Dulce M. D´az-Monta˜ o a,∗ , Marie-Line D´ lia b , ı n e a , Pierre Strehaiano b Mirna Estarr´ n-Espinosa o
CIATEJ-Centro de Investigaci´ n y Asistencia en Tecnolog´a y Dise˜ o del Estado de Jalisco, Av. Normalistas 800, o ı n Col. Colinas de la Normal, 44270 Guadalajara, Jalisco,Mexico b Laboratoire de G´ nie Chimique-CNRS UMR 5503, Ecole Nationale Sup´ rieure des Ing´ nieurs en Arts Chimiques et Technologiques, e e e Site de Basso Cambo BP 1301, 5 rue Paulin Talabot, 31106 Toulouse Cedex 01, France
Abstract Five yeast strains isolated from agave juice were studied for their fermentative and aromatic capacity. The experiments were performed using agave juicesupplemented with ammonium sulphate, as is commonly done in tequila distilleries. Three strains classiﬁed as Saccharomyces cerevisiae showed high biomass and ethanol production, as well as higher ethanol tolerance than those classiﬁed as Kloeckera africana and Kloeckera apiculata, which showed scarce growth. The results suggest that Kloeckera strains were affected by nutritional limitation and/or toxiccompounds present in agave juice. Agave juice analyses showed a lower amino acid content than those reported in grape juice. S. cerevisiae strains produced predominantly amyl and isoamyl alcohols, n-propanol, 2-phenyl ethanol, succinic acid, glycerol, methanol, isoamyl acetate, ethyl hexanoate, acetaldehyde and isobutanol, whereas Kloeckera strains showed a high production of acetic acid, 2-phenylethyl acetate and ethyl acetate. The methanol concentration was signiﬁcantly different among the yeasts studied. The diversity between three S. cerevisiae strains were higher for the aromatic proﬁle than for genetic level and kinetic parameter. On the other hand, the diversity of Kloeckera yeasts were lower than Saccharomyces yeasts even when belonging to two different species.
Keywords: Agave;Saccharomyces; Kloeckera; Alcoholic fermentation; Tequila
1. Introduction Tequila is a Mexican alcoholic beverage distilled from the fermentated juice of cooked Agave tequilana Weber (blue variety). Production is strictly regulated such that only beverages produced from A. tequilana Weber (blue variety) cultivated in a protected region of Mexico can be labeled with the guarantee of origin (NormaOﬁcial Mexicana, 2005). The tequila process involves multiple steps: upon harvest, fermentable sugars are obtained from heads of the agave plant by steaming, milling and pressing. During the steaming process, the polysaccharides (inulin) are hydrolyzed into a mixture of sugars which mainly consist of fructose. Besides the hydrolysis of inulin during the cooking process of agave, many volatile com-Corresponding author. Tel.: +52 333 3 45 52 00x1305; fax: +52 333 3 45 52 45. E-mail address: email@example.com (D.M. D´az-Monta˜ o). ı n
pounds mainly Maillard compounds are produced, principally 5-hydroxymethyl furfural . In some tequila distilleries, fermentation occurs spontaneously while in others, the agave juice is inoculated using commercial or indigenous yeast cultures, oftenSaccharomyces cerevisiae. In spontaneous juice fermentation, Lachance  reported that Torulaspora delbrueckii, Hanseniaspora spp., and Kluyveromyces marxianus progressively gave way to S. cerevisiae, Zygosaccharomyces bailii, Candida milleri and Brettanomyces spp. After fermentation, the agave juice fermented is distilled twice and then diluted to obtain a ﬁnal alcohol content of 38% (v/v). Theproduct is aged in white oak barrels for 2–12 months to obtain “reposado” or “a˜ ejo” n tequila, respectively. A complete description of the production process was reported by Cede˜ o . n During alcoholic fermentation, the yeast cells produce primarily ethanol and CO2 as well as many ﬂavor compounds as secondary products. Existing studies report the aromatic proﬁle of tequila [4–6]. Benn...