鲈鱼（Lateolabrax japonicus）蛋白抗氧化肽的酶解制备及结构鉴定

Enzymatic Preparation and Structural Identification of Antioxidant Peptides from Bass Protein

摘要:

为了制备具有较强抗氧化活性的鲈鱼蛋白肽段，采用风味蛋白酶、复合蛋白酶、Alcalase 2.4 L、中性蛋白酶及木瓜蛋白酶等5种商业蛋白酶在其最适宜作用条件下水解鲈鱼鱼肉蛋白，比较分析了鲈鱼酶解产物的水解度（DH）、蛋白回收率和抗氧化活性（氧自由基吸收能力和ABTS自由基清除能力），用超高压液相色谱串联质谱（UPLC-MS/MS）分离鉴定具有较好抗氧化活性酶解产物的肽段结构，并进一步合成肽段验证其活性。结果发现：鲈鱼蛋白的木瓜蛋白酶酶解产物不仅具有最高的DH和蛋白回收率，分别为25.61%和78.71%，同时具有最强的抗氧化活性，其ORAC值为783.56 μmol TE/g，TEAC值为734.55 μmol TE/g。采用UPLC-MS/MS分析鉴定木瓜蛋白酶酶解产物，共得到9条肽段，其中3条肽段具有显著抗氧化活性，分别是EYGTVVVFQ，HRDRLCVVQ和GGGAGMLLK，且EYGTVVVFQ抗氧化活性最强，其ORAC值和TEAC值分别为为1.43 μmol TE/ μmol和2.34 μmol TE/ μmol。本研究结果为深入研究开发鲈鱼抗氧化肽和海洋蛋白资源提供了更多理论基础。

Abstract:

In order to prepare the bass protein peptide with strong antioxidant activity, bass proteins were hydrolyzed by flavorzyme, protamex, alcalase 2.4 L, neutrase and papain under appropriate condition. The degree of hydrolysis (DH), protein recovery rate and antioxidant activity (oxygen radical absorbance capacity and 2,2‘-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radical scavenging capacity) of the hydrolysate of bass proteins were determined by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The peptide structure of the hydrolyzed product with good antioxidant activity was analyzed and identified, and the peptide was further synthesized to verify its activity. The results showed that the papain hydrolysate of bass protein not only exhibited the highest DH (25.61%) and protein recovery rate (78.71%) among five hydrolysates,but also revealed the most potent ORAC and TEAC values of 783.56 and 734.55 μmol TE/g, respectively. UPLC-MS/MS analysis was applied for structural identification of papain hydrolysate. A total of 9 peptides were found by searching in the Uniprot, and 3 of them showed antioxidant activity, which were EYGTVVVFQ, HRDRLCVVQ and GGGAGMLLK. EYGTVVVFQ possessed the most potent ORAC and TEAC values of 1.43 and 2.34 μmol TE/μmol, respectively. This study provided theoretical guidance for the development and utilization of bass antioxidant peptides as well as marine proteins.