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Effects of Darkness and Light Spectra on Nutrients and Pigments in Radish, Soybean, Mung Bean and Pumpkin Sprouts

Fresh sprouts are an important source of antioxidant compounds and contain useful phytonutrients in the human diet. Many factors, such as the time of germination and types of light, influence the physiological processes and biosynthetic pathways in sprouts. The effect of red, blue and white light vs. dark conditions on the quality parameters in different sprout species after 5 d of germination was evaluated. Total ascorbate, soluble proteins, sugars, phenolic compounds, and pigments, such as carotenoids, chlorophylls, and anthocyanins, were investigated in radishes, soybeans, mung beans, and pumpkin sprouts. The light treatments increased the contents of vitamin C and the various pigments in all sprouts, conversely, they increased the soluble proteins and sugars, including d-glucose, d-fructose and sucrose, in soybeans and pumpkins, respectively. The dark treatment prevented the decrease in dry matter due to the lighting, while the red light induced an increase in polyphenols in soybean. These results suggest that the nutritional content of different sprouts grown under different light conditions depend on the dark or specific spectral wavelength used for their growth. The manuscript may increase the knowledge on light use for the industrialized food production aiming at preserving the phytonutrient content of vegetables, increasing the consumer health, or developing tailored diets for specific nutritional needs.


Introduction
In recent years, the use of sprouts, particularly in vegan and vegetarian diets, has become a topic of interest to model the "Mediterranean diet". Careful consumers take advantage of introducing fresh plant foods into their diets, such as sprouts, that are rich in important nutrients, easy to digest and low cost. The sprouts originate through the germination of seeds in a process in which physical, chemical and metabolic changes occur and where proteins and polysaccharides are converted into amino acids and sugars. In some legumes, germination results in an increase and decrease in vitamins and anti-nutritional factors, respectively [1], such as an increase in antioxidant compounds [2] and angiotensin converting enzyme [3], which has been observed. Shah et al. [4] reported that the process of germination improved the nutritional value of mung beans in terms of a higher concentration of nutrients, reduced phytic acid, and improved protein content and ascorbic acid. In addition, the important functional properties of the sprout nutrients have attracted attention as potential health-promoting functional foods [5]. Several potential anti-hypertensive, anti-hyperlipidemic, or antidiabetic compounds have also been identified in germinating seeds [5,6]. The sprouts of mung beans and soybeans have been shown to be more effective for an anti-hypertensive diet [7]. The sprouts of cruciferous plants, such as radishes and broccoli, contain high levels of CoQ10, which is considered to be a potential anti-hypertensive and anti-hyperlipidemic compound [8]. The sprouts showed morphological differences. The soybean and mung bean sprouts in WL had fleshy cotyledons that were also present when the young leaves appeared, while in radishes and pumpkins, the cotyledons changed into photosynthetic leaflets.

Determination of Dry Matter Content
The dry matter was determined in five-day sprouted seeds. For the analysis, lots of 30 random sprouts of each sample were weighed (fresh matter) as quickly as possible to limit the losses through evaporation and dried at 105 ± 2 °C in an oven until a constant weight was observed (approximately 24 h). The dry matter content was expressed as a percentage of the ratio of constant weight following drying and the initial fresh matter.

Anthocyanin Content
The anthocyanin content was determined as reported in [19]. Briefly, each sprout sample (5 g) was cut into pieces and incubated at 65 °C for 2 h with 20 mL of a solution containing 98% methanol and 0.24 M HCl. After centrifugation at 4500× g for 10 min, the anthocyanin content was measured spectrophotometrically at both 530 nm and 657 nm. The formula A530 − 0.25 × A657 that corrects the absorbance for the chlorophyll degradation product was utilized.

Determination of the Chlorophyll and Carotenoid Contents
The chlorophyll and carotenoid contents were determined as described by Lichtenthaler [20] with some modifications. Five grams of fresh material was homogenized with 10 mL of absolute acetone and 3 mg of Na2CO3 and centrifuged at 20,000× g for 15 min. The absorbance of the supernatant was measured spectrophotometrically at 645 and 662 nm, respectively, for chlorophyll a and b and at 470 nm for the carotenoids. The total chlorophyll and carotenoid content was calculated using Lichtenthaler's equations.

Vitamin C Content
Five grams of fresh tissue was homogenized with ten volumes of cold 5% (w/v) metaphosphoric acid in a porcelain mortar. The homogenate was centrifuged for 15 min at 20,000× g, and the supernatant was collected for the analysis of L-ascorbic acid (AsA) and L-dehydroascorbic acid (DHA) as described in [21].

Soluble Protein Assay
At various times, 5 g of sprouts was ground by pestle and mortar in 15 mL of extraction medium containing 100 mM potassium phosphate buffer pH 7.0, 0.5 M sorbitol, 1 mM EDTA and 0.05% (w/v) cysteine. After centrifugation (20,000× g, 20 min, 2 °C), the soluble fraction was desalted by dialysis against 50 mM Tris-HCl, pH 7.8. This desalted fraction was used to quantify the protein content with a Protein Assay kit from Bio-Rad (Hercules, CA, USA) with bovine serum albumin as the standard [22]. The reproducibility of Bio-Rad kit, expressed as coefficient of variation (%CV), is 2% approximately; the lower limit of detection for protein molecular weight is 3000 to 5000 daltons. The sprouts showed morphological differences. The soybean and mung bean sprouts in WL had fleshy cotyledons that were also present when the young leaves appeared, while in radishes and pumpkins, the cotyledons changed into photosynthetic leaflets.

Determination of Dry Matter Content
The dry matter was determined in five-day sprouted seeds. For the analysis, lots of 30 random sprouts of each sample were weighed (fresh matter) as quickly as possible to limit the losses through evaporation and dried at 105 ± 2 • C in an oven until a constant weight was observed (approximately 24 h). The dry matter content was expressed as a percentage of the ratio of constant weight following drying and the initial fresh matter.

Anthocyanin Content
The anthocyanin content was determined as reported in [19]. Briefly, each sprout sample (5 g) was cut into pieces and incubated at 65 • C for 2 h with 20 mL of a solution containing 98% methanol and 0.24 M HCl. After centrifugation at 4500× g for 10 min, the anthocyanin content was measured spectrophotometrically at both 530 nm and 657 nm. The formula A 530 − 0.25 × A 657 that corrects the absorbance for the chlorophyll degradation product was utilized.

Determination of the Chlorophyll and Carotenoid Contents
The chlorophyll and carotenoid contents were determined as described by Lichtenthaler [20] with some modifications. Five grams of fresh material was homogenized with 10 mL of absolute acetone and 3 mg of Na 2 CO 3 and centrifuged at 20,000× g for 15 min. The absorbance of the supernatant was measured spectrophotometrically at 645 and 662 nm, respectively, for chlorophyll a and b and at 470 nm for the carotenoids. The total chlorophyll and carotenoid content was calculated using Lichtenthaler's equations.

