Improving the enzymolysis efficiency of potato protein by simultaneous dual-frequency energy-gathered ultrasound pretreatment: Thermodynamics and kinetics

Accepted Manuscript Improving the enzymolysis efficiency of potato protein by simultaneous dualfrequency energy-gathered ultrasound pretreatment: thermodynamics and kinetics Yu Cheng, Yun Liu, Juan Wu, Ofori Donkor Prince, Ting Li, Haile Ma PII: DOI: Reference:

S1350-4177(17)30043-3 http://dx.doi.org/10.1016/j.ultsonch.2017.01.034 ULTSON 3528

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

14 November 2016 23 January 2017 23 January 2017

Please cite this article as: Y. Cheng, Y. Liu, J. Wu, O.D. Prince, T. Li, H. Ma, Improving the enzymolysis efficiency of potato protein by simultaneous dual-frequency energy-gathered ultrasound pretreatment: thermodynamics and kinetics, Ultrasonics Sonochemistry (2017), doi: http://dx.doi.org/10.1016/j.ultsonch.2017.01.034

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1 2

Improving the enzymolysis efficiency of potato protein by simultaneous

3

dual-frequency energy-gathered ultrasound pretreatment: thermodynamics and

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kinetics

5 6

Yu Cheng*, Yun Liu, Juan Wu, Ofori Donkor Prince, Ting Li and Haile Ma

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

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Zhenjiang, Jiangsu 212013, China

9

10

11

*

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[email protected] (Y.Cheng).

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Tel.: 0086-150-51149622. Fax: 0086-511-88780201.

To whom correspondence should be addressed:

14 15 16 17

(Summited to Ultrasonics Sonochemistry)

18 19 20 21 1

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Abstract

23

The thermodynamics and kinetics of traditional and simultaneous dual frequency

24

energy-gathered ultrasound (SDFU) assisted enzymolysis of potato protein were

25

investigated to get the knowledge of the mechanisms on the SDFU’s promoting

26

efficiency during enzymolysis. The concentration of potato protein hydrolysate and

27

parameters of thermodynamic and kinetic during traditional and SDFU assisted

28

enzymolysis were determined. The results showed that potato protein hydrolysate

29

concentration of SDFU assisted enzymolysis was higher than traditional

30

enzymolysis at the hydrolysis time of 60 min (p < 0.05) whereas not significantly

31

different at 120 min (p > 0.05). In some cases, SDFU assisted enzymolysis took less

32

hydrolysis time than traditional enzymolysis when the similar conversion rates of

33

potato protein were obtained. The thermodynamic papameters including the energy

34

of activation (Ea), enthalpy of activation (∆H), entropy of activation (∆S) were

35

reduced by ultrasound pretreatment while Gibbs free energy of activation (∆G)

36

increased little (1.6%). Also, kinetic papameters including Michaelis constant (KM)

37

and catalytic rate constant (kcat) decreased by ultrasound pretreatment. On the

38

contrary, reaction rate constants (k) of SDFU assisted enzymolysis were higher than

39

that of traditional enzymolysis (p < 0.05). It was indicated that the efficiency of

40

SDFU assisted enzymolysis was higher than traditional enzymolysis in a limited

41

time. The higher efficiency of SDFU assisted enzymolysis was related with the

42

decrease of Ea and KM by lowering the energy barrier between ground and active

43

state and increasing affinity between substrate and enzyme.

44 45

Keywords: Potato protein; Ultrasound; Enzymolysis; Kinetic; Thermodynamic 2

46

1. Introduction

47

Potato protein has been considered as a high quality protein resource [1, 2]. It has

48

exhibited potential functionality for food application [3, 4]. Therefore, many

49

researchers have been trying to produce potato protein with good functionality for its

50

application in food industry [5-7]. However, at present, most of commercial potato

51

proteins were not dissolved in water because of the high temperature denaturation

52

during processing. The low solubility of potato proteins has limited its application as

53

food ingredient. To take use of these potato proteins, enzymatic hydrolysis has been

54

used to improve the solubility of potato protein [8, 9]. Moreover, as potato protein

55

hydrolysate has showed in vitro bioactivities such as antioxidant[9-11]and

56

antihypertension [12, 13], release of functional and bioactive peptides from potato

57

protein using different enzymes was of interest [14-16]. Again, potato protein

58

hydrolysate, which showed better bioactivity than potato protein, was demonstrated

59

to be able to retard the lipid oxidation [9, 17, 18] and protein oxidation [19] in the

60

food model system. However, little has been done to improve the efficiency of the

61

enzymatic hydrolysis and enhance the conversion rate of insoluble potato protein to

62

soluble potato protein hydrolysate.

63

Enzymolysis has been used to modify the properties of food proteins in many

64

researches[20-24]. To make the proteins more sensitive to enzymolysis, several

65

physical pretreatment methods have been used including heating, high pressure,

66

ultrasound, microwave [25-27]. As one of the most used pretreatment methods,

67

ultrasound pretreatment was able to change the molecular structure of proteins [28] 3

68

to decline the activation energy and Gibbs free energy of reaction[29, 30].

69

Meanwhile, ultrasound pretreatment also had positive effect on the kinetics of

70

protein hydrolysis with enzymes by increasing the initiate velocity and decreasing

71

Michaelis

72

pretreatment has been successfully used to improve bioactive peptides release and

73

the enzymolysis efficiency of proteins [28] such as alcalase based enzymolysis of

74

wheat germ protein[29], corn protein[30] and wheat gluten[31]. In these researches,

75

all the proteins were byproduct of starch industry with low solubility. Also,

76

commercial potato protein was byproduct of starch industry with low solubility.

77

Thus, we hypothesized that ultrasound pretreatment was able to lower the activation

78

energy and Michaelis constant and enhance initiate velocity of potato protein

79

hydrolysis with alcalase. Ultrasound pretreatment may thus improve the efficiency of

80

potato protein hydrolysis with alcalase.

81

Therefore, the aim of our research was to make it clear if ultrasound pretreatment

82

can promote the enzymolysis efficiency of commercial potato protein and find out

83

the effect of ultrasound pretreatment on the enzymolysis thermodynamics and

84

kinetics of commercial potato protein. Simultaneous dual-frequency energy-gathered

85

ultrasound (SDFU) was used in the present research.

