Enzymolysis kinetics and structural characteristics of rice protein with energy-gathered ultrasound and ultrasound assisted alkali pretreatments

Ultrasonics Sonochemistry 31 (2016) 85–92

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Enzymolysis kinetics and structural characteristics of rice protein with energy-gathered ultrasound and ultrasound assisted alkali pretreatments Suyun Li a,b,c, Xue Yang a, Yanyan Zhang a, Haile Ma a,⇑, Wenjuan Qu a, Xiaofei Ye a, Rahma Muatasim a, Ayobami Olayemi Oladejo a,d a

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, China School of Food and Biological Engineering, Zhengzhou University of Light Industry, 5 Dongfeng Road, Zhengzhou, Henan 450002, China c Collaborative Innovation Center of Food Production and Safety, Henan Province, 5 Dongfeng Road, Zhengzhou, Henan 450002, China d Department of Agricultural and Food Engineering, University of Uyo, P.M.B 1017, Uyo 520003, Nigeria b

a r t i c l e

i n f o

Article history: Received 29 October 2015 Received in revised form 9 December 2015 Accepted 9 December 2015 Available online 10 December 2015 Keywords: Ultrasound Enzymolysis Kinetics Rice protein Ultrasound assisted alkali pretreatment

a b s t r a c t This research investigated the structural characteristics and enzymolysis kinetics of rice protein which was pretreated by energy-gathered ultrasound and ultrasound assisted alkali. The structural characteristics of rice protein before and after the pretreatment were performed with surface hydrophobicity and Fourier transform infrared (FTIR). There was an increase in the intensity of fluorescence spectrum and changes in functional groups after the pretreatment on rice protein compared with the control (without ultrasound and ultrasound assisted alkali processed), thus significantly enhancing efficiency of the enzymatic hydrolysis. A simplified kinetic equation for the enzymolysis model with the impeded reaction of enzyme was deduced to successfully describe the enzymatic hydrolysis of rice protein by different pretreatments. The initial observed rate constants (Kin,0) as well as ineffective coefficients (kimp) were proposed and obtained based on the experimental observation. The results showed that the parameter of kin,0 increased after ultrasound and ultrasound assisted alkali pretreatments, which proved the effects of the pretreatments on the substrate enhancing the enzymolysis process and had relation to the structure changes of the pretreatments on the substrate. Furthermore, the applicability of the simplified model was demonstrated by the enzymatic hydrolysis process for other materials. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction With the improvement of people living standard and social rhythm speeding up, many people are in the sub-health state. Functional food can improve this state, which relies on the biological activity of food. Rice protein is a kind of functional food, which is hydrolyzed by enzymolysis has been largely studied because of their activity polypeptide, preventing lifestyle-related diseases by their hypertension, antioxidant, hypoallergenic activity [1–3]. The controlled enzymolysis process can make the isolation of peptides. However, traditional enzymolysis has many disadvantages such as the low degree of hydrolysis (DH) and the long time of hydrolysis. As a result, many studies have been devoted to developing methods to improve the conversion rate of substrate and reduce the enzymolysis time [4–6]. The use of ultrasound assisted enzymolysis of proteins is a well known technology, because it is attributed ⇑ Corresponding author. E-mail address: [email protected] (H. Ma). http://dx.doi.org/10.1016/j.ultsonch.2015.12.005 1350-4177/Ó 2015 Elsevier B.V. All rights reserved.

to mechanical and thermal effects enacted by cavitation [7,8]. Many authors reported that ultrasound can improve the protein properties [9], enhance the enzyme activity [10], extract the polysaccharides [11]. Recently, the interest of food researcher has turned to change the design of the ultrasonic equipments in improving functional properties of food materials. The equipments of dual-frequency, single-frequency [12], ultrasonic horn and ultrasonic bath [13] and sweep frequency [14] were compared the efficacy of various acoustic and hydrodynamic cavitation. In our experiments, the apparatus of the single-frequency energygathered ultrasound equipment (SFEGU) was developed by our team and has many advantages. ‘‘Energy-gathered” is realized by avoiding the waste of energy, due to the direction of the ultrasound wave contrary to that of the solution [15]. In addition this kind of the machine belongs to the low frequency high-energy ultrasound in the kHz range relative to the MHz range [10]. Therefore the SFEGU can improve functional properties of food materials using minor energy.

