Ultrasonics Sonochemistry 20 (2013) 14081413

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Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Enzymolysis reaction kinetics and thermodynamics of defatted wheat

germ protein with ultrasonic pretreatment
Wenjuan Qu a, Haile Ma a,b,, Bin Liu a, Ronghai He a, Zhongli Pan a, Ernest Ekow Abano a,c
a
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, China
b
Key Laboratory for Physical Processing of Agricultural Products, Zhenjiang, Jiangsu 212013, China
c
Agricultural Engineering Department, University of Cape Coast, Cape Coast, Ghana

a r t i c l e i n f o a b s t r a c t

Article history: This research explores the mechanism of ultrasonic pretreatment on enzymolysis of defatted wheat germ
Received 3 December 2012 protein (DWGP). The enzymolysis reaction kinetics and thermodynamics were studied after ultrasonic
Received in revised form 15 April 2013 pretreatments using a probe-type sonicator and an ultrasonic cleaning bath, and the results were com-
Accepted 29 April 2013
pared with traditional enzymolysis. The results showed that both the traditional and ultrasonic pre-
Available online 9 May 2013
treated enzymolysis t well to rst-order kinetics. Both the temperature and ultrasound had a positive
effect on the enzymolysis of DWGP, with temperature playing a dominant role. Under the optimized con-
Keywords:
ditions of DWGP concentration of 1% (w/v), Alcalase concentration of 2000 U/g, time of 10 min and tem-
Wheat germ protein
Polypeptides
perature of 50 C, both the probe and cleaning bath ultrasonic pretreated enzymolysis showed high
Ultrasound polypeptide concentrations (231.019 and 231.320 lg/mL) and low energy requirements. In comparison
Kinetics with traditional enzymolysis, these methods signicantly increased the reaction rate constant (k) by
Thermodynamics 166.7% and 144.4%, 92.9% and 85.7%, 28.0% and 28.0%, 16.1% and 12.9% at 20, 30, 40 and 50 C, and
decreased the activation energy (Ea), enthalpy of activation (DH), Gibbs free energy of activation (DG)
and entropy of activation (DS) by 68.6% and 62.4%, 74.1% and 67.5%, 34.3% and 31.2%, 1.4% and 1.3%. It
can be concluded that ultrasonic pretreatment of DWGP can remarkably improve the enzymolysis ef-
ciency and consequently leads to the production of higher polypeptide yield.
2013 Elsevier B.V. All rights reserved.

1. Introduction that ultrasound increased the enzyme specic activity due to

Wheat germ, rich in protein content (30%), is a potential pro-
tein resource for producing biological polypeptides [1]. Tradi-
tional polypeptide production methods include autolysis,
fermentation and enzymatic hydrolysis [24], in which the
enzymolysis method is usually used due to moderate reaction
conditions and high specicity for substrates [5,6]. However,
traditional enzymolysis exerts some limitations in practical appli-
cations such as low conversion rate of the substrate, low utiliza-
tion rate of the enzyme, and low enzymolysis efciency [7]. This
is mainly due to low contact frequency between enzyme and sub-
strate, and non suitable protein structure. Thus, there is a great
demand for developing more efcient enzymolysis methods to
overcome these shortcomings.
Recently, there are studies conducted on enzyme structural
changes and enzymolysis efciency improvements using an ultra-
sound technology [8,9]. For example, Ma et al. [10] have reported
Corresponding authors. Address: School of Food and Biological Engineering,
Jiangsu University, 301 Xuefu Roadm, Zhenjiang, Jiangsu, 212013, China.
E-mail address: mhl@ujs.edu.cn (H. Ma).
Alcalase surface structural change by increasing the number of
tryptophan and reducing the number of random coil. Shah and
Gupta [11] have indicated that ultrasonic pretreatment enhanced
the enzyme specic activity and therefore accelerated the en-
zyme-catalyzed reaction. These changes are attributed to high
pressures, temperatures and shear forces generated by the ultra-
sonic wave, which can result in increased contact frequency be-
tween substrate and enzyme, and advantageously change
enzyme structure [1215]. Presently, the ultrasound technology
has adopted two kinds of ultrasounds: a cleaning bath and a
probe ultrasound. The cleaning bath ultrasound is performed in
an ultrasonic cleaning bath equipped with the upper and the low-
er at ultrasound emitters and shows the advantage of uniform
distribution of ultrasonic energy in the sample solution. The
probe ultrasound is done through a probe ultrasound emitter sub-
merged below the sample solution in a breaker in order to con-
centrate ultrasonic energy at one place. However, no research
has been reported on the theoretical and practical application of
ultrasonic pretreatment on enzymolysis reaction of defatted
wheat germ protein (DWGP). Therefore, the effects of two kinds
of ultrasonic pretreatments on the enzymolysis reaction are very
important to be studied.

