Beer Spoilage Lactic Acid Bacteria Research Articles

Development of Culture-Independent Detection Method for Beer Spoilage Lactic Acid Bacteria

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.

Applications of the Third-Generation DNA Sequencing Technology to the Detection of Hop Tolerance Genes and Discrimination of Saccharomyces Yeast Strains

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 .

Study of Beer Spoilage Lactobacillus nagelii Harboring Hop Resistance Gene horA

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.

Preceding Subculture Conditions Affect Growth Characteristics of Beer Spoilage Lactic Acid Bacteria in Quality Control Culture Media: Comparative Study on Hard-to-Culture and Culturable Secundilactobacillus (Lactobacillus) paracollinoides Strains

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.

Beer spoilage lactic acid bacteria from craft brewery microbiota: Microbiological quality and food safety

Craft beer is more susceptible to microbial spoilage because it does not have a pasteurization or filtration process, with lactic acid bacteria (LAB) being the most common beer spoilage microorganism. The aim of this study was to isolate LAB in a craft brewery and their characterization from a food safety and microbiological quality perspective, with a special focus on their abilities to produce biogenic amines (BA) and spoil the beer. The results of 60 monitored points inside the craft brewery showed that LAB associated with the craft brewing processes belonged to Lactobacillus, Pediococcus, and Leuconostoc genera, and most of them were detected in the filling area, which can lead to secondary contamination. Two isolates of L. brevis showed the most significant beer spoilage ability because they could grow in more acidic conditions, at a higher hop and alcohol content, and they displayed horA, horC, and hitA genes, which spoiled the vast majority of the tested beers. In addition, the aforementioned L. brevis isolates showed the highest BA production.

Malt and wort bio-acidification by Pediococcus acidilactici HW01 as starter culture

In a previous study, indigenous bacteriocin-producing Pediococcus acidilactici HW01 was isolated from malt. The bacterium was chosen as it is properly adapted to the malting conditions, and it showed a strong antagonistic effect against beer-spoilage lactic acid bacteria (LAB) in the previous study. Strain HW01 was added to the malting process for bio-acidification and bio-preservation of LAB. Changes in the microorganisms, enzymes, pH, total soluble nitrogen, free amino nitrogen, colour, filtration time and viscosity of wort were compared with the control. The LAB starter culture improved several characteristics of malt modification, such as microbiological stability, viscosity and filtration time. Especially, the starter culture protected the malt from the beer-spoilage bacteria of the genus Pseudomonas, by producing several acids and antimicrobial substances. The potential of using HW01 strains for bio-acidification and the bio-preservation of LAB are discussed.

PCR Analysis Methods for Detection and Identification of Beer-Spoilage Lactic Acid Bacteria.

Polymerase chain reaction (PCR) analysis enables rapid and accurate detection of beer-spoilage lactic acid bacteria (LAB). Hop resistance genes, horA and horC, are utilized as genetic markers to determine the spoilage ability of LAB strains. PCR analysis of horA and horC, combined with multiplex PCR methods of 12 beer-spoilage species, enables simultaneous and comprehensive detection easily and inexpensively.

Hop bitter acids inhibit carbohydrate metabolism, enhance biogenic amine metabolism and alter L‐malic acid, glutamic acid and arginine metabolism of Lactobacillus brevis 49

SummaryBeer contains only limited amounts of readily fermentable carbohydrates and amino acids. Beer spoilage lactic acid bacteria have to come up with metabolic strategies in order to deal with selective nutrient content. The research was performed to investigate the influence of iso‐α‐acids on the metabolism of organic acids, biogenic amines (BAs), off‐flavour compounds, carbohydrates and amino acids of Lactobacillus brevis 49. Only glyoxylic acid and ethyl formate in de man, rogosa, sharpe broth was consumed, and multiple organic acids, BAs and off‐flavour compounds were produced. By supplementing a series of concentrations of hop iso‐α‐acids, consumption of L‐malic acid, glutamic acid and arginine and generation of BAs were found benefit for bacteria to develop hop‐resistance. In the metabolism of carbohydrates, glucose was preferred over maltose and maltriose, and hops significantly inhibited the utilisation of carbohydrates. The results provide comprehensive information of metabolites of L. brevis 49 under various concentrations of hop stress.

