Release of deuterated nonenal during beer aging from labeled precursors synthesized in the boiling kettle.

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.     

Ferulic acid release and 4-vinylguaiacol formation during brewing and fermentation: indications for feruloyl esterase activity in Saccharomyces cerevisiae

The release of ferulic acid and the subsequent thermal or enzymatic decarboxylation to 4-vinylguaiacol are inherent to the beer production process. Phenolic, medicinal, or clove-like flavors originating from 4-vinylguaiacol frequently occur in beer made with wheat or wheat malt. To evaluate the release of ferulic acid and the transformation to 4-vinylguaiacol, beer was brewed with different proportions of barley malt, wheat, and wheat malt. Ferulic acid as well as 4-vinylguaiacol levels were determined by HPLC at several stages of the beer production process. During brewing, ferulic acid was released at the initial mashing phase, whereas moderate levels of 4-vinylguaiacol were formed by wort boiling. Higher levels of the phenolic flavor compound were produced during fermentations with brewery yeast strains of the Pof(+) phenotype. In beer made with barley malt, ferulic acid was mainly released during the brewing process. Conversely, 60-90% of ferulic acid in wheat or wheat malt beer was hydrolyzed during fermentation, causing higher 4-vinylguaiacol levels in these beers. As cereal enzymes are most likely inactivated during wort boiling, the additional release of ferulic acid during fermentation suggests the activity of feruloyl esterases produced by brewer's yeast.     

Modification of the levels of polyphenols in wort and beer by addition of hexamethylenetetramine or sulfite during mashing.

The effects of addition of hexamethylenetetramine (HMT) or sulfite during mashing on the polyphenol content and oxidative stability of wort and beer have been evaluated in a series of laboratory mashings and pilot brews. HMT reduced the concentration of catechin, prodelphinidin B-3, and procyanidin B-3 in wort and beer, whereas the concentration of ferulic acid was unaffected. Sulfite had only a minor effect on the concentration of phenolics in wort and beer. Addition of HMT or sulfite during mashing increased the oxidative stability of the beer slightly as judged by the tendency of formation of radicals (ESR spin trapping technique), although sensory analysis gave identical flavor acceptance scores to beers produced from untreated and HMT-treated wort and lower scores to beer from sulfite-treated wort. No difference in the oxidative stability of the differently treated sweet worts could be detected as judged by the rate of formation of radicals. HMT addition during mashing has thus been demonstrated to be a valuable experimental tool to control the level of polyphenols in wort and for producing brews with various levels of polyphenols from a single malt.     

Influence of the brewing process on furfuryl ethyl ether formation during beer aging

In beer, the development of a solvent-like stale flavor is associated with the formation of furfuryl ethyl ether. The synthesis rate of this important flavor compound is proportional to the concentration of furfuryl alcohol in beer. This study shows that furfuryl alcohol in beer is mainly formed by Maillard reactions initiated during wort boiling and malt production. A mechanism for its formation from alpha-(1,4)-oligoglucans and amino acids in wort and beer is proposed. During wort boiling, a quadratic relationship was found between the wort extract concentration, on the one hand, and the increase of furfuryl alcohol and furfural, on the other. The reduction of furfural by yeast during fermentation further increases the furfuryl alcohol content. In pale beers, the furfuryl alcohol concentration is essentially determined by the thermal load on wort during brewing operations. In dark beers, a considerable fraction of furfuryl alcohol may, however, come from the dark malts used. These results lead to important practical conclusions concerning the control over furfuryl ethyl ether in beer.     

Release and evaporation of volatiles during boiling of unhopped wort

The release and evaporation of volatile compounds was studied during boiling of wort. The observed parameters were boiling time, boiling intensity, wort pH, and wort density. The effect of every parameter was discussed and approached chemically, with an eye on beer-aging processes. The results indicated that pH highly influenced the release of flavor compounds and that the formation of Strecker aldehydes was linear with boiling time. However, because of evaporation of volatiles, information about the applied thermal load on wort is lost when using a volatile heat load indicator. The thiobarbituric acid (TBA) method, which includes the nonvolatile precursors of volatile aging compounds, proved to be a more reliable method to determine all kinds of heat load on wort. Finally, it was discussed how the obtained insights could help to understand the mechanism of beer aging.     

3-methylthiopropionaldehyde as precursor of dimethyl trisulfide in aged beers

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.     

