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Lambic beer is the oldest style of beer still being produced in the Western world using spontaneous fermentation. Gueuze is a style of lambic beer prepared by mixing young (one year) and older (two to three years) beers. Little is known about the volatiles and semi-volatiles found in commercial samples of gueuze lambic beers. SPME was used to extract the volatiles from nine different brands of lambic beer. GC-MS was used for the separation and identification of the compounds extracted with SPME. The pH and color were measured using standard procedures. A total of 50 compounds were identified in the nine brands. Seventeen of the 50 compounds identified have been previously identified. The compounds identified included a number of different chemical groups such as acids, alcohols, phenols, ketones, aldehydes, and esters. Ethyl acetate, 4-ethylphenol, and 4-ethylguaiacol are known by-products of the yeast, Brettanomyces, which is normally a spoilage microorganism in beer and wine, but important for the flavor characteristics of lambic beer. There were no differences in pH, but there were differences in color between the beer samples.
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beverages
Article
Acid and Volatiles of Commercially-Available
Lambic Beers
Katherine Thompson Witrick, Susan E. Duncan, Ken E. Hurley and Sean F. O’Keefe *
Department of Food Science and Technology, Virginia Tech, Blacksburg, VA 24061, USA;
kat.a.thompson@gmail.com (K.T.W.); duncans@vt.edu (S.E.D.); hurl@vt.edu (K.E.H.)
*Correspondence: okeefes@vt.edu; Tel.: +1-530-231-4437
Academic Editor: Ombretta Marconi
Received: 10 January 2017; Accepted: 6 October 2017; Published: 26 October 2017
Abstract:
Lambic beer is the oldest style of beer still being produced in the Western world using
spontaneous fermentation. Gueuze is a style of lambic beer prepared by mixing young (one year)
and older (two to three years) beers. Little is known about the volatiles and semi-volatiles found in
commercial samples of gueuze lambic beers. SPME was used to extract the volatiles from nine
different brands of lambic beer. GC-MS was used for the separation and identification of the
compounds extracted with SPME. The pH and color were measured using standard procedures.
A total of 50 compounds were identified in the nine brands. Seventeen of the 50 compounds
identified have been previously identified. The compounds identified included a number of different
chemical groups such as acids, alcohols, phenols, ketones, aldehydes, and esters. Ethyl acetate,
4-ethylphenol, and 4-ethylguaiacol are known by-products of the yeast, Brettanomyces, which is
normally a spoilage microorganism in beer and wine, but important for the flavor characteristics
of lambic beer. There were no differences in pH, but there were differences in color between the
beer samples.
Keywords: lambic beer; solid-phase microextraction; gas chromatography
1. Introduction
Lambic beer is one of the oldest styles of beer still being brewed today [
1
]. Eight lambic
breweries (Belle Vue, Boon, Cantillon, De Troch, Girardin, Lindemans, Mort Subite, Timmermans),
five blenders (De Cam, Drie Fonteinen, Hanssens, Oud Beersel, Tilquin), and two lambic breweries
located in West Flanders (Bockor, Van Honsebrouck) are currently producing and selling lambic beer.
However, the distribution of this type of beer is very limited within the United States [
2
]. Many lambic
brewers and blenders are in financial trouble because of the time required to produce lambic beer; they
can spend up to several years aging in casks or fermentation tanks before they are ready to be sold.
This causes breweries to hold onto hundreds of thousands of dollars’ worth of inventory while the
beer is aging. Another issue that arises from aging lambic beer is that the current tax system in place
forces brewers to pay taxes on their beer within a year of being produced. This is a problem since true
lambic beers are required to age for a minimum of one year. Oftentimes, brewers are in debt to the
government before the beer is even sold. The art and craft of making lambic beer is also dying, because
few people are willing to take the place of retiring brewers [
1
,
3
]. However, in the U.S. craft industry,
there has been a great deal of interest in complex fermented sour beers in the last few years.
Beer is a complex beverage system made up of volatile and semi-volatile compounds belonging
to a number of different chemical classes such as alcohols, ethyl esters, fatty acids, higher alcohol
acetates, isoamyl esters, carbonyl compounds, furanic compounds, terpenoids, C13-norisoprenoids,
and volatile phenols [
4
]. Many chemical compounds play important roles in the appearance, aroma,
flavor, and mouthfeel of alcoholic beverages. Consumers judge the quality, character, and acceptability
Beverages 2017,3, 51; doi:10.3390/beverages3040051 www.mdpi.com/journal/beverages
Beverages 2017,3, 51 2 of 12
of alcoholic beverages based upon visual, olfactory, and taste properties. The aroma profiles of beer
are composed of many different chemical compounds varying in concentrations and polarity [5].
Similar to other alcoholic beverages, beer is made up of a large number (~800) of volatile
and semi-volatile compounds; however, only ten to thirty are aroma active [
6
8
]. Different flavor
compounds can affect the aroma and flavor individually, synergistically, or antagonistically, and not all
compounds affect the aroma of a product equally. Some compounds enhance the background profiles,
while others contribute to the key aroma and flavor characteristics [
9
]. Compounds with the greatest
concentration do not always have the greatest influence on a product’s aroma. In actuality, compounds
with low concentrations often have the greatest influence on the aroma of a product [
10
]. What is
important is the concentration relative to the odor detection threshold in the beer matrix.
The goal of this study was to compare the chemical and volatile compositions of commercially
available lambic beers using GC-MS and HPLC. GC-MS was utilized to analyze the volatiles while
HPLC was used to quantify the acids.
