洋河大曲”白酒的香气物质

Flavour Fragr. J . 2006; 21: 333–342

Published online 12 May 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ffj.1621

Identification of aroma compounds in Chinese ‘Yanghe Daqu’ liquor by normal phase chromatography fractionation followed by gas chromatography/

olfactometry

Wenlai Fan and Michael C. Qian*

Department of Food Science and Technology Oregon State University, Corvallis, Oregon 97331, USA Received 15 December 2004; Revised 23 March 2005; Accepted 24 March 2005

ABSTR ACT:Aroma compounds in Chinese ‘Yanghe Daqu’ liquor were investigated with normal phase chromato-graphy fractionation followed by gas chromatography–olfactometry (GC–O). Yanghe Daqu liquor was extracted with

Freon 11, and then separated into acidic and neutral/basic fractions. The neutral/basic fraction was further separated into four fractions, using silica gel normal phase chromatography. Aroma compounds in each fraction were analysed by GC–O and GC–MS. The results showed that Yanghe Daqu liquor aroma was mainly contributed by esters and fatty acids.Among them, ethyl hexanoate (Osme value =15), ethyl butanoate (Osme value ≥14) were probably the most important esters, based on their Osme values. In addition, ethyl octanoate, 3-methylbutyl hexanoate and ethyl pentanoate could be important (Osme values ≥10). Acids with high Osme value included hexanoic, butanoic, 3-methylbutanoic and pentanoic acids. They contributed to sour, acid, cheesy and sweaty aromas. 1-Hexanol and 3-methylbutanol also had high Osme values. In addition, 4-ethylguaiacol, 4-methylguaiacol and 4-ethylphenol were identified in Yanghe Daqu liquor and they contributed to clove, sweet, smoky and goaty aromas. 1,1-Diethoxy ethane, 1,1-diethoxy-2-methylpropane and 1,1-diethoxy-3-methylbutane were identified and they gave fruity and floral aromas. Copyright © 2005 John Wiley & Sons, Ltd.KEY WORDS:

aroma; Chinese liquor; Yanghe Daqu liquor; GC–O; Osme; distillate

* Correspondence to: M. Qian, Department of Food Science and Technology,100 Wiegand Hall, Oregon State University, Corvallis, OR 97331-6602,USA.

E-mail: Michael.qian@oregonstate.edu

Introduction

Chinese liquor is one of the oldest distillates in the world.Compared with other spirits, such as vodka, whisky and brandy, Chinese liquor has a higher ethanol content (nor-mally 40–55% by volume). The annual production of Chinese liquor is approximately 4 millions metric tons.1

Chinese liquor is typically fermented from grains with ‘Daqu’ in the solid state or ‘Xiaoqu’ in the semi-solid state.2 Daqu and Xiaoqu are crude enzymes used for Chinese liquor fermentation. Daqu is typically made from wheat or mixture of wheat, barley and pea. The raw materials are milled, mixed with water, and pressed into a brick-shaped block, which is then incubated under controlled conditions for several months before being used to make Chinese liquor.

The grain used for Chinese liquor manufacturing is sorghum or a mixture of sorghum, wheat, rice, sticky rice and corn. The grain is ground and cooked, mixed with Daqu powder and then fermented for several months.After fermentation, the liquor is distilled out with steam.The distillate is aged for several years to develop the bouquet aroma.

Chinese liquor can be classified into five categories according to their aroma characteristics: strong aroma type, light aroma type, soy sauce aroma type, sweet honey type and miscellaneous type. Of these, the strong aroma type liquor accounts for about 70% of total liquor production in China. It has strong fruity, pineapple- and banana-like aromas.3

The volatile composition of Chinese liquors has been studied. Sixty-eight volatile components, including alcohols, esters, acids, acetals, ketones, aldehydes and heterocyclic compounds, have been identified in Chinese liquors.4,5 Very few studies report on aroma compounds in Chinese liquor. I t has been suspected that ethyl hexanoate could be very important to the aroma of Chinese strong aroma-type liquor, based on the cal-culated odour activity value.6

‘Yanghe Daqu’ liquor is one of the most famous Chinese strong aroma-type liquors. This liquor was first manufactured in the Tang dynasty and is now pro-duced at Yanghe Distillery Stock Co. Ltd. of Jiangsu province in China. It is made from a mixture of sorghum,wheat, corn, rice and sticky rice. Up to now, no studies have reported on the contribution of aroma compounds to the characteristic bouquet of this liquor. The objec-tive of this study was to identify the aroma compounds in Yanghe Daqu liquor by gas chromatography–olfactometry (GC–O).

