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 20690^T and L. para‐collinoides DSM 15502^T, 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.     

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.     

Comparison of Enrichment Media for Routine Detection of Beer Spoiling Lactic Acid Bacteria and Development of Trouble‐shooting Medium for Lactobacillus backi

The beer spoiling lactic acid bacteria (LAB) are known to have a substantial financial impact in the brewing industry and their rapid detection is essential. Thus more effective media for the cultivation of LAB in both routine quality control and special trouble‐shooting situations are needed. In this study, different media were tested for the routine detection of LAB at a commercial brewery. The results showed that the use of an enzyme controlled glucose delivery system, in combination with beer‐MRS medium, can significantly decrease the total analysis time. For more effective trouble‐shooting in contamination incidents a trouble‐shooting media, including the reducing agents L‐cysteine‐HCl and sodium bicarbonate, was developed. The presented medium was shown to improve the growth of beer spoiling L. backi and L. brevis, and is thereby suggested for faster detection of these strains at the breweries.     

Induction of Viable but Nonculturable State in Beer Spoilage Lactic Acid Bacteria

Strong beer spoilage strains Lactobacillus lindneri DSM 20692 and Lactobacillus paracollinoides JCM 11969^T were repeatedly subcultured in degassed beer and their culturability on MRS agar was examined. As a result, the two strains were found to show decreased culturability, suggesting that the prolonged contact with beer reduces the culturability of beer spoilage lactic acid bacteria (LAB). After 30 subcultures in degassed beer, both strains were subjected to sublethal heat treatment. As a consequence, L. lindneri DSM 20692 and L. paracollinoides JCM 11969^T were no longer detectable on MRS agar despite the presence of 460 viable cells, indicating that the viable but nonculturable (VNC) states were induced for both strains. Problematically, the heat treated VNC strains were shown to exhibit beer spoilage ability, suggesting that spoilage incidents can occur without detection by culture media. It was also shown that, once acquired, the VNC states are stably maintained in beer without further heat treatment. These results suggest the possibility that beer spoilage LAB strains remain hidden in pitching yeast and work‐in‐process products without detection. Furthermore L. lindneri DSM 20692 and L. paracollinoides JCM 11969^T in VNC states were successfully stored at ?80°C with 10% dimethyl‐sulfoxide as a cryoprotectant and reconstituted in degassed beer without losing VNC characteristics. Taken together, these findings show that valuable bioresources can be acquired from culturable beer spoilage LAB strains and maintained for long‐term storage as frozen culture stocks.     

A Review of Hop Resistance in Beer Spoilage Lactic Acid Bacteria

Hop bitter acids play a major role in enhancing the microbiological stability of beer. However, beer spoilage lactic acid bacteria (LAB) are able to grow in beer by exhibiting strong hop resistance. Recently two hop resistance genes, horA and horC, have been identified in beer spoilage Lactobacillus brevis ABBC45. The horA gene was shown to encode an ATP dependent multidrug transporter that extrudes hop bitter acids out of bacterial cells. In contrast, the product of the horC gene confers hop resistance by presumably acting as a proton motive force (PMF)‐dependent multidrug transporter. Strikingly, the homologs of horA and horC genes were found to be widely and almost exclusively distributed in various species of beer spoilage LAB strains, indicating these two hop resistance genes are excellent species‐independent genetic markers for differentiating the beer spoilage ability of LAB. Furthermore the nucleotide sequence analysis of horA and horC homologs revealed that both genes are essentially identical among distinct beer spoilage species, indicating horA and horC have been acquired by beer spoilage LAB through horizontal gene transfer. Taken collectively, these insights provide a basis for applying horA and horC to the species‐independent determination of beer spoilage LAB, including yet uncharacterized species. In addition to the hop resistance mechanisms mediated by multidrug transporters, proton translocating ATPase and the ATP production system were shown to contribute to the hop resistance mechanisms in beer spoilage LAB by generating PMF and ATP that are necessary for survival in beer.     

