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Synergistic Effect of Thermal Reaction and Vacuum Dehydration for Improving Xylose-Phenylalanine Conversion to N-(1-deoxy-Dxylulos-1-yl)-phenylalanine during an Aqueous Maillard Reaction Heping Cui, Khizar Hayat, Chengsheng Jia, Emmanuel DUHORANIMANA, Qingrong Huang, Xiaoming Zhang, and Chi-Tang Ho J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04448 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 8, 2018

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Journal of Agricultural and Food Chemistry

Synergistic Effect of Thermal Reaction and Vacuum Dehydration for Improving Xylose-Phenylalanine Conversion to N-(1-deoxy-D-xylulos-1-yl)-phenylalanine during an Aqueous Maillard Reaction

Heping Cui

†,‡

, Khizar Hayat §, Chengsheng Jia †, Emmanuel Duhoranimana †,

Qingrong Huang ‡, Xiaoming Zhang †*, Chi-Tang Ho ‡*

†

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, P. R. China.

‡

Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick 08901, NJ, USA.

§

Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, P. O. Box 2460, Riyadh 11451, Saudi Arabia.

Author information * Corresponding Author: Xiaoming Zhang & Chi-Tang Ho (1) Xiaoming Zhang, Ph.D., Professor Postal address: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, Jiangsu 214122, People’s Republic of China. E-mail: [email protected] (X. Zhang). Tel.: +86 510 85197217. Fax: +86 510 85884496. 1

ACS Paragon Plus Environment


1 2

(2) Chi-Tang Ho, Ph.D., Professor Postal address: Department of Food Science, Rutgers University, 65 Dudley Road,

3

New Brunswick 08901, NJ, USA. E-mail: [email protected]

4

Heping Cui, Ph.D.

5

Postal address: School of Food Science and Technology, Jiangnan University, Lihu

6

Road 1800, Wuxi,

Jiangsu

7

[email protected]

8

Khizar Hayat, Ph.D.

214122,

People’s Republic of China. E-mail:

9

Postal address: Department of Food Science and Nutrition, College of Food and

10

Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi

11

Arabia. E-mail: [email protected]

12

Chengsheng Jia, Ph.D.

13


14

Road 1800, Wuxi,

15

[email protected]

16

Emmanuel Duhoranimana, Ph.D.

17

Road 1800, Wuxi,

19

[email protected]

20

Qingrong Huang, Ph.D.

22 23

214122,



18

21

Jiangsu

Jiangsu

214122,


Postal address: Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick 08901, NJ, USA. E-mail: [email protected] 2


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Page 3 of 36


24

ABSTRACT

25

The synergistic effect of thermal reaction and vacuum dehydration on the

26

conversion of xylose (Xyl) and phenylalanine (Phe) to Maillard reaction intermediate

27

(MRI) was researched. The yield of the N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine

28

was successfully improved and increased from 13.62% to 47.23% through the method

29

combining thermal reaction and vacuum dehydration. A dynamic process was involved

30

in the transformation of Xyl and Phe (Xyl-Phe) to N-substituted D-xylosamine and

31

N-substituted D-xylosamine to N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine at the

32

initial stage of dehydration; then only the transformation of N-substituted D-xylosamine

33

to N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine at the final stage. Furthermore, the MRI

34

was prepared under the optimized conditions (temperature 90 °C and pH 7.4), and the

35

obtained MRI was characterized and confirmed by ESI-mass spectra and NMR.

36

WORDS:

Synergistic

effect;

Maillard

37

KEY

38

N-(1-deoxy-D-xylulos-1-yl)-phenylalanine; Transformation

reaction

intermediate;

39

40

Chemical compounds studied in this article

41

d-xylose (PubChem CID: 135191); l-phenylalanine (PubChem CID: 6140); Ethanol

42

(PubChem CID: 702); Deuteroxide (PubChem CID: 24602).

