Timely Addition of Glutathione for Its Interaction with Deoxypentosone

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Food and Beverage Chemistry/Biochemistry

Timely Addition of Glutathione for its Interaction with Deoxypentosone to Inhibit the Aqueous Maillard Reaction and Browning of Glycylglycine-Arabinose System Siyun Lu, Heping Cui, Huan Zhan, Khizar Hayat, Chengsheng Jia, Shahzad Hussain, Muhammad Usman Tahir, Xiaoming Zhang, and Chi-Tang Ho J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02053 • Publication Date (Web): 24 May 2019 Downloaded from http://pubs.acs.org on May 28, 2019

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Journal of Agricultural and Food Chemistry

Timely Addition of Glutathione for its Interaction with Deoxypentosone to Inhibit the Aqueous Maillard Reaction and Browning of Glycylglycine-Arabinose System

Siyun Lu†, Heping Cui†, Huan Zhan†, Khizar Hayat§, Chengsheng Jia†, Shahzad Hussain§, Muhammad Usman Tahir⊥, Xiaoming Zhang†,*, Chi-Tang HoͰ,* †

State Key Laboratory of Food Science and Technology, School of Food Science and

Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, China § Department

of Food Science and Nutrition, College of Food and Agricultural Sciences, King

Saud University, P. O. Box 2460, Riyadh 11451, Saudi Arabia ⊥

Department of Plant Production, College of Food and Agricultural Sciences, King Saud

University, P. O. Box 2460, Riyadh 11451, Saudi Arabia Ͱ

Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, New

Jersey 08901, USA 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 214122, Jiangsu, People’s Republic of China. E-mail: [email protected] (X. Zhang). Tel: +86 510 85197217 Fax: +86 510 85884496 (2) Chi-Tang Ho, Ph.D., Professor Postal address: Department of Food Science, Rutgers University, 65 Dudley Road, New

ACS Paragon Plus Environment


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Brunswick, New Jersey, 08901, United State. E-mail: [email protected] Siyun Lu, Master. Postal address: School of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi 214122, Jiangsu, People’s Republic of China. E-mail: [email protected] Heping Cui, Ph.D. Postal address: School of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi 214122, Jiangsu, People’s Republic of China. E-mail: [email protected] Huan Zhan, Ph.D. Postal address: School of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi 214122, Jiangsu, People’s Republic of China. E-mail: [email protected] Khizar Hayat, Ph.D., Professor Postal address: Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia. Email: [email protected] Chengsheng Jia, Ph.D., Associate Professor Postal address: School of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, Jiangsu 214122, People’s Republic of China. E-mail: [email protected] Shahzad Hussain, Ph.D., Professor Postal address: Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia. Email: [email protected] Muhammad Usman Tahir, Ph.D. 2


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Postal address: Department of Plant Production, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia. E-mail: [email protected]

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ABSTRACT

2

The inhibitory effects of glutathione (GSH) and oxiglutathione (GSSG) on Maillard

3

browning were compared, and it was clarified that free sulfhydryl was the key substance for

4

the inhibition. The Amadori rearrangement product (ARP) derived from glycylglycine (Gly-

5

Gly) and arabinose (Ara) was prepared by aqueous Maillard reaction and LC-MS/MS was

6

used to investigate the reaction products of GSH and purified ARP. Reaction between GSH

7

and deoxypentosone (DP) was found to alter the pathway of aqueous Maillard reaction, which

8

reduced the production of glyoxal, methylglyoxal and furfural, thereby inhibited the formation

9

of melanoidins. In order to determine the optimal conditions for browning inhibition, a

10

stepwise increase of temperature was used to prepare Maillard reaction products (MRPs).

11

Results showed that the optimum browning inhibitory effect was obtained by adding GSH

12

after Gly-Gly and Ara heating at 80 ℃ for 60 min.

13 14

KEY WORDS: glutathione; inhibit Maillard browning; Amadori rearrangement product;

15

deoxypentosone; reaction between glutathione and deoxypentosone

16 17

Chemical compounds studied in this article

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Glutathione (PubChem CID: 124886); D-arabinose (PubChem CID: 66308); Glycylglycine

19

(PubChem CID: 11163); Oxiglutathione (PubChem CID: 65359)

20

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INTRODUCTION

22

The Maillard reaction (nonenzymatic browning) involves the condensation reaction

23

between reducing sugars and amino acids or peptides, which usually occurs during food

24

production and storage.1 It is commonly divided into three stages. In the early stage, free amino

25

groups react with carbonyl groups to form a reversible Schiff base, which rearranges to stable,

26

covalently bonded Amadori or Heyns rearrangement products (ARP or HRP).2 The second

27

stage involves a series of reactions such as fragmentation, cyclization, Strecker degradation

28

and others. In the ultimate stage, intermediates are further converted to dark-brown colored

29

cross-linked polymers, called melanoidins.1, 3 The Maillard reaction has a great influence on

30

the color, odor, taste, nutritional value and the functional characteristics of food.4 Although

31

this reaction is to some extent desirable, too much browning can result in blackening,

32

formation of off-odors and off-tastes and nutritive loss, therefore, declining in the quality and

33

shelf-life of foods.5 Additionally, Maillard browning is not beneficial for pasteurized or

34

sterilized products such as milk powder and fruit juices.6 Therefore, it is of great interest to

35

search for appropriate antibrowning agents.

