Effects of Darkness and Light Spectra on Nutrients and Pigments In

antioxidants

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

Linda Mastropasqua, Nunzio Dipierro and Costantino Paciolla * Department of Biology, University of Bari “Aldo Moro”, Via E. Orabona 4, 70125 Bari, Italy; [email protected] (L.M.); [email protected] (N.D.) * Correspondence: [email protected]; Tel.: +39-080-544-3557

Received: 29 May 2020; Accepted: 23 June 2020; Published: 26 June 2020

Abstract: 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.

Keywords: sprouts; vitamin C; light treatments; antioxidants; phytonutrients

1. 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].

Antioxidants 2020, 9, 558; doi:10.3390/antiox9060558 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 558 2 of 12

The temperature, time of germination and light influence the biochemical and nutritional properties of the sprouts [9]. Significant differences in the polyphenol, flavonoid and phenolic acid content in legume seeds before and after germination in darkness at 30 and 40 ◦C for 5 d are reported [7]. The light induces many developmental and physiological responses in the life cycle of the plant, including germination. The wavelength, irradiance, photoperiod and direction of the light influence the growth of the plants. Red and blue light induce a greater increase in growth and development, as they increase the photosynthetic process and regulate various responses in higher plants [10–12]. The synthesis of vitamin C (l-ascorbic acid; AsA), an important antioxidant compound, is light dependent as observed in Avena [13], broccoli [14] and asparagus [15] post-harvest. In addition, blue light is efficient at inducing anthocyanin biosynthesis [16,17]. A diet rich in flavonoids, such as anthocyanins, decreases the rate of death from cardiovascular diseases, stimulates the responses of the immune system and reduces the inflammatory process. Dietary sprouts are generally grown in darkness and appear to be yellow in colour due to their chlorophyll deficiency and are utilized uncooked. When the sprouts are exposed to the light, the genes involved in the chlorophyll and carotenoid biosynthesis are upregulated with subsequent greening [18]. However, very little attention has been paid to the role that the light quality has on phytonutrients of dietary sprouts. Therefore, the aim of this work was to provide a more in-depth investigation of the effects of white (WL), blue (BL) and red (RL) lights, and darkness (D) on four types of sprouts usually utilized for food. In particular, qualitative parameters related to physiological and biochemical processes, such as soluble sugars (d-glucose, d-fructose and sucrose), starch, soluble proteins, chlorophyll a and b and carotenoids, and compounds related to human health, such as vitamin C, anthocyanins and total phenols, were investigated in radish, soybean, mung bean and pumpkin sprouts grown under different light treatments.

2. Materials and Methods

2.1. Plant Materials, Seed Germination and Light Treatments Seeds of soybeans (Glicine max L. Merr), mung beans (Vigna radiata L. Wilczek), radishes (Raphanus sativus L.) and pumpkins (Cucurbita moschata Duch) were purchased from Bivacchi Spa (Perugia, Italy). The seeds were sanitized with 0.5% sodium hypochlorite solution (v/v) for 15 min and then thoroughly washed for 5 min with distilled water to remove any trace of sodium hypochlorite and soaked in distilled water for 2 h. Two batches of seeds of each plant species (10 g) were placed on two layers of filter paper (Whatman Grade 1) into Petri dishes (12 cm diameter). The filter paper was moistened with 5 mL of distilled water once a day. The Petri dishes were incubated in a plant growth chamber at 24 1 C with 78 2% of relative air humidity under different light conditions with a 16 h photoperiod ± ◦ ± or in continuous darkness (D). White light (WL) was provided by a T8 L18W/835 Osram 26Ø fluorescent tube with a wavelength range of 400–700 nm, and three different peaks: blue at 440 nm, green at 545 nm, yellow at 580 nm and red at 610 nm. Blue light (BL) was provided by a T8 L18W/67 Osram 26Ø fluorescent tube with a wavelength range of 400–460 nm and a peak at 440 nm. Red light (RL) was provided by a T8 L18W/60 Osram 26Ø fluorescent tube with a wavelength range of 600–630 nm and a peak at 610 nm. The photosynthetic photon flux density for each light source at the Petri dish surface were 110 1 µmoL m 2 s 1 measured with a Photo-Radiometer, Model HD2302.0 (Delta OHM SRL, ± − − Pordenone, Italy) according to the manufacturer’s instructions. Five days after germination, when reaching commercial length, the sprouts were collected, washed with sterile water, dried with absorbent paper and analysed. For instance, a picture of the shoots used is shown in Figure1. Antioxidants 2020, 9, 558 3 of 12 Antioxidants 2020, 9, x FOR PEER REVIEW 3 of 12

