Food Science and Technology Research
Online ISSN : 1881-3984
Print ISSN : 1344-6606
ISSN-L : 1344-6606
Original papers
Characterization of Flavor Compounds in Rice-flavor baijiu, a Traditional Chinese Distilled Liquor, Compared with Japanese Distilled Liquors, awamori and kome-shochu
Xuan Yin, Yumiko Yoshizaki , Shugo Kurazono, Mina Sugimachi, Haruka Takeuchi, Xing-Lin Han, Kayu Okutsu, Taiki Futagami, Hisanori Tamaki, Kazunori Takamine
Author information
Keywords: Chinese distilled liquor, flavor, rice-flavor baijiu, shochu, volatile compounds, ethyl lactate
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2020 Volume 26 Issue 3 Pages 411-422

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Abstract

This study aimed to reveal the chemical and flavor profiles of rice-flavor baijiu by comparison with awamori and kome-shochu, traditional Japanese liquors. Rice-flavor baijiu is similar to awamori and kome-shochu with regard to ingredients and the fermentation starter. Of the 15 rice-flavor baijiu samples in this study, 3 had a light yellow to dark-brown color. Dark-brown samples had high glucose and low amino acid contents. Lactic acid was detected in all rice-flavor baijiu samples. Compared to awamori and kome-shochu, rice-flavor baijiu contained more acetic acid. Thirty-four volatile compounds in rice-flavor baijiu were identified and quantified. In all, 18 compounds in rice-flavor baijiu, 15 in awamori and 13 in kome-shochu had an odor activity value (OAV) of >1. Of these, 11 compounds showed a three-fold higher OAV in rice-flavor baijiu than in awamori and kome-shochu. Principal component analysis revealed that ethyl lactate is a key volatile compound that is distinct to rice-flavor baijiu.

Introduction

Baijiu is a traditional Chinese distilled liquor, and rice-flavor baijiu is mainly produced in southern China. Rice-flavor baijiu is made from rice as the sole ingredient and xiaoqu as the fermentation starter, which is comprised of rice powder and rice bran (Zheng and Han, 2016). A few types of microorganisms are present in xiaoqu, including Rhizopus sp., Mucor sp., and yeasts (Zheng and Han, 2016).

The rice-flavor baijiu manufacturing process involves semi-solid-state fermentation (semi-SSF) (Zheng and Han, 2016). SSF is performed using a solid matrix in the absence of free water. Briefly, after steaming the rice, a small amount of xiaoqu is added and mixed well (Fig. 1), and then the solid mixture is incubated for 20–30 h at room temperature. After incubation, water is added and the mixture is fermented in a liquid state for 6–8 days. Finally, the fermented mash is distilled in the liquid state.

Fig. 1.

Schematic diagram of manufacturing of rice-flavor baijiu, awamori and kome-shochu.

The manufacturing process of rice-flavor baijiu is very different from that of other types of Chinese baijiu (Fig. 1). Those types of Chinese baijiu, such as strong-flavor baijiu, sauce-flavor baijiu and light-flavor baijiu, are typically made of several cereals, such as sorghum, wheat and rice, and complex microorganisms from the natural mixed starter daqu. SSF is performed for several months in a mud pit or earthen jars (Zheng and Han, 2016). Many microorganisms, such as caproic acid-producing bacteria, live in mud pits and earthen jars (Xiong et al., 2010), and this complex microbial flora contributes to the flavor of the liquor (Wang et al., 2014a).

On the other hand, the manufacturing process of rice-flavor baijiu is similar to that of awamori and kome-shochu, two traditional Japanese distilled liquors. Awamori uses tane-koji as a fermentation starter, which is prepared from koji mold (Aspergillus luchuensis). Rice koji is a solid culture of koji mold grown on steamed rice for approximately 42 h. Rice koji, water and yeast seed culture are mixed and fermented in a liquid state for 10–12 days. After fermentation, the mash is distilled in the liquid state. Kome-shochu is made from rice and rice koji prepared from A. luchuensis (Fig. 1). It is produced according to the same method as for awamori, except that the main ingredients are added separately after the first fermentation.

Awamori flavor is typically expressed as koji-like, mushroom-like, fruity, and sweet flavors. These descriptions are also contained in the flavor wheel of awamori (Miyamoto, 2018). Kome-shochu has a characteristic fruity flavor (Hayashida, 1998). Isobutyl alcohol, 1-octen-3-ol, nerolidol and S-methyl thioacetate are the key volatile compounds in awamori compared to other Japanese shochu types (Fukuda et al., 2016). In particular, 1-octen-3-ol, which is derived from rice koji, imparts the mushroom-like flavor to awamori (Yoshizaki et al., 2010). Vanillin, the most studied volatile compound in awamori, contributes to the sweet aroma and is produced by biochemical and chemical reactions (Koseki et al., 1996; Koseki and Iwano, 1998). Firstly, ferulic acid present in the rice cell wall is liberated by a ferulic acid esterase produced by A. luchuensis during fermentation. Then, the ferulic acid is transferred to 4-vinylguaiacol (4-VG) by heating during distillation. Finally, 4-VG is converted to vanillin by chemical transformation via oxidation during aging. pH and alcohol concentration during aging influence the chemical transformation of ferulic acid to vanillin.

Rice-flavor baijiu has a characteristically sweet aroma and a clear aftertaste (Zheng and Han, 2016). Ethyl lactate and β-phenylethyl alcohol are key volatile compounds characteristic of rice-flavor baijiu compared to other types of baijiu (Jin et al., 2017). However, to date, no studies have investigated the flavors specific to rice-flavor baijiu in comparison with awamori and kome-shochu. Okinawa Prefecture in Japan, the main production center of awamori, and Guangdong and Guangxi provinces in China, the main production centers of rice-flavor baijiu, are geographically close. Therefore, rice-flavor baijiu is important for understanding the historical and technical relationships between Chinese and Japanese distilled liquors. Knowledge of the chemical and flavor profiles of rice-flavor baijiu will assist in understanding the similarities and/or differences between Chinese and Japanese distilled liquors.

