Influence of Yellow Light-Emitting Diodes at 590 nm on Storage of Apple, Tomato and Bell Pepper Fruit

The human body produces reactive oxygen species (ROS), such as superoxide anion radicals, hydroxyl radicals and hydrogen peroxide, which can be benefi cial in small amounts, but can lead to oxidative stress in larger amounts. To balance the ROS, there is the need for antioxidants in our diet (1). As a strong scavenger activity against free radicals is found in many plants worldwide, the intake of fruit and vegetables is associated with a lower risk of cancer and cardiovascular disease (2). ConISSN 1330-9862 scientifi c note


Introduction
The human body produces reactive oxygen species (ROS), such as superoxide anion radicals, hydroxyl radicals and hydrogen peroxide, which can be benefi cial in small amounts, but can lead to oxidative stress in larger amounts. To balance the ROS, there is the need for antioxidants in our diet (1). As a strong scavenger activity against free radicals is found in many plants worldwide, the intake of fruit and vegetables is associated with a lower risk of cancer and cardiovascular disease (2). Con-ISSN 1330-9862 scientifi c note doi: 10.17113/ft b.54.02. 16.4096 sumption of natural exogenous antioxidants, such as polyphenols, has protective eff ects against these diseases, which can be partly att ributed to several specifi c components: vitamins, fl avonoids, anthocyanins and other phenolic compounds (1).
Polyphenols are secondary metabolites that have aromatic rings and hydroxyl groups, and these compounds can be divided into phenolic acids, stilbenes, fl avonoids and lignans. The levels of polyphenols in plants vary depending on diff erent cultivars, soil composition, growth conditions, maturity state and postharvest treatments (3). Flavonoids are a group of polyphenolic antioxidants that have the capacity to transfer electrons to free radicals, to activate antioxidant enzymes, and to reduce α-tocopherol radicals. Flavonoids occur in foods of plant origin in different variations: fl avonols, fl avones, catechins, fl ava no nes, anthocyanidins and isofl avonoids (2,4). Previous studies have confi rmed that there are high levels of phenols and fl avonoids in the fruit of apple (Malus domestica 'Granny Smith') (5,6), tomato (Solanum lycopersicum L.) (7,8) and sweet pepper (Capsicum annuum) (9,10).
Light is one of the most important environmental factors for life, and its intensity and spectra can aff ect the physiological response of plants, including the range of accumulated phytochemicals, and their levels (11,12). Irradiation in visible and UV range can induce stress that evokes antioxidant defence system responses (13).
Exposure of fruit to light in orchard plays an important role in the development of fruits, accumulation of secondary metabolites and consequently the postharvest behaviour (14). Solar irradiation together with climatic conditions (15), harvest date, genotype and postharvest factors, as storage temperature, O 2 and CO 2 contents, signifi cantly infl uence postharvest behaviour of fruits and vegetables (16).
Nowadays more and more vegetables and fruits are produced in controlled environment with artifi cial light sources such as light-emitt ing diodes (LEDs) (17). Their usage in growth chambers and greenhouse is increasing due to numerous benefi ts, such as high energy-conversion effi ciency, small mass and volume, and relatively cool surfaces with minimum heating and long life expectancy. Furthermore, a major advantage is the ability to control their spectral composition, and therefore their wavelengths can be matched to plant photoreceptors (17)(18)(19).
Wider application of LEDs in agriculture has led to several studies where the eff ects of the light spectrum and quality on the preharvest (6) and postharvest physiology (11,20) were evaluated, oft en showing inconsistent results. Responses to diff erent light spectra can vary across plant species or even varieties (21) and are dependent on the maturity stage. It has been shown that white and red LEDs enhance the tomato yield (22), whereas blue LED light had positive eff ects on their growth and development (23,24). Similar eff ects have been observed in pepper plants, where supplemental irradiation with blue LED light resulted in bett er development of the stems, leaves and plant biomass (25). Light is not only the crucial factor for plant development during growth phase but can also infl uence the postharvest behaviour when fruits and vegetables are irradiated during storage. Some recent studies have shown that the application of LEDs during storage can preserve and even improve the nutritional quality of fruits (26) and vegetables (20,27,28). The results are nevertheless inconclusive and detailed analysis of LED light spectra on postharvest physiology is still lacking.
Yellow light (wavelength around 580 nm) is one of the least studied when the infl uence on plant physiology is evaluated. There are nevertheless some promising reports that irradiation with light in the range of 500-600 nm results in an increase of ascorbic acid and anthocyanin content (29) and that yellow light with wavelengths in the range of 580-600 nm may infl uence gene expression in plants during growth (30). However, there are no literature data available about the infl uence of irradiation with yellow LED light during storage on secondary metabolites of fruits and vegetables. The challenge to horticulturists and postharvest operators is to manipulate and manage light application to maximize yield and storability of fruits and vegetables. Therefore, the present study was carried out to investigate the contents of ascorbic acid (AAC), total phenolic compounds (TPC), total fl avonoids (TFC), chlorophylls, carotenoids and tocopherols, and the antioxidant potential (AOP) of the apple, tomato and red pepper fruit during 7 days of storage under LED light at a wavelength of 590 nm.

