vegetables are a good source of many phytochemicals with health-related activity, and their dietary consumption is associated with the reduction in the incidence of several human chronic inflammatory diseases [1
]. Both the profile and the amount of these phytochemicals are strongly affected by the genotype (different species/cultivar/landraces), by the environmental conditions during plant growth and by the different plant growth stages. Some authors have detected great variation of several antioxidant compounds, as such as glucosinolates, carotenoids, polyphenols and ascorbic acid, and of the related bioactivity of several landraces (LRs) and crop wild relatives (CWRs) of B. oleracea
, evidencing elite breeding materials useful for improving the nutraceutical traits of the products [5
Among the detected phytochemicals, polyphenols are a large class of organic compounds including several aromatic rings associated with different phenolic groups. Phenolics range from simple and single aromatic-ringed compounds, with a low molecular-weight, to large and complex tannins and derived polyphenols [7
]. They can be classified based on the number and arrangement of their carbon atoms in flavonoids (flavanols, flavones, flavan-3-ols, anthocyanidins, flavanones, isoflavones and others) and non-flavonoids (phenolic acids, hydroxycinnamates, stilbenes and others) and they are commonly found in plants as conjugated to sugars and organic acids [7
Polyphenols are often produced in response to biotic or abiotic stress [9
] and act as reducing agents, hydrogen donating antioxidants and singlet oxygen quenchers. The multifunctionality of polyphenols is due to their distribution in different tissues and organs of plants at different concentrations. The most widespread and different groups of polyphenols in Brassica
species are the flavonoids (mainly flavonols but also anthocyanins) and the hydroxycinnamic acids [10
]. Phenolic compounds play a role in different stages of cancer development, exerting their activity in regulating different signaling pathways involved in cell survival, growth and differentiation of the reproductive organs.
Vitamins are indispensable micronutrients for humans with antioxidant activity. Studies in vitro demonstrated vitamin (E and C) consumption decreases the risks of chronic diseases by acting as direct antioxidants and electron donors [12
Today, new natural products containing high levels of bioactive compounds consumed through food are being researched and studied. Sprouts, microgreens and baby leaves represent a growing market segment within vegetable products, mainly consumed raw for their high nutritional value and sensory traits [14
]. Candidate genotypes are expanding based on sensory and health criteria; however, currently, most of the species belong to Brassicaceae, Asteraceae, Chenopodiaceae, Lamiaceae, Apiaceae, Amarillydaceae, Amaranthceae, and Cucurbitaceae families [15
In the last few years, the request of seeds and sprouts of broccoli and of other Brassicaceae species has become increasingly popular among consumers interested in improving and maintaining their health status by changing dietary habits [16
The production and commercialization of sprouts are legally defined, and generally they are grown in dark conditions, without growing medium, fertilizers and agrochemicals [14
]. Studies carried out on broccoli sprouts exposed to low intensity and high intensity of ultraviolet A (UVA) or ultraviolet B (UVB) demonstrated an enhancement of bioactive compounds, suggesting their exploitation as a functional food with potential industrial applications [17
A new class of specialty salad crops exploited for their color- and flavor-enhancing phytonutrient content are the microgreens [18
]. They differ from sprouts for the fully developed cotyledons and the appearance of the first true leaf; they are defined as young and tender vegetables, with a species-dependent production cycle of 1–3 weeks from seed germination, and they are harvested at soil level [20
]. Compared to their mature-leaf counterparts, microgreens contain higher amounts of important phytonutrients (ascorbic acid, β-carotene, phylloquinone, etc.), minerals and lower nitrate levels [20
]. Microgreens have been proposed as ’super foods‘ for their favorable content in micronutrients and bioactive compounds, whose yield can be increased by modulating the blue light proportion in red and blue light-emitting diode lighting [23
]. Due to their phytonutrient compounds, a recent study supported the idea that microgreens could be considered as a resilient phytochemical factory for the dietary and psychological needs of crew members in orbital flights and platforms [26
]. They are thought to be a new food for solving malnutrition problems affecting over two-thirds of the world’s people living in countries of every economic status.
