Content uploaded by Slađana Žilić
Author content
All content in this area was uploaded by Slađana Žilić on Sep 24, 2018
Content may be subject to copyright.
Original article
Can the sprouting process applied to wheat improve the contents
of vitamins and phenolic compounds and antioxidant capacity of
the flour?
Sla dana
Zili
c,
1
* Zorica Basi
c,
2
Vesna Had
zi-Ta
skovi
c
Sukalovi
c,
3
Vuk Maksimovi
c,
3
Marijana Jankovi
c
1
& Milomir Filipovi
c
4
1 Department of Technology, Maize Research Institute, Slobodana Baji
ca 1, Belgrade-Zemun 11085, Serbia
2 Military Medical Academy, Institute of Hygiene, Crnotravska 17, Belgrade, Serbia
3 Institute for Multidisciplinary Research, University of Belgrade, Kneza Vi
seslava 1, Belgrade, Serbia
4 Department of Breeding, Maize Research Institute, Slobodana Baji
ca 1, Belgrade-Zemun 11085, Serbia
(Received 1 July 2013; Accepted in revised form 20 September 2013)
Summary The content of tocopherols, B vitamins, as well as free soluble, soluble conjugated and insoluble bound
phenolic compounds was determined in untreated, steeped and sprouted wheat grains. Antioxidant capac-
ity of whole wheat grains and their phenolic fractions was also evaluated. Sprouting significantly increased
the levels of tocopherols, niacin, riboflavin, as well as free and bound phenolic compounds improving
nutritional value and antioxidant capacity of wheat grains/flour. After sprouting for 5 days, the content
of total phenolics, flavonoids and ferulic acid calculated as the sum of its fractions was increased by 9.9,
30.7 and 21.6%, respectively. The content of a-, b+c- and d-tocopherols was increased for 3.59-fold, 2.33-
fold and 2.61-fold respectively, while the content of niacin, as predominant B vitamin, was increased for
1.19-fold after sprouting. The total antioxidant capacity of untreated, steeped and sprouted whole wheat
grains was 19.44, 20.37 and 22.70 mmol Trolox Eq/kg, respectively. Sprouted wheat, as a rich source of
bioavailable phytochemicals, should be used to improve the nutritional quality of food.
Keywords Antioxidant capacity, phenolics, sprouted wheat, steeped wheat, vitamins.
Introduction
Many epidemiological studies have demonstrated that
health beneficial effects of whole wheat were attributed
to the bioactive factors in bran, such as nondigestible
carbohydrates and phytochemicals (Jensen et al.,
2004). However, the importance of wheat has mainly
been attributed to its ability to be ground into flour
and semolina that form the basic ingredients of bread
and pasta, respectively, while bran is mainly used for
animal feeding. Due to low nutritional quality of
bread, its vitamin fortification has become mandatory
in many countries. In contrast to fortification, adding
of sprouted wheat flour is a natural way to increase
vitamin levels. Although sprouting has been known
for a very long time mainly in the Eastern countries,
in recent years, sprouted grains have become very pop-
ular and widely accepted as a functional food because
of their nutritious and health benefits. Hydrolytic
enzymes activated during the process of wheat sprout-
ing result in the degradation of starch and nonstarch
polysaccharides, as well as proteins, leading to an
increase in reducing sugars, soluble dietary fibres,
peptides and amino acids, as well as the release of the
insoluble phenolic compounds covalently bound to cell
wall polysaccharides (Hung et al., 2012). Further, the
biochemical processes, occurring during sprouting, can
generate bioactive components, such as riboflavin,
thiamine, biotin, pantothenic acid, niacin, vitamin C,
tocopherols and phenolic compounds, and also
increase their availability (Moongngarm & Saetung,
2010). Sprouting of wheat has been suggested as an
inexpensive and effective method to enhance the anti-
oxidant capacity of flour through the increase of low-
molecular weight antioxidants (Alvarez-Jubete et al.,
2010). Improving the hydrolytic enzymes efficiency,
sprouting supports the biochemical mechanisms in
humans and therefore can be considered as a kind of
predigestion that helps breaking down the high-molec-
ular complex compounds into their building blocks
that are considered to have many physiological
*Correspondent: Fax: +381 11 37 54 994;
e-mail: szilic@mrizp.rs
International Journal of Food Science and Technology 2014, 49, 1040–1047
doi:10.1111/ijfs.12397
©2013 Institute of Food Science and Technology
1040
benefits related to life-threatening diseases (Komatsu-
zaki et al., 2007). In contrast to the increase in bioac-
tive compounds, the baking performance of wheat
drastically decreases during sprouting, making the use
of sprouted wheat for baking restricted. Additionally,
due to the increased content of free amino acids and
reducing sugars, sprouted wheat can potentially
promote the Maillard reaction (Abderrahim et al.,
2012). On the other hand, the study of Hussain & Ud-
din (2012) indicated that sprouting improved wheat
flour functional properties such as water absorption
capacity that is important in the development of
ready-to-eat cereal foods.
