Analysis of copigmentation between organic acids and cyanidin 3-O-glucoside in blackberry wine

Abstract

Background

Cyanidin 3-O-glucoside (C3G) is the most abundant anthocyanin in nature. However, the copigmentation of organic acids with C3G has not been analyzed. In this study, the contents of organic acids and C3G in blackberry wine were determined, and the copigmentation between them was investigated.

Results

The addition of organic acids did not significantly change the molecular weight of the compounds in the solution. Regarding the copigmentation of organic acids with C3G, the organic acids altered the solution polarity. This led to the rotation of the B ₋ ring plane of C3G, restored the shielding color intensity, and enhanced the color rendering strength of the C3G solution. Hydrophobic and hydrogen bonding forces are the main intermolecular forces that affect their copigmentation. This study enriched copigmentation of organic acids on anthocyanins. Moreover, it provides a reference to produce high-quality fruit wine and reduce environmental pollution caused by perishable fruits.

Highlights

• •During copigmentation, C3G did not form covalent bonds with organic acids.
• •Fermentation with organic acid can boost the color intensity of blackberry wine.
• •Organic acids can alter the structure of C3G, causing copigmentation effect.

1. Introduction

Berries are rich in minerals, vitamins, and polyphenolic compounds, which can supplement the human body with a variety of beneficial substances (de Souza et al., 2014). Among these, polyphenolic compounds such as anthocyanins, flavan-3-ols, and flavanols play important roles in preventing and delaying chronic diseases related to oxidative stress, such as cardiovascular and cerebrovascular diseases, diabetes, and cancer (Brownmiller et al., 2008).

Blackberries (Rubus sp.) are important sources of natural antioxidants, rich in anthocyanins, tannins, and phenolic acids, offering health benefits such as anti-inflammatory, antioxidant, and free radical scavenging properties (Wang & Lin, 2000). However, owing to their high- water content and fragile tissues, blackberries are highly perishable and must be consumed within a few days after harvesting, because they are prone to spoilage and loss (Temocico et al., 2008). Brewing the blackberries into wine not only preserves the abundant polyphenolic compounds in the fruits, which can reduce the economic losses caused by fruit spoilage owing to untimely consumption, but also meets the modern preference for low-sugar diets with its unique flavor and low sugar content (Johnson et al., 2016).

The winemaking process of blackberry is usually similar to that of grape wine; however, the browning during its aging process is far more severe than that of red wine, greatly restricting its development (Wu et al., 2024). The browning phenomenon in aging of wine is attributed to anthocyanin content reduction in the wine over time, causing the red color to gradually fade and eventually leading to browning. Anthocyanin instability is caused by various factors such as light, oxygen, temperature, and pH (Del Giovine & Bocca, 2003). Currently, the copigmentation phenomenon between organic acids and anthocyanins, which can maintain the stability of anthocyanins in wine, has become a research focus. In red wine, the copigmentation mechanism of malvidin 3-glucoside (M3G) and organic acids has been widely studied (Boulton, 2001). This can be attributed to three aspects. First, organic acids change the pH of the wine, which helps to enhance the stability of substances in the wine (Cristea et al., 2021). Second, the interaction between organic acid molecules and the M3G plane serves to enhance color of the wine (Han & Xu, 2015). Third, glycosylation and acetylation reactions occur between glycosides and organic acids with M3G, resulting in the formation of new types of anthocyanin (Carvalho et al., 2010). However, for its copigmentation with organic acids, C3G, as the most abundant pigment in fruits including blackberries, mulberries, strawberries, and cherries (Ponder et al., 2021), has not yet been comprehensively researched.

This study aimed to thoroughly explore the copigmentation between organic acids and C3G in blackberry wine, to provide innovative insights for reducing the browning phenomenon during the aging of fruit wines, and to offer theoretical guidance for reducing environmental pollution caused by the rotting of uneaten fruits.

2. Materials and methods

2.1. Materials and chemicals

Frozen blackberry berries of the ‘Hull’ variety was purchased from Zhongliang Organic Vegetable and Fruit Food Co., Ltd. (Nanjing, China). SDS, Guanidine hydrochloride, NaCl, urea, Malic acid, Succinic acid, Acetic acid, Tartaric acid, Lactic acid, Citric acid, Sodium hydroxide, and Hydrochloric acid were of analytical grade. Methanol, acetonitrile, ethanol, and formic acid were HPLC grade. K₂S₂O₅, pectinase (60 ku·ml⁻¹), sugar are food grade. Saccharomyces cerevisiae (LAlVIN DV10, Lallemand) were used. C3G (HPLC grade) was purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (China). The water used to prepare all samples and standards was purified using the Synergetic system (Merck Millipore, Germany).

