Alessandro Alberto Casazza

Effects of polyphenol extract from olive pomace on anoxia-induced endothelial dysfunction.

Anoxia modulates the expression of molecules associated with endothelial dysfunction and vascular diseases. Polyphenols have potent antioxidant properties due to their ability to modulate genes involved in oxidative tissue damage. In this study, we investigated the effect of polyphenol extract from olive pomace (PEOP) and its main constituents, Tyrosol and Oleuropein, on endothelial cells subjected to anoxia by evaluating the expression of molecules critical for endothelial function, proliferation and migration, and the signaling pathway involved. EAhy926 human endothelial cells were exposed to anoxic stress in the presence or absence of PEOP. Anoxia increased the nitric oxide (NO) level and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNFα). These effects were prevented by PEOP treatment in a dose-dependent manner. Moreover, PEOP prevented the proliferation and migration associated with anoxia in EAhy926 cells, down-regulated the levels of matrix metalloproteinase (MMP)-2, MMP-9 and membrane type-1 MMP (MT1-MMP) and increased tissue MMP inhibitor-1 (TIMP-1) expression. Purified Oleuropein or Tyrosol restored to a basal level the anoxia-induced expression of MMP-9 and partially of MMP-2. The expression of TNFα was reduced by both polyphenols in a dose-dependent manner, but more efficiently by Tyrosol. Conversely, Oleuropein and Tyrosol had no significant effects on iNOS, COX-2 and TIMP-1 expression when used at the concentration found in PEOP. PEOP induced a time-dependent phosphorylation of p38 MAPK and ERK1/2 and inhibited anoxia-induced NF-κB activation. PEOP treatment restores the endothelial functions that are impaired by anoxia by regulating the expression of genes involved in proteolysis, angiogenesis and inflammation more efficiently than the single purified components. Therefore, the combined use of polyphenols, as in PEOP, could represent a powerful tool for the treatment and chemoprevention of endothelial dysfunction-associated vascular diseases.

Grape by-products: extraction of polyphenolic compounds using supercritical CO2 and liquid organic solvent - a preliminary investigation.

This research focussed on the exploitation of grape by-products as a source of polyphenolic compounds, which are of interest to the food, pharmaceutical and cosmetic industries. In particular, two substrates were tested: Pinot Noir grape skins and grape seeds. Pinot Noir grape skins were extracted by supercritical CO2 added with ethanol as modifier at constant temperature (45 °C) and at variable pressure (200, 300, 400 or 500 bar). The supercritical extraction kinetics of polyphenolic compounds was obtained. Grape seeds were extracted by combining supercritical (at 40 °C and 500 bar and using CO2 or CO2 added with ethanol as modifier) with liquid ethanol extraction. The supercritical technique seemed not to be really effective in extracting polyphenolic compounds; it can be anyway utilized to selectively extract grape seed oil by avoiding any solvent contamination of the matrix which can be further extracted for the recovery of polyphenolic compounds by means of liquid organic solvent.

Combined effect of starter culture and temperature on phenolic compounds during fermentation of Taggiasca black olives

The influence of two operative parameters on the fermentation process of table olives from Taggiasca cultivar were investigated. Laboratory scale fermentations were performed using Lactobacillus plantarum as the only starter and in combination with Saccharomyces cerevisiae at three different temperatures (23, 30 and 37°C). Control tests used for each trial were fermented only by indigenous microflora. pH and phenolic compounds were monitored in the brine and olive flesh during the fermentation. Higher temperatures (37°C) enhanced notably the release of phenolic compounds in the brine. High performance liquid chromatography (HPLC) analysis of brines evidenced the complete hydrolysis of oleuropein after 100 days of fermentation at 37°C for all treatments. The antioxidant power of the extracts was linearly correlated to their polyphenol contents. The results confirmed the efficiency of treatments compared with the control tests for debittering process of table black olives. Phenolic compounds in the brines can be then extracted and used in food, cosmetic and pharmaceutical industries.

