Enzymatic browning in fresh-cut products represents a significant quality deterioration issue impacting commercial value and consumer acceptance. This study investigated 3-mercapto-2-butanone, a naturally occurring thiol compound with established food safety approval, as a novel anti-browning agent. Concentration-response experiments demonstrated that 3-mercapto-2-butanone at 50 μL/L achieved optimal browning inhibition in fresh-cut potato shreds, maintaining excellent color after 4 days while also preventing browning in tobacco leaf pulp, confirming broad cross-species efficacy. Transcriptomic analysis revealed biphasic molecular responses: immediate modulation of phenylalanine metabolism and phenylpropanoid biosynthesis pathways, followed by sustained antioxidant enhancement and metabolic reorganization. Direct enzymatic assays confirmed potent polyphenol oxidase (PPO) inhibition, with complete activity suppression at the optimal concentration. Mechanistic studies demonstrated practical preservation of phenolic substrates while preventing quinone intermediate accumulation during enzymatic reactions. Molecular modeling revealed competitive binding at the PPO active site, with thiol groups forming stable copper coordination complexes that displaced natural substrates and prevented enzymatic oxidation. These findings establish 3-mercapto-2-butanone as a multi-target browning inhibitor operating through direct enzyme inhibition, transcriptional modulation of protective pathways, and competitive substrate displacement. The compound's established safety profile as a food flavoring agent, combined with superior efficacy compared to conventional inhibitors, positions it as a promising commercial alternative for fresh-cut produce preservation.
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