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Optimization of calcium chloride concentration for removal of polysaccharide contamination during plant DNA isolation
Frnčová, Ekaterina ; Šlosárová, Katarína (referee) ; Fialová, Lenka (advisor)
The greatest difficulty in isolating DNA is the presence of contaminants that cause side effects. Polysaccharides are the most common contaminants in fruits. They can distort the results in spectrophotometric determination of purity or act as inhibitors in PCR analysis together with other substances (for example, proteins or phenolic substances). The aim of this work was to investigate the influence of different concentrations of calcium chloride on the process of DNA isolation. In the experimental part, DNA from the apple was isolated using different concentrations of calcium chloride. The isolation was carried out four times, and each time the sample was adjusted in different ways. It was found that the isolation method used works only with a sample that has been lyophilized. Isolation of DNA from fresh fruit provided very low yields. Probably, this was due to the large water content in the sample, and the proportion of the solid component was smaller. Subsequently, PCR analysis and electrophoresis were performed to determine the amplifiability of the isolated DNA. Two sets of primers with different specificity were used for this analysis. Amplifiability was confirmed only when using primers specific to apple DNA when using 100 mM solution of CaCl2. Other samples have been amplifiable using both types of primers. Probably, samples isolated using a 100 mM solution of CaCl2 had a larger amount of inhibitors that do not affect all PCR reactions equally, which may also indicate a small effectiveness of this amount of CaCl2.
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Testing and optimization of protocols for removal of contaminants during DNA isolation from hibiscus blossoms
Brabcová, Martina ; Kubalová, Michaela (referee) ; Fialová, Lenka (advisor)
The Roselle (Hibiscus sabdariffa) is a widely used plant in food industry, especially in tea production. The hibiscus flower is rich in polyphenolic substances, which are a major problem in DNA isolation. This work deals with the modification of the CTAB isolation protocol to ensure concentration and purity of DNA sufficient for its amplification. DNA isolated according to the CTAB protocol did not have sufficient concentration and purity for amplification due to high contamination with polyphenolic substances. A procedure with double incubation of plant tissue in CTAB buffer containing 3% polyvinylpyrrolidone (PVP) was proposed to remove it. These isolates were successfully amplified. The effect of PVP concentration (1-5%) in CTAB buffer on the concentration and purity of isolated DNA was also investigated. PCR results showed that double incubation with CTAB buffer had a greater effect on the removal of polyphenolic compounds than changing the PVP concentration. Subsequently, the modified isolation protocol was also successfully used to isolate amplifiable DNA from commercial tea blends.
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Testing of primers for real-time PCR-HRM analysis of fruit products containing one fruit species
Boháčová, Barbora ; Dzurendová, Simona (referee) ; Fialová, Lenka (advisor)
Determining the authenticity of fruit products, which are often counterfeited by substituting part of a more expensive fruit with a cheaper but botanically similar fruit, is a current topic in the food industry. A prime example is the dilution of apricot products with peach puree. This study focuses on testing specific primers AGS18 and PdCass to reveal the true proportion of apricot content in model products mixed with peach puree using PCR analysis followed by HRM analysis. Testing of these primers for authenticity determination revealed limited utility of AGS18 primers due to the formation of small amount or no specific products during reactions with fruit DNA. PdCass primers required PCR condition optimization, but subsequently, specific DNA sequences from fruit leaves could be amplified in sufficient quantities. PCR analysis of DNA from model products with PdCass primers provided specific products in samples with apricot content of 70% or less. HRM analysis of samples and calculation of GCP did not distinguish purees with 0%, 10%, and 30% apricot content. However, purees with 50% and 70% apricot content exhibited significantly different melting curves compared to other samples.
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Utilization of PCR technique for identification of probiotic bacteria in daily hygiene product
Horobets, Yuliia ; Fialová, Lenka (referee) ; Smetana, Jan (advisor)
Probiotic bacteria have traditionally been used in the food industry, but their applications have now expanded to the prevention and treatment of various diseases. Increasing evidence supports the efficacy of bacteria of the genus Lactobacillus in the oral cavity has led to the application of probiotic strains of this genus in mouthwashes and other personal care products. In this bachelor thesis, DNA was isolated by two methods, then quantified spectrophotometrically and amplified via conventional PCR. The results of the polymerase chain reaction were detected by gel electrophoresis. The presence of probiotic bacteria species Lactobacillus delbrueckii, Lactobacillus plantarum and Lactobacillus pentosus was confirmed.