2. Materials and Methods
2.1. Chemicals
Ethyl isobutyrate (99% purity), ethyl butyrate (99% purity), ethyl 2-methylbutyrate (99% purity),
ethyl isovalerate (99% purity), isobutyl alcohol, iso-amyl alcohol, styrene, nonyl aldehyde (95% purity),
ethyl caprylate (99% purity), n-pentadecane (99% purity), decanal, ethyl nonanoate, 1-octanol
(99% purity), isobutryic acid (99% purity), mono-ethyl succinate (95% purity), ethyl undecanoate
(97% purity), 1-decanol (99% purity), ethyl dodecanoate (98% purity), hexanoic acid (99% purity),
n-nonanoic acid (97% purity), decanoic acid (99% purity), lauric acid (99.5% purity), and (
)-ethyl
lactate were purchased from Fisher Scientific (Pittsburg, PA, USA) and used as standards. Acetic
acid with a concentration of 0.150 g/L and L-lactic acid with a concentration of 0.204 g/L were
purchased from R-Biopharm AG (Darmstadt, Germany). Octanoic and hexanoic acids at concentrations
of 5 mg/10 mL were purchased from Fluka Analytical (Sigma-Aldrich, St. Louis, MO, USA).
Isobutyric acid with a concentration of 5 mg/10 mL was purchased from Acros Organics (Geel,
Belgium). Replicate bottles of gueuze lambic beer samples were purchased from local wine and beer
stores in Blacksburg, VA and Athens, GA. The brands were Cuvee Reneégueuze lambic (LK23JGC
2975 23 November 2012), Oude Gueuze Vieille (30 October 2026 L8304), Hanssens Artisan, Cantillon
Gueuze 100% Lambic Bio (3 December 2010 Bottled), 3-Fonteinen (Bottled 23 February 2006), Gueuze
Girardin (XO179), Oude Gueuze Boon (Best before 26 January 2025), Gueuze Boon (02 December 2025),
and Cantillon-Classic Gueuze (13 November 2009 bottled).
2.2. Sample Preparation
The purchased beer was stored at room temperature before analysis. Beer was degassed using
an ultrasonic bath (Model FS20, Fisher Scientific) for 10 min to facilitate a sample measurement.
After the beer was sonicated, it was filtered using a 5 mL syringe with a 0.45
µ
m filter (Fisherbrand
MCE, mixed cellulose ester, Cat 09-719B).
2.3. pH Measurement
pH was measured in triplicate for all bottles of beer immediately after the bottles were opened.
The pH measurements were conducted using an Accumet XL20 probe which was calibrated before use
(Fisher Scientific, Pittsburg, PA, USA).
2.4. Color
Color was measured with the official AOAC 976.08 method using a scanning spectrophotometer
(Shimadzu model UV-2550, Columbia, MD, USA) [11].
Beverages 2017,3, 51 3 of 12
2.5. Solid Phase Microextraction (SPME)
The extraction and concentration of volatile compounds in commercially available gueuze beer
was performed by solid phase microextraction. An SPME fiber (50/30
µ
m DVB/Carboxen/PDMS,
Supelco, Bellefonte, PA, USA) was exposed to the headspace above 4 mL of gueuze beer in 10 mL
headspace vials with a Teflon-lined silicone septa (Chromacol, Fisher Scientific) for 30 min at 40
C with
an agitation speed of 250 rpm. Samples were equilibrated at 40
C for two minutes prior to exposing
the fiber. An AOC-5000 Plus (Shimadzu Scientific, Columbia, MD, USA) SPME autosampler was used
for the automation of extraction and injection. Volatile compounds were desorbed for five minutes
in the injection port of a QP2010 Ultra (Shimadzu, Columbia, MD, USA) gas chromatograph-mass
spectrometer. The injection port was set to 250
C, and all injections were made in splitless mode
using a narrow bore, deactivated glass insert. Volatile compounds were separated using a nonpolar
SHRXI-5MS column (Shimadzu; 30 m
×
0.25 mm i.d.
×
0.25
µ
m film thickness) with He as the carrier
gas at a flow rate of 2.0 mL/min (linear velocity 53.8 cm/sec). The GC oven temperature program was
35
C held for 5 min and then increased to 225
C at a rate of 6
C /min. Once the final temperature
of 225
C was reached, it was maintained for 10 min. The MS was maintained at 200
C and the
sample mass was scanned in the range of 40–800 amu. GCMS was performed to identify the volatile
compounds present in commercial samples of gueuze. Peaks were identified using a standardized
retention time (retention index values, RI) and the fragmentation spectra of standards and the Wiley
2010 mass spectral library.
Identification
RI and Odor. Volatile compounds were identified based upon their RI values using both polar
(DB-Wax) and nonpolar (DB-5) columns (30 m
×
0.25 mm i.d., 0.25
µ
m film; J&W, Folsom, CA, USA).
The RI values were compared to literature values. Aliphatic hydrocarbon standards were analyzed in
the same manner on both the DB-5 and DB-Wax columns to calculate RI:
RI = 100N+ 100n(tRa tRn)/(tR(N+n)tRN )
Nis the carbon number of the lowest alkane and nis the difference between the carbon number
of the two n-alkanes that are bracketed between the compound; t
Ra
,t
Rn
, and t
R(N+n)
are the retention
times of the unknown compound, the lower alkane, and the upper alkane, respectively.
2.6. High Performance Liquid Chromatography (HPLC)
An analysis of acids was conducted using an Agilent 1100 Series LC (Agilent Technologies, Santa
Clara, CA, USA) with a degasser, quaternary pump, autosampler, thermostated column oven, and
a diode array detector (DAD). A 5
µ
m 250 mm
×
4.6 mm (i.d.) Nucleosil phenyl (C
6
H
5
) column
(Macherey-Nagel, Bethlehem, PA, USA) was used at 20
C. The mobile phase consisted of 10 mM
aqueous phosphate buffer at pH 2.5. The wavelength range of 200–400 nm was recorded using the
DAD and used for spectral analysis. The flow rate was 1.0 mL/min and the injection volume was
5
µ
L. External standard curves for acetic and L-lactic acid were made at 200–1200 mg/L concentrations
in beer.
