LWT - Food Science and Technology 99 (2019) 262–267

Contents lists available at ScienceDirect

LWT - Food Science and Technology

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

Evaluating of quality of rice bran protein concentrate prepared by a T

combination of isoelectronic precipitation and electrolyzed water treatment
Masanori Watanabea,∗, Chikako Yamadab, Isamu Maedac, Charin Techapund, Ampin Kuntiyad,
Noppol Leksawasdid, Phisit Seesuriyachand, Thanongsak Chaiyasod, Shinji Takenakae,
Tadahiko Shionof, Kozo Nakamurag, Shujiro Endoh
a
Graduate School of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, Yamagata, 997-8555, Japan
b
Graduate School of Nutritional Sciences, Nagoya University of Arts and Sciences, Nisshin, Aichi, 470-0196, Japan
c
Graduate School of Agricultural Science, Utsunomiya University, 350 Minemachi, Utsunomiya, 321-8505, Japan
d
Bioprocess Research Cluster, The School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University, Mae-Hea, Mueang, Chiang Mai, 50100, Thailand
e
Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657–8501, Japan
f
Hiroshima Prefectural Food Technology Research Center, 12-70 Hijiyamahon-machi, Minami-ku, Hiroshima, 732-0816, Japan
g
Department of Bioscience and Biotechnology, Graduate School of Agriculture, Shinshu University, Minamiminowamura, Nagano, 399–4598, Japan
h
Sanwa Yushi Co., Ltd., 4-1-2 Hitoichi-machi, Tendo, Yamagata, 994-0044, Japan

ARTICLE INFO ABSTRACT

Keywords: A combination of isoelectronic precipitation and electrolyzed water treatment (IP-EWT) process was use for
Defatted rice bran simultaneous recovery of protein and phosphorus compounds from heat-stabilized defatted rice bran (HSDFRB).
Isoelectric precipitation The highest protein content (65.1 w/w%) recovery ratio (over 50%) of protein concentrate were attained by
Electrolyzed water two-stage protein extraction with IP-EWT. Analysis of amino-acid composition demonstrated excellent amino
Rice protein
acid value (76.6%) in the protein fraction than those of commercial cereals such as rice (61%), wheat (39%), and
Allergenic proteins
corn (31%). In addition, according to SDS-PAGE and immunoblotting analysis, neither rice allergenic protein nor
heavy metals were detected in either HSDFRB or protein fractions extracted by IP-EWT. These results suggest
that as an eco-friendly process, this combined (IP-EWT) process is suitable for practical recovery of highly
concentrated and safe protein from HSDFRB without using enzymes or chemicals such as organic solvents,
buffering agents, and surfactants.

1. Introduction
Celiac disease, namely, a disorder in genetically susceptible persons,
is caused by ingestion of gluten in products containing wheat, rye, and
barley (Rubio-Tapia, Ludvigsson, Brantner, Murray, & Everhart, 2012).
The disease causes inflammation of the small intestine and serious in-
testinal damage, which make it difficult to efficiently absorb several
important nutrients (Lazaridou, Duta, Papageorgiou, Belc, & Biliaderis,
2007). The effective way to treat celiac disease is to eliminate gluten
from the diet of susceptible persons, who must follow a gluten-free diet
throughout their lives (Tsatsaragkou, Gounaropoulos, & Mandala,
2014).
It is known that rice protein is gluten-free and recognized as nu-
tritionally superior to other proteins such as soybean, corn and wheat,
especially in regard to its reported hypoallergenicity and anti-cancer
activity (Fabian & Ju, 2011). Its protein efficiency ratio (1.6–1.9) is
comparable to that of casein (2.5) (Fabian & Ju, 2011). Producing
protein from rice bran has thus been extensively studied. Physico-
chemical treatments, including solvent extraction (Chen & Houston,
1970; Lew, Houston, & Fellers, 1975), compressed-hot-water extraction
(Wiboonsirikul, Kimura, Kanaya, Tsuno, & Adachi, 2008), and enzy-
matic treatments using endoprotease, exoprotease, xylanase, phytase,
amylase, and carbohydrase (Fabian & Ju, 2011), are currently being
used for effectively extracting protein from rice bran. However, these
methods consume large amounts of energy, use harmful chemicals, and
incur high labor costs. Therefore, the rice-processing industry has to
consider commercialization in regard to extracting proteins from rice
bran while keeping energy consumption and production costs low
(Watanabe, Maeda, Koyama Nakamura & Sasano, 2015).
In addition, heat-stabilized defatted rice bran (HSDFRB), namely,
defatted rice bran with low polar solvent, approximately 100% de-
fatted, is known as the most suitable and interesting resource for rice

