Influence of Light Exposure during Cold Storage of Minimally Processed Vegetables ( Valeriana sp . )

Minimally processed vegetables are sensitive to leaves discoloration and quality deterioration, and these negative aspects can aect their shelf life. During the manufacturing processing,Valeriana leaves were submitted to dierent dipping acid solutions and then were stored at cold temperature in dark and in light to study the inuence of the light exposure on their quality in terms of total free acidity, dry matter percentage, colour, water activity, total bacterial count, antioxidant components, and total antioxidant activity. e results suggest that dark storage is recommended for a better physicochemical quality of Valeriana leaves. In particular, light inuenced signicantly the browning index and hue angle with a signicance level of p< 0.01 and p< 0.05, respectively, whereas both exposure conditions aected total bacterial count and pH (p< 0.01). Dipping treatments had an eect on pH, aw, and total bacterial count, while the storage time inuenced with highly signicant dierences the most of the studied parameters, except for the antioxidant compounds.


Introduction
e quality of minimally processed or ready-to-eat vegetables depends mainly on external and internal parameters.Quality changes are due to the product quality at the harvesting and storage conditions during the postharvest period.External parameters, such as the colour, are very important in the leafy vegetables because of the appearance and the capacity to promote the purchasing by the consumer [1].
Usually, leafy vegetables are sold within about 7 days after postharvest treatment, and packaging is aimed to maintain external colour, reducing browning of tissue and improving shelf life.In particular, when the typical green colour of leafy tissues was lost, browning and degradation of carotenoids, chlorophyll, and total phenols take place.Moreover, the yellowing of tissue is a natural discoloration due to the senescence of the leaves successive to the light exposure of the product.For these reasons, the quantication of total pigments and phenols is also very important to determinate the antioxidant activity of vegetables.Moreover, phenolic compounds show variable evolution during the storage, without signi cantly reducing their content up to an advanced storage [1].Recently, the e ect of light exposure on quality and physiology of fresh fruits and vegetables was studied [2][3][4][5][6][7].One of the most important parameters during the storage is temperature: 4 °C is considered useful to preserve the quality of leafy vegetables, as reported in the literature [8,9].Furthermore, speci c pretreatments with acids, such as citric and ascorbic acids, during the leafy dipping process, can improve shelf life and minimise the colour changes.Zhu et al. [10] found that the combination of ascorbic and citric acids was indeed e ective in slowing down the rate of enzymatic browning.In particular, citric acid (C 6 H 8 O 7 ) is of interest for this purpose, since it is inexpensive; so it is widely used as an acidulant agent in food industry [11].It also proved to promote the retention or even the enhancement of various physical properties of foods, such as colour and texture [10].
e objective of this study is to investigate the in uence of light exposure and darkness on physical and chemical qualitative characteristics during the cold storage by monitoring the colour changes (tissue browning/yellowing), the antioxidant activity, and the hygienic state of the product during the storage.

Materials.
Fresh leaves of Valeriana (Valeriana locusta L.) were purchased from a local market, stored at 4 °C, and successively subjected to different treatments by dipping solutions in a factory (COF SpA, Vibo Valentia) prior to spin-dry and package.e samples were named as follows: T1, control sample dipped in tap water for 5 min; T2, sample dipped in 1% (w/v) citric acid; and T3, sample dipped in 0.5% (w/v) citric acid and 0.5% (w/v) ascorbic acid.e samples were packaged (125 g of weight) in conventional polypropylene antifog bags (25 cm × 20 cm of size, 35 μm of thickness, OTR: 1600 cm 3 •m −2 24 h −1 •atm −1 according to ASTM D3985, and WVTR: 6 g•m -2 24 h −1 according to ASTM F1249) and immediately transported to the laboratory at Mediterranea University of Reggio Calabria where they were stored at 4 °C in darkness and light in fluorescence tubes that simulated the commercial exposure of minimally processed vegetables.e fluorescent lights were obtained from three lights (8 W and 430 lm for each light) installed in the storage thermoincubators (950 lux measured by means of an ITT Metrix instrument, Annecy, France).e distance between the sample and light was about 75 cm with a similar illuminance for all samples during storage.e physicochemical and microbiological analyses were performed at zero, three, seven, ten, fourteen, and sixteen days over the recommended seven days by the producer.Two replicates were done for each of the three treatments.
e quality of vegetables dipped in acid solutions was compared with that of vegetables dipped in water.

