Effect of the Addition of Protein Hydrolysates from Grape Seed Meal Residue to Red Wines in Warm Regions in the Stabilization Stage

LWT - Food Science and Technology 205 (2024) 116554 Available online 26 July 2024 0023-6438/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/bync/4.0/). Effect of the addition of protein hydrolysates from grape seed meal residue to red wines in warm regions in the stabilization stage Ana Bel´ en Mora-Garrido, M. Luisa Escudero-Gilete * , M. Lourdes Gonz´ alez-Miret , Francisco J. Heredia , María Jesús Cejudo-Bastante Food Colour and Quality Laboratory, Facultad de Farmacia, Universidad de Sevilla, 41012, Sevilla, Spain ARTICLE INFO Keywords: Grape seed protein hydrolysates Differential tristimulus colorimetry Polyphenolic compounds Copigmentation Warm climate ABSTRACT The effect of adding protein hydrolysates from defatted grape seed meals, a residue of the grape pomace industry, on the improvement of color stabilization of Syrah wines in warm climates was evaluated. Factors such as the type of raw material, the hydrolysis time, and the doses of protein hydrolysate were assessed. Hydrolysates were added to the wines at the stabilization stage and scrutinized over five months focusing on the polyphenolic composition, copigmentation and polymerization, as well as color by differential tristimulus colorimetry. The addition of 3 g/L protein hydrolysates from 1-h hydrolysis depicted a color stabilization effect, leading to higher values of chroma (C* ab ), phenolic compounds (benzoic acids and flavan-3-ols), and copigmentation, although assuming a slightly more aged-related tonality. These data obtained provide an interesting insight, not reported so far, on the use of this oenological alternative to avoid common color losses of red wines elaborated in warm climate. 1. Introduction Nowadays, one of the main purposes of industries is to reduce their overall impact to help fight climate change, an important mainstay of European and international policies. In this sense, reusing by-products is one of the tools of significant current interest to reduce the waste they generate, which implies additional economic benefits for industry through circular production (Dwyer, Hosseinian, &Rod, 2014). The wine industry generates enormous amounts of grape pomace annually, which are currently processed and reused by the grape pomace industry to obtain some interesting products, such as alcohols, natural colorants, tartaric acid, and grape seed oil, among others (Mora-Garrido, Cejudo-Bastante, Heredia, &Escudero-Gilete, 2022). Tons of defatted grape seed meal (DGSM) are generated after the extraction of grape seed oil, commonly used as biomass and animal feed. Due to its protein fraction (approximately 7–10% (w/w)), this undervalued residue has been currently suggested as a potential by-product of the grape pomace industry (Mora-Garrido et al., 2022). The significant increase in temperatures and the number of hours of light per year in summer in warm climate zones, provoked by the advance of climate change, causes phenolic and technological maturation not to coincide at the time of grape harvest, in contrast to what happens in colder viticulture zones (Mira de Ordu˜ na, 2010). This fact causes color instability of red wines over time (L´ opez, S´ anchez, Díaz, Ramírez, &Morales, 2007). Consequently, the color of wines decreases after months of storage in bottles or barrels due to copigmentation processes being hampered by the imbalance between pigments and copigments (Boulton, 2001). There are numerous studies in which this color loss is counteracted by adding phenolic rich products to red wine, such as oak wood chips during fermentation (Gordillo, Cejudo-Bastante, Rodríguez-Pulido, Gonz´ alez-Miret, &Heredia, 2013), tannins, or seeds or pomace from white or red grape varieties (Bautista-Ortín, Martínez-Cutillas, Ros-García, L´ opez-Roca, &G´ omez-Plaza, 2005;Canals et al., 2008; Cliff, Stanich, Edwards, &Gao et al., 2013; Gordillo et al., 2014). Aromatic amino acids, peptides and some proteins have been described to enhance the chemical and color stability of grape anthocyanins (Chung, Rojanasasithara, Mutilangi, &McClements, 2017; Qian et al., 2023). Under this assumption and focusing on the stabilization of red wines color, the protein fraction of the grape seed could be of great interest for oenology due to its great techno-functional features for modulating the quality properties of red wines, such as appearance, * Corresponding author. Food Colour &Quality Laboratory, Facultad de Farmacia, Universidad de Sevilla, 41012, Sevilla, Spain. E-mail addresses: [email protected] (A.B. Mora-Garrido), [email protected] (M.L. Escudero-Gilete), [email protected] (M.L. Gonz´ alez-Miret), [email protected] (F.J. Heredia), [email protected] (M.J. Cejudo-Bastante). Contents lists available at ScienceDirect LWT journal homepage: www.elsevier.com/locate/lwt https://doi.org/10.1016/j.lwt.2024.116554 Received 14 March 2024; Received in revised form 23 July 2024; Accepted 25 July 2024 LWT 205 (2024) 116554 2 color, and stability (Gazzola et al., 2014;Gazzola, Vincenzi, Marangon, Pasini, &Curioni, 2017), while avoiding the current legal restrictions because it is a grape-derived product. Previous studies have evaluated the effect of the addition of vegetal protein hydrolysates on the color of wines (Tschiersch, Nikfardjam, Schmidt, &Schwack, 2010). Furthermore, Cejudo-Bastante et al. (2016) proposed the addition of enzymatic protein hydrolysates from grape seeds prior to fermentation as an oenological strategy to stabilize the color of wines produced in warm regions, resulting in red wines with higher values of chroma and copigmentation percentage, without significantly changing the hue. In addition, recent investigations support the use of grape seed proteins as co-pigmenting agents and color stabilizers in red wines by demonstrating the chemical interaction between 7S globulin from grape seeds and malvidin-3-O-glucoside, the main pigment in red wine (Chamizo-Gonz´ alez, Gordillo &Heredia, 2021). Based on the promising results obtained by using grape seed protein as a color stabilizer, Cejudo-Bastante, Oliva-Sobrado, Gonz´ alez-Miret, and Heredia (2022) optimized the production of wine-soluble protein hydrolysates from defatted grape seed meal by enzymatic hydrolysis, a solvent-free and environmentally friendly alternative process. As a further step towards stabilizing the color of red wines in warm regions using grape-derived protein by-product, the main objective of this work was to evaluate the effect of the addition of protein hydrolysates of DGSM from the industrial grape pomace processing. Our interest focused on the study of the polyphenolic composition (benzoic acids, flavan-3-ols, hydroxycinnamic acid derivatives (HACD), flavonols, and anthocyanins), copigmentation and polymerization, and the chromatic characteristics of the resulting wines applying differential tristimulus colorimetry. For the study, three variability factors were considered, related to raw material (type of grape seed meal: DGSM from red and white grape variety), the method of obtaining (hydrolysis time: 1 and 4 h) and the winemaking application (protein hydrolysate dose: 1 and 3 g/L). This is the first attempt to add these protein-grape products in the stabilization stage of winemaking, as well as consider the aforementioned variables; therefore, this study hopes to lead to important advanced forwards the color stabilization of red wines from warm climate. 