Novel Saccharomyces cerevisiae variants slow down the accumulation of staling aldehydes and improve beer shelf-life.

Beer quality generally diminishes over time as staling compounds accumulate through various oxidation reactions. Here, we show that refermentation, a traditional practice where Saccharomyces cerevisiae cells are added to beer prior to bottling, diminishes the accumulation of staling aldehydes. However, commonly used beer yeasts only show a limited lifespan in beer. Using high-throughput screening and breeding, we were able to generate novel S. cerevisiae hybrids that survive for over a year in beer. Extensive chemical and sensory analyses of the two most promising hybrids showed that they slow down the accumulation of staling aldehydes, such as furfural and trans-2-nonenal and significantly increased beer flavor stability for up to 12 months. Moreover, the strains did not change the original flavor of the beer, highlighting their potential to be integrated in existing products. Together, these results demonstrate the ability to breed novel microbes that function as natural and sustainable anti-oxidative food preservatives.

The influence of storage conditions on the chemistry and flavor of hoppy ales.

Understanding the factors which lead to the (in)stability of the chemistry and sensory of hoppy beer styles such as India Pale Ales (IPAs) has become a major concern for many brewers. Therefore, the evolution of several volatiles under different storage conditions (room temperature, cold storage, forced aging) was investigated in eleven hoppy ales and one lager which were commercially produced in Germany. Compared to the lager, the fresh ales contained higher initial aldehyde levels. Furthermore, the distribution of lipid oxidation and Maillard reaction products differed from those typically found in lager beer. Upon storage, significant increases of some staling aldehydes were observed. Interestingly, the concentrations of some hop aroma volatiles like terpenoids (i.e. linalool and geraniol) were relatively stable throughout storage and counteracted "oxidized" impressions in ales high in these compounds. In comparison, hop aroma in some ales was driven by less stable volatiles such as esters.

Comprehensive Structural Analysis of the Bitter Components in Beer by the HPLC-Assisted Crystalline Sponge Method.

Trans-iso-α-acid is one of the main contributors to the bitter taste of fresh beer and is known to transform into various derivatives during beer aging. However, structural characterization of the derivatives has been a challenging task because of the formation of too many components. Herein, we report that most of the transformation products of trans-iso-α-acid, isolated in this study in only small quantities by HPLC, can be structurally analyzed with the crystalline sponge method. Thirteen compounds, including eight that were previously unreported, have been successfully isolated and analyzed with complete assignment of their absolute configuration. This provides an improved understanding of the chemical transformations that occur during beer aging.

A Comprehensive Evaluation of Flavor Instability of Beer (Part 2): The Influence of De Novo Formation of Aging Aldehydes.

Flavor instability of beer is affected by the rise of aroma-active aldehydes during aging. Aldehydes can be either released from bound-state forms or formed de novo. This second part of our study focused on the de novo formation of aldehydes during the Maillard reaction, Strecker degradation, and oxidation reactions. Key precursor compounds for de novo pathways are free amino acids. This study varied the potential for reactions by varying free amino acid content in fresh beer using different proteolytic malt modification levels (569-731 mg/100 g d. m. of soluble nitrogen) of the used malt in brewing trials. Overall, six pale lager beers were produced from three malts (different malt modification levels), each was made from two different barley varieties and was naturally and forcibly aged. It was found that higher malt modification levels in fresh beer and during beer aging increased amino acid and dicarbonyl concentrations as aging precursors and Strecker aldehyde contents as aging indicators. Dicarbonyls were degraded during aging. Advanced glycation end products as possible degradation products showed no consistent formation during aging. Therefore, Strecker reactions were favored during beer aging. No alternative oxidative formation of Strecker aldehydes from their corresponding alcohols could be confirmed. Along with the preceding part one of our investigation, the results of this study showed that de novo formation and release occur simultaneously. After 4 months of natural aging, aldehyde rise is mainly accounted for by de novo formation.

The Influence of Proteolytic Malt Modification on the Aging Potential of Final Wort.

