Key compounds contributing to the fruity aroma characterization in Japanese raw soy sauce

Abstract

In order to clarify the aroma characteristics of raw soy sauce (RS), the application of gas chromatography−olfactometry analysis to the aroma concentrate from a RS revealed 76 aroma peaks, of which 25 peaks showed fruit-like aromas. Furthermore, the head space aromatic compounds of RS were analyzed with 32 peaks detected. Ethyl 2-methylpropanoate, ethyl butanoate, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, and ethyl 4-methylpentanoate were detected with higher flavor dilution factor (FD factor) than other aroma compounds by aroma extract dilution analysis. Quantitative analysis suggested that these compounds were common in all RS samples tested in this study, and were present at higher concentrations than their perception thresholds. The concentrations and the FD factors of these compounds were significantly decreased during the heating of the RS. Fruitiness is one of the key aroma characteristics of RS and the ethyl esters identified in this study are the key components contributing to this distinct aroma.

Graphical abstract

Fruitiness is one of the key aroma characteristics of raw soy sauce and the ethyl esters identified in this study are the key components contributing to this distinct aroma.

Key words:

• raw soy sauce
• ethyl ester
• fruit-like aroma

Soy sauce is a traditional fermented seasoning common in East Asia and is widely used in Japan on a daily basis. Furthermore, soy sauce is also a popular seasoning in the Western world because of its intense umami taste and characteristic aroma. In addition, “Washoku” has been placed on the list of intangible cultural heritage elements of UNESCO.¹⁾

Japanese soy sauce is produced by fermenting boiled soybeans and roasted wheat for six to eight months. After maturation, the mixture is pressed to separate the solution and residue. Then the solution is heated for pasteurization and to adjust the color and aroma.²⁾ On the other hand, a membrane filtration technology was developed for removing micro-organisms from the solution in 2010. And thus, as a new type of soy sauce, the popularity of this new raw soy sauce (RS, the solution treated by membrane filtration) has been growing in recent years.

The aroma of soy sauce is one of the most important factors in determining its quality as well as its marketability to consumers. More than 300 flavor components have been identified in Japanese soy sauce and most of them are formed by microbes during the long period of aging and fermentation.^3–5) Previous studies have demonstrated that soy sauce possesses caramel-like, roasted and burnt aroma characteristics, and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), 2 or 3-methylbutanal, 3-hydroxy-4,5-dimethyl-2(5H)-furanone (HDMF), 3-(methylthio)propanal (methional), and 4-ethyl-2-methoxyphenol (4EG) are the key aroma components in soy sauce.^6,7) To date, there have been only a few studies of the aroma compounds in RS. Recently, Kaneko et al.⁸⁾ reported that HEMF, HDMF and methional were the key aroma compounds in RS and that the burnt-aroma phenolic compounds were significantly increased during heat treatment of RS. In our previous works, we have reported that the flowery 2-phenylethanol and volatile thiols, such as 2-methyl-3-furanthiol and 2-furanmenthanethiol,⁹⁾ are also key aroma compounds as important as HEMF¹⁰⁾ in RS.

Heat treatment of soy sauce causes a clear change in the aroma, which is correlated with changes in the concentrations of important aroma compounds, especially for compounds with a low boiling point.^6,10) The results of gas chromatography−olfactometry (GC–O) in our previous works suggested that heat-treated soy sauce (HS) has a fruit-like aroma characterization.^10,11) Typically, the intensity of the fruit-like aroma decreases during heating and regular soy sauce is heated at the end of the production process during pasteurization. Production of RS lacks the final heating process and as a result, the aroma characteristics of RS are not yet clear. Therefore, we anticipate that RS will give a strong, fruit-like aroma characterization, and this aroma characterization is likely to be significantly stronger than in HS (regular soy sauce) and would be weakened during the heating process.

The aim of the present investigation was to clarify the aroma characteristics in RS by solvent-assisted flavor evaporation (SAFE) and solid phase micro-extraction (SPME) analysis, to identify the key aroma compounds in the head space of RS by aroma extract dilution analysis (AEDA) and to clarify the contribution of these key aroma compounds to RS and HS by quantitative analysis.

