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Obligate anaerobic beer spoilage bacteria have been a menace to the brewing industry for several decades. Technological advances in the brewing process aimed at suppressing aerobic spoilers gave rise to problems with obligate anaerobes. In previous studies, the metabolic spectrum of Pectinatus and Megasphaera species has been described, but their metabolism in the beer environment remains largely unknown. We used high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GCMS) to further characterize beer spoiled by 30 different strains from six beer-spoiling species of Pectinatus and Megasphaera (P. cerevisiiphilus, P. frisingensis, P. haikarae, M. cerevisiae, M. paucivorans, and M. sueciensis). We detected differences in carbohydrate utilization and the volatile organic compounds (volatilome) produced during beer spoilage by all six species. We were able to show that glycerol, one of the basic components of beer, is the common carbon source used by all strains. It appears that this carbon source allows for anaerobic beer spoilage by Pectinatus and Megasphaera despite the spoilage-preventing intrinsic barriers of beer (iso-α-acids, ethanol, low pH, scarce nutrients); thus, extrinsic countermeasures are key for prevention. [ABSTRACT FROM AUTHOR]

Beer, with its low pH, presence of hop acids, alcohol content, and limited nutrient availability, presents a hostile environment for most bacteria. However, Levilactobacillus brevis remains a significant spoilage organism in the brewing industry. This study examines the impact of high hydrostatic pressure (HHP) on the inactivation and sublethal injury of Lb. brevis KKP 3574 in beer and wort. The results indicate that applying HHP at 400 MPa for 5 min effectively inactivates Lb. brevis, achieving up to a 7 log CFU/mL reduction in bacterial counts in beer, with no detectable sublethal injuries in beer samples. In contrast, in 10% wort, a sublethal injury level of 1.1 log CFU/mL was observed following the same HHP treatment. Furthermore, this study reveals a differential response of Lb. brevis cells depending on their growth phase; cells in the logarithmic growth phase are more susceptible to HHP, showing greater reduction in viability compared to those in the stationary phase. The survival dynamics of sublethally injured cells during refrigerated storage are also explored, with no regeneration observed in beer samples treated at pressures of 400 MPa or higher. These findings underscore the potential of HHP as a robust method for enhancing the microbiological safety and stability of beer while minimizing the risk of spoilage due to sublethally injured bacterial cells. This study provides crucial insights into optimizing HHP parameters to ensure product quality in the brewing industry. [ABSTRACT FROM AUTHOR]

Why was the work done: To measure the heat resistance of three vegetative bacteria in buffer (pH 4), alcoholic and alcohol-free beer. To verify that Pediococcus acidilactici ATCC 8042 and Lactobacillus brevis BSO 566 are as heat resistant as previously reported and to establish if Enterococcus faecium NRRL B-2354 could have application in validation studies for the pasteurisation of beer. How was the work done: The heat resistance of L. brevis, P. acidilactici and E. faecium in buffer, alcoholic and alcohol-free beer was determined using two approaches - capillary test tubes and flask method. What are the main findings: E. faecium was the most heat resistant microorganism in all three liquids. D values were significantly greater, and z-values were similar or significantly greater than the corresponding values of L. brevis and P. acidilactici. Why is the work important: E. faecium is used in the food industry as a pathogenic surrogate for the validation of thermal and nonthermal processes. The work reported here suggests that E. faecium can also be used for the validation of pasteurisation of beer. Its high z-value suggests that at higher pasteurisation temperatures (>65°C) it may be more resistant than yeast ascospores and could therefore be used as an indicator for flash pasteurisation. [ABSTRACT FROM AUTHOR]

Obligate anaerobic beer spoilage bacteria have been a menace to the brewing industry for several decades. Technological advances in the brewing process aimed at suppressing aerobic spoilers gave rise to problems with obligate anaerobes. In previous studies, the metabolic spectrum of Pectinatus and Megasphaera species has been described, but their metabolism in the beer environment remains largely unknown. We used high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GCMS) to further characterize beer spoiled by 30 different strains from six beer-spoiling species of Pectinatus and Megasphaera (P. cerevisiiphilus, P. frisingensis, P. haikarae, M. cerevisiae, M. paucivorans, and M. sueciensis). We detected differences in carbohydrate utilization and the volatile organic compounds (volatilome) produced during beer spoilage by all six species. We were able to show that glycerol, one of the basic components of beer, is the common carbon source used by all strains. It appears that this carbon source allows for anaerobic beer spoilage by Pectinatus and Megasphaera despite the spoilage-preventing intrinsic barriers of beer (iso-α-acids, ethanol, low pH, scarce nutrients); thus, extrinsic countermeasures are key for prevention.

Beer, with its low pH, presence of hop acids, alcohol content, and limited nutrient availability, presents a hostile environment for most bacteria. However, Levilactobacillus brevis remains a significant spoilage organism in the brewing industry. This study examines the impact of high hydrostatic pressure (HHP) on the inactivation and sublethal injury of Lb. brevis KKP 3574 in beer and wort. The results indicate that applying HHP at 400 MPa for 5 min effectively inactivates Lb. brevis, achieving up to a 7 log CFU/mL reduction in bacterial counts in beer, with no detectable sublethal injuries in beer samples. In contrast, in 10% wort, a sublethal injury level of 1.1 log CFU/mL was observed following the same HHP treatment. Furthermore, this study reveals a differential response of Lb. brevis cells depending on their growth phase; cells in the logarithmic growth phase are more susceptible to HHP, showing greater reduction in viability compared to those in the stationary phase. The survival dynamics of sublethally injured cells during refrigerated storage are also explored, with no regeneration observed in beer samples treated at pressures of 400 MPa or higher. These findings underscore the potential of HHP as a robust method for enhancing the microbiological safety and stability of beer while minimizing the risk of spoilage due to sublethally injured bacterial cells. This study provides crucial insights into optimizing HHP parameters to ensure product quality in the brewing industry.

To determine factors affecting the growth of the genus Pectinatus in beer, P. frisingensis ABBC437 was inoculated into 15 different beer samples. Although the pH, alcohol content, and bitterness unit of the 15 beer samples were adjusted to similar values within tolerance, growth kinetics as expressed by OD600 (optical density measured at a wavelength of 600 nm) were considerably different among the beer samples, indicating the presence of "as yet" unidentified factor(s) affecting the growth of P. frisingensis in beer. Chemical compositions of the beer samples were determined, and multiple regression analysis was conducted to predict the OD600 value. As a result, iron was suggested as one of the factors that affects the growth of P. frisingensis in beer because the additions of iron to the beer samples showed enhanced growth of P. frisingensis in a dose-dependent manner. Stimulatory effects of growth by the addition of iron were also observed for P. cerevisiiphilus and P. haikarae. Additionally, effects of diatomaceous earth treatment on the iron concentrations of the beer samples were evaluated. Iron concentrations varied, depending on the brand of diatomaceous earth, and the growth of P. frisingensis in beer showed a high correlation coefficient (0.924, p < 0.01) with the iron concentration in the beer samples. From these results, iron can be considered as an important factor for the growth of the genus Pectinatus in beer and selecting a low iron concentration diatomaceous earth is an effective way to improve the stability of beer against the risk of spoilage by the genus Pectinatus. [ABSTRACT FROM AUTHOR]

