Quantification and Distribution of Thiols in Fermented Grains of Sauce-Aroma Baijiu Production Process

Five volatile thiol compounds (methanethiol, ethanethiol, 2-mercapto-1-ethanol, 2-furfurylthiol, and 2-methyl-3-furanethiol) in fermented grains of sauce-aroma baijiu were determined using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The samples were pre-treated using a modified QuEChERS method. 4,4′-Dithiodipyridine (DTDP) derivatization reaction improved the detectability and stability of volatile thiol compounds. From the end of the first round to the end of the seventh round of fermentation and different fermentation states from the fifth round of fermented grains of the sauce-aroma baijiu production process were analyzed. The results showed that the concentrations of methanethiol (67.64–205.37 μg/kg), ethanethiol (1.22–1.76 μg/kg), 2-furfurylthiol (0.51–3.03 μg/kg), and 2-methyl-3-furanthiol (1.70–12.74 μg/kg) were increased with the number of fermentation rounds. Methanethiol, 2-furfurylthiol, and 2-methyl-3-furanthiol increased during fermentation and distillation in the fifth round. Fermentation and distillation were important stages for their widespread production. After distillation, there were still a large number of volatile thiol compounds in the fermented grains. The thermal reaction was of great significance in the formation of these thiols.


Introduction
Volatile thiol compounds in alcoholic beverages (wine, baijiu, beer, etc.) are important contributors to sensory aroma. These thiols with low thresholds exhibit powerful and characteristic odors [1,2] that can contribute to either pleasant or unpleasant aromas in alcoholic beverages, depending on their nature and concentration [3][4][5]. Such compounds include 2-methyl-3-furanethiol (odor thresholds: 0.0048 µg/L in 46% ethanol-water [6], roasted meat, fried), 2-furfurylthiol (0.1 µg/L, coffee, roasted sesame seeds), ethanethiol (0.8 µg/L, onion, rubber), methanethiol (2.2 µg/L, burned rubber, gasoline), etc. In the last decade, volatile thiols, which are important for sensory qualities, have gained widespread interest in baijiu [7][8][9]. The concentrations of these volatile thiol compounds are well above their low odor thresholds in baijiu [10]. However, an excessive concentration of thiols results in an abnormal smell that gives sauce-aroma baijiu its pickle-like off-odor [11,12]. This abnormal smell eventually degrades the quality of baijiu. Balancing the concentrations of these potentially off-odor compounds in alcoholic beverages is currently a major challenge. Therefore, determining the concentration of thiols and gaining insight into their production during the fermentation process will be beneficial for achieving a balance of thiols in alcoholic beverages.
Low concentrations, poor stability, and the complexity of the matrix make the qualitative and quantitative analysis of volatile thiol compounds with various odor flavors

Fermented Grains of Baijiu
The production of sauce-aroma baijiu was carried out by feeding only two batches of fresh grains. After the first round of distillation, the grains after distillation were spread on the ground to cool and mixed with daqu powder, and distillates were collected from the first round of distillation, ready for the second round of heap fermentation and alcoholic fermentation. Seven iterations of the fermentation processes were carried out, from heap fermentation to alcoholic fermentation. From the end of the first round to the end of the seventh round of fermentation, fermented grains of baijiu from Chinese sauce-aroma baijiu-producing regions (Maotai Town, Guizhou Province) were collected. Additionally, initial grains, heaped grains, fermented grains, distilled grains, and samples from the fifth round of fermented grains of baijiu were collected from another batch. Fermented and distilled grains were selected from the upper, middle, and bottom layers. All samples were collected from 2020 to 2021 and stored at −20 • C for further analysis. Triplicates for each grain sample were used.

Derivatizing Reagent
The reagent preparation procedure was as follows: 220 mg of DTDP were sonicated in a mixture of 20 mL of water and 100 µL of concentrated HCl (37% w/w). To the dissolved DTDP solution, Milli-Q water was added to a final volume of 100 mL. Aliquots were then stored at -20 • C until needed [23].