43

3



44

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INTRODUCTION

45

Maillard reaction is a cascade of complex and competitive reactions involving

46

amino compounds and reducing sugar during food processing leading to structural

47

changes and flavor formation of food. 1-3 The important Maillard reaction intermediates

48

(MRIs) known as Amadori or Heyns rearrangement products (ARPs or HRPs) occur in

49

the early stage of Maillard reaction, condensation of aldose with amine occurs, leading

50

to the formation of a labile N-substituted amino sugar such as N-substituted

51

glycosylamine and N-substituted fructosamine.

52

addition, the Schiff base of glycosylamine (or fructosamine) rearranges via

53

1,2-eneaminol which leads to the glycation products of ARPs or HRPs 6. These MRIs

54

widely exist in both wholefood and processed foods,

55

considered as potential natural food ingredients;

56

prospect was proposed.

57

minimal processing conditions has become an attractive approach to generate authentic

58

flavor profiles resonating with consumers’ demand for more naturalness,

59

control of reaction steps to generate the stable MRIs adapted to the thermal processes is

60

required. 16 Flower-like flavor compounds were formed during the heat treatment of the

61

ARP derived from xylose and phenylalanine (Xyl-Phe), and this ARP could be used for

62

fresh process flavors of bakery foods

63

derived ARP and there is a need for its characterization at a molecular level. 12, 13

64 65

11-14

4, 5

Specifically, after the nucleophilic

9-11

7, 8

they are consequently

and their extensive application

Especially, for the trend that using traditional cooking and

15

a good

16

. Thus, it is significant to prepare the Xyl-Phe

Water is a common solvent for Maillard reaction and Maillard flavorings production. 17-19

However, previous researchers have reported that the ARP hydrolysis could occur 4


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20, 21

66

in aqueous solution during heating, generating free sugar and amino acid.

67

to increase the ARP yield, in this study, vacuum dehydration combined with thermal

68

reaction was employed to improve the transformation from N-substituted D-aldosamine

69

to the ARP derived from Xyle-Phe (N-(1-deoxy-D-xylulos-1-yl)-phenylalanine). The

70

ARP derived from Xyl and Phe was synthesized through two stages: (i) refluxing at a

71

constant temperature in aqueous solution; (ii) and continued thermal reaction with

72

vacuum dehydration treatment. When the vacuum dehydration was conducted after the

73

time when the ARP optimally formed, the dehydration process would result in the

74

secondary reaction during the excessive reaction. Thus, the reaction time of stage (i)

75

was shortened from the optimized time, to maintain the total preparation time equal to

76

the optimized time. The change in concentration of N-substituted D-aldosamine and the

77

conversion of Xyl-Phe to ARP during the thermal reaction combined with vacuum

78

dehydration was researched. Synergistic effect of thermal reaction and vacuum

79

dehydration

80

N-(1-deoxy-D-xylulos-1-yl)-phenylalanine was proposed. Additionally, the relationship

81

between the ARP yield and the water content during dehydration was evaluated, and the

82

mechanism of N-(1-deoxy-D-xylulos-1-yl)-phenylalanine yield improvement was

83

discussed. Furthermore, the molecular structure of the ARP derived from Xyl and Phe

84

was characterized to support the effectiveness of ARP preparation through the

85

synergistic effect of thermal reaction and vacuum dehydration.

for

improving

xylose-phenylalanine

86

87

MATERIAL AND METHODS 5


In order

conversion

to


88

Chemicals. l-phenylalanine, d-xylose, anhydrous ethanol and deuteroxide were

89

purchased from Sigma-Aldrich Chemical Co. Ltd (Shanghai, China). Formic acid,

90

acetonitrile, 2-(2,3,4-trihydroxybutyl)-quinoxaline (97%) and o-phenylenediamine were

91

obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Standard ARP

92

(N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine) (98%) was prepared in our lab.

