Abstract
The majority of the World’s cocoa production originates from the West African countries of Ivory Coast, Ghana and Nigeria. In these countries, cocoa is a crop of great socio-economic importance as it is often the main source of income for families in the rural cocoa growing regions. Being the principal raw material for chocolate production, good quality cocoa beans are in high demand on the World market as a prerequisite for the production of high quality chocolates and other confectionary products. To produce good quality cocoa suitable for chocolate production, it is essential that the beans undergo fermentation and drying processes, during which biochemical reactions lead to the formation of cocoa specific flavour precursors. During subsequent roasting, these precursors are transformed into a wide array of aroma compounds as a result of complex Maillard and Strecker degradation reactions. Despite the importance of a properly conducted fermentation process, poor post-harvest practises, in combination with the unpredictable spontaneous nature of the fermentations, often results in sub-optimal flavour development. Our understanding of the microorganisms responsible for carrying out the fermentation of cocoa has greatly increased during the last decade. To overcome the inherited variability of spontaneous fermentations, this detailed insight into the microbial ecology has led to the development of defined inoculation cultures encompassing yeast and bacterial strains with beneficial functional properties.
With the object ive of improving the volatile aroma and sensory properties of Ghanaian Forastero cocoa, the present Ph.D. study investigates the use of two defined mixed starter cultures encompassing strains of Acetobacter pasteurianus and Lactobacillus fermentum in combination with either a commercially available aromatic strain of Pichia kluyveri or a pectinolytic strain of Kluyveromyces marxianus. Fermentations were conducted in an experimental small-scale tray setup at the Cocoa Research Institute of Ghana during the main crop of 2011/12. Using a combination of culture-dependent and culture-independent molecular techniques, the growth and survival of the two yeast inoculation cultures was verified at strain level, whilst the bacterial inoculum was identified at species level. Aroma profiling was conducted using dynamic headspace gas chromatography-mass spectrometry for identification and relative quantification of volatile compounds present in roasted and un-roasted cocoa liquors, as well as in finished chocolates. Sensory analyses of un-conched chocolate and finished chocolate was performed using a panel of un-trained judges and ordinary consumers. Furthermore, the present study describes the impact of acetic acid concentration on the generation of flavour precursors and volatile aroma compounds in cocoa beans subjected to incubation in acetic acid buffers.
(GTG)5-based rep-PCR fingerprinting in combination with 26S rRNA (D1/D2 region) and actin gene sequencing revealed that during the first 12 hours of fermentation, the yeast communities of both inoculated and spontaneous fermentations were dominated by Hanseniaspora opuntiae and Hanseniaspora thailandica, with the latter being described for the first time in relation to cocoa fermentation. After 24 hours, the inoculated strains of Pichia kluyveri and Kluyveromyces marxianus were dominating the fermentation - composing 39.8% and 51.3% of the total yeast population, respectively. Chromosome Length Polymorphism among yeast populations belongingto the inoculation species was determined using Pulsed Field Gel Electrophoresis. While all K. marxianus isolates were found to be identical to the inoculation strain, four strains of P. kluyveri were identified, with the inoculation strain composing ~88% of the population.
The volatile aroma profile of chocolates made from cocoa beans inoculated with P. kluyveri
contained significantly higher concentrations of phenylacetaldehyde compared to a spontaneously fermented control, whereas inoculation with K. marxianus led to significantly higher concentrations of benzyl alcohol, benzyl acetate, phenethyl alcohol, and phenethyl acetate. When compared to chocolates made from conventionally heap and tray fermented cocoa, chocolates from the experimental small scale tray setup generally contained less volatile compounds. The considerably lower pH (~0.5 pH units) of the experimental cocoa is thought to impair the reactivity of the flavour precursors and reduce the formation of aroma compounds during roasting. The choice of fermentation technique therefore seemed to have a greater influence on the quantitative composition of volatile aroma compounds than the use of starter cultures.
Sensory profiling described the conventional heap and tray fermented chocolates as sweet with cocoa and caramel flavours, whilst the inoculated chocolates were characterised as fruity, acid and bitter with berry, yoghurt and balsamic flavours. Despite differences in the volatile aroma profile, the inoculated chocolates could not be distinguished from a spontaneously fermented control in a triangle test by a panel of un-trained judges. Arguably, using a panel of judges trained in chocolate tasting might help to reveal the finer nuances of the samples.
Although incubation of fresh cocoa beans for 48 hours at 45°C in a 1.5% acetic acid solution
produced cocoa with a comparable content of free amino acids and reducing sugars as
conventionally heap and tray fermented samples, aroma analysis of the roasted cocoa liquors
showed great variation between the incubated and conventionally fermented samples. While the fermented samples displayed the most diverse composition of volatile compounds, the incubated samples completely lacked essential cocoa aroma compounds such as pyrazines and terpene alcohols. A microbial fermentation process therefore seems essential for developing the full complexity of compounds which characterises cocoa aroma.
