Brewery Environment Research Articles

Insights on Producing and Copacking Special Products in a Brewery Environment

Insights on Producing and Copacking Special Products in a Brewery Environment

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.

Megasphaera paucivorans sp. nov., Megasphaera sueciensis sp. nov. and Pectinatus haikarae sp. nov., isolated from brewery samples, and emended description of the genus Pectinatus

Seven unidentified strictly anaerobic, Gram-negative, non-spore-forming bacteria from spoiled beer or the brewery environment were characterized. Based on 16S rRNA gene sequence analyses, all strains were affiliated to the Sporomusa sub-branch of the class 'Clostridia'. Three of the strains were non-motile cocci, on average 1.5 x 1.2 microm or 1.2 x 1.0 microm, occurring mainly singly or in pairs. They shared nearly identical (>99 %) 16S rRNA gene sequences, being most closely related to the species of the Megasphaera-Anaeroglobus group (< or =93.9 % similarity). According to DNA-DNA hybridization results, the coccoid strains represented two genospecies, neither of which was related to any of the recognized Megasphaera species. Several phenotypic characteristics and/or DNA G+C content also differentiated the strains from each other and from their closest relatives. The other four novel strains were motile, slightly curved to helical rods, 0.6-0.8 x 3-50 microm or more in size. They shared identical 16S rRNA gene sequences and ribofragment patterns. The highest 16S rRNA gene similarity was found between these isolates and Pectinatus cerevisiiphilus ATCC 29359T (95.6 %) and Pectinatus frisingensis ATCC 33332T (93.6 %). The novel strains also differed from recognized Pectinatus species in their sugar utilization, proteolytic activity, catalase activity, antibiotic resistance and temperature tolerance. The results suggest that the bacteria belong to three novel species, for which the names Megasphaera paucivorans sp. nov. (type strain VTT E-032341T = DSM 16981T), Megasphaera sueciensis sp. nov. (type strain VTT E-97791T = DSM 17042T) and Pectinatus haikarae sp. nov. (type strain VTT E-88329T = DSM 16980T) are proposed.

Application of Ribotyping and rDNA Internal Space Analysis (RISA) for Assessment of Microflora in Brewery Environments

The applications of ribotyping and rDNA internal space analysis (RISA) were evaluated for assessment of microflora in brewery environments. For this purpose, approximately 2,000 strains isolated in our breweries were analyzed by ribotyping, and the database was constructed using all the data acquired. The strains that displayed unidentifiable ribopatterns were presumptively identified by 16S rDNA analysis, and novel ribopatterns were registered in our database as Custom ID patterns. As a result, it was demonstrated that ribotyping is capable of discriminating the brewery isolates on the strain level and well suited to the detailed analysis of microflora in brewery environments. We have also successfully accumulated an enormous database of the strains isolated from the brewery environments. We further evaluated RISA as an inexpensive and rapid alternative to ribotyping. As a consequence, RISA was shown to distinguish strains broadly on the species level, which is sufficient for the practical assessment of brewery isolates. Because this technique has an advantage in terms of cost and time required for analysis, RISA was also considered to complement ribotyping as a prescreening method. On the basis of these results, we have developed a reliable system for assessing the microflora in brewery environments by combining ribotyping and RISA.

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 20690T and L. para-collinoides DSM 15502T, 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.

Lactobacillus paracollinoides sp. nov., isolated from brewery environments.

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 genus Lactobacillus and are most closely related to Lactobacillus collinoides (approximately 99% similarity). The novel strains could be differentiated from L. collinoides on the basis of DNA-DNA relatedness, differences in beer-spoilage ability and the inability to utilize D-fructose. These isolates represent a novel species, for which the name Lactobacillus paracollinoides sp. nov. is proposed. The type strain is LA2T (=DSM 15502T=JCM 11969T).

Characterization of Pectinatus and Megasphaera Strains by Automated Ribotyping

A total of 32 Pectinatus and Megasphaera strains, isolated from spoiled beer or brewery environments and identified by conventional methods, were analysed by the automated RiboPrinter® System. One strain from each ribotype was further subjected to partial 16S rDNA sequencing to confirm the ribotyping results. The restriction enzyme EcoRI was used in ribotyping of Pectinatus strains. Eight strains, identified by conventional tests as P. cerevisiiphilus, generated five different ribotypes. The strains of three types were considered to be members of P. cerevisiiphilus, but the strains of two types were most probably members of a new species within the genus Pectinatus. The 24 strains identified by conventional tests as P. frisingenis generated nine different ribotypes. The similarity between the ribotypes was rather low, but all these strains obviously belonged to the same species. Thirteen Megasphaera cerevisiae strains were analysed with three restriction enzymes EcoRI, Pstl and PvuII and four, six and three different ribotypes, respectively, were generated resulting in seven different combinations. The best discrimination among these strains was obtained with Pstl. According to these results 12 of 13 brewery strains were considered to be M. cerevisiae, but one strain most probably represented a new species within the genus Megasphaera. During the work, 14 RiboPrint® patterns of Pectinatus, five of Megasphaera, two of Selenomonas and two of Zymophilus were created with EcoRI. In addition seven patterns of Megasphaera were created with Pstl and four with Pvull. All these identification patterns (genetic fingerprints) were saved at the database of VTT for future use.

Isolation of Novel Beer-Spoilage Bacteria from the Brewery Environment

Two strains of beer-spoilage bacteria, BS-1 and EM-63, were isolated from the environment (from drainage) in our breweries. Strain BS-1 belonged to a homo-fermentative species of the genus Lactobacillus. The partial sequence of 16S rDNA indicated that strain BS-1 was related to Lactobacillus coryniformis. However, the fermentation pattern of the sugars was different from that of L. coryniformis and other species of Lactobacillus, because of the narrow fermentation spectrum, glucose, mannose, and fructose. Strain EM-63 was a Gram-negative, strictly anaerobic rod-shaped bacterium with no flagella. The GC content of DNA was 41%, and the cell wall peptidoglycan was directly cross-linked mesodiaminopimelic acid. The major fermentation acids were succinic acid and D-lactic acid in the culture media, while propionic acid was neither produced in culture media nor in beer. These characteristics indicated that strain EM-63 did not belong to the genus Pectinatus, known as Gram-negative, rod-shaped, beer-spoilage bacteria, or the genus Zymophilus or Selenornonas, known as Gram-negative, rod-shaped, pitching yeast contaminants. On the basis of the physiological characteristics and genetic analysis, we conclude that these strains are novel beer-spoilage bacteria.

The Rapid Identification by Protein Fingerprinting of Yeast and Bacterial Brewery Contaminants

A simple, rapid procedure is presented for extraction and solublization of cellular proteins from bacteria or yeast that, when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gives a protein fingerprint pattern specific to the gene expression of that particular isolate. Visual cross-referencing of protein patterns of unknown isolates with known references can be used to quickly discern their relationship to one another. Protein fingerprints of brewing ale and lager yeasts, non-brewing yeasts, and a variety of bacteria are presented. Possible applications of this technique in a brewery environment are also discussed.

THE DETECTION AND IDENTIFICATION OF PEDIOCOCCUS AND MICROCOCCUS IN BREWERIES, USING A SEROLOGICAL METHOD

A commercially-available antiserum for the identification of group D strains of Streptococcus contains antibodies which react with Pediococcus and Micrococcus, and provides a convenient method to detect these genera in yeast, wort and beer. After suitable adsorption it can be used to distinguish between Pediococcus and Micrococcus and, within each genus, to differentiate between groups normally found in a brewery environment and those from other sources.