Temperature-Dependent Olive Pomace Extraction for Obtaining Bioactive Compounds Preventing the Death of Murine Cortical Neurons.

High-pressure and temperature extraction (HPTE) can effectively recover bioactive compounds from olive pomace (OP). HPTE extract obtained by extracting OP with ethanol and water (50:50 v/v) at 180 °C for 90 min demonstrated a pronounced ability to preserve intracellular calcium homeostasis, shielding neurons from the harmful effects induced by N-methyl-d-aspartate (NMDA) receptor (NMDAR) overactivation, such as aberrant calpain activation. In this study, the extraction temperature was changed from 37 to 180 °C, and the extracts were evaluated for their antioxidant potency and ability to preserve crucial intracellular Ca2+-homeostasis necessary for neuronal survival. Additionally, to verify the temperature-induced activity of the extract, further extractions on the exhausted olive pomace were conducted, aiming to identify variations in the quality and quantity of extracted phenolic molecules through HPLC analysis. The results revealed a significant increase in bioactive compounds as a function of temperature variation, reaching 6.31 ± 0.09 mgCAE/mL extract for the extraction performed at 180 °C. Subsequent extraction of the exhausted residues yielded extracts that remained active in preventing calcium-induced cell death. Moreover, despite increased antiradical power, extracts re-treated at 180 °C did not display cell protection activity. Our results indicate that the molecules able to maintain physiological Ca2+-homeostasis in murine cortical neurons in conditions of cytotoxic stimulation of NMDAR are wholly recovered from olive pomace only following extraction performed at 180 °C.

A Bioactive Olive Pomace Extract Prevents the Death of Murine Cortical Neurons Triggered by NMDAR Over-Activation.

We have recently demonstrated that bioactive molecules, extracted by high pressure and temperature from olive pomace, counteract calcium-induced cell damage to different cell lines. Here, our aim was to study the effect of the same extract on murine cortical neurons, since the preservation of the intracellular Ca2+-homeostasis is essential for neuronal function and survival. Accordingly, we treated neurons with different stimuli in order to evoke cytotoxic glutamatergic activation. In these conditions, the high-pressure and temperature extract from olive pomace (HPTOPE) only abolished the effects of N-methyl-d-aspartate (NMDA). Particularly, we observed that HPTOPE was able to promote the neuron rescue from NMDA-induced cell death. Moreover, we demonstrated that HPTOPE is endowed with the ability to maintain the intracellular Ca2+-homeostasis following NMDA receptor overactivation, protecting neurons from Ca2+-induced adverse effects, including aberrant calpain proteolytic activity. Moreover, we highlight the importance of the extraction conditions used that, without producing toxic molecules, allow us to obtain protecting molecules belonging to proanthocyanidin derivatives like procyanidin B2. In conclusion, we can hypothesize that HPTOPE, due to its functional and nontoxic properties on neuronal primary culture, can be utilized for future therapeutic interventions for neurodegeneration.

Microencapsulation of Theobroma cacao L. waste extract: optimization using response surface methodology.

The cocoa extract (Theobroma cacao L.) has a significant amount of polyphenols (TP) with potent antioxidant activity (AA). This study aims to optimise microencapsulation of the extract of cocoa waste using chitosan and maltodextrin. Microencapsulation tests were performed according to a Box-Behnken factorial design, and the results were evaluated by response surface methodology with temperature, maltodextrin concentration (MD) and extract flowrate (EF) as independent variables, and the fraction of encapsulated TP, TP encapsulation yield, AA, yield of drying and solubility index as responses. The optimum conditions were: inlet temperature of 170 °C, MD of 5% and EF of 2.5 mL/min. HPLC analysis identified epicatechin as the major component of both the extract and microparticles. TP release was faster at pH 3.5 than in water. These results as a whole suggest that microencapsulation was successful and the final product can be used as a nutrient source for aquatic animal feed. Highlights Microencapsulation is optimised according to a factorial design of the Box-Behnken type. Epicatechin is the major component of both the extract and microcapsules. The release of polyphenols from microcapsules is faster at pH 3.5 than in water.

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.

From laboratory- to industrial-scale plants: Future of anaerobic digestion of olive mill solid wastes.

In this review, the main properties of olive mill solid waste, the primary by-product of olive oil production, and its feasibility as a feedstock for anaerobic digesters operating at laboratory-, pilot- and industrial-scales are discussed in detail. Nutrient addition and thermal pretreatments were found to have the potential to address the challenges arising from the high carbon-to-nitrogen ratio, the low pH, and the high concentration of phenolic compounds. Furthermore, anaerobic co-digestion with different organic feedstocks has been identified as one of the most promising options to solve the aforementioned problems and the seasonality nature of olive waste, while improving the efficiency of anaerobic treatment plants that operate throughout the whole year. The insights generated from this study show co-digestion with wastes from animal farming to be the most environmentally and economically sustainable method for improving anaerobic digestion processes with olive mill solid waste.

