# White wine lees as a source of antioxidants: Insights into their chemical profile

Luca Garcia, Benjamin Poulain, Alice Douliez, Eloise Naud, Josep

Valls-Fonayet, Claudia Nioi

► To cite this version:

Luca Garcia, Benjamin Poulain, Alice Douliez, Eloise Naud, Josep Valls-Fonayet, et al.. White wine lees as a source of antioxidants: Insights into their chemical profile. Food Chemistry, 2025, 494, pp.146118. 10.1016/j.foodchem.2025.146118. hal-05273047

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Submitted on 22 Sep 2025

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fermentation. Dubourdieu and Lavigne-Cruege (Dubourdieu & Lavigne-Cruège, 2004) demonstrated that ageing on lees preserves high GSH levels in white wines, enhancing their redox stability. Furthermore, studies by Jaehrig et al. (Jaehrig et al., 2008) showed that hydrolysis of mannoproteins and cell wall proteins leads to the release of antioxidant peptides. More recently, a study on the antioxidant metabolome of white wines revealed an increase in sulphur (—S) and nitrogen (—N) compounds in wines aged on lees, likely due to the release of antioxidant peptides (Romanet et al., 2020). The protein content, rich in peptides and amino acids, possesses antioxidant activity through its ability to chelate metals, specifically those containing histidine and glutamic acid (Elias et al., 2008). Additionally, sulfhydryl-containing peptides, such as glutathione, have the potential to scavenge free radicals and deactivate the singlet oxygen (Foti & Amorati, 2009). The nucleophilic thiol function in these compounds allows o-quinones trapping, preventing the polymerisation reactions of polyphenols and thus preventing the browning of white wines (Nikolantonaki et al., 2014).

Regarding yeast cell wall polysaccharides, β-glucans, may have the capacity to chelate the metal ions Fe²⁺ and Cu²⁺ through the formation of coordination bonds (Pristov et al., 2011). The same metal chelation behaviour has been described for organic acids by the formation of coordination bonds with Fe²⁺ (Danilewicz, 2014).

Polyphenols, which are adsorbed on yeast walls, can play a major role in the antioxidant capacity of wine lees. Polyphenols are primary antioxidants in oenological matrices, reacting through multiple mechanisms. They have the property of chelating metal ions and therefore inhibiting the formation of free radicals formed by metal catalysis (Hudson & Lewis, 1983). They act as ROS inhibitors and scavengers, and also have the property of quenching singlet oxygen (Ávila et al., 2001). Furthermore, due to their nucleophilic nature, polyphenols are able to react with the quinones formed during wine oxidation (Ma & Waterhouse, 2018).

Although previous studies have identified various antioxidant compounds in wine lees, assessing their overall antioxidant potential based on composition remains challenging, and the specific contribution of each compound to antioxidant activity is not well defined. Despite the diversity of antioxidant families present, no study has yet established a comprehensive link between the chemical composition of a large set of white wine lees and their antioxidant capacity.

Therefore, this article has the following objectives: (i) to provide a comprehensive chemical composition of white wine lees using both global and targeted approaches, (ii) to measure the free radical scavenging capacity of white wine lees and (iii) to establish the link between the composition of white wine lees and their free radical scavenging activity.

# 2. Materials and methods

## 2.1. Chemicals

Ultrapure water (Purite Type I Ultrapure Water System, Veolia, France) was used for sample preparation. HPLC grade ethanol (EtOH), methanol, chloroform and sodium carbonate was purchased from VWR (France). The 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Trolox, Gallic acid, and Phenol were purchased from ThermoFisher Scientific (France). 2,4,6-Tri(2-Pyridyl)-s-Triazine (TPTZ), Folin-Ciocalteu reagent, EDTA, 5,5-dithio-bis-(acide 2-nitrobenzoïque (DTNB) hydrochloric acid, standards of trans-Resveratrol, 1-Glutathione reduced (GSH) and oxidised (GSSG) were purchased from Sigma-Aldrich (France). Sulfuric acid (98 %) was purchased from Supelco (France). UHPLC-HRMS chromatographic analysis was performed with LC-MS grade acetonitrile, deionized ultrapure water and formic acid (Optima, Fisher Chemical, France). Polyphenol standards were purchased from Extrasynthese (France) and included flavonols (taxifolin, quercetin, quercetin 3-glucoside, quercetin 3-glucoronide, quercetin 3-rhamnoside), phenolic acids (caffeic acid, gallic acid, coutaric acid and caftaric acid), flavanols and procyanidins

(catechin, epicatechin, gallocatechin, epicatechin-3-O-gallate, B1, and B2) and trans-ε-viniferin. Other stilbenoids (trans-piceid, trans-piceatannol, trans-ω-viniferin, pallidol, parthenocissin A, α-viniferin, δ-viniferin, miyabenol C, hopeaphenol, isohopeaphenol) were obtained in the laboratory.

## 2.2. Wine lees samples

A set of 41 white wine lees samples were collected after alcoholic fermentation (AF) from wines produced at various estates within the Bordeaux appellation region. The details of the grape varieties, yeast strain, vintage were provided in the supplementary data (Table S1). The liquid-solid suspension of lees was centrifuged and the solid pellet was then freeze-dried in the laboratory, ground and stored in a dark environment at room temperature prior to analysis to preserve the bioactivity of samples as described by Poulain et al. (2024).

