Beer-spoilage Lactobacillus Research Articles

Formation of viable, but putatively non-culturable (VPNC) cells of beer-spoilage lactobacilli growing in biofilms

Bacteria belonging to the genus Lactobacillus are the most commonly-found beer-spoilage microorganisms in breweries. This work compares the ability of biofilm and planktonic cells of beer-spoilage Lactobacillus sp. to enter a viable, but putatively non-culturable (VPNC) state. Planktonic and biofilm cells of five beer-spoilage species of Lactobacillus were incubated at 26 °C in the lager beer with sub-culturing. Both biofilm and planktonic cells could be induced to enter the VPNC state, however, the state-transition time is variable: it took a minimum of 63 days for the biofilm cells to enter the VPNC state, while the time for planktonic cells was 119–217 days. The presence of VPNC cells in non-culturable biofilms was further confirmed by PCR coupled with propidium monoazide pretreatment that amplified genes 16S rDNA and horA. Taken together, this study provides evidence that Lactobacillus biofilm cells can enter the VPNC state at a rate faster than the planktonic cells under stressful conditions of the beer (low pH, high concentrations of hop bitter compounds and ethanol, and limited carbon/nitrogen sources) in the brewing process.

A novel horA genetic mediated RCA detection of beer spoilage lactobacillus

As one of the most popular beverage, beer has strong resistance to bacteria except for lactic acid bacteria (LAB), as its strong viability in beer. Contaminating LAB results in beer spoilage. HorA gene carried by beer spoilage lactobacillus confers hop resistance and is associated with the growth of Lactobacillus in beer, thus can be used as a perfectly valid genetic marker for the detection of beer spoilage Lactobacillus strains. This study applied genome and bioinformatics analyses, together with genetic alignment and comparison techniques to build a rolling circle amplification (RCA) detection method targeted on horA for beer spoilage Lactobacillus. The horA genetic mediated RCA method showed rapid, sensitive and specific on beer spoilage lactobacillus detection.

Transcriptomic analysis on the formation of the viable putative non-culturable state of beer-spoilage Lactobacillus acetotolerans.

Lactic acid bacteria (LAB) are the most common beer-spoilage bacteria regardless of beer type, and thus pose significant problems for the brewery industry. The aim of this study was to investigate the genetic mechanisms involved in the ability of the hard-to-culture beer-spoilage bacterium Lactobacillus acetotolerans to enter into the viable putative non-culturable (VPNC) state. A genome-wide transcriptional analysis of beer-spoilage L. acetotolerans strains BM-LA14526, BM-LA14527, and BM-LA14528 under normal, mid-term and VPNC states were performed using RNA-sequencing (RNA-seq) and further bioinformatics analyses. GO function, COG category, and KEGG pathway enrichment analysis were conducted to investigate functional and related metabolic pathways of the differentially expressed genes. Functional and pathway enrichment analysis indicated that heightened stress response and reduction in genes associated with transport, metabolic process, and enzyme activity might play important roles in the formation of the VPNC state. This is the first transcriptomic analysis on the formation of the VPNC state of beer spoilage L. acetotolerans.

ビール混濁性を示すLactobacillus paracollinoidesおよびL. lindneriからの難培養株の取得

Beer spoilage Lactobacillus paracollinoides and L. lindneri strains were repeatedly subcultured in degassed beer and the culturability of these Lactobacillus strains was periodically examined on MRS agar. As a consequence, L. paracollinoides JCM 11969T and LA 4 progressively exhibited diminished culturability on MRS agar, and after the 70 th and 60 th subcultures, respectively, most of the viable cells were no longer detectable on the agar. Similarly, L. lindneri DSM 20692 and HC 92 gradually lost culturability, and after the 70 th and 40 th subcultures, respectively, these strains exhibited viable but nonculturable (VNC) characteristics on the agar. These results suggested that the prolonged adaptation to beer and related environments reduces the culturability of beer spoilage Lactobacillus strains on detection media, posing a threat to the brewing industry. Strikingly, the majority of nonculturable cells, as counted by esterase staining, showed beer spoilage ability, suggesting that microbiological incidents can occur without the detection of beer spoilage lactic acid bacteria on culture media. Taken collectively, these findings indicate that the risk presented by these microorganisms should be nullified by the use of sufficiently robust quality assurance approaches to ensure product stability.

