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2 2:303 100 A
c 0:25 10; 000
2
where A = absorbance at 500 nm; c = protein concentration (g/ml).
ESI min
A
0
10
A
0
A
10
3
2.4.3. Foaming properties
Foaming properties of the hydrolysates with different DHs,
including foaming capacity and foam stability, were determined
according to the method of Sathe and Salunkhe (1981) with slight
modications. 200 ml of 10 mg/ml hydrolysate solutions in 250 ml
cylinder were adjusted to pH 2.0, 4.0, 7.0 and 10.0 and homoge-
nised using homogenizer (IKA, Germany) at a speed of
10,000 rpm for 1 min to incorporate the air at 25 C. The total vol-
ume after whipping was read immediately and used to calculate
the foaming capacity, based on the following equation (Sathe &
Salunkhe, 1981):
Foaming capacity %
A B
B
100% 4
where A is the volume after whipping (ml) and B is the volume be-
fore whipping (ml).The total volume of the whipped sample was re-
corded after standing for 10 min at 25 C, and used to calculate the
foam stability:
Foam stability %
A B
B
100% 5
where A is the volume after standing (ml) and B is the volume be-
fore whipping (ml).
2.4.4. Differential scanning calorimetry measurements
The net heat energy (enthalpy, DH) and the onset (T
onset
) and
maximum (T
max
) temperatures for endothermic transitions of the
hydrolysates with different DHs, as a function of temperature were
determined using DSC (differential scanning calorimeter, Innity
Series F5010, Instrument Specialists, Inc., Spring Grove, IL, USA).
2.5 mg of each sample were hermetically sealed in an aluminium
pan and scanned from 30 to 150 C at a heating rate of
10 C/min. Temperature calibrations were performed prior to mea-
surements according to the manufacturer and an empty pan was
used as reference. The averages of six measurements were used
to report the results.
2.5. Statistical analysis
All determinations were carried out at least three times. Data
were analysed by analysis of standard deviations and variances
using DPS V7.05 software (Data Processing System, Zhejiang Uni-
versity, China). The software has been developed to execute a
range of standard numerical analyses and operations used in
experimental design, statistics and data mining (Tang & Zhang,
2013).
3. Results and discussion
3.1. Structures of the hydrolysates with different DHs prepared by
Protamex and Alcalase
3.1.1. Zeta potential observation
Zeta potentials of the hydrolysates with different DHs prepared
by Protamex and Alcalase are shown in Table 1. The zeta potentials
of the samples hydrolysed by both enzymes increased with
increase of DH. Generally, a high absolute value of zeta potential
generates a repulsive electrostatic force between the molecules,
which is a key property of an aggregation resistant suspension.
Therefore, it can be reasonably inferred that an increase of electro-
static repulsive forces between the hydrolysate molecules would
favour an increase of their solubility and other solubility-related
functional properties.
Besides, although the specicities of Protamex and Alcalase for
peptide bonds adjacent to certain amino acid residues were quite
different (Khantaphant & Benjakul, 2008), the difference of pro-
duced zeta potential after the hydrolysis of surimi processing by-
products by the two enzymes was not signicant (p > 0.05). Thus,
both Protamex and Alcalase could be efcient enzyme choices for
preparing hydrolysates from surimi processing by-products.
3.1.2. Surface hydrophobicity
Surface hydrophobicity of the hydrolysates with different DHs
prepared by Protamex and Alcalase was measured (Table 2).
Changes in surface hydrophobicity mainly inuence the interfacial
properties of the hydrolysates. The results showed that the surface
hydrophobicity of the hydrolysates was signicantly affected by
DH (p < 0.05). Calderon de la Barca, Ruiz-Salazar, and Jara-Marini
(2000) reported that proteolysis, due to shortening of peptide
chains, is accompanied by gain or loss of hydrophobicity, which
mainly depends on the nature of the hydrolysed protein and
molecular weight of the formed peptides. In the present work,
enzymatic hydrolysis by Protamex was accompanied by a decrease
of surface hydrophobicity. The possible reason is that peptides re-
leased from protein in surimi processing by-products gradually
adopted a conformation with hydrophilic groups exposed outward,
while surface hydrophobicity of hydrolysates prepared by Alcalase
increased along with the increasing DH. Liu, Kong, Xiong, and Xia
(2010) found that enzymatic hydrolysis of porcine plasma protein
by Alcalase was coupled with a decrease of surface hydrophobicity.
Probably the difference of protein nature could explain the oppo-
site tendency.
