Elicitor Activity of Low-Molecular-Weight Alginates Obtained by Oxidative Degradation of Alginates Extracted from Sargassum muticum and Cystoseira myriophylloides.

Alginates extracted from two Moroccan brown seaweeds and their derivatives were investigated for their ability to induce phenolic metabolism in the roots and leaves of tomato seedlings. Sodium alginates (ALSM and ALCM) were extracted from the brown seaweeds Sargassum muticum and Cystoseira myriophylloides, respectively. Low-molecular-weight alginates (OASM and OACM) were obtained after radical hydrolysis of the native alginates. Elicitation was carried out by foliar spraying 20 mL of aqueous solutions (1 g/L) on 45-day-old tomato seedlings. Elicitor capacities were evaluated by monitoring phenylalanine ammonia-lyase (PAL) activity, polyphenols, and lignin production in the roots and leaves after 0, 12, 24, 48, and 72 h of treatment. The molecular weights (Mw) of the different fractions were 202 kDa for ALSM, 76 kDa for ALCM, 19 kDa for OACM, and 3 kDa for OASM. FTIR analysis revealed that the structures of OACM and OASM did not change after oxidative degradation of the native alginates. These molecules showed their differential capacity to induce natural defenses in tomato seedlings by increasing PAL activity and through the accumulation of polyphenol and lignin content in the leaves and roots. The oxidative alginates (OASM and OACM) exhibited an effective induction of the key enzyme of phenolic metabolism (PAL) compared to the alginate polymers (ALSM and ALCM). These results suggest that low-molecular-weight alginates may be good candidates for stimulating the natural defenses of plants.

Exploring the Diversity of Red Microalgae for Exopolysaccharide Production.

Microalgae constitute a remarkable biological diversity but a limited number of them have been the object of study for their ability to produce exoplysaccharides (EPS). Among them, the red marine microalgae Porphyridium or Rhodella produce sulphated EPS, exhibiting some biological activities with potential interest in the pharmaceutical and cosmetic industries. EPS from Porphyridium and Rhodella being relatively similar in their composition, it has long been considered that all the red microalgae produced similar EPS and no attention was paid to other red microalgae. The objective of our work was then to explore the diversity of red microalgae for the production of EPS, focusing in this first step on the screening of the strains for their ability to produce EPS and preliminary structural characterization. The study was conducted with 11 microalgae strains belonging to the proteorhodophytina subphylum. All microalgae were able to produce EPS, released in the culture medium (strains belonging to Porphyridiophyceae and Rhodellophyceae classes) or remaining bound to the cells (strains from Stylonematophyceae class). The analysis of monosaccharides composition was found significantly different, with for instance high levels of glucuronic acids in the EPS from C. japonica and N. cyanea, but also strong differences in the sulphation degrees of polymers (between 1.2 and 28.7% eq. SO4).

Potential of Exopolysaccharide from Porphyridium marinum to Contend with Bacterial Proliferation, Biofilm Formation, and Breast Cancer.

Exopolysaccharide (EPS) from marine microalgae are promising sources of a new generation of drugs. However, lot of them remain to be discovered and tested. In this study, EPS produced by Porphyridium marinum and its oligomers prepared by High Pressure Homogenizer have been tested for different biological activities, i.e., antibacterial, anti-fungal and antibiofilm activities on Candida albicans, as well as for their effects on the viability of murine breast cancer cells. Results have shown that all EPS samples present some biological activity. For antibacterial and antibiofilm activities, the native EPS exhibited a better efficiency with Minimum Inhibitory Concentration (MIC) from 62.5 µg/mL to 1000 µg/mL depending on the bacterial strain. For Candida albicans, the biofilm formation was reduced by about 90% by using only a 31.3 µg/mL concentration. Concerning breast cancer cells, lower molar masses fractions appeared to be more efficient, with a reduction of viability of up to 55%. Finally, analyses of polymers composition and viscosity measurements were conducted on all samples, in order to propose hypotheses involving the activities caused by the intrinsic properties of polymers.

Induction of Natural Defenses in Tomato Seedlings by Using Alginate and Oligoalginates Derivatives Extracted from Moroccan Brown Algae.

