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LWT - Food Science and Technology 63 (2015) 992e1000

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LWT - Food Science and Technology


journal homepage: www.elsevier.com/locate/lwt

Characterization and stability of bioactive compounds from soybean


meal
Fabricio de Oliveira Silva, Daniel Perrone*
rio de Bioqumica Nutricional e de Alimentos, Chemistry Institute, Federal University of Rio de Janeiro, Av. Athos da Silveira Ramos 149, CT, Bloco A,
Laborato
sala 528A, 21941-909, Rio de Janeiro, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 22 December 2014
Received in revised form
12 April 2015
Accepted 13 April 2015
Available online 23 April 2015

In the present study we characterized soybeans and soybean meals, which is the high protein co-product
of soybean oil extraction, produced at industrial or laboratory scale. Additionally, for the purpose of
future metabolic and bioactivity in vivo studies, we conducted, for the rst time, a 6-month stability
study of soybean meal dry extracts. Soybean meal extracts were obtained either by ethanol or a ternary
solvent mixture composed of water, ethanol and ethyl acetate (40:40:20). Soybean meals presented 43%
higher protein content, from 29% to 101% higher bioactive compounds contents and 52% higher antioxidant capacity than soybeans. High moisture thermal procedure employed during soybean meal
processing led to a 13-fold increase in aglycone isoavones contents, which could affect the bioavailability of isoavones in this residue. In addition to a 29-fold higher extraction yield, bioactive compounds
showed higher stability in ternary solvent mixture extracts in comparison to ethanol, independently of
the sample or storage conditions. We concluded that dry soybean meal extracts are suitable materials for
performing long-term in vivo studies, as these extracts were stable when stored at room temperature
unprotected from light for 180 days.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Soybean
Soybean meal
Isoavones
Soyasaponins
Stability

1. Introduction
Differently from Asian countries, soybean-based foods are not
widely consumed in Western countries, where soybean is most
commonly crushed and the oil is extracted and rened into foodgrade oil for frying and cooking. The co-product from this process
is a high-protein meal, known as soybean meal, mainly used as
animal feed. It may also be used for the production of soy protein
isolates and concentrates, which in turn are used as ingredients in
meat and bakery products, beverages, soups, infant formulas and
other food products (Aguiar et al. 2012), thus aggregating economic
value to this residue.
In addition to its content of high-quality protein, soybean meal
contains high contents of bioactive compounds, mainly isoavones
(Kao & Chen, 2006; Kao, Chien, & Chen, 2008) and soyasaponins
(Wang, Wang, Lu, Kao, & Chen, 2009). These bioactive compounds
from soybeans and its products are associated with lower prevalence of a number of chronic diseases, including cardiovascular

* Corresponding author. Tel.: 55 21 3938 8213.


E-mail addresses: silvafo@live.com (F.O. Silva),
(D. Perrone).
http://dx.doi.org/10.1016/j.lwt.2015.04.032
0023-6438/ 2015 Elsevier Ltd. All rights reserved.

danielperrone@iq.ufrj.br

diseases and certain types of cancer (Georgetti, Casagrande, Souza,


Oliveira, & Fonseca,, 2008).
Soybean meal may be used as a source of various bioactive
compounds for metabolic and bioactivity studies. However, papers
have focused on soybean meal as a source of isoavones (Wang
et al., 2009), while soyasaponins are usually overlooked, even
though this class of bioactive compounds may occur in higher
amounts than isoavones (Kao & Chen, 2006). Bioactivity studies
investigating the chronic effects of isoavones and soyasaponins
isolated from soybean meal in animals and humans should
consider the stability of these compounds during the whole intervention period, since it is known that isoavones and soyasaponins
contents and/or prole may be affected by exposure to high temperatures, oxygen and UV-light (Georgetti et al., 2008; Rostagno,
Palma, & Barroso, 2005). However, to the best of our knowledge,
the stability of bioactive compounds in soybean meal extracts has
not been yet investigated.
Therefore, the aim of this work was to chemically characterize
soybean meal samples, with an emphasis on isoavones and
soyasaponins, in order to promote its use as a food ingredient and
thus widen high added-value applications of this residue. Additionally, we aimed to investigate, for the rst time, the stability of