Isolation and Identification of Potential Beer-Spoilage Pediococcus inopinatus and Beer-Spoilage Lactobacillus backi Strains Carrying the horA and horC Gene Clusters

Four beer-spoilage strains, LA20, LA21, LA22 and LA23, were isolated from brewery environments. Based on the 16S rRNA gene sequence, LA20 was identified as Pediococcus inopinatus and the remaining three were identified as Lactobacillus backi. The homologs of horA and horC, the hop resistance genes originally identified in L. brevis ABBC45, were detected simultaneously in LA22 and LA23, while only a horA homolog and a horC homolog were found in LA20 and LA21 respectively. The 5.6 kb DNA regions containing the horA homolog in LA20 and LA22 were almost 99% identical with the corresponding region of ABBC45. Similarly the 8.2 kb regions containing the horC homolog in LA21 and LA22 were more than 99% identical with that of ABBC45. Interestingly the horA-containing 5.6 kb regions in LA20 and LA22 were found to be completely identical despite the distinct genus status. Coupled with the fact that LA20 and LA22 were isolated from the same sampling site, these results, taken collectively, reinforce our hypothesis that horA and horC genes were acquired by beer-spoilage species through horizontal gene transfer and confirm the usefulness of horA and horC as genetic markers for the species-independent determination of beer-spoilage ability in lactic acid bacteria.

Comparative Study of Two Plasmids, pRH45 and pRH 20690, Isolated from Beer-Spoilage Lactobacillus Brevis ABBC45 and L. lindneri DSM20690T

Southern blot analysis with beer-spoilage Lactobacillus lindneri DSM 20690T showed the presence of the homologue of horA, a hop-resistance gene originally identified in L. brevis ABBC45. The horA homologue found in L. lindneri DSM 20690T was carried by a plasmid designated pRH20690. This plasmid is similar in size to pRH45, a plasmid harboring horA gene in L. brevis ABBC45. The full sequencing analysis of pRH20690 revealed that this 13.0-kb plasmid is remarkably similar in the organization of the open reading frames (ORFs) to that of pRH45. The putative replication origin regions and the ORFs encoding a putative rep gene were found to be 99.0 and 100.0% identical, respectively, between the two beer-spoilage strains, indicating that these two plasmids share the same origin. It was further shown that the 6.9-kb DNA portions of the plasmids containing six putative ORFs are 99.4% identical in nucleotide sequence, suggesting that the hop-resistant Lactobacillus strains acquired horA by plasmid-mediated horizontal gene transfer. This hypothesis potentially provides brewers with a theoretical basis for applying trans-species genetic markers, such as horA, to complement the traditional species-specific approaches.

Genetic Characterization of Hop-Sensitive Variants Obtained from Beer-Spoilage Lactobacillus Brevis

Hop-sensitive variants were isolated from six different beer-spoilage strains of Lactobacillus brevis after the 15th subculture at 37°C. All the variants lost beer-spoilage ability, indicating that the loss of hop resistance is primarily responsible for the loss of beer-spoilage ability of the variants. The polymerase chain reaction and Southern blot analysis revealed that all variants lost the homologs of horA, a hop resistance gene originally identified in beer-spoilage L. brevis ABBC45. When the wild-type L. brevis strains were subcultured at 37°C with 200 μM hop compounds, the isolates obtained from the 15th subculture exhibited a higher level of hop-resistance ability than did the hop-sensitive variants. The horA homologs were also detected in the strains subcultured under these conditions. Further subcultures at 37°C with hop compounds did not cause the loss of hop resistance and horA homologs in any of the L. brevis strains, indicating that the loss of the horA homologs are at least partly responsible for the loss of hop resistance observed with the variants. It is also suggested that the horA homologs are carried as part of plasmids, accounting for the relatively unstable nature of hop-resistance ability of beer-spoilage L. brevis strains found in our study.