Vitamin C Content
Five grams of fresh tissue was homogenized with ten volumes of cold 5% (w/v) metaphosphoric acid in a porcelain mortar. The homogenate was centrifuged for 15 min at 20,000× g, and the supernatant was collected for the analysis of l-ascorbic acid (AsA) and l-dehydroascorbic acid (DHA) as described in [21].

Soluble Protein Assay
At various times, 5 g of sprouts was ground by pestle and mortar in 15 mL of extraction medium containing 100 mM potassium phosphate buffer pH 7.0, 0.5 M sorbitol, 1 mM EDTA and 0.05% (w/v) cysteine. After centrifugation (20,000× g, 20 min, 2 • C), the soluble fraction was desalted by dialysis against 50 mM Tris-HCl, pH 7.8. This desalted fraction was used to quantify the protein content with a Protein Assay kit from Bio-Rad (Hercules, CA, USA) with bovine serum albumin as the standard [22]. The reproducibility of Bio-Rad kit, expressed as coefficient of variation (%CV), is 2% approximately; the lower limit of detection for protein molecular weight is 3000 to 5000 daltons.

Soluble Carbohydrates and Starch Measurements
Five grams of sprouts from each treatment was cut into pieces and analysed. After hot extraction (80 • C) with ethanol (95%) and centrifugation at 4500× g for 10 min, the levels of mono-and di-saccharides (d-glucose, d-fructose and sucrose) were determined spectrophotometrically using a Megazyme kit (Sucrose/d-Fructose/d-Glucose Assay Kit; K-SUFRG, Megazyme, Ireland) according to the manufacturer's protocol. The reproducibility of the K-SUFRG is for D-Glucose %CV = 0.76, for D-Fructose %CV = 0.87, for Sucrose %CV = 0.24.
The alcohol insoluble fraction (pellet) was used to determine the starch content using a Megazyme kit (Total Starch Assay Kit AA/AMG; K-TSTA, Megazyme, Ireland) according to the manufacturer's protocol. Reproducibility value is %CV = 3%. The Total Starch Kit can accurately measure starch levels as low as 1% w/w.

Determination of the Polyphenol Contents
The fresh tissue was homogenized with a 30% ethanol solution in a 1:2 weight/volume ratio. The homogenate obtained was diluted at 1:6 with distilled water. The sample was incubated at 80 • C for 1 h in darkness and centrifuged at 7000× g for 10 min. For each sample, 0.3 mL of supernatant was utilized for analysis as described in [23]. The total phenolic content was determined according to a calibration curve of gallic acid. The results were expressed as mg of gallic acid equivalent (GAE) for 1 g of fresh matter.

Starch Qualitative Analysis
The seeds of soybeans and mung beans were soaked in distilled water for 2 h. Cotyledon transversal sections (15 µm thick) were cut with a DSK-1000 vibratome (Dosaka, Kyoto, Japan) and stained with Lugol solution (I 2 KI) diluted (Lugol:distilled water, 1:40) for 10 min. The sections were mounted on standard microscope slides and examined with a BX-40 light microscope Olympus and DP-21 digital camera (Olympus America Inc., Center Valley, Pennsylvania, PA, USA).

Statistical Analyses
The data reported are the average of at least three replicates from four independent experiments. A one-factor ANOVA was performed on the observed means of the compound content for each sample, and the significance of the different treatments (white, blue, and red light) within each sample with respect to the dark control was evaluated using Tukey's HSD test for multiple comparisons (p < 0.05).

Effect of Light Spectral Properties on Dry Matter and Soluble Proteins
Water was the primary component of the fresh matter in the sprout (represented by the apex, cotyledons, hypocotyl and root). However, differences in the dry matter occurred among the various sprouts that were treated differently. The highest value of dry matter was observed in those that were grown in darkness (Figure 2A). Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 12 lower metabolic activity for the low consumption of sugars and starch as observed. However, other components, such as cell wall polysaccharides, lipids and mineral salts, may contribute to the dry matter. Values represent the means of at least three replicates from four independent experiments. Identical letters over the columns indicate non-significant differences between the treatments within each sprout (Tukey's HSD test, p < 0.05). White light ( ); blue light, ( ); red light ( ), and dark ( ).
In the dry soybean seeds, the proteins represent the prominent type of storage accumulated in the cotyledons. In the soybean sprouts, the content of the soluble proteins was higher than that of the other sprouts after light exposure ( Figure 2B). The soluble protein content significantly increased (p < 0.05) in the soybean in all light treatments and in radish sprouts in the WL and RL compared to the dark. After five days of germination, the WL became the most effective, and the high presence of soluble proteins suggested the beginning of photosynthetic activity in the greening cotyledons of the soybean. No significant difference in the different light treatments with respect to the dark was observed in pumpkins, while there was a decrease in mung beans.

Effect of Light on Sugars and Starch
Light spectrum properties strongly influence the physiology, growth, development and phytochemical accumulation in planta, particularly for vegetables produced in controlled environments [25]. The common sugars are phytochemicals classified as primary metabolites. Total sugar content (D-fructose plus D-glucose plus sucrose) of the different sprouts in both the dark and the different light conditions is shown in Figure 3A. Values represent the means of at least three replicates from four independent experiments. Identical letters over the columns indicate non-significant differences between the treatments within each sprout (Tukey's HSD test, p < 0.05). White light ( Antioxidants 2020, 9, x FOR PEER REVIEW Values represent the mean at least three replications from four independent experiments. Identical letters over the colum indicate non-significant differences between the treatments within each segment (Tukey's HSD t p < 0.05).
In darkness, the total sugar content was significantly (p < 0.05) higher in mung bea soybeans; in particular, the value of the soybean was 55% lower when grown under WL darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with re the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germ the lipids are decomposed to succinate that in other cell compartments (mitochondria and are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose was th abundant sugar among those analysed, and it was 84% of the sum of the three sugars in d ( Figure 3A).
Interestingly, since it is not a reducing sugar, the sucrose could be transported across d and this might serve to allow for the growth and development of the soybean sprouts. The li also catabolized in the dark, in which the gluconeogenesis process in dark conditions allows etiolated seedlings to grow and develop in the absence of photosynthesis. In the soybean se storage tissues are poor in starch and rich in lipids that are probably converted into sucrose germination. After five days from germination, the persistence of the high sucrose content the dark and light conditions, highlights a surplus of sucrose, probably due to a limited or utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide sucrose 26% maximum of the total sugars, while both D-glucose and D-fructose are more abundant pumpkin sprouts, the increase in soluble sugars in the light may be due in part to th photosynthetic level for the presence of the great greening cotyledons. The significant decreas total sugar content in mung beans and soybeans could be correlated with higher consu ); blue light, ( Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 12 lower metabolic activity for the low consumption of sugars and starch as observed. However, other components, such as cell wall polysaccharides, lipids and mineral salts, may contribute to the dry matter. In the dry soybean seeds, the proteins represent the prominent type of storage accumulated in the cotyledons. In the soybean sprouts, the content of the soluble proteins was higher than that of the other sprouts after light exposure ( Figure 2B). The soluble protein content significantly increased (p < 0.05) in the soybean in all light treatments and in radish sprouts in the WL and RL compared to the dark. After five days of germination, the WL became the most effective, and the high presence of soluble proteins suggested the beginning of photosynthetic activity in the greening cotyledons of the soybean. No significant difference in the different light treatments with respect to the dark was observed in pumpkins, while there was a decrease in mung beans.

