Impact of Foliar Application of Winemaking By-Product Extracts in Tempranillo Grapes Grown Under Warm Climate: First Results

Abstract

The Tempranillo grape variety is the most widely cultivated red grape cultivar in Spain. However, it has been observed that this variety may not always exhibit the optimal colour properties in warm climates such as Andalusia (southern Spain). Several strategies have been proposed to enhance productivity and resilience to stress or improve the quality of both grapes and wines. The foliar application of agricultural plant extracts has been identified as a highly effective method of promoting the synthesis of secondary metabolites in the grapevine. The objective of the current research was to analyse the incidence of foliar application of different extracts from winemaking by-products (grape stems: GS, grape pomaces: GP and wine lees: WL) on Tempranillo grapevine leaves, studying the impact on the quality of the resulting wines. The oenological and colour characteristics of the wines were assessed to evaluate the impact of these extracts on wines during the winemaking process. The detailed composition of the wines revealed that GS and WL extracts led to significant differences in wine colour properties. Wines derived from grapes treated with both extracts exhibited heightened colour intensity, accompanied by discernible alterations in CIELab coordinates, with lower L* and higher a* and C*ab values in comparison to CT. These results are encouraging, and the foliar application of GS extracts at veraison appears to be a feasible alternative to enhance the colour of wines from red grape cultivars with colour difficulties in warm climates.

1. Introduction

The colour of wine is one of its key sensory attributes, and it is influenced by the extraction of pigments from grapes and technological processing during winemaking. The levels and composition of phenolic compounds in wines are influenced by the grapevine cultivar, environmental factors (including climate, soil conditions, or canopy management) and the technological processes employed [1].

The current focus of industry efforts and consumer preferences is on highly coloured wines. Consequently, Spanish warm climate regions have focused their production efforts on red wines with deep colour and hue. A wide range of environmental factors, including temperature, sunlight, and rainfall, have been demonstrated to significantly influence the yield of the vines and the quality of the berries [2]. Specifically, elevated summer temperatures have been shown to impact anthocyanin synthesis [3]. Furthermore, limited day–night temperature variation in summer or higher night temperatures [3,4] may exacerbate this situation.

In Andalusia, high temperatures and intense sunlight result in suboptimal conditions for the maturation of phenolic compounds. Consequently, the phenolic content of certain red grape varieties is found to be relatively low in comparison to those grown in colder climates [5]. This in turn leads to a reduction in the stability and intensity of the colour of the grapes [6,7].

Vineyards in southern Mediterranean regions are exposed to a combination of rising air temperatures, drought, and an increased frequency of extreme weather events (e.g., heatwaves) due to climate change. As most of the berry growth and ripening phases occur under these conditions, limitations in yield and quality are frequently observed.

In light of this scenario and vineyard sustainability objectives, novel strategies in vineyard management have been suggested. Among these strategies, the utilisation of biostimulant products has emerged as a promising and environmentally sustainable approach with the potential to diminish the reliance on synthetic fertilizers and pesticides in vineyards [8].

Biostimulants are defined as products composed of substances that promote physiological and biochemical processes in plants, thereby supporting their ability to adapt and maintain good agronomic yields and quality in adverse environmental conditions (including salinity, drought, high temperatures, and nutrient deficiencies). These substances are derived from a variety of sources, including humic substances, amino acids, and algae extracts [8]. However, due to the complexity of their composition, it is challenging to identify the specific active compounds [9]. In recent years, there has been a notable increase in the number of studies examining the use of biostimulants in grape production [10,11,12,13,14,15,16,17,18,19,20,21,22,23]. The most extensively explored effects triggered by biostimulant applications in grapevines are those related to defence mechanisms and the regulation of secondary metabolic pathways (including the biosynthesis of phenolic compounds [10,13,14,23]). The impact of foliar applications in vineyards on the synthesis of secondary metabolites in grape berries is dependent on several factors, including the specific substance applied, the dosage employed, the scheduling of applications, and the cultivar in question [17,18].

Alternative methods for obtaining biostimulants from agricultural by-products are currently under evaluation [19], such as vine shoot extracts [20]. Some studies have proposed a potential correlation between the application of phenolic compounds, whether synthetic [21,22] or natural [20], and the enhancement of grape and wine quality. Specifically, the utilisation of polyphenol-enriched biostimulants has been demonstrated to stimulate the phenylpropanoid pathway by inducing the activation of several enzymes, including polyphenol oxidase, lipoxygenase, phenylalanine ammonia lyase, and peroxidase (leading to the accumulation of phenolic compounds) [18,23]. In this regard, Ertani et al. [24] demonstrated that plant extracts from red grape skins, which are abundant in phenols, enhance the phenolic content of diverse crops, including maize, when employed as biostimulants. Furthermore, Sánchez-Gómez et al. [22] also evaluated the application of phenol-enriched vine shoot extracts as biostimulants on other grape varieties, which resulted in a notable increase in polyphenol and mineral content, as well as a change in the organoleptic profile of the wine resulting from the treated grapes.

Conversely, a considerable challenge confronting wine-producing regions concerns the development of sustainable solutions for the management of substantial volumes of waste generated during the harvest season. In this context, agroindustrial residues emerge as a substantial reservoir of bioactive compounds, including antioxidants and phenols, that can be utilised by farmers to enhance crop yield.

Spain, with its extensive vineyard area (954,734 ha in 2022, according to OIV statistics), is a prominent example of this. The winemaking process itself generates a range of solid by-products, representing up to 40% by weight, including grape stems from de-stemming, grape pomace from pressing, and wine lees from racking. Vineyard management also generates solid by-products, such as trimmed vine shoots from pruning. The potential applications of these solid by-products are receiving increased attention due to their promising properties and the growing environmental concerns that have emerged [25,26,27,28].

However, the economic value of these by-products is currently negligible, as they are often discarded or left in the field as organic matter [29]. The reuse of these by-products has the potential to serve as an alternative means of mitigating the environmental impact of winemaking and generating additional value through the commercial exploitation of extracts rich in beneficial compounds, such as phenolic compounds.