1350-4177/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ultsonch.2013.04.012
 W. Qu et al. / Ultrasonics Sonochemistry 20 (2013) 14081413
The objectives of this research were to (1) study the effects of
the probe and cleaning bath ultrasonic pretreatments on the enzy-
molysis reaction kinetics at different temperatures and times, (2)
determine the thermodynamic parameters of the probe and clean-
ing bath ultrasonic pretreated enzymolysis at different tempera-
tures, and (3) compare the reaction kinetic and thermodynamic
parameters of ultrasonic pretreated enzymolysis with that of tradi-
tional enzymolysis. This research was used to explain the mecha-
nism of ultrasound action on enzymolysis reaction of DWGP and
predict the polypeptide production, which can provide the theoret-
ical basis and technological support for further research in poly-
peptide production.
2. Materials and methods
2.1. Materials and reagents
Defatted wheat germ (DWG) was obtained from An-yang Man-
tianxue Food Manufacturing Co., Ltd (Henan, China). Alcalase
2.4LFG, with the activity of 2.670 U/g and recommended tempera-
ture of 50 C and pH value 9.0, was purchased from Novozymes Co.,
Ltd (Tianjing, China). Bovine serum albumin (BSA), diastase, so-
dium chloride (NaCl), sodium hydroxide (NaOH), hydrochloric acid
(HCl), sodium carbonate (Na2CO3), copper sulfate pentahydrate
(CuSO45H2O), sodium tartrate, sodium tungstate (Na2WO42H2O),
sodium molybdate (Na2MoO42H2O), phosphoric acid (H3PO4), lith-
ium sulfate (Li2SO4H2O), and liquid bromine were purchased from
Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All the re-
agents were of analytical grade. Folin-phenol reagent A was pre-
pared on the day of the test, which comprised of 0.185 M Na2CO3
in 0.098 M NaOH mixed with 0.393 mM CuSO45H2O in
0.694 mM sodium tartrate. Folin-phenol reagent B was prepared
according to the following procedures. A mixture of 100 g Na2WO4-
2H2O, 25 g Na2MoO42H2O, 700 mL DI water, 50 mL 85% H3PO4
and 100 mL HCl was distilled for 10 h in a Soxhlet apparatus. After
adding 150 g Li2SO4H2O, 50 mL distilled (DI) water, and a few
drops of liquid bromine, the mixture was open boiled in a water
bath (SHZ-88A, Taicang Laboratorial Equipment Factory, Jiangsu,
China) for 15 min and then cooled to room temperature (25 C).
The solution was diluted to 1000 mL using DI water and centri-
fuged (Xiangyi Inc., Changsha, Hunan, China) at room temperature
at 10,000 rpm for 5 min, and stored in a 4 C refrigerator until its
usage.
2.2. Methods
The whole experiment was set up according to the following
steps: First, the defatted wheat germ protein (DWGP) was pre-
pared with the method described in the Section 2.2.1. Second,
the DWGP solution was pretreated with two kinds of ultrasounds
according to the method described in the Section 2.2.2. Last, the
DWGP solutions with and without ultrasonic pretreatments were
hydrolyzed with Alcalase based on the method described in the
Section 2.2.3.
2.2.1. Defatted wheat germ protein (DWGP) preparation
The DWGP was prepared based on a reported method [16]. A
volume of 1000 mL DI water was added to 100 g DWG with parti-
cle size of 0.15 mm and then mixed with 1% (w/v) NaCl solution in
a water bath at 50 C for 30 min. Then the solution was adjusted to
pH value 9.0 using 1 M NaOH solution and stirred continuously for
30 min at 50 C. The liquid was separated from the solid by a low
speed centrifuge (LD5-10B, Beijing Lab Centrifuge Co., Ltd, Beijing,
China) operating at 5000 rpm at room temperature for 20 min.
Subsequently, the liquid was adjusted to pH value 6.5 using 1 M
1409