Effect of Light on Sugars and Starch
Light spectrum properties strongly influence the physiology, growth, development and phytochemical accumulation in planta, particularly for vegetables produced in controlled environments [25]. The common sugars are phytochemicals classified as primary metabolites. Total sugar content (D-fructose plus D-glucose plus sucrose) of the different sprouts in both the dark and the different light conditions is shown in Figure 3A.
); red light ( Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 12 lower metabolic activity for the low consumption of sugars and starch as observed. However, other components, such as cell wall polysaccharides, lipids and mineral salts, may contribute to the dry matter. In the dry soybean seeds, the proteins represent the prominent type of storage accumulated in the cotyledons. In the soybean sprouts, the content of the soluble proteins was higher than that of the other sprouts after light exposure ( Figure 2B). The soluble protein content significantly increased (p < 0.05) in the soybean in all light treatments and in radish sprouts in the WL and RL compared to the dark. After five days of germination, the WL became the most effective, and the high presence of soluble proteins suggested the beginning of photosynthetic activity in the greening cotyledons of the soybean. No significant difference in the different light treatments with respect to the dark was observed in pumpkins, while there was a decrease in mung beans.

Effect of Light on Sugars and Starch
Light spectrum properties strongly influence the physiology, growth, development and phytochemical accumulation in planta, particularly for vegetables produced in controlled environments [25]. The common sugars are phytochemicals classified as primary metabolites. Total sugar content (D-fructose plus D-glucose plus sucrose) of the different sprouts in both the dark and the different light conditions is shown in Figure 3A.  In darkness, the total sugar content was significantly (p < 0.05) higher in mung beans a soybeans; in particular, the value of the soybean was 55% lower when grown under WL than darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with respect the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germinati the lipids are decomposed to succinate that in other cell compartments (mitochondria and cytos are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose was the m abundant sugar among those analysed, and it was 84% of the sum of the three sugars in darkn ( Figure 3A).
Interestingly, since it is not a reducing sugar, the sucrose could be transported across distan and this might serve to allow for the growth and development of the soybean sprouts. The lipids also catabolized in the dark, in which the gluconeogenesis process in dark conditions allows for etiolated seedlings to grow and develop in the absence of photosynthesis. In the soybean seeds, storage tissues are poor in starch and rich in lipids that are probably converted into sucrose dur germination. After five days from germination, the persistence of the high sucrose content in b the dark and light conditions, highlights a surplus of sucrose, probably due to a limited or lack utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide sucrose was 26% maximum of the total sugars, while both D-glucose and D-fructose are more abundant. In pumpkin sprouts, the increase in soluble sugars in the light may be due in part to the h photosynthetic level for the presence of the great greening cotyledons. The significant decrease in total sugar content in mung beans and soybeans could be correlated with higher consumpti ).
The lighting caused a lower value in the dry matter than the darkness, and different types of light did not exhibit a significant difference. Soybeans showed the highest value in both the light and dark treatments followed by pumpkins, mung beans and radishes. The trend observed in all the sprouts, that there was more high dry matter in darkness and with the highest value in soybeans, was probably due to the abundant amount of storage compounds contained in the cotyledons of the shoot. Indeed, during the soybean germination, the storage compounds are mainly utilized in the sprouts grown in the light rather than in darkness. Blue, red and white light caused a larger utilization of storage with the subsequent loss of dry matter. This was probably due to a higher active metabolism under light conditions, as also reported by Chen and Chang [24], in which a significant loss of lipids occurred in soybean sprouts. On the other hand, the preservation in darkness of dry matter was probably due to a lower metabolic activity for the low consumption of sugars and starch as observed. However, other components, such as cell wall polysaccharides, lipids and mineral salts, may contribute to the dry matter.
In the dry soybean seeds, the proteins represent the prominent type of storage accumulated in the cotyledons. In the soybean sprouts, the content of the soluble proteins was higher than that of the other sprouts after light exposure ( Figure 2B). The soluble protein content significantly increased (p < 0.05) in the soybean in all light treatments and in radish sprouts in the WL and RL compared to the dark. After five days of germination, the WL became the most effective, and the high presence of soluble proteins suggested the beginning of photosynthetic activity in the greening cotyledons of the soybean. No significant difference in the different light treatments with respect to the dark was observed in pumpkins, while there was a decrease in mung beans.

