# Impact of a Microbial Cocktail Used as a Starter Culture on Cocoa Fermentation and Chocolate Flavor
## 3 Discussion  
In order to evaluate their influence on the fermentation of cocoa beans and on the final sensorial characteristics of produced chocolate, _S. cerevisiae_ UFLA CCMA 0200, _Lactobacillus plantarum_ CCMA 0238, and _Acetobacter pasteurianus_ CCMA 0241 were used as starter cultures for the cocoa PH15 fermentation. The organic compounds and microbial communities involved during the fermentation of non-inoculated and inoculated cocoa were analyzed. Furthermore, the sensorial characterization of chocolate (PH15 I Ch and PH15 NI Ch) produced from the fermented beans was evaluated. The PCR-DGGE analyses showed that the bacterial and yeast communities were different according to the fermentation process. This may be explained by the use of starter cultures that may have generated changes in the natural microbiota, as shown in Figure 1A,B. Species of LAB and AAB were identified in both fermentations. The _L. plantarum_ and _Fructobacillus pseudoficulneus_ species were the bacteria present in both fermentations. However, in PH15 I these species were only identified until in the middle of the fermentation period (72 h), but after this time, they seem not to be present. It is reported that the increase of ethanol concentration during cocoa fermentation inhibited _L. fermentum_ growth [15; 25]. This could explain the low population rate of _L. plantarum_ in PH15 NI. _Gluconobacter oxydans_ and _A. pasteurianus_ were AAB species identified in both fermentation processes. In addition, _A. pasteurianus_ (Figure 1A--bands 15, 16, 17, 18, 19, and 20) was present at all fermentation times of PH15 I, and this did not happen in the fermentation without inoculum. These species have been described in cocoa bean fermentation in Brazil, Ghana, and Indonesia [3; 16; 19; 20]. Figure 5: Temporal Dominance Sensory of chocolate produced from cocoa fermented beans PH15 NI (**A**) and PH15 I (**B**). Sensorial attributes: Acid (\(\bullet\)  
The LAB species _Leuconostoc_ sp., _Lactobacillus helveticus_ were detected in non-inoculated and inoculated fermentations, respectively. The _Zymomonas mobilis_, an ethanol strain producer, _Acetobacter_ sp., an AAB species, and _Bacillus_ sp. were only detected in non-inoculated fermentation (Figure 1A). The _Bacillus_ sp. present in PH15 I fermentation can be explained as the fermentation was not in aseptic conditions. _Bacillus_ sp. and filamentous fungi may participate in the spontaneous cocoa bean fermentation process after four or five days of fermentation [14; 16; 17; 20; 25]. Fingerprinting based on PCR-DGGE showed that the most common yeast _Saccharomyces cerevisiae_ was present during the fermentation in both fermentations. This fact indicated that this yeast may be a promising starter culture used for the cocoa fermentation process. Some works using _S. cerevisiae_ as a starter culture have been reported [4; 7], and concluded that yeast inoculation accelerated the fermentation process. The microbial activity and metabolites produced during the cocoa beans' fermentation leads to an increase of temperature and pH value [19]. Therefore, this may explain the temperature and pH increase in both fermentations, but in PH15 I there was a greater increase. According to the chemical results, carbohydrates were consumed faster in the inoculated assay (Figure 2B). This is likely due to the higher microorganism population in the inoculated assay than in the control. Further, higher ethanol concentrations (almost two times the concentration detected in the control) were observed in this assay (Figure 3A). However, this was not the case for acetic acid, similar to results previously described elsewhere [4]. However, in inoculated fermentation, acetic acid was detected in the cotyledon at the end of fermentation (Figure 3C). Sucrose was not detected in the fermentation probably because it was hydrolyzed into glucose and fructose when the pods were broken open, as previously described [4].