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The Effect of Light on Antioxidant Properties and Metabolic Profile of Chia Microgreens

The Effect of Light on Antioxidant Properties and Metabolic Profile of Chia Microgreens applied sciences Article The E ect of Light on Antioxidant Properties and Metabolic Profile of Chia Microgreens 1 2 1 1 1 Selma Mlinaric ´ , Vlatka Gvozdic ´ , Ana Vukovic ´ , Martina Varga , Ivan Vlašicek ˇ , 1 1 , Vera Cesar and Lidija Begovic ´ * Department of Biology, J. J. Strossmayer University of Osijek, Ulica cara Hadrijana 8/A, 31000 Osijek, Croatia; smlinaric@biologija.unios.hr (S.M.); avukovic@biologija.unios.hr (A.V.); mjelosek@biologija.unios.hr (M.V.); ivan.vlasicek@gmail.com (I.V.); vcesarus@yahoo.com (V.C.) Department of Chemistry, J. J. Strossmayer University of Osijek, Ulica cara Hadrijana 8/A, 31000 Osijek, Croatia; vgvozdic@kemija.unios.hr * Correspondence: lbegovic@biologija.unios.hr; Tel.: +385-31-399-936 Received: 30 July 2020; Accepted: 17 August 2020; Published: 19 August 2020 Featured Application: Exposition of dark-grown chia microgreens to lower light intensity increases the production of bioactive compounds and enhances their antioxidative activity. Therefore, illuminated chia microgreens have the potential to be included in human diet as well as raw seeds. Abstract: Chia (Salvia hispanica L.) is a one-year plant known as a source of nutrients that can be consumed in the diet in the form of seeds or sprouts. The purpose of this study is to investigate the e ect of illumination for 24 and 48 h on dark-grown chia microgreens. Total antioxidant capacity was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assays, along with the total phenolics, ascorbic acid and cellulose content, and chlorophyll and carotenoid concentrations. Fourier transform infrared spectroscopy (FTIR) was used to evaluate the biochemical composition and elucidate the changes in compound structures between dark-grown and illuminated chia microgreens. Analysis of the results showed that illumination significantly increased the content of all measured bioactive compounds as well as antioxidative capacity, especially 48 h after exposure to light. FTIR analyses supported structural and molecular changes in chia microgreens grown under di erent light regimes. Our results suggest that illumination has a positive e ect on the antioxidant potential of chia microgreens, which may present a valuable addition to the human diet. Keywords: Salvia hispanica; antioxidant activity; DPPH; polyphenolics; ascorbic; acid; carotenoids; light 1. Introduction Chia (Salvia hispanica L.) is a one-year-old plant (Lamiaceae) that is a native species in Mexico and Northern Guatemala, where it was bred as a cereal in pre-Columbian times. An adult plant grows up to 1 m in height and has versatile leaves that are 5 cm wide and 8 cm long, with white to purple flowers. The most important product of this plant is its seeds, which are 1–2 mm in diameter, oval, black, gray, or white, with black dots [1]. The seeds are used whole or in the form of flour or oil. Although they have a neutral taste, they are an interesting addition to diet because they form a sticky mucus in contact with water [2,3]. Whole seeds and sprouts are added to salads and beverages to improve the density, and for the same reason, flour can be added to yogurt. The cultivation of chia sprouts is very simple, and they have also been recently used in the human diet for their nutritive and antioxidative properties [4,5]. Appl. Sci. 2020, 10, 5731; doi:10.3390/app10175731 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 5731 2 of 13 Sprouts or microgreens are grown from the seeds of various kinds of grains, vegetables, nuts, and legumes. They have a high content of protein, calcium, magnesium, vitamin A, vitamin B, ascorbic acid, and vitamin E [6,7]. The use of microgreens is increasingly popular and they are considered the food of the future due to their relatively light weight, fast and easy growing, and high nutritional value [6]. Light intensity, quality, and duration significantly influence plant growth and development through morphogenesis, the functioning of the photosynthetic apparatus, and metabolic pathways [8]. Light conditions also have the potential to evoke di erent actions of the antioxidant system [9] and metabolic pathways [10,11]. Light can enhance the synthesis of di erent antioxidants such as ascorbic acid [11,12], polyphenols, carotenoids, chlorophylls, and other enzymatic and nonenzymatic compounds that help in preventing and balancing oxidative damages in plants [13]. However, di erent growth conditions can have an impact on the antioxidative and nutritive properties of plant species [6]. Sprouts used in diets are usually grown in dark conditions and consumed raw. They show a pale yellow color due to the lack of chlorophyll. After exposure to light, the expression of genes involved in chlorophyll and carotenoid biosynthesis is upregulated, leading to the change of color from pale yellow to green [14,15]. Di erent sprouts or microgreens are considered to be young plants of vegetables, grains, and herbs, with two fully developed cotyledons. Due to their antioxidant capacity and various bioactive compounds with antibacterial, anti-inflammatory, and other health beneficial properties, sprouts are considered “functional food” [16], which has recently been widely grown and used in the global food system. Fourier transform infrared spectroscopy (FTIR) is widely used for the identification of biomolecules, functional groups, types of bonding, and changes in molecular conformations. Due to its extensive applicability to the di erent kinds of tissues and a small amount of sample needed for analyses, this technique has found broad applications in the analyses of metabolic profiles of di erent plant species [17]. FTIR spectroscopy is used for the determination of antioxidant potential and health benefits in chia [18–20], as well as other medicinal plants [17]. Although there have been many reports on the connection between the e ect of light on nutritive properties and the antioxidant capacity of various microgreens, few studies have investigated the e ect of light on nutritional properties and bioactive compounds in chia microgreens under di erent light regimes. Therefore, the aim of this work is to determine the e ect of illumination on dark-grown chia microgreens by measuring total antioxidant capacity, the concentration of carotenoids and chlorophylls, total soluble phenolics, and ascorbic acid and cellulose content, as well changes in biochemical composition and structure of biomolecules by Fourier transform infrared spectroscopy (FTIR). 2. Materials and Methods 2.1. Plant Material and Light Treatment Chia seeds (Salvia hispanica L., producer Golden Sun, Trittau, Germany) were planted on two layers of moistened filter paper in glass jars (400 mL). In each jar, 1.5 g of seeds were planted. The jars were then wrapped in aluminum foil and perforated on the top to allow ventilation during growth. The jars were placed in a growth chamber at 22  1 C. Plants were grown for seven days and watered 2 1 every other day. Afterward, microgreens were exposed to constant light (100 mol photons m s ) for 24 and 48 h, and the dark-etiolated plants were considered as control. Three biological replicates (each group contained 1.5 g of seeds) of samples with three light treatments were collected and used for analyses. Etiolated microgreens were additionally protected from light by placing them in the cardboard box. Appl. Sci. 2020, 10, 5731 3 of 13 2.2. Determination of Chlorophyll and Carotenoid Content One hundred milligrams of fresh seedling tissue were ground in liquid nitrogen using mortar and pestle. The extraction of pigments was performed in pure acetone for 24 h at 20 C. Next day samples were centrifuged at 18,000 g for 10 min at 4 C. The absorbance was measured at 470, 645, and 662 nm using a spectrophotometer (Specord 40, Analytik Jena, Jena, Germany), and pure acetone was used as a blank. Carotenoid and chlorophyll content were determined according to Lichtenthaler [21]. 2.3. Total Soluble Polyphenols and Protein Content Approximately 500 mg of fresh seedling tissue was used for analyses. Plant material was ground in liquid nitrogen and extracted for 24 h at 20 C in 2.5 mL of 96% ethanol [6]. Measurement of total soluble polyphenol content was performed in the reaction mixture containing 100 L of ethanol extract, 700 L of distilled H O, 50 L of Folin–Ciocalteu reagent, and 150 L of sodium carbonate solution (200 g/L). Samples were incubated in a water bath for 60 min and 37 C, and absorbance was measured spectrophotometrically at 765 nm using gallic acid (GA) as a standard. Total polyphenol content was expressed as gallic acid equivalents per g of fresh weight (FW). Protein content was determined using the Bradford assay [22]. Briefly, 500 mg of ground tissue was extracted with 1 mL of 100 mM KP bu er, pH = 7.0. After 15 min of extraction on ice, samples were centrifuged at 18,000 g for 15 min and 4 C. Supernatants were used for protein content determination. Absorbance was measured spectrophotometrically using 1 mg/mL of bovine serum albumin (BSA) as a standard. Protein content was expressed as mg per g of fresh weight (FW). 2.4. Estimation of Total Antioxidant Capacity 2.4.1. DPPH Scavenging Activity DPPH scavenging activity was determined according to the Brand–Williams method [23], using the same extract for the determination of total soluble phenolic content. The reaction mixture contained 20 L of extract and 980 L of 0.094 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) previously dissolved in methanol. The reaction was carried out in the dark at 22 C for 15 min, with occasional shaking. The standard curve was prepared by dissolving 2.5 mg of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) in 10 mL of methanol. Absorbance was measured at 515 nm. Total antioxidant activity was expressed as the equivalents of Trolox per g of fresh weight (FW). 2.4.2. Ferric Reducing Antioxidant Power Assay (FRAP) Frozen tissue powder (200 mg) was extracted on ice with the addition of cold 80% EtOH for 15 min. After the extraction, samples were placed in a hot bath at 84 C for 30 min. The homogenate was centrifuged at 22,000 g and 4 C for 15 min. The supernatant was used for further analyses of antioxidant capacity by FRAP. The FRAP assay is based on antioxidants as electron-donors in reaction with a yellow ferric 2,4,6-tripyridyl-s-triazine (Fe III TPTZ) complex, resulting in a blue-colored ferrous form (Fe II TPTZ). The intensity of the blue color, which is proportional to the reducing power of the antioxidants, was monitored spectrophotometrically at 593 nm. The reaction mixture for the FRAP assay consisted of 0.5 mM TPTZ and 1 mM FeCl 6H O in acetate bu er (pH 3.6). The sample (10 L) 3 2 and reaction mixture (290 L) were added to the wells of microtiter plates, mixed, and incubated at room temperature. After incubation, absorbance at 593 nm was measured using a microplate reader (Tecan, Spark, Männedorf, Switzerland). Total antioxidant capacity was determined using a standard curve in which Trolox was used as a standard at a concentration range from 0.25 to 2 mM. The results are expressed in mol Trolox equivalents per 100 g of fresh weight (FW). Appl. Sci. 2020, 10, 5731 4 of 13 2.5. Ascorbic Acid Content Approximately 600 mg of fresh tissue was ground with mortar and pestle in liquid nitrogen and extracted in 10 mL of distilled water. The homogenates were centrifuged for 15 min at 3000 g at 4 C. The supernatant was used for the determination of ascorbic acid content [24]. The reaction mixture consisted of 300 L of aqueous extract, 100 L of 13.3% trichloroacetic acid, 25 L of deionized water, and 75 L of 2,4-dinitrophenylhydrazine (DNPH) reagent. The DNPH reagent was prepared by dissolving 2 g of DNPH, 230 mg of thiourea, and 270 mg of CuSO in 100 mL of 5 M H SO . 