Faiza Hammaz

Florence Charles

Rachel E Kopec

Charlotte Halimi

Salah Fgaier

Jawad Aarrouf

Laurent Urban

Patrick Borel
email: patrick.borel@univ-amu.fr

Temperature and storage time increase provitamin A carotenoid concentrations

Keywords: β-carotene, α-carotene, vitamin A deficiency, UV-C light, pulsed light, bioavailability. Chemical compounds studied Beta-carotene (PubChem CID: 5280489), Alpha-carotene

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1) Introduction

Vitamin A deficiency is still a major health issue in many developing countries [START_REF] Schmidhuber | The Global Nutrient Database: availability of macronutrients and micronutrients in 195 countries from 1980 to 2013[END_REF]. One strategy to fight this is to increase provitamin A carotenoid (proVA CAR) concentrations in fruits and vegetables commonly consumed in these countries.

This can be achieved by providing fruits and vegetable cultivars that are naturally rich in proVA CAR or genetically modified foods, such as golden rice and the super banana [START_REF] Tang | Golden Rice is an effective source of vitamin A[END_REF]Waltz, 2014), or by acting on environmental conditions to modify the nutrient and phytochemical composition of crops. Indeed, it has been shown that several pre-and post-harvesting practices (e.g. drought, UV radiation) can increase apparent concentrations of bioactives [START_REF] Atkinson | Enhancing beneficial antioxidants in fruits: a plant physiological perspective[END_REF], including proVA CAR [START_REF] Poiroux-Gonord | Health benefits of vitamins and secondary metabolites of fruits and vegetables and prospects to increase their concentrations by agronomic approaches[END_REF][START_REF] Saini | Significance of Genetic, Environmental, and Pre-and Postharvest Factors Affecting Carotenoid Contents in Crops: A Review[END_REF]. We specify "apparent" concentration because, in most cases, reports do not specify whether there was a true increase in concentration or if the plant structure modifications increased extraction of these compounds, leading to measurably higher concentrations during phytochemical quantification.

The orange carrot variety is one of the richest proVA CAR sources in the human diet and is widely consumed in several countries. Therefore, it is a very good candidate to identify environmental conditions that could increase its proVA CAR concentrations. In fact, it has been shown that both pre-and post-harvest factors can improve proVA CAR concentrations in carrots [START_REF] Seljasen | Quality of carrots as affected by pre-and postharvest factors and processing[END_REF]. Acting on pre-harvest factors, e.g. watering, has the advantage of being able to simultaneously modify the proVA CAR content of very large quantities of carrots. However, this can have consequences in terms of yield, and this may lead to an increase in the cost of sale. Therefore, we assume it will be easier for inhabitants of countries where there is still vitamin A deficiency, and who are mostly people with low income and low education, to modify some post-harvest factors of carrots stored by wholesalers, local retailors or even at home, than to advise/suggest to the cultivators to modify and control environmental conditions of their carrot culture. Therefore, we decided to focus on the effects of post-harvest factors on the concentration of proVA CAR in orange carrots.

There are several candidate post-harvest factors that could theoretically improve the proVA CAR concentration in carrots during storage [START_REF] Saini | Significance of Genetic, Environmental, and Pre-and Postharvest Factors Affecting Carotenoid Contents in Crops: A Review[END_REF]. Those which are, a priori, the simplest to use are storage time and storage temperature. Concerning storage time, it has been observed in several studies that carrot proVA CAR concentrations increase during the first two to three weeks of storage, then decrease [START_REF] Berger | Correlations of carotene with sensory attributes in carrots under different storage conditions[END_REF][START_REF] Brown | The effect of winter storage on the carotene content of carrot varieties[END_REF][START_REF] Howard | beta-carotene and ascorbic acid retention in fresh and processed vegetables[END_REF][START_REF] Imsic | Effect of Storage and Cooking on beta-Carotene Isomers in Carrots (Daucus carota L. cv. 'Stefano')[END_REF][START_REF] Lee | Changes in carotenoid content of carrots during growth and post-harvest storage[END_REF]. However, it is not known whether this increase is due to biosynthesis of proVA CAR during storage, or to better extractability of proVA CAR due to the slow degradation of the carrot matrix over time. Concerning the effect of storage temperature, there is surprisingly no clear conclusion. Although results presented in three studies suggest that it might modify proVA CAR concentration in carrots [START_REF] Berger | Correlations of carotene with sensory attributes in carrots under different storage conditions[END_REF][START_REF] Imsic | Effect of Storage and Cooking on beta-Carotene Isomers in Carrots (Daucus carota L. cv. 'Stefano')[END_REF][START_REF] Negi | Effect of low-cost storage and packaging on quality and nutritive value of fresh and dehydrated carrots[END_REF], it is not possible to conclude because these studies were not originally designed to answer this question. Ethylene and ambient oxygen concentrations are two additional candidate factors.

