Riboflavin applications to grapevine leaves and berries blue-light post-harvest treatments modifies grape anthocyanins and amino acids contents

Food Research International 122 (2019) 479–486

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Food Research International journal homepage: www.elsevier.com/locate/foodres

Riboflavin applications to grapevine leaves and berries blue-light postharvest treatments modifies grape anthocyanins and amino acids contents

T

E.P. Pérez-Álvareza, , R. Ruiz-Gonzálezb,c, , S. Nonellb, T. Garde-Cerdána ⁎

⁎

a

Grupo VIENAP, Instituto de Ciencias de la Vid y del Vino (CSIC, Gobierno de La Rioja, Universidad de La Rioja), Ctra. de Burgos, Km. 6., 26007 Logroño, Spain Institut Químic de Sarrià, Universitat Ramon Llull, Vía Augusta, 390.08017, Barcelona, Spain c Esencias Moles S.A. Avenida de Cataluña, 11. 08758 Cervelló, Barcelona, Spain b

ARTICLE INFO

ABSTRACT

Keywords: Vitamin B2 Irradiation Nitrogen compounds Phenolic compounds Vitis vinifera

Light is an energy source and key environmental factor for plants. Out of the different light wavelengths, bluelight is one of the most relevant spectral regions because of its relation to anthocyanins biosynthesis. Among the compounds present in grapes, anthocyanins determine their main organoleptic and healthy properties; while a minimum concentration of ammonium and amino acids is necessary for a desirable development of the alcoholic fermentation. Moreover, amino acids are precursors of several volatile compounds synthetized during the fermentation. The aim of this study was to assess the influence of riboflavin (vitamin B2) applications, at harvest and one week later,to grapevine leaves in combination with post-harvest blue-light irradiation on Tempranillo (Vitis vinifera L.) grape anthocyanins and amino acidscomposition. The combination of blue-light irradiation and two riboflavin doses as well as theseindividual factors affected both grape anthocyanins and amino acidsconcentrations. After one week of storage, anthocyanins concentration diminished when clusters were irradiated with blue-light; while for amino acids content, the trend to increase or decrease is dependent on the riboflavin dose applied in vines and the storage time.

1. Introduction

foods (Powers, 2003). Flavin derivatives, including riboflavin, are known to generate reactive oxygen species (ROS) (Gorner, 2007) and can induce oxidative stress upon ultraviolet (UV) or visible light exposure (Ahmad & Vaid, 2006). In humans, vitamin B2 deficiency is associated with health and nutritional problems due to its effect on key redox reactions to human metabolism (Powers, 2003). In plants, increased levels of riboflavin have been shown to exert eliciting capacity (Dong & Beer, 2000) via the induction of ROS and hormonal signaling transduction pathways, promoting the phenylpropanoid pathway (Liu et al., 2010; Taheri & Tarighi, 2011). Also, its role in enhancing disease resistancehas been described in grapevines (Boubakri et al., 2013). Besides, an increase of amino acids production was observed when riboflavin was applied to vineyard leaves (González-Santamaría, RuizGonzález, Nonell, Garde-Cerdán, & Pérez-Álvarez, 2018). Irradiation of plant tissues with blue and UV light causes oxidative stress which leads to important effects on phenolic metabolism (HuchéThélier et al., 2016). A previous work has described that, after being irradiated with UVB/UVC, the content of resveratrol in the grapes could be increased up to 3-fold, depending on storage temperature (0 vs 15 °C) (Cantos, García-Viguera, de Pascual-Teresa, & Tomás-Barberán, 2000). This knowledge has been applied to increase the stilbenes

Nitrogen is the most abundant soil-derived macronutrient in grapevine and plays essential roles in biological functions and processes both in grape and fermentative microorganisms (Bell & Henschke, 2005). Nitrogen concentration and composition in grapes and musts can potentially affect wine quality and its marketable value. Thus, manipulation of grapevine nutrition –for instance supplementing vineyard with nitrogen sources- has the potential to influence grape development and composition (Bell & Henschke, 2005). An increase in concentration of the major nitrogen compounds in grapes, i.e. free amino acids and ammonium, has been observed after nitrogen application in the vineyard (Bell & Henschke, 2005; Pérez-Álvarez, GardeCerdán, García-Escudero, & Martínez-Vidaurre, 2017). Moreover, while the amino acids concentration and composition can vary according to several factors like as the grape cultivar, site or seasonal conditions, the application of nitrogen to grapevines has a positive impact on the yeast assimilable nitrogen (YAN), which is the sum of the ammonium plus the primary amino acids found in grapes (Bell & Henschke, 2005). Riboflavin is a water-soluble vitamin (vitamin B2, Scheme 1)which is ubiquitously distributed in nature and found in a wide variety of

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Corresponding authors. E-mail addresses: [email protected] (E.P. Pérez-Álvarez), [email protected] (R. Ruiz-González).

https://doi.org/10.1016/j.foodres.2019.05.007 Received 27 August 2018; Received in revised form 1 May 2019; Accepted 3 May 2019 Available online 06 May 2019 0963-9969/ © 2019 Elsevier Ltd. All rights reserved.

