# Impact of a Microbial Cocktail Used as a Starter Culture on Cocoa Fermentation and Chocolate Flavor
## 1 Introduction  
The cocoa (_Thebroma cacao_ L.) supply chain for the production of chocolate is complex. It involves several post-harvest steps, which can determine the quality of the final product. In Brazil, cocoa production suffered a vast impact with the emergence of \"witches' broom\" disease [1; 2]. In order to recover the cocoa production, many disease-resistant hybrid plants, such as PH9, PH15, PH16, PS1030, PS1319, CCN51, CEPEC2002, CEPEC2004, and FA13, have been developed [3; 4].  
As a matter of consequence, different cocoa hybrids generate cocoa beans that produce chocolate with variable quality [5; 6; 7]. In this context, PH15 hybrid has great relevance due to high-productivity, adaptation, and resistance to some diseases, such as \"witches' broom\" and ceratocysts wilt [8; 9; 10].  
The fermentation of cocoa beans is a microbiological process with enzymatic activity and the development of chocolate flavor precursors [11; 12; 13]. This traditional process is spontaneous and uncontrolled. After opening of the cocoa pods, the beans are transferred to the area of fermentation and placed in heap or fermentation boxes. These methods are the most commonly used among the cocoa producer countries [14; 15].  
Yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) are the main microbial communities involved during cocoa fermentation. Yeast species are reported as the primary colonizers of cocoa fermentation. _Saccharomyces_, _Hansenispora_ (anamorph _Kloeckera_), and _Pichia_ are the prevalent genera found in cocoa fermentation in different countries. _Saccharomyces cerevisiae_ is particularly the most reported species in many fermentations [16; 17; 18; 19; 20].  
Simultaneously with the yeast growth, LAB colonize the cocoa mass and degrade the pulp's glucose into lactic acid and assimilate the citric acid also present in the pulp. Several studies concerning the microbial fermentation reported two LAB species as the most prevalent in this process: _Lactobacillus plantarum_ and _Lactobacillus fermentum_[19; 20; 21].  
Yeast populations, which are responsible for the ethanol production, decline together with the LAB populations. AAB dominates the process and are responsible to the exothermic reaction of ethanol conversion into acetic acid. _Aectobacter pasteurianus_ is the most frequent species of AAB found in cocoa fermentation, but other species, such as _Aectobacter aceti_, _Aectobacter ghanensis_, _Aectobacter fabarum_, _Gluconobacter oxydans_, and _Gluconobacter xylinus_, have also been reported in the literature [16; 17; 20; 22].  
Species of _Bacillus_ (e.g., _Bacillus subtilis_, _Bacillus megaterium_, and _Bacillus flexus_) may also grow during fermentation and can affect bean quality and cocoa flavor [16; 17; 23].  
Different compounds, such as alcohols (e.g., 2-methyl-1-propanol, 2-phenylethylanol, methanol), aldehydes (e.g., acetaldehyde, benzaldehyde), ketones (e.g., 2-pentanone, phenylmethyl ketone), esters (e.g., ethyl acetate, 2-phenylethyl acetate), and carboxylic acids (e.g., butanoic acid, nonanoic acid), are produced during fermentation, affecting the final flavor character in chocolate [13; 17; 24; 25; 26].  
The aim of this work was to use a cocktail of microorganisms as a starter culture on the fermentation of the ripe cocoa pods from PH15 cocoa hybrid, and evaluate its influence on the microbial communities present on the fermentative process, on both the volatile and non-volatile compounds produced during the fermentation, and to perform the chocolate sensorial characterization.