Ethanol Extraction of Polar Lipids from Nannochloropsis oceanica for Food,Feed, and Biotechnology Applications Evaluated Using Lipidomic Approaches

Nannochloropsis oceanica can accumulate lipids and is a good source of polar lipids, which are emerging as new value-added compounds with high commercial value for the food, nutraceutical, and pharmaceutical industries. Some applications may limit the extraction solvents, such as food applications that require safe food-grade solvents, such as ethanol. However, the effect of using ethanol as an extraction solvent on the quality of the extracted polar lipidome, compared to other more traditional methods, is not yet well established. In this study, the polar lipid profile of N. oceanica extracts was obtained using different solvents, including chloroform/methanol (CM), dichloromethane/methanol (DM), dichloromethane/ethanol (DE), and ethanol (E), and evaluated by modern lipidomic methods using LC-MS/MS. Ultrasonic bath (E + USB)- and ultrasonic probe (E + USP)-assisted methodologies were implemented to increase the lipid extraction yields using ethanol. The polar lipid signature and antioxidant activity of DM, E + USB, and E + USP resemble conventional CM, demonstrating a similar extraction efficiency, while the DE and ethanol extracts were significantly different. Our results showed the impact of different extraction solvents in the polar lipid composition of the final extracts and demonstrated the feasibility of E + USB and E + USP as safe and food-grade sources of polar lipids, with the potential for high-added-value biotechnological applications.     

Biobased Solvents for Pressurized Liquid Extraction of Nannochloropsis gaditana Omega-3 Lipids

To develop greener extraction alternatives for microalgae biomass, ultrasound assisted extraction (UAE) and pressurized liquid extraction (PLE) with different biobased solvents were investigated, demonstrating that both techniques are useful alternatives for algal lipid extraction. Specifically, Nannochloropsis gaditana lipids were extracted by UAE and PLE at different temperatures and extraction times with sustainable solvents like 2-Methyltetrahydrofuran (2-MeTHF) and its mixtures with ethanol and other alcohols. The best oil yields for both PLE and UAE of N. gaditana were achieved with the mixture of 2-MeTHF:ethanol (1:3), reaching yields of up to 16.3%, for UAE at 50 °C and up to 46.1% for PLE at 120 °C. Lipid composition of the extracts was analyzed by HPLC-ELSD and by GC-MS to determine lipid species and fatty acid profile, respectively. Different fractionation of lipid species was achieved with PLE and solvent mixtures of different polarity. Thus, for the extraction of glycolipids, ethanolic extracts contained higher amounts of glycolipids and EPA, probably due to the higher polarity of the solvent. The optimized method was applied to microalgae Isochrysis galbana and Tetraselmis chuii showing the potential of mixtures of biobased solvents like 2-methyl-THF and ethanol in different proportions to efficiently extract and fractionate lipids from microalgal biomass.     

Evaluation of the Antioxidant Activity of Cell Extracts from Microalgae

A growing market for novel antioxidants obtained from non-expensive sources justifies educated screening of microalgae for their potential antioxidant features. Characterization of the antioxidant profile of 18 species of cyanobacteria (prokaryotic microalgae) and 23 species of (eukaryotic) microalgae is accordingly reported in this paper. The total antioxidant capacity, accounted for by both water- and lipid-soluble antioxidants, was evaluated by the (radical cation) ABTS method. For complementary characterization of cell extracts, a deoxyribose assay was carried out, as well as a bacteriophage P22/Salmonella-mediated approach. The microalga Scenedesmus obliquus strain M2-1 exhibited the highest (p > 0.05) total antioxidant capacity (149 ± 47 AAU) of intracellular extracts. Its scavenger activity correlated well with its protective effects against DNA oxidative damage induced by copper(II)-ascorbic acid; and against decay in bacteriophage infection capacity induced by H₂O₂. Finally, performance of an Ames test revealed no mutagenic effects of the said extract.     

