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Characterization of cytotoxic effect of combined antimicrobial nanomaterials
Kozlíčková, Hana ; Fialová, Lenka (referee) ; Márová, Ivana (advisor)
This thesis deals with the study of the effects of combined nanomaterials on human skin cells. Pure antimicrobial substances, two types of liposomes enriched with antimicrobial substances, nanofibers with antimicrobial substances and, finally, four types of combined nanomaterials were analyzed from the point of view of cytotoxicity. The analysed active substances were eugenol, thymol, cavarcrol, curcumin, vitamin E and the antibiotics streptomycin and ampicillin. In the theoretical part of the work, the cell line of human keratinocytes, used in the experimental part of the work for cell tests, was characterized. Furthermore, individual active substances with an antimicrobial effect were described and the principles of biological effects were described, which include antimicrobial, antioxidant, cytotoxic and synergistic effects. Additionally, the theoretical part described individual nanomaterials, their preparation and usage in cosmetics and medicine. The experimental part was based on the characterization of prepared nanomaterials and on testing the influence of individual antimicrobial substances on the proliferation and viability of human HaCaT cells. Using the DLS method, the size of the prepared liposomes was measured and the effect of PHB and the type of active substance on their size was studied. MTT and LDH tests were chosen to test the cytotoxicity of individual substances. Furthermore, a scratch test was performed to monitor the effect of the investigated substances on proliferation and the rate of wound healing by cells. The last performed tests were immune response assays, in which were tested the samples for production of the human anti-inflammatory cytokines IL-6 and IL-8. In experimental part of this thesis, it was found that the prepared nanofibrous materials are safe for use in healthcare or cosmetics and, in the future, suitable to produce nanofibrous wound coverings enriched with antimicrobial substances, which can give them exceptional properties.
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Impact of honey enrichment on its antimicrobial activity by bioactive substances against Escherichia coli
ŠEBESTOVÁ, Lucie
The main aim of this bachelor's thesis is to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of both native honeys and honeys enriched with selected essential oils on the growth of Escherichia coli. In the theoretical part of the bachelor's thesis, bacteria Escherichia coli is briefly characterized, including its pathogenic strains and antibiotic resistance. Furthermore, honey is described, including its chemical composition and physiological effects. Additionally, essential oils are generally described, including their antimicrobial properties, along with a brief description of each selected oil. In the methodological part of the bachelor's thesis, procedures for preparing mediums, agar, inoculum, and the determination of their minimum inhibitory and bactericidal concentrations are described. MIC was measured spectrophotometrically in 96 well microtiter plates. MBC was determined by inoculation from the plate onto Mueller-Hinton agar followed by a 24hour incubation. A total of 3 native honeys were tested, which were then enriched with 8 selected essential oils. Essential oils with the addition of dimethyl sulfoxide (DMSO) were also tested. The results of the obtained data are statistically evaluated, graphically presented, and compared with scientific sources.
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Preparation of Cosmetic Product Based on Microbial Extracts
Reichertová, Klára ; Trachtová, Štěpánka (referee) ; Hudečková, Helena (advisor)
This thesis is focused on the preparation of a cosmetic product based on microbial extracts. The thesis is divided into two main parts, theoretical and practical. The theoretical part describes the types of cultured microorganisms, microbial metabolites, legislation and safety of cosmetic products, composition of human skin and last but not least the applied analyses. The experimental part deals with the use of waste products of the food industry, specifically coffee grounds as a substrate for culture media. Furthermore, the cultivation is studied with a focus on the gain of metabolites useful in the cosmetic industry, the analysis of these metabolites by GC/FID and HPLC/PDA. The work addresses the preparation of microbial extracts, their testing including determination of polyphenol and flavonoid content and characterization of antioxidant activity and antimicrobial activity tested on bacterial cultures of Esterichia coli and Staphylococus epidermidis. The application of the analysed extracts and their application to the final product is also addressed. The last chapter discussed in this thesis to test the final product using Visioscope PC–35, instrument and performing sensory analysis. The cultured strains were Phaffia rhodozyma CCY 77-1-1, Cystophilobasidium macerans CCY 10-1-2, Rhodosporidium toruloides CCY 062-002-004 Chlorella vulgaris CCALA 924. The biomass obtained by cocultivation of Phaffia rhodozyma CCY 77-1-1 and Chlorella vulgaris CCALA 924 as well as the biomass of these two microorganisms cultured separately were used for the preparation of microbial extracts.
