Amino Acid Profile of Must and Aromatic Potential of 30 Minor Grape Varieties Grown in Alcalá de Henares (Spain)

Abstract

Amino acid composition and nitrogen quantification in grape must are of great importance given their usefulness for varietal characterization, influence on fermentation processes and identification of the aromatic potential of musts. The objective of this work was to determinate the amino acid and nitrogen compound profiles of 30 minority varieties of potential use in winemaking, all grown in the same ampelographic collection in Alcalá de Henares. The concentrations of 31 amino acids in must were identified and quantified using high-performance liquid chromatography (HPLC) during four seasons (2020 to 2023), and the average values of total free amino acids (TAN), yeast assimilable nitrogen (YAN) and aromatic precursor nitrogen (APN) were calculated for each variety. ‘Tazazonal’, a minority red grape variety, was found to exhibit high concentrations of yeast assimilable nitrogen (YAN), total amino nitrogen (TAN) and ammonia nitrogen (APN), comparable to those observed in ‘Tempranillo’ and ‘Garnacha Tinta’. These two cultivars are representative of traditional Spanish red grapevine varieties and are among the most widely cultivated in the country. In the case of white varieties, ‘Albillo del Pozo’, ‘Pintada’ and ‘Verdejo Serrano’ showed higher concentrations of these parameters than ‘Malvar’ and ‘Airén’, which are also widely grown in Spain. The results revealed distinct amino acid profiles for each variety, enabling their classification and supporting the identification of variants within the autochthonous germplasm. This approach aimed to highlight minority varieties of potential interest for future studies, given their relevance to both regional and national viticulture.

1. Introduction

Nitrogen is a macronutrient that plays a fundamental role in many of the biological functions and processes of plants, particularly vines, and of fermentative micro-organisms (yeasts and malolactic bacteria). Therefore, the state of this macronutrient in the vine directly influences the quality of the grape. In grapes and, therefore, in the must, nitrogen is present in organic (proteins and amino acids) and inorganic (ammonium and ammonium salts) forms. Except for proline and hydroxyproline, all amino acids can be metabolized by yeasts under normal winemaking conditions [1,2,3]. The concentration of nitrogen in the vine and its availability in the form of amino acids depends on several factors, such as the variety and/or rootstock [3,4,5], location (climate and soil) [6,7,8,9,10] and season [11,12]; cultural practices, such as soil management [13,14,15], the training system [16,17], water status [18,19,20], shading and canopy temperature [21]; and the dose, timing and type of nitrogen supply [12,22,23,24]. This means that the nitrogen composition of the vine and its response is defined by genetic, environmental and cultural practice interactions. Amino acids are the most important nitrogen source for yeast assimilation; they are essential for the development and reproduction of the organism during alcoholic fermentation; moreover, the rate of fermentation depends on the quality and quantity of amino acids present in the must [25]. In general, Ala, Arg, Asp, Cit, Cys, Glu, Gly, His, Iso, Leu, Lys, Meth, Orn, Phen, Pro, Ser, Thr, Try, Tyr and Val are the individual amino acids usually found in higher amounts in grapes [6,25]. Amino acids serve as an energy source for both the yeasts that carry out alcoholic fermentation and the bacteria that carry out malolactic fermentation [6]. They are also involved in the formation of certain compounds that are precursors of aromas and flavors [26,27,28]. The nitrogen from grapes and musts, accessible to yeasts, is expressed as yeast assimilable nitrogen (YAN). The minimum requirement for the fermentation of musts is 150 mgN/L. YAN consists of ammonium and assimilable amino acids (all amino acids except proline and hydroxyproline). The nutritional value of amino acids is much higher than that of ammonium, so amino acids, particularly some of them, such as arginine, play a major role as a form of resistance nitrogen at the end of alcoholic fermentation [27]. Thus, yeast growth, fermentation kinetics and aroma metabolism are largely affected by the nitrogen status of the must, which must be manipulated by adding nitrogen into the cellar when its level is not optimal [29]. In addition, the role of yeasts in the development and expression of aromas, flavors and the mouthfeel of wine is practically defined, as is the impact of nitrogen on yeast flavor [26,30]. However, under certain conditions, some amino acids can contribute to the formation of undesirable compounds, such as ethyl carbamate, a substance considered potentially carcinogenic [29]. It has been reported that some amino acids (Asp, Ile, Leu, Phe, Thr, Tyr and Val) act as precursors in the formation of aromatic compounds during fermentation, hence the importance of calculating the level of aromatic precursor nitrogen (APN) by summing the concentrations of these amino acids (mg N/L). Previous research highlights the significance of amino acids in characterizing grapevine varieties, reflecting their genetic and metabolic compositions [31]. Studies consistently demonstrate the utility of amino acid profiling for varietal classification and the characterization of minority varieties [24,27,31,32,33]. These profiles also help establish typical parameters in wines from different regions.

Minority varieties are rare varieties that are generally, and almost exclusively, linked to a specific territory and, therefore, have good local adaptation, a characteristic that makes them useful tools for promoting the diversification of wine products in the face of conditions of varietal erosion caused by climate change in a given region [34,35,36,37]. Among the main changes, we can identify changes in the seasonality of phenology [38,39], production volume [35] and instability of oenological parameters in the must for vinification and in the wine [40,41]. Phenological and agronomic characterization of 34 Spanish grapevine varieties, 30 of them minority varieties, conserved and cultivated in the ‘El Encín’ grapevine germplasm bank of the Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario (IMIDRA) has recently been carried out. This work has identified the earliness of the main phenological stages, the correlation of technological and physiological ripening with the annual thermal variation, in relation to the historical thermal series from 1957 to 2019, and the oenological quality profile of the must of each variety [42]. Considering the importance of the amino acid content and distribution in grape must and its influence on fermentation dynamics and the formation of aromatic compounds, this study aims to advance the understanding of these minority grapevine varieties. Particular emphasis is placed on (1) determining the amino acid profiles of the studied varieties, (2) characterizing red and white varieties based on their amino acid compositions and (3) identifying those of greatest oenological interest according to the concentration of aroma precursor amino acids.