F.O. Silva, D. Perrone / LWT - Food Science and Technology 63 (2015) 992e1000

isoavones and soyasaponins in dry extracts obtained from soybean meal under different storage conditions, for future use in
long-term bioactivity studies.
2. Materials and methods
2.1. Standards and chemicals
Folin-Ciocalteau reagent, gallic acid, 2,20 -azino-bis-(2ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),
2,4,6-tris(2-pyridyl)-S-triazine (TPTZ), potassium persulfate, uorescein, 2,20 -azobis(2-methylpropionamidine) dihydrochloride
(AAPH)
and
()-6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid (Trolox) were purchased from SigmaeAldrich (St.
Louis, MO). Sodium carbonate, vanillin and aluminum chloride
were purchased from Spectrum Chemical Manufacturing Corp.
(Gardena, CA). Iron (II) sulfate was purchased from Merck (Darmstadt, Germany). Daidzin, glycitin, genistin, daidzein, glycitein,
genistein, soyasapogenol B and soyasaponins B-I, B-II and B-III
standards were purchased from Apin Chemicals (Abingdon, UK).
Saponin standard from soybean was purchased from Wako Pure
Chemical Industries (Osaka, JP). All solvents were HPLC grade from
Tedia (Faireld, OH). Milli-Q water (Millipore, Bedford, MA) was
used throughout the experiments.
2.2. Samples
Two sets of samples comprised of soybeans and soybean meal
were investigated. In both sets, samples were from the same batch.
One set was donated by a soybean oil crush industry, while the
other was produced in our laboratory using commercial soybeans
purchased in local markets.
Lab-scale soybean meal production was analog to industrial
processing, with adaptations. Seeds were cracked and the hulls
separated using an air stream. Dehulled soybeans were wet and
incubated at 60  C for 1 h in an oven. The sample was occasionally
water-sprayed to keep the beans wet. The seeds were then ground
in a domestic processor and dried for 5 h at 60  C in an oven. After
drying, the sample was additionally ground in an analytical mill
(IKA A11 basic S1) in order to maximize oil extraction. 20 g of
sample was placed in a cellulose cartridge (33  94 mm) and the
extraction procedure was conducted in a Soxhlet extractor for 7 h
using n-hexane. The meal was left in a fume hood until dry and
stored at room temperature.
2.3. Proximate composition
Moisture, ash, proteins and lipids were determined by the ofcial methods of the AOAC (2000). Total carbohydrates were determined by difference. Each sample was analyzed in triplicate.
2.4. Bioactive compounds analysis
Soybean and soybean meal samples were extracted with a
ternary solvent mixture (water:ethanol:ethyl acetate e 40:40:20).
Briey, 2 g of sample were extracted with 10 mL of the ternary
mixture in a vortex for 2 min. After centrifugation for 10 min at
1690  g, the supernatant was collected and the residue was reextracted twice as described above. The extraction procedure for
each sample was conducted in duplicate. These extracts were
analyzed for the determination of phenolic compounds, avonoids
and saponins by spectrophotometric assays, and isoavones and
soyasaponins by LC-DAD-MS.

993

2.4.1. Spectrophotometric assays


Total phenolic content was determined by the Folin-Ciocalteau
reagent assay, as described by Singleton, Orthofer, and Lamuelas (1999). Quantication was performed using a gallic acid
Ravento
calibration curve. Results were expressed as mg of gallic acid
equivalents per g on a dry weight basis (dwb) (mg GAE/g).
Total avonoid content was determined by the spectrophotometric assay described by Taie, El-Mergawi, and Radwan (2008).
Quantication was performed using a genistein calibration curve.
Results were expressed as mg of genistein equivalents per g dwb
(mg GE/g).
Total saponin content was determined by the vanillin-sulfuric
acid assay, as described by Shiau et al. (2009). Quantication was
performed using a calibration curve prepared with a commercial
mixture of saponins obtained from soybeans. Results were
expressed as mg of total saponins per g dwb.
The antioxidant capacity of the extracts was determined by
FRAP (Ferric Reducing Ability of Plasma) and TEAC (Trolox Equivalent Antioxidant Capacity) assays. The FRAP assay was performed
according to Benzie and Strain (1996) with slight modications. The
TEAC assay was performed according to Re, Pellegrini, and
Proteggente (1999) with slight modications.
Each sample was independently analyzed by each assay in
triplicate.
2.4.2. Isoavones and soyasaponins
Isoavones and soyasaponins were simultaneously analyzed by
LC-DAD-MS as described by Fonseca, Villar, Donangelo, and Perrone
(2014). The LC system (Shimadzu, Kyoto, Japan) comprised an LC10ADvp quaternary pump, a CTO-10ASvp column oven, an 8125
manual injector (Rheodyne) with a 20 mL loop and an SPD-M10Avp
diode array detector (DAD). This LC system was coupled to an
LCeMS 2010 mass spectrometer (MS) (Shimadzu, Kyoto, Japan)
equipped with an electrospray ion source. Chromatographic separation was achieved using a Kromasil C18 column (150  2.1 mm,
5 mm, 100 ) maintained at a constant temperature of 40  C. The LC
two-phase mobile system consisted of a gradient of water (eluent
A) and acetonitrile (eluent B), both added with 0.3 g/100 mL formic
acid, with a constant ow rate of 0.3 mL/min. Prior to injection, the
column was equilibrated with 15% B. After injection, this proportion
was modied to 23% B in 1 min, kept constant until 23 min and
increased to 50% B until the end of the 35 min run. Twenty min
intervals were used to re-equilibrate the column with 15% B. Extracts were ltered through a 0.45 mm PTFE lter unit.
Isoavones and soyasaponins were monitored by DAD between
190 and 370 nm and by MS using positive ionization, with a
nebulizer gas (N2) ow of 3.0 L/min, operated in the single ion
monitoring (SIM) mode. Identication of aglycone and glycosylated
isoavones was performed by comparison with retention time and
pseudomolecular ion of the respective standard. Identication of
soyasaponins was performed by comparison with retention time
and the most abundant ion of the respective standard ([MH] for
soyasaponin B-I and [M-sugar-H2O H] for both soyasaponins BII and B-III). Identication of compounds for which there were no
commercial standards available (malonylglucosilated and acetylglucosilated isoavones) was performed by the pseudomolecular
ion in the MS (Supplementary Figure 1AeE).
Quantication was performed by external standardization. Isoavones were quantied by their DAD peak areas at 250 nm. The
contents of malonylglucosilated and acetylglucosilated isoavones
were determined from the calibration curve of the corresponding
b-glucosylated isoavone. Soyasaponins (B-I, B-II and B-III) were
quantied by their DAD peak areas at 195 nm. Soyasaponins B-II
and B-III were quantied together, as it was not possible to chromatographically separate these compounds. Data were acquired by

