Food Chemistry 294 (2019) 216–223

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Gelling and emulsifying properties of soy protein hydrolysates in the T

presence of a neutral polysaccharide
⁎
José A. Lopes-da-Silva , Sónia R. Monteiro
QOPNA – Organic Chemistry, Natural and Agro-Food Products Research Unit, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

A R T I C LE I N FO A B S T R A C T

Keywords: Soy protein hydrolysates (SPH) with different degrees of hydrolysis (DH 0–16%) were obtained by varying the
Soy proteins time of hydrolysis with bromelain. The objective of this study was to evaluate how selected techno-functional
Hydrolysates properties (gelation, emulsification) of SPH were affected by the presence of a non-gelling polysaccharide. A
Galactomannans slight hydrolysis was beneficial to increase gel strength. Also, the emulsifying activity was improved for low DHs,
Protein-polysaccharide interactions
whereas hydrolysis was detrimental for emulsion stability. Under certain conditions the presence of the non-
Gelation
Emulsifying properties
gelling polysaccharide was beneficial to improve SPHs’ functional properties, but the effect was in general
complex and strongly dependent on both biopolymers’ concentration and molecular weight. Nevertheless, it was
demonstrated that by using SPH and galactomannan mixtures and controlling the biopolymers’ concentration
and molecular weight, improved functionalities can be obtained with useful applications in food formulation.

1. Introduction decrease in molecular mass, it is also expected an increase in both

Functionality of commercial soy proteins isolates is often compro-
mised due to their native high molecular weight and the high degree of
denaturation that usually accompanies these samples (Monteiro &
Lopes da Silva, 2018; Wagner, Sorgentini, & Añón, 2000). Controlled
enzymatic hydrolysis of soy proteins can improve not only the techno-
functionality of these proteins (Kuipers et al., 2005; Ortiz & Wagner,
2002; Tsumura et al., 2005; Luo et al., 2010) but also several of their
bioactive properties (Moure, Domínguez, & Parajó, 2006; Tsou, Lin,
Chao, & Chiang, 2012).
Depending on the hydrolysis mechanisms of proteases used, extent
of hydrolysis and environmental conditions, a wide diversity of pep-
tides can be obtained. With the increased use of peptides obtained by
partial hydrolysis of soy proteins in food formulations, there is also a
need to predict and understand what interactions these compounds may
establish with other food constituents, namely to avoid or minimize
possible negative effects, or to potentiate possible beneficial effects to
the consumer from a nutritional and / or functional point of view.
Partial enzymatic hydrolysis breaks peptide bonds that may cause
relevant changes in the secondary and tertiary protein structures
(Panyam & Kilara, 1996) accompanied by partial unfolding of the
protein and exposure of hydrophobic groups and other reactive amino
acids previously hidden within the interior of the molecule (Jung,
Murphy, & Johnson, 2005). Therefore, in addition to the obvious
availability of hydrophobic groups and number of ionizable groups
(Wouters, Rombouts, Fierens, Brijs, & Delcour, 2016), often leading to
an increase in interactions favoring aggregation (Foegeding & Davis,
2011).
Several proteases, including bromelain used in the present study,
were shown to induce aggregation of soy proteins (Fuke, Sekiguchi, &
Msuoka, 1985; Zhong, Wang, Xu, & Shoemaker, 2007), especially of the
glycinin fraction (Kuipers & Gruppen, 2008), with hydrophobic inter-
actions playing the main role in the aggregation process. If the ag-
gregation process occurs under controlled conditions and to a desirable
extent, this may be beneficial, namely to improve the gelling properties
of these proteins and/or to improve the viscoelastic properties of the
interfacial protein films, in case of their application in the stabilization
of dispersed systems. It is possible to hypothesize that these changes, in
addition to causing expected changes in protein functionality as men-
tioned above, may also play a relevant role in the interactions that these
proteins/peptides can establish with other food components, namely
polysaccharides. Currently this information is scarce although being of
great importance for the knowledge of the functional properties of the
real food systems where these hydrolysates can be potentially used. The
main objective of the present study is within this framework aiming to
contribute to the lack of knowledge in this area.
Protein-polysaccharide interactions have an important role on the
bulk and/or interfacial properties of multicomponent systems,

⁎
Corresponding author.
E-mail address: jals@ua.pt (J.A. Lopes-da-Silva).

