Enzymolysis kinetics of garlic powder with single frequency countercurrent ultrasound pretreatment

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Enzymolysis kinetics of garlic powder with single frequency countercurrent ultrasound pretreatment Liurong Huang a,b , Haile Ma a,b,∗ , Lei Peng a , Zhenbin Wang a,b , Qiaorong Yang c a b c

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China Jiangsu Provincial Key Laboratory for Physical Processing of Agricultural Products, Zhenjiang 212013, China School of Mechanical Engineering, Jiangsu University, Zhenjiang 212013, China

a b s t r a c t This research was to describe the enzymolysis process of garlic powder which was pretreated by single frequency countercurrent ultrasound. The kinetic constants for ultrasonic pretreated and traditional enzymolysis have been determined. Results showed the value of KM in ultrasonic pretreated enzymolysis decreased by 12.7% over the traditional enzymolysis, which indicates a fast reaction speed induced by ultrasound pretreatment. Degrees of hydrolysis (DH) in the enzymolysis of garlic powder were determined and kinetics of the reaction was considered in detail in relation to substrate concentration, enzyme concentration, reaction time and DH. Our results suggested a general kinetic equation for the enzymolysis model of ultrasonic pretreated garlic powder. For the model system, the calculated values agree well with the experimental data with average relative error of 4.42%. The proposed kinetic equation can be used to model the bio-reaction process of protein enzymatic hydrolysis and optimize the operation parameters for bioreactor design. © 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Ultrasound; Enzymolysis; Kinetics; Kinetic constants; Garlic; Enzymolysis model

1.

Introduction

Power ultrasound is a clean technology, which has been widely used in the laboratory or food processing factory to improve food physicochemical properties. The periodic mechanical motions of a probe can transfer ultrasonic energy into a fluid medium and lead to the formation of small rapidly growing bubbles (Mason, 1990). Collapse of these bubbles will generate high temperatures, pressures and shear forces at the probe tip, which can result in the changes of structure and activity of biologic molecules (Kadkhodaee and Povey, 2008; Jambrak et al., 2009). For example, several authors reported that high-energy ultrasound can cause the degradation of polysaccharides (Ying et al., 2011), change of enzyme activity (Ma et al., 2011), and damage of DNA (Furusawa et al., 2012). Garlic is one of the edible plants which have attracted particular attention of modern medicine because of the widespread health use around the world. To date, many favorable experimental and

clinical effects of garlic extract have been reported. These biological responses have been largely attributed to anticancer (Tsubura et al., 2011), antimicrobial (Yoshida et al., 1987) and antioxidant activities (Panpatil et al., 2013). Several studies also suggest that garlic contains some compounds with the functions of inhibiting angiotensin I-converting enzyme (ACE) and lowering the blood pressure on spontaneously hypertensive rats (SHRs) (Oboh et al., 2013; Hosseini et al., 2007). These are the characteristics of the direct extracts. Recently, we reported that the enzymolysis hydrolysates of garlic power had the highest ACE inhibitory activity among those of the multi-stage isolates. This result shows that the crude polysaccharide and water extracts of garlic also have certain ACE inhibitory activity (Peng et al., 2012). When garlic powder was treated by ultrasound and alcalase, the ACE inhibitory activity was increased by 170.9% over the water extracts (Peng et al., 2012). Combination of enzymatic hydrolysis and ultrasonic pretreatment has been confirmed be an effective way to improve the functional

∗ Corresponding author at: School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu Province, China. Tel.: +86 511 88790958; fax: +86 511 88790958. E-mail address: [email protected] (H. Ma). Received 5 September 2014; Received in revised form 19 September 2014; Accepted 20 October 2014 http://dx.doi.org/10.1016/j.fbp.2014.10.015 0960-3085/© 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

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Nomenclature a b DH e0 e E Ea Ed ES k2 k3 KM KS Kp P p r s0 s t

kinetic parameter (dimensionless) kinetic parameter (dimensionless) hydrolysis degree (%) initial enzyme activity in the reaction system, simplified as the volume of enzyme (␮L) enzyme activity in the reaction system, simplified as the volume of enzyme (␮L) free enzyme active form of enzyme inactive form of enzyme enzyme substrate complex reaction rate constant for proteolysis (s−1 ) reaction rate constant for inactivation (s−1 ) Michaelis–Menten constant (g/L) inhibition constant by substrate (g/L) inhibition constant by product (g/L) product product concentration (g/L) reaction rate for enzymolysis (g/L·s) initial substrate concentration (g/L) substrate concentration (g/L) reaction time (min)