994

F.O. Silva, D. Perrone / LWT - Food Science and Technology 63 (2015) 992e1000

LCMS solution software (Shimadzu Corp., version 2.00, 2000). Results were expressed as mg of compound per g dwb. Each sample
was analyzed in duplicate.
This chromatographic method was previously validated
(Fonseca et al., 2014) and was shown to be linear (R2 > 0.994 for all
analytes), reproducible (inter-day precision of 10.9%) and presented
satisfactory limits of quantication for both isoavones (<151.2 ng/
mL) and soyasaponins (<2178.6 ng/mL).
2.5. Stability of bioactive compounds from soybean meal extracts
For the stability test both meals were subjected to two different
extractions using as solvent either a ternary mixture (water:ethanol:ethyl acetate e 40:40:20) or ethanol. The former solvent
was selected due to its high efciency in extracting soy bioactive
compounds (unpublished results), while the latter is usually
employed to extract isoavones from soy products. 20 g of soybean
meal samples were extracted with 100 mL of solvent in an orbital
shaker (IKA KS 4000i control) at 500 rpm. Extraction time was
30 min and 60 min for the ternary solvent mixture and ethanol,
respectively. After centrifugation (1690  g, 15 min), the supernatant was collected and the residue was re-extracted twice as
described above. The extracts were concentrated in a rotary evaporator (Bchi V-700) until the solvent was removed. The concentrated extracts were freeze dried (Labconco Freezone 2.5) for 24 h
and stored in three different conditions: freezer (20  C); room
temperature (protected or unprotected from light). Total phenolic
compounds, total avonoids, total saponins, antioxidant capacity
(FRAP and TEAC) and isoavones and soyasaponins by LC-DAD-MS
were analyzed as described above after 15, 30, 60, 90, 120 and 180
days of storage after complete dissolution of the dry extract in
water and ltration through a 0.45 mm PTFE lter unit.
2.6. Statistical analysis
Data are expressed as mean standard deviation. Analysis of
variance (one-way ANOVA) followed by Tukey's post-test was used
for comparing means. Statistical analyses were performed using
GraphPad Prism software for Windows (version 5.04). Differences
were considered signicant when p < 0.05.
3. Results and discussion
3.1. Proximate composition
Experimental and industrial soybean samples showed equivalent proximate composition (Fig. 1A), on average 23.2, 33.7, 5.4 and
37.8 g/100 g dwb for lipids, proteins, ash and carbohydrate,
respectively, which are in accordance with typical values for soybeans (Joshi, Liu & Sathe, 2015). Soybean meal samples also showed
equivalent proximate composition, indicating that the conditions
used in our laboratory for soybean oil extraction were similar to
those employed in the industry. Soybean meal samples presented
on average 1.8, 48.2, 7.0 and 43.0 g/100 g dwb for lipids, proteins,
ash and carbohydrate, respectively, which are in accordance with
previous reports (Grieshop et al., 2003).
3.2. Total phenolics, avonoids, saponins and antioxidant activity
Total phenolics (TP), total avonoids (TF) and total saponins (TS)
contents were 57%, 29% and 32%, higher, respectively, in soybean
meal than soybeans samples (Fig. 1B). These higher contents are
due to the extraction of lipids, which concentrates the functional
components naturally present in the protein fraction.