Optimization of the QuEChERS Method
Acetonitrile is a frequent extractant used in QuEChERS [22]. The overall effectiveness of extraction was set using various extractants [24]. To ensure that the molecules of interest were correctly extracted from the matrix, the performance of various commonly used solvents was assessed. Acetonitrile, methanol, dichloromethane, ethanol, and ethyl acetate were commonly utilized in reported extraction processes. The aforementioned five typical solvents were chosen, but ethyl acetate cannot be directly injected. When ethyl acetate was used as an extractant, it was nitrogen-blown dried before being redissolved in 400 µL of methanol.
The d-SPE phase was the second stage of the QuEChERS method. Primary secondary amine (PSA), C 18 , and GCB are the most widely reported adsorbents. NH 2 and PSA have similar functions. They are used to clear lipids, organic acids, carbohydrates, fatty acids, and certain pigments. These sorbents are typically employed alone or in combination, and certain purification procedures can be avoided. To optimize the three purifying agents (NH 2 , GCB, and C 18 ) as samples for optimization, fermented grains of baijiu from the fifth fermentation stage were chosen. Figure 1 shows the entire process, from the pre-treatment to the injection stage. The sample was made up of 10 g fermented grains of sauce-aroma baijiu and 5 mL of water in a 50 mL centrifuge tube. It was then placed in an ice-water bath for 5 min using an ultrasonic cleaner. An ACN solution (10 mL) (stored at −20 • C) containing 10 µL 2phenylethanethiol (internal standard) at a concentration of 58.7 mg/L was added to the tube and mixed by vortex shaking. The samples were then prepared using the QuEChERS method. MgSO 4 (4 g), NaCl (1 g), sodium citrate tribasic dihydrate (1 g), and sodium citrate dibasic sesquihydrate (0.5 g) were added to the tube, which was immediately vortexed for 2 min, hand-shaken for 30 s, chilled in an ice-water bath, and centrifuged for 5 min at 4500 rpm and 4 • C. In an ice-water bath, the supernatant (5 mL) was transferred to a new 15 mL tube. Subsequently, EDTA-Na 2 (5 mg), 50% acetaldehyde (20 µL), and freshly thawed DTDP reagent (10 mM, 400 µL) were added to the tube. After 30 min, the tubes were then dried in a stream of nitrogen until the sample volume reached 400 µL [6]. The final solution was filtered through a 0.22 µm PTFE membrane before the UPLC-MS/MS analysis.

UPLC-MS/MS Analysis
UPLC-MS/MS analysis was performed using an ACQUITY UPLC system (binary solvent manager; Waters Corp., Milford, CT, USA). Chromatographic separation was performed on a C18 column (100 × 2.1 mm, 1.7 μm; Waters BEH C18). The column temperature was maintained at 40 °C. The liquid chromatography eluent consisted of a gradient of water (mobile phase A) and ACN (mobile phase B), both of which contained 0.1% formic acid. The program was performed as follows: 0-13 min, 15%-22% B; 13-14 min, 22-30% B; 14-18 min, 30-35% B; 18-18.5 min, 35-100% B; 18.5-21.5 min, 100% B; and 21.5-22 min, 100%-15% B. The flow rate was 0.3 mL/min, and the injection volume was 10 μL. A triple-quadrupole mass spectrometer (Xevo TQ-S; Waters Corp.) was used with an electrospray ionization source. The scanning mode was set to the positive ionization mode. The following mass spectrometry detection conditions were chosen: source temperature, 150 °C; desolvation temperature, 500 °C; and capillary voltage, 3 kV. The multiple reaction monitoring (MRM) mode was used for targeted compound analysis. The derivatization products were optimized via direct injection into LC-MS to determine the MRM conditions, and the optimized parameters of volatile thiols in baijiu were characterized by Yan Yan [6]. The derivatization reaction was a chemical reaction with a chemical dose ratio of 1:1, and the DTDP measured in the sample guaranteed excess derivatization. The retention times determined by reaction with the standard in the sample were the same. Thus, the content of the derivatization product might represent that of the original compound. The content was calculated based on the internal standard, and the peak area of the compound was determined in the MRM mode.
The retention times, parent ions, and daughter ions of the five volatile thiol compounds are displayed in Figure 2, and detailed information on the MRM method for the thiol compounds is displayed in Table

