*Article* **Improvement of Antioxidant Properties in Fruit from Two Blood and Blond Orange Cultivars by Postharvest Storage at Low Temperature**

**Lourdes Carmona 1,2, Maria Sulli <sup>3</sup> , Gianfranco Diretto <sup>3</sup> , Berta Alquézar 1,2, Mónica Alves 2,4 and Leandro Peña 1,2,\***


**Abstract:** Numerous studies have revealed the remarkable health-promoting activities of citrus fruits, all of them related to the accumulation of bioactive compounds, including vitamins and phytonutrients. Anthocyanins are characteristic flavonoids present in blood orange, which require low-temperature for their production. Storage at low-temperature of blood oranges has been proven to be a feasible postharvest strategy to increase anthocyanins in those countries with warm climates. To our knowledge, no studies comparing the effect of postharvest storage effect on phenylpropanoid accumulation in cultivars with and without anthocyanins production have been published. We have investigated the effect of postharvest cold storage in flavonoid accumulation in juice from *Citrus sinensis* L. Osbeck in two different oranges: Pera, a blond cultivar, and Moro, a blood one. Our findings indicate a different response to low-temperature of fruit from both cultivars at biochemical and molecular levels. Little changes were observed in Pera before and after storage, while a higher production of phenylpropanoids (3.3-fold higher) and flavonoids (1.4-fold higher), including a rise in anthocyanins from 1.3 ± 0.7 mg/L to 60.0 ± 9.4 mg/L was observed in Moro concurrent with an upregulation of the biosynthetic genes across the biosynthetic pathway. We show that postharvest storage enhances not only anthocyanins but also other flavonoids accumulation in blood oranges (but not in blond ones), further stimulating the interest in blood orange types in antioxidant-rich diets.

**Keywords:** antioxidants; blood oranges; flavonoids; anthocyanins

## **1. Introduction**

Nutraceuticals are phytochemical compounds found in vegetables and fruits, which are getting high consideration for their health-promoting effects when consumed with a certain frequency. Both fruits and vegetables are rich sources of polyphenols, including flavonoids, involved in reducing inflammation and oxidative stress related with chronic diseases, such as those derived from cardiovascular risks, and different types of cancer or diabetes [1–3]. Flavonoids, biosynthesised from the phenylpropanoid pathway (Figure 1), constitute the largest class of nutraceuticals in our diet [4]. Depending on their structure, flavonoids can be grouped into six main categories: flavones, flavonols, isoflavones, flavanones, flavanols, and anthocyanins. Among them, flavanones and anthocyanins present a higher antioxidant activity [5].

**Citation:** Carmona, L.; Sulli, M.; Diretto, G.; Alquézar, B.; Alves, M.; Peña, L. Improvement of Antioxidant Properties in Fruit from Two Blood and Blond Orange Cultivars by Postharvest Storage at Low Temperature. *Antioxidants* **2022**, *11*, 547. https://doi.org/10.3390/ antiox11030547

Academic Editors: Agustín G. Ausero and Noelia Tena

Received: 22 February 2022 Accepted: 11 March 2022 Published: 14 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

or inhibiting viral replication [20–22].

**Figure 1.** A schematic representation of the phenylpropanoid pathway. Red-bold labelled genes are those studied in this work. Gene names are abbreviated as follows: *PAL*, *phenylalanine ammonia-lyase*; *C3H*, *p-coumarate 3-hydroxylase*, *C4H*, *cinnamate 4-hydroxylase*; *4CL*, *4-hydroxy-cynnamoyl CoA ligase*; *CHS*, *chalcone synthase*; *CHI*, *chalcone isomerase*; *COMT*, *caffeic acid 3-O-methyltransferase*; *FNS*, *flavone synthase*; *F3H*, *flavanone 3-hydroxylase*; *F3′H*, *flavonoid 3′-hydroxylase*; *F3′5′H*, *flavonoid 3′5′-hydroxylase*; *GST, glutathione-S- transferase; OMTs*, *O-methyltransferases*; *FLS*, *flavonol synthase*; *DFR*, *dihydroflavonol 4- reductase*; *ANS*, *anthocyanidin synthase*; *UFGT*, uridine *diphosphate-glucose:flavonoid 3-O-glucosyltransferase* and *UGTs*, *O-methyltransferase*. **Figure 1.** A schematic representation of the phenylpropanoid pathway. Red-bold labelled genes are those studied in this work. Gene names are abbreviated as follows: *PAL*, *phenylalanine ammonialyase*; *C3H*, *p-coumarate 3-hydroxylase*, *C4H*, *cinnamate 4-hydroxylase*; *4CL*, *4-hydroxy-cynnamoyl CoA ligase*; *CHS*, *chalcone synthase*; *CHI*, *chalcone isomerase*; *COMT*, *caffeic acid 3-O-methyltransferase*; *FNS*, *flavone synthase*; *F3H*, *flavanone 3-hydroxylase*; *F3*0*H*, *flavonoid 3*0 *-hydroxylase*; *F3*0*5* 0*H*, *flavonoid 3*0*5* 0  *hydroxylase*; *GST, glutathione-S- transferase; OMTs*, *O-methyltransferases*; *FLS*, *flavonol synthase*; *DFR*, *dihydroflavonol 4- reductase*; *ANS*, *anthocyanidin synthase*; *UFGT*, uridine *diphosphate-glucose:flavonoid 3-O-glucosyltransferase* and *UGTs*, *O-methyltransferase*.

Citrus fruits are excellent sources of nutrients due to their abundance in vitamin C, sugars, dietary fibre, minerals and phytochemicals, including flavonoids [6–12]. The vital bioactivities of these secondary metabolites have made blood oranges (BO) to be used as traditional medicine in different Asian countries [13,14]. Flavones are found mainly in their peels, while flavanones are present in both peel and juice of oranges, mandarins, lemons and grapefruits [15]. Regarding anthocyanins, they are only accumulated in peel and juice of the BO, providing not only vivid colours but also a higher antioxidant activity to their pulp and juices [16–18]. In fact, it has been proposed that dietary anthocyanins are more effective antioxidants than vitamins E and C [19]. Other beneficial effects of anthocyanins include their anticancer activities, antiviral properties and protective effects against various metabolic, degenerative and cardiovascular diseases as well as eyesight

Anthocyanin production in BO is very dependent on cold-temperature, making their quality at harvest to fluctuate between geographical locations and seasons. In specific cold regions of China, Spain and Italy, BO develop optimal colour, while countries with tropi-Citrus fruits are excellent sources of nutrients due to their abundance in vitamin C, sugars, dietary fibre, minerals and phytochemicals, including flavonoids [6–12]. The vital bioactivities of these secondary metabolites have made blood oranges (BO) to be used as traditional medicine in different Asian countries [13,14]. Flavones are found mainly in their peels, while flavanones are present in both peel and juice of oranges, mandarins, lemons and grapefruits [15]. Regarding anthocyanins, they are only accumulated in peel and juice of the BO, providing not only vivid colours but also a higher antioxidant activity to their pulp and juices [16–18]. In fact, it has been proposed that dietary anthocyanins are more effective antioxidants than vitamins E and C [19]. Other beneficial effects of anthocyanins include their anticancer activities, antiviral properties and protective effects against various metabolic, degenerative and cardiovascular diseases as well as eyesight or inhibiting viral replication [20–22].

Anthocyanin production in BO is very dependent on cold-temperature, making their quality at harvest to fluctuate between geographical locations and seasons. In specific cold regions of China, Spain and Italy, BO develop optimal colour, while countries with tropical climates such as Sao Paulo (Brazil) or Florida (USA) yield BO with a very low pigmentation [23]. The number of hours of exposition to low-temperature has been proposed as a crucial factor to get a strong purple/red coloration in the fruit [24]. Storage at

either 4 or 8 ◦C has been reported to promote BO characteristic coloration because of the activation of anthocyanin biosynthetic genes [25–29], and by the increase in proteins related to anthocyanin biosynthesis, energy input, and other metabolic pathways associated with defence, oxidative and stress responses [30]. Storage at 9 ◦C in comparison to 4 ◦C has been demonstrated to be more effective for enrichment of anthocyanin production in BO [31] and an additional stress treatment (curing) further promotes anthocyanin production, increasing additionally flavonoids accumulation [32]. Low temperature conservation offers also the advantage of being the most frequently used technique to extend the postharvest life and preserve the quality of citrus fruits [33]. Additionally, in order to control pests, several countries require storage at low temperature for citrus fruits exportation to other markets [34].

The consumption of BO may be an important contribution to healthy diets [18,22]. Thus, juices and beverages flaunting red/purple orange in their composition increase their market value, owing to the established health-promoting potential of purple/red orange bioactive components [35]. Besides, increasing flavonoids accumulation in blond oranges by postharvest treatment can also improve the health-promoting properties of derived products or even of fruit consumption. To our knowledge, there is little information comparing the postharvest storage effect on phenylpropanoids accumulation in blond and BO. In this work, we investigated low-temperature storage effect on flavonoids and other phenylpropanoids in two selected cultivars of *Citrus sinensis* (L.) Osbeck, Pera (blond) and Moro (blood). Pera blond sweet orange is the most important citrus cultivar in Brazil (2nd worldwide citrus producer), constituting more than a third of the commercial acreage in São Paulo State [36]. Regarding BOs, Moro, characterised by yielding deeply purple-coloured fruits [17], is the most widely grown cultivar in Europe for food processing and other industrial applications, and the most common BO cultivar grown in the United States [36].

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Storage Conditions*

The effect of postharvest storage on fruit colour was determined in two different cultivars of *Citrus sinensis* L. Osbeck, Moro (blood type) and Pera (blond type) mature fruits (8 months after the bloom). Fruits were harvested from adult trees grown (13 years old) and grafted on citrumelo Swingle under standard cultivation in a commercial orchard in Maringá (21◦45053" S; 48◦28021.15" O, 540 m), Gavião Peixoto-Sao Paulo State (Brazil). The local climate is Cwa type (mountain subtropical), characterised by dry winter (<1230 mm total rainfall of the year) and a normal average air temperature of ≥17 ◦C and ≥28 ◦C in the coldest and in the warmest month, respectively [37]. Maringá soil type is classified as *latossolo vermelho* (Red-oxisol), dystrophic A moderate type, and soft-moderately wavy relief [38]. A total of 80 fruits were harvest from 15 trees planted at 7.0 m × 3.0 m spacing. Fruits were uniform in size and colour, as well as free of damage or external defects. Harvested fruits were stored for 45 days at 9 ◦C, and 90–95% relative humidity in constant darkness. At the time of harvest (zero) and after 15, 30 and 45 days of storage, pulp and juice samples were taken and stored at −80 ◦C until analysis. Three replicate samples of 5 fruits each per storage time were used. Pulp was separated with a scalpel, and immediately frozen in liquid nitrogen and ground into a fine powder. Juice was extracted with a domestic squeezer (Braun GmbH, Germany), filtered through a metal sieve with a pore size of 0.8 mm and frozen in liquid nitrogen.

#### *2.2. Determination of Internal Maturity Index, pH and Total Anthocyanin Quantification*

Titration with phenolphthalein was used to determine the juice acidity, and data were expressed as mg citric acid per 100 mL [31]. Briefly, 5 mL of centrifuged juice were diluted to 45 mL with water (Sigma) and supplemented with 5 drops of phenolphthalein. Acidity was evaluated by titration with NaOH (0.1 N) and expressed as mg of citric acid per 100 mL. The determination of soluble solids content (◦Brix) was estimated by refractometer, using an Atago® refractometer (Tokyo, Japan) as described by Carmona et al. [31]. Maturity index

was calculated and expressed as the ratio of ◦Brix/acidity. The pH value was measured with a pH-meter Gehaka® (Sao Paulo, Brasil) [31].