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Scheme 1. Graphical details of the steps involved in the proceedings of the study.

was mechanically tilled, and the rest of the management practices were similar in all treatments and they were made according to the standard practices for the region.

content of table grapes by delivering controlled UV irradiation pulses taking into account parameters such as irradiation power, time or distance (Cantos, Espín, Fernández, Oliva, & Tomás-Barberán, 2003; Cantos, Espín, & Tomás-Barberán, 2001; Guerrero, Puertas, Fernández, Palma, & Cantos-Villar, 2010). On the other hand, anthocyanins accumulation occurred upon non-UV or UV exposure in Arabidopsis thaliana (Bieza & Lois, 2001; Xu, Mahmood, & Rothstein, 2017) or suspensioncultured strawberry cells (Mori & Sakurai, 1999); whereas, its concentration in grapes remained unaltered upon UV-light exposure (Cantos et al., 2003). To our knowledge, the approach to exploit the combination of bluelightplus a photosensitizing nitrogenated molecule such as riboflavin has not been explored so far. The aim of the present work was to study the influence of riboflavin pre-harvest treatments to grapevine leaves and the post-harvest irradiation of the clusters with blue-light on grapes amino acids and anthocyanins contents (Scheme 1).

2.2. Grape post-harvest treatment with blue-light irradiation Grapes were manually harvested at their optimum technological maturity, i.e. when the weight of 100 berries remained constant and the probable alcohol reached 13 (% v/v). In the winery, harvested bunches for each replicate were separated into two lots of 5 kg each. One lot was irradiated with blue-light (λ) and the other was not exposed to light (non-λ). Grape samples were irradiated from the top(30 cm) with a Studio PAR 64 CAN RGBWA+UV Light Emitting Diode (LED) based lamp equipped with 12 LEDs of 12 W each (Cameolight, Neu-Anspach, Germany). Irradiation was performed for 2 min at 7.5 mW/cm2fluence in the blue-light channel (456 ± 20 nm) in order to obtain the best overlap with riboflavin spectra (Fig. 1a). Fluence rates were measured using a power meter. Bunches were kept at 15 °C in the absence of light for 7 days prior to further analysis.

2. Materials and methods 2.1. Vineyard description and pre-harvest treatments

2.3. Determination of grape anthocyanins content

A Tempranillo (Vitis vinifera L.) vineyard, located at Logroño, (La Rioja region), NorthernSpain (42°26′3418´´ N, 2°30′5307´´ W, 465 m above sea level), was used for this study. It was carried out in 2016. Vineyard soil properties and climate data were detailed in GonzálezSantamaría et al. (2018). Grapevines were planted in 1990 with 1.2 m and 2.30 m plant and row spacing, respectively. They were trained to a vertical shoot position system (VSP) on a double Cordon Royat and were spur pruned to 12 buds per grapevine. The experimental design consisted of a randomized complete block with three different treatments and three replications per treatment. Each replicate had three adjacent grapevines and between treatments, in each replicate, there were two grapevines considered buffer and excluded in sampling to avoid edge effects. A control and two different doses of riboflavin (Sigma-Aldrich, Madrid, Spain) were the studied treatments. Control plants were foliar sprayed with a water solution of Tween 80 (SigmaAldrich, Madrid, Spain) alone, used as the wetting agent (1 mL/L). Riboflavin stock solutions of 0.5 and 1 mM were prepared, with Tween 80 (1 mL/L). For each application, 200 mL/plant of each solution was sprayed over grapevine leaves, i.e. riboflavin doses were: 100 g/ha (Rf1) and 200 g/ha (Rf2). The treatments were applied to the grapevines twice, at veraison (> 50% of grapes colored) and one week later. The experimental vineyard had not been nitrogen-fertilized, the soil

Grape anthocyanins contents were determined using the method described by Portu, López-Alfaro, Gómez-Alonso, López, and GardeCerdán (2015). Briefly, 50 mL of aqueous methanol solution (50% v/v) was added to 50 g of each grape sample and the pH was adjusted to 2 with formic acid (Sigma-Aldrich, Madrid, Spain). Grape samples were then homogenized in an Ultra-Turrax T-18 (IKA, Staufen, Germany). Then, the samples were extracted twice using 50 mL of aqueous methanol solution, and both supernatants were collected. The extracts from each sample were filtered (0.22 μm, Easyprep, Quebec, Canada) and 10 μL were injected into the HPLC (Agilent 1260 Infinity, Palo Alto, CA, USA). The separation was achieved on a column Zorbax Eclipse XDB-C18 (Agilent). Detection was performed in a diode array detector (DAD). The treatments were carried out in triplicate, so the results for grape anthocyanins content correspond to the average of three analyses (n = 3). 2.4. Must amino acids determination by HPLC The method described by Garde-Cerdán et al. (2014) was used for the determination of free amino acids in the musts. Briefly, 5 mL of the sample was mixed with 100 μL of norvaline, and 100 μL of sarcosine, 480

