Antiproliferative Activity of Cyanophora paradoxa Pigments in Melanoma,Breast and Lung Cancer Cells

The glaucophyte Cyanophora paradoxa (Cp) was chemically investigated to identify pigments efficiently inhibiting malignant melanoma, mammary carcinoma and lung adenocarcinoma cells growth. Cp water and ethanol extracts significantly inhibited the growth of the three cancer cell lines in vitro, at 100 µg·mL⁻¹. Flash chromatography of the Cp ethanol extract, devoid of c-phycocyanin and allophycocyanin, enabled the collection of eight fractions, four of which strongly inhibited cancer cells growth at 100 µg·mL⁻¹. Particularly, two fractions inhibited more than 90% of the melanoma cells growth, one inducing apoptosis in the three cancer cells lines. The detailed analysis of Cp pigment composition resulted in the discrimination of 17 molecules, ten of which were unequivocally identified by high resolution mass spectrometry. Pheophorbide a, β-cryptoxanthin and zeaxanthin were the three main pigments or derivatives responsible for the strong cytotoxicity of Cp fractions in cancer cells. These data point to Cyanophora paradoxa as a new microalgal source to purify potent anticancer pigments, and demonstrate for the first time the strong antiproliferative activity of zeaxanthin and β-cryptoxanthin in melanoma cells.     

Cytotoxicity,Fractionation and Dereplication of Extracts of the Dinoflagellate Vulcanodinium rugosum,a Producer of Pinnatoxin G

Pinnatoxin G (PnTX-G) is a marine toxin belonging to the class of cyclic imines and produced by the dinoflagellate Vulcanodinium rugosum. In spite of its strong toxicity to mice, leading to the classification of pinnatoxins into the class of “fast-acting toxins”, its hazard for human health has never been demonstrated. In this study, crude extracts of V. rugosum exhibited significant cytotoxicity against Neuro2A and KB cells. IC₅₀ values of 0.38 µg mL⁻¹ and 0.19 µg mL⁻¹ were estimated on Neuro2A cells after only 24 h of incubation and on KB cells after 72 h of incubation, respectively. In the case of Caco-2 cells 48 h after exposure, the crude extract of V. rugosum induced cell cycle arrest accompanied by a dramatic increase in double strand DNA breaks, although only 40% cytotoxicity was observed at the highest concentration tested (5 µg mL⁻¹). However, PnTX-G was not a potent cytotoxic compound as no reduction of the cell viability was observed on the different cell lines. Moreover, no effects on the cell cycle or DNA damage were observed following treatment of undifferentiated Caco-2 cells with PnTX-G. The crude extract of V. rugosum was thus partially purified using liquid-liquid partitioning and SPE clean-up. In vitro assays revealed strong activity of some fractions containing no PnTX-G. The crude extract and the most potent fraction were evaluated using full scan and tandem high resolution mass spectrometry. The dereplication revealed the presence of a major compound that could be putatively annotated as nakijiquinone A, N-carboxy-methyl-smenospongine or stachybotrin A, using the MarinLit™ database. Further investigations will be necessary to confirm the identity of the compounds responsible for the cytotoxicity and genotoxicity of the extracts of V. rugosum.     

Gloeothece sp.—Exploiting a New Source of Antioxidant,Anti-Inflammatory,and Antitumor Agents

Bioactive lipidic compounds of microalgae, such as polyunsaturated fatty acids (PUFA) and carotenoids, can avoid or treat oxidation-associated conditions and diseases like inflammation or cancer. This study aimed to assess the bioactive potential of lipidic extracts obtained from Gloeothece sp.–using Generally Recognized as Safe (GRAS) solvents like ethanol, acetone, hexane:isopropanol (3:2) (HI) and ethyl lactate. The bioactive potential of extracts was assessed in terms of antioxidant (ABTS^•+, DPPH^•, ^•NO and O₂^•assays), anti-inflammatory (HRBC membrane stabilization and Cox-2 screening assay), and antitumor capacity (death by TUNEL, and anti-proliferative by BrdU incorporation assay in AGS cancer cells); while its composition was characterized in terms of carotenoids and fatty acids, by HPLC-DAD and GC-FID methods, respectively. Results revealed a chemopreventive potential of the HI extract owing to its ability to: (I) scavenge ^-NO^• radical (IC₅₀, 1258 ± 0.353 µg·mL⁻¹); (II) inhibit 50% of COX-2 expression at 130.2 ± 7.4 µg·mL⁻¹; (III) protect 61.6 ± 9.2% of lysosomes from heat damage, and (IV) induce AGS cell death by 4.2-fold and avoid its proliferation up to 40% in a concentration of 23.2 ± 1.9 µg·mL⁻¹. Hence, Gloeothece sp. extracts, namely HI, were revealed to have the potential to be used for nutraceutical purposes.     