UPLC-MS/MS Analysis
UPLC-MS/MS analysis was performed using an ACQUITY UPLC system (binary solvent manager; Waters Corp., Milford, CT, USA). Chromatographic separation was performed on a C18 column (100 × 2.1 mm, 1.7 µm; Waters BEH C18). The column temperature was maintained at 40 • C. The liquid chromatography eluent consisted of a gradient of water (mobile phase A) and ACN (mobile phase B), both of which contained 0.1% formic acid. The program was performed as follows: 0-13 min, 15%-22% B; 13-14 min, 22-30% B; 14-18 min, 30-35% B; 18-18.5 min, 35-100% B; 18.5-21.5 min, 100% B; and 21.5-22 min, 100%-15% B. The flow rate was 0.3 mL/min, and the injection volume was 10 µL. A triple-quadrupole mass spectrometer (Xevo TQ-S; Waters Corp.) was used with an electrospray ionization source. The scanning mode was set to the positive ionization mode. The following mass spectrometry detection conditions were chosen: source temperature, 150 • C; desolvation temperature, 500 • C; and capillary voltage, 3 kV. The multiple reaction monitoring (MRM) mode was used for targeted compound analysis. The derivatization products were optimized via direct injection into LC-MS to determine the MRM conditions, and the optimized parameters of volatile thiols in baijiu were characterized by Yan Yan [6]. The derivatization reaction was a chemical reaction with a chemical dose ratio of 1:1, and the DTDP measured in the sample guaranteed excess derivatization. The retention times determined by reaction with the standard in the sample were the same. Thus, the content of the derivatization product might represent that of the original compound. The content was calculated based on the internal standard, and the peak area of the compound was determined in the MRM mode.
The retention times, parent ions, and daughter ions of the five volatile thiol compounds are displayed in Figure 2, and detailed information on the MRM method for the thiol compounds is displayed in Table

Statistical Analyses
The UPLC-MS/MS data were processed using Mass Lynx V4.1. Statistical analysis was performed using SPSS software (Version 26.0; Chicago, IL, USA) and Microsoft Excel 2019. The drawings were completed using Origin Pro 2022b.

Optimization of the Extraction Solvent
The effect of extracting thiols from the fermented grains with five commonly used organic reagents was optimized. Methanethiol produced the best response, and the remaining four thiols were pre-treated and then detected in other organic reagents and could not even be detected or provided a bad response. Thus, Figure 3 demonstrates the effect of methanethiol on the extraction of various organic reagents. The abundance of methanethiol extracted from the fermented grains using acetonitrile was approximately 10 6 , while the detection after extraction using the rest of the organic reagents was approximately only 10 4 to 10 5 . Comparing the results of the other four thiols after extraction using different organic reagents, the results were the same as those using methanethiol.

Statistical Analyses
The UPLC-MS/MS data were processed using Mass Lynx V4.1. Statistical analysis was performed using SPSS software (Version 26.0; Chicago, IL, USA) and Microsoft Excel 2019. The drawings were completed using Origin Pro 2022b.

Optimization of the Extraction Solvent
The effect of extracting thiols from the fermented grains with five commonly used organic reagents was optimized. Methanethiol produced the best response, and the remaining four thiols were pre-treated and then detected in other organic reagents and could not even be detected or provided a bad response. Thus, Figure 3 demonstrates the effect of methanethiol on the extraction of various organic reagents. The abundance of methanethiol extracted from the fermented grains using acetonitrile was approximately 10 6 , while the detection after extraction using the rest of the organic reagents was approximately only 10 4 to 10 5 . Comparing the results of the other four thiols after extraction using different organic reagents, the results were the same as those using methanethiol.
The response of the extracted thiol compounds was the highest when acetonitrile was employed. Other extraction solvents resulted in poor extraction results, low abundance, and numerous heterogeneous peaks. This is because acetonitrile is a polar solvent with low lipophilicity, which allows good recovery of polar chemicals while reducing fat, wax, and pigment extraction compared to other solvents. Acetonitrile was selected as the final extraction solvent.

Selection of Clean-Up QuEChERS Extracts
To optimize the three purifying agents (NH 2 , GCB, and C 18 ) as samples for optimization, fermented grains of baijiu from the fifth fermentation stage were chosen. Mixes of GCB (20 mg), C 18 (120 mg), and NH 2 (120 mg) were chosen along with a blank control group [25] (Figure 4). Combination and separate adsorbents were used to extract volatile thiols from the solid materials. The extraction effects of five volatile thiols were considered together. The use of C 18 alone resulted in very poor detection of methanethiol, 2-furfuranethiol, and 2-mercapto-1-ethanol, and GCB alone resulted in very poor detection of 2-mercapto-1-ethanol. Furthermore, the use of NH 2 alone resulted in poor detection of 2-mercapto-1-ethanol, ethanethiol, and 2-methyl-3-furanethiol, and the combination of purifying agents resulted in very poor detection of 2-methyl-3-furanethiol.  The response of the extracted thiol compounds was the highest when acetonitrile was employed. Other extraction solvents resulted in poor extraction results, low abundance, and numerous heterogeneous peaks. This is because acetonitrile is a polar solvent with low lipophilicity, which allows good recovery of polar chemicals while reducing fat, wax, and pigment extraction compared to other solvents. Acetonitrile was selected as the final extraction solvent.