86

2. Materials and methods

87

2.1. Materials

88

Commercial potato protein powder which contains 60.4% (w/w) protein was

89

purchased from Ci Yuan Biotechnology Co., Ltd (Shanxi, China). Alcalase 2.4L FG,

constant

of

enzymolysis

reaction[29,

30].Therefore,

ultrasound

4

90

with the activity of 2.4 AU/g, was purchased from Novozymes Co., Ltd (Tianjing,

91

China). All other chemicals were of analytical grade and obtained from Sinopharm

92

Chemical Reagent Co. Ltd. (Shanghai, China). All solutions were prepared with

93

distilled deionised water.

94

2.2. Pretreatment of potato protein using ultrasound

95

Dual-frequency energy-gather ultrasound, which was developed by our research

96

team and manufactured by Meibo Biotechnology Co., Ltd (Zhenjiang, China), was

97

used in current research. The details of the equipment have been introduced in our

98

previous researches [32, 33]. In brief, the equipment consists of two ultrasonic

99

probes, controllers, a reaction vessel, a liquid circulating system and a temperature

100

controlling system. Each probe generates different frequencies and produces a

101

maximum output power of 300 W.

102

Potato protein suspensions with different protein concentration of 12.080, 18.120,

103

24.160, 30.200, and 36.240 g/L were prepared by dissolving different mass of potato

104

protein powder that passed through sieve with the size of 60-mesh in 800 mL

105

distilled deionised water. Those potato protein solutions were treated respectively

106

using ultrasound at the temperature of 25 ± 2 ºC. The temperature was controlled by

107

water bath. Ultrasonic pretreatment was conducted at dual frequency of 20/40 KHz

108

in simultaneous working mode. The pulsed on-time and off-time were 10 s and 3 s

109

respectively with the duration of 10 min and power density of 250W/L during

110

ultrasound processing. The control was carried out with a magnetic stirrer instead of

111

ultrasound at 25 ºC for 10 min. The ultrasound parameters including ultrasound 5

112

frequency, ultrasound time and ultrasound power were chosen according to the

113

single factor experiment.

114

2.3. Enzymolysis of potato protein

115

2.3.1 Different enzymolysis temperature

116

The potato protein suspensions (substrate concentration 24.160 g/L) pretreated with

117

ultrasound were poured into the double-jacketed beaker and preheated for 10 min to

118

rise up to certain temperatures (30, 40, 50 and 60 ºC). The suspensions were mixed

119

using a magnetic stirrer (KMO 2, IKA, Germany) at a speed of 300 r/min. And the

120

temperature was controlled by a digital cryogenic refrigeration circulator (WCR-P6,

121

WiseCircu, Korea). The hydrolysis of potato protein suspensions were carried out by

122

alcalase (0.320 mL, with mass density of 0.400 g/L in broth) with the hydrolysis time

123

of 120 min. During the hydrolysis, pH of suspensions was maintained at 8.0 by

124

addition of 2.0 M NaOH using an automatic potentiometric titrator (ZDJ-4A,

125

Instrument and Electronics Science Co., Ltd, Shanghai, China) at pH-stat mode. The

126

potato protein hydrolysate was withdrawn from the broths at different hydrolysis time,

127

and the pH of potato protein hydrolysate was adjusted to 7.0 following by boiling

128

hydrolysate for 10 min to inactivate the enzymes. Then the hydrolysate was

129

centrifuged at 9000 rpm for 10 min and the supernatant was stored at 4 ℃ for further

130

analysis and used within 24h. The concentration of potato protein hydrolysate during

131

hydrolysis was determined by Biuret method[34].The conversion rate (%) of potato

132

protein to hydrolysate was defined as concentration of potato protein hydrolysate in

133

the supernatant divided by the protein concentration of the original potato protein 6

134

suspension then multiplying by 100. The traditional enzymolysis was performed using

135

the same procedure without any ultrasonic pretreatment of the substrate.

136

2.3.2 Different substrate concentration

137

The potato protein suspensions pretreated with ultrasound at different substrate

138

concentration (12.080, 18.120, 24.160, 30.200, and 36.240 g/L) were hydrolyzed at

139

50 ºC as described under section 2.3.1. The traditional enzymolysis was performed

140

using the same procedure without any pretreatment of the substrate.

141

2.4. Thermodynamics of enzymolysis

142

2.4.1. Determination of reaction rate constant

143

The first-order kinetic model was used to analyze the reaction rate constant k of

144

enzymolysis[29, 30].The equation of first-order kinetic model based on substrate

145

consumption was given as:

146





= − 

(1)

147

The equation (1) was integrated and rearranged to its linear form:

148

ln  = − +  

149

where C0 and C indicate the initial concentration of potato protein (g/L) and

150

concentration of potato protein remained at a certain time during hydrolysis (g/L)

151

respectively; t indicates the hydrolysis time (min); k indicates the reaction rate

152

constant.

153

As the concentration of leftover substrate is difficult to obtain, it was determined

154

using the amount of peptides released from potato protein [29, 30]. In the condition

(2)

7

155

of experiment, C0 was substituted with C∞ while C

156

Then the equation (2) was rearranged to:

157

ln  −   = − + ln 

158

where Ct indicates the concentration of potato protein hydrolysate at a certain time

159

during hydrolysis (g/L); C∞ indicates the concentration of potato protein hydrolyate

160

(g/L) that was prepared by hydrolyzing potato protein at pH 8.0 and 50℃ for 10 h

161

using alcalase [30]. After 10 h of hydrolysis, little sodium hydroxide was added and

162

the ultimate concentration of potato protein hydrolysate was assumed to be equal to

163

the concentration of potato protein was able to be hydrolyzed. The reaction rate

164

constant k is able to be obtained from the slope of the straight line plotting by ln

165

(C∞-Ct) against t.

166

2.4.2. Determination of thermodynamic parameters

167

Arrhenius equation [35] was given to describe the relationship between reaction rate

168

constant k and temperature T as follow:

169

k = A 

170

The equation (4) was transformed and rearranged to:

171

lnk = − !" + #

172

where k indicates the reaction rate constant (1/min); A indicates the pre-exponential

173

factor (min-1); R indicates the universal gas constant (8.314 J·mol-1·K-1); T indicates

174

the Kelvin temperature (K); Ea indicates the activation energy (J/mol).

