*3.4. Total Polyphenols Content*

Total polyphenols content (TPC) was significantly higher at T<sup>10</sup> (4327 mg GAE kg−<sup>1</sup> DW) that at T<sup>20</sup> (4034 mg GAE kg−<sup>1</sup> DW) (Table 6), but with strong interactive effects with genotype and storage time. Indeed, while 'Eletta' showed no differences among the 2 thermal regimes, TPC was strongly promoted by the lowest thermal regime in 'Sugarland' (+20%), followed by 'Ottymo' (+9%) (Table 6). As regards its temporal trend, TPC significantly increased passing from S<sup>0</sup> to S<sup>7</sup> (+17%) then sharply declined at S<sup>14</sup> (−16%), with a steeper rise in the S0–S<sup>7</sup> period recorded at T<sup>10</sup> (+22%) than at T<sup>20</sup> (+12%) (Table 6). Moreover, the studied genotypes displayed different time-courses of TPC along the storage period, since 'Sugarland' proved the highest TPC rise passing from S<sup>0</sup> to S<sup>7</sup> (+37%) followed by the strongest decline at S<sup>14</sup> (−33%) (Figure 3A).

#### *3.5. Carotenoids Content*

Figure 2 shows the HPLC carotenoids profile extracted from cherry tomato 'Sugarland'. At harvest date, the level of lycopene ranged from 68.1 to 582.5 mg kg−<sup>1</sup> DW in 'Ottymo' and 'Sugarland', respectively, followed by β-carotene, which varied from 72.8 to 82.17 mg kg−<sup>1</sup> DW, in 'Ottymo and 'Eletta', respectively. Among the genotype tested, 'Eletta' proved the highest levels of both phytoene and phytofluene (54.2 and 50.7 mg kg−<sup>1</sup> DW, respectively). The levels determined in 'Sugarland' and 'Ottymo' varied from 31.0 to 38.2 mg kg−<sup>1</sup> DW, for phytoene and from 36.1 to 39.9 mg kg−<sup>1</sup> DW, for phytofluene, respectively.

The phytoene content of the studied genotypes proved different time courses among the 2 thermal regimes, as it significantly increased passing from S<sup>7</sup> to S<sup>14</sup> when the T<sup>20</sup> storage was considered (from 41.2 to 48.3 mg kg−<sup>1</sup> DW, +14%) (Table 6). Among the genotypes, 'Sugarland' proved the highest phytoene rise passing from S<sup>0</sup> to S<sup>7</sup> (from 36.2 to 46.1 mg kg−<sup>1</sup> DW, +28%), whereas in 'Ottymo' a significant increase was recorded between S<sup>7</sup> (35.1 mg kg−<sup>1</sup> DW) and S<sup>14</sup> (45.3 mg kg−<sup>1</sup> DW, +29%) (Figure 3B).


**Table 6.** Nutraceutical variables of cherry tomato as affected by the main factors.

Different letters among factor means indicate significance at Tukey's HSD test (*p* ≤ 0.05). Interaction values (*p* = 0.05) related to 'storage temperature × genotype' and 'storage temperature × storage time' are reported. NS: not significant.

β‐

−

−

−

−

−

 

 

 

 

 

≤

**Figure 2.** HPLC profile of carotenoids extracted from cherry tomato 'Sugarland' at harvest date (S<sup>0</sup> ).

 β‐ **Figure 3.** Total polyphenols (**A**), phytoene (**B**), phytofluene (**C**), lycopene (**D**) and β-carotene (**E**) content as affected by 'genotype × storage time' interaction. Black bars: S<sup>0</sup> ; grey bars: S<sup>7</sup> ; white bars S14.

‐

−

−

−

−

<sup>−</sup> −

−

Regarding phytofluene, the lowest storage temperature showed a depressive effect in 'Eletta' (in which it was reduced by 9%) and the opposite in 'Ottymo' (in which it increased by 10%) (Table 6). Phytofluene content proved also wider temporal oscillations at T20, as the initial value was reduced by 6.4 mg kg−<sup>1</sup> DW at S<sup>7</sup> (−9%), then increased by 6.4 mg kg−<sup>1</sup> DW at S<sup>14</sup> (+19%) (Table 6). Such temporal oscillations proved to be genotype-dependent too, since 'Eletta' showed the highest reduction passing from S<sup>0</sup> (50 mg kg−<sup>1</sup> DW) to S<sup>7</sup> (38.4 mg kg−<sup>1</sup> DW, <sup>−</sup>23%), then the sharpest rise at S<sup>14</sup> (44.5 mg kg−<sup>1</sup> DW, +16%) (Figure 3C).

