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Detection of Soybean Proteins in Fermented

Soybean Products by Using Heating Extraction


Naoki Morishita, Takashi Matsumoto, Fumiki Morimatsu, and Masatake Toyoda

Soybean is used in processed foods worldwide. Because soybean can cause adverse reactions in some atopic
patients, appropriate labeling regarding its content in processed foods is needed to better protect consumers. In the
previous study, we developed a reliable sandwich Enzyme Linked Immunosorbent Assay (ELISA) method with high
sensitivity and specificity for detecting soybean proteins by using antibody to Gly m Bd 30K, which was originally
characterized as a vacuolar protein with a molecular mass of 34 kDa in soybean. The ELISA displayed satisfactory
repeatability and reproducibility in an interlaboratory evaluation. However, it could not detect soybean protein in
fermented soybean products. We therefore developed an extraction method combined with a heating process to inhibit
soybean protein degradation by microbial proteolytic enzymes in fermented soybean products. This extraction method
enables the sensitive detection of soybean protein in fermented soybean products such as natto and miso. It was able to
detect with high-sensitivity soybean protein present at 10 g/g levels in model processed foods. This method is suitable
for quantifying soybean protein in processed foods without the degrading effects of microbial proteolytic enzymes. The
present extraction method can be used sensitively to monitor labeling systems in a reliable manner and should be useful
for the mandatory inspections required under Japanese regulations.

Abstract:

Keywords: Enzyme Linked Immunosorbent Assay (ELISA) kit, fermentation, food allergen, food safety, soybean

The extraction and ELISA methods that we developed enable sensitive detection of soybean protein
in soybean products, including fermented foods. These methods should be useful for reliable and sensitive monitoring of
product labeling systems and should help to solve the problem of insensitive in soybean labeling of processed foods.

Introduction
Food allergies are important diseases. The clinical manifestations of food allergies vary from mild symptoms such as oral allergy syndrome or mild urticaria to severe anaphylactic reactions
with fatal consequences. Approximately, 8% of children and 2%
of adults in industrialized countries have food allergies (Jansen
and others 1994; Ebisawa and others 2003). To prevent possible
life-threatening reactions, the only effective treatment is to strictly
avoid the consumption of these allergenic foods. We therefore
need sufficient information about potentially allergenic ingredients in food products. In 1999, the Joint FAOWHO Codex Alimentary Commission agreed to label 8 kinds of food ingredient
that have components known to be allergenic; these include soybean (FAO of the United Nations 1995). In Japan, the Ministry of
Health, Labor, and Welfare (MHLW) has enforced a labeling system for allergenic food materials since April 2002 (MHLW 2002).
In this system, labeling for 7 food ingredients (egg, milk, wheat,
buckwheat, peanuts, shrimp, and crab) is mandatory, and labeling
is recommended for 20 other food materials, including soybean
and walnut. Because of the almost unlimited uses for soybean and
the fact that the number of patients with soybean allergy has been
increasing, the most important of these recommended labelings is

MS 20131805 Submitted 12/3/2013, Accepted 3/12/2014. Authors Morishita,


Matsumoto, and Morimatsu are with Nippon Meat Packers. Inc., 3-3 Midorigahara,
Tsukuba, Ibaraki, 300-2646, Japan. Author Toyoda is with Jissen Womens Univ.,
1-1 Oosakaue 1-chome, Hino, Tokyo, 191-8510, Japan. Direct inquiries to author
Morishita (E-mail: n.morishita@nipponham.co.jp).
Author disclosures: None.
R

C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12461
Further reproduction without permission is prohibited

that for soybean (Savage and others 2010). The high functionality
and good processing properties of soybean have promoted its use
in processed foods (for example, tofu, soybean milk, meat alternatives, and fermented soybean products) worldwide. Fermented
soybean products such as soy sauce, miso, natto, and tempeh are
produced by fermentation processes using microorganisms. These
foods are traditional in Japan and other Asian countries (Golbitz 1995; Murooka and Yamshita 2008; Phromraksa and others
2008; Namgung and others 2010). Fermented soybean products
retain their allergenicity (Astwood and others 1996; Tsuji and others 1997; Ito and others 2005; Kobayashi 2005; Frias and others
2008; Phromraksa and others 2008; Inomata and others 2012).
Accordingly, to better protect the consumer, we need adequate
labeling information about the presence of soybean in processed
foods. In the previous study, we developed a reliable sandwich Enzyme Linked Immunosorbent Assay (ELISA) method with high
sensitivity and specificity for detecting soybean proteins by using antibody to Gly m Bd 30K, which has been characterized
as a vacuolar protein with a molecular mass of 34 kDa (Morishita and others 2008). The ELISA displayed sufficient repeatability and reproducibility in an interlaboratory evaluation (Sakai
and others 2010). However, it cannot detect soybean protein in
fermented soybean products (Morishita and others 2008). Other
studies aimed at detecting soybean protein by using ELISA have
been reported (Brandon and others 1991; Tsuji and others 1997;
Bando and others 1998; Moriyama and others 2005; Hei and others 2012). However, reliable measurement using these methods is
considered difficult in processed foods because of cross-reactivity
and low repeatability and reproducibility. In addition, these methods cannot detect soybean protein in fermented soybean products.

