Food Research International 35 (2002) 511–518

www.elsevier.com/locate/foodres

Hydrolysates of native and modified soy protein isolates:

structural characteristics, solubility and foaming properties
Sara E. Molina Ortiza,b, Jorge R. Wagnera,c,*
a
Centro de Investigación y Desarrollo en Criotecnologı´a de Alimentos (CIDCA). Facultad de Cs. Exactas,
UNLP. 47 y 116, La Plata (1900), Argentina
b
Comision de Investigaciones Cientı´ficas de la Provincia de Buenos Aires (CIC), La Plata, Argentina
c
Consejo Nacional de Investigaciones Cientı´ficas y Técnicas (CONICET), Buenos Aires, Capital Federal, Argentina

Received 24 May 2001; accepted 6 September 2001

Abstract
The effect of enzymatic hydrolysis on structure characteristics (surface aromatic hydrophobicity and molecular size) of both
native and modified soy protein isolates was studied. Effects on thermal behavior and functional properties (solubility and foam
formation and stabilization at pH 4.5) were also analysed. Hydrolysates were obtained by bromelain digestion at pH 8 of native (N)
and of thermally treated isolates at pH 7 (T7) and at pH 1.6 (T1.6). The differential effect of bromelain on the three isolates pro-
duced partially hydrolyzed structures, which exhibited an enhancement of their protein solubility (STCA and SpH 4.5). Bromelain
digestion was more effective on isolate T7 resulting hydrolysates with improved capacity to form foams at pH 4.5. The different
functional behavior at pH 4.5 of hydrolysates was explained through the changes in thermal behavior, surface aromatic hydro-
phobicity and molecular mass distribution. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Isolate soybean protein; Hydrolysates; Functional properties; Protein structure

1. Introduction solubility of soybean proteins, especially at high con-

Native soybean isolates are mainly composed of
native 7S and 11S globulins and show high solubility
(> 90%) at alkaline conditions (Thanh & Shibasaki,
1976). As already reported, the solubility of native soy
isolates at pH < 7 decrease to a minimum close to the
isoelectric point (pI=4.5; Pearson, 1983). The low
solubility of soy proteins in acidic media complicates
their utilization in foodstuffs of moderate acidity (citric
beverages, dressings, etc.), specially when the required
functional properties depend on solubility, e.g. foaming
and emulsifying properties (Walstra, 1989, Chap. 1).
Though thermal denaturation of soybean proteins
improves the aforementioned properties and water
absorption capacity, it does not beneficially affect the
solubility. Also, thermal denaturation reduces water
* Corresponding author. Tel.: +54-221-4254-853; fax: +54-221-
4254-853.
E-mail address: jrwagner@nahuel.boil.unlp.edu.ar (J.R. Wagner).
0963-9969/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0963-9969(01)00149-1
centrations (Sorgentini, Wagner, & Añón, 1995;
Wagner, Sorgentini, & Añón, 2000). Acidic treatment
(pH 2–3) in combination with a thermal treatment is
useful to increase the solubility through partial deami-
dation and mild hydrolysis of proteins (Matsudomi,
Sasaki, Kato, & Kobayashi, 1985). However, a sig-
nificant increase in protein solubility is only achieved at
considerably low pH and high temperatures at long
incubation times. An useful alternative to increase the
protein solubility of both native and modified soybean
isolates is through induced hydrolysis with proteases
(Adler-Nissen, 1976). Some of the enzymes employed in
the production of hydrolysates for food use were from
plant or microbial origin, as bromelain, papain or ficin
(Lahl & Braun, 1994).
The aim of this work was to study changes in protein
solubility at pH 4.5, surface aromatic hydrophobicity,
and foaming properties of three soy protein isolates
(native and denatured) subjected to bromelain hydro-
lysis. Both denaturation and hydrolysis degrees were
studied.
512 S.E. Molina Ortiz, J.R. Wagner / Food Research International 35 (2002) 511–518
2. Materials and methods
2.1. Materials
Bromelain (E.C.3.4.22.4) and bovine serum albumin
(Alb) were purchased from Sigma (Sigma Chemical, St
Louis, MO, USA). Ammonium salt of 1-anilino-8-
naphtalene-sulphonic acid (ANS) was purchased from
Aldrich Chemical Company (Milwaukee, USA).
