FRIN-05661; No of Pages 7

Food Research International xxx (2015) xxx–xxx

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Food Research International

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The ultrasound-treated soybean seeds improve edibility and nutritional

quality of soybean sprouts
Hui Yang a,b,c, Jinyan Gao b, Anshu Yang c, Hongbing Chen a,c,⁎
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
b
School of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047, China
c
Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China

a r t i c l e i n f o a b s t r a c t

Article history:
Received 11 November 2014
Received in revised form 11 January 2015
Accepted 11 January 2015
Available online xxxx
Chemical compounds studied in this article:
Gamma-aminobutyric acid (PubChem CID: 119)
Isoflavone (PubChem CID: 72304)
Daidzin (PubChem CID: 107971)
Genistein (PubChem CID: 5280961)
Daidzein (PubChem CID: 5281708)
Genstin (PubChem CID: 5281377)
Limited data are available concerning the physical and nutrient properties of soybean sprouts from the
ultrasound-treated seeds. In this study, soybean seeds were treated by ultrasound at different power levels
(0 W to 300 W), then germinated for 5 d in darkness. Morphological changes, protein patterns, amino acid con-
tents, gamma-aminobutyric acid (GABA) content, IgE-binding, lipoxygenase isozyme activity, trypsin inhibitor,
and isoflavone content of soybean sprouts were quantified. Results showed that ultrasound treatment increased
germination rate, sprout length, and GABA content of soybean sprouts. No significant change was observed in
protein patterns. The IgE-binding, lipoxygenase isozyme activity, and trypsin inhibitor content of soybean
sprouts showed a power-dependent decrease after the seeds were exposed to ultrasound. As for the isoflavone
content, daidzin and genstin contents markedly decreased, whereas, daidzein and genistein contents increased
compared with those of the untreated sample. The present work indicated that ultrasound treatment will be a
novel approach to improve the edibility and nutritional quality of soybean sprouts.
© 2015 Elsevier Ltd. All rights reserved.

Keywords:
Ultrasound
Edibility properties
Nutritional quality
Soybean sprouts

1. Introduction
Due to the increasing consumer demands for high quality food, new
safe and effective methods of food processing are being developed.
Ultrasound is an example of novel technology in the food industry and
is becoming increasingly appreciated (Arzeni et al., 2012). It is generally
considered safe, non-toxic, and environmentally friendly, which is a
major advantage over other techniques (Awad, Moharram, Shaltout,
Asker, & Youssef, 2012). Meanwhile, ultrasound could lead to a reduc-
tion of the processing time or decrease the financial costs (Cárcel,
García-Pérez, Benedito, & Mulet, 2012). The application of ultrasound
is related to many areas, such as enzyme inactivation, crystallization,
drying, degassing, extraction, filtration, homogenization, meat tenderi-
zation, oxidation and sterilization (De Gennaro, Cavella, Romano,
& Masi, 1999; Jambrak, Mason, Lelas, Paniwnyk, & Herceg, 2014;
Santacatalina, Garcia-Perez, & Benedito, 2011). Interestingly, ultrasound
could also stimulate seed germination, increasing the percentage of ger-
mination, and accelerating the growth of plants (Wang et al., 2012). The
⁎ Corresponding author at: 235 Nanjing Dong Road, Nanchang 330047, China. Tel.: +86
791 88334552; fax: +86 791 88333708.
E-mail address: chenhongbing@ncu.edu.cn (H. Chen).
most common interaction mechanisms involved in this case are the cav-
itation which causes heat and chemical effects. Moreover, acceleration
of the rate of influx or uptake of water into seeds by ultrasound can
also be caused by mechanical effects, i.e. shear stress developed by
eddies arising from shock waves. Although ultrasound treatment has
been applied to stimulate germination in many different types of plant
seeds, limited information is available about the effects of ultrasound
on the germinated seeds in the view of food science (Goussous,
Samarah, Alqudah, & Othman, 2010).
Soybean (Glycine max L. Merr.) plays an important role in food
consumption worldwide because of its high nutritional quality and
physicochemical functions (Natarajan, Luthria, Bae, Lakshman, &
Mitra, 2013). Among soybean products, soybean sprouts, which are
rich in dietary fiber, various nutrients, and bioactive components, are
the valuable dietary supplements in many parts of the world that may
promote health and well-being, particularly in rural areas in Asian
countries, where seasonal fruits and vegetables are not available all-
year-round (Mbithi, Van Camp, Rodriguez, & Huyghebaert, 2001).
Germination is considered as an inexpensive and effective technology
to improve the nutritional quality of soybean because this process trig-
gers a sequence of metabolic changes (Paucar-Menacho, Berhow,
Mandarino, Chang, & Mejia, 2010). It has been demonstrated that