36

Glutathione (γ-Glu-Cys-Gly, GSH), a tripeptide of L-glutamate, L-cysteine, and glycine,

37

can be commonly found intracellularly in bacteria, plants, and mammals and serves

38

multifaceted biological functions.7 It participates in transhydrogenation reactions associated

39

with the formation and maintenance of the sulfhydryl groups of other molecules. It provides

40

reduced ability for a variety of reactions and plays a critical part in the detoxification of

41

hydrogen peroxide and free radicals. In addition, as a component of the enzymatic pathway,

42

GSH possesses a good antioxidant potential.8 The biological significance of GSH is mainly 5



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43

related to its free sulfhydryl moiety of the L-cysteine residue, which confers special redox and

44

nucleophilic properties.9 Previous research has proved that GSH could prevent Maillard

45

browning of heated amino acid-reducing sugar mixtures.10 For example, GSH at a low

46

concentration could effectively inhibit the browning of a model system containing glucose-

47

glutamic acid and glucose–lysine.4, 11 In addition to its functional properties, GSH has good

48

flavor characteristics. Dunkel et al.12 demonstrated that GSH and its Maillard reaction products

49

(MRPs) had the mouthfulness-enhancing (kokumi) effect. Kuroda et al.13 confirmed that

50

although GSH had a weak taste in water, it greatly enhanced continuity, odor and taste

51

enrichment when appended to an umami solution. In addition, MRPs prepared from GSH and

52

sugar have strong flavor characteristics, thus it can add mouthfulness-enhancing effect both in

53

water and food.

54

A great deal of researches were carried out to explore the inhibitory mechanism of GSH

55

on enzymatic browning.14,

15

56

competitively inhibiting polyphenol oxidase, thereby forming stable colorless compounds.15

57

However, the mechanism of Maillard browning inhibition is still a challenging problem due

58

to the complexity of Maillard reaction when GSH is involved. Therefore, the objectives of this

59

study were to investigate the inhibitory mechanism of GSH on Maillard browning and

60

determine the optimal application conditions. The preparation of Maillard reaction products

61

using peptides, especially mixed peptides, can produce rich flavor compounds. However, few

62

reports on the preparation of ARP from peptides and sugars are available, thus a simple system

63

of dipeptide and pentose was used for theoretical research in this experiment. Firstly, the effect

64

of sulfhydryl on color inhibition was studied to clarify the role of GSH in the browning

GSH prevented the enzymatic browning of fresh fruits by

6


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65

inhibition. In order to determine in which stage of the Maillard reaction the GSH plays a role,

66

the effect of different addition amounts of GSH on the production amount of several

67

characteristic products in the different stages of Maillard reaction were determined. Then,

68

Aqueous Maillard reaction coupled with vacuum dehydration was used to prepare ARP

69

derived from glycylglycine (Gly-Gly) and arabinose (Ara). LC-MS/MS was applied to

70

investigate the reaction products derived from GSH and ARP, and the degradation rate of ARP

71

was further studied to reveal the mechanism of GSH inhibition on Maillard browning.

72

Furthermore, the Maillard reaction performed under stepwise increase of temperature was

73

used to study the inhibitory effect of GSH added at different times, and the optimal application

74

conditions of GSH were proposed for achieving the best browning inhibition.

75 76

MATERIAL AND METHODS

77

Chemicals. Glutathione, oxiglutathione, D-arabinose, and glycylglycine were purchased from

78

Sigma Chemical Co. Ltd (Shanghai, China). Formic acid, glyoxal, methylglyoxal, furfural,

79

sodium hydroxide, 3,4-hexanedione, methanol, acetonitrile, o-phenylenediamine, 4-(2-

80

hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), diethylenetriamine pentaacetic acid

81

(DTPA) and ammonium hydroxide were obtained from Sinopharm Chemical Reagent Co. Ltd

82

(Shanghai, China). Ultrapure water was acquired by purifying the demineralized water from

83

Milli-Q equipment (Millipore, Bedford, MA). ARP derived from glycylglycine and arabinose

84

was prepared in our lab (the purity was 98.15%).

85

Determination of GSH oxidation and thermal degradation at elevated temperature. GSH

86

solution (2.14 mg/mL) was heated at 110 °C for different times (0, 20, 40, 60, 80, 100, 120 7



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87

min) to determine the concentration changes of GSH and oxiglutathione (GSSG). An HPLC

88

with Ultraviolet (UV) system was used for the separation and analysis of GSH and GSSG. A

89

5 µm SunFireTM 150 × 4.6 mm C18 column (Waters Co., Milford, MA, USA) was used for

90

separation, the injection volume was 10 µL, detection was performed at 210 nm by UV

91

detector and flow rate was adjusted to 1 mL/min.16 A linear gradient from 5 to 45%

92

methanol/water (0.1% formic acid) over 15 min was used for analysis.

