Influence of Treatment with Natural Phytoregulators on Purple Carrots (Daucus carota L.) during Cold Storage

Abstract

In this work, we investigated the effect of natural phytoregulators (i.e., methyl jasmonate and abscisic acid) on quality physicochemical parameters, carotenoid and anthocyanin content and antioxidant activity of purple carrots in storage at 5 °C for 21 days. As a result, study of the natural evolution of fresh-untreated purple carrots in storage exhibited increase in carotenoids (from 1.41 to 3.79 mg EβC g⁻¹ DW) and stability of anthocyanins (2.18 vs. 2.23 mg ECGg⁻¹ DW) but significant loss of organoleptic quality. Treatment with methyl jasmonate and abscisic acid resulted in similar or even higher carotenoid content (1.61 and 2.15 mg EβC g⁻¹ DW for methyl jasmonate and abscisic acid, respectively) as compared with the value measured in fresh-untreated carrots before storage (1.41 mg EβC g⁻¹ DW). In contrast to carotenoids, anthocyanins and antioxidant activity mostly decreased with the treatments. However, physicochemical parameters indicating organoleptic quality improved considerably, which was meaningful considering their importance in terms of consumer acceptance. These results reflect the slowing-down effect of natural phytoregulators on spoilage of purple carrots over storage. Optimization of this approach is scheduled to minimize anthocyanin losses. It can therefore be an interesting approach to extending purple carrot shelf-life with no need for artificial preservatives.

1. Introduction

Carrot (Daucus carota L.) is one of the most widely cultivated vegetables in the world. Its consumption has progressively increased in the last few years thanks to its pleasant flavor and the health benefits related to its nutritional value. Carrot is an important source of carotenes, minerals, vitamins and dietary fiber [1]. Apart from the conventional orange-root carrots, there are materials with different colors owing to the accumulation of different carotenoids and anthocyanin pigments. In particular, black or purple carrots are rich in anthocyanins, which are known for their health-promoting properties. In general, diets including both pigment types, carotenoids and anthocyanins, have been associated with reduction in the risk of chronic pathologies such as cardiovascular, cancer, stroke and neurodegenerative diseases [2].

Nevertheless, the chemical composition of foods is often altered over the storage period, which affects both their sensorial characteristics and nutritional value. Carotenoids are usually degraded in the presence of oxygen, light, high temperatures and certain enzymes due to their highly unsaturated structure [3]. Also, anthocyanins are modified by pH, enzyme activity, oxygen and, in particular, high temperatures [4]. In relation to organoleptic properties of carrots, it is common to observe loss of water content and firmness, slimy texture, discoloration, bitterness and an oxidized odor [5]. For these reasons, it is essential to carefully select the storage conditions with attention to assuring sensory and health-related quality.

Several methods to store carrots exist, such as freezing, drying or canning. However, they bring about undesired modifications in sensorial quality as compared with fresh roots. Specifically, softer texture in frozen carrots has not yet been avoided with any freezing technique reported to date [6]. Also, discoloration and development of an off-flavor is still observed in dried carrots, whereas addition of salt, sugar and preservatives, together with a bizarre metallic taste, is currently the result of canning [7].

In this context, refrigeration is undoubtedly the most efficient method to store carrots. Nevertheless, the temperature has to be carefully controlled in order to achieve a high-sensorial quality product [8]. In this respect, it is interesting that milder temperatures (i.e., around 20 °C) have been demonstrated to offer advantages over cold temperatures, since they promote increase in carotenoids [9]. In addition, room temperature storage has been recommended rather than colder values to minimize anthocyanin degradation in black carrot juice [10]. On the other hand, it is evident that the temperature used to store carrots must always guarantee the absence of rooting and decomposition. In this regard, raw carrot shelf-life without refrigeration varies from 5 to 7 days. The use of cold temperature can prolong this period until 3 or 4 weeks at the most [11]. We intended to develop a procedure based on the use of refrigeration that enabled purple carrot shelf-life to be prolonged by means of preservation of organoleptic characteristics and, as much as possible, health-related compounds.

