Received: 30 April 2018 Revised: 30 June 2018 Accepted: 1 July 2018
DOI: 10.1111/jfpe.12865
OR I G I N A L A R T I C L E
Ultrasound assisted enzymolysis of sunflower meal protein:
Kinetics and thermodynamics modeling
Mokhtar Dabbour1,2 | Ronghai He1 | Benjamin Mintah1,3 | Yingxiu Tang1 | Haile Ma1
1School of Food and Biological Engineering,
Jiangsu University, Zhenjiang, China Abstract
2Department of Agricultural and Biosystems This study examined the effect of dual-frequency ultrasound (DFU) pretreatment on thermody-
Engineering, Faculty of Agriculture, Benha namics and kinetics of sunflower-meal protein (SMP) using alcalase to improve efficiency in
University, Benha, Egypt enzymolysis process. The concentration of hydrolyzed protein and kinetic parameters after tra-
3ILSI-UG FSNTC, Department of Nutrition and ditional pretreatment (control) were investigated and compared with DFU-assisted enzymolysis.
Food Science, University of Ghana, Accra,
Ghana The results indicated that DFU-pretreatment enhanced SMP-enzymolysis efficiency at different
Correspondence substrate and enzyme concentrations, temperature, and pH. Kinetics analysis showed DFU-
Ronghai He and Mokhtar Dabbour, pretreatment reduced the Michaelis constant by 11.29%, while the apparent breakdown rate
School of Food and Biological Engineering, constant increased by 1.96%, indicating DFU-pretreatment improved the affinity among sub-
Jiangsu University, 301 Xuefu Road, Zhenjiang
strate and enzyme. The rate constants for DFU-pretreatment were increased by 45.96, 26.92,
212013, China.
Email: heronghai1971@126.com 21.14, and 27.89% at 293, 303, 313, and 323 K, respectively (p < .05). On Arrhenius kinetics,
and DFU reduced the activation energy, enthalpy and entropy by 24.28, 26.13, and 9.10%, respec-
mokhtar.dabbour@fagr.bu.edu.eg
tively (p < .05). DFU had slight influence on Gibbs-free energy when temperature increased
Funding information
from 293 to 323 K.
Government of Jiangsu Province, Grant/Award
Number: 2016YFD0401401 Practical applications
The positive impact of DFU pretreatment of sunflower meal protein on enzymatic hydrolysis
kinetics makes this method suitable for use in the pharmaceutical and food process industries to
yield peptides from residues of oil industry.
1 | INTRODUCTION To overcome this problem, protein hydrolysis is normally used to
improve and expand the functional properties by enzymatic or chemical
Plant protein is a good and economic alternative for animal protein and modifications. However, the enzymes application provides a milder pro-
plays a dominant role in food formulations. Sunflower seeds are a sig- cess condition and permits for a selective hydrolysis of protein (Lahl &
nificant source of plant protein, which comprises about 20% of protein Braun, 1994). The enzymolysis process is generally used because of its
(Pickardt et al., 2009). Sunflower meal, a residue obtained in large reasonable reaction conditions and high specificity for substrates (Kida
amount after sunflower seed oil extraction, contains an important et al., 1995). Also, enzymolysis can enhance the nutritional properties
amount of protein (about 30–50%) which can be used as food ingredi- (Cui, Qian, Sun, & Zhao, 2016) and physicochemical characteristics
ents. Usually, this residue is used as animal feed, but the high protein (Sun, 2011) of proteins. However, conventional enzymolysis has many
content can increase the residue value if this protein is aimed to human disadvantages including low enzyme utilization, low substrate conver-
nutrition (Damodaran & Paraf, 1997). Defatted sunflower meal is sion and time-consuming (Qu et al., 2012), and low enzymatic efficacy
known to have high quality amino acid profile, but extraction process (Qu et al., 2013). These limitations are mostly attributed to the unsuita-
of industrial oil often affects the biological value of protein, reducing ble protein conformation, which makes it less suitable to attack by the
the bioavailability as well as protein solubility (Lomascolo, Uzan-Boukh- enzyme. Therefore, it is imperative to develop a more effective enzy-
ris, Sigoillot, & Fine, 2012; Ugolini et al., 2015), especially at higher tem- matic method to overcome the mentioned limitations.
perature and at pH value around the point of isoelectric (Damodaran & As a new physical pretreatment method, ultrasound technology is
Paraf, 1997), which limits its application in food processing. broadly used in assisted enzymatic hydrolysis (Qu et al., 2013) due to
J Food Process Eng. 2018;41:e12865. wileyonlinelibrary.com/journal/jfpe © 2018 Wiley Periodicals, Inc. 1 of 10
https://doi.org/10.1111/jfpe.12865
2 of 10 DABBOUR ET AL.