2.2. Main experimental instruments and equipment

Food juicer (Lnkaxss, HES-011, China); Nuclear magnetic (600 MHz, Bruker AVANCE III HD); pH meter (pHS-3E, Lei Magnetic Instrument Company); HPLC (LC-1260, MS G6420A, Agilent); Microplate reader (Infinite M200 Pro, NanoQuant).

2.3. Methods

2.3.1. Winemaking method for blackberry wine

The brewing method of blackberry wine is an adaptation of our established technique (Wu et al., 2021). Initially, thawed blackberries are pulped, and then, the pulp is treated with 100 mg·L⁻¹ K₂S₂O₅ and 0.2 ml·l⁻¹ pectinase enzymes to facilitate enzymatic digestion for 2 h at 20 °C–25 °C. Sucrose was added to achieve a soluble solid content of 24° Brix in the pulp, and 0.02 % active dried yeast previously activated in a 2 % sugar solution for 20 min, was incorporated. Fermentation occurred at 20 °C–25 °C for 15 days. Following fermentation, the blackberry wine was filtered through four layers of sterilized cotton gauze (121 °C for 30 min) and then supplemented with 50 mg·L⁻¹ K₂S₂O₅ and 0.25 g·L⁻¹ bentonite. The mixture was thoroughly combined and allowed to rest for 7 days at 20 °C–25 °C. Afterward, it was again mixed and held for an additional 7 days at the same temperature. Subsequently, the wine was cooled and stabilized at 2 °C to 0 °C for 7 days. The final product was filtered through a 0.21 μm filter, bottled in 750 ml brown glass bottles sealed with cork and a plastic cap, and aged in darkness at 20 °C–25 °C until analysis.

2.3.2. Analysis of organic acids in blackberry wine

The different samples were analyzed by filtering them through a 0.22 μm filter subsequently examined via liquid chromatography. The chromatography procedure employed Agilent Poroshell 120 EC-C₁₈ columns (2.7 μm, 100 × 3 mm) at 30 °C. The mobile phase consisted of acetonitrile (mobile phase A) and a 0.1 % formic acid solution (mobile phase B), with an injection volume of 5 μL and a flow rate of 0.3 ml·min⁻¹ (A: B = 10 %: 90 %). The quantification of various organic acids in blackberry wine was conducted based on the basis of standard curves established for each acid. The standard curves for malic acid, tartaric acid, citric acid, and lactic acid presented a linear range of 1 ˗ 10 g·L⁻¹, whereas those for succinic acid and acetic acid presented a linear range of 0–1 g·L⁻¹.

2.3.3. Chroma changes of blackberry wine fermented with added organic acids

Blackberry wine fermentation with different organic acids added was conducted in 3 ˗ L fermenter. Each fermenter was filled with 1.5 L of blackberry pulp. The fermenters were then divided into three groups, and different concentrations of tartaric, citric, and malic acids were added separately. In particular, 0, 2, 4, and 6 g of the corresponding organic acids were added to the fermenters in each group. The addition ratios of yeast, sugar, and K₂S₂O₅, along with the experimental details, were determined and carried out following the method discussed in Section 2.3.1. When the alcohol content of the blackberry wine exhibited negligible change, the fermented blackberry wine was centrifuged, and its chroma and anthocyanin content were analyzed. The experiment was repeated three times.

2.3.4. HPLC/mass spectrometry (MS) analysis

The identification of compounds in the samples was carried out as follows: HPLC/MS analysis of the samples utilized a liquid chromatography–MS system, specifically Xevo TQs (WAD0150) equipped with an ACQUITY UPLC BEH C₁₈ column (1.7 μm, 2.1 × 100 mm). The mobile phases consisted of H₂O with 0.1 % formic acid (solvent A) and acetonitrile (solvent B). The injection volume of the sample was 2 μL, and the flow rate was maintained at 0.3 ml·min⁻¹. The initial gradient consisted of the eluent at 5 % B for 0–6 min, which was increased to 8 % B for 6–12 min, before the eluent was returned to 5 % B until the end of the analysis. MS acquisition was performed in negative ionization mode across a mass-charge ratio (m/z) rang of 100–2000. MS/MS analysis was conducted with a collision energy of 30 ± 10 V. Instrument control, data processing, and analysis were performed via MassLynx (version 4.2).

2.3.5. Effects of functional groups on copigmentation

2.3.5.1. Effects of hydroxyl, aldehyde and carboxyl groups on copigmentation

To analyze the effects of hydroxyl, aldehyde and carboxylic groups in organic acids on C3G copigmentation, the following methods were employed. Solutions of ethanol, acetaldehyde, and acetic acid were prepared using deionized water at concentrations of 0, 20, 40, 60, 80, and 100 %, respectively. These solvents were then mixed with the C3G solution at a volume ratio of 1:1 (v/v). Following complete homogenization, the absorbance of the solution at 420 nm, 520 nm, and 620 nm was measured after it was left for 2 h at 20 °C in the dark. The roles of hydroxyl, aldehyde, and carboxyl groups in copigmentation were then analyzed.

