苦荞多肽的纯化、结构与体外抗氧化活性研究

Study on purification,structure and in vitro antioxidant activity of buckwheat peptides

为了探究苦荞多肽粗提液抗氧化活性的主要来源,需要进一步对苦荞多肽进行分离纯化,通过结构鉴定来分析抗氧化活性与多肽结构之间的关系。该研究以苦荞功能提取物废渣为原料提取苦荞粗蛋白辅以植物乳杆菌发酵法制备苦荞多肽,采用大孔吸附树脂、葡聚糖凝胶、液相色谱对苦荞多肽粗提液进行分离纯化,并以抗氧化活性为指标,筛选抗氧化活性较强的组分,利用液相色谱-质谱联用技术鉴定抗氧化多肽结构。结果表明,纯度不同的苦荞多肽抗氧化能力也不同,经过Sephadex G-15葡聚糖凝胶分离纯化得到的T-3组分ABTS和DPPH自由基清除能力最优,清除率分别为96%、94%。纯化后苦荞多肽的抗氧化活性显著高于粗提液(P<0.05),但是抗氧化活性并没有完全与苦荞多肽纯度呈正相关,通过液相色谱进一步分离纯化T-3组分后得到的组分T-3-1,苦荞多肽纯度虽然达到了98%,但是对DPPH和ABTS抑制率不再增强,反而稍有下降,抑制率分别为89%和90%。由质谱鉴定结果得到,主要抗氧化肽的氨基酸序列为苯丙氨酸-脯氨酸-酪氨酸Phe-Pro-Tyr(FPY)和酪氨酸-亮氨酸-脯氨酸-苯丙氨酸Tyr-Leu-Pro-Phe(YLPF)。研究结果以期为苦荞多肽的进一步开发利用提供一定的理论基础。

In order to investigate the main source of antioxidant activity of tartary buckwheat polypeptide crude extract,it was necessary to further isolate and purify buckwheat peptides and analyze the relationship between antioxidant activity and peptide structure by structural identification.In this study,the buckwheat polypeptide was prepared by using the waste residue of buckwheat functional extract as raw material to extract buckwheat crude protein supplemented by lactobacill plant fermentation method,and the coarse extract of buckwheat polypeptide was isolated and purified by macroporous adsorption resin,dextran gel and liquid chromatography,and the antioxidant activity was taken as the index,the components with strong antioxidant activity were screened,and the antioxidant polypeptide structure was identified by liquid chromatography-mass spectrometry.The results showed that tartary buckwheat polypeptides with different purities also had different antioxidant capacity.The ABTS and DPPH radical scavenging ability of T-3 components separated and purified by Sephadex G-15 dextran gel were the best,and the scavenging rates were 96%and 94%,respectively.The antioxidant activity of purified tartary buckwheat polypeptide was significantly higher than that of crude extract(P<0.05),but the antioxidant activity was not positively correlated with the purity of tartary buckwheat polypeptide.After further separation and purification of T-3 components by liquid chromatography,the component T-3-1 was obtained.Although the purity of Tartary buckwheat polypeptide reached 98%,the inhibition rate of DPPH and ABTS was no longer enhanced.The e inhibition rates were 89%and 90%,respectively.The amino acid sequences of the main antioxidant peptides were phenylalanine-proline-tyrosine(Phe-Pro-Tyr,FPY)and tyrosine-leucine-proline-phenylalanine(Tyr-Leu-Pro-Phe,YLPF).The results are expected to provide a theoretical basis for further development and utilization of tartary buckwheat polypeptides.