Trends in Food Science & Technology 112 (2021) 336–347

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Trends in Food Science & Technology

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

Insights into formation, detection and removal of the beany flavor in

soybean protein
Bei Wang a, Qiang Zhang b, Na Zhang c, Kathrine H. Bak d, Olugbenga P. Soladoye e,
Rotimi E. Aluko f, Yu Fu a, *, Yuhao Zhang a
a
College of Food Science, Southwest University, Chongqing, 400715, China
b
School of Biological and Food Engineering, Guangdong University of Petrochemical Technology, Maoming, 525000, China
c
Key Laboratory of Food Science and Engineering of Heilongjiang Province, College of Food Engineering, Harbin University of Commerce, Harbin, 150076, China
d
Institute of Food Safety, Food Technology and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210, Vienna, Austria
e
Food Processing Development Centre, Ministry of Agriculture and Forestry, Government of Alberta, Leduc, Alberta, T9E 7C5, Canada
f
Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada

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

Keywords: Background: Plant-based meat analogues (PBMA) are promising to address the issue of imbalance between global
Soybean meat supply and demand. PBMA can contribute to an environmentally friendly and sustainable food production.
Beany flavor As one of the main raw materials in PBMA, the naturally occurring beany flavor in soybean restricts their further
Plant-based meat analogues
development and consumer acceptance. To enhance the application of soybean as an ingredient in PBMA as well
Formation
Detection
as improve consumer acceptability, the beany flavor should be effectively eliminated.
Removal methods Scope and approach: In the present review, the main beany flavor compounds, their formation mechanism, the
interaction between soybean protein and off-flavor compounds, the detection methods, and the removal methods
were systematically reviewed. Notably, the main removal approaches for beany flavor of soybean can be divided
into three categories, namely biotechnological, physical and chemical methods. The different removal methods
of beany flavor in soybean protein were compared and discussed.
Key findings and conclusions: Application of biotechnological methods to eliminate beany flavor in soybean is
challenging. Although enzymatic method is safe and clean, its catalytic efficiency is relatively low. Chemical
modification methods possess food safety threats. By contrast, physical processing is relatively effective in
removing beany flavor, while heating may exert a negative impact on the functional properties of soybean
protein. Collectively, enzymatic modification combined with novel non-thermal processing as well as the opti­
mized texturization process of soybean protein is of great significance to elimination of beany flavor in PBMA.

1. Introduction Coronavirus Disease 2019 (COVID-19) pandemic and animal diseases

With the rapid growth of global population, there is an unprece­
dented increase in the demand for meat products. The current tradi­
tional animal husbandry cannot satisfy the rapidly increasing
consumption of meat products, which created daunting challenges for
the meat industry. Additionally, the imbalance between global supply
and demand for meat products has been further exacerbated by the
such as African swine fever and avian influenza (Wei et al., 2020). To
relieve the supply pressure on meat products, development of meat
analogues can serve as an effective way to solve the imbalance of pro­
duction and consumption of meat products. Meat analogues, a meat
substitute, can be produced by controllable industrial production
instead of the traditional meat industry involving a series of processes,
such as breeding, slaughtering, storage and transportation. The

Abbreviations: PBMA, plant-based meat analogues; SPI, soy protein isolate; COVID-19, coronavirus disease 2019; LOX, lipoxygenases; PUFA, polyunsaturated fatty
acids; HPETEs, hydroperoxy-eicosatetraenoic acids; LPS, lipohydrolases; HPL, hydroperoxide lyases; ADH, alcohol dehydrogenase; HxAc, hexyl acetate; SFE, su­
percritical fluid extraction; SAFE, solvent assisted flavor evaporation; SPME, solid phase microextraction; SDE, simultaneous decomposition and extraction; PA,
polyacrylate; CAR/PDMS, carboxen/polydimethylsiloxane; DVB/CAR/PDMS, divinylbenzene/Carboxen/polydimethylsiloxane; PDMS/DVB, polydimethylsiloxane/
divinylbenzene.
* Corresponding author.
E-mail address: fuy987@swu.edu.cn (Y. Fu).

https://doi.org/10.1016/j.tifs.2021.04.018
Received 15 November 2020; Received in revised form 20 January 2021; Accepted 7 April 2021
Available online 18 April 2021
0924-2244/© 2021 Elsevier Ltd. All rights reserved.
B. Wang et al. Trends in Food Science & Technology 112 (2021) 336–347

availability of meat analogues can effectively alleviate the negative
impact of environmental pollution and animal diseases encountered
during the traditional animal husbandry and muscle food processing
(Kumar et al., 2017). Meat analogues can be divided into two categories,
namely plant-based meat analogues (PBMA) and cultured meat (Ismail,
Hwang, & Joo, 2020). Cultured meat is the meat generated from in vitro
growth of stem cells, rather than from slaughtered animals. However,
cultured meat has some limitations for large-scale production and
application due to the high cost and technical requirements (Verbeke,
Sans, & Van Loo, 2015). By contrast, PBMA are mainly produced using
isolated plant proteins (esp. soybean protein). The isolated protein is
further processed by electrospinning, extrusion technology and 3D
printing to fabricate meat texture, followed by addition of flavoring and
colorants that mimic the flavor and color of meat (Chiang, Loveday,
Hardacre, & Parker, 2019; Godoi, Prakash, & Bhandari, 2016; Kumar
et al., 2019). To date, PBMA have been considered the most promising
substitute for meat. Compared with traditional meat processing, PBMA
has broad development prospects due to its traceability, environmental
friendliness and controllable quality. They are expected to provide an
effective solution to challenges encountered with animal welfare, global
public health, sustainable development and meat supply problems.
The proteins isolated from soybean (Glycine max) or pea (Pisum sat­
ivum) currently serve as the main raw materials for manufacture of
PBMA (Joshi & Kumar, 2015). Given soybean protein has a more
widespread application in production of PBMA, the present review
article focuses on its off-flavor compounds. Since soybean protein has an
unpleasant beany and grassy odor (Ravi, Taheri, Khandekar, & Millas,
2019), it affects the overall flavor of PBMA, which limits consumer
acceptability and purchase of the products as well as restricts the
development of PBMA as food products. Therefore, effective removal of
beany flavor in soybean protein to improve the overall flavor of PBMA is
worth further investigation. The beany flavor of soybean protein is
primarily due to polyunsaturated fatty acids (PUFA) derivative. These
compounds could be produced by PUFA degradation catalyzed by LOX
(lipoxygenase, EC 1.13.11.12) to yield hydroperoxyl derivatives, which
are further degraded into alcohols, aldehydes, ketones, acids, amines
and other volatile compounds with different thresholds of odor, thus
forming beany flavor (Roland, Pouvreau, Curran, van de Velde, & de
Kok, 2017).
At present, the supply and demand for meat products are experi­
encing unprecedented challenges. The presence of PBMA can provide a
possibility to solve such a serious issue. However, whether the beany
flavor can be effectively removed is the key point for consumers to
accept and recognize PBMA. Therefore, in this work, the type of off-
flavor compounds, their formation mechanism, detection methods and
removal strategies are reviewed. Besides, some future research di­
rections are also discussed. This article can provide a useful reference for
the research and development of PBMA.
2. The formation of off-flavors in soybean
2.1. Key odorants of beany flavor
Beany flavor is a combination of many volatile compounds produced

Table 1
Main off-flavor odorants and their odor thresholds (OTs) in water.
Odorants CAS OT/ppb Aroma attributes References

