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Preuzmi PDF 3.92 MB Utjecaj lipoksigenaze na sastav hlapljivih tvari u maslinovom ulju autohtonih dalmatinskih sorti Soldo, Barbara Doctoral thesis / Disertacija 2016 Degree Grantor / Ustanova koja je dodijelila akademski / stručni stupanj: University of Zagreb, Faculty of Science / Sveučilište u Zagrebu, Prirodoslovno-matematički fakultet Permanent link / Trajna poveznica: https://urn.nsk.hr/urn:nbn:hr:217:750629 Rights / Prava: In copyright Download date / Datum preuzimanja: 2021-10-07 Repository / Repozitorij: Repository of Faculty of Science - University of Zagreb PRIRODOSLOVNO-MATEMATIČKI FAKULTET Barbara Soldo UTJECAJ LIPOKSIGENAZE NA SASTAV HLAPLJIVIH TVARI U MASLINOVOM ULJU AUTOHTONIH DALMATINSKIH SORTI DOKTORSKI RAD Mentori: Prof. dr. sc. Maja Pavela-Vrančić Doc. dr. sc. Ivica Ljubenkov Zagreb, 2016. FACULTY OF SCIENCE Barbara Soldo INFLUENCE OF LIPOXYGENASE ON THE COMPOSITION OF VOLATILE COMPOUNDS IN OLIVE OILS OF AUTOCHTHONOUS DALMATIAN CULTIVARS DOCTORAL THESIS Supervisors: Professor Maja Pavela-Vrančić, PhD Assistant Professor Ivica Ljubenkov, PhD Zagreb, 2016. Ovaj je doktorski rad izrađen na Odjelu za kemiju Prirodoslovno-matematičkog fakulteta Splitu, pod vodstvom prof. dr. sc. Maje Pavele-Vrančić i doc. dr. sc. Ivice Ljubenkova. Zahvaljujem Mentorici prof. dr sc. Maji Paveli-Vrančić na susretljivosti, korisnim savjetima koji su omogućili da rad poprimi ovaj oblik. Mentoru doc. dr. sc. Ivici Lubenkovu, poznavaocu ulja i maslinarstva, od kojeg je i potekla ideja rada, na jednostavnom pristupu, smjernicama i potpori tijekom eksperimentalnog dijela i pisanja rada. Članovima povjerenstva prof. dr. sc. Tihani Žanić Grubišić, doc. dr. sc. Borisu Mildneru i doc. dr. sc. Jasmini Rokov Plavec na pažljivom čitanju i korisnim sugestijama koje su omogućile da ovaj rad poprimi svoj konačan oblik. Posebno hvala kolegama Odjela za kemiju i Odjela za biologiju Prirodoslovno-matematičkog fakulteta te kolegama i prijateljima iz Laboratorija za imunologiju i medicinsku genetiku Medicinskog fakulteta u Splitu, na ugodnoj radnoj atmosferi i podršci tijekom izrade ovog rada. Također, hvala i kolegama s Instituta za jadranske kulture i melioraciju krša, bez njih ne bi bilo ulja. Nije bilo lako, ali bez dragih i stručnih ljudi možda bi bilo i nemoguće. Od srca zahvaljujem kolegicama Viljemki Bučević Popović i Matildi Šprung. Najveća hvala mom suprugu, jednoj djevojčici i dječaku na ohrabrivanju i vjeri u uspjeh. I svakako veliko hvala našim roditeljima, na pomoći koju nesebično pružaju. § Sadržaj v __________________________________________________________________________________________________________________________________________________________________________________________________________________________________ Sadržaj SAŽETAK ............................................................................................................................ VIII ABSTRACT ............................................................................................................................ IX § 1. UVOD ............................................................................................................................... 1 § 2. LITERATURNI PREGLED .......................................................................................... 4 2.1. Lipoksigenaza ............................................................................................................................... 4 2.1.1. Lipoksigenaza u biljaka ............................................................................................................... 4 2.1.1.1. Unutarstanična lokalizacija LOX enzima ................................................................................... 4 2.1.1.2. Primarna i tercijarna struktura LOX enzima .............................................................................. 5 2.1.1.3. Vezno mjesto za supstrat ............................................................................................................. 7 2.1.1.4. Specifičnost prema supstratu……………………………………………………………………………8 2.1.1.5. Reakcijski mehanizam ................................................................................................................. 9 2.1.1.6. Podjela lipoksigenaza ................................................................................................................ 10 2.1.1.7. Hipoteze o pozicijskoj specifičnost LOX enzima ....................................................................... 12 2.1.1.8. Stereokemija reakcije ................................................................................................................ 13 2.2. Olea europea L. ........................................................................................................................... 