2. Materials and methods 2.1. Chemicals and solvents Ammonia, hydrochloric acid, and sodium carbonate were purchased (Panreac Química S.L.U., Barcelona, Spain). Folin-Ciocalteau reagent, sodium hydroxide, formic acid, malvidin-3-glucoside, (+)-catechin, (−)-epicatechin, gallic acid, caffeic acid, and quercetin were acquired (Sigma-Aldrich, Madrid, Spain). HPLC-grade acetonitrile was purchased (VWR International Eurolab S. L, Barcelona, Spain). Acetaldehyde was acquired (Merck, Darmstadt, Germany). Tartaric acid was purchased (LabKem, Barcelona, Spain). Ethanol, potassium metabisulphite, and sodium chloride were acquired (Panreac Química S.L.U., Barcelona, Spain). Enzymatic protein hydrolysates were produced using the endoprotease Alcalase®(subtilisin A) (Novozymes®, Bagsvaerd, Denmark). 2.2. Protein hydrolysates of grape seeds The protein hydrolysates of grape seed were obtained from the DGSM supplied by Alvinesa Natural Ingredients, S.A. (Daimiel, Ciudad Real, Spain), a grape seed pomace processing industry. Briefly, grape pomace is subjected to different processes in the industry to extract the compounds of interest (alcohol, tartaric salts, phenols, etc.), washed, destemmed, and dried. The grape seeds are then separated from the pomace, dried, ground and granulated for subsequent organic extraction of its oil. The resulting residue corresponds to defatted grape seed meal (DGSM), which is desolventized to remove residual hexane (Mor- a-Garrido et al., 2022). The extraction process followed by enzymatic hydrolysis was carried out to obtain protein hydrolysates. Two different types of DGSM were considered, one from Tempranillo red variety (R), and another mainly from Air´ en white grapes (W) (Air´ en/Tempranillo 75:25, respectively). The DGSMs were milled and stored in plastic containers in the dark at room temperature until use. Protein hydrolysates were obtained according to the method described by Cejudo-Bastante et al. (2022). Briefly, DGSM/water (20:80 (w/v)) were submitted to alkaline protein extraction in a bioreactor (Biorreactor Bio Console ADI 1025 Applikon Biotechnology, Inc.) at room temperature and controlled agitation and pH (180 rpm, pH 10.5) for 3 h. After centrifugation of the obtained protein extract (14880 g, 4 min, 4 ◦C), the precipitate was discarded, and the supernatant was concentrated in a rotary evaporator (70 ◦C and under vacuum conditions, up to pH about 8). The protein concentrate remained in a water bath for 5 min at 80 ◦C to inactivate its endogenous enzymes (Mokni Ghribi et al., 2015). Subsequently, the protein concentrate was subjected to enzymatic hydrolysis under alkaline conditions with 0.6 v/v Alcalase, under optimal conditions (50 ◦C and pH 8.5) (Parrado et al., 2008). Two different hydrolysis time (1 and 4 h) were tested, considering short (S) and long (L) hydrolysis time, respectively. After hydrolysis, the enzyme was deactivated by subjecting the hydrolysates to a temperature of 80 ◦C for 10 min (Mokni Ghribi et al., 2015). Subsequently, the pH was reduced to 3.5 (the normal pH of wine) with HCl (37%), separating the non-soluble peptides by centrifugation (33600 g, 20 min, 4 ◦C). The supernatants were concentrated in a vacuum rotary evaporator and finally lyophilized for subsequent addition to wine. Four different protein hydrolysates were obtained from the two types of DGSM used: two from red DGSM and two from mostly white DGSM. These protein hydrolysates were previously chemically characterized in detail by Mora-Garrido, Escudero-Gilete, Heredia, and Cejudo-Bastante (2024). 2.3. Winemaking Winemaking was carried out in the facilities of the Bodegas Nuestra Se˜ nora del Socorro (Rociana del Condado, Huelva, Spain). This study was performed with red wine from Vitis vinifera cv. Syrah grapes grown in the Condado de Huelva Designation of Origin (DO), in south-western Spain. Approximately 600 kg of grapes were destemmed and crushed, and the resulting material was distributed in 4 stainless steel tanks of 220 L each, for maceration/fermentation process (150 kg per tank). Daily for 10 days, a manual punch down of the content was carried out during skin maceration until the alcoholic fermentation was complete. The wine was then separated by removing the solid parts and poured into 50 L stainless steel tanks. Standard oenological parameters (pH, total and volatile acidity, and free and total SO 2 ) were analyzed during the stabilization period using the official methods established by the European Union (EU, 2003). The analyses were performed in triplicate. Six months after fermentation, when color loss is usually experienced in warm climates, different experimental micro-vinification based on the addition of protein hydrolysates were performed. The four different hydrolysates obtained were added to the experimental wine at two different doses (1 g/L and 3 g/L), similar to that used in the addition of other protein sources in winemaking (Gordillo, Chamizo-Gonz´ alez, Gonz´ alez-Miret, &Heredia, 2021;Guadalupe, Palacios, &Ayestar´ an, 2007). In total, nine different types of wine were produced: CW, control wine without protein hydrolysates addition; WL1 and WL3, wines added with protein hydrolysates from white DGSM and long hydrolysis time, at 1 and 3 g/L, respectively; WS1 and WS3, wines added with protein hydrolysates from white DGSM and short hydrolysis time, at 1 and 3 g/L, respectively; RL1 and RL3, wines added with protein hydrolysates from red DGSM and long hydrolysis time, at 1 and 3 g/L, respectively; RS1 and RS3, wines added with protein hydrolysates from red DGSM and short hydrolysis time, at 1 and 3 g/L, respectively. The micro-vinifications were performed in triplicate. Wines were supervised A.B. Mora-Garrido et al. LWT 205 (2024) 116554 3 for five months after adding the hydrolysates, as this is, approximately, the period spent in the steel tanks until final bottling. 