Introduction

Climate change is modifying environmental conditions in all wine-growing areas of the world. Its effects can be positive for vineyards located in cold areas, but for those located in hot and dry areas, such as in the Region of Murcia, the consequences can be negative, both for the volume of production and for the quality of grapes and wines.

High temperatures accelerate sugar accumulation and acid degradation in grapes compared to the rates observed under typical Mediterranean climate conditions (Mira de Orduña, 2010; Martínez-Moreno et al., 2023) and there is a growing imbalance between technological maturity and phenolic maturity as a consequence of this climate change. Therefore, the wine obtained from these grapes reaches a higher alcoholic degree, presents fewer organic acids (mainly malic), a higher pH and a lower anthocyanin content and, therefore, a lower colour (Resco et al., 2016; van Leeuwen & Destrac-Irvine, 2017), since the skins, and especially seeds remain phenolically immature.

In fact, the alcoholic content of wines has increased steadily since the eighties, and today it is normal to find red wines with 15 % ethanol. The alcohol concentration in wine is important for several reasons as ethanol is essential for the ageing, stability and organoleptic properties of wine (Pickering et al., 1998). However, a high ethanol content also presents a series of technical drawbacks such as I) problems in alcoholic fermentation, with slow development of microbiological activity due to inhibition or death of yeasts and stops in the process, II) problems in malolactic fermentation, due to the lower activity of Oenococcus oeni, which delays the stabilisation of the wines and increases some unfavourable sensory characteristics, III) lower volatility of some aromatic compounds that can lead to a lower aromatic complexity of these wines. To this, the inconveniences for the consumer must be added, such as excessive warming sensation during tasting, lack of fruit flavour and freshness (possibly due to the greater solubility of the volatile components of the wine that affects the threshold detection and the distribution in the headspace (Robinson et al., 2009)), excessive caloric content, negative effects of alcohol on human health, severe controls for driving vehicles and price increases in those countries whose taxes are linked to the ethanol content of the drink.

For all these reasons, there is clear evidence that wine consumers demand wines with a lower alcoholic content. Therefore, the studies of Mora et al. (2021) concluded that, when exploring young consumer attitudes to different red wines, they showed a preference for soft red wines (described as lower ratio total polyphenol index/polysaccharides), and with floral and fruity aromas and low alcohol content. The studies of Deroover et al. (2021) as well as the sector report published by Alimarket Gran Consumo (2020) highlight some trends that are beginning to consolidate in the wine sector, among them the demand for calmer wines (with less alcohol content), that will increase in response to a growing trend for healthier beverages.

One potential approach winemakers can use to reduce alcohol levels in wine is to harvest grapes with lower sugar content but to produce a high-quality wine with these grapes requires solving the problem of their low phenolic maturity at this stage, and especially the seeds, which will give wines with a high astringency and bitterness (Keller, 2020). However, it should not be forgotten that tannins are very important to assure wine colour stability through polymerisation with anthocyanins (Singleton & Trousdale, 1992) and this reduction in their concentration may affect the potential of these wines for ageing. We conducted a previous study (Bautista-Ortín et al., 2014) elaborating a Monastrell wine from well-ripen grapes where all the seeds were removed from the grapes, and the results were quite promising. The absence of seed tannins did not affect anthocyanin concentration, and the tannin concentration was 40 % lower than in a control wine. The copigmentation (CA) study showed that both wines had a similar extent of CA phenomena, although the colour intensity was higher when seed tannins were present. Moreover, the panellist evaluated the wines elaborated without seeds as being fruitier, less astringent and with an overall higher quality. However, these wines showed a high alcohol content, which does not align with current consumer preferences for lower-alcohol wines (Sekhon and Sun, 2024). For that, in this work Monastrell wines were elaborated from unripe grapes with seed total removal and their phenolic content, chromatic and sensorial characteristics were evaluated and compared with respect to those wines obtained of unripe and optimal ripe grapes in the presence of seeds after 6 months of ageing in the bottle to verify if this oenological practical may be an alternative to mitigate challenges posed by climate change and produce Monastrell wines with a lower alcohol content without compromising their quality.

