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

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

Roya Abka Khajouei, Javad Keramat, Nasser Hamdami, AlinaVioleta Ursu, Cedric Delattre, Céline Laroche, Christine Gardarin, Didier Lecerf, Jacques Desbrières, Gholamreza Djelveh, Philippe Michaud PII: DOI: Reference:

S0141-8130(18)31049-3 doi:10.1016/j.ijbiomac.2018.06.154 BIOMAC 9994

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

5 March 2018 22 May 2018 27 June 2018

Please cite this article as: Roya Abka Khajouei, Javad Keramat, Nasser Hamdami, AlinaVioleta Ursu, Cedric Delattre, Céline Laroche, Christine Gardarin, Didier Lecerf, Jacques Desbrières, Gholamreza Djelveh, Philippe Michaud , Extraction and characterization of an alginate from the Iranian brown seaweed Nizimuddinia zanardini. Biomac (2018), doi:10.1016/j.ijbiomac.2018.06.154

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

Roya Abka Khajoueia,b, Javad Keramata,∗, Nasser Hamdamia, Alina-Violeta Ursub, Cedric

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Delattreb, Céline Larocheb, Christine Gardarinb, Didier Lecerfc, Jacques Desbrièresd, Gholamreza

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Djelvehb, Philippe Michaudb

Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology,

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Isfahan 84156-83111, Iran

Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut Pascal, F-63000 Clermont-

Laboratoire Polymères Biopolymères Surface, CNRS FRE 3101, Université de Rouen, Bd Maurice de

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c

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Ferrand, France.

Broglie, 76821 Mont Saint Aignan Cedex, France.

Université de Pau et des Pays de l'Adour, IPREM, Helioparc Pau Pyrénées, 2 avenue P. Angot, 64053

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Pau cedex 9, France

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∗ Corresponding author:

Associate Professor Dr. J. Keramat E-mail addresses: [email protected] Tel: +98 313 3913380 Fax: +98-31-33912254

ACCEPTED MANUSCRIPT Abstract 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

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determine its structure and its physico-chemical properties. This alginate has a M/G ratio of 1.1,

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a molecular weight of 103 kDa, a polydispersity index of 1.22, and an intrinsic viscosity of 342

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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

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between 1 and 5 % (w/v) in water and 0.5 M NaCl were investigated indicating a Newtonian

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fluid type behaviour in water and a shear thinning fluid type behaviour in NaCl solutions.

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Keywords: Nizimuddinia zanardini, alginate, polysaccharide, antioxidant activity

ACCEPTED MANUSCRIPT 1. Introduction Macroalgae are source of many polysaccharides. Based on their pigments content they are divided into brown, green and red algae having specific polysaccharide contents. Among them, brown algae (Phaeophyceae) are source of alginates and/or fucans or fucoidans [1]. Brown algae

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(Phaeophyceae) possess a pigmentary equipment constituted by chlorophylls `a` and `c` masked

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by carotenes and xanthophylls. The phycoxanthin (a xanthophyll) is responsible for their brown

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color. The matricial polysaccharides extracted from Phaeophyceae are alginates, fucoidans (including hexouronoxylofucans), lichenan, and laminaran [2]. Fucoidan and laminaran are

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mostly interesting for their biological activities whereas alginates have a lot of usages in food

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and medical industries as thickening, emulsifier, stabilizing agents and as pharmaceutical additive [3,4]. Alginates are anionic polysaccharide that existences in mixer of salt forms (Na+,

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Mg2+ and Ca2+) in cell walls of brown algae makes the tissues flexible and strong [1,5]. They

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consist of (1,4) linked β-D-mannuronic acid (M) with 4C1 ring conformation and -L-guluronic

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acid (G) with 1C4 ring conformation, the two uronic acids being in pyranosic conformation. They offer three varying sequences identified as the blocks MG consisting of nearly equal proportion

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of both monomers with a high number of MG (or GM) dimmers, the blocks GG and the blocks MM [6,7]. M/G ratio, length and distribution of sequences depend on the algae species but also

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on their growth conditions and geographic origins. It also determines the chemical and physical properties of alginates [8]. Indeed, their gelling properties are dependent on guluronic acid content and explained by the structural features of GG blocks where selective alkaline earth metal multivalent cations and notably Ca2+ take place by chelation. This phenomenon is known and explained as the “egg-box” model. The formed gel is not soluble in water. However sodium alginate is a water-soluble polymer having generally pseudoplastic properties in solution [9]. The conversion of insoluble forms of alginate in the cell walls of algae to its sodium salt (soluble

ACCEPTED MANUSCRIPT form) is at the origin of their extraction and purification using acidic and alkaline solutions [10,11]. Polysaccharides have wide range of pharmacological activities and some of them are considered as antitumor, antihypertensive, immunomodulator as well as antioxidant agents. Their radical scavengers role can be used to inhibit oxidative damage in foods and improve the their

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nutritional quality [12,13]. Structural features of polysaccharides such as molecular weights,

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monosaccharidic composition, glycosidic branching, type, degree and position of functional

