Article Cite This: J. Agric. Food Chem. 2018, 66, 689−694
pubs.acs.org/JAFC
Characterization of the Potent Odorants Contributing to the Characteristic Aroma of Beijing Douzhi by Gas Chromatography− Olfactometry, Quantitative Analysis, and Odor Activity Value Jia Huang, Yuping Liu,* Wenxi Yang, Yingqiao Liu, Yu Zhang, Mingquan Huang, and Baoguo Sun Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University; Beijing Key Laboratory of Flavor Chemistry; and Beijing Laboratory for Food Quality and Safety, Beijing 100048, China ABSTRACT: Beijing douzhi (BD) is a traditional snack in Beijing, China, and it has been listed as a part of Beijing’s intangible cultural heritage. The potent odorants that contribute to the characteristic aroma of BD were investigated by analyzing the isolates from solvent-assisted flavor evaporation (SAFE) and simultaneous distillation−extraction. Using aroma extract dilution analysis based on gas chromatography−mass spectrometry and gas chromatography-olfactometry, 31 aroma-active compounds with flavor dilution (FD) factors ranging from 1 to 2187 were identified by comparison of their odor characteristics, MS data, and retention indices with those of reference compounds. To further determine their contribution to the aroma of BD, the odorants isolated using SAFE with FD factors ≥9 were quantified, and their odor activity values (OAVs; ratio of concentration to the respective odor threshold in water) were calculated. Eleven compounds were found to have OAVs ≥ 1, which indicated they were the potent odorants that contributed substantially to the characteristic aroma of BD. Among the 11 odorants, (E,Z)-2,6nonadienal, eugenol, methional, p-cresol, 1-octen-3-one, and 3-methylbutanoic acid were not previously identified in BD. KEYWORDS: Beijing douzhi, aroma-active compounds, GC-O, AEDA, quantitative analysis, OAV
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INTRODUCTION Beijing douzhi (BD, also called douzhi) is a traditional snack in Beijing1 and in 2007, it was listed as a part of Beijing’s intangible cultural heritage. It has a light gray color; a strong sulfurous, sour, cheese odor with a slight green, vegetable, cooked potato nuance; and a sour taste, but it is still enjoyed by a portion of the population of Beijing because of its nutritional value and health benefits. BD has some similarities with soybean milk; for example, both are liquid foods made from bean materials. However, there are some differences between them as well; for example, the raw materials used and the processing methods are different. Soybean milk is made from soybeans, while BD is made from mung beans. Soy milk is a drink produced by soaking soybeans, grinding them in water, boiling the mixture, and filtering out the residues. The preparation of BD is more complicated. First, high-quality mung beans are chosen and steeped in 25 °C water for 12 h; then, the soaked beans are rinsed with water, ground, and filtered to generate mung bean milk (MBM). MBM is then mixed with the sour slurry from the sedimentation step of the previous batch of BD. The sour slurry is added to provide the MBM with microorganisms and inoculate MBM to begin the fermentation. The fermented MBM is statically set for a specific length of time to separate the crude starch and the sour slurry. The sour slurry is further sedimented to produce sour douzhi, which is then cooked gently to obtain BD.2 Mung bean contains many ingredients of nutritional value, such as high levels of proteins, amino acids, dietary fibers, oligosaccharides, and polyphenols.3 When mung bean is processed into douzhi after fermentation, some of the beneficial components become easier to digest. BD is served as a snack in many restaurants in Beijing. © 2017 American Chemical Society
At present, the studies on BD have focused on the processing techniques,1,4 microbial and biochemical changes,2 dominant bacteria groups,5 and volatile organic components.6−8 Odor is a very important characteristic of foodstuffs. However, there are very few reports on the volatile compounds in BD. Miao et al.6 first reported the volatiles in BD. A total of 38 compounds were identified by gas chromatography−mass spectrometry (GCMS), including 13 alcohols, 4 aldehydes, 2 ketones, 8 organic acids, 3 esters, 2 phenols, 5 sulfur-containing compounds, and 1 nitrile. On the basis of only the odor characteristics of the identified constituents, the authors believed that the organic acids and sulfur-containing compounds were the constituents that gave BD its characteristic aroma. Lu et al.7,8 also isolated and analyzed the volatile constituents in BD, and their experimental results were almost the same as Miao’s. In these reports, headspace solid-phase microextraction (SPME) was the only pretreatment method used, and the potent odorants that contribute to the characteristic aroma of BD were not determined. Therefore, the objective of the present study is (i) to isolate the volatiles in BD by different pretreatment methods, (ii) to identify the aroma-active compounds in BD by gas chromatography−olfactometry (GC-O), (iii) to quantify those odorants, (iv) to calculate their OAVs (OAV indicates the ratio of the concentration of an odorant to its odor threshold9), and (iv) determine the potent odorants contributing to the characteristic aroma of BD. Received: Revised: Accepted: Published: 689