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The rice protein is mainly composed by glutelin (
80%), which is alkali-soluble [9]. Therefore ultrasound assisted alkali pretreatment can enhance mass transfer, increase contact frequency between substrate and enzyme [16]. In our previous study, the pretreatment of rice protein ultrasound assisted alkaline was investigated, and the enzymatic hydrolysis was also significantly enhanced. For an enzymolysis process, a kinetic model which may appropriately describe the enzymatic reaction is essential to the controlled hydrolysis and the design of the reactor. The classical Michaelis–Menten modeling has been used for enzyme kinetics of proteins with different enzymes. However, enzymatic hydrolysis of proteins is a highly complex process due to the different pretreatment to substrates and leading to the undefined nature of substrates, a heterogeneous reaction system and so on [17]. But no study has been reported on the kinetics of SFEGU assisted alkali enzymolysis for producing active peptides from different rice protein concentration and time. The aims of this study were to (1) further explore the structural changes of rice protein before and after pretreatment with SFEGUassisted and SFEGU-assisted alkali at a mild condition, including the comparison of surface hydrophobicity and Fourier transform infrared (FTIR), (2) deduce a simplified kinetic model which could indicate the efficiency of the different pretreatments, avoiding solving complicated ordinary differential equations and using many uniquely determined parameters, (3) the hydrolysis equation could predict the enzymatic hydrolysis process for other materials. 2. Materials and methods

Engineering Co., Ltd., Wuxi, China; Model FBTQ 2000), which was dipped to a depth of 2 cm in the rice protein suspension. The ultrasonic generator can deliver a maximum power of 300 W. This machine provides a continuous flow of raw materials by peristaltic pump and the direction of the solution moved counter currently to that of the ultrasound wave. The temperature of the reaction is kept constant by water bath. 2.2. Pretreatments of rice protein with SFEGU-assisted and SFEGU-assisted alkali The solution containing 32 g rice protein and 800 mL deionized water was stirred at 50 °C for 15 min. After pretreatment, the solution was divided into two equal parts. One part (400 mL) solution was centrifuged at 5030 (g) to remove the supernatant, and the precipitate was subjected vacuum freeze drying for 36 h to obtain the treated rice protein; the other part (400 mL) was used for enzymatic hydrolysis. This method was called method 1. The solution was prepared as mentioned previously. The difference is the solution was pretreated by ultrasound at 50 °C for 15 min. Based on our previous result, the optimal ultrasonic parameters were 58 W/L (power density), pulsed on-time 3 s and off-time 2 s and the frequency was set at 28 kHz. After ultrasound application, the solution was subjected to the same treatment as stated in method 1 above. This was named method 2. The rice protein solution was first adjusted to pH 8.0 using 1 M NaOH before ultrasound. Ultrasonic parameters were the same with the method 2. After ultrasound, the way of dealing with the solution was at the same with method 1. The ultrasound-alkali assisted pretreatment of rice protein was denoted as method 3.

2.1. Materials and ultrasonic equipment 2.3. Enzymatic hydrolysis and analysis The rice protein used in this study was purchased from Zhengzhou Tianshun food additives Co., Ltd. (Henan, China). The particle size of rice protein was 0.15 mm and crude protein content was 813 g/kg. Commercial Alcalase 2.4 L purchased from Novozymes Co., Ltd. (Tianjin, China) (activity of 23,400 U/mL) was used in the enzymatic hydrolysis. All reagents used in the experiment were of analytical grade. In our experiment the single-frequency energygathered ultrasound equipment (developed by our team, Fig. 1) was equipped with a 2 cm flat tip probe (Fanbo Biological

After pretreatment by three different methods, the solution was subjected to enzymatic hydrolysis. The equipment consisted of a pH meter (FE-20, Mettler Toledo Co., Shanghai, China), a digital thermostat water bath (DK-S26, JingHong experimental apparatus Co., Shanghai, China) and an impeller-agitator (JJ-1, Zhongda instrument Co., Jiangsu, China) at a speed of 100 rpm. Rice protein solution was stirred at 50 °C and 1 M NaOH was added to adjust the pH value to 8.5, then Alcalase enzyme (E/S = 1638 U/g) was

Fig. 1. Schematic diagrams of the single-frequency energy-gathered ultrasound equipment (SFEGU).

