Korean Journal of Food Preservation
The Korean Society of Food Preservation
ARTICLE

Determination of fusel oil content in various types of liquor distributed in Korea

Soo-Baek Lee, Jung-Ah Shin, Ki-Teak Lee*
Department of Food Science and Technology, Chungnam National University, Daejeon 34134, Korea
* Corresponding author. ktlee@cnu.ac.kr82-42-821-6729, 82-42-821-6721

© The Korean Society of Food Preservation. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Apr 26, 2017; Revised: May 23, 2017; Accepted: Jun 13, 2017

Abstract

This study was performed to analyze the content of 6 different fusel oils in 9 types of liquor distributed in domestic market. GC-FID method was employed for quantifying fusel oil (1-propanol, iso-butanol, 1-butanol, 2-butanol, iso-amyl alcohol, active amyl alcohol) levels in 260 liquor samples of liquor. Relative standard deviations (%) of intraand interday measurements were under 1.56 and 2.44%, respectively, while recovery rates (%) were 98.22-105.26% and 98.53-107.15%, respectively. Pretreatment method (filtering and centrifugation) of Takju did not affect analytic results. The average of total fusel oil contents in Yakju (39 types) and fruit wines (30 types) were 497.6 and 151.9 mg/L, showing Yakju contains more fusel oils than Takju or fruit wines. In fruit wines, iso-amyl alcohol was the major fusel oil component (at 6.8-249.0 mg/L). The highest content of fusel oil was found in foreign brandy, whereas the diluted Soju did not contain fusel oils. However, the average of total fusel oil contents was high at 764.5 mg/L in the three types of distilled Soju and iso-amyl alcohol content ranged from 114.2 to 421.0 mg/L. Domestic and foreign beers were similar in terms of their fusel oil compositions and contents. In conclusion, excluding the diluted Soju, the contents of total fusel oils ranged from 114.8 to 1447.3 mg/L in the monitored liquors.

Keywords: fusel oil content; gas chromatograph; flame ionization detector; liquor; monitoring

Introduction

Alcohols other than ethanol, aldehydes, organic acids, esters, and carbonyl compounds can be produced during the course of liquor fermentation. Of the higher alcohols, iso-amyl alcohol (3-methyl-1-butanol), active amyl alcohol (2-methyl- 1-butanol), iso-butanol (2-methyl-1-propanol), 1-propanol, 1-butanol, and 2-butanol are main components of fusel oils, and have been reported to be derived from specific amino acids. Generally, high fusel oil concentrations negatively affect the flavors liquors while, when present at low concentrations, fusel oils can improve the flavors of certain liquors. In addition, it is known that if fusel oils are consumed in large quantities, they can have adverse consequences on health (1-3).

To analyze fusel oil levels in liquors, gas chromatograph equipped with a flame ionization detector (FID) has been used by the Alcohol and Tobacco Tax and Trade Bureau (TTB) in the USA (4) and by the Commission of the European Communities in Europe (5). In most cases, the major compounds of fusel oil in liquor are 1-propanol, iso-butanol, 1-butanol, 2-butanol, iso-amyl alcohol, and active amyl alcohol. Although there is a colorimetric method given by the Korea National Tax Services, it is required to perform an instrumental analysis for quantification of fusel oil in liquors. Until now, limited results have been issued on fusel oil contents of various types of liquor distributed in Korea. Furthermore, it is needed to study a pretreatment method for determining fusel oil levels in liquor which contains suspended matters such as Takju.

In this study, an internal standard curve was prepared and linearity, precision, accuracy, limit of detection (LOD), and limit of quantitation (LOQ) were determined. In addition, recovery rates with respect to ethanol concentrations (5, 10, and 20%) and different sample matrices (Takju, Yakju, Cheongju, beer, fruit wine, brandy, and whiskey) were investigated. Finally, we determined the contents of fusel oil in various types of liquor currently being sold in Korean market.