Territrem and Butyrolactone Derivatives from a Marine-Derived Fungus Aspergillus Terreus

Seventeen lactones including eight territrem derivatives (1–8) and nine butyrolactone derivatives (9–17) were isolated from a marine-derived fungus Aspergillus terreus SCSGAF0162 under solid-state fermentation of rice. Compounds 1–3 and 9–10 were new, and their structures were elucidated by spectroscopic analysis. The acetylcholinesterase inhibitory activity and antiviral activity of compounds 1–17 were evaluated. Among them, compounds 1 and 2 showed strong inhibitory activity against acetylcholinesterase with IC₅₀ values of 4.2 ± 0.6, 4.5 ± 0.6 nM, respectively. This is the first time it has been reported that 3, 6, 10, 12 had evident antiviral activity towards HSV-1 with IC₅₀ values of 16.4 ± 0.6, 6.34 ± 0.4, 21.8 ± 0.8 and 28.9 ± 0.8 μg·mL⁻¹, respectively. Antifouling bioassay tests showed that compounds 1, 11, 12, 15 had potent antifouling activity with EC₅₀ values of 12.9 ± 0.5, 22.1 ± 0.8, 7.4 ± 0.6, 16.1 ± 0.6 μg·mL⁻¹ toward barnacle Balanus amphitrite larvae, respectively.     

Identification of the Major ACE-Inhibitory Peptides Produced by Enzymatic Hydrolysis of a Protein Concentrate from Cuttlefish Wastewater

The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC₅₀ = 76.8 ± 15.2 μg mL⁻¹) after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO) membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC₅₀ value of 58.4 ± 4.6 μg mL⁻¹. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC). Four fractions (A–D) with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR) followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC₅₀ values of the fractions ranged from 1.92 to 8.83 μg mL⁻¹, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions.     

Hunt for Palytoxins in a Wide Variety of Marine Organisms Harvested in 2010 on the French Mediterranean Coast

During the summer of 2010, 31 species including fish, echinoderms, gastropods, crustaceans, cephalopods and sponges were sampled in the Bay of Villefranche on the French Mediterranean coast and screened for the presence of PLTX-group toxins using the haemolytic assay. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for confirmatory purposes and to determine the toxin profile. The mean toxin concentration in the whole flesh of all sampled marine organisms, determined using the lower- (LB) and upper-bound (UB) approach was 4.3 and 5.1 µg·kg⁻¹, respectively, with less than 1% of the results exceeding the European Food Safety Authority (EFSA) threshold of 30 µg·kg⁻¹ and the highest values being reported for sea urchins (107.6 and 108.0 µg·kg⁻¹). Toxins accumulated almost exclusively in the digestive tube of the tested species, with the exception of octopus, in which there were detectable toxin amounts in the remaining tissues (RT). The mean toxin concentration in the RT of the sampled organisms (fishes, echinoderms and cephalopods) was 0.7 and 1.7 µg·kg⁻¹ (LB and UB, respectively), with a maximum value of 19.9 µg·kg⁻¹ for octopus RT. The herbivorous and omnivorous organisms were the most contaminated species, indicating that diet influences the contamination process, and the LC-MS/MS revealed that ovatoxin-a was the only toxin detected.     

