Abstract
Douchi is a traditional fermented soybean product originated in China and it has been consumed since ancient times as seasoning for food. Volatile components of three commercial douchies were extracted using a simultaneous steam distillation and extraction apparatus (SDE). The extracts were analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 131 compounds were identified, but only 25 components were common to all three brands. Major classes of compounds included esters (29), acids (18), alcohols (16), pyrazines (14), ketones (13), aldehydes (12), phenols (6), hydrocarbons (5), furans (5), sulphur-containing compounds (5), pyridines (4), pyrimidines (2), and miscellaneous compounds (2).
INTRODUCTION
Soybean and its products such as “natto” and “miso” in Japan, “tempe” in Indonesia, “thua-nao” in Thailand, “chungkuk-jang” in Korea, and “sufu,” “douchi,” and “soy sauce” in China, have been appreciated by consumers as health foods due to their valuable nutritional, unique flavour and taste, and medicinal attributes. Douchi is a traditional fermented soybean product that originated in China. This product has been consumed in China since ancient times as seasoning for food. There are three types of douchi that are fermented by Mucor, Bacteria or Aspergillus strains.
As the population consuming foods with added douchi is increasing, there is an increasing interest in its physiological properties. Previous studies have the antioxidant[Citation1 Citation–2] and anti-hypertensive[Citation3] activities of douchi. Douchi is made from soybeans by pretreatments (soaking followed by steaming) and a two-step fermentation process (pre-fermentation and post-fermentation), some of them include the subsequent drying process.[Citation4] Chemical components are changed and new compounds are generated during douchi fermentation by a series of complex biochemical reactions.[Citation4 Citation–5] During prefermentation, where douchi qu (Koji) is produced, some enzymes such as proteinase and amylase were produced by microorganisms. Some components of douchi, such as: protein, lipid and starch were converted to peptides, amino acids, fatted acids, reducing sugars, ethanol, and various aroma components by salt-tolerant microorganisms during post-fermentation.[Citation5]
Good quality douchi products should have the characteristic flavour, palatably soft texture, and a bright black colour. However, information on the volatile components of this fermented product is scarce. Therefore, the objective of this study was to determine and compare the volatile components in three types of commercial douchies. Douchi was extracted using a simultaneous steam distillation and extraction apparatus (SDE), and extracts were analyzed by gas chromatography-mass spectrometry (GC-MS).
MATERIALS AND METHODS
Samples
Three brands of douchi commonly available and consumed in China were chosen in this investigation. The place of manufacture, raw materials and types of three commercial douchies are listed in . The samples were collected directly from the douchi manufacturing factory (name not mentioned) in each province.
Table 1 Place of manufacture, raw materials and types of three commercial douchies
Chemical Analysis of Douchi
Official methods[Citation6] were used to determine other chemical compositions of douchi, including moisture (Vacuum Oven Method 925.09), protein (Method 955.04 using 6.25 as conversion factor), fat (Method 945.39), and ash (Method 924.05) contents. The reducing sugar content was measured using the dinitrosalicylic acid method.[Citation7]
Collection of Volatile Components
Volatile components of douchi were isolated according to the method described by Chung[Citation8] with some modifications. A Likens and Nickerson[Citation9] type simultaneous steam distillation and extraction (SDE) apparatus was used for the extraction of volatile components. Samples (40 g each) were placed in a 1-L round-bottom sample flask, and 0.3 mL of 2, 4, 6-trimethylpyridine [internal standard (I. S.)] aqueous solution at 10 μg/mL was added before extraction. Four hundred millilitres of distilled water was added to the sample flask. Distilled dichloromethane (50 mL) was used as the extraction solvent. Each extraction was carried out for 3 h after the distilled water started to boil in the sample flask. Extracts were initially concentrated by a gentle steam of nitrogen gas of 99.995% purity to 15 mL, dried with 2.3 g of anhydrous sodium sulfate, and further concentrated to 1 mL by a stream of nitrogen gas. Extracts were kept in a freezer (−20°C) until further analysis. SDE extracts were prepared in duplicate.
