Optimization of ultrasound assist extraction of polyphenols in cocoa beans / Optimización del proceso de extracción por ultrasonido de polifenoles en granos de cacao

Background: The determination of polyphenols in cocoa beans allows the establishing of antioxidant properties of great benefit to this product. Objectives: Optimization of the ultrasound-assisted extraction method to determine the content of polyphenols in cocoa beans. Methods: Two experiments design stages were carried out to optimize the ultrasound-assisted extraction process of polyphenols from the cocoa bean. In the first experiment design stage, the adequate extraction solvent was determined; for this purpose, five types of solvents were evaluated through a completely random design unrestricted (CRD). In the second experiment stage, a central composite design 22 + star point (with two central points) was used, which was evaluated using the response surface methodology to determine the influence of the temperature, time, and solute / solvent ratio. Results: The experiment found that acetone: water: acetic acid (70: 29.5: 0.5) mixturee, leads to a greater amount of total extracted phenols measured for the Folin-Ciocalteu method. Analysis of variance (ANOVA) found that six significant effects that influence the response variable (total phenols extracted). The main effects were of the three factors and three of their interactions. Conclusions: After the optimizing said factors, an optimal point was found: 39.3 ° C of temperature, 74.5 minutes, and 22.8 mL of solvent per gram of cocoa sample.

Antecedentes: La determinación de polifenoles en granos de Cacao permite establecer propiedades antioxidantes de gran valor agregado a este producto. Objetivo: Optimización del método de extracción asistida con ultrasonido para determinar el contenido de polifenoles en granos de Cacao. Métodos: Se realizaron dos etapas del diseño experimental para la optimización del proceso de extracción de polifenoles del grano de cacao por ultrasonido. En la primera etapa se determinó el solvente de extracción más adecuado evaluando cinco tipos de solventes, mediante un diseño completamente al azar sin restricciones (DCA). En la segunda etapa experimental, se usó un diseño central compuesto 22 + estrella (con dos puntos centrales), que se evaluó mediante la metodología de superficie de respuesta para determinar la influencia de los factores Temperatura, tiempo y relación soluto/solvente. Resultados: Se determinó que la mezcla acetona: agua: ácido acético (70:29.5:0.5) extrae la mayor cantidad de fenoles totales cuantificados por el método de Folin-Ciocalteu. Mediante la aplicación de un análisis ANOVA, se encontró que seis efectos significativos influyen sobre la variable de respuesta (fenoles totales), estos incluyen los efectos principales de los tres factores y tres de sus interacciones. Conclusiones: Después de la optimización de dichos factores se encontró un punto óptimo que corresponde a 39.3 °C de temperatura, 74.5 minutos y 22.8 mL de solvente por gramo de muestra de cacao.

Caracterización molecular de los microorganismos presentes durante el proceso fermentativo de los granos de cacao (Theobroma cacao) / Molecular characterization of microorganisms during cocoa-bean fermentation process (Theobroma cacao)

La fermentación de granos de cacao es un proceso espontáneo de post cosecha muy importante para el desarrollo de aroma y sabor a chocolate el cual involucra un sin número de actividades microbianas complejas. En el presente estudio se identifican los microorganismos presentes en granos de cacao antes, durante y después del proceso de fermentación aplicando dos métodos: el análisis de secuenciamiento de ADN y la espectrometría de masas MALDI TOF/TOF. Dentro del grupo de bacterias y levaduras predominantes identificadas por el primer método se encontro a Lactobacillus plantarum (29%), L. brevis (18%), Bacillus cereus (15%), Pediococcus acidilactici (12%), y Pichia kudriavzevii (100%). Asimismo se caracterizó por huella de masas las secuencias peptídicas más importantes de cada cepa identificada. Por otro lado, aplicando el segundo método, se identificaron 57 especies de microorganismos, siendo el 73.7% especies bacterianas y el 26.3% especies de levaduras. Adicionalmente se detectaron secuencias peptídicas de la proteína vicilina responsable del aroma característico de los granos de cacao fermentados y a la proteína albumina de 21KDa.