its special acoustic cavitation. The cavitation process produces high
pressures (about 50 MN/m2) and high temperatures (about 5,500 K)
in a short time, resulting in high intensity of shearing force, free radi-
cals and shock waves (Chandrapala, Oliver, Kentish, & Ashokkumar,
2012). Ultrasound pretreatment of plant proteins has been effectively
used to improve bioactivities and efficiency of enzymatic hydrolysis
(Ozuna et al., 2015), particularly from industrial residues such as rape-
seed protein (Jin, Ma, Wang et al., 2015b), wheat gluten (Zhang et al.,
2015) and potato protein (Cheng et al., 2017). This could be attributed
to the thermal and mechanical effects produced by sonic waves, thus
resulting in increased rate of mass transfer and improved contact
frequency between enzyme and substrate (Sangave & Pandit, 2004).
Additionally, the reaction rate and the substrates conversion rate are
improved (Jia et al., 2010; Ma et al., 2011). The protein molecular
structure can be modified by the ultrasound pretreatment (Ozuna
et al., 2015) to reduce the Gibbs free energy ΔG and activation energy
Ea of reaction (Jin, Ma, Qu, et al., 2015a). Moreover, ultrasound pre-
treatment has positive influence on the reaction kinetics of proteolysis
with enzymes by reducing Michaelis constant and improving the initial
velocity of enzymolysis reaction (Jin, Ma, Qu, et al., 2015a).
Knowledge of the hydrolysis reaction kinetics is important for
protein enzymolysis and for improving the utilization of plant proteins
in food products. However, little information on the mechanism and
effects of DFU pretreatment on the enzymolysis efficiency and the
FIGURE 1 The schematic diagram of DFU equipment (Dabbour
parameters of thermodynamics and kinetics of SMP are available. et al., 2018)
Consequently, the objective of this work was to examine the effect of
DFU pretreatment on enzymolysis of SMP under various enzyme and several frequencies (20, 28, 35, 40, and 50 kHz) at a power output of
substrate concentrations, temperatures and pH. Also, the influence of about 300 W. Furthermore, the circulation and temperature of the
DFU pretreatment on the kinetic and thermodynamic parameters of sample solution can be controlled by a liquid circulating system (two
SMP was explored. This work will be useful in describing the mecha- pumps) and a thermostatic bath, respectively, connected to the equip-
nism of DFU action on enzymatic hydrolysis reaction of SMP, which ment. The DFU was directly applied to the sample solutions. An ali-
can provide the scientific basis and technological backing for further quot of 1,000 mL SMP suspension with different substrate
research in peptide production. concentrations (4–20 g/L) was poured into the reaction vessel, and
the probes of ultrasound were submerged to 2.0 cm in the sample.
Based on our previous optimization study (Dabbour, He, Ma, &
2 | MATERIALS AND METHODS
Musa, 2018), the DFU experiments were conducted at the optimum
conditions for dual-frequency mode: power density (220 W/L), tem-
2.1 | Materials perature (45  2 
C), time (15 min), dual-frequency (20/40 kHz),
Sunflower meal (protein content 26.38% estimated by the Kjeldahl pump circulation speed (100 rpm), and pulsed off and on-time 2 and
method) was kindly obtained from Xinjiang Jinhai Oils Co., Ltd. 5 s, respectively. An experimental control (traditional enzymolysis)
(Xinjiang, China). It was milled into powder using a DFT-100A mill was carried out under the same parameters but without DFU pre-
(Wenling Lin Da Machinery Co., Ltd., Jiangsu, China) to realize the size treatment, employing an impeller-agitator at a speed of 100 rpm. All
of particles lower than 60-mesh size, then kept in a zip lock plastic experiments were run in triplicate.
bags at 4 
C for further processing. Alcalase 2.4 LFG (activity
150,000 U/mL and density of 1.17 g/mL) was purchased from Novo- 2.3 | Enzymolysis of SMP
zymes Biological Technology Co. Ltd. (China). Trichloroacetic acid
The enzymolysis apparatus comprises of an impeller-agitator (JJ-1,
(TCA) and other reagents used in this research were of analytical
Jintan Xichengxinrui instrument Co., Jiangsu, China) at a speed of
grade.
100 rpm, an electro-thermostatic bath (HH-6A, Zhongzheng equip-
ment manufacture Co. Ltd., Jintan, China), a digital pH meter (PB-10,
2.2 | DFU pretreatment Sartorius Scientific instruments, Beijing, China), and centrifuge (TGL-
DFU (Miebo Biotech. Co., Jiangsu, China) (Figure 1) pretreatment of 16, High-Speed Tabletop, Pioway, Nanjing, China). Following the con-
SMP solution was followed by enzymolysis. The DFU consists of con- trol and DFU pretreatments, the pH of sample solutions at different
trollers, a reaction vessel and two probes. Each probe produces protein concentrations (4, 8, 12, 16, and 20 g/L) were adjusted to 7.0,
DABBOUR ET AL. 3 of 10
8.0, and 9.0 using 1 mol/L NaOH after 15 min of preheating at given Thus, km and kA can be determined experimentally from the slope
temperatures (293, 303, 313, and 323 K), and the alcalase (1,000, and intercept, respectively, of a linear line by plotting 1V versus
1
i C
.