93

Preparation of MRPs. MRPs is a general term for products produced by a series of cascade

94

reactions of amino acids (or proteins) and reducing sugars at high temperatures.17 MRPs were

95

prepared according to the report of Liu et al.18 with some modification. A model system (5.2

96

g) was composed of Gly-Gly and Ara (in a molar ratio of 1:2). After they were completely

97

dissolved in deionized water (120 mL), the solution was divided into six parts, then the GSH

98

or GSSG with various concentrations (0, 5, 10, 20, 40 mg/mL) were added separately to the

99

solution. The pH of the solution was adjusted to 7.5 with NaOH (2 mol/L). Subsequently, the

100

solution was transferred to the temperature and pressure resistant bottles. Then the temperature

101

of the solution was raised to 110 °C immediately and held for 120 min in oil bath. After this

102

treatment, the solution was immediately cooled in ice bath to stop the reaction. Furthermore,

103

the stepwise increase of temperature method as reported by Huang et al.19 was used to

104

determine the optimal inhibition conditions. Firstly, the solution of Gly-Gly and Ara was

105

heated at 80 °C for different times (0-100 min), then GSH (10 mg/mL) was added to the

106

solution and the temperature was elevated to 110 °C for 120 min. Other parameters were

107

consistent with the above method.

108

Browning intensity measurement. The browning intensity of MRPs was monitored by their 8


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absorbance values at 420 nm (A420) with a UV-vis spectrophotometer (UV-1800, Shimadzu

110

Co., Shanghai, China). A420 indicated the accumulation of melanoidins in MRPs.20, 21 When

111

necessary, appropriate dilutions were made in order to acquire a suitable absorbance between

112

0.1-1.0.

113

Determination of Maillard reaction Intermediates in MRPs. MRPs were diluted to 25-

114

folds and the absorbance was measured at 294 nm (A294) with a UV-vis spectrophotometer.

115

The absorbance at 294 nm was used for monitoring the content of the colorless Maillard

116

reaction intermediates.22, 23 The control group was without Gly-Gly; the concentrations of the

117

intermediates produced by Maillard reaction of Ara and GSH were determined as well.

118

Determination of α‑dicarbonyl compounds in MRPs. One milliliter of MRPs with the

119

addition of 1 mL of derivatization agent (o-phenylenediamine (2%) was dissolved in the

120

HEPES (1 mol/L, pH 7.0) with 11 mmol/L DTPA) containing 20 µL of internal standard (3,4-

121

hexanedione, 0.96 mg/mL) and the solution was incubated under darkness at room temperature

122

for 2h. Then the reaction solution was filtered through a 0.22 μm membrane filter.24,

123

Afterwards, glyoxal (GO) and methylglyoxal (MGO) benzoquinoxaline derivatives generated

124

in MRPs were detected using HPLC by comparison with known standard benzoquinoxaline

125

derivatives.26 When the inhibitory effect at different times was measured, the relative

126

concentration was used for analysis and deoxypentosone (DP) were identified by the HPLC-

127

MS after derivatization due to the lack of standards. The relative value was calculated by C2/C1,

128

where C2 represented the peak area of α‑dicarbonyl compound derivatives, and C1 represented

129

the peak area of internal standard derivatives. A 5 µm SunFireTM 150 × 4.6 mm C18 column

130

was used for the separation of compounds. Samples were eluted using a step-wise gradient of

25

9



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methanol and water (0.1% formic acid) as follows: 0–10 min, 5% methanol; 10–20 min, a

132

linear gradient of 5% methanol to 30% methanol; and 20– 40 min, 30% methanol to 40%

133

methanol, in the end, using 100% methanol for elution from 40 to 60 min at a flow rate of l

134

mL/min. The column temperature was set at 35 °C and 25 µL of sample was injected into the

135

HPLC system. Spectral data from all peaks were recorded in the range of 200–800 nm, and

136

chromatograms were recorded at 315 nm for all α‑dicarbonyl benzoquinoxaline derivatives

137

detection.

138

Determination of furfural in MRPs. The concentration of furfural in MRPs was directly

139

performed on a HPLC system equipped with an Ultraviolet (UV)-detector and a SunFireTM

140

150 × 4.6 mm C18 column. The injection volume was 10 µL. The mobile phase flow rate was

141

set to 1.0 mL/min by linear gradient from 5% to 45% methanol/water over 15 min and the

142

wavelength of the UV detector was 284 nm. Furfural was quantified by an external standard

143

(furfural > 99.5%) procedure using a calibration curve.

144

Preparation of ARP by aqueous Maillard reaction coupled with vacuum dehydration.

