Food Science and Technology Research, 20 (2), 283_293, 2014Copyright © 2014, Japanese Society for Food Science and Technologydoi: 10.3136/fstr.20.283

http://www.jsfst.or.jp

*To whom correspondence should be addressed. E-mail: zhourqing@scu.edu.cn

IntroductionThe manufacturing procedures of spirits in the world such as

whisky, brandy, rum, vodka, cachaça, and Chinese spirits are constituted of three consecutive processes: fermentation, distillation and ageing. For these processes, distillation process is closely correlated with the quality and yield of the raw spirit. Alembic stills and column stills are applied to the distillation of brandy, whisky, and cachaça. The former has no trays or appreciable reflux and requires multiple distillation to achieve high-proof raw spirits, while the latter has a reflux column to decreases the concentration

of the congeners in raw spirits (Claus and Berglund, 2005). A distinct device called as Zeng-tong (Li et al., 2012) has been widely applied to the distillation of Chinese spirits for a long history. The Zeng-tong is composed of reboiler, taper barrel, condenser and other attachments, such as stream pipes, lid and tube type heat exchanger (shown in Figure 1A). Similar to fruit brandy (Claus and Berglund, 2005), the operation mode of Chinese spirits belongs to a batch distillation. The main production process of Chinese spirits is as follows (Figure 1B). Prior to the spirit distillation, the fresh grains and grain hull are mixed with the fermented grains to adjust

Short communication

Volatile Compounds of Raw Spirits from Different Distilling Stages of Luzhou-flavor Spirit

Jia Zheng1, Ru Liang

1, Jun Huang1, Rui-Ping Zhou

4, Zhe-Jun Chen4, Chong-De Wu

1, Rong-Qing Zhou1,2,3* and

Xue-Pin Liao1,2

1 Key Laboratory of Leather Chemistry and Engineering, Ministry of Education, and College of Light Industry, Textile & Food Engineering, Sichuan University, Chengdu 610065, China

2 National Engineering Laboratory for Clean Technology of Leather Manufacture, Sichuan University, Chengdu 610065, China3 National Engineering Research Center of Solid-State Brewing, Luzhou 646000, China4 Xufu distillery company, Yibin 644000, China

Received August 6, 2013 ; Accepted November 9, 2013

Distillation process is closely correlated with the quality and yield of the raw spirit. The volatile properties of raw spirits from three distilling stages (head, heart and tail) of Luzhou-flavor spirit were investigated based on GC and GC-MS analyses. A total of 71 compounds were identified and ester derivatives attributed the largest number and concentration of volatile compounds in three raw spirits. The total concentration of raw spirit was decreased from the head raw spirit (5105 mg/L) to the tail raw spirit (1843 mg/L). Many volatile compounds with high concentrations such as acetaldehyde, 1-hexanol, 2-methylbutanoic acid, hexanoic acid, and ethyl hexanoate were found to decrease during distillation. Odor active value (OAV) was used to evaluate the contribution of special compounds to the whole odor of the raw spirit. Ethyl hexanoate followed by ethyl butyrate, ethyl heptanoate, ethyl octanoate, and 3-methylbutanal were found to be the most potent odor-active compounds. The liquid-liquid extraction together with GC-MS and GC analyses could be a useful tool to characterize the volatile composition of different distilling cuts of Chinese spirit. The decreasing of total content of volatile compound was determined by the ester derivate because of its high volatility and content.

Keywords: volatile compound, raw spirit, distilling process, odor active value

J. Zheng et al.284

their titer acidity, moisture and bulk density. Then the mixture is gradually filled into the barrel with the vapor spilling out from the surface of layer and forms a specific packed bed column. The distilling process begins after the mixture fully fills the Zeng-tong. After the distillation completed, the distilled mixture is cooled and mixed with Daqu-starter for fermentation in the pit. At last the fermented grains are sequentially treated with the way described above. Therefore, the distillation process involves not only the condensing of alcohol and fractionating of the congeners, but also the gelatinization and liquification of starch in grains. The quality of raw spirit is generally controlled by dividing the distillates into several appropriate fractions or “cuts” (i.e discarding the head fraction, collecting the heart fraction and redistilling/discarding the tail fraction) (Scanavini et al., 2010). However, the aromatic stability and consistence of the raw spirit completely depend on the operator’s smell and taste. Till now, little information available concerning the effect of distillation process on the quality of raw spirit was reported.

Chinese spirits can be classified into several fragrance types including strong aroma (Luzhou-flavor), light aroma (Fen-flavor), sauce aroma (Maotai-flavor), sweet honey and miscellaneous type

according to their flavor characteristics and brewing technology. The volatile compositions of them have been previously studied, and more than seven hundreds components have been identified (Fan and Qian, 2006b; Zhu et al., 2007). Whereas, few attention was paid on the characteristics of volatile compounds in different distilling stages (head, heart and tail fraction). Furthermore, confirming the fingerprint of flavor compounds in these distillates may contribute to optimize the distillation process and increase the yield and distilling efficiency, and evaluate the influence of distillation process on the formation of harmful compounds. From the latest literatures, Rogelio et al. analyzed the role of batch rectification on the quality of Mexican tequila, and compared the copper and stainless steel alembics on the formation of volatile compounds (Rogelio et al., 2005). Bruno et al. investigated the influence of the configuration of the distilling system and procedure on the level of ethyl carbamate in cachaça (Bruno et al., 2007). Lukić et al. analyzed the change of concentrations of various volatile constitutions during the distillation process of fermented Muscat Blanc and Muškat ruža porečki grape marcs (Lukic et al., 2011). However, till now, little is known about the variation of the volatile compounds and their odor active values (OAV) in the

Fig. 1. (A) Diagram of the distillation system (Zeng-tong) for Luzhou-flavor spirit. (a): reboiler-equipped with a secondary stream distributor; (b): taper barrel-filled with fermented grains; (c): lid; (d): stream pipes; (e): tube type heat exchanger. (B) Brief manufacturing procedure of Chinese spirits.

