The global non-alcoholic beer (NAB) market is experiencing unprecedented growth due to a general trend in reduced alcohol consumption. Despite this growth, NABs often do not match their alcoholic counterparts in sensory quality, particularly in aroma complexity and “worty” notes. Yeasts can contribute to aroma not only by producing ethanol, esters, and higher alcohols, but also through biotransformation of bound hop- and malt-derived precursors, releasing low-threshold volatile thiols such as 3-mercaptohexan-1-ol (3MH), 3-sulfanyl-4-methylpentan-1-ol (3S4MP) and 4-mercapto-4-methylpentan-2-one (4MMP), which can impart tropical, guava, grapefruit and blackcurrant notes. Here, we screened 86 yeasts, including non-Saccharomyces species, via a multi-step screening strategy combining micro fermentation-based assessment of maltose utilisation, inability to produce volatile phenolic off-flavours via decarboxylation, biotransformation potential (assessed qualitatively via growth on cysteine as sole nitrogen source, indicative of β-lyase activity), and aromatic potential. Nine strains meeting these criteria were applied in wort fermentation trials. Strain selection to pilot-scale trials was determined by sensory analysis and chemical analysis, including direct quantification of thiol release via GC–MS/QQQ. Of these, Pichia membranifaciens and Saccharomycodes ludwigii were identified as most suitable for non-alcoholic IPA-style beer production, exhibiting enhanced fruity and floral aromas relative to a commercial maltose-negative Saccharomyces cerevisiae strain. In two pilot-scale trials using different hop varieties, and in one trial with the addition of thiol precursors, GC–MS/QQQ analyses combined with sensory evaluation showed that yeast strain selection and precursor supplementation both enhanced hop-derived aroma expression, particularly when dry hopping during active fermentation. Direct quantification confirmed that precursor supplementation increased the wort bound-thiol precursor concentration up to 2.3-fold, supporting precursor availability as a key driver of thiol release. These findings demonstrate that targeted, low-cost micro fermentation-based yeast screening, combined with hop and precursor management, provides a practical route to producing aromatic, hop-forward non-alcoholic beers with enhanced tropical and fruity character.

The present study investigated the effect of steeping solution containing a thiol- and amino-functionalization of hydrolyzed brewers' spent yeast hydrolysate (HBSY) on the enzymatic and nutritional profile of germinated barley. Thiol- and amino-functionalized HBSY (GSN) resulted in significantly elevated activities of α-amylase (35.5 U/g), β-amylase (41.2 U/g), protease (29.9 U/g), cysteine protease (12.6 U/g), lipase (4.8 U/g), and phytase (3.4 U/g) compared to control samples germinated in water (GW) (α-amylase: 23.5 U/g; β-amylase: 12.4 U/g; protease: 8.4 U/g; cysteine protease: 2.6 U/g; lipase: 2.1 U/g; phytase: 1.06 U/g). Higher cysteine protease activity was associated with increased β-amylase activity, consistent with a higher maltose concentration in GSN (2.92 g/L) than in the control (1.81 g/L). Comparative electrophoretic patterns and the branch-chain length distribution of amylopectin indicated that GSN is reflected more strongly in protease- and amylase-mediated hydrolysis, respectively. Steeping with GSN during a 96-h germination period produced nearly twice the γ-aminobutyric acid (GABA) content (2381.30 μg/g) compared with water steeping (1132.00 μg/g). Fortification with GSN resulted in significantly elevated levels of folic acid, niacin, riboflavin, γ-tocopherol, and minerals.

