Beer Fermentation Research Articles

Impact of wood species on microbial community composition, beer chemistry and sensory characteristics during barrel‐ageing of beer

SummaryBarrel‐ageing of conventionally fermented beers is becoming increasingly popular in recent years, but only very little is known about the underlying process. In this study, we show that wood species significantly affects the bacterial community composition, beer chemistry and sensory characteristics throughout 38 weeks of barrel‐ageing. Whereas the microbial communities of oak‐ and acacia‐aged beer became dominated by Pediococcus damnosus and Brettanomyces bruxellensis, beer aged in oak barrels also contained a large fraction of Acetobacter sp. (29.34%) and to a lesser extent Paenibacillus sp. (2.74%) that were almost undetected in acacia‐aged beer. Oak barrels also imparted substantial concentrations of eugenol, lactones and vanillin, while acacia‐aged beer contained high concentrations of total polyphenols and β‐glucan, which also translated into different sensory perceptions. Altogether, our results provide novel insights into the barrel‐ageing process of beer, and may pave the way for a new generation of beers with a noteworthy flavour complexity.

Wine Saccharomyces Yeasts for Beer Fermentation

Multiple studies in recent years have shown the potential of Saccharomyces wild yeasts to produce craft beers with new flavour profiles and other desirable properties. Yeasts isolated from food (wine, bread, kombucha…) have shown potential promise for application in brewing. The aim of this study is to evaluate the ability of 141 Saccharomyces yeast strains isolated from the Madrilenian agriculture (from grapes, must, wine, vineyard, and cellars) to produce a novel ale beer. Fermentation activity of the strains was compared against the commercial strain Saccharomyces cerevisiae Safale S-04. In addition to the other aspects such as melatonin production, thirty-three volatile compounds belonging to higher alcohols, esters, aldehydes/cetones, acids, lactones and phenolic groups, were analysed by GC for selection of the strains. Ten strains were finally chosen, among which the most relevant was the strain G 520 showing a higher production of esters, higher alcohols and acids compared with S-04. The apparent attenuation for this strain was lower than commercial strain, which translates into more residual sugars. Furthermore, G 520 was more capable of producing significantly higher amounts of melatonin studied by HPLC, as well as showing a higher antioxidant capacity. Consumer study showed that G 520 strain could be used to produce a potential beer that has a place in the current market.

The Effect of Sound Frequency and Intensity on Yeast Growth, Fermentation Performance and Volatile Composition of Beer.

This study investigated the impact of varying sound conditions (frequency and intensity) on yeast growth, fermentation performance and production of volatile organic compounds (VOCs) in beer. Fermentations were carried out in plastic bags suspended in large water-filled containers fitted with underwater speakers. Ferments were subjected to either 200–800 or 800–2000 Hz at 124 and 140 dB @ 20 µPa. Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used to identify and measure the relative abundance of the VOCs produced. Sound treatment had significant effects on the number of viable yeast cells in suspension at 10 and 24 h (p < 0.05), with control (silence) samples having the highest cell numbers. For wort gravity, there were significant differences between treatments at 24 and 48 h, with the silence control showing the lowest density before all ferments converged to the same final gravity at 140 h. A total of 33 VOCs were identified in the beer samples, including twelve esters, nine alcohols, three acids, three aldehydes, and six hop-derived compounds. Only the abundance of some alcohols showed any consistent response to the sound treatments. These results show that the application of audible sound via underwater transmission to a beer fermentation elicited limited changes to wort gravity and VOCs during fermentation.