HCl solution and then reacted with diastase added at 20 U/g DWGP
(protein content calculated by Kjeldahl Method) at 50 C for 3 h.
The solution was deposited by adjusting to pH value 4.0 using
1 M HCl solution. The deposited solid was separated from the li-
quid by centrifuging at 5000 rpm at room temperature for
20 min and then the DWGP powder by spray drying was stored
in a desiccator for further analysis.
2.2.2. Defatted wheat germ protein (DWGP) pretreatments
Prior to enzymolysis reaction, the DWGP was pretreated by two
kind of ultrasonic pretreatments using a probe-type sonicator and
an ultrasonic cleaning bath.
A 10 L volume of 2% (w/v) DWGP solution was put into a 15 L
ultrasonic cleaning bath equipped with the upper and the lower
at ultrasound emitters (distance of 7 cm). This ultrasonic cleaning
bath, which was manufactured by Jiangsu University, has function
modes of sweep frequency operation and pulsed operation. For
example, the sweep frequency operation refers to the sweep fre-
quency cycle of increasing period from 22 to 26 kHz and decreas-
ing period from 26 to 22 kHz with the same linear speed in the
form of an isosceles triangle. Therefore the sweep frequency is re-
ported as 24 2 kHz and an increasing period plus a decreasing
period is dened as a cycle time of the sweep frequency in this re-
search. The pulsed operation denotes that the ultrasound is gener-
ated in a pulsed mode with an on-time and an off-time. The DWGP
solution was sonicated at 20 C for 30 min under the following
conditions: power of 1200 W, combination frequency of
24 2 kHz and 68 2 kHz, cycle time of the sweep frequency of
100 s, and pulsed on-time and off-time of 500 and 10 s.
A 1667 mL volume of 2% DWGP solution was put into a 2000 mL
breaker and the sample solution was sonicated in a probe-type
sonicator (GA92-II DB, Shanghai Biotechnology Co., Wuxi, China)
with a probe ultrasound emitter (diameter of 2 cm) submerged
2 cm below the sample solution in the breaker. The sample solu-
tion was treated at 20 C for 10 min under the conditions: power
of 600 W, xed frequency of 20 kHz, and pulsed on-time and off-
time of 2 and 2 s. The control was submitted to the same process
and performed at the same conditions but without ultrasonic
pretreatment.
2.2.3. Defatted wheat germ protein (DWGP) enzymolysis reaction
Both the 2% DWGP solutions with and without ultrasonic pre-
treatments were diluted to 1% solutions prior to enzymolysis reac-
tion. Then 1% sample solutions subjected to probe and cleaning
bath ultrasonic pretreatments were separately adjusted to pH va-
lue 9.0 using 1 M NaOH solution and reacted with Alcalase added
at 2000 U/g DWGP in a water bath at 20, 30, 40, and 50 C for 2,
4, 6, 8, and 10 min, respectively. The solution in process of enzy-
molysis was controlled at pH value 9.0. After the hydrolysis, the
enzyme in hydrolysate was inactivated with the same volume of
6% trichloroacetic acid and then boiled in a water bath for
10 min. Then the hydrolysate was cooled down to 40 C, centri-
fuged from the solid at 10,000 rpm at room temperature for
15 min, and analyzed. The traditional enzymolysis was performed
using the same procedure with ultrasonic pretreated enzymolysis
of the control solution as the substrate.
2.2.4. Enzymolysis reaction kinetics
The rst-order kinetic model reported by Takahashi et al. [17]
was applied in the enzymolysis kinetics study of DWGP. The ki-
netic model was written as:
dC t
kC t 1
dt
where k is the total reaction rate constant involving the rate con-
stants of ultrasound ku and temperature kc (Eq. (4)), and Ct is the
1410 W. Qu et al. / Ultrasonics Sonochemistry 20 (2013) 14081413