Kombucha fermentation is a complex process infl uenced by many diff erent process parameters and the dynamics of the microorganisms present. Since no universally established standards exist for large-scale production of kombucha, manufacturers still need to acquire the knowledge to establish controllable and reproducible processes. The aim of this study was to investigate the extent to which the kombucha fermentation process can be infl uenced by varying the oxygenation regime and the initial pH value. For this purpose, co-culture fermentations with the yeast Zygosaccharomyces bailii and the acetic acid bacterium Komagataeibacter hansenii were carried out under defi ned conditions, varying the type and intensity of aeration using various fixed aeration rates and controlled oxygen saturations. The pH values were varied in ranges between 4.1 and 7.5. The fermentations were comprehensively characterized via online and offl ine analytics, so that both the processes and the products could be chemically, microbiologically, and sensorially evaluated over time. By splitting the process into a suffi ciently long anaerobic phase of 4 days followed by an active aeration phase at 0.1 vvm for three to four days, a suffi cient oxygen supply was achieved under the given conditions to adequately convert the ethanol produced by the yeast into acetic acid by the bacteria. Sensory properties of the products were not negatively aff ected by oxidation reactions. Compared to reference processes conducted under traditional, static fermentation conditions, a process acceleration was achieved with equivalent sensory quality but significantly reduced alcohol concentrations (<0.08 %vol vs. 0.47 - 0.50 %vol. after 7 days). This study provides an example of how to produce a kombucha product under defi ned and accelerated conditions that includes an aerobic phase, and which can be used as basis for further production process developments.

Consumers increasingly seek more complex and tropical flavors in their alcoholic beverages. In beer and wine, yeast can release glutathione and cysteine-bound thiols from hops and grapes enhancing their tropical and fruity aromas. This study aimed to enhance cider aroma by combining yeast strains, hop and apple varieties. Yeast strains were screened for the presence and functionality of the IRC7 gene encoding the β-lyase and low temperature. Two strains showed a combination of desirable aromatic characteristics and good low temperature fermentation performance. These were used to study the impact of different hop varieties and apple cultivars. Results showed that the apple variety has the most significant impact on both chemical and sensory properties of the cider. This study suggests that dry hopping and yeast selection are effective for enhancing aroma and increasing flavor diversity in cider production.

Strains of Saccharomyces cerevisiae were isolated from a traditionally produced Andean maize-based chicha from Ecuador and characterised with respect to their potential use in industrial beer brewing. Whole-genome sequencing revealed that the strains were related to the ‘French Guiana’ and ‘Mexican Agave’ S. cerevisiae clades, though the available evidence indicates that they belong to a previously undescribed population, and are thus unrelated to traditional European brewing strains. Small-scale screening for wort fermentation revealed two strains with brewing potential. These outperformed commercial reference brewing strains and had fermentation profiles and alcohol yields similar to those of diastatic S. cerevisiae strains of the Mosaic/Beer 2 group. Indeed, both strains possessed functional copies of the STA1 gene responsible for extracellular glucoamylase activity seen in diastatic members of the Beer 2 group. Sequence identity suggested that the same STA1 gene is shared by the chicha and Beer 2 strains despite their genomic dissimilarity, suggesting the possibility of ancient admixture. Pilot-scale brewing trials confirmed the wort fermentation potential of the chicha strains. Beers were characterised by high concentrations of fruity esters and the clove-like compound 4-vinylguaiacol, both of which are typical features of wheat beers and related styles. Sensory trials further confirmed the potential of these strains for brewing, with beers comparing favourably to one produced with a commercial wheat-beer strain under the same conditions. Apart from 4-vinylguaiacol, which is considered an essential flavour compound in wheat beers, no off-flavours were detected in test beers. Results highlight the value of assessing industrial brewing potential of non-European strains associated with traditional cereal-based fermentations.

Dry hopping of beer can result in unintended refermentation, also known as hop creep, because of intrinsic hop diastatic activity. The objective of the work described herein was to determine the enzymatic activity across 16 different hop cultivars grown in Germany in crop years 2019, 2020, and 2021. Optimized enzyme kit protocols were used to quantitate hop alpha-, beta-amylase, amyloglucosidase, and limit dextrinase activities, while a recently published method measured hop diastatic activity. Clear varietal distinctions exist, and hops of harvests 2019, 2020, and 2021 were subsequently classified into three groups depending on their enzymatic activity. With respect to different harvest years, the results imply an annual influence on the amylolytic activity of hops in principle, but more monitoring is needed. Processing methods such as pelletization and storage under different conditions showed a minimal impact on enzymatic activity. Based on further sampling from hops of the harvest 2022, it was observed that differences among hop fractions are pronounced, with the vegetative material and strig exhibiting higher enzymatic activity compared to the lupulin fraction.