∗
Corresponding author. Tel./fax: +81 235 282848.
E-mail address: mwata@tds1.tr.yamagata-u.ac.jp (M. Watanabe).

https://doi.org/10.1016/j.lwt.2018.09.059
Received 15 July 2018; Received in revised form 11 September 2018; Accepted 22 September 2018
Available online 23 September 2018
0023-6438/ © 2018 Elsevier Ltd. All rights reserved.
M. Watanabe et al. LWT - Food Science and Technology 99 (2019) 262–267

protein, because a huge amount of defatted rice bran has been produced Table 1
by oil-milling companies as a by-product. However, those companies Proximate composition of HSDFRB.
have not made an effective profit from that by-product in spite of Content g/100 g HSDFRB
adding natural food ingredients (such as dietary fiber and antioxidant
supplement) to it (Zhang, Wang, Zhang, & Zhang, 2014). Nutritional component Moisture content 5.5 ± 0.4
Protein 18.3 ± 1.5
In a previous study, aiming to develop a novel process that could
Fat 1.7 ± 0.2
simultaneously produce highly concentrated proteins and phosphorus Ash 14.6 ± 0.9
compounds without organic solvents and be applied to commercial use, Starch 20.9 ± 1.9
we developed a new technique of isoelectric precipitation combined Crude fiber 35.3 ± 2.6
with electrolyzed-water treatment (IP-EWT). Rice protein and phos-
Minerals Na 0.0086 ± 0.0004
phorus concentrates were prepared using acid and alkaline extraction,
P 3.23 ± 0.21
followed by the isoelectric precipitation (IP) and a washing process Fe 0.0117 ± 0.0007
with electrolyzed water (EW). The pH of EW can be simply adjusted by Ca 0.0642 ± 0.0032
using the quantitative ratios of acidic to alkali EW. Therefore, electro- K 2.39 ± 0.09
Mg 1.38 ± 0.05
lytes in the protein concentrate except for protein can be removed by
Cu 0.00078 ± 0.00003
using EW adjusted to pH values used in the IP process without having to Zn 0.00821 ± 0.00022
add acids or alkalis while keeping the precipitated protein resuspended. Mn 0.0244 ± 0.0014
The process was focused on in terms of protein content and efficiency of
desalination and protein purification without using enzymes or che- Functional component Inositol 0.055 ± 0.004
Phytic acid 9.52 ± 0.19
micals such as organic solvents, buffering agents, and surfactants. The
γ-amino butyric acid 0.042 ± 0.04
highest protein content (52.3 w/w%) was attained when machine de- Oryzanol 0.049 ± 0.03
fatted rice bran (oil-pressed rice bran, approximately 50% defatted) was Ferulic acid 0.35 ± 0.02
treated by the process (Watanabe, Maeda, Koyama, Nakamura, &
Sasano, 2015). HSDFRB: heat-stabilized defatted rice bran. Experiments were carried out at
However, until now, the ability to recover protein from HSDFRB by least in triplicate.
using isoelectric precipitation combined with electrolyzed-water treat-
ment (IP-EWT) has not been demonstrated. Besides, qualities such as
hypoallergenicity and nutritional value of recovered protein derived compounds had been precipitated was mixed with the rice-bran residue
from HSDFRB by IP-EWT also has not been clarified. on a high torque stirrer (SW-RS077, Nisshin Rika Co., Ltd., Tokyo,
In present study, we evaluated the protein-recovery ratio and con- Japan) at a setting of 500 rpm for an arbitrary time. NaOH was added to
centration of protein recovered from HSDFRB by isoelectric precipita- the mixture to dissolve the rice bran protein and restore it to the su-
tion combined with IP-EWT. In addition, amino-acid score and rice pernatant. The mixture was then centrifuged at 5000 g for 10 min at
allergenic protein (RA) and other allergenic proteins were detected by ambient temperature and the supernatant was collected as protein ex-