Materials and Methods

Materials and reagents

The fusel oil standards used in the study, that is, 1-propanol, iso-butanol, 1-butanol, 2-butanol, iso-amyl alcohol, and active amyl alcohol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Also, 3-pentanol used as internal standard was purchased from Sigma-Aldrich. Takju, which was used to check sample pretreatment recovery rates (i.e., filtering and centrifugation), and eight types of liquors (Takju, Yakju, Cheongju, beer, fruit wine, brandy, and whiskey), which were used to check recovery rates from different sample matrices, were purchased from the supermarkets in Seoul. To monitor the content of fusel oils, commercially available Takju (72 types), Yakju (39 types), fruit wines (30 types), diluted Soju (10 types), distilled Soju (3 types), foreign whiskey (12 types), brandy (9 types), domestic beer (39 types), and foreign beers (46 types) were used.

Pre-treatment of samples

For GC analysis of carbonated Takju and beer, carbonic acid was removed by repeated pipetting until no bubbles were generated. Takju was centrifuged (Hanil, HA-1000-3, Hanil Science Co., Daejeon, Korea) for 10 min at 3,000 rpm to remove suspended matter. Separately, 3-pentanol (50 mg) was placed in a 100 mL volumetric flask and then 10% ethanol solution was added to prepare a 3-pentanol (internal standard, IS) solution of 500 mg/L. Two milliliter of the supernatant of Takju obtained after centrifugation was mixed with the same volume of 3-pentanol solution (500 mg/L), and then it was vortexed for 1 min. This mixture was then filtered through a 0.50 μm syringe filter (PTFE, DISMIC-13JP, Tokyo Roshi Kaisha, Tokyo). In order to determine how filtering and centrifugation affected analytical results, we compared the values before and after the addition of each fusel oil standard with 100 mg/L concentration in Takju.

GC analysis of fusel oil

Samples were injected into a GC (Younglin 6100, Younglin, Anyang, Korea) combined with a flame ionized detector (FID). Separation of fusel oils was performed on the DB-624 column (60 m×0.25 mm×1.4 μm, Agilent Technologies, Santa Clara, CA, USA). Helium was used as a carrier gas at a flow rate of 0.7 mL/min. The oven temperature program was as follows: initial temperature of 40℃ for 5 min, increased by 10℃/min to final temperature of 250℃ and held for 10 min. The detector temperature was 280℃. The injector was set at 250℃ with split ratio of 100:1.

Preparation of calibration curves

Fusel oil contents were quantified by dividing the GC peak areas of six fusel oil standard materials at 10, 50, 100, 250, and 500 mg/L by areas of added internal standards (3-pentanol, 250 mg/L). Calibration curves of six fusel oil standards were plotted by setting the ratios between peak areas of fusel oils and peak areas of internal standard materials on the Y axis, and concentrations of fusel oil standards (10, 50, 100, 250, and 500 mg/L) on the X axis. Ratios of peak areas were obtained by averaging the results of five independent experiments.

Linearity, precision, and accuracy

The fusel oil standards (1-propanol, iso-butanol, 1-butanol, 2-butanol, iso-amyl alcohol, and active amyl alcohol) and the internal standard (3-pentanol) were dissolved in the 10% ethanol solution.

Intraday accuracy was determined by conducting experiments for three times in one day using the same conditions, and interday accuracy was obtained by conducting experiments on three separate days using the same conditions. Fusel oil standard materials were prepared by dissolving six types of fusel oils standards in the 10% ethanol aqueous solution to a concentration of 100 mg/L, whereas the internal standard was made up at 250 mg/L. Relative standard deviation (RSD%) was defined as shown below, and results are presented as averages and standard deviations.