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groups (hydroxyl, sulfate, amine, carboxyl, acetyl, phosphate and other) affect their antioxidant activity [14]. The molecular weight and M/G ratio of alginates play an important role on their

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scavenging ability on free radical. It was supposed that polysaccharides with low molecular

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weights would have more reductive hydroxyl group terminals (on per unit mass basis) to accept and eliminate the free radicals [15]. On the other hand, higher proportion of G blocks increases

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the antioxidant activity because the diaxial linkage in these blocks may cause a hindered rotation

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around the glycosidic linkage. As a result, flexibility of the G blocks increase, thus influencing the availability of hydroxyl groups in sodium alginate and the ability to donate H-atom [16]. The

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alginates have also the potential ability to inhibit the lipid peroxidation of phosphatidylcholine

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and linoleate liposomes, protect NT2 neurons against H2O2-induced neurotoxicity and stop free radical chain reactions [17, 18, 19].

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Nizamuddinia zanardinii (Schiffner) P.C. Silva is a marine brown algae (Phaeophyceae, Fucales) of Sargassaceae family and the sole type species (holotype) of the genus Nizamuddinia (Fig1). It grows in South-west Asia: the Arabian Sea coasts (Oman, Yemen and Pakistan), Persian gulf (Qatar) and in the Oman Sea and Persian gulf (Chabahar and Gheshm Island, Iran). There is only one species or infra specific name in the AlgaeBase at present and it was not explored in literature for its alginate content. It is the first report on extraction, structural characterization, shear flow properties evaluation of the alginate from this non exploited macroalgae.

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Seaweed material

Nizamuddinia zanardinii (Schiffner) P.C. Silva was collected on December 2015 along the Oman Sea in south of Iran (Chabahar bay: 25°20'53" N and 60°28'1" E). Taxonomic group of the

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seaweed was determined by the Off-Shore Fisheries Research Center. After cutting, it was

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2.2. Extraction and purification of alginates

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mesh and stored in sealed bags at room temperature.

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washed with fresh water, sun-dried at ambient temperature, milled, treated with a sieve of 1 mm

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Alginate was extracted using a method adapted from Calumpong et al. [20]. Twenty five gram of dried algae was soaked under steering at room temperature for 24 h in 800 mL of 2% (v/v)

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formaldehyde to remove phenolic compounds and pigments. After that, the insoluble fraction

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was washed 3 times with MilliQ water and supplemented by 800 mL of 0.2 M HCL before to be incubated at 60°C for 3 h under stirring (250 rpm). The suspension was then centrifuged (10000

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g for 20 min at 20°C) and pellets with alginic acid were washed 3 times with MilliQ water before

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to be socked in 3 % (w/v) Na2CO3 solution for 2.5 h at 60 °C. The mixture was centrifuged (10000 g for 30 min at 20°C) and the supernatant was precipitated with 3 volumes of ethanol 96

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% (v/v). Pellets containing sodium alginate were dissolved in MilliQ water and precipitated with ethanol as described above. This step was repeated two times before to collect and freeze dried the sodium alginate.

2.3. Chemical and biochemical analysis Moisture, proteins and total ash contents of Nizamuddinia zanardinii were evaluated by distillation in toluene, Kjeldahl analysis and calcinations at 550 °C, respectively [21]. Metal

ACCEPTED MANUSCRIPT cations extracted using HNO3 (65 %) were determined by inductive coupled plasma (ICP) instrument (PerkineElmer OptimaTM 7300DV ICP-OES). Fat content was determined using Soxhlet apparatus and soluble carbohydrates were quantified by phenol-sulfuric [22,23]. Protein contents of extracted alginates were evaluated using BioRad reagent adapted from

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Bradford using bovine serum albumin (BSA) as standard [24]. Acidic sugars were measured with

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m-hydroxydiphenyl and D-glucuronic acid as standard according to Blumenkrantz et al. and Van

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den Hoogen et al. [25, 26]. Neutral sugars were determined by resorcinol reagent in the presence of sulphuric acid with D-glucose as standard as described by Monsigny et al. [27]. Quantification

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of neutral sugars was done according to the corrective formula described by Montreuil et al. [28].

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Total phenolic compounds were quantified at A725 after Folin Ciocalteau method [29]. Results

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were expressed in gallic acid equivalents. All analyses were performed in triplicate.

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2.4. DPPH (diphenylpicrylhydrazyl) assay

The 2,2’-diphenyl-1-picrylhydrazyle (DPPH) radical scavenging efficiency of sodium alginate

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was determined using the method of Yamaguchi with some modifications [30]. Briefly, different

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concentrations of sodium alginate (0.01–10 mg/mL) were prepared. One mL of sample was added to 1 mL of DPPH solution (0.1 mM in 99% ethanol) and stirred. The mixture was

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incubated for 30 min in dark at room temperature and A517 nm was measured. Ascorbic acid was used as positive standard. The DPPH inhibition (%) was estimated by using Equation (1): (1) Where Asample and Acontrol are the absorbances at 517 nm of 1 mL of the sample (0-10 g/L) and 1 mL of distillated water with 1 mL of DPPH at 0.1 mM in ethanol respectively.