In this regard, there has been extensive investigation of the chemical composition of grape stems [30,31,32], grape pomaces [33,34,35,36], and wine lees [37,38,39] in terms of their phenolic content. Nevertheless, the exploitation of these potential active compounds in different applications requires their prior extraction.

Conventionally, such extracts are obtained using solid–liquid extraction and organic solvents. Green extraction methodologies aim to reduce energy consumption, allowing the use of alternative solvents and renewable natural products, and ensuring a safe and high-quality extract/product. Microwave-assisted extraction (MAE) has emerged as a highly efficient green extraction technique for obtaining polyphenol-enriched extracts from plant sources. Compared to other green extraction techniques such as ultrasound-assisted extraction or liquid-pressurised extraction, MAE offers important advantages. The efficiency of MAE is characterised by its ability to achieve higher extraction yields in a shorter time frame, a consequence of the effective heating and cell disruption induced by microwaves. This method also affords enhanced control over extraction parameters, resulting in improved reproducibility and scalability in industrial settings. Furthermore, MAE frequently involves a reduced volume of solvent, thereby aligning with sustainable practices by diminishing chemical consumption and waste generation [40].

In this study, three extracts of different winemaking by-products (grape stems: GS, grape pomaces: GP and wine lees: WL) were obtained by MAE and their application as possible plant biostimulants in grapevines grown under warm climate conditions was proposed. In order to achieve this objective, extracts of GS, GP, and WL by-products were applied to the foliage of Tempranillo grapevines at veraison, and the impact on the quality of the resulting wines was subsequently studied.

2. Materials and Methods

2.1. Winemaking By-Product Samples

Samples of winemaking by-products were obtained in accordance with conventional winemaking conditions from the Tempranillo grape variety in the experimental winery IFAPA—Centro Rancho de la Merced. Randomised selection was performed at the conclusion of the destemming, pressing, or racking stages in 2019, 2020, and 2021 vintages. Subsequently, the samples were refrigerated at 4 °C and oven-dried (60 °C for grape stems and grape pomaces) or lyophilised (wine lees) until reaching a constant weight (representing a loss ranging from 60% to 70% of the original weight). Thereafter, the samples were crushed in a milling device, mixed to obtain a homogeneous powder, and stored in vacuum-sealed bags at a controlled temperature until extraction.

2.2. Grapevines

The Tempranillo grape variety was selected due to its status as the most prominent red grape variety cultivated in Spain. The grapes from Vitis vinifera L. cv. Tempranillo used for the winemaking assays were cultivated in an experimental vineyard located in Jerez de la Frontera (IFAPA—Centro Rancho de la Merced, GPS: 36°43′46.1″ N 6°09′50.7″ W), in south-western Spain, under the climatic conditions typical of warm Spanish regions. The grapevines were spaced 2.30 × 1.20 m, grafted on 140R rootstocks, trained to a vertical trellis on a bilateral cordon, and managed using the standard viticultural practices of the region.

2.3. Extract Preparation

The extractions of samples from grape stems, grape pomaces, and wine lees were conducted using an Ethos One microwave digestion system (Milestone, Shelton, CT, USA) employing microwave technology. The power controller enabled the microwave radiation to be set at any level within the range of 0–100% of the nominal power (up to 1500 W). The time controller facilitated the adjustment of the microwave application duration over a broad range. The system is also equipped with a temperature controller that can monitor the temperature 20 times per second, enabling precise adjustment of microwave power output to the desired temperature. The final extraction conditions selected were as follows.

• For the extraction of grape stems, 10 g of ground sample, 80% ethanol in water as the extraction solvent, an extraction temperature of 25 °C, a system power of 750 W, and an extraction volume of 50 mL were selected. The extraction time was set to 15 min.
• For grape pomaces, 10 g of ground sample was selected, 50% ethanol in water was utilised as the extraction solvent, an extraction temperature of 100 °C, system power of 750 W, and an extraction volume of 50 mL were used. The extraction time was 30 min, with a preheating time of 5 min.
• For the extraction of wine lees, the procedure was repeated using 10 g of ground sample, 50% ethanol in water as the extraction solvent, an extraction temperature of 100 °C, system power of 750 W, and an extraction volume of 50 mL. The extraction time was reduced to 15 min, with a preheating time of 5 min.

Subsequent to the completion of the extraction process, the vessels were cooled at room temperature using an ice bath, with the exception of the grape stems. Subsequently, all the extracts obtained were subjected to a centrifugation process at 4000 rpm for a duration of 10 min in a Digicen 20-R centrifuge (Orto Alresa, Madrid, Spain). Thereafter, the extracts were concentrated under vacuum to a concentration of 5% ethanol, filtered under vacuum through a paper filter, and stored in refrigerated conditions at 4 °C until the day prior to field application.

2.4. Grapevine Treatments

Grape stem (GS), grape pomace (GP), and wine lees (WL) 5% ethanolic extracts were prepared using 0.05% (v/v) of the Agral adjuvant (Syngenta-España, Porriño, Spain). This is a surface-active wetting agent that is commonly utilised in foliar treatments. It is composed of an inert blend of polymers. The vineyards were treated with 250 mL of each formulation per plant by spraying locally over leaves, which were applied once at the 7th day post-veraison. This was in accordance with other reported foliar applications of biostimulants from phenol-enriched extracts of a comparable nature [22,41,42,43]. Moreover, the control sampling (CT) was conducted on 7 plants that were treated with water, 5% ethanol and the adjuvant, following the same protocol. The experimental plot was organised using a randomised complete block design (RCBD) consisting of three blocks of 30–36 vines each and four different foliar applications (GS, GP, WL, CT), with 7 vines per treatment and block (21 vines/treatment). Treatments were carried out when the environmental temperature was below 20 °C, between 7 and 9 a.m. The optimal harvest date was established after four weeks of monitoring the technological (sugar–acidity ratio) and phenolic ripening [44] (Table S1).

