Characterization of Key Odorants in Soy Sauce and Their Concentration Changes during Storage

Wang, Rui; Chang, Shiyu; Liang, Miao; Wu, Yajian; Xin, Runhu; Liu, Yuping; Chen, Liang; Zhou, Shangting

doi:https://doi.org/10.1155/2023/6673089

Journal of Food Quality

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Abstract Introduction Materials and Methods Results and Discussion Conclusion Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 6673089 | https://doi.org/10.1155/2023/6673089

Characterization of Key Odorants in Soy Sauce and Their Concentration Changes during Storage

Rui Wang,¹Shiyu Chang,¹Miao Liang,¹Yajian Wu,¹Runhu Xin,¹Yuping Liu,¹Liang Chen,²and Shangting Zhou²

Academic Editor: Antoni Szumny

Received13 Jun 2023

Revised31 Jul 2023

Accepted20 Sept 2023

Published28 Sept 2023

Abstract

To explore the key odorants and their concentration changes in high-salt liquid-state fermentation soy sauce (HLFSS) during storage, solvent extraction coupled with solvent-assisted flavor evaporation was applied to isolate the volatiles in HLFSS, and the volatiles were analyzed by gas chromatograph-mass spectrometry-olfactory and gas chromatograph-flame ionization detector-olfactometry combined with the aroma extract dilution assay. A total of 37 odor-active compounds with flavor dilution (FD) factors ranging from 1 to 1024 were identified, and 23 aroma components with odor activity values ≥1 were considered as key odorants. Methional, 3-methylbutanoic acid, phenethyl alcohol, sotolon, 4-hydroxy-2-methyl-5-ethyl-3(2H)-furanone (HEMF), and 4-hydroxy-2,5-dimethylfuran-3(2H)-one (HDMF) had the highest FD factors of 1024. The results of quantitative analysis through the internal standard curve showed that the concentrations of HEMF, HDMF, acetic acid, 1-hydroxy-2-butanone, acetol, and furfuryl alcohol varied greatly during storage, so these compounds might be used as the indicators to determine the shelf life of HLFSS. The recombination experiment also confirmed the important contribution of these compounds. This result will provide valuable information for understanding the flavor changes of HLFSS.

1. Introduction

Soy sauce (SS) originated from China with more than 3000 years of history, and its unique flavor was characterized by a potent umami, salty, and a characteristic aroma such as caramel-like and smoky-like [1], which could enhance the overall aroma of some kinds of dishes. SS is produced by microbial fermentation, enzymatic or nonenzymatic reactions using soybeans, wheat flour, salt, etc., as raw materials [2]. The production of SS in China has shown a gradual increase, with an annual output of 5.2 million tons, making the SS industry become one of the most prosperous industries in China [3]. According to the difference of the SS brewing process, it is divided into high-salt liquid-state fermentation SS (HLFSS) and low-salt solid-state fermentation SS (LSFSS) [4]. Because of HLFSS with some advantages, such as health protection functions, higher nutritional value, pleasant flavor, beautiful color, etc., the fermentation process of HLFSS occupies a leading position in the SS industry in China at present [5, 6].

The aroma of SS is an indispensable indicator to determine its quality and consumer acceptance in the market [5]. SS contains various amino acids, sugars, and other substances, providing a good foundation for the occurrence of Maillard reactions [7]. During storage, some reactions occur in SS, such as Maillard reaction, oxidation reaction, esterification reaction, etc., resulting in the changes of concentration of odor constituents slowly in the process, which in turn affects the overall aroma of the SS [8]. However, there are very few research studies on the content changes of the aroma-active components in SS during storage.

