Grain & Oil Science and Technology 4 (2021) 191–200

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Grain & Oil Science and Technology

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science-and-technology/

Detection and inactivation of allergens in soybeans: A brief review of recent

research advances
Lina Tokuna Mulalapele a,b, Jun Xi
⁎,a,b
a
College of Food Science and Engineering, Henan University of Technology, Zhengzhou 450001, China
b
Faculty of Agronomy, Department of Animal Science, University of Kikwit, B.P. 76, D.R.C., Kikwit

A R T I C L E I N F O A B S T R A C T

Article history: In the last decade, soybean allergies have been on the increases to such an extent that they have now
Received 31 March 2021 become a public health issue thus prompting more studies and researches on the topic. The allergenicity
Received in revised form 3 November of soybean is attributed to its protein fraction. The best way to prevent hypersensitive patients from
2021 ingesting allergenic compounds is to exclude such soybean allergens from their diet. As a result, it is es-
Accepted 3 November 2021 sential to provide detailed and reliable knowledge of food ingredients. Therefore, precise and reliable
approaches for detecting soybean allergens found in various food products must be used. The main
Keywords: way to reduce allergy risk is the identification of allergenic sites in food and their inactivation by various
Soybean food-processing methods. It has been reported that food processing may lead to the modification of con-
Soy allergens formational structure of the protein or protein distortion that inhibit the binding of immunoglobulin E
Detection (IgE) to epitopes on food allergens and also the mechanism of allergic reactions. Food processing tech-
Inactivation nologies employed for inactivating allergenic epitopes used thermal and nonthermal techniques. Cur-
Allergenicity rently, several detection methods including protein-based and DNA-based approaches using
analytical techniques such as enzyme-linked immunosorbent assay (ELISA), enzyme allergosorbent
test (EAST), radioallergosorbent test (RAST), lateral flow immunoassay (LFIA), immunoblotting, real-
time polymerase chain reaction (PCR), mass spectrometry and biosensors have been improved for iden-
tifying and quantifying these epitopes. This research focused on allergenic proteins of soybean, the most
modern approaches for detecting and quantifying these allergens, and finally, the various methods used
to inactivate these proteins and their effects on soy allergenicity.

1. Introduction ingestion of which causes adverse allergenic symptoms

Soybean is a legume whose seeds are rich in unsaturated
fatty acids, vitamins, minerals, and other nutrients. A soy-
based diet is generally recommended because of its ability
to lower plasma cholesterol levels and lipoprotein density
and to prevent cancer, diabetes mellitus, and obesity.
Therefore, the use of soy has increased significantly, espe-
cially in Europe and North America [1,2]. However, soy-
bean contains various anti-nutritional factors (ANFs), the
[3]. In recent years, soy allergy has become a public health
problem, and its occurrence has increased due to the high
consumption of soy products. Typical symptoms of soy al-
lergy include nausea, vomiting, diarrhea, runny nose,
cough, wheezing, weakness, gastrointestinal distress, dys-
pnea, cardiovascular or cutaneous symptoms, and other
vague symptoms, up to and including anaphylactic shock,
which can be life-threatening [4,5]. Ingestion of a small
amount of an allergenic food can trigger an allergic

⁎ Corresponding author.
E-mail address: xijunhnu@163.com (J. Xi).

http://dx.doi.org/10.1016/j.gaost.2021.11.001
2590-2598/© 2021 Henan University of Technology. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
L.T. Mulalapele, J. Xi
symptom in susceptible people. To safeguard the health of
those who are predisposed to allergic reactions because of
soy consumption, universal regulations for labeling aller-
genic ingredients in foods have been introduced in various
countries around the world. Ensuring that there are no “al-
lergen traces” or quantifying their presence would allow for
more accurate labeling, thus facilitating the management of
allergy risks. As a result, the labeling of foods containing al-
lergens has become of critical importance. To prevent aller-
gic reactions, it is necessary to avoid ingestion of foods
containing the triggering allergen [6,7]. Thus, methods to
detect and deactivate allergenic epitopes become impera-
tive. Food processing techniques can influence the aller-
genic potential of allergens. They have been found to
effectively reduce the allergenicity of foods by destroying
conformational and sequence epitopes, resulting in protein
aggregates, cross-links, or amino acid chain distortion
[8,9]. This paper presented latest methods for the detection
and deactivation of antigenic soy proteins and the effects of
processing on the inactivation of these antigenic proteins.
2. Soybean allergens and their characterization
At least 16 different proteins have been discovered in
soybean (Table 1). Storage protein β-conglycinin (7S) and
the storage protein glycinin (11S) are the two primary pro-
teins in soybean. These major seed storage proteins account
for 70%–80% of the total seed globulin fraction, and they
have been recognized as a source of dietary allergens for
humans. Studies have shown that the allergenicity of soy-
bean is mainly due to β-conglycinin, glycinin, Gly m Bd
Table 1
Soybean allergens.