2.3.5.2. Effect of the number of carboxyl groups on copigmentation

The copigmentation experiments were conducted according to the following procedure: Tartaric acid, succinic acid, malic acid, citric acid, acetic acid, and lactic acid were utilized as copigments. Each organic acid was prepared at a concentration of 200 mM. Use a few amounts of 1 M HCl or NaOH to regulate the pH of these organic acids to 3.5. The different kinds of co-pigments and C3G (0.01 M) solutions were fully mixed at 1:1 (v/v) and left for 2 h at 25 °C away from light. Their absorbance is measured at 420 nm, 520 nm, and 620 nm.

2.3.6. Effects of intermolecular forces on copigmentation of organic acids

The effect of intermolecular forces on the copigmentation of organic acids was studied using our previous method (Wu et al., 2023). Eight reagents were prepared: 0.3 M sodium chloride (S1),1.2 M sodium chloride (S2),1.2 M sodium chloride with 3 M urea (S3), 1.2 M sodium chloride with 2 M Guanidine hydrochloride (S4), 1.2 M sodium chloride with 20 % propanediol (S5), 3 M urea (S6), 20 % propanediol (S7), and 2 % SDS (S8). The C3G solution (0.01 mol·L⁻¹) was thoroughly mixed with each of the organic acid solution at a 1:1 (v/v), and allowed to stand for 2 h at 20 °C in the dark. The absorbances of these solutions at 420 nm, 520 nm, and 620 nm were subsequently measured.

2.3.7. Nuclear magnetic spectrum analysis of the solution

To prepare the NMR samples, 100 mg of C3G was dissolved in 2 ml of Dimethyl sulfoxide-d6 (DMSO‑d₆) and mixed thoroughly. Next, 500 μL of this C3G solution was combined with 50 μL of anhydrous acetic acid via a vortex mixer for 2 min. For comparison, the C3G solution containing 50 μL of DMSO‑d₆ was used as the control group for NMR analysis. The chemical shifts of ¹H and ¹³C in DMSO‑d₆ solvent were recorded via NMR at 300 K, with chemical shifts expressed in ppm relative to an internal reference. The ¹H spectrum was scanned 16 times, with a delay time of 2 s, and a spectral frequency of 600 MHz; the ¹³C spectrum was scanned 512 times with a delay time of 2 s and a spectral frequency of 150.92 MHz.

2.3.8. Molecular dynamics (MD) simulation

The method of MD was slightly modified from the approach of Rusishvili et al. (2019). In short, the structural changes in C3G at various organic acid concentrations were simulated over a period of 100 ns, used the GROMACS 19.6 software package, alongside the Amber99sb force field and the TIP3P explicit water model. The molecules were fully solvated in TIP3P water, within cubic box with 12 Å edges that accommodating 5 C3G molecules under periodic boundary conditions. The variations of Dihedral Angle between the plane of B – ring with A – ring and C – ring (P_AC) of C3G were analyzed under different conditions: 0, 50, 500, and 5000 organic acid molecules. Chloride ions were incorporated to ensure electrical neutrality within the systems. The constant temperature and pressure of the complex system were subsequently attained through a series of simulations, including energy minimization (steepest drop of 1000 kJ mol⁻¹·nm⁻¹), NVT (300 K, 100 ps), and NPT (1.0 bar, 500 ps). The volume was maintained as fixed, and velocity rescaling was employed to keep the average temperature stable (NVT ensemble). The findings from the MD simulation elucidate the conformational and energetic changes in C3G and organic acids during their interactions, thereby providing an essential foundation for understanding the copigmentation of C3G and organic acids.

2.3.9. Color evaluation

The anthocyanin content was analyzed by the pH differential method (Lee et al., 2005). The chroma of the solution is calculated as described by Jiang et al. (2021),

$$\mathrm{A}={\mathrm{A}}_{420}+{\mathrm{A}}_{520}+{\mathrm{A}}_{620}$$

$${\mathrm{A}}_{\text{yellow}}={\mathrm{A}}_{420}/\mathrm{A}$$

$${\mathrm{A}}_{\mathrm{red}}={\mathrm{A}}_{520}/\mathrm{A}$$

$${\mathrm{A}}_{\text{bule}}={\mathrm{A}}_{620}/\mathrm{A}$$

where A is the chromaticity of the solution and A₄₂₀, A₅₂₀, and A₆₂₀ are the absorbances of the solutions at 420 nm, 520 nm, and 620 nm, respectively.