2.2. Isolates preparation
Protein isolates were prepared from defatted soybean
flour (Santista Alimentos S.A., São Paulo, Brazil). Pro-
teins were extracted by stirring the flour in alkaline
water (1:10 w/v flour:water ratio) at 25
C and adjusting
the pH to 8 with 2 N NaOH. After 2 h of extraction,
samples were centrifuged at 10 000 g for 30 min at 4
C.
The pellet was discarded and the supernatant adjusted
to pH 4.5 with 2 N HCl. In this condition, an isoelectric
precipitate was obtained and was separated by cen-
trifugation at 5000 g for 5 min at 4
C. The isoelectric
precipitate was resuspended in water (5% dry matter
w/w). From this dispersion, native and modified isolates
were prepared as follows: N (native), pH was adjusted
to 8.0 with 1 N NaOH; T7 (thermally treated at pH 7),
pH was adjusted to pH 7.0 and then heated at 90
C
(water bath) for 30 min; T1.6 (thermally treated at pH
1.6), pH was adjusted to 1.6 with 1 M HCl and then
heated as for isolate T7 (Matsudomi et al., 1985). After
the treatments, all dispersions were freeze-dried. Isolate
N is composed of 7S and 11S proteins in a non-dena-
tured state (Sorgentini & Wagner, 1999). Total denat-
uration of 7S and 11S proteins in isolates T7 and T1.6
was demonstrated by DSC assays (thermograms with-
out endothermic peaks, total denaturation enthalpy of
 0 J g1).
2.3. Preparation of protein hydrolysates
Soy protein isolates (N, T7, T1.6) were dispersed in
0.01 M phosphate buffer pH 8.0 (30 mg ml1) and
mixed with bromelain (0.434 mg ml1; ratio iso-
Table 1
Values of protein solubility in water of isolates N, T7 and T1.6 at pH
4.5 and in 12.5% trichloroacetic acid
Isolate Solubility (%)a
pH 4.5 TCA 12.5%
N 8.2 0.6a 0.55 0.08a
T7 4.4 0.2b 0.62 0.10a
T1.6 10.3 2.0a 0.81 0.07b
a
For each column, different letters indicate significantly (P<0.05)
different values.
late:bromelain 4:1) in a stirred bath at 40
C for 0, 2, 6
and 12 h. At each time, the reaction mixture was divided
in two aliquots: one aliquot was stopped by the addition
of 18.8% trichloroacetic acid solution (volume ratio
TCA solution:aliquot 1:0.5, final concentration of TCA
was 12.5%) and the other was stopped with the addition
of 1 M HCl until pH 2.5. From the latter, one aliquot
was adjusted to pH 4.5 to determine protein solubility
and the rest was lyophilized. Samples at t=0 were con-
sidered as controls (Nc, T7c and T1.6c).
2.4. Determination of TCA-protein solubility
Aliquots corresponding to the reactions stopped by
addition of TCA were centrifuged at 4000 g for 5 min.
Soluble peptides were determined in the supernatants
according to Lowry (Lowry, Rosembroug, Lewis, &
Randall, 1951) and TCA-solubility (STCA) was calculated
as percent of total protein. The increment in solubility
(%
STCA) was calculated with respect to the corre-
sponding control at t=0 using the following equation:
%
STCA ¼ 100½ðSTCA Þt  ðSTCA Þ0 =ðSTCA Þ0
where t is the hydrolysis time.
2.5. Determination of protein solubility at pH 4.5
Aliquots corresponding to the reactions stopped at
pH 2.5 were adjusted to pH 4.5 with 1 M NaOH and
centrifuged 4000 g for 5 min. Soluble peptides were
determined in the supernatants according to Lowry
(Lowry et al., 1951). Protein solubility at pH 4.5 (SpH4,5)
was calculated as percent of total protein. The incre-
ment in protein solubility (%
STCA) was calculated
using a similar equation as described in Section 2.4.
2.6. pH–protein solubility profile
Protein solubility as a function of pH (3.5–8.0) was
determined on controls (Nc, T7c and T1.6c) and on
hydrolysates obtained at 12 h (Nh, T7h and T1.6h).
