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applied
sciences
Article
The Effect of Light on Antioxidant Properties and
Metabolic Profile of Chia Microgreens
Selma Mlinari´c 1, Vlatka Gvozdi´c 2, Ana Vukovi´c 1, Martina Varga 1, Ivan Vlašiˇcek 1,
Vera Cesar 1and Lidija Begovi´c 1,*
1
Department of Biology, J. J. Strossmayer University of Osijek, Ulica cara Hadrijana 8/A, 31000 Osijek, Croatia;
smlinaric@biologija.unios.hr (S.M.); avukovic@biologija.unios.hr (A.V.); mjelosek@biologija.unios.hr (M.V.);
ivan.vlasicek@gmail.com (I.V.); vcesarus@yahoo.com (V.C.)
2Department of Chemistry, J. J. Strossmayer University of Osijek, Ulica cara Hadrijana 8/A, 31000 Osijek,
Croatia; vgvozdic@kemija.unios.hr
*Correspondence: lbegovic@biologija.unios.hr; Tel.: +385-31-399-936
Received: 30 July 2020; Accepted: 17 August 2020; Published: 19 August 2020
Featured Application: Exposition of dark-grown chia microgreens to lower light intensity
increases the production of bioactive compounds and enhances their antioxidative activity.
Therefore, illuminated chia microgreens have the potential to be included in human diet as
well as raw seeds.
Abstract:
Chia (Salvia hispanica L.) is a one-year plant known as a source of nutrients that can be
consumed in the diet in the form of seeds or sprouts. The purpose of this study is to investigate the
effect of illumination for 24 and 48 h on dark-grown chia microgreens. Total antioxidant capacity
was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power
(FRAP) assays, along with the total phenolics, ascorbic acid and cellulose content, and chlorophyll and
carotenoid concentrations. Fourier transform infrared spectroscopy (FTIR) was used to evaluate the
biochemical composition and elucidate the changes in compound structures between dark-grown and
illuminated chia microgreens. Analysis of the results showed that illumination significantly increased
the content of all measured bioactive compounds as well as antioxidative capacity, especially 48 h after
exposure to light. FTIR analyses supported structural and molecular changes in chia microgreens
grown under different light regimes. Our results suggest that illumination has a positive effect on the
antioxidant potential of chia microgreens, which may present a valuable addition to the human diet.
Keywords:
Salvia hispanica; antioxidant activity; DPPH; polyphenolics; ascorbic; acid; carotenoids; light
1. Introduction
Chia (Salvia hispanica L.) is a one-year-old plant (Lamiaceae) that is a native species in Mexico and
Northern Guatemala, where it was bred as a cereal in pre-Columbian times. An adult plant grows
up to 1 m in height and has versatile leaves that are 5 cm wide and 8 cm long, with white to purple
flowers. The most important product of this plant is its seeds, which are 1–2 mm in diameter, oval,
black, gray, or white, with black dots [
1
]. The seeds are used whole or in the form of flour or oil.
Although they have a neutral taste, they are an interesting addition to diet because they form a sticky
mucus in contact with water [
2
,
3
]. Whole seeds and sprouts are added to salads and beverages to
improve the density, and for the same reason, flour can be added to yogurt. The cultivation of chia
sprouts is very simple, and they have also been recently used in the human diet for their nutritive and
antioxidative properties [4,5].
Appl. Sci. 2020,10, 5731; doi:10.3390/app10175731 www.mdpi.com/journal/applsci
Appl. Sci. 2020,10, 5731 2 of 13
Sprouts or microgreens are grown from the seeds of various kinds of grains, vegetables, nuts,
and legumes. They have a high content of protein, calcium, magnesium, vitamin A, vitamin B,
ascorbic acid, and vitamin E [
6
,
7
]. The use of microgreens is increasingly popular and they are
considered the food of the future due to their relatively light weight, fast and easy growing, and high
nutritional value [6].
Light intensity, quality, and duration significantly influence plant growth and development
through morphogenesis, the functioning of the photosynthetic apparatus, and metabolic pathways [
8
].
Light conditions also have the potential to evoke different actions of the antioxidant system [
9
]
and metabolic pathways [
10
,
11
]. Light can enhance the synthesis of different antioxidants such as
ascorbic acid [
11
,
12
], polyphenols, carotenoids, chlorophylls, and other enzymatic and nonenzymatic
compounds that help in preventing and balancing oxidative damages in plants [
13
]. However,
different growth conditions can have an impact on the antioxidative and nutritive properties of plant
species [6].
Sprouts used in diets are usually grown in dark conditions and consumed raw. They show a pale
yellow color due to the lack of chlorophyll. After exposure to light, the expression of genes involved in
chlorophyll and carotenoid biosynthesis is upregulated, leading to the change of color from pale yellow
to green [
14
,
15
]. Different sprouts or microgreens are considered to be young plants of vegetables,
grains, and herbs, with two fully developed cotyledons. Due to their antioxidant capacity and various
bioactive compounds with antibacterial, anti-inflammatory, and other health beneficial properties,
sprouts are considered “functional food” [
16
], which has recently been widely grown and used in the
global food system.
Fourier transform infrared spectroscopy (FTIR) is widely used for the identification of biomolecules,
functional groups, types of bonding, and changes in molecular conformations. Due to its extensive
applicability to the different kinds of tissues and a small amount of sample needed for analyses,
this technique has found broad applications in the analyses of metabolic profiles of different plant
species [
17
]. FTIR spectroscopy is used for the determination of antioxidant potential and health
benefits in chia [18–20], as well as other medicinal plants [17].