This study aimed to determine the characteristic flavor compounds of commercially available rice-flavor baijiu in comparison with awamori and kome-shochu. First, the concentrations of alcohol, sugar, amino acids, and organic acids in rice-flavor baijiu were measured by high-performance liquid chromatography (HPLC). Next, the concentrations of volatile compounds in rice-flavor baijiu were measured by gas-chromatography mass spectrometry (GC-MS) analysis in comparison with awamori and kome-shochu. Finally, the characteristic flavor compounds in rice-flavor baijiu were determined by principal component analysis of the quantitative results of volatile compounds of rice-flavor baijiu, awamori, and kome-shochu.

Materials and Methods

Liquor samples and chemicals    Fifteen different commercial rice-flavor baijiu were purchased from a local market in China (Table 1) on the basis of the company, place of production and sales price range. In addition, six and five different commercial awamori and kome-shochu were purchased, respectively, from a local market in Japan. Sodium hydroxide, glucose, fructose, sucrose, maltose, acetonitrile, and ethanol were acquired from Nacalai Tesque, Inc. (Kyoto, Japan). Bromothymol blue, neutral red, phenolphthalein, lactic acid, acetic acid, p-toluenesulfonic acid monohydrate, and bis-Tris were acquired from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). Ethylenediaminetetraacetic acid (EDTA) was acquired from Dojindo Laboratories (Kumamoto, Japan). Authentic standards for GC-MS were purchased from several companies as follows. Isobutyl alcohol, isoamyl alcohol, 1-hexanol, decanoic acid, dodecanoic acid, ethyl isovalerate, ethyl stearate, isoamyl caproate, isoamyl caprylate, isobutyl caprylate, phenylethyl acetate, phenylethyl butyrate, and 2-pentyl furan were purchased from FUJIFILM Wako Pure Chemical Corp. 1-Butanol, ethyl lactate, ethyl phenylacetate, 2-nonenal, and dimethyl trisulfide were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). β-Phenylethyl alcohol, ethyl isobutyrate, ethyl caprylate, ethyl caprate, ethyl myristate, ethyl palmitate, ethyl oleate, ethyl linoleate, isoamyl acetate, phenylethyl octanoate, and furfural were purchased from Sigma-Aldrich (Steinheim, Germany). Octanoic acid, ethyl caproate, ethyl laurate, ethyl salicylate, and ethyl benzoate were purchased from Nacalai Tesque, Inc.

Table 1. Sample informations used in this study.
Sample Name Types of liquor Region, Country Company Aging periods (years) Distillation type Alcohol† (%, v/v)
S1 Rice-flavor baijiu Guangxi, China A 5 Atmospheric* 35
S2 Rice-flavor baijiu Guangxi, China A Non aging* Atmospheric* 30
S3 Rice-flavor baijiu Guangxi, China B 18 - 49
S4 Rice-flavor baijiu Guangxi, China C 18 - 53
S5 Rice-flavor baijiu Guangxi, China D 5 - 49
S6 Rice-flavor baijiu Guangxi, China D - - 46
S7 Rice-flavor baijiu Guangxi, China D - - 48
S8 Rice-flavor baijiu Guandong, China E 5 Atmospheric* 48
S9 Rice-flavor baijiu Guandong, China E Non aging* Atmospheric* 52
S10 Rice-flavor baijiu Guandong, China F Non aging* - 49
S11 Rice-flavor baijiu Guandong, China F 10 - 54
S12 Rice-flavor baijiu Guandong, China F 5 - 48
S13 Rice-flavor baijiu Guandong, China G - - 47
S14 Rice-flavor baijiu Liaoning, China H - - 50
S15 Rice-flavor baijiu Liaoning, China H - - 54
A1 Awamori Okinawa, Japan <3* Atmospheric 30
A2 Awamori Okinawa, Japan <3* Mixture of vacuum and atmospheric 30
A3 Awamori Okinawa, Japan <3* Vacuum 20
A4 Awamori Okinawa, Japan <3* Atmospheric 30
A5 Awamori Okinawa, Japan <3* Mixture of vacuum and atmospheric 30
A6 Awamori Okinawa, Japan <3* Atmospheric 30
K1 Kome-shochu Kumamoto, Japan 3∼5 months* Vacuum 25
K2 Kome-shochu Kumamoto, Japan <3* Mixture of vacuum and atmospheric 25
K3 Kome-shochu Kumamoto, Japan 3~5* Atmospheric 25
K4 Kome-shochu Kumamoto, Japan <3* Vacuum 25
K5 Kome-shochu Kumamoto, Japan 3* Vacuum 25
†  Alcohol content of each sample was measured in our labolatory.

Awamori and Kome-shochu samples are made by all different companies.

Aging periods were described what was entered in to the package. “-” means we don't have any information about aging time.

*  These informations were confirmed with each company directly.

Total and volatile acidity of liquor samples    Total and volatile acidity values were determined following the official methods of the National Tax Administration Agency, Japan (Brewing Society of Japan, 2006), and it is stated that these methods are applicable to the fermented mash of shochu. Moreover, we applied these methods to the rice-flavor baijiu in this study. Total acidity was measured as titratable acidity. Briefly, 10 mL of each liquor sample was titrated against 0.1 N sodium hydroxide, with the indicator containing bromothymol blue and neutral red, until it turned light green. Volatile acidity was determined by titration of each liquor sample after distillation. Briefly, 100 mL of each liquor sample was distilled using a small distillation apparatus. Approximately 70 mL of the distillate was collected, and the volume was adjusted to 100 mL with deionized water. Further, 10 mL of this solution was titrated with phenolphthalein against 0.01 N sodium hydroxide until it turned light pink. Total acidity and volatile acidity were represented in the titration volume of 0.1 and 0.01 N sodium hydroxide, respectively.

