# White wine lees protein extract: A promising fining agent for red wine clarification

Luca Garcia, Alice Douliez, Benjamin Poulain, Lucie Gagnepain, Fanny Gallo, Romain Kapel, Sophie Beaubier, Claudia Nioi

► To cite this version:

Luca Garcia, Alice Douliez, Benjamin Poulain, Lucie Gagnepain, Fanny Gallo, et al.. White wine lees protein extract: A promising fining agent for red wine clarification. LWT - Food Science and Technology, 2025, 238, pp.118867. 10.1016/j.lwt.2025.118867. hal-05448130

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# White wine lees protein extract: A promising fining agent for red wine clarification

Luca Garcia<sup>a</sup>, Alice Douliez<sup>a</sup>, Benjamin Poulain<sup>a</sup>, Lucie Gagnepain<sup>a</sup>, Fanny Gallo<sup>b</sup>, Romain Kapel<sup>b</sup>, Sophie Beaubier<sup>b</sup>, Claudia Nioi<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Wine lees

Subcritical water extraction

Proteins

Fining agents

## ABSTRACT

This study investigates the potential of white wine lees, a protein- and polysaccharide-rich winemaking by-product, as a sustainable fining agent for red wine clarification. Protein-rich extracts were produced using subcritical water extraction (SWE) and optimized via Response Surface Methodology. Chemical and molecular analyses (SDS-PAGE and SEC) revealed that the extracts were enriched in low to medium-molecular-weight peptides, similar to commercial gelatin hydrolysates. The fining performance of the extract was assessed in red wine and compared to commercial products like whole lees, gelatin, and patatin. The lees extract significantly reduced turbidity (–64.4 %) and tannin content (–18.4 %) while preserving color stability, achieving performance comparable to gelatin and superior to patatin. Moreover, it effectively reduced wine astringency, with minimal impact on chromatic characteristics. These findings demonstrate that SWE-derived white wine lees extract is a promising alternative to conventional fining agents, supporting circular economy practices in the winemaking industry.

## 1. Introduction

Fining is an essential oenological practice widely used to improve wine clarity, polyphenolic stability, and sensory properties. It involves the removal of colloidal or insoluble particles and the modulation of the wine's organoleptic profile and stability through interactions between fining agents and wine components (Boulton et al., 1999; Marchal et al., 2002). Among the various fining agents, proteins are particularly valued for their ability to interact with phenolic compounds, especially tannins, through hydrophobic interactions and hydrogen bonding (Maury et al., 2001). These interactions lead to the formation of insoluble aggregates that precipitate, thereby reducing turbidity, modifying the phenolic structure, and improving sensory attributes, particularly by decreasing astringency and stabilizing red wine color (Gambuti et al., 2012; González-Neves et al., 2014; Karamanidou et al., 2011). Traditionally, fining agents have included proteins of animal origin such as gelatin, casein, and egg albumin (Boulton et al., 1999). While these agents are effective in modulating the phenolic stability and improving visual and sensory attributes, their use raises concerns related to food safety and consumer expectations. Specifically, milk and egg proteins are

recognized food allergens and may remain as residues in the final product, posing potential risks for sensitive individuals (Tolin et al., 2012). As a result, European regulations now require allergen labeling when residues exceed 0.25 ppm in wine (EU Regulation No. 579/2012). In parallel, consumer preferences are evolving towards natural, allergen-free, and vegan products. The global increase in vegetarian and vegan populations has significantly influenced food and beverage production practices, including wine. In response to this shift, the wine industry has begun exploring non-animal fining alternatives (Río Segade et al., 2020). Plant proteins derived from sources such as pea, potato (patatin), and lupin have been studied as viable alternatives (Gambuti et al., 2012; Maury et al., 2003). Particularly, Patatin, a glycoprotein extracted from potato tubers, has demonstrated significant fining efficacy by reducing astringency and modifying phenolic content, with minimal sensory impact (Gambuti et al., 2012). In addition, pea and lupin vegetable protein extracts showed fining performance comparable to gelatin in terms of reducing turbidity and the polyphenols responsible for the astringency of red wine (Granato et al., 2014; Maury et al., 2003). More recently, grape seed protein extracts have been presented as a viable alternative to the most common fining agents of animal and

<footnote><sup>a</sup> Univ. Bordeaux, Bordeaux INP, Bordeaux Sciences Agro, INRAE, OENO, UMR 1366, ISVV, F-33140, Villenave d'Ornon, France</footnote>

<footnote><sup>b</sup> Université de Lorraine, CNRS, LRGP, F-54000, Nancy, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: claudia.nioi@u-bordeaux.fr (C. Nioi).</footnote>
vegetable origin (Gazzola et al., 2017).

Another promising class of alternative fining agents is yeast protein extracts (YPE), which have gained attention for their effectiveness in wine clarification and phenolic modulation (Lochbühler et al., 2015; Mekoue Nguela et al., 2024). Derived from yeast biomass, these extracts are non-allergenic, biodegradable, and compatible with natural and vegan winemaking. Studies have shown that YPEs have a strong affinity for tannins and haze-forming compounds, comparable to that of animal proteins (Lochbühler et al., 2015; Mekoue Nguela et al., 2024). Despite growing interest in YPE, wine lees, and especially white wine lees, after winemaking remains largely underutilized as a functional oenological by-product. Wine lees are the sediment formed during and after alcoholic fermentation, composed mainly of yeast cells, grape solids, tartaric acid precipitates, and various organic compounds (Balmaseda et al., 2025). Although their composition varies depending on grape variety, fermentation conditions, and winemaking practices, white wine lees are particularly rich in proteins, mannoproteins, and polysaccharides (Garcia et al., 2025). Thanks to their ability to interact with polyphenols and colloids, these macromolecules show great potential as natural and sustainable alternatives to conventional fining agents.

To develop protein-rich extracts from white wine lees with functional properties favorable to wine chemical stability, we investigated and optimized a green extraction process using subcritical water. Subcritical water extraction (SWE) has recently emerged as a promising green technology for valorizing agro-industrial by-products such as wine lees (Poulain et al., 2024) and grape seeds (Breniaux et al., 2025). SWE has been successfully applied to extract proteins from soybeans, rice bran, and microalgae (Costa et al., 2023; Ndlela et al., 2012), often yielding hydrolysates rich in low-molecular-weight peptides with enhanced solubility and reactivity. Compared to conventional chemical extraction, SWE offers several advantages, including lower environmental impact, reduced use of solvents, and improved extraction efficiency (Zhang et al., 2020). Additionally, integrating statistical optimization tools such as Response Surface Methodology (RSM) enables precise control of extraction parameters (temperature, time, solid-to-liquid ratio), thereby maximizing protein yield.

To our knowledge, there are no articles dealing with the use of proteins extracts from wine lees as a fining agent in wines. Therefore, this study proposes a comprehensive approach to evaluate the potential of white wine lees as a source of oenological fining agents. To this end:

* a Response Surface Methodology (RSM) was applied to optimize subcritical water extraction and maximize protein yield;

* an in-depth characterization of the extracted proteins was conducted, particularly focusing on molecular size under optimal conditions;

* a practical fining trial on red wine was performed, comparing the effectiveness of the lees extract to that of commercial fining agents currently used in the industry.

