Deciphering the core microbes and their interactions in spontaneous Baijiu fermentation: A comprehensive review.

The spontaneous Baijiu fermentation system harbors a complex microbiome that is highly dynamic in time and space and varies depending on the Jiuqu starters and environmental factors. The intricate microbiota presents in the fermentation environment is responsible for carrying out various reactions. These reactions necessitate the interaction among the core microbes to influence the community function, ultimately shaping the distinct Baijiu styles through the process of spontaneous fermentation. Numerous studies have been conducted to enhance our understanding of the diversity, succession, and function of microbial communities with the aim of improving fermentation manipulation. However, a comprehensive and critical assessment of the core microbes and their interaction remains one of the significant challenges in the Baijiu fermentation industry. This paper focuses on the fermentation properties of core microbes. We discuss the state of the art of microbial traceability, highlighting the crucial role of environmental and starter microbiota in the Baijiu brewing microbiome. Also, we discuss the various interactions between microbes in the Baijiu production system and propose a potential conceptual framework that involves constructing predictive network models to simplify and quantify microbial interactions using co-culture models. This approach offers effective strategies for understanding the core microbes and their interactions, thus beneficial for the management of microbiota and the regulation of interactions in Baijiu fermentation processes.

Wild Patagonian yeast improve the evolutionary potential of novel interspecific hybrid strains for lager brewing.

Lager yeasts are limited to a few strains worldwide, imposing restrictions on flavour and aroma diversity and hindering our understanding of the complex evolutionary mechanisms during yeast domestication. The recent finding of diverse S. eubayanus lineages from Patagonia offers potential for generating new lager yeasts with different flavour profiles. Here, we leverage the natural genetic diversity of S. eubayanus and expand the lager yeast repertoire by including three distinct Patagonian S. eubayanus lineages. We used experimental evolution and selection on desirable traits to enhance the fermentation profiles of novel S. cerevisiae x S. eubayanus hybrids. Our analyses reveal an intricate interplay of pre-existing diversity, selection on species-specific mitochondria, de-novo mutations, and gene copy variations in sugar metabolism genes, resulting in high ethanol production and unique aroma profiles. Hybrids with S. eubayanus mitochondria exhibited greater evolutionary potential and superior fitness post-evolution, analogous to commercial lager hybrids. Using genome-wide screens of the parental subgenomes, we identified genetic changes in IRA2, IMA1, and MALX genes that influence maltose metabolism, and increase glycolytic flux and sugar consumption in the evolved hybrids. Functional validation and transcriptome analyses confirmed increased maltose-related gene expression, influencing greater maltotriose consumption in evolved hybrids. This study demonstrates the potential for generating industrially viable lager yeast hybrids from wild Patagonian strains. Our hybridization, evolution, and mitochondrial selection approach produced hybrids with high fermentation capacity and expands lager beer brewing options.

Impact of non-Saccharomyces yeasts derived from traditional fermented foods on beer aroma: Analysis based on HS-SPME-GC/MS combined with chemometrics.

In recent years, numerous studies have demonstrated the significant potential of non-Saccharomyces yeasts in aroma generation during fermentation. In this study, 134 strains of yeast were isolated from traditional fermented foods. Subsequently, through primary and tertiary screening, 28 strains of aroma-producing non-Saccharomyces yeast were selected for beer brewing. Headspace-solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) and chemometrics were employed to analyze the volatile flavor substances in beer samples fermented using these strains. Chemometric analysis revealed that distinct species of non-Saccharomyces yeast had a unique influence on beer aroma, with strains from the same genus producing more similar flavor profiles. Accordingly, 2,6-nonadienal, 1-pentanol, phenyl ethanol, isoamyl acetate, ethyl caprate, butyl butyrate, ethyl propionate, furfuryl alcohol, phenethyl acetate, ethyl butyrate, ethyl laurate, acetic acid, and 3-methyl-4 heptanone were identified as the key aroma compounds for distinguishing among different non-Saccharomyces yeast species. This work provides useful insights into the aroma-producing characteristics of different non-Saccharomyces yeasts to reference the targeted improvement of beer aroma.

Multipoint monitor of beer fermentation.