properties of protein (Jia et al., 2010; Jambrak et al., 2014; Chen et al., 2011). The use of ultrasound in bio-processing may break chemical bonds and lead to the opening up of the substrate surface to the action of enzymes (Yachmenev et al., 2009). In comparison with the extensive researches devoted to the optimization of ultrasonic and enzymolysis conditions, the kinetic processes of ultrasound-assisted enzymolysis are just reported by a few technologists (Márquez and Vázquez, 1999; Gonzàlez-Tello et al., 1994). Biotechnology reactions are usually carried out using fed-batch reactors for the production of manufacture. The optimization of batch bioreactors always depends on the kinetics of the processes. A good reaction model is capable of reproducing the kinetic behavior of a system over a wide range of operating conditions, thus providing a useful tool for the optimal reactor design. The objectives of this research were to (1) study the effects of optimal ultrasonic conditions on the kinetic constants of garlic enzymolysis, and (2) determine the hydrolysis curves of garlic catalyzed by alcalase in solution, which is modeled by a simple empirical equation from which kinetic parameters can be deduced. This research can provide the theoretical basis and technological support toward better understanding and designing enzymatic hydrolysis of other ultrasonic pretreated materials.

2.

Materials and methods

2.1.

Materials

Dehydrated garlic slices were purchased from Xinghua Vegetable Foods Co., Ltd. (Jiangsu, China) and had a protein content of 16.13% determined by Kjeldahl method (Kjeldahl, 1883). Alcalase with the activity of 1.5 × 105 U/mL was purchased from Wuxi Xuemei Enzyme Preparation Co., Ltd. (Wuxi, China). Sodium hydroxide (NaOH), hydrochloric acid (HCl), sodium carbonate (Na2 CO3 ), copper sulfate pentahydrate (CuSO4 ·5H2 O), sodium potassium tartrate (C4 H4 O6 KNa·4H2 O), Folin-phenol reagent B and bovine serum albumin (BSA) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the reagents were of analytical grade. Folin-phenol reagent A was comprised of 0.185 M Na2 CO3 , 0.098 M NaOH, 0.393 mM CuSO4 ·5H2 O and

0.694 mM C4 H4 O6 KNa·4H2 O, which was prepared on the day of the test.

2.2.

Ultrasonic pretreatment of garlic powder

Dehydrated garlic slices were screened with sieves of 0.25 mm openings to produce garlic powder. A mass of 100 g garlic powder was put into a 2000 mL beaker and mixed with 800 mL distilled water. Sample solutions were sonicated in a reconstructed single frequency countercurrent ultrasound processor, which was equipped with a 2 cm flat tip probe (Fanbo Biological Engineering Co., Ltd., Wuxi, China; Model FBTQ 2000). Based on our previous result, the optimal process conditions of single frequency countercurrent ultrasound obtained by Box–Behnken design were as follows: ultrasonic time of 72 min, initial temperature of 45 ◦ C and initial pH of 8.2 (Peng et al., 2012). Therefore, the sample solution was treated at 45 ◦ C for 72 min under these conditions: initial pH of 8.2, power of 410 W, fixed frequency of 20 kHz, circulating pump speed of 300 r/min and pulsed on-time and off-time of 3 and 2 s. The control was submitted to the same process and performed at the same conditions but without ultrasonic pretreatment.

2.3.

Enzymolysis reaction of garlic powder

After sonication, the garlic solution was diluted to different concentrations. A 100 mL volume of garlic solution was preincubated to 50 ◦ C for 10 min, and the reaction was started with addition of alcalase. The pH of the reaction for hydrolysis procedures was monitored by a pH meter and kept at the optimal pH 9.0 by adding drops of 1 M NaOH. After hydrolysis for different time, the enzyme in hydrolysate was inactivated with the same volume of 6% trichloroacetic acid and then boiled in a water bath for 10 min. Then the hydrolysate was cooled down to 40 ◦ C, centrifuged from the solid at 5000 rpm at room temperature for 15 min. The supernatant was collected for further analyses. The garlic powder without ultrasonic pretreatment was performed using the same enzymolysis procedure. The other hydrolysis conditions were described in Sections 2.4.1 and 2.5.1.

2.4. Test of effect of ultrasound on enzymolysis kinetic constants 2.4.1.

Enzymolysis reaction condition

Garlic was pretreated with ultrasound at power of 410 W for 72 min and then hydrolyzed by alcalase at substrate concentrations of 0.02, 0.04, 0.06, 0.08, 0.10 and 0.12 g/mL, respectively. The garlic without ultrasonic pretreatment was hydrolyzed at substrate concentrations of 0.01, 0.03, 0.05, 0.07, 0.09 and 0.12 g/mL, respectively. The other hydrolysis conditions were: pH, 9.0; temperature, 50 ◦ C; E/S ratio, 3000 U/g; hydrolysis time, 5 min.