#### *2.3. Flavonoids, Anthocyanins and Phenylpropanoid Extraction and Identification*

All compounds were extracted using 2 mg of Moro (blood type) or Pera (blond type) mature freeze-dried fruits pulp re-suspended in 20 mL of water (LC-MS Grade, LiChrosolv®, Merck, Darmstadt, Germany). Flavonoids and other phenylpropanoids were extracted as previously described by Carmona et al. [32], with the following modifications: 0.5 mL of each re-suspended pulp solution was shaken for 1 h at 20 Hz using a Mixer Mill 300 (Qiagen, Hilden, Germany) with 1.5 mL of methanol containing 0.3% formic acid, plus 2 µg/L of formononetin as the internal standard, and centrifuged at 15,000× *g* for 20 min (15 ◦C). The supernatants (0.4 mL) were filtered into vials for LC/MS analysis (Mini-UniPrep® syringeless filters with 0.2 µm pore size PTFE membrane, Whatman®, Maidstone, UK). LC/MS analysis was performed using an HPLC system equipped with a photodiode array detector (Dionex, ThermoFisher Scientific, Sunnyvale, CA, USA) coupled to a quadrupole-Orbitrap Q-exactive system (ThermoFisher Scientific, Sunnyvale, CA, USA). HPLC analysis was performed using a C18 Luna Column (Phenomenex, Aschaffenburg, Germany) (150 × 2.0 mm; 3 µm). Total of 5 µL of each extract were injected at a flow of 0.25 mL/min. Total run time was 32 min using an elution system running at 0.250 mL/min and consisting of (A) water (0.1% formic acid) and (B) acetonitrile: H2O 90:10 (0.1% formic acid). Gradient was 0 to 0.5 min 95/5%-A/B, 24 min 25/75%-A/B, and 26 min 5/95%-A/B. MS analysis of flavonoids and other phenylpropanoids was carried out with a heated electrospray ionization (HESI) source operating in positive and negative ion mode. Mass spectrometer parameters were as follows: sheath and aux gas flow rate set at 40 and 10 units, respectively; capillary temperature was at 250 ◦C, discharge current was set at 3.5 µA and S-lens RF level at 50. The acquisition was carried out with *m*/*z* 110–1600 Full MS scan range, and the following parameters: resolution 70,000, microscan 1, AGC target 1e6, and maximum injection time 50. Data were analysed using the Xcalibur 4.4 software (ThermoFisher Scientific, Sunnyvale, CA, USA). Metabolites were identified as M+H and M-H adducts, based on their accurate masses (*m*/*z*) and MS fragmentation, using both in house database and public sources (e.g., KEGG, ChemSpider, PubChem, MetaCyc, Metlin, Phenol-Explorer). Relative abundances of the investigated flavonoids and other phenylpropanoids were calculated as fold average and the standard deviation of integrated areas under the *m/z* peak of the adduct of each compound and the internal standard peak area (Fold/ISTD), calculated with the Xcalibur 4.4 software (ThermoFisher Scientific, Sunnyvale, CA, USA).

Anthocyanins were analysed in samples obtained by mixing 500 µL of each resuspended pulp solution with 500 µL 85:15 methanol:HCl (1 N) containing 0.3% formic acid and 2 µg/L of formononetin (Sigma-Aldrich, San Luis, MO, USA) as internal standard [39]. Samples were shaken for 12 h and subsequently centrifuged for 10 min at 15,300× *g* at 25 ◦C. Supernatants (0.25 mL) were transferred to new Eppendorf vials, dried by Speedvac concentrator, resuspended in 0.1 mL 75% methanol (plus 0.1% formic acid) and centrifuged (10 min at 15,300× *g* at 25 ◦C). Total of 70 µL of supernatant was transferred to HPLC vials for LC/MS analysis, and 10 µL of extract was injected to the HPLC-PDA/MS. The method used for separation performed with a C18 Luna column (Phenomenex, Aschaffenburg, Germany) (150 × 2.0 mm; 3 µm) was as previously described [39] and PDA detection was performed by an online Accela Surveyor photodiode array detector (PDA; ThermoFisher Scientific, Sunnyvale, CA, USA), acquiring continuously from 200 to 600 nm. Mass spectrometry analysis was performed using a quadrupole-Orbitrap Q-exactive system (ThermoFisher scientific, Sunnyvale, CA, USA) and ionization was carried out with a heated electrospray ionization (HESI) source operating in positive ion mode. The MS parameters used are as follows: nitrogen was used as sheath and auxiliary gas (45 and 15 units, respectively), capillary and vaporizer temperatures 30 ◦C and 270 ◦C, respectively, discharge current 4.0 KV, probe heater temperature at 370 ◦C, S-lens RF level at 50 V. The acquisition was carried

out in the 110–1600 *m/z* scan range, resolution 70,000, microscan 1, AGC target 1e6, and maximum injection time 50 [39]. In detail, MS analysis was performed using a first full scan with data-dependent MS/MS fragmentation in order to identify the anthocyanins in pulp extracts. Subsequently, a single ion monitoring (SIM) with targeted MS/MS fragmentation was applied to identify anthocyanins for which dd-MS/MS fragmentation was not successful, and to further validate the tentative identifications. Anthocyanins were analysed using Xcalibur 3.1 software (ThermoFisher Scientific, Sunnyvale, CA, USA) and identified as M+ adducts, based on their accurate masses (m/z) and MS fragmentation, compared with in house database and public sources (e.g., KEGG, MetaCyc, ChemSpider, PubChem, Metlin, Phenol-Explorer), as well as with comigration with available authentic standards (cyanidin 3-glucoside, peonidin 3-glucoside and dephinidin 3-glucoside) (Extrasynthese, Genay, France). Absolute amounts were measured as previously described [39] using the two most abundant fragments per compound, and data were normalised based on integrated peak areas of external calibration curves of previously described standards [39]. LOD (limit of detection) was estimated from signal-to-noise ratio (S/N) as described [40] and defined as signal intensity corresponding to three times of that noise, while LOQ (limit of quantification) was nine times of that noise. All data are presented as means and standard deviation of at least three independent biological replicates. All the chemicals and solvents used during both the procedures were of LC/MS grade.

## *2.4. Quantitative RT-PCR Analysis*

Plant material used for flavonoids and anthocyanins analysis was the same as used for total RNA isolation. Total RNA extraction, DNase treatment, cDNA synthesis and quantitative real-time PCR (qPCR) and relative gene expression were performed as described previously by Carmona et al. [27]. Briefly, qPCR was achieved with a StepOne Plus Real Time PCR System (Applied Biosystem, Waltham, MA, USA) and analysed using StepOne Software version 2.3 (Thermo Fisher, Valence, Spain). RT-PCR was carried out with 50 ng of total cDNA adding 6 µL of SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA) and 0.3 µM of gene specific primers in a total volume of 12 µL. The RT-PCR procedure consisted of 95 ◦C 10 min followed by 40 cycles at 95 ◦C 15 s and 60 ◦C 40 s. Primers sequences for analysing *phenylalanine ammonia-lyase* (*PAL*), *cinnamate 4-hydroxylase* (*C4H*), *4-hydroxy-cynnamoyl CoA ligase* (*4CL*), *chalcone synthases 1* and *2* (*CHSs*), *chalcone isomerase* (*CHI*), *flavonoid 3-hydroxylase* (*F3H*), *flavonoid 3*0*5* 0 *-hydroxylase* (*F3*0*5* 0*H*), *flavonol synthase* (*FLS*), *dihydroflavonol 4-reductase* (*DFR*), *anthocyanidin synthase* (*ANS*), *uridine diphos-phate-glucose:flavonoid 3-O-glucosyltransferase* (*UFGT*) and *glutathione-S-transferase* (*GST*) genes are described in Table S1. The relative expression between cold-treated and control samples (zero time of orange fruits) was determined by the method described by Livak et al. [41]. Values are presented as the mean of at least three independent analyses. Statistical analyses were performed using ANOVA.

#### **3. Results**

#### *3.1. Pulp and Juice Appearance and Quality Parameters in Moro and Pera Oranges after Storage*

The effect of postharvest storage on visual aspect, maturity index (MI), total flavonoid and anthocyanin contents were assessed in pulp and juice from Moro (blood type) and Pera (blond type) mature oranges subjected to postharvest storage at 9 ◦C (Figure 2 and Table 1). The visual aspect of pulp and juice was evaluated at the onset, at 30 and 45 days. No changes in colour were detected in pulp and juice from Pera during all the storage period, while a notable enhancement in the red/purple coloration was observed in those from Moro (Figure 2).

those from Moro (Figure 2).

**Figure 2.** Internal oranges appearance and colour of pulp (**up**) and juices (**down**) of Pera (**left**) and Moro (**right**) oranges from Sao Paulo (Brazil), during storage at 9 °C for 0, 30 and 45 days. **Figure 2.** Internal oranges appearance and colour of pulp (**up**) and juices (**down**) of Pera (**left**) and Moro (**right**) oranges from Sao Paulo (Brazil), during storage at 9 ◦C for 0, 30 and 45 days.

The effect of postharvest storage on visual aspect, maturity index (MI), total flavonoid and anthocyanin contents were assessed in pulp and juice from Moro (blood type) and Pera (blond type) mature oranges subjected to postharvest storage at 9 °C (Figure 2 and Table 1). The visual aspect of pulp and juice was evaluated at the onset, at 30 and 45 days. No changes in colour were detected in pulp and juice from Pera during all the storage period, while a notable enhancement in the red/purple coloration was observed in

No differences were found in MI and pH along the storage period in any of the two fruit types investigated (Table 1). Total flavonoids content was different between both types, being 1.4-fold higher in Moro than in Pera at the onset of the experiment (Table 1). **Table 1.** Maturity index, pH, flavonoids and anthocyanin content of Pera and Moro pulp during storage for 0, 30 and 45 days. Statistical analyses were performed using ANOVA and different letters indicate statistically significant different values (*p* ≤ 0.01) for a given time.


**Pera Moro 0 Days 30 Days 45 Days 0 Days 30 Days 45 Days Maturity index (MI)** 7.9 ± 0.7 a 7.9 ± 0.4 a 7.9 ± 0.6 a 9.5 ± 0.7 a 9.2 ± 0.2 a 9.4 ± 0.1 a **pH** 3.8 ± 0.1 a 3.8 ± 0.1 a 3.9 ± 0.0 a 3.6 ± 0.2 a 3.8 ± 0.1 a 3.7 ± 0.0 a **Total flavonoids** 22.4 ± 1.5 a 22.1 ± 1.5 a 22.4 ± 1.4 a 31.6 ± 3.3 b 28.2 ± 2.8 b 45.4 ± 2.8 c **Total anthocyanin content (mg/L)** - - - 1.3 ± 0.1 a 43.4 ± 4.2 b 60.0 ± 3.5 c *3.2. Accumulation of Hydroxycinnamates and Flavonoids (Non-Anthocyanins) in Moro and*  No differences were found in MI and pH along the storage period in any of the two fruit types investigated (Table 1). Total flavonoids content was different between both types, being 1.4-fold higher in Moro than in Pera at the onset of the experiment (Table 1). Moreover, no enhanced accumulation was detected in the blond cultivar during the storage period, while an increment of 1.4-fold was observed in Moro at the end of the storage. No anthocyanin presence was detected in Pera orange, while Moro fruit displayed a noticeable presence and considerable enhancement of anthocyanins from 1.3 ± 0.7 mg/L to 60.0 ± 9.4 mg/L under storage conditions.

#### *Pera Oranges during Postharvest Storage*  Contents of hydroxycinammic acids (HA) and main flavonoids were assessed in the *3.2. Accumulation of Hydroxycinnamates and Flavonoids (Non-Anthocyanins) in Moro and Pera Oranges during Postharvest Storage*

pulp of Pera and Moro fruit during 9 °C post-harvest storage (Figure 3 and Table S2). The profile of the eleven HA identified showed significant differences between both cultivars (Table S2). In general, the BO presented a higher HA content at the onset of the experiment, and a much higher accumulation along all the postharvest experimental period. For instance, the content of coumaric acid, precursor of both HA and flavonoids, was 4.9- and 13.6-fold higher in Moro than in Pera at the onset of the experiment and after 45 days, respectively (Figure 3 and Table S2). Contents of hydroxycinammic acids (HA) and main flavonoids were assessed in the pulp of Pera and Moro fruit during 9 ◦C post-harvest storage (Figure 3 and Table S2). The profile of the eleven HA identified showed significant differences between both cultivars (Table S2). In general, the BO presented a higher HA content at the onset of the experiment, and a much higher accumulation along all the postharvest experimental period. For instance, the content of coumaric acid, precursor of both HA and flavonoids, was 4.9- and 13.6-fold higher in Moro than in Pera at the onset of the experiment and after 45 days, respectively (Figure 3 and Table S2).

