# A two-step biocatalytic process to extract and to acylate anthocyanins: a sustainable approach for red grape pomace biorefining

Emelyne Jolly, Pascale Goupy, Angélique Claisse-Gainvors, Olivier Dangles, Diana Garcia-Bernet, Claire Dufour, Caroline Rémond

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## ► To cite this version:

Emelyne Jolly, Pascale Goupy, Angélique Claisse-Gainvors, Olivier Dangles, Diana Garcia-Bernet, et al.. A two-step biocatalytic process to extract and to acylate anthocyanins: a sustainable approach for red grape pomace biorefining. Applied Food Research, 2025, pp.101431. 10.1016/j.afres.2025.101431. hal-05333857

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Submitted on 6 Nov 2025

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# A two-step biocatalytic process to extract and to acylate anthocyanins: a sustainable approach for red grape pomace biorefining

Emelyne Jolly<sup>a</sup>, Pascale Goupy<sup>b</sup>, Angélique Claisse-Gainvors<sup>a</sup>, Olivier Dangles<sup>b,D</sup>,
Diana Garcia-Bernet<sup>c</sup>, Claire Dufour<sup>b</sup>, Caroline Rémond<sup>a,* D</sup>

## ARTICLE INFO

**Keywords:**
Grape pomace
Anthocyanins
Polyphenols
Pectinase
Extraction
Lipase
Acylation

## ABSTRACT

Grape pomace represents an abundant source of anthocyanins displaying coloring and health-promoting properties for food applications. At the industrial stage, the extraction of anthocyanins from grape pomace consumes solvents and/or sulfites, which are no longer desirable due to environmental issues and consumer demands. Furthermore, anthocyanins extracted from grape pomace are simple (non-acylated) O-glucosides lacking chemical stability. In this study, a two-step biocatalytic approach was developed to extract anthocyanins from grape pomace and esterify their glucosyl moiety. Different enzymes and conditions were compared for anthocyanin extraction. The highest amount of extracted anthocyanins was 1.44 g/kg fresh pomace after three sequential extraction steps with the enzyme Lysis Elite in water - ethanol (4:1). The purified anthocyanin extract was further acylated with N435 lipase in the presence of lauric acid or dihydroferulic acid, reaching acylation yields of 19.8 ± 0.7 % and 8.5 ± 1.7 %, respectively.

## 1. Introduction

Winemaking represents an important worldwide economic sector with 75 million tons produced annually (FAO, 2023). Grape pomace, the residual material left after the extraction of juice from Vitis spp. grapes, is rich in phenolic compounds, such as flavonoids, phenolic acids and tannins. Winemaking produces huge amounts of grape pomace mainly consisting of skins, seeds and stalks. Industrial processes have been developed for more than a century and important improvements in the last decades allowed grape pomace to be valorized through biorefining approaches, thus reducing their environmental impact according to circular economy principles. After extraction of a wide range of products, such as ethanol, grape seed oil, flavors, anthocyanins and tartrate, the remaining pomace is mainly composted or used for energy production through anaerobic digestion (Beres et al., 2017).

Among polyphenols, anthocyanins display red, purple and blue colors useful for food applications (e.g., the anthocyanin-rich food additive E163). In addition to their coloring property, anthocyanins display various beneficial biological properties, such as prebiotic, anti-inflammatory and antioxidant activities (Merecz-Sadowska et al.,

2023; X. Zhang, Yang, Wu & Weng, 2016). In nature, anthocyanins are mainly present in fruits, vegetables, flowers and grains. They display an aglycone moiety (chromophore) called anthocyanidin, which is generally represented by its main form under acidic conditions, i.e. a 2-phenyl-1-benzopyrylium (flavylium) cation typically hydroxylated at positions C4', C3, C5 and C7. Anthocyanidins are O-glycosylated to form anthocyanins. The sugar moieties can be O-acylated depending on biomass (Konczak & Zhang, 2004; Wallace & Giusti, 2015). This gives rise to a large natural diversity of anthocyanins. Depending on external factors, such as pH, light, temperature, water activity, transition metal traces and dioxygen, anthocyanin colors are relatively unstable (Dangles, 2024; Dangles & Fenger, 2018; Fenger, Moloney, Robbins, Collins & Dangles, 2019; Gérard et al., 2019). Color loss can be either reversible because of water addition to the flavylium cation (hydration), which yields a mixture of colorless hemiketal and chalcones in equilibrium, or irreversible, through a combination of hydrolytic and oxidative mechanisms, typically leading to the cleavage of the heterocycle (C-ring). Anthocyanins acylated by hydroxycinnamic acid residues, often present in dark vegetables, such as black carrot or red cabbage, have proven more stable than non-acylated anthocyanins

<footnote><sup>a</sup> Université de Reims Champagne-Ardenne, INRAE, FARE, UMR A 614, AFERE, Reims, France</footnote>

<footnote><sup>b</sup> INRAE, Avignon University, UMR408 SQPOV F-84000 Avignon, France</footnote>

<footnote><sup>c</sup> Université de Montpellier, INRAE, LBE F-11100 Narbonne, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: caroline.remond-zilliox@inrae.fr (C. Rémond).</footnote>
(Giusti & Wrolstad, 2003), as they typically adopt folded conformations in which the anthocyanidin and the phenolic residues develop Π-stacking interactions (Robbins, Collins, Kondo, Yoshida & Dangles, 2018; Trouillas et al., 2016). This phenomenon, known as intra-molecular copigmentation, at least partially inhibits water addition to the flavylium ion (hydration), thus raising the fraction of anthocyanin under its colored forms (flavylium ion + neutral and anionic bases depending on pH). More generally, any phenomenon drawing the anthocyanidin into a relatively hydrophobic environment is expected to be color-protecting, not only by limiting hydration, but also by restricting the contact between the oxidizable anthocyanidin and transition metal traces (typical autoxidation initiators) in the aqueous phase. For instance, O-acylation of the anthocyanin sugar moieties by fatty acids leads to amphiphilic derivatives with improved color stability that could expand the range of food and non-food applications to non-aqueous media. So far, this approach has been developed from pure individual anthocyanins (Cruz et al., 2017; Marquez-Rodriguez et al., 2021; Zhang et al., 2021), which of course is economically non-viable for industrial applications, and from blackcurrant or jaboticaba anthocyanin extracts. In addition, acylation with cinnamic acid and related acyl donors has been attempted with pure cyanidin glycosides (Fernandez-Aulis, Torres, Sanchez-Mendoza, Cruz & Navarro-Ocana, 2020; Yan et al., 2016) and blueberry anthocyanins (Liu et al., 2020). In grape pomace, anthocyanins are mainly composed of malvidin-, peonidin-, petunidin-, delphinidin-, and cyanidin-3-O-glucosides, malvidin-3-O-glucoside being by far the major contributor, along minor concentrations of 3-O-acetylglucoside and 3-O-p-coumaroylglucoside (Trikas, Melidou, Papi, Zachariadis & Kyriakidis, 2016; Zhao, Zhang, Zhang, He & Duan, 2020). A few studies have addressed O-acylation of pure malvidin-3-O-glucoside, but none in plant extracts (Cruz, Fernandes, Guimaraes, de Freitas & Mateus, 2016; Cruz et al., 2017).