By characterizing their amino acid profiles, we provide valuable information for viticulturists seeking to diversify cultivated germplasm. Furthermore, the study explores the unique oenological potential of these varieties, particularly with regard to their distinctive amino acid compositions and their contribution to the aromatic complexity of the resulting wines.

2. Materials and Methods

2.1. Plant Material

This work was carried out during four vintages, from 2020 to 2023. A total of 34 grapevine varieties were analyzed—16 white and 18 red, cultivated in the ‘El Encín’ Grapevine Variety Collection (

Table 1. List of varieties studied.

White Varieties	Id 1	Red Varieties	Id 1
Airén	Air	Azargón	Azg
Albillo del Pozo	APz	Benedicto	Ben
Aúrea	Aur	Cadrete	Cdt
Cagarrizo	Cgz	Crepa	Cpa
Castellana Blanca	CBl	Tazazonal	Taz
Hebén	Heb	Garnacha Tinta	GTi
Jarrosuelto	Jar	Granadera	Grn
Lucomol	Luc	Listán Prieto	LPt
Malvar	Mal	Morate	Mte
Marfileña	Mfñ	Rayada Melonera	RMe
Montonera	Mnr	Rubeliza	Rub
Pintada	Pin	Sanguina	San
Verdejo Serrano	Vse	Tempranillo	Tem
Salvador	Sal	Terriza	Ter
Tortozón	Tzn	Tinto Bastardo	TBd
Zurieles	Zur	Tinto de Navalcarnero	TNa
		Tinto Fragoso	TFg
		Tortozona Tinta	TTa

¹: Short identification name (Id).

) belonging to IMIDRA—in Alcalá de Henares, Madrid, Spain (40°310′ N, 3°170′ W, 610 m.a.s.l.). The vines were planted in 2002, grafted on Richter 110 rootstock and trained in a unilateral cordon, in a vertical trellis system and in a rectangular frame of 0.80 × 2.60 m. The vineyard is located on a fluvial terrace of the Henares River, with a semi-arid Mediterranean climate, characterized by an average annual temperature of 14 °C (1957–2023) and a water regime with an average annual rainfall of 400 mm, which is maintained with a backup automated irrigation system, as described in [42]. The vineyard comprises 30 minority varieties and the red varieties ‘Tempranillo’ and ‘Garnacha Tinta’, as well as the white varieties ‘Airén’ and ‘Malvar’. These were designated as reference varieties due to their regional significance and widespread cultivation at the national level.

2.2. Climatology

The climate data were obtained from the meteorological station located at the collection site (

Table 2. Minimum, maximum and average monthly temperatures (°C) from 2020 to 2023 and historical series (1957 to 2019).

Month	Minimum Average Temperature				Maximum Average Temperature				Average Temperature				Historical Serial (1957–2019)
	2020	2021	2022	2023	2020	2021	2022	2023	2020	2021	2022	2023	Min Average	Max Average	Average
January	1.46	−2.28	−2.22	−0.55	11.55	7.85	13.05	10.90	5.80	2.38	4.11	4.96	−0.15	10.37	5.11
Ferbuary	2.58	4.33	0.40	−1.53	16.22	14.73	16.12	13.44	9.07	9.36	7.73	5.40	0.55	12.50	6.53
March	5.02	2.58	5.30	3.80	16.66	16.79	14.02	18.58	10.58	9.70	9.62	11.13	2.64	16.23	9.43
April	8.59	6.52	5.13	5.50	18.47	18.17	18.10	24.13	13.39	12.06	11.59	15.38	4.82	18.33	11.58
May	11.35	8.88	10.60	9.00	26.01	24.10	27.41	23.98	18.80	16.76	19.73	16.64	8.25	23.06	15.66
June	13.31	13.85	14.71	15.46	28.88	28.95	32.16	29.09	21.69	21.85	24.33	22.20	12.54	28.91	20.73
July	18.17	15.41	18.98	17.47	35.76	33.01	37.35	35.00	27.63	24.82	28.97	26.93	15.04	32.90	23.97
August	16.20	16.59	18.67	17.68	32.83	34.17	34.69	35.46	24.81	25.57	27.10	27.08	14.72	32.50	23.61
September	13.15	13.55	12.83	14.05	27.38	26.24	27.55	26.22	20.32	19.46	20.44	19.88	11.86	27.64	19.75
October	6.28	7.70	11.38	11.13	19.49	22.41	24.46	22.86	12.69	14.54	17.53	16.47	7.81	20.63	14.22
November	5.48	1.51	5.30	5.49	15.84	13.84	16.09	15.60	10.09	7.24	10.64	10.26	3.08	14.13	8.60
December	1.57	2.12	5.73	0.16	10.78	13.58	12.77	11.83	6.30	7.26	9.02	5.35	0.54	10.78	5.66

and

Table 3. Monthly precipitation (mm) records during the four study seasons in the ampelographic collection ‘El Encín’, Alcalá de Henares, Madrid.