Aldehyde
(E)-2-hexenalab 6728-26-3 0.082 leafy Chen (2015)
(E)-2-heptenalab 18829-55- 0.013 soap, fat Navicha, Hua, Masamba, Kong, & Zhang (2018)
5
(E)-2-octenalab 2548-87-0 0.003 fatty, green, cucumber Kaczmarska et al. (2018)
(Z)-3-hexenalak 6789-80-6 0.00012 green Roland et al. (2017)
pentanalac 110-62-3 0.008 irritant Poliseli-Scopel, Gallardo-Chacón, Juan, Guamis, and Ferragut (2013)
hexanalac 66-25-1 0.0045 cut-grass, green Navicha et al. (2018)
heptanalac 111-71-7 0.003 dry fish Navicha et al. (2018)
nonanalac 124-19-6 0.001 green, fatty Navicha et al. (2018)
octanalac 124-13-0 0.0007 fatty, pungent (Navicha et al.,
2018bib_Navicha_et_al_2018abib_Navicha_et_al_2018abib_Navicha_et_al_2018a)
(E)-2-nonenalad 18829-56- 0.0004 beany, green Kaczmarska et al. (2018)
6
benzaldehydeac 100-52-7 0.003 almond Navicha et al. (2018)
(E, E) − 2, 4-nonadienalae 5910-87-2 0.0001 fatty Chen (2015)
(E, E) − 2, 4-heptadienalad 4313/2/4 0.00256 fatty, fishy Navicha et al. (2018)
(E, E)-2,4-decadienalae 25152-84- 0.000027 spices Chen (2015)
5
Pyrazine
2-isopropyl-3- 93905-03- 0.0008 pea-like, earthy Ravi et al. (2019)
methoxypyrazineef 4
Acid
acetic acidsad 64-19-7 180 sour Navicha et al. (2018)
Ketone
2-heptanonead 110-43-0 3 fragrance Navicha et al. (2018)
1-penten-3-onead 1629-58-9 0.0009 spicy, onion Roland et al. (2017)
1-octen-3-oneai 4312-99-6 0.000003 green-beany Chen (2015)
2-octanoneai 111-13-7 0.05 soapy, floral Navicha et al. (2018)
Furan
2-penthyfuranab 3777-69-3 0.0058 beany off-flavors Chen (2015)
2-ethylfuranbj 3208-16-0 0.0023 beany, earthy, malty, Poliseli-Scopel et al. (2013)
sweet
Alcohol
1-octen-3-olac 3391-86-4 0.007 mushroom Matsui et al. (2018)
1-pentanolab 71-41-0 0.1502 green, wax Yoo and Chang (2016)
1-penten-3-olab 616-25-1 0.3581 beany, green Kaczmarska et al. (2018)
hexanolab 111-27-3 0.5 beany, green Chen (2015)
3-methyl-1-butanolah 123-51-3 0.004 balsamic Chen (2015)

Note: aOdor thresholds in water from van Gemert. (2011); bFrom Zhu et al. (2015); cFrom Liao et al. (2020); dFrom Pang et al. (2019); eFrom Pollner & Schieberle
(2016); fFrom Wagner & Grosch (1998); hFrom Wang et al (2020); iFrom Xu et al. (2019); jFrom Giri et al. (2010); kFrom Jung et al. (2017).

