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Recombinant probiotics
Surá, Tereza ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Theoretical part of this thesis focuses on present state and research of recombinant probiotics and their use in food industry and health care. It also concentrates on their beneficial effects on the health of individuals. The experimental part focuses on the identification of specific bacterial strain in a probiotics food supplements. The DNA was isolated from these products by use of magnetic microparticles and obtained DNA was subsequently analysed through polymerase chain reaction.
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Probiotics and their use in food industry
Diado, Aleksandra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic bacteria are defined as live microorganisms, which when consumed in the determining quantities, have healthy and beneficial effects. Most of probiotics belongs to the genera Lactobacillus and Bifidobacterium. These and other genera of microorganisms are successfully used in industry, including food industry at present. Probiotics are used primarily in dairy products and food additives in food idustry. Probiotic bacteria, like other organisms, can be to identifie by PCR method that allows amplifying specific regions of DNA. Polymerase chain reaction was performed after DNA isolation from bacterial cultures of three strains using phenol extraction method. PCR specific for the domain Bacteria and genus-specific PCR were used for the confirmation of the presence of bacteria of the genus Lactobacillus.
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Real-time PCR and it´s use in food processing
Tomanová, Barbora ; Pravečková, Martina (referee) ; Trachtová, Štěpánka (advisor)
Polymerase chain reaction (PCR) is a method abundantly used in molecular diagnostics. PCR in real time or quantitative PCR (qPCR) is one of its modifications and thanks to its advantages it finds still wider utilization nowadays. It finds its use in the food-processing industry too with relatively precise detection, identification, and qualification of both desirable and undesirable components in food, which often brings considerable difficulties and leads to an intensive development of this method. In the experimental part a DNA was isolated from dairy product Bio Via Natur drink for its further processing by means of PCR and gain more detailed information about a bacterial composition using real-time PCR and HRM analysis.
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Testing of lactic acid bacteria Lactobacillus genus for bacteriocins production
Volecová, Veronika ; Němcová, Andrea (referee) ; Trachtová, Štěpánka (advisor)
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.
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The used of magnetic microparticles for isolation and prove of probiotic bacterial DNA in meat
Vašíček, Roman ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
This thesis deals with the isolation of probiotic DNA from meat products and its assesment by PCR methods. In this thesis is developed homogenization of samples of sausages with kopist, preparation of sausage cells lysates and isolation of DNA by using of magnetic microparticles. The DNA was isolated from sausage lysates by using magnetic microparticles. Isolated DNA was further amplified in genus and spesies-specific PCR methods. In tested products was proven presence of DNA of domain Bacteria, type Lactobacillus and Bifidobacterium. In one product was proven presence of species Lactobacillus acidophilus and Bifidobacterium animalis.
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PCR identification of nonpathogenic bacteria strains in cheeses
Jurečková, Nela ; Doušková, Dagmar (referee) ; Španová, Alena (advisor)
Different species of genus Bifidobacterium are part of human and animal intestinal flora. These bacteria have benefit effects and therefore they are used in foods and pharmaceutical products as probiotics. Cheese is now suitable as a probiotic matrix except yoghurts and fermentated milks. This diploma thesis was focused on optimalization of DNA isolation from bacteria of genus Bifidobacterium. Magnetic microparticles (P(HEMA-co¬-GMA)) were used for DNA isolation in presence of 8% polyethyleneglycol PEG 6000 and 5 M sodium chloride. Phenol extraction weas also used as an isolation method. Isolated DNA was used for amplification in domain, genus and species specific PCRs. Optimized method was tested for detection of bacteria of genus Bifidobacterium in experimentaly prepared probiotic cheeses. These cheeses contained potential probiotic bacteria from Laktoflóra collection. Bacteria were identified into species using species specific PCR. Species Bifidobacterium animalis was identified in all samples of probiotic cheeses.
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Sample preparation for DNA analysis from foods of plant origin
Silná, Renata ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The isolation of high quality DNA is nessecary for many molecular biology applications. However, plant DNA contains high amonts of polysaccharides, polyphenols and various secondary metabolites, which decrease yield and quality of isolated DNA. The aim of this study was preparation of samples and different food matrices for DNA isolation DNA by magnetic particles. It was about 5 species of vegetable and 10 species of processed plant food. Homogenization of samples was performed in CTAB buffer. Isolation of plant DNA was performed by magnetic particles covered with carboxyl groups. All DNAs were isolated in conventional PCR qualities using primers for 700 bp amplicons, in the case of heat processed products for 220 bp ampilicons and for real time PCR. The efficiancy of separation of magnetic particles with DNA by magnetic separator and magnetic needle was compared. It was find out that DNA of higher purity was isolated using magnetic needle. The micromethod of isolation of plant DNA from homogenates with CTAB with magnetic particles is suitable for different processed food.
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Isolation of PCR-ready DNA from probiotic products for baby nutrition
Mantlová, Gabriela ; Havlíková, Šárka (referee) ; Španová, Alena (advisor)
The aim of thesis is focused on isolation of DNA in quality for polymerase chain reaction (PCR) and the identification of probiotic bacteria. From six probiotic supplements for children were isolated PCR-ready DNAs using magnetic carriers P(HEMA-co-GMA). Isolated DNA was amplified by genus-specific and species-specific primers. DNAs of Lactobacillus, Bifidobacterium and Streptococcus genera were identified as: L. acidophilus, L. rhamnosus, L. casei, B. bifidum, B. longum ssp. longum, B. breve, B. longum ssp infantis, B. animalis and S. thermophilus. The identification corresponded with the data declared by the producers.
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Detection of selected fruit species in plant-based foodstuffs through instrumental analysis and methods of molecular biology
Ondruch, Petr ; Langová, Denisa (referee) ; Fialová, Lenka (advisor)
Food is often subject to adulteration, including fruit products containing strawberries. This work deals with the detection of strawberries in model and real fruit products, so that possible falsification can be detected. The aim of the work was the preparation of model and real fruit products, their analysis using selected instrumental and molecular-biological methods and mutual comparison of the results of these methods. Model mixtures of fruit products were prepared and commercial ones were purchased. The content of polyphenols and flavonoids was determined by UV-VIS spectrophotometry, which were characterized in more detail by HPLC. DNA was isolated from the fruit purees, suitable primers confirmed the amplifiability and formation of a specific product for strawberry, and this product was verified by electrophoretic control. In the instrumental part, certain substances were detected by extraction from model and commercial mixtures and the HPLC method, and the amount of selected substances was determined. The lowest amount of strawberry DNA that could be detected by selected primers in the PCR method was 0.7 ng /µl, in commercial mixtures this DNA could not be detected. In the HPLC method, the strawberry was detected in all samples by focusing on a particular fingerprint and the amount of pelargonidin-3-glucoside in the samples. The instrumental method can be considered more suitable in this case.
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Application of the method PCR-HRM analysis to identify bacteria in foods and food supplements
Šurková, Alice ; Illková, Kateřina (referee) ; Trachtová, Štěpánka (advisor)
Theoretical part of the thesis was focused on foods and food supplements containing microorganisms, especially bacteria. Furthermore, the thesis deals with methods for identification of the bacteria, primarily polymerase chain reaction (PCR). The thesis also includes real-time PCR and is specially focused on high resolution melting analysis (HRM). During the experimental part, the DNA sample was isolated from a chosen probiotics product using magnetic microparticles. The concentration of the DNA sample was determinate and DNA was subjected to PCR with subsequent detection PCR products by agarose gel electrophoresis. To the results specify HRM analysis was then performed.
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