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Fate of Hop and Fermentation Odorants in Commercial Belgian Dry-Hopped Beers over 2 Years of Bottle Storage: Key-Role of Oxidation and Hop Esterases
Abstract
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.
... Ethyl acetate (headspace technique) was found at concentrations ranging from 100 mg/L (LG) to 237 mg/L (RV), significantly higher than in traditional top fermentation beers (8-48 mg/L) and filtered gueuzes (33-67 mg/L) but similar to the levels found in unfiltered gueuzes (61-167 mg/L). [11] These values were expected in the presence of Brettanomyces, as mentioned by Snauwaert et al. [6] Conversely, isoamyl acetate, which is typically around 1-4 mg/L in top fermentation beers, [24] was found here at lower concentrations in both Rodenbach beers (0.3-0.4 mg/L). Probably because of a much lower level of Brettanomyces, a more usual value (1.73 mg/L) characterized the LG beer. ...
... Conversely, hexanoic acid was found at much higher concentrations (10-12 mg/L) than is usual in top fermented beers (2-4 mg/L), [24] and it was well above its odor threshold of 8 mg/L. ...
Red and brown Flemish sour beers form a distinct class of Belgian beers obtained by mixed (yeast/lactic bacteria) microbial fermentation and often resulting from blending a 1-to-2-year-old beer with a younger one to obtain a balance between acidic character and sweetness. A detailed composition in volatiles (phenols, lactones, esters, alcohols, acids, …) of three beers representative of the red and brown subcategories is presented. GC data were obtained after different extraction procedures, including solvent-assisted flavor evaporation (SAFE) and headspace. The first results showed the influence of Brettanomyces yeast on the phenol and ester contents. An efficient Brettanomyces activity in the red sour beers (especially in Rodenbach Vintage) was observed, favored by long maturation in wooden casks. This was organoleptically perceived by the horsey flavors brought by 4-ethylguaiacol and 4-ethylphenol, and the solvent-like ethyl acetate through esterase activity. The brown Flemish sour beer (produced in stainless steel fermenters) showed significantly more unreduced 4-vinylguaiacol and 4-vinylphenol, although traces of 4-ethylguaiacol and 4-ethylphenol were also detected (most probably here issued from torrefied malts, as suggested by the opposite substituted phenol/guaiacol ratio).
... 2-methylbutyl isobutyrate) down ( Figure 8A,B, Figure 5). These results agree with past work on ethyl esters [48,49] and hop related esters [5,6] and show that enzymatic (trans)esterification reactions can occur in unpasteurised ale whereas pasteurisation can reduce this effect. Overall, the current publication does not claim to be a detailed enzymatic investigation and at this point, a connection to hop or yeast related enzymes cannot be drawn. ...
... Famous for their very low thresholds (ng/L level) and pleasant flavors (fruity or floral odors for those with five or more carbon atoms), polyfunctional thiols (PFTs) [1][2][3] play a key role in the organoleptic profiles of hopped-forward beers [4,5] as well as wines. [6] A slight proportion of them can arise simply through solid-liquid extraction during late or dry hopping. ...
Polyfunctional thiols (PFTs) present in beers or in wines are known to be released by yeast, during fermentation, from bound forms originally found in raw materials. In the brewing field, huge amounts of S-conjugates have been evidenced in several dual-purpose hop varieties. Malt, however, being the major raw material of beer, could also be a significant contributor of PFTs to beer (cysteinylated, Cys- and glutathionylated, G- precursors of 3SHol already identified in a few samples of barley and malt). Forty-two barley malts from 2 to 1500 EBC and five other malted cereals were screened to characterize their thiol precursor profile (G- and Cys- as well as dipeptidic bound CysGly- and γGluCys- forms of 3SHol). First, it was confirmed that G-3SHol was ubiquitous reaching up to 320 µg/kg in some samples, whereas Cys-3SHol remained at a trace level of up to 13 µg/ kg. Moreover, for the first time, dipeptidic bound forms of 3SHol were evidenced in malt (up to 10 and 11 µg/kg for CysGly-3SHol and γGluCys-3SHol, respectively). In pale malts, the level of the CysGly- form was shown to be proportional to that of G-3SHol (in situ γGT during the malting process). This appeared to be no longer true for special malts (ranging from 5 to 45°EBC), whose CysGly-3SHol level correlated instead with Cys-3SHol (suspected chemical conversion from the dipeptide conjugate). As for γGluCys-3SHol, it was only found in the special barley malts (indicating another chemical break of the Cys-Gly bond, here on G-3SHol) and in malted rye, spelt, and wheat. No precursors were found in roasted malt.
... The SAFE extraction conditions were as follows: the water bath temperature was set at 40 • C, the pressure was kept below 1 × 10 −4 mbar, and the temperature of the apparatus body (Glasblaeserei Bahr, Manching, Germany) was 30 • C. Two hundred and fifty microliters of a solution containing 2-acetylthiophene at 10 mg/L (IST) were added to ground cocoa beans (50 g) mixed with 50 mL Milli-Q water to obtain an IST concentration of 50 µg/kg. Since the distillation involves cocoa powder, which cannot be introduced dropwise like beer samples, for instance [25], the entire solution was directly introduced into the vessel before applying a vacuum. After 45 min of distillation, the distillate was recovered in a liquid-nitrogen-cooled flask and then extracted three times with 16 mL of biodistilled dichloromethane. ...
Fine-grade cocoa beans are characterized by their great organoleptic quality. Brazilian cocoa producers increasingly privilege organoleptic quality over yield. Catongo and FL89, fine Brazilian cocoa genotypes from Bahia, are characterized by particular flavors (respectively, kerosene and whisky). The beans IMC67, PA121, and P7 from the state of Pará are genotypes that have high resistance to diseases. Solvent-assisted flavor evaporation (SAFE) aroma extraction was used here to identify and quantify the volatile compounds discriminating these genotypes. The results show that the kerosene aroma of Catongo is likely due to the presence, at high levels, of ethyl acetate and isobutyl acetate. On the other hand, ethyl benzoate, heptanoate, and octanoate, trans-2-nonenal, 1-octen-3-ol, and 3-methylbutanol could play a key role in the whisky notes of FL89. Heptan-2-ol, heptan-2-one, nonan-2-one, linalool (although still more concentrated in IMC67 from Pará state), benzaldehyde, and phenylacetaldehyde also discriminate these beans. Other compounds, although not discriminating, appear important in determining their aromatic quality. The PCA showed that cocoa from Pará state formed a cluster due to similar aromas, while FL89 was the most distinct among the genotypes. Beans from Brazil show great potential and diversity for the fine cocoa market.