Gas Chromatography-Mass Spectrometry (GC-MS)
Qualitative and quantitative analyses of extracts were carried out with a Trace TM 2000 gas chromatography (GC) (Thermo Quest Mass Lab Group, Manchester, UK) coupled with a Voyager mass selective detector (MSD) (Finnigan Corporation, Sunnyvale, CA, USA). Separation of volatile compounds was performed on a DB-Wax column (60 m length × 0.25 mm. i. d. × 0.25 μm df, Agilent Technologies, Palo Alto, CA, USA). One microlitre of each extract was injected in a splitless mode into the GC. GC oven conditions were initially at 40°C, held for 5 min; programmed at 5°C/min until 240°C, and held for 35 min; injector temperature was 220°C, and helium carrier gas flow was at 1 mL/min. MS conditions were as follows: ion source temperature, 240°C; ionization voltage, 70 eV; mass range, 29–540 amu; scan rate, 0.5 s/scans; and electron multiplier voltage, 350V.
Compound Identification and Quantification
Positive identification of a component was performed by comparison of its retention time and mass spectrum. Mass spectral identification was completed by comparing spectra with the mass spectra from the NIST Mass Spectral Search Program (version 1.7) and the WILEY Mass Spectral Search Program or by manual interpretation. Approximate concentrations of volatile compounds were calculated according to the internal standard method, using 2, 4, 6-trimethylpyridine and the Xcalibur Software (Vienna, VA).
Statistical Analysis
The mean values of assays in duplicate SDE extracts of each douchi brand were calculated. The SAS system (SAS for Windows 6.12, SAS Institute Inc., Cary, NC, USA) was used for statistical analysis. Duncan's multiple range tests were used to estimate the significant differences among the mean values at the 5% probability level.
RESULTS AND DISCUSSION
shows the data from chemical analysis of all three douchi samples. The wet basis ranges of moisture, protein, fat, ash and reducing sugar contents were 17.22–37.04, 25.87–44.84, 14.46–20.60, 5.81–14.32, and 2.24–2.94%, respectively. shows the volatile components identified in three samples. Brands A, B and C had a total of 80, 55, and 79 components, respectively. Combining the number of volatiles in all three douchi samples, 131 compounds were identified, of which only 25 were common in all brands. Volatiles belonged to 13 classes of compounds including esters (29), acids (18), alcohols (16), pyrazines (14), ketones (13), aldehydes (12), phenols (6), hydrocarbons (5), furans (5), sulfur-containing compounds (5), pyridines (4), pyrimidines (2), and miscellaneous compounds (2). Most of the compounds identified were previously found in soybean processed products such as sufu, miso, soy sauce, and natto.[Citation8,Citation10–17 According to the results obtained and the volatile compounds of other fermented soybean foods, pyrazines, esters, acids, and alcohols compounds are the important contributors to the characteristic flavour of douchies.
Table 2 Chemical analysis (wet %) of three commercial douchies.Footnote a, Footnote b
Table 3 Volatile components identified in three commercial douchies (dry weight basis)
The concentrations of volatile components in different brands of douchi were different (). The concentrations of alcohols, aldehydes, acids and esters in brand B were higher than those in brand A and C. However, the concentrations of pyrazines in brand A were much higher than those in the other two brands. Brands A and C are both fermented by Aspergillus strains, brand C has a characteristic flavour due to the addition of ginger. Brand B fermented by Mucor strains had high concentrations of alcohols, aldehydes, acids and esters because of the addition of alcohol at post-fermentation.[Citation12] It has been stated that there are many factors affecting the flavour and volatile compounds of douchi, such as raw materials, soaking time, cooking time, fermentation microorganisms, inoculation rate, fermentation temperature and the addition (such as salt, alcohol and ginger) at post-fermentation, among others.
Figure 1 Differences of concentration of various volatile fractions in three commercial douchies. (Misc. cpds.: miscellaneous compounds; and S-cpds.: sulfur-containing compounds).
ESTERS
Twenty-nine esters were detected in the douchi extracts. Esters were among the largest classes of compounds detected. Most esters have pleasant aromas and enhance douchi flavour quality.[Citation11] Seven compounds, phenylacetic acid methyl ester, phenylacetic acid ethyl ester, 2-phenethyl acetate, hexadecanoic acid methyl ester, hexadecanoic acid ethyl ester, 9,12,15-octadecatrienoic acid(Z, Z, Z) methyl ester, and ethyl 9-octadecenoate(E) were found in all three brands.
