Journal Pre-proof Modification of soy protein isolates using combined pre-heat treatment and controlled enzymatic hydrolysis for improving foaming properties Guijiang Liang, Wenpu Chen, Xuejiao Qie, Maomao Zeng, Fang Qin, Zhiyong He, Jie Chen PII:
To appear in:
Received Date: 4 May 2019 Revised Date:
9 February 2020
Accepted Date: 11 February 2020
Please cite this article as: Liang, G., Chen, W., Qie, X., Zeng, M., Qin, F., He, Z., Chen, J., Modification of soy protein isolates using combined pre-heat treatment and controlled enzymatic hydrolysis for improving foaming properties, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/ j.foodhyd.2020.105764. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Modification of soy protein isolates using combined pre-heat treatment and controlled enzymatic hydrolysis for improving foaming properties
3 4 5
Guijiang Liangab, Wenpu Chenac, Xuejiao Qieab,Maomao Zengab, Fang Qinab, Zhiyong He*ab, Jie Chen*ab
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122,
Abbott Nutrition Research & Development (ANRD), Singapore
Professor Jie Chen
Professor Zhiyong He
State Key Laboratory of Food Science and Technology, School of Food Science and
Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China
E-mail address: [email protected]
; [email protected]
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34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77
Abstract The foaming properties of soy protein hydrolysates modified with a combination of pre-heat treatment and controlled enzymatic hydrolysis by pepsin were investigated. Degree of hydrolysis, soluble protein percentage, peptide profile, molar mass distribution, surface properties at air-water interface and interfacial rheological properties of hydrolysates were measured to gain insights into the relationships between molecular composition and foaming properties. It was found that pre-heat treatment of soy protein at 55˚C for 30 min promoted the hydrolysis of hydrophilic acidic subunits and increased the ratio of 7S/11S. As result, it resulted in the best interfacial properties and foam capacity as well as stability amongst all treatments. Although pre-heat treatment above 65˚C before soy protein isolate hydrolysis increased degree of hydrolysis, higher proportion of large protein aggregates in the hydrolysates with increasing temperature deteriorated their foaming capacity and stability, which resulted in lower storage modulus (G') of the adsorbed layer and less surface excess concentration (Γ) compared to pre-heat treatment at 55˚C. Without pre-heat treatment, unfolded soy protein did not provide sufficient accessible sites for pepsin hydrolysis. As result, foam capacity and stability were inferior compared to 55˚C pre-heat treatment hydrolysates. This study provided the foundation for the application of soy protein isolate in aerated food products with desirable foaming properties.
Key words: Soy Protein, Pepsin, Hydrolysates, Foaming Capacity, Foaming Stability
2|P a g e
Food foams provide desirable textures and unique mouthfeel to many aerated foods,
such as meringues, cakes, bread, soufflés and marshmallow in which tiny air bubbles
are trapped in the food system (Campbell & Mougeot, 1999; Zeng et al., 2013). Food
foam is a thermodynamically unstable system where air phase is dispersed in a liquid
matrix, and thus requires external energy to generate and maintain its stability.
According to the thermodynamic dictum, phase separation rapidly occurs to minimize
the interfacial contact area and free energy in colloid dispersions with two immiscible
phases. Typically, surface active species with amphiphilic properties are added to
reduce interface tension between continuous and dispersed phases. Two common
surface active species, namely small molecule surfactants, such as monoglycerides,
and proteins have been used to lower surface tension at air-water interfaces and
impact foam stability (Patino, Niño, & Sánchez, 2003). They play a key role in
maintaining foam stability and preventing foam destruction caused by liquid drainage,
coalescence and disproportionation (Sadoc & Rivier, 2013). Traditionally, egg white
proteins and milk proteins e.g. sodium caseinate are commercial agents for the
desirable foaming properties (Nicorescu et al., 2011)The behaviors of those proteins
at air-water interfaces and their role on foaming capacity and stability have been
studied in previous research papers (Murray, 1998; Morris & Gunning, 2008).
Recently, soy protein isolate (SPI) has been considered as an alternative to or
extension of egg white protein and milk proteins because of its excellent nutritional
value and functionalities (Anderson, Anthony, Cline, Washburn, & Garner, 1999;
Dickinson & Matsumura, 1991). Soy protein isolate with exposed hydrophobic and
hydrophilic groups have been used as foaming agents (He et al., 2015). However,
intact soy protein isolate with less flexible biological structure limits surface
properties and foaming capacity (Wagner & Guéguen, 1999). Flexible protein
structure and rapid molecular adsorption at air-water interface are required for soy
protein as a good foam agent. Moreover, the formation of a cohesive viscoelastic
adsorbed protein layer via intermolecular interactions at air-water interfaces is 3|P a g e
necessary to keep foam stability (Dickinson, 2003). The interfacial behaviors of
adsorbed protein layer are impacted by many factors such as protein molecular
structure, protein size and molar mass, conformational structure, composition and
sequence of amino acids, and some environmental factors such as pH and ionic
strength (Foegeding, Luck, & Davis, 2006).
