Effects of Development Stage and Sodium Salts on the Antioxidant Properties of White Cabbage Microgreens

agriculture

Article Effects of Development Stage and Sodium Salts on the Antioxidant Properties of White Cabbage Microgreens

Antoanela Patras

Department of Sciences, Faculty of Horticulture, “Ion Ionescu de la Brad” University of Agricultural Sciences and Veterinary Medicine of Iasi, 3 Mihail Sadoveanu Alley, 700490 Iasi, Romania; [email protected] or [email protected]

Abstract: Microgreens, considered “superfood”, are easy to cultivate and very rich in health- promoting compounds, as antioxidants. White cabbage (Brassica oleracea var. capitata) microgreens contain high quantities of phenolics, which contribute together with other bioactive compounds to their important antioxidant properties. The present study analyses the effects of development stage

(5-, 7- and 9-days) and two sodium salts, NaCl and Na2SO4, in two concentrations (0.01 M and 0.1 M), on the antioxidant properties of white cabbage microgreens. Among the three development levels, the 5-day microgreens revealed the highest total phenolic content, DPPH radical scavenging and

total reducing capacities. Concerning the effects of sodium salts, 0.01 M NaCl and 0.1 M Na2SO4 determined higher total reducing capacity. Additionally, 0.01 M NaCl induced the highest DPPH radical scavenging capacity, while the most important total phenolics and anthocyanins contents were

obtained in case of 0.1 M Na2SO4. In conclusion, from the developmental point of view, the 5-day old microgreens present, globally, the best characteristics. Considering the elicitor effects of sodium

salts, 0.01 M NaCl and 0.1 M Na2SO4 generally determined the strongest antioxidant properties. The results could be used to develop new production technologies for antioxidant-enriched microgreens. Citation: Patras, A. Effects of Development Stage and Sodium Salts Keywords: Brassica oleracea; microplants; sprouts; phenolic content; health-promoting; salt stress; on the Antioxidant Properties of elicitor; sodium chloride; sodium sulphate; functional food White Cabbage Microgreens. Agriculture 2021, 11, 200. https:// doi.org/10.3390/agriculture11030200 1. Introduction Academic Editors: Gianluca Caruso, Nowadays, world population is subjected to severe trials in the context of COVID-19 László Baranyai and Vasile Stoleru pandemic threat. Today’s people, generally, have low immunity due to stress, pollution, sedentary lifestyle and unhealthy diet. Now, more than ever, it is to be taken into account Received: 31 January 2021 the possibility of improving humans’ immunity and general condition through health- Accepted: 23 February 2021 Published: 28 February 2021 promoting food. Microgreens and sprouts are considered such functional foods (products that offer health beneﬁts beyond their nutritional value) due to their high content in

Publisher’s Note: MDPI stays neutral antioxidants and other bioactive compounds [1,2]. Both microgreens and sprouts are with regard to jurisdictional claims in early stages of the plant development. Sprouts are germinated seeds, the very first stage published maps and institutional affil- of a plant; are grown basically on water and harvested at 2- to 7-days old; the whole iations. being edible: seed, root, and stem [3,4]. Microgreens are the immediate superior stage of development, the “cotyledon growth stage”, when the first couple of leaves appears; are grown in soil, peat moss or similar substrate and harvested at 5- to 21-days old, by cutting the above-soil part, which is edible: the stem and developing leaves [3,4]. This differentiation among the two types of products was clearly formulated the latest years. Copyright: © 2021 by the author. Licensee MDPI, Basel, Switzerland. Previously, most of the studies were treating both sprouts and microgreens, as sprouts. This article is an open access article For example, there are publications presenting results concerning 10-day “sprouts” of distributed under the terms and white and red cabbage [5], 11-day broccoli “sprouts” [6] or 12-day tronchuda cabbage conditions of the Creative Commons “sprouts” [7]. More than that, 4 varieties of Brassica oleracea “sprouts” were grown 9 days Attribution (CC BY) license (https:// on vermiculite, in growth chamber, and were harvested by cutting above the substrate [8]. creativecommons.org/licenses/by/ However, no matter if they are named microgreens or sprouts, it was repetitively proved 4.0/). by numerous studies that these edible seedlings contain superior quantities of antioxidants

Agriculture 2021, 11, 200. https://doi.org/10.3390/agriculture11030200 https://www.mdpi.com/journal/agriculture Agriculture 2021, 11, 200 2 of 10