The contents of bioactive compounds analyzed by spectrophotometric assays in both soybean and soybean meal samples were
equivalent, with the exception of TP. While industrial soybeans
showed a 12% higher TP content than commercial soybeans, the
industrial meal showed a 19% lower TP content than that obtained
in laboratory (Fig. 1B). This behavior may be related to differences
in stability of phenolic compounds present in soybean samples or
to other compounds that may interfere with the Folin-Ciocalteau
reagent assay. Differences between soybean samples might result
from minor phenolics, such as phenolic acids, mainly chlorogenic
and trans-cinnamic acids (Xu & Chang, 2008) that are also present.
No statistical difference was found between soybean samples
(on average 0.88 mg/g) and between soybean meal samples (on
average 1.11 mg/g) (Fig. 1B). Similar values were reported by
Malen
ci
c, Popovi
c, and Miladinovi
c (2007), ranging from 0.32 to
0.61 mg of rutin equivalents/g for different soybean cultivars. In
contrast, Taie et al. reported a much higher TF content (6.81 mg of
quercetin equivalents per g) for inorganic fertilized soybeans. These
differences are probably due to the specic absorptivity of chromophores generated by reacting these standards with AlCl3.
The average TS content for soybean samples (27.4 mg/g) (Fig. 1B)
were higher than those reported by Xu and Chang (2008) (20.7 mg/
g), but similar to those reported by the same group in a later study
(Xu & Chang, 2011) (25.0 mg/g). It is worth noting that although the
vanillin-sulfuric acid method is very sensitive, the reaction is not
specic for saponins and colored products may be formed from
phytosterols and other compounds like avonoids (Shiau et al.,
2009). Nevertheless, this method is widely used for screening of
saponins in food samples.
The antioxidant capacity (AC) measured by both FRAP and TEAC
assays were equivalent between soybean samples and between
soybean meal samples (Fig. 1C). Soybean samples showed, on
average, FRAP and TEAC values of 7.2 mmol Fe2/g and 11.5 mmol
Trolox/g, respectively. Soybean meal samples presented 42% and
63% higher FRAP and TEAC values, respectively, than soybean
samples, on average 10.3 mmol Fe2/g and 18.6 mmol Trolox/g. The
FRAP values found in the present study for soybean samples were
similar to those reported by Xu and Chang (2008) for 28 different
soybean cultivars grown in the North Dakota-Minesota region,
ia (2011)
ranging from 8.5 to 13.4 mmol Fe2/g. Bolanho and Bele
reported a higher TEAC value (7.4 mmol Trolox/g) for a soybean
sample than those found in the present study. In contrast, lower
FRAP and TEAC values were reported in the same study (Bolanho &
ia, 2011) for a commercial defatted soybean our sample
Bele
(9.5 mmol Fe2/g and 9.2 mmol Trolox/g, respectively) in comparison
to those found in the present study for the soybean meal samples.
3.3. Isoavones and soyasaponins by LC-DAD-MS
In the present study up to 12 isoavones were found in soybean
and soybean meal samples (Table 1). Soybean meal samples
showed a 49% higher total isoavones contents (on average
1.72 mg/g dwb) than soybean samples (on average 1.15 mg/g dwb).
There was no difference between experimental and industrial
soybean and soybean meal samples. However, industrial processing
led to a higher increase (84%) in total isoavones contents than
laboratory processing (22%). Isoavones contents in soybean samples in the present study are in accordance with literature data,
which reports a wide range of concentration, from 0.6 to 3.3 mg/g
(Sakthivelu et al., 2008; Wang & Murphy, 1994). For soybean meals,
the contents found in our samples were similar to those reported by
Kao and Chen (2002), which found up to 2.1 mg/g of total isoavones in a soybean our using different solvents and supersonic
or shaking extraction. Wu and Muir (2010) reported higher total
isoavones content in a commercial soybean our sample (3.0 mg/

F.O. Silva, D. Perrone / LWT - Food Science and Technology 63 (2015) 992e1000

995

Fig. 1. Proximate composition (A), contents of total phenolics (TP), avonoids (TF) and saponins (TS) (B), and antioxidant capacity (C) in commercial soybean ( ), experimental meal
( ), industry soybean ( ) and industry meal ( ) samples. Values expressed as mean standard deviation (n 3); different superscript letters indicates signicant difference
(ANOVA, Tukey's post-test; p < 0.05); GAE gallic acid equivalents; GE genistein equivalents; TE Trolox equivalents.