4 2 4 Blanks were made in parallel for each sample, as described above, without the addition of the DNPH reagent. Samples were incubated in a water bath for 60 min at 37 C. After incubation, the DNPH reagent was added to all blanks, and 500 L of 65% H SO was added to all samples. The absorbance 2 4 was measured at 520 nm. The concentration of ascorbic acid was obtained from a standard curve with known concentrations of ascorbic acid (2.5–20 g/mL). The content of ascorbic acid is expressed in mg per 100 g of fresh weight. 2.6. Determination of Crystalline Cellulose Content Chia microgreens were dried at 65 °C for 48 h, ground with mortar and pestle, and extracted four times at 80 °C using 80% ethanol. Crystalline cellulose content was determined according to Foster et al. (2010) using Updegra reagent [25]. One mL of Updegra reagent (acetic acid: nitric acid: water, 8:1:2 v/v) was added to the 70 mg of dry tissue. Afterward, samples were heated at 100 C for 30 min, centrifuged, and the pellet was washed with water and acetone and air-dried overnight. The pellet was hydrolyzed with 72% sulfuric acid. Crystalline cellulose content was determined using the colorimetric Anthrone assay for 96-well microtiter plates [26]. For standard curve preparation, glucose (1 mg/mL) and Anthrone reagent (2 mg/mL sulfuric acid) were used. Crystalline cellulose content was measured using a microplate reader (Tecan, Spark, Männedorf, Switzerland) and expressed as glucose equivalents. 2.7. Fourier Transform Infrared Spectroscopy (FTIR) FTIR was used to screen the dark-grown and illuminated chia seedlings. After grinding, 3 mg of dry tissue sample was mixed with 100 mg of KBr (spectroscopy grade, Merck, Darmstat, Germany). Each FTIR spectrum was recorded by 20 co-added scans at a resolution of 2 cm in the region of wave number (WN) 500 to 4000 cm (FTIR-8400S, Shimadzu, Tokyo, Japan). The spectra were baseline-corrected. 2.8. Statistical Analyses The mean values between the di erent groups (dark-grown, 24 h, and 48 h) were subjected to analyses of variance one-way ANOVA. Subsequently, a posthoc analysis was performed using Fisher ’s LSD test (the least significant di erence). The experiment was repeated three times, with five technical replicates. The results are presented as mean  standard deviation (SD) of 15 replicates. The analyses were performed with Statistica 13.1. (Tibco Software Inc., Palo Alto, CA, USA). 3. Results and Discussion 3.1. E ect of Illumination on Carotenoid and Chlorophyll Content Dark-grown chia microgreens showed an etiolated, pale phenotype with increased hypocotyl length. After exposure of the etiolated chia microgreens to light, a change of color from pale to green was evident after 24 h, indicating the beginning of chlorophyll synthesis and subsequent development of the photosynthetic apparatus. Similar findings were also reported by Paiva et al. [27], where the authors investigated chia germination and seedling development under di erent light regimes. These authors Appl. Sci. 2020, 10, 5731 5 of 13 found that when chia was germinated under constant-dark conditions, seedlings had the longest shoots, while under constant-light, the shoots were shorter. The concentrations of total chlorophyll and carotenoid content di ered significantly between dark-grown microgreens and microgreens exposed to light (Figure 1). Compared to dark-grown microgreens, the increase was observable 24 h after the treatment. Additionally, there was an evident increase in the concentration of both total chlorophyll (Figure 1a) and carotenoid content (Figure 1b) in chia microgreens 48 h after the treatment. Appl. Sci. 2020, 10, x 5 of 12 Figure 1. Total chlorophyll (a) and carotenoid (b) content in dark-grown chia microgreens and Figure 1. Total chlorophyll (a) and carotenoid (b) content in dark-grown chia microgreens and microgreens after exposure to light for 24 and 48 h. Data represent mean values from three experiments microgreens after exposure to light for 24 and 48 h. Data represent mean values from three with five replicates (n = 15). The error bars show standard deviation (SD). Di erent letters signify experiments with five replicates (n = 15). The error bars show standard deviation (SD). Different letters values that are statistically di erent at p  0.05 according to Fisher ’s LSD test. signify values that are statistically different at p ≤ 0.05 according to Fisher’s LSD test. The increase of photosynthetic pigments in treated chia microgreens indicates the association The increase of photosynthetic pigments in treated chia microgreens indicates the association between chlorophyll and carotenoid synthesis and the length of exposure to light. between chlorophyll and carotenoid synthesis and the length of exposure to light. As reviewed in Solymosi and Mysliwa-Kurdziel [28], chlorophylls are good antioxidants because of As reviewed in Solymosi and Mysliwa-Kurdziel [28], chlorophylls are good antioxidants their e ective scavenging activity of reactive oxygen species (ROS) and inhibition of lipid peroxidation. because of their effective scavenging activity of reactive oxygen species (ROS) and inhibition of lipid Chlorophylls are most commonly used as natural food colorants, but, recently, their bioactive peroxidation. Chlorophylls are most commonly used as natural food colorants, but, recently, their properties [29] have become more in focus as research has shown their emerging role as potential bioactive properties [29] have become more in focus as research has shown their emerging role as prebiotics in rebalancing gut microbiota in mice [30]. potential prebiotics in rebalancing gut microbiota in mice [30]. Dietary intake of carotenoids is associated with a reduced risk of degenerative diseases [31,32] and Dietary intake of carotenoids is associated with a reduced risk of degenerative diseases [31,32] stroke prevention [33], and this suggests their anti/pro-oxidant roles [34]. In our study, light caused an and stroke prevention [33], and this suggests their anti/pro-oxidant roles [34]. In our study, light increase of carotenoid content in chia seedlings and therefore increased their antioxidative properties. caused an increase of carotenoid content in chia seedlings an 2 d there 1 fore increased their antioxidative Lower light intensity in a range from 100 to 300 mol m s had a positive e ect on photosynthetic −2 −1 properties. Lower light intensity in a range from 100 to 300 µmol m s had a positive effect on processes [35], increased chlorophyll content [36], and more e ective light use [37] in plants. In this photosynthetic processes [35], increased chlorophyll content [36], 2 1 and more effective light use [37] in study, we used the light intensity of 100 mol photons m s , which generally has a beneficiary e ect −2 −1 plants. In this study, we used the light intensity of 100 µmol photons m s , which generally has a on the synthesis of di erent investigated bioactive compounds and enhances antioxidative properties beneficiary effect on the synthesis of different investigated bioactive compounds and enhances in chia microgreens. antioxidative properties in chia microgreens. 3.2. E ect of Illumination Total Phenolic and Protein Content 3.2. Effect of Illumination Total Phenolic and Protein Content The content of total phenolics increased after the illumination with respect to the dark-grown chia The content of total phenolics increased after the illumination with respect to the dark-grown microgreens. The analysis did not show the di erences between chia microgreens after exposure to chia microgreens. The analysis did not show the differences between chia microgreens after exposure light for 24 h and 48 h (Figure 2). to light for 24 h and 48 h (Figure 2). Different soluble phenolic content reported in numerous studies have shown that the mechanisms of polyphenol synthesis vary between plant species, raw seeds, and sprouts, and is also affected by different growth conditions, germination processes, and sprout developmental stages [7]. Gómez-Favela et al. [5] showed that the controlled germination process exhibited a higher content of bioactive compounds (among others, increased total phenolics) in flour from chia seeds. Compared with the content of phenolics in other sprouts, our results showed that the amount of phenolics in illuminated chia microgreens (24 and 48 h) was higher or similar [7,38,39]. These variations could be attributed to the different enzymatic activity of hydrolases and polyphenol oxidases [40], de novo synthesis of phenolics, differences in polymerization and oxidation processes, and degradation of free or bound phenolics [41,42]. The increase of phenolics observed after exposure to light could also be attributed to the development of defense mechanisms as a response to illumination, which activates different metabolic pathways to enhance antioxidant activity in etiolated sprouts. Therefore, illuminated chia microgreens represent a significant source of phenolics and can be considered as an alternative source in diets. Appl. Sci. 2020, 10, 5731 6 of 13 Appl. Sci. 2020, 10, x 6 of 12 Figure 2. Total phenolic content (a) and protein content (b) in dark-grown chia microgreens and Figure 2. Total phenolic content (a) and protein content (b) in dark-grown chia microgreens and microgreens after exposure to light for 24 and 48 h. Data represent mean values from three experiments microgreens after exposure to light for 24 and 48 h. Data represent mean values from three with five replicates (n = 15). The error bars show standard deviation (SD). Di erent letters signify experiments with five replicates (n = 15). The error bars show standard deviation (SD). Different letters values that are statistically di erent at p  0.05 according to Fisher ’s LSD test. signify values that are statistically different at p ≤ 0.05 according to Fisher’s LSD test. Di erent soluble phenolic content reported in numerous studies have shown that the mechanisms Under constant-dark conditions, protein content was significantly higher when dark-grown chia of polyphenol synthesis vary between plant species, raw seeds, and sprouts, and is also a ected microgreens and illuminated microgreens were compared (Figure 2b). Additionally, protein content by di erent growth conditions, germination processes, and sprout developmental stages [7]. did not differ between chia microgreens 24 and 48 h after the light treatment. Proteins represent Gómez-Favela et al. [5] showed that the controlled germination process exhibited a higher content of storage for germinating seeds, and, during the germination process, they are hydrolyzed by bioactive compounds (among others, increased