2.4. Data statistics

All the measurements were conducted in triplicate and the results are expressed as the mean ± standard deviations. One-way analysis of variance (ANOVA) was performed via SPSS software version 23.0 to identify significant differences, with a significance level stablished at p < 0.05.

3. Results and analysis

3.1. Organic acid contents in blackberry wine

The intense red, nearly black color of blackberries is closely associated with their composition and elevated levels of anthocyanins within fruit cells (Kaume et al., 2012). These anthocyanins are preserved in blackberry wine during fermentation, which contributes to its appealing red hue. Addressing the challenge of maintaining a stable red color of the wine body throughout the aging process is a pressing research focus. Table 1 presents the C3G and organic acid contents in blackberry wines at different aging stages.

Table 1.

The types and contents of organic acids in blackberry wines (g˖L⁻¹).

	Juice	Fresh wine	Aged wine(6 m)	Aged wine(12 m)
Tartaric acid	1.175 ± 0.029a	2.590 ± 0.113b	3.621 ± 0.504b	3.056 ± 0.223b
Malic acid	6.747 ± 0.235a	8.858 ± 0.287 ac	7.695 ± 0.286ab	7.934 ± 0.628bc
Citric acid	1.614 ± 0.014a	1.678 ± 0.036a	1.643 ± 0.114a	1.609 ± 0.008a
Succinic acid	0.037 ± 0.001a	0.104 ± 0.009c	0.093 ± 0.004bc	0.077 ± 0.021b
Acetic acid	0.762 ± 0.015a	0.567 ± 0.362a	0.776 ± 0.032a	1.139 ± 1.632b
2-Hydroxypropanoic acid	4.821 ± 0.240a	6.355 ± 0.337b	7.067 ± 0.466b	6.713 ± 0.422b
pH	3.117 ± 0.054a	3.197 ± 0.120b	3.203 ± 0.022b	3.323 ± 0.025c
C3G	0.108 ± 0.052a	0.048 ± 0.014b	0.023 ± 0.012c	0.002 ± 0.002d

Malic acid is the most important organic acid in blackberry juice and wine. Its content in blackberry juice and fresh blackberry wine is 6.75 g·L⁻¹ and 8.86 g·L⁻¹, respectively. This result is consistent with the research findings of Michelle and de Mejia (2012). Some studies have found that malic acid content in wine during aging can decrease by >70 % of its original value owing to microbial factors (Lamikanra, 1997). However, in this experiment, even after aging for 12 months, its content in blackberry wine still reached 7.93 g·L⁻¹. This may be because the acidity of blackberry wine is higher than that of grape-wine. Lactic acid content in the samples is second only to that of malic acid. Its contents in fruit juice, fresh wine, 6-month aged wine and 12-month aged wine were 4.82 g·L⁻¹, 6.36 g·L⁻¹, 7.07 g·L⁻¹, and 6.71 g·L⁻¹, respectively. This change is attributed to malolactic fermentation occurring during the winemaking process.

The citric acid content in the samples ranged from 1.64 g˖L⁻¹ to 1.71 g˖L⁻¹, ranking second only to malic and lactic acids. Some studies suggest that citric acid may be the most significant organic acid present in blackberry fruits. This is likely because of differences in blackberry varieties and ripeness (Famiani & Walker, 2009). In addition, the acetic acid content in blackberry wine aged for 12 months was higher than that in blackberry juice, which is likely owing to the oxidation of ethanol to acetic acid during the aging process (Bell-Parikh & Guengerich, 1999).

3.2. Changes in the molecular weight of C3G during copigmentation with organic acids

In studies concerning red wine, organic acids can acylate M3G, resulting in a copigmentation phenomenon. In addition, it has been reported that anthocyanins and organic acids can spontaneously form complexes (Babaloo & Jamei, 2018). C3G was the principal pigment in all blackberry samples, with its content ranging from 43.6 % to 95.2 % of the total pigment (averaging 82.9 %) (Torre & Barritt, 1977).

After mixing organic acids with C3G, the molecular weight of the resulting solution was determined, as shown in Fig. 1.

Fig. 1.

Molecular weight changes of different organic acids mixed with C3G solution.

A: Ethyl alcohol; B: Malic acid; C: Acetic acid.

When compared with the blank group, the mixtures of C3G with malic acid (Fig. 1B) and acetic acid (Fig. 1C) showed no obvious peaks. Therefore, in this experiment, the copigmentation of C3G and organic acids was not attributed to the acylation reaction between them.

In addition, because metal ions were not included in this experiment, any color changes associated with the chelation of C3G and metal ions were not examined. Considering the fact that there was no binding phenomenon between organic acids and C3G, alternative mechanisms must be explored to explain the copigmentation observed in the solution.

3.3. The chroma changes of blackberry wine fermented with added organic acids

Compared with the samples with no organic acids added (0 g·L⁻¹), the color intensity of blackberry wines fermented with different added organic acids increased in a dose-dependent manner. Among them, the color intensity of wine body with added citric acid showed the most increased (Table 2).