Volatile Compounds of Luzhou-flavor Raw Spirits 285

distillation process of Luzhou-flavor spirit.The aim of this study was to investigate the volatile compounds

in raw spirits from three distilling stages, and analyze the influence of distillation on the quality of raw spirit. The OAV was used to evaluate the contribution of the volatile compounds to the whole odor.

Methods and MaterialsSamples and chemicals Ethyl acetate (99.9%), ethyl lactate

(99.0%), ethyl butyrate (99.5%), ethyl hexanoate (99.0%), ethyl palmitate (99.0%), ethyl linoeate (99.0%), ethyl phenylacetate (98.0%), ethyl oleate (98.0%), methyl caprylate (99.0%), phenethyl alcohol (99.0%), furfural (98.0%), benzenacetaldehyde (99.0%), 4-ethylphenol (97.0%), 4-ethyl-2-methoxylphenol (98.0%), acetic acid (99.5%), butanoic acid (99.0%), hexanoic acid (98.0%), caprylic acid (98.0%), methanol (99.8%) and acetaldehyde (99.5%) were all purchased from Sigma-Aldrich (St. Louis, MO, USA), and all standards used were of GC purity. Other reagents were of analytical purity.

A total of three raw spirits belong to three distilling stages in the same batch were collected from Xufu distillery Co., Ltd., which is one of the typical Luzhou-flavor spirit manufacturing company in China. The distilling stages and related raw spirits were as follows: (1) head stage: about 1.5 kg raw spirit is collected from the pipe of tube type heat exchanger (e, Fig. 1) at the beginning of distillation, and this spirit is named as “head spirit”; (2) heart stage: along with the distillation, the heart raw spirit is collect within 30 min with special operation parameters (the spirit temperature: more than 30℃, outflow of spirit: approximately 2.5 kg/min), and this part of spirit is named as “heart spirit”; (3) tail stage: when the collection of heart spirit completed, the operators usually increase the steam reaching the maximum level to gelatinize and liquify the starch in grains and this stage will cost 45 min to 1 h, and the spirit collected at this stage is named as “tail spirit”. Before the analyses, a total of 500 mL samples of each distilling stage were putted into reagent bottles with grinding stopper, sealed and stored at room temperature until analysis.

Extraction of volatile compounds The volatile compounds in raw spirits were extracted based on the liquid-liquid extraction (LLE) method reported previously (Qian and Reineccius, 2002) with some modifications. Briefly, 10 mL raw spirit was transferred into roundflask and internal standard (methyl caprylate and capryl-ic acid) was added. Prior to the extraction of volatile compounds, the concentration of ethanol in raw spirit was adjusted to approxi-mately 14% by distilled water. The mixture was saturated with NaCl and adjusted to pH 11 with 10% NaOH. Then 50 mL anhy-drous diethyl ether was added into the pretreated raw spirit mixture to extract volatile compounds. The organic phase was transferred into the clean glass tube and labeled as “neutral fraction”. The aqueous phase was adjusted to pH 1.7 with 2 M H2SO4 and extract-ed by 50 mL anhydrous diethyl ether, and the extracted organic

phase was labeled as “acidic fraction”. All fractions were dried with 5 g of anhydrous Na2SO4, and concentrated the filtrate to 0.5 mL under the soft nitrogen. Each raw spirit was extracted and tested in three duplicate.

GC-MS analysis The neutral and acidic fraction were ana-lyzed on a Trace GC Ultra gas chromatograph-DSQ ΙΙ mass spec-trometer (Thermo Electron Corporation, Waltham, MA, USA) equipped with a HP-5MS capillary column (30.0 m × 0.25 mm i.d., 0.25 μm film thickness, Agilent, Santa Clara, CA, USA), respec-tively. GC analyses were performed under the following condi-tions: an inlet temperature of 250℃, split ratio of 10:1, and Helium (purity: 99.999%) carrier gas flow of 1 mL/min. The oven tempera-ture was kept at 40℃ for 5 min, followed by an increase of 5℃ / min to 200℃, and then programmed to 220℃ at 10℃/min, and held for 5 min. For mass spectrometer, the temperatures of the transfer line, quadruple and ionization source were of 250℃, 150℃, and 230℃, respectively. The mass spectrum was generated in the electron impact (EI) mode at 70 eV. Detection was carried out in the full scan mode in the range of m/z 35 ~ 400.

Each volatile compound was identified by comparison of their mass spectrum with the NIST05 spectrum database. Kováts retention indices (RI) of each compound were calculated by using C8 ~ C20 n-alkanes (Sigma-Aldrich, St. Louis, MO, USA) (Cates and Meloan, 1963). The identification of each volatile compound was additionally confirmed by comparison of their RI with the RI reported in previous literatures (RIL). The relative concentration of volatile compound (mg/L) to the internal standards (methyl octanoate and octanoic acid) was semi-quantified on the basis of comparing their peak areas to that of the internal standard on GC total ion chromatograms.

