Structural Characterization and Rheological and Antioxidant Properties of Novel Polysaccharide from Calcareous Red Seaweed

A novel sulfated xylogalactan (JASX) was extracted and purified from the rhodophyceae Jania adhaerens. JASX was characterized by chromatography (GC/MS-EI and SEC/MALLS) and spectroscopy (ATR-FTIR and 1H/13C NMR) techniques. Results showed that JASX was constituted by repeating units of (→3)-β-d-Galp-(1,4)-3,6-α-l-AnGalp-(1→)n and (→3)-β-d-Galp-(1,4)-α-l-Galp-(1→)n substituted on O-2 and O-3 of the α-(1,4)-l-Galp units by methoxy and/or sulfate groups but also on O-6 of the β-(1,3)-d-Galp mainly by β-xylosyl side chains and less by methoxy and/or sulfate groups. The Mw, Mn, Đ, [η] and C* of JASX were respectively 600 and 160 kDa, 3.7, 102 mL.g−1 and 7.0 g.L−1. JASX exhibited pseudoplastic behavior influenced by temperature and monovalent salts and highly correlated to the power-law model and the Arrhenius relationship. JASX presented thixotropic characteristics, a gel-like viscoelastic behavior and a great viscoelasticity character. JASX showed important antioxidant activities, outlining its potential as a natural additive to produce functional foods.


Monosaccharide Analysis
The GC/MS analysis (after acidic hydrolysis and derivatization) demonstrated that JASX was principally constituted of galactose (Gal, 73.06%), xylose (Xyl, 16.66%), glucose (Glc, 8.46%) and small amounts of glucuronic acid (GlcA, 1.81%). This result suggested that JASX was a highly sulfated xylogalactan as described in the literature for sulfated galactans/xylogalactans of the Corallinales order such as J. rubens and C. officinalis [10,12]. JASX's main constitutive monosaccharides were Xylp and Galp, with a Galp/Xylp ratio of 4.39. (Table 2). : intrinsic viscosity was measured by SEC Visco-DRI. Analyses were run in triplicate, and the relative standard deviations are less than 5%. g C*: critical overlap concentration was determined using the Williamson model.

Molar Mass and Macromolecular Characteristics of JASX
From the SEC-MALLS-Viscosity analysis (Table 2), JASX was characterized by a mass-average molecular mass (Mw) and a number-average molecular mass (Mn) of 600 × 10 3 and 160 × 10 3 g/mol, respectively. High Mw values were similar with previous research on sulfated polysaccharides obtained from some rhodophyceae, having values greater than 1 × 10 5 g/mol [5,26,27]. These findings remained lower than those reported for xylogalactans isolated from Corallinales order, such as J. rubens, B. orbigniana and C. officinalis [10,12]. The polydispersity index (Đ = Mw/Mn) value of 3.7 could be due to the presence of a weak short chain in the JASX structure, which decreased the Mn value and therefore slightly increased the polydispersity [14]. According to Khan et al. [28], polymer intrinsic viscosity [η] (mL.g −1 ), indicating the capacity of polymers to enhance the viscosity of fluids, depends on several physicochemical characteristics such as its molecular conformation, type and degree of ramifications, molar mass, and solvent properties. JASX exhibited [η] and hydrodynamic radius Rh values of 102 mL.g −1 and 17.2 nm, suggesting a flexible random coil conformation with a value of Mark-Houwink-Sakurada ([η] = K.Mw α ) exponent α ranging between 0.5 and 0.8 [4].

