The impact of flavonoid-phycocyanin covalent conjugation on pigment properties and stability

Phycocyanin is a naturally derived blue pigment comprising a protein–chromophore complex; its use in foods is limited by processing-induced color loss, primarily attributed to protein denaturation that alters chromophore conformation. Covalent protein–polyphenol grafting under alkaline conditions is well established; however, to our knowledge, it has not been applied to stabilize the color of protein-bound chromophores such as phycocyanobilin. In this study, we investigated five flavonoids—quercetin, rutin, kaempferol, nicotiflorin, and naringenin—chosen to represent different capacities for quinone formation, a determinant of alkaline conjugation. We hypothesized that covalent conjugation would improve color retention relative to non-covalent complexation. Among the tested flavonoids, only quercetin and rutin formed covalent conjugates, as shown by mass balance of bound flavonoids and decreases in free thiols/amines, consistent with catechol groups enabling alkaline conjugation. Covalent conjugates of quercetin and rutin improved color retention by 46 % and 21 %, respectively, after heating (72 °C, 30 s) compared with native phycocyanin, an effect not attributable to changes in denaturation temperature. During high-pressure processing (550 MPa, 5 min), rutin and quercetin conjugates also showed better color retention in phosphate buffer (10 mM, pH 7), while all samples remained stable in a pressure-resistant buffer. Antioxidant assays (ABTS, DPPH) indicated that both covalent and non-covalent complexes enhanced radical scavenging immediately after preparation; however, free quercetin precipitated during cold storage, reducing activity, whereas quercetin conjugates retained antioxidant capacity. These findings demonstrate that alkaline covalent conjugation with catechol-containing flavonoids can enhance the processing stability of phycocyanin's blue color while preserving antioxidant functionality.