Study of the Inhibitors of Cooked Off-Flavor Components in Heat

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Chemistry and Biology of Aroma and Taste

Study of the Inhibitors of Cooked Off-Flavor Components in Heat-Treated XiZhou Melon Juice DongSheng Luo, Xinxing Xu, Shuang Bi, Yuping Liu, and JiHong Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03398 • Publication Date (Web): 23 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019

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Journal of Agricultural and Food Chemistry

Study of the Inhibitors of Cooked Off-Flavor Components in Heat-Treated XiZhou Melon Juice Dongsheng Luo1, Xinxing Xu1, Shuang Bi1, Yuping Liu2, Jihong Wu1 1. College of Food Science and Nutritional Engineering, China Agricultural University; Key Laboratory of Fruit and Vegetable Processing, Ministry of Agriculture; National Engineering Research Center for Fruit and Vegetable Processing, Beijing 100083, China 2. Beijing Key Laboratory of Flavor Chemistry, Beijing Technology and Business University, Beijing 100048, China

1

Corresponding author. Tel/fax: +86-010-62737434-603. E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract: This research applied inhibitors to reduce the content of cooked off-flavor

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components (dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, and

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3-(methylthio)propanaldehyde) in heat-treated melon juice. The effects of glucose

4

oxidase (GOD) on the formation and release of these four volatile sulfur compounds

5

were also investigated. Results showed that GOD strongly inhibited the formation of

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the four compounds. In GOD-treated melon juice, S-methylmethionine was strongly

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protonated and not easily degraded into dimethyl sulfide. Moreover, the release of the

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dimethyl sulfide that did form was restrained by the hydrophobic interactions of

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gluconic acid and oxidation by hydrogen peroxide. In addition, gluconic acid (or

10

glucose) and hydrogen peroxide could form a stable complex with methionine in an

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acidic

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3-(methylthio)propanaldehyde, dimethyl disulfide, and dimethyl trisulfide by the

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Maillard reaction during heat processing.

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Key words: melon juice; cooked off flavor; inhibitor; glucose oxidase; mechanisms

matrix

and

thus

prevented

the

methionine


from

producing

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Introduction

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Melon juice, a common deep-processed product, is convenient to transport and store,

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helping to avoid postharvest loss of melon fruits.1 Volatile sulfur compounds (Figure

18

1), such as dimethyl sulfide (1), dimethyl disulfide (2), dimethyl trisulfide (3), and

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3-(methylthio)propionaldehyde (4), are major contributors to off flavors in

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heat-treated melon juice.2-4 The production of these compounds seriously degrades the

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flavor quality of melon juice. However, some heat treatments, like heat sterilization,

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which can kill and inactivate microorganisms and enzymes in juice, are very

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important for the safety and storage of melon juice. Therefore, controlling the

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development of the volatile sulfur compounds in heat-treated melon juice is necessary

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for the deep processing of melon fruits.

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S-methylmethionine (SMM) and methionine (Met) are the two main flavor precursors

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of the four volatile sulfur compounds that form in melon juice.4 However, these

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precursors are almost impossible to eliminate completely by common separation

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techniques (such as ultrafiltration), while guaranteeing the good quality of the melon

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juice. Moreover, they are micronutrient elements in juices and can't be removed easily.

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Thus, use of inhibitors is a practical method to improve the flavor quality of the

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heat-treated melon juice. Lermusieau, (2015) reported that the content of compound 1

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content in boiling wort could be reduced with an acid additives.5 Phenol compounds,

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especially, polyphenols can trap reactive dicarbonyls to control the development of

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off-flavor components through the Maillard reaction in ultrahigh heat-treated milk.6

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Cysteine, pyridoxine, and thiamine can scavenge free radicals (or reactive dicarbonyls)



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and compete to react with the amino acids commonly used as Maillard reaction

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inhibitors in the food industry.7 In addition, phenolic acid, ascorbic acid, and glucose

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oxidase (GOD) can restrain the Maillard reaction and heat degradation of SMM by

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reducing free radicals (or reactive dicarbonyls), and decreasing the dissolved oxygen,

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glucose content, and the pH values of the food matrix.8-10 Nevertheless, few reports on

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improving the flavor quality of juice using these inhibitors are currently available. The

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functional mechanisms of some inhibitors on the formation of compounds 1, 2, 3, and

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4 in juices during heat processing are unknown.