Figure 1. Sprouts of radishes (A), soybeans (B), mung beans (C) and pumpkinspumpkins (D) growngrown inin whitewhite lights (WL)(WL) 55 dd afterafter germination.germination. TheThe sproutssprouts showedshowed morphologicalmorphological didifferences.fferences. The soybean and mung beanbean sprouts sprouts in in WL WL had had fleshy fleshy cotyledons cotyledons that werethat alsowere present also present when the when young the leaves young appeared, leaves whileappeared, in radishes while in and radishes pumpkins, and pumpkins, the cotyledons the cotyledons changed into changed photosynthetic into photosynthetic leaflets. leaflets.

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

2.3. Anthocyanin Content

The anthocyanin content was determined as reported in [19]. [19]. Briefly, Briefly, each each sprout sprout sample (5 (5 g) was cut into pieces and incubatedincubated atat 6565 ◦°CC 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 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 0.25 A that corrects the spectrophotometrically at both 530 nm and 657 nm. The formula A530530− − 0.25 ×× A657657 that corrects the absorbance for the chlorophyll degradationdegradation productproduct waswas utilized.utilized. 2.4. Determination of the Chlorophyll and Carotenoid Contents 2.4. Determination of the Chlorophyll and Carotenoid Contents The chlorophyll and carotenoid contents were determined as described by Lichtenthaler [20] with The chlorophyll and carotenoid contents were determined as described by Lichtenthaler [20] some modifications. Five grams of fresh material was homogenized with 10 mL of absolute acetone with some modifications. Five grams of fresh material was homogenized with 10 mL of absolute and 3 mg of Na2CO3 and centrifuged at 20,000 g for 15 min. The absorbance of the supernatant acetone and 3 mg of Na2CO3 and centrifuged× at 20,000× g for 15 min. The absorbance of the was measured spectrophotometrically at 645 and 662 nm, respectively, for chlorophyll a and b and supernatant was measured spectrophotometrically at 645 and 662 nm, respectively, for chlorophyll a at 470 nm for the carotenoids. The total chlorophyll and carotenoid content was calculated using and b and at 470 nm for the carotenoids. The total chlorophyll and carotenoid content was calculated Lichtenthaler’s equations. using Lichtenthaler’s equations. 2.5. Vitamin C Content 2.5. Vitamin C Content Five grams of fresh tissue was homogenized with ten volumes of cold 5% (w/v) metaphosphoric acid inFive a porcelain grams of mortar. fresh tissue The homogenate was homoge wasnized centrifuged with ten forvolumes 15 min of at cold 20,000 5% (gw, and/v) metaphosphoric the supernatant × wasacid collectedin a porcelain for the analysismortar. ofThel-ascorbic homogenate acid (AsA) was andcentrifugedl-dehydroascorbic for 15 min acid at (DHA)20,000× as g, described and the insupernatant [21]. was collected for the analysis of L-ascorbic acid (AsA) and L-dehydroascorbic acid (DHA) as described in [21]. 2.6. Soluble Protein Assay 2.6. Soluble Protein Assay At various times, 5 g of sprouts was ground by pestle and mortar in 15 mL of extraction medium containingAt various 100 mMtimes, potassium 5 g of sprouts phosphate was ground buffer pHby pe 7.0,stle 0.5 and M mortar sorbitol, in 1 15 mM mL EDTA of extraction and 0.05% medium (w/v) cysteine.containing After 100 mM centrifugation potassium (20,000phosphateg, 20buffer min, pH 2 C),7.0, the0.5 solubleM sorbitol, fraction 1 mM was EDTA desalted and 0.05% by dialysis (w/v) × ◦ againstcysteine. 50 After mM Tris-HCl,centrifugation pH 7.8. (20,000× This desalted g, 20 min, fraction 2 °C), was the used soluble to quantify fractionthe was protein desalted content by dialysis with a Proteinagainst Assay50 mM kit Tris-HCl, from Bio-Rad pH 7.8. (Hercules, This desalted CA, USA)fraction with was bovine used to serum quantify albumin the protein as the standard content with [22]. 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. Antioxidants 2020, 9, 558 4 of 12

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.

