The effect of Pediococcus damnosus and Pediococcus pentosaceus on the growth of pathogens in minced meat.

The antibacterial effects of one strain of Pediococcus damnosus and two strains of Pediococcus pentosacaeus against Clostridium perfringens, Listeria monocytogenes, Salmonella infantis and Yersinia enterocolitica were investigated. Growth inhibition studies were conducted in juice from minced meat incubated at +6 degrees C and +15 degrees C for various periods after the inoculation with pediococci. Inhibitory effects were seen for all bacteria tested.

Detection of beer spoilage bacteria Megasphaera and Pectinatus by polymerase chain reaction and colorimetric microplate hybridization.

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 (> or = 5 x 10(3) colony forming units [cfu]/100 ml) and Pectinatus frisingensis (> or = 5 x 10(5) 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.

Screening and selection of lactic acid bacteria strains suitable for ensiling grass.

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.

Characterization of the brewery spoilage bacterium Obesumbacterium proteus by automated ribotyping and development of PCR methods for its biotype 1.

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: Automated ribotyping and PCR could improve microbiological quality control in breweries by facilitating the detection, identification and tracing of spoilage bacteria.

Immunological Characterization of Pectinatus cerevisiophilus Strains.

Eleven Pectinatus cerevisiophilus strains of brewery origin were classified serologically by gel diffusion precipitin tests, immunoelectrophoresis, and the fluorescent antibody staining technique. The Pectinatus strains could be assigned immunologically to three different groups. Groups I and III were found to be very closely related, and only some of the antisera used showed differences. The antisera against the strains belonging to group II contained a common group antigen. A strong precipitation band found near the antigen was shown to represent the interaction of the lipopolysaccharide antibody and the respective antigen.

Production and characterization of antibacterial compounds produced by Pediococcus damnosus and Pediococcus pentosaceus.

The broad-spectrum antibacterial activity exhibited by three Pediococcus strains isolated from beer was preliminarily characterized. Factors affecting the production rate of bacterial inhibitors were screened and the effects of simultaneous cultivation of Lactococcus and Pediococcus on the production of inhibitory substances were studied. The antibacterial activity against a range of Gram-negative test organisms was not affected by catalase or proteolytic enzymes and was extremely thermotolerant. Production of the inhibitors was maximal between pH 6 and pH 7. A growth medium containing unhopped end-fermented wort was beneficial for the production of inhibitors, particularly by the Pediococcus damnosus strain, and anaerobic growth conditions were preferable. The antagonistic activity against the Gram-negative test organism Salmonella infantis could be demonstrated after an incubation period of only 2 d if the Pediococcus and Lactococcus strains were incubated simultaneously as a mixed population.

Microbiological, biochemical, and electron microscopic characterization of a pectinatus strain.

A spoilage organism isolated from turbid beer is described. The bacterium was gram negative, catalase negative, strictly anaerobic, and rod shaped, having flagella only on one side of the cell. The main metabolic product was propionic acid. In addition acetic, succinic, and lactic acids and acetoin were formed. Malonate inhibited the production of propionic acid by the strain studied and by both Pectinatus and Propionibacterium strains. The guanine-plus-cytosine content of deoxyribonucleic acid was 36 mol%. Differences between this strain and Pectinatus strains were 2 to 5 percentage points. Immunofluorescent staining and gel diffusion precipitin tests revealed that the antigenic structure differed from those of Pectinatus strains. The isolated organism can, despite some differences, be regarded as belonging to the genus Pectinatus.

Phytate degradation by micro-organisms in synthetic media and pea flour.

AIMS: To screen micro-organisms for the ability to produce phytase enzyme(s) and to use promising strains for the fermentation of pea flour. METHODS AND RESULTS: Two methods using the indirect estimation of phytate degradation were evaluated and both shown to be inadequate. A third method, measuring the inositol phosphate (IP3-IP6) content directly during fermentation, was used instead of the indirect estimations of phytate degradation. In synthetic media, some strains required customized conditions, with no accessible phosphorus sources other than phytate, to express phytase activity. The repression of phytase synthesis by inorganic phosphorus was not detected during fermentation with pea flour as substrate and seemed to be less significant with a higher composition complexity of the substrate. None of the tested lactic acid bacteria strains showed phytase activity. CONCLUSIONS: The methodology for the phytase screening procedure was shown to be critical. Some of the screening methods and media used in previous publications were found to be inadequate. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper highlights the pitfalls and difficulties in the evaluation of phytase production by micro-organisms. The study is of great importance for future studies in this area.

Separation and characterization of two chemically distinct lipopolysaccharides in two Pectinatus species.

Lipopolysaccharides (LPS) from the type strains of the anaerobic beer spoilage bacteria Pectinatus cerevisiiphilus and P. frisingensis were extracted with the 5:5:8 volume ratio modification of the phenolchloroform-petroleum ether method (H. Brade and C. Galanos, Eur. J. Biochem. 122:233-237, 1982). Sequential precipitations of LPS with water and acetone from the phenol phase yielded LPS which differed in that water-precipitable material (LPS-H2O; 0.1 to 0.4% of the dry weight of the cells) was rough-type LPS, whereas acetone-precipitable material (LPS-Ac; 4.6 to 5.8% of the dry weight) contained both rough-type LPS and high-molecular-weight material resembling smooth LPS. The LPS were chemically characterized, and they contained D-glucosamine, 4-amino-4-deoxy-L-arabinose, 3-deoxy-D-manno-2-octulosonic acid, D-fucose, D-galactose, D-glucose, D-mannose, and phosphate. D-Fucose was present mostly in LPS-Ac, suggesting that it is a constituent of the O antigen. The major fatty acids were ester- and amide-linked (R)-3-hydroxytridecanoic and ester-linked undecanoic acids, with minor amounts of ester-linked tridecanoic and (R)-3-hydroxyundecanoic acids. The chemical compositions of LPS-H2O and LPS-Ac suggested that they differ not only in their smooth or rough nature but also in the structure of their core regions. This may explain their different precipitabilities from the extraction mixture. The extraction method was also shown to be applicable to the isolation of smooth-type LPS from Salmonella enterica serovar Typhimurium. Extraction of two Typhimurium strains carrying chemically different O antigens resulted in high yields (8% of the dry weight) of LPS. Strain SH2183, which contains the relatively hydrophobic O-4,5,12 antigen yielded almost exclusively LPS-Ac, whereas the LPS of strain SH5770, which has a hydrophilic O-6,7 antigen, was exclusively LPS-H2O. No fractionation to smooth and rough LPS occurred with the Typhimurium strains.