In this context, the exogenous application of natural phytoregulators has already been described to enrich plant-based foods in biologically active compounds such as carotenoids and anthocyanins [12,13,14]. Nonetheless, the effect of natural phytoregulators on dark-colored carrots has been scarcely studied in general [15,16] and, in particular, with regards to their deterioration during storage, no studies can be found in the literature up to now.

The aim of this research was to study the effect of the post-harvest treatment with natural phytoregulators in combination with cold temperatures on purple carrot quality in terms of both organoleptic and health-promoting properties. To that end, quality physicochemical parameters, content in carotenoids and anthocyanins and antioxidant activity were measured. Our final intention was to propose an approach enabling purple carrot shelf-life to be extended by means of natural preservative.

2. Materials and Methods

2.1. Chemicals and Reagents

HPLC-grade methanol (MeOH), acetone, ethyl acetate and acetonitrile were purchased by Macron Fine Chemicals (Radnor Township, PA, USA). Hexane and chloroform were obtained from LabScan (Bangkok, Thayland). Ultrapure water was obtained from a purification system (Macron Fine Chemicals, USA). 2,2-diphenyl-2-picrylhydrazil (DPPH), methyl jasmonate (MJ), abscisic acid (ABA), lanolin, Β-carotene, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), potassium chloride, sodium hydroxide (NaOH), sodium acetate anhydrous standards were all supplied by Sigma-Aldrich (Steinheim, Germany). Ethanol (EtOH), pentane, cyclohexane, acetic acid glacial and hydrochloric acid (HCl) were acquired from Scharlau Chemie S.A. (Barcelona, Spain) and lutein standard was obtained from Extrasynthese (Genay, France).

2.2. Samples

Fresh purple carrot roots (Purple Haze variety, Figure 1) were supplied by a local producer (Madrid, Spain). The variety, used for both fresh market and processing, was characterized by a dark purple color with an orange core and 3–5 cm in diameter. After reception, samples were split into three different groups. Carrots in the first group were immediately analyzed to be used as a reference (so-called fresh-untreated on day 0). Samples in the second group were stored at 5 °C in the dark for 21 days to assess the natural progress of purple carrots without treatment under the specific storage conditions used in this study (so-called fresh-untreated on day 21). Finally, carrot roots in the third group were first subjected to the treatments with MJ and ABA and subsequently stored as explained below (so-called MJ-treated and ABA-treated, respectively).

2.3. Treatments

For the experiments, the same size carrots with no sign of sprouts or any other damage were selected. The application of MJ and ABA was carried out at room temperature. To consider root-to-root variation, every treatment was performed in duplicate by using different roots for each phytoregulator. Based on our previous results, MJ and ABA were chosen to carry out the treatments for their higher effectiveness [13,17,18]. Approximately 25 mg of the phytoregulator (i.e., 0.2 mg g⁻¹) was mixed with lanolin paste, which was prepared by merging lanolin with distillated water (1:2 w/w). This mixture was applied on one half of the root by using a spatula (treated), while the other half was treated with lanolin paste without the phytoregulator (control). The four treated carrots (i.e., two for each phytoregulator) were stored at 5 °C in the absence of light for 21 days. After this time, the paste was carefully removed from the carrots using dry paper, and control and treated samples were analyzed. The whole experiment was repeated twice. Overall, this implies four different replicates for MJ and four for ABA.

2.4. Physicochemical Parameters

The physicochemical properties of fresh-untreated (on day 0 and on day 21) and treated (with MJ or ABA) purple carrots were studied by determining juiciness, moisture content, total soluble solid (TSS) content, acidity (as both pH and total titratable acidity, TAA) and maturity index. Prior to the actual measurements, juice was obtained from purple carrots by following this procedure: carrots were cut and put in a blender. The resulting paste was then centrifuged at 20 °C and 10,000 rpm for 15 min. The juice obtained was separated from the remaining paste by vacuum filtration, and the physicochemical parameters were immediately measured.

2.4.1. Juiciness

The juiciness of the samples was estimated from weight ratio percentage between the juice obtained and the carrot root used in its preparation.

2.4.2. Moisture Content

The moisture content of the purple carrots was calculated gravimetrically. Purple carrots were sliced and ground with a food processor. After that, the carrot powder obtained was dried in a conventional stove at 105 °C until constant weight. The weight difference indicated the moisture content.

