Further Elucidation of Beer Flavor Instability: The Potential Role of Cysteine-Bound Aldehydes Jeroen J. Baert,1 Jessika De Clippeleer, Barbara Jaskula-Goiris, Filip Van Opstaele, Gert De Rouck, Guido Aerts, and Luc De Cooman, KU Leuven, Department of Microbial and Molecular Systems (M2S), Cluster for Bioengineering Technology (CBeT), Laboratory of Enzyme, Fermentation and Brewing Technology (EFBT), Technology Campus Ghent, Gebroeders De Smetstraat 1, 9000 Ghent, Belgium research by Baert et al. in model systems (4) failed to show significant formation of imines under malt-, wort-, and beer-like production conditions. In contrast, binding of aldehydes with bisulfite, a well-known carbonyl scavenger, was clearly confirmed in the model solutions. The study of Baert et al. (4) also points to a type of bound-state aldehyde not yet reported in connection with beer flavor instability (i.e., 2-substituted 1,3-thiazolidine-4-carboxylic acids). Indeed, cysteine, an amino acid containing a sulfhydryl group, showed strong reactivity and binding toward typical aging aldehydes such as (E)-2-nonenal. Whereas saturated aldehydes such as nonanal are limited to monoadduct formation, α,β-unsaturated aldehydes such as (E)-2-nonenal may give rise to both monoand diadducts (Fig. 1). In this study, focus will be on the formation of bisulfite and cysteine adducts in model systems and, more specifically, on the subsequent release of aldehydes from these bound-state precursor forms. Based on findings described in literature, we hypothesize that oxidative losses of free bisulfite and free cysteine in aging beer shift the equilibria of the bound-state aldehydes, resulting in aldehydes being released from the dissociating adducts. Bisulfite has been utilized for decades in beer production for its antioxidant capacity (15,22), and it is well known that its concentration decreases over time during beer aging (16,17). More recently, the antioxidant activities of thiols (i.e., compounds containing sulfhydryl groups) have been investigated and linked to beer flavor stability (9,26,30,31). Lund and Andersen (23) found a positive correlation between sulfite content, thiol content, and oxidative stability by performing principal component analysis on 12 different beers. Although prooxidant properties were reported in one study (2), cysteine, in particular, may exhibit antioxidant behavior (18), potentially even stronger than glutathione (14); and, in aging beer, its concentration was seen to decrease over time (26). Furthermore, during beer storage, release of volatiles has been reported from cysteine-Sconjugates; for example, from S-3-(hexan-1-ol)-cysteine (7). In this study, after allowing for adduct formation, the compound 4-vinylpyridine (4VP), known for decades for its high reactivity toward thiols and used as an alkylating agent in peptide mapping (1,3,6,12,24,27), was added to the samples, either to reduce the amount of free cysteine in the case of cysteine-binding experiments, or by acting as a strong base, causing the release of free bisulfite in cases of bisulfite binding assays. As a result, bound-state aldehydes will shift into their free, volatile form, which can be quantified by headspace solid-phase microextraction (HS SPME), followed by gas chromatographic separation and mass spectrometric detection (GC-MS).
ABSTRACT J. Am. Soc. Brew. Chem. 73(3):243-252, 2015 The potential involvement of 2-substituted 1,3-thiazolidine-4-carboxylic acids in beer flavor stability was further investigated. The binding behavior of beer-aging aldehydes toward both cysteine and bisulfite was confirmed and compared in model solutions of varying pH values that are relevant to malting and brewing (pH 6.0, 5.2, and 4.4). It was found that binding of aldehydes increased with increasing pH, especially for binding to cysteine. Furthermore, a sample preparation approach was developed to release aldehydes from their bound-state. After binding between aldehydes and cysteine, the strong base 4-vinylpyridine (4VP) was added to the samples as a competitor of the aldehydes toward cysteine binding, and subsequent release of aldehydes was clearly observed. The same approach also resulted in a release of aldehydes from preformed bisulfite adducts. Sample treatment with 4VP was also applied to fresh pale lager beer, resulting in increased levels of free beer-aging aldehydes. Moreover, the presence of furfuralderived 1,3-thiazolidine-4-carboxylic acid in fresh pale lager beer was confirmed and quantified by a newly developed ultra-performance liquid chromatography-UV method. The insights gained in this study strengthen the hypothesis that 2-substituted 1,3-thiazolidine-4-carboxylic acids may play an important role in beer flavor stability or instability. Keywords: Aldehyde, Beer flavor stability, Bisulfite, Cysteine, Thiazolidine-4-carboxylic acid, 4-Vinylpyridine
Beer shows a limited shelf life compared with many other beverages, with several undesirable aging flavors arising upon storage. Aged beer flavor is strongly associated with “staling” or “aging” aldehydes (5), which are volatile compounds with low to very low and specific flavor threshold values. At beer bottling, the concentrations of various aging aldehydes are mostly below their respective flavor thresholds but, with increasing storage time, the aldehyde levels increase, giving the beer highly unpleasant aged flavors. Several major chemical and enzymatic pathways that contribute to aldehyde formation during malting and wort and beer production are unlikely to have a significant impact on aldehyde formation in packaged beer and, consequently, the mechanisms behind increasing aging aldehyde levels during packaged beer storage remain unclear (5). Several studies indicate that fresh beer contains boundstate aldehydes, formed during the wort and beer production processes. From this bound state, aldehydes might be released during beer storage, thus causing beer flavor instability (19–21,25,29,32). The different types of bound-state aldehydes that are most likely formed during wort and beer production, and that may give rise to free aldehydes during beer aging, are summarized in Figure 1. Imines, derived from the reaction of an aldehyde with the amino group (e.g., of amino acids, peptides, and proteins), have been proposed as a potential source of aging aldehydes (5). However,
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EXPERIMENTAL Chemicals Hexanal (≥95%, CAS 66-25-1), (E)-2-nonenal (≥95%, CAS 18829-56-6), furfural (≥99%, CAS 98-01-1), 2-methylpropanal (≥99%, CAS 78-84-2), 2-methylbutanal (≥95%, CAS 96-17-3), 3-methylbutanal (≥98%, CAS 590-86-3), methional (≥97%, CAS
Corresponding author. Phone: +32 9 265 86 13; Fax +32 9 265 87 24;
[email protected]
http://dx.doi.org/10.1094/ASBCJ-2015-0531-01 © 2015 American Society of Brewing Chemists, Inc.