Plant material
Apple, tomato and bell pepper fruit were subjected to LED light irradiation at the wavelength of 590 nm. Commercial varieties of apple (Malus domestica 'Granny Smith'), red tomato (Solanum lycopersicum L.) and bell pepper (Capsicum annuum) fruit were studied. The selected fruit were purchased from a supermarket in Ljubljana, Slovenia, in January 2014. The fruit were of commercial size, adequate colour, and at a physiologically mature stage. The purchased samples were immediately prepared for storage in LED light irradiation box, or in darkness as the control. All of the analyses were carried out before the LED light irradiation and aft er 7 days, i.e. at the end of the fruit storage.

Storage conditions
Half of the fruit used for storage (three fruits per group) were put into a LED light irradiation cardboard box at 10 °C for 7 days under constant lighting provided by yellow LEDs at a wavelength of 590 nm. The other half of the fruit (three fruits per group) represented the controls, which were stored in the dark under the same conditions. Each fruit group for the experimental samples was irradiated with six LEDs providing a total radian fl ux of 0.14 W on the irradiated surface. The LEDs were positioned approx. 15 cm from the fruit surfaces and the average irradiance on the fruit surface was 1.81 W/m 2 .

Sample preparation
For the analysis of apple fruit, only the skin of the fruit was used. Strips of apple skin approx. 15 mm wide were removed using a commercial hand peeler. Tomato and bell pepper samples (three fruits of each) included fruit skin and fl esh, without seeds approx. 1 cm thick, taken using a cork borer. Sampling procedure included only irradiated parts of the fruit, i.e. upper part of the fruit.
For the sample extraction, 6 g of fruit sample were homogenised with 18 g of metaphosphoric acid dissolved in distilled water (2 % by mass), using a T 25 Ultra-Turrax ® homogenizer (IKA ® -Werke GmbH&Co. KG, Staufen, Germany) at 13 500 rpm. The homogenised samples were fi ltered fi rst through a fi lter paper, and then through 0.45--μm fi lters (17-mm cellulose acetate (CA) syringe fi lter; Sartorius AG, Goett ingen, Germany). The samples were then stored at -80 °C until further analysis.

Ascorbic acid
Ascorbic acid content (AAC) determination was performed on an HPLC system (model 1260 Infi nity; Agilent Technologies, Santa Clara, CA, USA) using a diode array detector, with the wavelength set at 254 nm. The separation of ascorbic acid was carried out on a 100 mm×2 mm, i.d. 3 μm, Scherzo SM-C18 column (Imtakt, Kyoto, Japan), at a fl ow rate of 0.3 mL/min. The mobile phase consisted of water (A) and acetonitrile (B), both of which contained 0.3 % (by volume) formic acid. The following elution gradient was used for solvent B: 0-3 min, 0-10 %; 3-4 min, 10-100 % and 4-6 min, 100 %. The temperature of the column was maintained at 30 °C, and the temperature of the automatic sample feeder at 4 °C. AAC was calculated using an external standard, and expressed in mg of ascorbic acid per 100 g of fresh mass (FM).

