This diploma thesis deals with sweet substances from Stevia rebaudiana and with their determination in beverages. The ambition was to adjust the separation method for major steviol glycosides (stevioside and rebaudioside A) by HPLC so as to be fast and effective enough, too. In experimental part was successfully developed the separatin method, so it is possible to determine the steviol glycosides and conservation substances, specifically benzoic and sorbic acid (eventually their sodium or potassium salts). At first, for analysis was used monolithic column, on which stevioside and rebaudioside A weren´t separated. Other analyses were performed on column Ascentis express C18 (150 x 4.6; 2.7 µm). The most suitable separation method was performed by mobile phase made up with 29:71 v/v mixture of acetonitrile and acetate buffer (with pH 4.2). The flow rate was 0.28 ml/min and the column temperature was kept constant at 50 °C. The analysis of steviol glycosides and conservation substances with these condition was verified on the samples of real beverages.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Karolína Dalecká Supervisor: Ing. Martin Drastík, Ph.D. Title: SPE in steviol glycosides analysis In the first part of this diploma thesis, the solid phase extraction method is described. The topics like the workflow, usability of sorbents and their formats and interactions with analytes are covered. The second part of this thesis is focused on utilization of solid phase extraction on samples containing steviol glycosides. An overview of solid phase sorbents their forms; solvents used for extraction and elution of steviol glycosides is provided in the discussion section at the very end of this work. Taking into account the fact that stevia has been used more and more on the European market in recent years, and is found in more food and other products, the frequency of determination of steviol glycosides and the use of solid phase extraction is on rise. The most commonly extracted samples include food products with steviol glycosides used as sweeteners or extracts of dried stevia leaves. The main sorbents used for solid phase extraction include silica gel with octadecyl and divinylbenzene-co-vinylpyrrolidine copolymer. This type of extraction is most often connected with high...

Comparative studies of taste perception have found that primates may differ markedly in their sensitivity for substances perceived as sweet by humans. These findings raise questions about the reason that may underlie these differences in sweet-taste sensitivity between species. The aim of the present study was to assess the taste responsiveness of chimpanzees (Pan troglodytes verus) to seven substances tasting sweet to humans and to compare the results with those of other primate species. Using a two-bottle preference test (1 min) I found that the taste preference thresholds of the chimpanzees for five food-associated carbohydrates ranged between 20-30 mM for sucrose, 20-50 mM for fructose, 60-80 mM for glucose, 50-80 mM for maltose, and 30-80 mM for lactose. Taste preference thresholds for two steviol glycosides ranged from 0.04-0.05 mM for stevioside, and 0.03-0.05 mM for rebaudioside A. The chimpanzees displayed clear preferences for all sweet-tasting substances presented. In line with data obtained in other primates, the taste preference threshold of the chimpanzees for sucrose was lower compared to the other carbohydrates presented and the taste preference thresholds for stevioside and rebaudioside A were lower compared to sucrose. In general, the taste sensitivity of the chimpanzees fell into the range of data reported in other nonhuman primate species. Interestingly, the taste preference thresholds of the chimpanzees reported here are similar to the taste detection thresholds obtained in humans, despite the fact that the former are only a conservative approximation of an animal’s taste sensitivity. This suggests that chimpanzees may be as sweet-taste sensitive as humans.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Marcela Hollá Supervisor: Ing. Martin Drastík, Ph.D. Title of Diploma Thesis: Determination of steviol glycosides by HPLC A new HPLC method was developed and validated for simultaneous detemination of major steviol glycosides stevioside and rebaudioside A in food supplements. Separation took place in hydrophilic interaction chromatography mode on column with core-shell particles. The method was aplicated on analysis of steviol glycosides in products Valosun, SlaDIA, Solia and extract from dried stevia leaves. Isocratic separation was performed using Kinetex 2,6u HILIC 100A, (100 x 2,1 mm; 2,6 µm), Phenomenex analytical column with mobile phase consisted of acetonitrile/0,05 M ammonium formate adjusted with formic acid to pH=3 in ratio 90:10, with flow rate 0,7 ml/min, column temperature set at 30 řC, pressure 19,8 MPa, UV detection at 203 nm and injection volume 1 µl. We compared the results of the analyzes with content of stevioside glycosides declared by the manufacturer. The new developed method allows rapid analysis of food supplements and plant extract containig steviol glycosides. Key words: steviol glycosides, stevioside, rebaudioside A, Stevia rebaudiana Bertoni, high...