http://dx.doi.org/10.1016/j.foodres.2015.01.011
0963-9969/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Yang, H., et al., The ultrasound-treated soybean seeds improve edibility and nutritional quality of soybean sprouts, Food
Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.01.011
2 H. Yang et al. / Food Research International xxx (2015) xxx–xxx
certain undesirable constituents, such as trypsin, chymotrypsin,
lipoxygenase activity, phytic acid and oligosaccharides could be elimi-
nated or declined during germination in soybeans (Quinhone & Ida,
2015; Shi, Nam, & Ma, 2010). Moreover, the amounts of vitamins, phy-
tosterols, tocopherols, and isoflavones increase during this metabolic
process (Shi et al., 2010).
The aim of this study was to investigate the effect of ultrasound
treatment on physical properties and nutritional quality of soybean
sprouts. Briefly, soybean seeds were exposed to ultrasound then follow-
ed germination, the morphological changes, protein fraction composi-
tions, IgE-binding, anti-nutritional factors, and GABA and isoflavone
contents of soybean sprouts were assessed. The technical data would
be a strong support for the application of ultrasound as a novel approach
to promote the development of soybean sprouts as high quality food.
2. Materials and methods
2.1. Materials
Soybean cultivar ‘Dongnong 48’ was kindly provided by Dr Li
Wenbin (Key Laboratory of Soybean Biology (Ministry of Education),
Northeast Agricultural University, China). This cultivar has an average
weight of 22.41 g per 100 seeds, and contains 44.53% protein and
19.19% lipids. Linoleic acid, Nα-benzoyl-L-arginine 4-nitroanilide hydro-
chloride, trypsin, and GABA were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Bradford dye binding assay was supplied by Bio-
Rad (Bio-Rad, Hercules, CA). Daidzin, genistin, daidzein, and genistein
were purchased from Tauto Co. (Shanghai, China) with purity ≥ 99%.
All of the other chemicals were of analytical grade.
The sera from eight soybean-allergic patients (with an average age
of 24.9 years) were collected from the First Affiliated Hospital of
Guangxi Medical University (Nanning, China). Among these eight pa-
tients, seven were males and one was female. Their specific IgE levels
were 1.45, 1.92, 3.54, 4.53, 4.89, 4.95, 13.59 and 23.71 kU/L, respectively,
and quantified using a CAP-fluorescent enzyme immunoassay system
(Phadia, Uppsala, Sweden). Among the eight patients, two suffered
from asthma, and five manifested urticaria. A serum pool established
from eight soybean-allergic patients in equal volumes was used for
ELISA.
2.2. Ultrasound treatment and germination of soybean seeds
Soybean seeds were cleaned to completely remove impurities, in-
cluding broken and diseased seeds. These seeds were then dispersed
in distilled water in 500 mL glass flasks (wall thickness = 0.1 cm,
height = 11.5 cm, inner diameter = 9.0 cm) for ultrasound treatment.
Briefly, the flasks containing samples (100 seeds in 160 mL of water)
were put in the center of the ultrasonic bath (Ultrasonic, Model SB-
5200DTD, HF-Pk-power 300 W, 40 kHz, internal dimensions:
300 mm × 150 mm × 150 mm, Ningbo, China) filled with 5 L of water.
This volume was sufficient to apply ultrasound to the whole of each
sample. Subsequently, the seeds were exposed to 100, 200, and 300 W
for 30 min at 25 °C. The ultrasonic intensity was 0.35 W cm− 2,
0.54 W cm−2 and 1 W cm−2, respectively. Only one sample was sub-
jected to each sonication. Ice and chilled water were used during this
treatment to prevent excessive temperature rises, whereas water was
continually circulated in and out of the ultrasound bath. Afterward
these seeds were germinated in a climatic cabinet (model LHP-250H,
Shanghai, China) at 30 °C for 5 d in darkness. The seeds were sprinkled
with sterile distilled water every 12 h. Germination was performed in
triplicate for each sample, 100 seeds per replicate.
2.3. Morphological changes in soybean sprouts
Morphological changes in sprouts were determined as follows:
(1) germination rate: the number of germinating seeds divided by the
Please cite this article as: Yang, H., et al., The ultrasound-treated soybean seeds improve edibility and nutritional quality of soybean sprouts, Food
Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.01.011
total number of seeds, (2) sprout length: length of sprout was measured
from above the root to the base of the cotyledon, (3) root length: length
of root was measured from the root to the base of the hypocotyl,
(4) fresh weight per sprout: average weight of 30 fresh sprouts, and
(5) fresh weight per root: average weight of 30 fresh sprouts. These
morphological changes in each sample were determined in triplicate,
each sample was then stored at −20 °C until further analysis.
2.4. Analysis of crude protein, lipid, total sugar, and moisture
Crude protein, lipid, and moisture were determined in accordance
with the AOAC standard method. Total sugar content in the soybean
sprouts was determined on the basis of a previously described protocol
with slight modifications (Shi et al., 2010). In brief, 0.5 g of ground dry
