Identiﬁcation of yeast and bacteria involved in the mezcal fermentation of Agave salmiana
´ ´ P. Escalante-Minakata1, H.P. Blaschek2, A.P. Barba de la Rosa1, L. Santos1 and A. De Leon-Rodrıguez1
´ ´ ´ ´ ´ ´ ´ 1 Division de Biologıa Molecular, Instituto Potosino de Investigacion Cientıﬁca y Tecnologica, San Luis Potosı, Mexico2 Department of Food Science and Human Nutrition and Institute for Genomic Biology, University of Illinois, Urbana, IL, USA
Keywords 16S rDNA, fermentation, ITS rDNA, mezcal, phylogenetic analysis. Correspondence ´ ´ ´ ´ A. De Leon-Rodrıguez, Division de Biologıa ´ Molecular, Instituto Potosino de Investigacion ´ ´ Cientıﬁca y Tecnologica, Apartado Postal 3-74 ´ Tangamanga, 78231 San LuisPotosı, S.L.P., ´ Mexico. E-mail: firstname.lastname@example.org
Abstract Aims: To identify the yeast and bacteria present in the mezcal fermentation from Agave salmiana. Methods and Results: The restriction and sequence analysis of the ampliﬁed region, between 18S and 28S rDNA and 16S rDNA genes, were used for the identiﬁcation of yeast and bacteria, respectively. Eleven different microorganisms wereidentiﬁed in the mezcal fermentation. Three of them were the following yeast: Clavispora lusitaniae, Pichia fermentans and Kluyveromyces marxianus. The bacteria found were Zymomonas mobilis subsp. mobilis and Zymomonas mobilis subsp. pomaceae, Weissella cibaria, Weissella paramesenteroides, Lactobacillus pontis, Lactobacillus keﬁri, Lactobacillus plantarum and Lactobacillus farraginis. Conclusions: Thephylogenetic analysis of 16S rDNA and ITS sequences showed that microbial diversity present in mezcal is dominated by bacteria, mainly lactic acid bacteria species and Zymomonas mobilis. Pichia fermentans and K. marxianus could be micro-organisms with high potential for the production of some volatile compounds in mezcal. Signiﬁcance and Impact of the Study: We identiﬁed the community of bacteriaand yeast present in mezcal fermentation from Agave salmiana.
2007 ⁄ 1572: received 26 September 2007, revised 25 January 2008 and accepted 20 February 2008
Introduction Mezcal is a traditional Mexican distilled beverage produced from the fermented juices of the cooked agave ˜ plant core (‘pina’ in Spanish). The Agave salmiana is used in Mexico’s Altiplanoregion for the mezcal pro´ ´ duction (De Leon-Rodrıguez et al. 2006, 2008). The ‘pi˜ nas’ are cooked in stone ovens to hydrolyze the inulin into fructose. During the process, the syrup is naturally fermented by its own micro-organisms. The sugars are converted mainly into ethanol, esters and organic acids ´ ´ (De Leon-Rodrıguez et al. 2008). Therefore the taste and aroma of mezcal is determinedby the composition of a mixture of compounds produced during the fermentation and others coming from the agave plant (De ´ ´ Leon-Rodrıguez et al. 2006, 2008). The mezcal process is carried out by spontaneous fermentation, therefore to provide a homogeneous quality among batches is
recommended to initiate the fermentation using identical inocula. Many techniques have been previouslydeveloped for the identiﬁcation of micro-organisms. For instance, Lachance (1995) used microbiological methods to identify 13 different yeast species of the micro-organisms involved in the fermentation of tequila (spirit obtained from Agave tequilana weber). The molecular methods have allowed a rapid and accurate identiﬁcation of yeast and bacteria. Methods based on the analysis of restriction fragmentlength polymorphism (RFLP) of ribosomal RNA genes (5S, 5.8S, 18S and 26S rDNA) and the noncoding internal transcribed spacers (ITS) have been used by Esteve-Zarzoso et al. (1999). Flores-Berrios et al. (2006) used the ampliﬁed fragment length polymorphism (AFLP) technique to study the genetic yeast diversity, and showed the underlying relationship between the molecular proﬁles, strain origin...