**4. Discussion**

The fresh biomass of *S. minor*, *S. arvensis*, and *T. o*ffi*cinale* microgreens (Figure 1) ranged from 0.8 kg/m<sup>2</sup> (*S. minor*) to 2.4 kg/m<sup>2</sup> (*S. arvensis*) and was consistent with that reported by Bulgari et al. [46], Paradiso et al. [47], and Renna et al. [48] for microgreens of vegetable crop species. Kyriacou et al. [27] found that the microgreens of 10 different species produced over 3 kg FW/m2, but these authors

adopted a longer growth period, harvesting the microgreens at the second leaf stage. At the baby green stage, *S. arvensis* and *T. o*ffi*cinale* yield (about 3 kg FW/m2) was higher than that of cultivated species [28,49,50]. The fresh biomass of *S. minor* baby leaves was only 0.2 kg FW/m2. In this case, the increase in plant fresh weight from microgreens to baby leaves did not compensate for the lower plant density, suggesting that a later stage of harvest (i.e., more than 5–6 leaves) would have been more proper for *S. minor*.

Wild edible plants contain important amounts of non-nutrient compounds beneficial for health, such as carotenoids and phenolic compounds [51]. Healthy e ffects of these bioactive molecules are often associated with antioxidant activity, leading to the reduction in cardiovascular disease risk factors, the decrease of the incidence of cancer, and protection against a wide range of chronic diseases [52]. Besides the health benefits, carotenoids and anthocyanins influence the organoleptic quality of plant products (taste, aroma) and their visual appearance [27,53]. Together with chlorophylls, they are the main pigments contributing to leaf color, which is particularly important for leafy vegetables since it strongly conditions the evaluation by the consumer and, especially in produce like microgreens and baby leaves, should be uniform and intense [38,54].

Considering the microgreen stage, the three studied wild species showed usually higher or, sometimes, comparable chlorophyll, carotenoids and anthocyanin concentrations than those of most vegetable crop species analyzed in previous studies [25,27,46,47,55,56]. Nevertheless, under LED illumination some microgreens of Brassicaceae family showed even higher carotenoid amounts [57], and particularly high contents of total anthocyanins were measured by Samuoliene et al. [ ˙ 26] in the microgreens of 10 vegetable species. As reviewed by Saini et al. [17] and Di Gioia et al. [58], many studies have shown that baby greens are a good source of antioxidants. To our knowledge, no comparison between baby greens and microgreens of the same species has been carried out on this aspect yet. Among the species we analyzed, *S. minor* showed the highest anthocyanin amounts, and baby greens were richer in these compounds, as well as in carotenoids than microgreens (Table 2 and Figure 2). Considering that, in general, these phytochemicals increase during leaf development and reach the maximum level in mature leaves [59] this result is probably ascribable to the di fferent stage of the harvest of the two products. For the same reason, the lower content of carotenoids found in *S. minor* and *T. o*ffi*cinale* micro/baby greens in comparison with values reported in the literature for adult plants of these species [51] is reasonable.

Microgreens and baby greens of vegetable crops show very variable nitrate contents [27,28]. Such variability is due to the di fferent accumulation ability of the di fferent genotypes, but it is also strongly influenced by agronomic and environmental factors [9]. When microgreens were compared to adult plants of the same species grown in the same conditions, lower nitrate content was observed in microgreens [34]. Accordingly, in our study, the more mature stage (baby greens) of *T. o*ffi*cinale* contained more nitrate than the microgreen counterpart. Conversely, compared to nitrate content measured in *T. o*ffi*cinale* adult leaves collected in the wild [60], we found much higher values, probably due to higher nitrogen availability in the nutrient solution than in the uncultivated soil. In *S. minor* and *S. arvensis*, no di fferences were found between microgreens and baby leaves (Figure 2).

