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Uvod: Posljednjih se godina intenzivno istražuju nove metode izolacije eteričnih ulja, s ciljem povećanja njihovog prinosa. Ciljevi: Cilj ovog istraživanja bio je istražiti da li različiti predtretmani vodene destilacije mogu povećati prinos eteričnog ulja kadulje, ružmarina i lovora te utvrditi eventualne promjene u kemijskom sastavu eteričnih ulja. Metode: Vodenoj destilaciji predhodili su različiti predtretmani: klasična ekstrakcija refluksiranjem, ekstrakcija refluksiranjem uz dodatak enzima pektinaze, celulaze i ksilanaze te ultrazvučna ekstrakcija. Kemijski sastav dobivenih ulja analiziran je GC- MS i NMR analizom. Rezultati: Svi predtretmani povećali su prinos eteričnih ulja u usporedbi s negativnom kontrolom, pri čemu je kvaliteta ulja ostala nepromijenjena. No, predtretman ekstrakcijom refluksiranjem s dodatkom enzima nije povećao prinos ulja u odnosu na sami predtretman ekstrakcijom refluksiranjem (kontrola bez enzima). Ovo upućuje na to da je povećanje prinosa ulja uzrokovala ekstrakcija refluksiranjem, a ne enzimska aktivnost, i suprotno je navodima mnogih ranijih istraživanja. Međutim, u tim istraživanjima prinos ulja nakon predtretmana enzimima uspoređen s negativnom kontrolom (bez predtretmana), umjesto s kontrolom bez enzima. Zaključno, rezultati ovog istraživanja pokazali su da predtretmani ultrazvukom i ekstrakcijom refluksiranjem mogu biti korisni u proizvodnji eteričnih ulja navedenih biljaka, ali da primjena enzima u ovu svrhu treba biti dodatno istražena.

Biological degradation of lignocellulose-containing raw materials employs fungi, mainly belonging to the group of white-rot and brown-rot basidiomycetes, which express a broad spectrum of enzymes. This requires long application periods with the rate of fungal degradation that is too low for industrial use and consumes a fraction of the plant polysaccharides [1]. More convenient than fungal is enzymatic degradation, which is very selective and fast but expensive at a large scale. The enzymatic hydrolysis of the lignin, hemicellulose and cellulose content of lignocellulosic biomass to their monomeric sugars suitable in the production of bioethanol and other value-added biochemicals has been interfered in many ways and is a key step in the production of bioethanol. The hydrolysis mostly requires multiple enzymes with different abilities to degrade the complex lignocellulosic structure [2-4]. Specifically, a synergetic action of enzymes - cellulases, hemicellulases, lignases (ligninolytic enzymes) and most recently LPMO (lytic polysaccharide monooxygenase enzyme), is required for an effective deconstruction activity [5]. The goal of enzymatic hydrolysis is to depolymerize the polysaccharides in the water-insoluble solid fraction that remains after pretreatment. However, for the industrial utilization of the fungi and their enzymes more collaborative effort and synergy has to be attained between molecular and genetic biotechnologies, enzymologist, and bioprocess engineers. The aim of this study was to present current knowledge of the fungi involved in lignocellulose degradation with an overview of the various classes of lignocellulose-acting enzymes engaged in the pretreatment and saccharification step.

The objective of this work was to develop an integrated bioprocess system for ethanol production and separation from selected lignocellulosic raw material (sugar beet pulp) by using different bioreactors. For that purpose, a column and horizontal rotating tubular bioreactor (HRTB) were used. The second objective was to develop an efficient enzyme system for cellulose fraction degradation of lignocellulosic biomass based on the application of fungal lytic polysaccharide monooxygenase (LPMO), with the aim to support hydrolytic enzymes, and different electron donors present in the lignocellulose hydrolysates. LPMOs oxidatively degrade insoluble lignocellulose polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. Binding studies showed that the reduction of the LPMO’s active site increased the affinity and the maximum binding capacity for cellulose. The next step was related to an optimization of enzymatic sugar beet pulp hydrolysis using various commercial enzyme mixtures (Ultrazym AFP-L, Viscozyme L, Pectinase and Cellulase). Ultrazym AFP-L showed the highest efficiency and together with produced extracellular degradation system from fungus Neurospora crassa, was used for sugar beet pulp hydrolysis at a large scale. A three step process comprising the hydrolysation, fermentation and in-situ gas stripping with a vacuum assisted recovery system, was integrated and optimized to increase the ethanol production. Environmental sustainability of the integrated processes has been evaluated and used for calculation of environmental impacts based on mass and energy balances of the processes.

