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Enzymolysis kinetics, thermodynamics and model of porcine cerebral protein with single-frequency countercurrent and pulsed ultrasound-assisted processing
Ultrasonics Sonochemistry 28 (2016) 294–301
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Enzymolysis kinetics, thermodynamics and model of porcine cerebral protein with single-frequency countercurrent and pulsed ultrasoundassisted processing Ye Zou a,1, Yangyang Ding b,1, Weiwei Feng a, Wei Wang a, Qian Li a, Yao Chen c, Huiyu Wu d, Xintong Wang b, Liuqing Yang b,⇑, Xiangyang Wu c,⇑ a
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China School of Chemistry and Chemical Engineering, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China c School of the Environment and Safety, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China d School of Pharmacy, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China b
a r t i c l e
i n f o
Article history: Received 23 June 2015 Received in revised form 24 July 2015 Accepted 11 August 2015 Available online 11 August 2015 Keywords: Porcine cerebral protein Single-frequency countercurrent and pulsed ultrasound Enzymolysis Kinetics Thermodynamics Enzymolysis model
a b s t r a c t The present work investigated the enzymolysis kinetics, thermodynamics and model of porcine cerebral protein (PCP) which was pretreated by single-frequency countercurrent and pulsed ultrasound. The kinetic constants for ultrasonic pretreated and traditional enzymolysis have been determined. Results showed that the value of KM in ultrasonic PCP (UPCP) enzymolysis decreased by 9% over that in the traditional enzymolysis. The values of reaction rate constant (k) for UPCP enzymolysis increased by 207%, 121%, 62%, and 45% at 293, 303, 313 and 323 K, respectively. For the thermodynamic parameters, ultrasound decreased activation energy (Ea), change in enthalpy (DH) and entropy (DS) by 76%, 82% and 31% in PCP, respectively. However, ultrasound had little change in Gibbs free energy (DG) value in the temperature range of 293–323 K. Therefore, a general kinetic equation for the enzymolysis model of UPCP by a simple empirical equation was suggested. The experimental values fits with the enzymolysis kinetic model with a low average relative error (4%) confirmed that the kinetic model was accurate to reflect the enzymolysis process. The positive effect of single-frequency countercurrent and pulsed ultrasound in this study and application of the kinetic model may be useful for the release of bioactive peptides from meat processing by-products. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Bioactive peptides derived from various proteins by enzymatic hydrolysis are recognized as functional food ingredients in preventing lifestyle-related diseases by their antioxidant, antimicrobial, immunomodulatory activities [1–3]. The isolation of peptides is mainly based on the controlled enzymolysis process [4]. However, traditional enzymolysis has many disadvantages that arise mainly from the low contact frequency and the decreased enzyme activity. Therefore, research have been focused on developing methods to improve the utilization rate of enzyme and the conversion rate of substrate, as well as reduce the enzymolysis time [5–8]. Pig brain, nervous system and spinal cord, has the highest level of cholesterol (13.52–21.95 g kg1) and also the highest
⇑ Corresponding authors. 1
E-mail addresses: [email protected] (L. Yang), [email protected] (X. Wu). Contributed to this article equally and are co-first authors.
http://dx.doi.org/10.1016/j.ultsonch.2015.08.006 1350-4177/Ó 2015 Elsevier B.V. All rights reserved.
amount of phospholipids compared to other meat by-products. For this reason, the United States Department of Health recommends that limited amounts of these by-products be eaten, because of their associated health concerns [9]. However, there is a growing interest in the utilization and disposal of hydrolysates of porcine cerebral protein (PCP) which are used for the treatment of neural system diseases and prevents the age-dependent dementia [10], characterized by progressive loss of cognitive capability [11], and by pathological changes in the brain [12]. Indeed, one such product; Cerebrolysin (Ebewe Pharmaceutical, Austria), has been in clinical application for more than 40 years. It is known to be a mixture of 75% free amino acids and 25% short-chain peptides, probably only a part of the peptides are physiologically active [13]. The use of ultrasound energy in enhancing the enzyme hydrolysis of proteins is a well known technique [14,15] because it is attributed to mechanical and thermal effects enacted by cavitation, which can result in enhanced mass transfer, increased contact frequency between substrate and enzyme and so on [16–18].
Y. Zou et al. / Ultrasonics Sonochemistry 28 (2016) 294–301
Moreover, the collapse of cavitation bubbles generates high temperatures (approximately 5500 K) and high pressures (approximately 50 MPa) in very short time (3 104 s), which results in high intensity of shear forces and shock waves and turbulence [19]. Qu et al. have indicated ultrasound-assisted enzymolysis significantly increased the initial reaction rate by 60% and raised the conversion rate of protein by 35% at substrate concentration of 24 g/L [20]. Single-frequency countercurrent and pulsed ultrasound has many advantages which releases of uniform energy distribution, avoids the energy waste, resonates of treated material easily, generated smaller thermal effect in a pulsed mode with an on-time and an off-time cycle [21]. However, no research has been reported on the kinetics of single-frequency countercurrent and pulsed ultrasound-assisted enzymolysis for producing active peptides from different PCP concentration and times. The objectives of this research were to (1) study the effects of the single-frequency countercurrent and pulsed ultrasound pretreatment on the kinetic constants of PCP enzymolysis, (2) determine the reaction kinetic parameter k and thermodynamic parameters of ultrasonic PCP (UPCP) enzymolysis at different temperatures and times, and (3) determine the hydrolysis curves of UPCP catalyzed by alcalase in solution, which is modeled by a simple empirical equation from which kinetic parameters can be deduced. This model can predict the release of bioactive peptides from other meat processing by-products. 2. Materials and methods 2.1. The ultrasonic pretreatment of PCP Fresh porcine brains were purchased from a local market in Zhenjiang, China. After removing hematoma and leptomeninges (pia mater and arachnoid) and rinsing with saline, porcine brains were minced, and added portion-wise to 5 volumes (v/w) of boiling water. They were boiled for 10 min after addition of the final portion. PCP was extracted thoroughly by ethanol treatment with a mince:ethanol ratio of 1:2 at 70 °C for 30 min to obtain a degreased powder. The powder was then mixed with water to form a slurry [22]. The deposited solid was separated from the liquid by centrifuging at 4000g at room temperature for 15 min. It was then dried and stored in a desiccator for further analysis. Prior to enzymolysis, PCP were pretreated by ultrasound (UPCP) with a 2.0 cm flat tip probe operating in a pulsed on time and offtime of both 2 s. A probe ultrasonic reactor (SC-II, Chengdu Jiuzhou Ultrasonic Technology Co., LTD.) working with a single frequency of 20 kHz and a maximum power of 80 W was used in the ultrasonication experiments for 5 min [23]. 2% (w/v) PCP solution was prepared by dispersing a predetermined weight of sample into 250 mL of deionized water in a beaker and it was placed in a thermostatic water bath at different initial temperatures. The pretreated sample solution passed through the probe by countercurrent method which two peristaltic pumps used to keep the material solutions in a counter-current flow state. The pH of the solution was then corrected to 8.5 using 0.5 M NaOH [20]. 2.2. Enzyme hydrolysis UPCP solution obtained from Section 2.1 reacted with alcalase (E/S ratio, 2000 U/g). The traditional enzymolysis was prepared from PCP and alcalase using the same procedure with UPCP enzymolysis. The pH of the reaction solution was adjusted to 8.5 and then the solution was incubated in a water bath at 20, 30, 40 and 50 °C, respectively. During the whole period of hydrolysis, the pH was maintained at 8.5 by frequent addition of 0.5 M NaOH. At the end of the incubation period, the enzymatic hydrolysis was