In recent years, efforts have been made to improve
plant raw materials that are widely used as food ingre-
dients through the increase in the content of bioactive
compounds, as well as processing methods to achieve
a higher bioavailability of nutrients naturally present
in the plant materials. There are a number of detailed
studies showing the nutritional and antioxidant prop-
erties of sprouted wheat. However, studies on quantifi-
cation of B vitamins and characterisation of all three
differently bioavailable chemical forms of phenolic
compounds of steeped and sprouted wheat flours are
very limited. Therefore, in this study, we investigated
the effects of steeping and sprouting on the content of
bioactive components such as riboflavin, thiamine,
pyridoxine, niacin and tocopherols in wheat flours and
also how this approach affected the health considering
the development of phenolic phytochemicals in terms
of content and chemical form and contribution to the
total antioxidant capacity of whole wheat flours.
Material and methods
Plant materials, steeping and sprouting conditions
The grains of bread wheat genotype harvested in Ser-
bia in 2012 were used. The wheat grains were washed
with distilled water in the flow for 60 min with air
continuously bubbled through the system. The grains
were then immersed in distilled water (1:1.5, w/v) and
steeped for 24 h at room temperature (21 °C). After
steeping, the water was decanted. A portion of
imbibed wheat grains were transferred into a plastic
container and incubated in the dark for 5 days in an
incubator at 20 °C and humidity of 50%. The grains
steeped for 24 h and grains sprouted for 5 days were
air-dried at 55 °C for 8 h to remove the moisture of
approximately 11%. For the control sample, untreated
whole wheat grains were used. All samples were milled
into whole-grain flour using a Perten 120 lab mill (Per-
ten, Sweden) to a fine powder (particle size <500 lm).
Steeped and sprouted whole wheat grains were
produced in a mill company (Bread Line d.o.o., Bel-
grade-Zemun, Serbia).
Analytical procedures
Analysis of tocopherols
The content of tocopherols was determined by the
HPLC method (S
anchez-P
erez et al., 2000). A n-hex-
ane extraction was applied. The wheat flour sample
(1 g) was mixed with 10 mL of n-hexane, and the mix-
ture was rigorously shaken at 4 °C for 30 min. After
centrifugation at 7000 gfor 15 min, the upper layer
was separated and evaporated under N
2
. The dried
sample was then redissolved in 5 mL of methanol, vor-
texed, centrifuged at 5000 gfor 10 min, and the clean
upper layer was collected. A HPLC system with the
Waters M600 E pump, thermostat and Rheodyne 7125
injector was used. The separation of tocopherols was
performed on the Nucleosil 50-5 C18 column
(250 94 mm, i.d., 5 lm) at flow rate of 1.0 mL min
1
.
The mobile phase consisted of 95% methanol. The
detection was performed with the Shimadzu RF-535
fluorescence detector at an excitation wavelength of
295 nm and an emission wavelength of 330 nm. Identi-
fied peaks were confirmed and quantified by data
acquisition and spectral evaluation using the ‘Clarify’
chromatographic software. The content of tocopherols
is expressed as lg per g of d.m.
Analysis of thiamine (vitamin B
1
), riboflavin (vitamin B
2
)
and pyridoxine (vitamin B
6
)
The thiamine, riboflavin and pyridoxine were released
from wheat flour by alkaline and enzymatic hydrolysis
as described by S
anchez-Machado et al. (2004). Specif-
ically, 1 g of a finely ground wheat sample was treated
with 15 mL of 0.1 MHCl at 100 °C for 30 min. After
cooling to room temperature, sample was brought to
pH 4.3–4.7 with 2.5 Msodium acetate, then 1.25 mL
of a 10% aqueous solution of taka-diastase was added,
incubated in an oven at 45 °C for 4 h, filtered through
Whatman No. 41 paper and diluted to 25 mL with
Milli-Q water. For thiamine determination, it was
necessary to perform its oxidation to thiochrome. One
mL of the solution prepared as mentioned was treated
with 0.5 mL of a 1% solution of potassium ferricya-
nide in cold 20% aqueous NaOH, vortexed for 10 s
and used. All extracts were injected in the HPLC
system. The separation of B vitamins was performed
on the Nucleosil 50-5 C18 column (250 94 mm, i.d.,
5lm). The mobile phases were a 450 mL of metha-
nol +650 mL of 5 mMammonium acetate for B1 and
B2 vitamins, and a 250 mL of methanol +770 mL of
5m
Mhexanesulphonic acid for B6 vitamin. The flow
rate was set at 1.0 mL min
1
. The detection was
performed with the Shimadzu RF-535 fluorescence
detector at the excitation wavelength of 370, 450 and
286 nm, and at the emission wavelength of 430, 530
and 392 nm for vitamin B1, B2 and B6, respectively.
Identified peaks were confirmed and quantified by data
©2013 Institute of Food Science and Technology International Journal of Food Science and Technology 2014
Bioactive compounds in sprouted wheat S.
Z ili
cet al. 1041
acquisition and spectral evaluation using the ‘Clarify’
chromatographic software. The content of B vitamins
is expressed as lg per g of d.m.