Lignocellulose is a renewable resource that can be used for the sustainable production of platform chemicals or fuels. However, the recalcitrance of cellulose to hydrolytic depolymerization is a barrier to microbial and industrial utilization of lignocellulosic biomass. Cellulose is broken down to glucose by endo- and exo-acting glycosyl hydrolases which process the glucan chains in cellulose to soluble cellobiose moieties. In a process known as oxidative cellulose degradation, copper-dependent lytic polysaccharide monooxygenases (LPMO) cleave polymeric substrates, including crystalline cellulose, hemicelluloses and starch. It remains difficult to find how LPMOs orchestrate the complex reaction between copper reduction, co-substrate activation and substrate binding. To test the hypothesis that copper reduction increases binding of LPMO to cellulose, we studied the binding of oxidized and reduced LPMO to amorphous cellulose (PASC) and to microcrystalline cellulose (MC). Also, we aimed to quantify the substrate binding by measuring binding isotherms. The data demonstrate that copper reduction is a driver for substrate binding.

Sugar beet pulp (SBP) is a promising feedstock for the production of 2nd generation biofuels, but efficient enzymatic hydrolysis remains a key challenge ; therefore, new process designs and/or bioreactor designs are crucial to overcome this hurdle. In this regard, horizontal rotating tubular bioreactors (HRTB) offer the advantage of high substrate loadings while minimizing the space and energy demand compared with conventional stirred tank reactors. Here, a statistical approach is used to optimize the hydrolysis of sugar beet pulp in laboratory experiments, and it is shown that such a process can be implemented in a HRTB. Using the design of experiments (DOE) method, the reaction conditions of four commercial enzyme mixtures (Ultrazym AFP-L, Viscozyme L, Pectinase and Cellulase) was optimized for the degradation of SBP in small-scale experiments. Using Ultrazym AFP-L as the most efficient mixture, a 10 L scale conversion was performed in a HRTB. At a substrate loading of 135 g L−1 and optimized conversion parameters (enzyme load, pH and rotating speed of the reactor), 0.525 W dm−3 were needed to achieve solubilisation of 30% of the total mass of initial SBP after 24 h. DOE was found to be an easy-to-apply method that allowed optimizing the conditions for enzymatic hydrolysis of SBP, resulting in a higher sugar yield. The results could be transferred to an HRTB, which is a suitable system for enzymatic conversion and efficient saccharification of semi-solid or solid substrates with relatively low energy consumption.

The depolymerisation of complex biomass such as lignocellulose by organisms depends on a network of enzymatic and chemical reactions. Fungi that can grow on lignocellulosic materials are equipped with a complex enzymatic degradation system, including a broad variety of hydrolases and oxidoreductases. In 2010, a new class of enzymes was discovered, which carry out oxidative cleavage of polysaccharides. These enzymes, today known as lytic polysaccharide monooxygenases (LPMOs, CAZy family AA9) are copper-dependent oxidoreductases that can hydroxylate the C1 or C4 positions of glucose and thereby cleave the glycosidic bonds in cellulose. These “cellulase activity boosters” provide hydrolytic enzymes access to crystalline cellulose by introducing chain breaks. LPMOs, despite their importance in biotechnology, are difficult to investigate because of their complex heterogeneous reaction mechanism. An open question is how LPMOs orchestrate the complex reaction between copper reduction, co-substrate activation and substrate binding. It is generally accepted that the first reaction step of LPMO is the reduction of the active-site copper by small molecule reductants or by other enzymes (CDH, GDH). In this work, we address the substrate binding of oxidized and reduced LPMO-02916 from Neurospora crassa to phosphoric acid swollen cellulose (PASC) and microcrystalline cellulose (MC) to investigate the role of the copper centre for substrate binding.