i
2,000, 3,000, and 4,000 U/g) was added to start the enzymolysis
reaction. The time of hydrolysis was 90 min, and the pH value of solu-
2.7 | Enzymolysis reaction kinetics (k)
tions was maintained by continuous addition of 1 mol/L NaOH during
the enzymatic hydrolysis process. The first-order kinetic model was used to estimate the reaction rate
constant k of SMP (Kadkhodaee & Povey 2008). The equation of
2.4 | Determination of hydrolysis degree (DH) kinetic model was given as follows:
DH for SMP was estimated by the pH-stat method outlined by Adler- dCt ¼ −kC ð6Þ
dt t
Nissen (1987) as follows:
After integrating, kinetic model was described as:
DH ð%Þ¼ BNb ×100 ð1Þ
αMphtotal ln Ct ¼ −kt+ ln Ci ð7Þ
−1
where, B representing the amount of NaOH consumed (mL); Nb is the where, k representing the reaction rate constant (min ), and Ct is the
NaOH concentration (mol/L); α denotes the mean dissociation degree SMP concentration (g/L) at a given time t (min).
of the α-NH in sunflower protein, which is 0.885 for alcalase; M As it is difficult to estimate the decrement of SMP, the reactionp
denotes the mass of hydrolyzed protein (g); and htotal representing the rate can be obtained from the amount of peptides released by SMP.
total content of peptide bonds in the protein (7.50 mmol/g sunflower At specific pressure and temperature, Ci = Vu and Ct = (Vu − Vt). So,
protein) (Ren et al., 2015). Equation (7) was rearranged to:
lnðVu−VtÞ¼ −kt+ lnVu ð8Þ
2.5 | Determination of hydrolyzed protein
where, Vu represents the maximum peptide concentration (μg/mL),
concentration (Pc)
which was obtained under the optimal DFU pretreatment parameters
To evaluate the enzymolysis kinetic parameters of SMP, the concen- and the optimum enzyme conditions pH = 9.0 and 323 K for 5 hr and
tration of hydrolyzed SMP was estimated according to Galvao, Pinto, Vt denotes the peptide concentration (μg/mL) at a certain time t. The
Jesus, Giordano, and Giordano (2009) as follows: value of k was calculated from the slope by plotting ln(Vu − Vt) against
enzymolysis time (t).
Pc¼Ci ×DH×0:01 ð2Þ Compared with control (without ultrasonic), the ultrasonic
where, Pc representing the concentration of hydrolyzed SMP (g/L), assisted enzymolysis reaction can be influenced by the ultrasonic and
and Ci is the initial SMP concentration (g/L). thermal effect. Consequently, the k in ultrasonic assisted enzymolysis
was described as:
2.6 | Measurement of initial reaction rate and kinetic k¼ kc + ku ð9Þ
parameters
where, kc and ku represents the reaction rate constant induced by
The initial reaction rate (Vi) of both the DFU and control pretreat- thermal effect in control and ultrasound effect in ultrasound pre-
ments of SMP were determined according to Jin, Ma, Wang, treated enzymolysis (min−1), respectively.
et al. (2015b) with slight alterations.
¼PcV 5 min ð3Þ 2.8 | Enzymolysis thermodynamicsi 5
Arrhenius equation used in describing the relation between k and
where, Vi is the initial rate of reaction (g/L. min), and Pc representing
enzymolysis temperature T (Parkin, 2007) was determined as:
the concentration of hydrolyzed SMP (g/L) for the first 5 min.
The kinetic model reported by Schurr and McLaren (1965) was ¼ −Eak Ae RT ð10Þ
applied in the enzymolysis kinetics study of SMP to determine
where, A represents the pre-exponential factor (min−1), T represents
the kinetic parameters kA and km. The kinetic model was given as absolute temperature (K), R represents constant of universal gas
follows:
(8.314 J/mol K), and Ea represents the energy of activation (J/mol).
¼ kA Ec Ci ð Þ The Equation (10) was transformed and rewritten as follows:Vi 4km +Ci
ln k¼ Ea− + lnA ð11Þ
where, kA indicates the average value of apparent breakdown rate RT
constant (min−1), Ec represents the enzyme concentration (g/L), and The linear plot of ln(k) versus 1/T was used for the estimation
km representing the Michaelis constant (g/L). Linearization of the of Ea.
Equation (4) as follows: Eyring transition state theory (TST) was applied to obtain the
1 ¼ km 1 1 ð Þ changes in thermodynamic parameters (ΔG, ΔS, and ΔH) of the enzy-× + 5
Vi kA Ec Ci kA Ec molysis process according to (Parkin, 2007).