... Higher temperatures during the production of these malts lead to the formation of colourants and odorants through thermal reactions including the Maillard reaction and Strecker degradation (Gasior et al, 2020). Whereas the impact of specialty hops on the aroma of beer and the transfer of odour active compounds to beer have already been studied at a molecular level (Peacock et al, 1981;Lermusieau and Collin, 2003;Neiens and Steinhaus, 2018a;Reglitz et al, 2018;Silva Ferreira and Collin, 2021), corresponding studies with speciality malts are scarce. We recently investigated the role of caramel barley malt and roasted barley malt for the aroma of bottom-fermented beers (Féchir et al, 2021). ...
Top-fermented wheat beers are known for their unique aroma. However, the impact of speciality wheat malts on the aroma of these beers and the transfer of odour active compounds from malt to the beer has not been investigated in detail. Three beers were brewed with different malt composition. The grist for each beer contained 50% kilned barley malt and 50% different wheat malts-beer (1) kilned wheat malt, beer (2) kilned wheat malt and caramel wheat malt, and beer (3) kilned wheat malt and roasted wheat malt. The odour active compounds in the beers were identified by aroma extract dilution analysis and their individual impact on aroma was evaluated by quantitation and calculation of odour activity values (OAVs). The results were verified sensorially by comparing aroma reconstitution models with the original beers. Characteristic odour active compounds in the beer brewed with caramel wheat malt were earthy compounds 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-3,6-dimethylpyrazine, 2,3-diethyl-5-methylpyrazine, caramel-like compounds 4-hydroxy-2,5-dimethylfuran-3(2H)-one and maltol, and sotolon with a soup seasoning-like aroma. The aroma of the roasted wheat malt beer was characterised by smoky and phenolic compounds 2-methoxyphenol and 4-methylphenol. Important beer odorants were quantified in the malts to assess their transfer from malt to beer. The results suggest that direct transfer of the odour active compounds in beers was not significant and that they were formed and/or released during the brewing process, confirming earlier results with different barley malts and bottom-fermented beers.
... Chemical structures and overview of the maximal amount of S-conjugates and free thiols found in malt, hop, and beers.13,14,19,25 *Data exposed for the first time here. ...
Fermented beverages such as beer are known for their relatively long shelf life. However, the main factor limiting their shelf life is the qualitative changes that occur during storage. From the moment the beer is produced, its characteristics, such as taste, aroma, and colloidal stability undergo continuous change. The intensity of these changes depends on the type of beer, storage conditions, and length of storage. While some degree of ageing can have a positive influence on sensory characteristics of a beer, beer stalling is seen as a significant problem. As it is currently understood, beer ageing is mainly caused by the formation of stalling aldehydes. At the same time, compounds which bestow the beer its flavour, such as esters, terpenes, and iso-α-acids undergo qualitative and quantitative changes. As a result, aroma discriminants such as freshness, fruitiness or florality are often lost over time. In their place, aromas described as ribes, cardboard, bread-like, honey-like or sherry-like appear. The article aims to present the changes in beer sensorial, physicochemical, and microbiological characteristics during storage and the factors that affect beer quality during ageing The article also describes the variables which according to the current literature, may alter the flavour stability of a beer.
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 scope of this study was to investigate how incrementallyi ncreasing dosing rates for dry hoping up to 1500 g/hl with type 90 Cascade pellets affects selected chemical and physical characteristics of beer. To this end, 6 hl of pale ale was brewed on a pilot system. After primary fermentation, this base beer was split in 20 litre NC kegs in order to perform each dry hopping trial in triplicate. From these beers the variations of the following characteristics after dry hopping were investigated: bittering units, alpha acids and iso- alpha acids, humulinones, total polyphenols, flavanoids, iso-xanthohumol and xanthohumol, real extract, alcohol, pH value, foam stability (NIBEM and Steinfurth) and turbidity. In addition, the influence of the plant material from the hops on these selected characteristics was examined. For this purpose, dry hopping trials using 1000 g/hl of spent hop pellets sourced from hop extraction with CO2 were also performed on the same base beer.
As a result of our trials, significant effects were observed with regard to the decrease in iso-alpha acids and foam stability, as well as an increase in humulinones, the pH value, real extract and total polyphenols in the instances where type 90 pellets were used for dry hopping. In particular, the decrease in iso-alpha acids appears to be linked to the presence of hop plant material. In contrast to the changes mentioned above, either behaviours of certain compounds remained unchanged or no clear conclusion was able to be drawn concerning the influence of dry hopping on the following: flavanoids, iso-xanthohumol, xanthohumol and alcohol content as well as the turbidity and the bittering units determined analytically.
