Identification of predominant yeasts associated with artisan Mexican cocoa fermentations using culture-dependent and culture-independent approaches

Abstract

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.

Genome Diversity and Evolution in the Budding Yeasts (Saccharomycotina)

“…Such a global genome analysis has not yet been done on other populations, so it is not known whether the wild populations from China (Wang et al 2012) or those associated with other human activities (Cromie et al 2013; Arana-Sanchez et al 2015; Ludlow et al 2016) are clean lineages or mosaics, though admixture was detected using genome-wide analysis of variants using restriction site associated DNA sequencing (Cromie et al 2013; Ludlow et al 2016). It would be very interesting if some populations associated with human activity are mosaic with genetic combinations that make them useful for particular activities, such as bread making.…”

“…Some of the populations from China are close to those previously described (Liti et al 2009a), but others, particularly the ones from primeval forests (populations CHN I, II, and III in Figure 2) nearly doubles the known diversity. In addition, distinct populations of S. cerevisiae are being found, which are associated with other fermentation activities such as cacao (Cromie et al 2013; Arana-Sanchez et al 2015; Ludlow et al 2016) and coffee (Ludlow et al 2016) processing.…”

“…In addition a New Zealand (NZ) subpopulation of the wine population has been described (Gayevskiy and Goddard 2012). The original distinction of two domestication events leading to wine and sake fermentation (Fay and Benavides, 2005; Cromie et al 2013; Arana-Sanchez et al 2015; Ludlow et al 2016) has been extended as more populations have been sampled. These include wild populations from oak and related trees, populations associated with fruit trees, and populations associated with fermentation activities.…”

Tuning Chocolate Flavor through Development of Thermotolerant Saccharomyces cerevisiae Starter Cultures with Increased Acetate Ester Production

“…These precursors will act as the substrate for Strecker degradation and Maillard reactions during roasting and conching (4). Yeasts are present throughout the fermentation process, with maximal population sizes reached in the middle of the process (12)(13)(14)(15). Apart from the primary metabolites ethanol and CO 2 , yeast cells produce numerous secondary flavor-active metabolites, including aldehydes, carbonyl compounds, esters, fatty acids, higher alcohols, organic acids, phenols, and sulfur-containing compounds (16).…”

Untitled

“…Issatchenkia hanoiensis was originally isolated from insect frass in Viet Nam 39 it has also been reported from various fermented foods [40][41][42] . Bacillus cereus is a common soil bacterium that sometimes causes foodborne illness; some strains have been used as probiotics in animal feeds to reduce the risk of Salmonella contamination 43 .…”