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Fate of Bitter Compounds through Dry-Hopped Beer Aging. Why cis -Humulinones Should be as Feared as trans -Isohumulones?
Abstract
The evolution of isohumulones, main bitter compound of beers, through storage and its bitter impact has been studied for many years. With the uprising of highly hopped and, especially, dry-hopped beers during the last decade, other compounds such as humulones, cis-humulinones and hulupones have been shown to participate in both the sensory and measured bitterness of these beers. However, the fate of these compounds through aging is still unclear. The present work evaluated, by means of spectrophotometric and RP-HPLC-UV analyses, the evolution of bitterness units (BU), cis- and trans-isohumulones, humulones, cis-humulinones, hulupones, and tetrahydro-isohumulones in 21 Belgian dry-hopped beers after two years of storage. Besides the expected degradation of trans-isohumulones, it was observed an average decrease of 91% of humulones and 73% of cis-humulinones. Together, these results indicate a decrease of 18 – 43% of the calculated bitterness intensity of dry-hopped Belgian beers. The losses of cis-humulinones in dry-hopped beers bring seven new oxidation compounds, some of them very similar to those issued from trans-isohumulones. The bitterness impact of these compounds is yet to be determined.
... As a good radical scavenger of hydroxyl radicals (HO°) and other reactive oxygen species ROS (e.g., O 2°− , HOO° issued from metal-induced Fenton and Haber-Weiss reactions [12][13][14][15][16]), it can prevent volatile and non-volatile compounds to be oxidized during storage in regular beer. Polyphenols and bitter compounds are recognized to be particularly sensitive to degradation during beer storage, especially in dry hopped beers [17][18][19][20][21]. In NABLABs, we can suspect that they will be still less protected in the absence of radical scavengers. ...
... Isohumulones (co-, n-, and ad-analogues of cis-and trans-isomers) rapidly degrade in the presence of ROS and generally undergo autoxidation during storage [12,15,17]. trans-Isohumulones, much less stable than their isomer [17], mainly deteriorate by protoncatalyzed cyclization, leading to the harsh and lingering bitter-tasting tricyclohumols, tricyclohumenes, isotricyclohumenes, tetracyclohumols, epitetracyclohumols, and scorpiohumols [18,34,35]. cis-Humulinones can also be degraded, leading to similar cyclized derived products [35]. ...
... Then, flavan-3-ols and bitter compounds levels were determined by Reverse-Phase High-Performance Liquid Chromatography (HPLC) with ESI(-)-MS/MS and UV detections. The fate of these attributes was further briefly compared to similar data recently published for the top dry hopped fermentation beers [18,21]. ...
Nowadays, non-alcoholic (NAB) and low-alcoholic beers (LAB) still significantly suffer from staling defects when fresh, partially due to absence of ethanol as antioxidant. In the current work, the fate of flavan-3-ols (monomers, dimers, and trimers) and bitter compounds (isohumulones, humulinones, etc.) of 11 commercial NABLABs available on the Belgian market was monitored through one year of aging at 20 °C in the dark. Fresh NABLABs contained variable flavan-3-ols and bitter compounds levels (between 3.0–10.0 mg/L and 8.0–39.0 mg/L, respectively), depending on different technological processes used. Chill haze and color were also investigated as potential oxidation markers of fresh and aged beers. Surprisingly, contrary to conventional beers, the oligomers’ concentration (dimer and trimer procyanidins) exhibited a strong correlation (R2 = 0.95) with chill haze before aging, suggesting prematured oxidation of the samples. After a year of storage, significant degradation occurred as for regular dry hopped beers (process very sensitive to oxidation), only 27% remaining for flavan-3-ol dimers and an average 16% for trans-isohumulones. Oxidation risk appears here as the main weakness of NABLABs, which could be probably improved by spiking very efficient antioxidants.
... Perceived bitterness correlated well with the levels of bitter substances. Ferreira and Collin [46] analyzed Belgian dry-hopped beers after 2 years of storage at 20 °C. The average loss of iso-α-acids under these conditions was 25%, while the average losses of humulones and humulinones were as high as 91% and 73%, respectively. ...
... In a study by Jaskula et al. [12], beers dosed with hop polyphenols extract were characterized by a higher stability of iso-α-acids, which may be related to the antioxidant properties of these compounds. Šibalić et al. [47] report that different hop varieties have quantitatively different composition of polyphenol compounds, which may translate into different antioxidant capacity of the beer, and, as a result, its stability during storage [45][46][47][48]. ...
Beer has been enjoyed by consumers for years. Today, hops are inextricably associated with this beverage. Although they have been the subject of research for decades, knowledge of their bittering components and interactions during the beer production process is still incomplete. Current literature clearly indicates that the bitterness experienced in beer comes from a much wider range of compounds than just iso-α-acids. Although compounds that can be classified into β-acids, humulinones, hulupones, hard resins, and polyphenols are characterized by lower levels of bitterness and are present in hops in lower quantities than α-acids, they might determine, together with them, the final level of bitterness in beer. Unlike α-acids, the influence of compounds from these groups, their transformations, changes in their content during the beer production process and factors that affect their final concentration in beer have not yet been thoroughly studied. In case of α-acids, it is known that factors, such as chemical composition of wort, its extract and pH, amount of hops added and α-acids’ content, boiling time, and temperature at which hops were added influence the level of bitterness. This phenomenon is further complicated when dry hopping is used. Due to the presence of humulinones, polyphenols, and α-acids, a relatively simple spectrophotometric determination of IBU can give erroneous results. IBU determination, especially in dry-hopped beers, should be coupled with HPLC analysis, taking into account appropriate bitterness coefficients.