Dynamic Optimisation of Beer Fermentation under Parametric Uncertainty

Fermentation is one of the most important stages in the entire brewing process. In fermentation, the sugars are converted by the brewing yeast into alcohol, carbon dioxide, and a variety of by-products which affect the flavour of the beer. Fermentation temperature profile plays an essential role in the progression of fermentation and heavily influences the flavour. In this paper, the fermentation temperature profile is optimised. As every process model contains experimentally determined parameters, uncertainty on these parameters is unavoidable. This paper presents approaches to consider the effect of uncertain parameters in optimisation. Three methods for uncertainty propagation (linearisation, sigma points, and polynomial chaos expansion) are used to determine the influence of parametric uncertainty on the process model. Using these methods, an optimisation formulation considering parametric uncertainty is presented. It is shown that for the non-linear beer fermentation model, the linearisation approach performed worst amongst the three methods, while second-order polynomial chaos worked the best. Using the techniques described below, a fermentation process can be optimised for ensuring high alcohol content or low fermentation time while ensuring the quality constraints. As we explicitly consider uncertainty in the process, the solution, even though conservative, will be more robust to parametric uncertainties in the model.

Screening of the freeze‐drying protective agent for high‐quality milk beer yeast ( Kluyveromyces marxianus ) and optimization of freeze‐drying process conditions

Milk beer is a new type of milk beverage produced by two-step fermentation of lactic acid bacteria and yeast and created first in China. Yeast is the key strain of milk beer fermentation, which determines the flavor and quality of milk beer. In this study, the preparation process of high-quality yeast strain MJ1 active freeze-dried powder suitable for milk beer fermentation was optimized. Results showed that the optimal freeze-dried protective agent was used: sucrose 11.27%, mannitol 4.88%, maltodextrin 15.43%, skimmed milk powder 7.82%, and applying the optimal freeze-drying conditions: rotation speed 6,000 rpm, centrifugation time 15 min, the mixing ratio of centrifugal bacterial sludge and the protective agent is 1:1.5, the mixing equilibrium time is 30 min, the cell survival rate of strain MJ1 can reach 90.21% ± 1.04%. It laid the foundation for the preparation of active freeze-dried yeast powder and the industrial production of milk beer. Novelty impact statement (1) The formulation and conditions of the freeze-dried protectant of Kluyveromyces marxianus yeast strain MJ1 were optimized to improve the cell survival rate of the freeze-dried yeast powder, (2) the optimal storage conditions of the freeze-dried yeast powder of strain MJ1 were screened to provide theoretical reference for the long-term storage of freeze-dried yeast powder, and (3) the fermentation performance of the freeze-dried yeast powder of strain MJ1 was verified, which provided a reliable basis for the industrial production of milk beer.

Repurposing brewery contaminant yeast as production strains for low-alcohol beer fermentation.

A number of fungal isolates were recently obtained from a survey of the microbiota of multiple breweries and brewery products. Here, we sought to explore whether any of these brewery contaminants could be repurposed for beneficial use in beer fermentations, with particular focus on low-alcohol beer. There were 56 yeast strains first screened for the utilization of different carbon sources, ability to ferment brewer's wort, and formation of desirable aroma compounds. A number of strains appeared maltose-negative and produced desirable aromas without obvious off-flavours. These were selected for further scaled-up wort fermentations. The selected strains efficiently reduced wort aldehydes during fermentation, thus eliminating undesirable wort-like off-flavours, and produced a diverse volatile aroma profile. Two strains, Trigonopsis cantarellii and Candida sojae, together with a commercial Saccharomycodes ludwigii reference strain, were selected for 30-L-scale wort fermentations based on aroma profile and similarity to a commercial reference beer during sensory analysis using projective mapping. Both strains performed comparably to the commercial reference, and the T.cantarellii strain in particular, produced low amounts of off-flavours and a significantly higher amount of the desirable monoterpene alcohol trans-geraniol. The strain was also sensitive to common food preservatives and antifungal compounds and unable to grow at 37°C, suggesting it is relatively easily controllable in the brewery, and appears to have low risk of pathogenicity. This study shows how the natural brewery microbiota can be exploited as a source of non-conventional yeasts for low-alcohol beer production.

