Food Microbiology & Safety Control of Native Spoilage Yeast on Dealcoholized Red Wine by Preservatives Alone and in Binary Mixtures Marta S´ anchez-Rubio, Kamal Guerrouj, Amaury Taboada-Rodr´ ıguez, Antonio L´ opez-G´ omez, and Fulgencio Mar´ ın-Iniesta Abstract: In order to preserve a commercial dealcoholized red wine (DRW), a study with 4 preservatives and binary mixtures of them were performed against 2 native spoilage yeasts: Rhodotorula mucilaginosa and Saccharomyces cerevisiae. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for potassium sorbate, sodium benzoate, sodium metabisulfite and dimethyl dicarbonate (DMDC) were evaluated in DRW stored at 25 °C. MICs of potassium sorbate and sodium metabisulfite were 250 and 60 mg/kg, respectively for both target strains. However for sodium benzoate, differences between yeasts were found; R. mucilaginosa was inhibited at 125 mg/kg, while S. cerevisiae at 250 mg/kg. Regarding MFC, differences between strains were only found for sodium metabisulfite obtaining a MFC of 500 mg/kg for R. mucilaginosa and a MFC of 250 mg/kg for S. cerevisiae. Potassium sorbate and sodium benzoate showed the MFC at 1000 mg/kg and DMDC at 200 mg/kg. Regarding the effect of binary mixtures the Fractional Fungicidal Concentration Index (FFC i ) methodology showed that binary mixtures of 100 mg/kg DMDC/200 mg/kg potassium sorbate (FFC i = 0.7) and 50 mg/kg DMDC / 400 mg/kg sodium benzoate (FFC i = 0.65) have both synergistic effect against the 2 target strains. These binary mixtures can control the growth of spoilage yeasts in DRW without metabisulfite addition. The results of this work may be important in preserving the health of DRW consumers by eliminating the use of metabisulfite and reducing the risk of growth of R. mucilagosa, recently recognized as an emerging pathogen. Keywords: benzoate, dealcoholized wine, DMDC, metabisulfite, spoilage yeast Introduction The partial or total dealcoholization of wines to obtain low or free alcoholic drinks seems a good choice for market ex- pansion, mainly within the nutraceutical beverages segment (Belisario-S´ anchez and others 2009; Taboada-Rodr´ ıguez and others 2013). Both, low and free alcohol wine products represent a specialized but growing share of the wine industry (Demiglio and Pickering 2008). However, the microbial stability of these new beverages should be quite different in comparison with traditional wines due to the reduction of the ethanol content (Taboada-Rodr´ ıguez and others 2013). It is generally recognized that the spoilage yeasts most frequently found in food and beverages during the industrial processing or storage, are very tolerant to acid preservatives and/or osmotic stresses (Brul and Coote 1999). They can generally resist extreme conditions better than bacteria and they are often found in low pH products and products containing preservatives to such extent that bacteria cannot grow (Deak 2008). Yeasts are well known for their importance in wine production and by their role as spoilage microorganisms in wines and musts (Terrell and others 1993). In fact, in bottled dealcoholized wine the lower alcohol content JFDS-2016-2192 Submitted 12/30/2016, Accepted 6/23/2017. Authors S´ anchez-Rubio and Mar´ ın-Iniesta are with Grupo de Biotecnolog´ ıa de Alimentos (Food Biotechnology Group). Dept. de Tecnolog´ ıa de Alimentos, Nutrici´ on y Bromatolog´ ıa, Facultad de Veterinaria, Univ. de Murcia, Campus de Espinardo, 30100 Murcia, Spain. Author Guerrouj is with Inst. Sup´ erieur des Professions Infirmi` eres et des Tech- niques de Sant´ e, Al Hoceima, Morocco. Author Taboada-Rodr´ ıguez is with Desarrollos Bio-Tecno Alimentarios Ltd. (Debiotec) Virgen de las Maravillas 6, P01 B. 30009, Murcia, Spain. Author L´ opez-G´ omez is with Food Engineering and Agricultural Equipment Dept., Univ. Polit´ ecnica de Cartagena, Paseo Alfonso XIII, 48, 30203 Cartagena, Spain. Direct inquiries to author Mar´ ın-Iniesta (E-mail: fmarin@um.es). and the sugars added, to enhance its sensory attributes, can fa- cilitate re-fermentative processes (Taboada-Rodr´ ıguez and others 2013). These are carried out by different species of yeasts and negatively affect wine sensory quality (Fleet 2003; Comuzzo and others 2006). Sulfur dioxide and sulfiting agents as sodium (potassium) metabisulfite are widely used during the different steps of wine- making and storage, for their antimicrobial and their antioxidant properties. However the use of these additives is strictly con- trolled due to the risks for human health derived from their consumption. Even at low doses considerable percentage of con- sumers show intolerance or high sensitivity to sulfites (Mischek and Krapfenbauer-Cermak 2012; Garaguso and Nardini 2015; Guerrero and Cantos-Villar 2015). Some weak acids or their salts (sorbic and benzoic acid) and dimethyl dicarbonate (DMDC) could be a good alternative to sulfur dioxide and sulfiting agents in order to preserve dealco- holized red wines. In fact, the main preservatives used in the wine industry are sulfur dioxide and sorbate/benzoate salts (Divol and others 2005; Barata and others 2008). Although DMDC is an- other preservative legally authorized whose use is less common, it is starting to spread in the beverage industry (Costa and others 2008; Nieto-Rojo and others 2015; Zuehlke and others 2015). The possible mechanisms of inhibition of these preservatives have been explained (Brul and Coote 1999; Thomas 2004) and some recent studies make deeper the knowledge about them (Costa and others 2008; Orij and others 2009; Stratford and others 2009; Nieto-Rojo and others 2015). DMDC is different in comparison with others preservatives, because of the mechanism of action, the way for adding to bever- ages, and the quick and short effectiveness at low concentrations. Only during decomposition phase that DMDC has microbicide C  2017 Institute of Food Technologists R  doi: 10.1111/1750-3841.13817 Vol. 00, Nr. 0, 2017  Journal of Food Science 1 Further reproduction without permission is prohibited