Francisco Piñaga

Studies on acetate ester production by non-Saccharomyces wine yeasts

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.

Acetate ester formation in wine by mixed cultures in laboratory fermentations.

Two non-Saccharomyces wine yeast strains, Hanseniaspora guilliermondii 11104 and Pichia anomala 10590, selected as good producers of acetate esters when grown on synthetic microbiological medium, have been tested in wine fermentations as mixed cultures together with Saccharomyces cerevisiae. Wines produced using mixed cultures showed levels of acetaldehyde, acetic acid, glycerol and total higher alcohols within the ranges described for wine, whereas an increase in acetate ester concentrations was found. Ethyl acetate was the main ester produced, and isoamyl acetate and 2-phenylethyl acetate made up the next largest group of ester compounds in the wines analysed. H. guilliermondii 11104 was found to be a strong producer of 2-phenylethyl acetate in both pure and mixed cultures whereas S. cerevisiae was the best producer of ethyl esters. Mixed cultures did not influence ethyl ester levels at all.

Aroma improving in microvinification processes by the use of a recombinant wine yeast strain expressing the Aspergillus nidulans xlnA gene.

A recombinant wine yeast strain has been constructed expressing the gene coding for beta-(1,4)-endoxylanase from Aspergillus nidulans under the control of the yeast actin gene promoter. The resulting recombinant strain is able to secrete active xylanase enzyme into the culture medium. Wines obtained by microvinification with the control and the recombinant wine yeast strain did not differ in their physicochemical characteristics although an increase in fruity aroma was organoleptically detected in the wine produced by the recombinant yeast. Also, an increase in the concentration of some esters, higher alcohols and terpenes was observed in the case of the recombinant strain.

Measurement of alcohol acetyltransferase and ester hydrolase activities in yeast extracts

Abstract A method has been developed for measurement in vitro of the simultaneous activities alcohol acetyltransferase (AATase) and ester hydrolase (EHase) in yeast extracts taking into account the possibility of interaction between the two opposite activities or the rapid inactivation of the AATase activity. A mathematical model, including as parameters a first order kinetic constant corresponding to the EHase activity and the inactivation constant of AATase, is proposed for the evaluation of AATase activity. To determine ester concentrations, the Headspace-SPME-GC technique has been used. The method has been successfully applied to three yeast strains belonging to the species Pichia anomala, Pichia heedii and Saccharomyces cerevisiae and the corresponding parameters for AATase and EHase have been calculated.

Cell-Wall Degrading Enzymes in the Release of Grape Aroma Precursors

The potential use of cell-wall degrading enzymes, such as xylanase and endoglucanase, for the release of nonvolatile aroma precursors during maceration of Chenin grapes was evaluated. Aroma precursor levels in must from enzymatically macerated grapes were greater than those detected in must obtained from control grapes macerated in the absence of added enzymes. Similar observations were also made regarding the terpene content (specially •-terpineol and nerol) of must aglycon moities. Fermentation of musts from non-enzymatically and enzymatically macerated grapes resulted in wines of similar chemical characteristics, although a clear increase in fruity aroma was sensorially detected in wines derived from enzymatically macerated grapes.

Heterologous production in Saccharomyces cerevisiae of different Aspergillus nidulans xylanases of potential interest in oenology

Three Aspergillus nidulans endo-β-(1,4)-xylanases (X22, X24 and X34) have been produced in Saccharomyces cerevisiae laboratory strains. In fermenter cultivation, the resulting strains were able to secrete the heterologous xylanases when grown in minimal medium containing glucose as carbon source, yielding production levels between 0·5 and 1·0 U ml−1. From such a culture, the xylanases were purified in a single chromatographic step and then physico-chemically characterised. The yeast derived enzymes had the same enzymatic and biochemical properties as the fungal xylanases (ie molecular weights, pI values, pH and temperature optima, Km Michaelis constants and pH and temperature stabilities). The heterologously produced enzymes retained maximum activity in the presence of 10% (w/v) glucose or 10% (w/v) fructose, concentrations normally found in musts. However, ethanol at 12% (v/v) caused inhibitions of 20% for X22 and X24 and 48% for X34.