Identification of Cooked Off-Flavor Components and Analysis of Their

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Cite This: J. Agric. Food Chem. 2018, 66, 5612−5620

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*,† †

College of Food Science and Nutritional Engineering, China Agricultural University; Key Laboratory of Fruit and Vegetable Processing, Ministry of Agriculture; and National Engineering Research Center for Fruit and Vegetable Processing, Beijing 100083, China ‡ Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266101, China ABSTRACT: Cooked off-flavor components were identified, and their formation mechanisms were studied in heat-treated melon juices. When flavor dilution analysis methods and odor activity values were used to evaluate the cooked off-flavor in heattreated melon juice, four volatile sulfide compounds (VSCs) were identified as contributors to the cooked off-flavor: dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), dimethyl sulfide (DMS), and 3-(methylthio)propanal (MTP). The cooked off-flavor intensities of heated juices from thick-skinned melons were stronger than those in juices from thin-skinned melons. We conducted a comparative analysis of VSCs before and after heat treatment by adding unlabeled and labeled S-methylmethionine (SMM) and/or methionine (Met) to the original melon juices. DMS and MTP were formed from SMM and Met through nucleophilic substitution and Strecker degradation, respectively. DMDS and DMTS were partly formed through the oxidative degradation of methanethiol produced from Met. Moreover, SMM could accelerate degradation of Met by increasing the amount of dicarbonyl compounds during heat treatment. KEYWORDS: melon, heat treatment, off-flavor, precursors, formation mechanisms

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INTRODUCTION Melon (Cucumis melo L.) is an important commercial crop, and China’s melon production is approximately 40% of the global production.1 However, melon fruits harvested during summer suffer from high postharvest loss due to high levels of water, low acidity, and long-distance transport.2 In general, postharvest loss can be controlled by postharvest treatments, preservation technologies, and deep processing technologies. Melon juice, an important deep processing product of melon fruits with a high nutritional value,3 is conveniently transported and stored. Clear melon juices acquired by ultrafiltration without clarifying agents or high temperature processing have high stability and quality.4 Thus, in food processing, clear melon juices are significant for reducing postharvest loss and promoting growth in the melon market. Thermal sterilization techniques, such as high temperature short time sterilization (HTST), are widely used in the fruit juice industry to improve safety and extend shelf life. However, Chen, Pang, Nath, and Sun found that heat-treated melon juice exhibits a strong cooked off-flavor.5−8 The cooked off-flavors formed during heat treatment impede the development of deep processing melon products. Thus, the analysis of cooked offflavor components and formation mechanisms is crucial for improving the flavor quality of heated melon juice. Cooked off-flavor components in heated fruit and vegetable juices include major volatile sulfide compounds (VSCs),9,10 such as dimethyl sulfide (DMS). The mechanisms of DMS formation in some foods, including biological and chemical pathways, were discussed.9−11 Other cooked flavors related to VSCs in pineapple juice, dairy products, and beer during heat © 2018 American Chemical Society

treatment and fermentation, such as hydrogen sulfide (H2S), dimethyl trisulfide (DMTS), and 3-(methylthio)propanal (MTP), were also reported. These products are mainly formed from the degradation of cysteine (Cys) and methionine (Met).12−14 Reports on cooked off-flavors in foods are helpful to systematically study the cooked off-flavors in melon juice during heat treatment. The current study aimed to (1) identify the cooked off-flavor components in HTST clear melon juices, (2) identify the main precursors of cooked off-flavor components, and (3) analyze the transformation mechanisms of cooked off-flavor components from precursors in clear melon juice during heat treatment.

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MATERIALS AND METHODS

Chemicals. N-alkanes (C5−C30), methionine (Met, CAS: 59-51-8), S-methylmethionine (SMM, CAS: 4727-40-6), dimethyl sulfide (DMS, CAS: 75-18-3), dimethyl disulfide (DMDS, CAS: 624-92-0), dimethyl trisulfide (DMTS, CAS: 3658-80-8), and 3-(methylthio)propanal (MTP, CAS: 3268-49-3) were purchased from Sigma-Aldrich Co., Ltd. (Milwaukee, WI, USA) with purity >98%. Formic acid and acetonitrile were obtained from Merck & Co., Inc., (Kenilworth, NJ, USA) with purity >99%. The labeled standards (2H3-Met, 2H6-SMM, 2 H6-DMS, 2H6-DMDS, 2H6-DMTS, and 2H3-MTP) were obtained from Guangzhou PUEN Scientific Instrument Co., Ltd. (Guangzhou, China). The other reagents were analytical grade purity and purchased Received: Revised: Accepted: Published: 5612

February 25, 2018 May 8, 2018 May 10, 2018 May 10, 2018 DOI: 10.1021/acs.jafc.8b01019 J. Agric. Food Chem. 2018, 66, 5612−5620

