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Use of polymerase chain reaction (PCR) technique for identification of probiotic microorganisms in milk diary product
Horká, Markéta ; Fialová, Lenka (referee) ; Smetana, Jan (advisor)
In this bachelor thesis, probiotic bacteria and their beneficial effects on human health were described. Several methods that are used to identify microorganisms were also stated. At the beginning of the experimental part, DNA was isolated from a selected dairy product. DNA extraction was made in two ways, both of which provided sufficiently high-quality DNA. The prepared DNA was subjected to polymerase chain reaction using specific primers. The presence of bacteria of the genus Lactobacillus, Bifidobacterium and the species Lactobacillus acidophilus, which were declared by the yogurt manufacturer, was proved by electrophoresis on agarose gel.
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Detection of probiotic bacteria in milk diary food products using PCR technique
Krempaská, Vladimíra ; Fialová, Lenka (referee) ; Smetana, Jan (advisor)
Probiotic bacteria play an important role in a healthy lifestyle. They help the consumer maintain the balance of intestinal microflora and prevent the overgrowth of harmful bacteria. Accurate identification and characterization of these probiotic strains is essential for research and the food industry. For exact identification, the use of molecular biological methods is necessary, thanks to which it is possible to validate probiotic products for commercial use. In this bachelor thesis, the DNA of probiotic bacteria was isolated from available dairy products. Two methods of isolation were used to isolate bacterial DNA, both of them provided sufficiently concentrated and high-quality DNA for further analysis by polymerase chain reaction (PCR). The presence of the Bacteria domain, genera Lactobacillus and Bifidobacterium were proved. Finally, the presence of Lactobacillus acidophilus species was also detected in the products.
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Biofilm formation in probiotic cultures and its application in pharmacy
Ryšávka, Petr ; Obruča, Stanislav (referee) ; Vorlová, Lenka (referee) ; Márová, Ivana (advisor)
The work was comprehensively focused on the development of adhesive forms of probiotics in the form of a biofilm on combined carriers with a prebiotic component. The second part dealed with the influence of food on the multiplication and survival of selected types of probiotic bacteria. Subsequently, the effect of individualized probiotic supplements on changes in the human intestinal microbiome was monitored. Suitable adherent probiotic strains for biofilm formation were selected and tested. Methods have been introduced and different variants of carriers for culturing and binding bacteria have been tested. In vitro experiments verified the stability of biofilm stucture and its resistance to low pH, bile and antibiotics in comparison with the planktonic cell form. The antimicrobial effect of probiotic strains in the form of a biofilm was studied. The cultivation of the multispecies biofilm on the combined carrier was optimized and the stability of the biofilm and the final viability of probiotic bacteria were confirmed. Furthermore, the influence of various foods and beverages on the viability of probiotic bacteria was evaluated with emphasis on the simulation of passage through the gastrointestinal tract. Both models, solutions with standardised concentrations of alcohol, sugar, salts, proteins or different pH and different types of real foods and beverages were tested. The effect of food and beverages was tested on monocultures of Lactobacillus acidophilus, Bifidobacterium breve and on probiotic capsules containing a mixed culture of probiotic microorganisms. The survival of probiotics in various food matrices in the simulated gastrointestinal tract was quantitatively different. We managed to define foods suitable for supporting the multiplication of probiotic bacteria. A separate part of the work was focused on the targeted modulation of the intestinal microbiome by individualized probiotics that were prepared on the basis of molecular biological analyzes of the intestinal microbiome aimed at detecting the percentage of lactobacilli, bifidobacteria and phylum Firmicutes and Bacteroidetes. Personalized probiotic supplementation confirmed the positive effect of this approach on microbiome changes, especially on the increase of the content of lactobacilli, bifidobacteria and the overall diversity of the microbiome.
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Vliv konzumace probiotik a synbiotik na modulaci intestinální mikroflóry a produkci tyraminu
Kolářová, Miroslava
The purpose of the study was to compare the impact of 3 weeks of consumption of commercial yoghurt enriched with Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus acidophilus LA-5 (P), and the same yoghurt supplemented with prebiotic-inulin (S), on feacal bacterial counts of healthy adults. A randomized, parallel-group, crossover, open-label – DBPC intervention with 66 volunteers was carried out. The 3 week administration of both P and S yoghurt increased (P lower 0.01) counts of bifidobacteria and Lactobacillus acidophilus, and reduced (P lower 0.01) counts of Clostridium spp., Enterococcus spp. and E. coli in fecal samples, in comparison with the control croup. The consumption of probiotics and synbiotics yoghurt reduced (P lower 0.05) the counts of tyramine producing enterococci and E. coli among the faecal isolates. In the synbiotics group, counts of bifidobacteria and Lactobacillus acidophilus remained higher, and counts of Clostridium spp., Enterococcus spp. and E. coli lower (P lower 0.05) after 1 week wash out period, in comparison with the preadministration phase. It was concluded that the synbiotics yoghurt was superior to the probiotics product in maintaining the increased levels of beneficial bacteria and decreased counts of potentially patogens bacteria in the intestine.