2.7. Chemical Analysis of Lambic Beers
The Enology Service Laboratory at Virginia Polytechnic Institute and State University (Virginia
Tech) is a part of the Wine/Enology Grape Chemistry Group. This is a full service laboratory that was
able to aid in the chemical analysis of the commercially available lambic beer samples. The Enology
Service Laboratory analyzed the reducing sugars (grams/L of glucose and fructose) using AOAC
923.09, pH (AOAC 945.10), titratable acidity (AOAC 950.07), and volatile acidity (TTB variation method
of AOAC 964.08).
Beverages 2017,3, 51 4 of 12
2.8. Statistical Analyses
Statistical analyses were performed with SPSS software for Windows (version 18.0; SPSS Inc.,
Chicago, IL, USA). Statistical analysis of the data for pH, color, and quantified compounds was
performed by one-way analysis of variance with the linear model. Tukey-Kramer HSD was used
to compare the least square means of separation. Brands were considered significant at p< 0.05.
Mean values were reported ±standard deviation (SD).
3. Results and Discussion
3.1. pH
All of the lambic beers examined contained relatively high levels of organic acids. The pH values
of gueuze, kriek (sour cherries), and framboise (raspberries) were lower than typical American lagers.
The pH range previously reported for gueuze lambic beer is 3.20–3.51 [
12
]. The pH of American lagers
tends to range from 3.7 to 4.8 [
1
]. Gueuze has a lower pH than other beer styles because of the additional
microbial activity, resulting in the production of acetic, lactic, and other acids [
1
]. The presence of
acetic or lactic acid bacteria is common and expected in lambic beers; however, in typical beers,
these microorganisms are considered spoilage organisms. Lactic acid bacteria produces off-flavors
and aromas such as honey or sweet butterscotch provided by the vicinal diketones-2,3-butanedione
(diacetyl) and 2,3-pentanedione. Acetic acid bacteria can be hop-insensitive (growth not inhibited by
hop antimicrobial components), similar to lactic acid bacteria, and can be responsible for the ropiness
of beer [13].
The pH range observed for the nine commercial beers that we examined ranged from 3.23 to 3.62
(Table 1). Hanssens Artisan and 3-Fonteine had the lowest pH values of 3.23 and 3.24, respectively.
These samples also had the highest total acidity (Tables 1 and 3). Hanssens Artisan, which had the
lowest pH, also had the highest titratable acidity (TA) at 7.83 g/L, while 3-Fonteine had the second
highest TA at 5.71 g/L. A significant difference in pH was found between all of the beers (Table 1).
Table 1. Comparison of pH levels for Commercial Lambic (Gueuze) Beers.
Brand NSubset for Alpha = 0.05
1 2 3 4
Hanssens Artisan 12 3.23 ±0.01 A
3-Fonteinen 12 3.24 ±0.02 A
Oude Gueuze Boon 6 3.43 ±0.005 B
Cantillon 6 3.44 ±0.01 B
Oude Gueuze Ville 9 3.44 ±0.03 B
Girardin 12 3.44 ±0.02 B
Gueuze Boon 6 3.52 ±0.04 C
Cantillon Bio 9 3.53 ±0.01 C
Cuvee Renée 9 3.62 ±0.02 D
Means followed by same superscript are not significantly different at the 0.05 level using Tukey-Kramer HSD.
3.2. Color
Lambic (gueuze) beer can exhibit a wide range of colors, from golden yellow for young lambic
beer to light amber for older (two to three years) beer. Gueuze typically ranges in color from 8 to
13 degrees SRM (Standard Reference Method) [
1
,
13
]. The color for the beer analyzed ranged from
SRM 6.85 to 10.25 (Table 2). The table shows that there were significant differences in the sample color
(p< 0.05) between samples. A significant difference between the color of Oude Gueuze Vieille and
Girardin was observed. Oude Gueuze Vieille had a color value of 6.85 while Girardin had a color value
of 10.25. Table 2reports the color values of the individual experimental units. The data show that most
of the brands have similar SRM color values with the exception of Girardin. American lagers tend
to be lighter in color than lambic beers; American lagers ranging between 2 and 5 degrees SRM [
1
].
In lambic beers, little color comes from the unmalted wheat used in the mash. The majority of the
Beverages 2017,3, 51 5 of 12
color comes from the lengthy boiling of the wort producing Maillard reaction between amines and
sugars resulting in melanoidins and caramel [
1
,
2
]. Additional color formation comes directly from
the wooden casks themselves, either from the wood or from oxidation during the fermentation and
maturation process [
1
]. It is not unusual for wort used in lambic beer to be boiled for four or more
hours while 60 min is typical for an American lager.
Table 2. Color comparison for commercial lambic (gueuze) beers.
Brand NMeans
Oude Gueuze Vieille
4
6.85 ±1.21 a
Cantillon Bio
3
7.29 ±0.26 ab
Cantillon
2
8.09 ±0.49 ab
Oude Gueuze Boon
2
8.26 ±0.19 ab
3-Fonteinen
4
8.46 ±0.13 abc
Gueuze Boon
2
8.86 ±0.13 bc
Hanssens Artisan
4
9.06 ±0.33 bc
Girardin
2
10.26 ±0.15 c
Means followed by same superscript are not significantly different at the 0.05 level experiment-wise using
Tukey-Kramer HSD.