337
B. Wang et al.
during the growth and processing of soybean (Yoo & Chang, 2016). As
shown in Table 1, more than 20 volatile compounds have been reported
to be associated with the beany flavor. These compounds are mainly
divided into fatty aldehydes, fatty alcohols, fatty ketones, furans, furan
derivatives and aromatic compounds (Roland et al., 2017). Among these
off-flavor compounds, hexanal is considered to be the most important
one (Yu, Liu, Hu, & Xu, 2017). Some non-volatile compounds, including
flavonoids, saponins, phenolic acids and certain amino acids, can pro­
mote the formation of beany odorants (Roland et al., 2017). However, in
the current review, only volatile compounds will be emphasized.
Short-chain aliphatic carbonyl compounds produced by PUFA
degradation catalyzed via LOX are considered to be the key reason for
formation of beany flavor in soybean. These off-flavor compounds
usually exert a negative effect on soybean products due to their rela­
tively low odor thresholds. Hexanal and (Z)-3-hexenal are two repre­
sentative volatile compounds formed by PUFA degradation (Roland
et al., 2017). As shown in Table 1, hexanal has a cut-grass and green
smell and it is characterized by an extremely low threshold (0.0045 ppb)
in water. It is an essential odor in soybean derived from the enzymatic
oxidation of linoleic acid (Fig. 1). (Z)-3-Hexenal (green beany flavor) is a
significant contributor to soybean flavor and has an extremely low odor
threshold (0.00012 ppb), which originates from the oxidation of
α-linolenic acid (deMan, Finley, Hurst, & Lee, 2018).
Fig. 1. The potential formation pathway of beany flavor compounds.
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Trends in Food Science & Technology 112 (2021) 336–347
2.2. Formation mechanisms of beany flavor
The beany flavor compounds in soybean have been previously
investigated because of the negative effects on the sensory property of
the resultant formulated products. The formation pathways of beany
flavor encompass three main routes, including automatic oxidation,
photosensitized oxidation, and enzymatic oxidation (deMan et al.,
2018). Automatic oxidation is a free radical reaction initiated by singlet
oxygen produced from natural pigments. The highly reactive singlet
oxygen attacks H on the α-methylene adjacent to the double bond to
form the alkyl radical (R⋅), which further absorbs oxygen from the air to
generate peroxide radical (ROO⋅), thus forming hydroperoxides. Light
can catalyze automatic oxidation of unsaturated fatty acids (deMan
et al., 2018). Unlike automatic oxidation, photooxidation is a reaction in
which singlet oxygen directly attacks the double bond of unsaturated
fatty acids. Oxygen can bind directly to fatty acids, thus forming hy­
droperoxides. Singlet oxygen is more critical for photosensitive oxida­
tion than automatic oxidation. Therefore, singlet oxygen quenchers
(β-carotene and tocopherol) can limit the occurrence of photosensitive
reactions, while oxygen concentration exerts a little impact on photo­
sensitive oxidation (Hammond & White, 2011). Besides, the number and
distribution of unsaturated double bonds also have a significant effect on
photooxidation. Enzymatic oxidation means that PUFA with (Z, Z)-1,
4-pentadiene structure can be specifically catalyzed by LOX to form
hydroperoxyl derivatives. These compounds are further degraded to
produce aldehydes, alcohols, and other volatile compounds, leading to
B. Wang et al.
beany flavor (Wang, Guldiken, Tulbek, House, & Nickerson, 2020).
Among three pathways, LOX-catalyzed degradation of unsaturated fatty
acids represents the enzymatic degradation and acts as the most pre­
dominant way to produce beany flavor compounds in soybean (Roland
et al., 2017). Overall, three reaction mechanisms are different, but all
the common reaction products are hydroperoxyl derivatives, which are
odorless but unstable. Metal ions and light can catalyze the degradation
of hydroperoxyl derivatives, which further produces ketones, aldehydes,
and acids.
In general, the factors affecting the degradation of unsaturated fatty
acids can be divided into internal and external factors. The external ones
include ambient temperature, initiators, antioxidants, oxygen concen­
tration, and surface area contact with air. It is worth noting that oxygen
concentration is more significant in photosensitive oxidation than
automatic oxidation. Namely, the higher the oxygen concentration, the
faster the automatic oxidation reaction rate. The predominant internal
cause is the number of double bonds of unsaturated fatty acids (deMan
et al., 2018). By contrast, lipoxygenase is the limiting factor in terms of
enzymatic oxidation.
LOX belongs to the family of nonheme iron-containing enzymes that
can catalyze the deoxygenation of PUFAs such as linoleic acid (18:2) and
linolenic acid (18:3), yielding hydroperoxy-eicosatetraenoic acids
(HPETEs) in addition to other hydroperoxyl derivatives (Alhendi, Yang,
& Sarnoski, 2018). Therefore, LOX is the main factor responsible for
beany flavor development in soybean products. To date, three soybean
LOX isozymes have been found, namely LOX1, LOX2 and LOX3 (Yang,
Smyth, Chaliha, & James, 2016). LOX3 can be further divided into
LOX3a and LOX3b; however, they are considered the same isozyme
because of their similar properties (Axelrod, Cheesbrough, & Laakso,
1981). These three isozymes have high homology with a cylindrical
structure composed of 150 amino acids at the homologous N-terminal
sequence. This unique structure is necessary for Ca2+ binding, enzyme
activation and nuclear membrane placement of LOX and its isomers.
Also, this structure may be involved in maintaining the holistic stability
of protein (Zaragoza et al., 2019). There is a conserved domain con­
sisting of 40 amino acids at the C-terminal including 6 His and 2 Tyr,
which can bind to Fe3+ as the active center of enzyme, thus regulating
enzyme activity. Elimination of LOX in soybean means that PUFA cannot
be further oxidized to form odorants. As a result, the content of volatile
odorous compounds in soybean was significantly reduced. LOX is
essential for formation of several critical secondary metabolites,
including jasmonic acid, which plays an ideal role in various critical
physiological processes. As a plant growth regulator, jasmonic acid plays
an essential role in plant defense against insects and necrotizing fungi
(Zhang, Lei, Lu, Wang, & Wu, 2020). The LOX in soybean can also serve
as the appropriate protease inhibitors, which are essential for protecting
plants from insects and pests (Kumari et al., 2015). In addition, lip­
ohydrolases (LPS), hydroperoxide lyases (HPL), alcohol dehydrogenase
(ADH) and isomerase (ISO) are also involved in the fatty acid meta­
bolism of soybean (Fig. 1).
The formation mechanism of major volatile compounds from PUFA
degradation in soybean is illustrated in Fig. 1. Firstly, LOX can catalyze
the conversion of fatty acids to form hydroperoxyl derivatives (Chedea
et al., 2014). Subsequently, short-chain aliphatic aromatic compounds
are produced by the HPL-catalyzed reaction. These aldehydes can be
further reduced to the corresponding alcohols under the action of ADH
(deMan et al., 2018). Eventually, these volatile compounds may further
aggregate to elicit the soybean odor.
2.3. The interactions between volatile compounds and soybean protein
Both β-conglycinin (7S) and glycinin (11S) proteins are the main
components of soybean because they account for over 80% of the total
proteins (Peng et al., 2019). PUFA derivatives could interact soybean
proteins by two fashions (physical or chemical process). These off-flavor
compounds elicit beany flavor in the process of consumption or
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Trends in Food Science & Technology 112 (2021) 336–347
processing, which can exert a negative impact on the overall flavor
profile of PBMA. Based on the difference in chemical structure and
functional groups between protein and volatile compounds, the type of
interactions can be divided into reversible (non-covalent) and irrevers­
ible (covalent) ones. The reversible binding interactions mainly include
hydrogen bond, hydrophobic interaction and ionic bond. The interac­
tion between off-flavor compounds and –COOH, –SH, –NH2, and –OH
groups of protein is usually reversible. The irreversible binding is mainly
due to the formation of covalent bonds between the off-flavor compound
and -S-S-, –SH, –NH2 groups in proteins. Proteins with a high content of
Lys, Arg and Cys tend to have a higher flavor binding ability because of
covalent conjugations. However, Joshi and Kumar (2015) demonstrated
that 7S protein was the main protein type suitable for processing PBMA,
as 11S protein is more odorous due to the higher content of
sulfur-containing amino acids that enables stronger binding of volatile
compounds (Wang & Arntfield, 2017).
Most of the studies on the binding thermodynamic process of soy­
bean flavor compounds and soybean protein have been previously re­
ported in the early 1990s. At present, the Scatchard equation has been
commonly used to represent the thermodynamic process of interactions
between protein and flavor compounds (Guo, He, Wu, Zeng, & Chen,
2019).
v
[L]
= K(n − v) (1)
v
= K[L] (2)
n− v
where v is the number of moles of flavor compound bound per mole of
protein, [L] is the concentration of the free flavor compound (mol/L) in
the solution, n is the total number of binding sites in the protein, and K is
the intrinsic binding constant. The binding constant (K) and the number
of binding sites on the proteins (n) can be determined using the double
reciprocal fashion of the Scatchard equation, the so-called Klotz plot:
1 1 1
= +
v n nK[L]
(3)
K[L]
v= n (4)
1 + K[L]
The higher binding constant (K), the more tightly the flavor com­
pounds bind to a protein; the smaller the free energy (ΔG), the easier the
reaction occurs (Wang & Arntfield, 2017). To date, the mechanisms
responsible for interactions between proteins and flavor compounds
have not been fully established. However, the binding constant and re­
action free energy are normally restricted to food systems as well as the
conformation of protein-flavor compounds complex (Wang & Arntfield,
2017). The structural characteristics of protein and flavor compounds
lead to different intermolecular force among different groups, which