Article

Journal of Agricultural and Food Chemistry from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Ultrapure water was purified using a Milipore Milli-Q system. Melon Samples. Thirteen melons were purchased directly from six production areas of China, including thick- and thin-skinned melons, in August 2017. Hami (C. melo var. saccharinus, HM), Jiashi (C. melo var. inodorus, JS), Jinlong (C. melo var. recticulatus, JL), and Xizhoumi (C. melo var. reticulates, XZM) were from the Xinjiang Uigur Autonomous Region; Jinmi (C. melo var. recticulatus, JM) and Hetaomi (C. melo var. chandalak, HTM) were from the Inner Mongolia Autonomous Region; Lvbaoshi (C. melo var. makuwa, LBS) was from the Henan Province; Wangwen (C. melo var. reticulates, WW), Cuili (C. melo var. pangalo, CL), and Yangjiaomi (C. melo var. chinensis, YJM) were from the Shandong Province; Bailan (C. melo var. makuwa, BL) and Huanghemi (C. melo var. casaba, HHM) were from the Gansu Province; and Fenglei (C. melo var. makuwa, FL) was from Tianjin. BL, CL, FL, LBS, and YJM are thin-skinned melons, and their soluble solid contents were 8.5−9.7%. HM, JL, JM, JS, WW, XZM, HTM, and HHM are thick-skinned melons, and their soluble solid contents were 11.6− 13.5%. Each melon was about 60 kg and kept at room temperature to facilitate fruit ripening during storage (20 °C, 3 days, relative humidity about 45%). HTST Clear Juice Preparation. Melons were placed in an ice bath for 12 h and then squeezed using a juicer (GT6G7, Light Industry Machinery Factory, Zhejiang, China). The squeezed juice was centrifuged at 8000 rpm for 15 min at 4 °C, and the supernatant was used to prepare clear melon juice using an ultrafiltration unit (Nanjing Kaimi Technology Inc., Nanjing, China) in accordance with the methods reported by Zhao and co-workers.15 The clear melon juice was heat-treated using an HTST processing system (Armfield FT74, Hampshire, England). The juice was heated to 135 °C at a flow rate of 9.98 L/h and then held for 15 s at 135 °C.16 Subsequently, the HTST melon juice was immediately cooled to 20 °C by a cooler (FT74-20MKIII). Sample Preparation for Static Headspace Gas Chromatograph−Mass Spectrometry (SHS-GC). Cooked off-flavor components in HTST melon juices were examined using SHS-GC-MS and flavor dilution (FD) analysis techniques as reported by Zhou and co-workers.17 HTST melon juice (100 mL) was transferred to a 250 mL gastight glass jar and equilibrated at 40 °C for 30 min with agitation provided by a magnetic stir bar. Subsequently, 25, 10, 5.0, 2.5, 1.0, 0.5, 0.2, or 0.1 mL of headspace was collected from the incubated jar with a preheated (45 °C) gastight syringe (SGE International Pty Ltd., Australia) and then injected into the injection port of the gas chromatography system (7890B, Agilent Technologies, Inc., USA) equipped with a mass spectrometer (5975C, Agilent Technologies, Inc., USA) and an olfactometer (ODP2; Gerstel, Inc., Germany). The column effluent was split 1:1 between the MS and ODP2 using a deactivated fused silica column. Separations were performed on DB-WAX (30 m × 0.32 mm i.d.; 1 μm film; Agilent Technologies, Inc.) and DB-5 columns (30 m × 0.32 mm i.d.; 1 μm film; Agilent Technologies, Inc.). The GC oven conditions had ramped temperature programs for the two chromatographic columns. The conditions began at 35 °C for 1 min, increased to 150 °C at 3 °C/min, increased to 225 °C at 8 °C/min, and then held for 2 min. For the MS conditions, the mass spectra were operated in electronic impact mode (voltage, 70 eV), and the ion source temperature was 250 °C with full scanning from 30 m/z to 500 m/z at 1 s intervals. Components Identified by MS/Olfactometer. Odor-active components obtained from SHS-GC-MS were further identified using an olfactory detection port (Sniffer 9000; Brechbuhler, Schlieren, Switzerland) supported by the GC-MS mentioned above. Three trained panelists independently evaluated the effluence of the same sample from a sniff port and were then asked to report the aroma attributes, retention time, and odor intensity of the effluence that possessed cooked off-flavor in accordance with the methods reported by Luo.18 Until two panelists reported that they perceived no odor for a sample, the same procedures were repeated for the next smaller volume. The cooked off-flavor components were positively identified by comparing their retention index (RI), odor properties, and mass spectra with those of authentic