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3268-49-3), phenylacetaldehyde (≥98%, CAS 122-78-1), and benzaldehyde (≥98%, CAS 100-52-7) were purchased from Acros Organics (Geel, Belgium). Deuterated 2-methylbutanal (2-methylbutanal-d10, 100 atom %D, ≈200 mg in 1.5 mL of CDCl3) was synthesized upon ordering. Deuterated benzaldehyde (benzaldehyde-d6, 98 atom %D, CAS 17901-93-8) was purchased from Sigma-Aldrich (St. Louis). Aliquots of the purchased aldehydes were transferred to ethanol and stored at –25°C in amber glass screw-capped vials. L-Cysteine (≥97%), 4VP (≥95%, containing hydroquinone at 100 ppm), hydrochloric acid (37%), o-(2,3,4,5,6pentafluorobenzyl)hydroxylamine hydrochloride (derivatization grade for GC, 99%, CAS 57981-02-9), and 2-(furan-2-yl)-1,3thiazolidine-4-carboxylic acid were obtained from Sigma-Aldrich. Potassium disulfite (≥96%), disodium hydrogen phosphate dihydrate (≥99.5%), sodium dihydrogen phosphate dihydrate (≥98%), sodium hydroxide (pellets for analysis), ethanol (≥99.2%), and acetic acid (≥99%, for synthesis) were purchased from Merck (Darmstadt, Germany). Methanol (high-performance liquid chromatography grade) was purchased from Acros Organics. All water used was ultrapure type-1 grade (mQ, 18.2 MΩcm at 25°C), obtained from a Synergy 185 system from Millipore S.A. (Molsheim, France). Nitrogen gas (N28), helium (P0252), and methane (N46) were obtained from Air Liquide (Liège, Belgium). Beer Samples All commercial pale lager beers used in this study (beer A to beer E ) were purchased at a local supermarket, in glass bottles of either 250 or 330 mL. All brands originated from European breweries and were selected based on general popularity and sufficiently high stock turnover in the store to guarantee freshness. For at least one brand (beer D), rice was used as an adjunct, and for another brand (beer E), maize, as advertised by the respective companies. The alcohol content of the beers ranged from 5.0 to 5.2% ABV.
Quantification of Beer-aging Aldehydes Offline sample preparation. For experiments on model systems, buffer solutions were sparged with nitrogen gas in a Duran bottle with customized screw cap and transferred to an anaerobic cabinet (Whitley Anaerobic Workstation A100 model B; Don Whitley Scientific Ltd., West Yorkshire, UK), then placed under a nitrogen atmosphere at room temperature. After spiking of the appropriate compounds to the buffer, 4-mL aliquots were transferred to transparent 20-mL glass vials. The vials were closed with magnetic bimetal crimp caps with polytetrafluoroethylene (PTFE)/silicone septa (Sigma-Aldrich). Where applicable, heat treatment was applied by placing the samples in an incubator (B8054S; Termaks A.S., Bergen, Norway). An external calibration curve was used for aldehyde quantification in these model systems. Calibration standards were prepared by manually adding 40 µL of the respective aldehyde calibration stock solution, prepared in ethanol, into the vial through the septum via a gas-tight syringe. For aldehyde quantification in beer samples, the beer bottles were opened in the anaerobic cabinet and 4-mL aliquots were transferred to 20-mL glass vials, which were sealed with magnetic bimetal crimp caps. A standard addition approach was used for aldehyde quantification in beer, thus manually adding 40 µL of the respective aldehyde calibration stock solution into the vial containing the beer through the septum via a gas-tight syringe. Specific details regarding the composition and treatment of the samples per experiment are listed below. Assessment of interaction between nonanal or (E)-2-nonenal and cysteine, and subsequent treatment aimed at aldehyde release. Phosphate buffer (0.05 M, pH 4.4, 5.2, or 6.0) was spiked with nonanal and (E)-2-nonenal (1 µM each) (Fig. 2). Where applicable, cysteine was added to 500 µM, and all samples were placed at 50°C for 1 hr. Subsequently, where indicated, 4VP (purchased stock), NaOH (0.6 M), or HCl (0.3 M) was added to
Fig. 1. General overview of interactions between saturated (top) (e.g., nonanal) or α-unsaturated (bottom) (e.g., (E)-2-nonenal) aldehydes and cysteine, an amine, or bisulfite, respectively (4,5).