4 of 10 DABBOUR ET AL.

 
¼ kB T ΔGk exp ð12Þ 3 | RESULTS AND DISCUSSION−
h RT
where, k denotes the Boltzmann constant (1.38 × 10−23 J/K), 3.1 | Effects of DFU and control pretreatments onB
h denotes Planck constant (6.6256 × 10−34 J s), ΔG denotes Gibbs the SMP enzymolysis
free energy (J/mol), ΔH represents the activation enthalpy (J/mol), The effect of DFU pretreatment and traditional enzymolysis on the con-
and ΔS denotes the activation entropy (J/mol K). centrations of hydrolyzed protein at different concentrations of sub-
The Gibbs free energy ΔG was determined as follows:
strate and enzyme, temperatures and pH were shown in Figures 2–5. It
ΔG¼ΔH−TΔS ð13Þ was observed from Figure 2, the concentration of hydrolyzed protein
raised gradually with the increase of hydrolysis time and substrate
After combination the Equations (12) and (13), TST equation was concentration. The concentrations of hydrolyzed protein curves were
transformed and written as follow: characterized by the initial rapid rate followed by a fast reduction in
  rate for DFU pretreatment and traditional enzymolysis. However, the
k ΔH 1 k ΔS
ln ¼ × + ln B− + ð14Þ concentrations of hydrolyzed protein in control and DFU pretreat-
T R T h R
ments increased gradually during the first 15 min of hydrolysis, then
The ΔH and ΔS values were obtained from the slop and intercept, shown a slow improvement until achieving stabilization in the 20th
respectively, of the linear plot of ln k versus 1. min. This occurred particularly at the lowest substrate concentrationsT T
(4 and 8 g/L) and the rate of increase slowed down at higher concen-
trations of substrate (12–20 g/L). This denotes that high values of
2.9 | Determination of peptide concentration
substrate concentration demands more time to achieve equilibrium.
The concentration of peptide (μg/mL) of SMP hydrolysate was esti- Additionally, it was observed that at the first 15 min the rate of enzy-
mated using the Folin-phenol method (Ledoux & Lamy, 1986). The molysis for DFU assisted enzymolysis was much higher than that of
SMP hydrolysate was mixed with 15% TCA (C2HCl3O2) (volume ratio the control pretreatment, particularly at high substrate concentration
of 2:1) and incubated at room temperature for 30 min. Then it was (12–20 g/L). As well, the results demonstrated that DFU pretreatment
centrifuged at 4,000g for 10 min. The absorbance of peptide was ana- remarkably enhanced the concentration of hydrolyzed protein com-
lyzed at 680 nm using an UV-spectrophotometer (TU-1810, Pui Gen- pared with traditional enzymolysis particularly at the beginning of the
eral Instruments Co. Ltd., Beijing, China). Deionized water was used as hydrolysis. The curves of enzymatic hydrolysis at various concentra-
a blank instead of the sample. tions of substrate corroborates this obtained by Qu et al. (2012).
Figure 3 shows the enzymolysis curves at different temperatures.
It was concluded, from the curves, that increasing the enzymolysis
2.10 | Statistical analysis
temperature increased the concentration of hydrolyzed protein for
All experiments and analyses were repeated in triplicate. The results both DFU pretreatment and traditional enzymolysis. The DFU pre-
were described as mean  standard deviations. Analysis of variance treatment produced a noticeably higher hydrolyzed protein than the
(ANOVA) was conducted to measure the influence of DFU at the signifi- control pretreatment at all temperature points, especially at 313 and
cance level of p < .05 using Minitab 17.0 Software. The differences 323 K. The concentration of hydrolyzed protein prepared with DFU
between individual groups were analyzed by one-way ANOVA with pretreatment was enhanced (p < .05) by 21.29, 13.58, 8.89, and
Tukey’s pairwise comparison test. All calculation and graphs were pre- 6.05% when compared with the traditional pretreatment (i.e., without
sented with the Microsoft Excel 2016 and Origin Pro 8.0 Software, ultrasonic) at 293, 303, 313, and 323 K, respectively, during the
respectively. 90 min of hydrolysis time. This indicated that the DFU pretreatment
FIGURE 2 The concentrations of hydrolyzed protein in traditional and DFU assisted enzymolysis at various substrate concentrations (pH = 9.0,
Ec = 3,000 U/gprotein, and T = 323 K)
DABBOUR ET AL. 5 of 10
FIGURE 3 The concentrations of hydrolyzed protein in traditional and DFU assisted enzymolysis at various temperatures (pH = 9.0,
Ec = 3,000 U/gprotein, and Ci = 12 g/L)
was more efficient than traditional enzymatic hydrolysis when the did not show significant (p > .05) difference after enzymolysis time of
temperature increased from 293 to 323 K. The trend of the results 30 min at pH 8.0 and 9.0. Additionally, when the hydrolysis time
agreed with that obtained by Jin, Ma, Qu, et al. (2015a). increased to 60 min, the comparable findings were obtained (p > .05)
Figure 4 shows the concentration of hydrolyzed protein in both at pH 7.0. Hence, the results proved that DFU pretreatment could sig-
traditional enzymolysis and DFU pretreatment under various enzyme nificantly improve the hydrolyzed protein concentration at high pH of
concentrations. In the whole enzymatic hydrolysis process, the con- SMP compared with control (without DFU).
