Postharvest Biology and Technology 184 (2022) 111774
Available online 9 November 2021
0925-5214/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Postharvest UV radiation enhanced biosynthesis of flavonoids and carotenes in bell peppers
Noelia Castillejo, Lorena Martínez-Zamora, Francisco Art´es-Hern´andez *
Postharvest and Refrigeration Group, Department of Agronomical Engineering & Institute of Plant Biotechnology, Universidad Polit´ecnica de Cartagena, Cartagena, Murcia, 30203, Spain
A R T I C L E I N F O Keywords:
Capsicum annuum Ultraviolet B and C Radiation Rutin Antioxidants Shelf life
A B S T R A C T
UV abiotic stresses have beneficial effects in plants inducing the synthesis of secondary metabolites when low doses were applied. The aim of this study was to evaluate the effect of 6 kJ m−2 UV (B or C) and 6 + 6 kJ m−2 UV (B + C) on the main bioactive compounds of red bell peppers during a refrigerated shelf-life period. Changes in carotenoids, phenolics, and flavonoids were studied after 8 and 14 d at 7 ◦C, an after an additional retail sale period of 4 d at 18 ◦C. Physicochemical quality attributes were not affected by any UV treatment. Generally, UV treatments induced carotenoid accumulation, highlighting that after 14 d at 7 ◦C, UVB and UVC increased by 59
% the total carotenoid content, and UVB + C did it by 94 % compared to non-UV-treated peppers as control (CTRL). UVC, UVB, and UVB + C are good elicitors of the flavonoid biosynthesis with 42, 66, and 43 % increases just after treatment, respectively, compared to CTRL. This behaviour was enhanced in UVC and UVB + C treated peppers after 8 d at 7 ◦C (15 and 44 %, respectively) and after 14 d at 7 ◦C (146 and 137 %) regarding CTRL peppers, which was also shown after the retail period assayed of 4 d at 18 ◦C. In conclusion, a postharvest 6 kJ m- 2 UV-C treatment could be a great tool for increasing the accumulation of carotenoids and flavonoids in red bell peppers.
1. Introduction
Spain, with 715,000 tonnes per year, is the main producer and exporter of bell peppers in Europe and the fifth in the world after China, Mexico, Turkey, and Indonesia (FAO, 2021). For that, Capsicum annuum L. is one of the key ingredients of the Mediterranean Diet, which has become very important due to the high carotenoid and flavonoid con- tent, being a powerful food that fights against ageing and chronic dis- eases (Boeing et al., 2012; Morales-Soto et al., 2013; Wang et al., 2014).
However, bell peppers are very sensitive to biotic and abiotic damage, which can trigger several postharvest physiological changes in the fruit reducing its shelf life (Wang et al., 2018).
Light is a key factor which directly affects plant growth and synthesis of primary and secondary metabolites (Zhang et al., 2020). Indeed, photosynthesis can vary by changes in the light intensity and wave- length applied. Therefore, UV lighting (200–400 nm) can be used to induce the expression of antioxidant enzymes, which increase the biosynthesis of vitamins, flavonoids, phenolic acids, and carotenoids, among others (Dou et al., 2019; Moreira-Rodríguez et al., 2017).
Particularly, UV (B and C) radiation has been reported an hormetic
behaviour characterized by a favourable biological response under low doses (Cisneros-Zevallos and Jacobo-Vel´azquez, 2020; Schreiner et al., 2014). However, high UV doses can damage the plant tissues reporting a negative effect on the photosynthesis and the biological activity of fruit and vegetables (Guidi et al., 2016). UV-C provides more energy due to the shorter wavelengths and has been shown to reduce decay and alle- viate chilling injury in red peppers (Vicente et al., 2005). In this sense, Rodoni et al. (2015) showed slight increases in the content of phenolic compounds after 10 kJ m−2 UV-C in fresh-cut red peppers. Also, Pro- myou and Supapvanich (2012) reported significant increases of the total flavonoid content after 6.6 kJ m-2 UV-C and 6 d of storage at 12 ◦C in yellow bell peppers, which positively affected the content of antioxidant enzymes such as catalase and superoxide dismutase. Previously, Mah- davian et al. (2008) showed important increases in the rutin and quer- cetin content in leaves of Capsicum annuum L., even 3-fold higher than control plants, after 129 kJ m−2 UV-C applied during 14 d.
Recent findings have also shown an improvement of the biosynthesis of carotenoids and flavonoids in tomatoes (Pataro et al., 2015) and in Clerodendrum volubile (Adetuyi et al., 2020) after low doses of UV-C radiation. A combination of postharvest visible blue and UV-C lighting
* Corresponding author.
E-mail address: [email protected] (F. Art´es-Hern´andez).
Contents lists available at ScienceDirect
Postharvest Biology and Technology
journal homepage: www.elsevier.com/locate/postharvbio
https://doi.org/10.1016/j.postharvbio.2021.111774
Received 8 July 2021; Received in revised form 15 October 2021; Accepted 23 October 2021
has been shown promising results in habanero pepper (Capsicum chi- nense) by increasing chlorophyll, carotenoid, capsaicin, phenolic, and flavonoid content (P´erez-Ambrocio et al., 2018). However, UV RESPONSE LOCUS 8 (UVR8), as the main UV-B receptor, is in charge to regulate flavonoid biosynthesis and plant vegetative growth (Jenkins, 2009). Thus, UV-B radiation (280–315 nm) has initially been shown to improve the content in carotenoids and flavonoids as main antioxidant compounds in bell peppers (Le´on-Chan et al., 2017). In addition, a postharvest combination of UV-B and UV-C has been studied in bimi broccoli (Formica-Oliveira et al., 2017a) and peaches (Abdipour et al., 2019) obtaining promising results. Nevertheless, this combination has never been simultaneously studied in mature red bell peppers. Consid- ering the lack of knowledge about the simultaneous application of postharvest UV-B and UV-C light, we studied the effect of the post- harvest application of 6 kJ m−2 of UV lighting, both the single and combined UV-C and UV-B (6 kJ m−2 UV-C + 6 kJ m−2 UV-B), on the main bioactive compounds (flavonoids and carotenoids) of red bell peppers during their commercial shelf-life period.