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Thus, this study aimed to (1) screen for the optimal inhibitor that can simultaneously

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control the heat degradation of SMM and Met in melon juice; (2) analyze the effect of

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the main changed components in melon juices on the release of compound 1; and (3)

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explore the molecular mechanisms underlying the inhibition of the formation of

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compounds 1, 2, 3, and 4 during heat processing.

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Materials and Methods

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Chemicals

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N-alkanes (C5-C30), L-methionine (Met, CAS: 59-51-8), S-methylmethionine (SMM,

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CAS: 4727-40-6), dimethyl sulfide (1, CAS: 75-18-3), dimethyl disulfide (2, CAS:

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624-92-0), 3-(methylthio)propanaldehyde (4, CAS: 3268-49-3), dimethyl trisulfide (3,

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CAS: 3658-80-8), L-homoserine (HS, CAS: 672-15-1), homoserine lactone

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hydrochloride (HSL, CAS: 2185-03-7), dimethyl sulfoxide (DMSO, CAS: 67-68-5),

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gluconic acid (CAS: 526-95-4), and glucose (CAS: 50-99-7) were purchased from

58

Sigma–Aldrich Co., Ltd. (Milwaukee, WI, USA) with purity>98%. Ammonium


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hydroxide solution (purity>25%), formic acid (purity>99%), and acetonitrile

60

(purity>99%) were obtained from Merck & Co., Inc., (Kenilworth, NJ, USA).

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Glucose oxidase (GOD, 250 U/mg), hydrogen peroxide (w/w, 35%), sodium

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dihydrogen phosphate, and disodium hydrogen phosphate were purchased from

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Beijing Solarbio Science & Technology Co., Ltd (Beijing, China).

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Melon samples

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XiZhou melon (C. melo var. Reticulates, 50 kg) was purchased directly from Xinjiang

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Uigur Autonomous Region of China in June 2018. Reducing sugar content was about

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7.0% (w/w) and pH value was at 5.2–5.6.

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Sampling melon juice and model solutions

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Melon fruits were maintained in an ice bath for 12 h (to reduce the deterioration of

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their quality during processing) and then squeezed. The juice was centrifuged at

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11000 ×g for 10 min at 4 °C. Clear melon juice was prepared from the supernatant by

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using an ultrafiltration unit (pore diameter 50 nm).4

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The blank solution was an aqueous solution with a pH of 5.2 (adjusted by phosphates).

74

The glucose and gluconic acid solutions were 7 g glucose/100g blank solution and

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2.33 g gluconic acid/100g blank solution, respectively. The mixture of glucose and

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gluconic acid solution was 4.67 g glucose and 2.33 g gluconic acid/100g blank

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solution. The model solutions of compound 1 were 9.0 mg 1/L blank solution, glucose

78

solution, gluconic acid solution, or the mixture solution. The SMM and Met model

79

solutions were 7 mg SMM/L glucose solution and 70 mg Met/L glucose solution,

80

respectively.



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Inhibitor and heat treatment

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Clear melon juice (10 mL) and inhibitors (5 mg/5 U, ferulic acid, chlorogenic acid,

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rosmarinic acid, protocatechuic acid, gallic acid, epicatechin, epicatechin gallate, rutin,

84

quercetin, resveratrol, cysteine, thiamine, ascorbic acid, pyridoxamine, and GOD)

85

were added to a flask. The flask was sealed and shaken at 200 rpm for 160 min (30

86

°C).

87

a UHT/HTST processing system in accordance with the method reported by Luo et al.

88

(2018).4 Met or SMM model solutions were treated by GOD and heat treatment

89

following the method previously described.

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Determination of inhibition ratios

91

Sample (6 mL) and sodium chloride (3 g) were transferred into screw-cap headspace

92

vials (22.8 mL, Chromoptic, France). The detection of compounds 1, 2, 3, and 4 in

93

samples through headspace solid-phase microextraction-gas chromatography-mass

94

spectrometry (HS-SPME-GC-MS) was performed as described by Luo et al. (2018).4

95

A 50/30 m polydimethylsiloxane/divinylbenzene/carboxen coated SPME fiber (2 cm)

96

was exposed to vial headspace for 30 min at 40 °C with agitation at 100 rpm after 10

97

min of equilibration. Then, the SPME fiber was inserted into a GC-MS injection port

98

(7890B/5975C, Agilent Technologies, Inc., Santa Clara, CA, USA) at 250 °C for 5

99

min. Volatile components were separated on DB-5MS capillary columns (30 m × 0.25

Then, the samples were subjected to heat treatment (130 °C, holding for 3 s) using

100

mm i.d. × 0.25 m; J&W Scientific, Folsom, CA, USA).