centration of hydrolyzed SMP increased by increasing enzyme con-
centration. Nevertheless, the DFU pretreatment presented a relative
3.2 | Effect of DFU pretreatment and traditional
sharp increase in the first 15 min especially at the enzyme concentra-
enzymolysis on kinetic parameters and initial
tions of 3,000 and 4,000 U/g when compared with traditional enzy-
reaction rate
molysis. This could be attributed to ultrasonic assisted enzymolysis
stimulated molecular unfolding of protein (Gülseren, Güzey, Bruce, & Enzymolysis reaction kinetics study is highly complex. This can be
Weiss, 2007), which was facilitated by binding by the enzyme and attributed to the several peptide bonds involved in reaction and their
consequently improving the hydrolysis rate. The trend agreed with differing vulnerability to be attacked by enzymes during the hydrolysis
that obtained by Jin, Ma, Qu, et al. (2015a) for corn gluten meal. process (Gónzalez-Tello, Camacho, Jurado, Paez, & Guadex, 1994).
Enzymolysis process of SMP in DFU and traditional pretreat- Generally, the reaction rates of enzymolysis are described by
ments under various pH during the 90 min of hydrolysis time was Michaelis–Menten kinetic models (Barros & Malcata, 2004). The sig-
shown in Figure 5. The results indicated that the hydrolyzed protein nificance of Michaelis constant in hydrolysis reaction kinetics cannot
concentration treated by DFU and traditional before the hydrolysis be underrated. It is a parameter that is not dependent on the sub-
enhanced by increasing hydrolysis time. The increase rate in hydro- strate concentration and it is impartial to characterize the affinity
lyzed protein was quite slow for the pH 7.0. Nevertheless, for the among an enzyme and substrate in the enzymolysis reaction. A little
pH 8.0 and 9.0, the concentrations of hydrolyzed protein continued value of km indicates a rapid reaction (Tardioli, Sousa, Giordano, &
to increase quite linearly during the hydrolysis time of 90 min. At Giordano, 2005). To estimate the variation of kinetic parameters
same hydrolysis time and pH, the DFU pretreatment was found to be which was influenced by hydrolysis of ultrasonicated sunflower meal
higher than the traditional enzymolysis. However, DFU pretreatment protein, the Lineweaver–Burk equation (Equation (4)) was applied, and
FIGURE 4 The concentrations of hydrolyzed protein in traditional and DFU assisted enzymolysis at various enzyme concentrations (pH = 9.0,
Ci = 12 g/L, and T = 323 K)
6 of 10 DABBOUR ET AL.
FIGURE 5 The concentrations of hydrolyzed protein in traditional and DFU assisted enzymolysis at various pH (Ec = 3,000 U/gprotein,
Ci = 12 g/L, and T = 323 K)
the plots were given in Figure 6. The plots of 1/Vi against 1/Ci for tra- Michaelis constant km. Similar trend was reported by Ren, Ma, Mao,
ditional enzymolysis and DFU pretreatment indicated a good linear and Zhou (2014) and Wang et al. (2016).
regression with their determination coefficient (R2) values of .986 and The kinetic parameter kA denotes the apparent breakdown rate
.990, respectively. constant between SMP and enzyme. In our study, the kA value of
The calculated kinetic parameters (kA and km) are presented in DFU pretreatment was higher than traditional enzymolysis by 1.96%.
Table 1. The results show that the km value for DFU assisted enzymo- The increase of kA value (with DFU pretreatment) is due to the
lysis reduced by 11.29% compared with the traditional one (control). improved mixing of proteases and substrate under ultrasound irradia-
It was proved that DFU pretreatment of SMP decreased the Michaelis tion. Comparable results were reported in the previous studies
constant. The variation of km demonstrated that ultrasound pretreat- (Qu et al., 2012; Zhang et al., 2015) demonstrating increase in the
ment was able to enhance the enzymolysis efficiency of SMP. Since values of kA after ultrasound pretreatment. Other researchers, how-
km is usually used to describe the affinity of enzyme to the substrate, ever, have indicated that when samples were subjected to ultrasound
reduction of k confirmed increase in the affinity between SMP and pretreatment before enzymolysis process, the kA value decreasedm
alcalase. The reduction in k value for DFU pretreatment may be (Cheng et al., 2017). This conflicting result could be due to the variousm
attributed to the sonication haven partially modified the conformation processing conditions and ultrasonication modes used by other
of SMP by affecting the noncovalent interactions (e.g., hydrophobic researchers. In conclusion, DFU pretreatment (in the current study)
and electrostatic interactions, hydrogen bonds, loosening the protein enhanced the enzymolysis efficiency by improving the affinity and
tissue, and van der Waals forces) (Wang et al., 2016). Additionally, binding frequency among the enzyme alcalase and substrate.
