Adding value in barley malt rootlets as a source of 5’-phosphodiesterase:biochemical and thermodynamic evaluation of enzyme activity

thermostable 5’-phosphodiesterase (5’-PDE, EC 3.1.4.1) was extracted from barley (Hordeum distichum var. Rex) malt rootlets. The purification procedure comprised acetone precipitation, S-Sepharose cation-exchange and DEAE-Sepharose anion-exchange chromatography. The enzyme was purified 101-fold with a recovery of 22% and a specific activity of 81.9 U mg-1 protein, Optimum enzyme activity was obtained at 70 °C, and pH 8.9. The SDS-PAGE profiling of the purified protein exhibited molecular weight of 116 kDa and revealed three sub-unit fractions of 26, 43, and 56 kDa making up its active configuration. The kinetic constants Km and Vmax were determined as 0.25 mM and 0.816 mmol min-1, respectively. Thermodynamic studies showed that the thermal inactivation of purified barley malt rootlets 5’-PDE followed the first-order kinetics, indicating inactivation energy (Ed) of 134 kJ mol-1. The half-life (t1/2) at 70 °C was estimated as 169 min. Thermodynamic parameters ΔH*, ΔS* and ΔG* were determined as a function of temperature and were 131.15 kJ mol-1, 37.01 kJ mol-1 K-1 and 118.4 kJ mol-1, respectively. The purified enzyme has long half-life with 11 days at 0 °C, 37 hours at 4 °C and 11 hours at room temperature. These results provide useful information about the factors that affects the activity of barley malt rootlets 5’-PDE and suggests a good indication for application of this enzyme in pharmaceutical and food industry

New Trends in the Ethanol Production as a Biofuel

Povećanje emisije stakleničkih plinova, energetska ovisnost i nestabilnost isporuke energenata posljedica su ubrzane potrošnje fosilnih goriva zbog ubrzanog rasta svjetske populacije i industrijalizacije. Bioetanol je postao atraktivno zamjensko biogorivo jer se proizvodi iz obnovljivih sirovina i ekološki je prihvatljiv. Upotrebljava se kao pogonsko gorivo i to kao hidrirani (96 %) ili bezvodni (u mješavinama s benzinom). Bioetanol se proizvodi fermentacijom s kvascem S. cerevisiae ili nekim drugim mikroorganizmom iz ugljikohidrata kao što su jednostavni šećeri, škrob i celuloza. Nakon fermentacije, bioetanol se izdvaja i pročišćava destilacijom i dehidracijom. Sastav je propisan specifikacijama za primjenu u motornim gorivima. Uobičajeni usjevi, kao što su kukuruz, šećerna repa i trska, zasad su osnovne sirovine za proizvodnju bioetanola. Međutim, proizvodnja bioetanola iz ovih sirovina ne može zadovoljiti globalne potrebe za bioetanolom, zbog njihove primarne uloge u prehrani ljudi i životinja. Lignoceluloza je pogodna sirovina za proizvodnju bioetanola jer je široko rasprostranjena, obnovljiva i ne upotrebljava se u prehrani. Proizvodnja bioetanola iz lignoceluloznih sirovina je složen proces, koji se u mnogim aspektima razlikuje od proizvodnje iz šećernih ili škrobnih sirovina. U ovom radu prikazane su dosadašnje i nove tehnologije u proizvodnji bioetanola uključujući primjenu različitih sirovina, postupaka proizvodnje i radnih mikroorganizama. Nadalje, navedene su mogućnosti integracije osnovnih koraka u bioprocesima proizvodnje s ciljem povećanja produktivnosti i smanjenja troškova proizvodnje.Rapidly growing fossil energy consumption, due to population and industrial growth, has caused increasing greenhouse-gas emissions, growing energy dependency and supply insecurity. Bioethanol has become an attractive alternative biofuel because of its environmental benefits and the fact that it is made from renewable resources. Ethanol is widely used as transport fuel, pure hydrous ethanol or anhydrous ethanol in mixtures with gasoline (Fig. 1). Bioethanol is produced from carbohydrates such as sugar, starch and cellulose by fermentation with yeast S. cerevisiae or other microorganisms. Thereupon, ethanol is separated and purifyed by distillation-rectification-dehydration to meet fuel specifications. Currently, conventional crops such as corn or sugarcane are the main feedstock for bioethanol production. Bioethanol production from the sucrose-containing feedstock is simpler compared to the starchy materials and the lignocellulosic biomass due to an additional step – feedstock hydrolysis. The process of ethanol production from starchy materials includes the hydrolysis of starch to glucose using α-amylase (1,4-α-D-glucan-4-glucanohydrolase) and glucoamylase (1,4-α-D-glucanglucohydrolase). Finally, the glucose is fermented to ethanol by yeast cells. Enzymatic hydrolysis of starch and fermentation of glucose can be carried out in different process configurations, such as separate hydrolysis and fermentation (SHF), and simultaneous saccharification and fermentation (SSF, Fig. 2). In consolidated bioprocessing (CBP), the conversion of starch into ethanol is performed in one step without added enzymes. This process configuration has potential to lower the cost of biomass processing due to elimination of operating and capital costs associated with dedicated enzyme production. Current bioethanol production from corn and sugarcane is unable to meet the global demand for bioethanol, due to their primary value as livestock feed and human food. The lignocellulosic biomass such as agricultural wastes (corn stover, crop straws, husks and bagasse), herbaceous crops (switchgrass), woody crops, forestry residue, waste paper and other wastes (municipal and industrial) is favourable feedstock for bioethanol production. The major advantages of lignocellulosic biomass are its renewable and ubiquitous nature and its noncompetitiveness with food crops. Ethanol production from lignocellulosic feedstock is complex and comprises two steps prior to fermentation: biomass pretreatment (breaking down the structure of the lignocellulosic matrix) and cellulose hydrolysis (enzymatic hydrolysis of cellulose to glucose). The lignocellulosic hydrolysate contains not only hexoses, but also pentoses that are not assimilated by yeast S. cerevisiae. Furthermore, the lignocellulosic hydrolysate contains a broad range of compounds that inhibit the yeast’s cells. The composition of the inhibitors depends on the type of the lignocellulosic material, and the chemistry and nature of the pretreatment process. The pretreated cellulose can be enzymatically hydrolysed either prior or simultaneously with glucose fermentation (Fig. 3). The four main steps involved in the process of lignocellulosic bioethanol production (pretreatment, cellulose hydrolysis, hexoses and pentoses fermentation) can be arranged in various process configurations, including separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF) and consolidated bioprocessing (CBP, Fig. 3). Specific strains of bacteria and yeasts have been developed to ferment sugars released from lignocellulosic biomass and hydrolysed cellulose, through a selection of new strains and genetic engineering of traditional strains

