知母多糖复合酶提取工艺优化及其免疫活性

本文对知母多糖的提取工艺及体外免疫活性进行研究，为知母多糖的开发利用提供理论依据。试验以复合酶提取知母粗多糖，在正交试验确定复合酶比例的基础上，通过单因素实验和响应面优化试验，确定知母多糖的最佳提取工艺；采用DEAE-52纤维素层析柱进行分离得到4种多糖组分APSE-0、APSE-1、APSE-2和APSE-3。以RAW264.7巨噬细胞为研究模型，采用MTT法、Griess法测定知母多糖各组分对细胞增殖能力和一氧化氮（NO）释放量的影响。结果表明，最佳复合酶比例为:木瓜蛋白酶16000 U/g，纤维素酶1200 U/g，果胶酶1600 U/g。最优提取工艺为酶解时间2 h，液料比15:1（mL/g），酶解温度52 ℃，在此条件下得率为（10.58%±0.03%）。体外免疫活性实验结果证明，四种知母多糖组分均能明显促进RAW246.7巨噬细胞的增殖，APSE-0、APSE-2和APSE-3均可极显著诱导NO的释放，其中，APSE-2的免疫活性最强，可作为潜在的功能性食品或作为免疫力低下人群的膳食补充剂。剂。

Optimization of Multi-enzymatic Extraction of Polysaccharide from Anemarrhena asphodeloides Bunge and Its Immunomodulatory Activity

In this study, the extraction process and immune activity of polysaccharide from Anemarrhena asphodeloides Bunge were studied, in order to provide a theoretical basis for its development and utilization. Crude polysaccharide was obtained by complex enzyme method from Anemarrhena asphodeloides Bunge. On the basis of orthogonal experiment to determine compound enzyme ratio, the extraction conditions of Anemarrhena asphodeloides Bunge polysaccharide by enzyme complex were optimized by using response surface methodology and mono-factor experiments, and the optimum technological conditions were obtained. Four polysaccharide components, APSE-0, APSE-1, APSE-2 and APSE-3, were isolated and purified by DEAE-52 cellulose chromatography column. The effects of the polysaccharide fractions on the proliferation of RAW264.7 cells were determined by MTT assay, and the release capacity of nitrogen monoxide NO was determined by the Griess kit. The results showed that compound enzyme ratio was papain 16000 U/g, cellulose 1200 U/g, and pectinase 1600 U/g. The optimum extraction conditions were hydrolysis time of 2 h, liquid-solid of 15:1 (mL/g), temperature of enzymatic hydrolysis of 52 ℃. Under these conditions the extraction yield was (10.58%±0.03%). The results of in vitro immunoassay proved that four polysaccharides could significantly promote the proliferation of RAW246.7 cells, and APSE-0, APSE-2 and APSE-3 significantly induced NO production, among which APSE-2 showed the strongest immunoregulatory activity. APSE-2 could be explored as a potential source for immunomodulatory agents of the functional foods or dietary complementary for people with compromised immune system.