The reproducibility aspect of fresh and moderately aged late and dry hopped beers was previously reported in the papers part 1 [1] and part 2 [2] respectively. The present paper (part 3) focuses on the transfer of several compounds from hops to beer and their behaviour during beer ageing at different temperatures for 470 days. Transfer rates were found to be different for the examined substance groups and whether the hops were applied in the brewhouse (late) or during maturation (dry hopped): mono-and sesquiterpene hydrocarbons < 1 % in late hopped beers, up to 2 % in dry hopped beers; esters: 20 to 40 % in late hopped beers, 40-80 % in dry hopped beers; linalool: 60 % in late hopped beers, 80 % in dry hopped beers; sesquiterpene alcohols: 7-18 % in late hopped beers, 10-50 % in dry hopped beers. A late and a dry hopped beer were aged for 470 days at 0 °C, 4 °C, 20 °C and 30 °C and analysed for several fermentation by-products, ageing carbonyls and relevant hop aroma compounds. The concentration of acetates and ethyl esters decreased significantly at 20 °C and 30 °C, higher alcohols showed good stability. The concentrations of all Strecker degradation products and furfural started to increase at 4 °C, with pronounced increases at 20 °C and 30 °C. Monoterpenes proved to be relatively stable when stored at low temperatures (0 and 4 °C); however, at 20 °C and 30 °C, lost approximately 25 % and 50 % of their initial concentrations. The hop-derived carboxylic acid esters in the beers decreased by ca. 10 % at 0 °C. The relative losses increased dramatically to 60 to 70 % at 20 °C, and to more than 70 % at 30 °C. Only at 30 °C there were significant losses of total linalool. By contrast, the concentration of α-terpineol increased. Similar to linalool, a significant decrease of the β-citronellol concentration was observed at 30 °C. The racemization of the flavour-active R-linalool to the more inactive S-linalool was particularly evident at the higher temperatures.
The aromatic complexity of craft beers, together with some particular practices (use of small vessels, dry hopping, etc.), can cause more oxidation associated with pre-maturated colloidal instability, Madeira off-flavors, bitterness decrease, and aroma loss. As bottle refermentation is widely used in Belgian craft beers, the aim of the present work is to assess how this practice might impact their flavor. In fresh beers, key flavors were evidenced by four complementary techniques: short-chain fatty acids determination, esters analysis, XAD-2 extract olfactometry, and overall sensory analysis. In almost all of the fresh beers, isovaleric acid was the sole fatty acid found above its sensory threshold. Selected samples were further analyzed through natural aging at 20 °C. The presence of yeast in the bottle minimized the trans-2-nonenal released from Schiff bases and proved less deleterious than suggested by previous studies with regard to fatty acid release and ester decrease through aging. Furthermore, according to the yeast species selected, some interesting terpenols and phenols were produced from glucosides during storage.
Eighty-four individual hop samples were gathered over three harvest years to determine chemical factors in hops that serve as indicators of a hop's aroma potential during dry-hopping. Two public American hop varieties that are important to U.S. hop farmers and used by craft brewers globally, Cascade (n = 51) and Centennial (n = 33), were evaluated. Using a constant dry-hopping rate (3.8 g/L), significantly different aroma intensities and qualities were observed across the various samples of hops within each cultivar. Multiple linear regression analysis based on the concentrations of 16 hop oil analytes identified geraniol to be more effective than total oil content in predicting Cascade aroma quality and intensity in dry-hopped beer. Centennial hops differed from Cascade in that β-pinene was identified as being a more improved indicator of dry-hop aroma as compared to total oil content. In each hop variety, the single hop volatiles explained approximately 50 % of the variation in the sensory qualities of the dry-hopped beer, while total hop oil content explained less than 30 % of the same variation. These results suggest that the dry-hop aroma potential of different hop varieties is predicted by different hop volatiles and that total oil content is not the best indicator of a hop's dry-hop aroma intensity or quality.
In this study, hop-derived isobutyric esters (isobutyl isobutyrate, isoamyl isobutyrate, and 2-methylbutyl isobutyrate) and ethyl esters of branched-chain fatty acids (ethyl isobutyrate, ethyl isovalerate, and ethyl 2-methylbutyrate) have been focused on. The wort, green beer, and finished beer samples hopped with total 42 hop varieties were analyzed and compared. Of the three isobutyric esters, 2-methylbutyl isobutyrate is most dominant component. All isobutyric esters gradually decreased during fermentation. All ethyl esters of branched-chain fatty acids were almost absent in wort and gently increased during total fermentation period, except for the fermentation using Huell Melon hops. Surprisingly, the wort made by Huell Melon hops only contained ethyl isobutyrate and ethyl 2-methylbutyrate at relatively high levels. On the other hand, the concentrations of all three ethyl esters in the Ekuanot (HBC366) beer were at relatively high levels and ethyl isovalerate was most dominant component. In addition, it is suggested that a part of 2-methylbutyl isobutyrate (threshold in beer, 78 µg/L) could be transesterificated to ethyl isobutyrate (threshold in beer, 6.3 µg/L). The transesterification between these esters might be an important reaction for hop aromas in beer. From this study, it is concluded that hop-derived various esters including branched-chain structures might be very important contributors to hop varietal aromas, having two roles, their own fruity flavours and precursors of ethyl esters of branched-chain fatty acids.
(A preliminary report of some of this work was given at the 35th Congress of the European Brewery Convention, Porto, Portugal, 24-28 May, 2015. )
BACKGROUND
Although fatty acids have a beneficial effect on yeast growth during fermentation, their effect on foam and sensory stability of beer is negative. In general, long‐chain fatty acids originate from raw materials, whereas short‐chain acids are produced by yeast during fermentation. If the concentration of short‐chain fatty acids, especially isovaleric and butyric acid, overreaches a sensory threshold, then an unpleasant aroma, such as cheesy or sweaty feet, can be formed in beer.
RESULTS
The distribution of fatty acids, from the preparation of sweet wort to the final beer, was studied using chemometric evaluation. Differences were observed between the decoction and infusion system using four barley varieties. Attention was paid to the behavior of short‐chain fatty acids, namely isovaleric acid. The concentration of isovaleric acid in commercial beers brewed in infusion and decoction systems was approximately 1.4 and 1.0 mg L⁻¹, respectively. The same trend was observed in experimental samples (1.3 and 0.5 mg L⁻¹, respectively). This phenomenon was confirmed experimentally; based on the results, this possibly explains why, during the fermentation, isovaleric acid is coupled with the redox state of yeast cell, which is given by the wort composition (i.e. by the mashing process).