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Fig. 1. A)Normalized spectra of riboflavin (orange solid line) and spectra of blue-LED channel (blue dashed line). B) Singlet Oxygen production of RF in water upon excitation at 355 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

internal standards (Sigma-Aldrich, Madrid, Spain). This mixture was submitted to automatic pre-column derivatization with o-phthaldialdehyde (OPA Reagent, Agilent, Palo Alto, CA, USA) and with 9-fluorenylmethylchloroformate (FMOC Reagent, Agilent, Palo Alto, CA, USA) using an Agilent 1100 Series (Palo Alto, CA, USA) HPLC equipment. 10 μL of the derivatized sample were injected. All chromatographic separations were performed on a Hypersil ODS column (Agilent, Palo Alto, CA, USA). Detection was performed by fluorescence and DAD detectors. The treatments were carried out in triplicate, so the results for free amino acids concentration correspond to the average of three analyses (n = 3).

Regarding the effect of storage time without blue-light treatment (0 daysvs7 days, non-λ), in the control grapes, the concentration of cyanidin-3-acglc, peonidin-3-acglc, and peonidin-3-cmglc increased with the time (Table 1). Moreover, for Rf1 samples, only the content of delphinidin-3-acglc was higher after 7 days of storage, while the content of cyanidin-3-acglc, malvidin-3-acglc, peonidin-3-cmglc, and malvidin-3-cfglc was lower after 7 days of storage. No significant differences were observed due to storage time for Rf2 samples (Table 1). Finally, regarding the effect of storage time for the samples irradiated with blue-light (0 daysvs 7 days,λ), when grape samples were irradiated, the content of cyanidin-3-acglc, and peonidin-3-cmglc was higher in control samples at 7 days than at 0 days; however, malvidin-3acglc, and malvidin-3-cfglc concentration in control samples was higher at harvest than after 7 days of storage. For Rf1 samples, the content of petunidin-3-acglc, malvidin-3-acglc, cyanidin-3-cmglc, malvidin-3-ciscmglc, and malvidin-3-cfglc was higher at harvest than after one week of storage (Table 1). In the case of Rf2 grapes, three anthocyanins (peonidin-3-acglc, malvidin-3-cis-cmglc, and malvidin-3-cfglc) were also found in higher concentrations at harvest than after storage for a week. Apart from the aforementioned comparisons, data in Table 1 suggest that: i) Blue-light irradiation did not affect the content of total anthocyanins in grapes; ii) Rf application did not have any impact on the total anthocyanins concentration in grapes, neither at harvest nor after one week of storage; and iii) the combined use of Rf and blue-light did not increase the content of anthocyanins in grapes. Previous studies are found that UVB and UVC irradiation did not affect the anthocyanins content as compared to non-irradiated samples, either in grapes or in wines (Cantos et al., 2001; Cantos et al., 2003).

2.5. Statistical analysis Data sets were analyzed with SPSS Version 20.0 for Windows (Chicago, IL, USA) using the analysis of variance (ANOVA). The treatments were compared by a Duncan post hoc test at p ≤ .05. 3. Results and discussion 3.1. Effect of foliar application of riboflavin and blue-light on anthocyanins concentration in grapes Table 1 summarizes the anthocyanins concentrations in grapes upon different foliar treatments (C: control; Rf1: riboflavin at 100 g/ha; Rf2: riboflavin at 200 g/ha), at harvest (0 days), and one week after harvest (7 days), with berries irradiated with blue-light (λ) or not (non-λ). At harvest, the concentrations of delphinidin-3-acglc, cyanidin-3acglc, petunidin-3-acglc, peonidin-3-cmglc, and malvidin-3-cfglc were affected by riboflavin application. The highest content of delphinidin-3acglc was found in Rf2 samples; cyanidin-3-acglc and peonidin-3-cmglc concentrations were the highest in the case of the Rf1 samples; andpetunidin-3-acglcand malvidin-3-cfglc content was higher in the control and Rf1 samples than in Rf2 ones (Table 1). After seven days of storage at 15 °C, for grapes not irradiated (7 days, non-λ), cyanidin-3-acglc, petunidin-3-acglc,peonidin-3-acglc, malvidin-3-acglc, peonidin-3-cmglc, malvidin-3-cglc and total acylated anthocyanins concentrations were higher in control samples than in Rf2 ones, without significant differences with Rf1 samples. The control grapes showed the highest content of malvidin-3-trans-cmglc (Table 1). When grape samples were irradiated with blue-light, the concentrations ofpetunidin-3-acglc, delphinidin-3-cmglc and peonidin-3cmglc after a week of storage (7 days, λ) were higher in the control samples. Petunidin-3-glc, malvidin-3-glc, malvidin-3-cis-cmglc and total anthocyanins contents were higher in control samples than in Rf2 ones, without significant differences with Rf1 grapes. In addition, the lowest concentrations of peonidin-3-acglc, malvidin-3-acglc, petunidin-3cmglc, malvidin-3-trans-cmglc and total acylated anthocyanins were found in Rf2 samples (Table 1).

