Accepted Manuscript Title: Structural, physicochemical and antioxidant properties of sodium alginate isolated from a Tunisian brown seaweed Author: Sabrine Sellimi Islem Younes Hanen Ben Ayed Hana Maalej Veronique Montero Marguerite Rinaudo Mostefa Dahia Tahar Mechichi Mohamed Hajji Moncef Nasri PII: DOI: Reference:
S0141-8130(14)00692-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.10.016 BIOMAC 4664
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
11-8-2014 7-10-2014 8-10-2014
Please cite this article as: S. Sellimi, I. Younes, H.B. Ayed, H. Maalej, V. Montero, M. Rinaudo, M. Dahia, T. Mechichi, M. Hajji, M. Nasri, Structural, physicochemical and antioxidant properties of sodium alginate isolated from a Tunisian brown seaweed, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.10.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Structural, physicochemical and antioxidant properties of sodium alginate isolated from
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a Tunisian brown seaweed
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Sabrine Sellimi1*, Islem Younes1, Hanen Ben Ayed1, Hana Maalej1, Veronique Montero2,
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Marguerite Rinaudo3, Mostefa Dahia4, Tahar Mechichi1, Mohamed Hajji1 and Moncef Nasri1
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Nationale d’Ingénieurs de Sfax, B.P. 1173-3038 Sfax, Tunisie.
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: Laboratoire de Génie Enzymatique et de Microbiologie, Université de Sfax, Ecole
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Montpellier II, ENSCM, 8, rue de l’Ecole-Normale, 34296 Montpellier cedex, France.
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: Biomaterials Applications, 6, rue Lesdiguières 38000 Grenoble, France
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: Département de Biologie, Faculté des sciences de la nature et de la vie, Université de
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Djelfa, Algérie.
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: Laboratoire de Glycochimie et Reconnaissance Moléculaire, UMR 5032, Université
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Corresponding author. E-mail address: [email protected]. Tel. : +216 20 096 945
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Page 1 of 38
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Abstract An original sodium alginate from Tunisian seaweed (Cystoseira barbata) was purified
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and characterized by circular dichroism (CD) and ATR-FTIR spectroscopies. ATR-FTIR
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spectrum of C. barbata sodium alginate (CBSA) showed the characteristic bands of
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mannuronic (M) and guluronic acids (G). The M/G ratio was estimated by CD (M/G=0.59)
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indicating that CBSA was composed of 37% mannuronic acid and 63% guluronic acid. The
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analysis of viscosity of CBSA showed evidence of pseudoplastic fluid behaviour. The
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emulsifying capacity of CBSA was evaluated at different concentrations (0.25-3%),
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temperatures (25-100 °C) and pH (3.0-11.0). Compared to most commercial emulsifiers, the
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emulsion formulated by CBSA was found to be less sensitive to temperature changes and
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more stable at acidic pH. CBSA was examined for antioxidant properties using various
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antioxidant assays. CBSA exhibited important DPPH radical-scavenging activity (74%
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inhibition at a concentration of 0.5 mg/ml) and considerable ferric reducing potential.
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Effective hydroxyl-radical scavenging activity (82% at a concentration of 5 mg/ml) and
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potent protection activity against DNA breakage were also recorded for CBSA. However, in
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the linoleate-β-carotene system, CBSA exerted moderate antioxidant activity (60% at a
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concentration of 1.5 mg/ml). Therefore, CBSA can be used as a natural ingredient in food
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industry or in the pharmaceutical field.
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Keywords : Cystoseira barbata ; sodium alginate ; cicular dichroism ; ATR-FTIR ; viscosity ;
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emulsifying capacity ; antioxidant activity
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Page 2 of 38
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1. Introduction Phycocolloids are polysaccharides associated with the cell wall and intercellular
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spaces of some seaweed species [1]. The major structural polysaccharide of brown seaweeds
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is alginate, which usually exists in the cell wall and in the matrix as a mixture of all the
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cationic salt forms found in seawater. In its native state, alginate exists as calcium,
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magnesium and sodium salts of alginic acid, providing both strength and flexibility to the
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algal tissue [2]. The most important algal sources of alginate are Macrocystis pyrifera,
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Ascophyllum nodosum, Laminaria spp, Ecklonia maxima, Eisenia bicyclis, Lessonia
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nigrecans and Sargassum spp [3]. Alginate has been widely used in many fields such as cell
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immobilization, tissue engineering, microencapsulation of nutraceuticals and drugs, as well as
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in food applications as thickening, stabilizing and gelling agents and as alginate-based edible
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films and coatings for food products [3,4].