Analysis of niacin (vitamin B
3
)
Total niacin was extracted according to the modified
AOAC (1984) method. One g of wheat flour was
hydrolysed with 25 mL of 4% calcium hydroxide for
2 h in an autoclave at 8.27 kPa. Cooled hydrolysates
were centrifuged for 15 min at 7000 g. After the pH
was adjusted to 5.0–5.2 with 1% phosphoric acid and
diluted to 50 mL, the extracts were filtered through
0.45-lm membrane filters and injected in the HPLC
system consisting of Waters M600 E binary pumps,
thermostat and Rheodyne 7125 injector connected to
the Waters 2996 diode array detector (Waters, Mil-
ford, MA, USA). Niacin was separated on the LiChro-
CART 125-4 Purospher C-18 RP column
(125 94 mm, i.d., 5 lm) at a temperature of 20 °C
using an isocratic elution programme with a mobile
phase containing 50 mMphosphate buffer pH 3.0, at a
flow rate of 1 mL min
1
. Identified peaks were con-
firmed and quantified by data acquisition and spectral
evaluation using the ‘Clarify’ chromatographic soft-
ware. The content of niacin is expressed as lg per g of
d.m.
Extraction of free soluble, soluble conjugated and insoluble
bound phenolic compounds
Free soluble, soluble conjugated and insoluble bound
phenolic compounds in wheat samples were extracted
according to the procedure described by Antoine et al.
(2004). Twenty mL of acetone/methanol/water mixture
(7:7:6, v/v/v) was used to extract free and soluble con-
jugated phenolic compounds from 0.5 g of flour. Insol-
uble phenolic compounds in the residue and
conjugated phenolic compounds in the acetone/metha-
nol/water extract were released by alkaline hydrolysis
for 4 h at room temperature using 4 MNaOH before
extraction. After the pH was adjusted to 2.0 by 6 M
HCl, all the hydrolysates were extracted with ethyl
acetate and diethyl ether (1:1, v/v) for four times. Five
mL of combined extracts were evaporated under N
2
stream at 30 °C to dryness. The final residues were
redissolved in 1.5 mL of methanol. After filtering
through a 0.45-lm nylon filter, samples were kept at
40 °C prior to the HPLC analysis. Such prepared
methanolic solutions of phenolic fractions (phenolic
extract) were used for the analyses of phenolic acids,
total phenolic compounds, flavonoids and antioxidant
capacity.
Measurement of total phenolics content (TPC)
The total phenolic content was determined according
to Singleton et al. (1999) and expressed as mg of gallic
acid equivalent (GAE) per kg of d.m.
Measurement of total flavonoids content
The flavonoid content was determined according to
Eberhardt et al. (2000) and expressed as mg of cate-
chin equivalent (CE) per kg of d.m.
Analysis of individual phenolic acids
Quantification of phenolic acids was performed by the
HPLC system. Prior to injection, methanolic extracts
were filtered through 0.22-lm nylon syringe filters
(Phenomenex, Torrance, CA, USA). Filtered extracts
were injected in the Waters HPLC system consisting of
1525 binary pumps, thermostat and 717 +autosampler
connected to the Waters 2996 diode array detector
(Waters). Phenolic acids were separated on the Sym-
metry C-18 RP column (125 94 mm, i.d., 5 lm)
using the linear gradient elution programme with a
mobile phase containing solvent A (0.1% phosphoric
acid) and solvent B (acetonitrile), at a flow rate of
1 mL min
1
with the following gradient profile:
20 min from 10 to 22% B, 20 min with a linear rise to
40% B, 5 min reverse to 10% B and additional 5 min
equilibration time. The identification of phenolic acids
was accomplished by comparing the retention time
and absorption spectra of peaks in wheat samples to
those of standard compounds. The quantification of
phenolic acids was based on calibration curves built
for each of the compounds identified in the samples.
The content of phenolic acids is expressed as lg per g
of d.m.
Analysis of total antioxidant capacity (TAC)
Measuring the total antioxidant capacity of phenolic
fractions and wheat flour samples was done based on
QUENCHER method described by Serpen et al.
(2008) using 7 mMaqueous solution of ABTS (2,2-azi-
no-bis/3-ethyl-benothiazoline-6-sulphonic acid) with
2.45 mMK
2
O
8
S
2
as the stock solution. The working
solution of ABTS
+
was obtained by diluting the stock
solution in water/ethanol (50:50, v/v). The wheat flour
samples (10 mg), as well as phenolic extracts (500 lL
evaporated to dryness), were mixed with 20 mL of
ABTS
+
working solution, and the mixture was rigor-
ously shaken at 4 °C for 25 min. After centrifugation
at 6200 gfor 5 min (10 °C), the absorbance measure-
ment was performed at 734 nm. The total antioxidant
capacity was expressed as the Trolox equivalent anti-
oxidant capacity (TEAC) in mmol of Trolox per kg of
d.m.
Statistical analysis
The analytical data were reported as mean SD of at
the least two independent extractions. Significance of
differences between wheat samples were analysed by
the Fisher’s least significant differences test, after vari-
ance analysis for trials was set up according to the
©2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2014
Bioactive compounds in sprouted wheat S.
Z ili
cet al.1042
randomised complete block design. Differences with
P<0.05 were considered significant. Statistical evalua-
tion was carried out by MSTATC statistical software
(Michigan State University, East Lansing, MI, USA).