In microbiological quality control of final beer products, 100–400 ml beer samples are traditionally filtered on a membrane filter, followed by a lengthy culturing process in/on a laboratory medium that usually requires 3–14 days. This study aims to detect beer spoilage lactic acid bacteria (LAB) in a culture-independent fashion, using a direct polymerase chain reaction (PCR) approach. In order to detect a trace level of spoilage LAB in beer products, pressure cycling technology was evaluated to determine if this technology could improve the direct recovery of DNA from membrane filters for PCR detection. In this study, a mixed cellulose ester filter was adopted to allow a larger volume of beer to be filtrated. As a result of the optimization of the DNA extraction process that minimizes the loss of DNA and the effect of PCR inhibitors, 100 (1–10) cells/300 ml beer were successfully detected for beer spoilage LAB. Furthermore, a challenge test using 3,000 ml beer showed that 100 cells of Levilactobacillus (Lactobacillus) brevis were detectable without the negative effects of beer-originated PCR inhibitors, indicating that a trace level of contamination, corresponding to 1 cell in 300 ml of beer products, is detectable. It was also shown that the test was completed in approximately 8 hours. Therefore, this newly developed method is considered to be useful for the culture-independent direct detection of an extremely low level of beer spoilage LAB without a traditional culturing process. Supplemental data for this article is available online at at. [ABSTRACT FROM AUTHOR]

A novel beer-spoilage bacterium, Lactobacillus acetotolerans, has frequently been encountered in south China beer breweries, imparting an undesirable and unpalatable "buttery" taste and oily mouthfeel to beer. Understanding the genetic basis of spoilage bacterial adaptability to the beer microenvironment is crucial for the proper control of bacteria such as L. acetotolerans CN247 at an industrial scale. In this study, the complete genome of L. acetotolerans CN247 was sequenced using the PacBio and Illumina Miseq high-throughput sequencing technologies, and the relationship between genetic and physicochemical properties was analyzed. The genome size of L. acetotolerans CN247 was found to be 1.677 Mb, with a GC content of 37.55%. A total of 1,636 protein-coding genes were predicted, accounting for 88.96% of the genome. The average length of coding sequences was 912 bp. The L. acetotolerans CN247 strain was tolerant to high concentrations of acetic acid (6%) and showed varying degrees of preference for different beer components (i.e., sugars such as maltose, ribose, cellobiose, and mannitol). This strain also had a high tolerance for ethanol (14%) in the beer and was resistant to streptomycin, at a minimum inhibitory concentration of 256 μg/mL. Finally, genes associated with viable but non-culturable (VBNC) bacteria were found in the genome of L. acetotolerans CN247. This is the first study reporting the complete genome sequence of L. acetotolerans as a beer spoilage bacteria. These findings will contribute to the understanding of this bacterium's life cycle and help in the improvement of industrial contamination prevention practices. Supplemental data for this article is available online at https://doi.org/10.1080/03610470.2021.1997280. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To expand the applicability of the third-generation DNA sequencing technologies for the brewing industry, the new experimental protocols for quality control (QC) brewery laboratories have been developed, using the MinION system. Firstly, the hop tolerance genes, horA and horC, were amplified with multiplex PCR, and the PCR amplicons were sequenced by the MinION system. As a result, all of the beer spoilage lactic acid bacteria (LAB) strains carrying horA and/or horC were positively identified. Secondly, STA1, SbKEX2 and GEX1, were evaluated as diagnostic marker genes (DMGs) for discriminating Saccharomyces cerevisiae and S. pastorianus strains that possess essentially indistinguishable nucleotide sequences of the rDNA and internal transcribed spacer (ITS) regions. As a consequence of multiplex PCR specific to STA1, SbKEX2 and GEX1, these DMGs were shown to separate these hard-to-discriminate strains into four groups, namely top-fermenting ale yeast strains (S. cerevisiae), bottom-fermenting lager yeast strains (S. pastorianus), non-brewing S. cerevisiae wild yeast strains, and S. cerevisiae var. diastaticus. This study also demonstrated that the multiplexed PCR amplicons are independently recognized and correctly identified by the MinION system. Taken together, the newly developed methods using the MinION system are applicable to the identification and discrimination of important brewery microorganisms for microbiological QC. It was also shown that the concurrent applications of the rDNA-sequencing-based species identification and DMG approaches by the MinION system are useful for the breweries that now manufacture both traditional beers and non-traditional beverages. Supplemental data for this article is available online at https://doi.org/10.1080/03610470.2021.1939606. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) cause the majority of beer spoilage incidents in the brewing industry. Previous studies revealed that the hop resistance genes, horA and horC, are applicable to the species-independent determination of beer spoilage ability of LAB. However, novel beer spoilage LAB species are occasionally emerging, and their insights should be shared in the industry to prevent spoilage incidents by these unknown species. In this study, Lactobacillus nagelii ABBC668 strain, isolated from spoiled pilsner-type beer, was investigated, as L. nagelii has been poorly characterized as a beer spoiler. The beer spoilage ability and hop resistance genes of L. nagelii ABBC668 were examined. Results showed that L. nagelii ABBC668 harbored hop resistance gene horA and exhibited beer spoilage ability. Also, it was considered that horA harbored by ABBC668 was acquired via horizontal gene transfer and had an unstable nature compared to that of Lactobacillus brevis. The unstable nature may account for the less frequent encounter of beer spoilage L. nagelii strains in the brewing industry. Furthermore, ABBC668 showed similar motility to other L. nagelii strains. To our knowledge, this is the first report of L. nagelii as a beer spoilage species. [ABSTRACT FROM AUTHOR]

Beer spoilage lactic acid bacteria (LAB) often display hard-to-culture characteristics and fail to grow on conventional quality control (QC) laboratory media, while retaining growth capability in beer. This phenomenon is at least partly due to profound adaptation of spoilage LAB to the brewing environments, where multifactorial antibacterial hurdles and low temperature stress are encountered. In this study, changeable culturability of Secundilactobacillus (Lactobacillus) paracollinoides strains, JCM 11969T and JCM 15729, has been evaluated by comparing the variants obtained under various preceding subculture conditions. Sodium acetate substantially inhibited the growth of hard-to-culture S. paracollinoides variants, with smaller negative impacts for growth observed with yeast extract, peptone, and manganese sulfate. Growth inhibition was also observed at values higher than pH5.3 for hard-to-culture JCM 11969T variants and higher than pH5.0 for hard-to-culture JCM 15729 variants, respectively. In contrast, culturable S. paracollinoides variants exhibited sensitivities to low pH and grew well at pH 5.6 or even higher. Strikingly, the addition of sodium acetate led to increased colony forming units for culturable JCM 15729 strains, suggesting that sodium acetate exerts both positive and negative effects on the growth of S. paracollinoides strains. It has been also demonstrated that hard-to-culture and culturable states are reversible for S. paracollinoides JCM 11969T and JCM 15729, depending on the preceding subculture conditions. Taken together, it has been suggested that culturability of S. paracollinoides strains is easily changeable, and brewing microbiologists must ensure their culturability is maintained as observed upon primary isolation. [ABSTRACT FROM AUTHOR]