**Figure 3.** Fold change of the 4 main hydroxycinnamic acids identified in the pulp of Moro (●) and Pera (○) oranges during storage for 0, 15, 30 and 45 days. Data are expressed as the mean fold change ± SD of each sample as compared to the control Pera fruits sample (at harvest time). Asterisk indicates statistically significant different values (*p* ≤ 0.01) for each given time point when comparing cultivars. **Figure 3.** Fold change of the 4 main hydroxycinnamic acids identified in the pulp of Moro (•) and Pera (#) oranges during storage for 0, 15, 30 and 45 days. Data are expressed as the mean fold change ± SD of each sample as compared to the control Pera fruits sample (at harvest time). Asterisk indicates statistically significant different values (*p* ≤ 0.01) for each given time point when comparing cultivars.

A total of 76 flavonoids, including flavanones, flavonols and flavones were identified and measured during the storage period in juice from both fruit types (Figure 4 and Table S2). Among them, the flavonol class was the main group constituting 38.2% of the total, followed by flavones (35.5%) and flavanones (26.3%). Additionally, polymethoxylated derivatives of each class were identified, with polimethoxyflavones (PMFs) being the most represented. The initial flavonoids profile, their content and the accumulation patterns along storage displayed drastic differences between Moro and Pera. Eight flavonoid compounds present in Moro were not detected in Pera fruit either at the onset or after postharvest storage, such as isosakuranetin or the flavonols kaemferol and myricetin, while only two flavonoids (poncirin and natsudaidain) were not present at the beginning in Moro, but were found later and progressively increased with the storage (0.19 and 122.2 fold, respectively) (Table S2). Regarding the initial content of the individual flavonoids, 25 flavonoids showed low (<0.6-fold) accumulation in Moro than in Pera, as the flavanones naringenin or eriocitrin (0.60 an 0.12-fold, respectively). Conversely, 24 flavonoid compounds exhibited a higher content in Moro than in Pera at the onset, which varied between the 2.21-fold increase of the chrysoriol-8-C-glucoside (scoparin) and 202.4-fold enrichment of dihydrokaempherol (Table S2). Considering the content after the storage period, most of the identified flavonoids (54%) were more than two-fold enhanced in Moro compared to Pera, being among them the main precursors naringenin chalcone (206.4-fold) and the dihydroflavonoids dihydrokaemferol and dihydroquercetin (1602.8 and 239.8-fold, respectively). However, 19.7% of the identified flavonoids displayed less than 0.70-fold accumulation in Moro along storage, such as eriocitrin (0.43-fold) or methoxykaempferol-3-*O*-neohesperidoside (0.07-fold) (Figure 4 and Table S2). Individual flavonoids followed also a different accumulation profile during the storage at 9 °C depending on the cultivar. Whereas in Pera most of the identified flavonoids barely changed with the storage, the individual profile varied in Moro depending on each metabolite (Figure 5 and Table S2). In general, main citrus flavanones (isosakuranetin derivatives), flavones (apigenin, luteolin and their derivatives), and flavonols (quercetin, kampferol and their derivatives) increased along storage (1.2 up to 361.6-fold) in Moro fruit (Figure 5). Although other compounds such as 3,3′,4′,5,6,7,8-heptamethoxyflavone, nobiletin or sinensetin showed a decreasing profile (1.4, 2.2 and 2.9-fold, respectively), their content A total of 76 flavonoids, including flavanones, flavonols and flavones were identified and measured during the storage period in juice from both fruit types (Figure 4 and Table S2). Among them, the flavonol class was the main group constituting 38.2% of the total, followed by flavones (35.5%) and flavanones (26.3%). Additionally, polymethoxylated derivatives of each class were identified, with polimethoxyflavones (PMFs) being the most represented. The initial flavonoids profile, their content and the accumulation patterns along storage displayed drastic differences between Moro and Pera. Eight flavonoid compounds present in Moro were not detected in Pera fruit either at the onset or after postharvest storage, such as isosakuranetin or the flavonols kaemferol and myricetin, while only two flavonoids (poncirin and natsudaidain) were not present at the beginning in Moro, but were found later and progressively increased with the storage (0.19 and 122.2 fold, respectively) (Table S2). Regarding the initial content of the individual flavonoids, 25 flavonoids showed low (<0.6-fold) accumulation in Moro than in Pera, as the flavanones naringenin or eriocitrin (0.60 an 0.12-fold, respectively). Conversely, 24 flavonoid compounds exhibited a higher content in Moro than in Pera at the onset, which varied between the 2.21-fold increase of the chrysoriol-8-C-glucoside (scoparin) and 202.4-fold enrichment of dihydrokaempherol (Table S2). Considering the content after the storage period, most of the identified flavonoids (54%) were more than two-fold enhanced in Moro compared to Pera, being among them the main precursors naringenin chalcone (206.4-fold) and the dihydroflavonoids dihydrokaemferol and dihydroquercetin (1602.8 and 239.8-fold, respectively). However, 19.7% of the identified flavonoids displayed less than 0.70-fold accumulation in Moro along storage, such as eriocitrin (0.43-fold) or methoxykaempferol-3-*O*-neohesperidoside (0.07-fold) (Figure 4 and Table S2). Individual flavonoids followed also a different accumulation profile during the storage at 9 ◦C depending on the cultivar. Whereas in Pera most of the identified flavonoids barely changed with the storage, the individual profile varied in Moro depending on each metabolite (Figure 5 and Table S2). In general, main citrus flavanones (isosakuranetin derivatives), flavones (apigenin, luteolin and their derivatives), and flavonols (quercetin, kampferol and their derivatives) increased along storage (1.2 up to 361.6-fold) in Moro fruit (Figure 5). Although other compounds such as 3,30 ,40 ,5,6,7,8-heptamethoxyflavone, nobiletin or sinensetin showed a decreasing profile (1.4, 2.2 and 2.9-fold, respectively), their content was still higher in Moro than in Pera fruit along the storage period (Figure 5 and Table S2).

S2).

S2).

was still higher in Moro than in Pera fruit along the storage period (Figure 5 and Table

*Antioxidants* **2022**, *11*, x FOR PEER REVIEW 8 of 17

was still higher in Moro than in Pera fruit along the storage period (Figure 5 and Table

**Figure 4.** Fold change of naringenin chalcone (flavonoids precursor, in red colour) and the dihydroflavonoids (anthocyanins precursors, in orange colour) dihydrokampherol, dihydroquecetin and dihydromyricetin in the pulp of Moro (●) and Pera (○) oranges during storage for 0, 15, 30 and 45 days. Data are expressed as the mean fold change ± SD of each sample compared to the control Pera fruits sample (at harvest time). Asterisk indicates statistically significant different values (*p* ≤ 0.01) for each given time. **Figure 4.** Fold change of naringenin chalcone (flavonoids precursor, in red colour) and the dihydroflavonoids (anthocyanins precursors, in orange colour) dihydrokampherol, dihydroquecetin and dihydromyricetin in the pulp of Moro (•) and Pera (#) oranges during storage for 0, 15, 30 and 45 days. Data are expressed as the mean fold change ± SD of each sample compared to the control Pera fruits sample (at harvest time). Asterisk indicates statistically significant different values (*p* ≤ 0.01) for each given time. **Figure 4.** Fold change of naringenin chalcone (flavonoids precursor, in red colour) and the dihydroflavonoids (anthocyanins precursors, in orange colour) dihydrokampherol, dihydroquecetin and dihydromyricetin in the pulp of Moro (●) and Pera (○) oranges during storage for 0, 15, 30 and 45 days. Data are expressed as the mean fold change ± SD of each sample compared to the control Pera fruits sample (at harvest time). Asterisk indicates statistically significant different values (*p* ≤ 0.01) for each given time.

**Figure 5.** Fold change of representative flavonoids identified in the pulp of Moro (●) and Pera (○) oranges during storage for 0, 15, 30 and 45 days. Compounds belonging to flavanones class are represented in red colour, flavonols class in orange and the flavones in purple and light green (polymethoxyflavones subgroup). a. Sum of eriocitrin and neoeriocitrin. b. Sum of diosmin and neodiosmin. c . Including the naringenin chalcone. Data are expressed as the mean fold change ± SD of each sample compared to the control Pera fruits sample (at harvest time). Asterisk indicates statistically significant different values (*p* ≤ 0.01) for each given time. **Figure 5.** Fold change of representative flavonoids identified in the pulp of Moro (●) and Pera (○) oranges during storage for 0, 15, 30 and 45 days. Compounds belonging to flavanones class are represented in red colour, flavonols class in orange and the flavones in purple and light green (polymethoxyflavones subgroup). a. Sum of eriocitrin and neoeriocitrin. b. Sum of diosmin and neodiosmin. c . Including the naringenin chalcone. Data are expressed as the mean fold change ± SD of each sample compared to the control Pera fruits sample (at harvest time). Asterisk indicates statistically significant different values (*p* ≤ 0.01) for each given time. **Figure 5.** Fold change of representative flavonoids identified in the pulp of Moro (•) and Pera (#) oranges during storage for 0, 15, 30 and 45 days. Compounds belonging to flavanones class are represented in red colour, flavonols class in orange and the flavones in purple and light green (polymethoxyflavones subgroup). <sup>a</sup> . Sum of eriocitrin and neoeriocitrin. <sup>b</sup> . Sum of diosmin and neodiosmin. <sup>c</sup> . Including the naringenin chalcone. Data are expressed as the mean fold change ± SD of each sample compared to the control Pera fruits sample (at harvest time). Asterisk indicates statistically significant different values (*p* ≤ 0.01) for each given time.

#### *3.3. Accumulation of Anthocyanins in Moro Orange during Postharvest Storage*

Variations in anthocyanins composition and contents in Moro orange during postharvest storage were evaluated for 45 days (Figure 6). No anthocyanins were detected in Pera, while a total of 11 anthocyanins were identified in Moro pulp. At the onset of the study, cyanidin 3-*O*-glucoside (C3-glu) and cyanidin 3-(6"-malonyl)-glucoside (C3- (6M)-glu) were the most abundant anthocyanins in Moro, representing 61.1% and 24.9% of total anthocyanins, respectively. Storage at 9 ◦C promoted a progressive increase in

anthocyanins accumulation, with C3-glu and C3-(6M)-glu remaining as the most abundant ones, representing 27.7% and 49.4% of total anthocyanins at day 45, respectively (Figure 6). Altogether, cyanidin 3-rhamnoside (C3-rha), delphinidin 3-(6" malonyl)-glucoside (D3- (6M)-glu) and delphinidin 3-glucoside (D3-glu) accounted for 20.8% of total anthocyanins by day 45, in comparison with 9.4% at the onset (Figure 6). At 45 days of storage, the main anthocyanins experimented showed an increase of 5.2-, 4.5-, 3.6- and 3.5-fold for C3-(6M) glu, C3-rha, Peo3-(6M)-glu and D3-(6M)-glu, respectively. Other minor compounds also increased at the end of the storage period as it was observed for pelargonidin 3-glucoside and cyanidin 3-(ferulyl)glucoside (C3-Fe-glu) (5.3-fold) and cyanidin 3-*O*-sophoride (C3 sph) (2.3-fold). Other anthocyanin pigments that were not found initially, such as petunidin 3,5-glucoside (Pe3,5-glu) and petunidin 3-(6"-malonyl)-glucoside (Pet3-(6M)-glu), were detected at the end of storage (0.14 ± 0.00 and 0.12 ± 0.00 mg/mL, respectively). anthocyanins, respectively. Storage at 9 °C promoted a progressive increase in anthocyanins accumulation, with C3-glu and C3-(6M)-glu remaining as the most abundant ones, representing 27.7% and 49.4% of total anthocyanins at day 45, respectively (Figure 6). Altogether, cyanidin 3-rhamnoside (C3-rha), delphinidin 3-(6″ malonyl)-glucoside (D3- (6M)-glu) and delphinidin 3-glucoside (D3-glu) accounted for 20.8% of total anthocyanins by day 45, in comparison with 9.4% at the onset (Figure 6). At 45 days of storage, the main anthocyanins experimented showed an increase of 5.2-, 4.5-, 3.6- and 3.5-fold for C3-(6M) glu, C3-rha, Peo3-(6M)-glu and D3-(6M)-glu, respectively. Other minor compounds also increased at the end of the storage period as it was observed for pelargonidin 3-glucoside and cyanidin 3-(ferulyl)glucoside (C3-Fe-glu) (5.3-fold) and cyanidin 3-*O*-sophoride (C3 sph) (2.3-fold). Other anthocyanin pigments that were not found initially, such as petunidin 3,5-glucoside (Pe3,5-glu) and petunidin 3-(6″-malonyl)-glucoside (Pet3-(6M)-glu), were detected at the end of storage (0.14 ± 0.00 and 0.12 ± 0.00 mg/mL, respectively).