334W. FAN AND M. C. QIAN

Materials and Methods

Chemicals

Methyl hexanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, propanoic acid, heptanoic acid, octanoic acid and 1-octanol were purchased from Eastman (Rochester,NY, USA). Propyl hexanoate, pentyl hexanoate, 2-methylpropyl hexanoate, hexyl hexanoate and ethyl 2-methylpropanoate were obtained from K & K Labora-tories (Plainview, NY, USA). 2-Pentanol, 2-octanol, 1-heptanol and ethyl 2-hydroxypropanoate were obtained

from Matheson Coleman & Bell (East Rutherford, NJ,USA). Dimethyl sulphide and benzothiazole were supplied by TCI America (Portland, OR, USA). Ethyl benzoate was from EKC nc. (Rosemont, L, USA).Phenol was purchased from EMD Chemical I

nc.(Gibbstown, NJ, USA). Acetic acid, 2-methylpropanoic acid, butanoic acid, 3-methylbutanoic acid, pentanoic acid, 4-methylpentanoic acid, hexanoic acid, 2-methylpropanol, 1-butanol, 2-butanol, 3-methylbutanol,1-pentanol, 1-hexanol, 2-heptanol, 2-ethyl-1-hexanol, 1-nonanol, benzene ethanol, benzene acetaldehyde, furfural (2-furancarboxaldehyde), 2-pentanone, ethyl acetate, ethyl

butanoate, ethyl pentanoate, ethyl hexanoate, ethyl heptanoate, ethyl dodecanoate, ethyl 3-methylbutanoate,ethyl benzeneacetate, ethyl 3-phenylpropanoate, 3-methylbutyl hexanoate, butyl hexanoate, hexyl octanoate,hexyl acetate, diethyl butanedioate, 2-phenylethyl acetate,3-methylbutyl acetate, 1,1-diethoxyethane, guaiacol (2-methoxyphenol) and 4-ethylguaiacol (4-ethyl-2-methoxyphenol ) were from Sigma-Aldrich Co. (St.Louis, MO, USA).

Freon 11 (fluorotrichloromethane) of 99%+ purity was purchased from Aldrich Chemical Co. I

nc.(Milwaukee, WI, USA). Pentane and methanol were from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA). Diethyl ether was obtained from Burdick & Jackson (Muskegon,MI , USA). Anhydrous sodium sulphate was from EMD Chemicals Inc. (Gibbstown, NJ, USA). Sodium chloride,sodium bicarbonate and sulphuric acid were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Silica Gel 60, particle size 0.2–0.5mm (35–70mesh), was from EMD Chemical Inc. (Gibbstown, NJ, USA).Synthesis of 3-methylbutyl octanoate, heptyl hexanoate, phenylethyl hexanoate and pentyl 2-hydroxypropanoate

3-Methylbutyl octanoate was synthesized by mixing 600µl octanoic acid with 2ml 3-methylbutanol. Heptyl hexanoate and phenylethyl hexanoate were synthesized by mixing 2ml 1-heptanol or benzene ethanol with 600µl hexanoic acid. Pentyl 2-hydroxypropanoate was synthesized by mixing 2ml 1-pentanol with 600µl 2-hydroxypropanoic acid. The reactions were catalysed by

acid (1N H 2SO 4, 500µl) at 100°C for 1h. After cooling,the reaction mixture was mixed with 5ml saturated NaCl solution and the esters were extracted with 5ml Freon 11in a separating funnel; 1µl extract was injected into the GC–MS apparatus (split ratio, 100:1) for identification.Synthesis of acetals

Acetals were synthesized by reacting aldehydes and alcohols in acidic conditions.7 600µl 2-methylpropanal and 300µl 3-methylbutanal were mixed with 20ml ethanol, respectively; 2ml H 2SO 4 (1N ) was added. The mixture was stirred at 58°C for 1h. After cooling, the reaction mix was added with 50ml saturated NaCl solu-tion and the product was extracted with 10ml Freon 11in a separating funnel. 1µl of acetal solution was injected to GC–MS (split ratio, 100:1) for identification.

Yanghe Daqu Liquor Sample Preparation Daqu preparation

Daqu was prepared in the manufacturing facility of Yanghe Distillery Stock Co. Ltd, Jiangsu Province,China. Wheat (500kg), barley (400kg) and peas (100kg)were milled first and then mixed well. Water (35–40°C)was added to reach the moisture content of 38–40%(w/w). The paste was then pressed into a mould to form bricks 30cm long, 18.5cm wide and 6cm high. The bricks were put into a room for fermentation under controlled temperature, moisture, carbon dioxide and oxygen levels. At the beginning of fermentation, the temperature of the Daqu was kept at 25–30°C. The tem-perature rose slowly as fermentation progressed during the first 8 days because of microbial growth. When it reached 58°C, the temperature of the Daqu was held in the range 55–58°C for 10 days by ventilating the room and/or spraying cooling water in the fermentation room. The Daqu was then cooled slowly to 22–24°C in 12 days and the moisture content was reduced to 16–18% (w/w). The Daqu bricks were then transferred to another room where they were stored for 3 months before being used.

Yanghe Daqu liquor manufacturing

The liquors were made at the facility of Yanghe Dis-tillery Stock Co. Ltd., Jiangsu Province, China. Sorghum,wheat, rice, sticky rice and corn (total amount of 800kg)were milled, mixed with rice husk (140kg) and cooked through a proprietary process. Water was added to the cooked grains to adjust the moisture content to 55%(w/w). The cooked grains were cooled to 13–16°C and mixed with Daqu powder (200kg). The mixture was fermented in a special fermentor (3.4m length, 1.8m

with a layer of fermentation mud made of clay, spent

grain, bean cake powder and fermentation bacteria

(Clostridium sp.). The fermentation was carried out for

60 days at 28–32°C under anaerobic conditions. After

the fermentation, the liquor was distilled with steam. The

fore-run and the after-run part of the liquor (tails) were

discarded, and the middle-run (heart) was saved as fresh

distillate.