## 2. Materials and methods

### 2.1. Chemicals

Ultrapure water (Purite Type I Ultrapure Water System, Veolia, France) was used for sample preparation. Ethanol (EtOH) was purchased from VWR (France). Sulfuric acid (≥97 %), Sodium hydroxide, Mixed indicator 5 (M5), Hydrochloric acid 0.1 M, Bovine Serum Albumine (BSA), Hydrochloric acid 12 N, and Sodium metabisulfite were purchased from Sigma Aldrich (France). Catalyst Kjeldahl tablets Kjelcat Cu were purchased from Gerhardt (France).

HPLC-grade solvents (water and acetonitrile), Bradford reagent 5x concentrate (Serva Electrophoresis™), and phenol were obtained from Fisher Scientific (Geel, Belgium). Synthetic peptides used for column calibration were purchased from GeneCust (Dudelange, Luxembourg). Unless otherwise specified, all additional chemicals and reagents were of analytical grade and used without further purification.

### 2.2. Fining agents

Fining trials were conducted using three different materials: (i) raw white wine lees before extraction, (ii) the wine lees extract obtained under the optimal extraction conditions defined by the experimental design, and (iii) two commercial fining products for comparison with the lees extract. The commercial agents included one of animal origin, based on porcine gelatin (Gecoll®, Laffort Oenologie, France), and one of plant origin, based on potato protein (patatin) (Vegecoll®, Laffort Oenologie, France). Before their addition to wine, all fining agents were rehydrated in water at the appropriate concentrations, as described in Section 2.11.

### 2.3. White wine lees sample

A white wine lees matrix was collected from a winery in the "Pessac-Léognan" (Bordeaux) appellation in the 2023 vintage immediately after fermentation of a Sauvignon Blanc must. After recovery, the solid-liquid suspension was centrifuged (4200 RPM), and the pellet was washed 3 times with water to remove tartaric acid. The water suspension was centrifuged at 4200 RPM during 10 min between each washing steps. The pellet was then freeze-dried in the laboratory, ground using a lab-mixer Pulverisette 11 (Fritsch, Idar-Oberstein, Germany) and stored in a dark environment at room temperature before fining experiments and chemical analysis.

### 2.4. Preparation of white wine lees protein extract

#### 2.4.1. Subcritical water extraction (SWE)

Subcritical water extraction (SWE) was carried out using a 750 mL batch high-pressure autoclave (HPP, France) with a maximum operating pressure of 80 bars and a temperature of 250 °C. The autoclave was equipped with a temperature and stirring controller (HPP, France), as illustrated in Fig. S1. Extraction time (t, 5–15 min), temperature (T, 100–200 °C), and solid-to-liquid ratio (S/L, 0.5–5 %) were independent variables modulated during extractions as defined by the RSM design. Regarding the extraction time, the moment when the set temperature was reached was chosen as the start of the extraction. In all experimental runs, the extraction volume was 250 mL with a stirring speed of 600 RPM, and pressure was maintained at 30 bars with injection of nitrogen in the reactor (Azote 5.0, Messer, France). Following SWE, extracts were cooled by the internal cooler to a temperature of 60 °C before being centrifuged at 4200 RPM for 20 min at 4 °C (Eppendorf 5804 R, Eppendorf, France), and frozen at –24 °C for freeze-drying (Freezone 4.5 plus, Labconco, USA). The resulting extracts were dried and stored in the dark at room temperature for subsequent analysis.

#### 2.4.2. Experimental design and statistical analysis

Response surface methodology was used to assess the effects of extraction parameters and to optimize SWE, with the purpose of maximizing the extraction of proteins from white wine lees. Box Behnken design consisting of fifteen randomized runs, including four replicates at the central point to evaluate the standard deviations (Table S1).

The independent variables used for the design of experiments were extraction time (t) ($X_1$, 5–15 min), temperature (T) ($X_2$, 100–200 °C), and solid-to-liquid ratio (R) ($X_3$, 0.5–5.0 %). The response variables investigated were proteins extracted from white wine lees analyzed by the Bradford method ($Y_1$, g/100 g of lees DM). The response variables, as a function of factors, were fitted using the following second-order polynomial function (1):

$$Y = a_0 + a_1X_1 + a_2X_2 + a_3X_3 + a_{12}X_1X_2 + a_{13}X_1X_3 + a_{23}X_2X_3 + a_{11}X_1^2 + a_{22}X_2^2 + a_{33}X_3^2 \quad (1)$$

Where Y represents the response prediction, Xi and Xj are the independent variables affecting the response, and a0, ai, aii, and aij are the
regression coefficients for the intercept, linear, quadratic, and interaction effects.

The standard deviations of the experimental responses were evaluated by repeating the experiments at the center of the experimental domain (Xi = 0) four times. XLSTAT software (Version 2022.4.1, Addinsoft, France) was used to implement the experimental matrices, calculate the model coefficients, assess the significance of the regression, the validity of the model, and plot the contour lines.

To validate the reliability of the model developed through the Box-Behnken design, we compared the predicted values obtained from the regression equations with experimental values measured under the corresponding optimal conditions. Optimal extraction conditions were independently determined using the desirability function implemented in XLSTAT to maximize protein extraction yield (response variable). Predicted values were obtained by applying the optimized parameters to the corresponding regression models. Experimental validation was performed under the same conditions in triplicate, and results are expressed as mean ± standard deviation from three independent replicates. The close agreement between predicted and experimental values (t-test) supports the accuracy and predictive reliability of the developed models within the experimental domain.

## 2.5. Determination of dry matter

The dry matter (DM) content of wine lees and protein extracts was determined by gravimetric analysis. A known mass of sample (m₁ in mg) was placed into a pre-weighed aluminium dish (m₂ in mg) and dried in an oven at 110 °C for 24 h. After drying, the dish containing the sample was weighed again (m₃ in mg). The dry matter content was then calculated using the following equation (2):

$$DM (\%) = \frac{(m_3 - m_2)}{(m_1 - m_2)} \times 100 \qquad (2)$$

## 2.6. Protein quantification

### 2.6.1. Bradford assay

The Bradford assay has been used to optimize protein extraction by the recommendations of the OIV International Oenological Codex (OIV-OENO 452-2012). It was performed in a 96-well microplate (Breniaux et al., 2025). In each microplate well, 50 µL of water for the blank, reference solutions, and samples solubilized in water at 20 g.L⁻¹ were deposited. Then 200 µL of 2:7.5 diluted Bradford reagent were added. After 5 min of incubation at room temperature, absorbance was measured at 595 nm with a microplate spectrophotometer UV-Vis MultiSkan Sky High (ThermoFisher Scientific, France). All determinations were carried out in triplicate with 4 deposits per sample (n = 12). Bovine Serum Albumin (BSA) was used as a protein standard to prepare a calibration curve (0–100 mg.L⁻¹ in water, R² = 0.999), and the protein content was expressed as mg (BSA equivalent) per 100 g of DM.

