Roberto Zironi

Molecular Detection and Identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in Spoiled Wines

ABSTRACT In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B. anomalus. Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from other Brettanomyces spp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B. bruxellensis and B. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection of B. bruxellensis and B. anomalus in wines suspected to be spoiled by Brettanomyces spp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.

Volatile metabolites produced in wine by mixed and sequential cultures of Hanseniaspora guilliermondii or Kloeckera apiculata and Saccharomyces cerevisiae

SummarySecondary products in wines obtained by pure, mixed and sequential cultures of Saccharomyces cerevisiae, Hanseniaspora guilliermondii or Kloeckera apiculata were studied. Consistent differences in the composition were determined in wines fermented by sequential cultures. When S. cerevisiae was added to musts partially fermented by apiculate yeasts, its metabolism was significantly affected. In particular it synthesized high amounts of n-propanol and metabolized high quantities of acetoin, produced by apiculate yeasts

Glycerol and other fermentation products of apiculate wine yeasts.

Ninety‐six strains of apiculate wine yeasts were studied for their ability to produce glycerol, acetaldehyde, ethyl acetate, sulphur dioxide and hydrogen sulphide in synthetic medium. Hanseniaspora guilliermondii produced smaller quantities of glycerol, acetaldehyde and hydrogen sulphide than Kloeckera apiculata, whereas the production of ethyl acetate and sulphur dioxide was found to be similar. Strains characterized by different capacities and properties were found for both species. The existence of apiculate strains differing in secondary compound production is of technological interest, as these yeasts constitute potential flavour producers. Selected strains of apiculate yeasts might favour an enhanced flavour formation and yield desirable characteristics to the final product.

Modified Nondestructive Colorimetric Method To Evaluate the Variability of Oxygen Diffusion Rate through Wine Bottle Closures

Some modifications to a previous nondestructive colorimetric method that permits evaluation of the oxygen diffusion rate through wine closures were proposed. The method is based on the reaction of indigo carmine solution with oxygen and the tristimulus measurement of the consequent color change. Simplified preparation and measurement procedures were set up, allowing the analysis of a large number of samples simultaneously. The method was applied to the evaluation of the variability within the lot of 20 different types of stoppers (synthetic, produced by molding, and natural cork). The closures were tested at a storage temperature of 26 degrees C. With regard to oxygen permeability, the natural cork stopper showed a low homogeneity within the lot, especially during the first month after bottling, whereas the synthetic closure showed a greater steadiness in the performance. The limits of the colorimetric method were also analyzed, and three possible causes of degradation of the indigo carmine solution were identified: oxygen, light, and heat.

Antioxidant properties of different products and additives in white wine.

Different winemaking products (ascorbic acid, glutathione, yeast lees and a yeast autolysate) were tested in comparison with sulphur dioxide, concerning radical scavenging activity (measured by DPPH assay), oxygen consumption capacity and ability to reduce wine colour and predisposition to browning. Trials were performed in white wines and model solution. SO2 was the most active in reducing wine colour development. Fresh lees and ascorbic acid were very effective in oxygen and free radical scavenging, but they both induced browning during wine storage, the former, by releasing phenolic compounds. Glutathione was also able to scavenge DPPH in wine, but less effective against oxygen, and it induced browning during storage. Surprisingly, the yeast derivative preparation was the treatment that behave more similarly to sulphiting; it was very active in scavenging DPPH, and, even without modifying oxygen consumption rate, it protected quite well wine colour over an 8months storage time.

Effect of Different Lysis Treatments on the Characteristics of Yeast Derivatives for Winemaking

The effects of three preparation techniques on the oenological properties of a yeast autolysate were investigated: enzymatic autolysis, thermolysis, and the combination of a slow freezing-defrosting and mechanical disruption were carried out on a commercial formulation of active dry yeasts (Saccharomyces cerevisiae). The powders obtained by freeze drying, were characterized: volatile compounds were analyzed by SPME-GC with mass spectrometric (MS) and olfactometric detection (O); the release of colloids in winelike solution was studied by SDS-PAGE and size exclusion chromatography (SEC). Finally, the effects of the powders addition on the aroma composition of a white wine were investigated by SPME-GC-MS, SPME-GC-O, and sensory evaluation. The products obtained were quite different from each other. In particular, enzymatic autolysis led to higher contents of nonglycosilated soluble proteins in the powders and determined a higher retention of wine aroma compounds. On the contrary, thermal autolysate was richer in glycoproteins, and it was able to increase the wine aroma intensity; nevertheless, in the wines treated with such preparation, a slight yeastlike olfactory note was perceived.

