Group-specific PCR-RFLP and real-time PCR methods for detection and tentative discrimination of strictly anaerobic beer-spoilage bacteria of the class Clostridia.

The strictly anaerobic brewery contaminants of the genera Pectinatus, Megasphaera, Selenomonas and Zymophilus in the class Clostridia constitute an important group of spoilage bacteria of unpasteurised, packaged beers. The aim of this study was to develop and evaluate group-specific PCR methods to detect and differentiate these bacteria in beer. A group-specific primer pair targeting a 342-bp variable region of the 16S rRNA gene was designed and evaluated in end-point PCR with gel electrophoresis and in real-time PCR with SYBR Green I dye. Significant cross-reactions with DNAs from any of the forty-two brewery-related, non-target microbes or from real brewery samples were not detected in either PCR system. The group-specific end-point and real-time PCR products could be differentiated according to species/genus and spoilage potential using restriction fragment length polymorphism (KpnI, XmnI, BssHII, ScaI) and melting point curve analysis, respectively. In combination with a rapid DNA extraction method, the PCR reactions detected ca 10(0)-10(3) CFU per 25 ml of beer depending on the strain and on the PCR system. The end-point and real-time PCR analysis took 6-7 h and 2-3 h, respectively. Pre-PCR enrichment of beer samples for 1-3 days ensured the detection of even a single cultivable cell. The PCR and cultivation results of real brewery samples were mostly congruent but the PCR methods were occasionally more sensitive. The PCR methods developed allow the detection of all the nine beer-spoilage Pectinatus, Megasphaera, Selenomonas and Zymophilus species in a single reaction and their differentiation below group level and reduce the analysis time for testing of their presence in beer samples by 1-2 days. The methods can be applied for brewery routine quality control and for studying occurrence, diversity and numbers of the strictly anaerobic beer spoilers in the brewing process.

Lactobacillus plantarum and Pediococcus pentosaceus starter cultures as a tool for microflora management in malting and for enhancement of malt processability.

Lactobacillus plantarum VTT E-78076 (E76) and Pediococcus pentosaceus VTT E-90390 (E390) starter cultures were added to the steeping water of normal malting barley in order to balance the microbial community and to enhance malt processability. In this study, we also investigated the effects of lactic acid-acidified MRS-spent medium (MRS-LA) on malting performance. Malting trials with five different two-row barley varieties were carried out in 25 kg pilot scale. The starter cultures promoted yeast growth during malting and restricted the growth of harmful bacteria and Fusarium fungi. Furthermore, they had positive effects on malt characteristics. Reduction in wort viscosity and beta-glucan content and enhanced xylanase and microbial beta-glucanase activities were observed. Starter cultures notably improved lautering performance. Some of the beneficial effects were due to the lactic acid and low pH, as similar effects were obtained with MRS-LA. Starter cultures offer a tool for tailoring of malt properties.

Lipopolysaccharides of anaerobic beer spoilage bacteria of the genus Pectinatus--lipopolysaccharides of a Gram-positive genus.

Bacteria of the genus Pectinatus emerged during the seventies as contaminants and spoilage organisms in packaged beer. This genus comprises two species, Pectinatus cerevisiiphilus and Pectinatus frisingensis; both are strict anaerobes. On the basis of genomic properties the genus is placed among low GC Gram-positive bacteria (phylum Firmicutes, class Clostridia, order Clostridiales, family Acidaminococcaceae). Despite this assignment, Pectinatus bacteria possess an outer membrane and lipopolysaccharide (LPS) typical of Gram-negative bacteria. The present review compiles the structural and compositional studies performed on Pectinatus LPS. These lipopolysaccharides exhibit extensive heterogeneity, i.e. several macromolecularly and structurally distinct LPS molecules are produced by each strain. Whereas heterogeneity is a common property in lipopolysaccharides, Pectinatus LPS have been shown to contain exceptional carbohydrate structures, consisting of a fairly conserved core region that carries a large non-repetitive saccharide that probably replaces the O-specific chain. Such structures represent a novel architectural principle of the LPS molecule.

Cellular fatty acyl and alkenyl residues in Megasphaera and Pectinatus species: contrasting profiles and detection of beer spoilage.

SUMMARYThe strictly anaerobic Gram-negative beer spoilage bacteria Megasphaera cerevisiae, Pectinatus cerevisiiphilus and P. frisingensis were subjected to cellular fatty acid analysis, employing acid- and base-catalysed cleavage, gas chromatography and mass spectrometry. M. cerevisiae contained 12:0, 16:0, 16:1, 18:1, 17:cyc, 19:cyc, 12:0(3OH), 14:0(3OH) as the main fatty acids, and alk-1-enyl chains instead of acyl chains were detected to a considerable extent (14% of total fatty acids), indicating the presence of plasmalogens. The fatty acid pattern of M. cerevisiae was almost identical to that of M. elsdenii, the only species previously assigned to this genus. P. cerevisiiphilus and P. frisingensis yielded fatty acids that were heavily dominated by odd-numbered chains; 11:0, 15:0, 17:1, 18:cyc and 13:0(3OH) were the main fatty acids detected in both species. Alk-1-enyl chains with similar chain lengths were also found. Both Pectinatus species contained six different 3-hydroxy fatty acids with chain lengths between 11 and 15 carbons, 13:0(3OH) being dominant and the others accounting for generally less than 1% of total fatty acids. Among the minor components, an unsaturated 3-hydroxy fatty acid was detected which was shown to be 13:1(30H). In addition, fatty acid analysis was shown to be applicable to detection of bacterial contamination of beer.

Detection of bacterial contamination in starch and resin-based papermaking chemicals using fluorescence techniques.

Rapid fluorescence techniques were evaluated for the detection of bacterial contaminants in papermaking chemicals including starch and the resin-based sizes and starch slurries used in the paper industry. Viable and non-viable bacterial cells were visualised by fluorescent probes and detected by epifluorescence microscopy and flow cytometry. The best discrimination ability was obtained with the fluorescent probes LIVE/DEAD and SYBR Green, based on the staining of cellular nucleic acid, and ChemChrome V3, which demonstrated cellular enzymatic activity. The process samples had to be diluted and filtered before fluorescence staining and analysis because they were viscous and contained solid particles. Fluorescence microscopic counts of bacteria in highly contaminated process samples were similar to plate counts, but flow cytometric enumeration of bacterial cells in process samples yielded 2- to 10-fold lower counts compared with plate counts, depending on the consistency of the sample. The detection limits in flow cytometric analysis and in epifluorescence microscopy were 10(3)-10(6) cells ml(-1) and 10(5)-10(6) cells ml(-1), respectively. Intrinsic bacterial contamination was detectable with fluorescence techniques and highly contaminated process samples could be analysed with fluorescence methods.

Detection of Pectinatus Beer Spoilage Bacteria by Using the Polymerase Chain Reaction.

Anaerobic bacteria of the genus Pectinatus cause beer spoilage by producing off flavors and turbidity. In unpasteurized beer even a small initial amount of contamination is likely to lead to a defective product. Detection of Pectinatus by traditional microbiological techniques is time-consuming and not practical as a preventive product control measure. In this paper Pectinatus -specific primers capable of discriminating among other beer contaminants by polymerase chain reaction are described. The present procedure, which includes the isolation of DNA from the contaminated beer sample, the polymerase chain reaction, and the electrophoretic identification of the reaction products could be performed within 10 h. The detection level in inoculated beer samples was ca. 20 cells per ml. The technique therefore has a potential in routine product control.