2.4. HPLC-DAD analysis of polyphenolic compounds The phenolic profile of the wines was obtained by HPLC separation, identification and quantification of the different phenolic compounds using an Agilent 1200 chromatographic system, equipped with a quaternary pump, and UV–vis diode array detector, an automatic injector and the ChemStation software (Palo Alto, CA). Samples were prefiltered through a 0.45- μ m nylon filter and analyzed in triplicate. Individual anthocyanins were determined according to the method described by Heredia et al. (2010). Two solvents were employed: A, acetonitrile-formic acid-water (3:10:87) and B, acetonitrile-formic acid-water (50:10:40). The elution profile was as follows: 0–10 min, 94% A-6% B; 10–15 min, 70% A-30% B; 15–25 min, 60% A-40% B; 25–35 min, 55% A-45% B; 35–40 min, 50% A-50% B; 40–42 min, 40% A-60% B; 42–43 min, 94% A-6% B. The flow rate was 0.8 mL/min, and the injection volume was 50 μ L. A reversed phase column Zorbax C 18 (250 ×4.6 mm, 5 μ m particle size), thermostatted at 38 ◦C, was used. The UV–vis spectra were recorded from 200 to 800 nm with 2.0 nm bandwidth. The different individual anthocyanins were quantified at 525 nm, using malvidin-3-glucoside as standard. Individual flavan-3-ols, flavonols, hydroxycinnamic acid derivatives (HCAD), and benzoic acids were identified and quantified using the method developed by Gordillo, Cejudo-Bastante, Rodríguez-Pulido, Lourdes Gonz´ alez-Miret, and Heredia (2013). A volume of 50 μ L was injected into a Zorbax C 18 column (250 ×4.6 mm, 5 μ m particle size), conditioned at 40 ◦C, with a flow rate of 0.63 mL/min. Solvent A was acetonitrile-formic acid-water (3:10:87), and solvent B was acetonitrile-formic acid-water (50:10:40). The elution profile was as follows: 0–5 min, 94% A-6% B; 5–15 min, 89% A-11% B; 15–20 min, 80% A-20%B; 20–25 min, 77% A-23% B; 25–30 min, 74% A-26 % B; 30–35 min, 60% A-40% B; 35–38 min, 50% A-50% B; 38–46 min, 40% A-60% B; 46 min, 94% A-6% B. UV–vis spectra were recorded from 200 to 800 nm with 2.0 nm bandwidth. Quantification of the individual phenolic compounds was performed at 280 nm (flavan-3-ols, and benzoic acids), 320 nm (HCAD), and 360 nm (flavonols) by comparing the areas and the retention times with the standards of gallic acid and catechin, caffeic acid, and quercetin, respectively. The total content of each family was calculated as the sum of individual polyphenolic compounds identified by HPLC. Total phenolic concentration (TP) was analyzed by the Folin-Ciocalteau method detailed by Singleton &Rossi (Singleton &Rossi, 1965). 2.5. Copigmentation and polymerization of anthocyanins To determine the contribution of copigmentation to the total color of the wine at pH 3.6, the percentage of copigmented anthocyanins (% CA) and the percentage of polymerized anthocyanins (% PA) were calculated (Boulton, 1996). For this, different measurements of absorbance at 520 nm were made in an Agilent 8453 UV–vis spectrophotometer (Agilent Technologies, Palo Alto, CA). All measurements were taken in triplicate. The total color of the wine (A acet ) was obtained at pH 3.6, by adding 20 μ L of acetaldehyde to 2 mL of wine; after 45 min, the 520 nm absorbance was measured using a 2 mm path length glass cell. The color of wine without the effect of copigmented anthocyanin (A 20 ) corresponded to the absorbance of the wine diluted 1:20 (v/v) with a model wine solution (12% (v/v) ethanol, 5 g/L tartaric acid, 11.48 g/L sodium chloride, and adjusted to pH 3.6), measured using a 10 mm path length glass cell. A third measurement was made by adding 10 μ L of 2% SO 2 (w/v) to the wine samples adjusted at pH 3.6 (A SO2 ) in a 2 mm path length glass cell. All absorbances were converted to a 1 mm path length to be adequately applied in the following equations for the %CA and %PA calculations: % Copigmentation =[(A acet - A 20 ) / A acet ]×100 % Polymerization =(A SO2 / A acet )×100 2.6. Spectrophotometric color measurement An Agilent 8453 UV–vis spectrophotometer (Agilent Technologies, Palo Alto, CA) was used to perform color measurements. Readings were taken over the entire visible spectrum (380–770 nm) at constant intervals (Δλ =2 nm) in triplicate, using 2 mm path length glass cells and distilled water as a reference. The original software CromaLab©was employed to determine the CIELAB color parameters (L*, a*, b*, C* ab and h ab ) (Heredia, ´ Alvarez, Gonz´ alez-Miret, &Ramírez, 2004), following the recommendations of the Commission Internationale de l’Eclairage, CIE (CIE, 2004): the CIE 1964 10◦Standard Observer and the CIE Standard Illuminant D65. The color differences between pairs of samples were calculated as the Euclidean distance between two points in the three-dimensional CIELAB color space using the CIE 1976 color difference formula (ΔE* ab ): ΔE* ab =[(ΔL*) 2 +(Δa*) 2 +(Δb*) 2 ] 1/2 . In addition, the relative contributions of lightness (% ΔL), chroma (% ΔC) and hue (% ΔH), that makes a given color difference (ΔE* ab ) expressed as percentages, were calculated as follows: %ΔL=[(ΔL*) 2 /(ΔE* ab ) 2 ]×100 %ΔC=[(ΔC* ab ) 2 /(ΔE* ab ) 2 ]×100 %ΔH=[(ΔH) 2 /(ΔE* ab ) 2 ]×100 being ΔHmathematically deduced from: ΔH=[(ΔE* ab ) 2 −(ΔL) 2 − (ΔC) 2 ] 1/2 2.7. Sensory evaluation Wines after 5 months from the addition of the protein hydrolysates were sensorially evaluated. The objective was to assess the effect of treatments, thus, treated and untreated wines were evaluated together. The samples were evaluated in triplicate; therefore, 9 wines were sensorially analyzed per tasting session. A panel of 15 judges trained in the sensory analysis of wines (nine women and six men, aged between 28 and 60) participated in the wine tasting. The sensory analysis chamber was designed according to the UNE-ISO 4121:2006 standard. A constant volume of 30 mL of each wine was evaluated in tasting glasses at 18–20 ◦C (ISO 3591:1977). All evaluations were conducted from 10:30 a.m. to 1:00 p.m. Ordinal scales were used for the evaluation, according to 5 descriptors of the visual phase: lightness, color intensity, hue, limpidity and acceptability. They were asked to identify the specific descriptors and rate the perceived intensity of every sensory descriptor on a 10-point scale, where 0 is not perceived (absence) and 10 is the maximum intensity perceived. All sensory evaluations were performed according to the Spanish and International Standards (UNE-ISO 6658:2019). 2.8. Statistical analysis Statistica v.8.0 software (StatSoft Inc., 2007) was used to perform all statistical analyses. Univariate analysis of variance ANOVA was applied to establish statistical differences between the different samples. Additionally, a principal components analysis (PCA) was carried out to highlight the main contributors to variance. 