Although there have been many reports on the connection between the effect of light on nutritive
properties and the antioxidant capacity of various microgreens, few studies have investigated the effect
of light on nutritional properties and bioactive compounds in chia microgreens under different light
regimes. Therefore, the aim of this work is to determine the effect of illumination on dark-grown chia
microgreens by measuring total antioxidant capacity, the concentration of carotenoids and chlorophylls,
total soluble phenolics, and ascorbic acid and cellulose content, as well changes in biochemical
composition and structure of biomolecules by Fourier transform infrared spectroscopy (FTIR).
2. Materials and Methods
2.1. Plant Material and Light Treatment
Chia seeds (Salvia hispanica L., producer Golden Sun, Trittau, Germany) were planted on two
layers of moistened filter paper in glass jars (400 mL). In each jar, 1.5 g of seeds were planted. The jars
were then wrapped in aluminum foil and perforated on the top to allow ventilation during growth.
The jars were placed in a growth chamber at 22
±
1
◦
C. Plants were grown for seven days and watered
every other day. Afterward, microgreens were exposed to constant light (100
µ
mol photons m
−2
s
−1
)
for 24 and 48 h, and the dark-etiolated plants were considered as control. Three biological replicates
(each group contained 1.5 g of seeds) of samples with three light treatments were collected and used
for analyses. Etiolated microgreens were additionally protected from light by placing them in the
cardboard box.
Appl. Sci. 2020,10, 5731 3 of 13
2.2. Determination of Chlorophyll and Carotenoid Content
One hundred milligrams of fresh seedling tissue were ground in liquid nitrogen using mortar and
pestle. The extraction of pigments was performed in pure acetone for 24 h at
−
20
◦
C. Next day samples
were centrifuged at 18,000
×
gfor 10 min at 4
◦
C. The absorbance was measured at 470, 645, and 662 nm
using a spectrophotometer (Specord 40, Analytik Jena, Jena, Germany), and pure acetone was used as
a blank. Carotenoid and chlorophyll content were determined according to Lichtenthaler [21].
2.3. Total Soluble Polyphenols and Protein Content
Approximately 500 mg of fresh seedling tissue was used for analyses. Plant material was ground
in liquid nitrogen and extracted for 24 h at
−
20
◦
C in 2.5 mL of 96% ethanol [
6
]. Measurement of total
soluble polyphenol content was performed in the reaction mixture containing 100
µ
L of ethanol extract,
700
µ
L of distilled H
2
O, 50
µ
L of Folin–Ciocalteu reagent, and 150
µ
L of sodium carbonate solution
(200 g/L). Samples were incubated in a water bath for 60 min and 37
◦
C, and absorbance was measured
spectrophotometrically at 765 nm using gallic acid (GA) as a standard. Total polyphenol content was
expressed as gallic acid equivalents per g of fresh weight (FW).
Protein content was determined using the Bradford assay [
22
]. Briefly, 500 mg of ground tissue
was extracted with 1 mL of 100 mM KP buffer, pH =7.0. After 15 min of extraction on ice, samples were
centrifuged at 18,000
×
gfor 15 min and 4
◦
C. Supernatants were used for protein content determination.
Absorbance was measured spectrophotometrically using 1 mg/mL of bovine serum albumin (BSA) as a
standard. Protein content was expressed as mg per g of fresh weight (FW).
2.4. Estimation of Total Antioxidant Capacity
2.4.1. DPPH Scavenging Activity
DPPH scavenging activity was determined according to the Brand–Williams method [
23
],
using the same extract for the determination of total soluble phenolic content. The reaction
mixture contained 20
µ
L of extract and 980
µ
L of 0.094 mM DPPH (2,2-diphenyl-1-picrylhydrazyl)
previously dissolved in methanol. The reaction was carried out in the dark at 22
◦
C for 15 min,
with occasional shaking. The standard curve was prepared by dissolving 2.5 mg of Trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) in 10 mL of methanol. Absorbance was
measured at 515 nm. Total antioxidant activity was expressed as the equivalents of Trolox per g of
fresh weight (FW).
2.4.2. Ferric Reducing Antioxidant Power Assay (FRAP)
Frozen tissue powder (200 mg) was extracted on ice with the addition of cold 80% EtOH for
15 min. After the extraction, samples were placed in a hot bath at 84
◦
C for 30 min. The homogenate
was centrifuged at 22,000
×
gand 4
◦
C for 15 min. The supernatant was used for further analyses of
antioxidant capacity by FRAP. The FRAP assay is based on antioxidants as electron-donors in reaction
with a yellow ferric 2,4,6-tripyridyl-s-triazine (Fe III TPTZ) complex, resulting in a blue-colored ferrous
form (Fe II TPTZ). The intensity of the blue color, which is proportional to the reducing power of the
antioxidants, was monitored spectrophotometrically at 593 nm. The reaction mixture for the FRAP
assay consisted of 0.5 mM TPTZ and 1 mM FeCl
3·
6H
2
O in acetate buffer (pH 3.6). The sample (10
µ
L)
and reaction mixture (290
µ
L) were added to the wells of microtiter plates, mixed, and incubated at
room temperature. After incubation, absorbance at 593 nm was measured using a microplate reader
(Tecan, Spark, Männedorf, Switzerland). Total antioxidant capacity was determined using a standard
curve in which Trolox was used as a standard at a concentration range from 0.25 to 2 mM. The results
are expressed in µmol Trolox equivalents per 100 g of fresh weight (FW).