Concerns about nitrate accumulation in vegetables are mainly related to the fact that nitrate ingestion is thought to be a risk factor for stomach cancer [9]. That has brought the EU Commission to establish maximum nitrate levels allowed for the commercialization of some vegetables (spinach, lettuce, and rocket) ranging from 2000 to 7000 mg/kg FW (Regulation No 1258/2011). On the other hand, the association between the estimated intake of nitrate in the diet and stomach cancer has been recently rejected on the basis of the review of the epidemiological literature [10]. Moreover, di fferent authors have reported that a diet high in nitrate is beneficial to humans for cardiovascular and cerebrovascular health [61,62], in particular in older adults [63]. In our study, nitrate concentration was over 2000 mg/kg FW in all the analyzed samples, and in *S. arvensis* microgreens and *T. o*ffi*cinale* baby greens exceed 7000 mg/kg FW. If, on one hand, that can be considered a limitation for these products, on the other hand, it makes them possible candidates to provide dietary nitrate supplementation for some categories of people like the elderly.

Data available in the literature demonstrate that wild edible plants may be an excellent source of macro and microelements for humans. Wild greens usually contribute to the dietary intake of minerals more than wild fruits, and for Ca, Mg, Fe, and Mn, the provided amounts may even reach half of the recommended daily requirement [4]. In *S. arvensis*, *S. minor* and *T. o*ffi*cinale* micro/baby greens, analyzed in this study, these elements showed concentrations sometimes higher and sometimes lower than those reported in the literature for adult counterparts [4,64,65]. In previous studies, microgreens were found to contain lower Ca amount than adults in amaranth [30] and kale [36], while the contrary was found in lettuce [34], and broccoli grown on compost [66]. Among the three analyzed species, *S. arvensis* showed higher Ca concentrations than *S. minor* and *T. <sup>o</sup>*ffi*cinale*, and, at the baby green stage, exceeded 200 mg/100 g FW (Figure 3A), which is considered a good Ca content [4]. In all the three species, baby greens were richer in Ca than microgreens (Table 3 and Figure 3A), confirming the results of Waterland et al. [36] in kale. These authors found that kale baby greens contained also higher amounts of Mg and Fe than microgreens of the same species. In our study, baby greens were richer in Mg than microgreens only in *S. minor* (Figure 3B). This species, on average, showed much more Mg than *S. arvensis* and *T. o*ffi*cinale* (Table 3). That is not surprising, considering that among wild edible greens, *S. minor* is considered one of the richest Mg sources [4]. Furthermore, *S. minor* needed eight days more than *S. arvensis* and *T. o*ffi*cinale*to reach the baby leaf stage and the different growth period could have affected the mineral composition [24]. In comparison with microgreens [24,27,47] and baby greens [28,67] of many vegetable crop species, the wild greens grown in our study showed medium to low content as microgreens and medium to high content as baby greens for Ca and contained medium to high amounts of Mg at both stages of harvest. For Fe, according to what was observed by Waterland et al. [36] in kale, *S. minor* baby greens showed higher concentration than microgreens, while the opposite occurred in *T. o*ffi*cinale* (Figure 3C). It is interesting to notice that, considering the reviewed literature on wild greens [2,4,68], vegetable microgreens [24,30,34,46,47], and vegetable baby greens [67] of different species, *T. o*ffi*cinale* microgreens exceeded the Fe amount of any of them. Some differences among species were observed in P, Cu, Zn, and Mn concentrations, and for P and Mn also between stages of harvest (Table 3 and Figure 3D–F). For all the three species and both the stages, values were comparable (P and Zn) or higher (Mn and Cu) than those measured by other authors in vegetable microgreens [24,47] or baby greens [67]. Waterland at al. [36], noticed higher Zn amounts in kale baby leaves in comparison with the microgreen counterparts. Contrasting this, we did not observe differences in Zn concentration between the two stages of harvest.

