The Effects of Antioxidants on the Changes in Volatile Compounds in Heated Welsh Onions (Allium fistulosum L.) during Storage

Welsh onion (Allium fistulosum L.) is usually used to enhance the flavor characteristics of various foods. Volatile compounds in Welsh onions, including sulfur-containing compounds, may vary during heat process and storage. Accordingly, the changes in the volatile compounds in Welsh onions, subjected to heat and antioxidant (ascorbic acid and glutathione) treatments during storage, are investigated in the present study. The majority of sulfur-containing compounds in Welsh onions showed significant differences between the untreated Welsh onions and heated Welsh onions. During the heating of the Welsh onions, some sulfur-containing compounds, such as 2-methylthiirane, 1-(methyldisulfanyl)prop-1-ene, 1-[[(E)-prop-1-enyl]disulfanyl]propane, 1-(propyltrisulfanyl)propane, 1-[[(E)-prop-1-enyl]trisulfanyl]propane, and (methyltetrasulfanyl)methane, showed significant differences between the untreated and heated Welsh onions (p < 0.05). In addition, partial least square discriminant analysis (PLS-DA) was applied to discriminate the heated Welsh onion samples added with different antioxidants. The heated Welsh onion samples added with ascorbic acid was mainly associated with 2-phenylacetaldehyde, acetic acid, methylsulfanylmethane, prop-2-ene-1-thiol, undecan-2-one, and (2E,4E)-deca-2,4-dienal. Moreover, the key volatile compounds in the heated Welsh onion samples added with glutathione were 3-ethylthiophene, 1-(methyldisulfanyl)-1-methylsulfanylpropane, 1-methylsulfanylpentane, 2-prop-2-enylsulfanylpropane, and 1-propan-2-ylsulfanylbutane.

Some studies found that these sulfur-containing compounds in the Allium genus can be changed by processing methods, such as high-temperature heating, drying, roasting, crushing, and aging [2][3][4][5][6][7][8]. In addition, Allium sulfides can be produced by thermal decomposition during the heating process, and their structural changes can occur by thermal reactions [7]. Accordingly, organosulfur compounds in Welsh onions can be changed during processing and storage, leading to the chemical transformation of various volatile components that can significantly affect the flavor quality [9,10].
Several studies have been conducted to improve the shelf life and quality of Welsh onions [11]. The volatile compounds in Welsh onions were affected by oxidizing conditions, which are inevitable in food systems [12]. These oxidizing conditions can be caused by various factors, such as temperature, moisture, gas, and pH levels during processing and storage, which spontaneously create oxidizing agents [13]. Reactive oxygen species and radicals produced under oxidizing conditions react with them, and can then lead to the loss of their original properties, resulting in a deterioration in the quality of food systems [14]. Therefore, antioxidants can be used to maintain the quality properties of Welsh onions during storage.
Ascorbic acid, which prevents browning and improves the nutritional value, can be found in many food systems, either naturally or as an antioxidant additive [15]. In addition, ascorbic acid is an antioxidant that can be classified as a singlet oxygen or oxygen scavenger, and it can react with free radicals to remove it [16]. Glutathione, which is well known as an antioxidant, transforms itself into oxidized glutathione (glutathione disulfide, GSSG), which breaks the disulfide bond of the surrounding substances and reduces it to cysteine [17]. This oxidized glutathione (GSSG) undergoes a cycle that is reduced again by a reductase or chemical reaction [17]. As a non-enzymatic mechanism, glutathione turns into an oxidized form and prevents the oxidation of other substances, or forms disulfide by combining sulfur with itself to prevent oxidation [17].
Various studies have been conducted to investigate the changes in the volatile compounds in the Allium plant after the heat process [18,19]. However, to date, studies on the effects of antioxidants on the changes in the volatile compounds in heated Welsh onion have not been conducted. Therefore, the objectives of this study are to investigate the effects of heat treatment and the application of antioxidants on the changes in the volatile compounds in Welsh onions during storage.

