ARTICLE IN PRESS

LWT 40 (2007) 1215–1223

www.elsevier.com/locate/lwt

Rheological properties of soy protein hydrolysates obtained from

limited enzymatic hydrolysis
B.P. Lamsal, S. Jung, L.A. Johnson
Department of Food Science and Human Nutrition, and Center for Crops Utilization Research, Iowa State University, Ames, IA, USA
Received 23 February 2006; received in revised form 24 August 2006; accepted 31 August 2006

Abstract

Soy protein products hexane-defatted soy flour, extruded-expelled soy flour, soy protein concentrate and soy protein isolate, were
modified by using the enzyme bromelain to 2% and 4% degrees of hydrolysis (DH). Peptide profiles, water solubility, and rheological
properties including dynamic shear, large deformation, and apparent viscosities of resulting hydrolysates were determined. Protein
subunits profiles for the hydrolysed isolates and concentrates were extensively altered by the treatment while only minor changes were
observed for the hydrolysed flours. Water solubility profiles of all hydrolysates in the pH range of 3.0–7.0 were enhanced by hydrolysis.
For the unhydrolysed controls, the isolate had the highest storage modulus (G0 ), followed by the concentrate, the extruded-expelled flour
and the hexane-defatted flour. The hydrolysates retained some of their gelling ability even though the losses in storage modulus (G0 ) were
substantial. After heating step to 95 1C, the G0 values of all substrates at 25 1C decreased with increase in DH. Texture profile analyses of
the soy protein gels were also lower in hardness after hydrolysis. The Power Law model provided excellent fit to hydrolysate dispersions
flow ðR2 40:99Þ. Hydrolysis decreased the consistency coefficients of dispersion and increased flow behavior index resulting in thinner
dispersions. These results suggest that limited protease hydrolysis of various soy protein meals with bromelain produce soy protein
ingredients with modified rheological properties.
r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Soy proteins; Limited hydrolysis; Rheology; Gelation; Texture analysis

1. Introduction and breads (Pszczola, 2005). Soy proteins undergo harsh

processing conditions involving heat, shear, and exposure
Food proteins, both animal and plant proteins, play to acid, and may lose much of their functionalities.
critical roles in human nutrition. This traditional role aside, Limited or controlled enzymatic hydrolysis of soy
proteins in food formulations are increasingly expected to proteins could provide ingredients with desired or restored
perform functional roles that are important to consumer functionalities (Panyam & Kilara, 1996; Surowka, Zmud-
food acceptance. Food proteins possess physico-chemical zinski, & Surowka, 2004; Surowka, Zmudzinski, Fik,
properties that govern their performance and behavior in Macura, & Lasocha, 2004; Jung, Murphy, & Johnson,
food systems during processing, storage and consumption 2005). Ingredients that form strong gels and give high
that are collectively termed functional properties. Such viscosity are preferred for use in comminuted meat
properties also provide the basis for utilizing an ingredient products and gravies, while yogurts, soups, and infant
in food applications. Some examples of novel uses include food formulations require less viscous product mix and
use in snack and energy bars, hard pretzels, sport weaker gelling properties. The extent of proteolysis during
beverages, whole soy ingredients, egg replacements in enzymatic hydrolysis can be established with the degree of
bakery products, infant formulae, coating systems, tortillas hydrolysis (DH). Hydrolysates intended for nutritional
formulations, e.g., hypoallergenic hydrolysates, are exten-
sively hydrolysed, with 90% of peptides being o500 Da in
Corresponding author. Tel.: +1 515 294 2544; fax: +1 515 294 8181. molecular weight (Mahmoud, 1994). Hydrolysates in-
E-mail address: jung@iastate.edu (S. Jung). tended for use as nutritional supplements undergo slight

0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2006.08.021
 ARTICLE IN PRESS
1216 B.P. Lamsal et al. / LWT 40 (2007) 1215–1223