Pachydictyols B and C: New Diterpenes from Dictyota dichotoma Hudson

Two new diterpenoids, pachydictyol B (1a/1b) and C (2), were isolated from the dichloromethane extract of the marine brown alga, Dictyota dichotoma, collected from the Red Sea coast of Egypt, along with the known metabolites, pachydictyol A (3a), dictyol E (4), cis-africanan-1α-ol (5a), fucosterol (6), tetrahydrothiophen-1,1-dioxide and poly-β-hydroxybutyric acid. GC-MS analysis of the nonpolar fractions also indicated the presence of β-bourbonene and nonanal, along with three hydrocarbons and five fatty acids or their simple derivatives, respectively. GC-MS analysis of the unsaponifiable algal petroleum ether extract revealed the presence of a further eight compounds, among them 2,2,6,7-tetramethyl-10-oxatricyclo[4.3.0.1(1,7)]decan-5-one (7), N-(4-bromo-n-butyl)-piperidin-2-one (8) and tert-hexadecanethiol. Structures 1–6 were assigned by 1D and 2D NMR, mass spectra (EI, CI, HREI and HRESI) and by comparison with data from related structures. The crude algal extract was potently active against the breast carcinoma tumor cell line, MCF7 (IC₅₀ = 0.6 µg mL⁻¹); pachydictyol B (1a) and dictyol E (4) showed weak antimicrobial properties, and the other compounds were inactive. Pachydictyols B (1a) and C (2) demonstrated a weak and unselective cytotoxicity against twelve human tumor cell lines with a mean IC₅₀ of >30.0 µM.     

Haloferax mediterranei Cells as C50 Carotenoid Factories

Haloarchaea produce C50 carotenoids such as bacterioruberin, which are of biotechnological in-terest. This study aimed to analyze the effect of different environmental and nutritional conditions on the cellular growth and dynamics of carotenoids accumulation in Haloferax mediterranei. The maximum production of carotenoids (40 µg·mL⁻¹) was obtained during the stationary phase of growth, probably due to nutrient-limiting conditions (one-step culture). By seven days of culture, 1 mL culture produced 22.4 mg of dry weight biomass containing 0.18 % (w/w) of carotenoids. On the other hand, carbon-deficient cultures (low C/N ratio) were observed to be optimum for C50 bacterioruberin production by Hfx. mediterranei, but negatively affected the growth of cells. Thus, a two-steps process was evaluated for optimum carotenoids yield. In the first step, a nutri-ent-repleted culture medium enabled the haloarchaea to produce biomass, while in the second step, the biomass was incubated under osmotic stress and in a carbon-deficient medium. Under the conditions used, the obtained biomass contained 0.27% (w/w) of carotenoids after seven days, which accounts for 58.49 µg·mL⁻¹ of carotenoids for a culture with turbidity 14.0.     

Crypthecodinium cohnii Growth and Omega Fatty Acid Production in Mediums Supplemented with Extract from Recycled Biomass

Crypthecodinium cohnii is a marine heterotrophic dinoflagellate that can accumulate high amounts of omega-3 polyunsaturated fatty acids (PUFAs), and thus has the potential to replace conventional PUFAs production with eco-friendlier technology. So far, C. cohnii cultivation has been mainly carried out with the use of yeast extract (YE) as a nitrogen source. In the present study, alternative carbon and nitrogen sources were studied: the extraction ethanol (EE), remaining after lipid extraction, as a carbon source, and dinoflagellate extract (DE) from recycled algae biomass C. cohnii as a source of carbon, nitrogen, and vitamins. In mediums with glucose and DE, the highest specific biomass growth rate reached a maximum of 1.012 h⁻¹, while the biomass yield from substrate reached 0.601 g·g⁻¹. EE as the carbon source, in comparison to pure ethanol, showed good results in terms of stimulating the biomass growth rate (an 18.5% increase in specific biomass growth rate was observed). DE supplement to the EE-based mediums promoted both the biomass growth (the specific growth rate reached 0.701 h⁻¹) and yield from the substrate (0.234 g·g⁻¹). The FTIR spectroscopy data showed that mediums supplemented with EE or DE promoted the accumulation of PUFAs/docosahexaenoic acid (DHA), when compared to mediums containing glucose and commercial YE.     

