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Production of proteolytic enzymes by selected microorganisms
Pala, Martin ; Lichnová, Andrea (referee) ; Márová, Ivana (advisor)
Presented work was focused on study of microbial production of proteolytic enzymes used in many industrial applications. Bacterium Bacillus subtilis was used for laboratory production of microbial proteolytic enzymes. During cultivation production of biomass and proteolytic enzymes were studied influence of substrate type and stress factor application (osmotic shock, hydrogen peroxide and ethanol) was tested too. The highest concentration of biomass was measured in concentrated BM medium after 32 hours of cultivation. Biomass yield was 1.11 g/l. The highest protease activity (88.28 U/ml) was obtained in the same cultivation medium and time of cultivation. According to results of stress experiments it can be concluded that most of used stress factors exhibited a toxic effects to bacterial culture even at low concentrations.
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Characterization of waste rice substrates and their potential utilization by microorganisms
Kapar, Jiří ; Duroňová, Kateřina (referee) ; Márová, Ivana (advisor)
The aim of this bachelor thesis was to characterize some cereal substrates. Based on the results of group parameters the most suitable substrate for cultivation of model industrial microorganisms was proposed. Analysis of total phenolics, reducing sugars and total sugars was performed spectrophotometrically. Using high-performance liquid chromatography with refractometry index detection content of individual mono-and disaccharides was measured. Saccharides were extracted from cereal substrates by water and then acid hydrolysis was applied. Two rice substrates were then used as nutrition sources for cultivation of two model industrial microorganisms: yeast Rhodotorula glutinis and bacterium Bacillus subtilis. Before use to production media substrates were processed by chemical and enzyme hydrolysis and analyzed. Production of microbial biomass in media with modified waste substrates was evaluated.
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Study of Polycaprolactone Biodegradability
Chamradová, Ivana ; Omelková, Jiřina (referee) ; Voběrková, Stanislava (advisor)
Aliphatic polyesters represent a group of degradable polymeric materials. These materials found their utilization almost in biomedical applications and as packing materials. In the theoretical part, the literature review on enzymatic and microbial degradation research of poly(?-caprolactone)(PCL) is summarized . In the experimental part, the methodology of biodegradation testing of PCL was formed and a series of experiments with a Bacillus Subtilis and Aspergilus-Lipase was carried out. Polymeric samples of Mn = 19 300 the film form with 100 µm thickness and 1 cm diameter were subjected to enzymatic degradation. Samples with 3 cm diameter were exposed to microbial degradation by the "solution casting" method. Biodegradable process was interpreted on the basis of changes of the molecular weight, melting point, crystallinity and changes on the surface of polymeric materials.
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Study of elementary inactivation processes acting during sterilization of procaryotic systems in dielectric barrier discharge
Bittnerová, Zuzana ; Sťahel, Pavel (referee) ; Kozáková, Zdenka (advisor)
Presented master’s thesis is focused on the study of the elementary inactivation processes acting during sterilization of procaryotic systems in dielectric barrier discharge (DBD). Sterilization is an important biomedical and food-industry application and plasma sterilization is one of the methods, which are suitable for sterilization of heat and chemical sensitive materials. Biologically contaminated samples were treated in dielectric barrier discharge operated at atmospheric pressure. The discharge was generated in argon and in nitrogen. The plasma power density was 2725,93 mW cm–3 in argon and 2325,93 mW cm–3 in nitrogen. Gram positive bacteria Bacillus subtilis and gram negative bacteria Escherichia coli were used as a bioindicator. Bacteria were spread onto the surface of Whatman No.1 filtration paper. The influence of UV radiation, reactive species, heat and plasma discharge where the synergistic function of all of the agents was studied. Effects of UV radiation and temperature were studied separately. In order to separate the effect of UV radiation generated by DBD the quartz glass transmitting UV radiation was employed. During the plasma exposition selected samples were covered with the quartz window while other samples were directly exposed to the plasma. Covered samples were exposed to UV radiation and temperature (which cannot be eliminated), samples without quartz window were directly plasma exposed (treated). Results show that for covered samples the lower inactivation was reached than by the samples directly exposed to plasma. When studying the effect of temperature, the temperature between the DBD electrodes was measured by means of a thermocouple. Afterwards the samples were placed in an oven and exposed to the same temperature as was measured between the electrodes. By comparing the results of heat treated samples and plasma treated samples it can be assumed that the influence of the temperature during the sterilization process in DBD is very low. The discharge parameters were studied by means of the Optical Emission Spectroscopy. Plasma treated samples were assessed employing Scanning Electron Microscopy (SEM). Damage of Bacillus subtilis cell wall due to the effect of plasma was observed while no effect of plasma on the structure of filtration paper was detected.
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Characterization of specific proteins form selected animal products.
