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Metabolic, Biochemical, Mineral and Fatty acid profiles of edible Brassicaceae
microgreens establish them as promising functional food
Yogesh Pant, Maneesh Lingwan and Shyam Kumar Masakapalli*
School of Biosciences and Bioengineering, Indian Institute of Technology Mandi, Kamand-
175075
*For correspondence: Email: shyam@iitmandi.ac.in
Graphical Abstract
Highlights
Brassicaceae microgreens are rich in molecules with relevance to nutrition and health
The biochemical analysis supported the antioxidant nature of microgreens
Comprehensive metabolite profiles of edible microgreens of Brassica juncea (Mustard),
Brassica rapa subsp. chinensis (Pak Choi), Raphanus sativus (Radish Pink), and Raphanus
ruphanistrum (Radish white) using GC-MS are reported
Ionomics analysis using the Brassicaceae microgreens exhibited promising levels of
microminerals Fe, Mn, Mg, K, and Ca
Fatty acid profiles show promising levels of Linoleic acid and Oleic acid, which have health
relevance
Mustard
(Brassica juncea)Pak choi
(Brassica rapa subsp.
chinensis)
Radish pink
(Raphanus sativus)Radish white
(Raphanus
raphanistrum)
Na
Mg Mn
Ca
K
Fe
Zn
Mineral profiles
(ICP-MS)
Cu
Biochemical profiles
High polyphenols, Antioxidant
Total proteins, carbohydrates, lipids
Metabolite profiles
(GC-MS)
•Amino acids, Sugars
•Organic acids, Polyphenols
•Sugar alcohol, amines, Others
•Oleic acid (C18:1, ω-9)
•Linoleic acid( C18:2, ω-6)
•Terpenes
Fatty acid profiles
Brassicaceae microgreens- promising functional food to alleviate “Hidden hunger”
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Abstract
Hidden hunger due to micronutrient deficiencies affecting one in three people is a global
concern. Identifying functional foods which provide vital health beneficial components in
addition to the nutrients is of immense health relevance. Microgreens are edible seedlings
enriched with concentrated minerals and phytochemicals whose dietary potential as functional
foods needs evaluation. In this study, comprehensive biochemical, mineral, metabolic, and
fatty acid profiles of four Brassicaceae microgreens - mustard (Brassica juncea), pak choi
(Brassica rapa subsp. chinensis), radish pink (Raphanus sativus), and radish white (Raphanus
ruphanistrum) was investigated. The biochemical and mineral profiling confirmed their
promising nutritional and antioxidant nature and as excellent sources of minerals. Mineral
profiling using inductively coupled plasma mass spectrometry (ICP-MS) exhibited promising
levels of Fe, Mn, Mg, K, and Ca in microgreens. Gas chromatography-mass spectrometry (GC-
MS) based metabolite profiling highlighted a range of phytochemicals- sugars, amino acids,
organic acids, amines, fatty acids, phenol, and other molecules. Fatty acid profiling established
promising levels of Oleic acid (C18:1; Monounsaturated fatty acids- MUFA) and linoleic acids
(C18:2; omega-6 Poly unsaturated fatty acid- PUFA), which are health beneficial. It is
estimated that fresh microgreens (100 g) can meet about 20 % to 50 % recommended dietary
allowance (RDA) of macro- and micro-minerals along with providing useful fatty acids and
antioxidants. Overall, the study highlighted Brassicaceae microgreens as an excellent nutrient
source that can act as functional foods with promising potential to overcome "hidden hunger".
Keywords- Microgreens; Nutritional analysis; Fatty acid; Metabolomics; GC-MS; ICP-MS
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1. Introduction
Despite innovation in global food production, the demand is expected to continuously increase
by 35-56 % to feed an approximately 10 billion world population by 2050 (Dijk et al., 2021).
According to the Food and Agriculture Organization (FAO), 41.9 percent of the world's
population could not afford a healthy diet in 2019 (FAO, 2019). This includes the deficiency
in multiple micronutrients while consuming an energy-efficient diet or a minimum number of
calories, termed "hidden hunger." Identifying and adopting micronutrient-rich diets to existing
foods will immensely benefit global health. Young edible seedlings, now termed microgreens,
are being promoted as nutritionally promising candidates to address the problems of hidden
hunger and malnutrition (Bhaswant et al., 2023).
Microgreens are young seedlings grown in soil or in-vitro-controlled environmental conditions
from the seeds of vegetables and herbs, having two fully developed cotyledon leaves. Their
harvesting stage depends upon the growing conditions and species type, varying from 7-21
days after germination (Xiao, 2012). The major families contributing to microgreens include
Brassicaceae, Asteraceae, Apiaceae, Amaryllidaceae, Amaranthaceae, Cucurbitaceae,
Fabaceae, Poaceae, and Lamiaceae. They are known for both flavoring and nutrition, and
numerous studies have established their dietary importance in terms of mineral contents,
vitamins, antioxidants, etc. (Lester et al., 2010). Studies on twenty-five different varieties of
microgreens, such as arugula, celery, cilantro, radish, and amaranth, showed higher
concentrations (4 to 40 times) of nutrients, antioxidants, and vitamins than mature plants (Xiao
et al. 2012).