DPPH radical-scavenging activity
Antioxidant potential (AOP) of the samples was determined spectrophotometrically, as the 2,2-diphenyl-1--picrylhydrazyl (DPPH; Sigma-Aldrich, Darmstadt, Germany) free-radical-scavenging capacity, based on the modifi ed method of Brand-Williams et al. (31). For this analysis, a 560-μM DPPH solution was prepared in methanol. A volume of 400 μL of the solution was mixed with 1580 μL (for apple peel and bell pepper samples) or 1550 μL of methanol (for tomato samples), and 20 μL of the individual samples (or 50 μL of tomato samples) were added, followed by vortexing for 10 s (VWR VM-3000 mini vortexer; Henry Troemner LLC, Thorofare, NJ, USA). The absorbance was measured aft er 40 min of incubation at room temperature by spectrophotometer (Cecil Aurius Series CE 2021 UV/Vis; Cecil Instruments Limited, Cambridge, UK) at 520 nm, against methanol as the blank. Calibration was done through seven-point standard curves of Trolox (Sigma-Aldrich) (R 2 =0.9985), which ranged from 1.56 to 10.94 mg per L, and the AOP of the samples was determined in triplicate and expressed in μmol per L of Trolox equivalents (TE) per 1 g of FM.

Total phenolic content
Total phenolic content (TPC) was determined according to the Folin-Ciocalteu method described by Singleton and Rossi (32), with minor modifi cations. Apple peel and red bell pepper samples (20 μL) or 100 μL of the tomato samples were incubated with 1380 or 1300 μL, respective-ly of deionised water, and 300 μL of diluted Folin-Ciocalteu reagent (10 mL of Folin-Ciocalteu reagent; Merck, Darmstad, Germany, in 20 mL of deionised water). These were mixed in 2-mL microcentrifuge tubes using a vortex (10 s). Aft er 5 min of incubation, 300 μL of 20 % Na 2 CO 3 (Merck) were added to the mixtures, which were vortexed again (10 s). Aft er a further incubation at room temperature for 30 min, the absorbances of the mixtures were measured on a spectrophotometer at 765 nm, with deionised water as the blank. All of the samples were processed in triplicate. TPC was quantifi ed through calibration using gallic acid (Fluka, Buchs, Switzerland) as standard. The eight-point calibration curves ranged from 1.7 to 13.6 mg/L of gallic acid (R 2 =0.9988). TPC was expressed in mg of gallic acid equivalents (GAE) per 100 g of FM.
Total fl avonoid content Total fl avonoid content (TFC) was determined according to the method described by Lin and Tang (9). A volume of 250 μL of the samples was mixed with 750 μL of 95 % ethanol, 50 μL of AlCl 3 (Fluka), 50 μL of 1 mol/L CH 3 COOK (Sigma-Aldrich), and 1400 μL of deionised water. Aft er incubation at room temperature for 40 min, the absorbance of the reaction mixture was measured by a spectrophotometer at 415 nm, against deionised water as the blank. For the calibration, fi ve-point standard curves for quercetin were used (R 2 =0.9998), which ranged between 0.3 and 15.0 mg/L. TFC of the samples was determined in triplicate and expressed in mg of quercetin equivalents (QE) per 100 g of FM.

