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The aims of this work were to characterize the fermentation process of mezcal from San Luis Potosi, México and identify the yeasts present in the fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the first fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were identified, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identified yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identifications were confirmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, confirming the results obtained with classic techniques.

Mezcal is a traditional iconic Mexican distilled beverage obtained from varied species of agaves. Regardless of the area of production, the process always consists of five stages: harvesting the agaves, cooking, crushing, fermentation, and distillation. It is produced in a large area of Mexican territory, a large part of which is protected by the Denomination of Origin mezcal (DOM). Over time, the word mezcal has evolved from a generic name to a more specific term used to describe the agave-distilled beverages produced in the territory protected by the DOM under the Mexican official standard NOM-070-SCFI-2016 which defined Mezcal as a “Mexican distilled alcoholic beverage, 100% from maguey or agave, obtained by distillation of fermented juices with spontaneous or cultivated microorganisms, extracted from mature heads of maguey or cooked agaves, harvested in the territory covered by the DOM.” In the last 10 years, official production has increased, from <1 million liters in 2011 to almost 8 million liters. This substantial increase in production puts a lot of pressure on resources, in particular raw material, as part of the production is obtained from wild agave. On the other hand, it exposes tradition at risk by increasing production by modernizing production processes and sacrificing the artisanal aspect of this production. We consider appropriate to address the issue of sustainability in this context of great tradition and growing market demand. The article presents the relevant aspects of mezcal production, highlighting some particularities specific to certain production areas, it also addresses the problem of the official standard. A broad discussion is presented on the sustainability of artisanal processes, and the main points to be taken care of in this framework. Additionally, some elements considered as fundamental in the perspective of the design of a sustainable artisanal distillery are described. In summary, this article aims to review the current state of mezcal production, how sustainability may be addressed in a very artisanal process and what are the challenges of the production chain to satisfy an increase in demand without sacrificing the tradition and culture related to this iconic Mexican beverage.

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Aims:The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. Methods and Results: A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. Conclusions: A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. Significance and Impact of the Study: This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.