The Changes in the Volatile Compounds in Welsh Onions by Heat Treatment
The volatile compounds in untreated and heat-treated Welsh onion samples were analyzed using gas chromatography-mass spectrometry (GC-MS) combined with solidphase microextraction (SPME). A total of 81 identified volatile compounds in the untreated and heat-treated Welsh onion samples are listed in Table 1. Among these volatile compounds, sulfur-containing compounds, such as 2-methylthiirane (propylene sulfide); 1-(methyldisulfanyl)prop-1-ene; 1-[[I-prop-1-enyl]disulfanyl]propane (propI(E)-1-propenyl disulfide); 1-(propyltrisulfanyl)propane; 1-[[(E)-prop-1-enyl]trisulfanyl]propane; and (meth yltetrasulfanyl)methane (dimethyl tetrasulfide), were significantly increased (p < 0.05) after the heat treatment. (1) All volatile compounds are listed by the order of their RI values in chemical class. (2) Retention indices were determined using n-alkanes C 6 -C 30 as external standards. (3) Mean values of the relative peak area to that of the internal standard ± standard deviation (n = 3). (4) Identification of the compounds was based on the following: A, the mass spectrum and retention index agreed with those of the authentic compounds under the same conditions (positive identification); B, the mass spectrum and retention index were consistent with those from the NIST database (tentative identification); and C, the mass spectrum was consistent with that of W9N08 (Wiley and NIST) and manual interpretation (tentative identification). (5) The letter 'a' indicates a significant difference between the two samples in a row using the t-test (p < 0.05). (6) N.D. = not detected.
Some volatile compounds, such as propanoic acid; heptanoic acid; pent-1-en-3-ol; prop-1-ene; and 3-methylthiophene-2-carbaldehyde, were only detected in untreated Welsh onion samples. In addition, most of the aldehydes tended to decrease or disappear after heating. It can be considered that those aldehydes underwent thermal decomposition or oxidation during heating [18].