2.5. Winemaking Procedure

In order to evaluate the influence of the extracts on the quality of wines, grapes from each treatment (three replicates per treatment) were processed separately. For each treatment, three wines were obtained on a pilot scale (10 L stainless steel tanks). The Tempranillo grapes (15 kg per treatment and block) were harvested in August 2022, when the grape ripening parameters (according to the optimum technological sugar levels, pH, and acidity for Tempranillo grapes grown in the same warm climate zone) were reached [45,46,47]. The grapes from each of the selected vines were harvested in a heterogeneous manner, in accordance with good sanitary conditions. Any grapes exhibiting signs of insect damage, physical breakage, or other external damage were discarded. The grapes were then placed into 18 kg plastic boxes and transported to the experimental winery. Following this, the grapes were de-stemmed and crushed. A total of 50 mg/kg of SO₂ was added to each batch in the form of a 15% w/v liquid solution (Enartis, Trecate, Italy). pH in all batches was adjusted to 3.60 with tartaric acid (Enartis, Trecate, Italy). Alcoholic fermentation (AF) was started after yeasting (20 g/100 kg exogenous yeasts Saccharomyces cerevisiae var. cerevisiae, Enartis, Trecate, Italy). AF was developed as a conventional red winemaking process at controlled temperature (22 °C). The fermentation cap was punched down twice daily. Following the completion of the AF stage (10–12 days), the grape pomace was pressed, resulting in the production of a combination of free-run and pressed wines. These wines were then inoculated with 1 g/100 L Oenococcus oeni commercial lactic bacteria (Challenge Easy ML, Enartis, Trecate, Italy). The inoculated wines were then maintained at a temperature of 18 °C for a period of 15–20 days, during which malolactic fermentation (MLF) occurred. Upon the conclusion of the MLF process, the wines were subjected to a process of racking and cold stabilisation. Thereafter, the wines were bottled and stored for a period of 12 months. At each stage of the process, chromatic and other oenological parameters were determined.

2.6. Oenological Parameters

The parameters selected for the evaluation of the treatments in the final wines were studied according to the official methodologies proposed by the OIV (OIV, 2020) [48]: total acidity (OIV-MA-AS313-01), pH (OIV-MA-AS313-15), potassium (mg/L) (COEI-2-POTASS), ethanol (% v/v) (OIV-MA-AS312-01A), volatile acidity (g/L acetic acid) (OIV-MA-AS313-02), reducing sugars (g/L) (OIV-MA-AS311-01A), and organic acids (OIV-MA-AS313-04, OIV-MA-AS313-08, OIV-MA-AS313-05A, OIV-MA-AS313-10 and OIV-MA-AS313-07). Tannins, anthocyanins, and total polyphenol index (TPI) were measured by spectrophotometry according to Ribereau-Gayon’s 2006 [49] methodologies.

2.7. Colour and Colorimetric Measurements

Wine colour assays were performed by calculating the Glories colour index parameters (Glories [44]): colour intensity (CI) and hue or tint (H). Spectrophotometric absorbances were read at 420, 520, and 620 nm with a spectrophotometer (Lambda 25, Perkin-Elmer, Waltham, MA, USA) equipped with cuvettes with a path length of 1 mm. Colorimetric measurements were registered with a Konica-Minolta CM-3600d spectrophotometer (Osaka, Japan) using 2 mm-path-length glass cells and distilled water as a reference. The CIELab coordinates (L *, a *, b *, C *ab, and h* *ab) were determined using SpectraMagic v.3.61G software (Cyberchrome Inc., Minolta Co., Ltd., Osaka, Japan) and the CIE D65/10° illuminant/observer condition according to the OIV method.

2.8. Sensory Analysis

The descriptive sensory analysis was carried out by a panel of eight trained tasters from the staff of the IFAPA Rancho de la Merced Centre, with ages ranging from 35 to 65 years. The visual, olfactory, and taste profiles of wines were evaluated for different attributes according to the standard ISO 6564:1985 [50]. The panellists used a 10-point unstructured scale, ranging from 0 (no character) to 10 (very strong character), to evaluate the intensity of the attributes.

2.9. Statistical Software

The observations within each group were evaluated separately. Normal distribution for the dependent variables was confirmed using the Kolmogorov–Smirnov test (p > 0.05). Significant differences between variables were assessed by one-factor analysis of variance (ANOVA) and Tukey’s least significant difference test (LSD) considering 95% significance using Statistix software version 9.0 (Tallahassee, FL, USA). Finally, a multifactorial ANOVA was employed to evaluate the significant differences between the values assigned to each of the variables to be optimised, as well as to obtain the conditions that ensured the maximum concentration of these three parameters, which were different for each matrix.

3. Results and Discussion

The objective of this study was to investigate the potential of three different winemaking by-product extracts (grape stems—GS, grape pomaces—GP, wine lees—WL) as viticultural biostimulants when applied foliarly to Tempranillo grapevines. Figure 1 shows the experimental design used. The underlying hypothesis suggests that the implementation of these methodologies may potentially enhance the quality of the resulting wines, particularly in terms of their chromatic characteristics. Consequently, the study examined the effect of the treatment on grape quality at harvest and the quality of the resulting wines.

3.1. By-Product Extraction

First, an optimisation process was conducted to obtain extracts from grape stems, grape pomaces, and wine lees using microwaves. Specifically, three variables were studied for optimisation: the type of solvent (EtOH, EtOH/water 50:50 and EtOH/water 80:20), extraction temperatures (25, 40, 55, 70, 85 and 100 °C), and extraction times (1, 5, 10, 15 and 30 min). Total polyphenols, total flavonoids, and total flavanols were selected as response variables to be optimised. The total phenolic content (also known as total polyphenol index) of the extracts was spectrophotometrically measured at 280 nm, as described by Ribereau-Gayon et al. [49]. Total anthocyanins, total flavonoids, and total proanthocyanidins were additionally determined following Ribereau-Gayon’s [49], Yang et al.’s [51], and Treutter’s [52] methodologies, respectively.

A multifactorial ANOVA was employed to evaluate the significant differences between the values assigned to each of the variables to be optimised, as well as to obtain the conditions that ensured the maximum concentration of these four parameters considering a 95% level of confidence.