RSD (%)=the standard deviation of the response/mean×100

The recovery rates (%) were calculated. Results are the average values of independent experiments performed in triplicate. Recovery rate was defined as:

Recovery rate (%)=C F -C U /C A ×100

  • CF= Concentration of analyte measured in fortified test sample

  • CU= Concentration of analyte measured in unfortified test sample

  • CA Concentration of analyte added to fortified test sample

Limit of detection and Limit of quantitation

GC chromatograms were obtained by injecting the blank (10% ethanol aqueous solution). Peak areas were then obtained by integrating areas near the retention time of each fusel oil standard material and average of peak areas was obtained after five repetitions. Limits of detection (LOD) were obtained by multiplying average peak areas by three, while limits of quantitation (LOQ) were set as ten times average peak areas. LOD and LOQ values were then calculated using gradients and standard deviations using the equations below (6).

Limit of detection (LOD)=3.3×SD/S Limit of quantitation (LOQ)=10×SD/S

Where,

  • S= the slope of the standard curve,

  • SD= the standard deviation of the response

Recovery rates according to ethanol concentration and sample matrices

Since ethanol concentrations of the liquors differed, recovery rates were investigated using aqueous ethanolic solutions of different concentrations. The six fusel oil standard materials (25 mg each) were placed into 25 mL flasks, and volumes were adjusted with ethanol to a fusel oil standard concentration of 1,000 mg/L. Also, 62.5 mg of internal standard material (3-pentanol) was added to a 25 mL flask and its concentration adjusted to 2,500 mg/L with ethanol. The prepared six fusel oil standards and the 3-pentanol solution were taken as 2 mL into 25 mL vial, respectively, and 6 mL of ethanol was added to prepare diluted fusel oil standard solutions. Meanwhile, 5%, 10%, and 20% ethanol solutions were prepared and mixed with the fusel oil standard solutions to obtain a fusel oil standard concentration of 100 mg/L, and an internal standard concentration of 250 mg/L.

Recovery rates were determined for eight different types of liquors (Takju, Yakju, Cheongju, beer, fruit wine, wine, brandy, and whiskey). The six fusel oil standards at a concentration of 1,000 mg/L and 3-pentanol at 2,500 mg/L were prepared in flasks. Standards were aqueous 10% ethanol solutions. The prepared standard solutions of fusel oils (2 mL) were mixed with 2 mL of internal standard solution. These were then diluted to a standard material concentration of 100 mg/L, and an internal standard concentration of 250 mg/L. These standard materials were spiked into the eight liquor types and their recovery rates were determined. The experiment was repeated twice and recovery rates were calculated using:

 Total fusel oil value-sample fusel oil value/added fusel oil value by spiking×100

Monitoring of fusel oils in liquors

Analysis was carried out for Takju (72 types), Yakju (39 types), fruit wines (30 types), diluted Soju (10 types), distilled Soju (3 types), foreign whiskey (12 types), brandy (9 types), domestic beers (39 types), and foreign beers (46 types). Liquors containing more than 20% ethanol content (such as whiskey, brandy, and distilled Soju) were appropriately diluted before analysis.

Statistical analysis

Analysis was carried out twice and averages and standard deviations were calculated. The significances of differences were determined using Duncan's test in SAS Ver. 9.2 (SAS Institute Inc., Cary, NC, USA), and statistical significance was accepted for p<0.05.

Results and Discussion

Effect of pre-treatment (filtering and centrifugation) on results

Samples containing suspended matter (Takju) and fusel oil standard solution were filtered through a polytetrafluorethylene (PTFE) syringe filter. The results obtained are presented in Table 1. The contents of fusel oils in Takju before filtering were 82.83 mg/L (1-propanol), 96.48 mg/L (iso-butanol), 198.98 mg/L (iso-amyl alcohol), and 50.03 mg/L (active amyl alcohol), while values after filtering were 81.51 mg/L (1-propanol), 96.36 mg/L (iso-butanol), 199.80 mg/L (iso-amyl alcohol), and 53.17 mg/L (active amyl alcohol), which showed filtering did not significant affect results (p>0.05). The recovery rate of filtered 100 mg/L fusel oil standard solution was in the range of 102.26-109.62%. Because the recovery rates of the fusel oils were not significantly affected by filtering (p>0.05), it was considered that filtering as a pre-treatment did not affect fusel oil contents.