Materials and methods

1. Vineyard Site and Grape Samples

Monastrell red grapes were harvested from a vineyard in the Aceniche Valley, located within the D.O. Bullas, region of Murcia, Spain. The Monastrell vines, planted in 1990, are spaced 2.5 by 3.0 meters apart (1333 vines per hectare). The vineyard sits at an altitude of 850 meters above sea level and experiences hot summers and very cold winters, with occasional snowfall. The average annual temperature was 15.9 °C, and the annual precipitation was 336 mm. The vines are cultivated using a bush training system and are grown under rainfed conditions (a cultivation method in which vines depend entirely on natural rainfall, without any irrigation).

2. Wine Production (Microvinification)

Monastrell grapes with varying degrees of ripeness (12 and 14° Baumé) from the same plot were used to carry out the different vinifications. Once the grapes were harvested, they were transported to the experimental winery at the University of Murcia and placed in a refrigeration chamber for a few hours. Subsequently, the grapes were crushed and destemmed, distributed into 10-litre stainless steel tanks. Seed removal treatment was manually performed only in unripe grapes. The other two vinification processes, involving the presence of seeds, were also conducted with both unripe and ripe grapes. All vinifications were conducted in triplicate. Fermentation for all wines took place at 22 °C using Viniferm CT007 (Agrovin SA, Alcázar de San Juan, Ciudad Real, Spain) as the fermentation yeast at a dosage of 25 g/hL. Actimax Plus (25 g/hL, Agrovin SA, Alcázar de San Juan, Ciudad Real, Spain) and Actimax Natura (25 g/hL, Agrovin SA, Alcázar de San Juan, Ciudad Real, Spain) were also added to prevent fermentation stops.

During the 7 days of maceration, the cap was submerged twice daily. After the corresponding time, the must wine was racked and pressed. The free-run and press must wines were mixed and reintroduced into different tanks. After the completion of alcoholic fermentation (AF), the wines were racked, sulphited and cold stabilised for a month. After this period, the wines were racked again, sulphited and bottled in 375 mL bottles. Analysis of chromatic parameters, phenolic and aroma compounds and sensorial was carried out after 6 months of wine ageing in the bottle.

3. Analytical Determinations

3.1. Physicochemical parameters

The wines were characterised by measuring the alcohol content, pH and acidity according to the European Community methods (Regulation Commission, 1990).

3.2. Spectrophotometric analysis

The chromatic parameters were evaluated using a UV/visible spectrophotometer (Helios Alpha model from Thermo Spectronic, Cambridge, United Kingdom). The colour intensity (CI) of the undiluted wine was determined as the sum of absorbances at 620 nm, 520 nm and 420 nm according to Glories (1984).

3.3. Determination of tannins by HPLC

Determination of tannin total concentration and composition (mean apparent degree of polymerisation, percentage of galloylation and the percentage of epigallocatechin) was carried out by the phloroglucinolysis method according to the conditions proposed by Busse Valverde et al. (2010). The following equipment was used: a Waters 2695 HPLC system (Waters, Milford, MA) coupled to a Waters 2996 photodiode array detector and an Atlantis dC18 column (250 × 4.6 mm, 5 μm packing) protected with a guard column of the same material (20 mm × 4.6 mm, 5 μm packing). The injection volume was 10 μL. A water/formic acid mixture (98:2, v/v) was used as solvent A and acetonitrile/solvent A (80:20 v/v) as solvent B, at a constant flow rate of 0.8 mL/min and an oven temperature of 30 °C. Elution conditions were as follows: 100 % A for 5 min, linear gradient from 100 to 90 % A in 30 min and gradient from 90 to 80 % in 30 min, followed by washing and re-equilibration of the column.