3. Results and discussion Regarding enological parameters, Syrah wines performed an adequate development of the alcoholic and malolactic fermentations A.B. Mora-Garrido et al. LWT 205 (2024) 116554 4 (density 998 g/L and malic acid 0.25 g/L). Volatile acidity was below the limit established by the EU (1.2 g/L), and optimal values of the free and total sulfur dioxide content were reported (30 and 70.4 mg/L, respectively). Tables 1–3show the total and individual phenolic content of the wines, belonging to different phenolic families (anthocyanins, benzoic acids, flavan-3-ols, HACD, and flavonols), percentages of copigmentation and polymerization, and CIELAB parameters (L*, C* ab and h ab ), at one and five months after adding the protein hydrolysates. Fig. 1 shows the evolution of the CIELAB colorimetric parameters of all red wines. Several phenolic compounds have been identified in the wines studied, including anthocyanins (non-acylated, acetylated, and p-cou- maroylated derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin), benzoic acids (gallic acid), hydroxycinnamic acid derivatives (t-GRP, c-GRP, t-caftaric, t-coutaric, c-coutaric, p-coumaric, and caffeic acid), monomeric flavan-3-ols ((+)-catechin and (−)-epi- catechin), and flavonols (quercetin in their 3-glucuronide and glucoside forms, and myricetin, laricitrin, kaempherol, isorhamnetin, and syringetin in the 3-glucoside form). All of them are the expected and wellknown compounds normally present in red wines (Castillo-Mu˜ noz, G´ omez-Alonso, García-Romero, &Hermosín-Guti´ errez, 2007;Cejudo-- Bastante, P´ erez-Coello, &Hermosín-Guti´ errez, 2011;G´ omez-Alonso, García-Romero, &Hermosín-Guti´ errez, 2007;Gordillo, L´ opez-Infante, Ramírez-P´ erez, Gonz´ alez-Miret, &Heredia, 2010). The content of total and individual phenolics, as well as the percentages of copigmentation and polymerization, and the CIELAB colorimetric parameters of all wines were in agreement with other authors (Cejudo-Bastante et al., 2016). 3.1. Evolution of treated wines over time Regarding the evolution of wines over time (differences between one and five months after hydrolysates addition), the anthocyanin and phenolic compounds evolved as is typical for red wines during the stabilization period (Cliff, Stanich, Edwards, &Saucier, 2012). Briefly, the content of anthocyanins, both total and individual, and most individual flavonoid compounds decreased significantly (p<0.05) in all red wines (Tables 1 and 2). Similarly, a significant (p<0.05) general reduction in the amount of hydroxycinnamic acids was observed, except for caffeic acid (Table 2). On the contrary, the content of total flavan-3-ols, mainly due to (+)-epicatechin, increased over time. Furthermore, as expected, the percentage of polymerization increased significantly (p<0.05) over time in most of the studied wines, in parallel with the hue (h ab ) (Tables 1 and 3), as well as the percentage of copigmentation of the treated wines (Table 1). The values of C* ab (which can be related to chromatic intensity) improved significantly (p<0.05) in most of the wines evaluated, while the L* (lightness) values remained constant (except for RL1 and RL3 wines, which showed a significantly (p<0.05) increase over time (Fig. 1 and Table 3). Table 1 Means and standard deviations (n=3) of the total anthocyanins, benzoic acids, flavan-3-ols, HCAD, flavonols, and polyphenols (mg/L), and copigmented anthocyanins (%) and polymerized anthocyanins (%), of the studied wines, one (1 m) and five months (5 m) after the addition of the protein hydrolysates. Stage Total anthocyanins Total benzoic acids Total flavan-3- ols Total HCAD Total flavonols Total phenols (Folin- Ciocalteau) % CA % PA CW 1 m 327.44 ±13.22 96.47 ±4.19 52.76 ±6.07 59.29 ± 1.36 34.61 ±0.52 2321.65 ±90.95 4.12 ±1.44 75.44 ± 7.43 5 m 188.92 ±10.47 89.98 ±5.03 67.89 ±4.19 60.90 ± 1.51 31.52 ±1.06 2401.07 ±72.88 5.79 ±0.17 80.67 ± 5.15 WL1 1 m 308.83 ±10.48 98.29 ±1.74 50.82 ±2.86 58.89 ± 0.75 30.96 ±1.91 2459.85 ±63.25 6.78 ±0.28 72.00 ± 0.78 5 m 195.63 ±0.85 94.50 ±1.13 61.08 ±4.79 61.42 ± 1.31 31.17 ±1.85 2295.97 ±284.06 7.30 ±1.02 77.40 ± 1.00 WL3 1 m 262.29 ±13.06 95.75 ±6.71 50.26 ±0.53 58.78 ± 0.50 30.89 ±0.34 2782.55 ±35.65 4.95 ±0.39 75.00 ± 1.27 5 m 161.35 ±3.32 97.24 ±1.22 57.27 ±2.55 62.74 ± 0.86 32.30 ±2.18 2267.37 ±123.92 6.73 ±0.99 79.34 ± 1.08 WS1 1 m 296.53 ±5.11 100.27 ±0.18 52.46 ±0.54 57.25 ± 0.11 32.25 ±0.16 3386.60 ±31.10 1.36 ±0.18 71.24 ± 0.47 5 m 179.38 ±4.51 92.91 ±0.87 61.17 ±4.77 61.02 ± 0.94 30.55 ±1.58 2708.75 ±56.35 7.84 ±0.64 78.72 ± 2.05 WS3 1 m 281.18 ±6.53 88.43 ±0.35 51.83 ±0.34 57.50 ± 0.24 31.93 ±0.67 2780.20 ±0.30 3.08 ±0.38 71.23 ± 0.68 5 m 162.99 ±5.35 95.20 ±1.63 57.74 ±1.58 59.14 ± 3.56 30.79 ±2.32 2909.30 ±277.20 7.32 ±0.05 80.14 ± 2.78 RL1 1 m 318.28 ±9.02 103.93 ±1.76 52.63 ±0.97 56.97 ± 0.88 33.20 ±1.29 2831.80 ±4.90 2.83 ±0.05 70.45 ± 0.76 5 m 211.91 ±10.68 96.74 ±1.53 65.81 ±1.81 33.52 ± 1.28 32.72 ±2.25 2335.60 ±330.36 7.22 ±0.34 75.71 ± 0.78 RL3 1 m 303.89 ±12.69 107.31 ±0.75 53.47 ±0.67 58.20 ±029 32.20 ±0.23 3337.55 ±13.75 3.69 ±0.93 72.23 ± 1.41 5 m 205.24 ±10.33 99.72 ±1.79 68.78 ±4.06 34.96 ± 0.57 32.82 ±2.34 2262.20 ±204.49 9.83 ±0.47 79.73 ± 5.40 RS1 1 m 310.95 ±15.78 103.22 ±0.52 52.51 ±0.31 57.43 ± 0.11 32.85 ±0.27 3456.90 ±34.50 4.01 ±0.59 71.88 ± 2.11 5 m 198.11 ±13.79 96.82 ±2.09 67.13 ±1.51 60.30 ± 2.46 32.86 ±2.82 2594.80 ±326.84 9.25 ±0.42 75.37 ± 0.60 RS3 1 m 298.54 ±3.95 108.28 ±2.07 53.89 ±1.68 59.44 ± 0.81 33.21 ±0.84 2798.30 ±47.70 4.17 ±0.11 71.64 ± 0.73 5 m 180.82 ±10.73 100.97 ±2.04 61.73 ±1.18 50.45 ± 8.57 33.59 ±3.07 2951.60 ±29.00 11.36 ± 1.55 78.54 ± 2.23 HCAD, Hydroxycinnamic acid derivatives; CA, Copigmented anthocyanins; PA, Polymerized anthocyanins; CW, control wine without hydrolysates; WL1 and WL3, wines with addition of white DGSM protein hydrolysates, from long hydrolysis time, at 1 and at 3 g/L; WS1 and WS3, wines with addition of white DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L; RL1 and RL3, wines with addition of red DGSM protein hydrolysates, from long hydrolysis time, at 1 and 3 g/L; RS1 and RS3, wines with addition of red DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L. A.B. Mora-Garrido et al. LWT 205 (2024) 116554 5 Table 2 Mean concentration (mg/L) (and standard deviations) (n=3) of monomeric anthocyanins, benzoic acids, hydroxycinnamic acid derivatives (HACD), flavan-3-ols, and flavonols in wines, at one (1 m) and five months (5 m) after the addition of the protein hydrolysates. CW WL1 WL3 WS1 WS3 RL1 RL3 RS1 RS3 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m Monomeric anthocyanins Delphinidin-3-glucoside 11.04 10.37 11.71 10.47 11.01 9.80 11.57 10.29 11.35 9.78 11.73 10.76 11.55 10.61 11.68 10.55 11.70 10.17 (1.64) (0.18) (0.16) (0.07) (0.22) (0.07) (0.11) (0.02) (0.10) (0.20) (0.21) (0.21) (0.23) (0.21) (0.37) (0.34) (0.10) (0.34) Cyanidin-3-glucoside 8.12 8.11 8.18 8.15 8.12 7.99 8.16 8.08 8.18 8.11 8.26 8.25 8.26 8.24 8.22 8.20 8.21 8.15 (0.22) (0.16) (0.02) (0.00) (0.03) (0.05) (0.01) (0.02) (0.03) (0.03) (0.03) (0.06) (0.07) (0.04) (0.04) (0.09) (0.03) (0.02) Petunidin-3-glucoside 17.76 12.61 17.08 12.65 15.41 11.05 16.74 11.95 16.18 11.13 17.31 13.40 16.82 13.12 17.11 12.71 16.95 11.84 (0.53) (0.72) (0.37) (0.39) (0.51) (0.14) (0.25) (0.23) (0.28) (0.21) (0.35) (0.51) (0.50) (0.53) (0.73) (0.60) (0.16) (0.65) Peonidin-3-glucoside 20.24 13.69 19.08 14.02 16.99 12.18 18.56 13.19 18.16 12.34 20.57 14.98 19.35 14.49 19.76 14.33 18.95 13.15 (0.84) (0.57) (0.46) (0.17) (0.68) (0.17) (0.26) (0.27) (0.59) (0.41) (0.70) (0.49) (0.61) (0.50) (0.81) (0.70) (0.42) (0.75) Malvidin-3-glucoside 