have varying effects on the interaction between soybean protein and
flavor compounds. Aldehydes usually have a higher binding affinity to
proteins when compared to ketones. The binding affinity of alcohol is
three times higher than that of ketones. However, the binding affinity of
the same class of volatile compounds increases with the increase of
carbon chain. It has been suggested that butanol shows a higher binding
constant than hexanol (Wang & Arntfield, 2017). This indicates that
hydrophobic interactions between different binding sites have different
hindering effects on proteins.
The structure of soybean protein is also a major factor affecting
binding affinity. The flavor compounds can bind to proteins through
hydrophobic interactions. In this case, the factors that change protein
conformation and expose or destroy the internal hydrophobic region
will change the binding affinity towards flavor compounds. Therefore,
the targeted modification of soybean protein structure or regulation of
key external factors in the food system can effectively alter the binding
affinity between the protein and flavor compounds in order to remove
B. Wang et al.
beany flavor.
There are two major ways to change the structure of soy protein. The
first one is to modify soy protein by chemical or enzymatic methods to
change its binding affinity with flavor compounds. The binding ability of
soy protein to flavor compounds changed when malondialdehyde
(MDA) was added to a soybean protein solution. Besides, the hydro­
phobic interactions between soy protein isolate (SPI) and flavor com­
pounds such as hexanal and nonanal was significantly reduced, when
the MDA concentration exceeded 2.5 mM (Wang, Zhao, Qiu, & Sun,
2018). The binding affinity of SPI to vanillin and maltol was investigated
by equilibrium dialysis (ultrafiltration) technique combined with
protein-glutaminase (PG). The results showed that the binding affinity
of two flavor compounds (vanillin and maltol) to soybean protein at
25 ◦ C was decreased after deamination. It was suggested that the
binding mechanism was changed from hydrophobic inter­
action/covalent bond to weak van der Waals force or hydrogen bond
(Suppavorasatit & Cadwallader, 2012).
The changes in the food system factors such as temperature, pH and
salt concentrations can also alter the interactions between protein and
flavor compounds and effectively remove the beany flavor in soybean.
The interactions of SPI with flavor compounds at pH 3.0, 6.0 and 9.0
have been recently been investigated by Guo et al. (2019). The results
showed that soybean protein aggregation could be significantly influ­
enced by changing the pH of solution, while SPI structure could alter
through recombination of specific polypeptide subunits. Besides, the K
value of hexyl acetate (HxAc) was 630 M− 1 at pH 3.0, whilst K values
rose sharply for SPI (5400 M− 1 at pH6.0) and (1300 M− 1 at pH9.0), as
the interface between SPI subunits tightened. Also, temperature is an
important factor affecting the binding constant. Kühn, Considine, and
Singh (2008) studied the binding between 2-nonanone and whey protein
isolate at 80 ◦ C. The binding capacity for 2-nonanone showed a
decreased trend with increasing heating time. After heating for 80 min,
the binding energy of 2-nonanone was decreased by 20%. Moreover, it
has been reported that the binding rate of soybean protein and flavor
compounds could be effectively reduced when a third competitive
compound was present in the food system. The application of β-cyclo­
dextrin to remove unpleasant odorants has been previously reported for
soybean protein. Arora and Damodaran (2010) found that β-cyclodex­
trin can effectively inhibit the binding of soybean protein with 2-nona­
none. The higher β-cyclodextrin concentration, the better the inhibitory
effect on off-flavor. When the concentration of β-cyclodextrin in the
system was 6 mM, the binding rate of soybean protein to 2-nonanone
was remarkably reduced by 94%. Although the mechanisms of
protein-flavor compound interactions are not clear, the changes in the
altered binding constant contribute to the removal of soy protein beany
flavor. Microbial transglutaminase (mTGase), MDA and cyclodextrin are
commonly used reagents for structural modification of soybean protein,
which can interfere with the binding mechanisms of protein-flavor
compounds and show promise for removal of beany flavors.
3. Analytical methods for beany flavor characterization
3.1. Extraction of volatile compounds
The volatile compounds in food matrices are complex, low in content
and unstable. They can easily be affected by external conditions during
the extraction process. Furthermore, the selection of extraction methods
may directly affect the quality and quantity of volatile compounds.
Therefore, depending on the experimental design and sample charac­
teristics, selection of appropriate methods to extract volatile compounds
is the primary premise to investigate the off-flavor compounds in soy­
bean. At present, the main extraction methods of volatile compounds
responsible for beany flavor include supercritical fluid extraction (SFE),
solvent assisted flavor evaporation (SAFE), solid phase microextraction
(SPME), simultaneous decomposition and extraction (SDE).
SFE acts as a separation technology that can be used to extract and
340
Trends in Food Science & Technology 112 (2021) 336–347
separate volatile compounds using supercritical fluid as solvent at
temperature and pressure above the critical point. It can be utilized to
extract fatty acids from soybean, which can contribute to removal of
beany flavor to a certain extent (Ibáñez, Mendiola, & Castro-Puyana,
2016). The phospholipids that act as precursors of off-flavors in soy­
bean meal were extracted by SFE and determined to be approximately
2–3 μg/g, which was significantly lower than that of the control (5–6.5
μg/g) (Kumari et al., 2016). Also, this study pointed out that the trend of
thiobarbituric acid (TBA) concentration was similar to the varying
contents of phospholipids. TBA can be an indicator of off-flavor gener­
ated from lipid peroxidation (Kumari et al., 2016).
SAFE is a method that can rapidly isolate volatile compounds from
complex solvent extracts (Pua et al., 2020). During the extraction pro­
cess, the solvent vaporizes rapidly under a high vacuum condition, and
the volatile compounds and non-volatile compounds are separated. SPI
treated by this method tends to have less beany flavor, higher water
holding capacity and emulsifying stability, but lower solubility. It was
suggested that the method might be the most suitable for pea flour
extraction (Murat, Gourrat, Jerosch, & Cayot, 2012). Hexanal accounted
for 2.3% of the total relative amount by SAFE, which was a negligible
amount, in comparison with purge and trap extraction (17.6%). This fact
means that beany flavor compounds can, to some extent, be removed by
a proper extraction process extent.
SPME is a rapid, solventless alternative to the conventional extrac­
tion method, which integrates several operations including sample
collection, extraction, analyte enrichment and isolation from sample
matrix. It normally employs a polydimethylsiloxane/divinylbenzene
(PDMS/DVB) fiber coated with different adsorption coatings to carry out
the adsorption and desorption of volatile compounds. The type of SPME
fiber has been identified as the vital factor, which can exert an impact on
extraction of volatile compounds. Four types of SPME fiber, namely
PDMS/DVB, polyacrylate (PA), carboxen/polydimethylsiloxane (CAR/
PDMS), and divinylbenzene/carboxen/polydimethylsiloxane (DVB/
CAR/PDMS), have previously been compared and investigated (Xu, Jin,
Lan, Rao, & Chen, 2019). Generally, PDMS/DVB and PA had lower peak
areas of average 2.3 × 106 counts, while peak areas of DVB/CAR/PDMS
and CAR/PDMS doubled at 4.2 × 106 counts. In terms of the off-flavor
profile in soybean, DVB/CAR/PDMS and CAR/PDMS were more suit­
able for further research.
SDE is an extraction method that heats sample and extractant at the
same time so that the two are fully mixed in a closed vessel. This method
has been applied in sensory research for decades, because it can com­
plete the separation and concentration of volatile compounds at the
same time (Xu et al., 2016). However, the extractive by SDE can hardly
reflect the original information of samples due to the low extraction
yield of beany flavor odorants. For instance, SPME and SDE were
employed to compare the effect of the extracted volatile compounds on
natto. The results showed that the number of volatile compounds in
natto extracted by SPME and SDE were 42 and 31, respectively (Yu et al.,
2017).
At present, SPME is the most commonly used extraction method in
the research of beany flavor. This method is efficient, fast and
economical, and has high extraction efficiency for volatile compounds
with a low boiling point. However, the extracted compounds depend on
polarity of the coating fibers used. Therefore, it is necessary to optimize
it prior to application. The extraction rate of SAFE for those volatile
compounds with low solubility in water is higher than that of SPME. SDE
is effective in the field of food science, but the intermediate compounds
produced by heating may lead to failure to reflect the actual information
of the extract.
3.2. Detection of volatile compounds
Gas chromatography-mass spectrometry (GC-MS) is an analytical
method that combines the advantages of mass spectrometry and gas
chromatography to identify the different volatile compounds within
B. Wang et al.
sample. Gas chromatography can make use of the difference in partition
coefficients of different compounds in the stationary phase and mobile
phase to separate them according to the different elution times in the
chromatographic column. Mass spectrometry is based on the movement
of ions in the magnetic field to separate samples according to their
different mass to charge ratio (m/z), which is very effective for the
identification of compounds. In recent years, GC-MS has been widely
used in the study of soybean beany flavor (Chigwedere et al., 2019; Xu,
Xu, et al., 2019). SPME combined with GC-MS has been utilized to
characterize the effect of soaking time and conditions (aerobic and
anaerobic) in water on the contents of key beany flavor compounds
(Matsui, Takemoto, Koeduka, & Ohnishi, 2018). Compared with the