Proizvodnja bioetanola iz kukuruznih oklasaka

Bioetanol je proizveden procesom istodobne saharifikacije i fermentacije (eng. Simultaneous saccharification and fermentation, SSF) s kvascem Saccharomyces cerevisiae na enzimskom hidrolizatu predobrađenih kukuruznih oklasaka. Istraživan je učinak trajanja predhidrolize kukuruznih oklasaka na učinkovitost procesa proizvodnje bioetanola te je uspoređen s konvencionalnim procesom SSF. Dvije smjese komercijalno dostupnih enzima rabljene su za hidrolizu sirovine; prva je sadržavala Celluclast 1.5L (Sigma) i β-glukozidazu (Carl Roth), a druga smjesa Celluclast 1.5L (Sigma) i Viscozyme L (Sigma). Dva dana predhidrolize imala su pozitivan učinak na prinos bioetanola, dok je dulje trajanje predhidrolize smanjilo prinos bioetanola. Najveća koncentracija etanola od 41,24 g dm−3 postignuta je u procesu SSF uz dva dana predhidrolize koja je iznosila 63,15 % teoretskog iskorištenja izračunatog na neobrađeni kukuruzni oklasak. Najveća ukupna produktivnost procesa od 0,36 g dm−3 h−1 postignuta je konvencionalnim SSF-om uz enzimsku smjesu koja je sadržavala Celluclast 1.5L (Sigma) i Viscozyme L (Sigma). Koncentracija etanola iznosila je 36,6 g dm−3, što je 56,02 % teoretskog prinosa etanola na neobrađeni kukuruzni oklasak

Bacterial nanocellulose (BNC) biosynthesis, and antimicrobial activity of kombucha in fermented coconut water

