A Standardized Extract of Microalgae <i>Phaeodactylum tricornutum</i> (Mi136) Inhibit D-Gal Induced Cognitive Dysfunction in Mice

The microalgae Phaeodactylum tricornutum (PT) is distinguished by its rich nutrient profile, characterized by well-documented neuroprotective activities, including fucoxanthin (FX), a major carotenoid and polyunsaturated omega-3 fatty acids (n-3 PUFA). The current study aims to evaluate the protective effects of a standardized extract of PT (Mi136) containing 2% FX on cognitive function, oxidative stress, and inflammation parameters in a mouse model of accelerated aging. Seventy-two (72) male mice were randomly assigned to the blank control group (BC), negative control group (NC), and four similar microalgae extract of PT groups (branded as BrainPhyt™) with different human equivalent doses to evaluate potential dose-response effects. From day 01 to day 51, mice in the BC group were injected with a 0.9% normal saline solution, while mice in all other groups were subcutaneously injected with D-galactose (D-Gal) at a dose of 150 mg/kg once per day, five days per week. Results indicated that, for the three higher microalgae extract of PT dose groups, spatial cognitive function, swim latency, and step-through latency impairments induced by chronic D-Gal intoxication were significantly and fully inhibited, with mean values similar to those in the BC group during each day of testing. Similar benefits were observed in biochemical analysis, specifically regarding brain and plasma levels of lipid peroxidation, TNF-α, and IL-6 markers. These data underscore the positive effects of a standardized extract of PT containing 2% FX on cognitive function parameters such as spatial working memory, long-term memory, and short-term memory through the regulation of oxidative stress and inflammation pathways

Fucoxanthin and Polyunsaturated Fatty Acids Co-Extraction by a Green Process

By their autotrophic nature and their molecular richness, microalgae are serious assets in the context of current environmental and societal challenges. Some species produce both omega-3 long chain polyunsaturated fatty acids (PUFAs) and xanthophylls, two molecular families widely studied for their bioactivities in the fields of nutrition and cosmetics. Whereas most studies separately deal with the two families, synergies could be exploited with extracts containing both PUFAs and xanthophylls. The purpose of our work was to determine cost effective and eco-friendly parameters for their co-extraction. The effect of several parameters (solvent, solvent/biomass ratio, temperature, duration) were studied, using two microalgal species, the non-calcifying Haptophyta Tisochrysis lutea, and the diatom Phaeodactylum tricornutum, that presents a silicified frustule. Analyses of PUFAs and fucoxanthin (Fx), the main xanthophyll, allowed to compare kinetics and extraction yields between experimental protocols. Co-extraction yields achieved using 96% ethanol as solvent were 100% for Fx and docosahexaenoic acid (DHA) in one hour from T. lutea biomass, and respectively 95% and 89% for Fx and eicosapentaenoic acid (EPA) in eight hours from P. tricornutum. These conditions are compatible with industrial applications

Ex situ and in situ investigation of protein/exopolysaccharide complex in Porphyridium cruentum biomass resuspension

International audienceDuring the extraction processes of B-phycoerythrin from P. cruentum, the possibility of the occurrence of extraction-inhibiting B-phycoerythrin/exopolysaccharide (EPS) complexes was highlighted. The use of confocal laser scanning microscopy allowed the observation of the cells including their sheath and their chloroplast, the bound EPS matrix, and extracellular vesicles involving phycobiliproteins and EPS in P. cruentum biomass resuspensions. The observations were performed using autofluorescence and fluorescence marking.pH variations showed more impact than ionic strength increase induced by NaCl addition on the B-phycoerythrin content and the organization of vesicles. Namely, agglomeration of the vesicles could be observed at pH 8.5 without NaCl addition. With addition of 1 M NaCl, this phenomenon was prevented but the vesicles were not visibly dissociated for any pH value. These results point towards a precipitate rather than a coacervate, which are expected to have narrow stability conditions regarding pH and ionic strength.In an attempt to dissolve or break the vesicles and the extracellular matrix, successive macerations in water and use of high pressure homogenizer process in one-step or two-step treatments were applied to the resuspensions. The successive macerations could free the cells and the vesicles from the extracellular matrix, whereas the homogenizer treatment disrupted the extracellular matrix, the vesicle and a part of the cells. In our case, only the successive macerations showed a positive extraction since the high pressure treatment caused unwanted denaturation.It is concluded that more experiments in model conditions could help understand the nature of the interactions between phycobiliproteins and EPS leading to the formation of the vesicles