... Ferreira and Collin [24] reported on the ageing of dry-hopped beer. They compared the general properties of the beer and the content of bitter substances (isohumulones, humulones, humulinones and hulupones) of 21 dry-hopped beers when they were fresh and after two years of ageing (stored in dark conditions at 20 °C). ...
... Ferreira et. al. [24] stored Belgian dry-hopped commercial beers at 20 °C for 2 years and measured the losses of individual bitter substances. The losses reported by them are a good approximation of the values determined in this research (Table 5). ...
The changes in hop bitter substances in beer over the course of ageing and the associated decline in sensory quality have been the subject of numerous studies, but none of these have explored if bitter substances react differently during ageing based on whether the hops are added on the hot or on the cold side. To elucidate this a lager beer was brewed with a late aroma hop addition in the brewhouse and additionally dry hopped. Both beers were stored for two years at 4 °C, 22 °C and 30 °C. Overall, ageing the beers at 4 °C resulted in minor changes only. Based on the analysis of the beers aged at 22 °C and 30 °C, the following observations can be drawn: Starting with the cis-iso-α-acids via the bittering units, total iso-α-acids, trans-iso-α-acids and on to the α-acids, the losses of these substances increase significantly. Trans-iso-α-acids are 5 to 8 times more sensitive to degradation than the cis form. The behaviour of co-hulupone was comparatively stable in this regard. A marked difference between the homologs (co-, n-and ad-) of the iso-α-acids could not be found in both beers. However, all of the bitter substances in the dry-hopped beers showed less degradation due to ageing than those in the late-hopped beers. Whether the higher stability of the bitter substances in dry-hopped beer can be attributed to the changes in its composition through the addition of dry hops cannot be clearly established. Additional polyphenols added by dry hopping could potentially provide a "more stable environment" with a higher antioxidative potential. A distinct decrease in xanthohumol was observed; however, it was largely offset by an equivalent increase in isoxanthohumol. Late and dry hopped beers showed no differences with regard to the prenylflavonoids.
Descriptors: beer ageing, hop bitter substances, iso-α-acids, humulinones, hulupones, flavour stability
... trans-Isohumulones are known to be the most degraded fraction, given their propensity to be converted to tricyclohumols. For cis-isohumulones, oxidative degradation to alloisohumulones is the main concern [52,53]. For flavan-3-ols, it is now recognized that the oxidation of catechins to dehydrodicatechins increases color, while oligomer oxidation leads to colloidal instability and astringency [38]. ...
Even when fresh, non-alcoholic, and low-alcoholic beers (NABLABs) exhibit significant staling defects due to premature oxidation. In this study, the antioxidant power of eleven fresh commercial NABLABs was assessed by means of three different assays: the oxygen radical absorbance capacity (ORAC), the linoleic acid-induced oxidation (TINH), and the indicator time test (ITT). Only the first two assays, both involving radicalar degradations initiated by AAPH, were found to correlate with each other. NABLABs displayed lower ORAC values than conventional beers (on average, 6127 μmol eq. Trolox/L), except for three samples made with special-colored malts or dry-hopped. Dealcoholization was the step with the greatest impact on the ORAC value (up to a 95% loss) and on flavan-3-ols, sotolon, and polyfunctional thiols, while pasteurization strongly affected color, TBA, and Strecker aldehydes. ORAC assays applied to hop, alternative cereals, and various botanical ingredients indicated that mashing with red sorghum, dry hopping/spicing, and wood maturation could bring the antioxidant power of a NABLAB close to those of conventional beers. With an ORAC value not reached by any other tested botanical ingredient (5234 µmol eq. Trolox/g), African Vernonia amygdalina leaves (traditionally used for Rwandan Ikigage beers) emerged here as the best candidate.
... The primary degradation pathways for IAA are acid-catalyzed degradation, oxidation, and photooxidation. The difference in degradation rates has been used to develop a metric of beer flavor stability, the trans-to-cis ratio (t/c), which is the ratio of the sum of the trans isomers to that of the sum of the cis isomers [18]. For most freshly brewed conventionally hopped ales the IAA content is approximately 30% trans IAA and 70% cis IAA. ...
To investigate the chemical and sensorial impact of dry hopping time on typical pale ale, a standardized beer was produced and separated into ten vessels. Nine vessels were dry hopped, and one vessel remained un-hopped as a control. Impact of dry hopping contact time was investigated over 96 h. Polyphenols and iso-α-acid t/c ratio were analyzed in both Young and Aged beer samples. Total polyphenol content generally increased in both young and aged treatments compared to controls. Analysis of the t/c ratio suggests that both Young and Aged beers were chemically preserved to some degree after approximately 12 h at the given dry hopping rate regardless of age. Within the Aged beer trials, 96 h of dry hop contact yielded a significant increase in t/c ratio compared to all other Aged trials. This suggests that a 4-day dry hop regime may yield additional oxidative protection of iso-α-acids in beers stored unrefrigerated for 30 days. Descriptive analysis was also performed with an 8-person, trained panel; however, beers were sensorially distinguished by their aging time as opposed to their dry hopping time.
... Nevertheless, general trends include a decline in bitterness, fruity and floral notes, and an increase in off-flavors like cardboard, ribes, honey, caramel, sherry, and bread (Bamforth & Lentini, 2009;Barnette & Shellhammer, 2019;Dalgliesh, 1977;Ferreira & Collin, 2020;Vanderhaegen et al., 2006). Most of these changes are attributed to increasing levels of flavor-negative carbonyl compounds in the beer, such as (E)-2-nonenal, acetaldehyde, and 2-methylbutanal (Hashimoto & Eshima, 1979;Vanderhaegen et al., 2006). ...