3.4. Determination of phenolic compounds by HPLC

The separation of anthocyanins and vitisins in wine was carried out in a Waters Acquity Arc liquid chromatograph (Waters, Milford, MA, USA) equipped with a Waters 2998 diode array detector (Waters, Milford, MA, USA). The column used was a Poroshell120 EC-C18 core-shell column (150 mm × 2.1 mm, 2.7 μm, Agilent Technologies, Santa Clara CA, USA). The composition of the mobile phases was: 1 % formic acid in water (A) and 1 % formic acid in 1:1 (v/v) methanol:acetonitrile (B). The elution gradient started with 100 % A for 2 min, linear increase from 0 to 15 % B in 33 min, from 15 to 21 % B in 15 min and from 21 % to 30 % B in 20 min followed by washing and re-equilibration of the column. The column oven was maintained at 55 °C, the flow rate was 0.3 mL/min and the injection volume was 5 μL. Compound identification and quantification were performed at 520 nm using malvidin-3-glucoside chloride as an external standard.

3.5. Determination of volatile compounds

For the analysis of Monastrell wine volatile compounds, 10 mL of wine was added to a 30 mL headspace glass vial. A total of 4 grams of sodium chloride (NaCl) and 25 μL of internal standard (2-octanol, Sigma-Aldrich) were added to the same vial. The vial was tightly sealed with a PTFE-lined cap. The solution was homogenised with a vortex shaker (IKA, Konigswinter, Germany). Gas chromatographic determination of volatile analyses was performed using a Gas Chromatograph HP 5890 GC system coupled to an HP 5972 quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA). In total, 2 µL of extract was injected in split mode. An HP InnoWax (50 m × 0.32 mm, 0.25 μm) capillary column (Agilent) was used for the analysis. The injector temperature was programmed at 40 °C for 5 min, raised to 225 °C at 3 °C/min, and then held at that temperature for 5 min. The detector runs at electronic impact mode (70 eV), with an acquisition range (m/z) from 50 to 200 amu. Peak tentative identification was carried out by comparing mass spectra with those of the mass library (Wiley 6.0, Chichester, UK) and comparing the calculated retention indices with those published in the literature. Semiquantitative data were obtained by calculating the relative peak area (total ion count signal or that of selected fragments in the case of some coeluted compounds) in relation to that of the internal standard.

3.6. Sensory analysis

Wines were subjected to sensory evaluation using a descriptive analysis. Prior to the sensory analysis, the wine from the three different replications for each experience was pooled to have a representative sample and to avoid differences among the replications. A total of 12 staff members with high experience in wine sensory analysis participated. They had several training sessions to establish and define suitable descriptors for the formal evaluation of wines. Wine samples were presented in 40 mL aliquots and coded and served in standard wine glasses. The evaluations took place in a controlled room maintained at 20 °C, free from any odour, noise or distraction, with the booth illuminated with a white light.

4. Statistical analysis

The statistical analysis of the data was performed using the package Statgraphics Centurion XVI.3 (Statpoint Technologies, Inc., The Plains, VA, USA). Physicochemical parameters, phenolic and volatile compounds and sensory analysis data were processed using the variance analysis (ANOVA) (p ≤ 0.05). The differences between means were compared using Tukey’s test.

Results and discussion

1. Physicochemical parameters

The physicochemical characteristics of the wines (alcohol, pH and total acidity) are shown in Table 1. The 12° Baumé grape wines produced with seeds (CW12) and without seeds (WW12) did not exhibit significant differences in their physicochemical parameters. Both wines had an average alcohol content of 12.4 % vol. In contrast, the 14° Baumé grape wines (CW14) demonstrated a higher alcohol content of 14.6 %. The pH values were 3.43 for the CW12 and WW12 wines and 3.58 for the CW14 wines, while the total acidity was close to 5.9 g/L for the CW12 and WW12 wines and 5.59 g/L for the CW14 wines.