2. Materials and methods 2.1. Plant material
‘Angus’ bell peppers (Capsicum annuum L.), California type, seeds were provided by Syngenta Espa˜na S.A. (Torre-Pacheco, Murcia), which were grown in a greenhouse of the Southeast of Spain by T´arraga y Henarejos, S.L. (Murcia, Spain). Red bell peppers were harvested on June 10th, 2020, when the maturity index reached 15.7 ± 0.9. After harvesting, plant material was transported to the Universidad Polit´ecnica de Cartagena about 30 km. A total of 500 undamaged bell peppers were selected and washed for 2 min with water (2.5 L kg−1) at 7
◦C containing 5 % peracetic acid (Citrocide® PC, Citrosol, Valencia, Spain), rinsed for 1 min in tap water and then dried with paper (100 % cellulose). Subsequently, the UV treatments were applied as follows.
2.2. UV treatments
The UV treatments were performed in a reflective stainless-steel chamber as represented in Fig. 1. The radiation chamber, which is fully described by Formica-Oliveira et al. (2017b), was equipped with 6 UV-B unfiltered emitting lamps (TL 40 W/01 RS; Philips, Eindhoven, The Netherlands) and 9 UV-C unfiltered emitting lamps (TUV 36 W/G36 T8; Philips, Eindhoven, The Netherlands). Bell peppers were placed between the two lines of lamps at 17.5 cm above and below. The applied UV-B intensity of 8.10 ± 0.35 W m−2 was calculated as the mean of 20 readings on each side of the net using a LP 471 UVB (Delta OHM, Italy) radiometer. The applied UV-C intensity of 25.99 ± 0.81 W m−2 was calculated as the mean of 20 readings on each side of the net using a VLX 254 (Vilber Lourmat, Marne la Vallee, France) radiometer. These doses were applied trying to adapt this model to the industry, in where red peppers can be treated in the postharvest handling lines. The doses applied were previously determined to avoid UV damage to the pepper skin. The treatments and doses, were based on our previous unpublished experiments for bell peppers and those published (Formica-Oliveira et al., 2017b; Vicente et al., 2005) with other horticultural commodities as follows:
- CTRL: No UV treatment was used as control.
- UVC: the bell peppers received an effective dose of 6 kJ m−2 UV-C during 3 min 29 s, according to our prototype.
- UVB: the bell peppers received an effective dose of 6 kJ m−2 UV-B during 12 min 58 s.
- UVB þ C: the bell peppers received the sum of both treatments, 6 kJ m−2 UV-C + 6 kJ m−2 UV-B, simultaneously applied (3 min 29 s +12 min 58 s, respectively).
After UV treatments (Fig. 1), bell peppers were stored at 7 ◦C and 80
% relative humidity under darkness conditions simulating a short postharvest storage and commercial exportation within Europe. In this sense, after 8 and 14 d at 7 ◦C, an additional retail sale period of 4 d at 18
◦C was carried out to determine the main quality parameters in such sampling times.
Fig. 1. Schematic representation of the experimental design of the study.
2.3. Physicochemical quality determinations
Physicochemical analyses were carried out in quintuplicate, in where each replicate was constituted by five bell peppers, on each sampling day (N = 25) following the methods described by Martínez-Zamora et al.
(2021). Bell peppers were weighed (g) and weight losses (%) were calculated during refrigerated storage and retail sale. Colour was determined using the CIELab system (L*, a*, and b* coordinates) and colour differences (ΔE) throughout storage were calculated according to Torres-S´anchez et al. (2020). The equatorial and longitudinal caliber and flesh thickness of each fruit were measured using a caliper. For flesh thickness, the red bell peppers were cut in two halves. Firmness of each bell pepper was measured at room temperature with a cylinder of 8 mm diameter and a surface area of 2.01 cm2 compressing the fruit by 5 mm at a contact speed of 2 mm s−1, expressing these results in newtons (N).
After that, the total soluble solids content (TSS), pH, and titratable acidity (TA) of the obtained bell pepper juice were determined and the maturity index (MI) was calculated as the ratio of TSS/TA following the methods described by Martínez-Zamora et al. (2021).
After physicochemical analyses, five replicates per treatment and sampling time (the mixture of five fruits per replicate) were frozen in liquid nitrogen and stored at − 80 ◦C. Frozen samples were ground to fine powder prior to analyses using liquid N2 with a mincer (IKA, A 11 basic, Berlin, Germany) at 12,700 ×g for 10 s.
2.4. Extraction of bioactive compounds
One gram of mixed bell peppers powder was weighed in plastic tubes and mixed with 10 mL methanol (80 %) (N = 3). Samples were vigor- ously shaken for 1 h, at 4 ◦C, in darkness. After this time, the extracts were centrifuged at 3.220 × g for 10 min at 5 ◦C and the supernatant was collected and kept at -80 ◦C until further analysis were performed (Martínez-Zamora et al., 2021).
2.5. Total phenolic content and total flavonoid content
The total phenolic content (TPC) and the total flavonoid content (TFC) were determined as previously described by Martínez-Zamora et al. (2021). For TPC analysis, 19 μL sample extract were mixed with 29 μL of 1 N Folin–Ciocalteu reagent and 192 μL of 0.4 % Na2CO3 2 % NaOH. After 1 h incubation in darkness, the absorbance was measured at 750 nm using a microplate reader (Tecan Infinite M200, M¨annedorf, Switzerland). The TPC was expressed as g of Gallic Acid Equivalents (GAE) kg−1. Each extract was analysed in triplicate (N = 3), for that the total number of samples per treatment and sampling time was N = 9. For TFC analysis, 30 μL of extract were mixed with 80 μL of 20 g L−1 AlCl3. After shaking and 1 h incubation in darkness, absorbance was measured at 415 nm. The TFC was expressed as g of Rutin Equivalents (RE) kg−1. Three replicates were analysed, and each sample extract was analysed in triplicate (N = 9).