145

The ARP was prepared according to the reported procedure with some modifications.27

146

Specifically, Gly-Gly and Ara mixture (4.3 g) in the molar ratio of 2:1, dissolved in deionized

147

water (50 mL), then pH was adjusted to 7.5 by using NaOH aqueous solution (2 mol/L). The

148

mixed system was heated in water bath for refluxing at 80 °C for 45 min under normal

149

atmosphere, subsequently, a rotary evaporation was used to remove the water and the reaction

150

was performed under vacuum at 80 °C for 15 min. Finally, solid products were dissolved in

151

deionized water of the equal volume as before. The conversion of Gly-Gly and Ara to ARP

152

could be effectively promoted through dehydration.28 The yield of the ARP in this experiment 10


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was 47%.

154

Purification of ARP. A column chromatography was used for the purification of ARP, and

155

the exchange resin of Dowex 50WX4 ion (H+) was chosen as the filler. First, the mixed

156

solution of ARP and unreacted materials was loaded onto the column and eluted with distilled

157

water, then the eluent was collected and detected by HPLC-ELSD, the elution was carried on

158

until there was no unreacted Ara in the eluent. Subsequently, the ARP was eluted with 0.2

159

mol/L ammonium hydroxide. The Xbrige® BEH Amide (4.6 × 150 mm, 5 mm) column was

160

used for further analysis of ARP. Specific analysis conditions were as follows: injection

161

volume was 10 µL and the flow rate was set to 0.8 mL/min by linear gradient from 80% to

162

68% acetonitrile /water (0.1% formic acid) over 14 min.

163

The calibration curve (Y= 1.5634E+06X + 2.2785E+04, R² = 0.996) for HPLC analysis

164

of ARP was measured using the purified product. The Gly-Gly and Ara conversion to ARP

165

(N-(1-deoxy-α-D-arabinos-1-yl)-glycylglycine) was calculated as the percentage of the

166

measured molar concentration of N-(1-deoxy-α-D-arabinos-1-yl)-glycylglycine to the initial

167

molar concentration of Gly-Gly.

168

NMR analysis of ARP. After purification, the pure ARP (40 mg) was dissolved in D2O, then

169

the solution was totally transferred to an NMR tube. Spectra of 1H (400 MHz) and 13C (100

170

MHz) were both performed on a Bruker DRX 400 MHz spectrometer (Bruker Bio Spin,

171

Germany) equipped with a 5 mm PABBO probe and operated at 25 °C (298 K). Mestrenova

172

software (version 9.0.1, Mestrelab Research, Escondido, CA, USA) was used for analyzing

173

the result.

174

Determination of the cumulative concentration of ARP in MRPs. During the Maillard 11



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reaction, ARP was continuously generated while continuing to undergo degradation. The

176

concentration of ARP at a certain time was the cumulative amount at that moment. The

177

concentration of ARP during the Maillard reaction at 30, 60, 120 min was determined by

178

HPLC-ELSD. Standard curve of ARP was made according to the purified ARP.

179

LC-MS/MS analysis of the reaction products produced by GSH and ARP. GSH and pure

180

ARP was mixed in a molar ratio of 1:1 and dissolved in distilled water, then the solution was

181

heated at 110 °C for 10 min. The UPLC-ESI-MS/MS spectrum was obtained by mass

182

spectrometry (Waters Synapt MALDI Q-TOF MS, USA) using a Waters Acquity PDA

183

detector in positive ESI mode. To facilitate the protonation of the sample, water containing

184

0.1% formic acid was used as the mobile phase. A linear gradient from 5 to 90%

185

methanol/water (0.1% formic acid) over 17 min and BHE C18 (1.7 µm, 2.1 × 100 mm) column

186

were used for ultra-performance liquid chromatography (UPLC) analysis. The injection

187

volume was 1 µL, flow rate was 0.3 mL/min and column temperature was 45 °C. The

188

ionization conditions were as followed: cone voltage was 20 V, capillary voltage was 3.5 kV,

189

collision voltage was 6 V and detector voltage was 1.8 kV. Temperatures of source block and

190

desolvation were 100 °C and 400 °C. Sample scans were recorded in a range of m/z 50-1000.

191

The scanning time was 1 s and the delay between scans was 0.1 s. The desolvation and cone

192

gas flow were 700 and 50 L/h, respectively. Mass Lynx software (version 4.1, Waters, Milford,

193

MA, USA) (version 4.1, Waters, Milford, MA, USA) was used to analyze the data.

194

Statistical analysis. All measurements were prepared and analyzed three times. SPSS version

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19.0 (IBM, Armonk, NY) was used for all statistical analyses. The results were presented as

196

mean values ± standard deviations, p < 0.05 was considered significant. 12


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RESULTS AND DISCUSSION

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GSH oxidation and thermal degradation. At room temperature, GSH might be unstable and

200

easily oxidized to form a disulfide bond and giving rise to a compound called GSSG, and

201

oxidation rate increased at high temperature.29 With increasing the heating time, a decrease in

202

the concentration of GSH and an increase in GSSG was observed (Figure 1). After 2 h at

203

110 °C, the total decrease in GSH was 26.58 % out of which 18.98% of GSH was oxidized to

204

GSSG, and the remaining 7.6% of GSH might have undergone thermal degradation. These

205

results indicated that after 2 h of high temperature treatment, only a small portion of GSH was

206

oxidized and thermally degraded, and most of the rest in the system was still GSH, which

207

indicated that the oxidation and degradation of GSH would not be so severe during the

208

following experiments.

