horticulturae

Article
Evaluation of Growth, Yield and Bioactive Compounds of
Ethiopian Kale (Brassica carinata A. Braun) Microgreens under
Different LED Light Spectra and Substrates
Ruth Nyambura Maru 1 , John Wesonga 1 , Hiromu Okazawa 2 , Agnes Kavoo 1 , Johnstone O. Neondo 3 ,
Dickson Mgangathweni Mazibuko 4,5 , Sarvesh Maskey 2 and Francesco Orsini 6, *

1 School of Agriculture and Environmental Sciences, Jomo Kenyatta University of Agriculture and Technology,
Nairobi 00200, Kenya; [email protected] (R.N.M.); [email protected] (J.W.);
[email protected] (A.K.)
2 Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo 156-8502, Japan;
[email protected] (H.O.); [email protected] (S.M.)
3 Institute of Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, Nairobi 00200,
Kenya; [email protected]
4 Graduate School of Agro-Environmental Science, Tokyo University of Agriculture, Tokyo 156-8502, Japan;
[email protected]
5 School of Natural and Applied Sciences, University of Malawi, Zomba P.O. Box 280, Malawi
6 Department of Agricultural and Food Sciences (DISTAL), University of Bologna Alma Mater Studiorum,
40125 Bologna, Italy
* Correspondence: [email protected]

Abstract: Microgreens are innovative vegetable products whose production and consumption are
gaining popularity globally thanks to their recognized nutraceutical properties. To date, the effects
of lighting conditions and growing substrate on the performances of Brassica carinata microgreens
(indigenous to Africa) remain underexplored. The present study aimed at providing insights into the
Citation: Maru, R.N.; Wesonga, J.; influence of different lighting treatments provided by LEDs, namely monochromatic blue (B), red
Okazawa, H.; Kavoo, A.; Neondo, J.O.; (R), cool white (W) and a combination of three color diodes (B + R + W), and substrates (cocopeat,
Mazibuko, D.M.; Maskey, S.; Orsini, F. sand and cocopeat–sand mix (v/v) (1:1)) on the growth, yield and bioactive compounds of B. carinata
Evaluation of Growth, Yield and microgreens. Seeds were germinated in dark chambers and cultivated in growth chambers equipped
Bioactive Compounds of Ethiopian with LED lighting systems for 14 days under a fixed light intensity of 160 ± 2.5 µmol m−2 s−1 and
Kale (Brassica carinata A. Braun) photoperiod of 12 h d−1 . The best performances were associated with the spectrum that combined
Microgreens under Different LED
B + R + W LEDs and with substrate resulting from the cocopeat–sand mix, including the highest
Light Spectra and Substrates.
yield (19.19 g plant−1 ), plant height (9.94 cm), leaf area (68.11 mm2 ) and canopy cover (55.9%).
Horticulturae 2024, 10, 436. https://
Enhanced carotenoid and flavonoid contents were obtained with B + R + W LEDs, while the B LED
doi.org/10.3390/horticulturae10050436
increased the total amount of chlorophyll (11,880 mg kg−1 ). For plants grown under B + R + W
Academic Editors: László Balázs and LEDs in cocopeat, high nitrate levels were observed. Our results demonstrate that substrate and light
Gergő Péter Kovács
environment interact to influence the growth, yield and concentration of bioactive compounds of B.
Received: 13 March 2024 carinata microgreens.
Revised: 19 April 2024
Accepted: 20 April 2024 Keywords: African indigenous vegetables; healthy diets; light quality; functional foods; nutraceutical;
Published: 24 April 2024 phytochemical

Copyright: © 2024 by the authors. 1. Introduction

Licensee MDPI, Basel, Switzerland.
Microgreens are gaining attention and recognition as a new class of food due to
This article is an open access article
distributed under the terms and
their unique characteristics such as flavor, tenderness, color [1,2] and nutrient density [3].
conditions of the Creative Commons
Microgreens are young plants harvested shortly after the first true leaves emerge, usually
Attribution (CC BY) license (https:// between 7 and 21 days after sowing. They are harvested by cutting the stem just above the
creativecommons.org/licenses/by/ medium, or over the roots when soilless cultivation is adopted [4]. The harvested shoots
4.0/). are eaten raw, either alone or in mixed salads, or used as a garnish for dishes [2]. The

Horticulturae 2024, 10, 436. https://doi.org/10.3390/horticulturae10050436 https://www.mdpi.com/journal/horticulturae