Variations in anthocyanins composition and contents in Moro orange during postharvest storage were evaluated for 45 days (Figure 6). No anthocyanins were detected in Pera, while a total of 11 anthocyanins were identified in Moro pulp. At the onset of the study, cyanidin 3-*O*-glucoside (C3-glu) and cyanidin 3-(6″-malonyl)-glucoside (C3-(6M) glu) were the most abundant anthocyanins in Moro, representing 61.1% and 24.9% of total

*Antioxidants* **2022**, *11*, x FOR PEER REVIEW 9 of 17

*3.3. Accumulation of Anthocyanins in Moro Orange during Postharvest Storage* 

**Figure 6.** Composition of anthocyanins in the pulp of Moro blood orange during storage for 0, 15, 30 and 45 days. Data are expressed as the mean ± SD of each sample as compared to the control sample (Moro fruit at harvest time). Asterisk indicates statistically significant different values (*p* ≤ 0.01) for each given time. **Figure 6.** Composition of anthocyanins in the pulp of Moro blood orange during storage for 0, 15, 30 and 45 days. Data are expressed as the mean ± SD of each sample as compared to the control sample (Moro fruit at harvest time). Asterisk indicates statistically significant different values (*p* ≤ 0.01) for each given time.

#### *3.4. Gene Expression Ratio of Phenylpropanoid Biosynthetic Genes between Pera and Moro Orange Pulp during Postharvest Storage*

Transcript accumulation levels between Moro and Pera during postharvest storage were determined and compared as the ratio between relative quantification of Moro vs. Pera at the onset and each postharvest time (Figure 7). A total of 13 genes were evaluated: *PAL*, *C4H* and *4CL* involved in the initial steps of the general phenylpropanoid pathway, seven genes involved in the initial steps of flavonoids biosynthesis (*CHSs*, *CHI*, *F3H*, *F3*0*H*, *F3*0*5* 0*H*), three structural anthocyanin biosynthesis genes (*DFR*, *ANS* and *UFGT*) and one gene involved in the transport of the purple/red pigments to vacuoles (*GST*). In general, expression profile revealed that all genes presented a higher ratio at the onset and during the storage period in Moro vs. Pera, with the only exception of *FLS*. At the onset, *F3*0*5* 0*H* showed up to 168-fold higher expression in Moro than in Pera, and upstream genes *CHS1*

and *CHS2* were 20 and 28-fold higher, respectively. Anthocyanin structural gene expression presented also a positive ratio in Moro vs. Pera at the onset of storage, between 20 and 122-fold. During the storage period, the highest induction was shown for *CHS2* and *F3*0*5* 0*H* with a final ratio of 1915-fold and 1330-fold increase, respectively (Figure 7). *Antioxidants* **2022**, *11*, x FOR PEER REVIEW 11 of 17

**Figure 7.** Ratio of the relative expression of *phenylalanine ammonia-lyase* (*PAL*), *cinnamate 4-hydroxylase* (*C4H*), *4-hydroxy-cynnamoyl CoA ligase* (*4CL*), *chalcone synthases 1* and *2* (*CHSs*), *chalcone isomerase* (*CHI*), *flavonoid 3-hydroxylase* (*F3H*), *flavonoid 3'5'-hydroxylase* (*F3'5'H*), *flavonol synthase* (*FLS*), *dihydroflavonol 4-reductase* (*DFR*), *anthocyanidin synthase* (*ANS*), *uridine diphos-phate-glucose:flavonoid 3- O-glucosyltransferase* (*UFGT*) and *glutathione-S-transferase* (*GST*) in the pulp of Moro vs. Pera oranges during storage for 0, 15, 30 and 45 days. Blue, and orange bars indicate genes involved in the general phenypropanoids and flavonoids, respectively. Yellow and purple bars indicate genes involved in the flavonols and anthocyanins biosynthesis, respectively. Red bars belong to those genes involved in the anthocyanin transport. **Figure 7.** Ratio of the relative expression of *phenylalanine ammonia-lyase* (*PAL*), *cinnamate 4-hydroxylase* (*C4H*), *4-hydroxy-cynnamoyl CoA ligase* (*4CL*), *chalcone synthases 1* and *2* (*CHSs*), *chalcone isomerase* (*CHI*), *flavonoid 3-hydroxylase* (*F3H*), *flavonoid 3'5'-hydroxylase* (*F3'5'H*), *flavonol synthase* (*FLS*), *dihydroflavonol 4-reductase* (*DFR*), *anthocyanidin synthase* (*ANS*), *uridine diphos-phate-glucose:flavonoid 3-O-glucosyltransferase* (*UFGT*) and *glutathione-S-transferase* (*GST*) in the pulp of Moro vs. Pera oranges during storage for 0, 15, 30 and 45 days. Blue, and orange bars indicate genes involved in the general phenypropanoids and flavonoids, respectively. Yellow and purple bars indicate genes involved in the flavonols and anthocyanins biosynthesis, respectively. Red bars belong to those genes involved in the anthocyanin transport.

#### **4. Discussion**

Among fruits, citrus pulp, juice and by-products constitute one of the most important sources of phytonutrients, especially in countries where these fruits are extensively produced. In the last years, the beneficial health-promoting effects claimed for flavonoids has stimulated the interest for investigating these phytochemical compounds in citrus fruits [6,42–46]. Postharvest storage of the fruit at low temperature, combined or not with a stress (i.e., curing) treatment, has been proven to induce anthocyanins and other flavonoids accumulation in BO via stimulation of their biosynthesis [26,28,31]. However, to our knowledge, information regarding changes in the accumulation of specific flavonoid contents in blond and BO under postharvest storage conditions is limited. In this work, the effect of storage on flavonoids and other phenylpropanoids accumulation was investigated and compared in fruits from two important orange cultivars for juice production, Pera (blond) and Moro (blood).

Different responses to the storage at low-temperature were noticed in Pera and Moro fruits. While no visual changes were observed in the blond fruit pulp and juice, Moro fruit displayed a conspicuous enhancement of its purple/red coloration in the pulp and juice, as expected (Figure 2) [31,32]. Juice pH, which can influence anthocyanin by changing their coloration [47], did not vary along the storage period in our study (Table 1). Instead, the progressive darkening of BO pulp and juice could be associated with the increase of anthocyanins content along storage (Figure 2 and Table 1) [31]: from a low content (1.3 ± 0.7 mg/L) at harvest time, it increased 46-fold along storage period due to the cold induction effect (Table 1) [25,26,31,32]. Red/purple colour enhancement in BO during cold storage is related to the greater increase of all individual anthocyanins (Figure 6 and Table 1) [26,28,31,32]. C3-glu and C3-(6M)-glu have been described as the main anthocyanins in BO juice, together with D3-glu, Peo3-(6M)-glu and cyanidin 3-(6" dioxalyl) glucoside (C6D-glu) [48]. Accordingly, the two main anthocyanins found in Moro orange were also C3-glu and C3-(6M)-glu, followed by D3-glu and D3-(6M)-glu (Figure 6). In contrast, although no C6D-glu was detected, other anthocyanins such as C3-rha, C3-Feglu and C3-sph were accumulated in response to storage at low-temperature in Moro orange (Figure 6) [32]. Due to their electron-donating properties, anthocyanins are potent antioxidants [49]. In the case of BO, the antioxidant activity of anthocyanins was favourable for human health, with impact on some diseases, derived from their anti-inflammatory, anticancer, and antidiabetic properties, due to the prevention of oxidation and free-radical chain reactions [50]. The absence of anthocyanins accumulation in blond orange fruit has been widely documented [23], and is mainly due to the lack of expression in key positive transcriptional factors required for the production of these compounds. Similarly, anthocyanins were not detected in Pera fruit (Table 1).

The beneficial effect of low temperature storage on anthocyanin enhancement has been shown in fruits from other BO cultivars, and was related to a strong boost in the induction and expression of the initial genes of the phenylpropanoids biosynthetic pathway concomitant with the induction of anthocyanin structural genes, leading also to the higher accumulation of other flavonoids [31,32]. Regarding flavonoids, their content and composition in citrus fruits vary among species, cultivars and fruit organs [13,17,51–53]. Moreover, stressful temperatures alter the general phenylpropanoid metabolism in citrus fruits [54,55]. In agreement with that, very different accumulation patterns of the individual flavonoid and HA metabolites were observed in fruit from the blood and blond cultivars, the BO pulp being richer at the onset and during postharvest storage (Figures 2 and 3 and Table S2). A higher abundance of HA has been reported in BO fruit compared to that of blond cultivars, being Moro one of the BO types accumulating higher amounts of total HA [17,25]. Additionally, the evaluation of flavonoid profiles revealed a higher initial content and a progressive increment along the storage of the main flavonoids (non-anthocyanins) and anthocyanins only in BO fruit (Figures 4–6 and Table S2). Interestingly, storage promoted the enhanced accumulation of precursor substrates in BO, as an effect of a major request of precursor for flavonols and anthocyanins to respond to temperature stress [25,26,31,32]. Concordantly,

storage at low temperature induced an increased accumulation of the flavonoid (naringenin chalcone) and anthocyanin (dihydroflavonols) main precursor substrates in Moro in comparison with Pera fruit, concurrent with a higher induction of early phenylpropanoid genes expression (Figure 7) [28,31,32]. Taken together, these results support the higher substrate availability for downstream production of phenylpropanoids in Moro and a different regulation from the initial steps in Moro vs. Pera oranges (Figures 2–4 and Table S2) [32].

Flavonoids are potent inducers of antioxidant defence mechanisms in animal cells, by stimulation of different enzymes activity such as glutathione peroxidase, catalase, and superoxide dismutases, or by inhibition of the accumulation of other enzymes such as xanthine oxidase and also the lipid peroxidation as well as protecting other biomolecules, such as DNA from oxidation [43]. In citrus fruits, flavanones have been described as the predominant class of flavonoids, being flavonols the less representative group [51,56]. In our study, flavonoid profiles revealed that the flavonols and flavones classes were the main ones, being flavanones the third (Table S2). These differences might be determined by the different cultivars used [17,52,53], as is supported by the less flavanones accumulation in fruit from blond cultivars stored at low temperature when compared with that of BO cultivars (Table 1 and Table S2) [25]. In our study, although flavanones class was not so representative in the edible portion of the fruit at the onset, storage promoted the increase in the accumulation of hesperidin, hesperitin, naringenin, narirutin or isosakuranetin (and derivatives) (Figure 5 and Table S2). Flavanones from citrus fruits have been awarded important biological activities, as helping in cardiovascular and cancer risk prevention and avoiding the onset of oxidative stress involved in inflammatory damage due to their antioxidant potential [57,58]. The main studies of citrus flavanones antioxidant and anti-inflammatory properties focused on hesperidin and its aglycones (hesperitin), and narirutin [59–63]. In the case of isosakuranetin anti-oxidative activities, they have been related with potential free radical scavenging mechanisms [64]. Numerous studies describing the effects of naringenin on human health reported increasing antioxidant defences, scavenging reactive oxygen species, antiviral responses or exerting anti-atherogenic and anti-inflammatory effects [65]. Naringenin is predominantly found in the edible citrus part and, although is poorly absorbed by oral ingestion, a positive orange juice prebiotic effect due to its bioavailability has been shown [66]. In relation to flavones, compounds grouped into this class also showed a better enrichment in Moro than in Pera fruit under postharvest storage. Among them, luteolin, apigenin and nobiletin (including some glycosylated forms) were the most enhanced by 9 ◦C storage (Table S2). Citrus flavones have been proposed as the most suitable and capable compounds in terms of antioxidant and anti-inflammatory activities due to their substitutions groups [67]. In the case of nobiletin, it has been indicated to prevent obesity, hepatic steatosis, dyslipidemia, and insulin resistance [42]. Luteolin effects on activation of antioxidant enzymes involved in cancer prevention as well as cardio-protective effects have been reported, as well as properties in inhibiting the onset and development of inflammatory diseases as asthma [68–71]. Multiple other beneficial bioactivities of apigenin have been proposed on different types of cancer or interactions on gut microbiota [72]. Finally, compounds belonging to the flavonols class were the main groups of flavonoids identified and accumulated in Moro fruit stored at low temperature, mainly limocitrol and quercetin (and their derivatives) (Table S2). Recently, the high intake of flavonol in the diet has been associated to a reduction in the risk of developing Alzheimer dementia [73]. Among the flavonols, limocitrol presented the highest increment observed in our study (Table S2). This compound has been reported as one of the main flavonoids in finger citron (*Citrus medica* cv. sarcodactylis) and their strong antioxidation and antiaging activities have been indicated in both in vitro and in vivo studies [74]. Regarding quercetin and its derivatives, the second most induced flavonol by storage at 9 ◦C, it is considered as one of the most relevant antioxidant metabolites due to its chemical structure. Its involvement in lipid peroxidation prevention and tocopherol regeneration has been described, as well as in ischemia injury reduction by the induction of nitric oxide synthase [43]. Quercetin has anticarcinogenic and anti-inflammatory properties with antioxidant and free radical

scavenging effects. Moreover, other antimicrobial, antiviral, and biological effects, which include anti-inflammatory activity has been attributed to it [75,76].