In Vivo Nutritional Assessment of the Microalga Nannochloropsis gaditana and Evaluation of the Antioxidant and Antiproliferative Capacity of Its Functional Extracts

Nannochloropsis gaditana is a microalga with interesting nutritional and functional value due to its high content of protein, polyunsaturated fatty acids, and bioactive compounds. However, the hardness of its cell wall prevents accessibility to these components. This work aimed to study the effect of a treatment to increase the fragility of the cell wall on the bioavailability of its nutrients and functional compounds. The antioxidant and antiproliferative capacity of functional extracts from treated and untreated N. gaditana was assessed, and the profile of bioactive compounds was characterized. Furthermore, to study the effect of treatment on its nutrient availability and functional capacity, an in vivo experiment was carried out using a rat experimental model and a 20% dietary inclusion level of microalgae. Functional extracts from treated N. gaditana exhibited higher antioxidant activity than the untreated control. Furthermore, the treated microalga induced hypoglycemic action, higher nitrogen digestibility, and increased hepatic antioxidant activity. In conclusion, N. gaditana has interesting hepatoprotective, antioxidant, and anti-inflammatory potential, thus proving itself an ideal functional food candidate, especially if the microalga is treated to increase the fragility of its cell wall before consumption.     

Complementary Analytical Platforms of NMR Spectroscopy and LCMS Analysis in the Metabolite Profiling of Isochrysis galbana

This study was designed to profile the metabolites of Isochrysis galbana, an indigenous and less explored microalgae species. ¹H Nuclear Magnetic Resonance (NMR) spectroscopy and Liquid Chromatography-Mass Spectrometry (LCMS) were used to establish the metabolite profiles of five different extracts of this microalga, which are hexane (Hex), ethyl acetate (EtOAc), absolute ethanol (EtOH), EtOH:water 1:1 (AqE), and 100% water (Aq). Partial least square discriminant analysis (PLS–DA) of the generated profiles revealed that EtOAc and Aq extracts contain a diverse range of metabolites as compared to the other extracts with a total of twenty-one metabolites, comprising carotenoids, polyunsaturated fatty acids, and amino acids, that were putatively identified from the NMR spectra. Meanwhile, thirty-two metabolites were successfully annotated from the LCMS/MS data, ten of which (palmitic acid, oleic acid, α-linolenic acid, arachidic acid, cholesterol, DHA, DPA, fucoxanthin, astaxanthin, and pheophytin) were similar to those present in the NMR profile. Another eleven glycerophospholipids were discovered using MS/MS-based molecular network (MN) platform. The results of this study, besides providing a better understanding of I. galbana’s chemical make-up, will be of importance in exploring this species potential as a feed ingredient in the aquaculture industry.     

Polar Lipids Composition,Antioxidant and Anti-Inflammatory Activities of the Atlantic Red Seaweed Grateloupia turuturu

Grateloupia turuturu Yamada, 1941, is a red seaweed widely used for food in Japan and Korea which was recorded on the Atlantic Coast of Europe about twenty years ago. This seaweed presents eicosapentaenoic acid (EPA) and other polyunsaturated fatty acids (PUFAs) in its lipid fraction, a feature that sparked the interest on its potential applications. In seaweeds, PUFAs are mostly esterified to polar lipids, emerging as healthy phytochemicals. However, to date, these biomolecules are still unknown for G. turuturu. The present work aimed to identify the polar lipid profile of G. turuturu, using modern lipidomics approaches based on high performance liquid chromatography coupled to high resolution mass spectrometry (LC–MS) and gas chromatography coupled to mass spectrometry (GC–MS). The health benefits of polar lipids were identified by health lipid indices and the assessment of antioxidant and anti-inflammatory activities. The polar lipids profile identified from G. turuturu included 205 lipid species distributed over glycolipids, phospholipids, betaine lipids and phosphosphingolipids, which featured a high number of lipid species with EPA and PUFAs. The nutritional value of G. turuturu has been shown by its protein content, fatty acyl composition and health lipid indices, thus confirming G. turuturu as an alternative source of protein and lipids. Some of the lipid species assigned were associated to biological activity, as polar lipid extracts showed antioxidant activity evidenced by free radical scavenging potential for the 2,2′-azino-bis-3-ethyl benzothiazoline-6-sulfonic acid (ABTS^●+) radical (IC₅₀ ca. 130.4 μg mL⁻¹) and for the 2,2-diphenyl-1-picrylhydrazyl (DPPH^●) radical (IC₂₅ ca. 129.1 μg mL⁻¹) and anti-inflammatory activity by inhibition of the COX-2 enzyme (IC₅₀ ca. 33 µg mL⁻¹). Both antioxidant and anti-inflammatory activities were detected using a low concentration of extracts. This integrative approach contributes to increase the knowledge of G. turuturu as a species capable of providing nutrients and bioactive molecules with potential applications in the nutraceutical, pharmaceutical and cosmeceutical industries.     

