Currently, there is a growing interest in the use of probiotic products, and there are many of them in the market. With the growing interest, greater emphasis is placed on the identification of declared probiotic microorganisms. Precise identification of microbial composition is often a difficult task and it requires more advanced methods especially in the field of molecular diagnostics. The diploma thesis was focused on the verification of the presence od declared probiotic microorganisms in probiotic food supplements GS Laktobacily Forte 21, Biopron 9 Premium and Linex® Forte. DNA was isolated from the complex matrices by phenol extraction, commercial kit and magnetic carriers F79/L3-PLL in the quality suitable for PCR. Subsequently, the isolated DNA was amplified by real-time polymerase chain reaction using genus- and species-specific primers. The specific PCR product was subjected to agarose gel electrophoresis, whereas species identification was not always in compliance with the data declared by producers. The next part of the thesis was focused on polymerase chain reaction with high-resolution melting analysis to distinguish bacterial strains belonging to the Lactobacillus group and to identify probiotic microorganisms present in the complex matrices of the probiotic food supplements. Eight primer sets were tested (V1F HRM a V1R-HRM, CHAU-V3F a CHAU-V3R, CHAU-V6F a CHAU-V6R, LAC2 a LAC4, LAC1 a LAC2, P1V1 a P2V1, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT). Three primer pairs (V1F HRM a V1R-HRM, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT) were evaluated as the most suitable for distinguishing Lactobacillus bacterial strains.

Diploma thesis was focused on identification of lactic acid bacteria of species Lactococcus lactis and subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris using species and subspecies specific polymerace chain reaction (PCR). PCR method was used for identification of bacteria of species Lactococcus lactis in 10 samples of hard cheeses. The method of sample preparation was evaluated for hard cheeses with the aim to receive sufficient amount of cells for the preparation of crude cell lysates. Whole DNA in quality suitable for PCR was separated using magnetic microspheres P(HEMA-co-GMA) in the presence of polyethylenglycol (PEG 6000) and sodium chloride. DNA isolated by phenol extraction was used as control of DNA isolation. PCR was used to the analysis of 7 strains of Lactococcus lactis from Collection of dairy microorganisms Laktofora (CCDM). Altogether 5 or 2 strains were identified into subspecies Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris, respectively.

The rapid and accurate identification of the bacterium of the genus Lactobacillus, which are an important part of the normal gastrointestinal microflora and fermented dairy products are currently mainly used amplification methods. The aim of the study was to analyze the possibility of resolution of selected bacterial strains of the genus Lactobacillus, using the metod of polymerase chain reaction in the real time combined with high resolution melting curve analysis (qPCR HRM). It was tested five primers designed for qPCR-HRM analysis of lactic acid bacteria. The specificity of the primers was also verified simultaneously using bioinformatic analysis. On the basis of analysis of the DNA were selected as the most appropriate primers P1V1/P2V1, V3F/V3R and V6F/V6R. The suitability of the primers V3F/V3R and V6F/V6R was verified on a complex sample of food supplement from which the DNA was isolated using magnetic particles. The presence of bacteria of the genus Lactobacillus was performed using high resoluting melting analysis curves. The obtained results were in agreement with the information given by the manufacturer.

Probiotic food products - food additives contain different species of probiotic bacteria. Accurate species identification with their characteristics is very important from the view of products quality. Methods of DNA diagnostics are used for these purposes. In this thesis DNA was isolated from 4 probiotic products. The presence of bacterial of genus Lactobacillus and species L. acidophilus, L. casei, L. plantarum, L. rhamnosus were detected in three products by PCR. This information was in accordance with the data provided by the manufacturer. Two sets of primers were used for identification of species. Using other primers sequences of genes such as bsh, lai and odc were detected in DNA isolated from the products. Differences were estimated among products concerning the detection of lai gene Lactobacillus acidophilus.

Antimicrobial substances, or bacteriocins are substances produced by probiotic lactic acid bacteria. They have a positive effect on the gastrointestinal tract of humans and are especially suitable for the food, but also the pharmaceutical industry. The aim of the thesis was the molecular identification of lactic acid bacteria of the Lactobacillus genus, species and their subsequent inclusion PCR method. Using the PCR method were tested also genes responsible for the production of bacteriocins. To confirm the production of bacteriocins has been selected the microbiological method, agarose droplet spot-test. In the present study also included the bioinformatics part to assess the specificity and non-specificity of the primers using in Primer-BLAST program.