The Brassicaceae family is reported to be rich in antioxidants, phenolics, vitamins, minerals,
and other phytochemicals like glucosinolates with anti-inflammatory and anticarcinogenic
activity (Bell et al., 2017). It is shown that microgreens could fulfill children's dietary
requirements for minerals such as Ca, Mg, Fe, Mn, Zn, Se, and Mo (Pinto et al., 2015).
Furthermore, low potassium-containing microgreens were recommended for patients with
reduced kidney function (Renna et al., 2018). Studies in mice suggest that when microgreens
are supplemented with a high-fat diet, they can modulate weight gain and cholesterol
metabolism and may protect against cardiovascular diseases by preventing
hypercholesterolemia (Huang et al., 2016). These statements nominate microgreens to be
considered a "Superfood." However, there is still a need for a comprehensive study to evaluate
the antioxidant potential, phytochemical, and mineral (ionomics) profiling of Brassicaceae
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microgreens. In addition, it will be interesting to investigate fatty acids in Brassicaceae
microgreens; being a rich source of oils, the unsaturated fatty acids available in the microgreens
will be immensely relevant to health benefits.
This study is focused on biochemical analysis and comprehensive profiling of metabolites,
minerals, and fatty acids of four Brassicaceae microgreens - mustard (Brassica juncea), pak
choi (Brassica rapa subsp. chinensis), radish pink (Raphanus sativus), and radish white
(Raphanus ruphanistrum). The optimal in-vitro cultivation of microgreens under controlled
conditions was achieved for the study. The biochemical content of total proteins,
carbohydrates, lipids, phenols, and antioxidants is evaluated. The mineral concentrations,
metabolic and lipid profiles are investigated using sensitive analytical platforms of ICP-MS,
GC-MS, and GC-FAME, respectively. Finally, we compared these profiles with nutritional
significance, establishing the Brassicaceae microgreens as a promising functional food.
2. Material and methods
All the chemicals and reagents were purchased from Sigma Aldrich, unless mentioned in the
methods.
2.1 Plant Materials and growth conditions for Microgreens
Four commonly consumed microgreens from the family Brassicaceae were selected in this
study. The microgreen seeds of Brassica juncea (Mustard), Brassica rapa subsp. chinensis
(Pak Choi), Raphanus sativus (Radish Pink), and Raphanus ruphanistrum (Radish white) were
purchased from the local company "AllThatGrows" (https://www.allthatgrows.in/). Further
substrate, seed density, and germination parameters were investigated for the efficient growth
of these microgreens. In general, the seeds were sown in different combinations of coco-peat
and vermiculite in the ratios 1:0, 3:1, 1:1, and 1:3 in a square petri plate (144 cm2, HiMedia-
PW050) with adequate moisture content. These were kept in dark for three days at 22 ± 2 °C
temperature, 60 ± 5 % relative humidity (RH), and the germination percentage was calculated.
Finally, the emerged seedlings were transferred to light with a 16/8 hour light-dark cycle, after
which the shoots of microgreens (6-9 cm height) were harvested at the fully grown, two-leaf
stage (Day 7). The samples were quenched using Liquid N2 and stored at -80 oC for further
analysis.
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2.2 Nutritional Analysis in Microgreens
2.2.1 Estimation of total proteins
Total proteins were estimated according to the standard DC protein assay (Bio-Rad, catalog
number 500-0116). For protein extraction, 20 mg of fresh sample was weighed and crushed in
liquid nitrogen. Further, 200 µl of extraction buffer (40 mM Tris-HCl, 250 mM Sucrose, 10
mM EDTA) was added to each vial (Wu et al., 2014). The content was then vortexed for 20
sec and incubated in ice for 5 min. This step was repeated thrice for 15 min, followed by
centrifugation at 15,000 rpm for 20 min at 4˚C. Next, the supernatant was collected and used
for the assay. Next, 5 µl of this aliquot was taken, and 25 µl Reagent A' (an alkaline copper
tartrate solution) was added. Further, 200 µl of Reagent B (a dilute Folin Reagent) was added,
and the content was mixed slowly. After 15 min of incubation in the dark, the absorbance of
the sample was taken at 750 nm in a microplate reader. Total proteins were estimated against
BSA (Bovine serum albumin) standard curve.
