Procjena aktivnosti piruvat dekarboksilaze različitih sojeva industrijskoga kvasca in situ

Cytoplasmic pyruvate decarboxylase (PDC, EC 4.1.1.1) is one of the key enzymes of yeast fermentative metabolism. PDC is the first enzyme which, under anaerobic conditions, leads to decarboxylation of pyruvate with acetaldehyde as the end product. The aim of this study is to develop a suitable method for PDC activity assay in situ for different industrial yeast strains. Saccharomyces sp. and Debaryomyces sp. yeast strains grew in fermentative medium with 12 % of glucose. Enzymatic assay was conducted in cell suspension treated with digitonin as permeabilisation agent, and with sodium pyruvate as a substrate, at temperature of 30 °C. Metabolites of PDC pathway were detected using gas chromatographic (GC) technique. Various parameters like type and molar concentration of the substrate, minimal effective mass fraction of digitonin, cell concentration, reaction time and effect of pyrazole (alcohol dehydrogenase inhibitor) were monitored to optimize PDC enzymatic assay in situ. In the concentration range of yeast cells from 1⋅10^7 to 1⋅10^8 per mL, linear correlation between the produced acetaldehyde and cell density was noticed. Only pyruvate was the specific substrate for pyruvate decarboxylase. In the presence of 0.05 M sodium pyruvate and 0.05 % digitonin, the enzymatic reaction was linear up to 20 min of the assay. During incubation, there was no formation of ethanol and, therefore, pyrazole was not necessary for the assay.Piruvat dekarboksilaza (EC 4.1.1.1) jedan je od ključnih enzima u fermentativnom metabolizmu kvasca. To je prvi enzim koji u anaerobnim uvjetima dovodi do dekarboksilacije piruvata i nastanka acetaldehida. Svrha je ovog istraživanja bila razviti prikladnu metodu procjene aktivnosti piruvat dekarboksilaze različitih sojeva industrijskoga kvasca in situ. Sojevi vrsta Saccharomyces i Debaryomyces uzgojeni su u fermentacijskom mediju s 12 % glukoze. Procijenjena je aktivnost enzima u otopini stanica obrađenih digitoninom (koji povećava propusnost stanične membrane), uz dodatak natrijeva piruvata, pri temperaturi od 30 C. Razgradni metaboliti piruvata određeni su plinskom kromatografijom. Za procjenu optimalne aktivnosti piruvat dekarboksilaze in situ praćeni su razni parametri: tip i koncentracija supstrata, minimalna efektivna koncentracija digitonina, gustoća stanica, vrijeme reakcije i učinak pirazola (inhibitora alkohol dehidrogenaze). U rasponu koncentracije stanica kvasca od 1·10^7 do 1·10^8/mL zamijećena je linearna korelacija proizvedenog acetaldehida i gustoće stanica. Ustanovljeno je da je piruvat jedini specifični supstrat za enzim piruvat dekarboksilazu, te da je u prisutnosti 0,05 M natrijeva piruvata i 0,05 % digitonina enzimska reakcija imala linearni tijek prvih 20 minuta inkubacije. Tijekom inkubacije nije nastao etanol, pa je zaključeno da pirazol nije potreban u procjeni aktivnosti piruvat dekarboksilaze

The impact of Special Economic Zones on local labour markets in Poland

Studies conducted so far suggest that SEZs are not treated by local authorities as the main mechanism of job creation in a given region. The objective of this paper is to highlight potential mechanisms through which SEZs impact labour markets in poviats (counties) in Poland. To this end we conducted a comparative analysis of changes that had taken place in the labour market over the period 2004-2016 in two groups of poviats with the highest unemployment rate reported in 2004: with and without SEZs. The study does not allow us to unambiguously conclude that SEZs contributed to the improvement of labour market situation in poviats with the highest unemployment rate in Poland. That can be attributed to the fact that SEZs in Poland are highly fragmented as well as to SEZs investors being able to select locations for their investment projects in relatively better developed regions

Pyruvate Decarboxylase Activity Assay in situ of Different Industrial Yeast Strains