Appl. Sci. 2020,10, 5731 4 of 13
2.5. Ascorbic Acid Content
Approximately 600 mg of fresh tissue was ground with mortar and pestle in liquid nitrogen
and extracted in 10 mL of distilled water. The homogenates were centrifuged for 15 min at 3000
×
g
at 4
◦
C. The supernatant was used for the determination of ascorbic acid content [
24
]. The reaction
mixture consisted of 300
µ
L of aqueous extract, 100
µ
L of 13.3% trichloroacetic acid, 25
µ
L of deionized
water, and 75
µ
L of 2,4-dinitrophenylhydrazine (DNPH) reagent. The DNPH reagent was prepared
by dissolving 2 g of DNPH, 230 mg of thiourea, and 270 mg of CuSO
4
in 100 mL of 5 M H
2
SO
4
.
Blanks were made in parallel for each sample, as described above, without the addition of the DNPH
reagent. Samples were incubated in a water bath for 60 min at 37
◦
C. After incubation, the DNPH
reagent was added to all blanks, and 500
µ
L of 65% H
2
SO
4
was added to all samples. The absorbance
was measured at 520 nm. The concentration of ascorbic acid was obtained from a standard curve with
known concentrations of ascorbic acid (2.5–20
µ
g/mL). The content of ascorbic acid is expressed in mg
per 100 g of fresh weight.
2.6. Determination of Crystalline Cellulose Content
Chia microgreens were dried at 65
°C
for 48 h, ground with mortar and pestle, and extracted
four times at 80
°C
using 80% ethanol. Crystalline cellulose content was determined according to
Foster et al. (2010) using Updegraffreagent [
25
]. One mL of Updegraffreagent (acetic acid: nitric
acid: water, 8:1:2 v/v) was added to the 70 mg of dry tissue. Afterward, samples were heated at 100
◦
C
for 30 min, centrifuged, and the pellet was washed with water and acetone and air-dried overnight.
The pellet was hydrolyzed with 72% sulfuric acid. Crystalline cellulose content was determined using
the colorimetric Anthrone assay for 96-well microtiter plates [
26
]. For standard curve preparation,
glucose (1 mg/mL) and Anthrone reagent (2 mg/mL sulfuric acid) were used. Crystalline cellulose
content was measured using a microplate reader (Tecan, Spark, Männedorf, Switzerland) and expressed
as glucose equivalents.
2.7. Fourier Transform Infrared Spectroscopy (FTIR)
FTIR was used to screen the dark-grown and illuminated chia seedlings. After grinding,
3 mg of dry tissue sample was mixed with 100 mg of KBr (spectroscopy grade, Merck, Darmstat,
Germany). Each FTIR spectrum was recorded by 20 co-added scans at a resolution of 2 cm
−1
in the
region of wave number (WN) 500 to 4000 cm
−1
(FTIR-8400S, Shimadzu, Tokyo, Japan). The spectra
were baseline-corrected.
2.8. Statistical Analyses
The mean values between the different groups (dark-grown, 24 h, and 48 h) were subjected to
analyses of variance one-way ANOVA. Subsequently, a posthoc analysis was performed using Fisher’s
LSD test (the least significant difference). The experiment was repeated three times, with five technical
replicates. The results are presented as mean
±
standard deviation (SD) of 15 replicates. The analyses
were performed with Statistica 13.1. (Tibco Software Inc., Palo Alto, CA, USA).
3. Results and Discussion
3.1. Effect of Illumination on Carotenoid and Chlorophyll Content
Dark-grown chia microgreens showed an etiolated, pale phenotype with increased hypocotyl
length. After exposure of the etiolated chia microgreens to light, a change of color from pale to green was
evident after 24 h, indicating the beginning of chlorophyll synthesis and subsequent development of the
photosynthetic apparatus. Similar findings were also reported by Paiva et al. [
27
], where the authors
investigated chia germination and seedling development under different light regimes. These authors
Appl. Sci. 2020,10, 5731 5 of 13
found that when chia was germinated under constant-dark conditions, seedlings had the longest
shoots, while under constant-light, the shoots were shorter.
The concentrations of total chlorophyll and carotenoid content differed significantly between
dark-grown microgreens and microgreens exposed to light (Figure 1). Compared to dark-grown
microgreens, the increase was observable 24 h after the treatment. Additionally, there was an evident
increase in the concentration of both total chlorophyll (Figure 1a) and carotenoid content (Figure 1b) in
chia microgreens 48 h after the treatment.
Appl. Sci. 2020, 10, x 5 of 12
Figure 1. Total chlorophyll (a) and carotenoid (b) content in dark-grown chia microgreens and
microgreens after exposure to light for 24 and 48 h. Data represent mean values from three
experiments with five replicates (n = 15). The error bars show standard deviation (SD). Different letters
signify values that are statistically different at p ≤ 0.05 according to Fisher’s LSD test.
The increase of photosynthetic pigments in treated chia microgreens indicates the association
between chlorophyll and carotenoid synthesis and the length of exposure to light.