3.3. Titratable Acidity, Residual Sugar, Lactic Acid, Volatile Acidity, and Ethanol
Because of the high attenuation rate found in gueuze lambic beer, small to trace amounts of
reducing sugars were found (Table 3). In prior research [
14
], only trace amounts (0.8% w/v) were
reported. The amount of reducing sugars in the eight commercial beers tested ranged from 0.7% w/v
to 1.8% w/v. Beers sweetened with syrups tend to contain a higher percentage of reducing sugars
(2% w/v) because these beers tend to undergo a limited secondary fermentation and are quickly
filtered and pasteurized once the fermentation process is complete [
1
]. Cantillon and Boon both
contained the highest percentage of reducing sugars at 1.8% w/v, while Oude Artisan had the lowest
at 0.7% w/v. A gueuze that is called “Oude” is considered an old gueuze that has been allowed to
ferment for three years, unlike traditional gueuzes that are fermented for two years. The lactic acid
(g/L) measured for the lambic (gueuze) beers ranged between 3.67 and 17.47 g/L. Oude Artisan
contained the highest lactic acid at 17.47 g/L while Cantillon had the lowest at 3.67 g/L. The volatile
acidity for the lambic (gueuze) beer ranged from 3.97 g/L to 17.27 g/L, where Oude Artisan had the
highest volatile acidity, while Boon had the lowest. Volatile acidity refers to the organic acids (such as
acetic or butyric acids) that are more volatile or are more easily vaporized than non-volatile or fixed
acids. Total acidity (g/L) for the lambic (gueuze) beer ranged from 2.62 to 7.83 g/L with Oude Boon
exhibiting the lowest value and Oude Artisan having the highest value. Ethanol ranged from 5.64% to
7.16%. Ethanol concentration for gueuze beers has been previously reported to range between 4.25%
and 5.20% [14].
Table 3. Chemical measurements of lambic beer samples.
Name Sample Size RS % w/vTA (g/L) TA-Lactic Acid (g/L) VA (g/L) Ethanol %
Cantillon n= 1 1.8 3.29 3.67 12.59 5.64
Cantillon Bio n= 2 1.2 4.42 ±0.07 9.86 ±0.15 11.15 ±1.53 6.06
3 Fonteine n= 2 1.2 5.71 ±0.32 12.73 ±0.71 7.22 ±1.17 6.39
Girardin n= 1 .90 4.96 11.07 6.30 6.43
Boon n= 1 1.8 2.71 6.06 3.97 6.02
Oude Boon n= 1 1.2 2.62 5.85 4.92 7.16
Hansanns Artisan n= 2 0.70 ±0.14 7.83 ±0.91 17.47 ±2.03 17.27 ±1.65 5.66
Oude Gueuze Vieille n= 2 1.65 ±0.07 2.74 6.10 ±0.01 5.71 ±0.07 6.5
RS—residual sugar; TA—Total Acidity (g/L)—was calculated as lactic acid equivalent Lactic acid (g/L); VA—Volatile
acidity (g/L).
Beverages 2017,3, 51 6 of 12
3.4. Solid Phase Microextraction Analysis of Volatiles
SPME has been used as an extraction technique for volatile and semi-volatile compounds in
beer [
15
]. The DVB/CAR/PDMS SPME fiber was reported by Rodriques et al. [
4
] as able to provide
more complete volatile profiles, due to the wider range of volatile and semi-volatile compounds
detected. A study comparing SPME with continuous liquid-liquid extraction/solvent-assisted flavor
evaporation (CLLE/SAFE) for lambic beer reported that SPME recovered more esters but CLLE/SAFE
recovered a greater number of acid compounds [
16
]. Others have shown that SPME-GCMS is useful
for analysing volatiles in a wide range of beer styles [17].
3.5. Volatile and Semivolatile Compounds
A total of 50 aroma compounds were identified by SPME-GCMS using a combination of retention
index and mass spectral matching against library standards (Table 4). Compounds that could
not be identified by comparing their retention index values were marked as tentatively identified.
The compounds identified belonged to a number of different chemical groups (ketones, acids, alcohols,
and phenols).
Thirty-three of the 50 compounds identified have not been previously reported in gueuze lambic
beer. Seventeen compounds have been reported by both Van Oevelen et al. [
12
] and Spaepen et al. [
18
].
The compounds previously reported by Van Oevelen et al. [
12
] were acetic acid, lactic acid, butyric
acid, propionic acid, isobutyric acid, propanol, butanol, isobutanol, isoamyl alcohol, amyl alcohol,
phenethylalcohol, ethyl acetate, and ethyl lactate. Spaepen and his colleagues [
18
] reported finding
caproic (hexanoic) acid, caprylic (octanoic) acid, capric (decanoic) acid, isoamyl acetate, ethyl
caproate (hexanoate), ethyl caprylate (octanoate), ethyl caprate (decanoate), and phenethyl acetate.
Rossi et al. [
17
] used SPME to characterize the volatiles in different types of beer (lambic was
not analysed) and reported that volatile fingerprints could be characterized based on the type of
fermentation (top versus bottom) and style.
The major chemical classes that account for gueuze lambic beer were alcohols, acids, esters,
phenols, aldehydes, and sulfur compounds. The production of alcohols in beer is a result of
yeast metabolism [
19
]. Of the 50 compounds identified, eight were alcohols. Phenethyl alcohol,
isoamyl alcohol, and isobutanol have been previously reported [
12
] in lambic beer. The compounds
2-methyl-1-butanol [
20
], 1-hexanol [
21
], heptyl alcohol [
22
], 1-octanol [
22
], 2-nonanol [
21
], and
1-decanol [22] have been previously reported in beer, but not lambic beer.