15 2.2.1. Rasprostranjenost maslina ........................................................................................................ 15 2.2.2. Razvoj i zrenje ploda masline .................................................................................................... 15 2.3. Djevičansko maslinovo ulje .......................................................................................................... 18 2.3.1. Kemijski sastav djevičanskog maslinovog ulja ........................................................................ 18 2.3.2. Fizikalno-kemijske vrijednosti djevičanskog maslinovog ulja ............................................... 19 2.3.2.1. Slobodne masne kiseline ............................................................................................................ 20 2.3.2.2. Peroksidni broj .......................................................................................................................... 20 2.3.2.3. Apsorbancija u UV području ..................................................................................................... 20 2.4. Hlapljive tvari u djevičanskim maslinovim uljima .................................................................... 21 2.4.1. Lipoksigenazni put ..................................................................................................................... 21 2.4.2. Enzimi LOX puta ....................................................................................................................... 23 2.5. Utjecaj sorte i stupnja zrelosti plodova na aktivnost LOX enzima .......................................... 26 2.6. Utjecaj sorte i stupnja zrelosti na količinu ukupnih fenolnih spojeva u djevičanskim maslinovim uljima ...................................................................................................................... 27 2.7. Utjecaj ukupnih fenolnih spojeva na aktivnost LOX enzima i sastav hlapljivih tvari u djevičanskim maslinovim uljima .............................................................................................. 28 2.8. Utjecaj LOX enzima na sastav hlapljivih tvari u djevičanskim maslinovim uljima ............... 28 2.9. Utjecaj sorte i stupnja zrelosti na sastav hlapljivih tvari u djevičanskim maslinovim uljima .......................................................................................................................................... 29 § Sadržaj vi __________________________________________________________________________________________________________________________________________________________________________________________________________________________________ § 3. EKSPERIMENTALNI DIO ........................................................................................ 32 3.1. Materijali .................................................................................................................................... 32 3.1.1. Istraživane sorte ......................................................................................................................... 32 3.1.2. Pokusni nasadi maslina .............................................................................................................. 34 3.2. Metode rada ................................................................................................................................... 34 3.2.1. Određivanje stupnja zrelosti plodova maslina ........................................................................ 34 3.2.2.1. Priprema plodova za pročišćavanje LOX enzima ..................................................................... 36 3.2.2.2. Optimizacija pročišćavanja LOX enzima .................................................................................. 36 3.2.2.3. Određivanje koncentracije izoliranih proteina ......................................................................... 38 3.2.2.4. Priprema supstrata .................................................................................................................... 39 3.2.2.5. Reakcijska smjesa za ispitivanje aktivnosti LOX enzima .......................................................... 39 3.2.2.6. Izolacija produkata 9- i 13-Z,E-HPOD .................................................................................... 40 3.2.2.7. Kvantifikacija produkata 9- i 13-Z,E-HPOD ............................................................................ 40 3.2.4. Prerada plodova u ulje ............................................................................................................... 42 3.2.5. Analize ulja ................................................................................................................................. 42 3.2.5.1. Osnovni parametri kvalitete ulja ..............................................................................................
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