Results and discussion
The content of tocopherols (a,b+cand d), thiamine
(vitamin B
1
), riboflavin (vitamin B
2
), niacin (vitamin
B
3
) and pyridoxine (vitamin B
6
) in the wheat samples
is given in Table 1. Tocopherol contents in untreated
wheat grains were low, with 0.94 lgg
1
of a-tocoph-
erol, 1.71 lgg
1
of b+c-tocopherol and 1.26 lgg
1
of
d-tocopherol. a-Tocopherol concentration, quantified
in this study, was a fivefold lower than that
(4.37 lgg
1
) reported by Yang et al. (2001), but
threefold higher than that (0.3 lgg
1
) reported by
Seibold (1990). According to Lampi et al. (2008) in
genotypes rich in tocols, there was usually a high pro-
portion of b-tocotrienols and low proportion of a-
tocopherol. However, the content of a-, b+c-, as well
as d-tocopherol was significantly affected by sprouting.
The content of aforementioned tocopherols was
increased for 3.59-fold, 2.33-fold and 2.61-fold respec-
tively, in relation to that in untreated wheat grains.
These results are in agreement with Yang et al. (2001).
Although the wheat samples contain all tocopherol
forms, only a-tocopherol is the most biologically active
form of vitamin E because in biological systems it is
the major lipid-soluble chain-breaking antioxidant.
Besides, bioaccessibility of a-tocopherol from wheat
bread is very high, being 99.6%, and that from wheat
germ almost as good, being 53.29% (Reboul et al.,
2006). These results indicate that sprouted wheat
grains and their products could be important sources
of a-tocopherol.
Although wholegrain cereals are a rich source of
many vitamins, thiamine, riboflavin and niacin
(vitamins B
1
,B
2
,andB
3
) have been normally added to
white flour and other refined grains since the 1940s to
replace the nutrients that are removed with refining. In
untreated wheat sample, the 92% of total content of
group B vitamins (79.27 lgg
1
) was mainly repre-
sented by niacin (72.81 lgg
1
; Table 1). Unfortu-
nately, it should be noted that some diseases, such as
pellagra, occur because niacin in cereals, being incor-
porated in large molecules (probably glycoproteins), is
mainly present in a nutritionally unavailable form
(Combs, 2008). Prodanov et al. (1997) have reported
increments in available niacin brought about by
sprouting. According to our study, after sprouting, the
content of total niacin was increased by 19.13% than
in untreated wheat grains, while that in steeped wheat
grains was slightly reduced. Our results have confirmed
early research of Nason (1950) according to which the
increase occurred in the maize embryo while the endo-
sperm lost niacin. Tarr & Arditti (1982) have suggested
that tryptophan from the endosperm acts as a niacin
precursor when translocated to the embryonic axis.
Our results indicate that sprouting also significantly
improved the content of riboflavin by about 1.5-fold
compared with the untreated wheat grains. However,
in accordance with the results of Moongngarm &
Saetung (2010) for germinated brown rice, sprouting
of the wheat brought a slight reduction of thiamine
and pyridoxine contents probably due to leaching out
of water-soluble vitamins during the soaking. Thia-
mine and pyridoxine contents decreased from 5.81 and
0.38 lgg
1
of nontreated to 5.18 and 0.30 lgg
1
of
sprouted wheat grains, respectively (Table 1).
The changes in the total phenolic content (TPC) of
wheat during sprouting determined by the Folin–Cio-
calteu method are shown in Table 2. The present study
demonstrated that bound phenolics were a predomi-
nant fraction in untreated grains, and its content was
higher by about 2.12 and 1.24-fold as compared to
those of soluble free and conjugated phenolics, respec-
tively. The level of total soluble conjugated phenolics
in wheat grains was higher by about 1.72 times than
those of its total soluble free phenolics. The similar
phenolic fractions relation was obtained by Liyana-
Pathirana & Shahidi (2006) in whole grains of soft
white winter and hard red spring wheat. According to
our study, both steeping and sprouting significantly
(P<0.05) increased the content of total soluble
free and insoluble bound phenolic fractions, while
decreased the conjugated component. Food compo-
nents to exert their biological effects must be in some
form bioavailable. Compared with untreated wheat
grains, the content of free phenolic compounds, that
may be declared bioavailable, was increased by 5.7
and 28% after steeping for 24 h and sprouting for
Table 1 Content of tocopherols and B vitamins in whole untreated,
steeped and sprouted wheat grains/flours
Vitamins
Untreated
wheat
Steeped
wheat
Sprouted
wheat
E vitamins (lgg
1
)
a-Tocopherol 0.94 0.09
b
0.96 0.08
b
3.38 0.26
a
b+c-Tocopherol 1.71 0.05
b
1.51 0.08
b
3.98 0.26
a
d-Tocopherol 1.26 0.06
b
1.06 0.08
b
3.29 0.23
a
Total tocopherols 3.91 0.14
b
3.53 0.18
b
10.65 0.49
a
B vitamins (lgg
1
)
Niacin 72.81 1.69
b
70.87 1.99
b
86.74 1.74
a
Thiamine 5.81 0.17
a
5.73 0.14
a
5.18 0.14
b
Riboflavin 0.27 0.02
b
0.28 0.02
b
0.41 0.02
a
Pyridoxine 0.38 0.02
a
0.37 0.01
a
0.30 0.02
b
Means followed by the same letter within the same row are not signifi-
cantly different, according to Fisher’s last significance difference test
(P=5%).