209

Effects of GSH and GSSG on browning intensity of MRPs. MRPs were ordinarily prepared

210

at temperatures above 100 °C.26, 27, 30 Therefore, the Maillard reaction was conducted at 110 °C

211

to generate browning pigments. Browning occurring during the Maillard reaction, typically

212

increases absorbance values of the products at 420 nm (A420).31 With adding GSH or GSSH,

213

the A420 of the Gly-Gly and Ara system is shown in Figure 2, which indicates that A420 values

214

of MRPs were significantly affected by the addition amount of GSH or GSSG (p < 0.05). A420

215

of the system decreased significantly with the increased addition of GSH, however, with the

216

increased addition of GSSG, A420 decreased first and then showed a slow upward trend.

217

Overall, as long as GSH or GSSG was added, the A420 value of MRPs was lower than that of

218

control group derived from Gly-Gly and Ara with no GSSG or GHS added. Thereby, the 13



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addition of GSH or GSSG changed the reaction process, both of them would compete with

220

Gly-Gly and react with Ara. When low concentration of GSH or GSSG (5 mg/mL) was added

221

to the system, though part of the amino groups of GSH or GSSG may react with Ara, the

222

browning of reaction products of GSH or GSSG and Ara were lower than that of Gly-Gly and

223

Ara, so the whole color was lower. When the addition of GSSG increased (10, 20, 40 mg/mL),

224

the concentration of free amino groups in the system increased, so it is reasonable that the

225

reaction browning gradually increased in the system when GSSG involved in. However, the

226

corresponding increase in GSH led to the decline of browning. Therefore, compared the

227

effectiveness of GSH and GSSG in inhibiting browning and combined the structural

228

differences between them, it could infer that free sulfhydryl had a much higher inhibitory

229

effect on Maillard browning when compared to the free amino group (Figure 2). These results

230

are in good agreement with previous research of Friedman et al,10 who reported that amino

231

acids containing free sulfhydryl such as GSH, L-cysteine, and N-acetyl-L-cysteine were as

232

effective as sodium bisulfite to prevent Maillard browning of heated glucose and amino acid

233

systems.

234

Effect of GSH on the formation of characteristic products at different stages of Maillard

235

reaction. Through the above experiments, GSH has been verified to have inhibitory effect on

236

browning. An interaction between GSH and the browning precursors formed at different stages

237

was speculated to account for browning inhibition of GSH. To further determine when it works

238

during the Maillard reaction, different amounts of GSH were added to the Gly-Gly and Ara

239

system. Then several characteristic products at different stages of Maillard reaction in MRPs

240

were determined. First, as one of the important products during the early period of the Maillard 14


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241

reaction, APR was monitored in presence of GSH. Figure 3a shows that the accumulation of

242

ARP during the reaction at 110 °C was very low, when compared with Gly-Gly, its highest

243

yield was only 14.22%. When heated at high temperature, the ARP rapidly undergoes

244

dehydration and degradation reactions, and as the Maillard reaction proceeded, ketones and

245

heterocyclic compounds are produced. These products could be dehydrated and polymerized

246

to form melanoidins.27 This theory might account for the low accumulation of ARP at 110 °C.

247

When GSH was added to the system, the concentration of ARP increased after two hours.

248

With the increasing addition of GSH, the accumulation of ARP showed an upward trend. In

249

addition, comparing the three points of different reaction time (30, 60, 120 min) with the same

250

GSH dosage, there is no significant difference between 30min and 60min (P > 0.05) , however

251

from 60 to 120 min, the accumulation of ARP increased significantly (Figure 3a), which

252

indicated that the production of ARP was relatively higher than that of degradation. In

253

summary, comprehensive horizontal comparison and vertical comparison, the accumulation

254

of ARP increased from 60 min to 120 min at 110 °C, and the addition of GSH could increase

255

the accumulation of ARP. These results revealed that the participation of GSH affected the

256

Maillard reaction pathway of Gly-Gly and Ara. In order to clarify whether the addition of GSH

257

increased the formation of ARP or decreased the degradation of ARP, the following

258

experiments were carried out to determine the amount of intermediate compounds in MRPs

259

when GSH was involved in.

