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.     

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.     

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.     

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.     

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     

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.     

Detection of culturable and viable but non‐culturable cells of beer spoilage lactic acid bacteria by combined use of propidium monoazide and horA‐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     

Differentiation of mixed lactic acid bacteria communities in beverage fermentations using targeted terminal restriction fragment length polymorphism

Lactic acid bacteria (LAB) are an important group of bacteria in beer and wine fermentations both as beneficial organisms and as spoilage agents. However, sensitive, rapid, culture-independent methods for identification and community analyses of LAB in mixed-culture fermentations are limited. We developed a terminal restriction fragment length polymorphism (TRFLP)-based assay for the detection and identification of lactic acid bacteria and Bacilli during wine, beer, and food fermentations. This technique can sensitively discriminate most species of Lactobacillales, and most genera of Bacillales, in mixed culture, as indicated by both bioinformatic predictions and empirical observations. This method was tested on a range of beer and wine fermentations containing mixed LAB communities, demonstrating the efficacy of this technique for discriminating LAB in mixed culture.     

啤酒有害菌的PCR检测和SYBR Green实时PCR定量

啤酒有害菌是一些能在啤酒中存活并使啤酒的外观和风味发生改变的细菌,对其进行快速检测和定量是啤酒生产急待解决的问题。我们从华润雪花啤酒（中国）有限公司各工厂提供的样品中分离到28株啤酒有害菌,16S rDNA序列的系统进化分析表明,其中26个菌株属于乳杆菌属（Lactobacillus spp．）、1个菌株为明串珠菌属（Leuconostoc spp．）,1个菌株为片球菌属（Pediococcu sp．）。根据酒花（hop）抗性基因horA、horB和horC的保守序列设计了扩增这3个基因的PCR引物,用这些引物对28株啤酒有害菌进行了常规PCR检测,检出率分别为89％、79％和75％,用hor A—horC双引物进行检测,检出率为100％。用SYBR Green实时定量PCR技术,以horA基因为靶序列,建立了对啤酒有害菌的细胞数进行快速定量的新方法,用该方法测定的污染啤酒样品中有害菌的浓度与平板培养法相近。     

Selection and identification of protective culture for controlling Staphylococcus aureus in fresh Domiati like cheese

Antibacterial activity of forty lactic acid bacteria (LAB) isolates toward Staphylococcus aureus was evaluated. The selected strains were then used as protective culture in artificial contaminated Domiati like cheese with S. aureus. The effect of using these strains on physicochemical properties and overall acceptability of fresh cheese was evaluated. Depending on its antibacterial activity, three strains of Lactobacillus rhamnosus 130RZFAAU, 131RZFAAU, and 190RZFAAU were selected for cheese making. No negative sensory properties were observed by the panelists when LAB strains were used as a single culture in the fresh cheese making. The application of these strains as protective culture in artificial contaminated cheesemaking process give a positive results. S. aureus was detected in cheese samples by culture method and propidium mono azide–quantitative polymerase chain reaction method. The results recommended that the strain L. rahmnosus 131RZFAUU that used in this study has antimicrobial activity against S. aureus and could be used as protective culture for improving the safety of Egyptian soft cheese.

Practical applications

Detection of pathogenic bacteria by classical tests can take several days. It would be useful to have a rapid detection protocol to screen for the presence of Staphylococcus aureus in milk and cheese. Application of real‐time PCR in cheese is sufficient in characterization the S. aureus communities in raw milk and follow the dynamics of the entire populations in cheese. Recently, some scientific publications have shown that the naturally cheese microflora can efficiently prevent the growth of pathogenic or spoilage microorganisms. The control of spoilage and pathogens bacteria has been traditionally done by chemical additives, but the application of promising protective cultures, especially for traditionally cheeses made from raw milk, is limited. This work present some protective culture selected for controlling S. aureus in soft cheese. This work confirm the PMA‐q PCR method for detection live cells of S. aureus in cheese rapidly.     

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.     

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.     