Quantification of saccharides    Saccharides were quantified by injecting 10 µL of each liquor sample into a Prominence HPLC system (Shimadzu Corp., Kyoto, Japan) under the following conditions: LC-20AD pump (Shimadzu Corp.); 4 × 250 mm i.d. Cosmosil Sugar-D column (Nacalai Tesque, Inc., Kyoto, Japan); mobile phase, acetonitrile:water (3:1); flow rate, 1.0 mL/min; oven temperature 40 °C; detector, RID-10A refractive index detector (Shimadzu Corp.) (Okutsu et al., 2012). Standard curves were constructed using linear regression of the analyte peak areas versus the known concentrations of each saccharide.

Analysis of organic acids    Organic acids were quantified by injecting 10 µL of each liquor sample into a Prominence HPLC system (Shimadzu Corp.) under the following conditions: LC-20AD pump (Shimadzu Corp.); 8 × 300 mm i.d. Shim-pack SCR-102H HPLC column (Shimadzu Corp.); mobile phase, 4 mM p-toluenesulfonic acid monohydrate; flow rate, 0.8 mL/min; oven temperature 50 °C; detector, CDD-10A VP conductivity detector (Shimadzu Corp.) (Rahayu et al., 2017). A mixture of 4 mM p-toluenesulfonic acid monohydrate, 16 mM bis-Tris and 80 µM EDTA served as a post–column reaction solution at a flow rate of 0.8 mL/min. Standard curves were constructed using linear regression of the analyte peak areas versus known concentrations of each organic acid.

Analysis of amino acids    Concentrations of amino acids were determined using the Prominence HPLC system (Shimadzu Corp.) and an RF-10AXL fluorescence detector (Shimadzu Corp.) using the post-column fluorescence derivatization method (Rahayu et al., 2017). Amino acid separation was achieved using an 6 × 100 mm i.d. Shimadzu Shim-pack Amino-Na column (Shimadzu Corp.) at 60 °C with a flow rate of 0.6 mL/min using the amino acid mobile-phase Na-type kit (Shimadzu Corp.). The RF-10AXL fluorescence detector was set to an excitation/emission wavelength pair of 350/450 nm, and the reaction reagents were obtained from an amino acid reaction kit (Shimadzu Corp.) and maintained at a flow rate of 0.2 mL/min.

GC-MS with stir bar sorptive extraction    The liquor samples were diluted to 25% (v/v) alcohol by adding deionized water, then 10 mL of each liquor sample was transferred to a sample vial and a 15 mm stir bar coated with 0.5 mm polydimethylsiloxane was added (Twister, Gerstel K.K., Japan) (Rahayu et al., 2017). The sample was stirred on a magnetic stirrer at 1 200 rpm for 1 h at room temperature. The stir bar was removed, washed with deionized water, dried with a tissue and placed into a glass insert. Volatile compounds were desorbed from the stir bar using the following temperature program of the Gerstel TDS3 and Gerstel CIS4 thermal desorption system (Gerstel K.K.): 20 °C for 1 min and 60 °C per min to 260 °C (hold for 1 min). Meanwhile, a Gerstel CIS4 cryotrap (Gerstel K.K.) was set to −150 °C to cryofocus. After the desorptive program was completed, the cryotrap was heated to inject the volatile compounds into a gas chromatography (GC) analytical column: −150 °C for 1 min and 12 °C per second to 270 °C (hold for 2 min). The gas chromatography–mass spectrometry (GC-MS) system was equipped with a 0.25 mm × 60 m i.d. Inner Pure-WAX column with a 0.25 µm film thickness (GL Sciences Inc., Tokyo, Japan). Analyses were carried out with helium as the carrier gas at a flow rate of 1.0 mL/min using the following temperature program: 40 °C for 5 min and 3 °C per min to 240 µC. The retention index (RI) was determined via sample injection with a series of straight-chain alkanes (C5–C24) (SUPELCO Analytical, Bellefonte, PA, USA). Identification of volatile compounds was confirmed by comparing their mass spectra with the NIST05a mass spectral database and the RI values in the AromaOffice database (Nishikawa Keisoku Co., Ltd., Tokyo, Japan). Standard curves were produced from a pure authentic reagent in 25% (w/w) ethanol solution. The absolute calibration curve was derived from peak areas using selected ions (m/z) (Tables 1 and 2). The sample was diluted accordingly to determine the concentrations. The analysis was repeated three times for each sample.

