Impact of Different Raw Materials on Changes in Volatile Compounds during Moromi Fermentation †

: The composition and ratio of volatile compounds in soy sauce have a major impact on its organoleptic properties. Considering the important inﬂuence of long-term (3 months) moromi fermentation on the aroma formation of soy sauces from different materials (soybean, rice, black bean, wheat, wheat ﬂour and mungbean), the volatile compounds of 24 samples in total, taken from three different stages of moromi fermentation, were analyzed via solid phase microextraction coupled with gas chromatography–mass spectrometry (SPME–GC–MS). The results show a total of 77 volatile compounds, including acids (4), alcohols (14), phenols (6), aldehydes (12), esters (26), ketones (5), furan(one)s (5) and pyrazines (5), and the majority of the compounds were common. Among all samples, the highest number of volatile compounds (5528.58 ± 1308 µ g/L) was detected in the moromi made from the combination of soybean, black bean and wheat ﬂour on the ﬁrst month of fermentation, and the sample that had the lowest number of volatile compounds (63.25 ± 1.70 µ g/L) was detected in the moromi sample made from the combination of soybean and wheat ﬂour on day 0. During the three months of moromi fermentation, the relative contents of acids, alcohols, phenols, aldehydes, esters, ketones, furan(one)s and pyrazines changed gradually. Finally, the total presence of volatile compounds identiﬁed in the 24 samples increased from day 0 to 1 month and from month to month perfectly.


Introduction
Soy sauce is a dark brown liquid from fermented soybeans and a wheat blend or wheat flour, originating in China and brought to Cambodia by Chinese people who immigrated there long ago.Chinese Cambodians mainly produce soy sauce in Cambodia, and over 90% of Cambodians consume soy sauce [1,2].It is now Asian and Western countries' most widely recognized fermented soyfood [3].Soy sauce is consumed as a culinary ingredient rather than a preservative.Its peculiar flavor has a strong umami, salty and caramel-like character that complements various foods' savory taste and scent.On the other hand, the processes of soy sauce manufacture are related to the country of origin.For example, the Chinese style employs 80:20 and 70:30 soybean to wheat or wheat flour ratios, respectively, whereas the Japanese style uses equal proportions of each (ratio 50:50) [4].Koji is made with Aspergillus oryzae and is then fermented at a greater salt concentration (160-180 g/L NaCl) at a controlled or uncontrolled temperature.The fermentation of moromi is a complicated process.For 3-6 months of fermentation, flavor and microbes, such as lactic acid bacteria and yeasts, are added to the fermentation process [5].Although physicochemical properties, particularly formaldehyde nitrogen, are crucial in determining the quality of soy sauce by the China National Standard (GB18186-2000, fermented soy Biol.Life Sci.Forum 2023, 26, 103 2 of 12 sauce), volatile characteristics and distinct flavors related to the fermentation process are also important [6].The composition and ratio of volatile chemicals in soy sauce significantly impact its organoleptic properties [7].During the moromi fermentation process in traditional Chinese soy sauce, significant changes in acids, alcohols, esters, aldehydes, ketones and furans are examined [8].The most common and robust technique to analyze (and identify) volatile aromatic compounds is gas chromatography-mass spectrometry (GC-MS) [9].GC separates the volatile compounds in a sample, whereas the analyte is fragmented by GC-MS and identified according to its mass [10].Not only are soybeans and wheat used as materials to make soy sauce, but other kinds of beans, such as black beans, peas and rice, have also been used to make soy sauce [11].The volatile compounds in moromi from different materials have yet to be widely studied.
The primary purpose of this study was to evaluate and differentiate the number and concentration of the volatile compounds extracted and detected from soy sauces during the moromi fermentation of different materials when they are zero days, one month, two months and three months old.

Sample Preparation
Samples were directly prepared for Koji fermentation (3 days) and then soaked into a brine solution to continue as moromi fermentation when the first stage of the sample was taken on day zero (only when Koji was soaked into the brine).Samples were aged at room temperature for a month, two months and three months, which were when the samples' second, third and fourth stages were taken, respectively.Therefore, samples had to be filtered with cheesecloth and placed into a falcon.After that, samples were centrifuged at 5000 rpm with one accel at 4 • C for 20 min [12] and kept at a temperature of −20 • C for further experiments.