## 2.3. Dry matter content (DM)

The dry matter (DM) content of wine lees was determined following the standard ISO-638-1:2021 of the ISO 638-1:2021(E) (2021). Briefly, a mass (m₂ in g) of freeze-dried wine lees was placed in a previously weighed aluminum dish (m₁ in g). This dish was then placed in an oven at 105 °C for 24 h. After drying, the mass of the dish containing the sample is weighed (m₃ in g). The dry matter content of the wine lees is then calculated according to the following eq. (1):

$$DM (\%) = \frac{(m_3 - m_1)}{(m_2 - m_1)} \times 100 \qquad (1)$$

This value was used to normalize the concentrations of specific compounds to a dry weight basis, ensuring accuracy and comparability between samples of differing moisture content. All quantitative results presented in this study are expressed relative to the dry matter content of the analysed lees samples.

## 2.4. Trolox equivalent antioxidant capacity (TEAC)

The TEAC was measured by DPPH radical scavenging activity as described by Poulain et al. (2024). Briefly, a DPPH* stock solution (9.0 × 10⁻⁵ M) in ethanol (EtOH) was prepared. After that, 1.9 mL of this reagent was mixed with 0.1 mL of blank (EtOH/H₂O, 50/50, v/v) and Trolox standard and rehydrated dry wine lees at 10 g.L⁻¹ (in EtOH/H₂O, 50/50, v/v) in a 2 mL plastic vial. After 30 min of incubation in the dark at 25 °C, the samples were centrifugated at 12,000 rpm for 2 min. Then, 300 µL of solution (sample and DPPH solution mix) was put on a 96-well microplate (n = 4) and the absorbance was measured at 517 nm with a microplate spectrophotometer UV–Vis MultiSkan Sky High (Thermo Fisher Scientific Inc., Waltman, MA, USA). All measurements were carried out in triplicate. A calibration curve was obtained using Trolox concentrations from 0 to 150 mg.L⁻¹ in EtOH/H₂O (50/50, v/v) and the results were expressed as milligrams Trolox Equivalent per gram of wine lees dry matter (mg TE.g⁻¹ DM⁻¹).

## 2.5. Total polyphenols content (TPC)

The total polyphenols content (TPC) was determined using the Folin-Ciocalteu method adapted on dried wine lees by Poulain et al. (2024). A volume of 200 µL of blank (EtOH/H₂O, 50/50, v/v) and Gallic acid standard and rehydrated dry wine lees at a concentration of 5 g.L⁻¹ in EtOH/H₂O (50/50, v/v) and 800 µL of sodium carbonate solution (7.5 % w/v) was added to 1000 µL of 1/10 Folin-Ciocalteu reagent solution in a 2 mL plastic vial. Following a 30-min incubation in the dark at 25 °C, samples were centrifuged at 12,000 rpm for 2 min. After centrifugation, 200 µL of mixed solution were put on a 96-well microplate (n = 4) and the absorbance was measured at 760 nm with a microplate spectrophotometer UV–Vis MultiSkan Sky High (Thermo Fisher Scientific Inc.,
Waltman, MA, USA). Each measurement was carried out in triplicate. A Gallic acid calibration curve ranging from 0 to 200 mg.L⁻¹ was used and TPC was expressed as milligrams of Gallic acid equivalent per gram of wine lees dry matter (mg GAE.g⁻¹ DM).

## 2.6. Lipids content

Wine lees lipids were extracted following the methodology described by Xiao (2005). First, approximatively 750 mg of wine lees were mixed with 15 mL of chloroform/methanol (2:1, v/v) solution and stirred at room temperature for 1 h at 500 rpm. Then, the suspension was filtered using a glass frit vacuum filtration system (porosity 5). The solid retentate was rinsed with 5 mL of chloroform/methanol (2:1, v/v) solution and filtered again. The liquid filtrate was mixed with 5 mL of 0.8 % KCl solution (in water) in a PTFE centrifuge tube (Nalgene, France), vortexed and centrifuged at 3000 rpm for 5 min. The aqueous layer was removed, and the organic layer was extracted twice with 10 mL of methanol/aqueous 0.8 % KCl/chloroform (48:47:3, v/v/v). The organic layer was transferred into a weighed clean round bottom flask (W₁) and evaporated under vacuum with a rotary evaporator (R-114, BUCHI, France). The flask weight with residue is recorded (W₂) and lipid content can be calculated by the following formula (2):

$$ \text{Lipid content} = \frac{W_2 (\text{g}) - W_1 (\text{g})}{\text{Initial lees sample weight (g)}} \quad (2) $$

The results were expressed as mg of lipids per gram of wine lees dry matter (mg.g⁻¹ DM). Each measurement was carried out in triplicate.

## 2.7. Protein content

The protein content was determined using the Kjeldahl methodology. First, approximately 200 mg of freeze-dried wine lees were weighed into a Kjeldahl digestion tube. A catalytic mixture containing 5 g of potassium sulfate and 0.5 g of copper sulfate was added to each tube, along with 10 mL of concentrated sulfuric acid (H₂SO₄). The samples were digested at 400 °C for 2 h with a Turbotherm digestion unit (Gerhardt, Königswinter, Germany). After cooling, the digested solution was diluted with 50 mL water, followed by alkalization with 40 mL of 32 % sodium hydroxide (NaOH) and distilled using a Vapodest distillation unit (Gerhardt, Königswinter, Germany) into a receiving beaker containing 40 mL of 2 % boric acid solution with a mixed indicator (methyl red-bromocresol green). Titration was carried out with 0.1 N hydrochloric acid (HCl) until the endpoint was reached, indicated by a colour change. The total nitrogen content was calculated and converted to protein content using a conversion factor of 6.25 (Nielsen, 2017). All analyses were performed in triplicate, and results were expressed as milligrams of proteins per gram of wine lees dry matter (mg.g⁻¹ DM).