As reviewed in Solymosi and Mysliwa-Kurdziel [28], chlorophylls are good antioxidants
because of their effective scavenging activity of reactive oxygen species (ROS) and inhibition of lipid
peroxidation. Chlorophylls are most commonly used as natural food colorants, but, recently, their
bioactive properties [29] have become more in focus as research has shown their emerging role as
potential prebiotics in rebalancing gut microbiota in mice [30].
Dietary intake of carotenoids is associated with a reduced risk of degenerative diseases [31,32]
and stroke prevention [33], and this suggests their anti/pro-oxidant roles [34]. In our study, light
caused an increase of carotenoid content in chia seedlings and therefore increased their antioxidative
properties. Lower light intensity in a range from 100 to 300 µmol m−2 s−1 had a positive effect on
photosynthetic processes [35], increased chlorophyll content [36], and more effective light use [37] in
plants. In this study, we used the light intensity of 100 µmol photons m−2 s−1, which generally has a
beneficiary effect on the synthesis of different investigated bioactive compounds and enhances
antioxidative properties in chia microgreens.
3.2. Effect of Illumination Total Phenolic and Protein Content
The content of total phenolics increased after the illumination with respect to the dark-grown
chia microgreens. The analysis did not show the differences between chia microgreens after exposure
to light for 24 h and 48 h (Figure 2).
Different soluble phenolic content reported in numerous studies have shown that the
mechanisms of polyphenol synthesis vary between plant species, raw seeds, and sprouts, and is also
affected by different growth conditions, germination processes, and sprout developmental stages [7].
Gómez-Favela et al. [5] showed that the controlled germination process exhibited a higher content of
bioactive compounds (among others, increased total phenolics) in flour from chia seeds. Compared
with the content of phenolics in other sprouts, our results showed that the amount of phenolics in
illuminated chia microgreens (24 and 48 h) was higher or similar [7,38,39]. These variations could be
attributed to the different enzymatic activity of hydrolases and polyphenol oxidases [40], de novo
synthesis of phenolics, differences in polymerization and oxidation processes, and degradation of
free or bound phenolics [41,42]. The increase of phenolics observed after exposure to light could also
be attributed to the development of defense mechanisms as a response to illumination, which
activates different metabolic pathways to enhance antioxidant activity in etiolated sprouts. Therefore,
illuminated chia microgreens represent a significant source of phenolics and can be considered as an
alternative source in diets.
Figure 1.
Total chlorophyll (
a
) and carotenoid (
b
) content in dark-grown chia microgreens and
microgreens after exposure to light for 24 and 48 h. Data represent mean values from three experiments
with five replicates (n =15). The error bars show standard deviation (SD). Different letters signify
values that are statistically different at p≤0.05 according to Fisher’s LSD test.
The increase of photosynthetic pigments in treated chia microgreens indicates the association
between chlorophyll and carotenoid synthesis and the length of exposure to light.
As reviewed in Solymosi and Mysliwa-Kurdziel [
28
], chlorophylls are good antioxidants because of
their effective scavenging activity of reactive oxygen species (ROS) and inhibition of lipid peroxidation.
Chlorophylls are most commonly used as natural food colorants, but, recently, their bioactive
properties [
29
] have become more in focus as research has shown their emerging role as potential
prebiotics in rebalancing gut microbiota in mice [30].
Dietary intake of carotenoids is associated with a reduced risk of degenerative diseases [
31
,
32
] and
stroke prevention [
33
], and this suggests their anti/pro-oxidant roles [
34
]. In our study, light caused an
increase of carotenoid content in chia seedlings and therefore increased their antioxidative properties.
Lower light intensity in a range from 100 to 300
µ
mol m
−2
s
−1
had a positive effect on photosynthetic
processes [
35
], increased chlorophyll content [
36
], and more effective light use [
37
] in plants. In this
study, we used the light intensity of 100
µ
mol photons m
−2
s
−1
, which generally has a beneficiary effect
on the synthesis of different investigated bioactive compounds and enhances antioxidative properties
in chia microgreens.
3.2. Effect of Illumination Total Phenolic and Protein Content
The content of total phenolics increased after the illumination with respect to the dark-grown chia
microgreens. The analysis did not show the differences between chia microgreens after exposure to
light for 24 h and 48 h (Figure 2).
Appl. Sci. 2020,10, 5731 6 of 13
Appl. Sci. 2020, 10, x 6 of 12
Figure 2. Total phenolic content (a) and protein content (b) in dark-grown chia microgreens and
microgreens after exposure to light for 24 and 48 h. Data represent mean values from three
experiments with five replicates (n = 15). The error bars show standard deviation (SD). Different letters
signify values that are statistically different at p ≤ 0.05 according to Fisher’s LSD test.
Under constant-dark conditions, protein content was significantly higher when dark-grown chia
microgreens and illuminated microgreens were compared (Figure 2b). Additionally, protein content
did not differ between chia microgreens 24 and 48 h after the light treatment. Proteins represent
storage for germinating seeds, and, during the germination process, they are hydrolyzed by
proteases, which increases their availability for plant growth [6]. The soluble protein content,
however, also depends on the balance between synthesis and demand [6]. In a recent work by
Mastropasqua et al. [43], authors showed that exposure to light impacted protein synthesis in
soybean, mung bean, pumpkin, and radish sprouts differently. Namely, dark-grown mung bean
sprouts had significantly higher soluble protein content in comparison to corresponding sprouts
exposed to light, which is in accordance with our results. This decreased protein content in chia
microgreens after light treatment could be explained by the increased degradation of proteins due to
higher demand for developmental processes taking part in growing microgreens induced by light
and redirecting metabolic pathways towards the synthesis of other secondary metabolites and
components involved in photoprotection.