175

Also, thermodynamic parameters of the enzymolysis reaction can be obtained by

was substituted with ‘C∞ -Ct’.



(3)

(4)

(5)

8

176

Eyring equations [35], which are expressed as:

177

=

178

where k indicates the reaction rate constant (s-1); kB indicates the Boltzman constant

179

(1.38 ×1023 J/K); h indicates the Planck constant (6.6256 ×1034 J·s) ; R indicates the

180

universal gas constant (8.314 J·mol-1·K-1); T indicates the Kelvin temperature (K);

181

∆) indicates Gibbs free energy of activation (J/mol).

182

The Gibbs free energy of activation (J/mol) can be calculated by equation as follow:

183

∆) = ∆* − T∆,

184

where ∆H is the enthalpy of activation (J/mol); ∆S indicates the entropy of activation

185

(J·mol-1·K-1).

186

At solution, the enthalpy of activation can be by equation as follow:

187

-. = ∆* + RT

188

Entropy of activation is able to be obtained from the linear form of Eyring equations

189

yielded by combining the equation (6) and (7) as follow:

190

ln " = −

191

2.5. Kinetics of enzymolysis

192

2.5.1. Determinations of hydrolysis degree and concentration of protein hydrolyzed

193

The degree of hydrolysis (DH) was obtained by the equation developed by

194

Adler-Nissen[36]as follow:

195

DH =

196

where Nb indiates the concentration of NaOH (mol/L), B indiates the volume of

$% " &

∆(

 

$

∆0 !

& &6

(6)

2

(7)

(8)

× " + 

× 100 =

$% &

+

∆3 !



9: ×; <×=> ×&6

(9)

× 10

(10)

9

197

sodium hydroxide consumed (mL), Mp is the mass of protein to be hydrolyzed (g),

198

htot is the total number of peptide bonds in the protein substrate, which is 5.1 mmol/g

199

for potato protein[37]; α is the average degree of dissociation of the α-NH2 groups,

200

which is related with the pK of the amino groups at particular pH and temperature,

201

and α is 0.871 for alcalase.

202

Concentration of potato protein hydrolyzed was calculated according to the equation

203

proposed by Qu et al.[38] :

204

 =

205

where, C0 and Ct indicate the initial concentration of potato protein (g/L) and

206

concentration of potato protein hydrolyzed at a certain time during hydrolysis (g/L)

207

respectively; DH indicates the degree of hydrolysis (%).

208

2.5.2. Determination of initial reaction rate

209

The initial reaction rate (V0, g·L-1·min-1 )were estimated by loss of substrate Ct[38]

210

or accumulation of product[35].

211

2.5.3. Determination of kinetic parameters KM and kcat

212

The kinetic model described by Parkin[35] was used to determine the KM and kcat of

213

potato protein hydrolysis, which was expressed as:

214

A =

215

where V0 indicates the initial reaction rate (g·L-1·min-1); kcat indicates the catalytic

216

rate constant (min-1), representing the average value of apparent breakdown rate

217

constant of substrate and enzyme complex to product; ET indicates the concentration

? ×@0 2

$B  3 CD E3

(11)

(12)

10

218

of alcalase (g/L); KM is Michaelis constant (g/L).

219

The equation (12) was rearranged to the linear form as:

220

2 F?

CD

=$

G 

2

×3+$

2

(13)

B 

221

The values of KM and kcat were determined according to the slope and intercept of

222

linear line plotting 1/V0 against 1/S.

223

2.6. Statistical analysis

224

All experiments were repeated at least three times (n ≥ 3) at different time. During

225

independent replication trials, each sample was analyzed for triplicate. Data were

226

subjected to one-way analysis of variance (ANOVA) using the statistical software

227

DPS 9.5(Institute of Insect Science, Zhejiang University, Hangzhou, China). LSD’s

228

test was used to identify the differences (P < 0.05) between means.

229

230

3. Results and discussion

231

3.1. Effect of SDFU pretreatment on protein hydrolysate release

232

Several researches have demonstrated that ultrasound pretreatment was able to

233

improve the efficiency of enzymolysis of plant proteins from industry

234

byproducts[29-31].However, almost all the data of enzymolysis processes reported

235

in these researches was concentration of hydrolyzed proteins, and little was known

236

about the concentration of protein hydrolysate. Although concentration of

237

hydrolyzed proteins was very important in determined the kinetics of enzymolysis,

238

concentration of protein hydrolysate was useful in estimating the conversion or the 11

239

solubility of proteins with low solubility such as commercial potato protein. To know

240

the concentration changes of potato protein hydrolyate during the hydrolysis may

241

help to ultimate the potato protein better. In this research, the concentration of potato

242

protein hydrolyate prepared by alcalase during the hydrolysis time of 120 min in

243

traditional and SDFU assisted enzymolysis at different temperature and substrate

244

concentration was displayed in Fig. 1 and Fig.2 respectively.

245

For enzymolysis at different temperatures, it was indicated that increasing the

246

hydrolysis temperature increased the concentration of the potato protein hydrolysate

247

Increasing the concentration of the potato protein hydrolysate meant the increase in

248

conversion rate of potato protein. Although ultrasonic pretreatment was able to

249

improve the conversion of potato protein during hydroysis, the concentration of

250

protein hydrolysate prepared by traditional and SDFU assisted enzymolysis did not

251

show significant difference after hydrolysis time of 60 min at 40 ºC and after 90 min

252

at 50 ºC (p > 0.05). Nevertheless, when the enzymolysis temperature rose to 60 ºC,

253

the similar results were obtained after hydrolysis time of 30 min. As higher

254

temperature may lead to inactivate Alcalase, whose optimum temperature for

255

enzyme activity is suggested as 55-70 ºC by producer, we did not hydrolyze the

256

potato protein beyond 60 ºC. Moreover, the results of Benjakul and Morrissey[39]

257

showed that the temperature higher than 60 had negative effect on protein hydrolysis

258

catalyzed by alcalase.