All extractions were performed at 750 W in triplicate. The ratio of sample to solvent was fixed at 10 g/50 mL, and magnetic stirring was selected for the extraction at a medium speed. The values of main extraction variables in the experiment carried out are shown in Figure 2. For grape stems, a significant increase in the total phenolic content, total flavonoids, and total flavonols was observed when using 80% EtOH as a solvent. Furthermore, the optimal conditions for recovery for all compounds were determined to be 25 °C extraction temperature and 15 min extraction time. In the case of grape pomaces, the highest extraction efficiency was achieved when 50% EtOH was used as the solvent. Additionally, the results indicated that the highest amount of target compounds was achieved using higher temperatures and longer extraction times. Furthermore, regarding the extraction of wine lees, the optimal yield for all the studied compounds was achieved using 50% EtOH as the extracting solvent. The use of an extraction temperature of 100 °C resulted in the highest content of all compounds, including total anthocyanins and total flavonoids. The findings indicated that most of the phenolic compounds present in the sample were extracted within 15 min. An increase in the extraction time from 15 to 30 min led to the same levels of recovery as 15 min. Consequently, an extraction time of 15 min was selected for these samples.

Subsequently, an evaluation of the chemical composition of the proposed by-products extracts obtained under selected conditions was conducted (see

Table 1. Phenolic characterisation of winemaking by-products extracts applied on assayed Tempranillo grapes.

Parameter	GS	GP	WL
Total phenolic content *	50.0 ± 5.7 b	315.6 ± 14.9 a	23.2 ± 2.0 b
Total flavanols **	37.1 ± 4.2 b	131.8 ± 12.6 a	14.3 ± 1.1 b
Total flavonoids **	46.2 ± 5.9 b	165.3 ± 9.6 a	18.7 ± 0.9 c
Total anthocyanins ***	0.9 ± 0.2 b	65.4 ± 7.7 a	2.0 ± 0.4 b

GS: grape stem extract; GP: grape pomace extract; WL: wine lees extract. *: mg/L gallic acid equivalent, **: mg/L catechin equivalent, ***: mg/L malvidin-3-glucoside equivalent. Different letters suppose significant differences at 95% confidence level.

). As expected, grape pomace extracts showed the highest phenolic content of all assayed by-products, ranging from 23.2 to 315.6 mg/L gallic acid equivalent. Similar trends were also registered for total flavanols (ranging from 14.3 to 131.8 mg/L catechin equivalent), total flavonoids (ranging from 18.7 to 165.3 mg/L catechin equivalent), and total anthocyanins (ranging from 0.9 to 65.4 mg/L malvidin-3-glucoside equivalent). In contrast, the WL extract was the poorest among all extracts tested.

As previously mentioned, there is little research focused on the use of biostimulants from agricultural by-products, so comparison with the literature is limited. Samuels et al. [53] evaluated the influence of Ecklonia maxima seaweed extract as a biostimulant in Cabernet Sauvignon, observing an increase in polyphenol concentration of 1.52 mg/L (from 9.67 mg/L in the control to 11.19 mg/L in the biostimulant treatment). Pardo-García et al. [41] conducted a study on the utilisation of oak extract as a biostimulant in Monastrell grapes, observing an increase of 9 mg/L in the concentration of total polyphenols six months following malolactic fermentation in comparison to the control. In this regard, it can be concluded that the increase in total polyphenol observed in the treatments is commensurate with the range of concentrations reported in the literature for the use of natural biostimulants derived from by-products of the agricultural industry.

3.2. Oenological Parameters of Grapes at Harvest

Tempranillo grapes from each treatment (GS, GP, WL), including the control samples (CT), were harvested at the optimal maturation moment, with the oenological parameters described in

Table 2. Oenological parameters in Tempranillo grapes on harvest day.

Parameter	CT	GS	GP	WL
Yield (kg/plant)	6.05 ± 0.72	5.92 ± 1.02	5.82 ± 1.01	5.89 ± 1.02
Baumé degree	12.6 ± 0.3	12.7 ± 0.3	12.5 ± 0.2	12.6 ± 0.3
Total acidity (g/L tartaric acid)	4.20 a ± 0.09	3.91 b ± 0.11	4.19 a ± 0.10	4.30 a ± 0.13
pH	3.38 ± 0.02	3.40 ± 0.07	3.36 ± 0.03	3.40 ± 0.07
Sugar/total acidity index	5.2 ± 0.1	5.2 ± 0.1	4.9 ± 0.2	4.9 ± 0.2

CT: control untreated grapes; GS: grapevines treated with grape stem extract; GP: grapevines treated with grape pomace extract; WL: grapevines treated with wine lees extract. Different letters within a row mean significant differences according to Tukey’s test (p-value less than 0.05).

. Grapes were harvested when the sugar-to-total-acidity ratio (Cillis and Odifredi index) in control grapes was closer to 5, which was the acceptable threshold for grape maturation in this variety and region. All treated grapes showed similar maturation index values. No significant differences were also recorded for Baumé degree or pH, with the GS treatment showing the lowest value for total acidity (Table 2). The grape yield was not significantly affected by the application of any treatment vs. CT.

The lack of differences in the ripening dates of Tempranillo grapes across treatments can be attributed to several factors. Firstly, the treatments, derived from vineyard residues, may have exerted similar effects on the physiological processes of the vines, such as photosynthesis and nutrient translocation, resulting in comparable ripening timelines. Secondly, uniform climatic conditions during the growing season may have minimised external variability, thereby further harmonising the maturation process. Furthermore, as the initial physiological status of the vines, including vigour and fruit load, was consistent across treatments, the impact on ripening speed may have been negligible, which is also supported by the known ability of phenolic compounds as scavengers for the removal of reactive oxygen species.

3.3. Wine Composition During Winemaking and Ageing Stages

3.3.1. Oenological Parameters

The resulting wines exhibited characteristics and values comparable to those produced from grapes grown in a similar warm climate region [44,45,46,54]. Oenological parameters are shown in

Table 3. Oenological characteristics of pressed and bottled wines.

