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].
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 5 includes a comparison of 4-ethylphenol levels in commercial brands. The sensory detection threshold for 4-ethylphenol is reportedly 425 μg/L, and 4-ethylguaiacol has a sensory threshold of 100 μg/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).
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,
36,
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).
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 6 shows 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.