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

Selection of Psychrotolerant Microorganisms Producing Cold-Active Pectinases for Biotechnological Processes at Low Temperature

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, 'psychrotolerant' 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)

Impact of antagonistic yeasts from wine grapes on growth and mycotoxin production by Alternaria alternata

Aims: Alternaria alternata is a major contaminant of wine grapes, meaning a health risk for wine consumers due to the accumulation of toxic metabolites. To develop a successful biofungicide, the effectiveness of epiphytic wine grape yeasts against A. alternata growth and toxin production was assessed in vitro under temperature and aW conditions that simulate those present in the field. Methods and Results: The effect of 14 antagonistic yeasts was evaluated on growth and alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) production by three A. alternata strains in a synthetic medium with composition similar to grape (SN) at three temperatures (15, 25 and 30°C). All Metschnikowia sp. yeast strains evaluated completely prevented A. alternata growth and mycotoxin production at all temperatures in SN medium. Meanwhile, the growth inhibition exerted by Starmerella bacillaris yeast strains was higher at 30°C, followed by 25 and 15°C, being able to show a stimulating or inhibiting effect. Hanseniaspora uvarum yeast strains showed a growth promoting activity higher at 15°C, followed by 25 and 30°C. Even at conditions where A. alternata growth was stimulated by the S. bacillaris and H. uvarum yeasts, high inhibitions of mycotoxin production (AOH, AME and TA) were observed, indicating a complex interaction between growth and mycotoxin production. Conclusion: There is a significant influence of temperature on the effectiveness of biocontrol against A. alternata growth and mycotoxin production. Metschnikowia sp. strains are good candidates to compose a biofungicide against A. alternata. Significance and Impact of the Study: Among the different antagonistic yeasts evaluated, only Metschnikowia sp. strains were equally effective reducing A. alternata growth and mycotoxin at different temperatures underlining the importance of considering environmental factors in the selection of the antagonists.Fil: Prendes, Luciana Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Aplicadas a la Industria; ArgentinaFil: Merín, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Aplicadas a la Industria; ArgentinaFil: Zachetti, Vanessa Gimena Lourdes. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigación en Micología y Micotoxicología. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación en Micología y Micotoxicología; ArgentinaFil: Pereyra, Anabela. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigación en Micología y Micotoxicología. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación en Micología y Micotoxicología; ArgentinaFil: Ramirez, Maria Laura. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigación en Micología y Micotoxicología. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación en Micología y Micotoxicología; ArgentinaFil: Morata de Ambrosini, Vilma I.. Universidad Nacional de Cuyo. Facultad de Ciencias Aplicadas a la Industria; Argentin