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Practical Application:

Detection of soybean in fermented food . . .


We therefore developed a present extraction method that included
a heating process to detect soybean protein in fermented soybean
products. We showed that the present extraction method could
be used to detect soybean protein in fermented soybean products
because the microbial proteolytic enzymes in these products were
deactivated by the heating process.

Materials and Methods

and stored as the calibration standard solution for ELISA at 4 C.


All experiments were performed using this calibration standard
solution.
To prepare a standard solution for western blot, the initial extract
was mixed 1:1 with Laemmli buffer (Bio-Rad Laboratories, Inc.,
Hercules, Calif., U.S.A.) containing 2.5% 2-ME. The sample was
boiled at 100 C for 5 min and then cooled in flowing water. It
was stored at 4 C.

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Food materials
Preparation of model processed foods
Soybeans and commercial processed foods were purchased at
The best sources of information on the performance of methlocal supermarkets (Ibaraki, Japan) between 2009 and 2012.
ods used to detect allergens are model processed foods. Three
kinds of model processed food (hamburger steak, rice gruel, and
Chemicals and reagents
sweet adzuki bean soup) were prepared in accordance with the
Acetone, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), method used in a previous report (Morishita and others 2008).
hexane, 2-mercaptoehanol (2-ME), methanol, polyoxyethylene- To give a final protein concentration of 10 g/g (soybean prosorbitan monolaurate (Tween 20), sodium chloride (NaCl), tein weight/sample weight) in the model processed foods, the
sodium dodecyl sulfate (SDS), sodium hydrogen carbonate amount of soybean powder used to spike each model processed
(NaHCO3 ), sucrose, and sulfuric acid (H2 SO4 ) were purchased food was calculated. The individual ingredients were spiked with
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Bovine the soybean powder and then cooked according to the following
serum albumin (BSA) and phosphate-buffered salts (PBS) were method. Hamburger steak consists of pork, salt, sugar, ice water,
purchased from Sigma-Aldrich Co. (St. Louis, Mo., U.S.A.) and and soybean powder. The hamburger steak was packed in a retort
Takara Bio, Inc. (Shiga, Japan), respectively.
pouch (SP-1000K, Mita Rika Kogyo Co., Ltd., Osaka, Japan) and
cooked at 80 C for 20 min. Rice gruel consists of rice, sugar,
Protein extraction from food samples
water, and soybean powder. The rice gruel was packed in a retort
Protein was extracted from food samples in accordance with a pouch and cooked at 121 C for 10 min. Sweet adzuki bean soup
previously reported method (MHLW 2002; Morishita and others consists of adzuki beans, sugar, water, and soybean powder. The
2008; Sakai and others 2010). Food samples were homogenized sweet adzuki bean soup was packed in a retort pouch and cooked
in a food processor (IFM-700G, Iwatani Intl. Co., Osaka, Japan). at 100 C for 10 min. The cooked model processed foods were
Nineteen milliliters of extraction buffer (120 mM Tris-HCl [pH cooled by passing the retort pouch under flowing water and then
7.4], 0.1% BSA, 0.05% Tween 20, 2% SDS, 2% 2-ME) was added stored at 20 C.
to 1 g of food homogenate. The mixture was then extracted
for 16 h by shaking at 90 to 110 rpm. This extraction method ELISA