The Effects of Antioxidants on the Change in the Volatile Compounds in Heated Welsh Onions during Storage Periods
The volatile compounds in the heated Welsh onion samples added with antioxidants, such as ascorbic acid and glutathione, during storage periods are listed in Table 2. In total, 65 volatile compounds were identified: 2 acids, 5 alcohols, 16 aldehydes, 1 ester, 2 furans, 5 hydrocarbons, 3 ketones, and 31 sulfur-containing compounds (3 thiols; 7 sulfides; 6 disulfides; 4 trisulfides; 1 tetrasulfide; 8 cyclic polysulfides; and 2 others).
The abundances of most volatile compounds in heated Welsh onion samples tended to decrease according to the storage time. Most of the aldehydes showed such behavior; however, some of them, such as (E)-oct-2-enal and decanal, were not changed significantly in the sample added with glutathione, compared to the other samples during 3-7 days of storage time. In addition, benzaldehyde, (2E,4E)-hepta-2,4-dienal, and (2E,4E)-deca-2,4dienal were not detected in the samples added with glutathione during the storage periods.    (1) All volatile compounds were grouped in chemical classes and listed in the order of their RI values. ( 2) Retention indices were determined using n-alkanes C 7 -C 30 as external standards.
(3) Mean values of the relative peak area to that of the internal standard ± standard deviation. (4) 0: unstored Welsh onion samples; 1: sample after one day; 3: sample after three days; 5: sample after five days; 7: sample after seven days; Control: heat-treatment sample; Ascorbic acid: heat-treatment sample with ascorbic acid; and Glutathione: heat-treatment sample with glutathione. (5) The identification of the compounds was based on the following: A, the mass spectrum and retention index agreed with those of the W9N08 mass spectral database, and the literatures and authentic standards; B, the mass spectrum was identical with that of the W9N08 mass spectral database, and the retention index was consistent with that in the literature; and C, the mass spectrum was consistent with that of W9N08 (Wiley and NIST) and manual interpretation. (6) Significant differences (p < 0.05) between the heated welsh onion samples according to the storage periods using Duncan's multiple comparison test. (7) N.D. = not detected.
The contents of most sulfur-containing compounds, except for thiols, decreased significantly according to storage periods. The contents of some linear and branched polysulfides, such as 1-(methyldisulfanyl)prop-1-ene, 1-[(E)-prop-1-enyl]disulfanyl]propane, (methyltrisulfanyl)methane, 1-(propyltrisulfanyl)propane, 1-[(E)-prop-1-enyl]trisulfanyl]propane, and (methyltetrasulfanyl)methane, which were the most abundant volatile compounds in the heated Welsh onion samples, decreased with the storage periods. These are known to be the predominant compounds in Welsh onions, and can also be related to the flavor characteristics of Allium plants [19][20][21][22][23]. The disulfide bonds of sulfur compounds can be cleaved to produce thio radicals and these radicals are attached to the double bond of another disulfide molecule to form a polysulfide [25]. In addition, the loss of a hydrogen molecule from the alkylthio group can form branched sulfides. In contrast with these sulfur compounds, the contents of some sulfides, such as methylsulfanylmethane (dimethyl sulfide), 3-prop-2-enylsulfanylprop-1-ene (diallyl sulfide), and (methyldisulfanyl)methane (dimethyl disulfide), were relatively low and decreased according to the storage periods. These sulfides can significantly influence the odor characteristics of Welsh onions, due to the low thresholds of such odor notes (unique cooked onion, cooked cabbage, and garlic-like odor notes), ranging from 0.16 to 1.2 ppb [26].
However, cyclic sulfur compounds, such as 2,4-dimethylthiophene, 2,3-dimethylthiop hene, 2,5-dimethylthiophene, and 3,4-dimethylthiophene, slightly increased during the initial storage periods (1 and 2 days), possibly due to the rearrangement and cyclization of the fragments from other sulfur-containing compounds. In addition, 3,5-dimethyl-2-(methylthio)-thiophene was increased in the samples added with antioxidants (ascorbic acid and glutathione), according to the storage periods in this study, possibly because highly reactive radicals can attack the linear structure of compounds to form another ring structure [25].
The contents of most sulfur compounds showed a tendency to decrease in the heated Welsh onion samples without antioxidants, according to the storage periods. On the other hand, the thiols and sulfides showed a tendency to increase in the samples added with antioxidants at the initial periods of storage (up to 3 days), whereas they decreased at later periods of storage. Some sulfur compounds, such as 1-methylsulfanylpentane (amyl methyl sulfide) and 1-propan-2-ylsulfanylbutane (butyl isopropyl sulfide), were identified only in the samples added with glutathione, possibly because hydrogen sulfide released from glutathione can participate in the formation of these sulfur compounds by the polymerization and rearrangement of non-enzymatic mechanisms [27]. In addition, it can be assumed that, when a large amount of hydrogen sulfide was produced according to the storage periods, it would react with unstable free radicals to produce other compounds.
Partial least square discriminant analysis (PLS-DA) was conducted to investigate the effects of different antioxidants on the change in the volatile compounds in the heated Welsh onion samples during storage. Figure 1 shows the PLS-DA score plot for the comparison of the volatile compounds in heated Welsh onion samples added with antioxidants (ascorbic acid and glutathione). PLS (partial least square) component 1 (PLS 1) and PLS component 2 (PLS 2) explained 38.0% and 26.3% of the variance, respectively, and, hence, together explained 64.3% of the total variance. The parameters of the cross-validation modeling were component 3, R 2 X = 0.49, R 2 Y = 0.93, and Q 2 Y = 0.40. A permutation test involving 100 iterations was also conducted to validate the model, which yielded R 2 = 0.056 and Q 2 = −0.282.

Materials
Welsh onions (Allium fistulosum) were purchased from Nonghyup (Goyang-si, Gyeong gi-do, Korea). The Welsh onions were cultivated in Jangseong-gun, Jeollanam-do in South Korea (2020). All the samples were stored at 25 • C in a temperature and humid chamber (Han Baek Scientific Co., Bucheon-si, Korea), before they were analyzed. The solid-phase microextraction (SPME) fibers and holders were purchased from Supelco (Bellefonte, PA, USA), whereas the vial and screw caps (Ultraclean 18 mm) were purchased from Agilent Technologies (Santa Clara, CA, USA). β-damascone was purchased from Sigma-Aldrich (St. Louis, MO, USA). The mineral oil was purchased from Sigma-Aldrich (St. Louis, MO, USA). The methanol and water were purchased from J.T.Baker (Phillipsburg, NJ, USA). The antioxidants, L-ascorbic acid and L-glutathione, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The Preparation of the Welsh Onion Samples by Heating
A total of 800 g of washed Welsh onions and 800 g of purified water were ground in a blender, and 1.5 kg of Welsh onion juice was obtained. The Welsh onion sample was placed into a 1 L flask and heated in a 2 L oil bath. After preheating at 70 • C for 5 min, it was heated at 100 • C for 10 min.