	CT	GS	GP	WL
Pressing stage
Total acidity (g/L tartaric acid)	6.29 a ± 0.10	5.89 b ± 0.02	6.01 ab ± 0.03	5.90 b ± 0.02
pH	3.77 ± 0.03	3.83 ± 0.02	3.76 ± 0.02	3.83 ± 0.04
Tartaric acid (g/L)	2.34 ± 0.21	2.31 ± 0.14	2.25 ± 0.29	2.21 ± 0.17
Malic acid (g/L)	2.71 ± 0.23	2.53 ± 0.12	2.63 ± 0.08	2.65 ± 0.05
Potassium (mg/L)	1561 a ± 15	1470 b ± 52	1586 a ± 29	1761 c ± 52
Bottling stage
Ethanol (% v/v)	12.7 ± 0.2	13.2 ± 0.3	12.5 ± 0.3	13.0 ± 0.3
Total acidity (g/L tartaric acid)	5.44 ± 0.08	5.43 ± 0.03	5.37 ± 0.05	5.49 ± 0.11
pH	3.67 ± 0.02	3.65 ± 0.01	3.64 ± 0.02	3.66 ± 0.01
Volatile acidity (g/L acetic acid)	0.37 ± 0.04	0.36 ± 0.09	0.38 ± 0.07	0.39 ± 0.11
Glycerine (g/L)	8.26 ± 0.17	8.51 ± 0.23	8.11 ± 0.19	7.72 ± 0.29
Reducing sugars (g/L)	2.04 ± 0.252	2.04 ± 0.17	1.90 ± 0.16	2.38 ± 0.15
Acetic acid (g/L)	0.32 ± 0.02	0.31 ± 0.04	0.33 ± 0.02	0.35 ± 0.03
Citric acid (g/L)	0.21 ± 0.01	0.18 ± 0.00	0.14 ± 0.02	0.11 ± 0.01
Tartaric acid (g/L)	1.21 ± 0.08	1.24 ± 0.14	1.24 ± 0.17	1.11 ± 0.19
Malic acid (g/L)	0.14 ± 0.02	0.15 ± 0.03	0.13 ± 0.02	0.15 ± 0.01
Lactic acid (g/L)	2.02 ± 0.11	1.83 ± 0.07	1.99 ± 0.14	1.88 ± 0.16
Succinic acid (g/L)	1.46 ± 0.09	1.43 ± 0.13	1.41 ± 0.11	1.21 ± 0.15

CT: control untreated grapes; GS: grapevines treated with grape stem extract; GP: grapevines treated with grape pomace extract; WL: grapevines treated with wine lees extract. Different superscripts within the same row and stage mean significant differences among treatments according to the post hoc Tukey test (95% confidence).

. All the pressed wines presented values within the optimal range for quality red wines [49,55]. Significant differences between assays were found in total acidity, where the measured values for the GS and WL wines were significantly lower than the CT and GP ones. Given the absence of differences in tartaric and malic acids, these discrepancies may be attributed to the varying contribution of cations, particularly potassium, derived from the original grape stem and wine lees by-product extracts. In addition, the elevated potassium levels in WL wines may be partially attributed to the intrinsic potassium content of red wine lees, which are rich in minerals and organic components [56]. In the course of red wine fermentation, the presence of potassium and calcium tartaric salts derived from maceration with solid parts has been observed [57,58]. The subsequent precipitation of these salts is attributed to the increasing content of alcohol, which consequently results in a decrease in its solubility within wine [49]. After the alcoholic fermentation process, the concentration of the acids also decreases. In the case of malic acid, this is due to malolactic fermentation, i.e., the conversion of malic acid into lactic acid and CO₂ [59]. Another factor contributing to the reduction of organic acids, mainly tartaric acid, is the low temperature recorded during cold stabilisation [58], the step preceding bottling. Together, these processes explain why initial differences in total acidity observed during pressing did not persist after bottling. A similar trend was observed for total acidity results between the pressing and bottling stages for the same grape variety vinified using different technologies [44,60].

The term ‘volatile acidity’ is used to describe the presence of saturated fatty acids in wine, which are formed during standard alcoholic and malolactic fermentation processes. In red table wines, these acids typically range from 0.3 to 0.6 g/L (expressed as acetic acid). Therefore, the volatile acidity values registered indicated that all fermentation processes were successfully completed. No significant differences were found in either volatile acidity or the residual concentration of reducing sugars, glycerine, and organic acids (acetic, citric, tartaric, malic, lactic, and succinic acids).

3.3.2. Phenolic Content Evolution

The subsequent phase of the study involved an evaluation of the evolution of the phenolic content in the different stages of the Tempranillo red wine production process. All parameters had values in the suitable range for quality Tempranillo wines [60,61,62]. In addition, significant differences were found among assays for TPI and anthocyanin and tannin content at the pressing and subsequent stages (

Table 4. Phenolic content of must, pressed wines, bottled wines, and aged wines.

	CT	GS	GP	WL
Alcoholic fermentation onset
TPI	8.0 ± 0.2	10.4 ± 0.1	8.9 ± 0.2	11.3 ± 0.1
Anthocyanins (mg/L)	34 ± 2	35 ± 2	28 ± 3	39 ± 4
Tannins (g/L)	0.68 a ± 0.09	0.88 b ± 0.04	0.76 b ± 0.11	0.96 b ± 0.15
Pressing
TPI	47.1 ± 3.7	48.4 ± 2.9	46.2 ± 2.5	53.4 ± 3.8
Anthocyanins (mg/L)	413 ab ± 3	454 a ± 7	388 b ± 4	449 a ± 6
Tannins (g/L)	3.84 ± 0.18	4.09 ± 0.25	3.86 ± 0.39	4.36 ± 0.47
After malolactic fermentation
TPI	42.8 a ± 2.4	49.4 b ± 2.1	43.9 a ± 1.2	49.7 b ± 1.6
Anthocyanins (mg/L)	375 ab ± 5	417 b ± 4	340 a ± 5	389 b ± 7
Tannins (g/L)	3.85 ab ± 0.10	4.17 b ± 0.19	3.72 a ± 0.11	4.21 b ± 0.13
Bottling
TPI	42.9 a ± 1.1	51.2 b ± 2.8	42.0 a ± 1.0	48.9 b ± 1.3
Anthocyanins (mg/L)	339 a ± 7	397 b ± 6	311 a ± 8	370 b ± 9
Tannins (g/L)	3.78 a ± 0.09	4.34 b ± 0.21	3.45 a ± 0.03	4.09 b ± 0.21
12-month ageing
TPI	40.1 a ± 1.2	48.6 b ± 2.5	41.8 a ± 1.4	47.2 b ± 2.0
Anthocyanins (mg/L)	175 a ± 5	201 b ± 3	154 c ± 5	185 a ± 2
Tannins (g/L)	3.44 a ± 0.10	4.11 b ± 0.19	3.53 a ± 0.06	3.97 b ± 0.09

CT: control untreated grapes; GS: grapevines treated with grape stem extract; GP: grapevines treated with grape pomace extract; WL: grapevines treated with wine lees extract. TPI: total polyphenol index (O.D. 280 nm). Different superscripts within the same row and stage mean significant differences among treatments according to Tukey’s test (95% confidence).