Much like other living organisms, yeast cells have a limited lifespan, both in terms of the maximal length of time a cell can stay alive (chronological lifespan) as well as the maximal number of cell divisions it can undergo (replicative lifespan). Over the past years, intensive research revealed that the lifespan of yeast depends both on the genetic background of the cells as well as on environmental factors. Specifically, the presence of stress factors, reactive oxygen species and the availability of nutrients profoundly impact lifespan, and signaling cascades involved in the response to these factors, including the TOR and cAMP/PKA pathways, play a central role. Interestingly, yeast lifespan also has direct implications for its use in industrial processes. In beer brewing, for example, the inoculation of finished beer with live yeast cells, a process called “bottle conditioning” helps improve the product’s shelf life by clearing undesirable carbonyl compounds such as furfural and 2‐methylpropanal that cause staling. However, this effect depends on the reductive metabolism of living cells and is thus inherently limited by the cells’ chronological lifespan. Here, we review the mechanisms underlying chronological lifespan in yeast. We also discuss how this insight connects to industrial observations and ultimately opens new routes towards superior industrial yeasts that can help improve a product’s shelf life, and thus contribute to a more sustainable industry.
Abstract: Dry-hopping is the addition of hops to the wort on the cold side of the brewing process. Unlike standard hop additions, its main purpose is not to produce a characteristic bitterness but to extract as much of the hop essential oils as possible, which are largely lost in the standard hopping process. When dry-hopped, it is possible to obtain a beer with an aroma that is difficult to achieve when hops are used on the hot side of the brewing process. As a result, this process has become very popular in recent years, particularly in beers that belong to the ‘craft beer revolution’ trend. In addition, the usefulness of this process is increasing with the development of new hop varieties with unique aromas. This article presents the main components of hops, focusing on those extracted during the process. Changes in the composition of beer bittering compounds and essential oils resulting from this process are discussed. This paper presents the current state of the knowledge on the factors affecting the degree of extraction, such as hop dosage, the time, and temperature of the process. Issues such as process-related physicochemical changes, hop creep, low flavor stability, haze formation, and green flavor are also discussed.
At present, non-alcoholic (NAB) and low-alcoholic beers (LAB) exhibit major staling defects even when fresh, partly due to absence of ethanol as an antioxidant. In the present work, the aroma stability of eleven commercial NABLABs available on the Belgian market, issued from different technological processes, was assessed. NABLABs were investigated, both when fresh and after one year of storage at 20 °C in the dark. Six stale-odorant compounds were found above their perception threshold in aged NABLABs: sotolon, abhexon, methional, phenylacetaldehyde, dimethyltrisulfide and β-damascenone. Based on the chemical structure of the first four, it can be concluded that oxidation is the main issue for NABLABs aging. Yet, five of these usual staling defects of a six-month lager beer were already key-odorants in fresh NABLABs: dimethyltrisulfide, methional, and β-damascenone were above the thresholds in all samples, phenylacetaldehyde in 10 out of 11, and sotolon in 7. In conclusion, development of efficient antioxidants is needed to improve NABLABs acceptability.
Dry hopping imparts distinct aromas but also a series of non-volatile compounds suspected of causing flavor and physical instability during beer storage. In this work, color, chill haze, total polyphenols, total flavanoids, and flavan-3-ol monomers (catechin and epicatechin) and oligomers (procyanidin dimers and trimers) were monitored in five commercial pale-colored Belgian dry-hopped beers over 24 months of storage at 20 °C in the dark. Fresh dry-hopped beers contained unusually high levels of flavan-3-ol monomers (up to 6.6 mg/L) and oligomers (up to 14.1 and 10.2 mg/L dimers and trimers, respectively). The increase in color intensity during storage (up to 6.4°EBC) correlated with fresh beer monomer levels, while the oligomer content correlated with chill haze formation (up to 25.7°EBC). The evolution of these two physical attributes also correlated with the level of total polyphenols in the fresh beers. In a pilot-scale production, kettle hopping was shown to impart either monomers (early) or oligomers (late), while dry hopping promoted efficient extraction of both monomers and dimers (extraction yields of 62 and 74%, respectively). Dry hopping thus plays an important role in color and chill haze increase.
The aim of the present work was to compare levels of short chain fatty acids, esters, terpenoids and polyfunctional thiols in (mostly bottle-refermented) commercial Belgian dry-hopped beers before and after 2 years of storage at 20 °C (the usual best-before date in Belgium). Among the hop-derived volatiles, the terpenoids linalool and geraniol, the polyfunctional thiols 3SHol, 3SHA and 3S4MPol, and the esters ethyl isobutyrate, ethyl isovalerate and ethyl heptanoate (up to 499, 53, 0.2, 2, 3, 84, 63, and 19 µg/L, respectively) were found above their sensory thresholds in most fresh dry-hopped beers. The fermentation-derived esters reached concentrations similar to those previously reported for non-dry-hopped beers, with ethyl hexanoate and isoamyl acetate (up to 0.4 and 3.9 mg/L, respectively) often above their sensory thresholds. Except ethyl isovalerate (more than 85% still present), most hop odorants and fermentation esters showed degradation over the 2-year storage period: only 45%–70% of linalool, geraniol, and ethyl hexanoate and even less than 40% for polyfunctional thiols, ethyl isobutyrate, and ethyl heptanoate initial concentrations were detected after storage. How the dry-hopping process affects this degradation was further investigated in model media. Fermentation esters proved to be more strongly impacted in dry-hopped than in non-dry-hopped beers because of hop esterase activity. In addition to being aware of the need to avoid hop esterases, craft brewers are here advised to use bottle refermentation for its ability to regenerate some flavors and consume packaged oxygen. No deleterious effect of yeast, such as short chain fatty acid excretion, was evidenced.
