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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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Nucleic acid detection using magnetic microparticles
Pešková, Aneta ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
Modern and fast separation proces for nucleic acids isolation uses magnetic microparticles in routine laboratory practise. The comparision of DNA isolation efectivity by three types of magnetic microparticles P(HEMA-co-GMA) (A, B), PGMA and magnetic nanoparticles covered by poly-L-lysine (PLy) from probiotic products Pangamin was carried out. The quality and quantity of isolated DNA were verified by spektrofotometric measurments and gel electrophoresis. Possibility of amplifying of isolated DNA was verified by polymerase chain reaction (PCR) with specific primers for yeast and evaluated by agarose gel electrophoresis. The quantity of isolated DNA depended on the product and the type of magnetic particles. The highest amount of DNA was isolated by magnetic nanoparticles of poly-L-lysine (PLy) and magnetic microparticles P(HEMA-co-GMA) (A).
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PROBIOTIC GENES OF SIGNIFICANT LACTIC ACID BACTERIA IN FOOD
Konečná, Jana ; Ševčovičová,, Andrea (referee) ; Doškař, Jiří (referee) ; Španová, Alena (advisor)
Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol chloroform extraction and DNA precipitation in ethanol is time consuming and requires the use of toxic phenol. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solidphase DNA extraction. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed to the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this contribution, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.
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Foodborne Staphylococcus Aureus: Identification and Enterotoxin Production in Milk and Cheese.
Hrušková, Vendula ; Španová, Alena (referee) ; Kmet,, Vladimír (referee) ; Kaclíková, Eva (advisor)
Onemocnění z potravin (alimentární onemocnění) vyvolaná bakteriemi jsou stále aktuálním tématem v celosvětovém měřítku. Abychom zajistili výrobu zdravotně nezávadných potravin, je potřeba nových poznatků o virulenci patogenů, které by doplnily již známé skutečnosti o jejich růstu a přeživání v potravinách. Také potřebujeme vyvíjet rychlé a citlivé metody na detekci těchto patogenů. Dizertační práce popisuje metodu na detekci S. aureus v potravinách, která je založená na PCR v reálném čase ve spojení s namnožením v selektivním médium. Dále pojednává o vlivu environmentálních faktorů na růst S. aureus a tvorbu enterotoxinů v mléce a sýrech. Vyvinuli jsme rychlou a citlivou metodu na detekci S. aureus v potravinách s použitím selektivního namnožení a PCR v reálném čase. Nově vyvinutá metoda umožnila detekci S. aureus na druhý den od přijetí vzorku. Tato metoda může být použita jako rychlejší, citlivějsí a vysoce specifická alternativní metoda ke konvenční mikrobiologické metodě. Zkoumali jsme vliv tří různých teplot, 8°C, 12°C a 20°C na růst S. aureus a tvorbu enterotoxinu D v pasterizovaném mléce a na růst, expresi genu sed a tvorbu enterotoxinu D v tekutém médiu s extraktem z mozku a srdce (BHI). Experimenty byly prováděny v malých skleněných fermentorech po 6 dní. Genová exprese byla sledována pomocí qRT-PCR a tvorba enterotoxinu D byla měřena pomocí imunologické metody ELISA. Růstová křivka v BHI měla stejný průběh při 20°C a 12°C, ale v při 12°C začal růst se spožděním. Při 8°C nebyl pozorován žádný růst. Růst S. aureus v mléce byl ve srovnání s BHI menší. sed mRNA byla detekována při 20°C po 4 hodinách a při 12°C po 7 hodinách a produkce enterotoxinu se objevila v exponenciální fázi růstu. V mléce se produkce SED při 20°C a při 12°C objevila dříve, ale celkové množství vyprodukovaného SED bylo nižší než v BHI. Při 8°C nebyla pozorována žádná produkce SED stejně jako v BHI. Dále byl zkoumán společný vliv nízké teploty 12°C a přítomnosti kompetitivní doprovodné mikroflóry pocházející ze surového mléka na růst S. aureus a produkci enterotoxinu v pasterizovaném mléce. Byl pozorován inhibiční účinek na růst a produkci enterotoxinů a vliv kompetice byl výraznější než vliv nízké teploty. Produkce enterotoxinu byla nízká a odpovídala růstu. Snížením množství doprovodné mikroflóry a zvýšením inokula došlo pouze k nepatrnému zvýšení produkce enterotoxinu. V další fázi byly dva různé typy sýrů zaočkovány S. aureus za účelem simulace sekundární kontaminace při výrobě sýrů. Vzorky byly odebírány v průběhu 4 týdnů. Kritické faktory jako jsou kompetitivní mikrofóra nebo pH, které jsou zodpovědné za regulaci virulence S. aureus byly sledovány. Snažili jsem se rozlišit situace při kterých: (i) není pozorován růst, ale objevuje se produkce enterotoxinu a (ii) dochází k růstu ale bez produkce enterotoxinu.
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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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Plasmid DNA interactions with lanthanoide compounds
Gőghová, Sabína ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
In the theoretical part of bachelor thesis, literature was summarised on the isolation and purification of plasmid DNA, the application of lanthanides as non enzymatic nucleases and practical importance of lanthanide compounds in medicine. In the experimental part of the thesis, plasmid DNA pUC19 was isolated by the method of alkaline lysis. Isolated plasmid DNA was cleaved by neodymium trichloride at 70 °C in different intervals. Gel electrophoresis was used for evaluation of experiments.
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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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Identification of selected probiotic bacteria in food additives
Bubeníková, Lucia ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
Bifidobacteria and lactobacilli are gram-positive bacteria which belong to the lactic acid bacteria group. They are ordinary constituents of gastrointestinal microflora, play a pivotal role in human nutrition and health, prevent pathogen colonization and maintain normal mucosal immunity. In present days they are used in production of functional food products and pharmaceutical additives. Polymerase chain reaction (PCR) is used to the detection and identification of bacteria which belong to the genus of Bifidobacterium and Lactobacillus. Specific primers for each genus are used in PCR reactions for in vitro amplification of definite part of DNA. In this work, total DNA was isolated from three additives (Pangamin Bifi plus, Biopron Junior and Probiodom) by the method of phenol extraction and amplified using genus-specific PCR primers giving amplicons of 523 bp (Bifidobacterium) and 250 bp (Lactobacillus). Occurrence of bacteria of both genera was proved in all of the three tested probiotic products.
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The application of magnetic particle for DNA isolation from selekted probiotic products for children
Vozárová, Petra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
In the food industry, it is important to correctly identify the species of bacteria and thier properties so that they can be used as a probiotic in dietary supplements. This is performed using DNA diagnostics. In the experimental part, the DNA from four probiotic dietary supplements for children was isolated. Magnetic particles P(HEMA-co-GMA) were tested for isolation. Isolated DNA was amplified by PCR and the presence of DNA of genus Lactobacillus, Bifidobacterium and Bacillus was demonstrated in the products according to the data declared by the manufacturer. The presence of species L.acidophilus, B.animalis in accordance with the data on the product has been demonstrated by PCR with species specific primers. Using PCR, the presence of L.casei, which was declared by the manufacturer, has not been proven in one product at given experimental conditions.
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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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