Month	Precipitation (mm)				Average Relative Humidity (%)
	2020	2021	2022	2023	2020	2021	2022	2023
January	12.40	37.50	9.80	14.60	-	80.00	66.00	72.00
Ferbuary	0.00	64.10	5.00	0.60	75.00	74.00	60.00	58.00
March	63.20	0.00	88.00	17.90	70.00	59.00	71.00	58.00
April	83.40	120.50	41.10	7.50	76.00	65.00	61.00	42.00
May	42.20	20.00	1.20	36.50	55.00	54.00	45.00	48.00
June	27.70	57.50	2.70	114.40	43.00	47.00	35.00	57.00
July	2.00	21.80	4.20	0.00	33.00	37.00	30.00	34.00
August	12.80	39.10	16.40	0.00	38.00	38.00	36.00	31.00
September	35.60	43.40	32.00	119.40	47.00	61.00	49.00	68.00
October	59.50	85.00	21.40	137.20	65.00	63.00	60.00	68.00
November	48.00	21.00	40.80	41.60	80.00	70.00	75.00	83.00
December	20.20	26.70	127.30	42.20	77.00	78.00	86.00	83.00

). Daily records of precipitation (mm), relative humidity (%) and minimum, mean and maximum temperatures (°C) were collected in a 10 min series during the four years of study and compared with existing historical records between 1957 and 2019 at the same weather station [42].

2.3. Evaluation of Maturity Status

During each study year, grape maturity was monitored weekly from veraison to harvest by measuring the total soluble solids (°Brix). °Brix was measured using a digital refractometer (PR-101 Palette, ATAGO^®, Tokyo, Japan) to track ripening and determine the optimal harvest time. The pH was determined using a laboratory GLP meter equipped with temperature and pH electrodes (Sension+ PH31, HACH^®, Loveland, CO, USA), and the titratable acidity value (g/L of tartaric acid) was determined according to the method proposed by Rustioni et al., 2014 [43], calculated at the time of harvest [42]. Must samples (40 mL) were obtained by manually pressing 10 clusters, randomly selected from 10 vines per variety, and were stored at −20 °C for subsequent amino acid analysis.

2.4. Amino Acid Determination

Proteins and peptides were precipitated from the defrosted must by adding 5% (v/v) sulfosalicylic acid and filtering through a 0.22 µm pore filter, and the samples obtained (i.e., the deproteinized must) were frozen at −80 °C until analysis. The determination of 31 nitrogenous compounds (amino acids, amines and ammonia) in the must was carried out according to the protocol established by Valdés, Vilanova, Sabio and Benalte (2011) [44]. The quantification of amino acids, ammonia and nitrogen compounds was performed via ion-exchange chromatography (HPLC), followed by derivatization with post-column ninhydrin and photometric detection—at 440 nm for proline (Pro) and hydroxyproline (Hyp) and 570 nm for the rest of the amino acids—using a Biochrom Series 30 amino acid analyzer (Biochrom Ltd., Cambridge Science Park, Cambridge, UK). The calibration standard was prepared using amino acid standard solutions (Sigma A6407 for acidic and neutral amino acids and Sigma A6282 for basic amino acids). For each variety and nitrogen parameter, values were calculated as the average of the values obtained in each year of study. The total free amino acids (TAN) were calculated by summing the concentrations of all quantified free amino acids (mgN/L). The concentration of assimilable amino acids (AAN) was determined by subtracting the concentrations of hydroxyproline (Hyp) and proline (Pro) (mgN/L) from the TAN value. Yeast assimilable nitrogen (YAN) (mgN/L) was obtained by adding the ammonia values (mgN/L) to the AAN value. Aromatic precursor nitrogen (APN) was calculated by adding the amino acid concentrations of Asp, Ile, Leu, Phe, Thr, Tyr and Val (mgN/L). Sulphur-containing amino acid nitrogen (SAN) (mgN/L) was calculated from the contribution of Cys, Met and Tau.

2.5. Data Analysis

The winter of 2021 was unusually cold, characterized by a severe snowstorm followed by a prolonged cold spell in the Madrid region. These extreme weather events caused frost damage to grapevine tissues, affecting vegetative and reproductive development and resulting in high variability in physiological, agronomic and biochemical parameters throughout the growing season and at harvest. Due to this variability, must samples from 2021 were excluded from the analysis.

In order to minimize interannual variability in amino acid content, the relative contribution of each amino acid to total amino acid nitrogen (TAN) was calculated for each grapevine variety, which allowed for the characterization of its amino acid profile. Similarity relationships between vine varieties were assessed via cluster analysis based on these amino acid profiles. The hierarchical clustering method was used to generate similarity dendrograms, which allowed for the identification of groups of varieties with similar amino acid profiles. All calculations were performed using the Addinsoft XLSTAT Bases 2015 statistical package.

3. Results

3.1. Characterization of Varieties

As

Table 4. Average must parameters in minority varieties at harvest during the study period (2020–2023).