### 2.6.2. Total protein content by kjeldahl assay

Total protein content was also determined using the Kjeldahl method (Kjeldahl, 1883). Approximately 200 mg of sample was accurately weighed into a Kjeldahl digestion tube. A catalytic mixture consisting of 5 g of potassium sulphate and 0.5 g of copper sulphate was added to each tube, followed by 10 mL of concentrated sulfuric acid (H₂SO₄). Samples were digested at 400 °C for 2 h using a Turbotherm digestion unit (Gerhardt, Königswinter, Germany). After digestion and subsequent cooling, samples were diluted with 50 mL of water and alkalinized with 40 mL of 32 % sodium hydroxide (NaOH). The solutions were then 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). The distillates were titrated with 0.1 N hydrochloric acid (HCl). The total

nitrogen content was calculated and converted to protein content using a conversion factor of 6.25 for lees and potatoes protein and 5.5 for gelatin (Nielsen, 2017). All analyses were performed in triplicate, and results were expressed as milligrams of proteins per gram of dry matter (mg.g⁻¹ DM).

## 2.7. Total polysaccharide content

Total polysaccharide content (TP) was determined using the phenolsulfuric methodology as described by Snyman et al. (2021). Both the experimental and commercial fining agents were rehydrated in water at 1 g.L⁻¹ and subjected to sonication for 15 min to ensure homogeneous suspension. Then, in a 20 mL glass vial, 1 mL of sample solution or blank (water) or standard solution (Glucose:Mannose, 1:1) were mixed with 1 mL of 5 % (v/v) aqueous phenol solution, immediately followed by the addition of 5 mL of concentrated sulfuric acid. The reaction mixtures were then incubated at room temperature for 30 min. Then, 200 µL of solution was transferred to 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). All measurements were performed in triplicate. Quantification was based on a Glucose:Mannose (1:1) calibration curve ranging from 0 to 500 mg.L⁻¹. Results were expressed as milligrams of sugar equivalent per gram of dry matter (mg sugar eq. g⁻¹ DM).

## 2.8. Analysis of soluble protein by SDS-PAGE

The molecular weight distribution of soluble proteins in the samples was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), according to the method of Laemmli (1970). Prior to analysis, samples were diluted 1:1 with Laemmli buffer without β-mercaptoethanol (non-denaturing conditions) or diluted 1:1 with a solution of Laemmli buffer containing 5 % (v/v) β-mercaptoethanol and heated at 95 °C for 15 min. Two molecular weight standards were used: Precision Plus Protein Standards (10–250 kDa) and Polypeptide SDS-PAGE Standards (6.5–26.6 kDa) (Bio-Rad, Hercules, CA, USA).

Aliquots of 10 µL of each prepared sample (20 g.L⁻¹ in water) and marker were loaded onto a 5 % (w/v) polyacrylamide stacking gel and separated in a 15 % (w/v) polyacrylamide resolving gel. Electrophoresis was carried out at a constant current of 20 mA. Following migration, the gels were stained with Coomassie Brilliant Blue and destained overnight in 10 % (v/v) acetic acid solution. All electrophoresis reagents and equipment were purchased from Bio-Rad (Hercules, CA, USA).

## 2.9. Size exclusion chromatography profiles and molecular weight distributions

Molecular size distribution of solubilized compounds was assessed by size exclusion chromatography (SEC), as described by Beaubier et al. (2019), using a Superdex™ 30 Increase 10/300 GL column (fractionation range: 100–7000 g mol⁻¹; 10 × 300 mm, GE Healthcare, USA). Solutions of fining agents were prepared at 5 g.L⁻¹ in water with pH adjusted at 10.5 using 2 M NaOH. Then, 10 µL were injected into the column maintained at 35 °C, and elution was carried out isocratically at a flow rate of 0.5 mL min⁻¹ using a mobile phase consisting of water, acetonitrile, and trifluoroacetic acid (69.9:30:0.1, v/v/v).

UV detection was performed using a 1 cm path length cell, with spectra recorded from 190 to 400 nm. Chromatograms were monitored at 214 nm for protein detection, and the presence of nucleic acid and phenolic compounds was monitored at 256 nm and 325 nm, respectively.

The column was calibrated using a set of 20 synthetic peptides (GeneCust, Dudelange, Luxembourg) with known molecular weights ranging from 220 to 1890 g mol⁻¹. Peptides were eluted under the same chromatographic conditions. Retention times (TRx) were determined for each peptide using LabSolutions software (Shimadzu Corporation,
Japan), and a calibration curve was generated by linear regression (performed in Excel) between log-transformed molar mass (MMx) and retention time. The resulting equation (3) showed a good fit (R² = 0.87):

$$MM_x = 10^{-0.068 \times Tr_x + 4.93} \quad (3)$$

where MMx is the molar mass (g.mol⁻¹) and Trx is the retention time (min) of point 'x' on the chromatogram.

Molecular weight distribution profiles of the peptide fraction (retention times between 17 and 34 min) were obtained by plotting the cumulative peptide mass fraction against the corresponding molar mass values derived from the calibration curve. Integration of the chromatographic signals was performed according to the method of Bodin et al. (2015), and the resulting distribution was visualized as previously illustrated by Beaubier et al. (2021).

## 2.10. Zeta potential

The electrophoretic mobility of proteins in the lees extract and commercial fining agents was measured using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK), based on Laser Doppler Velocimetry. The corresponding zeta potential (ζ) values were then calculated from the mobility data using the Smoluchowski equation, which is commonly applied to aqueous systems (Sze et al., 2003). Protein extracts were prepared at concentrations of 80 mg.L⁻¹ for gelatin and patatin, and 200 mg.L⁻¹ for lees and lees extract, in a model wine solution composed of 12 % ethanol and 5 g.L⁻¹ tartaric acid, using ultrapure water (resistivity ≥18.2 MΩ cm). The solutions were sonicated in an ultrasonic bath for 10 min at room temperature (25 °C). The pH was then adjusted to between 3.0 and 4.0 b y the addition of 1 M NaOH.

Measurements were performed using folded capillary cells (DTS1070, Malvern). Each sample was analyzed in triplicate, and the mean zeta potential was calculated from the three replicates.

## 2.11. Fining experiments

One unfined red wine (vintage 2024) made from Cabernet-Sauvignon from the Bordeaux appellation region was used for the fining experiments. The main physicochemical characteristics are summarized in Table S2 (Supplementary data).

Aliquots of 90 mL of unfined wine were distributed into five experimental treatments, each performed in triplicate: a control (C) with water addition, gelatin (G) at 8 g hL⁻¹, and potato protein (P) at 8 g hL⁻¹ as recommended by the supplier. White wine lees (L) and the subcritical protein extract (E) were added at 20 g hL⁻¹, like to the classic concentration for yeast protein extracts (Fernandes et al., 2015). Following the addition of fining agents, the wines were stored for 7 days at 10 °C to allow the fining agents to settle, after which physicochemical analyses were performed on the clarified wines.

### 2.11.1. Classical oenological parameters

Classical oenological parameters (e.g., alcoholic percentage, pH, total and volatile acidity) were measured by Fourier-transform infrared spectroscopy - FTIR (WineScan, FOSS France, Nanterre, France).