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≈500 mM 4VP at pH 10 or 2, as part of the online sample preparation to both samples and the external calibration standards. Assessment of interaction between nonanal or (E)-2-nonenal and bisulfite, and subsequent treatment aimed at aldehyde release. Phosphate buffer (0.05 M, pH 4.4, 5.2, or 6.0) was spiked with nonanal and (E)-2-nonenal (1 µM each) (Fig. 3). Where applicable, bisulfite was added to 500 µM, and all samples were placed at 50°C for 1 hr. Subsequently, where indicated, 4VP (purchased stock), NaOH (0.6 M), or HCl (0.3 M) was added to ≈500 mM 4VP at pH 10 or 2, as part of the online sample preparation to both samples and the external calibration standards. Assessment of interaction between aging aldehydes and cysteine, and subsequent treatment with 4VP aimed at aldehyde release. Phosphate buffer (0.05 M, pH 4.4, 5.2, or 6.0) was spiked with hexanal, (E)-2-nonenal, furfural, 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, methional, phenylacetaldehyde, and benzaldehyde (1 µM each) (Fig. 4). Where applicable, cysteine was added to 500 µM, and all samples were placed at 50°C for 1 hr. Where indicated, 4VP was added to 500 mM as part of the online sample preparation to both samples as well as the external calibration standards. Assessment of interaction between aging aldehydes and bisulfite, and subsequent treatment with 4VP aimed at aldehyde release. Phosphate buffer (0.05 M, pH 4.4, 5.2, or 6.0) was spiked
with hexanal, (E)-2-nonenal, furfural, 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, methional, phenylacetaldehyde, and benzaldehyde (1 µM each) (Fig. 5). Where applicable, bisulfite was added to 500 µM, and all samples were placed at 50°C for 1 hr. Where indicated, 4VP was added to 500 mM as part of the online sample preparation to both samples as well as the external calibration standards. Assessment of 4VP addition to fresh commercial pale lager beer in relation to the release of aldehydes. Per beer brand, two different bottles were measured (Fig. 6). For each bottle, a standard addition calibration curve was prepared as such, and an additional standard addition calibration curve was prepared with 4VP added to 500 mM to each calibration standard. Online sample preparation. For sample treatments that included the addition of 4VP, NaOH, or HCl, the designated vial was automatically transferred from a cooled metal tray (7°C) to a heated agitator tray (30°C). Subsequently, 4VP, NaOH, or HCl addition was performed by a 500-µL 1750N CTC syringe (Hamilton Company, Bonaduz, Switzerland) mounted on a GCPAL autosampler (CTC Analytics AG, Zwingen, Switzerland). The sample was homogenized by shaking vigorously at 500 rpm for 2 min (cycles of 5 sec of shaking and 2 sec of rest), and it was subsequently transferred back to the cooled metal tray, where it rested for at least 6.5 hr before proceeding. The steps described here
Fig. 2. Assessment of interaction between nonanal or (E)-2-nonenal and cysteine, and subsequent treatment aimed at aldehyde release. Phosphate buffer (0.05 M, pH 4.4, 5.2, or 6.0) was spiked with nonanal and (E)-2nonenal. All samples were placed at 50°C for 1 hr before additional treatment; n = 3, error bars = standard error. A, Samples: ref. = samples without additional treatment, Cys = samples additionally spiked with Cys at onset of the reaction, and 4VP = samples additionally treated with 4vinylpyridine after the reaction. B, NaOH = samples additionally treated with NaOH to pH 10 after the reaction. C, HCl = samples additionally treated with HCl to pH 2 after the reaction.
Fig. 3. Assessment of interaction between nonanal or (E)-2-nonenal and bisulfite, and subsequent treatment aimed at aldehyde release. Phosphate buffer (0.05 M, pH 4.4, 5.2, or 6.0) was spiked with nonanal and (E)-2nonenal. All samples were placed at 50°C for 1 hr before additional treatment; n = 3, error bars = standard error A, Samples: ref. = samples without additional spiking, SO2 = samples additionally spiked with bisulfite at onset of the reaction, and 4VP = samples additionally treated with 4-vinylpyridine after the reaction. B, NaOH = samples additionally treated with NaOH to pH 10 after the reaction. C, HCl = samples additionally treated with HCl to pH 2 after the reaction.
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were omitted for samples that were not treated with 4VP, NaOH, or HCl. At the start of all analyses, the designated vial was automatically transferred from the cooled metal tray to the heated agitator tray. With the 500-µL syringe, 80 µL of aqueous internal standard working solution (prepared weekly) was added into the sample vial through the septum via a gas-tight syringe, resulting in 2-methylbutanal-d10 at 20 µg L–1 and benzaldehyde-d6 at 2 µg L–1 used as internal standards. The sample was subsequently homogenized by shaking vigorously at 500 rpm for 2 min (cycles of 5 sec of shaking and 2 sec of rest). HS SPME with on-fiber derivatization. A polydimethylsiloxane/divinylbenzene SPME fiber (Stableflex SPME fiber assembly; Supelco Analytical, Bellefonte, PA), loaded on a CombiPAL autosampler (CTC Analytics AG, Zwingen, Switzerland), was first positioned in the headspace of 10 mL of an aqueous solution of o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) at 1 g L–1. For each batch of samples, this stock was freshly prepared in a transparent 20-mL glass vial and placed in the 30°C agitator tray. For each sample, the fiber was loaded with PFBHA during 10 min of exposure to the headspace while shaking at 250 rpm in cycles of 5 sec of shaking and 2 sec of rest. The PFBHA-loaded fiber was subsequently exposed during 30 min to the sample’s headspace for extraction and derivatization of aldehydes, while shaking at 250 rpm in cycles of 5 sec of shaking and 2 sec of rest, at a temperature of 30°C. Possible carryover by the SPME fiber was prevented by conditioning it for 3 min at 250°C in the fiber-conditioning module prior to each new sample collection.