Table 1. Effect of sample pretreatments (filter ing and centrifugation) for analyzing fusel oils in liquor (unit: mg/L)
Sample Fusel oils Before sample pretreatment After filtering After centrifugation
Takju 1-Propanol 82.83±4.28 81.51±2.63 81.5±4.06
Iso-butanol 96.48±1.70 96.36±0.82 97.37±0.98
1-Butanol ND1) ND ND
2-Butanol ND ND ND
Iso-amyl alcohol 198.98±2.48 199.80±0.30 197.47±7.12
Active amylc alcohol 50.03±0.14 53.17±2.11 51.55±1.95
100 mg/L of fusel oil solution 1-Propanol 105.86±2.50 106.02±1.43 -2)
Iso-butanol 109.38±0.20 109.62±0.22 -
1-Butanol 103.76±5.05 106.16±2.12 -
2-Butanol 104.18±0.74 105.23±2.47 -
Iso-amyl alcohol 101.95±9.14 102.26±7.90 -
Active amyl alcohol 103.02±6.17 103.35±3.80 -

ND, under limit of detection.

-, experiment was not conducted.

Analysis was performed in duplicate and no significant differences were found among samples (before and after treatment) from Duncan test.

Download Excel Table

Centrifugation is an another method used to remove floating matter present in liquor. Fusel oil contents of Takju after centrifugation are presented in Table 1. The contents of fusel oils after centrifugation were 81.5 mg/L (1-propanol), 97.37 mg/L (iso-butanol), 197.47 mg/L (iso-amyl alcohol), and 51.55 mg/L (active amyl alcohol), and these did not differ significantly from results obtained before centrifugation (p>0.05). The analytical methods used in foreign countries were designed for liquors with less suspended matter, such as, beer, wine, brandy, and spirit, than Takju. Since floating matter causes column and injector problems during GC analysis, reproducibility can be negatively affected. In the present study, neither filtering nor centrifugation of Takju to remove suspended matter significantly affected fusel oil content results.

Precision, accuracy, limit of detection, and limit of quantitation

Calibration curves were prepared using the ratios of peak areas of the six standard materials at 10, 50, 100, 250, and 500 mg/L versus the internal standard, and coefficients of correlation (R2) were calculated. The R2 values of calibration curves were >0.99 for all six types of fusel oils examined, which is similar to that found for 1-propanol and iso-amyl alcohol at concentration ranges of 5-500 mg/L and 50-3,000 mg/L, respectively in a previous study (7). Therefore, the reliable result was expected in the concentration range 10-500 mg/L. GC-FID detects component with low boiling points first, and components with greater affinity for the packing material (stationary phase) move more slowly through thecolumn. Of the six types of fusel oils examined, 1-propanol eluted first with a retention time of 11.3 min.

Generally, when alcohols are separated by GC, a polar column with crossbond-polyethylene glycol is used, and it is difficult to separate iso-amyl alcohol and active amyl alcohol using such a column. However, DB-624 column (mid-polarity stationary phase, i.e. 6% cyanopropyl phenyl and 94% dimethyl polysiloxane) can improve the separation of these two components. Iso-amyl alcohol (16.0 min) had a slightly smaller retention time than active amyl alcohol (16.1 min). Relative standard deviations (RSD%) were calculated from average values and standard deviations of fusel oils in order to quantify intraday and interday precisions (Table 2). For intraday precisions, the RSDs% of 1-propanol, iso-butanol, 1-butanol, 2-butanol, iso-amyl alcohol, and active amyl alcohol were 1.07, 1.39, 1.36, 0.81, 0.99, and 1.56%, respectively. For interday precisions RSDs% range from 1.09 to 2.44%. In AOAC, a satisfactory RSD is cited to be 3.7% if analyte concentration is 1,000 mg/L, 5.3% for 100 mg/L, and 7.3% for 10 mg/L (6). Recovery rates were also calculated to determine intraday and interday accuracies. Intraday recovery rates ranged from 98.22% (1-propanol) to 105.26% (active amyl alcohol), and interday rates from 98.53% (1-propanol) to 107.15% (active amyl alcohol). Both precision and accuracy were higher than recommendation. The limits of detection (LOD) and limits of quantitation (LOQ) are presented in Table 2 and their LODs and LOQs fell in the ranges of 1.29-2.58 mg/L and 4.94-8.65 mg/L, respectively. Previously the LOD of fusel oils in a distilled liquor called Raki was in the range of 2-5 mg/L for 1-propanol, 2-butanol, and iso-amyl alcohol (7), which are similar to our results.