260

The products formed during intermediate stages of the Maillard reaction were determined

261

by measuring the ultraviolet absorption at 294 nm as described by Ajandouz.32 Figure 3b

262

shows that when GSH addition varied from 0 mg/mL to 10 mg/mL, A294 showed a downward 15



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263

trend. When the amount of GSH added was between 10 mg/mL and 20 mg/mL, A294

264

maintained at almost the same level. Moreover, as the addition of GSH continued to increase,

265

A294 increased rapidly. However, in the control group, Ara was allowed to react with different

266

amounts of GSH without Gly-Gly. In the case of excess ratio of sugar to GSH, the yield of the

267

colorless intermediate produced was very low. With increasing the amount of GSH, the A294

268

almost exhibited a linear upward trend. The increase in absorbance at 294 nm meant an

269

increased formation of colorless compounds, which could be the intermediate compounds of

270

the Maillard reaction.33 From the results, it could be inferred that in the Ara and Gly- Gly

271

system, a small amount of GSH would participate in the reaction, change the route of Maillard

272

reaction and effectively inhibit the formation of colorless intermediates. However, when high

273

concentration of GSH (>20 mg/mL) was added, excessive GSH would directly react with Ara,

274

the total colorless intermediate compounds in this system included the products of two

275

Maillard reaction systems (Ara-GSH or Ara and Gly-Gly), so resulting a significant increase

276

in A294.

277

GO and MGO, as important intermediates, it was of great significance to determine the

278

changes of them with the different addition of GSH. Quantitative analysis of α-dicarbonyl

279

compounds were carried out by HPLC after derivatization to quinoxalines. The concentrations

280

of GO and MGO changed with the added amount of GSH. According to Figure 3c, unlike the

281

production of ARP (Figure 3a), more the amount of GSH, less the concentrations of GO and

282

MGO in MRPs. Fiedler et al.34 investigated the relevance of several α-dicarbonyl compounds

283

with the melanoidin formation and their molecular size distribution, and clarified that short

284

chain α-dicarbonyl compounds were the direct precursors for carbohydrate-based melanoidins. 16


Page 17 of 37


285

They also pointed out that MGO showed the highest browning activity. Therefore, as key

286

compounds of the Maillard browning, the decrease of GO and MGO can explain the reduction

287

of browning value of the system. Based on the results (Figure 3c), there might be two possible

288

reasons for the reduced amounts of GO and MGO, one was the reduced formation of short

289

chain α‑dicarbonyl compounds, and the other could be the subsequent reaction of the short

290

chain α‑dicarbonyl compounds to reduce the amount of accumulation. However, if the second

291

case occurred, the content of melanoidins produced should increase and lead to a deeper

292

browning of the system. Combined with the previous results of A42O, the addition of GSH

293

reduced the browning degree of the system. Therefore, the first reason is more likely. In other

294

words, after the addition of GSH, the content of the α‑dicarbonyl compounds generated during

295

the Maillard reaction were reduced, and thus the degree of Maillard browning was

296

correspondingly lowered. When the dosage of GSH reached 40 mg/mL, although the

297

intermediate compounds generated increased, the content of GO and MGO produced by the

298

subsequent Maillard reaction were still reduced. It was not difficult to speculate that GSH had

299

a barrier effect, preventing the intermediate compounds to form short-chain α‑dicarbonyl

300

compounds.

301

Furfural as one of the important products of Maillard reaction, was similar to GO and

302

MGO, could react with amine compounds, then went further polymerization to form

303

melanoidins.35 Sugar was decomposed into furfural through two possible pathways:

304

caramelization and Maillard reaction.36 The caramelization reaction required a higher

305

temperature (> 120 ℃),37 so furfural was mainly produced by Maillard reaction under our

306

experimental conditions. In the Maillard reaction, furfural was generated primarily via 317



Page 18 of 37

307

deoxypentosone (3-DP) dehydration, followed by the release of intact amino acids.

308

As shown in the Figure 3d, the yield of furfural decreased significantly with the increasing

309

dosage of GSH. The content of furfural was closer to zero when the amount of GSH added

310

was 40 mg/mL. From these results, it can be concluded that GSH had a certain inhibitory effect

311

on the formation of furfural. Burton et al.38 reported that most of the rapid browning with

312

active nitrogen functional groups occurred on linear unsaturated aldehydes. When the

313

conjugated unsaturation was in the ring structure, such as furfural, the development of the

314

chromophore was slower. Although the browning potential of furfural was relatively low, the

315

reduction in its formation could also reduce the Maillard browning to some extent. Figures 3c

316

and Figure 3d reflected the same trends, which indicated that GSH changed the pathway of

317

Maillard reaction before GO, MGO and furfural were formed.

