VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

Results: Out of 210 samples of raw goat milk 9 samples showed thepresence of Listeria monocytogenes which confirmed by Micro- ID Listeriakit. All the isolates of Listeria monocytogenes were beta haemolytic andpositive for CAMP reaction. All the isolates were negative for phenylalanine deaminase, ornithine decarboxylase, lysine decarboxylase, malonateutilization and beta galactosidase tests. These were also negative for acidproduction from arabinose, D-xylose, mannitol, soluble starch and sucrosebut acid was produced in rhamnose, salicin, and trehalose. Hydrogen sulfideproduction was recorded in tripticase soy broth with lead acetate paper stripsbut negative with triple sugar iron agar. Out of 9 isolates of L.monocytogenes only one produced acid from lactose. In serotyping all theisolates were serotype 4b.Conclusion: The Results showed the presence of Listeria in goat milk whichmay act as source of infection in man. The use of Micro- ID Listeria kits forisolation of Listeria monocytogenes not only decrease the isolation time butalso its sensitivity is high. The problem of these kits is that before usingthem for haemolytic activity of the organism CAMP test should be done.FMP010Micro-Raman spectroscopy - A promising technique foridentification of milk-extracted pathogensS. Meisel* 1 , S. Stöckel 1 , M. Elschner 2 , F. Melzer 2 , P. Rösch 1 , J. Popp 1,31 Institute of Physical Chemistry, Friedrich-Schiller-University, Jena,Germany2 Institute of Bacterial Infections and Zoonoses, Friedrich-Löffler-Institute,Jena, Germany3 Institute of Photonic Technology, Jena, GermanyIn matters of quality assurance and hygienic standards, separation,concentration and detection of small numbers of microbial pathogensdirectly from milk without culture enrichment bear a big challenge for thefood industry. Additionally the current tense political situation induces aheightened risk of bioterroristic attacks in this manufacturing branch. Forexample, a deliberate attack with Brucella melitensis, causing the highlyinfective zoonotic disease brucellosis, on the milk-producing industry couldcause considerable damage on economy and public health. As a potentialbio-weapon this agent gained notoriety for being hardly detectable.This implies a necessity for a fast and reliable identification technique, butmicrobiological approaches are demanding and time-consuming so far. Alsoother more sophisticated and sensitive molecular genetic or immunologicalmethods are cost-intensive and pre-cultivation is still necessary. Herevibrational spectroscopy offers a promising opportunity, since it is nondestructiveand achieves high specificities [1]. Especially Ramanspectroscopy enables a striking reduction of preparation and analysis timesand has already verified its feasibility in this matter [2]. Since micro-Ramanspectra provide a fingerprint of the total molecular composition of singlecells, they inherently contain all information needed to accurately identifymicroorganisms down to subspecies level [3].In this contribution Brucella spp. and closely related species isolated fromspiked milk samples were identified successfully. For this purposeappropriate inactivation and extraction steps were developed with respect tothe compatibility towards Raman spectroscopic measurements. Abeforehand established database with Raman spectra of a various number ofGram-negative microorganisms together with chemometrical calculationslike linear discriminant analysis provide the basis for an identification withinhours.Acknowledgement: Funding of the research projects 13N9547 and13N9549 from the Federal Ministry of Education and Research, Germany(BMBF), is gratefully acknowledged.[1] Harz, M., et al (2009): Vibrational spectroscopy - a powerful tool for the rapid identification ofmicrobial cells at the single-cell level. Cytometry, Part A, 75A:104-113.[2] Rösch, P., et al (2003): The identification of microorganisms by micro-Raman spectroscopy.Journal of Molecular Structure 661:363-369.[3] Stöckel, S., et al (2010): Raman spectroscopic evaluation of different inactivation methods onbacterial endospores. Applied and Environmental Microbiology 76:2895-2907.FMP011Specific enumeration of Lactobacillus plantarum starterculture in the malolactic fermentation of Grauburgunderwhite wine using quantitative PCRS. Krauss* 1 , M. Huch 1 , M. Du Toit 2 , C. Franz 1 , G.