Interactions between yeast autolysates and volatile compounds in wine and model solution

The addition of a commercial yeast autolysate to a model solution of five typical wine aroma compounds (ethyl octanoate, linalool, 2-phenylethanol, β-ionone and octanoic acid) was investigated considering different variables, such as temperature, pH and the presence of highly concentrated natural volatile substances in wine (e.g. 3-methyl-1-butanol). The interactions of such compounds with both yeast walls and released colloids were studied using gas chromatography, with liquid-liquid extraction and solid-phase microextraction. The results were compared with those obtained by adding the commercial product to a white table wine, spiked with the five standard compounds. The data confirmed that yeast walls mainly bind less polar molecules: their loss in synthetic medium seemed to increase at higher pH values. Temperature and pH affected differently the interactions between yeast colloids and volatile compounds in wine and model solution: in complex solutions (as the addition of 3-methyl-1-butanol demonstrated) the interaction mechanisms could be influenced by competitive or other matrix-related effects, which can reduce the binding of single compounds, or even enhance their volatility.

Biotechnological Strategies for Controlling Wine Oxidation

Apart from the controversial positive effects of moderate wine consumption on human health, wine antioxidant capacity plays a key role in winemaking technology. From juice extraction to bottle storage, oxygen management is one of the most critical points for making quality wines. In the past, the protection of juice and wine from oxidations was based on the sole use of sulfur dioxide; more recently, the toxicity and the allergenic potential of this additive, together with the increased knowledge on wine oxidation mechanisms, have given rise to new biotechnological approaches and producing trends, leading to a significant reduction of sulfites in winemaking. The aim of this paper is to review the oxidation mechanisms of grape juice and wine and to discuss the opportunities to reduce as much as possible sulfur dioxide addition by a proper management of alcoholic and malolactic fermentation and by the supplementation of some important yeast nutritional factors (e.g., thiamine). The use of natural antioxidants complementing the activity of sulfites (i.e., ascorbic acid, glutathione, yeast lees, and yeast derivatives) is also discussed.

Influence of specific fermentation conditions on natural microflora of pomace in "Grappa" production

As reported in the European Community regulation, grappa is a spirit beverage made in Italy from marc that has been steam distilled or distilled after the addition of water. Grape marc from red grapes has already undergone alcoholic fermentation with the must and can be distilled immediately. Grape marc from white grapes does not contain ethanol but contains sugars that are fermented by spontaneous anaerobic fermentation during a storage period. The characteristic aroma of grappa consists of a large number of volatile compounds, which arise from various sources, the most important of which is yeast. Very few studies have been undertaken to characterize the natural populations of yeast during the fermentation of grape marc. The goal of this study was to understand how different pHs, temperatures and yeast starter cultures affect the growth and dynamics of yeast species involved in pomace fermentation, which could be the basis for improving the final quality of grappa production. We found that a temperature of 15°C has the greatest effect on improving the quality of the product. Unfortunately, due to the solid state of the grape marc and the impossibility of its mixing, it appears that acidification and the addition of yeast starter cultures during the silage period are not effective.

Recent advances in the process of flotation applied to the clarification of grape musts

Abstract Flotation applied to grape must and fruit juice clarification has been found during the last few years to be of considerable applied interest. The system allows the continuous treatment of a large quantity of raw must. It is possible to guide the liquid—solid separation up to the degree of limpidity desired, rationalising the use of fining agents. This process has been used together with the techniques of hyperoxygenation and cross‐flow filtration of must, and can thus be considered innovative both with regard to the process of elaborating stable wines and the production of new products, for example wines without sulphur dioxide.