According to the Regulation (EU) No. 1169/2011 on the provision of food information to consumers, foods can be considered significant sources of mineral elements if they contain, per 100 g, at least 15% of the reference values reported in the Annex XIII, and corresponding to (in mg): 120.0 (Ca), 56.3 (Mg), 105.0 (P), 2.10 (Fe), 0.15 (Cu), 1.50 (Zn), 0.30 (Mn), 0.0060 (Cr), 0.0083 (Se), and 0.0075 (Mo). The comparison between these amounts and data shown in Table 3, Table 4 and Figure 3, Figure 4 would indicate that micro/baby greens of the wild species analyzed in our study should be good sources of several minerals in the human diet. Nevertheless, for evaluating their contribution it cannot be disregarded that specialty produce, especially microgreens, are normally consumed in small amounts. Therefore, in order to avoid overestimations, in our study EDI% was calculated for a portion of 20 g (Table 5), which was considered quite a reasonable amount for the comparison between microgreens and baby greens. As reference values, RDI or AI as defined in the Materials and Methods section were considered. The largest contributions were observed for Cr, Mn, Mo, Mg, and Fe. For the latter, particularly noticeable was the EDI% of *T. o*ffi*cinale* microgreens (almost 20%). Intermediate EDI% values were noticed for Se and Cu, and the lowest for Zn, Ca, and P. Zinc and P data are consistent with the fact that leafy vegetables, either wild or cultivated, do not stand out by their P and Zn concentrations, and thus they are not generally recognized as good sources of these elements [4].

Minor elements (Cr, Se, Mo, Co, Al, Ni, As, Cd, and Pb) have been rarely measured in micro/baby greens. Molybdenum concentration in lettuce microgreens [34] was comparable to the values found in the microgreens of the wild species considered in our study but lower than those of *S. minor* and *T. o*ffi*cinale* harvested at the baby stage (Figure 4A). For Se, the wild greens, independently from the stage of harvest, showed higher amounts than those measured in lettuce microgreens [34], but *S. minor* and *T. o*ffi*cinale* were richer in this element than *S. arvensis* (Table 4). Xiao et al. [24] investigated Cd and Pb content of 30 vegetable microgreens of the Brassicaceae family, finding that these elements were under the limit of detection. Also, Paradiso et al. [47] observed that Pb was under the detection limit in some genotypes of microgreens belonging to Brassicaceae or Asteraceae, while in the same samples Cd concentration was about 10 times over the values observed in our study (Figure 4D). The species considered in our study resulted to contain Pb, and, in *S. minor* microgreens, the amount of this metal exceeded the ML of 30 μg/100 g FW recommended by the FAO/WHO *Codex Alimentarius* Commission for leafy vegetables [45]. Other heavy metals detected in the wild greens were Cr, Co, Al, Ni, and As. That was not surprising since ruderal species, like *S. minor*, *S. arvensis* and *T. <sup>o</sup>*ffi*cinale*, are well-known for their capability to accumulate contaminants, especially in leaves [8,12,13]. For example, Giacomino et al. [69] and Stark et al. [70] found potentially hazardous levels of Pb and As, respectively, in some samples of spontaneously growing *T. <sup>o</sup>*ffi*cinale*, and *S. arvensis* stood out among different wild species for Cd and Cr accumulation in both contaminated and not-contaminated soils [12,64]. In our study, microgreens and baby greens were grown in a controlled environment and hydroponically, using a nutrient solution prepared with distilled water, therefore it can be supposed that the detected trace elements derived from the mineral fertilizers used to prepare the nutrient solution [71,72] and from vermiculite used as growing medium [73]. However, since HRI values <1 are assumed to be safe in terms of population exposure to metals [43], HRI calculated for Fe, Cu, Mn, Cr, Se, Mo, Co, Ni, As and Cd considering a portion of 20 g, being far below 1 (Table 6), excluded health risks due to the consumption of micro/baby greens in relation to these elements. Health risks were excluded also for Al, whose ingestion was calculated on a weekly basis according to EFSA recommendation [44]. Even considering portions of 100 g, which are quite improbable for these products, HRI values would still be below 1 in most cases. Only *S. minor* baby greens and *T. o*ffi*cinale* microgreens would show HRI >1 for Mn and Cr, and for Co and Cr, respectively, if EDIBw was reported to 100 g product.