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Characterization of selected complex vitamin supplements
Bandarenka, Andrei ; Fialová, Lenka (referee) ; Márová, Ivana (advisor)
Modern society places great emphasis on a healthy lifestyle and balanced diet, leading to increased interest in products with high levels of vitamins, minerals, and other health-promoting substances. Consequently, there has been an increased interest in various dietary supplements, including so-called "greens" products. These supplements are designed to provide a concentrated source of nutrients from various green plants while also containing a high proportion of vitamins, minerals, antioxidants, and other bioactive compounds. The aim of this thesis is to characterize complex food supplements in terms of their composition and content of active ingredients to better understand their mechanisms of action and identify substances responsible for their health-promoting effects. The theoretical part of the work focuses on describing legislation of dietary supplements, including their characteristics and the evolution of interest in them over recent decades. Furthermore, the theoretical part addresses the description of basic components commonly found in supplements, such as vitamins, plants, algae, prebiotics, and probiotics. Finally, methods for determining these components and the most frequently used techniques for their analysis are described. In the experimental part, 12 different plant-based dietary supplements falling into the category known as "greens" were selected. The content of phenolic compounds, chlorophylls, carotenoids, and antioxidants were determined in these products. Additionally, the influence of these products on the growth of bacterial cultures and the metabolic activity of gram-negative and gram-positive bacteria was examined. Analysis of the vitamin C and B-group vitamins content in these supplements was also conducted. The test results showed that the examined supplements are a rich source of phenolic compounds, chlorophyll, antioxidants, and vitamin C. Furthermore, it was found that these products can be used as prebiotics in the diet. Based on the conducted tests, it was concluded that "greens" food supplements offer a wide range of health benefits and can be considered a useful contribution to a balanced diet and a healthy lifestyle. Practically, in all conducted tests, the products with a high matcha content achieved the best results, making this ingredient a key component for creating quality dietary supplements from plant-based ingredients.
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Optimizing the production of non-chocolate sweets with the addition of Filipendula ulmaria extract using various sweeteners
Malyszová, Markéta ; Diviš, Pavel (referee) ; Vítová, Eva (advisor)
The aim of this master's thesis was to optimize the production process of jelly candies using various alternative sweeteners and subsequently adding herbal extract of meadowsweet, achieving not only better nutritional properties but also enhanced sensory characteristics of the final product. Sensory analysis was utilized to determine the ideal combination of sweetener type and amount, which most closely aligns with consumer preferences for sweetness and texture, while maintaining a low energy component content. Based on these criteria, maltitol was selected as the sweetener in a 50% w/w solution. The study also focused on optimizing the addition of Filipendula ulmaria extract to the candies made with the "ideal" sweetener. Again, using sensory analysis, the optimal concentration of the extract and the type of gelling agent were selected to achieve the desired consistency, taste, and other sensory parameters that make the product attractive for consumers. Following these experiments, the ideal values were determined to be 50% w/w for sweetener, a 5 % addition of Filipendula ulmaria extract, and the use of gelatin as the gelling agent. Subsequently, a chemical analysis of the produced samples and meadowsweet extract was also carried out. Identification and quantification of volatile aromatic compounds were performed using the HS SPME GC-MS method, which identified 39 different substances, predominantly from the group of alcohols and aldehydes. To determine the total content of phenolic compounds, the Folin-Ciocalteu spectrophotometric method was used, and the pH differential method was employed to determine the content of anthocyanins. The total content of phenolic compounds in the extract amounted to 1418.16 ± 6.56 gml-1, and the total anthocyanin content was determined to be 249.19 ± 27.40 gml-1. Antioxidant activity was tested using two methods, TEAC and DPPH. The results of the antioxidant activity measured by the DPPH method were higher, confirming the greater robustness of this method. However, both tests confirmed the antioxidant activity of the tested samples. Finally, the antimicrobial activity of the samples was tested using the diffusion well method against strains of Bacillus cereus and Escherichia coli. The samples exhibited antimicrobial activity against both tested microorganisms, but showed more effective action against Bacillus cereus, which may be due to the simpler structure of their cell wall.