Pigments
The analysis of the pigments was carried out according to a method described previously (33). Pigments were extracted from approx. 200 mg of frozen fruits with 5 mL of ice-cold acetone in an ice bath, using T 25 Ultra-Turrax homogenizer (Ika-Labortechnik) for 30 s. All extraction procedures were performed in dim light. Acetone extracts were fi ltered through 0.2-μm Minisart SRP 15 fi lter (Sartorius Stedim Biotech GmbH, Goett ingen, Germany). Levels of the pigments lycopene, neoxanthin, violaxanthin, antheraxanthin, lutein, chlorophyll a, chlorophyll b, α-carotene and β-carotene were determined using an HPLC system (a Spherisorb ODS2 column, 250 mm×4.6 mm, i.d. 5 μm, with a Spherisorb ODS2 precolumn, 50 mm×4.6 mm, i.d. 5 μm; Alltech Associaties, Inc., Deerfi eld, IL, USA), with elution using acetonitrile/water/methanol (100:10:5, by volume) as mobile phase A, and acetone/ethyl acetate (2:1, by volume) as mobile phase B, at a fl ow rate of 1 mL/ min. The following linear gradient was used for solvent B: 0-18 min, 10-70 %, with a run time 30 min. The injection volume was 20 μL. The pigment compounds were monitored at a wavelength of 440 nm. The analysis was performed on a Surveyor HPLC system with a diode array detector (Thermo Finnigan, San Jose, CA, USA). Identification of compounds was achieved by comparing the retention times and the spectra as well as by the addition of standards. The mass fractions (in mg per g of FM) of the pigments were calculated by the external standard method, using the following standards: α-carotene, β-carotene, neoxanthin, violaxanthin, lutein, antheraxanthin and chlorophylls (DHI Water & Environment, Hørsholm, Den-mark), and lycopene (Sigma-Aldrich). All standards were highly purifi ed (at least 95 % pure). The solvents acetone, ethylacetate, methanol and acetonitrile were from Merck, all of HPLC grade. Water used for preparation of solutions and eluents was double-distilled and purifi ed with a Milli-Q water purifi cation system by Millipore (Bedford, MA, USA).

Tocopherol content
The method used for the analysis of tocopherols was described by Wildi and Lütz (34). Tocopherols were extracted from frozen samples with ice-cold acetone exactly as described for chloroplast pigments. Spectra-Physics HPLC system with a P4000 SpectraSYSTEM™ pump and an AS1000 SpectraSYSTEM™ automatic sample feeder (Thermo Fisher Scientifi c Inc.) was used. The tocopherols were passed through a Spherisorb ODS2 guard column (50 mm×4.6 mm, 5 μm), and separated on a Spherisorb ODS2 column (250 mm×4.6 mm, 5 μm). The temperature of the automatic sample feeder was 4 °C, and the column was at room temperature. Methanol was used as the mobile phase, at fl ow rate of 1 mL/min. The injection volume of the samples was 20 μL, with an analysis duration of 30 min. Excitation was determined at 295 nm and emission at 325 nm, with the tocopherol levels calculated against an external standard (Sigma-Aldrich). The tocopherol levels were expressed in μg per g of FM.

Surface colour
The surface colour of the fruit was measured using a colour-measurement device (Minolta CR-400, Minolta, Kyoto, Japan). At the beginning of the study and aft er 7 days of storage, L*, a* and b* were measured. L* represents the lightness of the colour, a* the position between red (+) and green (-), and b* the scale between blue (-) and yellow (+). These colour measurements were taken at four locations on each of three individual fruit samples (N=12).

Firmness
The force required to penetrate the apple fruit was measured at four locations on each peeled apple, using a 53200SP penetrometer (T.R. Turoni, Forlì, Italy). To determine the fi rmness of the tomato and bell pepper fruit, a Kramer shear cell (Stable Micro Systems, Godalming, Surrey, UK) was used. The fi rmness was measured in kg/ m 2 at the beginning of the study, before storage, and aft er 7 days of storage.

Statistical analysis
The data were analyzed according to the method of least squares, using a general linear model (GLM) procedure, SAS Soft ware v. 8.01 (35). All of the measurements were performed in triplicate (N=3). Diff erences at p<0.05 were considered as statistically signifi cant.

