Inﬂuence of Ripening Stage on Quality Parameters of Five Traditional Tomato Varieties Grown under Organic Conditions

: Consumers demand traditional, tasty tomatoes in contrast to new hybrid cultivars that have poor sensory characteristics. Some physicochemical parameters (total soluble solids, pH, titratable acidity, color and ﬁrmness), functional properties (vitamin C, lycopene, β -carotene and total antioxidant activity) and sensory characteristics of ﬁve traditional tomato varieties (T1-T5) in three ripening stages, grown under organic conditions, were evaluated. These were compared to the commercial hybrid ‘Baghera’, grown in the same conditions. Firmness of all varieties declined and the color parameters L* and H* decreased, whereas a* and a*/b* increased progressively with ripening. Lycopene also increased with ripening in all varieties, with the highest content being 132.64 mg kg − 1 fw for T4. All traditional tomato varieties were richer in lycopene than commercial ones in the two last ripening stages. Vitamin C content ranged widely between 27.33 and 267.27 mg kg − 1 fw. Among the traditional varieties, T2 (BGV003524) stood out due to its highest total soluble solids and vitamin C contents, total antioxidant activity, and H* and a*/b*; this variety was also the most appreciated by panelists. These traditional varieties could be an alternative to commercial cultivars, as they have good quality characteristics and can satisfy consumer demand for organic produce.


Introduction
Tomato (Solanum lycopersicum L.) is one of the most widely cultivated vegetables both for fresh and processed produce. In 2020, tomato production for fresh consumption was 186.8 million tons in the world, of which 4.3 million tons were produced in Spain, which is the second largest producer in Europe after Italy [1]. Commercial hybrids are usually employed in this crop; one of these is the cultivar 'Baghera', that is widely cultivated in Spain due to its early production, medium-size, uniformly colored fruits and resistance to the main diseases of this crop.
The widespread consumption of tomatoes makes them a major source of health-related compounds [2]. Tomato antioxidants include vitamin C and carotenoids, such as lycopene and β-carotene. These antioxidants play an important role in protecting cells and cellular components against oxidative damage [3,4]. Lycopene is the carotenoid responsible for the red color in tomatoes [5], and accounts for more than 80% of the total carotenoids in ripe tomatoes [6]. The reported benefits of lycopene include prevention of degenerative diseases, such as certain types of cancer [7,8] and cardiovascular diseases [9,10]. Tomato antioxidant concentrations are related to genotype, but also to factors such as ripening stage, cultivation practices and climatic environment [11,12].
The most important external criterion to assess ripeness in tomato is color, which is directly related to the perception of quality. Tomatoes are usually consumed at their maximum organoleptic quality, i.e., when red color appears but before excessive softening occurs [13].
There is a growing interest in promoting the survival of traditional tomato varieties, as consumers find them more tasty and healthy [14,15]. Organic horticulture represents a sustainable and alternative agricultural model with the potential to provide both environmental improvements and high quality output [16]. In many regions, traditional landraces of tomato are still cultivated on a small scale for local consumption [17], and they are sold at higher prices than modern hybrids, as they are greatly appreciated by consumers [15,18,19]. It is necessary to characterize the quality and nutritional attributes of traditional tomato varieties, because only some phenotypic descriptors are available [20,21].
The aim of this work was to assess variations in the physicochemical parameters and bioactive compounds of five traditional tomato varieties, grown under organic conditions, in three ripening stages, and to compare these with the commercial hybrid 'Baghera' produced in the same conditions.