## 2.8. Tartaric acid content

Tartaric acid concentration in wine lees was determined by K-TART enzymatic test (Megazyme, Ireland) and performed with a microplate spectrophotometer UV-Vis MultiSkan Sky High (Thermo Fisher Scientific Inc., Waltman, MA, USA) using spectrophotometric detection. Briefly, an aqueous solution of freeze-dried wine lees in water (10 g.L⁻¹) was ultra-sounded during 10 min and centrifugated at 12,000 rpm for 2 min. Then, 10 µL of the supernatant was mixed with the enzymatic reagents and after incubation (4 min), the absorbance was measured at 505 nm. A calibration curve was obtained using tartaric acid as the standard (0–5 g.L⁻¹ in H₂O) and the results were expressed as grams of Tartaric acid per gram of DM of extract (g.g⁻¹ DM).

## 2.9. Total polysaccharides content (TP)

Total polysaccharides content (TP) was determined using the phenol-sulfuric methodology (Snyman et al., 2021). Wine lees were

rehydrated at 1 g.L⁻¹ in water and sonicated during 15 min in order to have a homogeneous suspension. Then, in a 20 mL glass vial, 1 mL of this solution or blank (water) or standard solution (Glucose:Mannose, 1:1) was mixed with 1 mL of 5 % phenol solution in water directly following by 5 mL of concentrated sulfuric acid. After acid addition, the mixed solution was incubated at room temperature for 30 min. Then, 200 µL of solution was deposed on a 96-well microplate (n = 4) and the absorbance was measured at 490 nm with a microplate spectrophotometer UV-Vis MultiSkan Sky High (Thermo Fisher Scientific Inc., Waltman, MA, USA). Each measurement was carried out in triplicate. A Glucose/ Mannose (1:1) calibration curve ranging from 0 to 500 mg.L⁻¹ was used and total polysaccharides content was expressed as milligrams equivalent of sugar per grams of wine lees dry matter (mg sugar eq.g⁻¹ DM).

## 2.10. Total sulfhydryl groups (TSH)

The total sulfhydryl groups were quantified using the Ellmans' assay (DTNB assay) following the method of Tietze (1969), with slight modifications. First, wine lees were submitted to a maceration in a hydro-alcoholic solution (EtOH/H₂O, 50/50, v/v) at 50 g.L⁻¹ during 30 min. After centrifugation at 7500 rpm for 5 min, 500 µL of supernatant was evaporated to dryness using a RapidVap (Labconco, France). The dry extract obtained was then reconstituted in 500 µL of a 0.1 N HCl solution. Then, a buffer solution was prepared by combining 50 mM Tris and 3 mM EDTA, adjusting the pH to 8.0 with 0.1 N HCl. A stock solution of DTNB was then prepared by dissolving DTNB in the buffer and adjusting the pH to 8.0 using 0.1 N NaOH. Finally, 245 µL of the DTNB reagent and 5 µL of the sample or blank or standard were added to each well of a 96-well microplate, with four replicates per sample (n = 4). After a 10-min incubation at room temperature, the absorbance was measured at 412 nm with a microplate spectrophotometer UV-Vis MultiSkan Sky High (Thermo Fisher Scientific Inc., Waltman, MA, USA). A calibration curve was prepared using glutathione (GSH) in 0.1 N HCl solution ranging from 0 to 1000 mg.L⁻¹, and results were expressed as milligrams of glutathione equivalent per gram of wine lees dry matter (mg GSHE.g⁻¹ DM). All measurements were performed in triplicate.

## 2.11. Glutathione content by UHPLC-HRMS

The quantification of glutathione in the wine lees was carried out following the protocol described by Winstel et al. (2024). The analyses were performed using an UHPLC appliance consisting of a Vanquish system (Thermo Fisher Scientific, Les Ulis, France) with binary pumps, an autosampler and a heated column compartment. An Atlantis Premier BEH C18 AX column (150 × 2.1 mm, 1.7 µm, Waters, Guyancourt, France) was used for the stationary phase, with water (Eluent A) and acetonitrile (Euent B), containing both 0.1 % of formic acid as mobile phases. The flow rate was set at 350 µL.min⁻¹ and the injection volume was 5 µL. The temperature of the column chamber was set at 30 °C in forced air mode. For the screening and quantitative analysis, eluent B varied as follows: 0 min, 5%; 3.0 min, 5%; 6.3 min, 30%; 6.6 min, 98%; 8.1 min, 98%; 8.3 min, 5%; 10.5 min, 5%. An Exactive Orbitrap mass spectrometer equipped with a heated electrospray ionisation (HESI II) probe (both from Thermo Fisher Scientific, Les Ulis, France) was used. Results were expressed as micrograms of Glutathione Equivalent per gram of wine lees dry matter (µg GSHE.g⁻¹ DM).