2.2.2 Estimation of total lipid
Lipid extraction and quantification were done using the gravimetric approach (Bligh and Dyer
1959). Freshly harvested microgreens (1 gm) were crushed with liquid nitrogen, and 3 ml of
chloroform: methanol (1:2) was added. The samples were homogenized and centrifuged at
3,000 rpm for 5 min. The supernatant was collected separately, and 3 ml of chloroform:
methanol (1:2) with 0.8 ml of 1 % KCl was added to the pellet. The above centrifugation step
was repeated, and both supernatants were pooled together. Further, 2 ml of chloroform and 1.2
ml of 1 % KCl was added to it and vortexed, followed by centrifugation at 3,000 rpm for 5
min. Finally, the bottom layer was collected from the pooled lipid extract sample into a
previously weighed centrifuge tube and subjected to solvent evaporation. The final weight of
the tubes was recorded, and total lipids were calculated as an increase in weight.
2.2.3 Estimation of total carbohydrates
20 mg of fresh microgreens sample were ground in a pestle and mortar using liquid nitrogen.
Carbohydrate extraction was performed in 80 % ethanol (1 ml) (Bauer et al., 2022). The
samples were vortexed and centrifuged at 15,000 rpm for 10 min. The supernatant was
collected, and 4 ml of anthrone reagent (2 mg/ml in H2SO4) was added. The mixture was again
vortexed and placed in a heat block at 100 ˚C for 10 min. The tubes were allowed to cool at
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room temperature, and absorbance was recorded at 630 nm. Total carbohydrates were
calculated using glucose as standard.
2.2.4 Estimation of total phenolic compounds
Fresh microgreens (100 mg) were crushed in liquid nitrogen and mixed with 70 % acetone (2
ml) for phenols extraction. The samples were vortexed and centrifuged at 10,000 rpm for 10
min at 4 ˚C. The procedure was repeated with 1 ml of 70 % acetone, and the supernatants were
pooled. Next, 100 µl of this sample was mixed with 500 µl Folin-Ciocalteau reagent (10% v/v
in MiliQ water) and incubated for 5 min (Ainsworth and Gillespie 2007). The reaction was
initiated by adding 400 µl sodium carbonate (5 % w/v in water) and setting it for 20 min in the
dark at room temperature. Total soluble phenols were calibrated using Gallic acid as a standard
(concentration ranging from 20-100 µg/ml) after absorbance was measured at 765 nm with a
spectrophotometer against water as blank.
2.2.5 Determination of DPPH radical-scavenging activity
500 mg of fresh-weight of samples were taken and crushed in liquid nitrogen. 10 ml of 80 %
ethanol was added, and the content was centrifuged at 10,000 rpm for 15 min. The supernatant
was collected in a separate vial. 900 µl of DPPH solution (0.1 mM DPPH in 80 % ethanol) was
mixed with 100 µl of different concentrations of sample extract (five concentrations- 0.04 %
to 0.2 %). The reaction mixture was vortexed and kept at room temperature in the dark for 30
min. The decrease in absorbance was recorded at 515 nm (Lingwan et al., 2021). Inhibition
percent (I %) of the free radical DPPH• in microgreens samples was expressed as-
I % = (Acontrol - Asample) x 100/ Acontrol; where: Acontrol is the absorbance of blank DPPH solution;
Asample is the absorbance of samples. Finally, IC50 (50 % inhibitory concentration) was
calculated, and values were compared to a positive ascorbic acid standard. The less the IC50
value to ascorbic acid (0.12 µg/ml), the more it is antioxidant.
2.3 Elemental analysis through ICP-MS
Powdered microgreens (100 mg dry weight) were subjected to acid digestion with 3.2 ml nitric
acid and 800 µl of hydrogen peroxide (Zou et al., 2021). The samples were filtered using a 0.2-
micron filter and diluted five times with MilliQ water. Finally, the samples were subjected to
ICP-MS along with standards for quantification of the following elements- Na, Mg, K, Ca, Mn,
Fe, Cu, and Zn. The quantities were expressed in milligrams per gram dry weight (mg/g DW)
for macro minerals and microgram per gram dry weight for micro minerals (µg/g DW). Further,
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the recommended dietary allowance (RDA) % was calculated as the percent contribution of
minerals present in microgreens to the recommended levels by FSSAI.
2.4 Gas chromatography-mass spectrometry (GC-MS) based profiling of metabolites
Metabolite extraction was performed in lyophilized microgreens samples (25 mg) with 1.2 ml
of 80 % methanol (Lisec et al. 2006). 20 µl of Ribitol (0.01 % w/v) was added to each sample
as an internal standard for relative quantification. These were incubated in a thermomixer at 70
°C and 900 rpm for 5 min and centrifuged at 13,000 rpm for 15 min at 25 °C. Next, 50 µl of
supernatant was dried in a speed vac. for further derivatization step. 35 μl of pyridine
containing methoxyl amine hydrochloride (20 mg/ml) was added to each dried sample and
incubated at 37 °C, 900 rpm for 2 hours. Later, 49 μl of MSTFA (N-methyl-N- (trimethylsilyl)-
trifluoroacetamide) was added to the tubes and incubated for 30 min. The sample was
centrifuged at 13,000 g for 10 min, and the supernatant was transferred to new inserts (0.2 ml
volume) for GC-MS data acquisition (Masakapalli et al., 2014; Lingwan and Masakapalli,
2022).
