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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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Probiotic lactic acid bacteria in food products
Sásková, Denisa ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
In food industry molecular genetic methods based on DNA analysis are used for identification of probiotic microorganisms. An example of these methods is the polymerase chain reaction, in which specific fragments of DNA are amplified using short oligonucleotide primers. The teoretical part of this bachelor thesis follows probiotic bacteria, their features, beneficial health effects and use in food and clinical applications. The experimental part of the thesis focuses on an analysis of two probiotic dietary supplements. DNA was isolated from these products by method of phenol extraction. The use of PCR confirmed the presence of DNA of probiotic bacteria of genera Lactobacillus and Bifidobacterium.
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Application of real-time PCR in microbiological analysis of food-stuffs
Novotná, Eva ; Michaela, Kotianová (referee) ; Španová, Alena (advisor)
Probiotic lactic acid bacteria of genus Lactobacillus play an important role in the digestive tract of human, e.g. protect against pathogenic microorganisms. Lactobacilli are often present in various food products. Lactic acid bacteria of the genus Lactobacillus can be detected by polymerace chain reaction (PCR) using specific primers LbLMA1 and R16. The bacterial DNA was isolated from Actimel Natur probiotic product by the phenol extraction method. DNA was amplified using real time PCR. Specific PCR products were detected using fluorescent intercalaction dye SybrGreen. The specific PCR products were verified by melting curve analysis (Tm 85°C) and by agarose gel electrophoresis (PCR products of 250 bp was amplified).
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Identification of lactic acid bacteria in food products using amplification methods
Jurečková, Nela ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
The aim of the study was isolation of genomic DNA from 7 milk products (yogurts/ yogurt milk) in quality for use it in PCR. Magnetic microparticles (P(HEMA-co¬-GMA)) were used for the DNA isolation. DNA was reversibly adsorbed to the microspheres in the presence of 8% poly(ethylene glycol) PEG 6000 and 5 M sodium chloride. The adsorbed DNA was released from the microspheres in a low ionic strength TE buffer and use as DNA matrix in genus specific PCR Lactobacillus. The presence of DNA of genus Lactobacillus was detected in all analysed products.
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Identification of lactic acid bacteria in probiotic products (tablets) using amplification methods
Balogová, Petra ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Lactobacillus have been playing a crucial role in the fermentation processes for millenia. Their probiotic effects have been studied deeply and nowadays they are used in the production of fermented products and food adjunkt including probiotic preparations. This thesis was focused on bakteria of genus Lactobacillus in probiotic preparations (tablets). Polymerase chain reaction method (PCR) based on amplification of DNA was used to identify lactobacillus in seven probiotic preparations. As DNA matrices whole DNA recovered from tablets with the help of magnetic microparticles (P(HEMA-co¬-GMA) coated by carboxylic groups was used into a PCR mixture. Genus specific PCR products (250 bp) were detected with the help of gel electrophoresis on agaróse. DNA of genus Lactobacillus was proved in all but one product. In this product only probiotic bacteria were declared presence without family identification was proved. It contained different bakteria then bakteria of Genus Lactobacillus probably.
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Plasmide DNA Isolation from Bacteria and Transfection to HEK293 Cell Line
Karmazínová, K.
Isolation DNA is a one of the basic methods in molecular biology. There are several methods of DNA amplification and isolation. In this paper phenol-chloroform extraction of three plasmid types is used: Channelrhodopsin-2, ASAP and KIR. Seven plasmids were isolated in total. These plasmids are then validated using gel electrophoresis. Successfully isolated plasmids are then transfected to HEK293 and taken on confocal microscope 24 hours after transfection.
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PCR-based detection of hidden carriers of cataracts in dogs
FARKOVÁ, Barbora
The hereditary cataract is one of the most common eye disease in dogs. The expansion of this disease in the Staffordshire bullterrier breed has been so massive that in the Czech Republic was introduced the rule of mandatory testing of at least one of a breeding pair. This is a degenerative disease of the lens causing total blindness of the affected animal within three years. Since some time ago there are no more dogs affected by the disease in the Czech Republic, there are however still hidden carriers which need to be discovered to the complete extinction of the disease in the genome. The goal of this study was to test simple ways of collecting biological samples, try them in practice and to verify whether they are suitable for the DNA isolation and also to test an alternative method of molecular detection of this disease. In total there have been 23 buccal swabs collected from male and female Staffordshire Bullterrier examples. The detection of the hidden carriers of the hereditary cataract was carried out by PCR analysis with specific primers. The obtained amplicons were detected by both gel and chip electrophoresis and by using fragment analysis. This detection of the carriers was based on the presence of two amplicons (heterozygotes). I came to conclusion that to detect hidden carriers it is neccessary to use the fragment analysis because of the difference of only one base in the reference section of DNA. Neither gel nor chip electrophoresis does provide sufficiently high resolution and it is not possible to detect two fragments that differ only by one bp. As the most appropriate sampling method I have chosen the buccal smear by cytological brush followed by isolating the DNA by Chelex with purification of the sample subsequently.
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Methods of DNA extraction from the fresh papaya fruit and from the candied
Ovesná, Jaroslava ; Hodek, Jan
Aim the work is to provide a working procedure for extraction of amplifiable DNA form papaya fruits (Carica papaya), which are a poor source of DNA. Methods are optimised for DNA extraction from papaya fruits and stones and from candied papaya- these both were predicted as suitable commodities for an eventually screening of non-approved GM papaya in the market of Czech Republic. The methodology was prepared on basis of demand of reference laboratories of public service, which are involved in GMO analyses and also as a flow-work of Methodics for Detection of GM papaya 55-1 and 63-1 (Hodek, Ovesná, Pavlátová 2008) and scientific publication Detection of transgenic papaya lines: extraction protocol optimisation and verification of DNA quality by PCR assay (Ovesná, Hodek 2009).
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