https://doi.org/10.1016/j.foodchem.2019.05.039
Received 28 November 2018; Received in revised form 23 March 2019; Accepted 7 May 2019
Available online 08 May 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
J.A. Lopes-da-Silva and S.R. Monteiro
influencing texture and stability of complex food formulations (van de
Velde, de Hoog, Oosterveld, & Tromp, 2015). For biopolymer mixtures,
the entropy of mixing is usually less significant than the enthalpic drive
to segregation, namely for enough high molecular weights. In the ab-
sence of an electrostatic drive to association, as may be expected for a
mixture between a protein and a neutral polysaccharide, the enthalpic
interactions between unlike chains are less favourable than interactions
between polymer chains of the same type, typically leading to segre-
gation (Picullel, Bergfeldt, & Nilsson, 1995; Morris, 1998). Previous
studies have demonstrated the influence of the polysaccharide struc-
tural characteristics and molecular size on interactions developed with
proteins in bulk or at interfaces (De Jong & van de Velde, 2007;
Kontogiorgos, Tosh, & Wood, 2009), including soy proteins (Li et al.,
2009; Monteiro & Lopes da Silva, 2017), but much less is known re-
garding the effects of changes in the native protein structure.
Addition of protein hydrolysates is expected to influence the func-
tional properties of other proteins, as verified for the rheological
properties of doughs (Schmiele, Felisberto, Clerici, & Chang, 2017) and
for the functionality of gluten (Guo, Sun, Zhang, Wang, & Yan, 2018).
In the latter case, it was suggested that the interactions between soy
protein hydrolysates and wheat proteins occur through disulphide
bonds, causing disruption of glutenin polymerization and hampering
gluten network formation. Certain surface and non-surface active
polysaccharides (e.g., xanthan gum, HPMC) have shown to improve the
interfacial film properties of soy protein hydrolysates, although locust
bean gum showed little effect on surface pressure and viscoelasticity of
the interfacial soy protein films (Martínez, Sánchez, Ruiz-Henestrosa,
Patino, & Pilosof, 2007). Certain specific interactions between poly-
saccharide chains may also be affected by the presence of protein hy-
drolysates, due to the low molecular weight and charge of the poly-
peptides. For example, soy protein hydrolysates were shown to retard
starch retrogradation (Lian, Zhu, Wen, Li, & Zhao, 2013), although no
comparison was made with the effects of the native soy proteins.
In the present study, the influence of neutral polysaccharides on
selected techno-functional properties exhibited by soy hydrolysates was
studied, namely those related with solvent interactions (solubility),
solvent and protein–protein interactions in the bulk (gelation) or at
interfaces (emulsifying activity).
2. Materials & methods
2.1. Samples
Commercial soy protein isolate (SPI, SAMPROSOY 90 EG ®, 91%
protein, 1.1% fat, 3.7% ash), was kindly provided by Solae Bunge
(Brazil) and was used without further purification. Commercial locust
bean gum (LBG, HG M200, 7.1% protein, 0.8% fat, 0.9% ash) was
provided by Danisco Portugal - Industrias de Alfarroba (Faro, Portugal).
Purification of the commercial LBG sample and preparation of LBG
samples with different molecular weight, by controlled enzymatic hy-
drolysis, were performed as previously described (Monteiro & Lopes da
Silva, 2017). Briefly, the commercial LBG sample was solubilized in
water and the galactomannan recovered by precipitation in 80%
ethanol. Galactomannan samples with different molecular weights were
prepared by controlled enzymatic hydrolysis using an endo-β-manna-
nase from Aspergillus niger (Megazyme International Ireland, Wicklow,
Ireland) (molecular characteristics of these galactomannan samples are
shown as supplementary material, Table S1).
2.2. Preparation of soy protein hydrolysates
Partial hydrolysis of the commercial SPI was carried out using
bromelain (EC 3.4.22.4), an endopeptidase obtained from pineapple
