Original Paper Characterization of Flavor Compounds in Rice-Flavor Baijiu, a Traditional Chinese Distilled Liquor, Compared With

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Original paper Characterization of Flavor Compounds in Rice-flavor baijiu, a Traditional Chinese Distilled Liquor, Compared with Japanese Distilled Liquors, awamori and kome-shochu

1 1,2* 2 3 2 4 Xuan Yin , Yumiko Yoshizaki , Shugo Kurazono , Mina Sugimachi , Haruka Takeuchi , Xing-Lin Han , 2 1,2 1,2 1,2 Kayu Okutsu , Taiki Futagami , Hisanori Tamaki and Kazunori Takamine

1The United Graduate School of Agricultural Sciences, Kagoshima University, 1-21-24 Korimoto, Kagoshima City, 890-0065, Japan 2Education and Research Center of Fermentation Studies, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima City, 890-0065, Japan 3Graduate school of Agriculture, Kagoshima University, 1-21-24, Korimoto, Kagoshima City, 890-0065, Japan 4China National Research Institute of Food and Fermentation Industries, 24-6 Jiuxianqiao Road, Chaoyang District, Beijing 100096, China

Received November 14, 2019 ; Accepted January 16, 2020

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-flavorbaijiu .

Keywords: Chinese distilled liquor, flavor, rice-flavorbaijiu , shochu, volatile compounds, ethyl lactate

Introduction xiaoqu is added and mixed well (Fig. 1), and then the solid Baijiu is a traditional Chinese distilled liquor, and rice- mixture is incubated for 20–30 h at room temperature. After flavor baijiu is mainly produced in southern China. Rice-flavor incubation, water is added and the mixture is fermented in a baijiu is made from rice as the sole ingredient and xiaoqu as liquid state for 6–8 days. Finally, the fermented mash is the fermentation starter, which is comprised of rice powder and distilled in the liquid state. rice bran (Zheng and Han, 2016). A few types of microorganisms The manufacturing process of rice-flavor baijiu is very are present in xiaoqu, including Rhizopus sp., Mucor sp., and different from that of other types of Chinese baijiu (Fig. 1). yeasts (Zheng and Han, 2016). Those types of Chinese baijiu, such as strong-flavor baijiu, The rice-flavor baijiu manufacturing process involves sauce-flavor baijiu and light-flavor baijiu, are typically made of semi-solid-state fermentation (semi-SSF) (Zheng and Han, several cereals, such as sorghum, wheat and rice, and complex 2016). SSF is performed using a solid matrix in the absence of microorganisms from the natural mixed starter daqu. SSF is free water. Briefly, after steaming the rice, a small amount of performed for several months in a mud pit or earthen jars

*To whom correspondence should be addressed. E-mail: [email protected] 412 X. Yin et al.

Fig. 1. Schematic diagram of manufacturing of rice-flavorbaijiu , awamori and kome-shochu.

(Zheng and Han, 2016). Many microorganisms, such as caproic 1996; Koseki and Iwano, 1998). Firstly, ferulic acid present in acid–producing bacteria, live in mud pits and earthen jars the rice cell wall is liberated by a ferulic acid esterase produced (Xiong et al., 2010), and this complex microbial flora by A. luchuensis during fermentation. Then, the ferulic acid is contributes to the flavor of the liquor (Wanget al., 2014a). transferred to 4-vinylguaiacol (4-VG) by heating during On the other hand, the manufacturing process of rice-flavor distillation. Finally, 4-VG is converted to vanillin by chemical baijiu is similar to that of awamori and kome-shochu, two transformation via oxidation during aging. pH and alcohol traditional Japanese distilled liquors. Awamori uses tane-koji as concentration during aging influence the chemical a fermentation starter, which is prepared from koji mold transformation of ferulic acid to vanillin. (Aspergillus luchuensis). Rice koji is a solid culture of koji Rice-flavor baijiu has a characteristically sweet aroma and mold grown on steamed rice for approximately 42 h. Rice koji, a clear aftertaste (Zheng and Han, 2016). Ethyl lactate and water and yeast seed culture are mixed and fermented in a β-phenylethyl alcohol are key volatile compounds liquid state for 10–12 days. After fermentation, the mash is characteristic of rice-flavor baijiu compared to other types of distilled in the liquid state. Kome-shochu is made from rice and baijiu (Jin et al., 2017). However, to date, no studies have rice koji prepared from A. luchuensis (Fig. 1). It is produced investigated the flavors specific to rice-flavor baijiu in according to the same method as for awamori, except that the comparison with awamori and kome-shochu. Okinawa main ingredients are added separately after the first Prefecture in Japan, the main production center of awamori, fermentation. and Guangdong and Guangxi provinces in China, the main Awamori flavor is typically expressed as koji-like, production centers of rice-flavor baijiu, are geographically mushroom-like, fruity, and sweet flavors. These descriptions close. Therefore, rice-flavor baijiu is important for are also contained in the flavor wheel of awamori (Miyamoto, understanding the historical and technical relationships 2018). Kome-shochu has a characteristic fruity flavor between Chinese and Japanese distilled liquors. Knowledge of (Hayashida, 1998). Isobutyl alcohol, 1-octen-3-ol, nerolidol the chemical and flavor profiles of rice-flavor baijiu will assist and S-methyl thioacetate are the key volatile compounds in in understanding the similarities and/or differences between awamori compared to other Japanese shochu types (Fukuda et Chinese and Japanese distilled liquors. al., 2016). In particular, 1-octen-3-ol, which is derived from This study aimed to determine the characteristic flavor rice koji, imparts the mushroom-like flavor to awamori compounds of commercially available rice-flavor baijiu in (Yoshizaki et al., 2010). Vanillin, the most studied volatile comparison with awamori and kome-shochu. First, the compound in awamori, contributes to the sweet aroma and is concentrations of alcohol, sugar, amino acids, and organic produced by biochemical and chemical reactions (Koseki et al., acids in rice-flavor baijiu were measured by high-performance Flavor Compounds of Rice-flavor baijiu 413

