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Study of natural antimicrobial mixtures and their stabilization
Vejrostová, Petra ; Veselá, Mária (referee) ; Márová, Ivana (advisor)
Present diploma thesis is focused on natural antimicrobial substances. The aim of the experimental part of thesis was testing of antimicrobial activity of selected natural substances in free and encapsulated forms, preparation of particles and monitoring of their long-term stability. Various teas extracts (aqueous, ethanoic and DMSO) and aqueous herbs extracts were used as plant antimicrobial substances. Liposomes and chitosan particles with encapsulated aqueous teas extracts were prepared by ultrasonication. Encapsulation efficiency, size, stability and antioxidant activity for all particles were specified. Consequently, within a two months period, long-term stability for these particles was monitored. Spectrophotometric method for determining total polyphenols was used for evaluating encapsulation efficiency as well as for monitoring of long-term stability. The DLS method was used for determination of size and stability of particles. Antioxidation activity was monitored by using spectrophotometric method using ABTS. For testing antimicrobial activity of various types of teas, herbs extracts and their mixtures and combianitons with lysozyme and antibiotics, broth dilution methods were used while using both Gram positive and Gram negative bacterial test strains. Antimicrobial activity of prepared particles was also monitored, the antimicrobial activity was especially significant with the chitosan particles. Suitable combinations of natural substances whatever with antimicrobial or antioxidation effect either in free or encapsulated form could be used for applications for example in cosmetics industry, food processing or in medicine.
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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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Analysis of nicotin content in some products
Pražáková, Jana ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with the determination of nicotine in different products. The theoretical part summarizes review on nicotine, smoking and opportunities how to quit. In the practical part a method for the determination of nicotine by HPLC / PDA was optimized. As the most suitable stationary phase was selected a Kinetex 5u C18 100A 150 x 4.6 mm column, as the optimal mobile phase was chosen a pure methanol with a flow rate of 1 ml min-1 and a temperature of 25 °C. For the analysis of nicotine were chosen: 18 kinds of cartridges for electronic cigarettes, two kinds of nicotine gum, nicotine spray, nicotine pastilles, nicotine orodispersible film and ten species of classic cigarettes. For each type of product the most appropriate method for extracting nicotine and its subsequent analysis by HPLC / PDA was found. For tobacco 24 hour extraction in methanol and 10s ultrasound was selected. The nicotine spray and electronic cigarette refills without flavours were only diluted with methanol. Flavoured refills were first diluted by sodium hydroxide and then with methanol. For chewing gums, pastilles and nicotine film extraction with 5% sodium hydroxide was chosen. In this study also new experimental nicotine product was designed. Nicotine has been encapsulated in alginate-starch material to form small gel particles. As the most suitable medium for storage the water medium was determined.
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Preparation of encapsulated enzymes for cosmetics application
Bokrová, Jitka ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
Presented diploma thesis is focused on testing of an appropriate form of encapsulated enzymes intended for application in cosmetic and pharmaceutical industry. For encapsulation, proteolytic enzymes bromelain, papain and collagenase were used. These enzymes were encapsulated into alginate and chitosan microparticles prepared by an encapsulator and packed into liposomes. Encapsulation effectiveness was evaluated by analysis of total proteins. Particles stability was evaluated in model and real conditions by photometrical analysis of released proteins. Proteolytic activity of released enzymes in model and real conditions were observed too. Alginate and chitosan microparticles prepared by the encapsulator were found as an appropriate form of encapsulated enzymes designed to wound healing. Encapsulation effectiveness of these particles and stability in model conditions were good in comparison with liposomes. Hydrogel and water-oil emulsion were used for analysis of particles stability at real conditions. Hydrogel was found as a good option for preservation of particles as well as proteolytic enzyme activity. Emulsion made particles less stable and proteolytic activity of enzymes decreased rapidly. Encapsulation enables long-term stabilization of biologically active compounds as well as possibility of targeted transport and controlled releasing. Presented diploma thesis suggests possibilities of application encapsulated enzymes in designing more effective formulations for wound healing.
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Encapsulation of selected natural extract for food application.
Vyskočilová, Terezie ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with encapsulation of natural extracts. In the theoretical part the methods of encapsulation, materials for particle preparation, as well as application of encapsulation techniques in food industry were described. In experimental part selected natural extracts of propolis, green barley and probiotics were characterized. There substances were encapsulated into alginate and chitosan. In the total of 25 types of prepared particles long-term stability in some model physiological conditions as well as in four different model foods was evaluated. Additionally, stability of selected particles in several real milk-based products was followed too. The stability of particles was determined spectroptohometrically. In natural extract a content of polyphenols, proteins, chlorophylls, as well as total antioxidant activity were analysed. To analysis of probiotics optical and fluorescence microscopy were used. In propolis and green barley antimicrobial activity was tested too. Moreover, in the sample of propolis also cytotoxic assay was applied. Agar-chitosan was chosen as the best shell material for propolis due to its optimal stability in model physiological conditions as well as model foods. Liposomes were evaluated as unstable and were not recommended for further application. As the suitable shell material for powdered green barley starch-alginate (rate 1:4) and agar-chitosan were proposed, while the second one showed better stability for released proteins. Agar-chitosan shell material was usable for fresh green barley too. For probiotics encapsulation alginate or alginate-starch were chosen because of their porosity and possibility of nutrients diffusion. In real foods the best results were reached with application of probiotic particles into milk. Coencapsulation of powdered barley and probiotics did not confirm inhibition of culture growth. Neither the antimicrobial effect of propolis and barley nor the cytotoxic effect of propolis were confirmed.