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Alginate is a linear anionic copolymer of β-D-mannuronic acid (M) and α-L-guluronic
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acid (G) (1-4)-linked residues, arranged either in heteropolymeric (MG) and/or
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homopolymeric (M or G) blocks (Fig. 1) [4,5].
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Fig. 1
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The mannuronic acid forms β (1-4) linkages, then, M-block segments show linear and flexible
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conformation. The guluronic acid, differently, gives rise to α (1-4) linkages, introducing a
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steric hindrance around the carboxyl groups, then, the G-block segments provides folded and
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rigid structural conformations, responsible of a pronounced stiffness of the molecular chains
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[4]. In the presence of divalent cations such as calcium, a strong interaction with the COO-
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groups of guluronic acid from different chains is formed, giving rise to a water insoluble and
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thermo-irreversible three-dimensional network (gel), whose conformation is often
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named « egg box » [6].
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Alginates are typically described by their M/G ratio and average molecular weight,
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since these parameters are closely related to the functionality of the alginates [1]. Generally,
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the extraction and purification processes of alginates are based on the conversion from the
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insoluble form in the seaweed cell walls to the soluble one, normally the sodium salt,
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followed by successive dissolutions and precipitations to eliminate impurities [5,7,8].
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Cystoseira barbata is a brown seaweed abundant along the Tunisian seacoast used in a
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few number of studies. Recently, structural features and antioxidant activity of fucans
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(sulfated polysaccharides) isolated from C. barbata have been reported by Sellimi et al. [9].
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However, alginate extracted from the C. barbata seaweeds was not yet studied. Then, the
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aims of the present study were to extract and purify sodium alginate from C. barbata. The
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extracted sodium alginate (CBSA) was characterized by circular dichroism, ATR-FTIR and
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size exclusion chromatography. Finally, viscosity, emulsifying capacity and antioxidant
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properties of CBSA were evaluated.
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2. Materials and methods
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2.1. Reagents
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1,1-Diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxyanisole (BHA), linoleic
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acid, β-carotene, polyethylene oxide, ferrous sulfate heptahydrate (FeSO4. 7H2O),
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galacturonic acid and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma
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Chemical Co. (St. Louis, MO, USA). Arabic gum and alginate under sodium salt form were
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purchased, respectively, from Merck (Germany) and Alfa Aesar (Tianjin, China). All other
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chemicals, namely sodium carbonate (Na2CO3), potassium ferricyanide, sodium chloride,
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trichloroacetic acid (TCA), ferric chloride (FeCl3), D-deoxyribose, hydrogen peroxide (H2O2),
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thiobarbituric acid (TBA), L-mannitol, L-ascorbic acid, 3,5 dimethylphenol, anthrone reagent,
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Tween 80 and other solvents, were of analytical grade. The vegetable oils used (olive oil,
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sunflower oil, corn oil, soybean oil, ricin oil, almond oil and argan oil) were purchased from
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the local supermarket.
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2.2. Biological material
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A brown seaweed C. barbata collected from Kerkennah island (Sfax, Tunisia), in
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November 2012, was studied. The freshly collected seaweed fronds were washed thoroughly
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with tap water to remove all sand particles and epiphytes. Then, they were dried at room
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temperature for about 20 days away from sunlight or heat such as to preserve as much as
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possible the quality of the initial material including alginates. The dried samples were ground
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using a mixer grinder (Moulinex) and were sieved in a 0.2 mm mesh size. The powder was
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kept in a clean, dried, and well-sealed amber glass container to protect it from sunlight.
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2.3. Preliminary treatments
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The C. barbata powder (50 g) was depigmented and deffated sequentially with
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acetone-methanol (7:3, 500 ml) (twice) and chloroform (300 ml) (twice) for 24 h at 30 °C
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under constant stirring (250 rpm) [9]. The algal material was air-dried to yield depigmented
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and deffated algal powder (45 g).
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2.4. Extraction and purification of sodium alginate
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The extraction of alginate from C. barbata seaweed was carried out using high
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temperature alkaline extraction according to Davis et al. [10] with slight modifications.