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S. Li et al. / Ultrasonics Sonochemistry 31 (2016) 85–92

added. The pH of the solution was maintained constant by adding 0.5 M sodium hydroxide continuously during the enzymolysis process. The time taken for the process of enzymolysis was 100 min and took note of the alkali consumption. Eqs. (1) and (2) were used to calculate the yield of DH according to Adler-Nissen [18]:

DH ð%Þ ¼

a¼

h Nb  V  100  100 ¼ htot a  Mp  htot

2.4. Structural characterization 2.4.1. Surface hydrophobicity 1-anilino-8-naphthalene-sulfonate (ANS (Sigma Chemical Co., St. Louis, MO, USA)) as a fluorescence probe was used to measure the surface hydrophobicity of rice protein dispersions according to the method of Li et al. [19]. With slight modifications, the freeze-dried rice protein samples which were prepared by method 1–3 were all 2 mg/mL in 0.01 M phosphate buffer solution (PBS) at pH 8.0 and were centrifuged at 5030 (g) for 10 min. The protein concentration in the supernatant was determined by the Lowry method [20]. Each supernatant was diluted with the PBS to a final concentration of 0.5 mg/mL. Then 20 lL of ANS (8.0 mM in sample phosphate buffer) was added to 4 mL supernatant and mixed. The mixture was kept in the dark for 5 min at room temperature (25 ± 1 °C). The relative fluorescence intensity was measured by a Cary Eclipse spectrophotometer (Varian Inc., Palo Alto, USA) at excitation wavelength of 380 nm (slit 5.0 nm), emission wavelength 400–550 nm and 120 nm/min of scanning speed. The relative fluorescence intensity expressed the surface hydrophobicity of the 0.5 mg/mL protein concentration.

ð1Þ

10pHpK

ð2Þ

1 þ 10pHpK

where, Nb (mol/L) is the normality of NaOH, V (mL) is the volume of NaOH consumed during the experiment to keep the pH value constant, Mp (g) is the mass of protein to be hydrolyzed; htot (mmol/g) is the total millimoles of peptide bonds per gram in the protein substrate, which is 7.72 for rice protein; a is the average degree of dissociation of the a-amino groups which were released during hydrolysis and related with the pK and pH, a is calculated by Eq. (2), where pH and pK are the values at particular condition, which are 8.5 and 7 in this study, respectively. So the value of a is 0.969.

method 1(control)

Emission fluorescence intensity a.u.

350

509 nm

480 nm

method 2

300

method 3

250 200 150 100 50 0 400

420

440

460

480

wave length

500

520

540

nm

Fig. 2. Effects of ultrasound and ultrasound assisted alkali pretreatments on the emission fluorescence spectra of rice protein (using ANS as a fluorescence probe). Sonication parameters: ultrasonic power density 58 W/L and 28 kHz for 15 min (pulsed on-time 3 s and off-time 2 s) at 50 °C. Alkaline solution parameters: pH = 8.0. The protein concentration was 0.5 mg/mL in pH 8.0 phosphate buffer solution.

100 method 1(control) method 2

90 transmittance(%)

method 3 80 70 60 50 40 1900

1700

1500

1300

1100

900

700

500

-1

wave number(cm ) Fig. 3. Effects of ultrasound and ultrasound assisted alkali pretreatments on the Fourier transform infrared spectroscopy of rice protein. Sonication parameters: ultrasonic power density 58 W/L and 28 kHz for 15 min (pulsed on-time 3 s and off-time 2 s) at 50 °C. Alkaline solution parameters: pH = 8.0.

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2.4.2. FTIR spectroscopy of rice protein The FTIR spectroscopy of rice protein was measured by a spectrum instrument (Model Nicolet IS50, Thermo Nicolet Corporation, USA). 1 mg of the freeze-dried rice protein powder treated by method 1–3 were mixed with 200 mg of solid KBr powder, respectively. Using an agate mortar and pestle to make the mixture homogenize, the powder was pressed into pellets (1–2 mm thick) by 15 t hydraulic equipment. The range of scanning was in the wave number 4000–400 cm1 and 128 scans. The media (KBr) containing no protein was recorded as reference spectra under setting parameters. Analysis of the FTIR spectral data was done using OMNIC software.