total phenolics) in flour from chia seeds. Compared with proteases, which increases their availability for plant growth [6]. The soluble protein content, the content of phenolics in other sprouts, our results showed that the amount of phenolics in illuminated however, also depends on the balance between synthesis and demand [6]. In a recent work by chia microgreens (24 and 48 h) was higher or similar [7,38,39]. These variations could be attributed Mastropasqua et al. [43], authors showed that exposure to light impacted protein synthesis in to the di erent enzymatic activity of hydrolases and polyphenol oxidases [40], de novo synthesis of soybean, mung bean, pumpkin, and radish sprouts differently. Namely, dark-grown mung bean phenolics, di erences in polymerization and oxidation processes, and degradation of free or bound sprouts had significantly higher soluble protein content in comparison to corresponding sprouts phenolics [41,42]. The increase of phenolics observed after exposure to light could also be attributed exposed to light, which is in accordance with our results. This decreased protein content in chia to the development of defense mechanisms as a response to illumination, which activates di erent microgreens after light treatment could be explained by the increased degradation of proteins due to metabolic pathways to enhance antioxidant activity in etiolated sprouts. Therefore, illuminated chia higher demand for developmental processes taking part in growing microgreens induced by light microgreens represent a significant source of phenolics and can be considered as an alternative source and redirecting metabolic pathways towards the synthesis of other secondary metabolites and in diets. components involved in photoprotection. Under constant-dark conditions, protein content was significantly higher when dark-grown chia microgreens and illuminated microgreens were compared (Figure 2b). Additionally, protein 3.3. Effect of Illumination on Ascorbic Acid Content content did not di er between chia microgreens 24 and 48 h after the light treatment. Proteins repr Ascorbic esent storage acid (A for A) germinating is involved in seeds, numer and, ous during functions the in p germination lants, from pr ant ocess, ioxid they ativar e def e hydr ense olyzed and photosynthesis to growth by proteases, which increases regulat their ion [44]. L availability ightfor triplant ggers grseed owthge [6rminat ]. Theion a soluble nd inf protein luenccontent, es the biosynt however hes ,is of ascorbic also depends acon id [4 the 5]. H balance igher irr between adiance le synthesis vels can incre and demand ase the cont [6]. enIn t and acc a recent umul work ation by of A Mastr A in opasqua plants [et 12]. al. [43], authors showed that exposure to light impacted protein synthesis in soybean, mung Ascorbic bean, pumpkin, acid (AA)and cont radish ent signi sprouts ficandi tly var erently ied b . Namely etween dark , dark-gr -grown and own mungillum beanin spr ate outs d ch had ia mi significantly crogreens (F higher igure 3) soluble . It is clpr eaotein rly visi content ble thain t the le comparison ngth of ito llucorr minati esponding on is associated wi sprouts exposed th increa tosi light, ng the content of AA. which is in accordance Analysis of the re with our results. sults showed th This decr at there were statistic eased protein content ally sig in chia nificant d microgr iffer eens ences after (p light ≤ 0.05) bet treatment ween all thre could bee chia sample explained bys. the increased degradation of proteins due to higher demand for Lu developmental and Guo [11] pr ob ocesses tainedtaking similapart r resu inltgr s in owing munmicr g beogr an sp eens rout induced s grown byunder light di and fferent redir ecting light regi metabolic mes (24pathways and 12 h towar light a ds nd da the synthesis rk-grown) of , showi other n secondary g that the content of metabolites AA andincrea components sed due to the involved longer expos in photoprotection. ure to illumination. When raw seeds and sprouts, such as soybean, mung bean, cowpea, and buckwheat, were 3.3. E ect of Illumination on Ascorbic Acid Content compared, studies found that the AA content was lower in raw seeds [7]. The germination process significantly increased AA content in various edible seeds and sprouts. It was suggested that an Ascorbic acid (AA) is involved in numerous functions in plants, from antioxidative defense increase of AA content and its accumulation in sprouts is due to de novo synthesis [11]. Namely, the and photosynthesis to growth regulation [44]. Light triggers seed germination and influences the synthesis of AA depends on electron transport within the photosynthetic apparatus. Since AA also plays an important role in photoprotection, exposure of dark-grown chia microgreens triggered Appl. Sci. 2020, 10, 5731 7 of 13 biosynthesis of ascorbic acid [45]. Higher irradiance levels can increase the content and accumulation of AA in plants [12]. Ascorbic acid (AA) content significantly varied between dark-grown and illuminated chia Appl. Sci. 2020, 10, x 7 of 12 microgreens (Figure 3). It is clearly visible that the length of illumination is associated with increasing the content of AA. Analysis of the results showed that there were statistically significant di erences increased AA synthesis in order to prevent photodamage in etiolated plants and diminished the (p  0.05) between all three chia samples. production of reactive oxygen species [46]. Figure 3. Ascorbic acid (AA) content in dark-grown chia microgreens and microgreens after exposure Figure 3. Ascorbic acid (AA) content in dark-grown chia microgreens and microgreens after exposure to light for 24 and 48 h. Data represent mean values from three experiments with five replicates (n = 15). to light for 24 and 48 h. Data represent mean values from three experiments with five replicates (n = The error bars show standard deviation (SD). Di erent letters signify values that are statistically 15). The error bars show standard deviation (SD). Different letters signify values that are statistically di erent at p  0.05 according to Fisher ’s LSD test. different at p ≤ 0.05 according to Fisher’s LSD test. Lu and Guo [11] obtained similar results in mung bean sprouts grown under di erent light 3.4. Crystalline Cellulose Content regimes (24 and 12 h light and dark-grown), showing that the content of AA increased due to the Dietary fibers have a beneficial effect on the gastrointestinal system, and chia seeds are rich in longer exposure to illumination. dietary fibers [47]. Since cellulose presents one of the main components of dietary fibers, we When raw seeds and sprouts, such as soybean, mung bean, cowpea, and buckwheat, investigated the effect of light on cellulose content in chia microgreens, considering that germination were compared, studies found that the AA content was lower in raw seeds [7]. The germination can impact dietary fiber depending on the germination time as well as plant species [48]. process significantly increased AA content in various edible seeds and sprouts. It was suggested that The cellulose content in dark-grown chia microgreens and microgreens 24 h after light exposure an increase of AA content and its accumulation in sprouts is due to de novo synthesis [11]. Namely, did not show significant differences. On the other hand, a significant decrease in cellulose content the synthesis of AA depends on electron transport within the photosynthetic apparatus. Since AA was observed in microgreens 48 h after exposure to light (Table 1). also plays an important role in photoprotection, exposure of dark-grown chia microgreens triggered increased AA synthesis in order to prevent photodamage in etiolated plants and diminished the Table 1. The crystalline cellulose content in dark-grown chia microgreens and microgreens 24 and 48 production of reactive oxygen species [46]. h after exposure to light. 3.4. Crystalline Cellulose Content Dark-Grown 24 h 48 h 0.656 a 0.657 a 0.317 b Dietary fibers have a beneficial e ect on the gastrointestinal system, and chia seeds are rich in dietary fibers [47]. Since cellulose presents(±0.0 one8) (± of the main0.09)components (±0.02) of dietary fibers, we investigated the e ect of light on cellulose content in chia microgreens, considering that germination can impact Data represent mean values from three experiments with five replicates (n = 15) expressed as µg dietary fiber depending on the germination time as well as plant species [48]. crystalline cellulose per mg of dry weight. Different letters signify values that are statistically different The at p ≤ cellulose 0.05 according content to F in isher’ dark-gr s LSD te own st.chia Valumicr es in ogr theeens brackets represent and microgreens ± standard de 24 h after viation (SD) light exposur . e did not show significant di erences. On the other hand, a significant decrease in cellulose content was The germination process tends to increase dietary fiber content in different plant species such as observed in microgreens 48 h after exposure to light (Table 1). peas and amaranth, as well as others [49–51]. Gómez-Favela et al. [5] reported on lower dietary fiber The germination process tends to increase dietary fiber content in di erent plant species such as peas content found in chia sprouts, suggesting that the synthesis of dietary fibers such as cellulose, and amaranth, as well as others [49–51]. Gómez-Favela et al. [5] reported on lower dietary fiber content hemicellulose, and pectin are much slower in comparison to other sprouts in particular growth found in chia sprouts, suggesting that the synthesis of dietary fibers such as cellulose, hemicellulose, conditions. and pectin are much slower in comparison to other sprouts in particular growth conditions. It is known that dark-grown microgreens have a characteristic pale phenotype with excessive shoot elongation compared to microgreens growing in normal light conditions. Therefore, more cellulose content could be found in such etiolated shoots [52]. In our study, a decline of cellulose content in microgreens, 48 h after exposure to light, could be due to the reprogramming of metabolic pathways and redirection of developmental processes in pathways that are light-dependent, such as photosynthesis and sugar synthesis, [53] as well as general plant elongation rather than cellulose synthesis. Appl. Sci. 2020, 10, 5731 8 of 13 Table 1. The crystalline cellulose content in dark-grown chia microgreens and microgreens 24 and 48 h after exposure to light. Dark-Grown 24 h 48 h 0.656 a 0.657 a 0.317 b (0.08) (0.09) (0.02) Data represent mean values from three experiments with five replicates (n = 15) expressed as g crystalline cellulose per mg of dry weight. Di erent letters signify values that are statistically di erent at p  0.05 according to Fisher ’s LSD test. Values in the brackets represent  standard deviation (SD). It is known that dark-grown microgreens have a characteristic pale phenotype with excessive shoot elongation compared to microgreens growing in normal light conditions. Therefore, more cellulose content could be found in such etiolated shoots [52]. In our study, a decline of cellulose content in microgreens, 48 h after exposure to light, could be due to the reprogramming of metabolic pathways and redirection of developmental