At present, the reports on odor compound changes of SS focus on the changes under different heating conditions. Wang et al. [9] investigated the aroma changes of SS heated at higher temperature (220°C) and boiling (100°C) for a different time, and the results suggested that the concentration changes of target aroma-active components led to vary caramel-like/sweet, roasted/roasted nut-like, and spicy/burnt odors. Liang et al. [10] researched the changes on physicochemical properties, organic acids, and volatile components in SS heated at 125°C for 15 min, and the results showed that physicochemical properties and organic acids did not vary significantly, but the concentrations of volatiles rose more than 30%; the intensities of spicy, caramel-like, and fruity notes in heated SS were higher. Guo et al. [11] used liquid-liquid extraction combined with gas chromatograph-mass spectrometry-olfactory to analyze the volatile compounds in dark SS and explore the changes of the flavor characteristic from 0 days to 20 days, and they found that the aroma intensity scores of roasted and smoky notes increased. In addition, a metabolomic approach was used for investigating the odor changes of SS during storage, and the results obtained indicated the changes of concentrations of several key biomarkers resulted in the sensory decreases in fruity/grape and nutty/sesame notes and the increases in methional/potato note and astringent attributes [12].

Accelerated aging experiments are widely used to simulate the changes of food during long-term storage. Our previous study results showed that the overall flavor profile of SS kept at 37°C for one week was similar to that of SS stored at room temperature for three weeks [13]. In order to further explore the concentration changes of odor-active compounds in SS during a longer storage, accelerated aging experiments were also applied in the present study.

The purpose of this study is (i) to screen and identify the odor-active compounds in SS using gas chromatograph-flame ionization detector-olfactometry and gas chromatograph-mass spectrometry-olfactory combined with aroma extract dilution analysis; (ii) to quantitate the odor compounds identified by using the internal standard curve; (iii) to determine the key odorants by calculating odor activity values; (iv) to investigate the change law of aroma-active compounds in HLFSS during long-term storage.

2. Materials and Methods

2.1. HLFSS Samples

The same batch of HLFSS samples was obtained from Jiajia Foods Co., Ltd. The raw materials for HLFSS samples were water, organic defatted soybeans, sugar, salt, wheat, yeast extract, sodium glutamate, disodium 5′-ribonucleotide, and sucralose. Samples 1–9 represent HLFSS samples stored at 37 ± 1°C for 1–9 weeks, respectively. All HLFSS samples that have reached storage time were kept at 4°C refrigerator until analysis.

2.2. Chemicals

Furfuryl alcohol (98%), acetic acid (>99%), propionic acid (99%), isobutyric acid (99%), isobutanol (99%), 1-butanol (99.5%), maltol (99%), phenethyl alcohol (99%), guaiacol (99%), γ-butyrolactone (99%), ethyl vanillate (98%), methylpyrazine (98%), 3-methylbutanoic acid (99%), 3-methylvaleric acid (>98%), 4-methylpentanoic acid (99%), 2-acetylpyrrole (98%), methionol (98%), 2-acetylfuran (97%), 2,5-trimethylpyrazine (99%), methyl cyclopentenolone (99%), 4-ethyl-2-methoxyphenol (98%), 2,6-dimethylpyrazine (98%), and 2,3,5-trimethylpyrazine (98%) were bought from J&K Chemicals Ltd. (Beijing, China); 3-octanol (98%), benzoic acid (≥99%), phenylacetaldehyde (95%), 2 (5H)-furanone (98%), methional (98%), 1-hydroxy-2-butanone (98%) were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China); acetol (>80%), and 4-ethylphenol (>97%) were purchased from TCI Chemical Ltd. (Shanghai, China); acetoin (97%) and 2-ethyl-6-methylpyrazine (>98%) were obtained from Adamas Reagents Co., Ltd. (Shanghai, China); HDMF (98%), HEMF (97%), phenylacetic acid (95%), ethyl lactate (99.8%), and sotolon (97%) were supplied by Aladdin Reagents Co., Ltd. (Shanghai, China), Ark Pharm Inc. (Chicago, IL, USA), Key Organics (Cornwall, England), Minda Technology Co., Ltd. (Beijing, China), respectively. C6∼C30 n-alkanes were obtained from Aldrich Chemical Co., Ltd. (Shanghai, China); anhydrous sodium sulfate and dichloromethane were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).