Table 2.

The chroma changes of blackberry wine fermented with added organic acids.

	Blank group	Malic acid			Citric acid			Tartaric acid
		2 g	4 g	6 g	2 g	4 g	6 g	2 g	4 g	6 g
Color Intensity	2.574 ± 0.109	2.917 ± 0.031e	3.253 ± 0.096d	3.568 ± 0.098c	3.076 ± 0.049de	3.722 ± 0.244bc	4.266 ± 0.083a	3.200 ± 0.041de	3.613 ± 0.026c	3.991 ± 0.092ab
A520	1.720 ± 0.070f	1.960 ± 0.024e	2.208 ± 0.055d	2.423 ± 0.059c	2.071 ± 0.023de	2.531 ± 0.182c	2.969 ± 0.056a	2.158 ± 0.020de	2.440 ± 0.009c	2.748 ± 0.054b
Ayellow	0.298 ± 0.001a	0.293 ± 0.000b	0.288 ± 0.000 cd	0.288 ± 0.000 cd	0.291 ± 0.000bc	0.286 ± 0.004d	0.276 ± 0.001f	0.290 ± 0.000bcd	0.289 ± 0.000bcd	0.282 ± 0.000e
Ared	0.665 ± 0.004d	0.672 ± 0.003 cd	0.679 ± 0.003bc	0.679 ± 0.003bc	0.673 ± 0.003 cd	0.68 ± 0.005bc	0.696 ± 0.004a	0.675 ± 0.003 cd	0.675 ± 0.003 cd	0.689 ± 0.002ab
Abule	0.038 ± 0.004a	0.035 ± 0.004a	0.033 ± 0.003a	0.033 ± 0.003a	0.035 ± 0.004a	0.034 ± 0.002a	0.028 ± 0.004a	0.035 ± 0.003a	0.035 ± 0.003a	0.030 ± 0.003a
C3G (g/L)	20.417 ± 0.271e	21.820 ± 0.347de	23.512 ± 0.784 cd	24.191 ± 0.735bc	22.354 ± 0.823d	23.490 ± 0.595 cd	29.563 ± 0.097a	22.137 ± 0.508d	23.200 ± 0.359 cd	25.599 ± 0.486b
pH	3.167 ± 0.009e	3.130 ± 0.008d	3.100 ± 0.008 cd	3.067 ± 0.009c	3.120 ± 0.008d	3.077 ± 0.012c	3.010 ± 0.008b	3.077 ± 0.012c	3.010 ± 0.022b	2.937 ± 0.012a

After adding 2, 4, and 6 g of citric acid, the A₅₂₀ absorbance was 3.08, 3.72, and 4.27, respectively, which were higher than those of the blackberry wine fermented without added organic acids (2.58). However, the color intensity change of the samples with added malic acid was the smallest, with corresponding values of 2.92, 3.25, and 3.57, respectively. In the blackberry wine with added organic acids, the C3G content slightly increased, which might be because the acidic conditions were beneficial to the extraction of anthocyanins from the cell walls of blackberries (Chandrasekhar et al., 2012). In contrast, the differences in absorbance at A₄₂₀ and A₆₂₀ of all samples were not obvious. Therefore, adding organic acids before fermentation can improve the chroma of blackberry wine.

Notably, compared with citric acid, the pH value of blackberry wine with added tartaric acid (2 g, 4 g, and 6 g) decreased more significantly, reaching 3.08, 3.01, and 2.94, respectively. However, the color intensity of the wine body was lower than that of the wine with citric acid added. This indicates that, apart from the pH, other mechanisms are responsible for the copigmentation of organic acids and C3G. As far as we know, few studies report this copigmentation between organic acids and C3G without affecting molecular weight. We will analyze this copigmentation of organic acids and C3G as follows.

3.4. Effects of functional organic acids groups on the copigmentation

3.4.1. Effects of hydroxyl, aldehyde, and carboxyl groups on copigmentation

Hydroxyl (˗OH), aldehyde (˗CHO), and carboxylic (˗COOH) groups are the most common functional groups observed in various organic compounds. Ethanol, acetaldehyde, and acetic acid are representative examples that contain these functional groups (Scheme 1).

Scheme 1.

Molecular Conformation Diagram of the Compound

Analyzing the color changes of C3G in the presence of these compounds can clarify the role of specific functional groups in copigmentation. Fig. 2 shows the chromaticity changes of C3G solutions after being mixed with various concentrations of ethanol, acetaldehyde and acetic acid.

Fig. 2.

Influence of different functional groups on C3G copigmentation.