Horticulturae 2024, 10, 436 2 of 14

superiority of microgreens over other plant stages of the same plant species is attributed to
the germination process from dry seeds to growing plants which involves many metabolic
activities and de novo synthesis of nutrients [5]. Microgreens are mainly grown in indoor
hydroponic systems using different growing substrates and integrating supplemental
lighting [2].
Ethiopian kale (Brassica carinata A. Braun) is one of the indigenous African leafy vegeta-
bles (ALVs) that are rich in nutrients and health-promoting secondary plant metabolites [6]
with potential for use against non-communicable diseases (e.g., cancer). The leaves and
seeds of B. carinata are rich in nutrients with high concentrations of glucosinolates, espe-
cially 2-propenyl glucosinolate (sinigrin), as well as phenolic compounds. B. carinata has
been reported to reduce afb1-induced DNA damage [7]. B. carinata microgreens have been
shown to contain flavonoids, phenols, tannins, saponins, alkaloids and terpenoids but not
glycosides [8].
Growth substrate is critical in the production of microgreens as it is a major contrib-
utor to production costs [9]. Substrates will affect the growth, yield and environmental
sustainability of microgreen production [10]. Locally available and inexpensive substrates
that have good water-holding capacity and provide aeration are ideal for microgreen pro-
duction. Those derived from renewable resources and/or those that can be recycled are
to be preferred [11]. According to several authors, peat and peat-based mixes represent
the most used growing substrates for the production of microgreens because of their good
physicochemical properties, but coconut coir (also referred to as cocopeat) is common as
well [10–13]. However, these substrates are quite expensive, and when they are not locally
available, they require importation. The use of peat poses environmental concern due to its
continuous extraction which contributes to the emission of carbon dioxide. On the other
hand, cocopeat (derived from the coconut processing industry and its discarded fibers)
is a renewable resource and could be used as an alternative to peat [11]. However, it can
also be an expensive material and requires treatment for the removal of its concentrated
salts before use, which increases costs. Accordingly, the exploration of alternative sub-
strates or additives enabling a reduction in the amount of cocopeat needed may lead to the
identification of sustainable, cheaper and renewable growing substrates for microgreens.
Light is another major factor in plant growth and influences the development and
production of phytochemical and bioactive compounds [14]. Light quality (its composition
in the spectral regions), quantity (intensity), direction and duration (photoperiod) are vital
components in microgreen production. In plants such as lettuce, high light intensity results
in the production of high amounts of phenolics, anthocyanins and carotenoids, among
others, which could be beneficial to human health [15]. Regarding the effects of light on
microgreen growth, research results vary across studies and for different vegetable species.
For example, it has been found [16] that growth and phytochemical accumulation in Brassica
juncea and Brassica napus using different R and B ratios differed depending on the species.
The chlorophyll, carotenoid and soluble protein contents depended on photoperiod [17] in
other Brassica species. Artificial light sources such as light-emitting diodes (LEDs) have been
used as a source of supplemental lighting in controlled environments such as indoor spaces
and greenhouses in the production of microgreens [17]. B, R and W LEDs used alone or in
combination have been used to produce high-quality microgreens with various nutritional
benefits [17]. However, the influence of LED grow lights on B. carinata microgreens is
still unknown. In addition, it is unclear how plants respond to LEDs in combination with
substrates since most of the previous studies assessed either LEDs or substrates alone.
Therefore, this study aimed to investigate the influence of different LED lights and growing
substrates on the growth, yield and phytochemical content of B. carinata microgreens.
The results obtained from this study provide a baseline towards an understanding of the
influence of the interactions between the substrate and LEDs on quality traits and bioactive
accumulation of B. carinata microgreens.
Horticulturae 2024, 10, 436 3 of 14

2. Materials and Methods

2.1. Experimental Materials and Design
The experiment was conducted in a controlled environment in a locally fabricated walk-
in growth chamber at Tokyo University of Agriculture between April and October 2023. The
chamber was divided into four compartments using black opaque fabric to prevent light
interference. Each compartment measured 100 cm by 100 cm. In each compartment, an LED
fixture was placed 50 cm above the surface of the substrate. Ethiopian kale (Brassica carinata)
seeds used in the study were sourced from a commercial vendor in Kenya. A phytosanitary
certificate allowing entry of seeds to Japan was obtained from the Kenya Plant Health
Inspectorate Service (KEPHIS). B. carinata was identified by a taxonomist at JKUAT GoK
laboratories, and a voucher specimen (JMW/JKUAT/BOT/H001) is maintained at the
JKUAT herbarium.

2.2. Growing Environment

Seeds of B. carinata were sown and grown using three substrates under four LED light
spectra in a factorial experiment. The light spectra used were B (with a peak at 450 nm),
R (with a peak at 650 nm), W, and B + R + W (managed by having one light with three
diodes; B, R and W combined in the ratio of 1:1:1) LEDs in each compartment. The three
substrate types (cocopeat, sand and a mix of cocopeat and sand) and one LED light were
placed in each compartment to give a split plot design with light being the main plot factor
and substrate the subplot factor. There were three replicates for light spectra and twelve
for the substrate. The lights had a fixed light intensity of 160 ± 2.5 µmol m−2 s−1 , and a
12 h photoperiod was applied. The air temperature in the walk-in growth chamber was
set and maintained at 26 ± 2 ◦ C while relative humidity was maintained at approximately
60% during the experimental period. Temperature and relative humidity were monitored
using a data logger (HOBO, OnSet Data Logging Solutions, Bourne, MA, USA). There were
no nutrients supplied throughout the growing period. Irrigation was performed using
capillary wick technology [18].