stems (5–15 units/mg protein, ref. B5144, Sigma-Aldrich, St. Louis,
MO). The lyophilized enzyme was dissolved in 0.01 mol/L phosphate
buffer (pH 8) at 0.8 mg/mL. The commercial SPI was also dispersed in
217
Food Chemistry 294 (2019) 216–223
the same buffer (35 mg/mL) and mixed with the enzyme solution in
order to have an isolate:enzyme ratio of 4:1 (Ortiz & Wagner, 2002).
The hydrolysis was performed under stirring at 40 °C for 5, 15, 30, 60,
120 and 180 min. At each time the reaction was stopped by heating at
90 °C for 5 min and the sample neutralized to pH 7.0 with HCl 0.1 mol/
L. Each sample (soy protein hydrolysate (SPH)) was then deep frozen in
liquid nitrogen, lyophilized and stored at 4 °C for subsequent analysis.
An enzyme-free control sample was prepared under the same conditions
(SPH0).
2.3. Preparation of solutions
SPH dispersions were prepared in pure water (Milli-Q, Millipore
Corp., Bedford, MA) by stirring gently at 4 °C overnight (pH adjusted to
7.0 if needed). LBG samples were first dispersed at room temperature
for 1 h, followed by heating at 90 °C for 30 min under magnetic stirring,
and then centrifuged (24,400 g, 30 min, 20 °C) after cooling. All mix-
tures and dilutions for subsequent tests were prepared on a weight basis
from stock solutions of each biopolymer, 24 wt% and 1.2 wt% for SPH
and LBG solutions, respectively, adding water as necessary. Mixtures of
SPH and LBG solutions were prepared at room temperature, under
gentle stirring for 60 min. Sodium azide 0.02% (w/w) was used as a
preservative. All solutions were degassed under vacuum during 1 h
before testing.
2.4. Characterization of protein and polysaccharide samples
The degree of hydrolysis (DH, %) of soy protein hydrolysates (SPH),
the percentage of the total number of peptide bonds cleaved during
hydrolysis, was determined based on the quantification of the free
amino groups using the trinitrobenzene sulfonic acid (TNBS) assay as
described elsewhere (Spellman, McEvoy, O’Cuinn, & FitzGerald, 2003).
Absorbances were measured at 340 nm using a Jenway 6405 UV/Vis
spectrophotometer (Jenway Limited, Essex, England).
Protein solubility in water was determined by dispersing the sample
in pure water (Milli-Q, Millipore Corp., Bedford, MA) at 10 mg/mL,
under stirring at 20–22 °C for 1 h, and then centrifuging at 12,000 g for
15 min. Aliquots from the supernatant were taken for determination of
the contents of soluble protein according to the bicinchoninic acid assay
(Smith et al., 1985) using the BCA-1 kit for protein determination from
Sigma-Aldrich Co. (Sigma-Aldrich, St. Louis, MO). Protein solubility
was expressed as the ratio of soluble to total protein. Total protein
content of each SPH was determined by measuring the nitrogen content
by combustion analysis (LECO CHNS-932 Elementary Chemical Ana-
lyzer, LECO Corporation Saint Joseph, MI) and a nitrogen conversion
factor of 6.25.
Turbidity measurements were made at room temperature by mea-
suring the absorbance of the SPI and SPH dispersions, prepared in pure
water at 5 mg/mL (stirring for 1 h), at 600 nm, using the Jenway 6405
UV/Vis spectrophotometer.
Mannose-to-galactose (M/G) ratios, intrinsic viscosities and the re-
lative average molecular weight for the galactomannan samples were
determined as described elsewhere (Monteiro, Rebelo, da Cruz e Silva,
& Lopes da Silva, 2013; Tavares, Monteiro, Moreno, & Lopes da Silva,
2005). Briefly, M/G ratio was calculated with basis on the relative
amounts of mannose and galactose determined by gas–liquid chroma-
tography after hydrolysis with sulphuric acid, and derivatisation to
alditol acetates. The relative viscosities of galactomannan aqueous so-
lutions were determined by capillary viscosimetry at 25.0 ± 0.1 °C, the
intrinsic viscosities, [η], were calculated by extrapolating to infinite
dilution, using the combined Huggin’s and Kraemer’s equations, and the
relative average molecular weight was determined by gel permeation
chromatography.
J.A. Lopes-da-Silva and S.R. Monteiro Food Chemistry 294 (2019) 216–223