sprouts was mixed with 10 mL of double distilled water and 1 mL of
HCl. After heating for 20 min at 100 °C, the mixture was cooled and
diluted with distilled water, and total sugar content was determined
using anthrone–sulfuric acid method. Glucose was used as a calibration
standard at a linear concentration range of 0 mg/L to 100 mg/L.
2.5. Assessment of protein fraction compositions
2.5.1. Preparation of protein extracts
Sprouts were frozen in liquid nitrogen, and ground immediately to
fine powder with mortar and pestle. The powder was defatted with ac-
etone (20 mL/g of flour) for 4 h, shaken, filtered, and air-dried. The pow-
der was then subjected to extraction twice with Tris–HCl buffer (50 mM
pH 8.0) containing salt (150 mM NaCl). The extracts (1:10, w/v) were
stirred for 3 h at 4 °C, and centrifuged at 9000 g for 30 min to remove in-
soluble materials. The supernatants were dialyzed against distilled H2O
for 24 h at 4 °C by using a dialysis membrane (MWCO, 3500 Da) and
freeze-dried. The nitrogen content of the total proteins was determined
by Kjeldahl method.
2.5.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE)
Changes in protein patterns were analyzed by SDS-PAGE in a Bio-
Rad Mini protein II system (Bio-Rad, Hercules, CA) with 5% stacking
gel and 12% separating gel (Laemmli, 1970). The amount and volume
of the protein solutions transferred to each well were approximately
20 μg and 20 μL, respectively, and the gels were stained with Coomassie
Brilliant Blue R250. The images were recorded with a Gel Doc 1000/
2000 gel documentation system (Bio-Rad, Hercules, CA).
2.5.3. Determination of amino acid
The amino acid composition of each sample was determined after
acid hydrolysis was performed. The sample was placed in an ampoule
and mixed with 6 M HCl. After sealing the ampoule with nitrogen, the
sample was hydrolyzed at 110 °C for 24 h. The hydrolysate was evapo-
rated to remove HCl, filtered, and loaded in an amino acid analyzer
(Model S433D, Sykam Corp., Eresing, Germany) for amino acid analysis.
Tryptophan was determined by alkaline hydrolysis according to the
AOAC standard method. The results were expressed in g/100 g of
protein.
2.5.4. Determination of GABA content
The defatted soybean powder was soaked in 70% ethanol for 4 h,
evaporated and lyophilized to yield dried powder extract. The GABA
content was analyzed by HPLC as described in a previous work with
slight modifications (Hyun, Eom, Jeun, Han, & Kim, 2013). In brief,
approximately 100 mg of powder extract was dissolved in 1 mL of eth-
anol–water–triethylamine solution (4:4:2) and then 80 μL of ethanol–
water–triethylamine–phenylisothiocyanate solution (6:1:1:1) was
added. To induce the formation of phenylisothiocyanate–GABA, the
mixture was incubated at room temperature (25 °C) for 30 min. After
each sample was filtered using a Millipore membrane (0.22 μm), 10 μL
 H. Yang et al. / Food Research International xxx (2015) xxx–xxx
of each sample was injected into a column and analyzed in triplicate.
HPLC (SIL-20A, Shimadzu, Tokyo, Japan) with a column (C18,
4.6 mm × 250 mm, 4 μm) was eluted with a mixture of 80% solution A
(0.1 M sodium acetate buffer, pH 5.8) and 20% solution B (60% acetoni-
trile) at a flow rate of 0.8 mL/min. Column temperature was set at 40 °C
and UV detector was set at 254 nm.
2.6. ELISA
The IgE-binding of soybean protein was quantified by ELISA. A 96-
well ELISA microplate was coated overnight at 4 °C with 2 μg/well pro-
tein in phosphate-buffered saline (PBS). The wells were washed three
times with PBS containing 0.05% Tween 20 (PBST), and blocked with
3% gelatin in PBS for 1 h at 37 °C. After further washing with PBST, the
plate was incubated for 1 h at 37 °C with 100 μL/well of the pooled
patients' serum. The wells were washed again, and 100 μL/well of
anti-human IgE conjugate horseradish peroxidase (HRP) antibody
diluted at 1:5000 in PBS was added for another 1 h at 37 °C. After the
wells were further washed three times with PBST, the reaction was de-
veloped with 100 μL/well peroxidase substrate (o-phenylenediamine
and hydrogen) with an incubation of 15 min at 37 °C. The reaction
was stopped after 15 min by adding 2 M H2SO4, and optical density
was measured at 490 nm by using an ELISA reader (Bio-Rad, Hercules,
CA). Data were presented as the mean of triplicate measurements.
2.7. Determination of anti-nutritional factors
2.7.1. Extraction and estimation of lipoxygenase isozyme activity
The defatted sprout flour was stirred with phosphate buffer (0.2 M,
pH 6.8) for 20 min at 4 °C, and centrifuged at 9000 g for 15 min. The
obtained supernatant was used to assay lipoxygenase isozyme activity
according to a previously described procedure (Axelrod, Cheesbrough,
& Laakso, 1981). Lipoxygenase I activity was estimated using 0.2 M
boric acid borax buffer (pH 9.0) and 10 mM sodium linoleate as a
substrate. Lipoxygenase II + III activities were analyzed with 0.2 M
phosphate buffer (pH 6.8) and 10 mM sodium linoleate as a substrate.
The changes in absorbance were measured at 234 nm by using a UV
spectrophotometer (Lambda 25, PerkinElmer, USA). Each sample was