Structural Characteristics and Anticancer Activity of Fucoidan from the Brown Alga Sargassum mcclurei

Three different fucoidan fractions were isolated and purified from the brown alga, Sargassum mcclurei. The SmF1 and SmF2 fucoidans are sulfated heteropolysaccharides that contain fucose, galactose, mannose, xylose and glucose. The SmF3 fucoidan is highly sulfated (35%) galactofucan, and the main chain of the polysaccharide contains a →3)-α-l-Fucp(2,4SO₃⁻)-(1→3)-α-l-Fucp(2,4SO₃⁻)-(1→ motif with 1,4-linked 3-sulfated α-l-Fucp inserts and 6-linked galactose on reducing end. Possible branching points include the 1,2,6- or 1,3,6-linked galactose and/or 1,3,4-linked fucose residues that could be glycosylated with terminal β-d-Galp residues or chains of alternating sulfated 1,3-linked α-l-Fucp and 1,4-linked β-d-Galp residues, which have been identified in galactofucans for the first time. Both α-l-Fucp and β-d-Galp residues are sulfated at C-2 and/or C-4 (and some C-6 of β-d-Galp) and potentially the C-3 of terminal β-d-Galp, 1,4-linked β-d-Galp and 1,4-linked α-l-Fucp residues. All fucoidans fractions were less cytotoxic and displayed colony formation inhibition in colon cancer DLD-1 cells. Therefore, these fucoidan fractions are potential antitumor agents.     

Purification and Characterization of a Fucoidanase (FNase S) from a Marine Bacterium Sphingomonas paucimobilis PF-1

The Search for enzyme activities that efficiently degrade marine polysaccharides is becoming an increasingly important area for both structural analysis and production of lower-molecular weight oligosaccharides. In this study, an endo-acting fucoidanase that degrades Miyeokgui fucoidan (MF), a sulfated galactofucan isolated from the sporophyll (called Miyeokgui in Korean) of Undaria pinnatifida, into smaller-sized galactofuco-oligosaccharides (1000–4000 Da) was purified from a marine bacterium, Sphingomonas paucimobilis PF-1, by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose column chromatography, and chromatofocusing. The specific activity of this enzyme was approximately 112-fold higher than that of the crude enzyme, and its molecular weight was approximately 130 kDa (FNase S), as determined by native gel electrophoresis and 130 (S1), 70 (S2) and 60 (S3) kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of FNase S were pH 6.0–7.0 and 40–45 °C, respectively. FNase S activity was enhanced by Mn²⁺ and Na⁺ (115.7% and 131.2%), but it was inhibited by Ca²⁺, K⁺, Ba²⁺, Cu²⁺ (96%, 83.7%, 84.3%, and 89.3%, respectively), each at 1 mM. The K_m, V_max and K_cat values of FNase S on MF were 1.7 mM, 0.62 mg·min⁻¹, and 0.38·S⁻¹, respectively. This enzyme could be a valuable tool for the structural analysis of fucoidans and production of bioactive fuco-oligosaccharides.     

Generation of Internal-Image Functional Aptamers of Okadaic Acid via Magnetic-Bead SELEX

Okadaic acid (OA) is produced by Dinophysis and Prorocentrum dinoflagellates and primarily accumulates in bivalves, and this toxin has harmful effects on consumers and operators. In this work, we first report the use of aptamers as novel non-toxic probes capable of binding to a monoclonal antibody against OA (OA-mAb). Aptamers that mimic the OA toxin with high affinity and selectivity were generated by the magnetic bead-assisted systematic evolution of ligands by exponential enrichment (SELEX) strategy. After 12 selection rounds, cloning, sequencing and enzyme-linked immunosorbent assay (ELISA) analysis, four candidate aptamers (O24, O31, O39, O40) were selected that showed high affinity and specificity for OA-mAb. The affinity constants of O24, O31, O39 and O40 were 8.3 × 10⁸ M⁻¹, 1.47 × 10⁹ M⁻¹, 1.23 × 10⁹ M⁻¹ and 1.05 × 10⁹ M⁻¹, respectively. Indirect competitive ELISA was employed to determine the internal-image function of the aptamers. The results reveal that O31 has a similar competitive function as free OA toxin, whereas the other three aptamers did not bear the necessary internal-image function. Based on the derivation of the curvilinear equation for OA/O31, the equation that defined the relationship between the OA toxin content and O31 was Y = 2.185X − 1.78. The IC₅₀ of O31 was 3.39 ng·mL⁻¹, which was close to the value predicted by the OA ELISA (IC₅₀ = 4.4 ng·mL⁻¹); the IC₁₀ was 0.33 ng·mL⁻¹. The above data provides strong evidence that internal-image functional aptamers could be applicable as novel probes in a non-toxic assay.     