2.6. Individual phenolic acids and flavonoid content by U-HPLC One mL of the methanolic extract was filtered using 0.2 μm poly- tetrafluoroethylene membrane filters. An Ultra High Performance Liquid Chromatography (UHPLC) instrument (Shimadzu, Kyoto, Japan) equipped as described by Castillejo et al. (2021a) was used. Chro- matographic analyses were carried out following the method described by Castillejo et al. (2021a) with slightly modifications. The mobile phases used were 0.5 % acetic acid (A) and acetonitrile (B) and the solvent gradient changed as follows: 0 min, 100:0 (A:B); 20 min, 80:20;
30 min, 70:30; 40 min, 50:50; 50 min, 25:75; 60 min, 0:100; 62 min, 100:0; and a final conditioning cycle of 8 min with the initial conditions.
A volume of 10 μL of pepper extract was injected. Phenolic acids (gallic and caffeic acid) and flavonoids (catequin, epicatequin, rutin, quercetin, luteolin, naringenin, and apigenin) were identified by comparison of the
retention time with analytical standards supplied by Sigma. Each sample was analysed in triplicate (N = 3). The absorption spectra were recorded between 200 nm and 400 nm and the results were expressed as g kg−1. 2.7. Total antioxidant capacity
Total antioxidant capacity (TAC) was analysed by using 2,2- diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethyl- benzothiazoline-6-sulfonic acid (ABTS) assays according to Castillejo et al. (2021b). DPPH assay was performed by adding 194 μL of DPPH (700 μM) solution to 21 μL of bell pepper extract. After 30 min incu- bation in darkness, absorbance was measured at 515 nm. ABTS assay was performed by adding 200 μL of the activated ABTS solution (320 μM) to 11 μL of pepper extract in a 96-well plate and incubated for 20 min in darkness. The TAC by ABTS was measured by changes in absorbance at 414 nm. All data were expressed as g of Trolox Equiva- lents (TE) kg−1. Three replicates were analysed, and each sample extract was analysed in triplicate (N = 9).
2.8. Extraction and determination of total carotenoids
Samples preparation was performed according to Castillejo et al.
(2016). For that, 0.25 g of powder sample were weighed and mixed with 9 mL of hexane and 15 mL of a methanol/acetone dilution (1:2, v/v).
Samples were vigorously shaken for 4 h in darkness at 4 ◦C. After that, 25 mL of 1 M NaCl were added, and the absorbance of the supernatant was measured at 470 nm in quartz cells. The equations developed by Wellburn (1994) and described by Castillejo et al. (2016) were used to determine the total carotenoid content (TCC), which was expressed as mg carotenoid kg−1. Each sample was extracted and analysed in tripli- cate on each sampling day (N = 3).
2.9. Data analysis
The experiment was a two-factor (UV treatment × storage time) design, and it was subjected to analysis of variance (ANOVA) using the statistic software Statgraphics Plus software (v. 5.1. Statpoint Technol- ogies. Inc. Warrenton. VA. USA). P < 0.05 was assessed as statistically significant, and Tukey’s multiple range test was used to separate means.
In addition, other data analysis at a significance level of P ≤ 0.05 fol- lowed by a least significant difference (LSD) test was carried out, where
***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; T, CTRL or UV treatment; T, storage time.
3. Results & discussion
3.1. Physicochemical quality changes
The mean weight of bell peppers at harvest was 204.7 ± 37 g, reporting an equatorial caliber of 87.2 ± 8.2 mm, a longitudinal caliber of 87.6 ± 7.6 mm, and a flesh thickness of 5.8 ± 0.9 mm (Table 1). These parameters are within the category California type peppers. Weight losses after 8 d at 7 ◦C were 0.57 %, while after 14 d at 7 ◦C such losses increased up to 0.95 % (Table 2). In this sense, UV treatments did not negatively affect to weight losses and storage conditions were adequate due to low weight losses of less than 1%. Furthermore, a simulated commercial period (4 d at 18 ◦C after refrigerated storage) was sufficient to maintain low weight losses. These results agree with Andrade Cuvi et al. (2011) who showed 1.3 % weight losses after 21 d at 0 ◦C.
Regarding colour development, there were no differences among CIELab parameters (Table 2). In fact, ΔE was increased by UV treatments only on day 14 at 7 ◦C, which means that there were not colour differ- ences during the refrigerated storage period induced by any of the UV treatments. This behaviour can be also compared to previous results of Andrade Cuvi et al. (2011), who reported no colour changes during red pepper storage for 21 d at 0 ◦C after a dose of 10 kJ m−2 UV-C at harvest.
In addition, these results also agree with Kasim and Kasim (2018) who showed no colour changes in Capia pepper during 7 d at 5 ◦C after 4.46 and 8.93 kJ m−2 UV-B treatments at harvest. Our results show how UV, both single and combined, does not induce colour changes during a commercial shelf-life period.
Initial firmness of red peppers was 18.3 ± 2.8 N, which increased 12
% after 14 d at 7 ◦C (21.3 ± 3.5 N) and decreased again to 19.2 ± 3.8 N after the additional 4 d at 18 ◦C. This fact was probably due to the storage temperature and dehydration. As a matter of fact, although there were no differences among treatments, UVC showed a slightly higher firmness at the beginning and at the end of the refrigerated storage (14 d at 7 ◦C). In this way, also Rodoni et al. (2012) did not find differences after a postharvest 20 kJ m−2 UV-C treatment in mature green peppers.
Hence, applied UV treatments did not negatively affect firmness throughout the shelf life of red bell peppers.
Mean pH value of studied bell peppers was 5.0 ± 0.02 units, with no differences among treatments, neither sampling days. These values agree with Vicente et al. (2005), who reported similar values in red peppers under gradual UV-C treatments. Furthermore, there were not differences among the obtained data with regards to TSS and TA of the studied red peppers. At harvest, bell peppers showed 8.35 ± 0.2 % of TSS, which slightly decreased to 7.8 ± 0.1 after 14 d at 7 ◦C and 4 d at 18
◦C in CTRL and UVB samples. These results also agree with Kasim and Kasim (2018), who reported a decrease of the TSS during 49 d at 5 ◦C treated at harvest with 4.46 or 8.93 kJ m−2 UV-B. By contrast, UVC and UVB + C kept constant the initial TSS up to 14 d at 7 ◦C + 4 d at 18 ◦C.