Humulinone and hulupone extracts were prepared for evaluating their bitterness intensity in beer. Previously established methods for oxidizing α-and β-Acids followed by preparative liquid chromatography resulted in humulinone and hulupone extracts of 93.5 and 92.7% purity, respectively, as measured by HPLC. The humulinone and hulupone extracts were dosed into unhopped lager over a range of concentrations from 8 to 40 mg/L. Similarly, purified iso-α-Acids were dosed into unhopped beer at concentrations ranging from 6 to 30 mg/L. A nine-member trained panel scaled the bitterness intensity of all samples in five replicated testing sessions. Humulinones were found to be 66% as bitter as isoα-Acids (±13%), and hulupones were found to be 84% as bitter as iso-α-Acids (±10%). The results of this study suggest that humulinones and hulupones are more bitter than was previously suspected.
Time-resolved electron paramagnetic resonance (TREPR) data collected during the photodegradation of iso-a-acids (isohumulones), the principal bittering agents from hops in beer, are presented and discussed, and, from the data, the photophysics leading to free-radical production as the primary step in the photodecomposition of iso-alpha-acids towards the development of "skunky" beer are explained. During laser flash photolysis of iso-alpha-acids at 308 nm in toluene/methylcyclohexane (1:1), TREPR spectra exhibit net emissive signals that are strongly spin polarized by the triplet mechanism of chemically induced electron spin polarization. From two potential photochemically active sites, the TREPR data show that although the first site, an enolized beta-triketone, is the primary light-absorbing chromophore, an uphill intramolecular triplet energy transfer process leads to Norrish type I alpha-cleavage at a second site, an alpha-hydroxycarbonyl. The energy transfer mechanism is supported by additional TREPR experiments with chemically modified hop compounds. Structural parameters (hyperfine coupling constants, g factors, line widths) for the observed free radicals, obtained from computer simulations, are presented and discussed.
Although long renowned worldwide for its unique dry-hopped (DH) Trappist beer, Belgium did not develop this process for other brands until the last decade. Twenty-one commercial Belgian DH beers were investigated and compared with a few other typical Belgian beers whose production involves either late hopping or aged hop addition (Gueuze). Bitterness was determined by spectrophotometric measurements (isooctane extraction) and by reversed phase high performance liquid chromatographic with UV detector (RP-HPLC-UV) (simultaneous quantitation of humulones, cis-/trans-isohumulones, reduced isohumulones, humulinones, and hulupones). In dry-hopped Belgian beers, humulinones (found at concentrations up to 13.3 mg/L) were estimated to be responsible for up to 28% of their bitterness. As humulinones revealed to be gradually lost through boiling (22%), clarification (5%), and fermentation (14%), non-dry-hopped (NDH) beers often displayed levels below 1.7 mg/L. Even in Gueuze beers for which old, humulinone-containing hops are used, no humulinone was found. Contrary to humulones, which were detected up to 7.2 mg/L in DH beers, hulupones were found at less than 3 mg/L in all Belgian beer styles. Humulinones were not produced in the boiling wort from humulones (in contrast to hulupones, readily synthesized from lupulones) but were significantly solubilized from hop thanks to their hydrophilicity. Yet, while the co-form accounted for about 50% of the humulones, the n-form prevailed for humulinones. Some humulinone degradation products were evidenced by RP-HPLC-MS/MS, and as suggested by their retention time (RT), should be more polar than their precursors. Bottle refermentation emerged as an additional critical step of humulinone loss, explaining the low levels found even in some strongly DH beers.
The range of different nonvolatile constituents extracted from hops in highly hopped beers suggests that isohumulones may not be the sole contributor to beers' bitterness. Among brewers producing hop-forward beer styles there is concern that the Bitterness Unit (BU) is no longer an accurate predictor of beer bitterness. This study examined factors within the beer matrix that influence sensory bitterness perception in highly hopped beers. Over 120 commercial beers were evaluated using sensory and instrumental techniques. Chemical analysis consisted of the BU via spectrophotometry, hop acids via HPLC, total polyphenols via spectrophotometry, and alcohol content plus real extract via an Alcolyzer. Sensory analysis was conducted over two studies, and the beers' overall bitterness intensity were rated using a 0-20 scale. This study identified that the BU measurement predicts sensory bitterness with a nonlinear response, and it proposed an alternative approach to predicting bitterness based on isohumulones, humulinones, and ethanol concentrations. The study also revealed the importance of oxidized hop acids, humulinones, as a significant contributor to beer bitterness intensity.
Thirty-four commercial lager beers were analysed for their hop bitter acid, phenolic acid and polyphenol contents. Based on analytical data, it was evident that the beers had been produced using a range of different raw materials and hopping practices. Principal Components Analysis was used to select a sub-set of 10 beers that contained diverse concentrations of the analysed bitter compounds. These beers were appraised sensorially to determine the impacts of varying hop acid and polyphenolic profiles on perceived bitterness character. Beers high in polyphenol and hop acid contents were perceived as having ‘harsh’ and ‘progressive’ bitterness, whilst beers that had evidently been conventionally hopped were ‘sharp’ and ‘instant’ in their bitterness. Beers containing light-stable hop products (tetrahydro-iso-α-acids) were perceived as ‘diminishing’, ‘rounded’ and ‘acidic’ in bitterness. The hopping strategy adopted by brewers impacts on the nature, temporal profile and intensity of bitterness perception in beer.
