响应面优化混菌发酵蜂王幼虫制备抗氧化肽及其结构鉴定

Preparation of antioxidant peptides by response surface optimization of mixed fermentation from queen bee larvae and its structure identification

以蜂王幼虫冻干粉为原料,通过混菌发酵制备蜂王幼虫抗氧化肽并对其抗氧化活性、结构进行研究。以发酵液的多肽得率和蛋白酶活力为评价指标,通过单因素和响应面试验确定最佳发酵工艺,采用超滤对酶解粗多肽进行分离,测定了粗多肽的氨基酸组成以及各组分的体外抗氧化活性,并通过LC-MS/MS鉴定了抗氧化活性最好的超滤组分氨基酸序列。结果表明,混菌发酵蜂王幼虫制备抗氧化肽的最佳工艺为:枯草芽孢杆菌与黑曲霉的复配比为2.4∶1,接种量6.5%,发酵时间57.5 h,该条件下多肽得率为15.49%,发酵体系中蛋白酶活力为133.55 U/mL。蜂王幼虫粗多肽的抗氧化氨基酸和疏水氨基酸含量分别为58.11%和47.2%。超滤分离得到5个组分F_(1)~F_(5),其中分子质量<1 kDa的组分F_(1)具有最优的抗氧化活性,其清除DPPH自由基和ABTS阳离子自由基的半抑制浓度(half maximal inhibitory concentration,IC_(50))值分别为(1.06±0.01)、(0.165±0.004)mg/mL,铁还原力OD_(700)=0.389。组分F_(1)通过LC-MS/MS分离鉴定并与Mascot多肽数据库比对得到9个可能的肽段,其分子质量在600~1100 Da。研究结果为蜂王幼虫的抗氧化肽产品开发提供实验依据。

Lyophilized powder of queen bee larvae was used as raw material,the antioxidant peptides were prepared by mixed fermentation,and their antioxidant activity and structure were studied.Taking peptide yield and protease activity of the fermentation broth as evaluation indexes,the optimum fermentation process was determined by single factor and response surface experiments.The crude peptides were separated by ultrafiltration.The amino acid composition of crude peptides and the in vitro antioxidant activity of the components of crude peptides were studied.In addition,the amino acid sequence of ultrafiltration component with the best antioxidant activity was identified by LC-MS/MS.The results showed that the optimum fermentation conditions were as follows:the ratio of Bacillus subtilis to Aspergillus niger,2.4∶1;inoculation amount of 6.5%;fermentation time of 57.5 h.Under these conditions,the peptide yield was 15.49%,and the protease activity was 133.55 U/mL.The content of antioxidant amino acids and hydrophobic amino acids in the crude peptides of queen bee larvae was 58.11%and 47.2%.Five components F_(1)-F_(5) were obtained by ultrafiltration.Component F_(1) with molecular weight less than 1 kDa had the best antioxidant activity.Its IC_(50) values of DPPH free radical and ABTS cation ion scavenging rate was(1.06±0.01)and(0.165±0.004)mg/mL,respectively,and iron reducing power OD_(700)=0.389(2 mg/mL).The F_(1) was separated and identified by LC-MS/MS and compared with Mascot peptide database.Nine possible peptides with molecular weight ranging from 600 to 1100 Da were obtained.The results provide experimental basis for the development of antioxidant peptide products of queen bee larvae.