Nutritional and Functional Composition of Microgreens: A Comparison of Various Species ^†

Abstract

The objective of this work is to evaluate the nutritional and functional composition of microgreens of different species. Beet, pea, adzuki bean, popcorn, onion, and carrot were studied. The nutritional composition was determined according to the AOAC. The content of total phenolic compounds, flavonoids, chlorophylls, total carotenoids, and the in vitro antioxidant activity were evaluated by spectrophotometry. The results show that the aduki bean showed the highest levels of chlorophylls and carotenoids. For total phenolic compounds and flavonoids, onion and carrot micro vegetables had the highest content of these bioactive compounds that also showed the highest antioxidant activity. The results obtained show that micro vegetables are an excellent alternative to fresh foods, providing the scope and importance for the introduction of these vegetables into the diet, as they, sometimes, require long production cycles and more resources or are not usually accepted by consumers.

1. Introduction

In recent decades, society has shown a growing interest in healthy eating and lifestyles, demanding fresh, ready-to-eat foods with functional value [1]. Referring to this trend, micro-scale vegetables, including microgreens, have gained special attention as an alternative for consumption, due to their potential to diversify and improve the human diet and address microelement and nutrient deficiencies, as well as providing a high content of phytochemicals with functional properties. Nowadays, there is an increasing demand for regular consumption of these products, and some of them are sold on the market [2].

Microgreens are the seedlings of edible plants harvested between 7 and 14 days after planting, when the first true leaves start to emerge [3]. Shoots are harvested by cutting them just above the roots, and the vibrant colors, visual textures, and flavors make them appealing; they are commonly eaten raw in salads, soups, or sandwiches [1]. These micro-scale vegetables are ideal for indoor production and require less water, fertilizer, pesticides, and space for crop cultivation. They are part of the global movement toward controlled environmental agriculture [3,4]. The short harvest time for microgreens and the high market value open the opportunity to grow vegetables in a cheaper way, in small growing spaces [1,3], providing the scope and importance for the introduction of these vegetables into the diet, as they sometimes require long production cycles and more resources or are not usually accepted by consumers.

Many of the vegetables consumed as microgreens are well known for their health benefits. They have been reported as functional foods in diet-based disease prevention, that is, obesity, cardiovascular disease, type 2 diabetes mellitus, and cancer [3]. Brassica vegetables contain glucosinolate compounds, which have a potential effect against cancer, among other benefits. Apiaceae also have compounds that are beneficial to health, such as carotenoids, one of the most studied antioxidants against age-related chronic diseases and oxidative stress. Plants in the Alliaceae family have organosulfur compounds known to reduce coagulation/thrombosis and improve glucose–insulin homoeostasis [5]. Beetroot contains betalains antioxidants and has anti-inflammatory properties [6]. Other common microgreens are cereals, legumes like pea, and oilseeds rich in amino acids, organic acids, and fatty acids [2].

Therefore, it is important to consider not only the aspect of micro-scale vegetables, but also their nutritional composition and their bioactive compounds that could have health benefits. Prior studies have explored the nutritional benefits of microgreens, and limited comparative analyses have been conducted among multiple species under controlled conditions. The most commercial families and, therefore, the most studied microgreens include first brassicaceae (cabbage, broccoli, kale, arugula), then apiaceae (celery, parsley, carrot), and asteraceae (sunflower), and, finally, in a lower proportion, chenopodiaceae, lamiaceae, amarillydaceae, amaranthceae, and leguminaceae [1,3]. Although microgreens are recognized for their bioactive compounds, comparative studies analyzing multiple species under uniform conditions remain scarce. This work aims to evaluate the nutritional profiles and bioactive compounds of six microgreen species, including underexplored legumes and Alliums that are not usually studied, to provide comprehensive data for potential dietary and industrial applications.

2. Material and Methods

2.1. Microgreens Production

The selected species of study were beet (Beta vulgaris, cv. Green Top Bunching), pea (Pisum sativum cv. Onward), aduki bean (Vigna angularis), sunflower (Helianthus annuus), popcorn (Zea mays, cv. Honey Dew), onion (Allium cepa, cv. Valcatorce), and carrot (Daucus carota, inbreeding line). The seeds of each species were sown in growth trays (28 cm × 54.5 cm × 5 cm) in a mixture of cocopeat and perlite (v/v: 50%), and the trays were stored at a temperature of 25 °C and 60% humidity for 48 h in the dark. After seed germination, the photoperiod was set using an artificial light LED for 12 h per day. Trays were irrigated twice a day with a hand pump sprayer in the plant growth chamber (~0.4 L water so that the soil remained moist but not wet). Harvesting was carried out during the first hours of the day at a temperature of 24 °C by cutting microgreens from the root collar area with a sharp, sterile knife. After harvest, the plants were quickly frozen at −80 °C and freeze-dried for 72 h in a vacuum system. The resulting lyophilized material was ground into powder with a mortar and stored at room temperature in vacuum-sealed bags until analysis to prevent degradation. Samples were analyzed one week later all at once.