standard compounds. The RI values were calculated in accordance with the methods used by Song and co-workers.19 Quantitation by Headspace Solid-Phase Microextraction (HS-SPME). We used stable isotope dilution (SID) supported by HS-SPME-GC-MS to quantitate the cooked off-flavor components because of its high sensitivity and selectivity.17,20 A series of standard solutions of VSCs (both labeled and unlabeled) was prepared in methanol at the concentrations of 498.32 (2H6-DMS), 501.71 (DMS), 10.31 (2H6-DMDS), 10.07 (DMDS), 10.87 (2H6-DMTS), 10.46 (DMTS), 150.60 (2H3-MTP), and 151.18 μg/L (MTP). To prepare the calibration standards for each VSC, we transferred serial volumes of unlabeled VSC solution (2, 4, 8, 16, or 32 μL) and 4 μL of labeled VSC solution into a 25 mL screw cap headspace vial with 10 mL of distilled water and 5 g of sodium chloride. After vibration, the vial was equilibrated for 20 min at 40 °C with agitation. A 50/30 μm polydimethylsiloxane/divinylbenzene/carboxen coated fiber was then exposed to the headspace of the vial for 30 min. For desorption, the fiber was inserted into the GC injection port at 250 °C for 4 min. A selected ion monitoring mode was used in MS analysis during VSC quantitation.6 The quantitative analysis of ions for both unlabeled and labeled DMS, DMDS, DMTS, and MTP resulted in the values of 62, 68, 94, 100, 126, 132, 104, and 107. Detailed conditions of the GC-MS were as described in the previously noted analysis methods. VSCs in HTST juice were quantitated using SID-HS-SPME-GC-MS based on the procedure described above. Quantitation by an Ultraperformance Liquid Chromatography System Coupled to a Triple Quadrupole Mass Spectrometer (UPLC-MS/MS). Flavor precursors were quantitated using an UPLC-MS/MS (Milford, MA, USA).21 Separation was achieved using a C18 column (Acquity UPLC BEH C18 100 mm × 2.1 mm, 1.7 μm particle size). The solvent system consisted of 0.2% aqueous formic acid (A) and acetonitrile (B) with a gradient elution at a flow rate of 0.3 mL/min and was utilized with the following conditions: initial conditions A/B = 95:5 (1 min hold), linear gradient to 95% B for 1 min, 5% B for 1 min, and then switched back to initial conditions and re-equilibration for 2 min. The sample injection volume was 1 μL, and the column oven temperature and sample tray temperature were maintained at 40 and 4 °C, respectively. The electron spray ionization source was used in the positive mode by the multiple reaction monitoring mode with the following conditions: ion source capillary voltage and cone voltage were 3.34 and 3.5 kV, respectively, and 35 and 39 V, respectively, for SMM and Met. The desolvation temperature was 400 °C. The collision gas was argon (purity >99.999%). The optimized selected MS/MS transition pairs of precursor and product ions were SMM 164 > 102 (collision voltage, 12 V), 2H6-SMM 170 > 102 (collision voltage, 13 V), Met 150 > 104 (collision voltage, 10 V), and 2 H3-Met 153 > 107 (collision voltage, 11 V). A series of standard solutions of the sulfides (SMM, 2H6-SMM, Met, and 2H3-Met) was prepared in ultrapure water at concentrations of 10.58 (2H6-SMM), 10.71 (SMM), 50.52 (2H3-Met), and 50.69 μg/mL (Met). For the preparation of the calibration standards, 2, 4, 8, 16, or 32 μL of SMM (or Met) standard solution and 4 μL of 2H6-SMM (or 2H3-Met) standard solution were transferred into 25 mL screw cap propylene tubes with 10 mL of ultrapure water. After being agitated, the tubes were centrifuged at 8000 rpm for 15 min (4 °C). Supernatants were filtered using disposable sample filters (0.22 μm) and then analyzed via UPLC-MS/MS. The SMM and Met in the melon juices were analyzed in accordance with the procedure described above. Equations Used for GC-MS and UPLC-MS/MS Analysis. VSC and SMM/Met concentrations were determined in accordance with methods described by Rotsatchakul and co-workers and Kim and co-workers22,23 using MS response factors and quantitative ion areas for each compound relative to the internal standard:

C i = C is × fi × A i /A is where Ci and Ai are the concentration and quantitative ion peak area of compound i, respectively. Cis and Ais are the concentration and quantitative ion peak area of the labeled internal standard, respectively. f i is the MS response factor of compound i relative to labeled internal standards calculated using the standard curve. 5613

DOI: 10.1021/acs.jafc.8b01019 J. Agric. Food Chem. 2018, 66, 5612−5620

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Journal of Agricultural and Food Chemistry Table 1. Identification of Volatile Sulfide Compounds (VSCs) in Melon Juice after Heat Treatment retention indexab VSC DMS DMDS DMTS MTP

