In vitro fermentation of anthocyanins encapsulated with cyclodextrins: Release, metabolism and influence on gut microbiota growth
Introduction
Interest in anthocyanin health benefits has increased over the last several years. In vitro and in vivo studies have shown that anthocyanins may exert a wide range of biological activities such as antioxidant capacity, cardioprotective effects, anti-inflammatory properties, reduction in the risk of diabetes and inhibition of tumour cell growth, especially those in the colon (He, Giusti, 2010, Norberto et al, 2013, Zafra-Stone et al, 2007). Some researchers have also proposed that anthocyanins can influence health by modulating gut microbial community composition (Forester, Waterhouse, 2010, Hidalgo et al, 2012). In this regard, microbial activities have been related to different disease outcomes (Lozupone, Stombaugh, Gordon, Jansson, & Knight, 2012).
However, despite their health promoting effects, the use of anthocyanins has been hindered by their low chemical stability under physicochemical conditions they are exposed to after oral consumption by humans (Fleschhut, Kratzer, Rechkemmer, & Kulling, 2006). Limited available experimental evidence indicates that in the acidic conditions that prevail in the gastric compartment, anthocyanins occur under the red-coloured flavylium cations form (pH ~2). When they pass to the intestine and the pH shifts from acidic to close to neutral or mildly alkaline, anthocyanins are converted to an unstable form, blue quinoidal base, by the loss of protons (Woodward, Kroon, Cassidy, & Kay, 2009). As a consequence, such transformations contribute towards a low bioavailability of anthocyanins (Vitaglione et al., 2007).
Encapsulation may provide a robust means to stabilize anthocyanins and thus increase availability in the intestine. Different procedures have been used for food product encapsulation (Aceituno-Medina et al, 2015, Desai, Park, 2005, Martín et al, 2007). In particular, several materials may be considered as capsule matrices for anthocyanins, including maltodextrin, cyclodextrins (CDs), pullulan, glucan gel, curdlan, sodium alginate and pectin (Fernandes et al, 2013, Ferreira et al, 2009, Tonon et al, 2010).
Among the materials used to encapsulate anthocyanins, CDs offer some advantages. They have the capacity to protect bioactive food components from the deleterious conditions in the stomach and upper small bowel, allowing them to be liberated in the colon (Kosaraju, 2005) and thus boost their beneficial effects, e.g. inhibition of the growth of tumour cells (Tsukahara & Murakami-Murofushi, 2012). CDs possess macrocycles that present a torus-shaped structure with an adaptable hydrophobic cavity, which gives them the ability to form reversible inclusion complexes with a wide variety of organic compounds (often phenolic substances). Concerning anthocyanins, they include in their structure hydrophobic aromatic moieties and hydrophilic polar groups like hydroxyl groups. This amphiphilic character makes anthocyanins a good candidate for molecular inclusion with cyclodextrins (Dangles, Wigand, & Brouillard, 1992).
However, despite the potential benefits of encapsulation to increase bioavailability, its use has not been widely examined by food and nutritional researchers. In this line, most of the studies found in the literature evaluate anthocyanin encapsulation techniques mainly to protect them from thermal or pH degradation for use as natural food colourants (Ferreira et al., 2009). The vast majority of knowledge about targeted release of encapsulated molecules in the gut has been obtained from research concerning delivery of drugs (Kosaraju, 2005).
The present study aimed to evaluate the influence of the human gut microbiota on the degradation of CDs coverage and the subsequent release of anthocyanins. In addition, it includes an evaluation of bacteria–anthocyanin interactions using in vitro batch culture systems modelling the human colon. Changes in the faecal microbiota were evaluated using 16S rRNA-based fluorescence in situ hybridization (FISH), whereas the potential biological effects of anthocyanin intervention metabolic end products were assessed by short chain fatty acid (SCFA) analysis. Changes in anthocyanins and phenolic microbial metabolites were also monitored by HPLC and LC-MS analysis.
Section snippets
Chemicals
Standards of gallic acid and syringic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, Spain), ferulic acid was obtained from Koch-Light Laboratorie Ltd. (Colnbrook, Bucks, England) and cyanidin-3-glucoside, delphinidin-3-glucoside and malvidin-3-glucoside were supplied by Extrasynthèse (Genay, France). Formic, lactic, acetic, propionic and butyric acids were provided by Sigma-Aldrich Co. Ltd (Poole, Dorset, UK). β-CDs were supplied by Fluka (Madrid, Spain).
General chemicals
Encapsulation of anthocyanins with β-cyclodextrin
Three different concentrations of β-CD were evaluated for cyanidin-3-glucoside encapsulation. A clear change in the colour of the anthocyanin when β-CDs were added was seen. The colour loss was greater with higher concentration of β-CD used. Specifically, there was a 7% decrease in absorbance with the addition of β-CD 4 × 10−4 M, 10% with β-CD 5 × 10−3 M and 30% with β-CD 5 × 10−3 M. Similar results have been described in the literature by Fernandes et al. (2013).
In order to study the
Conclusions
These studies have shown that CDs ensure steady and sustained release of anthocyanins in the colon, which should improve their bioavailability in vivo. A bacterial interaction with the liberated anthocyanins was observed, which suggest that anthocyanins and their metabolites may exert a positive modulation of the intestinal bacterial population. Therefore, the results show the potential utility of encapsulating anthocyanins to allow them to exert their beneficial effect in the gut.
Acknowledgements
Financial support for this study was provided by Comunidad de Madrid (Projects ANALISYC-II S-2009/AGR1464). Dr. Gema Flores thanks CSIC for her JAE-Doc contract and European Molecular Biology Organization ASTF 326 - 2014 (EMBO) for a fellowship to carry on her experiments at University of Reading.
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