2.3. Growth Measurements

Growth was assessed at the end of the experiment (14 days after sowing) in terms of
height, leaf area and canopy cover. Ten plants were randomly selected from each subplot
and harvested for height and leaf area measurements. The plants were harvested by cutting
above the substrate. The individual height of each plant was measured using a ruler.
Leaf area values were estimated using ImageJ v.1.5 software [19]. Leaves from the ten
selected plants were spread on a clean white sheet of paper, and photographs were taken
against a ruler as a reference. Additionally, a square paper of known area (2 × 2 mm)
was included for verification of the measurements obtained. Canopy cover was estimated
using Canopeo software (version 1.1.7) [20]. This was done by taking aerial photographs
of all the above-ground plant materials. To achieve uniformity in all the photographs, a
30 cm distance from the camera to the treatment was maintained. The photographs were
processed with Canopeo software, and canopy cover was calculated as a percentage of the
total surface area.

2.4. Yield and Biomass Analysis

Yield and dry biomass were obtained by weighing the whole harvested microgreen
shoots 14 days after sowing (DAS). All above-ground parts including the leaves, stems and
cotyledons were harvested by cutting them at the base, and fresh weight (yield) and dry
biomass (after freeze drying at −41 ◦ C for 24 h) were weighed using a weighing balance.
The samples were further powdered and used for phytochemical analysis.
Horticulturae 2024, 10, 436 4 of 14

2.5. Phytochemical Analysis

2.5.1. Flavonoids
The estimation of total flavonoids in the sample was performed using the aluminum
chloride method. Rutin was used as the standard [21]. The sample (0.1 mL) and standards
were prepared in triplicates, vortexed and incubated for 5 min at room temperature. Af-
terward, 10% aluminum chloride was added, vortexed and incubated for 6 min at room
temperature. The absorbance was measured against the blank at 510 nm using a spectropho-
tometer (Shimadzu model UV-1601 PC, Kyoto, Japan). The standard curve was plotted,
and the total amount of flavonoids in the sample was expressed as mg of rutin equiva-
lent (RE)/g of dry weight of the sample. Equation (1) was used to compute flavonoids
(mg/100 g) from absorbance.

( As − Ab )
Flavonoids = 0.0001 ∗ ∗D (1)
0.0018 ∗ W
where Ab = absorbance of the blank, As = absorbance of the sample, D = dilution factor (30),
W = weight of the sample (g), 0.0018 is the slope of the standard curve and 0.0001 is the
factor for conversion to mg/100 g.

2.5.2. Carotenoids
Total carotenoids were extracted using acetone and analyzed using column chromatog-
raphy (Rodriguez- Amaya and Kimura, 2004; AOAC, 1996) and a UV spectrophotometer
(Shimadzu model UV-1601 PC, Kyoto, Japan) [22]. Approximately 0.08 g of dried sample
was weighed and ground in a mortar containing 10 mL of acetone, and extraction was
repeated until the residue turned colorless. Then, 25 mL of the extract was evaporated to
dryness using a rotary evaporator; the residue was dissolved in 10 mL of petroleum ether,
and the solution was introduced into a chromatographic column. Absorbance was read
at 450 nm in a UV-Vis spectrophotometer. Equation (2) was used to calculate carotenoids
(mg/100 g) from absorbance.

A
Carotenoids = 0.001 ∗ (2)
2592 ∗ W
where A = absorbance, W = weight of the sample (g) and 2592 is the absorption coefficient
of β-carotene in petroleum ether.

2.5.3. Nitrates
The nitrate content in the test samples was determined by the calorimetric method
using salicylic acid [22]. Samples of 0.3 g dry B. carinata were weighed and put in a test
tube. Hot (90–95 ◦ C) distilled water measuring 10 mL was added. The closed tubes were
placed in a water bath at 80 ◦ C and shaken for 30 min. The samples were then cooled
and centrifuged at 4500 rpm. Chlorophyll in the sample was removed by adding 0.5 g
MgCO3 to the supernatant and centrifuging it again. The supernatant containing the nitrate
extract was then treated with NaOH and a combination of salicylic acid and H2 SO4 . Nitrate
standards were prepared using a sodium nitrate calibration curve. Absorbance was read at
410 nm in a UV-Vis spectrophotometer (Shimadzu model UV-1601 PC, Kyoto, Japan). The
nitrate concentration was expressed on a dry weight basis (mg/100 g DW). Equation (3)
was used to calculate nitrates (mg/100 g) from absorbance.