processes in pathways that are light-dependent, such as photosynthesis and sugar synthesis, [53] as well as general plant elongation rather than cellulose synthesis. Appl. Sci. 2020, 10, x 8 of 12 3.5. E ect of Illumination on Antioxidant Capacity 3.5. Effect of Illumination on Antioxidant Capacity It has been shown that the germination process promotes antioxidant capacity in di erent It has been shown that the germination process promotes antioxidant capacity in different sprouts [4,7,10,54]. In our study, exposure to light generally increased the content of chlorophylls and sprouts [4,7,10,54]. In our study, exposure to light generally increased the content of chlorophylls and carotenoids, total polyphenolics, and ascorbic acid, which are part of the plant’s antioxidative system. carotenoids, total polyphenolics, and ascorbic acid, which are part of the plant’s antioxidative system. Accordingly, it could be expected that the antioxidant capacity in the analyzed chia microgreens Accordingly, it could be expected that the antioxidant capacity in the analyzed chia microgreens exposed to light will also be increased. exposed to light will also be increased. Light treatment caused an increase of total antioxidant capacity in chia microgreens measured by Light treatment caused an increase of total antioxidant capacity in chia microgreens measured 1, 1-diphenyl-2-picrylhydrazyl scavenging radical (DPPH) assay. A significant di erence was found by 1, 1-diphenyl-2-picrylhydrazyl scavenging radical (DPPH) assay. A significant difference was between dark-grown and chia microgreens exposed to light. There were no di erences in DPPH found between dark-grown and chia microgreens exposed to light. There were no differences in scavenging activity between microgreens exposed for 24 and 48 h (Figure 4a). DPPH scavenging activity between microgreens exposed for 24 and 48 h (Figure 4a). Figure 4. Antioxidant activity in dark-grown chia microgreens and microgreens after exposure to light Figure 4. Antioxidant activity in dark-grown chia microgreens and microgreens after exposure to for 24 and 48 h evaluated by DPPH scavenging activity (a) and FRAP assay (b). Data represent mean light for 24 and 48 h evaluated by DPPH scavenging activity (a) and FRAP assay (b). Data represent values from three experiments with five replicates (n = 15). The error bars show standard deviation mean values from three experiments with five replicates (n = 15). The error bars show standard (SD). Di erent letters signify values that are statistically di erent at p  0.05 according to Fisher ’s deviation (SD). Different letters signify values that are statistically different at p ≤ 0.05 according to LSD test. Fisher’s LSD test. In dark-grown chia microgreens, total antioxidative capacity measured by the FRAP assay was In dark-grown chia microgreens, total antioxidative capacity measured by the FRAP assay was the highest (Figure 4b). When di erent light treatments were compared, similar values were observed the highest (Figure 4b). When different light treatments were compared, similar values were in microgreens exposed to light for 24 and 48 h, whilst a significant decrease was observed 24 h after observed in microgreens exposed to light for 24 and 48 h, whilst a significant decrease was observed exposure to light in comparison to dark-grown chia microgreens. 24 h after exposure to light in comparison to dark-grown chia microgreens. The FRAP assay exhibited lower values in comparison with the DPPH assay. Other authors have also reported differences between obtained values when the two assays were used [4,55]. For instance, in 7-day-old chia sprouts exposed to 12 h/12 h (light/dark) cycles, DPPH scavenging activity was lower in comparison to the FRAP assay [54]. The reason for such discrepancies might be due to differences in the chemistry and sensitivity of these two assays. In a recently published study by Mitrović et al. [38], the authors reported that the DPPH assay is more suitable than the FRAP assay for evaluating the antioxidant activity of chia. This suggests that the obtained values of these assays are impacted by the species, germination processes, and growth conditions, as well as temperature and duration of light exposure. 3.6. Effect of Illumination on FTIR Spectra The FTIR spectra of the three chia samples under the study are depicted in Figure 5, and their main bands are summarized in Table 2. It can be observed that they are similar, in particular, the samples exposed to light for 24 and 48 h. Appl. Sci. 2020, 10, 5731 9 of 13 The FRAP assay exhibited lower values in comparison with the DPPH assay. Other authors have also reported di erences between obtained values when the two assays were used [4,55]. For instance, in 7-day-old chia sprouts exposed to 12 h/12 h (light/dark) cycles, DPPH scavenging activity was lower in comparison to the FRAP assay [54]. The reason for such discrepancies might be due to di erences in the chemistry and sensitivity of these two assays. In a recently published study by Mitrovic ´ et al. [38], the authors reported that the DPPH assay is more suitable than the FRAP assay for evaluating the antioxidant activity of chia. This suggests that the obtained values of these assays are impacted by the species, germination processes, and growth conditions, as well as temperature and duration of light exposure. 3.6. E ect of Illumination on FTIR Spectra The FTIR spectra of the three chia samples under the study are depicted in Figure 5, and their main bands are summarized in Table 2. It can be observed that they are similar, in particular, the samples Appl. Sci. 2020, 10, x 9 of 12 exposed to light for 24 and 48 h. Figure 5. FTIR spectra of dark-grown (a) chia microgreens and microgreens after exposure to light for Figure 5. FTIR spectra of dark-grown (a) chia microgreens and microgreens after exposure to light for −1 24 (b) and 48 (c) h. FTIR spectrum was recorded by 20 co-added scans at a resolution of 2 cm in the 24 (b) and 48 (c) h. FTIR spectrum was recorded by 20 co-added scans at a resolution of 2 cm in the −1 region of wave number (WN) 500 to 4000 cm . The spectra were baseline-corrected. region of wave number (WN) 500 to 4000 cm . The spectra were baseline-corrected. Table 2. Main bands in the FTIR spectra and their assignments of dark-grown chia microgreens and −1 A large number of peaks appeared in the region from 1000 to 3000 cm , indicating that chia microgreens after exposure to light for 24 and 48 h. microgreens have a rich chemical composition (proteins, lipids, carbohydrates) [18]. More precisely, −1 Dark-Grown 24 h 48 h the band located around 3290 cm represents N–H stretching vibrations that are caused by proteins Peak No. Assignments 1 1 1 Wavenumber (cm ) Wavenumber (cm ) Wavenumber (cm ) −1 [20]. The bands between 2800 and 3000 cm mainly represent C–H stretching vibrations that are I not assigned 889.9 890 889 −1 caused by lipids. The region between 1550 and 1700 cm are protein absorption bands, including II Polysaccharides 1062 1062 1063 −1 amide I and amide II. Additionally, the fingerprint region between 1000 and 1500 cm is where amide III Amide III region 1238 1239 1239 IV Amide III region 1457 1457 1457 II and the functional groups of nucleic acid and carbohydrates contribute to these absorption bands. V Amide II region 1540 1540 1540 VI Amide I region 1647 1647 1647 Table 2. Main bands in the FTIR spectra and their assignments of dark-grown chia microgreens and VII Fat content 2855 disappeared disappeared VIII Fat content 2926 2927 2925 microgreens after exposure to light for 24 and 48 h. IX Protein content 3269 3270 3271 48 h Dark-Grown 24 h Peak No. Assignments Wavenumber A large number of peaks appeared in the region−1from 1000 to 3000 cm−1 , indicating that chia Wavenumber (cm ) Wavenumber (cm ) −1 (cm ) microgreens have a rich chemical composition (proteins, lipids, carbohydrates) [18]. More precisely, I not assigned 889,9 890 889 the band located around 3290 cm represents N–H stretching vibrations that are caused by proteins [20]. II Polysaccharides 1 1062 1062 1063 The bands between 2800 and 3000 cm mainly represent C–H stretching vibrations that are caused by III Amide III region 1238 1239 1239 lipids. The region between 1550 and 1700 cm are protein absorption bands, including amide I and IV Amide III region 1457 1457 1457 V Amide II region 1540 1540 1540 VI Amide I region 1647 1647 1647 VII Fat content 2855 disappeared disappeared VIII Fat content 2926 2927 2925 IX Protein content 3269 3270 3271 In comparison with the other two samples, the dark-grown chia sample showed negligible −1 differences regarding the peak shifting in the region comprised between 1000 and 1700 cm . We −1 observed two well-pronounced bands in dark-grown chia microgreens around 2855 and 2926 cm . −1 Regarding the peak shifting, with the increment of light treatment, the band at about 2855 cm (dark- −1 grown microgreens) disappeared in illuminated microgreens (24 and 48 h), while bands at 2926 cm remain relatively stable in all chia samples. −1 Nine main bands were analyzed between 800 and 3500 cm in the spectra of the three chia −1 samples, and the band positions were relatively similar (with the exception of the band at 2855 cm ) to each other, indicating that structure was sensitive to treatments in the region of fats, vax, and lipids after 24 and 48 h of exposure to light [19,20]. Appl. Sci. 2020, 10, 5731 10 of 13 amide II. Additionally, the fingerprint region between 1000 and 1500 cm is where amide II and the functional groups of nucleic acid and carbohydrates contribute to these absorption bands. In comparison with the other two samples, the dark-grown chia sample showed negligible di erences regarding the peak shifting in the region comprised between 1000 and 1700 cm . We observed two well-pronounced bands in dark-grown chia microgreens around 2855 and 2926 cm . Regarding the peak shifting, with the increment of light treatment, the band at about 2855 cm (dark-grown microgreens) disappeared in illuminated microgreens (24 and 48 h), while bands at 2926 cm remain relatively stable in all chia samples. Nine main bands were analyzed between 800 and 3500 cm in the spectra of the three chia samples, and the band positions were relatively similar (with the exception of the band at 2855 cm ) to each other, indicating that structure was sensitive to treatments in the region of fats, vax, and lipids after 24 and 48 h of exposure to light [19,20]. 4. Conclusions Our findings show that growth conditions with the lower light intensity of 100 mol photons 2 1 m s evoked a positive e ect on total antioxidant capacity, synthesis of chlorophyll and carotenoids, total soluble phenolics, and ascorbic acid in dark-grown chia microgreens. Thus, the synthesis of bioactive compounds and the antioxidative potential of illuminated chia microgreens was improved. The DPPH assay was shown to be more sensitive in detecting antioxidative activity in comparison with FRAP. Therefore, we can conclude that chia microgreens could be considered a valuable supplement to the human diet, in addition to raw chia seeds and other popular microgreens. Present trends use di erent approaches in growing di erent sprouts and microgreens that are used for human consumption. 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The Effect of Light on Antioxidant Properties and Metabolic Profile of Chia Microgreens