and other bioactive compounds then their seeds and mature plants [1,6,9]. Some authors even name microgreens and sprouts as “superfood” [3,10]. Among edible seedlings of various families, those of cruciferous (including white cabbage) have the advantage of different classes of bioactive compounds, e.g., phenolics (the main antioxidants), vitamins, and the specific Brassicaceae constituents-glucosinolates and isothiocyanates. They have antioxidant, antibacterial, antiviral, antiinflammatory properties, improve the immunity and offer protection against cancers, cardio-vascular and degenerative diseases [11–18]. The phenolic compounds are very active in human body protection at the cellular level against the negative effects of free radicals [19]. More than that, it was recently noted that phenolics are “the most promising small molecules identified as coronavirus inhibitors” [20]. Microgreens and sprouts are easy to produce in a short period of time and the content in bioactive compounds depends on their development (number of days since sowing) [21] and can be affected by different treatments or technologies [22–24], e.g., a salt-enriched growth substrate. Hassini et al. (2017) observed that NaCl treatments slightly influenced the total phenolics, anthocyanins and flavonols of white and red cabbage 10-days “sprouts” [5]. Yuan et al., 2010 noticed also the change of antioxidant capacity and total phenolics of radish sprouts in presence of NaCl, and depending on the salt concentration, it was registered an improvement or, contrary, a diminution [25]. The general conclusion was that “adequate salt stress could be one useful way to enhance health-promoting compounds of plant food” [25]. The present study aims to investigate the possibilities to improve the health-promoting potential of white cabbage microgreens by harvesting at the most appropriate moment of their growth, or by using non-conventional conditions for germination in order to stimulate the biosynthesis of phenolic compounds, the main antioxidants of Brassicaceaes. It explores the effects of germination duration (5, 7 and 9 days) and two sodium salts—sodium chloride and sodium sulphate—administrated in two concentrations (0.01 M and 0.1 M), on the phenolic content and antioxidant properties reflected by the Folin-Ciocalteu total reducing capacity and DPPH radical scavenging capacity of white cabbage microgreens. The results can be used for the elaboration of new production technologies for antioxidant-enriched microgreens.

2. Materials and Methods 2.1. Production of Microgreens The experiment used white cabbage seeds of cultivar Mercado de Copenhague 2, from Amia Company (Otopeni, Romania), sterilized with 0.07% sodium hypochlorite and soaked 10 h in distilled water. Sprouting was performed in trays filled with a peat-based germination substrate (DSM 2w, from Kekkilä Garden Professional Substrate, Vantaa, Finland), in a climatic germination room (MRC PGI-500H, Holon, Israel). n = 200 seeds were sown in ditches of 7.5 ± 2.5 mm deep and 25 mm distance between rows. Sprouting conditions: first 60 h at 24 ◦C, humidity (RH) 80%, lighting 80 W with fluorescent lamps, and for the rest of time till harvest, at 22 ◦C, RH 80% and lighting 360 W. Microgreens were watered with distilled water and harvested at 5-, 7- and 9-day old. The effects of two sodium salts at 0.01 M and 0.1 M: sodium chloride (NaCl), and sodium sulphate (Na2SO4) were tested by replacing water with the same quantity of saline solution (110 ± 0.5 mL/tray/day) and by the direct administration on the substrate during 9 days, in 4 parallel assays, one for each concentration and each of them performed in triplicate and compared to untreated control. Treated microgreens were harvested at 9 days old.

2.2. Soluble Dry Matter (SDM) It was determined in the juice resulted from squeezing the whole microgreens at 19 ± 1 ◦C, using a Zeiss portable refractometer, and was expressed as Brix degrees (◦Bx). Agriculture 2021, 11, 200 3 of 10

2.3. Extraction Hydroethanolic extracts were obtained from whole microgreens, using 70% ethanol acidiﬁed with 0.01% hydrochloric acid in a two-steps protocol described by Patras et al. (2017). Brieﬂy, from each sample, 3.2 g of mixed microgreens were weighted to 4 decimal and grounded. Then, samples were extracted in presence of 15 mL ethanolic solvent for 30 min at 38 ± 1 ◦C on a water-bath under shaking. The obtained extract was centrifuged at 7000 rpm for 15 min. The remaining residue from centrifugation was re-extracted with 10 mL solvent for 15 min in the same conditions, followed by same centrifugation (15 min). The two supernatants, resulted from one sample, were mixed and stored at 4 ◦C prior to analysis [26].

2.4. Total Phenolic Content (TPC) The direct reading of extract’s absorbance at 280 nm, after adequate dilution, according to the method described by Ribéreau-Gayon et al. (2006), was used for the total phenolic content determination. The results are expressed as gallic acid equivalents per fresh weight (mg GAE/100 g FW) using a calibration curve with gallic acid [27].

2.5. Total Anthocyanins Content (TAC) The total content of anthocyanins was determined by pH differential method following the procedure described by Ribéreau-Gayon et al. (2006) with slight modiﬁcations. The pH of the extract was adjusted to 0.6 and 3.5, respectively, with adequate buffer solutions [27], as follows: 10 mL buffer was added to a mixture of 1 mL extract and 1 mL of 96% ethanol containing 0.1% HCl. Absorbances of both solutions were measured at 520 nm against a distilled water reference. The results were calculated from a calibration curve with cyanidin-3-glucoside, and reported as mg/100 g FW.

2.6. Total Reducing Capacity (TRC) The Folin-Ciocalteu method is a measure of the total reducing capacity [28], but also reﬂects the phenolic content of a liquid sample. A mixture of 0.1 mL extract, 7.4 mL distilled water, 0.5 mL Folin-Ciocalteu reagent and 2 mL 20% Na2CO3 was kept in the dark for 30 min. The measurement of absorbance was done at 750 nm, compared to a blank (same mixture, but replacing the extract with 0.1 mL distilled water). The results were expressed as mg GAE/100 g FW, calculated from a calibration curve with gallic acid.