Effect of Light on Sugars and Starch
Light spectrum properties strongly influence the physiology, growth, development and phytochemical accumulation in planta, particularly for vegetables produced in controlled environments [25]. The common sugars are phytochemicals classified as primary metabolites. Total sugar content (d-fructose plus d-glucose plus sucrose) of the different sprouts in both the dark and the different light conditions is shown in Figure 3A.
Antioxidants 2020, 9, x FOR PEER REVIEW 6 of 12 Values represent the means of at least three replications from four independent experiments. Identical letters over the columns indicate non-significant differences between the treatments within each segment (Tukey's HSD test, p < 0.05).
In darkness, the total sugar content was significantly (p < 0.05) higher in mung beans and soybeans; in particular, the value of the soybean was 55% lower when grown under WL than in darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with respect to the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germination, the lipids are decomposed to succinate that in other cell compartments (mitochondria and cytosol) are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose was the more abundant sugar among those analysed, and it was 84% of the sum of the three sugars in darkness ( Figure 3A).
Interestingly, since it is not a reducing sugar, the sucrose could be transported across distances and this might serve to allow for the growth and development of the soybean sprouts. The lipids are also catabolized in the dark, in which the gluconeogenesis process in dark conditions allows for the etiolated seedlings to grow and develop in the absence of photosynthesis. In the soybean seeds, the storage tissues are poor in starch and rich in lipids that are probably converted into sucrose during germination. After five days from germination, the persistence of the high sucrose content in both the dark and light conditions, highlights a surplus of sucrose, probably due to a limited or lack of utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide sucrose was the 26% maximum of the total sugars, while both D-glucose and D-fructose are more abundant. In the pumpkin sprouts, the increase in soluble sugars in the light may be due in part to the high photosynthetic level for the presence of the great greening cotyledons. The significant decrease in the total sugar content in mung beans and soybeans could be correlated with higher consumption. Values represent the means of at least three replications from four independent experiments. Identical letters over the columns indicate non-significant differences between the treatments within each segment (Tukey's HSD test, p < 0.05).
In darkness, the total sugar content was significantly (p < 0.05) higher in mung beans and soybeans; in particular, the value of the soybean was 55% lower when grown under WL than in darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with respect to the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germination, the lipids are decomposed to succinate that in other cell compartments (mitochondria and cytosol) are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose was the more abundant sugar among those analysed, and it was 84% of the sum of the three sugars in darkness ( Figure 3A).
Interestingly, since it is not a reducing sugar, the sucrose could be transported across distances and this might serve to allow for the growth and development of the soybean sprouts. The lipids are also catabolized in the dark, in which the gluconeogenesis process in dark conditions allows for the etiolated seedlings to grow and develop in the absence of photosynthesis. In the soybean seeds, the storage tissues are poor in starch and rich in lipids that are probably converted into sucrose during germination. After five days from germination, the persistence of the high sucrose content in both the dark and light conditions, highlights a surplus of sucrose, probably due to a limited or lack of utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide sucrose was the 26% maximum of the total sugars, while both D-glucose and D-fructose are more abundant. In the pumpkin sprouts, the increase in soluble sugars in the light may be due in part to the high photosynthetic level for the presence of the great greening cotyledons. The significant decrease in the Values represent the means of at least three replications from four independent experiments. Identical letters over the columns indicate non-significant differences between the treatments within each segment (Tukey's HSD test, p < 0.05).
In darkness, the total sugar content was significantly (p < 0.05) higher in mung beans and soybeans; in particular, the value of the soybean was 55% lower when grown under WL than in darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with respect to the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germination, the lipids are decomposed to succinate that in other cell compartments (mitochondria and cytosol) are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose was the more abundant sugar among those analysed, and it was 84% of the sum of the three sugars in darkness ( Figure 3A).
Interestingly, since it is not a reducing sugar, the sucrose could be transported across distances and this might serve to allow for the growth and development of the soybean sprouts. The lipids are also catabolized in the dark, in which the gluconeogenesis process in dark conditions allows for the etiolated seedlings to grow and develop in the absence of photosynthesis. In the soybean seeds, the storage tissues are poor in starch and rich in lipids that are probably converted into sucrose during germination. After five days from germination, the persistence of the high sucrose content in both the dark and light conditions, highlights a surplus of sucrose, probably due to a limited or lack of utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide sucrose was the 26% maximum of the total sugars, while both D-glucose and D-fructose are more abundant. In the pumpkin sprouts, the increase in soluble sugars in the light may be due in part to the high photosynthetic level for the presence of the great greening cotyledons. The significant decrease in the ), sucrose ( Antioxidants 2020, 9, x FOR PEER REVIEW 6 of 12 Values represent the means of at least three replications from four independent experiments. Identical letters over the columns indicate non-significant differences between the treatments within each segment (Tukey's HSD test, p < 0.05).
In darkness, the total sugar content was significantly (p < 0.05) higher in mung beans and soybeans; in particular, the value of the soybean was 55% lower when grown under WL than in darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with respect to the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germination, the lipids are decomposed to succinate that in other cell compartments (mitochondria and cytosol) are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose was the more abundant sugar among those analysed, and it was 84% of the sum of the three sugars in darkness ( Figure 3A).
Interestingly, since it is not a reducing sugar, the sucrose could be transported across distances and this might serve to allow for the growth and development of the soybean sprouts. The lipids are also catabolized in the dark, in which the gluconeogenesis process in dark conditions allows for the etiolated seedlings to grow and develop in the absence of photosynthesis. In the soybean seeds, the storage tissues are poor in starch and rich in lipids that are probably converted into sucrose during germination. After five days from germination, the persistence of the high sucrose content in both the dark and light conditions, highlights a surplus of sucrose, probably due to a limited or lack of utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide sucrose was the 26% maximum of the total sugars, while both D-glucose and D-fructose are more abundant. In the pumpkin sprouts, the increase in soluble sugars in the light may be due in part to the high photosynthetic level for the presence of the great greening cotyledons. The significant decrease in the In darkness, the total sugar content was significantly (p < 0.05) higher in mung beans and soybeans; in particular, the value of the soybean was 55% lower when grown under WL than in darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with respect to the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germination, the lipids are decomposed to succinate that in other cell compartments (mitochondria and cytosol) are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose was the more abundant sugar among those analysed, and it was 84% of the sum of the three sugars in darkness ( Figure 3A).
Interestingly, since it is not a reducing sugar, the sucrose could be transported across distances and this might serve to allow for the growth and development of the soybean sprouts. The lipids are also catabolized in the dark, in which the gluconeogenesis process in dark conditions allows for the etiolated seedlings to grow and develop in the absence of photosynthesis. In the soybean seeds, the storage tissues are poor in starch and rich in lipids that are probably converted into sucrose during germination. After five days from germination, the persistence of the high sucrose content in both the dark and light conditions, highlights a surplus of sucrose, probably due to a limited or lack of utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide sucrose was the 26% maximum of the total sugars, while both D-glucose and D-fructose are more abundant. In the pumpkin sprouts, the increase in soluble sugars in the light may be due in part to the high photosynthetic level for the presence of the great greening cotyledons. The significant decrease in the ); blue light ( OR PEER REVIEW 5 of 12 tivity for the low consumption of sugars and starch as observed. However, other as cell wall polysaccharides, lipids and mineral salts, may contribute to the dry ry matter content in the sprouts 5 d after germination following various treatments. he soluble proteins in the sprouts 5 d after germination following various treatments. nt the means of at least three replicates from four independent experiments. Identical columns indicate non-significant differences between the treatments within each s HSD test, p < 0.05). White light ( ); blue light, ( ); red light ( ), and dark ( bean seeds, the proteins represent the prominent type of storage accumulated in the soybean sprouts, the content of the soluble proteins was higher than that of the light exposure ( Figure 2B). The soluble protein content significantly increased (p an in all light treatments and in radish sprouts in the WL and RL compared to the ys of germination, the WL became the most effective, and the high presence of ggested the beginning of photosynthetic activity in the greening cotyledons of the ficant difference in the different light treatments with respect to the dark was kins, while there was a decrease in mung beans. n Sugars and Starch m properties strongly influence the physiology, growth, development and cumulation in planta, particularly for vegetables produced in controlled . The common sugars are phytochemicals classified as primary metabolites. Total uctose plus D-glucose plus sucrose) of the different sprouts in both the dark and conditions is shown in Figure 3A.
); red light ( R PEER REVIEW 5 of 12 vity for the low consumption of sugars and starch as observed. However, other s cell wall polysaccharides, lipids and mineral salts, may contribute to the dry matter content in the sprouts 5 d after germination following various treatments. e soluble proteins in the sprouts 5 d after germination following various treatments. the means of at least three replicates from four independent experiments. Identical columns indicate non-significant differences between the treatments within each SD test, p < 0.05). White light ( ); blue light, ( ); red light ( ), and dark ( ean seeds, the proteins represent the prominent type of storage accumulated in e soybean sprouts, the content of the soluble proteins was higher than that of the ight exposure ( Figure 2B). The soluble protein content significantly increased (p n in all light treatments and in radish sprouts in the WL and RL compared to the s of germination, the WL became the most effective, and the high presence of gested the beginning of photosynthetic activity in the greening cotyledons of the cant difference in the different light treatments with respect to the dark was ns, while there was a decrease in mung beans.