Soy protein subunits demonstrate diverse interfacial properties due to their
inherent structure and amino acids composition. For example, β-conglycinin (7S)
shows better foaming and emulsifying properties compared to glycinin (11S) because
β-subunit from β-conglycinin has a high proportion of hydrophobic amino acids such
as alanine, valine, leucine, and phenylalanine, which have a high tendency to interact
with hydrophobic phase at the interface (Nielsen, 1985; Lam & Nickerson, 2013).
Basic subunit with higher hydrophobicity in glycinin (11S) are buried and shielded by
the hydrophilic acid subunit in aqueous solution (Kuipers & Gruppen, 2008). Hence,
emulsifying properties of glycinin (11S) are inferior to β-conglycinin (7S).
To improve foaming and emulsifying properties of soy protein isolate, thermal
treatment and enzymatic treatment have been used to modify the soy protein structure
(Tsumura et al., 2005; Were, Hettiarachchy, & Kalapathy, 1997). Shao, Lin, & Kao
(2016) reported that thermal treatment to commercial soy protein isolate improved its
foam capacity and stability. According to Cui, Zhao, Yuan, Zhang, & Ren (2013), soy
protein hydrolysates made by 0.3% pepsin at pH 2 for 60 min had an improvement in
solubility, emulsification and foam stability. For enzymatic modification, the choice
of protease and control of degree of hydrolysis have great impacts on foaming
properties of SPI hydrolysates. According to Ortiz & Wagner (2002), SPI hydrolysates
processed with bromelain (pH 7) had a good foaming property at pH 4.5. As an
endopeptidase, pepsin has been used to improve the functionality and structure
characteristics of intact soy protein in the controlled condition (Cui, Zhao, Yuan,
Zhang, & Ren, 2013; Tsumura et al., 2005). Tsumura et al. (2005) found that SPI
hydrolysates with selective digestion of glycinin by pepsin exhibited better
whippability than other treatment. However, there are limited studies reported on the 4|P a g e
use of a combination of pre-heat treatment at different temperature with controlled
enzymatic hydrolysis to manipulate the compositions of hydrolysates. Moreover, the
impacts of the compositions of SPI hydrolysates with pre-heat treatment on the
surface properties are still unknown.
In this study, soy protein isolate was subjected to pre-heat treatment at various
temperatures prior to hydrolysis and the effects of pre-heat treatment on controlled
pepsin-driven hydrolysates and their interfacial properties were observed. Degree of
hydrolysis, soluble protein percentage, protein profile, molar mass distribution,
interfacial rheological properties and surface properties of the hydrolysates were
studied to gain insights into the relationship of peptides composition and surface
properties, which were used to understand their foaming properties. The purpose of
this study was to determine the optimum pre-heating temperature condition before
pepsin-driven hydrolysis for SPI hydrolysates with desirable foaming properties.
2.0 Materials and methods
2.1. Materials and chemicals
Soybeans were obtained from a local market (Wuxi, China). Pepsin (3000 U/mg) was
purchased from Sangon Biotech Co, Shanghai, China. Trinitro-benzene-sulfonic acid
(TNBS) solution and β-mercaptoethanol were purchased from Sigma-Aldrich (St.
Louis, USA). All chemicals used were of analytical grade unless otherwise specified.
Deionized water was used as the ingredient water.
2.2. Preparation of SPI hydrolysates
Soybeans were crushed and peeled into small particles, then degreased with n-hexane
to obtain defatted soy flour. SPI was prepared from defatted soy flour according to the
method provided by Puppo et al. (2004) with slight modifications. Soy flour was
dispersed into water and adjusted to pH 8 using 2M NaOH. The dispersion was stirred
for 2h at room temperature and then centrifuged for 20 min at 10000 x g at 4℃. The
supernatant was adjusted to pH 4.5 with 2M HCl and centrifuged (3300 x g, 10 min,
4℃) for the sediment. The obtained sediment was re-suspended with distilled water (1:
5, v/v) and adjusted to pH 7.0 with 2M NaOH before freeze drying. 5|P a g e
The samples above were reconstituted in water to make a solution with 7% (w/v)
protein with Biuret method given by Chang (2014). The solution was divided into 6
portions for different heat treatment. Portion 1 was not subjected to thermal treatment.