2.5. Gelation assessed by rheological measurements Table 1

Variation of the degree of hydrolysis (DH), solubility (S) and dispersion tur-
Gelation behavior was analyzed by oscillatory rheological tests, bidity (T) for the soy protein samples degraded to different extents with bro-
under small deformation amplitude, using a controlled-stress rheometer melain (pH 8, 40 °C). SPHx – Non-hydrolysed commercial soy protein isolate
(SPH0, substrate) and soy protein hydrolysates obtained after different reaction
(AR-1000, TA Instruments, New Castle, DE), fitted with a roughish
times.a
plate-plate geometry (diameter 40 mm, gap 1 mm). After transferring
the sample to the rheometer plate, the exposed sample surface was Sample Reaction time (min) DH (%) S (%) T (A600nm)
covered with a thin layer of mineral oil to avoid evaporation during the
SPH0 0 0 36.4 ± 2.9 A 0.93 ± 0.06 A
measurements. The dynamic tests were performed at an angular fre- SPH1 5 4.6 ± 0.9 A 72.7 ± 1.8B 0.78 ± 0.03B
quency of 2 rad/s and 0.3% strain, within the linear viscoelastic region, SPH2 15 8.7 ± 0.4B 80.2 ± 2.1C 0.64 ± 0.02C
and comprised temperature sweeps from 40 °C to 95 °C at 1 °C/min, and SPH3 30 11.3 ± 0.6C 85.1 ± 1.5 D 0.55 ± 0.03 D
cooling back to 20 °C after keeping the sample at 95 °C for 10 min. At SPH4 60 13.4 ± 0.3 D 78.4 ± 2.0C 0.65 ± 0.02C
SPH5 120 15.2 ± 0.5E 71.2 ± 1.4B 0.73 ± 0.04B
the end of selected tests and after a short equilibration period (30 min,
SPH6 180 16.1 ± 0.4E 62.7 ± 1.9E 0.80 ± 0.03B
20 °C), a frequency sweep was performed at 20 °C to analyze the gel
viscoelastic profile. Soy protein and hydrolysates were studied at 12 wt a
Mean ± (standard deviation) for triplicate measurements; different ca-
%, alone or in mixtures with the galactomannans at 0.3 wt%. All data pital letters along each column denote for significant differences (p < 0.05).
reported are the means of at least three replicate tests (three or more
different samples). (Jung et al., 2005; Lamsal, Jung, & Johnson, 2007). Quantitative dif-
ferences regarding the hydrolysis rate and the achieved degree of hy-
2.6. Emulsifying properties drolysis by comparing these results with previous studies (Ortiz &
Añón, 2000; Ortiz & Wagner, 2002) are not surprising, considering the
The emulsions were prepared by mixing 6 mL of 0.2 wt% SPH dis- expected influence of the proteins’ molecular state, namely the degree
persion (in the presence or not of 0.005 wt% galactomannan) in of denaturation and how the secondary and tertiary structure of these
0.05 mol/L Tris-HCl buffer (pH 7.5) with 2 mL of soybean oil, and then proteins were affected by the treatments that they previously under-
homogenized in a T25 Ultra-turrax homogenizer (IKA-Werke, gone during production. The commercial SPI under study revealed
Germany) for 1 min at aprox. 17,000 rpm. Emulsion aliquots (50 μL) absence of endothermic peaks for both 7S and 11S soy protein fractions
were taken immediately after preparation (t = 0) and after 10 min by DSC essays (results not shown), allowing to assume that the initial
(t = 10 min) and diluted 1:100 (v/v) in 0.1 wt% sodium dodecyl sulfate soy proteins were essentially denaturated. Higher extent of protein
(SDS) solution. The absorbance of diluted emulsions was read at denaturation was shown to promote access of bromelain to the soy
500 nm using a Jenway 6405 UV/Vis spectrophotometer (Jenway protein cleavage sites leading to improved hydrolysis (Lamsal et al.,
Limited, Essex, England). The emulsifying activity index (EAI) and the 2007).
emulsion stability index (ESI) were calculated accordingly to the fol- Protein functional properties, like gelling and emulsifying studied in
lowing equations (Wang et al., 2008): this work, are strongly dependent on protein solubility. Commercial soy
protein isolates typically exhibit low protein solubility in water and
m2 2 × 2.303 × A0 × DF
EAI ⎛⎜ ⎟⎞ = ≈ 41 × A0 high dispersions’ turbidity due to the high degree of denaturation re-
⎝ g ⎠ c × ∅ × (1 − θ) × 10000 (1) sulting from the thermal treatments and contact with organic solvents
A0 during the production procedures (Monteiro & Lopes da Silva, 2018;
ESI (min) = × 10 Wagner et al., 2000). Protein solubility in water and dispersions’ tur-
A0 − A10 (2)
bidity are also shown in Table 1. Solubility increased steeply with the
where A0 and A10 are the absorbances (500 nm) of the diluted emul- hydrolysis time, up to 30 min, reaching 85% for SPH3, while the solu-
sions at 0 and 10 min, DF is the dilution factor (1 0 0), c is the initial bility of the non-hydrolysed isolate (SPH0) was only 36%. This initial
protein concentration (0.15 g/100 mL), ϕ is the optical path (0.01 m) increase in solubility is attributed to the decrease of molecular weight
and θ is the fraction of oil used to prepare the emulsion (0.25). of the peptide chains and/or to the presence of more unfolded protein
structures and increased hydrophilic interactions with water, resulting
2.7. Statistical analysis from the partial enzymatic hydrolysis. However, there was a sub-
sequent decrease in solubility for longer hydrolysis times, which was
Data are presented as the mean and standard deviation of triplicated also followed by an increase in dispersion turbidity. Within this time
measurements, unless indicated otherwise. The data obtained were range, from 30 to 180 min of hydrolysis, the DH only increased slightly
statistically treated by variance analysis, while the means were com- (from 11 to 16%).
pared using t-tests, and values reported as significantly different at Turbidity decreased with the hydrolysis time up to 30 min, reaching
p < 0.05 (Statistica software v. 6.0, StatSoft Inc.). 0.55 for SPH3, and then increased reaching a value of 0.80 for SPH3.
Therefore, the initial increase in solubility due to the increase in DH is
3. Results and discussion close related to a decrease in protein aggregation, thus causing a pro-
nounced decrease in dispersion’s turbidity (See Fig. S1 as
3.1. Degree of hydrolysis and solubility Supplementary material, for a graphical representation of the changes in
protein solubility and dispersion’s turbidity with DH).
The degree of hydrolysis (DH) obtained for the soy protein samples The decrease in solubility for DH > 12% is likely associated to the
treated with bromelain (pH 8, 40 °C), for different reaction times, is increasing ability to undergo aggregation, despite the reducing in mo-
shown in Table 1. The observed degrees of hydrolysis were in the range lecular weight, essentially mediated by hydrophobic interactions, due
of 5–16% within the hydrolysis time range analysed (180 min). Al- to the expected higher exposure of hydrophobic groups that were in-
though DH increased with the elapse of the enzyme reaction, as ex- itially buried inside the globular protein structure (Panyam & Kilara,
pected, the increase was more pronounced within the first 30 min of the 1996; Jung et al., 2005). Aggregation may occur during the hydrolysis
reaction (DH 0–11%) and then slowed down reaching DH ∼ 16% after reaction (Inouye, Nakano, Asaoka, & Yasukawa, 2009; Kuipers, Alting,
3 hr of hydrolysis. & Gruppen, 2007) or even during lyophilization and storage of the
Similar hydrolysis profiles were observed for soy proteins treated samples (Lv, Guo, & Yang, 2009).
with this and other proteases, under similar experimental conditions