Detection of a Hop-Tolerance Gene horA Insertion Variant in Lactic Acid Bacteria That Results in a Truncated HorA Lacking the Walker B Motif Necessary for Transport Function

The hop-tolerance gene horA frequently found in beer-spoilage lactic acid bacteria (LAB) was investigated for sequence variability. Although the horA gene was found to have less sequence variability relative to the LAB hop-tolerance gene horC, a sequence insertion in horA in some isolates resulted in early truncation of HorA translation. This truncated HorA was found in LAB both capable and incapable of growth in beer. Protein modeling revealed that the truncated HorA may retain some capacity to bind and sequester hop iso-α-acids but lacks the transport function essential for moving hop compounds out of the cell. Sequence analysis of LAB plasmids that contain horA revealed a high level of conservation in all of the genes comprising the horA gene cassette. Assessing whether a LAB isolated in a brewery setting is capable of making a full-length protein with intact hop transport function or a truncated HorA requires redesign of commonly used horA polymerase chain reaction primers to target detection of horA both with and without the sequence insertion.

Isolation and characterization of bacteriocin-producing Pediococcus acidilactici HW01 from malt and its potential to control beer spoilage lactic acid bacteria

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.

Effect of bacteriocin-producing Pediococcus acidilactici K10 on beer fermentation

Beer is generally considered to be a beverage that has high microbiological stability. However, some undesirable lactic acid bacteria (LAB) can grow in beer and consequently spoil this beverage. In this study, bacteriocin-producing Pediococcus acidilactici K10 was used as a means of bio-acidifying the mash and reducing the spoilage LAB content of the beer. The K10 strain had antimicrobial activity against two beer spoilage LAB strains in wort and did not grow in a beer environment. The K10 strain was inoculated before the mashing step. The effect of K10 as a starter culture was investigated and compared with a control. As a result, filtration time was shortened by 17 min, alcohol content was increased by 137%, foam stability was increased by 156%, bitterness was increased by two bitterness units and there was a significant difference (p < 0.05) in aromatic and sour odour. The feasibility of using bacteriocin-producing LAB strain in beer brewing is envisaged. Copyright © 2016 The Institute of Brewing & Distilling

Detection of culturable and viable but non-culturable cells of beer spoilage lactic acid bacteria by combined use of propidium monoazide andhorA-specific polymerase chain reaction

Current methods of detecting beer spoilage lactic acid bacteria (LAB) are time-consuming and do not differentiate between viable and non-viable bacteria. In this study, a combination of the conventional polymerase chain reaction (PCR) and propidium monoazide (PMA) pretreatment has been described to circumvent the disadvantages. The horA-specific PMA-PCR described here identifies beer spoilage LAB based not on their identity, but on the presence of a gene that is shown to be highly correlated with the ability of LAB to grow in beer. The results suggest that the use of 20 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable, but putatively non-culturable (VPNC) Lactobacillus acetotolerans. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. acetotolerans cells was 1.5 µg/mL. The detection limit of established PMA-PCR assays was found to be 100 VPNC cells/reaction for the horA gene. Furthermore, the horA-specific PMA-PCR assays were subjected to 18 reference strains, representing 100% specificity with no false positive amplification observed. In conclusion, the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for the detection of potential beer spoilage LAB and efficiently discriminates between live and dead cells. Copyright © 2016 The Institute of Brewing & Distilling

The Occurrence of Beer Spoilage Lactic Acid Bacteria in Craft Beer Production.

Beer is one of the world's most ancient and widely consumed fermented alcoholic beverages produced with water, malted cereal grains (generally barley and wheat), hops, and yeast. Beer is considered an unfavorable substrate of growth for many microorganisms, however, there are a limited number of bacteria and yeasts, which are capable of growth and may spoil beer especially if it is not pasteurized or sterile-filtered as craft beer. The aim of this research study was to track beer spoilage lactic acid bacteria (LAB) inside a brewery and during the craft beer production process. To that end, indoor air and work surface samples, collected in the brewery under study, together with commercial active dry yeasts, exhausted yeasts, yeast pellet (obtained after mature beer centrifugation), and spoiled beers were analyzed through culture-dependent methods and PCR-DGGE in order to identify the contaminant LAB species and the source of contamination. Lactobacillus brevis was detected in a spoiled beer and in a commercial active dry yeast. Other LAB species and bacteria ascribed to Staphylococcus sp., Enterobaceriaceae, and Acetobacter sp. were found in the brewery. In conclusion, the PCR-DGGE technique coupled with the culture-dependent method was found to be a useful tool for identifying the beer spoilage bacteria and the source of contamination. The analyses carried out on raw materials, by-products, final products, and the brewery were useful for implementing a sanitization plan to be adopted in the production plant.