subjected to this experiment in triplicate.
2.7.2. Estimation of trypsin inhibitor content
The defatted sprout flour was extracted using NaOH (0.01 M) for 3 h
in constant stirring, and then centrifuged at 9000 g for 30 min at 4 °C.
The supernatant was used to assay trypsin inhibitor content by using
benzoyl DL-arginine para nitroanilide hydrochloride as a substrate
(Smith, Van Megen, Twaalfhoven, & Hitchcock, 1980). Absorbance was
determined at 410 nm by using a UV spectrophotometer (Lambda 25,
PerkinElmer, USA). Trypsin content was determined as follows, and
each sample was measured in triplicate.
trypsin inhibitor mg=of defatted sample
differential absorbance  dilution factor
¼
0:019  1000
2.8. Determination of isoflavone content
Isoflavone was extracted using a previously described method
(Phommalth, Jeong, Kim, Dhakal, & Hwang, 2008). Approximately
0.2 g of defatted sprout flour was added to 10 mL of 80% methanol
and incubated in a shaking incubator at 50 °C for 8 h. Afterward, the
samples were centrifuged at 4 °C for 20 min at 9000 g, and then filtered
using a 0.22 μm membrane. The obtained sample was subjected to iso-
flavone analysis by using HPLC (SIL-20A, Shimadzu, Tokyo, Japan)
equipped with a column (C18, 4.6 mm × 250 mm, 4 μm). A linear
HPLC gradient was prepared using methanol (solvent A) and water
Please cite this article as: Yang, H., et al., The ultrasound-treated soybean seeds improve edibility and nutritional quality of soybean sprouts, Food
Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.01.011
3
(solvent B) as the mobile phase. After loading 10 μL of sample, solvent
A was increased from 20% to 40% for 15 min. It was further increased
from 40% to 72% for another 15 min, and then reduced from 72% to
20% for 15 min. The solvent flow rate was 1.0 mL/min. Elution was
monitored by UV absorption at 254 nm. Isoflavone was identified by
comparing with the retention times of genuine standards, including
daidzein, genistein, daidzin and genistin.
2.9. Statistical analysis
The data were expressed as mean ± S.D. Statistical analysis was done
using SPSS software (version 11.5, SPSS Inc., Chicago, IL, USA). One-way
analysis of variance was performed, and differences between the sample
means were performed using the Tukey's test at α level of 0.05.
3. Results and discussion
3.1. Morphological changes in soybean sprouts
The efficacy of ultrasound on the morphological changes in soybean
sprouts is shown in Table 1. The germination rate of sprouts increased
after these sprouts were treated with ultrasound compared with that
of the control group (0 W), this parameter increased by 18.07% at
300 W. However, no significant difference (P ≤ 0.05) was observed
between 100 and 200 W. Germination rate possibly enhanced because
ultrasound vibration caused changes in the cellular ultrastructure, en-
zyme stability and cell growth, such as the structure, the deformability
and the permeability of membranes inside and outside the seed cells,
which could improve the release of enzymes from the cell wall and
biochemical metabolism (Chen, Liu, Yue, Meng, & Liang, 2013).
The average length of sprout increased with ultrasound treatment,
by contrast, the average length of root decreased. The average length
of sprout increased by 24.42% at 300 W and the average length of
root decreased by 20.41% compared with those of the control group.
These results suggested that some of the metabolic processes in the
ultrasound-treated seeds were altered, such as the endogenous hor-
monal balance of treated plant tissue or cells, resulting in abnormal
sprout and root length.
These results showed that ultrasound treatment is an efficient and
low-cost approach to change the morphological characteristics of soy-
bean sprouts. Ultrasound could also increase the edibility of soybean
sprouts and improve the acceptance for human consumption.
3.2. Changes in protein, lipid, total sugar and moisture contents of soybean
sprouts
The changes in chemical components, such as protein, lipid, total
sugar, and moisture contents of soybean sprouts are shown in Table 2.
The protein content of sprouts exposed to 0 W was 45.68%, but in-
creased to approximately 47.66% at 300 W. The lipid and sugar contents
of the sprouts showed an evident decrease as ultrasound power was in-
creased. The lipid and sugar contents decreased by 27.41% and 21.66% at
300 W, respectively, compared with those of the control group. The
moisture content of the sprouts increased from 81.42% to 87.26% after
these sprouts were treated with ultrasound. In fact, the degradation of
lipid and sugar during germination is a process that provides the energy
required for protein synthesis. This result indicated that ultrasound
treatment exhibited a beneficial effect on macro-nutrients of soybean
sprouts.
3.3. Protein fraction compositions
3.3.1. Electrophoresis profile of sprout protein
The protein patterns in soybean sprouts were analyzed by SDS-
PAGE. The electrophoretic patterns of the sprout protein were not
altered on the gels (Fig. 1). However, the intensities of some protein
4 H. Yang et al. / Food Research International xxx (2015) xxx–xxx