However, we chose not to study their effects because both have a negative impact on carrot organoleptic properties [START_REF] Carlton | Effects of ethylene & oxygen on production of a bitter compound by carrot roots[END_REF][START_REF] Kato-Noguchi | Effects of a low oxygen atmosphere on lactic fermentation in shredded carrot root tissues[END_REF][START_REF] Seljåsen | Effects of washing and packing on sensory and chemical parameters in carrots (Daucus carota L)[END_REF]. The last candidate factor we considered for improving the concentration of proVA CAR is treatment with artificial light. Indeed, it has been shown in several fruits and vegetables after harvest that different light treatments (i.e. UV-C, UV-B, high light, red light) with different time durations can significantly modify concentrations in several phytochemicals (Zhang, et al., 2015), including proVA CAR [START_REF] Poiroux-Gonord | Health benefits of vitamins and secondary metabolites of fruits and vegetables and prospects to increase their concentrations by agronomic approaches[END_REF]. For example, pulsed light successfully increased the concentration of proVA CAR in mango (de Almeida Lopes, Silva, Laurent, Charles, [START_REF] De Almeida Lopes | The influence of pulsed light exposure mode on quality and bioactive compounds of fresh-cut mangoes[END_REF][START_REF] Lopes | Low fluence pulsed light enhanced phytochemical content and antioxidant potential of 'Tommy Atkins' mango peel and pulp[END_REF]. While the mechanisms are not fully understood, it has been hypothesized that light irradiation can induce plants to produce antioxidant phytochemicals that participate in the defence against free radicals generated by these light radiations. However as previously suggested, this factor may also have an impact on the plant matrix, resulting in an increase in the extractability of antioxidants. Few studies are available concerning the effect of light treatments on proVA CAR concentrations specifically in carrots. Furthermore, in these studies the irradiation was performed on peeled or sliced carrots [START_REF] Aguiló-Aguayo | Impact of pulsed light on colour, carotenoid, polyacetylene and sugar content of carrot slices[END_REF][START_REF] Alegria | Freshcut carrot (cv. Nantes) quality as affected by abiotic stress (heat shock and UV-C irradiation) pre-treatments[END_REF] and proVA CAR quantification was performed on shredded carrots [START_REF] Aguiló-Aguayo | Impact of pulsed light on colour, carotenoid, polyacetylene and sugar content of carrot slices[END_REF].

Following these observations and hypotheses, the first step of this study consisted of assessing whether short exposure to light, provided either by UV-C irradiation or by pulsed light, can significantly increase proVA CAR concentrations in stored carrots. Then we tried to optimize the proVA CAR concentrations by varying the storage duration, the storage temperature, and the dose of artificial light. Finally, we assessed whether carrots with higher concentrations of proVA CAR resulting from modification of a post-harvest factor during storage had greater quantities of bioaccessible proVA CAR, i.e. micellarized proVA CAR following simulated gastrointestinal digestion, since this step is assumed to be a pre-requisite for proVA CAR absorption [START_REF] Borel | Factors affecting intestinal absorption of highly lipophilic food microconstituents (fatsoluble vitamins, carotenoids and phytosterols)[END_REF][START_REF] Desmarchelier | Overview of carotenoid bioavailability determinants: from dietary factors to host genetic variations[END_REF][START_REF] Reboul | Bioaccessibility of carotenoids and vitamin E from their main dietary sources[END_REF].

2) Materials and methods

Chemicals.

HPLC grade methanol, methyl tert-butyl ether (MTBE), dichloromethane, ethanol, nhexane, as well as HPLC grade H 2 O, were purchased from Carlo Erba reagents (Val de Reuil, France). HPLC standards including α-carotene, β-carotene and echinenone>95%, and digestive enzymes used in the in vitro digestion (i.e. pepsin 800-2.500 U/mg protein, pancreatin, and bile extract porcine), were obtained from Sigma Aldrich (France).

Assessment of the effect of UV-C and pulsed light treatments on proVA CAR concentrations.

In a first step, two irradiation devices, generating UV-C and pulsed light, were compared to evaluate their capacity to provoke an increase in proVA CAR concentrations in mature orange carrots (cultivar not specified) purchased from a local supermarket. UV-C treatments were performed in an enclosure system composed of 10 UV-C lamps (DSP UV-C tube, OSRAM HNL, 24 W) with an irradiation peak at 254 nm. Samples were placed on a quartz plate and irradiated with UV-C lamps located 20 cm above and below the samples. Each treatment lasted for 168 s to obtain a UV-C dose of 5 kJ/m 2 . Pulsed light was provided by Xenon lamps (Phoxène-Lumix S.R.L., Dardilly, France). The emitted spectrum ranged from UV-C to infrared. Whole carrots were placed onto the illumination area at 5 cm from the lamp and rotated manually (180°) in order to ensure both halves received even exposure to pulsed light. Each pulsed light flash lasted 500 µs, i.e. 2000 Hz frequency, and provided a dose of 5 kJ/m 2 on both carrot halves. After UV-C or pulsed light treatment, carrots were stored on nonhermetic plastic trays (above absorbent paper saturated with 20 mL of water) in a dark chamber at controlled temperature (i.e. 20 °C). The control batch was stored under the same conditions as the artificial light treated carrot batches. Sampling was performed after 4 days to evaluate the effect of both types of irradiation on the proVA CAR concentrations in the epidermis and the cortex of the carrots. Each testing batch included 5 carrots, and proVA CAR measurements were performed on each carrot sample three times, i.e. 3 technical replicates. Therefore, 15 measurements were performed per treatment condition.