This fact underlines a positive tendency to prolong storage after appli- cation of UVC and UVB + C treatments (Kasim and Kasim, 2018). From the other point of view, TA of red peppers was 0.53 ± 0.03 g citric acid 100 m L-1, which decreased by 4 % after 8 d at 7 ◦C + 4 d at 18 ◦C, and by 34 % after 14 d at 7 ◦C + 4 d at 18 ◦C. This pepper variety has similar TSS and TA than other variety reported by (Kus¸çu et al., 2016). In this way, maturity index was 15.7 ± 1.0 which increased to 22.6 ± 1.1 after 14 d at 7 ◦C + 4 d at 18 ◦C. This value could vary depending on the red pepper variety. As observed, although no differences were found among the UV treatments, the TA of bell peppers was inversely proportional to maturity index throughout the shelf-life simulation. UV treatments do not affect the main physicochemical parameters; however, the storage time and temperature were beneficial to increase the maturity index.
3.2. Bioactive compounds content and total antioxidant capacity TPC and TFC in red bell peppers can be related to their antioxidant capacity because they are secondary metabolites, which are linked with the plant’s defence system (Hamed et al., 2019). Regarding these results, an increase in phenolic accumulation is shown by UVB + C red peppers on 8 d 7 ◦C (77 %) and 14 d 7 ◦C (32 %), compared to CTRL (Table 3). In addition, UVB treated peppers showed 90 % higher TPC than CTRL ones after 8 d at 7 ◦C, while UVC also increased the biosynthesis of phenolics by 27 % on 8 d 7 ◦C + 4 d 18 ◦C compared to CTRL. In this sense,
although the TPC of untreated peppers was maintained during shelf life, it is important to remark that a higher phenolic accumulation was produced under UVB and UVB + C treatments after 8 d at 7 ◦C, while also UVC slightly increased the phenolic biosynthesis throughout the study compared to CTRL peppers.
Something similar occurs with flavonoids (Table 3). In fact, UVC and UVB + C showed the highest flavonoid accumulation, while CTRL and UVB reported the lowest values throughout the study. In this way, after 8 d at 7 ◦C + 4 d at 18 ◦C and 14 d at 7 ◦C + 4 d at 18 ◦C, UVC increased by 18 and 46 % the flavonoid biosynthesis while UVB + C did it by 65 and 31 %, respectively, compared to CTRL. By contrast, UVB did not posi- tively affect in this case the flavonoid accumulation in comparison with CTRL. In this sense, this fact may be produced since both radiations (UV- B and UV-C) could share the same photoreceptor (Jiang et al., 2012), which can justify that low doses of UV-C can act as light stimuli of the biosynthesis of phytochemicals, such as phenolics and flavonoids (Abdipour et al., 2019; Rabelo et al., 2020a,b).
Fig. 2 shows the TCC of UV-treated and untreated bell peppers during the storage and retail sale periods. CTRL samples reported 66.8 ± 3.2 g carotene kg−1 at harvest, which was increased by 19 % after 8 d at 7 ◦C and by 39 % after the supplementary 4 d at 18 ◦C. However, after 14 d at 7 ◦C, the TCC decreased to 49.9 ± 2.6 g carotene kg−1, which was sta- bilized again to 80.3 ± 5.3 g carotene kg−1. Furthermore, a significant trend was observed in UV-treated bell peppers. In fact, during the first shelf-life study, the carotenoid biosynthesis was exponentially increased by UV-C and UV-B, both single and combined. Firstly, just after the UV was applied at harvest, an increase of 21 % in the TCC was observed compared to CTRL. In this way, after 8 d at 7 ◦C, UV radiation improved this effect by 26 %, while after the supplementary 4 d at 18 ◦C it was increased by 29 % compared to CTRL. Likewise, after 14 d at 7 ◦C UVB and UVC increased the TCC by 59 % and UVB + C did it by 94 % compared to CTRL, while after the supplementary 4 d at 18 ◦C the TCC was slightly increased with the UVC and UVB + C treatments by 17 and 13 %, respectively, compared to CTRL.
In contrast to our results, Vicente et al. (2005) showed a decrease of the TCC in red peppers treated at harvest with 7 kJ m−2 UV-C and stored for 18 d at 10 ◦C. In this way, as previously described, UVR8 protein has been described as the main UV-B receptor, and its action spectrum also includes the UV-C region (Jiang et al., 2012), which suggests that they may share such receptor. This hypothesis can also explain that low doses of single UV-C can stimulate carotenoids among other phytochemicals (Abdipour et al., 2019; Rabelo et al., 2020a,b). In fact, the active form of UVR8, a UV specific photoreceptor of both wavelengths (UV-C and UV-B) directly interacts with COP1 and regulates HY5 gene expression, which also triggers the production of carotenoids, directly linked to photoprotection (Jenkins, 2014, 2009).
No differences were found among UV treatments regarding its TAC measured by ABTS and DPPH (Table 3) reporting at harvest, just after the UV treatments, 1.3 ± 0.1 g TE kg−1 by ABTS and 0.52 ± 0.07 g TE kg−1 by DPPH. In fact, lower doses than 6 kJ m-2 UV-C may also enhance Table 1
Initial characterization of red bell peppers after harvesting before the UV treatments were applied (n = 100).
Weight (g) Caliber (mm)
Thickness (mm)
Equatorial Longitudinal
204.7 ± 36.9 87.2 ± 8.2 87.6 ± 7.6 5.8 ± 1.0
the TAC and bioactive compounds in red peppers, as it was shown by Vicente et al. (2005) after the application of 1, 2, 3, and 7 kJ m-2 UV-C, which can explain a demonstrated hormesis of UV-C. Similarly to our study, Andrade Cuvi et al. (2011) did not show differences in the TAC measured by DPPH after 10 kJ m-2 UV-C at harvest. As well as Rodoni et al. (2012), who did not show differences after 20 kJ m-2 UV-C treat- ment in green fresh-cut bell peppers regarding TAC measured by DPPH and ABTS methods.
