Stable Benzacridine Pigments by Oxidative Coupling of Chlorogenic Acid with Amino Acids and Proteins: Toward Natural Product-Based Green Food Coloring

Abstract

The occasional greening of sweet potatoes and other plant tissues observed during cooking or other food processing has been shown to arise from the autoxidative coupling of chlorogenic acid (CGA, 5-caffeoylquinic acid) with amino acid components, leading to trihydroxybenzacridine pigments. To explore the potential of this reaction for food coloring, we report herein the optimized biomimetic preparation of trihydroxybenzacridine pigments from CGA and amino acids such as glycine and lysine, their straightforward purification by gel filtration chromatography, the UHPLC-MS/MS analysis of the purified pigment fraction, and a detailed characterization of the pH-dependent trihydroxybenzacridine chromophore. Similar green pigments were also obtained by analogous reaction of CGA with a low-cost protein, bovine serum albumin, and by simply adding CGA to chicken egg white (CEW) under stirring. Neither the purified pigments from amino acids nor the pigmented CEW exerted significant toxicity against two human cell lines, Caco-2 and HepG2, at doses compatible with common use in food coloring. Additions of the pure pigments or pigmented CEW to different food matrices imparted intense green hues, and the thermal stability of these preparations proved satisfactory up to 90 °C. The potential application of the greening reaction for the sensing of fish deterioration is also disclosed.

Lowering greening of cookies made from sunflower butter using acidic ingredients and effect on reducing capacity, tryptophan and protein oxidation

“…This higher acidity did not favour green pigment formation, as also illustrated by the the suppression of green derivatives measured at 680 nm (Table 1). The observed A max at 680 nm, in the lysine-chlorogenic acid adducts solution, was attributed to trihydroxy benzacridine (TBA) derivatives, consistent with observations in model systems (Iacomino et al, 2017), in our previous study (Liang & Were, 2018b), and that of cookies in the current study (Table 2, Fig. 1, supplementary materials Table S1).…”

Preliminary Observations on the Interactions Between Chlorogenic Acid and Select Amino Acids

“…Acidification of reaction mixtures containing the green pigment leads to a loss of that green color similar to what is discussed elsewhere. [7] However, the results presented in Figure 7 do suggest that the green pigment generated in the presence of TY was more acid-stable than the green pigment generated in the presence of Y. In addition, acidification of the reaction mixture resulting from the reaction between CGA and W, led to a loss of the absorbance at 700nm, but not to a loss of the absorbance at 550nm (see Figure 9, panel D).…”

“…This observation is in line with the observed pH effects on the green pigment obtained through the reaction between AA and CGA. [7,18] To illustrate the effect of the dilution with SEC solvent on the green pigment present in the mixtures following the reaction between CGA and Y or TY, the triplicate crude reaction mixtures were diluted twofold with either deionized water or SEC solvent. Figure 7 presents the average Vis spectra thus obtained.…”

“…The AA side chain is cleaved off and the amino functionality is left behind inside the benzacridine structure (circled in Figure 1). [6,7] Figure 1: Formation of benzacridine pigment from CGA and AAs as based upon [6,7]. Thus, apart from special cases like cysteine (C; highly reactive thiol that reacts with monomers of CGA), lysine (K; two types of primary amino groups) or proline (P; secondary amino group), the use of any type of  should lead to the same green pigment.…”

“…Catechols, when oxidized to their quinone form, can readily react with primary amino functionalities leading to Michael-type additions, Schiff base reactions or Strecker degradation reactions as reviewed by Yang et al [8] Reactions between catechols and AA have been discussed for: a) the interactions between DOPA and C leading to pheomelanin [9], b) the crosslinking interactions between polydopamine or DOPA units in select proteins [10] and c) the interactions between CGA and AA [6,7] as discussed in this report. Given the abundance and importance of catecholic compounds (catechol, pyrogallol, epinephrine, norepinephrine, dopamine, DOPA, caffeic acid, catechin) in the physiology of main lifeforms, it appears to this author that a broad, systematic study of the chemistry between AAs and catechols is lacking.…”