## 3 Results and Discussion  
### Fermentation Kinetics  
The particular anatomy of stool (_Dasylirion_ sp.) delivers a rich growth medium that promotes the viability of yeasts, bacteria, and filamentous fungi, which complete the spontaneous fermentation of the stool must [3]. Therefore, to better understand this interaction, the growth of the microbial population was analyzed during the spontaneous fermentation of stool must, as depicted in Figure 1. A figure that illustrates the relative abundance of fungi and yeasts cell counts during fermentation can be found in the Supplementary Material (Figure S1). It was observed that yeasts were the most abundant microorganisms, followed by lactic acid bacteria (LAB) and then filamentous fungi. The growth trends for yeasts and LAB were similar throughout the fermentation. Yeast counts reached a maximum growth (8.77 log10 CFU/mL) after day two of fermentation. Similarly, for LAB, maximum growth (6.12 log10 CFU/mL) was achieved after day two. After that, the cell growth of yeasts was steady until the end of the fermentation on day five, where their growth began to decline, whereas the viable cells of LAB began to decline after day three. The numbers of yeasts and LAB reached levels of 8.55 and 5.79 log10 CFU/mL, respectively, at the end of the fermentation. Our results showed that a symbiosis can exist among the microorganisms present in the spontaneous fermentation of sotol, which is in agreement with the observation presented in other studies of indigenous fermented beverages produced from _Agave_ spp., such as tequila, bacanora [13; 14], and mezcal [15]. In this context, spontaneous fermentation is a process originated and fulfilled by the combined action and/or succession of different species of yeasts, _Lactobacillus_ and fungi, that can thrive together in a complex microbial consortium [16; 17]. yeasts start by acting upon sugars and consuming available oxygen and micronutrients, leading to an anoxic microenvironment, which promotes the production of ethanol. The anoxic conditions support the growth of LAB that produce lactic acid, which, in combination with the available ethanol, is metabolized by acetic acid bacteria (ABA) to produce volatile acetic acid. These metabolites and further biochemical changes give rise to significant flavor precursors [17; 18]. The acidification of sotol must by LAB can also play a significant role in the process, as acidification can improve sugar extraction, fermentability, and nitrogen yield, improving color and flavor such as in acidic ales obtained by spontaneous fermentation [19]. The filamentous fungi presented a steady growth since day zero, suggesting that, when water was added to the sotol must (identified as day zero) after left to stand for two days, fungal growth was in an advanced stage. The maximum level of the filamentous fungi observed was 2.50 log10 CFU/mL, and after day three, the cell growth dropped, reaching a minimum level (1.15 log10 CFU/mL) at the end. Sotol filamentous fungi can play an important role in the viability of yeasts and LAB, as they are a source of fructotransferases [20] that act upon fructans, the natural occurring polysaccharides in sotol (_Dasylirion_ spp.) [21]. It is then likely that fungi throughout the production of fructotransferases participate in the initial phase of sotol must fermentation, hydrolyzing fructans found in the cellular structure and liberating fructose along with glucose, which aid the initial growth of yeasts and LAB. Additionally, fungi belonging to the genera _Aspergillus_ and _Penicillium_ have been reported to produce a combination of endo- and exo-inulinases, that release oligosaccharides along with fructose from inulin-rich plant extracts [22]. It is then important to perform further studies in order to evaluate the hydrolytic activities of fungal strains isolated from sotol plant. Figure 1 shows that the initial concentration of sugars was around 75.84 g/L; this increased to 118.1 g/L after 24 h due to the transfer of these from the sotol fibers to the liquid medium.