3.2. Riboflavin foliar applications and blue-light treatments influence on the grape amino acids concentration The significant differences among the treatments at harvest time (0 days) were previously measured and discussed (González-Santamaría et al., 2018). Briefly, riboflavin foliar application in the grapevines increased the content of amino acids in the grapes, especially when the lowest dose of riboflavin was applied (Table 2). After 7 days of storage, for the grapes not exposed to light (non-λ), the control samples presented the highest content of Ala, Thr, Ser, Trp, Val, Leu, Cit, Phe, Ile, Met, Lys, Orn, and total amino acids without Pro (Table 2). Meanwhile, Gln content was higher in control samples than in Rf1 ones, and the concentration of Tyr, and Gly was higher in control than in Rf2 grapes. Moreover, the lowest concentration of Cys was found in Rf1 grapes. After blue-light irradiation (λ) and 7 days of storage, the highest concentrations of most amino acids (Arg, Gln, GABA, Glu, Ala, Thr, His, Ser, Trp, Val, Cit, Tyr, Asn, Lys, Gly, total amino acids with and without Pro) and the lowest concentration of Pro were found in the samples 481

482

197 ± 16a, A, α 19 ± 2a, A, α 151 ± 9a, A, α 46 ± 5a, A, α 460 ± 23a, A, α 16 ± 2a, A, α 5.24 ± 0.02a, A, α 15 ± 1b, A, α 3.5 ± 0.2a, A, α 38 ± 1a, A, β 56 ± 4a, A, α 9.1 ± 0.4a, A, α 50 ± 3a, A α 21.7 ± 0.3a, A, α 11 ± 1a, A, α 228 ± 8a, A, α 6.3 ± 0.3b, A, β 1335 ± 57a, A, α 461 ± 14a, A, α 874 ± 53a, A, α

206 ± 41a, A, α 23 ± 4a, A, α 158 ± 26a, A, α 50 ± 10a, A, α 455 ± 41a, A, α 20 ± 1a, A, α 6.6 ± 0.7b, A, α 16 ± 1b, A, β 3.9 ± 0.5a, A, α 37 ± 2a, A, β 53 ± 6a, A, α 9.3 ± 0.5a, A, β 49 ± 2a, A, α 25.1 ± 0.7b, A, α 13 ± 1a, A, β 218 ± 7a, A, α 6.6 ± 0.3b, A, β 1351 ± 141a, A, α 457 ± 19a, A, α 893 ± 122a, A, α

Rf1 167 ± 22a, A, α 19 ± 3a, A, α 131 ± 14a, A, α 45 ± 7a, A, α 440 ± 64a, A, α 25 ± 2b, A, α 5.1 ± 0.2a, B, α 12 ± 1a, A, α 3.6 ± 0.1a, A, β 33 ± 3a, B, α 51 ± 6a, A, α 7 ± 1a, A, α 45 ± 6a, A, α 20 ± 2a, B, α 11.5 ± 0.3a, A, β 210 ± 22a, A, α 5.0 ± 0.3a, B, β 1273 ± 182a, A, α 429 ± 41a, A, α 844 ± 148a, A, α

Rf2 220 ± 54a, A 27 ± 8a, A 165 ± 35a, A 60 ± 21a, A 490 ± 95a, A 24 ± 3a, A 6.6 ± 0.6b, B 15.1 ± 0.3b, A 4.69 ± 0.09b, B 36 ± 2b, A 56 ± 5a, A 8.7 ± 0.6a, A 50.3 ± 0.4a, A 26.7 ± 0.2b, B 11.4 ± 0.3a, A 230 ± 3b, A 5.8 ± 0.1b, A 1437 ± 225a, A 476 ± 12b, A 961 ± 213a, A

C

non-λ

7 days

192 ± 12a, A 25 ± 2a, A 149 ± 2a, A 50 ± 3a, A 419 ± 15a, A 26 ± 1a, B 6.0 ± 0.4ab, A 13.6 ± 0.2ab, A 3.7 ± 0.2ab, A 33.8 ± 0.2ab, A 52 ± 2a, A 11 ± 2a, A 48 ± 2a, A 25 ± 1ab, A 11 ± 1a, A 211 ± 1a, A 7 ± 2ab, A 1283 ± 34a, A 449.0 ± 0.6ab, A 834 ± 34a, A

Rf1 186 ± 90a, A 20 ± 13a, A 142 ± 63a, A 47 ± 29a, A 419 ± 143a, A 22 ± 6a, A 4.8 ± 0.6a, A 11.8 ± 0.9a, A 3.3 ± 0.5a, A 29 ± 3a, A 50 ± 6a, A 7 ± 2a, A 43 ± 5a, A 19 ± 3a, A 10 ± 1a, A 201 ± 6a, A 4.2 ± 0.4a, A 1220 ± 358a, A 406 ± 20a, A 814 ± 338a, A