## 2.12. Characterization of polyphenols by UHPLC-HRMS

The quantification of polyphenols in the wine lees was performed as follows. Briefly, 50 mg of freeze-dried wine lees were extracted with 1 mL of methanol during 15 min in an ultra-sonic bath. Samples were centrifugated at 12,000 rpm for 5 min and the pellet was submitted to another extraction with 1 mL of methanol/water solution (50/50; v/v) during 15 min in an ultra-sonic bath. The supernatants were combined and centrifugated at 12,000 rpm during 10 min before injection. The
analyses were performed following the methodology of Pébarthé-Courrouilh et al. (2024) using an UHPLC Vanquish system (Thermo Fisher Scientific, Les Ulis, France) coupled to a Q-Exactive Plus mass spectrometer equipped with a heated electrospray ionisation (HESI I) probe (Thermo Fisher Scientific, Les Ulis, France). A Luna OmegaPolar C18 column (50 × 2.1 mm, 1.6 µm; Phenomenex, Torrance, CA, USA) was used as stationary phase, with water (Eluent A) and acetonitrile (Eluent B), containing both 0.1 % of formic acid as mobile phases. The flow rate was set at 0.5 mL.min⁻¹ and the injection volume was 1 µL. The temperature of the column chamber was set at 40 °C in forced air mode. The gradient elution was set as follows: 1 % B (0 min), 1–36 % B (0–7.5 min), 36–95 % B (7.5–8.5 min), isocratic 95 % B (8.5–10 min), 95–1 % B (10–10.25 min) and finally isocratic 1 % B (10.25–11.25 min). HRMS analyses main parameters were: Negative polarity; Spray voltage: 3 kV; Sheath gas: 45 a.u.; Auxiliary gas: 15 a.u.; Capillary temperature: 320 °C; Probe heater temperature: 250 °C; S-lens RF level: 100. Data were acquired in full-scan mode within the range of 70 to 1050 m/z with the following parameters: Resolution: 35,000; AGC target: 3e6, Maximum IT: 100 ms.

The sample injections followed a specific sequence. It started with three injections of a blank solvent sample, followed by three consecutive injections of a quality control sample (equivolume mix of each sample). Subsequently, wine lees samples were injected in a randomized manner. After every ten samples injections, a quality control sample was injected. Reproducibility of peak area was checked for key compounds, and all showed RSDs below 10 %. A representative UHPLC-HRMS total ion chromatogram extracted from 1.7 Freestyle software (Thermo Fisher Scientific, Les Ulis, France) is shown in the supplementary data (Fig. S1).

Quantification was performed by comparison to a calibration curve built with standards in the range of 0.033–16.94 mg.L⁻¹. The list of components and the performance of the method are given in the supplementary data (Table S2). All compounds were quantified with the corresponding standard, except procyanidin B4 (expressed as B2 equivalent), cis-caftaric acid (as trans-caftaric acid equivalent), cis-coutaric acid (as trans-coutaric acid equivalent), cis and trans-fertaric acid (as trans-coutaric acid equivalent) and cis-piceid (as trans-piceid equivalent). Results were expressed as micrograms per gram of wine lees dry matter (µg.g⁻¹ DM).

## 2.13. Statistical analysis

XLSTAT software (Addinsoft version 19.02, Paris, France) was used to performed PCA and correlation tests. Spearman correlation coefficient was employed to determine correlations between wine lees composition and antioxidant parameters. These tests were preceded by Shapiro tests to verify normality or not of the data.

# 3. Results and discussion

## 3.1. Composition and antioxidant capacity of white wine lees

A comprehensive analysis of the chemical composition and antioxidant capacity was performed. This section provides an overview of the different results of a representative set of 41 samples of white wine lees

(Table 1) with the detailed results available in Supplementary Table S3.

The mean value for the radical scavenging activity of wine lees is expressed as TEAC (mg TE.g⁻¹ DM) and determined using the DPPH method was 5.7 ± 2.8 mg TE.g⁻¹ DM. A significant variability in the antioxidant activity of wine lees was observed, with minimum and maximum values between 2.1 mg TE.g⁻¹ DM and 12.4 mg TE.g⁻¹ DM, respectively. The available literature data is limited; however, the observed antioxidant activity values are comparable to those reported by Zhijing et al. (2018) for white wine lees, which ranged from 5.8 to 11.1 mg TE.g⁻¹ DM.

The total polyphenol content (TPC) in wine lees was determined using the Folin-Ciocalteu method. On average, the white wine lees samples analysed contained 18.7 ± 6.8 mg GAE.g⁻¹ DM, with values ranging from 7.7 to 32.7 mg GAE.g⁻¹ DM (Table 1). These results were consistent with those of Zhijing et al. (2018), who reported TPC values between 3.1 ± 0.2 and 10.3 ± 0.4 mg GAE.g⁻¹ DM in white wine lees. The presence of polyphenols in wine lees results from wine and their adsorption onto yeast cell walls (Morata et al., 2005).

Lipids content, primarily associated with grape and yeast residues, revealed an average of 22.7 ± 11.2 mg.g⁻¹ DM with values ranging from 5.6 to 52.6 mg.g⁻¹ DM. Similar values were reported by Gómez et al. (2004), who characterised white Sherry wine lees and found an average lipid content of approximately 54.0 ± 4.6 mg.g⁻¹ DM.

