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Influence of PHA accumulation on resistance of bacteria against selected antibacterial drugs
Hrabalová, Vendula ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
The aim of this bachelor thesis was to study the effect of bactericidal drugs on bacteria from the genus C. necator H16 and its mutant genus PHB-4. The genus H16 shows ability to accumulate polyhydroxyalkanoates (PHA) in the form of granules while the genus PHB-4 lacks to show this ability. The theoretical part of the bachelor thesis is focused on the effect of antibiotics on bacteria in general and the determination of susceptibility of bacteria to antimicrobial substances. The effect of three specific antibiotics (nisine, streptomycin and penicillin) on both bacterial strains was tested in the experimental part. The viability of bacteria was determined by the spread plate method and flow cytometry. Agar diffusion test and broth microdilution test were used to test the susceptibility of bacteria. It was concluded that the accumulation of PHA decreases the tolerance of bacteria to antimicrobial substances because the genus C. necator H16 is more susceptible to streptomycin and penicillin then the strain C. necator PHB-4.
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Use of some encapsulation techniques to controlled release of active substances in food and cosmetics products.
Skoumalová, Petra ; Rittich, Bohuslav (referee) ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
The presented doctoral thesis is focused on preparation, characterization and application of organic micro- and nanoparticles as transport systems for active components and some their complex natural sources. Active component were packed into liposomes and polysaccharide particles. As active components were used caffeine, some drugs – clotrimazole and ibuprofen, further antioxidants and vitamins. Antimicrobial herbs and spices extract, antimicrobial peptides lysozyme, nisin and other antimicrobial ingredients were encapsulated too. Encapsulation of selected hydrolytic enzymes was tested, too. Particles were also used for encapsulation of probiotic strains Bifidobacterium breve and Lactobacillus acidophilus and prebiotic components. These prebiotics were co-encapsulated into capsules with probiotic cells. Natural extracts were encapsulated e.g. extracts of guarana, ginseng, goji, green barley, propolis, black, green and white tea, coffee, fruit and vegetable extracts. The efficiency of encapsulation was determined by HPLC/PDA and by spectrophotometry. Long-term stability of particles and amount of released component in model/real foods, in model cosmetic conditions and in a model physiological environment were monitored too. Size of prepared liposomes and polysaccharide particles was determined by dynamic light scattering and by light microscopy and electron microscopy, respectively. Stability of the particles was measured using a zeta potential. Also, analytical centrifugation was used to measurement of sedimentation velocity and stability of the prepared particles. The antimicrobial activity were tested using two Gram-positive (Bacillus subtilis, Micrococcus luteus), two Gram-negative (Escherichia coli, Serratia marcescens) bacteria and one fungal strains (Candida glabrata). For determining the antimicrobial properties of active component and prepared particles two the most widely used methods were used - agar diffusion method and broth dilution method. The viability of probiotic strains were performed using flow cytometry and fluorescence microscopy. Encapsulation of active component was successful in all types of particles. Liposome showed a very good long-term stability mainly in water conditions with neutral pH and polysaccharide particles were stable in acidic conditions. Prepared particles showed a very good stability in model stomach environment, while in model intestines environments particles were disintegrated and active component were released. Prepared particles with encapsulated caffeine as well as other tested antioxidants and vitamins could be used to modern types of energy drinks, food supplements and also for some cosmetics applications. Encapsulated antimicrobial components could be used for food application as well as for cosmetics and pharmaceutical application like antimicrobial wound formulation. Encapsulated enzymes can be used for controlled release of proteases in wound healing, as delivery systems in digestive tract and as a part of pharmaceutical preparative and food supplements for enzyme therapy. The study revealed that encapsulation of probiotics and also co-encapsulation of probiotics with prebiotics exhibited longer stability of particles and survival bacterial cells. So, prepared particles are suitable for use to food product with beneficial effects on the human body.