Some esters found in douchi extracts have been reported in other soy products. Phenylacetic acid ethyl ester and hexadecanoic acid ethyl ester identified in three samples were also detected in sufu[Citation8,Citation10] and miso.[Citation11] Ethyl acetate and ethyl propionate, which were commonly found in the aroma component of some oriental fermented products such as chungkun-jiang[Citation15] and miso,[Citation11] were also detected in douchi extracts. High-molecular-weight fatty acid esters, such as ethyl 9-octadecenoate(E) which is likely produced by the effect of fungal lipases on soybean lipids has been reported in sufu,[Citation8,Citation18] and also detected in douchi extracts.
Some other compounds such as methyl isobutyrate which is the major component of natto [Citation15] was not detected in douchi extracts. Having a highest number of components among all classes, with various unique odour characters of individual components, the esters could be an important class contributing to the flavour of the douchies.
Acids
Acids were the second largest class containing 18 compounds in douchi extracts. 3-Methylbutanoic acid, tetradecanoic acid and n-hexadecanoic acid were found in all three brands. Acetic acid, 2-methylpropanoic acid, hexanoic acid and 9,12-octadecadienoic acid(Z, Z) were only detected in brand B. 4-Methylpentanoic acid and 8,11,14-eicosatrienoic acid(Z, Z, Z) were found in brands B and C.
Some acids found in douchi have been reported in other soy products. Acetic acid, 2-methylpropanoic acid, 3-methylbutanoic acid, and hexanoic acid were found in douchi extracts, which also have previously been reported in soybean processed products such as sufu,[Citation10,Citation12] miso,[Citation11] soy sauce,[Citation14] and natto.[Citation15] Some other compounds such as 2-methylbutanoic acid, the major flavour component of natto[Citation17] was not found in three brands; instead its isomer 3-methylbutanoic acid was detected in three samples. 2-Methylpropanoic acid and 3-methylbutanoic acid were derived from valine and leucine degradation.[Citation19] With one of the highest numbers of components among all classes, and with some components having very high concentrations among other classes, GC/olfactometry are necessary to validate the effect of acids to the flavour of the douchies in subsequent experiments.
Alcohols
Alcohols were the third largest class containing 16 different compounds. Five kinds of alcohols, 2-methyl-1-propanol, 3-methyl-1-butanol, 2-furanmethanol, benzyl alcohol, and phenyl ethyl alcohol were found in all three brands. Some alcohols found in douchi extracts have been reported in other soy products. Most of the alcohols can impact the flavour of the douchi. 2-Methyl-1-propanol and benzyl alcohol identified in this work were also detected in sufu,[Citation8,Citation10,Citation12] miso,[Citation13] soy sauce,[Citation14] and natto.[Citation15] 3-Methyl-1-butanol was also found in sufu,[Citation10] soy sauce,[Citation14] and natto.[Citation15] 1-Pentanol and 1-octen-3-ol previously reported in soybean milk,[Citation20] sufu,[Citation8,Citation10] natto,[Citation17] and miso [Citation13] were also detected in douchi extracts.
Most alcohols detected in the douchi extracts have characteristic aromas. Ethanol has a pleasant odour and could be directly produced by metabolism of fermentable carbohydrates by starters and yeasts.[Citation21] 3-Methyl-1-butanol has fusel oil and whiskey-like odours[Citation10]; 1-pentanol has sweet and balsamic odours.[Citation8] 3-Octanol has nutty, herbaceous, melon and citrus aromas.[Citation8] 1-Octen-3-ol which has a beany odour is considered as an important contributor to the characteristic odour of sufu,[Citation8, Citation10] natto [Citation17] and miso.[Citation13] It is an enzymatic derivative of linoleic acid, which has been found much higher in the low-salt miso products than in other products.[Citation11] Having the highest concentration of components among all classes, with various unique odour characters of individual components, the alcohols could be an important class contributing to the flavour of the douchies.