2.7. 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.

2.8. 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.

2.9. 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 (I2KI) 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).

2.10. 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).

3. Results and Discussion

3.1. 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 (Figure2A). AntioxidantsAntioxidants 2020Antioxidants 2020, 9, 9x, FORx FOR 2020 PEER PEER, 9, REVIEWx FORREVIEW PEER REVIEW 5 of5 of12 12 5 of 12 lowerlower metabolic metaboliclower activitymetabolic activity for for activity the the low low for consum consumthe lowptionption consum of of sugars sugarsption andof and sugars starch starch andas as observed. starchobserved. as However,observed. However, otherHowever, other other components,components,components, such such as as cell cellsuch wall wall as polysaccharides, cellpolysaccharides, wall polysaccharides, lipid lipids sand and lipidmineral minerals and salts, salts,mineral may may salts,contribute contribute may contributeto to the the dry dry to the dry matter.matter. matter.Antioxidants 2020, 9, 558 AntioxidantsAntioxidants 2020, 92020, x FOR, 9, x PEER FOR PEERREVIEW REVIEW 5 of 12 6 of 126 of 12

FigureFigure 2. 2.(A (Figure)A Dry) Dry matter 2. matter ((AA)) Drycontent Dry content matter matter in in the content contentthe sprouts sprouts in in the5 the d5 sproutsd sproutsafte after germinationr germination5 5 d d afte afterr germination germination following following various following followingvarious treatments. treatments. various various treatments. treatments. (B()B Content) Content (ofB )of the Content the soluble soluble of of proteinsthe the proteins soluble soluble Figurein in theproteins proteins theFigure sprouts 3. sprouts (A in in)3. Contents5the the( Ad5 )daftersprouts sprouts Contentsafter germination of germination 5 5soluble d d ofafter after soluble germinationsugars— germinationfollowing following sugars—D-fructose, various followingvarious followingD-fructose, treatments. ( treatments. various various (), D-glucose treatments.), treatments. D-glucose ( (), sucrose ), sucrose ( ()—In )—In ValuesValues represent representValues the representthe means means of the of at meansatleast sproutsleast three ofsproutsthree at5 replic d least replicafter 5 dates three germinationafterates from replicatesreplic fromgermination four atesfour followiindependent fromindependent followi fourngfour various independentindependentng experiments. variousexperiments. treatments. treatments. experiments. Identical Identical White White light, Identical light, WL; blueWL; light,blue light, BL; red BL; light, red light, lettersletters over over letterslettersthe the columns overcolumnsover thethe indicate columns indicatecolumns RL,non-significant indicate non-significantindicate andRL, dark, and non-signiﬁcant non-significant dark, D. differences ( Bdifferences )D. Starch (B) di Starchﬀ contenterencesdifferencesbetween between content in between the the betweenin sproutstr theeatmentstr theeatments sprouts treatments the5 d afterwithintr 5eatmentswithin d germinationafter within each eachgermination within each following sprout each following various various treatments. treatments. sproutsprout (Tukey’s (Tukey’ssprout(Tukey’s HSD HSD(Tukey’s HSD test, test, test,p HSD

sugar content content thecontent protein content highest wassignificantly significantly content was valuesignificantly significantly insignificantly soybeans,increased increased (p < ( was0.05) (pincreasedp (

3.2. Effect of Light on Sugars and Starch lowerlower metabolic metabolic activity activity for for the the low low consum consumptionption of of sugars sugars and and starch starch as as observed. observed. However, However, other other components,components, such such as as cell cell wall wall polysaccharides, polysaccharides,Light lipid spectrum lipids sand and mineral properties mineral salts, salts, strongly may may contribute contribute influence to tothe the the physiology,dry dry growth, development and matter.matter. AntioxidantsAntioxidantsAntioxidantsAntioxidantsAntioxidantsphytochemical 2020 2020 2020, 2020,9 9 ,2020, , x 9x ,FOR, FORx9, , FOR 9x, PEERFOR xPEER accumulation FOR PEER PEER REVIEW REVIEWPEER REVIEW REVIEW REVIEW in planta, particularly for vegetables produced in66 controlledof of6 12of 126 12of6 of 12 12 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 Antioxidants 2020, 9, x FOR PEER REVIEW 6 of 12 dark and the different light conditions is shown in Figure3A.