Portion 2 was subjected to 55˚C for 30 min. Portions 3-6 were subjected to 65˚C,
75˚C, 85˚C, and 95˚C for 10 min. After thermal treatment, all the samples were
chilled in ice slurries, then were adjusted to pH 2.0 using 2M HCl and incubated at
37˚C for 30 min. Pepsin was added to each portion at enzyme: substrate ratio of 0.3%
(w/w) to initiate hydrolysis. Each portion was incubated at 37˚C for 60 min.
Hydrolysis was terminated by adjusting to pH 7 with 2M NaOH. The hydrolysates
were centrifuged (10000 x g, 10 min, 25℃) and the supernatant was sterilized for 20s
at 120˚C before freeze drying. The hydrolysates of portion 1-6 was referred as
control,SPH55, SPH65, SPH75, SPH85 and SPH95 respectively.
2.3. Soluble protein determination
All the hydrolysates were centrifuged at 10000 x g for 10 min and the content of
soluble protein in the supernatant was determined with Biuret method (Chang, 2014).
The percentage of soluble hydrolysates was calculated according to the equation
182 Soluble protein content in hydrolysate(g/mL)Volume of hydrolysate(mL) 183
× 100% (1)
Soluble protein %= Total protein(g)
2.4. Degree of hydrolysis (DH)
DH of the hydrolysates was determined with TNBS method given by Spellman,
McEvoy, O’cuinn, & FitzGerald (2003), with slight modifications. L-Leucine with
different concentrations was used to generate a standard curve at absorbance at 420
nm for the standard nitrogen content. The absorbance at 420 nm of the samples before
and after hydrolysis was measured. The values were substituted into the standard
curve to calculate the amino nitrogen content of the protein substrate.
DH values were calculated using the following formula (Harnedy et al., 2018)
193 DH% =
AN2 - AN1 Npb
6|P a g e
where AN1 is the amino nitrogen content of the protein substrate before hydrolysis
(mg/g protein), AN2 is the amino nitrogen content of the protein substrate after
hydrolysis (mg/g protein). Npb is the nitrogen content of the peptide bonds in the
protein substrate (mg /g protein). A value of 109.2 was used for soy protein.
2.5. Molar mass distribution
The molar mass distribution of the hydrolysates was measured by HPLC equipped
with a gel permeation chromatographic (GPC) column (Shodex Protein KW-84
column; 8 mm I.D X 30 cm, Shodex Co., Tokyo, Japan) and a Waters 2487 dual λ
absorbance detector (Waters Co., USA). The elution buffer consisted of 50mM
phosphate (pH 7.0) with 0.3M NaCl (flow rate: 1.0 mL/min). Before testing, all
samples were dissolved in ultrapure water and centrifuged to remove insoluble
portion (10000 x g, 25 ˚C, 10 min). After that, the protein concentration in all samples
was adjusted to 1% for further test. Bovine thyroglobulin (669 kDa), amylase (200
kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29
kDa) and cytochrome c (12 kDa) were used as markers.
2.6. Sodium dodecyl sulphate-polyarylamide gel electrophoresis (SDS-PAGE)
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed according to the method reported by Liu, Xiong, & Butterfield (2000),
with slight modifications. The hydrolysates were run using a mini-protein
electrophoresis system (Bio-Rad Laboratories, Hercules, Calif., U.S.A.). SDS-PAGE
was conducted on a discontinuous buffered system with 12% separating gel and 4%
stacking gel. Samples (2 mg/mL in buffer containing 0.0625M of Tris-HCl, 10%
glycerin, 2% SDS and 0.0025% bromophenol blue) were incubated for 1h at room
temperature with and without β-mercaptoethanol (5%, v/v). After incubation, all
samples were heated at 100℃ for 5 min. Aliquots (20 µL) of the prepared samples
were loaded onto the gels. All gels were scanned by a computing densitometer
(Molecular Imager ChemiDocXRS+, Bio-Rad, USA) Image Lab software (Bio-Rad,
USA) was used to integrate the intensities of bands.
2.7. Surface properties measurement 7|P a g e
Dynamic interfacial tension (γ) of the hydrolysates at air-water interface was
determined using a DCAT21 automated surface tension meter (Dataphysics, Berlin,
Germany). The critical micelle concentration (CMC) was automatically calculated
from surface tension (γ) of the samples. The surface excess concentration (Γ) and the
area occupied by each molecule (A) were calculated according to the Gibbs
adsorption in Equation: Γ=
where C is the concentration, R is the gas constant, and T is the absolute temperature.
