Beer-spoilage Bacteria Research Articles

Occurrence of Pectinatus and Megasphaera in the Major UK Breweries

The occurrence of beer spoilage bacteria belonging to the genera Pectinatus and Megasphaera in ten major UK breweries was investigated. The sampling points were selected from fermentation areas, beer conditioning areas and beer bottling and canning sites. Multiplex PCR methodology was used for detection of three Pectinatus and three Megasphaera species using species-specific primers. The presence of six Lactobacillus species was also examined. Overall, 117 samples were analysed from ten breweries; six samples were positive for the presence of Pectinatus species and three samples were positive for the presence of Megasphaera species, while 34 samples were positive for the presence of Lactobacillus species. Lactobacillus species appeared to be the major potential spoilage microorganisms. Although none of the actual beer samples were found to be positive for Pectinatus and Megasphaera species, their occurrence in aerobic brewery environments indicates sanitation problems and revealed the presence of highly established biofilms in some breweries.

125th Anniversary Review: Microbiological Instability of Beer Caused by Spoilage Bacteria

Beer has been generally recognized as a microbiologically stable beverage. However, microbiological incidents occasionally occur in the brewing industry. The microbiological instability of beer is often caused by bacteria consisting of four genera, Lactobacillus, Pediococcus, Pectinatus and Megasphaera. Lactobacillus and Pediococcus belong to the lactic acid bacteria (LAB), whereas Pectinatus and Megasphaera form a group of strict anaerobes that are known as intermediates between Gram-positive and Gram-negative bacteria. The frequencies of beer spoilage incidents caused by these four genera have been reported to exceed 90% in Europe and therefore Lactobacillus, Pediococcus, Pectinatus and Megasphaera are considered to be the principal spoilage agents in the brewing industry. Thus, this review consists of three parts involving these four genera. The first part describes spoilage LAB in alcoholic beverages with some emphasis on beer spoilage LAB. In this part, the emergence and evolution of these spoilage LAB is discussed, the insight of which is useful for developing quality control methods for these beverages. The second part is devoted to the hop resistance in beer spoilage LAB. This area of research is evolving rapidly and recent progress in this field is summarized. The third part concerns Pectinatus and Megasphaera. Although this group of beer spoilage bacteria has been described relatively recently, the incident reports in Europe increased in the early 1990s, reaching around 30% of spoilage incidents. Various aspects of Pectinatus and Megasphaera, ranging from their taxonomy and beer spoilage ability to detection and eradication methods are described.

Isolation and Characterization of Bacteriophages That Inhibit Strains of Pediococcus Damnosus, Lactobacillus Brevis, and Lactobacillus paraplantarum That Cause Beer Spoilage

The aim of this study was to isolate and characterize bacteriophages against Pediococcus and Lactobacillus strains that cause spoilage in brewing processes. A number of beer-spoilage bacteria were isolated from breweries and characterized by 16S rRNA typing. Five distinct P. damnosus phages and four Lactobacillus phages, which lysed both L. brevis and L. paraplantarum, were isolated from municipal sewage and farmyard slurries. A rapid DNA isolation method was used to isolate DNA of sufficient purity for restriction endonuclease digestion from de Man, Rogosa, and Sharpe (MRS) broth. Phages were analyzed using restriction digest and transmission electron microscopy and shown to be in the family Siphoviridae, with genomes ranging in size from 40 to 50 kb. All phages were characterized and shown to be distinct. This study identifies five novel phages against P. damnosus. Phages for this species are very rare in the scientific literature. Four novel phages against L. brevis and L. paraplantarum are also identified. These phages may have an application in the biocontrol of beer-spoilage bacteria.