DWGP concentration in reaction solution at a given time t min (lg/ 5.5

mL).
After integrating Eq. (1), kinetic model was expressed as:
5.4
ln C t kt ln C 0 2
where C0 is the initial DWGP concentration in reaction solution (lg/
mL). 5.3

ln (V -Vt)
As it is difcult to measure the decrement of DWGP, the reac-
tion rate can be determined by the increased amount of polypep-
tide released by DWGP. The rst-order kinetic model under the 5.2 20 C
o

reaction conditions t = 01, Ct = Q1Qt, and C0 = Q1 was written o

30 C
as: o
40 C
o
lnQ 1  Q t kt ln Q 1 3 5.1 50 C

where Q1 is the ultimate polypeptide concentration in the reaction

solution (lg/mL), and Qt is the polypeptide concentration in the 5.0
reaction solution at a given time t min (lg/mL). 0 1 2 3 4 5 6 7 8 9 10 11
The k and Q1 values can be determined experimentally from (a) Enzymolysis time (min)
the slope and intercept by plotting ln(Q1Qt) against t.
Compared with traditional enzymolysis, the ultrasonic pre- 5.5
treated enzymolysis reaction can be inuenced by the ultrasound
effect in addition to the thermal effect. Therefore, the k in ultra-
sonic pretreated enzymolysis was expressed as: 5.4

k kc ku 4
where kc is the reaction rate constant induced by thermal effect in 5.3
-Vt)

traditional enzymolysis, and ku is the reaction rate constant induced

by ultrasound effect in ultrasonic pretreated enzymolysis.
ln(V

5.2
o
2.2.5. Enzymolysis thermodynamics 20 C
o
Arrhenius equation was applied in describing the relationship 30 C
o
between reaction rate constant k and temperature T, which was 40 C
5.1 o
written as [18]: 50 C
Ea
k Ae RT 5
5.0
where A is the pre-exponential or collision factor (1/min), Ea is the 0 1 2 3 4 5 6 7 8 9 10 11
activation energy (J/mol), R is the universal gas constant (8.314 J/ (b) Enzymolysis time (min)
mol K), and T is the absolute temperature (K).
The plot of ln k against 1/T was used for the calculation of Ea. 5.5
Eyring transition state theory (TST) was used to understand the
effect of temperature on enzymolysis and also the macroscopic
changes observed in this study [10]. The TST equation was written 5.4
as:
   
kB T DG kB T DH DS
-Vt)

k exp  exp  6
h RT h RT R 5.3
ln(V

23
where kB is the Boltzmann constant (1.38  10 J/K), h is the
Planck constant (6.6256  1034 J/s), DG is the Gibbs free energy
of activation (J/mol), DH is the enthalpy of activation (J/mol), and 5.2
DS is the entropy of activation (J/mol K). o
After logarithm transformation, TST equation was expressed as: 20 C
o
    5.1
30 C
k DH 1 DS h o
ln    ln 7 40 C
T R T R kB o
50 C
The plot of ln (k/T) against 1/T was used for the calculations of
5.0
DG, DH, and DS. 0 1 2 3 4 5 6 7 8 9 10 11
(c) Enzymolysis time (min)
2.2.6. Determination of polypeptide concentration
The polypeptide concentration, lg/mL, was determined using a Fig. 1. The ln(Q1Qt) values versus times in traditional (a), probe (b) and cleaning
reported Folin-phenol colorimetric method [19]. Initially, a volume bath (c) ultrasonic pretreated enzymolysis at different temperatures. The regression
coefcients of the curves obtained at 20, 30, 40 and 50 C are 0.96, 0.95, 0.98 and
of 4 mL Folin-phenol reagent A was mixed with 0.5 mL sample in a
0.95 in (a), 0.96, 0.96, 0.96 and 0.99 in (b), and 0.96, 0.96, 0.96 and 0.99 in (c),
glass tube. The solution was incubated for 10 min at room temper- respectively.
ature, after which 0.5 mL Folin-phenol reagent B was added to each
glass tube, and the absorbance was read at 500 nm on a spectro- 30 min incubation. A blank was prepared by using DI water in
photometer (Unic 7200, Unocal Corporation, Shanghai, China) after place of the sample. Standards of 050 lg/mL BSA were assayed
 W. Qu et al. / Ultrasonics Sonochemistry 20 (2013) 14081413 1411

