Identification of Cooked Off-Flavor Components and Analysis of Their

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Chemistry and Biology of Aroma and Taste

Identification of Cooked Off-flavor Components and Analysis of Their Formation Mechanisms in Melon Juice during Thermal Processing DongSheng Luo, Xueli Pang, Xinxing Xu, Shuang Bi, Wentao Zhang, and JiHong Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01019 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 10, 2018

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Journal of Agricultural and Food Chemistry

Identification of Cooked Off-flavor Components and Analysis of Their Formation Mechanisms in Melon Juice during Thermal Processing *

Dongsheng Luo1, Xueli Pang2, Xinxing Xu1, Shuang Bi1, Wentao Zhang1, 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. Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao, 266101, China

*

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

ACS Paragon Plus Environment


1

Abstract: Cooked off-flavor components were identified, and their formation

2

mechanisms were studied in heat-treated melon juices. When flavor dilution analysis

3

methods and odor activity value were used to evaluate the cooked off-flavor in

4

heat-treated melon juice, four volatile sulfide compounds (VSCs) were identified as

5

contributors to the cooked off-flavor, as follows: dimethyl disulfide (DMDS),

6

dimethyl trisulfide (DMTS), dimethyl sulfide (DMS), and 3-(methylthio)propanal

7

(MTP). The cooked off-flavor intensities of heated juices from thick-skinned melons

8

were stronger than those in juices from thin-skinned melons. We conducted a

9

comparative analysis of VSCs before and after heat treatment by adding unlabeled

10

and labeled S-methylmethionine (SMM) and/or methionine (Met) to original melon

11

juices. DMS and MTP were formed from SMM and Met through nucleophilic

12

substitution and Strecker degradation, respectively. DMDS and DMTS were partly

13

formed through the oxidative degradation of methanethiol produced from Met.

14

Moreover, SMM could accelerate degradation of Met by increasing the amount of

15

dicarbonyl compounds during heat treatment.

16

Keywords: melon, heat treatment, off-flavor, precursors, formation mechanisms


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17

Introduction

18

Melon (Cucumis melo L.) is an important commercial crop and China’s melon

19

production is approximately 40% of the global production1. However, melon fruits

20

harvested during summer suffer from high postharvest loss due to high levels of water,

21

low acidity, and long-distance transport2. In general, postharvest loss can be

22

controlled by post-harvest treatments, preservation technology, and deep processing

23

technologies. Melon juice, an important deep processing product of melon fruits with

24

high nutritional value3, is conveniently transported and stored. Especially, clear melon

25

juices acquired by ultrafiltration avoid clarifying agents and high temperature

26

processing with high stability and quality4. Thus, in food processing, clear melon

27

juices are significant in reducing postharvest loss and promoting growth in the melon

28

market.

29

Thermal sterilization techniques, such as high temperature short time sterilization

30

(HTST), are widely used in the fruit juice industry to improve safety and to extend

31

shelf life. However, Chen, Pang, Nath, and Sun found that heat-treated melon juice

32

exhibit a strong cooked off-flavor5-8. The cooked off-flavors formed during heat

33

treatment impede the development of deep processing melon products. Thus, the

34

analysis of cooked off-flavor components and formation mechanisms is crucial for

35

improving the flavor quality of heated melon juice.

36

Cooked off-flavor components in heated fruit and vegetable juices are major volatile

37

sulfide compounds (VSCs)9-10, such as dimethyl sulfide (DMS). The mechanisms of

38

DMS formation in some foods, including biological and chemical pathways, were



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39

discussed 9-11. Other cooked flavors related to VSCs in pineapple juice, dairy products,

40

and beer during heat treatment and fermentation, such as hydrogen sulfide (H2S),

41

dimethyl trisulfide (DMTS), and 3-(methylthio)propanal (MTP), were also reported.

42

These products are mainly formed from the degradation of cysteine (Cys) and

43

methionine (Met), respectively12-14. Reports on cooked off-flavors in foods are helpful

44

to systematically study the cooked off-flavors in melon juice during heat treatment.

