LWT - Food Science and Technology 134 (2020) 110151

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

An innovative two-step enzyme-assisted aqueous extraction for the

production of reduced bitterness soybean protein hydrolysates with high
nutritional value
Xiaohong Tong a, Ziteng Lian a, Liming Miao a, Baokun Qi a, Shuang Zhang a, Yang Li a, c,
Huan Wang a, b, *, Lianzhou Jiang a, c, **
a
College of Food Science, Northeast Agricultural University, Harbin, 150030, China
b
Heilongjiang Beidahuang Green Health Food co. LTD, Heilongjiang, Jiamusi, 154000, China
c
Heilongjiang Institute of Green Food Science, Harbin, 150028, China

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

Keywords: Soybean protein hydrolysates (SPH), by-product of enzyme-assisted aqueous extraction (EAEP), were a prom­
Soybean protein hydrolysates ising source of natural nutraceutical. Nevertheless, the bitterness of SPH has limited their use as food ingredients
Bitterness or additives. The objectives of the present study were to use two-step enzyme-assisted aqueous extraction to
LC-MS/MS
produce SPH with reduced bitterness and high nutritional value (antioxidant activity and peptide nitrogen
Bioinformatics
levels). In this study, the optimum conditions for SPH production were obtained using Protex 6L (2 h) followed
High nutritional value
by Protease A 2SD (3 h) in two-step hydrolysis. The bitterness of SPH was positively correlated with the hy­
drophobic amino acids (Ala, Leu, Ile, Trp, Phe, Tyr) and hydrophobic peptide fractions. Additionally, the pro­
duced potential bitter peptides of SPH-6L were identified and predicted by LC-MS/MS and bioinformatics.
IceLogo plot and heat map revealed that Protease A 2SD tends to cleave Leu and Arg residues from the N-terminal
in bitter peptide to degrade bitter peptide. Thus, it could be concluded that the utilization of multi-enzyme
hydrolysis, could be a promising process for the production of SPH with low bitter and potential antioxidant
activity, which offers a promising approach for enhancing the usability and high-valued utilization of EAEP.

1. Introduction Previous research showed that bitterness of protein hydrolysates is

Soybean provides the largest source of plant protein to human diet
due to its superior functional and nutritional base (Nishinari, Fang, Guo,
& Phillips, 2014). Several in vitro and in vivo studies have demonstrated
that bioactive dietary soybean protein hydrolysates (SPH) or peptides
have potential functional properties and physiological activities, such as
antioxidative, antihypertensive, immunomodulatory (Singh & Vij,
2018). SPH is the main by-product of the enzyme-assisted aqueous
extraction processing (EAEP) (Liu, Gasmalla, Li, & Yang, 2016). The
EAEP is an environmentally friendly way to separate oil and protein
from soybeans, which gives 4 distinct fractions: skim (rich in protein
hydrolysates), residual fraction, free oil and cream (Liu et al., 2016;
Sekhon, Rosentrater, Jung, & Wang, 2018). Nevertheless, the formation
of bitter peptides in protein hydrolysates from EAEP limit consumer’s
acceptance and impedes their utilization as food ingredients.
due to the release of low molecular mass peptides containing hydro­
phobic amino acid residues (Kim & Lichan, 2006; Singh et al., 2020).
Numerous methods have been investigated to prevent, reduce and
eliminate the bitterness of protein hydrolysates, including chromato­
graphic removal, treatment with activated carbon, selective extraction
with alcohol and isoelectric precipitation to separate bitter peptides
(Saha & Hayashi, 2001). However, these methods would cause the loss
of some amino acid residues from hydrolysates (Fitzgerald & O’Cuinn,
2006). As an alternative, the bitterness can be masked by the addition of
polyphosphates, a-cyclodextrins, some specific amino acids (Sun, 2011).
Other methods such as cross-linking using transglutaminase, trans­
peptidation reactions in T-plastein reaction have also helped to reduce
the bitterness (Carvalho et al., 2019; Sun, 2011). Especially, the
peptidase-mediated debittering by exopeptidases (Fu et al., 2019; Hou,
Li, Zhao, Zhang, & Li, 2011).

* Corresponding author. College of Food Science, Northeast Agricultural University, Harbin, 150030, China.
** Corresponding author. College of Food Science, Northeast Agricultural University, Harbin, 150030, China.
E-mail addresses: whname@neau.edu.cn (H. Wang), jlzname@neau.edu.cn (L. Jiang).