©2013 Institute of Food Science and Technology International Journal of Food Science and Technology 2014
Bioactive compounds in sprouted wheat S.
Z ili
cet al. 1043
5 days, respectively. This result can be caused by the
hydrolysis of conjugated phenolic compounds that
contributes to increase the free phenolics content after
sprouting. As regards the 1.14-fold increase in insolu-
ble bound phenolic compounds found in sprouted
wheat compared with untreated, this result can be
explained by phenols biosynthesis de novo in the
embryonic axis of sprouted wheat grains. It is known
that bound wheat phenolics associated with the cell
walls may survive upper gastrointestinal tract digestion
and finally reach the colon, where colonic digestion by
intestinal microflora may release the bulk of them.
Thus, our results suggest that the majority of the
wheat phenolic compounds are those which can be
released in the colon to exert their healthful benefits
locally and beyond after absorption. Based on the
literature data, it can be concluded that the content of
different fractions of total phenolics is highly depen-
dent on sprouting conditions. Although our results are
consistent with the studies of Hung et al. (2012), these
authors reported reduction of waxy wheat bound
phenolic compounds after 12 and 24 h of sprouting,
but then a significant increase after 36 and 48 h. Yang
et al. (2001) suggested that wheat grains steeped for
24 h and then sprouted for 7 days would produce the
most desirable sprouts with respect to concentration of
antioxidants, such as phenolics.
The total flavonoid content (TFC) of untreated,
steeped and sprouted wheat is shown in Table 2. In
accordance with the study of Dinelli et al. (2009),
insoluble bound flavonoids were predominant fraction
in all samples. The content of total soluble flavonoids
(free +conjugated fractions) was low being 56.37,
48.33 and 58.48 mg CE kg
1
in untreated, steeped and
sprouted wheat grains, respectively. The obtained
range was slightly lower than the total soluble flavo-
noids contents of 10 durum wheat evaluated by Dinelli
et al. (2009). However, our results indicated that a sig-
nificant increase in the content of soluble free and
insoluble bound flavonoids, respectively by 48.77 and
38.49%, occurred after sprouting for 5 days.
Flavonoids are polyphenolic compounds that occur
ubiquitously in plant tissues in relatively high concen-
trations as sugar conjugates. They occur mostly in O-
glycosidic form with a number of sugars such as glu-
cose, galactose, rhamnose, arabinose, xylose and ruti-
nose. However, major cereal crops such as wheat
predominantly synthesise flavone-C-glycosides, which
are stable to hydrolysis (Brazier-Hicks et al., 2009). It
follows that can be assumed that a low degree of fla-
vone-C-glycosides hydrolysis may be reason for the
low values of conjugated flavonoids in all wheat sam-
ples, given that the applied spectrophotometric assay is
based on the aluminium chloride complex formation
with flavonoid aglycones. Among bioactive com-
pounds, flavonoids are particularly important in the
human diet as antioxidants and antiviral agents. Also,
the epidemiological studies have indicated that their
consumption is associated with a reduced risk of can-
cer and cardiovascular disease (Wang et al., 2012).
Four individual phenolic acids, ferulic, isoferulic,
p-coumaric and caffeic acid, were detected in the wheat
samples. The contents of soluble free, soluble conju-
gated, insoluble bound, as well as the total phenolic
acids calculated as the sum of these fractions, are
given in Table 3. As shown, ferulic acid was the major
compound, followed by isoferulic, p-coumaric and
caffeic acids. In addition, the insoluble bound form
Table 2 Content of free soluble, soluble conjugated and insoluble bound total phenolics (mg GAE kg
1
) and flavonoids (mg CE kg
1
)in
whole untreated, steeped and sprouted wheat grains/flours
Phenolics Phenolic fractions* Untreated wheat Steeped wheat Sprouted wheat
Total phenolics Free soluble 673.56 0.17
c
(20.65)
712.22 2.99
b
(23.58)
862.46 29.89
a
Soluble conjugated 1156.58 36.34
a
(35.47)
818.62 47.51
c
(27.09)
1094.96 6.64
b
(30.55)
Insoluble bound 1430.70 17.99
c
(43.87)
1490.67 23.34
b
(49.33)
1626.89 16.16
a
(45.39)
Total 3260.84 58.13
a
3021.51 67.86
b
3584.31 52.70
a
Total flavonoids Free soluble 18.41 0.06
c
(7.31)
20.69 0.22
b
(8.54)
27.39 0.84
a
(8.32)
Soluble conjugated 37.96 1.05
a
(15.08)
27.64 1.56
c
(11.41)
31.09 0.19
b
(9.45)
Insoluble bound 195.43 2.36
b
(77.61)
193.81 2.99
b
(80.04)
270.66 2.68
a
(82.23)
Total 251.80 4.11
b
242.14 5.41
c
329.14 4.77
a
Means followed by the same letter within the same row are not significantly different, according to Fisher’s last significance difference test
(P=5%).
*Values in parentheses represent the percentage contribution of each phenolic fraction to its total.
©2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2014
Bioactive compounds in sprouted wheat S.
Z ili
cet al.1044
was the major form of the phenolic acids in all wheat
samples. Several previous studies have found increases
in free and total phenolic acids of sprouted wheat
(Yang et al., 2001; Hung et al., 2011). In our study,
the germination process increased the content of feru-
lic acid compared with that in untreated wheat grains
by about 175, 88 and 19% in soluble free, conjugated
and insoluble bound phenolic fractions, respectively.
