Active films based on cocoa extract with antioxidant, antimicrobial and biological applications
Highlights
► Novel films of EVOH with flavanol-rich cocoa powder were successfully developed. ► Release of cocoa extract incorporated produced antioxidant activity in Caco-2 cells. ► Fraction obtained after in vitro gastrointestinal digestion presented antioxidant activity. ► A high concentration of cocoa extract did not have a cytotoxic effect on Caco-2 cells. ► Films produced total inhibition of growth against all microorganisms tested.
Introduction
The widespread incidence of foodborne diseases associated with microbial pathogens represents a threat to public health, and a challenge for the food industry. Pathogen microorganisms are generally eliminated by standard thermal treatments during processing, but these processes may impair the quality of the product and they do not prevent possible subsequent microbiological contamination. This can cause serious food poisoning, which in the case of children’s food can be very dangerous. It is possible to generate additional protection against deterioration and contamination of food with bioactive molecules used as additives. Nowadays there is increasing interest in the replacement of chemical additives with natural products with a low environmental impact. This has encouraged the use of natural antimicrobial and antioxidant compounds such as cocoa extract. Cocoa (Theobroma cocoa) has various applications in the food industry, mainly as a flavouring for chocolate manufacturing. Cocoa flavanols have gained additional attention because of their antioxidant capacity and their possible beneficial implications for human health (Katz, Doughty, & Ali, 2011). Because of its high percentage of anthocyanins and flavonoids, cocoa has a protective role against cardiovascular disease, oxygen-derived free radicals, and nitrogen chelation of metals (iron, copper, etc.). In vitro studies have shown that cocoa has anti-inflamamatory, antiallergic, antiviral, and anticancer activity (Katz et al., 2011).
The cocoa extract employed in this work was a flavanol-rich cocoa powder developed by a patented industrial process that preserves the major fraction of the original flavanol content (Cienfuegos-Jovellanos, Ibarra, Pasamar, Montanes, & Pons-Andreu, 2008). This product had eight times more epicatechin and procyanidin B2 content than conventional cocoa powder (Tomas-Barberan et al., 2007). Table 1 shows the total polyphenol and flavan-3-ol content of the cocoa powder used in this work (taken from (Quinones et al., 2010).
Novel structures with controllable morphology made from natural materials hold promise for several applications in pharmaceuticals, foods and biotechnology field (Patel, Nijsse, & Velikov, 2011). In recent years antimicrobial release systems have been employed mainly in pharmaceutical applications, while their use in food packaging is very restricted because antimicrobial agents have to be approved for application in direct contact with food. One of the main reasons for packaging of many foods is to prevent surface growth where a large portion of spoilage and contamination occurs (Shakeri, Shahidi, Beiraghi-Toosi, & Bahrami, 2011). Antimicrobial active packaging systems have been proposed to extend the shelf life and improve food safety (Suppakul, Miltz, Sonneveld, & Bigger, 2003). Antimicrobial films can be classified in 2 types: an antimicrobial agent that is effective against surfaces without migration, and an antimicrobial agent that migrates to the surface of the food (Suppakul et al., 2003). Nonmigratory active packaging offers the potential for improving food safety and quality while minimizing the migration of the active agent into food. The aim of release systems intended for food packaging applications is to transfer the bioactive agent from the polymeric matrix to the food in order to keep a predetermined concentration of the active compound in the packed food for a determined period of time (Buonocore et al., 2003).
Several works have studied the antimicrobial activity of cocoa powder (Busta and Speck, 1968, Percival et al., 2006), but few studies have focused on the properties of cocoa as an antimicrobial agent for food application (Pina-Perez, Silva-Angulo, Muguerza-Marquinez, Aliaga, & Lopez, 2009) and it has never been incorporated into films for active packaging to date. Furthermore, there are no studies that combine film production with biological applications such as antimicrobial and antioxidant activity.
In this work, new active films based on ethylene–vinyl alcohol copolymer, EVOH, and flavanol-rich cocoa extract have been developed. EVOH copolymer is a common packaging material mainly characterised by its low permeability to gases and flavours and excellent physical and optical properties. Despite being a synthetic material that comes from oil, this copolymer has the advantage of being biodegradable and biocompatible. It is composed of two different monomers linked to the same polymer chain, combining the hydrophobic nature of ethylene and the hydrophilic behaviour of polyvinyl alcohol. Since the hydroxyl groups (OH) are responsible for the gas barrier property, the greater the number of (OH), the lower the permeability (Catala & Gavara, 2001).
In this study, EVOH 29, containing 29% mol of ethylene, was selected because of its better processability as a coating. The release kinetics of antioxidants from the film into an aqueous media was monitored. The antioxidant effect and cytotoxicity of the compounds released from these films, as well as the gastrointestinal extract obtained after in vitro digestion of cocoa extract, were studied against human intestinal Caco-2 cells; a cell culture widely used as an in vitro model for the small intestine. Finally, antimicrobial activity of the films was studied against Gram-positive foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes) and Gram-negative pathogens (Escherichia coli, Salmonella enterica). In addition, the films were applied to an infant formula in order to reduce the great risk of microbial contamination, reducing its pathogenic flora and also extending its shelf life.
Section snippets
Chemicals and reagents
An ethylene–vinyl alcohol copolymer with a 29% ethylene molar content (EVOH) was kindly provided by The Nippon Synthetic Chemical Company (Osaka, Japan). Reagent-grade absolute ethanol, 1-propanol, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) were purchased from Sigma (Madrid, Spain).
Flavanol-rich cocoa extract (Table 1) was supplied by Natraceutical Group (Aldaya, Spain). It was developed by a patented industrial process (Cienfuegos-Jovellanos et al., 2008).
Water was obtained
Results and discussion
EVOH films of 25-μm-thick containing aqueous cocoa extract as antioxidant agent were successfully produced by casting. This technique has the advantage of reducing thermal degradation of the antioxidants with respect to conventional extrusion and can be compared to film coating techniques from a technological point of view. All these films were flexible, transparent, and without visual discontinuities. The films showed a slightly pink coloration that increased with the concentration of the
Conclusion
Novel films of EVOH with flavanol-rich cocoa powder were successfully developed. Sustained release of the bioactive agents produced an antioxidant and antimicrobial effect against foodborne pathogens. Studies of bioavailability and cytotoxicology on the films with cocoa extract showed that the digests presented antioxidant activity against oxidative stress induced by H2O2 in Caco-2 cells without any cytotoxic effect, even at a high concentration. These results are indicative that the films
Acknowledgements
The authors acknowledge the financial support of the Spanish Ministry of Science and Innovation, Projects AGL2006-02176, AGL2009-08776 and Fun-C-Food CSD2007-00063, and the C. L-d-D fellowship (FPU program). Karel Clapshaw provided assistance with the English language text.
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