A significant by-product of beer brewing is brewer's spent yeast (BSY), which is often discarded. However, it has been demonstrated that the feasibility of BSY valorization can be achieved through the extraction and isolation of a range of economically valuable components, including proteins, functional peptides and amino acids, vitamins, bioactive β-glucans, minerals, flavor compounds, and dietary fiber. In the present study, BSY valorization was conducted through hydrolysis with pepsin to obtain HBSY with varying degrees of hydrolysis (DH%). These hydrolysates were then thiolated and aminated using different reagents, including silanization, amidation, thiourea, and glutaraldehyde cross-linking. According to Ellman's reagents, the thiolation (SH; 104.16 mg/100 g for HBSY and 39.37 mg/100 g for BSY) and ninhydrin reagent, amination (NH) contents of BSY (16.66 mg/100 g) and HBSY (41.16 mg/100 g) in silanization were substantially higher than those of other treatments. FTIR depicted an absorption peak around 2048 cm-1 (~45%) and 2347-2438 cm-1 after thiolation, and an augmented N-H bond at 1033 cm-1 and 1625 cm-1 following amination. The amid bands I and II underwent significant changes following thiolation and amidation, which were confirmed by SEM images. CD spectroscopy revealed that the amount of α-helix reduced and converted to random coil structure. A positive correlation (R = 0.99, p < 0.05) was observed with %DH, thiol, and amino surface groups, whereas a negative correlation was observed between surface hydrophobicity (H 0) and thiol (R = -0.94, p < 0.05) and amino groups (R = -0.97, p < 0.05). The thiolated and aminated HBSY significantly enhanced emulsion (78.1% and 91.6%) and foam-forming abilities (83.5% and 98%), respectively, which were more pronounced at basic and acidic pH, respectively. Moreover, the antioxidant capacities (DPPH radical scavenging activity) substantially improved after thiolation (33.6%-79.1%) and amination (21.9%-51.7%) compared to BSY (5.6%-11.7%) and HBSY (12.9%-24.6%). Therefore, the thiol- and amino-functionalization of HBSY allows for the development of novel applications, specifically as a functional food ingredient and as a substrate for fermentation processes.

A persistent challenge in brewing is the efficient utilization of hop bitter acids, with about 50% of these compounds precipitating with trub during wort boiling. This study aims to uncover the correlation between the barley cultivar proteome and hop bitter acid utilization during wort boiling. Therefore, comparative experiments were conducted using two cultivars, Liga and Solist, with varying proteomes to identify specific proteins' role in hop bitter acids precipitation. High-performance liquid chromatography (HPLC) was used to measure hop bitter acid content, while liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify and identify proteins. The 107 protein groups, particularly enzymes linked to barley metabolic defense mechanisms, exhibited significant differences between the two cultivars. Results revealed significantly lower alpha- and iso-alpha-acid content in wort produced from the barley cultivar Liga. This study highlights the critical role of the barley proteome in optimizing process efficiency by enhancing hop utilization through barley cultivar selection.

Why was the work done: Yeast flocculation is an important parameter in brewery fermentation, as beer quality is affected by the onset and intensity of cell aggregation. Malt induced premature yeast flocculation (PYF) is described as a rapid decline of yeast cells in suspension during fermentation even in the presence of sufficient nutrients. Though occurring only sporadically, premature yeast flocculation can represent a significant risk to brewery operations. What are the main findings: This review considers the known and suspected mechanisms underlying both normal and abnormal flocculation. Particular effort is made to propose definitive methods for the detection of PYF and to summarise potential strategies to alleviate premature yeast flocculation, with emphasis on relevant approaches for industrial brewing. Why is the work important: Recent research has provided new insights to elucidate the mechanisms involved in premature yeast flocculation, though a complete understanding has yet be reached. Given the association of PYF with poor field conditions during barley growth, it may be expected that climate change will contribute to an increase in the frequency or severity of PYF. This review is intended to serve as a resource for further research on the topic as well as development of practical prevention or mitigation strategies.