Table 1. Sample informations used in this study.

Sample Aging periods Alcohol† Types of liquor Region, Country Company Distillation type Name (years) (%, v/v) S1 Rice-flavorbaijiu Guangxi, China A 5 Atmospheric* 35 S2 Rice-flavorbaijiu Guangxi, China A Non aging* Atmospheric* 30 S3 Rice-flavorbaijiu Guangxi, China B 18 - 49 S4 Rice-flavorbaijiu Guangxi, China C 18 - 53 S5 Rice-flavorbaijiu Guangxi, China D 5 - 49 S6 Rice-flavorbaijiu Guangxi, China D - - 46 S7 Rice-flavorbaijiu Guangxi, China D - - 48 S8 Rice-flavorbaijiu Guandong, China E 5 Atmospheric* 48 S9 Rice-flavorbaijiu Guandong, China E Non aging* Atmospheric* 52 S10 Rice-flavorbaijiu Guandong, China F Non aging* - 49 S11 Rice-flavorbaijiu Guandong, China F 10 - 54 S12 Rice-flavorbaijiu Guandong, China F 5 - 48 S13 Rice-flavorbaijiu Guandong, China G - - 47 S14 Rice-flavorbaijiu Liaoning, China H - - 50 S15 Rice-flavorbaijiu 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. liquid chromatography (HPLC). Next, the concentrations of Japan). Bromothymol blue, neutral red, phenolphthalein, lactic volatile compounds in rice-flavor baijiu were measured by gas- acid, acetic acid, p-toluenesulfonic acid monohydrate, and bis- chromatography mass spectrometry (GC-MS) analysis in Tris were acquired from FUJIFILM Wako Pure Chemical comparison with awamori and kome-shochu. Finally, the Corp. (Osaka, Japan). Ethylenediaminetetraacetic acid (EDTA) characteristic flavor compounds in rice-flavor baijiu were was acquired from Dojindo Laboratories (Kumamoto, Japan). determined by principal component analysis of the quantitative Authentic standards for GC-MS were purchased from several results of volatile compounds of rice-flavor baijiu, awamori, companies as follows. Isobutyl alcohol, isoamyl alcohol, and kome-shochu. 1-hexanol, decanoic acid, dodecanoic acid, ethyl isovalerate, ethyl stearate, isoamyl caproate, isoamyl caprylate, isobutyl Materials and Methods caprylate, phenylethyl acetate, phenylethyl butyrate, and Liquor samples and chemicals Fifteen different 2-pentyl furan were purchased from FUJIFILM Wako Pure commercial rice-flavor baijiu were purchased from a local Chemical Corp. 1-Butanol, ethyl lactate, ethyl phenylacetate, market in China (Table 1) on the basis of the company, place 2-nonenal, and dimethyl trisulfide were purchased from Tokyo of production and sales price range. In addition, six and five Chemical Industry Co., Ltd (Tokyo, Japan). β-Phenylethyl different commercial awamori and kome-shochu were alcohol, ethyl isobutyrate, ethyl caprylate, ethyl caprate, ethyl purchased, respectively, from a local market in Japan. Sodium myristate, ethyl palmitate, ethyl oleate, ethyl linoleate, isoamyl hydroxide, glucose, fructose, sucrose, maltose, acetonitrile, and acetate, phenylethyl octanoate, and furfural were purchased ethanol were acquired from Nacalai Tesque, Inc. (Kyoto, from Sigma-Aldrich (Steinheim, Germany). Octanoic acid, 414 X. Yin et al. ethyl caproate, ethyl laurate, ethyl salicylate, and ethyl Na-type kit (Shimadzu Corp.). The RF-10AXL fluorescence benzoate were purchased from Nacalai Tesque, Inc. detector was set to an excitation/emission wavelength pair of Total and volatile acidity of liquor samples Total and 350/450 nm, and the reaction reagents were obtained from an volatile acidity values were determined following the official amino acid reaction kit (Shimadzu Corp.) and maintained at a methods of the National Tax Administration Agency, Japan flow rate of 0.2 mL/min. (Brewing Society of Japan, 2006), and it is stated that these GC-MS with stir bar sorptive extraction The liquor methods are applicable to the fermented mash of shochu. samples were diluted to 25 % (v/v) alcohol by adding deionized Moreover, we applied these methods to the rice-flavor baijiu in water, then 10 mL of each liquor sample was transferred to a this study. Total acidity was measured as titratable acidity. sample vial and a 15 mm stir bar coated with 0.5 mm Briefly, 10 mL of each liquor sample was titrated against 0.1 N polydimethylsiloxane was added (Twister, Gerstel K.K., Japan) sodium hydroxide, with the indicator containing bromothymol (Rahayu et al., 2017). The sample was stirred on a magnetic blue and neutral red, until it turned light green. Volatile acidity stirrer at 1 200 rpm for 1 h at room temperature. The stir bar was determined by titration of each liquor sample after was removed, washed with deionized water, dried with a tissue