(about 90% peptides 45000 Da) or moderate (about 46% acquired from Iowa Soy Specialities (Vinton, IA). The
peptides 45000 Da) hydrolysis. crude protein contents for the substrate as determined in
Enzymatic hydrolysis of protein employs various pro- our laboratory were: SF, 54 g/100 g, EE, 49 g/100 g, SPC,
teases, bromelain being one such protease. Bromelain is 77 g/100 g, and SPI, 93 g/100 g on as is basis. Protein
an endoprotease chiefly used for muscle tenderization dispersibility index (PDI) was determined externally by
(Melendo, Beltran, & Roncales, 1997; Teran, 2003; Kolle, Eurofins Scientific Inc. (Des Moines, IA) and were 88, 68,
McKenna, & Savell, 2004), and producing acid-coagula- 71, and 11 for SF, EE, SPC, and SPI, respectively.
tion resistant milk (Christensen, Florin, & Harris, 2002).
Studies using bromelain to improve physicochemical and 2.2. Enzymatic hydrolysis
functional properties of proteins have focused on emulsion
formation and water-holding capacity (Karakaya & The food-grade enzyme bromelain, an endopeptidase
Ockerman, 2002), umami chicken flavor development derived from pineapple stems (Bio-Cat Inc., Troy, VA) was
(Maehashi, Matsuzaki, Yamamoto, & Udaka, 1999), soy used to carry out the hydrolysis. The enzyme activity was
protein solubility and foaming properties (Molina-Ortiz & specified as 2000 gelatin digestion units (GDU)/g protein.
Wanger, 2002), and controlling of gluten network forma- Hydrolysis was performed at 50 1C and pH 7.0. DH was
tion in low fat pastry products (Hart, 1999). controlled and determined by using the pH-Stat method
Native, globular proteins are generally resistant to (Adler-Nissen, 1986), which determines the %DH on the
enzyme hydrolysis due to their compact tertiary structures basis of the number of free titratable amino groups
that protect many of the peptide bonds (Adler-Nissen, produced by hydrolysis of peptide bonds. Compared to
1976). Denatured proteins, like most processed soy other methods, the pH stat method allows direct monitor-
proteins, have exposed peptide bonds available for enzy- ing of DH in real time. DH was calculated using the
matic cleavage. Enzymatic hydrolysis can be responsible following equation:
for (1) a decrease in hydrolysate molecular weight, (2) an
DH ¼ ½ðVNaOH  NNaOH Þ=ða  MP  htot Þ  100%,
increase in ionizable group number, and (3) exposure of
previously concealed hydrophobic groups (Panyam & where a is the degree of dissociation of a-amino groups,
Kilara, 1996). Changes in functional properties, including MP is the mass of protein (g), htot is the number of peptide
rheological characteristics, are a direct result of the above- bonds in the substrate (meqv/g protein), concentration of
mentioned changes. Structural modifications to the two the base (NaOH) was 2 mol/l, a value was 0.44, and htot
major soy protein components, namely glycinin and was 7.8 (Adler-Nissen, 1986).
b-conglycinin, by proteases (endo or exo-peptidases) Hydrolysis of the 10 g/100 g substrate dispersion was
dictate the resultant hydrolysate properties (Jung et al., carried out in a 250-mL temperature-controlled glass
2005). Gel hardness and fracturability of soy proteins are reactor with constant stirring. pH during hydrolysis was
attributed to glycinin, whereas b-conglycinin contributes to controlled by using a titrator (718 STAT Trition,
gel elasticity (Utsumi, Matsumura, & Mori, 1997). Brinkmann, Westbury, NY). Appropriate enzyme-to-sub-
Extensive literature is available on the enzymatic strate ratios were selected to reach 2% or 4% DH values,
modification of soy proteins from moderate to high DH wherein a plateau in DH over time was achieved. Freezing
by proteolytic enzymes and effects on hydrolysate func- the hydrolysates, which were later freeze-dried, quickly
tional properties (Kim, Park, & Rhee, 1990; Achouri, stopped enzymatic reaction. The unhydrolysed control
Zhang, & Shiying, 1998; Calderon-de-la Barca, Ruiz- dispersion was prepared at the same pH, temperature and
Salazar, & Jara-Maarini, 2000; Hrckova, Rusnakova, & reaction time as was necessary to achieve 4% DH and
Zemanovic, 2002; Tsumura et al., 2005). The effects of similarly freeze-dried. Crude protein contents of the freeze-
limited hydrolysis on rheological properties of soy proteins, dried hydroylsates were the same as those of the
however, are not understood. The aim of the present study corresponding substrates.
was to determine the rheological properties (gelation and
viscosity) of soy protein products, which have undergone 2.3. Sodium dodecyl sulfate polyacryalamide gel
limited protease hydrolysis (up to 4% DH). electrophoresis (SDS-PAGE)