[²H₃]-Methionol and [²H₃]-methional were prepared from [²H₃]-methyl iodide reacted with ethyl 3-mercaptopropionate to give ethyl 3-[²H₃]-methylthiopropionate, which was reduced by lithium aluminum hydride to obtain [²H₃]-methionol; [²H₃]-methional was formed by oxidizing [²H₃]-methionol with pyridinium chlorochromate [14]. [²H₃]-2,3,5-trimethylpyrazine was synthesized from[²H₃]- iodomethane through the reaction of 3-chloro-2,5-dimethylpyrazine with the Grignard reagents, under the catalytic agent of [1,3-bis-(diphenylphosphino) propane] nickel (II) chloride according to the reference [15].

2.3. Extraction of Volatiles from HLFSS

The volatiles were isolated based on the method used in our previous research, and some modifications were made [16]. HLFSS samples (100 mL) with dichloromethane (50 mL × 3) were extracted at room temperature by using an oscillator (ZWY-100H, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.) for 1 h, and then the extracts were centrifuged at 4°C, 8000 rmp by using a centrifugal (H1750R, Hunan Xiangyi Instrument Manufacturing Co., Ltd.); the organic phase was separated. Repeat the above operation 2 times. The resulting organic phase was distilled by solvent-assisted flavor evaporation (SAFE) (Edwards TIC Pumping Station from BOC Edwards, England) in a 40°C water bath, and the vacuum pressure was 5 w10⁻⁴ mbar. Anhydrous sodium sulfate (50 g) was added to the distillate obtained from SAFE to remove water, and then the distillate was placed in a −20°C refrigerator overnight.

Using Vigreux columns (50 cm × 1 cm; Beijing Jingxing Glassware Co., Ltd., Beijing, China) concentrated the distillate to about 3 mL at 50°C and then condensed to 1 mL under mild nitrogen flow. The obtained isolate was conducted to GC-MS-O and GC-FID-O analysis.

2.4. Gas Chromatograph-Mass Spectrometry-Olfactory (GC-MS-O) Analysis

The Agilent model 7890B gas chromatography combined with the Agilent 5977A mass spectrometer detector (MSD) and an olfactory detector port (OPD3, Gerstel, Germany) were used to analyze the volatiles in HLFSS. DB-WAX and HP-5 capillary columns (30 m × 0.25 mm, 0.25 μm film thickness) were utilized to analyze chromatographic separation of the volatiles in SS isolates. The oven temperature was held at 40°C for 2 min, then increased the temperature to 80°C at a rate of 8°C/min, then to 100°C at a rate of 4°C/min, and finally to 230°C at a rate of 6°C/min, and it was maintained at this temperature for 10 min. Helium was utilized as the carrier gas with a constant flow rate of 1.7 mL/min, the GC injector temperature was 250°C, and the injection volume was 1 μL (splitless mode), while the ion source temperature was 230°C. Mass spectra electronic impact (EI) ionization was 70 eV, with scanning range m/z 30–350. The temperature of quadrupole was set at 150°C. Nitrogen was used as a supplemental gas to ensure that the mass spectrometer and the olfactory detector could detect the signal simultaneously; the temperatures of transmission line and sniffing port were set at 250°C and 120°C, respectively, and moist air was introduced at the sniffing port to avoid dehydration of the nasal mucosa of the sniffers, which could affect the accuracy of the sniffing.

2.5. Gas Chromatograph-Flame Ionization Detector-Olfactometry (GC-FID-O) Analysis

GC-FID-O analysis was conducted on a gas chromatography (Agilent model 7890B) coupled with an FID (Agilent Technologies, Inc.) and an olfactory detector port (OPD3, Gerstel, Germany). A polar capillary column DB-WAX (30 m × 0.25 mm, 0.25 μm film thickness) was utilized for chromatographic separation of volatile components. The temperature procedure was consistent with Section 2.4. The effluent from the GC was split 1 : 1 between the FID and sniffing port. Helium was utilized as carrier gas with a constant flow rate of 1 mL/min, and the temperature of the sniffing port was 120°C, and the capillary linked to the FID was maintained at 280°C. Three sensory experts were selected for GC-FID-O analysis.