CONCLUSION
The formation of isovaleric acid is not only caused by microbiology infection or by oxidized hops, but also is influenced by the mashing process. © 2018 Society of Chemical Industry
Hops play a significant role in determining the aroma of beer. The essential oil of hops contains a large number of flavor-active components. Concentrations of essential oil constituents in beer depend on factors such as the time of hop addition in the brewing process and hop amount added. Generally, compound classes such as mono- and sesquiterpenes do not reach the threshold concentrations in the final product, but in dry-hopped beers after main fermentation they often do. Two factors that potentially cause decreased amounts of terpenoids in beer were investigated. In case of the non-polar compound β-myrcene, losses due to releases into the gas phase during standardized laboratory-scale fermentations were studied. Samples of industrially produced all malt wort (11.5 °P) were dry-hopped at pitching with Mosaic hops. Two yeast strains that are widespread in German beer production were used in trials, TUM 68 (S. cerevisiae) and TUM 34/70 (S. pastorianus). A method for dissolving fermentation gases in bubbling water columns was used. The hops, SPE-water extracts and beer samples were analyzed by several chromatographic systems using two different GC-FID, nanoLC-MS/MS, GC-MS and HS-GC-MS, respectively. Tendency was shown that higher temperatures at primary fermentation cause increased releases of aroma compounds into the gas phase, which was observed on model fermentations in previous studies. The reversible uptake of β-myrcene by yeast cells, identified in separate test series, was determined as being a highly effective factor decreasing amounts in beer systems. In bottled beers 100 million cells/ml led to decreased amounts of about 98–99 %. It was shown that solvent systems with similar properties to beers (5 % and 10 % ethanolic solution) are inadequate for re-dissolving compounds attached to yeasts. The absorbed amount in yeast therefore cannot contribute to the flavor of beer. Incomplete recovered amounts of β-myrcene even in pure ethanol suspensions indicate that there are strong bonds between yeast cells and the odor compound. Linalool, on the other hand, was not affected by the test conditions used.
The essential oil fraction of hops clearly plays a significant and important role in their contribution to the aroma of beer, yet the potential role of water-soluble flavor precursors in hops is much less certain. The study presented herein investigated a range of glycoside hydrolysis techniques on the aqueous extracts of " spent " hops obtained after supercritical CO 2 extraction of hop pellets. A range of enzyme treatments, acid hydrol-ysis, and yeast fermentations were compared with each other and against an untreated control by measuring the volatile aroma composition of the spent hop treatments using stir-bar sorptive extraction and gas chromatog-raphy–mass spectrometry. In general, the enzyme systems show promise for investigating the broad range of glycosides in hops and offer a similar scale of aglycone delivery as yeast fermentations, whereas acid hydroly-sis may offer a relatively quick gauge of total content of glycosidically bound terpene alcohols. Eighteen different hop varieties were examined for glycoside composition using one of the enzyme techniques. Selection was based on their genetic similarity or dissimilarity (pedigree), importance to the brewing community, and input from brewers, hop researchers , and hop breeders. Clear differences were apparent in the terpene alcohols following glycoside hydrolysis across these hop varieties, most notably with Columbus and Centennial hops. However, because of the high usage levels used in this study (50 g of spent hops/L of buffer), brewers are cautioned against overstating the importance of the glycoside fraction to the overall aroma of hops in beer.
Recently, brewers have dramatically increased the complexity and intensity of aromas in hop-forward beers by using diverse hopping regimes. For this study, the terpenoid content and sensory attributes of beers made using different hop additions were measured. Beers were brewed while varying two factors: hop cultivar (Simcoe and Hallertau Mittelfrüh) and timing of hop addition (60 min boil, 25 min whirlpool, or 48 h dry hopping). Additionally, the impact of yeast strain on treatment was investigated. Each treatment was compared with an unhopped control using stir-bar sorptive extraction GC-MS and descriptive sensory analysis. Multivariate statistical analysis showed relationships between instrumental and sensory techniques. Whirlpool additions produced beers with the highest concentrations of geraniol, linalool, and β-citronellol; beers brewed with highly aromatic Simcoe hops produced more intense and individually distinct aromas for each hopping regime compared with the Hallertau Mittelfrüh hopped beers. Conversely, beers brewed with Hallertau Mittelfrüh hops showed less intense aromas with less distinction between hopping regimes, except for the dry-hopped treatment, which was characterized by a more floral type of aroma than the other Hallertau Mittelfrüh treatments. This research shows that despite the popularity of dry hopping as an aroma hopping method, whirlpool additions can produce more intensely aromatic beers.
In previous studies, we found that the concentration of geraniol and β-citronellol in finished beer could be enriched depending on the initial geraniol content in the wort, as a result of single-hopped test-brewing by using geraniolrich hop. In this study, we tried to confirm the effect of blend-hopping using geraniol-rich Bravo hop on the concentration of monoterpene alcohols during fermentation. As a result, the additional Bravo hop, at only 0.4 g of hop/L dosage, could increase approx. 20 μg/L of geraniol and > 15 μg/L of β-citronellol in blend-hopped beers in comparison with single-hopped control beers. We have also previously demonstrated that the coexistence of linalool, geraniol, and β-citronellol could increase the average scores of Citrus by using the model solution (5 % v/v ethanol, carbonated). In this study, we observed same effect of increase of monoterpene alcohols on the change of flavour characters, first time in hopped beers. Furthermore, we found that there might be an additive effect between a volatile thiol (4-methyl-4-sulfanylpentan-2-one (4MSP)) and monoterpene alcohols (linalool and geraniol) and that only 1.2 ng/L of 4MSP was enough for this effect. In addition, as a result of model sensory evaluation, it was also found that the Tropical character could be maximized under coexistence of 4MSP and three monoterpene alcohols (linalool, geraniol, and β-citronellol). We assumed that the synergy among volatile thiols and monoterpene alcohols could contribute to such flavours in beers made from certain Flavour Hop varieties, containing both groups of compounds. This result is also useful for determining blend-hopping recipe based on a composition of flavour compounds of each hop variety.
There have always been beers with a hop aroma, especially in the Pilsner sector. Linalool with its fl oral character is often cited as an indicator substance for the corresponding sensory impression [1-4]. The rapid development of the craft brewing scene originating in the USA, not least through the re-discovery of dry hopping, has led to the appearance of numerous hoppy beers on the market. This trend includes not only the practically mandatorily dry hopped pale ales and India pale ales, but now also pilsners, wheat beers and other beer styles.