GC-MS-based analysis was performed using Agilent Technology GC, model no. 7890B with
a run time of 60 min in splitless mode using helium as carrier gas at a flow rate of 0.6 ml/min.
The program was set initially at 50 °C temperature for 1 min, increasing to 200 °C for 4 min at
the rate of 10 °C and finally to 300 °C at 5 °C/min for 10 min (Shree et al. 2019). The mass
spectra were processed through Metalign software for baseline correction. Further, analyzing
retention time and fragmentation patterns, metabolites corresponding to the peaks were
identified through MassHunter Qualitative Navigator software using NIST version 2.3, 2017,
and Fiehn Metabolomics 2013 libraries with an identity score of ≥70 %.
2.5 Lipid extraction and GC-FAME-based profiling
For lipid extraction and transesterification, fresh microgreens samples (50 mg) were crushed
in liquid nitrogen and saponified with 1 ml of saturated methanolic KOH at 100 °C for 30 min.
After 2 min incubation at room temperature, 2 ml of 5 % HCl prepared in methanol was added
to the extract and subjected to 80 °C for 10 min. Additionally, 1.25 ml solution of 1:1 n-hexane
and methyl tertiary-butyl ether was added, and the mixture was gently mixed. The tubes were
positioned upright for phase separation, and the top layer was collected and washed with 3 ml
of 1.2% KOH solution. Finally, a saturated NaCl solution was added to completely separate
the n-hexane phase containing Fatty acid methyl esters (FAMEs) (Woo et al., 2012). 1 µl of
these extracts were directly injected into the GC-MS equipped with HP-5 (30 m, 0.32 mm i.d.,
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0.25 μm) column. The method parameters include a run time of 40 min in splitless mode using
helium as carrier gas at a flow rate of 1 ml/min. The program was set initially at 50 °C
temperature for 1 min, increasing to 200 °C at 25 °C/min for 5 min and finally to 230 °C at 3
°C/min for 18 min.
2.6 Statistical and multivariate data analysis
The identified compounds and their respective abundances were subjected to multivariate
statistical analysis using MetaboAnalyst 5.0 online tool. Data pre-processing was performed
by normalization by the median, log transformation, and Pareto scaling. Finally, PCA
(Principal component analysis), PLS-DA (Partial least square discriminant analysis) plots, and
Heat maps were generated (Chong et al., 2019). The area of the internal standard, Ribitol, is
used to obtain the relative proportions of the peaks, leading to the calculation of fold changes
of metabolite/ peak levels among the treatments. Fundamental statistical methods were used to
determine the significance or non-significance of data using GraphPad Prism8 software.
3. Results and discussion
3.1 Optimal growth of Brassicaceae microgreens
Substrate, Seed density, and germination percentage were optimized for the efficient growth of
microgreens. Different ratios of Coco-peat: vermiculite (1:0, 3:1, 1:1, and 1:3) were tested as
substrates for microgreens. Based on the maximum number of seeds germinated, the 1:1 ratio
of coco-peat and vermiculite mixture was observed to be the best composition. The seeds of
selected microgreen seeds were categorized under three seed densities (low, medium, and high)
based on their relative sizes. Seed density of 2.5 g and 3 g for pakchoi and mustard, and 3.5 g
for radish pink and radish white per 144 cm2 (grown in square petri plate) was found to be
optimum. The germination percentage of four selected microgreens was between 86.5 ± 9 %
and 98 ± 4%. There were no significant differences among the microgreens since all germinated
efficiently on the cocopeat-vermiculite (1:1) substrate (Figure 1A). Microgreens can grow on
multiple substrates, jute mats, potting mixes, and hydroponically. Therefore, optimal substrate
combinations are vital for producing safe and high-yield microgreens, and a coco-peat and
vermiculite mixture could be considered.
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3.2 Biochemical analysis show higher nutritional and antioxidant nature of Brassicaceae
microgreens
The study of biochemical analysis of Brassicaceae microgreens shed light on their promising
nutritional and antioxidative potential. The total carbohydrates ranged between 32 and 86 mg/g
of fresh weight (FW) in the microgreens (Figure 1B). No apparent differences were found
among mustard, radish pink, and radish white microgreens and are similar to the levels present
in mature mustard leaves (U.S. Department of Agriculture, 2019). Pak choi microgreens
showed 2 to 4-fold higher carbohydrate levels than the others (U.S. Department of Agriculture,
2019; Goyeneche et al., 2015). The total proteins were observed to be ranging from 7 mg/g
FW to 17 mg/g FW in microgreens which were less than the total proteins reported in mature
leaves of mustard, pak choi, and radish, i.e., 15 to 40 mg/g FW (Goyeneche et al. 2015; U.S.