simultaneously with triplicate assays of the samples in order to
calculate the polypeptide concentrations of samples.
2.3. Statistical analysis
All experiments were conducted in triplicate samples. Data was
presented as mean. Analysis of variance (ANOVA) was performed
to compare the effect of ultrasound under the signicance level
of p < 0.05. All graphs and calculation were preformed with the
Origin Pro 7.5SR1 and Microsoft Ofce Excel 2003, respectively.
3. Results and discussion
3.1. Effect of ultrasonic pretreatment on enzymolysis reaction kinetics
The value of rate constant is an important parameter of the
chemical reaction kinetics, which is independent on the substrate
concentration. The change of enzymolysis of DWGP induced by
ultrasonic pretreatment will result in the change of rate constant.
Fig. 1 shows the plots of ln(Q1Qt) versus t in the traditional, probe
and cleaning bath ultrasonic pretreated enzymolysis at different
temperatures. It can be seen from Fig. 1(a) that the ln(Q1Qt) had
a good linear relationship with time at various temperatures, as re-
vealed by the correlation coefcients greater than 0.95. This result
depicted that the traditional enzymolysis process of DWGP obeyed
rst-order kinetics. Because the correlation coefcients in Fig. 1(b)
and (c) were greater than 0.96, the probe and cleaning bath ultra-
sonic pretreated enzymolysis also t well to rst-order kinetics.
In general, both the traditional and ultrasonic pretreated enzymol-
ysis obeyed rst-order kinetics within the temperature range stud-
ied. The reaction kinetic parameters k and Q1 were determined
from the slope and intercept by plotting ln(Q1Qt) against t, using
Origin Pro 7.5SR1, and are listed in Table 1.
It can be observed from Table 1 that the k value was the highest,
followed by kt and ku at all temperatures tested. The higher k than
kt at any temperature indicated that the type of ultrasound
whether the probe and cleaning bath did not signicantly im-
proved the reaction rate of DWGP enzymolysis compared with tra-
ditional enzymolysis. Similarly, Jia et al. [20] have found that the
ultrasonic pretreatment with power of 01800 W and time of
20 min can facilitate the enzymatic hydrolysis of DWGP and pro-
mote the release of polypeptides from DWGP. This is because the
intense high pressures, temperatures and shear forces generated
by ultrasound cavitation efcacies can change the DWGP and Alca-
lase structures [21]. The exposure of active center caused by ultra-
sound would make enzyme combine easily with DWGP and exhibit
higher reaction ability [22,23]. Liu et al. [24] have conrmed that
DWGP structure was changed by ultrasonic treatment with power
of 600 W and time of 10 min determined by FTIR and uorescence
spectra, and its structural changes were benecial to promote
high-efcient enzymatic hydrolysis. Jia et al. [25,26] have also
found that DWGP structure was altered by ultrasound treatments
using free sulfhydryl and disulde bond contents, ultraviolet spec-
trum, and uorescence spectra and therefore its physicochemical
properties changed. Moreover, Ma et al. [10] have reported that
the Alcalase structure determined by uorescence spectroscopy
and circular dichroism (CD) spectroscopy improved as a result of
ultrasound application, which positively affected its activity. In
addition, the higher kt than ku at all temperatures indicated that
temperature played a dominant role in both the probe and cleaning
bath ultrasonic pretreated enzymolysis although the rate constant
was improved by ultrasound application. It was also observed that
both k and kt increased as the temperature increased from 20 C to
50 C. Thus, it can be concluded that the thermal effect dominated
and positively inuenced the enzymolysis reaction of DWGP. This
is due to the ability of increase in temperature to increase the en-
zyme activity and collision frequency between enzyme and sub-
strate, making the enzyme reaction faster [27]. This conrmed
the report by Kyl-Puhju et al. [28] that there is a positive linear
correlation between enzyme activity and temperature. Compared
with traditional enzymolysis, the increasing rates of k in probe
ultrasonic pretreated enzymolysis at 20, 30, 40 and 50 C were
166.7%, 92.9%, 28.0% and 16.1%, respectively with corresponding
increasing rates of k in the cleaning bath ultrasound as 144.4%,
85.7%, 28.0% and 12.9%, respectively. Moreover, the ku decreased
with the increase of enzymolysis temperature, which was opposite
to k and kt. This indicated that ultrasound effects from the probe
and cleaning bath types contributed largely to the rate constant
at lower temperatures but very little at higher temperatures. The
decrease in ku is due to the decrease of gas solubility in the bulk
of uid and the increase of equilibrium vapor pressure of the sys-
tem as the temperature increased [29]. The variation of rate con-
stant ku with temperature was in agreement with the result
reported by Kadkhodaee and Povey [30]. The results showed that
ultrasound application at low temperatures impacted more impor-
tant advantage than that at high temperatures. It can be seen from
Table 1 that the Q1 value was not signicantly inuenced by the
ultrasonic pretreatment when compared with traditional enzymol-
ysis. By considering the enzymolysis efciency and polypeptide
production, the recommended method should be the ultrasonic
pretreated enzymolysis regardless of the type of ultrasound for
the DWGP concentration of 1% (w/v), Alcalase concentration of
2000 U/g, time of 10 min, and temperature of 50 C, which gave a
high polypeptide concentration of 231.019 lg/mL or 231.320 lg/
mL for the probe or cleaning bath ultrasound.
3.2. Effect of ultrasonic pretreatment on enzymolysis thermodynamics
The thermodynamic parameters Ea, and DG, DH and DS were
determined from the slopes and intercepts by plotting lnk against