Table 1
Morphological changes of soybean sprouts derived from ultrasound-treated seeds.

Parameters Different ultrasound power levels

0W 100 W 200 W 300 W

Germination percentage (%) 83 ± 1.00c 91 ± 0.00b 95 ± 0.60b 98 ± 1.10a

Sprout length (cm) 8.68 ± 0.63c 9.59 ± 0.49b 9.38 ± 0.42b 10.80 ± 0.40a
Root length (cm) 16.02 ± 0.46a 15.53 ± 0.91ab 15.17 ± 0.78ab 12.75 ± 0.32b
Fresh weight per sprout (g) 0.91 ± 0.01b 1.05 ± 0.03a 1.10 ± 0.01a 1.13 ± 0.03a
Fresh weight per root (g) 0.15 ± 0.00a 0.13 ± 0.01a 0.15 ± 0.01a 0.11 ± 0.02a

Values are expressed as averages ± standard deviation. Different characters in each row indicate significant (P ≤ 0.05) differences among samples treated under different ultrasound
power levels. Mean values in each row sharing the same letter are not significantly different (P N 0.05).

bands changed, indicating that the peptide chains of sprout protein 3.3.3. GABA content of soybean sprouts
were altered by ultrasound. GABA, a significant component of free amino acids, is widely distrib-
uted in plants and implicated in neurotransmission (Iimure et al., 2009).
The GABA content (ranging from 83.10 mg/100 g to 119.29 mg/100 g) of
3.3.2. Amino acid composition of soybean sprouts soybean sprouts at different ultrasound power levels is presented in
The changes in the amino acid content of soybean sprouts from ul- Fig. 2. GABA content continuously increased as ultrasound power was
trasound-treated seeds are recorded in Table 3. The results clearly increased, and the sample treated at 300 W was 43.39% higher than
showed that ultrasound treatment resulted in a considerable increase the untreated sample. This result showed that ultrasound can contrib-
in amino acid levels. ute to an increase in GABA content.
Among the non-essential amino acids (NEAA), Glu (10.59 g/100 g pro- A GABA generating system comprises glutamate, glutamate decar-
tein) presented the highest amount at 0 W followed by Asp (7.51 g/100 g boxylase (GAD), and GABA transaminase. GAD is the rate-limiting
protein) and Arg (5.42 g/100 g protein). Gly (2.72 g/100 g protein) and enzyme for GABA synthesis and GABA accumulation is mediated pri-
Ala (2.77 g/100 g protein) contributed the least amount of NEAA. marily via GAD, and there is a significantly positive correlation between
Among the essential amino acids (EAAs), Leu (4.82 g/100 g protein) the GABA content and GAD activity (Komatsuzaki et al., 2007; Shelp,
and Lys (3.92 g/100 g protein) yielded the highest levels. By contrast, Bown, & McLean, 1999). GAD activity increases in brown rice during
Met (0.92 g/100 g protein) and Cys (1.21 g/100 g protein) presented germination, and GABA is continuously produced (Oh, Soh, & Cha,
the lowest values. Ultrasound treatment resulted in a significant 2003). Likewise, ultrasound treatment before germination occurs may
decrease in Glu and Arg and a significant increase in Asp, Ala, His, Phe, enhance GABA content by activating GAD in the seeds. Similar studies
and Trp, no changes were observed in other amino acids. The total have investigated the effect of steeping on the GABA content of germi-
EAA content showed an increase of 18.28% at 300 W compared with nated grains. Guo et al. showed that the GABA content of germinated
that of the control group. In a previous study, the levels of sulfur soybean increases rapidly as soaking time is prolonged (Guo, Chen,
amino acids remain almost constant in germinated soybean seeds,
whereas, Asp content is increased compared with those of raw seeds
(El-Adawy, 2002). However, the application of high pressure treatment
to soybean seeds prior to germination caused a reduction in Glu and Ala
(NEAA) and in Trp, Met, and Cys (EAAs) compared with those in the
germinated seeds (Peñas, Gomez, Frias, Baeza, & Vidal-Valverde, 2011).
In general, two types of physiological processes, proteolysis or
amino acid metabolism, produce amino acids. Proteolysis could increase
amino acid concentration, while amino acid metabolism changes con-
centration of certain amino acids (Ueno et al., 2009). Ultrasound was
used to cause partial degradation of internal soybean cell structure
(Rokhina, Lens, & Virkutyte, 2009). Cellular membrane systems were
damaged in ultrasound-treated soybean. Thus, mass transfer in
ultrasound-treated soybean was accelerated, compared with that in
control soybean, which promoted enzymatic reactions, leading to the
increase in amino acid levels.

Table 2
Changes in protein, lipid, total sugar, and moisture contents of soybean sprouts after ultra-
sound treatment.