was defined as the previous extraction method. In contrast,
Polyclonal antibody against p34, the allergenic protein Gly m
with the present extraction method we used heating instead of Bd 30K, was prepared according to the method used in a preshaking. The present method was as follows. Nineteen milliliters vious report (Morishita and others 2008). Three hundred grams
of extraction buffer was added to 1 g of food homogenate and 5 of soybean was homogenized in 2 L of a 0.1-M Tris-HCl (pH
glass beads 5 mm in diameter to make the good dispersibility of the 8.6), 10 mM 2-ME mixture with a juicer (MX-X6, Matsushita
sample in the extraction buffer for heating process. The mixture Electric Industrial Co., Ltd., Osaka, Japan). The soybean same
was extracted by heating at different temperatures (25, 40, 60, 80, as the soybean used for to make the standard for the calibration
or 100 C) in a water bath for different times (5, 15, or 60 min), curve was used. The homogenate was centrifuged at 20000g to
with vortexing every 5 min. Food extracts produced by using the collect the oil-body pad. The oil-body pad was resuspended in
previous and the present methods were centrifuged at 3000g for a 5-time volume of 0.1 M Na2 CO3 for 1 h on ice, and the su20 min, and the supernatant was filtered through a filter paper (5A pernatant containing the protein was collected by centrifugation
Filter Paper, Toyo Roshi Kaisya, Ltd., Tokyo, Japan). The filtrate at 20000g. The p34 was purified by gel filtration chromatography
was collected as the food sample extract and used immediately for (Superdex 75, 26/90 cm, GE Healthcare UK Ltd.) equilibrated
analysis.
with 0.1 M Na2 CO3 and then used as an antigen to obtain the
antibody. The antigen was mixed 1:1 with Freunds complete adPreparation of calibration standard solutions
juvant and injected into Japanese white female rabbits as a 1st
To detect soybean proteins by using ELISA, a calibration stan- injection. After the 1st injection, injections of antigen and Fredard solution needs to be prepared. This solution was prepared in unds incomplete adjuvant were performed 4 more times at 2-wk
accordance with the official Japanese guideline (MHLW 2002). A intervals over a total period of 8 wk. One week after the final
300-mg sample of soybean powder was added to a 20-mL mixture injection, whole blood was collected. The antibody was purified
of 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 0.5% SDS, and 2% against the affinity-column-fixed antigen. The care and use of the
2-ME. The mixture was then shaken for 16 h at room temper- experimental animals followed the Ethical Guidelines of Aniature for extraction. The extract was centrifuged at 10000g for mal Care, Handling, and Termination prepared by Nippon Meat
30 min, and the supernatant was filtered through a 0.8-m mi- Packers.
A microtiter plate (F8 Maxisorp Nunc-Immuno module, Nunc,
crofilter paper (DISMIC-25CS, Toyo Roshi Kaisya Ltd.). The
protein content of the initial extract was assayed with a 2-D Roskilde, Denmark) was coated with the diluted polyclonal antiQuant Kit (GE Healthcare UK Ltd., Amersham, England). The body against p34 in 1 M NaHCO3 (pH 8.5) (100 L/well) and
initial extract was diluted to 50 ng/mL with 0.1 M PBS (pH incubated for 16 h at 4 C. After 2 washings with a washing buffer
7.4), 0.1% SDS, 0.1% 2-ME, 0.1% BSA, and 0.1% Tween 20 (100 mM PBS [pH 7.4], 1% Tween 20, 250 L/well), the plate
T1050 Journal of Food Science r Vol. 79, Nr. 5, 2014

Detection of soybean in fermented food . . .