Water dispersions (1% w/v) were gently stirred for 1 h
at room temperature. pH was adjusted to the desired
value with dilute solutions of HCl or NaOH. Disper-
sions were centrifuged at 10 000 g for 10 min at 4
C,
and protein content in supernatant was measured by the
Lowry method (Lowry et al., 1951). Protein solubility
was expressed as grams of soluble protein/100 g of
sample. All protein solubility determinations were con-
ducted in duplicate.
2.7. Electrophoresis
All electrophoretic runs were performed in gel mini-
slabs (Mini Protean II Model, Bio-Rad Laboratories,
 S.E. Molina Ortiz, J.R. Wagner / Food Research International 35 (2002) 511–518
Hercules, CA). Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) were performed by the
method of Laemmli (1970) as modified by Petruccelli
and Añón (1994). Samples were prepared by resus-
pending the TCA-precipitates of each isolate in sample
buffer. The soluble fraction obtained at pH 4.5 was
precipitated with TCA (volume ratio 18.8% TCA solu-
tion:aliquot 1:0.5, final concentration of TCA 12.5%)
and analysed by SDS-PAGE with b-mercaptoethanol.
2.8. Surface aromatic hydrophobicity (H0)
H0 was determined with the hydrophobicity fluores-
cence probe 1-anilino-8-naphtalene-sulphonate (ANS)
according to Hayakawa and Nakai (1985). Serial dilu-
tions in 0.01 M phosphate buffer were prepared with the
supernatants at pH 4.5. Dilutions were prepared at pH
4.5 to a final concentration of 0.01–1 mg ml1. Forty
microliters of ANS (8 mM) were added to 3 ml of each
dilution and the fluorescence intensity (FI) was mea-
sured at 365 nm (excitation) and 484 nm (emission)
using a Perkin-Elmer 2000 equipment (Perkin-Elmer,
Norwalk, CT, USA). The initial slope of the FI versus
protein concentration (mg ml1) plot (calculated by lin-
ear regression analysis) was used as an index of surface
aromatic hydrophobicity (H0).
2.9. Differential scanning calorimetry (DSC)
Twenty percent dispersions of lyophilized samples in
0.01 M phosphate buffer pH 7 were hermetically sealed
in aluminum pans. Samples were analysed at 10
C
min1 in a range of 20–120
C using DSC Polymer
Laboratories equipment (Rheometric Scientific, Weston
Road), using an empty double pan as a reference.
Transition temperatures and areas below the endother-
mic curves were measured to calculate the correspond-
ing thermal denaturation enthalpies (
H in Joules per
gram of dry weight). All runs were performed at least
twice.
2.10. Foaming properties
Assays were performed as described previously
(Wagner, Sorgentini, & Añón, 1996). N2 was sparged at
a flow rate of 1.70 ml s1 through 6 ml of 1.0 mg ml1
of sample (native and hydrolysates) in 0.1 M phosphate
buffer, pH 4.5. Bubbling was continued until a fixed
volume of foam (100 ml) was reached, or after a max-
imum elapsed bubbling time of 140 s. The initial rate of
liquid incorporation to the foam (v0, ml min1) and the
maximum volume of liquid incorporated to the foam
(Vmax, ml) were determined. The time for half-drainage
of the liquid that was incorporated to the foam at the
end of the bubbling period (t1/2, s) was also measured.
Determinations were performed in duplicate.
513
2.11. Statistical analysis
Data were analysed by analysis of variance (ANOVA)
and differences between means by the Fisher’s test
(Systat version 5.0). Significance was considered at
=0.05.
3. Results and discussion
3.1. Protein solubility and structural characteristics
Both protein solubility and foaming properties of soy
protein isolates and hydrolysates were studied at pH
4.5. This condition was selected because this is the pI of
the storage proteins in soybean seeds (Pearson, 1983)
and consequently, the point of lowest protein solubility.
Isolates N and T1.6 showed similar water protein
solubility at pH 4.5 (SpH4.5) but statistically (P < 0.05)
different to that of isolate T7 (Table 1). Isolate T7
showed the lowest SpH4.5 probably because of the
aggregation caused by the thermal treatment. Although
isolate T1.6 was also subjected to a thermal treatment,
the similar protein solubility to N may be a consequence
of deamidation and hydrolysis in acidic conditions
(Matsudomi et al., 1985; Wagner & Guéguen, 1995). On
the other hand, STCA was 10 times lower than SpH4.5.
