Effects of multi-frequency power ultrasound on the enzymolysis of corn gluten meal: Kinetics and thermodynamics study

Accepted Manuscript Effects of multi-frequency power ultrasound on the enzymolysis of corn gluten meal: kinetics and thermodynamics study Jian Jin, Haile Ma, Wenjuan Qu, Kai Wang, Cunshan Zhou, Ronghai He, Lin Luo, John Owusu PII: DOI: Reference:

S1350-4177(15)00124-8 http://dx.doi.org/10.1016/j.ultsonch.2015.04.031 ULTSON 2854

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

5 March 2015 27 April 2015 27 April 2015

Please cite this article as: J. Jin, H. Ma, W. Qu, K. Wang, C. Zhou, R. He, L. Luo, J. Owusu, Effects of multifrequency power ultrasound on the enzymolysis of corn gluten meal: kinetics and thermodynamics study, Ultrasonics Sonochemistry (2015), doi: http://dx.doi.org/10.1016/j.ultsonch.2015.04.031

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Effects of multi-frequency power ultrasound on the enzymolysis of corn gluten meal: kinetics and thermodynamics study† Jian Jina,b,*, Haile Maa,b, Wenjuan Qua,b, Kai Wanga, Cunshan Zhoua,b, Ronghai Hea,b, Lin Luoa,b, John Owusua,c

a

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang

212013, China b

Jiangsu Provincial Key Laboratory for Physical Processing of Agricultural Products, 301 Xuefu

Road, Zhenjiang 212013, China c

Department of Hospitality, School of Applied Science and Technology, Koforidua Polytechnic,

P.O. Box 981, Koforidua, Ghana *

Corresponding author.

Address: School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China. Tel.: +86 511 88790958; fax: +86 511 88780201. E-mail address: [email protected] (J. Jin)

†

Abbreviations: CGM, corn gluten meal; DH, degree of hydrolysis; MPU, multi-frequency power ultrasound; SFPU, sweeping frequency and pulsed ultrasound. 1

Abstract

The effects of multi-frequency power ultrasound (MPU) pretreatment on the kinetics and thermodynamics of corn gluten meal (CGM) were investigated in this research. The apparent constant (KM), apparent break-down rate constant (kA), reaction rate constants (k), energy of activation (Ea), enthalpy of activation (∆H), entropy of activation (∆S) and Gibbs free energy of activation (∆G) were determined by means of the Michaelis-Menten equation, first-order kinetics model, Arrhenius equation and transition state theory, respectively. The results showed that MPU pretreatment can accelerate the enzymolysis of CGM under different enzymolysis conditions, viz. substrate concentration, enzyme concentration, pH, and temperature. Kinetics analysis revealed that MPU pretreatment decreased the KM value by 26.1% and increased the kA value by 7.3%, indicating ultrasound pretreatment increased the affinity between enzyme and substrate. In addition, the values of k for ultrasound pretreatment were increased by 84.8%, 41.9%, 28.9%, and 18.8% at the temperature of 293, 303, 313 and 323 K, respectively. For the thermodynamic parameters, ultrasound decreased Ea, ∆H and ∆S by 23.0%, 24.3% and 25.3%, respectively, but ultrasound had little change in ∆G value in the temperature range of 293-323 K. In conclusion, MPU pretreatment could remarkably enhance the enzymolysis of CGM, and this method can be applied to protein proteolysis industry to produce peptides. 2

