**1. Introduction**

Cocoa beans (*Theobroma cacao*) are concentrated dietary sources of flavanols, a subclass of polyphenols, which are thought to be responsible for many of the bioactivities of cocoa [1–5]. (−)-epicatechin and (+)-catechin (which is epimerized to (−)-catechin during roasting) are the major monomeric flavanols in raw cocoa. Native flavanols in cocoa beans are approximately 58% procyanidins (PCs), or flavanol oligomers and polymers (monomeric residues linked via 4→β6 or 4→β8 bonds). These flavanols exhibit potent antioxidant and health-protective activities, including the modulation of oxidative stress and potentially reducing the risk of various chronic conditions, such as cardiovascular

disease (CVD), type II diabetes mellitus (T2D), and different forms of cancer. Additionally, cocoa beans contain other bioactives, such as lipids, fiber, lignins, melanoidins (after roasting), methylxanthines, and other complex compounds that have not been extensively characterized. Beans contain approximately 55% fat, 16% fiber, 10% protein, and 3% ash, depending on variety. The health benefits associated with dietary cocoa are likely due to multiple bioactive compounds and their interactions, rather than one compound or class of compounds, because of the complex composition of cocoa and the reactions that occur during cocoa processing [6].

After harvesting, cocoa beans undergo a series of processing steps, including fermentation, drying, roasting, winnowing, and various other processes that may include pressing or alkalization, to produce a final product, such as cocoa powder or chocolate. These processes strongly influence the chemical composition of the product, with fermentation resulting in approximately 0–70% loss of total polyphenols and roasting costing an additional 15–40% loss [7–9]. Additionally, non-enzymatic browning reactions occur between native cocoa polyphenols and mono- or polysaccharides, proteins, and amino acids to produce Maillard reaction products, most notably melanoidins [10]. The widely-accepted paradigm is that preservation of native flavanols is critical for retaining bioactivity [7,9,11]. However, it is possible that reactions occurring throughout processing may generate processing-derived compounds with novel activities, such as lignin-like complexes and melanoidins, potentially preserving or even enhancing certain bioactivities as compared to the raw cocoa bean [7,12–19]. The levels and activities of these large, complex, and diverse compounds in cocoa are largely unknown due to analytical challenges (such as lack of authentic standards, structural complexity and size of potential products), low bioavailability of large complex compounds, and the continued research focus on small monomeric flavanols.

In vivo, flavanols have highly variable systemic oral bioavailability, with absorption being inversely proportional to molecular weight and ~0% absorption for compounds ≥ tetramer [20–24]. Thus, the actions of these bioactives are likely to predominantly occur in the lumen and epithelium of the gastrointestinal (GI) tract, where the delivery of native flavanols and processing products is high and not subject to various barriers and metabolism/transport processes that reduce flavanol concentration and activity. Bioactivity exerted within the lumen and epithelium of the gut might play a key role in the mitigation and prevention of obesity and related conditions, such as T2D and CVD. Specifically, the inhibition of gut digestive enzymes to limit macronutrient digestion is a promising mode of bioactivity that does not require systemic bioavailability. Previous studies demonstrate that cocoa flavanols inhibit lipases, α-amylase, and α-glucosidase; α-glucosidase appears to be the most susceptible to cocoa inhibition [13,25]. α-glucosidase is a brush border enzyme that hydrolyzes starch and maltose into absorbable glucose [26]. The inhibition of α-glucosidase is a potential strategy for inhibiting or slowing blood glucose absorption in the context of glucose intolerance. Commercially available α-glucosidase inhibitors, such as acarbose, miglitol, and voglibose, come with high prices and various side effects (such as GI discomfort), therefore warranting the investigation of dietary flavonoids (from berries, red wine, green tea, cocoa, etc.) as potential inhibitors [16]. While isolated PCs are effective α-glucosidase inhibitors, we recently found that various cocoas were effective inhibitors of α-glucosidase, despite large reductions in native flavanols as a result of fermentation and roasting [13]. These results challenge the idea that losses of native flavanols inherently reduces all bioactivity of cocoa and, therefore, warrant further investigation into the factors that determine α-glucosidase inhibition. Specifically, the impact of cocoa processing on subsequent bioactivity and the identification of non-flavanol cocoa components with bioactivity are of interest.

Fermentation and roasting are the logical steps in cocoa processing to manipulate in order to produce a final product with variable flavanol composition. Sourcing cocoas with the same bean origin and known processing history is impractical if not impossible, due to poor bean traceability, poor documentation, and varying practices of fermentation. A controlled model pilot-scale fermentation using a common starting material is necessary to conduct research regarding the impact of fermentation and further processing on bean composition and subsequent bioactivity [27–29]. Therefore, the main objectives of this work were to (1) evaluate the effect of extremes in fermentation and roasting on the composition of cocoa beans and powder, (2) determine how fermentation and roasting affect α-glucosidase inhibitory activity of cocoa powder, and (3) identify the compositional factors and processing conditions that optimize α-glucosidase inhibitory activity of cocoa.

## **2. Materials and Methods**
