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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 probiotic Bifidobacterium strains in dairy products
Zovčáková, Monika ; Horák, Daniel (referee) ; Španová, Alena (advisor)
Lactobacilli are dominant bacteria of the vaginal flora. Lactobacillus-containing probiotics products are used for the treatement and profylaxis of bacterial urogenital infections. This work is focused on DNA identification and species identification of probiotic bacteria in 5 different vaginal tablets using molecular-genetic methods. Total DNA isolated from complex matrix of vaginal tablets was used for amplification in polymerase chain reaction. DNA was isolated from crude cell lysates by magnetic particles P(HEMA-co-GMA) and by method of phenol extraction. Identification of species of probiotic bacteria was verified using genus-specific and species-specific PCRs. Results of bacterial identification obtained by PCR were compared with declared specification given by producers. Bacteria of genus Lactobacillus were proved in all tablets whereus species identification was in accordance with the stated composition in 1 tablet only.
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Identification of selected genes in lactic acid bacteria
Kristová, Mária ; Vojtíšková, Marie (referee) ; Španová, Alena (advisor)
Lactic acid bacteria are natural habitants of human gastrointectinal tract. Among the most important are bacteria of genus Lactobacillus and genus Bifidobacterium that contain a lot of probiotic species. Probiotic species are used as food supplements. This work was focused on DNA separation from crude cell lysates of 4 food supplements using magnetic carrier P(HEMA-co-GMA) covered by carboxyl groups. DNA was reversible adsorbed to the carriers in the presence of PEG 6000 (16%) and NaCl (2 M) (final concentrations) and eluted into TE buffer. Lysis of cells from food supplements was performed by lysozyme, SDS and proteinase K. The amount of lysozyme was optimalized. Concentration of separated DNA was measured by spectrophotometric method. The amount of isolated DNA was suitable for PCR. Isolated DNA was used for PCR with universal primers, PCR specific for genus Lactobacillus and genus Bifidobacterium and for 9 different species-specific PCRs: Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei/paracasei, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium infantis. Amplicons were detected by agarose gel electrophoresis (1,8%). It was shown that DNA amplification methods are quick and precise for identification of studied species. The results of bacteria identification were compared with data provided by the manufacturer. In all food supplements, bacteria of genus Lactobacillus and Bifidobacterium were detected. However, only some species provided by manufacturer were identified by PCR in each tablet.
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Reversible immobilisation of DNA on newly designed magnetic carriers
Kubisz, Petr ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of work was an optimization of separation deoxyribonucleic acid (DNA) with the use of nucleic acid reversible adsorption to the surface of magnetic particles coated by functional groups. Six carriers were verificated for DNA isolation: P (HEMA-co-GMA) ox, F-kol B 30 ox, F-kol 77 ox, F-kol B100 ox, F-kol 135 ox, coated with carboxyl groups and Perovskit 439 (coated by silicone). Bacterial DNA was isolated by phenol extraction procedure, first. DNA was reversibly bond to magnetis carrier in the presence of high concentration of NaCl ( 5 M) and poly (ethylene glycol) (PEG 6000). The final PEG and NaCl concentrations of 16.0 % (w/v) and 2.0 M, respectively, were used.DNA was eluted into TE buffer. The quality of extracted DNA was checked by PCR amplification. It was found out that although different quantities of DNA were isolated, the quality of isolated DNA was always compatible with PCR. Nanoparticles Perovskit 439 had the best separative characteristics in comparison to the other magnetic carriers because highest amounts of DNA was isolated. However, next optimisation of DNA separation procedure is required for the use of studied microspheres in real samples.
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PCR identification of selected bacteria in cheeses
Gregušová, Barbora ; Burdychová, Radka (referee) ; Španová, Alena (advisor)
This work is focused on identification and species specification of a collection of 44 clostridial strains using molecular-genetic methods. DNA of bacteria isolated from late-blowing defected cheeses was used. The purified DNA was diluted to 10 ng/µl and its ability to be amplified was verified by PCR with universal primers. By genus-specific PCR was proved that DNA of all samples belongs to Clostridium genus. Based on species-specific PCR reactions, it was determined that 7 strains belong to C. butyricum and 12 strains belong to C. tyrobutyricum. 15 strains were positively detected in both species-specific PCRs and therefore identified as mixed cultures. Another 10 strains were not classified into tested species. Strains with the gene encoding the enzyme hydrogenase hydA were searched using PCR. Specific PCR products for this gene were detected in 30 strains of the analysed collection. Especially intense were the amplicons by all strains belonging to C. butyricum species.
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DNA isolation from probiotic lactic acid bacteria in food additives
Tvrdíková, Jana ; Vojtíšková, Marie (referee) ; Španová, Alena (advisor)
In this work the functionalised magnetic particles were tested with streptavidin to selective DNA isolation. The method of selective DNA isolation was tested by using DNA probiotic strain Lactobacillus paracasei subsp. paracasei CCDM 211/06. A test was done on the biotinyl oligonucleotic particles, which was immobilised by containing streptavidin and it was used like a DNA probe for isolation complementary DNA chain by means of DNA/DNA hybridization. The primer R 5´ bio and the biotinyl denatured specific PCR product were tested for species Lb. paracasei as a DNA probe. These following experimental conditions were optimized for selective DNA isolation: temperature and time of hybridization, amount of DNA and the release of DNA from microspheres. Isolation of DNA was verified by PCR with specific generic primers. The specific generic PCR product was amplified in extent 250 bp, which was detected by using electrophoresis in agarose gel. This optimized method was successfully used in selective isolation of DNA Lactobacillus from a complementary sample of supplementary food (BIFI pangamin).