Equipment
The key for this experiment was the gas chromatography-mass spectrometry (GC-MS) apparatus, Shimadzu GCMS-QP2010 Ultra with AOC-5000 (Kyoto, Japan).On the other hand, an SPME fiber 50/30 um DVB/CAR/PDMS, Stableflex (2 cm) 24 Ga, including its holder (bought from Sigma-Aldrich and Supelco, from Merck KGaA, Darmstadt, Germany), a 20 mL gas-tight glass vial with a PTFE septum in an aluminum cap, a magnetic heating stirrer with a stirring bar, micropipettes, a thermometer and a salt meter were used for the SPME extraction of the volatile compounds through a headspace.The column used in this study was SH-Rxi-5SilMS with a 30 m length, 0.32 mm diameter and 0.1 µm thickness.

SPME Extraction of Volatile Compounds
In the first stage, after thawing, each sample had to be checked for salt content with the salt meter (a few drops of soy sauce were put on the salt meter in the sensor section to measure its salt).Then, salt (sodium chloride: NaCl) was gradually weighted and adjusted to 25% of the 5 mL sample.Salt and 5 mL of the soy sample were then placed into a 20 mL vial, and the vial was covered with an aluminum cap (once a reagent was put into the vial, they were immediately capped to avoid contamination) [13].In the next step, 10 µL of the internal standard, 2-methyl-3-heptanone (it was first diluted with methanol before being added and had a final concentration obtained of 20 mg/L) [14], and 10 µL of 4-nonanol (with the concentration of 0.082 g/L) were added [15].In addition, a magnetic stirring bar was placed in the vial to balance the mixture with a magnetic stirrer in the following stage.The vial was then carefully and tightly closed.It was then submerged in water in a beaker set over a heating stirrer until the mixture level sank; as a result, the mixture was homogenized for 20 min at each absorption time and temperature condition.The calibrated fiber was injected into the headspace and inside the vial after being equilibrated (the fiber needle was thought not to have been in direct contact with the liquid phase of the mixture), and the extraction of volatile compounds was carried out at a specific temperature for a specific amount of time (40 • C, 40 min; in this study).

GC-MS Analysis of Volatile Compounds
Some equipment had to be calibrated before the extraction of volatile compounds via the SPME technique to prevent errors.The SPME fiber needed to be injected into the GC-MS injector port at 250 • C at least 30 min before use to ensure that there were no remaining compounds in the fiber.The GC-MS apparatus used in this work was made up of an SH-Rxi-5SilMS column with the following specifications: 30 m in length, 0.32 mm in diameter and a 1 m thick film (J&W Scientific, Folsom, CA, USA).A 26.6 mL/min flow of 99.999% pure helium gas was used, and the injection mode was split.The GC oven's temperature was maintained at 40 • C for 2 min before being raised to 250 • C at a rate of 5 • C/min and was maintained for 5 min.The ion source's mode was electron ionization, and its temperature was 230 • C with an electron voltage of 70 eV.The ions produced by ionization were scanned between 34 and 348 m/z during the study of volatiles.
Following the SPME extraction of volatile compounds from the soy sauce, the extracted analytes were desorbed by injecting the SPME fiber onto the GC injector port at 250 • C for 10 min.Once injected, a computer system attached to the GC-MS equipment was used to control the detection procedure.The GC-MS real-time analysis application ran the analysis of the volatile compounds for 59 min for each sample.Each sample was analyzed in triplicate.The identification of volatile compounds in this study was evaluated according to the mass spectra, depending highly on the measure of the similarity score being at least an 85% match to the NIST20 library.Then, each unknown compound was confirmed by calculating the retention indices (RI) using series n-alkane C7-C40.