Polysaccharides extracted from marine algae have attracted much attention due to their biotechnological applications, including therapeutics, cosmetics, and mainly in agriculture and horticulture as biostimulants, biofertilizers, and stimulators of the natural defenses of plants. This study aimed to evaluate the ability of alginate isolated from Bifurcaria bifurcata from the Moroccan coast and oligoalginates derivatives to stimulate the natural defenses of tomato seedlings. Elicitation was carried out by the internodal injection of bioelicitor solutions. The elicitor capacities were evaluated by monitoring the activity of phenylalanine ammonia-lyase (PAL) as well as polyphenols content in the leaves located above the elicitation site for 5 days. Alginate and oligoalginates treatments triggered plant defense responses, which showed their capacity to significantly induce the PAL activity and phenolic compounds accumulation in the leaves of tomato seedlings. Elicitation by alginates and oligoalginates showed an intensive induction of PAL activity, increasing from 12 h of treatment and remaining at high levels throughout the period of treatment. The amount of polyphenols in the leaves was increased rapidly and strongly from 12 h of elicitation by both saccharide solutions, representing peaks value after 24 h of application. Oligoalginates exhibited an effective elicitor capacity in polyphenols accumulation compared to alginate polymers. The alginate and oligosaccharides derivatives revealed a similar elicitor capacity in PAL activity whereas the accumulation of phenolic compounds showed a differential effect. Polysaccharides extracted from the brown seaweed Bifurcaria bifurcate and oligosaccharides derivatives induced significantly the phenylpropanoid metabolism in tomato seedlings. These results contribute to the valorization of marine biomass as a potential bioresource for plant protection against phytopathogens in the context of eco-sustainable green technology.

Enhanced growth and metabolite production from a novel strain of Porphyridium sp.

Microalgae are capable of generating numerous metabolites that possess notable biological activities and hold substantial promise for various industrial applications. Nevertheless, the taxonomic diversity of these photosynthetic microorganisms has not received thorough investigation. Using the 18S rRNA encoding gene, a recently discovered strain originating from the Tunisian coast (the governorate of Mahdia) was identified as a member of the Porphyridium genus. The growth response as well as the metabolite accumulation of Porphyridium sp. to different culture media (Pm, F/2, and Hemerick) was investigated over a period of 52 days. The highest biomass production was recorded with Pm medium (2 × 107 cell/mL). The apparent growth rates (µ) and the doubling time (Dt) were about 0.081 day-1 and 12.34 days, respectively. The highest chlorophyll a (0.678 ± 0.005 pg/cell), total carotenoids (0.18 ± 0.003 pg/cell), phycoerythrin (3.88 ± 0.003 pg/cell), and proteins (14.58 ± 0.35 pg/cell) contents were observed with F/2 medium. Cultivating Porphyridium sp. in both F/2 and Hemerick media yielded similar levels of starch accumulation. The Hemerick medium has proven to be the most suitable for the production of lipids (2.23% DW) and exopolysaccharides (5.41 ± 0.56 pg/cell).

Description of the Wild Strain Rhizobium rosettiformans DSM26376, Reclassified under Peteryoungia rosettiformans comb.nov., for Producing Glucuronan.

Glucuronan is a polysaccharide composed of ß-(1,4)-linked d-glucuronic acids having intrinsic properties and biological activities recoverable in many fields of application. Currently, the description of Sinorhyzobium meliloti M5N1CS mutant bacterial strain as the sole source of glucuronan makes it relevant to the exploration of new microorganisms producing glucuronan. In this study, the Peteryoungia rosettifformans strain (Rhizobia), was identified as a wild producer of an exopolysaccharide (RhrBR46) related to glucuronan. Structural and biochemical features, using colorimetric assays, Fourier infrared spectroscopy, nuclear magnetic resonance, high pressure size exclusion chromatography coupled to multi-angle light laser scattering, and enzymatic assays allowed the characterization of a polyglucuronic acid, having a molecular mass (Mw¯) of 1.85 × 105 Da, and being partially O-acetylated at C-2 and/or C-3 positions. The concentration of Mg2+ ions in the cultivation medium has been shown to impact the structure of RhrBR46, by reducing drastically its Mw¯ (73%) and increasing its DA (10%). Comparative structural analyses between RhrBR46 and the glucuronan from Sinorhyzobium meliloti M5N1CS strain revealed differences in terms of molecular weight, degree of acetylation (DA), and the distribution of acetylation pattern. These structural divergences of RhrBR46 might contribute to singular properties or biological activities of RhrBR46, offering new perspectives of application.