Composition and Quantitation of Microalgal Lipids by ERETIC 1H NMR Method

Accurate characterization of biomass constituents is a crucial aspect of research in the biotechnological application of natural products. Here we report an efficient, fast and reproducible method for the identification and quantitation of fatty acids and complex lipids (triacylglycerols, glycolipids, phospholipids) in microalgae under investigation for the development of functional health products (probiotics, food ingredients, drugs, etc.) or third generation biofuels. The procedure consists of extraction of the biological matrix by modified Folch method and direct analysis of the resulting material by proton nuclear magnetic resonance (¹H NMR). The protocol uses a reference electronic signal as external standard (ERETIC method) and allows assessment of total lipid content, saturation degree and class distribution in both high throughput screening of algal collection and metabolic analysis during genetic or culturing studies. As proof of concept, the methodology was applied to the analysis of three microalgal species (Thalassiosira weissflogii, Cyclotella cryptica and Nannochloropsis salina) which drastically differ for the qualitative and quantitative composition of their fatty acid-based lipids.     

Comparative Studies on the Characteristic Fatty Acid Profiles of Four Different Chinese Medicinal Sargassum Seaweeds by GC-MS and Chemometrics

Sargassum seaweeds produce abundant biomass in China and have long been used as herbal medicine and food. Their characteristic fatty acid (FA) profiles and related potential function in promoting cardiovascular health (CVH) have not been systematically investigated. In this study, FA profiles of four medicinal Sargassum were characterized using GC-MS. Principal component analysis was used to discriminate the four medicinal Sargassum, and orthogonal projection to latent structures discriminant analysis was carried out between the two official species HAI ZAO and between the two folk medicine species HAI QIAN. In all of the algae investigated, the major SFA and MUFA were palmitic and stearic acid, respectively, while the major PUFAs were linoleic, arachidonic, and eicosapentaenoic acid. S. fusiforme and S. horneri had higher concentrations of PUFAs. With respect to CVH, all of the studied species, particularly S. fusiforme, exhibited satisfactory levels such as PUFA/SFA ratio and n-6/n-3 ratio. Each species possesses a unique FA profile and is discriminated clearly. Potential key FA markers (between the two Chinese official species, and between the two folk species) are assessed. The study provides characteristic fatty acid profiles of four Chinese medicinal Sargassum and their related potential function in promoting CVH.     

High-EPA Biomass from Nannochloropsis salina Cultivated in a Flat-Panel Photo-Bioreactor on a Process Water-Enriched Growth Medium

Nannochloropsis salina was grown on a mixture of standard growth media and pre-gasified industrial process water representing effluent from a local biogas plant. The study aimed to investigate the effects of enriched growth media and cultivation time on nutritional composition of Nannochloropsis salina biomass, with a focus on eicosapentaenoic acid (EPA). Variations in fatty acid composition, lipids, protein, amino acids, tocopherols and pigments were studied and results compared to algae cultivated on F/2 media as reference. Mixed growth media and process water enhanced the nutritional quality of Nannochloropsis salina in laboratory scale when compared to algae cultivated in standard F/2 medium. Data from laboratory scale translated to the large scale using a 4000 L flat panel photo-bioreactor system. The algae growth rate in winter conditions in Denmark was slow, but results revealed that large-scale cultivation of Nannochloropsis salina at these conditions could improve the nutritional properties such as EPA, tocopherol, protein and carotenoids compared to laboratory-scale cultivated microalgae. EPA reached 44.2% ± 2.30% of total fatty acids, and α-tocopherol reached 431 ± 28 µg/g of biomass dry weight after 21 days of cultivation. Variations in chemical compositions of Nannochloropsis salina were studied during the course of cultivation. Nannochloropsis salina can be presented as a good candidate for winter time cultivation in Denmark. The resulting biomass is a rich source of EPA and also a good source of protein (amino acids), tocopherols and carotenoids for potential use in aquaculture feed industry.     

