Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation.

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12-24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)5-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.

Relationship of sensory and instrumental aroma measurements of dark chocolate as influenced by fermentation method, roasting and conching conditions.

The investigation was undertaken to establish a relationship between key odorants and perceived flavor attributes of dark chocolate as influenced by cocoa fermentation method, roasting and conching conditions, using multivariate data analysis in an attempt to use one variable to predict the other. Eight of the sixteen flavor attributes used by a trained sensory panel to describe and quantify the intensity of attributes in the samples were significantly different (p < 0.05). Roasting significantly reduced astringency in heap-fermented samples but the reduction in tray-fermented samples was not significant. Unconched samples were rated higher in banana attribute than conched samples. Multivariate data analytical tools, Principal Component Analysis (PCA) and Partial Least Squares (PLS) were used to investigate quantitative descriptive analysis and GC-O data and also to relate the two sets of data. PLS 1 models based on single sensory attributes gave better models than PLS2 models based on all sixteen sensory attributes. Ethyl-3-methylbutanoate (fruity, flowery); 2,5-dimethylpyrazine (popcorn); dihydro-2(3H)-furanone, (sweet); linalool oxide (sweet, flowery); benzaldehyde (earthy, nutty) and 2/3-methylbutanal (cocoa, roasted) modeled fruit attribute. It was also possible to model the attribute astringent from the aroma compounds 5-methyl-2-phenyl-2-hexenal (sweet, roasted cocoa), ethyl-3-methylbutanoate and pentyl acetate (green, cucumber). Since fruit attribute was higher in unconched samples and astringent higher in unroasted samples, it may be possible to use the levels of these important aroma compounds as indicators of the sensory attributes fruit and astringent.