基于黄鲫蛋白肽制备南美白对虾多酚氧化酶复合生物抑制剂的研究

Development of a half-fin anchovy(Setipinna taty)hydrolysate peptides based composite biololgical inhibitor for polyphenol oxidase derived from Litopenaeus vannamei

目的 以黄鲫蛋白肽(half-fin anchovy hydrolysate peptides,HAHp)为基料复配抗坏血酸和茶多酚制备南美白对虾多酚氧化酶(polyphenol oxidase, PPO)复合生物抑制剂(composite biological inhibitor, CBI)。方法 以南美白对虾PPO抑制率为检测指标,分别研究HAHp、抗坏血酸、茶多酚、柠檬酸和乙二胺四乙酸二钠盐(ethylenediaminetetraacetic acid disodium salt, EDTA-2Na)单独抑制PPO活性,选取HAHp、抗坏血酸和茶多酚进行三因素三水平正交优化实验优化CBI配方组成,通过酶促动力学研究CBI对南美白对虾PPO的抑制模式。结果 HAHp、抗坏血酸、茶多酚、柠檬酸和EDTA-2Na对南美白对虾PPO都有一定的抑制作用,且有一定的剂量依赖性,但单一抑制剂对PPO的抑制作用有限。HAHp与天然抗氧化剂抗坏血酸和茶多酚复配,经(L933)正交实验优化CBI的最佳配方为:0.7%HAHp、0.02%抗坏血酸和0.20%茶多酚。经测定CBI对南美白对虾PPO抑制率达到94.71%±0.46%,酶促动力学结果表明CBI是一种混合型抑制剂,通过降低南美白对虾PPO对底物亲和力实现抑制PPO活力。结论 以HAHp为基料复配抗坏血酸和茶多酚制备的CBI能够有效地抑制南美白对虾PPO活性,有望开发成一种防虾黑变的天然抑制剂,为南美白对虾天然保鲜剂的研究奠定了理论基础。

Objective To prepare a composite biological inhibitor(CBI)on polyphenol oxidase(PPO)of Litopenaeus vannamei using half-fin anchovy hydrolysate peptides(HAHp)as the basic substrate and combination of ascorbic acid and tea polyphenols.Methods Using Litopenaeus vannamei PPO inhibition rate as the indicator,the PPO inhibition rates of 5 individual inhibitors,namely HAHp,ascorbic acid,tea polyphenols,citric acid,and ethylenediaminetetraacetic acid disodium salt(EDTA-2Na),were determined,and HAHp,ascorbic acid and tea polyphenols were selected to optimize the formula composition of CBI through a three-factor and three-level of orthogonal experiment.Furthermore,the inhibitory mode of CBI on PPO of Litopenaeus vannamei was evaluated through enzymatic kinetics.Results All of these five individual inhibitors HAHp,ascorbic acid,tea polyphenols,citric acid,and EDTA-2Na had certain inhibitory effects on PPO,and showed dose-dependent manners to some extent.However,the inhibitory effect of individual inhibitor on PPO was limited.By combining HAHp with natural antioxidants of ascorbic acid and tea polyphenols,the optimal formula composition of CBI was optimized through L933 orthogonal experiment as follows:0.7%of HAHp,0.02%of ascorbic acid and 0.20%of tea polyphenol.The inhibition rate of CBI on PPO reached to 94.71%±0.46%.The enzymatic kinetics results showed that CBI should be a mixed inhibitor on PPO by reducing the affinity of Litopenaeus vannamei’PPO to substrate.Conclusion CBI prepared by combining ascorbic acid and tea polyphenols with HAHp as the base substrate can effectively inhibit the PPO activity of Litopenaeus vannamei,which can be expected to develop a natural melanosis inhibitor to inhibit shrimp melanosis,thus providing a theoretical basis for the development of natural preservative for Litopenaeus vannamei.