### 2.11.2. Turbidity

Wine turbidity was assessed using a 2100Q Turbidimeter (Hach Instrument, Szeged, Hungary) and was measured before and post-fining treatment. All measurements were performed in triplicate. Turbidity was expressed in nephelometric turbidity units (NTU).

### 2.11.3. Chromatic characteristics

Total color intensity (sum of absorbance values at 420, 520 and 620 nm), hue (ratio between absorbance values at 420 and 520 nm) and the CIELab color space parameters (L*, a*, b* and ΔE) were determined by

UV-visible spectrophotometry using a UV-2600i spectrophotometer (Shimadzu France, Marne la Vallée, France) according to reference method OIV-MA-AS2-07 B and OIV-MA-AS2-11 respectively as described in the “Compendium of International Methods of Analysis” published in 2009 b y the International Organisation of Vine and Wine (OIV).

### 2.11.4. Total monomeric anthocyanins (TA)

Total monomeric anthocyanin (TA) content was determined following the method described by Ribéreau-Gayon and Stonestreet (1965). Briefly, 1 mL of the wine sample was mixed with 1 mL of ethanol, acidified with 0.1 % HCl, and 20 mL of water acidified with 2 % HCl (w/w). From this mixture, 10 mL were distributed equally into two test tubes. To the first tube, 4 mL of distilled water was added (Tube 1), while the second received 4 mL of a 15 % sodium metabisulfite solution (Tube 2). After 15 min of reaction time, the absorbance of each solution was measured at 520 nm (A1 for tube 1 and A2 for tube 2) using a UV-2600i spectrophotometer (Shimadzu France, Marne la Vallée, France). All measurements were performed in triplicate. Total anthocyanin concentration was calculated (expressed as mg.L⁻¹) using the following equation (4):

$$TA (mg.L^{-1} \text{ of malvidin} - 3 - O - \text{glucoside}) = 875 \times (A1-A2) \quad (4)$$

### 2.11.5. Total tannins (TT)

Total tannins (TT) content was determined following the method described by Ribéreau-Gayon and Stonestreet (1966). Briefly, two test tubes were prepared, each with 4 mL of diluted wine sample (1/50) was mixed with 2 mL of water and 6 mL concentrated HCl. One of the tubes was heated at 100 °C for 30 min (1), the other was left at room temperature (2). After cooling, 1 mL of absolute ethanol was added to each test tube, and the absorbance at 550 nm was measured (A1 for tube 1 and A2 for tube 2) using a UV-2600i spectrophotometer (Shimadzu France, Marne la Vallée, France). All measurements were performed in triplicate. Total tannin content was calculated (expressed as g.L⁻¹) using the following equation (5):

$$TT (g.L^{-1} \text{ of cyanidin}) = 19.33 \times (A1-A2) \quad (5)$$

### 2.11.6. Gelatin index

The gelatin index was determined using the method described by Glories (1978). Briefly, the total tannin content was measured on the wine sample (C0) using the method described above. Then, 2.5 mL of a 70 g.L⁻¹ gelatin solution (Vegecoll, Laffort Oenologie, France) was added to 25 mL of the wine sample. The sample was then placed at room temperature for 3 days and centrifuged at 3000 RPM for 5 min, and the total tannins were then determined on the supernatant (C1). All measurements were performed in triplicate. Gelatin index was calculated using the following equation (6):

$$Gelatin index = \frac{C0-C1}{C0} \times 100 \quad (6)$$

## 2.12. Statistical analysis

The ANOVA tests were performed using XLSTAT software (Addinsoft version 19.02, Paris, France). Tukey test was carried out, and when p-values were <0.05, the data were considered significant.

# 3. Results and discussion

## 3.1. Subcritical water extraction of white wine lees protein extract

This study proposes the use of subcritical water extraction (SWE) for the valorization of white wine lees proteins. Several key parameters influencing the extraction of proteins in subcritical water, as measured by the Bradford assay, were studied: extraction temperature and time,
and the solid-liquid ratio. To identify the optimal conditions for SWE, Response Surface Methodology (RSM) was applied, enabling a more efficient experimental process by minimizing the number of required trials. Specifically, the Box-Behnken design, a widely used statistical tool for optimizing extraction parameters, was employed. The Box-Behnken design comprised 15 experimental runs, and the results of each experiment in protein were analyzed by the Bradford assay and are presented in Table S1 (Supplementary data). Based on the experimental results, an ANOVA was performed to develop a quadratic model (Equation (7)) for protein extraction and to predict outcomes across the range of SWE parameters, with (t) the extraction time, (T) the temperature and (R) the solid-liquid ratio.

$$ \begin{aligned} \text{Proteins g / 100 g DM} = & 1.45 + 0.032 \times t + 0.089 \times T + 0.023 \\ & \times R - 0.039 \times t \times t - 0.058 \times t \times T + 0.107 \times t \\ & \times R - 0.857 \times T \times T + 0.058 \times T \times R - 0.193 \\ & \times R \times R \end{aligned} \quad (7) $$

The statistical significance of the model was assessed using the F-test and corresponding p-values, as detailed in Table S3 (Supplementary data). According to this quadratic function, the quadratic term of the temperature (a2a2) was the most important factor with the most significant coefficient effect (Table S3, Supplementary data). The effect of temperature is therefore non-linear, indicating the presence of an optimum. For the other variables (e.g. extraction time and solid-liquid ratio) and respective interactions, no significant difference was observed at the 95 % confidence interval and therefore has minimal or no impact on protein extraction.

The quality of fit was assessed using the coefficient of determination (R²) and the adjusted R², which together offer a comprehensive view of model performance. The R² value was 0.956 while the corresponding adjusted R² value was 0.877 (Table S3, Supplementary data), indicating that the models provided a good representation of the experimental values.

To enhance clarity, response surface models for protein extraction were generated and are presented in Fig. 1. The 3D surface plots illustrate the relationships between protein concentration and pairs of extraction variables: (A) temperature and solid-liquid ratio, (B) extraction time and temperature. Only pairs involving temperature are presented, as temperature was the only parameter with a statistically significant effect.

Fig. 1A and B shows an increase in protein extraction at 1.46 g/100 g of lees DM until the temperature reaches a maximum around 155 °C and a rapid decrease after passing this temperature, regardless of the extraction time and the solid-liquid ratio confirming the ANOVA

analysis of the prediction model with the presence of an optimum.

This maximum extraction temperature of around 150 °C has already been observed in the literature for the extraction of proteins from soybean flakes and grape seeds using sub-critical water (Breniaux et al., 2025; Ndlela et al., 2012). The decrease in protein extraction capacity after this maximum temperature may be due to the increase in the protein hydrolysis reaction with the increase in temperature during SWE (Marcet et al., 2016; Poulain et al., 2024). Moreover, the Bradford assay method reacts specifically with proteins with molecular weights greater than 3 kDa (Lucarini & Kilikian, 1999), so the hydrolysis reactions during SWE at high temperature may have led to the release of non-quantifiable small peptides and therefore to a decrease in the concentration of high molecular weight proteins in our extracts. Neither extraction time or solid-liquid ratio had a significant effect on protein yield over the experimental range tested, suggesting a rapid equilibrium of extraction in our static system. Based on the experimental results, the optimal conditions predicted to maximize protein extraction with a desirability of 0.869 were 13 min of extraction at 155 °C with a solid-liquid ratio of 3.8 %. This would make it possible to extract 1.47 g of protein/100 g of DM from white wine lees.