2.4.2.

Kinetic equation

The classic Michaelis–Menten equation was applied in effect of ultrasonic pretreatment on enzymolysis kinetic constants of garlic powder. 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 1 1 KM × + = Vmax [S] Vmax V

(1)

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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].

2.4.3.

Determination of initial reaction rate

In order to calculate the initial reaction rate of enzymolysis, the polypeptide concentration (g/mL) was determined at enzymolysis time of 5 min. We confirmed that both the ultrasonic pretreated and traditional enzymolysis exhibited first-order reaction kinetics (data not shown) at short enzymolysis period of 5 min. The polypeptide concentration was determined using a reported Folin-phenol colorimetric method (Chen et al., 2003). Firstly, a volume of 4 mL Folin-phenol reagent A was mixed with 0.5 mL sample in a glass tube. The solution was incubated for 10 min at 25 ◦ C, after which 0.5 mL Folin-phenol reagent B was added to each glass tube, and the absorbance was read at 500 nm on a spectrophotometer (Unic 7200, Unocal Corporation, Shanghai, China) after 30 min incubation. A blank was prepared by using distilled water in place of the sample. BSA was assayed as standard for the calculation of polypeptide concentrations of samples. The initial reaction rate was express as: Initial reaction rate(g/mL s) =

polypeptide concentration(g/mL) 5 × 60(s)

(2)

When the relationship between the two mechanisms was considered, combination of Eq. (3) with Eq. (4) provides the ratio: −s0

k2 d(DH) = de k3 [E]

(5)

At steady state approximation, the mass balance for enzyme-substrate complex (ES) leads to the following equation: [ES] =

[E][S] KM

(6)

If enzyme is inhibited by substrate and product, the reactions are S + ES ⇔ SES E + P ⇔ EP, the kinetic equations for the processes are given by Eqs. (7) and (8) When KS = (k−4 /k4 ) and KP = (k−5 /k5 ): [SES] =

[EP] =

[S][ES] [S]2 [E] = KS KM KS

(7)

[E][P] KP

(8)

Since the enzyme present in the solution is either free or combined state, the total enzyme concentration would be expressed in the form at a given moment: e = [E] + [ES] + [SES] + [EP]

(9)

When Eqs. (6)–(8) were substituted into Eq. (9), expression for the free enzyme concentration can be simplified as Eq. (10) when [S] = s0 and [P] = p.

2.5. The enzymolysis kinetic model of ultrasonic pretreated garlic 2.5.1.

[E] =

The enzymolysis reaction condition

Garlic was pretreated with ultrasound at power of 410 W for 72 min and then hydrolyzed by alcalase. The experiments were divided into three groups according to the substrate concentrations of 60, 80 and 100 g/L. In each group, the addition of alcalase was 80, 160 and 400 ␮L, respectively. The hydrolysis temperature and pH value were fixed at 50 ◦ C and 9.0 respectively. The degrees of hydrolysis under different enzymolysis time (2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70 min) were investigated.

2.5.2.

Enzymolysis kinetic model

In the process of enzymolysis, enzyme first was adsorbed by substrate protein. Then the enzymes attached on the surface of the protein and cleaved the peptide bonds to produce polypeptides. The hydrolysis reaction can be expressed as: k2

E + S ⇔ ES−→E + P. The reaction rate will be determined by the irreversible stage and gives Eq. (3).

r = s0

d(DH) = k2 [ES] dt

(3)

e 1 + (s0 /KM ) + (s0 2 /KS KM ) + (p/KP )

(10)

If KM <
eKS KM KP KS KP s0 + KP s0 2 + KS KM p

[ES] =

(11)

es0 KS KP KS KP s0 + KP s0 2 + KS KM p

(12)

Insertion of Eq. (11) in Eq. (5) and simplification [E] = e gives −

d(DH) k2 (KS KP s0 + KP s0 2 + KS KM p) 1 = de k3 KS KM KP s0 e

(13)

Integration of Eq. (13) between the limits of the initial and final reaction conditions provides:



k3 KS KM KP s0 (DH) e = e0 exp − k2 (KS KP s0 + KP s0 2 + KS KM p)

 (14)

When Eq. (14) was substituted into Eq. (12) and then the obtained equation was inserted into Eq. (3), Eq. (15) can be yielded as following:

k3

If enzyme inactivation reaction is E + ES−→Ea + Ed + P, then the kinetic equation for this process is given by [17]:

−

de = k3 [E][ES] dt

d(DH) k2 e0 KS KP = dt KS KP s0 + KP s0 2 + KS KM p



(4)

× exp −

k3 KS KM KP s0 (DH) k2 (KS KP s0 + KP s0 2 + KS KM p)

 (15)

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y = 4434.5x + 9459.9

500000

Table 1 – Kinetic constants in ultrasonic pretreated and traditional enzymolysis.