45

The current study aimed to 1) identify the cooked off-flavor components in HTST

46

clear melon juices; 2) identify the main precursors of cooked off-flavor components;

47

and 3) analyze the transformation mechanisms of cooked off-flavor components from

48

precursors in clear melon juice during heat treatment.

49

Materials and Methods

50

Chemicals

51

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

52

CAS: 4727-40-6), dimethyl sulfide (DMS, CAS: 75-18-3), dimethyl disulfide (DMDS,

53

CAS:

54

3-(methylthio)propanal (MTP, CAS: 3268-49-3) were purchased from Sigma–Aldrich

55

Co., Ltd. (Milwaukee, WI, USA) with purity > 98%. Formic acid and acetonitrile

56

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

57

The labeled standards (2H3-Met, 2H6-SMM, 2H6-DMS, 2H6-DMDS, 2H6-DMTS, and

58

2

59

(Guangzhou, China). The other reagents were analytical grade purity, and purchased

60

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

624-92-0),

dimethyl

trisulfide

(DMTS,

CAS:

3658-80-8),

and

H3-MTP) were obtained from Guangzhou PUEN Scientific Instrument Co., Ltd


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61

water was purified using a Milipore Milli-Q system.

62

Melon samples

63

Thirteen melons were purchased directly from six production areas of China including

64

thick- and thinned-skin melons, in August 2017. Hami (C.melo var. saccharinus, HM),

65

Jiashi (C.melo var. inodorus, JS), Jinlong (C.melo var. recticulatus, JL), and

66

Xizhoumi (C.melo var. reticulates, XZM) were from Xinjiang Uigur Autonomous

67

Region; Jinmi (C.melo var. recticulatus, JM) and Hetaomi (C.melo var. chandalak,

68

HTM) were from inner mongolia Autonomous Region; Lvbaoshi (C.melo var.

69

makuwa, LBS) was from Henan province; Wangwen (C.melo var. reticulates, WW),

70

Cuili (C.melo var. pangalo, CL), and Yangjiaomi (C.melo var. chinensis, YJM) were

71

from Shandong province; Bailan (C.melo var. makuwa, BL) and Huanghemi (C.melo

72

var. casaba, HHM) were from Gansu province; and Fenglei (C.melo var. makuwa, FL)

73

was from Tianjin. BL, CL, FL, LBS, and YJM are thin-skinned melons and the

74

soluble solid contents were 8.5-9.7%. HM, JL, JM, JS, WW, XZM, HTM, and HHM

75

are thick-skinned melons and the soluble solid contents were 11.6-13.5%. Each melon

76

was about 60 kg and kept at room temperature to facilitate fruit ripening during

77

storage (20 °C, 3 days, relative humidity about 45%).

78

HTST clear juice preparation

79

Melons were placed in an ice bath for 12 h and then squeezed using a juicer (GT6G7,

80

Light Industry Machinery Factory, Zhejiang, China). The squeezed juice was

81

centrifuged at 8000 rpm for 15 min at 4 °C, and the supernatant was used to prepare

82

clear melon juice using an ultrafiltration unit (Nanjing Kaimi Technology Inc.,



83

Nanjing, China) in accordance with the methods reported by Zhao et al. (2016)15. The

84

clear melon juice was heat-treated using an HTST processing system (Armfield FT74,

85

Hampshire, England). The juice was heated to 135 °C at a flow rate of 9.98 L/h and

86

then held for 15 s at 135 °C16. Subsequently, the HTST melon juice was immediately

87

cooled to 20 °C by a cooler (FT74-20-MKIII).

88

Sample preparation for static headspace gas chromatograph-mass spectrometry

89

(SHS-GC)

90

Cooked off-flavor components in HTST melon juices were examined using

91

SHS-GC-MS and flavor dilution (FD) analysis techniques as reported by Zhou et al.

92

(2002)17. HTST melon juice (100 mL) was transferred to a 250 mL gas tight glass jar

93

and equilibrated at 40 °C for 30 min with agitation provided by a magnetic stir bar.