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Matěj Rücker Supervisor: Ing. Martin Drastík, Ph.D. Title: Stevia rebaudiana - food and pharmaceutical industry applications The first part of this thesis deals with the description of the plant and its substances, the most important are steviolglycosides, especially stevioside and rebaudioside A. In the following chapter, the toxicity and safety are evaluated, which is directly related to the use of stevia and steviolglycosides in food and pharmaceutical practice. In the next part, the utilization in the food industry and connected legislation is described. The harmfulness of the refined sugar is outlined as well. Application in the pharmacy is not so widespread, therefore this chapter deals primarily with the effects of steviolglycosides. Stevia rebaudiana Bertoni and steviolglycosides have great potential to replace sugar and other sweeteners. Due to their beneficial effects, they are very popular among researchers, so we can look forward to their widespread application in food and pharmaceutical industry in the near future. Key words: steviolglycosides, stevioside, rebaudiosede A, sugar, pharmaceutical industry, food industry

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SyfteSyftet med denna laborativa pilotstudie in vitro var att undersöka hur den mikrobiella sammansättningen och pH i den orala biofilmen påverkades vid upprepad exponering för Stevia (steviosid och rebaudiosid A), sackaros eller en kombination av reb A och sackaros. Ett ytterligare syfte var att studera om isolat av orala bakterier kunde metabolisera reb A i buljongkultur. Material och metodBiofilmer från tre personer blev exponerade för 0,025 % steviosid, 0,025 % reb A eller 10 % Todd Hewitt (TH) under 15 minuter tre gånger dagligen i fyra dagar. Artbestämning gjordes i början och slutet av försöken. Biofilmerna exponerades för 0,025 % reb A och 10 % sackaros eller en kombination av dessa varefter pH-värdet mättes. Vidare studerades 23 bakteriestammar avseende förmåga att metabolisera 0,025% respektive 0,5% reb A. ResultatStreptokocker dominerade i samtliga biofilmer vid försökets slut oavsett om de exponerats för steviosid, reb A eller enbart TH. En pH-sänkning kunde ses hos biofilmerna som enbart blivit exponerade för sackaros eller för en kombination av sackaros och reb A. Däremot kunde endast en marginell pH-sänkning observeras efter exponering för enbart reb A. Ingen av de enskilda bakteriestammar som studerades kunde metabolisera reb A.SlutsatsIngen större skillnad i biofilmens bakteriesammansättning kunde ses efter upprepad exponering för steviosid eller reb A i förhållande till TH.Upprepade exponeringar av reb A i kombination med sackaros gav en liknande pH-sänkning som enbart sackaros, dock sågs ingen pH-sänkning efter exponering för enbart reb A. Enskilda bakteriestammar kunde inte metabolisera reb A. / AimsIn this in vitro laboratory pilot study, the effect on the microbial composition and pH of the oral biofilms after repeated exposure to Stevia (stevioside and rebaudioside A), saccarose or a combination, was investigated. Another aim was to study whether isolate of oral bacteria were able to metabolise reb A in broth bacteria culture.Material and MethodBiofilms from three adult individuals were repeatedly exposed to 0,025% stevioside, 0,025% reb A or 10% Todd Hewitt (TH). Samples were taken at the beginning and at the end of the experiment for cultivation and bacterial identification.Biofilms were exposed to 0,025% reb A and 10% saccharose, either separately or combined, followed by a pH-measurement. Furthermore, 23 bacterial species were studied in regards to ability to metabolize reb A. ResultsAll biofilms were dominated by Streptococcus regardless of whether they were exposed to stevioside, reb A, or TH. A pH-decrease was seen in biofilms exposed to saccharose or a combination of saccharose and reb A. However, only minor changes were noted after exposure to reb A.None of the species studied were able to metabolize reb A.ConclusionNo major differences in the bacterial composition of the biofilms were seen after repeated exposure to stevioside or reb A compared to TH.No pH-decrease after repeated exposure to reb A was noted. Exposure to reb A combined with saccharose resulted in a pH-decrease similar to exposure to saccharose. The bacterial species tested in this study were not able to metabolise reb A.

