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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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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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Application of magnetic particles for isolation and purification of DNA
Němeček, Milan ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With a development of molecular biology methods it is an increasing interest in new procedures of DNA isolation of high quality. DNA isolation is performed on crude cell lysates by many techniques e.g. phenol extraction, salting out or adsorption on solid phase. Classical DNA isolation, such as phenol extraction is quite complicated and time consuming. New alternative methods of DNA isolation was development using reverse immobilizing DNA to a solid phase. Widespread is the use of the magnetic particles as carriers, which allow the isolation of DNA in high quality directly from crude cells lysates of complex samples. The current method of DNA adsorption onto the surface of magnetic particles does not provided sufficiently pure DNA for analysis of some comlex samples (e.g. food). Some inhibitors of the polymerase chain reaction (PCR) are apparently adsorbed onto the tube wall and the next step of DNA elution leads to their release into the solution and cpnsequent negative effect on quality of DNA (e.g. decreasing of PCR amplification). The principle of the developed procedure is design a device, which utilizes transfer of magnetic particles by paramagnetic newddle from one to another Eppendorf tube, in which further processing of the sample extends. Transfer of magnetic particles with DNA using needle prevents transmission of contaminating impurities. The proposed device allows to realize above-mentioned procedure. The functionality of the device being tested in the isolation of plasmid pUC19 DNA from crude lysates of E. Coli JM 109 (pUC19).
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Identification of probiotic lactobacili in dairy products
Dofková, Květoslava ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
The genus Lactobacillus is part of intestinal microbiota which constitution plays important role in health and well-being of the host. Probiotic properties of lactobacili have been studied in lot of studies and clinical effects (immune enhancement, prevention of diarrhoeal disease etc.) of some probiotic species have been documented. Therefore bacteria of genus Lactobacillus are used in the production of fermented dairy products and probiotic preparations. PCR method based on amplification of 16S – 23S rRNA intergenic spacer regions was developed for identification of Lactobacillus species presented in dairy products. Whole DNA obtained by phenol extraction from dairy product Actimel Natur was used in this work as template for PCR. Purified DNA was amplificated by genus-specific PCR. Bacteria of genus Lactobacilllus were detected due to the detection of specific PCR product. Using species-specific PCR probiotic species Lactobacillus casei was detected in the Actimel Natur in quantity that approximately corresponded to declared amounts.
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The use of magnetic microparticles for bacterial DNA isolation
Hrudíková, Radka ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was testing of two types of magnetic mikrosheres functionalised with –COOH groups for the isolation of bacterial DNA. Isolation of DNA was carried out from crude lysates of cells prepared from pure culture of Lactobacillus paracassei RL-10 in the presence of binding buffer with 2 M NaCl and 16% PEG 6000. The influence of RNA degradation by enzyme RNase A on the amount of isolated DNA was investigated. It was estimated that RNA degradation affects the amount of DNA isolated. The amount of DNA depended on the type of microparticles. Higher amounts of DNA were isolated using particles with higher content of carboxyl groups. DNA applicable in PCR was isolated using both types of microsheres. In next part of the work, microparticles functionalised with –NH2 groups were used to DNA isolation using electrostatic forces. It was shown that buffer with lower pH is suitable for DNA adsorption onto magnetic microparticles.
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Identification of microorganisms in cosmetic products with probiotics
Langová, Denisa ; Španová, Alena (referee) ; Trachtová, Štěpánka (advisor)
Probiotics products are an integral part of the current market. Products containing probiotics cultures are also cosmetic products. The first part of the study focuses on testing of bacterial survival abilities in the environment of preservatives presented in cosmetic products. Collection strains of genus Lactobacillus were used for these tests. Another part of the study focuses on isolation of bacterial DNA from probiotic cosmetic products Ryor, Yoghurt of Bulgaria, FeminaMed and Lactovit Activit in PCR-ready quality. DNA was isolated by fenol extraction and with magnetic particles. Presence of bacteria was proved by genus and species specific PCRs Lactobacillus. Species specific PCR for identification of Lactobacillus pentosus was optimalized. Species identification was in accord with data declared by producers.
