Gut Health Benefits of Natural and Alkali-Processed Cocoa (Theobroma cacao) with and without Inulin

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.