The fresh distillate was aged in a china jar (1000l,

sealed) for more than 3 years at 15–25°C. In this study,

one sample was made in 2001 and aged for 3 years,

while another sample was made in 1999 and aged for

5 years.

Yanghe Daqu Liquor Aroma Extraction and

Fractionation

Aroma extraction

Yanghe Daqu liquor sample (100ml, 70% ethanol by

volume) was diluted with 400ml boiled, deionized water

(boiled 5min, cooled to 10°C) according to the pro-

cedures described previously.8,9The diluted sample was

added with 80g analytical-grade sodium chloride, and

then extracted three times with 70ml aliquots of Freon

11 in a separatory funnel. All extracts were combined

and labelled as ‘extract 1’.

Acidic and neutral/basic fractionation

Boiled, deionized water (200ml, boiled 5min, cooled to

10°C) and analytical-grade sodium chloride (60g) were

added to ‘extract 1’ and mixed well. The aqueous phase

was adjusted to pH 9.0 with sodium bicarbonate solution

(10%), then separated in a separating funnel and saved.

The organic phase was labelled as ‘extract 2’.

The aqueous phase was adjusted to pH 2 with 2N

H

2SO

4

, and then extracted three times with 200ml

aliquots of freshly redistilled diethyl ether. The diethyl ether extracts were combined and dried with 10g anhy-drous sodium sulphate overnight. The extract was filtered. The filtrate was slowly concentrated to 2ml and then to 200µl with a stream of nitrogen. This concentrate was labelled as the ‘acidic fraction’ for further GC–O analysis.

‘Extract 2’ was dried over anhydrous sodium sul-phate, and then concentrated to 3ml with a stream of nitrogen. This fraction was labelled as the ‘neutral/basic fraction’.

Normal-phase liquid chromatography

A glass column (30cm × 1.5cm i.d.) packed with 5g of silica gel was washed with 100ml methanol, then 100ml diethyl ether, and then conditioned with 100ml pentane. The ‘neutral/basic fraction’ (3ml) was applied to the column. 50ml pentane:diethyl ether (98:2, fraction A), pentane:diethyl ether (95:5, fraction B), pentane:diethyl ether (90:10, fraction C) and diethyl ether (fraction D) were sequentially applied to elute the aroma compounds from the column at a flow rate of 3ml/min. All elutes were slowly concentrated to 10ml and then to 200µl with a stream of nitrogen for GC–O and GC–MS analysis.

GC–O Analysis

GC–O analysis was performed on a Hewlett-Packard 50 gas chromatograph equipped with a flame ioniza-tion detector (FI D) and an olfactometer. The column carrier gas was nitrogen at constant pressure (15 p.s.i., 2ml/min column flow, measured at 25°C). Half of the column flow was directed to the FID, while the other half was directed to the olfactometer. Samples were analysed on a DB-Wax column (30m ×0.32mm i.d., 0.25µm film thickness; J&W Scientific, Folsom, CA, USA) and a DB-5 column (30m × 0.32mm i.d., 1µm film thickness; J&W Scientific). A 1µl sample was injected into the GC. The split ratio was 1:1. The oven temperature was held at 40°C for 2min, then raised to 230°C at a rate of 6°C/min, and held at 230°C for 15min; injector and detector temperatures, 250°C.

Two panelists (one female and one male) were selected for the GC–O study. One panelist had more than 5 years of sensory analysis experience in Chinese liquor. Another was selected from students in the Department of Food Science and Technology at Oregon State University. Yanghe Daqu liquor fractions were served as training samples. The panelists were trained for 30h over a period of 15 days. They responded to the aroma intensity of the stimulus by using a 16-point scale ranging from 0 to 15;‘0’ was none, ‘7’ was moderate, while ‘15’ was extreme. The retention time, intensity value and aroma descriptor were recorded. Each fraction was replicated three times by each panelist. The Osme values for aroma intensity were averaged for the six analyses (two panelists, three times). When a panelist could not detect a aroma compound, the intensity was considered as zero in the averaging process.10,11

Retention Indices (RI)

Retention indices (RI) were calculated in accordance with a modified Kováts method.12 A standard mixture of paraffin homologues C5–C25 was prepared. The sample and the hydrocarbon standard mixture were co-injected into the GC and the retention times were used to calcu-late retention indices.336W. FAN AND M. C. QIAN

GC–MS Analysis

Capillary GC–MS was carried out using an Agilent GC 60-5973MSD. The sample was analysed on a DB-Wax column (30m ×0.25mm i.d., 0.50µm film thickness) and a DB-5 column (30m × 0.32mm i.d., 0.25µm film thickness). The oven and injector temperatures were iden-tical to those of GC–O analysis, described above. The column carrier gas was helium at a constant flow rate of 2ml/min. An Agilent 5973 Mass Selective Detector (MSD) was used for identification. The electron impact (EI) energy was 70eV, and the ion source temperature was set at 230°C. Mass spectra of unknown compounds were compared with those in the Wiley 275.L Database (Agilent Technologies I nc.). Positive identification was achieved by comparing the mass spectra, aromas and retention indices with those of the standards. Tentative identification was achieved by comparing aroma or mass spectrum only.