#### **5. Conclusions**

BO is an excellent source of natural antioxidant and bioactive compounds, promoting the interest of consumers and researchers in the recent years [6,7]. Many in vivo studies associate the beneficial health-effects of BO juice consumption in reduction of inflammatory processes related to its remarkable antioxidant power [18]. Protection against oxidative stress of flavonoids by induction of reactive oxygen and nitrogen species have been described to play a role as markers of different degenerative diseases [14,18,22]. All these protective bioactivities are likely due to the marked presence of phenolic acids, flavonoids and other phytochemicals in BO [35], although the contribution of each phytochemical in such antioxidant properties requires further research. In this study, storage at low temperature induced a great enrichment on anthocyanins and flavonoids accumulation levels in Moro orange, suggesting that all these compounds could be contributing to their higher antioxidant capacity. However, we did not observe a similar effect of cold storage in Pera fruit. Taken together, we show here that through a regular postharvest practise the content of not only anthocyanins, but also specific health-related flavonoids is enhanced in Moro blood orange pulp and juice (but not in the Pera blond orange counterpart), reinforcing the interest of blood orange to improve natural antioxidant diets. This work should further analyse flavonoids composition and content in other blood and blond orange cultivars to assess whether the drastic increases observed in flavonoids accumulation in Moro fruit during cold storage may be extended to blood-orange types or it is independent of anthocyanins production.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11030547/s1. Table S1: Primer sequences used for the quantitative RT-PCR analyses. Table S2: Fold changes of flavonoids identified in the pulp of Pera and Moro orange during storage at 9 ◦C for 0, 15, 30 and 45 days. Data are expressed as the mean fold change ± SD of each sample as compared to the control sample (harvest time of Pera fruit).

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

**Funding:** This work was supported by the São Paulo Research Foundation (FAPESP, Brazil) project FAPESP 2014/12616-9, Fundecitrus, PRIMA Foundation (Reference number: 2132) through 1.3. 1-2021 PROMEDLIFE and the Rosty-COST Actions CA18210 for networking. LC was funded by FAPESP grant (2014/23447-3).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data are contained within the article and supplementary material.

**Acknowledgments:** We appreciate the help of Citrosuco for allowing us accessing their orchards and using their fruits. The technical support of Tatiane M. Malara is also acknowledged.

**Conflicts of Interest:** The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### **References**


## *Article* **Preharvest Application of Phenylalanine Induces Red Color in Mango and Apple Fruit's Skin**

**Michal Fanyuk 1,2, Manish Kumar Patel <sup>1</sup> , Rinat Ovadia <sup>3</sup> , Dalia Maurer <sup>1</sup> , Oleg Feygenberg <sup>1</sup> , Michal Oren-Shamir <sup>3</sup> and Noam Alkan 1,\***


**Abstract:** Anthocyanins are secondary metabolites responsible for the red coloration of mango and apple. The red color of the peel is essential for the fruit's marketability. Anthocyanins and flavonols are synthesized via the flavonoid pathway initiated from phenylalanine (Phe). Anthocyanins and flavonols have antioxidant, antifungal, and health-promoting properties. To determine if the external treatment of apple and mango trees with Phe can induce the red color of the fruit peel, the orchards were sprayed 1 to 4 weeks before the harvest of mango (cv. Kent, Shelly, and Tommy Atkins) and apple fruit (cv. Cripps pink, Gala and Starking Delicious). Preharvest Phe treatment increased the red coloring intensity and red surface area of both mango and apple fruit that was exposed to sunlight at the orchard. The best application of Phe was 2–4 weeks preharvest at a concentration of 0.12%, while a higher concentration did not have an additive effect. A combination of Phe and the positive control of prohydrojasmon (PDJ) or several applications of Phe did not have a significant added value on the increase in red color. Phe treatment increased total flavonoid, anthocyanin contents, and antioxidant activity in treated fruit compared to control fruits. High Performance Liquid Chromatography analysis of the peel of Phe treated 'Cripps pink' apples showed an increase in total flavonols and anthocyanins with no effect on the compound composition. HPLC analysis of 'Kent' mango fruit peel showed that Phe treatment had almost no effect on total flavonols content while significantly increasing the level of anthocyanins was observed. Thus preharvest application of Phe combined with sunlight exposure offers an eco–friendly, alternative treatment to improve one of the most essential quality traits—fruit color.

**Keywords:** anthocyanin; phenylalanine; prohydrojasmon; flavonoids; mango; apples; preharvest; red color; fruit quality

## **1. Introduction**

Fruits and vegetables are lost or get a lower price due to poor appearance, quality problems, and consumer preferences [1]. Fruit is downgraded if it does not meet a very high standard of quality requirements, causing loss of profitability [2] and possibly food loss. Color and appearance, nutritional value, texture, and aroma are the main factors determining fruit and vegetable quality [3].

Fruit quality and maturity stage can often be indicated by the color of its skin [4]. The consumer prefers red-colored fruit. Therefore, the red color in fruit peel is a major contributor to the acceptance of fruit, which allows it to be priced higher and sold more easily [5]. Red color of fruit peel is important in terms of appearance and constitutes an advantage in resistance to pathogens and cold. For example, mango fruit exposed to

**Citation:** Fanyuk, M.; Kumar Patel, M.; Ovadia, R.; Maurer, D.; Feygenberg, O.; Oren-Shamir, M.; Alkan, N. Preharvest Application of Phenylalanine Induces Red Color in Mango and Apple Fruit's Skin. *Antioxidants* **2022**, *11*, 491. https:// doi.org/10.3390/antiox11030491

Academic Editor: Stanley Omaye

Received: 24 January 2022 Accepted: 26 February 2022 Published: 28 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

sunlight developed red color, had higher anthocyanin concentration in the fruit peel, and was more resistant to pathogenic fungi including *Colletotrichum gloeosporioides, Alternaria alternata*, and *Lasiodiplodia theobromae* in comparison with green fruit from the same tree [6,7]. Red-colored mango fruit (cv. Shelly) is more resistant to cold than green fruit and, therefore, could be stored for more extended periods [6,8].

Anthocyanins are the main compounds responsible for the red coloring of mango and apple fruit peel [9]. Anthocyanins and flavonoids are natural compounds synthesized via the phenylpropanoid pathway, which have antioxidant and antifungal qualities, as well as health-promoting properties for humans [7,10]. Anthocyanins and flavonoids synthesis is induced in response to biotic or abiotic stresses, such as light, temperature, drought, and pathogen attacks [11,12].

Phenylalanine (Phe), the precursor of the phenylpropanoid pathway, is an aromatic amino acid existing naturally in plants and derived from the shikimate pathway [13]. Flavonoids, including anthocyanins and lignin, are secondary metabolites of the phenylpropanoid pathway, which is one of the main plant defense mechanisms [14]. Application of Phe to petunia, *Arabidopsis,* and cut chrysanthemum flowers, increased their resistance to *B. cinerea* by activating the phenylpropanoid pathway and flavonoids with antifungal activity [15,16]. Similarly, applying phenylalanine pre- or postharvest reduces decay in mango, avocado, strawberry, and citrus fruit, which is caused by various pathogenic fungi [17].

Red color induction methods, including application of plant growth regulators, in fruit have been studied for many years. In particular, previous studies have shown that mango and apple fruit have stronger red pigmentation and higher anthocyanin levels when exposed to direct sunlight, when grown outside of the tree canopy, compared with fruit from the inner parts of the canopy [6,18]. Pre-harvest application of harpin proteins or inactivated yeast treatments also showed an enhanced red color and anthocyanin accumulation [19,20]. Moreover, it was found that exogenic application of Prohydrojasmon (PDJ), methyl jasmonate (MJ), or Abscisic acid (ABA), along with direct sunlight exposure, promotes the phenylpropanoid pathway in mango fruit and contributes to the accumulation of red skin on the fruit [4,21].

Mango (cv. Kent, Shelly, and Tommy Atkins) and apple (cv. Cripps pink, Gala, Starking Delicious, and Anna) are fruits known to have light-red fruit peel and were chosen for this study. Our research goal is to determine if preharvest Phe application to mango and apple orchards will induce the red color in the fruit peel. Preharvest application of Phe could provide a healthy, safe, and relatively cheap means to increase fruit red color, reduce postharvest decay, and improve the profitability of the cultivar.

#### **2. Materials and Methods**

#### *2.1. Plant Material, Preharvest Spray, Harvest, and Storage*

The orchards used for this study were twenty-year-old mango orchards (cv. Kent and Shelly and Tommy Atkins), in Kibbutz Ravid (32◦5100300 N 35◦2705200 E; elevation +165 m), 15-years old apple orchards (cv. Cripps pink, Starking Delicious, and Gala) in Merom Golan (33◦0705900 N 35◦4603300 E; elevation +977 m), and a 15-years old apple orchard cv. Anna was grown in Arugot (31◦4405 00 N 34◦4601500 E). Average-size mango trees (cv. Shelly and Kent) in the season of August 2020 (three trees per repetition, three repetitions per treatment) and apple trees (cv. Gala and Cripps Pink) in August 2020 and November 2020 (three trees per repetitions, four repetitions per treatment) were untreated (control) or sprayed with 0.12% phenylalanine (Phe) or with 0.2% prohydrojasmon (PDJ) on different weeks (1, 2, or 3) before harvest. The experiments were repeated on mango (cv. Shelly and Tommy Atkins) and apple (Cripps pink, Anna and Starking Delicious) fruit from June to November 2021. With the exception of 'Starking Delicious' apple cultivar, the mango and apple fruits were harvested from the outside of the tree canopy, which was exposed to direct sunlight. After harvest, control, and treated mango and apple fruits were transported (up to 2 h) to the ARO Volcani Center, Israel, and immediately stored at 12 ◦C and 2 ◦C for 21–28 days, respectively, followed by 7 days of shelf-life storage at 22 ◦C. Gala apple fruit was stored

at 2 ◦C for 2 months, followed by 13 days of shelf-life storage at 22 ◦C. The fruits were evaluated at harvest, after cold storage, and after shelf life.

#### *2.2. Measurements of Fruit Skin Color*

#### 2.2.1. Red Color Evaluation

We assessed the percentage of the red-colored surface area of each fruit from individual mango and apple fruits in each treatment at various time points (at harvest and after shelf life). Apple fruits were also evaluated after cold storage. Furthermore, the intensity of the red color was graded according to an index (0–5; 0—no red color, 1—faint red color, and 5—very intense red color). Data were collected and analyzed from 14 and 28 mango and apple fruits, respectively, per treatment.

#### 2.2.2. Chroma Measurement

The skin color (Hue) was measured at the reddest point of 14 and 26 mango and apple fruits, respectively, for each treatment using a CR-400/410 Chromometer (Konica Minolta, Osaka, Japan). The hue angle (h◦ ) measures color according to the wheel of colors, where 120◦ angle represents green color; 40◦–60◦ angle represents orange-yellow color and 0 ◦–40◦ represents red color. The transition between red color to green color is represented by a\* value, where value of +60◦ correlates to full red color and –60◦ correlates to full green color.