).

During winemaking and wine storage, the principal reactions that may occur are a dynamic process leading to increasing structural diversity in wines. They comprise copigmentation, cycloaddition, and condensation reactions, as well as hydrolyzation, oxidation, and polymerisation processes. Thus, anthocyanins could evolve into more stable structures because of several interactions with other wine molecules such as acetaldehyde, glyoxylic acid, and/or flavanol. Furthermore, they also undergo hydrolysis and polymerisation reactions, leading to the formation of colourless pigments [63,64,65]. These reactions play a significant role in the quality of wines, impacting parameters such as stability and ageing behaviour. Moreover, these compounds contribute to the organoleptic properties of wine, given their impact on parameters such as colour, astringency, and bitterness [65,66].

As can be seen in Table 4, significant differences in the total phenolic content (TPI) were detected after malolactic fermentation. This finding may be attributed to the previously reported transformation of phenolic compounds by lactic acid bacteria (LAB) [59]. This interaction is influenced by the strain of LAB and the type and concentration of phenolic compounds present in the wine, among other factors [67]. In addition, it has been established that wines with higher TPI exhibit a diminished evolution rate due to stabilisation reactions among different phenolic compounds [63,64,68]. This finding may provide a possible explanation for the significantly higher phenolic content of GS and WL treatments vs. the other assayed wines, which persisted throughout all subsequent stages, including the ageing process.

The influence of these treatments on anthocyanins was also evaluated. The anthocyanin content was found to be within the range observed in Tempranillo wines produced in the same climatic zone [44,45,46,54,60]. The application of GS and WL extracts to grapevines resulted in wines with a consistently higher total anthocyanin content throughout the winemaking process. The enhanced levels of anthocyanins detected in wines from GS and WL treatments are indicative of a direct correlation with colour stability and copigmentation effects, which are essential in the quality of red wine. GS-treated wines exhibited slower declines in anthocyanin levels during bottle ageing, maintaining significantly higher concentrations even after 12 months, compared to untreated wines. This finding suggests that the phenolic composition of GS extracts is more effective at promoting the stabilisation of anthocyanins, possibly by facilitating the formation of stable polymeric pigments, which are less prone to precipitation or degradation over time. In contrast, wines derived from GP-treated grapevines exhibited diminished anthocyanin content relative to CT in all stages.

As expected, the anthocyanin levels observed in the Tempranillo wines declined from the initial pressing stage to the bottle ageing stage. This decline was attributed to several different reactions, including partial removal and pigment precipitation (promoted by lactic bacteria during malolactic fermentation or by low temperatures in the stabilisation step) [63]. Other researchers [45,46,54,62] have documented this trend for red wines produced in regions characterised by warm climatic conditions, where significant anthocyanin losses have been observed after pressing.

At bottling, anthocyanin losses in CT and GP wines were almost double those in GS and WL (10% and 9% vs. 5% and 5%, respectively). Thus, for young red wines, the grapevines treated with GS and WL extracts led to wines whose anthocyanin content remained at higher levels than those of untreated ones.

A comparable trend was observed for tannins, which also declined in conventional red winemaking due to different reactions that occur during the storage and wine ageing process. Tannins found in wines made from grapes treated with GS and WL extracts exhibited higher levels in all cases than those derived from untreated grapes.

During ageing, all analysed compounds tended to decrease, especially for anthocyanins. Thus, following a 12-month ageing period, all parameters had undergone a progressive decline in all wines, as expected, showing comparable anthocyanin losses (approximately 50% vs. bottling). Nevertheless, wines derived from grapevines treated with GS extracts still exhibited a significantly higher content of anthocyanins, TPI, and tannins than CT. This allows for the production of more colourful wines. WL-treated grapes continued showing higher TPI and tannin levels than CT; however, their anthocyanin was found to be quite similar. In contrast, wines derived from GP-treated grapevines showed a diminished anthocyanin concentration, while no notable differences were observed between GP-treated and CT in terms of TPI or tannin levels.

The findings reported here are consistent with those of previous studies on the evolution of phenolic content in Tempranillo wines following bottling [54,61,69,70] and with the effects of analogous biostimulants, which exhibit a comparable phenolic nature [20,21]. The high extracted phenolic compound has a favourable impact on anthocyanin stability, facilitating copigmentation phenomena and promoting the formation of complex structures that enhance the red colour intensity and stability of the wine [71].

It thus appears that foliar application of GS and WL extracts may enhance the phenolic content, thereby limiting the loss of pigments and consequently improving the final colour of the wines. The use of GP extract, in contrast, did not yield any discernible benefit when compared to untreated grapevines.

3.3.3. Colour and CIELab Evolution

In order to assess the colour of the wine, the parameters of colour intensity and hue were considered (

Table 5. Evolution of colour parameters during winemaking, bottling, and bottle ageing.