The mean area occupied per molecules of the adsorbed surfactant, A, was calculated
according to A=
where N is Avogadro’s constant. The calculation was calculation by DCATSoftware.
concentration was adjusted to 2% for all measurements by Biuret method.
2.8. Interfacial rheology measurement
Measurements of the interfacial rheology of proteins (1%) at the air/water interface
were obtained using a HAAKE MARS ℃ rheometer (Thermo Scientific, Germany)
equipped with a Du Noüy ring (platinum ring，diameter 19.450 mm). The thickness of
the wire was 0.379 mm. 20 mL of the sample was placed in a beaker (50 mm in
diameter) and the ring was lowered to contact the surface. To increase repeatability
and avoid destruction of the platinum ring, the gap was zeroed with cone geometry
and then the gap was kept constant at the position of 48.723 mm. The inertia
determination and MSC (Micro Stress Control) calibration were performed for every
measurement. Dynamic time sweeps were performed within the linear region at a
strain amplitude of γ =1% and an angular frequency of ω =1 rad s-1 for 1 h. Dynamic
strain sweeps were carried out with an angular frequency of ω = 1 rad s-1 in the range
of = 0.1-100%, with frequency of ω =1 rad s-1 at 25 °C. 8|P a g e
2.9. Foaming properties
Foaming properties were evaluated according to the method given by Adebowale,
Schwarzenbolz, & Henle (2011). Protein solution (100 mL, 1% w/v) was placed in a
500 mL graduated cylinder and homogenized with a disperser homogenizer (T 18
basic ULTRA-TURRAX R, IKA Corp., Staufen, Germany) at 17,500 rpm for 2 min.
The foam volume was recorded at time zero (V0) and after 30 min (V30) of
Foaming capacity (FC) and foaming stability (FS) were calculated using Equation (5)
and (6), respectively and 100 is the initial volume of solutions (mL). FC (%) =
V0 - 100 100
FS (%) =
V30 - 100 V0 - 100
2.10. Statistical Analysis
All experiments and associated measurements were performed at least in triplicate.
Statistical analysis was performed using a two-way ANOVA (P< 0.05) by Statistix 9.0
(Statistix, Tallahassee, FL, USA).
3.0. Results and discussion 3.1 Degree of hydrolysis and soluble protein percentage
The effect of heating temperatures on the degree of hydrolysis and soluble protein
content of soy protein hydrolysates were examined. SPH95 had the highest degree of
hydrolysis while the control had the lowest degree of hydrolysis amongst all the
samples. As shown in Fig.1, increasing pre-treatment temperature before hydrolysis
resulted in higher degree of hydrolysis of SPI because the unfolding of protein
structure caused by thermal treatment favored enzymatic hydrolysis by making more
accessible sites available to enzymes (Lam & Nickerson, 2013). Pepsin has a
preference to cleave the peptides containing linkages with aromatic or carboxylic
L-amino acids (Worthington, 1993). Thermal treatment caused soy protein molecules 9|P a g e
to unfold and expose the sulphydryl and hydrophobic groups (Keerati-u-rai &
Corredig, 2009). Two distinct thermal transition peaks, ranging from 68-75°C and
85-93°C, have been identified for denaturation of β-conglycinin and glycinin
respectively (Scilingo & Añón, 1996; Renkema, Lakemond, Jongh, Gruppen, & Vliet,
2000). According to Achouri, Zhang, & Shiying (1998), pre-heat treatment of intact
soy protein isolate at 80°C for 10 and 30 min prompted unfolding of some fractions
and a gradual molecular dissociation, which facilitated enzymatic hydrolysis. The
subunits in β-conglycinin would dissociate when it was subjected to thermal treatment
(above 70°C), causing structural changes (Iwabuchi, Watanabe, & Yamauchi, 1991).
Protein solubility is crucial for protein functional properties such as emulsifying,
rheological and surface-active properties. In theory, controlled enzymatic hydrolysis
of soy protein would improve the solubility of protein due to decreasing molar mass
of protein and increasing number of charge groups (Panyam & Kilara, 1996).