Carotenoids,Phenolic Compounds and Tocopherols Contribute to the Antioxidative Properties of Some Microalgae Species Grown on Industrial Wastewater

This study aimed at investigating the potential of microalgae species grown on industrial waste water as a new source of natural antioxidants. Six microalgae from different classes, including Phaeodactylum sp. (Bacillariophyceae), Nannochloropsis sp. (Eustigmatophyceae), Chlorella sp., Dunaniella sp., and Desmodesmus sp. (Chlorophyta), were screened for their antioxidant properties using different in vitro assays. Natural antioxidants, including pigments, phenolics, and tocopherols, were measured in methanolic extracts of microalgae biomass. Highest and lowest concentrations of pigments, phenolic compounds, and tocopherols were found in Desmodesmus sp. and Phaeodactylum tricornuotom microalgae species, respectively. The results of each assay were correlated to the content of natural antioxidants in microalgae biomass. Phenolic compounds were found as major contributors to the antioxidant activity in all antioxidant tests while carotenoids were found to contribute to the 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical scavenging activity, ferrous reduction power (FRAP), and ABTS-radical scavenging capacity activity. Desmodesmus sp. biomass represented a potentially rich source of natural antioxidants, such as carotenoids (lutein), tocopherols, and phenolic compounds when cultivated on industrial waste water as the main nutrient source.     

Potential Anti-Obesity,Anti-Steatosis,and Anti-Inflammatory Properties of Extracts from the Microalgae Chlorella vulgaris and Chlorococcum amblystomatis under Different Growth Conditions

Microalgae are known as a producer of proteins and lipids, but also of valuable compounds for human health benefits (e.g., polyunsaturated fatty acids (PUFAs); minerals, vitamins, or other compounds). The overall objective of this research was to prospect novel products, such as nutraceuticals from microalgae, for application in human health, particularly for metabolic diseases. Chlorella vulgaris and Chlorococcum amblystomatis were grown autotrophically, and C. vulgaris was additionally grown heterotrophically. Microalgae biomass was extracted using organic solvents (dichloromethane, ethanol, ethanol with ultrasound-assisted extraction). Those extracts were evaluated for their bioactivities, toxicity, and metabolite profile. Some of the extracts reduced the neutral lipid content using the zebrafish larvae fat metabolism assay, reduced lipid accumulation in fatty-acid-overloaded HepG2 liver cells, or decreased the LPS-induced inflammation reaction in RAW264.7 macrophages. Toxicity was not observed in the MTT assay in vitro or by the appearance of lethality or malformations in zebrafish larvae in vivo. Differences in metabolite profiles of microalgae extracts obtained by UPLC-LC-MS/MS and GNPS analyses revealed unique compounds in the active extracts, whose majority did not have a match in mass spectrometry databases and could be potentially novel compounds. In conclusion, microalgae extracts demonstrated anti-obesity, anti-steatosis, and anti-inflammatory activities and could be valuable resources for developing future nutraceuticals. In particular, the ultrasound-assisted ethanolic extract of the heterotrophic C. vulgaris significantly enhanced the anti-obesity activity and demonstrated that the alteration of culture conditions is a valuable approach to increase the production of high-value compounds.     

Biological Activities and Chemical Composition of Methanolic Extracts of Selected Autochthonous Microalgae Strains from the Red Sea