2.4.3. Total Soluble Solids (TSS)

TSS was determined by applying the method described in the literature [19]. An Atago digital refractometer, model dbx-30 (Labexchange, Westwood, MA, USA), was used to perform the measurements. Just a drop of carrot juice was placed onto the refractometer. The result was obtained from the mean value of three measurements for each sample. The results were expressed as a percentage of brix degrees and grams of sucrose per dry weight (DW).

2.4.4. Acidity

Active acidity (or hydrogen concentration pH)

The active acidity was calculated by measuring the pH using a 913 pH-meter (Metrohm, Madrid, Spain). To that end, the corresponding electrode was just sunk into a 50 mL volume of purple carrot juice.

Total titratable acidity (TTA)

The determination was accomplished by acid–base titration. A 50 mL volume of juice was neutralized with NaOH solution (0.1 N). A pH of 8.2 was fixed to consider the endpoint for titration. The amount of base used until neutralization showed the acid content. The results were expressed as a percentage of grams of major organic acid and grams of organic acid per DW.

2.4.5. Maturity Index

The maturity index was estimated from the ratio between the TSS values and the TTA values. The numeric value of the ratio expresses the maturity index: the higher the numerical value, the more advanced the root will be in the post-harvest maturity stage.

2.5. Health-Related Quality

The carotenoid and anthocyanin content and the antioxidant activity via DPPH and FRAP assays were determined in fresh-untreated and treated purple carrot samples as detailed below.

2.5.1. Determination of Carotenoids

Extraction

Purple carrots were ground in a food processor. Isolation of carotenoids was carried out by following the procedure described elsewhere [20]. In brief, a 20 mL volume of acetone was added to 2 g of ground carrot. Then, the mixture was homogenized by using an Ultra-Turrax (T18 Digital, IKA, Staufen, Germany) for 5 min. After that, it was passed through a filter paper. Additional acetone was used to sweep the remaining extract until the sample became colorless. Subsequently, a 25 mL volume of hexane was added, and the mixture was shaken. Finally, the acetone phase was discarded, and the hexane layer was up to 50 mL. Total carotenoid content (TCC) and individual carotenoids were determined in the extracts obtained as explained below.

Total carotenoid content (TCC)

A spectrophotometer (Beckman Coulter DU-800 spectrophotometer, Barcelona, Spain) was used to accomplish the measurements. The absorbance of the extracts was registered at 485 nm and all the measurements were performed twice. TCC was determined by applying the extinction coefficient (E_1% = 2500) [21]. Results were expressed as µg of β-carotene equivalents (EβC) per g of DW.

Individual carotenoids by HPLC

The content of individual carotenoids in purple carrot extracts was determined by HPLC (Alliance Separation Module 2695, Waters, Mildford, CT, USA) equipped with an automatic injector and a photodiode array detector 996 (DAD, Waters, Mildford, CT, USA). The separation was carried out on a 3.9 mm × 150 mm reversed-phase Nova-Pak C₁₈ column (particle size 4 µm, Waters, Milford, CT, USA). The mobile phase consisted of acetonitrile–methanol–ethyl acetate (73:20:7, v/v/v), and isocratic mode was used. Based on the literature [22] and on the test of different flow rate values, the analyses were performed at 0.3 mL min⁻¹. The chromatographic signals were registered at 450 nm by using the software (Empower2 software, Waters, Mildford, CT, USA). Carotenoids (i.e., lutein and β-carotene) were identified by comparison with the retention time of the corresponding standards. In addition, the identification was verified with bibliographic information [22]. Quantitative determination of carotenoids was accomplished by using calibration curves of β-carotene. All analyses were performed in duplicate. The results were expressed as µg EβC per g of DW.

2.5.2. Anthocyanins

Extraction

Anthocyanins were extracted from fresh-untreated and treated purple carrots by following the procedure reported elsewhere [23]. In brief, 50 mL of EtOH:H₂O (1:1, v/v) containing 0.01% HCl (37% v/v) was added to 25 g of ground sample. Then, the mixture was stirred for 2 h at room temperature. The extract was purified by adding chloroform (2 × 25 mL), pentane (2 × 25 mL) and cyclohexane (2 × 25 mL). The aqueous fraction was properly separated, and the solvent was removed at 30 °C until a final volume of 25 mL. The extract obtained was stored in the dark at −20 °C. Each extraction was carried out in duplicate. Total anthocyanin content (TAC) and antioxidant activity via DPPH and FRAP assays were determined in the extracts obtained, as described below.