Plant Material
Tomato fruits were grown in open air under organic conditions in the south west of Spain, according to Spanish regulations, in one season. The study was carried out in an experimental field at the Scientific and Technological Research Center of Extremadura (CICYTEX) at an altitude of 217 m above sea level (38 • 51 N; 6 • 40 W). The climate is semi-temperate, with around 550 mm of rainfall and mild winters. The soil texture is sandy loam, pH 6.8, and low organic matter content. Plants of all cultivars were nursery produced and transplanted outdoors on the same date, in a randomized complete block design with three replications. The experimental unit was a plot of 15 m 2 . Trellising was used to grow tomato plants that were pruned to a single stem.
The traditional tomato varieties studied were T1, T2, obtained from the Centre for the Conservation and Improvement of Agrodiversity (COMAV, Valencia, Spain), T3, T4, and T5 from the Institute of Agricultural and Food Research and Development (IMIDA, Murcia, Spain). The commercial hybrid used in this study was 'Baghera' from (Clause, Portes lès Valence, France) (Table 1, Figure 1). Tomato samples were picked in a single harvest and visually classified at three different ripening stages: turning (turning), light-red (light-red) and red (red), according to the color ripeness chart established by the United States Standards for Grades of Fresh Tomatoes [22]. Ten fruits per variety and ripening stage were randomly selected and analyzed in the laboratory for total soluble solids content (TSS), titratable acidity (TA), pH, firmness and color. Firstly, color and firmness measurements were performed on whole tomatoes, and then the fruits were homogenized for analysis of TSS, TA and pH. In addition, 10 fruits per variety and ripening stage were packed and stored at −80 • C for carotenoids, vitamin C, and total antioxidant activity (TAA) analyses. Finally, a sensory evaluation was carried out with tomato samples in the light-red stage.

Firmness
For each variety and ripening stage, firmness was evaluated on ten fruits by a compression test using a TA.XT2i Texture Analyzer (Aname, S.L., Madrid, Spain). Measurements were taken at two opposite points on the equator of each fruit with a plane

Firmness
For each variety and ripening stage, firmness was evaluated on ten fruits by a compression test using a TA.XT2i Texture Analyzer (Aname, S.L., Madrid, Spain). Measurements were taken at two opposite points on the equator of each fruit with a plane compression plate (100 mm diameter), a speed of 2 mm s −1 and a fruit deformation of 2%. Maximum force (N) and slope of the force/deformation curve (N mm −1 ) were registered.

Color
Color measurements were performed on the surface of ten tomatoes per variety and ripening stage with a Konica Minolta CM-3500d Spectrophotometer (Aquateknica, S.A., Valencia, Spain). Reflectance measurements were made with the primary illuminant D65 and a circular measurement area of 30 mm diameter. Color readings were taken on two different positions of the equatorial region of each fruit. The CIEL*a*b* parameters were directly obtained and then Hue angle (H* = tan −1 (b*/a*)), Chroma (C* = (a* 2 + b* 2 ) 1/2 ) and a*/b* ratio were calculated.

Carotenoids
Lycopene and β-carotene were determined by HPLC, according to the method described by Sabio et al. [25] in an Agilent 1100 HPLC chromatograph (Agilent Technologies, Inc., Palo Alto, CA, USA) with an Agilent LiChrosorb RP-18 4.6 × 200 mm-10 µm column maintained at 30 • C and after extraction with acetone: ethanol: hexane. The flow rate was 1 mL min −1 , peaks were detected by DAD at 460 nm and quantification was carried out using lycopene and β-carotene standards solutions. Extractions were carried out in triplicate and the results were expressed as mg kg −1 fw.

Vitamin C
Vitamin C was assessed as ascorbic acid (AA) by HPLC with UV detection according to González-Cebrino et al. [16]. Fruits were sliced and homogenized using a domestic blender, and then mixed and extracted with EDTA/metaphosphoric solution (85%) on ice for 1 min (Omni Mixer Homogenizer, Omni International, Kennesaw, GA, USA). The extracted sample was filtered (Millipore 0.45 µm) and injected into a 1050 HPLC chromatograph (Agilent Technologies, Inc., Palo Alto, CA, USA) with an Agilent Zorbax SB-C8 4.6 × 250 mm-5µm column maintained at 30 • C. A buffer solution of 50 mM acetic acid/acetate (pH 4) was used as the mobile phase. The flow rate was 0.5 mL min −1 and detection was at 260 nm. Quantification was carried out using AA standards solutions. Analyses were carried out in triplicate and the results were expressed as mg of vitamin C kg −1 fw.