Stage	Assay	Parameter
		Colour Intensity	Hue	L*	a*	b*	C*ab	h*ab	ΔE*ab
Pressing	CT	11.17 ± 0.72	0.50 ± 0.01	57.96 ± 2.28	49.92 ± 2.00	−2.57 ± 1.23	49.99 ± 1.22	357.37 ± 0.55	-
	GS	11.42 ± 1.43	0.50 ± 0.01	60.21 ± 1.25	49.22 ± 1.86	−1.81 ± 0.88	50.51 ± 3.11	358.33 ± 1.57	2.48
	GP	10.75 ± 1.14	0.49 ± 0.01	60.17 ± 2.01	47.42 ± 2.24	−2.58 ± 1.03	45.96 ± 3.28	356.52 ± 0.99	3.34
	WL	11.49 ± 0.82	0.50 ± 0.01	60.23 ± 1.68	49.64 ± 1.75	−1.43 ± 1.11	50.12 ± 3.10	357.82 ± 0.94	2.54
Bottling	CT	6.47 ab ± 0.17	0.64 ± 0.12	63.86 ab ± 2.27	40.62 ab ± 1.19	0.89 ab ± 0.13	40.63 ab ± 1.22	1.26 ab ± 0.28	-
	GS	8.02 a ± 0.25	0.61 ± 0.10	56.33 a ± 1.16	48.54 a ± 1.91	2.03 a ± 0.65	48.58 a ± 2.56	2.39 b ± 0.55	10.99
	GP	5.52 b ± 0.13	0.67 ± 0.09	68.64 b ± 2.91	36.01 b ± 1.82	0.11 b ± 0.09	36.02 b ± 1.79	0.77 a ± 0.16	6.69
	WL	7.55 a ± 0.16	0.63 ± 0.15	56.51 a ± 1.02	48.45 a ± 2.31	2.06 a ± 0.50	48.49 a ± 2.06	2.43 b ± 0.67	10.80
12 months ageing	CT	8.45 ab ± 0.11	0.67 a ± 0.01	57.19 ab ± 1.38	40.27 ab ± 0.46	4.35 a ± 0.33	40.50 ab ± 0.35	6.19 a ± 1.02	-
	GS	9.75 b ± 0.19	0.68 a ± 0.00	54.40 a ± 1.77	41.67 b ± 0.31	7.40 b ± 0.65	42.32 b ± 0.88	10.11 b ± 0.65	7.62
	GP	7.58 a ± 0.14	0.71 b ± 0.00	61.60 b ± 1.30	36.41 a ± 0.64	5.35 a ± 0.58	36.81 a ± 0.40	8.38 ab ± 0.53	4.23
	WL	8.77 ab ± 0.10	0.72 b ± 0.01	56.90 ab ± 2.55	39.79 ab ± 0.42	6.94 b ± 0.25	40.39 ab ± 0.27	9.88 b ± 0.55	7.39

CT: control untreated grapes; GS: grapevines treated with grape stem extract; GP: grapevines treated with grape pomace extract; WL: grapevines treated with wine lees extract. Different superscripts within the same row and stage mean significant differences among treatments according to Tukey’s test (95% confidence).

), as these provide insights into the quality and stability of the colour. These parameters should be interpreted together. A higher colour intensity and a lower hue are indicative of superior wine colour quality.

In accordance with the typical behaviour observed in conventional red winemaking, the colour intensity of wines typically declines from the pressing stage to the bottling stage. This observation has been previously documented [44,72]. In contrast, during the ageing process, a decrease in colour intensity was observed, which was accompanied by an increase in colour saturation. This phenomenon is believed to be caused by the condensation and polymerisation of anthocyanins with other polyphenols [71,72,73]. Moreover, the presence of flavanols within the wine matrix has been demonstrated to be implicated in oxidative browning reactions [70,74].

For colour intensity, higher values than CT were observed at bottling for GS and WL wines, particularly for the GS treatment, with values ranging from 24 to 17% higher than CT, respectively. However, after 12 months of ageing, it was observed that only GS wines remained significantly higher in colour intensity than CT wines. The enhanced colour intensity values of wines obtained from GS-treated grapevines can be attributed to the consistent anthocyanin, tannin, and total phenolic concentrations present in GS wines following MLF. This higher phenolic content has the potential to facilitate the formation of more stable oligomers and polymeric pigments. Consequently, GS wines may be less susceptible to oxidative browning reactions associated with the ageing process.

The changes in hue (Table 5) are indicative of typical oxidation and browning processes during wine ageing. These changes align with well-documented phenomena where increased hue values correspond to a shift from red to orange–brown tones due to anthocyanin degradation and the formation of polymeric pigments through oxidation and condensation reactions [72]. In the wines treated with GP, higher hue values could be associated with lower anthocyanin concentrations and reduced copigmentation effects, rendering these wines more vulnerable to oxidation. Conversely, the WL treatment, despite showing higher hue values, also exhibited higher phenolic content, suggesting that its oxidative behaviour might be moderated by the antioxidant properties of certain phenolics present in the extract.

The results indicated that GS wines had the highest colour intensity/hue ratio at both the bottling and ageing stages. This can be attributed to the higher optical density values at 520 nm (red colour contribution: 0.50 vs. 0.44 in CT wines) and to their high TPI value, which provides the production of the highest quality and stable wines [75]. These findings correlate with the documented effects of other biostimulants of similar woody nature on other single-cultivar wines [41].

The evolution of CIELab colour during the winemaking process (Table 5) resulted in notable discrepancies among the wines from the bottling stage onwards. It was observed that the L* values were the highest for CT and GP wines in all steps, which led to the conclusion that the wine obtained through conventional and GP extract foliar applications were of a lighter colour and less saturated.

Significant differences were observed for the red–green component (a*) between the GS and WL wines and the CT wines at the time of bottling. The results demonstrated that the GS and WL wines presented a greater prevalence of reddish tones in comparison to the CT wines. Similarly, significant differences were observed for the b* coordinate (yellow–blue component), with GS and WL wines showing significantly higher values than CT and GP wines.

The 12-month ageing stage resulted in a slight decrease in the a* coordinate, indicating a slight decline in the treatments, with only the GS wines showing a significantly higher value in comparison to CT. Conversely, the b* coordinate presented an upward trend, in accordance with the anthocyanin–tannin condensation reactions previously described by Fulcrand, 2006 [68]. At this stage, significant differences were found for the b* coordinate between the GS and WL and the CT.

C *ab and h *ab coordinates, which are correlated with colour perception, showed downward and upward trends, respectively, from pressing to 12-month ageing. The h *ab angle showed no discernible variations among the samples at the pressing stage. However, notable differences were discerned at a subsequent stage.