93

Preparation of ARP. Model system (5 g) of Xyl and Phe mixture in a 2:1 relative molar

94

ratio of Xyl to Phe, was solubilized in 80 mL deionized water. The solution pH was then

95

adjusted to different values (5.9-7.9) with the NaOH solution at the concentration of 3

96

mol/L. The reaction was divided into two stages. Firstly, the solution was heated in a

97

water bath for refluxing at a constant temperature of 90 °C, and then the atmospheric

98

pressure inside the bottle was decreased to 25 mbar in 5 min using rotary evaporator

99

under vacuum (R-215, Büchi, Flawil, Switzerland). After dehydration at 90 °C for 15

100

min, the mixture was immediately cooled in ice water. The obtained solid product was

101

dispersed in anhydrous ethanol (40 mL), and rotary evaporated under vacuum at 30 °C

102

for 30 min (the vacuum pressure was above 0.07 MPa). The objective of sample

103

dispersion in anhydrous ethanol was to form azeotropic system of water-ethanol, so that

104

the residual water could be removed at low boiling point using rotary evaporation, to

105

stabilize the prepared N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine.

106

Moisture measurement. All the ingredients with known moisture content were mixed

107

in a certain ratio, thus, initial moisture of the system was accordingly calculated. The

108

weight loss of the system after rotary evaporation was roughly regarded as the decrease

109

of the water weight. The moisture content of the system was expressed as the weight 6


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Page 7 of 36


110

percentage of remained water to the solution.

111

Purification of the ARP. The preliminary purification of ARP was performed using the

112

method of Cui et al. 16. The ARP solution was dried by rotary evaporator at 45 °C, and

113

the obtained solid was mixed with 500 mL anhydrous ethanol. The undissolved

114

components were filtered off and the solution was dried using a rotary evaporator again,

115

then the remaining solid of ARP was dissolved in water. The obtained ARP was further

116

purified using the method of Davidek et al. 22 with slight modifications. A column filled

117

with Dowex 50WX4 ion exchange resin in H+-form was selected for purification of the

118

ARP. The column was firstly eluted with water till the eluent showed negative to

119

2,3,5-triphenyltetrazoliumchloride (TTC) test.

120

ammonium hydroxide (0.2 mol/L). The fractions containing the ARP were further

121

purified using semi-preparative RP-HPLC. The sample was eluted at 1.0 mL/min by

122

linear gradient from 2 to 100% acetonitrile/0.1% formic acid over 18 min; and C18

123

RP-HPLC (10 µm, 22 × 200 mm) column was used. The recovery percentage of the

124

purified ARP was 65%.

125

Analysis of ARP by HPLC. The method of Cui et al. 16 for ARP analysis was employed

126

for the analysis of ARP. The ARP was dissolved in water (80 mL). The resulted aqueous

127

solution was filtered using polyvinylidene fluoride filters (PVDF, 0.22 Millipore) before

128

injection into RP-HPLC system. UV detection was performed at 215 nm with a photo

129

diode-array detector 2996 (Waters, Milford, MA, USA). The elution conditions were

130

similar to those of semi-preparative RP-HPLC. The X-Select C18 RP-HPLC (3.5 µm, 4.6

131

× 150 mm) column was used for the analysis.

22

Then the ARP was eluted with

7



Page 8 of 36

132

The ARP in the sample was quantified based on calibration curve of purified

133

product (y = 1.1039 x﹣2.8301, R2 = 0.9971). The yield of ARP was calculated as the

134

percentage of the measured molar concentration of ARP to the initial molar

135

concentration of phenylalanine.

136

Mass

137

N-substituted

138

N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine

139

simultaneously existed in the sample, they were hardly separated through HPLC. Thus,

140

Maillard reaction intermediates were often analyzed using mass spectrometry.