Materials and methods

Soy sauce samples

Raw soy sauces sample A (ARS) and heat-treated soy sauce sample B (BHS) were purchased from a local market in Morioka, Japan. AR and BH were made by the same brewing company but had different lot numbers. Raw soy sauce sample N (NRS) was made by Kikkoman Corporation (Noda, Japan) for this study. Sample NHS was prepared by heating NRS at 80 °C for 30 min in a dry stirring bath. Eight RS samples (S1–S8) and eight HS samples (S9–S16) were purchased from a local market in Morioka. These RS and HS samples were made by four different brewing companies. S1and S9, S2 and S10, and S3 and S11 were made by companies A, B, and C, respectively. S4–S8 and S12–S16 were made by company D. All of the commercial soy sauces had different lot numbers.

Chemicals

Ethyl 2-methylpropanoate, ethyl (R/S)-2-methylbutanoate, ethyl 3-methylbutanoate, and ethyl 4-methylpentanoate were purchased from Ogawa & Company Ltd. (Chiba, Japan). Ethanol was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Ethyl 2-ethylbutanoate, ethyl butanoate, ethyl propanoate, and the other authentic compounds in this study were purchased from Kanto Chemicals (Tokyo, Japan).

Preparation of the aroma concentrates of RS and HS for GC–O by SAFE

The aroma concentrates of ARS and BHS were extracted and assayed by the method described in our previous study.¹²⁾ First, sodium sulfite (1 g) and 0.5 mL of ethyl acetate were added to 200 mL of a 40% (w/v) soy sauce solution. Second, the sample solution was stirred twice with 5 mL of dichloromethane at 1000 rpm for 5 min. This operation was repeated twice, and a total of 160 g of soy sauce sample was used for preparation of the aroma concentrate. The resulting organic phase was dried with anhydrous sodium sulfate and then distilled by SAFE (40 °C, <5.0 × 10⁻³ Pa). The soy sauce aroma concentrates were obtained by concentrating the organic phase with a 40 °C water bath and then concentrated to 25 μL under a nitrogen flow in a glass tube. Each of the obtained samples was then subjected to GC–O analysis.

Classification of odor compounds based on their odor qualities by GC–O

GC–O analyses were performed by three panelists (two females and one male). Panelists were instructed to limit the odor description of sensory perceptions to the descriptors defined by the study of Shimoda et al.¹³⁾ and Imamura et al.¹⁴⁾ Three panelists participated in an evaluation meeting to share common perceptions about the odor descriptions numerous times. More than 50 odor-active compounds were detected from the soy sauce samples by GC–O. In order to clarify the aroma characteristics of RS, the odor-active compounds were classified by a system of six odor evaluation items based on their odor qualities by the method described in our previous studies.^10,11) According to the results of the GC–O analysis and the reports of Kaneko et al.⁸⁾, Steinhaus et al.⁶⁾, and Imamura et al.¹⁴⁾, the system of six odor evaluation items included caramel-like/sweet, nutty/roasty, salty, spicy/burnt, fruity, and unpleasant.

AEDA of RS and HS by SPME

A 20 mL of sample (NRS or NHS) was loaded into a 50 mL glass vial. The vial was tightly capped and kept in a water bath at 40 °C for 10 min and then extracted for 30 min at the same temperature. A 75 μm carboxen/polydimethylsiloxane fiber (CAR/PDMS; Supelco) was used for volatile extraction. According to previous studies, CAR/PDMS fiber is able to adsorb volatiles from soy sauce or miso soup more efficiently than other fibers, giving higher intensities and widest range of volatiles.^15,16) The fiber was conditioned at 230 °C for 10 min before extraction to prevent any contamination. After extraction, the fiber was inserted into the injection port of the GC–O at 230 °C to desorb the analytes.