Oxidative stability in brewing refers to the ability of wort and beer to resist degradation by free radicals and reactive oxygen species. It is a critical quality aspect in beer production, as it affects both the shelf life and overall excellence of the final product. The catalytic role of iron, copper and manganese in radical-associated staling is widely acknowledged. In this context, the present study investigates the effectiveness of polyphenolic chelators (tannic acid, pomegranate and green tea extract) in enhancing oxidative wort stability by sequestering transition metals during mashing. The results, obtained from 12 comparable brews from two distinct pilot breweries, show that incorporating either pomegranate extract or tannic acid during mashing effectively lowers the levels of transition metals, specifically iron, in the brewhouse. Early mash addition of pomegranate extract (90 % ellagic acid) demonstrates the highest efficacy, with an almost 90 % decrease in iron levels and a nearly 80 % reduction in radical concentration, as measured in the final wort by ICP-OES and ESR spectroscopy, respectively. While the investigated chelators do not facilitate the removal of copper or manganese, their levels naturally decline during the brewing process. Chelator addition yields an average reduction of 40 - 60 % in total post-boil aldehydes. Furthermore, strong correlations are identified between iron levels, polyphenols and wort aldehydes after boiling, whereas only weak to moderate correlations with copper and manganese. Aldehydes levels, however, are greatly influenced by thermal stress throughout the brewing process. The findings suggest that natural chelators have the potential to enhance beer flavour stability by diminishing radical formation during brewing and lowering the amount of transition metals and aldehydes in the final product. However, further research is needed to fully understand the implications of these findings on beer stability, given the intricacy of staling.

Hop bitter acids are used in the brewing industry to give beer bitterness. However, much of this bitterness is lost during processing, specifically during the wort boiling step. One of the major causes might be the interaction with protein-protein complexes. Therefore, the aim of this study was to clarify the role of hop bitter acids in protein aggregate formation using a proteomic approach. The effect of hop addition on protein composition was analyzed by liquid chromatography-mass spectrometry/MS (LC-MS/MS), and further analyses were performed to characterize the wort before and after boiling. Addition of hop bitter acids yielded a change in wort protein profiles, and hop bitter acids were found to bind primarily to less abundant proteins which are not related to beer quality traits, such as foam or haze. Wort protein aggregate profiles were revealed, and findings from this study suggested the precipitation of particular proteins in the aggregates during boiling when hops were added.

Demand for low- or non-alcoholic beers has been growing in recent years. Thus, research is increasingly focusing on non- Saccharomyces species that typically are only able to consume the simple sugars in wort, and therefore have a limited production of alcohol. In this project, new species and strains of non-conventional yeasts were sampled and identified from Finnish forest environments. From this wild yeast collection, a number of Mrakia gelida strains were selected for small-scale fermentation tests and compared with a reference strain, the low-alcohol brewing yeast Saccharomycodes ludwigii . All the M. gelida strains were able to produce beer with an average of 0.7% alcohol, similar to the control strain. One M. gelida strain showing the most promising combination of good fermentation profile and production of desirable flavor active compounds was selected for pilot-scale (40 L) fermentation. The beers produced were matured, filtered, carbonated, and bottled. The bottled beers were then directed for in-house evaluation, and further analyzed for sensory profiles. The beers produced contained 0.6% Alcohol by volume (ABV). According to the sensory analysis, the beers were comparable to those produced by S. ludwigii , and contained detectable fruit notes (banana and plum). No distinct off-flavors were noted. A comprehensive analysis of M. gelida ’s resistance to temperature extremes, disinfectant, common preservatives, and antifungal agents would suggest that the strains pose little risk to either process hygiene or occupational safety.

