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-β-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.