White Varieties	°Brix ¹	CV ²	pH ¹	CV ²	Titratable Acidity ¹	CV ²	Red Varieties	°Brix ¹	CV ²	pH ¹	CV ²	Titratable Acidity ¹	CV ²
Air	20.70	0.07	3.66	0.03	3.98	0.11	Azg	19.50	0.05	3.72	0.05	2.88	0.31
APz	23.30	0.07	3.61	0.02	4.64	0.20	Ben	23.20	0.03	3.29	0.03	5.86	0.11
Aur	22.37	0.09	3.45	0.04	5.70	0.08	Cdt	22.60	0.05	3.58	0.06	3.50	0.22
CBl	22.50	0.04	3.23	0.03	5.63	0.05	Cpa	16.65	0.07	3.36	0.00	5.13	0.10
Cgz	21.47	0.06	3.60	0.04	4.35	0.06	GTi	22.27	0.03	3.38	0.03	4.42	0.26
Heb	21.40	0.06	3.68	0.09	4.58	0.09	Grn	19.50	0.02	3.52	0.01	4.50	0.08
Jar	23.37	0.07	3.46	0.05	4.73	0.30	LPt	23.30	0.02	3.92	0.01	2.73	0.01
Luc	22.80	0.05	3.59	0.07	5.10	0.28	Mte	21.63	0.05	3.43	0.04	5.50	0.14
Mal	22.10	0.04	3.63	0.07	3.56	0.05	RMe	22.10	0.06	3.42	0.04	5.15	0.24
Mfñ	21.55	0.06	3.96	0.08	3.26	0.54	Rub	22.55	0.07	3.57	0.00	3.75	0.19
Mnr	23.13	0.15	3.39	0.04	6.81	0.23	San	20.33	0.03	3.11	0.04	6.72	0.18
Pin	21.35	0.06	3.45	0.05	5.40	0.21	Taz	23.53	0.08	3.90	0.04	5.66	0.29
Sal	22.15	0.04	3.69	0.02	4.38	0.12	Tem	22.33	0.04	3.80	0.06	3.80	0.07
Tzn	22.50	0.00	3.57	0.01	4.64	0.21	Ter	17.95	0.07	3.45	0.07	4.95	0.41
Vse	23.00	0.06	3.52	0.04	4.94	0.26	TBd	24.30	0.03	3.54	0.09	4.00	0.18
Zur	21.77	0.03	3.69	0.01	3.35	0.11	TNa	20.50	0.04	3.80	0.00	3.63	0.15
							TFg	23.87	0.02	3.47	0.03	5.06	0.08
							TTa	22.43	0.07	3.34	0.03	5.82	0.26

¹: Average value of total soluble solids (°Brix), pH and titratable acidity (g tartaric acid /L) of the must between the 2020 and 2023 campaigns; ²: the coefficient of variation (CV).

displays, the °Brix of the must at harvest ranged from 20.70 to 23.37 °Brix in the white varieties and 16.65 to 24.30 °Brix in the red varieties. It is noteworthy that in ‘Jarrosuelto’ and ‘Albillo del Pozo’ (white varieties) and ‘Tinto Bastardo’, ‘Tinto Fragoso’, ‘Listán Prieto’ and ‘Benedicto’, the °Brix average exceeded 23 °Brix. Varieties such as ‘Castellana Blanca’, ‘Benedicto’, ‘Sanguina’ and ‘Tortozona Tinta’ have been identified with pH values below 3.5, in combination with suitable levels of soluble solids (°Brix) and titratable acidity. These values are considered favorable for ensuring microbiological stability and promoting optimal alcoholic fermentation performance.

3.2. Nitrogen Parameters

3.2.1. White Varieties

Figure 1a,b illustrate the concentration of nitrogen parameters averaged over the period 2020–2023 for the white grape varieties. Figure 1a displays the mean values of TAN, YAN and AAN for the analyzed grapevine white varieties, while the mean values of APN and SAN are shown in Figure 1b.

The TAN values ranged from 101.50 mgN/L in ‘Lucomol’ to 281.37 mgN/L in ‘Albillo del Pozo’, which also exhibited the highest AAN (240.71 mgN/L) and YAN (217.82 mgN/L) concentrations. In contrast, the lowest AAN and YAN contents were registered in ‘Tortozón’ (86.09 mgN/L) and ‘Lucomol’ (71.16 mgN/L), respectively. Overall, ‘Albillo del Pozo’ displayed higher nitrogen compound concentrations than the other white varieties (Figure 1a).

For APN, ‘Albillo del Pozo’ had the highest concentration (30.76 mgN/L), while ‘Lucomol’ had the lowest (8.37 mgN/L). The lowest SAN concentration was observed in ‘Airén’ (0.06 mgN/L), whereas ‘Albillo del Pozo’ again exhibited the highest value (2.15 mgN/L) (Figure 1b).

The maximum YAN value (240.71 mgN/L) corresponds to 85.55% of the estimated TAN (281.37 mgN/L), while the minimum YAN (86.09 mgN/L) and TAN (101.50 mgN/L) values are 84.82%. The minimum APN concentration (8.37 mgN/L) is 8.25% of the minimum TAN (101.50 mgN/L), while the maximum APN concentration (281.37 mgN/L) is 10.93% of the maximum TAN value (30.76 mgN/L) (Figure 1a,b). The variety with the highest ammonium concentration is ‘Verdejo Serrano’ (29.59 mgN/L), while the variety with the lowest ammonium concentration is ‘Tortozón’ (10.98 mgN/L) (Table S1).

3.2.2. Red Varieties

Figure 2a and Figure 3b present the mean values of the analyzed nitrogen fractions across grapevine varieties. In Figure 2a, the TAN values range from 83.06 mgN/L to 305.88 mgN/L, recorded for the varieties ‘Terriza’ and ‘Tazazonal’, respectively. Likewise, the AAN concentrations are lowest in ‘Terriza’ (66.88 mgN/L) and highest in ‘Tazazonal’ (240.83 mgN/L), and the YAN concentrations exhibit a similar pattern, with values ranging from a minimum of 80.99 mgN/L to a maximum of 270.24 mgN/L in the same varieties as observed for TAN and AAN. Figure 2b shows the APN and SAN concentrations. ‘Terriza’ exhibited the lowest APN concentration (7.05 mgN/L), whereas ‘Tempranillo’ had the highest (38.34 mgN/L), followed by ‘Terriza’ with the second-highest APN concentration (31.83 mgN/L). With respect to SAN, ‘Tazazonal’ exhibited the highest value for this nitrogen fraction, while ‘Tinto de Navalcarnero’ (1.04 mgN/L) and ‘Terriza’ (1.10 mgN/L) had the lowest.