In the concentration range of 0 % ˗ 10 %, the color intensity of the C3G solution containing ethanol and acetaldehyde gradually increased. This trend suggests that ethanol and acetaldehyde exert a modest copigmentation effect via hydrogen bonding (Goto & Kondo, 1991). In particular, the carbonyl group (˗CHO) in acetaldehyde and the hydroxyl group (˗OH) in ethanol form hydrogen bonds with the hydroxyl group of C3G. These results indicate that the presence of a single carbonyl (C=O) or hydroxyl group (˗OH) has a minimal copigmentation-promoting effect on C3G. By contrast, within the same concentration range, the colorimetric analysis of the C3G solution combined with acetic acid showed a significant increase, indicating that the carboxyl group (˗COOH), which contains both carbonyl and hydroxyl groups, substantially promotes copigmentation with C3G (Yawadio & Morita, 2007).

3.4.2. C3G copigmentation analysis by the carboxyl groups of organic acids

Citric acid, a tricarboxylic organic acid; malic, tartaric, and succinic acids, each possessing two carboxyl groups, and acetic acid and lactic acid, each containing one carboxyl group (Scheme 1), were analyzed for their copigmentation effects on C3G. In addition, hydrochloric acid, lacking a carboxyl group, was considered in this analysis. At the same pH, the copigmentation effects of different organic acids with C3G were observed, as shown in Fig. 3.

Fig. 3.

Copigmentation of organic acids on C3G.

TA:Tartaric acid; SA:Succinic acid; MA: Malic acid; CA:Citric acid; AA: Acetic acid; LA: Lactic acid; HCL: Hydrochloric acid; CK: C3G with unadjusted pH.

At equivalent concentrations and pH values, the color intensity value (2.55) of the C3G solution containing citric acid exceeded that of the solutions containing malic, tartaric, and succinic acids. This observation suggests that an increased number of carboxyl groups is correlated with a strong copigmentation effect. However, under these experimental conditions, acetic (2.67) and lactic (2.61) acids exhibited even more potent copigmentation effects than the other organic acids tested. These findings imply that the quantity of carboxyl groups is not the sole determinant influencing the copigmentation of anthocyanins.

Furthermore, at a consistent pH, no significant differences in the red tone of the C3G solution were detected among all the tested organic acids and hydrochloric acid. This result indicates that the copigmentation of organic acids is closely related to pH (Babaloo & Jamei, 2018).

3.5. Effects of the intermolecular forces of organic acids on the copigmentation

NaCl can disrupt ionic bonds. A concentration of 0.05 mol·L⁻¹ NaCl effectively disrupted nonspecific cross-linking, whereas the combination of 0.6 mol·L⁻¹ NaCl and 1.5 mol·L⁻¹ urea broke hydrogen bonds. Notably, urea and GuHCl disrupt noncovalent molecular forces, such as electrostatic interactions, hydrogen bonds, and hydrophobic interactions. In addition, propylene glycol has significant effects on the intermolecular forces in solution and is commonly utilized to enhance hydrogen bonding and electrostatic interactions while simultaneously reducing hydrophobicity by lowering the dielectric constant of the solvent. Moreover, sodium dodecyl sulfate creates unfavorable conditions for the formation of hydrogen bonds and hydrophobic interactions. Table 3 presents the changes in the absorbance ratio of red tone (Ar) upon the introduction of various interfering agents into the copigmented C3G solution.

Table 3.

Effects of intermolecular forces on copigmentation of C3G and organic acids (%).

Disturbing agent	0.15 M Nacl	0.6 M Nacl	0.6 M Nacl+1.5 M Urea	0.6 M Nacl+1 M Guanidine hydrochloride	0.6 M Nacl +10 % Propanediol	1.5 M Urea	10 % Propanediol	1 % SDS
Simplified code	S1	S2	S3	S4	S5	S6	S7	S8
Acetic acid	3.969 ± 0.157	4.275 ± 0.224	5.364 ± 0.230	19.056 ± 1.541	7.563 ± 0.619	−6.701 ± 0.474	−2.501 ± 0.236	19.551 ± 0.867
Succinic acid	1.806 ± 0.124	8.081 ± 0.375	3.781 ± 0.206	28.094 ± 1.231	7.471 ± 0.632	−5.507 ± 0.231	−3.794 ± 0.180	26.862 ± 1.575
Malic acid	−2.081 ± 0.164	−6.257 ± 0.918	−3.434 ± 0.157	12.514 ± 0.954	4.809 ± 0.619	−5.411 ± 0.596	5.530 ± 0.397	4.104 ± 0.237
Tartaric acid	−5.744 ± 0.752	−4.643 ± 0.387	−1.689 ± 0.205	5.452 ± 0.414	5.193 ± 0.207	0.643 ± 0.357	4.392 ± 0.348	4.627 ± 0.414
Citric acid	−0.802 ± 0.087	−3.421 ± 0.283	2.555 ± 0.094	5.481 ± 0.223	9.232 ± 0.306	0.687 ± 0.078	5.286 ± 0.292	8.956 ± 0.611
Average	−0.571	−0.393	1.315	14.119	6.854	−3.258	1.783	12.82

Different intermolecular forces exert different effects on the copigmentation of organic acids, with hydrophobic forces being the most influential. The addition of interfering agents (S4, S8,and S5) that affect the hydrophobic force increased the red tone of the copigmented C3G solution by 14.12 %, 12.82 %, and 6.85 %. This finding is consistent with previous results and supports the conclusions of this study (Eiro & Heinonen, 2002).