119.97 57.07 113.01 59.29 92.07 44.82 107.40 53.38 100.35 44.79 116.20 67.18 110.12 63.73 113.29 61.08 107.26 51.80 (5.60) (4.44) (4.15) (1.97) (5.63) (2.00) (1.84) (1.90) (3.00) (2.48) (3.47) (4.24) (5.38) (4.05) (7.01) (5.39) (1.43) (5.94) Petunidin-3-acetyl-glucoside 10.37 8.87 10.46 9.01 10.32 8.93 10.51 8.98 10.16 8.98 10.17 9.14 10.02 9.03 10.27 9.04 10.26 9.08 (0.28) (0.38) (0.06) (0.02) (0.04) (0.10) (0.05) (0.04) (0.10) (0.04) (0.07) (0.07) (0.06) (0.07) (0.07) (0.20) (0.11) (0.02) Peonidin-3-acetyl-glucoside 19.68 12.61 18.79 12.80 16.50 11.31 18.04 11.96 17.34 11.29 19.38 13.77 18.73 13.55 18.95 13.04 18.29 12.21 (0.68) (0.71) (0.57) (0.29) (0.67) (0.28) (0.28) (0.26) (0.33) (0.26) (0.47) (0.58) (0.65) (0.56) (0.76) (0.73) (0.24) (0.91) Malvidin-3-acetyl-glucoside 67.69 32.13 63.81 34.57 52.15 25.50 60.19 29.79 56.57 25.98 65.90 38.25 63.14 37.33 63.74 34.32 60.77 31.97 (3.24) (2.72) (2.57) (1.31) (3.38) (1.06) (1.56) (1.15) (1.57) (1.16) (2.23) (2.62) (3.18) (2.52) (3.88) (3.45) (0.91) (1.09) Petunidin-3-p-coumaroyl-glucoside 9.56 8.65 9.95 8.84 9.34 8.48 9.80 8.65 9.56 8.59 9.95 9.03 9.64 8.99 9.95 8.88 9.93 8.73 (1.21) (0.55) (0.18) (0.03) (0.17) (0.02) (0.03) (0.03) (0.06) (0.08) (0.14) (0.16) (0.19) (0.16) (0.22) (0.23) (0.11) (0.18) Peonidin-3-p-coumaroyl-glucoside 12.16 9.92 12.76 10.01 11.37 9.15 12.51 9.54 11.96 9.27 13.28 10.37 12.60 10.14 13.11 10.15 12.69 9.64 (2.85) (0.19) (0.52) (0.15) (0.38) (0.04) (0.20) (0.15) (0.12) (0.11) (0.39) (0.46) (0.43) (0.42) (0.42) (0.50) (0.22) (0.34) Malvidin-3-p-coumaroyl-glucoside 27.31 14.87 23.99 15.82 19.02 12.14 23.04 13.57 21.36 12.73 25.53 16.77 23.67 15.99 24.85 15.81 23.51 14.07 (1.61) (0.72) (1.58) (1.70) (1.43) (0.16) (0.65) (0.49) (0.55) (0.42) (1.15) (1.35) (1.46) (1.28) (1.48) (1.61) (0.73) (1.36) Benzoic acids Gallic acid 96.47 89.98 98.29 94.50 95.75 97.24 100.27 92.91 88.43 95.20 103.93 96.74 107.31 99.72 103.22 96.82 108.28 100.97 (4.19) (5.03) (1.74) (1.13) (6.71) (1.22) (0.18) (0.87) (0.35) (1.63) (1.76) (1.53) (0.75) (1.79) (0.52) (2.09) (2.07) (2.04) Monomeric flavan-3-ols (+)-catechin 35.39 32.86 33.05 29.86 31.72 26.75 34.60 26.61 32.79 31.07 34.59 31.17 33.82 34.55 34.68 35.17 35.74 34.01 (5.68) (1.18) (2.89) (1.27) (0.74) (0.47) (0.39) (2.39) (0.47) (2.94) (0.89) (1.73) (0.66) (3.89) (0.26) (1.75) (0.98) (1.09) (−)-epicatechin 17.36 35.02 17.76 31.22 18.55 30.52 17.85 34.56 19.04 26.67 18.04 34.64 19.64 34.22 17.84 31.97 18.15 27.72 (0.39) (3.61) (0.04) (3.82) (0.31) (2.11) (0.19) (2.62) (0.14) (2.52) (0.09) (0.12) (0.05) (0.78) (0.06) (2.49) (1.00) (1.26) HACD trans-GRP 0.83 0.32 0.83 0.17 0.64 0.49 0.55 0.08 0.47 0.22 0.49 0.10 0.42 0.15 0.54 0.09 0.65 0.12 (0.10) (0.06) (0.03) (0.01) (0.06) (0.01) (0.02) (0.00) (0.02) (0.02) (0.04) (0.00) (0.01) (0.01) (0.01) (0.01) (0.13) (0.01) cis-GRP 4.09 <QL 4.35 0.25 4.03 0.20 2.81 0.21 2.79 0.12 2.98 0.30 2.79 0.30 3.12 0.27 3.44 0.26 (0.38) (0.04) (0.01) (0.63) (0.00) (0.10) (0.01) (0.08) (0.05) (0.17) (0.00) (0.08) (0.01) (0.03) (0.01) (0.61) (0.01) trans-Caftaric acid 32.87 32.31 32.63 32.58 32.64 32.75 32.30 32.30 32.70 30.48 32.85 9.50 33.04 9.51 32.61 32.86 33.10 24.47 (0.46) (1.19) (0.36) (0.73) (0.17) (0.62) (0.03) (0.55) (0.07) (2.02) (0.44) (0.92) (0.10) (0.06) (0.14) (0.69) (0.33) (7.84) trans-Coutaric acid 14.14 14.17 14.02 14.43 14.00 14.78 14.12 14.23 14.30 13.70 14.24 3.69 14.72 3.68 14.20 14.35 14.67 5.29 (0.21) (0.47) (0.15) (0.49) (0.42) (0.21) (0.07) (0.24) (0.09) (1.01) (0.15) (0.30) (0.07) (0.46) (0.09) (0.32) (0.15) (1.95) cis-Coutaric acid 3.00 2.99 2.91 2.95 2.92 2.99 2.95 2.87 2.95 2.69 2.96 0.74 2.99 0.68 2.97 2.91 2.99 2.06 (0.02) (0.09) (0.02) (0.08) (0.11) (0.04) (0.02) (0.06) (0.03) (0.20) (0.03) (0.08) (0.01) (0.07) (0.04) (0.01) (0.02) (0.75) p-Coumaric acid 4.37 3.37 4.16 3.62 4.56 4.04 4.51 3.51 4.29 3.23 3.45 3.13 4.24 3.15 3.99 3.35 4.59 3.41 (0.24) (0.17) (0.22) (0.39) (0.35) (0.06) (0.07) (0.51) (0.13) (0.07) (0.11) (0.05) (0.05) (0.04) (0.10) (0.09) (0.13) (0.03) Caffeic acid n.d 7.73 n.d 7.43 n.d 7.49 n.d 7.81 n.d 8.69 n.d 16.06 n.d 17.48 n.d 6.47 n.d 14.84 (0.17) (0.20) (0.14) (0.10) (0.56) (0.04) (0.10) (1.36) (1.93) Flavonols Myricetin-3-glucoside 6.84 6.29 6.50 6.03 6.67 6.04 6.64 5.91 6.77 5.53 6.79 6.10 6.79 6.13 6.72 6.16 6.81 6.28 (0.77) (0.22) (0.41) (0.31) (0.34) (0.46) (0.08) (0.34) (0.52) (0.24) (0.26) (0.50) (0.30) (0.51) (0.05) (0.55) (0.19) (0.63) Quercetin-3-glucuronide 9.13 9.24 8.82 9.14 8.77 9.15 9.38 8.80 9.23 8.85 9.83 9.66 9.53 9.67 9.58 9.54 9.79 9.87 (1.15) (0.02) (0.56) (0.32) (0.08) (0.60) (0.03) (0.43) (0.05) (0.55) (0.48) (0.66) (0.21) (0.63) (0.09) (0.79) (0.25) (0.86) (continued on next page) A.B. Mora-Garrido et al. LWT 205 (2024) 116554 6 3.2. Effect on phenolic content of the addition of protein hydrolysates to red wines Although significant (p<0.05) differences between the control and treated wines were detected even from the first month after adding hydrolysates (Table S1), the discussion is focused on 5 months after hydrolysates addition since this is a common period for consider the bottling of wines and, consequently, their consumption. Table 4 reports the statistical analysis of wines with the addition of DGSM protein hydrolysates compared to the control wine after five months of stabilization time. The total anthocyanin content of all treated wines remained invariable and similar to control wines (CW), except for wines added with the highest amount of protein hydrolysates from white DGSM (WL3 and WS3), which displayed a significantly (p<0.05) lower content (Tables 1 and 4), mainly due to O-glucosides of monomeric anthocyanins, malvidin-3-acetyl-glucoside, and petunidin-3-p-coumaroyl-glucoside (Tables 2 and 4). This fact was already observed one month after the addition of the hydrolysates (Table S1). These results could be supported by the higher content of sulfur, aromatic, and hydrophobic amino acids present in protein hydrolysates from white DGSM (0.47 %, 24.07 % and 32.56 %, respectively) compared to that derived from red DGSM (0.32 %, 22.23 % and 26.13 %, respectively) (Mora-Garrido et al., 2024), because aromatic amino acids and peptides could establish interactions with anthocyanins (Chung et al;Ferrer-Gallego, Hern´ andez-Hierro, Rivas-Gonzalo, &Escribano-Bail´ on, 2011;Ferrer-Gallego et al., 2015; He et al., 2016) influencing their concentration in wine. Analogous to anthocyanin compounds behavior, WL3 and WS3 presented a significantly (p<0.05) lower amount of total flavan-3-ol compounds, and (+)-catechin and (−)-epicatechin, compared to CW, together with RS3 (Table 4 and S1). However, no differences in the percentage of polymerization were observed (Table 4). Ramos-Pineda et al. (2017) pointed out the existence of a cooperative behavior between (+)-catechin and (−)-epicatechin with proteins, thus certain peptides and amino acids present in WL3 and WS3 could alter the