<p>U okviru ove disertacije su ispitane mogućnosti primene savremenih postupaka za dobijanje ekstrakata rtanjskog čaja (Satureja montana L.).<br />U ekstraktima dobijenim primenom superkritičnog ugljendioksida (različitih gustina) utvrđeno je prisustvo karvakrola kao najzastupljenije komponente. Ekstrakt sa najvećim sadržajem karvakrola je dobijen pri uslovima 350 bar i 50 &deg;C (60,82%), dok je najveći prinos karvakrola ostvaren pri pritiscima 325 i 350 bar i temperaturi 60 &deg;C, i iznosio je 2,4 g/100 g droge. Primenom ugljendioksida na većim pritiscima se ostvaruje efikasnija ekstrakcija karvakrola iz droge, kao i proizvodnja ekstrakata sa većim sadržajima karvakrola. Druge komponente prisutne u ekstraktima u znatno nižim koncentracijama su: p-cimen, borneol, trans-kariofilen, kariofilen-oksid, &gamma;-terpinen i linalool.<br />Da bi se utvrdila mogućnost unapređenja prinosa ekstrakcije superkritičnim ugljendioksidom, kao i pobolj&scaron;anja kvaliteta ekstrakata, ispitan je uticaj različitih predtretmana biljnog materijala. Ustanovljeno je da predtretman vodom najznačajnije povećava prinos ekstrakcije (za 25%), dok je ultrazvučni predtretman najadekvatniji izbor za dobijanje ekstrakata sa najvećim sadržajem karvakrola (66,46%). Međutim, najveći prinos ekstrakcije karvakrola iz 100 g droge postignut je primenom etanola i vode kao predtretmana.<br />Ekstrakti dobijeni primenom subkritične vode (uz variranje temperature i vremena ekstrakcije) su hemijski okarakterisani i radi utvrđivanja uslova ekstrakcije pri kojima se postiže najveći kvalitet ekstrakata u pogledu sadržaja polifenolnih komponenti, kao i antioksidantne aktivnosti, primenjena je metoda odzivne povr&scaron;ine (RSM). Utvrđena je prednost primene subkritične vode u odnosu na klasičnu metodu ekstrakcije u pogledu sadržaja polifenolnih komponenti i antioksidantne aktivnosti.<br />Suvi ekstrakti S. montana dobijeni spray drying tehnologijom su&scaron;enja (sa različitim koncentracijama maltodekstrina) su ispitani u pogledu fizičkih i hemijskih osobina. Takođe, radi ispitivanja mogućnosti primene u vidu funkcionalnih ili prehrambenih proizvoda, izvr&scaron;ena je senzorna analiza dobijenih suvih ekstrakata, i analizirana je njihova farmakolo&scaron;ka aktivnost u smislu ACE inhibitornog i antioksidantnog delovanja.<br />Ustanovljeno je da je najniža koncentracija maltodekstrina (10%) najadekvatnija za dobijanje suvog ekstrakta S. montana sa najvećim sadržajem polifenolnih komponenti, sadržajem etarskog ulja i u pogledu antioksidantne i ACE inhibitorne aktivnosti. Takođe, suvi ekstrakt sa 10% maltodekstrina ispunjava i ostale fizičko-hemijske parametre, kojima se osigurava kako efikasno su&scaron;enje, tako i kvalitet ekstrakta.<br />In vivo aktivnost odabranih ekstrakata S. montana ispitana je na modelu hepatotoksičnosti indukovane primenom ugljentetrahlorida. Analiziran je uticaj ekstrakata na funkciju jetre, enzimske parametre oksidativnog stresa i serumske parametre oksidativnog o&scaron;tećenja izazvanog ugljentetrahloridom i ustanovljen je hepatoprotektivni učinak ekstrakata S. montana. Takođe, ispitano je dejstvo ekstrakata na rast Ehrlich-ovih ascitnih tumora implantiranih mi&scaron;evima, kao i na biohemijske parametre oksidativnog stresa u ćelijama tumora i utvrđeno je da je vreme aplikacije ekstrakata od izuzetnog značaja.</p> / <p>This dissertation investigates the possibilities of applying contemporary processes for obtaining extracts of winter savory (Satureja montana L.).<br />Carvacrol was determined to be the most abundant component in extracts acquired by using supercritical carbon dioxide (of various densities). Extract with the richest content of carvacrol was obtained under conditions of 350 bar and 50 &deg;C (60,82%), while the highest carvacrol yield was produced under 325 bar and 350 bar pressures and at a 60 &deg;C temperature reaching 2.4 g/100 g of dry weight. By applying carbon dioxide on higher pressures, a more efficient carvacrol extraction from herbal material is performed, alongside production of extracts with higher carvacrol content. Other components in the extracts present in significantly lower concentrations are the following: p-cymen, borneol, trans-caryophyllene, caryophyllene-oxide, &gamma;-terpinene and linalool.<br />In order to recognize the possibility of improving the extraction yield produced by employing supercritical carbon dioxide and enhancing the quality of extracts, the influence of several different pre-treatments was investigated. It was determined that the water pre-treatment raises the extraction yield the most (for 25%), while the ultrasonic pre-treatment was the most adequate choice for acquiring extracts with the highest content of carvacrol (66.46%). However, the highest carvacrol extraction yield from 100 g of dry weight was achieved by applying ethanol and water as pre-treatment.