As for the preparation of anthocyanin-rich extracts, it must be considered that these pigments are embedded within complex plant matrices, including cell walls and other cellular components. Breaking down these matrices to release anthocyanins without compromising their stability and properties can be challenging. While there have been significant advancements in anthocyanin extraction techniques, several challenges persist in the field. Classical extraction methods are related to maceration of anthocyanin-rich biomass in the presence of sulfites or solvents as ethanol (Castañeda-Ovando, Pacheco-Hernández, Páez-Hernández, Rodríguez & Galán-Vidal, 2009). These approaches suffer from the use of large amounts of these chemicals. Greener extraction methods for polyphenols, such as microwave heating, ultrasonication, and enzymatic methods, have been developed to limit the use of chemicals and achieve high extraction efficiency while maintaining anthocyanin stability (Panja, 2018; Panzella et al., 2020). As described in recent reviews, numerous studies deal with the extraction of anthocyanins from various plant biomasses (fruits, vegetables, food matrices) with a large panel of techniques, each displaying advantages and disadvantages (Constantin & Istrati, 2022; Moutinho, Gouvinhas, Domínguez-Perles & Barros, 2023; Panzella et al., 2020; Tena & Asuero, 2022). The objective of the present study was to develop a complete biocatalytic process from the extraction of grape pomace anthocyanins to their derivatization through acylation. Enzymatic acylation of a red grape pomace extract rich in anthocyanins has been explored with dihydroferulic acid or lauric acid to prepare anthocyanins bearing a phenolic acid or fatty acid residue, respectively.

# 2. Materials and methods

## 2.1. Materials

Red grape was obtained from the Distillerie Sud Languedoc (Sigean, Aude, France, harvested in September 2020). Grape pomace was stored under lyophilized form, as previous experimental studies showed that this is the best storage method for anthocyanin preservation. The water

contents for the fresh and lyophilized grape pomaces were 65.0 % and 12.4 %, respectively. Malvidin-3-O-glucoside or oenin (≥ 98 %) was from Extrasynthèse (Genay, France). Pectinex® Ultra SPL and Lysis Elite were purchased respectively from Sigma-Aldrich (Saint Quentin-Fallavier, France) and OenoFrance (Magenta, France). Lipase N435® was obtained from Strem Chemicals (Bischheim, France).

The substrates used to measure the enzymatic activities were p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-xylopyranoside, p-nitrophenyl-β-d-galactopyranoside (Sigma-Aldrich), methyl ferulate (Alfa Aesar) and xylans from beechwood (Carl Roth, Lauterbourg, France). HPLC-MS grade ethanol, methanol, ethyl acetate and formic acid were purchased from Carlo Erba Reagents (Val de Reuil, France). Folin-Ciocalteu reactant, sodium carbonate, sodium hydroxide, sodium acetate, potassium sodium tartrate tetrahydrate, potassium ferricyanide, formic acid, gallic acid, lauric acid, polygalacturonic acid, anhydrous 2-methyl-2-butanol (2M2B), 4Å molecular sieves and the TSK resin (Sigma Aldrich) were obtained from Sigma-Aldrich (Saint Quentin-Fallavier, France) and 3,5-dinitrosalicylic acid from Acros Organics (Illkirch, France). Dihydroferulic acid (3-(4-hydroxy-3-methoxyphenyl)propionic acid) was obtained from Fisher Scientific (Illkirch, France). Absolute ethanol was from VWR (Fontenay-sous-Bois, France).

## 2.2. Investigation of the enzymatic extraction of polyphenols and anthocyanins

Extraction assays were investigated with Pectinex® Ultra SPL or Lysis Elite enzymes in the presence of grape pomace (10 % w/v) in 50 mM sodium acetate buffer at pH 4.5. Extractions were carried out at 50 °C for 6 h under magnetic stirring in final volumes of 50 mL. Various enzyme loadings were tested: 1 %, 0.1 %, and 0.01 % (w proteins/w pomace).

To study the effect of extraction duration, assays were conducted under the same conditions with Pectinex® Ultra SPL or Lysis Elite at 0.01 % (w proteins/w pomace) at 50 °C under magnetic stirring in final volumes of 50 mL. Reactions were monitored during 24 h with sampling at 1 h, 6 h and 24 h.

To compare enzyme-assisted extraction with ultrasound-assisted extraction, the lyophilized grape pomace extract (10 g) was extracted by 20 mL of EtOH—H₂O (7:3) containing 2 % concentrated HCl in a US bath (Elmasonic P from Elma) operating at 37 Hz, 90 % of the max. power and at room temperature. After 5 min, the mixture was filtered and the residue extracted three more times under similar conditions, except for the second extraction step for which 40 mL of solvent were used to take into account the loss from rehydration. The combined filtrates were adjusted to 80 mL with acidified 70 % EtOH and 200 µL were added to 2 mL of the two buffers at pH 5.0 and pH 1.0 for the determination of anthocyanins by the colorimetric method as described below. Three independent repetitions of the US-assisted extraction were carried out.

## 2.3. Sequential enzymatic extraction of polyphenols and anthocyanins

Three successive extractions of 6 h with the same sample of 10 % grape pomace (w/v) in either pure buffer (50 mM sodium acetate at pH 4.5) or buffer-EtOH (4:1) were performed in the presence of Lysis Elite (0.01 %) at 50 °C under magnetic stirring in 50 mL reaction volumes. At the end of each extraction, samples were filtrated (Nitex nylon filter cloth, 1 µm, Dutscher France), then centrifugated (10 min, 1680 g, 4 °C). A volume of fresh buffer or buffer-ethanol (4:1), as well as Lysis Elite (0.01 %), were added onto the pomace solid residue recovered from the filtration. The operation was repeated three times. All experiments were performed in triplicate.

## 2.4. Separation of anthocyanins from other polyphenols and sugars

An extract produced from grape pomace with Lysis Elite (0.01 %
during 6 h at 50 °C) containing 17 g of sugars and 194 mg of total polyphenols, including 17.34 mg of anthocyanins, was purified onto an anionic TSK resin (160 mL) in order to obtain anthocyanin-rich fractions. After loading the extract, the resin was first washed with 50 mM sodium acetate buffer pH 4.5 (2 bed volumes), then with MeOH (3 bed volumes). One volume of acidified water (0.01 % formic acid) was added to the MeOH solution containing both anthocyanins and other polyphenols, and MeOH was evaporated under vacuum at 50 °C. Next, an equivalent volume of ethyl acetate was added to remove weakly polar polyphenols by liquid-liquid extraction. The operation was repeated twice. The anthocyanins recovered from the aqueous phase were lyophilized for the acylation step.

## 2.5. Enzymatic acylation of anthocyanins

The acylation of anthocyanins (4 mM in malvidin-3-O-glucoside equivalent) in 2-methyl-2-butanol (2M2B) was catalyzed by lipase N435 (4 %, w/v) in the presence of lauric acid or dihydroferulic acid with an anthocyanins/acyl donor molar ratio of 1/100. Reactions were performed in 1 mL final volumes in the presence of molecular sieves (10 %, w/v) at 50 °C during 72 h under magnetic stirring. At the end of the reaction, centrifugation (10 min, 1680 g) was performed to recover the liquid phase. The molecular sieves and the immobilized lipase were washed with EtOH (70 %, 1 mL) in order to recover the adsorbed anthocyanins. The washing was repeated 3 times with a centrifugation step (10 min, 1680 g) at the end of each washing. Native and acylated anthocyanins were quantified by UHPLC-DAD-MSn from both reaction media and washings. Acylation reactions were performed in triplicate.