Headspace Analysis.
Prior to opening the bags, headspace gas composition, expressed as oxygen and carbon dioxide percentages, was determined using the CheckPoint handheld gas analyser (PBI Dansensor Italia Srl, Milan, Italy).
e gas sample was taken with a needle inserted through a septum placed on the packages.

Preparation of Extracts.
After opening the bags, the leaves were homogenised in a common blender, and 5 g of the sample was added to 50 ml of distilled water.e contents were mixed in a stomacher and centrifuged at 5,000xg for 5 min.Supernatant solution was collected, and residues were reextracted.
e two supernatant solutions were filtered through a paper and combined for the chemical analyses.

Physicochemical Analysis.
For the extracts, titratable acidity, expressed as % of citric acid g −1 , and pH (with a pH meter Crison GLP, Barcelona, Spain) were measured according to the AOAC method [12].Dry matter (% d.m.) was evaluated by loss in weight in an oven at 70 °C to constant weight, and water activity (a w ) was measured by means of the AquaLab LITE (Decagon, Inc., Washington, USA) instrument.
Determination of colour was referred to the CIELAB colour space and performed for the parameters L * (lightness), ranging from 0 (black) to 100 (white), a * ranging from 60 (red) to −60 (green), and b * ranging from 60 (yellow) to −60 (blue).e colour space parameters were measured on ten points of the leaves surface by using a tristimulus colorimeter (model CM-700d, Konica Minolta, Osaka, Japan).e mean of the measurements and standard deviation were reported.
e hue angle (H °) describes the relative amounts of redness and yellowness where 0 °/360 °is defined for red/magenta, 90 °for yellow, 180 °for green, and 270 °for blue or purple, or intermediate colours between adjacent pairs of these basic colours [13,14].Hue angle was calculated from a * and b * values according to the following formula according to Wrolstad and Smith [15]: e browning index is an important parameter to define browning in the foods.It represents the purity of brown colour and is calculated using L * , a * , and b * according to Mohammadi et al. [16]: browning index(BI) � 100(x − 0.31) 0.17 where 2.5.Microbiological Analysis.Total bacterial count (TBC) was examined on the samples.At each sample time point (0, 3, 7, 10, 14, and 16 days), ten grams of Valeriana were diluted with the sterile Ringer's solution in a stomacher bag filter and homogenised in the BagMixer (Interscience, France) for 5 min, and decimal serial dilutions were prepared and plated on Petri plates.TBC was enumerated on PCA (Plant Count Agar) growth land (Oxoid) at 26 °C for 48 h and was expressed as log 10 CFU g −1 .

Total Phenolic Content and Antioxidant Activities.
Valeriana leaves were homogenised in a common blender, and 5 g of the sample was added to 25 mL of methanol : water (80 : 20, v : v), mixed, and then centrifuged at 10,000xg for 10 min according to the method of Zhan et al. [17] with some modifications.Supernatant solution was collected, and residues were reextracted.e two supernatant solutions were filtered through syringe filters (0.45 µm Chromafil RC-45/25) and combined for subsequent analyses.
e method of Singleton and Rossi [18] was followed to determine total phenolic content.100 µL of the extract was analysed spectrophotometrically at 760 nm after reaction with the Folin-Ciocalteu reagent.e results are reported as mg gallic acid g −1 .
e Trolox equivalent antioxidant capacity (TEAC) was determined using the ABTS + radical cation decolourisation assay, according to the method of Re et al. [19].25 µL of the sample extract reacted with 2975 µL of ABTS solution for 2 Journal of Food Quality 6 min in the dark.e absorbance was measured at 734 nm. e TEAC of extracts was expressed as µM•TE•g −1 .Also, DPPH radical-scavenging activity was determined according to the method of Brand-Williams et al. [20].50 µL of the sample extract reacted with 2950 µL of DPPH solution for 15 min in the dark.e absorbance was measured at 515 nm.
e antioxidant capacities of extracts were expressed as percentage of inhibition according to the following formula: where A t0 is the value of absorbance of DPPH solution at initial time, while A tend is the value of the absorbance measured after fifteen minutes.

Statistical Analysis.
All experiments were performed in duplicate.e effects of the light, treatment methods, and storage time were evaluated by statistical analysis of variance (one-way ANOVA and multivariate analysis) using the SPSS software (version 15).All data were presented as mean values and standard deviations.Tukey's multiple range test was used to evaluate differences among values, and the statistical significance was defined as p < 0.05.