Janhuba, Filip ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
The master's thesis is focused on study of specific protective proteins from animal products. Two different types of antimicrobial egg white proteins were studied in detail - antimicrobial protein ovotransferrin (conalbumin) and enzyme lysozyme. Ovotransferrin belongs to transferrin group of proteins and exhibits activities similar to milk protective protein lactoferrin. The main effects of ovotransferrin are antiviral, anticancer and immunomodulatory. Antimicrobial activity of ovotransferrin based on the possibility to bind iron is still a subject of interest. For comparison the second egg protein lysozyme (N-acetyl muramidglycan hydrolase) was used. Lysozyme is a hydrolytic enzyme which primary attack cell wall of bacteria. In the theoretical part of the thesis an overview of the specific antimicrobial proteins in selected animal products was introduced mainly focused on ovotransferrin and lysozyme. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial activity, protein concentration and purity. For quantitative analysis of total proteins, optimized Hartree – Lowry spectrophotometric method was used. For the determination of molecular weight and purity SDS-PAGE was used and stained by Coomassie Brilliant Blue G250 and silver. In experimental part the real sample of egg white was compared with samples of lyophilized antimicrobial proteins and therapeutical pills supplied by industrial partner. Protein composition and purity of these preparative has been determined. Antimicrobial activity of ovotransferrin was studied on cultures of G+ bacterium Bacillus subtilis and for comparison on G– E. coli. Ovotransferrin showed antimicrobial effect only at very high concentrations of about 75 mg/ml (Bacillus subtilis) and 50 mg/ml (E coli) even with addition of high amount (100 mM) of hydrogen carbonate ions. The inhibitory effect was most evident in liquid media. On the other hand, lysozyme exhibited significant inhibitory activity from 0.3 mg/ml on gram positive bacteria. Inhibitory effect on E. coli was not observed. Another part of study was focused on isolation of ovotransferrin from egg white using gel permeation chromatography on Sephadex G100. As mobile phases 0.1 M phosphate buffer and 0.05 M Tris-HCl buffer were tested. By SDS-PAGE the purity of ovotransferin comparing to standard was evaluated. Finally, the encapsulation of ovotransferrin and lysozyme was tested. Ovotransferrin and lysozyme was encapsulated into liposome and chitosan particles. Particles stability, distribution and average size distribution were studied by dynamic light scattering and zeta potential measurement. The stability of particles in the model physiological conditions was studied too.
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Aronia – the source of bioactive compounds
Štumarová, Klára ; Vespalcová, Milena (referee) ; Veselá, Mária (advisor)
The aim of this bachelor thesis is to research the possible antimicrobial activity of aqueous and ethanolic extracts of aronia berries and tea mixture containing aronia. Also, the antioxidant activity and the concentration of the biologically active substances - polyphenols, flavonoids and anthocyanins was determined. Theoretical part contains a summary of basic information about Aronia melanocarpa, chemical composition, use and health effects of substances contained in its fruits. The experimental part focuses on the antimicrobial activity of the extracts on microorganisms Bacillus subtilis, Serratia marcescens and Micrococcus luteus. Farther there is spectrophotometrical determination of antioxidant activity, polyphenolic compounds, flavonoids and anthocyanins. From the experiment results flow that the extracts have almost none antimicrobial ability except tea mixture extracted with hot water. This extract has inhibitory effect on M. luteus and growth supportive effect on B. subtilis. It has been found that aronia fruits and tea mixture are rich in polyphenols and flavonoids.
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Sea buckthorn – the source of natural compounds
Blašková, Daniela ; Vítová, Eva (referee) ; Veselá, Mária (advisor)
The main aim of the bachelor thesis was a research of common sea buckthorn, often denoted as "a plant of future". Botanic characteristics of the given bush, verification of biologically active substances and various concentration extract effects on selected kinds of microorganisms. In the theoretical part, the literature search is done, which discusses the characterization of the researched plant. The practical part deals with the application of selected methods of research, as follows: spectrophotometric determination of biologically active substances and microbiological verification of microbial activity on microorganisms, such as Bacillus subtilis, Serratia marcenscens and Micrococcus luteus. Considering the thesis results, it ensues that the given extracts do not show too great bacterial activity. Ex adverso, the extracts contain too big amount of determined active substances, in some cases.
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Comparison of sensitivity of Bacillus subtilis and Clostridium difficile against selected chemical ingredients.
Šlitrová, Barbora ; Ing.Veronika Holá, Ph.D. (referee) ; Omelková, Jiřina (advisor)
Sporicidal activity of disinfectants is tested on Bacillus subtilis spores according to ČSN EN 13704 standard. Until December 2007, 10 countries encountered outbreaks and 7 additional countries had endemic cases of Clostridium difficile-associated disease due to hypervirulent strain C. difficile PCR ribotype 027. This outbreak of CDAD with increased severity and mortality is spread as typical nosocomial infection. Clostridium difficile spores can persist on dry surfaces for 5 months. Therefore it is important to compare the effectiveness of disinfectants on both strains and find out whether Bacillus subtilis is the only proper microorganism for testing the sporicidal activity.
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Preparation of some application formula of antimicrobial substances
Krempaský, Kamil ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
The main aim of this study is to determine antimicrobial activity of herbal extracts and also encapsulate these extracts and lysozyme to liposomes. The extracts were prepared from above ground part of wormwood (Artemisia absinthium), aerial part of breckland thyme (Thymus serpyllum), the root of purple coneflower (Echinacea purpurea), liquorice (Glycyrrhiza glabra), corolla of pot marigold (Calendula officinalis) and berries of sea buckthorn (Hippophae rhamnoides). In all extracts antioxidant activity and the amount of total polyphenols and flavonoids was determined. All of these extracts and also lysozyme were used for encapsulation to liposomes. Prepared liposomes were then tested for their stability. Further, encapsulated as well as free extracts were tested for their antimicrobial activity on Escherichia coli, Serratia marcescens, Micrococcus luteus and Bacillus subtilis. Extract from Echinacea purpurea showed the highest antimicrobial activity.
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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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