GC analysis The specific low molecular volatile compounds (methanol and acetaldehyde) were quantified by GC analysis. Concentrations of methanol and acetaldehyde were analyzed using GC-FID (Agilent 6890A, Santa Clara, CA, USA) equipped with DB-WAX (30.0 m × 0.25 mm i.d., 0.25 μm film thickness, Agilent, Santa Clara, CA, USA). The GC analysis procedure was according to the procedure reported in Lukić et al. (2011).

Statistical analysis The content of volatile compound was expressed as mg/L. Analysis of variance (ANOVA) with Turkey’s test was performed to evaluate significant differences in volatile compounds from different distilling stages. Significance of difference was defined at p < 0.05 (n = 3). One-way ANOVA was conducted using SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA).

Results and DiscussionAccuracy of quantitative analysis based on GC-MS The

accuracy and reliability of final analyses results are tightly correlated with the sample preparation procedure. In some previous literatures, numerous sample preparation methods, such as LLE, solid-phase microextraction (SPME) and stir bar sorptive extraction

J. Zheng et al.286

(SBSE), coupled with GC-MS were used to determine volatile compounds in Chinese spirits and wines (Hernandez-Gomez et al.; 2005, Sanchez-Palomo et al., 2009; Fan and Qian, 2006a). LLE represented a better applicability for sample preparation in previous experiments because all volatile compounds (low, medium and high volatility) can be analyzed in one or more extraction steps (Hernanz et al., 2008; Caldeira et al., 2007). Prior to GC-MS detection, the main volatile compounds determined in previous literature (Fan and Qian, 2006a) were used to validate the accuracy of the detection method in the present research. The peak area ratios (neutral fraction/methyl caprylate and acidic fraction/caprylic acid) were used for the quantification of each compound. As shown in Table 1, good linearity of calibration curve (r > 0.9) was obtained, and extraction recoveries were higher than 90% for all compounds tested. It was suggested that methyl caprylate and caprylic acid used as the internal standards was suitable to semi-quantitatively or quantitatively evaluate the relative level of volatile compounds in distillates from different distilling stages.

Volatile compositions of different raw spirits In this study, the volatile compounds from the head, heart and tail raw spirit were extracted by LLE method and then detected using GC-MS and GC analyses. Typical total ion chromatograms of neutral and acidic fraction in GC-MS system are displayed in Figure 2. Figure 3 represents the total concentration of the volatile compounds. It is clear that the total concentration of volatile compounds significantly decreased with the distillation ( p < 0.05). The highest concentration was detected in head raw spirit (5105 mg/L), which was 1.4- and 2.8-fold higher than that in the heart (3611 mg/L) and tail raw spirits (1843 mg/L), respectively. The levels of esters and other compounds were sharply decreased from the head to tail raw spirit, whereas high levels of acids, alcohols, aldehydes and ketones were observed in the heart raw spirit ( p < 0.05).

The compounds identified, quantified and grouped by their affiliation with different chemical classes are revealed in Table 2. A total of 71 compounds composed of 10 alcohols, 34 esters, 10 acids, 5 aldehydes, 4 ketones, 2 phenols, 3 hydrocarbon compounds and 3 oxygen-containing compounds were identified and quantified. The raw spirit exhibited the ethanol contents ranging from 44% in the tail spirit to 75% (v/v) in the head spirit. Besides the ethanol, esters which accounted for 14% _ 61% of the total concentration were the largest group among these volatiles. The most abundant compounds were methanol , 2-pentanol , 3-methylbutanol, 1-hexanol, propanoic acid, hexanoic acid, ethyl butyrate, ethyl pentanoate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, acetaldehyde, 3-methylbutanal, 1,1-diethoxyethane and 1,1-diethoxyl-3-methylbutane.

Esters: Esters, the most numerous volatiles, are formed mostly through the esterification of alcohols with fatty acids during the fermentation, thermal distillation and maturing processes (Fan and Qian, 2005). A total of 34 esters including 24 ethyl esters and 10 other esters were quantified with the total concentration ranging from 260 mg/L to 3133 mg/L (Table 2). Esters especially ethyl esters represented the largest group in the number and concentration of volatile compounds identified. This result was coincided with the previous study (Fan and Qian, 2006a) which also reported that ethyl ester was the main group in esters. Ethyl hexanoate was considered to be the dominant compound in Luzhou-flavor spirit (Xu et al., 2010). In this study, ethyl hexanoate accounting for 36.7 _ 70.0% of the total esters was the most important ethyl ester and its concentration obviously decreased during the distillation process (P < 0.05). Meanwhile, the concentration of most esters was also significantly decreased from the head to tail raw spirit (P < 0.05), such as ethyl butyrate, pentanoate, hexanoate, nonanoate, palmitate, oleate and butyl

Table 1. Characteristics of calibration curves.

Standard materiala Standard curve R2 Validation rage(mg/L)

Recovery(%)

Detection limit(LOD, μg/L)d

Ethyl butyrate y=0 .0309x + 0 .457b 0 . 9899 10 .96 _ 1096 97 .85 0 .03Ethyl lactate y=0 .093x + 0 .9671b 0 . 9772 5 .40 _ 540 96 .52 0 .04

Ethyl hexanoate y=1 .8196x + 10 .458b 0 . 9874 21 .8 _ 21850 98 .12 0 .01Ethyl phenylacetate y=0 .0286x + 1 .1054b 0 . 8521 1 .29 _ 129 99 .11 0 .02Ethyl hexadecanoate y=0 .0063x + 0 .3924b 0 . 9475 10 .75 _ 1075 90 .42 0 .03