Malt-induced premature yeast flocculation (PYF) is a sporadic problem within the brewing industry. The use of PYF malts is concomitant with a number of negative impacts on beer quality, including incomplete fermentation and/or flavor defects. Although malt-induced PYF is widely acknowledged, actions taken so far have proved insufficient to solve the PYF-related issues. To limit the detrimental effects of PYF malts on beer production, an adaptive laboratory evolution (ALE) process was applied in this study to an industrial lager brewing yeast strain (TT02), in an attempt to generate variant strains with improved fermentation performance in PYF wort. Through a batch fermentation-based adaptation process, evolved variants were isolated and screened for their phenotypic and metabolic traits. The investigation focused mainly on the tendency to remain in suspension, fermentation capacity and final acetaldehyde concentration. We successfully obtained a variant (TT02-30 T) with improved fermentation properties. The improvement was seen in worts prepared from different types of PYF malt as well as normal malt. Furthermore, ALE of lager brewing yeast in PYF wort yielded a wide array of mutations. Several changes in the genomes (copy number variation in flocculin encoding gene FLO1 and a missense SNP in a putative mitochondrial membrane protein coding gene FMP10) of the variant strains relative to the original strain were observed. These could potentially contribute to the improved yeast suspension during fermentation. Importantly, mutational enrichment in genes related to ion binding in PYF-evolved strains suggests the involvement of the yeast ion transportation process in dealing with the PYF stress. Our study demonstrates the possibility of attenuating yeast sensitivity to PYF malts over time through adaptive laboratory evolution via spontaneous mutation.

Yeasts are ubiquitous in temperate forests. While this broad habitat is well-defined, the yeasts inhabiting it and their life cycles, niches, and contributions to ecosystem functioning are less understood. Yeasts are present on nearly all sampled substrates in temperate forests worldwide. They associate with soils, macroorganisms, and other habitats and no doubt contribute to broader ecosystem-wide processes. Researchers have gathered information leading to hypotheses about yeasts' niches and their life cycles based on physiological observations in the laboratory as well as genomic analyses, but the challenge remains to test these hypotheses in the forests themselves. Here, we summarize the habitat and global patterns of yeast diversity, give some information on a handful of well-studied temperate forest yeast genera, discuss the various strategies to isolate forest yeasts, and explain temperate forest yeasts' contributions to biotechnology. We close with a summary of the many future directions and outstanding questions facing researchers in temperate forest yeast ecology. Yeasts present an exciting opportunity to better understand the hidden world of microbial ecology in this threatened and global habitat.

Breeding and domestication have generated widely exploited crops, animals and microbes. However, many Saccharomyces cerevisiae industrial strains have complex polyploid genomes and are sterile, preventing genetic improvement strategies based on breeding. Here, we present a strain improvement approach based on the budding yeasts' property to promote genetic recombination when meiosis is interrupted and cells return-to-mitotic-growth (RTG). We demonstrate that two unrelated sterile industrial strains with complex triploid and tetraploid genomes are RTG-competent and develop a visual screening for easy and high-throughput identification of recombined RTG clones based on colony phenotypes. Sequencing of the evolved clones reveal unprecedented levels of RTG-induced genome-wide recombination. We generate and extensively phenotype a RTG library and identify clones with superior biotechnological traits. Thus, we propose the RTG-framework as a fully non-GMO workflow to rapidly improve industrial yeasts that can be easily brought to the market. Domesticated industrial yeast strains are sterile, which hampers to breed strains with novel properties. Here, the authors employ the genetics paradigm return-to-growth to induce genome wide recombination in two sterile polyploid industrial yeasts and identify clones with superior biotechnological traits.