ACCEPTED MANUSCRIPT 2. 5. Partial acid hydrolysis of alginate Partial acid hydrolysis of alginate from Nizimuddinia zanardini was done according to the modified procedure of Leal et al. [31]. One gram of alginate was solved in 100 mL of MilliQ water and after adding 3 mL of 3 M HCl, the solution was heated at 100 °C (reflux) for 20 min.

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After cooling, the mixture was centrifuged (3000 g, 20 min and 20°C). The supernatant was

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neutralized by addition of 1 M NaOH and then precipitated with 100 mL of ethanol. The

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precipitate was collected by centrifugation (10000 g, 20 min and 20°C), dissolved in 50 mL of MilliQ water and freeze dried (MG blocks, Fraction 1 (F1)). Pellets were heated at 100°C after

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supplementation of 100 mL of 0.3 M HCl for 2 h (reflux) and centrifuged at 10000 g during 20

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min at 20°C. Pellets were dissolved in 100 mL of MilliQ water and neutralized by addition of 1 M NaOH. pH was then adjusted to 2.85 adding 1 M HCl. Soluble and insoluble fractions were

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separated by centrifugation (10000 g, 20 min and 20°C). The supernatant was neutralized (1 M

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NaOH), dialyzed against MilliQ water for 48 h and freeze dried (MM blocks, F2). Pellets were

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dissolved in 100 mL of MilliQ water, dialyzed and freeze dried (GG blocks, F3).

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2.6. Total acid hydrolysis of alginate Complete hydrolysis of alginate was performed according to Chandía et al. [32]. 4.5 mL of

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formic acid (90% v/v) were added to 10 mg of alginate or its fractions from partial hydrolysis in sealed tube and heated at 100°C. Solutions were mixed every one hour during 6 hours, diluted with 20 mL of MilliQ water, heated at 100°C for 2 h (reflux) and concentrated to 300 μL under vacuum.

ACCEPTED MANUSCRIPT 2.7. Chromatography The native alginate and its blocks were analyzed by HPAEC-PAD after complete hydrolysis. Analysis were done on a Dionex ICS-3000 system consisting of a SP gradient pump, a pulsed amperometric detector with a gold working electrode, an Ag/AgCl reference electrode and

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Chromeleon version 6.5 (Dionex Corp., Sunnyvale, CA). All eluents were degassed under

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vacuum and maintained under helium flush. The system was equipped with a CarboPac PA-1

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guard column (4 mm×50 mm Dionex) and a CarboPac PA-1column (4 mm×250 mm). The eluent was composed of solutions A (100 mM NaOH) and B (1 M NaOAc in 100 mM NaOH)

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mixed in a linear gradient mode between 0 and 100% of B in A during 60 min at a flow rate of 1

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mL/min. 25 µL of each sample at 1 mg/mL were injected in the system. The mass average molar mass (Mw), number average molar mass (Mn), gyration radius (Rg),

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hydrodynamic radius (Rh) and intrinsic viscosity ([η]), were evaluated by high pressure size

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exclusion chromatography (HPSEC) coupled on line with three detectors: multi-angle laser light scattering (MALLS, DAWN-EOS from Wyatt Technology Corp., USA) filled with a Ga-As

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laser (λ = 690 nm) and a K5 cell (50 μL) (HELEOS II Wyatt Technology Corp., USA), a

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differential refractive index (DRI) (RID10A Shimadzu) and a viscosimeter (Viscostar II, Wyatt Technology Corp., USA). The SEC line consisted of an OHPAK SB-G guard column and two

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OHPAK SB 804 and 806 HQ columns (Shodex Showa Denko K.K., Japan) in series eluted by LiNO3 0.1 M filtered through 0.1 μm filter unit (Millipore) and degassed (DGU-20A3 Shimadzu, Japan) at 0.5 mL/min flow rate (LC10Ai Shimadzu, Japan). Samples were solubilized at 1 mg/mL in LiNO3 0.1 M during 24 h under stirring, filtered through 0.45µm (Millipore) and injected (500 µL) onto analytical line equipped with automatic injector (SIL-20A Shimadzu, Japan). Data were analyzed using the ASTRA 6.1.7.15 software package using Zimm order 1.

ACCEPTED MANUSCRIPT The concentration of eluted fractions has been determined according to a differential refractive index dn/dc of 0.14 mL/g.

2.8. Spectroscopy

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Fourier-Transform Infrared (FT-IR) measurements were carried out using a VERTEX 70 FT-IR

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instrument. Dried polysaccharide was dispersed on ATR A225 diamond. The IR spectra (50

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scans, 4 cm-1 resolution) were recorded at room temperature (referenced against air) in the wavenumber range of 500–4000 cm−1. Spectra were analyzed with OPUS 7.2 software. H analyses were performed at 80°C. Alginate was dissolved in D2O (50 g/L) prior to NMR

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analysis. NMR spectra were recorded on a Brucker Advance 400 (Germany), operating at 400.13

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2.9. Rheological measurements

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MHz (number of scans = 64). All the chemical shifts were in relative with Me4Si.