259

Wang[9] has demonstrated that potato protein hydrolysate prepared Alcalase at the

260

hydrolysis time of 1 h showed good antioxidant properties. In this research, the 12

261

conversion rate of potato protein pretreated with ultrasound increased to 38.8%,

262

54.7%, 74.4% and 92.9% at 30, 40, 50 and 60 ºC respectively at the hydrolysis time

263

of 60 min. Meanwhile, the concentration of protein hydrolysate prepared with

264

ultrasound pretreatment increased by 23.5%, 6.3% and 5.5% when compared with

265

that without ultrasound pretreatment at 30, 40 and 50 ºC respectively (p < 0.05).

266

Moreover, the concentration of protein hydrolysate prepared with ultrasound

267

pretreatment at the hydrolysis time of 45 min was demonstrated no significant

268

difference with that without ultrasound pretreatment at the hydrolysis time of 60 min

269

(p > 0.05). This indicated that ultrasound assisted enzymolysis was able to be more

270

efficient than traditional enzymolysis. Although other research[8] has been carried

271

out to improve the enzymolysis of potato protein using combined enzymes, the

272

hydrolysis time used was too long.

273

When compared with the hydrolysis time of 60 min, the concentration of protein

274

hydrolysate prepared with ultrasound pretreatment at the hydrolysis time of 120 min

275

increased by 20.7%, 23.4%, 19.2% and 9.5% at 30, 40, 50 and 60 ºC respectively (p

276

< 0.05). While the concentration of protein hydrolysate that was prepared without

277

ultrasound pretreatment increased by 33.9%, 25.2%, 23.6% and 9.2% respectively (p

278

< 0.05) in the same case. This suggested that potato protein pretreated without

279

ultrasound may convert faster than potato protein pretreated with ultrasound after a

280

certain time. And the changes may lead to loss of the significant difference in protein

281

hydrolysate concentration between traditional and SDFU assisted enzymolysis.

282

Since the temperature of 60 ºC is close to the optimum temperature of enzyme 13

283

activity, the high enzyme activity at 60 ºC may lead to the high conversion rate.

284

Almost of the potato protein was soluble after being hydrolyzed at 60 ºC for 120

285

min.

286

For enzymolysis at different substrate concentration, during the first hour of

287

hydrolysis, almost of the protein hydrolysate prepared by SDUF assisted

288

enzymolysis showed higher protein concentration than the ones prepared by

289

traditional enzymolysis when compared at same substrate concentration and at the

290

same hydrolysis time (p < 0.05). However, at the hydrolysis time of 120 min, protein

291

hydrolysate prepared by ultrasound assisted enzymolysis showed higher protein

292

concentration than traditional enzymolysis only at the substrate concentration of

293

30.20 g/L (p < 0.05). After 60 min of hydrolysis time, the conversion rate of potato

294

protein pretreated with ultrasound raised up to 93.2%, 83.1%, 74.4%, 70.6% and

295

63.9% at the substrate concentration of 12.08, 18.12, 24.16, 30.20 and 36.24 g/L

296

respectively. Although the conversion of potato protein to protein hydrolysate was

297

higher at low substrate concentration, the concentration of protein hydrolysate was

298

higher at high substrate concentration because more potato protein was used.

299

Moreover, the concentration of protein hydrolysate prepared with ultrasound

300

pretreatment increased by 3.8%, 4.1%, 5.5%, 7.5% and 5.9% at the substrate

301

concentration of 12.08, 18.12, 24.16, 30.20 and 36.24 g/L respectively when

302

compared with that without ultrasound pretreatment (p < 0.05). However, when

303

substrate concentration increased from 30.20 to 36.24 g/L, the difference of protein

304

conversion rate between SDFU assisted and traditional enzymolysis did not 14

305

increased but decreased from 7.5% to 5.9%. This suggested ultrasound assisted

306

enzymolysis may display little difference with traditional enzymolysis on protein

307

hydrolysate concentration if higher substrate concentration was used. The reason

308

may be explained that when all the enzyme were saturated with substrate at high

309

substrate concentration, the enzyme catalysis may transform to zero order reaction in

310

which reaction rate would be control by the concentration of enzyme.

311

The concentration of protein hydrolysate prepared with ultrasound pretreatment at

312

the hydrolysis time of 120 min increased by 14.2%, 13.5%, 19.2%, 12.5% and 14.9%

313

at the substrate concentration of 12.08, 18.12, 24.16, 30.20 and 36.24 g/L

314

respectively when compared with data at the hydrolysis time of 60 min (p < 0.05).

315

While the concentration of protein hydrolysate prepared without ultrasound

316

pretreatment increased by 18.9%, 17.2%, 23.6%, 23.6%, 22.7% in the same case (p

317

< 0.05). It came to the similar results with enzymolysis at different temperatures that

318

protein hydrolysate concentration between traditional and SDFU assisted

319

enzymolysis did not show significant difference after a certain time. The reasons

320

may be relative with the thermodynamics and kinetics of traditional and SDFU

321

assisted enzymolysis.

322

It was indicated that ultrasound assisted enzymolysis can used to improve the

323

hydrolysis of potato protein at suitable conditions. To get the reasons for such

324

situation and the knowledge of difference between traditional and SDFU assisted

325

enzymolysis, thermodynamics and kinetics of traditional and SDFU assisted

326

enzymolysis of potato protein was investigated. 15

327

3.2. Effect of SDFU pretreatment on reaction rate constant

328

As mentioned in methods above, Arrhenius and transition-state theory equations

329

were used to determine the thermodynamics parameters. Since reaction rate constant

330

is important variable in these equations and depended on the temperature, reaction

331

rate constant at the temperatures of 303, 313, 323 and 333K was estimated. The

332

concentration of potato protein hydrolysate during the initial 10 min of hydrolysis

333

which the data will fit linear regression model well was used to determine the

334

reaction rate constant. The plots of ln(C∞-Ct) versus time in traditional and

335

ultrasound assisted enzymolysis at different temperatures were displayed in Fig 3.

336

Reaction rate constants were obtained from the slope of the linear regression plots.

337

The reaction rate constants of traditional enzymolysis kt and that of ultrasound

338

assisted enzymolysis ku at different temperatures were demonstrated in Table 1. It

339

was showed that regression coefficient (R2) of all linear plots was more than 0.98.