Total Antioxidant Activity
Total antioxidant activity (TAA) was estimated in triplicate, according to the method established by Cano et al. [26] with some modifications. Extractions were carried out using 1 g of tomato homogenate diluted in 50 mM Na phosphate buffer (pH 7.5). Then, 20 µL of diluted homogenate was placed in a spectrometric cuvette and 1 mL of the radical cation ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) diammonium salt (Sigma-Aldrich, Madrid, Spain) was added. The reaction was monitored in a UV-2401 PC Shimadzu spectrophotometer (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA) at 730 nm, and the decrease in absorbance was determined after 20 min. The results were expressed as mg of Trolox 100 g −1 fw.

Sensory Evaluation
The sensory panel comprised eight trained judges from our institution, all of whom were regular consumers of fresh tomatoes. Each panelist was asked to rate the following sensory attributes: color, sourness, sweetness, juiciness, and consistency. The evaluation was carried out with samples in the light-red stage. Firstly, tomatoes were cut into slices of approximately 1 cm thickness and labeled with a random number. The sensory panel evaluated the intensity of the attributes on a 10-point scale, where 0 represented "no intensity" and 10 represented "strong intensity". No information about tomato varieties was provided to the panelists.

Statistical Analysis
The results were analyzed using SPSS 21.0 statistical software (SPSS Inc, Chicago, IL, USA) by one-way analysis of variance (ANOVA) by ripening stage and variety, and when differences were detected, mean values were compared with Tukey's test (p < 0.05). Correlations were performed using Pearson's correlation coefficient (r, p < 0.01) to identify significant interactions among studied variables.

Total Soluble Solids, Titratable Acidity and pH
The total soluble solid content, pH and tritatable acidity results are presented in Table 2, and acceptability and flavor indexes in Figure 2. TSS content increased with ripening for all varieties, showing the highest values at the last stage (red), except in T5, which tended to decrease with maturity. At turning and light-red, T2 had the highest TSS values, whereas in the red stage 'Baghera' showed the highest value together with T2, although the latter was without significant differences with T1 and T4 (Table 2). Ruiz et al. [19] also recorded a higher mean value for a commercial variety in the red stage. Although pH values showed differences among varieties, there was a narrow range of variation: T4 had the lowest value at turning, whereas the maximum was recorded for both T2 and T5 in the light-red stage. T2 and T3 showed a clear decrease in tritatable acidity with ripening, and together with T5, had the lowest TA values in the red stage. In most varieties, the Acceptability and Flavor Indexes were higher in the red stage. T5 and T1 had the highest values of the Acceptability Index at turning, and T2 at lightred. The latter also had the highest Flavor Index at all ripening stages ( Figure 2). For all varieties and ripening stages, Flavor Index values were higher than 0.85, which was de- In most varieties, the Acceptability and Flavor Indexes were higher in the red stage. T5 and T1 had the highest values of the Acceptability Index at turning, and T2 at light-red. The latter also had the highest Flavor Index at all ripening stages ( Figure 2). For all varieties and ripening stages, Flavor Index values were higher than 0.85, which was described by Hernández et al. [24] as the minimum required value for an acceptable flavor in tomato.

Firmness
It is known that firmness is a tomato quality parameter closely associated with ripening stage [27]. It can be seen in Table 2 and Figure 3 that the firmness of each tomato variety was strongly affected by the ripening stage, and force (N) and slope (Nmm −1 ) values exhibited a clear decrease as ripening progressed. Therefore, firmness is a useful criterion for assessing the ripening stage of tomato fruits. Although tomatoes harvested in the red stage could be considered the best-tasting tomatoes, these fruits are more fragile and can become easily damaged; thus it is better to harvest them at an earlier stage in order to extend their shelf-life and prevent damage from occurring during handling. Differences in maximum force (N) and slope (Nmm −1 ) were observed among the tomato varieties studied (Table 2, Figure 3). T2 and 'Baghera' recorded the highest firmness values at turning. In the red stage, all the traditional tomato varieties had similar or higher firmness values than the commercial hybrid 'Baghera'.