Among the ready-to-eat products, the baby leaf vegetable market has been growing and offering to consumers convenient, healthy and appealing items, which may contain useful bioactive compounds. The consumer demand for more convenient fresh food products led to a rapid growth in the fresh-cut industry, which became a multi-billion-dollar sector worldwide in the last decade [27
]. Fresh-cut vegetables can meet the consumer demands for their relationship among food, healthy lifestyle and convenience [29
The production of sprouts, microgreens and baby leaves from local varieties and wild edible species may provide novel and nutraceutical vegetables, which can satisfy the demand of modern consumers. Moreover, they represent further expressions of biodiversity in vegetable production, contributing to preserve and to increase the value of many vegetable LRs at risk of genetic erosion or extinction. The phytochemical composition of Brassicaceae varies considerably as a consequence of the plant growth stage and of the species considered [30
]. For these reasons, we investigated the content of bioactive compounds at three stages of plant growth (sprouts, microgreens and baby leaves) of a traditional Sicilian broccoli LR and of two commercial standard cultivars, one of broccoli and one of kale.
2. Materials and Methods
2.1. Plants Materials, Seed Morphological Characteristics and Germination Test
Seeds of the standard commercial cultivars of broccoli (Brassica oleracea L. var. italica Plenck) ‘Cavolo Broccolo Ramoso Calabrese’ (CR) and of kale (Brassica oleracea L. var. acephala) ‘Cavolo Lacinato Nero di Toscana’ (CL) were purchased by the S.A.I.S. S.p.A. seed company (Cesena, Italy). The Sicilian landrace of sprouting broccoli (Brassica oleracea L. var. italica Plenck) ‘Broccolo Nero’ (BN) belongs to the Di3A active genebank collection (BR 354, UNICT 4939).
The weight of 1000 seeds, number of seeds per gram, perimeter, longitudinal and transversal lengths were registered. The germination test was carried out at the laboratory of the Department of Agriculture, Food and Environment (Di3A) of Catania University (Italy). Four replicates of 50 seeds for each cultivar (cv) were placed in 90 mm Petri dishes, between two layers of filter paper (Whatman® no. 2), imbibed with 10 mL of distilled water. Petri dishes were placed in dark condition in five incubators (FTC 90E, Refrigerated Incubator, VELP Scientifica, Italy) set at constant temperatures of 5, 10, 15, 20 and 25 °C. At the end of the experiment, the following indices were calculated: germinability (%) = ((N/NT) × 100), where N is the number of germinated seeds and NT is the number of the total seeds utilized; Mean Germination Time (MGT) = ∑(ni × di)/n, where n indicates the number of germinated seeds at day i, d is the incubation period in days, and n is the number of the total seeds germinated.
2.2. Sprouts, Microgreens and Baby Leaves Production and Characterization
For the sprouts, microgreens and baby leaf production, seeds were sown in cellular trays placed in cold greenhouse under natural light (4.6 to 9.2 MJ·m−2·d−1) and temperature (15.4 ± 5.8 °C) conditions, from November to December 2017 at Catania (South Italy, 37°31′10″ N 15°04′18″ E; 105 m above sea level (m a.s.l.) using organic growing practices. We utilized the organic substrate Brill®semina bio (Geotec, Italy) to fill the cellular trays and we treated the baby leaves once by BTK ® 32 WG (Xeda, Italy) based on Bacillus thuringiensis sub. kurstaki for controlling Pieris brassicae. The plantlets were irrigated on the basis of the ordinary techniques. Plants were collected at the three plant growth stages analyzed: sprouts (germinated seeds without coats and roots), microgreens (plantlets with the first true leaf) and baby leaves (plantlets with almost three true leaves), weighted and stored immediately at −80 °C, and then freeze-dried for analyzing them.
Plants were characterized for the main morphological descriptors. Longitudinal and transversal lengths of the cotyledons, number of true leaves, if present, and the hypocotyl length were measured. The morpho-biometric parameters of sprouts, microgreens and baby leaves were acquired using the software IM50 v. 117 (Leica, Germany).