318

Analysis of the reaction products of GSH and ARP by LC-MS/MS. Based on the above

319

results (Figure 3) and Maillard reaction pathway,3 it was easy to infer that added GSH did not

320

play a role in the final stage of Maillard reaction, but in the middle stage of the reaction. This

321

result was similar to the cysteine inhibitory effect previously reported by Huang et al,19 who

322

reported that GO and MGO derived from DP reacted quickly and nonreversible with cysteine

323

to form browning pigments and supposed that addition of cysteine would be effective for

324

inhibiting browning only before the short chain α-dicarbonyl compounds are formed. In order

325

to further study the interaction between GSH and intermediates, ARP was prepared. According

326

to the method of Cui et al,28 ARP was prepared by thermal reaction coupled with vacuum

327

dehydration, the exchange resin was then used to obtain purified ARP. The structure of ARP

328

was identified by NMR. ARP can be easily reconstituted in aqueous medium, resulting in the 18


Page 19 of 37


329

formation of cyclic form due to tautomerism of ARP,27 so it was confirmed that the purified

330

product contained N-(1-deoxy-α-D-arabinos-1-yl)-glycylglycine and its cyclic isomer

331

(Supporting Information). These isomers coexist in aqueous media. Owing to the similar

332

structural and chemical properties, they could not be separated by the separation method

333

employed in this experiment. Specific NMR information of the ARP is presented in the

334

Supporting Information. In order to clarify the mechanism of GSH inhibiting Maillard

335

browning, the reaction products of GSH and ARP were analyzed by UPLC-MS/MS. In the

336

mass spectrometry result, no adduct compound of GSH and ARP was detected, however, the

337

adduct compound of GSH and DPs were detected. This is consistent with previous theory. In

338

the presence of sulfhydryl groups, aldehydes could competitively interact with sulfhydryl

339

groups to form thioacetals or thioketals, thereby inhibiting Maillard browning.10 In different

340

substances and different environments, the reactivity of aldehyde or ketone groups with -SH

341

or NH2 groups was different, moreover, this relative activity would determine the inhibitory

342

degree of Maillard browning.10 The mixed solution of GSH and ARP was acidic. Under acidic

343

conditions, the production of 3- DP was higher than 1-deoxypentosone (1-DP).2 Due to the

344

similarity of the structure, the fragments of the adduct compound formed by 3-DP or 1-DP

345

and GSH should be substantially the same. Therefore, take 3-DP as an example in the

346

following analysis of mass spectrometry. It is well known that the exact mass of GSH is 307.08,

347

while 3-DP has exact mass of 132.04. It could be found from the mass spectrum that m/z

348

440.14 was the parent ion of GSH and 3-DP adduct. The reactivity of the aldehyde group in

349

3-DP was higher than that of the ketone group. Accordingly, the addition reaction with the

350

aldehyde group was first considered. It was observed that adduct (parent ion with m/z = 440.14) 19



Page 20 of 37

351

continuously lost two molecules of water, which corresponded to the positive single charged

352

fragments with m/z 422.11 and 404.12 (Figure 4a). The mass spectra of the ion [M + H - H2O]

353

was the most abundant. The adduct product could break again and reform the positively

354

charged GSH of m/z 308.09. In addition, peptide bonds were prone to cleavage, forming a

355

variety of different ions. The detailed fragmentation is shown in Figure 4b based on the

356

structural feature of adduct and the mass spectrometry information.

357

DP, an intermediate formed in the mid-term of the Maillard reaction, has been identified

358

as a key precursor for the formation of melanoidins. It had a very high reactivity and was easy

359

to carry out subsequent reactions under high temperature. The formation of melanoidins were

360

parallel to the increase of DP.39 The activity of aldehyde group in DP was high and

361

preferentially reacted with sulfhydryl groups in GSH (Figure 4). This sulfur-harboring product

362

was relatively stable, and thus it could not be easily converted back to active unsaturated

363

α‑dicarbonyl compounds contributing to the Maillard browning. Under the circumstances of

364

high concentration of GSH, even if part of GSH would react with Ara, and the formation of

365

colorless intermediate compounds in the mixed system increase significantly (Figure 3b), but

366

once DP was formed, it would immediately undergo an addition reaction with excessive GSH.

367

In other words, when excessive GSH was added to the system, the degradation and

368

dehydration of DP were inhibited, so the production of glyoxal, methylglyoxal, and furfural

369

were all reduced (Figure 3c and 3d).

370

Analysis of the degradation of ARP. The degradation of the ARP will first produce 1-DP

371

and 3-DP.40 During the generation of DP, the amino acids are regenerated simultaneously.41

372

As shown in Figure 5, ARP degraded rapidly at high temperature, and the degradation rates of 20


Page 21 of 37


373

ARP with or without GSH addition at 110 °C were significantly affected by the reaction time

374

(p < 0.05). After 10 minutes of the reaction, the concentration of ARP in the system containing

375

GSH decreased slightly slower than that in the control group. Except for 0 min and 10 min,

376

the degradation rate of ARP had significant difference in the presence or absence of GSH at

377

the same time (p < 0.05). Combined with all the above-mentioned results, the reaction between

378

GSH and DP changed the pathway of the Maillard reaction and blocked the cascade reaction,

379

resulting in a slower degradation rate of ARP. The result can also explain the increased ARP

380

accumulation in the system with increasing GSH after being heated at 110 ℃. On the one hand,

381

GSH could react with DP, inhibiting the production of glyoxal, methylglyoxal and furfural,

382

resulting in reduced formation of melanoidins. On the other hand, the degradation of ARP and

383

the process of Maillard reaction slowed down together; thus, these two aspects together

384

achieved the effect of inhibiting Maillard browning.