A biorefinery is a facility that integrates biomass conversion processes and equipment to produce bio-products from biomass. Biorefineries will have to employ the best possible processes (for biomass cultivation, harvesting, storage and transport, pre-treatment, fermentation and bio-chemical conversion) to ensure efficient bio-based production. Nevertheless, numerous technical, strategic and commercial challenges have to be overcome to reinforce importance of bio-based production. Mathematical models can be used to simulate all aspects of bio-based production systems including the process design, production economics, supply logistics and environmental impacts. Those models can provide powerful tools to optimize biorefinery processes and evaluate its technical feasibility as well as economics and environmental impacts. In the presentation, examples of different mathematical models will be discussed, from the physical base model including kinetic, transport and diffusion equations to the statistical base model and data analyses. In the end, life cycle assessment analysis of the ethanol production from the corn stover will be provided as the example of modelling application in better understanding of the bio-based production environmental impact.

Cilj rada je prikazati utjecaj tjelesne aktivnosti na smanjenje pojavnosti srčanog udara te smrtnosti od posljedica istog i njezinu ulogu u liječenju i rehabilitaciji. Srčanožilne bolesti su značajan javnozdravstveni problem u svijetu te su u razvijenim zemljama glavni uzrok smrti. I u Republici Hrvatskoj (RH) srčanožilne bolesti predstavljaju vodeći javnozdravstveni problem, a prema podacima Hrvatskog zavoda za javno zdravstvo, srčani udar jedan je od najčešćih pojedinačnih uzroka smrti u nas. Dosadašnja istraživanja dokazala su da se kvalitetnim načinom života, odnosno promjenom načina života i kontrolom čimbenika rizika, kroz aktivnosti primarne i sekundarne prevencije, smanjuju obolijevanje i smrtnost od srčanožilnih bolesti. Tjelesna aktivnost je ključna mjera u prevenciji bolesti i očuvanju zdravlja, a temeljni je dio i u liječenju i rehabilitaciji nakon srčanog udara. Uključivanje u tjelesnu aktivnost nakon srčanog udara započinje organiziranim i nadziranim programima u specijaliziranim ustanovama, a nastavlja se ambulantno u skladu sa zdravstvenim statusom bolesnika. Tjelesna aktivnost je neizostavan dio strategije za unaprjeđenje zdravlja u svijetu i u nas. The aim of this paper is to show the impact of physical activity on reducing the incidence of myocardial infarction and death from its consequences and the role of physical activity in treatment and rehabilitation. Cardiovascular diseases are a significant public health problem in the world and are the main cause of death in developed countries. In Croatia, cardiovascular diseases are also the leading public health problem and according to data from the Croatian Institute of Public Health, a myocardial infarction is one of the most common single causes of death. Previous studies have proven that a healthier way of life, namely lifestyle changes and the control of risk factors, through the activities of primary and secondary prevention, reduces morbidity and mortality from cardiovascular disease. Physical activity is a key factor in preventing disease and maintaining health, but the treatment and rehabilitation after a myocardial infarction is also of fundamental importance. Engaging in physical activity after a myocardial infarction begins in organized and supervised programs in specialized institutions and continues on an outpatient care basis in accordance with the health status of the patient. Physical activity is an essential part of the strategy for improving health in the world and in Croatia.