Factorial design to clarify the role of post-harvest factors on proVA CAR concentrations.

To study the effect and the potential interactions between storage temperature, storage time, and dose of artificial light, a full factorial design was generated and tested using Trial Run 1.0 software (SPSS, Chicago, IL, USA) (Tyssandier, Lyan, & Borel, 2001). The design comprised 3 to 5 levels for each factor: 3 levels for storage time, i.e. 3, 6 and 10 days; 3 levels for storage temperature, i.e. 4, 20 and 30 °C; and 5 levels for artificial light energy, i.e. 0 (no pulsed light, control samples), 2, 5, 10 and 20 kJ/m 2 . According to the design, 45 randomized experiments, i.e. 45 batches of 5 carrots, were carried out.

The full factorial design experiment was conducted on the Daylance variety of orange carrots. Carrots were sorted and divided into 45 homogeneous batches, i.e. homogeneous in size, diameter, and color. Pulsed light experiments were conducted with the same equipment as described above, but with different energy doses per flash. The first untreated batch represented the control, the second batch was treated with 2 kJ/m 2 per flash; the third batch with 5 kJ/m 2 per flash, the fourth batch with 10 kJ/m 2 per flash; and the last batch with 20 kJ/m 2 per flash. Due to the setting, only the central part in length of the carrots (approximately 10 cm) was irradiated. After treatment, carrot batches were stored on non-hermetic plastic trays (above absorbent paper saturated with 20 mL of water) in a dark chamber at different temperatures, i.e. +4°C, +20°C and +30°C, and for different durations, i.e. 3, 6 or 10 days. At the end of each experiment, carrots were cut into 3 parts and the central part (10 cm) was stored at -80°C until further analysis. Note that we did not peel the carrots in this experiment and 3 extractions and proVA CAR measurements by HPLC were performed on each carrot (technical replicates).

Evaluation of the effect of high doses of pulsed light on the proVA CAR concentrations.

In this experiment, we used the Nantes carrot variety because Daylance, a winter variety, was no longer available. The pulsed light equipment was the same as described in the previous protocol and we used flashes of 20 kJ/m 2 . Carrots were treated with 1, 2, or 3 flashes to obtain 20, 40, or 60 kJ/m 2 , respectively. Non-treated carrots represented the control group.

After treatment, carrots were stored as previously described on non-hermetic plastic trays, and stored in the dark at 20°C for 3 or 7 days. As explained above, following each experiment nonpeeled carrots were cut in 3 parts and stored at -80°C until extraction and proVA CAR analysis in triplicate (technical replicates).

Measurement of proVA CAR bioaccessibility.

We compared two batches of carrots that had different proVA CAR concentrations because they were stored for different durations, i.e. 3 and 6 days. We used an in vitro digestion protocol adapted from [START_REF] Desmarchelier | The distribution and relative hydrolysis of tocopheryl acetate in the different matrices coexisting in the lumen of the small intestine during digestion could explain its low bioavailability[END_REF]. In summary, 2 g of carrot cut into small pieces were mixed with a meal consisting of 6.7 g mashed potatoes, 1.2 g ground beef, 200 µL of olive oil (all purchased from a local supermarket) and 32 mL of 0.9% aq. NaCl. The mixture was homogenized for 10 min at 37°C using gentle stirring (190 shakes per minutes). Then, 2.5 mL an artificial saliva solution was added, and the mixture further incubated for 10 min at 37 °C under gentle stirring. The pH was then adjusted to 4±0.02 with 1 M HCl. After the addition of 2 mL of pepsin solution, the mixture was incubated at 37°C for 30 min under gentle stirring. The pH was then adjusted to 6±0.02 with 0.9 M NaHCO 3 buffer before adding 9 mL of a pancreatin solution and 4 mL of a 10% bile solution. The mixture was further incubated for 30 min at 37°C using the same stirring. Aliquots of 4 mL of digesta were collected, and the remaining digesta centrifuged at 1200 x g for 1 h and 12 min at 10 °C. The recovered supernatant was filtered through a 0.8 µm and then a 0.22 µm syringe filter. The samples obtained were stored at -80 °C until proVA CAR extractions were made.

ProVA CAR extraction

Carrot proVA CAR (α-carotene and β-carotene) were extracted from 2 g of raw carrots first crushed with a knife mill (Grindomix GM 200, Retsh) under liquid nitrogen for 15 s and then homogenized in 50 mL of distilled water. A volume of 500 µL was taken to which 500 µL of echinenone (internal standard) diluted in ethanol was added. For micellar and digesta sample extractions, 2 mL sample was used. A double extraction with hexane was carried out (with two volumes of hexane per volume of the ethanol-sample mixture). After centrifugation at 1200 x g for 10 min at 4 °C, the hexane phases were pooled and evaporated under nitrogen gas until a dry film was obtained. Samples were redissolved in 200 µL of methanol/dichloromethane (65:35, v/v) for subsequent analysis by HPLC-DAD. Injection volumes of 50 µL (crushed carrots) and 100 µL (micelle samples and digestate samples) were used for HPLC analysis.

