Hop Flavor and Aroma

Beer Lightstruck Flavor: The Full Story DENIS DE KEUKELEIRE,1 ARNE HEYERICK,1 KEVIN HUVAERE,1,2 LEIF H. SKIBSTED,2 and MOGENS L. ANDERSEN2 1

Ghent University, Faculty of Pharmaceutical Sciences, Laboratory of Pharmacognosy and Phytochemistry, Ghent, Belgium; 2 Royal Veterinary and Agricultural University, Department of Food Science, Food Chemistry, Frederiksberg C, Denmark E-mail: denis.dekeukeleire@ugent.be Beer lightstruck flavor has been known for a long time. Lintner (1875) was the first to report on the formation of an offending taste and obnoxious odor in beer exposed to light, which he called lightstruck flavor (LSF). In the 1960s, Kuroiwa et al. (1961, 1963) suggested that the main constituent of the off-flavor was 3-methylbut-2-ene-1-thiol (MBT) derived from photodecomposition of isohumulones, the beer bittering principles, in the presence of a photosensitizer, namely riboflavin (vitamin B2). MBT, one of the most powerful flavor substances known (threshold of about 4 ng per liter of beer) (Irwin et al., 1993), is generally referred to as skunky thiol, because the odor resembles that of secretions of the anal glands of skunks (Mustela vison L.). Moreover, the Kuroiwa group established that the blue part of the visible spectrum (350–500 nm), in particular, is most efficient in generating LSF. This key finding was described in a formal mechanism, which involved light absorption by riboflavin, energy transfer to the isohumulones, Norrish Type I photocleavage within the excited isohumulones, release of a carbonyl radical (4-methylpent-3-enoyl) followed by decarbonylation to a stabilized dimethyl allyl radical (3-methylbut-2-enyl), and, finally, trapping by a thiol radical derived from “some” sulfur source (Fig. 1).

It is an inevitable fact: light leads to LSF, so beer must be protected from light. Indeed, a pale yellow beer absorbs some blue light, while a dark beer absorbs throughout the whole visible spectrum. Brown glass cuts off light around 500 nm; hence, beer in a brown bottle seems protected. On the other hand, green glass has a cut-off around 400 nm, indicating that the most energetic part of the visible spectrum, namely blue light, may penetrate a green glass; hence, protection against LSF is compromised.

PHOTOINDUCED OXIDATION OF ISOHUMULONES: KEY STEP IN THE FORMATION OF LSF The pivotal reaction in beer brewing is the isomerization of humulones, present in all hop varieties, to isohumulones, present in all beers (10–100 mg L 1) (Fig. 2). Humulones constitute a mixture of three analogs: so-called n-humulone (R = CH2CH(CH3)2), cohumulone (R = CH(CH3)2), and adhumulone (R = CH(CH3)CH2CH3). Due to the presence of two chiral centers in the isohumulones, each individual humulone gives rise to the formation of two isohumulones, called cis and trans, respectively. These stereochemical denotations refer to the spatial orientation of the tertiary alcohol group and the prenyl side chain relative to the five-membered ring. Orientation to the same face of the ring indicates a cis-configuration; an 1

2 Hop Flavor and Aroma: Proceedings of the 1st International Brewers Symposium

Fig. 1. Formal mechanism for formation of 3-methylbut-2-ene-1-thiol (MBT) (Data from Kuroiwa et al., 1961, 1963).

Fig. 2. Isomerization of humulones to isohumulones.

opposite orientation indicates a trans-configuration. The cis-compounds are more stable than the trans-compounds due to the trans-configuration of the two long side chains; hence, the cis-compounds predominate in the mixture of cis and trans. In normal brewing conditions, the ratio is 7:3 in favor of the cis-compounds, while the total yield of isohumulones is only 20–30%.