Lactobacillus species are the main spoilage bacteria in beer production and storage. The prerequisite for Lactobacillus species to grow in beer is to be able to resist the hop bitter compound iso-α-acid. This study examined the underlying iso-α-acid resistance mechanism of L. casei strain 2-9-5 through proteomic analysis. The significantly up-regulated proteins in L. casei 2-9-5 under 0.04% iso-α-acid are mainly involved in carbohydrate metabolism, energy generation, organic acids metabolism, and cell wall composition. Based on the proteomic analysis, it is proposed that organic acids may be important for L. casei 2-9-5 to resist iso-α-acid. The extracellular organic acids concentrations in L. casei 2-9-5 under iso-α-acid pressure were significantly higher than the control. Furthermore, additions of oxalic acid, tartaric acid, pyruvate, and ketoglutaric acid in the media were able to enhance the growth of L. casei 2-9-5 under iso-α-acid pressure. These phenomena were also observed in different Lactobacillus species, with different iso-α-acid resistance ability since the addition of organic acids also promoted the growth of these strains. Together, the data supports that organic acids are closely related to the resistance ability of Lactobacillus species towards iso-α-acid. [ABSTRACT FROM AUTHOR]

Beer draught lines are frequently contaminated with biofilm-forming microorganisms, which forces retailers to spend considerable time and money cleaning and replacing lines. In light of this financial burden, draught tubing composition was examined for its role in the prevention of biofouling in beer lines. Three types of draught tubing - vinyl, polyethylene, and nylon barrier - were inoculated with a combination of biofilm-forming microorganisms (Hafnia paralvei, Raoultella planticola, Pediococcus damnosus and Saccharomyces cerevisiae) and used to simulate a bar environment for sixteen weeks. Following simulation, the degree of biofouling in each draught line was determined by spectrophotometry and microscopy. Absorption values and fluorescence images showed that nylon barrier tubing was superior to the other lines at resisting biofilm maturation. These results suggest that tubing composition plays a significant role in the prevention of biofilm formation in beer draught lines and supports the adoption of nylon barrier tubing as an effective strategy against biofouling in a variety of applications. [ABSTRACT FROM AUTHOR]

When a beer is contaminated with unknown microorganisms, rapid and accurate identification of the contaminants and risk assessment of their spoilage capability are of critical importance for proper corrective actions. Molecular-based identification techniques have been used for years to identify beer-spoilage bacteria in the breweries. However, these techniques require expensive, advanced analytical instruments (e.g., DNA sequencer, MALDI-TOF MS, etc.), and advanced skills, hindering their practical use for routine microbiological testing in the field. Thus, the evaluation and development of alternative methods for microbial identification that are low-cost, but rapid and provide the same level of accuracy, are a necessity. One particular technology is the nanopore-based sequencer, MinION, developed by Oxford Nanopore Technologies (ONT). MinION is a low cost, portable real-time device for DNA and RNA sequencing. We developed and evaluated a MinION sequencer-based platform for beer-spoilage bacterial identification. The near full length 16S rRNA gene of a wide variety of beer-spoilage bacteria and those commonly found in the brewery environment, were sequenced using three different sequencing kits and analyzed using the EPI2ME cloud-based analysis platform and a consensus sequence-based approach. The methods successfully rapidly identified test-microorganisms at a species level, (approx. 3.5 h from DNA extraction to data analysis) and with a high accuracy, similar to Sanger sequencing (average per. identity > 98.5%). The MinION sequencer-based microbial identification platform was shown to be a powerful tool for on-field rapid and accurate beer-spoilage bacterial identification, which could revolutionize the way microbial identification is performed in a brewery QC laboratory. [ABSTRACT FROM AUTHOR]

Abstract Background Lactobacillus brevis is a member of the lactic acid bacteria (LAB), and strains of L. brevis have been isolated from silage, as well as from fermented cabbage and other fermented foods. However, this bacterium is also commonly associated with bacterial spoilage of beer. Results In the current study, complete genome sequences of six isolated L. brevis strains were determined. Five of these L. brevis strains were isolated from beer (three isolates) or the brewing environment (two isolates), and were characterized as beer-spoilers or non-beer spoilers, respectively, while the sixth isolate had previously been isolated from silage. The genomic features of 19 L. brevis strains, encompassing the six L. brevis strains described in this study and thirteen L. brevis strains for which complete genome sequences were available in public databases, were analyzed with particular attention to evolutionary aspects and adaptation to beer. Conclusions Comparative genomic analysis highlighted evolution of the taxon allowing niche colonization, notably adaptation to the beer environment, with approximately 50 chromosomal genes acquired by L. brevis beer-spoiler strains representing approximately 2% of their total chromosomal genetic content. These genes primarily encode proteins that are putatively involved in oxidation-reduction reactions, transcription regulation or membrane transport, functions that may be crucial to survive the harsh conditions associated with beer. The study emphasized the role of plasmids in beer spoilage with a number of unique genes identified among L. brevis beer-spoiler strains.

The growth inhibitions of four commercially available isomerized hop products, in which dihydro iso-α-acids, tetrahydro iso-α-acids, and hexahydro iso-α-acids are derived from natural iso-alpha-acids, against brewing-related microorganisms were studied. The tests were carried out in three media: liquid (broth, pH 6–7), solid (agar, pH 6–7) and beer (pH 4.2). The results indicate that Staphylococcus aureus (S. aureus) ZJ007 (G+) was the most sensitive strain to any hop product at the lowest concentration of 0.1 g/L in liquid and 0.5 g/L in solid. However, in liquid media, hexahydro iso-alpha-acids showed significant antibacterial potency on E. coli DH5a and Lactococcus species AB133 at 0.5 g/L (equating to 50 mg/L concentration). Interestingly, the growths of E. coli DH5α (G−) and wild Saccharomyces cerevisiae BY101 (wild yeast), respectively, in the solid media were inhibited by hexahydro and tetrahydro iso-alpha-acids (equating to 250–500 mg/L) and dihydro and iso-alpha-acids (750–1500 mg/L), and no effect on Saccharomyces cerevisiae (DM303). By the same means, the growths of Lactococcus species AB133 (possible hop resistant, G+) and Lactococcus lactis NZ9000 (hop-resistant G+, tolerated to iso-alpha-acids) were inhibited by hexahydro > tetrahydro iso-alpha-acids (equating to 500 mg/L) and not affected by dihydro and iso-alpha-acids. Overall, the hexahydro iso-alpha-acids are the most effective bacteriostat. The microscopy did not detect any strain in the inoculated and hopped beer (≥ 50 mg/L or BU) precipitates after fermentation. The GC–MS analysis showed insignificant aroma difference compared to the control beer (DM303 yeast only). For the implications, four isomerized hop products are more soluble and offer higher concentration in any neutral medium (pH 6–7) than their undissociated forms (pH ≤ 4). In the milder pH range, their antibacterial behavior is hexahydro > dihydro > tetrahydro ~ iso-alpha-acids. [ABSTRACT FROM AUTHOR]