Table 2. Relative standard deviation (RSD%), recovery rate (RR %), limit of detection (LOD), and limit of quantitation (LOQ) of the fusel oils
Fusel oils Intraday Interday LOD (mg/L) LOQ (mg/L)
RSD% RR (%) RSD% RR (%)
1-Propanol 1.07 98.22 2.44 98.53 2.58 8.65
Iso-butanol 1.39 104.98 1.6 106.62 1.5 5.59
1-Butanol 1.36 98.95 2.2 102.53 1.52 5.7
2-Butanol 0.81 102.94 1.31 102.83 1.54 5.95
Iso-amyl alcohol 0.99 101.55 1.09 103.03 1.47 5.04
Active amyl alcohol 1.56 105.26 1.5 107.15 1.29 4.94
Download Excel Table
Evaluation of recovery rates

Since various liquor types containing different ethanol concentrations are available, effect of the recovery rate in relation to the concentration of ethanol need to be investigated. Particularly, for the liquors with higher concentration of ethanol, these were diluted to around 20% for the analysis. The recovery rates of the six types of fusel oils with respect to ethanol concentration are presented in Table 3. Notably, the recovery rates for all fusel oils in the presence of 5, 10, and 20% ethanol were not significantly different (p>0.05).

Table 3. Recovery rate (%) and relative standard deviation (RSD%) of fusel oil solutions with different ethanol concentration
Fusel oils Recovery rate (%)
5% Ethanol 10% Ethanol 20% Ethanol Mean RSD%
1-Propanol 101.09±2.93 101.94±1.99 99.75±0.70 100.93±1.11 1.1
Iso-butanol 102.15±0.72 100.77±2.36 102.34±0.20 101.75±0.86 0.85
1-Butanol 99.45±0.88 99.36±1.19 100.92±1.05 99.91±0.88 0.88
2-Butanol 104.68±1.43 105.44±0.14 105.24±0.85 105.12±0.39 0.37
Iso-amyl alcohol 104.43±3.95 102.22±1.39 101.36±0.22 102.67±1.59 1.54
Active amyl alcohol 101.73±3.95 99.50±1.51 102.72±0.44 101.32±1.65 1.63
Download Excel Table

The recovery rate range of the fusel oils in 5% ethanol was 99.45-104.68%, in 10% ethanol was 99.36-105.44%, and in 20% ethanol was 99.75-105.24%. RSDs (%) were in the range of 0.37-1.63%. In case of 2-butanol, the average recovery rate was 105.12% with a RSD of 0.37%, which was somewhat higher than that of other fusel oil components. These results show that recovery rates of fusel oils were unaffected by ethanol concentrations in the 5-20% range. Recovery rates were also investigated for the six types of fusel oil for different sample matrices. For this purpose, we chose Takju, Yakju, Cheongju, beer, obtained before and after spiking with fusel oil standard solution are presented in Table 4. fruit wine, wine, brandy, and whiskey. The recovery rates Recovery rates in brandy was 99.57% for active amyl alcohol, and 92.67% for 1-propanol. The recovery rates of the other liquor types ranged from 106.91% (iso-amyl alcohol in beer) to 93.55% (iso-amyl alcohol in Takju).