Results and Discussion

Development of extraction method with a heating process


In previous research, we developed a reliable sandwich ELISA
method with high sensitivity and specificity for detecting soybean
proteins by using antibody to Gly m Bd 30K (Morishita and others
2008; Sakai and others 2010). The limit of detection of the ELISA
was 1 g/g. However, the ELISA could not detect soybean proteins in fermented soybean products, likely because of degradation
of the soybean proteins by microbial proteolytic enzymes remaining in the fermented products. Figure 1 shows the results of sodium
dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE)
analyses of natto and miso from which proteins were extracted
by using the previous extraction method (extraction for 16 h with
shaking at 90 to 110 rpm). A band of high molecular mass, including BSA with a molecular mass of 66 kDa, was not observed in
either the natto or the miso extract after application of the previous
extraction method (lane 2). Thus the soybean protein and BSA in
the extraction buffer were degraded into low-molecular-weight
peptides by the microbial proteolytic enzymes remaining in the
natto or miso after the 16 h extraction. The microbial proteolytic
enzymes likely inhibited the detection of soybean protein in the
fermented soybean products.
We developed the present extraction method to inhibit protein degradation by the microbial proteolytic enzymes. Generally,
heating, pH, and protease inhibitors are used to inhibit microbial
proteolytic enzymes. Because of utility and cost considerations,
we selected an extraction method that uses heating. To determine
the best heating temperature and time for inhibiting microbial
proteolytic enzymes, we examined different heating temperatures
and extraction times (Figure 1 and 2).
Bands of high molecular mass were observed in both natto and
Western blot
miso upon extraction using heating at 80 or 100 C (Figure 1).
Western blot using monoclonal antibody against p34 was per- On the other hand, no high-molecular-mass bands were detected
formed to detect p34 in fermented soybean products. Monoclonal antibody against p34 was obtained from Kansai Univ. of
Welfare Sciences (Ogawa and others 1991, 1993). The sample
solution was mixed 1:1 with Laemmli buffer (Bio-Rad Laboratories) containing 2.5% 2-ME. The sample was boiled at 100 C
for 5 min and then cooled in flowing water. The sample and a
molecular weight marker (Precision Plus Protein All Blue Standard, Bio-Rad Laboratories) were applied at 10 L/lane to 15%
separation gel (E-T15L, Atto Co., Tokyo, Japan) in a running
buffer (10 TrisglycineSDS buffer, pH 8.3, Bio-Rad Laboratories). Electrophoresis was performed at a constant electric current
of 20 mA/gel. The protein was blotted onto a PolyVinylidene
DiFluoride (PVDF) membrane (Hypond-P, Wako Pure Chemical
Industries, Ltd.) in a blotting buffer [a mixture of 10 Tris/glycine
buffer, pH 8.3 (Bio-Rad Laboratories), methanol, and distilled water at 1:2:7] by using a blotting system (Trans-Blot SD Cell, BioRad Laboratories). Blotting was performed at a constant electric
current of 2 mA/cm2 for 1 h. The membrane was blocked for 30
min at 25 C with 1 TBS (Bio-Rad Laboratories) containing
0.1% BSA and 0.05% Tween 20. After 3 washings with a washing buffer (1 TBS containing 0.05% Tween 20), the membrane
was shaken with a diluted monoclonal antibody against p34 in
blocking buffer for 1 h at 25 C. After 3 washings, the mem- Figure 1SDSPAGE analyses of natto and miso subjected to extraction by
brane was shaken with a diluted peroxidase-labeled anti-mouse using the previous method or to heating at different temperatures (25, 40,
IgG antibody (Envision System, Dako Japan, Inc., Tokyo, Japan) 60, 80, or 100 C) in a water bath for 15 min. (A) SDSPAGE of natto; (B)
in blocking buffer for 30 min at 25 C. After 3 washings, the SDSPAGE of miso. Lanes: 1, molecular weight markers (250, 150, 100,
membrane was detected with 3,3 -5,5 -tetramethylbenzidine (Ez 75, 50, 37, 25, 20, 15, 10 kDa); 2, previous method (nonheating extraction
for 16 h); 3, heating extraction at 25 C; 4, heating extraction at 40 C;
Westblue, Atto Co.). The reaction was stopped by adding distilled 5, heating extraction at 60 C; 6, heating extraction at 80 C; 7, heating
water.
extraction at 100 C. Arrow = BSA band (molecular weight 66 kDa).
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was blocked with a blocking buffer (100 mM PBS [pH 7.4], 1%


BSA, 5% sucrose, 200 L/well) for 3 h at 25 C. The blocking
buffer was removed, and then the plate was dried for 16 h at 25 C.
The food sample extract was diluted 1:19 in dilution buffer A (100
mM PBS [pH 7.4], 0.1% BSA, 0.1% Tween 20, 0.1% SDS, 0.1%
2-ME). The calibration standard solution was diluted to concentrations of 0.78, 1.56, 3.13, 6.25, 12.5, 25, and 50 ng/mL with
dilution buffer B (a mixture of dilution buffer A and extraction
buffer at 19:1). The diluted food sample extract and the calibration standard solution were added to the plate (100 L/well) and
incubated for 1 h at 25 C. All experiments were performed in
triplicate. After 5 washings, diluted biotin-labeled polyclonal antibody against p34 (100 L/well) was added to the plate and the
mixture was then incubated for 1 h at 25 C. After the plate has
been washed, a diluted horseradish peroxidase-conjugated streptavidin (Poly-HRP streptavidin N200, Thermo Fisher Scientific
Inc., Rockford, Ill., U.S.A.; 100 L/well) was added to the plate
and the mixture was incubated for 30 min at 25 C. After the plate
had been washed again, 3,3 -5,5 -tetramethylbenzidine (TMBE1000, Moss, Inc., Pasadena, Md., U.S.A.; 100 L/well) was added
and the plate was incubated for 20 min at 25 C. The reaction was
stopped by the addition 100 L of 0.25 M H2 SO4 . Absorbance was
measured with a plate reader (Sunrise 2000, Wako Pure Chemical Industries, Ltd.) at dominant and subdominant wavelengths of
450 and 620 nm, respectively. A 4-parameter logistic analysis was
used to calculate the concentration of the soybean soluble protein in the diluted food sample extract. The value of the soybean
soluble protein in the foods was converted by using a dilution
factor (400).