2.3. Polyphenol Analysis
The polyphenol compounds were analyzed in accordance with Soengas et al.’s method [31
] with some modifications. For the extraction, 60 mg of lyophilized powder was dissolved in 1.5 mL of a 1:1 v/v mixture of methanol and 0.06 N HCl. The mixture was vortexed and put in an ultrasonic bath for 10 min. After the thermal hydrolysis which was performed by putting samples in hot bath (80 °C for 1 h), the mixture was centrifuged (13,000 rpm, 10 min, 4 °C). The supernatant was stored at −20 °C and analyzed by HPLC Agilent 1200 series system equipped with a diode array detector (DAD). We utilized the analytical column Lichrospher 100RP-18 (240 × 4 mm i.d., particle size = 5 μm). The mobile phase contained water/acetic acid (90:10, v/v, A) and acetonitrile/acetic acid (90:10, v/v, B). Chromatography was performed with 0.6 mL/min flow rate and the following gradient program: 0–7 min 1% B, 7–20 min 30% B, 20–28 min 50% B, 28–33 min 50% B, 33–38 min 1% B, 38–48 min 1% B. The injection volume of sample was 30 μL. Polyphenols were detected by DAD monitoring the absorbance at 280, 310, 325, 350, 520 nm. Hence, the characterization of each phenol compound was based on its characteristic absorption spectra. Each sample was analyzed in triplicate. Quantification was based on the calibration curves of external standards, by comparing each compound through the absorption spectra for apigenin, caffeic acid, chlorogenic acid, cyanidin chloride, gallic acid, coumaric acid, kaempferol and sinapic acid; polyphenols concentrations were expressed in mg g−1
2.4. Ascorbic Acid Analysis
For the ascorbic acid (AA) measurements, the titration method reported by Thillmans et al. [32
] was used. Freeze-dried samples were treated by 3% metaphosphoric acid by shaking. Extracts after filtration on filter paper (size: 110 mm, medium filtration rate, particle retention: 5–13 μm) were titrated by 1.5 mM 2,6-dichlorophenol-indophenol (DCIP) water solution at room temperature. The ascorbic acid contents were quantified by comparing them with the standard curve obtained for the known ascorbic acid concentrations. Each sample was analyzed in triplicate, and the results were expressed as mg g−1
2.5. Folin–Ciocalteu Index
The Folin–Ciocalteu index (FCI) was calculated on methanolic extracts as described by Meda et al. [33
], with slight modifications. Twenty milligrams of freeze-dried material were dissolved in 0.5 mL of 80% ethanol (EtOH). The mixture was vortexed and put in ultrasonic bath for 10 min and then centrifuged (13,000 rpm, 10 min, 4 °C). The supernatant was collected and the remaining pellet was re-extracted as described above. The collected supernatant was pooled. Twenty microliters of the extract were diluted with 1580 μL of distilled water and 100 μL of Folin-Ciocalteu reagent and then we left it at room temperature for 5 min. The solution was then treated with sodium carbonate (20%, 300 μL) and incubated in the dark for 2 h at room temperature. The absorbance of the blue-colored solution was measured at 750 nm. Each sample was analyzed in triplicate. The total polyphenol content was expressed as gallic acid equivalents (GAE mg g−1
d.w.) after using a standard calibration curve obtained by the known concentrations of the gallic acid standard.
2.6. Antioxidant Activity
The antioxidant capacity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical quenching and peroxyl radical (ROO) scavenging methods. For the sample preparations, 100 mg of lyophilized powder was extracted by 1.5 mL of a 1:1 v/v mixture of ethanol (EtOH) and 0.06 N HCl, homogenized and centrifuged at 25,000 g for 5 min at 4 °C. The DPPH was measured using electronic paramagnetic resonance (EPR) with a MiniScope MS200 Magnettech (Berlin, Germany) following the protocol detailed in Picchi et al. [34
]. The antioxidant activity towards ROO was determined spectrophotometrically by measuring the enzymatic peroxidation of linoleic acid after the addition of lipoxygenase, as described in Lo Scalzo et al. [35
2.7. Statistical Analysis
The experiments were performed using a completely randomized design. The significance of differences between the main factors of morphometric characteristics was evaluated by one-way analysis of variance (ANOVA). Data were reported as mean ± standard error (S.E.). To evaluate the effect of the genotype and of the stage of growth, the results of the Folin–Ciocalteu index, ascorbic acid and antioxidant activity were analyzed through a multifactorial ANOVA. The mean values associated with the main factors as well as their interactions were evaluated using Tukey’s test (p < 0.05). All statistical analyses were performed using CoStat release 6.311 (CoHort Software, Monterey, USA).