https://doi.org/10.1016/j.lwt.2020.110151
Received 27 July 2020; Received in revised form 26 August 2020; Accepted 30 August 2020
Available online 3 September 2020
0023-6438/© 2020 Elsevier Ltd. All rights reserved.
X. Tong et al.
Protex 6L is an alkaline protease with endopeptidase activity, which
can provide rapid and efficient hydrolysis in the EAEP of oil and protein
(Liu et al., 2016; Moura & Johnson, 2009). Protease A 2SD is a fungal
neutral protease having high exo- and endo-peptidase activities (Fu
et al., 2019), exopeptidase plays a role in the debittering process, and Fu,
Liu, Hansen, Bredie, and Lametsch (2018) also observed that Protease A
2SD generate bovine muscle and porcine plasma hydrolysates with low
bitterness. Despite many attempts to debitter hydrolysates using
exopeptidase treatment, there is no report concerning the utilization of
multi-enzyme to debitter in EAEP, and the bitterness and nutritional
value (antioxidant activity and peptide nitrogen levels) of SPH produced
by EAEP have not been deeply investigated either. Previous studies have
shown that protein hydrolysates containing higher hydrophobic pep­
tides have higher antioxidant activity (Pownall, Udenigwe, & Aluko,
2010). During the debittering process, the degradation of hydrophobic
peptides has a negative effect on antioxidant activity. In this study, on
the basis of ensuring a high oil and protein extraction yields,
enzyme-based combinations (Protex 6L and Protease A 2SD) was used to
produce hydrolysates with good sensory properties, antioxidant activity,
and peptide nitrogen levels. The methods and software tools developed
by bioinformatics can understand the biological mass data, and bioin­
formatics tools can effectively understand the information about bitter
peptides (Iwaniak, Minkiewicz, Darewicz, & Hrynkiewicz, 2016).
Therefore, the bitter peptides in SPH-6L can be predicted by the bioin­
formatics database (BIOPEP database). The relationship between the
bitterness of resulting hydrolysates and the protease cleavage specificity
was elucidated by iceLogos.
2. Materials and methods
2.1. Materials
Full-fat soybean flakes were kindly provided by the Lanshan Group
Corporation (Liaocheng, China). Protease A 2SD (100,000 U/g) was
supplied by Amano Enzyme Inc. (Nagoya, Japan). Protex 6L (580,000
DU/g) from Bacillus licheniformis was purchased from Novozymes
Biotechnology Co., Ltd. (Beijing, China). Gel filtration standards
including thyroglobulin, bovine γ-globulin, chick ovalbumin, equine
myoglobin, and vitamin B12 were purchased from Bio-Rad Laboratories
(Hercules, CA, USA). 2,2′ -azinobis (3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt (ABTS), 2,2′ -Azobis (2-amidinopropane) dihy­
drochloride (AAPH), Trolox and fluorescein sodium salt were purchased
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All the other
chemicals and solvents were of analytical grade.
2.2. Preparation of soybean protein hydrolysates
Soybean protein hydrolysates (SPH) were prepared according to our
previous work (Zhang et al., 2018). Briefly, the extruded soybean
powder was mixed with deionized (DI) water at a ratio of 1:6, w/v. For
the one-step process, Protex 6L (0.5%, v/w; pH 8.0; 55 ◦ C) or Protease A
2SD (0.5%, v/w; pH 7.0; 50 ◦ C) was added and incubated at 70 rpm for 2
h. After incubation, the mixture was heated in boiling water for 10 min
to ensure enzyme inactivation. Then the mixture was centrifuged in a
GL-21M refrigerated centrifuge (Xiangyi Instrument Co. Ltd., Changsha,
China) at 8000 × g for 20 min at 4 ◦ C and the skim fraction was collected
and lyophilized (abbreviated as SPH-6L and SPH-A). In two-step hy­
drolysis, Protex 6L (0.5%, v/w; pH 8.0; 55 ◦ C) was applied in the first
hydrolysis stage for 2 h and was inactivated in boiling water bath for 10
min. Then, Protease A 2SD (0.5%, v/w) was added and the reaction was
at pH 7.0, 50 ◦ C. The proteolysis reaction was stopped at intervals of 1,
2, 3, 4, and 5 h, with the enzyme immediately inactivated by heat in
boiling water for 10 min. Then the mixture was centrifuged at 8000 × g
for 20 min at 4 ◦ C and the skim fraction was collected and lyophilized
(abbreviated respectively as SPH-1, SPH-2, SPH-3, SPH-4, SPH-5).
2
LWT 134 (2020) 110151
2.3. Degree of hydrolysis
The degree of hydrolysis (DH) of SPH was evaluated with the method
of the o-phthaldialdehyde (OPA) as described by Nielsen et al. (2010).
The samples (400 μL) were mixed with 3 mL of OPA reagent. The DI
water and the serine solution were taken as blank and standard solu­
tions, respectively. Absorbance was read at 340 nm (UV-1601 spectro­
photometer, Shimadzu, Kyoto, Japan) after the mixture had stood for 2
min and DH was calculated using the following equations:
ODsample − ODblank (0.9516 × V × 100)
Serine − NH2 = × (1)
ODstandard − ODblank (X × P)
where V is sample volume (L), X is sample weight (g), P is protein
content (%) of the sample.
(Serine − NH2 ) − β
h= (2)
a
where α is 0.97 and β is 0.342.
h
DH = × 100 (3)
htot
where h is the number of hydrolyzed bonds, htot is 7.8.
2.4. Extraction yield
The extraction yields of the oil and protein were determined ac­
cording to Jung and Mahfuz (2009).
[
C×W
]
Extraction Yield(%) = 100 × 1 − (4)
CS × W S
where CS is the concentration of oil or protein (%, g/100 g) in extruded
soybean powder, WS is the weight of extruded soybean powder (g, as is),
C is the concentration of oil or protein solids (%, g/100 g) in the insol­
uble fraction, and W is the weight of the insoluble fraction (g, as is).
2.5. Evaluation of peptide nitrogen and formaldehyde nitrogen levels
The formaldehyde titration method was used to determine the
formaldehyde nitrogen content of the supernatant (Wu et al., 2019). The
SPH solution (5 mL) was adjusted to pH 8.2 using 0.05 mol/L NaOH. A
volume of 10 mL of neutral formaldehyde was mixed with SPH solution
and titrated with 0.05 mol/L NaOH to a pH of 9.2. The calculation was as
follows:
Formaldehyde nitrogen (g / mL) = (V1 − V0 ) × C × 0.014 / 5 (5)
where V1 is the volume (mL) of 0.05 mol/L NaOH added for the second
time, V0 is the volume (mL) of 0.05 M NaOH added in the blank
experiment. C is the concentration of NaOH (mol/L), 0.014 is the mass
(g) of nitrogen equivalent to 1 mL NaOH (1 mol/L), 5 is the volume (mL)
of the sample.
The soluble nitrogen, peptide nitrogen of the samples was deter­
mined as described by Liu, Zhu, Peng, Guo, and Zhou (2016). The sol­
uble nitrogen content of the samples was evaluated using the
micro-Kjeldahl method. The peptide nitrogen content was determined
as “soluble nitrogen content -formaldehyde nitrogen content”. All the
results were expressed as the percentage on the basis of total nitrogen
content in samples, respectively.
2.6. Amino acid analysis
The total amino acid compositions were measured following the
procedure of Zhu, Chen, Tang, and Xiong (2008). Samples were hy­
drolyzed with 6 mol/L HCl at 110 ◦ C for 24 h in the sealed, evacuated