Semi-Quantification
The quantification of volatile compounds was calculated using the peak area ratio to the internal standard, and it was multiplied by the concentration of the internal standard.The semi-qualification of aldehydes, esters, ketone, phenols and Furan(one)s was calculated by using 2-methyl-3-heptanone as the internal standard, whereas acids, alcohols and pyrazines were calculated by using 4-nonanol as the internal standard [16].Semi-quantification was calculated using the following formula: UC conc.= UC peak area IS peak area × IS conc.
where UC conc.= Unknown compound concentration (µg /L); IS conc.= Internal standard concentration (µg /L); UC peak area = Unknown compound peak area; IS peak area = Internal standard peak area.

Volatile Compound Identification of 24 Soy Sauces
The identification of volatile components in the extracts was performed using GC-MS.Whereas the RI (retention index) of unknown compounds was calculated using GC retention index standards (hydrocarbons from straight-chain C7-C40) and compared to the RI of the standards or those reported in the published work, the majority of compounds were identified via comparison of their spectra [17].Six soy sauces were produced from different raw materials, and each sample was taken four times from different stages of moromi fermentation: day zero, one month, two months and three months.Thus, there were 24 samples in total that were examined.In this study, around 300 volatile compounds were observed in the 24 soy sauce samples using SPME-GC-MS methods.But not all those substances were retrieved to be examined or discussed.Most compounds were assumed to be rejected from semi-quantification since they had similarity scores below 85 and could not be confirmed with the retention index.As shown in Table 1, 77 of the 300 volatile compounds were quantified using internal standards (2-methyl-3-heptanone and 4-nonanol), belonging to eight compound groups.The eight groups were acids (4), alcohols (14), phenols (6), aldehydes (12), esters (26), ketones (5), furan(one)s (5) and pyrazines (5).

Alcohols
Alcohols can be formed through many pathways, like aldehydes [18].Most of the alcohols in soy sauce are produced during the fermentation process under aerobic conditions from sugars and amino acids [19].Alcohols may specifically arise during the fermentation process of the fermented soy sauce type at the moromi stage of soy sauce production.Alcohols can occur during the moromi stage either through spontaneous fermentation or through the addition of LAB and certain yeast species, as well as through the reduction of aldehyde molecules [20].Moreover, amino acid catabolic and biosynthetic pathways can decarboxylate and then decrease keto acids, equivalent to the alcohols, to make higher alcohols [21].A total of 15 alcohols were identified in the 24 soy sauces.Among these, 2-methyl-1-butanol and 1-hexanol were present in almost all samples tested; however, phenyl ethyl alcohol seemed to be the predominant alcohol.2-methy-1-propanol and 2-methyl-1-butanol are compounds that are mainly produced through the Ehrlich pathway during fermentation [14]; however, the breakdown of 2-methyl-1-butanal also results in the production of 2-methyl-1-butanol, which adds to the malty aroma.Moreover, 2-methyl-1propanol and 2-methyl-1-butanol were also found in Chinese soy sauces that were made via high-salt-diluted state fermentation in a previous study [19].2-ethyl-1-hexanol was found in each of the samples but was able to be identified in only 12 samples: SBW0, SWF0, SWFB0, SR2, SR0, SRW0, SW2, SW0, SRW1, SWFB2, SW1 and SWFB1.The highest concentration provided was 123.16 ± 22.51 µg/L in SBW0, followed by SWF0.Some alcohols are also produced via the oxidation of fat during Koji incubation; Aspergillus oryzae produces lipase to break fat long-chain molecules, which is why it is prone to fat oxidation and produces alcohols such as 1-hexanol, 3-octanol, 2-octen-1-ol and 1-octen-3-ol.The highest amount of 1-hexanol, 3-octanol and 1-octen-3-ol was 90.07-112.44 µg/L, 3.92-4.67µg/L and 158.58-177.30µg/L, respectively; thus, during moromi fermentation, the mixtures of the sample did not break down sufficiently to form a higher concentration of alcohol.But according to Gao et al. (2010), alcohol concentrations increase from day 0 to the first and second month, and they peak in the third month [8], which is similar to this study, as presented in Figure 1.

