Análises cinéticas e moleculares das leveduras dominantes da fermentação alcoólica em uma destilaria de Alagoas: um estudo de final de safra

Abstract

The dominant yeasts in distilleries are highly adaptable and resistant to ethanol and contaminants, and are widely used in fermentation processes. However, their permanence throughout the harvest is questioned. This study investigated the predominant yeasts during the last ten days of the 2023/2024 harvest at the Santa Clotilde mill (Rio Largo, Alagoas). Samples were taken from two vats a day, at the beginning (1-2h) and end of fermentation (8h), and Brix, pH and cell viability were analyzed. Isolation of colonies in WLN medium revealed 40 strains, of which 35 were recovered after reactivation. Plating on Nagai medium identified 10 respiratory deficient colonies and 12 mixed isolates, including the reference strain CAT-1, which showed normal characteristics. DNA extraction from the 35 predominant colonies and the initial yeast from the harvest enabled genetic analysis by PCR, using primers NL1 and NL4 to amplify e D1/D2 region of the 26S gene. At the same time, the permanence or replacement of the customized strain introduced at the beginning of the harvest was assessed, as well as molecular and metabolic variations, using the oligonucleotides Delta 12, Delta 21, YLL021WR, YLL021WF and (GTG)5. Fermentation capacity was tested using the multiple tube method with molasses must (MM15), sugarcane juice (MCC12) and sucrose (YPSac10), compared to the reference strain CAT-1 and the injection yeast C1. Six morphotypes showed superior performance and were selected for kinetic evaluation. Of these, four isolates showed higher yields than the CAT-1 reference strain and the C1 strain introduced at the start of the harvest. The kinetic evaluation determined the specific cell growth rate by absorbance. The results of the molecular analyses indicated detectable variations between the predominant isolates. Analyses with the Delta 12 and Delta 21 oligonucleotides revealed distinct patterns between the isolates and the reference strain. The (GTG)5 marker indicated significant polymorphisms, while YLL021WR and YLL021WF confirmed differences between the genetic profiles of the strains analyzed. Amplification of the D1/D2 region with NL1/NL4 demonstrated the genetic diversity present at the end of the harvest. The results indicated that the initial strain was partially replaced by environmental variants, showing the impact of the stressful conditions at the end ains with greater fermentation performance suggests the possibility of optimizing ethanol production, promoting greater efficiency and sustainability in the process.