<br />Extracts which were attained with subcritical water (by varying the temperature and time of extraction) were chemically characterized and Response Surface Method (RSM) was applied for the purposes of determining conditions of extractions under which the highest quality of extracts in terms of content of polyphenolic compounds and antioxidant activity is reached. The advantage of employing subcritical water over the classical method of extraction in respect of content of polyphenolic components and antioxidant activity was demonstrated.<br />Dry extracts of S. montana obtained by using the spray drying technology (with different concentrations of maltodextrine) were examined in terms of their physical and chemical characteristics. Also, in order to investigate the possibility of their use as functional or food products, sensory analysis of attained extracts was performed, and furthermore, their pharmacological activity in terms of the ACE inhibitory and antioxidant effect was analysed as well.<br />It was determined that the lowest concentration of maltodextrin (10%) was the most adequate for obtaining dry extracts of S. montana with the richest content of polyphenolic components and essential oil, and in respect of antioxidant and ACE inhibitory activities. Also, dry extract with 10% of maltodextrin meets all other physical-chemical parameters, which provide efficient drying and quality of extracts.<br />In vivo activity of selected extracts of S. montana was examined on a model of hepatotoxicity induced by applying carbon tetrachloride. The influence of extracts on liver function, enzyme parameters of oxidative stress, and serum parameters of oxidative damage caused by carbon tetrachloride was analysed and hepatoprotective effect of S. montana extracts was determined. In addition, the influence of extracts on the growth of Ehrlich ascite tumors implanted in mice was investigated, and their impact on biochemical parameters of oxidative stress in tumor cells as well, and it was determined that the time of application of extracts is of great significance.</p>

Les voies de valorisation actuelles ne permettent pas d'absorber la totalité des coproduits de l'industrie du bois (noeuds, écorces, souches), qui sont des matières riches en composés bioactifs tels que les polyphénols, préférentiellement extraits par des solvants comme l'éthanol ou le méthanol. Le fractionnement aqueux à l'aide d'extracteur bi-vis permet d'obtenir des extraits polyphénoliques à activité antioxydante. Après avoir déterminé l'extractabilité des polyphénols de chaque matière en eau subcritique, les paramètres d'extraction en bi-vis ont été optimisés de façon à d'atteindre des conditions de température et de pression caractéristiques de l'eau subcritique. La présence d'un marqueur spécifique dans les extraits étaye l'hypothèse selon laquelle l'extracteur bi-vis permet d'atteindre les conditions de l'eau subcritique. Les effets des extraits sur la biodégradabilité de pots horticoles dans lesquels ils ont été incorporés ainsi que sur la croissance de plantes mises en culture dans ces pots ont été déterminés. La valorisation de l'extrudat comme matériau pour le thermopressage en panneaux de particules a également été étudiée. / Actual exploitation procedures can not absorb all the wood by-products (knots, barks, stumps), which are potent sources of bioactive compounds such as polyphenols, preferentially extracted by solvents such as ethanol or methanol. Aqueous fractionation has been performed with twin-screw extractors to obtain polyphenolic extracts with antioxidant activity. After having determined the extractability of polyphenols from each byproduct with subcritical water, the parameters of twin-screw extraction were optimized in order to reach temperature and pressure conditions in subcritical water area. The presence of a specific marker in the extracts supports the hypothesis that a twin-screw extractor allows to reach subcritical water conditions. The effects of the extracts on the biodegradability of horticultural pots in which the extracts have been incorporated and on the development of the plants grown in these pots have been evaluated. The valorisation of the extrudate as raw material for thermopressing particle boards has also been studied.