During hop storage secondary metabolites, primarily bitter acids, terpene hydrocarbons and terpenoids, undergo qualitative and quantitative changes. One reaction that influences both - bitter properties and aroma - is the release of extremely flavour active short chain monocarboxylic acids by cleavage of the acyl-side chain of bitter substances. Short chain acids exhibit very intense unpleasant cheesy, sweaty, and rancid smells, however they are key precursors of highly positive flavourings oflate hopped beer. In the current paper a stable isotope dilution assay for precise quantification of short chain carboxylic acids was integrated into conventional hop oil analysis. Short chain acids, terpene hydrocarbons, terpenoids, and hop bitter substances where analyzed in fresh as well as in moderately and excessively aged hop samples.
When calculating the transfer rate of geraniol in dry hopped beers based solely on the initial value in hops,
there are tremendous fl uctuations of between about 40% and over 200%. Some varieties, including Cascade,
Hallertau Blanc and Polaris, contain considerable amounts of geranyl acetate in addition to geraniol. However,
since no trace of geranyl acetate can be found in the beer, even at high dosages, it can be concluded that at
least under these conditions (dry hopping before maturation and storage) geranyl acetate is hydrolyzed and
geraniol is released. Including the dosage of geranyl acetate into the calculation of the transfer rates, this
yields values of 36 % to 62%, a variation clearly within the limits of error analysis.
In a previous study, we have focused on biotransformation of hop-derived monoterpene alcohols (linalool, geraniol, beta-citronellol, nerol, and a-terpineol) and their contribution to the flavor of hopped beer. Geraniol showed a drastic decrease during the growth phase and a part of geraniol was converted to P-citronellol by yeast. P-Citronellol was almost absent in hop and wort and gradually increased during the total fermentation period. The concentrations of geraniol and beta-citronellol in finished beer could be enriched depending on the initial geraniol content in the wort by using a geraniol-rich hop. As a result of sensory evaluation, there was an additive effect among linalool, geraniol, and beta-citronellol, and the flavor impression became lime-like by coexistence of these three monoterpene alcohols. In this study, we compared the composition of monoterpene alcohols in various hops, including new flavor hop varieties. Of all screened hops, most of the U.S. hop varieties contained relatively large amount of geraniol. Cascade, Bravo, and Mosaic were screened as geraniol-rich flavor hops. By using these varieties, we brewed test beers under various hop-addition timings to compare the composition of three monoterpene alcohols and total hop flavor profiles in the finished beers. As a result, the content of geraniol in finished beer could increase by delaying the timing of hop addition, while the yeast growth phase was avoided. On the other hand, the P-citronellol contents in the finished beers made with the same hop variety (except Bravo) were present at almost the same levels, regardless of the timings of the hop addition, because of active generation of beta-citronellol during the storage period. In addition, interesting behaviors of monoterpene alcohols, especially P-citronellol, observed in this study, and the relationship between the timing of hop addition and the profile of various flavor compounds in beer are discussed.
Among the most important factors influencing beer quality is the presence of well-adjusted amounts of higher alcohols and esters. Thus, a heavy body of literature focuses on these substances and on the parameters influencing their production by the brewing yeast. Additionally, the complex metabolic pathways involved in their synthesis require special attention. More than a century of data, mainly in genetic and proteomic fields, has built up enough information to describe in detail each step in the pathway for the synthesis of higher alcohols and their esters, but there is still place for more. Higher alcohols are formed either by anabolism or catabolism (Ehrlich pathway) of amino acids. Esters are formed by enzymatic condensation of organic acids and alcohols. The current paper reviews the up-to-date knowledge in the pathways involving the synthesis of higher alcohols and esters by brewing yeasts. Fermentation parameters affecting yeast response during biosynthesis of these aromatic substances are also fully reviewed.
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.
Dry-hopping, which is the cold, aqueous extraction of hops into beer, has gained popularity in recent years as a method for achieving intense hop aroma and flavor in beer. With some brewers dry-hopping at rates up to 2 kg/hL (60 hL (50 bbl) of beer at a rate of 1,592 g/hL (4.125 lb/bbl). Approximately one-third of the dry-matter composition of hops was lost during dry-hopping regardless of hop variety; however, there was high retention of both a-acids (77% pilot scale, 52% industrial) and hop essential oil (51% pilot scale, 33% industrial). The oil remaining in the spent hops was enriched in hydrocarbon compounds and depleted in oxygenated compounds. These results indicate that spent dry-hops contain considerable brewing value and have the potential for re-use.
It is generally recognized within the brewing industry that hop aroma and flavor in beer changes as beer ages post-packaging. Lager beer staling has been studied extensively, while dry-hopped beer has received limited attention. This study evaluated the impact of dissolved oxygen on the sensory and hop volatile profiles of dry-hopped beer during storage. Commercially-brewed dry-hopped beer was dosed with oxygen in a controlled fashion to create beers with a range of dissolved oxygen concentrations from approximately 40 to 250 μg/L 40 to 250 µg/L and then stored under chilled (3 °C) or accelerated (30 °C) storage conditions. Projective Mapping (Napping®) generated unique Euclidian configurations for each panelist, and ultra-flash profiling enriched the configurations with sensory descriptors. Sensory results identified storage temperature, used as a proxy for aging duration, as having the greatest effect on aroma during storage. Dissolved oxygen concentration was observed to have a lesser, but significant, impact at both high and low storage temperatures after only two weeks of aging. Higher temperature and dissolved oxygen concentrations resulted in decreased tropical, citrus, and hoppy characteristics and the expression of malty, dried fruit, and cardboard aromas. Hop derived monoterpenes were not significantly affected by treatment temperature or dissolved oxygen, suggesting stale character expression from alternate sources such as lipid oxidation or Strecker aldehydes formation.
