|
|
Selective isolation of the genus Bifidobacterium bacteria from foods
Mizerovská, Lucie ; Šárka, Havlíková (referee) ; Rittich, Bohuslav (advisor)
Probiotic lactic acid bacteria (LAB) are very often used in food procesing industry, such as milk products, cheese and fermentsd salami production in nova days. In diploma thesis were tested symbiotic food supplements from different producers. Bacterial DNA was isolated from crude cell lysates of six food suplements by magnetic particles P(HEMA-co-GMA). PCR-ready DNAs were isolated. from all products The detection of Bifidobacterium bacteria identified by PCR was in agreement with those declared by the manufacturers. Magnetic particles with immobilized antibodies against Bifidobacterium were used in the next part of thesis. These particles were used for the isolation of target cells from two products with cell identification by genus specific PCR.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
|
|
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.
|
guest ::