Phenols
Five phenols were identified in this work.Out of the 24 samples, the phenols were not detected only in SBW0, and phenol concentrations of 455.037 ± 17.576 µg/L were found in SW3, except for 4-ethylphenol, which was not present in SWF2.However, 4-ethylphenol is also a significant phenol compound in soy sauces.It has been previously reported in Japanese, Korean and Thai soy sauces [22].These compounds are generated from the degradation of lignin glycoside in cereal bran during fermentation [21].
The enzyme peroxide can also produce 4-ethyl guaiacol, which causes aromatic amino acids to break down.When wheat bran is used for moromi fermentation, a microbe called Torulopsis transforms ferulic acid into 4-ethyl guaiacol, which can be thought of as one of the desirable volatile components for soy sauces [23].Soy sauces have a smoky aroma from 4-ethyl guaiacol, but these sauces also have spicy and sweet vanilla scents.4-ethyl-2methoxyphenol was also reported in Japanese raw and thermally treated soy sauces [24], according to [19].2,4-Di-tert-butylphenol was detected in 16 samples.It has bioactive and antifungal properties, and it is also found in rice and some plants [25].As shown in Figure 2, phenols in SBW1 contained the highest total concentration (1114.75µg/L), followed by SW2 (485.71µg/L); however, SWFB0 had the lowest concentration among all presented phenols in the soy sauces.

Acids
Microbial mechanisms in fermented soy sauces produce acids during the fermentation process, but in acid-hydrolyzed soy sauces, acids are byproducts of lipid destruction aided by heat [26].Among the 24 soy sauces, four acids were identified: 2-methyl-butanoic acid, 3-methyl-butanoic, acetic acid and octanoic acid.As shown in Figure 3, acids started to be detected from the first month of fermentation, and they were found ranging from 2.46 ± 0.1 µg/L to 148.78 ± 31.47 µg/L.The lowest concentration was 2.46 ± 0.1 µg/L.Acetic acid produced by lactic acid bacteria during fermentation gives a sour odor to soy sauce and contributes substantially to its aromatic profile.Acetic acid can react with alcohols to generate the corresponding acetate esters, which impart various fruity aromas [27].Acetic acid is one of the most vital acids in soy sauces [4].
From a previous study, the total semi-quantification of acids increased gradually from day 0 to the third month and slightly decreased after that [8].Therefore, the semiquantification of acids in this study seemed to be similar to the above-referenced research, except for the SBW sample, in which the concentration in SBW2 (1-month-old soy sauce) increased up to 148.78 ± 31.47 µg/L, which was the highest among all samples tested.This might have been caused by the formation of esters related to the metabolism of lipids by yeast, which provides many acids and alcohols that may undergo esterification to yield a variety of esters [26].
From a previous study, the total semi-quantification of acids increased gradually from day 0 to the third month and slightly decreased after that [8].Therefore, the semiquantification of acids in this study seemed to be similar to the above-referenced research, except for the SBW sample, in which the concentration in SBW2 (1-month-old soy sauce) increased up to 148.78 ± 31.47 µg/L, which was the highest among all samples tested.This might have been caused by the formation of esters related to the metabolism of lipids by yeast, which provides many acids and alcohols that may undergo esterification to yield a variety of esters [26].

Esters, Furan(one)s and Pyrazines
Esters also play essential roles as volatile compounds in soy sauce.Esters are mainly formed from the esterification of alcohols with fatty acids during fermentation [21].Furthermore, because most of the microorganisms found in moromi fermentation have active lipase systems that can break down triglycerides into free fatty acids and glycerol, monoglycerides and diglycerides, fatty acids are considered to be degradation products of soybean fat and significantly contribute to the flavor of soy sauce.However, only a small number of fatty acids were found in the initial research and in the presence of other fatty acids in the matching esters.In contrast, other fatty acids were absent, suggesting that most acids generated esters through an esterification reaction during moromi fermentation [8].Many esters were present in this study; 26 esters were detected.Ethyl acetate was the compound that was present the most, especially in SBW1, which had a concentration between 443.08 µg/L and 738.21 µg/L.