Due to high concentrations of ethyl butanoate, ethyl pentanoate and ethyl hexanoate in the pentane:diethyl ether (98:2) fraction, some peaks were obscured by these esters on both polar and non-polar columns. To facilitate GC–MS identification, this fraction was further analysed with a 20:1 split ratio. By reducing the sample load onto the column, ethyl 2-methylpropanoate, ethyl 3-methylbutanoate, 3-methylbutyl acetate, methyl hexanoate, hexyl acetate, 1,1-diethoxy-2-methylpropane and 1,1-diethoxy-3-methylbutane were chromatograph-ically separated from ethyl butanoate, ethyl pentanoate and ethyl hexanoate.

Results and Discussion

Although most of Chinese liquor consumed today has 40–55% alcohol content (by volume), traditional Yanghe Daqu liquor (70% ethanol by volume) was analysed in this study. To minimize the extraction of alcohols by Freon 11, the sample was diluted to 14% ethanol (by volume) with distilled and freshly boiled water according to the methods described by Ferreira et al.8and Ebeler et al.9

Fractionation

The aroma extract was first fractionated into acidic and neutral/basic fractions. The acidic fraction had a relatively simple composition, so it was analysed directly on a polar (DB-Wax) column. While the neutral/basic fraction was complex, it was difficult to resolve all of the aroma com-pounds and perform GC–O analysis reliably. To facilitate the GC and Osme analysis, the neutral/basic fraction was further separated into fraction A (pentane:diethyl ether, 98:2), fraction B (pentane:diethyl ether, 95:5), fraction C (pentane:diethyl ether, 90:10) and fraction D (diethyl ether) by silica gel chromatography, as described by Qian and Reineccius.13 These fractions were analysed on both polar (DB-Wax) and non-polar (DB-5) columns. Normal phase chromatographic fractionation separated volatile compounds based on their polarities. Although the groups were reasonably well separated, some com-pounds had very low sensory thresholds, and they could be detected by GC–O in another fraction although they were present at trace amount. In this study, most of the esters were in fraction A. Some esters, however, could also be detected by GC–O in fraction B, due to their low sensory thresholds. Hydroxyl esters were detected in frac-tions B and C due to the addition of the polar OH group on the molecule. Acetals were also detected in fraction A. Aldehydes and ketones were in fraction B. Phenols were separated to fractions B and C, while most of the alcohols were distributed in fractions C and D.

Acids

Acids seem to play important roles in Yanghe Daqu liquor aroma. Based on the Osme value, hexanoic and butanoic acids were probably the most important free fatty acids. The Osme values for these two acids were more than 12 in both liquors analysed on the polar column (Table 1). Both hexanoic and butanoic acids contributed to sweaty and rancid odours. I n addition, 3-methylbutanoic and pentanoic acids could be very im-portant because they all had relatively high Osme values (Osme value ≥10 in both samples). 3-Methylbutanoic and pentanoic acids were rancid, cheesy and sweaty. Acetic, 2-methylpropanoic and octanoic acids had Osme values of 7–10 in both samples. Acetic acid gave vinegar and acid odours. 2-Methylpropanoic and octanoic acids con-tributed sweaty, rancid and cheesy aromas. Heptanoic, propanoic and 4-methylpentanoic acids had lower Osme values in the 2001 liquor (Osme value = 6) but higher in the 1999 liquor (Osme values = 7–9). 4-Methylpentanoic acid was detected for the first time in Yanghe Daqu liquor; it had been reported previously in strawberry.14 I t was demonstrated that free fatty acids in Yanghe Daqu liquor were mainly formed during the fermentation process of grains by bacteria.3,6,15,16 Since the inside of the fermentor was coated with a layer of fermentation mud rich in butanoic and hexanoic acids bacteria, most of the free fatty acids were produced by these bacteria during the fermentation. Many other microorganisms could be introduced from raw materials and environment because the fermentation was performed in an open system.17t was postulated that acetic and propanoic acids were produced by acetic acid bacteria and propanoic acid bacteria during the fermentation. Yeasts can also produce butanoic, hexanoic, decanoic, octanoic and 3-methylbutanoic acids through amino acid degradation, fatty acid biosynthesis and oxidation of alcohols.12,18AROMA COMPOUNDS IN CHINESE YANGHE DAQU LIQUOR337 Table 1.Important aroma compounds of Yanghe Daqu liquor in acid fraction by Osme on DB-Wax column

RI RI Compounds Descriptor Basis of Osme value [reference]identification a

20011999

14731467 [32]Acetic acid Acid, vinegar MS, aroma, RI811 15611525 [13]Propanoic acid Vinegar MS, aroma, RI67 15851568 [33]2-Methylpropanoic acid Acid, rancid MS, aroma, RI810 16361623 [33]Butanoic acid Rancid, cheesy MS, aroma, RI1513 16861655 [33]3-Methylbutanoic acid Rancid, cheesy MS, aroma, RI1210 17531720 [20]Pentanoic acid Sweaty, rancid MS, aroma, RI1011 18244-Methylpentanoic acid Sweat, sour MS, aroma, RI69 18661854 [33]Hexanoic acid Sweaty, cheesy MS, aroma, RI1515 19761900 [13]Heptanoic acid Unpleasant MS, aroma, RI69 20912060 [33]Octanoic acid Sweat, cheese MS, aroma, RI812 a MS, compounds identified by MS spectra; aroma, compounds identified by the aroma descriptors; RI, compounds identified by comparison with pure standard; RIL, compounds identified by comparison with retention indices from the literature.