glass tubes. The hydrolysates were derivatized with phenyl
X. Tong et al.
isothiocyanate, followed by the performance of high-performance liquid
chromatography (HPLC, Thermo Fisher Scientific, San Jose, CA, USA)
equipped with a reversed-phase column (150 × 3 mm, 2.6 μm, Thermo
Fisher Scientific, San Jose, CA, USA). Alkaline hydrolysis was done to
determine tryptophan contents according to the method described by
Yust et al. (2004).
The free amino acid compositions were determined using the method
of You, Zhao, Regenstein, and Ren (2010) with a slight modification.
Briefly, SPH samples were mixed with 10% cold trichloroacetic acid to
precipitate peptides and/or proteins for 2 h, followed by centrifuging at
11,000 × g for 15 min. The supernatant was adjusted to pH 2.0 using
HCl aqueous solution and was filtered using a 0.45 μm microfiltration
membrane. The free amino acid compositions were derivatized and
determined by reference to the method of the total amino acid compo­
sitions. The calculation equation of peptide-bound amino acids was
calculated with equation (6). Take Asp as an example:
Peptide − bound Asp (g / 100 g Protein) = Total Asp − Free Asp (6)
Finally, Peptide-bound Asp (g/100 g Protein) was converted to
Peptide-bound Asp (g/100 g Total peptide-bound amino acids).
2.7. Hydrophobic peptide fractions
The hydrophobic peptide fractions (HPF) was determined according
to the method of Aspevik, Totland, Lea, and Oterhals (2016). Briefly, the
samples (5 g) were diluted in 50 mL of DI water and mixed with 100 mL
of 2-butanol. After shaking the mixture for 2 min, the mixture was
transferred to a separation funnel. After separation of 1 h, the upper,
butanol-rich extract was recovered. The hydrophobic peptide fraction
(HPF) was calculated using the following equation (7):
HPF(%) = 100 × m / ms (7)
where m is the mass (g) of protein in the 2-butanol phase and ms is the
mass (g) of protein in the samples.
2.8. Molecular weight distribution
The molecular weight (MW) distribution of the SPH was measured
using an HPLC system (Waters e2695, Milford, MA, USA). An Advan­
ceBio SEC column (300 × 4.6 mm, 2.7 μm, Agilent Technologies, Wil­
mington, DE, USA) was applied. HPLC was carried out with 0.1 mol/L
sodium phosphate buffer (pH 7.0) and eluted at a flow rate of 0.2 mL/
min. The eluent was monitored at 220 nm. The standards were thyro­
globulin (670 kDa), bovine γ-globulin (158 kDa), chick ovalbumin (44
kDa), equine myoglobin (17 kDa), and vitamin B12 (1.35 kDa) (Zhang
et al., 2018). The standard curve equation was y = − 0.1547x+6.2886
(R2 = 0.9990).
2.9. Sensory analysis
The bitterness of SPH was quantified following the method of Liu
et al., (2016). A sensory panel consisting of ten trained panelists (5 male
and 5 female, aged between 20 and 35 years) had been trained with
quinine standards over 1 month (1 h per session, four times a week). At
each sitting, panelists were first presented with solutions of 0, 1 × 10− 5,
2 × 10− 5, 3 × 10− 5, 4 × 10− 5, 5 × 10− 5, 6 × 10− 5 g/mL, which had been
scored as 0, 1, 2, 3, 4, 5, 6, respectively (0, no bitterness; 6, extremely
bitter). The standard condition for sensory evaluation was maintained at
room temperature for 0.01 g/mL SPH. In between each tasting,
non-sparkling mineral water and unsalted crackers were served for
palate cleansing.
2.10. Electronic tongue
In the present study, the taste sensing system SA402B (Insent Inc.,
3
LWT 134 (2020) 110151
Atsugi-chi, Japan) was used. The e-tongue consists of bitterness sensor
(SB2C00), astringency sensor (SB2AE1) and saltiness sensor (SB2CT0).
The 0.01 g/mL SPH was used for the electronic tongue measurements.
The reference solution was prepared by potassium chloride (2.2365 g)
and tartaric acid (0.045 g) mixed with 1000 mL DI water. The bitterness
was determined according to the method of Woertz, Tissen, Kleine­
budde, and Breitkreutz (2010). The results were expressed as the in­
tensity values of bitterness, bitterness aftertaste (aftertaste-B),
astringency, astringent aftertaste (aftertaste-A), and saltiness.
2.11. ABTS ⋅+ radical scavenging activity
The ABTS⋅+ radical scavenging ability was determined according to
the method of Re et al. (1999). The absorbance was measured at 734 nm
using a microplate reader (Tecan Infinite M200; Tecan Inc., Maennedorf,
Switzerland). The formula for calculating ABTS⋅+ radical scavenging
activity was using the following equation (8):
[ ( / )]
ABTS˙+ radical scavenging activity(%) = 1 − Asample Ablank × 100 (8)
where Asample is the absorbance of the sample; and Ablank is the absor­
bance of the blank.
2.12. Oxygen radical absorbance capacity (ORAC) assay
The oxygen radical absorbance capacity (ORAC) was determined by
modifying the method of Ou, Hampsch-Woodill, and Prior (2001). The
sample (0.2 mg/mL, 20 μl) and fluorescein working solutions were
added to 96-well microplate and allowed to react for 12 min at 37 ◦ C,
then 60 μL AAPH (40 mmol/L) was added. The 96-well microplate was
read (excitation wavelength of 485 nm and emission wavelength of 535
nm) every minute for 120 min using a microplate reader (Tecan Infinite
M200; Tecan Inc., Maennedorf, Switzerland). Buffer without the addi­
tion of sample was used for the blank and Trolox standards (0, 12.5, 25,
50, 75 and 100 μmol/L) were used for the calibration solutions. The
ORAC value was expressed as μmol/L Trolox equivalents/mg (μmol/L
TE/mg).
2.13. LC-MS/MS analysis
SPH samples were analyzed using an Easy-nLC 1000 UHPLC system
(Thermo Fisher Scientific, San Jose, CA, USA) coupled online to a Q
Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA,
USA). Samples were separated by C18-reversed phase column (150 mm
× 75 μm, 5 μm, Thermo Fisher Scientific, San Jose, CA, USA). The
mobile-phase eluent A was 0.1% formic acid (FA) in HPLC-grade water
and eluent B was 0.1% FA in 84% acetonitrile. The flow rate was fixed at
250 nL/min and increased the concentration of eluent B: from 4% to
50% (0–50 min), then from 50% to 100% (50–54 min), held at 100% for
6 min (54–60 min). Through survey scan (the range was 100–2300 m/z)
to dynamically choose the most abundant precursor ions for HCD frag­
mentation, and run in positive mode. MS/MS spectra were searched
using MAXQUANT engine (version 1.2.2.5) against Uniprot soybean
(include 85,644 sequences, downloaded at 20180706). Bioinformatics
can predict bitter peptides, therefore, sequence matching of identified
peptides using the BIOPEP database (http://www.uwm.edu.pl/bioch
emia/index.php/pl/biope) to predict bitter peptides. For peptide and
protein identification. The protease specificity cleavage sites were
identified using an iceLogo software (version 1.2) for amino acids at the
P3-P2-P1-P1ʹ-P2ʹ-P3ʹ positions (Colaert, Helsens, Martens, Vandekerck­
hove, & Gevaert, 2009).
2.14. Statistical analysis
All measurements were performed in at least three independent ex­
periments and results were expressed as mean ± standard deviation
X. Tong et al. LWT 134 (2020) 110151