94

Subsequently, 25, 10, 5.0, 2.5, 1.0, 0.5, 0.2, or 0.1 mL of headspace was collected

95

from the incubated jar with a preheated (45 °C) gas-tight syringe (SGE International

96

Pty Ltd, Australia) and then injected into injection port of the gas chromatography

97

system (7890B, Agilent Technologies, Inc., USA) equipped with a mass spectrometer

98

(5975C, Agilent Technologies, Inc., USA) and an olfactometer (ODP2; Gerstel, Inc.,

99

Germany). The column effluent was split 1:1 between the MS and ODP2 using a

100

deactivated fused silica column. Separations were performed on DB-Wax (30 m ×

101

0.32 mm i.d.; 1 µm film; Agilent Technologies, Inc.) and DB-5 columns (30 m × 0.32

102

mm i.d.; 1 µm film; Agilent Technologies, Inc.). GC oven conditions were as follows:

103

ramped temperature programs of the two chromatographic column was initially 35 °C

104

for 1 min; increased to 150 °C at 3 °C/min; and increased to 225 °C at 8 °C/min and


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105

held for 2 min. The MS conditions were as follows: mass spectra was operated in

106

electronic impact mode (voltage, 70 eV), and ion source temperature was 250 °C with

107

full scanning from 30 m/z to 500 m/z at 1 s intervals.

108

Components identified by MS/olfactometer

109

Odor-active components obtained from SHS-GC-MS were further identified using an

110

olfactory detection port (Sniffer 9000; Brechbuhler, Schlieren, Switzerland) supported

111

by the above-mentioned GC-MS. Three trained panelists independently evaluated the

112

effluence of the same sample from a sniff port and were then asked to report the

113

aroma attributes, retention time, and odor intensity of the effluence that possessed

114

cooked off-flavor in accordance with the methods reported by Luo (2017)18. Until two

115

panelists reported that they perceived no odor for a sample, the same procedures were

116

repeated for the next smaller volume. The cooked off-flavor components were

117

positively identified by comparing their retention index (RI), odor properties and mass

118

spectra with those of authentic standard compounds. The RI values were calculated in

119

accordance with the methods used by Song et al. (2013)19.

120

Quantitation by headspace solid-phase microextraction (HS-SPME)

121

We used stable isotope dilution (SID) supported by HS-SPME-GC-MS to quantitate

122

the cooked off-flavor components because of its high sensitivity and selectivity17, 20. A

123

series of standard solutions of VSCs (both labeled and unlabeled) was prepared in

124

methanol at the following concentrations: 498.32 (2H6-DMS), 501.71 (DMS), 10.31

125

(2H6-DMDS), 10.07 (DMDS), 10.87 (2H6-DMTS), 10.46 (DMTS), 150.60 (2H3-MTP),

126

and 151.18 µg/L (MTP). To prepare the calibration standards for each VSC, we



Page 8 of 36

127

transferred serial volumes of unlabeled VSC solution (2, 4, 8, 16, or 32 µL) and 4 µL

128

of labeled VSC solution into a 25 mL screw cap headspace vial with 10 mL of

129

distilled water and 5 g of sodium chloride. After vibration, the vial was equilibrated

130

for

131

polydimethylsiloxane/divinylbenzene/carboxen coated fiber was then exposed to the

132

headspace of the vial for 30 min. For desorption, the fiber was inserted into GC

133

injection port at 250 °C for 4 min. Selected ion monitoring mode was used in MS

134

analysis during VSC quantitation6. The quantitative analysis of ions for both

135

unlabeled and labeled DMS, DMDS, DMTS, and MTP resulted in the following

136

values: 62, 68, 94, 100, 126, 132, 104, and 107. Detailed conditions of GC-MS were

137

as described in the previously noted analysis methods. VSCs in HTST juice were

138

quantitated using SID-HS-SPME-GC-MS based on the procedure described above.