distillation. Briefly, 100 mL of each liquor sample was distilled and placed into a glass insert. Volatile compounds were using a small distillation apparatus. Approximately 70 mL of desorbed from the stir bar using the following temperature the distillate was collected, and the volume was adjusted to program of the Gerstel TDS3 and Gerstel CIS4 thermal 100 mL with deionized water. Further, 10 mL of this solution desorption system (Gerstel K.K.): 20 ℃ for 1 min and 60 ℃ was titrated with phenolphthalein against 0.01 N sodium per min to 260 ℃ (hold for 1 min). Meanwhile, a Gerstel CIS4 hydroxide until it turned light pink. Total acidity and volatile cryotrap (Gerstel K.K.) was set to −150 ℃ to cryofocus. After acidity were represented in the titration volume of 0.1 and the desorptive program was completed, the cryotrap was heated 0.01 N sodium hydroxide, respectively. to inject the volatile compounds into a gas chromatography Quantification of saccharides Saccharides were quantified (GC) analytical column: −150 ℃ for 1 min and 12 ℃ per by injecting 10 μL of each liquor sample into a Prominence second to 270 ℃ (hold for 2 min). The gas chromatography– HPLC system (Shimadzu Corp., Kyoto, Japan) under the mass spectrometry (GC-MS) system was equipped with a following conditions: LC-20AD pump (Shimadzu Corp.); 4 × 0.25 mm × 60 m i.d. Inner Pure-WAX column with a 0.25 μm 250 mm i.d. Cosmosil Sugar-D column (Nacalai Tesque, Inc., film thickness (GL Sciences Inc., Tokyo, Japan). Analyses Kyoto, Japan); mobile phase, acetonitrile:water (3:1); flow rate, were carried out with helium as the carrier gas at a flow rate of 1.0 mL/min; oven temperature 40 ℃ ; detector, RID-10A 1.0 mL/min using the following temperature program: 40 ℃ for refractive index detector (Shimadzu Corp.) (Okutsu et al., 5 min and 3 ℃ per min to 240 ℃ . The retention index (RI) was 2012). Standard curves were constructed using linear regression determined via sample injection with a series of straight-chain of the analyte peak areas versus the known concentrations of alkanes (C5–C24) (SUPELCO Analytical, Bellefonte, PA, each saccharide. USA). Identification of volatile compounds was confirmed by Analysis of organic acids Organic acids were quantified comparing their mass spectra with the NIST05a mass spectral by injecting 10 μL of each liquor sample into a Prominence database and the RI values in the AromaOffice database HPLC system (Shimadzu Corp.) under the following (Nishikawa Keisoku Co., Ltd., Tokyo, Japan). Standard curves conditions: LC-20AD pump (Shimadzu Corp.); 8 × 300 mm i.d. were produced from a pure authentic reagent in 25 % (w/w) Shim-pack SCR-102H HPLC column (Shimadzu Corp.); ethanol solution. The absolute calibration curve was derived mobile phase, 4 mM p-toluenesulfonic acid monohydrate; flow from peak areas using selected ions (m/z) (Tables 1 and 2). The rate, 0.8 mL/min; oven temperature 50 ℃ ; detector, CDD-10A sample was diluted accordingly to determine the VP conductivity detector (Shimadzu Corp.) (Rahayu et al., concentrations. The analysis was repeated three times for each 2017). A mixture of 4 mM p-toluenesulfonic acid monohydrate, sample. 16 mM bis-Tris and 80 μM EDTA served as a post–column Data analysis To identify which volatile compounds are reaction solution at a flow rate of 0.8 mL/min. Standard curves likely to contribute the most to the different characteristics of were constructed using linear regression of the analyte peak the liquor samples used in this study, principal component areas versus known concentrations of each organic acid. analysis (PCA) was performed using Ekuseru-Toukei 2008 Analysis of amino acids Concentrations of amino acids statistical software (Social Survey Research Information Co., were determined using the Prominence HPLC system Ltd., Tokyo, Japan). (Shimadzu Corp.) and an RF-10AXL fluorescence detector (Shimadzu Corp.) using the post–column fluorescence Results and Discussion derivatization method (Rahayu et al., 2017). Amino acid Chemical compositions and properties Descriptions of all separation was achieved using an 6 × 100 mm i.d. Shimadzu 15 rice-flavor baijiu samples selected for this study, including Shim-pack Amino-Na column (Shimadzu Corp.) at 60 ℃ with details regarding their manufacturers, price range and aging a flow rate of 0.6 mL/min using the amino acid mobile-phase periods, are shown in Table 1. Guangdong and Guangxi Flavor Compounds of Rice-flavor baijiu 415

