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...

The market of medicinal plants and herbs such as cilantro (Coriandrum sativum) and stevia (Stevia rebaudiana) has been growing due to the demand of the food, chemical and pharmaceutical industries. Leaves of C. sativum are used by many countries in the preparation of food and in folk medicine. The essential oil from the leaves has potential for use in the food industry as an antimicrobial agent and as a condiment to mask undesirable odors and flavors. Studies were conducted to evaluate the activity of the essential oil of cilantro against Leishmaniasis, and the results showed that the essential oil and its major compounds inhibited the growth of L. donovani in its different forms, indicating potential use of this plant for the production of new drugs against Leishmaniasis. From these results, another study was conducted to evaluate five materials of cilantro, a cultivar and four germplasm accessions from the North American Department of Agriculture regarding the quality of the raw material for the food and pharmaceutical industries. The materials with the highest potential to use were AMES 18596 and PI193770 for producing larger amount of dry biomass and the highest production of major compounds, which are desirable by industries. Stevia is a species with high potential to be consumed by diabetics and obese, it is a source of diterpene glycosides used as natural sweeteners. Sweeteners derived from stevia are ingredients in the production of foods, juices and soft drinks in Asia, Europe and in the Americas. The compounds responsible for the sweetness of stevia leaves are well characterized in the literature. However, good agronomic practices are still poorly studied. Some countries have worked on improving this culture and have achieved cultivars with higher yield. In the area of plant nutrition, calcium is one of the most important nutrients for the production of biomass and stevioside in stevia plants. Thus, a preliminary study was conducted to test the effect of calcium applied in nutrient solution in stevia yield, and stevioside and rebaudioside in hydroponic system. The leaf, stem and shoot biomass yield, by stevia plants were influenced by Ca rates in nutrient solutions and were maximal with 7.0, 5.7, 6.2 mmol L-1 of Ca, respectively. The production of stevioside and rebaudioside A was maximal with addition of 4.0 mmol of Ca L-1. / O mercado de plantas medicinais, condimentares e aromáticas tais como coentro (Coriandrum sativum) e estévia (Stevia rebaudiana) vêm crescendo devido à demanda das indústrias alimentícias, químicas e farmacêuticas. As folhas C. sativum são utilizadas por diversos países no preparo de alimentos e na medicina popular. O óleo essencial das folhas tem potencial para utilização na indústria de alimentos como agente antimicrobiano e como condimento para mascarar cheiros e sabores indesejáveis. Estudos foram realizados para avaliar a atividade do óleo essencial de coentro contra a leishmaniose, e os resultados revelaram que o óleo essencial e seus compostos majoritários inibiram o crescimento L. donovani em suas diferentes formas, indicando potencial de uso dessa planta para a produção de novos medicamentos contra Leishmaniose. A partir desses resultados, outro estudo foi realizado com o objetivo de avaliar cinco materiais de coentro, uma cultivar e quatro acessos do germoplasma do Departamento de Agricultura norte americano quanto à qualidade da matéria prima para as indústrias de alimentos e farmacêuticas. Os materiais variaram entre si quanto à produção de biomassa, acúmulo de nutrientes e produção de aldeídos alifáticos tais como (E)- 2-decenal, (E)- 2-dodecenal. Os materiais que apresentaram maior potencial de uso foram AMES 18596 e PI193770, pois, produziram maior quantidade de massa seca e produção de compostos majoritários, que são os desejáveis pelas indústrias. Quanto à estévia, essa espécie apresenta elevado potencial para ser usado por pessoas diabéticas e obesas, pois é fonte de diterpenos glicosídicos usados como adoçantes naturais. Os adoçantes derivados de estévia são usados na produção de alimentos, sucos e refrigerantes em diversos países asiáticos, europeus e no continente americano. Os compostos responsáveis pelo dulçor das folhas de estévia são bem caracterizados pela literatura, entretanto, as boas práticas agronômicas ainda são pouco estudadas. Alguns países trabalharam no melhoramento dessa cultura e conseguiram alcançar cultivares mais produtivas. Na área de nutrição, o cálcio está entre os nutrientes mais importantes para a produção de esteviosídeo e produção de biomassa de estévia. Assim, um estudo preliminar foi conduzido com o objetivo de estudar o efeito do cálcio aplicado em solução nutritiva na produção de estévia, e de esteviosídeos e rebaudiosídeos, em sistema hidropônico. A produção de folhas, caule e parte aérea por plantas de estévia foi influenciada por níveis de Ca na solução nutritiva e foram máximos com as doses de 7,0, 5,7, 6,2 mmol L-1 de Ca, respectivamente. A produção de esteviosídeo e rebaudiosídeo A foi máxima com a adição de 4,0 mmol L-1 de Ca.