Table 1
Reaction rate constants in traditional, probe and cleaning bath ultrasonic pretreated enzymolysis at different temperatures.

Type Temperature (C) k (1/min) kc (1/min) ku (1/min) Q1 (lg/mL)

Traditional enzymolysis 20 0.009 0.009 0 243.935
30 0.014 0.014 0 243.861
40 0.025 0.025 0 238.245
50 0.031 0.031 0 245.746
Probe ultrasonic pretreated enzymolysis 20 0.024 (166.7%)a 0.009 0.015 247.176
30 0.027 (92.9%) 0.014 0.013 244.057
40 0.032 (28.0%) 0.025 0.007 225.721
50 0.036 (16.1%) 0.031 0.005 231.019
Cleaning bath ultrasonic pretreated enzymolysis 20 0.022 (144.4%) 0.009 0.013 249.236
30 0.026 (85.7%) 0.014 0.012 245.550
40 0.032 (28.0%) 0.025 0.008 225.270
50 0.035 (12.9%) 0.031 0.004 231.320
a
The values in parenthesis are the increasing rates of reaction rate constant in ultrasonic pretreated enzymolysis compared with traditional enzymolysis.
1412 W. Qu et al. / Ultrasonics Sonochemistry 20 (2013) 14081413

-6.5 tional and ultrasonic pretreated enzymolysis implied that less

energy was needed for enzymolysis reaction and faster reaction
Traditional enzymolysis
rate between the DWGP and Alcalase. In addition, it was observed
Probe ultrasonic pretreated enzymolysis
-7.0 that Ea values of enzymolysis reaction with the probe and clean-
Cleaning bath ultrasonic pretreated enzymolysis
ing bath ultrasonic pretreatments decreased signicantly in com-
parison with traditional one and the decreasing rates were as
-7.5 high as 68.6% and 62.4%. This result further certied from the
ln k (1/s)

mechanism that ultrasound signicantly decreased the energy

barrier required for enzymolysis reaction. Therefore, ultrasonic
-8.0 pretreated enzymolysis ran more quickly than traditional one,
which was consistent with the phenomenon observed in the
experiment. The DH, DG, and DS values, showing a similar trend
-8.5
with Ea, were respectively reduced by 74.1% and 67.5%, 34.3% and
31.2%, and 1.4% and 1.3% for the probe and cleaning bath ultra-
sounds, which also can further explain that the enzymolysis reac-
-9.0
tion catalyzed by ultrasonic pretreatment can occur very swiftly.
A similar report has showed that the thermodynamic parameters
-9.5 (Ea, DH, DG, and DS) of Alcalase can be reduced by ultrasound
3.0 3.1 3.2 3.3 3.4 3.5 treatment and consequently leads to increase in the enzyme
-3
1/T10 (1/K) activity [10]. The large decrease in the thermodynamic parame-
ters can be attributed to the ultrasonically induced breakage of
Fig. 2. The relationship between ln k and 1/T in traditional, probe and cleaning bath hydrogen bonds, disruption of internal hydrophobic core, oxida-
ultrasonic pretreated enzymolysis. The regression coefcients of the curves are tive modication of amino acid residues, initiation of cross-link-
0.98, 0.99 and 0.98, respectively.
ing, and aggregation of proteins [10]. In addition, it was found
that the declines in both the DH and DS were benecial to the
DG decrease, whereas the change in DH had much more inuence
-12.5
on DG because of higher decreasing rate of DH than DS. In gen-
Traditional emzymolysis
eral, the results obtained clearly demonstrated that ultrasonic
Probe ultrasonic pretreated enzymolysis
pretreatment irrespective of the type was benecial for the enzy-
-13.0 Cleaning bath ultrasonic pretreated enzymolysis
molysis reaction of DWGP because of its low energy
ln(k/T) (1/s.K)