## 2.6. Quantitative analysis

### 2.6.1. Total sugars, proteins and enzymatic activity measurement

Total sugars were quantified according to the DNS method (Miller, 1959). Proteins in enzyme mixtures were quantified with the colorimetric Bradford method (Bradford, 1976). All the enzymatic activities were measured at 50 °C in 50 mM sodium acetate buffer at pH 4.5. Pectinase activity was measured by quantifying the reducing sugars released from polygalacturonic acid (0.5 %, w/v) in the presence of 3, 5-dinitrosalicylic acid at 540 nm (Bailey & Pessa, 1990). Xylanase activity was estimated by quantifying the reducing sugars produced from xylans (0.5 %, w/v) in the presence of potassium ferricyanide at 420 nm (Kidby & Davidson, 1973). The measurement of β-glucosidase, β-xylosidase and β-galactosidase activities was performed in the presence of 5 mM pNP-β-d-glucopyranoside, pNP-β-d-xylopyranoside and pNP-β-d-galactopyranoside, respectively. These activities were determined from the release of p-nitrophenol at 401 nm (Smaali, Rémond & O'Donohue, 2006). Feruloyl esterase activity was tested against methyl ferulate by quantifying the release of ferulic acid (Ralet, Faulds, Williamson & Thibault, 1994). Enzyme activities are expressed in International Unit (IU) corresponding to 1 µmol of product released per minute.

### 2.6.2. Polyphenol and anthocyanin quantification

Total polyphenol contents were quantified through the Folini-Ciocalteu method (Singleton, Orthofer & Lamuela-Raventós, 1999). The quantification of total anthocyanins was carried out through a colorimetric method adapted from a previously described protocol (Lee, Durst & Wrolstad, 2005). Each sample was prepared in 0.1 M HCl (pH 1.0) and in 50 mM sodium acetate buffer at pH 5.0. The absorbance of each sample was measured at 520 nm and 700 nm after 2 h of incubation at room temperature in the dark. The total anthocyanin content (expressed as malvidin-3-O-glucoside equivalent) was determined using the Eq. (1):

[Anthocyanin] in g/kg fresh grape pomace

$$ \frac{A \times MW}{\varepsilon \times W \times l} \times V \times DF \times CF \quad (1) $$

Where

$$ A = (A_{520nm} - A_{700nm})_{pH 1} - (A_{520nm} - A_{700nm})_{pH 5} $$

$$ MW = 493.43 \text{ g/mol for malvidin-3-O-glucoside} $$

V = reactional volume

DF = dilution factor of samples,

CF = conversion factor from dry weight to fresh matter

$$ \varepsilon = 25,000 \text{ M}^{-1} \text{ cm}^{-1} \text{ (molar absorption coefficient of malvidin-3-O-glucoside)} $$

W = weight of pomace

l = pathlength in cm

The recovery of total polyphenols and anthocyanins was expressed in g per kg of fresh grape pomace. With this grape pomace, multiplying by a CF of 0.4 allowed to convert contents in g per kg lyophilized matter into g per kg fresh grape pomace.

Anthocyanins from grape pomace and acylated anthocyanins were analyzed by UHPLC-DAD-MSn using an Acquity UPLC® system (Waters Corp., Milford, MA, USA) coupled simultaneously to both an UV-Vis diode array detector and a Bruker Daltonics HCT Ultra Ion Trap MS equipped with an electrospray ion source. Separations were carried out using a HSST3 C18 column (150 mm x 2.4 mm, i.d. 1.8 µm; Waters) at 35 °C. The mobile phase was constituted by a binary solvent system with water/formic acid (97/3, v/v, solvent A) and methanol/formic acid (97/3, v/v, solvent B) at a flow rate of 0.3 mL/min. The elution gradient was as follows: 0–25 min linear 8–30 % B; 25–28 min, linear 30–40 % B; 28–35 min, linear 40–65 % B; 35–42 min, linear 65–100 % B; 42–44 min, linear 100–8 % B; 44–48 min, isocratic 8 % B. To separate the different acylated anthocyanins, the gradient was extended starting from 35 min: 35–42 min, linear 65–85 % B; 42–44 min, linear 85–100 % B; 44–48 min, isocratic 100 % B; 48–50 min, linear 100–8 % B; 50–54 min, isocratic 8 % B.

Acylated samples in 2M2B (100 µL) were diluted with 30 µL of acetone and 70 µL of MeOH/H₂O (80/20, v/v). Anthocyanins and their acylated derivatives were analyzed in the positive electrospray ionization mode from m/z 100 to 1200. Conditions were as follows: capillary voltage of ± 4 kV, nitrogen flow rate at 8 L/min, desolvation temperature at 365 °C and nebulization pressure at 50 psi.

Data processing was performed with DataAnalysis software (Bruker Daltonics, Bremen, Germany).

Area at 530 nm were reported for the quantification of all the anthocyanins. Native anthocyanins from grape pomace and acylated anthocyanins were quantified using 6-point calibration curves run in triplicates with malvidin-3-O-glucoside concentrations in MeOH/H₂O (50/50, v/v) ranging from 99.04 to 693.30 µM.

Results are expressed as mean ± standard deviation (SD) on at least three replicates. The significance of the results related to and polyphenol and anthocyanin quantification was assessed with analyses of variance (ANOVA).

# 3. Results and discussion

## 3.1. Enzyme-assisted extraction of anthocyanins from grape pomace

Two commercial enzyme cocktails were tested for the extraction of anthocyanins from grape pomace. Both enzyme cocktails were selected due to their high pectinase and low debranching activities (Table S1). Extraction assays were performed with various Pectinex® Ultra SPL and Lysis Elite loadings (Table 1). With Pectinex® Ultra SPL, the maximal yield for anthocyanin extraction was reached for 0.1 and 0.01 %, whereas the maximal yield for total polyphenol extraction was obtained with 0.01 % loading (p < 0.001). For Lysis Elite treatment, the maximal yields for anthocyanin and total polyphenol extraction were achieved with 0.01 % enzyme loading (p < 0.001). Except for Pectinex® Ultra SPL 0.01 %, the presence of enzymes did not improve polyphenol extraction compared to the control reaction without enzyme (p < 0.001). Extraction performed by enzyme-free maceration led to 0.72 g anthocyanins/kg pomace, which is higher than the yields obtained with enzyme-
<table><caption>**Table 1**

Quantification of anthocyanins and total polyphenols from grape pomace extracts after 6 h of extraction. Values are Mean ± SD (n = 3).</caption><thead><tr><th>Treatment</th><th>Extracted anthocyanins*<br>(g/kg pomace)</th><th>Total extracted polyphenols**<br>(g/kg pomace)</th></tr></thead><tbody><tr><td>Pectinex® Ultra<br>SPL 1 %</td><td>0.29 ± 0.02</td><td>1.2 ± 0.1</td></tr><tr><td>Pectinex® Ultra<br>SPL 0.1 %</td><td>0.65 ± 0.04</td><td>2.4 ± 0.2</td></tr><tr><td>Pectinex® Ultra<br>SPL 0.01 %</td><td>0.69 ± 0.07</td><td>4.1 ± 0.3</td></tr><tr><td>Lysis Elite 1 %</td><td>0.31 ± 0.04</td><td>1.1 ± 0.1</td></tr><tr><td>Lysis Elite 0.1 %</td><td>0.69 ± 0.04</td><td>3.1 ± 0.3</td></tr><tr><td>Lysis Elite 0.01 %</td><td>0.94 ± 0.09</td><td>3.3 ± 0.4</td></tr><tr><td>No enzymes</td><td>0.72 ± 0.04</td><td>3.6 ± 0.4</td></tr></tbody></table>

