# Impact of a Microbial Cocktail Used as a Starter Culture on Cocoa Fermentation and Chocolate Flavor
## 2 Results  
### Culture-Independent Analysis with PCR-DGGE  
Analyses of the microbial communities on both inoculated and non-inoculated samples of the PH15 cocoa hybrid were performed by Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis (PCR-DGGE) for prokaryote (Figure 1A) and eukaryote (Figure 1B) microorganisms. Identification of DGGE bands are shown in Table 1.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Identification** & **Bands \({}^{\text{a}}\)** & **Similarity** & **Accession** & **Sample Found** \\ \hline
**Prokaryotes** & & & & \\ _Fructobacillus pseudoficultneus_ & 1, 5, 6 & 98 & AB498052.1 & PH15 NI, PH15I \\ _Lencomoco_ sp. & 2 & 97 & DC254391.1 & PH15 NI \\ _Lactobacillus plantarum_ & 3, 4, 8, 9 & 99 & K1327866.1 & PH15 NI, PH15I \\ _Lactobacillus hettericus_ & 7 & 99 & KP764179.1 & PH15I \\ _Zymomonas mobilis_ & 10 & 100 & CP003715.1 & PH15 NI \\ Uncultured bacterium & 11, 12, 13, 14 & 99 & LN875309.1 & PH15 NI, PH15I \\ _Aectobacter pasteurianus_ & 15, 16, 17, 18, 19, 20 & 100 & K4983001.1 & PH15 NI, PH15 NI \\ _Aectobacter_ sp. & 21 & 98 & KCP76965.1 & PH15 NI \\ _Gluconobacter oxydans_ & 22, 23, 24 & 99 & CP003926 & PH15 NI, PH15I \\ _Bacillus_ sp. & 25, 26 & 99 & JF309224 & PH15 NI \\ \hline \hline \end{tabular}
\end{table}
Table 1: Identification of the bands based on Basic Local Alignment Search Tool (BLAST) in comparison with those in GenBank as obtained by PCR-DGGE using universal primers for yeasts and bacteria.  
The bacterial and yeast communities changed according to different fermentation processes (inoculated or non-inoculated). The bacterial species _Glucobacter oxydans_ (bands 22-24), _Lactobacillus plantarum_ (bands 3, 4, 8, and 9), uncultured bacterium (bands 11-15), _Acetobacter pasteurianus_ (bands 15-20), and _Fructobacillus pseudoficulneus_ (bands 1, 5, and 6) were detected for both inoculated and non-inoculated fermentations.  
The species _Leuconstoc_ sp. (band 2), _Zymomonas mobilis_ (Band 10), _Acetobacter_ sp. (Bands 21), _Bacillus_ sp. (Bands 25 and 26) were detected in the non-inoculated fermentation. The _Lactobacillus helveticus_ (Band 7) was only detected in inoculated fermentation. The yeast species _Saccharomyces  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Identification** & **Bands a** & **Similarity** & **Accession** & **Sample Found** \\ \hline
**Eukaryotes** & & & & \\ _Hansenispora uvarum_ & 1, 2 & 99 & KC544511 & PH15 NI \\ _Saccharomyces cerevisiae_ & 3, 5, 6, 7, 8, 9, 10 & 99 & KT22954.1 & PH15 NI, PH15I \\ _Saccharomyces_ sp. & 4 & 98 & KU350335.1 & PH15 NI \\ _Thoebronica cao_ & 11 & 97 & J228327.17 & PH15 NI \\ _Rhodotorula uncilaginous_ & 12, 13 & 100 & HM588765 & PH15I \\ _Lentinula edodes_ & 14 & 98 & KM015456.1 & PH15I \\ _Pichia tluyveri_ & 15, 16, 17, 18, 20 & 99 & FM864201 & PH15 NI, PH15I \\ _Trichosporan ashii_ & 19 & 97 & JQ425402 & PH15I \\ \hline \end{tabular}  
* Bands are numbered as indicated on the DGGE gel. PH15 NI: PH15 non-inoculated; PH15I: PH15 inoculated.  
\end{table}
Table 1: _Cont._  
Figure 1: Changes in prokaryote (**A**) and eukaryotic (**B**) communities during fermentation of the PH15 cocoa hybrid without (PH15 NI) and with (PH15 I) inoculation, and fingerprints of the starter cultures (CCMA 0238, CCMA 0241, and CCMA 0200). The identities of the bands are presented in Table 1. Bands marked with numbers were excised, re-amplified, and sequenced.  
cerevisiae_ (Bands 3 and 5-10) and _Pichia kluyveri_ (Bands 15-18 and 20) were detected for both fermentations. _Saccharomyces_ sp. (Band 4), _Hanseniispora uvarum_ (Bands 1 and 2), and _Theobroma cacao_ (Band 11) were only detected in the non-inoculated fermentation: this was possible because universal eukaryote primers were used. In the inoculated fermentation _Rhodotorula mucilaginousa_ (Bands 12 and 13), _Trichospora asalhi_ (Band 19), and _Lentinua edodes_ (Band 14) were detected.