Cytoplasmic pyruvate decarboxylase (PDC, EC 4.1.1.1) is one of the key enzymes of yeast fermentative metabolism. PDC is the first enzyme which, under anaerobic conditions, leads to decarboxylation of pyruvate with acetaldehyde as the end product. The aim of this study is to develop a suitable method for PDC activity assay in situ for different industrial yeast strains. Saccharomyces sp. and Debaryomyces sp. yeast strains grew in fermentative medium with 12 % of glucose. Enzymatic assay was conducted in cell suspension treated with digitonin as permeabilisation agent, and with sodium pyruvate as a substrate, at temperature of 30 °C. Metabolites of PDC pathway were detected using gas chromatographic (GC) technique. Various parameters like type and molar concentration of the substrate, minimal effective mass fraction of digitonin, cell concentration, reaction time and effect of pyrazole (alcohol dehydrogenase inhibitor) were monitored to optimize PDC enzymatic assay in situ. In the concentration range of yeast cells from 1⋅10^7 to 1⋅10^8 per mL, linear correlation between the produced acetaldehyde and cell density was noticed. Only pyruvate was the specific substrate for pyruvate decarboxylase. In the presence of 0.05 M sodium pyruvate and 0.05 % digitonin, the enzymatic reaction was linear up to 20 min of the assay. During incubation, there was no formation of ethanol and, therefore, pyrazole was not necessary for the assay

Procjena aktivnosti piruvat dekarboksilaze različitih sojeva industrijskoga kvasca in situ

Cytoplasmic pyruvate decarboxylase (PDC, EC 4.1.1.1) is one of the key enzymes of yeast fermentative metabolism. PDC is the first enzyme which, under anaerobic conditions, leads to decarboxylation of pyruvate with acetaldehyde as the end product. The aim of this study is to develop a suitable method for PDC activity assay in situ for different industrial yeast strains. Saccharomyces sp. and Debaryomyces sp. yeast strains grew in fermentative medium with 12 % of glucose. Enzymatic assay was conducted in cell suspension treated with digitonin as permeabilisation agent, and with sodium pyruvate as a substrate, at temperature of 30 °C. Metabolites of PDC pathway were detected using gas chromatographic (GC) technique. Various parameters like type and molar concentration of the substrate, minimal effective mass fraction of digitonin, cell concentration, reaction time and effect of pyrazole (alcohol dehydrogenase inhibitor) were monitored to optimize PDC enzymatic assay in situ. In the concentration range of yeast cells from 1⋅10^7 to 1⋅10^8 per mL, linear correlation between the produced acetaldehyde and cell density was noticed. Only pyruvate was the specific substrate for pyruvate decarboxylase. In the presence of 0.05 M sodium pyruvate and 0.05 % digitonin, the enzymatic reaction was linear up to 20 min of the assay. During incubation, there was no formation of ethanol and, therefore, pyrazole was not necessary for the assay.Piruvat dekarboksilaza (EC 4.1.1.1) jedan je od ključnih enzima u fermentativnom metabolizmu kvasca. To je prvi enzim koji u anaerobnim uvjetima dovodi do dekarboksilacije piruvata i nastanka acetaldehida. Svrha je ovog istraživanja bila razviti prikladnu metodu procjene aktivnosti piruvat dekarboksilaze različitih sojeva industrijskoga kvasca in situ. Sojevi vrsta Saccharomyces i Debaryomyces uzgojeni su u fermentacijskom mediju s 12 % glukoze. Procijenjena je aktivnost enzima u otopini stanica obrađenih digitoninom (koji povećava propusnost stanične membrane), uz dodatak natrijeva piruvata, pri temperaturi od 30 C. Razgradni metaboliti piruvata određeni su plinskom kromatografijom. Za procjenu optimalne aktivnosti piruvat dekarboksilaze in situ praćeni su razni parametri: tip i koncentracija supstrata, minimalna efektivna koncentracija digitonina, gustoća stanica, vrijeme reakcije i učinak pirazola (inhibitora alkohol dehidrogenaze). U rasponu koncentracije stanica kvasca od 1·10^7 do 1·10^8/mL zamijećena je linearna korelacija proizvedenog acetaldehida i gustoće stanica. Ustanovljeno je da je piruvat jedini specifični supstrat za enzim piruvat dekarboksilazu, te da je u prisutnosti 0,05 M natrijeva piruvata i 0,05 % digitonina enzimska reakcija imala linearni tijek prvih 20 minuta inkubacije. Tijekom inkubacije nije nastao etanol, pa je zaključeno da pirazol nije potreban u procjeni aktivnosti piruvat dekarboksilaze