In summary, in the present work, no relevant changes were observed during shelf life in any sampling days, regarding the TAC measured by DPPH and ABTS assays. Indeed, only UVC increased 2-fold the TAC measured by DPPH compared to CTRL on 8 d 7 ◦C + 4 d 18 ◦C and 14 d 7
◦C, which agree with previous reports by Rabelo et al., 2020a,b.
Table 2
Physicochemical parameters evolution of UV treated red bell peppers during 8 or 14 d at 7 ◦C followed by a supplementary retail sale period of 4 d at 18 ◦C in each case.
UV treatment At harvest 8d7 ◦C 8d7
◦C+4d18 ◦C 14d7 ◦C 14d7
◦C+4d18 ◦C ΔE CTRL – 7.5 ± 3.0 7.7 ± 3.1 6.0 ± 3.2
B 6.6 ± 2.8
UVC – 10.5 ±
7.9 8.8 ± 6.3 10.4 ±
6.2 AB 9.0 ± 6.1
UVB – 11.4 ±
6.5 10.4 ± 6.9 11.2 ±
7.2 A 9.4 ± 7.1
UVB + C – 11.3 ±
5.2 10.6 ± 5.9 13.3 ±
5.8 A 9.2 ± 4.8 Weight losses (%)
CTRL – 0.53 ±
0.2 BC b 0.79 ± 0.3 A
a 0.89 ±
0.3 B a 0.52 ± 0.3 B
b
UVC – 0.35 ±
0.2 C b 0.80 ± 0.3 A
a 0.80 ±
0.4 B a 0.62 ± 0.3 B a
UVB – 0.78 ±
0.5 A b 0.50 ± 0.2 B
c 1.28 ±
0.4 A a 0.28 ± 0.1 C c
UVB + C – 0.65 ±
0.1 AB b 0.41 ± 0.2 B
c 0.80 ±
0.3 B ab 0.98 ± 0.3 A
a
Firmness (N)
CTRL 17.8 ± 2.4
AB b 17.5 ±
2.1 b 19.4 ± 3.4 ab 21.3 ±
3.5 AB a 19.2 ± 4.5 ab UVC 19.9 ± 3.2
A ab 17.7 ±
3.9 b 19.1 ± 3.5 ab 22.1 ±
3.4 A a 19.7 ± 3.7 ab UVB 16.9 ± 2.7
B b 19.1 ±
3.3 ab 18.7 ± 3.1 ab 19.0 ±
3.4 B ab 19.9 ± 3.7 a UVB + C 18.5 ± 3.0
AB ab 16.7 ±
2.6 b 18.7 ± 3.4 ab 19.9 ±
2.5 AB a 18.0 ± 3.2 ab Total Soluble Solid content (ºBrix)
CTRL 8.4 ± 0.2 a 7.7 ± 0.2
b 7.8 ± 0.2 b 7.8 ± 0.3
b 7.8 ± 0.2 b UVC 8.3 ± 0.2 7.7 ± 0.2 7.9 ± 0.1 7.8 ± 0.4 8.0 ± 0.5 UVB 8.3 ± 0.3 a 7.9 ± 0.1
ab 7.7 ± 0.3 b 7.9 ± 0.2
b 7.8 ± 0.1 b UVB + C 8.4 ± 0.1 7.9 ± 0.2 7.8 ± 0.3 8.3 ± 0.5 8.1 ± 0.2 Titratable Acidity (g citric acid 100 m L−1)
CTRL 0.54 ±
0.05 a 0.51 ±
0.02 a 0.51 ± 0.04
a 0.43 ±
0.01 B b 0.36 ± 0.01 c
UVC 0.53 ±
0.01 a 0.51 ±
0.02 a 0.52 ± 0.02
a 0.36 ±
0.02 B b 0.34 ± 0.03 c
UVB 0.52 ±
0.01 a 0.50 ±
0.02 a 0.54 ± 0.04
a 0.53 ±
0.05 A a 0.34 ± 0.02 b UVB + C 0.53 ±
0.04 a 0.53 ±
0.04 a 0.49 ± 0.01
a 0.43 ±
0.03 B b 0.36 ± 0.01 c Maturity Index (TSS/TA)
CTRL 15.6 ± 1.6
c 15.2 ±
0.5 c 15.3 ± 1.0
AB c 18.1 ±
0.6 A b 21.6 ± 0.9 a UVC 15.6 ± 0.6
bc 15.1 ±
0.5 c 15.4 ± 0.5
AB c 17.3 ±
1.6 AB b 23.5 ± 0.9 a UVB 16.0 ± 0.5
b 15.8 ±
0.9 b 14.3 ± 0.9 B
b 15.1 ±
1.4 B b 23.2 ± 1.5 a UVB + C 15.7 ± 1.1
c 15.1 ±
1.2 c 16.0 ± 0.7 A
c 19.6 ±
1.3 A b 22.2 ± 1.0 a Different capital letters denote significant differences (p < 0.05) among different treatments for the same sampling time. Different lowercase letters denote sign.
Table 3
Phenolic compounds (g gallic acid equivalents kg−1), flavonoids (g rutin equivalents kg−1), and total antioxidant capacity (g trolox equivalents kg−1) of UV treated red peppers of UV treated red bell peppers during 8 or 14 d at 7 ◦C followed by a supplementary retail sale period of 4 d at 18 ◦C in each case.
