CARATTERIZZAZIONE MOLECOLARE E FISIOLOGICA DI CEPPI DI SACCHAROMYCES CEREVISIAE ISOLATI DA FERMENTAZIONI SPONTANEE DI NEBBIOLO

The purpose of this study was the selection of autochthonous strains for production of a starter culture for the fermentation of Nebbiolo vine for Barolo. Spontaneous fermentations, conduced in small scale with two modalities (spontaneous and with the use of ¿pie de cuve¿) was the source for the sampling of about 100 strains of S. cerevisiae isolated on plates and identified with a PCR method. For the genetic characterization, we used a RAPD PCR molecular technique (Random Amplified Polymorphic DNA) with two different primers (M13 and D8635). Each strain characterized was tested for its physiological characteristics: the capacity of growth at different temperatures and in different conditions like the concentration of ethanol and sulphur dioxide. Also the production of hydrogen sulphide was tested. The growth was tested at 20°C, 25°C, 30°C and 37°C. Ethanol was tested at concentrations of 10%, 12%, 14%, 16% and sulphur dioxide at concentrations of 50 mg/l, 100 mg/l, 150 mg/l, 200 mg/l. Cluster analysis of elecrtophoretic profiles together with the results of the physiological tests identified the best candidates for the constitution of the enological starter. By the cluster analysis was shown that there is a close correlation between strains isolated on the same sampling day. The difference between strains, isolated the same day of each type of fermentation was minimal. After molecular and physiological characterization, was possible to identify a large number of strains that had medium characteristics, few strains totally unfit and some strains that could have the right characteristics for going to the next test: microvinifications. A lot of strains that had medium characteristic (good growth with sulphur dioxide and ethanol) had the problem of the large production of hydrogen sulphide. Many strains had excellent capacity of growing with ethanol or sulphur dioxide but few of those (4) had both of the characteristics. Three of those are able to produced medium levels of hydrogen sulphide and only one showed also a low production of this molecule. This strain could represent the most interesting subject for the microvinification.

Nella selezione dei ceppi ideali per l'utilizzo come starter, la determinazione di particolari caratteristiche fisiologiche rappresenta un parametro fondamentale. Dopo la determinazione delle caratteristiche di crescita (capacità e velocità) in condizioni di temperatura diverse, a diverse concentrazioni di etanolo e di anidride solforosa e dopo la determinazione della produzione di acido solfidrico su Biggy Agar, è stato possibile individuare alcuni ceppi dotati di buone caratteristiche generali, pochi con caratteristiche fortemente negative ed alcuni che possono rappresentare ottimi riferimenti per progredire con la selezione di starter mediante prove di microvinificazione. Molti dei ceppi dotati di caratteristiche genericamente positive, ovvero in grado di crescere in presenza di etanolo e anidride solforosa ed a temperature diverse, ma senza manifestare particolari velocità di crescita, sono caratterizzati da una grande produzione di acido solfidrico. Molti ceppi presentano un'ottima crescita in presenza di etanolo o di solforosa, ma solo 4 ceppi presentano le 2 caratteristiche contemporaneamente. Fra questi 4 ceppi, 3 sono caratterizzati da una media produzione di acido solfidrico ed uno da una produzione di livello più basso . Quest'ultimo potrebbe costituire il ceppo di maggiore interesse per successive prove di microvinificazione.