STCA was similar in N and T7 and significantly different
in T1.6 (P < 0.05). Adler-Nissen (1976) previously
reported that only peptides of very low molecular
weight remain soluble in 12.5% TCA. Consequently,
STCA may be higher in isolate T1.6 due to the presence
of small peptides produced by acid hydrolysis.
Bromelain digestion gradually increased protein solu-
bility at pH 4.5 in the three isolates. After 12 h of
hydrolysis, protein solubility increased until reach
values between 20 and 26%. The increment in protein
solubility as a function of time of hydrolysis
(%
SpH4.5) is shown in Fig. 1. Bromelain was more
effective on isolate T7 (> 400%), in which 7S and 11S
proteins were completely denatured (
Hd=0 J g1).
Although isolate T1.6 was also composed of totally
denatured proteins, these proteins were less accessible to
the protease possibly due to the presence of partially
deamidated polypeptides (Matsudomi et al., 1985). The
increment in protein solubility of the native hydrolysate
showed intermediate values between T7 and T1.6.
Fig. 2 displays SDS-PAGE gels of TCA insoluble
proteins from control (Nc and T7c) and hydrolysates (2,
6 and 12 h). Nc profile showed typical bands belonging
to the 7S fraction (a, a0 and b subunits) and 11S fraction
(AB subunit, A and B polypeptides) (Fig. 2a). Besides,
high molecular weight aggregates were observed at the
beginning of the gel line. Hydrolysates of N (Nh) were
composed of polypeptides generated by partial or total
hydrolysis of the control sample. The enzymatic
514 S.E. Molina Ortiz, J.R. Wagner / Food Research International 35 (2002) 511–518
hydrolysis produced a decrease in the high-molecular-
mass aggregates, a loss of a0 - and a-7S subunits and the
disappearance of bands corresponding to high-mole-
cular-weight peptides (594 kDa). On the other hand,
the intensity of bands of 43 and 20–30 kDa increased
during hydrolysis (Fig. 2a). A small amount of bands
were observed for isolate T7c probably due to the pre-
sence of a high proportion of insoluble aggregates
(Fig. 2b). The only visible bands corresponded to the
most hydrophilic subunits of 7S (a0 and a) and 11S (A).
Hydrolysis of T7 (T7h) produced the total dis-
appearance of a0 - and a-7S, and A-11S polypeptide and,
as a consequence of protease action on insoluble aggre-
gates, originated aggregates of high molecular mass
visible at the top of the gel. Bands of very low molecular
mass peptides were visible at the bottom of the gel.
SDS-PAGE performed in the presence of b-mercapto-
ethanol with the soluble fraction at the pI (and pre-
cipitated in TCA) allows analysing protein species
responsible for the protein solubility at pH 4.5. Fig. 3
shows the profiles obtained for hydrolysates and con-
trols corresponding to N isolates. At short times, bands
corresponding to a- and a0 -7S subunits were absent,
those corresponding to A-11S showed low intensity and
two polypeptides of molecular size of 25 and 28 kDa
became visible. The intensity of the bands correspond-
ing to these species increased with time, together with
the apparition of bands of lower molecular weight (16–
18 kDa), the total disappearance of A-11S and the
reduced intensity of bands corresponding to b-7S and
B-11S. These results indicated that the effect of bro-
melain on N was mainly on the hydrophilic fractions
(a0 -, a-7S and A-11S) that are consequently more
exposed in the native structures of globulin 7S and 11S.
As already reported, the effect of bromelain on N could
be explained through a ‘‘zipper’’ mechanism (Molina
Ortiz & Añón, 2000). The soluble fraction of T7h and
T1.6h at pH 4.5 was also composed of 7S and 11S sub-
Fig. 1. Effect of time of enzymatic hydrolysis on the increment of
protein solubility in water at pH 4.5 (
SpH4.5) for soy isolates N, T7
and T1.6.
SpH4.5 was expressed as a percentage respect to the value
corresponding to non-hydrolyzed isolate (see Section 2). The values
are the means of at least two determinations.
units either partially or totally hydrolyzed (SDS-PAGE,
data not shown). Regarding the SDS-PAGE profiles,
the increment in SpH4.5 of Nh, T7h and T1.6h (Fig. 1),
could be ascribed to the liberation of peptides of low
molecular weight and the increase in charge associated
to the cleavage of peptide bonds.