UV At harvest 8d7 ◦C 8d7
◦C+4d18 ◦C 14d7 ◦C 14d7
◦C+4d18 ◦C Total phenolic content
CTRL 9.6 ± 0.3
ab 7.8 ± 0.7
C c 8.5 ± 0.3 B bc 8.8 ± 0.4
B bc 10.5 ± 0.6 a
UVC 10.2 ± 0.8 10.7 ±
0.7 B 10.8 ± 1.2 A 11.0 ±
1.2 AB 11.0 ± 0.6 UVB 11.4 ± 1.0
b 14.8 ±
1.0 A a 9.2 ± 0.1 AB c 9.0 ± 0.4
B c 10.1 ± 0.6 bc UVB +
C 10.2 ± 2.0
b 13.8 ±
1.3 A a 10.9 ± 1.0 A
ab 11.6 ±
1.3 A ab 10.5 ± 0.2 ab Total flavonoid content
CTRL 1.5 ± 0.1 B
ab 1.1 ± 0.0
B c 1.7 ± 0.1 C a 1.3 ± 0.1
B b 1.3 ± 0.1 B b UVC 1.8 ± 0.1 A
a 1.5 ± 0.2
A b 2.0 ± 0.1 B a 1.9 ± 0.1
A a 1.9 ± 0.1 A a UVB 1.1 ± 0.1 C
c 1.2 ± 0.1
AB bc 1.8 ± 0.2 BC a 1.4 ± 0.1
B b 1.3 ± 0.1 B bc UVB +
C 1.4 ± 0.2 B
b 1.5 ± 0.1
A b 2.8 ± 0.1 A a 1.8 ± 0.2
A b 1.7 ± 0.1 A b Total Antioxidant Capacity (ABTS)
CTRL 1.3 ± 0.0
AB bc 1.1 ± 0.1
c 1.6 ± 0.1 a 1.5 ± 0.1
ab 1.1 ± 0.1 c UVC 1.4 ± 0.1 A
ab 1.2 ± 0.1
b 1.5 ± 0.0 a 1.6 ± 0.1
a 1.4 ± 0.1 ab UVB 1.2 ± 0.1 B
b 1.3 ± 0.2
ab 1.6 ± 0.1 a 1.4 ± 0.1
ab 1.3 ± 0.2 ab UVB +
C 1.2 ± 0.0 B
b 1.2 ± 0.1
b 1.7 ± 0.1 a 1.4 ± 0.2
ab 1.1 ± 0.2 b Total Antioxidant Capacity (DPPH)
CTRL 0.54 ±
0.04 a 0.48 ±
0.04 ab 0.26 ± 0.05 B
c 0.29 ±
0.01 B c 0.43 ± 0.04 b
UVC 0.60 ±
0.07 0.47 ±
0.02 0.50 ± 0.05 A
a 0.54 ±
0.03 A 0.52 ± 0.06
UVB 0.51 ±
0.10 0.46 ±
0.13 0.41 ± 0.04
AB a 0.40 ±
0.04 AB 0.39 ± 0.00 UVB +
C 0.44 ±
0.07 0.49 ±
0.05 0.37 ± 0.11
AB a 0.35 ±
0.11 B 0.37 ± 0.10 Different capital letters denote significant differences (p < 0.05) among different treatments for the same sampling time. Different lowercase letters denote sig- nificant differences (p < 0.05) among different sampling times for the same treatment. Absence of letters indicates that there are no significant differences (p
>0.05).
Fig. 2. Total carotenoid content of UV treated red bell peppers during 8 or 14 d of storage at 7 ◦C followed by a supplementary retail sale period of 4 d at 18
◦C in each case. T: treatment; t: time. ***: P < 0.001. *: significant differences compared to CTRL.
Recently, these authors studied the enhancement of phenolic com- pounds and vitamin C in acerola fruits after an exposition to 6 kJ m−2 UV-C at harvest.
3.3. Flavonoid biosynthesis
As one of the main bioactive compounds in red bell peppers, the TFC was improved by the UV treatments assayed in the present study (Table 3). Therefore, further analyses were conducted, and individual flavonoids content are shown in Table 4. As a summary, Fig. 2.A. shows the TFC as the sum of all the individual peaks identified as flavonoids, while Fig. 3.B. shows the content of the main flavonoids identified, as the sum of rutin and its derivatives in red peppers.
In this way, the TFC found at harvest in red bell peppers was 1.394 ±
78 mg flavonoid kg−1 (Fig. 3.A), without differences among UV treat- ments. The TFC was subsequently increased by 27, 39, and 25 % after 8 d at 7 ◦C in UVC, UVB, and UVB + C treated peppers, respectively, compared to CTRL. After an additional retail sale period of 4 d at 18 ◦C, UVB + C increased by 17 % the flavonoid accumulation in comparison with CTRL. This behaviour was also observed after 14 d at 7 ◦C with increases of 36 and 30 % in UVC and UVB + C treated peppers, respectively, regarding CTRL, which was preserved after the additional retail sale period of 4 d at 18 ◦C.
Rutin and its derivatives (Fig. 3.B) represent the 37 % of the total flavonoid compounds identified by UHPLC. In this way, UVC, UVB, and UVB + C showed to be a good abiotic stress elicitor reporting increases immediately after the UV treatment (at harvest) of 42, 66, and 43 %, respectively, compared to CTRL. This behaviour was enhanced after 8
Table 4
Individual flavonoid and phenolic acid (mg kg−1) content of UV treated red bell peppers during 8 or 14 d at 7 ◦C followed by a supplementary retail sale period of 4 d at 18 ◦C in each case.