The maximum YAN value (270.24 mgN/L) is 88.35% of the maximum TAN estimate (305.88 mgN/L), while the minimum YAN value (80.99 mgN/L) is 97.51% of the minimum TAN value (83.06 mgN/L). The minimum APN concentration (7.05 mgN/L) is 12.53% of the minimum TAN value (83.06 mgN/L), while the maximum APN concentration (38.34 mgN/L) is 8.49% of the maximum TAN value (305.88 mgN/L). The red variety with the lowest ammonium concentration is ‘Cadrete’ (10.38 mgN/L), while the highest is ‘Tempranillo’, with a concentration of 33.68 mgN/L (Table S2).

3.3. Amino Acid Profile

3.3.1. White Varieties

In white varieties, the following amino acids have been identified as the most abundant on the basis of their mean concentrations (mg N/L) across all analyzed varieties (mg N/L): L-Arg (67.41), Pro (36.45), Amm (21.90), Gaba (8.89), L-Ala (7.63), Glu (7.05), Thr (5.70), Ser (5.50), L-His (5.46) and Hypro (4.13). Conversely, the amino acids present in the lowest concentrations include Hylys (0.13), L-Met (0.13), Aaba (0.21), Pea (0.29), PhSer (0.51), Urea (0.52), Gly (0.56), Tyr (0.67), Baiba (0.82) and L-Orn (0.93). In general, the highest values were observed in Pro and L-Arg. The grapevine varieties with the highest Pro concentrations were ‘Albillo del Pozo’ (56.28 mgN/L), ‘Salvador’ (51.63 mgN/L) and ‘Castellana Blanca’ (47.49 mgN/L), while the lowest concentrations were recorded in ‘Marfileña’ (25.36 mgN/L), ‘Lucomol’ (27.36 mgN/L) and ‘Hebén’ (27.60 mgN/L). For L-Arg, the highest concentrations were observed in ‘Albillo del Pozo’ (103.46 mgN/L), ‘Aurea’ (91.70 mgN/L) and ‘Pintada’ (91.56 mgN/L), whereas the lowest concentrations were found in ‘Lucomol’ (28.56 mgN/L), ‘Tortozón’ (36.16 mgN/L) and ‘Airén’ (46.64 mgN/L).

‘Albillo del Pozo’ stands out for its high concentrations of Thr (13.21 mg N/L), Ser (10.61 mg N/L), L-Ala (15.41 mg N/L) and GABA (14.73 mg N/L) compared to the average concentrations of these amino acids across all analyzed varieties: Thr (5.70 mg N/L), Ser (5.50 mg N/L), L-Ala (7.64 mg N/L) and GABA (8.90 mg N/L), respectively.

Similarly, the concentration of Asn is particularly high in ‘Jarrosuelto’ (8.16 mg N/L) and ‘Zurieles’ (15.63 mg N/L) compared to the average concentration across all analyzed varieties (3.47 mg N/L). ‘Salvador’ also exhibits notably elevated levels of certain amino acids, such as L-Ala (12.91 mg N/L) and Ser (10.59 mg N/L), relative to their average concentrations across varieties: L-Ala (7.64 mg N/L) and Ser (5.50 mg N/L), respectively.

It should be noted that ‘Lucomol’ and ‘Tortozon’ are white varieties with the lowest concentration of amino acids at both general and specific levels.

It is noteworthy that ‘Verdejo Serrano’ exhibited the highest standard deviations for most of the analyzed amino acids, particularly for L-Arg (61.6), Pro (19.2), GABA (12.3), L-His (10.6), Val (8.3) and Asn (8.2). Additionally, with respect to the rest of the amino acids identified and quantified, Pro, L-Arg, GABA, L-Ala, Asn and L-His showed higher standard deviations in all analyzed varieties. All these values are presented in the Supplementary Materials (Table S1).

3.3.2. Red Varieties

The most abundant amino acids in red varieties were identified according to their average concentration (mgN/L): L-Arg: 66.28, Pro: 36.23, Amm: 22.58, Gaba: 7.92, Glu: 7.12, L-Ala: 6. 79, L-His: 5.27, Thr: 5.18, Ser: 4.74 and Hypro: 3.86; those with lower abundance are as follows: Baiba: 0.46, Urea: 0.48, Phser: 0.51, Gly: 0.56, Tyr: 0.65, L-Phe: 0.70, Citr: 0.85, B-ala: 0.93 and L-Lys: 0.99.

The highest Pro concentrations were observed in ‘Tazazonal’ (62.28 mgN/L), ‘Tinto Fragoso’ (58.64 mgN/L) and ‘Sanguina’ (56.51 mgN/L), while the lowest were found in ‘Terriza’ (13.91 mgN/L), ‘Crepa’ (16.41 mgN/L) and ‘Rayada Melonera’ (18.32 mgN/L). Similarly, ‘Tazazonal’ exhibited the highest L-Arg concentration (109.25 mgN/L), followed by ‘Rayada Melonera’ (98.75 mgN/L), ‘Garnacha Tinta’ (98.17 mgN/L) and ‘Tempranillo’ (94.13 mgN/L), whereas the lowest concentrations were recorded in ‘Terriza’ (32.20 mgN/L), ‘Azargón’ (35.60 mgN/L) and ‘Morate’ (39.05 mgN/L).