Keywords: Corn gluten meal; ultrasound; Enzymolysis; Kinetics; Thermodynamics

3

1. Introduction

Proteins are essential components of the diet for human nutrition as a source of amino acids (AAs) used for functional and tissue protein synthesis. It is estimated that the world has a shortage of millions of tons of proteins every year. Unfortunately, a large number of protein resources are underutilized. A typical example of these is corn gluten meal (CGM), a by-product of the corn wet-milling process, which contains 60%–71% protein [1], but has limited use in the human food industry due to its water insolubility. It was reported that the solubility of CGM can be increased by acid hydrolysis, alkaline hydrolysis or emzymolysis [2, 3]. Among all the hydrolysis methods, enzymolysis of proteins is regarded as the most appropriate method because of its controllable mild reaction conditions, large scale commercial availability, and high product quality [4-6]. Nevertheless, the traditional enzymolysis has many disadvantages such as low utilization rate of the enzyme, low conversion rate of the substrate, long enzymolysis time, and energy-extensive consumption [7, 8]. This is largely due to the unsuitable conformation of protein which makes it difficult for the enzyme to attack the enzyme-susceptible bonds of a protein. Therefore, developing a more efficient enzymolysis method to overcome the drawbacks above is in great demand. The ultrasonic technology, as a novel physical processing technology, has attracted much attention in assisted extraction of bioactive components [9-11], assisted enzymatic 4

hydrolysis [7, 8, 12, 13]and ultrasound-assisted freezing [14-16] because of its special acoustic cavitation effect that the collapse of cavitation bubbles generates high temperatures (approximately 5 500 K) and high pressures (approximately 50 MPa ) in very short time ( 3×10

-4

s ), which results in high intensity of shear forces and shock

waves and turbulence[17-19]. Some researchers have used the ultrasound to pretreat the substrate proteins or the enzyme before enzymolysis, and the results showed that the ultrasonic pretreatment could improve the bioactivity of target products as well as the reaction rate and the conversion rate of substrates significantly[7, 8, 20, 21]. The ultrasound frequency is one of the important parameters that affect the efficiency of sonochemical reactions [12, 22, 23]. Ren et al.[12] studied the effects of sweeping frequency ultrasound treatment on enzymatic preparations of ACE-inhibitory peptides from zein, they found that using 40 ± 2 kHz remarkably raised the degree of hydrolysis and ACE-inhibitory activity of the hydrolysates. However, few researches have focused on the effects of multi-frequency power ultrasound (MPU) on the enzymatic hydrolysis of CGM. Alcalase, which is an industrial- and food-grade enzyme produced by a selected strain of Bacillus licheniformis, and whose main component is subtilisin Carlsberg [24], has been used as a model enzyme to hydrolyze CGM [25-27]. In the current research, before CGM was hydrolyzed by Alcalase, the CGM was pretreated by MPU. According to the results of our previous study [28], the sweeping frequency and pulsed ultrasound (SFPU) 5

equipment (Fig.1a) was used in the present research. The sweeping frequency operation (f0 ± δ kHz) refers to the sweeping frequency cycle of increasing period from f0 - δ to f0 + δ kHz and decreasing period from f0 + δ to f0- δ kHz with the same linear speed in the form of an isosceles triangle (Fig.1b), and the pulsed operation indicates that ultrasound is generated in a pulsed mode with an on-time and an off-time cycle. Compared with classical ultrasound technology, the SFPU has many advantages such as uniform energy distribution and the ease of resonate of treated material. Moreover, the cavitation yield of multiple frequencies operation is higher because the implosion of the cavitation bubbles coming from lower frequency irradiation can provide new cavitation nuclei not only for itself, but also for the other ultrasound irradiation [29]. Accordingly, this paper investigated the effects of MPU pretreatment on the enzymatic hydrolysis of CGM under different substrate and enzyme concentrations, pH, and temperatures. Furthermore, the effects of MPU pretreatment on the kinetic and thermodynamic parameters of enzymolysis of CGM were studied.

2. Materials and methods 2.1. The ultrasonic pretreatment of CGM Raw material CGM which contains 15.4% starch and 58.6% protein was purchased from FenDa Starch Co. (Pizhou, Jiangsu, China), the part passed through a 70-mesh