Selection of Clean-Up QuEChERS Extracts
To optimize the three purifying agents (NH2, GCB, and C18) as samples for optimization, fermented grains of baijiu from the fifth fermentation stage were chosen. Mixes of GCB (20 mg), C18 (120 mg), and NH2 (120 mg) were chosen along with a blank control group [25] (Figure 4). Combination and separate adsorbents were used to extract volatile thiols from the solid materials. The extraction effects of five volatile thiols were considered together. The use of C18 alone resulted in very poor detection of methanethiol, 2-furfuranethiol, and 2-mercapto-1-ethanol, and GCB alone resulted in very poor detection of 2mercapto-1-ethanol. Furthermore, the use of NH2 alone resulted in poor detection of 2mercapto-1-ethanol, ethanethiol, and 2-methyl-3-furanethiol, and the combination of purifying agents resulted in very poor detection of 2-methyl-3-furanethiol.
It was discovered that skipping the purification phase was more effective than using adsorbents. This might be because the DTDP derivatization reaction worked well at pH ≥ 3.4 and reacted rapidly and completely [20]. However, the purifiers, particularly NH2, were alkaline in solution, and after adding the purification step, the pH of the extract It was discovered that skipping the purification phase was more effective than using adsorbents. This might be because the DTDP derivatization reaction worked well at pH ≥ 3.4 and reacted rapidly and completely [20]. However, the purifiers, particularly NH 2 , were alkaline in solution, and after adding the purification step, the pH of the extract became alkaline, causing the derivatives to become unstable and deteriorate in an alkaline environment. As a result, the ultimate option was to skip the purification step.

Validation of the Method
Thiol compound standard solutions of different concentrations were obtained by diluting the stock solutions in Milli-Q water. The different concentration levels were determined using UPLC-MS/MS under the pre-treatment settings described above. Calibration values were calculated by comparing the peak area ratio of each thiol component against various internal standard concentrations (2-phenylethanethiol). Good coefficients of determination (R 2 : 0.9900-0.9935) were observed between the concentration and the matching peak area of each thiol. The limits of detection (LODs) and limits of quantitation (LOQs) of the diluted thiol compounds were determined using continuous analysis. The LODs and LOQs were calculated when the signal/noise ratio (S/N) reached 3 and 10, respectively. Detailed information is presented in Table 1. became alkaline, causing the derivatives to become unstable and deteriorate in an alkaline environment. As a result, the ultimate option was to skip the purification step.

Validation of the Method
Thiol compound standard solutions of different concentrations were obtained by diluting the stock solutions in Milli-Q water. The different concentration levels were determined using UPLC-MS/MS under the pre-treatment settings described above. Calibration values were calculated by comparing the peak area ratio of each thiol component against various internal standard concentrations (2-phenylethanethiol). Good coefficients of determination (R 2 : 0.9900-0.9935) were observed between the concentration and the matching peak area of each thiol. The limits of detection (LODs) and limits of quantitation (LOQs) of the diluted thiol compounds were determined using continuous analysis. The LODs and LOQs were calculated when the signal/noise ratio (S/N) reached 3 and 10, respectively. Detailed information is presented in Table 1.  The accuracy of the method was assessed based on the recovery rate and evaluated by adding three spiked levels (high, medium, and low) to test the target compound in the fermented grains of baijiu. Samples from the fifth round of fermented grains were chosen to validate the research method. In repeated experiments, the samples were pre-treated using the previously reported QuEChERS method, as well as a derivatization reaction; the final solution was injected into the LC-MS/MS system for analysis. The recoveries of the five volatile thiol compounds in fermented grains of baijiu were 71.72-104.72%, and the intra-and inter-day relative standard deviations (RSDs) were 0.63-7.72% and 1.96-9.44%, respectively. The recoveries were within the acceptable range of 70-110% in terms of accuracy. Additionally, accuracy was determined, with RSD values less than 15%, supporting the accuracy of the method. The results are presented in Table 2. Table 2. Recovery and precision of the standard approach for derivatized thiols.