DSC studies on both native and hydrolysates were
performed in order to correlate structural changes with
thermal stability. Thermograms of native soy isolate (N)
presented two endothermic peaks with transition tem-
peratures of 77.6 1.2. and 90.5 1.6
C (Sorgentini &
Wagner, 1999), corresponding to the denaturation of 7S
and 11S globulins, respectively (total denaturation
enthalpy:  17 J g1). Thermograms of the control of N
(Nc) showed only one endothermic transition
(78.0 0.3
C,
H=0.84 0.02 cal g1) corresponding
to 7S. This result is due to total denaturation of 11S
when enzymatic action was stopped at pH 2.5 (Wagner
et al., 1996). Thermograms corresponding to Nh also
show one peak with a transition temperature similar to
Nc, but with a lower denaturation enthalpy at all times
of hydrolysis (
H 0.76 0.05; 0.70 0.04; 0.51 0.06
cal g1 for 2, 6 and 12 h of hydrolysis). On the basis of
the results obtained from DSC and SDS-PAGE, we
could conclude that the structural conformation of the
partially hydrolyzed 7S in Nh would be mainly stabi-
lized by b subunits (Fig. 3). This is more evident at 12 h
of hydrolysis, when the decrease in the intensity of the
band corresponding to b-7S correlates well with the
lowest decrease in
H. Isolates T7 and T1.6, and their
corresponding hydrolysates, did not show endotherms
due to the total denaturation of 7S and 11S fraction by
the heat and/or pH treatments.
Fig. 2. Electrophoretic patterns (SDS-PAGE) of total proteins from soy
isolates (a) N and (b) T7 at different times of enzymatic hydrolysis (0, 2, 6
and 12 h). a0 a and b, subunits of 7S globulin; A and B, acidic and basic
polypeptides of 11S globulin; AB, monomeric subunit of 11S globulin,
Lane MM, low MM markers with 14.4, 20.1, 30, 43, 67 and 94 kDa.
 S.E. Molina Ortiz, J.R. Wagner / Food Research International 35 (2002) 511–518 515

TCA protein solubility (STCA) was studied to follow than the non-hydrolyzed samples. Hydrolysate Nh
the liberation of peptides of low molecular size during showed a similar solubility profile to Nc, but with a
hydrolysis (Fig. 4). After bromelain hydrolysis, STCA wider range of minimum solubility (pH 4.3–5.8 with
increased in all cases, specially at short times (2 h). S15%). However, the increase in solubility of hydro-
Sample T7 was more susceptible to the bromelain lysates T7c and T1.6c was practically pH-independent
digestion in terms of %
STCA ( > 600% at 12 h). N and (S=14–22%). Comparing these profiles, it could be
T1.6 had lower %
STCA values (< 350%) with no sig-
nificant differences (Fig. 4).
Our results indicate that bromelain hydrolyzed more
easily T7 isolate, generating a gradual increase in the
amount of soluble proteins at pH 4.5 and peptides with
MW < 3000 Da, between 2 and 12 h of hydrolysis. The
same effect was observed on isolate N, but in a lesser
degree and at short times (2–6 h). However, this period
of time was enough for the protease to interact with the
external layer of globulins 7S and 11S. The proteolytic
action on T1.6 originated mainly small peptides after 2
h of hydrolysis.
Fig. 5 shows the pH–protein solubility profiles of the
three isolates (Fig. 5a) and the corresponding hydro-
lysates at 12 h (Fig. 5b). The protein solubility of Nc Fig. 4. Effect of time of enzymatic hydrolysis on the increment of
protein solubility in 12.5% TCA (
STCA) for soy isolates N, T7 and
showed a minimum at pH 4.3–4.6 (S< 1%), whereas at T1.6.
