Oxidative Conjugation of Chlorogenic Acid-Rich Extracts with Plant Proteins for the Production of a Natural Blue-Green Colorant

Developing natural food colorants remains challenging because suitable sources are limited, stability varies with temperature, pH, and light, and production costs are high. In parallel, valorizing plant-derived bioactive compounds has gained increasing attention as a strategy for developing sustainable food ingredients. Among these compounds, some phenolics, such as caffeic acid, can undergo oxidative reactions with amines in alkaline media, leading to the formation of trihydroxybenzacridine derivatives with intense blue-green coloration and high thermal stability. However, sustainable food-grade approaches for producing these compounds, as well as their stability and safety in food matrices, remain poorly explored. Therefore, this study aimed to develop a blue-green colorant by conjugating phenolic compounds from green coffee and yerba mate with almond proteins and to evaluate its color development, stability in different food matrices, and preliminary cytotoxicity. The colorant was produced through a sustainable six-step process involving extraction of phenolic compounds, production of a powdered extract, incorporation of the extract in the almond suspension, and reaction over time. The effects of extract concentration on the colorant production were evaluated based on the visual color formation over the reaction time, color parameters (L*a*b*), and the phenolic compound content analyzed by ultrahigh performance liquid chromatography (UHPLC) coupled to a photodiode array detector (PDA). The stability of the resulting colorant was subsequently investigated in water, yogurt, and milk during storage, while its cytotoxicity was evaluated in Vero cells using the MTT assay at different sample concentrations (0.5–10%). Color development increased progressively with reaction time, reaching maximum intensity between 24-48 h, particularly at higher extract concentrations. The formation of the blue-green color was characterized by increasingly negative a* and b* values, reaching -12 and -5 after 48 h of reaction, respectively. A reduction in the content of 3-caffeoylquinic acid, 5-caffeoylquinic acid, rutin, and other phenolic compounds in the extracts added to almond suspension after 48 h confirmed the involvement of phenolic compounds in oxidative conjugation. At the same time, residual chlorogenic acids indicated the retention of potentially bioactive phenolic compounds in the final product. During storage, the colorants showed good color stability in water and milk. However, color changes were observed in yogurt, probably due to its lower pH (4,4) and other components. Cell viability was maintained at all tested concentrations, whereas 0.1% Triton, used as a cell death control, markedly reduced cell viability. These results indicate no detectable cytotoxic effect of the colorant on Vero cells under the tested conditions. Overall, the results demonstrate the feasibility of producing a phenolic-based blue-green colorant from plant-derived, food-grade ingredients for use in neutral-pH food products.