Lycopene was significantly affected by the storage temperature, as it was lower at T<sup>10</sup> than at T<sup>20</sup> (445 vs. 488 mg kg−<sup>1</sup> DW), and this reduction was more marked for 'Eletta' (−12%) and 'Sugarland' (−6%) (Table 6). Moreover, T<sup>20</sup> promoted a sharper lycopene rise than T<sup>10</sup> passing from S<sup>0</sup> to S<sup>7</sup> (from 416 to 577 mg kg−<sup>1</sup> DW, +39%) followed by a milder decrease at S<sup>14</sup> (484 mg kg−<sup>1</sup> DW, <sup>−</sup>16%) (Table 6). All the studied cultivars showed a significant decrease in lycopene content between S<sup>7</sup> and S<sup>14</sup> (ranging from 119 to 221 mg kg−<sup>1</sup> DW in 'Ottymo and 'Eletta', respectively), with 'Ottymo' and 'Sugarland' proving also a higher lycopene increase between S<sup>0</sup> and S<sup>7</sup> (by 252 mg kg−<sup>1</sup> DW, on average) (Figure 3D).

β-carotene concentration proved to be not sensitive to the storage temperature, and was higher in 'Eletta' (94.0 mg kg−<sup>1</sup> DW) than in the other genotypes (81.9 mg kg−<sup>1</sup> DW, on average) and, over the storage period, increased up to 93.1 mg kg−<sup>1</sup> DW at S<sup>14</sup> (Table 6). However, such temporal increase was more marked in 'Eletta' within the S0–S<sup>7</sup> period (from 82.1 to 100.0 mg kg−<sup>1</sup> DW) and in 'Ottymo' in the S7–S<sup>14</sup> one (from 75.9 to 102.3 mg kg−<sup>1</sup> DW) (Figure 3E).

#### **4. Discussion**

The fruits stored at 10 ◦C showed a higher fruit weight and a lower dry matter content as compared to those stored at 20 ◦C, indicating that fruit transpiration and water loss were the main processes affected by storage temperature. As a consequence, at 20 ◦C tomatoes proved a higher loss of fruit firmness over time. The transpiration-driven softening of tomatoes during postharvest is a major problem, as it increases their susceptibility to damages along the distribution chain [32]. Moreover, fruit firmness is considered a key indicator of tomato freshness, able to influence the purchasing behavior of consumers [33]. However, despite cold storage is commonly practiced for reducing postharvest softening of tomatoes, the opposite effect can be found when too low storage temperatures are used, because of the tropical origin of the plant [8]. For this reason, storage temperature over 11–12 ◦C are advised for storing tomatoes, depending on fruit typology and ripening stage [32–35]. Nonetheless, the differences in terms of fruit weight and firmness we found among the 2 thermal regimes showed that storage at 10 ◦C was a suitable way to extend these main characteristics of tomato fruits. Among the studied cultivars, both 'Sugarland' (small-fruited) and 'Ottymo' (large-fruited) showed the highest fruit weight reduction during storage, consistent with their steeper rise in dry matter content. Differently, 'Eletta' (medium-fruited) proved the highest temporal stability in relation to both variables. Hence our results suggest that the genotypic attitude of cherry tomato to retain fruit weight and firmness during postharvest, is dependent from factors other than simply the fruit size (i.e., the ratio among berry volume and its external transpiring surface) [36], and likely due to the functional traits of the epicarp. Indeed, it has been reported that the dynamics of fruit water loss and consequent tissue collapse are influenced by genotypic differences in structural characteristics of the cuticle, whose alteration over time is an intrinsic feature of the genetically-programmed ripening process [37].