The Storage Conditions of the Heated Welsh Onion Samples
Each 20 mL of heated Welsh onion samples in a 50 mL amber laboratory bottle was stored at 25 • C in constant temperature and humidity chamber (Han Baek Scientific Co., Bucheon-si, Korea). The different antioxidants, L-ascorbic acid and L-glutathione, were added to the heated Welsh onion samples at a concentration of 0.05g/100g (w/w). The Welsh onion samples were sealed with a PBT screw cap and coated silicon PTFE gasket to prevent the loss of volatile compounds. During the storage periods, the samples were obtained at 0, 1, 3, 5, and 7 days.

The Extraction of Volatile Compounds Using SPME
A sample of Welsh onions (5 g) was placed in a 20 mL vial; β-damascone (1 mg/mLin β-damascone/methanol solvent mixture (1:200, v/v)) was added as an internal standard, and the vial was sealed with a screw cap. SPME was used to obtain the volatile compounds of Welsh onions. The sample was maintained at 60 • C for 30 min to reach a state of equilibrium. An SPME fiber coated with carboxen/polydimethylsiloxane (CAR/PDMS) was used to adsorb volatile compounds at 40 • C for 20 min, and desorption was executed at 230 • C in a GC injector for 5 min.

GC-MS Analysis
GC-MS analysis was performed using a 7890A series gas chromatograph (Agilent Technologies) and a 5975C mass detector (Agilent Technologies) equipped with a DB-5MS column (30 m length × 0.25 mm i.d. × 0.25 µm film thickness, J&W Scientific, Folsom, CA, USA). The GC oven temperature was programed as follows: the initial temperature was maintained at 40 • C for 6 min, raised to 170 • C at a rate of 5 • C/min and held for 3 min, and increased to 220 • C at a rate of 5 • C/min. The flow rate of helium, the carrier gas, was constant at 0.8 mL/min, whereas the mass spectra was obtained with a mass scan rage of 35-350 atomic mass units (a.m.u.) at a rate of 4.5 scans/s, and the electron impact (EI) mode was 70 eV. All the samples were prepared and analyzed in triplicate.

The Identification and Semi-Quantification of the Volatile Compounds
The identification of each volatile compound was positively confirmed by a comparison of the retention time and mass spectral data with those of the authentic standard compounds. When the standard compounds were not available, each volatile compound was identified on the basis of its mass spectral data using the NIST.08 and Wiley.9 mass spec-tral libraries and the retention index (RI) values from the previous literature [1,2,20,26,27]. The RI value of the volatile compounds was calculated with the n-alkanes from C 6 to C 30 as the external standards. The quantification of the volatile compounds was performed by comparing their peak areas with that of the internal standard compound on the total ion chromatogram of GC-MS.

The Statistical Analysis
The significance of the differences (p < 0.05) between the untreated Welsh onion samples and the heated Welsh onion samples were evaluated by Duncan's t-test using the SPSS program (version 12.0, Chicago, IL, USA). Multivariate statistical analysis, partial least square discriminant analysis (PLS-DA), was conducted using SIMCA-P (version 11.0, Umetrics, Umea, Sweden) to determine the key volatile compounds related to the discrimination of the heated Welsh onions added with antioxidants.

Conclusions
This study investigated the changes in the volatile compounds in Welsh onions by heat treatment and the effects of antioxidants (ascorbic acid and glutathione) on the changes in the volatile compounds during storage. Some sulfur-containing compounds, such The application of PLS-DA to the data sets of volatile compounds revealed that heated Welsh onion samples can be distinguished according to different antioxidants, such as ascorbic acid and glutathione. 3-ethylthiophene; 1-(methyldisulfanyl)-1-methylsulfanylpropane; 1-methylsulfanylpentane; 2-prop-2-enylsulfanylpropane; and 1-propan-2-ylsulfanylbutane were strongly correlated with the heated Welsh onion samples added with glutathione. On the other hand, 2-phenylacetaldehyde; acetic acid; methylsulfanylmethane; prop-2ene-1-thiol; undecan-2-one; and (2E,4E)-deca-2,4-dienal were related to the heated Welsh onion samples added with ascorbic acid. These findings indicate that the profiles of the volatile compounds of the heated Welsh onion samples added with different antioxidants can significantly change the volatile profiles during storage. These results can be used to improve the quality of Welsh onion-based products and develop processed foods.