2. Materials and methods SDS-PAGE was performed on unhydrolysed controls

and enzyme hydrolysates to determine the effects of the
2.1. Substrates enzyme treatments on the protein polypeptide profiles.
SDS-PAGE was carried out using a SDS-Tris-glycine
Four commercial soy protein products were used as buffer system with 4% stacking gels and 13% resolving gels
starting materials; a hexane-defatted soy flour (SF, (Mini Protean II Gel, Biorad Inc., Hercules, CA) following
Nutrisoy 7B), a soy protein concentrate (SPC, Acron S) methods of Jung et al. (2005). A low-range molecular
and a soy protein isolate (SPI, Profam 955), which were weight (MW) marker ranging from 66 to 6.5 kDa (M3913,
acquired from Archer Daniels Midland Co. (Decatur, IL) Sigma Chemical Co., St. Louis, MO) and laboratory-
and an extruded-expelled soy flour (EEF), which was purified b-conglycinin and glycinin were used as standards.
 ARTICLE IN PRESS
B.P. Lamsal et al. / LWT 40 (2007) 1215–1223 1217

Ten ml of protein in sample buffer and 5 ml of standards, offered by the gel during compression was taken as gel
both at 1 mg/ml concentration, were loaded per lane. hardness.

2.4. Solubility profile 2.7. Apparent viscosity

Solubility profiles of the protein hydrolysates in water Shear stresses developed with applied shear rates ranging
over the pH range of 3.0–7.0 were determined. Freeze-dried from 10 to 500 s1 for 10 g/100 g protein dispersions were
hydrolysates were suspended in deionized water at 1 g/100 g measured with the same cone-and-plate probe previously
protein concentration. The pH of the dispersion was described. These flow curves were modeled using the Power
adjusted to 3, 4, 5, 6, and 7 with 2 mol/l NaOH or 2 mol/l Law model:
HCl and stirred at room temperature for 1 h, readjusting
t ¼ Kgn ,
the pH if necessary. The dispersions were then centrifuged
at 10,000  g for 15 min at 20 1C (Avanti J-20, fixed-angle where t is shear stress, K is consistency coefficient, g is
rotor JLA 10.5, Beckman Coulter, Fullerton, CA). Crude shear rate, and n is flow behavior index.
protein content in the supernatant was determined by using Apparent viscosities were calculated at shear rates of 10
the Biuret method with bovine serum albumin (A-7906, and 500 s1 from the fitted parameters.
Sigma Chemical Co., St. Louis, MO) as standard.
Solubility was expressed as the percentage of original 2.8. Crude protein and moisture determination
protein present in the supernatant.
The nitrogen contents of the samples were determined
2.5. Steady shear rheology with a combustion-type nitrogen analyzer (Rapid
N-Analyzer, Elementar Americas, NJ). Nitrogen contents
A 20.0 ml 10 g/100 g protein dispersion in deionized were converted to crude protein contents using a factor of
water at pH 7 was prepared for each soy protein 6.25. Moisture contents were determined by drying the
hydrolysate. The dispersion was allowed to equilibrate samples in a forced-draft oven at 130 1C for 3 h.
overnight at 4 1C and stirred for 30 min at room
temperature before analysis. Degassing the dispersions 2.9. Statistical analysis
was done in 100-ml vacuum flasks. Small-amplitude
