Beer ethanol and iso-α-acid level affect microbial community establishment and beer chemistry throughout wood maturation of beer.

Sour beers produced by barrel-aging of conventionally fermented beers are becoming increasingly popular. However, as the intricate interactions between the wood, the microbes and the beer are still unclear, wood maturation often leads to inconsistent end products with undesired sensory properties. Previous research on industrial barrel-aging of beer suggests that beer parameters like the ethanol content and bitterness play an important role in the microbial community composition and beer chemistry, but their exact impact still remains to be investigated. In this study, an experimentally tractable lab-scale system based on an in-vitro community of four key bacteria (Acetobacter malorum, Gluconobacter oxydans, Lactobacillus brevis and Pediococcus damnosus) and four key yeasts (Brettanomyces bruxellensis, Candida friedrichii, Pichia membranifaciens and Saccharomyces cerevisiae) that are consistently associated with barrel-aging of beer, was used to test the hypotheses that beer ethanol and bitterness impact microbial community composition and beer chemistry. Experiments were performed using different levels of ethanol (5.2 v/v%, 8 v/v% and 11 v/v%) and bitterness (13 ppm, 35 ppm and 170 ppm iso-α-acids), and beers were matured for 60 days. Samples were taken after 0, 10, 20, 30 and 60 days to monitor population densities and beer chemistry. Results revealed that all treatments and the maturation time significantly affected the microbial community composition and beer chemistry. More specifically, the ethanol treatments obstructed growth of L. brevis and G. oxydans and delayed fungal growth. The iso-α-acid treatments hindered growth of L. brevis and stimulated growth of P. membranifaciens, while the other strains remained unaffected. Beer chemistry was found to be affected by higher ethanol levels, which led to an increased extraction of wood-derived compounds. Furthermore, the distinct microbial communities also induced changes in the chemical composition of the beer samples, leading to concentration differences in beer- and wood-derived compounds like 4-ethyl guaiacol, 4-ethyl phenol, cis-oak lactone, vanillin, furfural and 5-hydroxymethyl furfural. Altogether, our results indicate that wood-aging of beer is affected by biotic and abiotic parameters, influencing the quality of the final product. Additionally, this work provides a new, cost-effective approach to study the production of barrel-aged beers based on a simplified microbial community model.

Description of the temporal dynamics in microbial community composition and beer chemistry in sour beer production via barrel ageing of finished beers.

Currently, there is a strong interest in barrel ageing of finished, conventionally fermented beers, as a novel way to produce sour beers with a rich and complex flavour profile. The production process, however, remains largely a process of trial and error, often resulting in profit losses and inconsistency in quality. To improve product quality and consistency, a better understanding of the interactions between microorganisms, wood and maturing beer is needed. The aim of this study was to describe the temporal dynamics in microbial community composition, beer chemistry and sensory characteristics during barrel ageing of three conventionally fermented beers that differed in parameters like alcohol content and bitterness. Beers were matured for 38 weeks in new (two types of wood) and used (one type of wood) oak barrels. Beer samples were taken at the start of the maturation and after 2, 12 and 38 weeks. Microbial community composition, determined using amplicon sequencing of the V4 region of the bacterial 16S rRNA gene and the fungal ITS1 region, beer chemistry and sensory characteristics substantially changed throughout the maturation process. Likewise, total bacterial and fungal population densities generally increased during maturation. PerMANOVA revealed significant differences in the bacterial and fungal community composition of the three beers and across time points, but not between the different wood types. By contrast, significant differences in beer chemistry were found across the different beers, wood types and sampling points. Results also indicated that the outcome of the maturation process likely depends on the initial beer properties. Specifically, results suggested that beer bitterness may restrain the bacterial community composition, thereby having an impact on beer souring. While the bacterial community composition of moderately-hopped beers shifted to a dominance of lactic acid bacteria, the bacterial community of the high-bitterness beer remained fairly constant, with low population densities. Bacterial community composition of the moderate-bitterness beers also resembled those of traditional sours like lambic beers, hosting typical lambic brewing species like Pediococcus damnosus, Lactobacillus brevis and Acetobacter sp. Furthermore, results suggested that alcohol level may have affected the fungal community composition and extraction of wood compounds. More specifically, the concentration of wood compounds like cis-3-methyl-4-octanolide, trans-3-methyl-4-octanolide, eugenol and total polyphenols was higher in beers with a high alcohol content. Altogether, our results provide novel insights into the barrel ageing process of beer, and may pave the way for a new generation of sour beers.

Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains.

Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man-made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off-)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces-related phenolic off-flavors 4-ethylguaiacol and 4-ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off-flavor production. Sequence analysis of genes related to phenolic off-flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.

Changes in the hop-derived volatile profile upon lab scale boiling.

Hop terpenes might be oxidized during kettle boiling into more water soluble compounds that could contribute to 'hoppy' aroma of kettle hopped lager beers. Our current research proves that the boiling process induces significant changes in the hop oil volatile profile. The discrimination between volatile profiles of unboiled and boiled hop essential oil was evaluated via principal component and cluster analysis (PCA and CA). HS-SPME-GC-MS analysis revealed quantitative changes (e.g. increases in the levels of oxygenated α-humulene and ß-caryophyllene derivatives) as well as qualitative changes (i.e. detection of compounds, not found in unboiled hop essential oil) in the hop oil volatile profile upon boiling. Many of these compounds were previously found in lager beer and may therefore contribute to beer flavor. Interestingly, the analytical difference between unboiled and boiled hop essential oil proved to be more pronounced as the initial hop essential oil concentration used for boiling was increased. In addition, lager beers spiked with boiled hop oil were described as 'hoppy/spicy' during sensory evaluations. Therefore, the newly formed products and hop oil constituents that are characterized by an increased recovery after boiling, are candidate compounds for 'hoppy' aroma in real brewing practice.

Characterization of novel single-variety oxygenated sesquiterpenoid hop oil fractions via headspace solid-phase microextraction and gas chromatography-mass spectrometry/olfactometry.

The volatile composition of novel varietal oxygenated sesquiterpenoid hop oil fractions ("spicy essences") was characterized by headspace solid-phase microextraction in combination with gas chromatography-mass spectrometry. Oxygenated sesquiterpenes represent the major chemical compound class, accounting for at least 65% of the total volatile fraction. In addition to oxygenated sesquiterpenes, spicy hop essences consist of several ketones, sesquiterpene and monoterpene hydrocarbons, and a relatively high number of unidentified compounds. On the basis of their relative composition, spicy hop essences can be fully differentiated according to their varietal origin. Multidimensional gas chromatography in combination with time-of-flight mass spectrometry on spicy hop essence cv. Spalter Select further demonstrated the enormous complexity of this particular hop oil fraction. The aromagram obtained via gas chromatography-olfactometry comprised nine odor-active regions described in terms of "citrus", "green", "haylike", "earthy", "woody", and "spicy". 2-Undecanone, 2-tridecanone, γ-cadinene, α-calacorene, calarene, humuladienone, caryolan-1-ol, caryophyllene oxide enantiomers, and humulene epoxide II are tentatively identified in the odor-active zones.

Characterization of novel varietal floral hop aromas by headspace solid phase microextraction and gas chromatography-mass spectrometry/olfactometry.

In this study, headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) were optimized and implemented to investigate the volatile composition of novel floral hop essences prepared from four German aroma hop varieties. In total, 91 different constituents were assigned, which were further grouped into monoterpene hydrocarbons, esters, ketones, aldehydes, furans, and oxygenated and nonoxygenated sesquiterpenes. Most volatiles belong to the ester group, whereas the monoterpene hydrocarbon ß-myrcene appears to be the predominant compound in all hop oil preparations investigated. Furthermore, as demonstrated by principal component analysis, varietal floral hop essences are clearly discriminated on the basis of their characteristic volatile composition. Via GC-olfactometry on the floral essence variety Spalter Select, ß-myrcene and 2-undecanone were identified as the most potent odorants. Several hop oil constituents were reported for the first time as impact odorants of hop aroma.