218
J.A. Lopes-da-Silva and S.R. Monteiro
Fig. 1. Changes in viscoelastic moduli as a function of temperature for SPH
aqueous dispersions (12 wt%, pH 7.0), during the initial heating step (1 °C/min,
ω = 2 rad/s, γ = 0.3%). (■,□) SPH0; (●,○) SPH1; (♦,◊) SPH3; (▲,△) SPH5.
Filled symbols denote for the storage modulus (G') and open symbols for the
loss modulus (G'').
(Additional information on the microstructure of lyophilized SPH as
observed by SEM is provided as Supplementary material, § S.3.)
3.2. Gelation behavior in the presence of galactomannans
Small amplitude oscillatory shear tests were used to evaluate non-
destructively the soy protein thermal-induced gelation process. All
tested samples (SPHs alone or in the presence of the polysaccharide)
could form gels under the analysed experimental conditions. Changes in
viscoelastic moduli (G', storage modulus; G'', loss modulus) during the
initial heating step are illustrated in Fig. 1 for selected SPH samples.
During the heating step, an abrupt increase in both viscoelastic moduli
can be seen for a certain temperature (Fig. 1) which corresponds to the
onset of gelation, with storage modulus (G') increasing faster and be-
coming higher than the loss modulus.
For the SPHs with no added polysaccharide, the gelation tempera-
ture, defined as the temperature at which G' equals G'', was significantly
lower (p < 0.05) for the SPH with the lowest degree of hydrolysis
(SPH1, Tgel = 64.8 °C), but then increased as the DH also increased
(Table 2), reaching higher values than the non-hydrolysed sample for
SPHs with DH ≥ 11%: Tgel was 67.5, 74.5, 75.0 and 71.0 °C for SPH3,
SPH4, SPH5 and SPH6, respectively. Worth to note that the hydrolysates
showed already lower initial moduli values, probably associated to less
protein aggregation and higher solubility of these samples, as pre-
viously discussed (Table 1).
Slight hydrolysis is probably sufficient to alter protein conformation
and to decrease initial protein aggregation, making the protein chains
more available to associate and gel formation induced by the increase
in temperature without the (slight) decrease in molecular mass being
negatively reflected in the gelation process. The effect of protein hy-
drolysis upon its gelation capability is expected to be a complex balance
between decreasing the molecular weight and increasing the available
hydrophobic groups for further intermolecular interactions. For higher
degrees of hydrolysis, the gelling capacity decreases, as expected, due
to the predominant effect of decreasing molecular mass combined with
increased charge repulsion between the protein/peptide chains due to
the likely net charge increase after hydrolysis (Lamsal et al., 2007;
Panyam & Kilara, 1996).
The curves obtained for the thermal-induced gelation of the
219
Food Chemistry 294 (2019) 216–223
mixtures with the polysaccharide were qualitatively similar to those
shown in Fig. 1. The main quantitative differences due to the poly-
saccharide presence are displayed in Table 2. Gelation of the SPHs
occurred at lower temperature in the presence of the galactomannan
(Table 2). The most pronounced decrease was observed for SPH0, with
Tgel decreasing from 64.8 °C to 48.4 °C due to the presence of the
polysaccharide. Segregative interactions between the protein and the
non-gelling polysaccharide may have occurred and contributed to a
higher local protein concentration resulting from the occurrence of
phase separation between both biopolymers (Monteiro et al., 2013),
thus leading to lower gelation temperatures and, as further discussed, to
higher gel strength. For SPH0, the presence of galactomannan caused a
relative decrease in gelation temperature of about 25%, while for SPH6
the relative decrease was only 2% (Table 2). The influence of the
polysaccharide on the onset of gelation is clearly dependent on the SP
degree of hydrolysis, being less pronounced as the DH increases.
Changes in both viscoelastic moduli (G', G'') during the heating and
cooling steps were qualitatively similar to previous reported results for
soy proteins (Monteiro & Lopes da Silva, 2017; Renkema & van Vliet,
2002). Fig. 2 shows how G' changed with temperature, during the
heating and cooling steps, for representative SPH and SPH + LBS
samples. After the cooling step, samples were allowed to rest in situ, on
the rheometer measuring system, for 30 min at 20.0 °C. Then the gels’
viscoelastic characteristics were assessed by frequency sweep tests.
Table 2 also shows the values of G', at an intermediate frequency of
ω = 0.5 rad/s, the slope (n) for the power relation between G’ and os-
cillatory frequency, G' ∼ ωn, the phase lag (tan δ = G''/G', ω = 0.5 rad/
s) as a measure of the relative liquid-like and solid-like character of the
gel, also obtained from the frequency sweep tests at 20 °C, and the re-
lative changes on these rheological parameters due to the presence of
the galactomannan.
Generally, an increase in gel strength is observed during and at the
end of the gelation process induced by the temperature increase, evi-
denced by higher values of G'. The effect of the presence of galacto-
mannan is more pronounced for intermediate DH values (5–13%,
Table 2). For the highest DH values, the lower molecular weight of the
SPH, associated with higher intermolecular repulsion between the
peptide chains, will be responsible for a significant decrease of the
phase separation between the peptide and the polysaccharide, the main
factor responsible for the positive effect of the presence of the poly-
saccharide on the gelation of soy proteins and their hydrolysates, re-
sulting in weaker gels.
After cooling back to 20 °C and resting at this temperature for
30 min, the SPH gels (See Fig. S3 as Supplementary material, for ex-
amples of the mechanical spectra obtained for SPH-LBG gels) showed the
typically viscoelastic profile of soy protein gels (Monteiro et al., 2013;
Renkema & van Vliet, 2002), with G' higher than G'' over the whole
frequency range, low dependence of G' on angular frequency
(G’ ∼ ω0.1). Contrarily to what was previous observed (Lamsal et al.,
2007), for DH 2–4%, here we observed higher G’ values for the gels
obtained from the SPHs with 5 and 9% DH (Table 2), in agreement with
other studies (Fuke et al., 1985). The general viscoelastic profile is re-
tained by addition of the galactomannan, with a general increase of
both moduli and a tendency for a slight higher dependence of G' on
frequency (slope (n), G' ∼ ωn,). Significant increase (p < 0.05) in the
viscous character of the systems due to the presence of the non-gelling
high-molecular weight polysaccharide was observed for the mixtures
with SPH1 and SPH2 (higher tan δ values, Table 2). Those mixtures that
showed the higher storage modulus were also characterized by a more
liquid-like character, suggesting that the presence of the non-gelling
polysaccharide besides promoting more extensive interactions between
the protein chains, probably due to partial phase separation, also causes
increasing molecular mobility within the non-gelled phase, not parti-
cipating directly in the elastic gel network.
J.A. Lopes-da-Silva and S.R. Monteiro Food Chemistry 294 (2019) 216–223