The protein content, mainly linked to yeast presence, was determined using the Kjeldahl method. The average protein content was 205.4 ± 67.5 mg.g⁻¹ DM, with a wide range of 99.6 to 363.7 mg.g⁻¹ DM. These results are consistent with those reported by Bustamante et al. (2008), who, based on nitrogen content calculations, observed protein levels ranging from 107.5 to 373.1 mg.g⁻¹ DM, with an average of 220 mg.g⁻¹ DM. Similarly, Gómez et al. (2004) reported an average protein content of 151.1 ± 5.7 mg.g⁻¹ DM in their study of white Sherry wine lees, a value comparable to the average observed in this study.

The tartaric acid, present as tartaric precipitates and quantified after solubilisation, had an average content of 0.28 ± 0.12 g.g⁻¹ DM. A notable variability was observed, with values ranging from 0.04 to 0.45 g.g⁻¹ DM. These results were in agreement with those of others studies (De Iseppi et al., 2020; Gómez et al., 2004), confirming that tartaric precipitates constitute a significant proportion of the dry matter in white wine lees.

The total sulfhydryl content (TSH) in wine lees was quantified using the Ellman's assay (DTNB method) and the values ranged from 0.29 to 1.51 mg GSHE.g⁻¹ DM, with a mean of 0.61 ± 0.28 mg GSHE.g⁻¹ DM for all samples analysed.

Finally, due to the presence of grape and yeast residues, wine lees are also rich in polysaccharides, with an average content of 197.3 ± 51.8 mg.g⁻¹ DM. Similar to the other compounds analysed, high variability was observed, with values ranging from 67.9 mg.g⁻¹ DM to 391.6 mg.g⁻¹ DM. The polysaccharides present in wine lees are mainly beta-glucans, mannoproteins, oligosaccharides, chitin, pectins and cellulose (Gómez et al., 2004) and belonging to the class of dietary fibres. The same authors reported a dietary fibre content of 215.0 ± 3.5 mg.g⁻¹ DM for Sherry wine lees, which aligns with the value obtained in this study.

Following the analysis of all the compounds, a principal component analysis (PCA) was performed on the composition data. This approach

<caption>**Table 1**</caption>

Antioxidant activity (TEAC) and chemical composition of white wine lees (Total polyphenols content, Lipids, Proteins, Tartaric acid, Total Sulfhydryl Compounds and Total Polysaccharides): Contents Mean, Standard deviation, Minimum, Maximum, and Median contents values for a Set of 41 Samples of white wine lees.

<table><thead><tr><th>Variables</th><th>Units</th><th>Average</th><th>Standard deviation</th><th>Min</th><th>Max</th><th>Median</th></tr></thead><tbody><tr><td>TEAC</td><td>mg TE.g<sup>-1</sup> DM</td><td>5.7</td><td>2.8</td><td>2.1</td><td>12.4</td><td>4.9</td></tr><tr><td>TPC</td><td>mg GAE.g<sup>-1</sup> DM</td><td>18.7</td><td>6.8</td><td>7.7</td><td>32.7</td><td>17.51</td></tr><tr><td>Lipids</td><td>mg.g<sup>-1</sup> DM</td><td>22.7</td><td>11.2</td><td>5.6</td><td>52.6</td><td>20.4</td></tr><tr><td>Total proteins</td><td>mg.g<sup>-1</sup> DM</td><td>205.4</td><td>67.5</td><td>99.6</td><td>363.7</td><td>200.6</td></tr><tr><td>Tartaric acid</td><td>g TA.g<sup>-1</sup> DM</td><td>0.28</td><td>0.12</td><td>0.04</td><td>0.45</td><td>0.30</td></tr><tr><td>TSH</td><td>mg GSHE.g<sup>-1</sup> DM</td><td>0.61</td><td>0.28</td><td>0.29</td><td>1.51</td><td>0.56</td></tr><tr><td>Total polysaccharides</td><td>mg sugar eq.g<sup>-1</sup> DM</td><td>197.3</td><td>51.8</td><td>67.9</td><td>391.6</td><td>188.2</td></tr></tbody></table>
allows to identify sample or variable groupings, assess the proper dispersion of the data, and detect any outliers. Fig. 1 shows the loadings and scores plots in the space described by the 1st and 2nd factors (total explained variance of 67.29 %). The first factor is mainly related to the quantity of tartaric acid and yeast biomass (proteins and total polysaccharides) and sulfhydryl compounds. The second factor appears to be related to richness in polyphenols and lipids on the other side.

The PCA showed a widely distributed pattern of sample indicating a diverse dataset. Overall, the samples represented a good diversity, based on visual inspection, no obvious clustering according to grape variety or yeast strain was observed in the PCA space. Therefore, this large variability in composition provides a broad representation of the white wine lees matrix. In addition, our samples differed considerably in terms of antioxidant capacity, providing an opportunity to explore potential interactions among compositional parameters and to evaluate their individual and combined contributions to the antioxidant properties of the wine lees.

## 3.2. Analysis of correlation between global composition parameters and antioxidant capacity

To investigate the relationship between the antioxidant activity of white wine lees and their overall chemical composition, a Spearman correlation analysis was conducted. The correlations between TEAC and the global composition parameters are presented in Table 2. The full correlation matrix, including inter-variable correlations, is available in the supplementary data (Table S3).