This short communication is devoted to a fully-mechanized flow analysis system for the control of beer fermentation process. The developed system is based on microsolenoid flow controlling devices (valves and pumps) and a flow-through optoelectronic detector. All these components are powered and controlled by a Adruino-compatible microprocessor platform that creates an integrated, compact, and robust analytical tool. Multiplication of sample aspiration ports of the analytical system allows for simultaneous monitoring of several independently performed fermentation processes, as well as a single process at the different places of fermentation tank. To demonstrate its practical utility, the developed system has been applied for online and real-time monitoring of yeast propagation and distribution in beer worts in the course of various fermentation processes. Potentially, this flow analysis system can be easily expanded to the form of multianalyte monitor equipped with optoelectronic sensors and biosensors for the determination of other parameters and analytes.

Modifying flavor profiles of Saccharomyces spp. for industrial brewing using FIND-IT, a non-GMO approach for metabolic engineering of yeast.

Species of Saccharomyces genus have played an irreplaceable role in alcoholic beverage and baking industry for centuries. S. cerevisiae has also become an organism of choice for industrial production of alcohol and other valuable chemicals and a model organism shaping the rise of modern genetics and genomics in the past few decades. Today´s brewing industry faces challenges of decreasing consumption of traditional beer styles and increasing consumer demand for new styles, flavors and aromas. The number of currently used brewer's strains and their genetic diversity is yet limited and implementation of more genetic and phenotypic variation is seen as a solution to cope with the market challenges. This requires modification of current production strains or introduction of novel strains from other settings, e.g. industrial or wild habitats into the brewing industry. Due to legal regulation in many countries and negative customer perception of GMO organisms, the production of food and beverages requires non-GMO production organisms, whose development can be difficult and time-consuming. Here, we apply FIND-IT (Fast Identification of Nucleotide variants by DigITal PCR), an ultrafast genome-mining method, for isolation of novel yeast variants with varying flavor profiles. The FIND-IT method uses combination of random mutagenesis, droplet digital PCR with probes that target a specific desired mutation and a sub-isolation of the mutant clone. Such an approach allows the targeted identification and isolation of specific mutant strains with eliminated production of certain flavor and off-flavors and/or changes in the strain metabolism. We demonstrate that the technology is useful for the identification of loss-of function or gain of function mutations in unrelated industrial and wild strains differing in ploidy. Where no other phenotypic selection exists, this technology serves together with standard breeding techniques as a modern tool facilitating a modification of (brewer's) yeast strains leading to diversification of the product portfolio.

Hi-C metagenomics facilitate comparative genome analysis of bacteria and yeast from spontaneous beer and cider.

Sequence-based analysis of fermented foods and beverages' microbiomes offers insights into their impact on taste and consumer health. High-throughput metagenomics provide detailed taxonomic and functional community profiling, but bacterial and yeast genome reconstruction and mobile genetic elements tracking are to be improved. We established a pipeline for exploring fermented foods microbiomes using metagenomics coupled with chromosome conformation capture (Hi-C metagenomics). The approach was applied to analyze a collection of spontaneously fermented beers and ciders (n = 12). The Hi-C reads were used to reconstruct the metagenome-assembled genomes (MAGs) of bacteria and yeasts facilitating subsequent comparative genomic analysis, assembly scaffolding and exploration of "plasmid-bacteria" links. For a subset of beverages, yeasts were isolated and characterized phenotypically. The reconstructed Hi-C MAGs primarily belonged to the Lactobacillaceae family in beers, along with Acetobacteraceae and Enterobacteriaceae in ciders, exhibiting improved quality compared to conventional metagenomic MAGs. Comparative genomic analysis of Lactobacillaceae Hi-C MAGs revealed clustering by niche and suggested genetic determinants of survival and probiotic potential. For Pediococcus damnosus, Hi-C-based networks of contigs enabled linking bacteria with plasmids. Analyzing phylogeny and accessory genes in the context of known reference genomes offered insights into the niche specialization of beer lactobacilli. The subspecies-level diversity of cider Tatumella spp. was disentangled using a Hi-C-based graph. We obtained highly complete yeast Hi-C MAGs primarily represented by Brettanomyces and Saccharomyces, with Hi-C-facilitated chromosome-level genome assembly for the former. Utilizing Hi-C metagenomics to unravel the genomic content of individual species can provide a deeper understanding of the ecological interactions within the food microbiome, aid in bioprospecting beneficial microorganisms, improving quality control and improving innovative fermented products.

Refermentation and maturation of lambic beer in bottles: a necessary step for gueuze production.