Results and Discussion
Changes in atmosphere composition (O 2 % and CO 2 %) in all packaged samples are shown in Figure 1.During the storage, O 2 % was influenced significantly by light exposure (p < 0.05).Instead, CO 2 % was affected by treatment, with p < 0.01, and it showed statistical differences among samples by the post hoc test after three, seven, and ten days of storage (statistical data not shown).Up to ten days of storage, the changes in atmosphere composition could be achieved by natural tissues respiration.en, CO 2 clearly increased for tissues respiration, as confirmed by Alegria et al. [21], as well the bacterial growth.O 2 percentage decreased for up to ten days, and then, its concentration inside the packaging increased with percentages always lower than the initial one.is aspect cannot be necessarily related to tissues respiration, but it may be probably due to the gas permeability of the used packaging material because of a humidity change around the package after ten days of storage, as reported by Kader et al. [22]: temperature, relative humidity, and air movement around the package can influence the permeability of the film.No correlation between TBC of samples and O 2 concentration inside the packages was found.In fact, TBC was correlated after sixteen days with water activity as demonstrated by Pearson's correlation (r � 0.660 and p < 0.05 in dark condition and r � 0.928 and p < 0.05 in light condition).
e light exposure of the samples influenced TBC with a significance of p < 0.01 by multivariate statistical analysis, and also, highly significant differences were found among treatments and at each monitoring time from three days of storage in light (Table 1).e graph (Figure 2) shows both an acidic increase and a microbial increase during storage days.
After sixteen days of storage, the highest total bacterial count was found in dark conditions in the T1 sample with 6.75 ± 0.00 log 10 CFU g −1 and in light conditions in the T3 sample with 7.22 ± 0.00 log 10 CFU g −1 .
e sample, treated with citric acid (T2), stored in darkness, was so hygienically better than the same sample stored in light for the lowest TBC (5.93 ± 0.02 log 10 ).Multivariate statistical analysis demonstrated that treatments significantly influence TBC (p < 0.01) as also confirmed in the literature: citric acid, as the solute in washing solutions, contributes to contain the microbial growth in carrots [23].Ascorbic acid is instead more suggested to retain nutritive compounds, as carotenoids [24].
Changes in physicochemical parameters during the storage are given in Tables 2 and 3, respectively, in dark and light conditions.An increment in titratable acidity, expressed as percentage of citric acid, was observed during the time, with highly significant differences among the treatments at the final storage day in light condition (p < 0.01; Figure 2).As titratable acidity increased, the pH tended to decrease.e highest acidity was found in the untreated sample (T1) with 0.19 ± 0.02% of citric acid and pH of 6.06 ± 0.13 in darkness and 0.24 ± 0.00% of citric acid and pH of 6.07 ± 0.24 in light, respectively (Table 2).Light exposure did not affect the total acidity during the storage, but it showed a major effect on the pH values as confirmed by multivariate statistical analysis (p < 0.01).
In samples dipped in acid solutions, dry matter was higher compared with that in T1 after sixteen days of storage (6.78 ± 0.02% in T2 and 6.85 ± 0.17% in T3 in light condition).As reported in the literature by Hiranvarachat et al. [23], this is probably due to the properties of the weak citric acid, which loosen the cell wall structure.
e browning index and hue angle were calculated by using Equations ( 1)-(3), and the results are shown in Tables 2  and 3. e browning index measured on samples indicated a very slow rate of occurrence of enzymatic browning, as just reported by Kortey et al. [13] in a study on mushroom colour during storage.Moreover, the light exposure significantly influenced the BI (p < 0.01) and hue angle (p < 0.05) by multivariate analysis of variance.e trend of BI is illustrated in Figure 3 as the ratio between the value at the given storage time and the initial value (BI t /BI 0 ).e T2 sample, treated with citric acid, showed a decrease in terms of BI t /BI 0 .BI increased in the sample dipped in water (T1) with significant differences between darkness condition (38.30 ± 9.05) and light condition (41.30 ± 9.16) at the final storage time (p < 0.05).e storage in light involved different browning indexes in samples differently treated; in particular, it was observed after three and ten days (p < 0.05).Hue angle decreased during storage time in all samples without statistical differences.e decrease was expected in green leafy vegetables as in the lettuce surface studied by Peiser et al. [25].e hue angle range was within the 130 °region which indicates an apparent green colour.In the T1 sample, it tended to decrease during the time near the 90 °region: it suggests leaves yellowing at the end of storage, with 108.03 °± 5.20 in darkness condition and 109.43 °± 5.18 in light condition.
e TEAC and DPPH radical-scavenging activity were tested for treated samples stored in dark and light Journal of Food Quality conditions.e T3 sample manifested the highest antioxidant activity by TEAC after production probably to the combined effect of mixed acids in dipping (385.06 ± 20.26 µM•TE•g −1 ).No statistical differences were observed among treated samples at each monitoring storage time with the exception of samples stored after seven days (p < 0.01).Table 1 shows that the storage time influenced the TEAC parameter with significance (p < 0.01).In particular, it was  4 Journal of Food Quality demonstrated by one-way ANOVA during the storage in dark condition: only TEAC of the T2 sample stored in dark condition increased during storage time with significant differences (p < 0.05) (Table 2).e light exposure of the samples did not affect antioxidant activities as shown by multivariate analysis (Table 1).Storage time highly influenced DPPH radical-scavenging activity by multivariate analysis with p < 0.01 (Table 1).More precisely, the dark condition influenced this parameter during the time more than the light as demonstrated by oneway ANOVA (Table 2): during the storage, DPPH radicalscavenging activity increased in T2 with the highest value of 20.38 ± 3.27% at the end, while a decrease was observed in T3 after sixteen days with p < 0.05 in dark condition.Probably, presence of citric acid had influenced this qualitative parameter: so the treatment with citric acid (T2) is more suggested to improve Valeriana leaves' antioxidant capacity.
e light exposure of samples did not affect total phenolic content (about 1110 mg gallic acid kg −1 ), and it did not change with significance in samples during the storage except for the untreated sample (T1) stored in light condition (p < 0.05).In particular, the total phenolic content tended to increase during the storage days, particularly in light conditions with a final content of 1464.82 ± 151.77, 1404.95 ± 205.04, and 1378.91 ± 136.24 mg gallic acid kg −1 in T1, T2, and T3 samples, respectively.is amount of total phenolic compounds may also play an inhibitory effect on the browning appearance, as just reported by Ferrante et al. [26].Positive correlations between phenolic compounds and antioxidant activities (by TEAC and DPPH assay) were found only at time zero (r � 0.792 and p < 0.05 and r � 0.844 and p < 0.05, respectively, for the two assays), showing that the increase in TEAC of the T2 sample stored in darkness was not probably related to the phenolic content.erefore, these last compounds cannot be used to estimate the overall health status and probably also the antioxidant property of leafy vegetables during storage, as suggested by Ferrante and Maggiore [27], and that the antioxidant activities in the Valeriana species may be attributed to some other compounds than the phenolics, flavonoids, and tannins as reported by Jugran et al. [28].