After evidence of the cysteinylated precursors of 3-sulfanyl-4-methylpentan-1-ol (Cys-26) and 3-sulfanylhexan-1-ol (Cys-23) in hop, S-glutathione precursors (G-23 and G-26) were recently discovered in different dual-purpose hop varieties. As free 3-sulfanylpentan-1-ol (21) has also been detected in hop, the present work aimed to identify its potential precursors. The compounds S-3-(1-hydroxylpentyl)cysteine (Cys-21), and S-3-(1-hydroxylpentyl)glutathione (G-21) were first synthesized and characterized by NMR and HRMS. HPLC-ESI(+)-MS/MS evidenced both for the first time in hop. Both S-conjugates were further quantitated in six hop samples: the well-known Saaz, Amarillo, Citra, Hallertau Blanc, Nelson Sauvin, and Polaris. Like G-23, G-21 appeared ubiquitous to all varieties. Of all the samples investigated here, Citra (harvest 2017) emerged as the richest in G-21, with 18 mg per kg of dry matter. Cys-21 was found in all samples at much lower concentration (up to 0.2 mg per kg of dry matter in Polaris, harvest 2017). Model media spiked with Cys-21 or G-21 allowed to confirm that brewing yeast is able to release free 21 from them.
Enzymes in hops have recently been demonstrated to hydrolyze beer dextrins, produce fermentable sugars in finished beer, and pose significant safety and quality challenges for brewers. In an effort to mitigate beer refermentation following dry-hopping in the presence of yeast and the effect of "hop creep," brewers look to adjust recipes, selection of ingredients, and dry-hopping conditions. With phytochemical/secondary metabolite based flavor differences known to exist across hop cultivars, it was posited that there may also exist unique enzymatic activities based upon cultivar and that brewers may use these differences to guide their hopping practices. The enzymatic power of 30 hop cultivars was screened using specific enzyme assay kits (a-amylase, b-amylase, amyloglucosidase) on hops as well as quantifying via liquid chromatography the non-fermentable dextrin degradation and sugar production in beer dry-hopped with different hops. Differences across all cultivars were found and cluster analysis revealed groupings that were not based on pedigree, genetic makeup, or specific enzyme activities. The influence of growing and harvest practices, processing conditions, and presumably age post processing on hop enzymatic power warrants more investigation. These findings indicate that the choice of hop cultivar may influence the degree of refermentation in finished beer as a result of dry-hopping.
Bottom-fermented and top-fermented beers, both either late or dry hopped with Huell Melon hops, and respective reference beers without late or dry hopping were subjected to a comparative odorant screening by aroma extract dilution analyses. On the basis of differences in the FD factors, 14 odorants were identified as hop-derived. Among them were ethyl 2-methylpropanoate, methyl 2-methylbutanoate, ethyl 2-methylbutanoate, propyl 2-methylbutanoate, myrcene, linalool, and geraniol. Differences between late hopped, dry hopped, and reference beers were substantiated by quantitation. Results showed minimal transfer of myrcene from hops into beer. Moderate transfer was observed for propyl 2-methylbutanoate, geraniol, and linalool. Process-induced changes of ethyl 2-methylpropanoate, ethyl 2-methylbutanoate, and methyl 2-methylbutanoate were beyond a direct transfer from hops into beer, suggesting a formation from the corresponding hop-derived carboxylic acids by yeast. Spiking experiments revealed that particularly linalool and propyl 2-methylbutanoate contributed to the characteristic aroma of beers flavored with Huell Melon hops.
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.
Hops are natural ingredients used to impart bitterness and flavor to beer. Recently, new varieties of hops have attracted global research attention. The Sorachi Ace variety, especially, interests many craft brewers. This hop imparts characteristic varietal aromas, including woody, pine-like, citrus, dill-like, and lemongrass-like, to finished beers. Here, we investigated specific flavor compounds derived from Sorachi Ace using selectable one-dimensional or two-dimensional gas chromatography-olfactometry/mass spectrometry and head space-solid phase microextraction-gas chromatography-mass spectrometry. The results showed that a unique volatile compound, geranic acid, was present at a signifi-cant level only in the test beer brewed with the Sorachi Ace hop. Furthermore, sensory evaluation tech-niques revealed that geranic acid has very unique characteristics. This compound is not odor-active but functions as an enhancer for hop-derived terpenoids at subthreshold levels.
Dry hopping is a powerful practice for imparting a multitude of flavors into beer. In this study, the influence of ethanol content, temperature, dosage, and hop variety on the transfer of essential oil during dry hopping was examined on a laboratory scale. The dry hopping was performed with nonalcoholic beer and beer containing 5.0 and 8.1% ethanol at 1 and 20°C using the hop varieties Tettnanger, Cascade, Hallertau Blanc, and Eureka. The results showed that the basic beer, hop variety, and dry hopping regime influence the composition of hop essential oil constituents in dry-hopped beer. The increase of the basic beer ethanol content, and especially the rise in temperature, led to a significant increase in the proportion of monoterpenes such as β-myrcene among hop volatiles in dry-hopped beers. Increasing hop dosage led to higher proportions of alcoholic compounds (linalool). Furthermore, the transfer rates of particular volatile hop-derived substances correlated with their octanol-water partition coefficients (log KOW), which is a measure of the hydrophobicity of a compound, regardless of tested factors in dry hopping. Therefore, it is proposed that the log KOW could be a useful model for the prediction of transfer rates of hop oil flavor components in dry hopping. However, the transfer rates of the alcohols linalool, geraniol, α-terpineol, and 1-octen-3-ol were higher than the expected levels from the log KOW values. These compounds are reported present in bound form in hops and released during dry hopping.
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.
The volatiles isolated from samples of the special flavor hop varieties Huell Melon and Polaris and from the aroma hop variety Hallertau Tradition by solvent extraction and solvent-assisted flavor evaporation (SAFE) were subjected to a comparative aroma extract dilution analysis (cAEDA), which resulted in 46 odor-active compounds in the flavor dilution (FD) factor range of 16 to 2048. On the basis of high FD factors, myrcene, (3R)-linalool, and 2- and 3-methylbutanoic acid were confirmed as important variety-independent hop odorants. (1R,4S)-Calamenene was identified for the first time as odor-active compound in hops. Clear differences in the FD factors and their subsequent objectification by stable isotope dilution quantitation suggested that high concentrations of the esters ethyl 2-methylbutanoate, ethyl 2-methylpropanoate, and propyl 2-methylbutanoate cause the characteristic fruity, cantaloupe-like odor note in Huell Melon hops, whereas the fruity and minty odor notes in Polaris are associated with high amounts of 3-methylbutyl acetate and 1,8-cineole.