It was observed that the period of time between bottling and 12 months of ageing resulted in significantly higher values for the h *ab angle coordinate in GS and WL in comparison to CT wines. In contrast, GP and CT wines presented values that were similar to one another.

On the other hand, C *ab was significantly different in the bottling and ageing stages. Significantly higher values were observed in GS and WL wines in comparison to CT wines at both bottling and ageing stages. Therefore, the application of GS extracts to grapevines resulted in an increase in the colour intensity of the wines at all stages (lower L* and higher C *ab).

Lastly, a comprehensive assessment of total colour differences (ΔE *ab) between assayed treatments and CT was conducted during the winemaking process. ΔE *ab values exceeding 3 CIELab units were indicative of perceptible colour differences to the human eye [76]. The mean colour differences (∆E *ab) calculated between GS, GP, or WL vs. CT throughout the winemaking process were discernible to the human eye for all foliar treatments, particularly for GS and WL from the bottling stage (Table 5).

Given that the GS and WL ∆E *ab values remained consistently higher than those of CT, it can be concluded that the GS and WL treatments exert a significant impact on wine colour. Furthermore, the discrepancies in lightness (∆L *ab) and chroma (∆C *ab) between these treatments and CT were evaluated.

The findings revealed that the colour differences were quantitative, with mean values of ∆L *ab = −5.16 and −3.82, and ∆C *ab = 4.89 and 3.88 CIELab units for GS and WL in comparison to CT, respectively.

Therefore, both extracts (mainly GS, but also WL) resulted in young wines with a darker hue and more pronounced colour intensity compared to those produced from untreated grapevines. This correlates with the reported effects of other biostimulants of similar woody nature on other single-cultivar wines [41].

3.4. Sensory Analysis

Finally, a sensory analysis was carried out to evaluate the influence of the treatments carried out and the differences observed in consumer perception of these wines. All wines were evaluated favourably at bottling (Figure 3).

The L*, a*, and b* values provide further insights into the visual characteristics of the wines. Lower L* values in GS and WL wines indicate darker, more saturated colours, which were clearly discernible by the sensory panel, which assigned the highest value of hue and colour intensity. Similarly, higher a* (red–green component) and b* (yellow–blue component) values in these treatments reflect a more vibrant and balanced visual appearance, contributing to the perception of superior colour quality by the panellists. These findings suggest a direct correlation between the colorimetric parameters and sensory perceptions of colour intensity, balance, and overall visual appeal. The observed ∆E *ab values, which exceeded the threshold for perceptible colour differences, reinforce the sensory panel’s ability to discern and favour wines with lower L* and higher a* and b* values, such as those treated with GS and WL extracts.

In terms of flavour profiles, GS and WL wines also showed the best sensorial properties. GS wines were characterised by red and black fruit aromas, along with balsamic notes, while WL wines exhibited ripe fruit and spice attributes. These differences can be partially explained by the higher levels of anthocyanins and tannins in GS and WL treatments (Table 4), as these compounds contribute to both aromatic intensity and mouthfeel. A preliminary analysis suggests that higher anthocyanin levels are positively correlated with the perception of red fruit and colour intensity, while elevated tannin concentrations are linked to the sensory attributes of body and structure. Consequently, perceived quality was found to be positively correlated with anthocyanin and phenolic content. As expected, the GS and WL wines exhibited higher positive scores than the untreated wines.

Furthermore, the relationship between tannin levels and bitterness or astringency is noteworthy. GS wines, which exhibited the highest tannin levels, were rated as having the lowest astringency and bitterness, being less pronounced than in those with lower tannin content, such as CT wines. This observation suggests the possibility of tannin structure polymerisation or softening in these wines, which could potentially result in a reduction in the intensity of these harsh sensations.

In comparison to CT, GS also showed a greater number of favourable characteristics with respect to flavour. Conversely, CT presented higher values for bitterness and lower values for the remaining evaluated attributes. Furthermore, GS wines were found to exhibit the highest colour intensity and received high scores for complexity, persistence, and flavour intensity.

4. Conclusions

The findings of the current study highlight the potential of foliar applications of winemaking by-product extracts, specifically grape stems and wine lees, as viticultural biostimulants to enhance the chromatic and phenolic properties of Tempranillo grapes cultivated under warm climate conditions. The results demonstrate that both GS and WL treatments resulted in wines with higher anthocyanin and tannin concentrations, contributing to enhanced colour intensity and stability. The effects were particularly pronounced with GS extract, which maintained superior anthocyanin levels and colour stability, even after 12 months of ageing. The ability of GS to enhance pigment copigmentation and anthocyanin–tannin interactions likely explains its more consistent impact across the winemaking and ageing processes.

In contrast, grape pomace extract showed limited efficacy in improving phenolic content or colour stability, possibly due to its composition, which seems less effective in stimulating the endogenous synthesis of secondary metabolites or stabilising pigments during vinification. These findings highlight the specificity of biostimulant effects, which depend not only on their phenolic composition but also on their interaction with grapevine physiology and environmental conditions.

The utilisation of these extracts is consistent with sustainable viticultural practices by valorising winemaking by-products, reducing environmental impact, and providing an alternative to synthetic additives. These results suggest that GS extract, and to a lesser extent WL extract, are viable tools for mitigating the challenges associated with warm climate viticulture, where achieving optimal grape colour remains a significant issue.

The findings obtained thus far are encouraging, but further research is required to optimise the application protocols for these extracts, including the timing, dosage, and frequency, with a view to maximising their efficacy. Long-term studies across multiple vintages and grape varieties are necessary to validate these results and assess their consistency under varying climatic conditions. In addition, it is essential to explore the molecular mechanisms underlying the biostimulatory effects of GS and WL extracts, in order to provide valuable insights into their mode of action, thus enabling the development of more targeted viticultural strategies.

In light of the limited impact observed with GP extract, future studies should aim to refine its extraction processes or investigate its combination with other biostimulants to enhance its effectiveness. Finally, expanding this research to include sensory analyses with diverse consumer panels and commercial-scale trials will be essential for evaluating the market potential and broader applicability of these sustainable biostimulants in the wine industry.