Steady-Shear Flow Measurements of JASX
The flow curves (apparent viscosity (η) vs. shear rate ( )) of aqueous JASX solutions (0.25-2.0%, w/v) at 25 °C are shown in Figure 4A. JASX solutions in water presented non-Newtonian shear-thinning (pseudoplastic) behavior since η (Pa.s) decreased with increasing (s −1 ) from 0.001 to 1000 s −1 , suggesting that intermolecular entanglements of JASX in water tended to rise with increasing polymer concentration. This observation was consistent with previous findings obtained for other sulfated galactans and/or xylogalactans isolated from H. durvillei and G. birdiae and [1,22].
The Ostwald-de Waele (power-law) model was achieved to fit the rheological data of JASX (0.25-2.0%, w/v) at 25 °C. Results showed that JASX presented values of flow behavior index n lower than 1 (n < 1), confirming the pseudoplastic fluid property ( Table 3). The  (Table 2), JASX was characterized by a massaverage molecular mass (M w ) and a number-average molecular mass (M n ) of 600 × 10 3 and 160 × 10 3 g/mol, respectively. High M w values were similar with previous research on sulfated polysaccharides obtained from some rhodophyceae, having values greater than 1 × 10 5 g/mol [5,26,27]. These findings remained lower than those reported for xylogalactans isolated from Corallinales order, such as J. rubens, B. orbigniana and C. officinalis [10,12]. The polydispersity index (Ð = M w /M n ) value of 3.7 could be due to the presence of a weak short chain in the JASX structure, which decreased the M n value and therefore slightly increased the polydispersity [14]. According to Khan et al. [28], polymer intrinsic viscosity [η] (mL.g −1 ), indicating the capacity of polymers to enhance the viscosity of fluids, depends on several physicochemical characteristics such as its molecular conformation, type and degree of ramifications, molar mass, and solvent properties. JASX exhibited [η] and hydrodynamic radius R h values of 102 mL.g −1 and 17.2 nm, suggesting a flexible random coil conformation with a value of Mark-Houwink-Sakurada ([η] = K.M w α ) exponent α ranging between 0.5 and 0.8 [4]. γ (s −1 ) from 0.001 to 1000 s −1 , suggesting that intermolecular entanglements of JASX in water tended to rise with increasing polymer concentration. This observation was consistent with previous findings obtained for other sulfated galactans and/or xylogalactans isolated from H. durvillei and G. birdiae and [1,22]. These results could be linked to the increase in intermolecular distances by minimizing entanglements and interactions between JASX chains (thermal expansion phenomenon) [4]. The Arrhenius-Frenkel-Eyring equation (Section 3.6.2) was applied to verify the temperature dependency, which could be attributed to the conformational modification of the JASX backbone or to a less stable molecular equilibrium transition accompanied by the thermal expansion phenomenon. The Ostwald-de Waele (power-law) model was achieved to fit the rheological data of JASX (0.25-2.0%, w/v) at 25 • C. Results showed that JASX presented values of flow behavior index n lower than 1 (n < 1), confirming the pseudoplastic fluid property ( Table 3). The rise in polymer concentration decreased the n values and increased the coefficient of consistency (k). The coefficients of determination (R 2 ) values superior to 0.99 showed that JASX (0.25-2.0%, w/v) flow behavior in water was well described by the Ostwald-de Waele model [29].
The effects of adding monovalent salts (0.5 M NaCl) and increasing temperature (from 20 to 45 • C) on flow properties of JASX (1.0-2.0%, w/v) are presented in Figures 4B and 5A. From Figure 4B, JASX exhibited pseudoplastic properties, and the η decreased with adding 0.5 M NaCl solutions. According to Hentati et al. [29], the intermolecular electrostatic repulsions number and/or the junction zones complexity between JASX molecules decreased with adding monovalent cations, demonstrating that this polysaccharide adopted a more compact conformation due to its low uronic acid content and the lack of sulfate groups in its complex and highly branched backbone. The data shown in Table 3 confirmed that the Na + ions slightly decreased the pseudoplastic character of JASX solutions by decreasing k values and increasing n values. As illustrated in Figure 5B, the high values of Ea implied that JASX at 2.0% (w/v) was very sensitive to the temperature. Table 4 showed that the flow activation energy (Ea) values were dropped with the rising , and the η was decreased by rising temperature, suggesting then that the JASX in water flowed more readily under shearing [30]. These observations demonstrated that the rise in temperature caused (i) an increase in the energy dissipation motions of macromolecules, (ii) a breakdown of the weak energy bonds, and (iii) then a drop in the Ea values, showing that these results were a kind of non-Newtonian pseudoplastic fluid property for JASX [29].  Regarding temperature effect, the η decreased with the rise in temperature from 20 to 45 • C, and a non-Newtonian shear-thinning behavior (n < 1) was observed ( Figure 4A).
These results could be linked to the increase in intermolecular distances by minimizing entanglements and interactions between JASX chains (thermal expansion phenomenon) [4]. The Arrhenius-Frenkel-Eyring equation (Section 3.6.2) was applied to verify the temperature dependency, which could be attributed to the conformational modification of the JASX backbone or to a less stable molecular equilibrium transition accompanied by the thermal expansion phenomenon.
As illustrated in Figure 5B, the high values of E a implied that JASX at 2.0% (w/v) was very sensitive to the temperature. Table 4 showed that the flow activation energy (E a ) values were dropped with the rising . γ, and the η was decreased by rising temperature, suggesting then that the JASX in water flowed more readily under shearing [30]. These observations demonstrated that the rise in temperature caused (i) an increase in the energy dissipation motions of macromolecules, (ii) a breakdown of the weak energy bonds, and (iii) then a drop in the E a values, showing that these results were a kind of non-Newtonian pseudoplastic fluid property for JASX [29]. The dynamic oscillatory analyses were investigated on JASX solutions (1.0-2.0%, w/v) in Milli-Q water at 25 • C, and the frequency dependence of elastic modulus G (or storage modulus) and viscous modulus G (or loss modulus) are described in Figure 6A. Results showed that G and G values increased with rising polymer concentrations and angular frequencies (ω) (from 0.063 to 62.83 rad.s −1 ). JASX solutions exhibited typical gel-like behavior throughout the entire frequency range, with significant deformation when the G values were greater than the G values. [4,14,31]. The difference between G and G (gap) for JSAX (1.0-2.0%, w/v) increased with increasing ω from 0.063 to 62.83 rad.s −1 , indicating that JASX has high viscoelasticity with a higher elastic contribution to the gel structure [4,32].
The damping factor, also known as the dynamic mechanical loss tangent (tan δ = G /G ), is a characteristic parameter used to evaluate viscoelastic behavior. The tan δ values were inferior to 1 and confirmed the elastic behavior for JASX aqueous samples. The JASX energy was dissipated by an elastic flow, and the gel conformational equilibrium increased by favoring elastic (weak gel) behavior [32].
The Ostwald-de Waele model was used to describe the dependence of G and G moduli. The n values were used to determine the nature/type and strength of the gel; high n values n > 0 (n ~1) indicate viscous gel (physical gel), whereas low n values (zero) indicate covalent gel [29,32]. In addition, the values of n and n and k and k ones present an indicator of the nature of the polymer behavior, for n > n and k > k , the system behaves as a gel, while for n > n and k > k , the polysaccharide samples present a viscouslike fluid. Results showed that the n magnitudes were larger than n ones, meaning that G increased with higher rates than G (Supplementary Table S1). The k values were higher than those of k , confirming the weak gel behavior of JASX. Regarding the values of R 2 and R 2 , the Ostwald-de Waele model could be used to describe the viscoelastic behavior of JASX in Milli-Q water.