2. Tannin concentration and composition

The concentration and composition of tannins were measured by the phloroglucinolysis method (Table 2). The table shows the content of condensed tannins (TT), their degree of polymerisation (mDP) and the percentage of subunits of epigallocatechin (%EGC) and galloylation degree (%Gal). CW14 wines had the highest concentration of TT and mDP with a value of 539.90 mg/L and 7.57, respectively. As expected, the lowest tannin concentration was found in WWS12 wine. This wine showed the highest content of EGC with a percentage of 18.9 % while CW12 wine obtained the lowest EGC values at 15.2 %. Moreover, the CW12 wine had a higher galloylation percentage (proportion of galloylated flavanol subunits to total flavanol subunits) than CW14 and WWS12 with values of 3.43 %, 2.61 % and 1.87 %, respectively. This indicates that higher concentrations of seed tannin diffused into the CW12 wine more effectively than into CW14, likely due to the reduced lignification of the unripe seeds, which allows better tannin diffusion from the seeds to the must/wine, while the intensive lignification in ripe seeds makes them more waterproof (Cadot et al., 2006).

These results align with the different tannin compositions of the skins and seeds previously reported in the literature (Busse-Valverde et al., 2010; Rodríguez Montealegre et al., 2006). The elevated concentration of tannins in CW14 wine is likely attributable to the higher ethanol content, which may enhance the solubilisation of cell walls from the skin and seed cuticle facilitating tannin diffusion into the wine. Canals et al. (2005) also observed an increase in phenolic compounds and tannins in wines made from riper grapes. Moreover, tannins derived from seeds possess a lower mean degree of polymerisation (mDP) compared to those from skins (Guaita et al., 2017). Therefore, the removal of seeds in WSS12 wine resulted in a reduction in tannin content and a rise in mDP relative to CW12. This significant increase in mDP has also been previously reported in wines made with early seed removal from red varieties such as Merlot (Lee et al., 2008) or Gaglioppo (Guaita et al., 2017). Additionally, seed tannins generally contain higher levels of galloylated forms than those from skin (Souquet et al., 1996), which resulted in a significant decrease in the galloylation percentage of these compounds in WWS12 wines.

It is important to not forget that the concentration and composition of tannins play a crucial role in the sensory characteristics of red wines (Gawel, 1998), significantly influencing their quality (Gómez-Plaza et al., 2016). However, a high concentration of tannins, along with elevated levels of galloylation and low mDP values, can enhance the perception of astringency and bitterness (Vidal et al., 2003). These effects, however, may be moderated by the presence of (-)-epigallocatechin within the tannin structure (Chira et al., 2009; Fernández et al., 2007).

3. Concentration of anthocyanins and vitisins

Table 3 shows free and polymeric anthocyanin, vitisin and total anthocyanin concentrations in wines made from grapes with different ripening stages (CW12 and CW14) and wine made with total seed removal (WWS12) determined by HPLC. Regarding total anthocyanin content, no significant differences were observed between CW12 and WW12, while CW14 presented the highest concentration of these compounds. Other studies also found no differences in anthocyanin concentration when seeds were removed in Gaglioppo (Guaita et al., 2017) or Monastrell (Bautista-Ortín et al., 2014) wines, or even an increase in anthocyanin content in Merlot wines (Lee et al., 2008), although in this last study, the seeds were removed when the sugar content during fermentation was close to 10° Brix. The greater accumulation of these compounds in the skin together with a higher degradation of the cell walls when the grape ripening increases would explain the higher presence of anthocyanins in CW14 wines, as also reported in previous studies conducted by Bautista-Ortín et al. (2006) with the Monastrell variety.

CW14 wine exhibited the highest content of free anthocyanins (92.16 mg/L) followed by CW12, while the WWS12 presented the lowest concentration. However, in all wines malvidin-3-glucoside was the predominant anthocyanin, while other anthocyanins, such as malvidin-(6-acetyl)-3-glucoside, peonidin-(6-coumaroyl)-3-glucoside and malvidin-(6-coumaroyl)-3-glucoside were not detected in WWS12 wine, although they were present in CW12 and CW14 wines. Regarding vitisins, seed removal significantly increased the content of these compounds in the wine, almost doubling the values shown by CW12 and exceeding CW14’s concentration by 32 %. In all wines the malvidin-3-O-glucoside-pyruvic acid (Vitisin A) showed the highest concentration, reaching values of 10.11 mg/L for WWS12, 7.29 mg/L for CW14 and 5.55 mg/L for CW12. Additionally, a greater formation of vitisin B in CW12 was also observed compared to CW14, although WWS12 again exhibited the highest concentration of this compound. Notably, WWS12 wine had a significantly higher content of polymeric anthocyanins than the other two wines, while no differences were found between CW14 and CW12.