Inulin as prebiotic encapsulating agent for the production of spray-dried Hibiscus sabdariffa L. tea microcapsules.

Due to the high content of phenolics and anthocyanins of Hibiscus sabdariffa L. tea and the sensibility of these bioactive compounds, this work aimed to optimize the obtention of microcapsules by spray-drying, using inulin as a carrier agent. Using a Box-Behnken Design, the effects of inlet temperature (130, 150, and 170 °C), feed flow rate (5, 10, and 15 mL min-1), and inulin concentration (5, 10, and 15 g L-1) were evaluated. It was possible to obtain pale-rose, slightly sweet instant powders with good total polyphenol content (1.12 mgGAE g-1) and anthocyanins encapsulation efficiency (32.3-60.6%), besides moisture (4.61-17.79%) and water activity (0.221-0.501), indicating physico-chemical and microbiological stability of the microcapsules. A simultaneous optimization with the desirability function was performed to maximize all the response variables analyzed, and the optimum conditions of 5 g L-1 of inulin, inlet temperature of 170 °C, and feed flow rate of 83 mL min-1 were found.

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.

Repetitive non-destructive extraction of lipids from Chlorella vulgaris grown under stress conditions.

The aim of this study was the investigation of non-destructive lipid extraction from Chlorella vulgaris grown under stress conditions of nutrient limitation and salinity. To select a suitable solvent for extraction, the performances of decane, dodecane and hexadecane were tested based on their effect on lipid extraction and cell viability. The results showed that dodecane was the most suitable solvent for the extraction process. The concentration of extracted lipids from stressed cells was 2762.52 ± 11.38 mg L-1, i.e. a value 1.75 times higher than that obtained from unstressed cells. Long-term extraction was also evaluated with continuous dodecane recirculation during five-stage extraction and a recovery time of 24 h between the extraction steps, which yielded after the fifth extraction stage a total lipid amount as high as 9811.56 mg L-1. These results showed that non-destructive lipid recovery can be effectively performed by applying stress conditions and in repetitive extractions.

Bioactive compounds and antioxidant potential for polyphenol-rich cocoa extract obtained by agroindustrial residue.

Processing of cocoa (Theobroma cacao L.) beans responsible for agricultural exports leads to large amounts of solid waste that were discarded, however, this one presents high contents of metabolites with biological activities. The major objective of this study was to valorise cocoa agroindustrial residue obtained by hydraulic pressing for extract rich in antioxidants. For it, the centesimal composition of residue was investigated, the green extraction was carried out from the residue after, the bioactive compounds, sugar contents and screaming by HPTLC were quantified for extract. The extract has a total polyphenol content of 229.64 mg/g and high antioxidant activity according to ABTS 225.0 µM/g. HTPLC analysis confirmed the presence in the extract, residue of terpenes, sesquiterpenes, flavonoids and antioxidant activity. These results, as a whole, suggest that the extract from the cocoa residue has interesting characteristics to alternative crops with potential industrial uses.

Immobilization of Aspergillus ficuum tannase in calcium alginate beads and its application in the treatment of boldo (Peumus boldus) tea.

Tannase (tannin acyl hydrolase, E.C. 3.1.1.20) is an enzyme that catalyzes the hydrolysis of ester and depside linkages in hydrolysable tannins such as tannic acid, releasing gallic acid and glucose. It has several commercial applications in food industry, among which are gallic acid production, reduction of tannin content in fruit juices, and preparation of instantaneous tea. In this study we immobilized Aspergillus ficuum tannase in calcium alginate beads and then used it to treat boldo (Peumus boldus) tea. Such a technique allowed entrapping tannase with a 75% efficiency and appreciably increasing its thermal and pH stability compared with the free enzyme. Storage stability and reuse of the immobilized enzyme were very promising, in that about 60% of starting enzyme activity was retained after bead storage for 90 days at 4 °C or after six cycles of use. Boldo tea treatment with immobilized tannase for 120 min at 40 °C led to 31 and 60% removals of tannins and epigallocatechin gallate, an increase of about two orders of magnitude in gallic acid content, 56 and 109% increases in total flavonoids and epigallocatechin contents, a 42.8% increase in antioxidant activity and significant enhancements of tea color, clarity and pH.

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 Bold's 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 Bold's 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. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:279-284, 2016.

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.

Optimisation of phenolics recovery from Vitex agnus-castus Linn. leaves by high-pressure and temperature extraction.

To optimise recovery of phenolics from Vitex agnus-castus Linn., a non-conventional high-pressure (2-24 bar) and temperature (100-180°C) extraction method was used under nitrogen atmosphere with methanol as a solvent. Optimal temperature was between 100 and 140°C, and optimal extraction time was about one half that of conventional solid/liquid extraction at room temperature. Final yields of total polyphenols, total flavonoids, o-diphenols and anthocyanins extraction were 2.0, 3.0, 2.5 and 11-fold those obtained by conventional extraction.

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 µg(TE) L(-1) and 15.80 ± 0.62 µg(DPPH) µL(extract) (-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).