Table 2
Rheological parameters characterizing the gelation process of SPH (12 wt%) alone or in the presence of the galactomannan (0.3 wt%).a
Sampleb Gelation temperature (Tgel, min)c Average decrease in Tgeld G' (Pa)e (0.5 rad/s) increase in G'f Slopeg G' ∼ ω tan δh (0.5 rad/s) Average increase in tan δi

SPH0 64.8A 627A 0.096A 0.150A

+ LBG-1 48.4F 25% 738F 18% 0.102 0.156A 4%
SPH1 60.2B 1012B 0.095A 0.151A
+ LBG-1 51.4G 15% 2054G 103% 0.099 0.203E 34%
SPH2 63.2A 885C 0.095A 0.142B
+ LBG-1 51.8G 18% 1854H 110% 0.113 0.182C 28%
SPH3 67.5C 565A 0.098A 0.166C,D
+ LBG-1 59.8H 11% 1712H 203% 0.111 0.177C 7%
SPH4 74.5D 242E 0.103A 0.173C
+ LBG-1 65.2I 12% 1005I 415% 0.108 0.175C 1%
SPH5 75.0D 266E 0.100A 0.161D
+ LBG-1 68.6 J 9% 702F 264% 0.106 0.164D 2%
SPH6 71.0E 194E 0.098A 0.152A
+ LBG-1 69.3 J 2% 564A 191% 0.104 0.161D 6%

a
Mean ± (standard deviation) for triplicate measurements; different capital letters along each column denote for significant differences (p < 0.05).
b
SPHx – Non-hydrolyzed commercial soy protein isolate (SPH0, substrate) and soy protein hydrolysates obtained after different reaction times; LBG – locust bean
gum sample.
c
Gelation temperature defined as the temperature at which G' equals G''.
d
Relative decrease in gelation temperature due to the presence of the galactomannan.
e
Storage modulus (G', ω = 0.5 rad/s) obtained from the frequency sweep tests at 20 °C.
f
Relative increase due to the presence of the galactomannan.
g
Slope (n) for the power relation between G’ and oscillatory frequency, G' ∼ ωn, obtained from the frequency sweep tests at 20 °C.
h
Phase lag (tan δ = G''/G', ω = 0.5 rad/s) obtained from the frequency sweep tests at 20 °C.
i
Relative increase in tan δ due to the presence of the galactomannan.