2.2. Nutritional Composition Analysis

The nutritional composition was determined following the official analysis methods of the Association of Analytical Communities (AOAC) [7]: for the determination of moisture (AOAC 167.03), dry matter (AOAC 167.03), total minerals (AOAC 942.05), nitrogen (AOAC 984.13), total protein (calculation: N × 6.25), total fat (AOAC 920.39C), crude fiber (AOAC 962.09), and carbohydrates (by difference).

2.3. Functional Composition Analysis

2.3.1. Pigment Analysis

The determination of photosynthetic pigments was performed according to Lichtenhaler and Buschmann [8]. For this, 0.025 g of freeze-dried material was extracted with 10 mL of 80% acetone and sonicated in an ultrasonic bath for 10 min. After that, the extracted mixture was centrifuged for 10 min at 14,000 rpm. The absorbance was measured at three different wavelengths: 663, 646, and 440 nm through a spectrophotometer. Values were expressed as mg/100 g of fresh weight (fw). The calculations were made on the basis of the following equations:

$$\mathit{Chlorophyll}a, {\mathit{Chl}}_a=12.21 A_{663}-2.81 A_{646}$$

$$\mathit{Chlorophyll}b, {\mathit{Chl}}_b=20.13 A_{646}-5.03 A_{663}$$

$$Total chlorophylls, Chls=Chla+Chlb$$

$$Total carotenoids, Cars=4.69 A_{440}-0.268 \times (Total Chls)$$

2.3.2. Total Phenolic Content and Total Flavonoid Content

The freeze-dried material of each species (0.025 g) was extracted in 2 mL of methanol:acid water with hydrochloric acid to pH = 2 (70:30 v/v), vortexed for 30 s and sonicated for 30 min at 25 °C. After that, the extracts were centrifuged at 14,000 rpm for 10 min at 4 °C and filtered through a 0.22 μm nylon membrane.

The total phenol content (TPC) was quantified using the Folin–Ciocalteu method following the procedure previously described by Lemos et al. [9]. For this, 0.05 mL of extract was mixed with 2.45 mL of distilled water, 0.25 mL Folin solution, and 0.75 mL of Na₂CO₃ (10% w/v). After 3 min, a constant volume of 2.5 mL of distilled water was added and incubated for 30 min under dark conditions. The absorbance was measured at 765 nm. Each sample was measured against a blank of reagents, containing distilled water instead of the extract. Total phenolic content was determined with a linear calibration curve equation and is expressed as the mean of mg of gallic acid equivalents per 100 g of fresh weight (mg GAE/100 g fw).

The total flavonoid content (TFC) was determined according to the method developed by Zhishen et al. [10], combining 100 µL of methanol:acid water extract with 1.4 mL of distilled water and 75 µL of Na NO₂ (5% w/v). After 5 min of reaction, 150 µL of AlCl₃ (10% w/v) was added to the mixture. Then, after another 6 min of reaction, 500 µL of NaOH (1 M) and 775 µL of distilled water was added, and the absorbance at 510 nm was measured. TFC is expressed as mg of catechin equivalents per gram of fresh weight (mgECat/g fw).

2.3.3. Antioxidant Activity In Vitro

Antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, according to [8]. Briefly, 0.65 mL of the extract obtained as described above (Section 2.3) was mixed with 2.5 mL of DPPH solution (40 mg/L in methanol; Abs ~ 1.0 at 517 nm). The samples were measured (A_E) against methanol and methanol with a DPPH blank (A_B). The experiment was carried out three times, and the absorbance sample (A_o) was considered. An acid gallic solution (1 mM) was used as a reference (A_REF). Radical scavenging activity was calculated as the percentage of inhibition (I%), as follows:

I% = [(A_B − (A_E − A_o)/A_B)/(A_B − A_REF/_AB)] × 100

2.4. Statistical Analysis

Values are expressed as means ± standard deviation. Data were analyzed by analysis of variance (ANOVA) to test for significant differences. Means were compared by the Tukey’s test using the InfoStat-Statistical Software2022 (Córdoba, Argentina). The results were considered significant at p ≤ 0.05 unless otherwise specified.