Metabolic strategies of beer spoilage lactic acid bacteria in beer

Beer contains only limited amounts of readily fermentable carbohydrates and amino acids. Beer spoilage lactic acid bacteria (LAB) have to come up with metabolic strategies in order to deal with selective nutrient content, high energy demand of hop tolerance mechanisms and a low pH. The metabolism of 26 LAB strains of 6 species and varying spoilage potential was investigated in order to define and compare their metabolic capabilities using multivariate statistics and outline possible metabolic strategies. Metabolic capabilities of beer spoilage LAB regarding carbohydrate and amino acids did not correlate with spoilage potential, but with fermentation type (heterofermentative/homofermentative) and species. A shift to mixed acid fermentation by homofermentative (hof) Pediococcus claussenii and Lactobacillus backii was observed as a specific feature of their growth in beer. For heterofermentative (hef) LAB a mostly versatile carbohydrate metabolism could be demonstrated, supplementing the known relevance of organic acids for their growth in beer. For hef LAB a distinct amino acid metabolism, resulting in biogenic amine production, was observed, presumably contributing to energy supply and pH homeostasis.

Role of Plasmids in Lactobacillus brevis BSO 464 Hop Tolerance and Beer Spoilage

Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate.

An improved plate culture procedure for the rapid detection of beer-spoilage lactic acid bacteria

Lactic acid bacteria (LAB) are the most frequently encountered beer-spoilage bacteria, and they can render beer undrinkable owing to the production of lactic acid, diacetyl and turbidity. Three beer-spoilage strains, 2011–6, 2011–8 and 2011–11, were isolated from finished beers. Based on the 16S rRNA sequence analysis, these three isolates were identified as Lactobacillus acetotolerans. Only the horA homologue was detected in these strains, while the horC homologue was not detected. In addition, an improved plate culture method for the rapid detection of beer-spoilage LAB by the addition of catalase was evaluated. Supplementation with catalase enhanced the growth and colony sizes of the spoilage LAB investigated. These beer-spoilage bacteria, including some slowly growing strains, were detected within five days of incubation using the modified method. Taken together, the modified procedure could be a rapid countermeasure against beer-spoilage LAB, and it compared favourably with the conventional plate culture method. Copyright © 2014 The Institute of Brewing & Distilling

Beer Spoilage Lactic Acid Bacteria -A New Hypothesis on their Origin and Evolution-

[in Japanese]

Combination of an Immunomagnetic Separation Method and a Chromogenic Oligonucleotide Array for the Detection of Beer-Spoilage Lactic Acid Bacteria

For the brewing industry, the possibility of beer spoilage by lactic acid bacteria (LAB) or by wild yeasts is always a concern. Contamination by certain LAB, such as Lactobacillus brevis, L. casei, L. plantarum, Pediococcus claussenii, P. damnosus, and P. inopinatus, may result in the deterioration of beer quality. Thus, monitoring of these spoilage organisms during the brewing process is critical to quality control. When concentrations of spoilage organisms in the brewing samples are low and, thus, difficult to detect, collection and concentration of these spoilage organisms prior to the application of diagnostic methods are important. Although the membrane filtration method can be used even if filters of the correct pore sizes are chosen, it is still possible that some LAB cells will go through the membrane filters. Another drawback of membrane filtration is membrane fouling, which restricts the beer volume to be filtered. In this study, polyclonal antibodies against beer-spoilage bacteria were prepared by rabbit immunization. The antibodies were then conjugated to magnetic beads for the capture and concentration of major beer-spoilage LAB. The captured cells were subsequently discriminated to species level by a commercially available oligonucleotide array. The combination of immunomagnetic separation with an oligonucleotide array allowed the simultaneous and sensitive detection of several major beer-spoilage LAB species in the brewing samples within 8 hr. This method is accurate and time-saving and can be used for routine monitoring and control of the spoilage organisms during the beer brewing process.

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.

Evaluation of Reverse-Transcription PCR Detection of 16S rRNA and Tuf mRNA for Viable/Dead Discrimination of Beer-Spoilage Lactic Acid Bacteria

The aim of this study was to evaluate the feasibility of using 16S rRNA and elongation factor (tuf) mRNA as targets for selective detection of viable beer-spoilage lactic acid bacteria (LAB) by reverse-transcription PCR (RT-PCR). One-step real-time RT-PCR was applied to monitor the target transcripts in Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Three methods were compared for total RNA extraction. To study the correlation between viability and RNA detection, log-phase cultures were inactivated using brewery disinfectant or heating. RT-PCR, denaturing agarose gel electrophoresis, and cultivation were performed before and at various times after the inactivation treatments. A method combining bead-beating with traditional RNA purification provided good RNA yield from LAB. The 16S rRNA and tuf mRNA targets were detected in inactivated cells by RT-PCR throughout a 21-day storage period. Cultivable cells could not be recovered at any postinactivation time. The 16S rRNA degraded with time, and its degradation appeared to be initially faster after disinfection than after heating. The overall degradation patterns between the strains were similar. Our results indicate that tuf mRNA and 16S rRNA can be stable in LAB cells inactivated under conditions they may encounter during the brewing process. Hence, the RT-PCR analysis of these target transcripts may not be a reliable technique for selective detection of viable LAB in brewery samples.