Critical Overlap Concentration (C*) of JASX
The C* named critical overlap concentration denotes the boundary between dilute (non-entangled system) and semi-dilute (entangled network) media. It was calculated from the log-log plot of the ηsp (specific viscosity) vs. JASX concentrations (0.5-2.0%, w/v)

Critical Overlap Concentration (C*) of JASX
The C* named critical overlap concentration denotes the boundary between dilute (non-entangled system) and semi-dilute (entangled network) media. It was calculated from the log-log plot of the η sp (specific viscosity) vs. JASX concentrations (0.5-2.0%, w/v) in Milli-Q water ( Figure 6B). As illustrated in Figure 6B, the η sp increased with rising JASX concentrations and the slope break between the two linear segments allowed for evaluation of the experimental value of C* [29]. The C* value of JASX at 25 • C was estimated at 7.0 g.L −1 (0.7%, w/v), and the linear segment slopes below and above the C* were, respectively, 4.93 and 2.68. The theoretical C* of JASX is an inverse function of the [η] (mL.g −1 ) and can be calculated using the following equation: C * = k s /[η], where k s is a specific constant for each type of polysaccharide [4]. For JASX in water, the k s value was calculated at around 0.714 (with C* = 7.0 g.L −1 ), which was consistent with the literature data when k s = 0.5-4 for coil polysaccharides in water [33].