Department of Agriculture, 2019). The total lipids ranged between 3.9 mg/g FW and 7.5 mg/g
FW. No significant differences were observed between mustard and pak choi microgreens.
Also, both radish pink and radish white varieties had similar lipid content. However, the lipids
were less in mustard and pak choi than in radish greens (Figure 1B). Furthermore, these levels
were equivalent to the mature Brassicaceae leaves (Goyeneche et al., 2015; U.S. Department
of Agriculture, 2019).
The total polyphenols and DPPH scavenging activity of Brassicaceae microgreens established
their promising antioxidant potential (Figure 1B). The total polyphenol content (TPC) in
microgreens ranged from 1.85 mg to 3.33 mg GAE/g FW which were higher compared to the
whole mustard plant (3.5 mg/g dry weight) and mature leaves (17.7 mg/g dry weight) reported
in the literature (Sun et al., 2018). We observed promising antioxidant potential of
Brassicaceae microgreens as evidenced by DPPH radicle scavenging abilities with IC50 of
76.5 µg/ml to 161.5 µg/ml compared to the positive control ascorbic acid (IC50: 119.6 g/ml).
Comparatively, similar values were noted in other microgreens previously reported (Ghoora et
al., 2020). An antioxidant-rich diet reduces the risk of cardiovascular diseases, hypertension,
and diabetes (Alissa and Ferns, 2017). In addition, polyphenols and other antioxidants function
as scavengers and reduce oxidative stress, which helps manage chronic non-communicable
diseases (Urquiaga and Leighton, 2000). Overall, TPC was higher in microgreens than in
mature plants, which can directly correlate to healthy human nutrition.
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Figure 1. (A) 7 days old microgreens of Mustard, Pak choi, Radish Pink, and Radish white
grown on 1:1 cocopeat-vermiculite substrate. (B) Comparison of total carbohydrates, proteins,
lipids, polyphenols, and antioxidant activity in Brassicaceae microgreens expressed in mg g-1
fresh weight (FW). Error bars represent the standard deviation for n=3
3.3. Brassicaceae microgreens are an excellent source of minerals
Minerals are necessary for any organism to function correctly and are vital to the human diet.
They regulate physiological and biochemical activities, metabolism, and homeostatic balance
(Kyriacou et al., 2021). Here we evaluated the Brassicaceae microgreens as a potential source
of minerals (macro and micro) measured through ICP-MS analysis (Table 1). Macro minerals
Na and K were found in concentrations ranging from 17 to 27 mg/g DW and 36 to 61 mg/g
DW respectively. In comparison, Ca and Mg were present at 9 to 17 mg/g DW and 6 to 9 mg/g
DW. Among the microminerals, Fe has the highest concentration (277 to 1092 µg/g DW),
while Cu has the lowest concentration (7.5 to 10.4 µg/g DW). Additionally, Mn and Zn are
present in the range between 53.5 and 206.5 µg/g DW of microgreens. The data was compared
to the Food Safety and Standards Authority of India (FSSAI) 2020 recommendations for
recommended dietary allowance (RDA). It is known that fruit and vegetable intake supply
approximately 11% Na, 24% Mg, 35% K, 7% Ca, 21% Mn, 16% Fe, 30% Cu, and 11% Zn of
recommended RDA to the human body (Levander, 1990). From the elemental profile, it is clear
Mustard
Pak choi
Radish Pink
Radish White
0
20
40
60
80
100
Total carbohydrate
mg/g FW
✱✱✱
ns
✱✱✱
MustardPak choi
Radish pink
Radish white
Ascorbic acid
0
50
100
150
200
Antioxidant activity
IC 50 (µg/ml)
✱ ✱
Mustard
Pak choi
Radish pink
Radish white
0
2
4
6
8
Total Lipids
mg/g FW
ns
✱✱✱ ✱✱
Mustard
Pak choi
Radish Pink
Radish White
0
5
10
15
20
Total proteins
mg/g FW
ns
✱✱✱ ✱✱✱
Mustard
(Brassica juncea)
Pakchoi
(Brassica rapa
subsp. chinensis)
Radish White
Raphanus
raphanistrum
Radish Pink
Raphanus.
sativus
Mustard
(Brassica juncea)Pak choi
(Brassica rapa subsp.
