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Cocoa beans are the seeds from the fruit pods of the cocoa tree, Theobroma cacao L., which
is cultivated in plantations in tropical regions throughout the world, West Africa being the
major producing region of the world production of cocoa. In the pods the cocoa beans are
embedded in a mass of mucilaginous pulp and after removal of both beans and pulp from the
pods the first step in cocoa processing is a spontaneous fermentation. The methods of
fermentation vary from country to country and even adjacent farms within a country may
differ in their processing practices. The cocoa beans are either fermented in heaps, boxes,
baskets or trays. In Ghana, fermentation in heaps, varying in sizes from 20 to 1000 kg and
covered with banana or plantain leaves, is by far the most dominant method. During
fermentation, microbial activity leads to the formation of a range of metabolic end-products,
such as alcohols, acetic acid and other organic acids, which diffuse into the beans and cause
their death. This induces biochemical transformations within the beans that lead to formation
of precursors of the characteristic aroma, flavor, and colour, which are further developed
during drying and finally obtained during roasting and further processing into chocolate.
Earlier studies on the microbiology of cocoa bean fermentation have shown that yeasts,
filamentous fungi, lactic acid bacteria (LAB), acetic acid bacteria (AAB) and Bacillus species
might contribute to cocoa bean fermentation.
The present multiphasic study aimed to investigate the microbial biodiversity and population
dynamics of LAB and AAB involved in spontaneous Ghanaian cocoa bean heap
fermentations. The population dynamics of this microbiota were studied through a cultivation-
dependent (plating, isolation, and molecular characterization of the isolates) and cultivation-
independent (Polymerase chain reaction-denaturing gradient gel electrophoresis) approach.
Unfortunately, not many studies dealt with the isolation, identification and functional role of
LAB and AAB species involved in cocoa bean fermentation performed. The analysis of the
community dynamics was combined with an extensive metabolite target analysis to
characterize the metabolic capacity of the microorganisms present, both in the natural
environment and by means of a cocoa pulp simulation medium. Therefore, a variety of
chromatographic techniques was applied on the ecosystem as a whole and on pure cultures,
respectively, such as high performance liquid chromatography, high performance anion
exchange chromatography with pulsed amperometric detection or conductivity, liquid
chromatography coupled to mass spectrometry, gas chromatography, and gas chromatography
coupled to mass spectrometry. With this knowledge a starter culture was developed and tested
to explore the possibilities of a standardized cocoa bean fermentation process.
During this study, a new and fast identification method for AAB was developed.
Amplification of repetitive bacterial DNA elements through the polymerase chain reaction
(rep-PCR fingerprinting) using the (GTG)5 primer, referred to as (GTG)5-PCR fingerprinting,
was found to be a promising genotypic tool for rapid and reliable speciation of AAB. The method was evaluated with 64 AAB reference strains, including 31 type strains, and 132 isolates from Ghanaian, fermented cocoa beans. Most reference strains grouped according to their species designation, indicating the usefulness of this technique for identification to the species level. The present study of the spontaneous Ghanaian cocoa bean heap fermentation process revealed a limited biodiversity and targeted population dynamics of both LAB and AAB during fermentation. Four main clusters were identified among the LAB isolated, based on the (GTG)5-PCR fingerprinting: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. The (GTG)5-PCR fingerprinting allowed differentiation of only three clusters among the AAB identified: Acetobacter pasteurianus, Acetobacter senegalensis, and Acetobacter ghanensis. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment (cocoa pod surfaces, baskets, knives, etc.), were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Strains of Leuconostoc pseudomesenteroides and Enterococcus casseliflavus, which actually derived from hands and baskets, were only present at the start of the fermentation. Yeast depectinized the pulp and produced ethanol from sugars and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Citrate fermentation appeared to be of utmost importance. Its metabolism contributed to strain competitiveness, aroma precursor formation, and pH regulation of the natural environment. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate, and converted ethanol into acetic acid as well as lactate, mannitol, and acetate into CO2 and H2O. A newly proposed Weissella sp., “Weissella ghanensis”, was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Both farm and season did not influence species diversity or fermentation course. However, microbial variability occurred between heaps. In a next step, the influence of turning and environmental contamination on spontaneous cocoa bean heap fermentations performed in Ghana was studied. Different selective agar media were used to isolate AAB during cocoa bean fermentation to exclude possible selective isolation of A. pasteurianus. Only four clusters were found among the AAB isolates identified: A. pasteurianus, A. ghanensis, A. senegalensis, and a potential new Acetobacter lovaniensis-like species (“A. fabarum”). Their isolation was dependent on the composition of the agar medium used, acetic acid tolerance being an important selection criteria. Growth of A. pasteurianus was favored by heap turning. Two main clusters were identified among the LAB isolated, namely L. plantarum and L. fermentum, growth of the latter