Beer is a hostile environment for microorganisms to survive. However, some strains of lactic acid bacteria (LAB) have chosen this harsh environment for their ecological niche and evolved by incrementally acquiring various genetic elements to overcome a range of growth hurdles posed by beer. In fact, over 100 genes appear to be involved for one beer spoilage LAB strain to grow in and spoil beer. Although many of them are chromosomally encoded genes, an increasing number of brewery-specific genetic clusters have been also identified on plasmids, which are often horizontally shared by a variety of beer spoilage LAB species/strains. These plasmid-localized genetic factors were once represented by hop tolerance genes, such as horA, horC, and hitA, that were uncovered in the early days of hop tolerance research. But the recent evidence increasingly indicates that beer spoilage potential of LAB is conferred by multifactorial and complex adaptive responses that are related to a large number of defense mechanisms, including divalent cation homeostasis, cell envelope modification, oxidative stress response, and pH homeostasis. Many of these defense systems have been shown to be encoded on plasmids that are carried by beer spoilage LAB. In the past decade, new plasmid-encoded diagnostic marker genes (DMGs), such as gtfD15 and fabZ, have been additionally reported for the discrimination of beer spoilage potential of LAB strains. This review summarizes the recent progress in this area of research and provides some insights into the microbiological quality control (QC) tests adopted in brewery laboratories. [ABSTRACT FROM AUTHOR]

Until 30 years ago, only a limited number of bacterial and wild yeast species had been recognized as beer spoilers. However, the number of spoilage species have been rapidly increasing since then, reaching approximately 30 species for traditional beer categories. One trend is the continual emergence of new beer spoilage lactic acid bacteria. Many of these novel beer spoilage species are almost exclusively found in the brewing environments and are considered as beer-associated microorganisms. The second trend is the rise of strictly anaerobic beer spoilage bacteria, including Pectinatus and Megasphaera. Currently, close to 10 strictly anaerobic beer spoilage species have been reported. As a countermeasure, species-specific PCR systems have been adopted in breweries, and continually modified to keep up with the increasing number of beer spoilage species. Nevertheless, the circumstances are changing because of the rising trend of non-traditional beverages, as well as the rising use of non-traditional ingredients, in the brewing industry. These non-traditional beverages include non-alcoholic beers and flavored low alcoholic beverages, and spoilage species for these microbiologically unstable products are dramatically increasing in number. The conventional species-specific PCR can only handle the identification of 20–30 species of microorganisms, while novel methods that allow for a much broader microbial identification, such as DNA sequencing, are becoming more necessary. A few brewers are already evaluating the potential quality control applications of inexpensive third generation DNA sequencing technologies with the brewing industry. [ABSTRACT FROM AUTHOR]

Yeasts play a key role in the production of alcoholic beverages by fermentation processes. However, because of their continuous growth, they commonly cause spoilage of the final product. Herein, we introduce dual magnetic/light‐responsive self‐propelled microrobots that can actively move in a beer sample and capture yeast cells. The presence of magnetic nanoparticles on the surface of the microrobots enables their magnetic actuation under fuel‐free conditions. In addition, their photoactivity under visible‐light irradiation leads to an overall enhancement of their swimming and yeast removal capabilities. It was found that after the application of the microrobots into a real unfiltered beer sample, these micromachines were able to remove almost 100 % of residual yeasts. In addition, these microrobots could also be added at the initial step of the fermentation process without altering the final beer properties, such as alcohol level, color, and pH. This work demonstrates the potential of using externally actuated microrobots as an innovative and low‐cost solution for avoiding yeast spoilage in complex liquid environments, such as alcoholic beverages. Therefore, these autonomous self‐propelled microrobots open new avenues for future applications in the food industry. [ABSTRACT FROM AUTHOR]

Lactobacillus brevis beer-spoiling strains harbor plasmids that contain genes such as horA, horC, and hitA which are known to confer hop tolerance. The L. brevis beer-spoiling strain UCCLBBS124, which possesses four plasmids, was treated with novobiocin, resulting in the isolation of UCCLBBS124 derivatives exhibiting hop sensitivity and an inability to grow in beer. One selected derivative was shown to have lost a single plasmid, here designated UCCLBBS124_D, which harbors the UCCLBBS124_pD0015 gene, predicted to encode a glycosyltransferase. Hop tolerance and growth in beer were restored when UCCLBBS124_pD0015 was introduced in one of these hop-sensitive derivatives on a plasmid. We hypothesize that this gene modifies the surface composition of the polysaccharide cell wall, conferring protection against hop compounds. Furthermore, the introduction of this gene in trans in L. brevis UCCLB521, a strain that cannot grow in and spoil beer, was shown to furnish the resulting strain with the ability to grow in beer, while its expression also conferred phage resistance. This study underscores how the acquisition of certain mobile genetic elements plays a role in hop tolerance and beer spoilage for strains of this bacterial species. [ABSTRACT FROM AUTHOR]

Lactobacillus brevis is a lactic acid bacterium that is known as a food and beverage spoilage organism, and more specifically as a beer-spoiler. Phages of L. brevis have been described, but very limited data is available regarding temperate phages of L. brevis. Temperate phages may exert benefits to the host, while they may also be employed to combat beer spoilage. The current study reports on the incidence of prophage sequences present in nineteen distinct L. brevis genomes. Prophage induction was evaluated using mitomycin C exposure followed by genome targeted-PCR, electron microscopy and structural proteome analysis. The morphological and genome sequence analyses revealed significant diversity among L. brevis prophages, which appear to be dominated by members of the Myoviridae phage family. Based on this analysis, we propose a classification of L. brevis phages into five groups.

To meet demands for fresh flavor and unique taste from beer consumer, there is an increase in the popularity of craft beer, which is more susceptible to microbial contamination than the industry beer. A beer‐spoilage strain was isolated from craft beer and identified as Staphylococcus xylosus strain BS7. The isolate BS7 showed that high beer‐spoilage ability at low temperature (4°C), low pH (4.0) and high ethanol concentration (7.0%, v/v). Compared with the other known strains of S. xylosus, strain BS7 was resistant to hop compounds and had an evolutionary stability in hop resistance. Strain BS7 was able to grow quickly and utilizes nutrients in commercial beer, produces organic acids and biogenic amines, and changes beer flavor profile. These results suggest that S. xylosus strain BS7 is a beer‐spoilage strain with the danger, which can lead to the beer‐spoilage issues during craft beer production. [ABSTRACT FROM AUTHOR]

Lactobacillus brevis is a lactic acid bacterium that is known as a food and beverage spoilage organism, and more specifically as a beer-spoiler. Phages of L. brevis have been described, but very limited data is available regarding temperate phages of L. brevis. Temperate phages may exert benefits to the host, while they may also be employed to combat beer spoilage. The current study reports on the incidence of prophage sequences present in nineteen distinct L. brevis genomes. Prophage induction was evaluated using mitomycin C exposure followed by genome targeted-PCR, electron microscopy and structural proteome analysis. The morphological and genome sequence analyses revealed significant diversity among L. brevis prophages, which appear to be dominated by members of the Myoviridae phage family. Based on this analysis, we propose a classification of L. brevis phages into five groups. [ABSTRACT FROM AUTHOR]

Lactobacillus brevis is the most significant beer spoilage bacteria worldwide. It is found as a contaminant at all stages of brewing, including during primary and secondary fermentation, storage, filtration and the packaging process. In production with flash pasteurisation and subsequent hygienic filling, avoiding and tracing secondary contaminations is the key to a microbiologically stable product. However, L. brevis strains vary in their spoilage potential and can grow in many different beer types. This study presents a physiological test scheme for growth potential and biofilm formation in various media. It was determined that a large number of L. brevis strains can form biofilms as a first coloniser. The identification of the species alone is therefore not enough to be sure of the spoilage risk, which shows the need for a more in depth differentiation. DNA fingerprint techniques are crucial to differentiate isolates of this species at strain level. The rep‐PCR fingerprint system (GTG)5 was used to differentiate a selected collection of 20 isolates, which were characterised in growth and biofilm formation in various media. The data showed a high variation within the selected isolates. As second step, generated fingerprint clusters of L. brevis were traced back to contamination sources in a German brewery, revealing a high number of isolates with potentially varying growth, spoilage and biofilm potential. L. brevis being the demonstrator species, the PCR system used is a powerful and compatible tracing and troubleshooting tool for all kinds of spoilage bacteria in the brewing industry. © 2019 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