The physiological benefits of flaxseed against pathological disturbances, such as cancers and heart diseases, are mainly attributed to its high lignan content. This study (Experiment 1) examined the application of pressurized low polarity water (PLPW) for extraction of lignans, proteins and carbohydrates from defatted flaxseed meal. Key processing conditions included temperature (130, 160, 190°C), solvent pH (4, 6.5 and 9), solvent to solid ratio (S/S) (90, 150 and 210 mL/g) and introduction of co-packing material (0 and 3 g glass beads). The addition of 3 g glass beads as co-packing material facilitated extraction by enhancing surface contact between the liquid and solid thus shortening extraction time. Elevated temperature accelerated the extraction rate by increasing the solid diffusion coefficient thereby reducing the extraction time. The maximum yield of lignans (99 %) was obtained at temperatures ranging from 160°C to 190°C, with solvent volume of 180 mL (90 mL/g meal) at pH 9. Optimal conditions for protein extraction (70 %) were pH 9, extraction volume of 420 mL (210 mL/g meal) and 160°C. Total carbohydrates yield was maximized at 50% recovery at pH 4 and 160°C with 420 mL solvent (210 mL/g meal). Increased temperature accelerated extraction, thus reducing solvent volume and time to reach equilibrium. For the extraction of proteins, however, a temperature of 130-160°C is recommended, as proteins are vulnerable to thermal degradation due to heat decomposition. The effects of flow rate and geometric dimensions for extraction of lignans and other flaxseed meal bioactives were further investigated in Experiment 2, based on the variables optimized in the previous experiment. Defatted flaxseed meal was extracted with pH 9 buffered water with meal to co-packing glass beads ratio of 1:1.5 at 5.2 MPa (750 psi) and 180°C. The aqueous extracts were analyzed for lignan, protein and carbohydrate using HPLC and colorimetric methods. The optimal extraction yields for lignan, protein and carbohydrate were found at flow rates of 1 to 2 mL/min with bed depth between 20 and 26 cm and a S/S ratio of 40 to 100 mL/g. The combination of low flow rate and high bed depth allowed the use of lower S/S ratio with reduced total solvent volume consumption. This study also evaluated the mass transfer kinetics governing the process of lignan extraction from flaxseed meal in a fixed bed extraction cell. Diffusion of solute into the continuously flowing solvent was mainly responsible for the mass transfer mechanism as flow rate did not increase proportionally with the yield and rate of extraction. The extraction kinetics were studied on the basis of two approaches: Fick’s diffusion equation and a two-site exponential kinetic model. The proposed two-site exponential kinetic model corresponding to the two-stage extraction (rapid and slow phases) successfully described the experimental data. Diffusivities attained from Fick’s diffusion model ranged from 2 x 10-13 to 9 x 10-13 m2s-1 while mass transfer coefficients were between 4.5 x 10-8 and 2.3 x 10-7 ms-1 for extraction of lignans at 180°C, pH 9 with 1:1.5 meal to co-packing material ratio. / February 2007

The physiological benefits of flaxseed against pathological disturbances, such as cancers and heart diseases, are mainly attributed to its high lignan content. This study (Experiment 1) examined the application of pressurized low polarity water (PLPW) for extraction of lignans, proteins and carbohydrates from defatted flaxseed meal. Key processing conditions included temperature (130, 160, 190°C), solvent pH (4, 6.5 and 9), solvent to solid ratio (S/S) (90, 150 and 210 mL/g) and introduction of co-packing material (0 and 3 g glass beads). The addition of 3 g glass beads as co-packing material facilitated extraction by enhancing surface contact between the liquid and solid thus shortening extraction time. Elevated temperature accelerated the extraction rate by increasing the solid diffusion coefficient thereby reducing the extraction time. The maximum yield of lignans (99 %) was obtained at temperatures ranging from 160°C to 190°C, with solvent volume of 180 mL (90 mL/g meal) at pH 9. Optimal conditions for protein extraction (70 %) were pH 9, extraction volume of 420 mL (210 mL/g meal) and 160°C. Total carbohydrates yield was maximized at 50% recovery at pH 4 and 160°C with 420 mL solvent (210 mL/g meal). Increased temperature accelerated extraction, thus reducing solvent volume and time to reach equilibrium. For the extraction of proteins, however, a temperature of 130-160°C is recommended, as proteins are vulnerable to thermal degradation due to heat decomposition. The effects of flow rate and geometric dimensions for extraction of lignans and other flaxseed meal bioactives were further investigated in Experiment 2, based on the variables optimized in the previous experiment. Defatted flaxseed meal was extracted with pH 9 buffered water with meal to co-packing glass beads ratio of 1:1.5 at 5.2 MPa (750 psi) and 180°C. The aqueous extracts were analyzed for lignan, protein and carbohydrate using HPLC and colorimetric methods. The optimal extraction yields for lignan, protein and carbohydrate were found at flow rates of 1 to 2 mL/min with bed depth between 20 and 26 cm and a S/S ratio of 40 to 100 mL/g. The combination of low flow rate and high bed depth allowed the use of lower S/S ratio with reduced total solvent volume consumption. This study also evaluated the mass transfer kinetics governing the process of lignan extraction from flaxseed meal in a fixed bed extraction cell. Diffusion of solute into the continuously flowing solvent was mainly responsible for the mass transfer mechanism as flow rate did not increase proportionally with the yield and rate of extraction. The extraction kinetics were studied on the basis of two approaches: Fick’s diffusion equation and a two-site exponential kinetic model. The proposed two-site exponential kinetic model corresponding to the two-stage extraction (rapid and slow phases) successfully described the experimental data. Diffusivities attained from Fick’s diffusion model ranged from 2 x 10-13 to 9 x 10-13 m2s-1 while mass transfer coefficients were between 4.5 x 10-8 and 2.3 x 10-7 ms-1 for extraction of lignans at 180°C, pH 9 with 1:1.5 meal to co-packing material ratio.