immunoblotting analysis of the recovered protein concentrate. In par- tract and used to quantitatively analyzed. Protein was isoelectrically
ticular, as for practical food usage, quality and safety of protein derived precipitated from the protein extracts was adjusting the pH with acetic
from HSDFRB as a byproduct of rice-oil production was evaluated. acid within a pH range of 3.0–5.5. Crude rice protein was precipitated
by centrifugation at 5000 g for 10 min at ambient temperature and
2. Materials and methods washed with pH-adjusted electrolyzed water (EW) to remove water-
soluble impurities. Two-stage protein extraction from protein-extracted
2.1. Materials residue was also applied. The precipitate (protein-extracted residue, see
above) obtained after the protein had been extracted was mixed with
Defatted rice bran derived from brown rice (Oryza sativa, cv. arbitrary concentrations of NaOH solution on a high torque stirrer at a
Koshihikari, harvested in Niigata prefecture, Japan) was obtained from setting of 500 rpm for 1 h. In addition, the sample mixture was then
Sanwa-Yushi Co. Ltd. (HSDFRB; Yamagata, Japan), packaged in poly- ultrasonicated in an ultrasonicator (Model UP50H, Dr. Hielscher,
ethylene bags, and stored at 3 °C. The rice bran was pre-heated (heat- GmbH) for 2 min. After sonication, protein fractions were recovered
stabilized) at 110–125 °C before the defatting process (i.e., hexane ex- and purified from protein extracts by IP and EWT, respectively (see
traction). Rice oil was extracted from the heat-stabilized rice bran by above).
using hexane. Proximate composition of HSFRB indicated in Table 1. EW was prepared by using an electrolyzed-water ionizer (TK8051,
Panasonic Electric Works Co., Ltd., Shiga, Japan), which generated EW
2.2. Methods with different pHs by electrolysis with a tap-water system with volume
flow rate 2.5 l min−1. Four kinds of EW were used in the experiment:
2.2.1. Experimental procedures EW with pH 3 and oxidative-reduction potential (ORP) of +748 mV;
Rice protein and phosphorus concentrates were prepared by acid EW with pH 4 and ORP of +909 mV; EW with pH 5 and ORP of
and alkaline extraction, followed by isoelectric precipitation (IP), and +964 mV; and EW with pH 6 and ORP of +1025 mV.
washing with electrolyzed/distilled water (Watanabe et al., 2015).
Thirty grams of rice bran or a defatted-rice-bran sample and 300 ml
of deionized water (less than 2 μS cm−1) were placed in a vessel, and to 2.2.2. Quantification of protein of extracted protein concentrates
elute the phosphorus compounds from rice bran, pH was adjusted to 3.5 The protein contents in rice bran and extracted-rice-protein con-
by using a 6N HCl solution. After that, to separate eluted phosphorus centrates were determined by using a total-nitrogen analyzer (variMAX
compounds (supernatant) from rice bran residue (precipitate), the CN; Elementar Analysensysteme GmbH, Denmark) in compliance with
vessel was centrifuged at 5000 g for 10 min at ambient temperature. the improved Dumas method. The percentages of total-protein content
The supernatant was adjusted to pH 7.5 with 6N NaOH to precipitate were calculated from the output of the instrument by using a conver-
the phosphorus compounds and centrifuged at 5000 g for 10 min at sion factor of 5.95 (Tang, Hettiarachchy, Eswaranandam, & Crandall,
ambient temperature. The precipitated fraction containing the phos- 2003). Protein recovery ratio (%) from HSDFRB was calculated by the
phorus compounds was heat dried at 80 °C and stored in a desiccator for method proposed Watanabe et al. (2015).
later analysis. The supernatant obtained after the phosphorus

