Improvement of the Antioxidant Properties and Postharvest Life of Three Exotic Andean Fruits by UVC Treatment

1Facultad de Ciencias de la Ingenierı́a, Universidad Tecnológica Equinoccial, Campus Occidental, Av. Occidental y Mariana de Jesús, 170129 Quito, Ecuador 2Centro de Investigación y Desarrollo en Criotecnologı́a de Alimentos (CIDCA), CCT La Plata CONICET-UNLP, 47 Esq. 116, 1900 La Plata, Argentina 3Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata (UNLP), Calle 60 y 119, 1900 La Plata, Argentina 4Laboratorio de Investigación en Productos Agroindustriales (LIPA), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, Calle 60 y 119, 1900 La Plata, Argentina


Introduction
Despite the large diversity of fleshy fruit-bearing species, world fruit production focuses on a narrow group of tem perate and tropical species [1].However, a variety of novel and exotic fruits recognized for their sensorial and nutritional properties are currently produced and have a great potential to gain international markets [2].Among these, some native fruits of the Andean Region of South America, such as naran jilla (Solanum quitoense Lam.), uvilla (Physalis peruviana L), and mortiño (Vaccinium floribundum Kunth), may be included.Naranjilla is a round delicate and acidic berry (ca.6 cm diameter) produced in Ecuador and Colombia mainly for juice and pulp processing [3].Uvilla is a sweet, juicy, and bright yellow berry (1.2-2.5 cm diameter) characterized by a calyx which protects the whole fruit [4].Uvilla is appreciated locally as well as in some European countries for consump tion in both fresh and processed forms [5].Finally, mortino is a small wild berry (0.5-0.8 cm diameter) resembling traditional blueberries, consumed in traditional desserts and juices [6].Unfortunately, the information available regarding these fruits' physical, chemical, and antioxidant properties and response to storage is very limited [3,5,7].Although proper low temperature storage management is accepted to reduce fruit deterioration, finding supplemental postharvest treatments would be of great value to improve the postharvest shelf-life.
UV-C irradiation is an alternative as nonchemical tech nology that has been shown to increase the storability of several fruits through different mechanisms [8], act as a germicidal agent altering microorganisms DNA and affecting cell division [9], modulate fruit ripening [10,11], and induce array responses including the accumulation of phytoalexins, the reinforcement of the cell walls, and the induction of defensive enzymes and compounds [9,12].UV-C irradiation has been also reported to improve the shelf-life by reduction of fruit respiration [13], softening, and chilling injury [14] and to delay senescence [15,16].In addition, there is growing interest on in the effect of UV-C radiation on fruit antioxi dants [17].Previous works found that UV-C exposure elicited antioxidant accumulation in several fruits, such as table grape [18], strawberry [19], blueberry [20,21], and tomato [22].However, the relevance and extent of these responses are highly variable depending on the species considered and UV-C dose applied [17].
While promising results as prestorage treatment to retain quality and enhance the nutritional properties have been reported for UV-C treatment in traditional fruits [8,12,23], no studies have been conducted to date in uvilla, naranjilla, or mortiño.Ihe potential effect of UV-C treatments to preserve or increase their antioxidant capacity also remains to be determined.Thus, in this work we first selected a suitable UV-C dose for the three Andean fruit and then evaluated the influence of UV-C treatment on antioxidant and physicochemical properties during refrigerated storage to determine the potential of this strategy to supplement proper postharvest temperature management.

Plant Material. Fruits of naranjilla (Solarium quitoense
Lam.), uvilla (Physalisperuviana L), and mortiño ( Vaccinium floribundum Kunth) produced in Ecuador (cities of Puerto Quito, Machachi, and Cotacachi, resp.) were harvested at commercial maturity.Ihe harvest time was determined based on surface color for naranjilla (75% orange) and mortiño (75% purple) and on calyx color for uvilla (color change from green to yellow).Fruits were rapidly brought to the laboratory after harvest and selected for uniformity and lack of physical damage or decay symptoms.For uvilla, the calyx was previously removed.Fruits were then washed with chlorinated water (100 mg I, 1 NaClO, pH 7.0 for 3 min) and dried at room temperature.

