Seleção e caracterização molecular de leveduras não-convencionais quanto ao consumo de alfa-terpineol

Abstract

Many microbial species are acknowledged by their ability to provide distinct organoleptic properties to countless foods and drinks, especially alcoholic beverages. Non-conventional yeasts stand out in using different metabolic pathways to produce biomolecules that may be interesting from the biotechnological point of view. The present research aimed to analyze the potential consumption of a monoterpenic compound, α-terpineol, by non-conventional yeasts CMRP collection center. For this purpose, 23 fruity aroma-producing, non-conventional yeasts were selected from one of the collections belonging to the CMRP collection center. Thereafter, these strains were molecularly identified. Subsequently, the strains were tested for their tolerance to α-terpineol, by cultivating them in YM broth, at pH 5.0, for 48 h. Concentrations of 2.5 μL/mL, 5.0 μL/mL, 7.5 μL/mL and 10.0 μL/mL of a mixture of α-terpineol and ethyl alcohol were added in the broth. The yeasts that survived any of these concentrations were cultured again, but this time in mineral DP liquid culture medium. This technique aimed to assess their consumption of α-terpineol. The overall cultivation time was 48h, however samples were collected every 12 hours of cultivation, including the initial time of 0 hour. The samples collected were extracted with dichloromethane and the extracts were analyzed by thin layer chromatography, using toluene: ethyl acetate (93:7) as a mobile phase and p-anisaldehyde sulfuric acid as stain for further visualization of the spots. The size, color and intensity of the spots were observed and the Rf values were calculated. The yeasts species were identified as Clavispora lusitaniae, being it 12 strains, Rhodotorula mucilaginosa with 8 strains and the last 3 strains were identified as Lodderomyces elongisporus. Furthermore, according to the phylogenetic tree, two of these strains (C. lusitaniae and L. elongisporus) have greater genetic proximity to each other than to the yeast R. mucilaginosa. From the 23 yeasts, 6 of them were shown to be tolerant to the concentration of 2.5 μL/mL of α-terpineol, one of which also showed tolerance to the concentration of 10.0 μL/mL for 12 hours. The tolerant strains were identified as C. lusitaniae (n = 1), L. elongisporus (n = 2) and R. mucilaginosa (n = 3). The yeast that endured the highest concentration, R. mucilaginosa, however, resited only 12 hours in all concentrations. It is more likely that the defense mechanism of the yeast was not able to prevent further damage to its membrane as the time in contact with α-terpineol increased, it could also be that there was an enzymatic degradation to some level, or even that the death of the strain might be due to the production of toxic compounds by that same strain. The TLC results showed that the extracts from CMIB 46 (R. mucilaginosa) and CMIB 33 (L. elongisporus) could be interpreted as potentially promising results of the production of new compounds by the yeasts. Therefore, both yeasts were cultivated again, thus the analysis could be performed one more time. At last, the results for the consumption of α-terpineol by these two strains were rather inconclusive, mainly due to the low concentration of the samples. For future analysis, it is proposed the use of auxiliary techniques, such as gas chromatography coupled to mass spectrometer for greater sensitivity in the detection of these compounds.