Table 4. Recovery rate (%) of fusel oils from different types of liquor
Liquor 1-Propanol Iso-butanol 1-Butanol 2-Butanol Iso-amyl alcohol Active amyl alcohol
Takju 102.68 102.33 106.7 102.58 93.55 105.37
Yakju 96.5 97.56 101.21 98.43 96.8 103.28
Cheongju 94.56 97.11 102.28 101.48 104.96 103.94
Beer 100.21 100.74 106.75 100.77 106.91 103.9
Fruit Wine 105.23 99.35 105.87 101.01 100.12 98.88
Wine 95.12 102.43 102.13 104.75 101.82 104.51
Brandy 92.67 98.36 99.21 98.8 97.56 99.57
Whiskey 93.59 93.75 101.18 99.82 95.06 100.97
Download Excel Table
Monitoring of fusel oil in the liquors sold in Korea

The compositions of fusel oils in domestically available Takju (72 types), Yakju (39 types), fruit wines (30 types), diluted Soju (10 types), distilled Soju (3 types), foreign whiskey (12 types), foreign brandy (9 types), domestic beers (39 types), and foreign beers (46 types) were analyzed (Table 5). The average of the content of total fusel oils (1-propanol, iso-butanol, 1-butanol, 2-butanol, iso-amyl alcohol, and active amyl alcohol) in Takju was 299.3 mg/L. Twenty-three types of sterilized Takju (average 265.9 mg/L) had lower fusel oil contents than 49 types of ordinary Takju (on an average 315.0 mg/L) (data not shown). Notably, iso-amyl alcohol (45.8-345.5 mg/L) and iso-butanol (ND-429.0 mg/L) were higher as compared to others. In the case of Takju, as rice fermentation progresses, iso-amyl alcohol content increased as compared with other alcohols, and it contributes a flavor to the Takju (8). The average of total fusel oil contents in Yakju (39 types) and fruit wines (30 types) were 497.6 and 151.9 mg/L, showing Yakju contains more fusel oils than Takju or fruit wines. In fruit wines, iso-amyl alcohol was the major fusel oil component (6.8-249.0 mg/L). Also, 1-propanol and iso-butanol were also present at maximum levels of 125.9 and 143.1 mg/L, respectively. In a previous report, iso-amyl alcohol content was found to be relatively high in Bokbunja wines (9).

Table 5. Concentration ranges of the content of fusel oils in liquors obtained from the market (unit: mg/L)
Liquor Number 1-Propanol Iso-butanol 1-Butanol 2-Butanol Iso-amyl alcohol Active amyl alcohol Average of total fusel oil contents
Takju 72 Min 24.1 ND1) ND ND 45.8 13 299.3
Max 106.8 429 9.4 ND 345.5 136.3
Yakju 39 Min 19.1 26.3 ND ND 90.9 23.4 497.6
Max 239 273.9 13.4 2.5 302.8 120.2
Domestic beer 39 Min 11.7 3.8 ND ND 27.8 9.5 121.1
Max 47.2 48.5 ND ND 104.6 34.3
Foreign beer 46 Min 13.3 9.5 ND ND 33.3 12.3 114.8
Max 28.5 42.3 5.8 ND 84 32
Fruit wine 30 Min ND ND ND ND 6.8 ND 151.9
Max 125.9 143.1 ND ND 249 69.5
Diluted Soju 10 Min ND ND ND ND ND ND ND
Max ND ND ND ND ND ND
Distilled Soju 3 Min 51.6 79.2 ND ND 114.2 40.6 764.5
Max 197.2 391.9 11.8 ND 421 129.5
Foreign brandy 9 Min 84.2 127.1 ND ND 424 116 1447.3
Max 240.6 609.1 37.8 13 1213.8 323.7
Foreign whisky 12 Min 109.4 217.5 ND ND 167.3 73.1 1137.9
Max 350.1 637.2 21.6 ND 1026.3 492.6

ND, under limit of detection.

Analysis was performed in duplicate.