After cellulose, lignin is the second most abundant renewable biopolymer. It is the one of major component of lignocellulosic biomass. This complex aromatic heteropolymer is tightly cross-linked with other cell walls components, covalently bound to hemicellulose. Lignin consists of three phenylpropane monomer units also known as monolignols: sinapyl, coniferyl and p-coumaryl alcohol. The compact structure that is insoluble in water and other common solvents make lignin biodegradation a challenge. Biological lignin degradation, caused by oxidative enzymes and/or small molecular weight mediators or factors such as radical, is unspecific. Fungi produce a variety of extracellular enzymes involved in the conversion and degradation of lignin aromatic compounds. These enzymes are required to degrade large aromatic compounds to smaller subunits. In our experiment the crude extracts from white-rot fungi (Trichoderma reesei and Phanerochaete chrysosporium) and oxidative enzymes (CDH, GDH, GOx, laccase) were used for direct treatment of lignin solution with the aim to degrade lignin. Firstly, we dissolved lignin in 50mM NaOH by applying ultrasound. Initially, the pH of lignin solution was adjusted to 5.0 by 100mM H3PO4 and then inoculated with crude extracts and oxidative enzymes with (or without) inducers (FeCl2, MnCl2) and incubated for 48 hours. As a control we added H2O instead of enzymes. The resulting degradation was confirmed by UV/VIS spectrometric and size-exclusion chromatography (SEC) measurements on incubated and control samples. The increase in the absorbance of lignin after incubation with crude extracts and enzymes has been considered as a criterion for the extent of lignin biodegradation.

Zbog iscrpljenosti nalazišta nafte u bliskoj budućnosti se očekuje energetska kriza (kriza goriva) kao i kriza kemijskih sirovina koje se dobivaju iz nafte. Pretpostavlja se da će se u prvo vrijeme taj manjak kompenzirati kemikalijama i energentima proizvedenim iz obnovljivih šećernih, škrobnih i lignoceluloznih sirovina. S ciljem upotrebe lignoceluloznih sirovina za proizvodnju etanola potrebno je provesti proces njihove predobrade. Predobrada je važan korak u proizvodnji biogoriva i biokemikalija pri čemu se koriste različite biološke, kemijske i fizikalne metode s ciljem izdvajanja lignina i pripreme sirovine za enzimsku hidrolizu. Primjenom toplinske predobrade ostvarena je bolja razgradnja sirovine i veća koncentracija šećera koje kvasac, u daljnjim fazama procesa proizvodnje bioetanola, može metabolizirati. Uslijed djelovanja topline i mehaničke dekompresije dolazi do promjena u strukturi sirovine pri čemu polimerna vlakana lignocelulozih sirovina depolimeriziraju do jednostavnih šećera. Potpuna razgradnja lignocelulozne sirovine zahtjeva primjenu različitih vrsta pektinolitičkih, hemicelulolitičkih, lignolitičkih i celulolitičkih enzima. Stoga je nakon predobrade toplinom provedeno istraživanje enzimske hidrolize lignocelulozne sirovine (kukuruzni oklasci, pulpa šećerne repe, slama) s enzimima izdvojenim iz supernatanata fungi. Novine predloženog predtretmana temelje se na primjeni ekstracelularnog razradnog sustava dobivenog iz fungi sa ciljem poboljšanja razgradnje celulozne frakcije lignocelulozne sirovine, a nedavna istraživanja enzimske razgradnje lignoceluloznih sirovina otkrila su da je sinergističko djelovanje enzima ključno za uspješnu razgradnju. Za planiranje eksperimenata te za obradu i prikaz rezultata korišten je programski paket Statistica. Dobiveni rezultati prikazani su centralno kompozitnom metodom odzivnih površina (eng. Response Surface Methodology ; RSM) koja se koristi za modeliranje i analizu utjecaja nezavisnih varijabli (procesnih parametara) na promatrani odziv s ciljem poboljšanja, razvoja i optimiranja procesa. Potrebno je odabrati odgovarajući dizajn eksperimenta (eng. Design of experiments, DOE) ili plan pokusa kojim će se definirati eksperimenti koje je potrebno provesti, a provedbom pokusa u svim kombinacijama određenim dizajnom eksperimenta dobiva se odzivna ploha.