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Fermentation of different cereals by the probiotic bacteria Lactobacillus plantarum 299v
Hamalová, Sabina ; Španová, Alena (referee) ; Prof.Siv Ahrne (advisor)
Počet obyvatel trpících různými infekčními, zánětlivými a alergickými nemocemi stejně jako výskyt laktózové nesnášenlivosti a vysoké hodnoty krevního cholesterolu, má narůstající tendenci. Některé z těchto zdravotních problémů jsou způsobeny nevyváženou střevní mikroflorou. Probiotika jsou pak chápána (nejen) jako potravní komponenty, které přispívají k ustanovení mikrobiální rovnováhy (Parker, 1974) mezi zdraví prospěšnými a škodlivými bakteriemi. Z tohoto důvodu, terapie založená na podávání probiotik pacientům přitáhla zájem ze strany vědců. Vhodný probiotický kmen se pak volí v závislosti na požadovaném zdravotním účinku (příp. zdravotním problému, který má být probiotickou terapií léčen). Lactobacillus plantarum 299v již prokázal své blahodárné účinky na lidech a zároveň byla i potvrzena jeho zdravotní bezpečnost, díky čemuž může tato bakterie být kategorizována jako probiotický kmen (Probi AB, Sweden). I díky tomu je Lactobacillus plantarum 299v ve značné oblibě přidáván do mnoha fukčních potravin a prodáván na trhu pod různými jmény, probiotický nápoj ProViva je jedním takovým příkladem. Cílem této práce bylo studovat fermentační proces na žitném, ječmenném a sojovém substrátu pomocí kmene Lactobacillus plantarum 299v, přičemž zvýšená pozornost byla věnována právě soji a ječmeni jako potenciálně novým substrátům pro výše uvedenou bakterii. Hlavními záměry bylo zkoumání růstu a metabolické aktivity bakterie Lactobacillus plantarum 299v v asociaci s různými cereálními substráty, a později bylo studováno totéž také ve směsi fermentované cereální komponenty s běžně dostupným ovocným džusem. K tomu, aby se dosáhlo optimálních podmínek fermentace, je třeba vzít v úvahu několik aspektů. Hlavní role při konceptování nového fermentovaného produktu patří především zpracování a taktéž kompozici surového materiálu, růstové kapacitě a produktivitě bakteriální kultury a stabilitě finálního produktu během skladování (De Vuyst, 2000). Tyto parametry jsou důležité hlavně ze strany výrobců. Krom toho jsou tu ale i zákazníci, pro něž je přijatelnost produktu založena z velké části na organoleptických vlastnostech finálního probiotického produktu, tj. aromatu a chuti. Přítomnost a dostupnost různých jednotlivých nutrientů, která byla obsažena ve fermentačním médiu výsledkem rozdílných použitých cereálních substrátů, pravděpodobně vyústila v odlišnosti metabolických drah, což pak později mohlo způsobit rozdíly v organoleptických vlastnostech finálního produktu.
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Development of transient transfection protocol for HEK293 EBNA1 cells
Šmíd, Jiří ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Recombinant proteins belong to considerable biofarmaceutics products used in biomedical research and in the treatment of human disease. Recombinant protines can be produced by stable transfection in big amount or by faster transient transfection with smaller amounts. To provide regular biological activity, it is necessary for the protein to be properly folded and post-translationally modified. As these modifications can be accurately performed only in mammalian cells, they have become the major host for complex r-protein expression. In this thesis is described transient transfection HEK 293 EBNA1 cells with linear polyethylenimines. These cells has been adapted to suspension cultivation in serum free medium. The cells were transfected with pcDNA3.1, pCI, pEBSV1, pCEP4, pEAK8 a pcDNA5/FRT/TO plasmids, everyone contained repoter gene SEAP. Concentration of SEAP in cell culture supernatants were determined in order to compare efficiencies of individual transfections. DNA:PEI ratio was another factor which was optimised and two different PEIs were compared. Highest achieved expresion was 50 mg per litre with transfection in 24 well plate when DNA:PEI ratio was 1:5. Comparison of six different plasmids give the bigest expresion pCEP4/SEAP, in well plate as well as in scaled up system.
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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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Identification of lactic acid bacteria in fermented dairy products using amplification methods
Tycová, Martina ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Polymerase chain reaction (PCR) is molecular diagnostic method which allows the identification of lactic acid bacteria used in food industry. In this work species-specific PCR primers (targeted on highly conserved 16S rDNA region) were used for identification of bacteria of species Streptoccocus thermophilus in 10 randomly commercially accessible fermented milk products and for identification of species Streptococcus thermophilus in 25 lyophilisates collected in Culture Collection of Dairy Microorganisms Laktoflora (CCDM, Tábor, Czech Republic). The PCR products (968 bp) were detected using electrophoresis in 1,2 % agarose gel. Bacterial DNA was isolated from crude cell lysates by magnetic carriers P(HEMA co GMA) containing carboxyl groups. DNA was reversibly bind on their surface in the presence of high concentrations of poly(ethylene glycol) (PEG 6000) and sodium chloride. Phenol extraction of DNA was used as control. Streptococcus thermophilus strains were identificated using PCR in all analysed samples.
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