Molecular
weight Soybean allergens References
(kDa)
7.0 Gly m 1a; hull protein
7.0 Gly m 1b; hydrophobic protein; hull
protein
7.5 Gly m 2; hull protein
8.0 Gly m 3; profilin
12.0–15.0 2S-Globulin fraction
17.0 Kunitz trypsin inhibitor; 2S-globulin
20.0 Whey fraction
18.0–21.0 Glycinin G2; basic chain of glycinin;
11S-globulin
22.0 Gly m Bd 28 K; 7S globulin
28.0 Gly m Bd 30 K, P34; immunodominant
allergen
30.0–34.0 Whey fraction
29.0–31.0 Soy lectin; soybean agglutinin
32.0 Glycinin G1; acidic chain of glycinin;
11S-globulin
35.0–40.0 7S-Globulin fraction
Grain & Oil Science and Technology 4 (2021) 191–200
30 K (P34), and Gly m Bd 28 K [11,12]. This section de-
scribes these four antigenic soybean proteins.
2.1. β-Conglycinin
β-Conglycinin is a glycopeptide that is a component of 7S
globulin; it has a molecular weight of 150–200 kDa and
consists of α' (58–83 kDa), α (58–77 kDa), and β
(42–53 kDa) with isoelectric points of 4.90, 5.18, and
5.66–6.00, respectively, and all-cause allergies [13,14].
While they are homologous, the subunit (Gly m Bd 60 K)
is the main allergen to which humans are sensitive. Accord-
ing to the study, Gly m Bd 60 K was found bound to IgE in
25% of serum from people with atopic dermatitis [15].
2.2. Glycinin
Soybean glycinin (Gly m 6) is a component of 11S and
the primary storage protein for soybean seeds, accounting
for 19.5%–23.1% of soy protein content and 40% of total
globulin protein content. Glycinin can range in size from
320 to 360 kDa. It consists of basic chain peptides (BI, BII,
BIII, BIV, BV, BVI) and acidic chain peptides (AI, AII, AIII,
AIV, AV) linked by disulfide bonds without undergoing a
glycosylation reaction [16]. The acidic glycinin chain is
the primary source of allergic reactions in hypersensitive
patients. However, the basic glycinin chain can also bind
to IgE and elicit an allergy response. Helm's researches on
glycinin epitopes discovered eleven linear epitopes, four
of which were found to be immunodominant [17,18].
2.3. Gly m Bd 30 K (P34)
Gly m Bd 30 K, one of the most allergenic storage pro-
teins of soybean, is frequently identified by IgE in the
serum of people with soybean allergies and can cause atopic
dermatitis. The molecular weight is approximately 43 kDa,
and it contains 379 amino acids [19]. Several studies have
been conducted on Gly m Bd 30 K epitopes: for example,
[8,10]
Ogawa et al. [15] identified five major allergenic epitopes,
[8]
and Hiroshi et al. [20] subsequently analyzed two major
[8,10] epitopes and produced the corresponding monoclonal anti-
[8,10] body. Helm and colleagues [18] also examined the primary
[8,10] amino acids of the sensitized epitopes and discovered that
[10]
[10]
two alanine-substituted amino acids of the epitope did not
[8,10] respond to antibody.
[10] 2.4. Gly m Bd 28 K
[8,10]
Gly m Bd 28 K is another 7S soybean moiety that has
[10]
[8] been reported as an essential allergen of the asparagine gly-
[8] coprotein and consists of 220 amino acid residues present in
the oligomeric type. It is a low abundance protein (26 kDa)
[8,10]
belonging to the cupin superfamily with a β-barrel structure
192
L.T. Mulalapele, J. Xi
[12]. Xiang and co-workers [21] investigated and screened
an essential IgE binding site (S256-A270) for the 28 K pro-
tein and identified four major amino acid sites (Y260,
D261, D262, and K264) for the epitope.
3. Soybean allergen detection methods
Soybean has several potential applications in the food/
feed industries. However, allergenic proteins in soybean
limits the extent of its full utilization. Over years, a variety
of soy-containing foods have become available and are
commonly consumed in the markets [6]. Given the high
risk of soy allergy, techniques for the detection of soy and
its proteins in foods are necessary. Several technological ad-
vancements have been developed for epitope detection and
quantification in this latest century. The recently applied
methods for soybean allergens analysis mainly include im-
munological methods, mass spectrometry, polymerase
chain reaction, and biosensors. This section reviewed
methods mentioned above.
3.1. Immunoanalytical methods
Food allergens analysis requires sensitive and speci-
fic techniques. Immunoanalytical approach remains the
most commonly used method for the detection and quanti-
fication of hidden allergens in food. It is based on the use of
antibodies directed against allergens. Immunoanalytical
methods include enzyme-linked immunosorbent assay,
radio-allergosorbent test, enzyme-allergosorbent test, lat-
eral flow immunoassay, and immunoblotting. Among
them, enzyme-linked immunosorbent assay (ELISA) has
emerged as the best routine test for allergen detection.