Određivanje aktivnosti sukcinat-dehidrogenaze in situ u soju kvasca Saccharomyces cerevisiae reakcijom s plavom tetrazolijevom soli

A spectrophotometric method for determining succinate dehydrogenase (SDH) activity assay in azide-sensitive yeast Saccharomyces cerevisiae has been developed. The permeabilization of yeast cells by 0.05 % digitonin permitted to study yeast enzymatic activity in situ. The reduction of blue tetrazolium salt (BT) to blue tetrazolium formazan (BTf) was conducted in the presence of phenazine methosulphate (PMS) as an exogenous electron carrier, and sodium azide (SA) as an inhibitor of cytochrome oxidase (Cyt) pathway. Various factors such as type of substrate, BT concentration, cell number, temperature and time of incubation, and different Cyt pathway blockers were optimized. In earlier studies, dimethyl sulfoxide (DMSO) had been selected as the best solvent for extraction of BTf from yeast cells. The linear correlation between permeabilized yeast cell density and amount of formed formazan was evidenced in the range from 9·10^7 to 5·10^8 cells per sample solution. Below the yeast cell concentration of 10^7 the absorbance values were too low to detect formazans with good precision. This standarized procedure allows the estimation of SDH activity in whole cells, depending on vitality level of yeast populations. Significant increases of succinate dehydrogenase activities were observed in sequential passages as the result of the increase of activity of the strain and adaptation to cultivation conditions.U ovom je istraživanju razvijen spektrofotometrijski postupak određivanja aktivnosti sukcinat-dehidrogenaze u soju Saccharomyces cerevisiae osjetljivom na azid. Propusnost stanica kvasca za 0,05 %-tni digitonin omogućila je ispitivanje enzimske aktivnosti toga soja in situ. U prisutnosti fenazin-metosulfata (kao prijenosnika elektrona) i natrijeva azida (koji inhibira enzim citokrom-oksidazu) došlo je do redukcije plave tetrazolijeve soli u plavi tetrazolijev formazan. Optimirani su razni faktori koji utječu na reakciju, kao što su: vrsta supstrata, koncentracija plave tetrazolijeve soli, broj stanica kvasca, temperatura i vrijeme inkubacije, te razni blokatori citokrom-oksidaze. U prijašnjim je istraživanjima kao najbolje otapalo za ekstrakciju plavog tetrazolijevog formazana iz stanica kvasca odabran dimetilsulfoksid. U području od 9·10^7 do 5·10^8 stanica u otopini uzorka utvrđena je linearna korelacija između gustoće permeabiliziranih stanica kvasca i količine nastalog formazana. Pri količini manjoj od 10^7 stanica kvasca vrijednosti apsorbancije bile su premale za precizno utvrđivanje prisutnosti formazana. Ovaj standardni postupak omogućuje procjenu aktivnosti sukcinat-dehidrogenaze u cijelim stanicama, zavisno od vitalnosti kvasaca. Tijekom odvijanja pokusa ustanovljen je značajan porast aktivnosti sukcinat-dehidrogenaze zbog povećanja aktivnosti soja izazvane njegovom prilagodbom uvjetima uzgoja

Određivanje aktivnosti sukcinat-dehidrogenaze in situ u soju kvasca Saccharomyces cerevisiae reakcijom s plavom tetrazolijevom soli

