[Show abstract][Hide abstract] ABSTRACT: Journal of the Institute of Brewing Vol.115 Nr.3, 167 - 176 The aim of this study was to evaluate easy pre-PCR processing procedures to allow rapid and reliable detection of strictly anaerobic beer-spoilage bacteria throughout the brewing process by end-point and real-time PCR techniques. The efficiencies of the new procedures were evaluated using spiked brewery samples and specific PCRs for the target group bacteria. We found for the first time that the inclusion of 0.25% (w/v) bovine serum albumin (BSA) or 0.5% (w/v) polyvinyl pyrrolidone (PVP) in the end-point PCR mixture reduces the inhibiting effect of brewery sample extracts (3-10%, v/v) on PCR. Membrane filtration with a PVP or a sodium tri-polyphosphate-EDTA wash, and cross-flow filtration were the most promising new methods to reduce inhibitors from beer samples before cell lysis. Together with BSA, they allowed the analysis of 10% (v/v) of crude extracts instead of <3% (v/v). Moreover, we developed a one-hour procedure to prepare target DNA from process samples containing brewer's yeast. It involved removal of inhibitors by a twostep centrifugation followed by physical disruption of cells. The detection limit of the procedure was 101-103 CFU/mL. The developed procedures help to reduce the risk of partial or complete PCR failure due to inhibition and target DNA losses, with minimal sample handling.
[Show abstract][Hide abstract] ABSTRACT: 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.
International Journal of Food Microbiology 08/2008; 125(2):162-9. DOI:10.1016/j.ijfoodmicro.2008.03.042 · 3.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The efficiency of a novel strain of lactic acid bacteria inoculant (Lactobacillus plantarum VTT E-78076, E76) on the fermentation quality of wilted silage was studied. Furthermore, the possibility to improve aero-bic stability of silages by combining an inoculant and chemical preservatives was investigated. Two ex-periments were conducted with wilted timothy-meadow fescue herbage (dry matter 429 and 344 g kg -1) using six treatments. In experiment I, E76 (10 6 cfu g -1 fresh matter (FM)) was applied alone and in combina-tion with sodium benzoate (0.3 g kg -1 grass FM) or low rate of formic acid (0.4 l t -1 FM). In experiment II, E76 and a commercial inoculant were applied alone and in combination with sodium benzoate. Untreated silage and formic acid (4 l t -1 FM) treated silage served as negative and positive controls in both experi-ments. The effect of sodium benzoate and potassium sorbate in experiment I, on aerobic stability was tested by treating silages prior to aerobic stability measurements. The novel lactic acid bacteria inoculant was equally effective in improving fermentation quality as the commercial inoculant. However, the aerobic stability of both inoculated silages was poorer than that of formic acid treated or the untreated one in one of the experiments. The results suggested that antimicrobial properties of E76 were not effective enough to improve aerobic instability. One option to overcome this problem is to use chemical additives in combina-tion with the inoculants.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Agricultural and Food Chemistry 06/2006; 54(11):3840-51. DOI:10.1021/jf052979j · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study the ability of automated ribotyping to characterize Obesumbacterium proteus and Hafnia alvei, to design primers and to evaluate standard end-point and real-time PCR for the detection of O. proteus biotype 1 in beer and in brewers's yeast-containing samples.
Automated ribotyping was carried out using the standard method with EcoRI and PvuII. The digestions with both enzymes clearly differentiated O. proteus biotypes 1 and 2 and H. alvei. PCR primers were designed according to the 16S rRNA gene sequence of the O. proteus type strain. Two primer sets (Obs137-Obs558 and Obs137-Obs617) detected O. proteus biotype 1 and H. alvei but not O. proteus biotype 2 or other tested beer spoilage bacteria (40 species) in the end-point and real-time PCR, indicating their high specificity. The detection limit for O. proteus was 160-1600 CFU 100 ml(-1) beer in the end-point PCR reactions and < or =160 CFU 100 ml(-1) beer in the real-time PCR reactions. More cells (from 16 to 3200) were needed for detection in the presence of brewer's yeast cells.
Automated ribotyping is a useful tool to characterize and identify O. proteus and H. alvei isolates. The designed primers are suitable for the rapid detection of O. proteus biotype 1 and H. alvei in brewery samples by PCR.
Automated ribotyping and PCR could improve microbiological quality control in breweries by facilitating the detection, identification and tracing of spoilage bacteria.
[Show abstract][Hide abstract] ABSTRACT: A real-time PCR technique was applied for the quantification of trichothecene-producing Fusarium species (TMTRI assay) as well as the highly toxigenic Fusarium graminearum (TMFg12 assay) present in barley grain and malt. PCR results were compared to the amounts of trichothecenes detected in the
samples to find out if the PCR assays can be used for trichothecene screening instead of expensive and laborious chemical
analyses. DNA was extracted from ground kernels using a commercial DNA extraction kit and analysed in a LightCycler® system
using specific primers and fluorogenic TaqMan probes. Both naturally and artificially contaminated grains were analysed. The
TMTRI assay and the TMFg12 assay enabled the quantification of trichothecene-producing Fusarium DNA and F. graminearum DNA present in barley grain and malt samples, respectively. Both TaqMan assays were considered to be sensitive and reproducible.