Ethyl oleate y=0 .095x + 0 .9172b 0 . 8827 11 .00 _ 1100 91 .49 0 .01Ethyl phenylacetate y=0 .0711x + 1 .4449b 0 . 9457 5 .85 _ 580 99 .11 0 .01Phenylacetaldehyde y=0 .093x + 0 .9671b 0 . 9772 5 .40 _ 540 96 .52 0 .05

Butanoic acid y=0 .0789x + 0 .4133c 0 . 8791 12 .00 _ 1200 101 .45 0 .04Hexanoic acid y=_0 .001x + 0 .052c 0 . 9815 11 .62 _ 1162 106 .75 0 .03

a Standard materials were all purchased from Sigma-Aldrich (St. Louis, MO, USA), and five levels of concentration for each volatile compound, covering the concentration ranges expected, were tested in triplicate.b Standard curves were fitted with the ratio of volatile compound to methyl caprylate.c Standard curves were fitted with the ratio of volatile compound to caprylic acid.d Detection limit of each compounds was calculated under the condition of the standard signal/noise of base line = 3.

Volatile Compounds of Luzhou-flavor Raw Spirits 287

hexanoate. The similar pattern was also found in Muškat Ruža Porečki grape marcs and melon wine (Lukic et al., 2011; Hernandez-Gomez et al., 2005). Whereas ethyl lactate significantly decreased during the distilling process. It is possible that high flow

rate of vapor in the initial stage of distillation process promote the volatilization of these esters with high boiling point.

Traditionally, the content sequence of ethyl esters in commercial Luzhou-flavor spirit is ethyl hexanoate > acetate ≥

Fig. 2. Total ion chromatogram of volatile compounds in (A) neutral fraction and (B) acidic fraction using GC-MS. Compounds series: internal standard (IS), alcohols (AL), aldehydes (AD), acids (AC), esters (ES), ketones (KT), phenols (PH), hydrocarbon compounds (HC) and oxygen-containing compounds (OC).

Fig. 3. Total concentration and relative abundance of each kind of volatile compounds in the head, heart and tail raw spirits. Error bars indicated standard deviations (n = 3). Different letters indicate significant differences ( p < 0.05, ANOVA, Turkey’s test).

J. Zheng et al.288Ta

ble

2.

Volu

me

frac

tion

of e

than

ol (v

/v) a

nd q

uant

itativ

e co

ncen

tratio

n (m

g/L)

of v

olat

ile c

ompo

unds

in d

iffer

ent r

aw sp

irits

.

Num

ber

Com

poun

dsR

I HP-

5MS

Iden

tifica

tion1

Thre

shol

d2 (mg/

L)C

once

ntra

tion3 (m

g/L)

OAV

Hea

dH

eart

Tail

Hea

dH

eart

Tail

Volu

me

frac

tion

of e

than

ol74

.50

75.8

044

.00

Alc

ohol

s (10

)A

L1M

etha

nol

-St

d13

68a

±8

1214

a±

713

18a

±7

AL2

1-Pr

opan

ol<8

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S, R

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64.

4±

0.5

10.4

±1.

23.

3±

0.4

<1<1

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Met

hylp

ropa

nol

<800

MS,

RIL

16 (Q

ian

and

Wan

g, 2

005)

0.53

a±

0.04

0.83

a±

0.04

37.5

b±

0.94

<1<1

<1A

L42-

Pent

anol

<800

MS,

RIL

8.1

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ang,

200

5)21

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816

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67a

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193

2<1

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ethy

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anol

<800

MS,

RIL

30 (G

uth,

199

7)11

7b±

613

6a±

112.

63c

±3.

004

5<1

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1-Pe

ntan

ol<8

00M

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5 (G

iri e

t al.,

201

0b)

0.10

±0.

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1.58

±0.

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-<1

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1-H

exan

ol87

5M

S, R

IL2.

5 (Q

ian

and

Wan

g, 2

005)

16.9

±0.

832

.3±

2.9

22.1

±0.

97

139

AL8

2-Fu

rald

ehyd

e di

ethy

l ace

tal

1080

MS

0.07

±0.

060.

05±

0.01

ndA

L91-

Non

anol

1106

MS,

RIL

1 (Q

ian

and

Wan

g, 2

005)

1.44

b±

0.08

1.35

b±

0.06

0.22

a±

0.01

11

<1A

L10

Phen

ethy

l alc

ohol

1116

MS,

RIL

, Std

1 (Q

ian

and

Wan

g, 2

005)

1.80

a±

0.15

1.38

a±

0.17

8.71

b±

0.40

21

9A

cids

(10)

AC

1A

cetic

aci

d<8

00M

S, S

td20

0 (G

uth,

199

7)1.

73ab

±0.

042.

31b

±0.

260.

05a

±0.

02<1

<1<1

AC

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acid

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MS

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(Fer

reira

et a

l., 2

000)

44.2

b±

8.1

36.3

b±

1.7

11.7

a±

2.0

54

1A

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ethy

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ic a

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240.

14±

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4.88

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41±

0.42

21.

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9A

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ethy

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c ac

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7.71

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72.

72.

6A

C7

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c ac

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58a

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287.

98b

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.9c

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2A

C8

Hex

anoi

c ac

id10

37M

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(Gut

h, 1

997)

115b

±12

153c

±15

87.2

a±

3.4

1419

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Hep

tano

ic a

cid

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MS

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b±

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b±

0.69

9.12

a±

1.19

AC

10H

exad

ecan

oic

cid

1963

MS,

Std

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b±

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2.20

a±

0.49

0.55

a±

0.02

Est

ers (

34)

ES1

Ethy

l ace

tate

<800

MS,

RIL

, Std

7.5

(Gut

h, 1

997)

13.0

b±

4.2

13.8

b±

0.5

2.63

a±

0.12

1.5

1.6

<1ES

2Et

hyl b

utyr

ate

806

MS,

RIL

, Std

0.02

(Gut

h, 1

997)

123c

±9

19.5

b±

4.1

6.34

a±

0.36

6127

975

468

ES3

Ethy

l lac

tate

823

MS,

RIL

, Std

14 (F

erre

ira e

t al.,

200

0)30

.3b

±5.