A method for the quantitation of six polyfunctional thiols, 4-methyl-4-sulfanylpentan-2-one (4MSP), 3-sulfanyl-4-methylpentan-1-ol (3S4MP), 3-sulfanyl-4-methylpentyl acetate (3S4MPA), 3-sulfanyl-3-methylbutan-1-ol (3S3MB), 3-sulfanylhexan-1-ol (3SH), and 3-sulfanylhexyl acetate (3SHA), in hops and beer without organic mercury compounds was developed. The method employed specific extraction of thiols using a silver ion solid phase extraction (SPE) cartridge and gas chromatography-tandem mass spectrometry (GC-MS/MS). For all thiols analyzed, good linearity was achieved by adding thioglycerol as an analyte protectant. Recoveries for both hops (74–100%) and beer (79–113%) were acceptable, and the repeatability for both was also good (relative standard deviations of 2.8–8.4%). The limits of detection for the six polyfunctional thiols were below their odor thresholds in beer. The method was applied to quantitation of hops and beer flavored with thiol-containing hop varieties. Due to their detected levels and level variations in different beers, 4MSP and 3S4MP are thought to be important polyfunctional thiols for the characteristic flavor of hop varieties.
The dual-purpose hop varieties Amarillo, Citra, Hallertau Blanc, Mosaic, and Sorachi Ace were recently shown to contain unusually high amounts of some discriminating terpenoids, polyfunctional thiols, and precursors of the latter (cysteine and glutathione adducts). The present work aimed to investigate the terpenol glucoside fraction in hops and its potential contribution to beer after a dry hopping process. Terpenols were quantified by stir-bar sorptive extraction GC-MS in five pilot monovarietal dry-hopped beers. In all of them, linalool and geraniol were found above their sensory thresholds (72-178 and 7-57 μg/L, respectively, for a threshold of 8 μg/L for linalool and 4 μg/L for geraniol). β-Citronellol also exceeded its threshold when the Amarillo, Citra, or Sorachi Ace cultivars were used. The hop glucoside potential was analyzed by GC-MS after enzymatic degradation. A relative hydrolysis efficiency factor was applied to our data to take into account that the commercial P-glucosidase releases octan-1-ol, used here as an internal standard, 2.8 times more efficiently than geraniol. β-Glucosidase treatment caused the release of linalool, α-terpineol, β-citronellol, and geraniol from all five dual-purpose cultivars, but in much lower amounts than the corresponding free terpenols (0.6-28.6 mg/kg of aglycons versus 7.8-109.2 mg/kg of free forms). Further quantitative analyses focusing on more traditional aromatic and bitter hops are now needed to compare their glucoside fractions with those here investigated.
Monovarietal dry-hopped beers were produced with the dual-purpose hop cultivars Amarillo, Hallertau Blanc, and Mosaic. The grapefruit-like 3-sulfanyl-4-methylpentan-1-ol was found in all three beers at concentrations much higher than expected on the basis of the free thiol content in hop. Even cysteinylated precursors proved unable to explain our results. As observed in wine, the occurrence of S-glutathione precursors was therefore suspected in hop. The analytical standards of S-3-(4-methyl-1-hydroxypentyl)glutathione, never described before, and of S-3-(1-hydroxyhexyl)glutathione, previously evidenced in grapes, were chemically synthesized. An optimized extraction of glutathionylated precursors was then applied to Amarillo, Hallertau Blanc, and Mosaic hop samples. HPLC-ESI(+)MS/MS revealed, for the first time, the occurrence of S-3-(1-hydroxyhexyl)glutathione and S-3-(4-methyl-1-hydroxypentyl)glutathione in hop, at levels well above those reported for their cysteinylated counterparts. S-3-(1-Hydroxyhexyl)glutathione emerged in all cases as the major adduct in hop. Yet, although 3-sulfanylhexan-1-ol seems relatively ubiquitous in free, cysteinylated, and glutathionylated forms, the glutathione adduct of 3-sulfanyl-4-methylpentan-1-ol, never evidenced in other plants up to now, was found only in the Hallertau Blanc variety.
Since ten years, many studies conducted on beers showed an important impact of polyfunctionnal thiols of the aroma profiles. Among them, three thiols responsible for blackcurrant bud, passion fruit, citrus and rhubarb notes have been intensively studied: the 4-methyl-4-mercaptopentan-2-one (4MMP), the 3-mercaptohexan-1-ol (3MH) and its corresponding acetate. Their origin was very complex in beers since they probably came from odorless precursors present either in hops or in malts. Our work focused on the formal identification of thiol precursors in hops and on their quantification. By using pure synthetic standards and mass spectrometry characterization, we formally identified for the first time the occurrence of glutathionylated conjugates of 4MMP and 3MH and the cysteinylated conjugate of 4MMP in hops. First quantification results obtained on 10 hop varieties, showed that 3MH conjugates were more ubiquitous than 4MMP ones. Conjugates of 3MH occurred at very high level until 20 mg.kg-1 in Cascade hop, which was considerably higher than concentrations found in grapes. Then, we compared the proportion of bound and free thiol fractions and we demonstrated that more than 99 % of 3MH occurred as precursors in hops. On the contrary, free 4MMP fraction represented the most important source of 4MMP in hops.
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.
Glycosidically bound flavour compounds of hops are considered to contribute to the hop flavour in beer. Therefore, different hop varieties and hop products were analyzed for their glycoside content. During extraction with supercritical CO2almost all of the hop glycosides remain in spent hops. After ethanol extraction, glycosides could be detected in both the ethanol tannin extract and the ethanol pure resin extract. In all examined varieties an enrichment of the sensorially important glycosides of monoterpene alcohols (e.g. linalool) and nor-carotenoids could be observed in the ethanol pure resin extracts. Some of these glycosides could be detected in hopped beer, where they could act as precursors for the organoleptically active linalool. This could be confirmed by a rise in free linalool in a stored beer brewed with a re-extracted ethanol pure resin extract containing linalyl glycoside, compared to a beer brewed with a glycoside-free hop preparation. The strongly flavouractive ß-damascenone could also be liberated from hop glycosides, but hops seem not to be the only source for this ketone.
