Inhibition of tyrosinase-mediated enzymatic browning by sulfite and natural alternatives

T.F.M. Kuijpers

Research output: Thesisinternal PhD, WU


Although sulfite is widely used to counteract enzymatic browning, its mechanism has remained largely unknown. We describe a double inhibitory mechanism of sulfite on enzymatic browning, affecting both the enzymatic oxidation of phenols into o‑quinones, as well as the non‑enzymatic reactions of these o‑quinones into brown pigments. The non‑enzymatic step is inhibited by formation of addition products of sulfite and o‑quinones, sulfophenolics. Sulfonated derivatives of chlorogenic acid were found in sulfite‑treated potato juice, and their structure was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Sulfonation of chlorogenic acid was demonstrated to occur via tyrosinase‑catalyzed o‑quinone formation. Tyrosinase activity was also irreversibly inactivated, in a relative slow time‑dependent way. Simultaneous treatment of tyrosinase with sulfite and competitive inhibitors of tyrosinase did not result in irreversible inactivation, indicating that sulfite acts in the active site of tyrosinase. LC‑MS analysis of protease digests of sulfite‑treated tyrosinase indicated that inactivation occurred via covalent modification of a single amino acid residue in the active site, most likely a copper‑coordinating histidine residue, which is conserved in all PPOs.

As the use of sulfite is controversial, we investigated the effect of potential natural inhibitors of enzymatic browning. Two different polyphenol oxidases (PPOs) were used to screen 60 plant extracts for potential inhibitors. PPOs were found to respond differently to these extracts: an extract that inhibited one PPO could activate the other. This suggests that natural alternatives to replace more generic anti‑browning agents, such as sulfite, are PPO specific.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Gruppen, Harry, Promotor
  • Vincken, Jean-Paul, Co-promotor
Award date25 Oct 2013
Place of PublicationS.l.
Print ISBNs9789461736680
Publication statusPublished - 2013


  • browning
  • sodium sulfite
  • catechol oxidase
  • monophenol monooxygenase
  • enzyme inhibitors


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