odor attributea cooked corn cooked cabbage cooked onion cooked potato

DBWAX

DB5MS

flavor dilutiond BL e

HHM 100

CL

FL

HM

100

50

250

JS

JL

JM

HTM

LBS

XZM

250

100

50

100

50

250

WW

YJM

identificationc

250

50

RI, MS, OA RI, MS, OA RI, MS, OA RI, MS, OA

628

504

50

1069

736

5

5

2.5

5

1

1

1

1

5

5

1

2.5

2.5

1366

968

5

5

5

5

2.5

5

2.5

5

2.5

1

5

5

1

1440

906

100

50

25

25

10

5

100

5

50

100

50

25

25

SS, SS, SS, SS,

a

Odor attribute and retention index were obtained from www.flavornet.org and www.nist.gov, respectively. bRetention index detected by a DB-WAX and DB-5 capillary column. cOA: odor attribute. RI: retention index. SS: standard substance. MS: mass spectrometry according to NIST08 library. d The ratio of maximum injection volume (25 mL) to minimum injection volume (no perceived odor attribute for the sample). eVSC having a contribution to the cooked flavor as FD ≥ 5. Validation Test. A specified amount of unlabeled and labeled standard substances, namely, SMM, Met, or a mixture of SMM and Met, was added to 5 L of original juice (clear melon juice), and the juices were agitated at 4 °C for 30 min at 200 rpm. The juices containing exogenous SMM and Met (additive juices) and original juices were then treated using HTST. The concentrations of SMM, Met, and VSCs (both labeled and unlabeled) in the additive and original juices were measured before and after HTST by UPLC-MS/MS and HS-SPME-GC-MS in accordance with the methods described above. The rate of increase of SMM/Met (Ipre) in additive juices, degradation rate of SMM/Met (Dpre) in HTST juices, and rate of increase of VSCs (Ivsc) in HTST juices were calculated as follows:

prepared using thick-skinned melons (HM, JS, XZM, and WW) from Xinjiang and Shandong production areas demonstrated the greatest FD factor for DMS (FD ≥ 250), whereas the FD factors of DMDS and DMTS in juices from thin-skinned melons (BL, CL, and FL) from the Gansu and Tianjin production areas were generally greater than those in juices from thick-skinned melons. According to previous reports, the juice flavors are closely related to fruit varieties and origins because of the flavor precursors and food matrices affecting the formation and release of volatile components.25,26 Moreover, the different maturity of each variety can lead to differences of precursors and matrices. Therefore, the authors speculate that the different precursor contents and effect of matrices on the degradation of precursors and release of VSCs would result in the different cooked off-flavor intensities of melon juices during HTST. Preliminary assessment of cooked off-flavor intensity by summing FD factors of four VSCs of each melon revealed that YJM juice showed the lowest cooked off-flavor intensity (FD = 58.5), whereas JS and WW juice demonstrated the highest intensity (FD = 300) after HTST. As shown in Figure 1, the

Ipre = C pre‐aj/C pre‐ck I vsc = Cvsc‐aj/Cvsc‐ck Dpre = (C pre‐fresh − C pre‐htst)/C pre − fresh where Cpre‑aj and Cpre‑ck are the concentrations of SMM/Met in the additive juice and original juice before HTST, respectively. Cvsc‑aj and Cvsc‑ck are the concentrations of VSCs in the additive and original juices after HTST. Cpre−fresh is the concentration of SMM/Met in the additive juice or original juice before HTST, and Cpre−htst is the concentration of SMM/Met in the additive juice or original juice after HTST. Statistical Analysis. All experiments in this work were carried out in triplicate. The means and standard deviations of the chromatographic data were acquired from SPSS software (version 17.0, Chicago, IL, USA). A univariate analysis of variance (ANOVA) was used to test the variances of the additives on each volatile sulfide compound in the validation test section. Then, multiple comparisons using the Duncan test were carried out for ANOVA with significance (p < 0.05) among the above data.

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RESULTS AND DISCUSSION Analysis of Cooked Off-Flavor Components and Intensity. DMS, DMDS, DMTS, and MTP were identified as key contributors to the cooked off-flavor in all HTST melon juices, as shown in Table 1. These compounds are known to contribute to the cooked off-flavor in some foods such as milk, wort, and various wines.12,13,24 In HTST melon juices, DMS was described as a cooked corn-like odor by the panelists, with the highest flavor dilution (FD) factors (50−250). The FD factors of MTP were between 25 and 100 with potato odor notes. DMDS and DMTS exhibited the lowest FD factors (1−5) with cooked cabbage- and cooked onion-like odors, respectively. The intensity and duration of the off-flavor positively correlated with the magnitude of the FD factor. Thus, both DMS and MTP majorly contributed to the cooked off-flavor in HTST melon juices. Further analysis found that the HTST juices

Figure 1. Contribution of dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and 3-(methylthio)propanal (MTP) to the cooked flavor in different melon juices after heat treatment.

odor intensity of the four VSCs was reassessed based on the odor activity value (OAV, the ratio of the concentration to odor threshold). The total OAV of VSCs in each melon was approximately 5000 after HTST, especially for XZM juice (>25000). This finding indicates that VSCs strongly influence the overall 5614