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Identification of lactic acid bacteria in hard cheeses using amplification methods
Herzogová, Jitka ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
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.
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The application of magnetic particles for DNA isolation from cereal products
Starenkova, Anastasiia ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The thesis has been focused on micro method for isolation of PCR- ready DNA iusing magnetic particles from cereal products. Cereal biscuits and cereal products for babies were selected for the analysis. These were homogenized using plastic copist in lysis buffer with cetyltrimethylammonium bromide (CTAB). The homogenates were purified using chloroform- octanol mixture. The effect of isopropanol in the preparation of homogenates was tested, too. Homogenates were used for DNA isolation by magnetic particles. Two ways to isolate magnetic particles with bounded DNA (magnetic separator and magnetic needle have been tested. Isolated DNA was analyzed spectrophotometrically its concentration and purity were assessed. . After that, amplification of the DNA was tested in PCR. Two sets of primers specific for plant ribosomal DNA were used for their amplification. PCR products of expected length 700 bp and 220 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from seeds and cereal products using magnetic particles was in PCR-ready quality.
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Intestinal metabolism of bilirubin
Jirásková, Alena ; Branny, Pavel (advisor) ; Španová, Alena (referee) ; Pátek, Miroslav (referee)
CONCLUSIONS In this study we focused on the process of bilirubin reducfion catalyzed by an anaerobic intestinal bacterium C' peýingen.s. We aimed to undertake analysis of bile pigments metabolized by C. perfringens and their respective reduction products and to identify gene(s) encoding protein(s) involved in metabolism of bilirubin. Analysis of bile pigments metabolized by C. perfringens and their respective reduction products 1) The C. perfringens strain BRI isolated from neonatal stools reduces a variety of different bile pigments indicating that this broad substrate speciÍicity could be an effective tool for disposal of electrons produced in catabolic pÍocesseswithin thesebacteria. 2) The examined strain reduces UCB only to the level of urobilinogen. Other bacterial sfoains and species, absent in neonates, are presumed to be essential for catabolism to the level ofstercobilinogen. Identification of gene(s)involved in bilirubin metabolism 1) The C. perfringens strain BRl is resistant to the transformation of plasmid DNA mediatedby electroporation and thereforeit is not a candidate suitable for transposonmutagenesis. A transformab|e C. peýingens P90.2,2. strain was found to reduce bilirubin. Rapid and simple method suitable for electroporation of this strain was developed providing transformation...
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Effect of probiotic bacteria Lactobacillus paracasei subsp. paracasei on the development of acute colitis in BALB/c mice.
Součková, Martina ; Jílek, Petr (advisor) ; Španová, Alena (referee)
Inflammatory bowel disease - ulcerative colitis and Crohn's disease - are chronic relapsing intestinal disorders of unknown etiology. Acute ulcerative colitis was induced in BALB/c mice by 3 % dextran sulfate sodium (DSS) administrated in drinking water for 5 days. The effect of Lactobacillus paracasei subsp. paracasei RL14-P, a bacterium with potential probiotic influence, on the development of inflammation was evaluated. Lactobacillus was administrated intragastrically to mice on days 1, 4 and 8 and the administration continued for next 5 days during DSS treatment. Control groups received instead of lactobacillus MRSC medium or phosphate-buffered saline according to the same schedule. Clinical symptoms such as diarrhoea, rectal bleeding and loss in body weight were evaluated daily. Cytokine secretion (IL-6, IL-10 and TNF-α) was determined in supernatants of 48 h cultivated splenocytes, mesenteric lymph node lymphocytes and colon pieces by ELISA. The impairment of colon descendens was evaluated histologically. Lactobacillus paracasei subsp. paracasei RL14-P administrated mice remained healthy without signs of the intestinal inflammation. We did not find the shortening of colon (a sign of inflammed intestine) in lactobacillus-treated mice. We observed that administration of probiotic bacteria...
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