Enhancing the Microbiological Stability of Malt and Beer — A Review

While beer provides a very stable microbiological environment, a few niche microorganisms are capable of growth in malt, wort and beer. Growth of mycotoxin‐producing fungi during malting, production of off‐flavours and development of turbidity in the packaged product due to the growth and metabolic activity of wild yeasts, certain lactic acid bacteria (LAB) and anaerobic Gram negative bacteria, impact negatively on beer quality. It follows that any means by which microbial contamination can be reduced or controlled would be of great economic interest to the brewing industry and would serve the public interest. There has been an increasing effort to develop novel approaches to minimal processing, such as the exploitation of inhibitory components natural to raw materials, to enhance the microbiological stability of beer. LAB species, which occur as part of the natural barley microbiota, persist during malting and mashing, and can play a positive role in the beer‐manufacturing process by their contribution to wort bioacidification or the elimination of undesirable microorganisms. Other naturally occurring components of beer that have been valued for their preservative properties are hop compounds. It may be possible to enhance the antimicrobial activities of these compounds during brewing. Some yeast strains produce and excrete extracellular toxins called zymocins, which are lethal to sensitive yeast strains. Yeast strains resistant to zymocins have been constructed. Imparting zymocinogenic activity to brewing yeast would offer a defence against wild yeasts in the brewery. Thus, the antimicrobial properties of naturally occurring components of raw materials can be exploited to enhance the microbial stability of beer.     

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

Lactic acid bacteria are the most frequently encountered beer‐spoilage 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.     

Microbiological and Fermentative Properties of Baker's Yeast Starter Used in Breadmaking

This study assessed the levels of microbial contaminants in liquid, compressed and dry commercial baker's yeasts used as starters in breadmaking. Eumycetes, Enterobacteriaceae, total and fecal coliforms, Bacillus spp., and lactic acid bacteria (LAB), in particular enterococci, were quantified. Results obtained in this study highlighted that baker's yeast could represent a potential vehicle of spoilage and undesirable microorganisms into the baking environment, even if these do not influence the leavening activity in the dough, as ascertained by rheofermentometer analysis. Different microbial groups, such as spore‐forming bacteria and moulds, were found in baker's yeast starters. Moreover, different species of LAB, which are considered the main contaminants in large‐scale yeast fermentations, were isolated and identified by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNA sequencing. The most recurrent species were Lactobacillus plantarum, Enterococcus faecalis, and Enterococcus durans, isolated from both compressed and dry starters, whereas strains belonging to Leuconostoc and Pediococcus genera were found only in dry ones. Nested‐Polymerase Chain Reaction (Nested‐PCR) and Randomly Amplified Polymorphic DNA–PCR (RAPD‐PCR) were also used to highlight the biodiversity of the different commercial yeast strains, and to ascertain the culture purity.     

啤酒污染乳酸菌PCR引物的设计

根据细菌16s rDNA序列的特点，通过对啤酒污染菌16s rDNA序列进行分析，设计合成了针对啤酒污染乳酸菌的引物，在16srDNA基因水平证明了该引物对乳酸菌的特异性，该引物的特异性是PCR检测技术在啤酒厂推广应用的前提，同时反映了16s rDNA在微生物鉴定中所起的重要作用。     

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     

Craft Beer Microflora Identification Before and After a Cleaning Process

Although beer is a relatively safe product, growth of spoilage microorganisms can cause economic damage. The most effective way to prevent the spoilage of beer is to control contamination with adequate cleaning and sanitation. The aim of this work was to evaluate the microbial flora present both before and after a specific hygiene process was implemented during a brewpub's craft beer production. Various selective and differential culture media methods for the enumeration of beer‐spoilage species are available, but they are time consuming compared to modern techniques. The utilisation of accurate molecular methods, added to the routine microbiological analyses, allowed for the fast identification of common environmental contaminants of beer. A poor sanitation plan by the brewer resulted in microbial contamination of the brewpub. This result demonstrates the importance of good sanitation to avoid the presence of undesirable microorganisms in the product. A cleaning‐in‐place (CIP) method, in operation in many small breweries, could be utilised to prevent the occurrence of such brewery and beer microbial contaminants. In small breweries, the simplest CIP units consist of a single tank and a portable pump. This method requires no additional equipment besides a spraying ball, which is usually included in the design of most tanks.     