Chitosan/dextran multilayer microcapsules for polyphenol co-delivery

Polysaccharide-based nanostructured polymeric microcapsules were fabricated by the electrostatic layer-by-layer self-assembly technique and used to encapsulate mixtures of four different polyphenols in order to achieve their controlled release. The real-time fabrication of the dextran/chitosan multilayer was monitored by quartz crystal microbalance with dissipation monitoring, and the morphology of the nanostructured polymeric capsules was characterized by scanning electron microscopy. The polyphenol encapsulation was obtained by reversible permeability variation of the capsule shell in ethanol:water mixtures. The loading efficiency in different water:ethanol mixtures and the release rate in acidic conditions were characterized by UV spectroscopy and HPLC. The higher loading efficiency was obtained with an ethanol:water 35:65 phenolic solution, equal to 42.0±0.6%, with a total release of 11.5±0.7 mg of total polyphenols per 11.3 μL of microcapsules after 240 min of incubation in acidic environment. The results suggest that polysaccharide-based capsules can be successfully used to encapsulate and release low water-soluble molecules, such as polyphenols.

Production of Chlorella vulgaris as a source of essential fatty acids in a tubular photobioreactor continuously fed with air enriched with CO2 at different concentrations

To reduce CO2 emissions and simultaneously produce biomass rich in essential fatty acids, Chlorella vulgaris CCAP 211 was continuously grown in a tubular photobioreactor using air alone or air enriched with CO2 as the sole carbon source. While on one hand, nitrogen‐limited conditions strongly affected biomass growth, conversely, they almost doubled its lipid fraction. Under these conditions using air enriched with 0, 2, 4, 8, and 16% (v/v) CO2, the maximum biomass concentration was 1.4, 5.8, 6.6, 6.8, and 6.4 gDB L−1 on a dry basis, the CO2 consumption rate 62, 380, 391, 433, and 430 mgCO2 L−1 day−1, and the lipid productivity 3.7, 23.7, 24.8, 29.5, and 24.4 mg L−1 day−1, respectively. C. vulgaris was able to grow effectively using CO2‐enriched air, but its chlorophyll a (3.0–3.5 g 100gDB−1), chlorophyll b (2.6–3.0 g 100gDB−1), and lipid contents (10.7–12.0 g 100gDB−1) were not significantly influenced by the presence of CO2 in the air. Most of the fatty acids in C. vulgaris biomass were of the saturated series, mainly myristic, palmitic, and stearic acids, but a portion of no less than 45% consisted of unsaturated fatty acids, and about 80% of these were high added‐value essential fatty acids belonging to the ω3 and ω6 series. These results highlight that C. vulgaris biomass could be of great importance for human health when used as food additive or for functional food production.

Antioxidant activity and biological evaluation of olive pomace extract.

The antiradical power of the methanol extracts of olive pomace (Taggiasca cultivar) achieved by high-pressure–high-temperature reactor were investigated using ABTS•+ and DPPH• assays. The highest antioxidant activity was quantified at 90 min of contact time and 180°C of extraction temperature (64.19 ± 0.16 µgTE L−1 and 15.80 ± 0.62 µgDPPH µLextract −1). The extract with high-antioxidant power resulted to be effective to counteract key aspects of cellular oxidation sensitive mechanisms and inflammation associated to vascular diseases. A linear correlation (p < 0.05) between total polyphenol contents and antioxidant capacity was given by the ABTS•+ method (R 2 = 0.9184) and DPPH assay (R 2 = 0.7062).

Influence of TiO2 Nanoparticles on Growth and Phenolic Compounds Production in Photosynthetic Microorganisms

The influence of titanium dioxide nanoparticles (pure anatase and 15% N doped anatase) on the growth of Chlorella vulgaris, Haematococcus pluvialis, and Arthrospira platensis was investigated. Results showed that pure anatase can lead to a significant growth inhibition of C. vulgaris and A. platensis (17.0 and 74.1%, resp.), while for H. pluvialis the nanoparticles do not cause a significant inhibition. Since in these stress conditions photosynthetic microorganisms can produce antioxidant compounds in order to prevent cell damages, we evaluated the polyphenols content either inside the cells or released in the medium. Although results did not show a significant difference in C. vulgaris, the phenolic concentrations of two other microorganisms were statistically affected by the presence of titanium dioxide. In particular, 15% N doped anatase resulted in a higher production of extracellular antioxidant compounds, reaching the concentration of 65.2 and 68.0 mg gDB −1 for H. pluvialis and A. platensis, respectively.