10-Hydroxy-2-decenoic Acid as an Antimicrobial Agent in Draft Keg-Conditioned Wheat Beer

In nature, 10-hydroxy-2-decenoic acid (10-HDA), an important unsaturated fatty acid, is only available in royal jelly. Natural 10-HDA exhibits several biological activities, including anticancer activity, and provides protection against acute radiation injury. In this study, we identified a method for extraction of 10-HDA from royal jelly. The antimicrobial effect of 10-HDA on beer-spoilage bacteria and yeast was investigated by flow cytometry and bacteriostatic circle (paper assay) experiments. We also determined the minimum inhibitory concentration of 10-HDA using the double-dilution method. Our results showed that 10-HDA could significantly inhibit common microorganisms, such as Lactobacillus brevis and Pediococcus damnosus, found in beer but had no inhibitory effect on yeast. A novel approach for preparing draft keg-conditioned wheat beer was developed using top-fermenting yeast strain 303 and bottom-fermenting yeast strain 308. The beer was then kegged for secondary fermentation. Because living yeasts were still in the beer, pasteurization could not be used, and it was difficult to keep everything clean and sanitary during brewing and kegging processes. In this study, 10-HDA was added to the green beer during kegging. We confirmed that 10-HDA, acting as an antimicrobial agent, increased the sanitary aspects of the kegged beer and improved the microbiological stability and shelf life of the beer.

Rapid Screening for Gram-Negative and Gram-Positive Beer-Spoilage Firmicutes Using a Real-Time Multiplex PCR

Current methods for detection and identification of beer-spoilage bacteria can be time-consuming and may not encompass all beer-spoilage isolates due to targeting of specific species. As such, a rapid method that targets a broader spectrum of beer-spoilage bacteria is likely to be more efficient for initial detection of contamination. Building on our previous real-time PCR (rltPCR) that detects Firmicutes, we created a system that enables concurrent detection and differentiation of gram-negative and -positive brewery-associated Firmicutes. Our two previously described rltPCR hydrolysis probes, which are able to detect all bacteria and Firmicutes, were used in combination with a newly developed probe (GmNeg) that detects only gramnegative brewery-associated Firmicutes. In silico analysis performed to determine the specificity of the GmNeg probe predicted that the probe would detect all gram-negative brewery-associated Firmicutes. This was confirmed by rltPCR analysis of brewery-associated bacteria, with the GmNeg probe showing specificity for gram-negative Firmicutes but not for gram-positive Firmicutes or any non-Firmicutes. The sensitivity of this rltPCR system was 35 fg of DNA per reaction, corresponding to approx. 10–20 bacteria. This multiplex rltPCR will enable brewery quality control laboratories to rapidly screen for brewery-associated Firmicutes, with identification of a contaminant as either a gram-negative or -positive bacterium.

Effects of Lysozyme on the Microbiological Stability and Organoleptic Properties of Unpasteurized Beer

J. Inst. Brew. 116(1), 33–40, 2010 Lysozyme is an antimicrobial enzyme that could be applied to counteract those bacterial species which, due to their own metabolic activity, possess notable beer spoilage ability and lead to loss of beer quality. Experiments were carried out to assess lysozyme potential to prevent the growth of beer spoilage bacteria, and to verify the effect of lysozyme on the microbiological stability and sensory characteristics of unpasteurized beer. Eight replicates, all from the same lot of Italian beer, were treated with 0 and 100 ppm lysozyme. Microbiological analyses were conducted bimonthly to investigate the presence of spoilage bacteria. Sensory analyses were performed to determine whether there were any significant differences in sensory impressions between beers produced with and without lysozyme. Lysozyme exerted a strong inhibitory action on the lactic acid bacteria (LAB) present in the beer and was very stable throughout the shelf life. Sensory tests revealed no unfavourable influence on beer flavour when using lysozyme. Indeed, the shelf life of beer with added lysozyme proved to be extended. Even as late as 1 month after the expiry date it still met with the panellists’ approval. Lysozyme may be regarded as an effective agent for preventing microbiological contamination and prolonging the stability of unpasteurized beer.

