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V diplomskem delu je predstavljena biotehnološka proizvodnja karotenoidov in njihova možna aplikacija. Karotenoide ločimo glede na kemijsko strukturo na karotene in ksantofile. Lahko so sintetizirani v času normalne celične rasti (primarni karotenoidi) ali kot posledica neustreznih rastnih pogojev (sekundarni karotenoidi). V celici imajo zaščitno funkcijo (absorbirajo svetlobo, zadržujejo presežek energije med fotosintezo) in antioksidativno funkcijo (nevtralizacija prostih radikalov, preprečevanje oksidacije lipidov). Diplomsko delo je osredotočeno na mikroalge, ker nudijo nove možnosti in izzive ter se vedno več uporabljajo za pridobivanje pomembnih in uporabnih snovi. Obstajata dva načina kultivacije mikroalg: odprti in zaprti sistemi. V diplomskem delu je na kratko opisana tudi biosinteza karotenoidov ter načini povečanja biosinteze. Navedene in opisane so različne stopnje zaključnih procesov (ločevanje, sušenje, liza celic, ekstrakcija, čiščenje, obstojnost) in katere metode se lahko uporabljajo v ta namen. Karotenoide lahko uporabljamo v živilski industriji in v pripravi krme zaradi barvnih in prehranskih lastnosti oziroma kot aditive. Uporabljajo se tudi kot naravna barvila v kozmetiki in v farmacevtski industriji. Med novejše aplikacije sodi tudi vgrajevanje karotenoidov v polimerne produkte. The diploma thesis presents the biotechnological production of carotenoids and their possible application. Carotenoids are divided into two groups according to their chemical structure, namely carotenes and xanthophylls. They can be synthesized during normal cell growth (primary carotenoids) or due to inadequate growth conditions (secondary carotenoids). They have a protective function in the cell (they absorb light, retain excess energy during photosynthesis) and an antioxidant function (neutralization of free radicals, prevention of lipid oxidation). The thesis focuses on microalgae because they offer new opportunities and challenges and are increasingly used to obtain important and useful substances. There are two ways to cultivate microalgae, namely as an open and closed system. The diploma thesis also briefly describes the biosynthesis of carotenoids and ways to increase biosynthesis. Different stages of the downstream processes (separation, drying, cell lysis, extraction, purification, persistence) and the methods used for this purpose are listed and described. Carotenoids can be used in the food industry and in the preparation of feed due to their colour and nutritional properties or as additives. They are also used as natural dyes in the cosmetics and pharmaceutical industries. Recent applications include the incorporation of carotenoids into polymer products.

V raziskavi smo določali profil karotenoidov v cianobakteriji Arthrospira platensis in mikroalgi Chlorella vulgaris ter vpliv mlečnokislinske fermentacije na prisotnost karotenoidov in fenolnih spojin. Pripravili smo ekstrakte past biomas A. platensis in C. vulgaris v dveh različnih topilih (100-% aceton, kloroform : metanol = 1 : 2). V ekstraktih smo karotenoide ovrednotili s spektrofotometrično metodo in s tankoplastno kromatografijo. V raziskavi smo ugotovili, da se vsebnost karotenoidov in ustreznost topil, ki sta se uporabljali pri A. platensis in C. vulgaris, razlikujeta, saj je aceton dal boljše rezultate za vzorce A. platensis in mešanica kloroform : metanol za vzorce C. vulgaris. Iz kontrolne biomase A. platensis smo s 100-% acetonom ekstrahirali 4,37 ± 0,16 (mg/g suhe biomase) karotenoidov in iz fermentirane 4,37 ± 0,07 (mg/g suhe biomase) karotenoidov. Z mešanico kloroform : metanol smo iz kontrolne biomase A. platensis ekstrahirali 25,9 % manj karotenoidov in iz fermentirane biomase 13,3 % manj karotenoidov. Iz kontrolne in fermentirane biomase C. vulgaris smo s 100-% acetonom ekstrahirali skoraj 10-krat manj skupnih karotenoidov, in sicer 0,45 ± 0,01 (mg/g suhe biomase) in 0,52 ± 0,02 (mg/g suhe biomase). Tudi z mešanico kloroform : metanol smo ekstrahirali manj karotenoidov, tako iz kontrolne biomase C. vulgaris kot fermentirane, in sicer 2,27 ± 0,12 (mg/g suhe biomase) in 1,39 ± 0,02 (mg/g suhe biomase). S tankoplastno kromatografijo smo ločili mešanico pigmentov v ekstraktih iz past kontrolnih in fermentiranih biomas. Pri A. platensis smo uspeli ločiti 10-16 komponent več z uporabo mešanice 1 kot z uporabo drugih mešanic, medtem ko smo pri C. vulgaris uspeli ločiti 4-13 komponent več z uporabo mešanice 3 kot z uporabo preostalih mešanic topil. S Folin-Ciocalteujevo metodo smo določali tudi vsebnost fenolnih spojin. Fenolne spojine predstavljajo od najmanj 0,02 % do največ 0,44 % biomase. Ugotovili smo, da je koncentracija skupnih fenolnih spojin višja v fermentiranih vzorcih kot v kontrolnih ter da A. platensis na splošno vsebuje več karotenoidov in skupnih fenolnih spojin (SFS) kot C. vulgaris. In this research, we determined profiles of carotenoids in cyanobacteria