(SD). Statistical analysis was performed by one-way analysis of variance
(ANOVA) and Duncan’s multiple range test at p < 0.05 using SPSS 22.0
(SPSS Inc., Chicago, IL, USA).
3. Results and discussion
3.1. Extraction yield
Edible oil and protein can be simultaneously recovered in enzyme-
assisted aqueous extraction processing (EAEP) (Moura & Johnson,
2009). Oil extraction yield and protein extraction yield are important
indicators in EAEP. Table 1 summarized the extraction yield of oil and
protein of different enzyme treatments. Compared with SPH-6L (oil
extraction yield 84.62%; protein extraction yield 77.32%), the extrac­
tion yields of SPH-A (oil extraction yield 61.44%; protein extraction
yield 59.07%) was relatively low, which was not suitable for industrial
applications. From Fig. 1, the oil extraction yields had a positive cor­
relation (r = 0.99) with the protein extraction yields. A positive corre­
lation coefficient (r = 0.87 and r = 0.82) between extraction yield with
DH was found (Fig. 1). The protein bodies of soybean contain about 80%
of the total protein, and destruction of the protein bodies is the pre­
requisite for oil release. The oil body that stores oil is stabilized by
amphiphilic protein and phospholipid (Campbel et al., 2011). The DH
can be used as a proteolysis-monitoring parameter (Meinlschmidt,
Schweiggert-Weisz, Brode, & Eisner, 2016), hence, extraction yield
gradually increased as DH of the protein increased. Protex 6L is an
endopeptidase, which can effectively disrupt protein bodies and oil
bodies. During further hydrolysis, the exopeptidase in Protease A 2SD
degrades bitter peptides, while the endopeptidase in Protease A 2SD
further improves the extraction yield of oil and protein by breaking the
binding of protein bodies and oil bodies to oil. Therefore, the oil
extraction yield and protein extraction yield further increased with
prolonging the enzymatic hydrolysis time until 5 h.
3.2. Analysis of the relationship between bitterness and each index
In the process of EAEP, Protex 6L hydrolyzes protein from protein
bodies and the oil bodies, resulting in exposure of the bitter peptides.
The addition of Protease A 2SD degrades the bitter peptides. Accord­
ingly, impact of Protex 6L and Protease A 2SD on bitterness of SPH was
studied. The sensor outputs of bitterness aftertaste (aftertaste-B),
astringency and astringent aftertaste (aftertaste-A) were correlated to
the hydrophobicity of bitter peptides, which reflected the bitterness.
However, the instantaneous maximum intensity of bitter taste (the
sensor output of bitterness) has a greater effect on sensory evaluation
than aftertaste-B (Liu et al., 2016). According to the output from the
electronic tongue in Fig. 2, SPH-6L has the highest bitter scores, while
SPH-A has a relatively lower bitterness value. There was a positive
correlation (r = 0.98) between the electronic tongue and the sensory
analysis (Fig. 1). Similarly, Newman, Harbourne, O’ Riordan, Jacquier,
and O’ Sullivan (2014) also observed a high correlation coefficient
between the electronic tongue and the sensory analysis in dairy protein
hydrolysates. Through electronic tongue analysis and sensory analysis
(Fig. 2 and Table 1), the intensity value of bitterness was decreased with
increasing hydrolysis time. Since exopeptidases (amino- and carboxy­
peptidases) has a significant role in eliminating bitterness (Hou et al.,
2011), as expected, the Protease A 2SD reduced the bitterness due to its
exo-peptidase activities, which can selectively release N-terminal or C-
terminal amino acid residues to degrade bitter peptides. As shown in
Fig. 2, SPH-A has the lowest saltiness value (− 1.44), because Protease A
2SD is neutral protease, the least NaOH solution was added in the
enzymatic hydrolysis process.
The highest bitter intensity was achieved when using Protex 6L alone
(Fig. 2, Table 1), which is accordance with previous study (Meinlsch­
midt, Sussmann, Schweiggert-Weisz, & Eisner, 2016) demonstrating
that Protex 6L caused the highest bitterness to soybean protein isolate
(SPI), compared with other commercial proteases. It can be explained by
the fact that Protex 6L has the tendency to hydrolyze at hydrophobic
amino acid residues position. Therefore, the resulting peptides had hy­
drophobic amino acid residues at the C-terminal and cause a relatively
high bitterness (Ishibashi et al., 1987b; (Meinlschmidt et al., 2016a)
Meinlschmidt, Schweiggert-Weisz, Brode et al., 2016). When compound
enzymatic hydrolysis (Protex 6L and Protease A 2SD) was used, the
content of total HAAs in peptide of the samples were reduced compared
to SPH-6L (Table 2). A decrease in bitterness with a decreasing amount
of HHAs, and the correlations showed significant positive association (r
= 0.94 and r = 0.97) between bitterness and total HAAs (Fig. 1). Pep­
tides with hydrophobic amino acid residues play a major role in
bitterness, since the higher proportion of hydrophobic amino acids in
the peptide lead to the higher bitterness. The main amino acids are Trp,
Ile, Tyr, Phe, Pro, Leu and Val (in decreasing order), which create a
bitter taste (Egerton, Culloty, Whooley, Stanton, & Ross, 2017). In our
case, except for Pro and Val, the content of amino acids (Trp, Ile, Tyr,
Phe and Leu) in peptide decreased with the increasing of hydrolysis
time. Especially, the content of Phe and Leu decreased from 4.14% to
2.16%, and 7.51%–5.37%. As the content of Phe or Tyr in peptides
decreased, indicating less intense of bitterness (Ishibashi et al., 1987b;
Saha & Hayashi, 2001), and as shown in Table 2, the content of Tyr in
peptide was also reduced from 3.35% to 2.63%. It was reported that a
peptide containing Gly, Ala, Val, Leu, Tyr and Phe can bind to taste bud
and produce bitterness (Zhao, Schieber, & Gänzle, 2016). In this
experiment, for hydrophobic amino acids, the reduction in the content
of Ala, Leu, Ile, Trp, Phe and Tyr may lead to a reduction in bitterness.
The HPF had a positive correlation (r = 0.95) with total HAAs content in
peptides during hydrolysis process (Fig. 1). As shown in Table 1,
compared with SPH-A, the content of HPF in SPH-6L is relatively higher,
and the HPF decreased significantly (p ˂ 0.05) with increasing hydrolysis
time. Similarly, a positive correlation (r = 0.95 and r = 0.94) between
bitterness and HPF was observed (Fig. 1). This study indicates that the
bitterness of protein hydrolysates may be related to the accumulation of
hydrophobic peptide fractions. From previous analysis of amino acid
composition (Table 2) and HPF (Table 1), it is noted that the bitterness

