Discovery of a natural cyan blue: A unique food-sourced anthocyanin could replace synthetic brilliant blue

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Science Advances  07 Apr 2021:
Vol. 7, no. 15, eabe7871
DOI: 10.1126/sciadv.abe7871
  • Fig. 1 Color expression of four blue pigments and the structure of RCAs.

    (A) The UV-visible spectra depicted highlights λmax and its respective location on the electromagnetic (EM) absorbance spectra. λmax for FD&C Blue No. 1 is 630 nm, spirulina is 617 nm, RCAs at pH 8 are 608 nm, and the Al3+(P2)3 complex at pH 7 is 640 nm. The violet contribution (VC) of each blue, defined as the absorption in the 500- to 600-nm range (depicted in gray), is shown. Values and calculations for VC, i.e., area under the curve (AUC), are provided in sections S1 and S2. (B) The red flavylium cation of the main anthocyanins in red cabbage is depicted as P1 to P8 on the HPLC-diode-array-detection (DAD) trace (520-nm detection) (method S11.2). RCAs display a structural homology (red and blue arrows) where the common building blocks are a cyanidin chromophore (highlighted in red), sugars, and small acyl groups (highlighted in blue). The sophorose moiety is bound at position 3, and a glucose is bound at position 5. The key differentiation is acyl groups (p-coumaroyl, feruloyl, and sinapoyl) and their substitution patterns on sugar 1 (Glc-1) or sugar 2 (Glc-2) of the sophorose.

  • Fig. 2 Absorption spectrum and stability sinapoyl containing RCAs.

    (A) Cuvettes with the corresponding visible spectrum, 400 to 700 nm, for P2, P5, and P8 at pH 7 with and without Al3+. The different equivalences of Al are color-coded: black = 0 eq, red = 1/3 eq, and blue = 1 eq. The λmax values for 0 eq are 598 nm for P2, 596 nm for P5, and 598 nm for P8. Adding 1/3 eq Al3+ generates a λmax value of 640 nm for P2, 602 nm for P5, and 617 nm for P8. Adding 1 eq of Al3+ does not appear to affect λmax for P2, P5, or P8. (B) Stability (absorbance at λmax of aqueous solutions monitored over time) of anthocyanins P2, P5, and P8 at pH 7 over time with (solid line) and without Al3+ (dashed line). The addition of Al3+ enhances the stability of both P2 and P8; however, it has a minor effect on the stability of P5. P8 has an inherent additional stability because of diacylation but is still surpassed by the stability of P2 + Al3+. Once the Al3+(P2)3 complex is formed, it demonstrates a marked increase in stability that is thought to stem from the intermolecular interlocking of the P2 moieties (figs. S8.2 to S8.5).

  • Fig. 3 Proposed trimeric structure.

    (A) A proposed trimeric structure is shown in (B), where three P2 molecules form a propeller-like structure around the aluminum ion. NMR, MS, and Cotton effects of Al3+(P2)3 were confirmed (details provided in sections S7, S8, and S10). Computational modeling indicates that the large bathochromic shift observed stems mainly from the enhanced distortion of angle θ, highlighted in blue on the molecular structure. Inset shows the calculated effect of λmax as a function of θ angle distortion. The color of each circle is the average expected color, the size of the marker corresponds to average oscillator strengths, and error bars are computed as SD of λmax. The data for the figure correspond to cyanidin-3-glucoside in fully solvated water. Molecular structures were pruned by replacing the sugar with a methyl group and absorption spectra calculated with time-dependent density functional theory [6-311G(d,p) at B3LYP] (DFT-B3LYP) and fully detailed in section S10. (B) 3D representation and coordination of the anthocyanin P2 around the aluminum.

  • Fig. 4 Enzyme discovery and design for transforming RCAs into P2.

    (A) Structural similarities between RCAs facilitate enzymatic enhancements of P1 and P2. Selective hydrolysis cleaving the acyl group from Glc-1 (red highlight) transforms P3, P4, and P5 into P1, and P6, P7, and P8 into P2. Selectivity toward the acyl group on Glc-1, but not the acyl group on Glc-2 (blue highlight), is critical for P2 production. (B) HPLC chromatograms of RCAs treated with different enzymes: RCAS without enzyme (red, top), RCAs after incubation with the native 1AUR WT protein (blue, middle), and RCAs after incubation with mutant 1AUR M73H (black, bottom). See method S11.2 for reaction conditions. (C) Phylogenetic tree of enzymes tested for activity on RCAs. Black triangles indicate that enzyme had desired hydrolytic activity. Additional metadata are provided in the tree and corresponding legend. See method S11.1 for methodology. (D) (Top) Cartoon depiction of 1AUR WT with P6 in the active site (yellow with gray highlight), with the protein backbone shown in blue. The predicted molecular interactions of the native amino acid (bottom left) and designed mutation (bottom right) with P6 are illustrated. Dashed lines from the introduced histidine depict the predicted new hydrogen bonds between the enzyme and anthocyanin, which we hypothesize contributes to the enhanced activity.

  • Fig. 5 Product prototyping.

    (A) Visible absorption spectra of Al3+(P2)3 at pH 7 (solid lines) and RCAs at pH 8 (dashed lines) in saturated sugar syrup monitored over time. Over the course of 10 days, the absorbance at λmax of RCAs drops by 57%, indicating loss of cyanidin chromophore, whereas the Al3+(P2)3 complex shows remarkable stability with only 14% loss of absorbance at λmax over the course of 55 days. Experimental details are in section S13.3. (B) Sugar-coated lentils (top) using the four blue colorants were obtained under typical panning conditions. The colorants were FD&C Blue No. 1, spirulina, RCAs at pH 8, and Al3+(P2)3 at pH 7. The colorimetry data are plotted in a*b* space and L* values in the sidebar. The Al3+(P2)3 complex at pH 7 provides a very similar hue angle to FD&C Blue No. 1 (sections S1 and S13). (C) Sugar-coated lentils (top) where the colorant blends used were synthetic green, spirulina, red cabbage at pH 8, and Al3+(P2)3 at pH 7, with the latter three mixed with safflower (Saf) as the yellow component. Al3+(P2)3 at pH 7 with safflower provides a very similar hue angle to synthetic green (fig. S13.4 and table S13.4).

Supplementary Materials

  • Supplementary Materials

    Discovery of a natural cyan blue: A unique food-sourced anthocyanin could replace synthetic brilliant blue

    Pamela R. Denish, Julie-Anne Fenger, Randall Powers, Gregory T. Sigurdson, Luca Grisanti, Kathryn G. Guggenheim, Sara Laporte, Julia Li, Tadao Kondo, Alessandra Magistrato, Mícheál P. Moloney, Mary Riley, Mariami Rusishvili, Neda Ahmadiani, Stefano Baroni, Olivier Dangles, Monica Giusti, Thomas M. Collins, John Didzbalis, Kumi Yoshida, Justin B. Siegel, Rebecca J. Robbins

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    • Sections S1 to S13
    • References

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