As − Ab
Nitrates = 0.1 ∗ ∗D (3)
0.0078 ∗ W
where Ab = absorbance of the blank, As = absorbance of the sample, D = dilution factor (30),
W = weight of the sample (g), 0.0078 is the slope of the standard curve and 0.1 is the factor
for conversion to mg/100 g.
Horticulturae 2024, 10, 436 5 of 14

2.5.4. Chlorophyll
Chlorophyll was extracted using acetone and analyzed using column chromatography
(Rodriguez- Amaya and Kimura, 2004; AOAC, 1996) and a UV spectrophotometer (Shi-
madzu model UV-1601 PC, Kyoto, Japan) [23]. Approximately 0.08 g of a dry sample was
weighed and ground in a mortar containing 10 mL acetone. The extraction was repeated
until the residue turned colorless. An aliquot of 25 mL of the extract was evaporated to
dryness using a rotary evaporator, and the residue was dissolved in 10 mL of petroleum
ether. The solution was introduced into a chromatographic column, and absorbance was
read at 645 nm and 663 nm in a UV-Vis spectrophotometer. Chlorophyll content was
determined by computation from the absorbance using Equation (4).

D
Total Chlorophyll (mg/100 g)Chl A = 0.1 ∗ (7.12 ∗ A663 + 16.8 ∗ A645 ) ∗ (4)
W
where A = absorbance at indicated wavelength (645 or 663), D = dilution factor (25),
W = weight of the sample (g).

2.6. Statistical Analysis

Statistical analysis was performed using GenStat software, version 12.1. Growth
measurements (leaf area and plant height) were analyzed based on the individual values of
the 10 sampled plants from each subplot, while canopy cover, yield and dry weight were
analyzed at the subplot level. All data were subjected to two-way ANOVA, and significant
differences among means were determined by Tukey’s multiple comparison test at p < 0.05.

3. Results
3.1. Effect of LED Light and Substrate on Height, Leaf Area and Canopy Cover
The results from the ANOVA indicated that the interaction between substrates and
LED light treatments did not have a significant effect on plant morphological parameters.
However, height differed significantly in response to both different substrates and LED light
treatments (Table 1). The microgreens grown using monochromatic R were significantly
shorter compared to those grown using other LEDs. More specifically, microgreens grown
under monochromatic R were 8% shorter compared to those under monochromatic B.
Microgreens grown under B, W and B + R + W did not differ significantly in height.
Microgreens grown in either sand alone or cocopeat–sand mix were significantly taller (F
(3,108) = 3.92, p < 0.001) than those grown in cocopeat alone. Microgreens in cocopeat were
shorter than those in sand and cocopeat–sand mix by 8%.

Table 1. Effect of LED light and substrate on height, leaf area and canopy cover.

Treatment Height (cm) Leaf Area (cm2 ) Canopy Cover (%)

LED Lights
B 9.9 (0.16) a 57.62 (1.40) c 50.68 (4.51) a
R 9.2 (0.16) b 57.36 (1.46) c 44.45 (2.66) b
W 9.7 (0.18) a 63.43 (1.56) b 56.39 (2.85) a
B+R+W 9.8 (0.11) a 68.11 (1.96) a 55.15 (2.76) a
P 0.011 <0.001 <0.001
LSD0.05 0.39 4.32 5.87
F Value F (3,108) = 3.92 F (3,108) = 11.18 F (3,33) = 13.12
Substrates
Sand 9.8 (0.13) a 60.0 (1.36) b 56.0 (3.26) a
Cocopeat 9.2 (0.12) b 59.1 (1.44) c 47.1 (2.07) b
Horticulturae 2024, 10, 436 6 of 14

Table 1. Cont.

Treatment Height (cm) Leaf Area (cm2 ) Canopy Cover (%)

a a
Sand + Cocopeat 9.9 (0.14) 65.6 (1.66) 51.9 (3.394) ab
P <0.001 0.001 0.005
LSD0.05 0.34 3.74 5.08
F Value F (3,108) = 11.86 F (3,108) = 7.28 F (3,33) = 12.02
Mean separation by the Tukey test at the 5% significance level. Values in brackets are standard errors of means.
Values without a letter in common in a column within a factor are significantly different (p < 0.05).

Both substrate and LED treatment had a significant effect on leaf area (Table 1). Micro-
greens grown under B + R + W had significantly higher leaf area (68.11 mm2 ) compared
to microgreens grown under W (63.43 mm2 ) and both under monochromatic B and R
(57.62 mm2 and 57.36 mm2 ). Leaf area in the cocopeat–sand mix was significantly higher
by 22% (65.75 mm2 ) compared to microgreens produced using cocopeat alone (59.12 mm2 ).
Both the growing media and LED treatments had a significant effect on canopy cover.
Canopy cover values under B + R + W treatment were significantly higher (55.15%) than
those produced in monochromatic R (44.45%). On the other hand, microgreens in sand had
a significantly higher canopy cover (55.95%) compared to those in cocopeat (47.11%).

3.2. Effect of LED Light and Substrate on Yield and Dry Weight
The results from the ANOVA indicated that the interaction between substrates and
LED light treatments was not significant. Similarly, no significant differences in yield were
noted among LEDs. Regarding the effects of LEDs on dry weight, significant differences
were noted between monochromatic R and all other LEDs. No differences were noted
between B + R + W, W and monochromatic B LEDs (Table 2). Dry weight among the
substrates ranged from about 1.0 g (cocopeat) to 1.3 g (sand). Regarding the yield, significant
differences were found among the substrates but not the LED lights. The microgreen yield
in sand and cocopeat–sand mix differed significantly from cocopeat alone (p < 0.05). Sand
alone had a yield that was not significantly different from the cocopeat–sand mix.