assisted extractions, except for Lysis Elite 0.01 % for which the yield reached 0.94 g anthocyanins/kg pomace. Increasing the protein content in the medium may lower the fraction of polyphenols released as these compounds are known to display a high affinity for proteins (Le Bourvellec & Renard, 2012). Even if several studies described the use of enzymes, such as cellulases, pectinases, and xylanases, for disrupting the plant cell walls from various biomasses in order to facilitate the extraction of intracellular polyphenols, few concern grape pomace (Ferri et al., 2016, 2023). As a matter of fact, five commercial enzymes were applied onto red Merlot pomace under conditions quite similar to those used in the present study (10 % grape pomace, enzymes at 1 or 2 % (w/w), pH 4.5, 50 °C) (Ferri et al., 2023). Celluclast® cocktail was the most efficient with 30 mg total polyphenols extracted/g dry weight after 4 h of extraction. Additionally, the amounts of total polyphenols in the extract reached 21 and 25 mg /g dry weight after 2 h and 4 h of extraction with Pectinex® Ultra SPL (1 %). These recovery yields of total polyphenols are higher than those obtained in our study and could probably be explained by different initial contents of polyphenols in both red grape pomaces. Regarding the plant cell composition, the nature of the selected enzymes is of importance as it impacts both the quality and quantity of recovered polyphenols, especially anthocyanins. The low yields of anthocyanins for the enzyme-assisted extraction could be explained by the release of chemically unstable anthocyanin aglycones due to the β-glucosidase activity of the enzyme mixtures. Debranching enzymes, such as β-glucosidase, β-galactosidase and α-l-arabinosidase, have already been described to trigger deglycosylation of native anthocyanins (Koponen, Buchert, Poutanen & Torronen, 2008; Landbo & Meyer, 2001; Wrolstad, Wightman & Durst, 1994). The lower β-glucosidase activity within Lysis Elite (compared to Pectinex®

Ultra SPL) could be responsible for the higher extraction efficiency observed. Commercial enzymes were tested for the extraction of anthocyanins from bilberries and blackcurrants and led to different contents and compositions of anthocyanins in the extracts (Buchert et al., 2005). The use of Pectinex® Ultra SPL onto bilberries allowed increasing the total polyphenol extraction by 27 % (vs. the extraction without enzyme) after 2 h at 45 °C. By selecting an appropriate enzyme cocktail displaying low undesirable enzymatic activities and by screening for optimal enzyme loading, it is thus possible to limit anthocyanin degradation. In the present study, further experiments were conducted with 0.01 % enzyme loading.

Fig. 1 presents the effect of enzyme-assisted extraction duration on the recovery of total polyphenols and anthocyanins from grape pomace. Concerning total polyphenols, the extraction efficiency increased significantly from 1 h to 6 h with and without enzymes (p < 0.05). The highest yields of total polyphenols were obtained at 24 h but were not significantly improved by enzymes (p > 0.05). Whereas the efficiency of anthocyanin extraction was highly improved between 1 h and 6 h (p < 0.05), no further improvement was observed after 24 h of extraction for all the tested conditions (p > 0.05). Compared to the assay without enzymes, Pectinex® Ultra SPL increased the anthocyanin extraction by 2.2-fold after 1 h of extraction, whereas no improvement was observed after 6 h. The extraction of anthocyanins was improved by 1.3-fold after 6 h with Lysis Elite, in comparison to the assay without enzymes. The enzyme-assisted extraction of anthocyanins with Lysis Elite at 0.01 % loading during 6 h was selected for further assays. The impact of extraction duration has been reported to be closely related to the enzymatic cocktail used. Several studies reported that short extraction duration is preferable to avoid degradation of anthocyanins (Landbo & Meyer, 2001; Swer, Chauhan, Paul & Mukhim, 2016). Recently, a DoE (design of experiments) approach performed with red Merlot pomace extracted in the presence of five different enzymes showed that the incubation time had no significant impact on the extraction efficiency of polyphenols and anthocyanins, which was more dependent on the enzyme mixtures (Ferri et al., 2023).

A sequential enzyme-assisted extraction process was conducted with Lysis Elite (0.01 %) in order to evaluate if the grape pomace was completely depleted from anthocyanins after 6 h (Fig. 2). Sequential extractions were performed from the same grape pomace sample, removing the liquid extraction phase after 6 h and adding fresh buffer and enzyme for a further 6 h of extraction. This operation was repeated to obtain three extractions of 6 h each. Enzymatic assays were also tested in a buffer/ethanol mixture (4:1). The results obtained in the buffer indicated that 76.9 % of anthocyanins were extracted during the first extraction step in the presence of Lysis Elite, whereas 14.9 % and 8.2 %

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Effect of extraction duration on the quantity of recovered anthocyanins (colorimetric quantification) and polyphenols (Folin-Ciocalteu quantification) from grape pomace in the presence of 0.01 % Pectinex® Ultra SPL or Lysis Elite. Values are Mean ± SD (n = 3).</figcaption></figure>

<footnote>* Colorimetric titration, ** Folin-Ciocalteu titration.</footnote>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Effect of three sequential 6 h-long extractions (E1 to E3) on the recovery of total polyphenols (A) and anthocyanins (B) from grape pomace with or without Lysis Elite (LE, 0.01 %) in 50 mM sodium acetate buffer (pH 4.5) with or without EtOH (20 %). Values are Mean ± SD (n = 3). Polyphenols and anthocyanins were quantified by Folin-Ciocalteu and colorimetric titrations, respectively.</figcaption></figure>

were obtained after the second and third extraction steps, respectively. The same behavior was observed for extraction without enzyme with 74.8 %, 20.8 % and 4.4 % anthocyanins extracted after steps 1, 2 and 3, respectively. The total amount of anthocyanins obtained reached 1.21 g/kg fresh pomace for enzyme-assisted extraction vs. 0.96 g/kg fresh pomace without enzyme. Total polyphenol extraction reached 7.6 and 7.2 g/kg pomace after the 3 steps with and without enzyme, respectively.

In the presence of 20 % ethanol, a higher extraction efficiency for both anthocyanins and total polyphenols was achieved (compared to extraction in buffer alone) whether enzymes are present or not, in agreement with polyphenols being generally more soluble in hydro-alcoholic mixtures than in water alone (Baron et al., 2021; Tena & Asuero, 2022).

The first 6 h-long extraction allowed obtaining the highest amounts of anthocyanins and total polyphenols. After the three sequential extraction steps, the total amount of anthocyanins reached 1.44 g/kg fresh pomace in enzyme-assisted extraction, vs. 1.17 g/kg fresh pomace without enzyme. By comparison, US-assisted extraction in acidified EtOH-water (7:3) only led to 0.82 ± 0.04 g of total anthocyanins per kg fresh pomace, suggesting that maceration has to be prolonged when starting from lyophilized matter (see Section 2.2). After the three extraction steps, total polyphenol concentrations reached 14.7 and 14.2

g/kg fresh pomace with or without enzyme, respectively, these values being 2-fold higher than in absence of ethanol. The first extraction step provided the highest amounts of both polyphenols and anthocyanins. These results are consistent with those previously reported for a multi-step extraction process conducted on the pulp from three Champagne grape varieties (Mané et al., 2007). To prevent microbial contamination and facilitate storage, the grape pomace used in the present study was stored under lyophilized form. The freezing step of lyophilization is expected to trigger plant cell breakage, thus facilitating extraction. Indeed, freezing grape pomace has been reported to enhance the extraction of anthocyanins and tannins (Garcia, Santesteban, Miranda & Royo, 2011; Nicolle, Marcotte, Angers & Pedneault, 2019). Thus, freezing could be a relevant step for extraction at industrial level.