SpH4.5 was expressed as a percentage of the value obtained for
pH55.8 and < 4 there was a significant increase in the non-hydrolyzed isolate (see Section 2). The values are the means of
protein solubility (P < 0.05). This behavior was similar at least two determinations.
to the native isolate (N) (data not shown) but with
lower protein solubility ascribed to the treatment and
storage of the samples at acidic pH. Protein solubility of
T1.6c showed a minimum in a wide range of pH (4.0–
5.8), with a significantly lower protein solubility than
Nc at pHs 3.6 and 5.8. The protein solubility of T7c was
lower than Nc and T1.6c for all pH conditions, but
higher at pH46.5. Fig. 5b shows that all hydrolysates
had a significantly higher protein solubility (P < 0.05)

Fig. 3. Electrophoretic patterns (SDS-PAGE) in the presence of

2-mercaptoethanol (5% v/v) of pH 4.5 soluble proteins of soy isolate
N at different times of enzymatic hydrolysis (0, 2, 6 and 12 h). a0 , a
and b, subunits of 7S globulin; A and B, acidic and basic polypeptides
of 11S globulin; AB, monomeric subunit of 11S globulin, Lane MM,
low MM markers with 14.4, 20.1, 30, 43, 67 and 94 kDa.
Fig. 5. Protein solubility in water of samples as a function of pH. (a)
Soy isolates control at 0 h of hydrolysis: Nc, T7c and T1.6c. (b)
Hydrolysates at 12 h of hydrolysis: Nh, T7h and T1.6h. Solubility was
expressed as a percentage of the total protein in sample. The values are
the means of at least two determinations.
516 S.E. Molina Ortiz, J.R. Wagner / Food Research International 35 (2002) 511–518
concluded that between pH 4.5 and 5.8, the protein
solubility was significantly higher in order
T7h > T1.6h > Nh (P < 0.05), whereas at pH47 the iso-
late Nh showed the highest protein solubility followed
by T7h. T1.6h had the lowest protein solubility
(P < 0.05) in those conditions. These results suggest that
the soluble fractions of Nh might have protein partially
hydrolyzed with a high degree of secondary–tertiary
structure. On the other hand, the soluble fractions of
T7h and T1.6h would be mainly composed of small free
peptides that would not be affected by pH.
Our results show that the degree of hydrolysis and the
structural characteristics of the protein species released
by proteolysis were markedly different in the three iso-
lates. These results correlated with surface aromatic
hydrophobicity values obtained for the soluble fraction
at pH 4.5, before and after hydrolysis (Fig. 6).
The native isolate showed a high surface aromatic
hydrophobicity at pH 4.5 (H0=1100) in comparison
with pH 7 (H0=160; Wagner et al., 2000). It is possible
to infer that charge neutralization of 7S and 11S pro-
teins close to their pI resulted in an increase in hydro-
phobicity and consequently, only a small fraction
remained soluble. When N was hydrolyzed, the resulting
peptides easily exposed their hydrophobic regions at pH
4.5 increasing the H0 1.8-fold at 2 h and to almost dou-
ble the initial value at 12 h. These results in combination
with those of protein solubility, indicate that Nh had a
high proportion of soluble and hydrophobic proteins at
pH 4.5, which would confer surface properties.
Non-hydrolyzed T7 showed a H0=379 at pH 4.5.
This lower hydrophobicity in comparison to the native
isolate would be consequence of the aggregation of
proteins denatured at pH 7 by the thermal treatment. At
pH 4.5 only hydrophilic proteins would be soluble
(polypeptide A-11S, a and a0 -7S and small peptides;
Fig. 2). Hydrolysis of T7 originated less hydrophobic
species. Protease digestion on the hidden hydrophobic
regions of the protein aggregates would allow only the
release of highly soluble peptides. On the other hand,
Fig. 6. Surface aromatic hydrophobicity (H0) of samples at pH 4.5 as
a function of time of enzymatic hydrolysis. The values are the means
of at least two determinations.
hydrolysis of soluble proteins already present in T7 ori-
ginated smaller species with less hydrophobicity. Similar
results were obtained for T1.6 but with a lower value of
H0 in the non-hydrolyzed isolate (H0=100). These
could be a consequence of the combined effect of the
thermal treatment and acidic conditions resulting in
highly hydrophilic species. Our results would indicate
that the increase in charge, the destruction of glutamine
and/or asparagine and the low hydrophobicity might
limit the action of bromelain. Hydrolysis of bovine
serum albumin (Alb), a highly hydrophobic protein with
low molecular mass (MM=67 kDa, H0=3200), par-
tially reduced the hydrophobicity (Fig. 6). This behavior
could be explained on the basis of the loss in the struc-
ture with the generation of hydrophilic peptides of low
molecular mass with low affinity for ANS. The hydro-
lysis of T7 and T1.6 originated species with decreasing
hydrophobicity similar to the Alb. This similarity could
be ascribed to the action of the bromelain at pH 8 on
the soluble hydrophilic fractions of low molecular mass.