Quantification of ProVA CAR by HPLC-DAD

The proVA CAR (α-carotene and β-carotene) in the sample were identified due to consistent retention times and spectra of pure standards, and quantified at 450 nm. HPLC analyses were carried out using a gradient with the mobile phase consisting of (eluent A: methanol, eluent B: methyl tert-butyl ether and eluent C: H2O) [START_REF] Gleize | Simple and fast HPLC method for simultaneous determination of retinol, tocopherols, coenzyme Q(10) and carotenoids in complex samples[END_REF] at a flow rate of 1 mL.min -1 , on a YMC C30 column (250 mm x 4.6 mm; 5µm particle size) with a pre-column (5 µm particle size, 10 mm x 4 mm) and a constant temperature (35 °C) [START_REF] Gleize | Simple and fast HPLC method for simultaneous determination of retinol, tocopherols, coenzyme Q(10) and carotenoids in complex samples[END_REF].

Statistical analysis

Means ± SEM were obtained from 5 carrot samples. The value of each carrot sample was obtained from 3 technical replicates, i.e. extraction and HPLC measurement of 3 samples of the same carrot.

Statistical analyses of the factorial design study were based on the general linear model and used ANOVA. The relationships between the independent variables, i.e. storage time, storage temperature and doses of pulsed light, and the response variables, i.e. α-and β-carotene concentrations in carrots, were shown by surface response curves (SRC). These statistical analysis as well as the drawing of the SRC were performed by the Trial Run software (SPSS, Chicago, IL, USA).

The other results were compared as follows: unpaired Student's t-test to compare means obtained in two carrot batches, and ANOVA followed by post-hoc Tukey/Kramer tests to compare means obtained in more than 3 carrot batches. These statistical analyses were performed using StatView software version 5.0 (SAS Institute, Cary, NC, USA). In all cases, p<0.05 was considered statistically significant.

3) Results

Effect of UV-C and pulsed light treatments on α-carotene concentrations.

Figure 1 shows that UV-C treatment had no significant effect on α-carotene concentration either in the cortex (A) or in the epidermis (B). Conversely, pulsed light treatment significantly increased α-carotene concentration in the cortex tissue, +32% (p<0.05), but not in the epidermis.

Effect of UV-C and pulsed light treatments on β-carotene concentration.

Figure 1 shows that UV-C treatment had no significant effect on β-carotene concentration either in the cortex (C) or in the epidermis (D). Conversely, as observed for αcarotene, pulsed light treatment significantly increased β-carotene concentration in the cortex tissue, +26% (p<0.05), but not in the epidermis.

Effect of storage time and temperature on proVA CAR concentration.

Figures 2A and 2B

show the effect of storage time and storage temperature on the concentrations of αand β-carotene in the central part of non-peeled whole carrots. Note that the test of between-sample effects showed that both factors significantly and independently modified both proVA CAR concentrations ((p<0.03), Table 1). Furthermore, there were interactions between these two factors, which almost reached significance for α-carotene (p=0.052) and significant for β-carotene (p=0.008, Table 1). The surface response curves (SRC) suggest that the effect of temperature was greater than that of storage time. They also suggest that the effect of the two parameters were greater for β-carotene than for α-carotene.

Finally, note that the values shown after 10-day storage at 30 °C are only theoretical. Indeed, they were calculated by the experimental design software which extrapolated them from the experimental values. This is because most carrots started to rot at 30 °C after a few days, and thus we did not measure the proVA CAR concentrations in these samples.

Effect of storage time and pulsed light dose on proVA CAR concentrations.

Figures 2C and 2D

show the effect of both storage time and pulsed light energy on the concentrations of αand β-carotene in the central part of non-peeled whole carrots stored at 20 °C. Note that the test of between-sample effects showed that the dose of pulsed light significantly and independently modified both proVA CAR concentrations (p<0.005, Table 1).

Furthermore, there were interactions between the dose of pulsed light and the storage time, almost significant (p=0.061) for α-carotene and significant (p=0.005) for β-carotene (Table 1).

Concerning the SRC, the first observation is that, as observed in Figures 2A and2B, when no pulsed light was applied (front parts of the figures 2C and 2D SRC) the storage time increased both proVA CAR concentrations and this effect was higher for β-carotene than for α-carotene.

The second observation is that there was a significant and independent effect of the energy dose of pulsed light on both proVA CAR concentrations (p=0.001 and p=0.004 for α and β-carotene, respectively). Furthermore, this effect was not linear. Indeed, a distorted U-shaped curve was observed for β-carotene (Figure 2D) and a J-shaped curve for α-carotene (Figure 2C and2F. When no pulsed light was applied (front parts of the SRC), the storage time increased β-carotene concentration. This was not observed for α-carotene. There was a significant and independent effect of the energy dose of pulsed light on both proVA CAR concentrations (p=0.001 and p=0.004 for α and β-carotene, respectively).

Effect of high doses of pulsed light on proVA CAR concentrations.