Rf2

193 ± 23a, α 21 ± 4a, α 148 ± 13b, α 48.2 ± 0.3a, α 453 ± 35b, α 19 ± 2a, α 5.45 ± 0.02a, β 12.7 ± 0.4b, α 3.59 ± 0.02b, α 33.4 ± 0.8b, α 50 ± 1c, α 8.4 ± 0.8a, α 46.6 ± 0.3b, α 27 ± 1b, β 10.3 ± 0.2b, α 221 ± 1b, α 5.2 ± 0.5a, α 1306 ± 84b, α 443 ± 8b, α 863 ± 76a, α

C

λ

155 ± 2a, α 16 ± 2a, α 121.8 ± 0.5ab, α 38 ± 2a, α 408 ± 9ab, α 22.9 ± 0.2a, α 5.4 ± 0.7a, α 11.0 ± 0.5a, α 3.60 ± 0.02b, α 32 ± 2b, α 46 ± 2b, α 6.9 ± 0.7a, α 42 ± 3b, α 20.0 ± 0.1a, α 9.6 ± 0.3ab, α 217 ± 5b, α 4.7 ± 0.8a, α 1159 ± 6ab, α 420 ± 14b, α 739 ± 8a, α

Rf1

137 ± 21a, α 15 ± 3a, α 107 ± 15a, α 37 ± 10a, α 349 ± 42a, α 19 ± 2a, α 4.6 ± 0.2a, α 10.2 ± 0.6a, α 3.17 ± 0.08a, α 26.0 ± 0.6a, α 41.4 ± 0.5a, α 6.3 ± 0.4a, α 35.8 ± 0.8a, α 18 ± 2a, α 9.3 ± 0.3a, α 187 ± 8a, α 3.7 ± 0.2a, α 1009 ± 85a, α 365 ± 6a, α 644 ± 92a, α

Rf2

All parameters are listed with their standard deviation (n = 3). For each anthocyanin, time (0 or 7 days), and light treatment (no-λ or λ), values with different lowercase letters mean significant differences between C and riboflavin doses treatment (p ≤ 0.05). Capital letters mean significant differences between time (0 and 7 days) in samples (C, Rf1 and Rf2) without blue-light irradiation. Greek characters mean significant differences between time (0 and 7 days) in samples (C, Rf1 and Rf2) irradiated with blue-light (λ).Nomenclature abbreviations: glc, glucoside; acglc, acetylglucoside; cmglc, coumaroylglucoside; cfglc, caffeoylglucoside.

Delphinidin-3-glc Cyanidin-3-glc Petunidin-3-glc Peonidin-3-glc Malvidin-3-glc Delphinidin-3-acglc Cyanidin-3-acglc Petunidin-3-acglc Peonidin-3-acglc Malvidin-3-acglc Delphinidin-3-cmglc Cyanidin-3-cmglc Petunidin-3-cmglc Peonidin-3-cmglc Malvidin-3-cis-cmglc Malvidin-3-trans-cmglc Malvidin-3-cfglc Total Total acylated anthocyanins Total non acylated anthocyanins

C

0 days

Table 1 Anthocyanins contents(mg/Kg) in grapes from control (C) grapevines and from grapevines treated with riboflavin at two doses, 100 g/ha (Rf1) and 200 g/ha (Rf2), at harvest (0 days), and one week later (7 days) with berries irradiated with blue-light (λ) or not (non-λ).

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73.56 ± 8.50b, B,β

67.41 ± 5.25b, B, β

54.88 ± 6.81b, B, β

37.12 ± 1.03c, B, β

27.20 ± 2.11b, B, β

19.87 ± 3.69b, B, β

32.85 ± 3.96a, A, α

32.24 ± 5.19a, A, α

30.59 ± 2.18a, A, α

19.19 ± 2.06a, A, α

17.43 ± 1.56a, A, α

11.39 ± 1.93a, A, α

10.42 ± 1.58a, A, α

Serine (Ser)

Tryptophan (Trp) Valine (Val)

Leucine (Leu)

Aspartic acid (Asp) Citrulline (Cit)

33.95 ± 0.40b, B, β

83.90 ± 4.90c, B, β

40.19 ± 3.15a, A, α

109.35 ± 17.23b, B, β 85.16 ± 5.33b, B, β

391.77 ± 43.04b, B, β 260.14 ± 57.80a, A, α 331.00 ± 24.31b, B, β 96.57 ± 2.16b, B, β

Rf1

Threonine (Thr) Histidine (His)

45.84 ± 10.46a, A, α

50.10 ± 12.72a, A, α

Glutamic acid (Glu) Alanine (Ala)

Glutamine (Gln) GABA

Proline (Pro)

196.31 ± 39.98a, A, α 158.70 ± 13.23a, A, α 155.84 ± 55.94a, A, α 56.06 ± 16.51a, A, α

Arginine (Arg)