Fate of pesticides during beer brewing

The fates of more than 300 pesticide residues were investigated in the course of beer brewing. Ground malt artificially contaminated with pesticides was brewed via steps such as mashing, boiling, and fermentation. Analytical samples were taken from wort, spent grain, and beer produced at certain key points in the brewing process. The samples were extracted and purified with the QuEChERS (Quick Easy Cheap Effective Rugged and Safe) method and were then analyzed by LC-MS/MS using a multiresidue method. In the results, a majority of pesticides showed a reduction in the unhopped wort and were adsorbed onto the spent grain after mashing. In addition, some pesticides diminished during the boiling and fermentation. This suggests that the reduction was caused mainly by adsorption, pyrolysis, and hydrolysis. After the entire process of brewing, the risks of contaminating beer with pesticides were reduced remarkably, and only a few pesticides remained without being removed or resolved.     

Variability in the release of free and bound hydroxycinnamic acids from diverse malted barley (Hordeum vulgare L.) cultivars during wort production

Volatile phenols have long been recognized as important flavor contributors to the aroma of various alcoholic beverages. The two main flavor-active volatile phenols in beer are 4-vinylguaiacol and 4-vinylphenol. They are the decarboxylation products of the precursors ferulic acid and p-coumaric acid, respectively, which are released during the brewing process, mainly from malt. In this study, the variability in the release of free and ester-bound hydroxycinnamic acids from nine malted barley ( Hordeum vulgare L.) varieties during wort production was investigated. A large variability between different barley malts and their corresponding worts was observed. Differences were also found between free ferulic acid levels from identical malt varieties originating from different malt houses. During mashing, free hydroxycinnamic acids in wort are both water-extracted and enzymatically released by cinnamoyl esterase activity. Esterase activities clearly differ between different barley malt varieties. Multiple linear regression analysis showed that the release of ferulic acid during mashing did not depend only on the barley malt esterase activity but also on the amount of ester-bound ferulic acid initially present in the wort and on its endoxylanase activity. The study demonstrates the importance of selecting a suitable malt variety as the first means of controlling the final volatile phenol levels in beer.     

Behavior of myclobutanil, propiconazole, and nuarimol residues during lager beer brewing

Over a 4 month brewing process, the fate of three fungicides, myclobutanil, propiconazole, and nuarimol, was studied in the spent grain, brewer wort, and final beer product. Only the residual level of myclobutanil after the mashing step was higher than its maximum residue limit (MRL) on barley. A substantial fraction was removed with the spent grain in all cases (26-42%). The half-life times obtained for the fungicides during storage of the spent grains ranged from 82 to 187 days. No significant influence of the boiling stage on the decrease of the fungicide residues was demonstrated. During fermentation, the content reduction varied from 20 to 47%. After the lagering and filtration steps, no significant decrease (<10%) was observed in any of the residues. Finally, during storage of the beer (3 months), the amounts of fungicides fell by 25-50% of their respective concentrations in the finished beer.     

Influence of lipids in the generation of phenylacetaldehyde in wort-related model systems

The effect of lipids on the formation of the Strecker aldehyde phenylacetaldehyde during wort boiling was studied to determine the role that small changes in the lipid content of the wort have in the production of significant flavor compounds in beer. Wort was treated with 0-2.77 mmol per liter of glucose, linoleic acid, or 2,4-decadienal and heated at 60-98 degrees C for 1 h. After this time, the amount of the Strecker aldehyde phenylacetaldehyde increased in the samples treated with linoleic acid or decadienal but not in the samples treated with glucose. Thus, the amount of phenylacetaldehyde produced in the presence of linoleic acid was 1.1-2.5 times the amount of the Strecker aldehyde produced in the control wort, and this amount increased to 3.6-4.6 times when decadienal was employed. The higher reactivity of decadienal than linoleic acid for this reaction decreased with temperature and was related to the oxidation of linoleic acid that occurred to a higher extent at higher temperatures. The above results suggest that lipids can contribute to the formation of Strecker aldehydes during wort boiling and that changes in the lipid content of the wort will produce significant changes in the formation of Strecker aldehydes in addition to other well-known consequences in beer quality and yeast metabolism. On the other hand, because of the high glucose content in wort, small changes in its content are not expected to affect the amount of Strecker aldehydes produced.     

How low pH can intensify beta-damascenone and dimethyl trisulfide production through beer 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.     

Identification of a stale-beer-like odorant in extracts of naturally aged beer

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.     