Soy sauce samples were diluted two times stepwise with ultra-pure water, and each fraction was analyzed by GC–O. AEDA was performed three times for each sample by three panelists. The successful detection of each compound was defined as not less than two detections by all panelists, and the flavor dilution factor (FD factor) of each compound was determined as the maximum degree of dilution that could be detected.

GC–O and gas chromatography–mass spectrometry

GC–O analysis was conducted on a Shimadzu GC-17A instrument. For the DB-WAX capillary column (J&W; 60 m × 0.25 mm (i.d.), 0.25 μm film thickness), the oven temperature was maintained at 40 °C for the first 5 min, then increased to 200 °C at a heating rate of 3 °C/min, and then held constant at 200 °C for 10 min. The flow rate of the He carrier gas was 1.5 mL/min, linear speed was 26.5 cm/s, and the injection and flame ionization detection (FID) temperatures were 230 °C and 230 °C, respectively. The effluent from the GC column was split 1:1 between the FID and sniffing port. Compounds were detected on a Shimadzu QP-2010 GC- MS instrument, equipped with a DB-WAX capillary column (J&W; 60 m × 0.25 mm (i.d.), 0.25 μm film thickness). The oven temperature was programmed in a manner similar to that conducted for GC–O. The mass spectrometer was operated in electron impact (EI) mode with an ionization voltage of 70 eV, and the ion source temperature was set to 200 °C. Injection for gas chromatography–mass spectrometry (GC–MS) analysis was conducted in the splitless mode with a splitless time of 1 min.

Identification of the key aroma compounds in the head space of RS and HS

Each aroma compound in the head space of the soy sauce samples was identified by comparing its Kovats GC retention index (RI) and mass spectrum with the authentic compound by GC–MS, in addition to the comparison of its RI and odor quality with the authentic compound by GC–O.

Quantitative analysis of the ethyl esters in RS and HS samples by SPME–GC–MS

Two standard calibration curves were prepared by adding a known amount of pure chemical standard into a 20 mL soy sauce sample in a 50 mL glass vial along with an internal standard (ethyl 2-ethylbutanoate). For the standard calibration curve, at concentrations ranging from 0 to 1 μg/L, 0.5 μg/L of ethyl 2-ethylbutanoate and 0, 0.5, and 1.0 μg/L of ethyl 4-methylpentanoate were added to the sample mixture. For ranges from 1 to 60 μg/L, 5 μg/L of ethyl 2-ethylbutanoate and 0, 5, 10, 15, 30, and 60 μg/L of ethyl 2-methylbutanoate were added.

The ethyl esters were analyzed by GC–MS in the selected ion monitoring (SIM) mode. The ratio of the height of the selected ion of each compound (Table 1) to that of the internal standard and the added concentration of each compound, was used for the quantitative analysis of each compound. The concentrations of the various compounds were determined by calculation from the approximated curve using the linear least-squares method. The respective quantitative values of ethyl esters were determined by averaging the triplicate experiments.

Table 1. Selected ions for quantization of ethyl esters by GC–MS.

Compound	Characteristic ions (m/z)
	Quantifier	Qualifier
Ethyl 2-ethylbutanoate*	116	43, 71, 99, 115
Ethyl butanoate	116	43,71, 88, 101
Ethyl 2-methylpropanoate	116	71, 88, 89, 101
Ethyl 2-methylbutanoate	115	74, 85, 102, 130
Ethyl 3-methylbutanoate	115	57, 60, 85, 88
Ethyl 4-methylpentanoate	115	88, 99, 101, 129

* Internal standard compound for quantization.