In conclusion, the results of the present study show that the volatile aroma profile of chocolate can be influenced using starter cultures. However, the differences observed were too small to significantly change consumer perception of the chocolates. Arguably, using higher inoculation densities and a fermentation setup with better drainage properties could accentuate the impact of the cultures investigated.
With the object ive of improving the volatile aroma and sensory properties of Ghanaian Forastero cocoa, the present Ph.D. study investigates the use of two defined mixed starter cultures encompassing strains of Acetobacter pasteurianus and Lactobacillus fermentum in combination with either a commercially available aromatic strain of Pichia kluyveri or a pectinolytic strain of Kluyveromyces marxianus. Fermentations were conducted in an experimental small-scale tray setup at the Cocoa Research Institute of Ghana during the main crop of 2011/12. Using a combination of culture-dependent and culture-independent molecular techniques, the growth and survival of the two yeast inoculation cultures was verified at strain level, whilst the bacterial inoculum was identified at species level. Aroma profiling was conducted using dynamic headspace gas chromatography-mass spectrometry for identification and relative quantification of volatile compounds present in roasted and un-roasted cocoa liquors, as well as in finished chocolates. Sensory analyses of un-conched chocolate and finished chocolate was performed using a panel of un-trained judges and ordinary consumers. Furthermore, the present study describes the impact of acetic acid concentration on the generation of flavour precursors and volatile aroma compounds in cocoa beans subjected to incubation in acetic acid buffers.
(GTG)5-based rep-PCR fingerprinting in combination with 26S rRNA (D1/D2 region) and actin gene sequencing revealed that during the first 12 hours of fermentation, the yeast communities of both inoculated and spontaneous fermentations were dominated by Hanseniaspora opuntiae and Hanseniaspora thailandica, with the latter being described for the first time in relation to cocoa fermentation. After 24 hours, the inoculated strains of Pichia kluyveri and Kluyveromyces marxianus were dominating the fermentation - composing 39.8% and 51.3% of the total yeast population, respectively. Chromosome Length Polymorphism among yeast populations belongingto the inoculation species was determined using Pulsed Field Gel Electrophoresis. While all K. marxianus isolates were found to be identical to the inoculation strain, four strains of P. kluyveri were identified, with the inoculation strain composing ~88% of the population.
The volatile aroma profile of chocolates made from cocoa beans inoculated with P. kluyveri
contained significantly higher concentrations of phenylacetaldehyde compared to a spontaneously fermented control, whereas inoculation with K. marxianus led to significantly higher concentrations of benzyl alcohol, benzyl acetate, phenethyl alcohol, and phenethyl acetate. When compared to chocolates made from conventionally heap and tray fermented cocoa, chocolates from the experimental small scale tray setup generally contained less volatile compounds. The considerably lower pH (~0.5 pH units) of the experimental cocoa is thought to impair the reactivity of the flavour precursors and reduce the formation of aroma compounds during roasting. The choice of fermentation technique therefore seemed to have a greater influence on the quantitative composition of volatile aroma compounds than the use of starter cultures.
Sensory profiling described the conventional heap and tray fermented chocolates as sweet with cocoa and caramel flavours, whilst the inoculated chocolates were characterised as fruity, acid and bitter with berry, yoghurt and balsamic flavours. Despite differences in the volatile aroma profile, the inoculated chocolates could not be distinguished from a spontaneously fermented control in a triangle test by a panel of un-trained judges. Arguably, using a panel of judges trained in chocolate tasting might help to reveal the finer nuances of the samples.
Although incubation of fresh cocoa beans for 48 hours at 45°C in a 1.5% acetic acid solution
produced cocoa with a comparable content of free amino acids and reducing sugars as
conventionally heap and tray fermented samples, aroma analysis of the roasted cocoa liquors
showed great variation between the incubated and conventionally fermented samples. While the fermented samples displayed the most diverse composition of volatile compounds, the incubated samples completely lacked essential cocoa aroma compounds such as pyrazines and terpene alcohols. A microbial fermentation process therefore seems essential for developing the full complexity of compounds which characterises cocoa aroma.
In conclusion, the results of the present study show that the volatile aroma profile of chocolate can be influenced using starter cultures. However, the differences observed were too small to significantly change consumer perception of the chocolates. Arguably, using higher inoculation densities and a fermentation setup with better drainage properties could accentuate the impact of the cultures investigated.
| Original language | English |
|---|
| Publisher | Department of Food Science, Faculty of Science, University of Copenhagen |
|---|---|
| Number of pages | 175 |
| Publication status | Published - 2014 |