A. platensis and microalgae C. vulgaris, the effect of lactic acid fermentation on present carotenoids, and also the presence of phenolic compounds. Biomass paste extracts of A. platensis and C. vulgaris were prepared in two different solvents (100 % acetone, chloroform : methanol = 1 : 2) so that we can compare the results and see which solvent is giving better results. After extraction, we tried to determine carotenoids by various methods, such as spectrophotometry and thin-layer chromatography. In addition to carotenoids, the presence of phenolic compounds was also determined using the Folin-Ciocalteu method. In this study, we found that the carotenoid content and solubility of the solvents used differed between A. platensis and C. vulgaris, as one solvent gave better results for A. platensis samples and the other solvent for C. vulgaris samples. 4.37 ± 0.16 (mg/g d. m.) was extracted from unfermented biomass of A. platensis in 100 % acetone and 4.37 ± 0.07 (mg/g d. m.) from fermented biomass. 3.24 ± 0.53 (mg/g d. m.) was extracted from unfermented biomass of A. platensis in chloroform : methanol solvent mixture and 3.79 ± 0.25 (mg/g d. m.) of fermented biomass. When it comes to A. platensis we extracted 14–22 % more carotenoids with 100 % acetone than with chloroform : methanol solvent mixture. 0.45 ± 0.01 (mg/g d. m.) was extracted from unfermented biomass of C. vulgaris in 100 % acetone and 0.52 ± 0.02 (mg/g d. m.) from unfermented biomass. 2.27 ± 0.12 (mg/g d. m.) was extracted from unfermented biomass of C. vulgaris in chloroform : methanol solvent mixture and 1.39 ± 0.02 (mg/g d. m.) of fermented biomass. When it comes to C. vulgaris we extracted 2–4 % more carotenoids with chloroform : methanol solvent mixture than with 100 % acetone. The same was observed in thin layer chromatography by comparing the suitability of four solvent mixtures. In A. platensis a non-polar solvent mixture 1 separated 10-16 more components compared to any of the other mixtures used and in C. vulgaris a polar solvent mixture 3 separated 4-13 more components compared to other solvent mixtures. We also used Folin-Ciocalteu method to determine the content of phenolic compounds. Phenolic compounds represent from at least 0.2 % to the maximum of 0.44 % of biomass. Finally, we concluded that the concentration of total phenolic compounds and carotenoids is generally higher in fermented samples than in unfermented samples and that A. platensis generally contains more carotenoids and total phenolic compounds (TPC) than C. vulgaris.

V raziskavi smo določili vsebnost bioaktivnih snovi v sedmih različnih vrstah samoniklih travniških rastlin: navadni rman (Achillea millefolium L.), navadna česnovka (Alliaria petiolata Cav. & Grande), mehki osat (Cirsium oleraceum (L.) Scop.), navadni jajčar (Leontodon hispidus L.), veliki trpotec (Plantago major L.), navadna črnoglavka (Prunella vulgaris L.) in dišeča vijolica (Viola odorata L.). Rastlinski material smo nabrali v spomladanskem času, v enakih vremenskih razmerah, v razvojni fazi, v kateri te rastline nabirajo za prehrano. Izmerili smo vsebnost klorofilov a in b, tokoferolov in in karotenoidov: -karoten, -karoten, lutein, neoksantin, violaksantin, anteraksantin in zeaksantin. Vzorce smo analizirali s pomočjo visokoločljivostne tekočinske kromatografije (HPLC). Največjo povprečno vrednost skupnega klorofila in največjo povprečno vrednost karotenoidov smo izmerili v navadni česnovki, največjo povprečno vrednost skupnih tokoferolov pa v velikem trpotcu. Za boljšo predstavo o vsebnosti bioaktivnih snovi v preučevanih rastlinah smo primerjali naše rezultate z literaturnimi podatki za te rastline in za zelenjavo, ki jo uživamo na enak način (mladi listi). The aim of this study was to determine lipophilic antioxidants content in seven grassland plant species: Yarrow (Achillea millefolium L.), garlic mustard (Alliaria petiolata Cav. & Grande), cabbage thistle (Cirsium oleraceum (L.) Scop.), rough hawkbit (Leontodon hispidus L.), broadleaf plantain (Plantago major L.), common selfheal (Prunella vulgaris L.) and sweet violet (Viola odorata L.). Plant material was all harvested in spring, at same weather conditions, at developmental stage that is prefered from forager and consumer's standpoint. Chlorophyll a and b, tocopherols ( and  and carotenoids: lutein, neoxanthin, violaxanthin, antheraxanthin and zeaxanthin contents, were determined quantitatively. Analysis was conducted with a high performance liquid chromatography (HPLC) method. The highest average value of total chlorophyll and carotenoid contents was measured in garlic mustard, the highest average value of total tocopherol was measured in broadleaf plantain. To get a better perception of the bioactive compounds amount in studied plants, we compared results from our study to the ones from the literature for the same plant species and for other species that are being consumed in the same way as those from our study (leafy greens).

