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

Evaluation of the difference in the development of stored insect pests on organic litter: Poster

On July 2017 in a warehouse of pet food shop in Italy an infestation of Oryzaephilus surinamensis was found on a pallet of organic litter, near an infested pallet of dog’s pasta. In order to investigate the origin of the infestation, and to support the risk assessment by the pest control operator, one test was conducted at Agroblu Laboratory of Applied Entomology (LEAA) to observe the feasibility of development of O. surinamensis, Plodia interpunctella and Tribolium confusum, in a substrate of 2,5 g of organic litter and to compare it to a balanced diet substrate. The results showed that only T. confusum was able to develop with no statistical difference both on the breeding diet and the organic litter.On July 2017 in a warehouse of pet food shop in Italy an infestation of Oryzaephilus surinamensis was found on a pallet of organic litter, near an infested pallet of dog’s pasta. In order to investigate the origin of the infestation, and to support the risk assessment by the pest control operator, one test was conducted at Agroblu Laboratory of Applied Entomology (LEAA) to observe the feasibility of development of O. surinamensis, Plodia interpunctella and Tribolium confusum, in a substrate of 2,5 g of organic litter and to compare it to a balanced diet substrate. The results showed that only T. confusum was able to develop with no statistical difference both on the breeding diet and the organic litter

Evaluation of the attractiveness of an organic litter compared to breeding substrate: Poster

In a pet shop warehouse, stored food pest insects can develop in various preserved animal feeds (dog’s pasta, puffed rice, kibble). However, there is another commodity that is rarely considered, such as the organic litter which is composed of bran, flours and other residues of the screening of corn that may result attractive to the same pest insects. The purpose of this laboratory test was to evaluate the attractiveness of organic litters on Plodia interpunctella, Tribolium confusum, Oryzaephilus surinamensis in comparison with breeding substrate. The results confirmed that the test insects were attracted by the breeding substrate rather than by the organic litter.In a pet shop warehouse, stored food pest insects can develop in various preserved animal feeds (dog’s pasta, puffed rice, kibble). However, there is another commodity that is rarely considered, such as the organic litter which is composed of bran, flours and other residues of the screening of corn that may result attractive to the same pest insects. The purpose of this laboratory test was to evaluate the attractiveness of organic litters on Plodia interpunctella, Tribolium confusum, Oryzaephilus surinamensis in comparison with breeding substrate. The results confirmed that the test insects were attracted by the breeding substrate rather than by the organic litter

Dendritic Cells in the Cross Hair for the Generation of Tailored Vaccines

Vaccines represent the discovery of utmost importance for global health, due to both prophylactic action to prevent infections and therapeutic intervention in neoplastic diseases. Despite this, current vaccination strategies need to be refined to successfully generate robust protective antigen-specific memory immune responses. To address this issue, one possibility is to exploit the high efficiency of dendritic cells (DCs) as antigen-presenting cells for T cell priming. DCs functional plasticity allows shaping the outcome of immune responses to achieve the required type of immunity. Therefore, the choice of adjuvants to guide and sustain DCs maturation, the design of multifaceted vehicles, and the choice of surface molecules to specifically target DCs represent the key issues currently explored in both preclinical and clinical settings. Here, we review advances in DCs-based vaccination approaches, which exploit direct in vivo DCs targeting and activation options. We also discuss the recent findings for efficient antitumor DCs-based vaccinations and combination strategies to reduce the immune tolerance promoted by the tumor microenvironment

Full recycling of spent lithium ion batteries with production of core-shell nanowires//exfoliated graphite asymmetric supercapacitor

A novel process is reported which produces an asymmetric supercapacitor through the complete recycling of end-of-life lithium ion batteries. The electrodic powder recovered by industrial scale mechanical treatment of spent batteries was leached and the dissolved metals were precipitated as mixed metals carbonates. Nanowires battery-type positive electrodes were produced by electrodeposition into nanoporous alumina templates from the electrolytic baths prepared by dissolution of the precipitated carbonates. The impact of the different metals contained in the electrodic powder was evaluated by benchmarking the electrochemical performances of the recovered nanowires-based electrodes against electrodes produced by using high-purity salts. Presence of inactive Cu in the nanowires lowered the final capacitance of the electrodes while Ni showed a synergistic effect with cobalt providing a higher capacitance with respect to synthetic Co electrodes. The carbonaceous solid recovered after leaching was in-depth characterized and tested as negative electrode. Both the chemical and electrochemical characterization indicate that the recovered graphite is characterized by the presence of oxygen functionalities introduced by the leaching treatment. This has led to the obtainment of a recovered graphite characterized by an XPS C/O ratio, Raman spectrum and morphology close to literature reports for reduced graphene oxide. The asymmetric supercapacitor assembled using the recovered nanowires-based positive electrodes and graphite as negative electrodes has shown a specific capacitance of 42 Fg-1, computed including the whole weight of the positive electrode and recovered graphite, providing a maximum energy density of ∼9 Whkg−1 and a power density of 416 Wkg−1 at 2.5 mA cm-