Impact of different wort boiling temperatures on the beer foam stabilizing properties of lipid transfer protein 1

Beer consumers demand satisfactory and consistent foam stability; thus, it is a high priority for brewers. Beer foam is stabilized by the interaction between certain beer proteins, including lipid transfer protein 1 (LTP1), and isomerized hop alpha-acids, but destabilized by lipids. In this study it was shown that the wort boiling temperature during the brewing process was critical in determining the final beer LTP1 content and conformation. LTP1 levels during brewing were measured by an LTP1 ELISA, using antinative barley LTP1 polyclonal antibodies. It was observed that the higher wort boiling temperatures ( approximately 102 degrees C), resulting from low altitude at sea level, reduced the final beer LTP1 level to 2-3 microg/mL, whereas the lower wort boiling temperatures ( approximately 96 degrees C), resulting from higher altitudes (1800 m), produced LTP1 levels between 17 and 35 microg/mL. Low levels of LTP1 in combination with elevated levels of free fatty acids (FFA) resulted in poor foam stability, whereas beer produced with low levels of LTP1 and FFA had satisfactory foam stability. Previous studies indicated the need for LTP1 denaturing to improve its foam stabilizing properties. However, the results presented here show that LTP1 denaturation reduces its ability to act as a binding protein for foam-damaging FFA. These investigations suggest that wort boiling temperature is an important factor in determining the level and conformation of LTP1, thereby favoring satisfactory beer foam stability.     

Evidence of the glycation and denaturation of LTP1 during the malting and brewing process

The influence of malting and brewing processes on the chemical and structural modifications occurring on LTP1 was investigated by mass spectrometry and circular dichroism. Proteins were first purified from malt, and samples were collected at various steps of beer processing performed on two barley cultivars. The levels of LTP1 found in malt were not significantly different from the amounts in barley seed. However, in malt, both LTP1b, a post-translational form of LTP1, and a third isoform named LTP1c were isolated. Moreover, both of these proteins were found to be heterogeneously glycated but still exhibited an alpha-helix structure. Both glycated LTP1 and LTP1b were recovered during mashing. It was also shown that glycated LTP1 was unfolded during heat treatment of wort boiling, which is in agreement with the denatured form previously isolated from beer.     

Oxidative reactions during early stages of beer brewing studied by electron spin resonance and spin trapping

An electron spin resonance (ESR)-based method was used for evaluating the levels of radical formation during mashing and in sweet wort. The method included the addition of 5% (v/v) ethanol together with the spin trap alpha-4-pyridyl(1-oxide)- N- tert-butylnitrone (POBN) to wort, followed by monitoring the rate of formation of POBN spin adducts during aerobic heating of the wort. The presence of ethanol makes the spin trapping method more selective and sensitive for the detection of highly reactive radicals such as hydroxyl and alkoxyl radicals. Samples of wort that were collected during the early stages of the mashing process gave higher rates of spin adduct formation than wort samples collected during the later stages. The lower oxidative stability of the early wort samples was confirmed by measuring the rate of oxygen consumption during heating of the wort. The addition of Fe(II) to the wort samples increased the rate of spin adduct formation, whereas the addition of Fe(II) during the mashing had no effect on the oxidative stability of the wort samples. Analysis of the iron content in the sweet wort samples demonstrated that iron added during the mashing had no effect on the iron level in the wort. The moderate temperatures during the early steps of mashing allow the endogenous malt enzymes to be active. The potential antioxidative effects of different redox-active enzymes during mashing were tested by measuring the rate of spin adduct formation in samples of wort. Surprisingly, a high catalase dosage caused a significant, 20% reduction of the initial rate of radical formation, whereas superoxide dismutase had no effect on the oxidation rates. This suggests that hydrogen peroxide and superoxide are not the only intermediates that play a role in the oxidative reactions occurring during aerobic oxidation of sweet wort.     

Solid-phase microextraction (SPME) technique for measurement of generation of fresh cucumber flavor compounds

Investigations were carried out to determine whether flavor compounds characteristic for fresh cucumbers could be rapidly determined using a solid-phase microextraction (SPME) dynamic headspace sampling method combined with gas chromatography and flame ionization detection. Cucumbers were sampled, during blending, for fresh cucumber flavor compounds (E,Z)-2,6-nonadienal and (E)-2-nonenal. The GC was such that the two target compounds were separated and baseline-resolved. Relative standard deviations for analysis of both (E,Z)-2,6-nonadienal and (E)-2-nonenal using this SPME sampling method were +/-10%. Utility of the analytical method was demonstrated by determining the effect of heat treatments on the ability of cucumbers to produce these flavor impact compounds.     