UV-C Treatment Selection.
Fruits were placed under a bank of 4 UV-C lamps (Philips, TUV G30T8, 30W, USA; emission peak 254 nm) and irradiated at a distance of 30 cm for 10 or 15 min to reach 8 or 12.5 kJ m 2, respectively (UVP, UVX Radiometer, USA).To achieve uniform treatments throughout the surface, the uvillas and naranjillas were manually rotated while the mortiños were placed on a shaker (VWR International, VWR Microplate Shaker, USA).Similarly, mortiños without UV-C treatment (control) were shook About 200 g of treated fruits was then packed in (a) plastic trays covered with perforated PVC film for uvilla and naranjilla; (b) PET perforated clamshell boxes for mortiño.Samples were stored at 6°C and evaluated weekly for 21 days (d).For each fruit, six trays were prepared per storage time (0, 7, 14, and 21 d) and treatment (UV-C and control).At each sampling date, fruit weight loss and deterioration index were calculated as indicated below to select the most suitable UV-C dose.Untreated fruit packed and stored as previously described was used as control.The whole experiment was repeated twice using fruit from different harvests.

Deterioration Index (DI).
Fruit deterioration was deter mined by visual inspection using a four-point hedonic scale (1 = no damage, 2 = mild damage, 3 = moderate damage, and 4 = severe damage).The main symptoms assessed were pitting, dehydration, firmness loss, browning, and decay.The deterioration index (DI) was calculated according to the following equation: Di y (Damage level) x (No. of trays per level) (Total No. of trays evaluated) For each fruit, six trays were evaluated per storage time and treatment.

Weight Loss.
Each fruit tray was individually weighed during storage and weight loss relative to the initial value was calculated and expressed in percentage.Six trays were evaluated per storage time and treatment.

Optimal UV-C Treatment.
Fruits harvested and con ditioned as described in Section 2.2 were treated with 12.5 kJ m 2 of UV-C radiation, packed, and stored as previ ously described.For each fruit, six trays containing ca. 200 g of fruit were prepared per storage time (0, 7,14, and 21 d) and treatment (UV-C and control).At each sampling date fruit was immediately analyzed for physicochemical properties or frozen in liquid N2 and stored at -80°C until antioxidants analysis.The whole experiment was repeated twice using fruit from different harvests.

Effect of UV-C Treatment on Physicochemical Properties
2.4.1.Firmness.Firmness was assessed with a penetrometer (TR Turoni, Digital fruit firmness tester 53205, Italy).Tests were performed in the equatorial region of fruit employing a 5 mm diameter probe.Results were expressed as Newton (N).Five fruits per tray were randomly selected and evaluated for each storage time and treatment.

Soluble Solids Content (SSC).
Groups of five fruits for naranjilla or fifteen fruits for mortiño and uvilla were randomly selected per tray.Pulp was homogenized and juice was obtained using a blender (Philips, Mixer HR1363 Turbo, Brazil).Soluble solids content was determined with a hand refractometer (Boeco, B&C Hand refractometer 30103, Germany).Results were expressed in percentage (% w/w).For each fruit, four trays were evaluated per storage time and treatment.

Titratable Acidity and pH.
Five fruits for naranjilla or fifteen fruits for mortiño and uvilla were randomly selected per tray.Whole mortiños and uvillas and pulp tissue of naranjillas were frozen in liquid N2 and processed in a blender.About 15 g of the resulting powder was weighed and 100 mL of water was added.Ihe pH of the solution was measured with a pHmeter (Oakton, PC 510, USA).Acidity was determined by titration with 0.1 N NaOH until pH 8.2.Results were expressed as H+ meq.per kg fresh weight.For each fruit, four trays were evaluated per storage time and treatment.

