不同脱蛋白方法对青钱柳多糖的结构和抗氧化活性的影响

何思辰; 陈凌利; 陈慧; 严谨; 王文君

doi:10.13386/j.issn1002-0306.2022100170

不同脱蛋白方法对青钱柳多糖的结构和抗氧化活性的影响

Effect of Different Protein Removal Methods on the Structure and Antioxidant Activity of the Cyclocarya paliurus Polysaccharides

摘要

摘要: 青钱柳多糖具有较强的降血糖和抗氧化活性，脱除蛋白对青钱柳多糖的纯化至关重要。为探索最适合青钱柳多糖的除蛋白方式，本文采用了三种除蛋白方法（HCl法、TCA法、NaCl法）对青钱柳粗多糖进行处理，同时采用高效液相凝胶色谱法、离子色谱法、傅里叶红外光谱法对三种脱蛋白方式得到的多糖的结构和成分进行分析，接着通过测定其对DPPH自由基、ABTS⁺自由基和羟基自由基的清除率评价其抗氧化能力。结果表明：HCl法在pH2时，蛋白脱除率为73.48%，多糖保留率为74.86%；TCA法在TCA体积分数为2%时，蛋白脱除率为79.88%，多糖保留率为81.78%；NaCl法在NaCl体积分数为7%时，蛋白脱除率为47.90%，多糖保留率为82.51%。三种除蛋白方法处理后得到的多糖主要包括三个不同分子量范围的组分：500~750 kDa，100~130 kDa和30~40 kDa左右的部分，但其比例各不相同，TCA法处理的多糖在100~130 kDa占比最大，NaCl法处理的多糖在30~40 kDa占比最大。三种多糖均由8种单糖组成，但其摩尔比不相同，HCl法处理后多糖中阿拉伯糖、半乳糖和葡萄糖的含量最大，TCA法处理后多糖中糖醛酸的含量更高，NaCl法处理后多糖中的鼠李糖、木糖和甘露糖的含量最大。通过比较同浓度下三种多糖对自由基的清除能力发现，TCA法处理的多糖抗氧化能力最强，HCl法次之，NaCl法最弱。由研究结果表明，不同的除蛋白方法会对青钱柳多糖的结构和活性产生不同的影响，其中TCA法是一种较好的青钱柳多糖脱除蛋白的方式。

Abstract: Removal of proteins is essential for the purification of Cyclocarya paliurus polysaccharides (CPP), which have strong hypoglycemic and antioxidant activities. In order to explore the most suitable way of removing protein from the polysaccharides, three methods (HCl, TCA and NaCl) were used to treat the crude polysaccharides, and the structure and composition of the polysaccharides obtained by the three deproteinization methods were analyzed by high performance liquid gel chromatography, ion chromatography and Fourier infrared spectroscopy, and their antioxidant activity was determined by measuring the scavenging rate of DPPH radicals, ABTS⁺ radicals and hydroxyl radicals. The results showed that the protein removal rate of the HCl method was 73.48% and the polysaccharide residue rate was 74.86% at pH2. The protein removal rate of the TCA method was 79.88% and the polysaccharide residue rate was 81.78% at a TCA volume fraction of 2%. The protein removal rate of the NaCl method was 47.90% and the polysaccharide residue rate was 82.51% at a NaCl volume fraction of 7%. The molecular weight distribution of the polysaccharides treated by the three methods could be roughly divided into three parts, 500~750 kDa, 100~130 kDa and 30~40 kDa, but their proportions were different. The polysaccharides treated by the TCA method accounted for the largest percentage of 100~130 kDa compared with the three, and the polysaccharides treated by the NaCl method accounted for the largest percentage of 30~40 kDa. All three polysaccharides consisted of eight monosaccharides, but their molar ratios were different. Of the three deproteinization method, the HCl-treated polysaccharide had the highest content of arabinose, galactose and glucose, the TCA-treated polysaccharide had a higher content of glyoxylate, and the NaCl-treated polysaccharide had the highest content of rhamnose, xylose and mannose. The scavenging ability of the three polysaccharides against free radicals at the same concentration was compared, and the antioxidant ability of the polysaccharides treated with TCA method was the strongest, followed by HCl method and the weakest was polysaccharides treated by NaCl method. As shown by the results, different methods of protein removal have different effects on the structure and activity of CPP, among which TCA method is a better way to remove protein from CPP.