Most enzyme-assisted extraction processes have been conducted in buffered media to ensure efficiency and stability of glycosidases. Piazza et al. (Piazza, Romanini & Meini, 2023) recently described the enzymatic extraction of polyphenols from grape pomace in acetate buffer (50 mM, pH 5) mixed with ethanol (0 - 30 %, v/v) in the presence of cellulase, pectinase or tannase. For all enzymes, the maximal extraction yields reached 1.3 and 2.3 g total phenolic compounds/100 g dry weight in the presence of 20 % and 30 % EtOH, respectively, vs. 0.5 to 0.6 g/100 g dry weight in buffer alone, again confirming the favorable influence of EtOH.
The UHPLC analysis of a crude extract of grape pomace is presented in Figure S1 and the MS identification summarized in Table S2. The main peaks were identified as anthocyanins based on UV-vis spectrum, molecular mass and fragmentation pattern, as well as comparison with the literature (Núñez, Monagas, Gomez-Cordovés & Bartolomé, 2004; Trikas et al., 2016; Zhao et al., 2020). The five anthocyanins classically found in grapes were identified (peaks A1 to A5), namely 3-O-glucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin, the latter being the most abundant. Eight anthocyanins acylated with either acetic acid, p-coumaric acid or caffeic acid, were also identified in low levels (peaks A6 to A13), malvidin-3-O-(6'-O-p-coumaroylglucoside) being the most abundant acylated anthocyanin. In grape pomace (a winemaking co-product), anthocyanins are mainly located in the skin. As the grape pomace studied was a mix from different varieties of the Sigean area (Aude) in South France, its anthocyanin profile was not characteristic of genotype or agricultural practice.

## 3.2. Production of fractions rich in anthocyanins, in other polyphenols, and sugars

A two-step purification was developed to provide three fractions of decreasing polarity from the extract produced after a 6 h treatment with Lysis Elite: a sugar-rich fraction, a fraction enriched in anthocyanins, and a fraction containing the other polyphenols. The initial extract contained 17 g of sugars and 194 mg of total polyphenols, including 17.34 mg of anthocyanins. The recovery yields of anthocyanins, other polyphenols and sugars after each step are displayed in Table 2.

The first step was a chromatographic purification on an anionic TSK resin. It yielded 81.7 % of the sugars after elution with a sodium acetate buffer. Polyphenols were also recovered (9.4 %) and to a higher extent after elution with MeOH (27.0 %). The anthocyanin recovery after elution with MeOH reached 84.1 %. Various resins (Amberlite XAD-7HP, Amberlite XAD-4, Dowex 50Wx8) have been already reported for anthocyanin purification (Heinonen et al., 2016; Madhusudhan & Raghavarao, 2012), not necessarily requiring additional purification step. However, for enzymatic acylation of anthocyanins, other polyphenols, especially glycosides, are expected to compete with anthocyanins for the enzyme. To further enrich the extract in anthocyanins, a liquid-liquid extraction of the MeOH extract was performed in ethyl acetate - water (1:1). The anthocyanins were recovered in the aqueous phase with a yield of 72.3 %, whereas the other polyphenols were recovered in both aqueous and organic phases.

<caption>**Table 2**</caption>

Recovery of anthocyanins, total polyphenols and sugars from the crude grape pomace extract and from fractions obtained by chromatographic separation and liquid/liquid fractionation.

<table><thead><tr><th></th><th>Quantity (mg)</th><th>Recovery yield (%)</th></tr></thead><tbody><tr><td><strong>Extraction</strong> (0.01 % Lysis Elite, 50 °C, 6 h)</td><td></td><td></td></tr><tr><td>Polyphenols*</td><td>194.27</td><td>100</td></tr><tr><td>Anthocyanins**</td><td>17.34</td><td>100</td></tr><tr><td>Sugars***</td><td>17.00</td><td>100</td></tr><tr><td><strong>TSK purification</strong></td><td></td><td></td></tr><tr><td><strong>1) Buffer elution</strong></td><td></td><td></td></tr><tr><td>Polyphenols*</td><td>18.21</td><td>9.4</td></tr><tr><td>Sugars***</td><td>13.88</td><td>81.7</td></tr><tr><td><strong>2) Methanol elution</strong></td><td></td><td></td></tr><tr><td>Polyphenols*</td><td>52.52</td><td>27.0</td></tr><tr><td>Anthocyanins**</td><td>14.59</td><td>84.1</td></tr><tr><td><strong>Liquid-liquid fractionation</strong></td><td></td><td></td></tr><tr><td><strong>1) Water phase</strong></td><td></td><td></td></tr><tr><td>Polyphenols*</td><td>24.47</td><td>12.6</td></tr><tr><td>Anthocyanins**</td><td>12.53</td><td>72.3</td></tr><tr><td><strong>2) Ethyl acetate phase</strong></td><td></td><td></td></tr><tr><td>Polyphenols*</td><td>23.12</td><td>11.9</td></tr></tbody></table>

## 3.3. Enzymatic acylation of the purified anthocyanin fraction recovered from grape pomace

Reactions between the purified grape pomace extract and dihydroferulic acid or lauric acid (C12 fatty acid) as acyl donors were catalyzed by lipase N435. All the reactions were carried out in anhydrous 2M2B in the presence of molecular sieves to prevent the reverse reaction of hydrolysis. After 72 h of reaction, the molecular sieves were washed with EtOH to recover the fraction of adsorbed anthocyanins. Figures 4A and 4B showed the UHPLC-DAD-MSn profile of the reaction medium and Tables 3 summarize the mass data for acylated anthocyanins. After acylation by dihydroferulic acid five new peaks were detected at 530 nm (Fig. 3A). The major compound at R<sub>t</sub> 31.5 min, displaying a λ<sub>max</sub> at 533 nm and a parent ion at m/z 671, corresponded to malvidin-3-O-dihydroferuloylglucoside (AF5) (Table 3). A fragment ion at m/z 331 (malvidin) in MS<sup>2</sup> indicated that acylation had occurred on the glucose residue. The major product was coeluted with peonidin-3-O-dihydroferuloylglucoside (AF4), which exhibits a parent ion at m/z 641 and a fragment ion at m/z 301 in MS<sup>2</sup>. Delphinidin-, cyanidin- and petunidin-3-O-dihydroferuloylglucosides (AF1 to AF3) were eluted at respectively 29.6, 30.5 and 30.8 min. The presence of unreacted peonidin- and malvidin-3-O-glucosides pointed to a partial acylation under the selected conditions.

With lauric acid as the acyl donor, highly lipophilic derivatives were detected between 38.7 and 39.7 min. UHPLC-DAD-MSn analysis confirmed the acylation of all five anthocyanins (Fig. 3B). The major compound at R<sub>t</sub> 39.5 min (AL4), exhibiting a λ<sub>max</sub> at 541 nm, a parent ion at m/z 675 and a fragment ion at m/z 331, corresponded to malvidin-3-O-lauroylglucoside (Table 3). It was coeluted with peonidin-3-O-lauroylglucoside (AL5), which presented a parent ion at m/z 645 and a fragment ion at m/z 301. Delphinidin, cyanidin and petunidin 3-O-lauroylglucosides (AL1 to 3) were eluted at 38.7, 39.1 and 39.2 min, respectively.

Acylation must have occurred at the primary hydroxyl (C6-OH) of the d-glucopyranosyl group. This regioselectivity is largely reported for lipase N435 (Stevenson, Wibisono, Jensen, Stanley & Cooney, 2006). The acylation yield was calculated relative to the initial concentration in native anthocyanins (4 mM) in the reaction medium: 4.2 ± 0.7 % for the pool of anthocyanins acylated by dihydroferulic acid (residual peonidin- and malvidin-3-O-glucosides = 0.6 ± 0.3 %), 12.4 ± 0.5 % for the pool of anthocyanins acylated by lauric acid (residual

<caption>**Table 3**</caption>

Identification of the major anthocyanins acylated by dihydroferulic acid (AF) and by lauric acid (AL) (UHPLC-DAD-MSn analysis in the positive ion mode).