equilibrium of flavan-3-ol in wines (Gordillo et al., 2010). The total HACDs concentration decreased significantly (p<0.05) in Table 2 (continued) CW WL1 WL3 WS1 WS3 RL1 RL3 RS1 RS3 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m 1 m 5 m Quercetin-3-glucoside 5.49 3.32 5.19 3.42 5.15 3.54 5.48 3.24 5.39 2.81 5.64 3.31 5.39 3.54 5.67 3.56 5.68 3.66 (0.81) (0.15) (0.35) (0.21) (0.10) (0.29) (0.05) (0.20) (0.02) (0.49) (0.21) (0.31) (0.11) (0.29) (0.05) (0.34) (0.13) (0.39) Laricitrin-3-glucoside 3.29 3.46 3.12 3.37 3.06 3.36 3.14 3.32 3.12 3.21 3.23 3.48 3.08 3.45 3.19 3.50 3.23 3.53 (0.31) (0.19) (0.18) (0.16) (0.07) (0.23) (0.01) (0.17) (0.02) (0.25) (0.11) (0.24) (0.06) (0.24) (0.03) (0.26) (0.07) (0.26) Kaempherol-3-glucoside n.d 1.27 n.d 1.28 n.d 1.30 n.d 1.14 n.d 1.26 n.d 1.19 n.d 1.39 n.d 1.29 n.d 1.49 (0.16) (0.12) (0.14) (0.05) (0.13) (0.12) (0.21) (0.23) (0.07) Isorhamnetin-3-glucoside 3.97 3.60 3.60 3.63 3.56 4.11 3.80 3.67 3.68 4.03 3.85 3.97 3.67 3.87 3.83 3.96 3.83 3.88 (0.15) (0.17) (0.24) (0.35) (0.06) (0.27) (0.01) (0.20) (0.04) (0.46) (0.12) (0.21) (0.08) (0.32) (0.04) (0.30) (0.12) (0.49) Syringetin-3-glucoside 3.95 4.34 3.72 4.30 3.67 4.81 3.81 4.47 3.74 5.10 3.87 5.00 3.73 4.77 3.86 4.85 3.88 4.88 (0.26) (0.14) (0.18) (0.40) (0.04) (0.27) (0.01) (0.22) (0.02) (0.34) (0.12) (0.23) (0.08) (0.36) (0.04) (0.37) (0.10) (0.42) <QL, compound detected but below quantification limit; n.d., compound not detected; HCAD, Hydroxycinnamic acid derivatives; CW, control wine without hydrolysates; WL1 and WL3, wines with addition of white DGSM protein hydrolysates, from long hydrolysis time, at 1 and at 3 g/L; WS1 and WS3, wines with addition of white DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L; RL1 and RL3, wines with addition of red DGSM protein hydrolysates, from long hydrolysis time, at 1 and 3 g/L; RS1 and RS3, wines with addition of red DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L. Table 3 Mean values and standard deviations (n=3) of CIELAB parameters lightness (L*), chroma (C* ab ), and hue (h ab ), at one month (1 m) and five months (5 m) after the addition of protein hydrolysates. Stage L*C* ab h ab CW 1 m 75.35 ±0.21 21.62 ±0.13 9.37 ±0.28 5 m 74.67 ±0.22 22.87 ±0.07 16.16 ±0.76 WL1 1 m 76.06 ±0.97 21.54 ±0.68 9.00 ±1.09 5 m 74.86 ±0.48 22.81 ±0.50 17.27 ±0.45 WL3 1 m 73.70 ±0.17 23.40 ±0.76 8.56 ±0.57 5 m 73.65 ±0.31 23.33 ±0.93 20.89 ±0.46 WS1 1 m 75.86 ±0.46 21.78 ±0.25 8.67 ±0.40 5 m 75.11 ±0.27 22.93 ±0.14 15.99 ±0.45 WS3 1 m 75.26 ±0.15 22.59 ±0.18 11.03 ±0.64 5 m 75.54 ±0.61 23.92 ±0.19 22.94 ±0.67 RL1 1 m 76.42 ±0.09 20.52 ±0.11 15.26 ±0.22 5 m 76.72 ±0.05 20.89 ±0.02 19.67 ±0.31 RL3 1 m 76.13 ±0.44 20.16 ±0.19 17.25 ±1.04 5 m 77.37 ±0.11 20.33 ±0.05 20.21 ±0.60 RS1 1 m 75.37 ±0.83 22.12 ±0.55 13.10 ±0.26 5 m 75.63 ±0.52 22.59 ±0.05 19.79 ±0.97 RS3 1 m 75.17 ±0.43 22.64 ±0.26 12.36 ±0.31 5 m 74.90 ±0.40 23.97 ±0.40 22.32 ±0.38 CW, control wine without hydrolysates; WL1 and WL3, wines with addition of white DGSM protein hydrolysates, from long hydrolysis time, at 1 and at 3 g/L; WS1 and WS3, wines with addition of white DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L; RL1 and RL3, wines with addition of red DGSM protein hydrolysates, from long hydrolysis time, at 1 and 3 g/L; RS1 and RS3, wines with addition of red DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L. A.B. Mora-Garrido et al. LWT 205 (2024) 116554 7 wines with the addition of protein hydrolysates from red DSGM with respect to CW, regardless of the dose and the hydrolysis time (RL1, RL3, and RS3) (Table 4), fact that was already detected after the first month (Table S1). Considering the HACD individually, these wines illustrated significantly (p<0.05) lower concentrations of tartaric esters of hydroxycinnamic acids (t-caftaric, t-coutaric, and c-coutaric acids) compared to their respective acids. Moreover, a significantly (p<0.05) higher amount of c-GRP was also observed in all treated wines compared to CW, to the detriment of the lower content of t-GRP (Tables 2 and 4). Regarding benzoic acids, wines with the addition of hydrolysates from red DSGM (RL3 and RS3) at the highest amount displayed a significantly (p<0.05) higher concentration of gallic acid compared to CW, even at the first month after addition (Table S1). In agreement with Cejudo-- Bastante, Hermosín-Guti´ errez, and P´ erez-Coello (2012), and Cejudo-- Bastante, P´ erez-Coello, and Hermosín-Guti´ errez (2010), these findings led us to think that small-scale controlled reactions from different nature, namely hydrolysis and isomerization, could be occurring in treated wines, above all those derived from red DSGM. In the absence of differences in the flavonol profile (Tables 1, 2 and 4), it can be affirmed that there is no interaction between them and the amino acids and peptides added to the wines. Taking into account the similar amount of anthocyanins compared to CW, the significant (p<0.05) increase in the copigmentation percentages of wines due to the addition of protein hydrolysates from red DGSM (Tables 1 and 4) could be explained by the higher content of copigments, viz. the colorless gallic acid or the presence of other compounds with planar polarizable nuclei in the protein hydrolysates from red DGSM (Fernandes, Br´ as, Mateus, &de Freitas, 2014;Ozdal, Capanoglu, & Altay, 2013). Statistical study was performed considering independently the three factors: type of row material, hydrolysis time and dose applied to winemaking. As can be seen in Table 5, the main conditioning factors in the phenolic content of red wines was the type of defatted grape seed meal used to obtain protein hydrolysates, followed by the protein hydrolysates dose. Regarding the type of raw material, the addition of red DSGM protein hydrolysates provided wines with a significantly (p<0.05) higher amount of total and individual anthocyanins together with gallic acid, (+)-catechin, and some HACD (c-GRP and caffeic acid) (Tables 1, 2 and 5). Consistent with these findings, these wines also displayed significantly (p<0.05) higher percentages of copigmentation (Tables 1 and 5), which was in agreement with the higher content of anthocyanins and phenolic-derived copigments (gallic acid and (+)-catechin), or with the existence of other compounds with planar polarizable nuclei (Fernandes et al., 2014;Ozdal et al., 2013). Additionally, those wines showed a significantly (p<0.05) lower content of t-GRP and esters of HACD (Tables 2 and 5). Considering the dose, high amounts of protein hydrolysates (3 g/L) led to a significantly (p<0.05) higher amount of gallic acid in the resulting wines, which could act as copigment and had influenced in the increase in the percentages of copigmentation (Tables 1, 2 and 5). The hydrolysis time to obtain the protein hydrolysates did not have a remarkably influence on the phenolic composition of the resulting wines (Tables 1, 2 and 5). 