Effect of high hydrostatic pressure on the structure of the soluble protein fraction in Porphyridium cruentum extracts

International audienceHigh hydrostatic pressure (HHP) treatments are trending as “green” stabilization and extraction process. The extraction of B-phycoerythrin from microalgae is getting more and more interest due to its numerous potentialities in foods, cosmetics and medicine. Thus, the effects of high pressure on the structural characteristics of B-phycoerythrin extracted from Porphyridium cruentum are explored in this paper.Spectrophotometric methods allowed to measure B-phycoerythrin content (UV–visible) and gave an indication on the protein structure (fluorescence). Micro-DSC analysis and electrophoresis complemented this structural investigation for all the protein fractions of P. cruentum extracts.Applying high hydrostatic pressure treatments up to 300 MPa during 5 min had no significant effect on B-phycoerythrin content and structure in P. cruentum extracts. Nevertheless, conformational changes of the protein are suggested by fluorescence yield decrease at 400 MPa, and protein aggregation of B-phycoerythrin, observed by Micro-DSC and electrophoresis, occurred at 500 MPa.Industrial relevanceThe HHP process is an emerging technology for the microbiological stability of various food matrices, including the proteins of microalgae as natural colorant. The target pressure to stabilize is around 400 MPa. High hydrostatic pressure can be used on P. cruentum extracts up to 300 MPa without any change in protein structure, as the threshold of protein aggregation is observed at 400 MPa. The observed changes of the proteins structure after applying HHP above 400 MPa can have a strong impact at macroscopic scale on the food matrices: increase of turbidity, change of texture, stability of emulsion

Improving the triphenylphosphine/triphenylphosphine oxide (TPP/TPPO)‐based method for the absolute and accurate quantification by FTIR‐ATR of hydroperoxides in oils or lipid extracts

International audienceAbstract Research on natural sources of polyunsaturated fatty acids (PUFAs) for both food and nutraceutical prospects has significantly grown in recent years. Some plant oils and lipid extracts contain carotenoids, xanthophylls, sterols, and/or phenolic compounds that can provoke a lipid hydroperoxides (LOOHs) overestimation when quantifying them using nonselective colorimetric assays. Herein, we have optimized a mid‐infrared method using Fourier‐transform infrared spectroscopy coupled with attenuated total reflectance (ATR) to quantify LOOHs in oils or lipid extracts. This method is based on the conversion of triphenylphosphine (TPP) to triphenylphosphine oxide (TPPO) in the presence of hydroperoxides, with a direct assessment of TPPO levels following the formation of an oil film on the ATR crystal's surface, allowing for a low detection limit of 0.5 mmol of LOOH kg −1 . The concentration of oil and TPP, as well as the reaction time, were optimized. It was demonstrated that the presence of pigments, unsaponifiable compounds, phenolics, and/or PUFAs on oils, do not disrupt the analysis. Furthermore, the stoichiometry of the TPP/LOOH reaction was examined, confirming the reliability of the method in detecting various forms of hydroperoxides. An accelerated oxidation study was carried out and the hydroperoxide contents measured using TPP/TPPO were found to be comparable to those obtained using the ferric thiocyanate method. Our method offers a fast, simple, robust, and sensitive approach to accurately quantify hydroperoxides, regardless of the chemical composition of oil or lipid extracts. Practical Application : The hydroperoxide assay method outlined in this study allows for the rapid and straightforward detection of hydroperoxides in pure oil matrices. The method's elevated sensitivity facilitates the early identification of oxidation indicators in oils, especially those with significant carotenoid or xanthophyll contents since these compounds may affect the results when using methods based on colorimetric quantification of hydroperoxides. This accurate, precise, and reproducible approach requires only small quantities of test samples and chemical products, making it well suited to routine application in laboratories and industrial environments