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Methods for detection of antimicrobial properties of lactic acid bacteria
Vránová, Petra ; Němcová, Andrea (referee) ; Trachtová, Štěpánka (advisor)
Lactic acid bacteria are classified as probiotics producing substances that act against microorganisms. These antimicrobial substances include organic acids, carbon dioxide, hydrogen peroxide and bacteriocins. Currently, the focus is on bacteriocins, which are used in the food and pharmaceutical industries. The aim of this thesis is creating an overview of antimicrobial properties of lactic acid bacteria, methods of their determination and their applications in the food industry. The experimental part deals with identification of lactic acid bacteria present in selected samples of bacterial DNA. In addition, we tested for bacteriocin Gassericin K7A-producing gene. Molecular diagnostic methods such as real-time polymerase chain reaction (qPCR) and conventional polymerase chain reaction were used to distinguish bacterial DNA as well as the bacteriocin.
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Use and application of natural antimicrobial substances
Dzurická, Lucia ; Byrtusová, Dana (referee) ; Skoumalová, Petra (advisor)
The presented bachelor thesis is focused on the study of antimicrobial activity of plant substances in combination with bacteriocins and the possibility of creating new encapsulated form of supplement which could be used in food and cosmetic products. 5 plant materials were selected: acerola, cinnamon, cloves, orange peel and yacon syrup. Nisin was chosen as the most suitable bacteriocin. In the theoretical part the components with antimicrobial and antioxidant activity are described, such as the materials of plants and animal origin used in this work. Also the possibility of increasing the antimicrobial effect and possible use of natural antimicrobial substances were described. In experimental part, the aqueous extracts of selected plant materials were firstly spectrophotometrically characterized for the content of total polyphenols, flavonoids and antioxidants. Selected substances were encapsulated into liposomes and subsequently preparation of liposomes coated with chitosan was optimized. The particles were tested for size, short and long term stability, encapsulation efficiency and antioxidant activity. Furthermore, the antimicrobial activities of selected components in nonencapsulated and encapsulated form were observed against four strains of microorganisms (Escherichia coli, Micrococcus luteus, Serratia marcescens and Candida glabrata). Also the expected synergisms of the antimicrobial effects of extracts in combination with nisin and prepared particles were observed.
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Antimicrobial peptides and their application into foods
Janhuba, Filip ; Petrik, Siniša (referee) ; Márová, Ivana (advisor)
The presented thesis is focused on a study of antimicrobial peptides and their potential applications into foods. Two types of antimicrobial peptides were studied in detail - bacteriocin nisin and antimicrobial enzyme lysozyme. In theoretical part an overview of antimicrobial peptides used in food industry was introduced. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial peptides concentration. Commonly used spectrophotometric methods for quantitative analysis of total proteins as well as high performance liquid chromatography were tested. To determine the molecular weight and purity a modified SDS-PAGE with the addition of urea and tricine buffer was performed. Further, encapsulation of nisin into liposome was tested. Encapsulated particles were prepared by three different procedures: by ultrasonication, ethanol injection and TLE. Particles stability, distribution and size were studied by dynamic light scattering. Stability of encapsulated peptides in model conditions and model foods was studied too. Finally, antibacterial properties of the selected peptides using test system Bacillus subtilis were studied. Nisin already showed antimicrobial effect at low concentrations - about 1 µg/ml. The inhibitory effect of nisin was more evident in liquid cultures.
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Production and characterization of antimicrobial substances from lactic acid bacteria
Franeková, Eliška ; Smetana, Jan (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on the production of antimicrobial compounds using lactic acid bacteria, their characterization and testing of their antimicrobial activity. The theoretical part of this work deals with the characteristics of lactic acid bacteria and the bacteriocins they produce, the possibilities of bacteriocin production and the factors that influence it. In the experimental part of this work, lactic acid bacteria Lactococcus lactis subsp. lactis, Lactobacillus helveticus and Bifidobacterium bifidum were cultivated, their growth curves and total protein content in supernatants after cultivation were measured. Cell-free culture supernatants were prepared by lyophilization and their antimicrobial activity against gram-positive and gram-negative bacteria was determined. The antimicrobial activity of commercial antimicrobial peptide nisin and a commercial preservative obtained using lactic acid bacteria was also measured. The sample obtained from Bifidobacterium bifidum was selected as the most effective of the isolates. Its antimicrobial activity was further tested on polymeric sausage packaging materials. Antimicrobial substances produced by lactic acid bacteria can be used in the food industry as preservatives or as a part of antimicrobial packaging, and in the pharmaceutical industry in materials for antimicrobial wound dressings.