The aim of this study was to differentiate hop pellets by essential oil analysis. Volatile compounds of five aromatic cultivars (Styrie, Saaz, Lublin, Mount Hood, and Hallertau) and seven bitter cultivars (Northern Brewer, Nugget, Pride of Ringwood, Northdown, Galena, Target, and Challenger) were extracted with a Likens-Nickerson simultaneous solvent extractor. The extracts had a strong hop aroma that varied according to the type of hop. Approximately 100 compounds were separated by gas chromatography (GC) and identified by GC-mass spectrometry. An identification flowchart including seven terpenic compounds, four esters, and one methyl ketone was established to discriminate between fresh samples of the 12 investigated cultivars. High amounts of bergamotene and farnesene were found only in Saaz, Lublin, and Styrie samples. Quantification of 4-decenoic acid methyl ester and 3-methyl butyl isobutyrate proved a quick means of distinguishing non-European and European bitter hops from aromatic cultivars.
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.
Free terpenoids and both free and bound polyfunctional thiols were investigated in five selected dual-purpose hop cultivars. Surprisingly, the dual-purpose Sorachi Ace variety was found to contain higher amounts of farnesene (2101 mg/kg) than aromatic hops such as Saaz but only traces of 3-methylbutylisobutyrate, a compound that usually distinguishes all bitter varieties. All five cultivars investigated here showed an exceptional citrus-like potential explained by either monoterpenic alcohols or polyfunctional thiols. Among the monoterpenic alcohols, β-citronellol at concentrations above 7 mg/kg distinguished Amarillo, Citra, Hallertau Blanc, Mosaic, and Sorachi Ace from Nelson Sauvin and Tomahawk, two previously investigated dual-purpose hops, while linalool (312 mg/kg) and geraniol (211 mg/kg) remained good discriminating compounds for Nelson Sauvin and Tomahawk, respectively. Regarding polyfunctional thiols, higher amounts of 3-sulfanylhexyl acetate (27 μg/kg) characterized the Citra variety. Free 4-sulfanyl-4-methylpentan-2-one proved discriminant for Sorachi Ace, while the bound form is predominant in Nelson Sauvin. On the other hand, an S-conjugate of 3-sulfanylhexan-1-ol was found in Sorachi Ace at levels not far from those previously reported for Cascade, although the free form was undetected here. Both free and bound grapefruit-like 3-sulfanyl-4-methylpentan-1-ol (never evidenced before the present work) emerged as discriminating compounds for the Hallertau Blanc variety. The apotryptophanase assay also allowed us to evidence for the first time an S-conjugate of 2-sulfanylethan-1-ol.
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.
Although hop technology has been a substantial part of brewing science for the last 130 years, we are still far from claiming to know everything about hops. As hops are considered primarily as a flavour ingredient for beer, with the added benefit of having anti-microbial effects, hop research is focused on hops as a bittering agent, as an aroma contributor and as a preservative. Newer fields in hop research are directed toward the relevance of hops in flavour stability, brewing process utilisation, the technological benefits of hops in brewing as well as hops as a source of various substances with many health benefits. However the more we find out about the so-called “spirit of beer” the more questions emerge that demand answers. While hop research was only an ancillary research field for decades, during the last ten years more universities and breweries have determined that hops must play a meaningful role in their research efforts. This article gives an overview of the up-to-date knowledge on hop aroma, hop derived bitterness, and the role of hops in flavour stability as well as light stability. Hop research is a wide field, therefore in this review only selected topics are reviewed. Other research areas such as hops utilisation, the antifoam potential of hops, or the advances in knowledge pertaining to the physiological valuable substances of hops go beyond the scope of this article.
The medium chain length fatty acids that are excreted during fermentation are produced by synthesis and not by degradation. The fermentation of a wort supplemented with propionic acid (C3) or valeric acid (C5) leads to the excretion of nonanoic acid (C9) in addition to the usual even chain acids. C9 acid was not detected in the beer when the inoculated yeasts contained a high proportion of pentadecanoic acid (C16) and heptadecanoic acid (C17) or when the C17 acid was added to the wort, demonstrating that a degradative route is unimportant. The content of the medium chain length fatty acids in beer varies directly with their content in yeast; thus the fatty acid composition of the beer reflects changes in the content of these acids in yeast brought about by alteration in the supply of oxygen or by the addition of C3 acid to wort.
Sulphanylalkyl alcohols and their corresponding acetates were investigated in 14 commercial Belgian beers. Although it was the major peak at the pulsed-flame photometric detector, the empyreumatic 2-sulphanylethyl acetate was found at concentrations below its individual odour threshold, estimated at 40 µg/L (0–4 µg/L in most fresh beers, 5–12 µg/L in three fresh high-bitter beers). Both the Ehrlich pathway and hop constituents contribute to this content. In 11 of the investigated samples, synthesis of 2SE-A and 3-sulphanylpropyl acetate (roasted, burned) continued during the first three months of storage. Although below their individual thresholds, these compounds might interact by synergy with other aged flavours. As yeast was absent from most of the investigated bottles, chemical degradation of precursors is suspected. Copyright © 2012 The Institute of Brewing & Distilling
Le suivi de tanks de garde d’une production industrielle montre que les arômes du houblon se solubilisent dans la bière dès les deux premières semaines du processus de houblonnage à cru (cas du linalool, du myrcène et de l’acide 3-méthylbutanoïque). Par contre, trois semaines sont nécessaires pour la production de molécules odorantes issues de glycosides du houblon (4-vinylsyringol, citronellol, béta-damascénone). De nombreux arômes soufrés (thiols polyfonctionnels) aux odeurs de fruits exotiques sont également libérés. Le procédé de houblonnage à cru se révèle donc bien plus complexe qu’une simple solubilisation des huiles essentielles du houblon, surtout si la période de maturation est prolongée.
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.