Sinteza magnetiziranih biokatalizatora unakrsnim vezivanjem pektinaze s makromolekulama kefirana, za njihovu primjenu u postupku bistrenja soka od jabuke

Research background. Pectinase enzyme has become a valuable compound in beverage industry. One of the most significant concepts to overcome the drawbacks of using industrial enzymes is their immobilization. In the present study, magnetic chitosan microparticles were utilized as a substrate for pectinase immobilization. New methods of enzyme immobilization involve the use of non-chemical cross-linkers between the enzyme and the substrate. The aim of this study is to immobilize the pectinase enzyme using polyaldehyde kefiran as a macromolecular cross-linker on magnetic particles. Experimental approach. Pectinase was immobilized in four steps: relative oxidation of kefiran and its application as a cross-linker, production of magnetic iron(II) iron(III) oxide (Fe3O4) microparticles, coating of magnetic Fe3O4 microparticles with chitosan, and immobilization of the enzyme on the substrate, prepared by the use of oxidized kefiran cross-linker. Parameters such as cross-linking concentration, time and ratio of chitosan magnetic microparticles to enzyme were optimized. Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering, transmission electron microscopy, and vibrating sample magnetometer were used to identify the groups and investigate the structures. The biochemical properties (stability of enzyme activity at different pH, temperature and time), enzyme reusability, kinetic parameters (Km and νmax) and apple juice turbidity, using free and immobilized pectinase enzymes, were also measured. Results and conclusions. Cross-linker concentration, cross-linking time and the ratio of magnetic Fe3O4 microparticles with chitosan to enzyme were important factors in activity recovery of pectinase. FTIR analysis correctly identified functional groups in the structures. The results showed that after enzyme stabilization, the particle size and molecular mass, respectively, increased and decreased the magnetic saturation strength. According to the thermal kinetic study, the activity of the immobilized pectinase was higher than of its free form. The findings of this study indicate excellent stability and durability of the immobilized pectinase. Finally, a magnetic pectinase micro-biocatalyst was used to clarify apple juice, which reduced turbidity during processing. Novelty and scientific contribution. This study investigates the usage of kefiran oxidized as a new cross-linker for the immobilization of pectinase enzyme. Magnetic pectinase micro-biocatalyst has a good potential for industrial applications in the food industry, with high thermal stability.Pozadina istraživanja. Enzim pektinaza postao je vrijedan sastojak koji se koristi u proizvodnji sokova. Najbitniji korak u rješavanju problema primjene industrijskih enzima je njihova imobilizacija. U ovom su radu upotrijebljene mikročestice magnetiziranog kitozana kao supstrat za imobilizaciju pektinaze. Nove metode imobilizacije enzima uključuju primjenu vezivnih materijala koji ne stvaraju kemijske veze između enzima i supstrata. Svrha je ovoga rada bila imobilizirati pektinazu njezinim unakrsnim vezivanjem s polialdehidom kefiranom na magnetiziranim česticama. Eksperimentalni pristup. Pektinaza je imobilizirana u četiri koraka: djelomična oksidacija kefirana i njegova primjena kao sredstva za vezivanje, proizvodnja magnetiziranih mikročestica željezovog(II,III) oksida (Fe3O4), premazivanje magnetiziranih čestica Fe3O4 kitozanom, te imobilizacija enzima na supstratu pomoću oksidiranog kefirana kao vezivnog materijala. Optimirani su sljedeći parametri: koncentracija vezivnog materijala, trajanje postupka i omjer magnetiziranih mikročestica kitozana i enzima. Za identifikaciju funkcionalnih skupina i određivanje strukture uzoraka upotrijebljeni su: infracrvena spektroskopija s Fourierovom transformacijom (FTIR), dinamičko raspršenje svjetlosti, transmisijski elektronski mikroskop i vibrirajući magnetometar. Ispitana su biokemijska svojstva (stabilnost enzima pri različitim pH-vrijednostima, temperaturama i trajanju postupka) i mogućnost ponovne uporabe slobodnog i imobiliziranog enzima, kinetički parametri (Km i νmax) postupka, te zamućenost soka od jabuke. Rezultati i zaključci. Koncentracija oksidiranog kefirana, vrijeme vezivanja te omjer magnetiziranih mikročestica Fe3O4 s kitozanom i enzima bitni su čimbenici iskorištenja pektinaze. FTIR analizom su točno identificirane funkcionalne skupine uzoraka. Rezultati pokazuju da je nakon stabilizacije enzima veličina čestica povećala, a njihova molekularna masa smanjila magnetsku zasićenost. Studija termalne kinetike pokazala je da je aktivnost imobilizirane pektinaze bila veća od aktivnosti slobodne pektinaze. Dobiveni rezultati pokazuju odličnu stabilnost i iskoristivost imobilizirane pektinaze. Naposljetku, magnetizirani biokatalizator s pektinazom korišten je za bistrenje soka od jabuke, pri čemu se smanjila zamućenost soka tijekom prerade. Novina i znanstveni doprinos. U radu je ispitana primjena oksidiranog kefirana kao novog vezivnog spoja za imobilizaciju pektinaze. Magnetizirani biokatalizator s pektinazom imao je dobra svojstva za primjenu u prehrambenoj industriji i veliku toplinsku stabilnost