Sugars and Starch
properties strongly influence the physiology, growth, development and mulation in planta, particularly for vegetables produced in controlled he common sugars are phytochemicals classified as primary metabolites. Total ctose plus D-glucose plus sucrose) of the different sprouts in both the dark and nditions is shown in Figure 3A. In darkness, the total sugar content was significantly (p < 0.05) higher in mung beans and soybeans; in particular, the value of the soybean was 55% lower when grown under WL than in darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with respect to the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germination, the lipids are decomposed to succinate that in other cell compartments (mitochondria and cytosol) are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose was the more abundant sugar among those analysed, and it was 84% of the sum of the three sugars in darkness ( Figure 3A).
Interestingly, since it is not a reducing sugar, the sucrose could be transported across distances and this might serve to allow for the growth and development of the soybean sprouts. The lipids are also catabolized in the dark, in which the gluconeogenesis process in dark conditions allows for the etiolated seedlings to grow and develop in the absence of photosynthesis. In the soybean seeds, the storage tissues are poor in starch and rich in lipids that are probably converted into sucrose during germination. After five days from germination, the persistence of the high sucrose content in both the dark and light conditions, highlights a surplus of sucrose, probably due to a limited or lack of utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide sucrose was the 26% maximum of the total sugars, while both D-glucose and D-fructose are more abundant. In the pumpkin sprouts, the increase in soluble sugars in the light may be due in part to the high photosynthetic level for the presence of the great greening cotyledons. The significant decrease in the ). Values represent the means of at least three replications from four independent experiments. Identical letters over the columns indicate non-significant differences between the treatments within each segment (Tukey's HSD test, p < 0.05).
In darkness, the total sugar content was significantly (p < 0.05) higher in mung beans and soybeans; in particular, the value of the soybean was 55% lower when grown under WL than in darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with respect to the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germination, the lipids are decomposed to succinate that in other cell compartments (mitochondria and cytosol) are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose was the more abundant sugar among those analysed, and it was 84% of the sum of the three sugars in darkness ( Figure 3A).
Interestingly, since it is not a reducing sugar, the sucrose could be transported across distances and this might serve to allow for the growth and development of the soybean sprouts. The lipids are also catabolized in the dark, in which the gluconeogenesis process in dark conditions allows for the etiolated seedlings to grow and develop in the absence of photosynthesis. In the soybean seeds, the storage tissues are poor in starch and rich in lipids that are probably converted into sucrose during germination. After five days from germination, the persistence of the high sucrose content in both the dark and light conditions, highlights a surplus of sucrose, probably due to a limited or lack of utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide sucrose was the 26% maximum of the total sugars, while both d-glucose and d-fructose are more abundant. In the pumpkin sprouts, the increase in soluble sugars in the light may be due in part to the high photosynthetic level for the presence of the great greening cotyledons. The significant decrease in the total sugar content in mung beans and soybeans could be correlated with higher consumption. Generally, the different content of the sugars in the various sprouts suggested that the responses of sprout to light qualities were related to species or cultivars as well as the growth period and light intensity as just reported in literature [26,27]. Particularly, the lower sucrose content in soybean sprouts in WL compared to the BL was probably caused by its consumption in cell growth processes with a possible involvement of phototropins. Indeed, in BL, the hypocotyl was less grown than in WL (data not shown) where sucrose was utilized for a greater growth of the sprout. It is reported that BL rapidly and strongly inhibits hypocotyl elongation [28], reduces the plant height [29] and the BL-activation of phototropin (nph1) influences cryptochrome signaling leading to growth inhibition [28]. However, further detailed studies, comprising also the activities of sucrose-metabolism associated enzymes, including sucrose synthase, sucrose phosphate synthase and invertases [30], are needed for a better comprehension of the effects of selected spectral light on sprouts qualities and are in progress.
Red light is needed for starch accumulation and for the proper development of the photosynthetic apparatus [31]. The starch content after the different light conditions and dark treatment is shown in Figure 3B. The highest content was observed in mung beans (15 in darkness and 14 mg g −1 of fresh matter in BL, respectively). Soybeans had the next highest content with a value of approximately 3 mg g −1 of fresh matter in all the treatments. Low levels and no significant difference under different lighting conditions and darkness were observed in pumpkins and radishes.
Many starch grains were observed in transverse sections of the mung bean cotyledons (Figure 4, Panel A). In soybeans, although the cotyledons had been incubated with the reagent of Lugol for the same time utilized for mung beans, the material did not show the presence of starch grains, while numerous protein vacuoles were observed (Figure 4, Panel B). Interestingly, due to the low sugar and starch levels, the radishes and pumpkin sprouts could be utilized for low carbohydrate diets.
Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 12 Generally, the different content of the sugars in the various sprouts suggested that the responses of sprout to light qualities were related to species or cultivars as well as the growth period and light intensity as just reported in literature [26,27]. Particularly, the lower sucrose content in soybean sprouts in WL compared to the BL was probably caused by its consumption in cell growth processes with a possible involvement of phototropins. Indeed, in BL, the hypocotyl was less grown than in WL (data not shown) where sucrose was utilized for a greater growth of the sprout. It is reported that BL rapidly and strongly inhibits hypocotyl elongation [28], reduces the plant height [29] and the BLactivation of phototropin (nph1) influences cryptochrome signaling leading to growth inhibition [28]. However, further detailed studies, comprising also the activities of sucrose-metabolism associated enzymes, including sucrose synthase, sucrose phosphate synthase and invertases [30], are needed for a better comprehension of the effects of selected spectral light on sprouts qualities and are in progress. Red light is needed for starch accumulation and for the proper development of the photosynthetic apparatus [31]. The starch content after the different light conditions and dark treatment is shown in Figure 3B. The highest content was observed in mung beans (15 in darkness and 14 mg g −1 of fresh matter in BL, respectively). Soybeans had the next highest content with a value of approximately 3 mg g −1 of fresh matter in all the treatments. Low levels and no significant difference under different lighting conditions and darkness were observed in pumpkins and radishes.
Many starch grains were observed in transverse sections of the mung bean cotyledons ( Figure  4, Panel A). In soybeans, although the cotyledons had been incubated with the reagent of Lugol for the same time utilized for mung beans, the material did not show the presence of starch grains, while numerous protein vacuoles were observed (Figure 4, Panel B). Interestingly, due to the low sugar and starch levels, the radishes and pumpkin sprouts could be utilized for low carbohydrate diets.