Conclusion
In conclusion, the results of this study showed that the quality of Valeriana minimally processed vegetables can be improved by treatments at the processing.e samples were stored for sixteen days over the recommended seven days by the producer with a total bacteria count within the satisfactory limits of 10 6 log 10 CFU g −1 , although a dipping in 1% of citric acid is recommended to limit even more the bacterial growth.Most of the studied qualitative parameters were significantly affected by the time of storage, with the exception of the antioxidant activity and total phenolic content.e darkness storage should be preferred than light exposure for a better leafy colour.Treatments of Valeriana leaves with a mixture of citric and ascorbic acids (T3) compared with citric acid (T2) were not suggested to

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Figure 2 :Figure 1 :
Figure 2: Effect of dark (a) and light (b) conditions on total bacteria count and titratable acidity of Valeriana pretreated leaves stored for sixteen days.

Figure 3 :
Figure 3: Leaves discoloration expressed as the browning index (BI) in dark (a) and light (b) conditions.Data are the ratio between the value at a given storage time (t) and the initial value (0).

Table 1 :
Results of multivariate analysis for some physicochemical parameters of Valeriana leaves with respect to different variables and their interactions.Treatments (T) Storage time (St) Storage conditions (dark or light) (Sc) T * St T * Sc St * Sc T * Sc * St * n.s.* * n.s.* * * * * Significance at p < 0.01; * significance at p < 0.05; n.s.: not significant.

Table 2 :
Physicochemical parameters of pretreated leaves of Valeriana stored in dark condition.
Results are presented as the mean value ± standard deviation.n � 2; means within a row with different letters are significantly different by Tukey's post hoc test; * * significance at p < 0.01; * significance at p < 0.05; n.s.: not significant.
Results are presented as the mean value ± standard deviation.n � 2; means within a row with different letters are significantly different by Tukey's post hoc test; * * significance at p < 0.01; * significance at p < 0.05; n.s.: not significant.