提取方法对酵母甘露糖蛋白组分及免疫活性的影响

Effects of Extraction Methods on the Composition and Immunological Activities of Yeast Mannoproteins

摘要:

【目的】 以酵母细胞壁为原料，比较不同提取方法对酵母甘露糖蛋白的提取得率、组分的影响，并评价其抗炎活性。 【方法】 采用热水提取、碱提取、酶法提取3种方法获得酵母甘露糖蛋白，经乙醇沉淀分别得到酵母甘露糖蛋白MP-W、MP-B、MP-E，利用单糖组成、氨基酸组成、相对分子质量分布、傅里叶变换红外光谱分析其组分和化学结构，并采用细胞试验研究其抗炎活性。 【结果】 3种提取方法得到的酵母甘露糖蛋白的提取得率分别为9.7%、11.5%和23.1%，MP-E的提取得率显著高于MP-W和MP-B。3种酵母甘露糖蛋白均是由甘露糖、葡萄糖和氨基葡萄糖组成，MP-E中葡萄糖峰面积占比显著高于MP-W和MP-B。MP-E的总氨基酸质量分数、天冬氨酸（Asp）质量分数显著低于MP-W和MP-B，苏氨酸（Thr）质量分数显著高于MP-W和MP-B。MP-W、MP-B、MP-E的重均分子量（M_w）分别为103 493、106 579、118 286。傅里叶变换红外光谱显示，3种酵母甘露糖蛋白均是糖蛋白结构，主要官能团无显著差异。当质量浓度为150 μg/mL时，在增加巨噬细胞RAW264.7白细胞介素10 （IL-10）分泌与抑制肿瘤坏死因子-α（TNF-α）分泌方面，MP-E效果极显著优于MP-W（P<0.001）和MP-B（P<0.001）。 【结论】 3种提取方法得到的酵母甘露糖蛋白的主要官能团无显著差异，但酶法提取能显著提升酵母甘露糖蛋白的提取得率，且所得样品显示出更高的抗炎活性，可为酵母甘露糖蛋白的开发与应用提供理论依据。

Abstract:

[Objective] The extraction efficiency of different methods and the effects of these methods on the composition of mannoproteins extracted from yeast cell walls were compared. Furthermore, the anti-inflammatory activities of the extracted mannoprotein were evaluated. [Method] Three yeast mannoproteins (MP-W, MP-B, and MP-E) were extracted via high-temperature water, alkali, and enzyme-assisted extraction methods and purified via ethanol precipitation. The identification of monosaccharide composition and amino acid composition, distribution of relative molecular mass, and Fourier transform infrared spectroscopy were employed to analyze the composition and chemical structures of yeast mannoproteins. A cell experiment was carried out to examine the anti-inflammatory activity. [Result] The yields of yeast mannoproteins extracted by the three methods were 9.7%, 11.5%, and 23.1%, respectively. The yield of MP-E was significantly higher than those of MP-W and MP-B. The yeast mannoproteins obtained were composed of mannose, glucose, and glucosamine, and the peak area proportion of glucose in MP-E was significantly higher than those in MP-W and MP-B. MP-E had significantly lower mass fractions of total amino acids and aspartate (Asp) and significantly higher mass fraction of threonine (Thr) than MP-W and MP-B. The weight-average molecular weights of the extracted yeast mannoproteins were 103 493, 106 579, and 118 286, respectively. Fourier transform infrared spectroscopy showed that all the three yeast mannoproteins were glycoproteins, with no significant difference in the main functional groups. At the mass concentration of 150 μg/mL, MP-E extremely outperformed MP-W (P<0.001) and MP-B (P<0.001) in increasing interleukin-10 secretion and inhibiting tumor necrosis factor-α secretion in RAW264.7 cells. [Conclusion] There is no significant difference in the main functional groups of yeast mannoproteins obtained by the three extraction methods. However, the enzyme-assisted extraction method can significantly improve the yield of yeast mannoprotein, and the obtained yeast mannoprotein shows higher anti-inflammatory activity. The findings provide a theoretical basis for the development and application of yeast mannoproteins.