Izolacija in karakterizacija flavonoidov iz lupin citrusov

Citrus flavonoids are very interesting for food and pharmaceutical industry since they possess many antioxidant properties and biological activities. Mandarin peels represent an important source of hesperidin and polymethoxy flavones nobiletin and tangeretin. Pommelo peels represent an important source of naringin that can be used as precursor for naringin dihdydro-2,3-chalcone artificial sweetener. Since pommelo peels possess good antioxidant and antimicrobial properties, their extracts are very interesting products for food industry. Therefore isolation of flavonoid from mandarin and pommelo peels is an important topic of investigations. The investigation of the doctoral dissertation is divided in four parts. The first part is focused on the determination of the optimal conditions for the isolation of hesperidin and naringin from mandarin and pommelo peels by conventional extraction. The optimal conditions and the influence of extraction parameters are determined by Taguchi methodology. Very good isolation efficiency of hesperidin, 61.3 mg HES from 1 g of mandarin peels, was obtained by 70 % acetone solution and extraction conditions: 60 °C, 90 min, material to solvent ratio 1/50 g/mL and 3 stages of extraction. The highest amount of naringin, 32.8 mg NAR were isolated from 1 g of material at conditions: conventional extraction, 120 min, albedo, 60 % ethanol, material to solvent ratio 1/50 g/mL and 60 °C. The simple procedure of conventional extraction shows results comparable to more sophisticated methods such as extraction with microwave and ultrasound. Taguchi experimental design was proved to be an efficient methodology to determine the optimal conditions and the parameters that significantly influence product properties. The second part of the investigation focuses on the characterization of mandarin and pommelo peel extracts and their antioxidant properties. Radical scavenging activity against DPPH and antioxidant capacity of lipid soluble and water soluble compounds were determined for mandarin and pommelo extracts. Taguchi experimental design was applied for determination of the influence of extraction parameters on antioxidant properties. The optimal conditions to prepare extracts with high antioxidant properties were also determined. In the third part the investigation focuses on the concentration and separation of citrus flavonoids by extraction with supercritical fluids. Supercritical CO2 was applied for the separation of polymethoxylated flavones (PMF) and flavanone glycoside from mandarin peels. The optimal conditions and the parameter influence on the separation were determined by using Taguchi experimental design. The influence of supercritical fluid extraction parameters on material pre-treatment and isolation of flavonoids was studied. These investigations show that supercritical CO2 is a potential solvent for isolation and separation of PMF from mandarin peels. The fourth and last part of the investigation focuses on the concentration of flavonoids in extract solutions. Since membrane separation processes are very interesting concentration methods in industry, microfiltration, ultrafiltration, nanofiltration and revese osmosis were used for concentration of mandarin and pommelo peel extract solutions. Microfiltration and ultrafiltration could be used in the separation steps after extraction, since they did not influence the amount of dry material and valuable compounds in tested solutions. Reverse osmosis and nanofiltration were shown as useful methods for separation of solvent from extract solutions.Flavonoidi iz citrusov so zaradi svojih antioksidativnih lastnosti ter bioloških aktivnosti zelo zanimivi za prehrambeno in farmacevtsko industrijo. Lupine mandarin predstavljajo pomemben vir hesperidina in polimetoksi flavonov nobiletina in tangeretina. Lupine pomela predstavljajo pomemben vir naringina, ki ga je mogoče uparabiti kot izhodiščno spojino za pripravo naringin dihidro-2,3-kalkona, umetnega sladila. Ker ima lupina pomela zelo dobre antioksidativne in antimikrobne lastnosti, so ekstrakti lupin pomela zelo zanimivi za prehrambeno industrijo. Ker so flavonoidi citrusov zelo atraktivni, predstavlja izolacija flavonoidov iz lupin mandarin in pomela pomembno področje raziskav. Raziskave doktorske disertacije so razdeljene v štiri dele. V prvem delu doktorske disertacije raziskave obravnavajo določanje optimalnih pogojev izolacije hesperidina in naringina iz lupin mandarin in pomela s konvencionalno ekstrakcijo. S pomočjo eksperimentalne metodologije po Taguchi-ju smo določili optimalne pogoje izolacije ter vpliv procesnih parametrov na učinkovitost procesa izolacije. Zelo visoko učinkovitost izolacije hesperidina (61.3 mg HES iz 1 g lupin mandarin) smo dosegli s 70 % vodno raztopino acetona pri ekstrakcijskih pogojih: 60 °C, 90 min, razmerje material–topilo 1/50 g/mL, v 3 ekstrakcijskih stopnjah. Največ naringina, 32.8 mg NAR smo izolirali iz 1 g pomelovega albeda pri pogojih: konvencionalna ekstrakcija, 120 min, 60 % ethanol, , razmerje material–topilo 1/50 g/mL in 60 °C. S preprostim postopkom konvencionalne ekstrakcije smo dosegli primerljive rezultate glede na bolj sofisticirane metode oz. postopke kot je ekstrakcija z mikrovalovi oz. ultrazvokom. Eksperimentalni načrt po Taguchi-ju se je izkazal kot zelo učinkovita metodologija za določanje optimalnih pogojev kot tudi za določanje vpliva procesnih parametrov na lastnosti produktov. V drugem delu raziskav je bilo raziskovalno delo usmerjeno v karakterizacijo ekstraktov lupin mandarin in pomela z namenom določitve antioksidativnih lastnoti ekstraktov. V ta namen so bile uporabljene sledeče metode: določanje protiradikalskega delovanja ekstraktov na prosti radikal DPPH, določanje antioksidativne kapacitete v lipidih topnih komponent (Antioxidant Capacity of Lipid soluble compounds – ACL) ter določanje antioksidativne kapacitete v vodi topnih komponent (Antioxidant Capacity of Water soluble compounds – ACW). Za določanje vpliva ekstrakcijskih parametrov na antioksidativne lastnosti ekstraktov smo uporabli metodologijo po Taguchi-ju. Prav tako so bili določeni optimalni pogoji za pripravo ekstraktov z visokimi antioksidativnimi lastnostmi. V tretjem delu doktorske disertacije so raziskave osredotočene na koncentriranje in separacijo flavonoidov iz citrusov z uporabo ekstrakcije s superkritičnimi fluidi. Superkritični CO2 smo uporabili za separacijo polimetoksi flavonov (PMF) in flavanon glikozidov iz lupin mandarin. S pomočjo eksperimentalnega načrta po Tahuchi-ju smo določili optimalne pogoje separacije ter vpliv procesnih parametrov. Prav tako so bile izvedene raziskave na področju predobdelave materiala s superkritičnimi fluidi: določili smo vpliv predobdelave materiala na izolacijo hesperidina iz lupin mandarin. Navedene raziskave potrjujejo možnost uporabe superkritičnega CO2 za isolacijo in separacijo PMF iz lupin mandarin. Četrti, zdanji del raziskav, obsega študijo koncentriranja flavonoidov v raztopinah ekstraktov. Membranski separacijski procesi mikrofiltracija, ultrafiltracija, nanofiltracija in reverzna osmoza so bili uporabljeni za raziskave koncentriranja raztopin ekstraktov lupin mandarin in pomela. Procesa mikrofiltracije in ultrafiltarcije lahko uporabimo pri postopku ločevanja po ekstrakciji saj ne vplivata na vsebnost suhe snovi in aktivnih komonent v testnih raztopinah. Procesa reverzne osmoze ter nanofitracije predstavljata zelo uporabno metodo separacije topila iz raztopin ekstraktov