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Depigmented, defatted and dried seaweed powder (25 g) was treated twice at pH around 2.0
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with 500 ml of 0.1 M HCl for 2 h at 60 °C under constant stirring (250 rpm) to ensure the
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powder demineralization. The supernatant was eliminated by centrifugation (5000 rpm, 15
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min, 4 °C). The residue was washed with distilled water and then treated with 3% Na2CO3
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(pH = 11.0) at 60 °C for 2 h under constant stirring to solubilize the alginate in sodium salt
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form. Then, the supernatants were collected and precipitated with absolute ethanol (2v). This
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precipitate, recovered by centrifugation, is suspended in distilled water and acidified with 6 N
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HCl to pH < 3.0 to precipitate alginic acid. The precipitate formed was resuspended in
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distilled water and neutralized by an aqueous solution of 1 M NaOH to pH 7.5. At end, the
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sodium alginate was purified by a second precipitation with absolute ethanol. The precipitate
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obtained was resuspended in distilled water and freeze dried to yield C. barbata sodium
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alginate powder (CBSA).
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2.5. Chemical analysis
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Moisture and ash contents were determined according to the AOAC methods (927.05
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and 942.05, respectively) [11].
Uronic acids (UA) were determined following the
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colorimetric method using galacturonic acid as standard and 3,5 dimethylphenol as a reagent
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[12] and neutral sugars (NS) were measured by anthrone colorimetric method [13].
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2.6. Circular dichroism and ATR-FTIR spectroscopies
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Circular dichroism (CD) spectrum was recorded on Jasco model J-815 CD
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Spectrometer, using measurement range at 190-250 nm and sample concentration of 0.8
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mg/ml (800 ppm in distilled water). The CD spectrum is often reported in degrees of
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ellipticity (θ) expressed as millidegrees (mdeg). The ratio of peak height to trough depth was
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calculated using the equation described by Morris et al. [14].
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Peak/trough ratio = (θ trough - θ peak)/ θ trough
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Infrared (IR) spectra of sodium alginates were determined on a Fourier transform (FT)
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spectrophotometer (Perkin Elmer®, Spectrum™ 100, Singapore) equipped with attenuated
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total reflection (ATR) accessory containing a diamond/ZnSe crystal. An extra accessory plate
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for powdered samples with a conic awl was used without need for previous sample
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preparation. ATR-FTIR spectra were obtained in the 4000-600 cm-1 range at room
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temperature, using 10 scans and 4 cm-1 resolution.
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2.7. Capillary viscometry
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The dynamic viscosity measurements were carried out on a capillary viscosimeter
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(Ubbelohde) immersed in a thermostated bath with a precision of ± 0.1 °C. Solutions at
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different concentrations were prepared in 0.1 M NaCl. The measurements were carried out at
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25 °C according to the method of Haug and Larsen [15]. The intrinsic viscosity [η] was
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determined using Huggins equation (1) by extrapolating ηsp/c against concentration curve to
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zero, and averaging the value of the intercept.
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η sp/c = [η] + k1 × [η]² × c (1)
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ηsp = (η – η0)/ η0
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where ηsp, k1 and c were the specific visocsity, the Huggins constant and the concentration of
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the alginate solution expressed as g/ml. η and η0 were the absolute viscosities of the alginate
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solution and that of the solvent (0.1 M NaCl), respectively. The viscometric-average
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molecular weight (M) (Da) was determined using the corresponding Mark-Houwink equation
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(2) with K = 2 × 10-3 (ml/g) and a = 0.97 in 0.1 M NaCl solvent at 25 °C [16].
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M
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[η] = K × Ma (2)
2.8. Molecular weight distribution
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The weight-average molecular mass (Mw), the number-average molecular mass (Mn)
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and polydispersity (Mw/Mn) of 0.3% of CBSA, dissolved in HPLC grade water, were
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determined after elution with 0.1 M NaCl at 25°C in a High Performance Size Exclusion
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Chromatography (HPSEC) Waters Alliance GPCV2000 (USA) equipped with a Multi-Angle
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Laser Light Scattering (MALLS) detector from Wyatt (USA). The weight-average degree of
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polymerization DPw (DPw = Mw/m0 ; m0 = 198 represents the repeat unit in the sodium salt
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form expressed as g/mole) and the number-average degree of polymerization DPn (DPn =
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Mn/m0) were obtained [17].
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Prior to measurements, the apparatus was calibrated using HPLC grade toluene and
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normalized using polyethylene oxide (72 kDa) in 0.1 M NaCl. Before injection, the sample
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was filtered on a 0.45 µm pore membrane to eliminate dust particules. The concentration
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injected was 3 mg/ml, with an injection volume of 100 µl using a column TSK-G2000 SWXL
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(7.8 mm × 300 mm). The eluent used was 0.1 M NaCl at 25 °C elution temperature and a flow
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rate of 0.5 ml/min. The specific refractive index increment (dn/dc) adopted is equal to 0.155
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[18]. Data were collected from the refractive index detector (DRI) and MALLS and evaluated
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with the ASTRA software version 4.72.03.