C

0 days

12.69 ± 0.34ab, A, α

24.13 ± 4.16b, B, α

19.83 ± 1.71a, A, α

23.52 ± 1.44b, A, α

40.45 ± 2.06a, A, α

45.60 ± 1.434a, A, α

42.06 ± 0.61a, A, α

59.85 ± 2.52b, B, α

68.97 ± 5.26ab, B, α

16.40 ± 1.52a, A

14.20 ± 2.93a, A

35.95 ± 9.67b, A

38.40 ± 7.22b, A

48.23 ± 0.80b,B

49.23 ± 6.34b, A

56.46 ± 0.16a, B

62.44 ± 6.61b, B

52.61 ± 4.38b, A

72.41 ± 2.68a, A

59.73 ± 9.55a, A

69.45 ± 3.07ab, B, α 88.90 ± 1.73ab, A, α

186.11 ± 21.30b, A

180.23 ± 44.95a, A

284.56 ± 27.04a, A

C

204.26 ± 5.41a, B, β

291.02 ± 8.93ab, A, α 159.73 ± 1.57a, A, β

Rf2

non-λ

7 days

11.31 ± 0.37b, A

7.92 ± 0.18a, A

16.94 ± 1.28a, A

17.23 ± 1.61a, A

34.40 ± 0.73a, A

33.39 ± 2.27a, A

48.07 ± 3.14a, A

43.49 ± 1.28a, A

34.11 ± 2.69a, A

46.61 ± 4.04a, A

53.51 ± 9.66a, A

111.44 ± 11.70a, A

195.96 ± 21.87a, A

242.93 ± 8.94a, A

Rf1

10.61 ± 1.86b, A

9.60 ± 2.28a, A

16.47 ± 3.77a, A

16.99 ± 3.86a, A

33.28 ± 1.25a, A

29.68 ± 5.97a, A

42.82 ± 8.19a, A

39.81 ± 3.45a, A

34.86 ± 6.08a, A

54.69 ± 15.95a, A

45.34 ± 0.95a, A

119.86 ± 27.41ab, A

144.84 ± 8.14a, A

255.71 ± 8.13a, A

Rf2

11.48 ± 0.13a, α

7.91 ± 0.20a, α

18.00 ± 4.42ab, α

18.93 ± 3.08a, α

39.50 ± 1.73b, β

31.42 ± 2.22a, α

46.59 ± 0.14a, β

41.89 ± 1.22a, α

42.71 ± 1.78a, α

58.24 ± 0.49a, α

68.30 ± 0.59a, α

116.60 ± 2.48a, α

198.96 ± 9.81b, α

268.34 ± 7.14a, α

C

λ

409.85 ± 15.49b, β 97.21 ± 8.34a, α 152.55 ± 9.00b, α 123.80 ± 16.35b, β 66.65 ± 4.21b, α 76.23 ± 9.90b, α 66.75 ± 5.04b, α 53.77 ± 2.21b, β 43.68 ± 4.37b, α 49.71 ± 0.94c, β 28.26 ± 1.20b, α 26.14 ± 1.10b, β 12.51 ± 2.59a, α

Rf2

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9.04 ± 1.44a, α

8.70 ± 0.83a, α

13.71 ± 3.85a, α

14.81 ± 2.57a, α

34.66 ± 0.42a, α

25.03 ± 4.42a, α

41.39 ± 3.69a, α

39.24 ± 3.34a, α

33.41 ± 2.57a, α

53.79 ± 5.34a, α

46.92 ± 3.88a, α

85.42 ± 21.30a, α

175.08 ± 19.97b, α

237.54 ± 12.54a, α

Rf1

Table 2 Amino acids concentrations (mg/L) in musts from control (C) grapevines and from grapevines treated with riboflavin at two doses, 100 g/ha (Rf1) and 200 g/ha (Rf2), at harvest (0 days), and one week later (7 days) with berries irradiated with blue-light (λ) or not (non-λ).

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8.61 ± 1.29a, B, β 8.40 ± 0.90b, B, β

3.78 ± 0.71a, A, α 3.14 ± 0.79a, A, α 4.01 ± 0.35b, B, β 1774.04 ± 71.11b, B, β 1513.89 ± 128.92b, B, β

4.28 ± 1.39a, A, α 3.89 ± 0.53a, A, α

2.47 ± 0.31a, A, α 1.92 ± 0.37a, A, α 1.63 ± 0.36a, A, α 908.64 ± 179.76a, A, α 749.95 ± 166.53a, A, α

18.65 ± 1.98b, B, β

9.69 ± 1.59a, A, α

18.23 ± 0.71b, B, β

18.35 ± 0.87b, B, β

10.09 ± 0.51a, A, α

8.45 ± 2.01a, A, α

18.99 ± 0.89b, B, β

Rf1

10.14 ± 1.47a, A, α

C

0 days

2.92 ± 0.37a, A, α 1.90 ± 0.19a, A, α 2.26 ± 0.23a, A, α 1214.88 ± 21.19a, B, α 1055.15 ± 19.62a, B, α

4.26 ± 1.44a, A, α 6.48 ± 0.23b, B, α

11.18 ± 0.53a, A, α

12.51 ± 1.25a, A, α

10.56 ± 0.68a, A, α

12.35 ± 0.52a, B, β

Rf2

3.48 ± 0.40b, A 2.56 ± 0.11b, A 2.70 ± 0.08b, A 1243.96 ± 91.07a, A 1063.74 ± 46.12b, A