Initially, a significant positive correlation was observed between wines lees total polyphenol content (TPC) and their antioxidant activity expressed as TEAC ($ρ = 0.657$). This result was already highlighted in others studies for other natural matrices (Romero-Díez et al., 2018; Zhijing et al., 2018). A significant positive correlation was also observed between the antioxidant activity of white wine lees and their sulfhydryl

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

Spearman's correlation coefficients ($ρ$) between the global composition of white wine lees (Variables) and their antioxidants capacity (TEAC). ** represents significance at $p ≤ 0.01$ and * represents significance at $p ≤ 0.05$.

<table><thead><tr><td>Variables</td><td>TEAC</td></tr></thead><tbody><tr><td>TPC</td><td>0,657**</td></tr><tr><td>Lipids</td><td>0,000</td></tr><tr><td>Total proteins</td><td>0,245</td></tr><tr><td>Tartaric acid</td><td>-0,446**</td></tr><tr><td>TSH</td><td>0,528**</td></tr><tr><td>Total polysaccharides</td><td>0,217</td></tr></tbody></table>

compound content ($ρ = 0.528$). This result demonstrates a direct link between the presence of thiol function (-SH) compounds and their contribution to the antioxidant activity of wine lees. This finding is consistent with observations reported by Romanet et al. (2020) that observe an increase in sulphur-containing compounds (—S) in wine antioxidant metabolome during ageing on lees through the gradual release of hydrophilic and hydrophobic peptides with antioxidant activity.

Concerning tartaric acid content, a significant negative correlation was found with the antioxidant activity ($ρ = -0.446$). Tartaric acid, present in wine lees as potassium bitartrate, does not have antioxidant activity. However, an increase in the proportion of potassium bitartrate in the lees reduces the concentration of other components, some of which contribute to antioxidant activity. Therefore, wine lees with higher tartaric acid content exhibit lower antioxidant activity.

Regarding other compound families analysed, no correlation was observed between the lipid and their antioxidant activity ($ρ = 0.000$). This result could be related to the fact that the implemented analysis of antioxidant activity (TEAC), based on a free radical scavenging mechanism (antiradical activity), does not measure a direct interaction of

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** PCA (Biplot) analysis of lees composition data (samples and variables); Explained variance of 67.29 %.</figcaption></figure>
compounds with oxygen, a mechanism demonstrated in the antioxidant properties of the lipid fraction of wine lees (Fornairon-Bonnefond & Salmon, 2003). Additionally, no direct correlation was observed between the protein content of wine lees, determined from their nitrogen content, and their antiradical activity (ρ = 0.245). Although Romanet et al. (2020) highlighted the involvement of nitrogen compounds in the antioxidant metabolome of wines aged on lees, the quantification of these compounds by measuring total nitrogen does not seem to allow conclusions regarding their role in the antioxidant potential of wine lees. Finally, no correlation was observed between total polysaccharides content of wine lees and their antioxidant activity (ρ = 0.217). Jaehrig et al. (2008) demonstrated that yeast cell wall hydrolysis products, including polysaccharides, possess antioxidant activity, but also noted the relatively weak activity of these compounds compared to peptides released during yeast hydrolysis. Thus, while protein and polysaccharides are not devoid of antioxidant activity, their contribution alone does not sufficiently explain the antioxidant potential of white wine lees.

In conclusion, a positive correlation was established only between polyphenolic compounds and sulfhydryl compounds and white wine lees antiradical activity. Therefore, a more detailed investigation was conducted to identify the specific compounds contributing to the antioxidant capacity.

### 3.3. Targeted correlation between phenolic, sulfhydryl compounds and antioxidant capacity

A quantification of specific polyphenols subclasses (phenolic acids, flavanols, flavonols, stilbenoids) and glutathione (reduced and oxidised) as well-known sulfhydryl compounds by UHPLC-HRMS was performed (Table 3). For transparency, the compounds included in each subclass and the detailed results are provided in Supplementary Table S4 and S5.

The targeted polyphenols and glutathione derivatives were selected based on their known occurrence in white wines and therefore in white wine lees and their potential antioxidant activity, as described in previous studies (Romanet et al., 2023; Winstel et al., 2024; Ye et al., 2022).

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

Summary of phenolic subclasses (phenolic acids, flavanols, flavonols, stilbenoids) and Glutathione (Reduced and Oxidised) Contents. Mean, Standard deviation, Minimum, Maximum, and Median contents values for a Set of 41 Samples of white wine lees. N·D (Non detected).

<table><thead><tr><th>Variables</th><th>Units</th><th>Average</th><th>Standard deviation</th><th>Min</th><th>Max</th><th>Median</th></tr></thead><tbody><tr><td><strong>Phenolic compounds</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Total phenolic compositon</td><td>µg.<br>g<sup>-1</sup><br>DM</td><td>922.2</td><td>933.0</td><td>61.0</td><td>4610.0</td><td>699.3</td></tr><tr><td>Phenolic acids</td><td>µg.<br>g<sup>-1</sup><br>DM</td><td>888.5</td><td>925.7</td><td>47.1</td><td>4553.8</td><td>686.7</td></tr><tr><td>Flavanols</td><td>µg.<br>g<sup>-1</sup><br>DM</td><td>10.9</td><td>18.7</td><td>0.8</td><td>105.4</td><td>3.3</td></tr><tr><td>Flavonols</td><td>µg.<br>g<sup>-1</sup><br>DM</td><td>9.0</td><td>10.6</td><td>0.6</td><td>41.2</td><td>5.6</td></tr><tr><td>Stilbenoids</td><td>µg.<br>g<sup>-1</sup><br>DM</td><td>13.8</td><td>10.9</td><td>1.4</td><td>51.9</td><td>11.0</td></tr><tr><td><strong>Glutathione</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Reduced - GSH</td><td>µg.<br>g<sup>-1</sup><br>DM</td><td>38.4</td><td>144.9</td><td>N·D</td><td>683.0</td><td>1.8</td></tr><tr><td>Oxidised - GSSG</td><td>µg.<br>g<sup>-1</sup><br>DM</td><td>98.7</td><td>178.8</td><td>N·D</td><td>987.6</td><td>44.3</td></tr></tbody></table>