2.7. DPPH Radical Scavenging Capacity (RSC) The Brand-Williams assay with slight modiﬁcations was used in order to analyze the antiradical activity of microgreens’ extracts [29]. The reduction of the absorbance at 515 nm of a DPPH• methanolic solution (0.12 mM) in the presence of studied extracts (ratio extract:DPPH• = 1:2) was measured against a methanol reference, after 5 min of incubation in the dark. The control sample contains 70% ethanol acidiﬁed with 0.01% hydrochloric acid, which replaces the microgreens extracts and DPPH• methanolic solution (0.12 mM), in same ratio (1:2). The results of DPPH RSC were expressed as mg ascorbic acid/100 g fresh weight (mg ascorbic acid/100 g FW) from a calibration curve with ascorbic acid.

2.8. Statistical Analysis Three independent experiments were performed. The obtained data were represented as scatter plots (excepting the soluble dry matter), grouped by the growth stage factor (5, 7, and 9 days old) and treatment (9-day-old control/9-day-old treatment). Data were analyzed through the SPSS Statistics 21 (IBM) software, using one-way analysis of variance (ANOVA) and the post-hoc test Tukey Honestly Signiﬁcance Difference (HSD). The signiﬁcance level was α = 0.05. The factors are between-subjects. The degrees of freedom (df) for the between- groups estimate of variance, the F ratio and p-value are given in the legend of each graph. The between-groups estimate of variance forms the numerator of the F ratio. F represents the ratio between the mean square between-groups and the mean square within-groups. Agriculture 2021, 11, 200 4 of 10

Each mean square was calculated by dividing the sum of square by its degrees of freedom. The p-value represents the signiﬁcance of the F ratio. If the p-value is p ≤ α (p ≤ 0.05), the null hypothesis that all means are equal can be rejected and differences between some of the means are considered signiﬁcant.

3. Results and Discussion 3.1. Soluble Dry Matter During the growth of microgreens, the soluble dry matter (SDM) is significantly decreasing by 8.8% from 5 to 7 days and by 12.9% (from 3.41 to 2.97 ◦Bx) from 5 to 9 days (Table1). The sodium chloride is slightly, but significantly decreasing the SDM of 9-days microgreens at 0.01 M, while at 0.1 M, the change is not significant. The sodium sulphate is increasing the SDM. At 0.01 M Na2SO4 the augmentation is 2%, insignificant statistically, while at 0.1 M Na2SO4 was registered a strong increase by 68.7% compared to untreated 9-days samples (from 2.97 to 5.01 ◦Bx).

Table 1. Soluble dry matter (SDM), ◦Bx, of untreated (5-, 7- and 9-day) and 9-day treated (with NaCl

or Na2SO4) white cabbage microgreens.

Variant of White Cabbage Microgreens SDM (◦Bx) 5-days 3.41 ± 0.01 d untreated 7-days 3.11 ± 0.14 c 9-days 2.97 ± 0.15 b NaCl, 0.01 M 2.83 ± 0.15 a NaCl, 0.1 M 2.91 ± 0.01 a,b 9-days treated b,c Na2SO4, 0.01 M 3.03 ± 0.06 e Na2SO4, 0.1 M 5.01 ± 0.01 Data represent mean value of soluble dry matter ± standard deviation. Different letters denote signiﬁcant differences.

Similarly, a previous study found that 0.01 M NaCl decreased SDM in broccoli and cauliflower 9-day-old sprouts and 0.1 M Na2SO4 induced a powerful increase, comparable to present result [26]. Other previous studies found that salt stress enhanced the soluble dry matter and dry matter percent of cauliflower and broccoli mature florets [30,31]. Hassini et al. (2017) found that 0.15 M NaCl significantly increased the moisture percent of 10-days- old broccoli “sprouts”, which has the significance of a decrease of dry matter percent [22].

3.2. Total Phenolic Content Some authors consider the direct reading of extract’s absorbance at 280 nm, a simple, fast and reliable method for the estimation of total phenolic content (TPC), although over- estimation of the total phenolic content may occur due to the ability of other compounds to absorb the 280 nm UV-light, as proteins, or other compounds containing π conjugated systems with hydroxyl-phenolic groups [32,33]. Contrary, there are few phenolic compounds, such as cinnamic acids or chalcones, which do not show absorption features at 280 nm [33]. The total phenolic content (TPC) has considerable values in all evolution stages of cabbage microgreens (Figure1A) and under all treatments (Figure1B), being between 80 and 149 mg GAE/100 g FW. Its variation is similar to the evolution of soluble dry matter. TPC signiﬁcantly decreases during the growth of white cabbage, by about 3% from 5-days to 7-days and by 18.36% from 5- to 9-days (from 108.63 to 88.69 mg GAE/100 g FW). This diminution must be the result of the use of phenolic compounds in different anabolic processes of the young plant’s metabolism. Sousa et al. found important loses in phenolics during the germination of tronchuda cabbage (Brassica oleracea var. costata DC) from 2 to 12 days [7]. Studying 3-, 4-, 5- and 6-days lentil sprouts, Fouad and Rehab (2015) stated the maximum concentration of TPC in 5-days sprouts (increasing from seeds to 3, 4 and 5-days) and a signiﬁcant decrease in 6-days sprouts, which they considered to be due to the higher activity of polyphenol oxidase [21]. Similarly, Korus (2011) observed the increase Agriculture 2021, 11, x FOR PEER REVIEW 5 of 10

