0001.As the result,these two regression equations were highly significant, and could be good used to predict the degradation results.In addition ,the method of response surface was used to optimize the process conditions,and the optimum conditions was as follows: the content of enzyne was 7.70%, enzymolysis temperature was 55℃,the concentration of chitosan was 1.25%, enzymolysis time was 5 h, pH5.0. Under these conditions, the degradation of chitosan, the droop rate of viscosity of chitosan was 97.21%, the concentration of reducing sugar reached 2.47 mmol/L,and the effect was better than the single enzyme.The Sephadex G-100 was selected as the separation medium to separete hydrolysis liquid with the method of gel chromatography. Elution curve shows that the distribution of relative molecular weight of degradation products was 2.3×10~5～1.1×10~5 and 1.0×10~4～2.7×10~3. Compared with the elution curve of raw materials , the degradation effect was significant,and the relative molecular weight of majority products were under 1×10~4.更多还原