Total anthocyanin content (TAC)

TAC was determined by applying the pH differential method [24]. In brief, carrot extracts were diluted with two different buffer solutions: 0.025 M potassium chloride at pH 1 and 0.4 M sodium acetate at pH 4.5. The measurements were performed by using the same equipment as that used for TCC and at two different wavelengths (i.e., 520 and 700 nm). The results were expressed as µg of cyanidin-3-O-glucoside equivalents (EC3G) per g of DW. The absorbance was calculated by applying the Equation (1), for which the molar extinction coefficient (i.e., 26,900 L cm⁻¹) and the molecular weight of C3G (i.e., 449.4 g/L) were used.

Abs_t = (Abs_{520 nm} − Abs_{700 nm}) _{pH = 1} − (Abs_{520 nm} − Abs_{700 nm}) _{pH = 4.5}

(1)

2.5.3. Antioxidant Activity

The antioxidant activity of the samples was evaluated in terms of the free radical scavenging activity by using the DPPH assay [25] and in terms of the capacity to reduce ferric ion (Fe³⁺) to ferrous iron (Fe²⁺) by the FRAP assay [26]. A BioTek Synergy HT multi-mode microplate reader with BioTek’s Gen 5TM software (BioTek Instruments Inc., Winooski, VT, USA) and 96-well microplates was used for the measurements. The working DPPH^● reagent was prepared by dissolving DPPH^● standard in methanol (1000 µM), whereas the working FRAP reagent was made by mixing acetate buffer (0.3 M) at pH 3.6 with 2,4,6-tripyridyl-s-triazine (TPTZ) in HCl (40 mM) and FeCl₃·6H₂O (20 mM) at a ratio of 10:1:1. The same procedure was applied for both assays. A 290 µL volume of the reagent (i.e., DPPH^● or FRAP) was added to 10 µL of the sample in each well. Subsequently, the mixtures were incubated at 37 °C for 30 and 20 min in the dark, respectively. The absorbance was measured at 515 nm for the DPPH assay and at 593 nm for the FRAP assay. The values of absorbance provided by the DPPH^● or FRAP reagent solutions were used as a blank in each case. A Trolox standard curve was used to obtain the data, and all analyses were performed in duplicate. When necessary, extracts were diluted to be adapted to the linear range of the curve. Results were expressed as mg of Trolox equivalents per g of DW.

2.6. Statistical Study

An analysis of variance for TCC, TAC, DPPH and FRAP data was performed using the one-way analysis of variance (ANOVA) method. The results are presented as the average of all values obtained and the standard deviation (SD). Data obtained from fresh-untreated and treated samples were statistically compared. Comparisons of means were made by using the Fishers´ protected LSD. Differences were considered significant at p < 0.05.

3. Results and Discussion

3.1. Physicochemical Quality

Visual observation

Comparing fresh-untreated purple carrots on day 0 (Figure 2a) with fresh-untreated ones stored for 21 days (Figure 2b), whitening was apparent as a result of the natural evolution of the samples over storage. The reason for the color change was the formation of lignin as a healing process of wounded tissues [27]. In addition, visible softness and moisture loss were also apparent in untreated roots as compared with fresh carrots on day 0. However, treated samples, whatever the phytoregulator used, maintained very similar color and apparent firmness to that of fresh-untreated samples on day 0 (Figure 2c,d). A comparison between MJ- and ABA-treated carrots reflected brighter color and visually higher water content for the carrots exposed to ABA.

Physicochemical parameters

Figure 3 depicts the moisture content and juiciness of purple carrot samples. As observed, both parameters maintained reasonable steadiness in most samples. Specifically, moisture content always ranged between 86.3 and 88.4% and juiciness between 30 and 35%. No significant (p > 0.05) influence of storage or of elicitation was found. An exception was found in MJ-treated samples, which displayed significantly (p < 0.05) lower moisture content (i.e., 83.8%) and, in particular, juiciness (i.e., 15%).