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applied sciences Article The E ect of Light on Antioxidant Properties and Metabolic Profile of Chia Microgreens 1 2 1 1 1 Selma Mlinaric ´ , Vlatka Gvozdic ´ , Ana Vukovic ´ , Martina Varga , Ivan Vlašicek ˇ , 1 1 , Vera Cesar and Lidija Begovic ´ * Department of Biology, J. J. Strossmayer University of Osijek, Ulica cara Hadrijana 8/A, 31000 Osijek, Croatia; smlinaric@biologija.unios.hr (S.M.); avukovic@biologija.unios.hr (A.V.); mjelosek@biologija.unios.hr (M.V.); ivan.vlasicek@gmail.com (I.V.); vcesarus@yahoo.com (V.C.) Department of Chemistry, J. J. Strossmayer University of Osijek, Ulica cara Hadrijana 8/A, 31000 Osijek, Croatia; vgvozdic@kemija.unios.hr * Correspondence: lbegovic@biologija.unios.hr; Tel.: +385-31-399-936 Received: 30 July 2020; Accepted: 17 August 2020; Published: 19 August 2020 Featured Application: Exposition of dark-grown chia microgreens to lower light intensity increases the production of bioactive compounds and enhances their antioxidative activity. Therefore, illuminated chia microgreens have the potential to be included in human diet as well as raw seeds. Abstract: Chia (Salvia hispanica L.) is a one-year plant known as a source of nutrients that can be consumed in the diet in the form of seeds or sprouts. The purpose of this study is to investigate the e ect of illumination for 24 and 48 h on dark-grown chia microgreens. Total antioxidant capacity was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assays, along with the total phenolics, ascorbic acid and cellulose content, and chlorophyll and carotenoid concentrations. Fourier transform infrared spectroscopy (FTIR) was used to evaluate the biochemical composition and elucidate the changes in compound structures between dark-grown and illuminated chia microgreens. Analysis of the results showed that illumination significantly increased the content of all measured bioactive compounds as well as antioxidative capacity, especially 48 h after exposure to light. FTIR analyses supported structural and molecular changes in chia microgreens grown under di erent light regimes. Our results suggest that illumination has a positive e ect on the antioxidant potential of chia microgreens, which may present a valuable addition to the human diet. Keywords: Salvia hispanica; antioxidant activity; DPPH; polyphenolics; ascorbic; acid; carotenoids; light 1. Introduction Chia (Salvia hispanica L.) is a one-year-old plant (Lamiaceae) that is a native species in Mexico and Northern Guatemala, where it was bred as a cereal in pre-Columbian times. An adult plant grows up to 1 m in height and has versatile leaves that are 5 cm wide and 8 cm long, with white to purple flowers. The most important product of this plant is its seeds, which are 1–2 mm in diameter, oval, black, gray, or white, with black dots [1]. The seeds are used whole or in the form of flour or oil. Although they have a neutral taste, they are an interesting addition to diet because they form a sticky mucus in contact with water [2,3]. Whole seeds and sprouts are added to salads and beverages to improve the density, and for the same reason, flour can be added to yogurt. The cultivation of chia sprouts is very simple, and they have also been recently used in the human diet for their nutritive and antioxidative properties [4,5]. Appl. Sci. 2020, 10, 5731; doi:10.3390/app10175731 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 5731 2 of 13 Sprouts or microgreens are grown from the seeds of various kinds of grains, vegetables, nuts, and legumes. They have a high content of protein, calcium, magnesium, vitamin A, vitamin B, ascorbic acid, and vitamin E [6,7]. The use of microgreens is increasingly popular and they are considered the food of the future due to their relatively light weight, fast and easy growing, and high nutritional value [6]. Light intensity, quality, and duration significantly influence plant growth and development through morphogenesis, the functioning of the photosynthetic apparatus, and metabolic pathways [8]. Light conditions also have the potential to evoke di erent actions of the antioxidant system [9] and metabolic pathways [10,11]. Light can enhance the synthesis of di erent antioxidants such as ascorbic acid [11,12], polyphenols, carotenoids, chlorophylls, and other enzymatic and nonenzymatic compounds that help in preventing and balancing oxidative damages in plants [13]. However, di erent growth conditions can have an impact on the antioxidative and nutritive properties of plant species [6]. Sprouts used in diets are usually grown in dark conditions and consumed raw. They show a pale yellow color due to the lack of chlorophyll. After exposure to light, the expression of genes involved in chlorophyll and carotenoid biosynthesis is upregulated, leading to the change of color from pale yellow to green [14,15]. Di erent sprouts or microgreens are considered to be young plants of vegetables, grains, and herbs, with two fully developed cotyledons. Due to their antioxidant capacity and various bioactive compounds with antibacterial, anti-inflammatory, and other health beneficial properties, sprouts are considered “functional food” [16], which has recently been widely grown and used in the global food system. Fourier transform infrared spectroscopy (FTIR) is widely used for the identification of biomolecules, functional groups, types of bonding, and changes in molecular conformations. Due to its extensive applicability to the di erent kinds of tissues and a small amount of sample needed for analyses, this technique has found broad applications in the analyses of metabolic profiles of di erent plant species [17]. FTIR spectroscopy is used for the determination of antioxidant potential and health benefits in chia [18–20], as well as other medicinal plants [17]. Although there have been many reports on the connection between the e ect of light on nutritive properties and the antioxidant capacity of various microgreens, few studies have investigated the e ect of light on nutritional properties and bioactive compounds in chia microgreens under di erent light regimes. Therefore, the aim of this work is to determine the e ect of illumination on dark-grown chia microgreens by measuring total antioxidant capacity, the concentration of carotenoids and chlorophylls, total soluble phenolics, and ascorbic acid and cellulose content, as well changes in biochemical composition and structure of biomolecules by Fourier transform infrared spectroscopy (FTIR). 2. Materials and Methods 2.1. Plant Material and Light Treatment Chia seeds (Salvia hispanica L., producer Golden Sun, Trittau, Germany) were planted on two layers of moistened filter paper in glass jars (400 mL). In each jar, 1.5 g of seeds were planted. The jars were then wrapped in aluminum foil and perforated on the top to allow ventilation during growth. The jars were placed in a growth chamber at 22  1 C. Plants were grown for seven days and watered 2 1 every other day. Afterward, microgreens were exposed to constant light (100 mol photons m s ) for 24 and 48 h, and the dark-etiolated plants were considered as control. Three biological replicates (each group contained 1.5 g of seeds) of samples with three light treatments were collected and used for analyses. Etiolated microgreens were additionally protected from light by placing them in the cardboard box. Appl. Sci. 2020, 10, 5731 3 of 13 2.2. Determination of Chlorophyll and Carotenoid Content One hundred milligrams of fresh seedling tissue were ground in liquid nitrogen using mortar and pestle. The extraction of pigments was performed in pure acetone for 24 h at 20 C. Next day samples were centrifuged at 18,000 g for 10 min at 4 C. The absorbance was measured at 470, 645, and 662 nm using a spectrophotometer (Specord 40, Analytik Jena, Jena, Germany), and pure acetone was used as a blank. Carotenoid and chlorophyll content were determined according to Lichtenthaler [21]. 2.3. Total Soluble Polyphenols and Protein Content Approximately 500 mg of fresh seedling tissue was used for analyses. Plant material was ground in liquid nitrogen and extracted for 24 h at 20 C in 2.5 mL of 96% ethanol [6]. Measurement of total soluble polyphenol content was performed in the reaction mixture containing 100 L of ethanol extract, 700 L of distilled H O, 50 L of Folin–Ciocalteu reagent, and 150 L of sodium carbonate solution (200 g/L). Samples were incubated in a water bath for 60 min and 37 C, and absorbance was measured spectrophotometrically at 765 nm using gallic acid (GA) as a standard. Total polyphenol content was expressed as gallic acid equivalents per g of fresh weight (FW). Protein content was determined using the Bradford assay [22]. Briefly, 500 mg of ground tissue was extracted with 1 mL of 100 mM KP bu er, pH = 7.0. After 15 min of extraction on ice, samples were centrifuged at 18,000 g for 15 min and 4 C. Supernatants were used for protein content determination. Absorbance was measured spectrophotometrically using 1 mg/mL of bovine serum albumin (BSA) as a standard. Protein content was expressed as mg per g of fresh weight (FW). 2.4. Estimation of Total Antioxidant Capacity 2.4.1. DPPH Scavenging Activity DPPH scavenging activity was determined according to the Brand–Williams method [23], using the same extract for the determination of total soluble phenolic content. The reaction mixture contained 20 L of extract and 980 L of 0.094 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) previously dissolved in methanol. The reaction was carried out in the dark at 22 C for 15 min, with occasional shaking. The standard curve was prepared by dissolving 2.5 mg of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) in 10 mL of methanol. Absorbance was measured at 515 nm. Total antioxidant activity was expressed as the equivalents of Trolox per g of fresh weight (FW). 2.4.2. Ferric Reducing Antioxidant Power Assay (FRAP) Frozen tissue powder (200 mg) was extracted on ice with the addition of cold 80% EtOH for 15 min. After the extraction, samples were placed in a hot bath at 84 C for 30 min. The homogenate was centrifuged at 22,000 g and 4 C for 15 min. The supernatant was used for further analyses of antioxidant capacity by FRAP. The FRAP assay is based on antioxidants as electron-donors in reaction with a yellow ferric 2,4,6-tripyridyl-s-triazine (Fe III TPTZ) complex, resulting in a blue-colored ferrous form (Fe II TPTZ). The intensity of the blue color, which is proportional to the reducing power of the antioxidants, was monitored spectrophotometrically at 593 nm. The reaction mixture for the FRAP assay consisted of 0.5 mM TPTZ and 1 mM FeCl 6H O in acetate bu er (pH 3.6). The sample (10 L) 3 2 and reaction mixture (290 L) were added to the wells of microtiter plates, mixed, and incubated at room temperature. After incubation, absorbance at 593 nm was measured using a microplate reader (Tecan, Spark, Männedorf, Switzerland). Total antioxidant capacity was determined using a standard curve in which Trolox was used as a standard at a concentration range from 0.25 to 2 mM. The results are expressed in mol Trolox equivalents per 100 g of fresh weight (FW). Appl. Sci. 2020, 10, 5731 4 of 13 2.5. Ascorbic Acid Content Approximately 600 mg of fresh tissue was ground with mortar and pestle in liquid nitrogen and extracted in 10 mL of distilled water. The homogenates were centrifuged for 15 min at 3000 g at 4 C. The supernatant was used for the determination of ascorbic acid content [24]. The reaction mixture consisted of 300 L of aqueous extract, 100 L of 13.3% trichloroacetic acid, 25 L of deionized water, and 75 L of 2,4-dinitrophenylhydrazine (DNPH) reagent. The DNPH reagent was prepared by dissolving 2 g of DNPH, 230 mg of thiourea, and 270 mg of CuSO in 100 mL of 5 M H SO . 