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Browsing by Author "Lambert, Joshua D."

Now showing 1 - 5 of 5
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    Anti-Diabetic and Anti-Obesity Activities of Cocoa (Theobroma cacao) via Physiological Enzyme Inhibition
    Ryan, Caroline Mary (Virginia Tech, 2016-06-01)
    Fermentation and roasting of cocoa (Theobroma cacao) decrease levels of polyphenolic flavanol compounds. However, it is largely unknown how these changes in polyphenol levels caused by processing affect cocoa's anti-diabetic and anti-obesity bioactivities, such as inhibition of certain enzymes in the body. Polyphenol profiles, protein-binding abilities, presence of compounds termed oxidative polymers, and abilities to inhibit α-glucosidase, pancreatic α-amylase, lipase, and dipeptidyl peptidase-IV (DPP4) in vitro were compared between unfermented bean (UB), fermented bean (FB), unfermented liquor (UL), and fermented liquor (FL) cocoa extracts. Overall, there were significant decreases (p<0.05) in total polyphenols, flavanols, and anthocyanins between the two sets of unfermented and fermented cocoa extracts (CEs). All CEs effectively inhibited α-glucosidase (lowest IC50 = 90.0 ug/mL for UL) and moderately inhibited α-amylase (lowest IC50=183 ug/mL for FL), lipase (lowest IC25=65.5 ug/mL for FB), and DPP4 (lowest IC25=1585 ug/mL for FB) in dose-dependent manners. Fermentation and roasting of the samples affected inhibition of each enzyme differently (both processes enhanced α-amylase inhibition). Improved α-glucosidase and α-amylase inhibitions were correlated with presence of different classifications of oxidative polymers, suggesting that these compounds could be contributing to the bioactivities observed. Some α-glucosidase inhibition might be due to non-specific protein-binding. Improved DPP4 inhibition was strongly correlated to increased CE degree of polymerization. In conclusion, potential enzyme inhibition activities of cocoa were not necessarily negatively affected by the large polyphenol losses that occur during fermentation and roasting. Additionally, it is possible that more complex compounds could be present in cocoa that contribute to its potential anti-diabetic and anti-obesity bioactivities.
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    Development and Characterization of a Pilot-Scale Model Cocoa Fermentation System Suitable for Studying the Impact of Fermentation on Putative Bioactive Compounds and Bioactivity of Cocoa
    Racine, Kathryn C.; Lee, Andrew H.; Wiersema, Brian D.; Huang, Haibo; Lambert, Joshua D.; Stewart, Amanda C.; Neilson, Andrew P. (MDPI, 2019-03-19)
    Cocoa is a concentrated source of dietary flavanols—putative bioactive compounds associated with health benefits. It is known that fermentation and roasting reduce levels of native flavonoids in cocoa, and it is generally thought that this loss translates to reduced bioactivity. However, the mechanisms of these losses are poorly understood, and little data exist to support this paradigm that flavonoid loss results in reduced health benefits. To further facilitate large-scale studies of the impact of fermentation on cocoa flavanols, a controlled laboratory fermentation model system was increased in scale to a large (pilot) scale system. Raw cocoa beans (15 kg) were fermented in 16 L of a simulated pulp media in duplicate for 168 h. The temperature of the fermentation was increased from 25–55 °C at a rate of 5 °C/24 h. As expected, total polyphenols and flavanol levels decreased as fermentation progressed (a loss of 18.3% total polyphenols and 14.4% loss of total flavanols during fermentation) but some increases were observed in the final timepoints (120–168 h). Fermentation substrates, metabolites and putative cocoa bioactive compounds were monitored and found to follow typical trends for on-farm cocoa heap fermentations. For example, sucrose levels in pulp declined from >40 mg/mL to undetectable at 96 h. This model system provides a controlled environment for further investigation into the potential for optimizing fermentation parameters to enhance the flavanol composition and the potential health benefits of the resultant cocoa beans.
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    Evaluation of cocoa (Theobroma cacao) bean processing strategies to enhance alpha-glucosidase inhibitory activity of dietary cocoa
    Racine, Kathryn Claire (Virginia Tech, 2019-06-18)
    Cocoa beans (Theobroma cacao) are a highly concentrated source of dietary flavanols- bioactive compounds associated with the health protective properties of cocoa. Cocoa beans undergo processing steps, such as fermentation, roasting, winnowing, grinding, pressing, etc., to produce a final product with specific desirable sensory attributes. It is well established that these processing steps, specifically fermentation and roasting, result in dramatic degradation of cocoa's native flavanols, but it is possible that these processing steps may generate compounds with novel activities, potentially preserving or enhancing bioactivity. Raw unfermented cocoa beans were processed by way of a partial factorial approach to produce cocoa powders from the same batch of raw beans using various combinations of fermentation [unfermented, cool fermented (maximum 46°C), hot fermented (maximum 60°C))] and roasting [unroasted, cool roasted (120°C), hot roasted (170°C)]. To simulate cocoa fermentation in a highly controlled environment, a pilot-scale fermentation model system was employed to eliminate many external unknowns and ensure that the differences between our cocoa powders were due to our various treatments, rather than unknown factors occurring during fermentation and roasting. Low and high molecular weight fractions (8-10 kDa cutoff) were produced from cocoa powder extracts (CPE) of each treatment to quantify Maillard reaction products (MRP). A HILIC-UPLC MS/MS method was developed to more efficiently and sensitively quantify cocoa flavanols with high degrees of polymerization (DP) produced during processing. Overall, cocoa processing significantly (p<0.05) decreased the total phenolic and total flavanol concentrations of CPEs. Hot roasting had the greatest impact on native flavanol degradation yet produced CPEs with the highest mean degree of polymerization (mDP). All CPEs dose-dependently inhibited α-glucosidase enzyme activity, with cool fermented/cool roasted cocoa powder exhibiting the best inhibition (IC50 of 62.2 µg/mL). Increasing flavanol mDP was correlated with decreasing IC50 values, suggesting that the complex flavanols produced during processing enhance cocoa's bioactivity (or their production is associated with other products that enhance bioactivity). Alternatively, high molecular weight CPE fractions were correlated with increasing IC50 values, suggesting that MRPs interfere with enzyme inhibition or are associated with other products (polyphenols, macronutrients, etc.) that interfere with enzyme inhibition. Overall, the data presented within this work indicate that the components of processed cocoa powders are promising inhibitors of α-glucosidase, despite a significant reduction in native flavanol composition induced by processing, and moreover that fermentation and roasting conditions can positively influence the bioactivity of cocoa despite losses of native flavanols.