The dynamic changes in beer flavor are determined by its aging potential, which comprises of present free and bound-state aldehydes and their precursors. Rising flavor-active aging compounds cause sensory deterioration (flavor instability). These compounds are mainly formed upstream in the brewing process through the Maillard reaction, the Strecker degradation, or lipid oxidation. Wort boiling is an especially critical production step for important reactions due to its high temperature and favorable pH value. Amino acid concentration, as an important aging-relevant precursor, is variable at the beginning of wort boiling, mainly caused by the malt modification level, and can further influence the aging potential aging formation during wort boiling. This study investigated the effect of the proteolytic malt modification level on the formation of precursors (amino acids and dicarbonyls) and free and bound-state aldehydes during wort boiling. Six worts (malt of two malting barley varieties at three proteolytic malt modification levels) were produced. Regarding precursors, especially Strecker, relevant amino acids and dicarbonyls increased significantly with an enhanced malt modification level. Concentrations of free and bound aldehydes were highest at the beginning of boiling and decreased toward the end. A dependency of malt modification level and the degree of free and bound aldehydes was observed for 2-methylpropanal, 2-methylbutanal, and 3-methylbutanal. Generally, a higher proteolytic malt modification level tended to increase free and bound aldehyde content at the end of wort boiling. Conclusively, the aging potential formation during boiling was increased by an intensified malt modification level.

A Comprehensive Evaluation of Flavor Instability of Beer (Part 1): Influence of Release of Bound State Aldehydes.

Flavor instability of pale lager beer depends decisively on aroma-active aldehydes from the Maillard reaction, Strecker degradation, and lipid oxidation, which are formed in various oxidative and non-oxidative reactions. Therein, aldehydes can be formed de novo and be released from bound states to a free, aroma-active form during aging. During malting and brewing, proteolysis affects the amount of soluble nitrogen and thus flavor instability in different ways (e.g., precursors for de novo formation and binding agents for bound states). To isolate nitrogen-related aging processes, beers from malts (two barley varieties, three proteolytic malt modifications) were produced on a 50 L scale in part 1 of this study. Sensory analysis revealed increased flavor instability for beers with higher amounts of soluble nitrogen. Especially Strecker aldehydes significantly increased with malt modification. The release of bound state aldehydes revealed most free aldehydes in fresh beers and with higher malt modification. During aging, the equilibrium between free and bound state aldehydes shifted toward the free form. These results reveal a nitrogen-dependent bound pool of aldehydes that is depleted during aging and is responsible for aged aroma, especially in the early and medium stages of aging. Therefore, bound state aldehydes are indicators of the early-stage prediction of flavor instability already in a fresh condition.

Gas-Diffusion Microextraction (GDME) Combined with Derivatization for Assessing Beer Staling Aldehydes: Validation and Application.

In this work, a gas-diffusion microextraction (GDME) methodology was optimized and validated for the analysis of selected staling aldehydes (furfural (FURF), 2-methylpropanal (2-MP), 2-methylbutanal (2-MB), 3-methylbutanal (3-MB), and acetaldehyde (ACET)) during natural and forced aging of beer. The methodology was optimized considering time, temperature of extraction, and derivatizing agent. Using 4-hydrazinobenzoic acid (HBA) as a derivatizing agent, the performance of the method was evaluated by assessing several parameters such as detection limits (ranging from 1.2 to 1857.7 µg/L for 2-MB and ACET, respectively), quantification limits (ranging from 3.9 to 6192.4 µg/L for 2-MB and ACET, respectively), recoveries (higher than 96%), intraday and interday precisions (lower than 3.4 and 9.2%, respectively), and linearity (r2 ≥ 0.995). During beer aging, higher content of Strecker aldehydes and FURF were found, while no significant variations in ACET levels were observed. In general, the aldehydes content assessed for beers stored at 37 ± 1 °C for 7 and 14 days mimics that observed for beers stored at 20 ± 2 °C for 3 and 6 months, respectively. Lower temperatures of storage (4 ± 1 °C) delayed the development of staling aldehydes. Based on PCA analysis, the content of staling aldehydes and beer color were responsible for 91.39% of the variance among the analyzed samples, and it was demonstrated that these are key parameters to discriminate fresh from aged beers. The results herein presented showed that the proposed analytic methodology is a valuable strategy for the characterization and quantification of important staling aldehydes in beer with a potential application in the quality control of beer during storage.