Color
Most of the external color parameters decreased with ripening except a* and a*/b*, which increased (Table 3 and Figure 4). The decrease of L* with ripening reflected the darkening of the fruits with carotenoid synthesis, the degradation of chlorophylls and, consequently, the disappearance of greenness [28,29]. As in other studies [13,28], a* values increased from turning to red as a consequence of lycopene synthesis. C* is not a good indicator of tomato ripening because it is essentially an expression of the saturation of a single color, and as a result, different colors may have the same chroma values [13], as was the case for T4 and 'Baghera'.  Differences in maximum force (N) and slope (Nmm −1 ) were observed among the tomato varieties studied (Table 2, Figure 3). T2 and 'Baghera' recorded the highest firmness values at turning. In the red stage, all the traditional tomato varieties had similar or higher firmness values than the commercial hybrid 'Baghera'.

Color
Most of the external color parameters decreased with ripening except a* and a*/b*, which increased (Table 3 and Figure 4). The decrease of L* with ripening reflected the darkening of the fruits with carotenoid synthesis, the degradation of chlorophylls and, consequently, the disappearance of greenness [28,29]. As in other studies [13,28], a* values increased from turning to red as a consequence of lycopene synthesis. C* is not a good indicator of tomato ripening because it is essentially an expression of the saturation of a single color, and as a result, different colors may have the same chroma values [13], as was the case for T4 and 'Baghera'.
Among the studied varieties, there were differences in mean values for all color parameters. At turning, T2 revealed the lowest significant values for a*, C* and a*/b* ratio, and the highest significant values for L* and H*. The a * value was slightly negative (−2.87), corresponding to an under-ripe stage and associated with a major accumulation of chlorophylls. In contrast, at light-red, T2 showed the lowest values for L*, b*, C*, and H*, and the highest values for a* and a*/b* ratio. Finally at red, T2 presented the significantly highest value of a*/b* (1.74), indicating a more intense red color (Figure 4).    The ratio a*/b* and H* are often used as indicators of tomato color changes [13,30]. For all varieties, the a*/b* ratio and H* values showed differences among the three ripening stages. There was a progressive decrease in H*, whereas a*/b* ratio increased, as observed in other studies [28,29].

Carotenoids
For lycopene and β-carotene content, differences were found among the studied varieties ( Figure 5). Lycopene ranged from 6.57 mg kg −1 fw for T2 (turning) to 132.64 mg kg −1 fw for T4 (red). The highest significant lycopene content at turning and light-red was found for T5, whereas T4 was the variety with the highest concentration at red. All the traditional varieties showed higher lycopene content than the commercial hybrid 'Baghera' in the two last stages, although other authors found no differences between commercial and traditional varieties in the red stage [14]. Our tomato varieties showed higher carotenoid content than those reported by Pinela et al. [31] in local farmers' varieties from Portugal. Lycopene content varied significantly among tomato varieties [32] and can be also affected by environmental factors and agricultural techniques [11,33]. Roselló et al. [34] reported values from 2.72 to 73.77 mg kg −1 fw for five traditional varieties. In organic production, the average lycopene content has been reported at 56 mg kg −1 fw for 13 commercial varieties harvested at the fully ripe stage [30], and Murariu et al. [33] found that the lycopene content of red tomatoes ranged from 9.53 to 3.60 mg 100 g −1 fw depending on the genotype and the production system. The values obtained in this work were considerably higher than those reported by these authors, mainly in the red stage.
Lycopene content is an internal quality attribute associated with the color and ripening process of tomatoes [28,[35][36][37]. In the studied samples, the lycopene content increased with ripening, on average more than four-fold from the turning to red stages, reaching the maximum concentration in the red stage in all varieties ( Figure 5). Lycopene showed a strong Pearson's correlations with different color and firmness parameters. There were positive correlations with a* (0.832) and a*/b* ratio (0.772), and negative with L* (−0.799), H* (−0.801), force (−0.775) and slope (−0.811).
Although lycopene is the most abundant carotenoid in red-ripe tomatoes, β-carotene content was also evaluated. This carotenoid is important because of its pro-vitamin A activity, and it accumulates during tomato fruit ripening [38]. For the studied samples, β-carotene content varied from 5.66 to 16.75 mg kg −1 fw. T5 showed the highest content in all stages, but no significant differences were found with T1 at turning. Roselló et al. [34] reported β-carotene values from 1.18 to 8.76 mg kg −1 fw for five traditional varieties, and Pinela et al. [31] values from 0.30 to 0.51 mg 100 g −1 for Portuguese local varieties. The highest concentrations of β-carotene were found in all varieties at light-red, except for T2, which recorded the maximum level in the red stage, as Gautier et al. [39] reported for other varieties.