During the last decades, “advanced� hop products have come to the market. Modern hop technology has allowed isolation of pure humulones from hop extracts that are prepared using liquid or supercritical carbon dioxide. Humulones can be very efficiently isomerized in alkaline conditions to isohumulones (1a-c, 2a-c), which can be further reduced to dihydroiso-

De Keukeleire, Heyerick, Huvaere, Skibsted, and Andersen 3

Fig. 3. Formation of reduced isohumulones.

humulones (3a-c), commonly known as rhoisohumulones or “rho” (reduction of carbonyl using sodium borohydride), tetrahydroisohumulones (4a-c), commonly known as “tetra” (hydrogenation of the double bonds in the side chains using catalytic hydrogenation), and hexahydroisohumulones (5a-c) (combination of both reactions) (Verzele and De Keukeleire, 1991) (Fig. 3). In current brewing practice, dihydroisohumulones are used for brewing lightstable beers, while tetrahydroisohumulones are used for increased bitterness and for stabilization of the foam head. Hexahydroisohumulones find only occasional applications.

Isohumulones and derivatives possess the same chromophore, an enolized beta-tricarbonyl group with UV absorption characteristics in the UV-B region (280-320 nm). Consequently, photoreactions may be induced by direct absorption of UV-B light. Since the compounds are transparent in the visible spectrum, photoreactivity can occur only via intervention of a photosensitizer such as riboflavin (RF) or other flavins, which are yellow and, thus, absorb blue light. Flavins are present in beer at levels of a few hundred micrograms. It is clear that photodecomposition of isohumulones by direct irradiation (exposure to UV-B light) follows a reaction pathway different from the reaction course on indirect (photosensitized) irradiation.

The triplet-state energy of the isohumulones (about 300 kJ mol 1) is significantly higher than the triplet-state energy of RF (about 210 kJ mol 1); consequently, energy transfer from RF to isohumulones must be an energetically unfavorable, highly unlikely process. It oc-

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Fig. 4. Cyclic voltammograms of ferrocene (a), trans-isohumulones (b), dihydroisohumulones (c), and tetrahydroisohumulones (d) in their respective anionic forms (0.5 mM in MeCN).

Fig. 5. Experimental (upper trace) and simulated (lower trace) spin patterns of radicals derived from isohumulones (left) and dihydroisohumulones (right) after electrolysis and trapping by 5,5-dimethyl-1pyrroline-N-oxide (DMPO) under nitrogen.

curred to us that the key reaction step toward formation of LSF could be photooxidation of isohumulones by triplet-excited RF (3RF*), since 3RF* is a strong electron acceptor and is able to oxidize various organic substrates (Heelis et al., 1978; Heelis, 1982; Kino et al., 1998). Furthermore, it should be realized that structural similarities between isohumulones and derivatives can lead to comparable photoreactivities. In this respect, isohumulones and tetrahydroisohumulones possess the same alpha-hydroxyketo group (acyloin group) composed of the tertiary alcohol on the ring and the carbonyl in the acyl side chain, while dihydroisohumulones have a related alpha-diol group. Our strategy emphasized the duality in unraveling the mechanism of LSF, so our studies were split into two separate parts: the investigation of LSF under direct light exposure (UV-B) and under indirect (sensitized) irradiation (visible light). The aim was to identify excited states and intermediate radicals using appropriate techniques, such as electrochemical procedures, (time-resolved) electron paramagnetic resonance spectroscopy (EPR), and laser flash photolysis spectroscopy, thereby seeking confirmation by analysis and identification of photoreaction products (GC-MS, LC-MS, ESI-MS, NMR).

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Fig. 6. Linear plot of the pseudo-first-order rate constant (kobs) for decay of 3RF*, observed at 720 nm, as a function of the concentration of the potassium salts of isohumulones following a laser flash at 440 nm (pH 4.6 (}) and at pH 7 (â–˛)).

VISIBLE-LIGHT-INDUCED PHOTODECOMPOSITION OF ISOHUMULONES AND REDUCED ISOHUMULONES Our first seminal paper on LSF was published in 2001 (Burns et al.) and, during the forthcoming years, a series of seven papers (Heyerick et al., 2003, 2005; Huvaere et al., 2003, 2004a, 2004b, 2005, 2006) detailed all our studies on LSF that ultimately established the full mechanism of the formation of LSF. In daily practice, the visible-light-induced formation of LSF is by far the most important process, and that is the process we will focus on in this report.