3.1.1. Enzyme-linked immunosorbent assay (ELISA)
To achieve adequate sensitivity of the test and avoid
cross-reactivity of antibodies with homologous proteins,
ELISA requires antibodies with high specificity, high titer,
and high affinity. The assay uses well plate capture reagents
and enzyme-labeled antibodies as detector reagents for di-
rect or indirect detection. Enzyme markers are used to visu-
alize the change in the sample by colored reactions. The
intensity of the color represents the concentration of anti-
gens or antibodies in the sample. Competitive, sandwich
and indirect ELISA is performed depending on the specific-
ity of the assay [22].
3.1.1.1. Competitive ELISA (cELISA). The competitive format
is commonly used for lower analytes. It is based on the prin-
ciple of encountering two known and unknown ligands to
bind a small number of antibodies. The inhibitor antigen
is first coated onto a microtiter plate, and then the incu-
bated sample with labeled antibodies is added to the
wells. This simultaneity is due to the limited number of
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Grain & Oil Science and Technology 4 (2021) 191–200
antibodies needed to bind antigens. Xi and Shi [23] pre-
pared a recombinant protein by expressing a DNA fragment
of Gly m Bd 28 K in Escherichia coli BL21 (DE3) and a target
monoclonal antibody against that recombinant protein for
the detection of allergenic protein Gly m Bd 28 K using an
indirect competitive ELISA (icELISA). The assay showed
an IC50 (half-maximal inhibitory concentration) value of
3.19 μg/mL and a limit of detection (LOD) value of
1.1 μg/mL. In the same variant, a competitive ELISA
(cELISA) based on monoclonal antibodies recognition of
glycinin was performed. In this study, two monoclonal anti-
bodies (IgG2b and IgG2a) with high specificity were pro-
duced against glycinin. Among these, IgG2b was selected
for the development of the assay. An IC50 value of
1.7 ng/mL with a LOD value of 0.3 ng/mL and a standard
curve value of 0.3–11.2 ng/mL with high reproducibility
were registered [24].
3.1.1.2. Sandwich ELISA (sELISA). This method is com-
monly used to test complex samples. In this essay, an anti-
gen is detected between two pairs of antibodies. The
capture antibodies are first applied to the wells where
they bind to the antigen of the sample. The detector anti-
bodies that have been paired with the enzyme are then
added. The enzyme-substrate is applied in the form of a so-
lution. The result is a colored solution. A photometer can be
used to determine the intensity of the color. Hei and col-
leagues [25] analyzed 469 soybean seeds and seven soy-
bean products from different provenance and processing
techniques. Then 20 monoclonal antibodies (mAbs) were
selected out of 139 mAbs produced. Based on the specificity
study, only five mAbs presented high specificity with
β-glycinin. Among these, only one mAb 5C5 had a light
cross-reactivity with glycinin, thus, a sandwich ELISA was
developed using 5C5 Mab and Goat anti-rabbit IgG HRP-
conjugated as capture and detection antibodies respec-
tively. The test discovered a detection limit of 1.63 ng/mL
for a sandwich format and 6.4 ng/mL for an indirect com-
petitive format. Isabel Segura-Gil and colleagues [26] quan-
tified soybean in processed food using two ELISA formats.
The assay demonstrated a LOD of 0.90 ng/mL and
30 ng/mL for a sandwich and indirect competitive formats
respectively. The limit of quantification (LOQ) was
2.1 ng/mL for sandwich formats and 70 ng/mL for compet-
itive formats, with a working range of 2–15 ng/mL for sand-
wich formats and 10–3000 ng/mL for competitive formats.
Besides, another sandwich assay using the combination of
monoclonal antibodies directed against glycinin as coating
antibodies and rabbit anti-glycinin polyclonal antibodies
as detected antibodies was developed to detect the trace
amounts of glycinin in soybean products. The developed
method showed a high sensitivity to glycinin with LOD of
1.63 ng/mL [27].
L.T. Mulalapele, J. Xi
3.1.1.3. Indirect ELISA (iELISA). The method is performed
in two steps. First, an unlabeled primary antibody binds
specifically to the antigen. A secondary antibody conju-
gated to an enzyme, forming the signal from the chromo-
genic or fluorogenic substrate, then recognizes the
primary antibody. This method is widely used to quantify
the total antibody concentration in samples. Liu et al.
[28] synthetized a recombinant protein (P34) by using a
full-length cDNA sequence of P34 and introduced into the
prokaryotic expression vector pET-28a. The P34 protein
was expressed in E. coli BL21 (DE3). Based on the recombi-
nant protein (P34), a polyclonal antibody (pAB) against
P34 was prepared and showed high specificity to the P34
protein of the soybean meal. The obtained result showed
a recovery rate of less than 7.77%, indicating the best
result.