#### 2.2.3. Estimation of Chlorophyll, Anthocyanins, and Flavonoids in Fruit Skin

Fruit peel fluorescence was evaluated to measure chlorophyll (SFR\_R), flavonoid (FLAV), and anthocyanin (ANTH\_RG) signals using a Multiplex III fluorescence detector (Force A, Orsay, France), based on fluorescence signal ratios between excitations and emissions correlated to these compounds. Data were collected and evaluated from 14 and 26 fruits of each treatment in mango and apple fruit, respectively.

#### *2.3. Decay Evaluation*

Stem end rot (SER), side decay, and total rotten fruits of mango and total decay of apples were evaluated after shelf-life storage, and apples (cv. Gala) were also evaluated after cold storage. We evaluated decay incidence (percent of fruit) and severity (index 0–10; 0—no decay, 1—mild decay; 5—moderate decay; 10—severe decay) for each treatment per box.

#### *2.4. Extract Preparation*

Fruit peel samples from each treatment were harvested and stored at −80 ◦C. Samples were ground (IKA A11 basic, Germany) in liquid N2, and 0.5 g were transferred into methanol solution (70%) for extraction and kept on the shaker overnight (250–300 rpm). The extraction mixture was centrifuged at 4100 rpm for 20 min at 20 ◦C (NF 8000R, Nuve, Turkey), and the supernatant was collected. The extraction was repeated. The collected supernatant liquid was concentrated in a CentriVap Concentrator (Labconco, Kansas, MO, USA) at room temperature until the extraction solution reached a volume of 0.5 mL. The concentrated samples were centrifuged at 12,000 rpm for 10 min at 20 ◦C, diluted 1:4 with distilled water, and stored at 4 ◦C until further use.

#### 2.4.1. DPPH Assay

The free radical scavenging activity was measured by 2,20 - diphenyl-1-picrylhydrazyl (DPPH) assay as previously described [22,23] with slight modifications. The stock solution was prepared (0.24% in absolute methanol) and diluted with methanol until the absorbance was 0.98 ± 0.02 at 517 nm. For each treatment, the diluted extracts (1:4) of mango and apple peels were mixed with 1 mL of DPPH solution and incubated for 10 min at room temperature. The absorbance was measured at 517 nm, and scavenging activities were calculated according to the following equation:

$$\text{scacvenging activity (\%)} = \text{[}\frac{\text{OD}\_{517 \text{ control}} - \text{OD}\_{517 \text{ treatment}}}{\text{OD}\_{517 \text{ control}}}\text{]} \times \text{100 }$$

#### 2.4.2. Total Phenolic Content

Total phenolic content of mango and apple fruit peels was evaluated with 0.2 N Folin– Ciocalteu (FC) reagent, and gallic acid was used as a reference standard [24,25]. For each treatment, the diluted mango and apple peel extracts were mixed with 750 µL of FC reagent and incubated for 5 min. 500 µL of sodium carbonate (Na2CO3, 75 gL−<sup>1</sup> ) were added to the reaction mixture followed by 15 min incubation. The absorbance was measured at 760 nm and total phenolic content was calculated as mg gallic acid equivalent/g of fresh weight (gFW).

#### 2.4.3. Total Flavonoid Content

To determine the total flavonoid content, the diluted mango and apple peel extracts from each treatment were mixed with 200 µL of sodium nitrite (NaNO2, 5%) and incubated for 5 min at room temperature. Then, 200 µL of aluminum chloride (AlCl3, 10%) and 1.5 mL of sodium hydroxide (NaOH, 1 M) were added to the reaction mixture and absorbance was measured at 510 nm. The total flavonoid content was calculated as mg quercetin equivalent/gFW [26].

#### *2.5. Flavonoid and Anthocyanin Characterization (HPLC)*

Analysis was conducted on peels of mango (cv. Kent) and apple (cv. Cripps Pink) fruit after cold storage, to identify and quantify the flavonoids and anthocyanins in the fruit peel. 0.3 g of peel samples were used for extraction (2 mL, methanol:water:acetic acid, 11:5:1, *v/v*) [27]. Anthocyanins and flavonols were quantified as described by [28].

The experiment was conducted according to [29]. High-performance liquid chromatography (HPLC) (Shimatzu, Kyoto, Japan) equipped with an LC-10AT pump, an SCL-10A controller, and an SPD-M10AVP photodiode array detector. Extracts were loaded onto an RP-18 column (Vydac 201TP54) and separated at 27 ◦C with the following solutions: (A) H2O, pH 2.3, and (B) H2O:MeCN:HOAc (107:50:40), pH 2.3. Solutions were applied as a linear gradient from a ratio of 4:1 (A:B) to 3:7 over 45 min and held at a ratio of 3:7 for an additional 10 min at a flow rate of 0.5 mL/min. Anthocyanidins and flavonoids were identified by comparing both the retention time and the absorption spectrum at 250–650 nm with those of standard purified anthocyanins and flavonols (ChemFaces, Wuhan), China and Extrasynthase (Genay, France). Identification and quantification of anthocyanins and flavonols were done using reference standards, and concentrations were expressed as peak area/gram of FW.

#### *2.6. Statistical Analysis*

Data are represented as mean value ± standard error (SE). Multifactorial analysis of variance (One-way ANOVA, Tukey–Kramer HSD test) and Wilcoxon non-parametric comparison were performed using JMP (JMP Pro 15 software, SAS Institute, Cary, NC, USA). Different letters represent a statistically significant difference (*p* ≤ 0.05) among different treatments at the same time point.

#### **3. Results**

The effect of preharvest Phe spraying on the redness of mango (cv. Kent and Shelly) and apple (cv. Cripps Pink and Gala) fruit peel was evaluated in comparison to untreated control fruit and the positive control of PDJ treated fruit. Fruit treated with either Phe or PDJ at harvest and after shelf-life storage had a significantly higher red color intensity of the peel in mango and apple fruit compared to the control (Figures 1A, 2A, S1A and S2A).

The percentage of surface coverage of the red color in the mango (cv. Kent and Shelly) fruit peel at all evaluated time points was significantly higher in most treatments (Figures 1B and S1B). Phe and PDJ significantly increased the red color surface coverage in almost all treated apples (cv. Gala) and increased the red color surface in treated 'Cripps pink' apples (Figures 2B and S2B). Hue values at the reddest point on the fruit peel were evaluated at several time points (harvest, after CS, and after SL) for mango (cv. Kent and Shelly) and apple (cv. Cripps pink and Gala) fruit. In general, Phe and PDJ treatments decreased hue values (wheel of colors, where 120 is for green color; 0–40 represents red color) in almost all treated fruit (Figures 1C, 2C, S1C, S2C and S3A,C). This trend was especially apparent in mango (cv. Kent) and apple (cv. Gala) fruit, where hue values of treated fruit significantly decreased compared to control fruit both at harvest and after SL (Figures 1C, S2A and S3A). In mango (cv. Shelly), fruit peel color varied similarly at harvest from 70.3 in control to 43.9–21.2 in treated fruit at this time point (Figure S1C). In apple (cv. Cripps pink), even though treated apples had significantly lower hue values at their reddest point compared to the control, all the treated and untreated fruit were within the orange color range (Figures 2C and S3C). The Red-Green color range, which is represented by a\* value, is directly proportional to the red-green color intensity. Higher and positive a\* values correlate with more intense red color, whereas lower positive and negative values correlate with yellow and green color intensities, respectively. Phe and PDJ significantly increased a\* values in most mango (cv. Kent and Shelly) and apple (cv. Gala) treated fruit. a\* value of apple (cv. Cripps pink) also increased after Phe treatment compared to the control (Figures 1D, Figure 2D, S1D, S2D, S3B,D). *Antioxidants* **2022**, *11*, 491 6 of 13

**Figure 1.** Red color evaluation of 'Kent' mango fruit peel. 'Kent' mango orchards were sprayed with 0.12% phenylalanine (Phe) or 0.2% prohydrojasmon (PDJ), one or two weeks preharvest. The fruit was evaluated at harvest (T0), after cold storage (CS, 3 weeks at 12 °C), and after shelf life (SL, 7 days at 22 °C). (**A**) Red color intensity (index 0−5). (**B**) Red surface area (% of fruit coverage). (**C**) Color (Hue value of the reddest point). (**D**) Green-red color range (a\* value). (**E**) Representative pictures of 'Kent' mango boxes after shelf-life storage. Mean values and standard errors are **Figure 1.** Red color evaluation of 'Kent' mango fruit peel. 'Kent' mango orchards were sprayed with 0.12% phenylalanine (Phe) or 0.2% prohydrojasmon (PDJ), one or two weeks preharvest. The fruit was evaluated at harvest (T0), after cold storage (CS, 3 weeks at 12 ◦C), and after shelf life (SL, 7 days at 22 ◦C). (**A**) Red color intensity (index 0–5). (**B**) Red surface area (% of fruit coverage). (**C**) Color (Hue value of the reddest point). (**D**) Green-red color range (a\* value). (**E**) Representative pictures of 'Kent' mango boxes after shelf-life storage. Mean values and standard errors are presented. Statistical analysis was conducted for each time point separately (small or capital letters). Different letters represent a significant difference (*p* ≤ 0.05).

presented. Statistical analysis was conducted for each time point separately (small or capital letters).

Different letters represent a significant difference (*p* ≤ 0.05).

Different letters represent a significant difference (*p* ≤ 0.05).

**Figure 2.** Red color evaluation of apple (cv. Cripps pink) peel. Apple orchard was sprayed preharvest with 0.12% phenylalanine (Phe) and/or 0.2% prohydrojasmon (PDJ). The fruit was evaluated at 3-time points: at harvest, after cold storage (CS, 3 weeks at 2 ◦C), and after shelf life (SL, 7 days at 20 ◦C). (**A**) Red color intensity (index 0–5). (**B**) Red surface area (% of fruit coverage). (**C**) Color (hue value at the reddest point). (**D**) Green-red color range (a\* value at the reddest point). (**E**,**F**). Representative pictures of Cripps pink apple box after shelf-life storage. Mean values and standard errors are presented. Statistical analysis was conducted for each time point separately (small or capital letters). Different letters represent a significant difference (*p* ≤ 0.05).

**Figure 1.** Red color evaluation of 'Kent' mango fruit peel. 'Kent' mango orchards were sprayed with 0.12% phenylalanine (Phe) or 0.2% prohydrojasmon (PDJ), one or two weeks preharvest. The fruit was evaluated at harvest (T0), after cold storage (CS, 3 weeks at 12 °C), and after shelf life (SL, 7 days at 22 °C). (**A**) Red color intensity (index 0−5). (**B**) Red surface area (% of fruit coverage). (**C**) Color (Hue value of the reddest point). (**D**) Green-red color range (a\* value). (**E**) Representative pictures of 'Kent' mango boxes after shelf-life storage. Mean values and standard errors are presented. Statistical analysis was conducted for each time point separately (small or capital letters).

The results described above were experiments conducted on apple cultivars (cv. 'Anna,' 'Starking Delicious, and 'Cripps Pink') and mango cultivars (cv. 'Shelly' and 'Tommy Atkins') in the following year, which showed a similar trend (Tables S1–S3). The fruit treated preharvest with Phe showed a significant increase in red color intensity and red color surface of almost all treatments compared to control. Moreover, hue values generally decreased and a\* value was usually increased (Tables S1 and S2).

Preharvest application of Phe in various concentrations showed that an increase in Phe concentration better induced the red color until optimum results at 0.12% Phe (Tables S1, S2 and S6). Phe at 0.12% increased the red color area and intensity in the fruit peel significantly better compared to lower concentrations as Phe at 0.01%, which was applied to apple (cv. Starking Delicious and Cripps pink) and mango (cv. Shelly) orchards (Table S1). Higher concentration (0.24%) did not contribute to better results of red color accumulation (Tables S1–S3). In the examination of the best time to apply Phe preharvest, it seems that in most experiments 2 weeks preharvest led to the best induction of red peel color (Tables S1–S3). While in Starking apples the application of 4 weeks preharvest was optimal for inducing red color (Table S4). A combination of several applications usually did not further induce the red color (Tables S2–S4). This induction of red color by preharvest application of Phe was correlated to a small decrease in decay incidence and severity in mango fruit (cv. Kent, Shelly, and Tommy) and inconclusive results in apple fruit (Tables S6 and S7).