Effect of UV-C Treatment on Antioxidant Properties
2.5.1.Antioxidant Capacity.Groups of five naranjillas and fifteen mortiños and uvillas were randomly selected per tray.Whole mortiños and uvillas and pulp tissue of naranjillas were frozen in liquid N2 and processed in a blender.A portion of resulting powder was weighed (0.2,2, and 3 g for mortiño, uvilla, and naranjilla, resp.) and then homogenized with 6 mL ethanol.Ihe suspension was stirred for 30 min at 4°C and centrifuged at 6,000 xg for 10 min in a refrigerated centrifuge (Hermle, Z323R, Germany).Ihe supernatant was collected and used for antioxidants assays.
The antioxidant capacity (AC) was determined by the ABTS assay [24].Briefly, 10 pL of ethanolic extract was added to 1 mL ABTS'+ working solution (absorbance of 0.700 ± 0.02 at 734 nm), incubated for 6 min at room temperature and the absorbance at 734 nm was measured in a spectrophotometer (Thermo Scientific, Evolution 60S, USA).Trolox® was used as a standard and results were expressed as Trolox Equivalent Antioxidant Capacity (TEAC) in mg per kg fresh weight.Measures were done in triplicate and four extracts were evaluated per storage time and treatment.

Total Phenolics.
Total phenolics were evaluated with the Folin-Ciocalteu reagent [25].An aliquot of 100 pL of ethanolic extract prepared as described in Section 2.5.1 was transferred to a test tube containing 500 pL distilled water.Two hundred microlitres of 1 eq I, 1 Folin-Ciocalteu reagent was added and allowed to react for 3 min at 20°C.After that 400 pL 20% (m/v) Na2CO, in NaOH 0.1 N was pipetted into each test tube and water was added to a final volume of 3mL.After 90 min at 20°C, the absorbance at 760 nm was measured in a spectrophotometer.Catechin was used as standard and results were expressed in mg per kg fresh weight.Measurement was done in triplicate and four extracts were evaluated per storage time and treatment.

Flavonoids.
Flavonoids were tested according to Shin et al. [26] with minor modifications.An aliquot of 300 pL of ethanolic extract prepared as described in Section 2.5.1 was transferred to test tubes containing 400 pL distilled water and 50 pL 5% (w/v) NaNO2.After 5 min, 50 pL 10% (w/v) A1C1, was added and samples were incubated at 20°C for 10 min.Finally, 250 pL NaOH 1N was pipetted into each tube and samples were taken to a final volume of 1.25 mL with distilled water.The absorbance was measured immediately at 510 nm in a spectrophotometer.Catechin was used as standard and results were expressed in mg per kg fresh weight.Measurements were done in triplicate and four extracts were evaluated per storage time and treatment 2.5.4.Anthocyanins.Groups of fifteen mortiños were ran domly selected, frozen in liquid N2, and processed in a blender.Approximately 0.1 g of the resulting powder was added to a test tube containing 10 mL methanol: HC1 (99:1 v/v), stirred for 10 min and centrifuged at 6,000 xg for 10 min at 4°C.The supernatant was collected and the pellet was reextracted twice in the same conditions.The supernatants were pooled and brought to a final volume of 30 mL.The absorbance of the extract was measured at 540 nm in a spec trophotometer and results were expressed as mg equivalents of cyanidin-3-glucoside per kg fresh weight.Measurements were done in triplicate and four extracts were evaluated per storage time and treatment.
2.5.5.Carotenoids.Five naranjillas or fifteen uvillas were randomly selected.Whole uvillas and pulp tissue of naran jillas were frozen in liquid N2 and processed in a blender.Approximately 5 g of the resulting powder was transferred to a glass tube and homogenized with 5 mL petroleum ether and 5mL acetone.The suspension was stirred for 30 min at 4°C and centrifuged at 6,000 xg for 10 min at 4°C.The supernatant was collected and the pellet was reextracted as indicated above.The supernatants were pooled and 10 mL of water was added.The absorbance of the upper phase (petroleum ether) was measured at 450 nm in a spectrophotometer.Carotenoid content was expressed in mg /5-carotene per kg fresh weight.Four extracts were evaluated per storage time and treatment.
2.5.6.Ascorbic Acid.Ascorbic acid content was determined according to Georgé et al. [27].Whole uvillas and mortiños and pulp tissue of naranjillas were frozen in liquid N2 and processed.Approximately 3.5 g of the resulting powder for uvilla and naranjilla and lg for mortiño were added to a test tube containing 10 mL acetone: water (70: 30, v/v).The suspension was first stirred for 10 min and the supernatants were recovered by filtration (raw extracts, RE).Next a solid phase extraction (Oasis HLB, Waters, USA) of RE was carried out obtaining the washing extract (WE).Finally, the ascorbic acid present in WE was destroyed by heating at 85°C for 2 h and colorimetrically deduced by subtracting the Folin-Ciocalteu response of the heated washing extract (HWE) from that of the WE.The Folin-Ciocalteu reaction was per formed as described in Section 2.5.2.Results were expressed in mg ascorbic acid per kg fresh weight.Measurements were done in triplicate and four extracts were evaluated per storage time and treatment.