3.1.2. Enzyme-allergosorbent test (EAST) and radio-
allergosorbant test (RAST)
These tests are widely used in laboratory assays, but
they can also be used for allergenicity identification. Gen-
erally, the test employs antibodies obtained from the
blood serum of allergenic patients. The detection principle
is based on the use of allergens fixed to the solid phase, in
which a blood sample containing specific IgE antibodies to
the target allergen is added and cross-linked with it, al-
though, enzyme-allergosorbent test identification emp-
loyed enzymes conjugated to immunoglobulin antibodies
[29]. The tests need costly equipment, and as well as the
possibility of false-positive outcomes because of the high
concentration of IgE antibodies in serum and the chal-
lenge of standardizing these tests. In addition, RAST or
EAST require human serum for the analysis, that is not
easy. Although, many tests can be performed at the same
time. These methods seem not to be relevant for food al-
lergen detection. Ogawa and his co-workers [12] exam-
ined the serum of patients with atopic dermatitis by
RAST with commercial allergen disks containing soy-
beans, egg white, milk, wheat, rice. A RAST score more
than 2 were diagnosed as positive subjects. After perform-
ing the essay, eleven patients were diagnosed as negative
subjects and four as positive on the soybean disk. How-
ever, by immunoblot analysis, the 11 patients who did
not give the positive RAST response (<2) on the soybean
disk were shown to carry specific IgE antibodies for soy-
bean proteins, whereas the 4 RAST-positive patients
(≥2) gave no response. In other words, the detection of
specific IgE antibodies by the immunoblotting method
was more sensitive than that by RAST. In addition, an
enzyme-allergosorbent test using two human blood se-
rums of the patient with soybean allergy was performed
and shown detection limits of 0.8 and 12 mg/L soy
flake, respectively [30].
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3.1.3. Lateral flow immunoassay (LFIA)
The technique does not need complex and costly appara-
tus for substance detection. As a result, LFIA technology has
found applications in a variety of fields where its intrinsic
advantages of simplicity, rapidity, cost-effectiveness and
lack of equipment or technological experience are critical.
Trace analysis of allergens on surface is currently the most
common application of lateral flow testing in allergen con-
trol. They are, however, ideal for detecting allergen resi-
dues in food and are quicker and simpler to manipulate
than an ELISA test. Furthermore, some lateral flow testing
can detect proteins not only in raw materials but also in
processed food products. Lateral flow immunoassay works
similarly to enzyme-linked immunosorbent assays
(ELISA). In essence, these experiments run a liquid sample
over the surface of a pad containing reactive molecules
that provide a visual positive or negative response [31].
Wang et al. [32] designed a LFIA strip to detect soybean
protein (β-conglycinin). Antibodies against β-conglycinin
were produced and then a sandwich test combined with col-
loidal gold were performed. The assay demonstrated no
cross-reaction with others allergens. The limit of detection
registered was 1.66 mg/kg. Further, Xi and Yu [33] de-
signed the same assay at a single difference. They used
p-amino thiophenol combined with colloidal gold to en-
hance signals and obtained a test performance range be-
tween 160 ng/mL and 100 μg/mL. The assay showed high
sensitivity and can be used for allergen detection tests.
3.1.4. Immunoblotting/Western blot
Immunoblotting, usually called Western blot, is a popu-
lar common technique employed to detect specific proteins
in samples. The method relies on the use of sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to
isolate proteins present in a sample. Next, the isolated pro-
tein is placed into a membrane and there it binds to an anti-
body specific for the target protein. This technique can
screen low concentrations of target proteins because of the
high resolving power of gel electrophoresis and the high
sensitivity and specificity of the immunological test
[34,35]. Krishnan et al. [36] have investigated whether or
not the α' and β- subunits of β-conglycinin may also act as
potential allergens. To make it, the reactivity of these sub-
units and human sera from seven patients allergic to soy
were examined by immunoblotting. The analysis indicated
soy proteins which were found bind to IgE antibodies
from soy sensitive patients. Furthermore, the obtained re-
sult has confirmed that all the three (α, α' and β) subunits
of β-conglycinin were potential allergens to sensitive peo-
ple. Tsuji et al. [37] have purified and studied the character-
ization of Gly m Bd 28 K by SDS-PAGE and immunoblotting
as described above. Three hundred micrograms of 7S pro-
tein globulin were loaded in 5% polyacrylamide and
L.T. Mulalapele, J. Xi
electrophoresed on two polyacrylamide gels in sheets. After
analysis, the proteins on the gel were stained with
Coomassie Brilliant Blue R250. At the end, the purity of
the protein was obtained. Furthermore, purified proteins
were put into a nitrocellulose membrane and electro-
blotted. The results showed that only one spot could be de-
tected by immunoblot in contrast to that of SDS-PAGE
which shows deux more spots. In addition, You et al. [38]
developed a monoclonal antibody based on a competitive
ELISA (cELISA) to detect β-conglycinin. In this study, an
immublotting analysis was performed to evaluate the
immunoreactivity of prepared monoclonal antibodies.