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Preservative systems for natural cosmetics
Koblasová, Dana ; Veselá, Mária (referee) ; Zemanová, Jana (advisor)
The aim of this bachelor thesis is to evaluate the antimicrobial activity of naturally derived preservatives against selected microorganisms. Theoretical part is focused on description of the most used natural, naturally derived and synthetic preservatives, on the safety of preservatives and finally, the issue of contamination of cosmetic products by microorganisms. In experimental part, antimicrobial activity of 4 naturally derived preservatives was evaluated against selected microorganisms, which were bacteria Micrococcus luteus, Kocuria rosea, Bacillus cereus, Bacillus subtilis, Serratia marcescens and one yeast Candida vini. The tested preservatives were from MinaSolve company - E-LEEN Green A, which contains pentylene glycol and phenylpropanol, E-LEEN Green OR, which is composed of citrus extracts, citric acid, ascorbic acid and pentylene glycol, Green B, which contains benzoic acid, sodium benzoate and pentylene glycol and Hexam+, which is a mixture of pentylene glycol and hexamidin diisethionate. Antimicrobial activity was evaluated by agar diffusion method. The highest antimicrobial activity showed Hexam+, the lowest E-LEEN Green OR. E-LEEN Green A and Green B did not show any antimicrobial activity against any microorganism in the 1% tested concentration. The measured values of the inhibition zones show that the most resistant microorganisms from all the tested in this thesis are Bacillus cereus and Bacillus subtilis and the least resistant are Micrococcus luteus and Kocuria rosea.
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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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Antimicrobial materials based on nanostructures with enzymes
Jurová, Bronislava ; Vysoká, Marie (referee) ; Skoumalová, Petra (advisor)
This bachelor thesis is focused on the preparation and characterization of antimicrobial gels and coatings containing antimicrobial components and enzymes. The theoretical part deals with a brief description of antimicrobial substances, their structure and mainly enzymes with antimicrobial effect. The methods used in the testing are also listed here. The practical part deals with testing the antimicrobial effects of extracts from cinnamon and cloves, clove essential oil, lysozyme and bromelain against Micrococcus luteus, Serratia marcescens and Candida glabrata. Various types of extracts were prepared from the selected spices, namely aqueous and ethanol extracts in different percentages. The individual extracts were characterized and, according to the total proportion of phenolic substances, the best ones were selected for encapsulation in liposome particles and gels. Extracts and enzymes were also added to the hydrogel and non-fibrous covers. A solution of sodium alginate and chitosan was used to prepare non-fibrous covers. A broth dilution method was used to test the antimicrobial effects on the given microorganisms. Furthermore, nanofibers based on PHB were prepared using the forcespinning method. These nanofibers were enriched with clove essential oil and their antioxidant activity was monitored.
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Biologically active substances of chokeberry (Aronia spp.)