In synthetic matrices, degradation of 3-methylthiopropionaldehyde, the main precursor of dimethyltrisulfide in aged beer, is influenced by the presence of anti- and prooxidants. Surprisingly, the reducing power of the beer proved not to be a key determinant of dimethyltrisulfide production during beer aging. However, other properties of anti- and prooxidants must be taken into account, such as the ability to bind 3-methyl-thiopropionaldehyde (sulfitic adducts) or methanethiol (copper complexation). This binding leads, respectively, to a higher or lower dimethyltrisulfide level.
The use of different types of pre-isomerised hop extracts containing iso--acids, dihydroiso--acids or tetrahydro-iso--acids, is discussed in relation to bitterness consistency and flavour stability. The emphasis is put on own practical experiences in respect of the application of pre-isomerised hop extracts in beer preparation.
The bitter taste of beer originates from resins in hops (Humulus lupulus L.), which are classified into two sub-types (soft and hard). Whereas the nature and reactivity of soft-resin-derived compounds, such as α-, β- and iso-α-acids, are well studied, there is only a little information on the compounds in hard resin. Here, we prepared hard resin from stored hops and investigated its compositional changes in an experimental model of beer aging. The hard resin contained a series of α-acid oxides. Among them, 4'-hydroxyallohumulinones were unstable under beer storage conditions, and their transformation induced primary compositional changes of the hard resin during beer aging. The chemical structures of the products, including novel polycyclic compounds scorpiohumulinols A and B and dicyclohumulinols A and B, were determined by HRMS and NMR analyses. These compounds were proposed to be produced via proton-catalyzed cyclization reactions of 4'-hydroxyallohumulinones. Furthermore, they were more stable than their precursor 4'-hydroxyallohumulinones during prolonged storage periods.
Oxidation is usually recognized as the major cause of development of a stale flavor in beer. However, no significant difference in trans-2-nonenal concentration has been observed between oxygen-receiving and oxygen-free beers after aging. Moreover, 18O2 was not incorporated into the carbonyl fraction, indicating that the cardboard flavor in beer is not due to lipid oxidation in the bottle. As shown by adding deuterated nonenol to the pitching wort, nonenal oxidation and sulfitic adduct degradation were also inefficient pathways of trans-2-nonenal synthesis. On the other hand, wort nonenal linked to amino acids and proteins revealed to be able to release nonenal at the beer pH. The measured nonenal potential proved a good indicator of beer staling; therefore, UV spectroscopy was used to find which experimental conditions destabilize trans-2-nonenal precursors in wort. Laboratory-scale experiments confirmed that SO2 can reduce both lipid autooxidation and the nonenal potential rise while the wort is boiling.
Odorant polyfunctional thiols such as 4-sulfanyl-4-methylpentan-2-one, 3-sulfanylhexan-1-ol (3SHol), and 3-sulfanyl-3-methylbutan-1-ol (3S3MBol) were shown to be strongly degraded in Belgian special beers after 1 year of aging. Yet, during the first 3 months, they also appeared to be synthesized in the bottle, even in the absence of yeast. Fresh filtered lager beer was spiked with nonvolatile S-cysteine conjugates of 3SHol, 3S3MBol, and 3-methylbut-2-en-1-thiol (MBT) before aging (1 to 3 months at 20 degrees C or 5 days at 40 or 60 degrees C). Thiol-specific para-hydroxymercuribenzoic acid extracts were analyzed by a GC-pulsed flame photometric detector, and HPLC-electrospray ionization/MS/MS was used to quantify the undegraded cysteinylated precursors. S-Cysteine conjugates were chemically degraded in the beer, releasing their corresponding thiols. The conversions were low (<1%) but could explain the trace amounts of 3SHol and 3S3MBol produced in beer during the first months of storage. On the other hand, chemical degradation of Cys-MBT in beer (although reaching up to 33%) proved not to be efficient enough to release significant amounts of the skunky off-flavor. In this case, photooxidative degradation of isohumulone most probably remains the major synthesis pathway. Complementary analyses are now needed to determine how other beer constituents, such as dicarbonyls, might participate in cysteine adduct degradation.
4, 5-Dimethyl-3-hydroxy-2(5H)-furanone or sotolon is known to impart powerful Madeira-oxidized-curry-walnut notes to various alcoholic beverages. It has been much studied in oxidized Jura flor-sherry wines, aged Roussillon sweet wines, and old Port wines, in which it contributes to the characteristic "Madeira-oxidized" aroma of these beverages. No scientific paper describes how sotolon might be involved in the Madeira off-flavor found in aged beers. The specific extraction procedure applied here allowed us to quantify this lactone in 7 special beers, at levels sometimes well above its threshold (from 5 to 42 µg/L after 6, 12, 18 and 24 months of natural aging, while unquantifiable in fresh beer). Investigation of spiked beers led us to highlight the key role of pro-oxidants and acetaldehyde. Addition of ascorbic acid without sulfites should be avoided by brewers, as the former would intensify sotolon synthesis. Acetoin, a beer fermentation by-product also emerged as possible precursor in beer, when combined with serine.