to 7‐days and by 18.36% from 5‐ to 9‐days (from 108.63 to 88.69 mg GAE/100 g FW). This diminution must be the result of the use of phenolic compounds in different anabolic pro‐ cesses of the young plant’s metabolism. Sousa et al. found important loses in phenolics during the germination of tronchuda cabbage (Brassica oleracea var. costata DC) from 2 to Agriculture 2021, 11, 200 12 days [7]. Studying 3‐, 4‐, 5‐ and 6‐days lentil sprouts, Fouad and Rehab (2015) stated5 of 10 the maximum concentration of TPC in 5‐days sprouts (increasing from seeds to 3, 4 and 5‐days) and a significant decrease in 6‐days sprouts, which they considered to be due to the higher activity of polyphenol oxidase [21]. Similarly, Korus (2011) observed the in‐ of polyphenol oxidases’ activity in kale (also from Brassicaceae family) during seedlings crease of polyphenol oxidases’ activity in kale (also from Brassicaceae family) during seed‐ maturation [34]. A possible similar increase may be an explanation for the observed lings maturation [34]. A possible similar increase may be an explanation for the observed decrease of TPC during the growth of microgreens in the present study. decrease of TPC during the growth of microgreens in the present study.

Figure 1. Total phenolic contentFigure 1. (TPC, Total mgphenolic GAE/100 content g FW) (TPC, of mg white GAE/100 cabbage g FW) microgreens. of white cabbage (A). Influence microgreens. of growth: (A). In‐ untreated 5-, 7- and 9-day-oldfluence microgreens of growth: (α untreated= 0.05, between 5‐, 7‐ and groups 9‐days df‐ =old 2, microgreens F = 247.962, p (α == 0.000). 0.05, between (B). Influence groups of df NaCl = 2, F or Na2SO4 treatments on 9-day-old= 247.962, microgreensp = 0.000). (B ().α Influence= 0.05, between of NaCl groups or Na2 dfSO =4 4,treatments F = 1657.826, on 9p‐days= 0.000).‐old microgreens Above the data (α = points are displayed the mean0.05, between values and groups a letter: df = different 4, F = 1657.826, letters denotep = 0.000). significant Above the differences data points between are displayed values (Tukey the mean HSD test). Series 1, 2 or 3 fromvalues the and legend a letter: represent different the letters 3 individual denote experiments.significant differences between values (Tukey HSD test). Series 1, 2 or 3 from the legend represent the 3 individual experiments. The treatments, generally, increase the TPC, with only one exception: the natrium chlorideThe treatments, at small concentration generally, increase (0.01 M), the which TPC, diminishes with only one the TPCexception: of 9-days the untreatednatrium chlorideplants byat 9.8%small (whichconcentration represents (0.01 8.72 M), mg which GAE/100 diminishes g FW). the The TPC higher of 9‐days concentration untreated of plantsNaCl by increases 9.8% (which the 9-days represents control’s 8.72 TPC mg byGAE/100 11%. Similarly, g FW). The Yuan higher et al. concentration (2010) observed of NaClthat 0.01increases M NaCl the decreased 9‐days control’s the TPC TPC of 3- by 5-, 11%. 7-day Similarly, radish sprouts Yuan (anotheret al. (2010) vegetable observed from thatBrassicaceae 0.01 M NaClfamily), decreased but did the not TPC affect of 3‐ its5‐, antioxidant7‐day radish activity sprouts [ 25(another]. Same vegetable authors from found Brassicaceaethat 0.1 M family), NaCl increased but did thenot TPCaffect in its 3- antioxidant and 5-day radish activity sprouts. [25]. Same Hassini authors et al. found (2017) thatstated 0.1 thatM NaCl 0.15 increased M NaCl decreased the TPC in (non-significantly) 3‐ and 5‐day radish the TPCsprouts. of 10-days Hassini white et al. cabbage (2017) statededible that seedlings 0.15 M [NaCl5]. These decreased fluctuations (non‐significantly) of TPC in presence the TPC of of different 10‐days concentrations white cabbage of ediblesalt can seedlings be explained [5]. These by the fluctuations complexity of of TPC metabolic in presence processes of different in young concentrations plants. Phenolic of saltcompounds can be explained are generated by the complexity by the phenylpropanoid of metabolic processes pathway. in young The increase plants. ofPhenolic TPC in compoundspresence of are 0.1 generated M NaCl by and the both phenylpropanoid concentrations pathway. of Na2SO The4 could increase be theof TPC result in pres of the‐ enceactivation of 0.1 M of NaCl this and pathway. both concentrations More than that, of inNa present2SO4 could case, be the the decrease, result of the in presenceactivation of of0.01 this M pathway. NaCl, of More TPC measuredthan that, in by present absorbance case, at the 280 decrease, nm, could in presence be an artifact of 0.01 due, M notNaCl, to a ofreal TPC decrease measured of phenolicby absorbance content, at 280 but nm, to a destructioncould be an ofartifact some due,π conjugated not to a real systems decrease with ofhydroxyl-phenolic phenolic content, but groups to a which destruction became of unablesome π toconjugated absorb the systems 280 nm with UV-light. hydroxyl Other‐ phenolicauthors groups also noticed which that became “the phenolicunable to compounds absorb the in280 plants nm UV can‐light. be changed Other authors by salt stress,also noticedbut this that is critically“the phenolic dependent compounds on the in salt plants sensitivity can be changed of plants” by [salt25]. stress, Further but in-depth this is criticallystudies dependent at molecular on level the salt should sensitivity be performed of plants” in [25]. order Further to research in‐depth the studies implications at mo‐ of lecularvarious level concentration should be performed of NaCl in in the order chain to reactions research the of the implications phenylpropanoid of various pathway, concen‐ or trationin any of other NaCl plant in the processes. chain reactions of the phenylpropanoid pathway, or in any other plant processes.While the impact of NaCl on sprouts and microgreens was intensively analyzed, only scarcedWhile information the impact canof NaCl be found on sprouts concerning and microgreens the Na2SO 4wasinfluence. intensively In the analyzed, present only study, scarcedthe natrium information sulphate can stimulates be found theconcerning augmentation the Na of2SO TPC4 influence. at both concentrations, In the present study, with an theimpressive natrium sulphate increase, stimulates by more than the 68% augmentation (from 88.69 of to TPC 149.40 at both mg GAE/100 concentrations, g FW) inwith case an of 0.1 M Na2SO4 and 9.6% in case of 0.01 M Na2SO4 (Figure1B). Similar enhancement was registered in other Brassica edible seedlings (broccoli and cauliflower) in case of treatment with 0.1 M Na SO [26]. Other study analyzed the elicitor effect of sulfur (administrated as 2 4 K2SO4), not on phenolics, but on glucosinolates in broccoli sprouts [23]. Agriculture 2021, 11, x FOR PEER REVIEW 6 of 10