Steady-shear flow measurements were assessed using sodium alginate solutions with

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concentration ranging from 1.0 to 5.0 % (w/v) in MilliQ water or in 0.5 mol/L NaCl solution.

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Shear stress and apparent viscosity were recorded at shear rate in the range of 0.01 to 1000 s -1. An aqueous solution of 2.0 % (w/v) of sodium alginate in MilliQ water was used to evaluate the

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effect of pH on flow behaviour. All rheological determination were performed at 20°C using an AR-2000 rheometer (TA Instrument, Great Britain, Ltd) equipped with a Peltier temperature control system using two types of test geometry in steady state shear flow: aluminium standard-size double concentric cylinders geometry for diluted solutions measurements (1.0 to 3.0% (w/v)) and parallel plate geometry with plate diameter 50 mm for solution with concentrations higher than 3.0 % (w/v) and for solution at pH lower than 5.0.

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3. Results and discussion 3.1. Chemical composition of Nizimuddinia zanardini The chemical composition of Nizimuddinia zanardini was measured according to the procedure

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described in methods. Moisture content was 7.17 % whereas proteins, ash, fat, and soluble

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carbohydrates were 9.63 %, 25.2 %, 3.2 % and 14 % (w/w of dry matter) respectively. The

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protein and soluble carbohydrate values were in agreement with those of Yazdani and et al. who reported contents of 10 % and 15.4 % (w/w) when they analyzed the biochemical composition of

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the same macroalgae [33]. The ash content estimated in this study was higher compared to that

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of Yazdani and et al. (17.4 %) [33] and mainly composed of sodium (32.9 %), calcium (28.1 %), potassium (20.7 %) and magnesium (16.8 %). This difference can be due to the effect of

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geographical location and season of algae harvesting. The fat content (3.2 % w/w) was similar to

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the result of Attaran Fariman et al. who described amounts of fat in Nizimuddinia zanardini

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varying between 3.08 to 4.11 % (w/w) depending on season [34].

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3.1. Extraction yield and chemical analysis of alginate The matricial polysaccharides from the brown seaweed Nizimuddinia zanardini were extracted

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according to the procedure described in methods and the isolated fraction was characterized for its uronic acid, neutral sugar, proteins and phenolic compounds (Table 1). A yield of 24 % (w/w) was obtained and the biochemical characterization clearly identified a polymer of uronic acids poorly contaminated by proteins and phenolic compounds. This yield was high compared to those obtained by other authors after extraction of alginates from species belonging to Sargassaceae family as Nizimuddinia zanardini (Table 2). The low amounts of neutral sugars can be explained by the presence of residual neutral polysaccharides such as fucoidans, sulphated

ACCEPTED MANUSCRIPT fucans or glucans in the alginate extract of Nizimuddinia zanardini, as those detected by Yazdani and et al [33]. The detection of neutral sugars in alginate extract has been also reported by numerous authors. For example Sellimi et al. (2015) have quantified 9 % (w/w) of neutral sugars in an alginate extracted from Cystoseira barbata [40]. This first result led us to conclude that the

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process applied to this macroalgae led to the extraction of an alginate.

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3.2. Partial and complete acid hydrolysis

Total hydrolysis by formic acid was applied to the sodium alginate extracted from Nizimuddinia

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zanardini and the hydrolysate was analyzed by HPAEC to selectively detect uronic acids. The

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chromatogram showed significant signals at 17.28 and 17.58 min which were attributed to Lguluronic and D-mannuronic acids respectively. A M/G ratio of 1.2 was obtained by comparison

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of their areas indicating that the alginate from Nizimuddinia zanardini was richer in mannuronic

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acid.

Complete hydrolysis and HPAEC analysis was also applied to fractions obtained after partial

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hydrolysis of the isolated sodium alginate as described in methods. The total yield for these 3

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fractions was 65% (w/w). The first fraction obtained after 0.3 M HCl treatment was composed of heteropolymeric blocks (F1, MG block). The F2 (M blocks) and F3 (G blocks) fractions were

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homopolymeric as confirmed by their M/G ratios evaluated by HPAEC at respectively 1.9 and 0.3. The recovery yields of the F1, F2 and F3 fractions were 11, 32 and 22 % respectively. The comparison of areas of D-mannuronic and L-guluronic acids in F2 and F3 led to a M/G ratio of 1.1 .The analytical data obtained for total hydrolysis and partial hydrolysis were then similar. Comparing values obtained for M/G ratios of the alginate of Nizimuddinia zanardini (1.1 vs 1.2) with other alginates extracted from the Sargassaceae family (Table 2), showed it was similar to

ACCEPTED MANUSCRIPT those of alginate from Sargassum turbinarioides (0.94), Sargassum fluitans (1.18) and Sargassum vulgare (1.27).