139

Quantitation by ultra-performance liquid chromatography system coupled to a

140

triple quadrupole mass spectrometer (UPLC-MS/MS)

141

Flavor precursors were quantitated using an UPLC-MS/MS (Milford, MA, USA)21.

142

Separation was achieved using a C18 column (Acquity UPLC BEH C18 100 mm ×

143

2.1 mm, 1.7 µm particle size). The solvent system consisted of 0.2% aqueous formic

144

acid (A) and acetonitrile (B) with gradient elution at a flow rate of 0.3 mL/min and

145

was utilized as follows: initial conditions A:B = 95:5 (1 min hold), linear gradient to

146

95% B for 1 min, 5% B for 1 min, and then switched back to initial conditions and

147

re-equilibration for 2 min. The sample injection volume was 1 µL and the column

148

oven temperature and sample tray temperature were maintained at 40 °C and 4 °C,

20

min

at

40

°

C

with

agitation.


A

50/30

µm

Page 9 of 36


149

respectively. The electron spray ionization source was used in positive mode by

150

multiple reaction monitoring mode with the following conditions: ion source capillary

151

voltage and cone voltage were 3.34 and 3.5 kV and 35 and 39 V for SMM and Met.

152

Desolvation temperature was 400 °C. The collision gas was argon (purity>99.999%).

153

The optimized selected MS/MS transition pairs of precursor and product ions were as

154

follows: SMM 164>102 (collision voltage 12 v), 2H6-SMM 170>102 (collision

155

voltage 13 v), Met 150>104(collision voltage 10 v), and 2H3-Met 153>107(collision

156

voltage 11v).

157

A series of standard solutions of the sulfides (SMM, 2H6-SMM, Met, and 2H3-Met)

158

was prepared in ultrapure water at concentrations of 10.58 (2H6-SMM), 10.71 (SMM),

159

50.52 (2H3-Met), and 50.69 µg/mL (Met). For the preparation of the calibration

160

standards, 2, 4, 8, 16, or 32 µL of SMM (or Met) standard solution and 4 µL of

161

2

162

tubes (25 mL) with 10 mL ultrapure water. After being agitated, the tubes were

163

centrifuged at 8000 rpm for 15 min (4 °C). Supernatants were filtered using disposable

164

sample filters (0.22 µm) and then analyzed via UPLC-MS/MS. The SMM and Met in

165

melon juices were analyzed in accordance with the procedure described above.

166

Equations used for GC–MS and UPLC–MS/MS anlysis

167

VSC and SMM/Met concentrations were determined in accordance with methods

168

described by Rotsatchakul et al. (2008) and Kim et al. (2017)22-23 using MS response

169

factors and quantitative ion areas for each compound relative to the internal standard,

170

which is calculated as follows:

H6-SMM (or 2H3-Met) standard solution were transferred into a screw cap propylene

Ci = Cis × fi × Ai / Ais



171

where Ci and Ai are the concentration and quantitative ion peak area of compound i,

172

respectively. Cis and Ais are the concentration and quantitative ion peak area of the

173

labeled internal standard, respectively. fi is the MS response factor of compound i

174

relative to labeled internal standards calculated using the standard curve.

175

Validation test

176

A specified amount of unlabeled and labeled standard substances, namely, SMM, Met,

177

or a mixture of SMM and Met, was added to 5 L of original juice (clear melon juice),

178

and the juices were agitated at 4 °C for 30 min at 200 r/min. The juices containing

179

exogenous SMM and Met (additive juices) and original juices were then treated using

180

HTST. The concentrations of SMM, Met, and VSCs (both labeled and unlabeled) in

181

additive and original juices were measured before and after HTST by UPLC-MS/MS

182

and HS-SPME-GC-MS in accordance with the methods described in the previous

183

section. The rate of increase of SMM/Met (I-pre) in additive juices, degradation rate

184

of SMM/Met (D-pre) in HTST juices, and rate of increase of VSCs (I-vsc) in HTST

185

juices were calculated as follows:

186

I - pre = Cpre - aj / Cpre - ck

I - vsc = Cvsc − aj / Cvsc − ck

187

D − pre = ( Cpre − fresh − Cpre − htst ) Cpre − fresh

188

where Cpre−aj and Cpre−ck are the concentration of SMM/Met in additive juice and

189

original juice before HTST, respectively. Cvsc−aj and Cvsc−ck are the concentration of

190

VSCs in additive and original juices after HTST. Cpre−fresh is the concentration of

191

SMM/Met in additive juice or original juice before HTST, and Cpre−htst is the

192

concentration of SMM/Met in additive juice or original juice after HTST.