The quality of draught beer in 57 on‐trade licensed premises in 10 locations in the UK Midlands was assessed using a forcing test. Of 149 samples of standard lager ('SL', abv ≤ 4.2%), 44% were in the 'excellent' quality band compared with 16% of 88 samples of keg ale ('KA', abv ≤ 4.2%). Of the total of 237 samples, >90% were represented by two lager and two ale national brands. There were differences in the quality index (QI) between the brands, with lager SL3 having a QI of 84% compared with 72% for lager SL6, 71% for ale KA5 and 68% for ale KA1. The susceptibility of the four brands to spoilage was assessed using a challenge test with microorganisms taken from forced draught beer samples of the brands. Ale KA5 (challenge test QI = 87.5%) was the most resistant to spoilage followed by lager SL3 (81.3%), lager SL6 (75%) and ale KA1 (62.5%). Keg beers in accounts with a national cask beer quality accreditation had the same QI as those without accreditation. Analysis of price vs quality showed that the most expensive price band had the lowest quality. Draught beer quality declined as the number of dispense taps increased across the bar. It was also noted that dispense into branded half‐pint glasses had variable take‐up, with lager SL3 served in the correct branded glassware on 71% of occasions but only on 5% of occasions for lager SL6. None of the keg ales were served in correctly branded glassware. © 2018 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

Microbial spoilage of alcohol-free and low-alcohol beers, beer-mixed beverages, and soft drinks is most commonly caused by yeast. Yeast-related biofilms are therefore a serious problem in the production of these beverages. Fast detection of developing biofilms is a key factor to prevent subsequent spoilage of the product. For fast yeast detection, a new specific medium was developed and combined with real-time polymerase chain reaction (PCR) detection of characteristic beverage spoiling yeast species. The medium is based on MYPG broth (malt extract, yeast extract, peptone, glucose broth) with resazurin as a redox indicator for cell activity. The growth and biofilm potential of representative strains of commonly present beverage spoilage yeast species was evaluated using the developed medium. A novel real-time PCR detection system for Rhodotorula mucilaginosa, an early biofilm colonizer, was designed and successfully validated. Two field tests of the medium in combination with real-time PCR were performed. One test showed a differentiated hygienic status on a filler, and the other test tracked the contamination source of Saccharomyces cerevisiae var. diastaticus. Biofilm relevance of the strain set was proven. The modified MYPG proved to be highly sensitive when detecting yeasts. The detection of the selected target species directly in the medium was compatible and can provide detailed hygienic profiles when combined with additional information on the target species. This provides a fast detection method for yeast-related biofilms in brewery environments, differentiated hygienic monitoring, and makes it possible to troubleshoot contamination incidents. [ABSTRACT FROM AUTHOR]

Lactobacillus brevis is a major hop-resistance bacterium which poses significant challenge for the brewing industry, mainly due to the difficulty or incapability in detection by routine culturing methodology and its beer spoilage ability.This study aimed at investigating the VBNC state of a hop-resistance strain, L. brevis BM-LB13908. The culturable, total and viable numbers of L. brevis cells were calculated by MRS agar plate counting, acridine orange direct count (AODC) method and Live/Dead BacLight bacterial viability kit with fluorescence microscope. VBNC formation was induced by 189 ± 5.7 days under low-temperature storage or 27 ± 1.2 subcultures by continuous passage in beer, and VBNC cells induced by both strategies were recovered by adding catalase. In addition, insignificant difference in beer-spoilage ability was found in 3 states of L. brevis, including logarithmic growing, VBNC and recovered cells. This is the first study on the formation of VBNC state for L. brevis and beer-spoilage ability of both VBNC and recovered cells, which indicate L. brevis strain could cause beer spoilage without being detected by routine methodologies. The results derived from this study may support further study on L. brevis and other hop-resistance bacteria, and guidance on beer spoilage prevention and control, such as improvement for brewers on the microbiological quality control by using the improved culture method with catalase supplementation.

A commercially available lactic acid bacteria (LAB) reference standard (EZ-Accu ShotTMLactobacillus rhamnosus) was evaluated for its suitability to be used in the microbiological proficiency test (PT) schemes that are utilized within brewery microbiological quality systems and protocols. The test material was evaluated for homogeneity, stability, and culturability on several routinely used beer-spoiler detection media using membrane filtration (MF). Mean numbers of CFUs were calculated and subjected to statistical analysis. The coefficient of variation (CV) value in the homogeneity test (10.6%) showed that the test material was sufficiently homogenous, and the pellet-to-pellet variation was acceptable. The P-value in the stability and culturability test (p > 0.05) showed no statistically significant differences between variables, indicating that the microorganisms in the pellet would be stable during long storage and transport and that the culturability on different beer-spoiler detection media are comparable. In conclusion, this study suggests that EZ-Accu ShotTML. rhamnosus is suitable as a test material for microbiological PT schemes in breweries. [ABSTRACT FROM AUTHOR]

Background: Beer spoilage caused by wild yeast and bacteria is a major concern to both commercial and home brewers. Objective: To address this problem, Molecular Epidemiology Inc. (MEI, Seattle, WA) has developed a beer spoilage organism detection kit consisting of an enrichment media (BSE) and a multiplex PCR DNA dipstick that simultaneously detects these organisms within 2 h following enrichment. Methods: The kit was tested by using samples obtained from breweries located in the Greater Seattle area. Samples were spiked with the target microbes, when necessary, and used for assessing the performance characteristics of the DNA dipstick assay. Microbial enumerations were performed as per the standard microbiological plating methods. The suitability of the BSE medium to support the growth of beer spoilage microbes was compared with the industry-approved NBB-C medium (Dohler, Darmstadt, Germany). Results: Inclusivity (a panel of 50 isolates) and Exclusivity (a panel of 92 isolates) testing indicated that the dipstick assay can exclusively detect the indicated target beer spoilage microbes. When compared with the NBB-C medium (Dohler, Darmstadt, Germany) approved by the European Brewers Convention for beer spoilage organisms, the BSE medium supported faster growth of critical spoilage lactic acid bacteria such as Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Conclusions: The beer spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the brewing process, thus offering a convenient, cost effective, and faster test system for brewers interested in monitoring the quality of their product. [ABSTRACT FROM AUTHOR]