295
terminated by boiling the mixture for 10 min. Then the mixture of the protein and enzyme was centrifuged at 4000g for 15 min at 4 °C. The supernatant of hydrolysate was collected and stored at 4 °C. 2.3. Determination of polypeptide concentration The polypeptide concentration (103 g/L) was determined using the reported Folin-phenol colorimetric method as explained by Wenjuan Qu [24]. 4 mL Folin-phenol (reagent A) was mixed with 0.5 mL sample and incubated for 10 min at room temperature, after which 0.5 mL Folin-phenol (reagent B) was added, and the absorbance was read at 500 nm on a spectrophotometer (Unic 7200, Unocal Corporation, Shanghai, China) after 30 min incubation at room temperature. 2.4. Test of effect of ultrasound on enzymolysis kinetic constants 2.4.1. Enzymolysis reaction condition The traditional and UPCP enzymatic hydrolysis were performed at substrate concentrations of 7.48, 9.98, 14.97, 19.96 and 29.94 g/L, respectively. Each treatment was replicated three times. The other hydrolysis conditions were: pH, 8.5; temperature, 50 °C; E/S ratio, 2000 U/g; hydrolysis time, 5 min. 2.4.2. Michaelis–Menten constant and maximum initial velocity (KM and Vmax) The classic Michaelis–Menten equation was applied in effect of ultrasonic pretreatment on enzymolysis kinetic constants of porcine cerebral protein. In order to estimate the two constants KM and Vmax, the experimental data can be plotted according to the double-reciprocal transformation of Eq. (1):
1 KM 1 1 þ ¼ V V max ½S V max
ð1Þ
where V is the initial reaction rate (g/mL s), [S] is the initial protein concentration (g/mL), KM is Michaelis constant, and Vmax is the maximum initial velocity (g/mL s). The KM and Vmax values were determined by Eq. (1) from the slope and intercept by plotting 1/V against 1/S. In order to calculate the initial reaction rate of enzymolysis, the polypeptide concentration (g/mL) was determined at enzymolysis time of 5 min. The initial reaction rate was express as:
Vðg=mL sÞ ¼
Qt 5 60ðsÞ
ð2Þ
Qt is the polypeptide concentration in the reaction solution at a given time t min. 2.5. Test of effect of ultrasound on the kinetics and thermodynamics 2.5.1. Enzymolysis reaction kinetics The kinetic models of PCP were fitted to the first-order kinetics [23,25]. The kinetic model was written as:
dC t ¼ kC t dt
ð3Þ
where k is the total reaction rate constant, and Ct is the PCP or UPCP concentration in reaction solution at a given time t min (lg/mL). After integrating Eq. (3), the kinetic model was expressed as:
ln
Ct C0
¼ kt
ð4Þ
where C0 is the initial concentration of PCP, t is time. As it is difficult to measure the decrement of PCP, the reaction rate can be determined by the increased amount of polypeptide released by PCP.
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The first-order kinetic model under the reaction conditions t = 0 1, Ct = Q1 Qt, and C0 = Q1 was written as:
ln ðQ 1 Q t Þ ¼ kt þ ln Q 1
2.5.2. Enzymolysis thermodynamics The dependence of the constant rate k on temperature can be described by Arrhenius equation [26], which was written as:
Ea RT
ð6Þ
where A is pre-exponential or collision factor and Ea the activation energy (J mol1). R is the universal gas constant (8.314 J (mol K1)). The plot of ln k against T1 was used for the calculation of Ea. In order to understand the effect of temperature on enzymolysis and also the macroscopic changes observed in this study, the Eyring transition state theory (TST) was used [27].
kB T DG kB T DH DS ¼ exp exp þ k¼ h RT h RT R
ð7Þ
where T is the absolute temperature in K, kB is the Boltzmann constant (1.38 1023 J K1), h is the Planck constant (6.6256 1034 J s1), DG, DH and DS are the parameters of changes in Gibbs free energy (J mol1), enthalpy (J mol1) and entropy (J (mol K1)) for the ultrasonic pretreated enzymolysis, respectively. 2.6. The enzymolysis kinetic model of UPCP 2.6.1. The enzymolysis reaction condition The experiments were divided into three groups according to UPCP of 9.98, 14.97 and 19.96 g/L. In each group, the addition of alcalase was 0.09, 0.18 and 0.45 g/L, respectively. The hydrolysis temperature and pH value were fixed at 50 °C and 8.5, respectively. The degrees of hydrolysis under different enzymolysis time (2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80 and 90 min) were investigated. UPCP of 7.48 and 29.94 g/L was used to confirm the validity of the enzymolysis kinetic model. 2.6.2. Enzymolysis kinetic model The hydrolysis reaction can
be
expressed
briefly
as:
k2
E þ S () ES ! E þ P. The reaction rate will be determined by the irreversible stage and gives Eq. (8).
r ¼ s0 If
dðDHÞ ¼ k2 ½ES dt the
mechanism
ð8Þ of
enzyme
inactivation
reaction
is
k3
E þ ES ! Ea þ Ed þ P, then the kinetic equation for this process is as follow:
de ¼ k3 ½E½ES dt
ð9Þ
When the relationship between the two mechanisms was considered, combination of Eq. (8) with Eq. (9) provides the ratio:
s0
dðDHÞ k2 ¼ de k3 ½E
ð10Þ
If enzyme is inhibited by substrate and product, the reactions Ks
strate complex (ES) leads to the following equation:
ð5Þ
where Q1 is the final polypeptide concentration in the reaction solution (lg/mL), and Qt is the polypeptide concentration in the reaction solution at a given time t min (103 g/L). The k values were determined experimentally from the slope by plotting ln(Q1 Qt) against t. k is the effective (total) rate constant and ultrasonic kus is induced by ultrasound in UPCP enzymolysis.
k ¼ A exp
At steady state approximation, the mass balance for enzyme–sub-
Kp
are S þ ES ¢ SES and E þ P EP, Ks = ðk4 =k4 Þ and Kp = ðk5 =k5 Þ.
½S½ES ½S2 ½E ¼ Ks KMKs
ð11Þ
½EP ¼
½E½P K M ½ES½P ¼ Kp K p ½S
ð12Þ
½ES ¼
½E½S KM
ð13Þ
½SES ¼
The total enzyme concentration would be expressed in the form at a given moment:
e ¼ ½E þ ½ES þ ½SES þ ½EP
ð14Þ
When Eqs. (11)–(13) were substituted into Eq. (14), expression for the free enzyme concentration can be simplified as Eq. (15) when [S] s0 and [P] p.
½E ¼
e 1þ
½ES ¼ K M s0
s0 KM
ð15Þ
s2
þ K s K0 M þ Kpp
e þ 1 þ Ks0s þ KKpMsp0
ð16Þ
Combination of Eq. (16) with Eq. (10) provides
dðDHÞ k2 K s K p s0 þ K p s20 þ K M K s p 1 ¼ de e k3 K M K s K p s0 "
e ¼ e0 exp
k K K K s ðDHÞ 3 M s p 0 k2 K s K p s0 þ K p s20 þ K M K s p
ð17Þ # ð18Þ
When Eq. (17) was substituted into Eq. (16) and then the obtained equation was inserted into Eq. (8), Eq. (19) can be yielded as following: " # dðDHÞ k2 e0 K s K p k3 K s K M K p s0 ðDHÞ exp ¼ dt K s K p s0 þ K p s20 þ K s K p p k2 K s K p s0 þ K p s20 þ K s K M p
ð19Þ Eq. (19) can be simplified as:
DH ¼
1 lnð1 þ abtÞ b
where, a = k2e0KsKp/ K s K p s0 þ K p s20 þ K s K M p , k2 K s K p s0 þ K p s20 þ K s K M p .