Moreover, interference with the ionic forces decreased the solution's average red tone by 0.571 %. By contrast, the influence with hydrogen bonding resulted in an average increase in the solution's red tone by 1.32 %. These findings are consistent with the results presented in Section 3.4. The effects of different organic acids on the increase in red tone significantly varied. Ethanol and succinic acid exhibited strong hydrophobic effects; however, the hydrophobic effects observed with malic, tartaric, and citric acid solutions were less pronounced. Notably, the addition of acetic and succinic acid solutions, which eliminated the hydrophobic interaction, increased the red tone (A₅₂₀) and decreased the blue tone (A₄₂₀).

Thus, the order of the effects of molecular forces on the copigmentation interaction of organic acids with C3G is as follows: Hydrophobic forces > Hydrogen-bond forces > Ionic-bond forces.

3.6. Nuclear magnetic spectrum analysis of copigmented organic acids and anthocyanins

Compared with the blank sample, the addition of organic acids to the C3G solution resulted in ¹H and ¹³C NMR shifts in the analytical solution, highlighting the significant structural change in the anthocyanins, as shown in Fig. 4.

Fig. 4.

Chemical shift of ¹H when C3G and organic acids are copigmentation.

A: Chemical shift of ¹H of C3G; B: Chemical shift of ¹H with acetic acid.

The ¹H chemical shift of C3G shifted to a low field upon organic acid addition, indicating that the presence of these acids modifies the environment of the anthocyanins. This alteration in the environment can be attributed to various intermolecular forces, such as hydrophobic interactions, hydrogen bonding, and van der Waals forces. These enhanced intermolecular forces decrease the density of the electron cloud surrounding the hydrogen nucleus, resulting in a diminished shielding effect (Jameson, 1982). Further analysis of the ¹H chemical shifts of the C3G structures revealed that the A ˗ ring, C ˗ rings, and glycosides exhibited less pronounced shifts in the low-field compared to the B ˗ ring. This observation suggests that the B ˗ ring structure is more variable than that of the A ˗ ring and glycosides. The differing degrees of twisting in a compound two planes account for the observed low-field chemical shift in the B ˗ ring of C3G upon the adding organic acids (Kenneth, 1986).

Fig. 5 presents the changes in the ¹³C chemical shift of C3G upon the addition of acetic acid.

Fig. 5.

Chemical shift of ¹³C when C3G and organic acids are copigmentation.

A: Chemical shift of ¹³C of C3G; B: Chemical shift of ¹³C with acetic acid.

The ¹³C nucleus exhibited a smaller cyclogenetic ratio than the ¹H nucleus, resulting in a smaller change in ¹³C chemical shift of C3G than that of ¹H. The ¹³C chemical shifts of the A – ring and C – ring shifted to the low field by about 0.01 ppm, while the B – ring shifted about 0.03 ppm to the low-field on average. This finding indicates that the average change of all carbon atoms in the B – ring is greater than that in the A – ring and C – ring.

¹H and ¹³C NMR shift analyses provide compelling evidence that the addition of organic acids induces structural changes in anthocyanins. Moreover, the addition of organic acids causes a certain degree of rotation of the B ˗ ring plane of C3G relative to the large plane formed by the A ₋ ring and C ₋ ring (P_AC).

3.7. Molecular dynamics simulation of the copigmentation of organic acids

MD simulations were also used to evaluate the structural changes of C3G when copigmentation occurred between C3G and organic acids (Fig. 6).

Fig. 6.

Results of molecular dynamics simulation when C3G and organic acids were co-colored.

A: The box contains 5 C3G molecules and 0 malic acid molecules;

B: The box contains 5 C3G molecules and 50 malic acid molecules;

C: The box contains 5 C3G molecules and 500 malic acid molecules;

D: The box contains 5 C3G molecules and 5000 malic acid molecules.