Results and discussion

Odor evaluation of RS and HS by GC–O analysis

The aroma concentrates of ARS and BHS prepared by SAFE were used in the GC–O analysis. For the results of GC–O analysis, the odor evaluation of odor-active compounds and the number detected were used to evaluate the aroma characteristics of ARS and BHS by the system of six odor evaluation items. As shown in Table 2, 76 odor active compounds were detected from the aroma concentrate of ARS. Among them, 25 compounds were detected with a fruity aroma, followed by 22 compounds with a caramel-like/sweet aroma. In contrast, in the BHS, 62 odor active compounds were detected with 14 of them caramel-like/sweet aroma compounds and 12 having a fruity aroma. The total number of odor active compounds in ARS was greater than that the BHS, which is in agreement with our previous report.¹⁰⁾ The results of the system of six odor evaluation metrics suggested that the number of fruity aroma compounds were detected at a similar rate as caramel-like/sweet aroma compounds in ARS and BHS. In the past 50 years, most of the studies on the aroma compounds of Japanese soy sauce suggested that the caramel-like/sweet, nutty/roasty, and spicy/burnt were the most important odor characteristics, but the fruity aroma was never seen as a key component. Unlike the regular soy sauce, RS is not heated in the final process, and as a result, has a strong fruity aroma characteristic, which is unique to this new type of soy sauce.

Table 2. Numbers of volatile compounds based on their odor qualities in raw and heat-treated soy sauce by GC–O analysis.

Odor qualities^a	Raw soy sauce A	Heat-treated soy sauce B
Total^b	76	62
Caramel-like/sweet	22	14
Nutty/roasty	6	10
Salty	5	6
Fruity	25	12
Spicy/burnt	4	7
Unpleasant	14	13

^a Six odor qualities that aptly described the odor of soy sauce from the odor description proposed by Shimoda et al.¹³⁾ and Imamura et al.¹⁴⁾

^b Total numbers of volatile compounds in the aroma concentrate of raw and heat-treated soy sauce by GC–O analysis.

Identification of the key aroma compounds in the head space of RS

In general, fruity aroma compounds have lower boiling points and higher volatilities than other odorous compounds in soy sauce. Hence, in order to fully characterize the fruity aroma compounds in RS, further experimentation involving the head space aroma compounds is necessary. AEDA of NRS was performed by SPME-GC-O, and 32 compounds were detected (Table 3). Ethyl 2-methylbutanoate (No.11), exhibiting a fresh fruity note, was detected as having the highest FD factor of 2048, followed by ethyl 3-methylbutanoate (No.12, green, orange-like), methional (No. 19, cooked potato-like), ethyl 2-methylpropanoate (No.7, fresh fruity), ethyl 4-methylpentanoate (No. 14, green melon-like), having FD factors from 32 to 1024. HEMF, HDMF, and HMF, exhibiting caramel-like/sweet notes, were detected as having FD factors of 32, 4, and 8, respectively. The burnt aroma compounds, 4-ethyl-2-methoxyphenol and 2-methoxy-4-vinylphenol were both detected with FD factor of 2 for each. All of these compounds have previously been reported as aroma compounds in the commercial HS. In Table 3, although most of the key head space aroma compounds in NRS were common to NHS as well, different aroma characterization of two kinds of soy sauce was made due to the different contributions of the aroma compounds.

Table 3. Key aroma compounds in raw and heat-treated soy sauce.

No.	RI^a	Odor qualities	Compound^b	FD factor^c
				NRS^d	NHS^d
1	658	malty	actealdehyde^e	4	16
2	804	malty	2-methylpropanal	4	32
3	906	matly	2-methylbutanal	1	128
4	912	matly	3-methylbutanal	n.d	128
5	941	alcohol-like	ethanol	32	32
6	959	fruity	ethyl propanoate	4	1
7	963	fruity	ethyl 2-methylpropanoate	256	32
8	974	sour	unknown	n.d	8
9	991	gom-like	unknown	1	16
10	1035	fruity	ethyl butanoate	8	4
11	1050	fresh, fruity	ethyl 2-methylbutanoate	2048	256
12	1066	fruity	ethyl 3-methylbutanoate	1024	256
13	1170	fruity	2-heptanone^e	16	4
14	1182	fruity	ethyl 4-methylpentanoate	32	32
15	1216	malty	2-methyl-1-butanol	n.d	4
16	1222	malty	3-methyl-1-butanol	2	1
17	1296	mushroom-like	1-octen-3-one	32	4
18	1344	roasty	2,5-dimethylpyrazine	n.d	32
19	1456	cooked potato-like	methional	1024	2048
20	1470	potato-like	2-ethyl-3,5-dimethylpyrazine	16	16
21	1498	musty	2,3,5,6-tetramethylpyrazine^e	8	8
22	1645	sweat	phenylacetaldehyde	2	16
23	1668	sour	unknown	2	4
24	1672	roasty	2-furanmethanol	8	128
25	1866	burnt	2-methoxyphenol	16	32
26	1890	sweat	unknown	32	8
27	1922	sweat	2-phenylethanol	4	4
28	2029	burnt	4-ethyl-2-methoxyphenol	2	8
29	2039	caramel-like	4-hydroxy-2,5-dimethyl-3(2H)-furanone	4	8
30	2065	caramel-like	4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone	32	32
31	2137	caramel-like	4-hydroxy-5-methyl-3(2H)-furanone	8	16
32	2206	burnt	2-methoxy-4-vinylphenol	2	8