3.3. Effect on the color of red wines after the addition of protein hydrolysates With regard to CIELAB parameters of the treated wines compared to CW, the addition of 3 g/L protein hydrolysates from 1-h hydrolysis regardless of the type of grape seed (WS3 and RS3) exhibited an improvement in the intensity of the color in red wines, in the light of the significantly (p <0.05) higher values of chroma (C* ab ) (Fig. 1;Table 3), which is of particular importance in regions with warm climate. This trend was already exhibited from the first month (Tables 3 and 4 and S1). Similar behaviors were previously found in wines after the pre- Fig. 1. Evolution of CIELAB parameters (means, n =3) during the five months after the addition of the protein hydrolysates. CW, control wine without hydrolysates; WL1 and WL3, wines with addition of white DGSM protein hydrolysates, from long hydrolysis time, at 1 and at 3 g/L; WS1 and WS3, wines with addition of white DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L; RL1 and RL3, wines with addition of red DGSM protein hydrolysates, from long hydrolysis time, at 1 and 3 g/L; RS1 and RS3, wines with addition of red DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L. A.B. Mora-Garrido et al. LWT 205 (2024) 116554 8 fermentative addition of protein hydrolysates from grape seeds (Cejudo-Bastante et al., 2016). Although an increase in tonality was also observed with respect to CW in these wines, which remained within the range of tones of other red wines, even with lower values than those of aged wines (Cejudo-Bastante et al., 2016;Gordillo et al., 2013). In fact, after a sensory evaluation by an expert panel, based on lightness, color intensity, hue, limpidity and acceptability parameters, all red wines were well accepted (acceptability rates greater than 7 over 10). However, the addition of protein hydrolysates from red DGSM and 4-h hydrolysis, regardless of concentration (being the wines RL1 and RL3) caused a lightning effect compared to CW (Fig. 1,Table 3), which was evidenced by the significantly (p<0.05) higher values of L* and lower of C* ab (Table 4). The chromatic intensity of these wines was already significantly (p<0.05) lower than CW from the first month of the addition (Table 3 and S1). This fact could be due to the formation of agglomerates protein-phenolic compound, leading to their precipitation (Charlton et al., 2002;Cliff et al., 2012). Furthermore, significantly (p< 0.05) higher hue values (h ab ) were observed in these wines (Tables 3 and 4), finding that it could be related to the chemical reactions, contributing to obtain more evolved color wines. Wines with these characteristics have properties more similar to aged wines (Cejudo-Bastante, Hermosín-Guti´ errez, &P´ erez-Coello, 2011). In the case of color, similar influence was observed among wines according to the factors studied (type of raw material, dosage and hydrolysis time) (Table 5). Regarding the raw material, the type of DGSM significantly (p< 0.05) influenced the color properties, showing significantly (p<0.05) higher values of lightness (L*) and hue (h ab ) the wines with the addition of protein hydrolysates from red DGSM (Fig. 1), although they showed a significant (p<0.05) increase in the percentage of copigmentation (Fig. 1) (Tables 1, 3 and 5). Regarding the dose, the wines with the highest dose of hydrolysates (3 g/L) showed a significantly (p<0.05) higher percentage of polymerization of their anthocyanins, which could be in concordance with the increase in hue (Fig. 1) (Tables 1, 3 and 5). Considering the hydrolysis time, the wines with addition of less hydrolyzed proteins (1 h) resulted in significantly (p<0.05) higher values of chroma (C* ab ) and percentage of copigmentation (Fig. 1) (Tables 1, 3 and 5). 3.4. PCA analysis Based on the results obtained from ANOVA, non-supervised pattern recognition statistical analysis (Principal Component Analysis) was applied to data corresponding to five months after adding protein hydrolysates to red wines, since they could better exhibit the influence of Table 4 p-Values of comparison between wines with addition of peptide hydrolysates and control wine, after five months of the addition of the protein hydrolysates. WL1 WL3 WS1 WS3 RL1 RL3 RS1 RS3 Total monomeric anthocyanins 0.4496 0.0181* 0.4195 0.0394* 0.0751 0.2293 0.5330 0.2839 Delphinidin-3-glucoside 0.5852 0.0481* 0.6640 0.0834 0.2438 0.5495 0.6017 0.5536 Cyanidin-3-glucoside 0.3816 0.0012* 0.0636 0.2505 0.6221 0.5968 0.9524 0.2506 Petunidin-3-glucoside 0.9391 0.0186* 0.1515 0.0312* 0.4035 0.6557 0.9746 0.2136 Peonidin-3-glucoside 0.4566 0.0205* 0.2614 0.0841 0.0589 0.2871 0.3312 0.4382 Malvidin-3-glucoside 0.5316 0.0203* 0.3038 0.0300* 0.0667 0.2590 0.4960 0.3092 Petunidin-3-acetyl-glucoside 0.8273 0.4321 0.7032 0.7854 0.5174 0.8583 0.7360 0.8539 Peonidin-3-acetyl-glucoside 0.7167 0.1202 0.4097 0.1325 0.1945 0.2804 0.6867 0.4315 Malvidin-3-acetyl-glucoside 0.4157 0.0199* 0.4431 0.0443* 0.0339* 0.0592 0.2592 0.9340 Petunidin-3-p-coumaroyl-glucoside 0.6155 0.0163* 0.1395 0.1111 0.8443 0.8135 0.5484 0.2865 Peonidin-3-p-coumaroyl-glucoside 0.7601 0.0907 0.3847 0.1932 0.5040 0.7116 0.8371 0.3365 Malvidin-3-p-coumaroyl-glucoside 0.6383 0.1090 0.4401 0.2928 0.3918 0.7072 0.3777 0.4478 Total benzoic acids 0.4390 0.0500 0.9654 0.1052 0.0602 0.0017* 0.0714 0.0005* Gallic acid 0.4390 0.0500 0.9654 0.1052 0.0602 0.0017* 0.0714 0.0005* Total flavan-3-ols 0.0753 0.0084* 0.0632 0.0109* 0.4955 0.7107 0.6079 0.0681 (+)-catechin 0.3374 0.0198* 0.0233* 0.9169 0.6008 0.6018 0.5572 0.5166 (−)-epicatechin 0.0594 0.0372* 0.4111 0.0004* 0.2232 0.4641 0.0943 0.0025* Total HACD 0.9202 0.7359 0.9701 0.7524 0.0002* 0.0002* 0.9630 0.0163* trans-GRP 0.0002* 0.0002* 0.0002* 0.0005* 0.0002* 0.0002* 0.0002* 0.0001* cis-GRP 0.0001* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* 0.0002* trans-Caftaric acid 0.9984 0.9948 0.9986 0.8242 0.0002* 0.0002* 0.9578 0.0446* trans-Coutaric acid 0.9415 0.8338 0.9759 0.4423 0.0002* 0.0002* 0.9658 0.0001* cis-Coutaric acid 0.9031 0.9775 0.9682 0.5639 0.0002* 0.0002* 0.9587 0.0146* p-Coumaric acid 0.5982 0.0271* 0.7685 0.6815 0.8050 0.7040 0.8688 0.8817 Caffeic acid 0.9109 0.7513 0.9210 0.4266 0.0001* 0.0002* 0.3594 0.0002* Total flavonols 0.8599 0.9140 0.8729 0.7875 0.9239 0.9596 0.9802 0.9270 Myricetin-3-glucoside 0.9942 0.9872 0.9764 0.7850 0.9899 0.9793 0.9487 0.9928 Quercetin-3-glucuronide 0.9820 0.8738 0.9173 0.9956 0.6999 0.8365 0.5620 0.7467 Quercetin-3-glucoside 0.7126 0.8142 0.9331 0.7515 0.9583 0.6686 0.8708 0.7454 Laricitrin-3-glucoside 0.8842 0.9506 0.9742 0.9698 0.9213 0.9619 0.9784 0.9843 Kaempherol-3-glucoside 0.9269 0.9962 0.5589 0.9888 0.5203 0.9360 0.9920 0.5996 Isorhamnetin-3-glucoside 0.9150 0.5868 0.9640 0.3076 0.8078 0.7558 0.7733 0.8377 Syringetin-3-glucoside 0.8872 0.3588 0.6266 0.0627 0.2588 0.2973 0.3877 0.4070 Total phenols (Folin-Ciocalteu) 0.7477 0.8172 0.4029 0.1340 0.5996 0.8921 0.4607 0.1317 % CA 0.1825 0.2048 0.0913 0.2276 