Lactobacillus brevis is a major hop-resistance bacterium which poses significant challenge for the brewing industry, mainly due to the difficulty or incapability in detection by routine culturing methodology and its beer spoilage ability.This study aimed at investigating the VBNC state of a hop-resistance strain, L. brevis BM-LB13908. The culturable, total and viable numbers of L. brevis cells were calculated by MRS agar plate counting, acridine orange direct count (AODC) method and Live/Dead BacLight bacterial viability kit with fluorescence microscope. VBNC formation was induced by 189 ± 5.7 days under low-temperature storage or 27 ± 1.2 subcultures by continuous passage in beer, and VBNC cells induced by both strategies were recovered by adding catalase. In addition, insignificant difference in beer-spoilage ability was found in 3 states of L. brevis , including logarithmic growing, VBNC and recovered cells. This is the first study on the formation of VBNC state for L. brevis and beer-spoilage ability of both VBNC and recovered cells, which indicate L. brevis strain could cause beer spoilage without being detected by routine methodologies. The results derived from this study may support further study on L. brevis and other hop-resistance bacteria, and guidance on beer spoilage prevention and control, such as improvement for brewers on the microbiological quality control by using the improved culture method with catalase supplementation. [ABSTRACT FROM AUTHOR]

Current recommendations for beer pasteurisation are based on the study in 1951 by Del Vecchio and coworkers. In this work, 14 beer spoilage bacteria were screened for their ability to grow or survive in ale and stout together with the determination of their thermo tolerance at 60°C. Using a capillary tube method, the D‐value (decimal reduction time) and z‐value (temperature resistance coefficient) of the three thermo tolerant bacteria (Acetobacter pasteurianus, Lactobacillus brevis and Lactobacillus hilgardii) were determined. Validation of pasteurisation at a range of pasteurisation units (PU) in packaged product were performed in a tunnel pasteuriser. This study showed that eight of the 14 microorganisms were able to grow in both beer styles, whilst different thermo tolerances were observed amongst the spoilage bacteria. Effective pasteurisation of the selected microorganisms was achieved at significantly lower PU values than those recommended by the European Brewery Convention Manual of Good Practice. In package pasteurisation conducted at 1.6 PU resulted in greater than an 8‐log reduction in viable cell numbers, resulting in 'commercial sterility'. Although this study demonstrated that successful pasteurisation was achieved for vegetative cells at significantly lower PU values than those recommended, further studies are required to demonstrate the optimal level of pasteurisation for spore forming bacteria and for yeast. © 2018 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

The aim of this study was to characterize 29 Leuconostoc mesenteroides isolates obtained from a brewing process in order to determine their potential contributions to this process and the product. In order to achieve this aim different enzymatic activities, the exopolysaccharide (EPS) production, the antagonistic activity, the beer spoilage ability and the production of lactic acid and diacetyl in beer, were assessed. Ln. mesenteroides isolates showed weak acidifying activity, and only 28% of them produced decreases in pH higher than 0.5 units after a 24 h period of incubation. The isolates were also scarcely proteolytic and did not produce EPS. Results from enzymatic activity assay showed that some of the isolates produced β-galactosidase and α- and β-glucosidases while none produced β-glucuronidase. All the isolates inhibited the growth of both Escherichia coli and Staphylococcus aureus , although none of them inhibited Pediococcus acidilactici . When Lactobacillus brevis was used as an indicator, 44.8% of the isolates showed antagonistic activity. The beer-spoilage ability assay showed that only 5 of the 29 isolates were able to grow in beer but none of them harboured horA or horC genes. However, the growth of these isolates in beer did not produce an excess of D-lactic acid or diacetyl. These findings show that the presence of any of the characterized isolates during the brewing process or in the bottled beer would not have a negative impact. On the contrary, the findings reveal interesting and potentially beneficial effects. [ABSTRACT FROM AUTHOR]

We wanted to identify key factors influencing the extent of β-glucan production by Lactobacillus brevis TMW 1.2112, which has been isolated from viscous, spoiled beer and which could contribute to viscosity increases of spoiled beverages via exopolysaccharide (EPS) production. In this way, we analyzed the influence of different initial pH values and carbohydrate sources on growth of and slime/β-glucan formation by this strain. In a screening of 48 carbohydrates, 14 fermentable sugars which enabled growth were identified. These sugars were further investigated regarding their EPS formation-promoting properties. The hexose-based mono- and di-saccharides enabled slime formation, while all pentoses failed to cause any thickening effect. The strongest slime formation was observed upon growth on D-maltose, the weakest on D-fructose. A lower initial pH (4.3) caused significant higher viscosities than an initially higher one (pH 6.2). This effect was independent from the carbohydrate supplied. Although the thickening of nutrient media by L. brevis TMW 1.2112 strongly depended on the initial pH and the available carbon source, all isolated polysaccharides were exclusively composed of glucose moieties and exhibited highly similar elution profiles after separation via asymmetric flow field-flow fractionation independently of the provided carbon source. Our results suggest that the extent of β-glucan/slime formation by special L. brevis strains isolated from the brewery environment is strongly influenced by the initial pH and the availability of certain EPS formation-promoting sugars with maltose being the most favored carbohydrate for the regulated and directive biosynthesis of capsular β-glucan. [ABSTRACT FROM AUTHOR]

The aim of this investigation has been the design and validation of an oligonucleotide microarray in order to detect 17 different wine-spoilage microorganisms, i.e. 9 yeasts, 5 lactic bacteria and 3 acetic acid bacteria species. Furthermore, several strains belonging to these species has been found to produce undesirable compounds for wine consumers. Oligonucleotide probes specific for each microorganism were designed to target the intergenic spacer regions (ISR) between 18S-5.8S region for yeasts and 16S-ITS1 region for bacteria. Prior to hybridization the ISR were amplified by polymerase chain reactions using designed consensus primers. Each oligonucleotide-probes exclusively recognized its target without undesired aspecific cross-hybridizations. Under our experimental condition, the microarray assay analysis was able to detect the amount of DNA equivalent to 24 ( Saccharomyces cerevisiae ), 160 ( Lactobacillus brevis ) and 124 ( Gluconobacter oxydans ) cells, three species chosen as experimental models for the three studied microbial classes. Moreover, a novel procedure that allowed the extraction of genomic DNA from a mixture of eukaryotic and prokaryotic cells from contaminated wine was developed. The obtained results confirm that the microarray assay is able to detect specifically different spoilage microorganisms present in mixture in contaminated wines. For the first time the microarray methodology has been applied for the simultaneous identification of different mixed population of spoilage yeast and bacteria directly isolated from wine, thus indicating the practicability of oligonucleotide microarrays as a contamination control in wine industry. [ABSTRACT FROM AUTHOR]

A total of 13,802 samples over seven years were investigated using real‐time PCR for the presence of beer spoilage bacteria, providing a rare large‐scale overview of the incidence of individual species. At the same time, the isolation site (early stages of the brewing process = type I and late stages = type II) and the type of contaminated beer and intermediate products (bottom‐fermented or top‐fermented) were evaluated using chi‐squared analysis. The most frequently occurring species were Lactobacillus brevis (7 year average 41.9%), Lactobacillus (para‐)casei (10.4%) and Lactobacillus backii (9.5%). L. (para‐)casei was found at significantly higher rates in the early stages of the brewing process, whereas Lactobacillus lindneri, the Lactobacillus [L. (para‐)plantarum and L. coryniformis] and the Pectinatus (P. cerevisiiphilus, P. frisingensis and P. haikarae) groups and Megasphaera cerevisiae predominated in the later stages and in package (significance level 95%). On a significance level of 95%, Pediococcus damnosus, M. cerevisiae and L. lindneri together with the Pectinatus and Lactobacillus groups grew predominantly in bottom fermented samples. L. brevis, in contrast, was found most frequently in top fermented beers. Copyright © 2018 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