However, as extraction time and solid-liquid ratio had no significant impact on protein yield, these parameters were set to the shortest time (5 min) and highest ratio (5 %) to improve efficiency and industrial applicability. To validate the model and the choice of extractions conditions, extractions under the chosen conditions were carried out in triplicate. The experimental response value obtained was 1.34 ± 0.1 g/ 100 g DM. This gives us a relative standard error of 7.8 % between the predicted and experimental values, confirming a strong agreement between the model and the experimental data.

## 3.2. Analysis of fining agents

### 3.2.1. Macromolecule composition

The quantification of total protein and total polysaccharide contents in various wine fining agents revealed distinct compositional profiles (Table 1). Gelatin consisted almost entirely of proteins (881.7 mg g⁻¹), a value consistent with the literature, with protein levels above 80 % (Braga et al., 2007; Gazzola et al., 2017). This is consistent with its known use as a protein fining agent to reduce phenolic compounds such as tannins in wine matrices (González-Neves et al., 2014). Patatin also exhibited a high protein content (806.4 mg g⁻¹) with minor polysaccharide presence (19.7 mg g⁻¹), reflecting its origin from plant tuber glycoproteins (Gambuti et al., 2012) and its emerging role as a plant-based alternative to animal-derived fining agents. In contrast,

<figure><img src="image_3.png" /><figcaption>Fig. 1. Response surfaces obtained from the Box-Behnken design (A) effect of extraction temperature (°C), solid-liquid ratio (%), and (B) effect of extraction time (min), temperature on protein extraction.</figcaption></figure>
**Table 1**

Protein and polysaccharide contents of the fining agents used in this study. Mean concentrations ± standard deviation (n = 3) are presented. One-way ANOVA showed significant effect of the fining agent and p-values were calculated. Different letters indicate significant differences between samples for a given parameter (Tukey's test for α = 0.05).

<table><thead><tr><th></th><th>Total proteins (mg.g<sup>-1</sup>)</th><th>Total polysaccharides (mg.g<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Gelatin (G)</td><td>881.7 ± 0.4 a</td><td>0.00 ± 0.0 d</td></tr><tr><td>Patatin (P)</td><td>806.4 ± 1.4 b</td><td>19.7 ± 0.7 c</td></tr><tr><td>Lees (L)</td><td>375.5 ± 1.1 c</td><td>543.0 ± 6.6 b</td></tr><tr><td>Lees extract (E)</td><td>361.7 ± 4.1 d</td><td>663.0 ± 5.9 a</td></tr><tr><td>p-value</td><td>&lt;0.0001</td><td>&lt;0.0001</td></tr></tbody></table>

white wine lees and its extract showed lower protein levels (375.5 mg g⁻¹ and 361.7 mg g⁻¹, respectively) but were markedly enriched in polysaccharides (543.0 mg g⁻¹ for lees and 663.0 mg g⁻¹ for lees extract). These values are consistent with previous studies, as yeast cell walls are primarily composed of mannoproteins and β-glucans (Balmaseda et al., 2025; De Iseppi et al., 2021), and the concentrations of polysaccharides reported in wine lees matrices (Garcia et al., 2025) support this observation. However, the compositional similarity between lees and their extracts suggests that the extraction process may not have substantially altered the relative proportions of proteins and polysaccharides. The absence of protein enrichment in the extract compared to the total lees may be explained by the presence of low-molecular-weight peptides that, although not specifically quantified, still contribute to the overall nitrogen-based protein content. Altogether, lees and lees extracts appear to be composed of high concentrations of macromolecules such as proteins and/or polysaccharides that may contribute to their fining efficacy through interactions with wine colloids and phenolic compounds (Mekoue Nguela et al., 2016).

**3.2.2. Zeta potential (ζ) of fining agents**

Zeta potential is a key factor in determining the stability of colloidal dispersions, as it directly influences the balance between attractive van der Waals forces and repulsive electrostatic forces (Filippi et al., 2008). If the zeta potential is too small (typically less than about ±25 mV), the repulsive force will not be strong enough to overcome the van der Waals attraction between the particles, and they will begin to agglomerate (Filippi et al., 2008).

Fig. 2 shows the evolution of the zeta potential of fining agents in model wine solutions as a function of pH. The pH range has been set between 3.0 and 4.0, which are the classic pH values for wines. A general decrease in zeta potential with increasing pH is observed across all samples (slightly less for lees and lees extract), in agreement with trends reported in the literature (Malhotra & Coupland, 2004). The potato protein-based fining agent exhibited the most positive zeta potential values, starting above +15 mV at pH 3.0 and decreasing gradually to around +5 mV at pH 4.0. In contrast, gelatin showed the most

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Evolution of the zeta potential of different fining agent solutions in a model wine solution (12% ethanol and 5 g.L⁻¹ tartaric acid) between pH = 3 and 4.</figcaption></figure>

pronounced decrease in zeta potential with pH. This transition indicates a shift from positive to negative surface charge, reflecting the protein's amphoteric behaviour. This greater variation with pH may be due to the presence of ionizable side chains that respond sharply in this pH range. The untreated lees and the lees extract showed relatively stable and mildly zeta potentials throughout the pH range, remaining between approximately -5 and 0.5 mV respectively. This indicates lower surface charge density, possibly due to a more heterogeneous composition (mannoproteins, glucans, and residual polyphenols) and limited ionizable groups exposed at the surface. Importantly, at typical wine pH (~3.2–3.8), all fining agents display zeta potentials between -25 and +25 mV, which places them within the zone of electrostatic instability. In this range, proteins are more prone to aggregation, a desirable feature for fining applications, as it facilitates the formation of flocs that can remove haze-forming particles or phenolics from wine.

**3.2.3. Protein profile characterization by SDS-PAGE**

The protein composition of the lees and the subcritical protein extract was evaluated using SDS-PAGE and compared to gelatin and patatin. Fig. 3 presents the electrophoretic profiles obtained under both non-reducing and reducing conditions (with the addition of 2% β-mercaptoethanol).

For the commercial references, gelatin displayed poorly resolved bands, predominantly below 30 kDa, which corresponds to small proteins and peptides. Patatin showed a major band around 40 kDa, along with additional bands near the gel top and at approximately 25, 20, and 15 kDa under reducing conditions. These profiles are in line with previous reports (Gambuti et al., 2012; Gazzola et al., 2017).

For the white wine lees sample, electrophoresis revealed a diffuse smear rather than discrete bands, accompanied by stained material accumulating at the top of the gel in both conditions. This smear-like band is consistent with previous findings (Gazzola et al., 2017) and is likely due to the formation of protein-phenolic complexes, which impede proper migration through the gel matrix. Additional factors may include the presence of nucleic acids released during yeast autolysis or insoluble yeast-derived aggregates, both of which can interfere with protein resolution (Zhao & Fleet, 2003). In contrast, the electrophoretic profile of the lees-derived extract showed an intense band at the bottom of the gel, suggesting the presence of low-molecular-weight peptides (<1.4 kDa) as already observed for yeast protein extract (Lochbühler et al., 2015). This suggests the presence of protein fragments, potentially resulting from thermal processing, as previously observed under extraction conditions that include a heating step (De Iseppi et al., 2021; Poulain et al., 2024).

