Validation of spectrophotometric microplate methods for polyphenol oxidase and peroxidase activities analysis in fruits and vegetables

Abstract Enzymes polyphenol oxidase (PPO) and peroxidase (POD) play important roles in the processing of fruits and vegetables, since they can produce undesirable changes in color, texture and flavor. Classical methods of activity assessment are based on cuvette spectrophotometric readings. This work aims to propose, to validate and to test microplate spectrophotometric methods. Samples of apple juice and lyophilized enzymes from mushroom and horseradish were analyzed by the cuvette and microplate methods and it was possible to validate the microplate assays with satisfactory results regarding linearity, repeatability, accuracy along with quantitation and detection limits. The proposed microplate methods proved to be reliable and reproducible as the classical methods besides having the advantages of allowing simultaneous analysis and requiring a reduced amount of samples and reactants, which can beneficial to the study of enzyme inactivation in the processing of fruits and vegetables.

摘要 多酚氧化酶（polyphenol oxidase，PPO）与过氧化物酶（peroxidase，POD）在果蔬加工过程中发挥着重要作用，因其可引发色泽、质地与风味出现不良变化。传统酶活性评估方法基于比色皿分光光度读数。本研究旨在提出、验证并测试微孔板分光光度法。采用比色皿法与微孔板法对苹果汁样品以及蘑菇、辣根来源的冻干酶进行了分析，最终成功验证了微孔板检测法，其在线性、重复性、准确性以及定量限与检出限方面均取得了令人满意的结果。所提出的微孔板法与传统方法同样可靠且可重复，此外还具备可实现同步分析、所需样品与试剂量更少的优势，这将有益于果蔬加工过程中酶失活相关研究的开展。