Table 2. Standard curves of volatile compounds used in this study.
Validation range (µg/L) Standard curve r2
Isobutyl alcohol 980–29,000 y = 8.49×10−11x2 + 1.66×10−3x + 2.67×102 1
29,000–600,000 y = 8.11×10−12x2 + 1.06×10−3x + 1.41×104 0.9996
Isoamyl alcohol 1,690–49,800 y = 8.74×10−4x − 1.58×103 0.9997
49,800–746,000 y = 4.11×10−13x2 + 7.90×10−4x − 3.55×103
1-Butanol 47–470 y = 2.38×10−3 x − 2.30×102 0.9992
470–4,700 y = 3.06×10−3 x − 5.23×102 0.9976
4,700–24,000 y = 2.06×10−3 x + 1.02×104 0.9992
β-Phenylethyl Alcohol 5,000–50,000 y = 5.02×10−4 x + 8,15×102 0.9996
50,000–500,000 y = 8,18×10−4 x − 3.23×105 0.9996
1-Hexanol 7–180 y = 1.20×10−4x − 1.19 0.9999
180–3,800 y = 1.13×10−4x − 3.17 0.9997
Octanoic acid 12–60 y = 1.42×10−4 x − 1.27×10 0.9996
60–600 y = 1.20×10−4 x − 1.50 1
600–6,000 y = 1.63×10−4 x − 1.21×103 0.9936
Decanoic acid 0.8–9 y = 8.95×10−5 x − 5.38 0.9851
9–200 y = 5.72×10−5 x − 7.40×10 1
200–600 y = 3.36×10−5 x + 8.75×10 1
600–14,000 y = 2.18×10−5 x + 2.69×102 1
Dodecanoic acid 0.8–10 y = 2.26×10−5 x − 1.22 0.9928
10–30 y = 2.06×10−5 x − 1.28 0.9873
30–600 y = 1.13×10−5 x + 2.28×10 0.9983
600–7,300 y = 8.00×10−6 x + 3.43×102 0.9746
Ethyl isobutyrate 6–145 y = 7.99×10−5 x − 4.64 1
145–1,500 y = 1.15×10−4 x − 5.95×10 0.9992
1,500–6,650 y = 4.66×10−12x2 − 6.90×10−5x + 1.59×103 1
Ethyl isovalerate 1–20 y = 5.17×10−6 x + 4.68×10−1 0.9951
20–165 y = 5.05×10−6 x − 7.43 0.9877
Ethyl □actate 1,000–10,000 y = 1.56×10−3 x + 2.51×102 0.9998
10,000–100,000 y = 1.63×10−3 x + 1.66×103 0.9849
50,000–290,000 y = 2.10×10−3 x − 5.60×103 0.9997
Ethyl caproate 10–100 y = 2.23×10−6 x − 2.68 0.9978
100–1,000 y = 3.23×10−6 x − 4.64×10 0.9996
1,000–10,000 y = 1.28×10−15x2 + 2.91×10−6x − 1.19×102 0.9996
10,000–100,000 y = 5.02×10−15x2 − 8.36×10−6x + 8.73×103 0.9992
Ethyl caprylate 1–15 y = 4.69×10−7 x − 3.27×10−1 0.9999
15–300 y = 8.10×10−7 x − 1.23×10 1
300–4,800 y = 1.96×10−6 x − 5.18×102 0.9995
Ethyl caprate 1–15 y = 7.69×10−15x2 − 4.84×10−8x − 1.07 1
15–300 y = 5.35×10−16x2 + 2.66×10−7x − 1.12 0.9962
300–1,800 y = 1.49×10−6 x − 4.97×102 0.999
Ethyl laurate 1–35 y = 3.74×10−7 x − 5.65×10−1 0.9965
35–600 y = 1.56×10−14 x1.92 0.9985
600–9,500 y = 2.71×10−6 x − 5.26×102 0.999
Ethyl myristate 1–20 y = 9.15×10−7 x − 9.65×10−1 0.9999
20–130 y = 1.02×10−6 x − 3.37 1
130–2,000 y = 4.52×10−6 x − 3.52×102 0.9823
2,000–16,500 y = 1.91×10−4 x − 9.97×104 0.9985
Ethyl palmitate 0.2–2 y = 1.02×10−6 x − 1.28 0.9831
2–25 y = 2.35×10−6 x − 6.24 0.9847
25–400 y = 4.08×10−6 x − 2.53×10 0.9999
400–2,500 y = 2.40×10−5 x − 2.18×103 0.992
Ethyl stearate 0. 4–8 y = 6.46×10−6 x − 1.58 0.9433
8–50 y = 5.18×10−6 x + 3.34×10−1 1
50–320 y = 6.46×10−6 x − 1.58 0.9433
Ethyl oleate 0.3–5.5 y = 6.40×10−12x2 + 2.17×10−6x − 6.47×10−3 0.9949
5.5–120 y = 1.37×10−5 x − 3.83 0.9999
Ethyl linoleate 1–20 y = 2.65×10−6 x + 2.71×10−1 0.9993
20–700 y = 6.33×10−5 x − 2.53×102 0.9999
350–1,700 y = 5.60×10−5 x − 3.45×102 0.999
700–18,000 y = 1.31×10−4 x − 2.33×103 0.9942
Ethyl salicylate 0.05–0.3 y = 2.46×10−6 x − 7.76×10−3 0.9947
0.3–3.5 y = 1.39×10−6 x + 1.64×10−1 0.9992
Ethyl benzoate 1–20 y = 1.63×10−6 x + 3.88×10−1 0.9996
20–130 y = 1.90×10−6 x − 1.46 0.9632
Ethyl phenylacetate 0.1–0.5 y = 9.57×10−7 x + 2.75×10−2 0.9999
0.5–10 y = 3.57×10−7 x + 1.84×10−1 0.9989
Isoamyl acetate 1–20 y = 3.68×10−6 x − 1.56 0.9639
20–350 y = 3.55×10−6 x − 2.26×10 0.9769
350–8,000 y = 8.05×10−6 x − 3.99×102 0.989
Isoamyl caproate 0.1–0.5 y = 6.61×10−8 x − 2.06×10−3 0.9996
0.5–5 y = 4.15×10−8 x − 1.16×10−1 0.9829
Isoamyl caprylate 1–20 y = 2.92×10−7 x − 3.15×10−1 0.9999
20–400 y = 7.95×10−16x2 + 6.21×10−8x + 1.17 1
400–1,450 y = 1.22×10−6 x − 4.03×102 1
Isobutyl caprylate 0.1–0.5 y = 9.33×10−8 x + 7.22×10−2 0.9806
0.5–5 y = 4.20×10−8 x + 6.97×10−1 0.9646
Phenethyl acetate 8–15 y = 1.52×10−6 x + 7.90 0.9689
15–400 y = 5.19×10−6 x − 9.44 1
400–8,400 y = 4.75×10−15x2 + 2.77×10−6x + 1.50×102 1
Phenylethyl butyrate 1–20 y = 2.20×10−14x2 − 2.24×10−8x + 1.02 1
Phenylethyl octanoate 0.05–0.3 y = 3.56×10−7 x − 5.17×10−2 0.9999
0.3–3 y = 3.27×10−7 x − 2.42×10−2 0. 9871
3–30 y = 4.07×10−7 x − 1.10 0.9979
2-Pentyl furan 1–5 y = 9.65×10−7 x + 2.94×10−1 0.9995
5–100 y = 2.08×10−7 x + 2.78 0. 9986
Furfural 6–60 y = 5.39×10−5 x + 1. 23 0.9818
60–1,800 y = 2.18×10−4 x − 2.17×102 0.9983
1,800–36,500 y = 4.61×10−4 x − 2.89×103 0.999
2-Nonenal 5–25 y = 1.13×10−5 x + 1.36 0.9916
25–250 y = 1.04×10−5 x + 3.34 1
Dimethyl trisulfide 0.2–2 y = 4.76×10−6 x + 1.04×10−1 0.9922
1–11 y = −7.04×10−13x2 + 5.57×10−6x − 7.9×10−2 1