Changes in the Vitamin C and Total Phenolic Contents
The quality and intensity of light are reported to be effective at regulating the AsA level in plants [13]. The lighting caused a significant (p < 0.05) increase in the ascorbate total content (AsA + DHA) in all the sprouts with respect to the dark ( Figure 5A). Radishes contained the highest value, followed by mung beans and soybeans, while in pumpkins, the total content of ascorbate was lower in both the light and darkness. No significant difference was observed among the different light treatments for each type of sprout. Different dehydrated orthodox seeds did not contain AsA, although it is immediately produced after seed imbibition. It is reported that the AsA levels are low in dehydrated orthodox soybean seeds, and its strong increase during germination is due to the reactivation of its biosynthesis. In particular, the WL, BL and ultraviolet lights were more effective at activating Lgalactono-γ-lactone dehydrogenase, the enzyme that converts the substrate L-Galactono-1,4-lactone into ascorbate [32]. In this study, we observed an increase in the AsA in the WL and BL treatments.

Changes in the Vitamin C and Total Phenolic Contents
The quality and intensity of light are reported to be effective at regulating the AsA level in plants [13]. The lighting caused a significant (p < 0.05) increase in the ascorbate total content (AsA + DHA) in all the sprouts with respect to the dark ( Figure 5A). Radishes contained the highest value, followed by mung beans and soybeans, while in pumpkins, the total content of ascorbate was lower in both the light and darkness. No significant difference was observed among the different light treatments for each type of sprout. Different dehydrated orthodox seeds did not contain AsA, although it is immediately produced after seed imbibition. It is reported that the AsA levels are low in dehydrated orthodox soybean seeds, and its strong increase during germination is due to the reactivation of its biosynthesis. Antioxidants 2020, 9, 558 8 of 12 In particular, the WL, BL and ultraviolet lights were more effective at activating L-galactono-γ-lactone dehydrogenase, the enzyme that converts the substrate L-Galactono-1,4-lactone into ascorbate [32]. In this study, we observed an increase in the AsA in the WL and BL treatments. However, the RL also induced the AsA increase in soybeans. In all the sprouts, the content of reduced ascorbate was higher than the oxidized form highlighting that the biosynthesis was higher than its degradation. Alternatively, the rapid increase in the AsA content and the activity of the enzymes of the ascorbate system is a fine strategy of defence in orthodox herbaceous plants seeds to counteract the high ROS level that occurs during germination [33]. Interestingly, the AsA content was the highest in the radishes whose cotyledons became active photosynthetic tissues.
However, the RL also induced the AsA increase in soybeans. In all the sprouts, the content of reduced ascorbate was higher than the oxidized form highlighting that the biosynthesis was higher than its degradation. Alternatively, the rapid increase in the AsA content and the activity of the enzymes of the ascorbate system is a fine strategy of defence in orthodox herbaceous plants seeds to counteract the high ROS level that occurs during germination [33]. Interestingly, the AsA content was the highest in the radishes whose cotyledons became active photosynthetic tissues.
The polyphenols are considered "scavengers" of free radical species and preserve the cell membranes from oxidative damage, thus benefitting human health. It is reported that various types of light influence the polyphenol content differently, and the responses were dependent on the species. The red LED increased the phenolic compound contents in common buckwheat sprouts but not in Tartary buckwheat sprouts [34]. Sweet basil grown under blue LED showed a lower total phenol content than that grown under white LED [35]. Qian and colleagues [36] reported that the vitamin C and total phenolic compounds of Chinese kale sprouts were not sensitive to the blue and red LED lights, respectively. However, our data indicated that when compared to the dark, the treatment with solely RL increased (p < 0.05) the phenolic content, while in the other sprouts, no difference between light and dark appeared ( Figure 5B). Therefore, the light generally did not influence the biosynthesis of the total polyphenols in the various sprouts. Of note, soybeans, followed by radishes, had a higher content than mung beans and pumpkins.  in the radishes whose cotyledons became active photosynthetic tissues.

Changes in the Anthocyanins, Chlorophylls and Carotenoids After Light and Dark Treatment
The polyphenols are considered "scavengers" of free radical species and preserve the cell membranes from oxidative damage, thus benefitting human health. It is reported that various types of light influence the polyphenol content differently, and the responses were dependent on the species. The red LED increased the phenolic compound contents in common buckwheat sprouts but not in Tartary buckwheat sprouts [34]. Sweet basil grown under blue LED showed a lower total phenol content than that grown under white LED [35]. Qian and colleagues [36] reported that the vitamin C and total phenolic compounds of Chinese kale sprouts were not sensitive to the blue and red LED lights, respectively. However, our data indicated that when compared to the dark, the treatment with solely RL increased (p < 0.05) the phenolic content, while in the other sprouts, no difference between light and dark appeared ( Figure 5B). Therefore, the light generally did not influence the biosynthesis of the total polyphenols in the various sprouts. Of note, soybeans, followed by radishes, had a higher content than mung beans and pumpkins. Identical letters over the columns indicate non-significant differences between treatments within each segment (Tukey's HSD test, p < 0.05).

Changes in the Anthocyanins, Chlorophylls and Carotenoids After Light and Dark Treatment
) and l-dehydroascorbic acid (DHA, the high ROS level that occurs during germination [33]. Interestingly, the AsA content was the highest in the radishes whose cotyledons became active photosynthetic tissues. The polyphenols are considered "scavengers" of free radical species and preserve the cell membranes from oxidative damage, thus benefitting human health. It is reported that various types of light influence the polyphenol content differently, and the responses were dependent on the species. The red LED increased the phenolic compound contents in common buckwheat sprouts but not in Tartary buckwheat sprouts [34]. Sweet basil grown under blue LED showed a lower total phenol content than that grown under white LED [35]. Qian and colleagues [36] reported that the vitamin C and total phenolic compounds of Chinese kale sprouts were not sensitive to the blue and red LED lights, respectively. However, our data indicated that when compared to the dark, the treatment with solely RL increased (p < 0.05) the phenolic content, while in the other sprouts, no difference between light and dark appeared ( Figure 5B). Therefore, the light generally did not influence the biosynthesis of the total polyphenols in the various sprouts. Of note, soybeans, followed by radishes, had a higher content than mung beans and pumpkins. Identical letters over the columns indicate non-significant differences between treatments within each segment (Tukey's HSD test, p < 0.05). In darkness, the total sugar content was significantly (p < 0.05) higher in mu soybeans; in particular, the value of the soybean was 55% lower when grown und darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. Durin the lipids are decomposed to succinate that in other cell compartments (mitochondr are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose abundant sugar among those analysed, and it was 84% of the sum of the three sug ( Figure 3A).

