2种蛋白酶酶解曲拉干酪素条件优化及抗氧化性比较

以曲拉干酪素为原料、水解度为指标,在酶解时间、酶解温度、pH值、曲拉干酪素质量浓度、酶添加量单因素试验基础上,采用响应面试验对碱性蛋白酶和胰蛋白酶酶解工艺条件进行优化,并对2种酶解液的1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基、超氧阴离子自由基、羟自由基清除率,Fe2+、Cu2+螯合能力和还原力等抗氧化性指标进行比较。结果表明,碱性蛋白酶和胰蛋白酶分别在酶解时间3.8、2.5 h,酶解温度49.8、47.8℃,曲拉干酪素质量浓度60、35 g/L,pH 8.5、7.5,酶添加量140、2 900 U/g时水解度最大,为24.25%和13.57%。碱性蛋白酶解液超氧阴离子自由基清除率、Fe²⁺螯合能力显著低于胰蛋白酶解液(P<0.01);羟自由基清除能力高于胰蛋白酶解液(P>0.05);2种蛋白酶酶解液在酶解液质量浓度1～5 mg/mL时,Cu²⁺螯合能力、DPPH自由基清除率和还原力随质量浓度均呈上升趋势,Cu2+螯合能力低于Fe2+螯合能力(P>0.05),DPPH自由基清除率和还原力二者差异显著(P<0.01)。2种蛋白酶对酶解物抗氧化性指标影响不同,碱性蛋白酶酶解物抗氧化性相对较优。

Optimization of Hydrolysis Conditions of Qula Casein with Two Proteases and Comparative Antioxidant Activity of Hydrolysates

The enzymatic hydrolysis of Qula casein with either alkaline protease or trypsin was optimized based on degree of hydrolysis (DH). The effects of hydrolysis time, temperature, pH, substrate concentration and enzyme concentration on DH were investigated by one-factor-at-a-time method. Subsequently, response surface methodology was used to optimize four significant variables. The resulting hydrolysates were comparatively evaluated for their 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion and hydroxyl radical scavenging capacity, Fe2+ and Cu2+ chelating ability, and reducing power. The results showed that the optimal hydrolysis time, temperature, substrate concentration, pH and enzyme concentration were 3.8 h, 49.8 ℃, 60 g/L, 8.5 and 140 U/g for alkaline protease; and 2.5 h, 47.8 ℃, 35 g/L, 7.5 and 2 900 U/g for trypsin, yielding the maximum DH value of 24.25% and 13.57%, respectively. The alkaline protease hydrolysate had significantly lower superoxide anion radical scavenging capacity and Fe2+ chelating ability (P < 0.01) but higher hydroxyl radical scavenging capacity than the trypsin hydrolysate (P > 0.05). For both hydrolysates, Cu2+ chelating ability, DPPH radical scavenging capacity and reducing power increased with increasing concentration from 1 to 5 mg/mL, and Cu2+ chelating ability was lower than Fe2+ chelating ability (P > 0.05). Significant difference (P < 0.01) was found as far as DPPH radical scavenging capacity and reducing power were concerned. In summary, the two proteases had different impacts on the antioxidant activity of hydrolysates and the alkaline protease hydrolysate better antioxidant properties.