Several authors focused their attention on the optimization of the germination process in order to maximize the content in health-promoting compounds of the sprouts [36
]. Seeds can germinate at a wide range of temperatures, but the germinability is drastically reduced at extreme temperatures [38
]. Previous investigation showed a good germinability at 30 °C but with a higher MGT values in comparison to 25 °C [37
]. That has been observed in our study in which no germination occurred at 5 °C for all the genotypes analyzed.
The phenolic profile of vegetables can be influenced by intrinsic factors related to the plant growth stage and to genetic variability, also within the same species. In seeds, the differences between varieties and genotypes suggested that the genotype was the main factor of variation; in fact, our results show that the main content in polyphenols was shown for CL and CR. During the germination process, reactivation of seed metabolism takes place, promoting the hydrolysis of storage proteins and carbohydrates by the synthesis/accumulation of metabolites with health-promoting properties [39
According to Paśko et al. [16
], higher total phenolic content in sprouts compared to seeds suggest the synthesis of phenolic antioxidants during germination may occur. The strong correlation found between total polyphenol content and antioxidant activity also suggests that this content is a good predictor of the in vitro antioxidant activity. It is thought that seeds mainly act as a reservoir for the development of the sprouts [40
]. The PPH compounds increased significantly for the three cultivars considered, from the seeds to the baby leaves stage, showing significant differences among them (Figure 3
, Figure 4
and Figure 5
Some of the health-promoting factors may be present at a ten times higher level in sprouts than in mature vegetables [41
]. This is the case of the flavonoids content and of the other phenolic compounds that clearly contribute to the antioxidant potential [43
Germination determines significant changes in the phenolic content, mainly due to activation of endogenous enzymes and to the complex biochemical metabolism of seeds during this process [44
]. For this reason, germination may be a suitable process to obtain functional food with high antioxidant capacity. Sprouts are produced by the seed germination process and they represent an interesting novel food and a valuable alternative to increase the consumption of different seeds in human nutrition [45
]. Sprout phenolic composition depends on genotype as well as on numerous environmental factors, including temperature, light or dark condition, humidity and sprouting time [46
] and also water quality and salt content [42
With regard to the cvs analyzed, BN showed the highest increment of the kaempferol and apigenin from the seed to the baby leaves growth stage, whereas for CL and CR, we ascertained the increment of the PPH compounds, mainly represented by kaempferol, caffeoyl and chlorogenic, just after germination process with no significant differences among the three plant growth stages considered (Figure 4
, Figure 5
and Figure 6
According to the literature data, Brassica
plants are rich in anthocyanins, phenolic acids and flavanols, in particular, kaempferol and quercetin [47
]. In different studies conducted by Cartea et al. [11
] on qualitative flavonoid evaluation in B. napus
, quercetin and kaempferol glycosides were found as the major compounds in Brassica
samples, in full qualitative accordance of previous data [11
] with the here presented ones. Moreover, a quantitative survey by Velasco et al. [48
] on kale and leaf rape reported a total phenol amount of 3–5 mg g−1
d.w., very close to the here presented data in seeds (Table 5
), and lower than the data in vegetative parts reported in the present work, especially for BN samples (Figure 3
According to Pająk et al. [49
] and Velasco et al. [48
], a range of phenolic acids have been identified in sprouts (chlorogenic, gallic, sinapyl, ferulic, p-cumaric). Broccoli sprouts are very rich in phenolic compounds, more than commercial broccoli florets; among them, free phenolic acids, caffeic, chlorogenic and gallic acids occurred in the largest amounts; the main free phenolic acids found in broccoli sprouts were ferulic and sinapic acids [49
]. Elevated levels of flavonoids and phenolic acids, which are highly bioavailable, were observed in a study conducted by Gawlik-Dziki et al. [50
] on broccoli sprouts; this is in accordance with our results where sprouts of BN showed the highest content of kaempferol and apigenin derivates.