Table 2. Effect of LED light and substrate on yield and dry weight of Brassica carinata.

Treatment Yield (g) Dry Weight (g)

LED Lights
B 17.9 (1.94) a 1.2 (0.11) ab
R 16.0 (0.86) a 1.0 (0.06) c
W 18.8 (2.36) a 1.3 (0.17) a
B+R+W 19.5 (2.22) a 1.2 (0.10) ab
P 0.339 0.053
LSD0.05 3.73 0.23
F (3,33) 1.28 5.38
Substrates
Cocopeat 15.2 (1.75) b 1.0 (0.08) a
Sand 19.2 (1.54) a 1.3 (0.10) b
Cocopeat + sand 19.8 (1.76) a 1.2 (0.13) ab
P 0.013 0.016
LSD0.05 3.23 0.20
F (2,33) 11.29 13.14
Mean separation by the Tukey test at the 5% significance level. Values in brackets are standard errors of means.
Values without a letter in common in a column within a factor are significantly different (p < 0.05).

3.3. Effect of LED Light and Substrate on Phytochemical Content

Carotenoids: There were significant differences for carotenoids among LED lights (F
(3,24) = 1270.56250, p < 0.001), substrates (F (2,24) = 50.24509, p < 0.001) and their interactions
(F (6,24) = 1814.12864, p < 0.001). Microgreens under B + R + W light in cocopeat had the
highest carotenoid content (644.4 mg kg−1 DW). Under monochromatic B and R, more
 3.3. Effect of LED Light and Substrate on Phytochemical Content
Carotenoids: There were significant differences for carotenoids among LED lights (F
Horticulturae 2024, 10, 436
(3,24) = 1270.56250, p < 0.001), substrates (F (2,24) = 50.24509, p < 0.001) and 7their of 14
interactions (F (6,24) = 1814.12864, p < 0.001). Microgreens under B + R + W light in
cocopeat had the highest carotenoid content (644.4 mg kg−1 DW). Under monochromatic
carotenoids were
B and R, more found in sand
carotenoids werecompared
found in to cocopeat
sand and in
compared to the cocopeat–sand
cocopeat mix.
and in the Under
cocopeat–
W
sand mix. Under W and B + R + W, cocopeat had higher carotenoids relative to thosethe
and B + R + W, cocopeat had higher carotenoids relative to those in sand alone and in
cocopeat–sand
sand alone andmix
the (Figure 1A).
cocopeat–sand mix (Figure 1A).

Figure1.
Figure 1. Effect
Effect of
of LED
LED light
light on
on phytochemicals
phytochemicals ((A)
((A) carotenoids,
carotenoids, (B)
(B) flavonoids,
flavonoids, (C)
(C) chlorophyll
chlorophyll and
and
(D) nitrates) under different substrates (cocopeat + sand, sand and cocopeat). Bars represent
(D) nitrates) under different substrates (cocopeat + sand, sand and cocopeat). Bars represent standard
standard errors of means. Different letters indicate significant differences at p < 0.05.
errors of means. Different letters indicate significant differences at p < 0.05.

Flavonoids: Flavonoids similarly showed significant differences among LED lights (F

(3,24) = 100.7731207, p < 0.001), substrates (F (2,24) = 98.2264237, p < 0.001) and interactions
(F (6,24) = 105.0911162, p < 0.001). Monochromatic B and B + R + W had higher flavonoid
contents in sand than in cocopeat alone as well as in the cocopeat–sand mix. Under
Horticulturae 2024, 10, 436 8 of 14

monochromatic B in sand, flavonoids were 16.8% higher than in cocopeat and 32.4% higher
than in the cocopeat–sand mix. For B + R + W in sand, flavonoids were 11.5% higher
than in cocopeat and 12.0% higher than in the cocopeat–sand mix. Monochromatic R had
higher flavonoid contents in sand alone than in cocopeat alone by 4.6% but lower flavonoid
contents in sand alone than in the cocopeat–sand mix by 15.7%. Similarly, under W, sand
had 9.8% more flavonoids than cocopeat alone but less flavonoids by 6.3% than in the
cocopeat–sand mix (Figure 1B).
Total Chlorophyll: Total chlorophyll content differed significantly among LED light (F
(3,24) = 2690.467, p < 0.001) and substrates (F (2,24) = 6647.472, p < 0.001). In addition, the
interaction between substrate and lights was significant (F (6,24) = 2957.422, p < 0.001).
Except for W, total chlorophyll content under monochromatic B, R and B + R + W was
higher in sand compared to cocopeat. The highest total chlorophyll content (11,880 mg
kg−1 ) was observed under monochromatic B in sand while the lowest (3100 mg kg−1 ) was
under monochromatic B in cocopeat, a reduction of 73.9%. The chlorophyll content under
B + R + W was higher in sand by 26.1% compared to B + R + W in cocopeat substrate, while
for monochromatic R it was 34.5% higher in sand than in cocopeat (Figure 1C).
Nitrates: There were significant differences for nitrates among LED lights (F (3,24) =
1696.0669, p < 0.001), substrates (F (2,24) = 110.4731, p < 0.001) and interactions (F (6,24)
= 983.5374, p < 0.001). Microgreens under B + R + W in cocopeat had extremely higher
nitrates (966.2 mg kg−1 DW) compared to other treatments. Except under W and B + R +
W, nitrate contents were higher in sand than in cocopeat. Under monochromatic B, nitrate
content in sand was higher by 53.4% compared to cocopeat, while for monochromatic R it
was 30.3% higher compared to cocopeat (Figure 1D).