GC-MS. The pentafluorobenzyloximes (PFBOs), formed from the on-fiber derivatization of the aldehydes with PFBHA, were thermally desorbed from the solid phase by insertion of the fiber into the injector of a Focus GC (Thermo Fisher Scientific Inc., Waltham, MA) for 3 min at 250°C. The injector contained a narrow-bore glass liner with a volume of 0.5 mL. The split/splitless injection port was used in the split mode, with a split flow of 10 mL min–1 and a split ratio of 12. An Rtx-1 Crossbond 100% dimethyl polysiloxane capillary column (40-m length, 0.18-mm internal diameter, and 0.20-µm film thickness; Restek Corporation, Bellafonte, PA) was used, with the helium carrier gas set at a flow rate of 0.8 mL min–1. Upon injection, the GC oven temperature program initiated at 50°C for 2 min and increased at a rate of 6°C min–1 to 250°C. This final temperature was held for 5 min. The MS transfer line was set at 260°C. An ISQ single-quadrupole mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) was used for detection, operating in the negative chemical ionization mode, with the ion source temperature set at 185°C. The reagent gas was methane, at a flow rate of 1.5 mL min–1. The electron lens was set at 15 V, the electron energy at 70 eV, the emission current at 50 µA, and the detector gain at 3.00 × 105. One characteristic ion per PFBO, with a negative charge, was used for identification and quantification in selected ion monitoring mode. For furfural, the selected characteristic ion was [M-HF-NO]– and, for phenylacetaldehyde, the [M-HF-HCN]– fragment was selected; whereas, for all other marker aldehydes, the [M-HF]– ion was chosen.
Fig. 4. Assessment of interaction between aging aldehydes and cysteine, and subsequent treatment with 4-vinylpyridine (4VP) aimed at aldehyde release. Phosphate buffer (0.05 M, pH 4.4, 5.2, or 6.0) was spiked with a mixture of aldehydes. All samples were placed at 50°C for 1 hr; n = 3, error bars = standard error. Samples: ref. = samples without additional spiking, Cys = samples additionally spiked with cysteine at onset of the reaction, and 4VP = samples additionally treated with 4VP after the reaction.
Beer Flavor Instability: Potential Role of Cysteine-Bound Aldehydes Quantification of 2-(Furan-2-yl)-1,3-Thiazolidine-4Carboxylic Acid Initially, for compound identification and method optimization, 2-(furan-2-yl)-1,3-thiazolidine-4-carboxylic acid was spiked in concentrations of 0.5 to 3.2 mg L–1 to both eluent mixture and beer B, the latter degassed by sonication. The eluent mixture was composed of mQ water and methanol, in a 55:45 (v/v) ratio, to which acetic acid was added at 5 mL L–1, and the pH of the mixture was adjusted to pH 5.5 with sodium hydroxide. For quantification of 2-(furan-2-yl)-1,3-thiazolidine-4-carboxylic acid in commercial pale lager beer samples, the concentrations were calculated based on an external calibration curve of 2 to 77 mg L–1 prepared in phosphate buffer (0.05 M, pH 4.4). All samples were prepared by transferring aliquots of 1 mL to 2-mL screw-top vials with pre-slit PTFE/ silicone septa (Waters Corp., Milford, MA). Beers were always degassed by sonication, and two bottles were measured per brand, each in twofold. 2-(Furan-2-yl)-1,3-thiazolidine-4-carboxylic acid was quantified using an ACQUITY ultra-performance liquid chromatography (UPLC) device, equipped with a Sample Manager autosampler and a photo diode array (PDA) detector (Waters Corp.). The sample vials were kept inside the autosampler at 10°C before analysis. Of each sample, 5 µL was injected on an ACQUITY UPLC HSS C18 column (150-mm length, 2.1-mm internal diameter, and 1.8-µm particle size) (Waters Corp.), set at a column temperature of 45°C and at a flow rate of 0.1 mL min–1. Under these chromatographic conditions, the compound eluted at a retention time of 4.3 min, and was quantified with the PDA detector at 276 nm. The total sample run time was 6 min. Data processing was performed using the Empower 2 software (Waters Corp.).
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RESULTS AND DISCUSSION Binding and Release Studies on Nonanal and (E)-2-Nonenal in Model Systems As in the previous study by Baert et al. (4), a simplified model system was used to obtain insight in the binding behavior of beeraging aldehydes. Initial experiments were performed with the aldehydes (E)-2-nonenal and nonanal because of their structural similarity, except for the difference in saturation or unsaturation at the α,β-position. Reactions were carried out in phosphate buffer of pH 4.4, 5.2, and 6.0 in order to approach the pH levels of pale lager beer, wort, and pale lager malt, respectively. In a first experiment (Fig. 2A), the interaction between the model aldehydes and cysteine was assessed. Upon addition of cysteine, with a subsequent heat treatment of 1 hr at 50°C (the “Cys” assay), the recovery of free nonanal decreased strongly at all tested pH compared with the reference samples without cysteine spiking (“ref.” assay). Somewhat less binding between the aldehyde and cysteine was noticed at the lower pH (pH 4.4), which can be explained by acid-catalyzed enolization or hydration of the aldehyde hampering the nucleophilic addition to the carbonyl group. This pH effect is much clearer for the model aldehyde (E)-2-nonenal, where an apparent lack of binding is seen at pH 4.4, in contrast to almost full binding at pH 6.0. These results are in line with our previous findings (4). Upon reaction with cysteine, 2-substituted 1,3-thiazolidine-4-carboxylic acids are most likely formed from both aldehydes. For nonanal, this consists of a one-step reaction; whereas, for (E)-2-nonenal, a twostep reaction may be taking place (11), where the first step in-
Fig. 5. Assessment of interaction between aging aldehydes and bisulfite, and subsequent treatment with 4-vinylpyridine (4VP) aimed at aldehyde release. Phosphate buffer (0.05 M, pH 4.4, 5.2, or 6.0) was spiked with a mixture of aldehydes. All samples were placed at 50°C for 1 hr; n = 3, error bars = standard error. Samples: ref. = samples without additional spiking, SO2 = samples additionally spiked with bisulfite at onset of the reaction, and 4VP = samples additionally treated with 4VP after the reaction.