^a RI, retention index.

^b The compound was identified by comparing its RI and mass spectrum with the authentic compound by GC–MS in addition to comparison of its RI and odor quality with the authentic compound by GC–O.

^c FD factor, flavor dilution factor.

^d NRS, raw soy sauce sample N; NHS, NHS was prepared by heating NRS at 80 °C for 30 min in a dry stirring bath.

^e The compound was tentatively identified by GC–O analysis using DB-WAX columns by comparison of the mass spectrum of the authentic compound in data base.

Changes in FD factors of key aroma compounds in the head space of RS during heating

In order to further investigate the change of head space aroma in RS during heating, FD factors of aroma compounds in NRS and NHS were compared in Table 3. The comparative AEDAs suggest that the contribution of fruity ethyl esters such as ethyl 2-methylbutanoate (No.11), ethyl 3-methylbutanoate (No.12), and ethyl 2-methylpropanoate (No. 7) significantly decreased as a result of the heating process. These ethyl esters have low boiling points and most of them are likely lost by volatilization or hydrolysis during heating. In contrast, the FD factors of burnt or spicy phenolic compounds, such as 4-ethyl-2-methoxyphenol and 2-methoxy-4-vinylphenol, significantly increased during this process. These compounds were reported to be formed from their corresponding hydroxycinnamic acids by decarboxylation reactions.⁸⁾ San-Juan F et al.¹⁷⁾ reported that the phenolic compounds played a negative role in red wine fruitiness, and the fruity aroma of ethyl esters mixtures significantly decreased with a concentration of 50 μg/L of phenolic compounds. In general, the phenolic compounds in RS can be increased to 2–4 mg/L by heating for pasteurization.⁸⁾ Therefore, these results suggest that NRS had a stronger fruitiness than NHS, and the fruity aroma was weakened by heating for two conceivable reasons. The first is the loss of ethyl esters by volatilization or hydrolysis upon heating and the second is the masking effect from the generation of phenolic compounds. Methional was detected with a FD factor 1024 and 2048 in NRS and NHS, respectively. This compound is formed from methionine by heating.¹⁸⁾ In summary, on the basis of the results in Table 3, the head space aroma was significantly different between the RS and HS, and a fruity aroma was verified as a key characterization parameter for RS for the first time.

Quantification of ethyl esters

To determine changes in concentrations of ethyl esters in RS during the heating process, a quantitative analysis of ethyl esters exhibiting high FD factors ≥8 in commercial RS samples was performed by standard addition. Hence, two standard curves for ethyl esters assay are prepared by GC–MS in the SIM mode. At concentrations ranging from 0 to 1 μg/L, the regression equation was linear in the form y = 0.408x, r = 0.999 (y = the concentration of ethyl ester, μg/L; x represents the height of the ethyl ester peak/the height of the internal standard peak). And for ranges from 1 to 60 μg/L, y = 0.024x, r = 0.985. As shown in Table 4, irrespective of the manufacturer, ethyl butanoate, ethyl 2-methylpropanoate, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, and ethyl 4-methylpentanoate were detected in all commercial RS and HS samples. In addition, these compounds were present in all samples at considerably higher concentrations than their perception thresholds, and ethyl 2-methylbutanoate was determined to have the highest odor activity value (OAV) in all of soy sauce samples. OAVs determined herein were in agreement with the results of AEDA for the NRS. Furthermore, the concentrations of 5 ethyl esters in commercial RS samples were higher than those in commercial HS samples. Ethyl esters are usually produced by micro-organism metabolism during the fermentation process, and the concentrations of the 5 ethyl esters differed significantly depending on the manufacturer. Therefore, the generation of ethyl esters is extremely variable depending on fermentation conditions, such as temperature or micro-organism species.