Domain Adaptation and Federated Learning for Ultrasonic Monitoring of Beer Fermentation

Beer fermentation processes are traditionally monitored through sampling and off-line wort density measurements. In-line and on-line sensors would provide real-time data on the fermentation progress whilst minimising human involvement, enabling identification of lagging fermentations or prediction of ethanol production end points. Ultrasonic sensors have previously been used for in-line and on-line fermentation monitoring and are increasingly being combined with machine learning models to interpret the sensor measurements. However, fermentation processes typically last many days and so impose a significant time investment to collect data from a sufficient number of batches for machine learning model training. This expenditure of effort must be multiplied if different fermentation processes must be monitored, such as varying formulations in craft breweries. In this work, three methodologies are evaluated to use previously collected ultrasonic sensor data from laboratory scale fermentations to improve machine learning model accuracy on an industrial scale fermentation process. These methodologies include training models on both domains simultaneously, training models in a federated learning strategy to preserve data privacy, and fine-tuning the best performing models on the industrial scale data. All methodologies provided increased prediction accuracy compared with training based solely on the industrial fermentation data. The federated learning methodology performed best, achieving higher accuracy for 14 out of 16 machine learning tasks compared with the base case model.

CRISPR/Cas12a-assisted rapid identification of key beer spoilage bacteria

Beer spoilage bacteria present severe challenges for the beer fermentation industry. Here, we establish a rapid, specific and highly portable CRISPR/Cas12a-based key beer spoilage bacterial scanning method, dubbed CRISPR-Beer Scan. With the variable 16S rDNA segments of these major spoilage microbe species serving as targets, highly efficient and specific crRNAs were determined. By coupling with recombinase polymerase isothermal amplification, CRISPR-Beer Scan could detect as low as 10 copies of DNA targets. Indeed, trace amounts of target genome DNAs can be accurately identified within a mixture of genome DNAs conditioned in beer. Moreover, the extracted genome DNA samples can be conveniently distinguished through the visual fluorescent signals excited by blue light in 45 min with CRISPR-Beer Scan Method. Taken together, the CRISPR-Beer Scan represents a widely applicable microbe-monitoring platform for not only breweries, but also other industries.

Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching

Yeast breeding is a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. However, breeding of industrial brewing yeast can be challenging, as strains are typically sterile and have large complex genomes. To facilitate breeding, we used the CRISPR/Cas9 system to generate double-stranded breaks in the MAT locus, generating transformants with a single specified mating type. The single mating type remained stable even after loss of the Cas9 plasmid, despite the strains being homothallic, and these strains could be readily mated with other brewing yeast transformants of opposite mating type. As a proof of concept, we applied this technology to generate yeast hybrids with an aim to increase β-lyase activity for fermentation of beer with enhanced hop flavour. First, a genetic and phenotypic pre-screening of 38 strains was carried out in order to identify potential parent strains with high β-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for β-lyase activity. Selected phenolic off-flavour positive (POF +) hybrids were further sporulated to generate meiotic segregants with high β-lyase activity, efficient wort fermentation, and lack of POF, all traits that are desirable in strains for the fermentation of modern hop-forward beers. Our study demonstrates the power of combining the CRISPR/Cas9 system with classic yeast breeding to facilitate development and diversification of brewing yeast.Key points• CRISPR/Cas9-based mating-type switching was applied to industrial yeast strains.• Transformed strains could be readily mated to form intraspecific hybrids.• Hybrids exhibited heterosis for a number of brewing-relevant traits.

Brewing Efficacy of Non-Conventional Saccharomyces Non-cerevisiae Yeasts

Consumer demands for new sensory experiences have driven the research of unconventional yeasts in beer. While much research exists on the use of various common Saccharomyces cerevisiae strains as well as non-Saccharomyces yeasts, there exists a gap in knowledge regarding other non-cerevisiae Saccharomyces species in the fermentation of beer, in addition to S. pastorianus. Here, five distinct species of Saccharomyces from the UC Davis Phaff Yeast Culture Collection, as well as one interspecies hybrid from Fermentis, were chosen to ferment 40 L pilot-scale beers. S. kudriavzevii, S. mikatae, S. paradoxus, S. bayanus, and S. uvarum yeasts were used to ferment wort in duplicate pairs, with one fermenter in each pair receiving 10 g/L dry-hop during fermentation. Analytical measurements were made each day of fermentation and compared to controls of SafAle™ US-05 and SafLager™ W 34/70 for commercial brewing parameters of interest. Finished beers were also analyzed for aroma, taste, and mouthfeel to determine the flavor of each yeast as it pertains to brewing potential. All beers exhibited spicy characteristics, likely from the presence of phenols; dry-hopping increased fruit notes while also increasing perceived bitterness and astringency. All of the species in this study displayed great brewing potential, and might be an ideal addition to beer depending on a brewery’s desire to experiment with flavor and willingness to bring a new yeast into their production environment.