Twenty-three esters were detected using SPME GC-MS. In prior research, only seven have
been previously reported and they are ethyl acetate, lactate, butyrate, caproate, caprylate, caprate,
and phenethyl acetate [
12
,
18
,
23
]. An additional fifteen ethyl esters were detected using SPME.
These compounds are shown in Table 4.
Acids play a vital role in the aroma and flavor profiles of lambic beer. A total of seven acids were
identified using SPME GC-MS. The acids identified were acetic, lactic, isovaleric hexanoic, valeric,
octanoic, and decanoic acid [
12
,
18
]. With the exception of isovaleric and valeric acids, all have been
previously reported in gueuze lambic beer, often associated with the aged hops used. Isovaleric and
valeric acid, however, have been reported in other styles of beer [19].
Beverages 2017,3, 51 7 of 12
Table 4. Chemicals identified within the commercial brands.
Chemical LRI Confirmed Cuvee
Renee
Oude Gueuze
Vielle Cantillon Hanssens
Artisan
Cantillon
Bio
3
Fonteinen Girardin Oude
Boon Boon Compounds
Ethyl acetate 587 628 x x x x x x x x x ester
Propanoic acid 637 668 x x acid
Isoamyl alcohol 683 734 x x x x x x x x x alcohol
2-methyl-1-butanol 689 744 x x x x x x x x x alcohol
Isobutyl acetate 752 776 x x x x x x x x x esters
Ethyl isobutyrate 764 756 x x x x x x x x x ester
Ethyl butyrate 804 800 x x x x x x x x x ester
Furfural (2-furanal) 833 829 x x x x x x x heterocyclic aldehyde
Isovaleric acid (3-Methylbutanoic acid) 851 854 x x x x x x Acid
Butanoic acid, 2-methyl-, ethyl ester (ethyl 2-methyl butyrate) 853 846 x x x x x x x x x ester
Butanoic acid, 3-methyl-, ethyl ester 857 854 x x x x x x x x x ester
2-Furanmethanol 860 866 x x x x x x x furfuyl alcohol
Hexanol 873 880 x x x x x x x x alcohol
Isoamyl Acetate 880 876 x x x x x x x x ester
1-Butanol, 2-methyl-, acetate 882 880 x x x x x x x x x ester
Styrene 888 893 x x x x x x benzene
Lactic Acid 906 x x x acid
1-(2-furanyl)-Ethanone 910 910 x x ketone
5,5-Dimethyl-2(5H)-furanone 952 951 x x x x x x x ketone
Heptyl alcohol 953 962 x x x x x x x x alcohol
Ethyl isohexanoate 966 968 x x x x x x x x ester
1-Propanol, 3-(methylthio)- 977 978 x x x x sulfur
Hexanoic acid (Caproic acid) 986 1019 x x x x x acid
Hexanoic acid, ethyl ester 998 996 x x x x x x x x x ester
Isoamyl lactate 1067 ND x x x x x x x x x ester
Octanol 1070 1072 x x x x x x x x x alcohol
Heptanoic acid, ethyl ester 1097 1097 x x x x x x x x ester
2-Nonanol 1099 1098 x x x x x x x x alcohol
Nonanal 1102 1104 x x x x x x x aldehyde
Valeric Acid 1104 x acid
Isopentyl 3-methylbutyrate (Butanoic acid, 3-methyl-,
3-methylbutyl ester) 1104 1103 x x x x ester
Phenylethyl Alcohol 1110 1118 x x x x x x x x x alcohol
2-ethyl-hexanoic acid 1119 1129 x x x acid
Ethyl benzoate (Benzoic acid etyl ester) 1166 1170 x x x x x x x x x ester
4-ethylphenol 1166 1169 x x x x x x x x x phenolic
Octanoic acid 1180 1179 x x x x x x x x x acid
Octanoic acid, ethyl ester (ethyl caprylate) 1197 1198 x x x x x x x x x ester
Decanal 1204 1209 x x x x x x x x x aldehyde
Benzeneacetic acid, ethyl ester 1244 1244 x x x x x x x x x ester
Isopentyl hexanoate (Isoamyl caproate) 1249 1254 x x x x x x x x x ester
β-Phenethyl acetate (Acetic acid, 2-phenylethyl ester) 1255 1260 x x x x x x x x x ester
Decanol 1271 1272 x x x x alcohol
p-Ethylguaiacol 1278 1287 x x x x x x x x x phenol
Nonanoic acid, ethyl ester 1295 1297 x x x x x x x x ester
Decanoic acid 1367 1373 x x x x x x x acid
Ethyl 9-decenoate 1386 ND x x x x x x x ester
Decanoic acid, ethyl ester (Ethyl decanoate) 1394 1398 x x x x x x x x ester
Octanoic acid, 3-methylbutyl ester 1455 1450 x x x x x x x x ester
Ethyl dodecanoate 1594 1593 x x x x x x x ester
Acetic acid x x x x x x x x Acid
Isobutanol (1-Propanol, 2-methyl-) 647 x x x x x x alcohol
Beverages 2017,3, 51 8 of 12
External standard calibration curves prepared in distilled water were used to quantify isovaleric
acid (IVA), ethyl octanoate, 4-ethylphenol (4EP), 4-ethylguaiacol (4EG), ethyl caprylate, octanol, ethyl
undecanoate, and ethyl acetate (Table 5). Isovaleric acid has been previously reported in beer, but
not in lambic beers [
19
]. Isovaleric acid, 4-ethylphenol and 4-ethylguaiacol are key components in
the overall aroma of Brettanomyces [
24
].The concentration of isovaleric (3-methylbutyric acid) acid
for gueuze lambic beer ranged from 1.92 mg/L for Oude Gueuze Vieille–3.01 mg/L for Cuvee Renée.