Table 2. Standard curves of volatile compounds used in this study.

Validation range Standard curve r2 (μg/L) _ _ 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 ｌactate 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 416 X. Yin et al.

_ 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 provinces are famous brewing districts and the main production centers of awamori and kome-shochu, respectively. centers of rice-flavor baijiu. Therefore, 13 samples were Of the 15 rice-flavor baijiu samples, two samples (S14 and selected from these regions, while the remaining two samples S15) were characterized by a strong dark-brown color, and one selected were made by a company in Liaoning province, in sample (S5) had a yellow color, indicating that some rice-flavor northern China. Okinawa and Kumamoto Prefectures are baijiu samples are stored in barrels. Sugar was detected by famous brewing districts in Japan and the main production HPLC in five rice-flavor baijiu samples (S3, S4, S5, S14 and Flavor Compounds of Rice-flavor baijiu 417

Table 3. Analysis of spirits.

Rice-flavorbaijiu 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.

S15) (Table 3). Glucose constituted at least 95 % of all sugars T 10781.3-2006) (Yu, 2016). According to this standard, the (data not shown). S14 and S15 contained a high glucose level quality level is prescribed according to sensory evaluation, of >60 mg/mL, and these samples were produced by the same total acidity, total ester content, ethyl lactate content, company. Therefore, it is likely that a flavoring agent such as β-phenylethyl alcohol content and solid content. A >0.3 g/L (= caramel might be added to these liquor samples. 5 mM acetic acid) total acid content equivalent to acetic acid Amino acids were detected in eight samples; five contained indicates a high quality level. Therefore, a high level of acetic a low level of amino acids, while the remaining three samples acid is one of the characteristics of rice-flavorbaijiu. (S5, S14 and S15) had a high level of amino acids. These Quantitation by GC-MS GC-MS with stir bar sorptive amino acids may also be derived from a flavoring agent, in the extraction was used to identify and quantify 34 compounds same manner as for glucose. from the mass spectra and RIs as follows: 5 alcohols, 3 acids, Rice-flavor baijiu showed higher total acidity compared to 22 esters, 2 furans, 1 aldehyde, and 1 sulfuric compound (Table awamori and kome-shochu, with a wide total acidity range 4). The rice-flavor baijiu contained a wide concentration range among the 15 samples. There was an 8.4-fold difference of 18 volatile compounds with an odor activity value (OAV) of between the samples with the highest (S15) and lowest (S8) >1 (Table 5). Awamori and kome-shochu contained 15 and 13 total acidity. On the other hand, volatile acidity was markedly compounds with an OAV of >1, respectively. Of these, 11 lower than total acidity, with rice-flavor baijiu having slightly showed three-fold higher OAVs in rice-flavor baijiu compared higher volatile acidity compared to awamori and kome-shochu. to awamori and kome-shochu: ethyl isobutyrate, ethyl However, there was a large difference between total acidity and isovalerate, ethyl lactate, ethyl caproate, ethyl laurate, ethyl volatile acidity in rice-flavor baijiu. Therefore, acid myristate, ethyl palmitate, ethyl linoleate, 2-pentyl furan, compositions were analyzed by HPLC (Table 3). All 15 rice- 2-nonenal, and dimethyl trisulfide. flavor baijiu samples mainly contained lactic and acetic acids. The levels of ethyl isobutyrate, ethyl isovalerate and ethyl Although other acids were detected in S5, S14 and S15, the lactate depend on the concentrations of the organic acids levels were very low (data not shown). There was a 14-fold isobutyric acid, isovaleric acid and lactic acid, respectively, in difference between the highest (S15) and lowest (S8) levels of the fermented mash (Shen, 2003; Rahayu et al., 2017). Ethyl lactic acid, while there was a 4-fold difference between the lactate is a common and important compound in Chinese highest (S14) and lowest (S9) levels of acetic acid. Lactic acid liquor. Esterification of ethanol and lactic acid in Chinese level was correlated with total acidity level in all 15 rice-flavor liquor are carried out during fermentation (Cheng et al., 2018). baijiu samples, indicating that a high lactic acid level results in Lactic acid in rice-flavor baijiu mash is mainly produced by high total acidity in rice-flavor baijiu. Lactic acid is a non- molds and lactic acid bacteria in xiaoqu (Yin et al., 2020). In volatile acid, and it is expected that it is difficult to detect in contrast, citric acid is the main organic acid in awamori and distilled liquors. Thus, these results imply that the addition of kome-shochu mashes; the koji molds (A. luchuensis and A. lactic acid is a generally employed technique in the production luchuensis mut. kawachii) used in the making of shochu mainly of rice-flavor baijiu, while the addition of sugar is restricted. produce and secrete citric acid (Kadooka et al., 2019). The concentrations of lactic acid and glucose detected in rice- Therefore, the lactic acid content of awamori and kome-shochu flavor baijiu were higher than that of amino acids. The amino mashes is not high. These findings suggest that ethyl lactate is acids detected in five of the samples might originate from the a characteristic compound in rice-flavor baijiu compared to flavoring reagents and lactic acid as an impurity. awamori and kome-shochu. In China, there is a national standard for rice-flavor baijiu, Fatty acid ethyl esters such as ethyl caproate, ethyl laurate, which has been developed by the Standardisation ethyl myristate, ethyl palmitate and ethyl linoleate were Administration of the People’s Republic of China (SAC) (GB/ abundantly found in rice-flavor baijiu. The genus Rhizopus is 418 X. Yin et al.