However, there was a decrease of soluble protein as the temperature of pre-heat
treatment increased in this study. The lowest soluble protein percentage was observed
in SPH95. This was explained as follows. Thermal pre-treatment induced unfolding of
protein molecules in partial or total form prior to hydrolysis and provided accessible
sites to pepsin. Pepsin hydrolysis promoted the exposure of hydrophobic and
hydrophilic groups, which led to formation of aggregates and thus greater loss of
solubility of hydrolysates (Chen, Chen, Ren, & Zhao, 2011). The formation of more
insoluble aggregates was caused by the hydrophobic interaction among the subunits
of soy protein isolate (Utsumi, Damodaran & Kinsella, 1984). The samples with high
temperature have more hydrophobic groups. However, the complexes formed by
β-subunit from β-conglycinin and basic subunit from glycinin were soluble when the
temperatures were above 90°C (Scilingo, & Añón, 1996; Renkema, Lakemond, Jongh,
Gruppen, & Vliet, 2000).
3.2. Composition of SPI hydrolysates
The polypeptide profile of SPI hydrolysates after pre-heating at different temperatures
was analyzed by SDS-PAGE for both non-reducing and reducing conditions and 10 | P a g e
untreated soy protein isolate was included as the reference (Fig.2A and Fig.2B). As
shown in Fig. 2A and 2B, all subunits from β-conglycinin (α, α' and β) and glycinin
(acidic and basic subunit) were present in all samples. Compared to the reference, the
complex of acidic and basic subunit (AB) was missing due to enzymatic hydrolysis.
This finding agreed with the reported by Chen et al. (2019). Under non-reducing
condition, large protein aggregates were identified on the top of the stacking gel
(Fig.2A). The bands of these protein aggregates disappeared under reducing condition
(Fig.2B), which indicated the interactions among the polymer molecules were
disulfide (S-S) linkage. The densitometric analysis of the main bands in SDS-PAGE
was performed to understand the impact of pre-heat treatment on soy protein
composition of SPI hydrolysates (Table 1). As shown in Fig. 2A and Table 1, the
bands corresponding to β-conglycinin (α', α, β) became faint when the pre-heat
temperature was above 65°C. Nearly 50% of α' and α subunit were lost at 65°C and
around 75% were lost when the temperature reached 85°C. The reduction rate of
β-subunit was lower than α' and α. According to Yamauchi, Yamagishi, & Iwabuchi
(1991), thermal treatment induced the dissociation of β-conglycinin into its
constituent’s subunits. When the heat treatment temperature was higher than the
denaturation temperature of β-conglycinin (68-75°C), ɑ and α' subunits formed
soluble aggregates (Petruccelli & Anon, 1995). Moreover, pepsin may decompose
ɑ-subunit from β-conglycinin selectively (Cui, Zhao, Yuan, Zhang, & Ren, 2013). In
synergy, both procedures led to the loss of ɑ and α' subunits once the thermal
temperature was above 65°C. The slow reduction rate of β-subunit was due to the
presence of α' and α subunits that prevented thermal aggregation of β-subunit (He, et,
al., 2015). Pepsin had little effect on β-subunit at pre-heat treatment temperature of
55°C (Table 1 and Fig. 2). However, when the pre-heat treatment temperature was
elevated above 65°C, more hydrophobic regions in β-subunit were exposed, which
increased the chances of association with other hydrophobic regions. This association
was irreversible-intermolecular aggregation that formed the aggregates of different
molecular sizes (Yamauchi, Yamagishi, & Iwabuchi, 1991). For glycinin (11S), the 11 | P a g e
reduction of acid subunit was faster than basic subunit, because the basic subunits
with higher hydrophobicity were buried and shielded by hydrophilic acid subunits in
aqueous solution (Kuipers & Gruppen, 2008). During hydrolysis, acidic polypeptides
were decomposed into peptides easily because endoproteases such as pepsin has a
preference to attack acidic amino acids (Jung, Roussel-Philippe, Briggs, Murphy, &
Johnson, 2004). When pre-heat temperature reached 95°C and above, there was
formation of soluble complexes between β-subunit from β-conglycinin and basic
subunit from glycinin (Lakemond, de Jongh, Hessing, Gruppen, & Voragen, 2000), as
shown by the reduction in the amount of β-subunit and basic subunit in Table 1.
3.3. Molar mass distribution of SPI hydrolysates
The molar mass of SPI hydrolysates after pre-heat treatment at various temperatures
can be categorized into four subgroups: group A (larger than 669 kDa), group B (100
-669 kDa), group C (10-100 kDa) and group D (less than 10 kDa) (Fig.3a and Fig.3b).
The relative areas of these four subgroups were different with different pre-heat
treatment conditions. It was observed that higher temperature used in pre-heat
treatment (55-95°C) resulted in an increase of the proportion of large molecules
(group A) significantly, while a decrease of the proportion of small molecules (group
B, C, D) was observed. The MW distribution of SPI subjected to different heat
treatment was in agreement with the results of SDS-PAGE analysis (Fig. 3).