ð20Þ b = k3KsKMKps0/
2.6.3. Assessment of degree of hydrolysis (DH) The degree of hydrolysis (DH) was calculated according to the pH-stat method described by Adler-Nissen [28]:
DH ð%Þ ¼
h Nb B 100 ¼ htot a Mp htot
ð21Þ
where, Nb is the concentration of NaOH (mol/L), B is the volume of NaOH consumed (mL), Mp is the mass of protein to be hydrolyzed (g), htot is the total number of peptide bonds in the protein substrate, which is 7.6 mmol/g for porcine cerebral protein, a is the average degree of dissociation of the a-NH2 groups. 2.7. 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 significance level
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3. Results and discussion 3.1. Effect of single-frequency countercurrent and pulsed ultrasound pretreatment on enzymolysis kinetic constants The reaction rate constant is an important parameter of the chemical reaction kinetics, which is independent of the substrate concentration. KM represents the affinity between substrate and enzyme, which indicates a fast reaction speed when it is low. Vmax is the maximum reaction rate, which implies a high binding frequency between substrate and enzyme [29]. To determine the change of reaction rate constants which was induced by the enzymolysis of UPCP, the Michaelis–Menten kinetic and Lineweaver–Burk equation was used, and the plots obtained were presented in Fig. 1. It can be observed that 1/V had a good linear relationship with 1/S with correlation coefficients greater than 0.95. This result reveals that both the Vmax obeyed first-order kinetics within the substrate range studied. The reaction kinetic constants KM and Vmax determined from the slope and intercept are given in Table 1. It can be seen that the rate constant KM of UPCP enzymolysis decreased by 8.5% over the traditional enzymolysis. The lower KM indicates the higher affinity between PCP and alcalase in both ultrasonic pretreatments. The decrease in KM value for UPCP enzymolysis was possibly due to the fact that ultrasonic pretreatment had partly altered the conformation of the protein by affecting the non-covalent interactions including hydrogen bond, Van der Waals force, hydrophobicity, electrostatic interaction and loosened the protein tissue [30,31]. Besides, the particle size of proteins might have been reduced by ultrasound which induced the shearing force, shock waves, free radicals [32,33], resulting in enlargement of specific surface areas of PCP, and thus increased the contact areas between alcalase and matrix. The changes of Vmax in the ultrasonic pretreated and traditional
enzymolysis were very small, which implies the highest binding frequency was obtained when the alcalase was saturated by substrate. After all, ultrasound can accelerate the enzymolysis process of PCP. 3.2. Effect of single-frequency countercurrent and pulsed ultrasound pretreatment on reaction rate constant k The KM change in enzymolysis of UPCP will result in the change in the reaction rate constant (k). The value of k is an important parameter and can describe the extent of UPCP enzymolysis over the traditional one, which is mainly dependent on the temperature of reaction, solvent and catalyst. UPCP and traditional enzymolysis were investigated by hydrolyzing the protein at substrate concentration of 19.96 g/L, enzyme concentration of 1.96 g/L, pH 8.5 and temperature varied from 293 K to 323 K for 90 min. Fig. 2 displays the plots of ln(Q1 Qt) versus time in the traditional and UPCP enzymolysis at different temperatures. It can be seen that hydrolysis of untreated as well as UPCP obeyed the first order kinetics because both the correlation coefficients were greater than 0.95. The rate constants k and kus at different temperatures have been summarized in Table 2. It can be seen that k increased as the temperature increased from 20 °C to 50 °C. This can be attributed to 4.9
4.8
4.7
4.6
ln(Q
of P < 0.05. All graphs and calculation were preformed with the Origin Pro 8.0 and Microsoft Office Excel 2003, respectively.
50 °C 40 °C 30 °C 20 °C
4.5
4.4 3500
Ultrasonic pretreated cerebral protein enzymelysis Traditional enzymolysis
4.3 2
4
1/V (mL·s/g)
3000
6
8
10
(a) Time (min) 4.9
2500
4.8 2000
4.7 4.6
1500
1000 40
50
60
70
80
90
100
1/S (mL/g)
ln(Q
4.5 4.4
50 °C 40 °C 30 °C 20 °C
4.3
Fig. 1. Plots of the reciprocal of the initial reaction rate (1/V) versus the reciprocal of substrate concentration (1/S) in UPCP and traditional enzymolysis.
4.2 4.1 3
Table 1 Kinetic constants in traditional and UPCP enzymolysis.
Traditional enzymolysis UPCP enzymolysis
6
9
(b) Time (min)
KM
Vmax (g/mL s)
R2
0.895 0.819
0.0253 0.0250
0.9785 0.9769
Fig. 2. The ln(Q1 Qt) values versus times in the traditional (a) and UPCP (b) at different temperatures. The regression coefficients of the curves obtained at 20, 30, 40 and 50 °C are 0.98, 0.97, 0.98 and 0.95 in (a) and 0.98, 0.99, 0.97 and 0.98 in (b), respectively.
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Table 2 Reaction rate constants in traditional and UPCP enzymolysis at different temperatures.
a
k (min1)
Type
Temperature (°C)
Traditional enzymolysis
20 30 40 50
0.0139 ± 0.0006 0.0226 ± 0.0013 0.0342 ± 0.0026 0.0426 ± 0.0015
UPCP enzymolysis
20 30 40 50
0.0427 ± 0.0031 (207%)a 0.0499 ± 0.0023 (121%) 0.0554 ± 0.0042 (62%) 0.0618 ± 0.0037 (45%)
kus (min1)
R2Adj
– – – –
0.9921 0.9907 0.9823 0.9895
0.0288 ± 0.0016 0.0273 ± 0.0012 0.0212 ± 0.0011 0.0192 ± 0.0007
0.9788 0.9856 0.9712 0.9830
The values in parenthesis are the increasing rates of reaction rate constant in ultrasonic pretreated enzymolysis compared with traditional enzymolysis.
UPCP enzymolysis gave a high polypeptide concentration of 158.0 103 g/L while the other gave a polypeptide concentration of 119.8 103 g/L at 50 °C. According to previous reports, the formation of aggregates was due to the formation of non-covalent interactions between protein molecules, e.g., electrostatic and hydrophobic interactions [37]. The high power ultrasound may cut off the electrostatic and hydrophobic interactions between protein molecules which resulted in PCP particle breakage. Zhou et al. reported the altering of protein structure with free sulfhydryl and disulfide bond contents by ultrasound treatments [38]. The destructive nature of ultrasonic cavitation and the vibration of the ultrasonic waves can lead to physical weakening of the structure [39]. An increased apparent viscosity accompanied by an increase in the consistency coefficient have been related to changes in the binding capacity of water when hydrophilic parts of amino acids are opened toward water surroundings [40].
-6
lnk (1/s)
-7
-8
Traditional enzymolysis UPCP enzymolysis -9
-10 3.0
3.1
3.2
3.3
3.4
3.5
-3
1/T×10 ᧤.᧥ Fig. 3. The relationship between ln k and T1 in the traditional and UPCP enzymolysis. The regression coefficients of the curves are 0.99 and 0.95, respectively.
the enhancement of collision frequency between the substrate and enzyme at higher temperatures. k value in UPCP enzymolysis was higher than that in the traditional enzymolysis at all temperatures, which means the effect of single-frequency countercurrent and pulsed ultrasound pretreatment can facilitate enzymolysis. k of ultrasound pretreatment increased by 207%, 121%, 62% and 45% at the temperature of 293, 303, 313 and 323 K, respectively. Subhedar et al [34] found that the ultrasonic pretreatment at a frequency of 20 kHz and time of 30 min facilitated the enzymatic hydrolysis of carboxymethyl cellulose and promoted the release of glucose from carboxymethyl cellulose. However, kus decreased with the increase of enzymolysis temperature, which was opposite to k. The results indicated that ultrasound effects contributed largely to the rate constant at lower temperatures but very little at higher temperatures. The decrease in kus was due to the decrease of gas solubility in the bulk of fluid and a lower value for the ratio of specific heats of water vapor; the increase of equilibrium vapor pressure of the system and the formed cavitation bubbles as the temperature increased [35]. The effect of ultrasound appeared to be weak at higher temperatures, which was in agreement with results reported by Liu et al [36].