Phenol-specific extracts of twelve Belgian special beers were analyzed by gas chromatography hyphenated to olfactometry (AEDA procedure) and mass spectrometry (single ion monitoring mode). As guaiacol and 4-methylphenol revealed to be more concentrated in brown beers (> 3.5 and > 1.1 µg/L, respectively), they are proposed as specific markers of the utilization of dark malts. Analysis of 5 differently colored malts (5, 50, 500, 900, and 1500° EBC) allowed confirmation of high levels of guaiacol (> 180 µg/L; values given in wort, for 100% specialty malt) and 4-methylphenol (> 7µg/L) for chocolate and black malts only (versus respectively < 3µg/L and undetected in all other worts). Monitoring of beer aging highlighted major differences between phenols. Guaiacol and 4-methylphenol appeared even more concentrated in dark beers after 14 months of aging, reaching levels not far from their sensory thresholds. 4-Vinylphenols and 4-ethylphenols, on the contrary, proved to be gradually degraded in POF(+)-yeast-derived beers. Vanillin exhibited an interesting pattern: in beers initially containing less than 25 µg/L, the vanillin concentration increased over a 14-month aging period to levels exceeding its sensory threshold (up to 160 µg/L). Beers initially showing an above-threshold level of vanillin displayed a decrease during aging.
2-Sulfanyl-3-methylbutyl formate and acetate were synthesized without purification steps, quantified with a pulsed flame photometric equimolar detector, and characterized by comparison with commercially available 3-sulfanyl-3-methylbutyl formate and acetate (retention indexes, mass spectra, odour descriptors, and intensities). Both formates exhibited a typical ribes flavour, in contrast to both acetates, which were much more piquant. The sensorial threshold of 3-sulfanyl-3-methylbutyl formate was much lower (57 ng/l in beer, BE-GC-LoADS = 0.0006 ng) than those measured for the three other esters. Only 3-sulfanyl-3-methylbutyl formate was perceived at the sniffing port in beer extracts. Concentrations up to 1230 ng/l were measured in pilot beers after 1 month at 20°C, although the compounds are rarely detected in commercial beers with highly oxygen-protected bottling. Accelerated ageing in the presence of oxygen confirmed the key role of oxygen. Copyright © 2013 John Wiley & Sons, Ltd.
Age-induced decomposition of iso-α-acids, the main bittering principles of beer, determines the consistency of the beer bitter taste. In this study, the profiles of iso-α-acids in selected high-quality top-fermented and lager beers were monitored by quantitative high-performance liquid chromatography at various time intervals during ageing. The degradation of the iso-α-acids as a function of time is represented by the ratio, in percentage, of the sum of the concentrations of trans-isocohumulone and trans-isohumulone to the sum of the concentrations of cis-isocohumulone and cis-isohumulone. This parameter is relevant with respect to the evaluation of bitterness deterioration in aged beers. Trans-iso-α-acids having a shelf half-life of less than one year proved to be significantly less stable than cis-iso-α-acids, but it appears feasible to counteract degradation if a suitable beer matrix is available. The fate of the trans-iso-α-acids in particular adversely affects beer bitterness consistency. In addition to using hop products containing low amounts of trans-iso-α-acids, brewers may profit of the remarkable stability of tetrahydroiso-α-acids, even on prolonged storage, for the production of consistently bitter beers.
The transformation of α-acids [in hops (Humulus lupulus L.)] to iso-α-acids (in beer) during the brewing process is well known, but the occurrence and structure of the oxidized α-acids during hop storage are not well documented. Because an understanding of these oxidized compounds is essential to optimize the effects of oxidized hops on the quality of beer, we investigated the autoxidation products of humulone (a representative congener of α-acids) using a simplified autoxidation model. Among the oxidation products, tricyclooxyisohumulones A (1) and B (2), tricycloperoxyisohumulone A (3), deisopropyltricycloisohumulone (4), and the hemiacetal 5 of tricycloperoxyhumulone A (5') were isolated, and their structures were elucidated for the first time. The occurrence of compounds 1-4 in stored hops was verified using LC/MS/MS analysis. We also monitored the levels of compounds 1-4 during hop storage using LC/MS/MS analysis.
The preparation of humulinone by the oxidation of humulone affords in our hands a single substance, whilst the similar oxidation of cohumulone leads to a single cohumulinone. The physical and chemical properties of humulinone and cohumulinone suggest that they may be identical with the compounds of m.p. 70–71° C. and m.p. 104–105° C., respectively, described by Verzele & Govaert.
For decades, MBT (3-methyl-2-buten-1-thiol) is known as the compound responsible for the lightstruck off-flavour in beer. This leads many brewers to adapt the procedures by using reduced hop extracts. Unfortunately, other off-flavours including onion-defects often characterize these “light stable” beers. In the present work, a commercial lager beer which did not contain isohumulones (blend of dihydroisoalpha acids; bottled in clear glass) was submitted to various natural aging. Whereas no MBT-defect (skunky-like) was detected by sensorial analyses, a strong “onion-like” off-flavour was evidenced in the samples exposed to light. GC-PFPD and GC-O analyses of global (XAD) and thiol specific (pHMB) extracts allowed us to identify 2-sulphanyl-3-methylbutanol (2S3MBol) as the key-off-flavour (AEDA Flavour Dilution = 32–1024 for 2S3MBol while only 8–64 for MBT). 2S3MBol revealed to be synthesized from 3-methyl-2-buten-1-ol (MBOH) found in hop extracts. The involved radicalar mechanism is strongly enhanced by light. Although reduced hop extracts improve light stability regarding MBT, aroma-extracts give rise to strong onion-like off-flavours in presence of light. The concentration of the hop allylic precursor should be monitored in commercial hop extracts.
