Incorporation of Leaf Chemical Constituents into Tobacco Brown Pigments

The percent incorporation of chlorogenic acid, chlorogenic acid oxidase (CAO), peroxidase (PRO) and Fraction I protein into the non-dialyzable fraction of acid-precipitated and acid-soluble brown pigments of tobacco was studied by use of ¹⁴C-precursors and appropriate substrates in the in vitro synthesis of brown pigments. The in vitro synthesized pigments showed the same gel permeation profiles as the brown pigments isolated from air-cured leaves. The incorporation of chlorogenic acid was correlated with its quantity in the reaction mixture, and a significant amount of incorporation appeared in the acid-soluble pigments which also correlated with browning intensity. The amount of protein fractions containing CAO and PRO affected the percent incorporation of radioactivity, but this was independent of chlorogenic acid concentration. In addition to the loss of CAO and PRO during the formation of brown pigments, the degradation of proteins, primarily Fraction I protein, occurred in the reaction mixture. The degradation products of Fraction I protein were recovered in both brown pigments. Results indicate that chlorogenic acid oxidation is the major factor for browning, and the degradation products of Fraction I protein and CAO and PRO contribute to the protein moiety of the pigments.