A simple cultural method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines

AIMS: The development of a simple and reliable procedure, compatible with routine use in wineries, for the presumptive detection of Brettanomyces/Dekkera from wine and wine-environment samples. METHODS AND RESULTS: The method of detection of these yeasts employs a selective enrichment medium. The medium contains glucose (10 g l(-1)) as carbon and energy source, cycloheximide (20 mg l(-1)) to prevent growth of Saccharomyces, chloramphenicol (200 mg l(-1)) to prevent growth of bacteria and p-coumaric acid (20 mg l(-1)) as the precursor for the production of 4-ethyl-phenol. After the inoculation with wine, the medium is monitored by visual inspection of turbidity and by periodic olfactive analysis. Contaminated wines will develop visible turbidity in the medium and will produce the 4-ethyl-phenol off-odour, which can be easily detected by smelling. CONCLUSIONS: A selective enrichment liquid medium was developed to differentially promote the growth and activity of Brettanomyces/Dekkera. The method is simple to execute, employing a simple-to-prepare medium and a periodic olfactive detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of the procedure make it particularly applicable in a wine-making environment thus presenting important advantages to the wine industry.     

Growth and fermentation behaviour of Brettanomyces/Dekkera yeasts under different conditions of aerobiosis

Brettanomyces/Dekkera yeasts grow in wine and their presence is often associated with spoiling activity. In this report, we investigated on the influence of different conditions of aerobiosis on growth and fermentation behaviour of these spoilage yeasts in wine. Results showed that in all conditions tested the Brettanomyces strain consumed all sugars, taking wine fermentation to completion. Strict-anaerobic conditions limited the growth of Brettanomyces. Both anaerobiosis (using a fermentation trap) and strict anaerobiosis did not negatively affect the principal by-products of fermentation whereas semi-anaerobiosis caused an increase of acetic acid, acetaldehyde and ethyl acetate that negatively affected the fermentation profile of resulting products.     

Kluyveromyces wickerhamii killer toxin: purification and activity towards Brettanomyces/Dekkera yeasts in grape must

Brettanomyces/Dekkera yeasts have been identified as part of the grape yeast flora. They are well known for colonizing the cellar environmental and spoiling wines, causing haze, turbidity and strong off-flavours in wines and enhancing the volatile acidity. As the general practices applied to combat Brettanomyces/Dekkera yeasts are not particularly appropriate during wine ageing and storage, a biological alternative to curtailing their growth would be welcomed in winemaking. In this study, we investigated the Kluyveromyces wickerhamii killer toxin (Kwkt) that is active against Brettanomyces/Dekkera spoilage yeasts. Purification procedures allowed the identification of Kwkt as a protein with an apparent molecular mass of 72 kDa and without any glycosyl residue. Interestingly, purified Kwkt has fungicidal effects at low concentrations under the physicochemical conditions of winemaking. The addition of 40 and 80 mg L(-1) purified Kwkt showed efficient antispoilage effects, controlling both growth and metabolic activity of sensitive spoilage yeasts. At these two killer toxin concentrations, compounds known to contribute to the 'Brett' character of wines, such as ethyl phenols, were not produced. Thus, purified Kwkt appears to be a suitable biological strategy to control Brettanomyces/Dekkera yeasts during fermentation, wine ageing and storage.     

Biotechnology, physiology and genetics of the yeast Pichia anomala

The ascomycetous yeast Pichia anomala is frequently associated with food and feed products, either as a production organism or as a spoilage yeast. It belongs to the nonSaccharomyces wine yeasts and contributes to the wine aroma by the production of volatile compounds. The ability to grow in preserved food and feed environments is due to its capacity to grow under low pH, high osmotic pressure and low oxygen tension. A new application of P. anomala is its use as a biocontrol agent, which is based on the potential to inhibit a variety of moulds in different environments. Although classified as a biosafety class-1 organism, cases of P. anomala infections have been reported in immunocompromised patients. On the other hand, P. anomala killer toxins have a potential as antimicrobial agents. The yeast can use a broad range of nitrogen and phosphor sources, which makes it a potential agent to decrease environmental pollution by organic residues from agriculture. However, present knowledge of the physiological basis of its performance is limited. Recently, the first studies have been published dealing with the global regulation of the metabolism of P. anomala under different conditions of oxygenation.     

Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts

Aims: The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. Methods and Results: A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4‐β‐glucosidase activity, but a strain dependent β‐glucanase activity was observed. Some Brettanomyces species did show 1,4‐β‐glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Conclusions: Pronounced exo‐β‐glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. Significance and Impact of the Study: The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo‐β‐glucanase activity. Higher activities can be found in Brettanomyces species with β‐glucosidase activity.