摘要:
优化复合酶(纤维素酶-果胶酶-木瓜蛋白酶)提取石榴幼果总黄酮的工艺,并测定其抑制α-葡萄糖苷酶活性。以总黄酮得率为评价指标,在单因素实验基础上,D-最优混料设计优化复合酶配比,正交试验设计对料液比、介质pH、酶解温度、酶解时间进行优化,并以PNPG为底物测定总黄酮对α-葡萄糖苷酶的抑制活性。结果显示:复合酶最优配比为:纤维素酶44.2%、果胶酶31.6%、木瓜蛋白酶24.2%,最佳提取条件为:料液比1:18 (g/mL)、介质pH5.0、酶解温度50 ℃、酶解时间4.0 h,总黄酮得率为3.38%,其浓度为1.5 mg/mL时,对α-葡萄糖苷酶抑制率达到63.9%,抑制作用的IC50为1.059 mg/mL,在浓度0.15~1.5 mg/mL范围内,石榴幼果总黄酮浓度与其对α-葡萄糖苷酶抑制效果之间呈现一定的正相关关系,其抑制机理属于可逆性抑制和非竞争性抑制。该方法可为石榴幼果总黄酮的提取和应用提供一定的科学依据。
Abstract:
To optimize the complex enzymatic(cellulose-pectinase-papain)extraction of total flavonoids from pomegranate fruitlets and to determine its anti-α-glucosidase activity.with the yield of total flavonoids as an evaluation index,D-optimal mixture design was applied to optimize the complex enzymeratio,and orthogonal experimental design was adopted in the optimization of solid/liquid ratio,medium pH,enzymatic hydrolysis temperature and enzymatic hydrolysis time based on the single factor test,then the anti-α-glucosidase activity was determined with PNPG as substrate. The results showed that optimal ratio of enzyme was determined to be 44.2% for cellulose,31.6% for pectinase and 24.2% for papain,to obtain a 3.38% of total flavonoids yield,the optimal conditions were determined to be 1:18 (g/mL)for solid/liquid ratio,5.0 for medium pH,50 ℃ for enzymatic hydrolysis temperature and 4.0 h for enzymatic hydrolysis time,the substrate inhibition rate reached 63.9% on α-glucosidase and the inhibitory effect of IC50 was 1.059 mg/mL at the centration was 1.5 mg/mL.There was a dose-response relationship between the total flavonoids and anti-a-glucosidaseactivity in the range of 0.15~1.5 mg/mL of concentration,the inhibition mechanism was reversible inhibition and non competitive inhibition. This method can provide ascientific basis for the extraction and application of total flavonoids from pomegranate fruitlets.
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