Satokari, Reetta

Helsingin yliopiston digitaalinen arkisto

HeIsncIN YLIoPISTo - HnmncToRS I.JNIVERSITET _ UNIVERSITY oT HBI.SINTIMuito tietojâ - Ovriga uppgifter - Additional informationSupervisors: Professor Atte von Wright and Docent Auli HaikaraSäilytyspaikkâ - Föryüingsställe - Wherc depositedLibrary of the Division of the General MicrobiologyAvainsanat - Nyckelord - Keywordsbeer spoilage bacteria, PCR, molecular biological typing methodsTiivistelm¿i - Referat - AbsFactBeer is an unfavourable environment for microbes, but nevertheless some bacteria are able to grow in it andcause defects in the product quality. Lactic acid bacteria (LAB) and strictly anaerobic bacteria ofthe generaPectinatus and Megasphaera are among the most prevalent beer spoilage organisms. Pediococci, mainlyPediococcus damnosus, cause buttery flavour (diacetyl) and ropiness in beer. Inctobacillus brevis andInctobacillus lindneri are among the most important beer spoilage lactobacilli causing acidity and turbidity intothe product. Pectinatus spp. and Megasphaera cerevisiae spoil beer by causing turbidity and producingmetabolic end-products, which give beer particularly unpleasant off-flavour and odour.Detection of beer spoilage bacteria is currently based on cultivation techniques, which are time-consuming andnon-specific. MoreoveL, the detection of Pectinatus and Megasphaera is complicated due to the anaerobicconditions required for their cultivation. In this study, a detection assay based on polymerase chain reaction(PCR) was developed for the specific detection of L. brevis, L. lindneri, Pectinatus spp. and M. cerevisiae inbeer. The species/genus-specific primers were designed/targeted on the 165 rDNA and they showed goodspecifîcity for their target organisms. A sample treatment method applicable for routine quality control workwas set up for the detection of bacteria in beer. To provide the adequate sensitivity (< 10 cfu/ bottle ofbeer) apre-enrichment step was included into the assay. In the detection ofstrictly anaerobic beer spoilers, considerabletime saving was achieved with the PCR assay compared to the presently used cultivation techniques. Some timesaving was also achieved in the detection oflactobacilli.In order to predict the spoilage potential of a bacterial contaminant it must be identified. The identification ofmany LAB by traditional microbiological methods and biochemical characterization is cumbersome and time-consuming. In this work, the suitability of ribotyping technique for the identification of pediococci wasevaluated- Genetic fingerprinting was performed with an aut,omated equipment RiboPrinterR (Qualicon, USA).Altogether twenty-seven stràins representing six Pediococcøs species were ribotyped and some heterogeneitywas found in the ribotypes within one species. Automated, highly søndardized ribotyping was found to be asuitable tool for the rapid identification of pediococci. However, due to the genotypic heterogeneity withinspecies it is essential that the identification fingerprint library is large and each species is represented by severalshains.Further, three molecular techniques, randomly amplified polymorphic DNA (RAPD), ribotyping and pulsedfietd gel electrophoresis (PFGE) were compared in typing of closely related lactobacilli strains. Twenty-fourstrains of the Lactobacillus casei grotp were used in the study. All three typing methods showed gooddiscrimination capacity and differentiated lactobacilli below species level. PFGE was found to be the mostdiscriminative method, followed by ribotyping and RAPD, In brewing microbiology different typing methodscan be applied e. g. for studying contamination routes ofspoilage bacteria.Työn laji - Arbetets a¡t - lævelLicentiate thesisOppiaine - Uiroämne - SubjectGeneral microbiologyTyön nimi - Arbetets titel - TitleNucleic acid-based techniques for the detection and typing of beer spoilagerbacteriaTekijä - Författare - AuthorSatokari, ReettaTiedekunta./Osasto - FakultelSektion - FacultyFaculty of ScienceAika - Datum - Month and yearAueust 1999Sivumäãrä - Sidoantal- Number of pages45 + appendexes I-VIåitos - Institution - DepârtmentDepartment of Biosciences,Division of General Microbiolosv

Nucleic acid-based techniques for the detection and typing of beer spoilage bacteria

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Helsingin yliopiston digitaalinen arkisto