6

screen (particle size of 0.21 mm) was used as experimental material. Different volume (100, 150, 200, 300, 400 mL) of CGM suspensions with the same content (5%, w/v) of protein were sealed in high pressure resistance bags and pretreated with the SFPU equipment (internal dimensions 362 mm × 294 mm × 502 mm, Shangjia Biotechnology Co., Wuxi, Jiangsu, China) of which the maximum output acoustic power was 600 W per single plate determined by the calorimetric method [30] at the temperature of 30 ºC using water as medium. The pretreatment conditions were obtained from our previous study and as follows: combination of ultrasonic sweeping frequency (28 ± 2) kHz (upper plate) and (68 ± 2) kHz (bottom plate), temperature of the solution (30 ± 2) ºC, pulsed on-time 10 s and off-time 3 s, cycle time of the sweeping frequencies 500 ms, duration 40 min and power density 80 W/L. The control was carried out with a magnetic stirring instead of ultrasound at 30 ºC for 40 min at a speed of 100 r/min. 2.2. Enzymolysis of CGM The enzymolysis apparatus consisted of a digital thermostat water bath (DK-S26, Jinghong experimental apparatus Co., Shanghai, China), a pH meter (FE-20, Mettler Toledo Co., Shanghai, China) and an impeller-agitator (JJ-1, Zhongda instrument Co., Jintan, Jiangsu, China ) at a speed of 100 r/min. The ultrasonic pretreated protein solutions were diluted to 500 mL to obtain different substrate protein concentrations (5.84, 7.30, 11.68, 14.60 and 23.36 g/L), after 10-min preheating at certain temperature 7

(20, 30, 40 and 50 ºC), the pH of the solution was adjusted to a certain value (7.0, 7.5, 8.0, 8.5, 9.0 and 9.5) and the Alcalase (0.59, 0.73, 1.17, 1.46 and 2.34 g/mL) with an activity of 23 400 U/mL and mass density of 1.17 g/mL was added to start the enzymolysis. The enzymatic hydrolysis time was 90 min, during which the pH was maintained by continuous addition of 0.5 M NaOH. 2.3. Determinations of degree of hydrolysis and hydrolyzed protein concentration The degree of hydrolysis (DH) was calculated according to the pH-stat method described by Adler-Nissen [31]: DH (%) =

h htot

=

N b × B ×100 α × M p × htot

(1)

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 9.2 mmol/g for corn protein, α is the average degree of dissociation of the α-NH2 groups. In order to describe the enzymolysis kinetics of CGM, the hydrolyzed protein concentration was determined by the following equation proposed by Qu et al.[32]: Ct = C0 × DH × 001 .

(2)

Where, Ct is the hydrolyzed protein concentration at a given time t min (g/L), C0 is the initial protein concentration (g/L), DH is the degree of hydrolysis.

8

2.4. Determination of initial reaction rate and kinetic parameters KM and kA The initial reaction rate (V0, g/L*min-1) were measured by the method described by Jia et al.[20] and the kinetic parameters KM, kA were determined using the following kinetic model developed by Schurr and McLaren [33]: K 1 1 1 = M × + V0 k A E0 C0 k A E0

(3)

Where, KM is the apparent constant analogous to Michaelis-Menten constant (g/L), kA is an average value of apparent breakdown rate constant (1/min), representing binding frequency between substrate and enzyme, E0 is the enzyme concentration (g/L), C0 is the substrate concentration (g/L). The slope and intercept of the line 1/V0 against 1/C0 are KM/(kAE0) and 1/(kAE0), respectively. 2.5. Determination of the reaction rate constant k In order to determine the reaction rate constant k, the first-order kinetic model described by Kadkhodaee and Povey [34] was applied. The kinetic model was written as: dCt = − kCt dt

(4)

After integrating, the model can be expressed as:

lnCt = −kt + lnC0

(5)

Where, Ct is the concentration of protein at a given time t (g/L), C0 is the initial 9

concentration of protein (g/L), t is the hydrolysis time (min), k is the reaction rate constant. As it is difficult to measure the decrement of the protein, the reaction rate can be reflected by the increase in the amount of peptides released by CGM. Under a certain temperature and pressure, C0 = Q∞ and Ct = (Q∞－Qt). Therefore, Eq. (5) can be written as:

ln(Q∞ − Qt ) = −kt + ln Q∞

(6)

