Kunming University of Science and Technology, College of Chemistry and Engineering Kunming, Yunnan 650500, China.
Peptides. 2012 Nov;38(1):13-21. doi: 10.1016/j.peptides.2012.08.014. Epub 2012 Aug 29.
To obtain hydrolysates with high degree of hydrolysis (DH) and scavenging radical activity, tilapia skin gelatin (TSG) was hydrolyzed by properase E and multifect neutral. The optimum hydrolysis condition of each enzyme was determined using the orthogonal experiment, and double-enzyme hydrolysis was further applied. The results showed the tilapia skin gelatin hydrolysate (TSGH) obtained by progressive hydrolysis using multifect neutral and properase E had the highest DH and hydroxyl radical scavenging activity. The IC(50) values of TSGH on scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide anion radical (·O(2)) and hydroxyl radical (·OH) activities were also determined. TSGH was further purified using gel filtration chromatography, ion exchange chromatography, and RP-HPLC. The peptides were identified using nano-LC-ESI mass spectrometry. Finally, two antioxidant peptides were identified and the amino acid sequences were Glu-Gly-Leu (317.33 Da) and Tyr-Gly-Asp-Glu-Tyr (645.21 Da), respectively. The IC(50) values of two peptides on hydroxyl radical scavenging activities were 4.61 μg mL(-1)and 6.45 μg mL(-1), respectively. Therefore, the results demonstrated that the hydrolysates of TSG prepared by multifect neutral and properase E could serve as a source of peptides with high antioxidant activity. It provided a scientific basis for the preparation of antioxidant peptides.
为了获得水解度（DH）高且具有自由基清除活性的水解产物，使用蛋白酶 E 和多酶中性蛋白酶对罗非鱼皮明胶（TSG）进行水解。通过正交实验确定了每种酶的最佳水解条件，并进一步进行了双酶水解。结果表明，通过多酶中性蛋白酶和蛋白酶 E 逐步水解得到的罗非鱼皮明胶水解产物（TSGH）具有最高的 DH 和羟基自由基清除活性。还测定了 TSGH 清除 1,1-二苯基-2-苦基肼（DPPH）自由基、超氧阴离子自由基（·O(2)）和羟基自由基（·OH）的 IC(50)值。使用凝胶过滤色谱、离子交换色谱和反相高效液相色谱对 TSGH 进行进一步纯化。使用纳升电喷雾质谱对肽进行鉴定。最后，鉴定出两种抗氧化肽，其氨基酸序列分别为Glu-Gly-Leu（317.33 Da）和 Tyr-Gly-Asp-Glu-Tyr（645.21 Da）。两种肽对羟基自由基清除活性的 IC(50)值分别为 4.61 μg mL(-1)和 6.45 μg mL(-1)。因此，结果表明，多酶中性蛋白酶和蛋白酶 E 制备的 TSG 水解产物可以作为具有高抗氧化活性的肽的来源。这为抗氧化肽的制备提供了科学依据。