21.

16a

±0.

267.

00a

±0.

642

<1<1

ES4

Ethy

l 2-m

ethy

lbut

anoa

te85

3M

S, R

IL0.

001

(Gut

h, 1

997)

0.28

b±

0.05

0.41

c±

0.05

0.07

a±

0.00

280

410

70ES

5Et

hyl 3

-met

hylb

utan

oate

857

MS,

RIL

0.00

3 (G

uth,

199

7)0.

81b

±0.

010.

59b

±0.

090.

08a

±0.

0327

019

326

.7ES

6Et

hyl p

enta

noat

e90

4M

S, R

IL0.

005

(Tak

eoka

et a

l., 1

990)

90.8

b±

12.5

44.8

b±

2.5

4.29

a±

0.08

1816

089

5085

8ES

7Et

hyl 3

-met

hylp

enta

noat

e92

9M

S,R

IL1.

07b

±0.

13b

0.49

a±

0.02

0.20

a±

0.02

ES8

Ethy

l 4-m

ethy

lpen

tano

ate

969

MS

1.82

b±

0.81

1.36

ab±

0.05

0.61

a±

0.04

ES9

Ethy

l 2-m

ethy

lpen

tano

ate

972

MS,

RIL

0.07

±0.

010.

59±

0.09

ndES

10Et

hyl h

exan

oate

1005

MS,

RIL

, Std

0.00

5 (G

uth,

199

7)25

78c

±62

1453

b±

319

1a±

251

5766

2907

2438

286

ES11

Ethy

l 2-h

ydro

xy-4

-met

hylp

enta

noat

e10

61M

S3.

07b

±0.

230.

65a

±0.

011.

06a

±0.

17

Volatile Compounds of Luzhou-flavor Raw Spirits 289

Tabl

e 2.

Vo

lum

e fr

actio

n of

eth

anol

(v/v

) and

qua

ntita

tive

conc

entra

tion

(mg/

L) o

f vol

atile

com

poun

ds in

diff

eren

t raw

spiri

ts.

Num

ber

Com

poun

dsR

I HP-

5MS

Iden

tifica

tion1

Thre

shol

d2 (mg/

L)C

once

ntra

tion3 (m

g/L)

OAV

Hea

dH

eart

Tail

Hea

dH

eart

Tail

ES12

Ethy

l 5-m

ethy

lhex

anoa

te10

65M

S0.

63±

0.02

0.47

±0.

020.

89±

0.14

ES13

Ethy

l hep

tano

ate

1100

MS,

RIL

0.00

2 (G

iri e

t al.,

201

0b)

84.3

c±

4.2

57.1

b±

6.1

5.30

a±

0.01

4216

028

535

2650

ES14

Ethy

l ben

zoat

e11

73M

S, R

ILnd

0.15

±0.

01nd

ES15

Ethy

l oct

anoa

te11

99M

S, R

IL0.

002

(Gut

h, 1

997)

81.2

c±

2.2

72.8

b±

5.3

6.63

a±

0.24

4059

036

390

3315

ES16

Ethy

l ben

zene

acet

ate

1248

MS,

RIL

, Std

0.15

5 (G

iri e

t al.,

201

0b)

0.45

±0.

050.

19±

0.00

0.63

±0.

052.

91.

24.

1ES

17Et

hyl n

onan

oate

1297

MS,

RIL

1.61

b±

0.05

1.64

b±

0.10

0.22

a±

0.02

ES18

Ethy

l ben

zenp

ropa

noat

e13

52M

S1.

18±

0.11

0.78

±0.

072.

46±

0.26

ES19

Ethy

l dec

anoa

te13

97M

S, R

IL0.

2 (T

ao a

nd Z

hang

, 201

0)3.

00b

±0.

023.

48b

±0.

320.

28a

±0.

0315

171

ES20

Ethy

l dod

ecan

oate

1597

MS

2.12

b±

0.20

1.83

b±

0.20

0.31

a±

0.01

ES21

Ethy

l tet

rade

cano

ate

1796

MS

4.19

b±

0.49

3.00

a±

0.25

a1.

04a

±0.

25ES

22Et

hyl p

alm

itate

1996

MS,

Std

2 (Q

ian

and

Wan

g, 2

005)

32.8

b±

1.0

22.4

b±

0.4

6.41

a±

0.20

ES23

Ethy

l lin

olea

te>2

000

MS,

Std

17.1

c±

2.7

9.59

b±

1.29

2.79

a±

0.69

ES24

Ethy

l ole

ate

>200

0M

S, S

td9.

67c

±1.

305.

76b

±0.

671.

82a

±0.

48ES

25H

exyl

ace

tate

1018

MS,

RIL

1.5

(Cam

po e

t al.,

200

6)3.

11b

±0.

080.

75a

±0.

010.

59a

±0.

072

<1<1

ES26

3-M

ethy

lbut

yl b

utan

oate

1059

MS

1.71

b±

0.17

1.18

a±

0.03

0.89

a±

0.03

ES27

3-M

ethy

lbut

yl 2

-met

hoxy

acet

ate

1072

MS

0.48

±0.