In tomato, the ethylene-driven ripening and senescence lead to the alteration of the carbon substrates content [38], as they are energy-requiring processes whose kinetic is influenced by the ambient temperature [24]. In our experiment, reducing sugars content, the ratio SSC/TA and fruit pH were not affected by the storage temperature, proving instead to be genotype-dependent. Despite their higher increase in dry matter, 'Sugarland' and 'Ottymo' highlighted the steepest drop in reducing sugars content at the end of storage period (by 19%, on average), denoting within the 10–20 ◦C range

a temperature-insensitive acceleration of their autocatalytic metabolism. This demonstrates that no chilling disturbance in reducing sugars metabolism occurred in the experiment [34]. To this end, while the cultivars did not show appreciable pH variations during storage, 'Ottymo' proved the highest SSC/TA reduction over time, denoting its lowest suitability to keep unchanged the taste peculiarities. Indeed, the SSC/TA ratio is a pivotal organoleptic descriptor, as it is related to the overall balance in the perceived sweetness (SSC) and sourness (TA) of tomatoes [39].

Color is one of most important and widely used parameters to define the quality of tomato and tomato products [40]. When fresh tomato fruits are concerned, it is linked to fruit ripeness and firmness and is generally associated by consumers to tomatoes eating quality. In the present experiment, we used an array of chromatic variables summarizing the main color modifications occurring in tomato epicarp. Chroma, (*a*\*/*b*\*)<sup>2</sup> and tomato color index have been related to quality traits of tomato [41,42], whereas ∆*E*\*ab has been successfully used to monitor the quality maintenance of potato sticks during refrigerated storage [43]. All these variables showed a certain variability among the studied cultivars, with two of them, namely Chroma and ∆*E*\*ab, increasing at T10, overall indicating a higher deviation toward more vivid fruit colors. In particular, after 14 days of storage, a higher reduction of Chroma was recorded at 20 ◦C, a condition which matched the strongest decrease in fruit weight and firmness experienced by the studied cultivars. 'Sugarland' and 'Ottymo' proved higher ∆*E*\*ab variations during storage. According to Dattner and Bohn [44], independently from the deviation formula, two colors can be optically distinguished if ∆*E* > 1. The ∆*E*\*ab differences attained by 'Sugarland' and 'Ottymo' (2.47 units, on average) and 'Eletta' (1.43) indicate for the former cultivars a higher perceivable color deviation along the storage period, consistent with their higher qualitative decline in terms of fruit weight and turgor.

When phytochemical composition was concerned, total polyphenols, lycopene and β-carotene contents found in our experiment were substantially in line with those reported by Fernandes et al. [45] for cherry tomato 'Moscatel RZ' grown in hydroponic or semi-hydroponic systems. On the other hand, phytoene and phytofluene contents were very similar to those found in cherry tomato by Mapelli-Brahm et al. [46]. Plant polyphenols are a large group of phytochemicals involved in the regulation of plant growth, reproduction and response to the environmental stressors [47]. From a nutraceutical viewpoint, they have strong antioxidant properties probably implicated in the decreased incidence of cardiovascular diseases and certain forms of cancer [48]. Both thermal regimes promoted a bell-shaped postharvest trend of TPC, consisting in their sharp rise at S7, followed by a decrease at S14, this last indicating the onset of metabolic senescence processes [49]. However, such increase was more marked at 10 ◦C, suggesting the occurrence of a cold-adaptive response in up-regulating the polyphenols expression during postharvest storage. Indeed, several phenolic compounds typically accumulate in plant cells subjected to cold stress, as they contribute to the homeostasis of cold-induced reactive oxygen species (ROS) and to enhance the thickness of the cell wall, so preventing lipid peroxidation and cell collapse [47]. This would explain the best retention of fruit firmness recorded at 10 ◦C, indicating at the same time, the improvement of tomato phenolic profile as a benefit induced by a mild cold stress. Thus, although polyphenols have not been considered a priority target in tomato breeding programs, our results suggest that they could represent a sensitive target for improving the functional profile of the tomato, mostly during postharvest cold storage.

Regarding the carotenoid fraction, we recorded variable effects, resulting from different time-course response to storage temperature and duration. Lycopene displayed a bell-shaped temporal trend too since, under both storage temperatures, this carotenoid sharply increased at S<sup>7</sup> then declined at S14. This trend substantially differed from that of β-carotene which continuously increased until S14, so confirming the higher stability of its postharvest accumulation in tomato [50]. According to Rodriguez-Amaya [51], carotenoids accumulation can continue during postharvest transport or storage, provided that the integrity of the fruit is maintained, so preserving the enzymatic activity responsible for carotenogenesis. Lycopene plays a paramount function in protecting the photosynthetic apparatus and plant lipid membranes, as its acyclic polyene structure (11 conjugated double bonds) increases its