Vitamin C
Vitamin C content ranged between 27.33 for T2 (turning) and 267.27 mg kg −1 fw for T1 (red), with differences depending on variety and ripening stage (Table 4). et al. [34] reported values from 2.72 to 73.77 mg kg −1 fw for five traditional varieties. In organic production, the average lycopene content has been reported at 56 mg kg −1 fw for 13 commercial varieties harvested at the fully ripe stage [30], and Murariu et al. [33] found that the lycopene content of red tomatoes ranged from 9.53 to 3.60 mg 100 g −1 fw depending on the genotype and the production system. The values obtained in this work were considerably higher than those reported by these authors, mainly in the red stage. Lycopene content is an internal quality attribute associated with the color and ripening process of tomatoes [28,[35][36][37]. In the studied samples, the lycopene content increased with ripening, on average more than four-fold from the turning to red stages, reaching the maximum concentration in the red stage in all varieties ( Figure 5). Lycopene showed a strong Pearson's correlations with different color and firmness parameters. There were positive correlations with a* (0.832) and a*/b* ratio (0.772), and negative with L* (−0.799), H* (−0.801), force (−0.775) and slope (−0.811).
Although lycopene is the most abundant carotenoid in red-ripe tomatoes, β-carotene content was also evaluated. This carotenoid is important because of its pro-vitamin A activity, and it accumulates during tomato fruit ripening [38]. For the studied samples, βcarotene content varied from 5.66 to 16.75 mg kg −1 fw. T5 showed the highest content in all stages, but no significant differences were found with T1 at turning. Roselló et al. [34] reported β-carotene values from 1.18 to 8.76 mg kg −1 fw for five traditional varieties, and Pinela et al. [31] values from 0.30 to 0.51 mg 100 g −1 for Portuguese local varieties. The highest concentrations of β-carotene were found in all varieties at light-red, except for T2, which recorded the maximum level in the red stage, as Gautier et al. [39] reported for other varieties. At turning both T5 and T1 revealed the highest significant concentrations (176.22 and 175.05 mg kg −1 fw, respectively), at light-red 'Baghera' and T2 (187.43 and 183.73 mg kg −1 fw, respectively), and at red T1 (267.27 mg kg −1 fw). These values were higher than those found by Adalid et al. [2], who reported concentrations of between 51 and 183 mg kg −1 for 15 traditional varieties, mainly at a pink stage. For organic tomatoes grown in open air, the vitamin C content observed by Murariu et al. [33] ranged from 11.61 to 29.21 mg 100 g −1 fw in the red stage.
As ripening progressed, changes in vitamin C content were observed in the studied varieties (Table 4), in contrast to the results of Raffo et al. [40], who indicated some ascorbic acid variation for cherry tomatoes during ripening. In T1, T4 and T5, vitamin C content decreased slightly from turning to light-red stage and then increased at red. On the other hand, in T2, T3 and 'Baghera' vitamin C accumulated during the first stages and then decreased at red, according to other authors [41]. A positive Pearson's correlation between vitamin C and lycopene (0.642) and a negative with H* (−0.608) were found in the studied tomato samples.