A spectrophotometric method for determining succinate dehydrogenase (SDH) activity assay in azide-sensitive yeast Saccharomyces cerevisiae has been developed. The permeabilization of yeast cells by 0.05 % digitonin permitted to study yeast enzymatic activity in situ. The reduction of blue tetrazolium salt (BT) to blue tetrazolium formazan (BTf) was conducted in the presence of phenazine methosulphate (PMS) as an exogenous electron carrier, and sodium azide (SA) as an inhibitor of cytochrome oxidase (Cyt) pathway. Various factors such as type of substrate, BT concentration, cell number, temperature and time of incubation, and different Cyt pathway blockers were optimized. In earlier studies, dimethyl sulfoxide (DMSO) had been selected as the best solvent for extraction of BTf from yeast cells. The linear correlation between permeabilized yeast cell density and amount of formed formazan was evidenced in the range from 9·10^7 to 5·10^8 cells per sample solution. Below the yeast cell concentration of 10^7 the absorbance values were too low to detect formazans with good precision. This standarized procedure allows the estimation of SDH activity in whole cells, depending on vitality level of yeast populations. Significant increases of succinate dehydrogenase activities were observed in sequential passages as the result of the increase of activity of the strain and adaptation to cultivation conditions.U ovom je istraživanju razvijen spektrofotometrijski postupak određivanja aktivnosti sukcinat-dehidrogenaze u soju Saccharomyces cerevisiae osjetljivom na azid. Propusnost stanica kvasca za 0,05 %-tni digitonin omogućila je ispitivanje enzimske aktivnosti toga soja in situ. U prisutnosti fenazin-metosulfata (kao prijenosnika elektrona) i natrijeva azida (koji inhibira enzim citokrom-oksidazu) došlo je do redukcije plave tetrazolijeve soli u plavi tetrazolijev formazan. Optimirani su razni faktori koji utječu na reakciju, kao što su: vrsta supstrata, koncentracija plave tetrazolijeve soli, broj stanica kvasca, temperatura i vrijeme inkubacije, te razni blokatori citokrom-oksidaze. U prijašnjim je istraživanjima kao najbolje otapalo za ekstrakciju plavog tetrazolijevog formazana iz stanica kvasca odabran dimetilsulfoksid. U području od 9·10^7 do 5·10^8 stanica u otopini uzorka utvrđena je linearna korelacija između gustoće permeabiliziranih stanica kvasca i količine nastalog formazana. Pri količini manjoj od 10^7 stanica kvasca vrijednosti apsorbancije bile su premale za precizno utvrđivanje prisutnosti formazana. Ovaj standardni postupak omogućuje procjenu aktivnosti sukcinat-dehidrogenaze u cijelim stanicama, zavisno od vitalnosti kvasaca. Tijekom odvijanja pokusa ustanovljen je značajan porast aktivnosti sukcinat-dehidrogenaze zbog povećanja aktivnosti soja izazvane njegovom prilagodbom uvjetima uzgoja

Succinate Dehydrogenase Activity Assay in situ with Blue Tetrazolium Salt in Crabtree-Positive Saccharomyces cerevisiae Strain

A spectrophotometric method for determining succinate dehydrogenase (SDH) activity assay in azide-sensitive yeast Saccharomyces cerevisiae has been developed. The permeabilization of yeast cells by 0.05 % digitonin permitted to study yeast enzymatic activity in situ. The reduction of blue tetrazolium salt (BT) to blue tetrazolium formazan (BTf) was conducted in the presence of phenazine methosulphate (PMS) as an exogenous electron carrier, and sodium azide (SA) as an inhibitor of cytochrome oxidase (Cyt) pathway. Various factors such as type of substrate, BT concentration, cell number, temperature and time of incubation, and different Cyt pathway blockers were optimized. In earlier studies, dimethyl sulfoxide (DMSO) had been selected as the best solvent for extraction of BTf from yeast cells. The linear correlation between permeabilized yeast cell density and amount of formed formazan was evidenced in the range from 9·10^7 to 5·10^8 cells per sample solution. Below the yeast cell concentration of 10^7 the absorbance values were too low to detect formazans with good precision. This standarized procedure allows the estimation of SDH activity in whole cells, depending on vitality level of yeast populations. Significant increases of succinate dehydrogenase activities were observed in sequential passages as the result of the increase of activity of the strain and adaptation to cultivation conditions

Effect of Microencapsulation by Spray-Drying and Freeze-Drying Technique on the Antioxidant Properties of Blueberry (Vaccinium myrtillus) Juice Polyphenolic Compounds

Blueberry juice with high polyphenol concentration was spray- or freeze-dried using different coating materials: HP-β-cyclodextrin and β-cyclodextrin. The quality of the obtained powders was characterised by their anthocyanin content, total polyphenols and antioxidant capacity. SEM was used for monitoring structures and size (2–20 μm) of the microparticles. The losses of total phenolic compounds during spray-drying reached 76–78% on average, while these of anthocyanins about 57%. Freeze-dried powders showed better retention values of anthocyanins, which was about 1.5-fold higher than for the spray-dried counterparts. All blueberry preparations studied were characterised by very high radical scavenging activity