Data analysis    To identify which volatile compounds are likely to contribute the most to the different characteristics of the liquor samples used in this study, principal component analysis (PCA) was performed using Ekuseru-Toukei 2008 statistical software (Social Survey Research Information Co., Ltd., Tokyo, Japan).

Results and Discussion

Chemical compositions and properties    Descriptions of all 15 rice-flavor baijiu samples selected for this study, including details regarding their manufacturers, price range and aging periods, are shown in Table 1. Guangdong and Guangxi provinces are famous brewing districts and the main production centers of rice-flavor baijiu. Therefore, 13 samples were selected from these regions, while the remaining two samples selected were made by a company in Liaoning province, in northern China. Okinawa and Kumamoto Prefectures are famous brewing districts in Japan and the main production centers of awamori and kome-shochu, respectively.

Of the 15 rice-flavor baijiu samples, two samples (S14 and S15) were characterized by a strong dark-brown color, and one sample (S5) had a yellow color, indicating that some rice-flavor baijiu samples are stored in barrels. Sugar was detected by HPLC in five rice-flavor baijiu samples (S3, S4, S5, S14 and S15) (Table 3). Glucose constituted at least 95% of all sugars (data not shown). S14 and S15 contained a high glucose level of >60 mg/mL, and these samples were produced by the same company. Therefore, it is likely that a flavoring agent such as caramel might be added to these liquor samples.

Table 3. Analysis of spirits.
Rice-flavor baijiu Awamori Kome-shochu
mean max min mean max min mean max min
Total acidity 13.8 37 4.4 0.71 1.45 0.10 0.33 0.91 0.09
Volatile acidity 1.70 2.8 0.45 - - - - - -
Glucose (mM) 62 386 0 - - - - - -
Amino acid (µM) 296 1978 0.9 - - - - - -
Lactic acid (mM) 7.4 23.8 1.7 nd nd nd nd nd nd
Acetic acid (mM) 5.0 10.5 2.6 0.85 1.70 nd 0.55 1.49 nd

nd, not detected. “-” means not to measure.

Amino acids were detected in eight samples; five contained a low level of amino acids, while the remaining three samples (S5, S14 and S15) had a high level of amino acids. These amino acids may also be derived from a flavoring agent, in the same manner as for glucose.

Rice-flavor baijiu showed higher total acidity compared to awamori and kome-shochu, with a wide total acidity range among the 15 samples. There was an 8.4-fold difference between the samples with the highest (S15) and lowest (S8) total acidity. On the other hand, volatile acidity was markedly lower than total acidity, with rice-flavor baijiu having slightly higher volatile acidity compared to awamori and kome-shochu. However, there was a large difference between total acidity and volatile acidity in rice-flavor baijiu. Therefore, acid compositions were analyzed by HPLC (Table 3). All 15 rice-flavor baijiu samples mainly contained lactic and acetic acids. Although other acids were detected in S5, S14 and S15, the levels were very low (data not shown). There was a 14-fold difference between the highest (S15) and lowest (S8) levels of lactic acid, while there was a 4-fold difference between the highest (S14) and lowest (S9) levels of acetic acid. Lactic acid level was correlated with total acidity level in all 15 rice-flavor baijiu samples, indicating that a high lactic acid level results in high total acidity in rice-flavor baijiu. Lactic acid is a non-volatile acid, and it is expected that it is difficult to detect in distilled liquors. Thus, these results imply that the addition of lactic acid is a generally employed technique in the production of rice-flavor baijiu, while the addition of sugar is restricted. The concentrations of lactic acid and glucose detected in rice-flavor baijiu were higher than that of amino acids. The amino acids detected in five of the samples might originate from the flavoring reagents and lactic acid as an impurity.

In China, there is a national standard for rice-flavor baijiu, which has been developed by the Standardisation Administration of the People's Republic of China (SAC) (GB/T 10781.3-2006) (Yu, 2016). According to this standard, the quality level is prescribed according to sensory evaluation, total acidity, total ester content, ethyl lactate content, β-phenylethyl alcohol content and solid content. A >0.3 g/L (= 5 mM acetic acid) total acid content equivalent to acetic acid indicates a high quality level. Therefore, a high level of acetic acid is one of the characteristics of rice-flavor baijiu.

