Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL⁻¹, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC₅₀ of 2.41 µg·mL⁻¹. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.     

Isolation,Characterization and Bioactive Properties of Alkali-Extracted Polysaccharides from Enteromorpha prolifera

Four new purified polysaccharides (PAP) were isolated and purified from the Enteromorpha prolifera by alkali extraction, and further characterization was investigated. Their average molecular weights of PAP-1, PAP-2, PAP-3, and PAP-4 were estimated as 3.44 × 10⁴, 6.42 × 10⁴, 1.20 × 10⁵, and 4.82 × 10⁴ Da, respectively. The results from monosaccharide analysis indicated that PAP-1, PAP-2, PAP-3 were acidic polysaccharides and PAP-4 was a neutral polysaccharide. PAP-1 and PAP-2 mainly consist of galacturonic acid, while PAP-3 and PAP-4 mainly contained rhamnose. Congo red test showed that no triple helical structure was detected in the four polysaccharides. The antioxidant activities were investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), Superoxide, and 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical assay. In vitro antitumor activities were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PAP-1 exhibited relatively stronger antioxidant activities among the four polysaccharides in a dose-dependent manner. At a concentration of 1.00 mg/mL, the antioxidant activities of PAP-1 on the DPPH radical scavenging rate, superoxide anion radical scavenging rate, and ABTS radical rate at 1.00 mg/mL were 56.40%, 54.27%, and 42.07%, respectively. They also showed no significant inhibitory activity against MGC-803, HepG2, T24, and Bel-7402 cells. These investigations of polysaccharides provide a scientific basis for the use of E. prolifera as an ingredient in functional foods and medicines.     

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.     

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.     

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.     

Determination of the Extraction,Physicochemical Characterization,and Digestibility of Sulfated Polysaccharides in Seaweed—Porphyra haitanensis

The aim of the study was to extract Porphyra haitanensis polysaccharides (PHPs) using the water extraction and alcohol precipitation methods and explore their antioxidant activity and physicochemical properties. The single-factor and Box-Behnken response surface methodologies were used to optimize the extraction of polysaccharides from Porphyra haitanensis. Our results showed that the polysaccharide yield was as high as 20.48% with a raw material to water ratio of 0.04, and extraction time of 3 h at 80 °C. The extraction rate observed was similar to the actual extraction rate, thus proving the reliability of the optimization model. The extracted polysaccharides primarily consisted of galactose, glucose, and fucose in the molar ratio 76.2:2.1:1, respectively. The high performance gel permeation chromatography (HPGPC) results showed that the molecular weight of the PHPs obtained was 6.3 × 10⁵ Da, and the sulfate content was 2.7 mg/mL. Fourier infrared spectroscopy was used to analyze the functional groups and structures of the polysaccharides. The effect of concentration, temperature, and pH on the apparent viscosity of the PHPs solution were studied using rheology experiments, which revealed that PHPs were a “non-Newtonian fluid” with shear-thinning behavior. The viscosity of the PHPs gradually increased with increasing sugar concentration, and decreased with increasing temperature, acidity, and alkalinity. Detection of the antioxidant activity of OH*, DPPH*, and ABTS* revealed that the scavenging activity of ABTS* was higher than that of OH* and DPPH* in the concentration range of 1–5 mg/mL. In the experiments of simulating gastric juice and alpha amylase in vitro, it was found that PHPs can better resist digestion of alpha amylase, and have better resistance than fructooligosaccharide (FOS), so PHPs have potential prebiotic activity. These findings demonstrate the potential of PHPs for use in the food and cosmetic industries.     

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.     

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.     