The accumulation of the ferulic acid was due to the
phenolic biosynthesis and hydrolysis of polyphenolic
compounds bound to cell walls, as reported by Yang
et al. (2001). The increase in the free ferulic acid
content found in sprouted wheat suggests an improved
bioavailability and a higher antioxidant potential.
However, the contents of free isoferulic as well as
bound caffeic acid were decreased by sprouting.
Here, the QUENCHER method with the ABTS
reagent was used to determine the antioxidant capacity
of phenolic fractions (Table 4) and whole untreated,
steeped and sprouted wheat grains (Fig. 1). Total anti-
oxidant capacity of free and bound phenolics corre-
lates very well with their contents in phenolic extracts
of untreated, steeped and sprouted wheat samples
(r
2
=0.72 and r
2
=1, P<0.05, respectively). In our
study, after sprouting, antioxidant capacity of free,
conjugated and bound phenolic was increased by 6.8,
2.3 and 9.2%, respectively, in relation to that in phe-
nolic extracts of untreated wheat grains (Table 4).
These results are in agreement with those reported by
Table 3 Content of free soluble, soluble conjugated and insoluble bound phenolic acids (lgg
1
) in whole untreated, steeped and sprouted
wheat grains/flours
Phenolic acids Phenolic acid fractions* Untreated wheat Steeped wheat Sprouted wheat
Ferulic Free soluble 11.08 0.22
b
(1.82)
2.93 0.05
c
(0.44)
30.5 0.42
a
(4.13)
Soluble conjugated 34.69 0.69
c
(5.71)
38.53 1.01
b
(5.75)
65.22 0.33
a
(8.83)
Insoluble bound 561.45 5.61
c
(92.46)
628.48 0.69
b
(93.87)
670.05 3.35
a
(90.75)
Total 607.22 5.64
c
669.94 6.11
b
738.32 4.43
a
Isoferulic Free soluble 4.92 0.10
a
(2.89)
2.31 0.06
b
(1.49)
1.66 0.01
c
(0.91)
Soluble conjugated 19.84 0.39
b
(11.68)
15.51 0.61
c
(9.98)
34.36 0.58
a
(18.88)
Insoluble bound 145.14 2.87
a
(85.43)
137.64 2.11
b
(88.53)
145.48 2.89
a
(79.94)
Total 169.90 3.01
b
155.47 2.57
c
181.99 3.26
a
p-Coumaric Free soluble n.d. (0.00) 0.31 0.01
(1.36)
n.d. (0.00)
Soluble conjugated 1.50 0.02
b
(7.60)
1.63 0.06
a
(7.13)
n.d. (0.00)
Insoluble bound 18.24 0.30
c
(92.40)
20.91 0.46
b
(91.51)
39.73 0.79
a
(100.0)
Total 19.74 0.39
c
22.85 0.52
b
39.73 0.79
a
Caffeic Free soluble n.d. (0.00) n.d. (0.00) n.d. (0.00)
Soluble conjugated n.d. (0.00) n.d. (0.00) n.d. (0.00)
Insoluble bound 5.53 0.13
b
(100.0)
7.11 0.25
a
(100.0)
4.79 0.39
c
(100.0)
Total 5.53 0.13
b
7.11 0.25
a
4.79 0.39
c
n.d., not detected.
Means followed by the same letter within the same row are not significantly different, according to Fisher’s last significance difference test
(P=5%).
*Values in parentheses represent the percentage contribution of each phenolic acid fraction to its total.
Table 4 ABTS radical-scavenging capacity (mmol Trolox Eq kg
1
)
of phenolic fractions from whole untreated, steeped and sprouted
wheat grains/flours
Fractions
Untreated
wheat
Steeped
wheat
Sprouted
wheat
Free soluble 11.61 0.19
b
11.17 0.03
c
12.40 0.36
a
Soluble conjugated 10.85 0.27
ab
10.67 0.60
b
11.10 0.13
a
Insoluble bound 24.66 0.11
b
24.61 0.86
b
26.93 0.45
a
Total 47.12 0.57
b
46.45 1.53
b
50.43 1.02
a
Means followed by the same letter within the same row are not signifi-
cantly different, according to Fisher’s last significance difference test
(P=5%).
©2013 Institute of Food Science and Technology International Journal of Food Science and Technology 2014
Bioactive compounds in sprouted wheat S.