<table><thead><tr><th>Peak number</th><th>R<sub>t</sub> (min)</th><th>λ<sub>max</sub> (nm)</th><th>[M]<sup>+</sup>(m/z)</th><th>MS<sup>2</sup> fragment (m/z)</th><th>Proposed structure</th></tr></thead><tbody><tr><td>AF1</td><td>29.6</td><td></td><td>643</td><td>nd</td><td>Delphinidin-3-O-dihydroferuloylglucoside</td></tr><tr><td>AF2</td><td>30.5</td><td></td><td>627</td><td>nd</td><td>Cyanidin-3-O-dihydroferuloylglucoside</td></tr><tr><td>AF3</td><td>30.8</td><td></td><td>657</td><td>317</td><td>Petunidin-3-O-dihydroferuloylglucoside</td></tr><tr><td>AF4</td><td>31.4</td><td></td><td>641</td><td>301</td><td>Peonidin-3-O-dihydroferuloylglucoside</td></tr><tr><td>AF5</td><td>31.5</td><td>533</td><td>671</td><td>331</td><td>Malvidin-3-O-dihydroferuloylglucoside</td></tr><tr><td>AL1</td><td>38.7</td><td></td><td>647</td><td>nd</td><td>Delphinidin-3-O-lauroylglucoside</td></tr><tr><td>AL2</td><td>39.1</td><td></td><td>631</td><td>287</td><td>Cyanidin-3-O-lauroylglucoside</td></tr><tr><td>AL3</td><td>39.2</td><td></td><td>661</td><td>317</td><td>Petunidin-3-O-lauroylglucoside</td></tr><tr><td>AL4</td><td>39.5</td><td>541</td><td>675</td><td>331</td><td>Malvidin-3-O-lauroylglucoside</td></tr><tr><td>AL5</td><td>39.7</td><td></td><td>645</td><td>301</td><td>Peonidin-3-O-lauroylglucoside</td></tr></tbody></table>

<footnote>* Folin-Ciocalteu titration, ** Colorimetric titration, *** DNS titration.</footnote>
<figure><img src="image_6.png" /><figcaption>Fig. 3. UHPLC profile at 530 nm of the purified extract acylated by dihydroferulic acid (A) and lauric acid (B).</figcaption></figure>

malvidin-3-O-glucoside = 1.9 ± 0.2 %). However, a large fraction of acylated anthocyanins remained adsorbed on lipase and molecular sieves. Indeed, extensive washing allowed the determination of much higher overall yields of acylated anthocyanins: 8.5 ± 1.7 % with dihydroferulic acid, 19.8 ± 0.7 % with lauric acid.

Several studies have described the enzymatic acylation of anthocyanin extracts from various biomasses, such as blackberry, blueberry and blackcurrant, which mainly contain cyanidin-3-O-glucoside and delphinidin-3-O-glucoside (Leonarski, Cesca, de Oliveira & Zielinski, 2023). After extraction and purification of cyanidin-3-O-glucoside from blackberry, acylation catalyzed by CALB lipase was conducted in 2M2B in the presence of various aliphatic fatty acids (C4 to C12) (Guimaraes, Mateus, de Freitas & Cruz, 2018). The yields of acylation were comprised between 47 % (C4) and 25 % (C12). With malvidin-3-O-glucoside acylation yields by C4 to C16 fatty acids (catalysis by CALB lipase) were in the range 40 to 22 %, thus showing a decrease with increasing fatty acid chain length (Cruz et al., 2017). This is consistent with a previous work with oleic acid (C18), which led to malvidin-3-O-oleoylglucoside in 21.2 % yield (Cruz et al., 2016).

As for phenolic acyl donors, acylation of a blueberry extract (a mixture of anthocyanidin galactosides and glucosides) catalyzed by lipase N435 in the presence of 4-hydroxy- and 3,4-dihydroxyphenylpropionic acids (Stevenson et al., 2006) led to yields in the range 27 to 47 %. In another case, cyanidin-3-O-glucoside was acylated by methylsalicylate under catalysis by lipase N435 with a 84 % yield (Yan et al., 2016). As largely described for the lipase-catalyzed acylation of

monosaccharides, esters are typically better acyl donors (trans-esterification) than free acids (Jocquel, Muzard, Plantier-Royon & Rémond, 2021). In the present study the conditions of enzymatic acylation were not optimized, as the objective was to evaluate the possible production of acylated anthocyanins directly from grape pomace. Higher acylation yields might be obtained with esters as acyl donors and shorter acylation duration.

## 4. Conclusions

Our study demonstrates the possibility to use a two-step biocatalytic process for the extraction and acylation of anthocyanins from grape pomace. Concerning the extraction step, the choice of the enzyme, its loading, as well as the reaction conditions, are crucial to achieve a high yield of extraction with the goal of limiting anthocyanin deglycosylation. In our work, Lysis Elite in low concentration (0.01 %) proved promising to improve anthocyanin extraction (vs. control without enzyme). Three successive enzymatic extraction steps allowed the complete recovery of anthocyanins from freeze-dried grape pomace. Other polyphenols and some sugars were co-extracted and recovered through a simple two-step purification by elution on an anionic resin and liquid-liquid fractionation. The purified anthocyanin extract was then acylated with either lauric acid or dihydroferulic acid. The yields of lipase-catalyzed acylation were rather modest and will have to be improved in future works. However, they compare favorably with analogous works starting from extracts instead of individual
anthocyanins. The mixtures of acylated anthocyanins prepared in this work are expected to adopt specific conformations and/or enhanced affinity for oil-water interfaces. As such, they could prove more stable in food systems for development as colorings.

Altogether our results highlight the interest of a complete bio-
catalytic approach for a higher-value valorization of the anthocyanins
from grape pomace, thus offering better incomes to the distillery sector.

Ethics statement

This study does not involve any studies with animal or human
subjects

CRediT authorship contribution statement

**Emelyne Jolly:** Methodology, Investigation, Writing – original draft.
**Pascale Goupy:** Methodology, Investigation, Supervision, Writing – original draft, Writing – review & editing.
**Angélique Claisse-Gainvors:** Writing – review & editing, Methodology, Conceptualization.
**Olivier Dangles:** Writing – review & editing, Validation.
**Diana Garcia-Bernet:** Writing – review & editing, Supervision, Conceptualization, Funding acquisition, Methodology, Resources, Writing – original draft.
**Claire Dufour:** Writing – review & editing, Supervision, Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Validation, Writing – original draft.
**Caroline Rémond:** Writing – original draft, Supervision, Project administration, Conceptualization, Funding acquisition, Methodology, Resources, Validation, Writing – review & editing.

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.

Acknowledgements

This work has been granted by 3BCAR and Qualiment® Carnot In-
stitutes in the frame of their 2020 call for projects. 3BCAR and Quali-
ment® are supported by ANR (agreements #20-CARN 0001 01 and #20
CARN 0026-01). FARE is supported by the Grand Reims through
financial support of the Chaire AFERE.

Supplementary materials

Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.afres.2025.101431.

Data availability

Data will be made available on request.

Reference

Baron, G., Ferrario, G., Marinello, C., Carini, M., Morazzoni, P., & Aldini, G. (2021). Effect of Extraction Solvent and Temperature on Polyphenol Profiles, Antioxidant and Anti-Inflammatory Effects of Red Grape Skin By-Product. *Molecules*, *26*(18), 5454. https://doi.org/10.3390/molecules26185454

Beres, C., Costa, G. N. S., Cabezudo, I., da Silva-James, N. K., Teles, A. S. C., ... Suely, P. (2017). Towards integral utilization of grape pomace from winemaking process: A review. *Waste Management*, **68**, 581–594. https://doi.org/10.1016/j.wasman.2017.07.017

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. *Analytical biochemistry*, **72**, 248–254.