‘Tempranillo’ displayed high concentrations of all analyzed amino acids, particularly L-Ala (16.57 mgN/L), GABA (13.33 mgN/L), L-His (13.53 mgN/L), Glu (11.33 mgN/L), Val (9.62 mgN/L), Asn (8.68 mgN/L), Thr (9.88 mgN/L) and Ser (9.96 mgN/L),) exceeding the average value across all varieties (4.74 mgN/L). ‘Tazazonal’ exhibited a similar but less pronounced trend, with notable concentrations of L-Ala, GABA, L-His, Glu, Val and Thr. ‘Garnacha Tinta’ also had elevated levels of GABA (10.77 mgN/L), L-His (8.35 mgN/L) and Asn (15.81 mgN/L). Conversely, varieties such as ‘Terriza’, ‘Crepa’, ‘Cadrete’ and ‘Tinto de Navalcarnero’ registered particularly low amino acid concentrations, both overall and individually.

Standard deviations were highest in ‘Tempranillo’ for L-Arg (61.6), Ser (4.2), GABA (8.4), I-Ile (7.0), L-Leu (7.1), Val (10.0), L-Ala (5.3) and Asn (10.5). Additionally, amino acids such as Asn, L-Ala, GABA, L-His, L-Arg and Pro showed greater variability compared to the others (Table S2).

3.4. Classification of Varieties by Amino Acids

The dendrograms (Figure 3) illustrate the similar relationships between the different white (Figure 3a) and red (Figure 3b) grape varieties, based on the proportion (%) of each amino acid profile with respect to TAN. This hierarchical analysis has made it possible to identify groups of varieties by their similarity in one or more elements of their amino acid profiles (Figure 4 and Figure 5). Varieties are grouped into three clusters according to their degree of similarity, with lower links indicating greater similarity between them. The length of the horizontal branches reflects the dissimilarity between the clusters, providing a clear visualization of the relationships between different varieties.

3.4.1. White Varieties

Hierarchical analysis of white grapevine varieties revealed similarity patterns among certain varietal groups. Clustering indicated comparable amino acid profiles, suggesting possible shared physiological adaptations. This distribution may help identify varieties with potentially similar enological characteristics, with applications in varietal selection and winemaking strategies (Figure 3a and Figure 4).

The groups are mainly characterized by the different contributions of Pro and Arg to TAN. Group 1 consists of the varieties ‘Tortozón’ (Tzn), ‘Castellana Blanca’ (CBl), ‘Airen’ (Air) and ‘Lucomol’ (Luc). Their characteristic amino acid profile is displayed in Figure 4. The contribution of Pro to TAN ranged from 26.49% to 29.66% and Arg from 28.14% to 36.32% in these varieties. In addition, this group is characterized by high contents of Glu (6.22%), GABA (5.70%) and L-Ala (4.19%), while Citr (0.08%), L-Met (0.02%) and HyLys (0.00%) are the least abundant amino acids. The most representative variety within this group is ‘Castellana Blanca’ (CBl), as its amino acid contribution percentages closely align with the group’s average values. As Figure 3a displays, group 2 includes the varieties ‘Montonera’ (Mnr), ‘Albillo del Pozo’ (APz), ‘Salvador’ (Sal), ‘Verdejo Serrano’ (Vse), ‘Marfileña’ (Mfñ), ‘Zurieles’ (Zur) and ‘Jarrosuelto’ (Jar) by degree of similarity. In these varieties, the contributions of Pro (16.02% to 23.93%) and Arg (32.08% to 40.52%) to TAN were lower and higher than in group 1, respectively. The group is also characterized by relatively high average levels of Gaba (5.58%), L-Ala (4.37%) and Thr (3.52%), while Aaba (0.12%), L-Met (0.11%) and Hylys (0.08%) contribute the least. The most representative variety of this group is APz, as its amino acidic profile closely matches the group’s average. Finally, ‘Malvar’ (Mal), ‘Cagarrizo’ (Cgz), ‘Pintada’ (Pin), ‘Hebén’ (Heb) and ‘Aurea’ (Aur) belong to group 3. The typical amino acid profile of this group is shown in Figure 4. In the amino acid profile of the varieties included in this group, the highest contributions of Arg (42.17% and 46.22%) and the lowest of Pro (0.00% to 20.37%) to TAN were observed. This group is distinguished by higher average levels of Glu (3.98%), L-Ala (3.94%) and GABA (3.81%), while Aaba (0.13%), HyLys (0.09%) and L-Met (0.04%) have the lowest contributions. The most representative variety is Mal, as its amino acid composition aligns closely with the group’s average.

3.4.2. Red Varieties

In red grapevine varieties, hierarchical analysis revealed greater diversity in the relative composition of free amino acids. Although some groups shared similar profiles, a broader dispersion among varieties was observed, which may reflect higher genetic heterogeneity or differential responses to agroclimatic conditions, including vintage effects. These differences have relevant implications for must quality and the assessment of fermentation potential (Figure 3b and Figure 5).

Group 1 includes the varieties ‘Benedicto’ (Ben), ‘Azargón’ (Azg), ‘Listán Prieto’ (LPt), ‘Sanguina’ (San), ‘Tinto Fragoso’ (TFg) and ‘Morate’ (Mte), in order of similarity. According to the typical amino acid profiles showed in Figure 5, the contribution of Pro to TAN in these varieties ranges from 23.36% to 28.64%, and for Arg, from 25.88% to 38.92%. In addition, this group is characterized by high average levels of GABA (5.14%), Glu (4.54%) and L-Ala (4.01%), while Aaba (0.14%), L-Met (0.07%) and HyLys (0.03%) are the least abundant. The most representative variety is Ben, as its amino acid profile closely matches the group’s average.

