Influence of the Anthocyanin and Cofactor Structure on the Formation Efficiency of Naturally Derived Pyranoanthocyanins

Abstract

Pyranoanthocyanins are anthocyanin-derived pigments with higher stability to pH and storage. However, their slow formation and scarcity in nature hinder their industrial application. Pyranoanthocyanin formation can be accelerated by selecting anthocyanin substitutions, cofactor concentrations, and temperature. Limited information is available on the impacts of the chemical structure of the cofactor and anthocyanin; therefore, we evaluated their impacts on pyranoanthocyanin formation efficiency under conditions reported as favorable for the reaction. Different cofactors were evaluated including pyruvic acid, acetone, and hydroxycinnamic acids (p-coumaric, caffeic, ferulic, and sinapic acid) by incubating them with anthocyanins in a molar ratio of 1:30 (anthocyanin:cofactor), pH 3.1, and 45 °C. The impact of the anthocyanin aglycone was evaluated by incubating delphinidin, cyanidin, petunidin, or malvidin derivatives with the most efficient cofactor (caffeic acid) under identical conditions. Pigments were identified using UHPLC-PDA and tandem mass spectrometry, and pyranoanthocyanin formation was monitored for up to 72 h. Pyranoanthocyanin yields were the highest with caffeic acid (~17% at 72 h, p < 0.05). When comparing anthocyanins, malvidin-3-O-glycosides yielded twice as many pyranoanthocyanins after 24 h (~20%, p < 0.01) as cyanidin-3-O-glycosides. Petunidin- and delphinidin-3-O-glycosides yielded <2% pyranoanthocyanins. This study demonstrated the importance of anthocyanin and cofactor selection in pyranoanthocyanin production.

Keywords: 10-catechyl-pyranoanthocyanins; accelerated formation; hydroxyphenyl-pyranoanthocyanins; naturally derived pigments.

Figure 1

Comparing the PACN formation after 72 h of anthocyanin (saponified black carrot, sBC) incubation with caffeic acid (CA), acetone, or pyruvic acid (PA). Chromatograms show the max plot in the 475–520 nm range. Table shows the wavelength of maximum absorption in the visible range (λ_vis-max), mass per charge ratio (m/z) of the main ion and its aglycone, and tentative identity (ID).

Figure 2

Pyranoanthocyanin yield (%PACN) with caffeic acid (CA) and pigment remaining (%) after incubation with different cofactors for 24, 48, and 72 h at 45 °C with cyanidin-glycosides (from saponified black carrot, sBC). Different Greek letters show significant differences among time points at a 0.05 level. Different letters show statistically significant differences among cofactors at the same time point at a 0.05 level. Results are expressed as means ± standard error (n = 3). ACN: anthocyanin, PA: pyruvic acid.

Figure 3

Comparing the pyranoanthocyanin formation after 72 h of anthocyanin (saponified black carrot, sBC) incubation with caffeic acid (CA), p-coumaric acid (pCA), ferulic acid (FA), and sinapic acid (SA). Chromatograms show the max plot in the 475–520 nm range. Table shows the wavelength of maximum absorption in the visible range (λ_vis-max), mass-per-charge ratio (m/z) of the main ion and its aglycone, and tentative identity (ID). C3XyGlGa: cyanidin-3-O-xylosyl-glucosyl-galactoside, C3XyGa: cyanidin-3-O-xylosyl-galactoside.

Figure 4

Pyranoanthocyanin yield (%PACN) with different hydroxycinnamic acids and pigment remaining (%) after incubation for 24, 48, and 72 h at 45 °C with cyanidin-glycosides (from saponified black carrot, sBC). Different Greek letters show significant differences among time points at a 0.05 level. Different letters show significant differences among cofactors at the same time point at a 0.05 level. Asterisks (*) and (**) indicate significant differences against the cofactor-free control (sBC) at a 0.05 and 0.01 level, respectively. Results are expressed as means ± standard error (n = 3). pCA: p-coumaric acid. CA: caffeic acid, FA: ferulic acid, SA: sinapic acid, Eq: equation.

Figure 5

Anthocyanins from fractions with different aglycones and PACN formation after 24 h of 45 °C incubation with caffeic acid (CA). Chromatograms show the max plot in the 475–520 nm range. Table shows the wavelength of maximum absorption in the visible range (λ_vis-max), mass-per-charge ratio (m/z) of the main ion and its aglycone, and tentative identity (ID).

Figure 6

Pyranoanthocyanin yield (%PACN) and pigment remaining (%) after incubation of anthocyanin fractions with different aglycones with caffeic acid (CA) for 24 h at 45 °C. Different letters show significant differences among different fractions at a 0.05 level. Results are expressed as mean ± standard error (n = 3). ACN: anthocyanin, Dp: delphinidin-derivatives fraction, Cy: cyanidin-derivatives fraction, Pt: petunidin-derivatives fractions, Mv: malvidin-derivatives fraction, Eq: equation.