Antifungal activities of extracts produced by liquid fermentations of Chilean Stereum species against Botrytis cinerea (grey mould agent)

Extracts obtained from liquid mycelial fermentations of Chilean species of the Stereum genus showed antifungal activity against Botrytis cinerea. Thirty-six strains were evaluated in vivo and in vitro assays, 25 belonging to Stereum hirsutum (Sh) and 11 to Stereum rameale (Sr). Two types of extracts were obtained: EtOAc-extract (liquid phase) and MeOH-extract (mycelial phase). Plate diffusion assay showed that EtOAc-extracts were more active than MeOH-extracts. S. hirsutum included 4 strains with the highest antifungal activity (Sh134-11, Sh144-11, Sh152-11, Sh155-11) and S. rameale highlighted with two strains (Sr25-11, Sr27-11). Effects on the mycelial growth of B. cinerea showed that EtOAc-extracts produced by S. hirsutum (Sh134-11, Sh152-11) inhibited from 1000 μg mL⁻¹, reaching 67 and 49%, respectively. At 2000 μg mL⁻¹, these strains inhibited nearly 80% of mycelial growth. EtOAc-extract of Sh134-11 was more effective to control the sporogenesis, inhibiting in 100% the sporulation at 500 μg mL⁻¹. Assays showed that Sh134-11 and Sh152-11 exhibited a minimal fungicidal concentration (MFC) of 50 and 100 μg mL⁻¹ respectively and minimal inhibitory concentration (MIC) at 20 μg mL⁻¹. EtOAc-extracts of Sr25-11 and Sr27-11 showed MFC value at 100 μg mL⁻¹ and MIC at 20 and 50 μg mL⁻¹, respectively. Strawberries treated with 1000 μg mL⁻¹of Sh134-11 and Sr25-11 reached 82 and 72% of decay inhibition, respectively. Treatments with 2000 μg mL⁻¹ showed a decay inhibition of 90% approximately. In vivo tests are in accordance with the results obtained in vitro assays, confirming the efficacy of Sh134-11 and Sr25-11 to control B. cinerea. Differences in antifungal activities observed in the different strains suggested that the ability to produce bioactive compounds is not homogenously distributed among S. hirsutum and S. rameale. Our study would suggest that submerged fermentations of Chilean S. hirsutum strain Sh134-11 produce extracts, which could be used as possible biofungicides and an alternative to synthetic fungicides.     

Biochemical Properties of a New Polysaccharide Lyase Family 25 Ulvan Lyase TsUly25B from Marine Bacterium Thalassomonas sp. LD5

Marine macroalgae, contributing much to the bioeconomy, have inspired tremendous attention as sustainable raw materials. Ulvan, as one of the main structural components of green algae cell walls, can be degraded by ulvan lyase through the β-elimination mechanism to obtain oligosaccharides exhibiting several good physiological activities. Only a few ulvan lyases have been characterized until now. This thesis explores the properties of a new polysaccharide lyase family 25 ulvan lyase TsUly25B from the marine bacterium Thalassomonas sp. LD5. Its protein molecular weight was 54.54 KDa, and it was most active under the conditions of 60 °C and pH 9.0. The K_m and k_cat values were 1.01 ± 0.05 mg/mL and 10.52 ± 0.28 s⁻¹, respectively. TsUly25B was salt-tolerant and NaCl can significantly improve its thermal stability. Over 80% of activity can be preserved after being incubated at 30 °C for two days when the concentration of NaCl in the solution is above 1 M, while 60% can be preserved after incubation at 40 °C for 10 h with 2 M NaCl. TsUly25B adopted an endolytic manner to degrade ulvan polysaccharides, and the main end-products were unsaturated ulvan disaccharides and tetrasaccharides. In conclusion, our research enriches the ulvan lyase library and advances the utilization of ulvan lyases in further fundamental research as well as ulvan oligosaccharides production.     