3.3. Effects of single-frequency countercurrent and pulsed ultrasound pretreatment on thermodynamics parameters Ea, DG, DH and DS Activation energy is the minimum amount of energy required to convert a normal stable molecule into a reactive molecule and it reflects the rapidity of chemical reactions [41]. The thermodynamic parameters Ea, and DG, DH and DS were determined from the slopes and intercepts by plotting ln k against T1 from Fig. 3 and ln (k/T) against T1 from Table 3, respectively. The Ea values of most reactions ranged from 40 kJ/mol to 400 kJ/mol. The lower Ea (<40 kJ/mol) from the traditional and two enzymolysis pretreated by ultrasound implied that less energy was needed for the enzymolysis reaction and faster reaction rate between the PCP and alcalase. In addition, it was observed that Ea values of enzymolysis reaction with UPCP decreased significantly in comparison with the traditional one and the rates decreased by 76%. The greatly reduced Ea value by ultrasound pretreatment indicates that ultrasound significantly decreased the energy barrier required for enzymolysis reaction, which is consistent with the phenomenon observed in the experiment. The DH, DS and DG values, showing a similar trend with Ea values, were respectively reduced by 82%, 31%, and 1% for UPCP enzymolysis when compared with the traditional enzymolysis, respectively. DH decreased dramatically, attributed to the ultrasonically induced rupture of hydrogen bonds stabilizing the protein and enzyme at ground state and the distraction of the internal hydrophobic core, both of which leads to alteration in the protein structure. DS represents the variation in the
Table 3 Thermodynamic parameters in traditional and UPCP enzymolysis at different temperatures.
a
Type
Ea (103 J mol1)
DH (103 J mol1)
DS (J (mol K1))
DG (103 J mol1)
Traditional enzymolysis UPCP enzymolysis
29.928 7.282 (76%)a
27.443 4.862 (82%)
220.033 289.157 (31%)
95.246 93.965 (1%)
The values in parenthesis are the decreasing rates of thermodynamic parameters in ultrasonic pretreated enzymolysis compared with traditional enzymolysis.
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extent of local disordering between transition state and the ground state. The decrease in the DS can be attributed to the oxidative modification of amino acid residues and initiation of crosslinking and aggregation which leads to the increase in enzymolysis activity [42]. DG had little change for ultrasonic treatment.
25
0.09 g/L 0.18 g/L 0.45 g/L
20
Table 4 Values of the kinetic parameters a and b obtained from UPCP enzymolysis. s0 (g/L)
e0 (g/L)
a
b
R2
9.98 9.98 9.98 14.97 14.97 14.97 19.96 19.96 19.96
0.09 0.18 0.45 0.09 0.18 0.45 0.09 0.18 0.45
0.887 1.069 1.616 0.806 0.967 1.375 0.796 0.886 1.160
0.1894 0.1721 0.1612 0.1838 0.1665 0.1691 0.1720 0.1654 0.1760
0.9967 0.9995 0.9995 0.9976 0.9992 0.9967 0.9993 0.9974 0.9963
DH (%)
15
10
5
0 0
20
40
60
80
100
(a) Enzymolysis time (min) 20
0.09 g/L 0.18 g/L 0.45 g/L
DH (%)
15
Fig. 5. Change trend of kinetic parameter a as the change of e0/s0.
10
5
0 0
20
40
60
80
100
(b) Enzymolysis time (min) 20
0.09 g/L 0.18 g/L 0.45 g/L
16
Therefore, the DH and DS can explain that the enzymolysis reaction catalyzed by ultrasonic pretreatment can occur very swiftly. Subhedar et al. [34] reported the change in thermodynamic parameters of cellulose after ultrasonic irradiation where DH, DS and DG were reduced by 68%, 37.3% and 1.3%, respectively. Moreover, Qu et al. [24] also report similar results on the change in thermodynamic parameters of alcalase enzyme after defatted wheat germ protein ultrasonic irradiation, where DH, DS and DG were reduced by 74.1%, 34.3% and 1.4%, respectively. These values compares favorably with that obtained in the present work. In general, the results obtained clearly demonstrated that UPCP was more beneficial for the enzymolysis reaction because of its lower energy requirements. 3.4. The enzymolysis kinetic model of UPCP
DH (%)
12
8
4
0 0
20
40
60
80
100
(c) Enzymolysis time (min) Fig. 4. Hydrolysis curves with different e0/s0 ratios at substrate concentration of (a) 9.98, (b) 14.97 and (c) 19.96 g/L in UPCP enzymolysis.
Learning from kinetic modeling can be augmented given the precise and accurate model parameters, which should be estimated using substrate protein and enzyme concentrations. Fig. 4 shows the change of DH in the enzymolysis process of UPCP under different substrate protein and enzyme concentrations and time. Values of a and b for different initial substrate protein and enzyme concentrations were calculated from nonlinear regression analysis. The value of a increased when the initial enzyme concentration increased while parameter b did not exhibit any trend. The average value of parameter b can be considered as 0.173 (Table 4). A straight line was obtained by the change trend of kinetic parameter a as the change of e0/s0 (Fig. 5). The linear regression analysis on the figure shows a correlation coefficient of 0.9898 and yields the expression:
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References
DH (%)
14
s0=7.48 g/L, e0=0.09g/L, Calculated values s0=7.48 g/L, e0=0.09g/L, Experimental data s0=19.96 g/L, e0=0.36g/L, Calculated values s0=19.96 g/L, e0=0.36g/L, Experimental data s0=29.94 g/L, e0=0.45g/L, Calculated values s0=29.94 g/L, e0=0.45g/L, Experimental data
7
0
0
40
80
Enzymolysis time (min) Fig. 6. Hydrolysis curves for different initial enzyme and substrate concentrations and calculated values fitted to Eq. (23) during enzymolysis time of 90 min.
a ¼ 0:02021
e0 þ 0:7044 s0
ð22Þ
Insertion of Eq. (10) and parameter b of 0.173 in Eq. (8), and the kinetic model for the enzymolysis of UPCP was obtained as following:
e0 DH ¼ 5:78 ln 1 þ 3:4946 þ 0:1219 t s0
ð23Þ
The above Eq. (23) agrees with Eq. (20) deduced from the proposed mechanism, which imply the relationship of DH, enzyme concentration, substrate concentration and enzymolysis time. To confirm the validity of the enzymolysis kinetic model and the kinetic parameters, three groups of experiments were done under different initial enzyme and substrate concentrations. The agreement between calculated values of DH versus enzymolysis time and the experimental data is shown in Fig. 6. The experimental values fitting with the enzymolysis kinetic model with a low average relative error (4%) confirmed that the kinetic model was accurate to reflect the enzymolysis process.
4. Conclusion In the present study, single-frequency countercurrent and pulsed ultrasound had a significant effect on the kinetic parameters of the enzymolysis reaction compared to traditional enzymolysis, in which KM was lower by 9%. The reaction rate constants for UPCP enzymolysis were significantly higher than that of the traditional enzymolysis at different temperatures especially at a low temperature. This accounted for the kus as well as lower activation energy (Ea), enthalpy (DH) and entropy of activation (DS). A suitable model of reproducing the kinetic behavior of the system under study was applied to hydrolysis reaction of UPCP, which allowed the calculation of the necessary kinetic parameters from experiments. The positive effects of single-frequency countercurrent and pulsed ultrasound in this study and application of the kinetic model may be useful for the release of bioactive peptides from meat processing by-products. Acknowledgments This work was supported financially by Graduate Innovative Projects in Jiangsu Province (CXZZ12-0701). The authors are thankful to Dr. Samuel Jerry Cobbina for language polishing.