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193

Statistical analysis

194

All experiments in this work were carried out in triplicate. The means and standard

195

deviations of the chromatographic data were acquired by using SPSS software (v17.0,

196

Chicago, IL, USA). A univariate analysis of variance (ANOVA) was used to test the

197

variances of the additives on each volatile sulfide compound in the validation test

198

section. Then, the multiple comparisons using the Duncan test were carried out for

199

ANOVA with significance (p25000). This finding indicates that VSCs strongly

231

influence the overall flavor27. Similar to the results of FD analysis, juices prepared

232

from thick-skinned melons generated strong cooked off-flavor and YJM juice

233

exhibited the lowest cooked off-flavor intensity according to total OAVs of four

234

VSCs. Further analysis found that DMS contributed to the cooked off-flavor of all

235

melon juices exceeding 50%, especially in thick-skinned melon juices. DMTS and

236

DMDS affected the cooked off-flavor more highly in juices from thin-skinned melons


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237

than in juices from thick-skinned melons. These results showed that varieties and

238

origins affect the cooked off-flavor intensity of melon juices.

239

The highest cooked off-flavor intensity was found in XZM juice according to OAVs

240

rather than in JS or WW juices based on FD analysis, thereby demonstrating that the

241

FD factor of DMS in XZM juice was greatest if the stepwise dilution of headspace

242

volume was continued. Additionally, in HTST juices (BL, CL, and FL), the OAV of

243

DMTS was higher than that of DMDS, but both exhibited the same (or low) FD

244

factors. This finding suggested the volatile components or the volatile components

245

and matrixes that influence the release of VSCs may interact, and such interaction

246

may change the odor intensity of odorants with small OAV or FD factor28. For these

247

reasons, the OAV and FD factor were considered as complementary methods for the

248

identification of VSCs in HTST melon juices.

249

Relations between VSCs and precursors

250

Four VSCs in 13 melon juices were first quantitated to analyze the relations between

251

the concentrations of precursors and VSCs in HTST original juices. As shown in

252

Table 1, the concentration of DMS (468.76–8604.82 µg/L) was higher than that of

253

MTP (31.21–431.11 µg/L), DMDS (4.14–23.43 µg/L), and DMTS (1.09–13.12 µg/L)

254

in all HTST juices. All VSCs were present at levels above their odor thresholds and

255

contributed to the cooked off-flavor27, 29. Moreover, HTST juices from thick-skinned

256

melons exhibited higher concentration of DMS but lower concentration of DMDS and

257

DMTS than juices from thin-skinned melons. The maximum MTP content was found

258

in BL juice, but no relationship was found between the level of MTP and melon



259

varieties. The quantitative data were consistent with the FD and OAV results, as

260

shown in Tables 1 and 2, which indicated that DMS and MTP were the two most

261

crucial cooked off-flavor components in HTST melon juices, especially in

262

thick-skinned melon juices. For thin-skinned melon juices, DMTS was an important

263

cooked off-flavor component, even though its levels were lower than those of DMDS

264

because of its extremely low odor threshold (0.01 µg/L in water).