Where Qt is the concentration of peptides at a given time t (g/L), Q∞ is the ultimate concentration of peptides released by CGM (g/L), which was obtained from the enzymolysis experiment conducted under pH 9.0 at 50 ºC for 10 h [21, 35]. The k value can be determined from the slope by plotting ln (Q∞－Qt) against t. For ultrasound pretreatment,

k = kt + kup

(7)

Where, kt is reaction rate constant induced by thermal effect in traditional enzymolysis, and kup is the reaction rate constant induced by ultrasonic effect. 2.6. Determination of thermodynamic parameters Ea, ∆H, ∆S, and ∆G The relationship between the constant rate k and temperature can be described by Arrhenius equation: ln k = −

Ea + ln A RT 10

(8)

Where, k is the reaction rate constant (1/min), A is the pre-exponential factor (1/min), R is the universal gas constant (8.314 J/mol*K-1), T is the Kelvin temperature (K), Ea is the activation energy (J/mol), which can be calculated from the slope of the plots of lnk against 1/T.

According to Swati et al. [36] and transition state theory, the changes in enthalpy of activation (∆H), entropies of activation (∆S), and Gibbs free energy (∆G) of the enzymolysis can be calculated with the help of the following equations:

∆H = Ea − RT

ln

(9)

k ∆H 1  k B ∆S  =− × +  ln +  T R T  h R 

∆G = ∆H − T ∆S

(10) (11)

Where, kB is the Boltzman constant (1.38 × 10-23 J/K), h is the Planck constant (6.6256 × 10-34 J*s), ∆G is the Gibbs free energy of activation (J/mol), ∆H is the enthalpy of activation (J/mol), ∆S is the entropy of activation (J/mol*K-1). 2.7. Statistical analysis All the experiments were carried out in triplicate and the results were expressed as mean ± standard deviation (SD). Analysis of variance and Tukey’s test were performed under the significance level of P < 0.05 with the aid of the statistical software SPSS 18.0 (IBM Corporation, NY, USA), and the graphs were created in OriginPro8.5 (OriginLab 11

Corporation, MA, USA).

3 Results and discussion 3.1. Effects of MPU pretreatment on the enzymolysis of CGM Fig. 2 to Fig. 5 show the hydrolyzed protein concentrations in traditional and MPU pretreatment under various substrate concentrations, enzyme concentrations, pH and temperatures during an enzymolysis time of 90 min. As can be seen from Fig. 2(a) and (b), typical hydrolysis curves were obtained in which the hydrolyzed protein concentration profiles were characterized by an initial fast rate followed by a rapid decrease in rate for both traditional and ultrasound-assisted enzymolysis. The hydrolysis rate for MPU pretreatment in the first 20 min was much higher than that for traditional one, especially at higher substrate concentration (11.68, 14.6 and 23.36g/L). The results also indicated MPU pretreatment remarkably improved the hydrolyzed protein concentration compared to traditional enzymolysis especially at the beginning of the enzymolysis period. The enzymolysis process of CGM at different substrate concentrations was in agreement with the results reported by Qu et al.[32]. Fig. 2(a) and (b) Fig. 3 shows the hydrolyzed protein concentration in both traditional (a) and MPU pretreatment enzymolysis (b) under different enzyme concentrations. The hydrolyzed

12

CGM protein concentration increased with increasing enzyme concentration during the whole enzymolysis process. However, the MPU pretreatment showed a relative sharp increase in the first 20 min when the enzyme concentration was higher than 1.46 g/L when compared with traditional enzymolysis. This is mainly due to the fact that ultrasound pretreatment induced molecular unfolding of protein [37], which was beneficial to be attacked by enzyme. Fig. 3(a) and (b) The hydrolyzed protein concentration in both traditional enzymolysis and MPU pretreatment under different pH was shown in Fig. 4. The hydrolyzed protein concentration increased with increasing pH in the first 10 min for both traditional enzymolysis and MPU pretreatment. After 30-min hydyolysis, as far as MPU pretreatment was concerned, the hydrolyzed protein concentration in hydrolysate at pH 9.5 was the same as that at pH 7.5, and the ultimate concentration did not change significantly (P > 0.05) at pH 8.0, 9.0, and 9.5. However, the hydrolyzed protein concentration in traditional enzymolysis at pH 8.5 and 9.0 showed no significant (P > 0.05) difference. On the other hand, the final hydrolyzed protein concentration at pH 7.5 was 3.79 g/L for MPU pretreatment, while the concentration at pH 8.5 was 4.03 g/L for traditional enzymolysis, representing 5% increment of the latter over the former. Actually, a large quantity of salt was produced during the peptide production process, meaning much resource must be put into the desalting process [38]. Therefore, MPU pretreatment 13