340

The plots were able to fit into the linear model well. Therefore, our hypothesis using

341

first order model to calculate reaction rate constant was reasonable. As increasing the

342

temperature will raise up Brownian movement of molecular, both reaction rate

343

constants kt and ku increased as the reaction temperature. Although kt rose faster than

344

ku in the same temperature interval, ku were higher than kt when compared in the

345

same temperature (p < 0.05). When compared with kt, ku was increased by 55.2%,

346

41.6%, 16.1% and 14.3% at 303, 313, 323 and 333K, respectively. It was suggested

347

that increasing the reaction temperature would reduce the difference of reaction rate

348

between traditional and ultrasound assisted enzymolysis. That may be one of the 16

349

reasons why protein hydrolysate concentration between traditional and SDFU

350

assisted enzymolysis would be no significant difference after a certain time. As the

351

hydrolysis time went by, the concentration of substrate would decrease. If the

352

substrate of SDFU assisted enzymolysis was consumed faster than traditional

353

enzymolysis, the reaction rate of ultrasound assisted enzymolysis would lower than

354

traditional enzymolysis after a certain time according to the relation between

355

reaction rate (v), reaction rate constant (k) and substrate concentration [S] which is

356

expressed as v=k[S].

357

3.3. Effects of SDFU pretreatment on the thermodynamic parameters

358

After reaction rate constants were obtained, lnk against 1/T in both traditional

359

enzymolysis and SDFU assisted enzymolysis were plotted. The activation energy Ea

360

were calculated from the slope of the linear regression plots of lnk against 1/T (Fig.

361

4). Enthalpy of activation ∆H, entropy of activation ∆, and Gibbs free energy ∆G

362

were calculated by equation (7) to (9). The results of the thermodynamic parameters

363

Ea, ∆H, ∆, and ∆G were showed in Table 2. As the value of ∆H, ∆, and ∆G in

364

the small range of temperature exhibited little difference, average value of ∆H, ∆,

365

and ∆G was used. It was showed that Ea, ∆H, ∆, of traditional enzymolysis were

366

higher than that of SDFU assisted enzymolysis (p < 0.05). When compared with

367

traditional enzymolysis, thermodynamic parameters Ea, ∆H and ∆, of enzymolysis

368

with SDFU pretreatment reduced by 19.2%, 20.4% and 1.6%. However, Gibbs free

369

energy of SDFU assisted enzymolysis increase by 1.6% when compared with

370

traditional enzymolysis. 17

371

Activation energy is the energy barrier between ground state and activated state of

372

substrate during converting substrate to product. As ultrasound pretreatment

373

decreased the activation energy of enzymolysis, potato protein pretreated with

374

ultrasound was easier and more efficient to reach activated state.

375

Positive values of enthalpy changes indicated endothermic nature of the hydrolysis

376

reaction. Since ∆G = ∆H − T∆S, decreasing the enthalpy was able to decrease the

377

Gibbs free energy. Also, lower enthalpy meant lower energy cost in converting

378

substrates to products. Negative values of entropy changes indicated lowering of

379

entropy during the hydrolysis. Changing the disordering in the reaction system by

380

combining the enzyme and substrate and by converting enzyme-substrate complex

381

from ground state to active state may be the reasons for that. The absolute value of

382

entropy suggested that potato protein pretreated with ultrasound may be easier to

383

combine with alcalase.

384

Althougth Gibbs free energy ∆ G and activation energy Ea belong to different

385

theories, they have the similar meaning in thermodynamic. In enzyme catalysis,

386

Gibbs free energy will be the energy barrier between ground state of complex of

387

substrate binding with enzyme and activated state of the complex of substrate

388

binding with enzyme. It was not surprised that the Gibbs free energy in SDFU

389

assisted enzymolysis was little higher than traditional enzymolysis. Because change

390

of Gibbs free energy during enzymolysis was considered as the sum of minimum

391

free energy change from free substrate to product ∆)" and free energy change of

392

substrate binding to enzyme ∆)3[35]. Ultrasound has been turned out to decrease 18

393

KM and improve the affinity of substrate and enzyme [29, 30]. The increase in

394

affinity would lead to increasing the ∆)3. Thus ∆G in SDFU assisted enzymolysis

395

may be little higher than traditional enzymolysis. Therefore, it suggested that SDFU

396

assisted enzymolysis of potato protein may decrease KM and increase the association

397

of potato protein to alcalase in present research.

398

3.4. Effect of the SDFU pretreatment on initial reaction rate of enzymolysis

399

The initial reaction rate of enzymolysis was first determined according to the

400

reported method by substrate decreasing[38]. The concentration of hydrolyzed

401

potato protein during hydrolysis was showed in Fig. 4. It has been suggested initial

402

velocity could be determined using the data in the early steady state phase of the enzymolysis

403

because formation of product or disappearance of substrate might display a linear relation with

404

time during this period [40]. It was also showed that formation of product or disappearance of

405

substrate verse time fitted linear model well when the consumption of initial substrate

406

concentrate was no more than 10% [40]. Our data in Fig. 5 showed that substrates at different

407

concentration consumed during the initial 10 min were all lower than 2 g/L for both the potato

408

protein pretreated with and without ultrasound. It indicated that the consumption of initial

409

substrate concentrate was less than 5%. Therefore, the slope of linear regression model of

410

hydrolyzed potato protein concentration versus time during the initial 10 min was

411

considered as initial reaction rate. The initial reaction rate of traditional enzymolysis

412

and SDFU assisted enzymolysis at different substrate concentration was showed in

413

Table 3-(a). The regression model exhibited good linear relationship as regression

414

coefficient (R2) of all linear plots was at the range of 0.988 to 0.998. Therefore, the 19

415

data of the initial reaction rate obtained was reasonable. The results showed that

416

increasing the substrate concentration was able to increase the initial reaction rate (p

417

< 0.05). However, the initial reaction rate was not significantly different between the

418

substrate concentration of 24.160 and 30.200g/L for both traditional and SDFU

419

assisted enzymolysis. Morevoer, when the substrate concentration was at the level of

420

24.160, 30.200 and 36.240 g/L, the initial reaction rate of ultrasound assisted

421

enzymolysis did not show significant difference with that of traditional enzymolysis

422

at the same substrate concentration. The initial reaction rate of ultrasound assisted

423

enzymolysis was 1.127 and 1.075 times higher than that of traditional enzymolysis at

424

the substrate concentration of 20 and 30 g/L respectively.

