The process of cocoa fermentation is a very important step for the generation or aromatic compounds, which are attributable to the metabolism of the microorganisms involved. There are some reports about this process and the identification of microorganisms; however, there are no reports identifying the yeasts involved in a Mexican cocoa fermentation process using molecular biology techniques, including restricted fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). The aim of this study was to identify the main yeast species associated with Mexican cocoa fermentations employing culture-dependent and -independent techniques achieving two samplings with a 1 year time difference at the same site. Isolation of the microorganisms was performed in situ. Molecular identification of yeast isolates was achieved by RFLP analysis and rDNA sequencing. Total DNA from the microorganisms on the cocoa beans was utilized for the DGGE analysis. Bands from the DGGE gels were excised and sequenced. Nineteen isolated yeasts were identified (al specie level), three of which had never before been associated with cocoa fermentations worldwide. The detected predominant yeast varied from one technique to another. Hanseniaspora sp. resulted dominant in DGGE however Saccharomyces cerevisiae was the principal isolated species. In conclusion, the culture-dependent and -independent techniques complement each other showing differences in the main yeasts involved in spontaneous cocoa fermentation, probably due to the physiological states of the viable but non culturable yeasts. Furthermore important differences between the species detected in the two samplings were detected.
Various consortia of yeasts and bacteria involved in the natural fermentation process of tequila have been identified, particularly non-Saccharomyces yeasts. This study evaluates the fermentative capacity of two non-Saccharomyces yeasts (isolated from traditional mezcal fermentation): Kluyveromyces marxianus (DU3) and Pichia kluyveri (GRO3), and assesses their production of volatile compounds. The values found are compared with those of the same attributes of a Saccharomyces cerevisiae (AR5) isolated from tequila fermentation. The fermentations were performed in two different media, agave juice (JA) and a semisynthetic medium (M11). The study also compared free and immobilized yeast fermentations in the JA medium in order to evaluate the potential benefits of immobilization on the yeast behaviour. This study demonstrated the potential of non-Saccharomyces yeasts, which fermented the agave juice in the same manner as S. cerevisiae but with higher ester production. Furthermore, K. marxianus produced more higher alcohols than S. cerevisiae. This could lead to tequila with different aroma profiles. Results were different in the synthetic medium, thus showing sensitivity to the composition of the medium. No significant differences between yeast fermentations with free and immobilized cells were detected, except for ethanol yield.
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