FigureFigure 2. 2.(A ()A Dry) Dry matter matter content content in in the the sprouts sprouts 5 d5 dafte after germinationr germination following following various various treatments. treatments. (B()B Content) Content of of the the soluble soluble proteins proteinsFigure Figurein Figurein the FiguretheFigure sproutsFigure 3.3. sprouts 3. ((A AFigure3.() A) 3. 3.Contents (Contents)5A (Contents(d5A) dafter3.Contents)) Contentsafter(A )germination ofofContents germination of solublesoluble ofsoluble ofof soluble soluble solubleof sugars— sugars— soluble followingsugars— following sugars— sugars—sugars— sugars—DD-fructose,-fructose,D various-fructose, Dvariousd-fructose,D-fructose,D-fructose,-fructose, treatments. (( treatments. ( ( ( ), ),(( D),D),-glucose -glucoseD), d-glucose), ),-glucoseD D-glucoseD-glucose-glucose (( ( ( ( ),),(), sucrose),sucrose sucrose ),sucrose), sucrose),sucrose sucrose ( ( (( )—In ( )—In)—In( )—In)—In sprouts)—In )—In ValuesValues represent represent the the means means of of at atleastsprouts sproutsleastsprouts threesprouts threesprouts5 5 5 d d replic5d after after replicsproutsd after5 after5d ates d germinationaftergermination germinationatesafter germination5 from d germination fromafter germination four germinationfour followingfollowi followiindependent followiindependent followi followingng following various variousng variousng experiments. variousng experiments.various varioustreatments. treatments. treatments. treatments. treatments. treatments. Identical IdenticalWhite White White White White White light, light, light,light, light, light,WL; light,WL; WL;WL; WL;blueWL; blue WL; blueblue blue bluelight, light, blue light, light, light, BL; light,BL; BL; BL; red redBL; red BL; red light, light,red light,redlight, light, light, light, RL, RL, and dark, D. (B) Starch content in the sprouts 5 d after germination following various treatments. lettersletters over over the the columns columns indicate indicate RL,RL,non-significant RL,non-significant and RL,and RL,andand dark, anddark, anddark, dark, dark, D.dark,D. D. differences( (D.B B D.differences)( )BD.Starch ( StarchB)( B Starch)()B StarchStarch) Starchcontent contentbetween content between contentcontent content in in inthe the inthe sprouts in sproutsthetr the sproutstheeatmentstr eatmentssprouts sprouts sprouts 5 5 d 5d after d afterwithin5 5 after5dwithin d d aftergermination germinationafterafter germination each germinationeach germination following following following following following various various various various various various treatments. treatments. treatments. treatments. treatments. treatments. White light ( ); blue light ( ); red light ( ), and dark ( ). Values represent the means of sproutsprout (Tukey’s (Tukey’s HSD HSD test, test, p

carbohydrate carbohydrate diets. diets.

FigureFigure 4. Light 4. Light micrograph micrograph of of transversal transversal sectionsection of th thee mung mung bean bean cotyledon; cotyledon; arrow arrow indicates indicates the the starchstarch grains grains (Panel (Panel (A )).(A Light)). Light micrographs micrographs ofof transversaltransversal section section of of the the soybean soybean cotyledon; cotyledon; arrow arrow indicates the protein vacuoles (Panel (B)). indicates the protein vacuoles (Panel (B)).