oscillatory shear tests were performed with a dynamic General linear model, PROC GLM, in SAS system
rheometer (RS 150 Haake, Karlsruhe, Germany) using a (Version 8.2, SAS Institute, Inc., Cary, NC) was used to
cone-and-plate probe (60 mm  21) and 0.105 mm gap. Two compare the means at po0:05.
to 3 ml of hydrolysate dispersion was loaded and excess
sample was wiped off the bottom plate. A thin layer of 3. Results and discussion
silicone oil and lubricated O-ring was put around the
bottom plate edge to prevent drying. Oscillatory strain of 3.1. Soy protein hydrolysis
1% was applied at 0.1 Hz frequency while the dispersion
was heated in situ from 25 to 95 1C (2 1C min1), held for Fig. 1 shows typical progression of hydrolysis (DH) with
3 min, and cooled to 25 1C at 2 1C min1. Evolution of time for soy protein substrates when hydrolysed with
storage modulus (G0 ) with temperature was monitored. At bromelain. The appropriate enzyme-to-substrate (E/S)
least three measurements were performed for duplicate ratios were identified to produce a plateau at 2% or 4%
dispersions prepared for each hydrolysate and mean values DH. The reaction progressed rapidly for the first 15 min
reported. The stock dispersion was kept stirring during and then relatively slowly over time reaching a plateau.
measurement period. This exponential reaction was typical of protease hydro-
lysis (Kim et al., 1990). The no-enzyme control substrates
2.6. Texture profile analysis (TPA) displayed DH of 0.5%, 0.35%, 0.07%, and 0.12% DH for
SF, EEF, SPC, and SPI, respectively. This DH was due to
Fifteen ml of a 10 g/100 g protein dispersion at pH 7.0 the solubilization of the proteins with time, which
was poured into three glass bottles (2.5 cm ID  4.8 cm decreased the pH, therefore the DH value of the controls
height) and tightly closed. They were shaken for 20 min, did not reflect any proteolytic activity (Jung et al., 2005).
immersed in a 96 1C water bath for 30 min, chilled in a The reaction times to reach 2% and 4% DH with
20 1C water bath for 30 min, and stored overnight at 4 1C. appropriate E/S ratio (g enzyme per g protein) for all
Gels were equilibrated to room temperature for 2 h before substrates are presented in Table 1. For SF and EEF, 2%
compression with an acrylic probe (1.2 mm ID  35 mm DH was achieved in less than one-half the time with about
height) in a texture analyzer (TA-XT2, Texture Technol- one-half the amount of enzyme than used to achieve 4%
ogies Corp., Scarsdale, NY). The uniaxial compression at DH. For SPC and SPI, the times needed to reach 2% DH
the rate of 0.5 mm/s was applied twice to the depth of were more than those needed to reach 4% DH when E/S
12 mm (37% deformation). The maximum resistance ratio was reduced to less than one-half.
 ARTICLE IN PRESS
1218 B.P. Lamsal et al. / LWT 40 (2007) 1215–1223

4.5 changes for SF and EEF (Fig. 2). The protein subunit
bands present in all unhydrolysed control samples, namely
4
subunits of b-conglycinins, and acidic and basic subunits of
3.5 glycinin, almost entirely disappeared in SPI and SPC
hydrolysates at 2% and 4% DH. On the other hand, most
Degree ofhydrolysis, %