may have great superiority in producing peptides with low content of salt by means of reducing the reaction pH of the traditional enzymolysis. Fig. 4(a) and (b) Fig. 5 exhibits the hydrolyzed protein concentration in both traditional enzymolysis and MPU pretreatment at various temperatures. The higher the temperature, the faster the reaction rate is. As can be seen from Fig. 5(a) and (b), the MPU pretreatment is obviously superior to the traditional enzymolysis at every temperature point especially at the temperature of 40 and 50 ºC. Having subjected to a 20-min hydrolysis, the hydrolyzed protein concentrations for MPU pretreatment were 1.94 and 2.47 g/L while those for traditional enzymolysis were 1.46 and 1.96 g/L at temperature of 40 and 50 ºC, and the peptides yield were improved by 32.9% and 26.0%, respectively. Fig. 5(a) and (b) 3.2. Effect of MPU pretreatment on kinetic parameters KM and kA It is generally accepted that serine protease such as Alcalase, when acting on peptide bonds, follow Michaelis–Menten kinetic [31, 39, 40]. To determine the kinetic parameters for both traditional enzymolysis and MPU pretreatment, the Lineweaver-Burk equation (Eq. (3)) was used, and the plots obtained were presented in Fig.6. As seen from the figure, the 1/V0 was linear to 1/C0 in both traditional enzymolysis and MPU pretreatment, with correlation coefficients of 0.959 and 0.967, respectively. The 14

estimated constants (KM and kA) were given in Table 1. 1/KM represents the affinity between enzyme and substrate, the KM value obtained for MPU pretreatment decreased by 26.1% compared with that for the control (traditional enzymolysis), indicating an improvement in the affinity between enzyme and substrate protein. In addition, the kA value increased by 7.3% after the irradiation of ultrasound, implying higher binding frequency between enzyme and CGM. Qu et al. [32] used SFP ultrasound with frequency of 24 ± 2 kHz to pretreat wheat germ protein, and the results showed that the kA value increased by 66.7% while the KM value decreased by 6.9% in comparison with traditional enzymolysis. Song et al. [7] utilized ultrasonic bath (40 kHz, 0.64 W/cm2) to pretreat enzyme used for degradation of untanned solid leather waste, and found out that the value of kA for the enzymatic hydrolysis of the untanned solid leather waste increased by 18% but the KM was almost unchanged. The variety of substrate protein and the differences in ultrasound parameters may explain the difference in the values of KM and kA. The decrease in KM value 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, hydrophobic and electrostatic interaction [41, 42], and also by disrupting the secondary structure of proteins, resulting in the exposure of more cleavage sites to the binding sites of the enzyme which can be certified by the increase in the kA value found in the present study. On the other hand, the shearing force, shock waves, free radicals which are induced by ultrasound might have crushed the 15