Buchert, J., Koponen, J. M., Suutarinen, M., Mustranta, A., Lille, M., Torronen, R., & Poutanen, K. (2005). Effect of enzyme-aided pressing on anthocyanin yield and profiles in bilberry and blackcurrant juices. *Journal of the Science of Food and Agriculture*, **85**(15), 2548–2556. https://doi.org/10.1002/jsfa.2284

Castañeda-Ovando, A., Pacheco-Hernández, M. d. L., Páez-Hernández, M. E., Rodríguez, J. A., & Galán-Vidal, C. A. (2009). Chemical studies of anthocyanins: A review. *Food Chemistry*, **113**(4), 859–871. https://doi.org/10.1016/j.foodchem.2008.09.001

Constantin, O. E., & Etrati, D. I. (2022). Extraction, Quantification and Characterization Techniques for Anthocyanin Compounds in Various Food Matrices—A Review. *Horticulturae*, **8**(11), 1084. https://doi.org/10.3390/horticulturae8111084

Cruz, L., Fernandes, I., Guimarães, M., de Freitas, V., & Mateus, N. (2016). Enzymatic synthesis, structural characterization and antioxidant capacity assessment of a new lipophilic malvinid-3-glucoside-oleic acid conjugate. *Food & Function*, **7**(6), 2754. https://doi.org/10.1039/C6FO00466K, 2762.

Cruz, L., Guimarães, M., Araújo, P., Évora, A., de Freitas, V., & Mateus, N. (2017). Malvinid 3-Glucoside-Fatty Acid Conjugates: From Hydrophilic toward Novel Lipophilic Derivatives. *Journal of Agricultural and Food Chemistry*, **65**(31), 6513–6518. https://doi.org/10.1021/acs.jafc.6b05461

Dangles, O. (2024). Anthocyanins as Natural Food Colorings: The Chemistry Behind and Challenges Still Ahead. *Journal of Agricultural and Food Chemistry*, **72**(22), 12356–12372. https://doi.org/10.1021/jacs.jafc.4c01050

Dangles, O., & Fenger, J. A. (2018). The Chemical Reactivity of Anthocyanins and Its Consequences in Food Science and Nutrition. *Molecules*, **23**(8). https://doi.org/10.3390/molecules23081970

FAO. (2023). Agricultural production statistics 2000-2022. FAOSTAT Analytical Briefs, 79. Rome.

Fenger, J. A., Moloney, M., Robbins, R. J., Collins, T. M., & Dangles, O. (2019). The influence of acylation, metal binding and natural antioxidants on the thermal stability of red cabbage anthocyanins in neutral solution. *Food & Function*, **10**(10), 6740–6751. https://doi.org/10.1039/C9FO01884K

Fernandez-Aulis, F., Torres, A., Sanchez-Mendoza, E., Cruz, L., & Navarro-Ocana, A. (2020). New acylated cyanidin glycosides extracted from underutilized potential sources: Enzymatic synthesis, antioxidant activity and thermostability. *Food Chemistry*, **309**, Article 125796. https://doi.org/10.1016/j.foodchem.2019.125796

Ferri, M., Bin, S., Vallini, V., Fava, F., Michelini, E., Roda, A., ... Tassoni, A. (2016). Recovery of polyphenols from red grape pomace and assessment of their antioxidant and anti-cholesterol activities. *New Biotechnology*, **33**(3), 338–344. https://doi.org/10.1016/j.nbt.2015.12.004

Ferri, M., Lima, V., Zappi, A., Fernando, A. L., Melucci, D., & Tassoni, A. (2023). Phytochemicals Recovery from Grape Pomace: Extraction Improvement and Chemometric Study. *Foods*, **12**(5), 959.

Garcia, S., Santesteban, L. G., Miranda, C., & Royo, J. B. (2011). Variety and storage time affect the compositional changes that occur in grape samples after frozen storage. *Australian Journal of Grape and Wine Research*, **17**(2), 162–168. https://doi.org/10.1111/j.1755-0238.2011.00134.x

Gérard, V., Ay, E., Morlet-Savary, F., Graff, B., Galopin, C., Ogren, T., ... J.. J. (2019). Thermal and Photochemical Stability of Anthocyanins from Black Carrot, Grape Juice, and Purple Sweet Potato in Model Beverages in the Presence of Ascorbic Acid. *Journal of Agricultural and Food Chemistry*, **67**(19), 5647–5660. https://doi.org/10.1021/jacs.jafc.9b01672

Giusti, M., & Wrolstad, Ronald. (2003). Acylated anthocyanins from edible sources and their applications in food systems. *Biochemical Engineering Journal*, **14**, 217–225. https://doi.org/10.1016/S1369-703X(02)00221-8

Guimaraes, M., Mateus, N., de Freitas, V., & Cruz, L. (2018). Improvement of the Color Stability of Cyanidin-3-glucoside by Fatty Acid Enzymatic Acylation. *Journal of Agricultural and Food Chemistry*, **66**(38), 10003–10010. https://doi.org/10.1021/acs.jafc.8b03536

Heinonen, J., Farahmandazad, H., Vuorinen, A., Kallio, H., Yang, B., & Sainio, T. (2016). Extraction and purification of anthocyanins from purple-fleshed potato. *Food and Bioproducts Processing*, **99**, 136–146. https://doi.org/10.1016/j.fbp.2016.05.004

Jocquel, C., Muzard, M., Plantier-Royon, R., & Rémond, C. (2021). An Integrated Enzymatic Approach to Produce Pentyl Xylisosides and Glucose/Xylose Laurate Esters From Wheat Bran. *Front Bioeng Biotechnol*, **9**, Article 647442. https://doi.org/10.3389/fbioe.2021.647442

Kidby, D. K., & Davidson, D. J. (1973). A convenient ferricyanide estimation of reducing sugars in the nanomole range. *Anal Biochem*, **55**(1), 321–325. https://doi.org/10.1016/0003-2697(73)90323-0

Konczak, I., & Zhang, W. (2004). Anthocyanins—More Than Nature’s Colours. *BioMed Research International*, **(5)**, Article 307613. https://doi.org/10.1155/S1110724304407013, 2004.

Koponen, J. M., Buchert, J., Poutanen, K. S., & Torronen, A. R. (2008). Effect of pectinolytic juice production on the extractability and fate of bilberry and black currant anthocyanins. *European Food Research and Technology*, **227**(2), 485–494. https://doi.org/10.1007/s00217-007-0745-2

Landbo, A. K., & Meyer, A. S. (2001). Enzyme-assisted extraction of antioxidative phenols from black current juice press residues (Ribes nigrum). *Journal of Agricultural and Food Chemistry*, **49**(7), 3169–3177. https://doi.org/10.1021/jf001443p

Le Bourvellec, C., & Renard, C. M. (2012). Interactions between polyphenols and macromolecules: quantification methods and mechanisms. *Crit Rev Food Sci Nutr*, **52**(3), 213–248. https://doi.org/10.1080/10408398.2010.499808

Lee, J., Durst, R. W., & Wrolstad, R. E. (2005). Effects of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. *J AOAC Int*, **88**(5), 1269–1278. https://doi.org/10.1093/jaoac/88.5.1269

Leonarski, E., Cesca, K., de Oliveira, D., & Zielinski, A. A. F (2023). A review on enzymatic acylation as a promising opportunity to stabilizing anthocyanins. *Critical Reviews in Food Science and Nutrition*, **63**(24), 6777–6796. https://doi.org/10.1080/10408398.2022.2041541
Liu, J., Zhuang, Y., Hu, Y., Xue, S., Li, H., Chen, L., & Fei, P. (2020). Improving the color stability and antioxidation activity of blueberry anthocyanins by enzymatic acylation with p-coumaric acid and caffeic acid. *LWT, 130*, Article 109673. https://doi.org/10.1016/j.lwt.2020.109673

Madhusudhan, M. C., & Raghavarao, K. S. M. S. (2012). Extraction and purification of Anthocyanins from red cabbage. *Food and Bioproducts Processing, 90*, 615–623. https://doi.org/10.1016/j.fbp.2012.07.004