When the cube box only contains C3G molecules, chloride ions, and water, the dihedral angles ꞷ1 (C12˗C11˗C13˗C14) and ꞷ2 (C12˗C11˗C13˗C15) are −105.07° and 103.04°, respectively. This indicates that the B – ring plane of C3G tends to be coplanar with P_AC (Rusishvili et al., 2019). At a C3G to organic acid ratio of 1:10 in the box, the dihedral angles ꞷ1 and ꞷ2 shift to −113.07° and 94.25°, respectively. This suggests that the plane of B – ring slightly rotates clockwise. When the C3G to organic acid ratios is 1:100, the two dihedral angles are −119.84° and 87.51°; when the ratio is 1:1000, the two dihedral angles are −126.74° and 80.88°. This change indicates that the plane of B – ring rotates clockwise by a larger angle with respect to P_AC as the concentration of organic acids increases.

Therefore, the copigmentation involves an organic acid induced change in the polarity of C3G solution, which causes the B – ring plane of C3G to rotate from a relative coplane with P_AC to an angle with P_AC due to hydrophobic and hydrogen-bond interactions. This interference in planar arrangement restores the shielded chromaticity of C3G, increasing the solution chromaticity and red hue.

4. Discussion

In the study of fruit wine, the mechanism of copigmentation between other pigment and anthocyanin is one of the focuses of researchers. Although the proportion of C3G in nature is several times that of M3G (Kayesh et al., 2013), the existing literature shows that there has been scant research on the copigmentation between copigments and C3G. From the molecular structure perspective, M3G and C3G exhibit differences solely in the B ˗ ring. In M3G, the R₁ and R₂ positions are occupied by methoxy groups. Conversely, in C3G, the R₁ position is a hydroxyl group and the R₂ position is a hydrogen atom (Scheme 2).

Scheme 2.

Structure of anthocyanins

Extant research has revealed that the hydroxyl group on the B ˗ ring of anthocyanins is less stable than the methoxy group (Liu et al., 2014). Therefore, enhancing the stability of C3G holds special significance in the research and brewing processes of fruit wine. This not only contributes to a profound understanding of the copigmentation between pigments and C3G, but also potentially exerts a positive influence on the quality and stability of fruit wine.

Moreover, in winemaking, adding organic acids is a common and relatively safe approach. This method not only improves the stability of C3G but also causes the B ˗ ring plane of C3G to rotate relative to P_AC. Theoretically, this rotation increases the steric hindrance for anthocyanin polymerization and reduces the formation of polymeric pigments. Han and Xu (2015) suggested that polymeric pigments can endow wines with stable color. However, it is believed that pigment polymerization increases the molecular weight of monomeric phenols. And once polymeric pigments bind to proteins, they are more likely to form wine haze, thereby reducing the color density of the wine.

Most wine color theories, apart from considering the impact of pH on color, focus on the formation of pigments with new molecular weights, such as the glycosylation and acylation of anthocyanins and the formation of polymeric pigments. According to the experimental results, we propose that in a solution with a relatively neutral pH, the plane of B – ring of anthocyanins is coplanar with P_AC. Here, the free monomeric anthocyanins, owing to differences in position and structure, reflect less of the anthocyanin characteristic color under light. As the solution acidity increases, the B – ring plane rotates relative to P_AC, causing more light of the anthocyanin – characteristic color to be reflected, and thus increasing the solution's chromaticity. This proposal complements the organic acid copigmentation mechanism. Nevertheless, many aspects remain unexplored. For example, what are the specific differences in how different organic acids affect C3G stability and pigment polymerization, and what are the rules of change in the anthocyanin molecular structure across various fruit-wine systems. Future research could focus on these areas to perfect the pigment – related theory system in fruit wine and provide more robust technical support for the development of the fruit – wine industry.

5. Conclusions

In summary, the copigmentation between organic acids and C3G is inextricably linked to the structural changes in C3G. Organic acids can alter the polarity of the solution. Owing to hydrophobic forces and hydrogen bonds, the plane of B ring of C3G rotates clockwise and gradually becomes perpendicular to the large plane formed by the A – ring and C – ring. As a result, the shielded color of C3G is restored, leading to a reddish coloration in the solution. Moreover, the influence of the carboxyl group on copigmentation is more significant than that of the hydroxyl group and aldehyde group. This mechanism provides insights into the commercialization of fruit juices and wines from perishable fruits and presents a viable solution to mitigate the environmental pollution caused by excessive fruit waste.

Funding

Financial support has been provided by the Jiangsu Agriculture Science and Technology Innovation Fund, China [grant number CX (22) 2026 and Financial support has been provided by the National Natural Science Foundation of China (Grant No. 31801531).

CRediT authorship contribution statement

Gang Wu: Writing – review & editing, Writing – original draft, Software. Lijun Yu: Investigation, Funding acquisition. Shuang Wu: Formal analysis, Data curation. Peng Li: Resources, Project administration. Caie Wu: Resources, Methodology, Conceptualization. Ying Wang: Visualization, Validation, Supervision, Formal analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.