3
of the a0 , a, and b subunits of b-conglycinin were
2.5 hydrolysed in EEF and SF. Glycinin subunits were largely
intact in EEF and SF at 2% DH, but acidic subunits were
2 hydrolysed to some extent at 4% DH. The basic subunits
1.5 of glycinin, however, were not hydrolysed in EEF, and SF,
which was expected because these subunits reside in the
1 interior of the undenatured glycinin complex and therefore
0.5
are less exposed to enzymatic attack. This observation
agreed with the findings of Lakemond, de Jongh, Hessing,
0 Gruppen and Voragen (2000), who observed that the
0 5 10 15 20 25 30 35 40 45
enzyme clostripain did not affect the basic polypeptides of
Time, min
the soy glycinin complex, whereas the acidic polypeptides,
Fig. 1. Typical progression of degree of hydrolysis with time for soy which predominantly face outside the complex, were
protein substrates (shown: extruded-expelled soy flour) when acted upon degraded by the enzyme at ionic strengths below 0.5.
by bromelain. Symbols: (&) 4% DH, (B) 2% DH, and (n) unhydrolysed These results confirm that the extent of substrate denatura-
control.
tion plays a major role in enzymatic hydrolysis (Adler-
Nissen, 1976; Jung, et al., 2005). Hydrolysis of ethanol-
Table 1 washed SPC (71 PDI), however, showed almost complete
Mean ðn ¼ 2Þ hydrolysis reaction times and enzyme-to-substrate (E/S) degradation of protein subunits. This hydrolysis behavior
ratios for each soy protein substrate can be explained by the fact that this particular SPC had
Soy protein Degree of E/S ratio (%) Time to plateau denatured glycinin and b-conglycinin as shown by Jung et
substrate hydrolysis (%) (min) al. (2005). EEF and SF hydrolysates showed new bands at
approximately 25 and 30 kDa, whereas SPI showed very
SF 4 0.51 65a
faint bands appearing below 20 kDa. Small peptides
2 0.24 23d
EEF 4 0.27 39b appeared at the bottom of the gel for all hydrolysates
2 0.12 21.5d suggesting the presence of peptides o6:5 kDa.
SPC 4 0.37 39.5b
2 0.17 64a
SPI 4 0.61 22d
3.3. Water solubility profiles
2 0.14 34.5c

Reaction times sharing same superscript are not significantly different Fig. 3 shows water solubility profiles for soy protein
ðpo0:05Þ. SF: soy flour; EEF: extruded-expelled soy flour; SPC: soy hydrolysates at 2% and 4% DH and corresponding
protein concentrate; and SPI soy protein isolate. The unhydrolysed
unhydrolysed controls. The solubility profiles for all
control for a given substrate was treated for the longest hydrolysis reaction
time without enzyme added. Enzyme-to-substrate ratio: g of enzyme per g hydrolysates were the typical U-shaped curves with
of protein  100 (%). solubility being higher on either side of isoelectric point
(around pH 4.5 for soy proteins). The unhydrolysed
controls at pH 7 had protein solubilities close to their
The extent of protein denaturation (i.e., PDI value) PDI values, except for SPC. Hydrolysis up to 4% DH
probably affected the reaction time to reach a given DH. increased their solubilities of all substrates compared to
SPI had the lowest PDI value (i.e. higher extent of control samples (2% and 4% DH hydrolysates being
denatured protein), which seems to have promoted similar in solubility). The percentages of increased solubi-
cleavage sites access of bromelain leading to 4% DH in lity for the 4% DH hydrolysates at pH 7 over the
about 22 min. For SF, which had the highest PDI value corresponding unhydrolysed control were 360, 225, and
(i.e., less protein denaturation), 4% DH was attained after 36 for SPI, SPC, and EE, with SF showing no change. The
65 min reaction time. Longer reaction times for higher PDI increased solubility of the hydrolysates over the unhydro-
soy substrates have also been reported by Henn and Netto lysed control were attributed to the production of soluble
(1998) and Jung et al. (2005). peptides (Tsumura et al., 2005) and increased number of
exposed ionizable amino and carboxyl groups (Panyam &
3.2. Hydrolysate peptide profiles Kilara, 1996) during hydrolysis, which in turn depended on
the degree of denaturation (Adler-Nissen, 1976). Therefore,
Modifications to the peptide profiles were substrate- it was not surprising that SPI with the lowest PDI had the
specific, with major changes for SPI and SPC and minor highest increase in solubility. The increased solubility of the
 ARTICLE IN PRESS
B.P. Lamsal et al. / LWT 40 (2007) 1215–1223 1219