Figure 4 represents TSS (% °Brix) (a) and acidity, expressed as TTA (%) and pH (b). As seen, TSS increased significantly (p < 0.05) in fresh-untreated purple carrots from day 0 (i.e., °Brix 5.0%) to day 21 (i.e., °Brix 5.65%). This reflects an increase in sugar content in carrot roots during the 21-day cold storage. The enhancement of sugars during ripening and storage has already been described in tubers [28,29] and other vegetables [30]. It is attributed to hydrolysis of polysaccharides into oligosaccharides and monosaccharides. It can also be due to the degradation of pectin substances into simple sugars occurring over ripening [31]. The increase in TSS with storage diminished significantly (p < 0.05) with elicitation, regardless of the phytoregulator used (i.e., 5.25% for MJ and 5.45% for ABA). As seen in the figure, no significant (p > 0.05) differences were found between both phytoregulators. Since TSS increment is related to ripening, the reduction in the TSS increment with elicitation indicates that treatment with MJ or ABA is effective in delaying spoilage and, hence, extending purple carrot shelf-life.

From Figure 4b, TTA, measured as % malic acid, increased significantly (p < 0.05) in fresh-untreated purple carrots with storage (i.e., from 0.08% on day 0 to 0.12% on day 21). The increase of TTA with storage here observed supports findings earlier published by other researchers [28,29]. It is most like due to the formation of acids by oxidation of reducing sugars or by breakdown of pectic substance, which can be in turn converted into organic acids or simple sugars, as previously commented. Regarding treatment effect, the trend found for both phytoregulators was similar. They both reduced TTA increase during the post-harvest period (i.e., 0.10% for MJ-treated carrots and 0.07% for ABA-treated carrots), although it is interesting to point out that only ABA impact was statistically significant (p < 0.05). In fact, ABA-treated carrots provided TTA values similar to those measured in fresh-untreated carrots on day 0 (i.e., 0.07% and 0.08%, respectively). The slowdown of TTA increase over storage as a consequence of elicitation supports the hypothesis above mentioned about the delaying effect of MJ and ABA on the decay rate.

It is also important to mention that the changes in TTA had a positive correlation with changes in pH values, which increased significantly (p < 0.05) from 5.9 in fresh-untreated carrots on day 0 to 6.12 on day 21, whereas the treatment with MJ and ABA resulted in values comparable to that of the fresh-untreated samples on day 0 (i.e., 6.01 and 6.04, respectively). These results are in accordance with those elsewhere reported [28]. Since acidity and pH are inversely proportional to each other, it is believed that protons are released from organic acid hydrolysis in such a way that the lower the acid content in the sample, the higher the hydrogen concentration.

Figure 5 depicts the maturity index of purple carrot samples. Since maturity index is a sugar–acid (TSS:TTA) ratio, it decreased significantly (p < 0.05) in fresh-untreated samples during cold storage (i.e., 62% on day 0 vs. 49% on day 21). This patter is a direct result of the marked increase in TTA measured in fresh-untreated samples throughout the storage period. It is also interesting that the decrease in maturity index was not affected by MJ elicitation. In fact, no significant (p > 0.05) differences between MJ-treated samples and fresh-untreated carrots on day 21 were measured (i.e., 49% vs. 51%, respectively). On the contrary, the exposition of purple carrots to ABA resulted in a preservation of their maturity index over storage as compared with that of fresh-untreated carrots on day 0 (i.e., 75% vs. 62%, respectively).

In view of these results, the post-harvest elicitation of purple carrots by using ABA as a natural phytoregulator was effective in preserving moisture content, juiciness, sugar content and organic acid content throughout cold storage for 21 days. Considering that sugar and organic acid contents determine the tasting quality of a product, elicitation with ABA seems a recommendable approach to preserve physicochemical quality of purple carrots during 21-day cold storage. Additionally, ABA treatment also provided satisfactory results in terms of visual appearance. Although studies in this respect have not been performed with purple carrots, a comparison with bibliographic reports on traditional orange carrots [6,7] suggests that ABA treatment in conjunction with low temperatures is able to overcome the limitations of freezing, drying and canning as a preservation method in terms of sensorial quality. On the other hand, the exogenous application of ABA on the roots during the post-harvest period is a simple, economic and reproducible approach with no need for an exhaustive control, as is the case for the use of refrigeration alone [8].