Results and Discussion
Recent studies have confi rmed the infl uence of LED light irradiation on plant growth and development. In the present study, we investigated the eff ects of yellow LED light (590 nm) on several biochemical compounds and quality parameters of apple, tomato and red bell pepper fruit. Our data show diff erences between the control (stored in the dark) and LED light-irradiated fruit for most of the studied parameters. The mechanisms of the infl uence of LED light on plant metabolism have been well studied in the plants grown in glasshouses, but they have not been studied in any great extent during fruit storage aft er harvest.
The data from the analyses of AAC, AOP, TPC and TFC of the control (dark storage) and LED light-irradiated fruit are given in Table 1. The eff ects of the yellow LED light under the controlled conditions on the compounds analysed in apple peel, tomato and bell pepper fruit varied considerably.
As shown in Table 1, there was higher AAC in the LED light-irradiated apple peel, tomato and red bell pepper fruit compared to the control samples stored in the dark, although these AAC diff erences did not reach statistical signifi cance. A previous investigation reported higher AAC in tomato fruit when blue light (450 nm) was applied (19). Kim  Samuolienė et al. (13) reported that supplemental LED light colours at 380 and 595 nm had negative eff ects on AAC in romaine baby leaf lett uce during the growth period. Relatively high standard deviations were observed among the individual fruit samples in the present study, which might have arisen from diff erent factors, including cultivar, genotype, growing season, harvest conditions, storage, and environmental conditions, such as location, growing temperature, soil and light (37)(38)(39).
Similarly, there was higher AOP in all of the LED light-irradiated samples; indeed, the diff erences in AOP were statistically signifi cant compared to the controls for apple peel and red bell pepper fruit (13). In the previously mentioned study on romaine baby leaf lett uce during the growth period signifi cantly higher AOP was also reported when supplemental green light (530 nm) was used, compared to yellow light in the present study (590 nm). On the other hand, the same authors reported lower AOP when LED light at 455 and 470 nm was applied.
Again, as for AAC and AOP, there was higher TPC in all of the LED light-irradiated samples, with statistically signifi cant diff erences observed for apple peel and tomato fruit. In comparison with the data of the present study, other studies have shown slightly lower TPC values (but higher TFC; see also below) in the fruit of several pepper cultivars (40). Samuolienė et al. (13) again reported that supplemental orange light (622 nm) enhanced the synthesis of phenolic compounds in romaine baby leaf lett uce during the growth period. They also reported in another study (21) that supplemental LED light at 505 nm promoted signifi cant increases in TPC, but on the other hand, LED light at 535 nm resulted in signifi cantly lower TPC. Also in baby leaf lett uce, Li and Kubota (11) showed an increase in TPC aft er exposure to LED light from 600 to 700 nm. Using LED light at 470 nm, Kim et al. (36) reported signifi cantly higher TPC in strawberry fruit, and as reported by Zhan et al. (41), storage of broccoli under fl uorescent light increased TPC.
Here, lower TFC was observed in LED light-irradiated apple peel, tomato and red bell pepper fruit samples, as compared to the controls, although again, these diff erences were not statistically signifi cant. In a previous study, use of a wavelength near the λ max of fl avonoids (i.e. UV-A light at 380-320 nm) increased TFC in radish sprouts, parsley and Indian spinach (42). Also, Harbaum--Piayda et al. (43) investigated preharvest UV-B irradiation (at 280-380 nm) of pak choi (Brassica campestris L. ssp. chinensis var. communis) and demonstrated a twofold increase in TFC compared to the non-irradiated control.
The pigments and tocopherols in the individual samples here showed diff erent responses to these yellow LED light conditions during storage. We analysed 12 diff erent pigments in these samples, the data for which are shown in Table 2.