The physiological benefits of flaxseed against pathological disturbances, such as cancers and heart diseases, are mainly attributed to its high lignan content. This study (Experiment 1) examined the application of pressurized low polarity water (PLPW) for extraction of lignans, proteins and carbohydrates from defatted flaxseed meal. Key processing conditions included temperature (130, 160, 190°C), solvent pH (4, 6.5 and 9), solvent to solid ratio (S/S) (90, 150 and 210 mL/g) and introduction of co-packing material (0 and 3 g glass beads). The addition of 3 g glass beads as co-packing material facilitated extraction by enhancing surface contact between the liquid and solid thus shortening extraction time. Elevated temperature accelerated the extraction rate by increasing the solid diffusion coefficient thereby reducing the extraction time. The maximum yield of lignans (99 %) was obtained at temperatures ranging from 160°C to 190°C, with solvent volume of 180 mL (90 mL/g meal) at pH 9. Optimal conditions for protein extraction (70 %) were pH 9, extraction volume of 420 mL (210 mL/g meal) and 160°C. Total carbohydrates yield was maximized at 50% recovery at pH 4 and 160°C with 420 mL solvent (210 mL/g meal). Increased temperature accelerated extraction, thus reducing solvent volume and time to reach equilibrium. For the extraction of proteins, however, a temperature of 130-160°C is recommended, as proteins are vulnerable to thermal degradation due to heat decomposition. The effects of flow rate and geometric dimensions for extraction of lignans and other flaxseed meal bioactives were further investigated in Experiment 2, based on the variables optimized in the previous experiment. Defatted flaxseed meal was extracted with pH 9 buffered water with meal to co-packing glass beads ratio of 1:1.5 at 5.2 MPa (750 psi) and 180°C. The aqueous extracts were analyzed for lignan, protein and carbohydrate using HPLC and colorimetric methods. The optimal extraction yields for lignan, protein and carbohydrate were found at flow rates of 1 to 2 mL/min with bed depth between 20 and 26 cm and a S/S ratio of 40 to 100 mL/g. The combination of low flow rate and high bed depth allowed the use of lower S/S ratio with reduced total solvent volume consumption. This study also evaluated the mass transfer kinetics governing the process of lignan extraction from flaxseed meal in a fixed bed extraction cell. Diffusion of solute into the continuously flowing solvent was mainly responsible for the mass transfer mechanism as flow rate did not increase proportionally with the yield and rate of extraction. The extraction kinetics were studied on the basis of two approaches: Fick’s diffusion equation and a two-site exponential kinetic model. The proposed two-site exponential kinetic model corresponding to the two-stage extraction (rapid and slow phases) successfully described the experimental data. Diffusivities attained from Fick’s diffusion model ranged from 2 x 10-13 to 9 x 10-13 m2s-1 while mass transfer coefficients were between 4.5 x 10-8 and 2.3 x 10-7 ms-1 for extraction of lignans at 180°C, pH 9 with 1:1.5 meal to co-packing material ratio.

