Influence of Nucleophilic Amino Acids on Enzymatic Browning Systems

In the present study the enzymatic oxidation of gallic acid and catechin catalyzed by nashi pear polyphenol oxidase (PPO) in the presence of the amino acids lysine, arginine, or cysteine was investigated for polyphenol-amino acid adducts. HPLC analyses revealed the formation of two novel dihydrobenzothiazine carboxylic acid derivatives (8-(3',4'-dihydro-2 H-chromene-3',5',7'-triol)-3,4-dihydro-5-hydroxy-2 H-benzothiazine-3-carboxylic acid and 7-(3',4'-dihydro-2 H-chromene-3',5',7'-triol)-3,4-dihydro-5-hydroxy-2 H-benzothiazine-3-carboxylic acid) from 2'-cysteinyl catechin and 5'-cysteinyl catechin in cysteine incubations, respectively. In contrast, arginine and lysine did not lead to any amino acid adducts. Target compounds were separated by high-performance countercurrent chromatography and preparative HPLC and unequivocally characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Mechanistic incubations starting from the catechin-cysteine adducts showed that both catechin and PPO are crucial components in the formation of the dihydrobenzothiazines. The cysteine incubations showed a red-brown coloration, which coincided with formation and degradation of the dihydrobenzothiazines finally leading to the formation of high-polymeric melanins. Therefore, these compounds might be the key intermediates to understand development of color during cysteine-driven enzymatic browning reactions.