Table 1
Isoavones and soyasaponins contents (mg/g dwb) in soybean and soybean meal experimental and industrial samples.a
Compound

MS identication

Experimental

m/z

Ion

Soybean

Soybean meal

[MH]
[MH]
[MH]

0.34 0.03a
0.07 0.01b
0.46 0.03a
0.87 0.08a

0.06
0.03
0.09
0.17

[MH]
[MH]
[MH]

0.28 0.02a
NDb
0.03 0.00b
0.31 0.03a

0.27 0.00a
ND
0.03 0.00b
0.30 0.00a

0.27 0.01a
ND
0.05 0.00a
0.32 0.01a

0.24 0.01a
ND
0.04 0.00a
0.28 0.02a

[MH]
[MH]
[MH]

0.03 0.00b
ND
0.03 0.00b
0.06 0.01b

0.03 0.00b
ND
0.01 0.00b
0.03 0.00b

0.02 0.00b
ND
0.00 0.00b
0.03 0.00b

0.05
0.03
0.21
0.28

[MH]
[MH]
[MH]

0.04 0.00b
0.01 0.00d
0.02 0.00c
0.07 0.01c
1.31 0.12b

0.36
0.36
0.37
1.09
1.60

0.00b
0.00c
0.00c
0.00c
0.03b

0.09
0.09
0.08
0.26
1.84

0.01c
0.00b
0.00b
0.01b
0.08a

[MH]
[M-sugar-H2O H]

1.13 0.06b
0.46 0.03d
1.60 0.03c

2.51 0.11a
0.78 0.00b
3.29 0.11b

Isoavones
b-glucosylated forms
Daidzin
417
Glycitin
447
Genistin
433
Sub total
Malonyl glucosylated forms
Daidzin
519
Glycitin
549
Genistin
535
Sub total
Acetyl glucosylated forms
Daidzin
459
Glycitin
489
Genistin
475
Sub total
Aglycones
Daidzein
255
Glycitein
285
Genistein
271
Sub total
Total
Soyasaponins
B-I
944
B-II BIII
423
Total
a
b

Industrial

0.00c
0.00c
0.00c
0.01c

0.01a
0.01a
0.02a
0.03a
0.04a

Soybean

0.17
0.08
0.26
0.51

0.05
0.05
0.04
0.14
1.00

0.01b
0.00b
0.00b
0.01b

1.42 0.10b
0.55 0.01c
1.96 0.11c

Soybean meal

0.34
0.15
0.53
1.02

0.01a
0.01a
0.02a
0.04a

0.00a
0.00a
0.02a
0.02a

2.95 0.16a
0.91 0.00a
3.86 0.16a

Values expressed as mean standard deviation (n 2); means in the same row with different online letters are signicantly different (ANOVA, Tukey's post-test; p < 0.05).
Not detected.

996

F.O. Silva, D. Perrone / LWT - Food Science and Technology 63 (2015) 992e1000

Fig. 2. Aglycone ( ), acetylglucosilated ( ), malonylglucosilated ( ), and b-glucosilated ( ) isoavones relative content from soybean and soybean meal experimental
and industrial samples.

g) using a ternary mixture of methanol, acetonitrile and water as


solvent. Kao et al. (2008) also reported higher total isoavones
contents (up to 3.3 mg/g) than those found in the present study for
a soybean meal sample subjected to supercritical carbon dioxide
extraction at different pressures and temperatures.
Despite the equivalent total isoavones contents between
experimental and industrial soybean meals, LC-DAD-MS analysis
revealed different proles between these samples (Fig. 2). Industrial soybean meal sample presented a b-glucoside and aglycone
isoavones prole similar to that of the corresponding soybean,
representing 53% and 14%, on average, of total isoavones,
respectively. In contrast, experimental soybean meal sample presented a malonyl and acetylglucoside isoavones prole similar to
that of the corresponding soybean, representing 20% and 3%, on
average, of total isoavones, respectively. While industrial processing of soybeans for oil production resulted in a 48% decrease in

