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Function of antimicrobial proteins in albumen of precocial birds
Krkavcová, Eva ; Kreisinger, Jakub (advisor) ; Kratochvíl, Lukáš (referee)
Antimicrobial proteins contained in the albumen represent maternal effects, including the non- genetic component allocated into the egg during its oogenesis. Especially for species, whose broods are exposed to environmental influences until completation, these proteins play a crucial role in the viability of embryos due to their potential to influence the risk of microbial infection, which is considered one of the main causes of reduced hatchability. Also, it is assumed that these proteins, beacause of their specific traits, may influence phenotype of chicks, especially its size and immunity in the early postembryonal stage. In my thesis I focused on three antimicrobial proteins of avian egg white - avidin, lysozyme and ovotransferrin, which vary in their antimicrobial activity. For a better understanding of causal relationships between the concentrations of these proteins in the albumen and their effect on hatching success or offspring phenotype, a series of manipulation experiments and correlative measurements were performed. These experiments were held on the eggs of two precocial species - Japanese Quail (Coturnix japonica) and Mallard (Anas platyrhynchos). Our results indicate a crucial role of antimicrobial proteins in reducing the risk of bacterial infection and their natural concentration...
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Molekulárna charakterizácia vybraných obranných faktorov v čeľadi Lumbricidae
Mančíková, Veronika ; Bilej, Martin (advisor) ; Král, Jiří (referee)
Earthworms belonging to oligochaete annelids have been a model for comparative immunology for over 40 years. They possess various defense mechanisms efficiently recognizing and responding to non-self substances. Among these there are molecules with many biological activities including cytolytic, antimicrobial and proteolytic. This work is aimed to compare the immunological features of two closely related earthworm species Eisenia andrei and Eisenia fetida. Due to many morphological and life cycle similarities they have been, until recently, regarded as members of subspecies. Interestingly, their natural habitat varies considerably, and it was of particular interest to investigate how these environmental differences affect the features of innate immunity of both species. Key words: annelids, innate immunity, Eisenia andrei, Eisenia fetida, CCF, fetidin, lysenin, lysozyme
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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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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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Preparation of PCR-ready DNA
Čuta, Robert ; Vojtíšková, Marie (referee) ; Rittich, Bohuslav (advisor)
In these days are probiotic lactic acid bacteria (LAB) very often used in food processing industry, such as milk products, cheese and fermented salami production. As well as the food conservation agent. Except of the industry usage of the LAB there are microbiological aspects. Identification of the bacterial species methods based on the isolation and amplification of DNA are very often used last few years. Diploma thesis deal with the bacterial cell of Lactobacillus species lysis and it´s optimalization. At first it was tested the optimal concentration of lysozyme (3mg/ml, 5mg/ml, 10mg/ml) and the exposure times (3, 5 a 10 hours). Another testing was aimed to the use and suitability of washing powder to the bacterial cell of Lactobacillus species lysis. I tested the Amway washing powder optimal concentration (1%, 2%, 3% a 4%). Four of another comercial washing powders were tested too. All these tests were performed at the pure Lactobacillus bacterial culture. To ensure the results I tested the washing powders at the real food matrix (Acidified milk, yogurt mango, yogurt white). All the methods were evaluated at the amplification method PCR with specific primers for the Lactobacillus genus. The DNA isolation was performed with the paramagnetic microsperes P(HEMA-co-GMA) and the amount of the DNA was quantified spectrophotometrically. The PCR products detection was performed with the agarose gel electrophoresis.
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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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Characterization of organic nanoparticles with encapsulated antimicrobial peptides
Vejrostová, Petra ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on characterization of particles containing encapsulated antimicrobial enzyme lysozyme. The theoretical part deals with characterization of antimicrobial peptides and their description. Further part of review was focused on lysozyme, the selected representative used in this thesis, its structure, mechanism of action and possible usage. In the experimental part the Hartree-Lowry method was used for lysozyme detection, determination of encapsulation efficiency and for detecting the amount of lysozyme released after incubation in model physiological environment and in model foods. In process of encapsulation the highest amount of lysozyme was packed into 1% chitosan particles, manually prepared alginate particles and into liposomes. During study of stability of particles in model foods as the least stable manually prepared chitosan particles were found. The released lysozyme exhibited changes probably caused by its degradation. The highest influence on particles proved 3% acetic acid. During studying the stability of the particles in artificial digestive fluids as the most unstable manually prepared chitosan particles were found, while alginate particles were the most stable. The thesis also deals with changes in antimicrobial activity of encapsulated lysozyme in prepared particles and after its application into the model environments. A gram-positive bacteria Bacillus subtilis was used in order to test the antimicrobial activity. Antimicrobial tests showed that after encapsulation antimicrobial activity of lysozyme was substantially decreased in most samples. Size and stability of prepared particles was tested using dynamic light scattering.
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Preparation and testing of application forms for natural antimicrobial substances
Vajglová, Klára ; Bokrová, Jitka (referee) ; Márová, Ivana (advisor)
The goal of this bachelor thesis was the study of antimicrobial and antioxidant effects of some herbal extracts: cloves, nettle, sage, chamomile, rosemary, elderberry, lavender and also lysozyme. Further, herbal extracts effect on Gram positive and Gram negative bacteria were compared. In the theoretical part were briefly characterized lysozyme, polyphenols, flavonoids and selected strains of microorganisms. In the experimental section characterization of herbs with antimicrobial and antioxidant effect was proved. Particles containing herbal extracts were prepared and encapsulation efficiency was determined. In some of tested herbs encapsulation efficiency of up to 80% was reached. Stability of the prepared liposomes was tested in the model digestive conditions and after 1 week and 1 month incubation period in model foods. Antimicrobial activity was tested on selected representatives of Gram negative and Gram positive bacteria. Gram positive bacteria were more sensitive to herbal extracts and liposomes containing encapsulated herbs, while lysozyme exhibited higher effect to Gram negative bacteria. Prepared particles with encapsulated antimicrobial extracts are especially suitable for use in the food industry.
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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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