Measurement of Fermented Grains of Baijiu
For this study, the QuEChERS binding derivatization pre-treatment approach was used for fermented grains. Five volatile thiol compounds were identified in the fermented grains of baijiu. Figure 5 (Table S2). The concentrations of four thiols (methanethiol, ethanethiol, 2-furfurylthiol, and 2-methyl-3-furanthiol) increased with the number of fermentation rounds. This implies that, between the fermentation rounds, volatile thiols may accumulate in the preceding round, resulting in an increase in the content of the next round. It was reported that the concentrations and odor thresholds in 46% ethanol-water of five thiols of 229-513 and 2.2 µg/L for methanethiol, 6.7-32.1 and 0.8 µg/L for ethanethiol, 0.03-0.08 and 130 µg/L for 2-mercapto-1-ethanol, 11.2-37.8 and 0.1 µg/L for 2-furfurylthiol, and 1.0-2.5 and 0.0048 µg/L 2-methyl-3-furanthiol, respectively, were determined in the sauce-aroma baijiu [6]. The concentrations of these volatile thiol compounds were closely correlated with their counterparts in the raw materials during baijiu fermentation.
The concentration of volatile thiol compounds in sauce-aroma baijiu increased as the number of rounds increased, but it was not clear at which stage in the production of a single round the most volatile thiol compound accumulated. Therefore, the content of volatile thiol compounds in the fifth round of fermented grains (initial, heaped, fermented, and distilled grains) was explored. The results are shown in Figure 6. The error lines of the fermented and distilled grains represent the differences in the concentrations of thiols at different locations. The average concentration of thiols in the initial, heaped, fermented, and distilled grains were 65.48, 65.00, 103.78, and 143.19 µg/kg for methanethiol, respectively; 1.15, 1.15, 1.17, and 1.21 µg/kg ethanethiol, respectively; 1.39, 1.53, 1.13, and 1.10 µg/kg for 2-mercapto-1-ethanol, respectively; 0.58, 0.59, 1.40, and 1.97 µg/kg for 2-furfurylthiol, respectively; 1.17, 0.95, 4.07, and 6.35 µg/kg for 2-methyl-3-furanthiol, respectively (Table S3). While the concentration of ethanethiol did not vary significantly, that of 2-mercapto-1-ethanol decreased during the production process. The concentrations of the other three thiols (methanethiol, 2-furfurylthiol, and 2-methyl-3-furanthiol) increased during fermentation and distillation. The thermal reaction was found to be of great significance for the formation of several thiols. This also explained why, after the fifth round of distillation, there was still a relatively great number of thiols in the fermented grains. The accumulation of precursor substances during the fermentation and accumulation of residual sulfides between rounds led to this phenomenon. Volatile thiol compounds appeared at locations during the entire distillation process of baijiu: one was extracted into the round of baijiu through ethanol extraction, the other was a large amount of residue in the fermented grains, and the third was in other environments. Thus, the fermentation and distillation processes, where the increase in volatile sulfides is higher, are important stages for their widespread production. The stacking process was not the primary stage in the production of these three thiol compounds.
Foods 2023, 12, x FOR PEER REVIEW 9 of 14 2-furfurylthiol, and 2-methyl-3-furanthiol) increased with the number of fermentation rounds. This implies that, between the fermentation rounds, volatile thiols may accumulate in the preceding round, resulting in an increase in the content of the next round. It was reported that the concentrations and odor thresholds in 46% ethanol-water of five thiols of 229-513 and 2.2 μg/L for methanethiol, 6.7-32.1 and 0.8 μg/L for ethanethiol, 0.03-0.08 and 130 μg/L for 2-mercapto-1-ethanol, 11.2-37.8 and 0.1 μg/L for 2-furfurylthiol, and 1.0-2.5 and 0.0048 μg/L 2-methyl-3-furanthiol, respectively, were determined in the saucearoma baijiu [6]. The concentrations of these volatile thiol compounds were closely correlated with their counterparts in the raw materials during baijiu fermentation. The concentration of volatile thiol compounds in sauce-aroma baijiu increased as the number of rounds increased, but it was not clear at which stage in the production of a single round the most volatile thiol compound accumulated. Therefore, the content of volatile thiol compounds in the fifth round of fermented grains (initial, heaped, fermented, and distilled grains) was explored. The results are shown in Figure 6. The error lines of the fermented and distilled grains represent the differences in the concentrations of thiols at different locations. The average concentration of thiols in the initial, heaped, fermented, and distilled grains were 65.48, 65.00, 103.78, and 143.19 μg/kg for methanethiol, respectively; 1.15, 1.15, 1.17, and 1.21 μg/kg ethanethiol, respectively; 1.39, 1.53, 1.13, and 1.10 μg/kg for 2-mercapto-1-ethanol, respectively; 0.58, 0.59, 1.40, and 1.97 μg/kg for 2-furfurylthiol, respectively; 1.17, 0.95, 4.07, and 6.35 μg/kg for 2-methyl-3-furanthiol, respectively. While the concentration of ethanethiol did not vary significantly, that of 2-mercapto-1-ethanol decreased during the production process. The concentrations of the other three thiols (methanethiol, 2-furfurylthiol, and 2-methyl-3-furanthiol) increased during fermentation and distillation. The thermal reaction was found to be of great significance for the formation of several thiols. This also explained why, after the fifth round of distillation, there was still a relatively great number of thiols in the fermented grains. The accumulation of precursor substances during the fermentation and accumulation of residual sulfides between rounds led to this phenomenon. Volatile thiol compounds appeared at locations during the entire distillation process of baijiu: one was extracted into the round