Thixotropic Properties of JASX
Solutions of several hydrocolloids are well known for their time-dependent properties, meaning that the η varies with shearing time [3]. Contrary to rheopexy, thixotropy is an ordinary property for non-Newtonian solutions where the η decreases with shearing time action [34]. According to Razmkhah et al. [30], the hysteresis loop method and the estimation of the gap values between the up and down curves were used to characterize the viscosity-time relationship of JASX (0.5-2.0%, w/v) in Milli-Q water at 25 • C. From Figure 6C, the hysteresis loops of JASX solutions presented thixotropic characteristics since the up and down curves were different [30]. The thixotropic properties tended to rise with the rise in JASX concentrations (0.5 to 2.0%, w/v), consequently suggesting that the time dependence and the damage of polymer macromolecules were stronger [3,34]. Authors such as Ma et al. [3] and Hentati et al. [14,29] demonstrated that the decrease in η with shearing time was primarily caused by the pseudoplastic behavior, the alignments disturbances of the polymer chain alignment disturbances, and disentanglement-entanglement processes.

DPPH Radical-Scavenging Activity
The DPPH radical-scavenging activity of JASX concentrations (0-1.0 mg.mL −1 ) was evaluated. Ascorbic acid and butylated hydroxyanisole (BHA) were used as positive control. Results in Figure 7A showed increasing DPPH antioxidant activity with increasing polymer concentration.
Lajili et al. [6] demonstrated that sulfated galactan extracted from the Pheophyceae Laurencia obtusa exhibited significant anti-DPPH ability (IC 50 = 24 ± 5 µg.mL −1 ) close to that of ascorbic acid (17 ± 3 µg.mL −1 ) and quercetine (18 ± 2 µg.mL −1 ). According to Yang et al. [31], the anti-DPPH capacity of sulfated polysaccharide fractions (F1 and F2) obtained from the red algae Corallina officinalis were 15.2% at 1.2 mg.mL −1 . At 0.6 mg.mL −1 , JASX showed important anti-DPPH power (~25.0%) but remained inferior to the one measured for polysaccharide extracted from G. carticata (73%) at the same concentration [35]. However, the JASX antiradical activity (84.41% at 1.0 mg.mL −1 ) was higher than those described for Undaria pinnitafida (30-35%) polysaccharide fractions (S1 and S2) at 2.2 mg.mL −1 [36]. From the literature, DPPH radical-scavenging activity of red algal polysaccharides is highly related to their uronic sugar and 3,6-α-L-AnGalp content. Aside from carboxylic groups, sulfate and methyl groups, double bonds, and their conjugation to hydroxyl groups (-OH) and ketonic groups (C=O) (as in the case of ascorbic acid) also play a substantial role in their antioxidant abilities [37]. According to Abad et al. [37], the presence of two consecutive active (-OH) groups in the polymer structure may also play a role in the scavenging capacity of sulfated polysaccharides (particularly carrageenan) via the typical H-abstraction reaction with free radicals. The low molar masses of polysaccharides influenced the antioxidant power [20], which can be correlated to their reduced sugar content, the availability of functional groups and their hydrogen-donating capacity.
Mar. Drugs 2022, 20, x 13 of 21 the scavenging capacity of sulfated polysaccharides (particularly carrageenan) via the typical H-abstraction reaction with free radicals. The low molar masses of polysaccharides influenced the antioxidant power [20], which can be correlated to their reduced sugar content, the availability of functional groups and their hydrogen-donating capacity.