Signifi cant increases were detected of lutein (14.8 %), chlorophyll b (17.9 %), chlorophyll a (21.6 %) and β-carotene (31.2 %) mass fractions between the control and the LED light-irradiated samples of apple peel. Although violaxanthin and neoxanthin contents were higher in the LED light-irradiated apple peel, these diff erences were not signifi cant. While this light exposure induced synthesis or slowed down the degradation of the above-mentioned pigments, a decrease in the tocopherol level was observed, although it did not reach statistical signifi cance. Wu et al. (18) irradiated pea seedlings with blue LED light (465-470 nm) and reported an increase in total chlorophyll content. Furthermore, they also irradiated pea seed- Nine pigments were detected in the tomato fruit, and their decreasing order of abundance in the control was: lycopene>α-tocopherol>β-carotene>chlorophyll a>lu tein> γ-tocopherol>violaxanthin>chlorophyll b>neoxanthin. The most abundant pigments were (in μg per g of FM): lycopene 475.60, followed by α-tocopherol 116.45 and β-carotene 75.64. The least represented tomato pigments were violaxanthin, chlorophyll b and neoxanthin, with mass fractions <5 μg per g of FM.
LED light irradiation resulted in an increase of only the neoxanthin content in the tomato fruit (34.9 %), although this was not signifi cant. However, signifi cant decreases in the LED light-irradiated tomato samples were observed in β-carotene (7.7 %), γ-tocopherol (26.7 %) and chlorophyll b (65.6 %). The lycopene, α-tocopherol, lutein and violaxanthin mass fractions also decreased, but these diff erences were not signifi cant. Using diff erent LED light wavelengths (i.e. 650-660 nm), Dhakal and Baek (20) reported an increase in lycopene in mature green tomato fruit. Li and Kubota (11) also showed an increase in xanthophyll and β-carotene in baby leaf lett uce as a result of LED light irradiation at 476 nm, along with a decrease in xanthophyll, β-carotene and chlorophyll content aft er LED light irradiation at 734 nm.
Signifi cant increases of α-tocopherol (12.4 %), chlorophyll a (16.5 %), β-carotene (31.2 %), lutein (46.3 %) and γ-tocopherol (62.8 %) mass fractions were observed in LED light-irradiated red bell pepper fruit. The only pigment that was present at lower mass fraction aft er the LED light irradiation was chlorophyll b, with a signifi cant 32.8 % decrease, compared to the control. Gangadhar et al. (44) reported that blue LED light enhanced the synthesis of chlorophyll a and chlorophyll b in chilli pepper fruit, while the combination of red and blue LED light resulted in the highest carotenoid levels. Samuolienė et al. (13) also reported a decrease in tocopherol content in the romaine baby leaf lett uce under LED light conditions. The colour readings for the apple, tomato and red bell pepper fruit samples are summarised in Table 3.
Here, compared to the controls, LED light irradiation had no signifi cant eff ects on any of the colour parameters determined for the fruit (L*, a* and b*), although there was a tendency to accelerate yellow colour development of apple fruit, and red colour development of red bell pepper fruit. Dhakal and Baek (20) reported that red light (at 650-660 nm) enhanced red colour development of tomato fruit.
Firmness is an important factor for postharvest storage, and it is also important from a nutritional point of view (45). Fig. 1 shows the fi rmness of apple, tomato and red bell pepper fruit. Here, there was a signifi cant diff erence between the apple fruit exposed to LED light, with a 10 % lower fi rmness, and the control. Although lower fi rmness was also seen of red bell pepper fruit and a slightly increased fi rmness of tomato fruit, this was signifi cant (Fig. 1). Dhakal and Baek (20) reported that tomato fruit irradiated with blue light (at 440-450 nm) had higher levels of fi rmness than those irradiated with red light (at 650-660 nm) or kept in darkness.  Further studies are now needed to take into account, in particular, the wavelength and power output of the LED light irradiation.

Conclusions
In the present study, the focus was on some bioactive compounds and their change aft er the exposure to yellow LED light irradiation. The statistically signifi cant eff ects of the yellow LED light on the irradiated apple peel and red bell pepper fruit during storage were found for antioxidant potential compared to the control samples stored in the dark. Also, there were signifi cant diff erences obtained in total phenolic content in LED light-irradiated apple peel and tomato fruits. The analysis of pigments and tocopherols showed signifi cantly higher mass fractions of lutein, chlorophyll a, chlorophyll b and β-carotene in LED light-irradiated samples of apple peel, and of β-carotene, α-tocopherol, γ-tocopherol, chlorophyll a and lutein in LED light-irradiated red bell pepper fruits. The fi rmness of LED light-irradiated apple fruit was also signifi cantly lower. The eff ects of yellow LED light on postharvest processes are not well elaborated in literature. We have shown that yellow LED light irradiation aff ects mass fractions of ascorbic acid, total phenolics, total fl avonoids, pigments, tocopherols and antioxidant potential, thus increasing the nutritional value of food.