The Group 2 consists of ‘Tortozona Tinta’ (Tna), ‘Granadera’ (Grn), ‘Cadrete’ (Cdt), ‘Terriza’ (Ter), ‘Garnacha Tinta’ (GTi), ‘Tinto Bastardo’ (TBd), ‘Tempranillo’ (Tem), ‘Tazazonal’ (Taz) and ‘Crepa’ (Cpa), ordered by similarity. Their typical amino acidic profile is shown in Figure 5 and is characterized by high average levels of Arg (32.84% to 44.70%), Pro (14.90% and 22.25%), GABA (4.58%), Glu (4.42%) and L-Ala (3.98%), while Aaba (0.13%), L-Met (0.11%) and HyLys (0.02%) contribute the least. Grn is the most representative variety, with amino acid contributions closely aligned with the group’s average.

The varieties Rub, RMe and TTa form group 3. These varieties are distinguished from the others by an amino acid profile (as shown in Figure 5) that is characterized by higher percentages of Arg, ranging from 49.65% to 53.52%. Additionally, the group is characterized by average levels of Glu (4.39%), L-Ala (2.93%) and GABA (2.90%), while Aaba (0.15%), Hylys (0.05%) and L-Met (0.03%) are the least abundant. The most representative variety is Rub, as its amino acid composition closely matches the group’s average.

4. Discussion

In this study, amino acid profiles for minority varieties were determined over the 2020, 2022 and 2023 seasons. To the best of our knowledge, this is the first time the amino acid profile of many of these minority varieties has been determined.

Since the quantity and amino acid profile are highly influenced by edaphoclimatic and cultivation factors [45], it is essential to consider the edaphoclimatic characteristics of the study area when interpreting the obtained data. Thus, each year, these temperatures exceeded the historical monthly averages recorded between 1957 and 2019. Additionally, extreme seasonal variations due to climate change were observed, including milder and shorter winters that were −2 to 3 °C warmer than usual, along with a tendency for summer temperatures to occur earlier than their historical seasonal average. Moreover, the extension of typical summer temperatures indicates that summers not only start earlier but also last longer than usual, with thermal sensations reflecting this shift. In July and August, temperatures were recorded at 3 to 5 °C above the historical average. Finally, as mentioned above, the severe winter snowstorm that followed affected the biochemical parameters of that year and probably the vegetative and reproductive development in subsequent years. In addition, high temperatures and extreme heat waves during the summer seasons of 2022 and 2023 further influenced these parameters. Since only the thermal variation was considered as a factor modifying the physiological expression of the plant material, since all the varieties are grown under the same semi-arid conditions characteristic of the region, within the same vineyard.

Given the importance of nitrogenous parameters, it is worth highlighting the high values of TAN, found in the white variety ‘Albillo del Pozo’ and in the red ‘Tazazonal’. (1701 and 1810.57, respectively when the contents are expressed in mg/L). These values registered were higher than those observed in other widely cultivated white and red varieties across different locations in Spain, such as ‘Airen’ in Albacete (1600 mg/L), ‘Cigüente’ in Badajoz (1627 mg/L), ‘Verdejo’ in Valladolid (1385 mg/L), ‘Tempranillo’ in Badajoz and ‘Garnacha’ in Logroño (746 mg/L). On the other hand, an interesting aspect of the results shown in Figure 1a and Figure 2a is the standard deviation of the TAN values across the different grape varieties. Due to the interannual variation, it provides insights into the resilience of each variety to annual meteorological variations. Notably low standard deviation values were observed for ‘Heben’ (81.72), ‘Salvador’ (119.48) and ‘Tortozón’ (73.59) among the white varieties and for ‘Sanguina’ (28.35) and ‘Terriza’ (68.52) among the red varieties, indicating greater stability under variable climatic conditions [46,47].

It is also important to note that in general, the varieties with high TAN levels also exhibited high YAN values (Figure 2a). The importance and quantity of YAN lies in the potential that this nitrogenous compound has to modify the rate of fermentation and the composition of the wine to be made [44]. The authors refer to a threshold between 140 and 150 mgN/L [27] as the minimum necessary to complete the fermentation of musts harvested with normal sugar levels. Values below these thresholds suggest delays and the risk of a stop in the fermentation period, causing high concentrations of sulfur compounds that develop undesirable aromas in the wine [48]. Regarding the white varieties, APz stands out for its high YAN concentration (240.71 mgN/L), followed by Pin (205.43 mgN/L), and the values of Mfñ (139.47 mgN/L), Heb (150.12 mgN/L), CBl (151.78 mgN/L) and Mal (153.74 mgN/L) are close to those estimated for the effective start of fermentation; also, particularly low YAN concentrations were registered in Luc and Taz, below 100 mgN/L.

In the red varieties, it is noteworthy that high values were found in Taz. Conversely, the values found in TNa, Mte, TTa and TBd are close to those found in Merlot (around 111 mgN/L). It should be noted that the quantities of the varieties considered as reference varieties—Tem and GTi (270.24 mgN/L and 261.53 mgN/L, respectively)—were higher than those obtained by other authors for the same variety in another locations (175 and 187 mgN/L) [49,50].

Given that various authors have highlighted the variations in YAN caused by edaphoclimatic causes and cultivation factors, such as water supply, foliar applications, fertirrigation and the use of elicitors, among others [18,21,45,46,50], it is important to note that all cultivars were grown under identical edaphoclimatic conditions. These findings underscore the significance of the varietal factor in YAN values. Additionally, they may provide preliminary insights into winemaking strategies (e.g., the necessity of nutrient additions to the must) that could be applied to the minority varieties investigated.