3.2. Foaming properties
Changes in structural properties (MM and surface
aromatic hydrophobicity) as a result of hydrolysis
would originate specific surface characteristics/func-
tional properties. Fig. 7 shows, as an example, the pro-
file of formation and destabilization of foam prepared
with T7c and its corresponding hydrolysate at 12 h
(T7h). The first part of the curve (a) is closely related to
the protein capacity to intake liquid to the foam. The
other part (b), is related to the destabilization of the
foam due to liquid drainage. If an isolate unable to form
foams (T7c) is hydrolyzed (T7h), it is possible to obtain
peptides soluble at pH 4.5 which can migrate to the
surface and hence form stable foams (Fig. 7). Bovine
serum albumin was used as a standard in order to
Fig. 7. : Volume of liquid incorporated into the foam as a function of
time. Assays were performed with isolate T7c (control at 0 h of
hydrolysis) and hydrolysate T7h (at 12 h of hydrolysis) dispersions at
pH 4.5. Left of dotted line (zone a): foam formation; right of dotted
line (zone b) foam liquid drainage.
 S.E. Molina Ortiz, J.R. Wagner / Food Research International 35 (2002) 511–518
Table 2
Foaming properties of soy isolates (N, T7 and T1.6) and bovine serum albumin (Alb) at time of bromelian hydrolysis th=0 and 12 ha
Isolate Foaming properties
th (h) v0 (ml s1)
N 0 0.029 0.015
12 0.135 0.002
T7 0 0.010 0.002
12 0.167 0.005
T1.6 0 0.114 0.016
12 0.134 0.004
Alb 0 0.150 0.030
12 0.170 0.010
a
Assays were performed with sample solutions at 1.0 mg ml1 in 0.1 M phosphate buffer pH 4.5. Parameters v0, Vmax (foam formation) and t1/2
(foam stabilization) as described in Section 2.
compare and analyse the foaming properties of all iso-
lates (Nc, T7c and T1.6c) and the corresponding
hydrolysates at 12 h (Nh, T7h and T1.6h; Table 2).
Bromelain did not modify markedly the foaming para-
meters of bovine serum albumin (Alb). This protein has
good properties to form and to stabilize foams at pH
4.5, which could be related to its low molecular size and
its high surface aromatic hydrophobicity. Bromelain
action on Alb produced only a slight increment in
values of foaming parameters v0, Vmax and t1/2 (13.0, 1.8
and 11.6%, respectively), results that correlated with the
high surface aromatic hydrophobicity observed in
hydrolysates of Alb (Fig. 6). Non-hydrolyzed N and T7
isolates do not form good foams, and the hydrolysis for
12 h improved the foam formation to values of v0 and
Vmax close to those obtained for the Alb. However,
foam stability (t1/2) for hydrolysates of N and T7 never
reached the standard albumin level. T7 markedly
improved its foaming properties after hydrolysis, result
that would be explain by the high increment in protein
solubility (higher
SpH4.5 and
STCA, Fig. 1 and 4).
Nevertheless, hydrolysate of N presented a lower incre-
ment in foam formation capacity but exhibited high
foam stability, probably by its high hydrophobicity that
allowed formation of a stable film at the interface. Iso-
late T1.6, which had better foaming properties than
non-hydrolyzed N and T7 samples, showed a less pro-
nounced improvement in their surface behavior after
enzymatic treatment. These results correlate with the
structural properties described for the isolates and the
corresponding hydrolysates, since foam formation is
related to the size of the proteins or peptides that can
migrate to and remain at the interface. The results cor-
responding to the structural studies of isolates and
hydrolysates suggest that protein solubility has a greater
impact than surface aromatic hydrophobicity on the
ability of the hydrolysates to form and stabilize foams.
517
Vmáx (ml) t1/2 (s)
0.40 0.05 0
2.57 0.10 52.0 2.0
0.28 0.02 8.0 1.0
4.37 0.24 45.5 4.5
1.68 0.07 30.0 5.0
3.70 0.05 49.0 3.0
5.54 0.04 87.0 4.0
5.64 0.01 97.5 2.5
4. Conclusions
Enzymatic hydrolysis of a native (N) and two mod-
ified (T7 and T1.6) soybean isolates, which presented
differences in denaturation and aggregation degrees,
was studied. At pH 4.5, all isolates exhibited low protein
solubility and low capacity to form and stabilize foams.