Ferric-Reducing Power
The ferric ion-reducing ability assay measures the electron donating power of an antioxidant. Because of the presence of reducing agents (polysaccharides), the Fe 3+ /ferricyanide complex (ferric iron form) is reduced to the ferrous iron form (Fe 2+ ). The A 700 nm of the resulting blue-green solution is proportional to the amount of Fe 2+ present. As a result, increased absorbance indicates greater ferric ion-reducing power. The reducing power of JASX, BHA and ascorbic acid increased with increasing concentrations (from 0 to 1.0 mg.mL −1 ) to obtain respectively reducing activities of 76.69, 98.64 and 100% at 1.0 mg.mL −1 ( Figure 7B). Lajili et al. [6] indicated that sulfated polysaccharide from the Pheophyceae L. obtusa showed IC 50 of 92 ± 2 µg.mL −1 , which was slightly lower than those of ascorbic acid (62 ± 8 µg.mL −1 ) and quercetine (83 ± 4 µg.mL −1 ). According to Qu et al. [38] and Hentati et al. [20], the FRAP capacity of polysaccharides was related to their sulfation rate, molecular mass, content of hydroxyl and carboxylic groups of uronic acid residues. Figure 7C depicts the ferrous ion-chelating activity of J. adhaerens sulfated xylogalactan. Through Fenton-type reactions, ferric iron (Fe 3+ ) can be reduced to active Fe 2+ and oxidized again, producing hydroxyl radicals [39]. The results showed that the JASX presented important concentration-dependent chelating capacity at 1.0 mg.mL −1 (78.52%), while EDTA ferrous chelating ability was 99.50% at the same concentration. The ferrous ionchelating power enregistrated for JASX was higher than that reported by Alves et al. [40] using sulfated polysaccharides from Hypnea musciformis, which had 8.0% ferrous ionchelating ability at 5.0 mg.mL −1 . The result of 65.13% (at 0.9 mg.mL −1 ) was higher than the result of 62.46% (at 2.0 mg.mL −1 ) found by Alencar et al. [41] for sulfated polysaccharides produced from Gracilaria caudata. This ion-chelating capacity may be explained by the nucleophilic nature of the free electrons of hydroxyl and sulfate groups present in the polysaccharide chemical structure [20].

Marine Seaweed Collection
Rhodophyceae Jania adhaerens was harvested in August 2018 from Tabarka (36 • 57 36.7 N 8 • 45 30.3 E, northern Tunisia). Macroalgae were washed with sea water (3 times), then with distilled water (3 times), and dried at 55 ± 1 • C for 11 days using a drying oven. The dried seaweeds were ground into a fine powder using a mechanical blender (Moulinex, France) and sieved with a mesh size of 0.3 mm. All chemicals were analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).

Extraction and Purification of Jania adhaerens Sulfated Xylogalactan (JASX)
For depigmentation, 100 g of algal powder was treated sequentially with acetone (99.5%) and ethanol (96%) for 24 h under stirring (400 rpm) at 25 • C. The depigmented biomass was then dried for 24 h at 50 ± 1 • C. The polysaccharide extraction was carried out according to the protocol proposed by Fenorodosoa et al. [1] with some modifications.
The depigmented powder (50 g.L −1 ) was dissolved in ultrapure water at 90 • C for 5 h under reflux and stirring (500 rpm). The mixture was successively treated using a fine mesh strainer, filtered through glass filters of porosity 2 (40-100 µm) and then 3 (16-40 µm). The permeate was centrifuged (12,000× g, 30 min, 20 • C), and the supernatant was collected before being precipitated with three volumes of −20 • C cold ethanol (96%) over 12 h at 4 • C with gentle stirring (250 rpm). The cold ethanolic precipitation and washing steps were repeated five times (using the same method) to remove salts (with conductimetry control). Samples were collected after centrifugation (8000× g) for 15 min at 4 • C and the final pellet was dissolved in 5-fold of milli-Q water and then freeze-dried. Finally, it was finely crushed and called Jania adhaerens sulfated xylogalactan (JASX).