Pyrazines
The pyrazines, important contributors to the characteristic odour of douchi, could be generated naturally during the aging process by the condensation of aminoketones formed through the Maillard reaction and Strecker degradation.[Citation8] Among the 14 pyrazines identified, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine and trimethylpyrazine could be found in all three brands. Pyrazines are also the major components of natto.[Citation17] Methylpyrazine, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, trimethylpyrazine, tetramethylpyrazine and 2-ethyl-6-methylpyrazine which have previously been detected in natto and liquid cultures of Bacillus subtilis[Citation16,Citation22 Citation–23] and sufu[Citation8] were also detected in douchi extracts. Many higher molecular weight alkylpyrazines are known to be present in coffee and roasted peanut volatiles. Pyrazines have a nutty aroma, especially alkylpyrazines. 2,5-Dimethylpyrazine, trimethylpyrazine and 2-ethyl-6-methylpyrazine contribute to natto odour.[Citation16] Tetramethylpyrazine has a sweet, musty, chocolate, cocoa, lard and burnt note.[Citation24]
Ketones
Altogether thirteen ketones were detected in douchi extracts. Among them, only 1-(2-furanyl)-ethanone could be detected in all three brands. 1-Octen-3-one, acetoin, 2-pentadecanone and dihydro-5-pentyl-2(3H) furanone were detected in brands A and C. 3-Octanone and acetophenone were only found in brand A. 1-(1H-pyrrol-2-yl)-ethanone and 2-decanone were only identified in brand B.
Some ketones found in douchi have also been reported in other soy products. 1-(2-Furanyl)-ethanone detected in the pyrolysis products of N-acetyglucosamine[Citation25] was also detected in three samples. Acetoin which is the major volatile compounds in natto[Citation17] was identified in brands A and C, which has a pleasant and cream-like note. 3-Octanone was found in brand A, which also has been reported in soybean processed products, such as soybean milk, textured soy protein[Citation20,Citation26] and fermented soybean by Bacillus subtilis.[Citation27] 1-(1H-pyrrol-2-yl)-ethanone identified in brand B was present in sufu.[Citation8,Citation10,Citation12] Ketones could be generated by fungal enzymatic actions or by Maillard reaction in sufu processing[Citation8] and have characteristic aromas. 3-Octanone has a floral, green, herbaceous and fruity-like note.[Citation8] 1-Octen-3-one has mushroom flavour.[Citation24]
Aldehydes
Among 12 aldehydes, furfural, benzaldehyde, 5-methyl-2-furancarboxaldehyde, phenylacetaldehyde and 5-methyl-2-phenyl-2-hexenal were found in all three brands. 3-Methylbutanal was only identified in brand A. Pentanal and 2,4-decadienal(E, E) were only found in brand B. 3-Methyl-2-butenal, nonanal and 4-methyl-2-phenyl-2-pentenal were only detected in brand C.
Some aldehydes found in douchi extracts have been reported in other soy products. Benzaldehyde identified in this work was reported in sufu,[Citation8,Citation10] miso,[Citation11,Citation13] natto,[Citation17] and soy sauce.[Citation14] Phenylacetaldehyde found in douchi extracts was previously reported in sufu,[Citation10] miso,[Citation11] soy sauce,[Citation14] and the unflavoured textures soy protein.[Citation26] 5-Methyl-2-phenyl-2-hexenal was reported in sufu. [Citation10] 3-Methylbutanal found in this work was also detected in sufu[Citation12] and miso.[Citation11] These compounds could be produced by lipid oxidation and degradation.[Citation26] Some other compounds such as hexanal was not found in all three douchi brands, whereas it has high concentration in sufu.[Citation8] Hexanal contributes a beany aroma to both the soybean milk and textured soy protein.[Citation20,Citation26]
Most aldehydes detected in the douchi extracts have characteristic aromas. Benzaldehyde has almond, aromatic and sweet notes.[Citation24] Phenylacetaldehyde has a harsh and hawthorn aroma when diluted.[Citation28] Furfural has a special spine note and exists in cooked coffee.[Citation24] 3-Methylbutanal has strongly medicinal-herb-like, fruity and nutty notes when the concentration is low.[Citation24] Pentanal has strongly fruity and nutty notes when the concentration is low.[Citation24] 2,4-Decadienal(E, E) has an intensely greenish aroma, which exist in peanut oil and fried potatoes.[Citation24] 4-Methyl-2-phenyl-2-pentenal and 5-methyl-2-phenyl-2-hexanal both have cocoa aroma.[Citation24] Nonanal has orange and rose aromas when diluted.[Citation24]
Phenols
Six important savory compounds, 2-methoxyphenol, 4-methoxyphenol, phenol, 4-ethylguaiacol, 4-ethylphenol, and 4-vinylguaiacol were detected in douchi extracts. Except 4-methoxyphenol, other phenols are also found in sufu,[Citation8,Citation10,Citation12,Citation27] miso,[Citation11,Citation13] and soy sauce.[Citation14] 4-Ethylguaiacol, 4-ethyl-phenol and 4-vinylguaiacol found in brands A and C were also the major components of miso.[Citation13]
4-Ethylphenol identified in douchi extracts is believed to be generated from the degradation of lignin glycoside during fermentation,[Citation30] while 4-vinylguaiacol characterizing the cooked soybean, together with 2-methoxyphenol were found in this work and thought to be the thermal degradation products of lignin-related phenolic carboxylic acids.[Citation8,Citation10]
Hydrocarbons
Five hydrocarbons identified in douchi extracts were only found in brand C. Zingiberene is a principal flavour compound of ginger, which is one of raw materials of brands C. It is suggested that zingiberene is to be an important flavour-contributing compound in brand C.