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Characterization of beverages and food supplements containig caffeine
Patočková, Klára ; Pekař, Miloslav (referee) ; Márová, Ivana (advisor)
This master thesis is focused on characterization of some energy drinks and formulation of their modified composition. The theoretical part was focused on summary of energy drinks and some of their natural components - guarana, goji as well as on techniques of encapsulation. In the experimental part concentrations of caffeine, vitamin C, polyphenols and carbohydrates in commercially purchased drinks was evaluated. Subsequently, three kinds of particles (particles with caffeine, particles with guarana and particles with guarana and goji) by 6 different techniques were prepared. The efficiency of encapsulation was determined by HPLC/UV-VIS (caffeine, vitamin C) and by spectrophotometry (phenolics). Stability of the particles was followed in real food and in a model physiological environment of artificial digestive juices. Size of prepared particles was determined by dynamic light scattering. In this work it was found that particles with best activity and stability were obtained by encapsulator. For manual particles the best results were found in the liposome particles. Liposomes exhibited the smallest particle size, the highest efficiency encapsulation, excellent stability in real food and juices and the best value of zeta potential.
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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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Possibilities of encapsulation of particular types of macromolecules and bacteria
Kapar, Jiří ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
Presented diploma thesis is focused on testing encapsulation methods of enzymes and probiotic bacteria. In the theoretical part a summary of different encapsulation techniques used in food industry is given. Further, materials for encapsulation, above all polysaccharides are presented. Next, some procedures of encapsulation of biopolymers and microorganisms – mainly enzymes and probiotic cultures are discussed. In the experimental part methods for preparation of several types of particles based on polysaccharides and liposomes are introduced. Particles were used for encapsulation of selected hydrolytic enzymes and probiotic strains Bifidobacterium breve a Lactobacillus acidophilus. The encapsulation effectiveness was evaluated by analysis of total proteins and enzyme activities. Particles sizes and their stability in water, in selected model foods and model body fluids were observed, too. According to results obtained in this work it was found that encapsulation of enzymes into polysaccharide particles were succesfull in all types of particles (encapsulation effectivness was more than 50 %). Polysaccharide particles showed a very good stability in body fluids as well as in model foods. As the most suitable materials for enzymes encapsulation chitosan and liposomes were found. Polysaccharide particles were used also for the encapsulation of microorganisms. The stability of particles with lactic acid bacteria was similar to particles containig enzymes, very good stability was verified aslo in model foods and model body fluids. Encapsulation enables long-term stabilization of biologically active compounds as well as posibility of their transport and controlled releasing in gastrointestinal tract. Encapsulation of probiotic bacteria could preserve their viability and long-term survival until the product expiration date. Thus, encapsulation is one of the most promissing procedures for production of foods and food suplements of great quality and high additional value.
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Encapsulation of natural antioxidants
Štindlová, Jitka ; Obruča, Stanislav (referee) ; Márová, Ivana (advisor)
In theoretical part of this diploma thesis the basic properties of antioxidants, especially anthocyanins and phenolic compounds are described, as well as the basic features and principles of free radicals formation. The theoretical part also describes some possible ways of encapsulation of antioxidants into polysaccharide and lipid particles. In the experimental part basic characteristics of extracts from selected lyophilized fruits and vegetables (carrots, apples and mixed berries) are described. As group parameters of plant extracts the total antioxidant activity, content of flavonoids and phenolics, carotenoids, anthocyanins and ascorbic acid were determined. In experimental part also various encapsulation techniques were tested, encapsulation effectiveness of each technique was evaluated and the stability and size of the created particles were determined. As the best encapsulation method in terms of encapsulation efficiency in most of samples/parameters ethanol injection was found. On the other hand the particles prepared by ethanol injection are relatively unstable in terms of zeta potential, which is followed by their tendency to aggregate. As the most stable particles prepared by thin layer evaporation (TLE) and reverse phase thin layer evaporation (RP-TLE) were evaluated. Particles prepared by TLE, RP-TLE and chitosan-alginate particles exhibited a negative charge, while particles prepared by ethanol injection stayed uncharged and chitosan particles have a positive charge.
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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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