Statistical Analysis.
Results were analyzed by ANOVA, the factors being storage time and treatment.Both main factors and their interaction were analyzed.Ihe means were compared on a Fisher test at a level of significance of P < 0.05.The lower rate of dehydration in treated fruit was related with the prevention of the loss of fruit turgor and firmness, by maintaining integrity of the tissue and cellular membrane [28],

Results and Discussion
Positive treatment results on quality retention were also observed in other treated fruits.UV-C exposure has been shown to slow-down ripening and fungal development in strawberry [29] and extended the shelf-life of mango by reducing the symptoms of visual damage and decay [30].Similarly, UV-C retained the quality of tomato [22,31] and blueberry [21] through the control of weight loss, ripening, and microbial growth.Overall, our results show that the UV-C treatments are effective to reduce deterioration in uvilla and naranjilla and to control the weight loss in mortiño, especially at the higher dose assayed.Thus, the UV-C treatment with a 12.5 kJ m 2 dose was selected to further evaluate the effects of UV-C radiation on fruit physicochemical and antioxidant properties of the Andean fruit evaluated here.

Firmness.
No firmness determinations were conducted in mortiño given the limited size of this berry and that the limiting factor for postharvest storage was surface dehydra tion.For the remaining fruit, in contrast to uvilla in which UV-C treatment had no effects on firmness loss, a marked delay in naranjilla softening was observed (Table 1).It is worth noting that at the last sampling date UV-C treated naranjilla showed a similar firmness values than control fruit stored just for 7 days (Table 1).Improved firmness retention has been also reported in other UV-C treated products such as peaches [13], tomato [15,31,32], and strawberry [29].The delay of softening by UV-C radiation has been widely related to the inhibition of an array of degrading enzymes involved in cell wall disassembly [33].For other climacteric fruit such as cherry tomato, Bu et al. [15] proposed that the inhibition of ethylene production by UV-C treatment maybe responsible for maintaining lower expression levels of genes coding for cell wall degrading enzymes which are known to be activated by this hormone, such as pectin methylesterase, polygalacturonase, and endoglucanase.