species being favored by heap turning. No differences in biodiversity of LAB and AAB were seen for fermentations carried out at the farm’s and factory’s sites, indicating the cocoa pods’ surfaces as the main inoculum for spontaneous cocoa bean heap fermentation. Further, the environmental factor did not influence the fermentation course of the cocoa beans significantly. Besides the microbiological and biochemical approach of the spontaneous Ghanaian cocoa bean heap fermentation, a cut test, chocolate production, and sensory analysis were performed on the beans from the spontaneous cocoa bean heap fermentations studied. Although the same microorganisms were involved in different heaps, carried out at different farms or in different seasons, heap fermentation temperatures and microbial metabolite concentrations were different. This could be due to the heterogeneity and size of the heaps, but mainly to microbial variability. Indeed, these differences caused pronounced effects on the flavor of chocolates made from the corresponding dried, fermented cocoa beans. For instance, a too high acetic acid concentration in the dried, fermented cocoa beans as a result of an enhanced acetic acid bacterial activity in the surrounding cocoa pulp, resulted in a sour-tasting chocolate. As turning of the heaps enhanced growth of AAB and hence the fermentation temperature, the production of acetic acid was higher for turned heaps as compared to heaps that were not turned. This in turn gave a sour taste to chocolate made from these beans, as revealed through sensory analysis. Also, the polyphenol and alkaloid contents of cocoa beans were crop- and heap-dependent. Polyphenols, theobromine, and caffeine contribute to astringency and bitterness of cocoa and chocolate. However, there was a serious drop of polyphenol and alkaloid levels during cocoa bean fermentation and a further fall of polyphenol levels during drying of fermented cocoa beans. Diffusion of soluble polyphenols, in particular epicatechin, and alkaloids, in particular theobromine, out of the bean cotyledons during fermentation, polyphenol oxidation, and condensation of polyphenols and proteins were responsible for a decrease of their levels and hence the bitter taste of the final chocolates. The cut test revealed well-fermented cocoa beans in all cases, but did not guarantee chocolates with the same or good taste. To define an appropiate starter culture formulation, in vitro fermentations were carried out in a cocoa pulp simulation medium to study microbial growth and metabolite production of the LAB and AAB present during Ghanaian cocoa bean heap fermentation. A selection was made out of the available pool of LAB and AAB cocoa isolates. Small-scale fermentations were done to determine fast growing strains that are ethanol-tolerant and tolerant to low pH, able to metabolize citric acid (LAB) or to oxidize ethanol to acetic acid (AAB). After large scale fermentation tests and extensive kinetic analyses the strains L. plantarum 80, L. fermentum 222, and A. pasteurianus 386B were chosen to be used as a starter culture for cocoa bean heap fermentations. Different starter culture mixes were tested during six cocoa bean heap fermentations performed in Ghana. Cocoa bean heap fermentations were performed in a natural (farmer’s plantation) and artificial (factory’s site) environment. The heap fermentations were not inoculated with yeasts, but a natural yeast fermentation was observed. Although the cocoa pod surfaces were not decontaminated, in all the cases the starter culture was able to outgrow the natural microbiota and dominate the entire fermentation course. The dominance of the starter culture may be ascribed to a faster degradation of citric acid and sugars, reflecting the higher end-concentrations of organic acids in pulp and beans. This study shows possibilities for starter cultures to be used in cocoa bean heap fermentations to come to a reliable and controlled fermentation that can consistently produce high quality cocoa and chocolate. To conclude, the present multiphasic study indicates that successful cocoa bean heap fermentation requires a succession of microbial activities, i.e. pectin degradation and ethanol formation by yeasts, citrate and sugar fermentation into lactic acid, acetic acid, and mannitol by LAB, and ethanol, lactate, and mannitol oxidation by AAB. There is almost no influence of the season and farmer’s plantation, although microbial variability influences chocolate flavor. Whereas turning of heaps accelerates fermentation speed and favors AAB growth, it does not necessarily favor the flavor of the fermented beans and the concomitant chocolate, at least for small heaps. Concerning LAB and AAB, species diversity is restricted; certain strains of L. plantarum, L. fermentum, and A. pasteurianus dominated the fermentation course. In addition, fermenting cocoa beans are a source of new LAB and AAB species, such as the newly described Weissella ghanensis and Acetobacter ghanensis. Although thought differently in the past, LAB do play an important role during the cocoa bean fermentation process, as citric acid fermentation by LAB plays a pH regulatory role, the lactic and acetic acids produced control microbial contamination, and the lactate and mannitol produced are beneficial for the growth of AAB. Finally, it has been shown that bacterial fermentation (microbial species and fermentation metabolites) has an impact on the flavor of chocolate made of the corresponding dried fermented cocoa beans, as revealed by the application of the competitive strains L. plantarum 80 and/or L. fermentum 222 plus A. pasteurianus 386B. Hence, the choice of the starter culture (microbial species and size of the inoculum) will be crucial to control the fermentation process and to steer taste and flavor of chocolate products obtained thereof.

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