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]

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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

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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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Introduction

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

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

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

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

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

23

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

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

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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

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market.

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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

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

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

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

34

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

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improving the flavor quality of heated melon juice.

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

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

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

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

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

41

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

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

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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.

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

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

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

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precursors in clear melon juice during heat treatment.

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Materials and Methods

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Chemicals

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

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

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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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water was purified using a Milipore Milli-Q system.

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Melon samples

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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HTST clear juice preparation

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

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

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

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

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

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

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

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

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

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Sample preparation for static headspace gas chromatograph-mass spectrometry

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(SHS-GC)

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

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

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

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

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

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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

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

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(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

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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:

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

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

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

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Components identified by MS/olfactometer

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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),

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

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

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

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

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for

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

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

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

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

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

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

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

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

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Quantitation by ultra-performance liquid chromatography system coupled to a

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triple quadrupole mass spectrometer (UPLC-MS/MS)

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

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

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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

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

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

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

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

20

min

at

40

°

C

with

agitation.

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50/30

µm

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

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

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

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

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

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

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

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voltage 11v).

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

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

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

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

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2

162

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

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

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

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

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

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

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

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

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which is calculated as follows:

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

Ci = Cis × fi × Ai / Ais

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

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

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

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relative to labeled internal standards calculated using the standard curve.

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Validation test

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

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

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

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

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

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

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

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

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

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juices were calculated as follows:

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I - pre = Cpre - aj / Cpre - ck

I - vsc = Cvsc − aj / Cvsc − ck

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

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

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

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

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

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

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Statistical analysis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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identification of VSCs in HTST melon juices.

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Relations between VSCs and precursors

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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

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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

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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

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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

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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

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

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

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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),

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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

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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

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

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

278

finding suggests that Met may only generate MTP during HTST.

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

282

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

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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

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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

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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

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30-31

.

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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.

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TOC graphic

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