Finally, given the importance of fermentation-derived volatile compounds in the aroma of white wines, it is noteworthy that ‘Albillo del Pozo’ exhibited an APN value of 30.76 mg N/L, which is substantially higher than that of the reference varieties ‘Airén’ (9.78 mg N/L) and ‘Malvar’ (15.44 mg N/L). As APN represents the sum of the primary amino acid precursors of volatile compounds responsible for the fermentation aroma, grape varieties with higher APN levels are expected to produce white wines with enhanced aromatic intensity.

In general, all amino acids were in the standard range of concentrations found for these compounds [27,47]. Although different amino acid profiles were obtained for various varieties, certain common traits were observed across all of them. Regardless of whether they were white or red varieties, and in agreement with previous studies that reported their prevalence at all stages of berry ripening, proline and arginine were the most abundant amino acids [1,7]. Since proline cannot be utilized by yeast, as mentioned below, it is important to consider its contribution to total assimilable nitrogen (TAN). In white varieties such as CBl, Luc, Tzn and Air, its contribution is higher (exceeding 26.00%), whereas in Zur, Mfñ and Heb, lower concentrations are observed (<16.00%). In red varieties, the proportion of proline is greater than 30.00% in Mte and TFg, while it remains below 15.00% in Gti and RMe. Furthermore, studies have reported that high concentrations of this amino acid can provide information on the degree of water stress experienced by the vine [18]. Therefore, these findings may provide an initial assessment of the drought resistance of these varieties [45].

Regarding L-Arg, the contribution to TAN, in general, is higher in the red (around 40.00%) than in the white varieties (close to 38.00%), including Rub, RMe and TTa (red varieties) and Aur, Cgz and Mal (white varieties); however, Ar did not reach 30% in Luc and Mte. It is very important to confirm that they follow the trend of other studies in terms of a higher concentration among the rest of the amino acids [5,45].

The Pro-to-Arg ratio is commonly used to classify grape varieties according to their ability to accumulate either one or the other of these two amino acids [27,32]. As can be deduced from the proline–arginine content (Tables S1 and S2) the Pro-to-Arg ratio was higher than 1 only in the white variety ‘Lucomol’ (1.00) and in the red varieties ‘Tinto Fragoso’ (1.04), ‘Azargón’ (1.31) and ‘Morate’ (1.36). Therefore, according to the results, with this exception, the rest of the minority varieties investigated are an arginine accumulator variety, since they accumulate a greater proportion of assimilable nitrogen (Arg) instead of non-assimilable nitrogen (Pro). With respect to the Pro and Arg contents reported in certain white grape varieties, such as ‘Airén’, ‘Albariño’, ‘Chenin blanc’, ‘Gewürztraminer’, ‘Godello’, ‘Muscat gordo’, ‘Sauvignon blanc’ and ‘Treixadura’, and red, such as ‘Syrah’, ‘Merlot’, ‘Garnacha’ and ‘Pinot Noir’, they behaved as Arg accumulator varieties, whereas ‘Chardonnay’ and ‘Semillon’ behaved as Pro accumulator varieties [50]. Among the white varieties, the highest values of these amino acids were found in group 2 (APz, Mfñ, Mnr, Sal, Vse and Zur). Notably, APz, Vse and Mnr exhibited the highest contribution of Thr, while the highest levels of L-Ile, L-Leu and Val were recorded in Vse. Regarding the red varieties, the highest contribution of aroma precursor amino acids was found in Cdt, Cpa, Grn, GTi, Taz, TBd, Tem, Ter and TNa, which belong to group 2, particularly in Grn, Tem and Taz. Ter and TNa exhibited the highest contribution of aroma precursor amino acids, with Grn, Tem and Taz showing the maximum values for Thr and Val. Meanwhile, Cpa and Cdt had the highest levels of Asp within the group. In general, the varieties from group 2 are of particular interest for potential evaluation in terms of vinification and the development of aromatic profiles during the winemaking process. These varieties may serve as promising candidates for further studies on their potential in shaping aromatic profiles. In contrast, Tzn and Luc were identified as the white varieties with the lowest APN values, falling into group 1, which includes those with the lowest amino acid contributions among the evaluated varieties.

Among the groups created for the identification of similarities in the amino acid profiles of red varieties, the high concentrations of Tem, in addition to other nitrogen compounds (YAN and TAN) and amino acids in particular, correspond to those identified by other authors [50] and studies evaluating the potential of the variety under different irrigation treatments and their effect, among others, on the expression of general values of amino acid precursors of aromas [45].

In general, although the estimated values of each amino acid in each variety and the nitrogen parameters follow a trend within the normal estimates regarding a characterization, it is important to note that the amino acid composition in the must is determined mainly by genetic factors and that they suffer modifications due to the action of environmental and cultural management factors.

5. Conclusions

The determination of the amino acid profiles of 30 minority grape varieties (14 red and the rest white), grown under identical edaphoclimatic conditions during four consecutive vintages, confirms the varietal character of these profiles. Proline and arginine were the predominant amino acids found in all the samples, depending on the variety. Cluster analysis allowed for the grouping of varieties based on similarities in their amino acid profiles. These clusters were defined primarily by the relative contributions of proline and arginine to the total amino acid content. In addition, the different levels of other amino acids—particularly those considered to be precursors of fermentation-derived aroma compounds—will assist in the development of tailored winemaking strategies for each variety.