the relative content of malonylglucoside isoavones and a 5.3-fold


increase in the relative content of acetylglucoside forms, the
experimental conditions used in our laboratory led to a drastic
reduction (84%) of b-glucoside isoavones relative content and a
12.9-fold increase in the relative content of the aglycone forms. It
has already been reported that isoavones prole changes greatly
depends on thermal processes employed during food preparation,
including cooking, baking, oven-drying and roasting (Georgetti
et al., 2008). Low moisture thermal processing was reported to
cause the conversion of malonyl to acetylglucoside isoavones.
Therefore, soybean meal roasting usually applied after industrial oil
extraction (Pananun, Montalbo-Lomboy, Noomhorm, Grewell, &
Lamsal, 2012) probably explains the isoavone prole change
observed in the industrial soybean meal sample. In contrast, high
moisture thermal processing leads to hydrolysis of b-glucosides
isoavones to aglycones, probably due to an increase in b-glycosidase activity (Lima & Ida, 2014). This phenomenon may explain the
observed shift in isoavone prole during the production of soybean meal under laboratory conditions, as soybeans were
constantly water-sprayed during drying prior to oil extraction.
The experimental soybean meal sample, which is the richest in
aglycone forms (1.09 mg/g) (Table 1), might be a more bioavailable
source of isoavones because these forms are absorbed faster and
in greater amounts than glucosides in human gastrointestinal tract
(Aguiar et al., 2012; Okabe, Shimazu, & Tanimoto, 2011). Even
though industrial processing increased total isoavones contents to
a higher extent, high moisture thermal processing, such as the
procedure employed in our laboratory, as well as fermentation
processes of soybean meal (Okabe et al., 2011), could be used to
improve functional value of this co-product, broadening its use in
food products with high isoavones bioavailability.
In the present study, we investigated the contents of three group
B soyasaponins and their respective aglycone, sapogenol B.
Although the industrial and experimental soybean samples showed
equivalent soyasaponins contents (on average of 1.78 mg/g), the
industrial soybean meal sample presented a 17% higher content of
these compounds than the experimental one (Table 1). The major

Fig. 3. Stability of bioactive compounds and antioxidant capacity (FRAP and TEAC) of dry experimental soybean meal extract obtained with ethanol ( ) or ternary mixture ( ) and
of dry industrial soybean meal extract obtained with ethanol ( ) or ternary mixture ( ) during storage at 20  C (freezer) (
) and room temperature (protected [
] and
unprotected from light [
]) for 180 days.

F.O. Silva, D. Perrone / LWT - Food Science and Technology 63 (2015) 992e1000

997

Fig. 4. Stability of isoavones and soyasaponins of dry experimental soybean meal extract obtained with ethanol ( ) or ternary mixture ( ) and of dry industrial soybean meal
extract obtained with ethanol ( ) or ternary mixture ( ) during storage at 20  C (freezer) (
) and room temperature (protected [
] and unprotected from light [
])
for 180 days.

soyasaponin in all samples was soyasaponin B-I, which corresponded to 72% of total soyasaponins in both soybean samples and
to 76% in both soybean meal samples. Soyasaponins B-II and B-III
accounted together for the remainder of soyasaponins content.
Soyasapogenol B was not found in any sample.
Berhow, Kong, Vermillion, and Duval (2006) reported total
soyasaponin contents ranging from 4.73 to 5.75 mg/g for soybeans
and from 5.86 to 6.58 mg/g for defatted soy ours. As observed in
the present study, the major soyasaponin in all samples analyzed by
Berhow et al. (2006) was soyasaponin I. Although the contents
reported by Berhow et al. (2006) were up to 2.2-fold higher than
those found in the present study, it should be noted that those
authors analyzed 13 soyasaponins, including soyasaponin bg,
which was the second most abundant soyasaponin in their

samples. Considering only the contents of the soyasaponins


analyzed in the present study (B-I, B-II and B-III), the contents
found in our samples were up to 79% higher than those reported by
Berhow et al. (2006). Unfortunately, we could not analyze soyasaponin bg in our samples due to the lack of a commercial standard.
3.4. Bioactive compounds stability
Stability of bioactive compounds and antioxidant capacity
evaluated by spectrophotometric assays during storage of extracts
at 20  C (freezer) and at room temperature (protected and unprotected from light) are depicted in Fig. 3. Stability of isoavones
and soyasaponins analyzed by LC-DAD-MS at the same conditions
are depicted in Fig. 4. Ternary mixture extraction yield (on average

998
Table 2
Contents of bioactive compounds and antioxidant capacity at t 0 and t 180 days in dry soybean meal (experimental and industrial) extracts (obtained with ethanol or ternary solvent mixture) during storage at 20  C (freezer)
and room temperature (protected and unprotected from light).a
Experimental

Industrial

Ethanol

Ternary

Ethanol

t 0 t 180 days

t 0 t 180 days

t 0 t 180 days

RTlight
11.1 10.3
Total phenolicsb
(mg GAE/g)
13.6 10.3* (24%)
Total avonoidsb
(mg GE/g)
b
71.5 52.5* (30%)
Total saponins
(mg/g)
74.7 35.1* (53%)
FRAPb
(mmol Fe2/g)
103.1 39.8* (61%)
TEACb
(mmol Trolox/g)
c
10.4 8.6
Total isoavones
(mg/g)
c
b-glucosides
1.0 1.2* (20%)
(mg/g)
0.6 0.4* (33%)
Malonylglucosidesc
(mg/g)
0.1 0.09
Acetylglucosidesc
(mg/g)
c
8.6 6.9
Aglycones (mg/g)
Soyasaponinsc
12.7 7.5* (41%)
(mg/g)