impressive increase, by more than 68% (from 88.69 to 149.40 mg GAE/100 g FW) in case of 0.1 M Na2SO4 and 9.6% in case of 0.01 M Na2SO4 (Figure 1B). Similar enhancement was registered in other Brassica edible seedlings (broccoli and cauliflower) in case of treatment 2 4 Agriculture 2021, 11, 200 with 0.1 M Na SO [26]. Other study analyzed the elicitor effect of sulfur (administrated6 of 10 as K2SO4), not on phenolics, but on glucosinolates in broccoli sprouts [23].

3.3. Total Anthocyanins Content 3.3. TotalAnthocyanin Anthocyanins pigments Content belong to the class of phenolic compounds, subclass flavo‐ noids.Anthocyanin They can exist pigments in very belong small to quantities the class of in phenolic some varieties compounds, of white subclass cabbage flavonoids. [35], but Theythey canare existcharacteristic in very small for the quantities red cabbage. in some Our varieties research of found white cabbagevery small [35 amounts], but they of are an‐ characteristicthocyanins (between for the red 0.28–0.71 cabbage. mg/100 Our research g FW) foundin all untreated very small microgreens amounts of anthocyaninsof the studied (betweenwhite cabbage 0.28–0.71 cultivar mg/100 (Mercado g FW) in de all untreatedCopenhague microgreens 2). The content of the studied of total white anthocyanins cabbage cultivar(TAC) was (Mercado analyzed de Copenhague due to their 2). importance The content in of a total healthy anthocyanins diet and (TAC)their contribution was analyzed to duethe toantioxidant their importance capacity in of a healthythe plant diet extract. and their During contribution the growth to theof white antioxidant cabbage capacity micro‐ ofgreens the plant was extract.registered During an insignificant the growth increase of white of cabbage TAC from microgreens 0.28 mg/100 was g registeredFW (at 5‐days an insignificantmicrogreens), increase to 0.43 of and TAC to from0.71 mg/100 0.28 mg/100 g FW g at FW 7‐ (atand 5-days respectively, microgreens), 9‐days‐ toold 0.43 (Figure and to2A). 0.71 All mg/100 the applied g FW atsodium 7- and salts respectively, increased 9-days-old the anthocyanins (Figure2 A).content All the compared applied to sodium 9‐days saltsuntreated increased control, the anthocyanins especially the content NaCl 0.01 compared M and tothe 9-days Na2SO untreated4 0.1 M. But control, only especiallyin the case theof NaClthe highest 0.01 M sodium and the sulphate Na2SO4 0.1concentration, M. But only inthe the difference case of the from highest the control sodium is sulphate statisti‐ concentration,cally significant. the differenceThe TAC in from treated the control samples is statistically was between significant. 0.72 and The 2.81 TAC mg/100 in treated g FW samples(Figure was2B). betweenIn other 0.72previous and 2.81 research, mg/100 it was g FW also (Figure found2B). a Insimilar other influence previous research,of the same it wastreatments also found on acauliflower similar influence sprouts of [26]. the sameSome treatments other authors on cauliflower showed that sprouts saline [ 26excess]. Some and otherother authors osmotic showed stress, such that salineas water excess deficiency, and other and osmotic flooding stress, stress, such may as waterinduce deficiency, anthocya‐ andnins flooding accumulations stress, may [36–38]. induce anthocyanins accumulations [36–38].