Four lipid-rich microalgal species from the Red Sea belonging to three different genera (Nannochloris, Picochlorum and Desmochloris), previously isolated as novel biodiesel feedstocks, were bioprospected for high-value, bioactive molecules. Methanol extracts were thus prepared from freeze-dried biomass and screened for different biological activities. Nannochloris sp. SBL1 and Desmochloris sp. SBL3 had the highest radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl, and the best copper and iron chelating activities. All species had potent butyrylcholinesterase inhibitory activity (>50%) and mildly inhibited tyrosinase. Picochlorum sp. SBL2 and Nannochloris sp. SBL4 extracts significantly reduced the viability of tumoral (HepG2 and HeLa) cells with lower toxicity against the non-tumoral murine stromal (S17) cells. Nannochloris sp. SBL1 significantly reduced the viability of Leishmania infantum down to 62% (250 µg/mL). Picochlorum sp. SBL2 had the highest total phenolic content, the major phenolic compounds identified being salicylic, coumaric and gallic acids. Neoxanthin, violaxanthin, zeaxanthin, lutein and β-carotene were identified in the extracts of all strains, while canthaxanthin was only identified in Picochlorum sp. SBL2. Taken together, these results strongly suggest that the microalgae included in this work could be used as sources of added-value products that could be used to upgrade the final biomass value.     

Bioactive Lipids of Marine Microalga Chlorococcum sp. SABC 012504 with Anti-Inflammatory and Anti-Thrombotic Activities

Microalgae are at the start of the food chain, and many are known producers of a significant amount of lipids with essential fatty acids. However, the bioactivity of microalgal lipids for anti-inflammatory and antithrombotic activities have rarely been investigated. Therefore, for a sustainable source of the above bioactive lipids, the present study was undertaken. The total lipids of microalga Chlorococcum sp., isolated from the Irish coast, were fractionated into neutral-, glyco-, and phospho-lipids, and were tested in vitro for their anti-inflammatory and antithrombotic activities. All tested lipid fractions showed strong anti-platelet-activating factor (PAF) and antithrombin activities in human platelets (half maximal inhibitory concentration (IC₅₀) values ranging ~25–200 μg of lipid) with the highest activities in glyco- and phospho-lipid fractions. The structural analysis of the bioactive lipid fraction-2 revealed the presence of specific sulfoquinovosyl diacylglycerols (SQDG) bioactive molecules and the HexCer-t36:2 (t18:1/18:1 and 18:2/18:0) cerebrosides with a phytosphingosine (4-hydrosphinganine) base, while fraction-3 contained bioactive phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules. These novel bioactive lipids of Chlorococcum sp. with putative health benefits may indicate that marine microalgae can be a sustainable alternative source for bioactive lipids production for food supplements and nutraceutical applications. However, further studies are required towards the commercial technology pathways development and biosafety analysis for the use of the microalga.     

Exploring the Mangrove Fruit: From the Phytochemicals to Functional Food Development and the Current Progress in the Middle East

Nowadays, the logarithmic production of existing well-known food materials is unable to keep up with the demand caused by the exponential growth of the human population in terms of the equality of access to food materials. Famous local food materials with treasury properties such as mangrove fruits are an excellent source to be listed as emerging food candidates with ethnomedicinal properties. Thus, this study reviews the nutrition content of several edible mangrove fruits and the innovation to improve the fruit into a highly economic food product. Within the mangrove fruit, the levels of primary metabolites such as carbohydrates, protein, and fat are acceptable for daily intake. The mangrove fruits, seeds, and endophytic fungi are rich in phenolic compounds, limonoids, and their derivatives as the compounds present a multitude of bioactivities such as antimicrobial, anticancer, and antioxidant. In the intermediary process, the flour of mangrove fruit stands as a supplementation for the existing flour with antidiabetic or antioxidant properties. The mangrove fruit is successfully transformed into many processed food products. However, limited fruits from species such as Bruguiera gymnorrhiza, Rhizophora mucronata, Sonneratia caseolaris, and Avicennia marina are commonly upgraded into traditional food, though many more species demonstrate ethnomedicinal properties. In the Middle East, A. marina is the dominant species, and the study of the phytochemicals and fruit development is limited. Therefore, studies on the development of mangrove fruits to functional for other mangrove species are demanding. The locally accepted mangrove fruit is coveted as an alternate food material to support the sustainable development goal of eliminating world hunger in sustainable ways.     