starch-protein cross-linkage [43] and reduced the particle size of proteins [44, 45], resulting in enlargement of specific surface areas of CGM, and thus increased the contact areas between Alcalase and matrix. Our previous study [28] showed that MPU pretreatment had altered the molecular conformation as well as the microstructures of CGM and its major protein fractions. Fig. 6 Table 1 3.3. Effects of MPU pretreatment on reaction rate constant k From above, the ultrasonic pretreatment can facilitate enzymolysis of CGM, but to what extent is still unknown. Thus, we investigated the effect of ultrasound on the reaction rate constant (k) by hydrolyzing the protein at substrate concentration of 14.6 g/L, enzyme concentration of 1.46 g/L, pH 9.0 and temperature varied from 293 K to 323 K for 90 min, and the plots between 2-10 min were taken into account. Fig.7 shows plots of ln (Q∞－Qt) versus t in traditional enzymolysis (a) and MPU pretreatment (b). As can be seen from Fig. 7, the ln (Q∞－Qt) had a good linear relationship with time at various temperatures because the correlation coefficients were greater than 0.99 and 0.96 for traditional enzymolysis and MPU pretreatment, respectively. Therefore, using the first-order kinetics to depict both the traditional enzymolysis and MPU pretreatment within the temperature range was suitable. The reaction rate constant k was determined 16

from the slope by plotting ln (Q∞－Qt) against t, and the results were shown in Table 2. As seen from the Table, the higher the temperature, the greater the rate constant k. When increasing the temperature from 293 to 313 K, the rate constant k of both traditional enzymolysis and MPU pretreatment obey the Van 't Hoff rule that the reaction rate constant k is 2~4 times of the primary. This might be due to the enhancement of the enzyme activity and higher collision frequency between the enzyme and protein at higher temperature. Compared with traditional enzymolysis, the reaction rate constant k of MPU pretreatment were increased by 84.8%, 41.9%, 28.9% and 18.8% at the temperature of 293, 303, 313 and 323 K, respectively. Additionally, the rise ratio of reaction rate decreased while the reaction rate constant induced by ultrasonic (kup) increased as the enzymolysis temperature increased. Kadkhodaee and Povey [34] used 30 kHz probe ultrasound to inactivate α-amylase from Bacillus amyloliquefacience, they reported kup decreased with the rise of temperature. This contradictory result might be due to the fact that the temperature they reported was the ultrasonic parameter (20-80 ºC) while the ultrasonic temperature of the present study was fixed at 30 ºC on average. On the other hand, the hydrophobic interaction increases with increasing temperature [46], thus the hydrophobic groups unfolded by ultrasound [13, 28, 37, 47] will rearrange more intensively to reach the minimum energy state. During the conformational rearrangement course [48-50], the contact frequency between protein and enzyme might be increased, as a consequence, the kup increased with temperature rise. 17

Fig. 7(a) and (b) Table 2 3.4. Effects of MPU pretreatment on the thermodynamic parameters The thermodynamic parameter activation energy (Ea) which represents the amount of energy required from non-activation molecules into activation molecules can reflect the rapidity of chemical reactions. The Ea values of most reactions range from 40 kJ/mol to 400 kJ/mol and the reaction will rapidly come to completion if the value is lower than 40 kJ/mol [8, 21]. By plotting ln k versus 1/T (Fig. 8), Ea was obtained from the slope of the Arrhenius plots and the results were presented in Table 3. The Ea values were 49.07 and 37.78 kJ/mol for traditional enzymolysis and MPU pretreatment respectively, indicating that ultrasonic pretreatment reduced the energy barrier required for the enzymolysis reaction by 23.0%. Consequently, MPU assisted enzymolysis proceeded rapidly than the traditional one, and this was verified in the experiment. Fig. 8 Table 3 The enthalpy of activation (△H), entropy of activation (△S), Gibbs free energy of activation (△G) at each experimental temperature were calculated according to Eqs.(9)~(11) and the results were presented in Table 3. Positive values of enthalpy indicate endothermic nature of the hydrolysis reaction. In addition, these values indicate 18

that it requires relative lower enthalpy of activation at higher temperature. Compared with traditional enzymolysis, the △H of MPU pretreatment was decreased by 24.3% on average in the temperature range of 293-323 K. Entropy (S) is a physical quantity that describes the local disordering in the reaction system. As can be seen from Table 3, the entropy of activation (△S) increased with increasing the temperature. By comparison, the