Table 2.Important aroma compounds of Yanghe Daqu liquor in four fractions by Osme on DB-Wax column a

RI RI Compounds Descriptor Basis of Osme value [reference]identification b

20011999

A B C D A B C D

Esters

1885 [33]Ethyl acetate Pineapple MS, aroma, RI118107 962955 [33]Ethyl 2-methylpropanoate Fruity, sweet MS, aroma, RI

10381043 [33]Ethyl butanoate Pineapple MS, aroma, RI14815753 10661075 [33]Ethyl 3-methylbutanoate Apple MS, aroma, RI10468 11241128 [33]3-Methylbutyl acetate Banana, fruity MS, aroma, RI75

11331141 [33]Ethyl pentanoate Apple MS, aroma, RI137154 11911204 [34]Methyl hexanoate Floral, fruity MS, aroma, RI87

12311238 [33]Ethyl hexanoate Fruity, floral, sweet MS, aroma, RI151065151055 12701270 [20]Hexyl acetate Floral, fruity MS, aroma, RI8274 13191324 [35]Propyl hexanoate Pineapple, sweet MS, aroma, RI96

13421342 [35]Ethyl heptanoate Fruity MS, aroma, RI88

13491342 [7]Ethyl 2-hydroxypropanoate Fruity MS, aroma, RI444 13551357 [35]2-Methylpropyl hexanoate Sweet, apple MS, aroma, RI66

14091402 [36]Butyl hexanoate Fruity, pineapple MS, aroma, RI910

14251422 [7]Ethyl 2-hydroxy-3-methylbutanoate Fruity MS, aroma, RIL669 14341439 [33]Ethyl octanoate Fruity MS, aroma, RI139157 14601452 [7]3-Methylbutyl hexanoate Fruity, apple, green MS, aroma, RI1415

15091493 [36]Pentyl hexanoate Fruity MS, aroma, RI76

15331530 [7]Ethyl nonanoate Fruity, rose-like MS, aroma, RI36

15441544 [7]Ethyl 2-hydroxyhexanoate Floral, jasmine MS, aroma, RIL5988 16111600 [36]Hexyl hexanoate Apple, peach MS, aroma, RI8465 16341634 [33]Ethyl decanote Fruity, grape MS, aroma, RI86

16521654 [33]3-Methylbutyl octanoate Fruity, pineapple MS, aroma, RI86

16701662 [33]Ethyl benzoate Herbal, fruity MS, aroma, RI76

Esters

Thirty-four esters were identified in the neutral/basic frac-tions of both the 2001 and the 1999 liquors. Among these esters, ethyl esters and hexanoate esters dominated (Tables 2 and 3). Esters were mainly eluted out in frac-tion A and some in fraction B. Ethyl hexanoate had a fruity, floral and sweet aroma. The Osme value of this compound was the highest among the aroma compounds identified in Yanghe Daqu liquor, with a value of 15 for both samples, as analysed by the polar column, in frac-tion A and a high value in fraction B; this compound could be extremely important to Yanghe Daqu liquor aroma. Ethyl hexanoate had been reported to be the most abundant ester in Chinese strong aroma-type liquors, having a concentration as high as 1.5–3.0g/l.3,6,19 Since its threshold is a quite low,20 it could be very important to all strong aroma-type liquors. Ethyl butanoate could also be very important, and it had an Osme value of 15 for the 1999 sample and 14 for the 2001 sample in fraction A, as analysed on the DB-Wax column. I t contributed a pineapple odour. Ethyl pentanoate, ethyl octanoate and338W. FAN AND M. C. QIAN

Table 2.(Continued)

RI RI Compounds Descriptor Basis of Osme value [reference]identification b

20011999

A B C D A B C D

16771682 [35]Diethyl butanedioate Fruity, sweet MS, aroma, RI8757 1703Heptyl hexanoate Fruity MS, aroma, RI67

17821785 [35]Ethyl benzeneacetate Rosy, honey MS, aroma, RI8757 18031804 [7]Hexyl octanoate Green, fruity MS, aroma, RI87

18121851 [33]2-Phenylethyl acetate Rosy, honey MS, aroma, RI77 18361840 [7]Ethyl dodecanoate Leaf MS, aroma, RI77

18801878 [35]Ethyl 3-phenylpropanoate Floral, fruity MS, aroma, RI67 171901 [7]Ethyl 3-methylbutyl butanedioate Fruity, sweet, honey MS, aroma, RIL7