EEF SF SPI SPC

MW
(kDa) β-con Gly M C 2% 4% C 2% 4% M C 2% 4% C 2% 4%

66

44
36 A
29
24 B
20

14.2
6.5

Fig. 2. Peptide profiles for the unhydrolysed control and bromelain-modified soy protein substrates at 2% and 4% DH. MW, molecular weight; b-con, b-
conglycinin; Gly, Glycinin; M, MW Marker; C, control, A, Gly acidic, and B, Gly basic subunits; EEF, extruded-expelled soy flour; SF, hexane-defatted
soy flour, SPI, soy protein isolate; and SPC, soy protein concentrate.

SF SPI
100
100
80
80
Solubility (%)
Solubility (%)

60 60

40 40

20 20

0 0
2 3 4 5 6 7 8 2 3 4 5 6 7 8
pH pH

EEF SPC
100 100

80 80
Solubility (%)
Solubility (%)

60 60

40 40

20 20

0 0
2 3 4 5 6 7 8 2 3 4 5 6 7 8
pH pH

Fig. 3. Water solubility profiles of control (n), and 2 (’), and 4% DH (~) bromelain hydrolysates from soy flour, SF (top left); soy protein isolate, SPI
(top right); extruded-expelled soy meal, EEF (bottom left); and soy protein concentrate, SPC (bottom right).

SPC hydrolysate was attributed to the denaturation state is useful in controlling texture and mouthfeel in various
of the glycinin and b-conglycinin, as previously reported. food applications such as comminuted meats and sausages,
yoghurts and puddings, products in which soy proteins are
3.4. Storage modulus (G0 ) and gelation temperature used. Fig. 4 shows representative evolution of storage
modulus (G0 ) with heating and cooling of 10 g/100 g soy
Storage modulus (G0 ) and loss modulus (G00 ) are two protein hydrolysate dispersion.
viscoelastic parameters indicating gel strength. After During heating, storage modulus G0 was small and
heating beyond a certain temperature, G0 values rise almost constant until a certain temperature was reached, at
dramatically due to increased aggregation of proteins, which it rapidly increased indicating transition from a
indicating stronger and more elastic gels. This information liquid-like state (sol) to a solid-like state. This temperature
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1220 B.P. Lamsal et al. / LWT 40 (2007) 1215–1223

7000 Table 2
Gel storage moduli for 10 g/100 g soy protein dispersions during heating
and cooling
6000
Soy protein Degree of Tgel, 1C Storage modulus (G0 ), kPa
substrate hydrolysis (%) mean  SE
5000
Storage modulus, G', Pa

95 1C  SE, 25 1C  SE,
Heating Cooling
4000
SPI Control 82.570.3cd 5.671.5b 3747120a
Cooling 2 – 0.0870.01d 5.470.9d
3000 4 – 0.270.02d 4.670.3d
SPC Control 80.670.4cde 36.677.3a 176.3730.4b
2 79.170.7e 0.770.2cd 12.073.4d
2000 4 85.270.4ab 0.770.1cd 6.170.8d
SF Control 83.073bc 4.270.6bc 38.071.8c
1000 Tgel 2 79.570.8de 0.970.1cd 6.070.3d
4 77.971.2ef 0.770.0cd 5.070.4d
EEF Control 86.470.3a 4.470.5bc 94.0722.2c
0 2 75.770.9f 1.370.b1cd 6.270.3d
20 30 40 50 60 70 80 90 100 4 79.171.4e 1.270.1cd 5.470.3d
Temperature, °C
SE: standard error of mean. Means sharing the same superscript in a given
Fig. 4. Representative storage modulus (G0 ) and temperature curve for column are not significantly different at po0:05.
10% w/w soy protein dispersions during heating and cooling.