3.1.3. Molecular weight distribution
The calibration curve of ve standard substances on a TSKgel
G2000SWXL column is shown in Fig. 1 and this was obtained to
Table 1
Zeta (f) potential of the hydrolysates with different DHs prepared by Protamex and
Alcalase.
Sample f Potential (mV)
Protamex DH 10% 22.5 (1.1)
b
Protamex DH 20% 26.9 (0.8)
ab
Protamex DH 30% 29.8 (3.1)
a
Alcalase DH 10% 24.0 (1.3)
b
Alcalase DH 20% 26.1 (2.4)
ab
Alcalase DH 30% 29.2 (0.9)
a
Data show mean values (SD) for ve replicates.
The different letters in columns indicate signicant difference at p < 0.05.
Table 2
Surface hydrophobicity of the hydrolysates with different DHs prepared by Protamex
and Alcalase.
Sample Surface hydrophobicity
Protamex DH 10% 565.5 (4.8)
a
Protamex DH 20% 265.4 (5.2)
c
Protamex DH 30% 225.1 (3.8)
d
Alcalase DH 10% 229.8 (3.1)
d
Alcalase DH 20% 262.1 (2.4)
c
Alcalase DH 30% 393.1 (3.5)
b
Data show mean values (SD) for three replicates.
The different letters in columns indicate signicant difference at p < 0.05.
Y. Liu et al. / Food Chemistry 151 (2014) 459465 461
interpret the results. The elution patterns (chromatograms not
shown), corresponding to the hydrolysates prepared by Protamex
and Alcalase with different DHs, displayed 67 major elution peaks
at 220 nm. Comparing with standard molecular weights and con-
sidering the exclusion limit of the column, the major elution peaks
corresponded to Mws of >150,000, 2468, 1360, 799, 502, 387, 226
and 138 Da, respectively. Relative proportions (%) of each peak are
presented in Table 3. The hydrolysate with DH 10%, treated by
Protamex, had the highest proportion of larger molecules
(Mw > 150,000 Da), which was about 8.3%. Those of others ranged
from 3.4% to 6.9%. Although the proportion of the larger molecules
was lower, it is speculated that they were crucial to the function-
ality (such as the interfacial and gelling properties). The hydroly-
sate with DH 10%, treated by Protamex also contained peptides
with Mw of 2468 Da, and the relative proportion reached 20.6%.
Besides, the molecular weights of peaks of all the other hydroly-
sates were all lower than 1400 Da. These results suggested that
hydrolysis by Protamex and Alcalase yielded a wide variety of pep-
tides. Some studies have demonstrated that molecular weights of
hydrolysates were closely related to the solubility (Dong et al.,
2008; Gbogouri et al., 2004). Lee, Shimizu, Kaminogawa, and
Yamauchi (1987) supported the conclusion that there is an opti-
mum molecular size for peptides to be good emulsiers. Slizyte
et al. (2009) further found that the hydrolysates, which had the
highest amount of peptides in the molecular weight range
80,0001000 Da, displayed best emulsifying properties. Therefore,
the peptides prepared by Protamex and Alcalase can also be con-
sidered as potential functional agents in food.
The present results also show that molecular weights of the
hydrolysates with the same DH, produced by Alcalase, were gener-
ally lower than those by Protamex, which could be associated with
the higher activities of the former (Klompong et al., 2007).
3.2. Functional properties of the hydrolysates with different DHs
prepared by Protamex and Alcalase
3.2.1. Solubility
The solubilities of the hydrolysates with different DHs, prepared
by Protamex and Alcalase in the pH range of 210, are shown in
Fig. 2. The solubilities increased from about 10% for raw material
(data not shown) to more than 65% over a wide pH range. The in-
crease of solubility was positively correlated with DH. And the dif-
ference of solubility for the hydrolysates prepared by Protamex at
different DHs was signicant (p < 0.5). Characterisation of molecu-
lar structures testied that the zeta potential, molecular size and
surface hydrophobicity of protein hydrolysates were affected by
the enzymatic hydrolysis and DH. The results of solubility indi-
cated that the degradation of large protein molecules to smaller
peptides was critical for marked increase of solubility. Gbogouri
et al. (2004) and Dong et al. (2008) also reported that the solubility
of the hydrolysates increases with the protein fraction with lower
molecular mass, and the smaller peptides from proteins are ex-
pected to have proportionally more polar residues, with the ability
1.0E+02
1.0E+03
1.0E+04
1.0E+05
6 8 10 12 14
Elution time min
M
w
(
D
a
)
Fig. 1. Calibration curve of standard substances on TSKgel G2000SWXL column at
220 nm. Five standard substances were bovine serum albumin (Mw: 67,000 Da),
peroxidase (Mw: 40,200 Da), ribonuclease A (Mw: 13,700 Da), glycine tetramer
(Mw: 246 Da) and p-aminobenzoic acid (Mw: 137.14 Da), respectively.