216521 [7]Phenylethyl hexanoate Fruity MS, aroma, RI6869

2315Ethyl 2-hydroxy-3-phenylpropanoate*Smoky MS, aroma6666 Alcohols

10221000 [13]2-Butanol Fruity MS, aroma, RI65 10901085 [37]2-Methylpropanol Wine, solvent MS, aroma, RI53 11181116 [37]2-Pentanol Fruity, alcoholic MS, aroma, RI7494 11431151 [20]1-Butanol Pungent, alcoholic MS, aroma, RI 12101219 [33]3-Methylbutanol Rancid, nail polish MS, aroma, RI128118 14581457 [38]1-Heptanol Green, fruity MS, aroma, RI95106 121252 [39]1-Pentanol Fruity, balsamic MS, aroma, RI75 13191333 [32]2-Heptanol Fruity MS, aroma, RI77 13621359 [33]1-Hexanol Floral, green MS, aroma, RI138105 14181416 [7]2-Octanol Fruity MS, aroma, RI7545 14951487 [37]2-Ethyl-1-hexanol Rosy, green MS, aroma, RI667 15581566 [40]1-Octanol Green MS, aroma, RI7585 16601658 [7]1-Nonanol Green MS, aroma, RI58 19361931 [33]Benzene ethanol Rosy, honey MS, aroma, RI5563

Phenols

18621882 [33]Guaiacol Spicy, clove, animal MS, aroma, RI6556 19561960 [7]4-Methylguaiacol Smoky MS, aroma, RIL7975 20112000 [20]o-Cresol Phenol MS, aroma, RIL554 20152004 [39]Phenol Phenol MS, aroma, RI6754 20302025 [33]4-Ethylguaiacol Clove, spicy MS, aroma, RI8787 20932077 [20]p-Cresol Phenol, animal MS, aroma, RIL8565 21882142 [33]4-Ethylphenol Smoky MS, aroma, RIL88103 24222422 [18]4-Vinylphenol Smoky MS, aroma, RIL5555 Ketones and aldehydes

970966 [39]2-Pentanone Fruity MS, aroma, RI32 14141388 [41]3-Octen-2-one Mushroom, earthy MS, aroma, RIL68 14661475 [33]Furfural Almond, sweet MS, aroma, RI87 1212 [20]Benzene acetaldehyde Floral, rosy MS, aroma, RI76

Acetals

1900 [37]1,1-Diethoxyethane Fruity MS, aroma, RI1110

9701,1-Diethoxy-2-methylpropane Fruity MS, aroma, RI67

10741062 [7]1,1-Diethoxy-3-methylbutane Fruity MS, aroma, RI109

Sulphur compounds

9435 [32]Dimethyl sulphide Cooked onion, sulphur MS, aroma, RI3657 19471958 [42]Benzothiazole Smoky, rubber MS, aroma, RI7485

Unknowns

1378Unknown Fruity, herbal n.i.57

1515Unknown Green n.i.75 1721Unknown Woody, fruity n.i.5576 1978Unknown Woody, burning n.i.57

1994Unknown Smoky, animal n.i.665

2126Unknown Phenol, burning n.i.87

2342Unknown Goaty, smoky n.i. 5 4

a A, pentane:diethyl ether, 98:2; B, pentane:diethyl ether, 95:5; C, pentane:diethyl ether, 90:10; D, diethyl ether fraction.

b MS, compounds identified by MS spectra; aroma, compounds identified by the aroma descriptors; RI, compounds identified by comparison to pure stand-ard; RIL, compounds identified by comparison with retention indices from the literature; n.i., not identified.

*Tentatively identified.

AROMA COMPOUNDS IN CHINESE YANGHE DAQU LIQUOR339 Table 3.Important aroma compounds of Yanghe Daqu liquor in four fractions by Osme on DB-5 column a

RI RI Compounds Descriptor Basis of Osme value [reference]identification b

20011999

A B C D A B C D

Esters

754770 [33]Ethyl 2-methylpropanoate Fruity, sweet MS, aroma, RI86107 800812 [33]Ethyl butanoate Sweet, fruity MS, aroma, RI1586615756 826803 [43]Ethyl 2-hydroxypropanoate Fruity MS, aroma, RI5958 8548 [33]Ethyl 3-methylbutanoate Apple MS, aroma, RI96119 878886 [33]3-Methylbutyl acetate Fruity MS, aroma, RI79

904908 [33]Ethyl pentanoate Fruity MS, aroma, RI139148 9231 [34]Methyl hexanoate Green, fruity MS, aroma, RI8777 975968 [28]Ethyl 2-hydroxy-3-methylbutanoate Fruity MS, aroma, RIL6566 10051004 [33]Ethyl hexanoate Ester, fruity MS, aroma, RI151284151055 10171014 [20]Hexyl acetate Floral, fruity MS, aroma, RI88124

1061Ethyl 2-hydroxyhexanoate*Floral, jasmine MS, aroma4778 1075Pentyl 2-hydroxypropanoate Floral, fruity MS, aroma, RI57 11001097 [41]Propyl hexanoate Fruity MS, aroma, RI79

11071100 [20]Ethyl heptanoate Fruity MS, aroma, RI79

11541137 [44]2-Methylpropyl hexanoate Fruity, woody MS, aroma, RI67

11761179 [33]Ethyl benzoate Floral MS, aroma, RI75

11881182 [28]Diethyl butanedioate Wine, fruity MS, aroma, RI6888 11941192 [41]Butyl hexanoate Fruity MS, aroma, RI713

