响应面法优化松茸多糖酶法提取工艺及其体外抗氧化性分析

Response Surface Analysis for the Optimization of Extraction Process for Polysaccharides from Tricholoma matsutake and Antioxidant Activity of the Extracted Polysaccharides

为确定复合酶(纤维素酶、果胶酶、中性蛋白酶)提取松茸多糖的最佳工艺,并对其体外抗氧化活性进行初步研究,在单因素试验的基础上,以料液比、pH值、酶解时间和酶解温度为影响因素,利用Box-Behnken方法进行四因素三水平试验设计,以多糖提取率为响应值,进行响应面分析;分别用邻苯三酚自氧化法和对DPPH自由基的清除作用测定松茸多糖的体外抗氧化性。结果表明:多糖提取的最佳工艺为料液比1∶40(g/mL)、pH 5、酶解温度35℃、酶解时间71 min,松茸多糖的提取率预测值为7.06%,验证值为6.95%,与预测值相对误差为1.56%;松茸多糖对超氧阴离子自由基和DPPH自由基具有较强的清除作用,其IC50值分别为0.565 mg/mL和0.454 mg/mL。因此,复合酶法提取松茸多糖高效、简单,可用作松茸多糖的提取工艺;松茸多糖具有明显的体外抗氧化活性。

Objective: To develop an optimized enzymatic method for extracting polysaccharides from Tricholoma matsutake with a combination of cellulase, pectinase and neutral protease and to evaluate the antioxidant activity in vitro of the extracted polysaccharides. Methods: On the basis of single-factor experiments, a four-factor, three-level Box-Behnken design was utilized for the mathematical modeling of polysaccharide yield as a function of material/water ratio, medium pH and hydrolysis time and temperature. This was followed by response surface analysis of the developed mathematical model. The antioxidant activity of Tricholoma matsutake polysaccharides was determined by pyrogallol autoxidation and free radical-scavenging assay. Results: The optimum condition for the extraction of Tricholoma matsutake polysaccharides were found to be hydrolysis at 35 ℃ for 71 min with a material/solvent ratio of 1:40(g/mL) at pH 5. Under these conditions, the model-predicted and experimentally measured values of polysaccharide yield were 7.06% and 6.95%, respectively, revealing a relative error of only 1.56% between them. The Tricholoma matsutake polysaccharides had a strong capacity to scavenge superoxide anion(IC50 = 0.565 mg/mL) and DPPH free radical(IC50 = 0.454 mg/mL). Conclusion: The proposed extraction method is efficient, simple and applicable for the extraction of polysaccharides form Tricholoma matsutakes and the extracted polysaccharides have significant antioxidant capacity in vitro.