3.3. Changes3.3. Changes in the in Vitaminthe Vitamin C andC and Total Total Phenolic PhenolicContents Contents The qualityThe quality and and intensity intensity of light of light are are reported reported to to be be eff effectiveective at at regulating regulating thethe AsAAsA level in plants [13]. [13]. The lighting caused a significant (p < 0.05) increase in the ascorbate total content (AsA + DHA) The lighting caused a significant (p < 0.05) increase in the ascorbate total content (AsA + DHA) in all in all the sprouts with respect to the dark (Figure 5A). Radishes contained the highest value, followed the sproutsby mung with beans respect and soybeans, to the dark while (Figure in pumpkins,5A). Radishes the total contained content of theascorbate highest was value, lower followed in both by mungthe beans light and and soybeans,darkness. No while significant in pumpkins, difference the was total observed content ofamong ascorbate the different was lower light in treatments both the light and darkness.for each type No of significant sprout. Different difference dehydrated was observed orthodox among seeds thedid dinotff erentcontain light AsA, treatments although it for is each type ofimmediately sprout. Di producedfferent dehydrated after seed imbibition. orthodox It seeds is reported did not that contain the AsA AsA, levels although are low in it dehydrated is immediately producedorthodox after soybean seed imbibition. seeds, and its It strong is reported increase that duri theng AsAgermination levels areis due low to inthe dehydrated reactivation of orthodox its soybeanbiosynthesis. seeds, and In itsparticular, strong increase the WL, duringBL and germination ultraviolet lights is due were to themore reactivation effective at of activating its biosynthesis. 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. AntioxidantsAntioxidants 2020 2020, ,9 9, ,x x FOR FOR PEER PEER REVIEW REVIEW 88 of of 12 12

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Interestingly, Interestingly, In all the sprouts,the the AsA AsA the content content content was was of reduced the the highest highest inin the theascorbate radishes radishes waswhose whose higher cotyledons cotyledons than the oxidizedbeca becameme active formactive highli photosynthetic photosyntheticghting that the tissues. tissues. biosynthesis was higher than its TheThedegradation. polyphenolspolyphenols Alternatively, areare consideredconsidered the rapid “scavengers”increase“scavengers” in the AsA ofof free freecontent radicalradical and the speciesspecies activity andand of the preservepreserve enzymes thethe of cellcell the ascorbate system is a fine strategy of defence in orthodox herbaceous plants seeds to counteract membranesmembranes from from oxidative oxidative damage, damage, thus thus benefitting benefitting human human health. health. It It is is reported reported that that various various types types the high ROS level that occurs during germination [33]. Interestingly, the AsA content was the highest ofof lightlight influenceinfluence thethe polyphenolpolyphenol contentcontent differently,differently, andand thethe responsesresponses werewere dependentdependent onon thethe in the radishes whose cotyledons became active photosynthetic tissues. Antioxidantsspecies. The2020 red, 9, 558 LED increased the phenolic compound contents in common buckwheat sprouts8 of but 12 species. TheThe red polyphenols LED increased are considered the phenolic “scavengers” compound of contentsfree radical in commonspecies and buckwheat preserve sproutsthe cell but not in Tartary buckwheat sprouts [34]. 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Therefore,Therefore, was higherthethe lightlight than generallygenerally its degradation. diddid notnot components,red LED such lights, as cellrespectively. wall polysaccharides, However, our lipiddata sindicated and mineral that when salts, comparedmay contribute to the dark,to the the dry components,influenceinfluence the thesuch biosynthesis biosynthesis as cell wall of of polysaccharides,the the total total polyphenols polyphenols lipid sin in and the the various mineralvarious sprouts. sprouts.salts, may Of Of note, contributenote, soybeans, soybeans, to the followed followed dry Alternatively,treatment the with rapid solely increase RL increased in the ( AsAp < 0.05) content the phenolic and the content, activity while of the in enzymes the other of sprouts, the ascorbate no matter.matter.byby radishes, radishes, had had a a higher higher content contentAntioxidantsAntioxidants than than mung 2020mung ,2020 9, xbeans beans ,FOR 9, x FORPEER and and PEER REVIEWpumpkins. pumpkins. REVIEW 6 of 126 of 12 systemdifference is a ﬁne between strategy light of defence and dark in orthodoxappeared herbaceous(Figure 5B). plantsTherefore, seeds the to light counteract generally the did high not ROS level thatinfluence occurs the during biosynthesis germination of the total [33 ].polyphenols Interestingly, in the the various AsA content sprouts. was Of note, the highestsoybeans, in followed the radishes whoseby cotyledons radishes, had became a higher active content photosynthetic than mung beans tissues. and pumpkins.