Pullulan Production Using Molasses and Corn Steep Liquor as Agroindustrial Wastes: Physiochemical, Thermal and Rheological Properties

Background and Objective: Pullulan is a microbial exopolysaccharide with wide uses in various industries. The aim of this study was to investigate pullulan production from agro-industrial wastes and study of pH, molasses concentration and corn steep liquor concentration as independent variables and yield of pullulan as response. Material and Methods: Briefly, 5% (v v-1) of the inoculation media (yeast extract 3 g, malt extract 3 g, peptone 5 g and sucrose 10 g per liter of distilled water), including Aureobasidium pullulans were added into media, containing 100 ml of molasses (100, 150 and 200 g l-1) and various corn steep liquor concentrations (20, 40 and 60 ml l-1) at adjusted pH (4.5, 5.5 and 6.5). After extraction and separation of the biomass using centrifuge, two folds of the supernatant volume of cold ethanol were added to the samples and stored at 4 °C for 24 h. After centrifuging, pullulan was dried and analyzed using Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis and rheological tests. Results and Conclusion: Findings revealed that the maximum production yield (18.29 g l- 1 ±0.10) was achieved under optimum fermentation conditions (pH of 5.3, molasses concen-tration of 165 g l-1 and corn steep liquor concentration of 43 ml l-1). Then, physiochemical and thermal properties of the pullulan under the highlighted conditions were investigated. Pullulan included 78.8% solubility with no hygroscopicity. Furthermore, structural analysis using Fou-rier transform infrared and X-ray diffraction verified presence of pullulan with an amorphous structure in the supernatant. The exopolysaccharide included acceptable thermal stability and gel-like behavior; in which, the elastic component was predominant based on the results of thermogravimetric analysis and rheological properties, respectively

Kontinuirani postupak bistrenja soka od obične žutike pomoću pektinaze imobilizirane oksidiranim polisaharidima