乙酰化蛋黄果多糖及其抗氧化活性研究

蛋黄果（Lucuma nervosa A. DC）乙酰化修饰多糖的抗氧活性优于未修饰多糖,通过分析乙酰化蛋黄果多糖的乙酰化度、红外光谱和形貌特征,阐明引起抗氧活性提升的原因。试验采用超声波辅助法提取蛋黄果果肉粗多糖,用三氯乙酸法除去蛋白质得到多糖。选取3份0.50 g多糖加入20% NaOH溶液溶解,分别加入1、3、5 mL乙酸酐,得到3种取代度（0.237±0.05、0.275±0.07、0.228±0.04）的乙酰化蛋黄果果肉多糖,评价其清除DPPH自由基、OH自由基和ABTS自由基的能力。结果表明,乙酸酐添加量由少到多时,蛋黄果多糖乙酰基取代度先显著升高,后趋于平缓下降。乙酰化蛋黄果多糖的红外光谱显示了3417 cm^-1（-OH）、2950 cm^-1（-CH）处的伸缩振动和1636 cm^-1处的（-OH）转动振动,1738 cm^-1处出现酯基C=O的伸缩振动吸收峰,表明乙酰化已经成功接入。扫描电镜结果显示,未经乙酰化的蛋黄果多糖表面相对光滑平整,呈片状结构;乙酰化修饰多糖分子间交联增强,构象发生了变化,变为表面形状不规则,出现很多空隙且表面粗糙。3种乙酰化蛋黄果多糖中DHG-Ac2（3 mL醋酐）清除效果最好,在浓度1.0 mg/mL时,DPPH清除能力、ABTS自由基清除率、OH自由基清除率分别为91.37%、58.73%、56.36%,分别高于蛋黄果多糖10.0%、43.7%、25.3%。这是引入的乙酰基能活化多糖链中的异头碳,使多糖的供氢能力提升,继而提升乙酰化蛋黄果多糖的抗氧化作用。本研究为海南产蛋黄果多糖的深入开发与抗氧化应用提供了基础研究数据。     

The Structure-Activity Relationship between Marine Algae Polysaccharides and Anti-Complement Activity

In this study, 33 different polysaccharides were prepared to investigate the structure-activity relationships between the polysaccharides, mainly from marine algae, and anti-complement activity in the classical pathway. Factors considered included extraction methods, fractionations, molecular weight, molar ratio of galactose to fucose, sulfate, uronic acid (UA) content, linkage, branching, and the type of monosaccharide. It was shown that the larger the molecular weights, the better the activities. The molar ratio of galactose (Gal) to fucose (Fuc) was a positive factor at a concentration lower than 10 µg/mL, while it had no effect at a concentration more than 10 µg/mL. In addition, sulfate was necessary; however, the sulfate content, the sulfate pattern, linkage and branching had no effect at a concentration of more than 10 µg/mL. Moreover, the type of monosaccharide had no effect. Laminaran and UA fractions had no activity; however, they could reduce the activity by decreasing the effective concentration of the active composition when they were mixed with the active compositions. The effect of the extraction methods could not be determined. Finally, it was observed that sulfated galactofucan showed good anti-complement activity after separation.     

Inhibition of SARS-CoV-2 Virus Entry by the Crude Polysaccharides of Seaweeds and Abalone Viscera In Vitro

Much attention is being devoted to the potential of marine sulfated polysaccharides as antiviral agents in preventing COVID-19. In this study, sulfated fucoidan and crude polysaccharides, extracted from six seaweed species (Undaria pinnatifida sporophyll, Laminaria japonica, Hizikia fusiforme, Sargassum horneri, Codium fragile, Porphyra tenera) and Haliotis discus hannai (abalone viscera), were screened for their inhibitory activity against SARS-CoV-2 virus entry. Most of them showed significant antiviral activities at an IC50 of 12~289 μg/mL against SARS-CoV-2 pseudovirus in HEK293/ACE2, except for P. tenera (IC50 > 1000 μg/mL). The crude polysaccharide of S. horneri showed the strongest antiviral activity, with an IC50 of 12 μg/mL, to prevent COVID-19 entry, and abalone viscera and H. fusiforme could also inhibit SARS-CoV-2 infection with an IC50 of 33 μg/mL and 47 μg/mL, respectively. The common properties of these crude polysaccharides, which have strong antiviral activity, are high molecular weight (>800 kDa), high total carbohydrate (62.7~99.1%), high fucose content (37.3~66.2%), and highly branched polysaccharides. These results indicated that the crude polysaccharides from seaweeds and abalone viscera can effectively inhibit SARS-CoV-2 entry.     

