【作者】 杨洋；

【导师】 杨鑫；

【作者基本信息】 哈尔滨工业大学 ， 食品科学与工程， 2022， 硕士

【摘要】 壳寡糖(chitosan oligosaccharide,COS)由于聚合度低,具有较高的水溶性而被广泛应用。本文对壳寡糖的酶法制备及其理化特性进行研究,对酶解产物进行除酶,并探究了除酶方法对产物还原糖、DP 2～6寡糖含量的影响。充分利用了甲壳类动物中含量丰富的壳聚糖资源,为壳寡糖酶法制备工业化提供参考基础。利用价格低廉的纤维素酶、胃蛋白酶及木瓜蛋白酶三种非专一性酶制备壳寡糖,通过测定酶解产物还原糖含量及DP 2～6寡糖含量确定酶解p H、酶解温度、酶解时间、酶添加量4个因素对酶解效果的影响:(1)对于纤维素酶,其最适p H为5.0,最适酶解温度为45℃,最适酶解时间为2.5 h,最适酶添加量为60 U/mg壳聚糖。所得产物还原糖含量为221 mg,DP 2～6寡糖含量为103 mg。且所得酶解产物中含有COS 2～COS 6,说明纤维素酶对壳聚糖具有内切、外切作用,是一种由内切酶与外切酶组成的复合酶。(2)对于胃蛋白酶,其最适p H为5.5,最适酶解温度为50℃,最适酶解时间为2 h,最适酶添加量为120 U/mg壳聚糖。产物还原糖含量为66 mg,DP 2～6寡糖含量为11 mg,且以COS 3为主,说明胃蛋白酶降解壳聚糖以外切为主。(3)对于木瓜蛋白酶,最适p H为5.5,最适温度为50℃,最适酶解时间为1.5 h,最适酶添加量为160 U/mg壳聚糖。还原糖含量为24 mg,DP 2～6寡糖含量为13 mg。且产物也以COS 3为主,说明木瓜蛋白酶与胃蛋白酶相同,降解壳聚糖时以外切作用为主。三种酶在不同条件下降解壳聚糖,所得产物中还原糖含量及DP 2～6寡糖含量不同,对不同工艺下所得酶解产物的抑菌活性进行研究,发现酶解产物抑菌活性随DP 2～6寡糖含量的升高而增强。MTT试验发现三种酶解产物均无细胞毒性。分别利用TCA法及沸水浴法对三种非专一性酶降解产物进行除酶,测定两种方法处理前后三种非专一性酶降解产物中还原糖含量及DP 2～6寡糖含量、单位质量产物中还原糖及DP 2～6寡糖占比(纯度)及产物中残留酶含量。发现在除酶过程中,两种除酶方法对还原糖及DP 2～6寡糖均会造成损耗。与沸水浴法除酶后产物相比,TCA法在除酶过程中对还原糖及DP 2～6寡糖损耗较少。而沸水浴法对还原糖及DP 2～6寡糖损耗较多,但产物纯度较高,且酶脱除率较高。通过对沸水浴法除酶后的产物抑菌活性及细胞毒性研究,发现三种非专一性酶降解壳聚糖所得产物具有良好的抑菌作用,且对机体正常细胞无毒性。更多还原

【Abstract】 Chitosan oligosaccharide(COS)has been widely used due to its low degree of polymerization and high water solubility.In this paper,the enzymatic preparation of chitooligosaccharides and its physicochemical properties were studied,and the enzyme removal method of the enzymatic products was explored.The effect of the enzyme removal method on the reducing sugar and DP 2–6 oligosaccharide content of the products was also explored.The chitosan resource which is rich in content in nature,especially in crustaceans,is fully utilized,and a theoretical basis is provided for realizing industrialization of the enzymatic preparation of chitosan oligosaccharide.Three non-specific enzymes including cellulase,pepsin and papain with low price were used to prepare chitosan oligosaccharide.The effects of four factors,namely enzymolysis p H,enzymolysis temperature,enzymolysis time and enzyme addition amount,were determined by measuring the reducing sugar content of enzymolysis products and the content of DP 2–6 oligosaccharides:(1)For cellulase,the optimal p H was 5.0,the optimal enzymolysis temperature was 45℃,the optimal enzymolysis time was 2.5 h and the optimal enzyme addition amount was 0.60 g.The reducing sugar content of the obtained product was 221 mg,and the oligosaccharide content of DP 2–6 was 103 mg.And that obtain enzymolysis product contains COS 2～COS 6,so that cellulase has endo-and exo-cutting effect on chitosan,and is a composite enzyme consisting of endoenzyme and exonuclease.(2)For pepsin,the optimal p H was 5.5,the optimal temperature was 50℃,the optimal enzymolysis time was 2 h,and the optimal enzyme addition was3.60 g.The reducing sugar content of the products was 66 mg,and the oligosaccharide content of DP 2–6 was 11 mg,mainly COS 3,indicating that the hydrolysis of chitosan by pepsin was dominated by exo-digestion.(3)For papain,the optimal p H was 5.5,the optimal temperature was 50℃,the optimal enzymolysis time was 1.5 h,and the optimal enzyme addition was 0.80 g.The reducing sugar content of the products was 24 mg,and the oligosaccharide content of DP 2–6 was 13 mg.Besides,COS 3 was also used as the main product,indicating that papain was the same as pepsin,and exocytosis was dominant during the degradation of chitosan.The three enzymes degraded chitosan under different conditions,and the reducing sugar content and DP 2–6 oligosaccharide content in the obtained products were different.The antibacterial activity of the enzymolysis products obtained under different processes was studied,and it was found that the antibacterial activity of the enzymolysis products was enhanced with the increase of DP 2–6 oligosaccharide content.MTT assay showed that none of the three enzymatic hydrolysates was cytotoxic.The TCA method and boiling water bath method were used to remove the enzymes from the three non-specific enzyme degradation products,respectively.The reducing sugar content and DP 2–6 oligosaccharide content of the three non-specific enzyme degradation products,the proportion(purity)of reducing sugar and DP 2–6 oligosaccharide per unit mass product and the residual enzyme content in the products before and after treatment with the two methods were determined.It was found that the two enzyme removal methods caused the loss of reducing sugar and DP 2–6 oligosaccharides in the enzyme removal process.Compared with the products after enzyme removal by boiling water bath method,the reducing sugar and DP 2–6 oligosaccharides were less consumed by TCA method in the enzyme removal process.The boiling water bath method consumed more reducing sugars and DP 2–6 oligosaccharides,but the product purity was higher and the enzyme removal rate was higher.The antibacterial activity and cytotoxicity of the products after the enzyme was removed by boiling water bath method were studied.The results showed that the products obtained by degrading chitosan with three non-specific enzymes had good antibacterial effect and no toxicity to normal cells of the body.更多还原