Quantitation by GC-MS    GC-MS with stir bar sorptive extraction was used to identify and quantify 34 compounds from the mass spectra and RIs as follows: 5 alcohols, 3 acids, 22 esters, 2 furans, 1 aldehyde, and 1 sulfuric compound (Table 4). The rice-flavor baijiu contained a wide concentration range of 18 volatile compounds with an odor activity value (OAV) of >1 (Table 5). Awamori and kome-shochu contained 15 and 13 compounds with an OAV of >1, respectively. Of these, 11 showed three-fold higher OAVs in rice-flavor baijiu compared to awamori and kome-shochu: ethyl isobutyrate, ethyl isovalerate, ethyl lactate, ethyl caproate, ethyl laurate, ethyl myristate, ethyl palmitate, ethyl linoleate, 2-pentyl furan, 2-nonenal, and dimethyl trisulfide.

Table 4. Volatile compounds in rice-flavor baijiu, awamori, and kome-shochu detected by GC-MS.
Compounds Odor description RI Identification method CAS No Quantification ion
Alcohol Isobutyl alcohol Fusel, alcohol 1092 MS, RI, STD 78-83-1 43
Isoamyl alcohol Alcohol, harsh, bitter 1209 MS, RI, STD 123-51-3 55
1-Butanol Medicinal, alcohol 1143 MS, RI, STD 71-36-3 56
β-Phenylethyl alcohol Floral, rose 1872 MS, RI, STD 60-12-8 91
1-Hexanol Vegetal, herbaceous 1339 MS, RI, STD 111-27-3 56
Acid Octanoic acid Sweet, chees 2026 MS, RI, STD 124-07-2 60
Decanoic acid Fatty, unpleasant 2233 MS, RI, STD 334-48-5 73
Dodecanoic acid Dry, metallic, laurel oil 2450* MS, RI, STD 143-07-7 73
Ester Ethyl isobutyrate Fruity, strawberry 945 MS, RI, STD 97-62-1 43
Ethyl isovalerate Fruity 1060 MS, RI, STD 108-64-5 88
Ethyl lactate Fruity, lactic, raspberry 1354 MS, RI, STD 97-64-3 45
Ethyl caproate Fruity, floral 1221 MS, RI, STD 123-66-0 88
Ethyl caprylate Pineapple, pear, floral 1419 MS, RI, STD 106-32-1 88
Ethyl caprate Fruity, fatty, solvent 1620 MS, RI, STD 110-38-3 88
Ethyl laurate Sweet, floral, fruity, cream 1830 MS, RI, STD 106-33-2 88
Ethyl myristate - 2030 MS, RI, STD 124-06-1 88
Ethyl palmitate Fatty, rancid, fruity, sweet 2235 MS, RI, STD 628-97-7 88
Ethyl stearate - 2442* MS, RI, STD 111-61-5 88
Ethyl oleate - 2460* MS, RI, STD 111-62-6 55
Ethyl linoleate - 2505* MS, RI, STD 544-35-4 67
Ethyl salicylate - 1774 MS, RI, STD 118-61-6 120
Ethyl benzoate Fruity 1635 MS, RI, STD 93-89-0 105
Ethyl phenylacetate Rose, honey 1753 MS, RI, STD 101-97-3 91
Isoamyl acetate Banana 1116 MS, RI, STD 123-92-2 43
Isoamyl caproate Pineapple, cheese 1451 MS, RI, STD 2198-61-0 70
Isoamyl caprylate Sweet, light fruity, cheese, cream 1645 MS, RI, STD 2035-99-6 70
Isobutyl caprylate - 1541 MS, RI, STD 5461-06-3 127
Phenylethyl acetate Floral, rose 1783 MS, RI, STD 103-45-7 104
Phenylethyl butyrate Fruity 1793 MS, RI, STD 103-52-6 104
Phenylethyl octanoate - 2345 MS, RI, STD 5457-70-5 104
Furan 2-Pentyl furan Green bean-like 1222 MS, RI, STD 3777-69-3 81
Aldehyde Furfural Bread, sweet 1431 MS, RI, STD 98-01-1 95
2-Nonenal Green 1512 MS, RI, STD 2463-53-8 70
Sulfric compound Dimethyl trisulfide Cooked onion 1355 MS, RI, STD 3658-80-8 126
*  Because RI was slightly out of the range, these RIs were estimated by adapting the conversion formula from retention time to RI prepared in this study.