RTdark

Freezer

11.1

10.3

10.4* (24%) 8.6* (36%)


50.9* (32%) 71.4
36.2* (52%) 32.8* (56%)

RTlight

RTdark

Freezer

12.4 15.9* (28%)

17.7* (43%)

13.1

7.3 8.0* (11%)

9.4* (30%)

6.7

225.0 190.9* (15%) 230.8


71.4 92.4* (30%)

RTlight
4.8 4.4
11.0 5.8* (47%)

172.2* (23%) 43.9 29.6

Ternary
t 0 t 180 days
RTdark

Freezer

RTdark

Freezer

4.7

4.9

10.5 13.2* (26%)

12.9* (23%)

12.0* (15%)

7.5* (32%)

7.3* (34%)

10.0 11.0* (10%)

10.8* (8%)

10.1

27.3* (38%) 36.7

RTlight

253.1 234.6

216.4* (14%) 200.4* (21%)

104.8* (47%) 78.5* (10%)

48.0 34.1* (29%) 42.4* (10%) 30.6* (36%)

49.4* (52%) 62.3* (40%) 105.0 49.8* (53%)

95.6

86.9

71.7 24.4* (66%) 11.3* (84%) 39.4* (45%) 109.5 107.5

11.0

10.0

5.3 7.3* (38%)

9.2* (76%)

6.1

6.2 5.7

5.9

6.3

5.6 8.9* (59%)

8.7* (57%)

8.1* (46%)

1.3* (33%)

1.1

0.7 1.1* (49%)

1.5* (106%)

0.9* (29%)

3.9 3.9

4.1

4.1

3.6 6.1* (67%)

6.0* (64%)

5.5* (53%)

0.5* (21%)

0.6

1.1 1.3* (13%)

1.6* (46%)

1.5* (30%)

0.3 0.1* (50%)

0.2* (24%)

0.3

0.6 0.9* (41%)

0.8* (33%)

0.8* (39%)

0.1

0.1

0.1 0.1

0.2* (90%)

0.1

0.3 0.3

0.3

0.3

0.4 0.6* (43%)

0.6* (43%)

0.5* (33%)

9.1
7.9* (38%)

8.1
7.6* (41%)

5.8* (78%)
18.3* (39%)

4.8* (47%)
15.5

1.6 1.3
9.0 7.1

1.3
6.9

1.5
6.1* (32%)

0.8 1.3* (48%)


15.3 20.4* (33%)

1.3* (48%)
22.0* (44%)

1.2* (47%)
18.9* (24%)

3.3 4.7* (44%)


13.2 13.9

91.6 113.0* (23%) 111.9* (22%) 99.9* (9%)


78.03* (29%) 117.0

a
Means at t 180 days with a superscript asterisk are signicantly different from their corresponding sample at t 0 (ANOVA, Tukey's post-test; p < 0.05). Relative change in relation to t 0 is shown in parenthesis.
GAE gallic acid equivalents; GE genistein equivalents.
b
Spectrophotometric analysis.
c
LC-DAD-MS analysis.