Global Biochemical Composition of JASX
Carbohydrates neutral and uronic contents were evaluated according to Dubois et al. [42], Monsigny et al. [43] and Blumenkrantz and Asboe-Hansen [44]. The sulfation content was evaluated by the turbidimetric method BaCl 2 /gelatin [45]. The 3,6-α-D-AnGalp concentration was estimated using the method of Yaphe and Arsenault [46] (D-Fru as standard). The content of pyruvic acetal was quantified by the method of Sloneker et al. [47]. Water soluble proteins were estimated using Coomassie Brilliant Blue G-250 method [48]. Total phenolic compounds were quantified by the method of Folin-Ciocalteu using gallic acid as standard [49]. The conductivity was converted into NaCl concentration by assuming that 2 mS.cm −1 was equivalent to 1.0 g.L −1 of NaCl. Every measurement was performed three times (n = 3).

Determination of Monosaccharide Composition by GC/MS
First, 15 mg of JADX was dissolved in 1.5 mL of trifluoroacetic acid (TFA, 2M) at 120 • C for 90 min to release the monosaccharides. Polysaccharides were stirred time to time during the hydrolysis. The obtained preparation was then evaporated under nitrogen stream at 60 • C for 3 h. The monosaccharide derivatization was carried out for 2 h at 30 • C using BSTFA: TMCS (99:1) method as described by Pierre et al. [50,51]. After The different stages of temperature increase were carried out according to the method described by Hentati et al. [14]. The ionization step was carried out by EI (70 eV) with the trap temperature set at 150 • C and the target ion set at 40-800 m/z. The injector temperature was set to 250 • C, the split ratio to 50:1, and the helium pressure to 8.8 psi. 3.6. Rheological Investigations 3.6.1. Rheological Measurements JASX solutions at concentrations ranging from 0.25 to 2.0% (w/v) were made by soaking dried samples in Milli-Q water or 0.5 M NaCl solutions for 4 h (at room temperature) and gently stirring until full dissolution. After that, samples were kept at 4 • C for 48 h to achieve a fully water-swelling polymer (biopolymer hydration) and to remove bubbles.
Rheological measurements on JASX solutions were carried out with a rheometer AR-2000 (TA Instrument, Great Britain, Ltd. New Castle, DE, USA ) fitted with a 40 mm cone-plate geometry (54 microns gap) and a Peltier heating system for precise control. To prevent solvent evaporation during rheological analysis, samples were covered with a thin layer of hexadecane (oil film) after structure recovery and temperature equilibration (15 min). The TA Instrument Rheology Advantage software was used to collect and analyze the data (V5.7.0).

Steady-Shear Flow Measurements
The cone-plate geometry was used to investigate the steady-shear flow properties of JASX solutions (0.25-2.0%, w/v) at 25 • C over the shear rate ( . γ) range from 0.001 to 1000 s −1 . The shear stress (τ) and dynamic viscosity (apparent viscosity, η) were measured as a function of . γ in different conditions (in Milli-Q water and in 0.5 M NaCl) by applying the following model (Equation (1)): To fit the experimental rheological data of JASX, the Ostwald-de Waele model (powerlaw model) (Equation (2)) was used. where τ: the shear stress (Pa); k: the consistency index (Pa.s n ); . γ : the shear rate (s −1 ) and n: the flow behavior index (dimensionless), which takes the values >1, 1 and <1 for plastic, Newtonian and pseudoplastic fluid behaviors, respectively.
The temperature-dependent viscosity of JASX (1.0-2.0% (w/v)) was measured at shear rates ranging from 1.0 to 1000 s −1 in the temperature range of 20 to 45 • C using the Arrhenius-Frenkel-Eyring model (Equation (3)): where η: the apparent viscosity (Pa.s); A: the proportionality constant (Pa.s); T: the absolute thermo-dynamical temperature (K); R: the universal gas constant (kJ.mol −1 ·K −1 ) and E a : the flow activation energy (kJ.mol −1 ). The Williamson model (Equation (4)) was used to calculate the critical overlap concentration (C*) of JASX aqueous solutions (0.25-2.0%, w/v) at 25 • C, which represents the limit between dilute (non-entangled system) and semi-dilute (entangled network) regimes. C* was calculated using a log-log plot of specific viscosity (η sp ) versus polysaccharide concentration.