2.6. Qualitative Analysis

All of odor-active components in HLFSS were positively identified by comparing their mass spectra, the retention indices (RIs), and aroma characteristics with those of standard compounds. The RIs were calculated on the basis of the retention time of C₆–C₃₀ hydrocarbon [17].

2.7. Quantitative Analysis

2.7.1. Internal Standard Curve Method

After adding 3-octanol (300 μL, 10⁻⁴ g/mL) as an internal standard into 100 mL HLFSS samples, then the volatiles were extracted according to the method described previously. For the standard calibration curve, a series of mixed solution of internal standard and standard chemicals (five different concentrations) were analyzed by GC-MS-O in the selected ion monitoring (SIM) mode under the same conditions mentioned in Section 2.4. Each standard curve was obtained by plotting the ratios of the peak areas of the standard to the peak areas of the internal standard and its concentration ratio. The concentrations of the volatiles were calculated from the approximated curve using the linear least-squares method. The quantitative results were obtained by averaging data of the triplicate experiments.

2.7.2. Stable Isotope Dilution Assays (SIDA)

The separation of HLFSS volatiles was conducted as mentioned previously (Section 2.4) after the addition of the isotope-labeled standards. To calculate the response factors, the solutions containing the labeled and unlabeled standards with five different concentrations (1 : 5, 1 : 3, 1 : 1, 3 : 1, and 5 : 1) were analyzed by GC-MS-O under the same procedure as above; the SIM mode was used. The quantitative results were obtained by averaging the triplicate experiment data.

2.8. Aroma Extract Dilution Analysis (AEDA)

The concentrates of the HLFSS sample were twice diluted (1 : 2, 1 : 4, 1 : 8, 1 : 16…1 : 1024) with dichloromethane and then analyzed with GC-FID-O until no odorant was perceived by all panelists at the sniffing port. The maximum dilution of each odor-active compound that at least two team members could perceive was recorded as the flavor dilution (FD) factor. AEDA was performed twice at least for each HLFSS sample by all panelists.

2.9. The Calculation of Odor Activity Value (OAV)

The OAV of each odorant with aroma activity is calculated as a ratio of the component concentration in SS to its threshold in water [18–20]. The compound with the OAVs ≥ 1 manifests that it contributed significantly to the overall aroma.

2.10. Sensory Evaluation

A total of 10 volunteers (6 women and 4 men, average age of 25) were recruited from the Beijing Key Laboratory Flavor Chemistry Beijing Technology and Business University. All volunteers were well in sensory evaluation of food and could correctly distinguish aromas of compounds. The six evaluation odor attributes were defined as follows: guaiacol for smoky aroma, methional for cooked-potato aroma, HEMF for caramel-like aroma, butanol for alcoholic aroma, 3-methyl-1-butanol for malty aroma, and 2-phenylethanol for floral aroma. Sensory experiments were conducted to evaluate the intensities of each aroma attribute of the HLFSS samples on a ten-point liner scale (0 is imperceivable, 10 is strongly perceivable). Sensory tests were carried out in a room temperature at 21°C, and each sample was placed in a 40 mL sealed glass vial, labelled with any three digits. The average score of all panelists was the score for each attribute.

2.11. Aroma Recombination Test

The recombination experiments were conducted by merging 23 key odorants with OAVs ≥ 1 in the odorless matrix (including 8 g monosodium glutamate, 17 g table salt, 0.35 g I + G, 0.012 g sucralose, 0.12 g potassium sorbate, and 75 mL odorless water). The recombination sample (RS) and the original sample (OS) were evaluated by the method mentioned previously (0 is not perceivable, 10 is strong perceivable). The average score of all panelists was the score for each attribute.

2.12. Statistical Analysis

All results were performed as the mean value ± standard deviation (SD) in triplicate. All obtained data were using Microsoft Office Excel 2021. Heatmaps were drawn by TB tool, and origin 2021b (Origin-Lab, Northampton, MA, USA) was used to gain the radar chart. One-way analysis of variance (ANOVA) and Duncan’s multiple-range tests were conducted by using the statistical package SPSS software; the results were significant when .