**3.2.4. Molecular weight distribution by Size-Exclusion Chromatography (SEC)**

To gain further insight into the molecular characteristics of the proteinaceous fractions and compare the lees-derived extracts with conventional fining agents, Size-Exclusion Chromatography (SEC) was performed. The chromatograms recorded at 214 nm, corresponding to peptide bond absorbance, are shown in Fig. 4A. Additional wavelengths were assessed to evaluate the presence of potential interfering compounds: 260 nm for nucleic acids and 325 nm for phenolic compounds (data not shown).

Under the applied chromatographic conditions, native proteins eluted in the dead volume (~15 min), followed by a broad range of peptides eluting between 17 (polypeptides) and 34 min (dipeptides). Free amino acids appeared at the end of the elution window. It is worth noting that absorbance in the amino acid region may be influenced by nucleic acids, which absorb strongly at 260 nm.

The lees sample was largely insoluble under the conditions used, likely due to the presence of yeast-derived aggregates. No signal was detected at 214 nm, despite matching protein concentrations with other samples, indicating that the protein content was not accessible to the chromatographic matrix. However, absorbance at 260 nm suggested the
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** SDS-PAGE profiles of fining agents (lees, lees-derived extract, gelatin, and potato protein) under non-reducing (NRC) and reducing conditions (RC). Molecular Weight Markers of proteins and polypeptides, in kDa, are shown on the left.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Size Exclusion Chromatograms of the initial lees, the lees-derived extract, and the commercial gelatin and patatin, at 214 nm (A) and the molecular weight distributions of lees-derived extract and gelatin of the peptide fraction (B). Chromatographic system: Superdex™ 30 Increase 10/300 GL column, solvent: water/ acetonitrile/TFA (69.9/30/0.1), flow rate of 0.5 mL min⁻¹.</figcaption></figure>

presence of nucleic acids, probably released from yeast cell lysis (Charpentier et al., 2005), further supporting the idea of insoluble cellular debris and complex aggregates preventing effective separation.

In contrast, the SEC profile of the lees extract revealed a broad peptide distribution across the elution range, a pattern characteristic of protein hydrolysates. Only a minor peak at 15 min (corresponding to intact proteins) was observed, representing less than 1 % of the total 214 nm signal. Beyond 37 min, the UV signal overlapped with nucleic acid interference, as evidenced by corresponding absorbance at 260 nm. Importantly, no absorbance was detected at 325 nm, indicating the absence of phenolic compounds and suggesting that proteins and peptides in the extract were not complexed or masked by phenolics. These results corroborate the SDS-PAGE findings and confirm that the extraction process yielded a hydrolysate-like material, rich in small, soluble peptides and indicative of extensive protein breakdown and cell lysis.

SEC profiles of the commercial references provided complementary insights. Patatin showed a dominant peak in the dead volume (intact protein) and minor polypeptide signals, aligning well with SDS-PAGE

data. Gelatin, however, presented a broad peptide distribution, typical of a hydrolysate rather than a structured protein isolate (Beaubier et al., 2019). The absence of a distinct native protein peak and the wide range of peptide sizes reflect its heterogeneous nature, derived from partial hydrolysis of collagen. No significant signal at 260 or 325 nm was observed for either reference, confirming the absence of nucleic acids or phenolic interference.

To quantitatively compare the peptide size distributions, the cumulative molar mass distribution was calculated from the 214 nm SEC signal for the two peptide-rich samples: the lees extract and gelatin (Fig. 4B). This analysis allowed determination of the median molar mass (Mw50), as well as the 10th and 90th percentiles (Mw10 and Mw90), offering a detailed characterization of the peptide population.

Both samples displayed similar molecular weight distributions, with a median molar mass of approximately 2000 g mol⁻¹, corresponding to peptides of around 17 amino acid residues. A subtle difference was observed in the high molecular weight tail of the distribution: 10 % of peptides in gelatin exceeded 5000 g mol⁻¹, while this threshold was closer to 4000 g mol⁻¹ for the lees extract.
Overall, SEC results highlight the importance of the molecular weight distribution in fining performance. The dominance of peptides (rather than proteins) likely enhances solubility and provides accessible functional groups capable (e.g., carboxyl, amine, and hydrophobic residues) of interacting with tannins or haze-forming proteins via hydrogen bonding, hydrophobic interactions, and electrostatic forces (Maury et al., 2001). The enrichment in low-molecular-weight fractions may also contribute to improved clarification efficiency due to increased surface area and mobility in solution. The enriched peptide profile of the subcritical water white wine lees extract suggests promising potential as a fining agent, offering performance similar to that of gelatin, while representing a sustainable and wine-derived alternative. It is therefore important to test the fining properties of this extract in red wine and compare it with commercial products.

### 3.3. Study of performance and effect of the fining agents in wine

The fining performance of the optimized protein extract from white wine lees obtained by SWE was compared to the lees before extraction as well as to commercial fining products of animal and vegetable origin (gelatin and patatin). It was proposed to evaluate the performance of raw wine lees as fining agents, given their known ability to adsorb phenolic compounds such as anthocyanins and condensed tannins (Mazauric & Salmon, 2005). Additionally, the aim was to assess the potential of SWE as a method to enhance the fining properties of wine lees.

An unfined red wine (crude wine) was treated according to the protocol described in section 2.11, and physico-chemical and chemical parameters were measured before and after fining. The results are given in Table 2.

#### 3.3.1. Turbidity

The primary objective of a fining agent is to clarify the wine (reduce turbidity). After fining, the turbidity of the wine was compared with that recorded before treatment (Table 2). Spontaneous settling was observed in the control sample (without the addition of proteins), with a 53 % reduction in initial turbidity, but the application of fining agents led to further substantial improvements.

Gelatin was the most effective, reducing turbidity to 3.08 ± 0.2 NTU, representing an 83.3 % decrease relative to the control. These performances have already been described in the literature (Gazzola et al., 2017; González-Neves et al., 2014; Mekoue Nguela et al., 2024). SEC analysis (Fig. 4) showed that gelatin presented a broad, hydrolysate-like peptide profile, with a wide range of low-to mid-molecular-weight peptides. This molecular heterogeneity likely increases surface reactivity and facilitates the formation of large, flocculable aggregates. The wine lees extract also demonstrated strong fining performance, reducing turbidity by 64.4 % compared to the control.