3.4. SPI hydrolysate surface properties
As shown in Table 2, increasing pre-heat treatment temperature led to a decrease in
the surface excess (Γ) and an increase of molecular area (A) at the air-water interface.
Pre-heat treatment did not impact on the surface tension and CMC of hydrolysates at
the air-water interface as shown in Table 2. The value of CMC and surface tension of
the control sample are similar to the results reported by Li et al. (2016). Compared to
the control, pre-heating temperature at 55°C resulted in an increased surface excess (Γ)
from 0.84 µmol/m2 to 1.22 µmol/m2. However, Table 2 shows surface excess (Γ)
progressively decreased from 1.22 µmol/m2 to 0.45 µmol/m2, when pre-heating
temperature was increased from 55°C to 95°C. SPH55 achieved a maximum surface 12 | P a g e
excess (Γ) and a minimum molecular area (A) amongst all samples, indicating the
amount of adsorbed SPH55 molecules was more than other hydrolysates with a
smallest occupied molecular area at air-water interface. With 55°C pre-heat treatment
and subsequent hydrolysis, the high ratio of hydrophobic subunits such as β-subunits
and basic subunits in hydrolysates favored the rapid adsorption at air-water interface,
because at the air-water interface, the hydrophobic subunits can be positioned to form
more interactions with the air phase to attain a more thermodynamically stable state,
thus indicating a good interfacial activity. Additionally, improved flexible molecular
structure by hydrolysis and more 7S compositions in the hydrolysates would
contribute to better interfacial activities. According to Shao & Tang (2014), higher
extent of protein denaturation and/or aggregation exhibited a higher surface excess (Γ)
compared to intact SPI. However, pre-heat treatment above 65°C increased the
amount of larger molecular aggregates at the expense of hydrophobic subunits, which
reduced the amount of absorbed protein at air-water interface with a lower trend of
surface excess (Γ).
3.5. Interfacial rheology measurement
The formation, structural reorganization and mechanical properties of protein layer at
the interface can be monitored by interfacial shear rheology (Qiao, Wang, Shao, Sun,
& Miller, 2015). The adsorption kinetics and characteristics of the film formed by soy
protein hydrolysates at air-water interface were obtained by interfacial rheology. Fig.4
records the change of storage modulus G' and loss modulus G'' during time sweep as
the function of pre-heat treatment temperature. The rates of film formation were
expressed by the slope (K) by building the linear model of G' as the function of time
(0 to 10 min). By comparing K values of all samples, SPH55 K value (0.34) in Fig.4B
is the highest amongst all samples. K value demonstrates a descending trend with
increasing pre-heat treatment temperature. SPH55 molecules migrated and formed a
gel-like film at the fastest rate compared to the other samples. SPH55 molecules can
also unfold, change structure conformation, interact with neighboring molecules and
form a two-dimensional viscoelastic gel, as was indicated by storage modulus G' 13 | P a g e
being higher than loss modulus G'' during the whole time sweep. With time going, the
increasing gap between G' and G'' demonstrated the strength of adsorbed SPH55 film
and more “gel-like” properties. The interfacial storage modulus G' was nearly one
order magnitude higher than loss modulus G'' and this trend did not change
remarkably in the whole measurement range, which proved that the higher ratio of
β-subunits and basic subunits in hydrolysates favored the formation of gel structure
by the association between molecules (Patino, Ortiz, Sánchez, Niño, & Añón, 2003).
Moreover, the highest storage modulus G' of SPH55 observed in all samples showed
that its film had the best mechanical properties against draining, coalescence and
rupturing. All samples except SPH95 demonstrated “solid-like” properties for
adsorbed protein layers at air-water interface (G' > G'') and no interfacial gel transition
(no cross-over between G' and G'') during the time sweep. The adsorbed protein layer
of SPH95 exhibited “fluid-like” properties at the beginning of the sweep and it took
some time to get cross-over between G' and G'' at air-water interface, indicating the
SPH95 protein molecules needed time to form a layer with some “solid-like”
characteristics. As discussed in Section 3.2, the loss of β-subunit and basic subunit
during the treatment can explain this difference.
The fracture mechanism and structure strength of the films formed by SPI
hydrolysates were investigated by strain sweeps (Fig.5). All the samples exhibited
similar trend whereby a plateau in a regime of linear viscoelasticity at the low strain
was observed followed by a rapid drop at the high strain due to some fractures in the
absorbed protein layer structure. The drop point of the dynamic storage modulus G' is
the point where the destroyed and newly formed bonds were balanced. The interfacial
layers formed by the control were relatively fragile compared to other sample. The
reason is that the control molecules without pre-heat treatment possessed a more rigid
structure and the protein was not unfolded, so the film was weak against external
strain sweeps. The unfolded protein molecules induced by pre-heat treatment
contributed to structural strength of the films.