FigureFigure 2. 2. ( A(A) )Dry Dry matter matter content content in in the the sprouts sprouts 5 5d dafte after rgermination germination following following various various treatments. treatments. FigureFigureFigure 5.5. ((AA 5.)) Content(ContentA) Content ofof totaloftotal total ascorbate—ascorbate— ascorbate—lLL-ascorbic-ascorbic-ascorbic acid acid (AsA, (AsA,(AsA, ) )and) andand L-dehydroascorbicl -dehydroascorbicL-dehydroascorbic acid acid acid Figure 5. (A) Content of total ascorbate—FigureFigure 3. ( AL3.-ascorbic) (ContentsA) Contents acid of soluble (AsA,of soluble sugars— ) sugars—and DL-fructose,-dehydroascorbicD-fructose, ( ( ), D acid-glucose), D -glucose ( ( ), sucrose ), sucrose ( ( )—In )—In (B(B) Content) Content(DHA, of of the the soluble )—Insoluble the proteins sproutsproteins 5in din theafter the sprouts germinationsprouts 5 5d dafter fo afterllowing germination germination various treatments. following following White various various light, treatments. treatments. WL; blue (DHA,(DHA,(DHA, )—In )—In )—In the the the sproutssprouts sprouts 5 5 d dd after after germination germination germination fo fo followingllowingllowing various various various treatments. treatments. treatments. White White White light, light, light, WL; WL; blue WL;blue ValuesValues represent representlight, BL; the redthe means light,means RL,of of at andat least least sproutsdark, three sproutsthree D. 5 replic( dBreplic ) after 5Total dates afterates germination polyphenolic from fromgermination four four independentfollowi independentcontent following in various ngthe experiments. variousexperiments.sprouts treatments. treatments.5 d afterIdentical Identical White the White light, light, WL; WL; blue blue light, light, BL; redBL; light,red light, bluelight, light, BL; BL;red redlight, light, RL, RL, and and dark, dark, D. D.(B ()B Total) Total polyphenolic polyphenolic content content in in the the sprouts sprouts 5 dd afterafter thethe lettersletterslight, over over germinationBL; the redthe columns light,columns of various RL, indicate indicate and treatments. dark, non-significantRL, non-significant andD.RL, White ( andBdark,) Total lightdark, D. differences ((differencespolyphenolic BD.) Starch ( );B )blue Starch content betweenlight between content ( in the );thethe inredin tr sprouts the trtheeatmentslighteatments sprouts sprouts ( 5 d ),within after 5 within and d5 afterd germinationdark aftereach eachgermination ( the following following various various treatments. treatments. germination of various treatments. White light ( ); blue light ( ); red light ( ), and dark ( sproutsproutgermination (Tukey’s (Tukey’s ). Values ofHSD HSD various representtest, test, ptreatments. p< < 0.05). the0.05). mean WhiteWhite White WhiteWhite of light lightat light leastlightlight light (( ( thr ( ( );ee ); blue ); blue ); replicates );blue blue blue light light, light, light light ( from( (( ( ); four );); red ); red red ); redindependent lightred light light light light ( ( ( ( ( ( ),), experiments. ), and and ), and ), ),and anddarkand dark dark dark darkdark (( ( ( (). ( ). Values ). Values represent represent the meansthe means of of ). Values represent the mean of at least three replicates from four independent experiments. Values). ). ). IdenticalValues represent representletters the over mean thetheof columnsmean atat least ofleast indicateat threeat least threeleast replicatesnon-sign three thrreplicationsee replications ificantreplicates from differences fourfrom fromfrom independentfour between fourfour independen independentindependen treatments experiments.t experiments. withint experiments.experiments. Identical each Identical Identical letters letters over over the columnsthe columns Identical letters over the columns indicate non-significant differences between treatments within each lettersIdenticalsegment over letters the (Tukey’s columnsover the HSD indicatecolumns test, pindicate < non-signiﬁcant indicate 0.05).indicate non-significant non-sign non-significant diﬀificanterences differences differences between differences between treatmentsbetween between the treatments within treatmentsthe treatments each within segmentwithin eachwithin each each segment segment (Tukey’s (Tukey’s HSD HSD test, test, segment (Tukey’s HSD test, p < 0.05). InIn the(Tukey’ssegment the dry dry soybean HSD(Tukey’s soybean test, HSDseeds,p seeds,< 0.05). test, the the p

Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 12 Antioxidantsthe 2020 total, 9, x polyphenols FOR PEER REVIEW in the various sprouts. Of note, soybeans, followed by radishes,9 had of 12 a higher content than mung beans and pumpkins. The various pigments examined in this work include the anthocyanins, whose synthesis is a The various pigments examined in this work include the anthocyanins, whose synthesisprocess is that a is light-regulated and includes numerous steps starting from a phenylalanine precursor. process that3.4. Changesis light-regulated in the Anthocyanins, and includes Chlorophylls numerous and steps Carotenoids starting from After Lighta phenylalanine and Dark Treatment Severalprecursor. plant species form anthocyanins in the light, while others do so in the dark, but their synthesis Several plant species form anthocyanins in the light, while others do so in the dark, but their synthesis TheAntioxidants various 2020, pigments9, x FOR PEER examined REVIEW in this work include the anthocyanins, whoseand content synthesis9 of 12rapidly is a increases when exposed to light [37]. The inductive effect of light on anthocyanin and content rapidly increases when exposed to light [37]. The inductive effect of light on anthocyanin process that is light-regulated and includes numerous steps starting from a phenylalaninesynthesis precursor. was observable in all the sprouts (Figure 6A). The anthocyanin content was significantly (p synthesis was observableThe various in all pigments the sprouts examined (Figure in this6A). work The anthocyanininclude the anthocyanins, content was whose significantly synthesis (p is a Several plant species form anthocyanins in the light, while others do so in the dark,< but 0.05) their higher synthesis in mung beans and soybeans in the various light conditions with the respect to the dark < 0.05) higherprocess in mung that beans is light-regulated and soybeans and in includes the various numero lightus stepsconditions starting with from the a phenylalanine respect to the precursor. dark and content rapidly increases when exposed to light [37]. The inductive effect of lightand on had anthocyanin the highest increase in mung beans. The BL followed by the WL were more effective than and had the highestSeveral plant increase species in formmung anthocyanins beans. The in BL the followed light, while by others the WL do so were in the more dark, effectivebutthe their RL, synthesisthan while in pumpkins and radishes, a significant (p < 0.05) increase was observed only in the BL synthesisand content was observable rapidly increases in all when the sprouts exposed (Figure to light 6[37].A). The The inductive anthocyanin effect contentof light on was anthocyanin significantly the RL, while in pumpkins and radishes, a significant (p < 0.05) increase was observed onlyand in the WL BL compared to the dark. Indeed, the BL is reported to significantly induce the anthocyanin (p < 0.05)synthesis higher was in observable mung beans in all andthe sprouts soybeans (Figure in the 6A). various The anthocyanin light conditions content was with significantly the respect (p to the and WL compared to the dark. Indeed, the BL is reported to significantly induce the anthocyanin dark< and 0.05) had higher the in highest mung beans increase and soybeans in mung in beans. the various The BLlight followed conditions by with the the WL respect wereaccumulation to more the dark effective in Arabidopsis seedlings [16], Chinese bayberry fruit [38], apple fruit [39] and post- accumulation in Arabidopsis seedlings [16], Chinese bayberry fruit [38], apple fruit [39] and post- thanand the RL,had whilethe highest in pumpkins increase andin mung radishes, beans. a The significant BL followed (p < 0.05)by the increase WL were was more observedharvest effective onlystrawberry than in the fruit [40]. Alternatively, anthocyanin accumulation in young tissues, such as harvest strawberry fruit [40]. Alternatively, anthocyanin accumulation in young tissues, such as BL andthe WLRL, while compared in pumpkins to thedark. and radishes, Indeed, a thesignificant BL is reported (p < 0.05) toincrease significantly was observed induceepicotyls only the in anthocyanin the and BL young leaves, is a common event in plants. As these organs lack morpho-anatomical AntioxidantsAntioxidants 2020 2020, 9, x9 ,FOR x FOR PEER PEER REVIEW REVIEW epicotyls and young leaves, is a common event in plants.5 of5 of12As 12 these organs lack morpho-anatomical accumulationand WL compared in Arabidopsis to theseedlings dark. Indeed, [16], Chinesethe BL is bayberryreported to fruit significantly [38], apple induce fruit [39thecomplexity,] andanthocyanin post-harvest the high presence of anthocyanins could prevent environmental stress, such as high complexity, theaccumulation high presence in Arabidopsis of anthocyanins seedlings [16],could Chinese prevent bayberry environmental fruit [38], applestress, fruit such [39] as and high post- strawberry fruit [40]. Alternatively, anthocyanin accumulation in young tissues,lighting such as [41]. epicotyls Indeed, anthocyanins that absorb wavelengths like those of chlorophyll b play an lowerlower metabolic metabolic activity activity for for the the low low consum consumlightingptionption [41]. of of sugarsharvest Indeed,sugars andstrawberry andanthocyanins starch starch asfruit as observed. observed. [40].that Alteabsorb However,rnatively, However, wavelengths anthocyaninother other like accumulati those ofon chlorophyll in young tissues, b play such an as and young leaves, is a common event in plants. As these organs lack morpho-anatomicalauxiliary complexity, role in photo-protecting the plant tissues. components,components, such such as as cell cell wall wall polysaccharides, polysaccharides,auxiliary lipid rolelipidsepicotyls inands photo-protectingand mineral mineraland young salts, salts, leaves, maythe may plant contributeis contributea common tissues. to eventto the the dryin dry plants. As these organs lack morpho-anatomical the high presence of anthocyanins could prevent environmental stress, such as high lighting [41]. matter.matter. AntioxidantsAntioxidants 2020, 9 ,2020 x FOR, 9, complexity,PEERx FOR REVIEW PEER REVIEWthe high presence of anthocyanins could prevent environmental stress,6 of 12 such 6 of 12 as high Indeed,lighting anthocyanins [41]. Indeed, that anthocyanins absorb wavelengths that absorb like wavelengths those of chlorophylllike those of bchlorophyll play an auxiliary b play an role in photo-protectingauxiliary role thein photo-protecting plant tissues. the plant tissues.

