Antioksidativna aktivnost ferementiranih i nefermentiranih esktrakata iz otpada nastalog pri proizvodnji kave

Coffee pulp contains natural antioxidants like hydroxycinnamic acids, most of which are covalently linked to the cell wall. These compounds can be released by fermentation or enzymatic processes. In this study, the antioxidant properties of fermented and nonfermented coffee pulp have been evaluated. Coffee pulp was fermented by solid-state fermentation using the fungus Aspergillus tamarii. Fermented and nonfermented samples of coffee pulp were extracted with aqueous methanol followed by alkaline hydrolysis. In both cases, the total polyphenol concentration was quantified by Folin-Ciocalteu method, then hydroxycinnamic acids were concentrated using ethyl acetate and quantified by HPLC. The antioxidant properties of samples were determined by radical monocation of 2,2’-azinobis-( 3-ethylbenzothiazoline-6-sulphonic acid) [ABTS]·+: the antioxidant activity was determined by kinetic parameters known as ED50, tED50 and antiradical efficiency (AE). Fermented extracts containing free hydroxycinnamic acids showed better antiradical activity against [ABTS]·+ than the other nonfermented ones. There were no significant differences in the total content of polyphenols in fermented and nonfermented coffee pulp, but the content of total hydroxycinnamic acids was higher in the nonfermented coffee pulp extracts (47.1 g/kg) than in the fermented coffee pulp (30.9 g/kg). Nevertheless, the fermentation process increased the fraction of free hydroxycinnamic acids (47 %) and consequently decreased those covalently linked to the cell wall. The results of the antioxidant activity assays could be explained by the presence of free hydroxycinnamic acids. Fermented coffee pulp assays showed that free hydroxycinnamic acids were metabolised by A. tamarii. This study shows the potential of using coffee pulp as a natural source of antioxidants.Otpad nastao pri proizvodnji kave sadržava prirodne antioksidanse, kao što su hidroksicinamične kiseline, od kojih je većina kovalentno vezana za staničnu stijenku. Takvi se spojevi mogu osloboditi fermentacijom ili pomoću enzima. U ovom su radu istražena antioksidativna svojstva fermentiranih i nefermentiranih esktrakata, pri čemu je fermentacija provedena s pomoću plijesni Aspergillus tamarii na čvrstoj podlozi od otpada nastalog pri proizvodnji kave. Fermentirani i nefermentirani spojevi esktrahirani su vodenom otopinom metanola, nakon čega je provedena njihova alkalna hidroliza. U oba je slučaja koncentracija ukupnih polifenola određena Folin-Ciocalteu metodom, a zatim su hidroksicinamične kiseline koncentrirane pomoću etil acetata i analizirane HPLC-om. Antioksidativna su svojstva uzoraka, tj. vrijednosti ED50 i tED50 te antiradikalni učinak, određena pomoću radikala 2,2\u27-azinobis(3-etilbenzotiazolin-6-sulfonske kiseline) [ABTS].+. Fermentirani su ekstrakti sadržavali slobodne hidroksicinamične kiseline i imali su bolju antioksidativnu aktivnost s obzirom na [ABTS].+ od nefermentiranih ekstrakata. Nije bilo bitne razlike u koncentracijama ukupnih polifenola u fermentiranim i nefermentiranim ekstraktima, ali je udio hidroksicinamičnih kiselina bio veći u nefermentiranim (47,1 g/kg) nego u fermentiranim ekstraktima (30,9 g/kg). Fermentacija je povećala udjel slobodnih (na 47 %), a smanjila udjel vezanih hidroksicinamičnih kiselina. Zaključeno je da je antioksidativna aktivnost ekstrakata ovisila o udjelu slobodnih hidroksicinamičnih kiselina, koji se povećao nakon fermentacije otpada nastalog pri proizvodnji kave s pomoću A. tamarii. Time je potvrđeno da se postupak može primijeniti za ekstrakciju prirodnih antioksidanasa

Aktivnost feruloil esteraze proizvedene fermentacijom na čvrstoj podlozi od otpada nastalog pri proizvodnji kave