DOI: 10.1021/acs.jafc.8b01019 J. Agric. Food Chem. 2018, 66, 5612−5620

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Journal of Agricultural and Food Chemistry flavor.27 Similar to the results of FD analysis, juices prepared from thick-skinned melons generated a strong cooked off-flavor and YJM juice exhibited the lowest cooked off-flavor intensity according to total OAVs of four VSCs. Further analysis found that DMS contributed to the cooked off-flavor of all melon juices exceeding 50%, especially in thick-skinned melon juices. DMTS and DMDS affected the cooked off-flavor in juices from thin-skinned melons more highly than in juices from thickskinned melons. These results showed that varieties and origins affect the cooked off-flavor intensity of melon juices. The highest cooked off-flavor intensity was found in XZM juice according to OAVs rather than in JS or WW juices based on FD analysis, thereby demonstrating that the FD factor of DMS in XZM juice was greatest if the stepwise dilution of headspace volume was continued. Additionally, in HTST juices (BL, CL, and FL), the OAV of DMTS was higher than that of DMDS, but both exhibited the same (or low) FD factors. This finding suggested the volatile components, or the volatile components and matrices, that influence the release of VSCs may interact, and such interactions may change the odor intensity of odorants with a small OAV or FD factor.28 For these reasons, the OAV and FD factor were considered as complementary methods for the identification of VSCs in HTST melon juices. Relations between VSCs and Precursors. Four VSCs in 13 melon juices were first quantitated to analyze the relations between the concentrations of precursors and VSCs in HTST original juices. As shown in Table 1, the concentration of DMS (468.76−8604.82 μg/L) was higher than those of MTP (31.21− 431.11 μg/L), DMDS (4.14−23.43 μg/L), and DMTS (1.09− 13.12 μg/L) in all HTST juices. All VSCs were present at levels above their odor thresholds and contributed to the cooked off-flavor.27,29 Moreover, HTST juices from thick-skinned melons exhibited a higher concentration of DMS but a lower concentration of DMDS and DMTS than juices from thinskinned melons. The maximum MTP content was found in BL juice, but no relationship was found between the level of MTP and melon varieties. The quantitative data were consistent with the FD and OAV results, as shown in Tables 1 and 2, which indicated that DMS and MTP were the two most crucial cooked off-flavor components in HTST melon juices, especially in thick-skinned melon juices. For thin-skinned melon juices, DMTS was an important cooked off-flavor component, even though its levels were lower than those of DMDS because of its extremely low odor threshold (0.01 μg/L in water). The concentrations of SMM and Met in 13 melon juices were then measured in the original juices before HTST. All of the melon juices contained Met and SMM, thereby implying that these compounds are potential precursors of VSCs (Table 1). The highest concentration of SMM was found in XZM juice (18.36 μg/mL), followed by JS juice (13.35 μg/mL), and the lowest concentration was found in YJM juice (4.71 μg/mL), which was consistent with the concentration of DMS measured in the three melons (Table 1). However, except for the three melons mentioned above, the apparent contradiction between the concentrations of DMS and the distribution of SMM in thick- and thin-skinned melons suggests that some factors affect the conversion of SMM into DMS during heat treatment. Similarly, the highest concentration of Met was found in BL (69.19 μg/mL), and the lowest concentration was found in LBS (13.93 μg/mL). These results were consistent with the concentration of MTP but inconsistent with the concentrations of DMDS and DMTS among the melons. This finding suggests that Met may generate only MTP during HTST.

The contradictions between the concentrations of precursors and VSCs may have arisen from different degradation rates of the precursors, because the degradation of SMM and Met in different melons will be seriously affected by the food matrix, including precursors, dissolved oxygen, reducing sugars, amino acids, and acidity.25,26,30,31 Degradation rate was calculated by quantitating SMM or Met before and after HTST to avoid the matrix influence between different melons (Table 2). The trend of SMM/Met degradation was consistent with the amount of DMS/MTP formed without regard to the influence on VSC release by the matrix. This finding demonstrated the effect of the matrix on SMM/Met degradation and illustrated the apparent contradiction between the concentration of precursors and concentration of VSCs in the melons. However, no relationship was found between Met and DMDS and DMTS in HTST melon juice. This suggests that SMM and Met may be the precursors of DMS and MTP, whereas other precursors for DMDS and DMTS are present. MTP is known to be a Maillard aldehyde, mainly generated from Met through Strecker degradation, which can be oxidized into DMDS and DMTS during heat processing.32 Thus, DMDS and DMTS might be generated from Met when MTP was present in HTST melon juices. It has been proposed that DMDS and DMTS may only be partially generated from thermal degradation of Met, whereas some were derived from unknown precursors, such as intermediate products of Met metabolism or enzyme action during the temperature rise of the melon juice, thereby leading to no specific connection between them.33−36 In the current work, we did not explore the unknown precursors and related enzymes of DMDS and DMTS due to their small contribution to the cooked off-flavor for most of the HTST melon juices. Identification of Precursors. The degradation rates of SMM and Met were analyzed in the original and additive juices containing exogenous SMM or Met after HTST because of their great influence on the formation of VSCs. No obvious differences between the degradation rates of different melon varieties were found (Figure 2). Thus, the rate of increase of SMM or Met in the additive juices before HTST will be similar to that of the VSCs after HTST if they were corresponding precursors. Based on the results, the relevance analysis was carried out to identify the precursors of VSCs by comparison of SMM and Met with VSCs in additive juice and original juice after HTST. The two-way multivariate analysis of variance of VSCs and SMM and Met are summarized in Figure 3. No significant interaction (p < 0.05) between SMM and Met on the formation of DMS, DMDS, and DMTS was found, thereby indicating that the formation of these three VSCs may be influenced by only one of the two proposed precursors. The addition of SMM led to an extremely significant increase of DMS (p < 0.01) in the additive juice after HTST. Moreover, a similar rate of increase of SMM (Figure 2) and DMS (Figure 3) was found in additive juice. Those results showed that SMM may be the unique precursor of DMS. No significant effect (p > 0.05) was found between SMM and DMDS and DMTS, thereby indicating that SMM posed a weak (or no) effect on formation of these two VSCs. By contrast, Met addition significantly (p < 0.01) increased DMDS and DMTS in additive juices after HTST, indicating that Met may be the common precursor of DMDS and DMTS. Nevertheless, the obvious differences in the rate of increase of Met (Figure 2) and the increases of DMDS and DMTS in additive juice (Figure 3) suggested that other precursors for 5615