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Transport of charged and neutral particles across the model biomembranes
Parisová, Martina ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
This work was focused on the preparation of model stabilized phospholipid membranes formed on porous polycarbonate carrier. 1,2-dipalmitoyl-sn-glycero-3-phosphocholin was used for their formation in hydrophilic pores of polycarbonate carrier. For characterization of the formation of phospholipid layers, their changes and a study of transport processes, electrochemical impedance spectroscopy and voltammetry were used. Transport of cadmium and copper ions was studied in the presence and in the absence of ionophore calcimycin which was incorporated into the formed of phospholipid membrane. Because these ions are often bound in complexes with various substances, such as low molecular weight organic acids (LMWOAs), this work was also focused on the transport of copper and cadmium ions across the model phospholipid membranes in the presence of malic acid, citric acid and oxalic acid at different pH. Besides the use of ionophore, some pilot experiments were performed to realize the transfer of copper ions using two peptides, nisin and transportan 10. Formation of phospholipid membranes and the transport processes were characterized by two proposed electric equivalent circuits which correspond to the covered and to the uncovered polycarbobate carrier. Keywords: Phospholipids, Membranes, Ionophore, Peptid....
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Use and application of natural antimicrobial substances
Dzurická, Lucia ; Byrtusová, Dana (referee) ; Skoumalová, Petra (advisor)
The presented bachelor thesis is focused on the study of antimicrobial activity of plant substances in combination with bacteriocins and the possibility of creating new encapsulated form of supplement which could be used in food and cosmetic products. 5 plant materials were selected: acerola, cinnamon, cloves, orange peel and yacon syrup. Nisin was chosen as the most suitable bacteriocin. In the theoretical part the components with antimicrobial and antioxidant activity are described, such as the materials of plants and animal origin used in this work. Also the possibility of increasing the antimicrobial effect and possible use of natural antimicrobial substances were described. In experimental part, the aqueous extracts of selected plant materials were firstly spectrophotometrically characterized for the content of total polyphenols, flavonoids and antioxidants. Selected substances were encapsulated into liposomes and subsequently preparation of liposomes coated with chitosan was optimized. The particles were tested for size, short and long term stability, encapsulation efficiency and antioxidant activity. Furthermore, the antimicrobial activities of selected components in nonencapsulated and encapsulated form were observed against four strains of microorganisms (Escherichia coli, Micrococcus luteus, Serratia marcescens and Candida glabrata). Also the expected synergisms of the antimicrobial effects of extracts in combination with nisin and prepared particles were observed.
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Influence of PHA accumulation on resistance of bacteria against selected antibacterial drugs
Hrabalová, Vendula ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
The aim of this bachelor thesis was to study the effect of bactericidal drugs on bacteria from the genus C. necator H16 and its mutant genus PHB-4. The genus H16 shows ability to accumulate polyhydroxyalkanoates (PHA) in the form of granules while the genus PHB-4 lacks to show this ability. The theoretical part of the bachelor thesis is focused on the effect of antibiotics on bacteria in general and the determination of susceptibility of bacteria to antimicrobial substances. The effect of three specific antibiotics (nisine, streptomycin and penicillin) on both bacterial strains was tested in the experimental part. The viability of bacteria was determined by the spread plate method and flow cytometry. Agar diffusion test and broth microdilution test were used to test the susceptibility of bacteria. It was concluded that the accumulation of PHA decreases the tolerance of bacteria to antimicrobial substances because the genus C. necator H16 is more susceptible to streptomycin and penicillin then the strain C. necator PHB-4.
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Methods for detection of antimicrobial properties of lactic acid bacteria
Vránová, Petra ; Němcová, Andrea (referee) ; Trachtová, Štěpánka (advisor)
Lactic acid bacteria are classified as probiotics producing substances that act against microorganisms. These antimicrobial substances include organic acids, carbon dioxide, hydrogen peroxide and bacteriocins. Currently, the focus is on bacteriocins, which are used in the food and pharmaceutical industries. The aim of this thesis is creating an overview of antimicrobial properties of lactic acid bacteria, methods of their determination and their applications in the food industry. The experimental part deals with identification of lactic acid bacteria present in selected samples of bacterial DNA. In addition, we tested for bacteriocin Gassericin K7A-producing gene. Molecular diagnostic methods such as real-time polymerase chain reaction (qPCR) and conventional polymerase chain reaction were used to distinguish bacterial DNA as well as the bacteriocin.
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