Table 1
The degree of hydrolysis (DH), extraction yields, formaldehyde nitrogen, peptide nitrogen, hydrophobic peptide fractions (HPF) and sensory analysis of soybean
protein hydrolysates.
Sample DH Oil extraction yields (%) Protein extraction yields (%) formaldehyde nitrogen peptide nitrogen HPF Bitterness intensity
f d d g a a
SPH-6L 13.37 ± 0.44 84.62 ± 0.43 77.32 ± 0.31 12.45 ± 0.07 76.90 ± 0.04 6.97 ± 0.08 5.1 ± 0.31a
SPH-A 8.96 ± 0.22g 61.44 ± 0.47e 59.07 ± 0.55e 9.60 ± 0.19e 70.43 ± 0.25e 4.73 ± 0.12f 0.5 ± 0.53e
SPH-1 18.13 ± 0.06e 87.77 ± 0.39c 78.48 ± 0.42c 14.09 ± 0.17f 75.90 ± 0.32b 6.42 ± 0.10b 4.2 ± 0.42b
SPH-2 21.78 ± 0.06d 89.73 ± 0.36b 79.26 ± 0.44b 17.42 ± 0.05d 73.62 ± 0.10c 6.02 ± 0.10c 2.1 ± 0.31c
SPH-3 23.32 ± 0.13c 92.56 ± 0.43a 80.64 ± 0.15a 19.43 ± 0.09c 71.82 ± 0.23d 5.39 ± 0.06d 0.9 ± 0.31d
SPH-4 25.68 ± 0.46b 92.65 ± 0.28a 80.80 ± 0.16a 23.98 ± 0.13b 67.58 ± 0.20f 4.90 ± 0.08e 0.8 ± 0.42d
SPH-5 27.18 ± 0.33a 92.51 ± 0.15a 80.89 ± 0.26a 28.80 ± 0.07a 62.84 ± 0.19g 4.72 ± 0.02f 0.9 ± 0.31d