UV Day of analysis Gallic acid
RT: 12.8 min 280 nm
Catechin RT: 16.4 min 280 nm
Epicatechin RT: 17.7 min 280 nm
Caffeic acid RT: 20.0 min 280 nm
Caffeic acid-dv RT: 20.4 min 280 nm
Caffeic acid-dv RT: 21.3 min 280 nm CTRL
At harvest 83 ± 4 AB 182 ± 7 AB ab 200 ± 12 ab 97 ± 4 AB ab 97 ± 1 AB ab 106 ± 6 ab
8d7 ◦C 94 ± 12 154 ± 19 b 176 ± 20 b 85 ± 9 b 87 ± 8 b 90 ± 11 b
8d7 ◦C+4d18 ◦C 79 ± 13 B 202 ± 8 a 222 ± 8 a 109 ± 4 a 107 ± 4 a 116 ± 4 AB a
14d7 ◦C 76 ± 3 B 198 ± 5 a 214 ± 8 a 104 ± 2 a 107 ± 3 a 111 ± 3 ab
14d7 ◦C+4d18 ◦C 89 ± 17 182 ± 21 ab 200 ± 21 ab 101 ± 10 ab 99 ± 10 ab 108 ± 12 ab
UVC
At harvest 106 ± 9 A 206 ± 4 A 217 ± 4 106 ± 2 A 109 ± 2 A 111 ± 4
8d7 ◦C 117 ± 9 188 ± 16 200 ± 16 99 ± 9 101 ± 7 109 ± 10
8d7 ◦C+4d18 ◦C 101 ± 12 B 190 ± 6 202 ± 4 100 ± 3 102 ± 3 111 ± 4 AB
14d7 ◦C 107 ± 4 A 203 ± 8 219 ± 10 107 ± 3 107 ± 3 112 ± 3
14d7 ◦C+4d18 ◦C 97 ± 9 195 ± 13 215 ± 15 105 ± 7 104 ± 6 117 ± 9
UVB
At harvest 77 ± 11 B 162 ± 14 B 187 ± 19 88 ± 7 B 89 ± 7 B 99 ± 4
8d7 ◦C 88 ± 24 193 ± 14 215 ± 23 104 ± 8 104 ± 8 108 ± 13
8d7 ◦C+4d18 ◦C 80 ± 4 B 192 ± 13 205 ± 15 103 ± 7 106 ± 7 105 ± 7 B
14d7 ◦C 88 ± 7 B 185 ± 7 202 ± 5 97 ± 3 101 ± 3 102 ± 3
14d7 ◦C+4d18 ◦C 105 ± 5 182 ± 18 199 ± 19 101 ± 9 101 ± 9 102 ± 12
UVB þ C
At harvest 61 ± 12 B c 184 ± 19 AB 192 ± 15 96 ± 11 AB 98 ± 9 AB 108 ± 12
8d7 ◦C 88 ± 10 b 190 ± 21 195 ± 15 98 ± 7 98 ± 8 104 ± 5
8d7 ◦C+4d18 ◦C 130 ± 7 A a 205 ± 11 223 ± 23 111 ± 5 112 ± 7 126 ± 13 A
14d7 ◦C 88 ± 5 B b 193 ± 20 197 ± 16 102 ± 10 105 ± 9 115 ± 14
14d7 ◦C+4d18 ◦C 101 ± 9 b 201 ± 4 211 ± 8 111 ± 5 106 ± 3 118 ± 7
UV Day of analysis Quercetin-dv
RT: 31.1 min 330 nm
Quercetin RT: 31.9 min 330 nm
Quercetin-dv RT: 32.9 min 330 nm
Luteolin RT: 36.7 min 330 nm
Naringenin RT: 37.9 min 330 nm
Apigenin RT: 40.3 min 330 nm CTRL
At harvest 112 ± 4 AB ab 114 ± 2 AB ab 115 ± 0 AB ab 91 ± 4 AB ab 28.4 ± 0.8 AB ab 1.00 ± 0.04
8d7 ◦C 96 ± 12 b 99 ± 12 b 106 ± 12 b 79 ± 10 b 24.5 ± 2.7 b 1.07 ± 0.34
8d7 ◦C+4d18 ◦C 126 ± 5 a 129 ± 5 a 137 ± 4 a 101 ± 4 a 32.5 ± 1.0 a 1.22 ± 0.43
14d7 ◦C 124 ± 3 a 127 ± 4 a 131 ± 3 a 100 ± 3 a 30.8 ± 1.3 a 0.87 ± 0.19
14d7 ◦C+4d18 ◦C 113 ± 12 ab 116 ± 13 ab 120 ± 14 ab 90 ± 10 ab 29.3 ± 3.3 ab 0.69 ± 0.09
UVC
At harvest 123 ± 3 A 126 ± 4 A 134 ± 5 A 101 ± 3 A 30.2 ± 0.5 A 0.87 ± 0.11
8d7 ◦C 113 ± 11 116 ± 10 124 ± 7 92 ± 8 28.2 ± 2.8 1.25 ± 0.26
8d7 ◦C+4d18 ◦C 113 ± 3 115 ± 3 125 ± 4 91 ± 3 30.4 ± 1.0 1.16 ± 0.13
14d7 ◦C 122 ± 4 126 ± 4 132 ± 5 100 ± 3 31.3 ± 1.2 0.88 ± 0.26
14d7 ◦C+4d18 ◦C 118 ± 7 121 ± 7 127 ± 8 95 ± 5 31.5 ± 2.3 1.11 ± 0.12
UVB
At harvest 100 ± 9 B 104 ± 9 B 110 ± 9 B 82 ± 8 B 25.7 ± 2.3 B 1.07 ± 0.15
8d7 ◦C 120 ± 9 123 ± 9 129 ± 12 97 ± 7 30.2 ± 2.1 1.24 ± 0.29
8d7 ◦C+4d18 ◦C 119 ± 8 121 ± 8 127 ± 9 95 ± 6 31.0 ± 2.1 0.91 ± 0.21
14d7 ◦C 115 ± 5 119 ± 4 124 ± 4 94 ± 4 28.9 ± 0.8 1.06 ± 0.11
14d7 ◦C+4d18 ◦C 113 ± 11 116 ± 11 119 ± 12 91 ± 8 29.6 ± 2.6 0.77 ± 0.04
UVB þ C
At harvest 110 ± 13 AB 113 ± 13 AB 122 ± 14 AB 88 ± 10 AB 27.6 ± 2.2 AB 0.87 ± 0.14
8d7 ◦C 112 ± 11 115 ± 11 120 ± 10 91 ± 9 28.7 ± 2.8 1.28 ± 0.05
8d7 ◦C+4d18 ◦C 123 ± 6 126 ± 7 137 ± 8 100 ± 5 32.7 ± 2.1 1.35 ± 0.30
14d7 ◦C 117 ± 13 120 ± 13 127 ± 12 94 ± 10 30.1 ± 2.6 0.97 ± 0.25
14d7 ◦C+4d18 ◦C 122 ± 3 125 ± 3 130 ± 4 97 ± 3 31.7 ± 1.1 1.15 ± 0.33
Different capital letters denote significant differences (p < 0.05) among different treatments for the same sampling time. Different lowercase letters denote significant differences (p < 0.05) among different sampling times for the same treatment. Absence of letters indicates that there are no significant differences (p > 0.05).
d at 7 ◦C + 4 d at 18 ◦C by UVC and UVB + C (15 and 44 %, respectively) and after 14 d at 7 ◦C + 4 d at 18 ◦C (146 and 137 %, respectively) in comparison with CTRL red peppers. In addition, UVB red peppers showed increased by 42 % the rutin accumulation after 14 d at 7 ◦C, but there was not found a tendency throughout the experiment.