265

The concentrations of SMM and Met in 13 melon juices were then measured in

266

original juices before HTST. All the melon juices contained Met and SMM, thereby

267

implying that these compounds are potential precursors of VSCs (Table 1). The

268

highest concentration of SMM was found in XZM juice (18.36 µg/mL), followed by

269

JS (13.35 µg/mL), and the lowest concentration was found in YJM (4.71 µg/mL),

270

which was consistent with the concentration of DMS measured in the three melons

271

(Table 1). However, except for the above mentioned three melons, the apparent

272

contradiction between the concentrations of DMS and the distribution of SMM in

273

thick- and thin-skinned melons suggest that some factors affect the conversion of

274

SMM into DMS during heat treatment. Similarly, the highest concentration of Met

275

was found in BL (69.19 µg/mL), and the lowest concentration was found in LBS

276

(13.93 µg/mL). These results were consistent with the concentration of MTP but

277

inconsistent with the concentrations of DMDS and DMTS among the melons. This

278

finding suggests that Met may only generate MTP during HTST.

279

The contradictions between the concentrations of precursors and VSCs may have

280

arisen from different degradation rate of precursors, because the degradation SMM


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281

and Met in different melons will be seriously affected by food matrix, including

282

precursors, dissolved oxygen, reducing sugars, amino acid, and acidity25-26,

283

Degradation rate was calculated by quantitating SMM or Met before and after HTST

284

to avoid the matrix influence between different melons (Table 2). The trend of

285

SMM/Met degradation was consistent with the amount of DMS/MTP formed without

286

regard to the influence on VSC release by the matrix. This finding demonstrated the

287

effect of matrix on SMM/Met degradation and illustrated the apparent contradiction

288

between the concentration of precursors and concentration of VSCs in the melons.

289

However, no relationship was found between Met and DMDS and DMTS in HTST

290

melon juice. This suggests that SMM and Met may be the precursors of DMS and

291

MTP, whereas other precursors for DMDS and DMTS are present.

292

MTP is known to be a Maillard aldehyde, mainly generated from Met through

293

Strecker degradation, which can be oxidised into DMDS and DMTS during heat

294

processing32. Thus, DMDS and DMTS might be generated from Met when MTP was

295

present in HTST melon juices. It has been proposed that DMDS and DMTS may only

296

be partially generated from thermal degradation of Met, whereas some were derived

297

from unknown precursors, such as intermediate products of Met metabolism, or

298

enzyme action during the temperature rise of melon juice, thereby leading to no

299

specific connection between them33-36. In the current work, we did not explore the

300

unknown precursors and related enzymes of DMDS and DMTS due to their small

301

contribution to cooked off-flavor for most of HTST melon juices.

302

Identification of precursors


30-31

.


303

The degradation rates of SMM and Met were analyzed in original and additive juices

304

containing exogenous SMM or Met after HTST because of their great influence on the

305

formation of VSCs. No obvious differences between the degradation rates of different

306

melon varieties were found (Figure 2). Thus, the rate of increase of SMM or Met in

307

additive juices before HTST will be similar with that of VSCs after HTST if they

308

were corresponding precursors.

309

Based on the above results, the relevance analysis was carried out to identify the

310

precursors of VSCs by comparison of SMM and Met with VSCs in additive juice and

311

original juice after HTST. The two-way multivariate analysis of variance of VSCs and

312

SMM and Met are summarized in Figure 3. No significant interaction (p102

%

0 0.00

25000 20000

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

4b DMS

DMDS

DMTS

MTP

CK

15000 10000 5000 0 5.00

10.00

15.00

20.00

25.00

30.00

35.00

25000 20000 15000

Ion 62.00

10000

Ion 68.00

2H -SMM 6

5000 0 5.00

10.00

15.00

20.00

25.00

30.00

35.00

25000 Ion 94.00 Ion 97.00 Ion 100.00

20000 15000

Ion 104.00

Ion 126.00 Ion 129.00 Ion 132.00

Ion 107.00 2H -Met 3

10000 5000 0 5.00

10.00

15.00

20.00

25.00

30.00

35.00

Note: Rt is the retention time of SMM/Met on liquid chromatogram; QIR is quantitation ion pair; 2

H6-SMM and 2H3-Met are hydrogen labeled SMM and Met, respectively.

Figure 4 a and b.


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Figure 5.



TOC graphic


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Identification of Cooked Off-Flavor Components and Analysis of Their

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