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Stevia rebaudiana is a perennial plant, native to South America; it has natural sweeteners (steviol glycosides) on their leaves free of calories, very sweet and could be replaced by sucrose without presenting problems to diabetic people diet. Stevia cultivation is widespread all over the world under different weather conditions and the environmental factors could affect the stevia yield. The aims of this study were to evaluate dry weight and sweetener concentration in Stevia rebaudiana leaves during different periods in the year to determinate the best time for harvesting and to verify the influence of environmental conditions in the sweeteners yield. The first trial was carried out during 2012/2013 agricultural year. In total 120 plants were selected and divided in six groups with 20 plants each. Twelve harvests were performed and the higher yield was reached in December at the first harvest for groups 3, 4 and 2 (292.4; 285.2 and 206.7 g m-2, respectively). The sweetener concentrations have differentiated between the date of the harvest and the compounds analysed. The highest concentrations of stevioside (12.16% and 11.36%) and rebaudioside C (2.95% e 1.95%) were found during the harvest in January while the sweetener rebaudioside A, the highest concentrations were 7.01%; 6.16% and 6.15% in December, February and March, respectively. The heighest stevioside concentrations (per cent) (12.16% and 11.36%) and rebaudioside C (2.95% and 1.95%) were found to the harvest done in January, whereas to rebaudioside A, the heighest concentration were in December (7.01%); February (6.16%) and March (6.15%). Photoperiod has influenced the number of days for each harvest as well as the sweetener concentration on the leaves. The second trial seven genotypes were tested by plants already established on the field. The higher yield of dry mass was found during the harvest in March and the sweetener concentrations have decreased to the second harvest comparing to the first one. The highest sweetener concentrations of stevioside in the first harvest were with the genotypes C1, C4 and C8 (11.24; 11.14 and 10.32%, respectively) and genotype C6 has presented the highest rebaudioside A and C concentrations (8.94 and 1.96% respectively). / Originária da América do Sul, Stevia rebaudiana é uma planta arbustiva perene que possui edulcorantes naturais (glicosídeos de esteviol) com alto poder adoçante em suas folhas, livre de calorias, podendo substituir a sacarose sem apresentar problemas na dieta de pessoas diabéticas. O cultivo de estévia é difundido em muitos países e os fatores climáticos podem influenciar significativamente o resultado de produtividade desta cultura. Os objetivos deste estudo foram avaliar massa seca e concetração de edulcorantes em folhas de estévia (Stevia rebaudiana) em diferentes períodos do ano para determinar a melhor época para a colheita e, verificar a influência dos fatores climáticos na quantidade dos edulcorantes. Os experimentos foram realizados nos anos agrícolas 2011/2012 e 2012/2013. No primeiro experimento 120 plantas foram divididas em seis grupos de 20 plantas cada. Doze cortes foram realizados, sendo a maior produtividade alcançada no mês de dezembro nos primeiros cortes dos grupos 3, 4 e 2 (292,4; 285,2 e 206,7 g m-2, respectivamente). A concentração dos edulcorantes nas folhas de estévia variou entre os períodos dos cortes e entre os compostos analisados. As concentrações mais altas de esteviosídeo (12,16% e 11,36%) e rebaudiosídeo C (2,95% e 1,95%) foram nas colheitas realizadas em janeiro, enquanto que para o rebaudiosídeo A (7,01%; 6,16% e 6,15%), as maiores porcentagens foram alcançadas nos meses de dezembro, fevereiro e março. O fotoperíodo influenciou o número de dias para o corte realizado bem como a concentração de edulcorantes nas folhas. No segundo experimento foram testados sete genótipos selecionados a partir de plantas já estabelecidas. A maior produtividade de massa seca foi encontrada no corte realizado no mês de março e, as concentrações dos edulcorantes diminuíram no segundo corte em todos os genótipos. As porcentagens mais altas de esteviosídeo no primeiro corte foram nos genótipos C1, C4 e C8 (11,24; 11,14 e 10,32%, respectivamente) e, o genótipo C6 apresentou as maiores concentrações de rebaudiosídeo A e C (8,94 e 1,96% respectivamente).