263
M. Watanabe et al. LWT - Food Science and Technology 99 (2019) 262–267

2.2.3. Electrophoretic analysis and amino-acid composition of extracted

protein concentrates derived from HSDFRB
Protein was purified by using a protein purification kit (ISOGEN-LS;
NIPPON GENE Co., Ltd., Toyama, Japan) in accordance with the
manufacturer's instructions. The extracted and purified proteins were
re-suspended in a Laemmli (Laemmli, 1970) sample buffer containing
mercaptoethanol, heated for several minutes to denature the proteins,
and centrifuged at 15,000 rpm for 5 min. The supernatant (protein
sample) was loaded onto 14% acrylamide gel and electrophoresed with
an electrophoresis system (type AE-6530; ATTO Co., Ltd., Japan).
Protein bands were observed by staining the gel with a solution con-
taining final concentration of 0.25% Coomassie Brililantblue R250,
45% 2-propanol, and 10% acetic acid, and destaining in a solution of
5% methanol and 7% acetic acid. The electrophoretic mobility of the
proteins derived from the solid particles was compared with that of the
marker protein (Low Molecular Weight; GE-Healthcare Co., Ltd., USA)
to determine the molecular mass of the proteins. Amino-acid compo-
sition was determined by an amino-acid analyzer (JEOL JLC-500/V2,
Japan) according to the procedure of Chang, Lee, and Brown (1986).

