Abstract
Tequila and mezcal are both traditional Mexican liquors that are produced from cooked Agave spp. must fermentation and usually rely on spontaneous or pure Saccharomyces cerevisiae strain inoculation. In order to contribute to the rational selection of yeast starters for tequila and mezcal productions, we tested a collection of 25 yeasts originally isolated from mezcal musts, spanning 10 different yeast species. These strains were first characterized in a semi synthetic medium (labeled as M2, having 90 g/L fructose and 10 g/L glucose of initial hexoses) at 48 h of culture, observing a differential pattern in the consumption of sugars and productivity. Selected Saccharomyces strains left around 10 g/L of fructose and showed higher fermentation performance. However, some non-Saccharomyces strains, specifically from Torulospora (Td), Kluyveromyces (Km), and Zygosaccharomyces (Zb) genera, consumed almost all the sugar (i.e., Km1Y9 with <5 g/L) and had a high productivity of ethanol. In general, all Saccharomyces strains presented a high production of ethyl-butyrate, ethyl-decanoate, and ethyl-hexanoate with peaks of 10, 38, and 3 μg/L, respectively. In addition, some Kluyveromyces and Torulospora strains showed a high production of phenyl ethyl acetate (i.e., Km1D5 with up to 1400 μg/L); isoamyl acetate (i.e., Km1D5 and Td1AN2 with more than 300 μg/L), and hexyl acetate (i.e., Td1AN2 with 0.3 μg/L). Representative strains of the most productive genera (Saccharomyces, Torulospora, and Kluyveromyces) were selected to evaluate their fermentative performance and survival in a mixed culture on a medium based on Agave tequilana must, and their population kinetics was characterized using specific fluorescent in situ hybridization (FISH) probes in a qualitative and semi-quantitative analysis during fermentation. We observed that the mixture ratios of 0.1:1:1 or 1:1:1 (Saccharomyces:Kluyveromyces:Torulospora), maintained good fermentation productivities, with alcohol yields above 0.45 g/g, and allowed a high survival rate of the non-Saccharomyces strains during the fermentation process. Finally, mixed inoculum fermentations on A. tequilana must medium, including different Saccharomyces strains and the finally selected Torulospora and Kluyveromyces strains, showed the best production parameters in terms of ethanol, carbon dioxide, glycerol, and acetic acid values, as well as improved volatile metabolite profiles as compared to the pure cultures. All these data were used to propose a methodology of selection of strains to be used as a pure or mixed starter for tequila and mezcal fermentations, with high primary metabolite productivity and desired aromatic profile.
Highlights
The aroma and flavor of alcoholic beverages obtained from Agave spp. plants are probably the most complex in the liquor world, due to the fact that they are the result of many volatile compounds that may be contained in the raw material and that vary among Agave species (Vera Guzmán et al, 2009)
All tested yeast species were able to produce ethanol, with S. cerevisiae strains being the most productive, ranging from 25 to 30 g/L as expected, but some of the non-Saccharomyces strains were able to produce around 30 g/L such as the K. marxianus (Km1Y9), T. delbrueckii (Td1AN9) as well as Z. bailii (Zb3Y1) and C. lusitaniae (Cl4Y4) during this fermentation time
We aimed to characterize the application potential of some of the yeasts found in mezcal from Tamaulipas (De la Torre-González et al, 2017), first by pure strain inoculation on a semi synthetic medium resembling Agave must hexose initial concentrations, and we found that all the strains, but mainly Saccharomyces, Torulospora, Kluyveromyces, Clavispora, and Zygosaccharomyces were the ones with the best fermentative performances, and were good producers of volatile compounds, as previously observed for some of these genera (Arellano et al, 2008; Lopez-Alvarez et al, 2012; PáezLerma et al, 2013; Nolasco-Cancino et al, 2018)
Summary
The aroma and flavor of alcoholic beverages obtained from Agave spp. plants (commonly known as Agave or maguey) are probably the most complex in the liquor world, due to the fact that they are the result of many volatile compounds that may be contained in the raw material and that vary among Agave species (Vera Guzmán et al, 2009). A very specific group of yeasts were obtained from Tamaulipas (Mexico) mezcal fermenting musts (De la Torre-González et al, 2017), which have been characterized in terms of their stress tolerance, when fermenting on high fructose contents (Oliva Hernández et al, 2013; De la Torre-González et al, 2016; Vergara-Álvarez et al, 2019) This is important due to the fact that cooked musts of Agave spp. plants contain, as fermentable sugar, a high fructose content (90%), fructose oligosaccharides (FOS), and toxic Maillard compounds, furfural, methanol and saponins, among others (Díaz-Montaño et al, 2008; Arroyo-López et al, 2009; Amaya-Delgado et al, 2013; Nava-Cruz et al, 2014), explaining in part why some of our yeasts have been recently characterized as genomically belonging to a separate, specific domestication event, as compared to other S. cerevisiae strains worldwide (Peter et al, 2018). In this study we evaluated the fermentative performance of 25 Saccharomyces and nonSaccharomyces mezcal strains on a semi synthetic (M2) medium, to assess and screen the strains, and on an Agave tequilana must-based medium for the selected yeasts, by using pure and mixed cultures to gain information to propose a minimum characterization methodology to select a starter for Agave must (mainly, tequila and mezcal) fermentative applications
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