141

research, purified ARP, N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine and N-substituted

142

D-xylosamine in the reaction solution were analyzed using Q-TOF MS spectrometry

143

(Waters Synapt Q-TOF MS, USA). N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine and

144

N-substituted D-xylosamine in the samples were quantified based on calibration curve

145

of purified ARP, as the ARP was stable to be prepared and purified, additionally

146

N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine and N-substituted D-xylosamine appeared

147

in the same peak in the chromatogram. The UPLC-ESI-MS system with positive ESI

148

mode mass spectra was used to obtain the spectra and a Waters Acquity PDA detector

149

was used. The ionization conditions were as follows: capillary voltage 3.5 kV, cone

150

voltage 20 V, and extractor voltage 7 V. The source block temperature and the

151

desolvation temperature were 100 °C and 400 °C, respectively. The cone gas flow was

152

adjusted to 50 L/h. For the analysis of N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine and

153

N-substituted D-xylosamine in the reaction solution, MS detection was in multiple

Spectrometry

analysis

of

N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine,

D-xylosamine

and and

When

deoxyosone. N-substituted

8


D-xylosamine

23

In this

Page 9 of 36


154

reaction monitoring (MRM) mode performed with the m/z 316, 298 for N-substituted

155

D-xylosamine, and the m/z 298, 280 for N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine.

156

For the analysis of purified ARP, MS detection was in full scan mode over the range of

157

m/z 20-1000 with 1 s scanning time and an inter-scanning delay of 0.1 s. The

158

quantification was based on the identification of the peaks of Phe (m/z 166 and 120) and

159

N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine (m/z 298 and 280). Furthermore, to

160

facilitate protonation of N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine required for the

161

detection by ESI+-MS, water containing 0.1% formic acid was used as an eluent. The

162

CSH C18 (1.7 µm, 2.1 × 100 mm) column was used for Ultra Performance Liquid

163

Chromatography (UPLC) analysis. Analytical UPLC conditions on LC-MS were as

164

follows: flow rate was 0.3 mL/min, and UV detector was set at 200-600 nm. The

165

samples were analyzed by linear gradient from 2 to 100% methanol /0.1% formic acid

166

over 20 min by directly injecting the sample. The samples were analyzed using an

167

autosampler, and the injection volume was 1 µL. Data were acquired using Mass Lynx

168

software (version 4.1, Milford, MA, USA).

169

Additionally, the analysis of deoxyosone was performed using the method of 24

170

Kocadaǧlı et al. with some modification.

Briefly, the sample of Maillard reaction

171

solution (100 µL) was mixed with 0.05 mol/L o-phenylenediamine solution (200 µL).

172

The mixture was placed in dark at 25 °C for 2 h before being filtered using

173

polyvinylidene fluoride filters (PVDF, 0.22 Millipore). Then the resulted solution was

174

injected into HPLC system. The HPLC column was LiChrospher C18 (250 mm × 4.6

175

mm, 5 µm, and the UV detection was performed at 320 nm. The samples were analyzed 9



176

using an autosampler, and the injection volume was 10 µL. Mobile phase A was 0.1%

177

formic acid aqueous solution, while mobile phase B was methanol. The flow rate of the

178

mobile phase was 1 mL/min, and the samples were analyzed by linear gradient: 0-35

179

min, B 35%-70%; 35-40 min, B 70%-35%, 40-50 min, B 35%. The ionization

180

conditions were as follows: capillary voltage 3.5 kV, cone voltage 20 V, and extractor

181

voltage 7 V. The source block temperature and the desolvation temperature were 100 °C

182

and 400 °C, respectively; the cone gas flow was adjusted to 50 L/h. MS detection was in

183

full scan mode over the range of m/z 20-1000 with 1 s scanning time and an

184

inter-scanning delay of 0.1 s. The quantification was based on the identification of the

185

peaks of deoxyosone derivative with o-phenylenediamine (m/z 205 and 187) and the

186

calibration curve of 2-(2,3,4-trihydroxybutyl)-quinoxaline. Data were acquired using

187

Mass Lynx software (version 4.1, Milford, MA, USA).