4. Discussion
4.1. Effect of LED Light and Substrate on Height, Leaf Area and Canopy Cover
In recent years, several scientific reports addressed the role of light in stimulating
specific plant photoreceptors, allowing plants to be manipulated to produce desirable phy-
tochemicals and nutrients. Lighting systems for indoor farming can therefore be designed
to maximize growth, control morphology and optimize yield [24]. This study established
that B. carinata grown under monochromatic B were significantly taller compared to those
grown using a monochromatic R source. Such a result is surprising since it is commonly
acknowledged that monochromatic B decreases hypocotyl elongation. For example, the
stem length of baby lettuce decreased by 33% when a supplemental B treatment was pro-
vided [25]. Furthermore, lettuce grown using an increased ratio of red radiation had an
increased shoot height and shoot/root ratio compared to that grown using a blue light
source [26]. Inconsistencies in results on the effect of different spectral regions across plant
species and phenological stages have been acknowledged as a gray area requiring further
research [27]. Monochromatic B and B in combination with far-red light were found to
increase mustard (Brassica juncea) and arugula (Eruca sativa) microgreen elongation (as
defined as plant height) [28]. The results presented herein suggest that sand alone or the
cocopeat–sand mix had better growth than cocopeat, indicating that these substrates pro-
vided a better growing environment. This could be due to the physiochemical properties
such as low water retention capacity allowing good aeration as compared to cocopeat
which could have retained excessive moisture potentially leading to anoxia conditions.
Similarly, ref. [29] reported that using cocopeat-based mixes with other coarser materials
such as burnt rice hull improved the growth of Celosia cristata.
The present research also found that B + R + W and white light resulted in better
yield performances than monochromatic red or blue. This was previously associated with
synergistic effects of the different spectral regions. Red light combined with varying ratios
of blue has been reported to enhance the growth characteristics of lettuce, spinach, kale,
basil and sweet pepper compared to red light alone [27]. Similarly, leaf area among other
growth parameters of lettuce increased with an increase in the proportion of red light in
combination with blue [30]. For leaf area and canopy cover, B + R + W LED in the ratio
Horticulturae 2024, 10, 436 9 of 14

of 1:1:1 and cocopeat–sand mix enhanced the leaf growth of B. carinata microgreens. In
this study, a cocopeat-based substrate (cocopeat–sand mix) showed increased leaf area of
B. carinata microgreens. Similar results showed that cocopeat-based substrate increased
plant growth, yield, nutritional, biochemical composition and antioxidant activity of vari-
ous microgreen species [31]. These positive effects were attributed to enhanced nutrient
acquisition, water retention and root development.

4.2. Effect of LED Light and Substrate on Yield and Biomass

Yield is an important parameter in microgreen production because microgreens are
sold on a fresh weight basis [32]. One of the limiting factors in microgreen production
continues to be low yield due to various elements [33]. Microgreen yield can be affected
by seed quality [34], growing media [35], and light quality and intensity [36], among other
factors. In our study, both substrate and light quality significantly affected the yield and
dry matter accumulation for B. carinata. Notably, the yield of microgreens varied across
the different light spectra used, being highest under W. The results obtained are similar
to those reported in the literature where fresh weight, which was used as a measure of
yield, responded differently in plants grown using different light spectra. On the other
hand, in the experiments presented herein, the increase in yield also depended on the
substrate used. For B. carinata microgreens, a higher yield was recorded in sand alone or
in the cocopeat–sand mix. In previous research comparing different substrates, the yield
of sunflower microgreens was significantly affected by the type of substrate used [12].
Dry mass yield is a good indicator of crop productivity and photosynthetic efficiency [37]
in microgreens. In our study, the highest dry matter accumulation was in microgreens
grown using W. Conversely, microgreens grown in cocopeat using R had the lowest dry
matter accumulation. Therefore, a significant effect resulting from substrate was noticed
in our trial indicating the importance of substrate and lighting on the yield of B. carinata
microgreens. Other studies on dry matter assessment of microgreens seem to indicate
interspecies variability. For example, ref. [38] found differences in dry mass accumulation
within W and R for broccoli, cabbage and radish microgreens.