Palkovská, Kateřina ; Pořízka, Jaromír (referee) ; Vítová, Eva (advisor)
This master thesis deals with preparation of extracts from fruits of aronia, optimization of conditions for extraction and subsequent characteristics of the obtained optimal extract. In the theoretical part information about the botanical characteristics of Aronia melanocarpa and about its utilization are mentioned. Furthermore, the chemical composition of aronia fruits is described and the main biologically active substances contained in the plant are described. At the end of the theoretical part extraction and separation techniques, determination methods of antioxidant, antimicrobial activity and determination of aromatic active compounds are described. The experimental part describes the optimization of maceration so that the macerate contains as much polyphenolic substances as possible, as well as aromatic active substances. 40% ethanol, at a temperature of 60 ° C, with a weight solvent ratio of 1: 5, for 40 minutes proved to be optimal for the maceration of aronia fruits. The total polyphenol content of the optimal extract was determined to be 5237.2 ± 40.7 mgGAEkg-1 and the antioxidant activity by TEAC to be 512.5 ± 5.04 gml-1. 24 aromatic active compounds were identified in the extract, with the largest percentage being monoterpenes and aldehydes. Furthermore, it was found that the extract did not show antimicrobial activity against the microorganisms used (Bacillus cereus, Escherichia coli and Candida glabrata). The obtained results confirm that aronia extracts are an important source of many biologically active compounds and can be used in food as a natural source of nutritionally and sensory valuable substances.
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PREPARATION AND APPLICATION OF SOME BIOPOLYMERS, NANOPARTICLES AND NANOFIBRES FOR COSMETICS AND FOOD
Bokrová, Jitka ; Pekař, Miloslav (referee) ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
The presented doctoral thesis is focused on preparation of nanoparticles and nanofibers with natural active ingredient and testing their biological effects. Modern types of application forms were prepared from biomaterials based on one or more natural polymers. Chitosan particles were prepared from cross-linked polymer using ultrasonication. A mixture of soy lecithin and cholesterol was used for preparation of liposomes. Poly-3-hydroxybutyrate was used for preparation of combined liposomes, too. All liposome particles were prepared by ultrasonication. Nanofibers were obtained from polyhydroxybutyrate using electrospinning. Mixtures of low-molecular antioxidants obtained by extraction from natural sources were used as active ingredients. Different types of teas, barks, herbs, spices, fruits and vegetables were selected as sources of natural antioxidants. Total phenolic and flavonoid content and total antioxidant activity of extracts were determined using spectrophotometrical methods. Obtained natural extracts were subsequently used for encapsulation. Prepared application forms were characterized in terms of their physicochemical properties. Particle size was monitored by dynamic light scattering. Colloidal stability of particles in suspension was determined using zeta potential. Spectrophotometry was used to evaluate the efficiency of encapsulation of active compounds into particles. The morphology of the new type of combined PHB liposomes was monitored by electron microscopy. Chromatography was used for quantification of individual components of particles. Morphology of nanofibers and incorporation of active agent into their structure were monitored using FTIR-ATR spectroscopy and electron microscopy. Afterwards, antimicrobial, cytotoxic and genotoxic effects of preparations were evaluated. It was found that the most suitable types of extracts for liposome preparation are aqueous and lipid extracts of natural antioxidants. Prepared particles showed excellent stability and good encapsulation efficiency. The study confirmed that incorporation of polydroxybutyrate into liposome structure does not reduce neither the colloidal stability of the particle, nor the efficiency of encapsulation process. Antimicrobial and antimycotic effect of preparations against model microorganisms Micrococcus lutues, Serratia marcescens and Candida glabrata was detected. It was found that process of encapsulation increases the inhibitory effect of natural extracts of antioxidants. The safety of preparations was assessed using two human cell cultures: epidermal keratinocytes and HaCaT cell line. Assays of cell viability and plasma membrane integrity were used to determine cytotoxicity of preparations. Low toxicity of liposome particles was confirmed by a series of cytotoxic tests. Obtained data showed that association of phospholipid with PHB polymer does not cause a significant increase in cytotoxicity in human skin cells. Genotoxicity testing on model procaryotic organism confirmed zero genotoxic potential of preparations. The new type of combined particles and polymeric fibers cant thus be used as a carrier for active ingredients, complex natural extracts, antimicrobial agents and many others.
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