Hydroxycinnamic acids (HAs) have a potential application in the food and pharmaceutical industry because they are rich in phenolics. Feruloyl esterases release phenolic compounds from plant cell walls. Coffee pulp is rich in HAs linked to polysaccharides. A solvent extraction of free HAs was performed with aqueous methanol (80 %). A response surface methodology was applied to optimise the extraction of these compounds from coffee pulp, and the best results were obtained at 56 °C for 34 min. Alkaline and acid hydrolyses were performed to evaluate the content of linked HAs. Treated (extracted) coffee pulp was used to produce feruloyl esterases in solid-state fermentation by Aspergillus tamarii V12307, previously selected by a hydrolysis plate assay. Different dilutions of a culture medium were added to the coffee pulp, and the diluted medium with half the nutrients allowed for higher CO2 production. A specific growth rate (μCO2 ) of 0.25 h^–1 and a lag phase (tlag) of 14.3 h were observed under the selected conditions. Finally, enzymatic activities were 14.0 and 10.8 nkat per g of dried matter when methyl and ethyl ferulate were used as substrates, respectively. Productivities (9.3 and 7.2 nkat per g of dried matter per day, respectively) were higher when compared to other studies carried out in solid-state fermentation. Utilisation of coffee pulp for enzyme production improves the added value of this abundant by-product of the coffee industry.Hidroksicinamične se kiseline mogu upotrijebiti u prehrambenoj i farmaceutskoj industriji, jer su bogate fenolima, koje enzim feruloil esteraza oslobađa iz staničnih stijenki biljaka. Otpad koji nastaje pri proizvodnji kave bogat je hidroksicinamičnim kiselinama vezanim za polisaharide. Ekstrakcija tih spojeva vodenom otopinom metanola (80 %) optimirana je pomoću metode odzivnih površina, a najbolji su rezultati postignuti pri 56 °C tijekom 34 minute. Alkalnom je i kiselom hidrolizom procijenjen udio vezanih hidroksicinamičnih kiselina. Pomoću odabranoga soja Aspergillus tamarii V12307 proizvedena je feruloil esteraza fermentacijom na čvrstoj podlozi od otpada nastalog pri proizvodnji kave. Otpadu su dodana različita razrjeđenja podloge za uzgoj, pri čemu je proizvedeno više CO2 primjenom podloge koja sadržava 50 % hranjiva. Pritom je specifična brzina rasta (μCO2) bila 0,25 h-1, a lag je faza (tlag) iznosila 14,3 h. Uporabom metil ferulata kao supstrata postignuta je aktivnost enzima od 14 nkat/g suhe tvari i produktivnost od 9,3 nkat/g suhe tvari po danu, dok je pomoću etil ferulata dobivena aktivnost enzima od 10,8 nkat/g suhe tvari i produktivnost od 7,2 nkat/g suhe tvari po danu. Produktivnost je procesa bila veća nego u prijašnjim istraživanjima. Primjenom otpada nastalog pri proizvodnji kave u proizvodnji enzima povećala se dodana vrijednost tog nusproizvoda

Nuclear expression of Rac1 in cervical premalignant lesions and cervical cancer cells

<p>Abstract</p> <p>Background</p> <p>Abnormal expression of Rho-GTPases has been reported in several human cancers. However, the expression of these proteins in cervical cancer has been poorly investigated. In this study we analyzed the expression of the GTPases Rac1, RhoA, Cdc42, and the Rho-GEFs, Tiam1 and beta-Pix, in cervical pre-malignant lesions and cervical cancer cell lines.</p> <p>Methods</p> <p>Protein expression was analyzed by immunochemistry on 102 cervical paraffin-embedded biopsies: 20 without Squamous Intraepithelial Lesions (SIL), 51 Low- grade SIL, and 31 High-grade SIL; and in cervical cancer cell lines C33A and SiHa, and non-tumorigenic HaCat cells. Nuclear localization of Rac1 in HaCat, C33A and SiHa cells was assessed by cellular fractionation and Western blotting, in the presence or not of a chemical Rac1 inhibitor (NSC23766).</p> <p>Results</p> <p>Immunoreacivity for Rac1, RhoA, Tiam1 and beta-Pix was stronger in L-SIL and H-SIL, compared to samples without SIL, and it was significantly associated with the histological diagnosis. Nuclear expression of Rac1 was observed in 52.9% L-SIL and 48.4% H-SIL, but not in samples without SIL. Rac1 was found in the nucleus of C33A and SiHa cells but not in HaCat cells. Chemical inhibition of Rac1 resulted in reduced cell proliferation in HaCat, C33A and SiHa cells.</p> <p>Conclusion</p> <p>Rac1 is expressed in the nucleus of epithelial cells in SILs and cervical cancer cell lines, and chemical inhibition of Rac1 reduces cellular proliferation. Further studies are needed to better understand the role of Rho-GTPases in cervical cancer progression.</p