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volves an addition of cysteine to the α,β-unsaturation, followed by a second addition to the carbonyl group (Fig. 1). When samples similar to the Cys assay were additionally spiked with 4VP after the heat treatment for binding but before HS SPME GC-MS analysis (Fig. 2A, Cys 4VP assay), a pronounced increase in aldehyde recovery was seen compared with the Cys samples. This result indicates that 4VP reacts with free cysteine, thereby shifting the equilibrium of aldehyde-bound cysteine to the free aldehyde form, making extraction and quantification possible. Control samples without cysteine but additionally treated with 4VP after the reaction time (4VP assay) yielded aldehyde recoveries similar to those of the reference samples. Addition of 4VP, which is a relatively strong base, resulted in a significant increase in the sample’s pH (pH 9.1 ± 0.0). To make sure this shift in pH is not the cause of aldehyde release, Cys samples were treated after the reaction time with sodium hydroxide instead of 4VP (Fig. 2B). However, this treatment (Cys NaOH assay, sample pH 10.4 ± 0.0) did not result in increased aldehyde recoveries. On the contrary, the interaction between the aldehyde and cysteine was even intensified, most likely due to an increased nucleophilic character of the sulfhydryl group (pKa 8.3) (28), and the amino group of cysteine (pKa 10.8) (28) at pH 10.4. Therefore, we concluded that the increased aldehyde recoveries observed in the Cys 4VP assays are the result of competitive binding of added 4VP with free cysteine, and not of the increase in pH. Instead of 4VP or sodium hydroxide, hydrogen chloride was also added to a series of Cys samples after the reaction time, in order to decrease the samples’ pH (Fig. 2C, Cys HCl assay, sample pH 2.1 ± 0.0). No effect of this treatment was found compared with the Cys samples, indicating that, under the applied condi-
tions, acidification is not a useable approach for aldehyde release from cysteine adducts. A second experiment was performed, completely analogous to the first, with the sole exception that cysteine was replaced by bisulfite as nucleophile for reaction with the model aldehydes nonanal and (E)-2-nonenal. Addition of bisulfite, followed by heat treatment (1 hr at 50°C) (the SO2 assay), resulted in a very strong decrease in the recovery of free nonanal at all tested pH (Fig. 3A). No significant effect of pH on binding of the strong nucleophile bisulfite was found for this aldehyde. For (E)-2-nonenal, however, a very strong trend was seen. A higher pH (pH 6.0) clearly results in more binding between (E)-2-nonenal and bisulfite. The above findings on bisulfite binding are comparable with results obtained in our previous study (4). Analogous to the cysteine binding experiments, bisulfite adduct samples were spiked with 4VP before HS SPME GC-MS analysis (Fig. 3A, SO2 4VP samples). From the high recoveries of free nonanal after treatment with 4VP, it can be concluded that practically all bisulfite-bound nonanal is released upon 4VP addition. On the other hand, for (E)-2-nonenal, increases in recovery were only limited after spiking with 4VP. Starting from nonanal, only a bisulfite monoadduct can be formed by addition of bisulfite to the carbonyl group whereas, for (E)-2-nonenal, two molecules of bisulfite may react with one molecule of aldehyde (Fig. 1). According to Dufour et al. (10), addition of bisulfite to an α,β-unsaturated aldehyde first takes place at the carbonyl group, followed by a second, irreversible addition to the α,β-carbon-carbon (C=C) double bond. Once the second reaction has taken place, the bisulfite added to the carbonyl group can still be released but returning to the free aldehyde is highly unlikely due to the stable binding of
Fig. 6. Assessment of 4-vinylpyridine (4VP) addition to fresh commercial pale lager beer in relation to the release of aldehydes; n = 2 calibration curves, error bars = standard error. Samples: ref. = beer samples without additional spiking and 4VP = beer samples additionally treated with 4VP.