of baijiu through ethanol extraction, the other was a large amount of residue in the Both the fermentation and distillation processes contributed to the formation of the three thiols to some extent. Therefore, it was necessary to focus on the possible pathways of thiols during both processes. Their presence in alcoholic beverages may be due to enzymatic and non-enzymatic reactions. Enzymatic reactions involve the degradation of sulfur-containing amino acids, formation of fermentation products, and metabolism of some sulfur-containing compounds. Non-enzymatic reactions involve thermochemistry and other chemical reactions of sulfur compounds during brewing and storage. Different pathways for the formation of volatile thiol compounds might occur in parallel during baijiu processing. Cystine, cysteine [26], methionine [27,28], glutathione [29], and thiamine [30] are sources of various sulfur compounds. Their degradations occur via enzymatic or nonenzymatic pathways, and their decompositions cause the formation of additional sulfur compounds [31].
The addition of volatile sulfur compounds to the yeast's early fermentation stage revealed the complexity of biological and chemical pathways involved in the formation of methanethiol [32]. Methionine [33] and cysteine [34] are the precursors of methanethiol. The Maillard reaction between amino acids and reducing sugars played a key role in the formation pathways of 2-furfurylthiol (FFT) and 2-methyl-3-furanthiol (2M3F). In the Maillard reaction, 4-hydroxy-5-methyl-3(2H)-furanone (NF) and 2-furfural can be generated from ribose using Amadori compounds [35]. 2-Furfural was also one of the main products produced by the reaction of pentose in the presence of amino acids. Hydrogen sulfide (H 2 S) is a Strecker degradation product of cysteine. 2-Furfural and NF are important precursors for the formation of FFT and 2M3F, respectively. In the presence of H 2 S, the hexose degradation products, 2-oxopropanal and hydroxyacetaldehyde, can lead to the formation of FFT and 2M3F. Possible pathway formations in the fermented grains are shown in Figure 7a. 2-Oxopropanal reacts with H 2 S to form mercapto-2-propanone. Depending on the side of the nucleophilic attack, the reaction of mercapto-2-propanone with hydroxyacetaldehyde may lead to the formation of 2M3F or FFT. Mercapto-2-propanone reacts with hydroxyacetaldehyde to form 4,5-dihydroxy-3-mercapto-2-pentanone intermediate product, which is cyclized and eliminated by two water molecules to form 2M3F. In contrast, the methyl nucleophile of mercapto-2-propanone attacks hydroxyacetaldehyde, forming 4,5-dihydroxy-1-mercapto-2-pentanone, which may produce FFT after the cyclization and elimination of water [36]. Possible pathway formations in the fermented grains are shown in Figure 7b. In the case of other sulfur precursor deficiencies, thiamine can act as a sulfur precursor to form 2M3F. Thiamine reacts to form the intermediate 5-hydroxy-3mercaptopentan-2-one, which undergoes cyclization, dehydration, and oxidation to form 2M3F [37,38]. Detailed information on its formation pathway is presented in Figure 7c. Lcysteine reacts with pentose (ribose, xylose, or arabinose) [39,40] and hexose (glucose) [31] to produce 2-furfurylthiol, 2-methyl-3-furfurylthiol, and other compounds. A variety of bacterial activities, producing β-lyase enzymatic activity, have been shown to degrade cysteine-furfural conjugates to generate 2-furylthiol [26]. The possible pathway formations in the fermented grains are shown in Figure 7d. fermented grains, and the third was in other environments. Thus, the fermentation and distillation processes, where the increase in volatile sulfides is higher, are important stages for their widespread production. The stacking process was not the primary stage in the production of these three thiol compounds. Both the fermentation and distillation processes contributed to the formation of the three thiols to some extent. Therefore, it was necessary to focus on the possible pathways of thiols during both processes. Their presence in alcoholic beverages may be due to enzymatic and non-enzymatic reactions. Enzymatic reactions involve the degradation of sulfur-containing amino acids, formation of fermentation products, and metabolism of some sulfur-containing compounds. Non-enzymatic reactions involve thermochemistry and other chemical reactions of sulfur compounds during brewing and storage. Different pathways for the formation of volatile thiol compounds might occur in parallel during baijiu processing. Cystine, cysteine [26], methionine [27,28], glutathione [29], and thiamine [30] are sources of various sulfur compounds. Their degradations occur via enzymatic or nonenzymatic pathways, and their decompositions cause the formation of additional sulfur Fermented grains contain large amounts of sugars, amino acids, and thiamine; therefore, volatile thiols may be produced during the production of baijiu through the various reaction pathways (Figure 7). The improved method enabled an effective and accurate determination of thiols in fermented grains, which was important for studying the formation pathways of thiols produced during solid-state fermentation, thereby facilitating the division of quality control of baijiu production into different important stages. diate 5-hydroxy-3-mercaptopentan-2-one, which undergoes cyclization, dehydration, and oxidation to form 2M3F [37,38]. Detailed information on its formation pathway is presented in Figure 7c. L-cysteine reacts with pentose (ribose, xylose, or arabinose) [39,40] and hexose (glucose) [31] to produce 2-furfurylthiol, 2-methyl-3-furfurylthiol, and other compounds. A variety of bacterial activities, producing β-lyase enzymatic activity, have been shown to degrade cysteine-furfural conjugates to generate 2-furylthiol [26]. The possible pathway formations in the fermented grains are shown in Figure 7d.