Isohumulones and reduced isohumulones (dihydroisohumulones, tetrahydroisohumulones) in their undissociated forms were subjected to electrochemical oxidation in acetonitrile, but surprisingly, none of the compounds were electroactive. In contrast, we found that the corresponding anions (resulting from proton abstraction from the enolized beta-tricarbonyl chromophore, which is common to all substrates) were readily oxidized and showed prominent oxidation waves in the cyclic voltammograms (Fig. 4; Ep of about 1.4 V). This feature has profound consequences, since isohumulones and reduced isohumulones with pKa values around 3 occur predominantly as salts in lager beers (pH usually between 4.2 and 4.4). Furthermore, the resulting radical intermediates proved to be highly reactive, since the oxidations were irreversible. The similar heights of the anodic waves with respect to ferrocene oxidation indicated that isohumulones and reduced isohumulones were oxidized in a one-electron process suggesting oxidation of a common species. The common species refers to the enolized beta-tricarbonyl group, which is typical of all compounds studied. It can be concluded that (reduced) isohumulones are prone to undergo one-electron oxidation in the presence of suitable electron acceptors.

Next, incipient radicals were investigated by EPR spectroscopy, but low concentrations prevented direct detection. Consequently, spin traps were applied, namely 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). With DMPO, both oxygen- and carbon-centered radicals were identified in the spin-trapped adducts. A double triplet with hyperfine coupling constants aN ~ 13.2 G and aH ~ 8.9 G (line width ~ 1.6 G) for isohumulones and aN ~ 13.1 G and aH ~ 9.5 G (line width ~ 2.6 G) for dihydroisohumulones showed the parallelism (Fig. 5). Furthermore, MNP spin trapping of radicals from isohumulones and dihydroisohumulones suggested addition of a triacylmethyl radical as a result of electron release from the beta-tricarbonyl group (aN ~ 13.6 G and aH ~ 15.3 G, respectively).

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RF was then introduced, but solubility problems favored the use of flavin mononucleotide (FMN) in subsequent experiments. Both compounds exhibit very similar spectroscopic properties as well as photoreactivities. Laser flash photolysis spectroscopy gave insight into the behavior of 3FMN* in the presence of (reduced) isohumulones. The absorption maximum of FMN at 720 nm is attributed exclusively to 3RF* or 3FMN*, since its decay is stimulated by the addition of appropriate triplet quenchers. Salts of isohumulones and reduced isohumulones affected the decay of 3RF* considerably. Bimolecular rate constants for the reaction of isohumulones with 3RF* at pH 4.6 and pH 7.0 were expected to differ but did not (Fig. 6). Indeed, the molecules prevail largely as salts at these pH values in view of the fact that the pKas are around 3. Strikingly, the reaction rate constants for isohumulones and reduced isohumulones varied only between 1.7 and 2.3 Ă— 108 dm3 mol 1 s 1, suggesting comparable behavior. It should be noted that these constants are very high and nearly diffusioncontrolled, thereby indicating that 3RF*-induced oxidation is extremely efficient.

Next, radicals originating in the interaction between 3RF* and (reduced) isohumulones were detected by EPR spectroscopy following spin trapping by MNP under nitrogen. A similar signal (aN ~ 14.5 G Âą 0.5 G) was observed for all compounds, confirming the notion that radicals arise from a reactive moiety common to isohumulones and reduced isohumulones, confirming the findings from electrochemical oxidation (Fig. 7). It was apparent from kinetic analysis that the reactivities of isohumulones and tetrahydroisohumulones were quite

Fig. 7. Experimental (upper trace) and simulated (lower trace) spin patterns of radicals derived from isohumulones (left), dihydroisohumulones (middle), and tetrahydroisohumulones (right) after photooxidation by triplet-excited flavin mononucleotide and subsequent spin trapping by 5,5-dimethyl-1pyrroline-N-oxide (DMPO) under nitrogen.

Fig. 8. Plot of the rate constants for decay of triplet-excited flavin mononucleotide measured at 720 nm (kobs) as a function of varying concentrations of trans-isohumulones, isohumulones, tetrahydroisohumulones, cis-tetrahydroisohumulones, and dihydroisohumulones.

De Keukeleire, Heyerick, Huvaere, Skibsted, and Andersen 7

similar, while dihydroisohumulones appeared slightly less reactive (Fig. 8). Moreover, transisohumulones interacted slightly stronger with 3FMN* than cis-isohumulones.

CONCLUSIONS 1. Radicals formed on photochemical oxidation by triplet-excited riboflavin or flavin mononucleotide are similar to radicals observed on electrochemical oxidation of isohumulones and reduced isohumulones.