The production of gueuze beers through refermentation and maturation of blends of lambic beer in bottles is a way for lambic brewers to cope with the variability among different lambic beer batches. The resulting gueuze beers are more carbonated than lambic beers and are supposed to possess a unique flavor profile that varies over time. To map this refermentation and maturation process for gueuze production, a blend of lambic beers was made and bottled, whereby one of them was produced with the old wheat landrace Zeeuwse Witte. Through the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high-throughput sequencing of bacterial and fungal amplicons, in combination with metabolite target analysis, new insights into gueuze production were obtained. During the initial stages of refermentation, the conditions in the bottles were similar to those encountered during the maturation phase of lambic beer productions in wooden barrels, which was also reflected microbiologically (presence of Brettanomyces species, Pediococcus damnosus, and Acetobacter lambici) and biochemically (ethanol, higher alcohols, lactic acid, acetic acid, volatile phenolic compounds, and ethyl esters). However, after a few weeks of maturation, a switch from a favorable environment to one with nutrient and dissolved oxygen depletion resulted in several changes. Concerning the microbiology, a sequential prevalence of three lactic acid bacterial species occurred, namely, P. damnosus, Lentilactobacillus buchneri, and Lactobacillus acetotolerans, while the diversity of the yeasts decreased. Concerning the metabolites produced, mainly those of the Brettanomyces yeasts determined the metabolic profiles encountered during later stages of the gueuze production.IMPORTANCEGueuze beers are the result of a refermentation and maturation process of a blend of lambic beers carried out in bottles. These gueuze beers are known to have a long shelf life, and their quality typically varies over time. However, knowledge about gueuze production in bottles is scarce. The present study provided more insights into the varying microbial and metabolite composition of gueuze beers during the first 2 years of this refermentation and maturation process. This will allow gueuze producers to gain more information about the influence of the refermentation and maturation time on their beers. These insights can also be used by gueuze producers to better inform their customers about the quality of young and old gueuze beers.

Fruit beers, beers with or without a co-fermentation step with fruits.

Belgium is known for its traditional lambic beer productions, obtained through spontaneous fermentation and maturation in wooden barrels. Lambic beer is also used to make fruit lambic beers, such as Kriek beer. Despite fruit beer being an old beer type, dating back to the second half of the seventeenth century, no research has been performed on lambic beer-fruit co-fermentation processes. Further, these beers get competition from market-driven, sweet, (fruit-)flavored ones without the co-fermentation step. This paper will first discuss a new, general fruit beer classification, going from sour fruit beers produced through co-fermentation to sweet ones without a co-fermentation step. Second, a state-of-the-art of the scarce literature on the microbiology and metabolomics of lambic beer-fruit co-fermentation processes will be given.

The initial composition and structure of microbial community determined the yield and quality of Baijiu during the spontaneous fermentation / La composición inicial y la estructura de la comunidad microbiana determinaron el rendimiento y la calidad del Baijiu durante la fermentación espontánea

The microbiota during pit mud fermentation is a crucial factor in Baijiu brewing since it determines the yield and flavor. However, the impact of the microbial community during the initial fermentation stage on Baijiu quality remains uncertain. Herein, high-throughput sequencing was employed to investigate the microbial diversities and distribution during Baijiu fermentation in individual pit mud workshops at both initial and late stages. During the initial fermentation stage, the bacterial community exerted a more pronounced effect on Baijiu quality than the fungal community. And the high-yield pit mud workshop exhibited lower richness and evenness, as well as greater Bray-Curtis dissimilarity during Baijiu fermentation. Lactobacillus was the dominant genus and biomarker in high-yield pit mud, and it constituted the only genus within the bacterial association network during the late fermentation stage. Fungal communities tended to maintain a simple association network with selected core species. Based on the correlation network, Rhizopus and Trichosporon were identified as biomarkers in Baijiu fermentation process. Together, Lactobacillus and Rhizopus could serve as bio-indicators for Baijiu quality during the initial fermentation stage. Therefore, these findings provided novel insights into microbiota interactions during fermentation and the impact of initial microbiota on final Baijiu quality.(AU)

Analysis of the bacterial and fungal populations in South African sorghum beer (umqombothi) using full-length 16S rRNA amplicon sequencing.