Table 4. Volatile compounds in rice-flavor baijiu, awamori, and kome-shochu detected by GC-MS.

Identification Quantification Compounds Odor description RI CAS No method 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.

recognized as a good lipase producer, and its lipase is used in Ethyl caproate is an important flavor compound in Chinese many biotech applications (Yu et al., 2016). On the other hand, liquors (Shen, 2003) and is generally produced by yeast. Aspergillus oryzae, a popular koji mold, does not produce a Although yeast is commonly employed as a microorganism for large amount of lipase in solid culture (Ohnishi et al., 1994). rice-flavor baijiu, awamori and kome-shochu, the yeast strain Therefore, it is suggested that long-chain fatty acid levels in has a greater influence on the formation of ethyl caproate than rice-flavor baijiu mash are higher compared to awamori and fermentation conditions such as aeration and fermentation kome-shochu, and that these ethyl esters are also produced by temperature (Piendl and Geiger, 1980). Yeasts synthesize ethyl yeast. Moreover, all of the rice-flavor baijiu samples in this caproate via two pathways: from caproic acid and ethanol by study had a higher alcohol level compared to the awamori and esterase, and from caproyl-CoA and ethanol by alcohol kome-shochu samples (Table 2). Therefore, long-chain fatty acyltransferase (Liu et al., 2004). The synthesis of ethyl acid ethyl esters are readily soluble and are found in large caproate in brewing is limited by the abundance of caproic acid quantities in Chinese liquors. in the fermentation mash. Therefore, differences in the yeast Flavor Compounds of Rice-flavor baijiu 419 - - 1 1 2 1 1 2 1 2 1 1 2 1 1 3 4 4 2 2 2 2 3 5 1 2 3 6 1 7 8 3 8 1 Ref . , - - 39 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> et al 4.6 8.6 1.9 3.2 4.8 1.7 5.7 122 3.9 1.5 1,470 3,320 % (w/w) Kome-shochu - - 11 16 99 41 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 110 3.2 1.0 7.3 6.4 635 1.6 5.5 0.2 7.6 . OAV Awamori - - 11 91 16 27 14 10 33 21 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 1> 4.8 8.1 1.1 2.3 1.1 1.3 8.0 255 1,110 4,910 % (w/w) ethanol), [3] Ferreira ., 2008; odor threshol in 12 kome-shochu et al , and ., 1988; odor threshold in water. Rice-flavor baijiu et al - - 3 5 2 1 0 0 15 30 awamori 200 500 500 500 870 450 575 100 125 800 250 961 , 5,000 8,000 7,200 1,400 40,000 30,000 10,000 15,000 15,000 14,000 14,000 14,100 (μg/L) baijiu Odor threshold tr tr tr tr % (w/w) alcohol, [6] Li nd 19 nd nd nd 54 nd nd nd nd nd 78 nd nd nd nd nd nd nd 4.7 240 297 1.3 0.9 0.7 573 3,988 Min 66,233 24,649 142,027 21 13 nd 59 16 14 13 10 nd 15 nd 185 207 6.0 6.2 1.7 3.1 3.0 3.8 2.1 1.0 4.0 2.3 % (w/w) ethanol), [8] Buttery 1,643 1,183 5,356 7,646 2,035 ., 1972; odor threshold in synthetic wine (11 Max 23,666 39,336 35,081 31,048 422,564 351,014 et al Kome-shochu 97 13 72 nd 16 nd nd 381 295 5.0 7.4 5.0 1.2 1.5 1.0 0.8 1.6 1.6 2.9 2.7 0.6 982 1.0 1.0 1.5 3.1 9,645 7,343 6,635 1,144 3,672 31,901 Mean 183,592 257,265 tr tr tr tr tr Tr nd 54 10 nd 60 nd nd nd nd nd nd nd nd nd nd 454 342 101 183 7.1 1.9 655 1.2 2,152 1,774 ., 2005; odor threshold in sake (15 Min 79,231 40,455 251,755 et al tr 53 35 18 nd 84 352 613 917 804 1.2 1.1 1.6 4.7 3.6 0.5 1.5 1.4 1.4 8.8 2.2 0.22 0.43 8,931 2,820 1,529 7,426 2,133 5,151 1,422 1,747 Max 210,516 393,797 108,734 Awamori tr 11 30 19 nd 14 777 168 242 550 320 0.6 0.8 1.6 3.4 2.8 0.4 1.3 1.0 0.2 705 3.3 1.5 0.04 0.36 5,160 1,188 4,475 1,270 2,955 1,364 72,604 Mean 126,280 337,228 ., 2001, [5] Isogai tr tr tr tr nd nd 43 54 12 15 21 nd nd nd nd nd nd nd nd nd 15 nd nd nd nd nd et al 191 3.0 2.2 2.1 Min 27,663 38,761 18,760 93,568 ., 2014b; odor threshold in synthetic Chinese liquor (46 baijiu 37 10 25 26 94 10 970 103 289 3.1 1.5 3.6 846 2.9 7.0 et al 2,011 9,330 1,081 3,321 2,867 3,946 5,857 8,431 1,596 2,765 3,273 2,437 Max 19,391 564,045 634,175 456,898 641,297 202,446 171,874 48 24 15 10 20 315 436 538 359 0.8 5.7 997 0.6 1.6 322 2.3 1.7 8.0 325 4.3 Rice-flavor 3,873 1,168 1,339 1,366 2,221 2,891 1,159 4,630 80,707 24,536 15,623 Mean 190,194 323,628 379,079 % (w/w) ethanol), [4] Zea The concentration (μg/L) and odor-active values of volatile compounds in rice-flavor The concentration (μg/L) and odor-active Table 5.Table g/L tartaric acid at pH 3.2, [7] Wang Wang 3.2, [7] tartaric acid at pH g/L Isobutyl alcohol Isoamyl alcohol 1-Butanol Alcohol β-Phenylethyl 1-Hexanol Octanoic acid Decanoic acid Dodecanoic acid Ethyl isobutyrate