The absence of seed tannins in the WW12 wine compared to CW12 and CW14 wines may have promoted that the anthocyanins were more readily available for the ongoing formation of low molecular mass pigments (vitisins) and polymeric pigments resulting in condensation products with tannins or even among them alone as it was reported by Bindon et al. (2014a). These same authors indicated although these pigments formed only by anthocyanins are more bleachable by bisulfite than those formed with tannins, they contribute a strong 520 nm absorbance at wine pH and moreover could be important intermediates in the pathway for pigmented polymer formation in wine, which includes downstream condensation reactions with tannins and/or other polyphenols. Bindon et al. (2014b) reported that the formation kinetics of wine polymeric pigments during ageing are not necessarily dependent upon tannin concentration, as the evolution of tannin pigmentation was similar in wines with both low and high anthocyanin and tannin concentrations. However, some studies suggest that enhanced grape skin tannin extraction during vinification can lead to higher overall wine tannin concentration and increased polymeric pigment formation (Cortell et al., 2005; Cortell et al., 2007; Ristic et al., 2010). Therefore, further studies are needed to understand the role of absent seed tannins in the incorporation of skin tannins into pigmented polymers.

4. Colour intensity

The colour intensity of the studied wines is depicted in Figure 1. The wine produced with seeds removed prior to maceration (WWS12) showed the highest colour intensity (13.2), followed by the wine made from riper grapes (CW14). In contrast, the lowest colour intensity was observed in the wine produced with seeds from less ripe grapes (CW12). The increased colour intensity in WWS12 wine may be attributed to its higher content of both low and high polymerisation pigments, which positively impact the wine´s colour, enhancing its colour intensity (Cheng et al., 2023). While Lee et al. (2008) reported no visually noticeable differences in colour between control wines and those with seed removal, their findings showed control wines to be slightly lighter (larger L* value) and more orange. However, the results of this study demonstrate clear differences in colour intensity, underscoring the influence of seed removal and grape ripeness on wine colouration.

Figure 1. Color Intensity (CI) spectrophotometrically measured on the Monastrell red wines.

5. Aroma compounds

To date, few works have been published regarding the aroma profile of seed-removed red wines. In this research, the aroma composition of Monastrell wines elaborated with grapes at different maturity levels, with and without seeds, was analysed after 6 months of bottle ageing. More than 80 volatile compounds were identified (Table S1). The volatile compounds were classified into 5 chemical groups: alcohols, esters, aldehydes and ketones, terpenes and acids (Table 4). Alcohols and esters, followed by acids represented the aroma compounds at higher concentrations. The CW14 wine, made from riper grapes, exhibited higher concentrations of alcohols, esters and terpenes, while CW12 wine, made from less ripe grapes, had higher concentrations of aldehydes, ketones and acids. As expected, the wine from the ripest grapes (CW14) had higher levels of aroma compounds, as grape ripening naturally leads to an increase in alcohols, esters and terpenes in the corresponding wines (Torchio et al., 2016). These results are very similar to those reported by Gu et al. (2022), which observed similar increases in alcohols, esters and terpenes across different red wine varieties as grapes ripened. Interestingly, although CW14 wine showed a significant increase in esters compared to CW12, the seedless WWS12 wine had an even higher ester concentration than its control (CW12) and closely resembled the riper CW14 wine. Esters, known for their fruity aromas, are produced during fermentation through the esterification of alcohols and fatty acids by yeast metabolism (Yilmaztekin et al., 2015). The observed increase in esters in the WWS12 wine may be attributed to the higher concentration of fatty acids present in this wine. Fatty acids are well-established precursors to aromatic esters, with higher concentrations of fatty acids leading to increased ester formation (Bertrand, 1981). Additionally, the manual removal of seeds during the WWS12 treatment likely facilitated the release of fatty acids into the wine, further promoting ester synthesis. Previous research has demonstrated that different pre-fermentation techniques significantly influence fatty acid concentrations in the must (Chen et al., 2023). Specifically, these studies reported a notable increase in fatty acid levels when techniques such as destemming and crushing were employed. Hence, the results obtained in this study suggest that seed removal during vinification of less ripe grapes (which typically have a lower alcohol content) can enhance fruity aromas, thereby improving wine quality. The higher concentration of acids in CW 12 wines compared to CW 14 wines may be due to variations in the grape must composition between the two, reflecting differences in grape maturity and chemical profile (Schreier, 1979)

