超滤联用中压柱层析分离咸鸭蛋清抗氧化肽

Separation of antioxidant peptides from salted duck egg white by ultrafiltrationcombined with medium-pressure column chromatography

为了提高咸鸭蛋清的综合利用,制备咸鸭蛋清抗氧化肽。该研究以脱盐咸鸭蛋清粉为原料,以自由基清除能力为指标,筛选出最适蛋白酶并确定水解条件。再通过超滤联用中压柱层析技术依次对水解粗肽进行分级和纯化。最后,鉴定所得活性肽纯度及序列。结果表明酸性蛋白酶为最适蛋白酶,其水解最优条件为酶解温度45℃、底物质量浓度为30 g/L、酶添加量为2×10^(4) U/g、水解时间为6 h。该条件下水解液DPPH自由基清除率达94.23%,水解度达33.52%。水解液经超滤和中压柱层析得最优组分UF-43,纯度达91.97%,其自由基清除能力较未分离的水解液提高约5倍。经高效液相串联二级质谱测定,其氨基酸序列为Phe-Gly-Ser-Val,分子质量为408 Da。因此,利用超滤联用中压柱层析技术可实现小分子活性肽分级和纯化。

To improve the comprehensive utilization of salted duck egg white,the antioxidant peptides of salted duck egg white were prepared.Firstly,the optimal protease was screened and hydrolysis conditions were determined using desalted salted duck egg white powder as raw material and free radical scavenging ability as the index.Secondly,the hydrolyzed peptides were successively classified and purified by ultrafiltration combined with medium-pressure column chromatography.Finally,the purity and sequence of the obtained active peptide were identified.Results showed that acid protease was the best protease,and the optimal hydrolysis conditions were as follows:hydrolysis temperature of 45℃,substrate concentration of 3%,enzyme dosage of 2×10^(4) U/g,and hydrolysis time of 6 h.Under these conditions,the DPPH free radical scavenging rate and hydrolysis degree of hydrolysate was 94.23%and 33.52%,respectively.The optimal component UF-43 was obtained by ultrafiltration and medium-pressure column chromatography with a purity of 91.97%,and its free radical scavenging ability was about 5 times higher than that of unseparated hydrolysate.Its amino acid sequence was Phe-Gly-Ser-Val by high-performance liquid chromatography coupled with tandem mass spectrometry(HPLC-MS/MS),and its molecular weight was 408 Da.Therefore,ultrafiltration combined with medium-pressure column chromatography can be used to classify and purify small molecule active peptides.