425

Reaction rate in enzymalysis can be also calculated by accumulation of the product

426

during hydrolysis. Thus concentration of the product potato protein hydrolysate was

427

used to determine the initial reaction rate. The slope of linear regression model (R2 in

428

the range of 0.980 to 0.991) of potato protein concentration hydrolysate against time

429

during the initial 10 min was considered as initial reaction rate of traditional

430

enzymolysis. While the slope of linear regression model (R2 in the range of 0.978 to

431

0.998) of potato protein concentration hydrolysate versus time during the initial 8

432

min was considered as initial reaction rate of SDFU assisted enzymolysis. The data

433

during initial 8 min was used in SDFU assisted enzymolysis because regression

434

coefficient in that case was higher and able to make the linear regression model more

435

reasonable. The results were showed in Table 3-(b). When compared traditional and

436

SDFU assisted enzymolysis, the difference of the initial reaction rate was not 20

437

significantly different at the same substrate concentration of 18.120, 24.160 and

438

36.240 g/L (p > 0.05). On the contrary, the initial reaction rate showed significant

439

difference at the same substrate concentration of 12.080 and 30.200 g/L (p< 0.05).

440

These were similar to the results got by substrate loss except at the substrate

441

concentration of 30.200 g/L. Otherwise, initial reaction rate at substrate

442

concentration of 30.200 and 36.240 g/L was higher than other substrate

443

concentrations in SDFU assisted enzymolysis, whereas only at 36.240 g/L in

444

traditional enzymolysis. This was similar to the results got by substrate loss that

445

increasing the substrate concentration was able to increase the initial reaction rate.

446

It was not surprised that the results of initial reaction rate did not consistence with

447

the results of reaction rate constant at the substrate concentration of 24.160 g/L at

448

50 °C. Because the initial reaction rate showed was apparent reaction rate and was

449

also related with changes of substrate. It was suggested that conversion rate constant

450

of enzyme-substrate complex to products in our research in traditional enzymolysis

451

may be faster than ultrasound assisted enzymolysis.

452

3.5. Effect of SDFU pretreatment on enzymolysis kinetic parameters KM and kA

453

To determine the kinetic parameters for both traditional enzymolysis and SDFU

454

pretreatment, the Lineweaver–Burk equation (Eq. (11)) was used. The initial rates

455

obtained by substrate consumption and product accumulation were both used to

456

determine the KM and kA. The plots of 1/V0 versus 1/S0 for traditional and ultrasound

457

assisted enzymolysis was showed in Fig. 6. The regression coefficient (R2) of linear

458

regression plots was 0.990 and 0.987 for traditional and ultrasound assisted 21

459

enzymolysis where the initial rates obtained by substrate consumption. While the

460

regression coefficient (R2) of linear regression plots was 0.978 and 0.963 for

461

traditional and ultrasound assisted enzymolysis where the initial rates obtained by

462

product accumulation. It indicated that our date was able to describe the enzymolysis

463

kinetics well. Nevertheless, the linear models got using initial rates obtained by

464

substrate consumption were better than that by product accumulation.

465

As showed in Table 4, the kinetic parameters KM and kcat were calculated by the

466

slope and intercept of the above linear models. It was proved that SDFU assisted

467

enzymolysis of potato protein decrease Michaelis constant KM. The results showed

468

that KM of ultrasound assisted enzymolysis got from substrate consumption and

469

product accumulation reduced by 37.5% and 36.5% respectively when compared

470

with traditional enzymolysis. Since KM was usually used to represent the apparent

471

affinity of substrate to the enzyme, decreasing of KM demonstrated increasing of the

472

apparent affinity between potato proteins with alcalase. It may explain the reason

473

why Gibbs free energy in SDFU assisted enzymolysis was little higher than

474

traditional enzymolysis. The decrease in KM value for SDFU assisted enzymolysis

475

was possibly due to the fact that ultrasonic pretreatment had partly altered the

476

conformation of the potato proteins and surface properties of materials[31-33, 41].

477

The change of KM indicated that pretreatment with ultrasound was able to promote

478

the enzymatic hydrolysis efficiency of potato protein. However, the breakdown

479

constant of ultrasound assisted enzymolysis kcat was lower than traditional

480

enzymolysis in our research. That was different with the results got by other 22

481

researches in which ultrasound increased the breakdown constant when using

482

alcalase to hydrolyze other proteins [29-31]. However, that was able to explain our

483

results why concentration of protein hydrolysate did not show significant difference.

484

The possible mechanism of SDFU pretreatment improved the enzymolysis efficiency

485

of potato protein was outline in Fig 7. SDFU pretreatment made the association of

486

potato protein and alcalase easier according to the activation free energy change of

487

binding. This resulted in the lower KM of SDFU assisted enzymolysis. At the early

488

stage of hydrolysis, the lower KM of SDFU assisted enzymolysis lead to higher

489

accumulation amount of enzyme-substrate complex. It enhanced the conversion rate

490

of potato protein even though the kcat was lower for SDFU assisted enzymolysis.

491

Therefore, the concentration of protein hydrolysate was higher in ultrasound assisted

492

enzymolysis. However, as the substrate was consumed faster, the diffusion of

493

enzyme to new substrate in SDUF assisted enzymolysis may slow down and

494

accumulation

495

accumulation amount of enzyme-substrate complex in traditional enzymolysis

496

decreased more slowly. Meanwhile, the higher kcat resulted in increasing the

497

conversion rate of potato protein of traditional enzymolysis. After a certain time, the

498

concentration of protein hydrolysate came to the same level for traditional and

499

SDFU assisted enzymolysis. Furthermore, combination and breakdown of substrate

500

and enzyme became balance in both SDFU assisted and traditional enzymolysis, and

501

potato protein conversion was conducted at the similar rate. Then the concentration

502

of protein hydrolysate did not show significantly difference during the hydrolysis

amount

of

enzyme-substrate

complex

decreased.

Whereas

23

503

after that time. As temperature went higher to 60 °C, the activity and diffusion rate of

504

enzyme increased faster, the balance appeared at a short time.