Antioxidant Bioactivity of Extracts from Beach Cast Leaves of Posidonia oceanica (L.) Delile

The marine environment is a generous source of biologically active compounds useful for human health. In 50 years, about 25,000 bioactive marine compounds have been identified, with an increase of 5% per year. Peculiar feature of algae and plants is the production of secondary metabolites, such as polyphenols, synthesized as a form of adaptation to environmental stress. Posidonia oceanica is a Mediterranean endemic and dominant seagrass and represents a biologically, ecologically and geologically important marine ecosystem. Within this study, methanolic and ethanolic extracts were generated from fresh and dried Posidonia oceanica leaves, with the aim to employ and valorize the beach cast leaves. The best yield and antioxidant activity (polyphenols content equal to 19.712 ± 0.496 mg GAE/g and DPPH IC50 of 0.090 µg/µL.) were recorded in 70% ethanol extracts (Gd-E4) obtained from leaves dried for two days at 60 °C and ground four times. HPLC analyses revealed the presence of polyphenols compounds (the most abundant of which was chicoric acid) with antioxidant and beneficial properties. Bioactive properties of the Gd-E4 extracts were evaluated in vitro using fibroblast cells line (HS-68), subjected to UV induced oxidative stress. Pre-treatment of cells with Gd-E4 extracts led to significant protection against oxidative stress and mortality associated with UV exposure, thus highlighting the beneficial properties of antioxidants compounds produced by these marine plants against photo damage, free radicals and associated negative cellular effects. Beach cast leaves selection, processing and extraction procedures, and the in vitro assay results suggested the potentiality of a sustainable approach for the biotechnological exploitation of this resource and could serve a model for other marine resources.     

Variability in Macro- and Micronutrients of 15 Commercially Available Microalgae Powders

The nutrient composition of 15 commercially available microalgae powders of Arthrospira platensis, Chlorella pyrenoidosa and vulgaris, Dunaliella salina, Haematococcus pluvialis, Tetraselmis chuii, and Aphanizomenon flos-aquae was analyzed. The Dunaliella salina powders were characterized by a high content of carbohydrates, saturated fatty acids (SFAs), omega-6-polyunsaturated fatty acids (n6-PUFAs), heavy metals, and α-tocopherol, whereas the protein amounts, essential amino acids (EAAs), omega-3-PUFAs (n3-PUFAs), vitamins, and minerals were low. In the powder of Haematococcus pluvialis, ten times higher amounts of carotenoids compared to all other analyzed powders were determined, yet it was low in vitamins D and E, protein, and EAAs, and the n6/n3-PUFAs ratio was comparably high. Vitamin B₁₂, quantified as cobalamin, was below 0.02 mg/100 g dry weight (d.w.) in all studied powders. Based on our analysis, microalgae such as Aphanizomenon and Chlorella may contribute to an adequate intake of critical nutrients such as protein with a high content of EAAs, dietary fibers, n3-PUFAs, Ca, Fe, Mg, and Zn, as well as vitamin D and E. Yet, the nutritional value of Aphanizomenon flos-aquae was slightly decreased by high contents of SFAs. The present data show that microalgae are rich in valuable nutrients, but the macro- and micronutrient profiles differ strongly between and within species.     

Antioxidant,Antimicrobial and Genotoxicity Screening of Hydro-alcoholic Extracts of Five Serbian <Emphasis Type="Italic">Equisetum</Emphasis> Species

The hydro-alcoholic extracts of five Equisetum species, E. arvense L., E. sylvaticum L., E. fluviatile L., E. palustre L. and E. telmateia Ehrh., growing-wild in Serbia were evaluated for their genotoxicity, antimicrobial activity, antioxidant capacity and the results related to the total phenol content and HPLC flavonoid profiles. The total phenol content was 92–349 μmol expressed as equivalents of chlorogenic acid per g of dried plant material. Main identified compounds were kaempferol-, quercetin- glycosides and caffeic acid derivatives. E. telmateia extract showed the greatest antioxidant capacity. Almost all tested microorganisms demonstrated some degree of sensitivity to the examined extracts. All tested extracts at 62.5 μg/ml showed higher incidence of micronucleus formation than in the control sample. The obtained data allowed mutual comparison of examined species and their assessment as possible sources of antioxidants, antimicrobials and/or genotoxic substances.     