Z ili
cet al. 1045
Hung et al. (2011). The results of Lee et al. (2005)
showed that flavonoid aglycones had greater antioxi-
dant activities than their glucosides when the potency
was assessed using the LDL oxidation assay. As
mentioned, in our study, the total antioxidant capacity
of whole grains of wheat samples was determined with
the direct measurement procedure that skips all time-
consuming solvent extraction and hydrolysis steps. As
both soluble and insoluble compounds simultaneously
come into contact with the ABTS radical cations, the
direct procedure is able to measure accurately the total
antioxidant capacity of whole wheat grains. Our results
indicate that sprouting significantly improved the anti-
oxidant capacity of whole wheat grains. Although
other antioxidant compounds such as tocopherols, car-
otenoids, proteins, vitamin C, sugars and lignans might
increase the antioxidant capacity of cereals grains
(
Zili
cet al., 2011), the fact is that the antioxidant
capacity of untreated, steeped and sprouted whole
wheat grains when compared with the sum of antioxi-
dant capacity of phenolic fractions in the samples was
considerably lower. So, the total antioxidant capacity
of untreated, steeped and sprouted whole wheat grains
was 19.44, 20.37 and 22.70 mmol Trolox Eq kg
1
,
respectively (Fig. 1), while the sum of antioxidant
capacity of phenolic fractions in the samples was
47.12, 46.45 and 50.43 mmol Trolox Eq kg
1
, respec-
tively (Table 4). Furthermore, our results indicate that
wheat grain phenolic compounds especially in bound
form, as dominant, exert lower antioxidant capacity in
comparison with its hydrolysed and isolated free forms
in analysed extracts. This behaviour could probably be
explained by the chemical nature and reactivity of the
aforementioned compounds present in the wheat grain.
The individual compounds can suffer polymerisation
and other reactions that promote important structural
changes and as a consequence, variations in their anti-
oxidant capacity. For instance, when the degree of
phenolic compounds polymerisation exceeds a critical
value, the reduced availability of its hydroxyl groups
and the increased molecular complexity promotes a
decrease in antioxidant capacity (Pinelo et al., 2004).
Conclusions
The present study shows that sprouting enhances the
nutritional value of whole wheat flours through
biosynthesis of tocopherols, niacin and riboflavin in
the embryonic axis. In addition to other antioxidants,
enhancement in phenolic compounds caused by either
biosynthesis de novo or release of these compounds by
induced enzymatic hydrolysis during sprouting
contributed to the increase in antioxidant capacity of
whole wheat grains/flour. Although whole sprouted
flour is a negative quality parameter with regard to
standard baking processes, it should be taken into con-
sideration for human diets as a food ingredient that
could provide positive health effects. Interests in incor-
porating bioactive ingredients such as phenolic antioxi-
dants and vitamins from sprouts into popular foods
such as bread have grown rapidly, due to the increased
consumer health awareness. Common dietary com-
pounds, such as bread, in the form of functional
foods, can deliver a high concentration of antioxidants
that may play a role in protection from diseases, such
as cancer, cardiovascular diseases and degenerative
diseases (Arts & Hollman, 2005).
Acknowledgments
This work was funded by the Ministry of Education,
Science and Technological Development of the Repub-
lic of Serbia (Grants No. TR-31069) and supported by
COST action FA1005 (Infogest).
References
Abderrahim, F., Huanatico, E., Repo-Carrasco-Valencia, R., Arri-
bas, S.M., Gonzalez, M.C. & Condezo-Hoyos, L. (2012). Effect of
germination on total phenolic compounds, total antioxidant capac-
ity, Maillard reaction products and oxidative stress markers in can-
ihua (Chenopodium pallidicaule). Journal of Cereal Science,56,
410–417.
Alvarez-Jubete, L., Wijngaard, H., Arendt, E.K. & Gallagher, E.
(2010). Polyphenol composition and in vitro antioxidant activity of
amaranth, quinoa buckwheat and wheat as affected by sprouting
and baking. Food Chemistry,119, 770–778.
Antoine, C., Peyron, S., Lullien-Pellerin, V., Abecassis, J. & Rouau,
X. (2004). Wheat bran tissue fractionation using biochemical mark-
ers. Journal of Cereal Science,39, 387–393.
Arts, I.C.W. & Hollman, P.C.H. (2005). Polyphenols and disease
risk in epidemiologic studies. The American Journal of Clinical
Nutrition,81, 317S–325S.
17
18
19
20
21
22
23
24
Untreated Steeped 24 h Sprouted 5 days
TEAC, mmol trolox Eq. kg–1
a
b
c
Figure 1 Total antioxidant capacity of whole untreated, steeped
and sprouted wheat grains/flours obtained by the QUENCHER
method with ABTS. The vertical bars represent the standard devia-
tion of each data point. Bars with different letters are statistically
significantly different, according to Fisher’s last significance differ-
ence test (P=5%).
©2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2014
Bioactive compounds in sprouted wheat S.
Z ili
cet al.1046
Association of Official Analytical Chemists. (1984). Official Methods
of Analysis. Washington, DC: AOAC.
Brazier-Hicks, M., Evans, K.M., Gershater, M.C., Puschmann, H.,
Steel, P.G. & Edwards, R. (2009). The C-glycosylation of flavo-
noids in cereals. Journal of Biological Chemistry,284, 17926–17934.
Combs, G.F. (2008). The Vitamins: Fundamental Aspects in Nutrition
and Health. Ithaca, NY: Elsevier Academic Press.
Dinelli, G., Carretero, A.S., Di Silvestro, R. et al. (2009). Determina-
tion of phenolic compounds in modern and old varieties of durum
wheat using liquid chromatography coupled with time-of-flight
mass spectrometry. Journal of Chromatography A,1216, 7229–7240.
Eberhardt, M.V., Lee, C.Y. & Liu, R.H. (2000). Antioxidant activity
of fresh apples. Nature,405, 903–904.