New types of antimicrobial compounds produced by Lactobacillus plantarum.

New types of antimicrobial compounds were identified in the culture filtrate of Lactobacillus plantarum VTT E-78076. Activity was detected in the low molecular mass fraction separated by gel chromatography. This fraction totally inhibited the growth of the Gram-negative test organism, Pantoea agglomerans (Enterobacter agglomerans) VTT E-90396. Characteristic compounds from this fraction were identified by GC/MS-analysis and the identification was confirmed using pure commercial reference compounds in identical chromatographs and in antimicrobial tests. The active fraction included benzoic acid (CAS 65-85-0), 5-methyl-2,4-imidazolidinedione (CAS 616-03-5, methylhydantoin), tetrahydro-4-hydroxy-4-methyl-2H- pyran-2-one (CAS 674-26-0, mevalonolactone) and 3-(2-methylpropyl)-2,5-piperazinedione (CAS 5845-67-0, cyclo(glycyl-L-leucyl)). These compounds in concentrations of 10 ppm inhibited growth of the test organism by 10-15% when acting separately, but 100% when all were applied together with 1% lactic acid. The inhibition was 40% by 1% lactic acid alone. The compounds were also active against Fusarium avenaceum (Gibberella avenacea) VTT-D-80147. The inhibition was 10-15% by separate compounds in concentrations of 10 ppm and maximally 20% in combinations. Fungal growth was not inhibited by lactic acid. Inhibition by unfractionated Lact. plantarum culture filtrate was 37% and by the low molecular mass fraction, 27%.

Antifungal activities of two Lactobacillus plantarum strains against Fusarium moulds in vitro and in malting of barley.

AIMS: The Lactobacillus plantarum strains VTT E-78076 (E76) and VTT E-79098 (E98) were studied for their antifungal potential against Fusarium species. METHODS AND RESULTS: In vitro screening with automated turbidometry as well as direct and indirect impedimetric methods clearly showed Lact. plantarum cell-free extracts to be effective against Fusarium species including Fusarium avenaceum, F. culmorum, F. graminearum and F.oxysporum. However, great variation in growth inhibition was observed between different Fusarium species and even between strains. The antifungal potential of Lact. plantarum E76 culture, including cells and spent medium, was also examined in laboratory-scale malting with naturally contaminated two-rowed barley from the crops of 1990-96. The growth of the indigenous Fusarium flora was restricted by the addition of Lact. plantarum E76 to the steeping water. However, the antifungal effect was greatly dependent on the contamination level and the fungal species/strains present on barley in different years. CONCLUSIONS: Lactobacillus plantarum strains E76 and E98 had a fungistatic effect against different plant pathogenic, toxigenic and gushing-active Fusarium fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study indicates that Lact. plantarum strains with known and selected characteristics could be used as a natural, food-grade biocontrol agent for management of problems caused by Fusarium fungi during germination of cereals.

Malt sprout extract medium for cultivation of Lactobacillus plantarum protective cultures.

AIMS: The aim was to develop a cheap cereal-based alternative medium for the large-scale production of biopreservative Lactobacillus plantarum VTT E-79098. We examined the effect of growth medium and pH control on the cell yield of Lact. plantarum E-79098 and the antimicrobial activity of the cell-free extracts. METHODS: Fermentations using a novel Malt Sprout Extract Medium (MSE) were performed with different pH regimes. The antimicrobial activity of the cell-free extracts against Pantoea agglomerans VTT E-90396 and Fusarium avenaceum VTT D-80147 was assessed with automated turbidometry. SIGNIFICANCE AND IMPACT OF THE STUDY: When compared with MRS, the MSE medium cultures produced equal growth yields of Lact. plantarum VTT E-79098 and enhanced antimicrobial potential against the Gram-negative bacterium P. agglomerans and a Fusarium fungus. The MSE medium can be used as a low-cost alternative to MRS for producing high cell yields and good antimicrobial activity of Lact. plantarum.

Sequence of the gene encoding the 16S rRNA of the beer spoilage organism Megasphaera cerevisiae.

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.

Protein modification by fermentation: effect of fermentation on the potential allergenicity of pea.

The effect of fermentation on components of potential significance for the allergenicity of pea was analyzed. Pea flour was fermented with three lactic acid bacteria, Pediococcus pentosaceus, Lactococcus raffinolactis, and Lactobacillus plantarum, and two fungi, Rhizopus microsporus, var. oligosporus and Geotrichum candidum. Residual antigenicity against antipea antibodies was reduced to 10% by the three lactic acid bacteria and R. microsporus. Reactions to anti-pea profilin and anti-Bet v 1 were still detectable after fermentation. The contents of lectin and pea protease inhibitor were not reduced by the microorganisms.