Mezcal as a Novel Source of Mixed Yeasts Inocula for Wine Fermentation

Mezcal yeasts were evaluated for their potential as grape-juice fermenters, characterizing their fermentation performance, both in terms of primary and volatile metabolites. Experiments were first carried-out in a semi-synthetic medium and then on grape juice, and population dynamics of the chosen mixed inoculum was assessed in grape juice. Accordingly, we initially tested 24 mezcal yeasts belonging to ten different species, and chose those that were more productive and stress tolerant for the mixed (dual) inoculum, having a final selection of three Saccharomyces cerevisiae strains (plus Fermichamp, a commercial wine strain) and three non-Saccharomyces strains, belonging to Kluyveromyces marxianus, Torulaspora delbrueckii, and Zygosaccharomyces bailii species. For the combination S. cerevisiae/T. delbrueckii (Sc/Td) mixed inoculum, we observed increasing isoamyl alcohol and phenyl ethyl acetate concentrations, as compared with the use of individual Saccharomyces strains, which resulted in a fruitier aroma profile. Alcohol final concentration was in average lower for the Sc/Td inoculum (fermentation power, FP, 13.6) as compared with the individual mezcal Saccharomyces strains (FP 14.3), and it was the highest when Td was co-cultured with the commercial strain (FP 14.6). Overall, our results show the feasibility of using yeasts isolated from mezcal as a novel source of inoculum for wine-type fermentation

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

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.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

The effect of hexose ratios on metabolite production in Saccharomyces cerevisiae strains obtained from the spontaneous fermentation of mezcal

Mezcal from Tamaulipas (Me´xico) is produced by spontaneous alcoholic fermentation using Agave spp. musts, which are rich in fructose. In this study eight Saccharomyces cerevisiae isolates obtained at the final stage of fermentation from a traditional mezcal winery were analysed in three semisynthetic media. Medium M1 had a sugar content of 100 g l-1 and a glucose/fructose (G/F) of 9:1. Medium M2 had a sugar content of 100 g l-1 and a G/F of 1:9. Medium M3 had a sugar content of 200 g l-1 and a G/F of 1:1. In the three types of media tested, the highest ethanol yield was obtained from the glucophilic strain LCBG-3Y5, while strain LCBG-3Y8 was highly resistant to ethanol and the most fructophilic of the mezcal strains. Strain LCBG-3Y5 produced more glycerol (4.4 g l-1) and acetic acid (1 g l-1) in M2 than in M1 (1.7 and 0.5 g l-1, respectively), and the ethanol yields were higher for all strains in M1 except for LCBG-3Y5, -3Y8 and the Fermichamp strain. In medium M3, only the Fermichamp strain was able to fully consume the 100 g of fructose l-1 but left a residual 32 g of glucose l-1. Regarding the hexose transporters, a high number of amino acid polymorphisms were found in the Hxt1p sequences. Strain LCBG-3Y8 exhibited eight unique amino acid changes, followed by the Fermichamp strain with three changes. In Hxt3p, we observed nine amino acid polymorphisms unique for the Fermichamp strain and five unique changes for the mezcal strains

Cinética de crecimiento de hongos filamentosos: morfometría de los micelios de A. niger y G. fujikuroi y su posible utilización en la predicción de la tasa específica de crecimiento

Utilizando relaciones establecidas para la descripción del crecimiento microbian0 y fúngico, se derivó una expresión que, utilizando los parámetros morfométricos de una hifa: 1anp;ity-l crítica (E) y diámetro (Dh); y los de la colonia; velooidad de exknsibn radial (uF), loq$ud de sus hifas distales (hJ, predice satisfactoriamente el valor de la tasa específica de crecimiento (p) de la biomasa. La expresión derivada fue la siguiente Se investigó el crecimiento, bajo diferentes condiciones nutricionales, de Gibberella fujikumi en cultivo líquido, y de Aspergillus niger en cultivo superfioial,,para comparar dos mohos de diferente velocidad de crecimiento, cultivados en medios de diferente naturaleza. Esto con el fin de conocer el rango de validez de la expresión propuesta. Para el crecimiento superficial de A. niger, se observó que, a menor concentración de glucosa, la longitud crítica (k) del túbulo germinal era mayor. El diámetro hifal (Dh) estuvo inversamente correliacionado con la concentración de gliucosa, y la velocidad de extensión radial (uí) tuvo un comportamiento parabólico, con un máximo a la concentración de glucosa de 120 g/L. Se encontró que el proceso de elongación sigue una cinética de saturación tipo Monod (b= 0.30 I 0.035h-', Ki= - 132 f 52 g/L). .* ... 1 La longitud crítica del túbulo germinal (E) fue menor que la longitud alcanzada por las hifas del borde de la colonia madura (h4 por lo que se puede concluir que el volumen de citoplasma que contribuye ai crecinaiento en cada estadio es difemnte, siendo mayor para el caso de la colonia madura. El crecimiento en líquido de G. fujikurwi presentó una morfología filamentosi dispersa. La longitud crítica (k) mostró (ai igual que con A. niger) valores crecientes a menor concentración de glucosa. Sin embargo, la velocidad de extensión radial fue la misma a las diferentes composiciones de medio utilizadas, y la longitud de las hifas distales (Lv) mayor a menor concentración de glucosa. El diámetro hifal (Dh) en cambio, fue mayor al aumentar la cantidad de glucosa del medio. Los parámetros que describieron la tasa específica de crecimiento de los cultivos fueron: la velocidad de extensión radial (ur), la longitud promedio de las hifas distalcs (I."), la longitud crítica de ramificación (k) , y el diiunetro promedio de las hifas (Dd . El hecho de que la expresión propuesta fuera adecuada para ambas clases de hongos, y para diferente tipo de medio, se interpretó como un indicio de que la derivación realizada tomaba en cuenta, efectivamente, los fenómenos que mejor describen el aumento de biomasa de un cultivo fúngico. Esto se corroboró por el hecho de que, aún y cuando los hongos elegidos presentaban compurtamientos diferentes en su morfología, se predijo adecuadamente el valor de la tasa específica de crecimiento, para cada uno, y a diferentes condiciones nutricionaíes. Este trabajo presenta una nueva metodología para cuantificar la tasa específica de crecimiento de cultivos fúngicos. Se basa en la determinación morfométrica de los micelios, durante la fase de germinación y el crecimiento vegetativo