Beer Flavor Instability: Potential Role of Cysteine-Bound Aldehydes bisulfite to the α,β-unsaturation. As a consequence, bisulfite diadducts are unlikely to be a source of free aldehydes during beer aging, whereas (E)-2-nonenal to which only the first addition took place may have an impact on beer flavor stability due to the lower stability of the monoadduct. In particular, at pH 6.0, irreversible (E)-2-nonenal-bisulfite binding is shown by comparing the results of the SO2 4VP assay with the SO2 samples (Fig. 3A). These findings suggest that, at pH 6.0, most of the (E)-2-nonenal transformed into bisulfite adducts and is present in diadducts, thus bound irreversibly. Control samples without bisulfite but with 4VP addition after the reaction time (4VP samples) showed aldehyde recoveries similar to the reference samples. To assess whether or not the release of aldehydes was caused by the increased pH after 4VP addition, samples were also spiked after the reaction time with sodium hydroxide instead of 4VP to a pH of ≈10. Whereas full release of nonanal was seen upon NaOH addition (SO2 NaOH assay), strikingly, (E)-2-nonenal showed binding compared with the SO2 samples (Fig. 3B). It is known that, from a pH above 7, significant dissociation of bisulfite adducts at the carbonyl group will occur (16), which is confirmed by the pronounced release of nonanal at a pH of ≈10. The same mechanism is applicable to (E)-2-nonenal; however, upon addition of the strong base NaOH, irreversible binding of bisulfite to
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the C=C double bond will take place (10), which explains the low recovery of (E)-2-nonenal in the SO2 NaOH assay. Addition of hydrogen chloride after the reaction time (Fig. 3C, SO2 HCl assay) shows that acidification to a pH of ≈2 is not a suitable approach for aldehyde release from bisulfite adducts. From the results described above, a general trend can be seen, regardless of the nucleophile (cysteine or bisulfite) added to the reaction mixture. A lower pH results in less aldehyde binding, and this trend is far more clear with the α,β-unsaturated aldehyde (E)-2nonenal than with the saturated aldehyde nonanal. Because the reaction comprises two components, the potential effect of pH on both reactants should be considered. First, there is the effect of the pH on the nucleophile. Within the applied pH range (pH 4.4 to 6.0), pH will have little effect on the reactivity of both the sulfhydryl group (pKa 8.3) and the amino group (pKa 10.8) of cysteine, as well a minimum effect on the reactivity of bisulfite, which is mainly present in its HSO3– form (16). Second, the effect of pH on the aldehyde reactivity needs to be taken into account. In general, protonation of the oxygen atom of the carbonyl group will take place more readily at a more acidic pH (pH 4.4), which will, in turn, cause enolization to some extent of the carbonyl group and, thus, less reactivity toward addition of the nucleophile (28). Moreover, at a more acidic pH, simple addition of a water mole-
Fig. 7. Ultra-performance liquid chromatography chromatograms (top, measured at 276 nm) and UV spectra (bottom) of 2-(furan-2-yl)-1,3-thiazolidine4-carboxylic acid A, in a model solution and B, in beer B, a fresh commercial pale lager beer. Analyses of separate samples with increasing levels of spiked 2-(furan-2-yl)-1,3-thiazolidine-4-carboxylic acid are stacked (see detail), visualizing the associated increase in peak intensity.
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cule may give rise to the dihydrate form of the carbonyl group. However, under the applied conditions of pH, saturated carbonyl compounds such as nonanal will be mainly present in their keto form (8). For the α,β-unsaturated aldehyde (E)-2-nonenal, the chemistry behind significantly reduced formation of adducts at the lower pH is somewhat more complex, because resonance forms must be taken into account as well. Obviously, the enol form of an aldehyde containing conjugated double bonds is stabilized by resonance (28), making the expected amount of (E)-2nonenal in its enol form higher compared with its saturated counterpart nonanal under identical reaction conditions. Consequently, because nucleophilic addition of cysteine or bisulfite takes place with the keto form, (E)-2-nonenal will be less prone to the addition reaction than nonanal. Moreover, in particular for α,β-unsaturated aldehydes, protonation of the oxygen atom of the carbonyl group in an acidic environment will lead to pronounced enolization and hydration. This explains the dramatic decrease in adduct formation between (E)-2-nonenal and either cysteine or bisulfite at pH 4.4. Although, based on pH and pKa values, it is chemically easy to explain why the addition of NaOH to the samples enhances binding of nonanal and (E)-2-nonenal to cysteine and why nonanal is released from its bisulfite adduct upon addition of NaOH, it is more complex to understand why treatment with NaOH leads to a pronounced formation of (E)-2-nonenal bisulfite adducts instead of aldehyde release. Again, typical resonance of (E)-2-nonenal has to be taken into account. Under alkaline conditions, no significant protonation of the oxygen atom of the carbonyl group will take place, enolization and hydration will thus be limited, and resonance structures exposing high reactivity toward nucleophilic addition will make a significant contribution to the overall resonance hybrid. Also, at a pH of ≈10.4, bisulfite will be mainly present in its very reactive dianionic form, making it even more prone to nucleophilic attack. Binding and Release Studies on Key Aging Aldehydes in Model Systems The experimental setup used for assessment of binding of nonanal and (E)-2-nonenal to cysteine and bisulfite, and subsequent release by addition of 4VP, was further applied on a series of typical beer-aging aldehydes. All parameters (e.g., pH and concentrations) and sample handling (preparation, pretreatment, and analysis) were similar to the experiments described above. When looking at the interaction between the aldehydes and cysteine (Fig. 4) and comparing the Cys samples with their corresponding reference samples, it becomes clear that the electrophilic character of the aldehyde determines the extent to which cysteine binding occurs, as has been reported by Baert et al. (4). Hexanal, the only saturated linear aliphatic aldehyde included in this experiment, shows strong cysteine binding at all tested pH. In this type of aldehyde, the double bond of the carbonyl group is strongly polarized due to the large difference in electronegativity between the oxygen and the carbon atom, and nucleophilic addition takes place readily within the applied range of pH. Similar TABLE I Quantification of 2-(Furan-2-yl)-1,3-Thiazolidine-4-Carboxylic Acid in Five Commercial Pale Lager Beersa Beer
Concentration (µg L–1)
Beer A Beer B Beer C Beer D Beer E a
Standard deviations are indicated (n = 4).