2.2.4. Immunoassay of extracted-protein concentrates derived from

HSDFRB Fig. 1. Effect of extraction temperature and pI value of IP-EWT on protein
To detect RAs (rice allergens) by immunoblotting analysis, mouse content (A) and recovery ratio (B) of protein concentrates extracted from
monoclonal antibody (25B9), specific to RAs and horseradish perox- HSDFRB. Bars indicate standard deviations (n = 3). Incubation was carried out
idase (HRP)-conjugated anti-mouse IgG (Jackson Immuno Research at arbitrary temperature (purple bar: 20 °C; blue bar: 30 °C; green bar: 40 °C;
Inc., PA, USA) were used. RAs are products of a multigene family and yellow bar 50 °C; red bar: 60 °C) for 2 h in 500-ml conical flask containing 30 g
have sequence similarity in relation to their amino-acid level from 70 to of rice bran and 300-ml NaOH solution.
95%, especially near the 1 C-terminus. 25B9 recognizes this site
(Alvarez et al., 1995); thus, the monoclonal antibody can direct all RAs.
RAs were analyzed by SDS-PAGE with 15%-acrylamide gel as men-
tioned previously and electrically transferred onto PVDF (poly vinyli-
dene difluoride) membrane according to the method of Towbin et al.
(Towbin, Staehelin, & Gordon, 1979) with a blotting system (type WSE-
4020; ATTO Co., Ltd., Japan). The blotted PVDF membrane was stained
with anti-RA antibody (25B9) and HRP-conjugated anti-mouse IgG
subsequently visualizing RAs by soaking membrane in EZ West Blue
(ATTO Co., Ltd., Japan) solution (Yamada et al., 2006).

3. Results and discussion

3.1. Effect of extraction temperature and pI value of IP-EWT on protein

content and recovery ratio of extracted protein concentrates derived from
HSDFRB

The effects of temperature of protein extraction and pI value of IP-

EWT on content and recovery ratio of the rice-bran protein after the
phosphorus compounds removed were investigated first. In the in-
vestigation, extraction time and sodium-hydroxide concentration were
fixed to 2 h and 1.5 w/v%, respectively. As shown in Fig. 1A, the pro-
tein content of the protein fraction extracted from HSDFRB tends to
decrease with increasing pI value of IP-EWT for all extraction tem-
peratures. Moreover, the optimal temperature of protein extraction was
50 °C for every pI value of IP-EWT. The effect of extraction temperature
and pI value of IP-EWT on recovery ratio is shown in Fig. 1B. It is clear
from the figure that the highest recovery ratio (30.3%) was attained
when extraction temperature of protein and pI value of IP-EWT were
50 °C and 3.5, respectively.
3.2. Effect of NaOH concentration and EWT on protein content and
recovery ratio of protein concentrates extracted from HSDFRB
To elucidate the effect of sodium-hydroxide concentration on solu-
bilization of HSDFRB protein in IP and IP-EWT, the effects of sodium-
hydroxide concentration on content and recovery ratio of the rice-bran
protein after the phosphorus compounds removed was investigated
next. In the investigation, extraction time and temperature were 2 h and
Fig. 2. Effect of NaOH concentration and EWT on protein content (A) and
recovery ratio (B) of protein concentrates extracted from HSDFRB. Bars
indicate standard deviations (n = 3). Incubation was carried out at 50 °C for 2 h
in 500-ml conical flask containing 30 g of rice bran and 300-ml NaOH solution.
50 °C, respectively. As shown in Fig. 2A, as sodium-hydroxide con-
centration in protein extraction decreases, protein content of protein
fraction recovered from HSDFRB decreases. The highest protein content
(65.1 w/w%) of the protein concentrate derived from HSDFRB was
attained by using a simultaneous recovery process of protein and
phosphorus compounds that includes IP-EWT under sodium hydroxide
concentration of 0.5 w/v%. This protein content exceeds those achieved
with reported protein concentrate (30–40 w/w%) derived from
HSDFRB (Prakash, 1996). It was reported that a protein concentrate
prepared by wet alkaline extraction (not including phosphorus re-
moval) of defatted rice bran contained 34–48% protein (which re-
presents 38–40% of the starting protein), 8–12% fat, and 25–30%