(a) 600

-t/ln(1-DH/100)

500 400 300 200

method 1 method 2

100

method 3

0 0

20

40

60

80

100

120

t/min

The enzymatic hydrolysis of rice proteins is a highly complex procedure owing to the following factors: (1) the reaction is conducted in a heterogeneous system, (2) the undefined nature of substrates, (3) modulated thermal inactivation of the enzyme and substrate or product inhibition [17,21–23]. Also based on Michaelis–Menten model, but using a different mathematical deduction in this study, a new kinetic model is constructed to describe the enzymatic hydrolysis in a heterogeneous system. The hydrolysis reaction of Michaelis–Menten model can be expressed briefly as:

(b) 600

500

-t/ln(1-DH/100)

400

k1

300

k1

6% by method 3 4% by method 2 4% by method 3

100

0 0

20

40

60

80

k2

E þ S ¢ ES ! E þ P, where E, S, ES and P represent the enzyme,

6% by method 2 200

100

120

t/min

(c) 700 600 500 -t/ln(1-DH/100)

2.5. Development of the kinetic model

the substrate, the enzyme–substrate complex and the product, respectively. The rate constants k1 and k2 refer to the forward reactions and k1 refers to the reverse reaction. The following assumptions in the model are made: (1) the adsorption of enzymes on the particle surface is rapid in comparison with the enzymatic reactions, (2) the impeded factors including the mass-transfer resistance, the product inhibition and non-reactive enzymes are looked as decay coefficient which have correlation with time, (3) changes which happen in the structure of solid substrate resulted from the pretreatment are reflected in the rates of reaction for the corresponding k1, k1, and k2. With the assumption, [ES] is at a quasi-steady-state and

400

d½ES ¼ 0: dt

300

The reaction rate of [ES] can be formulated by the equation as follow:

E/S=702 U/g

d½ES ¼ k1 ½E½S  k1 ½ES  k2 ½ES dt

E/S=1170 U/g

200

ð3Þ

E/S=1638 U/g 100

ð4Þ

(3) and (4)

0 0

20

40

60

80

100

120

t/min Fig. 4. Plot of t/ln(1  DH/100) versus time (t). (a) The effect of ultrasound and ultrasound assisted alkali pretreatments on enzymatic hydrolysis for 4% (w/v) rice protein. (b) The effect of the amount of substrate (4% (w/v) and 6% (w/v)) on enzymatic hydrolysis with ultrasound and ultrasound assisted alkali pretreatments. (c) The effect of enzyme loading on enzymatic hydrolysis (substrate 4% (w/v)).

can get ½ES ¼

k1 ½E½S ½E½S k1 þ k2 ¼ and km ¼ k1 þ k2 km k1

ð5Þ

where km is the Michaelis constant [24]. Based on the Michaelis– Menten equation and Eq. (5), the rate of the product can be determined by Eq. (6),

d½p k2 ¼ k2 ½ES ¼ ½E½S dt km

ð6Þ

Table 1 Values of kin,0, kimp, a and R2 for the effects of ultrasound and ultrasound assisted alkali pretreatments on enzymatic hydrolysis of rice protein. Substrate

Method

4%, 4%, 4%, 4%, 4%, 6%, 6%,

Method Method Method Method Method Method Method

(E/S = 1638 U/g) (E/S = 1638 U/g) (E/S = 1638 U/g) (E/S = 702 U/g) (E/S = 1170 U/g) (E/S = 1638 U/g) (E/S = 1638 U/g)

1 2 3 1 1 2 3

Kin,0 (min1)

a (min1)

R2 of fitting

Kimp (min1) at 50 min

Kimp (min1) at 100 min

0.0078 ± 0.0003 0.0179 ± 0.0022 0.0227 ± 0.0031 0.0067 ± 0.0005 0.0071 ± 0.0005 0.0144 ± 0.0015 0.0166 ± 0.0006

0.0291 ± 0.0165 0.0693 ± 0.0162 0.0913 ± 0.0164 0.0325 ± 0.0153 0.0297 ± 0.0151 0.0583 ± 0.0165 0.0674 ± 0.0164

0.9981 0.9967 0.9976 0.9959 0.9967 0.9967 0.9995

0.024 0.031 0.033 0.025 0.024 0.030 0.031

0.015 0.017 0.018 0.015 0.015 0.017 0.017

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S. Li et al. / Ultrasonics Sonochemistry 31 (2016) 85–92