The market of medicinal plants and herbs such as cilantro (Coriandrum sativum) and stevia (Stevia rebaudiana) has been growing due to the demand of the food, chemical and pharmaceutical industries. Leaves of C. sativum are used by many countries in the preparation of food and in folk medicine. The essential oil from the leaves has potential for use in the food industry as an antimicrobial agent and as a condiment to mask undesirable odors and flavors. Studies were conducted to evaluate the activity of the essential oil of cilantro against Leishmaniasis, and the results showed that the essential oil and its major compounds inhibited the growth of L. donovani in its different forms, indicating potential use of this plant for the production of new drugs against Leishmaniasis. From these results, another study was conducted to evaluate five materials of cilantro, a cultivar and four germplasm accessions from the North American Department of Agriculture regarding the quality of the raw material for the food and pharmaceutical industries. The materials with the highest potential to use were AMES 18596 and PI193770 for producing larger amount of dry biomass and the highest production of major compounds, which are desirable by industries. Stevia is a species with high potential to be consumed by diabetics and obese, it is a source of diterpene glycosides used as natural sweeteners. Sweeteners derived from stevia are ingredients in the production of foods, juices and soft drinks in Asia, Europe and in the Americas. The compounds responsible for the sweetness of stevia leaves are well characterized in the literature. However, good agronomic practices are still poorly studied. Some countries have worked on improving this culture and have achieved cultivars with higher yield. In the area of plant nutrition, calcium is one of the most important nutrients for the production of biomass and stevioside in stevia plants. Thus, a preliminary study was conducted to test the effect of calcium applied in nutrient solution in stevia yield, and stevioside and rebaudioside in hydroponic system. The leaf, stem and shoot biomass yield, by stevia plants were influenced by Ca rates in nutrient solutions and were maximal with 7.0, 5.7, 6.2 mmol L-1 of Ca, respectively. The production of stevioside and rebaudioside A was maximal with addition of 4.0 mmol of Ca L-1. / O mercado de plantas medicinais, condimentares e aromáticas tais como coentro (Coriandrum sativum) e estévia (Stevia rebaudiana) vêm crescendo devido à demanda das indústrias alimentícias, químicas e farmacêuticas. As folhas C. sativum são utilizadas por diversos países no preparo de alimentos e na medicina popular. O óleo essencial das folhas tem potencial para utilização na indústria de alimentos como agente antimicrobiano e como condimento para mascarar cheiros e sabores indesejáveis. Estudos foram realizados para avaliar a atividade do óleo essencial de coentro contra a leishmaniose, e os resultados revelaram que o óleo essencial e seus compostos majoritários inibiram o crescimento L. donovani em suas diferentes formas, indicando potencial de uso dessa planta para a produção de novos medicamentos contra Leishmaniose. A partir desses resultados, outro estudo foi realizado com o objetivo de avaliar cinco materiais de coentro, uma cultivar e quatro acessos do germoplasma do Departamento de Agricultura norte americano quanto à qualidade da matéria prima para as indústrias de alimentos e farmacêuticas. Os materiais variaram entre si quanto à produção de biomassa, acúmulo de nutrientes e produção de aldeídos alifáticos tais como (E)- 2-decenal, (E)- 2-dodecenal. Os materiais que apresentaram maior potencial de uso foram AMES 18596 e PI193770, pois, produziram maior quantidade de massa seca e produção de compostos majoritários, que são os desejáveis pelas indústrias. Quanto à estévia, essa espécie apresenta elevado potencial para ser usado por pessoas diabéticas e obesas, pois é fonte de diterpenos glicosídicos usados como adoçantes naturais. Os adoçantes derivados de estévia são usados na produção de alimentos, sucos e refrigerantes em diversos países asiáticos, europeus e no continente americano. Os compostos responsáveis pelo dulçor das folhas de estévia são bem caracterizados pela literatura, entretanto, as boas práticas agronômicas ainda são pouco estudadas. Alguns países trabalharam no melhoramento dessa cultura e conseguiram alcançar cultivares mais produtivas. Na área de nutrição, o cálcio está entre os nutrientes mais importantes para a produção de esteviosídeo e produção de biomassa de estévia. Assim, um estudo preliminar foi conduzido com o objetivo de estudar o efeito do cálcio aplicado em solução nutritiva na produção de estévia, e de esteviosídeos e rebaudiosídeos, em sistema hidropônico. A produção de folhas, caule e parte aérea por plantas de estévia foi influenciada por níveis de Ca na solução nutritiva e foram máximos com as doses de 7,0, 5,7, 6,2 mmol L-1 de Ca, respectivamente. A produção de esteviosídeo e rebaudiosídeo A foi máxima com a adição de 4,0 mmol L-1 de Ca.

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...