Phenolic compounds and vitamin C are the major antioxidants of Brassica
vegetables. The content of vitamin C among Brassica
vegetables varies significantly among and within each species and interspecific entity [1
]. The cause of the reported variations in vitamin C content might be related to the different genotypes studied [51
]. In particular, in the study by Vallejo et al. [52
], the content of vitamin C was very irregular among the genotypes analyzed; the content ranged from 43.1 mg per 100 g fw in Lord (commercial cultivar) to 146.3 mg per 100 g fw in SG-4515 (experimental cultivar). In our work, the highest content in vitamin C was found in BN microgreens. Vitamin C is an important water-soluble dietary antioxidant, which significantly decreases the adverse effects of the free radicals can cause oxidative damage to macromolecules such as lipids, DNA and proteins, which are in turn implicated in chronic diseases, like cardiovascular disease, stroke, cancer, neurodegenerative diseases and cataractogenesis [53
The average levels of DPPH and ROO scavenging activity were highest in sprouts and tended to decrease with growth (Figure 7
). In particular, as regards to the DPPH quenching activity, CR and CL sprouts showed higher values compared to microgreens and baby leaves, and a similar trend was observed for ROO antioxidant activity, even if with different results depending on the cultivars. Our findings partially agree with the results of Ebert et al. [54
], who found that the antioxidant activity for amaranth sprouts was much higher than for microgreens, but not for the fully expanded leaves plant growth phase. Indeed, higher levels of the Folin–Ciocalteu index were found in sprouts (Figure 2
), while the highest level in AA was found in microgreens (Figure 6
) and single polyphenols usually increased in baby leaves, particularly for BN (Figure 3
). These results suggest the presence of other antioxidants than kaempferol and apigenin or AA in Brassica sprouts, which may enhance the antioxidant capacity of the sprouts compared to the microgreens and the baby leaves. Interestingly, the Sicilian landrace BN of broccoli was distinguished for the highest ROO scavenging activity both at the stage of sprouts and of microgreens, but not at the stage of baby leaves, for which its value significantly decreased. On the other hand, for CL and CR, the ROO antioxidant capacity of the baby leaves increased in comparison to microgreens, being finally higher by around 20% compared to the BN baby leaves.
The activities carried out provide additional data related to the novel foods proposed, such as sprouts, microgreens and baby leaves. B. oleracea crops, and in this case broccoli and kale, are shown to represent good sources of antioxidant compounds, such as polyphenols and ascorbic acid, confirmed also by their high antioxidant capacities. The cultivars utilized showed different polyphenol profiles among them and also in relation to the plant growth stages proposed, correspondent to the three novel foods investigated.
The total polyphenols showed the highest values for the sprouts of both the two cvs of broccoli utilized. The Sicilian landrace of ‘Broccolo Nero’ showed the highest amount of kaempferol and apigenin in late growth stages, whereas the former represents the main polyphenol compound for the other cultivars of broccoli and of kale studied. Among the plant growth stages analyzed, the baby leaves showed high values of kaempferol and apigenin. The ascorbic acid varied significantly both in relation to the cultivar and to the plant growth stage and the highest value was observed for the microgreens of ‘Broccolo Nero’. The antioxidant capacity showed in general the highest values for the sprouts for all the three cultivars analyzed.
The experimental factors analyzed offer the opportunity to improve the knowledge related to these novel foods of interest for diversifying the vegetable items by the exploitation of the B. oleracea landraces. Of interest are the data of the antioxidant traits acquired for implementing the nutraceutical and organoleptic traits of the novel foods proposed for improving the horticultural organic food supply chains.