½S0  ¼ ½S þ ½P

(a) 25

ð8Þ

then Eq. (7) can be changed as

15

where defined

DH/%

20

  ½P ½S ¼ ½S0   ½P ¼ ½S0  1  ½S0 

method 1

  d½P k2 k2 DH ¼ ½E½S ¼ ½E½S0  1  dt 100 km km

method 3 fitted method 1 fitted method 2

5

0 20

40

60

80

100

t/min

dDH k2 ½E0  ½E ½E ¼ ð100  DHÞ ¼ kin;0 ð100  DHÞ dt km ½E0  ½E0 

an important parameter of the enzyme catalytic reaction kinetics. It depends on the initial accessibility and activity of the enzyme to the solid substrate as well as the efficiency of the pretreatment on the substrate. During the course of enzymatic reaction, the degradation of rice protein is slow because it is insoluble and poorly accessible to enzymes so that the activity of an enzyme is impeded and the rate of hydrolysis is gradually reduced [25]. In addition, product inhibition and thermal inactivation of enzyme [26] also should be considered. Therefore the total impeded factors can be represented

25

20

15 DH/%

4% by method 2 4% by method 3

kimp

by the scheme, E ƒƒƒ! Eief , where Eief refers to the ineffective enzyme. kimp refers to impeded coefficient. The amount of Eief transformed from E is dependent on [E], thus the rate of impeded reaction of the enzyme can be expressed as a first-order equation with the kimp during the enzymatic hydrolysis.

6% by method 2 10

6% by method 3 fitted 4% by method 2 fitted 4% by method 3 fitted 6% by method 2

5

fitted 6% by method 3

d½Eief  ¼ kimp ½E dt

0 20

40

60

80

100

t/min

(c)

20

ð13Þ

Based on the results of Bansal [27], Yang [25] and the experimental observation, kimp can be expressed as

kimp ¼ 4%,e/s=702 U/g 4%,e/s=1170 U/g 4%,e/s=1638 U/g fitted 4%,e/s=702 U/g fitted 4%,e/s=1170 U/g fitted 4%,e/s=1638 U/g

25

ð12Þ

where the initial observed rate constant kin,0 is kin;0 ¼ k2k½Em0 , which is

(b)

0

ð11Þ

Eq. (11) can be also written as Eq. (12)

fitted method 3

0

ð10Þ

Combined Eq. (6), Eqs. (9)–(11) can be deduced

method 2

10

½P DH ¼ ½S0  100

ð9Þ

2a 1 þ at

ð14Þ

Eq. (14) shows that the opportunity for an enzyme to act on the substrate would be decreased with time. Taking a material balance for the enzyme gives

½E0  ¼ ½E þ ½ES þ ½Eief 

ð15Þ

By taking differentiation with respect to time for Eq. (15), it gives Eq. (16).

10

d½E d½ES d½Eief  þ þ ¼0 dt dt dt

DH/%

15

5

ð16Þ

In the Eq. (15) [E0] is the initial concentration of the enzyme and

0 0

20

40

60

80

100

t/min Fig. 5. Comparison of predicted and experimental DH. (a) The effect of ultrasound and ultrasound assisted alkali pretreatments on enzymatic hydrolysis for 4% (w/v) rice protein. (b) The effect of the amount of substrate (4% (w/v) and 6% (w/v)) on enzymatic hydrolysis with ultrasound and ultrasound assisted alkali pretreatments. (c) The effect of enzyme loading on enzymatic hydrolysis (substrate 4% (w/v)). Curves: the predicted DH were calculated using Eq. (21).

According to the law of conservation of mass, Eq. (7) can be got

½S0  ¼ ½S þ ½ES þ ½P

ð7Þ

where [S0] is the initial concentration of the substrate. Because [ES] is at a quasi-steady-state and k2  k1, [ES] can be negligible and Eq. (7) can be simplified as

a constant, so d½Edt0  ¼ 0, [E] is the concentration of the active enzyme, [Eief] is the concentration of the ineffective enzyme and [ES] is the concentration of the enzyme–substrate complex. The equation for the changes of the enzyme concentration is given by Eqs. (3), (13) 2a and (16) when kimp ¼ 1þ at



d½E 2a ¼ dt ½E 1 þ at

ð17Þ

Integration of Eqs. (17) and (18) is obtained.