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Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Enzymolysis kinetics, thermodynamics and model of porcine cerebral protein with single-frequency countercurrent and pulsed ultrasoundassisted processing Ye Zou a,1, Yangyang Ding b,1, Weiwei Feng a, Wei Wang a, Qian Li a, Yao Chen c, Huiyu Wu d, Xintong Wang b, Liuqing Yang b,⇑, Xiangyang Wu c,⇑ a
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China School of Chemistry and Chemical Engineering, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China c School of the Environment and Safety, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China d School of Pharmacy, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China b
a r t i c l e
i n f o
Article history: Received 23 June 2015 Received in revised form 24 July 2015 Accepted 11 August 2015 Available online 11 August 2015 Keywords: Porcine cerebral protein Single-frequency countercurrent and pulsed ultrasound Enzymolysis Kinetics Thermodynamics Enzymolysis model
a b s t r a c t The present work investigated the enzymolysis kinetics, thermodynamics and model of porcine cerebral protein (PCP) which was pretreated by single-frequency countercurrent and pulsed ultrasound. The kinetic constants for ultrasonic pretreated and traditional enzymolysis have been determined. Results showed that the value of KM in ultrasonic PCP (UPCP) enzymolysis decreased by 9% over that in the traditional enzymolysis. The values of reaction rate constant (k) for UPCP enzymolysis increased by 207%, 121%, 62%, and 45% at 293, 303, 313 and 323 K, respectively. For the thermodynamic parameters, ultrasound decreased activation energy (Ea), change in enthalpy (DH) and entropy (DS) by 76%, 82% and 31% in PCP, respectively. However, ultrasound had little change in Gibbs free energy (DG) value in the temperature range of 293–323 K. Therefore, a general kinetic equation for the enzymolysis model of UPCP by a simple empirical equation was suggested. The experimental values fits with the enzymolysis kinetic model with a low average relative error (4%) confirmed that the kinetic model was accurate to reflect the enzymolysis process. The positive effect of single-frequency countercurrent and pulsed ultrasound in this study and application of the kinetic model may be useful for the release of bioactive peptides from meat processing by-products. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Bioactive peptides derived from various proteins by enzymatic hydrolysis are recognized as functional food ingredients in preventing lifestyle-related diseases by their antioxidant, antimicrobial, immunomodulatory activities [1–3]. The isolation of peptides is mainly based on the controlled enzymolysis process [4]. However, traditional enzymolysis has many disadvantages that arise mainly from the low contact frequency and the decreased enzyme activity. Therefore, research have been focused on developing methods to improve the utilization rate of enzyme and the conversion rate of substrate, as well as reduce the enzymolysis time [5–8]. Pig brain, nervous system and spinal cord, has the highest level of cholesterol (13.52–21.95 g kg1) and also the highest
⇑ Corresponding authors. 1
E-mail addresses: [email protected] (L. Yang), [email protected] (X. Wu). Contributed to this article equally and are co-first authors.
http://dx.doi.org/10.1016/j.ultsonch.2015.08.006 1350-4177/Ó 2015 Elsevier B.V. All rights reserved.
amount of phospholipids compared to other meat by-products. For this reason, the United States Department of Health recommends that limited amounts of these by-products be eaten, because of their associated health concerns [9]. However, there is a growing interest in the utilization and disposal of hydrolysates of porcine cerebral protein (PCP) which are used for the treatment of neural system diseases and prevents the age-dependent dementia [10], characterized by progressive loss of cognitive capability [11], and by pathological changes in the brain [12]. Indeed, one such product; Cerebrolysin (Ebewe Pharmaceutical, Austria), has been in clinical application for more than 40 years. It is known to be a mixture of 75% free amino acids and 25% short-chain peptides, probably only a part of the peptides are physiologically active [13]. The use of ultrasound energy in enhancing the enzyme hydrolysis of proteins is a well known technique [14,15] because it is attributed to mechanical and thermal effects enacted by cavitation, which can result in enhanced mass transfer, increased contact frequency between substrate and enzyme and so on [16–18].
Y. Zou et al. / Ultrasonics Sonochemistry 28 (2016) 294–301
Moreover, the collapse of cavitation bubbles generates high temperatures (approximately 5500 K) and high pressures (approximately 50 MPa) in very short time (3 104 s), which results in high intensity of shear forces and shock waves and turbulence [19]. Qu et al. have indicated ultrasound-assisted enzymolysis significantly increased the initial reaction rate by 60% and raised the conversion rate of protein by 35% at substrate concentration of 24 g/L [20]. Single-frequency countercurrent and pulsed ultrasound has many advantages which releases of uniform energy distribution, avoids the energy waste, resonates of treated material easily, generated smaller thermal effect in a pulsed mode with an on-time and an off-time cycle [21]. However, no research has been reported on the kinetics of single-frequency countercurrent and pulsed ultrasound-assisted enzymolysis for producing active peptides from different PCP concentration and times. The objectives of this research were to (1) study the effects of the single-frequency countercurrent and pulsed ultrasound pretreatment on the kinetic constants of PCP enzymolysis, (2) determine the reaction kinetic parameter k and thermodynamic parameters of ultrasonic PCP (UPCP) enzymolysis at different temperatures and times, and (3) determine the hydrolysis curves of UPCP catalyzed by alcalase in solution, which is modeled by a simple empirical equation from which kinetic parameters can be deduced. This model can predict the release of bioactive peptides from other meat processing by-products. 2. Materials and methods 2.1. The ultrasonic pretreatment of PCP Fresh porcine brains were purchased from a local market in Zhenjiang, China. After removing hematoma and leptomeninges (pia mater and arachnoid) and rinsing with saline, porcine brains were minced, and added portion-wise to 5 volumes (v/w) of boiling water. They were boiled for 10 min after addition of the final portion. PCP was extracted thoroughly by ethanol treatment with a mince:ethanol ratio of 1:2 at 70 °C for 30 min to obtain a degreased powder. The powder was then mixed with water to form a slurry [22]. The deposited solid was separated from the liquid by centrifuging at 4000g at room temperature for 15 min. It was then dried and stored in a desiccator for further analysis. Prior to enzymolysis, PCP were pretreated by ultrasound (UPCP) with a 2.0 cm flat tip probe operating in a pulsed on time and offtime of both 2 s. A probe ultrasonic reactor (SC-II, Chengdu Jiuzhou Ultrasonic Technology Co., LTD.) working with a single frequency of 20 kHz and a maximum power of 80 W was used in the ultrasonication experiments for 5 min [23]. 2% (w/v) PCP solution was prepared by dispersing a predetermined weight of sample into 250 mL of deionized water in a beaker and it was placed in a thermostatic water bath at different initial temperatures. The pretreated sample solution passed through the probe by countercurrent method which two peristaltic pumps used to keep the material solutions in a counter-current flow state. The pH of the solution was then corrected to 8.5 using 0.5 M NaOH [20]. 2.2. Enzyme hydrolysis UPCP solution obtained from Section 2.1 reacted with alcalase (E/S ratio, 2000 U/g). The traditional enzymolysis was prepared from PCP and alcalase using the same procedure with UPCP enzymolysis. The pH of the reaction solution was adjusted to 8.5 and then the solution was incubated in a water bath at 20, 30, 40 and 50 °C, respectively. During the whole period of hydrolysis, the pH was maintained at 8.5 by frequent addition of 0.5 M NaOH. At the end of the incubation period, the enzymatic hydrolysis was