Because results presented in Figures 2B and2C showed that the maximal effect of pulsed light was observed 3 days after treatment with the maximal dose used in this experiment, we decided to assess whether higher doses of pulsed light can further increase the concentrations in proVA CAR. Therefore, pulsed light doses of 20, 40, and 60 kJ/m 2 were applied and carrots were stored up to 7 days to assess whether we again observed an increase of proVA CAR after 3 days and a decrease between 3 and 7 days. The results of this experiment are shown in Figure 3. Note that the effects of the 60 kJ/m 2 dose are not shown because carrot epidermis appeared burnt 3 days after this treatment and thus we did not continue to make experiments with this condition. Conversely to what was observed in the previous experimental design (Figures 2B and2C), irradiating another variety of carrots with 20 kJ/m 2 pulsed light did not significantly modify proVA CAR concentrations after 3-day storage. There was also no effect of the higher dose of pulsed light (40 kJ/m 2 ) on these concentrations. Similar results were observed for α-carotene but with a lesser amplitude, as previously described in the experimental design.

Bioaccessibility of proVA CAR in carrots stored under different environmental conditions.

Figure 4 shows that the bioaccessibility of proVA CAR from carrots stored for 6 days at 20 °C, with greater concentrations in proVA CAR than carrots stored 3 days at the same temperature (Figures 2A to 2D), was higher than that in carrots stored for 3 days, i.e. 30.3% vs.

21.2% for α-carotene (p=0.03) and 26.9% vs. 18.4% for β-carotene (p=0.059), respectively.

4) Discussion

In the first part of this study, we aimed to assess whether a short treatment with two artificial light sources, i.e. UV-C and pulsed light, can significantly increase proVA CAR concentrations in whole intact carrots stored in a temperate environment, i.e. 20 °C. Because data from the literature suggest that both UV-C and pulsed light irradiation can increase the concentration of several phytochemical species in various fruits and vegetables (Fgaier, de Almeida Lopes, de Oliveira Silva, Aarrouf, & Urban, 2019; Urban, Charles, de Miranda, &

Aarrouf, 2016), we started by comparing the effect of these two irradiation methods. Because there are few studies on the effects of artificial light on proVA CAR content in whole carrots, and because data obtained in other crops have observed effects at very different energy levels, i.e. between 0.6 and 30 kJ/m² (Aguiló-Aguayo, Gangopadhyay, Lyng, Brunton, & Rai, 2017; [START_REF] Lopes | Low fluence pulsed light enhanced phytochemical content and antioxidant potential of 'Tommy Atkins' mango peel and pulp[END_REF][START_REF] Pataro | The influence of post-harvest UV-C and pulsed light treatments on quality and antioxidant properties of tomato fruits during storage[END_REF], we chose an intermediate energy dose, i.e. 5 kJ/m 2 . The results obtained herein clearly show that UV-C treatment failed to modify the concentrations in our targeted micronutrients, i.e. α-and β-carotene. We acknowledge that we assessed the effect of only one dose of UV-C, i.e. 5 kJ/m 2 , which was taken from previous studies showing that it led to an increase in CAR concentration in tomato [START_REF] Bravo | Effects of postharvest UV-C treatment on carotenoids and phenolic compounds of vine-ripe tomatoes[END_REF][START_REF] Liu | Effects of UV-C, red light and sun light on the carotenoid content and physical qualities of tomatoes during post-harvest storage[END_REF], and it is possible that other doses would have given different results. Indeed, a lower dose of UV-C (0.78 kJ/m 2 ) increased proVA CAR content in carrots (Alegria, Pinheiro, Duthoit, Gonçalves, Moldão-Martins, & Abreu, 2012), but peeled carrots were treated, and the proVA CAR content was measured in shredded carrots that were first stored at 0 °C and then at 5 °C. In short, these conditions were very different from the conditions described herein that aimed to mimic what could happen in retail supermarkets. In agreement with a recent study [START_REF] Aguiló-Aguayo | Impact of pulsed light on colour, carotenoid, polyacetylene and sugar content of carrot slices[END_REF], we observed that pulsed light treatment induced a significant increase in apparent proVA CAR concentration, thus we decided to focus on this light treatment in the full factorial design. Note that we specified the apparent concentration because we could not tell the difference between a true increase of proVA CAR concentration and an increase of proVA CAR extractability due to an effect of pulsed light on the food matrix facilitating extraction [START_REF] Lyan | Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method[END_REF][START_REF] Serino | HPLC assay of tomato carotenoids: validation of a rapid microextraction technique[END_REF]. The mechanisms that could explain these two potential phenomena are discussed later.

Nevertheless, the fact that pulsed light led to an increase in proVA CAR concentration in the cortex tissue but not in the epidermis is noteworthy. Indeed, since carrots are generally peeled before consumption, prior pulsed light treatment would still be an effective way to increase their proVA CAR content.