chinensis)
Radish pink
(Raphanus sativus)Radish white
(Raphanus
ruphanistrum)
Mustard
Pak choi
Radish pink
Radish white
0
1
2
3
4
Total polyphenols
mg GAE/g FW
✱✱✱
✱
✱✱✱
✱✱✱
(A)
(B)
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that all the selected Brassicaceae microgreens are excellent sources of macro and micro
minerals. Consuming mustard microgreens (about 10 grams DW) can fulfill 50 % RDA of iron
(Table 1). Overall, these Brassicaceae microgreens can provide 10-20 % of the RDA for
macronutrients and 4-50 % of the requirement for micronutrients, depending on the mineral
type and species. The analysis confirmed that the levels of Fe, Mn, Mg, K, Ca, and Na in
Brassicaceae microgreens were higher when compared with the mineral content of mature wild
edible parts of mustard, pak choi, and radish (Filho et al., 2018, Kyriacou et al., 2021,
Mezeyova et al., 2022). High levels of minerals have also been reported in other Brassicaceae
microgreens, such as arugula, broccoli, and red cabbage, compared with their mature leaves
(Johnson et al., 2021; Supplementary table S2). In addition, calcium and magnesium, critical
elements in the human diet, were higher in all the Brassicaceae microgreens studied (Armesto
et al., 2019).
Many of these macro and micro minerals are commonly deficient in the population of both
developed and developing countries, the symptoms of which are not always immediately
visible. Hence, including Brassicaceae microgreens in the diet which are observed to be dense
in minerals, can assist in meeting daily needs and overcoming "hidden hunger."
Macro-minerals (mg/g DW)
RDA
(mg per day),
by FSSAI
Mustard
RDA %*
Pak choi
RDA %*
Radish
pink
RDA %*
Radish
white
RDA %*
Na
2000
20.7 ± 1.44
10
26.3 ± 1.33
13
20.9 ± 1.99
10
18.2 ± 1.03
9
Mg
385
7.3 ± 0.52
19
8.6 ± 0.38
22
6.6 ± 0.70
17
6.9 ± 0.43
18
K
3500
56.3 ± 4.0
16
58.3 ± 3.1
17
52.9 ± 5.3
15
38.7 ± 2.5
11
Ca
1000
14.4 ± 1.5
14
16.9 ± 0.64
17
10.4 ± 0.98
10
9.4 ± 0.56
9
Micro-minerals (µg/g DW)
RDA
(µg per day)
Mn
4000
115.7 ± 8.8
29
88.5 ± 4.2
22
51.2 ± 5.1
13
50.4 ± 3.1
13
Fe
19000
1021 ± 71.9
51
266.4 ± 11.5
13
624.6 ± 66.1
31
293.6 ± 18.8
15
Cu
2000
7.4 ± 0.9
4
9.6 ± 0.8
5
8.0 ± 0.6
4
6.7 ± 0.8
3
Zn
17000
135.9 ± 10.3
8
196.2 ± 10.3
12
136.3 ± 13.9
8
118.9 ± 6.9
7
* Percent RDA (%) met after consuming 10 g DW of Brassicaceae microgreens [compared
with RDA 2020; FSSAI].
Table 1- Different macro and micro minerals concentrations in Brassicaceae microgreens
measured using ICP-MS. The estimated RDA % of minerals met via dietary source on
consuming 10 g DW of microgreens daily (equivalent to 100 g FW) is tabulated and described
as the percent contribution of minerals present in Brassicaceae microgreens to recommended
dietary allowance (RDA) values approved by FSSAI 2020.
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3.4. GC-MS-based metabolite profiling shows microgreens are a rich source of bioactive
compounds
The aqueous methanol soluble metabolite profiles of Brassicaceae microgreens using GC-MS
captured sugars, amino acids, fatty acids, organic acids, and bioactive compounds, including
various polyphenols, sugar alcohols, and amines. (Figure 2, Supplementary Table S1). Among
the sugars and sugar alcohols, Fructose, glucose, meso-erythritol, and threitol were
predominant (Supplementary Figure S1). Other sugars identified are mannose, arabinose,
glycerol, erythritol, glucopyranoside, and myoinositol. The amino acids; alanine, valine,
leucine, isoleucine, glycine, serine, threonine, aspartic acid, glutamic acid, phenylalanine,
asparagine, glutamine, lysine, and tyrosine were identified. These include essential amino
acids, which can be of nutritional relevance. In addition, the branched-chain amino acids valine,
leucine, and isoleucine, generally recommended in protein supplements for athletes, could be
interesting. Myristic acid, palmitic acid, linolenic acid, and stearic acid are among the identified
fatty acids. Organic acids found in microgreens, such as lactic acid, glycolic acid, citric acid,
malic acid, amino-butanoic acid, glyceric acid, and butenedioic acid, may aid in human
digestion (Nguyen and Kim, 2020). The metabolite profiles also revealed the presence of
polyphenols and amines of nutritional relevance in all the Brassicaceae microgreens. These
include sinapinic acid, oxoproline, and hydroxylamine. In addition, amino furanone and an
unknown metabolite at a retention 22.52 min were also reported. Overall, the metabolite
profiles of microgreens showed several small molecules belonging to sugars, amino acids, fatty
acids, organic acids, polyphenols, sugar alcohols, and amines that have nutritional significance.