Ethyl isovalerate Ethyl lactate Ethyl caproate Ethyl caprylate Ethyl caprate Ethyl laurate Ethyl myristate Ethyl palmitate Ethyl stearate Ethyl oleate Ethyl linoleate Ethyl salicylate Ethyl benzoate Ethyl phenylacetate Isoamyl acetate Isoamyl caproate Isoamyl caprylate Isobutyl caprylate Phenethyl acetate Phenylethyl butyrate Phenylethyl octanoate 2-Pentyl furan Furfural 2-Nonenal Dimethyl trisulfide 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. [2] Salo Odor threshold abtained from references: [1] Guth, 1997; odor in water/ethanol (90+10, w,w), 2000; odor threshold in synthetic wine (11 ethanol containing 5 Alcohol Acid Ester Furan Aldehyde Sulfric compound 420 X. Yin et al. 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 Fig. 2. PCA biplot (scores [A] and loading [B]) of the is thought that the rice-flavor baijiu and awamori mashes have concentrations of volatile compounds in the liquor samples used greater enzyme activity compared to kome-shochu. Therefore, in the study. Filled triangles: rice-flavor baijiu samples; crosses: awamori samples; open circles: kome-shochu samples. PCA, the amino acid level during fermentation may differ, impacting principal component analysis 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 principal components (PC1 and PC2) correlated positively with 1-butanol, ethyl caprylate, isoamyl acetate and phenylethyl the diversity of rice-flavor baijiu and the differences between acetate. Isoamyl acetate and phenylethyl acetate are produced Chinese and Japanese distilled liquors, respectively. In in yeast cells by alcohol acetyl-transferases (AATases; EC particular, ethyl lactate and isoamyl alcohol were important 2.3.1.84) with alcohols and acetyl–coenzyme A (acetyl-CoA) compounds that were distinct between rice-flavor baijiu, and as substrates. AATase activity and the expression level of the awamori and kome-shochu. Isobutyl alcohol, β-phenylethyl AATase gene ATF1 are inhibited by unsaturated fatty acids alcohol, and ethyl caproate contributed to distinct types of rice- (Fujii et al., 1997). The high fatty acid content of rice-flavor flavor baijiu. Rice-flavor baijiu types made by the same baijiu mash is attributed to lipase production by Rhizopus sp., company were plotted in close proximity; for example, S1 and as previously described. Therefore, the AATase of yeast might S2, S5-S7, S8 and S9, S10–S12, and S14 and S15. Differences be more strongly inhibited in rice-flavor baijiu mash compared according to aging period were small. Therefore, this indicates to awamori and kome-shochu. that the flavor of rice-flavor baijiu strongly depends on the PCA of volatile compounds PCA results revealed diversity manufacturing company. among the 15 rice-flavor baijiu samples (Fig. 2), while all six In this study, it was revealed that the high levels of acetic awamori samples and most of the five kome-shochu samples acid, lactic acid, and ethyl lactate in commercial rice-flavor were grouped into the same cluster. The first and second baijiu were important to distinguish from commercial awamori Flavor Compounds of Rice-flavor baijiu 421 and kome-shochu. In the future, it will be necessary to meaning of vanillin in awamori. Nippon Jyozokyokai shi, 93, investigate the relationship between these key compounds and 510‒517. (In Japanese). the characteristic production process of each liquor. Li, H., Tao, Y.-S., Wang, H., and Zhang, L. (2008). Impact odorants of Chardonnay dry white wine from Changli country (China). Eur. Acknowledgments This work was supported by JSPS Food Res. Technol., 227, 287‒292. KAKENHI (grant No. 18K02188). Lilly, M., Bauer, F. F., Lambrechts, M. G., and Pretorius, I. S. (2006). The effect of increased yeast branched-chain amino acid References transaminase activity and the production of higher alcohols on the Äyräpää, T. (1965). The formation of phenethyl alcohol from flavor profiles of wine and distillates.FEMS Yeast Res., 6, 726‒743. 14C-labelled phenylalanine, J. Inst. Brew., 71, 341‒347. Liu, S.Q., Holland, R., and Crow, V.L. (2004). Esters and their Brewing Society of Japan (2006). “Fourth revision official methods of biosynthesis in fermented dairy products: a review. Int. Dairy J., 14, national tax administration agency.” Japan: (Chapter 3). 923‒945. Buttery, R.G., Turnbaugh, J.G., and Ling, L. C. (1988). Contribution of Long, K., Zhao, Z.-K., Ma, Y.-Y., and Yang, J.