385

Determining the critical conditions for achieving the optimal browning inhibition of

386

GSH. Based on the reaction between GSH and one of the intermediates (DP), the addition

387

time of GSH was critical to inhibit the browning formation during reaction. Under high

388

temperature, the reaction rate is too fast and it is difficult to capture the best time, so stepwise

389

increase of temperature was used to determine the optimal inhibition conditions. Results

390

(Figure 6a) showed that A420 exhibited firstly a decreasing and then increasing trend. When

391

GSH was added at 60 min, the final A420 of MRPs was the lowest. These results are similar to

392

some previous researches.19, 27 At the same time, the trend of A294 and α‑dicarbonyl compounds

393

in MRPs were consistent with A420 (Figure 6a, 6b). These results revealed that GSH added at

394

this time would slow down the process of Maillard reaction and finally reduce the production 21



Page 22 of 37

395

of melanoidins. Figure 6c shows that an increase in reaction time leads to a higher formation

396

of DP. In the early stage of low temperature reaction, DP has not been produced in large

397

quantities. When GSH was added at this time, it was highly probable that most of the GSH

398

first reacted with Ara, and the Maillard reaction between GSH and Ara was the main reaction.

399

When ARP degraded to form DP, the content of free sulfhydryl in the system was relatively

400

low, and major DP continued to proceed to the subsequent stage of Maillard reaction, therefore,

401

the inhibitory effect was relatively weak. Figure 6d shows that the content of GO and MGO

402

increased greatly when the reaction was carried out for 80 min. Although DP in the system

403

has been generated in large quantities at this time, the partial DP had undergone an irreversible

404

degradation reaction. When GSH was added at this time, the downward reaction of DP that

405

occurred could not be inhibited, so GSH could not prevent these products from further reacting

406

to form chromogenic substances, hence, the color of the system was still deepening. In

407

summary, when Gly-Gly and Ara reacted at 80 °C for 60 min, the derived DP had been formed

408

and accumulated to a large amount (Figure 6c), whereas, the GO and MGO generated in the

409

system were still low (Figure 6d), adding GSH at this time would most effectively inhibit

410

Maillard browning.

411 412

ABBREVIATIONS

413

GSH, glutathione; GSSG, oxiglutathione; MRPs, Maillard reaction products; Gly-Gly,

414

glycylglycine; Ara, arabinose; ARP, Amadori rearrangement product; DP, deoxypentosone;

415

3-DP, 3-deoxypentosone; 1-DP, 1-deoxypentosone; GO, glyoxal; MGO, methylglyoxal

416 22


Page 23 of 37


417

FUNDING SOURCES

418

This research was supported by the National Natural Science Foundation of China (31671826),

419

National Key R&D Program of China (2017YFD0400105), and the National first-class

420

discipline program of Food Science and Technology (JUFSTR20180204). This research was

421

also supported by Deanship of Scientific Research at King Saud University through research

422

grant No (RG-1440-020).

423 424

SUPPORTING INFORMATION DESCRIPTION

425

Statement on the manuscript’s significance

426 427

Conflict of Interest

428

The authors declare no conflict of interest.

429

23



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430

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Zhang, X. Controlled formation of flavor compounds by preparation and application of

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541

29



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542

FIGURE CAPTIONS

543

Figure 1. The oxidation of GSH to GSSG and the degradation of GSH at 110 °C for 120 min.

544

Figure 2. Effect of different additions of GSH and GSSG on the browning (A420) of final

545

Maillard reaction products at 110 °C for 120 min.

546

Figure 3. Effect of different addition amount of GSH on the formation of characteristic

547

products at different stages of Maillard reaction. (a) ARP ， (b) colorless intermediate

548

compounds (A294), (c) α‑dicarbonyl compounds, (d) furfural.

549

(a: reacted at 110 °C for 30, 60, 120 min, respectively; b, c, d: reacted at 110 °C for 120 min)

550

Figure 4. LC-MS/MS spectrum (a), fragmentation of GSH and 3-deoxypentosone adducts in

551

LC-MS/MS spectrum (b).

552

Figure 5. Comparison of degradation of ARP with and without GSH addition at 110 °C.

553

Figure 6. Inhibition of Maillard reaction by adding GSH after different reaction time (0–

554

100min) at first mild reaction step.

555

(a, b: first mild reaction step: Gly-Gly and Ara were reacted at 80 °C for different times;

556

elevated temperature reaction step: GSH was added into system and reacted at 110 °C for 120

557

min; c, d: first mild reaction step: Gly-Gly and Ara were reacted at 80 °C for different times)

558

30


Page 31 of 37


FIGURES

Figure 1

31



Page 32 of 37

Figure 2

32


Page 33 of 37


Figure 3

33



Page 34 of 37

Figure 4 (a)

(b)

34


Page 35 of 37


Figure 5

35



Page 36 of 37

Figure 6

36


Page 37 of 37


TOC graphic

37


Timely Addition of Glutathione for Its Interaction with Deoxypentosone

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