Detection of soybean in fermented food . . .


Table 1Detection of soybean protein in model processed foods.
Previous extraction method
Food
Hamburger steak
Rice gruel
Sweet adzuki bean soup

Present extraction method

Mean (g/g)

Recovery (%)

Mean (g/g)

Recovery (%)

Present method / previous method (%)

10.3
10.5
10.4

103.0
104.8
103.7

9.8
9.5
8.9

98.0
94.8
89.0

95.1
90.5
85.8

Detection of soybean protein in 3 kinds of model processed food (hamburger steak, rice gruel, and sweet adzuki bean soup) by using the present and previous extraction methods.
The model processed foods contained soybean protein at approximately 10 g/g. Mean = average concentration (soybean protein weight/food weight) of soybean proteins.
Recovery = mean/10 (g/g).

in natto or miso upon extraction using heating at 25 or 40 C.


Temperatures of 80 C or more therefore likely inhibited the
degradation of soybean protein by microbial proteolytic enzymes.
Degradation of soybean protein in natto was stronger than in miso.
We speculated that this difference in degradation was due to differences in the fermentation process used to make natto and miso.
Microbial proteolytic enzymes remain in natto, because heating is
not performed to destroy microorganisms after fermentation.
We performed SDSPAGE analyses of natto and miso after
extraction by heating at 80 C in a water bath for different times
(5, 15, or 60 min) (Figure 2). Bands of high molecular mass were
detected in both natto and miso after extraction for 15 or 60 min.
These results suggested that the suitable heating time for extraction
was 15 min or more. Accordingly, the heating conditions chosen
to optimize the inhibition of microbial proteolytic enzymes were
80 C for 15 min. We therefore set the heating temperature for
the extraction to 80 C and the time to 15 min. This protocol was
called the present extraction method.

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Figure 2SDSPAGE analyses of natto and miso subjected to extraction


by heating at 80 C in a water bath for different times (5, 15, or 60 min).
(A) SDSPAGE of natto; (B) SDSPAGE of miso. Lanes: 1, molecular weight
markers (250, 150, 100, 75, 50, 37, 25, 20, 15, 10 kDa); 2, heating for
5 min; 3, heating for 15 min; 4, heating for 60 min. Arrow = BSA band
(molecular weight 66 kDa).

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Table 2Detection of soybean protein in commercial processed


foods.
Previous
extraction
method
Food
Roasted soybean flour
Soybean milk
Yuba (bean curd skin)
Soybean boiled in water
Tofu
Freeze-dried tofu
Fried tofu
Soy pulp

Present
Present
extraction method / previous
method
method

Mean (mg/g) Mean (mg/g)


104.6
41.7
58.6
51.4
43.5
330.0
105.8
19.6

104.8
42.5
50.2
48.0
34.0
318.2
98.1
23.8

(%)
100.2
101.9
85.6
93.4
78.2
96.4
92.7
121.2

Detection of soybean proteins in 8 kinds of commercial food by using the present and
previous extraction methods. Mean = average concentration (soybean protein
weight/food weight) of soybean proteins. Recovery = mean/10 (g/g).