is usually taken as gelation temperature (Tgel) and EEF. Heat-set gels produced by non-hydrolysed SPI were
corresponds to the temperature at which G0 increases and the strongest (highest G0 value), followed by SPC, EEF and
becomes greater than the background noise, which is one SF. Although the hydrolysates retained some gelling
of the common methods of detecting the gelling point in ability, hydrolysis of up to 4% DH caused important
the absence of a crossover between G0 and G00 (Matsumura losses in gel strengths for all the substrates, ranging
& Mori, 1996; Ould Eleya & Gunasekaran, 2002). Gelation between 6- and 75-fold compared to the controls. The
temperature is usually above the denaturation temperature reduced hydrophobicity of protein hydrolysates caused by
and denaturation is a prerequisite for heat-induced gelation enzymatic hydrolysis (Fan et al., 2005; Jung et al., 2005)
of globular proteins (Nagano, Hirotsuka, Mori, Kohyama, and the drop in sulfhydryl exchange reactions during
& Nishinari, 1992; Utsumi et al., 1997; Ould Eleya & gelation (Fan et al., 2005) may explain the inferior gel
Gunasekaran, 2002). Heat denaturation opens up buried forming abilities of the hydrolysates. In addition, increased
hydrophobic patches inside protein molecules and facil- charge repulsion between peptides due to net charge
itates subsequent peptide associations and formation of the increase upon hydrolysis may have occurred and con-
gel network structure. Storage modulus further increases tributed to the decreased gelling ability (Panyam & Kilara,
during subsequent cooling, which is seen in Fig. 4. Such an 1996).
increase during cooling, called gel reinforcement (Ould The G0 values at 25 1C were similar for both 2% and 4%
Eleya & Gunasekaran, 2002), is typical of protein gels and DH hydrolysates for a given substrate. This result was
generally attributed to consolidation of attractive forces expected, as there were no dramatic differences in the
such as van der Waals and hydrogen bonding between peptide profiles of the 2% and 4% DH hydrolysates.
proteins within gel primary network. Although some newer polypeptides formed in the 25 and
Table 2 shows Tgel, and G0 values during heating and 30 kDa molecular weight range for EEF and SF, they did
cooling of hydrolysate dispersions. The Tgel values for not affect the final gel strength because the hydrolysates
untreated substrates ranged between 80 and 86 1C, which is from all four substrates had similar G0 values at 25 1C.
within the reported range for soy proteins (Nagano et al.,
1992; Nagano, Akasaka, & Nishinari, 1994; Utsumi et al., 3.5. Texture profile analysis (TPA)
1997). b-conglycinin starts denaturing at around 65 1C and
peaks at 70 1C, whereas glycinin denaturation starts at A typical force vs. time curve during uniaxial compres-
around 80 1C and a gel network begins to form (Yamauchi, sion of gels is shown in Fig. 5 (top) and was used to
Yamagishi, & Iwabuchi, 1991). While onset of gelation evaluate the impact of enzyme treatment on gel hardness
coincides with onset temperature of denaturation of (Fig. 5, bottom). The SF control gels were the hardest
b-conglycinin and glycinin solutions, onset of gelation is followed by EEF, SPC, and SPI controls. This result
greater than the denaturation temperature in soy protein suggested that when SF control was heated for 30 min, it
preparations (Kang & Lee, 2005). formed a gel having the highest hardness. This result might
The Tgel values for the 2% and 4% DH hydrolysates be due to interactions between proteins and carbohydrates
were slightly lower than for the control samples of SF and (oligosaccharides and fiber) that are in a higher amount in
 ARTICLE IN PRESS
B.P. Lamsal et al. / LWT 40 (2007) 1215–1223 1221

