Auli Haikara

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.

Real-time PCR for Quantification of Toxigenic Fusarium Species in Barley and Malt

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.

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.

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.

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.

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.

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.

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 brewerss yeast‐containing samples.

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.