Insights into chemical and sensorial aspects to understand and manage beer aging using chemometrics.

Beer chemical instability remains, at present, the main challenge in maintaining beer quality. Although not fully understood, after decades of research, significant progress has been made in identifying "aging compounds," their origin, and formation pathways. However, as the nature of aging relies on beer manufacturing aspects such as raw materials, process variables, and storage conditions, the chemical profile differs among beers. Current research points to the impact of nonoxidative reactions on beer quality. The effect of Maillard and Maillard intermediates on the final beer quality has become the focus of beer aging research, as prevention of oxidation can only sustain beer quality to some extent. On the other hand, few studies have focused on tracing a profile of whose compound is sensory relevant to specific types of beer. In this matter, the incorporation of "chemometrics," a class of multivariate statistic procedures, has helped brewing scientists achieve specific correlations between the sensory profile and chemical data. The use of chemometrics as exploratory data analysis, discrimination techniques, and multivariate calibration techniques has made the qualitatively and quantitatively translation of sensory perception of aging into manageable chemical and analytical parameters. However, despite their vast potential, these techniques are rarely employed in beer aging studies. This review discusses the chemical and sensorial bases of beer aging. It focuses on how chemometrics can be used to their full potential, with future perspectives and research to be incorporated in the field, enabling a deeper and more specific understanding of the beer aging picture.

Forced into aging: Analytical prediction of the flavor-stability of lager beer. A review.

Despite years of research, sensory deterioration during beer aging remains a challenge to brewing chemists. Therefore, sensorial and analytical tools to investigate aging flavors are required. This review aims to summarize the available analytical methods and to highlight the problems associated with addressing the flavor-stability of beer. Carbonyls are the major contributors to the aroma of aged pale lager beer, which is especially susceptible to deterioration. They are formed via known pathways during storage, but, as recent research indicates, are mainly released from the bound-state during aging. However, most published studies are based on model systems, and thus the formation and breakdown parameters of these adducts are poorly understood. This concept has not been previously considered in previous forced-aging analysis. Only weak parallels can be drawn between forced and natural aging. This is likely due to the different activation energies of the chemical processes responsible for aging, but may also be due to heat-promoted release of bound aldehydes. Thus, precursors and their binding parameters must be investigated to make appropriate technological adjustments to forced-aging experiments. In combination with sophisticated data analysis, the investigation of volatile indicators and non-volatile precursors can lead to more reliable predictions of flavor stability.

Analysis of 100-Year-Old Beer Originated from the Czech Republic.

Three bottles of different beers were found in 2015 during a reconstruction of the brewery of the Raven Trading s.r.o. company in Záhlinice, Czech Republic. Thanks to good storage conditions, it was possible to analyze their original characteristics. All three bottles contained most probably lager type beer. One beer had sulfuric and fecal off-flavors; it was bright with the original extract of 10.3° Plato. The second beer, with an original extract of 7.6° Plato, was dark and very acidic, resembling Lambic. DNA analysis proved the presence of Dekkera bruxellensis, which corresponded to its chemical profile (total acidity, FAN, ethyl acetate, total esters). The third beer contained traces of carbon dioxide bubbles, was light brown and slightly bitter, with an original extract 10.4° Plato. Because it obviously underwent a natural aging process, sweetness, honey, and fruity off-flavors were detected and transformation products of iso-α-acids were found.

Chemical Characterization of Beer Aging Products Derived from Hard Resin Components in Hops (Humulus lupulus L.).