Figure 2. Total anthocyaninsFigure content 2. (TAC) Total anthocyanins of white cabbage content microgreens. (TAC) of white (A). Influence cabbage ofmicrogreens. growth: untreated (A). Influence 5-, 7- and of 9-day-old microgreens (α = 0.05,growth: between untreated groups 5‐ df, 7‐ =and 2, F 9 =‐day 2.721,‐oldp microgreens= 0.144). (B). ( Influenceα = 0.05, between of NaCl groups or Na2SO df4 =treatments 2, F = 2.721, p = on 9-day-old microgreens (α0.144).= 0.05, ( betweenB). Influence groups of NaCl df = 4, or F Na = 13.586,2SO4 treatmentsp = 0.000). on Above 9‐day the‐old data microgreens points are (α displayed= 0.05, between the mean values and a letter: differentgroups letters df = 4, denote F = 13.586, significant p = 0.000). differences Above the between data points values are (Tukey displayed HSD the test). mean Series values 1, 2 or and 3 a from the legend represent theletter: 3 individual different experiments. letters denote significant differences between values (Tukey HSD test). Series 1, 2 or 3 from the legend represent the 3 individual experiments. 3.4. Total Reducing Capacity 3.4. Total Reducing Capacity The values of total reducing capacity (TRC) of white cabbage are summarized in FigureThe3A,B. values During of total the maturation reducing capacity of microgreens, (TRC) of itwhite was cabbage noticed a are continuous summarized decrease in Fig‐ ofure TRC 3A, from B). During 5- to 7- the and maturation 9-days, with of statisticallymicrogreens, significant it was noticed differences. a continuous This evolution decrease is of similarTRC from to TPC 5‐ to (D280) 7‐ and variation. 9‐days, with The most statistically important significant diminution differences. was registered This evolution from 5- to is 7-daysimilar old to plants, TPC (D280) by 17.99% variation. from The 101.08 most to 82.90important mg GAE/100 diminution g FW, was followed registered by from a second 5‐ to diminution7‐day old plants, from 7-by to 17.99% 9-days, from representing 101.08 to 82.90 an additional mg GAE/100 2.55% g FW, decrease, followed from by 82.90a second to 80.32diminution mg GAE/100 from 7 g‐ to FW. 9‐days, representing an additional 2.55% decrease, from 82.90 to 80.32Concerning mg GAE/100 the g effects FW. of treatments, two of them determined strong augmentations of TRC: 0.01 M NaCl-by 42.73%, leading to the highest TRC value, which is 114.64 mg GAE/100 g FW, and 0.1 M Na2SO4-by 41.88%, up to a TRC of 113.96 mg GAE/100 g FW. Besides, the difference between these two highest values is not significant statistically. The other applied sodium salts treatments determined slight decreases of TRC, by 3.98% in case of 0.1 M Na SO , and by 13.4% in case of 0.1 M NaCl. Other authors found that NaCl 2 4 in 3 concentrations (0.01, 0.05 and 0.1 M) diminished the reducing capacity measured by ferric reducing antioxidant power assay (FRAP) and the trolox equivalent antioxidant Agriculture 2021, 11, 200 7 of 10

Agriculture 2021, 11, x FOR PEER REVIEW 7 of 10

capacity (TEAC) of 5-days broccoli sprouts, while the Folin-Ciocalteu method revealed increases for all 3 NaCl treatments [39].

