ORIGINAL ARTICLE Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment V. Fabrizio 1 , I. Vigentini 2 , N. Parisi 3 , C. Picozzi 2 , C. Compagno 2 and R. Foschino 2 1 Centro di Ricerca, Formazione e Servizi della Vite e del Vino, Riccagioia S.C.p.A., Torrazza Coste (PV), Italy 2 Department of Food, Environmental and Nutritional Sciences, Universit a degli Studi di Milano, Milan, Italy 3 Co.Pro.Vi. Societa’ Cooperativa, Casteggio (PV), Italy Significance and Impact of the Study: Brettanomyces/Dekkera bruxellensis is the main yeast involved in red wine spoilage that occurs during ageing in barrel, generating considerable economic losses. Current sanitization protocols, performed using different chemicals, are ineffective due to the porous nature of the wood. The thermal inactivation of D. bruxellensis cells by hot water treatment proves to be effica- cious and easy to perform, provided that the holding time at the killing temperature takes into account the filling time of the vessel and the time for the heat penetration into the wood structure. Keywords Brettanomyces, decimal reduction time, Dekkera, thermal inactivation, wine, z-value. Correspondence Roberto Foschino, Department of Food, Envi- ronmental and Nutritional Sciences, Universit a degli Studi di Milano, Via G. Celoria, 2-20133 Milan, Italy. E-mail: roberto.foschino@unimi.it 2015/0599: received 24 March 2015, revised 6 May 2015 and accepted 7 May 2015 doi:10.1111/lam.12444 Abstract Cell suspensions of four Dekkera bruxellensis strains (CBS 2499, CBS 2797, CBS 4459 and CBS 4601) were subjected to heat treatment in deionized water at four different temperatures (55Á0, 57Á5, 60Á0 and 62Á5°C) to investigate their thermal resistance. The decimal reduction times at a specific temperature were calculated from the resulting inactivation curves: the D-values at 55Á0°C ranged from 63 to 79Á4 s, at 57Á5°C from 39Á6 to 46Á1 s, at 60Á0°C from 19Á5 to 20Á7 s, at 62Á5°C from 10Á2 to 13Á7 s. The z-values were between 9Á2 and 10Á2°C, confirming that heat resistance is a strain-dependent character. A protocol for the sanitization of 225 l casks by immersion in hot water was set up and applied to contaminated 3-year-old barrels. The heat penetration through the staves was evaluated for each investigated temperature by positioning a thermal probe at 8 mm deep. A treatment at 60°C for an exposure time of 19 min allowed to eliminate the yeast populations up to a log count reduction of 8. Introduction The negative role of Brettanomyces bruxellensis (the ana- morph of Dekkera bruxellensis species) has recently assumed increasing importance in winemaking since this spoilage yeast is considered the main hazard for red wines aged in wood (Suarez et al. 2006; Schifferdecker et al. 2014). The current incidence of the defect is not easy to be determined, partly because some oenologists are reluc- tant to admit the problem and partly because early con- tamination is difficult to detect. However, analytical data for Italian wines (Agnolucci et al. 2009; Campolongo et al. 2010) estimate that about 15% of the products is involved in the phenomenon, as the levels of volatile phe- nols in the investigated samples were higher than the thresholds of perception. Actually, the so-called ‘Brett’ defect in wine originates by the strain-specific capacity of B. bruxellensis (Vigentini et al. 2008) to produce volatile phenols through sequential enzymatic steps (Oelofse et al. 2008) in which the hydroxy-cinnamic acids, that are nat- urally present in the grape berries, are transformed in vinyl-phenols and ethyl-phenols. In particular, 4-ethyl- phenol and 4-ethyl-guaiacol give rise to disagreeable aro- mas, classified as ‘animal’ or ‘pharmaceutical’ notes, when their concentrations in wine are higher than 230 and 50 lgl À1 respectively (Suarez et al. 2006; Benito et al. 2009). Godoy et al. (2008) and Tchobanov et al. (2008) firstly and partially have purified a coumarate decarboxyl- ase and a vinyl-phenol reductase; the role of these enzymes was then confirmed through the experiments of Letters in Applied Microbiology 61, 186--191 © 2015 The Society for Applied Microbiology 186 Letters in Applied Microbiology ISSN 0266-8254