E 1 ¼ E0 ð1 þ atÞ2

ð18Þ

Eq. (18) is combined with Eq. (12) to give as following

dDH 1 ¼ kin;0 ð100  DHÞ dt ð1 þ atÞ2

ð19Þ

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S. Li et al. / Ultrasonics Sonochemistry 31 (2016) 85–92

Table 2 Predicted and experimental yields of DH using ultrasound and ultrasound assisted alkali pretreatments on enzymatic hydrolysis for 4% (w/v) rice protein and the error of prediction. Method

Hydrolysis time

Predicted

a b

Error of predictionb (%)

DH (%) a

Experimental

Method 1 (without unsound)

10 min 20 min 40 min 60 min 100 min

5.8361 9.3461 13.3614 15.5924 17.9942

5.60 9.79 13.36 15.50 18.05

4.22 4.53 0.01 0.60 0.31

Method 2 (with ultrasound)

10 min 20 min 40 min 60 min 100 min

10.0332 13.9328 17.2892 18.7972 20.2063

11.12 13.77 16.83 18.36 20.60

9.77 1.18 2.73 2.38 1.91

Method 3 (with ultrasound assisted alkali)

10 min 20 min 40 min 60 min 100 min

11.1892 14.8411 17.7317 18.9618 20.0754

12.04 14.79 17.14 18.67 20.40

7.07 0.35 3.45 1.56 1.59

The predicted DH were calculated from the Eq. (22). of DHExperimental yield of DH Error of prediction ð%Þ ¼ Predicted yield  100. Experimental yield of DH

Table 3 Values of kin,0 (min), a (min1), kimp and R2 for the model applied in other hydrolysis situations of the literatures. Substrate

Kin,0 (min1)

a (min1)

R2 of fitting

Kimp (min1) at 70 min

Refs.

S = 60 g/L, e = 80 lL S = 60 g/L, e = 160 lL S = 60 g/L, e = 400 lL s = 9.98 g/L, e = 0.09 g/L s = 9.98 g/L, e = 0.18 g/L s = 9.98 g/L, e = 0.45 g/L

0.0217 0.0260 0.0350 4.3476 4.1662 3.7914

0.0717 0.0799 0.0946 148.63 129.27 96.606

0.9971 0.9973 0.9976 0.9967 0.9967 0.9967

0.0238 0.0242 0.0248 0.0192 0.0198 0.0209

[36] [36] [36] [37] [37] [37]

Eq. (19) is solved to give the equation

ln 100  lnð100  DHÞ ¼

kin;0 t 1 þ at

ð20Þ

Eq. (20) is rearranged as



t 1 a
¼ þ t DH kin;0 kin;0 ln 1  100

ð21Þ

Eq. (21) is used to correlate the experimental results by plotting a and t/ln (1  DH/100) versus t, and the slope and intercept are kin;0 1 , kin;0

respectively.

3. Results and discussion 3.1. Effects of ultrasound and ultrasound assisted alkali pretreatments on the surface hydrophobicity of rice protein The emission fluorescence spectra of the pretreatments by method 2 and 3 were obtained using ANS as a fluorescence probe (Fig. 2).The intensity of fluorescence spectrum can indicate the protein surface hydrophobicity, which is mainly attributed to the Trp, Tyr and Phe residues, particularly the Trp residue [28,29]. As shown in Fig. 2, the fluorescence peak (450–550 nm) intensity of pretreated rice protein increased greatly compared with the control (method 1). This phenomenon showed that method 2 and 3 could destroy partial hydrophobic interactions of protein molecules, which induced more molecular unfolding of protein, causing more hydrophobic groups to expose outside of protein molecule. In addition, the peak value of rice protein pretreated by method 3 decreased relative to that of pretreatment by method 2 and had occurred ‘‘red shift”, suggesting that the residues of hydrophobic

groups decreased but increased soluble small molecules coursed by synergistic damages of ultrasound and alkali. The changes might have been attributed to the effect of ultrasound in alliance with alkali on the protein structure and made the protein denaturation, thus an increase in pH increased the unfolding of the protein [30]. This observation corroborated with a previous report [31] on the whey protein isolate denaturation of pH, which showed that different pH solvent resulted in the unfolding of the major whey protein components leading to protein aggregation. Therefore the rice protein pretreated by method 2 and 3 was conducive to enzymolysis and were also verified by the variation of Kin,0 in the kinetics model. 3.2. Effects of ultrasound and ultrasound assisted alkali pretreatments on the functional groups of rice protein The changes of peak position about amide I (1700–1600 cm1), II (1530–1550 cm1) and III (1260–1300 cm1) are often used to analyze the transformation of protein structure [32]. This study analyzed the effects of different ultrasound pretreatments on the functional groups of rice protein using FTIR spectrum. The results showed that amide I and II peak positions had changed slightly (Fig. 3). The peak position of amide I had shifted from 1640.3 cm1 (method 1) to 1640.8 cm1 (pretreated by method 2) and 1641.2 cm1 (pretreated by method 3). An increase in wave number revealed that part of b-sheet in rice protein had turned into a-helix or random coil. In respect of amide II, the peak positions had moved from 1533.4 cm1 (method 1) to 1532.5 cm1 (pretreated by method 2 and 3). This phenomenon (decreasing in wave number) might reveal that part of random coil had turned into b-sheet in rice protein. Positions altering in amide I and II elaborated that the secondary structure of rice protein structure had

