(57c) Implementation of a Mixed Integer Linear Programming Approach to Establish De Novo Synthesis Routes of Antioxidants Derived from the Fermentation of Theobroma Cacao Seeds

Cacao (Theobroma cacao) is a plant of South American origin rich in nutrient contents widely commercialized around the globe because of the production of chocolate. Chocolate is a highly demanded product due to its organoleptic characteristics and nutritional value. In order to obtain this product, cacao seeds covered by a mucilaginous pulp of the fruit are fermented in boxes in a traditional way. This process lasts about 6 to 7 days and begins whit the pulp degradation and consequent formation of the flavor, odor and color precursors which, strongly influence the chocolate’s quality. Three main types of microorganisms found in the fermentation process have been proven to be essential to obtain of high-quality chocolate: yeasts, acetic and lactic acid bacteria. Among the nutritional compounds and organoleptic properties than make cacao a very desirable product with a high added value, the most important are: antioxidants and astringent, bitter and sweet flavors. Considering the nutritional and economic importance of the compounds found in fermented cacao seeds, a need to deeply understand and determine the metabolic routes associated with the biochemical reactions they are involved in arises. Then this work aimed to propose novel routes to synthesize antioxidants derived from enzymes present in the fermentation process of Theobroma cacao seeds that could be used to construct a complete process of production in bioreactor using heterologous expression. To gain in the knowledge of those products, it is proposed to create a catalogue of the metabolic routes associated with the synthesis of both nutritional and organoleptic compounds.

METHODS

First, we determined the optimal reactant and product combination with the overall stoichiometry of overall reactions proposed for obtain the naringenin flavonoid; a. from glucose, b. the amino acid L-tyrosine and c. L-phenylalanine and d. shikimate, to obtain the maximum achievable yield for all the possible bioconversions. This was carried out considering the thermodynamic restrictions. Metabolites and reactions required database were obtained based on reported metabolic reconstructions of individual species involved of the fermentation process and the complete KEGG database was also used. All reactions were considered reversible in the databases. Substrates and products were obtained using the polyphenol composition of the seed and an antioxidant predictor respectively. Then, it was identified the routes and reactions that satisfy the identified overall stoichiometry taking into account the free energy change of the reactions and minimizing the amount of reactions required to the pathways. Subsequently, general reactions were generated to produce naringenin compounds, and the catalog of metabolic pathways associated with the synthesis of nutritional and organoleptic compounds of fermentation were prepared.

RESULTS

By means of both the algorithm and the catalogue, eleven metabolic routes for each of the proposed general reactions that are involved in the synthesis of the most relevant antioxidants were obtained from glucose, L-tyrosine, L-phenylalanine and shikimate, and later, from naringenin. The catalogue made easier the search for compounds involved not only in the fermentation process of cacao, but in any other fermentation process in food industry.

CONCLUSION

Due to the economic and nutritional importance of chocolate, the identification of the routes of synthesis of precursor compounds, organoleptic and nutritional characteristics, helps in the design of high quality products, not only in the chocolate industry but also in any other product Fermented industry.