Conjugation of Coffee Phenolics With Milk Bioactive Peptides: Characterization of Coffee and Apple Phenolics and Evaluation of Polyphenol Oxidase as a Natural Biocatalyst.

Abstract

This study aimed to characterize phenolic compounds from green coffee beans and apple juices and to evaluate polyphenol oxidase (PPO) from different apple cultivars as a natural biocatalyst for phenolic-milk bioactive peptide conjugation. Phenolic profiling by HPLC-MS analysis showed that chlorogenic acids were the major phenolic compounds in green coffee beans, with 5-CQA being predominant. Among the tested apple cultivars, Braeburn exhibited the highest PPO activity (6262.4 ± 462.6 U/L) and a moderate chlorogenic acid content. BioZate1 (BZ1) and Glycomacropeptide (GMP) peptides were modified under alkaline and PPO-mediated conditions. A significant reduction in free CQA levels was observed after 24 h, particularly in alkaline-treated samples. MALDI-TOF-MS and RP-HPLC analyses provided evidence consistent with the formation of peptide-CQA conjugates, with alkaline modification yielding the highest bound phenolic content (14.95 μg/mg protein for ABZ1 and 10.14 μg/mg for AGMP). These findings indicate that apple-derived PPO can promote coffee phenolic conversion under the tested conditions and support its potential as a natural biocatalytic system for peptide-phenolic modification. However, the lower CQA-associated fraction observed with PPO-mediated treatment compared with alkaline treatment, together with the absence of a heat-inactivated PPO control, limits definitive attribution of the observed modification specifically to PPO or confirmation of covalent conjugation. Further structural investigations are therefore required to establish the nature and specific sites of peptide-phenolic interactions. Moreover, the developed modification system may provide a basis for future studies on the functional and technological properties of the modified peptides.