After SDS-PAGE, the separated protein was transferred
electrophoretically from gel to a 0.45 μm nitrocellulose
membrane. The result showed that the prepared monoclo-
nal antibodies were able to recognize β-conglycinin and
could be employed for detecting of this protein in food
samples.
3.2. Mass spectrometry (MS)
Over the past decade, mass spectrometry has become a
widely used and valued method for the detection of
foodborne allergens. It shows good sensitivity, even if the
food allergens to be analyzed have undergone changes as
a result of treatments or transformations. This fact reduces
the probability of false negative results in MS, unlike immu-
nological tests, which tend to give false positive results due
to the cross-reaction of antibodies with homologous pro-
teins. In fact, mass spectrometry has indeed solved these
problems. Furthermore, the technique does not require the
use of antibodies and allows simple multiplex testing. How-
ever, MS uses expensive equipment and requires an active,
trained personnel to carry out the test. Houston and col-
leagues [39] used a quantitative proteomic approach to de-
tect allergenic proteins in 20 commercial soybean varieties.
Further, the work of Jia et al. [40] on the quantification of
Gly m 5.0101 in soybean and soybean products showed a
consistent recovery of 103.43%–113.13% with a detection
limit of 0.48 ng/mL. The assay allowed the detection of
even low allergen doses.
3.3. Polymerase chain reaction (PCR)
This method does not directly identify the protein but
identifies the sequence of the DNA that codes for the aller-
genic protein, therefore, it is an indirect detection method.
The method is performed in three steps: denaturation, hy-
bridization, and expansion. The method is based on the
principle of exponential production (amplification) of spe-
cific DNA fragments. As the reaction progresses, more
DNA fragments are amplified and quantified after each
cycle. To obtain a better result, PCR can be combined with
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Grain & Oil Science and Technology 4 (2021) 191–200
an ELISA assay. In recent years, real-time PCR (RT-PCR)
has become popular for greater quantitative detection. Un-
like traditional PCR, where amplification products are mea-
sured at the end of the reaction, RT-PCR uses a fluorescent
probe to quantify the fluorescence emitted during the reac-
tion as an indicator of the number of DNA sequences gener-
ated in real-time during each cycle [41]. Its success lies in
its ability to monitor the amplification of a target sequence
in real-time using fluorescent labels. It also enables the pre-
cise quantification of nucleic acids, even when the starting
material is used at very low concentrations. This method,
however efficient, has some limitations. The targeted DNA
may not be allergen specific, the process requires sophisti-
cated analytical equipment and calibrators, and there is
the possibility of DNA contamination. Costa et al. [42] de-
veloped a RT-PCR method coupled with a fluorescent hy-
drolysis probe to detect soybean allergen in processed
meat products. The assay showed a detection limit of
9.8 mg/kg in pork meat and in both raw and thermally
processed samples. Besides, Gryson et al. [43] used PCR to
identify soy components in bread and obtained the best re-
sults with a lower detection limit. Mayer and colleagues
[44] also performed a droplet digital-PCR assay in which
they used chloroplast DNA instead of nuclear DNA for the
quantification of the soybean allergen and reported a LOD
value of 0.16 mg/kg with a LOQ of 0.63 mg/kg. The assay
performed well compared to the results of other researchers
who called for a lower detection limit.
3.4. Biosensors
Biosensors are analytical tools used in various fields to
identify changes in a variety of biological targets, including
antibodies, metabolites, immune systems, nucleic acids, tis-
sues, and cells. A biosensor typically consists of three parts:
a bioreceptor that recognizes the analyte, a signal generator
that generates and converts a signal for further analysis, and
a signal detection system. To produce diagnostic data ef-
fects, the device stores, displays, and analyzes the output
signals. These devices are used in various fields ranging
from medical to biological, agricultural, and food process-
ing applications, such as identification of allergens and ad-
ditives in food, pesticides, and pathogenic species, due to
their excellent flexibility in terms of receptors, materials,
and transduction modes. They can be listed as electrochem-
ical, optical, magnetic, mass sensitive, calorimetric, electro-
mechanical methods, etc. [45,46]. The excellent results
obtained by Yuan and his colleagues [47,48] after studying
the antigenicity of three different food allergens showed
that the use of biosensors could be employed as an efficient
method for food allergen detection. However, the validity
of the method needs to be improved to avoid reliability
problems.
L.T. Mulalapele, J. Xi
4. Processing technologies and their influence on soy-
bean antigenicity
Food processing generally improves the physical and or-
ganoleptic properties of foods as well as their shelf life.
They have an impact on food preservation, detoxification,
and allergenicity. On the other hand, food processing can
lead to a loss of allergenicity, have no effect on allergenicity,
or lead to the formation of neoallergens [49]. In this section,
the different techniques used in soybean processing and
their effects on soybean allergenicity were reviewed.