A new bioenergetic and thermodynamic approach to batch photoautotrophic growth of Arthrospira (Spirulina) platensis in different photobioreactors and under different light conditions.

Photobioreactor configuration, mode of operation and light intensity are known to strongly impact on cyanobacteria growth. To shed light on these issues, kinetic, bioenergetic and thermodynamic parameters of batch Arthrospira platensis cultures were estimated along the time at photosynthetic photon flux density (PPFD) of 70μmolm(-2)s(-1) in different photobioreactors with different surface/volume ratio (S/V), namely open pond (0.25cm(-1)), shaken flask (0.48cm(-1)), horizontal photobioreactor (HoP) (1.94cm(-1)) and helicoidal photobioreactor (HeP) (3.88cm(-1)). Maximum biomass concentration and productivity remarkably increased with S/V up to 1.94cm(-1). HoP was shown to be the best-performing system throughout the whole runs, while HeP behaved better only at the start. Runs carried out in HoP increasing PPFD from 40 to 100μmolm(-2)s(-1) revealed a progressive enhancement of bioenergetics and thermodynamics likely because of favorable light distribution. HoP appeared to be a promising configuration to perform high-yield indoor cyanobacterial cultures.

Recovery of phenolic compounds from grape seeds: effect of extraction time and solid–liquid ratio

The aim of this research was to study the recovery of phenolic compounds from grape seeds, by-products from winemaking industries, using ethanolic solid–liquid extraction. For such a purpose, the combined effects of the extraction time (9, 19 and 29 h) and the solid–liquid ratio (0.10, 0.20 and 0.30 gdw mL−1), were investigated (where dw = dry waste). Results demonstrated that Pinot Noir seeds had high levels of both total polyphenols (73.66 mgGallic Acid Equivalent gdw−1) and flavonoids (30.90 mgCatechin Equivalent gdw−1), being the optimum extraction time 19 h approximately. The main phenolic compounds analysed with high performance liquid chromatography were catechin and quercetin with a maximum extraction yield obtained at 29 h (362.23 and 339.35 mg/100 gdw, respectively). Concentration of the polyphenols and their antiradical powers are demonstrated to have a significant linear correlation.

Chlorella vulgaris as a lipid source: Cultivation on air and seawater-simulating medium in a helicoidal photobioreactor

The freshwater microalga Chlorella vulgaris was cultured batchwise on the seawater‐simulating Schlösser medium either in a 1.1‐L‐working volume helicoidal photobioreactor (HeP) or Erlenmeyer flask (EF) as control and continuously supplying air as CO2 source. In these systems, maximum biomass concentration reached 1.65 ± 0.17 g L−1 and 1.25 ± 0.06 g L−1, and maximum cell productivity 197.6 ± 20.4 mg L−1 day−1 and 160.8 ± 12.2 mg L−1 day−1, respectively. Compared to the Bolds Basal medium, commonly employed to cultivate this microorganism on a bench‐scale, the Schlösser medium ensured significant increases in all the growth parameters, namely maximum cell concentration (268% in EF and 126% in HeP), maximum biomass productivity (554% in EF and 72% in HeP), average specific growth rate (67% in EF and 42% in HeP), and maximum specific growth rate (233% in EF and 22% in HeP). The lipid fraction of biomass collected at the end of runs was analyzed in terms of both lipid content and fatty acid profile. It was found that the seawater‐simulating medium, despite of a 56–63% reduction of the overall biomass lipid content compared to the Bolds Basal one, led in HeP to significant increases in both the glycerides‐to‐total lipid ratio and polyunsaturated fatty acid content compared to the other conditions taken as an average. These results as a whole suggest that the HeP configuration could be a successful alternative to the present means to cultivate C. vulgaris as a lipid source.