affinity for singlet oxygen and radical scavenging activity beyond the other carotenoids [52]. For this reason, it has been reported that oxidation is the main cause for lycopene degradation [14]. This could partly explain the depressive effect on lycopene content we recorded upon storing tomatoes in a stressing, ROS-inducing environment (10 ◦C). In this view, it is interesting to note the contrasting effect of cold storage on tomato compositional traits, resulting in a higher polyphenols accumulation in case of a lower lycopene content. This suggests the existence of a fine tuning among different classes of compounds in response to cold stress. However, by comparing the temporal trend of lycopene with that of its colorless precursors phytoene and phytofluene, clear time-dependent temperature effects on carotenogenesis where noticeable. Indeed, at S7, the highest lycopene content recorded at 20 ◦C matched the strongest reduction of both phytoene and phytofluene. In other words, the lowest the lycopene concentration the highest the accumulation of its precursors and vice versa. This implies that reduced transformation kinetics of both phytoene and phytofluene represented the earliest metabolic constraints recorded in response to the imposed cold stress. According to Dumas et al. [53] the over-expression of phytoene desaturase (leading to lycopene synthesis by desaturating both phytoene and phytofluene) is the most important upstream metabolic step in increasing the lycopene content of tomato fruits at harvest. Our results bear this out in postharvest conditions too, as they indicate that, under mild cold stress storage conditions, desaturation of phytoene and phytofluene represents the earliest metabolic bottleneck in lycopene synthesis of cherry tomatoes, hence a possible priority target to modulate the postharvest evolution of their nutraceutical profile. On the other hand, to which extent this implies a mid-term modification of the overall nutraceutical profile of tomato represents an interesting topic, taking into account that, despite they are not effective antiradicals as lycopene, phytoene and phytofluene are among the prevailing carotenoids found in human plasma and tissues, and their bioaccessibility following gastro-intestinal digestion of tomato juice has been found ~3–4 fold higher than that of lycopene [54,55].

Among the studied cultivars 'Sugarland' proved the highest lycopene and total polyphenols content, whereas 'Eletta' overcame the other cultivars for phytoene and phytofluene. Excepting β-carotene, which over time increased more sharply in 'Eletta' and 'Ottymo', these differences were still noticeable at the end of the storage period, regardless of the storage temperature. This highlights, beyond the environmental influence, the existence of a strong genetic component determining the stoichiometric relationships among lycopene and its precursors. Unravelling the possible interactive effects among these three carotenoids in generating the antioxidative health benefits [16,49] will allow for a better orientation of breeding programs toward the most convenient phytochemical evolution of tomatoes during refrigerated storage.

### **5. Conclusions**

The results of the present experiment highlighted complex postharvest modifications of cherry tomatoes in response to the studied factors. By storing them under mild stressing conditions (10 ◦C) it was possible to improve the stability over time of carpometric traits (mainly fruit weight, firmness and Chroma) having commercial relevance, without alterations of compositional traits related to taste perception (reducing sugars content, SSC/TA and pH). Moreover, when compared to 20 ◦C, storing at 10 ◦C boosted the accumulation of total polyphenol and, at least in the short term (within 7 days of storage), the concentration of both phytoene and phytofluene, probably inhibiting their enzymatic desaturation leading to lycopene. This suggests their possible usefulness in modulating the nutraceutical evolution of cold stored cherry tomatoes during postharvest. This idea is reinforced by the stable varietal differences we found in terms of stoichiometric relationships among lycopene, phytoene and phytofluene. Regarding the varietal attitude to postharvest storage, the stability over time of fruit weight, dry matter content, SSC/TA and ∆*E*\*ab proved to be highly discriminant among cultivars, indicating the lowest ability of 'Ottymo' and 'Eletta' to maintain their fruit peculiarities over time. Thus, our results suggest the use of these variables to screen for cherry tomato germplasm suited to periods of postharvest storage.

**Author Contributions:** Conceptualization, R.P.M., M.D. and E.A.; methodology, R.P.M., M.D. and S.B.; validation, F.G., R.P.M. and E.A.; formal analysis, M.D. and S.B.; investigation, M.D., R.P.M. and S.B.; data curation, M.D. and S.B.; writing—original draft preparation, R.P.M., E.A. and M.D.; writing—review and editing, F.G., C.L. and B.F.; supervision, F.G., C.L. and B.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