Esters, Furan(one)s and Pyrazines
Esters also play essential roles as volatile compounds in soy sauce.Esters are mainly formed from the esterification of alcohols with fatty acids during fermentation [21].Furthermore, because most of the microorganisms found in moromi fermentation have active lipase systems that can break down triglycerides into free fatty acids and glycerol, monoglycerides and diglycerides, fatty acids are considered to be degradation products of soybean fat and significantly contribute to the flavor of soy sauce.However, only a small number of fatty acids were found in the initial research and in the presence of other fatty acids in the matching esters.In contrast, other fatty acids were absent, suggesting that most acids generated esters through an esterification reaction during moromi fermentation [8].Many esters were present in this study; 26 esters were detected.Ethyl acetate was the compound that was present the most, especially in SBW1, which had a concentration between 443.08 µg/L and 738.21 µg/L.
Esters formed from the esterification of alcohols with fatty acids during moromi fermentation.As shown in Figure 4, SBW1 contained the highest total semi-quantification (775.84 µg/L) of esters identified.However, esters found in the 1-month and 3-month samples were agreeably increased, so esterification increased extensively and formed more esters.
From a previous study, the total semi-quantification of acids increased gradually from day 0 to the third month and slightly decreased after that [8].Therefore, the semiquantification of acids in this study seemed to be similar to the above-referenced research, except for the SBW sample, in which the concentration in SBW2 (1-month-old soy sauce) increased up to 148.78 ± 31.47 µg/L, which was the highest among all samples tested.This might have been caused by the formation of esters related to the metabolism of lipids by yeast, which provides many acids and alcohols that may undergo esterification to yield a variety of esters [26].

Esters, Furan(one)s and Pyrazines
Esters also play essential roles as volatile compounds in soy sauce.Esters are mainly formed from the esterification of alcohols with fatty acids during fermentation [21].Furthermore, because most of the microorganisms found in moromi fermentation have active lipase systems that can break down triglycerides into free fatty acids and glycerol, monoglycerides and diglycerides, fatty acids are considered to be degradation products of soybean fat and significantly contribute to the flavor of soy sauce.However, only a small number of fatty acids were found in the initial research and in the presence of other fatty acids in the matching esters.In contrast, other fatty acids were absent, suggesting that most acids generated esters through an esterification reaction during moromi fermentation [8].Many esters were present in this study; 26 esters were detected.Ethyl acetate was the compound that was present the most, especially in SBW1, which had a concentration between 443.08 µg/L and 738.21 µg/L.
the furan group was mostly found in the 3-month soy sauces and showed the highest total concentration in SW3; however, furans were also found on day 0.This might have happened as a result of roasting raw materials for the Koji stage [19].In addition, furans were produced during moromi fermentation and slightly increased on the following date [8]; this is why, in the first month of fermentation, furans were also able to be observed but could not be identified, as they had low similarity and peak area.Pyrazines have essential characteristics resulting from the presence of two nitrogen atoms.The Maillard reaction between saccharide and amino residues and the ambient temperature reaction of microbial metabolites can produce pyrazines [29].Many pyrazines were detected in the samples, such as methylpyrazine, 2,3-dimethylpyrazine, 2,5dimethylpyrazine, 2-ethyl-3-methylpyrazine and 2,6-dimethylpyrazine.As shown in Figure 6, in these samples, pyrazines were only found in nine samples, including ten pyrazines in SR, SRW, SRW0 and SW.