S. Li et al. / Ultrasonics Sonochemistry 31 (2016) 85–92

taken place transformation. The changes in the secondary structure content might have two possible models. The first model suggested the changes of amide could be ascribed to amide group turning into carboxyl, which cause negative charge increase and relatively reduce the amount of intramolecular hydrogen bond. Therefore the effects of electrostatic repulsion are enhanced correspondingly [33]. The second model proposed that might be mainly attributed to the fact that the ultrasonic treatment broke the interactions between the local sequences of amino acids and between the different parts of the protein molecule, such as the disulfide bonds [34]. Generally, the method 2 and 3 pretreatments could weak the interaction of rice protein and reduced the internal disulfide bonds and polar hydrogen bonding. Combined with the results from circular dichroism (CD) spectroscopy and amino acid analysis [35], those changes of secondary structure can significantly improve the solubility in water of rice protein so as to promote the degree hydrolysis and increase Kin,0 of the enzymatic hydrolysis.

(a) 300

-t/ln(1-DH/100)

250

200

150 80µL 160µL

100

400µL 50

0 0

10

20

30

40

50

60

70

91

80

t/min

(b)

3.3. Calculation of kinetic constants

30

25

DH/%

20

fitted 80µL

15

fitted 160µL fitted 400µL

10

80µL 160µL

5

400µL

0 0

10

20

30

40

(c)

50

60

70

80

t/min

600

-t/ln(1-DH/100)

500 400 300 0.09g/L 0.18g/L

200

0.45g/L 100 0 0

20

40

60

80

100

t/min

(d) 20 18 16 14 DH/%

12 fitted 0.09g/L

10

fitted 0.18g/L 8

fitted 0.45g/L

6

0.09g/L

4

0.18g/L

2

0.45g/L

The experimental results of the DH were used to correlate the kinetic constants by the plots of t/ln(1  DH/100) versus time (t) for effects of different ultrasound pretreatments, amounts of substrate and enzyme loading, as shown in Fig. 4(a)–(c), respectively. The values of kin,0, kimp, a and R2 from the data fitting were got (Table 1), and the high R2 values showed that the model was good fit for the experimental results. For 4% (w/v, E/S = 1638 U/g) pretreated by method 2 and method 3 (both with ultrasound), kin,0 and Kimp at 50 min were higher than those pretreated by method 1 (without ultrasound). The results suggested that the exposed substrate was increased because of the pretreatment making the enzyme more accessible to the substrate and resulting in a larger kin,0. In addition, kin,0 for pretreated by method 3 (0.0227 min1) was much larger than that for pretreated by method 2 (0.0179 min1), however values of kimp at 50 min were almost the same (0.031 min1 for method 2 and 0.033 min1 for method 3), indicating that the process of hydrolysis reaction was dominated by the initial rate. The value kin,0 of method 3 was larger than that of method 2, which agreed with the changes of surface hydrophobicity and secondary structure about rice protein pretreated by different methods (Figs. 2 and 3). For different substrate loadings (4% and 6% (w/v)) and the same ratio of enzyme to substrate (E/ S = 1638 U/g) in enzymatic hydrolysis with ultrasound (method 2), the values of kin,0 and kimp at 50 min for 4%, were 0.0179 min1 and 0.031 min1, and those for (6%) were 0.0144 min1 and 0.030 min1. These two values were decreased with the increase of the substrate concentration, implying that the reaction was impeded by the mass-transfer resistance due to a large amount of rice protein. After ultrasound assisted alkali pretreatment, the mass-transfer resistance was dramatically reduced, as verified by kin,0 and kimp at 50 min for 4% being 0.0227 min1 and 0.033 min1, and those for 6% being 0.0166 min1 and 0.031 min1. As far as enzyme loading concerned, the values of kin,0 and kimp at 50 min for 4% (E/S = 702 U/g) were 0.0067 min1 and 0.025 min1, and those for 4% (E/S = 1170 U/g) were 0.0071 min1 and 0.024 min1, and those for 4% (E/S = 1638 U/g) were 0.0078 min1 and 0.024 min1, displaying that the slower initial rate was resulted from less active enzyme for lower enzyme loading. 3.4. Comparison of the predicted and experimental yields of DH

0 0

20

40

60

80

100

t/min

Fig. 6. Verification of the model with the experimental data adopted from Huang et al. [36] (Fig. 4(a) and (b)) and Zou [37] (Fig. 4(c) and (d)). (a) and (c) Plot of t/ln (1  DH/100) versus time (t). (b) and (d) Comparison of predicted curves by this model and experimental data.