This study aimed to investigate the contamination degree and quality control of seven breweries from the Yangtze River Delta region, China. A total of 205 spoilage bacteria strains were isolated and no wild yeast was observed. The vast majority of the spoilage bacteria belonged to Lactobacillus, Lactococcus, and Leuconostoc species and 28.8% of them could grow in beer. Among the seven sampling points, fermented beer, finished beer, and equipment contained larger amounts of spoilage bacteria. The most abundant of the spoilage bacteria that could grow in beer existed in fermented beer and the number of spoilage bacteria was significantly reduced in the finished beer. The equipment in fermentation and packaging workshops showed high percentages of spoilage bacteria that could grow in beer. This indicated that more attention should be paid in controlling the spoilage bacteria in the fermentation and packaging processes. The hop tolerance related gene analysis showed that the majority of spoilage bacteria contained at least one of the HorA, HorC and HitA genes. None of these genes were observed in the two spoilage bacteria strains that could grow in the beer. This information could be useful for breweries in the Yangtze River Delta region for removing risks in order to produce high-quality products in good microbial condition. [ABSTRACT FROM AUTHOR]

Despite several hurdles, which hinder bacterial growth in beer, certain bacteria are still able to spoil beer. One type of spoilage is characterized by an increased viscosity and slimy texture caused by exopolysaccharide (EPS) formation of lactic acid bacteria (LAB). In this study, we characterize for the first time EPS production in a beer-spoiling strain (TMW 1.2112) of Lactobacillus brevis , a species commonly involved in beer spoilage. The strain’s growth dynamics were assessed and we found an increased viscosity or ropiness in liquid or on solid media, respectively. Capsular polysaccharides (CPS) and released EPS from the cells or supernatant, respectively, were analyzed via NMR spectroscopy and methylation analysis. Both are identical β-(1 → 3)-glucans, which are ramified with β-glucose residues at position O 2. Therefore, we assume that this EPS is mainly produced as CPS and partially released into the surrounding medium, causing viscosity of e.g. beer. CPS formation was confirmed via an agglutination test. A plasmid-located glycosyltransferase-2 was found as responsible for excess β-glucan formation, chromosomal glucanases were proposed for its degradation. The glycosyltransferase-2 gene could also be specifically identified in beer-spoiling, slime-producing Lactobacillus rossiae and Lactobacillus parabuchneri strains, suggesting it as promising marker gene for the early detection of β-glucan-producing Lactobacilli in breweries. [ABSTRACT FROM AUTHOR]

Abstract: The quality of draught beer is important to consumers but can be inconsistent, ranging from excellent through to unacceptable. The few, dated studies of draught beer quality have focused on the number of microorganisms that are present in the product. Work reported here suggests that this approach has its limitations and fails to relate to beer quality post‐dispense. An alternative approach using the long‐established ‘forcing’ method provides a better but still retrospective assessment of draught beer quality. Samples post dispense are ‘forced’ by static incubation at 30°C for four days and beer quality is ranked by the measurement of absorbance at 660 nm. The increase in absorbance reflects the growth of beer spoilage microorganisms present in the beer at dispense. Four quality bands are proposed, where quality is described as excellent (absorbance increase of <0.3), acceptable (0.3–0.6), poor (0.6–0.9) and unacceptable (>0.9). The method is straightforward, requires no special skills and enables, for the first time, the robust quantification of draught beer quality. It is anticipated that the method will have widespread application in the measurement and improvement of the quality of draught beer. Copyright © 2017 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

As one of the most popular beverage, beer has strong resistance to bacteria except for lactic acid bacteria (LAB), as its strong viability in beer. Contaminating LAB results in beer spoilage. HorA gene carried by beer spoilage lactobacillus confers hop resistance and is associated with the growth of Lactobacillus in beer, thus can be used as a perfectly valid genetic marker for the detection of beer spoilage Lactobacillus strains. This study applied genome and bioinformatics analyses, together with genetic alignment and comparison techniques to build a rolling circle amplification (RCA) detection method targeted on horA for beer spoilage Lactobacillus. The horA genetic mediated RCA method showed rapid, sensitive and specific on beer spoilage lactobacillus detection. [ABSTRACT FROM AUTHOR]

Beer-spoilage-related lactic acid bacteria (BSR LAB) belong to multiple genera and species; however, beer-spoilage capacity is isolate-specific and partially acquired via horizontal gene transfer within the brewing environment. Thus, the extent to which genus-, species-, or environment- (i.e., brewery-) level genetic variability influences beer-spoilage phenotype is unknown. Publicly available Lactobacillus brevis genomes were analyzed via BlAst Diagnostic Gene findEr (BADGE) for BSR genes and assessed for pangenomic relationships. Also analyzed were functional coding capacities of plasmids of LAB inhabiting extreme niche environments. Considerable genetic variation was observed in L. brevis isolated from clinical samples, whereas 16 candidate genes distinguish BSR and non-BSR L. brevis genomes. These genes are related to nutrient scavenging of gluconate or pentoses, mannose, and metabolism of pectin. BSR L. brevis isolates also have higher average nucleotide identity and stronger pangenome association with one another, though isolation source (i.e., specific brewery) also appears to influence the plasmid coding capacity of BSR LAB. Finally, it is shown that niche-specific adaptation and phenotype are plasmid-encoded for both BSR and non-BSR LAB. The ultimate combination of plasmid-encoded genes dictates the ability of L. brevis to survive in the most extreme beer environment, namely, gassed (i.e., pressurized) beer. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the occurrence of sporeforming bacteria in different types of beers (n = 163) and to assess the presence of hor genes in the isolates. Additionally, the study aimed to evaluate the fate of five representative sporeforming bacteria harboring horA and horC genes in alcohol-free and lager beers. Two hundred and sixty (n = 260) sporeforming bacteria belonging to eight different genera were isolated from beers, i.e., Bacillus (n = 118), Paenibacillus (n = 89) and Brevibacillus (n = 41), Lysinbacillus (n = 6), Cohnella (n = 3), Rummeliibacillus (n = 1), Alicyclobacillus (n = 1), and Anoxybacillus (n = 1), respectively. A predominance of members within the Bacillus cereus sensu lato (n = 72; 27.1%), followed by B. megaterium (n = 18; 7%), P. validus (n = 16; 6.1%), P. humicus (n = 13; 5%), P. alginolyticus (n = 13; 5%) and Br. brevis (n = 13; 5%) was observed in beer samples analyzed. Only 5% (n = 14) out of 260 sporeforming bacterial isolates recovered from beers harbored one or both horA and horC genes. Only one (0.3%) isolate, i.e., Bacillus cereus sensu lato (identified as B. thuringiensis LMQA 206) presented both horA and horC genes. None of the five bacterial sporeforming strains harboring horA or horC genes inoculated was able to grow in the beers throughout the storage period studied, and no spoilage was detected. The results of this study indicated a widespread occurrence of sporeforming bacteria in several types of beers from different brands, highlighting that measures should be taken to reduce the occurrence of sporeforming bacteria considering stability and safety concerns. [ABSTRACT FROM AUTHOR]