Detection of soybean proteins in model processed foods


and commercial processed foods by using the present
extraction method
We developed a present extraction method that used heating to
inhibit the degradation of soybean protein in fermented soybean
products by microbial proteolytic enzymes. The conditions of this
present method were 80 C for 15 min. In contrast, in previous
research, we developed a highly sensitive and specific ELISA using the previous extraction method, namely extraction for 16 h
with shaking at 90 to 110 rpm. The extraction time used in the
present method was therefore much shorter than that used in the
previous one. In Japan, the threshold for food allergen labeling in
processed foods has been set at 10 g/g (MHLW 2002). Therefore, the present extraction method needed to be able to sensitively
detect the relevant soybean protein at 10 g/g levels in processed
foods (Matsuda and others 2006). To evaluate the capacity of the
present extraction method, we performed a recovery study using incurred model processed foods (Table 1). The recoveries of
soybean protein by using the previous extraction method were
103.0%, 104.8%, and 103.7% in hamburger steak, rice gruel, and
sweet adzuki bean soup, respectively. The recoveries of soybean
protein using the present extraction method were 98.0%, 94.8%,
and 89.0% in hamburger steak, rice gruel, and sweet adzuki bean
soup, respectively. The correlation between the results of the previous and present extraction methods was 85.8% to 95.1%. Thus
the present extraction method had good extraction ability that
was equal to that of the previous method. In addition, the present
extraction method shortened the extraction time from 16 h to
15 min.
To evaluate the usefulness of the present method, 8 kinds of
commercial processed food that included highly concentrated
soybean were subjected to extraction by using the previous and
present methods; the extracts were then analyzed by using ELISA
(Table 2). The correlation between the results of the previous and

Detection of soybean in fermented food . . .


Table 3Detection of soybean protein in fermented soybean peh, douchi, and tofu-yoh were higher with the present method
products.
than with the previous one (Table 3). The results with the present
Previous
extraction
method

Present
extraction
method

Present
method/previous
method

Mean
(g/g)

Mean
(g/g)

(%)

Product
Soybean miso
Rice miso
Barley miso
Raw miso
Soy sauce
Natto
Crushed natto
Tempeh
Tianmianjiang
Douchi
Tofu-yoh
Soybean milk yoghurt

322.2
2550.8
474.5
743.3
nd
0.8
0.7
46436.0
2056.8
2.5
4.9
26184.0

4022.4
6795.6
2347.6
4223.2
nd
1402.0
835.6
67420.0
1987.6
13079.2
11294.0
26856.0

>1000.0
266.4
494.8
568.2

>1000.0
>1000.0
145.2
96.6
>1000.0
>1000.0
102.6

method were equal to those with the previous one in the case of
tianmianjiang and soybean milk yogurt. Soybean proteins could
be not detected in soy sauce with either method. These results
showed that the present method was able to detect soybean proteins sensitively in most fermented soybean products.
We examined the results of western blot analyses of fermented soybean products using monoclonal antibody against p34
(Figure 3). With the present method, bands of p34 were detected
in all fermented soybean products except soy sauce, though that
of p34 were detected very slightly in natto and crashed natto.
In contrast, with the previous extraction method the western

Detection of soybean protein in 12 kinds of fermented soybean product by using the


previous and present extraction methods. Mean = average concentration (soybean
protein weight/food weight) of soybean proteins; Recovery = mean/10 (g/g); nd =
not detected (<0.03 g/g);
Soybean miso = miso fermented with malted soybean; rice miso = miso fermented
with malted rice; barley miso = miso fermented with malted barley; raw miso =
unheated miso; crushed natto = natto made from ground soybeans; tianmianjiang =
sweet soybean paste; douchi = fermented black soybeans; tofu-yoh = fermented tofu.

Detection of soybean protein in fermented soybean


products by using the present extraction method
Previous studies have detected soybean proteins by using ELISA
based on antibodies to soybean proteins such as the trypsin inhibitor 7S-conglycinin and 11S glycinin (Hitchcock and others
1981; Ravestein and Driedonks 1986; Brandon and others 1991;
Koppelman and others 2004). However, to our knowledge there
have been no reports of the successful detection of soybean proteins
in fermented soybean products. Hei and others (2012) constructed
a sandwich ELISA based an antibody to -conglycinin, but this
method could not detect sufficient soybean protein in fermented
soybean products. Tsuji and others (1995, 1997) constructed a
sandwich ELISA method based on a monoclonal antibody to p34,
but this method could not detect soybean protein in miso, natto,
or soy sauce. We speculated that the microbial proteolytic enzymes in fermented soybean products cause the degradation of
soybean proteins, because neither of these methods used heating
as part of the extraction process. On the other hand, Moriyama
and others (2005) developed a sandwich ELISA based an antibody
to -conglycinin and heating extraction for 5 min. Their method
was able slightly to detect soybean proteins in natto. We consider
that this poor detection ability was due to the short heating time
used in the extraction.
We developed our present method so as to inhibit the degradation of soybean proteins by the microbial proteolytic enzymes in fermented soybean products. To evaluate the usefulness of the method, we subjected 12 kinds of fermented
soybean product to extraction by using the previous and
present methods, and we analyzed the results by using ELISA
(Table 3) and western blot analysis with a monoclonal antibody
against p34 (Figure 3). The concentrations of soybean proteins
detected in 4 kinds of miso and in natto, crushed natto, tem-