Data are presented as the mean ± standard deviation, n = 3. Different superscripts within the same column indicate significant differences (p < 0.05). SPH-6L: soybean
protein hydrolysates (Protex 6L for 2 h); SPH-A: soybean protein hydrolysates (Protease A 2SD for 2 h); SPH-1, SPH-2, SPH-3, SPH-4, SPH-5: soybean protein hy­
drolysates (Protex 6L for 2 h, Protease A 2SD for 1 h, 2 h, 3 h, 4 h, 5 h).

4
X. Tong et al.
Fig. 1. Heatmap summarizing correlation coefficients between different indicators. Red represents a positive correlation, blue represents a negative correlation, the
depth of color reflects the magnitude of the correlation coefficient.
Fig. 2. Radar distribution results of sensor responses of soybean protein hy­
drolysates obtained by the electronic tongue. SPH-6L, SPH-A,
SPH-1, SPH-2, SPH-3, SPH-4, SPH-5.
SPH-6L: soybean protein hydrolysates (Protex 6 L for 2 h); SPH-A: soybean
protein hydrolysates (Protease A 2SD for 2 h); SPH-1, SPH-2, SPH-3, SPH-4,
SPH-5: soybean protein hydrolysates (Protex 6L for 2 h, Protease A 2SD for 1 h,
2 h, 3 h, 4 h, 5 h).
was reduced with increasing hydrolysis time.
The MW distribution of SPH is shown in Fig. 3. We observed mainly
four peptide fractions with the MW above 10 kDa, 5–10 kDa, 1–5 kDa,
and below 0.5 kDa. As the hydrolysis time increased, the MW of SPH
gradually decreased. In this study, the contents of peptide fractions
above 10 kDa and 5–10 kDa decreased respectively from 34.31% to
29.73% in SPH-6L to 12.89% and 9.71% in SPH-5 due to Protease A 2SD
destroyed large molecular weight proteins or peptides. This trend is
consistent with the change of DH (Table 1). It was reported that the
5
LWT 134 (2020) 110151
soybean peptides (1–4 kDa) induced the most bitterness (Cho, Unkles­
bay, Hsieh, & Clarke, 2004). It was also possible that the bitterness of
protein hydrolysates is associated with hydrophobic bitter peptides (MW
less than 1 kDa) (Aspevik et al., 2016). Our results were inconsistent
with previous studies, the peptides with MW less than 1 kDa and 1–5
kDa increased (Fig. 3), the sensory evaluation showed that the bitterness
decreased as the time of two-step hydrolysis increased, which due to
high exo- and endo-peptidase activity of Protease A 2SD. The Protease A
2SD degraded more proteins and hydrolyzed large molecular weight
peptide into small peptides or free amino acids (Fu et al., 2020). Such
low MW peptides-rich dietary is believed to have high nutritional value
and high potential bioactivity, such as antioxidative and ACE-inhibitory.
(Singh & Vij, 2018). These findings also confirmed the Protease A 2SD is
specific for hydrophobic amino acids of bitter peptides, which led to
further degradation of the bitter peptide and produced small peptides
and free amino acids. The bitterness value of SPH obtained by
multi-enzymatic hydrolysis was low.
3.3. Evaluation of soluble nitrogen, formaldehyde nitrogen and peptide
nitrogen levels
As shown in Table 1, a hydrolysis time-dependent effect was
observed for formaldehyde and peptide nitrogen levels. The formalde­
hyde nitrogen content of the sample obtained by complex enzymolysis
was increased with increasing time (Table 1). The formaldehyde nitro­
gen content reflects the exo-activity of the Protease A 2SD. From Table 1,
treatment by Protease A 2SD hydrolysis induced a decrease of peptide
nitrogen level. The peptide nitrogen contents of SPH-4 and SPH-5 were
67.58% and 62.84%, respectively (less than 70%). The amino acid
composition and digestibility determine the quality of protein for health,
small peptides are absorbed more rapidly than an equivalent amount of
free amino acids after ingestion of protein (Egerton et al., 2017). Pro­
tease A 2SD treatment time had an adverse effect on the peptide nitro­
gen, so the Protease A 2SD hydrolysis time should be controlled within 3
h.
X. Tong et al. LWT 134 (2020) 110151

Table 2
Amino acid composition (g/100 g Total peptide-bound amino acids) of soybean
protein hydrolysates.
Peptide- Sample
bound
SPH- SPH-A SPH-1 SPH-2 SPH-3 SPH-4 SPH-5
amino
6L
acids