010.

05±

0.01

0.04

±0.

01ES

28Pr

opyl

hex

anoa

te10

96M

S, R

ILnd

5.75

b±

0.81

b0.

67a

±0.

04ES

29B

utyl

hex

anoa

te11

34M

S0.

7 (T

akeo

ka e

t al.,

199

0)0.

52b

±0.

030.

50b

±0.

030.

09a

±0.

01<1

<1<1

ES30

Isob

utyl

hex

anoa

te11

53M

S2.

97b

±0.

152.

42b

±0.

140.

26a

±0.

04ES

31D

ieth

yl su

ccin

ate

1184

MS

0.70

±0.

040.

29±

0.00

0.55

±0.

01ES

32H

exyl

but

anoa

te11

93M

S20

.2b

±0.

517

.7b

±1.

01.

70a

±0.

41ES

33Is

open

tyl h

exan

oate

1252

MS

1.52

a±

0.06

11.8

b±

1.0

1.15

a±

0.05

ES34

Pent

yl h

exan

oate

1289

MS,

RIL

1.97

b±

0.01

1.94

b±

0.13

0.22

a±

0.02

Ket

ones

(4)

KT1

2-Pe

ntan

one

<800

MS

0.01

(Qia

n an

d W

ang,

200

5)nd

nd0.

21±

0.03

--

26K

T22-

But

anon

e<8

00M

S80

(Qia

n an

d W

ang,

200

5)0.

05b

±0.

010.

04a

±0.

010.

08c

±0.

00<1

<1<1

KT3

2-H

epta

none

894

MS

0.00

1 (Q

ian

and

Wan

g, 2

005)

0.57

c±

0.05

0.51

b±

0.00

0.06

a±

0.01

5750

6K

T44-

Met

hyl-3

-hep

tano

ne93

4M

S0.

02±

0.01

nd0.

03±

0.02

Ald

ehyd

es (5

)A

D1

Ace

tald

ehyd

e-

Std

1.2

54.9

b±

0.3

4.58

a±

0.10

3.83

a±

0.05

454

3A

D2

3-M

ethy

lbut

anal

<800

MS

0.00

035(

Qia

n an

d W

ang,

200

5)37

.9c

±2.

529

.4b

±1.

53.

18a

±0.

0410

8285

8400

090

85A

D3

1,1-

Die

thox

yeth

ane

<800

MS,

RIL

0.05

(Gut

h, 1

997)

48.3

b±

4.7

40.9

b±

1.1

2.97

a±

0.01

965

820

60A

D4

Furf

ural

838

MS,

RIL

,Std

14.1

(Fer

reira

et a

l., 2

000)

4.06

b±

0.21

1.60

a±

0.16

0.02

a±

0.00

<1<1

<1A

D5

Ben

zene

acet

alde

hyde

1044

MS,

RIL

1(C

ampo

et a

l., 2

006)

0.38

a±

0.02

0.30

a±

0.01

1.98

b±

0.21

<1<1

<1

J. Zheng et al.290

lactate (or lactate > acetate) > butanoate > pentanoate, and the appropriate content range of ethyl hexanoate in typical Luzhou-flavor spirit range from 1200 mg/L to 2800 mg/L according to the China National Standard (GB10781.1-2006, Strong flavor Chinese spirit). Result of this study showed that the highest level of ethyl hexanoate was monitored in the head spirit (2578 mg/L), and ethyl pentanoate, butanoate and palmitate also represent higher content in the head raw spirit than these in other samples. Furthermore, the tail spirit represented the lowest content of ethyl esters especially ethyl hexanoate (191 mg/L). In this way, only the heart raw spirit showed the most acceptable content of ethyl hexanoate (1453 mg/L) because the blending process could increase the total content of ethyl hexanoate.

Acids: Acids were the second largest group detected in this study. In the spirit samples, 10 acid derivates were identified (Table 2), and 9 of them were also indentified in previous studies (Fan and Qian, 2006a). The total concentration decreased from 239 mg/L in the heart raw spirit to 167 mg/L in the tail raw spirit. Based upon their concentration percentage, hexanoic acid was the major acid in all spirits (accounting for approximately 50% of total acids). A sharp decrease of the concentration was noted in propanoic acid and palmitic acid from the head to tail raw spirit ( p < 0.05), which was deviated with that obtained by Lukić (Lukic et al., 2011). Li et al., (2012) found that high flow rate of vapor could increase the volatility of hexanoic acid. Combined with the decreasing tendency of ethyl lactate, it assumed that the excessive high flow rate of vapor at the head stage lead to the acids streamed out too early.

Alcohols: Alcohols are major products of fermentation of sugars and amino acid (Silva et al., 1996; Wondra and Berovic, 2001). Among 10 alcohols detected, methanol, 2-methylpropanol, 2-pentanol, 3-methylbutanol and 1-hexanol were considered to be the predominant alcohols on account of their high content. Approximately equal concentrations of methanol were detected from the head raw spirit to tail spirit, which showed a similar pattern in the Muškat ruža porečki grape marc (Lukic et al., 2011). It was reported that methanol is produced by the hydrolysis of pectic substances (Mangas et al., 1995). High level of methanol in raw spirits maybe explained by the intense liquefaction of grains especially the grain hull during the distilling process. The concentration of 2-pentanol significantly decreased from the head to tail spirit ( p < 0.05). High concentrations of 1-propanol, 3-methylbutanol and 1-hexanol were monitored in the head and heart raw spirits, and this was agreement with the previous finding (Leaute, 1989). Probably high volatility of these low boiling point compounds such as 1-hexanol and 1-propanol leaded to high content in the heart raw spirit. As it documented that 1-hexanol is considered to be a positive affection on the aroma of the distillate when the content of which up to 20 mg/L (Apostolopoulou et al., 2005). It suggested that the quality of the heart raw spirit may superior to others. The aromatic higher alcohol-phenethyl alcohol (8.70 mg/L) exhibited an increasing trend from the head to tail raw

Tabl

e 2.