188

NMR analysis of ARP. The purified ARP powder was dissolved in deuteroxide (500

189

µL), and the mixture was then transferred to a 5 mm NMR tube for NMR analysis. The

190

analysis was performed on a Bruker DRX 400 MHz spectrometer (Bruker Bio Spin,

191

Ettlingen, Germany) equipped with a 5 mm PABBO probe and operated at 25 ºC (298

192

K). Data were obtained using MestReNova software (version 9.0.1, Mestrelab Research,

193

Escondido, CA, USA).

194

Statistical Analysis. The results were presented as mean values ± standard deviation.

195

Each reaction was done for three times and each sample was analyzed in triplicate.

196

SPSS version 19.0 (IBM, Armonk, NY, USA) was used for all statistical analyses.

197

Bivariate Pearson correlation analysis among the parameters tested was also conducted 10


Page 10 of 36

Page 11 of 36


198

using SPSS. Duncan’s multiple range test was used, and p < 0.05 was considered as

199

significant.

200

201

RESULTS AND DISCUSSION

202

Effect of dehydration on ARP yield. According to Nursten (Chapter 2, Paragraph 8, 9)

203

25

, low temperatures could facilitate the initial stage of Maillard reaction rather than the

204

degradation of ARP. Thus, in this study, a temperature (80 °C) below the normal

205

Maillard reaction procedure (usually 100-180 °C) was used for ARP preparation, with

206

or without dehydration. The yields of N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine at

207

different dehydration temperature are shown in Fig. 1.

208

Without dehydration, the yield of ARP increased up to 13.62% at 80 °C with

209

extended reaction time (Fig. 1). The optimal yield of ARP prepared by dehydration at

210

50 °C was 13.78%, which was similar to that prepared at 80 °C without dehydration

211

(13.62%) (Fig. 1). However, the yield of ARP prepared by only dehydration at 50 °C

212

without the first reaction stage (reaction time t = 0 min) reached 7.45% and was higher

213

than that obtained after reacting at 80 °C for 20 min of reaction without dehydration

214

(Fig. 1). These results indicated that dehydration for 15 min increased the yield of ARP.

215

Moreover, dehydration at 80 °C increased the optimal yield to 20.21%, which was much

216

higher than the optimal yield obtained with dehydration at 50 °C (13.78%) (Fig. 1).

217

These results demonstrated that dehydration was important for yielding the ARP from

218

Xyl-Phe during the Maillard reaction in aqueous medium, and the increase of

219

temperature could further improve the yield. Thus, in order to improve the ARP yield, 11



Page 12 of 36

220

an increased temperature was needed for efficient dehydration in the following research.

221

When the ARP was prepared at 80 °C without dehydration, the yield of

222

N-(1-deoxy-D-xylulos-1-yl)-phenylalanine was only 13.62%, and N-substituted

223

D-aldosamine, the precursor of N-(1-deoxy-D-xylulos-1-yl)-phenylalanine, was

224

observed

225

N-(1-deoxy-D-xylulos-1-yl)-phenylalanine preparation. It was probably because that

226

N-substituted D-aldosamine was labile in aqueous medium that it could either

227

disintegrate to regenerate the original sugar and amino acid or dehydrate to complete

228

Amadori rearrangement. 3, 21

229

Improvement of ARP yield by vacuum dehydration synergistically with thermal

230

reaction. The optimal yield of ARP during the reaction at 80 °C without dehydration

231

was 13.62%, and the optimal yield during the reaction at 80 °C and dehydration at

232

50 °C was 13.77% (Fig. 1). These results indicated that the yield increased by only

233

1.10% through the dehydration at low temperature. Results, showed that the optimal

234

yield of Xyl-Phe to ARP during the reaction at 90 °C without dehydration was 15.15%

235

(Fig. 2a), which has increased by 11.23% compared to the optimal yield during the

236

reaction at 80 °C without dehydration. However, the optimal yield during the reaction at

237

80 °C and dehydration at 80 °C was 20.21% (increased by 48.38% compared to the

238

optimal yield during the reaction at 80 °C without dehydration), and the optimal yield