Isolation, Identification, and Characterisation of Beer-Spoilage Lactic Acid Bacteria from Microbrewed Beer from Victoria, Australia

J. Inst. Brew. 116(1), 14–22, 2010 Lactic acid bacteria are the most frequently encountered beerspoilage bacteria, and they may render beer undrinkable due to the production of lactic acid, diacetyl, and turbidity. Microbrewed beer is typically sold unpasteurised, leaving it more susceptible to spoilage by lactic acid bacteria. In this study, the incidence of lactic acid bacteria in bottled microbrewed beer from Victoria, Australia was investigated. A total of 80 beers from 19 breweries were screened for lactic acid bacteria. Almost 30% contained culturable lactic acid bacteria, and many had lactic acid levels well above the flavour threshold. Ethanol, hops, and the pH levels of the beers were not predictors for spoilage in the beers examined, and contamination appeared to be more closely linked to the source brewery. The 45 lactic acid strains isolated from these beers were identified by RAPD-PCR, with Lactobacillus brevis being the most frequently isolated species. All isolates were capable of spoiling beer and contained putative hop resistance genes. At typical beer levels, pH and ethanol had no effect on the growth of the particular spoilage bacteria isolated in this study.

Detection of Beer-spoilageLactobacillus brevisstrains by Reduction of Resazurin

Lactobacillus brevis is the most common beer-spoilage bacteria found in breweries. Due to its high prevalence and biodiversity, it is necessary to differentiate between the strains based on their hop tolerance. Forcing tests are often conducted for different types of beer, which can vary in cereal base, fermentation type, ethanol- and hop content. These conventional tests are considered to be the safest way to determine the ability of a given strain to cause beer-spoilage, but they are very time consuming and costly. Since NADH 2 is used as cofactor by many cellular dehydrogenases, this study used the reduced form of intracellular nicotinamide adenine dinucleotide (NADH 2 ) as an indicator for microbial metabolic activity and thus ability to spoil beer, in order to reduce the time required to conduct the forcing tests for beer production. Beer-spoiling L. brevis strains were detected among other strains in beer samples within two days (of sampling).

Amplification Facilitators and Pre-Processing Methods for PCR Detection of Strictly Anaerobic Beer-Spoilage Bacteria of the Class Clostridia in Brewery Samples

The aim of this study was to evaluate easy pre-PCR processing procedures to allow rapid and reliable detection of strictly anaerobic beer-spoilage bacteria throughout the brewing process by end-point and real-time PCR techniques. The efficiencies of the new procedures were evaluated using spiked brewery samples and specific PCRs for the target group bacteria. We found for the first time that the inclusion of 0.25% (w/v) bovine serum albumin (BSA) or 0.5% (w/v) polyvinyl pyrrolidone (PVP) in the end-point PCR mixture reduces the inhibiting effect of brewery sample extracts (3–10%, v/v) on PCR. Membrane filtration with a PVP or a sodium tri-polyphosphate-EDTA wash, and cross-flow filtration were the most promising new methods to reduce inhibitors from beer samples before cell lysis. Together with BSA, they allowed the analysis of 10% (v/v) of crude extracts instead of <3% (v/v). Moreover, we developed a one-hour procedure to prepare target DNA from process samples containing brewer's yeast. It involved removal of inhibitors by a two-step centrifugation followed by physical disruption of cells. The detection limit of the procedure was 101-103 CFU/mL. The developed procedures help to reduce the risk of partial or complete PCR failure due to inhibition and target DNA losses, with minimal sample handling.

Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria

The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population.