-S. Cho 11 Max Rubner Institute, Safety and Quality of Fruit and Vegetables,Karlsruhe, Germany2 Institute for Wine Biotechnology, University of Stellenbosch, StellenboschSout Africa, South AfricaThe Lactobacillus plantarum B188 strain isolated from wine wasinvestigated for use as a starter culture in the malolactic fermentation ofGrauburgunder white wine. In order to identify this strain amongautochthonous wine lactic acid bacteria, a method was developed which wasbased on quantitative PCR. For this, the qPCR primers needed to target astrain-specific sequence, which would allow discrimination between thisstrain and other Lactobacillus plantarum strains that might be present in thefermentation. Lactobacillus plantarum B188 was determined to contain aplasmid that could serve as a specific target sequence for qPCR. A randomlyamplified polymorphic DNA (RAPD) PCR was performed using primerLB2 and the plasmid from strain B188 as a template. The randomlyamplified fragments were cloned into a TA cloning vector and sequenced. ADNA fragment which did not reveal homology to other DNA sequences inthe GenBank databank was used as a target for strain-specific qPCR. A 50 LGrauburgunder white wine fermentation was started in the autumn of 2010with approx 10 8 cfu/ml three days following the start of the alcoholicfermentation. The lactic acid bacteria counts were performed in regularintervals using plate counting on de Man, Rogosa and Sharpe (MRS) agar.The microbial counts were assessed in paralell using quantitative PCR. TheqPCR results were correlated to microbial counts with the aid of apreviously established standard curve that was generated from DNA ofknown amounts of live Lactobacillus plantarum B188 bacterial cells. As thebackground lactic micropopulation in the wine fermentation was negligible,as determined by a control fermentation without starter culture, we couldshow that the viable plate counts correlated well with quantitative PCRenumeration of the L. plantarum B188 strain.FMP012A novel Muroendopeptidase from Steptomycesalbidoflavus DSM 40233 and its Application in GrowthInhibition of Wine-relevant BacteriaE. Gasser*, P. Pfeiffer, H. KönigInstitute of Microbiology and Wine Research, Johannes-Gutenberg-University, Mainz, GermanyIn Germany, winemaking has long tradition. The wine-related microbiotasuch as yeasts, lactic acid bacteria and acetic acid bacteria can largelyinfluence the wine quality. Some are well known for their off-flavour orbiogenic amine production, which can lead to wine spoilage. Therefore it isnecessary to inhibit the growth of undesired microbes. This is done so far bysulphuring or the addition of lysozyme. However, these measures are limitedin their efficiency. Sulphuring can cause problems with the alcoholicfermentation because of the partial or total growth inhibition of the wineyeasts. Lysozyme does not attack many wild type strains of lactic acidbacteria due to modifications of their cell wall composition. Furthermorethese treatments may interfere with the health of the consumer. Sulfur maylead to incompatibility ans lysozyme may cause allergic reactions. In thisstudy an alternative method will be described. Bacteria of the genusStreptomyces are able to produce hydrolytic exoenzymes. A novelmuroendopeptidase from the supernatant of Streptomyces albidoflavus wasisolated by cation exchange chromatography and gel filtration. Thebiochemical characteristics of the enzyme were determined. Under theconditions of winemaking such as low pH and low temperature values it isrelatively stable and it exhibits high lytic activity against wine-relatedorganisms. N-terminal sequencing showed a similarity with ametalloprotease of the family M23 from Streptomyces albus J1074. Theexact cleavage site of this protease in the cell wall of Micrococcus luteuswas determined. In terms of winemaking, the use of this enzyme is muchmore effective than the use of lysozyme due to the higher lytic activity. It ismuch more stable and lysis a broader spectrum of target microbes.spektrum | Tagungsband 2011