There is a need to profile microorganisms which exist pre-and-post-production of umqombothi, to understand its microbial diversity and the interactions which subsequently influence the final product. Thus, this study sought to determine the relative microbial abundance in umqombothi and predict the functional pathways of bacterial and fungal microbiota present. Full-length bacterial 16S rRNA and internal transcribed spacer (ITS) gene sequencing using PacBio single-molecule, real-time (SMRT) technology was used to assess the microbial compositions. PICRUSt2 was adopted to infer microbial functional differences. A mixture of harmful and beneficial microorganisms was observed in all samples. The microbial diversity differed significantly between the mixed raw ingredients (MRI), customary beer brew (CB), and optimised beer brew (OPB). The highest bacterial species diversity was observed in the MRI, while the highest fungal species diversity was observed in the OPB. The dominant bacterial species in the MRI, CB, and OPB were Kosakonia cowanii, Apilactobacillus pseudoficulneus, and Vibrio alginolyticus, respectively, while the dominant fungal species was Apiotrichum laibachii. The predicted functional annotations revealed significant (p < 0.05) differences in the microbial pathways of the fermented and unfermented samples. The most abundant pathways in the MRI were the branched-chain amino acid biosynthesis super pathway and the pentose phosphate pathway. The CB sample was characterised by folate (vitamin B9) transformations III, and mixed acid fermentation. Biotin (vitamin B7) biosynthesis I and L-valine biosynthesis characterised the OPB sample. These findings can assist in identifying potential starter cultures for the commercial production of umqombothi. Specifically, A. pseudoficulneus can be used for controlled fermentation during the production of umqombothi. Likewise, the use of A. laibachii can allow for better control over the fermentation kinetics such as carbohydrate conversion and end-product characteristics, especially esters and aroma compounds.

Effect of Extraction Time on the Yield, Chemical Composition, and Antibacterial Activity of Hop Essential Oil Against Lactic Acid Bacteria (Lactobacillus brevis and Lactobacillus casei) Beer Spoilage.

Hop essential oil (EO) generates interest for its antioxidant and antimicrobial properties, in addition to the volatile compounds that are responsible for the hop aroma in beer. Thus, the objective of this study was to evaluate the chemical composition, EO yield, and antibacterial activity of hop essential oil from hops of the Chinook variety against lactic acid bacteria (Lactobacillus brevis and Lactobacillus casei) at different times of extraction. EO extraction was performed by hydrodistillation at different times. By analyzing the chemical composition by gas chromatography and mass spectrometry, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined. The major compounds of hop EO were α-humulene, ß-myrcene, and ß-caryophyllene, and the extraction yields were 0.67, 0.78, and 0.85% mass of EO per mass of hops pelletized hops (m/m), for extractions of 90, 180, and 300 min, respectively. The EO obtained in 90 min was efficient against L. casei at 2.5 mg/mL (MIC) and 5.0 mg/mL (MBC), and the 300 min one against L. brevis at 2.5 mg/mL (MIC) and 25 mg/mL (MBC). The antibacterial activity was affected by the chemical makeup of the oil, revealing that the hop EO extracted in 300 min was the most efficient among the other extraction times.

Production and characterization of anthocyanin-rich beer from black wheat by an efficient isolate Saccharomyces cerevisiae CMS12.

Beer is the world's third most popular fermented beverage. It is typically made from malted barley. Tropical countries must import barley from temperate countries for brewing, which is an expensive process. Therefore, it is critical to investigate alternative possible substrates for beer production in order to meet the growing demand for high-nutritional-quality beer. The current study involves the creation of a fermented beverage from anthocyanin-rich black wheat with the help of yeast, Saccharomyces cerevisiae CMS12, isolated from fruit waste. Characterization (UV, HPLC, NMR, FTIR, and ICPMS) was then performed, as well as a comparative study with white (amber) wheat beer. Further, process parameters optimization included initial sugar concentration, inoculum size, and pH. Black wheat wort contained 568 mg GAE/L total phenolic content, 4.67 mg/L anthocyanin concentration, 6.8% (v/v) alcohol content, and a pH of 4.04. The sensory analysis revealed that black wheat beer was more acceptable than white wheat beer. The developed fermented beverage has enormous commercialization potential.

Alginate encapsulation improves probiotics survival in carbonated sodas and beers.