Preharvest application of Phe increased the antioxidant activity in mango (cv. Kent and Shelly) and apple (cv. Cripps pink) fruit peels (Figures 3A,D and S4A). Phe also increased total phenolics and flavonoid content in most treatments in mango (cv. Kent and Shelly) and apple (cv. Cripps pink) fruit peels (Figures 3B,C,E,F and S4B,C). Trends of total phenolic and flavonoid contents correlate to the level of antioxidant activity in mango fruit (cv. Kent and Shelly) both after CS and after SL, and in apple (cv. Cripps pink) after SL (Figures 3 and S4). In both mango cultivars (cv. Kent and Shelly), combined treatment of

PDJ 2w + Phe 2w had the highest antioxidant activity (Figures 3A and S4A). However, in apples (cv. Cripps pink), Phe 2w and Phe 3w treatments had the highest antioxidant activity at harvest and after CS and SL, respectively (Figure 3D). The highest quantity of flavonoid content in mango (cv. Kent) was measured in the PDJ 2w treatment and PDJ 2w + Phe 2w treatment in mango (cv. Shelly) (Figures 3C and S4C). Quantification of total phenolic content showed a similar trend in 'Kent' mango and 'Shelly' mango after CS (Figures 3B and S4B). In apple (cv. Cripps pink), total phenolics and flavonoid content were highest in Phe 2w or 3w treatments, similar to its antioxidant evaluation (Figure 3). However, in apples (cv. Cripps pink), Phe 2w and Phe 3w treatments had the highest antioxidant activity at harvest and after CS and SL, respectively (Figure 3D). The highest quantity of flavonoid content in mango (cv. Kent) was measured in the PDJ 2w treatment and PDJ 2w + Phe 2w treatment in mango (cv. Shelly) (Figures 3C and S4C). Quantification of total phenolic content showed a similar trend in 'Kent' mango and 'Shelly' mango after CS (Figures 3B and S4B). In apple (cv. Cripps pink), total phenolics and flavonoid content were highest in Phe 2w or 3w treatments, similar to its antioxidant evaluation (Figure 3).

**Figure 2.** Red color evaluation of apple (cv. Cripps pink) peel. Apple orchard was sprayed preharvest with 0.12% phenylalanine (Phe) and/or 0.2% prohydrojasmon (PDJ). The fruit was evaluated at 3-time points: at harvest, after cold storage (CS, 3 weeks at 2° C), and after shelf life (SL, 7 days at 20° C). (**A**) Red color intensity (index 0−5). (**B**) Red surface area (% of fruit coverage). (**C**) Color (hue value at the reddest point). (**D**) Green-red color range (a\* value at the reddest point). (**E,F**). Representative pictures of Cripps pink apple box after shelf-life storage. Mean values and standard errors are presented. Statistical analysis was conducted for each time point separately

Preharvest application of Phe increased the antioxidant activity in mango (cv. Kent and Shelly) and apple (cv. Cripps pink) fruit peels (Figures 3A,D and S4A). Phe also increased total phenolics and flavonoid content in most treatments in mango (cv. Kent and Shelly) and apple (cv. Cripps pink) fruit peels (Figures 3B,C,E,F and S4B,C). Trends of total phenolic and flavonoid contents correlate to the level of antioxidant activity in mango fruit (cv. Kent and Shelly) both after CS and after SL, and in apple (cv. Cripps pink) after SL (Figures 3 and S4). In both mango cultivars (cv. Kent and Shelly), combined treatment of PDJ 2w + Phe 2w had the highest antioxidant activity (Figures 3A and S4A).

(small or capital letters). Different letters represent a significant difference (*p* ≤ 0.05).

*Antioxidants* **2022**, *11*, 491 7 of 13

**Figure 3.** Effect of preharvest treatment with 0.12% Phe or 0.2% PDJ on antioxidant, phenolic, and flavonoid content in mango (cv. Kent) and apple fruit (cv. Cripps pink) at harvest, after cold storage (CS), and shelf-life (SL). Antioxidant activity, total phenolic, and total flavonoid content were evaluated from mango peels (cv. Kent) (**A**−**C**), apple (cv. Cripps pink) (**D**−**F**) peels. Mean values and standard errors are presented. Statistical analysis was conducted for each time point separately **Figure 3.** Effect of preharvest treatment with 0.12% Phe or 0.2% PDJ on antioxidant, phenolic, and flavonoid content in mango (cv. Kent) and apple fruit (cv. Cripps pink) at harvest, after cold storage (CS), and shelf-life (SL). Antioxidant activity, total phenolic, and total flavonoid content were evaluated from mango peels (cv. Kent) (**A**–**C**), apple (cv. Cripps pink) (**D**–**F**) peels. Mean values and standard errors are presented. Statistical analysis was conducted for each time point separately (small or capital letters). Different letters represent significant differences (*p* ≤ 0.05).

(small or capital letters). Different letters represent significant differences (*p* ≤ 0.05). The fluorescence of chlorophyll, flavonoids, and anthocyanins in the fruit peel was evaluated at the reddest point of all mango (cv. Kent and Shelly) and apple (cv. Cripps pink and Gala) fruit. Chlorophyll fluorescence decreased in almost all the Phe treated The fluorescence of chlorophyll, flavonoids, and anthocyanins in the fruit peel was evaluated at the reddest point of all mango (cv. Kent and Shelly) and apple (cv. Cripps pink and Gala) fruit. Chlorophyll fluorescence decreased in almost all the Phe treated mango (cv. Kent and Shelly) and apple (cv. Cripps pink and Gala) fruits, and almost all treatments presented statistical significance (Figures 4A,D, S5A,D and S6A,D).

mango (cv. Kent and Shelly) and apple (cv. Cripps pink and Gala) fruits, and almost all treatments presented statistical significance (Figures 4A,D, S5A,D and S6A,D). 'Kent' mango had a significant increase in flavonoid fluorescence in all Phe treatments compared to the control both after harvest and after SL (Figures 4B and S6B). Similarly, 'Shelly' mango also showed an increase in flavonoids in all Phe treatments at harvest and after CS, however, after SL storage, no difference between the treatments was observed (Figure S6B). Apple (cv. Cripps pink and Gala) showed increased flavonoid fluorescence in treated fruit at harvest, while flavonoid fluorescence did not increase in treated fruit after SL storage (Figures 4F, S5E and S6E). Phe and PDJ increased anthocyanin fluorescence in all treated fruit. The highest level of anthocyanins was detected mainly in Phe 2w and Phe 3w and their combination with other treatments in both mango (cv. Kent and Shelly) and apple (cv. Cripps pink) after SL. Apple (cv. Gala) presented the highest level of anthocyanins in Phe 1w treatment and its combinations (Figures 4C,F, S5C,F and S6C,F).

S6C,F).

**Figure 4.** Effect of preharvest treatment of Phe and PDJ on chlorophyll, anthocyanin, and flavonoids fluorescence. Mango (cv. Kent, **A−C**) and apple (cv. Cripps pink, **D−F**) orchards were treated 1−3 weeks (1W, 2W, 3W) preharvest with 0.12% Phe or 0.2% PDJ and chlorophyll, anthocyanin, and flavonoids fluorescence at the reddest point of the fruit peel was analyzed at 3-time points: after harvest, after cold storage (CS), and after shelf-life (SL). (A, D) Chlorophyll fluorescence (SFR\_R). (**B,E**) Flavonoids fluorescence (FLAV). (**C,F**) Anthocyanin fluorescence (ANTH\_RG). Mean values and standard errors are presented. Statistical analysis was conducted for each time point separately (small and capital letters). Different letters represent significant differences (*p* ≤ 0.05). **Figure 4.** Effect of preharvest treatment of Phe and PDJ on chlorophyll, anthocyanin, and flavonoids fluorescence. Mango (cv. Kent, **A**–**C**) and apple (cv. Cripps pink, **D**–**F**) orchards were treated 1–3 weeks (1W, 2W, 3W) preharvest with 0.12% Phe or 0.2% PDJ and chlorophyll, anthocyanin, and flavonoids fluorescence at the reddest point of the fruit peel was analyzed at 3-time points: after harvest, after cold storage (CS), and after shelf-life (SL). (**A**,**D**) Chlorophyll fluorescence (SFR\_R). (**B**,**E**) Flavonoids fluorescence (FLAV). (**C**,**F**) Anthocyanin fluorescence (ANTH\_RG). Mean values and standard errors are presented. Statistical analysis was conducted for each time point separately (small and capital letters). Different letters represent significant differences (*p* ≤ 0.05).

'Kent' mango had a significant increase in flavonoid fluorescence in all Phe treatments compared to the control both after harvest and after SL (Figure 4B and S6B). Similarly, 'Shelly' mango also showed an increase in flavonoids in all Phe treatments at harvest and after CS, however, after SL storage, no difference between the treatments was observed (Figure S6B). Apple (cv. Cripps pink and Gala) showed increased flavonoid fluorescence in treated fruit at harvest, while flavonoid fluorescence did not increase in treated fruit after SL storage (Figure 4F, S5E and S6E). Phe and PDJ increased anthocyanin fluorescence in all treated fruit. The highest level of anthocyanins was detected mainly in Phe 2w and Phe 3w and their combination with other treatments in both mango (cv. Kent and Shelly) and apple (cv. Cripps pink) after SL. Apple (cv. Gala) presented the highest level of anthocyanins in Phe 1w treatment and its combinations (Figures 4C,F, S5C,F and

Quantification and identification of metabolites in mango (cv. Kent) and apple (cv. Cripps pink) fruits treated with Phe or PDJ after cold storage was performed using HPLC analysis (Figure 5). In apple (cv. Cripps pink) peel, three types of compounds were detected related to the phenylpropanoid pathway: anthocyanins, flavonols, and dihydrochalcones, while in mango peel only anthocyanins and flavonols were detected (Figure 5). Both apple and mango peels from fruit treated with Phe had significantly higher levels of anthocyanins in the sample, compared to the control (Figure 5A,D). In mango, the anthocyanins content increased by 11.7 fold in the Phe 2w treatment. While the highest levels of anthocyanins and flavonols in apple (cv. Cripps pink) peel were observed in Phe 1w treatment, with an increase in anthocyanins and flavonols by 9.3 and 3.3 folds, respectively, compared to control (Figure 5). These levels of increase suggest a major shift in biosynthetic activity. Similarly, the total amount of dihydrochalcones detected in Phe treated apple samples was higher compared to the control (Figure 5C). Quantification and identification of metabolites in mango (cv. Kent) and apple (cv. Cripps pink) fruits treated with Phe or PDJ after cold storage was performed using HPLC analysis (Figure 5). In apple (cv. Cripps pink) peel, three types of compounds were detected related to the phenylpropanoid pathway: anthocyanins, flavonols, and dihydrochalcones, while in mango peel only anthocyanins and flavonols were detected (Figure 5). Both apple and mango peels from fruit treated with Phe had significantly higher levels of anthocyanins in the sample, compared to the control (Figure 5A,D). In mango, the anthocyanins content increased by 11.7 fold in the Phe 2w treatment. While the highest levels of anthocyanins and flavonols in apple (cv. Cripps pink) peel were observed in Phe 1w treatment, with an increase in anthocyanins and flavonols by 9.3 and 3.3 folds, respectively, compared to control (Figure 5). These levels of increase suggest a major shift in biosynthetic activity. Similarly, the total amount of dihydrochalcones detected in Phe treated apple samples was higher compared to the control (Figure 5C). Unlike mango, apple peel samples contained a significantly higher amount of flavonols in Phe treated fruit compared to the control (Figure 5B,E).