△S of MPU pretreatment was decreased by 25.3% on average, this decrease in △S indicated that the enzyme and CGM were more orderly distributed after ultrasound irradiation. △G increased from temperature 293 K to 323 K in both traditional enzymolysis and MPU pretreatment (Table 3), ultrasound decreased △G slightly because the decrease in △G was dependent on the declines in both △H and △S. Ma et al.[21] pretreated Alcalase with energy-gathered ultrasound (20 kHz, 80 W, 4 min), and the results showed that the thermodynamic parameters Ea, ∆H, ∆S and ∆G reduced by 70.0%, 75.8%, 34.0% and 1.3%, respectively. Meanwhile, spectroscopic analysis revealed that the ultrasonic treatment had increased the number of tryptophan on Alcalase surface slightly, increased the number of α-helix by 5.2%, and reduced the number of random coil by 13.6%. These decreases in Ea, △H, △S and △G can be attributed to the ultrasonically induced breakage of non-covalent bonds which stabilize the protein structure at ground state, to the exposure of the internal hydrophobic core, to the oxidative modification of amino acid residues [8], and to its acoustic cavitation on the molecular spatial conformation and microstructure of the protein [28, 37] as well as on 19

the starch granules cross-linking with protein [43].

4. Conclusions

MPU pretreatment can accelerate the enzymolysis of CGM under different enzymolysis conditions, viz. substrate concentration, enzyme concentration, pH, and temperature. Moreover, ultrasound pretreatment may have great superiority in producing peptides with lower content of salt by decreasing the optimal reaction pH. The results of kinetics and thermodynamics analysis showed that MPU pretreatment increased the affinity between enzyme and CGM, and increased reaction rate constants (k) as well as reduced activation energy (Ea), enthalpy of activation (△H), entropy of activation (△S), and free energy of activation (△G). In general, MPU pretreatment of CGM before proteolysis an efficient and environmentally friendly method for producing peptides, and the methodology and framework presented in this paper can be applied to enzymatic hydrolysis of other proteins, cellulose biomass and so on.

Acknowledgements

The authors wish to acknowledge the supports from the grant (2013AA102203) of the Project of National 863 Plan of China, National Natural Science Foundation of China (31471698

and

31301423),

Research-Innovation

Program

(CXZZ13-0695)

for

Postgraduates in General Universities of Jiangsu Province, Natural Science Foundation of 20

Jiangsu Province (BK2012708), Key University Science Research Project of Jiangsu Province (12KJA550001), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Figure captions Fig.1 The sweeping frequency and pulsed ultrasonic (SFPU) equipment (a) and frequency variation curve for sweeping model (b), f0 is the central frequency, δ is the sweeping amplitude which was found as 2 kHz in the present study. Fig.2 The hydrolyzed protein concentrations in traditional enzymolysis (a) and MPU pretreatment (b) at different substrate concentrations (E0 = 1.46 g/L, pH = 9.0, T =50 ºC) Fig.3 The hydrolyzed protein concentrations in traditional enzymolysis (a) and MPU pretreatment (b) at different enzyme concentrations (C0 = 14.6 g

net protein/L,

pH = 9.0, T

=50 ºC) Fig.4 The hydrolyzed protein concentrations in traditional enzymolysis (a) and MPU pretreatment (b) at different pH (C0 = 14.6 g net protein/L, E0 = 1.46 g/L, T =50 ºC) Fig.5 The hydrolyzed protein concentrations in traditional enzymolysis (a) and MPU pretreatment (b) at different temperatures (C0 = 14.6 g

net protein/L,

E0 = 1.46 g/L, pH =

9.0,) Fig.6 The plots of the reciprocal of the initial reaction rate (1/V0) versus the reciprocal of the protein concentration (1/C0) in traditional enzymolysis and MPU pretreatment. Fig.7 The relationship curves between ln (Q∞－Qt) and reaction time (t) for traditional enzymolysis (a) and MPU pretreatment (b). The regression coefficients (R2) of the curves obtained at 293, 303, 313, and 323 K for traditional enzymolysis are 0.9921, 0.9989, 0.9960, and 0.9955, respectively; the regression coefficients (R2) of the curves obtained at 293, 303, 313, and 323 K for MPU pretreatment are 0.9686, 0.9803, 0.9896, and 0.9961, respectively. Fig.8 The fitting curves by plotting ln k against 1/T.