Soluble Solids Content, Acidity, and pH. At harvest,
uvilla presented the highest level of SSC (ca.13.9-14.1%),followed by mortiño (9.8-10%) and naranjilla (7.2-7.3%)(Table 1).During storage, SSC showed moderate changes in naranjilla and mortiño, whereas it increased markedly in uvilla.Gutierrez et al. [34] also found an increase in SSC during uvilla storage and related this with the climacteric behavior and ripening of fruit.Likewise, Valdenegro et al. [35] reported a constant increase in SSC during uvilla ripening through 14 days of shelf-life period.Interestingly, UV-C treated uvilla showed higher SSC after 14 and 21 days than nonirradiated fruit.However, the nature of this increase is uncertain since the presence of starch in this fruit has not been determined and further studies will be necessary.
Titratable acidity was near 328, 379, and 190 meq Kg 1 in uvilla, naranjilla, and mortiño, respectively.No major changes were observed due to UV-C treatment for both acidity and pH (Table 1).Similarly, no changes in these attributes were found in UV-C treated strawberry [36], grapefruit [37], and blueberry [38].Overall, results show that UV-C treatment caused no negative changes in fruit tasterelated attributes and indeed favored the SSC accumulation in uvilla.

Antioxidant Capacity (AC).
Ihe TEAC value at harvest was around 5and 7-fold higher in mortiño (6,800 mg kg :) than in uvilla (1,500 mgkg ') and naranjilla (870 mg kg4), respectively (Figure 2).Mortiño showed a very high AC, which was even higher than that reported for other Vaccinium species [6,39].Though lower than in mortiño, the AC of uvilla and naranjilla are comparable with those found in tree tomato [40] and higher than those of common subtropical fruit, such as citrus [41].
Significant losses of AC were found in the three fruits studied during storage (Figure 2); however, the effect of the UV-C treatment varied markedly depending on the species considered.Treated uvilla showed higher AC than the control fruit (P < 0.05) immediately after treatment and during storage (Figure 2(a)).The nature of the response suggests that UV-C radiation elicited antioxidant accumulation.In accordance, an increase of the antioxidant activity after UV-C radiation was found in other fruit such as strawberry [42], table grape [18], and blueberry [20].Genes coding for key regulatory steps of the phenylpropanoid pathway, such as phenylalanine ammonia-lyase (PAL), have been shown to be upregulated in response to UV-C radiation in some but not all fruit [33,43,44].Downstream enzymes involved in flavonoid synthesis, such as chaicone synthase, chaicone isomerase, and dihydroflavonol reductase, were also induced in response to UV-C elicitation [45,46].UV-C treatment did not trigger the accumulation of antioxidants in mortiño but was effective to slow-down the loss of AC during storage (P < 0.05).Consequently, after both 14 and 21 days, UV-C treated mortiño maintained higher AC than control fruit (Figure 2(c)).Losses of antioxidants have been shown to increase due to loss of cell compartmenta tion.This has been particularly important in phenolic-rich fruit in which tissue disruption may facilitate the access of polyphenol oxidases and peroxidases to their substrates [13].The increased stability on fruit AC in this case may have been due to improved maintenance of tissue integrity as reflected in a lower DI and weight loss (Figure 1(c)).
Finally, UV-C treatment did not exert appreciable benefits (P < 0.05) on the AC of naranjilla (Figure 2(b)).Instead, at long storage times slightly lower AC was recorded in treated fruit.These results point to a very diverse response to UV-C irradiation among the fruit studied.No benefits in AC are observed in treated naranjilla, while UV-C exposure may be exploited as a prestorage treatment to prevent degradation or to induce antioxidant accumulation in mortiño and uvilla, respectively, contributing to a good quality and high human nutrition.