3.3. Effect of Illumination on Ascorbic Acid Content
Ascorbic acid (AA) is involved in numerous functions in plants, from antioxidative defense and
photosynthesis to growth regulation [44]. Light triggers seed germination and influences the
biosynthesis of ascorbic acid [45]. Higher irradiance levels can increase the content and accumulation
of AA in plants [12].
Ascorbic acid (AA) content significantly varied between dark-grown and illuminated chia
microgreens (Figure 3). It is clearly visible that the length of illumination is associated with increasing
the content of AA. Analysis of the results showed that there were statistically significant differences
(p ≤ 0.05) between all three chia samples.
Lu and Guo [11] obtained similar results in mung bean sprouts grown under different light
regimes (24 and 12 h light and dark-grown), showing that the content of AA increased due to the
longer exposure to illumination.
When raw seeds and sprouts, such as soybean, mung bean, cowpea, and buckwheat, were
compared, studies found that the AA content was lower in raw seeds [7]. The germination process
significantly increased AA content in various edible seeds and sprouts. It was suggested that an
increase of AA content and its accumulation in sprouts is due to de novo synthesis [11]. Namely, the
synthesis of AA depends on electron transport within the photosynthetic apparatus. Since AA also
plays an important role in photoprotection, exposure of dark-grown chia microgreens triggered
Figure 2.
Total phenolic content (
a
) and protein content (
b
) in dark-grown chia microgreens and
microgreens after exposure to light for 24 and 48 h. Data represent mean values from three experiments
with five replicates (n =15). The error bars show standard deviation (SD). Different letters signify
values that are statistically different at p≤0.05 according to Fisher’s LSD test.
Different soluble phenolic content reported in numerous studies have shown that the mechanisms
of polyphenol synthesis vary between plant species, raw seeds, and sprouts, and is also affected
by different growth conditions, germination processes, and sprout developmental stages [
7
].
Gómez-Favela et al. [5] showed that the controlled germination process exhibited a higher content of
bioactive compounds (among others, increased total phenolics) in flour from chia seeds. Compared with
the content of phenolics in other sprouts, our results showed that the amount of phenolics in illuminated
chia microgreens (24 and 48 h) was higher or similar [
7
,
38
,
39
]. These variations could be attributed
to the different enzymatic activity of hydrolases and polyphenol oxidases [
40
], de novo synthesis of
phenolics, differences in polymerization and oxidation processes, and degradation of free or bound
phenolics [
41
,
42
]. The increase of phenolics observed after exposure to light could also be attributed
to the development of defense mechanisms as a response to illumination, which activates different
metabolic pathways to enhance antioxidant activity in etiolated sprouts. Therefore, illuminated chia
microgreens represent a significant source of phenolics and can be considered as an alternative source
in diets.
Under constant-dark conditions, protein content was significantly higher when dark-grown
chia microgreens and illuminated microgreens were compared (Figure 2b). Additionally, protein
content did not differ between chia microgreens 24 and 48 h after the light treatment. Proteins
represent storage for germinating seeds, and, during the germination process, they are hydrolyzed
by proteases, which increases their availability for plant growth [
6
]. The soluble protein content,
however, also depends on the balance between synthesis and demand [
6
]. In a recent work by
Mastropasqua et al. [43],
authors showed that exposure to light impacted protein synthesis in soybean,
mung bean, pumpkin, and radish sprouts differently. Namely, dark-grown mung bean sprouts had
significantly higher soluble protein content in comparison to corresponding sprouts exposed to light,
which is in accordance with our results. This decreased protein content in chia microgreens after
light treatment could be explained by the increased degradation of proteins due to higher demand
for developmental processes taking part in growing microgreens induced by light and redirecting
metabolic pathways towards the synthesis of other secondary metabolites and components involved
in photoprotection.
3.3. Effect of Illumination on Ascorbic Acid Content
Ascorbic acid (AA) is involved in numerous functions in plants, from antioxidative defense
and photosynthesis to growth regulation [
44
]. Light triggers seed germination and influences the
Appl. Sci. 2020,10, 5731 7 of 13
biosynthesis of ascorbic acid [
45
]. Higher irradiance levels can increase the content and accumulation
of AA in plants [12].
Ascorbic acid (AA) content significantly varied between dark-grown and illuminated chia
microgreens (Figure 3). It is clearly visible that the length of illumination is associated with increasing
the content of AA. Analysis of the results showed that there were statistically significant differences
(p≤0.05) between all three chia samples.
Appl. Sci. 2020, 10, x 7 of 12
increased AA synthesis in order to prevent photodamage in etiolated plants and diminished the
production of reactive oxygen species [46].
Figure 3. Ascorbic acid (AA) content in dark-grown chia microgreens and microgreens after exposure
to light for 24 and 48 h. Data represent mean values from three experiments with five replicates (n =
15). The error bars show standard deviation (SD). Different letters signify values that are statistically
different at p ≤ 0.05 according to Fisher’s LSD test.
3.4. Crystalline Cellulose Content
Dietary fibers have a beneficial effect on the gastrointestinal system, and chia seeds are rich in
dietary fibers [47]. Since cellulose presents one of the main components of dietary fibers, we
investigated the effect of light on cellulose content in chia microgreens, considering that germination
can impact dietary fiber depending on the germination time as well as plant species [48].