Research background. Barberry juice is a rich source of bioactive compounds and shows different health properties such as antioxidant and anticancer activities. Clarification, as the removal process of suspended material, is an important step in the production of fruit juice due to its significant effect on the appearance, flavour and commercialisation of juice. Pectinase is the most important enzyme applied in juice clarification that breaks down the pectin polymer structure and reduces the undesirable turbidity. Pectinase immobilisation is a way to overcome free enzyme drawbacks such as instability, high cost, the difficulty of recovery and recyclability. Also, continuous clarification process which is highly preferred in fruit juice industry is not possible without enzyme immobilisation. Experimental approach. Pectinase enzymes were immobilised on the functionalised glass beads (glass bead with (3-aminopropyl)triethoxysilane) by glutaraldehyde, polyaldehyde derivatives of pullulan and kefiran and the barberry juice was clarified in the batch and continuous processes in a packed bed reactor (PBR). Also, the effect of clarification on the physicochemical and antioxidant properties of the barberry juice samples was evaluated. Results and conclusions. The optimum conditions for clarification in the PBR were: flow rate 0.5 mL/min, temperature 50 °C and treatment time 63 min. Clarification led to a decrease in turbidity, pH, total soluble solid content, viscosity, total phenolic content and antioxidant activity of the juice samples. Also, this process increased the clarity, acidity, reducing sugar concentration and the lightness parameter of the barberry juice. The greatest effect of clarification on the studied properties of barberry juice was related to the pectinase immobilised by the polyaldehyde of kefiran in the continuous process and both new cross-linkers (polyaldehyde derivatives of pullulan and kefiran) immobilised the enzyme better than the common cross-linker (glutaraldehyde). Novelty and scientific contribution. For the first time, barberry juice was clarified with pectinase immobilised by polyaldehyde derivatives of pullulan and kefiran and the obtained results showed that the pectinase immobilisation by these new cross-linkers was much more efficient than by the glutaraldehyde as a common cross-linker. These findings can be of use for an industrialised production of fruit juices.Pozadina istraživanja. Sok od obične žutike bogat je bioaktivnim spojevima te ima razna ljekovita svojstva, poput antioksidacijskih i antikancerogenih. Bistrenje je postupak uklanjanja materijala iz suspenzije i važan je korak u proizvodnji sokova jer bitno utječe na izgled, okus i ekonomsku isplativost soka. Pektinaza je najvažniji enzim koji se koristi za bistrenje sokova, a razgrađuje polimernu strukturu pektina i time smanjuje zamućenost soka. Imobilizacijom pektinaze uklanjaju se nedostaci slobodnog enzima, kao što su nestabilnost, visoka cijena, malo iskorištenje i slaba ponovna iskoristivost. Osim toga, kontinuirani postupak bistrenja, koji se najviše koristi u proizvodnji sokova, nije moguće provesti bez imobilizacije enzima. Eksperimentalni pristup. Pektinaza je imobilizirana pomoću glutaraldehida te polialdehidnih derivata pululana i kefirana na staklenim kuglicama prevučenim (3-aminopropil)trietoksisilanom, a sok od obične žutike bistren je šaržnim ili kontinuiranim postupkom u reaktoru s nasutim slojem nosača. Osim toga, ispitana su fizikalno-kemijska i antioksidacijska svojstva soka. Rezultati i zaključci. Optimalni uvjeti za bistrenje soka u reaktoru s nasutim slojem nosača bili su: protok 0,5 mL/min, temperatura 50 °C i trajanje postupka 63 min. Postupkom bistrenja smanjila se zamućenost soka, snizila njegova pH-vrijednost i smanjili ukupan udjel topljive tvari, viskoznost, ukupan udjel fenola te antioksidacijska aktivnost. Osim toga, povećali su se bistroća, kiselost, koncentracija reducirajućih šećera te parametri boje soka. Svojstva soka najviše su se poboljšala bistrenjem pomoću pektinaze imobilizirane polialdehidom kefirana u kontinuiranom postupku. Oba derivata polialdehida, pululan i kefiran, imobilizirali su enzim bolje od uobičajeno korištenog glutaraldehida. Novina i znanstveni doprinos. U ovom je radu po prvi put proveden postupak bistrenja soka od obične žutike pomoću pektinaze imobilizirane derivatima polialdehida pululana i kefirana. Dobiveni rezultati pokazuju da je ovako imobilizirana pektinaza bitno učinkovitija od one imobilizirane pomoću glutaraldehida, te se može upotrijebiti u industrijskoj proizvodnji voćnih sokova

Extraction and purification matairesinol bioactive lignan of arizonica cypress (cupressus arizonica)

Extraction and purification matairesinol bioactive lignan of arizonica cypress (cupressus arizonica) A. Sedaghat1, A. Abdulkhani 2 1. M. Sc, Department of wood and paper science, Faculty of Natural resources, University of Tehran 2. Assistant Professor, Department of wood and paper science, Faculty of Natural resources, University of Tehran Matairesinol lignan (MR) of arizonica cypress was isolated, characterized and purified. Isolated was made using a hexane pretreatment for removing the lipophilic moieties followed by an ethanol-water (9:1 v/v) treatment by soaking the wood flour in the solvents. Also matairesinol, was purified with potassium acetate. Identification of extracts were performed in all stages by means of gas chromatography- mass spectrometry (GC/MS). Results showed that amount of MR lignan in the original ethanol extract, sediment and clear solution after centrifugation was 11.3, 32.59 and 40.44% respectively.... ...... .... .... .... .. ..... .... .... ... .......................................................................................................................... ......... .............................. ..... ...................................................................................................... Keywords: arizonica cypress, gas chromatography- mass spectrometry (GC/MS), wood knot, lignan, matairesinol (MR), extractive