Polyphenol oxidase mediated substrate selectivity underlies enzymatic browning in the edible insect Antheraea pernyi...

Antheraea pernyi pupa is highly susceptible to enzymatic browning during processing, yet the endogenous phenolic substrates involved and the substrate selectivity of PPO remain poorly understood. In this study, LC-MS/MS, initial rate analysis, and untargeted metabolomics were integrated to investigate browning mediated by PPO and dependent on substrate type in A. pernyi pupa. Fourteen endogenous compounds related to phenolic metabolism and browning reactions were detected, among which taxifolin, caffeic acid, and chlorogenic acid showed clear browning potential mediated by PPO. Taxifolin exhibited the strongest catalytic response across the tested concentration range, indicating that PPO activity depended on substrate type. It also showed the highest browning activity across the tested pH range and reached its maximum at pH 6, whereas the caffeic acid and chlorogenic acid systems showed their highest activity at pH 5 and declined markedly as pH increased. After tissue disruption, visible browning developed rapidly, while total phenolic content decreased from 0.01032 to 0.00768 mg GAE/g within 2 h, corresponding to an overall reduction of 25.6%; the most pronounced decrease (18.3%) occurred within the first 0.5 h. Untargeted metabolomics further revealed clear substrate-dependent metabolic divergence and identified key oxidation-associated metabolites, including hydroquinone, pyrocatechol, 3,4-dihydroxy-L-phenylalanine, and 5,6-dihydroxyindole-2-carboxylic acid. The browning process was also associated with perturbation of pathways related to amino acid metabolism. These results indicate that browning in A. pernyi pupa is initiated by selective oxidation of susceptible endogenous substrates and then extended through continued chemical evolution centered on quinones. This work provides a mechanistic basis for targeted browning control during the processing of A. pernyi pupa.