Ricin B lectin-like proteins of the microsporidian Encephalitozoon cuniculi and Anncaliia algerae are involved in host-cell invasion.

Microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. The infection process requires a step of prior adherence of Microsporidia to the surface of host cells. A few studies demonstrated the involvement of proteins containing a ricin-B lectin (RBL) domain in parasite infection. In this study Anncalia algerae and Encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing RBL-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. Three proteins named AaRBLL-1 and AaRBLL-2 from A. algerae and EcRBLL-1 from E. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural RBL domain despite a significant amino acid sequence divergence. The production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. Adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both E. cuniculi and A. algerae, strongly suggesting that these proteins are involved in the infection process.

Influence of the sulfate content of the exopolysaccharides from Porphyridium sordidum on their elicitor activities on date palm vitroplants.

Given the increasing interest that is being paid to polysaccharides derived from algae as plant natural defense stimulators, the degree of sulfation of exopolysaccharides produced by P. sordidum for inducing defense responses in date palm vitroplants was investigated. Firstly, the culture parameters of P. sordidum were optimized to maximize the amount of sulfate in EPS using a Box-Behnken experimental design and the elicitor effects of two EPS which differ in the sulfation degrees were compared. Results demonstrated that the concentrations of NaCl, NaNO3 and MgSO4 set at 28, 0.54 and 16.31 g/L, respectively yielded the best sulfate contents. To elucidate defense-inducing activities in date palm vitroplants, EPS with the highest sulfate content (EPS1) were prepared for comparison with those obtained under standard conditions (EPS0). A fucoidan extracted from Cystoseira compressa was used as positive control and MgSO4 as negative control. Both EPS and the fucoidan displayed H2O2 accumulation and expression of PR1, SOD, PAL and WRKY genes. Interestingly, EPS1 was significantly more bioactive than EPS0 and the fucoidan suggesting that the elicitor activity is positively correlated with the sulfate groups content of this polysaccharide.

Optimization of Exopolysaccharides Production by Porphyridium sordidum and Their Potential to Induce Defense Responses in Arabidopsis thaliana against Fusarium oxysporum.

Polysaccharides from marine algae are one novel source of plant defense elicitors for alternative and eco-friendly plant protection against phytopathogens. The effect of exopolysaccharides (EPS) produced by Porphyridium sordidum on elicitation of Arabidopsis thaliana defense responses against Fusarium oxysporum was evaluated. Firstly, in order to enhance EPS production, a Box-Behnken experimental design was carried out to optimize NaCl, NaNO3 and MgSO4 concentrations in the culture medium of microalgae. A maximum EPS production (2.45 g/L) higher than that of the control (0.7 g/L) was observed for 41.62 g/L NaCl, 0.63 g/L NaNO3 and 7.2 g/L MgSO4 concentrations. Structurally, the EPS contained mainly galactose, xylose and glucose. Secondly, the elicitor effect of EPS was evaluated by investigating the plant defense-related signaling pathways that include activation of Salicylic or Jasmonic Acid-dependent pathway genes. A solution of 2 mg/mL of EPS has led to the control of fungal growth by the plant. Results showed that EPS foliar application induced phenylalaline ammonia lyase and H2O2 accumulation. Expression profile analysis of the defense-related genes using qRT-PCR revealed the up-regulation of Superoxide dismutases (SOD), Peroxidase (POD), Pathogenesis-related protein 1 (PR-1) and Cytochrome P450 monooxyge-nase (CYP), while Catalase (CAT) and Plant defensin 1.2 (PDF1.2) were not induced. Results suggest that EPS may induce the elicitation of A. thaliana's defense response against F. oxysporum, activating the Salicylic Acid pathway.