A bacteriocin-producing lactic acid bacterium was isolated from malts. The isolate was identified as Pediococcus acidilactici by morphological, API 50 CHL kit and 16S rRNA analysis, and designated as strain HW01. The antimicrobial activity was maintained over a broad pH range (pH 2–11). Proteolytic enzymes and α-amylase treatments inactivated the bacteriocin activity; while catalase, heat, detergents, and solvents did not. The molecular weight of the crude bacteriocin was approximately 6 kDa by SDS-PAGE. The bacteriocin production phenotype (Bac + ) was linked to a 9.8 kb plasmid and was not increased by co-culture with an indicator microorganism. Bacteriocin HW01 inhibited several Gram-positive bacteria, including health-threatening microorganisms ( Staphylococcus aureus, Streptococcus mutans, Lactobacillus curvatus, Listeria monocytogenes, and L. innocua ). Particularly, Pediococcus damnosus and P. claussenii , which were isolated from spoiled beer, were strongly antagonised by the bacteriocin. P. acidilactici HW01 has tolerance to hop compounds but is unable to grow in a simulated beer environment. The potential of the HW01 strain, as a starter culture in brewing, to control beer spoilage lactic acid bacteria, is discussed. [ABSTRACT FROM AUTHOR]

Objective The present study aimed at investigating the capability of L. plantarum strain BM-LP14723 to enter into and recover from the viable but nonculturable (VBNC) state and to cause beer spoilage. Methods VBNC state was induced by incubating in beer with subculturing or low temperature treatment. Culturable, total, and viable cells numbers were assessed by MRS agar plate counting, acridine orange direct counting, and Live/Dead BacLight bacterial viability kit, respectively. Organic acids concentrations were measured by reversed-phase high performance liquid chromatography. Results VBNC L. plantarum cells were detected after 189 ± 1.9 days low temperature treatment or 29 ± 0.7 subcultures in beer. The VBNC L. plantarum retained spoilage capability. Addition of catalase is an effective method for the recovery of the VBNC L. plantarum cells. Conclusion L. plantarum strain BM-LP14723 is capable of entering into and recovery from the VBNC state and maintained spoilage capability. The current study presented that beer-spoilage L. plantarum can hide both in breweries and during transporting and marketing process and thus lead to beer-spoilage incidents. [ABSTRACT FROM AUTHOR]

Objectives This study aimed to investigate the spoilage capability of Lactobacillus lindneri during the induction and resuscitation of viable but nonculturable (VBNC) state. Methods L. lindneri strain was identified by sequencing the PCR product (amplifying 16S rRNA gene) using ABI Prism 377 DNA Sequencer. During the VBNC state induction by low temperature storage and beer adaption, total, culturable, and viable cells were assessed by acridine orange direct counting, plate counting, and Live/Dead BacLight bacterial viability kit, respectively. Organic acids and diacetyl concentration were measured by reversed-phase high performance liquid chromatography and head dpace gas chromatography, respectively. Results VBNC state of L. lindneri was successfully induced by both beer adaption and low temperature storage, and glycerol frozen stock was the optimal way to maintain the VBNC state. Addition of catalase was found to be an effective method for the resuscitation of VBNC L. lindneri cells. Furthermore, spoilage capability remained similar during the induction and resuscitation of VBNC L. lindneri . Conclusions This is the first report of induction by low temperature storage and resuscitation of VBNC L. lindneri strain, as well as the first identification of spoilage capability of VBNC and resuscitated L. lindneri cells. This study indicated that the potential colonization of L. lindneri strain in brewery environment, formation and resuscitation of VBNC state, as well as maintenance in beer spoilage capability, may be an important risk factor for brewery environment. [ABSTRACT FROM AUTHOR]

Beer is generally considered a stable product owing to its intrinsic 'unfavourable' conditions (hops, alcohol, low oxygen, etc.) that inhibit the growth of pathogenic microorganisms. However spoilage microorganism such as Lactobacillus brevis, Pediococcus damnosus, Acetobacter aceti, Zymomonas mobilis and various wild yeasts (e.g. Brettanomcyes spp.) can have significant detrimental effects on the organoleptic properties of the final product. The presence of essential vitamins, such as thiamine and riboflavin, can help to enhance the growth of these microorganisms, accelerating the rate of spoilage. The presence of thiamine had a noticeable effect on the lactic acid productivity of L. brevis and P. damnosus, acetaldehyde productivity of Z. mobilis and acetic acid production of Brettanomyces spp., while riboflavin enhanced 2,3-pentanedione production by P. damnosus and Brettanomyces spp. Copyright © 2017 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

To meet consumer demands for fresher and healthier foods, the pasteurization of beer has largely been replaced by membrane filtration methods. Research on beer spoilage caused by hop-resistant bacteria is thus of great interest. In this context, we have isolated a beer-spoilage strain of bacteria from turbid bottled beer treated by membrane filter sterilization, which we identified as Bacillus cereus strain 3012. Strain 3012 was able to produce acids and bioamines, resulting in a change in the flavor profile of the beer. Unlike other known strains of B. cereus, this isolate was resistant to hop compounds present in the beer and was in an evolutionarily stable state in terms of hop resistance. Our results suggest that this hop-resistant B. cereus strain 3012 is a foodborne pathogen with the potential to cause beer-spoilage incidents in the brewing industry. [ABSTRACT FROM AUTHOR]

Beer-spoiling ability, survival after 14 weeks of incubation in 2 beer types under 2 temperature regimes and the presence of spoilageassociated horA, horC and hitA genes in the genome were studied in 69 strains of lactic acid bacteria. Beer spoilage was determined in 12 (48 %) of 25 strains of Lactobacillus brevis, 2 (25 %) of 8 strains of L. plantarum, 7 (26 %) of 27 strains of L. paracasei subsp. paracasei, and in 1 (33 %) of 3 strains of Leuconostoc spp. Survival in beer without adverse effects on its quality was more widely spread among the strains (L. brevis 89 %, L. plantarum 75 %, L. buchneri 100 %, L. paracasei subsp. paracasei 82 %, L. lactis 66 %, Leuconostoc spp. 33 %). In the strongest beer spoiler L. brevis, beer spoilage was observed only in 2 strains containing horC and in 6 strains containing the combinations horA/hitA and horA/horC/hitA; 4 strains spoiled beer without having any of these genes. Beer spoilage, detected in a total of 10 strains of all other species, was not associated with the presence of these genes.Beer-spoiling ability, survival after 14 weeks of incubation in 2 beer types under 2 temperature regimes and the presence of spoilageassociated horA, horC and hitA genes in the genome were studied in 69 strains of lactic acid bacteria. Beer spoilage was determined in 12 (48 %) of 25 strains of Lactobacillus brevis, 2 (25 %) of 8 strains of L. plantarum, 7 (26 %) of 27 strains of L. paracasei subsp. paracasei, and in 1 (33 %) of 3 strains of Leuconostoc spp. Survival in beer without adverse effects on its quality was more widely spread among the strains (L. brevis 89 %, L. plantarum 75 %, L. buchneri 100 %, L. paracasei subsp. paracasei 82 %, L. lactis 66 %, Leuconostoc spp. 33 %). In the strongest beer spoiler L. brevis, beer spoilage was observed only in 2 strains containing horC and in 6 strains containing the combinations horA/hitA and horA/horC/hitA; 4 strains spoiled beer without having any of these genes. Beer spoilage, detected in a total of 10 strains of all other species, was not associated with the presence of these genes.