Changes in the Anthocyanins, Chlorophylls and Carotenoids After Light and Dark Treatment
Interestingly, since it is not a reducing sugar, the sucrose could be transported a and this might serve to allow for the growth and development of the soybean sprouts also catabolized in the dark, in which the gluconeogenesis process in dark condition etiolated seedlings to grow and develop in the absence of photosynthesis. In the soy storage tissues are poor in starch and rich in lipids that are probably converted into germination. After five days from germination, the persistence of the high sucrose the dark and light conditions, highlights a surplus of sucrose, probably due to a lim utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide s ); blue light ( Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 12 lower metabolic activity for the low consumption of sugars and starch as observed. However, other components, such as cell wall polysaccharides, lipids and mineral salts, may contribute to the dry matter. In the dry soybean seeds, the proteins represent the prominent type of storage accumulated in the cotyledons. In the soybean sprouts, the content of the soluble proteins was higher than that of the other sprouts after light exposure ( Figure 2B). The soluble protein content significantly increased (p < 0.05) in the soybean in all light treatments and in radish sprouts in the WL and RL compared to the dark. After five days of germination, the WL became the most effective, and the high presence of soluble proteins suggested the beginning of photosynthetic activity in the greening cotyledons of the soybean. No significant difference in the different light treatments with respect to the dark was observed in pumpkins, while there was a decrease in mung beans.

Effect of Light on Sugars and Starch
Light spectrum properties strongly influence the physiology, growth, development and phytochemical accumulation in planta, particularly for vegetables produced in controlled environments [25]. The common sugars are phytochemicals classified as primary metabolites. Total sugar content (D-fructose plus D-glucose plus sucrose) of the different sprouts in both the dark and the different light conditions is shown in Figure 3A.  In the dry soybean seeds, the proteins represent the prominent type of storage accumulated in the cotyledons. In the soybean sprouts, the content of the soluble proteins was higher than that of the other sprouts after light exposure ( Figure 2B). The soluble protein content significantly increased (p < 0.05) in the soybean in all light treatments and in radish sprouts in the WL and RL compared to the dark. After five days of germination, the WL became the most effective, and the high presence of soluble proteins suggested the beginning of photosynthetic activity in the greening cotyledons of the soybean. No significant difference in the different light treatments with respect to the dark was observed in pumpkins, while there was a decrease in mung beans.

Effect of Light on Sugars and Starch
Light spectrum properties strongly influence the physiology, growth, development and phytochemical accumulation in planta, particularly for vegetables produced in controlled environments [25]. The common sugars are phytochemicals classified as primary metabolites. Total sugar content (D-fructose plus D-glucose plus sucrose) of the different sprouts in both the dark and the different light conditions is shown in Figure 3A. In darkness, the total sugar content was significantly (p < 0.05) higher in mung soybeans; in particular, the value of the soybean was 55% lower when grown under W darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content wit the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During g the lipids are decomposed to succinate that in other cell compartments (mitochondria a are converted in sugars, primarily sucrose. In soybeans, the di-saccharide sucrose wa abundant sugar among those analysed, and it was 84% of the sum of the three sugars ( Figure 3A).
Interestingly, since it is not a reducing sugar, the sucrose could be transported acros and this might serve to allow for the growth and development of the soybean sprouts. Th also catabolized in the dark, in which the gluconeogenesis process in dark conditions all etiolated seedlings to grow and develop in the absence of photosynthesis. In the soybea storage tissues are poor in starch and rich in lipids that are probably converted into suc germination. After five days from germination, the persistence of the high sucrose cont the dark and light conditions, highlights a surplus of sucrose, probably due to a limited utilization. In contrast, in radishes, mung beans and pumpkins, the di-saccharide sucro ). Values represent the mean of at least three replicates from four independent experiments. Identical letters over the columns indicate non-significant differences between treatments within each segment (Tukey's HSD test, p < 0.05).
The polyphenols are considered "scavengers" of free radical species and preserve the cell membranes from oxidative damage, thus benefitting human health. It is reported that various types of light influence the polyphenol content differently, and the responses were dependent on the species. The red LED increased the phenolic compound contents in common buckwheat sprouts but not in Tartary buckwheat sprouts [34]. Sweet basil grown under blue LED showed a lower total phenol content than that grown under white LED [35]. Qian and colleagues [36] reported that the vitamin C and total phenolic compounds of Chinese kale sprouts were not sensitive to the blue and red LED lights, respectively. However, our data indicated that when compared to the dark, the treatment with solely RL increased (p < 0.05) the phenolic content, while in the other sprouts, no difference between light and dark appeared ( Figure 5B). Therefore, the light generally did not influence the biosynthesis of the total polyphenols in the various sprouts. Of note, soybeans, followed by radishes, had a higher content than mung beans and pumpkins.