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    Flavanol Polymerization Is a Superior Predictor of α-Glucosidase Inhibitory Activity Compared to Flavanol or Total Polyphenol Concentrations in Cocoas Prepared by Variations in Controlled Fermentation and Roasting of the Same Raw Cocoa Beans
    Racine, Kathryn C.; Wiersema, Brian D.; Griffin, Laura E.; Essenmacher, Lauren A.; Lee, Andrew H.; Hopfer, Helene; Lambert, Joshua D.; Stewart, Amanda C.; Neilson, Andrew P. (MDPI, 2019-12-11)
    Raw cocoa beans were processed to produce cocoa powders with different combinations of fermentation (unfermented, cool, or hot) and roasting (not roasted, cool, or hot). Cocoa powder extracts were characterized and assessed for α-glucosidase inhibitory activity in vitro. Cocoa processing (fermentation/roasting) contributed to significant losses of native flavanols. All of the treatments dose-dependently inhibited α-glucosidase activity, with cool fermented/cool roasted powder exhibiting the greatest potency (IC50: 68.09 µg/mL), when compared to acarbose (IC50: 133.22 µg/mL). A strong negative correlation was observed between flavanol mDP and IC50, suggesting flavanol polymerization as a marker of enhanced α-glucosidase inhibition in cocoa. Our data demonstrate that cocoa powders are potent inhibitors of α-glucosidase. Significant reductions in the total polyphenol and flavanol concentrations induced by processing do not necessarily dictate a reduced capacity for α-glucosidase inhibition, but rather these steps can enhance cocoa bioactivity. Non-traditional compositional markers may be better predictors of enzyme inhibitory activity than cocoa native flavanols.
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    Gut Health Benefits of Natural and Alkali-Processed Cocoa (Theobroma cacao) with and without Inulin
    Essenmacher, Lauren Alexis (Virginia Tech, 2020-06-22)
    Chronic conditions such as obesity, inflammatory bowel disease (IBD), and colitis are associated with gastrointestinal (GI) inflammation and compromised GI barrier integrity. Cocoa may be a potential dietary strategy to mitigate gut-related conditions and been shown to elicit anti-inflammatory, antioxidant, and prebiotic effects. Alkali treatment of cocoa was once thought to reduce its bioactivity, but new evidence suggests it may enhance cocoa's health properties, through the formation of new, potentially bioactive high molecular weight compounds. Inulin, a fructose-containing plant polymer, exerts prebiotic effects and has also been investigated in the mitigation of IBD. This study aims to 1) investigate effects of alkali processing on gut health related bioactivity and phytochemical composition of cocoa and 2) evaluate potential additive benefits of combining cocoa and inulin. Polyphenolic and flavanol compounds in natural cocoa, alkalized cocoa, and inulin powders were characterized using Folin-Ciocalteu (total polyphenols) and 4-dimethylaminocinnamaldehyde (total flavanols) assays, thiolysis , and HILIC UPLC-MS/MS. Treatments of cocoa and inulin were made in 1:2 cocoa:inulin and 1:4 cocoa:inulin mixtures for both natural and alkalized cocoas. Cocoa mixtures, in addition to both cocoa powders and inulin alone, were subjected to an in-vitro digestion to generate material for an in-vitro fecal fermentation. Samples collected from the fermentation at 0, 6, 12, and 24 hours were analyzed via HPLC-MS for microbial metabolites, applied to HT-29 colon cancer cells to assess anti-inflammatory activity, and applied to a florescence assay measuring PLA2 inhibitory activity. The alkalized cocoa powder was found to have a significantly lower concentration of total polyphenols and total flavanols, as well as a lower mDP, suggesting that alkalization may affect larger procyanidins more than smaller flavanol compounds. Inulin enhanced the inhibition of the PLA2 enzyme and enhanced the IL-8 anti-inflammatory properties of cocoa, although the trends were weak. Overall, we did not see any clear, significant effects of alkalization or the addition of inulin to cocoa's colonic metabolite formation or its gut bioactivity in vitro. However, we have demonstrated that colonic fermentation of cocoa may have a negative effect on its bioactivity in vitro. Future research should further explore flavanol DP and bioactivity, fiber's interaction with polyphenols, colonic metabolism of cocoa, and cocoa's gut health effects in vivo.