-G. (2013). Progresses volatile to rice aroma. J. Agric. Food Chem., 36, 1006‒1009. of researches on Rhizopus for liquor-making. Modern Food Sci. Cheng, W., Zhang, J., Pan, T.Q., Li, N., Wu, L.H., Han, X., Xue, X.J., Technol., 29, 443‒447. and Peng, B. (2018). Application of Angel aroma-producing active Machida, M. (2002). “Advances in applied microbiology.” vol. 51, dry yeast in the production of qingxiang xiaoqu bajiu by solid-state London: Academic Press, Chapter Progress of Aspergillus oryzae fermentation. Liquor-making Sci. Technol., 7, 83‒91. (In Chinese) Genomics. Ferreira, V., López, R., and Cacho, J.F. (2000). Quantitative Miyamoto, M. (2018). The awamori flavor wheel. Nippon Jyozokyokai determination of the odorants of young red wines from different shi, 113, 536‒543. (In Japanese). grape varieties. J. Sci. Food Agric., 80, 1659‒1667. Ohnishi, K., Yoshida, Y., and Sekiguchi, J. (1994) Lipase production Fujii, T., Kobayashi, O., Yoshimoto, H., Furukawa, S., and Tamai, Y. of Aspergillus oryzae. J. Ferment. Bioeng., 77, 490‒495. (1997). Effect of aeration and unsaturated fatty acids on expression Okutsu, K., Yoshizaki, Y., Takamine, K., Tamaki, H., Ito, K., and of the Saccharomyces cerevisiae alcohol acetyltransferase gene. Sameshima, Y. (2012). Development of a heat-processing method Appl. Environ. Microbiol., 63, 910‒915. for koji to enhance its antioxidant activity. J. Biosci. Bioeng., 113, Fukuda, H., Han, J., and Yamada, O. (2016). Characteristics of the 349‒354. volatile composition of commercially available awamori. Nippon Piendl, A. and Geiger, E. (1980). Technological factors in the Jyozokyokai shi, 111, 261‒270. (In Japanese). formation of esters during fermentation, Brewers Digest, 55, 30‒35. Guth, H. (1997). Quantitation and sensory studies of character impact Rahayu, Y.Y.S., Yoshizaki, Y., Yamaguchi, K., Okutsu, K., Futagami, odorants of different white wine varieties. J. Agric. Food Chem., 45, T., Tamaki, H., Sameshima, Y., and Takamine, K. (2017). Key 3027‒3032. volatile compounds in red koji-shochu, a Monascus-fermented Hayashida, Y. (1998). Breeding shochu yeast for flavor improvement product, and their formation steps during fermentation. Food Chem., of rice shochu (vacuum type). Nippon Jyozokyokai shi, 93, 504‒509. 224, 398‒406. (In Japanese). Salo, P., Nykänen, L., and Suomalainen, H. (1972). Odor thresholds Hu, X.-L., Du, H., and Xu, Y. (2015). Identification and quantification and relative intensities of volatile aroma components in an artificial of the caproic acid-producing bacterium Clostridium kluyveri in the beverage imitating whisky. J. Food Sci., 37, 394‒398. fermentation of pit mud used for Chinese strong-aroma type liquor Shen, Y.F. (2003). Study on the formation mechanism of four main production. Int. J. Food Microbiol., 214, 116‒122. kinds of ethyl esters in the fermentation of liquors. Liquor-making Isogai, A., Utsunomiya, H., Kanda, R., and Iwata, H. (2005). Changes Science and Technology, 5, 28‒30. in the aroma compounds of sake during aging. J. Agric. Food Chem., Wang, C.D., Chen, Q., Wang, Q., Li, C.H., Leng, Y.Y., Li, S.G., Zhou, 53, 4118‒4123. X.W., Han, W.J., Li, J.G., Zhang, X.H., and Li, Y.Z. (2014a). Long- Jin, G., Zhu, Y., and Xu, Y. (2017). Mystery behind Chinese liquor term batch brewing accumulates adaptive microbes, which fermentation. Trends Food Sci. Technol., 63, 18‒28. comprehensively produce more flavorful Chinese liquors. Food Res. Kadooka, C., Izumitsu, K., Onoue, M., Okutsu, K., Yoshizaki, Y., Int., 62, 894‒901. Takamine, K., Goto, M., Tamaki, H., and Futagami, T. (2019). Wang, X., Fan, W., and Xu, Y. (2014b). Comparison on aroma Mitochondrial citrate transporters CtpA and YhmA are required for compounds in Chinese soy sauce and strong aroma type liquors by extracellular citric acid accumulation and contribute to cytosolic gas chromatography-olfactometry, chemical quantitative and odor acetyl coenzyme A genedation in Aspergillus luchuensis mut. activity values analysis. Eur. Food Res. Technol., 239, 813‒825. kawachii, Appl. Environ. Microbiol., 85, e03136-18 Xiong, L., Hu, Y., Liu, J., Yuan, J., and Gao, J. (2010). Research on Koseki, T. and Ito, Y., Furuse, S., Ito, K., and Iwano, K. (1996). caproic acid bacteria isolation from fermentation pit mud and its Conversion of ferulic acid into 4-vinylguaiacol, vanillin and vanillic induced mutation breeding. J. Sichuan Univ. Sci. Eng., 23, 324‒327. acid in model solutions of shochu. J. Ferment. Bioeng., 82, 46‒50. (In Chinese). Koseki, T. and Iwano, K. (1998). A mechanism for the formation and Yin, X., Yoshizaki, Y., Ikenaga, M., Han, X.L., Okutsu, K., Futagami, 422 X. Yin et al.