Mané, C., Souquet, J. M., Ollé, D., Verriés, C., Véran, F., Mazerolles, G., & Fulcrand, H. (2007). Optimization of Simultaneous Flavanol, Phenolic Acid, and Anthocyanin Extraction from Grapes Using an Experimental Design: Application to the Characterization of Champagne Grape Varieties. *Journal of Agricultural and Food Chemistry, 55*(18), 7224–7233. https://doi.org/10.1021/jf071301w

Marquez-Rodriguez, A. S., Guimarães, M., Mateus, N., de Freitas, V., Ballinas-Casarrubias, M. L., Fuentes-Montero, M. E., & Cruz, L. (2021). Disaccharide anthocyanin delphinidin 3-O-sambubioside from Hibiscus sabdariffa L.: Candida antarctica lipase B-catalyzed fatty acid acylation and study of its color properties. *Food Chemistry, 344*, Article 128603. https://doi.org/10.1016/j.foodchem.2020.128603

Merecz-Sadowska, A., Sitarek, P., Kowalczyk, T., Zajdel, K., Jęcek, M., Nowak, P., & Zajdel, R. (2023). Food Anthocyanins: Malvidin and Its Glycosides as Promising Antioxidant and Anti-Inflammatory Agents with Potential Health Benefits. *Nutrients, 15*(13), 3016. https://doi.org/10.3390/nu15133016

Miller, G. L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. *Analytical Chemistry, 31*(3), 426–428.

Moutinho, J., Gouvinhas, I., Domínguez-Perles, R., & Barros, A. (2023). Optimization of the Extraction Methodology of Grape Pomace Polyphenols for Food Applications. *Molecules, 28*(9), 3885. https://doi.org/10.3390/molecules28093885

Nicolle, P., Marcotte, C., Angers, P., & Pedneault, K. (2019). Pomace limits tannin retention in Frontenac wines. *Food Chemistry, 277*, 438–447. https://doi.org/10.1016/j.foodchem.2018.10.114

Núñez, V., Monagas, M., Gomez-Cordovés, M. C., & Bartolomé, B. (2004). Vitis vinifera L. cv. Graciano grapes characterized by its anthocyanin profile. *Postharvest Biology and Technology, 31*(1), 69–79. https://doi.org/10.1016/S0925-5214(03)00140-6

Panja, P. (2018). Green extraction methods of food polyphenols from vegetable materials. *Current Opinion in Food Science, 23*, 173–182. https://doi.org/10.1016/j.cofs.2017.11.012

Panzella, L., Moccia, F., Nasti, R., Marzorati, S., Verotta, L., & Napolitano, A. (2020). Bioactive Phenolic Compounds From Agri-Food Wastes: An Update on Green and Sustainable Extraction Methodologies. *Front Nutr, 7*, 60. https://doi.org/10.3389/fnut.2020.00060

Piazza, D. M., Romanini, D., & Meini, M. R. (2023). High-efficiency novel extraction process of target polyphenols using enzymes in hydroalcoholic media. *Appl Microbiol Biotechnol, 107*(4), 1205–1216. https://doi.org/10.1007/s00253-023-12386-7

Ralet, M. C., Faulds, C. B., Williamson, G., & Thibault, J. F. (1994). Degradation of feruloylated oligosaccharides from sugar-beet pulp and wheat bran by ferulic acid esterases from Aspergillus niger. *Carbohydr Res, 263*(2), 257–269. https://doi.org/10.1016/0008-6215(94)00177-4

Robbins, R., Collins, T., Kondo, T., Yoshida, K., & Dangles, O. (2018). Red cabbage anthocyanins: The influence of D-glucose acylation by hydroxycinnamic acids on their structural transformations in acidic to mildly alkaline conditions and on the

resulting color. *Dyes and Pigments*, 158. https://doi.org/10.1016/j.dyeig.2018.05.057

Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). 14]Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. *In Methods in Enzymology, 299* pp. 152–178). Academic Press.

Smaali, I., Rémond, C., & O'Donohue, M. J. (2006). Expression in Escherichia coli and characterization of beta-xylosidases GH39 and GH-43 from Bacillus halodurans C-125. *Appl Microbiol Biotechnol, 73*(3), 582–590. https://doi.org/10.1007/s00253-006-0512-5

Stevenson, D. E., Wibisono, R., Jensen, D. J., Stanley, R. A., & Cooney, J. M. (2006). Direct acylation of flavonoid glycosides with phenolic acids catalysed by Candida antarctica lipase B (Novozym 435 (R)). *Enzyme and Microbial Technology, 39*(6), 1236–1241. https://doi.org/10.1016/j.enzmictec.2006.03.006

Swer, Tanya L., Chauhan, Komal, Paul, Prodyut K., & Mukhim, C. (2016). Evaluation of enzyme treatment conditions on extraction of anthocyanins from Prunus nepalensis L. *International Journal of Biological Macromolecules, 92*, 867–871. https://doi.org/10.1016/j.ijbijacm.2016.07.105

Tena, N., & Asuero, A. G. (2022). Up-To-Date Analysis of the Extraction Methods for Anthocyanins: Principles of the Techniques, Optimization, Technical Progress, and Industrial Application. *Antioxidants, 11*(2), 286. https://doi.org/10.3390/antiox11020286

Trikas, E. D., Melidou, M., Papi, R. M., Zachariadis, G. A., & Kyriakidis, D. A. (2016). Extraction, separation and identification of anthocyanins from red wine by-product and their biological activities. *Journal of Functional Foods, 25*, 548–558. https://doi.org/10.1016/j.jff.2016.06.033

Trouillas, P., Sancho-García, J. C., De Freitas, V., Gierschner, J., Otyepka, M., & Dangles, O. (2016). Stabilizing and Modulating Color by Copigmentation: Insights from Theory and Experiment. *Chemical Reviews, 116*(9), 4937–4982. https://doi.org/10.1021/acs.chemrev.5b00507

Wallace, T. C., & Giusti, M. M. (2015). *Anthocyanins. Adv Nutr, 6*(5), 620–622. https://doi.org/10.3945/an.115.009233

Wrolstad, R. E., Wightman, J. D., & Durst, R. W. (1994). Glycosidase Activity of Enzyme Preparations Used in Fruit Juice Processing. *Food Technology*, 48, 90–98.

Yan, Z., Li, C., Zhang, L., Liu, Q., Ou, S., & Zeng, X. (2016). Enzymatic Acylation of Anthocyanin Isolated from Black Rice with Methyl Aromatic Acid Ester as Donor: Stability of the Acylated Derivatives. *Journal of Agricultural and Food Chemistry, 64* (5), 1137–1143. https://doi.org/10.1021/acs.jafc.5b05031

Zhang, P., Liu, S., Zhao, Z., You, L., Harrison, M. D., & Zhang, Z. (2021). Enzymatic acylation of cyanidin-3-glucoside with fatty acid methyl esters improves stability and antioxidant activity. *Food Chem, 343*, Article 128482. https://doi.org/10.1016/j.foodchem.2020.128482

Zhang, X., Yang, Y., Wu, Z., & Weng, P. (2016). The Modulatory Effect of Anthocyanins from Purple Sweet Potato on Human Intestinal Microbiota in Vitro. *Journal of Agricultural and Food Chemistry, 64*(12), 2582–2590. https://doi.org/10.1021/acs.jafc.6b00586

Zhao, X., Zhang, S. S., Zhang, X. K., He, F., & Duan, C. Q. (2020). An effective method for the semi-preparative isolation of high-purity anthocyanin monomers from grape pomace. *Food Chemistry, 310*, Article 125830. https://doi.org/10.1016/j.foodchem.2019.125830