2. Formation of radicals derived from isohumulones is the initial step in the decomposition to a 3-methylbut-2-enyl radical, which is a key intermediate in the pathway leading to the LSF in beer.

3. Tetrahydroisohumulones are equally photoreactive as isohumulones, but remarkably, dihydroisohumulones are also oxidized by triplet-excited riboflavin or flavins. This result contradicts the commonly held belief that these compounds are light-proof.

THE MECHANISM OF SULFUR INCORPORATION REVEALED It is clear that sulfur-containing beer constituents such as proteins, polypeptides, or amino acids should be suitable sulfur sources to deliver the sulfur that is eventually incorporated in MBT. However, the mechanism of this pivotal final reaction step in the photoinduced formation of LSF, namely the intervention of a sulfhydryl radical, remained elusive until recently. We used relevant sulfur-containing amino acids and derivatives in model systems involving flavin-mediated photooxidation. Highly reactive, short-lived intermediates were analyzed using laser flash photolysis spectroscopy and spin trapping with subsequent

Fig. 9. Transient absorption of triplet-excited flavin mononucleotide sodium salt (3FMN*) in water, observed at 720 nm, in the presence of varying concentrations of S-methylcysteine (a: 0 mM, b: 1 mM, c: 2 mM, d: 3 mM, e: 4 mM) at pH 7.0. Inset: Linear plot of the pseudo-first-order rate constants (kobs) for the decay of 3FMN* as a function of the concentration of S-methylcysteine.

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Fig. 10. Experimental (upper) and simulated (lower) electron paramagnetic resonance (EPR) signals of spin adducts resulting from photooxidation of sulfur-containing amino acids by triplet-excited flavin mononucleotide at pH 7.0 in water and subsequent trapping by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Left: cysteine, right: S-methylcysteine.

EPR spectroscopy. Furthermore, photoreactions were the subject of a comprehensive product analysis by mass spectroscopy in order to provide support for the existence of the proposed reaction intermediates (Huvaere et al., 2006).

In order to establish the feasibility of generating sulfhydryl radicals by photooxidation, the kinetics of the interaction of sulfur-containing amino acids and derivatives with 3FMN* were investigated by laser flash photolysis spectroscopy. Increasing concentrations of the substrates proportionally affected the decay rate of 3FMN*, as shown for S-methylcysteine in Figure 9. As a result, the bimolecular rate constants for the interaction could be determined from the slope of the linear plot of the pseudo-first-order rate constants (observed by transient absorption spectroscopy at 720 nm) as a function of the concentration of sulfur-containing compounds. The values (expressed as 106 dm3 mol 1 s 1) were 328.6 (pH 4.2) and 120.8 (pH 7.0) for S-methylcysteine and 1.4 (pH 4.2) and 0.5 (pH 7.0) for cysteine. The much higher reactivity of S-methylcysteine with respect to that of cysteine reflects the stability of the respective radicals being formed (Cardoso et al., 2004).

Next, EPR was applied to model systems (FMN and substrates) that were irradiated at 440 nm in the presence of spin traps. Figure 10 shows the experimental and simulated patterns of spin adducts (DMPO) of cysteine and S-methylcysteine. For cysteine, two adducts were detected: a major adduct (80.5%), with coupling constants of aN (G) ~ 15.2 and aH (G) ~ 17.2 (cysteinyl addict), and a minor adduct (19.5%), with coupling constants of aN ~ 15.0 G and aH ~ 15.7 G (sulfhydryl adduct). S-methylcysteine, on the other hand, gave only one adduct with coupling constants of aN ~ 15.3 G and aH ~ 18.0 G (methylthio adduct). These results were nicely corroborated by headspace GC-MS analysis of the model systems (including isohumulones as well), leading to the identification of the major volatile analytes, which proved to be MBT for cysteine and S-methyl-MBT for S-methylcysteine (Fig. 11).

The characterization of this reaction step was the missing link in the unraveling of the mechanism of visible-light-induced formation of LSF. Hence, the overall mechanism has now been fully established.

CONCLUSIONS 1. Amino acids such as cysteine do not directly absorb visible light, but clearly, tripletexcited flavins may interact with sulfur-containing amino acids via electron abstraction, thus furnishing the corresponding sulfur-centered radicals.