Table 4. Quantitative analysis of ethyl esters in commercial soy sauces.

Compound	OT^b (μg/L)	Raw soy sauce^a			Heat-treated soy sauce^a
		Concentrationc, μg/L			Concentration^c, μg/L
		Max^d	Min^d	Ave^d (OAVe)	Max	Min	Ave(OAV)
Ethyl butanoate	20	9.6 (±0.4 ^f)	0.7 (±0.1)	3.6 (<1))	7.4 (±1.0)	<0.2	2.6 (<1))
Ethyl 2-methylpropanoate	15	50.3 (±1.4)	10.7(±0.2)	27.7 (1.8)	43.6(±0.6)	1.3(±0.1)	15.5 (1)
Ethyl 2-methylbutanoate	1	48.5(±1.2)	4.5 (±0.4)	14.5 (14.5)	31.6(±6.9)	0.4(±0.1)	9.3 (9)
Ethyl 3-methylbutanoate	3	65.6 (±1.8)	5.1 (±0.6)	19.9 (6.6)	43.9(±4.7)	0.7(±0.1)	15.1 (5)
Ethyl 4-methylpentanoate	0.75	16.3 (±0.4)	<0.2	3.3 (4.4)	1.5 (±0.7)	<0.2	0.9 (1)

^a Eight commercial raw and eight commercial heat-treated soy sauce were made by four different brewing companies, and these samples had different lot numbers.

^b OT, odor threshold in dilute water/alcohol solution.

^c Mean value in triplicate experiments.

^d max, min, ave, Maximum, minimum and average concentrations detected in 4 samples, respectively.

^e OAV, odor activity value.

^f SD, standard deviation.

A total of 5 ethyl esters in NRS and NHS, which had the same lot number, were quantified. As shown in Table 5, the concentrations of 5 ethyl esters significantly decreased during the heating process. These results suggest that ethyl esters were significantly degraded, and the fruit-like aroma characteristic lost during the heating process. The fruitiness is one of the key aroma characterizations of RS, and in order to control the quality of RS, further research is necessary to clarify mechanisms of the generation of fruit-like aroma compounds in RS, such as ethyl esters.

Table 5. Quantitative analysis of the 5 ethyl esters in raw soy sauce by heating.

Compound	NRS^a	NHS^a	Ratio
	Concentration^b, μg/L (SD^c, μg/L)	Concentration^b, μg/L (SD^c, μg/L)
Ethyl butanoate	5.6 (±0.5)	0.5 (±0.01)	11.2
Ethyl 2-methylpropanoate	40.7 (±2.8)	11.2 (±1.1)	3.6
Ethyl 2-methylbutanoate	22.9 (±1.2)	6.9 (±0.3)	3.3
Ethyl 3-methylbutanoate	30.5 (±2.4)	10.5 (±0.4)	2.9
Ethyl 4-methylpentanoate	0.8 (±0.1)	<0.2	>4

^a NRS, raw soy sauce sample N; NHS, NHS was prepared by heating NRS at 80 °C for 30 min in a dry stirring bath.

^b Mean value in triplicate experiments.

^c SD, standard deviation.

Author contributions

Qi Meng (QM) and Etsuko Sugawara designed the experiment. QM performed the experiments and wrote the manuscript. All authors contributed to the development of the manuscript. All authors have read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.