Extraction of biological materials using subcritical and supercritical fluids

Tehnologije s superkritičnimi fluidi omogočajo pridobivanje produktov, z lastnostmi, ki jih je s klasičnimi postopki zelo težko ali celo nemogoče doseči. Visokotlačni procesi, kot je na primer postopek superkritične ekstrakcije, predstavljajo alternativo za izolacijo snovi iz G. lucidum z visoko biološko aktivnostjo. V prvem delu doktorske disertacije smo s pomočjo klasične ter superkritične ekstrakcije iz medicinske gobe G. lucidum skušali izolirati komponente z biološko aktivnostjo. Ekstrakcije smo izvedli iz zasnov trosnjakov t.i. primordijev ter iz zrelih trosnjakov gobe G. lucidum. Osredotočili smo se na »in vitro« biološke aktivnosti ekstraktov. Ekstraktom smo določili antioksidativno aktivnost po dveh različnih metodah. Prav tako smo določali še celokupne fenole, celokupne polisaharide ter proteine. Določili smo tudi inhibicijo encima acetilholinstearaze (AChE). Ekstraktom, ki smo jih pridobili s pomočjo superkritične ekstrakcije s CO2 smo določili »in vitro« protitumorsko aktivnost na HUVEC (Human Umbilical Vein Endothelial Cells - endotelijske celice iz popkovnične vene) ter na CaCo-2 (Caucasian colon adenocarcinoma - rakave celice debelega črevesa) celičnih linijah. V drugem pa smo se osredotočili na raziskovanje faznih ravnotežij β-karotena v subkritičnih in superkritičnih fluidih. V primeru ekstrakcije karotenoidov je potrebno le te iz rastlinskega tkiva hitro izolirati, da se izognemo oksidativni in encimski degradaciji. Glavni predstavnik karotenoidov je β-karoten. Nahaja se v številnih rastlinskih in živalskih tkivih, zato se ga da izolirati iz tkiv tudi s pomočjo uporabe SCF. Za načrtovanje visokotlačnih kemijskih procesov, kot je na primer ekstrakcija s SCF, je poznavanje in hkrati tudi razumevanje faznih ravnotežij ključnega pomena. Naš namen je bil v prvi vrsti določiti ravnotežne topnosti čistega β-karotena v sub- in superkritičnih fluidih (CO2 ter propanu). V nadaljevanju pa smo raziskovali fazna ravnotežja trikomponentnega sistema β-karotena – triolein – SC-CO2 v določenem tlačnem območju in pri različnih temperaturah. Dobljene rezultate o topnosti smo korelirali s Chrastilovim topnostnim modelom.Nowadays the technology of supercritical fluids allows producing products, that is very difficult or almost impossible to achieve by the conventional methods. High-pressure processes such as the process of supercritical extraction represent an alternative to the isolation of substances from the G. lucidum with the high biological activity. In the first part of the dissertation the conventional and supercritical extraction of the medical mushroom Ganoderma lucidum are presented. The biological active compounds from G. lucidum were thus isolated. Extractions were performed from immature fruiting body, so-called primordia, and from mature fruiting body. The biological activity of the extracts was determined "in vitro". The antioxidant activity was determined by the free-radical method and the antioxidant values of Trolox and ascorbic acid were obtained by Photochem method. The amount of total phenols, total polysaccharides, proteins and inhibition of antiacetycholinesterase (AChE) were also investigated. The cytotoxic activity of G. lucidum extracts obtained by supercritical extraction with CO2 was tested on HUVEC (human umbilical vein endothelial) and CaCo-2 (human colon adenocarcinoma) cell lines. In the second part of the dissertation the phase equilibria of β-carotene in sub- and supercritical fluids is reported. To avoid the oxidative and enzymatic degradation of carotenoids during the extraction process it is necessary to isolate them as fast as possible from the plant tissue. One of the most common carotenoids is β-carotene. It is located in a number of plant and animal tissues and can be isolated from the tissues by using supercritical fluids as solvent. Solubility or phase equilibrium data are very important for designing new high-pressure processes where supercritical fluids are used as solvents. The work presented here focused primarily on the equilibrium solubility of pure β-carotene in supercritical CO2 and subcritical propane. Additionally, the phase equilibrium of the ternary system of β-carotene – triolein – supercritical CO2 at different pressures and temperatures was investigated. Afterwards the obtained solubility data was correlated with the density based model purposed by Chrastil