Green compressed fluid technologies for the extraction of bioactive compounds from Porphyridium cruentum in a biorefinery approach

Trabajo presentado al 17th European Meeting on Supercritical Fluids (EMSF) y al 7th European Meeting on High Pressure Technology, celebrados en Ciudad Real (España) del 8 al 11 de abril de 2019.[Introduction]: Microalgae are promising microorganisms that can play a key role in bio-based economy, since they may serve as a continuous and reliable source of safe natural products. Porphyridium cruentum is a red microalga rich in bioactive compounds, such as proteins, polysaccharides, polyunsaturated fatty acids and pigments. This microalga is able to accumulate great amounts of zeaxanthin and β-carotene, with beneficial physiological functions, such as anti-cancer, anti-diabetic, and aged-related macular degeneration. However, in order to improve the sustainability and economic feasibility of the biomass production process, it is necessary to obtain other high value compounds from the same biomass, following a biorefinery approach. Indeed, P. cruentum can accumulate up to 2% (w/w) of phyocoerythrin, which is a protein commonly used as a fluorescence-based indicator in biomedical investigations and also as a natural colorant in the food and cosmetic industries. Furthermore, phyocoerythrin may have therapeutical value since some researchers demonstrated its immunomodulatory and anti-cancer activities. In the present study, two new biorefinery approaches for the extraction of bioactive compounds from P. cruentum microalgae have been developed using pressurized green solvents with different techniques, such as pressurized liquid extraction (PLE) and high pressure processing (HPP), in order to obtain high added-value extracts. The extractions were carried out in two sequential stages: (1) pressurized pure water at 25 ºC, and (2) pressurized pure ethanol at high temperatures. The residual biomass after each extraction was used as raw material for the following extraction. The first step was carried out in order to obtain phycoerythrin-enriched extracts, while the second step was performed with the main objective of extracting carotenoids. All extracts were chemically characterized using spectrophotometric and chromatographic methods.[Results and discussion]: The strategy has been selected considering the extraction of phycoerythrin in the first step, to avoid its thermal degradation and with the main aim of recovering carotenoids in the second step. For this purpose, carotenoids extraction was independently optimized and, later on, integrated in a complete biorefiney process. For the optimization of the carotenoids extraction, pressurized ethanol extractions were carried out at 100 bar and five different temperatures from 50 ºC to 150 ºC for 20 min. These results were compared with the values obtained from a conventional extraction with ethanol (24 h, 500 rpm, atmospheric pressure, 20 ºC). All extraction yields varied between 3.12 and 11.36 %, increasing with temperature due to an improvement in the mass transfer from the sample to the extraction solvent, as a result of an increase in the vapor pressure of the compounds and a decrease in the viscosity of the solvent. According to the carotenoids content, all the pressurized extractions were significantly higher than the conventional extraction. Results indicate that the highest content of carotenoids was obtained at 125 ºC, so it was selected as the extraction temperature for PLE. However, no significant differences between 70-150 ºC were found. Then, the extracts from the biorefinery process using PLE (100 bar, 20 min) were obtained. In the first step, aqueous extracts were obtained at 25 ºC and 20 min, with high amounts of phycoerythrin (up to 25 mg/g extract); and the second step considered optimum conditions selected previously (ethanol, 125 ºC, 20 min), achieving high carotenoids content with no significant differences compared to the optimization step. HPLCMS characterization confirmed zeaxanthin and β-carotene as the major carotenoid compounds. Alternatively, a second biorefinery process was evaluated using ultra-high pressure (up to 6000 bar). Taking into account the results obtained in the previous optimization and the working conditions allowed by the equipment, extraction temperatures were set at 25 ºC (for water) and 70 ºC (for ethanol). Furthermore, three different pressures were evaluated: 1000, 3000 and 6000 bar. For comparison purposes, time was fixed at 20 min. Extraction yields and phycoerythrin content in the first step were above the 7% and 30 mg/g extract, respectively, higher than the values obtained using PLE. The higher the pressure, the higher the phycoerythrin content; nevertheless, results indicates that at the highest pressure (6000 bar), not only breakage of the cell wall was observed but also a degradation of the protein itself. Moreover, the protein purity values obtained at 1000-3000 bar are higher than 3, which are also higher than the obtained using PLE. Recovery of carotenoids in the second HPP step using ethanol as extracting solvent provide a new process alternative that should be studied in depth in order to select the best biorefinery approach.[Conclusions]: In this study, two biorefinery approaches were described for the first time to extract bioactives compounds from microalga P. cruentum using GRAS – generally recognized as safe – solvents and pressurized fluid technologies. Extractions were performed in two sequential steps using (1) water and (2) ethanol, considering the residue of the first extraction step as the raw material for the second extraction. Step 1 provided the extract enriched in phycoerythrin, while step 2 provided the extract enriched in carotenoids, mainly zeaxanthin and β-carotene. These biorefinery approaches carried out using technologies considered as green, such as PLE and HPP, could be considered sustainable processes.Authors thank projects ABACUS (Algae for a Biomass Applied to the production of added value compounds, grant agreement No 745668, funded by the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 research and innovation programme), AGL2017-89417-R (MINECO, Spain), ILINK+1096 (CSIC, Spain) and Natural Sciences and Engineering Research Council of Canada (NSERC, #05356-2014) for financial support. M.B. acknowledges MINECO for a “Juan de La Cierva-Formación” postdoctoral grant FJCI-2016-30902.Peer reviewe