239

during the reaction at 90 °C and dehydration at 90 °C was 47.23% (increased by

240

211.75% compared to the optimal yield during the reaction at 90 °C without

241

dehydration) (Fig. 1 and 2a). Both of the yield growth rates were much higher than the

in

heated

Xyl-Phe

aqueous

12


solution

during

Page 13 of 36


242

improvement by either increasing the temperature or combining with dehydration at a

243

low temperature. These results revealed that synergistic effect of thermal reaction and

244

vacuum dehydration for improving the ARP yield existed during the reaction. Hence,

245

the ARP yield could be improved through the adjustment of temperature and vacuum

246

during the Maillard reaction in aqueous medium.

247

According to previous studies, the activation energy of Amadori arrangement (33.5

248

kJ·mol-1) was found to be slightly greater than that of Schiff base formation (27.3

249

kJ·mol-1) and dehydration (31.3 kJ·mol-1) in Maillard reaction of Phe, which indicated

250

that the ARP generation was more sensitive to temperature and favored by high

251

temperature.

252

rising temperature, which would probably improve Amadori arrangement

253

theory might account for the synergistic effect of thermal reaction and vacuum

254

dehydration for improving Xyl-Phe conversion to the ARP.

21

Particularly under low moisture conditions, dehydration was favored by 20

. This

255

With dehydration at 80 and 90 °C, the yield of ARP increased firstly and then

256

decreased with increasing the reaction time (Fig. 1 and 2a). When the reaction

257

temperatures were 80, 90 and 100 °C, the optimal reaction time was 60, 40 and 30 min,

258

respectively (Fig. 1 and 2a). This result showed that the optimal reaction time for ARP

259

preparation was decreased when the reaction temperature was increased, which would

260

be an advantage in the industrial production of ARP. However, the optimal yield of

261

ARP was 47.23% when the reaction was at 90 °C, which was higher than that obtained

262

after the reaction at 100 °C (45.28%). It suggested that higher temperature expedited the

263

formation of ARP, meanwhile overreaction led to the disintegration of accumulated 13



Page 14 of 36

7, 20, 26

264

ARP. These results are in accordance with some previous researches,

265

reported that the reaction rate at the intermediate stage of Maillard reaction was

266

enhanced when reaction temperature increased above 90 °C, which resulted in the

267

formation of 1-deoxyglucosone, furfural, methylglyoxal, and others. Additionally,

268

increasing temperature could also promote the degradation of original sugar or amino

269

acid (the sugar or amino acid in reactants), and their reaction with the products at

270

intermediate stage of Maillard reaction.

271

the reaction and dehydration when the effect of initial pH on the Xyl-Phe conversion to

272

ARP was evaluated.

7, 18, 20, 26

which

Accordingly, 90 °C was selected for

273

pH 7.4 showed the optimal improvement in ARP yield, compared with the other

274

pHs in the pH range 5.9-7.9 (Fig. 2b). This result was similar to previously reported

275

findings.

276

for generating ARP. The Maillard reaction was initiated by the nucleophilic attack of the

277

amino group on the sugar to form a Schiff base, which is a precursor of ARP. 30 Besides,

278

reverse Amadori rearrangement was accelerated in aqueous solution heated during

279

alkalization,

280

condition.

281

under alkaline conditions, facilitating the secondary reactions.

282

excessive protonation of amine group led to a decrease in glycation of amino acids at

283

low pH levels, which was not favorable to ARP generation. 3, 9, 33 Furthermore, the yield

284

of original sugar formed from ARP degradation was found to decrease at pH > 7,

285

whereas, a relatively neutral pH was favorable to ARP generation.

27, 28

29

According to Mossine et al, 29 protonation of reducing sugar is necessary

29, 31

while the reverse rearrangement could be inhibited under neutral

In addition, the rate of intermediate and final stages could be increased

14


32

On the other hand,

30

Page 15 of 36


286

Therefore, overtime reaction, excessively high temperature and pH were adverse to

287

improve the yield of ARP, and the optimal reaction conditions based on synergistic

288

effect of thermal reaction and vacuum dehydration were evaluated as follows: reaction

289

time of 40 min, reaction and dehydration temperature of 90 °C and the initial pH 7.4.