Group-specific PCR-RFLP and real-time PCR methods for detection and tentative discrimination of strictly anaerobic beer-spoilage bacteria of the class Clostridia

The strictly anaerobic brewery contaminants of the genera Pectinatus, Megasphaera, Selenomonas and Zymophilus in the class Clostridia constitute an important group of spoilage bacteria of unpasteurised, packaged beers. The aim of this study was to develop and evaluate group-specific PCR methods to detect and differentiate these bacteria in beer. A group-specific primer pair targeting a 342-bp variable region of the 16S rRNA gene was designed and evaluated in end-point PCR with gel electrophoresis and in real-time PCR with SYBR Green I dye. Significant cross-reactions with DNAs from any of the forty-two brewery-related, non-target microbes or from real brewery samples were not detected in either PCR system. The group-specific end-point and real-time PCR products could be differentiated according to species/genus and spoilage potential using restriction fragment length polymorphism ( KpnI, XmnI, BssHII, ScaI) and melting point curve analysis, respectively. In combination with a rapid DNA extraction method, the PCR reactions detected ca 10 0–10 3 CFU per 25 ml of beer depending on the strain and on the PCR system. The end-point and real-time PCR analysis took 6–7 h and 2–3 h, respectively. Pre-PCR enrichment of beer samples for 1–3 days ensured the detection of even a single cultivable cell. The PCR and cultivation results of real brewery samples were mostly congruent but the PCR methods were occasionally more sensitive. The PCR methods developed allow the detection of all the nine beer-spoilage Pectinatus, Megasphaera, Selenomonas and Zymophilus species in a single reaction and their differentiation below group level and reduce the analysis time for testing of their presence in beer samples by 1–2 days. The methods can be applied for brewery routine quality control and for studying occurrence, diversity and numbers of the strictly anaerobic beer spoilers in the brewing process.

Identification of novelhorA-harbouring bacteria capable of spoiling beer

An ATP-binding cassette (ABC) multi-drug resistance (MDR) gene was found in 4 Gram-positive bacterial isolates of environmental origin and found capable of spoiling beer. The bacteria isolated were Bacillus cereus, Bacillus licheniformis, Paenibacillus humicus, and Staphylococcus epidermidis; all of which were previously unappreciated as beer-spoilage bacteria. The MDR gene found in these bacteria has less than 37% similarity to known ABC MDR proteins described for Bacillus and Staphylococcus, and this is the first finding of an ABC MDR gene in the genus Paenibacillus. The sequenced region of the gene was translated and compared phylogenetically with the closest GenBank matches of the respective species and the closest GenBank matches overall. The ABC MDR proteins from these isolates were found to cluster among known sequences of HorA, sharing 99.5% identity within the sequenced region. In the beer-spoilage-associated genera Lactobacillus and Pediococcus, the presence of the MDR gene horA correlates with the ability to grow in beer. As the unique horA-harbouring isolates described here are capable of growing in beer, it is likely that the presence of the horA gene likewise confers hop resistance to these organisms.

Application of Multiplex PCR to the Detection of Beer-Spoilage Bacteria

Multiplex PCR methods were developed to detect 12 species of beer-spoilage bacteria that belong to the genera Lactobacillus, Pectinatus, Pediococcus, and Megasphaera. For microbiological quality control in breweries, we divided these species into three groups and evaluated a multiplex PCR method to simultaneously detect beer-spoilage species belonging to each group. Subsequently, the primer designs and blending ratios for each multiplex PCR analysis were optimized to satisfy the performance required for practical applications by breweries. As a result, each PCR method showed sufficient specificity to the target species and exhibited identical sensitivity with corresponding species-specific simplex PCR methods. In addition, none of the three multiplex methods showed a false-positive reaction to the nonspoilage bacterial strains frequently detected in breweries. Furthermore, artificial positive control DNAs were designed from λ-phage genomic DNA sequences to assure the performance of the PCR tests. Consequently, only one PCR test tube was required to ensure that all primer pairs in a multiplex PCR method were fully functional, instead of using multiple test tubes for separate confirmation of the respective primer pairs. Taken together, these multiplex PCR analyses enabled us to reliably and comprehensively detect beer-spoilage bacteria more easily and inexpensively.