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     

THE USE OF CONTROLLED LEVELS OF ACTIDIONE FOR BREWING AND NON-BREWING YEAST STRAIN DIFFERENTIATION

The variability of results using actidione media for the identification of beer spoilage bacteria has been shown to be due to the heat sensitivity of this reagent. Critical evaluation shows that with a particular brewery yeast, media containing 0·06 p.p.m. allow the growth of a single brewing strain but inhibit others. This permits the rapid classification of yeast strains in this pitching yeast. In a further brewery, media containing 0·16 p.p.m. differentiate brewing yeast strains from beer spoilage yeasts and this permits the quantitative assessment of these for process control at all stages of brewing. In a particular instance where a culture yeast strain is not inhibited at 0·16 p.p.m. but is p-aminobenzoic acid dependent, a differential medium incorporating this low level of actidione and omitting the vitamin allows the identification of beer spoilage yeasts in process control. These results illustrate the potential of this approach for microbiological control in specific brewery problems.     

Microbiological Analyses of Dry and Slurry Yeasts for Brewing

Classical microbiological methods in association with molecular methods (DNA amplification, Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) were used. These methods, developed to rapidly analyze microbial communities on the basis of sequence‐specific separation of DNA amplicons, allowed the detection of DNA differences in the amplicons tested and the identification of the strains analyzed by the comparison of unknown sequences with sequences of known species. TGGE allowed the comparison of the different Saccharomyces cerevisiae strains used in brewing while DGGE allowed the identification of lactic acid bacteria (LAB) in beer. These methods are a reliable tool for fast comparison of strains of Saccharomyces cerevisiae collected from different craft breweries where they were used as starters to check the presence of possible yeast contaminants in the brewing process and for rapid LAB identification.     

Pediococcus damnosus strains isolated from a brewery environment carry the horA gene

Beer is recognized as a safe beverage, owing to its excellent microbiological stability provided by its components, especially iso‐α‐acids from hop and ethanol which have antimicrobial activity. Despite these unfavourable conditions for bacteria, some lactic acid bacteria (LAB) can cause beer spoilage. Resistance to hop compounds is caused, in part, by the product of genes like horA . Understanding how LAB adapts to hop compounds as well as quick detection of these microorganisms is necessary to ensure high‐quality beverages produced by the brewing industry. In this work, we searched for the presence of two main hop‐resistance genes, horA and ORF5, and determined the capacity of four strains of Pediococcus damnosus isolated from a brewery environment to grow in the presence of increasing concentrations of iso‐α‐acids. All strains were able to grow in increasing concentrations of iso‐α‐acids up to 150 μg mL⁻¹. This amount is 10 times greater than the concentration in average beer. Genetic amplification of genes associated with hop‐resistance, demonstrated the presence of horA , but not ORF5 in all tested strains. This communication represents the first report of the presence of horA gene in bacteria isolated from breweries in our country. Copyright © 2017 The Institute of Brewing & Distilling     

Sake and Beer Spoilage Lactic Acid Bacteria — A Review

Japanese rice wine, sake, is a traditional alcoholic beverage in Japan. Similar to the case with beer, sake is known to be microbiologically stable and most microorganisms fail to grow in sake. This is principally due to its high ethanol concentration that reaches approximately 20% (v/v) in undiluted sake products and 15% (v/v) in finished products. Despite the high level of ethanol content, spoilage incidents occasionally occur in sake, due to the presence of highly ethanol‐tolerant lactobacilli, known as hiochi‐bacteria. Hiochi‐bacteria are generally composed of two groups of lactobacilli, hiochi‐lactobacilli and true hiochi‐bacilli. The former group of lactobacilli is less ethanol‐and heat‐tolerant, and therefore rarely poses a problem to sake products. In contrast, the true hiochi‐bacilli exhibit extraordinarily high ethanol tolerance and cause spoilage incidents in sake, conferring acidity and off‐flavors, such as diacetyl, in spoiled products. From a taxonomic standpoint, the true hiochi‐bacilli mainly consist of two Lactobacillus species, L. fructivorans and L. homohiochi. The strains of true hiochi‐bacilli prefer sake‐like environments, and the presence of ethanol and mevalonic acid, in combination with low pH milieu, is essential or stimulatory for the growth of these bacteria. Interestingly, the type strain of L. fructivorans does not show such characteristics, suggesting the true hiochi‐bacilli are profoundly adapted to sake environments. Although beer spoilage lactic acid bacteria do not have close taxonomic relationships with true hiochi‐bacilli, there are striking similarities between these two groups of spoilage lactic acid bacteria. In this review, unique features of sake and beer spoilage lactic acid bacteria are discussed in comparative terms.     