290

Relationship between the water content and Xyl-Phe conversion to intermediates

291

during dehydration with thermal reaction. Since the synergistic effect of thermal

292

reaction and vacuum dehydration was crucial on improving the ARP yield, the Maillard

293

reaction with vacuum dehydration was proposed as an effective method to prepare ARP

294

in aqueous medium, and the yield was increased from 13.62% to 47.23% (Fig. 1 and 2).

295

The dehydration with thermal treatment was critical during the intermediates (both of

296

N-substituted

297

generation, and great influence of dehydration extent on the total intermediate

298

(including

299

N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine) yield was observed (Fig. 1). The decrease

300

of water content resulted in the change of water activity (aW), which had a great

301

influence on the kinetics of ARP formation and degradation, especially change of aW

302

from 0.86 to 0.96.

303

stability, and the reducing sugar degradation probably occurred at a high aW.

304

the effect of moisture change on intermediates generation during the dehydration with

305

thermal reaction was evaluated.

D-xylosamine

and

N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine)

N-substituted

34

D-xylosamine

and

Additionally, higher aW had an unfavorable influence on the ARP 34

Thus,

306

During the dehydration, the moisture content of the reaction system sharply

307

decreased with dehydration time and after 20 min it reached zero percent (Fig. 3). 15



Page 16 of 36

308

Meanwhile, the yield of ARP also increased up to the climax at also 20 min dehydration

309

time (Fig. 3). The Pearson’s correlation was applied to see the relationship between the

310

water content and the yield of ARP, and strong negative correlation was observed in the

311

results (r = -0.902, p < 0.01). These results revealed that dehydration facilitated the ARP

312

yield, and the optimal dehydration time was 20 min. Additionally, in aqueous medium,

313

an

314

N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine occurred during the Maillard reaction (eq.

315

1).

equilibrium

Phe + Xyl 316

k1 k-1

among

Xyl-Phe,

N-substituted D-xylosamine

N-substituted

k2 + H2O

D-xylosamine

and


(eq. 1)

317 318

Based on the observed concentration of the intermediates at equilibrium, k1, k2 and k-1

319

were calculated as 0.018, 20.48 and 55.87, respectively, according to the method

320

reported in previous study.

321

N-substituted

322

N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine formation, was rate-limiting;

323

rate-limiting effect of N-substituted D-xylosamine formation was more remarkable with

324

the changes in reaction temperature.

325

reported research on the kinetic significance of the Schiff base reversion in the aqueous

326

Maillard reaction model of a phenylalanine-glucose.

327

conversion to N-substituted D-xylosamine was important for the improvement of

328


329

N-substituted D-xylosamine was improved at the reaction and dehydration temperature

35

The k-1 and k2 were much greater than k1, suggesting that

D-xylosamine

formation,

25

but

not

the 21

and this

These results were similar to the previous

35

Therefore, increasing Xyl-Phe

formation;

16


and

the

formation

of

Page 17 of 36


330

of 90 °C, which probably further accelerated Amadori arrangement.

331

Change of the intermediates concentration during the dehydration with thermal

332

reaction. Before the dehydration, the yield of ARP was only 10.03% (Fig. 3). By using

333

UPLC-MS/MS, the concentrations of N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine and

334

N-substituted D-xylosamine were measured as 58.92 mmol/mol and 39.28 mmol/mol,

335

respectively (Fig. 4). However, the concentration of N-substituted D-xylosamine

336

dropped to zero after 20 min dehydration (Fig. 4), and the concentration of

337

N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine increased by 425%. According to the

338

Pearson’s

339

concentration showed strong positive correlation with dehydration time (r = 0.913, p

Synergistic Effect of a Thermal Reaction and Vacuum Dehydration on

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