Total Antioxidant Activity (TAA)
TAA values showed differences depending on variety and ripening stage (Table 4), ranging from 28.05 to 85.41 mg Trolox 100 g −1 fw, corresponding to T2 at turning and light-red, respectively. The highest TAA values were found in T4 at turning (69.83 mg Trolox 100 g −1 fw), in T2 at light-red (85.41 mg Trolox 100 g −1 fw) and in both T4 and T2 at red. On the other hand, the commercial hybrid 'Baghera' showed the lowest values in the light-red and red stages, although without significant differences with T1 and T3.
Ascorbic acid, together with phenolic compounds, represents the main water-soluble antioxidants in tomato [40], but it must be taken into account that the antioxidant properties of tomatoes largely depend on their lycopene content [37]. Significant correlations between lycopene content and antioxidant activity have been previously reported [38,42]. However, in our study, no such significant correlation was observed.
Most of the tomato varieties did not show differences in TAA values as ripening advanced, as reported by Cano et al. [42]. T2 and T5 exhibited an increase from the turning to the red stage, with the highest value at light-red. In general, higher TAA levels were observed in tomatoes harvested at light-red, as found by Raffo et al. [40].
There was also a negative Pearson's correlation (−0.617) between H* and TAA, as previously described for lycopen and vitamin C, so H* could be used as a good indicator of functional quality in tomato.

Sensory Evaluation
As far as we know, very few sensory analyses of traditional tomato varieties have been carried out. The results of the sensory evaluations carried out with the tomato varieties in the light-red stage are depicted in Figure 6. There were significant differences for color, sweetness and juiciness. However, no significant differences were found for sourness and consistency. T4 showed the best value for sourness (7.22) and T2 for consistency (8.16). With respect to color, T3 was the best considered variety (8.16), while no significant differences were found with T1, T2 and 'Baghera'. The highest average score in juiciness corresponded to T2 (8.12), without significant differences with T1, T3 and T4. There was also a negative Pearson's correlation (−0.617) between H* and TAA, as previously described for lycopen and vitamin C, so H* could be used as a good indicator of functional quality in tomato.

Sensory Evaluation
As far as we know, very few sensory analyses of traditional tomato varieties have been carried out. The results of the sensory evaluations carried out with the tomato varieties in the light-red stage are depicted in Figure 6. There were significant differences for color, sweetness and juiciness. However, no significant differences were found for sour ness and consistency. T4 showed the best value for sourness (7.22) and T2 for consistency (8.16). With respect to color, T3 was the best considered variety (8.16), while no significant differences were found with T1, T2 and 'Baghera'. The highest average score in juiciness corresponded to T2 (8.12), without significant differences with T1, T3 and T4. T5 was the variety that was least appreciated by panelists. In contrast, T2 achieved the highest scores for sourness and sweetness, which is in accordance with its high values of AI and FI, since these indexes are related to sweetness, sourness and flavor perception T5 was the variety that was least appreciated by panelists. In contrast, T2 achieved the highest scores for sourness and sweetness, which is in accordance with its high values of AI and FI, since these indexes are related to sweetness, sourness and flavor perception.

Conclusions
Variety and ripening stage have a strong influence on the physicochemical variables and bioactive compound contents of tomatoes. In general, the traditional varieties showed similar physicochemical characteristics and Acceptability and Flavor Indices to those of 'Baghera', although in the red stage, they presented concentrations of lycopen, vitamin C and TAA higher than those 'Baghera'. As expected, the firmness of all the varieties declined and the color parameters L* and H* decreased with ripening, whereas a* and a*/b* increased progressively.
In the turning stage, T2 together with 'Baghera' showed the highest values of TSS and firmness, while T1 and T5 stood out in terms of their lycopene and vitamin C contents. In the light-red stage, T2 was notable for its highest TSS and vitamin C content, as well as TAA, whereas T5 presented the greatest carotenoid content. In the red stage, T4 exhibited remarkable levels of lycopene, vitamin C and TAA. The traditional tomato T2 (at light-red) was the variety that was most appreciated by panelists for sweetness and sourness, two major descriptors related to the final taste of tomatoes.
In summary, most of the traditional studied varieties had good quality characteristics and could meet the increasing consumer demand for tasty and healthy food, since they also presented higher concentrations of bioactive compounds at full ripeness.