Parameters Different ultrasound power levels

0W 100 W 200 W 300 W

Protein (%) 45.68 ± 0.30b 47.52 ± 0.57ab 47.61 ± 0.49a 47.66 ± 0.43a
Lipid (%) 11.53 ± 0.15a 9.94 ± 0.08b 9.69 ± 0.03b 8.37 ± 0.12c
Sugar (%) 12.14 ± 1.24a 11.45 ± 1.43ab 11.34 ± 1.57ab 9.51 ± 0.73b
Moisture (%) 81.42 ± 1.78b 83.99 ± 1.05ab 88.63 ± 0.96a 87.26 ± 1.24ab

Values are expressed as averages ± standard deviation. Different characters in each row Fig. 1. SDS-PAGE profiles of the sprout protein from ultrasound-treated seeds. (M, stan-
indicate significant (P ≤ 0.05) differences among samples treated under different ultra- dard protein marker; lane 1, soybeans were treated with ultrasound at 0 W; lane 2, soy-
sound power levels. Mean values in each row sharing the same letter are not significantly beans were treated with ultrasound at 100 W; lane 3, soybeans were treated with
different (P N 0.05). ultrasound at 200 W; lane 4, soybeans were treated with ultrasound at 300 W).

Please cite this article as: Yang, H., et al., The ultrasound-treated soybean seeds improve edibility and nutritional quality of soybean sprouts, Food
Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.01.011
 H. Yang et al. / Food Research International xxx (2015) xxx–xxx 5

Table 3
Amino acid content of soybean sprouts.

Amino acids Different ultrasound power levels

(g/100 g)
0W 100 W 200 W 300 W

Non-essential amino acids

Asp 7.51 ± 0.28b 6.92 ± 0.21b 6.94 ± 0.22b 20.41 ± 0.19a
Glu 10.59 ± 0.10a 10.23 ± 0.14ab 9.92 ± 0.20b 8.75 ± 0.15c
Ser 3.33 ± 0.08a 3.31 ± 0.11a 3.31 ± 0.10a 3.67 ± 0.13a
Gly 2.72 ± 0.02b 3.07 ± 0.13a 3.01 ± 0.13a 2.42 ± 0.17c
Arg 5.42 ± 0.31a 4.59 ± 0.14a 4.59 ± 0.12a 3.31 ± 0.12b
Ala 2.77 ± 0.10b 3.94 ± 0.08a 3.98 ± 0.12a 3.98 ± 0.12a
Pro 3.30 ± 0.16a 2.41 ± 0.12a 2.47 ± 0.15a 3.02 ± 0.19a

Essential amino acids

His 1.82 ± 0.11b 2.74 ± 0.03a 2.74 ± 0.02a 2.43 ± 0.05a
Val 3.01 ± 0.09b 3.65 ± 0.12a 2.12 ± 0.08c 3.36 ± 0.10ab
Met 0.92 ± 0.00c 2.16 ± 0.14b 3.62 ± 0.21a 0.98 ± 0.01c
Cys 1.21 ± 0.03b 1.27 ± 0.05b 3.63 ± 0.09a 1.21 ± 0.09b
Ile 2.70 ± 0.08b 3.65 ± 0.13a 3.65 ± 0.02a 3.08 ± 0.05b
Leu 4.82 ± 0.19b 6.01 ± 0.13a 6.05 ± 0.17a 4.59 ± 0.01b
Phe 3.03 ± 0.12b 3.33 ± 0.09a 3.33 ± 0.09a 3.33 ± 0.10a
Fig. 3. IgE-binding of sprout protein from ultrasound-treated seeds. Values are expressed
Tyr 2.41 ± 0.03b 2.76 ± 0.03a 2.76 ± 0.07a 2.45 ± 0.00b
as averages ± standard deviation. Different characters (a–c) on the top of the column in-
Lys 3.92 ± 0.08b 6.64 ± 0.11a 6.37 ± 0.11a 3.62 ± 0.10b
dicate significant (P ≤ 0.05) differences among samples treated under different ultrasound
Thr 2.49 ± 0.10b 3.30 ± 0.15a 3.33 ± 0.15a 2.45 ± 0.07b
power levels.
Trp 1.71 ± 0.02b 1.99 ± 0.09ab 2.26 ± 0.11a 2.27 ± 0.09a