Main Antioxidants Compounds.
The main antioxidant groups prevailing on the three fruits studied were markedly different (Table 2).Ascorbic acid was present at high level in uvilla and naranjilla (523 and 472rngkg ', resp.) as opposed to mortiño where it was 3-4-fold lower (Table 2).Carotenoid content was comparable in naranjilla and uvilla (ca.16 mg kg 1) mainly due to the contribution of /5-carotene which has been identified as the major compound within this antioxidant group in both fruits [3,4].Naranjilla and uvilla also had similar contents of total phenolics; however, the major class of compounds differed.Flavonoids were the most abundant subgroup of phenolics in naranjilla, as opposed to uvilla where they were just 30% of this group (Table 2).As expected, total phenolics in mortiño were 10-fold higher than in uvilla and naranjilla, mainly due to contribution of anthocyanins (Table 2).In accordance, Vasco et al. [6] found that anthocyanins accounted for about 67% of mortiño phenolics.
To characterize the responses to UV-C radiation, we also analyzed the changes in each antioxidant group during storage.UV-C treatment induced ascorbic acid accumulation in uvilla just after the treatment.However, the differences with the control lasted until day 7. Flavonoids were not elicited in this fruit by UV-C and decreased with slight variations between control and treated uvilla during storage (Table 2).Interestingly, higher levels of phenolic compounds and carotenoids were found in UV-C treated uvilla just after the photochemical treatment.Alothman et al. [10] also found a rapid increase of total phenolics and flavonoid compounds just after UV-C exposure in banana and guava.Carotenoids were induced by UV-C exposure in yellow bell pepper [28] and carrot [47].Results show that uvilla antioxidants are particularly responsive to elicitation by UV-C radiation.
In mortiño ascorbic acid, total phenolic compounds or flavonoids were slightly enhanced immediately after UV-C exposure (Table 2).Moreover, the UV-C treated berries retained a slight but higher content than control fruit dur ing storage.Flavonoids, particularly anthocyanins, showed higher stability in UV-C treated mortiño after long-term storage.These results are in line with the report for blueberry by Nguyen et al. [21] who described an increase of phenolic and anthocyanin compounds after UV-C treatment and a reduced degradation rate during refrigerated storage.

Conclusions
UV-C radiation improved quality retention of uvilla and naranjilla and to a lesser extent in mortiño.Treatment with 8 and 12.5 kJ m 2 resulted in similar effects in naranjilla and mortiño.In contrast, the highest UV-C dose was more beneficial in uvilla and also effectively reduced the weight loss in mortiño.Consequently, this dose was selected to further evaluate the physicochemical and antioxidant properties during storage.UV-C treatment was highly effective to delay softening in naranjilla and increased the SSC in uvilla.At long storage times, UV-C treated uvilla and mortiño maintained higher AC than untreated fruit.The increase of AC found in uvilla could result from the activation of the biosynthesis pathway of phenolic compounds in response to UV radia tion.Instead, in mortiño the treatment markedly improved anthocyanin stability at long storage times presumably by maintaining of the tissue integrity.Overall, results indicate that short prestorage UV-C exposure maybe an effective non chemical approach to supplement low temperature storage, maintain quality, and extend the postharvest life of Andean naranjilla, uvilla, and mortiño fruit.

3. 1 .
UV-C Dose Selection.Peel pitting was the most promi nent visual symptom of quality loss in uvilla (Figure l(al)), whereas softening and peel browning were the main causes of deterioration in naranjilla (Figure l(a2)).Weight loss and
) and l(c2))> while for mortiño the treatment with 12.5 kJ m 2 was especially effective to delay weight loss (P < 0.05) in berries stored for over 2 weeks (Figure l(c3)).

Table 1 :
Firmness, soluble solids content (SSC), titratable acidity, and pH in control and UV-C treated (12.5kJ m 2) uvilla, naranjilla, and mortiño during storage at 6°C for 0, 7,14, or 21 d.For each fruit, different letters between treatments show differences according to a Fisher test (P < 0.05).The mean ± standard error is shown.

Table 2 :
Ascorbic acid, total phenolics, flavonoids, anthocyanins, and carotenoids in control and UV-C treated (12.5 kJ m 2) uvilla, naranjilla, and mortiño stored at 6°C for 0, 7,14, or 21 d.For each fruit, different letters between treatments show differences according to a Fisher test (P < 0.05).The mean ± standard error is shown.