2

R = 0.9984

Traditional enzymolysis Ultrasonic pretreated enzymolysis

1/V (mL·s/g)

400000

KM Traditional enzymolysis Ultrasonic pretreated enzymolysis

300000

0.106 0.104

200000 100000

y = 3952.6x + 9604.3 2

R = 0.9991

0 -20

0

20

40

60

80

100

1/S (mL/g) Fig. 1 – Plots of the reciprocal of the initial reaction rate (1/V) versus the reciprocal of substrate concentration (1/S) in ultrasonic pretreated and traditional enzymolysis.

If a = (k2 e0 KS KP /KS KP s0 + KP s0 2 + KS KM p), b = (k3 KS KM KP s0 / k2 (KS KP s0 + KP s0 2 + KS KM p), therefore, Eq. (15) can be simplified as: d(DH) = a exp[−b(DH)] dt

(16)

After logarithm transformation, Eq. (16) was expressed as: DH =

2.5.3.

1 ln(1 + abt) b

(17)

Assessment of degree of hydrolysis (DH)

DH was calculated from the amount of alkali (NaOH) added to keep the pH constant during the hydrolysis as given below (Adler-Nissen, 1979): DH(%) =

h BNb × 100 = × 100 htot ˛Mp htot

(18)

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 rate constant KM of ultrasonic pretreated enzymolysis was decreased by 12.7% over the traditional enzymolysis. The lower KM indicates the higher affinity between garlic protein and alcalase. The changes of Vmax between the ultrasonic pretreated and traditional enzymolysis are very small, which implies the highest binding frequency was obtained when the alcalase was saturated by substrate. After in all, ultrasound can accelerate the enzymolysis process of garlic. Ultrasonic pretreatment might break chemical bonds of polysaccharide and protein in garlic cell walls. The breakage may increase the surface hydrophobicity and loosen the protein tissue of garlic, which facilitate the release of bioactive factors with ACE inhibitory activity from garlic powder during enzymatic hydrolysis (Lourenc¸o da Costa et al., 2007). These results further certified the reaction mechanism that single frequency countercurrent ultrasound can accelerate the enzymolysis process of garlic protein and therefore ultrasonic pretreated enzymolysis had higher enzymolysis efficiency than traditional enzymolysis, which was in accordance with the results of wheat germ protein pretreated with SFP ultrasound reported by Qu et al. (2012). To clarify the ultrasonic mechanism, the polysaccharide and protein will be purified for further investigation.

3.2. where B is the NaOH consumed (mL) to keep the pH, Nb is the concentration of the NaOH (mol/L), ˛ is the average degree of dissociation of the ␣-NH2 groups in protein substrate (0.99 at 50 ◦ C and pH 9.0), Mp is the mass of hydrolyzed protein (g), htot is the total number of peptide bonds in garlic and was assumed to be 8.2 mmol/g determined by the amino acid composition. All experiments were carried out in duplicate (figures show the average values of the experiments).

3.

0.47 0.41

Vmax (g/mL s)

Results and discussion

3.1. Effect of ultrasonic pretreatment on enzymolysis kinetic constants The reaction rate constant is an important parameter of the chemical reaction kinetics, which is independent on the substrate concentration. Vmax is the maximum reaction rate, which implies a high binding frequency between substrate and enzyme. KM represents the affinity between substrate and enzyme, which indicates a fast reaction speed when it is low (Briggs and Haldane, 1925). The change of enzymolysis of garlic induced by ultrasonic pretreatment will result in the change of reaction rate constant. The plots of the reciprocal of the initial reaction rate (1/V) versus the reciprocal of the protein concentration (1/S) in the enzymolysis of ultrasonic pretreated and untreated garlic were displayed in Fig. 1. It can be observed that 1/V had a good linear relationship with 1/S with correlation coefficients

The calculation of model parameters

Fig. 2a shows the changes of DH in enzymolysis process of garlic powder at substrate concentration of 60 g/L. Values of a and b for different initial enzyme concentrations were calculated from nonlinear regression analysis and the results are shown in Table 2. It can be observed that value a increased when the initial enzyme concentration increased. However, parameter b did not exhibit any trend when the initial enzyme concentration changed and its values lied within a very small range. Fig. 2b and c shows the hydrolysis curves for substrate concentrations of 80 g/L and 100 g/L, respectively. All the values of the kinetic parameter b (Table 2) did not present either any dependence on initial enzyme concentration and substrate concentration. The average value of parameter b can be considered 0.1883. In contrast, value a decreased at substrate concentration of 80 g/L and 100 g/L when compared with that obtained at 60 g/L. This result shows that alcalase can be inhibited by high concentration of garlic protein.