Aldehydes and Ketones
Twelve aldehydes and five ketones were identified in the 24 soy sauces.These arose mainly from the raw materials and the fermentation procedure.The most critical aldehydes were 2-methylbutanal and 3-methylbutanal, which showed the highest concentrations in SBW1 (71.9 ± 31.177 µg/L and 16.8 ± 53.535 µg/L, respectively).2-methylbutanal, which gives a malty aroma, is a crucial aromatic compound in Japanese and Korean soy sauces [26].Two aromatic aldehydes, benzaldehyde and benzeneacetaldehyde, were both detected.Benzaldehydes were detected and identified in almost all samples, whereas benzeneacetaldehydes were not found on day 0 of SW.As shown in Figure 7, benzaldehyde was seen in SBW1 with 3197 µg/L; this amount was higher than that of benzaldehyde from other samples because these compounds came from black beans [30].Ketone is one of the essential smells in soy sauce.During fermentation, some amino acids break down and produce ketones, and some ketones form from the oxidation of alcohol [31].
the furan group was mostly found in the 3-month soy sauces and showed the highest total concentration in SW3; however, furans were also found on day 0.This might have happened as a result of roasting raw materials for the Koji stage [19].In addition, furans were produced during moromi fermentation and slightly increased on the following date [8]; this is why, in the first month of fermentation, furans were also able to be observed but could not be identified, as they had low similarity and peak area.Pyrazines have essential characteristics resulting from the presence of two nitrogen atoms.The Maillard reaction between saccharide and amino residues and the ambient temperature reaction of microbial metabolites can produce pyrazines [29].Many pyrazines were detected in the samples, such as methylpyrazine, 2,3-dimethylpyrazine, 2,5dimethylpyrazine, 2-ethyl-3-methylpyrazine and 2,6-dimethylpyrazine.As shown in Figure 6, in these samples, pyrazines were only found in nine samples, including ten pyrazines in SR, SRW, SRW0 and SW.

Aldehydes and Ketones
Twelve aldehydes and five ketones were identified in the 24 soy sauces.These arose mainly from the raw materials and the fermentation procedure.The most critical aldehydes were 2-methylbutanal and 3-methylbutanal, which showed the highest concentrations in SBW1 (71.9 ± 31.177 µg/L and 16.8 ± 53.535 µg/L, respectively).2-methylbutanal, which gives a malty aroma, is a crucial aromatic compound in Japanese and Korean soy sauces [26].Two aromatic aldehydes, benzaldehyde and benzeneacetaldehyde, were both detected.Benzaldehydes were detected and identified in almost all samples, whereas benzeneacetaldehydes were not found on day 0 of SW.As shown in Figure 7, benzaldehyde was seen in SBW1 with 3197 µg/L; this amount was higher than that of benzaldehyde from other samples because these compounds came from black beans [30].Ketone is one of the essential smells in soy sauce.During fermentation, some amino acids break down and produce ketones, and some ketones form from the oxidation of alcohol [31].

Aldehydes and Ketones
Twelve aldehydes and five ketones were identified in the 24 soy sauces.These arose mainly from the raw materials and the fermentation procedure.The most critical aldehydes were 2-methylbutanal and 3-methylbutanal, which showed the highest concentrations in SBW1 (71.9 ± 31.177 µg/L and 16.8 ± 53.535 µg/L, respectively).2-methylbutanal, which gives a malty aroma, is a crucial aromatic compound in Japanese and Korean soy sauces [26].Two aromatic aldehydes, benzaldehyde and benzeneacetaldehyde, were both detected.Benzaldehydes were detected and identified in almost all samples, whereas benzeneacetaldehydes were not found on day 0 of SW.As shown in Figure 7, benzaldehyde was seen in SBW1 with 3197 µg/L; this amount was higher than that of benzaldehyde from other samples because these compounds came from black beans [30].Ketone is one of the essential smells in soy sauce.During fermentation, some amino acids break down and produce ketones, and some ketones form from the oxidation of alcohol [31].Five ketones were detected in the samples: acetoin, 2-heptanone, 3-octanone, butyrolactone and benzophenone.As shown in Figure 8, ketones found in SBW1 and SRW0 had total concentrations of 63.29 ± 21 µg/L.1 and 51.9 ± 0.71 µg/L, respectively.

All Volatile Compounds Included
The volatile compounds found in this study have been reported in many previous works of traditional fermented soy sauce [17].However, the number of the identified volatile compounds in this work gradually increased from month to month, as shown in Figure 9.Moreover, during the moromi fermentation of the six types of samples, between 11 and 17 volatile compounds were found on day 0 of fermentation, and about 30 to around 40 volatile compounds were discovered in the 3-month-old samples.Furthermore, during the liquid fermentation, the mixture of soy sauces started breaking down from day to day, which is why many more compounds were present on the following date, even though some compounds that were present on day 0 were degraded but produced newer compounds (which caused the aroma of soy sauce to be more interesting).The more unique volatile compounds, such as acids and aldehydes produced by microbial mechanisms during fermentation, rose interestingly [8].