requirements.
-13.5
4. Conclusions

-14.0 The results showed that both the traditional and ultrasonic pre-
treated enzymolysis t well to rst-order kinetics. Ultrasound sig-
nicantly promoted the reaction rate of DWGP enzymolysis at
-14.5 various temperatures especially at a low temperature but no sig-
nicant effect on ultimate polypeptide concentration. Compared
with traditional enzymolysis, the rate constants of probe and
-15.0 cleaning bath ultrasonic pretreated enzymolysis were respectively
3.0 3.1 3.2 3.3 3.4 3.5 increased by 166.7% and 144.4%, 92.9% and 85.7%, 28.0% and 28.0%,
1/T10 (1/K)
-3
16.1% and 12.9% at 20, 30, 40 and 50 C. The thermal effect played a
dominant role in both the probe and cleaning bath ultrasonic pre-
Fig. 3. The relationship between ln (k/T) and 1/T in traditional, probe and cleaning
treated enzymolysis compared with ultrasound effect. Both the
bath ultrasonic pretreated enzymolysis. The regression coefcients of the curves are
0.97, 0.99 and 0.96, respectively. probe and cleaning bath ultrasounds showed signicant positive
effects on the thermodynamics of DWGP enzymolysis when com-
pared with traditional enzymolysis, in which the Ea, DH, DG, and
1/T from Fig. 2 and ln (k/T) against 1/T from Fig. 3, respectively DS were respectively reduced by 68.6% and 62.4%, 74.1% and
and are displayed in Table 2. Usually Ea, the amount of energy re- 67.5%, 34.3% and 31.2%, 1.4% and 1.3%. In general, both the probe
quired form non-activation molecules into activation molecules, and cleaning bath ultrasonic pretreated enzymolysis were efcient
reects the rapidity of chemical reactions, which represents a fast methods for producing polypeptides from DWGP. Under the rec-
reaction speed when it is low and a slow speed when high [31]. ommended DWGP concentration of 1% (w/v), Alcalase concentra-
The Ea values of most reactions range from 40 kJ/mol to 400 kJ/ tion of 2000 U/g, time of 10 min and temperature of 50 C, these
mol. If the value is lower than 40 kJ/mol, the reaction will com- methods showed high polypeptide concentrations (231.019 and
plete very rapidly. The lower Ea (<40 kJ/mol) from both the tradi- 231.320 lg/mL) and low energy requirements.

Table 2
Thermodynamic parameters in traditional, probe and cleaning bath ultrasonic pretreated enzymolysis.

Type Ea (103 J/mol) DH (103 J/mol) DS (J/mol K) DG (103 J/mol)

Traditional enzymolysis 34.256 31.699 209.9 96.379
Probe ultrasonic pretreated enzymolysis 10.762 (68.6%)a 8.205 (74.1%) 281.916 (34.3%) 95.077 (1.4%)
Cleaning bath ultrasonic pretreated enzymolysis 12.865 (62.4%) 10.309 (67.5%) 275.358 (31.2%) 95.160 (1.3%)
a
The values in parenthesis are the decreasing rates of thermodynamic parameters in ultrasonic pretreated enzymolysis compared with traditional enzymolysis.
 W. Qu et al. / Ultrasonics Sonochemistry 20 (2013) 14081413
Acknowledgements
The authors wish to extend their appreciation to the National
Natural Science Foundation of China (31071502), the National Pub-
lic Welfare Industry (Agriculture) Science and Technology Special
of China (201303071), the Natural Science Foundation of Jiangsu
Province (BK2012708), and the Priority Academic Program Devel-
opment (PAPD) of Jiangsu Higher Education Institutions for their
nancial support toward the study.
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