Application of innovative extraction techniques of bioactive compounds from tobacco waste

Cilj ovog istraživanja bio je ispitati utjecaj četiri inovativne tehnike ekstrakcije: ekstrakcija potpomognuta ultrazvukom (UAE), ekstrakcija potpomognuta mikrovalovima (MAE), superkritična \(CO_{2}\) ekstrakcija (\(SC-CO_{2}\)) te ekstrakcija vodom u supkritičnom stanju (SWE) na iskorištenje i sadržaj bioaktivnih spojeva u duhanskom otpadu (refabrikat, prašina i rebro) koji nastaje tijekom industrijske prerade lista. U ekstraktima su primjenom tekućinske i/ili plinske kromatografije određeni isparljivi spojevi, fenolni spojevi te nikotin, a primjenom spektrofotometrijskih metoda određena je antiradikalna aktivnost i sadržaj ukupnih fenola. U SC-\(CO_{2}\) ekstraktima određen je i masnokiselinski sastav primjenom plinske kromatografije s plameno-ionizacijskim detektorom. Na temelju dobivenih rezultata uočen je značajan utjecaj primijenjenih procesnih parametara na promatrane odzive (iskorištenje ekstrakcije, sadržaj klorogenske kiseline i nikotina). Primjenom metode odzivnih površina određeni su optimalni parametri svake pojedine tehnike ekstrakcije. Od ispitivanih tehnika ekstrakcije, primjenom UAE dobiven je najveći sadržaj klorogenske kiseline, dok se MAE pokazala pogodnom za ekstrakciju nikotina. Primjenom SWE dobiveni su ekstrakti bogati fenolnim spojevima i nikotinom, no primjena viših temperatura (200-250 °C) uzrokovala je nastanak degradacijskih produkata. \(SC-CO_{2}\) ekstrakcija pokazala se pogodnom za ekstrakciju nepolarnih spojeva (isparljivih spojeva i masnih kiselina), gdje je identificicran nikotin (1,6 %), a dominantna masna kiselina bila je palmitinska kiselina (21,84 %). Od promatrana tri otpada, refabrikat se pokazao najbogatijim bioaktivnim spojevima u usporedbi s listom duhana kao polaznom sirovinom. U konačnici, da bi se u potpunosti iscrpio biljni materijal, pristupilo se valorizaciji refabrikata kroz kombinaciju dvije ekstrakcijske tehnike (\(SC-CO_{2}\) i SWE). Dvostupanjska ekstrakcija dala je najbolje rezultate, te predstavlja inovativan pristup valorizaciji ove vrste otpada istovremeno dajući maksimalno iskorištenje ekstrakcije (74,05 %), uz znatno veći zastupljenosti nikotina (4,67 %) i fenolnih spojeva: klorogenske kiseline (0,74 %), neoklorogenske kiseline (0,46 %), rutina (1,03 %).The aim of this study was to determine the influence of four innovative extraction techniques: ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), supercritical \(CO_{2}\) extraction (\(SC-CO_{2}\)) and subcritical water extraction (SWE) on the yield and content of bioactive compounds in tobacco waste extracts. Three fractions of tobacco waste, generated during the industrial processing of leaves (scrap, dust and midrib), were used for the extractions and were compared with the leaf as the starting material. Volatile compounds, phenolic compounds and nicotine were determined in the extracts using liquid and/or gas chromatography. Additionally, total phenol content and antioxidant activity were determined using spectrophotometric assays. In \(SC-CO_{2}\) extracts, the fatty acid composition was determined using gas chromatography with a flame ionization detector. Based on the obtained results, a significant influence of the applied process parameters on the responses (extraction yield, chlorogenic acid content and nicotine) was observed. Using the response surface methodology, the optimal parameters of each extraction technique were determined. Of the four extraction techniques observed, UAE yielded the highest chlorogenic acid content, while MAE proved to be suitable for nicotine extraction. Using SWE, extracts rich in phenolic compounds and nicotine were obtained, but due to the application of higher temperatures (200-250 °C), the formation of degradation products was observed. \(SC-CO_{2}\) showed high selectivity for the extraction of non-polar compounds, particularly targeted fatty acids (the dominant was palmitic acid) and volatile compounds (nicotine was the most abundant). Among the three wastes observed, the scrap proved to be the richest in bioactive compounds. In order to obtain completely exhaust waste material, a two-stage extraction process (\(SC-CO_{2}\) + SWE) was conducted on the scrap. With twostage extraction (\(SC-CO_{2}\) + SWE), the highest extraction efficiency (74.05%) and significantly higher abundance of nicotine (4.67%) and of phenolic compounds (chlorogenic acid (0.74%), neochlorogenic acid (0.46%) and rutin (1.03%)) were obtained. Therefore, two-stage extraction (\(SC-CO_{2}\) + SWE) represents an innovative approach to the valorization of tobacco waste