4.3. Effect of LED Light and Substrate on Phytochemical Content

4.3.1. Carotenoids
Microgreens grown using B + R + W and in cocopeat had higher amounts of carotenoids.
This is consistent with previous observations on the effect of light treatments on carotenoid
accumulation in plants, where the R + B combination increased carotenoid accumulation
in lettuce, spinach and pepper [27], while in kale and basil, carotenoid accumulation was
increased under monochromatic B. Earlier studies also demonstrated that R/B combina-
tions positively influenced carotenoid accumulation in lettuce [26]. Conversely, however,
enzymatic activities involved in the metabolic pathways of carotenoid pigments were
largely increased under monochromatic B, resulting in higher carotenoid accumulation in
Chinese cabbage [25]. For Brassica sprouts, carotenoid transcription of biosynthesis genes,
namely PSY, βLCY and βOHASE1, was enhanced by a higher B percentage compared to
R [39], therefore increasing the carotenoid accumulation in the sprouts. Similar results
were associated with a combined spectrum (resulting from the integration of blue, red and
amber diodes) that enhanced the transcription of a gene involved in carotenoid biosynthesis
(PSY), leading to higher carotenoid accumulation in various Brassica plants [40]. In the
present study, the results are consistent, as the treatment B + R + W often presented higher
amounts of carotenoids. Such findings corroborate the concept that combined light spectra
are superior to monochromatic B or R light supply. On the sand substrate, carotenoids
were higher under monochromatic R and monochromatic B. We hypothesize that these two
spectra may have boosted photosynthesis, and therefore leaf transpiration, a scenario that
could have led to drought stress ultimately inducing carotenoid biosynthesis and accumu-
lation. Further studies on water retention in sand (compared to other substrates) and how
it influences carotenoid accumulation are needed to provide a conclusive explanation.
Horticulturae 2024, 10, 436 10 of 14

4.3.2. Flavonoids
Flavonoids are important plant compounds that are produced as a result of stress
to prevent DNA damage [41]. Light quality triggers different transcriptional genes that
are used for the biosynthesis of flavonoids and could cause differences in the levels of
flavonoid accumulation in plants [42]. In the current study, both monochromatic B and
B + R + W enhanced the accumulation of flavonoid content in B. carinata microgreens
grown on sand and cocopeat substrates, just as monochromatic R and W did in those
grown on the cocopeat–sand mix. An earlier study indicates that monochromatic B highly
influenced the accumulation of flavonoids by modulating the phenylpropanoid pathway, a
pathway in which most plant secondary metabolites are synthesized [43]. The adoption
of R/B combinations at low intensities was formerly found to increase the accumulation
of flavonoids in lettuce [44]. This could have resulted from the influence of different R/B
ratios on the phenylalanine ammonia lyase (PAL), chalcone synthase (CHS) and other
enzymes involved in the flavonoid biosynthesis, ultimately leading to the accumulation of
flavonoids [45]. For Scrophularia kakudensis, ref. [46] reported that flavonoid accumulation
was higher in monochromatic B and R than in W. Furthermore, these effects of light were
also influenced by the substrate used (although different from those adopted in this study).
While monochromatic B enhanced flavonoid accumulation in cocopeat and sand, R and W
enhanced the same phytochemical in cocopeat–sand mix. These subtle differences point
toward a substrate–light interaction, as also previously hypothesized [47].

4.3.3. Chlorophyll
Besides its role as photosynthetic pigment, total chlorophyll content is also one of the
key indicators of quality in vegetables, as the green color indicates freshness, which leads
to product acceptability or rejection by consumers. In microgreens, vivid and intense colors
are particularly appreciated and tend to influence consumer preference [48]. Chlorophylls
represent part of the light-harvesting complex and therefore play a significant role in
photosynthesis. As reported in the literature, significant genotypic variations were observed
for chlorophyll content in microgreens, with their level also being highly dependent on the
lighting conditions [2,49]. In the present study, monochromatic B increased chlorophyll
biosynthesis and accumulation in plant tissues. The role of B in boosting chlorophyll
accumulation was evidenced in previous studies thanks to both increased photosynthetic
efficiency and a concentration factor (e.g., as a consequence of lower leaf extension as
compared with spectra with a higher R fraction) [49,50]. Blue light improves the expression
of genes such as MgCH, GluTR and FeCH, involved in chlorophyll biosynthesis, while red
light may lead to a reduction in 5-aminolevulinic acid, a tetrapyrrole precursor required for
chlorophyll synthesis [51]. Furthermore, when a monochromatic R, a monochromatic B
and a combination of R and B ratio (with R/B = 6) were alternatively applied to Chinese
cabbage, a lower chlorophyll content was associated with monochromatic R, as a result of
reductions in the synthesis of chlorophyll precursors including ALA, Proto IX, Mg-Proto IX
and protochlorophyllide [51]. In an analysis of the effect of the tested substrates, higher
chlorophyll content was observed in B. carinata grown using sand compared to those grown
using cocopeat, which could have contributed to the higher yield observed for the same
treatments. The use of sand for microgreen production is not common. Elsewhere, the use
of sand as a substrate is reported as an additive to another substrate [35]. The effects of
sand as a microgreen substrate may thus require some further investigation, e.g., by using
different mixture combinations.