Asp 11.36 12.70 11.92 12.19 12.56 12.82 13.65

± ± ± ± ± ± ±
0.01g 0.01c 0.02f 0.12e 0.02d 0.03b 0.04a
Thr 4.27 4.53 4.19 4.19 4.12 3.94 4.65
± ± ± ± ± ± ±
0.08c 0.03b 0.03d 0.01d 0.03e 0.03f 0.02a
Ser 5.70 6.00 5.98 6.19 5.91 6.20 6.39
± ± ± ± ± ± ±
0.09g 0.08d 0.01e 0.09c 0.02f 0.12b 0.02a
Glu 19.94 20.70 21.30 21.74 22.00 22.69 23.10
± ± ± ± ± ± ±
0.07g 0.12f 0.02e 0.01d 0.02c 0.03b 0.03a
Gly 4.35 3.73 4.57 4.74 4.96 5.04 3.66
± ± ± ± ± ± ±
0.07e 0.02f 0.05d 0.08c 0.12b 0.09a 0.08g
Ala 3.72 3.65 3.65 3.67 3.54 3.57 3.40 Fig. 3. The molecular weight distributions of soybean protein hydrolysates.
± ± ± ± ± ± ± ＜ 1 kDa, 1–5 kDa, 5–10 kDa, ＞10 kDa. Data are
0.01a 0.01c 0.04c 0.07b 0.04e 0.09d 0.07f presented as the mean ± standard deviation, n = 3.SPH-6L: soybean protein
Cys 1.99 1.94 2.06 2.03 2.01 2.15 2.17 hydrolysates (Protex 6 L for 2 h); SPH-A: soybean protein hydrolysates (Pro­
± ± ± ± ± ± ± tease A 2SD for 2 h); SPH-1, SPH-2, SPH-3, SPH-4, SPH-5: soybean protein
0.05f 0.03g 0.16c 0.02d 0.01e 0.03b 0.01a hydrolysates (Protex 6 L for 2 h, Protease A 2SD for 1 h, 2 h, 3 h, 4 h, 5 h).
Val 4.08 4.28 4.20 4.22 4.22 4.34 5.05
± ± ± ± ± ± ±
0.05f 0.03c 0.09e 0.01d 0.02d 0.04b 0.02a 3.4. Antioxidant activity analysis
Met 1.71 1.98 1.76 1.64 1.65 1.80 1.78
± ± ± ± ± ± ±
The antioxidant activity of SPH was shown in Fig. 4. The
0.19e 0.02a 0.02d 0.02g 0.08f 0.01b 0.07c
Ile 4.65 4.45 4.60 4.64 4.68 4.54 4.29
ABTS⋅+radical scavenging capacity and the ORAC value of SPH-6L were
± ± ± ± ± ± ± 50.19% and 71.65 μmol/L TE/mg, respectively. Fig. 1 shows a positive
0.02b 0.09f 0.09d 0.04c 0.05a 0.03e 0.01g correlation (r = 0.99) between ORAC value and ABTS⋅+ radical scav­
Leu 7.51 6.10 6.37 6.25 6.14 5.55 5.37 enging activity. The antioxidant activity of hydrolysates increased after
the addition of Protease A 2SD, however, the antioxidant activity
± ± ± ± ± ± ±
0.04a 0.05e 0.01b 0.02c 0.21d 0.01f 0.03g
Tyr 3.35 2.45 3.32 3.13 3.16 2.99 2.63 decreased with the reaction processing, which indicated that too much
± ± ± ± ± ± ± enzymatic treatment was negative for sample’s antioxidant activity.
0.06a 0.05g 0.02b 0.22d 0.09c 0.02e 0.07f With the addition of Protease A 2SD, the antioxidant activity of SPH was
Phe 4.14 3.33 3.11 2.67 2.62 2.07 2.16
improved in the early stage of hydrolysis (Fig. 4), which can be
± ± ± ± ± ± ±
0.06a 0.23b 0.01c 0.09d 0.13e 0.05g 0.17f
explained as the following two reasons. Firstly, hydrolysis produce
His 2.69 2.82 2.67 2.69 2.69 2.64 2.48 smaller fractions with MW < 1 kDa (from 8.95% to 19.98%) and 1–5
± ± ± ± ± ± ± kDa (from 27.01% to 57.42%) (Fig. 3), since most of the antioxidative
0.11b 0.11a 0.05c 0.03b 0.12b 0.09d 0.04e peptides derived from food sources have molecular weights ranging
Lys 7.15 7.36 7.11 7.01 7.05 6.98 6.34
from 0.5 to 1.8 kDa (Samaranayaka & Li-Chan, 2011). Secondly, the
± ± ± ± ± ± ±
0.07b 0.05a 0.08c 0.02e 0.09d 0.23f 0.12g negatively charged amino acids (NCAAs) in peptides increased duo to
Arg 7.59 8.01 6.76 6.41 6.52 5.99 6.26 hydrolysis (Table 2), since NCAAs can provide electrons to quench free
± ± ± ± ± ± ± radicals (Udenigwe & Aluko, 2011). The presence of high HAAs contents
0.17b 0.05a 0.04c 0.01e 0.15d 0.09g 0.03f can enhance the solubility of hydrolysates in lipids, resulting in an in­
Pro 4.73 5.14 5.34 5.54 5.19 5.76 5.99
± ± ± ± ± ± ±
crease in antioxidant activity (Pownall et al., 2010). However, Protease
0.14g 0.01f 0.01d 0.20c 0.21e 0.01b 0.01a A 2SD caused an HAAs decrease in peptide (Table 2), so the antioxidant
Trp 1.07 0.83 1.09 1.05 0.98 0.93 0.63 activity of SPH decreased, as excessive hydrolysis. For ABTS⋅+ radical
± ± ± ± ± ± ± scavenging activity and ORAC value, SPH-2 (56.92% and 76.76 μmol/L
0.09b 0.08f 0.04a 0.23c 0.13d 0.05e 0.06g
TE/mg) and SPH-3 (57.81% and 74.03 μmol/L TE/mg) displayed strong
Total 36.95 34.15 35.5 34.84 34.19 33.70 33.47
HAAs ± ± ± ± ± ± ± capacity of antioxidant activity. SPH-A has low DH (Table 1) and low
(%) 0.06a 0.12e 0.21b 0.22c 0.12d 0.09f 0.13g content of peptides less than 5 kDa (Fig. 3). Hence, the antioxidant ac­
Total 31.30 33.40 33.22 33.93 34.56 35.51 36.75 tivity of SPH-A was relatively low, where ABTS⋅+ radical scavenging
NCAAs ± ± ± ± ± ± ± activity was 29.78% and ORAC value was 43.67 μmol/L TE/mg (Fig. 4).
(%) 0.12g 0.06f 0.14e 0.02d 0.06c 0.08b 0.08a

Data are presented as the mean ± standard deviation, n = 3. Different super­

scripts within the same row indicate significant differences (p < 0.05). Com­
bined total of hydrophobic amino acids (HAAs): Ala, Val, Leu, Ile, Pro, Trp, Met,
Phe, Tyr, Cys (Pownall et al., 2010). Negatively charged amino acids (NCAAs):
Asx and Glx.SPH-6L: soybean protein hydrolysates (Protex 6 L for 2 h); SPH-A:
soybean protein hydrolysates (Protease A 2SD for 2 h); SPH-1, SPH-2, SPH-3,
SPH-4, SPH-5: soybean protein hydrolysates (Protex 6L for 2 h, Protease A
2SD for 1 h, 2 h, 3 h, 4 h, 5 h).
3.5. LC-MS/MS analysis and protease cleavage specificity
In the present work, the P3-P2-P1-P1ʹ-P2ʹ-P3ʹ indicates the positions
upstream or downstream of the cleavage site. The relative abundances
and frequency of the amino acid at the P3-P2-P1-P1′ -P2′ -P3’ positions in
iceLogos and heat map annotate the cleavage site specificity for Protex
6L and Protease A 2SD (Fu et al., 2018). After Protex 6L hydrolysis for 2
h, then hydrolyzed using Protease A 2SD for 3 h, high oil and protein
extraction yields were obtained, and SPH-3 has low bitterness value,

6
X. Tong et al. LWT 134 (2020) 110151

Fig. 4. Antioxidant activity of soybean protein hydrolysates. Data are presented as the mean ± standard deviation, n = 3. SPH-6L: soybean protein hydrolysates
(Protex 6L for 2 h); SPH-A: soybean protein hydrolysates (Protease A 2SD for 2 h); SPH-1, SPH-2, SPH-3, SPH-4, SPH-5: soybean protein hydrolysates (Protex 6L for 2
h, Protease A 2SD for 1 h, 2 h, 3 h, 4 h, 5 h).