3.2. Health-Related Quality

Table 1. Total content of carotenoids (TCC) and anthocyanins (TAC) in fresh-untreated purple carrots and purple carrots treated with MJ and ABA.

	Fresh-Untreated		MJ Treatment		AA Treatment
	On Day 0	On Day 21	Control	Treated	Control	Treated
TCC (mg EβC g−1 DW)	1.41 ± 0.03 a	3.79 ± 0.05 b	1.59 ± 0.02 a	1.61 ± 0.03 a	2.06 ± 0.04 c	2.15 ± 0.03 c
TAC (mg ECGg−1 DW)	2.23 ± 0.05 a	2.18 ± 0.04 a	0.43 ± 0.02 b	0.37 ± 0.01 b	0.26 ± 0.01 b	0.30 ± 0.02 b

Different letters between samples in the same row indicate differences at p < 0.05.

shows TCC, expressed as mg EβC g⁻¹ DW, and TAC, expressed as mg EC3G g⁻¹ DW, in fresh-untreated purple carrots on day 0 and day 21 and in MJ- and ABA- treated samples, including their corresponding controls. Data are expressed as mean values (n = 2) ± SD. Different letters between purple carrot samples indicate differences at p < 0.05. From the table, it is interesting that TCC values in fresh-untreated purple carrots on day 0 (i.e., 1.41 mg EβC g⁻¹ DW) were similar to those reported in the literature for the typical orange-rooted carrots (i.e., 1.29 mg g⁻¹ DW) [32]. As was also observed, TCC increased significantly (p < 0.05) in fresh-untreated carrots after cold storage (from 1.41 mg EβC g⁻¹ DW on day 0 to 3.79 mg EβC g⁻¹ DW on day 21). Other authors have also found an increase in typical orange carrot β-carotene during the first 2 or 21 days of refrigerated storage [33,34,35,36]. However, the reason why this increase occurs is not well known. It is speculated that either biosynthesis of carotenoids continues in storage or extractability of β-carotene improves because of the slow degradation of the carrot matrix. In fact, it is possible that both mechanisms are at play in conjunction. On the one hand, carotenoid biosynthesis after harvest has been suggested in many climacteric fruits and tuberous vegetables other than carrots, e.g., tomatoes or sweet potatoes. On the other, it is known that the carrot matrix is disaggregated with time, and it is reasonable to believe that this disaggregation enables carotenoids to be released. In any case, it is obvious that the degradation of carotenoids is always slower than any of the two mechanisms described above. From a nutritional standpoint, the results are interesting regardless of the mechanism involved. There are several factors contributing to the accumulation of carotenoids in fresh-untreated purple carrots during the three-week cold storage, such as relative humidity or chilling stress. In this respect, various pre- and post-harvest factors (i.e., high light, salinity, drought, cultivation practice, etc.) have been reported to positively modulate the enhanced biosynthesis of carotenoids [37]. However, all these studies also describe decrease in carotenoid content in longer storage periods and highlight the extreme importance of the storage conditions.

As also seen in Table 1, TAC in fresh-untreated purple carrots was steadily maintained in storage (from 2.23 mg EC3G g⁻¹ DW on day 0 to 2.18 mg EC3G g⁻¹ DW on day 21). As no bibliographic studies about the storage effect on anthocyanin-rich carrots have been carried out on purple carrot to our knowledge, no comparison with the literature could be properly accomplished. However, works on black carrot juice have revealed the degradation of anthocyanins in a cold environment in such a way that storage at 20 °C is recommendable for longer shelf-life [7]. Supporting this study, the decrease in anthocyanin content as a result of the refrigeration has also been observed by other researchers in plant-based foods other than purple carrots [38].

Regarding treatment effect, the same trend was observed for TCC and TAC. In all cases, for both MJ and ABA, the comparison between those treated with their corresponding controls showed no significant differences (p > 0.05). This reflects the transference of the phytoregulator at some level from the treated half to the control half of the root. However, the statistical comparison of treated purple carrots by using any phytoregulator with fresh-untreated carrots on day 21 exhibited a significant (p < 0.05) drop in both TCC and TAC. This once more confirms the hypothesis on the slowdown of the decay process as a result of the treatments and, therefore, the longer preservation of the sensory quality.