Cocoa beans fermentation is a spontaneous process of post-harvest very important for the development of chocolate aroma and flavor, which involves a number of complex microbial activities. In this work, we identify the microorganisms present in cocoa beans before, during and after the fermentation process, applying DNA sequencing analysis and MALDI TOF / TOF mass spectrometry. With the first method, the predominant bacteria and yeast identified were Lactobacillus plantarum (29%), L. brevis (18%), Bacillus cereus (15%), Pediococcus acidilactici (12%), and Pichia kudriavzevii (100%). The most important peptide sequences of each identified strain by mass fingerprint were characterized too. By the second method, 51 species of microorganisms being 73.7% bacterial species and 26.3% yeast species were identified. Additionally peptide sequences responsible Vicilin protein characteristic aroma of the fermented cocoa beans and the albumin protein of 21KDa were detected.

Diversity of yeasts during fermentation of cocoa from two sites in the Brazilian Amazon

The purpose of this study was to identify the yeasts involved in spontaneous fermentation of cocoa from the Brazilian Amazon region. The fermentation process was carried out experimentally with cocoa seeds from two sites (Medicilândia and Tucumã), State of Pará, northern Brazil, during a six-day period. Totals of 44 yeasts were isolated from Medicilândia and 29 from Tucumã. Molecular identification was carried out by sequencing the D1/D2 region fragment of the rRNA 26S gene, expanded with universal primers for the NL1GC and LS2 eukaryotes. Pichia manshurica and Saccharomyces cerevisiae were identified in Medicilândia and five yeast species (Pichia fermentans, P. kudriavzevii, P. manshurica, S. cerevisiae and Zygosaccharomyces bailii) were identified in Tucumã. The results showed that P. manshurica and S. cerevisiae may have potential for use as starter cultures in future studies to improve the quality of cocoa seeds fermented in the Brazilian Amazon region. (AU)

A proposta deste estudo foi identificar as leveduras envolvidas na fermentação espontânea de cacau da Amazônia brasileira. A fermentação foi realizada em Medicilândia e Tucumã, Pará, Brasil, durante 6 dias. Em total foram obtidos 44 isolados de leveduras de Medicilândia e 29 de Tucumã. A identificação molecular foi realizada por sequenciamento do fragmento da região D1/D2 do gene rRNA 26S, amplificado com primers universais para eucariotos NL1GC e LS2. Em Medicilândia, foram identificadas Pichia manshurica e Saccharomyces cerevisiae. Em Tucumã foram identificadas cinco espécies (Pichia fermentans, P. kudriavzevii, P. manshurica, S. cerevisiae e Zygosaccharomyces bailii). Os resultados sugerem que P. manshurica e S. cerevisiae podem ter potencial para uso como culturas starter em estudos futuros, para melhorar a qualidade das sementes de cacau fermentadas na Amazônia brasileira.(AU)

Assessment of the Diversity of Lactic Acid Bacteria Involved in Cocoa Fermentation of Six Main Cocoa Producing Regions of Côte d’ivoire

Aims: The variability of lactic acid bacteria (LAB) species involved in cocoa bean fermentation would cause inconsistency in the quality of cocoa. The aims of this study is to investigate the physicochemical parameters of cocoa bean fermentation in order to assess the activity and the molecular diversity of LAB involved in cocoa fermentation from six other regions of Côte d’Ivoire. Place and Duration of Study: Laboratory of Biotechnology, UFR Biosciences, University Félix Houphouet-Boigny (Côte d’Ivoire), between October 2016 and September 2017. Methodology: Spontaneous heap fermentations were conducted in six cocoa producing regions during 6 days. Physicochemical analysis of cocoa mass such as temperature, pH, titratable acidity and reducing sugars were carried out. In addition, LAB isolation was performed using plate culture on MRS medium and their fermentative type as well as their profile were determined. In addition, LAB species were determined by restriction profile analysis of the 16S gene. Results: a total of 568 LAB were isolated from cocoa fermentation. Biochemical and morphological identification of these germs revealed the clear dominance of the bacilli form (81.16%) and the heterofermentative type (over 80%) with facultative heterofermentative type recording more than half (54.4%) of the isolated population. Their molecular identification by sequencing the hypervariable zone of the 16S rDNA gene of a few representatives from each restriction group revealed 08 species with a predominance of Lactobacillus plantarum (76.76%) and Leuconostoc mesenteroides (15.31%) associated with minority species. This species diversity could be exploited for selecting appropriate starter cultures. Conclusion: This diversity of LAB species could be responsible for the variability of cocoa quality in Côte d’Ivoire.