Developing new products with kernel partial least squares model inversion

In recent years, data-driven approaches (e.g., latent variable model) have excited the development of new products and the control of product quality. To derive an input space within which raw material properties or initial operating conditions can yield the required product quality, previous researchers developed a linear latent variable model and derived the solution of inputs through PLS model inversion. In this research, a novel method based on kernel partial least squares (KPLS) model inversion is proposed for product design of nonlinear processes and an input domain is derived. Constraints on the model inputs or outputs and on the KPLS model are discussed, and solutions are provided based on an optimization framework and the Monte Carlo sampling method. The effectiveness of the method is demonstrated by numerical simulation and a beer fermentation experiment, and the KPLS model inversion method outperforms the PLS model inversion method in terms of the precision.

METALS AND POLYCYCLIC AROMATIC HYDROCARBONS (PAHs) IN BEER: A REVIEW

The major cations in beer are potassium, sodium, magnesium, and calcium with the anions, chloride, sulphate, nitrate and phosphate. The minor ones include iron, copper, zinc, manganese, lead, arsenic and phosphorous. These metals are important in beer fermentation because they supply the appropriate environment for yeast growth and influence yeast metabolism. Copper and iron are involved in beer conditioning and ageing through reactions resulting in the formation of reactive oxygen species which readily oxidize organic compounds present in beer, changing the quality of foaming and flavour stability of beer. Small amounts of nitrogen-containing constituents are present and phosphorous is associated with some of these. Only trace amounts of sulphur-containing compounds are present but some of these are very potent flavouring agents. Beer is also subject to PAHs contamination due to the drying and roasting of barley. PAHs have been detected in different types of beverages such as tea, coffee, distilled drinks, wine, and fruit juice. Although PAHs are usually found in low concentrations in foods and beverages, they are one of the main factors that contribute to the onset of cancer in humans. This review covers the determination of levels of metals and polycyclic aromatic hydrocarbons in beer. The review was designed to cover the sources of metals and PAHs in beer as well as functions of metals and methods for the determination of metals and PAHs in beer. It was also designed to give insight to the health concerns of beer consumers.

Mixed-Culture Metagenomics of the Microbes Making Sour Beer

Mixed microbial cultures create sour beers but many brewers do not know which microbes comprise their cultures. The objective of this work was to use deep sequencing to identify microorganisms in sour beers brewed by spontaneous and non-spontaneous methods. Twenty samples were received from brewers, which were processed for microbiome analysis by next generation sequencing. For bacteria, primers were used to amplify the V3-V4 region of the 16S rRNA gene; fungal DNA detection was performed using primers to amplify the entire internal transcribed spacer region. The sequencing results were then used for taxonomy assignment, sample composition, and diversity analyses, as well as nucleotide BLAST searching. We identified 60 genera and 140 species of bacteria, of which the most prevalent were Lactobacillus acetotolerans, Pediococcus damnosus, and Ralstonia picketti/mannitolilytica. In fungal identification, 19 genera and 26 species were found, among which the most common yeasts were Brettanomyces bruxellensis and Saccharomyces cerevisiae. In some cases, genetic material from more than 60 microorganisms was found in a single sample. In conclusion, we were able to determine the microbiomes of various mixed cultures used to produce beer, providing useful information to better understand the sour beer fermentation process and brewing techniques.