Isovaleric acid was found in six of the nine commercial beers (Cuvee Renée, Oude Gueuze Vieille,
Cantillion, Cantillion Bio, Girardin, and Oude Boon). When comparing the means for all the brands,
no difference was found for IVA. Both 4-ethylphenol and 4-ethylguaiacol are known by-products of
the yeast species Brettanomyces. Neither compound, however, has been quantified for lambic beers.
The concentration of 4-ethylphenol ranged from 0.28 mg/L to 1.13 mg/L. Cuvee Renée had the highest
concentration of 4-ethylphenol at 1.13 mg/L and it was found at 0.28 mg/L for both Girardin and
Oude Boon. Table 5includes a comparison of 4-ethylphenol levels in commercial brands. The sensory
detection threshold for 4-ethylphenol is reportedly 425
µ
g/L, and 4-ethylguaiacol has a sensory
threshold of 100
µ
g/L [
25
,
26
]. The 4-ethylguaiacol concentration ranged from 0.52 mg/L to 5.77 mg/L.
Oude Boon was found to have the lowest concentration of 4EG within the commercial brands, while
Cuvee Renée had the highest concentration of 4EG at 5.77 mg/L (Table 5). When 4-ethylphenol is in
the presence of 4-ethylguaiacol, the sensory threshold for 4-ethylphenol is lower [
27
]. The ratio of 4EP
to 4EG is most often reported as 10:1. The ratio, however, can vary between regions and wines [
5
,
25
].
Little is known about the ratio of 4EP:4EG in lambic beers, but in our samples, we obtain a ratio of
0.33:1.
Ethyl octanoate (ethyl caprylate) was the fourth compound quantified. Ethyl octanoate has been
previously reported in the literature as being found in lambic beer. The concentration of ethyl octanoate
found within the literature was reported to be 0.16–0.59 mg/L [
23
]. Ethyl octanoate was found within
all nine commercial brands tested. The concentration of ethyl octanoate ranged from 1.36 mg/L for 3
Fonteinen to 5.72 mg/L for Cantillion. When comparing the means, a difference was found between
the different brands (Table 5).
Octanol has been previously reported in beer [
22
,
28
,
29
], but never specifically lambic beers.
The concentration of octanol ranged from 0.025 mg/L to 0.084 mg/L. Oude Boon, Boon, and Cantillion
Bio all had a concentration of 0.025 mg/L, while Hanssens Artisan had the highest concentration of
0.084 mg/L (Table 5). Ethyl undecanoate has been reported in wine [
30
], brandy [
31
], whiskey [
32
],
cognac [
33
], and rum [
34
], but not beer. Ethyl undecnaote was detected in four of the nine brands.
The range for ethyl undecanoate was 8.6 mg/L to 46.02 mg/L. Cantillion Bio had the lowest
concentration of ethyl undecanoate at 8.6 mg/L, Oude Gueuze Vieille was next at 16.72 mg/L,
Cantillion was third at 28.87 mg/L, and Cuvee Renée had the highest at 46.02 mg/L. (See Table 5).
Ethyl acetate is one of the twenty-seven compounds previously identified in lambic beer [
12
,
23
].
Ethyl acetate was identified in all of the commercial brands of lambic beers. The highest concentration
of ethyl acetate previously reported in the literature for lambic beer was 539.8 mg/L. The average
concentration for refermented gueuze was 60.9–167 mg/L, while filtered gueuze ranged from 33.4 to
67.6 mg/L [
12
]. The concentration of ethyl acetate in the commercial lambic beers ranged from 11.82 to
66.89 mg/L. Boon had the lowest concentration of ethyl acetate and Hanssens Artisan had the highest
concentration (Table 5).
Beverages 2017,3, 51 9 of 12
Table 5. Quantification of Compounds for Commercial Lambic Beers.
Compound mg/L Cuvee Renée Oude Gueuze Vielle Cantillon Hanssens Artisan Cantillon Bio 3 Fonteinen Girardin Oude Boon Boon
Isovaleric acid 3.01 ±1.02 1.92 ±0.06 2.15 ±0.0 2.94 ±0.21 2.95 ±0.37 2.3 ±0.23 —
Ethyl octanoate 5.67 ±1.53 A2.68 ±1.88 AB 5.72 ±1.89 A2.74 ±1.24 BCD 4.52 ±1.54 ABC 1.36 ±1.32 D2.22 ±1.32 CD 1.66 ±1.32 D1.62 ±1.86 D
4-Ethyl phenol 1.13 ±0.02 E0.57 ±0.02 G1.08 ±0.08 EF 0.57 ±0.07 G0.96 ±0.06 F0.44 ±0.03 H0.28 ±0.02 I0.28 I0.32 HI
4-Ethyl guaiacol 5.77 ±0.08 J1.06 ±0.09 L2.44 ±0.07 K1.36 ±0.31 L2.1 ±0.23 K0.99 ±0.06 LM 1.08 ±0.15 L0.52 ±0.01 M0.97 LM
Octanol 0.041 O0.031 ±0.01 O0.052 ±0.01 O0.084 ±0.01 N0.025 O0.034 ±0.01 O0.031 ±0.01 O0.02 O0.02 O
Ethyl undecanoate 46.0 ±9.83 P16.3 ±5.78 QR 28.8 ±1.7 PQ 8.6 ±1.0 R— — —
Ethyl Acetate ND 22.3 ±0.95 V28.4 ±1.09 U66.9 ±4.36 S46.9 ±0.29 T22.1 ±0.75 V21.4 ±2.04 V17.0 ±0.22 VW 11.8 ±0.22 W
Means followed by same superscript are not significantly different at the 0.05 level experiment-wise using Tukey-Kramer HSD. ND signifies not detected.