4 2 4 Blanks were made in parallel for each sample, as described above, without the addition of the DNPH reagent. Samples were incubated in a water bath for 60 min at 37 C. After incubation, the DNPH reagent was added to all blanks, and 500 L of 65% H SO was added to all samples. The absorbance 2 4 was measured at 520 nm. The concentration of ascorbic acid was obtained from a standard curve with known concentrations of ascorbic acid (2.5–20 g/mL). The content of ascorbic acid is expressed in mg per 100 g of fresh weight. 2.6. Determination of Crystalline Cellulose Content Chia microgreens were dried at 65 °C for 48 h, ground with mortar and pestle, and extracted four times at 80 °C using 80% ethanol. Crystalline cellulose content was determined according to Foster et al. (2010) using Updegra reagent [25]. One mL of Updegra reagent (acetic acid: nitric acid: water, 8:1:2 v/v) was added to the 70 mg of dry tissue. Afterward, samples were heated at 100 C for 30 min, centrifuged, and the pellet was washed with water and acetone and air-dried overnight. The pellet was hydrolyzed with 72% sulfuric acid. Crystalline cellulose content was determined using the colorimetric Anthrone assay for 96-well microtiter plates [26]. For standard curve preparation, glucose (1 mg/mL) and Anthrone reagent (2 mg/mL sulfuric acid) were used. Crystalline cellulose content was measured using a microplate reader (Tecan, Spark, Männedorf, Switzerland) and expressed as glucose equivalents. 2.7. Fourier Transform Infrared Spectroscopy (FTIR) FTIR was used to screen the dark-grown and illuminated chia seedlings. After grinding, 3 mg of dry tissue sample was mixed with 100 mg of KBr (spectroscopy grade, Merck, Darmstat, Germany). Each FTIR spectrum was recorded by 20 co-added scans at a resolution of 2 cm in the region of wave number (WN) 500 to 4000 cm (FTIR-8400S, Shimadzu, Tokyo, Japan). The spectra were baseline-corrected. 2.8. Statistical Analyses The mean values between the di erent groups (dark-grown, 24 h, and 48 h) were subjected to analyses of variance one-way ANOVA. Subsequently, a posthoc analysis was performed using Fisher ’s LSD test (the least significant di erence). The experiment was repeated three times, with five technical replicates. The results are presented as mean  standard deviation (SD) of 15 replicates. The analyses were performed with Statistica 13.1. (Tibco Software Inc., Palo Alto, CA, USA). 3. Results and Discussion 3.1. E ect of Illumination on Carotenoid and Chlorophyll Content Dark-grown chia microgreens showed an etiolated, pale phenotype with increased hypocotyl length. After exposure of the etiolated chia microgreens to light, a change of color from pale to green was evident after 24 h, indicating the beginning of chlorophyll synthesis and subsequent development of the photosynthetic apparatus. Similar findings were also reported by Paiva et al. [27], where the authors investigated chia germination and seedling development under di erent light regimes. These authors Appl. Sci. 2020, 10, 5731 5 of 13 found that when chia was germinated under constant-dark conditions, seedlings had the longest shoots, while under constant-light, the shoots were shorter. The concentrations of total chlorophyll and carotenoid content di ered significantly between dark-grown microgreens and microgreens exposed to light (Figure 1). Compared to dark-grown microgreens, the increase was observable 24 h after the treatment. Additionally, there was an evident increase in the concentration of both total chlorophyll (Figure 1a) and carotenoid content (Figure 1b) in chia microgreens 48 h after the treatment. Appl. Sci. 2020, 10, x 5 of 12 Figure 1. Total chlorophyll (a) and carotenoid (b) content in dark-grown chia microgreens and Figure 1. Total chlorophyll (a) and carotenoid (b) content in dark-grown chia microgreens and microgreens after exposure to light for 24 and 48 h. Data represent mean values from three experiments microgreens after exposure to light for 24 and 48 h. Data represent mean values from three with five replicates (n = 15). The error bars show standard deviation (SD). Di erent letters signify experiments with five replicates (n = 15). The error bars show standard deviation (SD). Different letters values that are statistically di erent at p  0.05 according to Fisher ’s LSD test. signify values that are statistically different at p ≤ 0.05 according to Fisher’s LSD test. The increase of photosynthetic pigments in treated chia microgreens indicates the association The increase of photosynthetic pigments in treated chia microgreens indicates the association between chlorophyll and carotenoid synthesis and the length of exposure to light. between chlorophyll and carotenoid synthesis and the length of exposure to light. As reviewed in Solymosi and Mysliwa-Kurdziel [28], chlorophylls are good antioxidants because of As reviewed in Solymosi and Mysliwa-Kurdziel [28], chlorophylls are good antioxidants their e ective scavenging activity of reactive oxygen species (ROS) and inhibition of lipid peroxidation. because of their effective scavenging activity of reactive oxygen species (ROS) and inhibition of lipid Chlorophylls are most commonly used as natural food colorants, but, recently, their bioactive peroxidation. Chlorophylls are most commonly used as natural food colorants, but, recently, their properties [29] have become more in focus as research has shown their emerging role as potential bioactive properties [29] have become more in focus as research has shown their emerging role as prebiotics in rebalancing gut microbiota in mice [30]. potential prebiotics in rebalancing gut microbiota in mice [30]. Dietary intake of carotenoids is associated with a reduced risk of degenerative diseases [31,32] and Dietary intake of carotenoids is associated with a reduced risk of degenerative diseases [31,32] stroke prevention [33], and this suggests their anti/pro-oxidant roles [34]. In our study, light caused an and stroke prevention [33], and this suggests their anti/pro-oxidant roles [34]. In our study, light increase of carotenoid content in chia seedlings and therefore increased their antioxidative properties. caused an increase of carotenoid content in chia seedlings an 2 d there 1 fore increased their antioxidative Lower light intensity in a range from 100 to 300 mol m s had a positive e ect on photosynthetic −2 −1 properties. Lower light intensity in a range from 100 to 300 µmol m s had a positive effect on processes [35], increased chlorophyll content [36], and more e ective light use [37] in plants. In this photosynthetic processes [35], increased chlorophyll content [36], 2 1 and more effective light use [37] in study, we used the light intensity of 100 mol photons m s , which generally has a beneficiary e ect −2 −1 plants. In this study, we used the light intensity of 100 µmol photons m s , which generally has a on the synthesis of di erent investigated bioactive compounds and enhances antioxidative properties beneficiary effect on the synthesis of different investigated bioactive compounds and enhances in chia microgreens. antioxidative properties in chia microgreens. 3.2. E ect of Illumination Total Phenolic and Protein Content 3.2. Effect of Illumination Total Phenolic and Protein Content The content of total phenolics increased after the illumination with respect to the dark-grown chia The content of total phenolics increased after the illumination with respect to the dark-grown microgreens. The analysis did not show the di erences between chia microgreens after exposure to chia microgreens. The analysis did not show the differences between chia microgreens after exposure light for 24 h and 48 h (Figure 2). to light for 24 h and 48 h (Figure 2). Different soluble phenolic content reported in numerous studies have shown that the mechanisms of polyphenol synthesis vary between plant species, raw seeds, and sprouts, and is also affected by different growth conditions, germination processes, and sprout developmental stages [7]. Gómez-Favela et al. [5] showed that the controlled germination process exhibited a higher content of bioactive compounds (among others, increased total phenolics) in flour from chia seeds. Compared with the content of phenolics in other sprouts, our results showed that the amount of phenolics in illuminated chia microgreens (24 and 48 h) was higher or similar [7,38,39]. These variations could be attributed to the different enzymatic activity of hydrolases and polyphenol oxidases [40], de novo synthesis of phenolics, differences in polymerization and oxidation processes, and degradation of free or bound phenolics [41,42]. The increase of phenolics observed after exposure to light could also be attributed to the development of defense mechanisms as a response to illumination, which activates different metabolic pathways to enhance antioxidant activity in etiolated sprouts. Therefore, illuminated chia microgreens represent a significant source of phenolics and can be considered as an alternative source in diets. Appl. Sci. 2020, 10, 5731 6 of 13 Appl. Sci. 2020, 10, x 6 of 12 Figure 2. Total phenolic content (a) and protein content (b) in dark-grown chia microgreens and Figure 2. Total phenolic content (a) and protein content (b) in dark-grown chia microgreens and microgreens after exposure to light for 24 and 48 h. Data represent mean values from three experiments microgreens after exposure to light for 24 and 48 h. Data represent mean values from three with five replicates (n = 15). The error bars show standard deviation (SD). Di erent letters signify experiments with five replicates (n = 15). The error bars show standard deviation (SD). Different letters values that are statistically di erent at p  0.05 according to Fisher ’s LSD test. signify values that are statistically different at p ≤ 0.05 according to Fisher’s LSD test. Di erent soluble phenolic content reported in numerous studies have shown that the mechanisms Under constant-dark conditions, protein content was significantly higher when dark-grown chia of polyphenol synthesis vary between plant species, raw seeds, and sprouts, and is also a ected microgreens and illuminated microgreens were compared (Figure 2b). Additionally, protein content by di erent growth conditions, germination processes, and sprout developmental stages [7]. did not differ between chia microgreens 24 and 48 h after the light treatment. Proteins represent Gómez-Favela et al. [5] showed that the controlled germination process exhibited a higher content of storage for germinating seeds, and, during the germination process, they are hydrolyzed by bioactive compounds (among others, increased total phenolics) in flour from chia seeds. Compared with proteases, which increases