Hung, P.V., Hatcher, D.W. & Barker, W. (2011). Phenolic acid com-
position of sprouted wheats by ultra-performance liquid chroma-
tography (UPLC) and their antioxidant activities. Food Chemistry,
126, 1896–1901.
Hung, P.V., Maeda, T., Yamamoto, S. & Morita, N. (2012). Effects
of germination on nutritional composition of waxy wheat. Journal
of the Science of Food and Agriculture,92, 667–672.
Hussain, I. & Uddin, M.B. (2012). Optimization effect of germina-
tion on functional properties of wheat flour by response surface
methodology. International Research Journal of Plant Sciences,3,
31–37.
Jensen, M.K., Koh-Banerjee, P., Hu, F.B. et al. (2004). Intakes of
whole grains, bran, and germ and the risk of coronary heart disease
in men. The American Journal of Clinical Nutrition,80, 1492–1499.
Komatsuzaki, N., Tsukahara, K., Toyoshima, H., Suzuki, T., Shi-
mizu, N. & Kimura, T. (2007). Effect of soaking and gaseous treat-
ment on GABA content in germinated brown rice. Journal of Food
Engineering,78, 556–560.
Lampi, A.-M., Nurmi, T., Ollilainen, V. & Piironen, V. (2008). Toc-
opherols and tocotrienols in wheat genotypes in the HEALTH-
GRAIN diversity screen. Journal of Agricultural and Food
Chemistry,56, 9716–9721.
Lee, C.H., Yang, L., Xu, J.Z., Yeung, S.Y.V., Huang, Y. & Chen,
Z.-Y. (2005). Relative antioxidant activity of soybean isoflavones
and their glycosides. Food Chemistry,90, 735–741.
Liyana-Pathirana, C.M. & Shahidi, F. (2006). Importance of insolu-
ble-bound phenolics to antioxidant properties of wheat. Journal of
Agricultural and Food Chemistry,54, 1256–1264.
Moongngarm, A. & Saetung, N. (2010). Comparison of chemical
compositions and bioactive compounds of germinated rough rice
and brown rice. Food Chemistry,122, 782–788.
Nason, A. (1950). The distribution and biosynthesis of niacin in
germinating corn. American Journal of Botany,37, 612–623.
Pinelo, M., Manzocoo, L., Nu~
nez, M.J. & Nicoli, M.C. (2004).
Interaction among phenolics in food fortification. Negative syner-
gism on antioxidant capacity. Journal of Agricultural and Food
Chemistry,52, 1177–1180.
Prodanov, M., Sierra, I. & Vidal-Valverde, C. (1997). Effect of
germination on the thiamine, riboflavin and niacin contents in
legumes. European Food Research and Technology,205,48–52.
Reboul, E., Richelle, M., Perrot, E., Desmoulins-Malezet, C., Pirisi,
V. & Borel, P. (2006). Bioaccessibility of carotenoids and vitamin
E from their main dietary sources. Journal of Agricultural and Food
Chemistry,54, 8749–8755.
S
anchez-Machado, D.I., L
opez-Cervantes, J., L
opez-Hern
andez, J.
& Paseiro-Losada, P. (2004). Simultaneous determination of thia-
mine and riboflavin in edible marine seaweeds by high-performance
liquid chromatography. Journal of Chromatographic Science,42,
117–120.
S
anchez-P
erez, A., Delgado-Zamerreno, M.M., Bustamante-Rangel,
M. & Hern
andez-Mendez, J. (2000). Automated analysis of vita-
min E isomers in vegetable oils by continuous membrane extrac-
tion and liquid chromatography-electrochemical detection. Journal
of Chromatography A,881, 229–241.
Seibold, R.L. (1990). Cereal Grass: What’s in it for you? Lawrence,
KS: Wilderness Community Education Foundation.
Serpen, A., G€
okmen, V., Pellegrini, N. & Fogliano, V. (2008). Direct
measurement of the total antioxidant capacity of cereal products.
Journal of Cereal Science,48, 816–820.
Singleton, V.L., Orthofer, R. & Lamuela-Raventos, R.M. (1999).
Analysis of total phenols and other oxidation substrates and an-
tioxidants by means of Folin-Ciocalteu reagent. Methods in Enzy-
mology,299, 152–178.
Tarr, J.B. & Arditti, J. (1982). Niacin biosynthesis in seedlings of
Zea mays. Plant Physiology,69, 553–556.
Wang, W.-X., Si, H. & Zhang, Z. (2012). Quantitative structure –
activity relationship study on antitumour activity of a series of
flavonoids. Molecular Simulation,38,38–44.
Yang, F., Basu, T.K. & Ooraikul, B. (2001). Studies on germination
conditions and antioxidant contents of wheat grain. International
Journal of Food Sciences and Nutrition,52, 319–330.
Zili
c, S., Had
zi-Ta
skovi
c
Sukalovi
c, V., Dodig, D., Maksimovi
c, V.,
Maksimovi
c, M. & Basi
c, Z. (2011). Antioxidant activity of small
grain cereals caused by phenolics and lipid soluble antioxidants.
Journal of Cereal Science,54, 417–424.
©2013 Institute of Food Science and Technology International Journal of Food Science and Technology 2014
Bioactive compounds in sprouted wheat S.
Z ili
cet al. 1047