Figure 3Western blot analyses of fermented soybean products subjected


to extraction by using the previous and present methods. (A) Western blot
results after application of the previous method; (B) Western blot results
after application of the present method. Lanes: 1, molecular weight markers
(250, 150, 100, 75, 50, 37, 25, 20, 15, 10 kDa); 2, standard solution of
soybean proteins; 3, soybean miso; 4, rice miso; 5, barley miso; 6, raw miso;
7, soy sauce; 8, natto; 9, crushed natto; 10, tempeh; 11, tianmianjiang; 12,
douchi; 13, tofu-yoh; 14, soybean milk yoghurt. Arrow = p34 band.
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present extraction methods was 78.2% to 121.2%. In commercial


processed foods consisting mainly of soybean, the present extraction method had good sensitivity that was equal to that of the
previous method.

Detection of soybean in fermented food . . .


blot did not detect p34 in any of the fermented soybean products except tempeh, tianmianjiang, and soybean milk yoghurt.
We consider that the soy sauce band was not detected with
either method because the soybean proteins in the soy sauce
were degraded completely into amino acids and peptides during fermentation (Kobayashi 2005). These results showed that
the present extraction method inhibits the degradation of soybean proteins by microbial proteolytic enzymes and thus enables
the detection of soybean proteins in most fermented soybean
products.

Conclusion
The present extraction method combined with heating is a
sensitive, and useful way of detecting soybean protein in processed foods and fermented soybean products. This method
is suitable for quantifying soybean protein in processed foods
without the effects of microbial proteolytic enzymes. The proposed extraction and ELISA method should be useful in sensitively monitoring labeling systems in a reliable manner and
thus for the mandatory inspections required under Japanese
regulations.

Acknowledgments
We thank Dr. Tadashi Ogawa of Kansai Univ. of Welfare Science
for providing the monoclonal antibody against p34. We also thank
Aiko Yagi, Shiori Ito, Aiko Kubota, Yuki Suwa, Nanako Yanai,
Sonoko Wada, Erika Naito, Kaori Ito, Yuki Nogami, and Emi
Matsunaga for their valuable discussions.

Author Contributions
N. Morishita collected test data and drafted the manuscript. T.
Matsumoto and F. Morimatsu discussed the results. M. Toyoda
designed the study, collected test data, and discussed the results.

References
Astwood JD, Leach JN, Fuchs RL. 1996. Stability of food allergens to digestion in vitro. Nat
Biotechnol 14:126973.
Bando N, Tsuji H, Hiemori M, Yoshizumi K, Yamanishi R, Kimoto M, Ogawa T. 1998.
Quantitative analysis of Gly m Bd 28K in soybean products by a sandwich enzyme-linked
immunosorbent assay. J Nutr Sci Vitaminol 44:65564.
Brandon DL, Bates AH, Friedman M. 1991. ELISA analysis of soybean trypsin inhibitors in
processed foods. Adv Exp Med Biol 289:32137.
Ebisawa M, Ikematsu K, Imai T, Tachimoto H. 2003. Food allergy in Japan. Allergy Clin
Immunol Int 15:2147.
Food and Agriculture Organization of the United Nations, World Health Organization (FAO).
1995. Report of FAO technical consultation on food allergens. November 1314, Rome
(Italy).

T: Toxicology &
Chemical Food Safety

T1054 Journal of Food Science r Vol. 79, Nr. 5, 2014

Frias J, Song YS, Martinez-Villaluenga C, Gonzalez de Mejia E, Vidal-Valverde C. 2008.