Dynamic Viscoelastic Properties
The oscillatory (dynamic) frequency sweeps for JASX (1.0-2.0%, w/v) were evaluated in a constant strain of 20% (or in linear viscoelastic range) at 25 • C using the cone-plate geometry over angular frequency (ω) ranging from 0.063 to 62.83 rad.s −1 (0.01 to 10 Hz). The storage or elastic modulus G , the loss or viscous modulus G and the loss tangent or damping factor (tan δ = G /G ) as a function of ω were continuously determined during the rheological analysis. The power-law model (Equations (5) and (6)) was used to describe the frequency dependence of the moduli G and G : where k and k : the specific constants; n and n : the frequency exponents and ω: the angular frequency (rad.s −1 or Hz).

DPPH Radical-Scavenging Activity
The method of Kirby and Schmidt [52] was used to evaluate the DPPH (2,2diphenyl-1-picrylhydrazyl) radical-scavenging ability of JASX. Briefly, 500 µL of samples (0-1.0 mg.mL −1 ) was mixed with ethanol (99%, 375 µL) and a DPPH diluted solution in ethanol (125 µL, 0.02% (w/v)). The mixture was homogenized, and then, the A 517 of samples were measured after incubation at 25 • C during 30 min in the dark. BHA and ascorbic acid were used as positive standards, and then, the DPPH scavenging activity was calculated using Equation (7).
where A control : the absorbance of the control reaction (absence of polysaccharide); A blank : the absorbance of JASX samples (except the DPPH solution); A sample : the absorbance of JASX in DPPH solution.

Ferric-Reducing Power
The Ferric-reducing activity of JASX was determined according to the method of Yildirim et al. [53]. First, 0.5 mL of polysaccharide solutions (0-1.0 mg.mL −1 ) were mixed with 1.25 mL of phosphate buffer (0.2 M, pH = 6.6) and 1.25 mL of potassium ferricyanide (1% (m/v)). Then, the mixture was incubated for 30 min at 50 • C, treated with 1.25 mL of trichloroacetic acid (10% w/v) and centrifuged 3000 g during 10 min. Next, 1.25 mL of the supernatant was thoroughly mixed with 1.25 mL of ultrapure water and 0.25 mL of a 0.1% (w/v) ferric chloride solution. After incubation (10 min, 25 • C), the A 700 was measured. Butylated hydroxyanisole (BHA) and ascorbic acid were used as standards, and then, the FRAP capacities were calculated using Equation (8). All the experiments were performed in triplicate.

Ferric reducing ability
where A 0 : the absorbance of a 66 µM Prussian blue solution measured in the same reaction medium free of reducing component (A 0 = 0.8) and A sample : the absorbance of JASX samples.
Ferrous ion chelating activity (%) =   A control − A sample − A blank A control   × 100 (9) where the absorbance of the control (without sample), the absorbance of the blank (without Ferrozine) and finally the absorbance of the extract respectively represented A control , A blank and A sample . The positive control test was performed by EDTA (ethylenediaminetetraacetic acid).

Conclusions
The main goals of this paper were to determine the structural features and the rheological properties and to evaluate the antioxidant activities of a sulfated xylogalactan produced from the red seaweed J. adhaerens. The structural analyses revealed that JASX (M w = 6.0 × 10 5 Da) was mainly composed of an agar-like sulfated xylogalactan with a repeating backbone of (→3)-β-D-Galp-(1,4)-3,6-α-L-AnGalp-(1→) n and (→3)-β-D-Galp-(1,4)-α-L-Galp-(1→) n substituted on O-2 and O-3 of the α-(1,4)-L-Galp units by methoxy and/or sulfate groups but also on O-6 of the β-(1,3)-D-Galp mainly by β-xylosyl side chains and less by methoxy and/or sulfate groups. The rheological investigations showed that JASX solutions exhibited a shear-thinning behavior influenced by temperature and adding salts. JASX presented thixotropy properties and a critical overlap concentration C* close to 7.0 g.L −1 . The dynamical viscoelastic properties showed a gel-like viscoelastic behavior (G > G ) with a great viscoelastic character. The physicochemical and antioxidant properties of JASX are at least as good as other polysaccharides from red marine seaweeds currently used for their functional properties as hydrocolloids.