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.

We have isolated a hop-sensitive variant of the beer spoilage bacterium Lactobacillus lindneri DSM 20692. The variant lost a plasmid carrying two contiguous open reading frames (ORF s) designated horB L and horC L that encode a putative regulator and multidrug transporter presumably belonging to the resistance-nodulation-cell division superfamily. The loss of hop resistance ability occurred with the loss of resistance to other drugs, including ethidium bromide, novobiocin, and cetyltrimethylammonium bromide. PCR and Southern blot analysis using 51 beer spoilage strains of various species of lactic acid bacteria (LAB) revealed that 49 strains possessed homologs of horB and horC. No false-positive results have been observed for nonspoilage LAB or frequently encountered brewery isolates. These features are superior to those of horA and ORF 5, previously reported genetic markers for determining the beer spoilage ability of LAB. It was further shown that the combined use of horB/horC and horA is able to detect all 51 beer spoilage strains examined in this study. Furthermore sequence comparison of horB and horC homologs identified in four different beer spoilage species indicates these homologs are 96.6 to 99.5% identical, which is not typical of distinct species. The wide and exclusive distribution of horB and horC homologs among beer spoilage LAB and their sequence identities suggest that the hop resistance ability of beer spoilage LAB has been acquired through horizontal gene transfer. These insights provide a foundation for applying trans-species genetic markers to differentiating beer spoilage LAB including previously unencountered species.Beer has been recognized as a beverage with high microbiological stability. Among the beer spoilers, several species of lactic acid bacteria (LAB) are reported to be responsible for approximately 70% of spoilage incidents caused by microorganisms (2, 3). For this reason, species-specific identification methods based on PCR have been widely evaluated for potential applications to microbiological quality control (6,19,41,42,44). Although species-specific PCR tests are rapid and reasonably accurate, there are two problems for applying this approach to the quality control of breweries.One problem is that the species-specific method is unable to distinguish intraspecies differences between beer spoilage strains and nonspoilage strains (9,26,28,35). Hop compounds added to confer bitter flavor are reported to exert an antibacterial effect by acting as proton ionophores and dissipate transmembrane pH gradient, which prevents gram-positive bacteria, including most LAB, from growing in beer (24,25,27,28,40). Hop resistance ability has been known as the distinguishing character of beer spoilage strains of LAB and nonspoilage strains typically exhibit hop resistance ability considerably weaker than that of beer spoilage strains belonging to the same species (1,9,28,34,35). The presence of nonspoilage strains within a beer spoilage species inevitably leads to false-positive results as long ...

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 ethanoland 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.

Aims:  To determine whether horC confers beer‐spoilage ability and to evaluate the validity of horC as a trans‐species genetic marker for differentiating the beer‐spoilage ability of lactic acid bacteria (LAB). Methods and Results:  Hop‐sensitive Lactobacillus brevis ABBC45cc was transformed with an expression plasmid, pHYchorBC, containing putative multidrug resistance gene horC and its putative regulator horB, and the transformant was designated as ABBC45cc/pHYchorBC. As a control, ABBC45cc was transformed with pHYchorB that contains horB, and the transformed strain was designated as ABBC45cc/pHYchorB. As a result of beer‐spoilage assay of these transformants, ABBC45cc/pHYchorBC exhibited beer‐spoilage ability, whereas ABBC45cc/pHYchorB did not. Furthermore ABBC45cc/pHYchorBC showed higher hop resistance than ABBC45cc/pHYchorB, accounting for the differences in beer‐spoilage ability observed between the two transformants. ABBC45cc/pHYchorBC also exhibited higher resistance to various structurally unrelated drugs, compared with ABBC45cc/pHYchorB. Conclusions:  horC was shown to confer hop resistance and beer‐spoilage ability on ABBC45cc by presumably encoding a multidrug transporter. Significance and Impact of the Study:  The finding that horC plays an important role in hop resistance and beer‐spoilage ability supports the validity of horC as a trans‐species genetic marker for differentiating the beer‐spoilage ability of LAB.

Aims:  To develop a detection medium for hard‐to‐culture beer‐spoilage lactic acid bacteria (LAB). Methods and Results:  Four hard‐to‐culture beer‐spoilage strains of LAB, belonging to Lactobacillus paracollinoides and Lactobacillus lindneri, have been obtained by repeatedly subculturing the wild‐type strains in beer. To develop a countermeasure against these hard‐to‐culture beer‐spoilage LAB, a beer‐based medium was modified. As a consequence, the supplementation of a small amount of de Man Rogosa Sharpe medium was found to enhance the growth of hard‐to‐culture beer‐spoilage LAB strains obtained in this study. In addition, sodium acetate was shown to improve the selectivity of this beer‐based medium. Further comparative study was performed with five other media widely used for the detection of beer‐spoilage LAB in the brewing industry. This study revealed that the newly developed medium, designated advanced beer‐spoiler detection (ABD) medium, possessed superior sensitivity for hard‐to‐culture beer‐spoilage LAB and comparable sensitivity with easy‐to‐culture beer‐spoilage LAB. Moreover, ABD medium was found to suppress the growth of nonspoilage micro‐organisms, and thereby allow the selective growth of beer‐spoilage LAB. Conclusions:  Advanced beer‐spoiler detection medium is considered as an effective tool for comprehensive detection of beer‐spoilage LAB in breweries. Significance and Impact of the Study:  The detection by ABD medium can be used as an indicator for differentiating the beer‐spoilage ability of LAB without further confirmatory tests in breweries.