the SF sample, and/or the native state of the proteins in the and glycinin and b-conglycinin interact non-covalently
SF and EEF samples. Denaturation of the proteins would with each other to form composite aggregates during gel
indeed affect the gel characteristics of the substrate. formation (Utsumi et al., 1997). Different hydrolysis of
Glycinin is related to hardness and fracturability of soy both glycinin and b-conglycinin, as observed in the SDS
gels, whereas b-conglycinin contributes to their elasticity PAGE, certainly explained the changes occurring in the gel
(Utsumi et al., 1997; Kang & Lee, 2005). The basic hardness of the hydrolysates. There was, however, no clear
polypeptides of glycinin preferentially associate with the trend in the gel hardness depending on the DH level or
b-subunits of b-conglycinin via electrostatic interactions, nature of the substrate. Gels prepared from 4% DH
hydrolysates were significantly ðpo0:05Þ less hard than the
unhydrolysed controls and 2% DH hydrolysates for SF
2.5 and EEF, the highest decrease being observed for SF. The
Hardness
2 hardness of the 2% DH SPC increased significantly
1.5 compared to the control whereas no gel was obtained with
Force, N

1 the 2% DH SPI. The reduced gel-forming ability, i.e.

0.5 reduced gel-hardness, may be related to reduced protein–
0 protein interactions and low surface hydrophobicity of the
-0.5 0 0.25 0.5 0.75 1 1.25 1.5 hydrolysate (Babiker, 2000; Fan et al., 2005) and/or
-1
Time, min increased charge repulsion between hydrolysed peptides
(Panyam & Kilara, 1996).
2.5
a
3.6. Apparent viscosity
2.0
Gel Hardness,N

The Power Law parameters (consistency coefficient K,

1.5
and flow behavior index, n), and calculated apparent
viscosities at 10 and 500 s1 shear rates for bromelain soy
1.0
b bc hydrolysates are shown in Table 3. The Power Law
c
d
d
d
e
provided excellent fit for the dispersion flow curves (shear
0.5
ef f
g
stress vs. shear rate data, R2 40:99). The K value for the
unhydrolysed control dispersion was highest for SPI,
0.0
followed by SPC, EEF, and SF. The viscosities of the
Control

Control

Control

Control
2% DH

4% DH

2% DH

4% DH

2% DH

4% DH

4% DH

unhydrolysed controls at 10 s1 were 1.68, 1.55, 1.07, and

0.08 for SPI, SPC, EEF, and SF, respectively, and followed
SF EEF SPC SPI the same trend as consistency coefficient. This was expected
because the consistency coefficient is related to apparent
Fig. 5. Typical texture profile analyses plot for soy protein gels (top) and
gel hardness comparison (bottom). Error bars indicate standard error of viscosity. Upon hydrolysis, the K values and apparent
mean. Values sharing the same letters are not statistically different viscosities followed similar trends (i.e., decreased values
ðpo0:05Þ. The 2% DH SPI hydrolysate did not produce a standing gel. with increased DH) resulting in thinner dispersions. This

Table 3
Mean ðn ¼ 6Þ Power Law parameters and apparent viscosity for 10% w/w soy protein hydrolysate dispersions

Soy substrate Degree of hydrolysis (%) Power law parameters Apparent viscosity (Pa s)

K (Pa sn) n 10 s1 500 s1

SPI Control 7.5872.8a 0.3570.06g 1.55a 0.11c

2 0.0370.0d 0.8870.01b 0.03d 0.02gh
4 0.0370.0d 0.8670.0b 0.02d 0.01h
SPC Control 4.9270.56b 0.5070.02f 1.54a 0.22a
2 0.4670.0d 0.6670.0e 0.21c 0.06d
4 0.4070.01d 0.6670.01e 0.18cd 0.05de
SF Control 0.1270.0d 0.8470.0bc 0.08cd 0.04def
2 0.0270.0d 0.9270.03a 0.02d 0.01h
4 0.0270.0d 0.9370.03a 0.02d 0.01h
EEF Control 3.0670.08c 0.5470.0f 1.07b 0.18b
2 0.2670.1d 0.7170.05d 0.13cd 0.05ef
4 0.170.01d 0.8070.02c 0.06cd 0.03fg

Means sharing the same superscript are not significantly different at po0:05. SE: standard error of mean; K: consistency coefficient; and n: flow behavior
index.
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