The cellulose content in dark-grown chia microgreens and microgreens 24 h after light exposure
did not show significant differences. On the other hand, a significant decrease in cellulose content
was observed in microgreens 48 h after exposure to light (Table 1).
Table 1. The crystalline cellulose content in dark-grown chia microgreens and microgreens 24 and 48
h after exposure to light.
Dark-Grown 24 h 48 h
0.656 a 0.657 a 0.317 b
(±0.08) (±0.09) (±0.02)
Data represent mean values from three experiments with five replicates (n = 15) expressed as µg
crystalline cellulose per mg of dry weight. Different letters signify values that are statistically different
at p ≤ 0.05 according to Fisher’s LSD test. Values in the brackets represent ± standard deviation (SD).
The germination process tends to increase dietary fiber content in different plant species such as
peas and amaranth, as well as others [49–51]. Gómez-Favela et al. [5] reported on lower dietary fiber
content found in chia sprouts, suggesting that the synthesis of dietary fibers such as cellulose,
hemicellulose, and pectin are much slower in comparison to other sprouts in particular growth
conditions.
It is known that dark-grown microgreens have a characteristic pale phenotype with excessive
shoot elongation compared to microgreens growing in normal light conditions. Therefore, more
cellulose content could be found in such etiolated shoots [52]. In our study, a decline of cellulose
content in microgreens, 48 h after exposure to light, could be due to the reprogramming of metabolic
pathways and redirection of developmental processes in pathways that are light-dependent, such as
photosynthesis and sugar synthesis, [53] as well as general plant elongation rather than cellulose
synthesis.
Figure 3.
Ascorbic acid (AA) content in dark-grown chia microgreens and microgreens after exposure
to light for 24 and 48 h. Data represent mean values from three experiments with five replicates
(n =15).
The error bars show standard deviation (SD). Different letters signify values that are statistically
different at p≤0.05 according to Fisher’s LSD test.
Lu and Guo [
11
] obtained similar results in mung bean sprouts grown under different light
regimes (24 and 12 h light and dark-grown), showing that the content of AA increased due to the
longer exposure to illumination.
When raw seeds and sprouts, such as soybean, mung bean, cowpea, and buckwheat,
were compared, studies found that the AA content was lower in raw seeds [
7
]. The germination
process significantly increased AA content in various edible seeds and sprouts. It was suggested that
an increase of AA content and its accumulation in sprouts is due to de novo synthesis [
11
]. Namely,
the synthesis of AA depends on electron transport within the photosynthetic apparatus. Since AA
also plays an important role in photoprotection, exposure of dark-grown chia microgreens triggered
increased AA synthesis in order to prevent photodamage in etiolated plants and diminished the
production of reactive oxygen species [46].
3.4. Crystalline Cellulose Content
Dietary fibers have a beneficial effect on the gastrointestinal system, and chia seeds are rich in
dietary fibers [
47
]. Since cellulose presents one of the main components of dietary fibers, we investigated
the effect of light on cellulose content in chia microgreens, considering that germination can impact
dietary fiber depending on the germination time as well as plant species [48].
The cellulose content in dark-grown chia microgreens and microgreens 24 h after light exposure
did not show significant differences. On the other hand, a significant decrease in cellulose content was
observed in microgreens 48 h after exposure to light (Table 1).
The germination process tends to increase dietary fiber content in different plant species such as peas
and amaranth, as well as others [
49
–
51
]. G
ó
mez-Favela et al. [
5
] reported on lower dietary fiber content
found in chia sprouts, suggesting that the synthesis of dietary fibers such as cellulose, hemicellulose,
and pectin are much slower in comparison to other sprouts in particular growth conditions.
Appl. Sci. 2020,10, 5731 8 of 13
Table 1.
The crystalline cellulose content in dark-grown chia microgreens and microgreens 24 and 48 h
after exposure to light.
Dark-Grown 24 h 48 h
0.656 a 0.657 a 0.317 b
(±0.08) (±0.09) (±0.02)
Data represent mean values from three experiments with five replicates (n =15) expressed as
µ
g crystalline cellulose
per mg of dry weight. Different letters signify values that are statistically different at p
≤
0.05 according to Fisher’s
LSD test. Values in the brackets represent ±standard deviation (SD).
It is known that dark-grown microgreens have a characteristic pale phenotype with excessive shoot
elongation compared to microgreens growing in normal light conditions. Therefore, more cellulose
content could be found in such etiolated shoots [
52
]. In our study, a decline of cellulose content in
microgreens, 48 h after exposure to light, could be due to the reprogramming of metabolic pathways and
redirection of developmental processes in pathways that are light-dependent, such as photosynthesis
and sugar synthesis, [53] as well as general plant elongation rather than cellulose synthesis.
3.5. Effect of Illumination on Antioxidant Capacity
It has been shown that the germination process promotes antioxidant capacity in different
sprouts [
4
,
7
,
10
,
54
]. In our study, exposure to light generally increased the content of chlorophylls and
carotenoids, total polyphenolics, and ascorbic acid, which are part of the plant’s antioxidative system.
Accordingly, it could be expected that the antioxidant capacity in the analyzed chia microgreens
exposed to light will also be increased.