Beverages 2017,3, 51 10 of 12
3.6. Organic Acids
The organic acids present in beer play important roles in aroma and taste. First, organic acids are
one of the primary groups of compounds that contribute to the sourness. All organic acids have their
own characteristic flavor, aroma, and taste [
35
37
]. Citric acid possesses a fresh acid flavor, which is
very different from that of malic acid, while succinic has both a salty and bitter flavor in addition to its
sourness. Second, acids can help protect beer from harmful microorganisms by decreasing the pH [
36
].
Third, the organic acids present in beer can aid in prolonging the shelf life by providing the beer with
a strong buffering capability [
36
,
38
]. Acetic acid has a flavor threshold of 200 ppm, while lactic acid
has a flavor threshold of 400 ppm [26,39].
Acetic and L-lactic acid were found in varying concentrations within different styles of lambic
beer [
39
]. It has been reported that the concentration of lactic can be as high as 10,000 mg/L for lactic
in ropy lambics and 1200 mg/L for acetic lambics [
12
].The comparison of acetic and lactic acid found
in commercial lambic beer can be found in Table 6. In comparison to gueuzes, ales and lagers have a
much lower concentration of acetic and lactic acid. Ales and lagers normally contain anywhere from
60 to 140 ppm acetic acid. The concentration of acetic acid in gueuze beer can range between 500 and
1500 mg/L. The concentration of acetic acid in the commercial samples ranged from 723 mg/L for
Oude Boon to 1642 mg/L for Hanssens Artisans. There was no difference in acetic acid concentration
between the different brands (p> 0.05).
Table 6. Comparison of Acids within Commercial Brands of Lambic Beer.
Brand Acetic Acid (mg/L) * Lactic Acid (mg/L)
Cuvee Renee 916 ±12.02 2557 ±8.26
Oude Gueuze Villie 1019 ±211.43 1094 ±13.54
Cantillion 1224 ±1.41 1417 ±184.25
Hanssens Artisan 1642 ±847.82 1389 ±55.64
Cantillon Bio 1473 ±79.21 1658 ±890.05
3 Fonteinen 1204 ±129.78 1294 ±61.2
Girardin 1499 ±109.53 1403 ±96.95
Oude Boon 723 ±8.48 1228 ±14.12
Boon 1137 ±13.44 995 ±18.55
* No difference was found between the brands.
The concentration of lactic acid in gueuze beer can range between 1500 and 3500 mg/L, while
typical American lagers tend to have much lower concentrations, around 40–150 ppm [
1
]. Table 6shows
a comparison of means for lactic acid. The concentration of lactic acid ranged from 1098 to 2979 mg/L.
Cantillon Bio had the highest level of lactic acid at 2979 mg/L followed by Cuvee Renée at 2563 mg/L.
Oude Gueuze Villie had the lowest concentration of lactic acid at 1098 mg/L. Based upon the
comparison of means, Cantillon Bio is significantly different from Girardin, Cantillion, Hanssens
Artisans, 3-Fonteinen, Boon, Oude Boon, and Oude Gueuze Viellie. Cuvee Renée was found not to be
significantly different from any of the other brands.
The origins of the volatiles in gueuze lambic include raw materials, and chemical/microbial
changes occurring during fermentation and aging. Spitaels et al. [
40
,
41
] reported a consistent
core microbial population including Pediococcus damnosus,Dekkera anomala,Dekkera bruxellensis,
Saccharomyces cerevisiae, and S. pastorianus during production to just D. bruxellensis after production.
Brettanomyces and Dekkera are used interchangeably with Dekkera most often used to describe the spore
form of the yeast. Extended aging was associated with higher levels of ethyl lactate and lower levels of
isoamyl acetate and ethyl decanoate with aging. Thus, the differences in some of the volatiles that we
have observed in our samples could represent production or age differences.
Beverages 2017,3, 51 11 of 12
4. Conclusions
In this study, the volatile and semi-volatile compounds of nine commercial brands of lambic
(gueuze) beer were identified using SPME-GC/MS and HPLC. A total of 50 volatile and semi-volatile
compounds were identified in the nine commercial brands. Of the 50 compounds identified, seventeen
of them have been previously identified in the literature. Ethyl acetate was found at 11.8–66.9 mg/L
and 4EG at 0.52–5.77 mg/L. Acetic and lactic acids were identified and quantified using HPLC with
ranges observed from 723–1642 mg/L and 995–2557, respectively. The results show the range of typical
values for volatile aroma compounds and acids in Belgian lambic beers and can help clarify the reasons
for some of the variation in flavor characteristics observed in commercial products.
Acknowledgments:
Funding for this work was provided in part by the Virginia Agricultural Experiment Station
and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of agriculture.
Author Contributions:
K.T.W., S.E.D. and S.F.O. conceived and designed the experiments; K.T.W. and K.E.H
performed the experiments; K.T.W. and S.F.O. analyzed the data; K.T.W. and S.F.O. wrote the paper.
Conflicts of Interest:
The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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... [13,14] In beer, 4-ethylphenol (medicinal) is found in lower concentrations than 4-ethylguaiacol (clove, spicy). [49,50] With the exception of the 100 mg/L beer, the concentration of 4-EP was found to be above the threshold value (0.10 mg/L) within the control, 50 mg/L, and 75 mg/L sample, which ranged from 0.27 to 0.45 mg/L. Similar results were also seen with 4-EG. ...
