In vitro Evaluation of a Functional Chickpea and Dark Chocolate- based Snack: Insights into Antioxidant, Anti-inflammatory, Immunomodulatory and Microbiota-modulating Properties

In recent years, interest in plant-based diets has grown, both for their environmental sustainability and for the beneficial health properties of the bioactive compounds. Legumes are a valuable source of protein and fibre, but their sensory properties are not fully appreciated by consumers. The formulation of legumes as snacks and the addition of dark chocolate not only improves their palatability, but also provides polyphenols and antioxidant compounds, combining taste pleasure with potential health benefits although the mechanisms by which this combination of foods influences human health is still limited. In this context, this doctoral thesis explored the nutritional, biological and technological potential of an innovative snack made from puffed chickpeas and dark chocolate - produced by the Fertitecnica Colfiorito company - studying its effects at different biological levels and evaluating sustainable approaches to the extraction of bioactive compounds. In a first step, the nutrigenomic, antioxidant and anti-inflammatory activities of the snack were evaluated using in vitro models an of optimised human intestinal epithelium (Caco-2) and macrophages (THP-1). Total polyphenol content (TPC) and antioxidant activity were measured after in vitro digestion. Caco-2 epithelia and THP-1 were pre-treated with high or low concentrations of digests. Inflammation was induced for 3h by Lipopolysaccharides (LPS) and Interleukin-1β (IL-1β). Transepithelial electrical resistance (TEER) was measured to assess barrier integrity. Gene expression related to tight junctions and inflammation was analysed using quantitative polymerase chain reaction (qPCR). Since the intestinal barrier and the immune system play a fundamental role in defining the composition and activity of the gut microbiota, to gain additional insight into the diet- immunity-microbiota axis, the impact of the snack on microbiota activity and composition was evaluated using an in vitro colonic fermentation model. Finally, from a technological and sustainability perspective, the application of Deep Eutectic Solvents (DES) as a “green” method for extracting bioactive compounds from food matrices was explored. After optimisation of the extraction method, the antioxidant activity of these molecules and cytotoxicity on cells was evaluated. TPC and antioxidant activity of the extracted compounds were evaluated. Cytotoxicity analyses were conducted using Caco-2 through the 3-(4,5-Di-2-yl)-2,5-ditetrazolium bromide (MTT) assay. Based on the results obtained, the chocolate and snack digests showed the highest antioxidant capacity. Treatment with all digests resulted in an increase in barrier integrity. In addition, chickpea upregulated tight junction gene expression and, both with chocolate, reduced IL-1β expression in Caco-2 cells. In THP-1 cells, these treatments increased expression of the Macrophage Mannose Receptor (CD206) and Interleukin-10 (IL-10), confirming an anti-inflammatory and immunomodulatory action. The microbiota, after treatment with the snack, significantly increased the production of short chain fatty-acids (SCFAs), suggesting a positive impact on microbial metabolism. Moreover, microbiota analysis showed that incubation time, but not treatment, significantly shaped community structure, with only a few fermentative genera consistently increasing alongside SCFAs production. Finally, DESs effectively extracted bioactive compounds, preserving and enhancing their antioxidant properties. TPC and antioxidant activity evaluated in the snack formulation were mainly attributable to the chocolate component. MTT showed that at the lowest concentrations (≤1% v/v), no cytotoxic effects were observed. Overall, the results obtained demonstrate that the chickpea and dark chocolate snack is a promising prototype for a functional food, capable of exerting antioxidant, anti- inflammatory and prebiotic effects, while integrating sustainable technological strategies for the valorisation of bioactive compounds.