Mezcal is a distillate produced by spontaneous fermentation of the must obtained from stalks of Agave spp. plants that are cooked and pressed. Agave must contains a high amount of fructose and phenolic compounds, and fermentation usually occurs under stressful (and uncontrolled) environmental conditions. Yeasts capable of growing under such conditions usually display advantageous biological and industrial traits for stress tolerance such as flocculation. In this study, seven Saccharomyces cerevisiae strains isolated from mezcal must were exposed to temperatures ranging between 10 and 40 °C, and to different sugar sources (fructose or glucose). Yeasts grown in fructose increased their stress tolerance, determined by colony count in a microdrop assay, under low temperature (10 °C) compared to the growth at 40 °C on solid cultures. The most stress-tolerant mezcal strain (Sc3Y8) and a commercial wine (Fermichamp) strain, used as control, were grown under fermentation conditions and exposed to long-term temperature stress to determine their performance and their potential for flocculation. Compared to glucose, fermentation on fructose increased the metabolite accumulation at the end of culture, particularly at 40 °C, with 2.3, 1.3 and 3.4 times more glycerol (8.6 g/L), ethanol (43.6 g/L) and acetic acid (7.3 g/L), respectively. Using confocal microscopy analysis, we detected morphological changes such as aggregation and wall recognition at the level of budding scars in yeast, particularly in the Sc3Y8 strain when it was exposed to 40 °C. The analysis confirmed that this mezcal strain was positive for flocculation in the presence of Ca2+ ions. Analysis of FLO1, FLO5 and FLO11 gene expression implicated in flocculation in both Saccharomyces strains showed a strong transcriptional induction, mainly of the FLO5 gene in the mezcal Sc3Y8 strain.; Mezcal je destilat koji se proizvodi spontanom fermentacijom mošta dobivenog kuhanjem stabljika biljke Agave spp. i prešanjem dobivene smjese. Mošt agave sadržava velike količine fruktoze i fenolnih spojeva, a fermentacija se najčešće odvija pri stresnim i nekontroliranim uvjetima. Kvasci koji mogu rasti u takvim uvjetima obično imaju povoljna biološka i industrijska svojstva koja ih čine otpornijim, poput svojstva flokulacije. U ovom je radu sedam sojeva kvasca Saccharomyces cerevisiae izoliranih iz mošta agave izloženo temperaturama od 10 do 40 °C i uzgojeno u podlogama u prisutnosti fruktoze ili glukoze. Brojanjem kolonija u tzv. „microdrop“ testu potvrđeno je da su kvasci koji su rasli u podlozi s dodatkom fruktoze imali veću otpornost na stres uzrokovan niskom temperaturom (10 °C), u usporedbi s kvascima uzgojenim pri 40 °C. Soj kvasca koji je najbolje podnio stres (Sc3Y8) i komercijalni vinski kvasac (Fermichamp) uzgojeni su u tekućoj podlozi za fermentaciju i dugotrajno izloženi toplinskom stresu, radi određivanja njihove sposobnosti flokulacije. Tijekom fermentacije u podlozi s fruktozom povećalo se nakupljanje metabolita na kraju procesa, osobito pri 40 °C, te je proizvedeno 2,3 puta više glicerola (8,6 g/L), 1,3 puta više etanola (43,6 g/L) i 3,4 puta više octene kiseline (7,3 g/L) nego tijekom fermentacije u podlozi s glukozom. Pomoću konfokalnog mikroskopa utvrđene su morfološke promjene u kvascu, kao što su agregacija stanica i prisutnost ožiljaka na stijenci nastalih uslijed pupanja kvasca, osobito u soju Sc3Y8 izloženom temperaturi od 40 °C. Potvrđeno je da ovaj soj kvasca za proizvodnju pića mezcal flokulira u prisutnosti iona kalcija. Analizom ekspresije gena FLO1, FLO5 i FLO11 uključenih u regulaciju flokulacije potvrđeno je da je došlo do indukcije transkripcije u oba soja kvasca S. cerevisiae, naročito gena FLO5 u soju Sc3Y8.