3. Results and Discussion

The time from seedling to harvesting mature microgreens in the second stage of true leaves differed considerably between the species examined. The growth period for onion and carrot was generally longer than for the other species. Significant differences (p ≤ 0.05) in the nutritional value and caloric intake were found among the studied species, with carrot and onion microvegetables distinguishing themselves for their mineral, calcium, and potassium content and crude fiber, as shown in Figure 1. For example, K and Na were found to have the highest concentrations in onion, carrot, and aduki bean microgreens; however, the onion exhibited the highest Mg content and popcorn the lowest Na/K ratio, detailed in

Table 1. Energy and mineral content (per 100 g fw) of microgreens of different species.

	Pea	Beet	Aduki Bean	Sunflower	Popcorn	Onion	Carrot
Energy, Kcal	30	11	90	34	23	104	132
Sodium, mg	86.3	155.7	229.4	12.9	7.4	98.7	99
Potassium, mg	242.4	419.4	37.3	110.4	151.4	691.2	1144
Calcium, mg	83.3	76.0	63.9	45.1	11.9	872.2	1864
Magnesium, mg	51.4	50.1	63.9	67.3	19.9	252.1	133

. In sunflower microgreens, we found lower values than those reported by Ghoora et al. [11]; nerveless, the values of the mineral content of carrots and onions were much higher than those reported by the same authors [11].

A noteworthy genotypic variation was observed in terms of microgreen pigmentation expressed as chlorophyll and carotenoid concentrations. Regarding the pigment content, the adzuki bean microgreens showed significantly higher values (p ≤ 0.05) of chlorophylls and carotenoids than other species (1298.73 mg/100 g fw, 0.30 mg/100 g, respectively), as shown in Figure 2a,b. Suathong et al. [12] reported a higher chlorophyll content for green peas than our results; however, these values are similar to our results found in aduki bean. In addition, the chlorophyll content in this legume is higher than the values reported for leafy microvegetables, such as lettuce or chicory [13]. On the other hand, in reports published by other authors, in most of the species of commercial microgreens, the carotenoid content has a higher range than the range values that we found in this work [11,14,15], probably due to the extraction method or the genotype studied.

For total phenolic compounds and flavonoids, carrot microvegetables had the highest content of these bioactive compounds (891.34 mg/100 g fw of total phenols) which was significantly different from that of the onion (p ≤ 0.05), the second microgreen with a high content of phenols that had approximately half of the content of TPC of the carrot (445.68 mg/100 g fw). On the other hand, beet was the species with the highest content of flavonoids, as shown in Figure 2c. The range values of TPC found in these microvegetables are similar to those found in other species reported by Xiao et al. [16] and Alturner et al. [14], such as Dijon mustard, basil, red amaranth, lentils or alfalfa; however, beet TPC values are lower than those reported by [16].

The carrot and onion microvegetables also had the highest in vitro antioxidant capacity, with 85% and 95%, respectively, as shown in Figure 3, and were significantly different from the rest of the species studied (p ≤ 0.05); this could be related to the high content of TPC of these microvegetables, as well as carotenoids. However, the aduki bean, a legume with a high content of bioactive compounds, did not have a good antioxidant capacity. Rozali and Rahim [17] reported that the antioxidant activity of corn was 25% lower than the percentage of inhibition that we found, although their TPC values were much higher than our results.

4. Conclusions

Research related to some of the micro-scale vegetable species studied in this work is, up to the present day, almost non-existent. The results obtained show that microvegetables are an excellent alternative to fresh foods, with a high nutritional and functional value. Carrot and onion microgreens, due to their high phenolic content and antioxidant activity, emerge as superior candidates for functional food applications. On the other hand, the aduki bean legume could also be taken into account as a functional food, as it shows the highest levels of chlorophylls and carotenoids. Therefore, it is worth promoting the consumption of microgreens among consumers and restaurants, as these microvegetables have beneficial health effects and do not need any preparation prior to consumption, unlike adult plants. Further studies are required to explore the impact of different cultivation methods on the microgreens’ bioactive compound content and to evaluate their shelf-life and consumer acceptance.