3. Results and Discussion

HLFSS and its volatile isolate were evaluated to obtain an idea of the overall aroma of SS and check whether the extraction method was suitable. The results revealed that the isolate had almost the same odor characteristic with SS, which indicated that the odor compounds contributing to the overall odor profile had been separated successfully from SS. Both of them had smoky, cooked-potato, caramel, alcoholic, malty, and floral notes.

3.1. Identification of Odor-Active Compounds in HLFSS by Combining FD Factors

A total of 37 aroma-active compounds were identified and displayed in Table 1, including seven acids (Nos.: 13, 16, 17, 21, 24, 25, 36), six alcohols (Nos.: 1, 2, 3, 6, 20, 28), five furans (Nos.: 15, 23, 32, 33, 35), five pyrazines (Nos.: 4, 7, 8, 11, 12), four ketones (Nos.: 5, 10, 26, 29), three esters (Nos.: 9, 19, 37), three phenols (Nos.: 27, 31, 34), two sulfur-containing substances (Nos.: 14, 22), one aldehyde (Nos.: 18), and one heterocyclic compound (Nos.: 30), and most of the compounds were identified in the previous study [16, 21].

3.1.1. Acids

Seven acids were determined in those HLFSS samples, and these odorants were mostly described as unpleasant aroma during GC-O analyses. Acetic acid was a very essential volatile constituent with an FD factor of 64, which was mainly generated by the metabolism of by lactic acid bacteria [22]. In addition, other acids may be the result of degradation of amino acids or may be the products of oxidative degradation of fatty acids [16, 23]. 3-Methylbutanoic acid (FD value = 256) was usually described as an unpleasant, intolerable, or pungent smell, which contributed significantly to the overall flavor, and it was mainly produced by the oxidation reaction of amino acids and corresponding aldehyde intermediates [24].

3.1.2. Alcohols

Alcohols mainly exist in the early stage of fermentation in SS [25], which has an effect on the formation of the overall flavor of SS. The concentrations of 3-methylbutanol and phenylethyl alcohol were higher than those of the four alcohols identified. They were generated through the Ehrlich pathway, in which amino acids as precursors conduct deamination, decarboxylation, and reduction to yield alcohols with one less carbon atom than the corresponding precursors, such as 3-methylbutanol from leucine and phenethyl alcohol from phenylalanine [26, 27]. Moreover, phenylethyl alcohol (floral note) with the FD factor of 256 was an important aroma-active compound, which had great contribution to SS aroma.

3.1.3. Furans

HEMF and HDMF, as representatives of furans with caramel-like odor, had been identified as crucial aroma-active compounds in SS in the previous study [20], which not only have high aroma intensity but also have the ability to improve aroma, enhance sweetness, and neutralize saltiness [28]. Moreover, the formation of HEMF was influenced by diverse factors, such as glucose concentration, yeast species, and fermentation temperature [21, 26]. The FD factors of HDMF, HEMF, and sotolon were all 1024, so these components were considered to be important aroma-active components in HLFSS and had a great impact on the unique flavor of HLFSS.

3.1.4. Pyrazines

A total of five pyrazines were detected in this study. 2,5-Dimethylpyrazine had a higher FD factor of 128 of them, followed by 2,6-dimethylpyrazine and 2,3,5-trimethylpyrazine with an FD factor of 16. Usually, pyrazines release an attractive nutty aroma, which is mainly produced during the fermentation process through Maillard reaction and heat sterilization [27].

3.1.5. Ketones

Four ketones including acetoin, 1-hydroxy-2-butanone, methylcyclopentenolone, and maltol were detected; 1-hydroxy-2-butanone had a higher concentration of 9148–18617 μg/L, followed by maltol with a concentration of 5449–5790 μg/L. Ketones mainly came from three sources, that is, raw materials, microbial metabolism, and Maillard reaction [16]; for example, methylcyclopentenolone and maltol could be formed from Maillard reaction; acetoin and 1-hydroxy-2-butanone were generated from microbial metabolism [29].