control (soaking in water for 0 min), the concentration of all compounds
reported in the study dramatically increased by soaking for 30 min
under aerobic conditions (Fig. 2). Specifically, for the same treatment,
the approximate concentration of hexanal (52 nmol/g) and hexanol (60
nmol/g) were almost 26 times and 10 times higher than that of the
control. However, when the air was replaced by nitrogen, an interesting
phenomenon was observed that all the contents of volatile compounds
declined significantly except for 1-octen-3-ol. As a result, an appropriate
conclusion can be drawn that 1-octen-3-ol in soybean was not produced
by LOX catalysis, which indeed needs oxygen. Thus, GC-MS detection
technology could effectively characterize the changes in off-flavor
compounds of soybean protein during processing.
Gas chromatography-olfactometry (GC-O) serves as an effective
method for the separation and identification of active aroma compo­
nents by combining the powerful separation ability of GC with human
keen sense of smell (Song & Liu, 2018). To analyze the aroma profile of
Japanese soy sauce, 36 active odorants were identified by GC-O coupled
with aroma extraction dilution analysis. Ethyl-2-methylpropanoate,
ethyl 2-methylbutanoate, and ethyl 3-methylbutanoate were consid­
ered as the most intense aroma-active components (Feng et al., 2015).
This method can be used to analyze the different beany flavor
Fig. 2. Formation of volatile compounds after hydration of soybean seed powder. (A) Chromatograms obtained after the addition of water (top, 0 min) and 30 min
after the addition of aerated (middle, 30 min) or deaerated (bottom, 30 min anaerobic) water. (B) Amounts of volatiles detected at 0 min (white bar), 30 min with
aerated water (grey bar), and 30 min with deaerated water (black bar). Readapted from Matsui et al. (2018) with permission from Elsevier, copyright 2018.
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compounds produced by various Bacilli (1-M3M, 2-M1L, 2-M4M,
9-M1M, 10-Mi6M) to determine the strain with the best sensory
acceptance (Yoon, Chang, & Lee, 2013).
The combination of GC-O and GC-MS, GC-O-MS can accomplish the
rapid detection of aroma active compounds and identification of their
chemical structures (Song & Liu, 2018). The electronic nose is an aroma
detection technology with bionic olfactory function. It is mainly
composed of sensor array and recognition pattern method, which can
reflect the overall information of the sample aroma (Yan et al., 2015).
Ravi et al. (2019) employed electronic nose technology to identify and
characterize the volatile components of five soybean varieties, which
have a higher potential yield. Among different soybean varieties, alde­
hydes accounted for the highest proportion in the variety UA5414RR,
esters in UA5014C and JTN5503, and sulfur compounds in JTN511 and
JTN5203.
Taken together, GC-O can effectively identify the active volatile
compounds in food, but the qualitative analysis of volatile compounds
has some limitations. By comparing the retention index (RI) and odor
characteristics between the detected compounds and the standard
samples, only preliminary identification of the compounds can be ach­
ieved. Therefore, GC-MS is still needed to obtain the structural infor­
mation on these aromatic active compounds. GC-O-MS can effectively
combine the advantages of GC-MS and GC-O, but its application range is
relatively limited. However, compared with GC-O, this technology can
be used to determine the quality and quantity of aroma compounds, and
provide holistic sample information.
4. Removal methods of beany flavor
Currently, there are three main approaches for removal of soybean
beany flavor, including biotechnological methods, physical methods and
chemical methods summarized in Fig. 3. Biotechnological methods
mainly focus on breeding high-quality soybean varieties without LOX, or
B. Wang et al.
Fig. 3. The summary of some common methods used for removal of
beany flavor.
treating soybean with hormones to reduce activities of LOX and HPL,
thus reducing the production of beany flavor compounds. Physical
methods mainly use radiation, heating, high-frequency electromagnetic
fields and other physical means to reduce the activity of fatty acid
synthase, or addition of flavor enhancer to cover the beany flavor (Yu
et al., 2017). Chemical methods include the addition of metal-reducing
agents or dehydrogenases to convert fatty aldehydes into the corre­
sponding acids. In addition, LOX activity can be reduced by adjusting pH
to remove the beany flavor in soybean (Table 2).
4.1. Biotechnological methods
Biotechnological methods to remove beany flavor mainly encompass
genetic engineering, enzymatic treatment, fermentation and
germination.
4.1.1. Genetic engineering
Genetic engineering as a method mainly refers to breeding soybean
varieties without LOX using molecular biotechnology. Having estab­
lished that the degradation of unsaturated fatty acids catalyzed by LOX
is the most predominant factor for production of beany flavor com­
pounds, the genetic engineering method may serve as the most effective
way to remedy the problem. It has been reported that the contents of
hydroperoxyl derivatives and hexanal produced by soybean without
LOX1, LOX2 or LOX3 isozymes were much lower than that of common
soybean varieties (Yang et al., 2016). The amount of hexanal in LOX-free
samples was approximately one-fifth of that in the control. Besides, the
nutritional value and quality of soybean varieties without LOX can be
also improved. Esteves, Martino, Oliveira, Bressan, and Costa (2010)
compared the chemical constituents of LOX2 and LOX3 deficient soy­
bean varieties (UFV-116) and conventional varieties (OCEPAR-19). It
was found that UFV-116 contained more essential amino acids, soluble
amino acids, total amino acids, and higher Ca, K, and Mg than those of
OCEPAR-19.
However, it worth noting that there are some limitations for the
application of genetic engineering methods. On the one hand, LOX plays
a physiological role in soybean, which are highly important to protect
plants from pests. Therefore, the complete elimination of LOX isozymes
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in soybean may have adverse effects on plant defense mechanism
(Kumari et al., 2015). On the other hand, in addition to LOX, oxidative
rancidity of unsaturated fatty acids can also produce beany flavor in
soybean products. Seventy genotypes of soybean seed were used to
characterize the correlation between LOX and soymilk characteristics,
which demonstrated no significant correlation between LOX-free soy­
bean raw material and soymilk flavor (Ma et al., 2015).
4.1.2. Enzymatic treatment
Enzymatic hydrolysis aims to remove beany flavor compounds in
soybean protein, such as hexanol, amyl alcohol, heptanol and hexanal
by converting the volatile compounds to the corresponding acids via
highly specific proteases, such as alcohol dehydrogenase and aldehyde
dehydrogenase. Beany flavor of soybean protein can be removed by
enzymatic treatment (Sethi, Tyagi, & Anurag, 2016). The processed
protein can also produce a certain flavor, which fundamentally improves
the flavor of soybean-based food. Due to the strong specificity of en­
zymes, they have no impact on other nutrients in soybean. It has been
shown that compared with homogenization alone, the beany flavor
score of fermented soybean milk prepared by the enzyme Celluclast 1.5L
(1.2% v/v and 3 h treatment) was remarkably lower (2.5) than that of a
single homogenization treatment (4.8) when measured on a 1 (weak) to
9 (strong) attribute scale (Rosenthal et al., 2003). In addition, the
inhibitory effect of enzyme concentration on bean curd was different.
Rosenthal et al. (2003) showed that when the concentration of Celluclast
1.5L was 1.6%, the odor attribute score of soybean milk was lowest
(1.8), which was significantly lower than that of the control (4.5).
Phospholipase A2 (PLA2) and cyclodextrin have been employed to
reduce beany odorant precursors by hydrolysis (Zhu & Damodaran,
2018). The results showed that the degradation efficiency of PLA2 was
75.7% at pH 8.0 and 43 ◦ C for 3 h, while the degradation efficiency of
α-cyclodextrin combined with PLA2 was 95.3% under the same condi­
tions. This indicates that the enzymatic method in combination with
α-cyclodextrin treatment can effectively remove the beany flavor.
Phospholipids and free fatty acids are precursors of beany flavor sub­
stances. Phospholipids can be hydrolyzed in the presence of PLA2, while
some free fatty acids can be generated. These free fatty acids are
enveloped by cyclodextrin to produce a molecular inclusion complex,
which further reduces beany odor (Lee, Kim, Lee, Ramachandraiah, &
Hong, 2020; Zhu & Damodaran, 2018). By contrast, enzymatic hydro­
lysis combined with filtration (20-μm pore size tissue) treatment could
partly reduce beany flavor by approximately 20% in native soybean
(Rosenthal et al., 2003).
4.1.3. Fermentation
Microbial fermentation refers to the process of transforming raw
materials into products through specific metabolic pathways under
suitable conditions. Microbial fermentation is widely used to improve
the flavor (El Youssef et al., 2020; Mäkinen, Wanhalinna, Zannini, &
Arendt, 2016) and nutritional value of legumes (Cao, Green-Johnson,
Buckley, & Lin, 2019).
Off-flavor compounds can be completely removed by microbial
fermentation. For instance, the content of hexanal in untreated samples
was 17.9 ppm, while it could be hardly detected in soybean milk fer­
mented by Lactobacillus and Streptococcus (Blagden & Gilliland, 2006).
On the other hand, some positive aroma properties of soybean were
enhanced by extra volatile compounds produced during the fermenta­
tion process, whereas beany flavor of soybean could be limited in the
same way. Taking a proper case, the volatile compounds of soybean
fermented by Bacillus subtilis and Rhizopus oligosporus differed from the
control. To be specific, there was a significant decline of aldehydes by
5–11%, which was closely associated with beany flavor during the
manufactory process; however, 1.35–4.87% of pyrazine unrecorded in
the control was identified at the end of the same period. Pyrazine could
serve as a flavor enhancer of nutty attributes and roasted food (Ho,
Zheng, & Li, 2015).
B. Wang et al. Trends in Food Science & Technology 112 (2021) 336–347