Also, it is interesting to point out the results on TCC obtained from ABA treatment. From Table 1, although ABA treatment values decreased significantly (p < 0.05) with respect to those measured in fresh-untreated samples on day 21, the contents were still significantly (p < 0.05) higher than those on day 0 (2.15 mg of EβC g⁻¹ DW in ABA-treated carrots vs. 1.41 mg of EβC g⁻¹ DW in carrots on day 0). The potential of ABA as a phytoregulator is also supported by the fact that the organoleptic characteristics of ABA-treated carrots after storage were still adequate from a commercial standpoint.

A comparison of the results found here with bibliographic data on conventional orange carrots indicates that ABA treatment provides steadier carotenoid levels than other preservation methods such as freezing and refrigeration, which cause the significant loss of carotenoids [39,40,41]. In contrast, dehydration and canning have been demonstrated to have minimal effects on carotenoid levels, despite their adverse effects on physicochemical quality [40].

As far as anthocyainins are concerned, since no studies on dark-colored carrots similar to the one performed here have been reported so far, no bibliographic contrast could be accomplished. However, there is evidence that any processing in an aqueous medium causes the loss of water-soluble components such as anthocyanins. Therefore, freezing, canning and all drying processes including previous freezing would also bring about anthocyanin losses.

Table 2. Carotenoid content by HPLC-DAD in fresh-untreated purple carrots and purple carrots treated with MJ and ABA.

Carotenoids	Fresh-Untreated		MJ Treatment	ABA Treatment
	On Day 0	On Day 21
LUTEIN (mgEβC g−1 DW)	0.23 ± 0.01 a	0.68 ± 0.03 b	0.18 ± 0.01 a	0.16 ± 0.02 a
β-CAROTENE (mgEβC g−1 DW)	4.87 ± 0.07 a	28.16 ± 0.10 b	2.06 ± 0.05 c	4.93 ± 0.09 a

Different letters between samples in the same row indicate differences at p < 0.05.

represents the HPLC-DAD analysis of carotenoids in fresh-untreated (on day 0 and day 21) and treated (with MJ and ABA) purple carrot samples. Data are expressed as mg EβC g⁻¹ DW (n = 3) ± SD. Different letters between purple carrot samples indicate differences at p < 0.05. Quantification was carried out by interpolating the results of the samples in a calibration curve of β-carotene whose concentrations ranged from 10 µg mL⁻¹ to 300 µg mL⁻¹ (r² = 0.9897). As can be seen in Table 2, lutein and β-carotene were the carotenoids identified in the extracts, the latter being the major one. Some authors have also reported minor concentrations of α-carotene in certain dark-colored carrot cultivars [32,42]. However, its presence depends on the specific cultivar and part of the carrot-root used in the analysis (i.e., core, cortex or a mixture). In general, the darker the cultivar is, the lesser the amount of α-carotene. Similarly, the closer to the core, the lesser the amount of α-carotene [32]. For this reason, the absence of α-carotene is not surprising. In fact, reports in the literature have determined lutein and β-carotene as the main carotenoids present in black-purple carrots [43]. From Table 2, the concentrations of lutein and β-carotene estimated in fresh-untreated purple carrots on day 0 (i.e., 0.23 mg EβC g⁻¹ DW and 4.87 mg EβC g⁻¹ DW, respectively) are in accordance with data described for dark carrot cultivars [42,43]. A comparison among the samples revealed, in general, a similar trend for individual carotenoids as that of TCC. Fresh-untreated purple carrots on day 21 exhibited an increase in lutein and β-carotene with respect to fresh samples on day 0 (i.e., 0.68 and 28.16 mg EβC g⁻¹ DW on day 21 vs. 0.23 and 4.87 mg EβC g⁻¹ DW on day 0). Also, MJ-treated carrots did not show significant (p < 0.05) differences in the content of lutein and β-carotene with respect to fresh-untreated samples on day 0 (i.e., 0.18 and 2.06 mg EβC g⁻¹ DW in MJ-treated vs. 0.23 and 4.87 mg EβC g⁻¹ DW in fresh samples on day 0). In contrast to the results on TCC, ABA-treated purple carrots unexpectedly showed lutein and β-carotene contents similar to those estimated in fresh-untreated samples on day 0 (i.e., 0.16 and 4.93 mg EβC g⁻¹ DW in ABA-treated vs. 0.23 and 4.87 mg EβC g⁻¹ DW in fresh samples on day 0). This is probably due to the determination of additional analytes other than carotenoids together with lutein and β-carotene in the TCC assay.