DOI: 10.1021/acs.jafc.8b01019 J. Agric. Food Chem. 2018, 66, 5612−5620

5616

11.81 ± 2.93

12.12 ± 2.36

D-Met (%)a

a

49.79 ± 8.41

69.19 ± 8.58

Met

3.30 ± 1.06

34.24 ± 6.83

37.86 ± 5.74

7.68 ± 2.54

7.50 ± 2.37

42.98 ± 8.35

34.21 ± 8.77

7.34 ± 2.87

250.73 ± 25.23

10.74 ± 2.74

52.51 ± 8.95

62.71 ± 9.42

13.35 ± 4.03

394.74 ± 35.73

3.24 ± 1.12

5.81 ± 1.85

2.30 ± 1.03

12.10 ± 3.06

7.00 ± 1.42

45.21 ± 8.56

62.12 ± 9.68

7.60 ± 2.24

7.43 ± 2.53

40.86 ± 9.27

51.26 ± 8.77

6.90 ± 2.14

7.06 ± 2.49

34.35 ± 9.36

40.92 ± 7.73

6.29 ± 2.62

274.76 ± 26.38 138.01 ± 18.79 124.34 ± 21.05

2.29 ± 1.04

4.14 ± 1.73

D-SMM: degradation rate of SMM. D-Met: degradation rate of Met. bThe mean and standard deviation. cStandard curve.

3.99 ± 1.23

36.30 ± 6.48

48.52 ± 7.36

38.59 ± 6.04

SMM (%)a

D-

8.83 ± 2.84

6.29 ± 2.37

7.48 ± 2.26

SMM

84.90 ± 16.39

3.11 ± 1.03

4.53 ± 2.04

4.67 ± 40.28

13.93 ± 3.16

52.46 ± 10.21

6.90 ± 2.24

44.92 ± 9.17

1.71 ± 0.52

13.03 ± 2.67

3.54 ± 1.47

8.16 ± 3.11

YJM

9.39 ± 2.28

3.56 ± 1.25 12.73 ± 2.79

22.76 ± 4.66 61.74 ± 9.42

47.52 ± 40.28 67.62 ± 8.64

18.36 ± 3.25

SCc

1.09 ± 0.25

9.38 ± 3.23

5.78 ± 2.28

17.90 ± 3.29

57.38 ± 8.68

4.71 ± 1.08

y = 2.0892x − 0.0855; R2 = 0.9996

36.47 ± 6.73

y = 1.2814x + 0.0887; R2 = 0.9998

y = 0.6497x − 0.0173; R2 = 0.9936

y = 0.8702x − 0.1298; R2 = 0.9623

y = 1.1004x − 0.1167; R2 = 0.9745

468.76 ± 39.26 y = 1.0622x − 0.3365; R2 = 0.9897

32.23 ± 4.13 357.80 ± 31.65 31.21 ± 6.02

4.70 ± 2.12

5.37 ± 1.28

85.28 ± 13.04

1.67 ± 0.62

3.42 ± 1.27

431.11 ± 239.46 ± 32.11 22.32

MTP

10.53 ± 2.83

23.43 ± 3.42

4.27 ± 1.21

LBS

13.12 ± 2.67

HTM

9.60 ± 2.14

JM

DMTS

11.44 ± 2.17

JL

17.61 ± 3.85

JS

17.20 ± 3.72

HM

DMDS

FL

WW

CL 3172.70 ± 123.48

HHM

XZM

BL

585.48 ± 939.63 ± 1363.94 ± 91.75 545.18 ± 38.81 2007.12 ± 84.63 6219.76 ± 211.26 1577.14 ± 99.63 638.37 ± 45.47 985.52 ± 70.39 874.67 ± 68.93 8604.82 ± 40.28 62.54 267.38

sulfides

DMS

contentb (μg/L)

Table 2. Content of Volatile Sulfide Compounds (VSCs), S-Methylmethionine (SMM), and Methionine (Met) and the Standard Curve in Different Melon Varieties (n = 3)

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.8b01019 J. Agric. Food Chem. 2018, 66, 5612−5620