Antioxidant Activity of Fermented and Nonfermented Coffee (Coffea arabica) Pulp Extracts

Coffee pulp contains natural antioxidants like hydroxycinnamic acids, most of which are covalently linked to the cell wall. These compounds can be released by fermentation or enzymatic processes. In this study, the antioxidant properties of fermented and nonfermented coffee pulp have been evaluated. Coffee pulp was fermented by solid-state fermentation using the fungus Aspergillus tamarii. Fermented and nonfermented samples of coffee pulp were extracted with aqueous methanol followed by alkaline hydrolysis. In both cases, the total polyphenol concentration was quantified by Folin-Ciocalteu method, then hydroxycinnamic acids were concentrated using ethyl acetate and quantified by HPLC. The antioxidant properties of samples were determined by radical monocation of 2,2’-azinobis-( 3-ethylbenzothiazoline-6-sulphonic acid) [ABTS]·+: the antioxidant activity was determined by kinetic parameters known as ED50, tED50 and antiradical efficiency (AE). Fermented extracts containing free hydroxycinnamic acids showed better antiradical activity against [ABTS]·+ than the other nonfermented ones. There were no significant differences in the total content of polyphenols in fermented and nonfermented coffee pulp, but the content of total hydroxycinnamic acids was higher in the nonfermented coffee pulp extracts (47.1 g/kg) than in the fermented coffee pulp (30.9 g/kg). Nevertheless, the fermentation process increased the fraction of free hydroxycinnamic acids (47 %) and consequently decreased those covalently linked to the cell wall. The results of the antioxidant activity assays could be explained by the presence of free hydroxycinnamic acids. Fermented coffee pulp assays showed that free hydroxycinnamic acids were metabolised by A. tamarii. This study shows the potential of using coffee pulp as a natural source of antioxidants

Feruloyl Esterase Activity from Coffee Pulp in Solid-State Fermentation

Hydroxycinnamic acids (HAs) have a potential application in the food and pharmaceutical industry because they are rich in phenolics. Feruloyl esterases release phenolic compounds from plant cell walls. Coffee pulp is rich in HAs linked to polysaccharides. A solvent extraction of free HAs was performed with aqueous methanol (80 %). A response surface methodology was applied to optimise the extraction of these compounds from coffee pulp, and the best results were obtained at 56 °C for 34 min. Alkaline and acid hydrolyses were performed to evaluate the content of linked HAs. Treated (extracted) coffee pulp was used to produce feruloyl esterases in solid-state fermentation by Aspergillus tamarii V12307, previously selected by a hydrolysis plate assay. Different dilutions of a culture medium were added to the coffee pulp, and the diluted medium with half the nutrients allowed for higher CO2 production. A specific growth rate (μCO2 ) of 0.25 h^–1 and a lag phase (tlag) of 14.3 h were observed under the selected conditions. Finally, enzymatic activities were 14.0 and 10.8 nkat per g of dried matter when methyl and ethyl ferulate were used as substrates, respectively. Productivities (9.3 and 7.2 nkat per g of dried matter per day, respectively) were higher when compared to other studies carried out in solid-state fermentation. Utilisation of coffee pulp for enzyme production improves the added value of this abundant by-product of the coffee industry