October 18, 2017 December 18, 2017 December 20, 2017 December 20, 2017 DOI: 10.1021/acs.jafc.7b04839 J. Agric. Food Chem. 2018, 66, 689−694
Article
Journal of Agricultural and Food Chemistry
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currents in the 33−350 mass range. The experiments were performed in triplicate. C7−C40 n-alkanes were evaluated under the same chromatographic conditions to allow the RIs of the detected compounds to be calculated. Gas Chromatography−Olfactometry−FID. GC-O/FID was carried out using an Agilent 7890 GC equipped with a FID (Agilent) and an olfactory detection port (ODP series 3, Gerstel). The effluent was split between the FID and ODP in a 1:2 ratio by volume at the end of the capillary by a T-type splitter and uncoated deactivated fused silica capillaries. The portion sent to the FID was held at 280 °C, and that sent to the ODP was held at 200 °C. The samples (1 μL) were analyzed on a DB-WAX column (Agilent, dimensions of 30 m × 0.25 mm, 0.25 μm film). The conditions for GC analysis were the same as those used for GC-MS analysis. The effluent sent to the ODP was enclosed within a stream of humidified air. During GC-O analysis, four trained panelists (one male and three females) from Beijing Key Laboratory of Flavor Chemistry at Beijing Technology & Business University evaluated the odor of the effluent from the sniffing port. As an odor was detected, the retention time and the odor characteristic were recorded. Analyses were conducted three times by each panelist. Aroma Extract Dilution Analysis (AEDA). For AEDA, the extract of the volatile compounds was diluted stepwise with dichloromethane to obtain serial dilutions of 1:3, 1:9, 1:27, 1:81, ..., and 1:2187. Aliquots (1 μL) of each sample were analyzed by GC−O using a DB-WAX column under the GC conditions described above until no odorant could be detected. The flavor dilution (FD) factor of each odorant represents the maximum dilution in which the aroma compound could be perceived.9 Each odorant was identified by comparing its odors, retention index (RI), and mass spectra with those of authentic compounds. Quantitation of Aroma Compounds. Odorants That Gave Peaks in the GC-MS Chromatogram Were Quantified by GC-MS. BD (100 mL) was spiked with 2-octanol as an internal standard to a final concentration of approximately 152 μg/L, and then 50 mL of dichloromethane was added to the solution. The mixture was shaken in a shaker for 1 h at room temperature, and the organic phase was then separated and collected. The extraction procedure was repeated two more times. The combined extracts were subjected to SAFE distillation to eliminate nonvolatiles, and then the distillate obtained was dried over anhydrous sodium sulfate overnight and then filtered. The distillate was concentrated using a Vigreux column (50 cm × 1 cm) to approximately 5 mL at 45 °C and then was further concentrated to 0.5 mL using a gentle nitrogen stream. The GC-MS conditions were set as above, and the analysis was carried out on a DBWAX column. Selective ion monitoring (SIM) mass spectrometry was used to quantify the aroma-active compounds. The solutions of the mixture of internal standard and reference compounds at different concentrations were prepared and analyzed by GC-MS. The standard curves were prepared by plotting the ratio of the peak areas of the reference compound relative to 2-octanol against their concentration ratio. Concentrations of Odorants That Did Not Give Peaks in the GCMS and GC-O/FID Chromatograms Were Estimated by GC-O. Though large volumes of the samples were used, the concentrations of some of the aroma-active compounds in the isolates were so low that there was no signal response on GC-MS and GC-O/FID. Attempts were made to estimate their concentrations by GC-O. First, their limits of detection (LODs) were determined by analyzing the corresponding reference compounds by GC-O/FID and taking the value when the signal-to-noise ratio of the GC chromatogram was 3.12 Next, solutions of the odorants at concentrations equal to their LODs were prepared; the solutions were analyzed by GC-O, and the FD factors of the aroma-active compounds in the solutions were determined. Finally, the concentrations of the aroma-active compounds in the isolate and in BD were tentatively estimated from the FD factor from the compound in the solution as well as in the isolate from BD.