Probiotic functionalization of non-dairy beverages has been garnering interest to provide dairy-sensitive populations with greater probiotic product varieties. The addition of probiotics into popularly consumed beverages-carbonated sodas and beers, presents an interesting challenge as the presence of acidic pH, hops-derived compounds, and ethanol have highly deleterious effects. Herein, alginate encapsulation was proposed to improve probiotics viability within sodas and beers. Three probiotics, namely Lacticaseibacillus rhamnosus GG, Escherichia coli Nissle 1917, and Bifidobacterium longum were encapsulated in alginate spheres and exposed to Coca-Cola, 7-Up, Tiger Beer, and Guinness under refrigerated, room temperature and simulated gastric fluid conditions. Results demonstrate that alginate encapsulation significantly improved the viabilities of all three probiotics in various beverages and conditions. Refrigerated storage better preserved probiotic viabilities and reduced the formation of the probiotic metabolic by-product, L-lactate, than at room temperature storage. Findings here could provide beverage manufacturers with a novel way to develop probiotic-sodas and probiotic-beers through encapsulation.

New insights into the role of key microorganisms and wooden barrels during lambic beer fermentation and maturation.

Belgian lambic beers are still produced through traditional craftsmanship. They rely on a spontaneous fermentation and maturation process that is entirely carried out in wooden barrels. The latter are used repetitively and may introduce some batch-to-batch variability. The present systematic and multiphasic study dealt with two parallel lambic beer productions carried out in nearly identical wooden barrels making use of the same cooled wort. It encompassed a microbiological and metabolomic approach. Further, a taxonomic classification and metagenome-assembled genome (MAG) investigation was based on shotgun metagenomics. These investigations provided new insights into the role of these wooden barrels and key microorganisms for this process. Indeed, besides their role in traditionality, the wooden barrels likely helped in establishing the stable microbial ecosystem of lambic beer fermentation and maturation by acting as an inoculation source of the necessary microorganisms, thereby minimizing batch-to-batch variations. They further provided a microaerobic environment, which aided in achieving the desirable succession of the different microbial communities for a successful lambic beer production process. Moreover, these conditions prevented excessive growth of acetic acid bacteria and, therefore, uncontrolled production of acetic acid and acetoin, which may lead to flavor deviations in lambic beer. Concerning the role of less studied key microorganisms for lambic beer production, it was shown that the Acetobacter lambici MAG contained several acid tolerance mechanisms toward the harsh environment of maturing lambic beer, whereas genes related to sucrose and maltose/maltooligosaccharide consumption and the glyoxylate shunt were absent. Further, a Pediococcus damnosus MAG possessed a gene encoding ferulic acid decarboxylase, possibly contributing to 4-vinyl compound production, as well as several genes, likely plasmid-based, related to hop resistance and biogenic amine production. Finally, contigs related to Dekkera bruxellensis and Brettanomyces custersianus did not possess genes involved in glycerol production, emphasizing the need for alternative external electron acceptors for redox balancing.

Rapid detection of beer spoilage bacteria based on label-free SERS technology.

Beer spoilage bacteria have been a headache for major breweries. In order to rapidly identify spoilage bacteria and improve the sensitivity and signal-to-noise ratio of bacterial SERS detection, the label-free SERS technique was used as a starting point, and we found eight bacteria species that led to beer spoilage. The impact of AgNP concentration and AgNP and bacterial binding time on the final results were thoroughly investigated. To maximize the increase in the SERS signal, an aluminized chip was created. We merged the t-SNE reduced dimensional analysis algorithm, and SVM, KNN, and LDA machine learning algorithms to further investigate the effect of the approach on the final identification rate. The results demonstrate that SERS spectra had an increased intensity and signal-to-noise ratio. The machine learning classification accuracy rates were all above 90%, indicating that the bacteria were correctly classified and identified.

Non-Saccharomyces yeasts for beer production: Insights into safety aspects and considerations.