Dental caries affects people worldwide. The oral microflora has been found to be less responsible than the carbohydrate diet, which is considered the main cause of dental caries. Carbohydrates from sucrose are processed by oral bacteria and acids are produced as a byproduct. High sugar intake is a risk factor for systemic health and for overall health of the oral cavity. Artificial sweeteners are considered a healthy alternative for people who prefer the sweet taste but renounce sucrose. In the United States there are several artificial sweeteners approved by the Food and Drug Administration. Among these, stevia is the only natural sweetener. Stevia is extracted from the Stevia rebaudiana plant. Steviol glycosides are extracted from the stevia leaf and refined to get the best taste quality. There are many types of steviol glycosides that are extracted from the stevia plant and the most important steviol glycosides will be discussed in the thesis. Scientific research (PubMed, EMBASE), appropriate websites (Colgate, Pure Circle Stevia Institute), the World Health Organization, HealthInfo Clinical Advisers and vectorstock as well as books in English and in Russian (The Stevia Deception: The Hidden Dangers of Low Calorie Sweeteners By Bruce Fife, and Puti Izbavlenie of Bolezne by Neumyvakin) were consulted. Electronic searches were limited to publications in the past five years. The scope of the thesis is to review the basic statistics on dental caries, the types of caries and the number of people affected, discuss the advantages and benefits of stevia relative to other artificial sweeteners, review the safety of stevia including side effects and potential as an addictive sugar substitute and lastly, to assess and recommend which form of stevia is most effective against dental caries.

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

La consommation excessive de sucres conduit à l’augmentation de désordres métaboliques, tels que l’obésité et le diabète. Les consommateurs souhaitent une alimentation plus saine à base de produits d’origine naturelle. Stevia rebaudiana, l’herbe sucrée du Paraguay, accumule dans ses feuilles des glycosides de stéviol (SGs) considérés comme des édulcorants naturels intenses, dont le marché est en pleine expansion au niveau mondial. Dans ce contexte, la société Oviatis implante une filière BIO de Stevia rebaudiana en Nouvelle-Aquitaine. Les objectifs de cette thèse CIFRE sont de caractériser la variabilité phénotypique d’une collection de cette espèce en vue de la mise en place future d’un programme de sélection. Les composantes du rendement, de biomasse foliaire, de quantité et qualité des SGs et de la réponse à Septoria sp. ont été finement décrites en condition de production pluriannuelle et multi-sites ou en conditions contrôlées. Ces travaux ont permis de 1) développer des outils de phénotypage métabolique, pathologique et au champ, 2) évaluer la variabilité phénotypique de cette collection de Stevia rebaudiana en condition de production et identifier les descripteurs de cette variabilité, 3) identifier les facteurs ontogéniques, abiotiques et culturaux impliqués dans la variabilité de cette collection, 4) évaluer la variabilité de réponses face à la septoriose. Ces résultats permettent d’identifier les critères de sélection de cette espèce pour une production BIO en Nouvelle Aquitaine, ainsi que des génotypes d’intérêt. Ils constituent les bases de la mise en place d’un programme d’amélioration variétale. / Obesity and diabetes are consequences of metabolic disorders due to excessive sugar consumption. Consumers want a healthier diet with natural products. Stevia rebaudiana, the Paraguayan sweet herb, accumulates steviol glycosides (SGs) into its leaves. Considered as intense natural sweeteners, SGs represent a global market constantly increasing. In this context, Oviatis establishes a complete organic sector in the Nouvelle-Aquitaine region. The aims of this CIFRE thesis are to characterise the phenotypic variability within a collection of genetic resources, in order to implement a breeding program. Yield components, leaves biomass, SGs quality and quantity and responses against Septoria sp. were described in perennial and multi-site field conditions or in controlled conditions. This work allowed to: 1) develop tools for metabolic, pathology field-phenotyping, 2) evaluate the phenotypic variability of this Stevia rebaudiana collection in field conditions and identify variability descriptors, 3) identify ontogenic, abiotic and cultural factors implicated into phenotypic variability, and 4) evaluate response variability against septoria disease. These results led to the identification of genotypes of interest, as well as selection criteria for the organic production in Nouvelle-Aquitaine, which are the basis for the implementation of a breeding program.