T., Tamaki, H., andTakamine, K. (2020) Manufactural impact of the China light industry Press Ltd., Appendix (In Chinese). solid-state saccharification process in rice-flavor baijiu production,J. Yu, X.-W., Xu, Y., and Xiao, R. (2016) Lipases from the genus Biosci. Bioeng., 129, 315‒321. Rhizopus: Characteristics, expression, protein engineering and Yoshizaki, Y., Yamato, H., Takamine, K., Tamaki, H., Ito, K., and application. Prog. Lipid Res., 64, 57‒68. Sameshima, Y. (2010). Analysis of volatile compounds in shochu Zea, L., Moyano, L., Moreno, J., Cortes, B., and Medina, M. (2001). koji, sake koji, and steamed rice by gas chromatography-mass Discrimination of the aroma fraction of sherry wines obtained by spectrometry, J. Inst. Brew., 116, 49‒55. oxidative and biological ageing. Food Chem., 75, 79‒84. Yu, Q.W. (2016). “Chuantong Baijiuniangzaojishu (Traditional baijiu Zheng, X.W. and Han, B.Z. (2016). Baijiu, Chinese liquor: History, brewing technology) (7th ed.).” ISBN 978-7-5019-7422-1, Beijing: classification and manufacture.Journal of Ethnic Foods, 3, 19‒25.