Values are expressed as averages ± standard deviation. Different characters in each row
indicate significant (P ≤ 0.05) differences among samples treated under different ultra-
sound power levels. Mean values in each row sharing the same letter are not significantly
different (P N 0.05).
Song, & Gu, 2011). Chung et al. found that steeping conditions before
waxy hull less barley grain is germinated significantly increase GABA
content as germination time is prolonged (Chung, Jang, Cho, & Lim,
2009). Our study indicated that germinated soybean treated with ultra-
sound can be used as a good source of GABA-enriched foods.
3.4. IgE-binding of sprout protein
ELISA was performed to assess the IgE-binding of protein from soy-
bean sprouts, and the results are shown in Fig. 3. Sprout protein showed
a sharp decrease in IgE-binding after ultrasound treatment was per-
formed. The sample treated at 300 W yielded the highest decrease in
IgE-binding potency (51.39%). However, no significant difference
(P ≤ 0.05) was observed among sprouts exposed to ultrasound power
of 0 W to 200 W treatments. The results suggested that ultrasound
treatment may cause changes in major soybean allergens during
germination, thereby degrading allergens into peptides and amino
acids. As a result, the structures of their epitopes are disrupted or
some epitopes are eliminated. Therefore, the allergenicity of sprout
protein is decreased.
Previous studies reported that ultrasound can change the struc-
ture of native protein by altering protein conformation, disrupting sec-
ondary structures, forming new intra/intermolecular interactions, and
rearranging disulfide bonds (Owen & Simons, 1957; Pavlovskaya,
McClements, & Povey, 1992). Similar results have also indicated that
ultrasound treatment significantly enhances the efficiency of enzyme
treatment to reduce IgE-binding by 50% in peanut protein (Li, Yu,
Ahmedna, & Goktepe, 2013), and high-intensity ultrasound can be
administered to reduce the allergenicity of shrimp (Li, Lin, Cao, &
Jameel, 2006).
Our study suggested that ultrasound could be utilized as a novel pro-
cessing tool to reduce the allergenicity of sprout-based food, such as
soybean. On the basis of these findings, we recommend further studies
such as clinical confirmation to test ultrasound-treated soybeans in
soybean-allergic individuals.

3.5. Determination of anti-nutritional factor

3.5.1. Lipoxygenase isozyme activity of soybean sprouts

Fig. 2. GABA content in soybean sprouts from ultrasound-treated seeds. Values are
expressed as averages ± standard deviation. Different characters (a–d) on the top of the
column indicate significant (P ≤ 0.05) differences among samples treated under different
ultrasound power levels.
Lipoxygenase (EC1.13.11.12) in soybean is present in three forms of
isozymes, namely, Lx-I, Lx-II, and Lx-III (Axelrod et al., 1981). Fig. 4(A)
shows the changes in the activities of Lx-I and Lx-II + III of the sprouts.
Ultrasound treatment (100–300 W) could decrease the activities of Lx-I
and Lx-II + III, and the maximum reduction of 36.22% in Lx-I and 55.57%
in Lx-II + III was visible at 300 W compared with those of the control
group. This behavior was observed possibly because ultrasound waves
caused the violent collapse of small bubbles or voids in cells, and severe
shear stresses may influence the lipoxygenase enzyme activity (Islam,
Zhang, & Adhikari, 2014).
Lipoxygenase is an important factor in the generation of odor and
flavor compounds from lipid and deteriorates the palatability of soy-
bean food. Our findings suggested that the substantial odor and flavor
scores of ultrasound-treated sprouts were improved. Our work was
similar to those of other investigators. For instance, Jadhav and Gogate
found that an increase in ultrasound intensity results in a decrease in
lipase enzyme activity (Jadhav & Gogate, 2014). Chemat and Khan
reported that 75% to 85% of lipoxygenase isozyme was inactivated in
ultrasound-treated soybean (Chemat & Khan, 2011).

Please cite this article as: Yang, H., et al., The ultrasound-treated soybean seeds improve edibility and nutritional quality of soybean sprouts, Food
Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.01.011
6 H. Yang et al. / Food Research International xxx (2015) xxx–xxx

Fig. 4. The activity of lipoxygenase isozyme (A) and trypsin inhibitor (B) contents of soybean sprouts derived from ultrasound treated seeds. Values are expressed as averages ± standard
deviation. Different characters (a–c) on the top of the column indicate significant (P ≤ 0.05) differences among samples treated under different ultrasound power levels.