295
terminated by boiling the mixture for 10 min. Then the mixture of the protein and enzyme was centrifuged at 4000g for 15 min at 4 °C. The supernatant of hydrolysate was collected and stored at 4 °C. 2.3. Determination of polypeptide concentration The polypeptide concentration (103 g/L) was determined using the reported Folin-phenol colorimetric method as explained by Wenjuan Qu [24]. 4 mL Folin-phenol (reagent A) was mixed with 0.5 mL sample and incubated for 10 min at room temperature, after which 0.5 mL Folin-phenol (reagent B) was added, and the absorbance was read at 500 nm on a spectrophotometer (Unic 7200, Unocal Corporation, Shanghai, China) after 30 min incubation at room temperature. 2.4. Test of effect of ultrasound on enzymolysis kinetic constants 2.4.1. Enzymolysis reaction condition The traditional and UPCP enzymatic hydrolysis were performed at substrate concentrations of 7.48, 9.98, 14.97, 19.96 and 29.94 g/L, respectively. Each treatment was replicated three times. The other hydrolysis conditions were: pH, 8.5; temperature, 50 °C; E/S ratio, 2000 U/g; hydrolysis time, 5 min. 2.4.2. Michaelis–Menten constant and maximum initial velocity (KM and Vmax) The classic Michaelis–Menten equation was applied in effect of ultrasonic pretreatment on enzymolysis kinetic constants of porcine cerebral protein. In order to estimate the two constants KM and Vmax, the experimental data can be plotted according to the double-reciprocal transformation of Eq. (1):
1 KM 1 1 þ ¼ V V max ½S V max
ð1Þ
where V is the initial reaction rate (g/mL s), [S] is the initial protein concentration (g/mL), KM is Michaelis constant, and Vmax is the maximum initial velocity (g/mL s). The KM and Vmax values were determined by Eq. (1) from the slope and intercept by plotting 1/V against 1/S. In order to calculate the initial reaction rate of enzymolysis, the polypeptide concentration (g/mL) was determined at enzymolysis time of 5 min. The initial reaction rate was express as:
Vðg=mL sÞ ¼
Qt 5 60ðsÞ
ð2Þ
Qt is the polypeptide concentration in the reaction solution at a given time t min. 2.5. Test of effect of ultrasound on the kinetics and thermodynamics 2.5.1. Enzymolysis reaction kinetics The kinetic models of PCP were fitted to the first-order kinetics [23,25]. The kinetic model was written as:
dC t ¼ kC t dt
ð3Þ
where k is the total reaction rate constant, and Ct is the PCP or UPCP concentration in reaction solution at a given time t min (lg/mL). After integrating Eq. (3), the kinetic model was expressed as:
ln
Ct C0
¼ kt
ð4Þ
where C0 is the initial concentration of PCP, t is time. As it is difficult to measure the decrement of PCP, the reaction rate can be determined by the increased amount of polypeptide released by PCP.
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The first-order kinetic model under the reaction conditions t = 0 1, Ct = Q1 Qt, and C0 = Q1 was written as:
ln ðQ 1 Q t Þ ¼ kt þ ln Q 1
2.5.2. Enzymolysis thermodynamics The dependence of the constant rate k on temperature can be described by Arrhenius equation [26], which was written as:
Ea RT
ð6Þ
where A is pre-exponential or collision factor and Ea the activation energy (J mol1). R is the universal gas constant (8.314 J (mol K1)). The plot of ln k against T1 was used for the calculation of Ea. In order to understand the effect of temperature on enzymolysis and also the macroscopic changes observed in this study, the Eyring transition state theory (TST) was used [27].
kB T DG kB T DH DS ¼ exp exp þ k¼ h RT h RT R
ð7Þ
where T is the absolute temperature in K, kB is the Boltzmann constant (1.38 1023 J K1), h is the Planck constant (6.6256 1034 J s1), DG, DH and DS are the parameters of changes in Gibbs free energy (J mol1), enthalpy (J mol1) and entropy (J (mol K1)) for the ultrasonic pretreated enzymolysis, respectively. 2.6. The enzymolysis kinetic model of UPCP 2.6.1. The enzymolysis reaction condition The experiments were divided into three groups according to UPCP of 9.98, 14.97 and 19.96 g/L. In each group, the addition of alcalase was 0.09, 0.18 and 0.45 g/L, respectively. The hydrolysis temperature and pH value were fixed at 50 °C and 8.5, respectively. The degrees of hydrolysis under different enzymolysis time (2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80 and 90 min) were investigated. UPCP of 7.48 and 29.94 g/L was used to confirm the validity of the enzymolysis kinetic model. 2.6.2. Enzymolysis kinetic model The hydrolysis reaction can
be
expressed
briefly
as:
k2
E þ S () ES ! E þ P. The reaction rate will be determined by the irreversible stage and gives Eq. (8).
r ¼ s0 If
dðDHÞ ¼ k2 ½ES dt the
mechanism
ð8Þ of
enzyme
inactivation
reaction
is
k3
E þ ES ! Ea þ Ed þ P, then the kinetic equation for this process is as follow:
de ¼ k3 ½E½ES dt
ð9Þ
When the relationship between the two mechanisms was considered, combination of Eq. (8) with Eq. (9) provides the ratio:
s0
dðDHÞ k2 ¼ de k3 ½E
ð10Þ
If enzyme is inhibited by substrate and product, the reactions Ks
strate complex (ES) leads to the following equation:
ð5Þ
where Q1 is the final polypeptide concentration in the reaction solution (lg/mL), and Qt is the polypeptide concentration in the reaction solution at a given time t min (103 g/L). The k values were determined experimentally from the slope by plotting ln(Q1 Qt) against t. k is the effective (total) rate constant and ultrasonic kus is induced by ultrasound in UPCP enzymolysis.
k ¼ A exp
At steady state approximation, the mass balance for enzyme–sub-
Kp
are S þ ES ¢ SES and E þ P EP, Ks = ðk4 =k4 Þ and Kp = ðk5 =k5 Þ.
½S½ES ½S2 ½E ¼ Ks KMKs
ð11Þ
½EP ¼
½E½P K M ½ES½P ¼ Kp K p ½S
ð12Þ
½ES ¼
½E½S KM
ð13Þ
½SES ¼
The total enzyme concentration would be expressed in the form at a given moment:
e ¼ ½E þ ½ES þ ½SES þ ½EP
ð14Þ
When Eqs. (11)–(13) were substituted into Eq. (14), expression for the free enzyme concentration can be simplified as Eq. (15) when [S] s0 and [P] p.
½E ¼
e 1þ
½ES ¼ K M s0
s0 KM
ð15Þ
s2
þ K s K0 M þ Kpp
e þ 1 þ Ks0s þ KKpMsp0
ð16Þ
Combination of Eq. (16) with Eq. (10) provides
dðDHÞ k2 K s K p s0 þ K p s20 þ K M K s p 1 ¼ de e k3 K M K s K p s0 "
e ¼ e0 exp
k K K K s ðDHÞ 3 M s p 0 k2 K s K p s0 þ K p s20 þ K M K s p
ð17Þ # ð18Þ
When Eq. (17) was substituted into Eq. (16) and then the obtained equation was inserted into Eq. (8), Eq. (19) can be yielded as following: " # dðDHÞ k2 e0 K s K p k3 K s K M K p s0 ðDHÞ exp ¼ dt K s K p s0 þ K p s20 þ K s K p p k2 K s K p s0 þ K p s20 þ K s K M p
ð19Þ Eq. (19) can be simplified as:
DH ¼
1 lnð1 þ abtÞ b
where, a = k2e0KsKp/ K s K p s0 þ K p s20 þ K s K M p , k2 K s K p s0 þ K p s20 þ K s K M p .
