响应面优化鱿鱼须脱皮液胶原肽酶解工艺及抗氧化活性

Optimization of Enzymatic Hydrolysis of Collagen-Rich Waste Liquid from Squid Tentacle Peeling for Preparing Antioxidant Peptides Using Response Surface Methodology and Their Antioxidant Activities

目的:研究秘鲁鱿鱼须脱皮液胶原肽酶解工艺及抗氧化活性。方法:以水解度和1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率为指标,利用单因素试验和响应面法对其酶解工艺进行优化,并采用2,2’-联氮-二(3-乙基苯并噻唑啉-6-磺酸)二铵盐(2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt,ABTS)自由基与·OH清除率及亚铁还原能力评价其酶解产物抗氧化活性以及通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)和凝胶排阻色谱分析其分子质量分布。结果:最适酶解工艺为酶解温度50℃、初始pH 7.4、底物蛋白质质量分数3.2%、酶解时间3.7 h、酶添加量3 000 U/g。在此条件下,水解度和酶解产物DPPH自由基清除率分别为(37.23±0.08)%和(43.61±0.09)%,对ABTS^+·和·OH清除率的IC_(50)分别为0.37 mg/m L和0.41 mg/m L,且表现出较强的亚铁还原能力;SDS-PAGE显示最适酶解工艺下,蛋白质基本被水解为小分子多肽;凝胶排阻色谱分析可知,酶解产物分子质量在1~5 k D之间。结论:酶解产物具备较强的抗氧化活性,可作为抗氧化活性物质基料,充分开发利用。

Objective： To investigate the optimum parameters for enzymatic hydrolysis of collagen-rich waste liquid from squid（Dosidicus gigas） tentacle peeling and evaluate the antioxidant activity of its hydrolysate. Methods： The hydrolysis parameters were optimized using one-factor-at-a-time method and response surface methodology based on 1,1-diphenyl-2-picrylhydrazyl（DPPH） radical scavenging activity and degree of hydrolysis（DH）. The antioxidant activity of hydrolysate was evaluated by using scavenging rate of 2,2＇-azinobis-（3-ethylbenzothiazoline-6-sulfonic acid）diammonium salt radical（ABTS＋·） and hydroxyl radical scavenging and ferric reducing/antioxidant power（FRAP） assays and the molecular weight distribution was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and gel filtration chromatography. Results： The optimal hydrolysis parameters were determined as follows： hydrolysis temperature, 50 ℃; substrate（protein） content, 3.2%; hydrolysis time, 3.7 h; enzyme dosage, 3 000 U/g; and initial pH, 7.4, resulting in a DH value of（37.23 ± 0.08） % and a scavenging rate of DPPH radical of（43.61 ± 0.09） %, respectively. The half inhibitory concentrations for ABTS^＋· and hydroxyl radical scavenging ability（IC50） of the hydrolysate obtained were 0.37 and 0.41 mg/m L, respectively. Moreover, the product showed a strong reducing capacity. SDS-PAGE analysis showed that most collagens in the waste liquid were hydrolyzed into small peptides with molecular weight of 1-5 k D as determined by gel exclusion chromatography. Conclusion： The hydrolysate derived from waste liquid from squid tentacles peeling has strong antioxidant activity and could be used as a functional seafood condiment and nutritional supplement for further development and utilization.