【作者】 许怡；

【导师】 吕晓玲； 刘丹；

【作者基本信息】 天津科技大学 ， 食品加工与安全（专业学位）， 2020， 硕士

【摘要】 甘草酸是甘草的主要成分,是一种天然甜味剂,甜度约是蔗糖的170倍,单葡萄糖醛酸甘草次酸的甜度约是甘草酸的5倍,具有更高倍的甜度,且甜味的持久性长,具有重要的研究价值。近年来,生物转化甘草酸制备单葡萄糖醛酸甘草次酸成为研究热点。本论文围绕β-葡萄糖醛酸苷酶的来源、提取工艺、酶学性质、单葡萄糖醛酸甘草次酸的酶法制备及分离纯化工艺,展开系统的研究。主要研究结果如下:首先以p-NPG以及甘草酸作为特异性底物,筛选出猪小肠来源的β-葡萄糖醛酸苷酶作为本研究的最佳生物催化剂,并对猪小肠β-葡萄糖醛酸苷酶的提取工艺进行了实验研究。通过常规提取工艺的单因素实验,确定最佳提取条件:0.1 mol/L pH 5磷酸盐缓冲溶液,浸提时间5 h,料液比1:8。按照最佳条件进行反复三次提取,通过冷冻干燥后得到猪小肠β-葡萄糖醛酸苷酶粗酶粉(得率为55%,酶活为23.41 U/g)。对该酶的酶学性质的研究表明,其最适温度为55℃,最适pH为6,在55℃以下和pH 4.5～6.5范围内,具有很好的热稳定性和pH稳定性。以p-NPG为底物时,动力学常数分别为Km=5.67×10-3 mol/L,Vmax=11.39 mmol/(L·min);以甘草酸为底物时,动力学常数分别为Km=8.12×10-3 mol/L,Vmax=4.59 mmol/(L·min)。在此基础上进行了酶法转化甘草酸生成单葡萄糖醛酸甘草次酸的工艺条件优化:底物与酶的体积比为4:1,反应温度55℃,反应时间10 h,甘草酸的转化率为97.21%。将酶转化液进行离心,然后对酶转化液沉淀中的单葡萄糖醛酸甘草次酸进行提取,提取条件如下:料液比为1:17.5(m:v),水浴温度为55℃,提取时间为5 h。甘草酸转化生成单葡萄糖醛酸甘草次酸的生成得率为58%。最后,对所得单葡萄糖醛酸甘草次酸粗品进行分离纯化,先经过酸沉醇溶,然后利用大孔吸附树脂纯化。大孔吸附树脂纯化工艺条件如下:大孔吸附树脂采用NKA-9,上样液浓度2mg/mL,上样液pH7.0,上样液流速2BV/h;洗脱液为70%乙醇溶液,洗脱液体积收集2-3BV,洗脱液流速1BV/h。单葡萄糖醛酸甘草次酸的纯度达到52.45%。本课题以甘草酸为底物,通过生物酶转化法制备单葡萄糖醛酸甘草次酸,为单葡萄糖醛酸甘草次酸的生物活性研究提供科学研究材料,对单葡萄糖醛酸甘草次酸的工业化生产具有积极作用。更多还原

【Abstract】 Glycyrrhizic acid is the main component of licorice.As a natural sweetener,glycyrrhizic acid has a sweetness of about 170 times that of sucrose.Glycyrrhetic acid 3-O-mono-β-D-glucuronide is about 5 times sweeter than glycyrrhizic acid.It has a higher degree of sweetness and a longer lasting sweetness.In recent years,the use of biological enzymes to convert glycyrrhizic acid into glycyrrhetic acid 3-O-mono-β-D-glucuronide has become a research hotspot.In this paper,the source and extraction process of β-glucuronidase were studied.The preparation,separation and purification of glycyrrhetic acid 3-O-mono-β-D-glucuronide were also studied.The main findings are as follows:Firstly,porcine intestinal-derived β-glucuronidase was obtained as the best biocatalyst for this study by using p-NPG and glycyrrhizic acid as specific substrates.A single factor experiment was then performed on the extraction of theβ-glucuronidase process.The optimal extraction conditions were determined by single factor experiment of conventional extraction process:0.1 mol/L pH 5 phosphate buffer solution,extraction time 5 h,ratio of material to liquid 1:8.The extraction was repeated three times according to the optimal conditions,and the porcine intestinalβ-glucuronidase crude enzyme powder was obtained by freeze-drying(yield was 55%,and the enzyme activity was 23.41 U/g).The optimum temperature of the enzyme is 55℃,the optimum pH is 6,and it has good thermal stability and pH stability in the range of 55 ℃ and below and pH 4.5 to 6.5.When p-NPG was used as the substrate,the kinetic constants were Km=5.67×10-3 mol/L and Vmax=11.392 mmol/(L·min).When glycyrrhizic acid was used as the substrate,the kinetic constants were Km=8.12×10-3 mol/L and Vmax=4.59 mmol/(L·min).On the basis of this,the enzymatic conversion of glycyrrhizic acid to glycyrrhetic acid 3-O-mono-β-D-glucuronide was carried out.The process conditions were as follows:the volume ratio of substrate to enzyme was 4:1,the reaction temperature was 55℃,and the reaction time was 10 h.The conversion of glycyrrhizic acid was 97.21%.The monogluconic acid glycyrrhetinic acid in the reaction solution was extracted under the following conditions:the ratio of material to liquid was 1:17.5(m:v),the temperature was 55 ℃,and the extraction time was 5 h.The yield of glycyrrhetic acid 3-O-mono-β-D-glucuronide is 58%.Finally,the crude glycyrrhetic acid 3-O-mono-β-D-glucuronide was purified by macroporous adsorption resin.The purification conditions of the macroporous adsorption resin are as follows:the macroporous adsorption resin is NKA-9,the concentration of the sample solution is 2 mg/mL,the sample solution is pH 7.0,the flow rate of the sample solution is 2 BV/h,and the eluent is 70%ethanol solution.The eluent volume was collected at 2-3 BV and the eluent flow rate was 1 BV/h.The purity of monoglucuronide glycyrrhetinic acid reached 52.45%.In this study,glycyrrhizic acid was used as a substrate to prepare glycyrrhetic acid 3-O-mono-β-D-glucuronide by bio-enzyme conversion method,which provided scientific research materials for the bioactivity study of glycyrrhetic acid 3-O-mono-β-D-glucuronide.更多还原