Table 5. The concentration (µg/L) and odor-active values of volatile compounds in rice-flavor baijiu, awamori, and kome-shochu.
Rice-flavor baijiu Awamori Kome-shochu Odor threshold (µg/L) OAV
Mean Max Min Mean Max Min Mean Max Min Rice-flavor baijiu Awamori Kome-shochu Ref
Alcohol Isobutyl alcohol 190,194 564,045 27,663 126,280 210,516 79,231 183,592 422,564 66,233 40,000 4.8 3.2 4.6 1
Isoamyl alcohol 323,628 634,175 38,761 337,228 393,797 251,755 257,265 351,014 142,027 30,000 11 11 8.6 1
1-Butanol 3,873 9,330 nd 5,160 8,931 2,152 9,645 23,666 3,988 5,000 1> 1.0 1.9 2
β-Phenylethyl Alcohol 80,707 456,898 18,760 72,604 108,734 40,455 31,901 39,336 24,649 10,000 8.1 7.3 3.2 1
1-Hexanol 315 1,081 nd 30 53 nd 97 185 nd 8,000 1> 1> 1> 1
Acid Octanoic acid 1,168 3,321 43 1,188 2,820 454 381 1,643 19 15,000 1> 1> 1> 2
Decanoic acid 1,339 2,867 54 777 1,529 342 295 1,183 nd 15,000 1> 1> 1> 1
Dodecanoic acid 436 970 12 168 352 54 13 21 4.7 7,200 1> 1> 1> 2
Ester Ethyl isobutyrate 1,366 3,946 191 242 613 101 72 207 tr 15 91 16 4.8 1
Ethyl isovalerate 48 103 15 19 35 10 5.0 13 nd 3 16 6.4 1.7 1
Ethyl lactate 379,079 641,297 93,568 4,475 7,426 nd nd nd nd 14,000 27 1> 1> 2
Ethyl caproate 24,536 202,446 21 550 917 183 7,343 35,081 240 5 4,910 110 1,470 1
Ethyl caprylate 2,221 5,857 3.0 1,270 2,133 60 6,635 31,048 297 2 1,110 635 3,320 1
Ethyl caprate 2,891 8,431 2.2 320 804 7.1 1,144 5,356 54 200 14 1.6 5.7 3
Ethyl laurate 538 1,596 tr 11 18 Tr 16 59 1.3 500 1.1 1> 1> 4
Ethyl myristate 1,159 2,765 tr 0.6 1.2 nd 7.4 16 tr 500 2.3 1> 1> 4
Ethyl palmitate 15,623 171,874 tr tr tr nd 5.0 14 tr 14,000 1.1 1> 1> 2
Ethyl stearate 24 289 nd nd nd nd 1.2 6.0 nd 500 1> 1> 1> 2
Ethyl oleate 359 2,011 nd 14 84 nd 1.5 6.2 nd 870 1> 1> 1> 2
Ethyl linoleate 4,630 19,391 nd 0.8 1.1 nd 1.0 1.7 0.9 450 10 1> 1> 2
Ethyl salicylate 0.8 3.1 nd 0.04 0.22 nd 0.8 3.1 nd - - - - -
Ethyl benzoate 15 37 nd 1.6 1.6 tr 1.6 3.0 nd 575 1> 1> 1> 3
Ethyl phenylacetate 5.7 10 2.1 3.4 4.7 1.9 1.6 3.8 nd 100 1> 1> 1> 5
Isoamyl acetate 997 3,273 nd 2,955 5,151 1,774 3,672 7,646 78 30 33 99 122 1
Isoamyl caproate 0.6 1.5 nd 0.36 0.43 tr 2.9 13 nd 1,400 1> 1> 1> 2
Isoamyl caprylate 10 25 nd 2.8 3.6 tr 2.7 10 0.7 125 1> 1> 1> 3
Isobutyl caprylate 1.6 3.6 nd 0.4 0.5 tr 0.6 2.1 nd 800 1> 1> 1> 6
Phenethyl acetate 322 846 15 1,364 1,422 655 982 2,035 573 250 1.3 5.5 3.9 1
Phenylethyl butyrate 2.3 2.9 tr 1.3 1.5 1.2 1.0 1.0 tr 961 1> 1> 1> 7
Phenylethyl octanoate 1.7 7.0 nd 1.0 1.4 nd 1.0 4.0 nd - - - - -
Furan 2-Pentyl furan 8.0 26 nd 0.2 1.4 tr 1.5 2.3 nd 1 8.0 0.2 1.5 8
Furfural 325 2,437 nd 705 1,747 nd nd nd nd 14,100 1> 1> 1> 3
Aldehyde 2-Nonenal 20 94 nd 3.3 8.8 nd 3.1 15 nd 0 255 41 39 8
Sulfric compound Dimethyl trisulfide 4.3 10 nd 1.5 2.2 nd nd nd nd 0 21 7.6 1> 1

Method of indentification: MS, mass spectrum comparison using NIST05a library; RI: retention index in agreement with literature value; STD, confirmed by authentic standards. nd, not detected. tr, trace.

Odor threshold abtained from references: [1] Guth, 1997; odor threshold in water/ethanol (90+10, w,w), [2] Salo et al., 1972; odor threshold in synthetic wine (11% (w/w) ethanol), [3] Ferreira et al. 2000; odor threshold in synthetic wine (11% (w/w) ethanol), [4] Zea et al., 2001, [5] Isogai et al., 2005; odor threshold in sake (15% (w/w) alcohol, [6] Li et al., 2008; odor threshol in 12% (w/w) ethanol containing 5 g/L tartaric acid at pH 3.2, [7] Wang et al., 2014b; odor threshold in synthetic Chinese liquor (46% (w/w) ethanol), [8] Buttery et al., 1988; odor threshold in water.

The levels of ethyl isobutyrate, ethyl isovalerate and ethyl lactate depend on the concentrations of the organic acids isobutyric acid, isovaleric acid and lactic acid, respectively, in the fermented mash (Shen, 2003; Rahayu et al., 2017). Ethyl lactate is a common and important compound in Chinese liquor. Esterification of ethanol and lactic acid in Chinese liquor are carried out during fermentation (Cheng et al., 2018). Lactic acid in rice-flavor baijiu mash is mainly produced by molds and lactic acid bacteria in xiaoqu (Yin et al., 2020). In contrast, citric acid is the main organic acid in awamori and kome-shochu mashes; the koji molds (A. luchuensis and A. luchuensis mut. kawachii) used in the making of shochu mainly produce and secrete citric acid (Kadooka et al., 2019). Therefore, the lactic acid content of awamori and kome-shochu mashes is not high. These findings suggest that ethyl lactate is a characteristic compound in rice-flavor baijiu compared to awamori and kome-shochu.