FigureFigure 2. 2.(A ()A Dry) Dry matter matter content content in inthe the sprouts sprouts 5 d5 afted after germinationr germination following following various various treatments. treatments.

(B()B Content) Content of ofthe the soluble soluble proteins proteins Figurein inthe the Figuresprouts 3. sprouts (A) 3. Contents5 d(5A afterd) afterContents germinationof germination soluble of soluble sugars— following following sugars—D-fructose, various variousD-fructose, treatments. ( treatments. ),( D-glucose ), D-glucose ( ),( sucrose ), sucrose ( )—In( )—In Figure 6. (A) Level of anthocyanins in the sprouts 5 d after germination following various treatments; ValuesValues represent represent the the means means of ofat atleastsprouts least three threesprouts 5 Figurereplicd afterreplic 5ates d 6.germinationates after Figure( Afrom )from LevelgerminationFigure four 6. four (of A followi6.independent )anthocyanins (independent LevelA) Levelfollowing of various anthocyaninsof nganthocyaninsexperiments. inexperiments. various thetreatments. sprouts treatments. in in Identical the the Identical 5White sproutsdsprouts after Whitelight, germination5 5 d d af WL; afterlight,ter germination blue germination WL; following light, blue BL;followinglight, following variousred BL; light, various red treatments; various light, treatments; treatments;( B) Carotenoid content in the sprouts 5 d after germination following various treatments. White light (B) Carotenoid content in the sprouts 5 d after germination following various treatments. White light lettersletters over over the the columns columns indicate indicate RL,non-significant non-significant andRL, dark,( andB) Carotenoid D.dark, differences( Bdifferences()B D.Starch) Carotenoid (B content) Starchcontent between between in content content inthe the sproutsthe sproutsin intr eatmentsthetr theeatments 5sprouts sproutsd 5 afterd afterwithin within5germ 5 d dgermination after after inationeach each germination germination following following following following various various treatments.various various treatments. treatments. treatments. White light White( light); blue light ( ); red light ( ), and dark ( ). (C) Contents of chlorophyll a ( ) and b ( ); blue light ( ); red light ( ), and dark ( ). (C) Contents of chlorophyll a ( ) and b sproutsprout (Tukey’s (Tukey’s HSD HSD test, test, p

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; Figure6C). Among the di fferent 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 (Figure6B). 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].

4. 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 2 1 from the seed. In this study, after dark or light treatments to moderate irradiance (110 µmol m− s− ), 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.

Author Contributions: Conceptualization, C.P. and L.M.; methodology, N.D.; data curation, C.P. and L.M.; writing—Original draft preparation, L.M. and C.P.; writing—Review and editing, C.P.; visualization, L.M.; supervision, C.P.; funding acquisition, C.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by University of Bari “Aldo Moro” (Italy), grant number H95E10000710005. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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