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Probiotics for children's nutrition
Pokorná, Martina ; Němcová, Andrea (referee) ; Skoumalová, Petra (advisor)
This Diploma thesis deals with probiotic bacterias for children nutrition. It proposes a probiotic food supplement with a probiotic blend composed of a strain of the genus Lactobacillus and of the genus Bifidobacterium, which would be most suitable for infant consumers. The theoretical part is focused on probiotics, constitution of the children´s ganstrointestinal tract and screening of probiotic and gelatine supplements which are already sold on the market. In the experimental part, probiotic bacteria were subjected to model digestion, whereby mixtures of strains having the lowest reduction in viability after digestion were blend based on the results. The blend of Bifidobacterium breve CCM 7825T, Bifidobacterium longum CCM 4990 and Lactobacillus casei CCM 4798 was chosen as the most suitable blend of the lowest viability reduction. PCR in real time was demonstrated the presence of all strains added to the blend after model digestion. Finally, from the data was suggested a more comprehensive probiotic food supplement in the form of an alginate-agar gummy-bear for children over three years of age. The probiotic food supplement was subjected to sensory analysis too. The proposed probiotic food supplement also contained Chlorella and Spirulina active compounds and higher content of omega 3 and 6 fatty acids in hemp oil.
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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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Formation of biofilm by probiotic bacteria and its processing to solid drug form.
Grossová, Marie ; Rittich, Bohuslav (referee) ; Buňka, František (referee) ; Márová, Ivana (advisor)
The aim of present work is cultivation of probiotic bacteria L. acidophilus, B. breve and B. longum in such a way that the culture forms cells clusters or comprehensive biofilm on the variety of free carriers. Biofilm formation of L. acidophilus on the silica from point of view bile and acid tolerance in gastrointestinal tract was studied. While the number of living cells in planktonic form (planktonic form) at pH 1 fell by 30 %, the viability of the biofilm cells was maintained to 90 % under the same environmental conditions. The biofilm culture showed also the protection against environment contained bile. Furthermore, the possibilities of drying procedures of biofilm cultures used as commercial technologies in pharmaceutical industry were studied. The comparison of freeze-drying and fluidization bed drying showed, that freeze-drying is more suitable method, which is able to achieve higher amount of viable cells after drying than fluidization bed drying. The effectivity of freeze-drying method is dependent on the selection of suitable cryprotective medium. In this case, about 90 % higher viability after freeze drying was achieved in comparison with fluidization bed drying. Finally, the industrial processing of probiotic strains into the solid dosage form was studied. Tablets should be produced at hardness between 70 and 90 N and water activity of tablet mixture can be maintained below 0.3. Consequently, the drying step of the tablets in a hermetically closed space with at least 10 % of silica gel must be ensured. Thereafter, the tablets contain (5.4 ± 0.7)109 viable cells after 6 months of drying process. Capsule production technology has no significant effect on the cell‘s viability during production. The triplex blistering foil for primary blistering of probiotic capsules was chosen. The triplex foil, which has low values of water vapour transition rate (0.07 g H2O / (m2 × day) and oxygen transition rate (0.01 cm3/m2 × day), was chosen. Other studied blistering foils commonly used in the pharmaceutical industry are not suitable for long storage of solid dosage forms contained probiotics.
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New cryoprotectives in preservation of potentially probiotic bacteria of Bifidobacterium genus using freeze-drying procedure
Vrabcová, Petra ; Killer, Jiří (advisor) ; Jakub, Jakub (referee)
The probiotic bacteria of the genus Bifidobacterium are especially used in the dairy and pharmaceutical industry. They are present in the form of viable cells in pro(syn)biotic fermented milk, dried and freeze-dried products for its positive effects on the health. Lyophilization represents one of the widely used methods for long-term preservation of bifidobacteria. The lyophilization process is challenging, but very needed, and for this reason it is still necessary to explore new cryo(lyo)protective substances, which protect the microbial cultures more effectively before the negative effects of cryopreservation and lyophilization.