4.3.4. Nitrates
Nitrates are among the main compounds that may negatively affect food safety. Veg-
etables can accumulate nitrates which are associated with harmful effects on human health,
with toxic effects of methemoglobinemia and the possibility of causing an endogenous
formation of carcinogenic N-nitroso compounds. Accumulation of nitrates in vegetables
may vary depending on the species, the substrate used for production or the stage of
Horticulturae 2024, 10, 436 11 of 14

plant growth at harvest. Several studies reported that microgreens recorded lower lev-
els of nitrates compared to their mature counterparts [52,53]; therefore, microgreens are
commonly considered safe to consume within a healthy diet. As reported earlier, lighting
conditions can influence the accumulation of nitrates in vegetables, thus affecting their
quality [52]. Regarding the substrates, the result contrasts with what was reported by two
studies that evaluated microgreens grown on different substrates and found significantly
lower concentrations of nitrates in microgreens grown using cocopeat substrate [2,10]. In
our case, cocopeat showed a higher nitrate content compared to the other substrates. This
could possibly be because of the differences in the lighting sources during cultivation.
Notably, no such results have been reported for microgreens, and this assumption could be
further investigated.

4.4. Interactive Effects of Light and Substrate on Phytochemicals

The current study reports some significant interactions between lighting treatments
and substrate composition. For example, the interaction between cocopeat and B + R + W
and the interaction between sand and B enhanced the production of all phytochemicals
investigated here. Further, the cocopeat–sand mix and R exhibit a strong interaction except
in the accumulation of carotenoids. This suggests that the effect of light was dependent
on the substrate. No such results have been previously reported for microgreens. Possibly,
the cause of these interactive effects may be associated with either reflective or absorptive
attributes of the substrates. This could be better studied, e.g., by measuring the light
intensity in a sealed box with light turned on and only one substrate at a time. The incident
radiation could be absorbed or reflected depending on the substrate, leading to differences
in lighting conditions experienced by the microgreens. Sand for instance is known to
have the capacity to cause light scattering [54], while cocopeat due to its color and texture
would be expected to absorb light. The light absorption and reflection are further affected
by moisture content, which varies across different substrates. It will be good to test this
assumption to understand the mechanisms involved in the noted interactive effects.

5. Conclusions
This study aimed to investigate the influence of LED light quality and different
substrates on the growth, yield and accumulation of selected bioactive compounds of
Brassica carinata microgreens. Our results demonstrate that substrate and light environment
interact to influence the growth, yield and concentration of bioactive compounds of B.
carinata microgreens, enabling improved cultivation strategies. A combination of various
light spectra (B + R + W) offers a better chance of obtaining higher yields and better-quality
B. carinata microgreens. A combination of cocopeat with sand is a viable alternative to
cocopeat considering the additional benefits of lower costs and ubiquitous availability
of sand. Further studies are needed to elucidate media-related physical and biochemical
dynamics that could potentially influence how different lighting systems lead to the varied
accumulation of phytochemicals. Since B. carinata microgreens have not been extensively
studied (compared to other species), such exploratory studies should first focus on the
most commonly studied microgreen taxa. Such an understanding would help to describe
the specific influence of the interactions between substrates and LED ratios on the quality
traits (nutritional value, color, texture, taste, etc.) of microgreens.

Author Contributions: Conceptualization, R.N.M. and J.W.; methodology, R.N.M. and J.W.; vali-
dation, A.K. and J.O.N.; formal analysis, R.N.M., J.W. and D.M.M.; investigation, R.N.M.; D.M.M.,
S.M. and H.O.; resources, F.O., H.O. and J.W.; data curation, R.N.M.; writing—original draft prepara-
tion, R.N.M., J.W., D.M.M. and H.O.; writing—review and editing, R.N.M., J.W., H.O., A.K., J.O.N.,
D.M.M., S.M. and F.O.; supervision, J.W., H.O., A.K. and J.O.N.; project administration, J.W. and H.O.;
funding acquisition, F.O., H.O. and J.W. All authors have read and agreed to the published version of
the manuscript.
Horticulturae 2024, 10, 436 12 of 14

Funding: The research leading to this publication has received funding from the European Union’s
Horizon Europe research and innovation program under grant agreement No. 101083790 (project
InCitis-Food). Part of the work was supported by the Africa ai Japan project under the JICA capacity-
building program at JKUAT.
Data Availability Statement: The original data presented in the study are openly available in AMS
Acta repository, at https://doi.org/10.6092/unibo/amsacta/7688.
Acknowledgments: The authors acknowledge the Inter-University Exchange Program of Tokyo
University of Agriculture and Jomo Kenyatta University of Agriculture and Technology (JKUAT) and
further mention the JASSO scholarship and Tokyo University of Agriculture for logistical support.
Conflicts of Interest: The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict of interest.

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