The enzymatic hydrolysis of isolates increased their
protein solubility (STCA and SpH 4.5). The soluble frac-
tion of the three hydrolysates showed differences in
molecular mass, surface aromatic hydrophobicity and
pH–protein solubility profile. These hydrolysates, with
better protein solubility and foam properties at acidic
pH than the native and modified isolates, could be used
for preparation of foodstuffs of moderate acidity.
Acknowledgements
The authors acknowledge the financial support from
the Secretarı́a de Ciencia y Técnica (SECyT) and the
Universidad Nacional de La Plata, Argentina. J. R.
Wagner is member of Consejo Nacional de Investiga-
ciones Cientı́ficas y Técnicas (CONICET).
References
Adler-Nissen, J. (1976). Enzymatic hydrolysis of proteins for increased
solubility. Journal of Agricultural and Food Chemistry, 24(6), 1090–
1093.
Hayakawa, S., & Nakai, S. (1985). Relationships of hydrophobicity
and net charge to the solubility of milk and soy proteins. Journal of
Food Science, 50, 486–491.
Laemmli, U. K. (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, 227, 680–685.
Lahl, W. J., & Braun, S. D. (1994). Enzymatic production of protein
hydrolysates for food use. Food Technology, 48(10), 68–71.
Lowry, O. H., Rosembroug, H. J., Lewis, A., & Randall, R. J. (1951).
518 S.E. Molina Ortiz, J.R. Wagner / Food Research International 35 (2002) 511–518
Protein measurement with the folin phenol reagent. Journal of Bio-
logical Chemistry, 193, 265–275.
Matsudomi, N., Sasaki, T., Kato, A., & Kobayashi, K. (1985). Con-
formational changes and functional properties of acid-modified soy
protein. Agricultural and Biological Chemistry, 49, 1251–1256.
Molina Ortiz, S. E., & Añón, M. C. (2000). Analysis of products,
mechanisms of reaction and some functional properties of soybean
hydrolysates. Journal of the American Oil Chemists’ Society, 77(12),
1293–1301.
Pearson, A. M. (1983). Soy proteins. In B. J. F. Hudson (Ed.), Devel-
opments in food proteins—2 (pp. 67–108). Essex: Applied Science
Publishers.
Petruccelli, S., & Añón, M. C. (1994). Relationship between the
method of obtention and structural and functional properties of soy
protein isolates. 1. Structural and hydration properties. Journal of
Agricultural and Food Chemistry, 42, 2161–2169.
Sorgentini, D. A., & Wagner, J. R. (1999). Comparative study of
structural characteristics and thermal behavior of whey and isolate
soybean proteins. Journal of Food Biochemistry, 23(5), 489–507.
Sorgentini, D. A., Wagner, J. R., & Añón, M. C. (1995). Effects of
thermal treatment of soy protein isolate on the characteristics and
structure. Function relationship of soluble and insoluble fractions.
Journal of Agricultural and Food Chemistry, 43, 2471–2479.
Thanh, V. H., & Shibasaki, K. (1976). Major proteins of soybean
seeds. A straightforward fractionation and their characterization.
Journal of Agricultural and Food Chemistry, 24, 1117–1121.
Wagner, J. R., & Guéguen, J. (1995). Effects of dissociation, deami-
dation, and reducing treatment on structural and surface active
properties of soy glycinin. Journal of Agricultural and Food Chem-
istry, 43, 1993–2000.
Wagner, J. R., Sorgentini, D. A., & Añón, M. C. (1996). Thermal and
electrophoretic behavior, hydrophobicity, and some functional
properties of acid-treated soy isolates. Journal of Agricultural and
Food Chemistry, 44, 1881–1889.
Wagner, J. R., Sorgentini, D. A., & Añón, M. C. (2000). Relation
between solubility and surface aromatic hydrophobicity as an indi-
cator of modification during preparation processes of commercial
and laboratory prepared soy protein isolates. Journal of Agricultural
and Food Chemistry, 48(8), 3159–3165.
Walstra, P. (1989). Principles of foam formation and stability. In
A. J. Wilson (Ed.), Foam: physics, chemistry and structure (pp. 1–
15). London: Springer-Verlag.