Biocidal efficacy of nitrogen (anoxic atmosphere) applied in operational condition to stored hazelnuts against pest insects at different stages of development.: Poster

Recently, a test was conducted in Italy for the evaluation of the biocidal efficacy of Nitrogen saturation (anoxic conditions). One application was carried out in a controlled atmosphere cell of a logistic center specialized in receiving, storing and shipping foodstuffs. The cell, circa 3682 m3 volume, with capacity of 752 big bags of fresh shelled hazelnuts on 4 height levels was saturated with Nitrogen (99,9%) and maintained at 15-18°C for 21 days. Five test species of insects Plodia interpunctella, Cadra cautella, Corcyra cephalonica, Tribolium confusum, Oryzaephilus surinamensis were observed at different development stages (egg, larva, adult). The target species were sorted in special biotest and inserted in the big bags to simulate an infestation. At the end of the exposure period the biotests were collected and analyzed. The treatment resulted sufficient to achieve a total control on eggs of Lepidoptera test species only. This result confirmed and integrates the available information in literature that showed the need of a longer minimum exposure period for total control of common stored pest insects.Recently, a test was conducted in Italy for the evaluation of the biocidal efficacy of Nitrogen saturation (anoxic conditions). One application was carried out in a controlled atmosphere cell of a logistic center specialized in receiving, storing and shipping foodstuffs. The cell, circa 3682 m3 volume, with capacity of 752 big bags of fresh shelled hazelnuts on 4 height levels was saturated with Nitrogen (99,9%) and maintained at 15-18°C for 21 days. Five test species of insects Plodia interpunctella, Cadra cautella, Corcyra cephalonica, Tribolium confusum, Oryzaephilus surinamensis were observed at different development stages (egg, larva, adult). The target species were sorted in special biotest and inserted in the big bags to simulate an infestation. At the end of the exposure period the biotests were collected and analyzed. The treatment resulted sufficient to achieve a total control on eggs of Lepidoptera test species only. This result confirmed and integrates the available information in literature that showed the need of a longer minimum exposure period for total control of common stored pest insects

Phagocytosis of Astaxanthin-Loaded Microparticles Modulates TGFβ Production and Intracellular ROS Levels in J774A.1 Macrophages

Radiation-induced fibrosis is a serious long-lasting side effect of radiation therapy. Central to this condition is the role of macrophages that, activated by radiation-induced reactive oxygen species and tissue cell damage, produce pro-inflammatory cytokines, such as transforming growth factor beta (TGFb). This, in turn, recruits fibroblasts at the site of the lesion that initiates fibrosis. We investigated whether astaxanthin, an antioxidant molecule extracted from marine and freshwater organisms, could help control macrophage activation. To this purpose, we encapsulated food-grade astaxanthin from Haematococcus pluvialis into micrometer-sized whey protein particles to specifically target macrophages that can uptake material within this size range by phagocytosis. The data show that astaxanthin-loaded microparticles are resistant to radiation, are well-tolerated by J774A.1 macrophages, induce in these cells a significant reduction of intracellular reactive oxygen species and inhibit the release of active TGFb as evaluated in a bioassay with transformed MFB-F11 fibroblasts. Micro-encapsulation of bioactive molecules is a promising strategy to specifically target phagocytic cells and modulate their own functions

Effects of Combination Treatments with Astaxanthin-Loaded Microparticles and Pentoxifylline on Intracellular ROS and Radiosensitivity of J774A.1 Macrophages

Radiation-induced fibrosis (RIF) is a serious, yet incurable, complication of external beam radiation therapy for the treatment of cancer. Macrophages are key cellular actors in RIF because of their ability to produce reactive oxidants, such as reactive oxygen species (ROS) and inflammatory cytokines that, in turn, are the drivers of pro-fibrotic pathways. In a previous work, we showed that phagocytosis could be exploited to deliver the potent natural antioxidant astaxanthin specifically to macrophages. For this purpose, astaxanthin encapsulated into \u3bcm-sized protein particles could spe- cifically target macrophages that can uptake the particles by phagocytosis. In these cells, astaxanthin microparticles significantly reduced intracellular ROS levels and the secretion of bioactive TGF\u3b2 and increased cell survival after radiation treatments. Here we show that pentoxifylline, a drug currently used for the treatment of muscle pain resulting from peripheral artery disease, amplifies the effects of astaxanthin microparticles on J774A.1 macrophages. Combination treatments with pentoxifylline and encapsulated astaxanthin might reduce the risk of RIF in cancer patients