4.1. Thermal processing
The thermal processing of food is the most common tech-
nique in food and feeds production. It is further divided into
dry heating and moist heating. Typical dry thermal treat-
ments applied to soybeans are roasting, infrared, microwave
radiation, while the moist thermal treatment methods are
extrusion and steam heating. In general, heat treatment is
used in industry to improve digestibility, ensure good micro-
biological quality and develop a wide range of aromas and
colors. Heat treatment of foods involves various changes in
some protein attributes such as denaturation, peptide bond
hydrolysis, disulfide bond reconstruction and interaction
with other components, especially carbohydrates. A typical
example of this reaction that occurs during roasting, which
involves changes in the amine groups of proteins by reduc-
ing sugars producing glycated proteins known as enhanced
glycation endings [1,19]. Thermal processing destroyed
also secondary and tertiary structures of proteins by
disrupting or exhibiting the conformational epitope, as a re-
sult, the ability of IgE to bind antigenic sites decreases [8].
Processed soybean proteins are widely used in food industry
to enhance specific functional properties such as improved
texture, moisture and fat retention, foaming properties and
emulsification [1].
Thermal processing of soy product is commonly used to
inactivate anti-nutritional components such as trypsin in-
hibitors, enhancing protein digestibility and reducing aller-
gen immunoreactivity in soy products. Thermal processing
can cause unfolding of proteins, exposing hydrophobic
and sulfhydryl groups located in the interior of the mole-
cule, which can result in irreversible protein aggregation,
thus leading to a decrease in solubility [9]. Yin et al. [50] re-
ported that by increasing the temperature during soybean
extrusion, H and S-S bonds are partially broken between
protein molecules, and the original spatial arrangement
spreads, resulting in the loss of molecular structure, which
reduces antigenicity. Thermal treatment of glycinin results
in aggregation as well as the loosening of the secondary
structure, leading to a reduction of sensitive bonds [51].
Moreover, at 100 °C, the conformational structure of the
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Grain & Oil Science and Technology 4 (2021) 191–200
β-conglycinin epitope is destroyed, but not its linear struc-
ture. Furthermore, thermal treatment of β-conglycinin re-
duces its sensitizing capacity by changing its structure via
protein aggregation with a loss of disulfide bonds [52].
4.2. Non-thermal processing
Non-thermal processing of foods is an important alterna-
tive to thermal processing of foods for reasons such as pres-
ervation of organoleptic properties and dietary qualities.
This method leads to a change in conformational structure
by deployment or aggregation [51]. In this section, non-
thermal processing methods for soybean were discussed.
4.2.1. High-pressure processing/high hydrostatic pressure
In the food industry, high pressure or high hydro-
static pressure is used to inactivate microorganisms, food-
degrading enzymes, and even allergenic proteins in food.
Pressures ranging from 100 to 800 MPa, disrupt hydropho-
bic groups, lead to depolymerization of proteins and de-
stroy non-covalent bonds that contribute to structural
changes in proteins and reduce their immunogenicity
[53]. After high pressure processing ≥300 MPa,
β-conglycinin is denatured. At 400 MPa, the quaternary
structure of β-conglycinin was identified, and more –SH con-
tents were observed with the increase of processing time
[54]. Moreover, Tang and co-workers [55] found that the
conformation of glycinin changes under pressure is greater
than or equal to 300 MPa. Penas and co-workers [56]
treated soybean and tofu extracts under high-pressure pro-
cessing of 300 MPa, 40 °C for 15 min and found that after
processing, the electrophoretic pattern does not change,
but the number of protein bands with starch becomes low.
Furthermore, Li and colleagues [57] reported that very
high static pressure treatment effectively changes the antige-
nicity of soy protein isolate in infant formula and affects its
functional properties, including water retention, emulsify-
ing activity, and foaming.
4.2.2. Irradiation with pulsed ultraviolet light
This method is used for food treatment and preservation
and involves sterilization of food, which increases shelf life
and food quality. The device consists of a charging capacitor
that gradually emits short pulses of light, when connected
to a food sample, the light interacts with food molecules
and induces changes in protein conformation through pho-
tochemical, photothermal, and photophysical reactions that
affect protein antigenicity [58]. Ultraviolet light destroys
protein completely and causes oxidation of the disulfide
bonds (S–S) to form sulfhydryl groups. Foods that are ex-
posed to UV light for long periods of time undergo various
transformations, including depolymerization of starch, oxi-
dation of unsaturated fatty acids to form peroxidized
L.T. Mulalapele, J. Xi
components, aggregation of proteins, and distortion of pro-
teins that damage the bonds of IgE to allergens. Yang and
his colleagues [59] have conducted some researches on
the impact of pulsed ultraviolet light on the antigenicity of
soybean extracts. Proteins were exposed under pulsed ultra-
violet light for 2, 4 and 6 min and then analyzed by SDS-
PAGE. An additional indirect ELISA was performed for IgE
binding. At the end of the test, they have found that the al-
lergenicity of soy decreased significantly due to the aggre-
gation mechanism and also, IgE binding was also decreased.