Monitoring Spoilage Bacteria and Wild Yeasts in Eastern Chinese Breweries

Based on 100 isolated strains of beer-spoilage bacteria and 22 wild yeast strains, this article investigates the contamination degree assessment and quality control procedures of seven breweries in eastern China. Samples were collected from five main points in each brewery: brewing water and wort; fermented beer; seed yeasts; equipment, air, and surroundings; and finished beer. All of the isolates were identified to the genus or species level by comparing their 16s rDNA, and species of wild yeast were identified as non-Saccharomyces and Saccharomyces. Determination of microorganism growth in degassed commercial lager beer indicated that approx. 80% of the bacteria had the ability to contaminate beer. This study of the occurrence of contamination in eastern Chinese breweries indicates that monitoring spoilage microorganisms is important for enhancing the sanitation quality control procedures in these plants.

HorA-Specific Real-Time PCR for Detection of Beer-Spoilage Lactic Acid Bacteria

Beer-spoilage bacteria have long been a problem for brewers. Among the most problematic beer spoilers are several species of the Gram-positive genera Lactobacillus and Pediococcus. Current methods of detecting and identifying these organisms are time-consuming and do not differentiate between bacteria capable of spoiling beer and benign bacteria. The horA-specific real-time polymerase chain reaction (rPCR) described here identifies beer-spoilage organisms based not on their identity, but on the presence of a gene that we show to be highly correlated with the ability of an organism to grow in beer. The horA hop-resistance gene has been shown to be associated with beer spoilage by isolates from four Lactobacillus spp. and one Pediococcus sp. We document the presence of the horA gene in one additional genus and 11 additional species, with many of these bacteria commonly found as beer spoilers. The use of horA-specific rPCR allows for a substantial reduction in the time required for detection of potential beer spoilage bacteria and efficiently discriminates between those organisms that have the horA gene (highly likely to spoil beer) and those organisms that do not have the gene (much less likely to spoil beer).

Identification of Beer-Spoilage Microorganisms Using the Loop-Mediated Isothermal Amplification Method

A new method for identifying beer-spoilage microorganisms was developed using a unique nucleic acid amplification method. Loop-mediated isothermal amplification (LAMP), developed by Eiken Chemical Co., Ltd., reacts under isothermal conditions and produces large amounts of DNA. The LAMP method requires a set of four specifically designed primers and a DNA polymerase with strand-displacement activity. The amplification products are stem-loop DNA structures with several inverted repeats of the target and cauliflower-like structures with multiple loops. After the LAMP reaction, white precipitates identified as magnesium pyrophosphate are found in the reaction mixture, which confirms that DNA was amplified. We designed primers specific to four representative beer-spoilage bacteria, optimized the conditions for LAMP, and developed an identification kit containing all four primers in one reaction mixture. The kit was used to identify isolated colonies. The kit is simple, specific, cost-effective, and applicable for practical use.

Application of Rep-PCR fingerprint in rapid identification of beer-spoilage bacteria

The application potential of rep-PCR in typing beer-spoilage isolates was studied. The effects of different factors, including DNA templates and primers, on the quality and reproducibility of fingerprints were investigated. The CATB protocol was shown to be the feasible method for DNA extraction. Primers BOXA1R and (GTG) 5 were used in rep-PCR, and the PCR products were sequenced to identify strains isolated from two breweries. Rep-PCR fingerprint profiles were obtained using GelCompar II software. Cluster analysis showed that the isolates belonging to Lactobacillus brevis, L. buchneri, L. casei/paracasei, and L. plantarum are divided into 2 or 3 subgroups. In addition, the two rep-PCR fingerprint profiles complemented each other in typing these isolates. By combining the similarity coefficient cut-off (SCC) of species, 9 unknown isolates were rapidly identified using both fingerprint databases. The results indicate that rep-PCR is a simple, reliable, and promising method for the rapid identification of beer-spoilage bacteria.