Production of biogenic amines by lactic acid bacteria and bifidobacteria isolated from dairy products and beer

The aim was to monitor production of eight biogenic amines (BAs) (histamine, tyramine (TYR), tryptamine, putrescine, cadaverine (CAD), phenylethylamine, spermine and spermidine) by selected 81 lactic acid bacteria (LAB) strains: Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Tetragenococcus and Bifidobacterium. The tested LAB and bifidobacteria were isolated from dairy products and beer. The decarboxylase activity of the micro‐organisms was studied in growth medium after cultivation. The activity was monitored by HPLC after the pre‐column derivatisation with dansylchloride. Fifty LAB showed decarboxylase activity. Thirty‐one strains produced low concentrations of CAD (≤10 mg L^?1). Almost 70% of beer isolates generated higher amounts of TYR (≤3000 mg L^?1). Most of the tested LAB demonstrated decarboxylase activity. The above micro‐organisms can contribute to the increase of content of BAs in dairy products or beer and thereby threaten food safety and health of consumers. Production of BAs even by the representatives of some probiotic strains (Bifidobacterium and Lactobacillus rhamnosus) was detected in this research. This study has also proved that contaminating LAB can act as sources of higher amounts of CAD and TYR in beer.     

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     

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.     

Anti-Helicobacter pylori activity of fermented milk with lactic acid bacteria

BACKGROUND: Ten strains of lactic acid bacteria (LAB) were investigated for their anti‐Helicobacter pylori effects. The bactericidal activity and organic acid content in spent culture supernatants (SCS) from fermented milk were measured. In addition, the exclusion effect of SCS against H. pylori infection of human gastric epithelial AGS cells was assayed. RESULTS: Three LAB strains, LY1, LY5 and IF22, showed better anti‐Helicobacter effects than the other strains. There were no significant differences in the bactericidal activity of LAB strains between original SCS, artificial SCS and SCS treated by heating or protease digestion. However, neutralised SCS lost this activity. These results suggest that the anti‐H. pylori activity of SCS may be related to the concentration of organic acids and the pH value but not to protein components. In the AGS cell culture test, both fermented LY5‐SCS and artificial LY5‐SCS significantly reduced H. pylori infection and urease activity (P < 0.05). CONCLUSION: In this study, in vitro methods were used to screen potential probiotics with anti‐H. pylori activity. This may provide an excellent and rapid system for studying probiotics in the functional food and dairy industries. Copyright © 2011 Society of Chemical Industry     

啤酒污染乳酸菌PCR引物的设计与验证

根据细菌16SrDNA序列的特点,对啤酒中常见污染乳酸菌16SrDNA序列进行分析,设计合成了针对啤酒污染乳酸菌的特征引物。并用该引物对从啤酒厂分离到的7种乳酸菌进行了检测,PCR结果表明该引物能够准确检测到啤酒中常见的乳酸菌。     

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.     

Detection of Beer‐spoilage Lactobacillus brevis strains 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 NADH₂ is used as cofactor by many cellular dehydrogenases, this study used the reduced form of intracellular nicotinamide adenine dinucleotide (NADH₂) 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).     

High‐Throughput Isolation of Bacteriocin‐Producing Lactic Acid Bacteria,with Potential Application in the Brewing Industry

Lactic acid bacteria (LAB) were isolated from malted cereals by means of a high‐throughput screening approach and investigated for antimicrobial activity against a range of beer‐spoiling bacteria. Putative bacteriocin‐producing strains were identified by 16S rRNA analysis and the inhibitory compounds were partially characterized. Following determination of the inhibitory spectra of the strains, an unspeciated Lactobacillus sp. UCC128, with inhibitory activity against a range of beer‐spoiling strains was subjected to further characterization. A bacteriocin was purified from this strain and analyzed by mass spectrometry to determine the weight of the protein. The result indicated that the bacteriocin was highly similar to pediocin AcH/PA‐1 from Pediococcus acidilactici. The bacteriocin‐producers identified in this study have the potential to be used in the brewing industry to enhance the microbiological stability of beer in conjunction with hurdles already in place in the brewing process.