4.49 ± 0.24b, A 6.14 ± 1.41a, A

14.79 ± 2.09b, A

16.33 ± 2.44b, A

20.49 ± 6.48b, A

16.02 ± 5.81b, A

C

non-λ

7 days

751.00 ± 98.30a, A

2.44 ± 0.15a, A 1.45 ± 0.14a, A 2.06 ± 0.41ab, A 946.96 ± 142.04a, A

2.39 ± 0.12a, A 4.07 ± 0.14a, A

8.28 ± 0.49a, A

12.02 ± 0.55ab, A

9.17 ± 0.59a, A

7.47 ± 0.52a, A

Rf1

757.15 ± 96.10a, A

2.09 ± 0.12a, A 1.17 ± 0.60a, A 1.77 ± 0.06a, A 901.99 ± 87.96a, A

4.07 ± 0.35b, A 3.93 ± 0.34a, A

8.60 ± 1.80a, A

10.13 ± 1.33a, A

8.73 ± 2.09a, A

6.93 ± 0.94a, A

Rf2

2.55 ± 0.39a, α 1.87 ± 0.88a, α 2.21 ± 0.04b, α 1021.97 ± 13.4a, α 823.02 ± 23.20a, α

6.09 ± 0.53b, α 3.13 ± 0.15a, α

9.40 ± 1.08b, α

10.63 ± 0.42a, α

9.53 ± 2.10a, α

7.71 ± 0.97ab, α

C

λ

683.69 ± 59.40a, α

1.83 ± 0.74a, α 1.01 ± 0.53a, α 1.47 ± 0.38a, α 858.76 ± 79.38a, α

3.17 ± 0.68a, α 3.28 ± 1.07a, α

6.67 ± 0.26a, α

9.46 ± 1.55a, α

7.60 ± 1.84a, α

5.53 ± 0.70a, α

Rf1

4.19 ± 0.21b, α 2.82 ± 0.60a, α 3.41 ± 0.09c, β 1294 ± 55.58b, α 1197.1 ± 47.24b, α

7.57 ± 0.50b, α 5.80 ± 0.21b, α

12.07 ± 0.73a, α 15.74 ± 0.08b, α 10.35 ± 0.63b, α

15.79 ± 1.41b, α 9.41 ± 0.14b, α

Rf2

All parameters are listed with their standard deviation (n = 3). For each amino acid, time (0 or 7 days), and light treatment (no-λ or λ), values with different lowercase letters mean significant differences between C and riboflavin doses treatment (p ≤ 0.05). Capital letters mean significant differences between time (0 and 7 days) in samples (C, Rf1 and Rf2) without blue-light irradiation. Greek characters mean significant differences between time (0 and 7 days) in samples (C, Rf1 and Rf2) irradiated with blue-light (λ). *Nomenclature abbreviations: Total aas, total amino acids; Total aas without Pro, total amino acids without proline.

Total aas without Pro

Methionine (Met) Cysteine (Cys) Asparagine (Asn) Lysine (Lys) Ornithine (Orn) Glycine (Gly) Total aas*

Phenylalanine (Phe) Isoleucine (Ile) Tyrosine (Tyr)

Table 2 (continued)

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treated with the highest riboflavin dose (Rf2). In the case of Trp, Met, Cys, and Gly, their content was the lowest in the grapes treated with the lowest dose of riboflavin (Rf1). Moreover, Leu and Phe concentration was higher in Rf2 than in Rf1 samples, without significant differences with the control grapes. Regarding the effect of storage time in the samples without bluelight treatment (0 daysvs 7 days, non-λ), for the control grapes no significant differences were found in the most of the amino acids concentration, except for Thr, His, and Trp, which concentration was higher after 7 days of storage (Table 2). However, in the case of the lowest dose of riboflavin (Rf1), all amino acids and total amino acids with and without Pro were found in higher concentration at harvest (0 days) than after 7 days of storage, with the exception of Pro, Lys, and Orn, which did not show significant differences (Table 2). On the other hand, the content of Gln, GABA, Ala, Thr, Asp, Phe, Asn, total amino acids and total amino acids without Pro in Rf2 samples was higher at harvest than after one week of storage. Finally, regarding the effect of storage time for the samples irradiated with blue-light (0 daysvs 7 days,λ), in control grapes, the amino acids content was similar independent of the time, with the exception of His and Trp, which concentration was higher after 7 days of storage than at harvest (Table 2). In the case of Rf1 samples, all amino acids and total amino acids with and without Pro were found in higher concentrations at harvest (0 days) than after one week of storage (7 days), with the exception of Pro, Lys, and Orn content, which did not show significant differences (Table 2). In the case of Rf2 samples, the content of Pro, Gln, and Phe was higher at harvest than after 7 days of storage; while Arg, GABA, His, Trp, Leu, and Gly concentration was lower at harvest than after 7 days of storage, without significant differences for the rest of the amino acids (Table 2). From these results, several conclusions can be made: i) Blue-light alone causes a diminution of most amino acids concentration plus an overall decrease of total amino acids concentration in the control grapes; ii) riboflavin treatments in the absence of light also resulted in a notorious diminution of the amino acids concentration one week after harvest. No significant differences between Rf doses could be addressed; and iii) the combined use of Rf and blue-light has resulted in contrasting results depending on the riboflavin dose. At the lowest Rf dose, the total amino acids content was halved one week post-harvest as compared to the concentration values obtained at harvest time; at the highest Rf dose, however, the total amino acids content presented in 7 days post-harvest samples was increased as compared to the values obtained at harvest. Despite a marked effect of the riboflavin applications on the amino acids concentrations in grapes, these results point out to a highly complex effect whose outcome relies on the combination of factors, such as riboflavin concentration, storage time, and blue-light irradiation. Despite not being fully conclusive, these results provide further insight into the potential role of riboflavin treatments on grape in order to adapt amino acids concentration. Further studies would be necessary to deal more closely with parameters such as kinetics, the temperature of storage and the compounds concentration evolution. The use of physiological/biochemical markers together with the measurement of ROS in situ would be also future ideas to consider.

