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Consumption of cocoa and dark chocolate products has been associated with positive health outcomes including reduced onset of cardiovascular disease, inflammation, diabetes, obesity, and platelet disorders. Cocoa polyphenols, putatively responsible for these beneficial activities, are highly impacted by cocoa variety, agronomic effects and processing history. However, the difference in polyphenol concentration and composition between cocoa products originating from different hybrid clones (selected for high yield) or from different fermentation conditions is not fully understood. Detailed polyphenol characterization including determination of total polyphenol and total procyanidin concentrations, and qualitative and quantitative analysis of (mean) degree of polymerization was conducted. Significant differences in total polyphenol and procyanidin concentrations were observed between five genetic clones grown by the USDA-ARS Cocoa Germplasm Repository located in Mayagüez, Puerto Rico. To facilitate cocoa fermentation research in laboratories distant from cocoa harvesting sites, a laboratory-scale cocoa fermentation model system was developed in this study. This model system used dried, unfermented, cocoa beans and simulated pulp medium as the starting material. The model system supported growth of the essential succession of cocoa fermenting microorganisms and generated similar chemical changes to those observed in on-farm cocoa fermentation. Using this model system, the impact of inoculation with proprietary yeast strains Saccharomyces cerevisiae Lev F and Saccharomyces cerevisiae Lev B on cocoa polyphenol concentration and composition was evaluated. Inoculation with both yeast strains resulted in increased fermentation rate and Lev B inoculation resulted in higher total polyphenol and procyandin contents at the end of fermentation. Overall, the present work addressed the influence of cocoa variety selection and fermentation process conditions on the composition and concentration of polyphenols. These findings will contribute to continued efforts to develop cocoa products with optimized bioactivity and maximum disease preventative effects.

Procyanidins (PCs) have been extensively investigated for their potential health protective activities, but the prospective bioactivities are limited by their poor bioavailability. The majority of the ingested dose remains unabsorbed and reaches the colon where extensive microbial metabolism occurs. The objectives of these studies are to better understand the roles and activities of PCs in the lower gastrointestinal tract. First, a new high-throughput Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry method was developed to efficiently analyze PCs and an extensive profile of their microbial metabolites. This method is sufficiently sensitive and effective in simultaneously extracting and measuring native PCs and their microbial metabolites in biological samples. Furthermore, administration of grape seed extract increased the expression of gut junction protein occludin and reduced levels of fecal calprotectin, which suggests an improvement of gut barrier integrity and a potential modulation of endotoxemia. Additionally, chronic supplementation of the diet with flavanols did not increase colonic tissue accumulation of PCs or their microbial metabolites over a 12 week feeding study. This was the first long-term study of its kind, and the results indicate that we still do not fully understand the outcome of ingested flavanols in the colon during chronic exposure rather than acute doses. Lastly, new understanding of the microbial metabolism of PCs in the colon has been reached by studying the colon as 4 segments, rather than as a complete unit as previous studies have done. Data show that a gradient is established along the length of the colon for both PCs and their metabolites, with PCs reaching highest concentrations within 3 h after ingestion, while metabolites reach maximum concentrations anywhere form 3-18 h after ingestion. Moreover, data indicate the progressive, step-wise degradation of PCs into small metabolites throughout the length of the colon. Overall, there is greater understanding of the colonic metabolism of dietary PCs derived from GSE and cocoa, the accumulation of these compounds, and their effect on gut permeability. Future work will build off of these novel studies, and will continue to advance the understanding of the health benefits of dietary PCs.

Flavanols are polyphenols that are known to have many beneficial effects on the body. Cocoa is a major source of these flavanols. However, research on the potential anti-obesity and anti-diabetic health benefits of cocoa flavanols is lacking in the literature. Furthermore, the effect that the size of these flavanols has on the extent of its beneficial properties is also unclear. The objective of this study was to evaluate the dietary effects of cocoa flavanols on the onset of obesity, insulin resistance and impaired glucose tolerance and to determine the impact that the size of these compounds has on the magnitude of this effect. Cocoa extract was fractionated into a monomer-, an oligomer-, and a polymer-rich fraction. Six groups (n=9) of C57BL/6J mice were fed either a control low-fat diet, a control high-fat diet, or a high-fat diet supplemented with 25 mg/kg*BW of cocoa extract or one of the three cocoa fractions. After 12 weeks on these diets, the oligomer-rich fraction proved to be most effective in preventing weight gain, fat mass accumulation, elevated fasting blood glucose and impaired glucose tolerance in diet-induced obese mice. This is the first long-term feeding study to examine the relative activities of cocoa constituents on diet-induced obesity and insulin resistance.