Eight flavonols were detected in mango (cv. Kent) peel: quercetin-3-*O*-galactoside (26.1 min, Qu-gal), quercetin-3-*O*-glucoside (27.2 min, Qu-glc), quercetin-3-*O*-xyloside (28.4 min, Qu-xyl), quercetin-3-*O*-arabinopyranoside (29.6 min, Qu-arap), quercetin-3- *O*-arabinofuranoside (30.4 min, Qu-araf), quercetin-3-*O*-rhamnoside (31.8 min, Qu-rha), kaempferol-3-*O*-glucoside (32.8 min, Ka-glc) and unknown compound (47.1 min) (Figure 5E). The ratios between the different flavonols within each treatment were similar, indicating that the treatment did not change the synthesis of individual compounds (Figures 5E and S7). In apple peels, seven flavonols were revealed (Figures 5B and S8). The chromatogram As opposed to mango (cv. Kent) peel analysis, several minor changes in composition levels were detected between treated fruit to control in apple (cv. Cripps pink) peel. For example, quercetin-3-*O*-galactoside (Qu-gal, 26.4 min) and quercetin-3 glucoside (Qu-glc, 27.6 min) showed a slightly higher level in treatments compared to the control, while quercetin-3-rhamnoside (Qu-rha, 32.3 min) and quercetin 3-xyloside

(Qu-xyl, 28.8 min) showed lower levels in treated fruit compared to control. Most of the other compounds had a more or less similar ratio to the control (Figure 5B). Unlike mango, apple peel samples contained a significantly higher amount of flavonols in Phe treated fruit compared to the control (Figure 5B,E).

**Figure 5.** Quantification of metabolites using HPLC in mango and apple fruit peel. Mango (cv. Kent) and apple (cv. Cripps pink) were sprayed with 0.12% phenylalanine (Phe) or 0.2% prohydrojasmon (PDJ) one or two weeks preharvest. Apple (**A**−**C**) and mango (**D**,**E**) peels were analyzed after 3 weeks of cold storage at 2 °C and 12 °C, respectively. (**A**,**D**) Anthocyanin content in apple and mango, respectively. (**B**,**E**) Flavonol content in apple and mango, respectively. (**C**) Dihydrochalcones in apple. All the values expressed as Peak area/gFW (×106). Mean values and standard errors are presented. Different letters represent significant differences for total content (*p* < 0.05). **Figure 5.** Quantification of metabolites using HPLC in mango and apple fruit peel. Mango (cv. Kent) and apple (cv. Cripps pink) were sprayed with 0.12% phenylalanine (Phe) or 0.2% prohydrojasmon (PDJ) one or two weeks preharvest. Apple (**A**–**C**) and mango (**D**,**E**) peels were analyzed after 3 weeks of cold storage at 2 ◦C and 12 ◦C, respectively. (**A**,**D**) Anthocyanin content in apple and mango, respectively. (**B**,**E**) Flavonol content in apple and mango, respectively. (**C**) Dihydrochalcones in apple. All the values expressed as Peak area/gFW (×10<sup>6</sup> ). Mean values and standard errors are presented. Different letters represent significant differences for total content (*p* < 0.05).

Eight flavonols were detected in mango (cv. Kent) peel: quercetin-3-*O*-galactoside (26.1 min, Qu-gal), quercetin-3-*O*-glucoside (27.2 min, Qu-glc), quercetin-3-*O*-xyloside (28.4 min, Qu-xyl), quercetin-3-*O*-arabinopyranoside (29.6 min, Qu-arap), quercetin-3-*O*arabinofuranoside (30.4 min, Qu-araf), quercetin-3-*O*-rhamnoside (31.8 min, Qu-rha), kaempferol-3-*O*-glucoside (32.8 min, Ka-glc) and unknown compound (47.1 min) (Figure 5E). The ratios between the different flavonols within each treatment were similar, indicating that the treatment did not change the synthesis of individual compounds (Figures 5E and S7). In apple peels, seven flavonols were revealed (Figures 5B and S8). The chromatogram As opposed to mango (cv. Kent) peel analysis, several minor changes in composition levels were detected between treated fruit to control in apple (cv. Cripps pink) peel. For example, quercetin-3-*O*-galactoside (Qu-gal, 26.4 min) and quercetin-3 glucoside (Qu-glc, 27.6 min) showed a slightly higher level in treatments compared to the control, while quercetin-3-rhamnoside (Qu-rha, 32.3 min) and quercetin 3-xyloside (Quxyl, 28.8 min) showed lower levels in treated fruit compared to control. Most of the other compounds had a more or less similar ratio to the control (Figure 5B). Mango contained only two anthocyanins: cyaniding-3-*O*-β-D-galactoside (11.3 min, Cy-gal) and 7-*O*-methylcyanidin 3-*O*-β-D-galactopyranoside (20.9 min, MCy-gal) (Figure. Mango contained only two anthocyanins: cyaniding-3-*O*-β-D-galactoside (11.3 min, Cy-gal) and 7-*O*-methylcyanidin 3-*O*-β-D-galactopyranoside (20.9 min, MCy-gal) (Figure 5D). 7-*O*-methylcyanidin 3-*O*-β-D-galactopyranoside was the main anthocyanin compound in mango fruit (Figure 5D and Figure S7). Anthocyanins apple (cv. Cripps pink) samples contained four cyanidin derivatives: cyanidin 3-galactoside (11.4 min, Cygal), and three unknown derivatives of cyanidin (18.0 min, 22.7 min, and 24.1 min) were detected. According to the literature, the most abundant peak in apple peels is cyanidin galactoside which has been identified before in 'Cripps Pink' [30,31]. The other cyanidin derivative peaks (Cy2, Cy3 and Cy4) in our study on apple peels could probably be designated as cyanidin 3-arabinoside, cyanidin 3-glucoside, cyanidin 3-xyloside [32,33]. All treatments presented similar ratios between the anthocyanin compounds, with cyanidin 3-galactoside being the main derivative detected (Figure 5). Dihydrochalcones, which were detected in apple peel as phloretin derivatives, also had a similar ratio in the control and treated fruit, including both phloridzin (35.1 min) and an unknown derivative of phloretin (31.7 min) (Figure 5C). The phloretin derivative could probably be designated as phloretin 2 0 -xyloglucoside [34]. In summary, it seems that preharvest Phe treatment increases the biosynthesis of flavonoids, anthocyanins, and dihydrochalcones, while keeping a similar ratio of the compounds as in the control (Figure 5).

5D). 7-*O*-methylcyanidin 3-*O*-β-D-galactopyranoside was the main anthocyanin

#### **4. Discussion**

Red color is important for the marketability of fruit. Redder fruit can help decrease food loss and also contribute to customer health [10]. Red color of mango, apple, and other fruit peels is associated with the secondary metabolites named anthocyanins. In this work, we evaluated the effect of preharvest Phe application as a relatively cheap and eco-friendly method [17] on the induction of the red color of mango (cv. Kent, Tommy Atkins, and Shelly) and apple (cv. Starking Delicious, Gala and Cripps Pink) fruit peel exposed to direct sunlight. The main finding of this research is that preharvest application of phenylalanine in combination with sunlight radiation, results in the induction of anthocyanin biosynthesis, enhanced red color of the mango and apple fruit peel, and an increase in antioxidant activity.

Phenylalanine, a naturally occurring aromatic amino acid derived from the shikimate pathway [13], is the precursor for the phenylpropanoid biosynthetic pathway, where anthocyanins are among the secondary metabolites of the downstream pathway, which contribute to the accumulation of red color in the fruit peel [9]. Phe treatment increased flavonols and fragrance related to upstream of the phenylpropanoid pathway in various flowers but did not lead to the accumulation of anthocyanins and red color in flowers as well as Arabidopsis, tomato, chrysanthemum and petunia leaves [15,16,35]. Similarly, postharvest application to various fruits increased flavonols but did not increase the red color of fruit [17].

Flavonoid and phenylpropanoid pathways are defense mechanisms in the plant which are tightly regulated and induced in response to biotic or abiotic stress, that is, pathogens or sunlight [11,12]. Thus, Phe application induces flavonols production but does not increase the production of anthocyanins unless the downstream of the pathway is induced. Therefore, in this work, we show that preharvest treatment with phenylalanine on mango or apple fruit accompanied by sunlight radiation led to an induction of red color coverage and intensity of the peel. The increase in red coloration occurred in fruit on the outer side of the canopy, due to sunlight exposure. Indeed, sunlight radiation is known to induce the phenylpropanoid pathway in various plants, including mango and apple fruit [6,18,21,36].

The current study also applied prohydrojasmon (PDJ), an analog of Jasmonic acid phytohormone as a positive control, after having been described as effective in red color induction of the fruit peel [29]. Although Phe showed similar results as did PDJ, the mode of action of Phe seems to be as a precursor of phenylpropanoid pathway and not in a hormone-like manner. Phenylalanine also has an economic advantage that makes it more affordable compared to PDJ.

Previous studies reported that preharvest treatment of mango fruit (cv. Kent, Shelly, and Maya) with PDJ and ABA exposed to sunlight, increased the red color intensity and coverage of the fruit peel [21,29]. Similarly, both in apple and mango fruit, the red color surface coverage and intensity of the peel have increased due to the preharvest application of phenylalanine in combination with sunlight (Figures 1 and 2). Shafiq and Singh have shown that phenylalanine at a concentration of 0.01% which was applied about 4 weeks preharvest increased the red color and anthocyanins of 'Cripps Pink' apples [37]. Here, various concentrations of Phe on both mango and apple fruit were tested on a larger scale in both mango and apples and at different years, finding that a higher concentration of 0.12% was much more efficient in inducing red color than the application of 0.01% of Phe (Tables S1, S3 and S4). It seems that increase concertation up to 0.12% led to an increase in red color in mango and apple peel (Tables S1 and S2).

The intensity of the red color, a\*, and hue value measurements showed that Phe treated fruit at 0.12% had a redder and more intense red peel color, whereas the fruit in the control group had a lighter intensity red color varying from orange to yellow colors (Figures 1 and 2). Similarly, preharvest applications of Methyl Jasmonate (MJ) and prohydrojasmon (PDJ) were reported to increase a\* values and reduce hue values in mango fruit (cv. Mahachanok) [4,21].

The phenylpropanoid pathway is responsible for the synthesis of flavonoids and anthocyanins [38]. Preharvest treatment with Phe significantly increased anthocyanin levels both in mango (cv. Kent) and in apple (cv. Cripps pink) fruit, while flavonoid levels were either increased or unchanged (Figures S5 and S6). Further phenylpropanoid metabolic analysis was done by HPLC, which showed a significant increase in total anthocyanin and flavonols levels compared to control in apple (cv. Cripps pink) and in anthocyanin levels in mango (cv. Kent) with almost no effect on the compound composition (Figure 5). As a comparison, mango fruit exposed to sunlight showed an increase of the same flavonoids in comparison to fruit from the inside of the tree canopy [8]. Due to the increase in flavonols and anthocyanins, which have antioxidative properties, it has been observed that preharvest application with phenylalanine also improved antioxidant activity both in mango and apple fruit (Figures 3–5). Indeed, a positive correlation between total phenolic and flavonoid content and the level of antioxidant activity was found in different apple varieties [39].

#### **5. Conclusions**

Preharvest treatment of Phenylalanine in combination with sunlight radiation increased the phenylpropanoid biosynthesis pathway, leading to an increase in the coverage and intensity of red-colored peel fruit in various mango and apple cultivars. Phe application increased the phenolic and flavonoid contents due to the activation of the phenylpropanoid pathway, which in combination with sunlight radiation led to the biosynthesis of anthocyanins that directly contribute to the red color of the peel and have health benefits. This study provides an effective new method of Phe spray at the orchard to improve the appearance of the fruit and its health benefits.

#### **6. Patents**

Provisional Patent Application No. 63/134,403 and 63/164,051, titled "Methods for improving fruit quality."

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox11030491/s1.

**Author Contributions:** Conceptualization, N.A.; methodology, M.F. and M.K.P.; validation, N.A.; formal analysis, M.F., M.K.P., R.O., D.M. and O.F.; resources, N.A. and M.O.-S.; data curation, M.F., R.O., D.M. and O.F.; writing—original draft preparation, M.F.; writing—review and editing, M.F., M.K.P., R.O. and N.A.; supervision; project administration; funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Copia and PostBoost, grant number 430-0875-Y-88.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article or supplementary material .

**Acknowledgments:** We thank Yigal Elad for his advices. We thank Copia Company, Galit Sharabani, Eyal Cohen and Ohad Zuckerman for their consult and support. A special thanks for Amos Ovadia for conducting all the field experiment.

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

#### **References**