Biochemical Characterization of a Bifunctional Enzyme Constructed by the Fusion of a Glucuronan Lyase and a Chitinase from Trichoderma sp.

Bifunctional enzymes created by the fusion of a glucuronan lyase (TrGL) and a chitinase (ThCHIT42) from Trichoderma sp. have been constructed with the aim to validate a proof of concept regarding the potential of the chimera lyase/hydrolase by analyzing the functionality and the efficiency of the chimeric constructions compared to parental enzymes. All the chimeric enzymes, including or nor linker (GGGGS), were shown functional with activities equivalent or higher to native enzymes. The velocity of glucuronan lyase was considerably increased for chimeras, and may involved structural modifications at the active site. The fusion has induced a slightly decrease of the thermostability of glucuronan lyase, without modifying its catalytic activity regarding pH variations ranging from 5 to 8. The biochemical properties of chitinase seemed to be more disparate between the different fusion constructions suggesting an impact of the linkers or structural interactions with the linked glucuronan lyase. The chimeric enzymes displayed a decreased stability to temperature and pH variations, compared to parental one. Overall, TrGL-ThCHIT42 offered the better compromise in terms of biochemical stability and enhanced activity, and could be a promising candidate for further experiments in the field of fungi Cell Wall-Degrading Enzymes (CWDEs).

Structural features and rheological behavior of a water-soluble polysaccharide extracted from the seeds of Plantago ciliata Desf.

A water-soluble polysaccharide (PSPC) was extracted from the seeds of Plantago ciliata Desf., a spontaneous Algerian Saharan plant by a hot aqueous extraction then purified by successive ethanolic precipitations. The final extraction yield for PSPC was close to 18.6% (w/v). PSPC was then investigated regarding its global composition, structural features and rheological properties. PSPC is a neutral arabinoxylan, composed of a ß-(1,3)/ß-(1,4)-d-xylan backbone with side chains of Xylp, and Araf residues attached in O-2 and O-3 positions. The macromolecular characteristics of PSPC in water was determined by SEC/MALLS, with a high molecular weight (Mw) of 700 kDa, a low polydispersity index (PDI) of 1.47 and an intrinsic viscosity [η] close to 157 mL/g. PSPC showed a pseudoplastic behavior in semi-dilute media and the critical overlay concentration (C*) was estimated around 0.32-0.37% (w/v). This current research has supplied original structural information on a new arabinoxylan which could be particularly useful as a novel source of soluble fiber belonging to psyllium.

Characterization of a new exopolysaccharide produced by Halorubrum sp. TBZ112 and evaluation of its anti-proliferative effect on gastric cancer cells.

In the present study, we aimed to extract, purify, analyze monosaccharide composition of  exopolysaccharide (EPS) produced by Halorubrum sp. TBZ112 (KCTC 4203 and IBRC-M 10773) and also to evaluate its possible antiproliferative activity against human gastric cancer (MKN-45) cell line and its biocompatibility effect on normal cells using human dermal fibroblast (HDF) cell line. Average molecular weight and monosaccharide composition were determined by high-pressure size exclusion chromatography (HPSEC) with multi-angle laser light scattering (MALLS) and high-pressure anion exchange chromatography (HPAEC), respectively. Fourier transform infrared (FTIR) spectroscopy was used for the partial characterization of the EPS. The EPS effect on the cell proliferation and viability of MKN-45 and HDF cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion, respectively. Strain TBZ112 excreted 480 mg.l-1 of the EPS under optimal growth conditions. The EPS had a molecular weight of 5.052 kDa and was a heteropolysaccharide containing ten moieties mainly composed of mannose (19.95%), glucosamine (15.55%), galacturonic acid (15.43%), arabinose (12.24%), and glucuronic acid (12.05%). No significant difference of the EPS treatments on the proliferation activity of MKN-45 and HDF cells were observed (P > 0.05). For the first time, the EPS from Halorubrum sp. TBZ112, an extremely halophilic archaeon related to Halorubrum genus, was isolated and chemically characterized. The EPS from Halorubrum sp. TBZ112 possesses a relatively low molecular weight and might be applied as a biocompatible compound. More investigations are needed to determine other biological activities of the EPS along with further details of its chemical structure.