3.3. Spectroscopic analyses

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The FT-IR spectra of sodium alginate from Nizimuddinia zanardini and its 3 fractions isolated

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after partial hydrolysis are given in the Fig. 2. A wide band at 3260 cm−1 and a weak band at

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2925 cm-1 were assigned to hydrogen bonded (O–H and C–H) stretching vibrations, respectively. Two strong absorptions were observed at 1599 and 1409 cm−1. They were attributed to

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asymmetric and symmetric stretching vibrations of carboxylate groups (O–C–O) [41, 42, 43].

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Two bands at 1296 and 1124 cm−1 were assigned to C–C–H and O–C–H deformations and C–O stretching respectively. Two peaks at 1083 and 1027 cm−1 were related to (C–O) stretching

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vibrations of mannuronic acids and C–O (and C–C) stretching vibrations of pyranose rings of

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guluronic acids respectively [32, 44]. The anomeric or fingerprint region of sodium alginate (950–750 cm−1) was related to vibration of uronic acid residues. The spectrum showed bands at

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947, 902.5, 811 and 778 cm−1 in this region [32, 44]. The FT-IR spectrum of F1 (heteropolymeric

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fraction) showed a specific band at 966 cm−1 not detected in the spectra of the two homopolymeric fractions [44]. Two bands at 889 and 813 cm−1 were assigned to mannuronic and

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guluronic acids respectively and attributed to C-H deformation of β-mannuronic acid and stretching vibrations of C–O with contributions from stretching of C–C and deformation vibrations of C–C–O of mannuronic and guluronic acids [31, 44]. The spectrum of F2 contained a band at 946 cm−1 indicative of uronic acid presence by the C–O stretching vibration. The band at 880 cm-1 was attributed to C1–H deformation vibration of β-D-mannuronic acid and that at 817 cm-1 confirmed the presence of mannuronic acid [31, 45]. The spectrum of F3 contained a band at 948 cm−1 (also detected in the F2 spectrum) and three bands at 904, 808 and 779 cm-1. These

ACCEPTED MANUSCRIPT three signals were assigned respectively to asymmetric ring vibrations of anomeric C-H, C–OH, C–C–H and O–C–H deformation vibrations of α-L-guluronic acid with contributions of the bending deformation vibration of the C–O–C glycosidic linkage in homopolymeric blocks [31].

H NMR spectroscopy is a reliable method to determine monad values (FM and FG) and diad

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3.4. H NMR spectroscopy

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frequencies (FGG, FMM, FMG, and FGM) of an alginate [46]. The M/G ratio of sodium alginate from Nizimuddinia zanardini was calculated comparing signal areas of HG-1 (IA), HM-1+HGM-5

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(IB) and HG-5 (IC) (Fig. 3) using the equations(2)-(4) described by Grasdalen et al. (1979) [47].

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FG=IA / (IB+IC) (2) FM= 1 – FG (3)

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M/G = (1-FG) /FG (4)

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The η parameter was expressed by the equation 5 [47]. η=FGM/(FM+FG) (5)

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The values obtained for the M/G ratio of extracted sodium alginate from Nizimuddinia zanardini

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were 1.11.This result is matching with that obtain using total hydrolysis and analysis of uronic acids by HPAEC (1.1 Vs 1.2). The η parameter (0.232) was < 1 showing the abundance of M

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homopolymeric blocks.

The diad sequences were calculated by using following equations: FGG=IC/ (IB + IC) (6) FGG +FGM =FG (7) FMM +FMG =FM (8) The results obtained (Table 2) confirmed that as alginates from some species of Sargassaceae family (vulgare and turbinaroides), the alginate from Nizimuddinia zanardini was composed of

ACCEPTED MANUSCRIPT substantially higher levels of FM (0.53) compared to FG (0.47). These results were correlated with substantially higher levels of MM blocks compared to GG ones as the level of FMG was very low. The low amount of alternating blocks (MG and GM) was in agreement with alginates from S.

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vulgare and latifolum [10, 35, 16].

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3.5. Molecular weight distribution

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The Molecular weight distribution was determined using SEC-MALLS analysis. The weightaverage molecular weight (Mw), number-average molecular weight (Mn), gyration radius (Rg),

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and Rh of extracted sodium alginate from Nizimuddinia zanardini were calculated at 1.03×105

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g/mol, 0.84×105 g/mol, 26.2 nm and 17.3 nm respectively. According to the literature, molecular weight of alginates from brown algae can be between 0.32 and 4.0×105 g/mol for commercial

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products [1]. The average molecular weight of alginate from Nizimuddinia zanardini was in

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accordance with those of other members of Sargassaceae family (Table 3). Intrinsic viscosity was determined at 342 mL/g. This value is lower than those of sodium alginates from Sargassum

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species which are in the range of 410–1520 mL/g (Table 3). The polydispersity index (Ip) was

zanardini (Fig. 4).