12051201 [33]Ethyl octanoate Fruity MS, aroma, RI115135 12451248 [20]Ethyl benzeneacetate Rosy, honey MS, aroma, RI9866 12511238 [44]3-Methylbutyl hexanoate Fruity, apple, green MS, aroma, RI69

12571265 [33]2-Phenylethyl acetate Rosy, honey MS, aroma, RI67 12881285 [45]Pentyl hexanoate Fruity MS, aroma, RI78

12961224 [46]Ethyl nonanoate Fruity MS, aroma, RI77

13491354 [28]Ethyl 3-phenylpropanoate Fruity MS, aroma, RI77 13851384 [11]Hexyl hexanoate Apple, peach MS, aroma, RI107109 13951399 [33]Ethyl decanoate Green, fruity MS, aroma, RI87

14451449 [33]3-Methylbutyl octanoate Fruity, pineapple MS, aroma, RI77

1460Ethyl 2-hydroxy-3-phenylpropanoate*Goaty, smoky MS, aroma7555 1482Heptyl hexanoate Fruity MS, aroma, RI68

1563Ethyl 3-methylbutyl butanedioate*Fruity, sweet, honey MS, aroma57 15801577 [47]Hexyl octanoate Green, fruity MS, aroma, RI73

15931596 [28]Ethyl dodecanoate Leaf, fruity MS, aroma, RI610

13Phenylethyl hexanoate Floral, fruity MS, aroma, RI7567

Alcohols

745753 [33]3-Methylbutanol Rancid, fusty, nail polish MS, aroma, RI9796 759766 [48]1-Pentanol Fruity, balsamic MS, aroma, RI5 887888 [33]1-Hexanol Floral, green MS, aroma, RI106126 911906 [32]2-Heptanol Fruity MS, aroma, RI109 9973 [28]1-Heptanol Green, fruity MS, aroma, RI7484 1004984 [47]2-Octanol Fruity MS, aroma, RI53 10331032 [41]2-Ethyl-1-hexanol Rosy, green MS, aroma, RI94 10751072 [40]1-Octanol Fruity MS, aroma, RI5496 11591146 [33]Benzeneethanol Rosy, honey MS, aroma, RI4454 11731156 [47]1-Nonanol Green MS, aroma, RI46

Phenols

980980 [20]Phenol Phenol MS, aroma, RI76 11681074 [20]p-Cresol Medicine, phenol MS, aroma, RIL6566 11811188 [33]4-Ethylphenol Goaty, smoky MS, aroma, RI77127 11951191 [49]4-Methylguaiacol*Smoky, unpleasant MS, aroma, RIL5597 12811288 [33]4-Ethylguaiacol Clove, spicy MS, aroma, RIL7797 Ketones and aldehydes

829853 [33]Furfural Fruity MS, aroma, RI78 10401036 [50]3-Octen-2-one Mushroom, earthy MS, aroma, RIL76 10451047 [20]Benzeneacetaldehyde Fruity MS, aroma, RI67

Acetals

719726 [28]1,1-Diethoxyethane Fruity MS, aroma, RI911

8591,1-Diethoxy-2-methylpropane Fruity MS, aroma, RI98

9552 [28]1,1-Diethoxy-3-methylbutane Fruity MS, aroma, RI1013340W. FAN AND M. C. QIAN

Table 3.(Continued)

RI RI Compounds Descriptor Basis of Osme value [reference]identification b

20011999

A B C D A B C D

Sulphur compounds

12281267 [42]Benzothiazole Roasted MS, aroma, RI87 Unknowns

1116Unknown Fruity n.i.8

1322Unknown Unpleasant n.i.87

1339Unknown Acid, sour n.i.4

1505Unknown Smoky, goaty n.i.34

1566Unknown Smoky, chemical, spicy n.i. 6

a A, pentane:diethyl ether, 98:2; B, pentane:diethyl ether, 95:5; C, pentane:diethyl ether, 90:10; D, diethyl ether fraction.

b MS, compounds identified by MS spectra; aroma, compounds identified by the aroma descriptors; RI, compounds identified by comparison with pure standard; RIL, compounds identified by compared with retention indices from the literature; n.i., not identified.

*Tentatively identified.

3-methylbutyl hexanoate could also be important aroma compounds. Their Osme values were more than 10 in both the 2001 and the 1999 liquors in fraction A, as determined on the DB-Wax column. Ethyl pentanoate, ethyl octanoate and 3-methylbutyl hexanoate were fruity, apple and green aromas.

Many other esters, including ethyl heptanoate, ethyl decanoate, methyl hexanoate, propyl hexanoate, butyl hexanoate, hexyl hexanoate, hexyl octanoate, hexyl ac-etate, ethyl 2-methylpropanoate, ethyl 3-methylbutanoate and 3-methylbutyl octanoate, had medium to strong odours (Osme values = 7–10). They contributed to sweet, fruity, apple and pineapple aromas and could be import-ant to Yanghe Daqu liquor aroma.