Structural characterization and antioxidant activity of water-soluble polysaccharides from the Tunisian brown seaweed Cystoseira compressa.

A fucoidan (CCF) and a sodium alginate (CCSA) were extracted and purified from the Tunisian brown seaweed Cystoseira compressa. CCF was a highly sulfated heterogalactofucan composed of α-(1→3), α-(1→4)-linked l-Fucp as main backbone which could be highly branched (31.84%) at O-3 and O-4 positions of α-(1→4)-l-Fucp and α-(1→3)-l-Fucp by terminal monosaccharides and side chains such as terminal α-l-Fucp, terminal ß-d-Galp, ß-d-Galp-(1→3)-α-l-Fucp and ß-d-Galp-(1→4)-α-l-Fucp. The ratio of α-(1→3)/α-(1→4) linkages was estimated at 3.86:1. CCSA was characterized by HPAEC-PAD, GC/MS-EI, ATR-FTIR, and 1H-NMR. The M/G ratio was M/G = 0.77, indicating that CCSA respectively contained 44% and 56% of mannuronic and guluronic acids. The values of FGG, FMM, FGM (or FMG) blocks as well as the parameter η were estimated. The two polysaccharides exhibited effective antioxidant activities by ferrous ion chelation, ferric ion reduction and DPPH radical-scavenging, outlining their potentials as natural additives.

Rheological and functional properties of asafoetida gum.

The asafoetida gum was extracted and purified from oleo-gum-resin of Ferula assa foetida root and characterized by high pressure anions exchange chromatography after acidic hydrolysis. It was composed of Gal:Ara:Rha:GlcA with the ratio 11.5:5.0:2.1:1.0. This monosaccharide composition was found similar to that of a commercial Arabic gum which exhibited a Gal:Ara:Rha:GlcA ratio of 11.7:5.4:3.2:1.0. As the Arabic gum is currently used for its emulsifying properties, the two gums were evaluated for their functional and rheological behaviors. Surface and interfacial tensions values were lower for asafoetida gum compared to Arabic gum. Critical micelle concentration was achieved at concentrations of 0.5% w/w and 1% w/w for asafoetida and Arabic gums, respectively. Values of emulsion capacity, emulsion stability and foaming properties were considerably higher for asafoetida gum in contrast to emulsion activity index that was lower than that of Arabic gum. As those of Arabic gum, solutions of asafoetida gum (2-30% w/w) exhibited Newtonian flow behavior at shear rates between 1 and 500 s-1. Apparent viscosities of Arabic and asafoetida gums were close and logically decreased by increasing temperature (10-80 °C). Higher viscosities were achieved at higher pH and CaCl2 concentrations.

Extraction and characterization of an alginate from the Iranian brown seaweed Nizimuddinia zanardini.

Sodium alginate from Nizimuddinia zanardini (an Iranian brown algae) was extracted with acid and alkaline solutions, partially and totally hydrolyzed and analyzed for its biochemical composition. 1H NMR spectroscopy, SEC-MALLS, HPAEC and FT-IR were performed to determine its structure and its physico-chemical properties. This alginate has a M/G ratio of 1.1, a molecular weight of 103 kDa, a polydispersity index of 1.22, and an intrinsic viscosity of 342 mL/g. Its antioxidant activity was tested by DPPH radical scavenging showing its potential for food preservation. Rheological properties of solutions of this alginate with concentrations between 1 and 5% (w/v) in water and 0.5 M NaCl were investigated indicating a Newtonian fluid type behaviour in water and a shear thinning fluid type behaviour in NaCl solutions.

Structural characterization and rheological behavior of a heteroxylan extracted from Plantago notata Lagasca (Plantaginaceae) seeds.