3.6. Foaming capacity and foaming stability
14 | P a g e
In soy protein hydrolysates dispersions, when air was incorporated into protein liquid
dispersion, the formation of a cohesive air-water interfacial film in rapid rate was a
prerequisite for desirable foaming capacity. Fig.6 showed that SPH55 displayed the
best foaming capacity and stability amongst all the samples. There was a significant
drop in the foaming capacity and stability when the pre-heat temperature was above
75°C. SPH55 molecules rapidly adsorbed at air-water interface with more exposed
hydrophobic subunits such as β-subunit and basic subunit (Table 1) and higher
proportion of small molecule peptides (Fig.2). The adsorption of SPH55 molecules
may not undergo a molecular unfolding step. As a result, the formation of adsorbed
protein film at air-water interface was accelerated (Davis & Foegeding, 2007), which
agreed with the results shown as the highest value of slope (K) in Section 3.5. As
discussed in Section 3.2, 55°C pre-heat treatment altered the ratio of 7S/11S in soy
protein molecules (Table 1) because pre-heat treatment promoted the hydrolysis of
acidic subunit and the increase of ratio of hydrophobic residues such as β-subunits
and basic subunit in the hydrolysates, which facilitated the initial anchoring of SPH55
molecules to the air phase and formed a high viscoelastic film. Moreover,
pepsin-driven hydrolysis produced a certain amount of small molar mass peptides,
which also contributed to foaming capacity because of its flexible molecular structure,
small molar mass and amphiphilic properties. According to Damodaran (2005), an
effective foaming agent requires protein molecules to be adsorbed at the air-water
interface rapidly. As indicated in Fig 3, the proportion of small molar mass peptides
(less than 10 kDa) was the second largest among all samples. Although higher
pre-treatment temperature (above 65°C) would accelerate the exposure of more
enzyme accessible sites compared to SPH55, the hydrophobic subunits formed larger
molecular aggregates with increasing DH and hydrophobicity (Kuipers & Gruppen,
2008). Intact soy protein structure or large molecular aggregates under high
temperature treatment (above 65°C) diffused slowly to the air-water interface,
indicating as decreasing foaming capacity.
Foam destabilization is caused by liquid drainage, disproportionation with growing of 15 | P a g e
larger bubbles and coalescence (Talansier et al., 2009). Good viscoelastic and
mechanical properties of film with favorable intermolecular interactions can resist a
high Laplace pressure in small bubbles against the rupture of foam. SPH55 provided
the most stable foams compared with other samples (Fig.6), indicating that pre-heat
treatment temperature had a good correlation with the strength of the interfacial film.
Combination of 55°C pre-heat treatment with pepsin-driven hydrolysis was desirable
for maintaining stable foam (Fig.6). When more hydrophobic subunits were present at
air-water interface, they were sufficient to enable the formation of a “gel-like” layer
via the formation of intermolecular polymers and prevent the collapse of air bubbles.
Additionally, adsorbed protein layers created disjoining pressure to increase the
thickness of liquid film and prevented liquid drainage in bubbles (Damodaran, 2005).
The disjoining pressure is the development of an osmotic pressure between the bulk
phase and the lamella fluid (Damodaran, 2005). When two bubbles approach each
other, higher surface excess (Γ) concentration would lead to disjoining pressure.
SPH55 has the highest surface excess (Γ) concentration at air-water interface,
indicating it had the highest disjoining pressure amongst all the samples. As a result,
water molecules would migrate from low-pressure area to high-pressure area, which
prevented liquid drainage when two bubbles approach.
In this study, the composition of soy protein hydrolysates can be manipulated through
a combination of pre-heat treatment and enzymatic treatment driven by pepsin to
prepare a foaming agent with good foaming properties. The results demonstrated that
55°C pre-heat treatment promoted the exposure of more accessible sites for pepsin
compared to the control. At the same time, increasing ratio of β-subunits and basic
subunits in SPH55 led to more surface excess (Γ) concentration and contributed to the
mechanical strength of the adsorbed protein film at the air-water interface. Moreover,
high ratio of 7S/11S of SPH55 and the presence of high ratio of small molecule
peptides facilitated its molecular flexibility and rapid adsorption at the interface. All
of them contributed to foaming capacity and stability. The aggregates in hydrolysates 16 | P a g e
induced by high temperature treatment (above 65°C) deteriorated foaming capacity
and stability. This finding provides useful insight regarding the relationship of soy
protein composition and its foaming properties.