Isolation of Hop-Sensitive Variants from Beer-Spoilage Lactobacillus Brevis Strains

To elucidate the mechanisms underlying the hop-resistance ability of beer-spoilage lactic acid bacteria, we attempted to isolate hop-sensitive variants from beer-spoilage Lactobacillus brevis. As a result, the repeated subcultures at 37°C were found to cause the hop resistance of six beer-spoilage L. brevis strains to be lost. In contrast to the wild-type strains, the hop-resistance ability of these variants was no longer inducible, as indicated by the inability to grow in DeMan-Rogosa-Sharpe (MRS) media supplemented with progressively higher concentrations of hop compounds. The diminished adaptability to hop compounds was comparable with that of the originally non-spoilage L. brevis strain, JCM1059T. When the variants were inoculated in beer and incubated anaerobically at 25°C, the viability of the strains was lost within one week, whereas the wild-type strains were able to grow during the same period of incubation. From these observations, it has been concluded that the variants obtained in this study lost the beer-spoilage ability. The biochemical characterization of the variants revealed narrower carbohydrate utilization profiles in some of the strains, notably with regard to the ability to ferment maltose.

Genetic characterization of non-spoilage variant isolated from beer-spoilage Lactobacillus brevis ABBC45.

To characterize the non-spoilage variant obtained from beer-spoilage Lactobacillus brevis ABBC45C and to identify a potential genetic marker capable of discriminating beer-spoilage L. brevis strains from non-spoilers. A non-spoilage variant was obtained from beer-spoilage L. brevis ABBC45C by repeatedly subculturing the strain at 37 degrees C. Genetic characterization of the variant revealed that 12,605 bp portion of one plasmid, designated pRH45II, was lost in the variant. The sequence analysis indicates the presence of 12 ORFs in the deleted region of pRH45II. The PCR and Southern hybridization study revealed that the homologues of ORF5 found in the deleted region were present in all of the beer-spoilage L. brevis strains examined in this study. In contrast, the homlogues appeared to be absent in non-spoilage L. brevis strains. The presence or absence of ORF5 homologues was found to be highly correlated with the beer-spoilage ability of L. brevis strains, indicating this ORF is potentially a useful genetic marker capable of differentiating beer-spoilage strains among L. brevis. A non-spoilage variant was successfully isolated from beer-spoilage L. brevis ABBC45C. This study could facilitate the understanding of mechanisms underlying beer-spoilage ability of L. brevis.

Genetic characterization and specific detection of beer-spoilage Lactobacillus sp. LA2 and related strains.

Lactobacillus sp. LA2 (DSM15502) and related strains (LA2 group) possess strong beer-spoilage ability. The 16S rDNA sequence of LA2 strain is virtually indistinguishable from that of L. collinoides, generally considered to be nonbeer-spoilage bacteria. The aim of this study was to identify the genetic marker to distinguish between Lactobacillus sp. LA2 group and L. collinoides and to provide a rapid means of identifying beer-spoilage strains belonging to Lactobacillus sp. LA2 group. The 16-23S rDNA intergenic spacer (ITS) regions of Lactobacillus sp. LA2 and L. collinoides JCM1123T were sequenced to identify a genetic marker to distinguish between the two groups. As a result, 300 and 500 bp ITS regions of Lactobacillus sp. LA2 were found to be almost identical with those of L. collinoides JCM1123T. Sequence comparison analysis between Lactobacillus sp. LA2 and L. collinoides JCM1123T revealed that the two contiguously located nucleotides are absent in both ITS regions of Lactobacillus sp. LA2. Based on the sequence difference, we have designed specific PCR primers with a minor modification to the primer sequence that can differentiate between beer-spoilage Lactobacillus sp. LA2 group and nonbeer-spoilage L. collinoides. The PCR-based method has been developed to identify Lactobacillus sp. LA2 group, providing a rapid and sensitive means of determining the beer-spoilage ability of detected bacterial strains. The substitution of one nucleotide, located at the third position to the 3'-end in the primer sequence, enhanced the specificity of the PCR method while retaining sufficient sensitivity. The nucleotide gap identified in this study appeared to serve as a useful genetic marker that can differentiate 12 beer-spoilage Lactobacillus sp. LA2 group strains from its close relatives that exhibit no beer-spoilage ability.