Table 2
Summary of recent studies on removal methods of beany flavor.
Category Method Material & Reagent Treatment Observation Reference

Biology Enzyme Protamex pH 6.0, 45 C for 40 min

◦
Beany flavor reduced around by Yoo and Chang (2016)
70%.
Phospholipase A2 pH 8.0, 43 C for 3 h
◦
The efficiency of removal of Zhu and Damodaran
phospholipids was 75.7%. (2018)
Fermentation Bacillus subtilis and Fermentation at 30 ◦ C for 3 days Increase in the number of Chukeatirote et al.
Rhizopus oligosporus compounds. (2017)
Genetic Soybean The varieties lacking of LOXs The sweet and fresh odor were Yang et al. (2016)
engineering enhanced.
Germination Soybean Germination at 22 ◦ C for 72 h, relative Flavor acceptability increased. Kaczmarska et al.
humidity 50–60% (2018)
Chemistry Reverse micelles 2-Ethylhexyl, sodium Reverse micelles treatment Relative peak area of hexanal Zhao, Zhang, Liu,
sulfosuccinate and reduced around by 5%. Zhang, and Ao (2018)
bicinchoninic acid
Physics Cyclodextrin β-Cyclodextrin 60 ◦ C, 0.75% β-cyclodextrin Beany flavor reduced by around Shi et al. (2017)
57%.
Phospholipase A2 and SPI (50 g/l), PLA2 (2 μkat of PLA2/g) SPI, The efficiency of removal of Zhu and Damodaran
cyclodextrin Cyclodextrin concentration (10 mM) pH 8.0, phospholipids was 95.3%. (2018)
at 43 ◦ C for 3 h
β-Cyclodextrin ISP (5%, w/w) and β-cyclodextrin (4%, w/ Relative peak area of hexanal was Lee et al. (2020)
w), heated to 90 ◦ C and incubated in a water reduced by around 4%.
bath at 90 ◦ C for 15 min, cooled to 10 ◦ C.
Flavor enhancer Monosodium glutamate The flavor enhancers, monosodium The improved amplitude. Milani et al. (2014)
monohydrate and disodium glutamate monohydrate and disodium 5-
5-inosinate, inosinate.
Heat – 121 ◦ C, heating for 3 min Peak area of hexanal reduced by Yoo and Chang (2016)
around 99%.
– 121 ◦ C, autoclaved for 40 min No hexanal was detected. Chukeatirote et al.
(2017)
– Boiling for 3 h No hexanal was detected. Chukeatirote et al.
(2017)
– Heating at 110 C for 60 min
◦
Hexanal was reduced around by Navicha et al. (2018)
26%.
– 110 ◦ C, roasting for 60 min Beany flavor reduced around by Navicha, Hua,
25%. Masamba, Kong, &
Zhang (2018)
– 45 ◦ C, storage for 48 weeks The major unsaturated fatty acids Prabakaran et al.
were reduced. (2018)
– 110 C, 600 s
◦
The efficiency of removal of LOX Jiang et al. (2018)
was 90%.
Pulsed electric – 400 Hz, 2 ls, 42kv/cm, 25 ◦ C Inactivation of soybean LOX was Li et al. (2008)
fields 88%.
Radio frequency – Radio frequency treatment for 210s The efficiency of removal of LOX Jiang et al. (2018)
treatment was 94.3%.
Supercritical – The vessel heater: 30 C, electrical heat
◦
The residual PLS and the TBA were Kumari et al. (2016)
carbon dioxide exchanger: 75 ◦ C, CO2 supply pressure:150 reduced.
extraction bar, 45 g/min for 45 min
Other Biotic elicitor Salicylic acid 0.1 mM Salicylic acid as elicitors on off- LOX and HPL activity of high off- Kumari et al. (2015)
methods treatments flavor soybean varieties flavor cultivar were reduced by 2.3
and 2.4 times, respectively.
Employment of Polyphenol Chlorogenic acid, 0.1 mg/L Chlorogenic acid exerted a Zhang et al. (2019)
antioxidants remarkable in vitro inhibitory effect
on soy LOX.

4.1.4. Germination heat treatment and non-heat treatment.

The activity of LOX can be significantly decreased after soybean
germination. However, some studies have shown that germination has
no obvious effect on reducing the beany flavor of soybean, but it has a
positive effect on improving the overall flavor. In order to study the
effect of germination on volatile compounds of soybean, Kaczmarska,
Chandra-Hioe, Frank, and Arcot (2018) analyzed volatile compounds by
GC-O and showed that the olfactory intensity of (E)-2-nonanal and
(E)-2-octenal were significantly decreased after soybean germination,
while the intensity of 1-penten-3-ol increased. Compared with the con­
trol, the intensity of beany flavor of germinated soybean remained un­
changed, but the sweet and baked flavor attributes were significantly
enhanced. Overall, germination can exert a positive effect on soybean
flavor.
4.2. Physical methods
The commonly used physical methods can be mainly divided into
4.2.1. Heating
Heating is the most common method for removal of beany flavor
during soybean processing. Heat treatment of soybean can effectively
reduce the activity of LOX, thus reducing the production of beany flavor
compounds (Navicha, Hua, Masamba, Kong, & Zhang, 2018a). In
addition, the volatile compounds such as pyrazine and alkylated pyr­
azine produced during the heat treatment also have a certain masking
effect on the beany flavor (Ma, Boye, Azarnia, & Simpson, 2016).
Maillard reaction often occurs during food processing and storage, and
plays a vital role in development of food flavor (Fu, Zhang, Soladoye, &
Aluko, 2020). Therefore, Maillard reaction products have been
commonly used as flavor enhancers in foods (Yu et al., 2018). Soybean
peptides and xylose were heat at 80, 100, 120 and 140 ◦ C for 2 h at
pH7.6 to prepare Maillard reaction products (Zhang et al., 2018). The
obtained Maillard reaction products were heat-treated with cysteine at
different temperatures. The results showed that a total of 63 sulfur