Bakterijska nanoceluloza (BNC) je obnovljiva prirodna polimerna sirovina koju karakteriziraju izvrsna svojstva poput visoke kristaliničnosti i stupnja polimerizacije, hidrofilnosti, kiralnosti, biorazgradivosti, te sposobnosti stvaranja različitih morfologija polukristaliničnih vlakana. U ovom je radu istraživan uzgoj kombuche u aerobnim uvjetima tijekom 14 dana fermentacije u kokosovoj vodi na sobnoj temperaturi, pri različitim koncentracijama saharoze. Praćene su promjene pH vrijednosti, koncentracije etanola, octene, glukonske i mliječne kiseline, kao i prinos BNC. Istraživana je kinetika otpuštanja vode iz sintetizirane BNC, a rezultati su potvrdili da su uzorci pogodni za zacjeljivanje rana na koži. Antimikrobna aktivnost kombuche je testirana na bakterijama Escherichia coli, Staphylococcus aureus, Salmonella typhimurium i Bacillus subtilis, te kvascu vrste Candida albicans. Uočena je antimikrobna aktivnost fermentirane kokosove vode na bakterije, no ne u potpunosti i na kvasac C. albicans.Bacterial nanoellulose (BNC) is a sustainable natural polymeric raw material characterized by exciting properties such as hydrophilicity, chirality, biodegradability, broad chemical-modifying capacity, and the formation of different semicrystalline fiber morphologies. This work studies cultivation of kombucha in aerobic conditions over a period up to 14 days of fermentation in coconut water, at room temperature with different sucrose concentrations added. Changes in pH value, ethanol, acetic acid, gluconic acid, and lactic acid concentrations were observed, as well as yield of BNC. The water release rate (WRR) were investigated, and the results supported its anticipated use as antimicrobial wound dressing material. The antimicrobial activity of kombucha was tested against bacteria Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Bacillus subtilis, as well as yeast Candida albicans. Antimicrobial activitiy of coconut water was observed in the fermented samples against all the investigated bacteria strains, but not completely against yeast Candida albicans

Nutritive value and antioxidant activity of wild edible mushroms Albarellus per-caprae and Armilaria mellea

Ovo istraživanje namijenjeno je određivanju prosječnog kemijskog sastava, udjela bioaktivnih spojeva i antioksidacijske aktivnosti jestivih samoniklih gljiva Albarellus per-caprae i Armilaria mellea. Rezultati određivanja kemijskog sastava su pokazali da su gljive bogat izvor bjelančevina i ugljikohidrata te da imaju male količine masti, a dobar su izvor energije. Koncentracije pet bioaktivnih spojeva (askorbinska kiselina, β-karoten, likopen, ukupni fenoli i flavonoidi) određene su ekstrakcijom plodišta gljiva u vrućoj vodi i metanolu. Tri komplementarna kemijska ispitivanja; reducirajuća snaga, uklanjanje slobodnih radikala (DPPH) i sposobnost keliranja iona željeza korišteni su za određivanje antioksidacijskih svojstava ekstrakata. Najveće vrijednosti antioksidacijskog kapaciteta određene su u ekstraktima A. mellea s vrućom vodom (9,01 μM TE/g s.tv. u reducirajućoj snazi, 65% u sposobnosti uklanjanja slobodnih radikala, i 85% u sposobnosti keliranja). Vrijednosti EC50 za tri različita ispitivanja antioksidanta bile su između 1,63 mg/ mL i 6,86 mg/mL. Zbog ovih karakteristika, jestive samonikle gljive A. per-caprae i A. mellea mogu biti dio dobro uravnotežene prehrane i izvor bioaktivnih sastojaka.This study is designed for the determination of proximate chemical composition, contents of bioactive compounds, and antioxidant activity of wild edible mushrooms Albarellus per-caprae and Armilaria mellea. The obtained results of chemical composition revealed that the mushrooms were rich source of proteins and carbohydrates, low in fat content, and provides a good source of energy. Concentrations of five bioactive compounds (ascorbic acid, β-carotene, lykopene, total phenols, and flavonoids) are determined in hot water and methanolic extracts of mushrooms fruit bodies. Three complementary chemical assays: reducing power, free radical scavenging (DPPH), and chelating ability for ferrous ions were used to screen the antioxidant properties of extracts. The highest antioxidant capacity values were found for hot water extracts of A. mellea (9.01 μM TE/g d.w. in reducing power, 65% in scavenging ability, and 85% in chelating ability). EC50 values for three different antioxidant assays were between 1.63 mg/mL to 6.86 mg/mL. Due to these characteristics, wild edible mushrooms A. per-caprae and A. mellea could be used in well-balanced diets and as a source of bioactive compounds