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Figure 2. Overlapping total ion chromatograms (TICs) of seven days old Brassicaceae
microgreens from retention time (A) 12 min to 19 min and (B) 20 to 35 min, covering all the
identified metabolites range. Ribitol was used as an internal standard (IS). The metabolites
identified correspond to the number as follows- 1. Lactic Acid; 2. Glycolic acid; 3. Alanine; 4.
Hydroxylamine; 5. Valine; 6. Benzoic Acid; 7. Ethanolamine; 8. Leucine; 9. Glycerol; 10. 4-
Dimethylamino-2(5H)-furanone; 11. Isoleucine; 12. 2-Butenedioic acid; 13. Glycine; 14.
Glyceric acid; 15. Nonanoic acid; 16. Serine; 17. Threonine; 18. Aspartic acid; 19. Timonacic;
20. Malic acid; 21. 3-Amino-2-piperidone; 22. Methyl L-alaninate; 23. Erythritol; 24.
12345
6
710
11
12345
6
7
10
11
12
3
45
6
7
9
10
11
12345
6
7
10
11
8
9
8
9
9
12
12
12
12
13
13
13
13
14
14
14
14
12’
12’
12’
12’
16
16
16
16
17
17
17
17
18
18
18
19
19
19
19
20
20
20
20
21
21
21
21
22
22
22
22
23
25
25
25
25
24
24
24
24
23
23
23
12 13 14 15 16 17 18 19
Relative abundance
Retention time (min)
Mustard
Pak choi
Radish pink
Radish white
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
26
26
26
26
27
27
27
27
28
28
28
28
29
29
29
29
30
30
30
31
(IS)
31
(IS)
31
(IS)
31
(IS)
32
32
32
32
33
33
33
33
34
34
34
34
35
35
35
36
36
36
36
37
37
37
37
38
38
38
38
39
39
39
39
40
40
40
40
41
41
41
41
42
42
42
42
43
43
43
43
44
44
44
44
45
45
45
45
46
46
46
46
47
47
47
47
48
48
48
48
Relative abundance
Retention time (min)
Mustard
Pak choi
Radish pink
Radish white
(A)
(B)
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Oxoproline; 25. 4-Aminobutanoic acid; 26. Glutamic acid; 27. Phenylalanine; 28. Asparagine;
29. Arabinose; 30. Ribono-1,4-lactone; 31. Ribitol; 32. meso-Erythritol; 33. Glutamine; 34.
Threitol; 35. Metabolite@22.52; 36. Citric acid; 37. Myristic acid; 38. Fructose; 39. Mannose;
40. Lysine; 41. Glucose; 42. Tyrosine; 43. Glucopyranoside; 44. Palmitic Acid; 45. Myo-
Inositol; 46. α-Linolenic acid; 47. Stearic acid; 48. Sinapinic acid. The spectra in red, green,
dark blue, and sky blue correspond to mustard, pak choi, radish pink, and radish white,
respectively.
3.5. Multivariate statistical analysis showed an overall variation among the microgreen
species
The variations in the metabolite profiles among the Brassicaceae microgreens were further
captured via Multivariate statistical analysis. Principal component analysis (PCA) showed
distinct clusters of microgreens represented by the first two principal components (PCs), where
PC1 and PC2 explained 65.4% and 12.9% of the variance in metabolite profiles, respectively
(Figure 3A). Additionally, the relative abundances of identified metabolites among the
microgreens were presented in bar graphs (Supplementary figure S2). This confirms that the
composition of soluble metabolites among the microgreens is distinct, which could contribute
to different attributes such as taste, color, smell, texture, etc., along with other parameters.
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15
Figure 3. Microgreens from the Brassicaceae family have distinct metabolic profiles. A)
Principal Component Analysis (PCA) plots depict the variation among the Brassicaceae
microgreens. B) The heat map shows the metabolic response to microgreens, characterized as
sugars, amino acids, organic acids, amines, fatty acids, and other phytochemicals. For each of
the observed metabolites, the average relative abundance (n=4) is shown as a set of color-coded
groups, from red (+3, high) to green (-3, low). The numbers against each metabolite correspond
to the designated peaks observed in the GC-MS spectra depicted in Figure 2.
The heat map exhibited the response of microgreens' metabolites, classified as sugars, amino
acids, organic acids, amines, fatty acids, and other phytochemicals. Results indicate that the
metabolite profiles of mustard and pak choi are comparable with higher levels of identified
sugars, organic acids, and fatty acids. Additionally, radish pink and radish white responded
nearly similar with higher levels of all amino acids except aspartic and glutamic acids. The
amines, hydroxylamine, oxoproline, and methyl alaninate were detected in relatively high
amounts in mustard, pak choi, and radish pink, whereas radish white had low levels in their
microgreens.
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Lastly, metabolite profiling also detected significant levels of polyphenol sinapinic acid in the
microgreens, with elevated levels in mustard and pak choi, followed by radish white and radish
pink.