Table 3
Relative proportion (%) of each molecular weight in the hydrolysates with different DHs prepared by Protamex and Alcalase.
Mw (Da)
>150,000 2468 1360 799 502 387 226 138
Protamex DH 10% 8.3 20.6 29.5 17.0 5.9 12.8 3.5
Protamex DH 20% 4.9 43.4 22.1 17.4 8.2 2.5
Protamex DH 30% 4.7 40.6 27.3 11.6 10.9 3.1
Alcalase DH 10% 6.9 40.8 22.8 5.8 5.1 11.6 6.1
Alcalase DH 20% 4.7 41.7 18.8 2.5 9.6 13.7 7.4
Alcalase DH 30% 3.4 45.5 23.0 4.6 3.2 9.2 9.8
: Not detected.
(a)
60
70
80
90
100
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0
pH
S
o
l
u
b
i
l
i
t
y
(
%
)
DH 10% DH 20% DH 30%
(b)
60
70
80
90
100
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0
pH
S
o
l
u
b
i
l
i
t
y
(
%
)
DH 10% DH 20% DH 30%
Fig. 2. Solubility of the hydrolysates with different DHs prepared by Protamex and
Alcalase as inuenced by pHs. (a) Samples prepared by Protamex; (b) samples
prepared by Alcalase. Bars represent standard deviations from triplicate
determinations.
462 Y. Liu et al. / Food Chemistry 151 (2014) 459465
to form hydrogen bonds with water. The hydrolysates prepared by
Alcalase at lower DH showed higher solubility. This lends further
support to the nding that molecular weights of that hydrolysate
were generally lower.
Some previous studies showed that the surface hydrophobicity
of peptides was another crucial inuence on the solubility of pro-
tein hydrolysate (Gbogouri et al., 2004; Klompong et al., 2007).
However, in the present study the effects of surface hydrophobicity
on the solubility were less important than were the smaller size
and charge group of the peptides produced during the hydrolysis
process. For example, the surface hydrophobicity of the samples
hydrolysed by Alcalase increased with increase of DH, while the
solubility also increased.
The solubilities of protein hydrolysates prepared by both
enzymes were relatively lower at about pH 4.0. The results of
SEC-HPLC evidenced that some protein and/or peptides with high
molecular weight (Mw) remained after hydrolysis. Such molecules
could precipitate at this pH, which was close to the isoelectric
point (pI) of sh proteins.
Due to the high solubility of the hydrolysates over a wide pH
range, it was presumed that the hydrolysates with different DHs,
prepared by Protamex and Alcalase, were good sources of protein
and appropriate for many functional applications.
3.2.2. Emulsifying properties
EAI (m
2
/g) and ESI (min) of the hydrolysates with different DHs,
prepared by Protamex and Alcalase as inuenced by pHs are shown
in Table 4. The hydrolysate with DH 10%, treated by Protamex,
exhibited the best emulsifying properties among all the samples
(p < 0.05). Mutilangi, Panyam, and Kilara (1996) found that higher
solubility accompanied the higher EAI of hydrolysates. In the pres-
ent study, the hydrolysate with DH 10% prepared by Protamex
showed lower solubility than did other samples. Thus, a higher
proportion of larger molecular weight peptides and higher surface
hydrophobicity, which were veried by the above characterisation
of molecular structures, played more important roles in the emul-
sifying properties once the solubility of the sample reached a cer-
tain value. The results also showed that EAI and ESI of
hydrolysates, prepared by both enzymes, decreased with increas-
ing DH, and the difference of the emulsifying properties for the
hydrolysates prepared by Protamex was signicant (p < 0.05) with
increasing DH. Both EAI and ESI of the hydrolysates with DH 20%
and 30%, prepared by Alcalase, were better than those of samples
with the same DH prepared by Protamex (p < 0.05). Thus, the size
and molecular weight of the hydrolysates played the most signi-
cant roles in the emulsifying properties of the present hydroly-
sates. Lee et al. (1987) also reported that there was an optimum
molecular size for peptides to be good emulsiers. Moreover, the
solubility, surface hydrophobicity and amino acid composition of
the hydrolysates prepared by different enzymes may also be vital
factors in governing the emulsifying properties.
When considering the effect of pH on EAI and ESI, the worst EAI
and ESI were found at pH 4.0. It is probable that the pH value was
close to the isoelectric point (pI) of sh proteins; therefore, some
large molecules of the hydrolysates precipitated or the net charges
of the large molecules were reduced, which led to the decrease of
emulsifying properties. Similar results were also found in the study
of Klompong et al. (2007).