Figure 3. Total reducing capacityFigure 3. (TRC, Total mg reducing GAE/100 capacity g FW) (TRC, of white mg GAE/100 cabbage g microgreens. FW) of white (cabbageA). Influence microgreens. of growth: (A). untreated 5-, 7- and 9-day-oldInfluence microgreens of growth: (α = 0.05, untreated between 5‐, groups7‐ and 9 df‐day = 2,‐old F = microgreens 363.932 p = 0.000). (α = 0.05, (B). between Influence groups of NaCl df or = 2, F Na2SO4 treatments on 9-day-old= 363.932 microgreens p = 0.000). ((αB).= Influence 0.05, between of NaCl groups or Na df2SO = 4,4 treatments F = 3816.965, on p9‐=day 0.000).‐old microgreens Above the data (α = points are displayed the mean0.05, values between and groups a letter: df different= 4, F = 3816.965, letters denote p = 0.000). significant Above differencesthe data points between are displayed values (Tukey the mean HSD test). Series 1, 2 or 3 fromvalues the and legend a letter: represent different the 3letters individual denote experiments. significant differences between values (Tukey HSD test). Series 1, 2 or 3 from the legend represent the 3 individual experiments. Comparing the results obtained for TPC (measuring the absorbance at 280 nm) with thoseConcerning for TRC (Folin-Ciocalteu the effects of treatments, assay), similar two of evolution them determined is observed strong (through augmentations both meth- ofods) TRC: during 0.01 theM NaCl growth‐by of42.73%, microgreens leading and to followingthe highest the TRC 0.1 value, M Na2 whichSO4 treatment. is 114.64 Thismg GAE/100similarity g is FW, normal, and 0.1 as bothM Na methods2SO4‐by can41.88%, be used up to for a theTRC estimation of 113.96 ofmg total GAE/100 phenolic g FW. con- Besides,tent. However, the difference the treatment between with these 0.01 two M highest NaCl induced values is a not completely significant different statistically. behavior: The otherthe increase applied of sodium TRC and salts decrease treatments of TPC. determined This may slight suggest decreases the presence of TRC, of antioxidantsby 3.98% in case(e.g., of ascorbic 0.1 M Na acid),2SO4, or and other by 13.4% compounds in case (even of 0.1 otherM NaCl. phenolics, Other authors e.g., cinnamic found that acids NaCl and inchalcones) 3 concentrations which react (0.01, with 0.05 Folin-Ciocalteu and 0.1 M) diminished reagent but the do reducing not absorb capacity light atmeasured 280 nm and by ferrica concomitant reducing depletionantioxidant of power some phenolicsassay (FRAP) or other and compoundsthe trolox equivalent containing antioxidantπ conjugated ca‐ pacitysystems, (TEAC) as aromatic of 5‐days glucosinolates. broccoli sprouts, Due towhile this typethe Folin of situations,‐Ciocalteu it method is necessary revealed to apply in‐ creasesmultiple for assays: all 3 NaCl for the treatments detection [39]. of phenolic compounds, reducing capacity and antioxi- dantComparing activity and the it isresults strongly obtained recommended for TPC (measuring that based on the the absorbance obtained results,at 280 nm) to follow with thosein-depth for TRC studies (Folin in‐ orderCiocalteu to elucidate assay), similar which areevolution the compounds is observed generated/depleted (through both meth in‐ ods)presence during of saltsthe growth and the of exact microgreens mechanisms and involved. following the 0.1 M Na2SO4 treatment. This similarity is normal, as both methods can be used for the estimation of total phenolic con‐ tent.3.5. DPPHHowever, Radical the Scavengingtreatment with Capacity 0.01 M NaCl induced a completely different behavior: the increaseThe radical of TRC scavenging and decrease capacity of TPC. (RSC) This is may one suggest of the the most presence important of antioxidants antioxidant (e.g.,properties. ascorbic In acid), case of or white other cabbage, compounds it is mainly (even other due to phenolics, the phenolic e.g., compounds cinnamic acids [35], evenand chalcones)if other antioxidants which react as with vitamins Folin (especially‐Ciocalteu vitaminreagent but C), glucosinolatesdo not absorb light and isothiocyanates at 280 nm and aor concomitant minerals, contribute depletion toof it.some The phenolics results of or the other DPPH compounds RSC analyses containing are summarized π conjugated in systems,Figure4A,B. as aromatic glucosinolates. Due to this type of situations, it is necessary to apply multipleDuring assays: the for maturation the detection of white of phenolic cabbage compounds, microgreens reducing no significant capacity change and antioxi was no-‐ dantticed. activity It was and registered it is strongly a slight recommended decrease from that 5-day based to 7-day on the samples, obtained similar results, to the to follow results inpreviously‐depth studies observed in order for broccoli to elucidate sprouts which [26 ].are This the decrease compounds is justified generated/depleted by the obtained in presencediminution of salts of the and total the phenolic exact mechanisms content and involved. total reducing capacity, and also, may be due to a diminution of vitamin C level, which was previously reported in broccoli sprouts by 3.5.Pérez-Balibrea DPPH Radical et al.Scavenging [40,41]. From Capacity 7 days to 9 days, the RSC of cabbage microgreens registered an insignificant augmentation, but the obtained value was inferior to the DPPH RSC The radical scavenging capacity (RSC) is one of the most important antioxidant prop‐ of 5-day microgreens (Figure4A). As consequence, the 5-day-old microgreens presented erties. In case of white cabbage, it is mainly due to the phenolic compounds [35], even if the highest antioxidant characteristics among the studied developmental stages. However, other antioxidants as vitamins (especially vitamin C), glucosinolates and isothiocyanates the length of 5-day microgreens was smaller than of 9-day-old (data not shown), and was or minerals, contribute to it. The results of the DPPH RSC analyses are summarized in preferred to test the effects of sodium salts on bigger size microgreens. Figure 4A, B.

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Figure 4. DPPH radical scavengingFigure 4. DPPH capacity radical (DPPH scavenging RSC, mg capacity ascorbic (DPPH acid/100 RSC, g mg FW) ascorbic of white acid/100 cabbage g FW) microgreens. of white (A). Inﬂuence of growth: untreatedcabbage microgreens. 5-, 7- and 9-day-old (A). Influence microgreens of growth: (α untreated= 0.05, between 5‐, 7‐ and groups 9‐day df‐old = 2, microgreens F = 1.618, p(α ==