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A much-anticipated revision of a benchmark resource, written by a renowned author, professor, and researcher in food flavors, Flavor Chemistry and Technology, Second Edition provides the latest information and newest research developments that have taken place in the field over the past 20 years. New or expanded coverage includes: Flavor and the Information Age. Food/Flavor interactions. Flavoring materials and flavor potentiators. Changes to food flavors during processing. Off-Flavors in foods. Performance of flavors during processing and storage. Applications of flavorings in processing. One of the many highlights of the new edition is the chapter on food/flavor interactions and flavor release in the mouth. Addressing one of the hottest topics in flavor today, the chapter presents current knowledge on criical issues such as why low-calorie foods do not taste as good as their full-calorie counterparts. The greatest changes in the book have been made to the chapter on food applications. The author supplies a compelling explanation of how flavors interact with basic food components and how these perform during processing and storage. The chapter on flavor production has been updated to include the latest information on the controlled release of flavorings. Actively involved in flavor research for 35 years, author Gary Reineccius is an award-winning flavor chemist. Drawing on his years of academic and practical experience, he focuses on the technology of flavors and applications in processing to provide a complete overview of the field.
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A solid-phase microextraction (SPME) headspace sampling technique has been applied to the gas chromatographic (GC) analysis of fruit-flavored malt beverages. The procedure provides an alternative to direct headspace, solvent extraction, and purge and trap methods for the monitoring of volatile components. The sampling technique is readily adapted to most capillary gas chromatographic systems with flame ionization detectors (FID). Over 40 components were identified by mass spectroscopy (MS) and monitored by GC-FID to evaluate 14 products containing raspberry, cherry, apple, and apricot flavors. Control of sample temperature and volume as well as SPME fiber position were important factors in obtaining consistent responses required for quantitation. Further application of this technique to monitor volatiles in unflavored beers and malt beverages is apparent.
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Organic acids in malt play an important role in determining the pH and buffering capability of wort. The organic acids content of commercial malts and factors influencing this content were investigated with the intention of better controlling organic acids in the required range. The content and proportion of each organic acid in different commercial malts varied greatly. Analysis of the correlation between organic acids and corresponding malt specifications indicated that malic acid in malts had a high positive correlation with Kolbach index (0.961), and pyruvic acid showed good negative correlation (-0.869) with acetic acid in malts. The difference in patterns of change for seven organic acids (pyruvic, malic, lactic, acetic, citric, fumaric, and succinic) during steeping, germinating, and kilning are also described in detail. One-at-a-time experiments confirmed that barley variety, malting conditions, and microorganisms were mainly responsible for the great variety in organic acids content in final malts, which may be very useful information for helping brewers to improve malt quality and satisfy brewery requirements.
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Chemometrics is the application of principles of measurement science and multivariate mathematics and statistics to efficiently extract maximum useful information from data. It can be applied to sensory, chemical, and biological measurements and typically is applied when multiple measurements are made on a set of samples. Exploratory data analysis (EDA) is often used to simplify and gain better understanding of large, complicated data sets. EDA can also be used to determine how many fundamental properties are represented in a data set and the extent to which measurements are redundant. Pattern recognition (PARC) can be used to identify the cultivar or growing area of a raw material or the brand or production plant in which a product was made from its pattern of analytical results. Advanced PARC procedures can detect adulteration or be used for multivariate quality assurance or quality control. Empirical modeling has many applications, including development of analytical methods, discerning the relationships between product composition and sensory properties, developing knowledge of relationships between molecular structure and biological properties, and developing control algorithms for unit operations or processes.
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The microbiota involved in lambic beer fermentations in an industrial brewery in West-Flanders, Belgium, was determined through culture-dependent and culture-independent techniques. More than 1300 bacterial and yeast isolates from 13 samples collected during a one-year fermentation process were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by sequence analysis of rRNA and various protein-encoding genes. The bacterial and yeast communities of the same samples were further analyzed using denaturing gradient gel electrophoresis of PCR-amplified V3 regions of the 16S rRNA genes and D1/D2 regions of the 26S rRNA genes, respectively. In contrast to traditional lambic beer fermentations, there was no Enterobacteriaceae phase and a larger variety of acetic acid bacteria were found in industrial lambic beer fermentations. Like in traditional lambic beer fermentations, Saccharomyces cerevisiae, Saccharomyces pastorianus, Dekkera bruxellensis and Pediococcus damnosus were the microorganisms responsible for the main fermentation and maturation phases. These microorganisms originated most probably from the wood of the casks and were considered as the core microbiota of lambic beer fermentations. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Lambic is a beer style that undergoes spontaneous fermentation and is traditionally produced in the Payottenland region of Belgium, a valley on the Senne River west of Brussels. This region appears to have the perfect combination of airborne microorganisms required for lambic's spontaneous fermentation. Gueuze lambic is a substyle of lambic that is made by mixing young (approximately 1 year) and old (approximately 2 to 3 years) lambics with subsequent bottle conditioning. We compared 2 extraction techniques, solid-phase microextraction (SPME) and continuous liquid–liquid extraction/solvent-assisted flavor evaporation (CCLE/SAFE), for the isolation of volatile compounds in commercially produced gueuze lambic beer. Fifty-four volatile compounds were identified and could be divided into acids (14), alcohols (12), aldehydes (3), esters (20), phenols (3), and miscellaneous (2). SPME extracted a total of 40 volatile compounds, whereas CLLE/SAFE extracted 36 volatile compounds. CLLE/SAFE extracted a greater number of acids than SPME, whereas SPME was able to isolate a greater number of esters. Neither extraction technique proved to be clearly superior and both extraction methods can be utilized for the isolation of volatile compounds found in gueuze lambic beer.Practical ApplicationThis work compares 2 common flavor extraction techniques for the analysis of flavor compounds present in gueuze lambic beer and characterizes the flavor compounds present. The results provide information important for the production and characterization of gueuze lambic beer that will be useful for brewers interested in this unique style.