3.2.3. Foaming properties
Foaming capacity (%) and foam stability (%) of the hydrolysates
with different DHs, prepared by Protamex and Alcalase as inu-
enced by pHs, are shown in Table 5. The hydrolysate with DH
10% prepared by Protamex also exhibited the best foaming proper-
ties among all the samples (p < 0.05). As DH increased, the hydrol-
ysates prepared by both enzymes displayed a lower foaming
capacity and foam stability (p < 0.05). The results were in agree-
ment with Klompong et al. (2007) and Van der Ven, Gruppen, De
Bont, and Voragen (2002). These authors also stressed that high
molecular weight peptides are generally positively related to foam
stability of protein hydrolysates, and surface hydrophobicity of un-
folded proteins has also been shown to positively correlate with
foaming characteristics.
The foaming properties of the hydrolysates were also affected
by pH value. For foaming capacity and foam stability, the lowest
value was found at pH 4.0 for all the samples, which coincided with
the precipitation of the large protein molecules at their isoelectric
pH. Klompong et al. (2007) found that foaming capacity of protein
hydrolysate of yellow stripe trevally, prepared by Alcalase and Fla-
vourzyme, decreased at very acidic or alkaline pH due to the repul-
sion of peptides (via ionic repulsion). But, in the present study,
higher foaming properties were found at pH 2.0, while the solubil-
ities of the samples were lower at pH 2.0 than those at pH 7.0 and
10.0 (Fig. 2). Thus, it seems that the effects of composition and net
charge of peptides, in hydrolysates produced in the present study,
on the foaming properties outweighed that of solubility.
The hydrolysates with DH 20% and 30% prepared by Alcalase,
exhibited foam properties superior to those of samples with the
same DH prepared by Protamex (p < 0.05). Possibly, the differences
of the surface hydrophobicity, size and charge of peptides could ex-
plain the difference in the foam properties.
3.2.4. Thermal properties
Table 6 compares the onset (T
onset
) and maximum (T
max
) tem-
peratures for endothermic transitions, as well as the net heat en-
ergy (enthalpy, DH) required for the reaction to occur.
Hydrolysates prepared by Protamex exhibited an endotherm with
T
max
shifted to lower temperatures. A shift to lower transition
temperature signied destabilization of protein structure and
therefore led to lower energy required to denature the proteins.
Interestingly, the T
max
values of hydrolysates prepared by
Protamex were higher than those prepared by Alcalase, which
Table 4
Emulsifying activity index (EAI, m
2
/g) and emulsion stability index (ESI, min) of the hydrolysates with different DHs prepared by Protamex and Alcalase as inuenced by pHs.
EAI (m
2
/g) ESI (min)
pH pH
2.0 4.0 7.0 10.0 2.0 4.0 7.0 10.0
Protamex DH 10% 67.7 (4.4)
a
49.7 (3.2)
a
54.9 (2.5)
a
89.7 (2.9)
a
24.0 (2.1)
a
15.6 (1.9)
a
25.3 (2.2)
a
19.7 (1.6)
a
Protamex DH 20% 39.0 (2.2)
cd
31.4 (3.3)
bc
39.2 (4.5)
b
61.4 (2.1)
c
14.1 (1.1)
b
12.7 (2.0)
ab
18.7 (1.0)
bc
13.6 (1.7)
bc
Protamex DH 30% 32.4 (4.3)
d
28.1 (3.4)
bc
33.9 (3.7)
b
58.3 (4.0)
c
13.4 (2.4)
b
10.5 (1.4)
b
17.7 (1.7)
c
12.4 (2.0)
c
Alcalase DH 10% 57.6 (4.2)
ab
34.7 (3.6)
b
36.2 (3.7)
b
75.6 (3.9)
b
20.8 (2.3)
a
13.6 (1.4)
ab
23.2 (1.7)
ab
17.2 (2.0)
ab
Alcalase DH 20% 47.7 (4.6)
bc
31.7 (3.1)
bc
34.9 (2.7)
b
71.6 (2.8)
b
15.0 (1.4)
b
11.8 (2.3)
ab
19.6 (1.5)
bc
14.7 (1.9)
bc
Alcalase DH 30% 37.9 (2.1)
cd
23.3 (3.1)
c
30.2 (2.0)
b
61.0 (2.6)
c
14.2 (1.2)
b
11.5 (1.3)
ab
18.2 (2.5)
bc
14.0 (1.1)
bc
Data show mean values (SD) for three replicates.
The different letters in columns indicate signicant difference at p < 0.05.