MATERIALS AND METHODS
Materials. BD (with a water content of 95%) was purchased from the Beijing Ciqikou douzhi restaurant, which is one of the more famous douzhi restaurants in Beijing. Before extraction, the sample was kept at 4 °C in a cooler. Chemicals. Acetic acid (99.7%), butanoic acid (99%), 2,3butanedione (98%), 1-butanol (99.5%), dimethyl trisulfide (98%), 4ethyl-2-methoxyphenol (98%), eugenol (98%), 2-furylmethanol (98%), 3-methylbutanoic acid (99%), 3-methyl-1-butanol (99%), methional (95%), phenylethyl alcohol (99%), propionic acid (99%), vanillin (99%), and 2-octanol (99%, internal standard) were purchased from J&K Chemical Ltd. (Beijing, China). Hexanoic acid (98%), 2methylpropionic acid (99%), and pentanoic acid (99%) were purchased from Amethyst Chemicals (Beijing, China). Dimethyl disulfide (98%), 4-ethylphenol (97%), (E,Z)-2,6-nonadienal (95%), and hexanal (95%) were supplied by TCI (Shanghai, China). Benzeneacetaldehyde (95%), 1-octen-3-one (95%), and 2-pentylfuran (98%) were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). (E,E)-2,4-Decadienal (90%) and 4-hydroxy-2,5-dimethyl3(2H)-furanone (98%) were purchased from Aladdin Reagents Co., Ltd. (Shanghai, China). 1-Hexanol (99%) and (Z)-3-hexenol (98%) were obtained by Beijing Peking University Zoteq Co., Ltd. (Beijing, China). Methanethiol (2000 μg/mL in toluene) was supplied by AccuStandard (New Haven, CT). Benzeneacetic acid (95%) was supplied by Key Organics (Cornwall, England). p-Cresol (99%), dichloromethane, and anhydrous sodium sulfate were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Dichloromethane was freshly distilled before experiments. C7−C40 normal alkanes (solvent: hexane) for calculating the retention indices (RIs) were purchased from O2si Smart Solutions (Charleston, SC). Isolation of the Volatiles by Direct Solvent Extraction and Solvent-Assisted Flavor Evaporation (DSE-SAFE). BD (100 mL) was extracted with dichloromethane (50 mL × 3) by stirring vigorously for 1 h × 3 at room temperature, and the resulting extracts were mixed together. To separate the volatile fraction from the nonvolatiles, the combined extracts were subjected to high-vacuum distillation using SAFE (Edwards TIC Pumping Station from BOC Edwards, England).10 The obtained distillate was dried over anhydrous sodium sulfate overnight and then filtered. The obtained distillate was concentrated using a Vigreux column (50 cm × 1 cm) (Beijing Jingxing Glassware Co., Ltd., China) to approximately 5 mL at 45 °C, and then it was further concentrated to 0.3 mL using a gentle nitrogen stream. This concentrate was used for GC-MS and GC-O analyses. Isolation of the Volatiles by Simultaneous Distillation− Extraction (SDE). BD (100 mL) and a stir bar were placed in a 250 mL round-bottom flask which was immediately attached to the SDE apparatus (Beijing Jingxing Glassware Co., Ltd., China). Dichloromethane (50 mL) was added to a 100 mL round-bottomed flask attached to the other side of the SDE apparatus. The BD was heated in oil bath at 110 ± 1 °C for 3 h, and the solvent was heated in water bath at 45 ± 1 °C.11 After extraction, the extracts were dried over a small amount of anhydrous sodium sulfate and filtered. The extracts were concentrated to approximately 5 mL using a Vigreux column (50 cm × 1 cm) at 45 °C and then further concentrated to 0.3 mL using a gentle nitrogen stream. This concentrate was also analyzed by GC-MS and GC-O. Gas Chromatography−Mass Spectrometry. An Agilent 7890B GC was coupled by a heated transfer line (230 °C) to a 5977a mass spectrometer (Agilent Technologies, United States). The DSE-SAFE and SDE isolates were analyzed on a DB-WAX capillary column (30 m × 250 μm i.d. × 0.25 μm, Agilent Technologies) and HP-5 column (30 m × 250 μm i.d. × 0.25 μm, Agilent Technologies), respectively. The carrier gas was helium at a constant flow rate of 1 mL/min to the column. The initial oven temperature was 40 °C, which was held for 2 min, increased to 80 °C at 8 °C/min, increased to 100 °C at 4 °C/ min, increased to 230 °C at 6 °C/min, and finally held for 5 min. The injection port was in splitless mode. The mass detector was operated at 150 °C in electron impact mode at 70 eV. The ion source temperature was 230 °C. MS data were acquired on a 3 min solvent delay. The chromatograms were recorded by monitoring the total ion 690
DOI: 10.1021/acs.jafc.7b04839 J. Agric. Food Chem. 2018, 66, 689−694
Article
Journal of Agricultural and Food Chemistry Table 1. Aroma Compounds Identified by Gas Chromatography-Olfactometry in Beijing Douzhi FD factorb
RI a
no.
aroma compd.
odor quality
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
methanethiol 2,3-butanedione dimethyl disulfide hexanal 1-butanol 3-methyl-1-butanol 2-pentylfuran 1-octen-3-one 1-hexanol (Z)-3-hexenol dimethyl trisulfide acetic acid methional propionic acid 2-methylpropionic acid (E,Z)-2,6-nonadienal butanoic acid benzeneacetaldehyde 2- and 3-methylbutanoic acid 2-furylmethanol pentanoic acid (E,E)-2,4-decadienal hexanoic acid phenylethyl alcohol 4-ethyl-2-methoxyphenol 4-hydroxy-2,5-dimethyl-3(2H)-furanone p-cresol eugenol 4-ethylphenol vanillin benzeneacetic acid
garlic buttery, sweet sulfurous, cabbage green malty, solvent-like malty green mushroom green green sulfury sour, vinegar cooked potato sour sour cucumber sour floral cheese-like bready, coffee sour oily, fatty sour floral clove caramel phenolic sweet, clove phenolic, smoky vanilla-like honey, sweaty
DB-WAX
HP-5
SAFE
SDE
714 977 1075 1081 1137 1203 1227 1296 1346 1377 1378 1431 1447 1508 1516 1582 1610 1636 1658 1666 1724 1805 1834 1907 2022 2024 2071 2161 2166 2559 2566