Monitoring Glucose in Fermented Beer by an Electrochemical Sensor Based on Graphene Oxide Decorated by Silver Nanoparticles

Monitoring Glucose in Fermented Beer by an Electrochemical Sensor Based on Graphene Oxide Decorated by Silver Nanoparticles

The Impact of Lactic and Acetic Acid on Primary Beer Fermentation Performance and Secondary Re-Fermentation during Bottle-Conditioning with Active Dry Yeast

The presence of high concentrations of organic acids is known to adversely affect the efficiency and quality of ethanol fermentation. The growing popularity of sour beers warranted the exploration of strain-specific performance under optimal and suboptimal conditions similar to those found in sour beer production. The focus of this study was on the performance of select active dried yeast strains under artificially acidified conditions. Nine common brewing strains of active dried yeast were assessed based upon overall fermentation performance and their ability to metabolize maltotriose and maltose between 0.0% w/w − 1.0% w/w lactic acid and 0.0% w/w − 0.5% w/w acetic acid. A single strain of active dried yeast specifically selected and bred for bottle conditioning environments was assessed based upon its ability to metabolize glucose, and carbonate artificially acidified finished beer between 0.0%−1.6% w/w lactic acid and 0.0%−1.0% w/w acetic acid. This study confirmed the suitability of active dry brewing yeast for sour beer fermentations that meet or exceed the typical organic acid concentrations encountered in sour wort. The majority of the selected strains performed well in sour wort containing < 0.4% w/w lactic acid or < 0.1% w/w acetic acid. The importance of strain selection became apparent at concentrations exceeding these reported values, with two strains displaying almost no change in fermentation capabilities across the range of organic acid concentrations. Bottle conditioning remained unhindered by lactic acid up to 1.6% w/w, while acetic acid concentrations at and above 0.4% w/w significantly hindered bottle conditioning.

Effect of Immobilization Support and Fermentation Temperature on Beer and Fermented Milk Aroma Profiles

The food industry increasingly produces wastes like coconut and peanut shells. In addition, low temperature fermentation is always a challenge. Therefore, in the present study, a sustainable exploitation of these by-products is proposed through the production of carriers for immobilized cells of yeast and bacteria. The immobilized cells, after thermally drying, were evaluated for their efficiency in beer and milk fermentations respectively, in various fermentation temperatures and storage for up to three months. The beers and fermented milks were evaluated for their aroma and the results showed products of high quality. Coconut shells resulted in better products with increased fruity ester content in fermented milks and reduced dimethyl sulfite and vicinal diketones and increased ratio of esters to alcohol in beers. These results reveal the possibilities of immobilized cells in coconut and peanut shells for application in food industry, however, more research is needed to evaluate their effect on sensory characteristics and possible prebiotic and probiotic potential especially in the case of fermented milks.

Wild Ecuadorian Saccharomyces cerevisiae Strains and Their Potential in the Malt-Based Beverages Industry

Ecuador is a biodiverse country where a wide range of Saccharomyces cerevisiae yeast is present. Ecuador also holds a rich tradition of brewing corn-based beer. Genetics and domestication of S. cerevisiae throughout thousands of years have resulted in a wide range of aromas and flavors produced during fermentation. A total of 94 yeast isolates were studied; RFLP analysis of the isolates was used for grouping the strains according to their genotype. A group of 15 yeasts characterized by six different RFLP profiles was chosen. Fermentations were carried out at room temperature (∼ 22 °C) to determine the fermented beers’ chemical and organoleptic characteristics. Significant differences between the yeast isolates were found by means of HPLC analysis and sensorial evaluation of the fermentation products. Moreover, fourteen out of fifteen isolates were found to be potentially useful in the production of different beer styles.