Other flavonoids and phenolic acids were identified (Table 4). The main phenolic acids identified were: gallic acid and caffeic acid and its derivatives while identified flavonoids were: catechin, epicatechin, quercetin and its derivatives, luteolin, naringenin, and apigenin (Morales-Soto et al., 2013).
In general, no differences were found among UV treatments regarding the synthesis of minor flavonoids and phenolic acids. Never- theless, it is remarkable that UV-C radiation positively affected the accumulation of gallic acid, catechin, caffeic acid and its derivatives, quercetin, and its derivatives, luteolin, and naringenin, immediately after treatment, which was not maintained throughout the shelf life assayed.
Particularly, only gallic acid experimented an accumulation after UV applications. UVC samples showed the highest values at harvest (~28 % compared to CTRL) and after 14 d at 7 ◦C (~41 % compared to CTRL), while UVB + C did it after 8 d at 7 ◦C + 4 d at 18 ◦C (~65 % compared to CTRL).
Thus, only the biosynthesis of the main flavonoid and its derivatives was positively stimulated after UV treatments in red bell peppers, especially under UVC and UVB + C treatments (Fig. 2.B). This behaviour can be justified, as previously described, by the fact that both radiations (UV-B and UV-C) may share the same photoreceptor, UVR8 protein, whose action spectrum includes both wavelengths (Jiang et al., 2012), although further studies should clearly elucidate such hypothesis. This could be due because the active form of UVR8, a UV specific photore- ceptor of both wavelengths (UV-C and UV-B), directly interacts with COP1 and regulates HY5 gene expression, which also triggers the pro- duction of flavonoids which are directly linked to photoprotection, as carotenoids do (Jenkins, 2014, 2009). This theory can also explain that low UV-C doses can stimulate the biosynthesis of bioactive compounds (Abdipour et al., 2019; Rabelo et al., 2020a,b), specially rutin and its derivatives, as main flavonoids found in red bell peppers, as well as the simultaneous combination of UV-B and UV-C. In fact, stress caused by high doses of UV lighting can enhance the production of reactive oxygen species (ROS) damaging DNA. In this sense, some flavonoids are effec- tive scavengers of ROS and absorbers of UV radiation (Falcone Ferreyra et al., 2012), inducing therefore the flavonoid biosynthesis in plants. In fact, UV is strongly absorbed by tryptophan amino acid residues in the UVR8 photoreceptor, which leads to its monomerization. UVR8 and COP1 form a complex that accumulates in the nucleus of the vegetal cells. In this way, the UVR8− COP1-SPA complex stabilizes bZIP tran- scription factor HY5 promoting the activity of different R2R3 MYBs for the transcription of specific flavonoid biosynthesis genes (Clayton et al., 2018; Jenkins, 2014; Logemann et al., 2000; Zoratti et al., 2014), which has been recently demonstrated in Gingko biloba leaves under 7.14 and
21.42 kJ m−2 UV-B (Zhao et al., 2020). Therefore, our hypothesis based on the enhancement of the biosynthesis of flavonoids by UV-C (6 kJ m−2) or the simultaneous application of UV-B + UV-C (6 + 6 kJ m−2), is focused on sharing the same photoreceptor (UVR8).
4. Conclusions
Studied postharvest UV treatments did not affect the physicochem- ical quality of bell peppers. However, a great elicitor response was observed as an abiotic stress with a great accumulation of several bioactive compounds, mainly after 6 kJ m−2 UVC, but also when it was simultaneously applied with 6 kJ m−2 UVB (UVB + C). In this sense, the carotenoid biosynthesis was enhanced by ~30 % under all UV treat- ments assayed throughout a shelf-life period, especially by UVC and UVB + C (59 and 94 %, respectively). Similarly, UVC and UVB + C enhanced the flavonoid accumulation by 36 and 30 %, respectively, after the shelf-life period assayed. Indeed, rutin and its derivatives, which represented 37 % of the TFC, showed increases of 42, 66, and 43
% under UVC, UVB, and UVB + C treatments, respectively. Therefore, a postharvest UV-C treatment has shown an hormetic effect in red bell peppers, as well as if it is combined with UV-B, greatly enhancing the accumulation of the main health promoting compounds throughout a shelf-life period.
Author contributions
Conceptualization and methodology: Francisco Art´es–Hern´andez (F.
A.H) and Lorena Martínez-Zamora (L.M.Z); performed the experiments:
Noelia Castillejo (N.C) and L.M.Z..; investigation: F.A.H., L.M.Z. and N.
C.; software: L.M.Z.; validation: F.A.H., L.M.Z. and N.C.; resources: F.A.
H.; data curation: N.C.; writing—original draft preparation + review and editing: F.A.H., L.M.Z. and N.C.; visualization: F.A.H.; supervision:
F.A.H.; project administration: F.A.H.; funding acquisition: F.A.H.
Funding
This research received no external funding.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
Noelia Castillejo contract was funded by a predoctoral grant (FPU16/
04763) from the Spanish Ministry of Education. Lorena Martínez- Zamora contract has been co-financed by the European Social Fund and the Youth European Initiative under the Spanish Seneca Foundation (21322/PDGI/19). Authors thanks Henarejos y T´arraga, S.L. and the Asociaci´on Eco-innovadora Agrícola de la Regi´on de Murcia for the Fig. 3. Total flavonoid (A) and rutin (B) content of UV treated red bell peppers during 8 or 14 d of storage at 7 ◦C followed by a supplementary retail sale period of 4 d at 18 ◦C in each case. T: treatment; t: time. n.s.: no significant differences. ***: P < 0.001. *: significant differences compared to CTRL.