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.

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.

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.

We have isolated a hop-sensitive variant of the beer spoilage bacteriumLactobacillus lindneriDSM 20692. The variant lost a plasmid carrying two contiguous open reading frames (ORF s) designatedhorB_LandhorC_Lthat 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 ofhorBandhorC. No false-positive results have been observed for nonspoilage LAB or frequently encountered brewery isolates. These features are superior to those ofhorAand ORF 5, previously reported genetic markers for determining the beer spoilage ability of LAB. It was further shown that the combined use ofhorB/horCandhorAis able to detect all 51 beer spoilage strains examined in this study. Furthermore sequence comparison ofhorBandhorChomologs 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 ofhorBandhorChomologs 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.

Three novel strains isolated from brewery environments are described. These strains were Gram-positive, facultatively anaerobic, heterofermentative rods that did not exhibit catalase activity. Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that these strains belong to the genusLactobacillusand are most closely related toLactobacillus collinoides(approximately 99 % similarity). The novel strains could be differentiated fromL. collinoideson the basis of DNA–DNA relatedness, differences in beer-spoilage ability and the inability to utilized-fructose. These isolates represent a novel species, for which the nameLactobacillus paracollinoidessp. nov. is proposed. The type strain is LA2^T(=DSM 15502^T=JCM 11969^T).