Extracellular Polymeric Substances Produced by the Thermophilic Cyanobacterium Gloeocapsa gelatinosa: Characterization and Assessment of Their Antioxidant and Metal-Chelating Activities

Cyanobacteria, particularly thermophilic strains, represent an important potential source of EPSs, harboring structural complexity that predicts diverse and specific bioactive potential. The thermophilic cyanobacteria Gloeocapsa gelatinosa, isolated from a natural hot source in Ain Echfa (Tunisia), was cultivated in a cylindrical reactor, and the production of biomass and EPSs was investigated. Results revealed that the strain is amongst the most efficient EPSs producers (0.89 g L⁻¹) and that EPSs production was not correlated with the growth phase. EPSs were sulfated heteropolysaccharides containing carbohydrates (70%) based on nine different monosaccharides, mainly mannose (22%), and with the presence of two uronic acids. EPSs were formed by two polymers moieties with a molecular weight of 598.3 ± 7.2 and 67.2 ± 4.4 kDa. They are thermostable in temperatures exceeding 100 °C and have an anionic nature (zeta potential of −40 ± 2 mV). Atomic force microscopy showed that EPSs formed multimodal lumps with 88 nm maximum height. EPSs presented high water holding capacity (70.29 ± 2.36%) and solubility index (97.43 ± 1.24%), and a strong bivalent metal sorption capacity especially for Cu²⁺ (91.20 ± 1.25%) and Fe²⁺ (75.51 ± 0.71%). The antioxidant activity of G. gelatinosa EPSs was investigated using four methods: the β-carotene-bleaching activity, DPPH assays, iron-reducing activity, and metal-chelating activity. EPS has shown high potential as free radicals’ scavenger, with an IC₅₀ on DPPH (0.2 g L⁻¹) three-fold lower than ascorbic acid (0.6 g L ⁻¹) and as a metal chelating activity (IC₅₀ = 0.4 g L⁻¹) significantly lower than EDTA. The obtained results allow further exploration of the thermophilic G. gelatinosa for several biotechnological and industrial applications.     

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.     

Enzymatic Degradation of Gracilariopsis lemaneiformis Polysaccharide and the Antioxidant Activity of Its Degradation Products

Gracilariopsis lemaneiformis polysaccharides (GLP) were degraded using pectinase, glucoamylase, cellulase, xylanase, and β-dextranase into low-molecular-weight polysaccharides, namely, GPP, GGP, GCP, GXP, and GDP, respectively, and their antioxidant capacities were investigated. The degraded GLP showed higher antioxidant activities than natural GLP, and GDP exhibited the highest antioxidant activity. After the optimization of degradation conditions through single-factor and orthogonal optimization experiments, four polysaccharide fractions (GDP1, GDP2, GDP3, and GDP4) with high antioxidant abilities (hydroxyl radical scavenging activity, DPPH radical scavenging activity, reduction capacity, and total antioxidant capacity) were obtained. Their cytoprotective activities against H₂O₂-induced oxidative damage in human fetal lung fibroblast 1 (HFL1) cells were examined. Results suggested that GDP pretreatment can significantly improve cell viability, reduce reactive oxygen species and malonaldehyde levels, improve antioxidant enzyme activity and mitochondria membrane potential, and alleviate oxidative damage in HFL1 cells. Thus, the enzyme degradation of GLP with β-dextranase can significantly improve its antioxidant activity, and GDP might be a suitable source of natural antioxidants.     

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.     

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.     

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.