Proizvodnja pektinaza, aktivnih pri niskim temperaturama, pomoću psihrotolerantnih mikroorganizama

In winemaking, low temperatures are favourable for the production and retention of flavour and colour components, requiring the use of cold-active enzymes. For this reason, \u27psychrotolerant\u27 microorganisms have been isolated and selected based on their ability to produce pectinolytic enzymes with satisfactory activity at low temperatures. Different mature grape varieties with designation of origin were sampled from the region of San Rafael (Mendoza, Argentina), and pectinolytic bacterial, fungal and yeast strains were isolated. The pectinolytic activity was measured by cup-plate assay, quantification of released reducing sugars and viscosity reduction of pectin solution. Two bacteria (Bacillus sp. SC-G and SC-H) and two yeast strains were selected for their good pectinase activity at low temperatures. Among them, the strain with the highest activity, Bacillus sp. SC-H, was selected. According to their 16S rRNA profiles, Bacillus sp. SC-G and SC-H can be classified as members of Bacillus subtilis. Among the assayed techniques, the rotary evaporation was found to be the most appropriate to obtain enzymatic extracts with highest activity. The optimal conditions for the enzymatic activity were 30 °C and pH=5.0 for the concentrated extract, and 45 °C and pH=6.0 for the filtered supernatant. The concentrated extract presented good activity at 3 °C, confirming that it was a cold-active enzyme. Natural extraction and enzymatic preparation were used to extract pigments and polyphenols from Malbec grapes. Better results were obtained for the enzymatic extract with regard to index, shade, CIELab coordinates, CIELab colour differences and polyphenols (measured using Folin-Ciocalteu).Da bi se dobili što boji okus i boja vina, u vinarstvu se primjenjuju enzimi aktivni pri niskim temperaturama. Izolirani su psihrotolerantni mikroorganizmi sposobni da sintetiziraju pektinolitičke enzime aktivne pri niskim temperaturama. Zrelo grožđe različitih sorata s oznakom podrijetla prikupljeno je s područja San Rafael (Mendoz, Argentina), pa su izolirani pektinolitički sojevi bakterija, kvasaca i plijesni. Njihova je učinkovitost ispitana utvrđivanjem aktivnosti pektinaze na agaru (tzv. „cup-plate assay“), određivanjem udjela reducirajućih šećera što nastaju djelovanjem enzima i praćenjem smanjenja viskoznosti otopine pektina. Odabrana su dva soja bakterija (Bacillus sp. SC-G i SC-H) i dva soja kvasaca što luče pektinaze aktivne pri niskim temperaturama. Najveću aktivnost imao je soj Bacillus sp. SC-H. Određivanjem profila 16S rRNA utvrđeno je da su obje bakterije sojevi Bacillus subtilis. Najučinkovitiji enzimski ekstrakti dobiveni su u rotacijskom isparivaču. Optimalni uvjeti za aktivnost enzima bili su: 30 ºC i pH=5,0 za koncentrirani ekstrakt, te 45 ºC i pH=6,0 za filtrirani supernatant. Koncentrirani je ekstrakt imao dobru aktivnost i pri 3 °C, što potvrđuje njegovu učinkovitost pri niskim temperaturama. Prirodnom i enzimskom ekstrakcijom iz grožđa sorte Malbec izdvojeni su pigmenti i polifenoli. Enzimska je ekstrakcija bila učinkovitija, što potvrđuju indeks boje, CIELab koordinate, razlika boje prema CIELab sustavu i udjel polifenola određen Folin-Ciocalteu metodom