Cyanobacterial Toxic and Bioactive Peptides in Freshwater Bodies of Greece: Concentrations,Occurrence Patterns,and Implications for Human Health

Cyanobacterial harmful algal blooms represent one of the most conspicuous waterborne microbial hazards in aquatic environments mostly due to the production of toxic secondary metabolites, mainly microcystins (MCs). Other bioactive peptides are frequently found in cyanobacterial blooms, yet their concentration and ecological relevance is still unknown. In this paper we studied the presence and concentration of cyanobacterial peptides (microcystins, anabaenopeptins, anabaenopeptilides) in 36 Greek freshwater bodies, using HPLC-DAD, ELISA, and PP1IA. Microcystins were found in more than 90% of the samples investigated, indicating that microcystin-producing strains seem to also occur in lakes without blooms. Microcystins MC-RR, MC-LR, and MC-YR were the main toxin constituents of the bloom samples. Anabaenopeptin A and B were predominant in some samples, whereas anabaenopeptolide 90A was the only peptide found in Lake Mikri Prespa. The intracellular concentrations of anabaenopeptins produced by cyanobacterial bloom populations are determined for the first time in this study; the high (>1000 µg·L⁻¹) anabaenopeptin concentration found indicates there may be some impacts, at least on the ecology and the food web structure of the aquatic ecosystems. The maximum intracellular MC values measured in Lakes Kastoria and Pamvotis, exceeding 10,000 µg·L⁻¹, are among the highest reported.     

Optimisation of Healthy-Lipid Content and Oxidative Stability during Oil Extraction from Squid (Illex argentinus) Viscera by Green Processing

Green extraction was applied to Argentinean shortfin squid (Illex argentinus) viscera, consisting of a wet pressing method including a drying step, mechanic pressing, centrifugation of the resulting slurry, and oil collection. To maximise the oil yield and ω3 fatty acid content and to minimise the oil damage degree, a response surface methodology (RSM) design was developed focused on the drying temperature (45–85 °C) and time (30–90 min). In general, an increase of the drying time and temperature provided an increase in the lipid yield recovery from the viscera. The strongest drying conditions showed a higher recovery than 50% when compared with the traditional chemical method. The docosahexaenoic and eicosapentaenoic acid contents in the extracted oil revealed scarce dependence on drying conditions, showing valuable ranges (149.2–166.5 and 88.7–102.4 g·kg⁻¹ oil, respectively). Furthermore, the values of free fatty acids, peroxides, conjugated dienes, and ω3/ω6 ratio did not show extensive differences by comparing oils obtained from the different drying conditions. Contrary, a polyene index (PI) decrease was detected with increasing drying time and temperature. The RSM analysis indicated that optimised drying time (41.3 min) and temperature (85 °C) conditions would lead to 74.73 g·kg⁻¹ (oil yield), 1.87 (PI), and 6.72 (peroxide value) scores, with a 0.67 desirability value.     

Overexpression and Characterization of a Novel Thermostable β-Agarase YM01-3, from Marine Bacterium Catenovulum agarivorans YM01T

Genome sequencing of Catenovulum agarivorans YM01^T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01^T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60 °C and had a K_m of 3.78 mg mL⁻¹ for agarose and a V_max of 1.14 × 10⁴ U mg⁻¹. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.     