101

The temperature program was initially held for 2 min at 35 °C, then increased to 150

102

°C

at the rate of 4 °C /min, increased to 250 °C at the rate of 10 °C /min, and held for 5


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103

min. MS was performed in electronic impact mode (70 eV). The ion source

104

temperature was 250 °C with selected ion monitoring mode (SIM).

105

The compounds 1, 2, 3, and 4 were positively identified in reference to the National

106

Institute of Standards and Technology mass spectrometer library (match quality

107

>80%), retention index (504, 736, 906, and 968), and standard substances.

108

The change of contents of compounds 1, 2, 3, or 4 in samples was evaluated using the

109

precise peak areas of each compound’s characteristic ion acquired by SIM, given the

110

identical detector response factor of each sulfur compound. The characteristic ions of

111

compounds 1, 2, 3, and 4 are 62, 94, 126, and 104, respectively.4

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The inhibition ratios for compounds 1, 2, 3, and 4 in heat-treated melon juice were

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calculated as follows:

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Inhibition ratio (%) 1

Ats 100% As

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where Ats and As are the peak areas of the characteristic ions of 1, 2, 3, or 4 in treated

116

and untreated melon juice, respectively, after heat treatment.

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Analysis of SMM/Met and degradation products

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The SMM or Met model solutions (1 mL) after treatment were purified using a

119

Cleanert-PEP SPE column (60 mg/3 mL, Phenomenex and Agela Technologies, CA,

120

USA) after centrifugation (11000 ×g, 10 min).11 The eluent from SPE column was

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filtered with disposable sample filters (0.22 m) and collected for analysis with an

122

ultra performance liquid chromatography system coupled to a triple quadrupole mass

123

spectrometer (UPLC-MS/MS) (Waters ACQUITYI-CLASS, Waters Co., Milford,

124

MA, USA). Separation was performed using the Acquity UPLC BEH C18 column



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125

and Acquity UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 m particle size).

126

The solvent system for the C18 column consisted of 0.1% aqueous formic acid (A)

127

and acetonitrile (B) with gradient elution at a flow rate of 0.4 mL/min. The system

128

was used as follows: 0– /R2

129

3.0–1 R2

130

was used in positive mode through full scanning mode and multiple reaction

131

monitoring mode. The ion source capillary voltage and cone voltage were 3.5 kV and

132

35 V, respectively. The optimized selected MS/MS transition pairs of the precursor

133

and product ions were as follows: Met 150>133 and 150>104 (collision voltage 9 and

134

10 V), SMM 164>102 (collision voltage 12 V), homoserine 120>74 (HL, collision

135

voltage 11 V), and homoserine lactone 102>74 (HSL, collision voltage 10 V).

136

The solvent system for the amide column consisted of 0.1% aqueous ammonia (A)

137

and acetonitrile (B) with gradient elution at a flow rate of 0.2 mL/min and was used as

138

follows: 0– /R2

139

5.1–0 /R2

140

by full scanning mode (50–1000 m/z) and daughter mode (collision voltage 12 V).

141

The other MS parameters were the same as those described by Luo et al. (2018).4

142

Compounds were identified and quantified through comparison with the retention

143

times, MS spectra, and MS/MS fragmentation patterns of standard substances. The

144

contents of SMM, HS, and HSL in the model solutions were expressed in terms of the

145

peak areas of their ion pairs.

146

Measurement of hydrogen peroxide (H2O2) in GOD-treated melon juice

1% B; 1.0– BR2

95%–1% B; and 3.1–0 /R2

1%–95% B; 1.5–1 /R2

95% B;

1% B. The electron spray ionization source

90%–60% B; 2.0–B /R2

60% B; 5.0–B R2

60%–90% B; and

90% B. The electron spray ionization source was used in negative mode


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H2O2 in GOD-treated melon juice was detected according to the method described by

148

Zhao et al., (2019) with minor modification.12 GOD-treated melon juice (processing

149

time at 20, 40, 60 80, 100, 120, 140, or 160 min, 6 g) was blended with precooled

150

acetone (6 mL). The mixture was shaken for 3 min and then centrifuged at 11000 ×g

151

at 4 °C for 5 min. Then, the supernatant (1 mL) was mixed with 20 mmol/L titanium

152

sulfate (0.1 mL) and ammonia (0.2 mL). The mixture was centrifuged at 11000 ×g at

153

4 °C for 5 min. After removal of the supernatant, the residue was dissolved in 1 mol/L

154

sulfuric acid (3 mL). The mixture was shaken for 3 min and then centrifuged at 11000

155

×g at 4 °C for 5 min. The absorbance of the supernatant was measured at 415 nm. For

156

the calibration curve, a series of H2O2 standard solutions was prepared in ultrapure

157

water at concentrations of 2.03, 4.11, 8.23, 16.15, 32.06, and 64.27 mmol/L

158

(y=0.61x+0.052, r2=0.9859).