The attractive flavor of beer changes rapidly upon storage and limits the shelf life of this beverage. This degradation is due to several factors that may be associated with the vulnerability of iso-alpha-acids to the light and the oxidation of iso-alpha-acids. These compounds are responsible for the beer bitterness and its characteristic flavor. Apart from adequate beer packaging, there are several methods which can minimize the degradation of iso-alpha-acids: addition of phenolic compounds with antioxidant properties, addition of pure stereoisomers cis-iso-alpha-acids or their reduced species, or use of riboflavin-binding proteins.
The impact of alpha-acids on the bitterness intensity of lager beer was investigated using a trained panel and a psychophysical test with a consumer panel. A trained panel evaluated samples with and without alpha-acids to offer initial analysis on aroma and bitterness intensity. Following the trained panel test, a triangle test comparing an unhopped lager with and without 14 ppm alpha-acids was presented to more than 100 consumers for psychophysical evaluation. Both panels found no significant difference between the samples. Furthermore, the statistical similarity of the control and the 14 ppm alpha-acids samples was validated due to the size of the psychophysical test. This confirmed that alpha-acids at levels as high as the solubility limit in beer contribute negligibly to the overall bitterness of lager beer.
α-Acids and β-acids, two main components of hop resin, are known to be susceptible to oxygen and degraded during hop storage, although the oxidation products in stored hops have not been fully identified. In this study, we developed a high-performance liquid chromatography (HPLC) analysis method suitable for separation and quantification of the oxidation products. This HPLC analysis clearly proved, for the first time, that humulinones and hulupones are major products in oxidized hops. We are also the first to identify novel 4'-hydroxy-allohumulinones, suggested to be oxidative products of humulinones, by means of NMR spectroscopy and high-resolution mass spectrometry. Using the developed analytical method, changes in α- and β-acids and their oxidation products during hop storage were clearly revealed for the first time.
Time-resolved electron para- magnetic resonance (TREPR) data col- lected during the photodegradation of iso-a-acids (isohumulones), the princi- pal bittering agents from hops in beer, are presented and discussed, and, from the data, the photophysics leading to free-radical production as the primary step in the photodecomposition of iso-a- acids towards the development of "skunky" beer are explained. During laser flash photolysis of iso-a-acids at 308 nm in toluene/methylcyclohexane (1:1), TREPR spectra exhibit net emis- sive signals that are strongly spin polar- ized by the triplet mechanism of chemi- cally induced electron spin polarization. From two potential photochemically active sites, the TREPR data show that although the first site, an enolized b- triketone, is the primary light-absorbing chromophore, an uphill intramolecular triplet energy transfer process leads to Norrish type I a-cleavage at a second site, an a-hydroxycarbonyl. The energy transfer mechanism is supported by additional TREPR experiments with chemically modified hop compounds. Structural parameters (hyperfine cou- pling constants, g factors, line widths) for the observed free radicals, obtained from computer simulations, are present- ed and discussed.
For the first time, quantitative LC-MS/MS profiling of 56 hop-derived sensometabolites contributing to the bitter taste of beer revealed a comprehensive insight into the transformation of individual bitter compounds during storage of beer. The proton-catalyzed cyclization of trans-iso-α-acids was identified to be the quantitatively predominant reaction leading to lingering, harsh bitter tasting tri- and tetracyclic compounds such as, e.g. the cocongeners tricyclocohumol, tricyclocohumene, isotricyclocohumene, tetracyclocohumol, and epitetracyclocohumol, accumulating in beer during storage with increasing time and temperature. The key role of these transformation products in storage-induced trans-iso-α-acid degradation was verified for the first time by multivariate statistics and hierarchical cluster analysis of the sensomics data obtained for a series of commercial beer samples stored under controlled conditions. The present study offers the scientific basis for a knowledge-based extension of the shelf life of the desirable beer's bitter taste and the delay of the onset of the less preferred harsh bitter aftertaste by controlling the initial pH value of the beer and by keeping the temperature as low as possible during storage of the final beverage.
The iso-alpha-acids, the major contributor to bitter beer taste, is well-known to strongly degrade during beer aging. The storage of beer in brown glass bottles revealed a strong depletion of the trans-configured isomers in a highly specific manner, whereas the corresponding cis-iso-alpha-acids seemed to be hardly affected. In comparison, storage of beer in polyethylene terephthalate bottles, which are known to be permeable to oxygen, induced a drastic degradation of both isomers independent of their cis/trans configuration. To investigate the chemical transformation of iso-alpha-acids under oxidative storage conditions, suitable model experiments were performed, and the reaction products that formed were identified as previously not reported hydroperoxy- and hydroxyl-allo-iso-alpha-acids by means of one-/two-dimensional NMR and liquid chromatography-mass spectrometry experiments; for example, cis- and trans-configured hydroperoxy-alloisohumulone as well as the corresponding hydroxy-alloisohumulones were generated upon oxidation of cis- and trans-isohumulone independent of their cis/trans configuration. In addition, the oxidation products formed from the various iso-alpha-acid congeners were quantitatively determined in a series of beer samples stored under defined conditions. For the first time, these data help to understand the molecular mechanism involved in the autoxidative degradation of iso-alpha-acids in beer.