The application of non-Saccharomyces yeasts in beer as a natural tool for innovation, to create different aroma profiles and flavoured non-alcoholic beers, has attracted great interest from both researchers and commercial brewers. As a result, a higher diversity of non-Saccharomyces yeasts for beer production is expected on the market in the coming years. However, the safe use of non-Saccharomyces yeasts has not been broadly investigated and no guidance for the safety assessment of yeasts is published. The fundamentals of a safety assessment include an accurate taxonomic species identification using up-to date methods, along with a literature study regarding the yeast species in question. The strain-specific safety concerns that should be assessed involve pathogenic potential, antifungal resistance, production of biogenic amines and possible allergic reactions. However, yeast safety assessment is in its infancy compared to bacterial safety assessment and research is needed to set cut-off values for antifungal resistance, identify potential virulence genes and validate screening tools to assess yeast strains. Finally, the individual breweries are responsible for the safety related to the process in which yeasts are applied and throughout the shelf life of the beer. The application of non-Saccharomyces yeasts for industrial beer production is promising in terms of defining new prototypes and developing healthier and safer beers, but only if good food safety measures, i.e., both for the strain and the production process, are in place throughout the food value chain. In this way, the ancient role of yeasts in making beverages safer and thereby improving food safety is emphasized.

Use of yeast biocapsules as a fungal-based immobilized cell technology for Indian Pale Ale-type beer brewing.

Immobilized cell technologies (ICT) have been used in wort fermentation, beer maturation, or production of alcohol-free or low-alcohol beer. The purpose of ICT is to restrict intact cells to a specific location while allowing biological function. It improves cell stability, operational flexibility, and control in brewing, as well as ease in executing continuous operations. We investigated the use of yeast biocapsules for Indian Pale Ale (IPA) type beer wort fermentation, a novel ICT in brewing. Yeast biocapsules are a spherical yeast immobilization system in which yeast cells are encapsulated and connected to the hyphae of an inactivated hollow filamentous fungus pellet. Fermentations with yeast encapsulated in alginate beads, as the standard immobilization practice, and in free (non-immobilized) forms were carried out in parallel. We found that yeast biocapsules are a better option for cell reutilization than alginate beads, but worse for beer must clarity. Beer brewed with yeast biocapsules differed in concentration for five volatile compounds (acetaldehyde, diacetyl, ethyl acetate, 1,1-diethoxyethane, and isoamyl alcohol) and three sensory characters (persistency of the foam, malt, and yeast character). KEY POINTS: • Yeast biocapsules were investigated for beer wort fermentation • Biocapsules improve cell reutilization but are limited for beer clarification • Beer brewed with biocapsules is chemically different than conventional beer • Most sensory features did not differ between biocapsule and control beer.

Stuck or sluggish fermentations in home-made beers: Beyond the surface.

In the last several years, the popularity of homebrewed beers has skyrocketed. However, this type of product is extremely vulnerable to microbial deterioration. Twelve homemade beers, some characterized by defects or stuck fermentation, were analysed by using a polyphasic approach encompassing culturomics and culture-independent techniques to better understand mechanisms that drive microbiota evolution throughout production and to highlight determinants responsible for crowning with success. Two sour beers, one apple-flavoured ale, two Italian grape ales, and seven standard ales were sampled. Microbiological characterization was obtained by plating on nine different media coupled with High-throughput sequencing analysis of fungal and bacterial communities by targeting ITS1-2 and the V3-V4 regions of the 16S rRNA, respectively. Total microflora on PCA largely varied among samples, ranging from <102 CFU/mL up to around 107 CFU/mL often reflecting yeast counts on WL and LM. LAB population's levels on MRS and SDBm did not overlap, with the counts on the latter being even 5 Log CFU/mL greater. Acetic Acid bacteria were retrieved in Sour beers, as well as in one IGA, even though acetic acid was not detectable by HPLC in this last sample. Brettanomyces spp. were only found in sour beers, as expected, whereas Enterobacteriaceae were never counted. A total of 63 yeasts were randomly isolated from countable plates. Saccharomyces cerevisiae and Wickerhamomyces anomalus were the most frequently isolated species. In many cases, Interdelta analysis biotyping of S. cerevisiae isolates consistently allowed the detection of the starter strain. By HST S. cerevisiae dominated the mycobiota in four samples, even if in one of them residual maltose and ethanol contents suggested a stuck fermentation. W. anomalus was found to be the dominant species in two beers. Fifty-five LAB cultures were isolated and identified. Pediococcus damnosus was the only species retrieved in sour beers and two Ales, while Levilactobacillus brevis was found in two Ale samples. HTS did not confirm this result in one Ale sample since the genus Panotea spp. accounted for over 90 % of the microbiota. Enterobacteriaceae which were never counted dominated the microbiome of two Ale beers. Biogenic amines content largely varied with three Ale samples greatly contaminated. Based on chemical and microbiological outcomes only one beer ASAle out of 12 could be considered acceptable. Furthermore, the widespread presence of LAB by culturomics and Enterobacteriaceae by HTS raises concerns about the final products' safety.