Fresh cucumber flavor in refrigerated pickles: comparison of sensory and instrumental analysis

The ability of nonacidified, refrigerated pickled cucumbers to produce the fresh cucumber flavor impact compounds (E,Z)-2,6-nonadienal and (E)-2-nonenal declined during storage. Production of these compounds decreased as the pH of refrigerated cucumbers was reduced. Despite the fact that the concentrations of (E,Z)-2,6-nonadienal and (E)-2-nonenal generated were over 10(5)-fold greater than the threshold levels, it was possible for a sensory panel to consistently detect differences in the intensity of fresh cucumber flavor, provided the pH difference between samples was 1 unit or greater. The presence of spices did not interfere with the ability of panelists to detect differences in fresh flavor intensity. There was a linear correlation between sensory scores and the amount of (E,Z)-2,6-nonadienal produced by cucumbers equilibrated at different pH levels.     

Oxidative degradation of lipids during mashing

Although hardly any polyunsaturated fatty acids (PUFAs) are present in the endproduct, the ingredients used for the production of beer contain a high concentration of PUFAs, such as linolic and linolenic acid. These compounds are readily oxidized, resulting in the formation of lipid-derived products that reduce the taste and quality of beer enormously. During mashing relatively high amounts of PUFAs are exposed to atmospheric oxygen at a relatively high temperature. This makes mashing a critical step in the brewing process with regard to the formation of lipid-derived off-taste products. F1 phytoprostane (PPF1) changes in antioxidant capacity and monohydroxy fatty acids (OH-FAs) were used as markers for the detection of oxidative damage to fatty acids during mashing. The pattern of OH-FA formation indicates that enzymatic oxidation of PUFAs is more important than nonenzymatic oxidation during the mashing process. Nevertheless, substantial nonenzymatic radical formation is evident from the increase of specific OH-FAs and PPF1s. It was found that a low oxygen tension reduces oxidative damage and gives a high antioxidant capacity of the mashing mixture. This indicates that mashing should be done under low oxygen pressure.     

Fate of xanthohumol and related prenylflavonoids from hops to beer

The fate of three prenylated flavonoids of the chalcone type, xanthohumol, desmethylxanthohumol, and 3'-geranylchalconaringenin, was monitored with LC/MS-MS from hops (Humulus lupulus L.) to beer in two brewing trials. The three prenylchalcones were largely converted into their isomeric flavanones, isoxanthohumol, prenylnaringenins, and geranylnaringenins, respectively, in the boiling wort. Losses of prenylflavonoids were due to incomplete extraction from the hops into the wort (13-25%), adsorption to insoluble malt proteins (18-26%), and adsorption to yeast cells (11-32%) during fermentation. The overall yield of xanthohumol, after lagering of the beer and largely in the form of isoxanthohumol, amounted to 22-30% of the hops' xanthohumol. About 10% of the hops' desmethylxanthohumol, completely converted into prenylnaringenins, remained in the beers. 3'-Geranylchalconaringenin behaved similarly to desmethylxanthohumol. Solubility experiments indicated that (1) malt carbohydrates form soluble complexes with xanthohumol and isoxanthohumol and (2) solubility does not dictate the isoxanthohumol levels of finished beers.     

Brewing Performance of Malted Lipoxygenase‐1 Null Barley and Effect on the Flavor Stability of Beer

We examined the malting and brewing performances of a lipoxygenase‐1 (LOX‐1) null line of barley (Hordeum vulgare L.). The LOX‐normal malt and the LOX‐null malt were prepared from F₄ populations derived from a single cross. We could not observe any major differences in the general malt characteristics between the two malts. A brewing trial was performed using these malts. The analysis of the wort and beer revealed that the absence of LOX‐1 had little effect on the general characteristics of the wort and beer. In contrast, beer made from the LOX‐null malt showed reduced levels of beer‐deteriorating substances, trans‐2‐nonenal (T2N), and trihydroxyoctadecenoic acid (THOD). In the sensory evaluation, well‐trained panel members recognized the significant superiority of the aged LOX‐null beer in terms of staleness. These results show that the LOX‐1 null barley line can be effectively used to improve the flavor stability of beer without changing the other important beer qualities.