Ce travail a porté sur l'extraction de substances naturelles de sous-produits de la vigne en mettant en œuvre des procédés "verts" tels que l'extraction par eau sous-critique et la purification par filtration membranaire. Ces procédés représentent une alternative à l'extraction par solvant, traditionnellement utilisée dans la production de substances bio-sourcées. La majeure partie de cette étude a été menée sur des marcs de raisin de cépages variés, l'extraction a été optimisée et comparée sur la base du rendement, de la composition chimique et de l'activité antioxydante des extraits obtenus. De tous les cépages testés, les extraits de Dunkelfelder ont présenté l'activité antioxydante la plus élevée et la concentration en familles de molécules polyphénoliques la plus importante. En outre, ce marc de raisin de Dunkelfelder a été utilisé comme modèle afin d'optimiser les différents paramètres du procédé tels que la température, la pression et le temps de séjour hydraulique. Après la phase d'extraction par eau sous-critique, les extraits obtenus se sont révélés riches en de nombreuses familles de molécules. Ainsi, une étape de purification des composés cibles avant usage industriel s'est révélée indispensable. Le couplage de l'extraction par eau sous-critique avec des procédés membranaires représente une solution innovante pour la purification de ces extraits. Des essais de filtration tangentielle de l'extrait ont été menés avec onze membranes d'ultrafiltration (100 kDa à 2 kDa) et neuf membranes de nanofiltration (1000 Da à 150 Da). Le suivi du procédé s’est appuyé sur une détermination des paramètres opératoires optimisés et sur la détermination des coefficients de rétention des différentes familles des macro et micromolécules. Les résultats obtenus ont démontré que l'utilisation des technologies membranaires pourrait dans le futur, constituer une innovation technologique pour la purification des composés bio-actifs / This work has dealt with extraction of natural substances from winery by-products using "green" processes such as extraction by subcritical water and purification by membrane processes. These processes are an alternative to solvent extraction traditionally used in the natural products industry. Main part of the work was done on different grape pomace, extraction was optimized and compared in terms of yield, chemical composition, and antioxidant activity of extracts. Dunkelfelder extracts exhibited the strongest antioxidant activity and comparison of chemical compositions of the different extracts indicated. Furthermore this Dunkelfelder grape pomace was used as model in order to optimize the different process parameters such as temperature, pressure and hydraulic retention time. After the subcritical water extraction, extracts produced were found to be rich in several families of molecules. An essential purification step of target compounds prior to industrial use was indispensable. Coupling the subcritical water with membrane processes offers an innovative solution for the purification of these extracts. Thereby, the extract was assayed in a cross-flow apparatus against eleven membranes of ultrafiltration (100 to 2 kDa) and nine membranes of nanofiltration (1000 to 150 Da). The monitoring of the process was carried out by determining performance parameters and retention coefficients of different families of macro and micromolecules. The results obtained have demonstrated that the use of membrane technologies could bring innovative changes in the recovery of bioactive compounds for future industries.

L’Oxydation en Voie Humide (OVH) est un procédé dont l’objectif est de dégrader la matière organique contenue dans les eaux usées à l’aide d’une réaction d’oxydation. L'OVH fonctionne à haute pression (10 – 30 MPa) et haute température (373 – 613 K) avec l’oxygène de l’air comme oxydant. Les procédés OVH sont mis en œuvre dans des réacteurs de type colonne à bulles, permettant de maximiser le temps de passage du liquide et le transfert de matière de l’oxygène du gaz vers le liquide, élément clé de l’efficacité du procédé. L’absence de données expérimentales et de corrélations, nécessaires pour l’estimation des paramètres gouvernant le transfert de matière dans les colonnes à bulles fonctionnant dans les conditions d’OVH, est donc pénalisante. Des mesures de rétention de gaz, de diamètre de bulle et d’aire interfaciale en eau claire et en présence d’un polluant (phénol), en conditions non réactives et réactives, ont montré des effets prépondérants de la vitesse superficielle de gaz, de la concentration en polluant et de la saturation du gaz par la vapeur d’eau. La bulle primaire, formée au niveau du distributeur de gaz, et le diamètre de la colonne sont des paramètres essentiels pour l’optimisation du procédé. La mesure du coefficient de transfert de matière, par une nouvelle méthode chimique utilisant l’oxydation du phénol, a montré que celui-ci dépend principalement du diamètre des bulles et des vitesses du gaz et du liquide. Ce travail expérimental est complété par l’établissement de corrélations des paramètres d’intérêt dans les conditions d’OVH, éléments pertinents pour le dimensionnement de procédés OVH fonctionnant en continu. / Wet Air Oxidation (WAO) is a process dedicated to degrade the organic matter contained in wastewater with the help of an oxidation reaction. WAO works at high pressure (10 – 30 MPa) and high temperature (373 – 613 K) using oxygen from air as oxidiser. WAO processes are carried out in bubble column reactors in order to maximise both the liquid residence time and the mass transfer of oxygen from the gas to the liquid, one of the key element of the process. The lack of available data and correlations, needed for the prediction of the parameters governing mass transfer in bubble columns working in WAO conditions, is then penalising. Measurements of gas holdup, bubble diameters and interfacial area in clear water and in presence of a pollutant (phenol), in reactive and non-reactive conditions, show major effects of superficial gas velocity, phenol concentration and saturation of the gas by water vapor. The primary bubble, formed at the gas distributor, and the column diameter, are important design parameters for the process optimisation. Measurement of the mass transfer coefficient by a new chemical technique using phenol oxidation shows that it depends primarily on the bubble diameter and on the gas and liquid velocities. This experimental work is completed by the development of correlations of the key parameters in WAO conditions, providing relevant elements for the design of WAO units working in continuous mode.