their availability for plant growth [6]. The soluble protein content, the content of phenolics in other sprouts, our results showed that the amount of phenolics in illuminated however, also depends on the balance between synthesis and demand [6]. In a recent work by chia microgreens (24 and 48 h) was higher or similar [7,38,39]. These variations could be attributed Mastropasqua et al. [43], authors showed that exposure to light impacted protein synthesis in to the di erent enzymatic activity of hydrolases and polyphenol oxidases [40], de novo synthesis of soybean, mung bean, pumpkin, and radish sprouts differently. Namely, dark-grown mung bean phenolics, di erences in polymerization and oxidation processes, and degradation of free or bound sprouts had significantly higher soluble protein content in comparison to corresponding sprouts phenolics [41,42]. The increase of phenolics observed after exposure to light could also be attributed exposed to light, which is in accordance with our results. This decreased protein content in chia to the development of defense mechanisms as a response to illumination, which activates di erent microgreens after light treatment could be explained by the increased degradation of proteins due to metabolic pathways to enhance antioxidant activity in etiolated sprouts. Therefore, illuminated chia higher demand for developmental processes taking part in growing microgreens induced by light microgreens represent a significant source of phenolics and can be considered as an alternative source and redirecting metabolic pathways towards the synthesis of other secondary metabolites and in diets. components involved in photoprotection. Under constant-dark conditions, protein content was significantly higher when dark-grown chia microgreens and illuminated microgreens were compared (Figure 2b). Additionally, protein 3.3. Effect of Illumination on Ascorbic Acid Content content did not di er between chia microgreens 24 and 48 h after the light treatment. Proteins repr Ascorbic esent storage acid (A for A) germinating is involved in seeds, numer and, ous during functions the in p germination lants, from pr ant ocess, ioxid they ativar e def e hydr ense olyzed and photosynthesis to growth by proteases, which increases regulat their ion [44]. L availability ightfor triplant ggers grseed owthge [6rminat ]. Theion a soluble nd inf protein luenccontent, es the biosynt however hes ,is of ascorbic also depends acon id [4 the 5]. H balance igher irr between adiance le synthesis vels can incre and demand ase the cont [6]. enIn t and acc a recent umul work ation by of A Mastr A in opasqua plants [et 12]. al. [43], authors showed that exposure to light impacted protein synthesis in soybean, mung Ascorbic bean, pumpkin, acid (AA)and cont radish ent signi sprouts ficandi tly var erently ied b . Namely etween dark , dark-gr -grown and own mungillum beanin spr ate outs d ch had ia mi significantly crogreens (F higher igure 3) soluble . It is clpr eaotein rly visi content ble thain t the le comparison ngth of ito llucorr minati esponding on is associated wi sprouts exposed th increa tosi light, ng the content of AA. which is in accordance Analysis of the re with our results. sults showed th This decr at there were statistic eased protein content ally sig in chia nificant d microgr iffer eens ences after (p light ≤ 0.05) bet treatment ween all thre could bee chia sample explained bys. the increased degradation of proteins due to higher demand for Lu developmental and Guo [11] pr ob ocesses tainedtaking similapart r resu inltgr s in owing munmicr g beogr an sp eens rout induced s grown byunder light di and fferent redir ecting light regi metabolic mes (24pathways and 12 h towar light a ds nd da the synthesis rk-grown) of , showi other n secondary g that the content of metabolites AA andincrea components sed due to the involved longer expos in photoprotection. ure to illumination. When raw seeds and sprouts, such as soybean, mung bean, cowpea, and buckwheat, were 3.3. E ect of Illumination on Ascorbic Acid Content compared, studies found that the AA content was lower in raw seeds [7]. The germination process significantly increased AA content in various edible seeds and sprouts. It was suggested that an Ascorbic acid (AA) is involved in numerous functions in plants, from antioxidative defense increase of AA content and its accumulation in sprouts is due to de novo synthesis [11]. Namely, the and photosynthesis to growth regulation [44]. Light triggers seed germination and influences the synthesis of AA depends on electron transport within the photosynthetic apparatus. Since AA also plays an important role in photoprotection, exposure of dark-grown chia microgreens triggered Appl. Sci. 2020, 10, 5731 7 of 13 biosynthesis of ascorbic acid [45]. Higher irradiance levels can increase the content and accumulation of AA in plants [12]. Ascorbic acid (AA) content significantly varied between dark-grown and illuminated chia Appl. Sci. 2020, 10, x 7 of 12 microgreens (Figure 3). It is clearly visible that the length of illumination is associated with increasing the content of AA. Analysis of the results showed that there were statistically significant di erences increased AA synthesis in order to prevent photodamage in etiolated plants and diminished the (p  0.05) between all three chia samples. production of reactive oxygen species [46]. Figure 3. Ascorbic acid (AA) content in dark-grown chia microgreens and microgreens after exposure Figure 3. Ascorbic acid (AA) content in dark-grown chia microgreens and microgreens after exposure to light for 24 and 48 h. Data represent mean values from three experiments with five replicates (n = 15). to light for 24 and 48 h. Data represent mean values from three experiments with five replicates (n = The error bars show standard deviation (SD). Di erent letters signify values that are statistically 15). The error bars show standard deviation (SD). Different letters signify values that are statistically di erent at p  0.05 according to Fisher ’s LSD test. different at p ≤ 0.05 according to Fisher’s LSD test. Lu and Guo [11] obtained similar results in mung bean sprouts grown under di erent light 3.4. Crystalline Cellulose Content regimes (24 and 12 h light and dark-grown), showing that the content of AA increased due to the Dietary fibers have a beneficial effect on the gastrointestinal system, and chia seeds are rich in longer exposure to illumination. dietary fibers [47]. Since cellulose presents one of the main components of dietary fibers, we When raw seeds and sprouts, such as soybean, mung bean, cowpea, and buckwheat, investigated the effect of light on cellulose content in chia microgreens, considering that germination were compared, studies found that the AA content was lower in raw seeds [7]. The germination can impact dietary fiber depending on the germination time as well as plant species [48]. process significantly increased AA content in various edible seeds and sprouts. It was suggested that The cellulose content in dark-grown chia microgreens and microgreens 24 h after light exposure an increase of AA content and its accumulation in sprouts is due to de novo synthesis [11]. Namely, did not show significant differences. On the other hand, a significant decrease in cellulose content the synthesis of AA depends on electron transport within the photosynthetic apparatus. Since AA was observed in microgreens 48 h after exposure to light (Table 1). also plays an important role in photoprotection, exposure of dark-grown chia microgreens triggered increased AA synthesis in order to prevent photodamage in etiolated plants and diminished the Table 1. The crystalline cellulose content in dark-grown chia microgreens and microgreens 24 and 48 production of reactive oxygen species [46]. h after exposure to light. 3.4. Crystalline Cellulose Content Dark-Grown 24 h 48 h 0.656 a 0.657 a 0.317 b Dietary fibers have a beneficial e ect on the gastrointestinal system, and chia seeds are rich in dietary fibers [47]. Since cellulose presents(±0.0 one8) (± of the main0.09)components (±0.02) of dietary fibers, we investigated the e ect of light on cellulose content in chia microgreens, considering that germination can impact Data represent mean values from three experiments with five replicates (n = 15) expressed as µg dietary fiber depending on the germination time as well as plant species [48]. crystalline cellulose per mg of dry weight. Different letters signify values that are statistically different The at p ≤ cellulose 0.05 according content to F in isher’ dark-gr s LSD te own st.chia Valumicr es in ogr theeens brackets represent and microgreens ± standard de 24 h after viation (SD) light exposur . e did not show significant di erences. On the other hand, a significant decrease in cellulose content was The germination process tends to increase dietary fiber content in different plant species such as observed in microgreens 48 h after exposure to light (Table 1). peas and amaranth, as well as others [49–51]. Gómez-Favela et al. [5] reported on lower dietary fiber The germination process tends to increase dietary fiber content in di erent plant species such as peas content found in chia sprouts, suggesting that the synthesis of dietary fibers such as cellulose, and amaranth, as well as others [49–51]. Gómez-Favela et al. [5] reported on lower dietary fiber content hemicellulose, and pectin are much slower in comparison to other sprouts in particular growth found in chia sprouts, suggesting that the synthesis of dietary fibers such as cellulose, hemicellulose, conditions. and pectin are much slower in comparison to other sprouts in particular growth conditions. It is known that dark-grown microgreens have a characteristic pale phenotype with excessive shoot elongation compared to microgreens growing in normal light conditions. Therefore, more cellulose content could be found in such etiolated shoots [52]. In our study, a decline of cellulose content in microgreens, 48 h after exposure to light, could be due to the reprogramming of metabolic pathways and redirection of developmental processes in pathways that are light-dependent, such as photosynthesis and sugar synthesis, [53] as well as general plant elongation rather than cellulose synthesis. Appl. Sci. 2020, 10, 5731 8 of 13 Table 1. The crystalline cellulose content in dark-grown chia microgreens and microgreens 24 and 48 h after exposure to light. Dark-Grown 24 h 48 h 0.656 a 0.657 a 0.317 b (0.08) (0.09) (0.02) Data represent mean values from three experiments with five replicates (n = 15) expressed as g crystalline cellulose per mg of dry weight. Di erent letters signify values that are statistically di erent at p  0.05 according to Fisher ’s LSD test. Values in the brackets represent  standard deviation (SD). It is known that dark-grown microgreens have a characteristic pale phenotype with excessive shoot elongation compared to microgreens growing in normal light conditions. Therefore, more cellulose content could be found in such etiolated shoots [52]. In our study, a decline of cellulose content in microgreens, 48 h after exposure to light, could be due to the reprogramming of metabolic pathways and redirection of developmental processes in pathways that are light-dependent, such as photosynthesis and sugar