Light treatment caused an increase of total antioxidant capacity in chia microgreens measured by
1, 1-diphenyl-2-picrylhydrazyl scavenging radical (DPPH) assay. A significant difference was found
between dark-grown and chia microgreens exposed to light. There were no differences in DPPH
scavenging activity between microgreens exposed for 24 and 48 h (Figure 4a).
Appl. Sci. 2020, 10, x 8 of 12
3.5. Effect of Illumination on Antioxidant Capacity
It has been shown that the germination process promotes antioxidant capacity in different
sprouts [4,7,10,54]. In our study, exposure to light generally increased the content of chlorophylls and
carotenoids, total polyphenolics, and ascorbic acid, which are part of the plant’s antioxidative system.
Accordingly, it could be expected that the antioxidant capacity in the analyzed chia microgreens
exposed to light will also be increased.
Light treatment caused an increase of total antioxidant capacity in chia microgreens measured
by 1, 1-diphenyl-2-picrylhydrazyl scavenging radical (DPPH) assay. A significant difference was
found between dark-grown and chia microgreens exposed to light. There were no differences in
DPPH scavenging activity between microgreens exposed for 24 and 48 h (Figure 4a).
Figure 4. Antioxidant activity in dark-grown chia microgreens and microgreens after exposure to
light for 24 and 48 h evaluated by DPPH scavenging activity (a) and FRAP assay (b). Data represent
mean values from three experiments with five replicates (n = 15). The error bars show standard
deviation (SD). Different letters signify values that are statistically different at p ≤ 0.05 according to
Fisher’s LSD test.
In dark-grown chia microgreens, total antioxidative capacity measured by the FRAP assay was
the highest (Figure 4b). When different light treatments were compared, similar values were
observed in microgreens exposed to light for 24 and 48 h, whilst a significant decrease was observed
24 h after exposure to light in comparison to dark-grown chia microgreens.
The FRAP assay exhibited lower values in comparison with the DPPH assay. Other authors have
also reported differences between obtained values when the two assays were used [4,55]. For
instance, in 7-day-old chia sprouts exposed to 12 h/12 h (light/dark) cycles, DPPH scavenging activity
was lower in comparison to the FRAP assay [54]. The reason for such discrepancies might be due to
differences in the chemistry and sensitivity of these two assays. In a recently published study by
Mitrović et al. [38], the authors reported that the DPPH assay is more suitable than the FRAP assay for
evaluating the antioxidant activity of chia. This suggests that the obtained values of these assays are
impacted by the species, germination processes, and growth conditions, as well as temperature and
duration of light exposure.
3.6. Effect of Illumination on FTIR Spectra
The FTIR spectra of the three chia samples under the study are depicted in Figure 5, and their
main bands are summarized in Table 2. It can be observed that they are similar, in particular, the
samples exposed to light for 24 and 48 h.
Figure 4.
Antioxidant activity in dark-grown chia microgreens and microgreens after exposure to light
for 24 and 48 h evaluated by DPPH scavenging activity (
a
) and FRAP assay (
b
). Data represent mean
values from three experiments with five replicates (n =15). The error bars show standard deviation
(SD). Different letters signify values that are statistically different at p
≤
0.05 according to Fisher’s
LSD test.
In dark-grown chia microgreens, total antioxidative capacity measured by the FRAP assay was
the highest (Figure 4b). When different light treatments were compared, similar values were observed
in microgreens exposed to light for 24 and 48 h, whilst a significant decrease was observed 24 h after
exposure to light in comparison to dark-grown chia microgreens.
Appl. Sci. 2020,10, 5731 9 of 13
The FRAP assay exhibited lower values in comparison with the DPPH assay. Other authors have
also reported differences between obtained values when the two assays were used [
4
,
55
]. For instance,
in 7-day-old chia sprouts exposed to 12 h/12 h (light/dark) cycles, DPPH scavenging activity was lower
in comparison to the FRAP assay [
54
]. The reason for such discrepancies might be due to differences in
the chemistry and sensitivity of these two assays. In a recently published study by Mitrovi´c et al. [
38
],
the authors reported that the DPPH assay is more suitable than the FRAP assay for evaluating the
antioxidant activity of chia. This suggests that the obtained values of these assays are impacted by
the species, germination processes, and growth conditions, as well as temperature and duration of
light exposure.
3.6. Effect of Illumination on FTIR Spectra
The FTIR spectra of the three chia samples under the study are depicted in Figure 5, and their main
bands are summarized in Table 2. It can be observed that they are similar, in particular, the samples
exposed to light for 24 and 48 h.
Appl. Sci. 2020, 10, x 9 of 12
Figure 5. FTIR spectra of dark-grown (a) chia microgreens and microgreens after exposure to light for
24 (b) and 48 (c) h. FTIR spectrum was recorded by 20 co-added scans at a resolution of 2 cm−1 in the
region of wave number (WN) 500 to 4000 cm−1. The spectra were baseline-corrected.
A large number of peaks appeared in the region from 1000 to 3000 cm−1, indicating that chia
microgreens have a rich chemical composition (proteins, lipids, carbohydrates) [18]. More precisely,
the band located around 3290 cm−1 represents N–H stretching vibrations that are caused by proteins
[20]. The bands between 2800 and 3000 cm−1 mainly represent C–H stretching vibrations that are
caused by lipids. The region between 1550 and 1700 cm−1 are protein absorption bands, including
amide I and amide II. Additionally, the fingerprint region between 1000 and 1500 cm−1 is where amide
II and the functional groups of nucleic acid and carbohydrates contribute to these absorption bands.