good antioxidant activity, and high peptide nitrogen content. The most
used enzyme in EAEP is Protex 6L. Thus, we screened out the SPH-6L
and SPH-3 samples for LC-MS/MS analysis. The restriction enzyme
cleavage site of Protex 6L was observed, and the debittering mechanism
of Protease A 2SD on the Protex 6L hydrolysates was analyzed.
The P1 position was enriched with Tyr, Ala, Leu, Lys, and the P1′
position was enriched with Arg (Fig. 5a). From Table 1, the nitrogen in
the Protex 6 L protein hydrolysates (SPH-6L) was mainly in the form of
peptide nitrogen. Therefore, there are many peptides in SPH-6L with
Tyr, Ala, Leu, Lys (C-terminal) and Arg (N-terminal), indicating high
bitterness, since most bitter peptides have hydrophobic amino acids or
Lys at the C-terminal and Arg at the N-terminal (Cho, 2000; Ishibashi,
Arita, et al., 1987). Similar results have also been reported by Liu et al.,
(2016) who observed that the hydrolysates prepared by Protex 6L had
the highest bitter taste because of hydrophobic amino acids located at
the C-terminal of short peptides. It was reported that when hydrophobic

Fig. 5. The iceLogo plots and heat map of SPH-6L (a) and SPH-3 (b). SPH-6L: soybean protein hydrolysates (Protex 6L for 2 h); SPH-3: soybean protein hydrolysates
(Protex 6L for 2 h, Protease A 2SD for 3 h).

7
X. Tong et al. LWT 134 (2020) 110151

amino acid residues were located at N- or C-terminal in peptides, the Table 3

bitterness of peptides was stronger (Ishibashi et al., 1987b). The Identification of the major potential soybean bitter peptides in SPH-6L.
commonly used enzyme in EAEP is Protex 6L and SPH-6L has the Amino acid sequence Mass (Da) Leading razor protein
strongest bitterness value, therefore, bitter peptides in SPH-6L were
AFPGSAKDIENLIK 1501.814 Q9FZP9
identified by LC-MS/MS and predicted by bioinformatics. By using the YVVNPDNNENLRLITL 1885.9898 O22120
BIOPEP database (Minkiewicz, Iwaniak, & Darewicz, 2019), the bolded RRPSYTNGPQEIY 1579.7743 P04776
and italicized sequence was reported as part of the bitter peptide. The SLENQLDQMPRRF 1632.8042 P04776
bitter peptides predicted in Table 3 contain sequences that have been VVNPDNNENLRLITL 1722.9264 O22120
RIDPELEKEIGAK 1496.8199 I1M005
reported in known bitter peptides sequences, and the N- or C-terminal is ENQLDQMPRRF 1432.6881 P04776
HAA. Bioinformatics prediction reduces time and costs for screening SLLNALPEEVIQHTFNLK 2065.1208 P04776
processes and provides a basis for a better understanding of the bitter NQLDQMPRRF 1303.6455 P04776
taste of EAEP hydrolysates (García-Moreno et al., 1987). RAELSEQDIFVIPA 1586.8304 O22120
LVPPQESQRR 1208.6626 Q549Z4
Compared with Fig. 5a, the content of Leu and Arg at the P1 position
IIDTNSLENQLDQMPRRFYLA 2536.2744 P04776
in Fig. 5b is higher, and after hydrolysis of Protease A 2SD, the content of GINAENNQRNFLA 1459.7168 O22120
Leu and Arg in the peptide decreased significantly, indicating that the IVRNLQGENEEEDSGAIVTVK 2299.1656 Q549Z4
two-step hydrolysis mainly produced free Leu or Arg, instead of peptides KLHENIARPS 1163.6411 Q9SB11
rich in Leu, or Arg residues at the C-terminal. This can explain that LSIVDMNEGALLLPHFNSK 2097.0929 O22120
AGANSLLNALPEEVIQH 1774.9214 P04776
Leucine aminopeptidases was present in Protease A 2SD and played a RVSDDEFNNY 1257.5262 P01070
significant role in debittering. L-leucine aminopeptidases produced by NALEPDHRVESE 1394.6426 Q9SB11
manufactured of Aspergillus oryzae has been commonly used for LAGNPDIEHPETM 1422.6449 Q7GC77
reducing bitterness in foods cleaving L-leucine residues from the N-ter­ GKHQQEEENEGGSIL 1653.7594 P04776
EQGGEQGLEYVVF 1453.6725 Q7GC77
minal (Meinlschmidt, Schweiggert-Weisz, & Eisner, 2016). There were a
large number of peptides with N-terminal Arg in SPH-6L, which may Bolded and italicized sequence was reported as part of the bitter peptide in
produce bitterness. It can be inferred that Protease A 2SD may to excise BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep).SPH-6L:
the Arg of the N-terminal of the peptide, lead to a decrease in perceived soybean protein hydrolysates (Protex 6L for 2 h).
bitterness. According to the supplier description, Protease A 2SD spe­
cifically hydrolyze at Gln, Met, Cys, Arg, Ser, Thr, Ala, Lys, Phe, Leu. public or published. There are no conflicts of interest.
Many bitter peptides predicted in Table 3 contain Leu and Arg at the
N-terminal. Therefore, Leu aminopeptidase and Arg aminopeptidase Acknowledgements
may present in Protease A 2SD and play an important role in the deb­
ittering process. This work was financially supported by Heilongjiang Province “Tens
of Millions” Project Science and Technology Major Special Projects (No.
4. Conclusions 2019ZX08B01), the “Young Talents” Project of Northeast Agricultural
University (No.19QC29), Major Science and Technology Innovation
The study aimed to investigate the relationship of bitterness with the Projects in Shandong Province(No.2019JZZY010722), National Natural
characteristics of peptide which was treated with sequential hydrolysis Science Foundation of China(No.31801579), Science Foundation Proj­
of Protex 6L and Protease A 2SD. Compared with the high bitterness ect of HeilongjiangProvince (No. JC2018009).
value of SPH-6L, the protein hydrolysates generated with Protex 6L and
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