Fig. 2. Storage modulus (G'/Pa) as a function of temperature during temperature sweep experiments (heating and cooling at ω = 2 rad/s, 0.3% strain, 1 °C/min) for
SPH (12 wt% protein, filled symbols) and SPH-LBG-1 dispersions (in the presence of 0.3 wt% galactomannan, open symbols). Only representative curves are shown
for (A) SPH0, (B) SPH2, and (C) SPH4. Arrows indicate the direction of the temperature variation.

3.2.1. Additional insights on concentration and polysaccharide molecular
weight effects
Gelation of selected SPHs was evaluated in the presence of different
concentrations of galactomannans and in the presence of galacto-
mannans with different molecular weights.
We have previously demonstrated that incompatibility between soy
proteins and galactomannans originates phase-separated networks
(Monteiro et al., 2013; Monteiro & Lopes da Silva, 2017), the extent of
the phase separation process and the magnitude of the resulting effects
being strongly dependent on concentration of both biopolymers and on
the polysaccharide molecular weight. Despite the positive effect of the
presence of galactomannan on the gelation of SPHs, previously ob-
served and discussed (§ 3.2), it is expected that above a certain con-
centration and/or molecular mass of the polysaccharide, the gelation
will be significantly hampered. This hypothesis was in fact confirmed,
with the negative effect of the presence of polysaccharide being de-
pendent on the stiffness of the SPH gel, i.e. on the SPH concentration.
Regarding the effect of the polysaccharide molecular weight, Fig. 3
shows examples of the storage modulus (G') and loss angle (tan δ)
obtained for gels after the 30 min resting at 20 °C. For 12 wt% protein,
addition of 0.3 wt% galactomannan with decreasing molecular weight
leads to a less pronounced effect on the gel stiffness (Fig. 3A and B). No
significant differences were observed in gel stiffness (G') or viscous
character (tan δ) between SPH alone or in the presence of the ga-
lactomannan with the lowest molecular weight (LBG-3), regardless of
the DH of the SPH. However, a very different behaviour was observed
by lowering the protein concentration and simultaneously slightly in-
creasing the galactomannan concentration (Fig. 3C and D). For a lower
protein concentration than that previously discussed (§ 3.2), the SPHs
still showed gelling capability under the studied conditions but ex-
hibited weaker gel structures. The most pronounced increase in gel
stiffness was observed by addition of the galactomannan with lower
molecular weight. Addition of galactomannan with higher molecular
weight caused a significant decrease in gel stiffness and a clear increase
in its viscous character, especially for SPHs with higher degree of hy-
drolysis (illustrated in Fig. 3 (C, D) for the SPH5 sample). We suggest
that increasing the molecular weight of the polysaccharide causes a
higher degree of de-mixing and more extensive phase separation

220
J.A. Lopes-da-Silva and S.R. Monteiro
Fig. 3. Storage modulus (G'/Pa) and loss angle (tan δ), measured at 0.5 rad/s (from frequency sweeps at 20 °C, 0.3% strain). (A) 12 wt% SPH1 + 0.3 wt% LBG; (B)
12 wt% SPH5 + 0.3 wt% LBG; (C) 10 wt% SPH1 + 0.4 wt% LBG; (D) 10 wt% SPH5 + 0.4 wt% LBG.
between the biopolymers, such that gelation is significantly compro-
mised, especially for more fragile SPH networks.
The effect of polysaccharide concentration was also studied (See
Fig. S4 as Supplementary material), for two different molecular weights
of both biopolymers. For a 10 wt% protein, the relative changes on G'
(measured at 0.5 rad/s for gels after 30 min at 20 °C) due to the pre-
sence of the galactomannan at different concentrations clearly show a
maximum beyond which gelation is hampered. This critical con-
centration increases as the SPH molecular weight increases. A weaker
SPH gel can accommodate a lower amount of non-gelling poly-
saccharide before gel stiffness being negatively affected. For the same
SPH network, increasing the galactomannan molecular weight caused
this critical concentration to decrease.
3.3. Emulsifying properties
Emulsifying activity index (EAI) and emulsion stability index (ESI),
for the native soy protein isolate and hydrolysates, alone or in the
presence of galactomannans with two different molecular weights, are
shown in Fig. 4.
For the SPH alone, the emulsifying activity was significantly higher
for the SPH with DH 5–9%, compared with the control sample, but for
221
Food Chemistry 294 (2019) 216–223
higher DH the EAI decreased for similar values as the non-hydrolyzed
sample. The emulsifying activity of a protein is expected to depend on a
complex set of factors, including the protein’s amphiphilic properties
and interfacial activity, protein solubility and mobility, i.e. the ability
of the individualized protein molecules to move quickly to the inter-
face. For our samples, the increase in EAI for the low DHs is likely
related to the observed increasing in protein solubility and unfolding of
the protein chains. An increase in protein surface hydrophobicity was
related to an increased emulsifying activity index (Qi, Hettiarachchy, &
Kalapathy, 1997), but other studies have not shown any direct relation
between the two parameters, suggesting that the decrease of the EAI
with the hydrolysis degree of the soy protein hydrolysates was related
to a decrease in protein solubility (Rickert, Johnson, & Murphy, 2004).
‘Excessive’ enzymatic hydrolysis (DH > 11%) caused a further de-
crease in the SPHs’ emulsifying capability, what might be attributed not
only to the decrease of size of the protein chains but also to a significant
decrease in surface hydrophobicity (Chen, Chen, Ren, & Zhao, 2011;
Jung et al., 2005).
The presence of the polysaccharide was effective for improving
emulsifying properties of soy protein hydrolysates. The galactomannan
significantly increases (p < 0.05) both EAI and ESI, an effect that was
more pronounced as the molecular weight of the polysaccharide
J.A. Lopes-da-Silva and S.R. Monteiro Food Chemistry 294 (2019) 216–223