4,704 ± 59 7,939 ± 55 4,486 ± 28 4,650 ± 28 4,341 ± 66
results are obtained when a methyl group is present at the 3-position, as in case of 3-methylbutanal. However, when present at the 2-position, a methyl group causes a clear decrease in cysteine binding, in particular at lower pH, as can be seen for 2-methylpropanal and especially 2-methylbutanal. The presence of the carbon atom of the methyl group, being more electronegative than hydrogen, causes an inductive effect, thereby reducing the aldehyde’s electrophilicity. More specifically, the presence of a methyl group at position 2 (α-position) of the aldehyde renders the α-hydrogen slightly more acidic, thereby enhancing acid catalyzed enolization of the carbonyl group and, thus, decreasing reactivity toward nucleophilic addition. When a conjugated system, including the carbonyl group, is present in the aldehyde, it is even less prone to cysteine binding, especially at a lower pH. Delocalization of the electrons due to resonance decreases the electrophilicity of the carbon atom of the carbonyl group and, as mentioned before, significant enolization and hydration of α,β-unsaturated aldehydes may occur at acidic pH. (E)-2-nonenal, for example, does not seem to interact with cysteine at pH 4.4 whereas, at pH 5.2 and especially at pH 6.0, strong binding is found. In furfural, the conjugated system is further extended, including the carbonyl group and the heterocyclic furan ring, and clear binding is only noticed at pH 6.0. For benzaldehyde, also showing a fully conjugated system, similar results are obtained. Cysteine binding is also found for phenylacetaldehyde, containing a benzene ring but no conjugated system in relation to the carbonyl group, and for methional, containing a thioether group. Although, for these aldehydes, the pH effect is less clear, strongest binding was again observed at the highest pH (pH 6.0). As to release of aldehydes, in general, it is found that, regardless of the applied pH and the aldehyde involved, aldehydes are set free from their cysteine-bound state by addition of 4VP to such an extent that recoveries are comparable with those of the reference samples. This finding is an incentive to extrapolate our methodology to more complex matrices, in particular beer, aiming at release of aging aldehydes from their potentially preformed cysteine-bound state. The interactions between the aging aldehydes and bisulfite (Fig. 5) show similarities but also some differences compared with interactions with cysteine. Strong decreases in recovery of hexanal, 3-methylbutanal, 2-methylbutanal, 2-methylpropanal, phenylacetaldehyde, and methional are noticed when comparing the SO2 samples with the reference samples. The extent of interaction again depends on the aldehyde (e.g., strongest binding with hexanal and 3-methylbutanal, somewhat less binding with 2-methylbutanal and 2-methylpropanal) but, surprisingly, no pH effect was found. This can be ascribed to the stronger nucleophilic character of bisulfite versus cysteine (i.e., more binding to the carbonyl group will shift equilibria away from other resonance structures like the enol form). With (E)-2-nonenal, however, decreased bisulfite binding is noticed at lower pH, as was also found for cysteine binding. Again, resonance typical for conjugated unsaturated aldehydes offers the explanation for this phenomenon. Furfural appears to interact only minimally at pH 6.0, which is due to the presence of the extended conjugated double bond system, including the furan ring, and to the presence of the oxygen atom in the heterocyclic ring, which further aids in reducing the electrophilic nature of the aldehyde. The results obtained on (E)-2-nonenal and furfural confirm that the presence of a conjugated double-bond system decreases the electrophilicity and, thus, the aldehyde’s reactivity toward bisulfite. However, bisulfite showed more reactivity toward benzaldehyde compared with furfural and (E)-2-nonenal, and no difference in binding of benzaldehyde was seen at different pH levels. Although, for benzaldehyde, there will be some reduction in electro-
Beer Flavor Instability: Potential Role of Cysteine-Bound Aldehydes philicity of the carbonyl group, resonance forms showing the intact conjugated double-bond system in the six-membered ring will prevail and, consequently, reactivity of the carbon atom of the carbonyl group toward the strong nucleophilic bisulfite will be relatively high. It is possible that the effect of pH on bisulfite binding to aldehydes could be elucidated more clearly by applying a different composition of the model solutions. For instance, in a 1:1 bisulfite/methional molar ratio, Gijs et al. (13) noticed a clear effect of pH (namely, an increasing interaction of methional with bisulfite with decreasing pH), whereas this was not found in our 500:1 molar ratio model; rather, the opposite would be expected from the insights obtained here. Nevertheless, we believe that a molar ratio of 500:1, as used in our model solutions, is more representative for the potential reactions taking place during the brewing process and beer aging; and, thus, we wish to conclude that the effect of pH on bisulfite binding is more or less negligible within a beer context for all aldehydes tested here, except for (E)-2-nonenal and furfural. Addition of 4VP clearly gives rise to the release of aldehydes (Fig. 5) and, except for (E)-2-nonenal, the results obtained suggest that all bound aldehydes were again set free upon 4VP treatment. For the α,β-unsaturated aldehyde (E)-2-nonenal, bisulfite addition to the C=C double bond is known to be irreversible, which explains lower recoveries for this aldehyde upon treatment of the samples with 4VP. Apparently, under the applied conditions, practically all of the (E)-2-nonenal is bound in irreversible bisulfite adducts, in contrast