3.3.

Model validation

A straight line was obtained by plotting parameter a against e0 /s0 (Fig. 3). The linear regression analysis on the figure shows a correlation coefficient of 0.9898 and yields the expression: a = 1.661

e0 + 3.029 s0

(19)

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Table 2 – Values of the kinetic parameters a and b for ultrasonic pretreated enzymolysis of garlic. s0 (g/L)

a

b

R2

4.962 7.698 13.555 4.581 6.156 11.662 4.253 5.824 10.229

0.1897 0.1939 0.1792 0.1894 0.1864 0.1857 0.1877 0.1884 0.1945

0.9902 0.9909 0.9904 0.9916 0.9914 0.9944 0.9914 0.9930 0.9937

e0 (␮L)

60 60 60 80 80 80 100 100 100

80 160 400 80 160 400 80 160 400

Fig. 4 – Hydrolysis curves for different initial enzyme and substrate concentrations and calculated values fitted to Eq. (20) during time of 70 min. Insertion of this Eq. (19) and parameter b of 0.1883 in Eq. (17), and the kinetic model for the enzymolysis of ultrasonic pretreated garlic was obtained as following:





DH = 5.3099 ln 1 + 0.3128

Fig. 2 – Hydrolysis curves for different initial enzyme concentrations at substrate concentration of (a) 60 g/L, (b) 80 g/L and (c) 100 g/L in ultrasonic pretreated enzymolysis during time of 70 min. 16 14

y = 1.661x + 3.029

12

R = 0.9898

a

10 8 6 4 2 0 0

1

2

3

4

5

e 0/s 0 (μL/g/L)

6

7

8

Fig. 3 – Variations of kinetic parameters a for different e0 /s0 values.

(20)

The above Eq. (20) agrees with Eq. (17) deduced from the proposed mechanism, which imply the relationship of DH, enzyme concentration, substrate concentration and enzymolysis time. To confirm the validity of the kinetic model and the kinetic parameters, three groups of experiments were done under different initial enzyme and substrate concentrations. The adequacy between the calculated values of hydrolysis degree versus time and the experimental data is shown in Fig. 4. The model values (calculated values) agree with the experimental data with average relative error of 4.42%. The strong correlation between the experimental and the calculated results confirmed that the kinetic model was accurate to reflect the enzymolysis process.

4.

2

 

e0 + 0.5704 t s0

Conclusion

We evaluated the effects of ultrasonic pretreatment on kinetic constants of garlic protein hydrolysis by alcalase. It was found that the initial reaction rate can be significantly enhanced by ultrasonic pretreatment. In order to represent the hydrolysis rate of enzyme-catalyzed hydrolysis of proteins mathematically, a simple empirical rate equation was applied to express hydrolysis curves. Much information can be obtained by researching empirical hydrolysis curves under different conditions. The proposed kinetic equation has been used to describe the kinetics of enzymatic hydrolysis for other animal and vegetable proteins. However, the reaction mechanism

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can be different when some substrate or product inhibitions take place. Hemoglobin hydrolysis (Márquez and Vázquez, 1999) and whey protein hydrolysis (Gonzàlez-Tello et al., 1994) were modeled without substrate-inhibited or productinhibited reactions. So the value of kinetic parameter a in Eq. (17) depends on different factors which include not only the reaction rate constant but also the concentrations of enzyme and substrate. A good reaction model is capable of reproducing the kinetic behavior of a system over a wide range of operating conditions, thus providing a useful tool for the optimal reactor design. In this investigation, a suitable model was applied to hydrolysis reaction of ultrasonic pretreated garlic, which allows the calculation of the necessary kinetic parameters from a few experiments. The positive effect of ultrasound and application of the kinetic model may be useful for the release of bioactive peptides from garlic in the food industry.

Acknowledgments This work was supported by Innovation Fund for Jiangsu Agricultural Science and Technology [CX(12)3084], Jiangsu Provincial Technology Support Program – Agricultural Part (BE2012393) and Program of Jiangsu Provincial Six Big Talents Summit.

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Please cite this article in press as: Huang, L., et al., Enzymolysis kinetics of garlic powder with single frequency countercurrent ultrasound pretreatment. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.10.015