483 484 485 486
Conflict of interest
The authors state that there are no conflicts of interest regarding publication of this article.
490 491 492
The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant No. 31471583) and National First-class discipline program of Food Science and Technology (Grant No. JUFSTR20180201).
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List of Figures
Fig.1 Degree of hydrolysis and percentage of soluble protein hydrolysate. Data are
means (n=3) ± standard deviation; Values with different superscript letter are
different P < 0.05. Abbreviation: DH: degree of hydrolysis, PSP: percentage of
soluble protein. 55: SPH55, 65: SPH65, 75: SPH75, 85: SPH85, 95: SPH95. A-F is
used for DH and a-e is used for PSP.
Fig.2 SDS-PAGE patterns of hydrolysates subjected to different temperature pre-heat
treatment. Samples for SDS-PAGE were without β-mercaptoethanol (A) or with
β-mercaptoethanol (B). MW: molar mass marker (Da). β-Conglycinin: α' (86 kDa), ɑ
(66 kDa), and β (51kDa); Glycinin: A, acidic subunit (34-43 kDa), and B, basic 21 | P a g e
subunit (17-26 kDa). Abbreviation: 55: SPH55, 65: SPH65, 75: SPH75, 85: SPH85,
95: SPH95, C: Control, SPI: Untreated soy protein isolate as reference.
653 654 655 656 657 658 659
Fig.3 Molar mass distribution of hydrolysates (a) High performance size exclusion chromatographic profiles of hydrolysates (A: > 669 kDa; B: 100– 669 kDa; C: 10– 100 kDa; D: <10 kDa). The equation of the standard curve was y =-0.4708x+6.6162; R2= 0.9856. (b) Relative areas (%) of the four protein fractions (A, B, C, and D) of hydrolysates. Abbreviation: 55: SPH55, 65: SPH65, 75: SPH75, 85: SPH85, 95: SPH95.
Fig. 4 The time evolution of the interfacial storage modulus G' and loss modulus G''
of layer at the air/water interface obtained from the time sweep. Abbreviation: A)
Control; B) SPH55; C) SPH65; D) SPH75; E) SPH85; F) SPH95.
Fig.5 Strain dependence of interfacial elastic modulus G' of hydrolysates at the
air/water interface. Abbreviation: 55: SPH55, 65: SPH65, 75: SPH75, 85: SPH85, 95:
668 669 670 671 672 673 674 675 676
Fig.6 Foaming capacity and stability of SPI hydrolysates. Data are means (n=3) ± standard deviation.Values with different superscript letter are significantly different P<0.05. Abbreviation: 55: SPH55, 65: SPH65, 75: SPH75, 85: SPH85, 95: SPH95. A-E is used for foaming capacity and a-e is used for foaming stability.
22 | P a g e
CRediT author statement
Guijiang Liang: Conceptualization, Methodology, Software， Formal analysis，Writing original draft Wenpu Chen：Data curation, Writing- Original draft preparation， Investigation Xuejiao Qie： Writing - Review & Editing, Visualization Maomao Zeng: Writing - Review & Editing, Resources Fang Qin: Investigation, Resources Zhiyong He: Supervision Jie Chen: Supervision
Table 1 Relative Compositions (%) of SPI hydrolysates
-βME (Non-reducing condition) 7S
+βME (Reducing condition)
*β-Conglycinin: α' (86 kDa), ɑ (66 kDa), and β (51kDa); Glycinin: A, acidic subunit (34-43 kDa), and B, basic subunit (17-26 kDa).
Table 2 Surface properties of hydrolysates Sample
(mg/ml) a a a a a a
47.89±0.90 46.40±1.41 47.05±1.35 46.60±1.26 46.67±0.76 47.24±2.17
Surface excess Γ (mol/m )
a a a a a a
1.22±0.23 0.99±0.27 0.88±0.29 0.62±0.22
Molecular area A(nm2) ab
1.97±0.34 a 1.35±0.15
* Suface excess (Γ) and molecular area (A), were calculated from the critical micelle concentration (CMC) and suface tension (γ). The experiments were performed in three replicates. The result were expressed as mean values (n=3) ± standard deviation. Values with different superscript letter are significantly different at P<0.05.
The hydrolysate with pre-heat treatment at 55 °C had the highest ratio of 7S/11S.
β subunit and basic subunits contributed to the desirable foaming properties.
Heat treatment (above 65°C) deteriorated hydrolysates' foaming capacity and stability.
Conflict of interest The authors state that there are no conflicts of interest regarding publication of this article.