Increased Rate of Yeast Cultivation from Packaged Beer with Environmentally Relevant Anaerobic Handling.

Beer production necessitates oxygen exclusion for the proper packaging and aging of the beer. Standard operating procedures, including those for quality testing, involve culturing microbes from packaged beer exposed to atmospheric oxygen, despite the generalized fact that packaged beer is an anaerobic environment. Our research goal was to apply an environmentally relevant culturing approach to improve yeast cultivation from bottled beer by attempting to ameliorate transplant shock. This is applicable to uniquely scrutinous quality assurance/control objectives and/or to grand cultivation goals, such as ancient beer samples. Although yeasts have the genetic capacity of oxygen protection, their epigenetic/biochemical states within anaerobic packaging may not adequately protect all cells from reactive oxygen species (ROS) at the moment of opening. Soon after opening, beer yeasts were found to be catalase negative, indicating deficient protection from at least one ROS. The general reduction/inhibition of growth was observed when the beer yeast was exposed to ROS in media, and atmospheric bottle opening was found to expose beer yeast to significantly increased levels of ROS. Our primary finding is that different oxygen handling methodologies (aerobic/microaerophilic/anaerobic) significantly impact the viable Saccharomyces yeast recovery rates of Bamberger's Mahr's Bräu Unfiltered Lager. Immediate anaerobic handling improved cultivation success rates, with significantly higher colony forming units (CFU)/mL being cultured, and reduced the volume of beer required to recover viable yeast. Aerobic standard operating procedures have mainly been developed to harvest yeast on large volumetric samples and/or samples with high viable cell numbers, but these procedures may be suboptimal and may underrepresent potential viable cell numbers. IMPORTANCE Procedures of beer production and packaging exclude oxygen to create a shelf-stable anaerobic environment, within which any viable organisms are stored. However, standard methodologies to cultivate microbes from such environments generally include opening in an oxygenated atmosphere. This study applies environmentally relevant culturing methods and compares the yeast recovery rates of beers handled in various oxygen conditions. When beer bottles were opened in anoxic conditions, higher colony counts were obtained, so a smaller volume of beer was required to recover viable cells. The yeast in beer, stored anaerobically, may not be biochemically prepared to fully protect cells from oxygen at the moment of opening. Negative catalase activity showed beer yeasts' vulnerabilities to reactive oxygen. Atmospheric opening may reduce viability, causing the underreporting of viable cells. Anaerobic opening could increase the odds of successfully detecting/cultivating viable cell(s) that are present, which is pertinent to uniquely stringent quality screens and ambitious culturing attempts from rare samples.

Identification of spoilage microflora in draught beer using culture-dependent methods.

AIMS: To determine whether the culture-dependent spoilage microflora found in draught beer are influenced by beer style. METHODS AND RESULTS: Four beer styles-lager, ale, stout and cask ale - were sampled twice from five different public houses (accounts) in four different locations. The microbiological quality of the dispensed beers was determined by a culture-dependent method ('forcing'), measuring the increase in turbidity after incubation at 30°C. The quality of draught beer varied from 'excellent' to 'poor' with cask beer samples having a higher Quality Index (90%) with keg ale the lowest (67.5%). With PCR amplified DNA (ITS1, ITS4, 16S rRNA primers) and blast identification of microflora, 386 colonies from agar plates were identified with 28 different micro-organisms from five genera of yeast and six of bacteria. Seven micro-organisms were found in all beer styles with Brettanomyces bruxellensis, B. anomalus and Acetobacter fabarum representing 53% of the identified micro-organisms. A subsequent, limited study using PALL multiplex PCR GeneDisc technology on forced samples (without selection on plates) suggests that draught beer microflora is qualitatively broader. It is noteworthy that the microflora of spoilt draught beer resembles that involved in the production of Belgian Lambic sour beers. CONCLUSIONS: Draught beer was of variable quality. Culture-dependent analysis suggests that species of Brettanomyces and Acetobacter are core microflora with some micro-organisms being associated with beer style. SIGNIFICANCE AND IMPACT OF THE STUDY: The microbiological quality of draught beer is important both commercially and to the consumer. Here, we report the core and diverse microflora found in different styles of draught beer using culture-dependent methods.