505

In conclusion，SDFU pretreatment was able to promote the enzymolysis efficiency

506

of commercial potato protein in the way of increasing the concentrate of potato

507

protein hydrolysate and shorten the hydrolysis time. The increase in the efficiency

508

was related with the decrease of the thermodynamic parameters including Ea, ∆H

509

and ∆, and kinetic parameters KM. These lowered the energy barrier between

510

ground state and atctive sate of substate-enzyme complex and made the association

511

of substate and enzyme easier and more efficiency. Nevertheless, SDFU assisted

512

enzymolysis only superior to traditional enzymolysis in a limited time. Our

513

hypothesis that the balance between the association effect of KM and breakdown

514

effect of kcat on complex of substrate and enzyme may lead to the loss of significant

515

difference in potato protein hydrolysate concentration between traditional and SDFU

516

assisted enzymolysis after a certain time. The effect of SDFU pretreatment on the

517

structure of potato protein and surface properties of potato protein particles will be

518

investigated in the future to explain the reasons for SDFU induced changes of

519

thermodynamic and kinetic parameters.

520

521

Acknowledgement

522

The study was supported, in part, by the National Natural Science Foundation of

523

China (Grants No. 31301422), the Natural Science Foundation of Jiangsu Province

524

(Grants No. BK20130494), the National High Technology Research and 24

525

Development Program of China (Grants No. 2013AA102203), Senior Personnel

526

Program (Grants No.11JDG051) and Young Backbone Teachers Program of Jiangsu

527

University.

528

Notes

529

The authors declare no competing financial interest.

530

531

532

533

534

535

536

537

538

539

540

541

542

25

543

Reference

544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585

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648

649

650

651

652

653

654

655

656

657

658 28

659

Figure Captions

660

Fig 1 The concentration of potato protein hydrolysate during (a) traditional and (b)

661

simultaneous dual-frequency energy-gathered ultrasound assisted enzymolysis at

662

different temperatures with substrate and enzyme concentration of 24.160 and 0.400

663

g/L respectively.

664

Fig 2 The concentration of potato protein hydrolysate during (a) traditional and (b)

665

simultaneous dual-frequency energy-gathered ultrasound assisted enzymolysis at

666

different substrate concentrations at 50 ºC.

667

Fig 3 Linear transformation plots of first order kinetic model for (a) traditional and

668

(b) simultaneous dual-frequency energy-gathered ultrasound assisted enzymolysis at

669

different temperatures with substrate and enzyme concentration of 24.160 and 0.400

670

g/L respectively.

671

Fig 4 Linear fitting curves of lnk against 1/T respectively for traditional and

672

simultaneous dual-frequency energy-gathered ultrasound assisted enzymolysis at

673

different temperatures with substrate and enzyme concentration of 24.160 and 0.400

674

g/L.

675

Fig 5 The consumption concentration of potato protein during (a) traditional and (b)

676

simultaneous dual-frequency energy-gathered ultrasound assisted enzymolysis at

677

different substrate concentrations at 50 ºC.

678

Fig 6 Double reciprocal linear transformation plots of initial reaction rate obtained

679

by (a) substrate loss or (b) product accumulation versus substrate concentration in

680

traditional and simultaneous dual-frequency energy-gathered ultrasound assisted

681

enzymolysis.

682

Fig 7 The outline of possible mechanism in SDFU pretreatment improving the

683

enzymolysis efficiency of potato protein in limited time. 29

684 685 686 687

Table 1 The reaction rate constant for traditional and simultaneous dual-frequency energy-gathered ultrasound assisted enzymolysis at different temperatures

T(K) 303 313 323 333

Traditional enzymolysis kt (1/min) 0.0072±0.0006h 0.0104±0.0004f 0.0209±0.0012d 0.0405±0.0013b

R2

Ultrasound assisted enzymolysis ku (1/min)

R2

0.984±0.006 0.990±0.003 0.995±0.002 0.995±0.004

0.0111±0.0009g 0.0147±0.0006e 0.0243±0.0008c 0.0462±0.0023a

0.995±0.004 0.987±0.010 0.991±0.002 0.997±0.003

688 689

30

690 691 692

Table 2 Thermodynamic parameters for traditional and simultaneous dualfrequency energy-gathered ultrasound assisted enzymolysis Ea △H △S (kJ·mol-1) (kJ·mol-1) (J·mol-1·K-1) Traditional enzymolysis Ultrasound assisted enzymolysis

46.329 37.437

43.685 34.793

-194.946 -198.046

△G (kJ·mol-1) 62.036 63.013

693 694

31

695 696 697 698 699

Table 3 Initial reaction rate of traditional and simultaneous dual-frequency energy-gathered ultrasound assisted enzymolysis of potato protein at different substrate concentrations obtained by (a) substrate consumption and (b) product accumulation.

Traditional enzymolysis

Ultrasound assisted enzymolysis

Substrate concentration (g·L-1) 12.080 18.120 24.160 30.200 36.240 12.080 18.120 24.160 30.200 36.240

Initial reaction rate (g·L-1·min-1) (a)

(b)

0.0952 ± 0.0017F

0.295 ± 0.002h

0.1155 ± 0.0057D

0.385 ± 0.014fg

0.1342 ± 0.0001

B

0.462 ± 0.006

de

0.1372 ± 0.0051

B

0.523 ± 0.044

bcd

0.1478 ± 0.0070

A

0.513 ± 0.042

bc

0.1073 ± 0.0030

E

0.365 ± 0.043

g

0.1242 ± 0.0008

C

0.436 ± 0.009

ef

0.1379 ± 0.0032

B

0.493 ± 0.064

cde

0.1392 ± 0.0007

B

0.590 ± 0.022

a

0.1502 ± 0.0041A

0.564 ± 0.032ab

700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 32

727 728 729 730 731

Table 4 Kinetic parameters (KM, kA) of traditional and simultaneous dualfrequency energy-gathered ultrasound assisted enzymolysis obtained by (a) substrate consumption and (b) product accumulation (E0 is 0.400 g·L-1). kA (min-1)

KM (g·L-1) Traditional enzymolysis Ultrasound assisted enzymolysis

(a)

(b)

(a)

(b)

13.726

25.578

0.510

1.850

8.572

16.224

0.459

1.681

732 733 734

33

30

Concentration of Protein Hydrolysate (mg/mL)

Concentration of Protein Hydrolysate (mg/mL)

735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778

Traditional enzymolysis 30 oC o 40 C 50 oC 60 oC

25

20

15

10

5

0 0

20

40

60

80

Hydrolysis Time (min)