Pyrones, biologically active compounds, are fundamental moieties of many naturally occurring compounds, as well as many synthetic compounds. They have numerous applications in medicine as pharmaceuticals, in agriculture as pesticides, and are often used as photosensitizers. Derivative of γ-pyrone 2, 6-di(p-tolyl)-4H-pyran-4-one was previously prepared by microwave-assisted decarboxylation of 6-p-tolyl-3-p-toluyl-dihydro- pyran-2, 4-dione. Exposure of solution of compound 1 in dichloromethane to UV radiation (λ = 256 nm) led to changes in its absorption spectrum. The appearance of isosbestic points suggests equilibrium between at least two species in solution: compound 1 and one or more photoproducts. From the reaction mixture a product of photochemical rearrangement 4, 6-di(p-tolyl)- pyran-2-one (2) was isolated, which was confirmed by IR, 1H-NMR, 13C-NMR spectra. Absorption and emission spectra of compound 2 have been studied and compared to corresponding spectra of compound 1. These spectra give information about the molecular electronic transitions, essential for determination of reaction mechanisms and prediction of the curse of photoreaction. Further on, DFT calculations were made to get more insight on the mechanism of this photochemical rearrangement. Structural characteristics of the isolated photoproduct 2 were also compared to that those of unsubstituted α-pyrone.

Rast kultura životinjskih stanica odvija se u složenim medijima za uzgoj koji se sastoje od hranjivih tvari poput soli, aminokiselina, vitamina, glukoze te seruma. Serum je najskuplja komponenta medija za uzgoj životinjskih stanica te među ostalim sadrži komponente koje nisu uvijek kemijski karakterizirane. Zbog toga je razvoj medija bez dodatka seruma potaknuo interes za primjenom proteinskih hidrolizata dobivenih iz tkiva životinja, mlijeka, kvasca ili biljaka. Hidrolizati proteina dobivaju se enzimskom, kiselinskom ili mikrobnom hidrolizom biološkog materijala, a sadrže različite hranjive tvari (npr. aminokiseline, oligopeptide, lipide, elemente u tragovima, vitamine i minerale) i ostale spojeve koji potiču proliferaciju i utječu na povećanje aktivnosti stanica. Cilj ovog rada bio je ispitati utjecaj dodatka proteinskih hidrolizata kvasca, pšeničnog glutena i soje na proliferaciju stanične linije CCO u UC mediju bez dodatka seruma. Ispitivanjem utjecaja dodatka navedenih hidrolizata proteina u koncentraciji od 4 g/L utvrđeno je da je najveći učinak na rast CCO stanica imao hidrolizat pšeničnog glutena. Koncentracije pšeničnog glutena od 8 i 12 g/L imale su inhibicijski učinak na rast CCO stanica. Dodatak frakcija hidrolizata pšeničnog glutena od 5 i 10 kDa nije imao stimulativni učinak na rast CCO stanica. Kombinacija hidrolizata pšeničnog glutena i soje (1+3 g/L) pokazala je najbolji učinak na proliferaciju CCO stanica. Daljnjim istraživanjima potrebno je ispitati mogući stimulativni utjecaj drugih biljnih hidrolizata na proliferaciju CCO stanica kao i mehanizme njihovog djelovanja.

Fenolni spojevi su relativno velika grupa organskih spojeva koji u svojoj strukturi imaju jedan ili više benzenskih prstenova. Razgradnja ovih spojeva, izoliranih iz biljnih materijala ili dobivenih tijekom različitih industrijskih procesa, slabo je istražena. Metabolički kapacitet bakterija mliječne kiseline obuhvaća i pregradnju fenolnih spojeva u reakcijama oksidoredukcije, dekarboksilacije i hidrolize. Negativni učinci fenolnih spojeva podrazumijevaju: narušavanje strukture i funkcije citoplazmatske membrane stanica bakterija mliječne kiseline, snižavanje pH vrijednosti i denaturaciju proteina u citoplazmi, gubitak iona kalija iz stanice te smrt stanice zbog nesmetanog prolaza otopljenih molekula iz okoline stanice u stanicu i u obrnutom smjeru. Neke vrste bakterija mliječne kiseline iz roda Lactobacillus i Oenococcus mogu se primjeniti u obradi otpadnih voda koje sadrže fenolne spojeve, npr. otpadnog materijala iz proizvodnje vina i maslinovog ulja.