264
M. Watanabe et al.
starch (Connor, Saunders, & Kohler, 1976). We previously reported that
the highest protein content (52.3 w/w%) of protein concentrate derived
from machine-defatted rice bran (approximately 50% defatted) was
attained in comparison with full-fat rice bran by using a process (in-
cluding IP-EWT) for simultaneously recovering protein and phosphorus
compound (Watanabe et al., 2015). Besides, phosphorus removal before
recovering protein effectively decreased the insoluble form of phos-
phorus compounds in purified protein concentrate (Watanabe et al.,
2015). These findings suggest that recovery of high quality (protein
content) protein concentrate might be attained by using HSDFRB and
DFRB with high defatted level (low lipid-content value) as feed stock
for recovering protein concentrate and removing phosphorus before
recovering protein. The effect of NaOH concentration in protein ex-
traction by IP-EWT on protein-recovery ratio is shown in Fig. 2B.
Highest recovery ratio (33.1%) was attained with sodium-hydroxide
concentration of 0.8 w/v%. Hence, 2 h was selected as the standard
time for protein extraction throughout the experiment from the view-
point of reducing process energy in IP-EWT.
3.3. Protein extraction from protein extracted HSDFRB residue
A lower percentage of extracted protein from HSDFRB than from
non-heat-stabilized rice bran has been reported (Tang et al., 2003).
Protein yield in the supernatant fraction of FFU (full-fat unstabilized)
bran was higher than that in DF (defatted) and FFS (full-fat-stabilized)
brans, indicating higher extractability of protein from the FFU bran
(Anderson & Harmeet, 2001). In addition, the soluble protein content
varied with the level of processing of the protein-extraction phase.
Sonication produced high soluble-protein concentration in the FFU,
FFS, and DF samples. The effect of sonication on protein extractability
is greater than that of colloid milling (Anderson & Harmeet, 2001).
Therefore, to improve the recovery yield of protein concentrate from
HSDFRB, protein was extracted from protein-extracted HSDFRB (re-
sidue) as well as from HSDFRB under arbitrary NaOH concentration by
using ultrasonication. As shown Fig. 3A, the lowest concentration of
protein extracted from residue is attained under sodium-hydroxide
concentration of 0.8 w/v%. Moreover, on the high-sodium-hydroxide-
concentration (strong alkaline) side higher than 0.8 w/v%, protein
content only increase slightly. On the low-sodium-hydroxide-
Fig. 3. Effect of NaOH concentration for protein content (A) and recovery
ratio (B) of protein concentrates extracted from protein-extracted
HSDFRB. Bars indicate standard deviations (n = 3). Incubation was carried out
at 50 °C for 2 h in 500-ml conical flask containing 30 g of rice bran and 300-ml
NaOH solution.
265
LWT - Food Science and Technology 99 (2019) 262–267
concentration (weak alkaline) side, however, protein content increase
steeply. The highest protein content (57.4%) was attained under so-
dium hydroxide concentration of 0 w/v%. In addition, as shown in
Fig. 3B, protein-recovery ratio from residue also increases on the low-
sodium-hydroxide-concentration (weak alkaline) side.
Rice-bran protein is usually obtained through alkaline treatment be-
cause alkaline solutions (pH > 7) can enhance the solubility of rice
protein by accelerating denaturation and hydrolysis of rice protein (Cao,
Wen, Li, & Gu, 2009). Besides, alkaline treatment extends the protein's
tertiary structure, thereby allowing water molecules to penetrate inner
protein bodies and accelerate the solubility of RBP after high-pH treat-
ment (Wang et al., 2015). However, the sodium-hydroxide concentration
that gives the highest protein concentration and recovery ratio in protein
extraction differ in the case of protein-extracted HSDFRB (second ex-
traction, Fig. 2A and B) compared with HSDFRB (first extraction, Fig. 1A
and B) in spite of the same protein source. Related to that finding, ni-
trogen solubility of rice-bran protein is reduced by the effect of higher salt
concentration on ionic strength (Bera & Mukherjee, 1989). The effect of
ionic strength on protein solubility was understood as the effectiveness of
salting out of various ions in regard to globular proteins. The effectiveness
is much greater for anions given in decreasing order as
SO42− > HPO42− > CH3COO− > Cl− > Br− > I− > SCN− and
for cations given in decreasing order as
Li+ > Na+ ∼ K+ > NH4+ > Mg2+ (Curtis & Lue, 2006). It is con-
sidered that mineral content of protein-extracted HSDFRB is lower than
that of HSDFRB. This is because the minerals and some of the proteins of
HSDFRB are already removed (extracted) by IP-EWT (Watanabe et al.,
2015). Therefore, it is plausible that protein extractability from protein-
extracted HSDFRB is mainly stimulated by the effect of reducing the ef-
fectiveness of salting-out compared with the case of HSDFRB. Hence, the
sodium-hydroxide concentration that gives the highest protein con-
centration and recovery ratio in protein extraction of protein-extracted
HSDFRB might be shifted lower than that in the case of non-extracted
HSDFRB.
The effects of sonication on protein concentration and recovery
ratio of extracted protein concentrates from protein extracted HSDFRB
by IP-EWT are shown in Fig. 4. Maximum protein-recovery ratio was
obtained after two times of intermittent sonication (18.7%, Fig. 4A).
This increase of protein-recovery ratio might be attributed to such as
Fig. 4. Effect of sonication on protein concentration (A) and recovery ratio
(B) of protein concentrates extracted from protein-extracted HSDFRB by
IP-EWT.Bars indicate standard deviations (n = 3).
M. Watanabe et al. LWT - Food Science and Technology 99 (2019) 262–267