to the other α,β-unsaturated aldehydes (i.e., furfural and benzaldehyde). The latter aldehydes do not seem to yield significant amounts of bisulfite adducts at a C=C double bond, presumably due to the presence of a fully conjugated aromatic ring structure. Release Studies on Aging Aldehydes in Pale Lager Beer Upon confirmation in model solutions that addition of 4VP gives rise to aldehyde release from both bisulfite- and cysteinebound states, the sample preparation technique was applied to beer samples. Using a standard addition approach, typical aging aldehydes were quantified in five fresh European commercial pale lager beers with and without 4VP (4VP assay and ref. assay, respectively) spiking to the beer samples before analysis. The overall results clearly point to release of the majority of aging aldehydes upon 4VP addition to beer (Fig. 6). In some cases, however, no significant effect was observed, and even decreased levels of methional (all beers) and phenylacetaldehyde (beer E) were seen after 4VP addition. No explanation for these decreases can be provided at this point. Benzaldehyde could not be reliably quantified in this experiment. Although, at this stage, it is not clear whether the observed increases in levels of aging aldehydes upon 4VP addition to beer are due to release from bisulfite adducts or cysteine adducts, sample pretreatment of fresh beers with 4VP may have potential to predict the aldehyde content of aged beers, which would be a convenient tool for brewers in estimating flavor stability. Detection of 2-(Furan-2-yl)-1,3-Thiazolidine-4-Carboxylic Acid in Pale Lager Beer Because addition of 4VP gives rise to release of aldehydes from both bisulfite and cysteine adducts, as was seen in the model solution experiments, release of aldehydes in beer could be attributed to both as well. The presence of bisulfite adducts in beer is generally assumed but 2-substituted 1,3-thiazolidine-4-carboxylic acids were not yet found in beer. Therefore, we looked for the presence of thiazolidine-4-carboxylic acids, in particular 2-(furan-2-yl)-1,3thiazolidine-4-carboxylic acid, the reaction product of cysteine and furfural, in the same five commercial pale lager beers used above.
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By applying UPLC for separation, and a PDA detector, we were able to detect a chromatographic peak of this compound dissolved in mobile phase (Fig. 7A), due to the aromatic furan ring present in the structure. Moreover, the same peak was also found in the examined pale lager beer samples (Fig. 7B), confirming the presence of this compound in beer. Subsequent standard addition experiments (i.e., adding increasing amounts of the compound to the beer samples and using resulting peak areas for calibration) allowed us to quantify the endogenous 2-(furan-2-yl)-1,3thiazolidine-4-carboxylic acid concentration. For these specific pale lager beers, levels ranged from 4.3 to 7.9 mg L–1 (Table I). Because furfural is present as a thiazolidine-4-carboxylic acid in fresh pale lager beer in such relatively high concentrations, one may also expect other beer-aging aldehydes to be present in a cysteine-bound state, especially because the other marker aldehydes were found to bind cysteine more efficiently than furfural. Future research will focus on the investigation of a potential link between beer flavor instability and the 2-substituted 1,3-thiazolidine-4-carboxylic acid levels in general, or the 2-(furan-2-yl)-1,3thiazolidine-4-carboxylic acid level in particular. CONCLUSIONS From our work in model solutions, it becomes clear that beeraging aldehydes react with both cysteine and bisulfite. The extent to which binding occurs is dependent on the electrophilicity of the specific aldehyde and, in many cases, on the pH of the reaction mixture, a higher pH leading to more binding. However, interaction of aldehydes with bisulfite seems, in general, less pH dependent than interaction with cysteine. After allowing for binding, addition of 4VP to the model solutions resulted in the release of aldehydes from cysteine and bisulfite adducts, to an extent suggesting that practically all preformed bound aldehydes are set free again. Only (E)-2-nonenal bisulfite adducts showed a minor increase in the level of free (E)-2-nonenal upon 4VP addition, which can be explained by the irreversible bisulfite adduct formation mechanism of this α,β-unsaturated aldehyde. Addition of 4VP to commercial pale lager beers resulted in increased levels in free aldehydes, especially those aldehydes known for their strong increases during beer aging. The presence of 2-(furan-2-yl)-1,3-thiazolidine-4-carboxylic acid was confirmed in commercial pale lager beers and its concentration proved to be in the range of milligrams per liter. Other 2-substituted 1,3-thiazolidine-4-carboxylic acids might be present in beer in significant amounts as well. Because aldehydes are released from both bisulfite adducts and cysteine adducts, the addition of 4VP to beer could be a valuable tool for determining bound-state aldehydes in fresh samples and, thus, for prediction of beer flavor instability. ACKNOWLEDGMENTS We thank the Agency for Innovation by Science and Technology (IWT, Flanders, Belgium) for financial support. LITERATURE CITED 1. Amons, R. Vapor-phase modification of sulfhydryl groups in proteins. FEBS Lett. 212(1):68-72, 1987. 2. Andersen, M. L., Outtrup, H., and Skibsted, L. H. Potential antioxidants in beer assessed by ESR spin trapping. J. Agric. Food Chem. 48(8):3106-3111, 2000. 3. Andrews, P. C., and Dixon, J. E. A procedure for in situ alkylation of cystine residues on glass fiber prior to protein microsequence analysis. Anal. Biochem. 161(2):524-528, 1987. 4. Baert, J. J., De Clippeleer, J., De Cooman, L., and Aerts, G. Explor-
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