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.     

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.     

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).     

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。     

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.     

The impact of different hop compounds on the growth of selected beer spoilage bacteria in beer

Beer spoiling lactic acid bacteria are a major reason for quality complaints in breweries around the world. Spoilage by a variety of these bacteria can result in haze, sediment, slime, off-flavours and acidity. As these bacteria occur frequently in the brewing environment, using certain hop products that inhibit the growth of these spoilers could be a solution to prevent problems. To investigate the impact of seven different hop compounds (α-acids, iso-α-acids, tetrahydro-iso-α-acids, rho-iso-α-acids, xanthohumol, iso-xanthohumol and humulinones) on the growth of six major beer spoilage bacteria (Lactobacillus brevis. L. backi, L. coryniformis, L. lindneri, L. buchneri, Pediococcus damnosous), two concentrations (10 and 25 mg/L) of each hop substance were added to unhopped beer. The potential growth of the spoilage bacteria was investigated over 56 consecutive days. A comparison of the results shows a strong inhibition of growth of all spoilage bacteria at 25 mg/L of tetrahydro-iso-α-acids closely followed by α-acids as the second most inhibitory substance. The results showed a high resistance of L. brevis to all hop compounds as well as an inhibition of L. coryniformis and L. buchneri at low concentrations of most hop components. In comparison with the control sample, L. lindneri showed increased growth in the presence of some hop compounds (rho-iso-α-acids, xanthohumol, iso-xanthohumol, humulinones). © 2020 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

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

Beer 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.     

EMA-PCR法快速检测啤酒中腐败短乳杆菌

将叠氮溴乙锭(ethidium bromide monoazide,EMA)与聚合酶链式反应(polymerase chain reaction,PCR)技术相结合,以酒花耐受基因hor C为靶基因,用啤酒腐败短乳杆菌基因组DNA作为模板进行扩增。结果发现,在前处理过程中加入EMA,当终质量浓度小于20μg/m L时,对活的短乳杆菌中靶基因的扩增没有明显抑制作用;而当EMA终质量浓度为1.0μg/m L时可有效抑制10~5 CFU/m L短乳杆菌死细胞的扩增。本实验建立的EMA-PCR检测方法的灵敏度为10~4活细胞/m L酒液样品。验证实验结果表明,在13株乳酸菌中,建立的hor C特异性EMA-PCR能有效检测到其中的全部5株啤酒污染菌,同时可区分这5株菌的活/死细胞混合体系,降低检测过程中的假阳性。     

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.     

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