159

Detection of dimethyl sulfoxide (DMSO) in model solution

160

H2O2 solution (100 L) and compound 1 glucose model solution (100 mL) were

161

transferred into a 150 mL screw cap glass vial. The sealed vial was shaken at 200 rpm

162

for 128 min. Then, the mixture (processing time at 1, 2, 4, 8, 16, 32, 64, or 128 min)

163

was centrifuged at 11000 ×g for 5 min. The supernatant was filtered with disposable

164

sample filters (0.22

165

chromatography equipped with a diode array detector (DAD) (HPLC, Agilent 1260,

166

Agilent Technologies, Inc., Santa Clara, CA, USA).13 Samples were separated with

167

the Venusil MP C18 column (4.6 mm × 250 mm). The solvent system consisted of

168

aqueous and acetonitrile (6:94). The flow rate was 1.0 mL/min, and column

m). DMSO was analyzed by high-performance liquid



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169

temperature was 35 °C. For the calibration curve, a series of standard solutions of

170

DMSO were prepared in ultrapure water at concentrations of 3.125, 12.5, 50, 200, and

171

800 mg/L (y=0.26x-0.038, r2=0.9937).

172

Determination of partition coefficients of dimethyl sulfide (1)

173

Different amounts of compound 1 model solution (0.5, 1, 2, 3, 4, or 5 mL) were

174

transferred into a 22.8 mL screw cap headspace vial with phase ratios of 45.6 to 4.56

175

(according to the model solution volumes). The samples were analyzed by

176

HS-GC/MS.14 The headspace vial was equilibrated for 120 min at 35 °C. After

177

shaking, a 500 L sample of the headspace was withdrawn with a 2.5 mL thermostatic

178

gastight syringe, and preheated to 45 °C on a Gerstel autosampling device (Mülheim

179

an der Ruhr, Germany). The syringe was inserted into the GC-MS injection port at a

180

rate 0.5 mL/s. The injection port was held at 250 °C with a split ratio of 1:3. The

181

conditions of the GC and MS were the same as those described above

182

(HS-SPME-GC/MS). Partition coefficients of compound 1 in different model

183

solutions were calculated via the method developed by Ettre et al. (1993)15: 1 A

184

1 liq i

fi C

1 fi Ciliq

kg/m

185

By plotting 1/A against , this equation gives a linear relationship between 1/A and ,

186

as follows:

187

1 A

a

b*

188

where kg/m is the partition coefficient between the gas and the matrix (namely, b/a), A

189

is the chromatographic peak area of compound 1, fi is the detector response factor,

190

Ciliq is the initial concentration of the compound in the vial, and


is the ratio between

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the headspace (Vg) and matrix (Vl) volumes.

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Analysis of interaction between compound 1 and glucose or gluconic acid

193

The changes in energy during the interaction of compound 1 and glucose or gluconic

194

acid were determined with an isothermal titration calorimeter (ITC-200 MicroCal, GE,

195

Northampton, USA).16 Compound 1, glucose, and gluconic acid solutions (11.21, 0.56,

196

and 0.56 mmol/L, respectively) were prepared in 1% propylene glycol aqueous

197

solution (v/v) at pH 5.2 (adjusted by phosphates). The solutions were degassed by

198

ultrasound for 5 min and filtered with disposable sample filters (0.22 m). The sample

199

cell was filled with 300 L glucose or gluconic acid solution and titrated with 50

200

of compound 1 model solution placed in the stirring syringe. Experiments were set up

201

with 24 consecutive injections (2.02 L) with a duration of 10 s each, at intervals of

202

150 s, a stirring speed of 300 rpm, and temperature fixed at 25 °C.

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Statistical analysis

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All experiments were conducted in triplicate. Data analysis was performed using

205

SPSS software (v17.0, Chicago, IL, USA). When differences between treatments

206

(peak area of 1, 2, 3, 4, HS, HSL and SMM with a 95% confidence interval) were

207

statistically significant, means were compared through Duncan’s multiple range test at

208

the significance level of p0.05) but SMM content obviously changed (p

Study of the Inhibitors of Cooked Off-Flavor Components in Heat

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