Conclusions
The efficient extraction of thiol compounds during sample preparation was demonstrated using QuEChERS and citrate buffer. Liquid chromatography-tandem mass spectrometry and DTDP derivatization procedures were combined to identify thiols in the fermented grains. Thiols could be derivatized in the MRM mode with good sensitivity and reproducibility using UPLC-MS/MS. This study found that the concentrations of methanethiol, ethanethiol, 2-furfurylthiol, and 2-methyl-3-furanethiol in fermented grains of sauce-aroma baijiu increased as the number of fermentation rounds increased. Therefore, further analysis will focus on later fermentation rounds for the study of thiol regulation. Volatile thiols had high concentrations in fermented grains after fermentation and distillation. Both fermentation and distillation processes promoted the formation of methanethiol, 2-furfurylthiol, and 2-methyl-3-furanethiol. The fermentation stage may contribute to its precursor substances, which form volatile thiols again during the distillation stage. Thus, more attention can be paid to distillation than just the fermentation process for the regulation of these thiols.
This study was advantageous in analyzing the content of these thiol compounds in baijiu liquor by determining their levels during fermentation and distillation to control the risk of off-odors. This will aid in the real-time monitoring of the alcoholic beverage production process and improve its quality.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/foods12142658/s1: Table S1: The detailed information of the MRM method for the thiol compounds, Table S2: The concentration of five thiols in fermented grains of sauce-aroma baijiu from the first to seventh rounds, and Table S3: The concentration of five thiols in fermented grains during the whole fifth round.

Data Availability Statement:
The data used to support the findings of this study can be made available by the corresponding author upon request.