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Journal of Agricultural and Food Chemistry

found in HTST melon juice. The labeled VSCs formed from the labeled additive juices (2H6-SMM juice and 2H3-Met juice) during heat treatment were further studied. SMM, Met, and VSCs (unlabeled and labeled) in melon juice were identified (Figure 4). Compared with that of original juice, the 2H6-DMS (Ion 69) detected in 2H6-SMM juice after HTST showed that the cleavage of carbon−sulfur bonds within SMM resulted in DMS formation. This finding also indicates that SMM is the precursor of DMS in melon juice. Combined with the results of relevance analysis, we confirm that SMM is the unique precursor of DMS in melon juice. The formation mechanism of DMS in an acid environment (pH 2−5) is different from those in an alkaline environment (pH > 7).42 Nonetheless, the reaction rate under alkaline conditions was much higher than that under acid conditions.38 The values of clear juice from the 13 different melons were pH 5.5−6.5, suggesting that both mechanisms may be active. The lack of 2H3-DMS (Ion 65) in 2 H6-SMM juice suggests that no free methyl or methanethiol formed from SMM, indicating that SMM cannot transform into Met by demethylation in melon juice during HTST. Although Wilson proposed that SMM can degrade into Met by demethylation at high temperatures,24 no reports describing the mutual transformation of SMM and Met in fruit juices were found at present. Except at high peak intensity, no labeled DMDS, DMTS, or MTP were found in 2H6-SMM juice compared with original juice. This demonstrates that SMM is capable of increasing the concentrations of MTP by promoting Met degradation instead of transforming into them. The authors speculated that SMM promoted Met degradation by increasing the content of dicarbonyl compounds (such as methylglyoxal and glyoxal), which are the important reactants for Strecker degradation.43 SMM increased dicarbonyl compound concentration because of two possible reasons: (1) Amddery products from SMM and homoserine can produce dicarbonyl compounds during heat treatment44 or (2) SMM and homoserine (free amino acids) can accelerate degradation of reducing sugar resulting in the formation of dicarbonyl compounds.45 Five labeled VSCs, including 2H3-DMDS (Ion 97), 2H6-DMDS (Ion 100), 2H3-DMTS (Ion 129), 2H6-DMDS (Ion 132), and 2 H3-MTP (Ion 107), were found in 2H3-Met juice after HTST, demonstrating that Met can generate DMDS, DMTS, and MTP in melon juice during heat treatment. According to previous reports, MTP resulting from the Strecker degradation of Met decomposes into methanethiol, which can be further oxidized into DMDS and DMTS at high temperatures.32,41 Nevertheless, sulfur donors, such as hydrogen sulfide or elemental sulfur from cysteine and thiamine in melon juice, are required for the formation of DMTS.46,47 In the current study, although methanethiol was not identified in this set of experiments, 2H3-DMDS and 2H3-DMTS, which formed in 2H3-Met juice, showed free methanethiol produced from labeled and unlabeled MTP. Thus, cysteine and thiamine were also suggested as the indirect sulfur donors.11,46,47 Labeled DMS was not detected, indicating that the concentration of DMS was not influenced by the addition of Met to melon juice after HTST. This finding was consistent with our relevance analysis results, suggesting that Met cannot transform into DMS due to a lack of methyl donors. The labeled additive test further demonstrates that SMM and Met are the unique precursors of DMS and MTP and that Met is also the precursor of DMDS and DMTS. In addition, several other compounds potentially generate DMDS and DMTS in melon juice during heat treatment. For example, the intermediate products of Met (1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one)

Figure 2. Rates of increase and degradation of S-methylmethionine (SMM) and methionine (Met) in original juice and additive juice.

Figure 3. Rate of increase of dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and 3-(methylthio)propanal (MTP) in additive juice after HTST.

DMDS and DMTS may still be present in melon juice as the above analysis indicates.33−37 Met addition could not significantly (p > 0.05) increase DMS, implying Met cannot degrade into DMS or facilitate DMS precursor degradation in clear melon juice. Some reports have demonstrated that DMS can be produced from Met during heat treatment,13,38 but the methyl donors, such as pectin, an important reactant for DMS formation from Met, may have been eliminated during the preparation of clear juice by ultrafiltration.39,40 Met could transform into MTP, by the Maillard reaction or the retro-Michael reaction, during heat processing.41 In the current work, Met addition significantly increased the concentrate of MTP in additive juice (p < 0.01) after HTST. Meanwhile, a similar rate of increase of Met (Figure 2) and of MTP (Figure 3) was found in additive juice. These results meant that if Met was the precursor of MTP in melon juice, it was a unique one. However, SMM addition could also significantly (p < 0.05) affect the formation of MTP, suggesting that SMM can transform into MTP or can promote the degradation of their precursors. In combination with the effect of Met on MTP and the significant interaction between SMM and Met (p < 0.05), SMM probably accelerates the degradation of Met in clear melon juice during HTST. Formation Mechanisms. Relevance analysis revealed that SMM and Met were the possible precursors of the four VSCs 5617

DOI: 10.1021/acs.jafc.8b01019 J. Agric. Food Chem. 2018, 66, 5612−5620

Article

Journal of Agricultural and Food Chemistry

Figure 4. (a and b) The content of S-methylmethionine (SMM), methionine (Met), and volatile sulfide compounds (VSCs) (labeled and unlabeled compounds) in melon juice after HTST.