Plantago notata (Plantaginaceae) is a spontaneous plant from Septentrional Algerian Sahara currently used by traditional healers to treat stomach disorders, inflammations or wound healing. A water-soluble polysaccharide, called PSPN (PolySaccharide fraction from Plantago Notata), was extracted and purified from the seeds of this semi-arid plant. The structural features of this mucilage were evaluated by colorimetric assays, Fourier transformed infrared spectroscopy (FT-IR), gas chromatography coupled to mass spectrometry (GC/MS) and 1H/13C Nuclear Magnetic Resonance (NMR) spectroscopy. PSPN is a heteroxylan with a backbone composed of ß-(1,3)-d-Xylp and ß-(1,4)-d-Xylp highly branched, through (O)-2 and (O)-3 positions of ß-(1,4)-d-Xylp by various side chains and terminal monosaccharides such as α-l-Araf-(1,3)-ß-d-Xylp, ß-d-Xylp-(1,2)-ß-d-Xylp, terminal Xylp or terminal Araf. The physico-chemical and rheological analysis of this polysaccharide in dilute and semi diluted regimes showed that PSPN exhibites a molecular weight of 2.3×106g/mol and a pseudoplastic behavior.

Improvement of exopolysaccharide production by Porphyridium marinum.

With the aim to optimize the production of exopolysaccharide (EPS) by Porphyridium marinum, cultures in photobioreactors were conducted on a modified Provasoli medium (P) and compared to a new medium (Pm) with an elemental composition of N0.0205S0.0597P0.005. Cultivation on this medium allowed the increase of EPS concentration up to 2.5gL(-1), without modification of the EPS productivity (0.096gL(-1)) and EPS structure. In a second time, photosynthetic activity of the strain was monitored as a function of irradiance and temperature, allowing improvement of kinetic parameters of growth and EPS production. A semi-continuous culture, carried out with the Pm medium, an optimal irradiance and temperature of respectively 360µmolphotonsm(-2)s(-1) and 28°C led to an EPS process productivity of 0.031gh(-1) instead of 0.020gh(-1) in batch culture.

Dextranase immobilization on epoxy CIM(®) disk for the production of isomaltooligosaccharides from dextran.

Endodextranase D8144 from Penicillium sp. (EC 3.2.1.2.) was immobilized on an epoxy-activated monolithic Convective Interaction Media (CIM(®)) disk in order to produce isomaltooligosaccharides (IMOS) from Dextran T40 in a continuous IMmobilized Enzymes Reactor (IMER). Enzymatic parameters and structure of IMOS were studied for free and immobilized enzymes. The immobilization efficiency of endodextranase D8144 was about 15.9% (w/w) and the real specific activity was close to 6.5 U mg enz(-1). The Km values (4.8 ± 0.2 g L(-1)) for free and immobilized enzymes were the same, showing the absence of diffusional limitation. Moreover, specific patterns of DPs (Degrees of Polymerization) distributions were observed during the enzymatic hydrolysis by HPAEC-PAD (High Pressure Anion Exchange Chromatography-Pulsed Amperometric Detection). Thus, sought-after sizes of IMOS (DPs 8-10) were generated all over the hydrolysis. Finally, the results showed the high stability of this IMER since a relative enzymatic activity about 78% was measured after 5400 volumes column.

Enzymatic degradation and bioactivity evaluation of C-6 oxidized chitosan.

C-6 oxidized chitosan was produced from chitosan by performing selective oxidation with NaOCl and NaBr using 2,2,6,6-tetramethylpiperidine-1-oxy radical (TEMPO) as catalyst. Endocellulase, Celluclast 1.5 L, Glucanex(®), Macerozyme R-10, hyaluronidase, hyaluronate lyase, red scorpionfish chitinase, glucuronan lyase and a protein mix from Trichoderma reesei were used to degrade the C-6 oxidized chitosan. Glucanex(®), the crude extract from T. reesei IHEM 4122 and Macerozyme R-10 validated the enzymatic degradation through final hydrolysis yields of the derivative respectively close to 36.4, 20.3 and 12.9% (w/w). The best initial reaction velocity (2.41 U/mL) was observed for Glucanex(®). The antileishmanial activity of the derivative was evaluated against Leishmania infantum LIPA 137. The antibacterial activities against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were also tested. Results showed an antileishmanial activity (IC50: 125 µg/mL) of the obtained derivatives against L. infantum LIPA 137.