Fatty acid ethyl esters such as ethyl caproate, ethyl laurate, ethyl myristate, ethyl palmitate and ethyl linoleate were abundantly found in rice-flavor baijiu. The genus Rhizopus is recognized as a good lipase producer, and its lipase is used in many biotech applications (Yu et al., 2016). On the other hand, Aspergillus oryzae, a popular koji mold, does not produce a large amount of lipase in solid culture (Ohnishi et al., 1994). Therefore, it is suggested that long-chain fatty acid levels in rice-flavor baijiu mash are higher compared to awamori and kome-shochu, and that these ethyl esters are also produced by yeast. Moreover, all of the rice-flavor baijiu samples in this study had a higher alcohol level compared to the awamori and kome-shochu samples (Table 2). Therefore, long-chain fatty acid ethyl esters are readily soluble and are found in large quantities in Chinese liquors.

Ethyl caproate is an important flavor compound in Chinese liquors (Shen, 2003) and is generally produced by yeast. Although yeast is commonly employed as a microorganism for rice-flavor baijiu, awamori and kome-shochu, the yeast strain has a greater influence on the formation of ethyl caproate than fermentation conditions such as aeration and fermentation temperature (Piendl and Geiger, 1980). Yeasts synthesize ethyl caproate via two pathways: from caproic acid and ethanol by esterase, and from caproyl-CoA and ethanol by alcohol acyltransferase (Liu et al., 2004). The synthesis of ethyl caproate in brewing is limited by the abundance of caproic acid in the fermentation mash. Therefore, differences in the yeast strain and fermentation conditions among rice-flavor baijiu, awamori and kome-shochu might affect the level of ethyl caproate in each liquor. Furthermore, caproic acid-producing bacteria, such as Clostridium kluyveri, are well known to contribute to the production of ethyl caproate in Chinese liquors (Hu et al., 2015). Therefore, caproic acid-producing bacteria might be the reason for the large amount of ethyl caproate in rice-flavor baijiu. However, no research on these microbes in rice-flavor baijiu manufacturing has been conducted to date. Future studies are required to reveal the relationship between minor microbes and the flavor of rice-flavor baijiu.

β-Phenylethyl alcohol has been reported as a characteristic compound in rice-flavor baijiu (Jin et al., 2017). It has a rose-like odor and is found in important aroma compounds in various alcoholic beverages (Lilly et al., 2006). In this study, levels of β-phenylethyl alcohol in rice-flavor baijiu and awamori were almost identical; however, the levels differed >2.5-fold compared to kome-shochu. β-Phenylethyl alcohol is produced from yeast by two pathways during fermentation: by degradation of phenylalanine to alcohol in the Ehrlich pathway, and from phenylpyruvate during phenylalanine synthesis from carbohydrates (Äyräpää, 1965). The level of β-phenylethyl alcohol is controlled by the amino acid level of the liquor mash via either pathway.

The manufacture of kome-shochu differs from that of rice-flavor baijiu and awamori (Fig. 1). Rice-flavor baijiu and awamori are produced from saccharified rice or rice koji, while kome-shochu is produced from steamed rice that is added to the liquor mash after five days of fermentation. The saccharified rice and rice koji contain various proteases from the mold in the fermentation starter (Long et al., 2013; Machida, 2002). It is thought that the rice-flavor baijiu and awamori mashes have greater enzyme activity compared to kome-shochu. Therefore, the amino acid level during fermentation may differ, impacting the alcohol level of the liquors.

The volatile compounds with a three-fold higher OAV in awamori or kome-shochu compared to rice-flavor baijiu were 1-butanol, ethyl caprylate, isoamyl acetate and phenylethyl acetate. Isoamyl acetate and phenylethyl acetate are produced in yeast cells by alcohol acetyl-transferases (AATases; EC 2.3.1.84) with alcohols and acetyl-coenzyme A (acetyl-CoA) as substrates. AATase activity and the expression level of the AATase gene ATF1 are inhibited by unsaturated fatty acids (Fujii et al., 1997). The high fatty acid content of rice-flavor baijiu mash is attributed to lipase production by Rhizopus sp., as previously described. Therefore, the AATase of yeast might be more strongly inhibited in rice-flavor baijiu mash compared to awamori and kome-shochu.

PCA of volatile compounds    PCA results revealed diversity among the 15 rice-flavor baijiu samples (Fig. 2), while all six awamori samples and most of the five kome-shochu samples were grouped into the same cluster. The first and second principal components (PC1 and PC2) correlated positively with the diversity of rice-flavor baijiu and the differences between Chinese and Japanese distilled liquors, respectively. In particular, ethyl lactate and isoamyl alcohol were important compounds that were distinct between rice-flavor baijiu, and awamori and kome-shochu. Isobutyl alcohol, β-phenylethyl alcohol, and ethyl caproate contributed to distinct types of rice-flavor baijiu. Rice-flavor baijiu types made by the same company were plotted in close proximity; for example, S1 and S2, S5–S7, S8 and S9, S10–S12, and S14 and S15. Differences according to aging period were small. Therefore, this indicates that the flavor of rice-flavor baijiu strongly depends on the manufacturing company.

Fig. 2.

PCA biplot (scores [A] and loading [B]) of the concentrations of volatile compounds in the liquor samples used in the study. Filled triangles: rice-flavor baijiu samples; crosses: awamori samples; open circles: kome-shochu samples. PCA, principal component analysis

In this study, it was revealed that the high levels of acetic acid, lactic acid, and ethyl lactate in commercial rice-flavor baijiu were important to distinguish from commercial awamori and kome-shochu. In the future, it will be necessary to investigate the relationship between these key compounds and the characteristic production process of each liquor.

Acknowledgments    This work was supported by JSPS KAKENHI (grant No. 18K02188).

References
 
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