reducing particle size and/or increases of protein extractability of rice

bran (Anderson et al., 2001; Prakash & Ramanatham, 1994). Moreover,
as shown in Fig. 4B, maximum protein content (51.5%) of the protein
fraction was attained after one sonication. From two times of inter-
mittent sonication and more, protein content of the protein fraction
decreased with increasing number of sonication.
Consequently, due to the combination of (sodium-chloride-con-
centration-controlled) two-stage IP-EWT for HSDFRB and protein-ex-
tracted HSDFRB and ultra-sonication for protein-extracted HSDFRB,
protein content and total recovery ratio from HSDFRB of approximately
50% were attained. These results suggest that the combination of IP-
EWT and ultra-sonication can effectively recover protein concentrates
with higher content and recover protein from HSDFRB with higher
efficiency.

3.4. Amino-acid composition of protein concentrates extracted from Fig. 5. SDS-PAGE and immunoblotting analysis of protein concentrate derived
HSDFRB from HSDFRB. Protein concentrates were extracted from HSDFRB by IP-EWT
under respective pH conditions and subjected to the analysis. The gel sheets
were stained CBB (Panel A), and blotted membranes were immunostained with
The amino-acid compositions of protein concentrates extracted from
monoclonal antibody 25B9 specific for the 14–16 kDa allergen (panel B).
HSDFRB prepared by the combination of IP-EWT and ultra-sonication
and original HSDFRB are listed in Table 2. Glutamic acid, aspartic acid,
leucine, and arginine are contained as the main amino-acid components The protein concentrate used exhibits a protein band at less than
of the extracted protein concentrates. All of the limiting essential amino 14 kDa, 14–20 kDa, 20–29 kDa, 29–45 kDa and 45–66 kDa. The pattern
acids (namely, Thr, Val, Met, Ile, Leu, Phe, His, Lys and Trp) are also of protein bands (RB, HSDFRB, and protein concentrates) correspond
contained. These amino-acid profiles of protein correspond to other fairly well to the respective reported rice-protein constituents
reported protein concentrates derived from RB (untreated), acid-stabi- (Watanabe et al., 2011). Therefore, protein concentrates might be in-
lized RB (acid and heat-treated) and HSDFRB (Fabian & Ju, 2011; cluded in the original rice-bran protein such as glutelin, prolamin, al-
Prakash, 1996; Connor et al., 1976). Besides, the essential amino acid bumin, and globulin (Fabian & Ju, 2011).
index of the extracted protein concentrates and HSDFRB are 76.6 and The immunochemical relationship among RB, HSDFRB, and protein
83.0 (%), respectively, which meets the requirement for a two-year-old concentrates derived from HSDFRB by IP-EWT were investigated by
child recommended by the FAO/WHO/UNU (Tang et al., 2003). using monoclonal antibodies (Yamada et al., 2006). Protein fractions
Moreover, extracted protein concentrates and HSDFRB have more ba- were separated by SDS gel electrophoresis, transferred onto nitro-cel-
lanced amino acid composition than those of major cereals such as rice lulose sheets, and immunostained by using monoclonal antibody (see
(61%), wheat (39%) and corn (31%) due to its higher contents of lysine “Materials and Methods”). As shown in Fig. 5B, only in the allergen
and sulphur-containing amino acids (OECD, 2004). protein (14 kDa) as a positive control, the RB fraction is stained.
However, the fraction of HSDFRB and protein concentrates are not
stained, indicating that HSDFRB and protein concentrate derived from
3.5. Protein profile and immunoassay of extracted protein concentrates HSDFRB might be rice allergen-free protein source. In the process of
derived from HSDFRB rice-oil production from rice bran, rice bran was pre-heated (heat-sta-
bilized) at 110–125 °C before defatting (see “Materials and methods”).
The SDS-PAGE pattern of protein in RB, HSDFRB, and protein It is possible that some protein (including the rice allergen protein in
concentrate extracted from HSDFRB by IP-EWT are shown in Fig. 5A. HSDFRB) was denaturated or digested by the heat-stabilizing process,
so allergen activity decreased at the same time. These results suggest
Table 2
that protein concentrate derived from HSDFRB by IP-EWT might be
Amino acid composition of extracted protein concentrates derived from
applicable for food and nutrition usage as an allergen-free and safer
HSDFRB and HSDFRB.
protein source compared with rice bran.
Amino acid Extracted protein concentrate from HSDFRB HSDFRB
(mg/g-N)
4. Conclusion
Asp 534.9 ± 14.3 539.6 ± 15.2
Thr 233.2 ± 8.2 223.7 ± 7.9
Ser 274.3 ± 7.6 250.0 ± 8.9 In this study, it was demonstrated that isoelectronic precipitation
Glu 850.4 ± 17.2 720.6 ± 19.3 and electrolyzed water treatment (IP-EWT) enable simultaneous re-
Pro 274.3 ± 5.6 256.6 ± 4.1 covery of protein and phosphorus compounds from heat-stabilized de-
Gly 315.5 ± 4.2 315.8 ± 4.6
Ala 384.1 ± 5.8 352.1 ± 7.4
fatted rice bran (HSDFRB). Besides, the supernatant obtained after the
Val 370.3 ± 3.5 309.3 ± 2.9 phosphorus compounds had been precipitated can be reused as the
Cys 35.6 ± 0.8 128.3 ± 1.8 extraction solvent of protein within IP-EWT. The highest protein con-
Met 137.1 ± 2.1 102.0 ± 3.5 tent (65.1 w/w%) and highest recovery ratio (over 50%) of protein
Ile 246.9 ± 3.3 197.4 ± 3.8
concentrate were attained by the synergistic effect of this two-stage
Leu 493.8 ± 8.7 394.8 ± 6.9
Tyr 205.8 ± 4.2 148.1 ± 4.1 protein extraction and IP-EWT. Analysis of amino-acid composition in
Phe 301.8 ± 5.3 246.7 ± 3.7 the protein fraction demonstrated excellent amino acid value (76.6%)
His 246.9 ± 7.3 177.6 ± 5.2 which value is higher than those of major cereals such as wheat and
Lys 260.6 ± 6.6 299.4 ± 4.1 corn. In addition, neither rice allergenic protein nor heavy metals in
Trp 97.4 ± 2.1 75.6 ± 1.9
Arg 480.0 ± 9.2 437.6 ± 8.8
either HSDFRB or protein fractions extracted from IP-EWT were de-
tected by SDS-PAGE and immunoblotting analysis. These results suggest
HSDFRB: heat-stabilized defatted rice bran. Experiments were carried out at that as an eco-friendly, the newly developed, combined process (IP-
least in triplicate. EWT) is suitable for practical recovery of highly concentrated and safe