Effect of high hydrostatic pressure on extraction of B-phycoerythrin from Porphyridium cruentum: Use of confocal microscopy and image processing

The aim of the study was to extract B-phycoerythrin from Porphyridium cruentum while preserving its structure. The high hydrostatic pressure treatments were chosen as extraction technology. Different methods have been used to observe the effects of the treatment: spectrophotometry and confocal laser scanning microscopy followed by image processing analysis. Image processing led to the generation of masks used for the identification of three clusters: intra, extra and intercellular. All methods showed that high hydrostatic pressure treatments between 50 and 500 MPa failed to extract B-phycoerythrin from Porphyridium cruentum cells. The fluorescence emission was negatively impacted by high hydrostatic pressure treatment from 400 MPa for the extracellular and intercellular cluster and from 500 MPa for the intercellular cluster. These results suggest that high pressure treatments could induce the denaturation of B-phycoerythrin in all clusters but with different intensities depending on the cluster

Protective Action of Ostreococcus tauri and Phaeodactylum tricornutum Extracts towards Benzo[a]Pyrene-Induced Cytotoxicity in Endothelial Cells

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Bioactive Oxylipins Profile in Marine Microalgae

International audienceMicroalgae are photosynthetic microscopic organisms that serve as the primary food source in aquatic environments. Microalgae can synthesize a wide variety of molecules, such as polyunsaturated fatty acids (PUFAs) of the omega-3 and omega-6 series. Oxidative degradation of PUFA due to radical and/or enzymatic conversion leads to the formation of oxylipins, which are compounds known for their bioactive properties. In the present study, we aim to profile oxylipins from five microalgae species grown in 10-L photo-bioreactors under optimal conditions. During their exponential phase, microalgae were harvested, extracted and analyzed by LC-MS/MS to determine the qualitative and quantitative profile of oxylipins for each species. The five different selected microalgae revealed a high diversity of metabolites, up to 33 non-enzymatic and 24 enzymatic oxylipins present in different concentrations. Taken together, these findings highlight an interesting role of marine microalgae as a source of bioactive lipids mediators, which we hypothesize have an important function in preventive health measures such as amelioration of inflammation. The rich mixture of oxylipins may display advantages to biological organisms, especially by providing for human health benefits including antioxidant, anti-inflammatory, neuroprotective or immunomodulator activities. Some oxylipins are also well known for their cardiovascular properties