Monoclonal Antibodies Showing Surface Reactivity with Lactobacillus and Pediococcus Beer Spoilage Bacteria

Five monoclonal antibodies (Mabs) were isolated based upon reactivity with both a Lactobacillus and a Pediococcus beer spoilage isolate. Immunoblot analysis and Proteinase K treatment both suggested that none of the antigens recognized by the Mabs were proteins. Since the Mabs were known to bind surface-accessible antigenic sites on Gram-positive bacteria, it was proposed that lipoteichoic acid (LTA) was the antigen recognized. Each of the Mabs was purified and conjugated with horseradish peroxidase. Isolation of LTA, followed by direct antibody binding and antibody competition enzyme immunoassays, confirmed this. To determine whether the five Mabs were binding the same site on the LTA, the ability of each Mab to inhibit the binding of each of the enzyme-conjugated Mabs was determined. The data obtained allow us to propose that the five Mabs recognize three spatially distinct epitopes on the bacterial LTA. As the Mabs were found to bind to most beer spoilage Lactobacillus and Pediococcus isolates tested, these Mabs could prove useful in rapid detection of these Gram-positive beer-spoilage bacteria.

Application of a bioluminescence method for the beer industry: sensitivity of MicroStar-RMDS for detecting beer-spoilage bacteria. Rapid Microbe Detection System.

We set up the original operating conditions of the MicroStar-Rapid Microbe Detection System (RMDS) to suppress false positives, which have kept this system from practical. The detection limit of our system was between 6.3 x 10(-16) mol and 3.1 x 10(-16) mol in terms of the amount of ATP, which is approximately equal to the ATP content of one yeast cell or 50 lactic acid bacteria cells. The detection time and the detection count were compared between the RMD method and the conventional plate count method (C.P.C. method) using 23 test samples of beer-spoilage Lactobacillus brevis. Judging from the detection time and detection count, 16-24 hours of cultivation for the RMD method corresponded to 40-96 hours of cultivation for the C.P.C. method. The RMD method reached a useful level for our practical use at the point of sensitivity.

Characterization of Lactobacillus spp. by Ribotyping

Some species of lactobacilli are known to be serious beer-spoilage bacteria. Therefore, there have been several reports about detection of beer-spoilage Lactobacillus spp. (4,9-13,15). In addition to species discrimination, strain typing has been necessary for epidemiological study. Ribotyping is one method of strain typing and is used for the characterization of the restriction fragment length polymorphism. Furthermore, this method has proved to be a useful molecular epidemiological technique (1). There have been several reports about ribotyping of a number of pathogens, such as Listeria monocytogenes (2,14), Vibrio anguillarum (7), and V. cholerae (3). Previously, we reported the application of this molecular-typing for the beer-spoilage bacteria Pectinatus spp. (6). There have also been some reports about ribotyping of Lactobacillus spp. (5,8,10,16). However, little attention has been given to whether ribotyping can be used to discriminate between species and subspecies. In this study, we attempt to establish ribotypes of beer-spoilage Lactobacillus spp., and whether ribotyping of Lactobacillus spp. can be used for species-subspecies discrimination and epidemiological study.

Two Novel Beer-Spoilage Lactobacillus Species Isolated from Breweries

Two novel species of the beer-spoilage Lactobacillus bacteria were isolated from the brewing environment. One species, of which LA-2 is a representative strain, is an obligately heterofermentative Lactobacillus sp. with a strong beer-spoilage ability. The other species, of which LA-6 is representative, is a facultatively heterofermentative Lactobacillus sp. with a weak beer-spoilage ability. The carbohydrate utilization pattern, ribotype, and DNA-DNA hybridization analysis revealed that these novel strains were not related at the species level to any Lactobacillus spp. examined in this study. However, the 16S ribosomal DNA sequence of LA-2 was found to exhibit 99.5% similarity with that of the L. collinoides gene, indicating that these species are related. We have also developed primer sets for the detection of the LA-2 and LA-6.

Effect of the ethanol content of beer on the heat resistance of a spoilage Lactobacillus

D‐values of a heterofermentative beer spoilage lactobacillus were measured at 55°C, 60°C and 65°C in beers containing <0·05% to 4·4% v/v ethanol. Z‐values for the different beers varied between 9·17 and 12·13°C. At each temperature an increase in ethanol reduced the measured D‐value. The maximum, 5·01 min was observed in alcohol‐free beer (<0·05%) at 55°C and the minimum, 0·31 min, at 60°C and 65°C in beer containing 4·4% ethanol. D‐values could be increased by prior growth in the presence of ethanol. They could be reduced by adding ethanol to alcohol‐free beer or by increasing its hop extract content. The implications for the pasteurization of low‐alcohol beers are discussed.