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B. Wang et al.
compounds were detected in the samples, including 51 heterocyclic
sulfur compounds. Sulfur-containing heterocyclic compounds typically
had a lower odor threshold and significantly contributed to the savory,
beany, baked, and boiled flavors of various foods. Habinshuti et al.
(2019) investigated the sensory properties of Maillard reaction products
derived from soybean proteins. The sensory evaluation showed that
soybean protein-derived Maillard reaction products exhibited higher
umami and caramel traits, in comparison with the soybean protein
control.
In general, heat treatment can be wet, semi-dry or dry heat treat­
ment. Boiling and steaming are the main treatments of wet heat and
semi-dry heat treatment. Each method can directly inactivate LOX, but
also have some different results. Chukeatirote, Eungwanichayapant, and
Kanghae (2017) employed a boiling method as well as high-pressure
steam to treat soybean, followed by the use of GC-MS/MS to detect
the volatile compounds. The percentage of alcohols, aldehydes and fu­
rans (18.78, 9.92 and 4.46%) in high-pressure steam was higher than
that of boiling (2.71, 0.83 and 1.32%). By contrast, the concentrations of
ketones (9.57%), acids and esters (35.68%) in boiling sample were
higher than that of high-pressure steam (0.4 and 30%). Aldehydes can be
removed more efficiently by wet heat treatment.
Generally, dry heat treatment includes conventional as well as
innovative processing methods. Hot air was previously employed as a
heat-conducting approach to destroy the activity of LOX. Navicha, Hua,
Masamba, Kong, and Zhang (2018b) reported that soybean was roasted
by hot air box, and sensory properties of the processed soybean products
at different temperatures and times were evaluated by a 15-point scale
(0: none, 15: extremely high). The results showed that the beany flavor
score of sample heated at 110 ◦ C for 60 min was 5.92, which was much
lower than that of the control (7.90). Prabakaran et al. (2018) studied
the changes in fatty acid stability and quality of soybean flour under
different storage temperatures and times. The results showed that the
content of unsaturated fatty acids in soybean flour stored at 45 ◦ C was
much lower than that at 4 and 20 ◦ C for the same storage period.
Conventional heating consumes a long time to achieve the desired
effect due to a slow heat conduction speed, while novel heating
methods, such as microwave and radio frequency (RF), have attracted
increasing interest in recent years due to the high efficiency. It has been
reported that microwave could inactivate LOX, and protein digestibility,
metabolic nitrogen and intestinal hydrolyzed protein were subsequently
increased. Since microwave heating does not involve water removal, the
loss of water-soluble vitamins in soybean flour is less; meanwhile,
different levels of microwave radiation power can affect the flavor
profiles of soybean protein. In other words, the higher the microwave
power applied, the lower the content of volatile compounds formed. RF
is an innovative heating technology based on capacitor dielectric heat­
ing. Jiang et al. (2021) studied the effects of RF heating on the pro­
cessing quality and physicochemical properties of soybean products, and
found that the inactivation rate of LOX was 93.4% after treatment for
285 s, while the content of hexanal was decreased by 1109.52 g/L to
7.64 g/L after 5 min. Compared with conventional heat treatment, RF
treatment can transfer energy quickly and evenly in food raw materials,
and inactivate endogenous enzymes more effectively, thus significantly
reducing the processing time. The inactivation rate of LOX was 94.30%
after RF treatment for 210 s. However, the LOX inactivation rate of
conventional heat treatment was only 3.08% due to the slow speed of
heat conduction (Jiang et al., 2018).
Overall, heat treatment can have a positive effect on flavor in soy­
bean. However, due to the integrity of soybean structure and the sta­
bility of fatty acids and proteases, soybean heated at a high temperature
for a long time to achieve enzyme inactivation is required, which in turn
can lead to denaturation of the high-quality proteins, decreased solu­
bility and loss of essential amino acids, thus reducing the nutritional
value of soybean products.
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4.2.2. Non-thermal treatment
Non-thermal treatment includes radiation treatment, high-frequency
electromagnetic field treatment and flavor masking. Non-thermal
treatment can eliminate the risk of protein denaturation caused by
heating to a certain extent. Gamma irradiation can effectively improve
the antioxidant properties of soybean by inactivating LOX activity.
Tewari, Kumari, Vinutha, Singh, and Dahuja (2015) irradiated soybean
with a low dose of gamma-rays (0.25 kGy), which significantly reduced
LOX activity. Soybean varieties with a darker color of seed coat were
more sensitive to gamma-ray irradiation.
Pulsed electric fields (PEF) is a non-thermal food preservation
method, which is commonly used in food sterilization. The PEF tech­
nology has been shown to exert a significant effect on LOX activity in
soybean. The stronger the parameters (pulse frequency and pulse
width), the higher the inactivation degree of LOX in soybean. The
maximum inactivation rate of LOX in soybean was 88% at a pulse fre­
quency of 400 Hz and a pulse width of 2 μs under the action of PEF at 42
kV/cm (Li, Chen, Liu, & Chen, 2008). Compared with traditional heat­
ing, PEF cannot only kill microorganisms and inactivate enzymes, but
also retain the taste, color, texture, vitamins, nutrients and
heat-sensitive functional components of food to a large extent.
In order to reduce the influence of beany flavor in soybean, cyclo­
dextrins, sugar and flavor enhancer are usually added to cover the off-
flavor of soybean during the manufacturing process. β-Cyclodextrin is
a cyclic carbohydrate composed of seven glucose units. β-Cyclodextrin
and other cyclodextrins can form water-soluble inclusion complexes
with insoluble and non-polar compounds of soybean, which can effec­
tively mask or reduce the unpleasant flavor of soybean (Damodaran &
Arora, 2013). Zhu and Damodaran (2018) compared the removal effects
of phospholipids (the precursors of soybean off-flavors) by addition of
α-, β-, and γ-cyclodextrins. The results showed that the three kinds of
cyclodextrins could effectively remove soybean odor precursors under
the same pretreatment conditions, and the removal rate of α-cyclodex­
trin precursor was more than 95%. Lee et al. (2020) indicated that the
addition of β-cyclodextrin played a significant role in reducing the
content of key beany flavor compounds (1-octen-3-ol, benzaldehyde,
hexanal and 2-heptaneone) in soybean. Although cyclodextrin can
effectively remove the beany flavor of soybean, the solubility of soybean
protein decreases after treatment, which indicates that the treatment
causes some structural changes to soybean protein.
Pre-extrusion aromatization has been employed by Milani, Menis,
Jordano, Boscolo, and Conti-Silva (2014) to process SPI using volatile
compounds (isovaleraldehyde, ethyl butyrate and butyric acid) and
flavoring agents (monosodium glutamate monohydrate and 5-inosi­
nate). Aroma intensity and sensory acceptability of the processed
products were previously studied. The results showed that SPI treated
with volatile compounds and flavor enhancers had ideal physical
properties, volatile retention and higher sensory acceptability than the
control under the conditions of 30% moisture and 170 ◦ C (Milani et al.,
2014).
4.3. Chemical methods
For the chemical removal of LOX and beany flavor compounds in
soybean, pH adjustment, extraction by organic solvent and addition of
reducing agents are employed. However, chemical methods are not
extremely efficient and often need to be combined with other methods.
LOX activity of soybean is highly pH-sensitive. Accordingly, pH of
solution can be adjusted by adding acid or alkali to deviate from the
optimal pH of LOX in order to inhibit its activity and thus reduce off-
flavor compounds. When soybean protein was extracted by boric acid
buffer solution (pH10.0), the content of beany flavor compounds was
100 counts of relative peak area. However, the content was much higher
(1700 counts) at pH 6.8. The results suggest that flavor attributes can be
optimized pH adjustment (Iassonova, Johnson, Hammond, & Beattie,
2009). Organic solvent can effectively leach the binding lipids and
B. Wang et al.
remove the beany flavor-producing compounds (Zha, Yang, Rao, &
Chen, 2019). However, organic solvents can exert a negative impact on
the functional properties of proteins when removing the beany flavor
compounds and their aroma precursors (Wang et al., 2020). Chemical
reagents can remove the beany flavor of soybean to a certain extent, but
its safety limitations concerning solvent residues in the food remain to
be further considered. In addition, the removal of beany compounds by
organic solvents is also not environmentally friendly.
4.4. Other methods
4.4.1. Employment of antioxidants
It has been shown that antioxidants in soybean can combine with
LOX to effectively reduce enzyme catalytic efficiency. Redrejo-
Rodriguez, Tejeda-Cano, del Carmen Pinto, and Macı ́as (2004) studied
the relationship between the structure and electrostatic properties of
