双酶分步水解制备卵黄高磷蛋白磷酸肽的实验研究

Experimental research on preparation of phosvitin phosphopeptide by double-enzyme fractional hydrolysis

为了研究开发持钙特性显著的卵黄高磷蛋白磷酸肽,以蛋黄粗蛋白为实验原料,优化双酶分步水解制备卵黄高磷蛋白磷酸肽的3种酶系组合方式的最佳技术参数。研究发现:若采用胰蛋白酶一步酶解方式的适宜条件为底物浓度为1%,加酶量5%,pH值8.0,水解温度50℃,水解时间4h;若采用胰蛋白酶+中性蛋白酶双酶分步水解的最佳技术参数为加酶量1.4%,pH值8.0,水解温度40℃,水解时间120min;若采用胰蛋白酶+Alcalase酶双酶分步水解的最佳技术参数为加酶量5%,pH值9.0,水解温度40℃,水解时间90min。采用胰蛋白酶+Alcalase酶双酶分步水解制备卵黄高磷蛋白磷酸肽的水解度最高18.12%,得率最高11.49%,N/P摩尔比最低13.91,被确定为卵黄高磷蛋白磷酸肽制备最佳酶系组合方式。

To study the phosvitin phosphopeptides（PPP） binding calcium notablely, the raw protein of yolk was used as the material. The optimized technological parameters of double-enzyme fractional hydrolysis with three kinds of enzyme system to prepare PPP were investigated. The optimized conditons using trypsase with one step enzymolysis were that： concentration of substrate 1%, enzyme dosage 5%, pH value 8.0, temperature of hydrolysis 50 ℃, time of hydrolysis 4 h. The optimized conditions using trypsase and dispase by doubleenzyme fractional hydrolysis were that： concentration of substrate 1.4%, pH value 8.0, temperature of hydrolysis 40 ℃, time of hydrolysis 120 min. The optimized conditions using trypsase and Alcalase by double-enzyme fractional hydrolysis were that concentration of substrate 5%, pH value 9.0, temperature of hydrolysis 40 ℃, time of hydrolysis 90 min. It was found that the optimzed enzyme system to prepare PPP was double-enzyme fractional hydrolysis using trypsase and Alcalase, of which the degree of hydrolysis reached 18.12%, the yield 11.49% and the N/P molar ratio 13.91.