L’alimentation des pays industrialisés, souvent trop riche en glucides et en lipides, a engendré une augmentation marquée de l’obésité et favorisé le risque de diabète. Face à ce constat, l’industrie agroalimentaire s’est efforcée de développer des produits allégés. Au sucre, ont ainsi été substitués les édulcorants acaloriques, molécules au goût sucré non métabolisées par le corps humain. Ces édulcorants sont souvent obtenus par voie de synthèse chimique comme l’aspartame, l’acésulfame K ou encore le sucralose. S’ils sont autorisés dans la confection de produits alimentaires, la toxicité de ces édulcorants et leur impact sur la santé humaine nourrissent cependant les débats au sein de la communauté scientifique, ainsi des solutions alternatives sont envisagées. Stevia rebaudiana Bertoni est une plante originaire du Paraguay. Son intérêt principal réside dans le fait qu’elle contient des molécules acaloriques au goût sucré, les glycosides de stéviol, dont les plus connus sont le rébaudioside A et le stévioside. Les études effectuées sur ces diterpènes glycosilés tendent à souligner leur innocuité. Ils présenteraient même des vertus thérapeutiques, comme des propriétés anti-hypertensive ou antidiabétique, et semblent ainsi être une alternative prometteuse au sucre et aux édulcorants artificiels. Notre étude s’est orientée sur l’extraction aqueuse des glycosides de stéviol des feuilles de Stévia. A l’échelle laboratoire, nous avons pu mettre en évidence que la cinétique d’extraction des molécules cibles est principalement limitée par la diffusion des solutés dans la matrice solide végétale. Il n’y aurait pas de réaction chimique, de gonflement de la matière ou de modification de la porosité pendant le processus d’extraction. Nous avons également pu souligner que la préparation de la matière (séchage et broyage) et les conditions opératoires (température, ratio liquide-solide et agitation) sont des facteurs prépondérants sur l’efficacité d’extraction des édulcorants. A l’échelle pilote, les résultats obtenus en systèmes discontinu et continu ont pu mettre en exergue des influences similaires de la préparation de la matière et des conditions opératoires ; nous avons de plus pu démontrer que la qualité du mélange des phases solide et liquide était un facteur clé à l’efficacité de l’extraction. Fort de ces résultats nous avons pu développer un procédé d’extraction en système continu, adapté à l’extraction des glycosides de stéviol des feuilles de Stévia, et combiné à une étape de séparation solide-liquide. Au travers de nos différentes expériences, nous avons pu décrire précisément son fonctionnement et optimiser l’extraction afin de pouvoir envisager une transposition à l’échelle industrielle. / Industrialized countries food, often too rich in carbohydrates and fat, has resulted in an important increase in obesity and risk of diabetes. Given this fact, the food industry tries to develop diet products. Sugar was substituted by calorie-free sweeteners, sweet-tasting molecules non-metabolized by the human body. These sweeteners are often obtained by chemical synthesis such as aspartame, acesulfame K or sucralose. Even if they are allowed in the manufacture of food products, the toxicity of these sweeteners and their impact on human health raise debate within the scientific community, so alternatives are being considered. Stevia rebaudiana Bertoni is a plant native from Paraguay. Its main interest lies in the fact that it contains calorie-free sweet tasting molecules, steviol glycosides, of which the most common are rebaudioside A and stevioside. Studies of these glycosylated diterpenes tend to highlight its safety. They even present therapeutic characteristics, such as anti-hypertensive or anti-diabetic properties, and thus seem to be a promising alternative to sugar and artificial sweeteners. Our study focused on the aqueous extraction of steviol glycosides from Stevia leaves. At thelaboratory scale, we were able to demonstrate that extraction kinetics of target molecules are mainly limited by diffusion of solutes in the plant solid matrix. There would be no chemical reaction, swelling of the plant material, or modification of the porosity during the extraction process. We have also shown that the preparation of the plant material (drying and milling) and the operating conditions (temperature, liquid-solid ratio and agitation) are overriding factors on the sweeteners’ extraction efficiency. At the pilot scale, the results obtained in batch and continuous systems could highlight similar influences with the preparation of the plant material and the operating conditions. We have also shown that the quality of the mixing of solid and liquid phase was a key factor in extraction efficiency. With these results we have developed a continuous extraction process, suitable for the extraction of steviol glycosides from Stevia leaves, which has combined with a step of solid-liquid separation. Through our experimentation we have accurately described its operation and optimized the extraction in order to consider a transposition to the industrial scale.

This thesis deals with the sorting and use of sweeteners in the production of fruit spreads. The theoretical part focuses on the sorting of sweetening agents and their classification. Furthermore, it presents procedures of production of fruit spreads and their assortment. The main goal is to compare sensory attributes of jams that I produced according to an assigned recipe, which is described in the practical part of this thesis. These jams were made from regional fruits (strawberries, apricots, sour-cherries) and sweetened with both natural (sucrose, sorbitol, Steviol glycosides) and artificial (sucralose) sweetening agents. The control group of this research were the students and employees of the University of South Bohemia in České Budějovice in the age of 24-60 years. The evaluators filled in a sequence test protocol and a questionnaire for consumers. The results of the sensory analysis were processed into tables and graphs with the aid of statistical methods.