3.1.6. Esters

A total of three esters were identified, namely, ethyl lactate, γ-butyrolactone, and ethyl vanillate. Most of the esters exhibited a fruit-like aroma; they were generally formed by metabolism of yeast or by esterification reactions of organic acid with alcohols [30]. Among three esters, ethyl lactate had the higher FD value of 16 and it was widely found in all kinds of alcoholic drinks with a fruity odor [31]. Moreover, few studies have reported the presence of ethyl vanillate in SS, which have been reported to exist in wines [32]. Ethyl vanillate had a low aroma intensity and was not easily detected by olfaction.

3.1.7. Phenols

A total of three phenols were sniffed during GC-O analyses in this study, including 4-ethyl-2-methoxyphenol, guaiacol, and 4-ethylphenol, which exhibited strong smoky aromas. The FD factor of guaiacol and 4-ethylphenol was 256, and that of 4-ethyl-2-methoxyphenol was 512, so these compounds were considered to be important aroma-active compounds and were crucial for the overall flavor enhancement of HLFSS [20, 33]. Research had shown that increasing the proportion of wheat flour in raw materials could raise the concentration of guaiacol in SS [34].

3.1.8. Sulfur-Containing Compounds

Sulfur-containing constituents were mainly derived from the decomposition of amino acids containing sulfur in raw materials [35]; although their peak areas were relatively small, their influence on the flavor of the SS should not be underestimated. These compounds often had an aroma similar to that of onions, potatoes, or garlic [36]. The FD factor of both methionol and methional was 256 with a cooked potato-like, and they had been identified in fermented alcoholic beverages such as Qingke Jiu and wine [37, 38]. Normally, methionol and methional have significantly lower thresholds; therefore, they could be sniffed even at a low concentration.

3.1.9. Aldehydes

Only phenylacetaldehyde with a pleasant floral and honey aroma was identified as an odor-active compound in this study. In the AEDA experiment, it reached an FD factor of 128, which indicated it had a large contribution to the aroma of SS. Phenylacetaldehyde could be produced through amino acid degradation, so it could be speculated that the formation of this aldehyde might be related to the fermentation process and protein metabolism [21].

3.1.10. Others

Except for the odorants above, the remnant compound only included 2-acetylpyrrole. Usually, pyrroles have the higher thresholds, so they have the insignificant aroma characteristics at low concentrations and are not easily detected by GC-O analyses. The threshold of 2-acetylpyrrole in water can reach 58000 μg/L [18], so it not easily sniffed; in this study, the FD factor of 2-acetylpyrrole (nutty) was less than 2.

3.2. Quantitative Analysis of Odor-Active Compounds in HLFSS during Storage

To investigate the changes on concentrations of odor-active compounds during storage, the odorants in the samples with different storage time were isolated and quantitated and the related data and the results obtained are shown in Tables 2 and 3 and Figure 1. From the results, it could be seen that the relative contents of furans, acids, alcohols, ketones, and sulfur-containing substances were higher among the odor-active compounds of the brewing SS samples, accounting for about 44.40%, 35.19%, 13.54%, 3.9%, and 1.29%, respectively. The compound with the highest concentration was acetic acid, which could reach 238250–257825 μg/L, followed by HEMF (207441 − 103763 μg/L), HDMF (101325–157806 μg/L), acetol (46319–54261 μg/L), butanol (22204 − 19536 μg/L), and 1-hydroxy-2-butanone (18617 − 9148 μg/L).

3.2.1. Aroma-Active Compounds with Increased Content during Storage in HLFSS

The concentration of acetic acid, propionic acid, isobutyric acid, 3-methylbutanoic acid, 3-methylvaleric acid, and 4-methylpentanoic acid rose during storage. This is because most of the microorganisms in SS have an active lipase system. Although the HLFSS is sterilized before leaving the factory, the enzymes in HLFSS are not completely inactivated. These enzymes can promote the degradation of amino acids and fat acid occurring in HLFSS [23], thereby increasing the concentration of some acids. Acetic acid (238250–257825 μg/L) continued to increase during storage in HLFSS, and it varied greatly throughout the storage process, so its content change could be used as an indicator to determine the storage time of HLFSS.

