Improved Stability of Blue Colour of Anthocyanins from Lycium ruthenicum Murr. Based on Copigmentation

Abstract

Natural blue food colourant is rare. The aim of this work was to screen compounds from the common copigments that could improve the blue tones of anthocyanins (ACNs) and to investigate the effect of different copigments on the colour stability of anthocyanins in neutral species. International Commission on Illumination (CIE) colour space, UV, IR, NMR, atomic force microscopy (AFM) and computational chemistry methods were utilised to evaluate ACNs from Lycium ruthenicum Murr. (LR), which is complexed with food additives and biological agents. The results indicate that Pro-Xylane (PX), Ectoin (ECT) and dipotassium glycyrrhizinate (DG) enhance the blue colour of the ACNs. ACNs-PX presents a colour close to Oxford Blue and has a surface height of 2.13 ± 0.14 nm and slightly improved stability. The half-life of ACNs-DG is improved 24.5-fold and had the highest complexation energy (-50.63/49.15) kcal/mol, indicating hydrogen bonds and π-π stacking forces enhance stability. These findings offer a new perspective for anthocyanin utilisation as a blue colourant and contribute to the large-scale application of LR.

Keywords: Lycium ruthenicum Murr.; anthocyanins; blue colour; copigmentation; stability.

Figure A1

HPLC spectrum of Lycium ruthenicum Murr. (LR) and p3cr5g. The eluted gradient: 0–20 min, 10–17% A; 20–25 min, 17% A; 25–55 min, 17–25%; 55–60 min, 25–90% A. Mobile phases, A: acetonitrile, B: 0.6% trifluoroacetic acid in water. (A) HPLC spectrum of LR extracts at 520 nm; (B) HPLC spectrum of LR extracts at 280 nm; (C) HPLC analysis spectrum of prepared p3cr5g at 280 nm.

Figure A2

¹H NMR spectrum of p3cr5g.

Figure A3

¹³C NMR spectrum of p3cr5g.

Figure A4

The effect of concentration of compounds on colour differences in anthocyanin solution. (A) ACNs−PX; (B) ACNs−ECT; (C) ACNs−DG. Compared with the group at 0 mg/mL, * p < 0.05, ** p < 0.01.

Figure A5

The effect of concentration of compounds on colour differences in anthocyanin solution. (A) Anthocyanins−Nicotinamide (ACNs−NTM); (B) Anthocyanins−Allantoin (ACNs−ALT); (C) Anthocyanins−Ergothioneine (ACNs−EGT); (D) Anthocyanins−α−arbutin (ACNs−α−ABT); (E) Anthocyanins−β−arbutin (ACNs−β−ABT); (F) Anthocyanins−Inositol (ACNs−IST); (G) Anthocyanins−Ceramide (ACNs−CRM); compared with the group at 0 mg/mL, * p < 0.05, ** p < 0.01.

Figure A6

Visual display and quantitative values of the blue contribution of anthocyanin complex.

Figure A7

Scheme of the relevant molecular structure of p3cr5g in the transversion of protonation and deprotonation.

Figure A8

Non−covalent interaction for the anthocyanin (A7 species) compound−binary complex by IGMH analysis. (A) p3cr5g−PX; (B) p3cr5g−ECT; (C) p3cr5g−DG.

Figure 1

The optimisation of concentrations and reaction time of anthocyanin colour. (A) The effect of concentrations of anthocyanins on International Commission on Illumination (CIE) parameters; (B) the effect of reaction time of anthocyanins on CIE parameters; (C) the effect of concentrations of anthocyanins on colour differences; (D) the effect of reaction time of anthocyanins on colour differences. Groups with different letters indicate significant differences, p < 0.05; the same letters indicate non−significant differences, p > 0.05.

Figure 2

The effect of concentration of compounds on the colour appearance of anthocyanins. (A) Anthocyanins−Pro−Xylane (ACNs−PX); (B) Anthocyanins−Ectoine (ACNs−ECT); (C) Anthocyanins−Dipotassium glycyrrhizinate (ACNs−DG).

Figure 3

The stability evaluation of anthocyanin complex at 40 °C storage within 168 h. (A). The colour swatch; (B) the colour changes; (C) the absorbance changes; I: ACNs (0.4 mg/mL); II: ACNs (0.4 mg/mL)−PX (12.0 mg/mL); III: ACNs (0.4 mg/mL)−ECT (120.0 mg/mL); IV: ACNs (0.4 mg/mL)−DG (120.0 mg/mL). Groups with different letters indicate significant differences, p < 0.05; the same letters indicate non−significant differences, p > 0.05.

Figure 4

The spectrum of anthocyanin complex. (A) FTIR spectra of anthocyanin complex; (B) ¹H NMR spectra of petunidin−3−O−coumaroylrutinoside−5−O−glucoside−Pro−Xylane (p3cr5g−PX) at different molar ratios; (C) ¹H NMR spectra of petunidin−3−O−coumaroylrutinoside−5−O−glucoside−Ectoine (p3cr5g−ECT) at different molar ratios; (D) ¹H NMR spectra of petunidin−3−O−coumaroylrutinoside−5−O−glucoside−Dipotassium glycyrrhizinate (p3cr5g−DG) at different molar ratios.

Figure 5

Atomic force microscope topography of anthocyanin compound−binary complex. (A) p3cr5g; (B) p3cr5g−PX; (C) p3cr5g−ECT; (D) p3cr5g−DG; three−dimensional images (upper) and two−dimensional images (lower) are presented.

Figure 6

Non−covalent interaction for the anthocyanin (A_4′ species) compound−binary complex by IGMH analysis. (A) p3cr5g−PX; (B) p3cr5g−ECT; (C) p3cr5g−DG.