5. Sensory analysis

The sensory evaluation of seedless Monastrell wines is shown in Figure 2. Significant differences were noted for all descriptors except colour intensity. The WWS12 wines displayed higher tonality scores (tonality evolved from violet-purple to ruby-garnet) compared to CW14, with tasters perceiving a more violet/purple colour in the seedless wine. Additionally, both CW14 and WWS12 wines received similar, and significantly higher, aroma quality scores compared to CW12 wines. This trend extended to mouthfeel attributes such as taste quality and persistence. In terms of aroma descriptors, WWS12 wine scored the highest in fruitiness and, the lowest in vegetal aromas, which are often associated with lower-quality red wines. Similar observations were made by Cliff et al. (2012), who found that grape seed extract suppressed fruity aromas. Furthermore, this increase in fruitiness score is consistent with the significant increase in esters in the WWS12 wine, reaching values similar to those obtained in CW14 (Table 4). Regarding taste description, the WWS12 wine showed the lowest astringency scores, followed by CW14. The WWS12 wine also exhibited less bitterness than CW14, aligning with previous findings in red wines with seed removal (Bautista-Ortín et al., 2014; Canals et al., 2008). The reduced bitterness in wines from unripe grapes likely stems from lower levels of galloylated phenolics, which interact more strongly with bitter taste receptors due to their lipophilic nature (VanderWeide et al., 2022), as well as increased levels of (-)-epigallocatechin in the tannin structure (Chira et al., 2009; Fernández et al., 2007). Moreover, the WWS12 wine significantly improved the persistence and harmony scores, achieving values comparable to the CW14 wine. Overall, seed removal during vinification had a marked impact on the sensory attributes of the resulting wines, particularly enhancing aroma and taste perceptions, leading to a more balanced and pleasant sensory experience.

Figure 2. Descriptive sensory analysis of the Monastrell red wines studied.

Conclusion

The study demonstrates that removing seeds during vinification significantly impacts the wine's colour, phenolic content and sensory profile. The seedless wine (WWS12) showed reduced tannin levels, increased epigallocatechin and decreased astringency and bitterness compared to the wines made with seeds (CW12 and CW14). This absence of seeds facilitated the development of polymeric pigments and vitisins, enhancing colour intensity in WWS12, likely due to the increased availability of anthocyanins and their interactions, which contribute to the formation of stable colour compounds during winemaking. Additionally, seed removal promoted fruity aromas, supported by the higher ester content, creating a sensory experience comparable to that of wines made from fully ripe grapes (CW14). This technique also improved flavour persistence and harmony, suggesting seed removal could enhance the balance and quality of wines made from underripe grapes. These findings emphasise the importance of adapting winemaking techniques to grape ripeness and conditions, with seed removal offering a potential strategy to elevate wine quality when using grapes not fully ripen. Further research is needed to explore the role of skin tannins and anthocyanins or anthocyanins alone in polymeric pigment formation and how these wines' aromatic profiles evolve over time. Moreover, as the complete removal of seeds is a technique that poses significant challenges in commercial wineries, future research should explore innovative solutions, including the development of specialised equipment, to make this process more feasible and efficient.