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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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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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Molecular identification of probiotic bacteria in milk products form commercial yoghurt cultures
Horňan, Samuel ; Fialová, Lenka (referee) ; Smetana, Jan (advisor)
Lactic acid bacteria are considered as an important group of bacteria with probiotic effects, which are being widely used in the food industry or pharmacology. Identification and characterization of important probiotic strains play an essential role in the validation of probiotic products for commercial purposes. Their identification using molecular-biology techniques (most commonly PCR method) is one of the standard tools in commercial operations and services. The aim of this bachelor thesis is a literature review of probiotics and probiotic strains as well as a summary of current knowledge about the use of molecular biology techniques for identification of these bacteria with probiotic properties in dairy products. The experimental part of this work verifies the presence of probiotic bacteria declared on selected commercial dairy products using the polymerase chain reaction (PCR) method.
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Authenticity of natural plant component in cosmetics products
Kubalová, Michaela ; Fialová, Lenka (referee) ; Němcová, Andrea (advisor)
The purpose of this thesis was to study the authenticity of selected natural ingredients in cosmetic products. These were specifically cosmetic products that contained citruses, mint or lavender. Commercially available isolation kits were used for DNA isolation. The presence of plant origin DNA was verified by PCR method using primers specific for the ITS2 region of plants. The presence of limonene, a significant allergen contained in said plants, was determined in the samples by PCR method using primers for limonene synthase. At the same time, its presence was verified by HPLC method. In addition, two primers were tested for lavender and monitored for their efficacy, with no significant difference in the usage.
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Detection of probiotic bacteria in milk diary food products using PCR technique
Krempaská, Vladimíra ; Fialová, Lenka (referee) ; Smetana, Jan (advisor)
Probiotic bacteria play an important role in a healthy lifestyle. They help the consumer maintain the balance of intestinal microflora and prevent the overgrowth of harmful bacteria. Accurate identification and characterization of these probiotic strains is essential for research and the food industry. For exact identification, the use of molecular biological methods is necessary, thanks to which it is possible to validate probiotic products for commercial use. In this bachelor thesis, the DNA of probiotic bacteria was isolated from available dairy products. Two methods of isolation were used to isolate bacterial DNA, both of them provided sufficiently concentrated and high-quality DNA for further analysis by polymerase chain reaction (PCR). The presence of the Bacteria domain, genera Lactobacillus and Bifidobacterium were proved. Finally, the presence of Lactobacillus acidophilus species was also detected in the products.
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Influence of matrix type on the authentication of foodstuffs containing fruits
Kopková, Pavlína ; Strečanská, Paulína (referee) ; Fialová, Lenka (advisor)
Certain types of food, mainly the more expensive ones, are often adulterated to reduce their manufacturing price. However, this reduces their quality and can also have a negative impact on the health of the consumer. Children's fruit products are also targeted by fraudulent producers, where the declared fruit is most often replaced by a cheaper version. This work focuses on the detection of adulterated foods using various analytical methods, in particular PCR. The theoretical part focuses on the issue of food adulteration, the analytical methods used for detecting adulteration, and also on mango and banana which are determined in this work. The aim of this thesis was to determine what effect the type of matrix has on the determination of fruit components in food by PCR. Three types of matrix were used for this purpose - fruit puree, smoothie, and bars. An important task was to optimize the DNA isolation to achieve adequate purity and concentration of DNA. Then, the amplifiability of the obtained DNA was verified. The DNA isolates were then analyzed by multiplex PCR with primers specific for mango and banana. The results were verified by agarose gel electrophoresis. Subsequently, it was possible to determine that the fruit component in bars and fresh smoothies was the most easily analyzed by PCR and, on the contrary, the determination was problematic for puree. The instrumental part was focused on the determination of phenolic compounds in the products by HPLC. For this purpose, optimization of the extraction of phenolic compounds was necessary. This method was able to detect the presence of mangoes in all samples.
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