The aim of the diploma thesis was evaluation of sensoric attributes of blueberry and current jams sweetened by selected Steviol glycosides and their comparing with other sweeteners. For evaluation of sensoric attributes were used rebaudioside A, stevioside, aspartame, erythritol and sugar beet. Stevia rebaudiana var. Bertoni is a persistent plant, which contains sweetening Steviol glycosides, which have a use in food processing. They are available in the area of EU indicated as E960 since 2011. The most used Steviol glycosides are stevioside and rebaudioside A, which are demarcated with a high sweetness, which could be 200-400 times higher than saccharose. The usage of sweeteners is legislatively allowed, which means it is also allowed to do sensoric analyse and detect perception of added substances in food. These analyses evaluate food with the help of sense - perception. Methodical part of the work contains procedure, used methods for gaining evaluation of data from a sensoric analyse after the degustation of jams, which has taken place with the presence of ten judges - an academic students in the working compartment of University of South Bohemia. Respondents entered their evaluation into protocols, which were prepared in advance. The results of diploma thesis are evaluated by the method of Kramer, which is used for this kind of analyse. The critical scale between 20 and 40 with the value of probability P = 95% was defined according to this method. Evaluation is described with the help of graphs and tables, added with a wordily describtion. Conclusion of analyse of diploma thesis is, that the usage of Steviol glycosides is not most suitable way of sweetening jams. Acceptable valuations of jams containing Steviol glycosides were proven in parameters of colour and aroma. The reasons for less successful evaluation of Steviol glycosides could be due to bad pH of food or badly chosen dosing, without combination of other kinds of sweeteners. The best sensory characteristics traditionally showed sugar beet and aspartame followed by rebaudioside A and stevioside, which were placed on the third and forth place from five used sweeteners. The part of the diploma thesis was an evaluation of questionnaire, where the result was, that asked people preferred fruit ingredient of jams (type of fruit, amount) not their way of sweetening while choosing a jam.

The steviolglycosides are the natural, sweet substances from Stevia rebaudiana Bertoni. It affect human health positively and its sweetness is 300x stronger than the sweetness of sucrose. That's why it's used to sweetening the commercial products. Because of its potential properties it's good to have an appropriate method to isolate it. High performance liquid chromatography ( HPLC) is based on the separation of analyte between two immiscible phases with high pressure pump and appropriately chosen stationary phase in the column. Than the analytes come out from the column in different retention times. This master´s thesis follows up selection of the best HPLC system for isolation of the main steviolglycosides and its analysis in commercial products. In the theoretical part of the thesis is described the origin, basic characteristics, botanical description, cultivation and affect to the human health of Stevia rebaudiana Bertoni and its use in food industry. There are also concisely characterized the sweet substances contained in the plant, so called steviolglycosides. Than there are given the theoretical basics of high performance liquid chromatography, HPLC instrumentation and its specific applications at sleviolglycosides with the basic chromatographic parameters. The object of the first experimental part was to research the optimum conditions for time and separation effective chromatographic analysis and select the best chromatographic system for isolation of steviolglycosides. In the second experimental part, I have compared and defined the main steviolglycosides (stevioside, rebaudioside A) in nine selected products, commercially available in Czech republic, with the best chromatographic method. In these products was the contain of the stevioside or rebaudioside A confirmed or refused.