The bitter taste of beer originates from resins in hops (Humulus lupulus L.), which are classified into two subtypes (soft and hard). Whereas the nature and reactivity of soft-resin-derived compounds, such as α-, ß-, and iso-α-acids, are well studied, there is only a little information on the compounds in hard resin. For this work, hard resin was prepared from stored hops and investigated for its compositional changes in an experimental model of beer aging. The hard resin contained a series of α-acid oxides. Among them, 4'-hydroxyallohumulinones were unstable under beer storage conditions, and their transformation induced primary compositional changes of the hard resin during beer aging. The chemical structures of the products, including novel polycyclic compounds scorpiohumulinols A and B and dicyclohumulinols A and B, were determined by HRMS and NMR analyses. These compounds were proposed to be produced via proton-catalyzed cyclization reactions of 4'-hydroxyallohumulinones. Furthermore, they were more stable than their precursor 4'-hydroxyallohumulinones during prolonged storage periods.

Occurrence of Theaspirane and its Odorant Degradation Products in Hop and Beer.

In model oxidized media, six theaspirane-derived compounds were identified by gas chromatography-high resolution mass spectrometry: 4-hydroxy-7,8-dihydro-ß-ionone, 6-hydroxy-7,8-dihydro-α-ionone, dihydrodehydro-ß-ionone, two monoepoxides, and a derived alcohol. Only 4-hydroxy-7,8-dihydro-ß-ionone and dihydrodehydro-ß-ionone have been described previously in the literature. Investigation of hop revealed five of these compounds in free form together with theaspirane (especially in the Mosaic variety), while the Citra and Amarillo hop varieties emerged as very interesting for the release of theaspirane, 4-hydroxy-7,8-dihydro-ß-ionone, and dihydrodehydro-ß-ionone from glucoside precursors. For the first time, theaspirane, 4-hydroxy-7,8-dihydro-ß-ionone, 6-hydroxy-7,8-dihydro-α-ionone, and both monoepoxides were found in a fresh commercial top fermentation beer (only theaspirane, 4-hydroxy-7,8-dihydro-ß-ionone, and dihydrodehydro-ß-ionone have recently been mentioned as Gueuze constituents).

Microbiota and metabolites of aged bottled gueuze beers converge to the same composition.

Gueuze beers are prepared by mixing young and old lambic beers and are bottle-refermented spontaneously for aging. The present study analyzed the microbiota and metabolites present in gueuze beers that were aged between a few months and up to 17 years. Yeasts were cultivated from all beers sampled, but bacteria could not be grown from beers older than 5 years. Lactic acid and ethyl lactate concentrations increased steadily during aging, whereas ethanol concentrations remained constant. The concentrations of isoamyl acetate and ethyl decanoate decreased during the aging process. Hence, ethyl lactate and ethyl decanoate can be considered as positive and negative gueuze beer-aging metabolite biomarkers, respectively. Nevertheless, considerable bottle-to-bottle variation in the metabolite profiles was found, which hindered the generalization of the effects seen during the aging of the gueuze beers examined, but which illustrated the unique character of the lambic beers. The present results further indicate that gueuze beers are preferably aged for less than 10 years.

Investigations on the Maillard reaction of dextrins during aging of Pilsner type beer.

Although Maillard reaction plays a pivotal role during preparation of food, only few investigations concerning the role of carbohydrate degradation in beer aging have been carried out. The formation of Maillard specific precursor structures and their follow-up products during degradation of low molecular carbohydrate dextrins in the presence of proline and lysine was studied in model incubations and in beer. Twenty-one α-dicarbonyl compounds were identified and quantitated as reactive intermediates. The oxidative formation of 3-deoxypentosone as the precursor of furfural from oligosaccharides was verified. N-Carboxymethylproline and N-formylproline were established as novel proline derived Maillard advanced glycation end products. Formation of N-carboxymethylproline and furfural responded considerably to the presence of oxygen and was positively correlated to aging of Pilsner type beer. The present study delivers an in-depth view on the mechanisms behind the formation of beer relevant aging parameters.

Guaiacol and 4-methylphenol as specific markers of torrefied malts. Fate of volatile phenols in special beers through aging.

Phenol-specific extracts of 12 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 were 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 five 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 (>7 µg/L) for chocolate and black malts only (versus respectively <3 µg/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 <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.