ultrasound assisted enzymolysis unfolds protein molecules, reduces The initial reaction rates (Vi) for DFU pretreatment and traditional
the particle size of SMP, and exposes active sites to enzyme action enzymolysis under various substrate concentrations are presented in
(Subhedar & Gogate, 2014) and thus resulted in the decrease in the Table 1. The value of Vi for both methods increased by increasing sub-
strate concentrations. The results show that DFU pretreatment at
20 g/L substrate concentration indicated the highest Vi value
(0.190  0.0027 g/L min). DFU pretreatment is clearly superior to the
control (traditional enzymolysis) at every substrate concentrations.
The Vi of DFU pretreatment increased by 8.79, 8.59, 4.60, 3.41, and
6.32% compared with that of the traditional enzymolysis at 4, 8,
12, 16, and 20 g/L, respectively (p < .05). So, these results prove that
DFU assisted enzymolysis significantly affected the hydrolysis reac-
tion rate. That means, DFU pretreatment before proteolysis can accel-
erate the enzymolysis of SMP. This phenomenon is consistent with
the results of Jia et al. (2010) and Cheng et al. (2017). This improve-
ment in enzymolysis of SMP may be due to the mechanical and cavita-
tion effects of DFU, which induces less agglomeration and easier
dispersion of enzyme.
3.3 | Effects of DFU pretreatment and traditional
enzymolysis on reaction rate constant k
FIGURE 6 The reciprocal plots of the initial reaction rate (1/Vi)
versus the reciprocal of the substrate concentration (1/C ) in DFU As mentioned above, Arrhenius and TST equations were applied toi
pretreatment and traditional enzymolysis calculate the parameters of thermodynamics. The constant rate (k) is
DABBOUR ET AL. 7 of 10
important factor in these equations and depended on the hydrolysis
temperature. Thus, we studied the changes in the k values induced by
traditional enzymolysis and DFU pretreatment by hydrolyzing the
SMP (12 g/L), enzyme concentration (3,000 U/gprotein = 0.281 g/L),
at different temperatures (293–323 K), pH 9.0 ,and the linear plots
between 3 and 15 min were taken into consideration. The plots of
ln(Vu − Vt) versus enzymolysis time in DFU and traditional pretreat-
ments at various temperatures are shown in Figure 7. The plots, both
DFU assisted enzymolysis and control (without ultrasonic) curves for
sunflower meal protein, indicated a good linear relationship with
hydrolysis time at different temperatures. The linearity is good
because the determination coefficients (R2) are higher than .92 and
.95 for DFU pretreatment and control, respectively. Then, our hypoth-
esis applying the first-order kinetics model to determine the constant
rate was suitable. The k values were obtained from the slope of the
linear plots versus enzymolysis time. The rate constants of DFU pre-
treatment ku and traditional enzymolysis kc at various temperatures
are demonstrated in Table 2. The value of rate constant k increased
gradually as enzymolysis temperature increased from 293 to 323 K,
for traditional and DFU pretreatments (Table 2). Consequently, the
results indicated that the thermal impact was dominant and had a pos-
itive influence on the enzymolysis reaction of SMP. This increase in k
may be due to the improvement of alcalase activity and higher colli-
sion frequency among enzyme and substrate at higher temperature
(Ma et al., 2011). Additionally, the higher kc values than ku at different
temperatures indicated that the temperature played a vital role in
DFU pretreatment although the values of reaction rate k were
enhanced by DFU application. Compared with control (traditional
enzymolysis), the reaction rate k of DFU pretreatment were increased
(p < .05) by 45.96, 26.92, 21.14, and 27.89% at 293, 303, 313, and
323 K, respectively. Comparable results were reported in previous
studies (Kadkhodaee & Povey, 2008; Subhedar & Gogate, 2014). The
rate constant k values for DFU pretreatment significantly increased
over the traditional enzymolysis group, suggesting that ultrasonication
improved the alcalase activity; therefore, enzymolysis efficiency was
significantly enhanced in the DFU compared with control.
3.4 | Effects of DFU pretreatment and traditional
enzymolysis on the thermodynamic parameters
Activation energy strongly influences enzymolysis kinetics which rep-
resents the least energy needed to begin chemical reactions. It needs
to alter stable molecules into reactive molecules, which reflects the
rapidity and rate of chemical reactions (Qu, Pan, & Ma, 2010).