Respuestas metabólicas al estrés de levaduras de importancia industrial

The production of metabolites and biomass on yeasts is performed usually under stressing conditions, mainly the ones concerning dissolved oxygen, osmotic pressure, temperature, pH and toxic compounds, amongst others. Due to their unicellular nature, yeasts usually rely on changes in their physiology and composition to deal with environmental stresses. In this review we analyzed the metabolic responses used by yeasts during industrial processes such as wine production, as well as research performed to elucidate their common physiological responses, and stressing the findings on Saccharomyces cerevisiae, which is by far the most important yeast from the industrial point of view.La producción de metabolitos y biomasa de levaduras a nivel industrial está generalmente sujeta a condiciones estresantes de cultivo, principalmente en cuanto a la concentración de oxígeno disuelto, presión osmótica, temperatura, pH y compuestos tóxicos como el etanol. Al ser las levaduras organismos unicelulares, los mecanismos para enfrentar estas situaciones de estrés se basan en cambios fisiológicos y de composición, así como toda una gama de respuestas de su metabolismo. En este trabajo se hace una revisión de las respuestas metabólicas en levaduras durante la producción a escala industrial de bebidas como el vino, así como las investigaciones básicas realizadas para elucidar las respuestas fisiológicas comunes usadas por estos microorganismos, con especial énfasis en la levadura Saccharomyces cerevisiae, que es por mucho la más importante a nivel industrial

Addition of abscisic acid increases the production of chitin deacetylase by Colletotrichum gloeosporioides in submerged culture

International audienceThe activity of chitin deacetylase from Colletotrichum gloeosporioides was studied by the addition of phytohormones (gibberellic acid, indole acetic acid, and abscisic acid) and amino sugars (glucosamine and N-acetyl glucosamine) in culture media. Abscisic acid exerted a positive and significant effect on enzyme production with 9.5-fold higher activity (1.05 U mg protein(-1)) than the control (0.11 U mg protein(-1)). Subsequently, this phytohormone was used in batch culture with higher chitin deacetylase activity being found at acidic pH (3.5) than at neutral pH (7). Furthermore, the highest activity was determined at the acceleration growth phase. The chitin deacetylase production was ascribed to the lag phase within the spore germination process and germ tube elongation instead of during the formation of appressoria, as evidenced by the scanning electronic microscopy results. Therefore, more inoculum and medium containing abscisic acid were added to the fed-batch culture, resulting in a significant increase in chitin deacetylase activity (3.64 U mg protein(-1)). The addition of abscisic acid led to changes in the acetylation degree of chitin extracted from the cell wall of C. gloeosporioides, with lower degree of acetylation (DA of 75.6%) than that determined with the culture without abscisic acid (DA of 90.6%)

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Biotecnologí

<i>Pyricularia</i>’s Capability of Infecting Different Grasses in Two Regions of Mexico

The genus Pyricularia includes species that are phytopathogenic fungi, which infect different species of Poaceae, such as rice and sorghum. However, few isolates have been genetically characterized in North America. The current study addresses this lack of information by characterizing an additional 57 strains of three grasses (Stenotaphrum secundatum, Cenchrus ciliaris and Digitaria ciliaris) from two distant regions of Mexico. A Pyricularia dataset with ITS sequences retrieved from GenBank and the studied sequences were used to build a haplotype network that allowed us to identify a few redundant haplotypes highly related to P. oryzae species. An analysis considering only the Mexican sequences allowed us to identify non-redundant haplotypes in the isolates of C. ciliaris and D. ciliaris, with a high identity with P. pennisetigena. The Pot2-TIR genomic fingerprinting technique resulted in high variability and allowed for the isolates to be grouped according to their host grass, whilst the ERIC-PCR technique was able to separate the isolates according to their host grass and their region of collection. Representative isolates from different host grasses were chosen to explore the pathogenic potential of these isolates. The selected isolates showed a differential pathogenic profile. Cross-infection with representative isolates from S. secundatum and C. ciliaris showed that these were unable to infect D. ciliaris grass and that the DY1 isolate from D. ciliaris was only able to infect its host grass. The results support the identification of pathogenic strains of Pyricularia isolates and their cross-infection potential in different grasses surrounding important crops in Mexico