Since previous studies have suggested that storage time [START_REF] Brown | The effect of winter storage on the carotene content of carrot varieties[END_REF][START_REF] Howard | beta-carotene and ascorbic acid retention in fresh and processed vegetables[END_REF][START_REF] Imsic | Effect of Storage and Cooking on beta-Carotene Isomers in Carrots (Daucus carota L. cv. 'Stefano')[END_REF][START_REF] Lee | Changes in carotenoid content of carrots during growth and post-harvest storage[END_REF] and storage temperature [START_REF] Berger | Correlations of carotene with sensory attributes in carrots under different storage conditions[END_REF][START_REF] Imsic | Effect of Storage and Cooking on beta-Carotene Isomers in Carrots (Daucus carota L. cv. 'Stefano')[END_REF][START_REF] Negi | Effect of low-cost storage and packaging on quality and nutritive value of fresh and dehydrated carrots[END_REF], can modulate proVA CAR concentrations in carrots, we also aimed to assess the interaction effects between pulsed light and these factors on proVA CAR concentrations. The results confirmed that pulsed light treatment can significantly increase the concentrations of the two proVA CAR in whole carrots. They also showed that the two other candidate factors can significantly and independently increase these concentrations.

Concerning the effect of the storage temperature, we observed that proVA CAR concentrations increased with temperature, i.e. between 4 °C and 30 °C for β-carotene and between around 9 °C and 30 °C for α-carotene. Although several studies have been devoted to the evaluation of the effect of carrot storage duration on proVA CAR concentrations, only two studies tested different storage temperatures [START_REF] Berger | Correlations of carotene with sensory attributes in carrots under different storage conditions[END_REF][START_REF] Imsic | Effect of Storage and Cooking on beta-Carotene Isomers in Carrots (Daucus carota L. cv. 'Stefano')[END_REF]. [START_REF] Berger | Correlations of carotene with sensory attributes in carrots under different storage conditions[END_REF] found a decrease of proVA CAR concentrations during storage at -18 °C and -25 °C and an increase at 4 °C and 20 °C. However, data was not provided to facilitate comparison of the increase at 4 °C and 20 °C for the same storage time. Imsic and colleagues [START_REF] Imsic | Effect of Storage and Cooking on beta-Carotene Isomers in Carrots (Daucus carota L. cv. 'Stefano')[END_REF] observed a higher increase at 20 °C as compared to 4 °C, but the storage times were different, i.e. 14 and 3 days, respectively. Thus, to our knowledge, this is the first time that a significant positive relationship has been observed between the storage temperature and the concentration of proVA CAR in carrots. Note that we did not measure the effect of storage temperature at 30 °C because carrots started to rot (see Results), thus the values shown in the SRC were calculated by the software. Nevertheless, it is likely that the optimal storage temperature to maximize the proVA CAR concentrations is higher than 20 °C, but it must be balanced with the risk of rotting and the decrease in organoleptic properties [START_REF] Seljåsen | Effects of washing and packing on sensory and chemical parameters in carrots (Daucus carota L)[END_REF], which increase with the storage temperature. Our observations suggest that the optimal temperature is between 20 °C and 30°C, depending on other post-harvest conditions, e.g. air humidity and storage duration.

The full factorial design experiment showed that proVA CAR concentrations increase with storage time, with 10 days as the longest duration tested, in agreement with several studies [START_REF] Berger | Correlations of carotene with sensory attributes in carrots under different storage conditions[END_REF][START_REF] Brown | The effect of winter storage on the carotene content of carrot varieties[END_REF][START_REF] Imsic | Effect of Storage and Cooking on beta-Carotene Isomers in Carrots (Daucus carota L. cv. 'Stefano')[END_REF][START_REF] Lee | Changes in carotenoid content of carrots during growth and post-harvest storage[END_REF]. Furthermore, between these publications, this effect has now been observed in at least 9 different carrot varieties, and thus we consider this to be a well demonstrated phenomenon. Nevertheless, it is important to state that this increase is transitory because, in most studies, it was followed by a continuous decrease in the concentration in proVA CAR when the storage was extended. The storage time at which this decrease starts varies among studies, from 7 days [START_REF] Berger | Correlations of carotene with sensory attributes in carrots under different storage conditions[END_REF] to 2 weeks [START_REF] Howard | beta-carotene and ascorbic acid retention in fresh and processed vegetables[END_REF] and even 21 days [START_REF] Imsic | Effect of Storage and Cooking on beta-Carotene Isomers in Carrots (Daucus carota L. cv. 'Stefano')[END_REF]. We hypothesize that these differences are due to other post-harvest conditions, e.g. storage temperature or humidity, as well as to different carrot varieties.