Several hydroxyl fatty acid esters were iden-tified in this study. Ethyl 2-hydroxyhexanoate, ethyl 2-hydroxypropanoate and ethyl 2-hydroxy-3-methylbutanoate had floral, jasmine and fruity aromas. These compounds had been identified in freshly distilled Calvados and Cognac.7Diethyl butanedioate was also identified, and it contributed to a fruity and sweet aroma. Several aromatic esters were detected in Yanghe Daqu liquor. Ethyl benzoate gave a floral aroma, while ethyl benzeneacetate and 2-phenylethyl acetate had a rosy and honey-like aroma.

Esters were mostly formed through esterification of alcohols with fatty acids during the fermentation and ageing process. Fan et al.21reported that Daqu had not only high hydrolase but also high esterase activities. The esterases could be very active during the fermenta-tion process and catalyse ester synthesis.

Ester formation can be influenced by many factors, such as fermentation temperature, oxygen availability and fermentation strains. Higher temperature leads to great loss of esters, due to increased rates of hydrolysis and volatilization, while a lower fermentation temperature favours the formation of short-chained esters.22,23I n the fermentation process of cooked grain, a relatively low temperature was maintained, thus favouring the formation of short-chained esters.

Higher alcohols

Alcohols were separated mainly into fraction C and some in fraction D. 1-Hexanol and 3-methylbutanol had Osme values >10 in fraction C on both the polar and non-polar columns. 1-Hexanol contributed to floral and green aroma notes. 3-Methylbutanol imparted a nail polish aroma.

1-Pentanol, 2-pentanol, 1-heptanol, 2-heptanol, 1-octanol and 2-octanol could also be important aroma compounds because they had medium Osme values (7–10). 1-Heptanol imparted a green and fruity odour. 1-Pentanol, 2-pentanol, 2-heptanol contributed to fruity odours. 1-Octanol and 2-octanol provided green and floral notes. In addition, benzene ethanol was detected in both liquors, although it had a low Osme value. It gave rosy and honey-like aromas.

Higher alcohols could be formed during the fermenta-tion, under aerobic conditions from sugar and under anaerobic conditions from amino acids.24Since the raw materials (sorghum, rice, sticky rice, wheat and corn) are rich sources of amino acids, higher alcohols can be con-verted from amino acids by yeast via the Ehrlic metabolic pathway.25 Small amounts of higher alcohols could also be made by yeast, through reduction of corresponding aldehydes.

Phenols

Eight phenols were detected in fractions B and C on a polar column, while five phenols were identified on a non-polar column. Guaiacol (2-methoxyphenol), 4-methylguaiacol (4-methyl-2-methoxyphenol) and

AROMA COMPOUNDS IN CHINESE YANGHE DAQU LIQUOR 341

4-ethylguaiacol (4-ethyl-2-methoxyphenol) had Osme values >6 for both liquors on the polar column. These compounds contributed to strong clove, spicy and smoky odours. Phenol, o -cresol (2-methylphenol), p -cresol (4-methylphenol), 4-ethylphenol and 4-vinylphenol were also identified in Yanghe Daqu liquor. These compounds contributed medicinal and animal odours. Phenolic compounds belong to the secondary plant constituents,derived from lignin degradation.26Acetals

Three acetals were identified in the neutral/basic fraction.1,1-Diethoxyethane had a strong fruity aroma. I t co-eluted with ethyl acetate on the polar column, but was separated on the non-polar column. 1,1-Diethoxy-2-methylpropane and 1,1-diethoxy-3-methylbutane gave fruity odours. These compounds had been reported in distillates, Chinese liquor, whisky and brandy.19,24,27Acetals were formed from alcohols and aldehydes in the presence of excess alcohols.28Sulphur compounds

Two sulphur compounds were identified in Yanghe Daqu liquor. They were detected in the fractions B and C on the polar column. Dimethyl sulphide had a cooked onion aroma, while benzothiazole gave a smoky and rubbery aroma. Dimethyl sulphide came from the degradation of sulphur-containing amino acids.29 Benzothiazole could be produce by fungi during the fermentation process.30Benzothiazole and those phenolic compounds identified could be the main compounds responsible for the smoky and toasty aromas in the Yanghe Daqu liquor.Ketones and aldehydes

Only a few aldehydes and ketones were identified in Yanghe Daqu liquor. Furfural (2-furancarboxaldehyde)gave almond and sweet aromas. 2-Pentanone was fruity.3-Octen-2-one imparted an earthy and mushroom aroma.Benzeneacetaldehyde contributed to a floral aroma and it was probably formed by yeast.31

Conclusions

Normal phase chromatography is very useful for separat-ing complex aroma extract before GC–O and GC–MS analysis. More than 70 aroma compounds were identified in Yanghe Daqu liquors by GC–O after the normal phase separation. Based on the Osme data, esters and acids were probably very important compounds to Yanghe Daqu liquor aroma. I

n addition, alcohols, phenolic compounds, aldehydes, ketones, sulphur-containing com-pounds and acetals could make contributions to the

aroma. Although more quantitative work and sensory evaluation are needed to confirm the results, the findings in this study will help to the further understanding of the key aroma compounds in Yanghe Daqu liquor.

Acknowledgements —The authors are grateful for the financial support of ‘Project 333’ of Jiangsu Province, the Department of Education of Jiangsu Province and Jiangsu Yanghe Distillery Stock Co. Ltd.

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