costs and contribute to sustainable and eco-friendly agriculture. The second step includes the application of blue-light via the LED source. The recent development of LED technologies presents an enormous potential for improving plant growth and making systems more sustainable (Darko, Heydarizadeh, Schoefs, & Sabzalian, 2014). In this respect, growth chambers and greenhouses are gradually being equipped with lighting technologies such as LED, which are substituting high-pressure sodium lamps. Among its advantages, LEDs can cover and control both fluence and wavelength requirements for different treatments (Darko et al., 2014; Islam et al., 2012) as it is in our case, where riboflavin absorption band was adequately matched by one of the irradiation channels of our LED source (Fig. 1a). Thus, the proposed combined treatment is price/quality/sustainably balanced and arguably not too complicated nor demanding in human labor means. Exposure to blue-light has been reported to increase the production of bioactive compounds, which, for fruit products and vegetables, can imply benefits for human health and overall improvement of wine quality. We have explored the effect of our treatment on anthocyanins, which had been described to be promoted by blue-light treatments in lettuce and kale (Carvalho & Folta, 2014; Li & Kubota, 2009), although not in our experimental design. Some other nutritionally important metabolites could be analyzed in future studies following this approach including chlorogenic acid (compound that decreases the risk of diabetes type II) or aliphatic glucosinolate (anticancer compound). Both metabolites have been reported to increase upon blue-light exposure in tomato and basil (for chlorogenic acid; Taulavuori, Julkunen-Tiitto, Hyöky, & Taulavuori, 2013) and sprouting broccoli (for aliphatic glucosinolate, among others; Kopsell, Sams, Barickman, & Morrow, 2014). Favourable effects of light quality on human health can also consist of decreased levels of unwanted components such as oxalate or nitrate, both reported to decrease when exposed to blue-light in vegetables such as lettuce or spinach (Ohashi-Kaneko, Takase, Kon, Fujiwara, & Kurata, 2007). The effect of the storage time on amino acids evolution (lowering) has been an unexpected result, but relevant to report. Other works have described that the days after the treatment can have a dismal effect on the overall outcome of the treatment. Thus, Cantos et al.(2000, 2001) showed how resveratrol content in grape skins increased dramatically two weeks after treatment especially for UVC, but also for UVB and even in the untreated samples. In some sense, oxidative stress induced a faster biosynthesis of several molecules of study including quercetin and caffeic acid derivatives. In this same study, the storage temperature effect was also evaluated. This phenomenon was also observed for stilbenes in different grape types (Guerrero et al., 2010). Interestingly, fluctuations depending on the time of measurement could be reported. Further studies would be necessary to assess the time effect both for aa and anthocyanins in our method. The temperature of storage, lamp power and the use of irradiation cycles would be potential alternatives for a better understanding of the system. 4. Conclusions The results point out that the impact on grape composition of the different treatments carried out in this work is highly complex and depends not only on the individual factors, but on its combination. The overall results might indicate that there is a cross-regulation between the combined treatment of photosensitizing Rf in combination with blue-light and content of anthocyanins and amino acidsin grapes and their evolution during the storage. Combined treatment resulted in an overall decreased in the content of anthocyanins in grapes after one week of storage, whereas the concentration of amino acids in the samples depended on the riboflavin dose applied and on the storage time.

3.3. Overall discussion and outlook This study opens a new pathway towards the use of a novel approach, which includes a two-step process. The first step consists of the supplementation in the grape leaves at veraison of riboflavin as an organic nitrogen source which is readily available, good for human health (Powers, 2003) and cheap when comparted to other elicitors with reported effects on grape (such as methyl jasmonate) as a consequence of the widespread and natural origin of Vitamin B2 (riboflavin). Moreover, the foliar application presents several advantages, such as a quick application and an efficient assimilation, which reduce 485

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Acknowledgements

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