F.O. Silva, D. Perrone / LWT - Food Science and Technology 63 (2015) 992e1000

Component

F.O. Silva, D. Perrone / LWT - Food Science and Technology 63 (2015) 992e1000

21.0%) was much higher than that obtained with ethanol (on
average 1.4%). Moreover, extraction solvent was the parameter with
the most prominent effect on bioactive compounds and antioxidant
capacity stability. In general, ethanolic extracts showed lower stability than those obtained with the ternary mixture, independently
of the sample (experimental or industrial) or the storage
conditions.
While TP, TF and FRAP increased after 180 days of storage (8%e
47%) in extracts obtained with the ternary mixture, corresponding
ethanolic extracts presented a constant behavior for TP and a
decrease in TF and FRAP (10%e56%) (Table 2). Although TS and TEAC
were unstable for most samples and conditions, a higher decrease
(from 30% to 84%) was observed for ethanolic extracts in comparison to those obtained with the ternary mixture (from 14% to 53%)
(Table 2).
Total isoavones behaved similarly to TP, showing stability in
ethanolic extracts regardless of the sample and storage conditions.
Ethanolic extracts obtained from the experimental soybean meal
sample stored at room temperature showed an average decrease of
27% in malonylglucoside isoavones accompanied by an identical
average increase in b-glucoside isoavones. Ethanolic extracts obtained from the industrial soybean meal sample stored in the same
conditions also showed a decrease in malonylglucoside isoavones
(on average 37%) (Table 2), but the corresponding increase in bglucoside forms was only observed until 120 days and not at the
end of the storage period. Similarly, Rostagno et al. (2005) reported
that during short term storage (7 days) of dry extracts obtained
with ethanol:water (50:50) malonyl forms were the most susceptible to degradation, yielding b-glucoside forms. In our study, isoavones contents and prole of all ethanolic extracts stored
at 20  C remained unchanged during the whole storage period
(Table 2), corroborating with Rostagno et al. (2005). These authors
concluded that temperature was the single most important factor
to affect isoavones stability and suggested that extracts should be
kept at temperatures lower than 10  C and protected from light to
avoid degradation. Nevertheless, Xu, Wu, and Godber (2002) reported that b-glucoside forms could resist to heat-induced
decomposition at temperatures near 100  C in the absence of
other factors, such as enzymes and only became unstable when
heating temperatures were increased above 135  C.
Surprisingly, total isoavones contents increased (38%e76%) in
extracts obtained with the ternary mixture after 180 days of storage. This increase, however, did not signicantly modify the isoavone prole of these extracts, as the concentration of all
isoavones forms increased similarly (from 13% to 90%) (Table 2). It
is known that isoavones may establish different types of interactions with soy proteins, especially glycinin and b-conglycinin,
because of their diverse polarity and hydrophobicity as well as their
~o
 n, 2010).
ability to form hydrogen bonds (Speroni, Milesi, & An
During long-term storage, structure of soy proteins such as glycinin
might change (Saio, Kobayakawa, & Kito, 1982), due to a decrease in
free sulfhydryl groups and an increase in disulfhydryl groups.
Moreover, the hydrogen bondings within the glycinin molecule
might decrease because of an increase in b-helix structure and a
decrease in the b-sheet structure (Hou & Chang, 2004). These
structural changes might have decreased the interaction of isoavones with soy proteins during storage, therefore increasing
their extractability. In addition, the aforementioned stability of
isoavones in ethanolic extracts (Table 2) strengthens this hypothesis and might be explained by the solubility of soy proteins in
this solvent, which is lower than in the ternary mixture, which
~ o, Prabhakaran, & Scholtz, 2004).
contains 40% water (Pace, Trevin
In fact, protein contents in the extracts obtained with the ternary
mixture (7.62 g/100 g) were 1.8 times higher than those of ethanolic
extracts (4.17 g/100 g).

999

In general, soyasaponins contents increased (24%e39%) in extracts obtained with the ternary mixture, whereas decreased (32%e
41%) in ethanolic extracts. Although these compounds are generally
considered stable at room temperature for short time periods (up to
60 min) (Heng et al., 2006), saponins from Aspargus racemosus dry
powder stored for 12 months at room temperature were highly
degraded (up to 50%) (Madan, Yadav, & Tyagi, 2005). The observed
increase in soyasaponins B-I, B-II and B-III contents in extracts
obtained with the ternary mixture may be related to the degradation of the corresponding DDMP conjugated soyasaponins, bg, ba
and gg, respectively. The conversion of soyasaponin bg to soyasaponin B-I during storage and extraction has been reported in peas
(Daveby, man, Betz, & Musser, 1998). Additionally, the hypothesis
raised to explain the increase of isoavones in these extracts could
also explain the increase in soyasaponins, as these compounds also
form very tight linkages to soy proteins (Potter, Jimenez-Flores,
Pollack, Lone, & Berber-Jimenez, 1993).
Although the ternary mixture led to higher extraction yields and
longer stability of bioactive compounds, it may not be practical,
from an industrial point of view, to produce extracts using this
solvent, due to higher energy consumption for solvent recycling.
Nevertheless, the use of the ternary mixture is feasible when
considering the small-scale production of extracts intended for
biological studies.
4. Conclusion
Our results suggest that soybean meals would be more interesting as an ingredient than soybeans themselves for the development of functional foods, since the former showed higher
protein and bioactive compounds contents as well as higher antioxidant capacity than the latter. Moreover, high moisture thermal
processing of soybeans is a procedure that could be employed to
improve the functional value of the obtained co-product as it seems
to yield meals with potentially higher isoavones bioavailability.
Soybean meal extracts for long-term bioactivity studies should
be preferably obtained using a ternary mixture composed of water,
ethanol and ethyl acetate (40:40:20) as this solvent led to higher
extraction yields and stability of bioactive compounds. For this
purpose, these extracts may be stored at room temperature unprotected from light for at least 180 days.
Acknowledgments
The authors gratefully thanks CAPES (Brazil) for nancial support and Mr. Silva's scholarship and UFRJ, CNPq (461464/2014-4)
and FAPERJ (Brazil) (E-26/110.420/2012 and E-26/102.974/2012) for
nancial support.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.lwt.2015.04.032.
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