**1. Introduction**

Microgreens are a new class of small, fresh, edible vegetables considered as a good nutritional source because of their high mineral and bioactive compound content. The meaning of microgreen refers to immature greens harvested at soil level between the first and third week after sowing, when the cotyledon is fully developed and the first true leaves have emerged [1–3], being different from both baby leaf (cut greens for salads) [4] and sprouts (germinated seeds with entire roots) [5]. Microgreens can be produced from many vegetables, herbaceous plants, aromatic herbs, grains and wild species [6–8], and possess distinctive organoleptic properties, such as color, shape, texture and taste [2,8,9].

These new and young vegetables are a versatile, nutritive and sustainable crop from cultivation to consumption. They can be adapted to different agronomic practices to obtain a final product which is of high organoleptic and nutritional quality [1]. Likewise, growing conditions (soil, compost, hydroponic) directly affect the plant growth and the levels of phytonutrients and minerals [5,10].

In this sense, soilless or hydroponic is based on the use of nutrient solution rather than soil for crop production, reducing fertilizer and water resources as well as the use of pesticides [11]. According to Weber [10], a higher amount of minerals was obtained in broccoli microgreens compared to the mature vegetable using about 200 times less water, 94% less time and without applying fertilizer, pesticides or energy-demanding transport. Besides the possibility of saving natural resources and chemicals, the production and consumption of microgreens have additional advantages, turning these products into a new, healthy, and environmentally-friendly vegetable option. For instance, the containerized production in an industrial, local or home scale implies that the final consumer can harvest them just at the moment of being used, and their consumption only without roots generates much less waste than adult vegetables [1,10,12].

In addition, microgreens have been considered as healthy foods because of their general higher levels of phytochemicals with respect to their mature counterparts [2,6,7,12]. In this context, a recent review has defined microgreens as a new food for the 21st century attributing them a potential role as anti-inflammatory, anti-carcinogenic, anti-obesogenic and anti-atherosclerotic [5]. In contrast to the grea<sup>t</sup> amount of nutrients expected to obtain health benefits, Renna et al. [13] developed chicory and lettuce microgreens with a reduced potassium content to be consumed by chronic kidney disease patients. Also, microgreens have been proposed as ideal food for people with a vegetable-based diet such as vegans or vegetarians, and even for space crew members due to their limited access to food diversity [14].

It is known that *Brassica* vegetables, at the mature stage, contain beneficial nutrients for human health [15], and available data reveal that their intake reduces the risk of chronic diseases [16]. Probably this is the reason why among the di fferent species used to obtain microgreens, the *Brassicaceae* family is one of the most widely grown to date [2]. Nevertheless, information in the literature about *Brassicaceae* microgreens is limited regarding the concentrations of the antioxidant bioactive compounds and minerals that were examined in this work. There are some studies on this subject in broccoli [7,10,17–20], kale [19–21], mustard [6,8,9,20,22–24] and radish [6,8,20,24–26] microgreens. However, the health-related e ffects of bioactive compounds of a food depend not only on their content and the amount consumed, but also on their bioavailability. Although in vivo assays are the gold standard for this purpose, these studies are expensive, lengthy, and have some ethical concerns. In turn, in vitro digestion allows one to estimate the bioaccessibility (the total amount of a food compound in soluble form and released from the solid food matrix that is available for absorption) [27], a prerequisite of bioavailability.

The aim of the present study was to evaluate the content and for the first time the bioaccessibility of the main antioxidant bioactive compounds (ascorbic acid, total carotenoids, total isothiocyanates, total anthocyanins, total soluble polyphenols), total antioxidant capacity, as well as macro- (K, Ca, Mg) and oligoelements (Fe, Zn) provided by the four studied hydroponic *Brassicaceae* microgreens: broccoli, kale, mustard and radish.

#### **2. Materials and Methods**

#### *2.1. Plant Material and Sample Preparation*

Four microgreen species belonging to the *Brassicaceae* family were evaluated in this study: broccoli (*Brassica oleracea* L. var. *italica* Plenck), green curly kale (*Brassica oleracea* var. *sabellica* L.), red mustard (*Brassica juncea* (L.) Czern.) and radish (*Raphanus sativus* L.). Mustard and radish cultivar seeds were purchased from CN Seeds Ltd. (Cambridgeshire, UK) and kale and broccoli from Rocalba S.A. (Huesca, Spain) and Intersemillas S.A. (Valencia, Spain), respectively.

Microgreens were produced by the Agronomic Innovation Center (CIAM) of Grupo Alimentario Citrus Company (Valencia, Spain) at the end of August 2017. A hydroponic system was created by placing substrates of pine tree fibers (12 cm × 12 cm × 0.4 cm) on plastic trays. Two seeding densities were selected: 3.8 seeds cm<sup>−</sup><sup>2</sup> for broccoli and kale and 2.8 seeds cm<sup>−</sup><sup>2</sup> for mustard and radish. The sown substrates were moistened with water and introduced into a growth chamber at 18 ◦C and 90% relative humidity (RH) until the germination of the seeds. Then, they were moved into an unheated greenhouse where no artificial light treatment was applied. The incidence of natural light at this time of year provided a daily average of 18 ◦C and 61% RH. The following nutritive solution expressed as mmol/L for each component was applied daily: NO3 − (5.3), H2PO4 −2 (1.5), SO4 −2 (4.4), HCO3 − (0.5), Cl− (5.3), K<sup>+</sup> (1.5), Ca+<sup>2</sup> (6.3), Mg+<sup>2</sup> (1.3) and Na<sup>+</sup> (3.1). An average fertigation value of 20.4 l m<sup>−</sup><sup>2</sup> per day from June to September 2017 was recorded. No phytosanitary treatment was used.

Nine days after seeding for radish and 7 days for broccoli, kale and mustard, the microgreens were transported in plastic trays (58 cm × 39 cm) from CIAM to the University of Valencia (UV). They were fertigated just before being moved in order to maintain good humidity conditions during the 30 min period of transportation. In our laboratory at the UV, a total of 40 trays were received (8 for kale, 12 for mustard and 10 for broccoli and radish). For each microgreen, approximately 400 g were harvested as close as possible to the root using sterilized scissors. Next, a pool was made to homogenize each microgreen sample, and then they were randomly divided into several replicates. Fresh microgreens were used immediately for ascorbic acid analysis and the rest of the collected samples were weighted inside aluminum containers before freezing at −80 ◦C. Frozen microgreens were lyophilized for 48 h (Sentry 2.0 Virtis SP Scientific, Philadelphia, PA, USA) and maintained in a desiccator until constant weight to obtain dry weight (DW) percentage (4.76 ± 1.43, 4.71 ± 1.49, 4.25 ± 1.36 and 4.91 ± 1.55 for broccoli, kale, mustard and radish, respectively) in accordance with the range 3.9–8.1% described in previous studies on these *Brassicaceae* microgreens [6–8,19]. Next, samples were ground into a fine powder in a grinder (Super Junior "S" Moulinex, Alençon, France) and stored at −20 ◦C for subsequent experiments.