The mechanisms that could explain the effect of storage time and storage temperature on proVA CAR concentrations have not yet been identified, but two hypotheses have been suggested (Figure 5). The first one is that the cell wall of the carrot matrix is gradually disaggregated by enzymes, e.g. cellulases and hemicellulases, during storage [START_REF] Marx | Effects of thermal processing on trans-cisisomerization of beta-carotene in carrot juices and carotene-containing preparations[END_REF] and this disaggregation allows the CAR to be better extracted from the carrot matrix in the first stage of extraction, which most often consists of using organic solvents for proVA CAR removal from the vegetable matrix [START_REF] Lyan | Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method[END_REF]. It is further reasonable to hypothesize that this enzymatic disaggregation is enhanced by higher temperatures, which would also explain the positive effect of temperature on proVA CAR concentrations. However, the fact that proVA CAR concentration first increased during storage then decreased [START_REF] Berger | Correlations of carotene with sensory attributes in carrots under different storage conditions[END_REF][START_REF] Howard | beta-carotene and ascorbic acid retention in fresh and processed vegetables[END_REF][START_REF] Imsic | Effect of Storage and Cooking on beta-Carotene Isomers in Carrots (Daucus carota L. cv. 'Stefano')[END_REF] suggests that another mechanism is involved. Other molecules can be degraded during storage, especially antioxidants that are the more labile. Therefore, there is likely a time when the CAR degradation rate becomes higher than the extractability "boost" provided by matrix disaggregation that ultimately improves proVA CAR concentrations, explaining this biphasic curve. The second hypothesis to explain the increase of proVA CAR concentrations as a function of time and temperature is that proVA CAR are still being synthesized in the carrots during storage. Indeed, CAR biosynthesis after harvest has been suggested in many climacteric fruits, e.g. tomatoes or mangoes, as well as in tuberous vegetables, such as sweet potatoes. The hypothesis that carrots continue to biosynthesize proVA CAR [START_REF] Rodriguez-Concepcion | Biosynthesis of carotenoids in carrot: an underground story comes to light[END_REF] after harvest is supported by two observations. First, there is a continuous increase of β-zeacarotene and ɤ-carotene, which are precursors of β-carotene in the biosynthetic pathway, in carrots during storage [START_REF] Lee | Changes in carotenoid content of carrots during growth and post-harvest storage[END_REF]. Secondly, treatment of carrots with 2-(4chlorophenylthio)triethylamine HCl, which is a pesticide that inhibits CAR synthesis, results in reduction of proVA CAR concentration [START_REF] Lee | Changes in carotenoid content of carrots during growth and post-harvest storage[END_REF]. Therefore, there are arguments supporting both hypotheses, and in fact both mechanisms may be at play concomitantly (Figure 5).

Regardless of the mechanism(s) involved, the net result is equally of interest from a nutrition perspective. Indeed, greater extractability of proVA CAR would likely lead to a higher quantity of bioaccessible CAR [START_REF] Desmarchelier | Overview of carotenoid bioavailability determinants: from dietary factors to host genetic variations[END_REF], and greater proVA CAR synthesis would also lead to more bioaccessible proVA CAR. This is the reason why we have compared the quantity of bioaccessible proVA CAR in carrots that exhibited different apparent proVA CAR concentrations in the last part of this study.

The interactions between the dose of pulsed light and the storage time (Figures 2C and2D) was intriguing. Although the highest dose of pulsed light led to a dramatic increase of proVA CAR concentrations after 3 days, these high concentrations significantly decreased upon the storage time. We hypothesize that pulsed light treatments induced the production of free radicals in the carrots and the carrots responded by synthesizing antioxidant phytochemicals to protect their tissues from free radical induced damage [START_REF] Pataro | The influence of post-harvest UV-C and pulsed light treatments on quality and antioxidant properties of tomato fruits during storage[END_REF]Urban, Sari, Orsal, Lopes, Miranda, & Aarrouf, 2018). Then, these newly synthesized antioxidants, including the proVA CAR, were eliminated by free radicals. Another hypothesis could be that pulsed light treatment modified the carrot matrix leading to a better extractability of the proVA CAR. Nevertheless, this hypothesis is less likely because it implies that the matrix modifications induced by the initial treatment with pulsed light were gradually repaired by the carrot during storage, which seems unlikely.

Results of the full factorial design experiment raised new questions about the effect of pulsed light treatment on proVA CAR concentrations. Indeed, they suggest that higher doses of pulsed light might further increase proVA CAR concentrations, especially during short storage times. Therefore, we performed a third experiment where we compared the effect of higher doses of pulsed light, i.e. 40 and 60 kJ/m 2 , than the dose demonstrating maximal effect in the full factorial design, i.e. 20 kJ/m 2 . Unfortunately, this third experiment did not reproduce the effect observed at 20 kJ/m 2 on proVA CAR concentrations after 3-days storage. No effect was observed at 40 kJ/m 2 either. Finally, 60 kJ/m 2 pulsed light led to a burning of carrot epidermis.

We hypothesize that our inability to reproduce the results obtained in both the first experiment and in the full factorial design was due to the difference in cultivars used. Cv. Daylance was used in the full factorial design, while cv. Nantes was used in the third experiment (as explained in the Material & Methods section). Furthermore, it has been shown that cv. Nantes carrots respond to light by decreasing proVA CAR concentration [START_REF] Llorente | Illuminating colors: regulation of carotenoid biosynthesis and accumulation by light[END_REF] while there is no data for the cv. Daylance cultivar.

Therefore, pulsed light treatment may not necessarily be effective, and/or its effect might only be transitory, as suggested by the results of the full factorial design. Taken together, we would not yet advise the use of this external stimulant to improve proVA CAR concentrations in stored carrots. Nevertheless, the data obtained suggest that this factor has a great potential to improve proVA CAR concentrations in stored carrots, and further studies are required to obtain a reproducible and lasting effect.