Table 3. Antioxidant activity by DPPH and FRAP assays in fresh-untreated purple carrots and purple carrots treated with MJ and ABA.

Assay	Fresh-Untreated		MJ Treatment		ABA Treatment
	On Day 0	On Day 21	Control	Treated	Control	Treated
DPPH (mg Trolox g−1 DW)	12.24 ± 0.06 a	12.71 ± 0.05 a	4.73 ± 0.02 b	4.09 ± 0.03 b	4.25 ± 0.02 b	4.89 ± 0.02 b
FRAP (mg Trolox g−1 DW)	17.57 ± 0.08 a	30.03 ± 0.07 a	7.38 ± 0.02 b	6.02 ± 0.02 b	6.37 ± 0.04 b	7.02 ± 0.03 b

Different letters between samples in the same row indicate differences at p < 0.05.

represents the antioxidant activity in terms of free radical scavenging activity, measured by DPPH assay, and the capacity to reduce ferric ion (Fe³⁺) to ferrous iron (Fe²⁺), measured by the FRAP assay. Data are expressed as mean values (n = 2) ± SD. Different lowercase letters between purple carrot samples indicate differences at p < 0.05. Comparing fresh-untreated samples on day 0 with day 21, the free radical scavenging activity did not vary significantly (p > 0.05) throughout the whole storage period (12.24 mg Trolox g⁻¹ DW vs. 12.71 mg Trolox g⁻¹ DW), whereas the ferric reducing power showed a significant (p < 0.05) increase in the values obtained (17.57 mg Trolox g⁻¹ DW on day 0 vs. 30.03 mg Trolox g⁻¹ DW on day 21).

DPPH values were directly related to TAC results. This indicates that anthocyanins were the main contributors to the free radical scavenging activity. From the results on fresh-untreated samples, it can be stated that the antioxidant activity of purple carrot root was not affected by the 21-day cold storage. Concerning the treated samples, no significant (p > 0.05) differences were established between the treated samples and their corresponding controls, whatever the phytoregulator. Furthermore, the application of both MJ and ABA resulted in a significant (p < 0.05) decrease in the antioxidant activity as compared to fresh-untreated samples using either assay.

4. Conclusions

Storage of fresh-untreated purple carrots at 5 °C for 21 days resulted in a three times higher content of carotenoid, as well as in the stability of anthocyanin content and antioxidant activity. However, sensorial quality in terms of sugar and organic acid contents and visual appearance showed evident decay of the roots after the storage time. In fact, decrease in the maturity index from 62% to 49% was observed. The treatment of purple carrots with ABA enabled spoilage rate during the post-harvest period to be reduced, which was reflected in a maturity index of 75%. As a consequence, organoleptic properties were preserved over the whole storage period and, therefore, purple carrot shelf-life was extended. However, the natural increase in carotenoids initially observed in untreated carrots was diminished from three times to barely double in ABA-treated samples. Similarly, anthocyanins decreased from 2.23 mg ECGg⁻¹ DW in fresh-untreated samples to 0.30 mg ECGg⁻¹ DW in ABA-treated ones instead of maintaining steadiness. Since sensorial quality is imperative to ensure the commercial value of foodstuffs, the aim now is to keep organoleptic characteristic improvement, increasing, in turn, the antioxidant activity with a focus on obtaining a long-lasting functional food. For that purpose, further study, including combination of ABA with other natural phytoregulators (i.e., salicylic acid and/or ethanol), together with a deeper optimization process of the storage conditions, is scheduled. The final intention is to propose exogenously applied natural phytoregulators as a tool for extending purple carrot shelf-life in storage with no need for artificial preservatives.