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measured at 1.22 indicating the homogeneity of the alginate extracted from Nizimuddinia

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The degree of polymerization (DP) was calculated at 589.32 with the equation (9). DP = 2 + (Mw/175.37) (9)

Where Mw is the molecular weight of alginate and 175.37 g/mol is the molecular weight of one monomer (G or M) [16]. From [η], the Mark-Houwink-Sakurada exponent “a” was calculated to have access to polymer conformation (eq 10). (10)

ACCEPTED MANUSCRIPT Where “k” and “a” are constant parameters giving conformational information’s for a defined polysaccharide-solvent pair. A linear random coil polysaccharide has a Mark-Houwink-Sakurada exponent between 0.5 and 0.8. On the contrary the stiffness is characterized by values of “a” above 0.8. The “a” exponent of the alginate from Nizimuddinia zanardini was calculated as egal

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to 0.98 (and K = 0.00113). This value is conformed to those of other alginates always above 0.8,

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(11)

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Another exponent “b” can be deduced from the equation (11).

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consequence of the length and stiffness of the hydrated alginate macromolecules in solution [48].

It gives also access to information on the conformation of the alginates. It should be around 1 for

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rod conformation, between 0.5 and 0.6 for coil conformation and around 0.33 for a sphere [49]. Moreover “b” is known to be dependent of the degree of M/G ratio and distribution along a

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sodium alginate chain. The “b” exponent value of the alginate from Nizimuddinia zanardini was

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0.64 led us to conclude that this alginate has a conformation between rod and coil, explained by the low molecular weight and higher proportion of flexible MM blocks compared to GG ones.

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The Rg/Rh ratio can also be used to describe the polysaccharide conformation in solution, sphere

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conformation (Rg/Rh=0.77), flexible linear chains (Rg/Rh = 0.82) and stiff rods (Rg/Rh >2)) [50]. It was found at the value of 1.47 for the sodium alginate from Nizimuddinia zanardini in

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accordance with information given by “a” and “b”exponents.

3.6. Rheology The viscous properties of alginate solutions were determined by steady-shear flow at shear rate ( ) from 0.01 to 1000 s-1. The curve modelisation was achieved using the power law (Ostwaldde-Waele relationship) (eq 12) (12)

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is the shear rate (s-1), k, the consistency index (Pa.sn) and n the

flow behaviour index (for liquid exhibiting Newtonian behaviour n=1, for pseudo plastic or shear-thinning fluid n<1 and for swelling plastic or shear-thickening fluid n>1). The relation between shear stress τ and apparent viscosity μ is done by equation 13.

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(13)

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Table 4 shows the effect of alginate concentration and solvent on consistency index and flow

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behaviour index. The values of flow behaviour index (n) for solutions of sodium alginate in

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water are closed to 1.0 meaning that the polymer exhibited almost Newtonian or very low shear thinning behaviour at concentrations from 1.0 to 5.0 % (w/v). This result was in agreement with

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observations of Rioux et al. (2007) for sodium alginate extracted from three brown seaweeds (Ascophyllum nodosum, Fucus vesiculosus and Saccharina longicruris) but for lower

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concentration (0.25 to 1.75% w/v)[51]. Note that in the majority of the publications describing

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the rheology of diluted and concentrated solutions of sodium alginate in water describe typical shear thinning behaviour almost in the same range of shear rate. This phenomenon can be

PT

explained by the alignment of alginate molecules in the flow [52]. The same tendency of flow

CE

index decreasing with increasing of weight fraction of alginate was described by Mancini et al. [53] for some commercial alginates. The flow behaviour index of sodium alginate was decreased

AC

in NaCl 0.5 mol/L NaCl solutions and by increasing polymer concentration (Table 4). For all concentrations n was lower than 1.0 showing a non-Newtonian shear thinning behaviour. The consistency index k increased with concentration independently of the nature of the solvent. The viscosity of sodium alginate depends on ionic strength of the solution. For lower concentrations of alginate (1.0 to 3.0%, w/w) and shear rate superior to 1.0 s-1, the viscosity decreased with ionic strength due to the progressive screening of the electrostatic repulsions between chains of polymers that reduce the overall coil dimension [54].

ACCEPTED MANUSCRIPT Fig. 5A and 5B plotted the apparent viscosity ( ) vs shear rate (

for solutions of sodium

alginate from Nizimuddinia zanardini in MilliQ or in 0.5 mol/L NaCl solution. Sodium alginate had a thickening effect and increasing polysaccharide concentration determine the viscosity rising. However, the shear rate increasing induces significant modification of the viscosity only

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for sample dissolved in NaCl.

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The influence of pH on the Ostwald parameters for a 2.0% (w/v) alginate solution is presented in

CR

Table 5 and Fig.6. The initial pH value of the native alginate solution was close to 7.0 and the

US

flow index decrease with pH decreasing. The Fig. 6 shows that solution at pH equal to 7.0 had a Newtonian behaviour (confirmed by the flow index close to 1.0, Table 5), an intermediary

AN

behaviour between Newtonian and non-Newtonian shear thinning at pH 6.0 and 5.0 and a clearly shear thinning behaviour at lower pH (4.5 and 3.0) was observed. Below a pH value of 3.0,

M

alginic acid precipitated. This viscosity enhancement at low pH is explained as a result of

ED

electrostatic repellence suppressing, enhancement of intermolecular hydrogen bonds and entanglements leading to complex structures [55]. At pH values from 5.0 to 3.0, the hydrophobic

PT

segments in the alginate chains increase, and the hydrophilic segments decrease. Conversely,

CE

small viscosity changes at pH 7.0 to 5.0 could be explained by the lack of electrostatic repellence

AC

due to low intermolecular interactions.