Other Compounds
2-Pentylfuran found in brands A and C is also detected in other soybean processed products. 2-Pentylfuran, the major component of natto,[Citation17] has the characteristic green bean note.[Citation8] Sulfur-containing compounds had a significant contribution to the aroma in foods.[Citation8,Citation29] Five kinds of sulfur-containing compounds were identified in douchi extracts. Dimethyl disulphide was found in brands A and B, dimethyl trisulphide and 3-methylthiopropanal were detected in brands A and C. Dimethyl disulphide was previously reported in miso[Citation11] and soy sauce,[Citation14] and it is one of the major flavour components of natto.[Citation17] 3-Methylthiopropanal, found in douchi extracts, was also found in sufu and reported to have a meaty and soy sauce-like flavour.[Citation10]
Four pyridines were identified in three brands, but only 3-phenylpyridine and pyridine were previously reported in other soybean products such as sufu,[Citation8,Citation10] miso[Citation11] and natto.[Citation15] Pyridines could be produced by the Maillard reaction.[Citation29] Two pyrimidines were identified in three brands, but they were not previously reported in other soybean products.
Among the miscellaneous compounds, only pyrrole and indole found in douchi extracts were previously found in other soybean processed products, such as sufu and miso.[Citation8,Citation10,Citation13] Pyrrole can be the products of Maillard reaction, which has a nutty, sweet and ethereal odour.[Citation10] Indole has an unpleasant, cadaverous, fecal and putrid note when in high concentration, but floral note when in low concentration.[Citation24] Indole could have an undesirable effect on the flavour of the douchies.
In summary, the presence of the volatile components in douchi extracts was partly due to the fungal actions on the soybean after their inoculation. Proteases and lipases produced by the fungi during the incubation periods generate large amounts of peptides, amino acids and fatty acids.[Citation5] This provides a pool of substrates for further biological and chemical reactions. During the post-fermentation of douchi, salt is added to douchi to provide the salty taste and also to control the microbial growth and enzyme activity. The ethanol impacts on the formation of volatile components, particularly esters in sufu.[Citation12] Present results also showed that brand B had high concentrations of esters, supposedly because of the addition of alcohol at post-fermentation.
From the results obtained, the concentrations of volatile components in different brands of douchi were different, such differences were likely due to some variations in the proprietary processing steps used by each manufacturer. Most of the pyrazines compounds identified were previously found in soybean processed products such as sufu, miso, soy sauce, and natto, this class of compounds together with other common components probably contribute more to the characteristic background flavour of all brands investigated. Less predominant groups including esters, acids and alcohols might serve an important role in the flavour of douchies.
The present investigation is concerned with the volatile components, particularly the common ones, without giving much consideration to their actual contribution to the aroma of the douchies. Subsequent experiments involving aroma extract dilution analysis (AEDA) and GC/olfactometry are necessary to screen out those important odorous components characterizing the common aroma in douchies.
CONCLUSIONS
One-hundred thirty-one combined volatile compounds were identified in three types of steamed commercial douchi samples. Thirteen classes of compounds were detected in these samples including hydrocarbons, alcohols, aldehydes, ketones, acids, esters, pyrazines, furans, sulfur-containing compounds, phenols, pyridines, pyrimidines, and miscellaneous compounds. Most of the identified compounds were previously reported in soybean processed products such as sufu, natto, soy sauce, and miso. The role of each of these compounds on the odour of douchi will require further investigation.
ACKNOWLEDGMENTS
This study was conducted within the framework of the collaborative research project between Japan and China titled “Development of sustainable production and utilization of major food resources in China” supported by Japan International Research Center for Agricultural Sciences (JIRCAS). This work was also supported by National Functional Food Project in the Eleventh Five-year Plan of the People's Republic of China (No.2006BAD27B09).
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