Total polyphenol composition, which corresponds to the sum of the different classes of polyphenols in white wine lees (phenolic acids, flavanols, flavonols, stilbenoids), was measured on average at 922.2 µg.g⁻¹ DM with values ranging from 61.0 to 4610.0 µg.g⁻¹ DM (Table 3). These results are consistent with the literature (Ye et al., 2022) and in particular with the distribution of the different sub-classes where phenolic acids represent the main sub-classes of phenolic compounds in the lees of white wines compared with flavanols, flavonols and stilbenes. This distribution may be due to the fact that phenolic acids are highly concentrated in the grape berry pulp and are readily released during the crushing step (Teixeira et al., 2013).

Reduced glutathione levels ranged from 0 (not detected) to 683 µg. g⁻¹ DM (Table 3). However, only 2 of the 41 wine lees had concentrations greater than 100 µg.g⁻¹ DM, with the majority of samples containing concentrations of around 10 µg.g⁻¹ DM (Table S3). However, a higher concentration of GSSG was observed, with an average of 98.7 µg. g⁻¹ DM, which could be explained by the fact that the wine lees were not properly protected from oxidation after collection from the winery (Winstel et al., 2024). For both GSH and GSSG, the concentrations are of the same range as those reported in the literature (Winstel et al., 2024).

Then, a Spearman correlation analysis was conducted to assess the relationships between specific compounds and the antioxidant capacity of the wine lees. The results revealed significant and varied correlations, as detailed in Table 4. The full correlation matrix, including inter-variable correlations, is available in the supplementary data (Table S3).

Total phenolic composition measured by UHPLC-HRMS showed a strong positive correlation with free radical scavenging capacity (ρ = 0.871), highlighting the central role of polyphenols as major contributors to the intrinsic antioxidant potential of white wine lees. This strong correlation reflects the ability of polyphenols to donate electrons and stabilize free radicals via delocalised π-electron systems (Rice-Evans, 1995). Total phenolic content therefore serves as an overall indicator of antioxidant potential, as it includes the combined effects of various subclasses of polyphenolic compounds. When we closely consider the different subclasses of polyphenols quantified, phenolic acids showed the highest correlation coefficient (ρ = 0.805), suggesting their predominant role in the antioxidant activity observed by DPPH assay. This may be explained by the fact that in terms of concentration found in the lees of white wines, these are the most abundant polyphenols (Table 3 and Table S5), compared to the other classes. Indeed, the concentration varies between 47.1 and 4553.8 µg.g⁻¹ DM with an average of 888.5 µg. g⁻¹ DM. In terms of reactivity, it was shown that hydroxycinnamic acids isomers of caftaric and coumaric acids have strong antiradical properties (Alonso et al., 2002). The ability of hydroxycinnamic acids to trap the DPPH radical depends on the number of hydroxyl groups on the benzene ring and the ortho substitution with the electron-donating methoxy group, which increases the stability of the phenoxy radical (Kikuzaki et al., 2002).

Flavanols (ρ = 0.621) and flavonols (ρ = 0.469) also showed positive correlations, although with moderate to weaker strengths compared to phenolic acids. For these two families of polyphenols, the flavonoid-type skeleton (C₆-C₃-C₆) and in particular the B ring (catechol or pyrogallol

<caption>**Table 4**</caption>

Spearman's correlation coefficients (ρ) between the targeted phenolic and sulfhydryl compounds and the antioxidants capacity. ** represents significance at p ≤ 0.01 and * represents significance at p ≤ 0.05.

<table><thead><tr><th>Variables</th><th>TEAC</th></tr></thead><tbody><tr><td>Total phenolic composition</td><td>0.871**</td></tr><tr><td>Phenolic acids</td><td>0.805**</td></tr><tr><td>Flavanols</td><td>0.621**</td></tr><tr><td>Flavonols</td><td>0.469**</td></tr><tr><td>Stilbenoids</td><td>0.243</td></tr><tr><td>GSH</td><td>0.231</td></tr><tr><td>GSSG</td><td>0.356*</td></tr></tbody></table>
type) contributed to their antioxidant activity through the presence of a hydroxyl group that stabilises the semi-quinone radical by resonance (Kilmartin et al., 2001). However, as their presence in wine lees are much lower than phenolic acids, respectively 10.9 and 9 µg.g⁻¹ DM on average for flavanols and flavonols compared with 888.5 µg.g⁻¹ DM (Table 3), their effects on antioxidant capacity are therefore less significant (Psarra et al., 2002). In contrast, stilbenoids showed a relatively low correlation (ρ = 0.243), indicating a limited contribution to free radical scavenging capacity under the experimental conditions. Stilbenoids, such as resveratrol, are known for their unique bioactive properties, often related to antioxidant activity (Waffo Teguo et al., 1998). However, their low abundance in white wine lees may reduce their overall impact. In addition, it is possible that stilbenoids act synergistically with other antioxidants, as has been shown for flavanols (Saucier & Waterhouse, 1999), meaning that their individual contribution may not fully be accounted for by correlation analysis.