Characterization of the brewery spoilage bacterium Obesumbacterium proteus by automated ribotyping and development of PCR methods for its biotype 1

To study the ability of automated ribotyping to characterize Obesumbacterium proteus and Hafnia alvei, to design primers and to evaluate standard end-point and real-time PCR for the detection of O. proteus biotype 1 in beer and in brewers's yeast-containing samples. Automated ribotyping was carried out using the standard method with EcoRI and PvuII. The digestions with both enzymes clearly differentiated O. proteus biotypes 1 and 2 and H. alvei. PCR primers were designed according to the 16S rRNA gene sequence of the O. proteus type strain. Two primer sets (Obs137-Obs558 and Obs137-Obs617) detected O. proteus biotype 1 and H. alvei but not O. proteus biotype 2 or other tested beer spoilage bacteria (40 species) in the end-point and real-time PCR, indicating their high specificity. The detection limit for O. proteus was 160-1600 CFU 100 ml(-1) beer in the end-point PCR reactions and < or =160 CFU 100 ml(-1) beer in the real-time PCR reactions. More cells (from 16 to 3200) were needed for detection in the presence of brewer's yeast cells. Automated ribotyping is a useful tool to characterize and identify O. proteus and H. alvei isolates. The designed primers are suitable for the rapid detection of O. proteus biotype 1 and H. alvei in brewery samples by PCR. Automated ribotyping and PCR could improve microbiological quality control in breweries by facilitating the detection, identification and tracing of spoilage bacteria.

The flagellin gene and protein from the brewing spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis

Flagellin genes from the anaerobic Gram-negative beer-spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were sequenced and the flagellin proteins initially characterized. Protein microsequencing led to the design of two degenerate PCR primers that allowed the P. cerevisiiphilus flagellin gene to be partially sequenced. A combination of PCR and Bubble PCR was then used to sequence the flagellin genes of three isolates from each species. Cloning and gene expression, followed by immunoblotting, confirmed the gene identities as flagellin. Analysis of the gene sequences revealed proteins similar to other bacterial flagellins, including lengths of 446 or 448 amino acids, putative sigma 28 promoters, and a termination loop. Antibody binding studies with isolated flagella correlated with gene sequence comparisons, with both indicating that the P. cerevisiiphilus isolates studied are very similar but that the P. frisingensis isolates show greater variation. Purified flagellins were found to be glycosylated, probably through an O linkage. Phylogenetic analysis revealed greater diversity within the flagellin sequences than within the 16S rRNA genes. Despite the Gram-negative morphology of Pectinatus, this genus proved most closely related to Gram-positive Firmicutes.

Study on ATP Production of Lactic Acid Bacteria in Beer and Development of a Rapid Pre-Screening Method for Beer-Spoilage Bacteria

Three beer-spoilage strains of lactic acid bacteria (LAB), Lactobacillus brevis ABBC45, L. lindneri DSM 20690T and L. para-collinoides DSM 15502T, exhibited strong ATP-yielding ability in beer. To investigate energy sources, these beer-spoilage strains were inoculated into beer. After the growth of the strains in beer, utilized components were determined by high performance liquid chromatography (HPLC). As a result, it was shown that citrate, pyruvate, malate and arginine were consumed by beer-spoilage LAB strains examined in this study. The four components induced considerable ATP production even in the presence of hop compounds, accounting for the ATP-yielding ability of the beer-spoilage LAB strains observed in beer. We have further examined the ATP-yielding ability of other strains of bacteria in beer. Beer-spoilage bacteria, including Pectinatus frisingensis and P. cerevisiiphilus, showed strong ATP-yielding abilities, whereas species frequently isolated from brewery environments exhibited low ATP-yielding abilities. Although some of the nonspoilage LAB strains produced substantial amount of ATP in beer, the measurement of ATP-yielding ability was considered to be useful as a rapid pre-screening method for potential beer-spoilage bacteria isolated from brewery environments.