0.274). (B). Influence of NaCl0.05, or Nabetween2SO4 treatments groups df = on 2, 9-day-old F = 1.618, microgreensp = 0.274). (B ().α Influence= 0.05, between of NaCl groups or Na2 dfSO =4 4,treatments F = 31.515, on 9‐ p = 0.000). Above the data pointsday‐old are microgreens displayed the (α mean= 0.05, values between and groups a letter: df different = 4, F = 31.515, letters denotep = 0.000). significant Above the differences data points between values (Tukey HSDare test). displayed Series 1,the 2 ormean 3 from values the and legend a letter: represent different the 3 letters individual denote experiments. significant differences between values (Tukey HSD test). Series 1, 2 or 3 from the legend represent the 3 individual experiments. All the 4 applied treatments determined increases of DPPH RSC of 9-day cabbage microgreens,During the but maturation only in case of white of NaCl cabbage at both microgreens concentrations, no significant the augmentations change was were no‐ ticed.statistically It was significantregistered a compared slight decrease to untreated from 5 control.‐day to 7 The‐day highest samples, antioxidant similar to activity the results was previouslyobtained in observed case of 0.01 for M broccoli NaCl treatment. sprouts [26]. Thus, This 0.01 decrease M NaCl is induced justified an by enhancement the obtained by diminution9.14% and 0.1 of the M NaCl, total phenolic by 7.11%. content The highest and total DPPH reducing RSC under capacity, the treatment and also, may with be 0.01 due M toNaCl a diminution is correlated of withvitamin the C highest level, TRCwhich and was can previously occur simultaneously reported in broccoli with the sprouts decrease by of PérezTPC measured‐Balibrea et at al. 280 [40,41]. nm. The From important 7 days antioxidant to 9 days, the activity RSC isof due cabbage to the microgreens increased content reg‐ isteredof all antioxidants, an insignificant including augmentation, also vitamin but C,the glucosinolates, obtained value or was even inferior other phenolics, to the DPPH e.g., RSCcinnamic of 5‐days acids microgreens and chalcones, (Figure which 4A). are As not consequence, detected by reading the 5‐day the‐old absorbance microgreens at 280 pre nm.‐ sentedMore the highest than that, antioxidant it was noticed characteristics that 0.01 Mamong NaCl the had studied no negative developmental influences stages. on the However,microgreens the development, length of 5‐ comparedday microgreens to untreated was samples,smaller than and theirof 9‐ sensorialday‐old properties(data not shown),are acceptable and was for preferred consumers. to test The the previous effects of studies sodium on salts NaCl on influence bigger size are microgreens. contradictory. GuoAll et al. the studied 4 applied the influencetreatments of determined NaCl on the increases sprouts ofof broccoli DPPH RSC and foundof 9‐day that cabbage 0.16 M microgreens,NaCl significantly but only increased in case antioxidant of NaCl at activity both concentrations, and TPC, while the 0.04 augmentations M and 0.08 M were NaCl statisticallydecreased themsignificant [42]. Hassinicompared et al.to untreated (2017) discovered control. The that highest DPPH RSCantioxidant and TPC activity in 10- wasdays obtained broccoli in sprouts case of strongly 0.01 M decreasedNaCl treatment. in presence Thus, of 0.01 0.15 M M NaCl NaCl induced [22]. Another an enhance study‐ mentof sodium by 9.14% chloride and 0.1 effect, M NaCl, but on by artichoke 7.11%. The leaves, highest showed DPPH that RSC DPPH under RSCthe treatment enhances withcontinuously 0.01M NaCl when is salinitycorrelated increases with the and highest the maximum TRC and was can obtained occur simultaneously at the highest level,with thewhich decrease was 0.2 of MTPC NaCl measured [43]. at 280 nm. The important antioxidant activity is due to the increasedThe sodiumcontent sulphateof all antioxidants, at tested concentrations including also increasedvitamin C, insignificantly glucosinolates, the or DPPH even otherRSC (Figurephenolics,4B). e.g., More cinnamic than that, acids it and inhibited chalcones, the germination which are not and detected the development by reading the of absorbancemicrogreens. at 280 nm. More than that, it was noticed that 0.01 M NaCl had no negative influences on the 4. Conclusions microgreens development, compared to untreated samples, and their sensorial properties are acceptableConcerning for theconsumers. development The previous stage, the studies 5-day-old on NaCl microgreens influence proved, are contradictory. globally, the Guobest et antioxidant al. studied properties. the influence Comparing of NaCl theon the effects sprouts of sodium of broccoli salts, and 0.01 found M NaCl that and 0.16 0.1 M M NaClNa2SO significantly4, generally, increased determined antioxidant the strongest activity antioxidant and TPC, properties.while 0.04 M Excepting and 0.08 theM NaCl right decreasedchoice of thethem harvest [42]. Hassini time, the et germination al. (2017) discovered under adequate that DPPH sodium RSC stressand TPC may in become 10‐days a broccoliway to increase sprouts thestrongly phenolic decreased content in and presence antioxidant of 0.15 capacityof M NaCl microgreens, [22]. Another improving study of sodiumtheir health-promoting chloride effect, but properties. on artichoke The present leaves, results showed could that beDPPH used RSC to develop enhances innovative contin‐ uouslytechnologies when salinity for the increases growth of and antioxidant-enriched the maximum was obtained microgreens, at the but highest further level, complex which wasstudies 0.2 M are NaCl needed [43]. to elucidate all the changes induced in plants.

Funding: This research received no external funding. Institutional Review Board Statement: Not applicable.

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Informed Consent Statement: Not applicable. Data Availability Statement: Data are available from the author. Acknowledgments: The author would like to thank the Horticultural Research Center of the Univer- sity of Agricultural Sciences and Veterinary Medicine (UASVM) of Ia¸sifor research infrastructure opportunity and the Francophone University Agency (AUF) for the research equipment of the Laboratory for the Analysis of Antioxidant Bioactive Compounds of UASVM of Ia¸si. Conﬂicts of Interest: The author declares no conﬂict of interest.

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