4.2.3. Fermentation
Fermentation is an ancient technique used by ancient
civilizations to preserve food. Nowadays, it is used to im-
prove the digestibility, nutritional value, sensory quality,
and preservation of foods, but also because of its beneficial
effects on reducing allergenicity due to the alteration or de-
struction of linear and conformational epitopes during pro-
tein hydrolysis. During fermentation, microorganisms
develop enzymes that act on food substrates in the absence
of oxygen, resulting in changes in appearance, taste, and nu-
trient composition. The microorganisms interact with the
food during fermentation and degrades antigenic proteins
into small molecules, resulting in a looser protein structure.
These reactions reduce the antigenicity of soy allergens and
thus the IgE binding potential. Yang et al. [60] used a solid
phase fermentation made of a combination of Lactobacillus
casei, yeast, and Bacillus subtili to study the antigenicity of
soybean meal. The authors registered a degradation of soy-
bean proteins in lower molecular weight polypeptides in
which apart of allergenic fragments of β-conglycinin and
glycinin have been destroyed.
4.2.4. Enzymatic hydrolysis
Soy allergens could be indigestible in vivo, so improving
the hydrolysis technique in vitro using enzymes such as tryp-
sin, pepsin, chymotrypsin, and other bacterial or fungal en-
zymes will be very important [61]. During hydrolysis,
enzyme interacts with proteins and breaks down the anti-
genic protein into small molecules leading to the loosening
of the protein structure. As a result, the molecular weight is
reduced, and the immunogenicity of the food proteins is
subsequently reduced [62]. Yang and co-workers [63] per-
formed an assay on fermentation-assisted alkalase hydroly-
sis process. In this study, four microorganisms were used to
ferment soy meal. In addition, ferment soymeal was hydro-
lyzed with alkalase. The result showed higher levels of hy-
drolysis from 10.68% to 17.39% with concomitant loss of
allergenicity of soy meal. Hydrolysis promotes intestinal ab-
sorption by improving solubility and antioxidant function.
Protease-treated soy protein (Gly m Bd 30 K) in particular,
is effective in reducing the allergenicity of soy proteins
[64]. In addition, ultra-high pressure-treated soybean
197
Grain & Oil Science and Technology 4 (2021) 191–200
whey protein combined with enzyme hydrolysis process re-
duces the antigenicity of Gly m 1 [65].
4.2.5. Glycosylation/glycation
Glycosylation of proteins via the Maillard-type polysac-
charide reaction has become widespread in recent years.
The process promotes the functional properties of the
protein-polysaccharide conjugate. The study by Xu et al.
[66] proved that glycosylation affects protein aggregation
of soybean. Van de Lagemaat et al. [67] have studied
Maillard reaction of soybean protein isolates glycated with
fructose or fructo-oligosaccharide in powder and liquid sys-
tems before and after glycosylation. After processing, the
authors have found that the content of primary free amino
groups of soybean protein isolates in powder system were
reduced to 85% while in liquid system was 96% and the re-
sult of SDS-PAGE analysis showed some changes in 7S and
11S structures of soybean protein isolates glycated with
fructo-oligosaccharide. In addition, in a certain heat tem-
perature, the glycosylation of soybean protein isolates in-
duces the alteration of its surface hydrophobicity, net
charge and its thermal stability, which may affect aggre-
gation and resulting in its antigenicity changes via an ap-
propriate Maillard reaction conditions. For example, the
greatest decrease in term of immunoreactivity of soybean
protein isolates was discovered after the heat treatment at
95 °C in a liquid system with a high concentration of carbo-
hydrates. By a direct ELISA, the same authors have found
that the allergenicity of soybean protein isolates glycated
were reduced by 90% contrary to unglycated proteins. Al-
though the method can significantly reduce the antigenicity
of soybean, but there are still some improvements related to
the optimization of Maillard reaction conditions and char-
acterization of glycated products Table 2.
5. Conclusions and future trends
More than 16 allergenic proteins have been discovered
in soybeans. Among these proteins, β-conglycinin 7S and
glycinin 11S storage proteins are considered to be the
most allergenic and represent 70%–80% of the total frac-
tion of the globulin of the seed. Apart from the two main
proteins quoted previously, we add to the list, Gly m Bd
30 K also known as P34 and Gly m Bd 28 K. Studies have
shown that these four proteins mainly cause the allergenic-
ity of soybean [11]. Therefore, the need to develop simple,
less expensive, accessible, reproductive, and specific
methods in the detection and quantification of soybean al-
lergen proteins in foods is now a priority. In latest years,
the detection methods that are mainly used are immunolog-
ical method, mass spectrometry, polymerase chain and bio-
sensors. Among these, immunological methods based on
enzyme-linked immunosorbent assay and lateral flow
L.T. Mulalapele, J. Xi Grain & Oil Science and Technology 4 (2021) 191–200

Table 2
Summary of soy processing method and their allergenicity outcomes.
