Flocculation and Expression of FLO Genes of a Saccharomyces cerevisiae Mezcal Strain with High Stress Tolerance

Abstract

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.

Rational Selection of Mixed Yeasts Starters for Agave Must Fermentation

“…In terms of resistance to stress, data suggest that it is not only the presence of fructose as the main fermentable sugar but also that of toxic compounds present on the Agave must, as furfural and vanillin (data not shown), that affects the viability and metabolic performance of the strains, as has been also observed by Arrizon et al (2006) for other Agave yeast strains. This clearly requires further investigation, but from the data obtained here and our previous work (Oliva Hernández et al, 2013;De la Torre-González et al, 2016Vergara-Álvarez et al, 2019) with these mezcal strains, the selection of Agave must isolated strains (Saccharomyces and non-Saccharomyces) to be used as inoculum for tequila and mezcal (as the main liquors produced from Agave spp. plants, but there are others) would need to be tested at 30 • C to assess that are be able to grow on agar plates containing 500 g/L of fructose, to be able to grow on yeast extract and peptone agar plates containing 8% ethanol and fructose as a carbon source.…”

“…Hexyl acetate was only detected for a Torulaspora (Td1AN2) strain. From all these data, plus previous works on tolerance to osmotic, oxidative, and temperature stresses (Oliva Hernández et al, 2013;De la Torre-González et al, 2016;Vergara-Álvarez et al, 2019), we selected a group of three S. cerevisiae mezcal strains (plus Fermichamp as fructophilic control) and the best performing strains of the Kluyveromyces (Km1Y9), Torulaspora (Td1AN9) and Zygosaccharomyces (Zb3Y1) genera, although it is worth mentioning that some other promising strains are still being investigated.…”

“…Tequila and mezcal liquors are produced from cooked Agave must fermentation, which usually depends on spontaneous or pure S. cerevisiae strain inoculation (Cedeño, 1995;Díaz-Montaño et al, 2008). The yeasts used in this work were obtained from mezcal musts and have been selected for having a higher tolerance to stress, particularly for osmotic, oxidative, and temperature unfavorable conditions (De la Torre-González et al, 2016;Vergara-Álvarez et al, 2019). In this study, 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 also were good producers of volatile compounds, as previously observed for some of these genera (Arellano et al, 2008;Lopez-Alvarez et al, 2012;Páez-Lerma et al, 2013;Nolasco-Cancino et al, 2018).…”

“…Fortunately, an increasing number of reports are slowly unraveling the microbial consortia complexities of Agave fermented beverages (Lachance, 1995;Narváez-Zapata et al, 2010;Verdugo Valdez et al, 2011;Páez-Lerma et al, 2013, Kirchmayr et al, 2017Nolasco-Cancino et al, 2018). 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, particularly, 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.…”

<i>Saccharomyces cerevisiae</i> como modelo de ensino de toxicologia a estudantes do ensino médio

“…Os genes FLO1, FLO5, FLO9, FLO10 e FLO11 codificam para a flocculina, uma família de proteínas da parede celular que produzem agregação reversível assexuada de células [33,34]. Além da expressão destes genes, sob condições de estresse, alterações nas interações hidrofóbicas que ocorrem na parede celular podem aumentar a capacidade de agregação de leveduras, levando a uma mudança adaptativa [35].…”