(IMCS Second Place Best Paper Award) Acetoin Biosensor Based on Acetoin Reductase-Modified Field-Effect Sensor Applied for Acetoin Detection in Beer Samples

Introduction In food industry, the use and dosing of flavoring substances is an important aspect during fabrication processes. Acetoin and diacetyl are flavoring agents which can be found in many food products, like yogurt, cheese, milk, wine and beer [1]. In beer, acetoin/diacetyl is a critical parameter for the maturation degree of the beverage during fabrication. They are formed during the first phase of beer fermentation and getting reduced during the later phase of fermentation and the maturation. Both can have a negative effect on the beer flavor, when they are above a certain concentration. Typical acetoin concentrations in beer are in the range of 10-50 µM. Measuring the acetoin/diacetyl concentration during the production can serve as a quality control and unnecessary maturation time could be avoided [2]. Until now, the quantitative determination of the acetoin/diacetyl concentration is mostly done “off-line” by gas chromatography. Therefore, a sample has to be sent to an external laboratory, which takes time till results are achieved, or the brewery needs an own laboratory, whereby enormous costs arise and trained staff is needed. Because of that, we recently developed a biosensor for the detection of acetoin based on an enzyme-modified electrolyte-insulator-semiconductor (EIS) sensor, which was characterized in buffer solutions [3]. In this work, the novel biosensor was applied for acetoin measurements in beer samples for the first time. Materials and Methods As sensor structures, capacitive Al-p-Si-SiO2-Ta2O5 field-effect sensors have been used, schematically shown in Fig. 1(a). The Ta2O5 transducer surface was modified with a novel acetoin reductase by means of cross-linking. The acetoin reductase (from B. clausii DSM 8716T) catalyzes the reduction of acetoin to 2,3-butanediol, while NADH serves as a cofactor [4]. The fabricated acetoin biosensors have been electrochemically characterized by capacitance-voltage- and constant-capacitance (ConCap) measurements in buffer solution (0.2 mM Tris-HCl) with varying acetoin concentration and beer samples, spiked with different amounts of acetoin. Results and Conclusions The acetoin sensitivity was investigated in beer solutions and the biosensors showed a linear behavior in the concentration range between 10 µM and 90 μM with a sensitivity of ca. 30 mV/dec, see Fig. 1(b). A detailed description of experiments and the obtained results will be presented and discussed. Acknowledgments The authors thank the ministry of innovation, science and research of the state of North-Rhine-Westphalia for financial support (funding program FH-Struktur 2016).

Biotechnological exploitation of Saccharomyces jurei and its hybrids in craft beer fermentation uncovers new aroma combinations

Hybridisation is an important evolutionary mechanism to bring about novel phenotypes and may produce new hybrids with advantageous combinations of traits of industrial importance. Within the Saccharomyces genus, Saccharomyces jurei is a newly discovered species and its biotechnological potential has not yet been fully explored. This yeast was found to be able to grow well in unhopped wort and at low temperatures, qualities necessary in good candidates for fermented bevarages. Here, we analysed its fermentation and aroma profile and created novel non-GMO hybrids between S. jurei and S. cerevisiae ale yeasts to develop new starter strains with interesting flavours for the craft brewing and beverage industry in general. Pilot beer fermentations with specific hybrids showed a good fermentation performance, similar to the ale parent strain, while eliminating the hyper-attenuation characteristic and a more complex flavour profile. This study exploits the genetic diversity of yeasts and shows how inter-specific hybridisation and clone selection can be effectively used in brewing to create new products and to eliminate or increase specific traits.

Genome comparison of three lager yeasts reveals key genes affecting yeast flocculation during beer fermentation.

Yeast flocculation plays an essential role in industrial application. Appropriate flocculation of yeast cells at the end of fermentation benefits the cell separation in production, which is an important characteristic of lager yeast for beer production. Due to the complex fermentation environment and diverse genetic background of yeast strains, it is difficult to explain the flocculation mechanism and find key genes that affect yeast flocculation during beer brewing. By analyzing the genomic mutation of two natural mutant yeasts with stronger flocculation ability compared to the parental strain, it was found that the mutated genes common in both mutants were enriched in protein processing in endoplasmic reticulum, membrane lipid metabolism and other pathways or biological processes involved in stress responses. Further functional verification of genes revealed that regulation of RIM101 and VPS36 played a role in lager yeast flocculation under the brewing condition. This work provided new clues for improving yeast flocculation in beer brewing.