This level of clarification is comparable to that reported for yeast protein extracts (Gaspar et al., 2019; Lochbühler et al., 2015). Lees extract SEC profile revealed a rich peptide distribution comparable to the gelatin peptide size distribution (Fig. 4), which provides higher solubility of these fragments that enhanced mobility and interaction potential with haze-forming compounds. Patatin, on the other hand, led to a more moderate turbidity reduction (−34.6 %). This result is somewhat unexpected, as potato protein has previously been reported to exhibit fining capacity comparable to gelatin (Gambuti et al., 2012; Gazzola et al., 2017). However, this discrepancy may be related to the specific chemical characteristics of the wine matrix used in this study, as well as to the dosage applied, which could influence protein-polyphenol interactions. However, its SEC and SDS-PAGE profile showed a dominant intact protein peak (around 40 kDa) with limited peptide presence. This globular structure may limit flexibility and aggregation potential, reducing efficacy. Whole lees showed the least fining capacity (−18.5 %), consistent with its insolubility observed in the SEC conditions. No protein signal was detected at 214 nm, suggesting that the proteins remained embedded in insoluble yeast aggregates, which likely hindered effective interaction with wine colloids. These results confirm that gelatin and lees extract significantly outperform spontaneous settling, while patatin and whole lees exhibit moderate but still significant clarifying activity.

#### 3.3.2. Chromatic characteristic

The variation of chromatic characteristics due to fining was determined by spectrophotometry (Table 2). The application of different fining agents resulted in measurable variations in the colorimetric parameters of the red wine treated. Compared with the control, color intensity decreased slightly, particularly with gelatin (−5.6 %) and lees extract (−2.9 %), reflecting a moderate reduction in pigment concentration, probably due to the adsorption of monomeric and polymerised anthocyanins or the reduction of copigments families such as flavanols and phenolics acids that can increase the color intensity of young red wine (González-Neves et al., 2014). Similar results have been observed in previous studies comparing different fining agents, particularly gelatin and yeast protein extracts (Karamanidou et al., 2011; Mekoue Nguela et al., 2024). In contrast, patatin and lees had no statistical impact on color intensity, suggesting a lower selectivity for coloured pigments, as observed in the study of Gambuti et al. (2012). Under current experimental conditions, fining treatments have had no statistical effect on the hue of the wine. When considering the impact on CIELAB color space parameters, L* (luminosity) values increase slightly for all treatments, with increases of up to +2.6 % and +1.6 % for gelatin and extract, respectively, reflecting a visually brighter wine, which is consistent with the decrease in color intensity. The a* parameter (red intensity) decreased markedly with gelatin (−3.1 %) and extract (−3.1 %).

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

Analytical characteristics of crude wine, control, and treated with the fining agents: gelatin at 8 g.hL-1, patatin at 8 g.hL-1, white wine lees and the subcritical protein extract were added at 20 g.hL-1. Mean concentrations ± standard deviation (n = 3) are presented. One-way ANOVA showed significant effect of the fining agent and p-values were calculated. Different letters indicate significant differences between samples for a given parameter (Tukey's test for α = 0.05).

<table><thead><tr><th></th><th>Turbidity (NTU)</th><th>Total monomeric anthocyanins (mg.L<sup>-1</sup>)</th><th>Total tannins (g.L<sup>-1</sup>)</th><th>Color intensity</th><th>Hue</th><th>L*</th><th>a*</th><th>b*</th><th>ΔE</th><th>Gelatin index</th></tr></thead><tbody><tr><td>Crude wine Control</td><td>39.8 ± 0.2 a<br>18.4 ± 0.6 b</td><td>207.4 ± 3.0 a<br>206.5 ± 2.3 a</td><td>3.9 ± 0.0 a<br>3.9 ± 0.1 a</td><td>14.2 ± 0.2 a<br>14.4 ± 0.1 a</td><td>0.8 ± 0.0 a<br>0.8 ± 0.0 a</td><td>64.1 ± 0.2 c<br>63.6 ± 0.3 d</td><td>34.5 ± 0.3 b<br>35.2 ± 0.3 a</td><td>4.2 ± 0.6 b<br>5.0 ± 0.1 a</td><td>64.1 ± 1.9 a<br>64.1 ± 0.8 a</td><td>64.1 ± 1.9 a<br>64.1 ± 0.8 a</td></tr><tr><td>Gelatin</td><td>3.08 ± 0.2 f</td><td>195.7 ± 1.0 c</td><td>3.1 ± 0.06 d</td><td>13.6 ± 0.1 c</td><td>0.8 ± 0.0 a</td><td>65.3 ± 0.2 a<br>63.9 ± 0.1 cd</td><td>34.2 ± 0.2 b<br>34.8 ± 0.2 ab</td><td>5.0 ± 0.2 a<br>5.3 ± 0.1 b</td><td>1.2 ± 0.2 a<br>1.2 ± 0.1 a</td><td>54.2 ± 1.2 c<br>62.1 ± 1.1 a</td></tr><tr><td>Patatin</td><td>12.0 ± 0.91 d</td><td>201.2 ± 2.7 ab</td><td>3.3 ± 0.1 c</td><td>14.4 ± 0.1 a</td><td>0.8 ± 0.0 a</td><td>64.0 ± 0.1 cd</td><td>34.5 ± 0.1 b</td><td>5.3 ± 0.1 a</td><td>1.2 ± 0.1 a</td><td>54.6 ± 0.6 c</td></tr><tr><td>Lees extract</td><td>15.0 ± 0.6 c</td><td>203.6 ± 2.8 ab</td><td>3.65 ± 0.0 b</td><td>14.4 ± 0.0 a</td><td>0.8 ± 0.0 a</td><td>64.7 ± 0.1 b</td><td>34.1 ± 0.1 b</td><td>5.1 ± 0.0 a</td><td>1.2 ± 0.1 a</td><td>54.6 ± 0.6 c</td></tr><tr><td></td><td>6.6 ± 0.1 e</td><td>197.8 ± 3.2 bc</td><td>3.2 ± 0.0 d</td><td>14.0 ± 0.0 b</td><td>0.8 ± 0.0 a</td><td>64.7 ± 0.1 b</td><td>34.1 ± 0.1 b</td><td>5.1 ± 0.0 a</td><td>1.2 ± 0.1 a</td><td>54.6 ± 0.6 c</td></tr><tr><td>p-value</td><td>&lt; 0.0001</td><td>0.001</td><td>&lt; 0.0001</td><td>&lt; 0.0001</td><td>0.026</td><td>&lt; 0.0001</td><td>0.001</td><td>0.002</td><td>0.94</td><td>&lt; 0.0001</td></tr></tbody></table>
‰), compared with a limited reduction for patatin (−1.4 ‰) and lees (−2.1 ‰). The effect of the different fining agents on b*coordinate (yellow indicator) was not significant when compared to control, according to the effect on hue. This evolution following fining may be attributed to the selective removal of specific compounds and anthocyanin families, which affected the relative distribution of individual anthocyanins and derived pigments. Despite these statistically significant variations, the differences in overall color (ΔE) between the treatments and the control remain below 3 in all cases, a threshold generally considered imperceptible to the human eye (Martínez et al., 2001).

## 3.4. Polyphenols

The application of fining agents induced significant changes in the phenolic profiles of the red wine (Table 2). However, after spontaneous decantation (control), no statistical differences with the crude wine were observed, so there was no precipitation of phenolic matter (anthocyanins and tannins) without the addition of a fining agent.

