鲣鳔蛋白抗氧化酶解物制备工艺

Methodology of the preparation of enzymatic hydrolysates with antioxidant activity of skipjack tuna (Katsuwonus pelamis) swim bladder protein

为有效提高鲣鳔蛋白的附加值,研究以DPPH自由基清除率为抗氧化活性评价指标,采用蛋白酶酶解制备活性多肽的工艺,选用菠萝蛋白酶、复合蛋白酶、碱性蛋白酶、木瓜蛋白酶、胃蛋白酶、胰蛋白酶、中性蛋白酶7种酶在各自最适的条件下酶解,筛选出复合蛋白酶为最适用酶,通过单因素实验分别研究加酶量、溶液初始p H、酶解温度和时间对酶解物抗氧化活性的影响,在此基础上,根据响应面法优化鲣鳔抗氧化酶解物的制备工艺。结果显示,最佳酶解工艺条件为加酶量8.53 U/mg,p H 5.54,温度50.03°C,时间5.07 h。此外,利用超滤法对最佳条件下制备的酶解物进行初步分级,得到分子质量分别为大于10 000 u、3000~10 000 u和小于3000 u的3段组分,且这3段组分对DPPH自由基的半抑制浓度IC50值分别为0.64、0.52和0.37 mg/m L。研究表明,最优条件下制备的酶解物的DPPH清除率达72.00%,与模型预测值71.60%接近,且其中小于3000 u的组分具有较强的DPPH自由基清除活性。

In order to obtain higher added-value of skipjack tuna（Katsuwonus pelamis） swim bladder, compound proteinase was selected for enzymatic hydrolysis of skipjack swim bladder from seven enzymes including bromelaine, compound proteinase, alkaline proteinase, papain, pepsin, trypsin and dispase. With the DPPH radical scavenging rate of hydrolysates as evaluating index, single-factor experiment was employed to investigate the effects of enzyme dosage, initial p H, reaction temperature and time on the antioxidant activity of hydrolysates separately. On the basis of the above observations of single-factor experiment, the optimum conditions for preparing antioxidant hydrolysates of skipjack tuna swim bladder were explored through response surface methodology（ RSM）. The results showed that, the optimal hydrolysis conditions were as follows： 8.53 U/mg of enzyme dosage, 5.54 of initial solution p H, 50.03 ℃ of reaction temperature and 5.07 h of reaction time.Furthermore, the hydrolysates were primarily sorted into three fractions with different molecular weight above10 000 u, 3000 to 10 000 u and 0 to 3000 u by using ultrafiltration. The inhibitory concentration 50%（IC50） values to DPPH radical of three fractions were 0.64, 0.52 and 0.37 mg/m L, respectively. It is demonstrated that DPPH radical scavenging rate of the hydrolysates could be up to 72.00% under these optimal conditions, which was in good agreement with the predicted value of 71.60%, and that fraction below 3000 u of the hydrolysates possesses the strongest effect. Preliminary results indicate that the hydrolysates of skipjack swim bladder could be an effective ingredient for antioxidant products such as health food or cosmetics.