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Meanwhile, the averages of total fusel oil contents in domestic beers (39 types) and foreign beers (46 types) were similar at 121.1 mg/L and 114.8 mg/L, respectively, and iso-amyl alcohol was the major fusel oil component. Beers contained lower fusel oil levels than other liquor types. In a previous report, iso-butanol concentration in beer was 5.5-22 mg/L, and the propanol concentration was 8.7-23 mg/L (10). In another report (11), 2-methyl- and 3-methyl-l-butanol (active amyl and iso-amyl alcohol) contents in beers were in the range of 51-62 mg/L. Major fusel oil components were similar to that of the present study.

Meanwhile, the six types of fusel oil components were not detected at all in diluted Soju. However, the average of total fusel oil contents was high at concentration of 764.5 mg/L among three types of distilled Soju and iso-amyl alcohol content ranged from 114.2 to 421.0 mg/L. In case of the distilled liquors produced from sugarcane, fusel oil component concentrations, such as, those of n-propyl alcohol (1-propanol) and iso-amyl alcohol, were markedly dependent on liquor type (12), which is probably caused by different grain feedstocks and fermentation conditions. The averages of total fusel oil contents were very high in whiskey (1,137.9 mg/L) and brandy (1,447.3 mg/L), which to some extent might be associated with the unique flavors of these drinks during the aging (13). Notably, iso-amyl alcohol concentrations were much higher than those of other fusel oil components (brandy up to 1,213.8 mg/L, and whiskey up to 1,026.3 mg/L). In all liquor types examined, 1-butanol and 2-butanol contents were much lower than other fusel oil components.

Acknowledgments

This research was supported by a grant (15162MFDS004) from Ministry of Food and Drug Safety in 2015.

References

1.

Ayrapaa T. 1971; Biosynthetic formation of higher alcohols by yeast. Dependence on the nitrogenous nutrient level of the medium. J Inst Brew. 77:266-276.

2.

Hazelwood LA, Daran JM, van Maris AJA, Pronk JT, Dickinson Jr. 2008; The ehrlich pathway for fusel alcohol production a century of research on Saccharomyces cerevisiae metabolism. Appl Environ Microbiol. 74:2259-2266

3.

Lachenmeier DW, Haupt S, Schulz K. 2008; Defining maximum levels of higher alcohols in alcoholic beverages and surrogate alcohol products. Regul Toxicol Pharm Col. 50:313-321

4.

US Department of Treasury. 2014; Alcohol and Tobacco Tax and Trade Bureau method (SSD TM 200): Capillary GC analysis of fusel oils and other components of interest scope and application. Washington DC, USA p. 1-8

5.

Commission of the European Communities. 2000; Commission Regulation (EC) No. 2870/2000: Community reference methods for the analysis of spirits drink. European Union Off J Eur Commun. L333:2046.

6.

AOAC. 2012; Appendix Guidelines for Standard Method Performance Requirements in Official Methods of Analysis. 19th edAssociation of Official Analytical Chemists. Rockville, MD, USA: p. 3-12.

7.

Anli RE, Vural N, Gucer Y. 2007; Determination of the principal volatile compounds of Turkish Raki. J InstBrew. 113:302-309

8.

Park HJ, Lee SM, Song SH, Kim YS. 2013; Characterization of volatile components in Makgeolli, atraditional Korean rice wine, with or without pasteurization during storage. Molecules. 18:5317-5325.

9.

Lim JW, Jeong JT, Shin CS. 2012; Component analysis and sensory evaluation of Korean black raspberry (Rubuscoreanus Mique) wines. Int J Food Sci Technol. 47:918-926

10.

Buckee GK. 1992; Determination of the volatile components of beer. J Inst Brew. 98:78-79

11.

Meilgaard MC. 1982; Prediction of flavor differences between beers from their chemical composition. J Agric Food Chem. 30:1009-1017

12.

Nonato EA, Carazza F, Silva FC, Carvalho CR, Cardeal ZL. 2001; A headspace solid-phase microextraction method for the determination of some secondary compounds of Brazilian sugar cane spirits by gas chromatography. J Agric Food Chem. 49:3533-3539

13.

Schreier P, Drawert F, Winkler F. 1979; Composition of neutral volatile constituents in grape brandies. J AgricFood Chem. 27:365-372