3.7. Antioxidant activity

Antioxidant capacity of polysaccharides such as alginates is an important characteristic for applying them in food and pharmaceutics [56-59]. According to previous reports, functional groups of polysaccharides such as sulfate, amino, hydroxyl and carboxyl groups, availability of hydroxyl groups, monosaccharide composition, molecular hydrogen bonds and molecular weight affect on the antioxidant activity [60-63] and alginates are described in literature as strong free

ACCEPTED MANUSCRIPT radical scavenging activity [64,14]. The DPPH method was used to determine the free radical scavenging activity of sodium alginate from Nizimuddinia zanardini and the results were compared with ascorbic acid used as standard (Fig. 7). The results showed that there is direct relation between concentration and antiradical activity of sodium alginate. Ascorbic acid, used as

T

standard, had higher radical scavenging activity than that of sodium alginate at the same

IP

concentrations. Sari-Chmayssem et al (2016) described higher antioxidant activity of G block

CR

compared to M ones. The diaxial linkage in the G blocks may cause a hindered rotation around the glycosidic linkage. As a result, flexibility of the G blocks increases, influencing the

US

availability of hydroxyl groups in sodium alginate and their ability to donate H-atom [16]. The

AN

antioxidant activity of sodium alginate from Nizimuddinia zanardini at 2 mg/mL was 66.4 %. This result was lower than values obtained with alginates exhibiting higher G blocks amounts

M

like alginate of Sargassum vulgare (74.29 %) with M/G=0.78 [16]. Moreover,the M/G ratio of

ED

sodium alginate from Cystoseira barbata (a Tunisian seaweed) was reported 0.59 and this alginate exhibited important DPPH radical-scavenging activity (74% inhibition at a

PT

concentration of 0.5 mg/ml), higher than that obtained with the alginate from Nizimuddinia

CE

zanardini[40]. Previous findings also revealed, a scavenging capability on DPPH radicals at a dosage of 5 mg/ml (62%) with the alginates isolated from Turbinaria conoides that is lower of

AC

the alginate of Nizimuddinia zanardini M/G ratio (1.39) [65]. Antioxidant activity depends also on molecular weight of alginate as some authors published an increasing of antioxidant properties of alginates by decreasing their molecular weights. Note also that all these results from literature not always take into account the purity of extracted alginates as some other antioxidant compounds such as phenolic ones may be co-extracted with alginates [66, 67]. Inhibition capacity (50%) of sodium alginate was calculated at 0.9 mg/mL against 0.005 mg/mL for ascorbic acid.

ACCEPTED MANUSCRIPT 4. Conclusion Nizimuddinia zanardini, a brown seaweed of Sargassaceae family, was collected from the Oman sea in south of Iran. Its sodium alginate was extracted and purified. This acidic polysaccharide was characterized by HPAEC, SEC-MALLS, 1H-NMR and FT-IR. To our knowledge, this study

T

is the first characterization of sodium alginate from Nizimuddinia zanardini. The M/G ratio value

IP

of this sodium alginate, obtained by HPAEC and 1H-NMR was between 1.1 and 1.2 and high

CR

values of homopolymeric block M (η<1) was found. This result was in agreement with other results reported for alginates extracted from macroalgae of the Sargasseae family [35]. Its

US

molecular weight and polydispersity index were evaluated using SEC-MALLS at 1.03×105

AN

g/mol and 1.22 and its conformation was between rod and coil. The rheological study of this alginate concluded that the solvent used to prepare sodium alginate solution has an impact on the

M

fluid type behaviour (rather Newtonian for alginate in water and shear thinning for alginate in

ED

sodium chloride solutions). The apparent viscosity increases with polymer concentration for both solvent type solutions. At pH below 5.0, the alginate solution behaviour is strongly shear

PT

thinning with very low flow index (n=0.441±0.017 at pH 4.5 and n=0.343±0.011 at pH 3.0). The

CE

sodium alginate showed good antioxidant properties at concentration 2 % (w/v) and higher using DPPH radical-scavenging activity assay. A mechanism of the anti-DPPH radical activities can be

AC

through the donation of hydrogen to break chain reactions or scavenging free radicals and capture of anomeric hydrogen from monosaccharides [68].

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AC

CE

PT

ED

M

543–552.