quercetin, catechin, taxifolin and luteolin against LOX activity. Cate­
chins and other flavonoids can closely bind to the secondary structure of
LOX, thus effectively reducing the catalytic efficiency of LOX. The
inhibitory efficiency is related to the structures of molecule and the
characteristics of the lowest unoccupied molecular orbital of the frontier
orbit. Zhang et al. (2019) investigated the binding constants of six
polyphenols (chlorogenic acid, caffeic acid, sinapic acid, ferulic acid,
naringenin and chrysin) by ultrafiltration liquid chromatography–mass
spectrometry (UF-LC-MS). The results showed that the binding constant
of chlorogenic acid was the highest. Chlorogenic acid and LOX in soy­
bean were used for an in vitro inhibitory test and an ultrafiltration
combined test. The results further revealed that polyphenols had an
excellent LOX inhibitory activity in soybean, and the binding ability was
positively correlated with inhibitory activity.
4.4.2. Biotic elicitor treatments
Biological induction treatment can improve the content of antioxi­
dant substances in soybean, which can also effectively reduce the off-
flavor in soybean protein. Biotic elicitors are a class of chemical mole­
cules of biological origin capable of inducing secondary metabolite
production (Naik & Al-Khayri, 2016). When a small amount of biotic
elicitors are added to the living cell system, they can promote the
biosynthesis of specific compounds and induce physiological changes in
plants, resulting in the increase of secondary metabolites, such as pig­
ments, flavonoids, antioxidants and other related compounds. Kumari
et al. (2015) demonstrated that exogenous salicylic acid could signifi­
cantly inhibit LOX and HPL activities in the course of soybean growth,
resulting in reduced off-flavor in soybean. During the processing of
soybean, soybean seeds treated with different concentrations of bio­
logical inducers such as chitosan and jasmonic acid can significantly
reduce the residue of phospholipids and thiobarbituric acid (Kumari
et al., 2016). Biological induction treatment can improve the content of
antioxidant substances in soybean, which can also effectively reduce the
off-flavor in soybean protein. Biotic elicitors are a class of chemical
molecules. When a small amount of elicitors are added to the living cell
system, they can promote the biosynthesis of specific compounds and
induce physiological changes in plants, resulting in the increase of sec­
ondary metabolites, such as pigments, flavonoids, antioxidants and
other related compounds. Kumari et al. (2015) demonstrated that
exogenous salicylic acid could significantly inhibit LOX and HPL activ­
ities in the course of soybean growth, resulting in reduced off-flavor in
soybean. During the processing of soybean, soybean seeds treated with
different concentrations of biological inducers such as chitosan and
jasmonic acid can significantly reduce the residue of phospholipids and
thiobarbituric acid (Kumari et al., 2016).
Overall, different methods have their advantages at different stages
of soybean processing, among which physical methods (especially
heating) are the most widely used and the most effective at present, but
protein functionality will be impaired. The LOX-free soybean varieties
produced through biotechnology serve as the most effective way of
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Trends in Food Science & Technology 112 (2021) 336–347
removing beany flavor in soybean. Although some soybean varieties
without LOX have been selected, these varieties are quite expensive and
have certain limitations in promotion and popularization. In addition,
the safety of genetically modified soybean is still to be further evaluated.
Chemical methods may provide few choices but need to be further
developed, as safety issue is a major limitation for removal of beany
flavor in soybean.
5. Conclusion and future trends
Based on available scientific facts as discussed in the preceding
sections, it is imperative to find an efficient method to remove beany
flavor and simultaneously retain the functional properties of soy protein
for the development of PBMA. At present, the removal methods for
beany flavor in soybean mainly focus on two aspects with the first being
LOX inactivation. The second one is to eliminate or cover up the beany
flavor caused by the accumulation of volatile compounds catalyzed by
LOX through fermentation; organic solvent reagent extraction and other
methods are used. LOX activity, the production mechanism of beany
flavor compounds and the interactions between protein and flavor
compounds constitute future research directions to properly understand
and overcome soybean beany flavor.
Since different methods for removing beany flavor have their ad­
vantages, the combination of different kinds of removal methods may
provide an opportunity. As the most important enzyme in the formation
of beany flavor, LOX needs to be treated at high temperature for a long
time in order to be completely inactivated. However, high temperature
can cause protein denaturation, followed by the reduction of solubility
and impaired functional properties. Besides, LOX plays a key role in the
physiological activity of soybean, and the complete removal may pro­
voke some negative impacts on physiological functions during soybean
seed development (Kumari et al., 2015). It has been suggested that heat
treatment of soybean can inactivate LOX and inhibit the production of
beany flavor compounds. Therefore, the integration of two methods may
provide new ideas for removal of beany flavor in soybean. It has been
demonstrated that microwave heating and radio frequency heating can
effectively inhibit the LOX activity of soybean in a very short time,
which is conducive to the removal of off-flavor in soybean protein (Jiang
et al., 2021; Phuhongsung, Zhang, & Bhandari, 2020) and to avoidance
of the influence of long-term heating on the functional properties of
soybean protein.
As the molecular structures of different proteins vary, their binding
affinity with flavor compounds is also different. By changing the struc­
ture of binding sites in a protein, the binding constant of off-flavor
compounds can be effectively affected. Therefore, the structural modi­
fication of soybean protein for production of PBMA may also provide
some new perspectives for removal of beany flavor. At present, MDA,
ethanol or isopropanol have been used to modify soy protein or to
extract beany flavor compounds. After treatment, the binding affinity of
soy protein with flavor compounds was altered. However, these reagents
may cause food safety threats. Furthermore, once the protein was
modified or hydrolyzed directly by enzymatic treatment, the flavor
profiles of protein hydrolysates were changed, and the binding affinity
of protein hydrolysate with flavor substances was significantly reduced
(Nakada et al., 2018). Hence, the enzymatic method can effectively
modify soybean protein and avoid the food safety issues caused by
chemical modification.
Despite the different approaches for removal of beany compounds
from soybean, the beany flavor may be still present in PBMA. Therefore,
processing technology for texturization of soy protein, which contrib­
utes to generation of meat flavor and cover-up of beany flavor, may
effectively solve this problem. The preparation of meat flavors using
sulfur compounds, reducing sugars and amino acids has been widely
applied in the food industry. Eric et al. (2013) established a soybean
protein isolate-xylose-cysteine system, and investigated the sensory
properties of Maillard reaction products derived from SPI. The results
B. Wang et al.
showed that the samples had obvious meat flavor properties, and
sulfur-containing furan derivatives played an important role in the for­
mation of meat flavor of soybean protein hydrolysates. In addition, the
Maillard reaction products of SPI had higher free glutamic acid contents
than the control, which resulted in better taste quality. Song et al. (2013)
also pointed out that after Maillard reaction of SPI, the resultant sample
exhibited an obvious meat flavor. The addition of meat-flavor essence or
Maillard reaction products are promising to control and enhance the
flavor of PBMA.
It is of great significance to effectively control the formation of beany
flavor compounds while preventing the denaturation of soy protein and
maintaining its nutritional value and functional properties. Non-thermal
inactivation of LOX was carried out before soybean was crushed, and
then soybean protein was directly modified or hydrolyzed to change the
structure of soybean protein. In the process of texturization processing of
PBMA, reducing sugars, e.g. xylose, can be added to induce Maillard
reaction and optimize flavor. There is great potential in exploring the
integrated method to remove the beany flavor compounds in soybean
protein. Besides, β-cyclodextrin is able to form inclusion complexes with
a variety of organic compounds. Therefore, it is also a promising method
for removal of beany flavor in soybean protein.
Declaration of competing interest
The authors declare no conflicts of interest.
Acknowledgments
The authors acknowledge the funding support by Innovation Pro­
gram for Chongqing’s Overseas Returnees (cx2019072) and Funda­
mental Research Funds for the Central Universities (SWU 019009).
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