Acid and Volatiles of Commercially-Available Lambic Beers

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.


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 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][7][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.

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 µm filter (Fisherbrand MCE, mixed cellulose ester, Cat 09-719B).

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).

Color
Color was measured with the official AOAC 976.08 method using a scanning spectrophotometer (Shimadzu model UV-2550, Columbia, MD, USA) [11].

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 µm 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 µm 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 µm 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: N is the carbon number of the lowest alkane and n is 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.

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 µm 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 µL.External standard curves for acetic and L-lactic acid were made at 200-1200 mg/L concentrations in beer.

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).

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).

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).Means followed by same superscript are not significantly different at the 0.05 level using Tukey-Kramer HSD.

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 2 reports 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 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.Means followed by same superscript are not significantly different at the 0.05 level experiment-wise using Tukey-Kramer HSD.

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].RS-residual sugar; TA-Total Acidity (g/L)-was calculated as lactic acid equivalent Lactic acid (g/L); VA-Volatile acidity (g/L).[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].

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).
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-3.01mg/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 0.16-0.59mg/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).Means followed by same superscript are not significantly different at the 0.05 level experiment-wise using Tukey-Kramer HSD.ND signifies not detected.

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).

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 11.8-66.9mg/L and 4EG at 0.52-5.77mg/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.

Table 1 .
Comparison of pH levels for Commercial Lambic (Gueuze) Beers.

Table 3 .
Chemical measurements of lambic beer samples.

Table 4 .
Chemicals identified within the commercial brands.

Table 5 .
Quantification of Compounds for Commercial Lambic Beers.

Table 6 .
Comparison of Acids within Commercial Brands of Lambic Beer.