Production and Characterization of Astaxanthin Nanoparticles

There is increasing interest on behalf of consumers and the food industry for the enrichment of common food with health-promoting bioactive molecules. Clinical studies have demonstrated that tangible health benefits may derive from the intake of bioactive compounds, in the prevention of dietary related pathologies such as diabetes, cancer, obesity and cardiovascular diseases. The beneficial effect is usually given by the presence in food of peculiar molecules such us carotenoids, polyphenols, polyunsaturated fatty acids and bioactive peptides, to cite a few. Unfortunately, these compounds display high susceptibility to environmental conditions such as light, extreme pH and temperature, and to standard food manufacturing processes. They can also account for undesirable flavors, colors or affect final product stability and appearance, thus rendering their presence in the product an issue rather than a useful addition. The addition of nutrients in small quantities into a food system may not significantly affect its proprieties, but the high amounts, often required to meet certain health claims and benefits, might bring to a product with a poorly acceptable sensory profile and a scarce stability. In particular, lipophilic bioactive ingredients display a major challenge. Besides their limited solubility in most of the foods and beverages, they are characterized by high susceptibility to oxidation and by a lower adsorption through the gastrointestinal tract in comparison to more hydrophilic substances, meaning a scanty bioavailability. Hence, there is a pressing need for the production of edible delivery systems or carriers that could efficiently encapsulate, protect and improve the handling of lipophilic molecules. The objective of this thesis was to develop a system suitable for the encapsulation of lipophilic molecules, capable of: a) protecting the ingredient from the surrounding environment (extreme pH, heat, UV light, oxygen); b) preserving its functionality (e.g. antioxidant activity); c) reducing the impact on the organoleptic level; d) improving the bioavailability of the encapsulated molecules. This last point in particular could be achieved by using sub-cellular delivery systems referred to as nanoparticles or nanocarriers, which may potentially enhance the transport across the intestinal wall. To this purpose, astaxanthin was chosen as a model bioactive compound. Astaxanthin is a keto-carotenoid that displays several biological activities, such as high antioxidant capacity, that may contribute to the prevention of degenerative pathologies like diabetes, cancer, cardiovascular diseases and chronic bowel disease. However, like all carotenoids it is characterized by a strong lipophilic character that makes its inclusion in many types of aqueous-based foods and beverages rather a challenge. This aspect is the main cause of its poor absorption through the human intestinal mucosae. Moreover, astaxanthin is labile to common processing conditions such as the presence of light and oxygen, extreme pH and high temperatures. For these reasons a lot of efforts have been put in these past years to find suitable solutions for its protection and manipulation. In order to develop the suitable encapsulate, in chapter 2 an emulsification and solvent-evaporation technique was used as encapsulation approach; whey proteins were selected as the matrix to envelope the core constituted by an astaxanthin-enriched oleoresin derived from Haematococcus pluvialis, a microalgae representing the main natural source of the carotenoid. The process was optimized by varying crucial parameters and the stability of the nanoparticles was tested in different conditions. This analysis highlighted a better performance of the encapsulated molecule in comparison to the starting oleoresin. Good release properties during in-vitro simulated digestion and the increase of the solubility in water were observed. In chapter 3, the study was focused on the research for plant alternative proteins as encapsulating matrices in order to satisfy the increasing interest of the consumers for substitutes of animal-deriving ingredients. This allowed to identify pea protein isolate as a valid candidate for the development of a vegetarian/vegan-allergen free nanocarrier. Finally, Chapter 4 dealt in depth with the antioxidant properties displayed by the astaxanthin nanoparticles through in vitro colorimetric assay and by the development of a cell-based assay. The encapsulates showed higher antioxidant capacity in comparison to the oleoresin. The uptake of the nanoparticles was studied in cell model systems through confocal laser microscopy and flow cytometry that indicated a probable energy-dependent mechanism.