Pyrazines were mainly produced through Maillard reaction, and they were considered to be the main volatiles in acid-hydrolyzed SS formed after being heated [1]. The concentrations of 2-methylpyrazine, 2,6-dimethylpyrazine, and 2-ethyl-6-methylpyrazine also went up during storage, probably due to more precursors formed, which favored their formation.

Except that furfuryl alcohol was formed by microbial fermentation during the production [39], some reducing sugars could react with amino acids to generate furfural, which could be further converted into furfuryl alcohol, leading to an increase in its concentration [11]. It should be noted that the concentration of furfuryl alcohol (1918–6953 μg/L) varied greatly throughout the storage process, and its content changes could be also used as an indicator to determine the storage period of HLFSS. In addition, the concentration of 3-methyl-1-butanol also rose; its formation was associated with the degradation of leucine [40], which could degrade during storage, resulting in increasing in its concentration.

The formations of furans were related to Maillard reaction to a certain extent [1]. Among five furans, the concentrations of 2-acetylfuran and HDMF increased during storage, especially that of HDMF exhibited significant changes (101325–157806 μg/L), so the content change of HDMF could be used as an indicator for determining the storage period of HLFSS. Phenylacetaldehyde was present at the lower level (69–132 μg/L) in HLFSS, and its concentration tended to increase during storage. It had been found in many fermented soy foods, and its concentration usually peaked during the later stages of SS fermentation. Long-term storage would lead to an increase in phenylacetaldehyde content, which was related to the degradation of phenylalanine.

Moreover, the concentrations of 2-acetylpyrrole, acetol, guaiacol, and γ-butyrolactone also increased during storage. Among them, acetol (46319–54261 μg/L) had a significant change in concentration throughout the storage process, which could be used as an indicator to determine the different storage periods of HLFSS.

3.2.2. Aroma-Active Compounds with Decreased Content during Storage in HLFSS

The contents of 4-ethyl-2-methoxyphenol and 4-ethylphenol decreased during storage, and Wang et al. [9] also found that as heating time was prolonged, their concentrations in SS went down. Maybe they reacted with other compounds to form new compounds. Among all alcohols, the levels of 2-phenylethanol (from 7338 μg/L to 5890 μg/L) and butanol (from 22204 μg/L to 19536 μg/L) declined during storage. The reason was that both of them could react with organic acids to give ester compounds, and they could be oxidized into aldehyde and organic acid, which made their concentrations decease. From the results obtained previously, it could be seen that the content of phenylacetaldehyde increased indeed.

HEMF, with a strong caramel-like note, had the higher concentration (207441−103763 μg/L) among odor-active compounds identified. HEMF could be generated not only by the biosynthesis pathway of yeasts but also by Maillard reaction [28]. However, HEMF contained active groups in its structure, so it could take place degradation reaction during storage, which led to a decrease in its concentration. Changes in the concentrations of HEMF during storage could be used as an indicator to determine different storage periods for HLFSS. In addition, the contents of 1-hydroxy-2-butanone (from 18617 μg/L to 9148 μg/L) and acetoin (from 2765 μg/L to 2307 μg/L) dropped because they easily took place oxidation or polymerization reactions to form diketone or dimmer compounds. Because of the great change on the concentration of 1-hydroxy-2-butanone, these changes could be used as an indicator to determine the storage period of HLFSS.

Esters were widely present in fermented foods; their formations were closely related to the yeast metabolism [23]. In the production process of SS, yeast could degrade some precursors into alcohols and acids, which took place esterification reaction to give ester compounds [27]. Among the three esters detected, the concentration of ethyl vanillate (from 206 μg/L to 132 μg/L) in HLFSS decreased during storage, which might be due to the hydrolysis of ethyl vanillate. Furthermore, the contents of phenylacetic acid and sotolon also showed a gradual decrease during storage.