The use of labeled nonenal enabled the demonstration that the appearance of the cardboard flavor in finished beer comes from lipid auto-oxidation during wort boiling and not from lipoxygenasic activity during mashing. Free trans-2-nonenal produced by linoleic acid auto-oxidation in the kettle disappears, owing to retention by wort amino acids and proteins. This binding linkage protects trans-2-nonenal from yeast reduction but is reversible, allowing release of the compound at lower pH during aging. Labeled trans-2-nonenal is detected after aging when deuterated precursors form in the boiling kettle. The amount of alkenal released correlates with the concentration of reversible associations in the pitching wort. This work brings new illumination to the formation of trans-2-nonenal and overturns many previous hypotheses. It also explains why a reduction in the beer pH intensifies the cardboard flavor.
Hop S-methylcysteine sulfoxide has previously been postulated as the precursor of dimethyl trisulfide (DMTS) in beers. The present data point to 3-methylthiopropionaldehyde, the Strecker aldehyde issued from methionine, as another potential precursor in aged beers. Spiking either fresh beer or wort before boiling leads in all cases to higher levels of DMTS after storage. Moreover, special malts with a high level of 3-methylthiopropionaldehyde also favor polysulfide synthesis. A higher pH should increase this onion-like off-flavor, whereas a low pH is unfortunately known to enhance the cardboard flavor of aged beers. 3-methylthiopropanol, issued from yeast reducing activity, can be considered as an additional DMTS source during aging.
Flavor quality is of major importance to the consumer, but the flavor characteristics of beer appear to deteriorate greatly with time, at a rate depending on the composition of the beer and its storage conditions (notably pH). Prior to identifying the influence of pH on the development of the most intense staling flavors found in aged lager beers, the corresponding key flavor compounds were determined by aroma extract dilution analysis. In addition to trans-2-nonenal, beta-damascenone seems at least as important in the flavor of aged beer. Ethyl butyrate, dimethyl trisulfide, 2-acetylpyrazine, 3-(methylthio)propionaldehyde, 2-methoxypyrazine, maltol, gamma-nonalactone, and ethyl cinnamate are also relevant to the sensory profile of aged beer. Upon aging, a beer having a higher pH produces less beta-damascenone, because acid-catalyzed glycoside hydrolysis is decreased. On the other hand, it produces more 3-(methylthio)propionaldehyde, owing to Strecker degradation of methionine. Raising the beer pH additionally causes the release of 3-(methylthio)propionaldehyde from sulfitic adducts. These adducts, more stable at a lower pH, protect the aldehyde against premature oxidation to 3-(methylthio)propionic acid, thus making it available for dimethyl trisulfide formation during aging.
Recently, it was reported that furfuryl ethyl ether is an important flavor compound indicative of beer storage and aging conditions. A study of the reaction mechanism indicates that furfuryl ethyl ether is most likely formed by protonation of furfuryl alcohol or furfuryl acetate followed by S(N)2-substitution of the leaving group by the nucleophilic ethanol. For the reaction in beer, a pseudo-first-order reaction kinetics was derived. A close correlation was found between the values predicted by the kinetic model and the actual furfuryl ethyl ether concentration evolution during storage of beer. Furthermore, 10 commercial beers of different types, aged during 4 years in natural conditions, were analyzed, and it was found that the furfuryl ethyl ether flavor threshold was largely exceeded in each type of beer. In these natural aging conditions, lower pH, darker color, and higher alcohol content were factors that enhanced furfuryl ethyl ether formation. On the other hand, sulfite clearly reduced furfuryl ethyl ether formation. All results show that the furfuryl ethyl ether concentration is an excellent time-temperature integrator for beer storage.
For a long time, beer staling has been a prime concern in brewery research. Yet, to improve flavor stability, better knowledge of all chemicals involved is still needed. From our aroma extract dilu-tion analyses (AEDA) applied to naturally aged lager beers emerged an old-beer-like odorant at RICP-SIL 5 CB = 1532 and RIFFAP = 2809, with a FD value close to that of trans-2-nonenal (the well-known cardboard off-flavor found in aged beers). Specific phenol extraction, GC cold trapping, and mass spectrometry (electron impact and chemical ionization) enabled us to identify it as 4-vinylsyringol. Although already mentioned in some fresh beers, this compound had never been highlighted as involved in the aging process of lager beers.
Besides Maillard reactions, structural rearrangements of flavan-3-ol monomers cause color changes in beer during storage. Acetone/water-soluble fractions (70/30, v/v) of three lager beers of the same batch, differently stabilized before bottling in glass or poly(ethylene terephthalate) (PET) bottles, were monitored by normal-phase HPLC-ESI(-)-MS/MS over a 1-year period of storage at 20 degrees C. In parallel, beer color was monitored by the European Brewery Convention assay. The evolution of color was similar in the silica gel-filtered beer to that in identically bottled and stored poly(vinylpolypyrrolidone)-treated samples, despite the high flavanoid dimers content of the former. On the other hand, color evolved more rapidly in the PET bottle, suggesting a key role of oxygen. The kinetics was still increased in model media containing (+)-catechin, while no color was detected when normal-phase HPLC-fractionated dimers or trimers were investigated. (+)-Catechin emerged as the precursor of less polar products, characterized by a yellow-brown color. MS/MS enabled us to identify these products as issued from the oxidation and intramolecular additions of dehydrodicatechin B4. Similar structures were found in aged beers spiked with (+)-catechin. Beer storage in the absence of oxygen and at low temperature is recommended so as to minimize the synthesis of such pigments.
Humulinone Formation in Hops and Hop Pellets and Its Implications for Dry Hopped Beers
- J P Maye
- R Smith
- J Leker
Hop Oxidative Transformations and Control of Beer Bitterness
- A J Rehberger
- L H Bradee