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

Enzimska ekstrakcija hidroksicinamičnih kiselina iz otpada nastalog pri proizvodnji kave

Ferulic, caffeic, p-coumaric and chlorogenic acids are classified as hydroxycinnamic acids, presenting anticarcinogenic, anti-inflammatory and antioxidant properties. In this work, enzymatic extraction has been studied in order to extract high value-added products like hydroxycinnamic acids from coffee pulp. A commercial pectinase and enzyme extract produced by Rhizomucor pusillus strain 23aIV in solid-state fermentation using olive oil or coffee pulp (CP) as an inducer of the feruloyl esterase activity were evaluated separately and mixed. The total content (covalently linked and free) of ferulic, caffeic, p-coumaric and chlorogenic acids was 5276 mg per kg of coffee pulp. Distribution was as follows (in %): chlorogenic acid 58.7, caffeic acid 37.6, ferulic acid 2.1 and p-coumaric acid 1.5. Most of the hydroxycinnamic acids were covalently bound to the cell wall (in %): p-coumaric acid 97.2, caffeic acid 94.4, chlorogenic acid 76.9 and ferulic acid 73.4. The content of covalently linked hydroxycinnamic acid was used to calculate the enzyme extraction yield. The maximum carbon dioxide rate for the solid-state fermentation using olive oil as an inducer was higher and it was reached in a short cultivation time. Nevertheless, the feruloyl esterase (FAE) activity (units per mg of protein) obtained in the fermentation using CP as an inducer was 31.8 % higher in comparison with that obtained in the fermentation using olive oil as the inducer. To our knowledge, this is the first report indicating the composition of both esterified and free ferulic, caffeic, p-coumaric and chlorogenic acids in coffee pulp. The highest yield of extraction of hydroxycinnamic acids was obtained by mixing the produced enzyme extract using coffee pulp as an inducer and a commercial pectinase. Extraction yields were as follows (in %): chlorogenic acid 54.4, ferulic acid 19.8, p-coumaric acid 7.2 and caffeic acid 2.3. An important increase in the added value of coffee pulp was mainly due to the extraction of chlorogenic acid.Ferulinska, kafeinska, p-kumarinska i klorogena kiselina pripadaju u hidroksicinamične kiseline, koje imaju antikarcinogena, protuupalna i antioksidativna svojstva. U radu je ispitana enzimska ekstrakcija hidroksicinamičnih kiselina iz otpada nastalog pri proizvodnji kave. Upotrijebljene su, zasebno i u smjesi, komercijalna pektinaza te enzimski esktrakt dobiven fermentacijom na čvrstoj podlozi od maslinovog ulja ili otpada nastalog pri proizvodnji kave (da bi se potaknula aktivnost feruloil esteraze), s pomoću Rhizomucor pusillus 23aIV. Ukupni je udio kovalentno vezanih i slobodnih hidroksicinamičnih kiselina bio 5276 mg/kg otpada. Od toga je bilo: 58,7 % klorogene; 37,6 % kafeinske; 2,1 % ferulinske i 1,5 % p-kumarinske kiseline. Većina je hidroksicinamičnih kiselina bila kovalentno vezana za staničnu stijenku, i to: 97,2 % p-kumarinske; 94,4 % kafeinske; 76,9 % klorogene i 73,4 % ferulinske kiseline. Pomoću udjela kovalentno vezanih hidroksicinamičnih kiselina izračunat je prinos ekstrahiranog enzima. Maksimalni je udio fermentacijom proizvedenog CO2 postignut na čvrstoj podlozi od maslinovog ulja, i to za kraće vrijeme nego na podlozi od otpada nastalog pri proizvodnje kave, iako je feruloil esteraza proizvedena primjenom ove druge podloge pokazala aktivnost veću za 31,8 %. Prema spoznajama autora ovo je prvo istraživanje u kojem su određeni udjeli vezanih i slobodnih hidroksicinamičnih kiselina u otpadu nastalom pri proizvodnji kave. Najveći je prinos ekstrakcije hidroksicinamičnih kiselina postignut primjenom komercijalne pektinaze i enzimskog ekstrakta. Pritom je izdvojeno 54,4 % klorogene; 19,8 % ferulinske; 7,2 % p-kumarinske i 2,3 % kafeinske kiseline, pa je zaključeno da je ovaj postupak dobar za ekstrakciju klorogene kiseline

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

Enzymatic Extraction of Hydroxycinnamic Acids from Coffee Pulp

Ferulic, caffeic, p-coumaric and chlorogenic acids are classified as hydroxycinnamic acids, presenting anticarcinogenic, anti-inflammatory and antioxidant properties. In this work, enzymatic extraction has been studied in order to extract high value-added products like hydroxycinnamic acids from coffee pulp. A commercial pectinase and enzyme extract produced by Rhizomucor pusillus strain 23aIV in solid-state fermentation using olive oil or coffee pulp (CP) as an inducer of the feruloyl esterase activity were evaluated separately and mixed. The total content (covalently linked and free) of ferulic, caffeic, p-coumaric and chlorogenic acids was 5276 mg per kg of coffee pulp. Distribution was as follows (in %): chlorogenic acid 58.7, caffeic acid 37.6, ferulic acid 2.1 and p-coumaric acid 1.5. Most of the hydroxycinnamic acids were covalently bound to the cell wall (in %): p-coumaric acid 97.2, caffeic acid 94.4, chlorogenic acid 76.9 and ferulic acid 73.4. The content of covalently linked hydroxycinnamic acid was used to calculate the enzyme extraction yield. The maximum carbon dioxide rate for the solid-state fermentation using olive oil as an inducer was higher and it was reached in a short cultivation time. Nevertheless, the feruloyl esterase (FAE) activity (units per mg of protein) obtained in the fermentation using CP as an inducer was 31.8 % higher in comparison with that obtained in the fermentation using olive oil as the inducer. To our knowledge, this is the first report indicating the composition of both esterified and free ferulic, caffeic, p-coumaric and chlorogenic acids in coffee pulp. The highest yield of extraction of hydroxycinnamic acids was obtained by mixing the produced enzyme extract using coffee pulp as an inducer and a commercial pectinase. Extraction yields were as follows (in %): chlorogenic acid 54.4, ferulic acid 19.8, p-coumaric acid 7.2 and caffeic acid 2.3. An important increase in the added value of coffee pulp was mainly due to the extraction of chlorogenic acid