I det här projektet extraherades värdefulla komponenter från äpple-pomace som är en restprodukt från cidertillverkning. Restprodukten var tillhandahållen av en svensk cidertillverkare. Extraktionen fokuserades mot pektin och fenolföreningar som uppvisar antioxiderande aktivitet. Extraktionsprocessen designades ur ett miljövänligt perspektiv, inga skadliga kemikalier användes. Fenolära substanser extraherades initialt från pomacen med en vattenlösning innehållande 50% etanol. Därefter studerades kompositionen och den antioxiderande aktiviteten i dessa extrakt. Fokuseringen i projektet låg främst på pektin. Pektin extraherades med subkritiskt vatten vid tre olika pH (3,5 och 7) samt vid två skilda temperaturer (120°C och 140°C) i antingen 5, 10 och 15 minuters sekvenser. Pektinet karakteriserades med avseende på extraktionsutvinningsgrad, sockerkomposition, molekylvikt och antioxiderande aktivitet. Resultaten jämfördes för att undersöka effekten av pH, temperatur och extraktionstid. Gelningsegenskapen hos olika pektin studerades som ett bevis på koncept i ett empiriskt experiment där hög-viskösa vätskor bestående av 5% pektin och 60% sackaros framställdes. Extraherade fenolföreningar har potential att fungera som naturligt utvunna antioxidanter i kosmetiska produkter och pektin kan tänkas användas som reologimodifierare i vattenbaserade formuleringar med låga pH-värden utan behov av kemiska modifieringar. / In this project, apple pomace from a Swedish cider making factory as a by-product was used as a raw material to extract valuable compounds. The extraction was focused on pectin and phenolic compounds with antioxidant activity. For the extraction procedures environmentally friendly processes were chosen without using any harsh chemicals. Phenolic compounds wereinitially extracted from the pomace using 50% aqueous ethanol and then the composition, total phenolic content and antioxidant activity were studied in these extracts. The main focus was on pectins, which were extracted by subcritical water at three different pH conditions (pH 3, 5 and 7) and two different temperatures (120°C and 140°C) in 5-, 10- and 15 minute sequences. Then the pectins were characterized in terms of extraction yield, sugar composition, molecular weight and antioxidant activity and the results were compared in terms of the effect of pH, temperatur eand extraction time. The gelling properties of the different pectins were also studied as a proof of concept in an empirical experiment, where highly viscous liquids were obtained at 5% pectinand 60% sucrose content. In summary, the extracted phenolic compounds have potential to function as naturally derived antioxidants in cosmetics and the pectin may be used as a rheology modifier in water-based formulations of low pH without any additional chemical modifications.

Enzymes and subcritical water can be used for the extraction of hemicelluloses from cereal by-products, making the processes eco-friendly. The polysaccharides extracted from cereal by-products can be used as matrices for development of materials for various applications. This includes bio-based materials such as films and hydrogels, which offer alternatives to existing materials produced from petrochemicals. The polymeric structure of cereal hemicelluloses contains functional groups which enable the modification of their structure by cross-linking, resulting in the formation of hydrogels. This project aims to use subcritical water extraction (SWE) to extract arabinoxylans (AXs) from corn and rye bran meanwhile the enzymatic treatment is done for purifying the samples during both pre- and post-treatment. AXs were further crosslinked by enzyme (laccase) for hydrogel preparation. During the whole project, the characterization included moisture and yield determination, starch and protein content which were tested using a spectrophotometer, monosaccharide content was analyzed by high performance anion exchange chromatography followed by pulsed amperometric detection (HPAEC-PAD) and phenolic acid content was quantified by high performance liquid chromatography (HPLC). The pretreatment for destarching and SWE process was successful. The result showed that arabinoxylans form corn bran were having higher content of arabino substituents, arabino toxylans ratio and ferulic acid content than rye samples. The enzymatic crosslinking could form strong gels in the condition that the AXs had high ferulic acid content. In terms of forming strong hydrogels or to improving the properties of AXs gel, the pre- and post-treatment should be optimized to increase the purity of the extracted feruloylated AX content. / Enzymer och subkritiskt vatten kan användas för extraktion av hemicellulosa från spannmålsbiprodukter, vilket gör extraktionen miljövänlig. Polysackariderna extraherade från spannmålsprodukter kan användas som matriser för utveckling av material för diverse applikationer. Detta inkluderar biobaserade material som filmer och hydrogeler, där petrokemikalier kan ersättas som råvara. Den polymera strukturen hos spannmålshemicelluloser innehåller funktionella grupper som möjliggör formation av tvärbindningar vilket resulterar i bildandet av hydrogeler. Syftet med detta projekt är extraktion av arbinoxylaner (AXs) från majs och rågkli genom att använda subkritiskt vatten-extraktion (SWE) där rening under för- och efterbehandling utförs enzymatiskt. AX modifierades därefter enzymatiskt (laccas) med tvärbindningar för hydrogelframställning. Under hela projektet karakteriserades hydrogelen utifrån fuktinnehåll, bestämmelse av utbyte, stärkelse och proteininnehåll som testades med en spektrofotometer, monosackaridhalten analyserades med högpresterande anjonsutbyteskromatografi följt avpuls-amperometrisk detektion (HPAEC-PAD), samt kvantifierades fenolsyrahalten med högupplöst vätskekromatografi (HPLC). Resultatet visade att arabinoxylaner från majskli hade högre innehåll av arabinosubstituenter, där förhållandet mellan arabino och xylans, samt arabino och ferulsyra innehållet var högre än för rågproverna. Den enzymatiska tvärbindningen kunde bilda starka geler i det tillståndet där AX hade en hög ferulsyrahalt. När det gäller att bilda starka hydrogeler eller att förbättra egenskaperna hos AXs-gel, bör för-och efterbehandlingen optimeras för att öka renheten fördet extraherade feruloylerade AX-innehållet.