Linearity of the assays was 4–5 log units when pure Fusarium DNAs were tested. The amount of Fusarium DNA analysed with the TMTRI-trichothecene assay could be used for estimation of the deoxynivalenol (DON) content in barley
grain. Furthermore, the TMFg12 assay for F. graminearum gave a good estimation of the DON content in north American barley and malt samples, whilst the correlation was poor among
Finnish samples. DON content and the level of F. graminearum DNA were found to be naturally low in Finnish barleys.
European Journal of Plant Pathology 01/2006; 114(4):371-380. DOI:10.1007/s10658-006-0001-9 · 1.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Barley (Hordeum vulgare L.) that is infested with Fusarium head blight (FHB, ‘scab’) is unsuitable for malting and brewing because it may contain mycotoxins and has unacceptable malting quality. Fungal proteinases are apparently often involved in plant-microbe interactions, where they degrade storage proteins, but very little is known about the enzymes that the fungi produce in the infected grain. We have shown previously that one plant pathogenic fungus, Fusarium culmorum, produced subtilisin- and trypsin-like enzymes when grown in a cereal protein medium. To establish whether these proteinases were also synthesized in FHB-infested barley in vivo, field-grown barley was infested as the heads emerged. Extracts were prepared from the grain as it developed and matured and their proteolytic activities were measured with N-succinyl-Ala-Ala-Pro-Phe p -nitroanilide and N-benzoyl-Val-Gly-Arg p -nitroanilide. The heavily infested barleys contained both subtilisin- and trypsin-like activities. These enzymes reacted with antibodies prepared against each of the two F. culmorum proteinases, indicating that those produced in the laboratory cultures and in the field-infested barley were the same. The presence of these proteinases correlated with the degradation of specific buffer-soluble proteins in the infested grains. These enzymes readily hydrolyzed barley grain storage proteins (C- and D-hordeins) in vitro. The presence of these Fusarium proteinases in the barley indicates that they probably play an important role in the infestation, but exactly how and when they function is not clear.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Industrial Microbiology and Biotechnology 05/2003; 30(4):239-44. DOI:10.1007/s10295-003-0046-0 · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Anaerobic bacteria of the genera Megasphaera and Pectinatus cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. A polymerase chain reaction (PCR) method and a colorimetric microplate hybridization assay were developed for the rapid and specific detection of Megasphaera cerevisiae and Pectinatus spp. A biotinylated primer pair was designed for the amplification of a 403 base pair (bp) fragment of the M. cerevisiae 16S rRNA gene and a primer pair from literature was used for the amplification of an 816 bp fragment of Pectinatus 16S rRNA gene. Amplified PCR products were analyzed by the colorimetric microplate hybridization method in which a biotinylated PCR product was captured by streptavidin and hybridized with a digoxigenin-labelled oligonucleotide probe. In the final step an enzyme-linked antibody and a colorimetric reaction were utilized. A simple and rapid sample treatment was set up for the PCR detection of contaminants in beer. Detection of M. cerevisiae (≥5·103 colony forming units [cfu]/100 ml) and Pectinatus frisingensis (≥5·105 cfu/100 ml) in beer was successful, but the sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.
International Journal of Food Microbiology 01/1999; 45(2-45):119-127. DOI:10.1016/S0168-1605(98)00154-8 · 3.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cultivation methods for the detection of bacteria in beer, although simple and sensitive, are time-consuming and lack specificity. In this study, the sensitivity of a polymerase chain reaction (PCR) assay consisting of an easy sample treatment and specific primers for Lactobacillus lindneri, L. brevis, Megasphaera cerevisiae, and Pectinatus spp. was improved by pre-enrichment. A pre-enrichment broth supporting the growth of lactobacilli and anaerobic beer spoilers better than currently used media was formulated. For determination of the pre-enrichment times needed for PCR detection of these contaminants, artificially contaminated beer samples were mixed with the pre-enrichment broth and incubated anaerobically at 30°C. Low levels of lactobacilli (≤10 CFU/100 ml) were detected after 1-3 days, Pectinatus spp. after 2-4 days, and M. cerevisiae after 2-3 days of pre-enrichment, depending on the strain and the alcohol content of the beer. After the pre-enrichment, the PCR analysis took <8 hr. The time saving compared to corresponding conventional methods requiring up to several weeks is marked, especially for L. lindneri, M. cerevisiae, and Pectinatus spp. Moreover, the assay described allows species- or genus-level detection of the most harmful beer spoilage bacteria in finished beer and is sensitive and simple enough for routine work.
Journal of the American Society of Brewing Chemists 01/1999; 57(3):99-103. · 0.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of food protection 11/1997; 60(12):1571-1573. · 1.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 16S ribosomal RNA gene from the beer-spoilage organism, Megasphaera cerevisiae was polymerase chain reaction (PCR)-amplified and sequenced. Analysis confirmed the phylogenetic position of M. cerevisiae as a sister taxon of Megasphaera elsdenii, within the obligately anaerobic, Gram-negative cocci. The sequence obtained should facilitate the development of DNA probes for early detection of this spoilage organism.
Journal of Industrial Microbiology 09/1995; 15(2):67-70. DOI:10.1007/BF01569801