Table 2. Main bands in the FTIR spectra and their assignments of dark-grown chia microgreens and
microgreens after exposure to light for 24 and 48 h.
Peak No. Assignments Dark-Grown
Wavenumber (cm−1)
24 h
Wavenumber (cm−1)
48 h
Wavenumber
(cm−1)
I not assigned 889,9 890 889
II Polysaccharides 1062 1062 1063
III Amide III region 1238 1239 1239
IV Amide III region 1457 1457 1457
V Amide II region 1540 1540 1540
VI Amide I region 1647 1647 1647
VII Fat content 2855 disappeared disappeared
VIII Fat content 2926 2927 2925
IX Protein content 3269 3270 3271
In comparison with the other two samples, the dark-grown chia sample showed negligible
differences regarding the peak shifting in the region comprised between 1000 and 1700 cm−1. We
observed two well-pronounced bands in dark-grown chia microgreens around 2855 and 2926 cm−1.
Regarding the peak shifting, with the increment of light treatment, the band at about 2855 cm−1 (dark-
grown microgreens) disappeared in illuminated microgreens (24 and 48 h), while bands at 2926 cm−1
remain relatively stable in all chia samples.
Nine main bands were analyzed between 800 and 3500 cm−1 in the spectra of the three chia
samples, and the band positions were relatively similar (with the exception of the band at 2855 cm−1)
to each other, indicating that structure was sensitive to treatments in the region of fats, vax, and lipids
after 24 and 48 h of exposure to light [19,20].
Figure 5.
FTIR spectra of dark-grown (
a
) chia microgreens and microgreens after exposure to light for
24 (
b
) and 48 (
c
) h. FTIR spectrum was recorded by 20 co-added scans at a resolution of 2 cm
−1
in the
region of wave number (WN) 500 to 4000 cm−1. The spectra were baseline-corrected.
Table 2.
Main bands in the FTIR spectra and their assignments of dark-grown chia microgreens and
microgreens after exposure to light for 24 and 48 h.
Peak No. Assignments Dark-Grown
Wavenumber (cm−1)
24 h
Wavenumber (cm−1)
48 h
Wavenumber (cm−1)
I not assigned 889.9 890 889
II Polysaccharides 1062 1062 1063
III Amide III region 1238 1239 1239
IV Amide III region 1457 1457 1457
V Amide II region 1540 1540 1540
VI Amide I region 1647 1647 1647
VII Fat content 2855 disappeared disappeared
VIII Fat content 2926 2927 2925
IX Protein content 3269 3270 3271
A large number of peaks appeared in the region from 1000 to 3000 cm
−1
, indicating that chia
microgreens have a rich chemical composition (proteins, lipids, carbohydrates) [
18
]. More precisely,
the band located around 3290 cm
−1
represents N–H stretching vibrations that are caused by proteins [
20
].
The bands between 2800 and 3000 cm
−1
mainly represent C–H stretching vibrations that are caused by
lipids. The region between 1550 and 1700 cm
−1
are protein absorption bands, including amide I and
Appl. Sci. 2020,10, 5731 10 of 13
amide II. Additionally, the fingerprint region between 1000 and 1500 cm
−1
is where amide II and the
functional groups of nucleic acid and carbohydrates contribute to these absorption bands.
In comparison with the other two samples, the dark-grown chia sample showed negligible
differences regarding the peak shifting in the region comprised between 1000 and 1700 cm
−1
.
We observed two well-pronounced bands in dark-grown chia microgreens around 2855 and 2926 cm
−1
.
Regarding the peak shifting, with the increment of light treatment, the band at about 2855 cm
−1
(dark-grown microgreens) disappeared in illuminated microgreens (24 and 48 h), while bands at
2926 cm−1remain relatively stable in all chia samples.
Nine main bands were analyzed between 800 and 3500 cm
−1
in the spectra of the three chia
samples, and the band positions were relatively similar (with the exception of the band at 2855 cm
−1
)
to each other, indicating that structure was sensitive to treatments in the region of fats, vax, and lipids
after 24 and 48 h of exposure to light [19,20].
4. Conclusions
Our findings show that growth conditions with the lower light intensity of 100
µ
mol photons
m−2s−1
evoked a positive effect on total antioxidant capacity, synthesis of chlorophyll and carotenoids,
total soluble phenolics, and ascorbic acid in dark-grown chia microgreens. Thus, the synthesis of
bioactive compounds and the antioxidative potential of illuminated chia microgreens was improved.
The DPPH assay was shown to be more sensitive in detecting antioxidative activity in comparison with
FRAP. Therefore, we can conclude that chia microgreens could be considered a valuable supplement
to the human diet, in addition to raw chia seeds and other popular microgreens. Present trends
use different approaches in growing different sprouts and microgreens that are used for human
consumption. Further investigations should be undertaken to reveal the best practices in growing chia
microgreens, as well as the effect of light on the regulation of molecular mechanisms involved in the
synthesis of bioactive compounds.
Author Contributions:
Conceptualization, S.M. and L.B.; methodology, V.G., A.V., and M.V.; formal analysis, I.V.,
V.G., A.V., and M.V.; writing—original draft preparation, S.M. and L.B.; writing—review and editing, S.M., L.B.,
and V.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors wish to thank Ksenija Doboš for valuable technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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