The activation energy values of most reactions varied from 40 to
400 kJ/mol. The reaction will be completed very quickly if the activa-
tion energy value is less than 40 kJ/mol (Qu et al., 2013). After rate
constants (k) were obtained, ln(k) versus 1/T for DFU pretreatment
and traditional enzymolysis (control) were plotted. The activation
energy (Ea) were obtained from the slope of the curves (Figure 8a) and
the results were presented in Table 3. The values of Ea were 23.89
and 31.49 kJ/mol for DFU and control pretreatments, respectively.
The results show that the enzymolysis reaction required less energy
after DFU and control pretreatments and subsequently leads to faster
reaction between the alcalase and SMP. DFU pretreatment reduced
TABLE 1 Effect of traditional enzymolysis and DFU pretreatment on kinetic parameters and initial reaction rate
Initial reaction rate (g/L min)
Enzymolysis methods kmk E (min)
1
k E (L min/g) k (g/L) k (min
−1
m A ) 4 (g/L) 8 (g/L) 12 (g/L) 16 (g/L) 20 (g/L)
A c A c
Traditional enzymolysis 32.37  1.915 4.11  0.259 7.88  0.466 0.50  0.032 0.083  0.0002b 0.117  0.0006b 0.145  0.0015b 0.170  0.0015b 0.178  0.0003b
DFU pretreatment 28.25  1.416 4.04  0.192 6.99  0.351 0.51  0.024 0.091  0.0009a 0.128  0.0012a 0.152  0.002a 0.176  0.0015a 0.190  0.0027a
Increase percentage (%) – – −11.29 1.96 8.79 8.59 4.60 3.41 6.32
Note: Ec = 0.486 g/L.
Within a column, the mean values with different letters show significant difference (p < .05). Means  SD (n = 3).
8 of 10 DABBOUR ET AL.
FIGURE 7 The ln(Vu − Vt) values versus enzymolysis time (t) for DFU pretreatment and traditional enzymolysis at different temperatures with
enzyme and substrate concentrations of 3,000 U/g and 12 g/L, respectively
Ea by 24.28% (p < .05) compared with the traditional enzymolysis. distribution of enzyme and SMP in the reaction process after DFU
Thus, DFU assisted enzymatic hydrolysis proceeded speedily than the pretreatment, which resulted from enhancement of the binding affin-
traditional one, which was experimentally verified. The reduction in Ea ity among substrate and enzyme. Moreover, negative ΔS values indi-
after sonication was reported previously (Ma et al., 2011; Qu et al., cate the decrease of entropy in the enzymolysis process. The ΔG
2013), confirming that ultrasonic pretreatment may facilitate the values increased by increasing the temperature (293–323 K) for both
enzymatic hydrolysis process. DFU pretreatment and traditional enzymolysis (Table 3). Compared
Moreover, the variations induced by DFU pretreatment and tradi- with control, the values of ΔG for DFU pretreatment were decreased
tional enzymolysis in the enthalpy (ΔH), entropy (ΔS), and Gibbs free by 1.30, 1.05, 0.80, and 0.56% when the temperature increased from
energy (ΔG) of the enzymolysis reaction were determined according 293 to 323 K, respectively. The decrease ratio of ΔG reduced with
to the Eyring equation. The linear plot of ln(k/T) versus 1/T is shown increasing enzymolysis temperature. Furthermore, the increase rate in
in Figure 8b, the ΔS and ΔH values were estimated from the intercept ΔG of DFU pretreatment was higher when compared with the
and slope of the linear plots, respectively. The values of ΔG at various traditional enzymolysis group. Similar trend was reported by Jin, Ma,
temperature were determined from Equation (13). The parameters of Qu, et al. (2015a). Nevertheless, the values of ΔG were positive,
thermodynamic ΔH, ΔS, and ΔG results were presented in Table 3. demonstrating that the hydrolysis reaction was nonspontaneous.
Dramatic reduction in ΔH signifies that DFU pretreatment modified Generally, the changes in ΔH and ΔS caused by DFU pretreatment
the structure of protein by disruption of hydrophobic interactions, sta- resulted from increased hydrolysis efficacy following ultrasound
bilizing the relationship between protein and enzyme, oxidative alter- assisted enzymolysis.