Hydrolysis of Fucoidan by Fucoidanase Isolated from the Marine Bacterium,Formosa algae

Intracellular fucoidanase was isolated from the marine bacterium, Formosa algae strain KMM 3553. The first appearance of fucoidan enzymatic hydrolysis products in a cell-free extract was detected after 4 h of bacterial growth, and maximal fucoidanase activity was observed after 12 h of growth. The fucoidanase displayed maximal activity in a wide range of pH values, from 6.5 to 9.1. The presence of Mg²⁺, Ca²⁺ and Ba²⁺ cations strongly activated the enzyme; however, Cu²⁺ and Zn²⁺ cations had inhibitory effects on the enzymatic activity. The enzymatic activity of fucoidanase was considerably reduced after prolonged (about 60 min) incubation of the enzyme solution at 45 °C. The fucoidanase catalyzed the hydrolysis of fucoidans from Fucus evanescens and Fucus vesiculosus, but not from Saccharina cichorioides. The fucoidanase also did not hydrolyze carrageenan. Desulfated fucoidan from F. evanescens was hydrolysed very weakly in contrast to deacetylated fucoidan, which was hydrolysed more actively compared to the native fucoidan from F. evanescens. Analysis of the structure of the enzymatic products showed that the marine bacteria, F. algae, synthesized an α-l-fucanase with an endo-type action that is specific for 1→4-bonds in a polysaccharide molecule built up of alternating three- and four-linked α-l-fucopyranose residues sulfated mainly at position 2.     

Complex Toxin Profile of French Mediterranean Ostreopsis cf. ovata Strains,Seafood Accumulation and Ovatoxins Prepurification

Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 μg·kg⁻¹ of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 μg·kg⁻¹ in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell⁻¹. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.     

Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37–40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca²⁺, Mn²⁺, Zn²⁺ or Ni²⁺, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.     

Production of Calcaride A by Calcarisporium sp. in Shaken Flasks and Stirred Bioreactors

Increased interest in marine resources has led to increased screening of marine fungi for novel bioactive compounds and considerable effort is being invested in discovering these metabolites. For compound discovery, small-scale cultures are adequate, but agitated bioreactors are desirable for larger-scale production. Calcarisporium sp. KF525 has recently been described to produce calcaride A, a cyclic polyester with antibiotic activity, in agitated flasks. Here, we describe improvements in the production of calcaride A in both flasks (13-fold improvement) and stirred bioreactors (200-fold improvement). Production of calcaride A in bioreactors was initially substantially lower than in shaken flasks. The cultivation pH (reduced from 6.8 to <5.4), carbon source (sucrose replacing glucose), C/N ratio and nature of mycelial growth (pellets or filaments) were important in improving calcaride A production. Up to 4.5 mg·g⁻¹ biomass (85 mg·L⁻¹) calcaride A were produced in the bioreactor, which was only slightly less than in shaken flasks (14 mg·g⁻¹, 100 mg·L⁻¹). The results demonstrate that a scalable process for calcaride A production could be developed using an iterative approach with flasks and bioreactors.     

Production,Characterization, and Antioxidant Activity of Fucoxanthin from the Marine Diatom Odontella aurita

The production, characterization, and antioxidant capacity of the carotenoid fucoxanthin from the marine diatom Odontella aurita were investigated. The results showed that low light and nitrogen-replete culture medium enhanced the biosynthesis of fucoxanthin. The maximum biomass concentration of 6.36 g L⁻¹ and maximum fucoxanthin concentration of 18.47 mg g⁻¹ were obtained in cultures grown in a bubble column photobioreactor (Ø 3.0 cm inner diameter), resulting in a fucoxanthin volumetric productivity of 7.96 mg L⁻¹ day⁻¹. A slight reduction in biomass production was observed in the scaling up of O. aurita culture in a flat plate photobioreactor, yet yielded a comparable fucoxanthin volumetric productivity. A rapid method was developed for extraction and purification of fucoxanthin. The purified fucoxanthin was identified as all-trans-fucoxanthin, which exhibited strong antioxidant properties, with the effective concentration for 50% scavenging (EC₅₀) of 1,1-dihpenyl-2-picrylhydrazyl (DPPH) radical and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical being 0.14 and 0.03 mg mL⁻¹, respectively. Our results suggested that O. aurita can be a natural source of fucoxanthin for human health and nutrition.