synthesis, [53] as well as general plant elongation rather than cellulose synthesis. Appl. Sci. 2020, 10, x 8 of 12 3.5. E ect of Illumination on Antioxidant Capacity 3.5. Effect of Illumination on Antioxidant Capacity It has been shown that the germination process promotes antioxidant capacity in di erent It has been shown that the germination process promotes antioxidant capacity in different sprouts [4,7,10,54]. In our study, exposure to light generally increased the content of chlorophylls and sprouts [4,7,10,54]. In our study, exposure to light generally increased the content of chlorophylls and carotenoids, total polyphenolics, and ascorbic acid, which are part of the plant’s antioxidative system. carotenoids, total polyphenolics, and ascorbic acid, which are part of the plant’s antioxidative system. Accordingly, it could be expected that the antioxidant capacity in the analyzed chia microgreens Accordingly, it could be expected that the antioxidant capacity in the analyzed chia microgreens exposed to light will also be increased. exposed to light will also be increased. Light treatment caused an increase of total antioxidant capacity in chia microgreens measured by Light treatment caused an increase of total antioxidant capacity in chia microgreens measured 1, 1-diphenyl-2-picrylhydrazyl scavenging radical (DPPH) assay. A significant di erence was found by 1, 1-diphenyl-2-picrylhydrazyl scavenging radical (DPPH) assay. A significant difference was between dark-grown and chia microgreens exposed to light. There were no di erences in DPPH found between dark-grown and chia microgreens exposed to light. There were no differences in scavenging activity between microgreens exposed for 24 and 48 h (Figure 4a). DPPH scavenging activity between microgreens exposed for 24 and 48 h (Figure 4a). Figure 4. Antioxidant activity in dark-grown chia microgreens and microgreens after exposure to light Figure 4. Antioxidant activity in dark-grown chia microgreens and microgreens after exposure to for 24 and 48 h evaluated by DPPH scavenging activity (a) and FRAP assay (b). Data represent mean light for 24 and 48 h evaluated by DPPH scavenging activity (a) and FRAP assay (b). Data represent values from three experiments with five replicates (n = 15). The error bars show standard deviation mean values from three experiments with five replicates (n = 15). The error bars show standard (SD). Di erent letters signify values that are statistically di erent at p  0.05 according to Fisher ’s deviation (SD). Different letters signify values that are statistically different at p ≤ 0.05 according to LSD test. Fisher’s LSD test. In dark-grown chia microgreens, total antioxidative capacity measured by the FRAP assay was In dark-grown chia microgreens, total antioxidative capacity measured by the FRAP assay was the highest (Figure 4b). When di erent light treatments were compared, similar values were observed the highest (Figure 4b). When different light treatments were compared, similar values were in microgreens exposed to light for 24 and 48 h, whilst a significant decrease was observed 24 h after observed in microgreens exposed to light for 24 and 48 h, whilst a significant decrease was observed exposure to light in comparison to dark-grown chia microgreens. 24 h after exposure to light in comparison to dark-grown chia microgreens. The FRAP assay exhibited lower values in comparison with the DPPH assay. Other authors have also reported differences between obtained values when the two assays were used [4,55]. For instance, in 7-day-old chia sprouts exposed to 12 h/12 h (light/dark) cycles, DPPH scavenging activity was lower in comparison to the FRAP assay [54]. The reason for such discrepancies might be due to differences in the chemistry and sensitivity of these two assays. In a recently published study by Mitrović et al. [38], the authors reported that the DPPH assay is more suitable than the FRAP assay for evaluating the antioxidant activity of chia. This suggests that the obtained values of these assays are impacted by the species, germination processes, and growth conditions, as well as temperature and duration of light exposure. 3.6. Effect of Illumination on FTIR Spectra The FTIR spectra of the three chia samples under the study are depicted in Figure 5, and their main bands are summarized in Table 2. It can be observed that they are similar, in particular, the samples exposed to light for 24 and 48 h. Appl. Sci. 2020, 10, 5731 9 of 13 The FRAP assay exhibited lower values in comparison with the DPPH assay. Other authors have also reported di erences between obtained values when the two assays were used [4,55]. For instance, in 7-day-old chia sprouts exposed to 12 h/12 h (light/dark) cycles, DPPH scavenging activity was lower in comparison to the FRAP assay [54]. The reason for such discrepancies might be due to di erences in the chemistry and sensitivity of these two assays. In a recently published study by Mitrovic ´ et al. [38], the authors reported that the DPPH assay is more suitable than the FRAP assay for evaluating the antioxidant activity of chia. This suggests that the obtained values of these assays are impacted by the species, germination processes, and growth conditions, as well as temperature and duration of light exposure. 3.6. E ect of Illumination on FTIR Spectra The FTIR spectra of the three chia samples under the study are depicted in Figure 5, and their main bands are summarized in Table 2. It can be observed that they are similar, in particular, the samples Appl. Sci. 2020, 10, x 9 of 12 exposed to light for 24 and 48 h. Figure 5. FTIR spectra of dark-grown (a) chia microgreens and microgreens after exposure to light for Figure 5. FTIR spectra of dark-grown (a) chia microgreens and microgreens after exposure to light for −1 24 (b) and 48 (c) h. FTIR spectrum was recorded by 20 co-added scans at a resolution of 2 cm in the 24 (b) and 48 (c) h. FTIR spectrum was recorded by 20 co-added scans at a resolution of 2 cm in the −1 region of wave number (WN) 500 to 4000 cm . The spectra were baseline-corrected. region of wave number (WN) 500 to 4000 cm . The spectra were baseline-corrected. Table 2. Main bands in the FTIR spectra and their assignments of dark-grown chia microgreens and −1 A large number of peaks appeared in the region from 1000 to 3000 cm , indicating that chia microgreens after exposure to light for 24 and 48 h. microgreens have a rich chemical composition (proteins, lipids, carbohydrates) [18]. More precisely, −1 Dark-Grown 24 h 48 h the band located around 3290 cm represents N–H stretching vibrations that are caused by proteins Peak No. Assignments 1 1 1 Wavenumber (cm ) Wavenumber (cm ) Wavenumber (cm ) −1 [20]. The bands between 2800 and 3000 cm mainly represent C–H stretching vibrations that are I not assigned 889.9 890 889 −1 caused by lipids. The region between 1550 and 1700 cm are protein absorption bands, including II Polysaccharides 1062 1062 1063 −1 amide I and amide II. Additionally, the fingerprint region between 1000 and 1500 cm is where amide III Amide III region 1238 1239 1239 IV Amide III region 1457 1457 1457 II and the functional groups of nucleic acid and carbohydrates contribute to these absorption bands. V Amide II region 1540 1540 1540 VI Amide I region 1647 1647 1647 Table 2. Main bands in the FTIR spectra and their assignments of dark-grown chia microgreens and VII Fat content 2855 disappeared disappeared VIII Fat content 2926 2927 2925 microgreens after exposure to light for 24 and 48 h. IX Protein content 3269 3270 3271 48 h Dark-Grown 24 h Peak No. Assignments Wavenumber A large number of peaks appeared in the region−1from 1000 to 3000 cm−1 , indicating that chia Wavenumber (cm ) Wavenumber (cm ) −1 (cm ) microgreens have a rich chemical composition (proteins, lipids, carbohydrates) [18]. More precisely, I not assigned 889,9 890 889 the band located around 3290 cm represents N–H stretching vibrations that are caused by proteins [20]. II Polysaccharides 1 1062 1062 1063 The bands between 2800 and 3000 cm mainly represent C–H stretching vibrations that are caused by III Amide III region 1238 1239 1239 lipids. The region between 1550 and 1700 cm are protein absorption bands, including amide I and IV Amide III region 1457 1457 1457 V Amide II region 1540 1540 1540 VI Amide I region 1647 1647 1647 VII Fat content 2855 disappeared disappeared VIII Fat content 2926 2927 2925 IX Protein content 3269 3270 3271 In comparison with the other two samples, the dark-grown chia sample showed negligible −1 differences regarding the peak shifting in the region comprised between 1000 and 1700 cm . We −1 observed two well-pronounced bands in dark-grown chia microgreens around 2855 and 2926 cm . −1 Regarding the peak shifting, with the increment of light treatment, the band at about 2855 cm (dark- −1 grown microgreens) disappeared in illuminated microgreens (24 and 48 h), while bands at 2926 cm remain relatively stable in all chia samples. −1 Nine main bands were analyzed between 800 and 3500 cm in the spectra of the three chia −1 samples, and the band positions were relatively similar (with the exception of the band at 2855 cm ) to each other, indicating that structure was sensitive to treatments in the region of fats, vax, and lipids after 24 and 48 h of exposure to light [19,20]. Appl. Sci. 2020, 10, 5731 10 of 13 amide II. Additionally, the fingerprint region between 1000 and 1500 cm is where amide II and the functional groups of nucleic acid and carbohydrates contribute to these absorption bands. In comparison with the other two samples, the dark-grown chia sample showed negligible di erences regarding the peak shifting in the region comprised between 1000 and 1700 cm . We observed two well-pronounced bands in dark-grown chia microgreens around 2855 and 2926 cm . Regarding the peak shifting, with the increment of light treatment, the band at about 2855 cm (dark-grown microgreens) disappeared in illuminated microgreens (24 and 48 h), while bands at 2926 cm remain relatively stable in all chia samples. Nine main bands were analyzed between 800 and 3500 cm in the spectra of the three chia samples, and the band positions were relatively similar (with the exception of the band at 2855 cm ) to each other, indicating that structure was sensitive to treatments in the region of fats, vax, and lipids after 24 and 48 h of exposure to light [19,20]. 4. Conclusions Our findings show that growth conditions with the lower light intensity of 100 mol photons 2 1 m s evoked a positive e ect on total antioxidant capacity, synthesis of chlorophyll and carotenoids, total soluble phenolics, and ascorbic acid in dark-grown chia microgreens. Thus, the synthesis of bioactive compounds and the antioxidative potential of illuminated chia microgreens was improved. The DPPH assay was shown to be more sensitive in detecting antioxidative activity in comparison with FRAP. Therefore, we can conclude that chia microgreens could be considered a valuable supplement to the human diet, in addition to raw chia seeds and other popular microgreens. Present trends use di erent approaches in growing di erent sprouts and microgreens that are used for human consumption. 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Published: Aug 19, 2020

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