Immunoreactivity and amino acid content of fermented soybean products. J Agric Food
Chem 9:99105.
Golbitz P. 1995. Traditional soyfoods: processing and products. J Nutr 125:570S2S.
Hei W, Li Z, Ma X, He P. 2012. Determination of beta-conglycinin in soybean and soybean
products using a sandwich enzyme-linked immunosorbent assay. Anal Chim Acta 13:628.
Hitchcock CHS, Bailey FJ, Crimes AA, Dean DAG, Davis PJ. 1981. Determination of soya
proteins in food using an enzyme-linked immunosorbent assay procedure. J Sci Food Agric
32:15765.
Inomata N, Nomura Y, Ikezawa Z. 2012. Involvement of poly ( -glutamic acid) as an allergen
in late-onset anaphylaxis due to fermented soybeans (natto). J Dermatol 39:40912.
Ito M, Kato T, Matsuda T. 2005. Rice allergenic proteins, 1416 kDa albumin and alphaglobulin, remain insoluble in rice grains recovered from rice miso (rice-containing fermented
soybean paste). Biosci Biotechnol Biochem 69:113744.
Jansen JJ, Kardinaal AF, Huijbers G, Vlieg-Boerstra BJ, Martens BP, Ockhuizen T. 1994.
Prevalence of food allergy and intolerance in the adult Dutch population. J Allergy Clin
Immunol 93:44656.
Kobayashi M. 2005. Immunological functions of soy sauce: hypoallergenicity and antiallergic
activity of soy sauce. J Biosci Bioeng 100:14451.
Koppelman SJ, Lakemond CM, Vlooswijk R, Hefle SL. 2004. Detection of soy proteins
in processed foods: literature overview and new experimental work. J AOAC Int 87:
1398407.
Matsuda R, Yoshioka Y, Akiyama H, Aburatani K, Watanabe Y, Matsumoto T, Morishita N,
Sato H, Mishima T, Gamo R, Kihira Y, Maitani T. 2006. Interlaboratory evaluation of two
enzyme-linked immunosorbent assay kits for the detection of egg, milk, wheat, buckwheat,
and peanuts in foods. J AOAC Int 89:16008.
Ministry of Health, Labor and Welfare of Japan. 2002. Notification. Nr. 106001.
Morishita N, Kamiya K, Matsumoto T, Sakai S, Teshima R, Urisu A, Moriyama T, Ogawa
T, Akiyama H, Morimatsu F. 2008. Reliable enzyme-linked immunosorbent assay for the
determination of soybean proteins in processed foods. J Agric Food Chem 27:681824.
Moriyama T, Machidori M, Ozawa S, Maebuchi M, Urade R, Takahashi K, Ogawa T,
Maruyama, N. 2005. A novel enzyme-linked immunosorbent assay for quantification of soybean -conglycinin, a major soybean storage protein, in soybean and soybean food products.
J Nutr Sci Vitaminol 51:349.
Murooka Y, Yamshita M. 2008. Traditional healthful fermented products of Japan. J Ind Microbiol Biotechnol 35:7918.
Namgung HJ, Park HJ, Cho IH, Choi HK, Kwon DY, Shim SM, Kim YS. 2010. Metabolite profiling of doenjang, fermented soybean paste, during fermentation. J Sci Food Agric
30:192635.
Ogawa T, Bando N, Tsuji H, Okajima H, Nishikawa K, Sasaoka K. 1991. Investigation of the
IgE-binding proteins in soybeans by immunoblotting with the sera of the soybean-sensitive
patients with atopic dermatitis. J Nutr Sci Vitaminol 37:55565.
Ogawa T, Tsuji H, Bando N, Kitamura K, Zhu YL, Hirano H, Nishikawa K. 1993. Identification
of the soybean allergenic protein, Gly m Bd 30K, with the soybean seed 34-kDa Oil-bodyassociated protein. Biosci Biotech Biochem 57:10303.
Phromraksa P, Nagano H, Boonmars T, Kamboonruang C. 2008. Identification of proteolytic
bacteria from Thai traditional fermented foods and their allergenic reducing potentials. J Food
Sci 73:M18995.
Ravestein P, Driedonks RA. 1986. Quantitative immunoassay for soya protein in raw and
sterilized meat products. J Food Technol 21:1932.
Sakai S, Adachi R, Akiyama H, Teshima R, Morishita N, Matsumoto T, Urisu A. 2010. Enzymelinked immunosorbent assay kit for the determination of soybean protein in processed foods:
interlaboratory evaluation. J AOAC Int 93:2438.
Savage JH, Kaeding AJ, Matsui EC, Wood RA. 2010. The natural history of soy allergy. J Allergy
Clin Immunol 125:6836.
Tsuji H, Okada N, Yamanishi R, Bando N, Kimoto M, Ogawa T. 1995. Measurement of
Gly m Bd 30K, a major soybean allergen, in soybean products by a sandwich enzyme-linked
immunosorbent assay. Biosci Biotech Biochem 59:1501.
Tsuji H, Okada N, Yamanishi R, Bando N, Ebine H, Ogawa T. 1997. Fate of major soybean
allergen, Gly m Bd 30K, in rice-, barley, and soybean-koji miso (fermented soybean paste)
during fermentation. Food Sci Technol Int Tokyo 3:1459.