<p>У оквиру ове докторске дисертације изведено је<br />испитивање различитих екстракционих поступака за<br />изоловање апигенина из цвета камилице, као и евалуација<br />биолошке активности добијених екстраката. Полазни<br />биљни материјал сачињавале су две групе латица<br />камилице: ферментисане и неферментисане (нативне).<br />Екстракција ферментисаних цветова је извођена применом<br />ултразвучне екстракције користећи етанол као екстрагенс,<br />а добијени екстракти су се одликовали изузетно високим<br />садржајем апигенина. Оптимизација екстракције је била<br />изведена применом методе одзивне површине. Применом<br />електрон-спин резонанце испитана је антирадикалска<br />активност екстраката. Додатно, фармаколошка вредност<br />добијених екстраката је потврђена и одређивањем њиховог<br />антимикробног и антипролиферативног потенцијала.<br />Нативни цветови камилице су екстраховани применом<br />различитих екстаркционих техника: микроталасне,<br />ултразвучне, Soxhlet екстракције као и екстракције<br />субкритичном водом. Eкстрaкција водом у субкритичном<br />стању се показала супериорнијом у односу на све остале<br />технике у погледу садржаја укупних фенола и флавоноида.<br />У циљу добијања екстраката са максималним садржајем<br />апигенина изведена је оптимизација овог екстракционог<br />процеса. Изоловање чистог апигенина је изведено из<br />екстракта добијеног под оптималним екстракцијом<br />условима (однос дрога:растварач 1:30, брзина мешања 3 Hz,<br />притисак 45 bar, температура 115&ordm;C, време 30 мин,<br />концентрација модификатора 0,001 М) применом поступка<br />колонске хроматографије на стубу полиамида. Хемијски<br />профил као и садржај појединачних полифенолних<br />компонената у екстрактима добијеним на различитим<br />притисцима, температурама и уз присуство модификатора<br />различитих концентрација одређен је применом UHPLCDAD-<br />HESI-MS/MS. У свим анализираним екстрактима<br />детектован је велики број полифенолних компонената, док<br />је апигенин у свима био доминантно једињење. Садржај<br />апигенина у екстракту добијеном под оптималним<br />екстракционим условима је износио 1.700,34 mg/kg.<br />Применом седам различитих тестова извршена је<br />евалуација антиоксидативног и антирадикалског<br />потенцијала екстраката. Антимикробни потенцијал<br />екстраката је одређен за осам различитих микробних<br />линија. in vitro тестовима испитана је способност<br />инхибиције &alpha;-амилазе, &alpha;-глукозидазе и тирозиназе.<br />Деловањем на раст три хистолошки различите ћелијске<br />линије, испитана је антипролиферативна активност<br />екстраката добијених субкритичном водом.<br />Антимотилитетна активност обе групе екстраката<br />(ферментисаних и неферментисаних цветова) одређена је у<br />in vitro условима.</p> / <p>U okviru ove doktorske disertacije izvedeno je<br />ispitivanje različitih ekstrakcionih postupaka za<br />izolovanje apigenina iz cveta kamilice, kao i evaluacija<br />biološke aktivnosti dobijenih ekstrakata. Polazni<br />biljni materijal sačinjavale su dve grupe latica<br />kamilice: fermentisane i nefermentisane (nativne).<br />Ekstrakcija fermentisanih cvetova je izvođena primenom<br />ultrazvučne ekstrakcije koristeći etanol kao ekstragens,<br />a dobijeni ekstrakti su se odlikovali izuzetno visokim<br />sadržajem apigenina. Optimizacija ekstrakcije je bila<br />izvedena primenom metode odzivne površine. Primenom<br />elektron-spin rezonance ispitana je antiradikalska<br />aktivnost ekstrakata. Dodatno, farmakološka vrednost<br />dobijenih ekstrakata je potvrđena i određivanjem njihovog<br />antimikrobnog i antiproliferativnog potencijala.<br />Nativni cvetovi kamilice su ekstrahovani primenom<br />različitih ekstarkcionih tehnika: mikrotalasne,<br />ultrazvučne, Soxhlet ekstrakcije kao i ekstrakcije<br />subkritičnom vodom. Ekstrakcija vodom u subkritičnom<br />stanju se pokazala superiornijom u odnosu na sve ostale<br />tehnike u pogledu sadržaja ukupnih fenola i flavonoida.