Y. Liu et al. / Food Chemistry 151 (2014) 459465 463
suggested that the former showed higher thermal stability than
the latter.
The enthalpy change of the hydrolysates, prepared by both en-
zymes, decreased with the increase of DH. The net DH indicates
cumulative effects of endothermic events, such as the breakdown
of hydrogen bonds and exothermic phenomena (Murray, Arnteld,
& Ismond, 1985). And the DH represents the extent of ordered
structure of a protein (Arnteld & Murray, 1981). Thus the data
suggested that the extent of the ordered structure of the samples
gradually decreased with increasing DH, which was in agreement
with the results of SEC-HPLC.
4. Conclusion
Structural and functional properties of sh protein hydrolysates
from surimi processing by-products, prepared by Protamex and
Alcalase with different DHs, were evaluated. The results reveal that
structures and functional properties of the hydrolysates were
determined by the DH and by the enzyme type employed. The
structurefunctionality relationships revealed by this study sug-
gest that it might be possible to select specic forms of enzymatic
hydrolysis to meet different application needs.
Acknowledgements
This research project was funded by Natural Science Foundation
of China (Project No. 31101214 and 31201427), Science Founda-
tion of Hunan Province, PR China (Project No. 13JJ4054), and the
Ministry of Science and Technology Support Program, PR China
(Project No. 2012BAD31B08).
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Table 5
Foaming capacity (%) and foam stability (%) of the hydrolysates with different DHs prepared by Protamex and Alcalase as inuenced by pHs.
Foaming capacity (%) Foam stability (%)
pH pH
2.0 4.0 7.0 10.0 2.0 4.0 7.0 10.0
Protamex DH 10% 58.1 (1.4)
a
40.3 (3.3)
a
46.2 (2.5)
a
50.5 (2.9)
a
97.5 (1.6)
a
80.6 (2.4)
a
88.6 (1.7)
a
90.0 (2.9)
a
Protamex DH 20% 40.2 (3.4)
bc
30.1 (3.5)
bc
34.7 (2.7)
b
36.2 (2.0)
bc
81.0 (3.2)
cd
74.5 (1.3)
bc
79.9 (2.7)
b
81.2 (1.0)
b
Protamex DH 30% 29.4 (3.2)
d
22.1 (3.3)
c
27.5 (2.5)
c
30.2 (2.1)
c
72.1 (2.4)
e
70.7 (1.4)
c
72.5 (1.7)
c
73.3 (2.0)
c
Alcalase DH 10% 48.2 (3.0)
b
32.0 (2.3)
ab
36.1 (1.6)
b
40.6 (4.1)
b
87.5 (3.0)
b
78.6 (1.4)
ab
80.6 (2.9)
b
85.0 (1.0)
ab
Alcalase DH 20% 46.5 (5.0)
b
32.8 (3.2)
ab
34.0 (2.0)
b
36.0 (2.8)
bc
87.0 (2.0)
bc
77.5 (1.8)
ab
75.9 (2.1)
bc
83.3 (2.0)
b
Alcalase DH 30% 36.6 (3.2)
cd
28.3 (3.3)
bc
32.1 (2.5)
bc
33.0 (3.0)
bc
78.1 (1.0)
de
76.7 (2.3)
ab
71.5 (1.5)
c
81.3 (1.9)
b
Data show mean values (SD) for three replicates.
The different letters in columns indicate signicant difference at p < 0.05.
Table 6
The onset (T
onset
) and maximum (T
max
) temperatures for endothermic transitions and
a net heat energy (enthalpy, DH) required for these transitions for the hydrolysates
with different DHs prepared by Protamex and Alcalase.
Sample T
onset
(C) T
max
(C) DH (J/g)
Protamex DH 10% 76.7 (2.2)
a
105.2 (5.1)
a
148.5 (2.7)
a
Protamex DH 20% 73.7 (3.5)
ab
102.4 (4.6)
a
127.6 (4.2)
b
Protamex DH 30% 68.2 (4.3)
ab
100.9 (3.8)
a
102.5 (3.6)
d
Alcalase DH 10% 56.8 (4.4)
c
95.8 (3.1)
a
119.0 (3.3)
c
Alcalase DH 20% 64.4 (2.8)
bc
94.0 (5.7)
a
113.4 (1.2)
c
Alcalase DH 30% 56.3 (5.6)
c
96.1 (2.6)
a
110.6 (2.8)
cd
The T
onset
, T
max
, and DH were determined from differential scanning calorimetry
(DSC) thermograms (gures not shown).
Data show mean values (SD) for six replicates.
The different letters in columns indicate signicant difference at p < 0.05.
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