Vo

lum

e fr

actio

n of

eth

anol

(v/v

) and

qua

ntita

tive

conc

entra

tion

(mg/

L) o

f vol

atile

com

poun

ds in

diff

eren

t raw

spiri

ts.

Num

ber

Com

poun

dsR

I HP-

5MS

Iden

tifica

tion1

Thre

shol

d2 (mg/

L)C

once

ntra

tion3 (m

g/L)

OAV

Hea

dH

eart

Tail

Hea

dH

eart

Tail

Phen

ols (

2)PH

14-

Ethy

lphe

nol

1175

MS,

RIL

, Std

1.01

(Giri

et a

l., 2

010b

)0.

52b

±0.

080.

18a

±0.

051.

92c

±0.

174

114

PH2

4-Et

hyl-2

-met

hoxy

phen

ol12

82M

S, R

IL, S

td0.

033(

Ferr

eira

et a

l., 2

000)

1.27

a±

0.06

0.55

a±

0.12

6.51

b±

1.06

5825

256

Hyd

roca

rbon

com

poun

ds (3

)H

C1

n-Pr

opyl

benz

ene

954

MS,

RIL

0.09

±0.

010.

10±

0.04

ndH

C2

2-M

ethy

lnap

htha

lene

1295

MS,

Std

0.30

±0.

040.

32±

0.07

0.33

±0.

07H

C3

1-M

ethy

lnap

htha

lene

1313

MS,

Std

0.18

a±

0.01

0.23

b±

0.08

0.21

ab±

0.10

Oxy

gen-

cont

aini

ng c

ompo

und

(3)

OC

11,

1-D

ieth

oxy-

3-m

ethy

lbut

ane

958

MS,

RIL

47.8

c±

5.3

32.0

b±

1.4

1.19

a±

0.03

OC

2(2

,2-D

ieth

oxye

thyl

)ben

zene

1327

MS

1.02

±0.

160.

90±

0.04

1.16

±0.

09O

C3

Hex

anoi

c ac

id, a

nhyd

ride

1370

MS,

RIL

0.47

b±

0.06

0.07

a±

0.01

0.10

a±

0.01

1 MS,

com

poun

ds w

ere

iden

tified

by

MS

spec

tra; R

IL, c

ompo

unds

wer

e id

entifi

ed b

y a

com

paris

on w

ith th

e re

tent

ion

inde

x fr

om th

e lit

erat

ures

; Std

, com

poun

ds w

ere

iden

tified

by

a co

mpa

rison

with

the

rete

ntio

n in

dex

from

the

pure

stan

dard

.2 T

he re

fere

nce

from

whi

ch th

e od

or th

resh

old

had

been

take

n w

as g

iven

in p

aren

thes

es.

3 The

con

cent

ratio

ns o

f vol

atile

com

poun

ds w

ere

repr

esen

ted

as m

ean

valu

e of

trip

licat

e sa

mpl

es ±

stan

dard

dev

iate

s (m

ean

± SD

). D

iffer

ent l

ette

rs in

dica

te si

gnifi

cant

diff

eren

ces (

p <

0.0

5, A

NO

VA,

Turk

ey’s

test

). ‘n

d’ m

eans

not

det

erm

ined

.

Volatile Compounds of Luzhou-flavor Raw Spirits 291

spirit, which was in accordance with the result of Lukić and Apostolopoulou (Lukic et al., 2011; Apostolopoulou et al., 2005). Additionally, 2-methylpropanol also exhibited the highest concentration of 37.5 mg/L in the tail raw spirit.

Aldehydes and ketones: Most aldehydes are probably metabo-lites of bacteria (Lachenmeier and Sohnius, 2008), and ketones are formed by the autoxidation of fatty acids, especially unsaturated fatty acids (Grosch, 1982). Furfural is formed during the distilla-tion due to dehydration of fermentable sugars (pentoses) caused by heating in acid conditions and/or Maillard reaction (Mangas et al., 1995). A total of 4 ketones and 5 aldehydes were observed, and the highest levels of aldehyde and ketone were determined in the heart raw spirit, followed by the head and the tail raw spirit. However, the previous literature observed a significant decrease in concentra-tion during the distillation (Apostolopoulou et al., 2005). It as-sumed that this was also owing to the high flow rate of vapor. Ac-etaldehyde, 3-methylbutanal and 1,1-diethoxyethane were considered to be dominant aldehydes in raw spirit upon their high concentration, and this agreed with these reported in previous stud-ies (Fan and Qian, 2005; Kim et al., 2009). A significantly de-c r e a s e d p a t t e r n o f a c e t a l d e h y d e , 3 - m e t h y l b u t a n a l , 1,1-diethoxyethane and furfural was represent from the head to tail raw spirit ( p < 0.05), assumedly depending on their relatively low boiling points (103 _ 209℃) and the solubility in alcohol. Acetal-dehyde is originated from the spontaneous or microbially mediated oxidation in distillates (Qian and Wang, 2005). In this study, the acetaldehyde in raw spirits may from the spirit fermentation pro-cess of raw material because the raw spirits were not stored before sampling.