3.5.2. Trypsin inhibitor content of soybean sprouts
Trypsin inhibitor is an anti-nutritional factor that affects protein
digestibility (Peace, Sarwar, & Touchburn, 1992). The trypsin inhibitor
content of soybean sprouts is presented in Fig. 4(B). Trypsin inhibitor
content decreased after the sprouts were treated with ultrasound. Ultra-
sound treatment at 300 W resulted in a 98.78% decrease in trypsin
inhibitor content which was higher than that of the control group. It
suggested that ultrasound treatment may promote the use of trypsin
inhibitor as an energy source during germination.
Although previous works focused on the trypsin inhibitor content of
germinated soybean seeds, studies have yet to be conducted regarding
the effect of ultrasound treatment on the trypsin inhibitor content of
sprouts (Owen & Simons, 1957; Pavlovskaya et al., 1992). However, a
similar observation on dry seeds has been reported. Huang et al. con-
cluded that ultrasound could inactivate trypsin inhibitor by inducing a
reduction in disulfide bonds and by altering structural conformations
(Huang, Kwok, & Liang, 2008).
3.6. Isoflavone content of soybean sprouts
Isoflavone, a phytoestrogen, elicits positive effects on human health
(Brouns, 2002; Shi et al., 2010). The isoflavone contents of soybean
sprouts are presented in Table 4. After ultrasound treatment was per-
formed, daidzin and genstin contents markedly decreased whereas
daidzein and genistein contents significantly increased compared with
those of the untreated sample. At 300 W, daidzin and genstin contents
decreased by approximately 79.62% and 70.95%, respectively. By con-
trast, daidzein and genistein contents increased by approximately
39.13% and 96.91%, respectively, compared with those of the untreated
sample.
Daidzin and genstin are β-glycoside forms; daidzein and genistein
are the aglycone forms of isoflavone (Quinhone & Ida, 2015). Studies
have shown that β-glucosidase activity may be closely related to the
total content of isoflavone and its isomeric forms (Ribeiro et al., 2006).
Soybean contains two endogenous isoforms of β-glucosidase that can
hydrolyze β-glycoside isoflavones to their aglycone forms (Chiarello
et al., 2006). These enzymes could be activated by ultrasound treatment
before germination occurred.
An increasing number of reports in the literature concern degrada-
tion during application of ultrasound in food processing (Pingret,
Fabiano-Tixier, & Chemat, 2013). In the case of food products rich in
lipids, the analysis tends to point to lipid degradation induced by cavita-
tion, although very few studies have tried to elucidate the mechanism
by which ultrasound energy degrades those compounds (Pingret et al.,
2012a). Unfortunately, we have not check the compound changes of
the ultrasonic soybean seeds, resulting in a limitation to explain our
data on nutrition quality of soybean sprouts. Meanwhile, ultrasound
used as a process has been successfully applied in the laboratory and
pilot plant scale. A larger reactor of 30 L extraction tank consisting of a
quadruple output of ultrasound at 25 kHz and 4 × 200 W has been
used for olive oil and polyphenol extractions (Achat et al., 2012;
Pingret, Fabiano-Tixier, Bourvellec, Renard, & Chemat, 2012), and it is
suggested that this ultrasound reactors are also suitable for enrichment
of edible oils with volumes of 100 to 1000 L and the extraction of 15 or
150 kg of dry plant material per time. Reasonably, we think it is possible
to increase productivity for soybean sprouts derived from the ultrasonic
soybean seeds. Nevertheless, further studies should be carried out to
evaluate the compound changes of soybean seeds after ultrasound
treatment and run out industrial trials or to scale-up laboratory
experiments.

Table 4 4. Conclusions
Isoflavone content in soybean sprouts from ultrasound-treated seeds.

Isoflavone Different ultrasound power levels

In conclusion, ultrasound treatment caused an increase in germina-
(mg/g) tion rate and sprout length of soybean sprouts. Ultrasound-treated
0W 100 W 200 W 300 W
soybean provided a potent source of biologically active compounds,
Daidzin 2.16 ± 0.05a 1.51 ± 0.03b 1.03 ± 0.03c 0.44 ± 0.02d such as isoflavone, and amino acids including GABA. Ultrasound treat-
Genstin 2.12 ± 0.07a 0.92 ± 0.02b 0.86 ± 0.03b 0.63 ± 0.05c
ment also improved edibility of sprouts. IgE-binding, lipoxygenase iso-
Daidzein 0.23 ± 0.02b 0.23 ± 0.02b 0.30 ± 0.02ab 0.32 ± 0.03a
Genistein 0.97 ± 0.05c 0.93 ± 0.03c 1.36 ± 0.03b 1.91 ± 0.07a zyme activity and trypsin inhibitor content of soybean sprouts showed
a significant power-dependent decrease of soybean sprouts after these
Values are expressed as averages ± standard deviation. Different characters in each row
indicate significant (P ≤ 0.05) differences among samples treated under different ultra-
seeds were exposed to ultrasound. The results indicated that ultrasound
sound power levels. Mean values in each row sharing the same letter are not significantly treatment is a useful approach to significantly improve the edibility and
different (P N 0.05). the nutritional quality of soybean sprouts. However, the potential

Please cite this article as: Yang, H., et al., The ultrasound-treated soybean seeds improve edibility and nutritional quality of soybean sprouts, Food
Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.01.011
 H. Yang et al. / Food Research International xxx (2015) xxx–xxx
mechanism of ultrasound treatment to improve the quality of soybean
sprouts should be further studied.
Abbreviations
GABA gamma-aminobutyric acid
NSI nitrogen soluble index
NEAA non-essential amino acids
EAA essential amino acid
PBS phosphate-buffered saline
GAD glutamate decarboxylase
Acknowledgments
The work was supported by the National High Technology Research
and Development Program of China (863 program, no. 2013AA102205),
the International Science & Technology Cooperation Program of China
(no. 2013DFG31380), the National Natural Science Foundation of
China (no. 31171716), the Key Program of Natural Science Foundation
of Jiangxi Province, China (no. 20133ACB20009), and the Research
Program of State Key Laboratory of Food Science and Technology (nos.
SKLF-ZZA-201302 and SKLF-ZZB-201302).
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