The last part of this study was dedicated to assess whether higher concentrations of proVA CAR induced by modifications of post-harvest factors during storage could lead to higher quantities of bioaccessible proVA CAR. In order to answer this question, we compared proVA CAR bioaccessibility in the carrots stored for 6 days at 20°C with the carrots stored for 3 days at the same temperature. The results obtained, i.e. the observation that the carrots stored 6 days had higher proVA CAR bioaccessibility than the carrots stored 3 days, confirm that higher apparent concentrations in proVA CAR translate in higher quantities of bioaccessible proVA CAR, although the mechanism(s) are not yet known.

In summary, pulsed light treatment, storage temperature and storage time can independently and additively improve apparent proVA CAR concentrations in carrots.

Nevertheless, in our experiments the effect of pulsed light was only transitory and nonreproducible in different carrot cultivars, indicating that further studies are required to overcome inconsistencies in these results. Currently, we suggest that the easiest and most sustainable way to improve the quantity of bioaccessible proVA CAR in whole stored carrots is to manipulate storage temperature and storage time. More precisely, we suggest that it is better to store whole carrots at the highest possible temperature to best improve apparent proVA CAR concentration. Obviously, the temperature should not be too high to avoid decreasing organoleptic properties, and avoid carrot rotting. Therefore, we recommend room temperature, e.g. 20 °C to 25 °C, rather than cool temperatures, e.g. 4 °C to 8 °C. Concerning the storage duration, these results suggest it is best to store the whole carrot between 1 and 2-3 weeks after harvest. Indeed, it has been shown that carrots were marketable for 18 days when stored between 17 °C and 21°C [START_REF] Negi | Effect of low-cost storage and packaging on quality and nutritive value of fresh and dehydrated carrots[END_REF]. Obviously, the longer the storage duration, the lower the storage temperature should be to avoid negative organoleptic effects and carrot spoilage. Urban, L., Sari, D. C., Orsal, B., Lopes, M., Miranda, R., & Aarrouf, J. (2018). UV-C light and pulsed light as alternatives to chemical and biological elicitors for stimulating plant natural defenses against fungal diseases. Sci. Hortic-Amsterdam, 235, 452-459. Waltz, E. (2014). Vitamin A Super Banana in human trials. Nature Biotechnology, 32(9), 857. Zhang, L., Ma, G., Yamawaki, K., Ikoma, Y., Matsumoto, H., Yoshioka, T., et al. (2015). Effect of blue LED light intensity on carotenoid accumulation in citrus juice sacs. Journal of Plant Physiology, 188, 58-63.

  figures 2C and 2D SRC). In fact, after 10 days of storage, α-carotene concentrations were

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Pairwise surface response curves of α-and β-carotene concentrations in stored

Figure 3 :

 3 Figure 3: Effect of pulsed light dose and storage time at 20 °C on α and β-carotene concentrations in stored carrots (cv. Nantes). D3: after 3-day storage. D3 after 20 kJ/m 2 : carrots irradiated with 20 kJ/m 2 pulsed light and then stored 3 days. D3 after 40 kJ/m 2 : carrots irradiated with 40 kJ/m 2 pulsed light and then stored 3 days. D7: after 7-day storage. D7 after

Figure 4 :

 4 Figure 4: ProVA CAR bioaccessibility in carrots stored either 3 or 6 days at 20 °C. ProVA

Figure 5 :

 5 Figure 5: Mechanistic hypotheses to explain the apparent increase in the concentration of

  R squared = 0.424 (adjusted R squared = 0.273). b R squared = 0.525 (adjusted R squared = 0.395). Statistical analyses were based on the general linear model and used ANOVA.Independent variables were storage time (days), storage temperature (°C) and doses of pulsed light (kJ). Response variables were α-and β-carotene concentrations in carrots.

	d x °C	1.303	3	0.434	4.094	0.008
	d x kJ	2.439	8	0.305	2.873	0.005
	°C x kJ	1.990	8	0.249	2.345	0.021
	d x °C x kJ	3.373	12	0.281	2.649	0.003
	Error	15.170	143	0.106
	Total	358.763	183
	Corrected total	31.908	182

a

Acknowledgments:

The authors thank Dr. Charles Desmarchelier for his very constructive critical reading of the manuscript. The authors thank also the Phoxène company of Dardilly, France, for supplying the pulsed light system used for organ treatments in this study.

Supporting information:

This project received funding from SFR Tersys, Avignon University, France.

Credit author statement:

Conflicts of interest:

None of the authors reported a potential conflict of interest.

i.e. α-and β-carotene, are represented by the abbreviation βC. A. In this scheme it is assumed that the different factors increase the biosynthesis of proVA CAR during storage. B. In this scheme it is assumed that the different factors accelerate the degradation of the plant matrix during storage, ultimately improving the bioaccessibility of the proVA CAR. C. In this scheme both mechanisms are involved. For example, an effect of the temperature on the plant matrix and time and temperature on biosynthesis is represented, but all possible combinations of effects both on the biosynthesis and on the plant matrix are possible.