Phenols: Phenols especially 4-ethyl-2-methoxyphenol (4-ethylguaiacol) were the thermal degradation products of lignin-related phenolic carboxylic acid (Zhang and Tao, 2009). The highest concentrations of 4-ethylphenol and 4-ethyl-2-methoxyphenol were monitored in the tail spirit, with the concentration of 1.92 mg/L and 6.51 mg/L, respectively. Similarly, these two phenols were also identified in Yanghe spirit (Fan and Qian, 2006b), Maotai spirit (Zhu et al., 2007) and whisky (Caldeira et al., 2007). High boiling point (217 and 236℃) could determine the concentration of them only emerging in the tail raw spirit because the last stage with higher flow rate and temperature comparing with other stages is always applied to gelatinize and liquefy the starch of grains.

Moreover, a total of 6 volatile compounds were identified. N-propylbenzene, 2-methylnaphthalene, 1-methylnaphthalene be-longs to hydrocarbon compound and (2,2-diethoxyethyl) benzene, 1,1-diethoxyl-3-methylbutane, and hexanoic acid, anhydride be-longs to oxygen-containing compound. The decreased tendency was monitored in 1,1-diethoxyl-3-methylbutane because of its low boiling point (156℃) and which has also been identified in other Chinese Luzhou-flavor liquor (Fan and Qian, 2005), aronia spirit and sparkling wines (Bosch-Fusté et al., 2007; Balcerek, 2010).

Due to their low concentration, the identification and classification of many compounds in these two groups (hydrocarbon and oxy-gen-containing compound) such as n-propylbenzene, 2-methyl-naphthalene, and 1-methylnaphthalene in raw spirit are still re-quired in the further analysis.

OAV of volatiles in different raw spirits The quantification data of volatile compounds in raw spirits from three distilling stages is listed in Table 2. It is obvious that ethyl hexanoate had the highest OAV, and its value decreased from the highest in the head spirit (515766) to the lowest in the tail spirit (38286). Previous studies also reported that ethyl hexanoate was the key volatile compound in most Luzhou-flavor spirits, such as Yanghe (Fan and Qian, 2005), Wuliangye and Jiannanchun (Fan and Qian, 2006a) based on its flavor dilution (FD) value, which contributed to the fruity, anise, apple, sweetish and pear odor (Jiang and Zhang, 2010). It suggested that ethyl hexanoate played the most important role in the whole odor feature of the raw spirit.

Most esters with high OAV detected had a typical “fruit” and “floral” descriptor, and contributed to fruity, sweet, apple, pineapple, and floral odors. For example, ethyl pentanoate (858 _ 18160), ethyl heptanoate (2650 _ 42160), and ethyl octanoate (3315 _ 40590) contributed to the odor of fruity, flora, sweet and pineapple (Fan and Qian, 2006a; Jiang and Zhang, 2010; Zhang et al., 2010). It was worth noting that 3-methylbutanal with low threshold value (0.35 μg/L) (Qian and Wang, 2005) may largely contributed to a characteristic aroma of apple, although the concentration was relatively low (3.18 mg/L-37.9 mg/L).

Meanwhile, the important aroma compounds (OAV ≥ 10) in-cluded 1-hexanol, hexanoic acid, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, ethyl benzenacetate, ethyl decanoate, 2-penta-none, 2-heptanone, 1,1-diethxoyethane 4-ethylphenol and 4-eth-yl-2-methoxylphenol. 1-Hexanol, with a floral and fruity odor (Ji-ang and Zhang, 2010), could be the dominant alcohol with the highest OAV. Hexanoic acid, one of main metabolites of Clostridi-um bacteria (Weimer and Stevenson, 2011), was also identified as the dominating volatile component in various raw spirits. Previous-ly, it was reported that 1,1-diethxoyethane was an important aroma compound in Yanghe spirit with the FD value more than 256 (Fan and Qian, 2005). In this study, 1,1-diethxoyethane also showed high OAV, assuming that a tight relationship existed between the raw spirit and commercial spirit. 4-Ethylphenol and 4-ethyl-2-me-thoxyphenol were the odor active compounds in many oriental fer-mentation condiments such as soy sauce and soybean paste, which had the odor of cooked soybean, smokey and phenolic odours (Lee and Ahn, 2009; Giri et al., 2010b; Giri et al., 2010a).

In addition, other volatile compounds such as propanoic acid, 2-methylbutanoic acid, ethyl acetate, ethyl butyrate, hexyl acetate and acetaldehyde with the OAV more than 1 may also contribute to the whole odor of the spirit. Ethyl acetate and ethyl butyrate represented the odor descriptions of green apple, strawberry, pineapple and sweet (Zhang et al., 2010; Vilanova et al., 2012).

J. Zheng et al.292

ConclusionThe volatile compounds of raw spirits collected from three

distilling stages of Luzhou-flavor spirit were detected using GC-MS and GC analyses. Results showed that the total concentration of volatile compounds was obviously decreased from the head spirit to tail spirit ( p < 0.05) without any research purely based on the different boiling points or solubility of the compounds (e.g. esters have higher volatility than alcohols). Ethyl hexanoate with the highest concentration and OAV was the most important odor active compound in each spirit. On the basis of high OAV, ethyl butyrate, ethyl heptanoate, ethyl octanoate, and 3-methylbutanal were also elucidated to be main contributors to the overall flavor of raw spirits. Results presented in this article may benefit to understand the changes and odorant contributions of volatile compounds in the distillation process of Luzhou-flavor spirit.

Acknowledgments This work was financially supported by the National Science Foundation of China (31171742).

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