六妹羊肚菌子实体多肽分离纯化及抗氧化活性

Isolation and Purification of Polypeptides from Morchella sextelata and Their Antioxidant Activity

采用碱提酸沉法获得六妹羊肚菌(Morchella sextelata)子实体蛋白质粗提物,以蛋白质酶解的水解度和粗多肽对DPPH自由基的清除率为指标,筛选蛋白酶进行酶解,所得粗多肽依次采用超滤法、DEAE-52纤维素层析法和Sephadex G-25层析法得到多肽,测定其抗氧化活性,通过二级质谱鉴定其氨基酸序列及相对分子质量,并测定不同温度、pH体外模拟胃液和肠液条件下多肽对DPPH自由基清除率的影响。结果表明:羊肚菌子实体蛋白质粗提物提取率为32.94%,其中蛋白质含量为78.3%;碱性蛋白酶酶解效果最好,纯化得到的SE-1对DPPH、ABTS和·OH自由基清除率分别为(86.7±1.75)%、(95.3±1.21)%和(80.6±1.35)%,具有较强的抗氧化活性;SE-1的氨基酸序列为Gly-Gly-Pro-Pro-Gly-Gly-Glu-Asp-Gly-Gly-Phe-Gly-Gly-Met,相对分子质量为1206;分别在25~55℃、pH4~8、体外模拟胃液和肠液消化处理2 h内,多肽对自由基的清除率较高。

Through alkaline extraction and acid precipitation,crude protein extract of Morchella sextelata fruiting bodies was obtained,and the extraction rate and protein content were 32.94%and 78.3%,respectively.Using degree of hydrolysis and DPPH scavenging rate of the resultant crude polypeptide as the indices,alkaline protease was then selected to digest the protein extract to yield crude polypeptide.The crude polypeptide was stepwise subjected to ultrafiltration DEAE-52 cellulose chromatography,and Sephadex G-25 gel filtration chromatography,and the resultant components of each step with high DPPH scavenging rate were used for the next purification.Eventually,two components SE-1 and SE-2 were obtained,and SE-1 had higher protein content and scavenging rates for DPPH,ABTS and·OH,reaching(86.7±1.75)%,(95.3±1.21)%and(80.6±1.35)%,respectively.The amino acid sequence of SE-1 was Gly-Gly-Pro-Pro-Gly-Gly-Glu-Asp-Gly-Gly-Gly-Phe-Gly-Gly-Met,and its relative molecular mass was 1206.Under in vitro simulated gastrointestinal digestion conditions,the DPPH scavenging rate of SE-1 was high at 25–55℃,pH4–8 within 2 h digestion.