Chemistry of color formation during rooibos fermentation.

Nonenzymatic oxidative degradation of aspalathin, a dihydrochalcone unique to green rooibos (Aspalathus linearis), resulted in formation of the characteristic red-brown color of processed rooibos tea. As recently reported, two colorless dimers were formed by oxidative coupling. Incubations of aspalathin showed further distinct signals. Isolation by multilayer countercurrent chromatography (MLCCC) followed by preparative high-performance liquid chromatography (HPLC) led to pure substances. Subsequent analysis by NMR and MS techniques identified a third colorless dimeric compound. In addition for the first time, two colored structures with dibenzofuran skeleton, (S)- and (R)-3-(7,9-dihydroxy-2,3-dioxo-6-ß-d-glucopyranosyl-3,4-dihydrodibenzo[b,d]furan-4a(2H)-yl) propionic acid, and their corresponding mechanistic precursors were unequivocally established. Color-dilution analysis revealed these compounds as the key chromophores of the incubated aspalathin solutions, ultimately being degraded to unknown, more stable tannin-like structures. Their mechanistic importance to color formation was further underlined by detection of the dibenzofurans also in fermented rooibos tea after trapping with o-phenylenediamine as their corresponding quinoxaline derivatives.

Oxidation of the dihydrochalcone aspalathin leads to dimerization.

Aspalathin and nothofagin are typical ingredients of unfermented rooibos (Krafczyk, N.; Glomb, M. A. J. Agric. Food Chem. 2008, 56, 3368). During oxidation these dihydrochalcones were degraded to higher molecular weight browning products under aerated and nonenzymatic conditions. In the early stages of browning reactions aspalathin formed two dimers. These two compounds were unequivocally established as atropisomers stemming from oxidative A to B ring coupling. Multilayer countercurrent chromatography (MLCCC) and preparative high-performance liquid chromatography (HPLC) were applied to obtain pure substances. The purity and identity of isolated dimers were confirmed by different NMR experiments, HPLC-DAD-MS, and HR-MS. In parallel to the formation of chromophores during the fermentation of black tea, the formation of aspalathin dimers implies an important mechanistic channel for the generation of color during the processing of rooibos.