266
M. Watanabe et al.
protein from HSDFRB without using enzymes or chemicals such as or-
ganic solvents, buffering agents, and surfactants. In addition, This IP-
EWT may applicable not only for rice protein but also for the other
protein containing unutilized resources and biomass as new eco-
friendly protein recovery/purification technology.
From the viewpoints of food and nutraceutical application, detailed
information such as solubility, forming and emulsifying properties are
necessary for practical application of protein recovered with the pro-
posed process. These approaches are currently being investigated.
Acknowledgement
This work was supported by Japan Science and Technology Agency
Adaptable and Seamless Technology Transfer Program through Target-
driven R&D (A-STEP) Grant Number AS262Z01572N, Faculty of Agro-
Industry, Chiang Mai University for funding (JRP.13012560), National
Research University – Chiang Mai University (NRU - CMU), and
National Research University – Office of Higher Education Commission,
Ministry of Education of Thailand (NRU - OHEC) was carried out as
collaborative research in the Core to Core Program (Advanced Research
Networks), which was supported by the Scientific Cooperation Program
of the Japan Society for the Promotion of Science (JSPS), the National
Research Council of Thailand, and the universities involved in the
program.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.lwt.2018.09.059.
References
Alvarez, A. M., Fukuhara, E., Nakase, M., Adachi, T., Aoki, N., Nakamura, R., et al.
(1995). Four rice seed cDNA clones belonging to the alpha-amylase/trypsin inhibitor
gene family encode potential rice allergens. Bioscience Biotechnology and Biochemistry,
59(7), 1304–1308.
Anderson, A. K., & Harmeet, S. G. (2001). Extractability of protein in physically processed
rice bran. Journal of the American Oil Chemists’ Society, 78(9), 969–972.
Bera, M. B., & Mukherjee, R. K. (1989). Solubility, emulsifying, and forming properties of
rice bran protein concentrates. Journal of Food Science, 54(1), 142–145.
Cao, X., Wen, H., Li, C., & Gu, Z. (2009). Differences in functional properties and bio-
chemical characteristics of congenetic rice proteins. Journal of Cereal Science, 50(2),
184–189.
Chang, K. C., Lee, C. C., & Brown, G. (1986). Production and nutritional evaluation of
high-protein rice flour. Journal of Food Science, 51(2), 464–467.
Chen, L., & Houston, D. F. (1970). Solubilization and recovery of protein from defatted
267
LWT - Food Science and Technology 99 (2019) 262–267
rice bran. Cereal Chemistry, 47, 72–79.
Connor, M. A., Saunders, R. M., & Kohler, G. O. (1976). Rice bran protein concentrates
obtained by wet alkaline extraction. Cereal Chemistry, 53, 488–496.
Curtis, R. A., & Lue, L. (2006). A molecular approach to bioseparations: Protein-protein-
salt interactions. Chemical Engineering Science, 61(3), 907–923.
Fabian, C., & Ju, Y. H. (2011). A Review on rice bran protein: Its properties and extraction
methods. Critical Reviews in Food Science and Nutrition, 51(9), 816–827.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature, 227(5259), 680–685.
Lazaridou, A., Duta, D., Papageorgiou, M., Belc, N., & Biliaderis, C. G. (2007). Effects of
hydrocolloids on dough rheology and bread quality parameters in gluten-free for-
mulations. Journal of Food Engineering, 79(3), 1033–1047.
Lew, E. J. L., Houston, D. F., & Fellers, D. A. (1975). A note on protein concentrate from
full fat rice bran. Cereal Chemistry, 52(5), 748–750.
OECD (2004). Series on the safety of novel food and feed No. 10. Consensus document on
compositional considerations for new variety of rice (Oryza sativa): Key food and feed
nutrients and anti-nutrients. Paris, France: OECD.
Prakash, J. (1996). Rice bran protein: Properties and food uses. Critical Reviews in Food
Science and Nutrition, 36(6), 537–552.
Prakash, J., & Ramanatham, G. (1994). Effect of stabilization of rice bran on the ex-
tractability and recovery of proteins. Nahrung, 38(1), 87–95.
Rubio-Tapia, A., Ludvigsson, J. F., Brantner, T. L., Murray, J. A., & Everhart, J. E. (2012).
The prevalence of celiac disease in the United States. American Journal of
Gastroenterology, 107(10), 1538–1544.
Tang, S., Hettiarachchy, N. S., Eswaranandam, S., & Crandall, P. (2003). Protein extrac-
tion from heat-stabilized defatted rice bran: II. The role of amylase, celluclast and
viscozyme. Journal of. Food Science, 68(2), 471–475.
Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: Procedure and some applications.
Proceeding of the National Academy of Sciences of the United States of America, 76(9),
4350–4354.
Tsatsaragkou, K., Gounaropoulos, G., & Mandala, I. (2014). Development of gluten free
bread containing carob flour and resistant starch. LWT-Food Science and Technology,
58(1), 124–129.
Wang, T., Liu, F., Wang, R., Wang, L., Zhang, H., & Chen, Z. (2015). Solubilization by
freeze-milling of water-insoluble subunits in rice proteins. Food & Function, 6(2),
423–430.
Watanabe, M., Ichinose, K., Sasano, K., Ozaki, Y., Tsuiki, T., Hidaka, H., et al. (2011).
Effect of enzymatic treatment on sedimentation and flocculation abilities of solid
particles in rice washing drainage and its relationship with protein profiles. Journal of
Bioscience and Bioengineering, 112(1), 67–70.
Watanabe, M., Maeda, I., Koyama, M., Nakamura, K., & Sasano, K. (2015). Simultaneous
recovery and purification of rice protein and phosphorus compounds from full-fat and
defatted rice bran with organic solvent-free process. Journal of Bioscience and
Bioengineering, 119(2), 206–211.
Wiboonsirikul, J., Kimura, Y., Kanaya, Y., Tsuno, T., & Adachi, S. (2008). Production and
characterization of functional substances from a by-product of rice bran oil and
protein production by a compressed hot water treatment. Bioscience Biotechnology and
Biochemistry, 72(2), 384–392.
Yamada, C., Yamashita, Y., Seki, R., Izumi, H., Matsuda, T., & Kato, Y. (2006). Digestion
and gastrointestinal absorption of the 14-16 kDa rice allergens. Bioscience
Biotechnology and Biochemistry, 70(8), 1890–1897.
Zhang, H. J., Wang, J., Zhang, B. H., & Zhang, H. (2014). Antioxidant activities of the
fractionated protein hydrolysates from heat stable defatted rice bran. International
Journal of Food Science and Technology, 49(5), 1330–1336.