ation of amino acid residues, and converting the enzyme-substrate
complex for ground state to active state (Ma et al., 2011; Cheng et al.,
2017). Furthermore, the positive ΔH value confirms endothermic 4 | CONCLUSIONS
nature of enzymolysis reaction and improved the hydrolysis process
by decreasing the enthalpy (ΔH) rate by 26.13% (p < .05). Additionally, DFU pretreatment noticeably promoted sunflower meal protein enzy-
low value of ΔH meant minimize energy cost in converting substrates molysis efficiency compared with control (without ultrasound) under
to products. The values of ΔS indicated a similar reduction trend such various hydrolysis conditions including substrate concentration, tem-
as Ea and ΔH. The ΔS value for DFU decreased by 9.10% (p < .05) perature, enzyme concentration, and pH. The increase of enzymolysis
compared with the traditional enzymolysis, indicating that DFU efficiency was related to the decrease of the kinetic parameter km and
assisted enzymolysis improved the affinity among the substrate and thermodynamic parameters Ea, ΔH, ΔS, and ΔG. These decreased the
enzyme. The reduction in ΔS was probably due to more orderly energy barrier among active and ground state of alcalase–substrate
TABLE 2 Reaction rate constants and R2Adj in DFU pretreatment and traditional enzymolysis at different absolute temperatures
Enzymolysis methods T (K) k (min−1) k (min−1) k (min−1c u ) R2Adj
Traditional enzymolysis 293 0.0127  0.0005f 0.0127  0.0005 0 .989
303 0.0209  0.0012e 0.0209  0.0012 0 .994
313 0.0332  0.0036c 0.0332  0.0036 0 .950
323 0.0411  0.0029b 0.0411  0.0029 0 .975
DFU pretreatment 293 0.0235  0.0013de 0.0127  0.0005 0.0108  0.0013 .988
303 0.0286  0.0042cd 0.0209  0.0012 0.007  0.0042 .920
313 0.0421  0.0022b 0.0332  0.0036 0.0089  0.0022 .989
323 0.0570  0.0034a 0.0411  0.0029 0.0159  0.0034 .986
Within a column, the mean values with different letters show significant difference (p < .05). Means  SD (n = 3).
DABBOUR ET AL. 9 of 10
FIGURE 8 The linear fitting curves by plotting (a) lnk versus 1/T (b) lnk/T versus 1/T for DFU pretreatment and traditional enzymolysis
TABLE 3 Thermodynamic parameters for traditional enzymolysis and DFU pretreatment
ΔG (kJ/Mol)
Enzymolysis methods Ea (kJ/mol) ΔH (kJ/mol) ΔS (J/mol K) 293 (K) 303 (K) 313 (K) 323 (K)
Traditional enzymolysis 31.51  0.377a 28.93  0.380a −215.95  1.236a 92.20  0.360 94.37  0.375 96.53  0.385 98.68  0.400
DFU pretreatment 23.86  0.286b 21.37  0.284b −237.56  0.923b 91.00  0.270 93.38  0.280 95.76  0.285 98.13  0.300
Decrease (%) −24.28 −26.13 −9.10 −1.30 −1.05 −0.80 −0.56
Within a column, the mean values with different letters show significant difference (p < .05). Means  SD (n = 3).
complex and made the correlation of alcalase and substrate more effi- taste characteristics of the resulting hydrolysates. International Journal
cient and easier and consequently lead to improve the affinity among of Food Science and Technology, 51(5), 1298–1304.
Dabbour, M., He, R., Ma, H., & Musa, A. (2018). Optimization of ultrasound
the sunflower meal protein and alcalase. Generally, DFU assisted
assisted extraction of protein from sunflower meal and its physico-
enzymolysis could enhance the SMP enzymatic hydrolysis, and this chemical and functional properties. Journal of Food Process Engineering,
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ACKNOWLEDGMENTS enzymatic hydrolyses of cheese whey. Journal of Food Engineering,
This research was supported by National Primary Research & 91(1), 109–117.
Gónzalez-Tello, P., Camacho, F., Jurado, E., Paez, M. P., & Guadex, E. M.
Development Plan (2016YFD0401401), Primary Research & Develop- (1994). Enzymatic hydrolysis of whey proteins: I kinetic models.
ment Plan of Government of Jiangsu Province (No. BE2016352, Biotechnology and Bioengineering, 44(4), 523–528.
BE2016355) and sponsored by Qing Lan Project. Gülseren, I., Güzey, D., Bruce, B. D., & Weiss, J. (2007). Structural and
functional changes in ultrasonicated bovine serum albumin solutions.
Ultrasonics Sonochemistry, 14, 173–183.
ORCID Jia, J., Ma, H., Zhao, W., Wang, Z., Tian, W., Luo, L., & He, R. (2010). The
Mokhtar Dabbour https://orcid.org/0000-0001-6109-9065 use of ultrasound for enzymatic preparation of ACE-inhibitory peptides
from wheat germ protein. Food Chemistry, 119, 336–342.
Ronghai He https://orcid.org/0000-0002-0904-0522 Jin, J., Ma, H., Qu, W., Wang, K., Zhou, C., He, R., … Owusu, J. (2015a).
Haile Ma https://orcid.org/0000-0002-3136-7927 Effects of multi-frequency power ultrasound on the enzymolysis of
corn gluten meal: Kinetics and thermodynamics study. Ultrasonics
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