Processing Processing Soy protein Comment Allergenicity References
category Methods fraction/Soy outcome
products
Thermal Heat treatment 1S-Glycinin Aggregation, loose of secondary structure and ↓ [51]
declination of sensitivity bonds.
7S-β-Conglycinin Destruction of conformational structure, protein ↓ [52]
aggregation, and reduction of disulfide bonds.
Extrusion 7S-β-Conglycinin Partly and secondary broke of H and SS of soy. ↓ [50]
Non-thermal High-pressure treatment 7S-β-Conglycinin Denaturation and change of subunits configuration. – [54]
/High hydrostatic 11S-Glycinin Conformation changes. – [55]
treatment Tofu No change of the electrophoretic pattern and decrease – [56]
of protein bands number with strength.
SPI1 Improvement of water retention capacity, emulsifying ↓ [57]
activity, and foaming.
Pulsed ultraviolet light Soy extracts Reduction of allergen levels and allergen activity. ↓ [59]
Fermentation SM2 Exhibition of higher degree of hydrolysis and loss of ↓ [60]
intensities in several major bands.
Enzyme hydrolysis SM2 Exhibition of higher degree of hydrolysis and loss of ↓ [63]
intensities in several major bands.
Gly m Bd 30 K Improvement of solubility and antioxidant function. ↓ [64]
Soybean whey ↓ [66]
protein (Gly m 1)
Glycosylation/Glycation 11S-Glycinin and Protein aggregation of soy. ↓ [67]
7S-β-conglycinin
Notes: 1SPI: Soybean protein isolate; 2SM: Soybean meal; ↓: Decrease; —: Nothing to report or none.

immunoassay seem to be commonly used because of their
high sensitivity, simplicity, short time of process and used
less expensive devices. We are expected that in the future,
the lateral flow immunoassay will become an immunologi-
cal method that people will refer for allergen detection be-
cause it has the asset of being cheaper and simpler
compared to the enzyme-linked immunosorbent assay,
and its detection principle is generally reduced at one
step, although the process requires high dose of
target allergens to set off an allergenic reaction. However,
some improvements related to the use of small amount of
target allergen and the increase of capacity of detecting sev-
eral allergens simultaneously are needed to enhance the re-
liability of the method.
In addition, another trend turned towards DNA based
method using polymerase chain reaction and biosensors
has also been noticed. DNA-based method is an indirect
technique using amplification of specific fragment of DNA
for allergen detection. This method reduces the risk of pro-
tein denaturation that occur during food processing and re-
ports less false results than immunoassays. In contrast to
previous mentioned methods, biosensors used various sig-
nal transduction techniques to detect allergens. The optical
biosensors are the most commonly employed and present
good sensitivity. However, this method still has problem re-
lated to validity. Thus, in the future, improvements related
to the development and validation of reliable methods is
needed.
As long as soy allergy is still a health public issue, pro-
cessing food is one of the alternatives for reducing the anti-
genicity of soybean proteins. Although, it is difficult to
completely eliminate the antigenicity of soy proteins
through processing but a good control and optimization of
processing conditions could be done. Soybean processing
technologies can be classified into two main groups: ther-
mal and non-thermal methods. The thermal methods gener-
ally result in protein modification or aggregation resulting
in losses of disulfide links which leads to reduction in reac-
tivity of IgE, but this may also have adverse effects on the
quality of soy. However, non-thermal methods appear to
do far much better in that regard. They can preserve the or-
ganoleptic and nutritional properties of soy while reducing
its allergenicity by changing conformational structure of
protein epitopes through protein's aggregates or crosslink,
and may also modify linear epitopes by division or deterio-
ration of fragmentation of amino acids. In the future, the
trend will turn towards non-thermal technologies to reduce
antigenicity of soy proteins but still needs some improve-
ments related to the processing conditions and non-
thermal process effects on functionality neo proteins.
In conclusion, several advances have been made in the
methods of detection and inactivation of soybean allergens
and that supposed to be continued. As mentioned above, in
future, the trend will rely on the development of fast, simple
and cheap methods which will allow the high detection of
soybean allergens and provide hypoallergenic foods.

198
L.T. Mulalapele, J. Xi
Author contributions
Lina Tokuna Mulalapele: Conceptualization, writing -
original draft preparation, reviewing; Jun XI: Supervision,
methodology, validation.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
Acknowledgement
This research was supported by the Natural Science Foun-
dation of China (NSFC, 32172310); Youth Key Teachers
from Henan University of Technology (21420043); the In-
novative Funds Plan of Henan University of Technology
(2020ZKCJ19). We would like to thank Raud Eucaristia
Mayoulou for his help in visualization and reviewing of this
paper.
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