ð20Þ b = k3KsKMKps0/
2.6.3. Assessment of degree of hydrolysis (DH) The degree of hydrolysis (DH) was calculated according to the pH-stat method described by Adler-Nissen [28]:
DH ð%Þ ¼
h Nb B 100 ¼ htot a Mp htot
ð21Þ
where, Nb is the concentration of NaOH (mol/L), B is the volume of NaOH consumed (mL), Mp is the mass of protein to be hydrolyzed (g), htot is the total number of peptide bonds in the protein substrate, which is 7.6 mmol/g for porcine cerebral protein, a is the average degree of dissociation of the a-NH2 groups. 2.7. 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 significance level
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3. Results and discussion 3.1. Effect of single-frequency countercurrent and pulsed ultrasound pretreatment on enzymolysis kinetic constants The reaction rate constant is an important parameter of the chemical reaction kinetics, which is independent of the substrate concentration. KM represents the affinity between substrate and enzyme, which indicates a fast reaction speed when it is low. Vmax is the maximum reaction rate, which implies a high binding frequency between substrate and enzyme [29]. To determine the change of reaction rate constants which was induced by the enzymolysis of UPCP, the Michaelis–Menten kinetic and Lineweaver–Burk equation was used, and the plots obtained were presented in Fig. 1. It can be observed that 1/V had a good linear relationship with 1/S with correlation coefficients greater than 0.95. This result reveals that both the Vmax obeyed first-order kinetics within the substrate range studied. The reaction kinetic constants KM and Vmax determined from the slope and intercept are given in Table 1. It can be seen that the rate constant KM of UPCP enzymolysis decreased by 8.5% over the traditional enzymolysis. The lower KM indicates the higher affinity between PCP and alcalase in both ultrasonic pretreatments. The decrease in KM value for UPCP enzymolysis was possibly due to the fact that ultrasonic pretreatment had partly altered the conformation of the protein by affecting the non-covalent interactions including hydrogen bond, Van der Waals force, hydrophobicity, electrostatic interaction and loosened the protein tissue [30,31]. Besides, the particle size of proteins might have been reduced by ultrasound which induced the shearing force, shock waves, free radicals [32,33], resulting in enlargement of specific surface areas of PCP, and thus increased the contact areas between alcalase and matrix. The changes of Vmax in the ultrasonic pretreated and traditional
enzymolysis were very small, which implies the highest binding frequency was obtained when the alcalase was saturated by substrate. After all, ultrasound can accelerate the enzymolysis process of PCP. 3.2. Effect of single-frequency countercurrent and pulsed ultrasound pretreatment on reaction rate constant k The KM change in enzymolysis of UPCP will result in the change in the reaction rate constant (k). The value of k is an important parameter and can describe the extent of UPCP enzymolysis over the traditional one, which is mainly dependent on the temperature of reaction, solvent and catalyst. UPCP and traditional enzymolysis were investigated by hydrolyzing the protein at substrate concentration of 19.96 g/L, enzyme concentration of 1.96 g/L, pH 8.5 and temperature varied from 293 K to 323 K for 90 min. Fig. 2 displays the plots of ln(Q1 Qt) versus time in the traditional and UPCP enzymolysis at different temperatures. It can be seen that hydrolysis of untreated as well as UPCP obeyed the first order kinetics because both the correlation coefficients were greater than 0.95. The rate constants k and kus at different temperatures have been summarized in Table 2. It can be seen that k increased as the temperature increased from 20 °C to 50 °C. This can be attributed to 4.9
4.8
4.7
4.6
ln(Q
of P < 0.05. All graphs and calculation were preformed with the Origin Pro 8.0 and Microsoft Office Excel 2003, respectively.
50 °C 40 °C 30 °C 20 °C
4.5
4.4 3500
Ultrasonic pretreated cerebral protein enzymelysis Traditional enzymolysis
4.3 2
4
1/V (mL·s/g)
3000
6
8
10
(a) Time (min) 4.9
2500
4.8 2000
4.7 4.6
1500
1000 40
50
60
70
80
90
100
1/S (mL/g)
ln(Q
4.5 4.4
50 °C 40 °C 30 °C 20 °C
4.3
Fig. 1. Plots of the reciprocal of the initial reaction rate (1/V) versus the reciprocal of substrate concentration (1/S) in UPCP and traditional enzymolysis.
4.2 4.1 3
Table 1 Kinetic constants in traditional and UPCP enzymolysis.
Traditional enzymolysis UPCP enzymolysis
6
9
(b) Time (min)
KM
Vmax (g/mL s)
R2
0.895 0.819
0.0253 0.0250
0.9785 0.9769
Fig. 2. The ln(Q1 Qt) values versus times in the traditional (a) and UPCP (b) at different temperatures. The regression coefficients of the curves obtained at 20, 30, 40 and 50 °C are 0.98, 0.97, 0.98 and 0.95 in (a) and 0.98, 0.99, 0.97 and 0.98 in (b), respectively.
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Table 2 Reaction rate constants in traditional and UPCP enzymolysis at different temperatures.
a
k (min1)
Type
Temperature (°C)
Traditional enzymolysis
20 30 40 50
0.0139 ± 0.0006 0.0226 ± 0.0013 0.0342 ± 0.0026 0.0426 ± 0.0015
UPCP enzymolysis
20 30 40 50
0.0427 ± 0.0031 (207%)a 0.0499 ± 0.0023 (121%) 0.0554 ± 0.0042 (62%) 0.0618 ± 0.0037 (45%)
kus (min1)
R2Adj
– – – –
0.9921 0.9907 0.9823 0.9895
0.0288 ± 0.0016 0.0273 ± 0.0012 0.0212 ± 0.0011 0.0192 ± 0.0007
0.9788 0.9856 0.9712 0.9830
The values in parenthesis are the increasing rates of reaction rate constant in ultrasonic pretreated enzymolysis compared with traditional enzymolysis.
UPCP enzymolysis gave a high polypeptide concentration of 158.0 103 g/L while the other gave a polypeptide concentration of 119.8 103 g/L at 50 °C. According to previous reports, the formation of aggregates was due to the formation of non-covalent interactions between protein molecules, e.g., electrostatic and hydrophobic interactions [37]. The high power ultrasound may cut off the electrostatic and hydrophobic interactions between protein molecules which resulted in PCP particle breakage. Zhou et al. reported the altering of protein structure with free sulfhydryl and disulfide bond contents by ultrasound treatments [38]. The destructive nature of ultrasonic cavitation and the vibration of the ultrasonic waves can lead to physical weakening of the structure [39]. An increased apparent viscosity accompanied by an increase in the consistency coefficient have been related to changes in the binding capacity of water when hydrophilic parts of amino acids are opened toward water surroundings [40].
-6
lnk (1/s)
-7
-8
Traditional enzymolysis UPCP enzymolysis -9
-10 3.0
3.1
3.2
3.3
3.4
3.5
-3
1/T×10 ᧤.᧥ Fig. 3. The relationship between ln k and T1 in the traditional and UPCP enzymolysis. The regression coefficients of the curves are 0.99 and 0.95, respectively.
the enhancement of collision frequency between the substrate and enzyme at higher temperatures. k value in UPCP enzymolysis was higher than that in the traditional enzymolysis at all temperatures, which means the effect of single-frequency countercurrent and pulsed ultrasound pretreatment can facilitate enzymolysis. k of ultrasound pretreatment increased by 207%, 121%, 62% and 45% at the temperature of 293, 303, 313 and 323 K, respectively. Subhedar et al [34] found that the ultrasonic pretreatment at a frequency of 20 kHz and time of 30 min facilitated the enzymatic hydrolysis of carboxymethyl cellulose and promoted the release of glucose from carboxymethyl cellulose. However, kus decreased with the increase of enzymolysis temperature, which was opposite to k. The results indicated that ultrasound effects contributed largely to the rate constant at lower temperatures but very little at higher temperatures. The decrease in kus was due to the decrease of gas solubility in the bulk of fluid and a lower value for the ratio of specific heats of water vapor; the increase of equilibrium vapor pressure of the system and the formed cavitation bubbles as the temperature increased [35]. The effect of ultrasound appeared to be weak at higher temperatures, which was in agreement with results reported by Liu et al [36].