<br />U cilju dobijanja ekstrakata sa maksimalnim sadržajem<br />apigenina izvedena je optimizacija ovog ekstrakcionog<br />procesa. Izolovanje čistog apigenina je izvedeno iz<br />ekstrakta dobijenog pod optimalnim ekstrakcijom<br />uslovima (odnos droga:rastvarač 1:30, brzina mešanja 3 Hz,<br />pritisak 45 bar, temperatura 115&ordm;C, vreme 30 min,<br />koncentracija modifikatora 0,001 M) primenom postupka<br />kolonske hromatografije na stubu poliamida. Hemijski<br />profil kao i sadržaj pojedinačnih polifenolnih<br />komponenata u ekstraktima dobijenim na različitim<br />pritiscima, temperaturama i uz prisustvo modifikatora<br />različitih koncentracija određen je primenom UHPLCDAD-<br />HESI-MS/MS. U svim analiziranim ekstraktima<br />detektovan je veliki broj polifenolnih komponenata, dok<br />je apigenin u svima bio dominantno jedinjenje. Sadržaj<br />apigenina u ekstraktu dobijenom pod optimalnim<br />ekstrakcionim uslovima je iznosio 1.700,34 mg/kg.<br />Primenom sedam različitih testova izvršena je<br />evaluacija antioksidativnog i antiradikalskog<br />potencijala ekstrakata. Antimikrobni potencijal<br />ekstrakata je određen za osam različitih mikrobnih<br />linija. in vitro testovima ispitana je sposobnost<br />inhibicije &alpha;-amilaze, &alpha;-glukozidaze i tirozinaze.<br />Delovanjem na rast tri histološki različite ćelijske<br />linije, ispitana je antiproliferativna aktivnost<br />ekstrakata dobijenih subkritičnom vodom.<br />Antimotilitetna aktivnost obe grupe ekstrakata<br />(fermentisanih i nefermentisanih cvetova) određena je u<br />in vitro uslovima.</p> / <p>In the frame of this thesis different extraction approaches for<br />apigenin isolation from chamomile ligulate flowers were<br />examined and biological activity of obtained extracts was<br />evaluated. Starting plant samples included fermented and<br />nonfermented (native) flowers.<br />Extraction of fermented flowers was performed by using<br />ultrasound-assisted extraction with ethanol. The concentration<br />of apigenin was high in obtained extracts. Optimization of the<br />extraction procedures was performed by response surface<br />methodology. Antiradical activity of observed extracts was<br />examined by electron-spin resonance spectroscopy.<br />Furthermore, pharmacological potential of obtained extracts<br />was confirmed by determining their antimicrobial and<br />antiproliferative activity.<br />Native chamomile flowers were extracted by different<br />extraction techniques: microwave, ultrasound, Soxhlet and<br />subcritical water extraction. Subcritical water extraction<br />showed to be superior in comparison to other applied techniques<br />in respect to total phenols and flavonoids content. Optimization<br />of the subcritical water extraction was directed to maximization<br />of apigenin content. Isolation of pure apigenin from extracts<br />obtained under optimal extraction conditions (sample-tosolvent<br />ratio 1:30, agitation rate 3 Hz, temperature 115&ordm;C,<br />pressure 45 bar, extraction time 30 min) was performed by<br />preparative chromatography. Chemical profiles and content of<br />individual polyphenolic components in extracts obtained at<br />different pressures, temperatures, and with different<br />concentrations of a modifier was determined by UHPLC-DADHESI-<br />MS/MS. In all analyzed extracts the great number of<br />polyphenolic components was detected while apigenin was the<br />dominant compound in all extracts. Content of apigenin in the<br />extract obtained under optimal extraction condition was<br />1,700.34 mg/kg. Antioxidant and antiradical potential of<br />extracts was evaluated according to different mechanisms.<br />Antimicrobial potential of extracts was determined against eight<br />different microbial strains. Ability of extracts to inhibit &alpha;-<br />amylase, &alpha;-glucosidase and tyrosinase was determined by in<br />vitro assays. Antiproliferative activity of subcritical water<br />extracts was defined by testing their influence on the growth of<br />three histologically different cell lines.<br />Anti-intestinal motility activity of both group of extracts (native<br />and fermented) was determined by in vivo experiments.</p>