Total monomeric anthocyanins measured by SO₂ bleaching showed moderate decreases ranging from −1.4 ‰ for lees modality, to −4.2 ‰ and −5.2 ‰ for lees extract and gelatin modality, respectively, compared to the control. This suggests a preferential removal of monomeric anthocyanins or weakly bound pigment complexes by peptide hydrolysates of proteins such as gelatin and lees extract, as shown in the study by Cosme et al. (2007), where the greater the rate of hydrolysis, the greater the reduction in monomeric anthocyanins (Francisco et al., 2021). The slight reduction in total anthocyanins by fining with patatin (−2.6 ‰) has already been shown in the literature (Gambuti et al., 2012). These compounds are primarily responsible for the red and purple hues in young red wines, and their partial removal is consistent with the slight observed decrease in color intensity and redness (a*) (Table 2).

Among the tested fining agents, gelatin exhibited the highest efficiency in reducing total tannin concentration (−20.9 ‰). This reduction is attributed to strong hydrophobic and hydrogen bonding between condensed tannins and proline-rich gelatin molecules, forming insoluble aggregates (Charlton et al., 1996; Maury et al., 2001). It has been shown that due to their low molecular weight peptide fraction, gelatin-based glues were the most selective and precipitated highly polymerised and galloylated tannins (Maury et al., 2001, 2003).

The lees extract significantly reduced tannins (−18.4 ‰), results similar to those obtained with gelatin and close to those obtained with commercial yeast protein extracts (Lochbühler et al., 2015). This can be explained by the similarity in proteins molecular size of these two fining agents (section 3.2). The lees extract is enriched in proteins derived from mannoproteins and in polysaccharide-rich cell wall fragments, with which polyphenols can interact to form aggregates due to strong interactions with yeast cytoplasmic constituents (Mekoue Nguela et al., 2024). Although it has been shown that the polysaccharidic structure of mannoproteins plays a major role in stabilizing aggregates (Mekoue Nguela et al., 2016), the use of subcritical water should have allowed these mannoproteins to be degraded, making the protein part more accessible and increasing interactions. Patatin, a protein of plant origin, also significantly reduced tannins (−13.9 ‰), though less effectively, probably due to structural differences affecting aggregation with tannins. The current results differ from those reported in the literature, where Gambuti et al. (2012) showed similar tannin reduction performance to gelatin. In these trials, the doses applied are higher (10, 20, and 30 g hL⁻¹) than in our tests (8 g hL⁻¹), which may explain the differences. Finally, the whole lees, before extraction, showed the lowest performance in terms of tannin removal (−5.3 ‰). This may be because the wine lees cell walls are essentially composed of mannoproteins and β-glucans (De Iseppi et al., 2021), which have a greater tendency to stabilize colloidal aggregates composed of polyphenols (Mekoue Nguela et al., 2016). These results confirm that gelatin and lees extract significantly impact the polyphenol compositions of red wine, following

patatin and whole lees, and can therefore play a role in the organoleptic properties of red wine.

### 3.4.1. Wine astringency by gelatin index

Astringency is a complex tactile sensation produced by the binding of polyphenols with saliva proteins, which then precipitate. This sensation is mainly caused by polyphenols (Haslam et al., 1988). In red wine, the oligomers and polymers of condensed tannins are still mainly responsible for the astringency of red wines.

The gelatin index is an *in vitro* method for assessing astringency by measuring the degree of precipitation of tannins by gelatin. Indeed, gelatin and salivary proteins are rich in proline (Bajec & Pickering, 2008). This method has been described as being correlated with astringency when the tannin concentration was less than 5 g.L⁻¹ (Goldner & Zamora, 2010), as is the case in our study (Table 2).

As shown in Table 2, no statistical differences were observed between crude wine and control, with a value of 64.1, which may highlight an astringent wine (Glories, 1978). As mentioned previously, the astringency of the wine is caused by the presence of condensed tannins, but as observed in section 3.4, there is no difference in the concentration of total tannins without the addition of a fining agent. However, the addition of a fining agent significantly reduced the astringency of the wine as measured by the gelatin index. Fining with gelatin and with white wine lees extracts were the most effective, with a reduction of 15.4 ‰ and 14.8 ‰, respectively, compared with the control, thereby reducing the astringency of the wine. This confirms the ability of gelatin to precipitate high molecular weight proanthocyanidins responsible for salivary protein aggregation (Maury et al., 2001). Similarly, lees extract, which has a similar protein composition to gelatin (Fig. 4), reduced total tannins (Table 2), thereby reducing the tannins responsible for the astringency of red wine, as described by Mekoue Nguela et al. (2024) for commercial yeast protein extract. However, the application of patatin reduced the astringency to a lesser extent (−10.2 ‰). These results are consistent with the reduction in total tannins observed in Section 3.4. However, they do not align with the findings of Gambuti et al. (2012), who reported a decrease in astringency following patatin addition, as measured by the Saliva Precipitation Index (SPI). This discrepancy may again be attributed to differences in the fining agent dosage used between the two studies. Finally, lees reduced the astringency measured by the gelatin index (−3.2 ‰), but the differences with the control were not statistically significant from the control wine. This may be because, although mannoproteins in lees are known to reduce astringency (de Andrade Bulos et al., 2023), in this case, they are not accessible and are not released into the wine as they might be during ageing on lees.

## 4. Conclusions

This study demonstrates the potential of white wine lees as a novel source of sustainable fining agents. By employing subcritical water extraction (SWE), a green and solvent-free technology, it was possible to recover protein-rich extracts under optimized conditions (155 °C, 5 min, 5 ‰ S/L ratio). Molecular analyses (SDS-PAGE and SEC) revealed that the SWE process generated a peptide-rich extract with a molecular profile closely similar to commercial gelatin hydrolysates, known for their effective fining performance.

When applied to red wine, the lees extract exhibited strong clarification capacity by reducing turbidity by over 60 ‰, significantly lowering tannin content and astringency measured by the gelatin index, while preserving desirable chromatic characteristics. These effects are comparable to those achieved with gelatin, the current benchmark fining agent, and superior to patatin, a widely used plant-based alternative. The extract fining efficacy is largely attributed to its enrichment in low and medium-molecular-weight peptides, which enhance solubility, mobility, and interaction potential with wine colloids and polyphenols. The extract also retains yeast-derived polysaccharides such as mannoproteins and β-glucans, which may synergistically stabilize colloidal
structures post-fining.

Beyond its oenological performance, the valorization of white wine lees through SWE supports key principles of sustainability and the circular economy. It enables the transformation of a waste material into a value-added ingredient, reducing reliance on animal-derived and allergenic fining agents. Future research should explore the specific composition of polyphenols and aromas as well as sensory impact of white wine lees extracts in a broader range of wine styles.

## CRediT authorship contribution statement

**Luca Garcia:** Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Alice Douliez:** Writing – review & editing, Validation, Methodology, Investigation, Formal analysis. **Benjamin Poulain:** Writing – review & editing, Methodology, Investigation, Conceptualization. **Lucie Gagnepain:** Methodology, Investigation. **Fanny Gallo:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Romain Kapel:** Writing – review & editing, Resources. **Sophie Beaubier:** Writing – review & editing, Methodology, Investigation, Formal analysis. **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-CE43-0003 of the French Agence Nationale de la Recherche.

We would also like to thank the French Institute of Vine and wine (IFV) and Emmanuel Vinsonneau for providing the red wine sample.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.lwt.2025.118867.

## Data availability

Data will be made available on request.

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