In this study, glutathione (GSH) showed a weak non-significant positive correlation with free radical scavenging capacity (ρ = 0.231). Despite this, sulfhydryl compounds and in particular GSH, are known to enhance the antioxidant metabolome of white wines through contact with lees (Romanet et al., 2023). In addition, GSH is an antioxidant containing a nucleophilic thiol function with the potential to trap reactive oxygen species (ROS) and regenerate oxidised polyphenols into their active reduced forms (Nikolantonaki et al., 2014). Thus, the weak correlation suggests that its contribution is minor compared with the polyphenols in terms of intrinsic antioxidant capacity. The low abundance of GSH in our wine lees may be a factor limiting its overall impact. However, the significant positive correlation with TSH compounds (part 3.2), which also includes glutathione, highlights the presence of other unknown sulfhydryl compounds (e.g. peptides containing cysteine) that may contribute to the antiradical capacity of white wine lees (Romanet et al., 2020). Furthermore, under oenological conditions, glutathione is released into the wine during prolonged contact with the lees, leading to autolysis of the yeast cells (Lavigne et al., 2007). Consequently, the fraction of free glutathione on the surface of the lees is low and has limited activity compared to polyphenols. In contrast, the significant negative correlation observed for GSSG (ρ = -0.356) suggests that the redox state of glutathione may affect its contribution to antioxidant activity. GSSG, the oxidised form of GSH, may act as a marker of oxidative stress rather than an active antioxidant. Its presence in higher concentrations could reflect oxidative degradation processes that reduce the overall antioxidant potential of white wine lees. In the presence of ROS, the thiol function of GSH releases an electron to neutralise the radical, converting it into GSSG by forming a disulfide bridge (covalent S-S bond), causing the thiol function to lose its nucleophilic character and therefore its antioxidant capacity (Foti & Amorati, 2009).

The results highlight the importance of polyphenols, particularly phenolic acids, as the main contributors to the antioxidant potential of white wine lees, thanks to their abundance and chemical properties.

## 4. Conclusions

The objective of this work was to provide new knowledge regarding the compounds responsible for the antioxidant capacity of white wine lees. In the present work, a combination of global methodologies (spectrophotometric, gravimetric) and targeted analysis by UHPLC-HRMS were used to obtain the complete chemical characterization of a set of 41 white wine lees obtained after alcoholic fermentation. The results obtained and the PCA analysis highlighted the intrinsic variability of the composition, regardless of the parameter studied (poly-saccharides, proteins, lipids, polyphenols, etc.), but also in terms of antioxidant potential (radical scavenging activity), with values ranging from 2.1 to 12.4 mg TE.g⁻¹ of dry matter. In order to study the impact of lees composition on the antioxidant potential, a Spearman correlation study was applied. The results showed the positive contribution of the total polyphenolic and sulfhydryl fractions (ρ = 0.657 and 0.528

respectively) highlighting the importance of compounds derived or adsorbed from yeast. Following these results, we targeted specific phenolic and sulfhydryl compounds find in white wines using UHPLC-HRMS. A study of the correlation of these compounds highlighted the importance of phenolic acids, particularly hydroxynaptic acids (ρ = 0.805) and, to a lesser extent, flavanols and flavonols (ρ = 0.621 and 0.469 respectively). The absence of a correlation for glutathione (ρ = 0.231) suggests the involvement of other antioxidant sulfhydryl compounds in the white wine lees. To our knowledge, no previous study has established a link between total composition (global and targeted by UHPLC-HRMS) and the antioxidant potential of white wine lees. These findings provide important insights into the bioactivity and antioxidant potential of white wine lees. In the future, the studies could be carried out to identify and isolate new compounds with anti-free radical properties using non-targeted approaches such as metabolomics or bioguided fractionation. From a valorisation perspective, this by-product is a promising source of high-value antioxidant compounds with potential applications in various industries.

## CRediT authorship contribution statement

**Luca García:** Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**Benjamin Poulain:** Writing – original draft, Methodology, Investigation, Formal analysis.
**Alice Douliez:** Writing – review & editing, Investigation, Formal analysis.
**Eloise Naud:** Investigation.
**Josep Valls-Fonayet:** Writing – review & editing, Methodology.
**Claudia Nioi:** Writing – review & editing, Validation, Supervision, Project administration, Funding acquisition, Conceptualization.

## 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 was supported by the ANR ValoLi project, grant ANR-21-43CE-0003 of the French Agence Nationale de la Recherche, the Region Nouvelle Aquitaine and the Biolaffort company. This work was supported by the Bordeaux Metabolome Facility (doi:10.15454/1.557241 2770331912E12) and the MetaboHUB (ANR-11-INBS-0010) project. We would also like to thank Gracie Boland for checking English grammar in this article.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2025.146118.

## Data availability

Data will be made available on request.

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