All Volatile Compounds Included
The volatile compounds found in this study have been reported in many previous works of traditional fermented soy sauce [17].However, the number of the identified volatile compounds in this work gradually increased from month to month, as shown in Figure 9.Moreover, during the moromi fermentation of the six types of samples, between 11 and 17 volatile compounds were found on day 0 of fermentation, and about 30 to around 40 volatile compounds were discovered in the 3-month-old samples.

All Volatile Compounds Included
The volatile compounds found in this study have been reported in many previous works of traditional fermented soy sauce [17].However, the number of the identified volatile compounds in this work gradually increased from month to month, as shown in Figure 9.Moreover, during the moromi fermentation of the six types of samples, between 11 and 17 volatile compounds were found on day 0 of fermentation, and about 30 to around 40 volatile compounds were discovered in the 3-month-old samples.Furthermore, during the liquid fermentation, the mixture of soy sauces started breaking down from day to day, which is why many more compounds were present on the following date, even though some compounds that were present on day 0 were degraded but produced newer compounds (which caused the aroma of soy sauce to be more interesting).The more unique volatile compounds, such as acids and aldehydes produced by microbial mechanisms during fermentation, rose interestingly [8].Furthermore, during the liquid fermentation, the mixture of soy sauces started breaking down from day to day, which is why many more compounds were present on the following date, even though some compounds that were present on day 0 were degraded but produced newer compounds (which caused the aroma of soy sauce to be more interesting).The more unique volatile compounds, such as acids and aldehydes produced by microbial mechanisms during fermentation, rose interestingly [8].

Conclusions
In conclusion, this study demonstrates the changes in volatile compounds during moromi fermentation.Long-term moromi fermentations are necessary for aroma formation.Based on the results, it was determined that the number of volatile compounds mostly steadily rose from day 0 to 1 month and from month to month.Alcohols increased from day 0 to 2 months, and most ester compounds increased from 2 months to 3 months.Additionally, given that most volatile compounds were produced during the early moromi fermentation stage, further research on optimizing the Koji-culturing process is crucial and is now being conducted to improve the flavor of soy sauce.There were 77 volatile compounds identified from 24 soy sauces, and they were detected via SPME-GC-MS and classified into eight groups of compounds, including acids (4), alcohols

( a )
Retention time of each compound after integration and identification.(b)Compounds selected by searching mass spectra with the NIST 20 library and at least 85% similarity, then confirmed with retention indices in the NIST 20 library. (c) Calculated retention indices from the experiment using series n-alkane C7-C40 standards.(d)Retention indices literature from NIST 20 library on semi-standard non-polar GC-MS column.n.d., not detected with GC-MS.

Figure 1 .
Figure 1.Total alcohol semi-quantification of each sample tested.

Figure 2 .
Figure 2. Total phenol semi-quantification of each sample tested.

Figure 3 .
Figure 3.Total acid semi-quantification of each sample tested.

Figure 4 .
Figure 4. Total ester semi-quantification of each sample tested.

Figure 3 .
Figure 3.Total acid semi-quantification of each sample tested.

Figure 4 .
Figure 4. Total ester semi-quantification of each sample tested.

Figure 4 .
Figure 4. Total ester semi-quantification of each sample tested.

Figure 7 .
Figure 7.Total aldehyde semi-quantification of each sample test.

Figure 8 .
Figure 8.Total ketone semi-quantification of each sample tested.

Figure 9 .
Figure 9.Total presence of volatile compounds in the 24 samples.

Figure 8 .
Figure 8.Total ketone semi-quantification of each sample tested.

Figure 7 .
Figure 7.Total aldehyde semi-quantification of each sample test.

Figure 8 .
Figure 8.Total ketone semi-quantification of each sample tested.

Figure 9 .
Figure 9.Total presence of volatile compounds in the 24 samples.

Figure 9 .
Figure 9.Total presence of volatile compounds in the 24 samples.

Table 1 .
Volatile compounds in SRW from day 0 to week 3.