3.3. Effects of single-frequency countercurrent and pulsed ultrasound pretreatment on thermodynamics parameters Ea, DG, DH and DS Activation energy is the minimum amount of energy required to convert a normal stable molecule into a reactive molecule and it reflects the rapidity of chemical reactions [41]. The thermodynamic parameters Ea, and DG, DH and DS were determined from the slopes and intercepts by plotting ln k against T1 from Fig. 3 and ln (k/T) against T1 from Table 3, respectively. The Ea values of most reactions ranged from 40 kJ/mol to 400 kJ/mol. The lower Ea (<40 kJ/mol) from the traditional and two enzymolysis pretreated by ultrasound implied that less energy was needed for the enzymolysis reaction and faster reaction rate between the PCP and alcalase. In addition, it was observed that Ea values of enzymolysis reaction with UPCP decreased significantly in comparison with the traditional one and the rates decreased by 76%. The greatly reduced Ea value by ultrasound pretreatment indicates that ultrasound significantly decreased the energy barrier required for enzymolysis reaction, which is consistent with the phenomenon observed in the experiment. The DH, DS and DG values, showing a similar trend with Ea values, were respectively reduced by 82%, 31%, and 1% for UPCP enzymolysis when compared with the traditional enzymolysis, respectively. DH decreased dramatically, attributed to the ultrasonically induced rupture of hydrogen bonds stabilizing the protein and enzyme at ground state and the distraction of the internal hydrophobic core, both of which leads to alteration in the protein structure. DS represents the variation in the
Table 3 Thermodynamic parameters in traditional and UPCP enzymolysis at different temperatures.
a
Type
Ea (103 J mol1)
DH (103 J mol1)
DS (J (mol K1))
DG (103 J mol1)
Traditional enzymolysis UPCP enzymolysis
29.928 7.282 (76%)a
27.443 4.862 (82%)
220.033 289.157 (31%)
95.246 93.965 (1%)
The values in parenthesis are the decreasing rates of thermodynamic parameters in ultrasonic pretreated enzymolysis compared with traditional enzymolysis.
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extent of local disordering between transition state and the ground state. The decrease in the DS can be attributed to the oxidative modification of amino acid residues and initiation of crosslinking and aggregation which leads to the increase in enzymolysis activity [42]. DG had little change for ultrasonic treatment.
25
0.09 g/L 0.18 g/L 0.45 g/L
20
Table 4 Values of the kinetic parameters a and b obtained from UPCP enzymolysis. s0 (g/L)
e0 (g/L)
a
b
R2
9.98 9.98 9.98 14.97 14.97 14.97 19.96 19.96 19.96
0.09 0.18 0.45 0.09 0.18 0.45 0.09 0.18 0.45
0.887 1.069 1.616 0.806 0.967 1.375 0.796 0.886 1.160
0.1894 0.1721 0.1612 0.1838 0.1665 0.1691 0.1720 0.1654 0.1760
0.9967 0.9995 0.9995 0.9976 0.9992 0.9967 0.9993 0.9974 0.9963
DH (%)
15
10
5
0 0
20
40
60
80
100
(a) Enzymolysis time (min) 20
0.09 g/L 0.18 g/L 0.45 g/L
DH (%)
15
Fig. 5. Change trend of kinetic parameter a as the change of e0/s0.
10
5
0 0
20
40
60
80
100
(b) Enzymolysis time (min) 20
0.09 g/L 0.18 g/L 0.45 g/L
16
Therefore, the DH and DS can explain that the enzymolysis reaction catalyzed by ultrasonic pretreatment can occur very swiftly. Subhedar et al. [34] reported the change in thermodynamic parameters of cellulose after ultrasonic irradiation where DH, DS and DG were reduced by 68%, 37.3% and 1.3%, respectively. Moreover, Qu et al. [24] also report similar results on the change in thermodynamic parameters of alcalase enzyme after defatted wheat germ protein ultrasonic irradiation, where DH, DS and DG were reduced by 74.1%, 34.3% and 1.4%, respectively. These values compares favorably with that obtained in the present work. In general, the results obtained clearly demonstrated that UPCP was more beneficial for the enzymolysis reaction because of its lower energy requirements. 3.4. The enzymolysis kinetic model of UPCP
DH (%)
12
8
4
0 0
20
40
60
80
100
(c) Enzymolysis time (min) Fig. 4. Hydrolysis curves with different e0/s0 ratios at substrate concentration of (a) 9.98, (b) 14.97 and (c) 19.96 g/L in UPCP enzymolysis.
Learning from kinetic modeling can be augmented given the precise and accurate model parameters, which should be estimated using substrate protein and enzyme concentrations. Fig. 4 shows the change of DH in the enzymolysis process of UPCP under different substrate protein and enzyme concentrations and time. Values of a and b for different initial substrate protein and enzyme concentrations were calculated from nonlinear regression analysis. The value of a increased when the initial enzyme concentration increased while parameter b did not exhibit any trend. The average value of parameter b can be considered as 0.173 (Table 4). A straight line was obtained by the change trend of kinetic parameter a as the change of e0/s0 (Fig. 5). The linear regression analysis on the figure shows a correlation coefficient of 0.9898 and yields the expression:
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References
DH (%)
14
s0=7.48 g/L, e0=0.09g/L, Calculated values s0=7.48 g/L, e0=0.09g/L, Experimental data s0=19.96 g/L, e0=0.36g/L, Calculated values s0=19.96 g/L, e0=0.36g/L, Experimental data s0=29.94 g/L, e0=0.45g/L, Calculated values s0=29.94 g/L, e0=0.45g/L, Experimental data
7
0
0
40
80
Enzymolysis time (min) Fig. 6. Hydrolysis curves for different initial enzyme and substrate concentrations and calculated values fitted to Eq. (23) during enzymolysis time of 90 min.
a ¼ 0:02021
e0 þ 0:7044 s0
ð22Þ
Insertion of Eq. (10) and parameter b of 0.173 in Eq. (8), and the kinetic model for the enzymolysis of UPCP was obtained as following:
e0 DH ¼ 5:78 ln 1 þ 3:4946 þ 0:1219 t s0
ð23Þ
The above Eq. (23) agrees with Eq. (20) deduced from the proposed mechanism, which imply the relationship of DH, enzyme concentration, substrate concentration and enzymolysis time. To confirm the validity of the enzymolysis kinetic model and the kinetic parameters, three groups of experiments were done under different initial enzyme and substrate concentrations. The agreement between calculated values of DH versus enzymolysis time and the experimental data is shown in Fig. 6. The experimental values fitting with the enzymolysis kinetic model with a low average relative error (4%) confirmed that the kinetic model was accurate to reflect the enzymolysis process.
4. Conclusion In the present study, single-frequency countercurrent and pulsed ultrasound had a significant effect on the kinetic parameters of the enzymolysis reaction compared to traditional enzymolysis, in which KM was lower by 9%. The reaction rate constants for UPCP enzymolysis were significantly higher than that of the traditional enzymolysis at different temperatures especially at a low temperature. This accounted for the kus as well as lower activation energy (Ea), enthalpy (DH) and entropy of activation (DS). A suitable model of reproducing the kinetic behavior of the system under study was applied to hydrolysis reaction of UPCP, which allowed the calculation of the necessary kinetic parameters from experiments. The positive effects of single-frequency countercurrent and pulsed ultrasound in this study and application of the kinetic model may be useful for the release of bioactive peptides from meat processing by-products. Acknowledgments This work was supported financially by Graduate Innovative Projects in Jiangsu Province (CXZZ12-0701). The authors are thankful to Dr. Samuel Jerry Cobbina for language polishing.
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