Changes in the Anthocyanins, Chlorophylls and Carotenoids After Light and Dark Treatment
The various pigments examined in this work include the anthocyanins, whose synthesis is a process that is light-regulated and includes numerous steps starting from a phenylalanine precursor. Several plant species form anthocyanins in the light, while others do so in the dark, but their synthesis and content rapidly increases when exposed to light [37]. The inductive effect of light on anthocyanin synthesis was observable in all the sprouts ( Figure 6A). The anthocyanin content was significantly (p < 0.05) higher in mung beans and soybeans in the various light conditions with the respect to the dark and had the highest increase in mung beans. The BL followed by the WL were more effective than the RL, while in pumpkins and radishes, a significant (p < 0.05) increase was observed only in the BL and WL compared to the dark. Indeed, the BL is reported to significantly induce the anthocyanin accumulation in Arabidopsis seedlings [16], Chinese bayberry fruit [38], apple fruit [39] and post-harvest strawberry fruit [40]. Alternatively, anthocyanin accumulation in young tissues, such as epicotyls and young leaves, is a common event in plants. As these organs lack morpho-anatomical complexity, the high presence of anthocyanins could prevent environmental stress, such as high lighting [41]. Indeed, anthocyanins that absorb wavelengths like those of chlorophyll b play an auxiliary role in photo-protecting the plant tissues.
Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 12 The various pigments examined in this work include the anthocyanins, whose synthesis is a process that is light-regulated and includes numerous steps starting from a phenylalanine precursor. Several plant species form anthocyanins in the light, while others do so in the dark, but their synthesis and content rapidly increases when exposed to light [37]. The inductive effect of light on anthocyanin synthesis was observable in all the sprouts ( Figure 6A). The anthocyanin content was significantly (p < 0.05) higher in mung beans and soybeans in the various light conditions with the respect to the dark and had the highest increase in mung beans. The BL followed by the WL were more effective than the RL, while in pumpkins and radishes, a significant (p < 0.05) increase was observed only in the BL and WL compared to the dark. Indeed, the BL is reported to significantly induce the anthocyanin accumulation in Arabidopsis seedlings [16], Chinese bayberry fruit [38], apple fruit [39] and postharvest strawberry fruit [40]. Alternatively, anthocyanin accumulation in young tissues, such as epicotyls and young leaves, is a common event in plants. As these organs lack morpho-anatomical complexity, the high presence of anthocyanins could prevent environmental stress, such as high lighting [41]. Indeed, anthocyanins that absorb wavelengths like those of chlorophyll b play an auxiliary role in photo-protecting the plant tissues. The light also influences the chlorophylls and carotenoids. These pigments increased to the different spectral ranges. After lighting, the radish had a higher content of total chlorophyll (chl a + chl b; Figure 6C). Among the different light treatments, a higher value of chlorophyll was observed  In darkness, the total sugar content was significantly (p < 0.05) higher in mung beans and soybeans; in particular, the value of the soybean was 55% lower when grown under WL than in darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with respect to the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germination, the lipids are decomposed to succinate that in other cell compartments (mitochondria and cytosol) ); blue light ( , the proteins represent the prominent type of storage accumulated in sprouts, the content of the soluble proteins was higher than that of the sure ( Figure 2B). The soluble protein content significantly increased (p ht treatments and in radish sprouts in the WL and RL compared to the ination, the WL became the most effective, and the high presence of e beginning of photosynthetic activity in the greening cotyledons of the rence in the different light treatments with respect to the dark was ); red light ( the proteins represent the prominent type of storage accumulated in prouts, the content of the soluble proteins was higher than that of the re ( Figure 2B). The soluble protein content significantly increased (p t treatments and in radish sprouts in the WL and RL compared to the ation, the WL became the most effective, and the high presence of eginning of photosynthetic activity in the greening cotyledons of the nce in the different light treatments with respect to the dark was ), and dark ( Antioxidants 2020, 9, x FOR PEER REVIEW 6 of 12 In darkness, the total sugar content was significantly (p < 0.05) higher in mung beans and soybeans; in particular, the value of the soybean was 55% lower when grown under WL than in darkness. In mung beans, the RL induced the 53% decrease in soluble sugar content with respect to the dark. In the soybean seeds, the lipids represent 22% of the dry matter [24]. During germination, the lipids are decomposed to succinate that in other cell compartments (mitochondria and cytosol) ). (C) Contents of chlorophyll a ( Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 12 The various pigments examined in this work include the anthocyanins, whose synthesis is a process that is light-regulated and includes numerous steps starting from a phenylalanine precursor. Several plant species form anthocyanins in the light, while others do so in the dark, but their synthesis and content rapidly increases when exposed to light [37]. The inductive effect of light on anthocyanin synthesis was observable in all the sprouts ( Figure 6A). The anthocyanin content was significantly (p < 0.05) higher in mung beans and soybeans in the various light conditions with the respect to the dark and had the highest increase in mung beans. The BL followed by the WL were more effective than the RL, while in pumpkins and radishes, a significant (p < 0.05) increase was observed only in the BL and WL compared to the dark. Indeed, the BL is reported to significantly induce the anthocyanin accumulation in Arabidopsis seedlings [16], Chinese bayberry fruit [38], apple fruit [39] and postharvest strawberry fruit [40]. Alternatively, anthocyanin accumulation in young tissues, such as epicotyls and young leaves, is a common event in plants. As these organs lack morpho-anatomical complexity, the high presence of anthocyanins could prevent environmental stress, such as high lighting [41]. Indeed, anthocyanins that absorb wavelengths like those of chlorophyll b play an auxiliary role in photo-protecting the plant tissues. The light also influences the chlorophylls and carotenoids. These pigments increased to the different spectral ranges. After lighting, the radish had a higher content of total chlorophyll (chl a + chl b; Figure 6C). Among the different light treatments, a higher value of chlorophyll was observed ) and b ( Antioxidants 2020, 9, x FOR PEER REVIEW The various pigments examine process that is light-regulated and in Several plant species form anthocyan and content rapidly increases when e synthesis was observable in all the sp < 0.05) higher in mung beans and soy and had the highest increase in mun the RL, while in pumpkins and radis and WL compared to the dark. Ind accumulation in Arabidopsis seedlin harvest strawberry fruit [40]. Alter epicotyls and young leaves, is a com complexity, the high presence of a lighting [41]. Indeed, anthocyanins auxiliary role in photo-protecting th The light also influences the c different spectral ranges. After light chl b; Figure 6C). Among the differe ) in the sprouts 5 d after germination following various treatments. White light, WL; blue light, BL; red light, RL, and dark, D. Values represent the means of at least three replicates from four independent experiments. Identical letters over the columns indicate non-significant differences between the treatments within each segment (Tukey's HSD test, p < 0.05).
The light also influences the chlorophylls and carotenoids. These pigments increased to the different spectral ranges. After lighting, the radish had a higher content of total chlorophyll (chl a + chl b; Figure 6C). Among the different light treatments, a higher value of chlorophyll was observed under the WL in all the sprouts, except for pumpkins. The highest increase in the carotenoids was in mung beans under the WL ( Figure 6B). The lowest level of chlorophylls and carotenoids was observed in darkness for various sprouts, with the minimal value in mung beans. Interestingly, the increase in carotenoids, which are pigments that are components of the light-harvesting complex, has a protective role for the chlorophylls subjected to photo-oxidation reactions [42,43].

Conclusions
The frequent use of sprouts in vegetable diets is closely related to food safety and the nutritional benefit of their consumption. It is well known that both time and light influence seed germination. Consequently, different growth conditions might change the quality of the relative sprouts developed from the seed. In this study, after dark or light treatments to moderate irradiance (110 µmol m −2 s −1 ), the quality-related parameters were differentially influenced in the four types of sprouts analysed after 5 d of germination.
Compared to the darkness, the WL, RL and BL preserved the contents of vitamin C, carotenoids, chlorophylls, and anthocyanins in all the types of sprouts, highlighting how the use of a specific spectral wavelength increases the content of these antioxidant compounds. Their increase is useful, as they might be involved in the complex mechanism to prevent the oxidation of biological cell membranes. Interestingly, an increase in polyphenols was induced only in soybeans and only by RL. On the other hand, darkness preserved the dry matter, whereas the light decreased it.
Minimally processed sprouts may benefit from lighting during seed germination to improve their quality. The positive effects on vitamin C, carotenoids, chlorophylls, and anthocyanins in all sprouts, the increased soluble proteins and sugars respectively in soybean and pumpkin seeds, the increase of polyphenols in soybeans, together with low-cost lighting, and ease of implementation towards other vegetables greatly contribute to the light application in industrialized production. This will eventually increase the production of sprouts with higher nutritional/health value tailored for people with specific nutritional needs. Further studies will be needed to understand the molecular mechanism through which light modulates the synthesis of phytonutrients and changes the nutritional content of sprouted seeds, which can be beneficial to human health.