26

Table 1 Kinetic parameters KM, kA for the traditional enzymolysis and MPU pretreatment a KM (g/L)

R2 Adj.

0.178±0.011

8.387±0.587

0.191±0.014

6.194±0.459

Enzymolysis methods

KM/(kAE0) (min)

1/(kAE0) (min*L/g)

kA (1/min)

Traditional enzymolysis

32.323±0.234

3.863±0.243

MPU pretreatment

22.244±0.072

3.601±0.255

(7.3%)

b

(-26.1%)

F Value

Prob. > F

0.9588

94.11

0.0023

0.9674

119.65

0.0016

a

E0 is 1.46 g/L.

b

The change rates of kinetic parameters in MPU pretreatment compared to traditional

enzymolysis.

27

Table 2 The reaction rate constant k for the traditional enzymolysis and MPU pretreatment * T (K)

Traditional enzymolysis 2

kt (1/min) 293 303 313 323 *

R

k (1/min)

Adj.

0.0031±0.0002a 0.9944 a

0.0072±0.0006 0.9989 a

0.0133±0.0003 0.9949 a

Ultrasound pretreatment

0.0197±0.0008 0.9960

0.0057±0.0003 (84.8%)b,

**

kup (1/min)

R2 Adj.

0.0026±0.0002

0.9715

0.0102±0.0004 (41.9%)

b

0.0030±0.0004

0.9803

0.0175±0.0003 (28.9%)

b

0.0039±0.0002

0.9892

0.0234±0.0007 (18.8%)

b

0.0037±0.0002

0.9959

Means ± SD (n=3). Within a row, means with different superscript letters are significantly

different (P < 0.05). **

The increase rates of initial reaction rate in MPU pretreatment compared to traditional

enzymolysis.

28

Table 3 Thermodynamic parameters for traditional enzymolysis and MPU pretreatment Enzymolysis methods

Traditional enzymolysis MPU pretreatment *

Ea (kJ/mol)

△S(J/mol* K-1)

△H (kJ/mol) △H 293 K

△H 303 K

△H 313 K

△H 323 K

△S 293 K

49.07±1.12b

46.63±1.12b

46.55±1.12b

46.46±1.12b

46.38±1.12b

-133.83±3.82b -132.59±3.70b

a

a

a

a

a

37.78±0.93 (-23.0%)

*

35.34±0.93

35.26±0.93

35.18±0.93

(-24.2%)

(-24.3%)

(-24.3%)

35.09±0.93 (-24.3%)

△S 303 K

a

-167.23±3.17 -166.89±3.06 (-25.0%)

a

(-25.9%)

△G(kJ/mol)

△S 313 K

△S 323 K

△G 293 K

△G 303 K

△G 313 K

△G 323 K

-132.84±3.58b

-134.71±3.47b

a

85.86

86.74

88.06

89.91

a

-166.77±2.97

-168.20±2.87

84.37

85.85

87.40

89.45

(-25.5%)

(-24.9%)

(-1.7%)

(-1.0%)

(-0.7%)

(-0.5%)

The decrease rates of thermodynamic parameters (Ea, ∆H, ∆S and ∆G) in MPU pretreatment compared to traditional enzymolysis. Means ± SD (n=3).

Within a column, means with different superscript letters are significantly different (P < 0.05).

29

Fig.1(a)

Fig.1(b)

Fig.2(a)

Fig.2(b)

Fig.3(a)

Fig.3(b)

Fig.4(a)

Fig.4(b)

Fig.5(a)

Fig.5(b)

Fig.6

Fig.7(a)

Fig.7(b)

Fig.8

Highlights: ⑴ MPU accelerated the CGM enzymolysis under different reaction conditions. ⑵ MPU increased the affinity between enzyme and CGM, accelerated the breakdown of CGM. ⑶ MPU increased reaction rate constant k significantly (P < 0.05). ⑷ MPU decreased the values of Ea, ∆H and ∆S (P < 0.05).

30