can produce DMTS during the storage of Japanese sake.37 Therefore, the authors speculate that the unknown precursors of DMDS and DMTS may be the intermediate products containing a methylthio group formed in methionine metabolism during the growth and postharvest of the melons, such as methylthionbose and α-keto-γ-methylthionbuyric acid, as reported by Gonda and co-workers and Kim and co-workers.35,36 In the current study, the formation mechanisms of DMS, MTP, and DMDS and DMTS, to a limited extent, in clear melon juice during heat treatment are summarized in Figure 5. SMM can be decomposed through a substitution reaction by water or intramolecular carboxyl group producing DMS, homoserine, or homoserine lactone in melon juice during HTST. Met first transforms into MTP through Strecker degradation, and then MTP degrades into methanethiol due to a carbon− sulfur bond rupture under high temperature. Methanethiol, a key intermediate product, can be oxidized into DMDS and

DMTS. Hydrogen sulfide and elemental sulfur are common sulfur donors occurring mainly from the thermal degradation of cysteine and thiamine in melon juice. SMM can increase the amount of Amddery products and accelerate the degradation of reducing sugars to yield a high concentration of active dicarbonyl compounds, which promote the degradation of Met. In addition, trisulfide can transform into disulfide by a disproportionation reaction at high temperature due to its high reactivity.48 In general, the cooked off-flavor intensity of thick-skinned melons was much higher than that of thin-skinned melons. DMS and MTP were the key cooked off-flavor components in both thick- and thin-skinned melon juices after heat treatment. However, DMDS and DMTS exhibited relatively high odor intensities in several thin-skinned melon juices. SMM can degrade into DMS through a substitution reaction, whereas Met transforms into MTP, DMDS, and DMTS through a chain reaction, including Strecker degradation, thermal degradation, 5618

DOI: 10.1021/acs.jafc.8b01019 J. Agric. Food Chem. 2018, 66, 5612−5620

Article

Journal of Agricultural and Food Chemistry

Figure 5. Proposed formation mechanism of volatile sulfide compounds of clear melon juice during heat treatment. (6) Pang, X. L. Identification and formation mechanism of predominant contributors to off-odors in thermally processed muskmelon juice. Doctoral Thesis, China Agricultural University: Beijing, China, 2015; pp 92− 100. (7) Nath, N.; Ranganna, S. Evaluation of a thermal process for acidified canned muskmelon (Cucumis Melo L.). J. Food Sci. 1977, 42, 1306−1310. (8) Sun, X. X.; Baldwin, E. A.; Plotto, A.; Manthey, J. A.; Duan, Y. P.; Bai, J. H. Effects of thermal processing and pulp filtration on physical, chemical and sensory properties of winter melon juice. J. Sci. Food Agric. 2017, 97, 543−550. (9) Averbeck, M.; Schieberle, P. Influence of different storage conditions on changes in the key aroma compounds of orange juice reconstituted from concentrate. Eur. Food Res. Technol. 2011, 232, 129−142. (10) Scherb, J.; Kreissl, J.; Haupt, S.; Schieberle, P. Quantitation of Smethylmethionine in raw vegetables and green malt by a stable isotope dilution assay using LC-MS/MS: Comparison with dimethyl sulfide formation after heat treatment. J. Agric. Food Chem. 2009, 57, 9091− 9096. (11) Attieh, J.; Djiana, R.; Koonjul, P.; Etienne, C.; Sparace, S. A.; Saini, H. S. Cloning and functional expression of two plant thiol methyltransferases: a new class of enzymes involved in the biosynthesis of sulfur volatiles. Plant Mol. Biol. 2002, 50, 511−521. (12) Zabbia, A.; Buys, E. M.; De Kock, H. L. Undesirable sulphur and carbonyl flavor compounds in UHT milk: a review. Crit. Rev. Food Sci. Nutr. 2012, 52, 21−30. (13) Scheuren, H.; Sommer, K.; Dillenburger, M. Explanation for the increase in free dimethyl sulphide during mashing. J. Inst. Brew. 2015, 121, 418−420. (14) Steinhaus, M.; Thomas, K.; Schieberle, P. Molecular Insights into Off-Flavor Formation during Pineapple Juice Processing. In Flavour Science. Proceedings from XIII Weurman Flavour Research Symposium. Ferreira, V., Lopez, R., Eds.; Academic Press: Amsterdam, The Netherlands, 2013; pp 87−90. (15) Zhao, L.; Wang, Y. T.; Hu, X. T.; Sun, Z. J.; Liao, X. J. Korla pear juice treated by ultrafiltration followed by high pressure processing or high temperature short time. LWT-Food Sci. Technol. 2016, 65, 283−289. (16) Jittanit, W.; Wiriyaputtipong, S.; Charoenpornworanam, H.; Songsermpong, S. Effects of varieties, heat pretreatment and UHT conditions on the sugarcane juice quality. Chiang Mai J. Sci. 2011, 38, 116−125. (17) Zhou, Q. X.; Wintersteen, C. L.; Cadwallader, K. R. Identification and quantitation of aroma-active components that contribute to the distinct malty flavor of buckwheat honey. J. Agric. Food Chem. 2002, 50, 2016−2021. (18) Luo, D. S.; Chen, J.; Gao, L.; Liu, Y. P.; Wu, J. H. Geographical origin identification and quality control of Chinese chrysanthemum flower teas using gas chromatography-mass spectrometry and

and oxidation. SMM is the only precursor of DMS, and Met is the unique precursor of MTP. Nevertheless, DMDS and DMTS may only be partially formed from Met (