Fig. 4. (A) Emulsifying activity index (EAI) and (B) emulsifying stability index (ESI) for soy protein samples alone (■) or in the presence of galactomannans with two
different molecular weights: (■) LBG-3 and (□) LBG-1 (0.2 wt% SPH, 0.005 wt% galactomannan, in the aqueous phase). Samples SPH0 to SPH6 denote for soy
protein hydrolysates obtained after 0, 5, 15, 30, 60, 120 and 180 min of hydrolysis with bromelain, respectively. Results shown are mean values and standard
deviations of triplicates (vertical bars). Different letters denote for significant differences (p < 0.05).

increased. mixture of peptides with a large dispersion of molecular mass, i.e, they
The stability of an emulsion will be more dependent on the ability of contain low molecular weight peptides as well as higher molecular
the protein to adsorb at the oil/water interface and form a stable vis- weight peptides and unhydrolyzed proteins, thus making their inter-
coelastic film, thus reducing the interfacial tension and stabilizing the facial effects more complex and probably strongly dependent on the
oil droplets against coalescence. Therefore, it will be strongly depen- hydrolysis mechanisms.
dent on the molecular mass of the protein, in addition to its hydro-
philic/hydrophobic characteristics. In general, increasing the soy pro-
4. Conclusions
tein DH decreased the emulsion stability. Similar behavior was
observed in the presence of the polysaccharide, although the stability
Soy protein hydrolysates obtained with bromelain showed gelling
index values were higher. The presence of the polysaccharide can
ability and good emulsion activity, but in general produced emulsions
compensate, in part, the negative effect of the protein hydrolysis upon
with lower stability than the nonhydrolyzed sample. Upon hydrolysis,
the emulsion stability. Worth to note that the SPHs are expected to be a
protein solubility increased initially and then decreased at high DH,

222
J.A. Lopes-da-Silva and S.R. Monteiro
showing a maximum at DH 11%. SPH with high DH were still able to
form gels, but clearly showed lower gel strengths due to the reduction
in the size of the protein molecules. The effect of the polysaccharide,
both its molecular weight and concentration, was more pronounced
upon the weaker three-dimensional soy protein networks obtained from
SPH with higher DHs. Nevertheless, both positive and negative effects
on gel strength and viscous character were found, depending on bio-
polymers’ molecular weight and concentration. Short hydrolysis times
(5–15 min) provided samples with the best emulsifying activity, both
for the soy protein hydrolysates alone or in the presence of the poly-
saccharide. Decreasing the protein or the polysaccharide molecular
weight was detrimental for the emulsifying stability. Techno-functional
properties of soy protein hydrolysates in the presence of galacto-
mannans is dependent on a complex set of factors, likely related to
phase-separation processes, namely concentration and molecular
weight of both biopolymers. The studied functional properties of soy-
bean proteins could be tailored by the concomitant partial enzymatic
hydrolysis and DH control plus the addition of neutral polysaccharides.
Declaration of interest statement
The authors declare no conflict of interest.
Acknowledgments
Thanks are due to FCT/MEC for the financial support to the QOPNA
research Unit (FCT UID/QUI/00062/2013), through national founds
and where applicable co-financed by the FEDER, within the PT2020
Partnership Agreement. Sónia Monteiro also thanks FCT for a PhD grant
(SFRH/BD/24335/2005). Authors also thank Solae Bunge (Brazil) for
kindly providing the soy protein isolate.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.05.039.
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