100

120

30

Ultrasound-assisted enzymolysis o

30 C o 40 C 50 oC 60 oC

25

20

15

10

5

0 0

20

40

60

80

100

120

Hydrolysis Time (min)

Figure 1

34

30

Concentration of Protein Hydrolysate (mg/mL)

Concentration of Protein Hydrolysate (mg/mL)

779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799

(a)Traditional emzymolysis 12.08 g/L 18.12 g/L 24.16 g/L 30.20 g/L 36.24 g/L

25

20

15

10

5

0 0

20

40

60

80

Hydrolysis Time (min)

100

120

30

(b)Ultrasound assisted emzymolysis 12.08 g/L 18.12 g/L 24.16 g/L 30.20 g/L 36.24 g/L

25

20

15

10

5

0 0

20

40

60

80

100

120

Hydrolysis Time (min)

Figure 2

35

3.2

3.2

3.0

3.0

ln(C∞ - Ct)

ln(C∞ - Ct)

800 801 802 803

2.8

2.8

(a)Traditional enzymolysis 303 313 323 333

2.6

K K K K

(b)Ultrasound assisted enzymolysis

2.4

2.4 0

804 805 806 807 808

303 K 313 K 323 K 333 K

2.6

2

4

6

8

Hydrolysis Time (min)

10

12

0

2

4

6

8

10

12

Hydrolysis Time (min)

Figure 3

36

-2.5

Traditional enzymolysis Ultrasound-assisted enzymolysis

-3.0

-3.5

y = −4.503 ×10 3 x + 10.761

R 2 = 0.962

-1

lnk (min )

809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825

-4.0

-4.5

y = −5.572 × 10 3 x + 13.970

R 2 = 0.979

-5.0

-5.5 0.0031

0.0032

0.0033

0.0034

1/T (K-1)

Figure 4

37

8.0

8.0

(a)Traditional enzymolysis

Concentration of Protein Hydrolyzed (g/L)

Concentration of Protein Hydrolyzed (g/L)

826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869

12.08 g/L 18.12 g/L 24.16 g/L 30.20 g/L 36.24 g/L

6.0

4.0

2.0

0.0

(b) Ultrasound-assisted enzymolysis 12.08 g/L 18.12 g/L 24.16 g/L 30.20 g/L 36.24 g/L

6.0

4.0

2.0

0.0 0

20

40

60

80

Hydrolysis Time (min)

100

120

0

20

40

60

80

100

120

Hydrolysis Time (min)

Figure 5

38

12

4.0

(a)

Traditonal enzymolysis Ultrasound-assisted enzymolysis

(b)

Traditonal enzymolysis Ultrasound-assisted enzymolysis 3.5

y = 67.459 x + 4.902

R 2 = 0.990

8

y = 46.724 x + 5.451

R 2 = 0.987

1/V0 (min. L.g-1)

10

1/V0 (min. L. g-1)

870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897

3.0

y = 27.650 x + 1 .081

R 2 = 0.985

2.5

2.0

6

y = 19.307 x + 1.190

R 2 = 0.965

1.5

4 0.02

0.03

0.04

0.05

0.06 -1

1/S0(L.g )

0.07

0.08

0.09

1.0 0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

1/S0(L.g-1)

Figure 6

898

899

900

901

902

903

904 39

905 [PPT] : Potato protein pretreated without SDFU [PPU] : Potato protein pretreated with SDFU

[Alc]:Alcalase

*: Transition state

[PPT-Alc] :Complex of [PPT] and [Alc] [PPU-Alc] :Complex of [PP U] and [Alc] △GT , GU : Activation free energy change of ground state to transition state

[PPT*-Alc] [PPU* -Alc]

△GT T, GUT: Minimum net activation free energy change for reaction to occur

KM-U > KM-T

△ GTS: Activation free energy change of binding [PPT ] and [Alc] △ GUS: Activation free energy change of binding [PPU ] and [Alc]

kcat-U < kcat-T

△GU

[PPU] + [Alc]

△GUT

t1

t0 [PPT] + [Alc]

[PPT-Alc] [PPU-Alc]

△ GT

△GTS

:Potato protein hydrolyate

CU>CT (p < 0.05)

:Potato protein pretreated with SDFU △ GUS

: Potato protein pretreated without SDFU

:Alcalase

CU: Concentration of PPH prepared using potato protein pretreated with SDFU

906

Balance of Affinity and Breakdown

△GTT

△GTS < △GUS

KM-U > KM-T

CT: Concentration of PPH prepared using potato protein pretreated without SDFU

t2

t3 CU=CT (p > 0.05)

KM -U, KM -T: Michaelis constant of SDFU-assisted and traditional enzymolysis kcat-U,k cat-T: Breakdown rate constantof SDFU-assisted and traditional enzymolysis

907 908

Figure 7

909

910

911

912

913

914

915

916

917

918

919 920 40

TOC Graph Concentration of Protein Hydrolysate (mg/mL)

921

[PPT*-Alc] [PPU*-Alc] △GTT △ GU

30

CU = CT

25

CU > C T 20

15 Hydrolysis Temperature

10

5

CU > CT

Concentration of Protein Hydrolysate (mg/mL)

△GUT

△GTS

[PPT] + [Alc]

△GT

[PPT-Alc]

△GUS

[PPU-Alc] △GTS < △GUS

KM-U > KM-T

922

30

20

40

60

80

100

120

Substrate concentration

U-24.16 g/L U-36.24 g/L T-24.16 g/L T-36.24 g/L

25

20

15

CU = CT 10

CU > CT

5

0 0

20

40

60

80

100

120

Hydrolysis Time (min)

KM-U > KM-T

t0

T-40 o C T-60 o C

0 0

[PPU] + [Alc]

U-40 oC U-60 oC

CU = CT

kcat-U < kcat-T

t1 CU > CT (p < 0.05)

Balance of Affinity and Breakdown

t2

t3 CU = CT (p > 0.05)

923

924 925

41

926

Highlights

927

(1) The efficiency of potato protein enzymolysis can be improved by simultaneous

928

929 930

931 932

dual frequency energy-gathered ultrasound (SDFU) pretreatment. (2) The thermodynamics and kinetics of traditional and SDFU assisted enzymolysis of potato protein was investigated. (3) The decrease of Ea and KM by SDFU pretreatment resulted in the higher enzymolysis efficiency of potato protein.

933 934

42