The equation for the predicted yields of DH (DHpred.) was derived from Eq. (21) as,

   kin;0  t DHpred ¼ 100  1  exp  1 þ at

ð22Þ

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S. Li et al. / Ultrasonics Sonochemistry 31 (2016) 85–92

The values of kin,0 and a from Table 1 were substituted into Eq. (22) to calculate DHpred with respect to t. The agreement between predicted curves of DH versus enzymolysis time and the experimental data was shown in Fig. 5. The predicted and experimental DH of ultrasound and ultrasound assisted alkali pretreatments on enzymatic hydrolysis for 4% (w/v) rice protein were shown in Table 2. Fig. 5 and Table 2 show that the experimental values are good fit with the enzymolysis kinetic mode for different pretreatment methods (<5%), except the cases of ultrasound and ultrasound assisted alkali pretreatments at 10 min of hydrolysis. The causes might be that the acceleration of enzymatic hydrolysis reaction in the early stage enlarges quickly comparing to without pretreatment to substrate and lead to the error increase. 3.5. Application of the kinetic model to other hydrolysis systems For verifying whether the model in this study can be applied to other hydrolysis cases, based on the experimental data of two hydrolysis systems with ultrasound in the literatures [36,37], kin,0 and kimp were got by using this model, respectively. The results were shown in the Table 3 and Fig. 6. The high R2 (>0.95) values of the correlation for estimating kin,0 and kimp and good fits of experimental data with the predicted curves can be obtained for the two systems. This shows that the simplified kinetic model might be applied to the pretreatment on the substrate and then enzymatic hydrolysis systems as well. 4. Conclusions Energy-gathered ultrasound and ultrasound assisted alkali pretreatments have effectively influenced on the enzymatic hydrolysis of rice protein by changing the surface hydrophobicity and secondary structure to enhance the degree of hydrolysis. The parameter of kin,0 in the kinetic model accounts for the initial activity and accessibility of enzyme to the substrate. After the different pretreatments, the value of kin,0 increases and proves that it has relation to the structures of the pretreatments on the substrate. The parameter of kimp suggests that the gradual loss of enzyme activity resulted from the impeded reaction in a heterogeneous system. The DH expression based on experiments only using two kinetic parameters in this model would be beneficial in controlling enzymatic hydrolysis and the designing for the bioreactor. Acknowledgments Great appreciation to the National Natural Science Foundation of China (31471698) and the Grant (2013AA102203) of the Project of National 863 Plan of China for providing financial support for this research. References [1] M. Shibasaki, S. Suzuki, H. Nemoto, T. Kuroume, Allergenicity and lymphocytestimulating property of rice protein, J. Allergy Clin. Immunol. 64 (1979) 259–265. [2] E.H. Jung, S. Ran Kim, I.K. Hwang, T. Youl Ha, Hypoglycemic effects of a phenolic acid fraction of rice bran and ferulic acid in C57BL/KsJ-db/db mice, J. Agric. Food Chem. 55 (2007) 9800–9804. [3] J. Zhang, H. Zhang, L. Wang, X. Guo, X. Wang, H. Yao, Isolation and identification of antioxidative peptides from rice endosperm protein enzymatic hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS/MS, Food Chem. 119 (2010) 226–234. [4] F.J. Izquierdo, E. Peñas, M.L. Baeza, R. Gomez, Effects of combined microwave and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins, Int. Dairy J. 18 (2008) 918–922. [5] M. Zeece, T. Huppertz, A. Kelly, Effect of high-pressure treatment on in-vitro digestibility of b-lactoglobulin, Innovative Food Sci. Emerg. Technol. 9 (2008) 62–69. [6] M. Galesio, J. Lourenço, D. Madeira, M. Diniz, J.L. Capelo, Unravelling the role of ultrasonic energy in the enhancement of enzymatic kinetics, J. Mol. Catal. B Enzym. 74 (2012) 9–15.

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