Investigation of Microbial Communities of Chinese Sourdoughs Using Culture‐Dependent and DGGE Approaches

Sourdough is typically characterized by the complex microbial communities mainly comprising of yeasts and lactic acid bacteria (LAB). The objective of this study was to explore the microbiota of Chinese traditional sourdoughs collected from different areas of China using culture‐dependent and denaturing gradient gel electrophoresis (DGGE) methods. A total of 131 yeasts, 2 molds, and 106 LAB strains were isolated and identified. Based on the culture‐dependent analysis, the populations of yeasts and LAB were at the level of 10⁵ to 10⁷ and 10⁶ to 10⁷ cfu/g, respectively. Similarly, the results of RT‐qPCR showed that the values of total yeasts and LAB populations were in the range of 10⁶ to 10⁷ and 10⁷ to 10⁸ copies/g, respectively. Using culture‐dependent method, a total of 7 yeasts, 2 molds and 7 LAB species were identified. Results showed that Saccharomyces cerevisiae and Lactobacillus plantarum were the predominant species among the yeasts and LAB microflora. Similarly, using PCR‐DGGE approach, 7 yeasts, 1 mold and 9 LAB species were detected. The yeast, S. cerevisiae, represented the predominant, while the yeast Candida tropicalis represented the subdominant species of the yeast community. Among the LAB community, Lactobacillus sanfranciscensis was the predominant species, while Lactococcus qarvieae, Enterococcus faecium, Lactobacillus delbrueckii and Enterococcus cecorum were among the less dominant species.     

INHIBITION OF BEER-SPOILAGE LACTIC ACID BACTERIA BY NISIN

149 strains of bacteria, mostly brewery contaminants able to spoil wort or beer, and 12 brewing strains of yeast (8 ale and 4 lager strains) have been screened using a well-test assay for sensitivity to the food preservative, Nisin (E234), Nisin inhibited growth of 92% of the gram-positive strains, predominantly lactic acid bacteria of the genera Lactobacillus and Pediococcus. In contrast, all 32 gram-negative strains tested, except 3 Flavobacter strains, were Nisin-resistant; in addition none of the brewing yeasts showed Nisin-sensitivity. Therefore. Nisin has potential applications in preventing spoilage of worts or beers by lactic acid bacteria.     

Beer enemy number one: genetic diversity,physiology and biofilm formation of Lactobacillus brevis

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     

Improved Enrichment Cultivation of Beer Spoiling Lactic Acid Bacteria by Continuous Glucose Addition to the Culture

Lactic acid bacteria (LAB) are known as predominant beer spoilers. They cause turbidity, acidity, gas formation and off‐flavors in beer by formation of side metabolites. Beer spoiling LAB have a substantial financial impact in the brewing industry making their rapid detection and identification essential. Despite the developed rapid diagnostic methods, the bottleneck in detection remains the lengthy enrichment cultivation step. This paper describes the applicability of a novel glucose auto delivery system, EnBase?, for the improved enrichment cultivation of beer spoiling LAB in MRS medium. By means of the applied system, glucose is slowly released into the culture during growth, which results in faster enrichment. Growth of Lactobacillus brevis DSM 20054^T and several beer spoiling LAB was accelerated resulting in up to a 300% increase in the cell density after 48 h of cultivation compared to the commonly used MRS medium. A test of naturally contaminated beer samples indicated that the addition of glucose by means of EnBase allows faster detection of LAB in breweries.