The aim of the thesis was to test the various mixtures of substances containing specific oligosaccharides, which have prebiotic properties, from the point of view of their potential cryo(lyo)protective effect of lyophilization process. For the purposes of the study, twelve bifidobacterial strains of human and animal origin were selected. As a potential cryo(lyo)protectives were used solutions on the basis of 5x concentrated TPY medium with addition of FOS, 10% fructooligosaccharides (FOS) and the solution on the basis of porcine gastric mucin. Number of viable cells was determined in lyophilisates enriched by the concrete solutions in the time intervals (30 days, 3 months and 6 months) after lyophilization and compared with the control lyophilized samples (fresh cultures without the additive solutions). The potential cryo(lyo)protective effectiveness of 5x concentrated TPY medium solution has been demonstrated only in B. boum, B. breve, B. longum subsp. infantis a Bombiscardovia coagulans, where decreased the number of viable cells about four orders of magnitude. The potential cryo(lyo)protective effectiveness in 10% FOS solution was found only in B. thermacidophilum subsp. porcinum and Bombiscardovia coagulans, where was found half the number of viable cells. The highest potential cryo(lyo)protective effectiveness was observed for a solution on the basis of porcine gastric mucin. The highest number of viable cells was demonstrated in B. animalis subsp. lactis, B. boum, B. longum subsp. infantis, B. longum subsp. longum, B. pseudolongum subsp. globosum and B. thermacidophilum subsp. porcinum lyophilisates using this potential cryo(lyo)protective solution.
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Fiber and its consumption on a hospital bed
KŘÍŽKOVÁ, Ivana
The theme of the bachelor thesis is Fiber and its consumption on a hospital bed. Dietary fiber is defined as a carbohydrate food ingredient that cannot be transformed by one´s own enzymes. Fiber was originally considered a ballast food component. Gradually, it was found that it is a part of food fermented by colonic bacteria. Fiber is substrate for these bacteria and they transform it into substances intended for human organism. Fiber can be divided into soluble and insoluble. Both types are essential for human nutrition. A recommended fiber intake for an adult is 30 g per day. In the practical part of the bachelor thesis, was used qualitative research method. The research sample consisted of hospital two-weeks menus. One diet was applied in the hospital facility in the Central Bohemian region and one in the South Bohemian region. Using a computer program "Nutriservis Professional" I calculated the fiber content in the individual meals. The findings were compared with the recommended daily intake published in the book called Referenční hodnoty pro příjem živin and S výživou po celý rok. I also calculated energy value, amount of proteins, fats and carbohydrates of the menus. The results were compared with the recommended values. As a research I interviewed the nutritional therapists in the hospital facilities about the patient´s diet and possible solutions to health problems caused by a lack of fiber. The interviews consisted of eight open questions focused on the fiber consumption, denying of foods containing fiber. The results of calculation previewed the menu at the first hospital facility that fiber deficiency occurs primarily in a liquid diet, while for rational diet, diabetic diet and for infants and older children the fiber content is sufficient. The menu of the other hospital contains insufficient amount of fiber, especially for diabetic diet or geriatic diet reducing fat. On the other hand, diabetic diet contains a plenty of fiber. The interviews that were supposed to find out how nutritional therapists approch the fiber issues in diets brought similar responses. The hospital in the Central Bohemian region include into the diet especially fruit, vegetable, whole grain products and legumes. Older patients ages constipation suffering from bowel obstuction, intestinal disease and flatulence frequently refuse fiber, especially legumes. If a patient has health problems caused be the lack of fiber nutritional therapists usually solve this problem by education and inclusion of fruit, vegetable and whole meal bread into the patient's diet. The hospital in the South Bohemian region frequently include into the diet potatoes, rice, fruit, legumes, vegetable and whole grain breads. Fiber is often rejected by older people and people suffering from diarrheal diseases. Health problems resulting from the lack of fiber are often solved by recommending by foods containing fiber or the nutritional supplements such as NutriFibre.
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The analysis of DNA isolated from different types of probiotic products using real-time PCR and HRM analysis
Sedláková, Lucie ; Rittich, Bohuslav (referee) ; Trachtová, Štěpánka (advisor)
The aim of this diploma thesis was to introduce real-time PCR with high-resolution melting analysis for Bifidobacterium species. Currently a small number of publication, dealing with identification of Bifidobacterium species using high-resolution melting analysis, is available. According to publications dealing with identification of lactic acid bacteria were selected primers P1V1 and P2V1, LAC1 and LAC2, LsppUPF and LsppUPR, V3F and V3R, V6F and V6R. Using this primers bacterial DNA was amplified by real-time PCR with high-resolution melting analysis. After evaluation of the measured results efficiency of selected primers was verified on DNA izolated from complex sample of probiotic product. After further optimisation real-time PCR with high-resolution melting analysis could be suitable using selected primers for Bifidobacterium species.
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