Antioksidansi su spojevi od širokog značaja za očuvanje zdravlja i sprječavanje negativnih učinaka potaknutih ubrzanim načinom života. Cilj ovog doktorskog rada bio je razvoj i primjena brzih i jednostavnih voltametrijskih metoda za određivanje oksidacijskih potencijala antioksidansa prisutnih u biljkama i životinjama. Spojevi su analizirani elektrokemijski, cikličkom i pravokutnovalnom voltametrijom te spektrofotometrijski. Primijenjene su voltametrijske tehnike imobiliziranih mikrokristala, mikrofilma i mikrokapi čime je omogućeno određivanje lipofilnih spojeva u vodenom mediju. Za potrebe razvoja ovih metoda, dio istraživanja bio je fokusiran na karakterizaciju standarda (kapsaicina, β-karotena, astaksantina i luteina), a u drugom dijelu ispitivala se primjena metoda u analizi namirnica biljnog i životinjskog porijekla (čili paprike, morski organizmi). Pomoću elektrokemijskih tehnika, u biljnim uzorcima (čili paprike) uspješno je kvantificirana ukupna količina kapsaicinoida. Razvijene tehnike omogućile su kvalitativnu analizu karotenoida u uzorcima riba, dok je u uzorcima škampa i kozica uspješno kvantificirana količina astaksantina. Antioxidants are versatile molecules that are used in maintaining human health and preventing adverse effects of fast paced life. Aim of this doctoral thesis was development and application of fast and easy-to-use voltammetric method for determination of oxidation potentials of antioxidants present in plant and animal samples. Compounds were analyzed with cyclic and square-wave voltammetry, and with spectrophotometry. Voltammetry of immobilized microparticles, microfilms and microdroplets allowed determination of lipophilic compounds in aqueous media. First part was focused on the characterization of standards (capsaicin, β-caroten, astaxanthin and lutein), and the second part on implementing this methods in analysis of foodstuff (chili peppers, marine organisms). By using electrochemical techniques, the total number of capsaicinoids in chili peppers was successfully quantified. In the analysis of fish samples, carotenoids were qualitatively determined, while the amount of carotenoid astaxanthin in shrimp samples was successfully quantified.

Zbrinjavanje krutog otpada u industriji voća i povrća važna je stavka jer u otpadu završi od 10 do 60 % sirovine. Nepravilno gospodarenje otpadom može dovesti do onečišćenja okoliša i stoga se mora zbrinuti na prihvatljiv način. U industiji voća i povrća otpad predstavljaju: sjemenke, kožica (kora), neiskorišteno usplođe, ljuska, lišće, peteljka. Sav taj otpad može se iskoristiti i u konačnici dovesti do povećanja industrijske profitabilnosti. Otpad se može zbrinuti kompostiranjem, gdje dobijemo kompost koji se koristi za poboljšanje teksture tla i dodatak hranjivih tvari, za dobivanje biogoriva u koja se ubrajaju biovodik, bioplin, peleti i bioetanol, za izdvajanje aktivnih biokomponenti ekstrakcijom kao što su prehrambena vlakna, karotenoidi i druge boje, proteini, antioksidansi. Taking care of solid waste in fruit and vegetables industry is important because 10 to 60% of raw material ends up in the waste. Incorrect management of the waste can lead to enviromental pollution and therefore it must be taken care of in accetable manner. In fruit and vegetables industry the waste present: seeds, peel, unused pericarp, shell, petiole leaves. All that waste can be used and ultimately bring to an increased industrial profitability. The waste can be disposed off by composting, where we get compost which is used for improving soil texture, and for addition of nutrients to obtain biofuels, which also include biohydrogen, biogas, pellets and bioethanol, to extract active biocomponent such as dietary fiber, carotenoids and other colors, proteins, antioxidants.