A Microencapsulation Method for Delivering Tetrodotoxin to Bivalves to Investigate Uptake and Accumulation

Most marine biotoxins are produced by microalgae. The neurotoxin tetrodotoxin (TTX) has been reported in many seafood species worldwide but its source is unknown, making accumulation and depuration studies in shellfish difficult. Tetrodotoxin is a water-soluble toxin and cannot be directly ingested by shellfish. In the present study, a method was developed which involved binding TTX to solid particles of humic acid and encapsulating them in agar-gelatin capsules. A controlled quantity of TTX-containing microcapsules (size range 20–280 μm) was fed to Paphies australis, a bivalve known to accumulate TTX in the wild. The TTX-containing microcapsules were fed to P. australis every second day for 13 days. Ten P. australis (including five controls fed non-toxic microalgae) were harvested after 7 days and ten after 13 days. Paphies australis accumulated TTX, reaching concentrations of up to 103 µg kg⁻¹ by day 13, exceeding the European Food Safety Authority recommended concentration of 44 μg kg⁻¹ in shellfish. This novel method will allow future studies to explore the effects, accumulation and depuration rates of TTX in different animals and document how it is transferred through food webs.     

Functional Feeds to Tackle Meagre (Argyrosomus regius) Stress: Physiological Responses under Acute Stressful Handling Conditions

Marine algae are recognised sources of bioactive compounds that have attracted great interest as nutritional supplements for aquaculture fish. Intensive rearing conditions often expose fish to husbandry-related stressors, rendering fish more susceptible to disease and reducing production yields. The present work evaluated the potential of two marine algae extracts (Fucus vesiculosus and Nannochloropsis gaditana) as nutritional supplements to mitigate stress effects in meagre (Argyrosomus regius) exposed to an acute handling stress (AS). A plant-based diet was used as a control, and three other diets were prepared, which were similar to the control diet but supplemented with 1% of each algal extract or a combination of the two extracts (0.5% each). The effects of supplemented diets on stress biomarkers, antioxidant enzyme activities, and immune response were analysed in fish exposed to AS after 4 weeks of feeding. Supplemented diets did not affect growth performance but the inclusion of F. vesiculosus promoted higher feed efficiency, as compared to the control group. Dietary algal extracts supplementation reduced plasma glucose levels, increased white blood cell counts, and reduced the expression of pro-inflammatory genes when compared with the control. N. gaditana supplementation led to a reduction in hepatic antioxidant enzyme activity and glutathione levels, while F. vesiculosus supplementation increased muscle glutathione reductase activity and reduced lipid peroxidation. These findings support the potential of algal extracts as nutraceuticals in aquafeeds to enhance the ability of fish to cope with husbandry-related stressful conditions and ultimately improve fish health and welfare.     

Effect of Solvent System on Extractability of Lipidic Components of Scenedesmus obliquus (M2-1) and Gloeothece sp. on Antioxidant Scavenging Capacity Thereof

Microalgae are well known for their biotechnological potential, namely with regard to bioactive lipidic components—especially carotenoids and polyunsaturated fatty acids (PUFA), well-known for therapeutic applications based on their antioxidant capacity. The aim of this work was to evaluate the influence of four distinct food-grade solvents upon extractability of specific lipidic components, and on the antioxidant capacity exhibited against both synthetic (2,2-diphenyl-1-picrylhydrazyl (DPPH^•) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS^+•)) and biological reactive species (O₂^•− and ^•NO⁻). A eukaryotic microalga (Scenedesmus obliquus (M2-1)) and a prokaryotic one (Gloeothece sp.) were used as case studies. Concerning total antioxidant capacity, the hexane:isopropanol (3:2) and acetone extracts of Sc. obliquus (M2-1) were the most effective against DPPH^• and ABTS^+•, respectively. Gloeothece sp. ethanol extracts were the most interesting scavengers of O₂^•−, probably due the high content of linolenic acid. On the other hand, acetone and hexane:isopropanol (3:2) extracts were the most interesting ones in ^•NO⁻ assay. Acetone extract exhibited the best results for the ABTS assay, likely associated to its content of carotenoids, in both microalgae. Otherwise, ethanol stood out in PUFA extraction. Therefore, profiles of lipidic components extracted are critical for evaluating the antioxidant performance—which appears to hinge, in particular, on the balance between carotenoids and PUFAs.