ACCEPTED MANUSCRIPT Tables Table 1.Yield and chemical characterization of sodium alginate from Nizimuddinia zanardini

Values (%, w/w)

Yield

24±0.8

Neutral sugars

15±0.4

Uronic acids

60±2

1.1±0.1

CE

PT

ED

M

AN

Phenolic compounds

AC

IP

CR

US 0.9±0.1

Proteins

T

Analytical parameters

ACCEPTED MANUSCRIPT Table 2. Compositional data of alginates extracted from genus of Sargassaceae. Species

Origin

Yield (%) M/G

Sargassum vulgare (SVHV)

Brazil

16.9

1.56

0.61

0.39

0.58

Sargassum vulgare (SVLV)

Brazil

16.9

1.27

0.56

0.44

0.55

Sargassum fluitans

Florida

21.1–24.5

1.18

0.54

0.46

Nizimuddinia zanardini

Iran

24.0

1.11

0.53

0.03

η

Ref. 0.12

35

0.01

0.43

0.04

35

0.36

0.36

0.28

0.72

36

0.47

0.058

0.416

0.232

T P

I R

this study

0.36

0.25

0.39

0.5

37

0.82

0.45

0.55

0.41

0.04

0.51

0.16

10

15.1–17.2

0.78

0.44

0.56

0.33

0.11

0.45

0.11

38

Lebanon

40.0

0.78

0.44

0.56

0.39

0.05

0.51

0.20

16

Cuba

21.1–24.5

0.52

0.34

0.66

0.25

0.18

0.57

0.40

36

England

18.0

0.31

0.24

0.76

0.07

0.34

0.59

0.93

39

Sargassum filipendula

Brazil

T P

D E

17.7

Sargassum muticum

0.474

FGG 0.36

C S

U N

A

FMG

0.52

Egypt

E C

C A

FMM

0.48

0.94

Sargassum latifolium

Sargassum fluitans

FG

M

Sargassum turbinarioides Grunow Madagascar10.0

Sargassumvulgare

FM

ACCEPTED MANUSCRIPT Table 3. The molecular weight of alginates extracted from genus of Sargassaceae family

Molecular weight (g/mol) [η] (mL/g)

Species

Ref.

1.94×105

410

35

Sargassum vulgare (SVHV)

3.30×105

690

35

Sargassum dentifolium

6.06×105

Sargassum asperifolium

7.34×105

Sargassum latifolium Sargassum turbinarioides.

IP 1520

10

4.16×105

870

10

AN

5.52×105

-

37

1.10×105

-

16

1.03×105

342

This study

CR

1260

US

ED

PT CE AC

10

M

Sargassum vulgare Nizimuddinia zanardini

T

Sargassum vulgare (SVLV)

ACCEPTED MANUSCRIPT Table 4. Consistency and flow behaviour index of solutions of sodium alginate from Nizimuddinia zanardini in MilliQ water and NaCl.

NaCl concentration Alginate concentration (%, w/v)

k

n

R2

T

(mol/L) 0.051±0.001 0.950±0.007 0.999

0.5

0.024±0.002 0.828±0.025 0.989

IP

0.0

CR

1.0

2.0 0.5

AN

0.0

AC

CE

PT

ED

4.0

0.062±0.006 0.878±0.025 0.990 0.091±0.001 0.976±0.003 0.999

0.5

0.218±0.018 0.845±0.023 0.990

0.0

0.207±0.020 0.963±0.003 0.999

0.5

0.397±0.029 0.814±0.021 0.992

0.0

1.039±0.051 0.940±0.013 0.997

0.5

2.579±0.016 0.789±0.018 0.993

M

3.0

5.0

0.064±0.005 0.995±0.002 0.999

US

0.0

ACCEPTED MANUSCRIPT Table 5. Influence of pH on the consistency and flow behaviour index of solutions of sodium

k

n

R2

7.0

0.064±0.005

0.995±0.002

0.999

6.0

0.067±0.006

0.895±0.024

0.991

5.0

0.068±0.005

0.899±0.021

IP

4.5

2.535±0.015

0.441±0.017

0.982

3.0

4.560±0.018

0.343±0.011

0.989

0.993

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pH

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alginate from Nizimuddinia zanardini at 2 % (w/v) in MilliQ water at several pH.

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Figure 1. Nizimuddinia zanardini (right) and its leaf (left).

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Figure 2. FT-IR spectra of sodium alginate from Nizimuddinia zanardini and its three fractions

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from partial hydrolysis.

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Figure 3. 1H NMR spectrum (80°C) of sodium alginate from Nizimuddinia zanardini

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Figure 4. SEC-MALLS chromatogram of sodium alginate from Nizimuddinia zanardini. (….)

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intrinsic viscosity, (- - - ) refractive index and (___) light scattering intensity at the angle 90◦.

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Figure 5. Apparent viscosity vs shear rate for solutions of different concentration of sodium

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alginate from Nizimuddinia zanardini at 20 °C in MilliQ water (A) or in 0.5M NaCl solution (B).

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different pH

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Figure 6. Apparent viscosity vs shear rate for 2.0 % (w/v) of sodium alginate solutions at

Figure 7. Scavenging effects on DPPH radical for (square symbol): sodium alginate, (circle square): ascorbic acid.

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Fig. 1

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Fig.6

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Fig.7