3.6. The fatty acid profile of Brassicaceae microgreens showed beneficial lipids
The FAMEs (fatty acid methyl esters) based profiling in Brassicaceae microgreens identified
six primary fatty acids (saturated and unsaturated). Saturated fatty acids include palmitic acid,
and stearic acid, whereas unsaturated fatty acids include oleic acid, linoleic acid, eicosenoic
acid, and erucic acid (Figure 4). Additionally, two terpenes, neophytadiene, and phytol, were
also observed. The relative abundances of palmitic acid, stearic acid, oleic acid, and linoleic
acid were similar among the microgreens. However, differences in the levels of erucic acid and
phytol were observed. Erucic acid was less in radish pink and highest in radish white.
The presence of essential fatty acids and terpenes identified through GC-MS FAMEs, i.e., oleic
acid, linoleic acid, neophytadiene, and phytol makes Brassicaceae microgreens nutritionally
rich (Figure 4,5). These unsaturated fatty acids are not synthesized in the human body and
should be involved in diet. They play several roles in human nutrition, including the building
block of the cell, cell membrane, hormone production, blood pressure regulation, inflammatory
responses, etc. (Chen and Liu, 2020). Besides the essential fatty acids, average erucic acid
levels were less than 5 % in all the Brassicaceae microgreens samples, which are considered
safe for human ingestion (Figure 5). Overall, the fatty acid profiles in microgreens are
encouraging, given that many are nutritionally essential.
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Figure 4. Brassicaceae microgreens have a beneficial lipid composition in their fatty acid
profile. The profiling using FAMEs (fatty acid methyl esters) revealed the presence of
saturated and unsaturated fatty acids. The orange, purple, dark green, and light green GC-
FAMES spectra of mustard, pak choi, radish pink, and radish white, respectively. Eicosane (IS)
was used as an internal standard at a concentration of 0.01 %.
Figure 5. Qualitative levels of primary fatty acids and terpenes in Brassicaceae
microgreens identified through GC-MS. The relative peak area abundance was normalized
Neophytadiene
Tetramethyl hexadecenol
Palmitic acid
Isophytol
Eicosane (IS)
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Eicosenoic acid
Erucic acid
Linoleic acid
Oleic acid
Phytol
Stearic acid
Retention time (min)
x107
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Abundance
Mustard
Pak choi
Radish pink
Radish white
Relative abundance
Retention time (min)
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18
with the internal standard Eicosane and expressed as mean ± standard deviation (n=3). The
abbreviation 'ns' denotes the absence of a statistically significant difference between the
replicates.
4. Conclusion
Microgreens are the edible young seedlings of various vegetables, herbs, and flowers consumed
at the two-leaf stage. These are reported to be rich in essential nutrients and other health
beneficial components for being considered as functional food. The current study evaluates
their dietary potential through biochemical, minerals, metabolite, and fatty acid profiles. The
in vitro cultivation under controlled growth conditions was best achieved for microgreens of
mustard (Brassica juncea), pak choi (Brassica rapa subsp. chinensis), radish pink (Raphanus
sativus), and radish white (Raphanus ruphanistrum). The suitable substrate composition for
optimum growth of Brassicaceae microgreens was optimized, and the 1:1 coco-peat and
vermiculite ratio was observed to be the best for their germination and growth. The biochemical
and mineral composition analysis (using ICP-MS) confirmed their promising nutritional,
antioxidant nature and as excellent sources of minerals. While the carbohydrate, lipids levels
in microgreens were comparable to mature Brassicaceae leaves, the total proteins were lower.
The total polyphenols in microgreens were higher than in mature plants, indicating that
including them in the diet can provide healthy human nutrition. Mineral profiling exhibited
promising levels of Fe, Mn, Mg, K, and Ca in microgreens that are of health relevance. Further,
when the mineral levels in Brassicaceae microgreens were compared to recommended dietary
allowance, it is clear that they can meet a significant proportion of daily nutrient needs and
assist in overcoming "hidden hunger." The GC-MS based metabolite profiles of microgreens
identified several small molecules belonging to sugars, amino acids, fatty acids, organic acids,
polyphenols, sugar alcohols, and amines with nutritional significance. The FAMEs-based
profiles show the presence of essential fatty acids, oleic acid, linoleic acid, and terpenes that
are considered nutritionally important. All these nutritional parameters show that the
Brassicaceae microgreens have health-beneficial role and can be used as excellent nutritive
sources as functional food. Recent studies have shown that UV-B irradiation in plants can
enhance their polyphenol levels. In future studies, such conditions can be optimized in
microgreens to develop biofortified food.
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ACKNOWLEDGMENT
SKM acknowledges Science and Engineering Research Board (SERB) Early career research
funding (File No: ECR/2016/001176). YP acknowledges the SERB, Ministry of Education, and
IIT Mandi for PhD fellowship.
Declarations
All the authors declare no conflict of interest.
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