Interspecies hybridization has been shown to be a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. Thanks to the popularity of heavily hopped ‘India Pale Ale’-style beers, there is an increased demand from brewers for strains that can boost hop aroma. Here, we explored whether hybridization could be used to construct strains with an enhanced ability to release hop-derived flavours through β-lyase activity, which releases desirable volatile thiols. Wild Saccharomyces strains were shown to possess high β-lyase activity compared to brewing strains, however, they also produced phenolic off-flavours (POF) and showed poor attenuation. To overcome these limitations, interspecies hybrids were constructed by crossing pairs of one of three brewing and one of three wild Saccharomyces strains (S. uvarum and S. eubayanus). Hybrids were screened for fermentation ability and β-lyase activity, and selected hybrids showed improved fermentation and formation of both volatile thiols (4MMP, 3MH and 3MH-acetate) and aroma-active esters compared to the parent strains. Undesirable traits (e.g. POF) could be removed from the hybrid by sporulation. To conclude, it was possible to boost the release of desirable hop-derived thiols in brewing yeast by hybridization with wild yeast. This allows production of beer with boosted hop aroma with less hops (thus improving sustainability issues).

Novel wine yeast strains have the potential to satisfy customer demand for new sensorial experiences and to ensure that wine producers have strains that can produce wine as efficiently as possible. In this respect, hybrid yeast strains have recently been the subject of intense research, as they are able to combine the favourable characteristics of both parental strains. In this study, two Saccharomyces “Kéknyelű” grape juice isolates were identified by species-specific PCR and PCR-RFLP methods and investigated with respect to their wine fermentation potential. Physiological characterization of the isolated strains was performed and included assessment of ethanol, sulphur dioxide, temperature and glucose (osmotic stress) tolerance, killer-toxin production, glucose fermentation ability at 16 °C and 24 °C, and laboratory-scale fermentation using sterile “Kéknyelű” must. Volatile components of the final product were studied by gas chromatography (GC) and mass spectrometry (MS). One isolate was identified as a S. cerevisiae × S. kudriavzevii hybrid and the other was S. cerevisiae. Both strains were characterized by high ethanol, sulphur dioxide and glucose tolerance, and the S. cerevisiae strain exhibited the killer phenotype. The hybrid isolate showed good glucose fermentation ability and achieved the lowest residual sugar content in wine. The ester production of the hybrid strain was high compared to the control S. cerevisiae starter strain, and this contributed to the fruity aroma of the wine. Both strains have good oenological characteristics, but only the hybrid yeast has the potential for use in wine fermentation.

In the first part of this work, a method for the quantification of the diastatic activity of hops using potato starch was developed and assay conditions were optimized. In this work, other substrates (corn starch, wheat starch, rice starch, maltodextrin, dextrin, and amylopectin) were tested for their suitability to serve as substrates using 17 different pelletized hop samples from crop 2020. In addition to potato starch, dextrin and maltodextrin were selected for further testing and results were compared to data from trials involving incubation of Pilsner beer with hops. Additionally, measuring hop alpha-amylase, beta-amylase, and amyloglucosidase activities with enzymatic test kits revealed that hop beta-amylase activity correlated highest with the diastatic activity as measured using potato starch (r = 0.829, p < 0.0001), followed by maltodextrin (r = 0.655, p = 0.0004) and beer (r = 0.578, p = 0.015). Potato starch results likewise showed the highest Pearson coefficient of correlation (r = 0.853, p < 0.001) and a coefficient of determination of R-2 = 0.727 with the release of fermentable sugars in beer. Maltodextrin results also showed a significant correlation with diastatic activity in beer, but at a lower level with r = 0.602, p = 0.010, while the dextrin correlation was insignificant at alpha = 0.05. Potato starch was therefore confirmed to be the most suitable substrate. Additional trials comparing enzymatic activities in unpelletized and pelletized hop samples showed that pelletizing has no considerable effect on hop enzymatic activity. Taken together, these data suggest that determining hop diastatic activity using potato starch appears to show the best results. This method can therefore be recommended for measuring the diastatic power of hops.