0.1406 0.0008* 0.0026* 0.0002* % PA 0.8859 0.8589 0.8773 0.6103 0.6140 0.8636 0.6824 0.6943 L*0.5682 0.0059* 0.5330 0.3244 0.0003* 0.0002* 0.0757 0.7488 C* ab 0.8687 0.4014 0.8645 0.0379* 0.0004* 0.0002* 0.7087 0.0426* h ab 0.0551 0.0002* 0.7474 0.0002* 0.0002* 0.0001* 0.0002* 0.0002* Asterisks denote significant differences at p<0.05 by ANOVA test (Student-Newman-Keuls). HCAD, Hydroxycinnamic acid derivatives; CA, Copigmented anthocyanins; PA, Polymerized anthocyanins; CW, control wine without hydrolysates; WL1 and WL3, wines with addition of white DGSM protein hydrolysates, from long hydrolysis time, at 1 and at 3 g/L; WS1 and WS3, wines with addition of white DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L; RL1 and RL3, wines with addition of red DGSM protein hydrolysates, from long hydrolysis time, at 1 and 3 g/L; RS1 and RS3, wines with addition of red DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L. A.B. Mora-Garrido et al. LWT 205 (2024) 116554 9 the addition of protein hydrolysates. Fig. 2 shows the PCA analysis performed to determine the main parameters that establish the differences between the red wines studied. Two significant principal components (PCs) were identified using the Kaiser criterion (eigenvalues >1), which explained 70.43% of the total variance. The first PC, PC-1, grouped the wines according to the type of grape seed meal used to obtain protein hydrolysates. Wines derived from the addition of white DGSM protein hydrolysates (W) and CW were located on the positive side of the axis, which contains total polyphenols, individual and total HACD, C* ab , and % PA, with a positive sign. On the contrary, wines with the presence of protein hydrolysates from red DGSM (R) showed negative signs in PC-1, where contribute the rest of the parameters studied (anthocyanins, flavan-3-ols, flavonols, % CA, and L*). PC-2 separated wines according to the dose of protein hydrolysate added. The wines with the addition of 3 g/L of hydrolysates were placed in the negative part of the axis, which were mainly characterized by the presence of flavonols and benzoic acids, together with C* ab ,h ab, and % CA. In contrast, wines with lower doses of hydrolysates (1 g/L), together with the control wine, were located on the positive side of the axis, being their phenolic content the main factor in this distribution. Finally, the hydrolysis time applied to obtain the protein hydrolysates did not have a defined pattern, therefore this factor was not influential in the differentiation of the wines (Table 5). 4. Conclusions This study is the first attempt at adding to wine soluble protein hydrolysates-based products from a residue of the grape pomace industry (defatted grape seed meal), in the stabilization period of winemaking. The implementation of wines with protein hydrolysates this residue could be a promising technique for the oenological industry as a non-animal alternative to traditional and non-grape derived stabilizers agents. Considering different factors related to the product, the obtaining methodology and the winemaking application, the results obtained suggest that the addition of 3 g/L of protein hydrolysate from 1-h hydrolysis, regardless of the type of grape seed (from white or red grape varieties), stabilizes color over time in red wines from warm climate regions. Although this research could provide new insights to improve the production of superior quality red wines made in warm climates, it would be needed further research to assess more accurately the effectivity of grape seed-derived protein hydrolysates as stabilization agents in winemaking. Funding sources This research was supported by MCIN/AEI/10.13039/ Table 5 ANOVA analysis applied to all the studied parameters. Type of grape seed meal Protein hydrolysates dose Hydrolysis time F p F p F p Total monomeric anthocyanins 6.4610 0.0062* 2.8330 0.0804 1.0810 0.3566 Delphinidin-3-glucoside 4.5200 0.0226* 4.0100 0.0327* 4.5550 0.0221 Cyanidin-3-glucoside 12.0000 0.0003* 1.0000 0.4014 0.4000 0.7009 Petunidin-3-glucoside 5.0930 0.0152* 2.9410 0.0738 1.2800 0.2980 Peonidin-3-glucoside 6.1160 0.0077* 3.4170 0.0510 0.8400 0.4452 Malvidin-3-glucoside 5.6315 0.0106* 3.4986 0.0479* 1.1247 0.3427 Petunidin-3-acetyl-glucoside 4.2000 0.0282* 0.7000 0.4977 0.4000 0.6545 Peonidin-3-acetyl-glucoside 6.9190 0.0047* 1.5840 0.2277 1.2740 0.2997 Malvidin-3-acetyl-glucoside 8.1220 0.0023* 1.8218 0.1853 1.1317 0.3405 Petunidin-3-p-coumaroyl-glucoside 7.0500 0.0043* 1.9400 0.1669 1.2700 0.2994 Peonidin-3-p-coumaroyl-glucoside 5.2550 0.0136* 2.6800 0.0908 0.6380 0.5376 Malvidin-3-p-coumaroyl-glucoside 4.3390 0.0258* 2.6265 0.0948 0.8114 0.4571 Total benzoic acids 10.0200 0.0008* 10.1600 0.0007* 1.9100 0.1715 Gallic acid 10.0200 0.0008* 10.1600 0.0007* 1.9100 0.1715 Total flavan-3-ols 12.4370 0.0002* 2.5540 0.1006 2.0300 0.1552 (+)-catechin 11.0260 0.0005* 0.4990 0.6140 0.6540 0.5300 (−)-epicatechin 2.6220 0.0952 5.9480 0.0086* 3.8660 0.0364* Total HACD 10.7948 0.0005* 0.7223 0.4968 2.6019 0.0968 trans-GRP 4.9587 0.0167* 5.9203 0.0088* 3.4940 0.0481* cis-GRP 59.8103 0.0000* 22.3932 0.0000* 24.5589 0.0000* trans-Caftaric acid 8.7612 0.0016* 0.8087 0.4582 3.2503 0.0580 trans-Coutaric acid 15.6418 0.0000* 1.5414 0.2363 1.3554 0.2785 cis-Coutaric acid 9.6368 0.0010* 0.9188 0.4138 2.7803 0.0838 p-Coumaric acid 4.6380 0.0208* 0.2520 0.7798 0.3220 0.7279 Caffeic acid 10.3395 0.0007* 1.9904 0.1605 1.5679 0.2310 Total flavonols 1.8070 0.1876 0.6780 0.5178 0.1130 0.8937 Myricetin-3-glucoside 1.2780 0.2986 0.2690 0.7667 0.2980 0.7453 Quercetin-3-glucuronide 4.1680 0.0292* 0.3350 0.7189 0.0710 0.9321 Quercetin-3-glucoside 1.2230 0.3135 0.5020 0.6119 0.1710 0.8441 Laricitrin-3-glucoside 1.6060 0.2234 0.0400 0.9606 0.0470 0.9539 Kaempherol-3-glucoside 0.7820 0.4698 3.4690 0.0490* 0.0734 0.9295 Isorhamnetin-3-glucoside 0.8080 0.4586 2.0700 0.1500 0.7920 0.4655 Syringetin-3-glucoside 2.2030 0.1342 2.5480 0.1011 1.4270 0.2614 Total phenols (Folin-Ciocalteau) 0.2380 0.7902 0.6050 0.5541 13.6650 0.0001* % CA 10.7386 0.0005* 3.5480 0.0462* 4.5550 0.0221* % PA 1.1100 0.3470 3.8600 0.0366* 0.0300 0.9737 L*8.4900 0.001851* 0.5700 0.5760 0.8000 0.4600 C* ab 3.4180 0.050974 0.4750 0.6280 5.3090 0.0131* h ab 4.0750 0.031219* 18.8320 0.0000* 2.5012 0.1050 Asterisks denote significant differences at p<0.05 by ANOVA test (Newman-Keuls test). HCAD, Hydroxycinnamic acid derivatives; CA, Copigmented anthocyanins; PA, Polymerized anthocyanins; CW, control wine without hydrolysates; WL1 and WL3, wines with addition of white DGSM protein hydrolysates, from long hydrolysis time, at 1 and at 3 g/L; WS1 and WS3, wines with addition of white DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L; RL1 and RL3, wines with addition of red DGSM protein hydrolysates, from long hydrolysis time, at 1 and 3 g/L; RS1 and RS3, wines with addition of red DGSM protein hydrolysates, from short hydrolysis time, at 1 and 3 g/L. A.B. Mora-Garrido et al.