3.2.3. Aroma-Active Compounds with Fluctuating Content during Storage in HLFSS

Sulfur-containing compounds had a low content in SS, but they had an important effect on the overall flavor. They were mainly produced by degradation of sulfur-containing amino acids or peptides in raw materials [3], and their threshold in water was very low [18], so these components were easily identified by GC-O analyses. The concentrations of methional and methionol fluctuated during storage; both of them could carry out interconversion under a certain condition, so it was speculated that there might be some dynamic equilibrium between them.

In addition, the concentrations of ethyl lactate, maltol, methylcyclopentenolone, 2(5H)-furanone 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, and isobutanol also showed a fluctuation trend during storage. The concentration of ethyl lactate (3715−3728 μg/L) was higher than other esters, which was consistent with previous studies [41]. Maltol was produced from Maillard reaction, and methylcyclopentenolone had been detected in the glucose-tyrosine model system and the glucose-histidine model system [42, 43]. It was presumed that these compounds might be in a state of transformation and formation during storage of HLFSS.

3.3. OAV

Among the 37 aroma-active compounds detected, 23 odorants had OAV ≥ 1, so they were identified as the key odorants of HLFSS (showed in Table 4 and Figure 2). In addition, the OAVs of 3-methylbutanoic acid and 4-methylpentanoic acid ranged from less than 1 to 1 during storage. HEMF (OAV: 90229–180383), methional (OAV: 6976–7931), and HDMF (OAV: 4544–7077) had relatively higher OAVs, and the OAVs of HEMF were the highest among all aroma compounds, so it could be concluded that this component should contribute greatly to the unique flavor of SS. Both methional (6976–7931) and methionol (44–48) exhibited potent cooked potato-like notes. The smoky aroma was a typical characteristic aroma of phenols; three phenols identified in the present research had an OAV > 1, and guaiacol (130–139) got a higher OAV. At the same time, the FD factors of the two sulfur-containing compounds mentioned previously and phenolic compounds were ≥256, indicating that these compounds played an essential role in the formation of the overall aroma of SS. The OAVs of 2-phenylethanol and phenylethyl aldehyde ranged from 11 to 21, which imparted a floral note to SS. Among the components with OAVs ≥ 1, 3-methyl-1-butanol (1193–1373), butanol (48−43), and isobutanol (22 − 19) had alcoholic aroma, and they were the main contributors to the alcoholic aroma of HLFSS.

3.4. Aroma Recombination Experiment

To investigate the contribution of aroma-active compounds with OAVs ≥ 1 to the overall aroma profile of SS, reconstitution experiments and sensory evaluation were conducted based on the detected key aroma components, and the results are shown in Figure 3. From Figure 3, it could be seen that the aroma profile of the recombinant sample (RS) was similar to that of the original sample (OS), and the scores for alcohol-like and floral attributes were closer. In addition, the RS had stronger intensities in malty, caramel-like, and smoky attributes compared to the OS; the reason might be that the matrix of HLFSS was more complex than that of the RS, resulting in the odorants in HLFSS that were not released. Therefore, it could be concluded that the RS better simulated the aroma profile of HLFSS.

4. Conclusion

GC-FID-O and GC-MS-O coupled with SE-SAFE were employed to investigate the flavor of HLFSS during storage. A total of 37 odor-active compounds were identified; their FD factors were measured through AEDA, and the FD factors of methional, 3-methylbutanoic acid, phenethyl alcohol, sotolon, HEMF, and HDMF were 1024. Twenty-three compounds with OAV ≥ 1 were considered as key odorants, and the recombination tests also confirmed the important contribution of these key odorants to HLFSS. The concentration of HEMF, HDMF, acetic acid, 1-hydroxy-2-butanone, acetol, and furfuryl alcohol vary greatly during storage, and the changes on content of these compounds could be used as the indicators to determine the storage time of HLFSS.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was funded from the Hunan Science and Technology Research Plan (2018GK5015).

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Copyright © 2023 Rui Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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