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2.9. Antioxidant activity
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2.9.1. DPPH assay
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The DPPH radical-scavenging activity of CBSA was determined by the method of
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Kirby and Schmidt [19], with some modifications. Briefly, a volume of 500 µl of CBSA at
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different concentrations (0.01-1 mg/ml) was added to 375 µl of 99% ethanol and 125 µl of
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DPPH solution (0.02% (w/v) in ethanol). The mixture was incubated for 60 min in the dark at
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room temperature. Scavenging capacity was measured spectrophotometrically (T70 UV-
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visible spectrometer PG Instruments Ltd, Japan) by monitoring the decrease of absorbance at
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517 nm. In its radical form, DPPH has an absorption band at 517 nm which disappears upon
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reduction by an antiradical compound. Lower absorbance of the reaction mixture indicated
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higher DPPH free radical-scavenging activity (expressed as percentage). BHA was used as
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positive standard. DPPH radical-scavenging activity was calculated as follows:
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DPPH radical-scavenging activity (%) =
A control + A blank - A sample
× 100
A control
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where A
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sample), A
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solution) and A sample is the absorbance of the CBSA with the DPPH solution. The experiment
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was carried out in triplicate and the results are mean values.
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2.9.2. Ferric-reducing activity
is the absorbance of the control reaction (containing all reagents except the
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is the absorbance of the CBSA (containing all reagents except the DPPH
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The reducing power of CBSA was determined by the method of Yildirim et al. [20].
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A 0.5 ml of CBSA solution at different concentrations (0.1-1.2 mg/ml) was mixed with 1.25
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ml of 0.2 M phosphate buffer (pH 6.6) and 1.25 ml of 1% (w/v) potassium ferricyanide. The
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mixture was incubated for 30 min at 50 °C. After incubation, 1.25 ml of trichloroacetic acid
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(10%, w/v) was added and the reaction mixture was centrifuged for 10 min at 3000 g. A 1.25
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ml aliquot of the supernatant from each sample mixture was mixed with 1.25 ml of distilled
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water and 0.25 ml of 0.1% (w/v) ferric chloride solution in a test tube. After 10 min, the
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absorbances of the resulting solutions were measured at 700 nm. BHA was used as standard.
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Values presented are the mean of triplicate analyses.
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2.9.3. ß-carotene-linoleic acid assay The ability of CBSA to prevent bleaching of ß-carotene was assessed as described by
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Koleva et al. [21]. A stock solution of β-carotene/linoleic acid mixture was prepared by
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dissolving 0.5 mg of β-carotene, 25 µl of linoleic acid and 200 µl of Tween 80 in 1 ml of
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chloroform. The chloroform was completely evaporated under vacuum in a rotary evaporator
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at 40 °C, then, 100 ml of distilled water were added and the resulting mixture was vigorously
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stirred. The emulsion obtained was freshly prepared before each experiment. Aliquots (2.5
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ml) of the β-carotene/linoleic acid emulsion were transferred to test tubes containing 0.2 ml of
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CBSA solution at different concentrations (0.05-1.5 mg/ml). Following incubation for 2 h at
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50 °C, the absorbance of each sample was measured at 470 nm. BHA was used as positive
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standard. The control tube contained no sample. Antioxidant activity in β-carotene bleaching
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model expressed as percentage was calculated with the following equation:
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ß-carotene-bleaching inhibition (%) = [1 – ((A0 – At) / (A’0 – A’t))] × 100
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where A0 and A’0 are the absorbances of the sample and the control, respectively, measured at
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time zero, and At and A’t are the absorbances of the sample and the control, respectively,
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measured after 2 h. Tests were carried out in triplicate.
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2.9.4. Hydroxyl radical-scavenging activity
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Hydroxyl radical-scavenging activity of CBSA was determined as described by
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Chung et al. [22] with slight modifications. Briefly, 0.2 ml of CBSA solution at different
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concentrations (0.25-5 mg/ml) was added to the reaction mixture containing 0.2 ml FeSO4 .7
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H2O (10 mM), 0.2 ml EDTA (10 mM) and 0.2 ml D-deoxyribose (10 mM). The volume was
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made up to 2 ml with phosphate buffer (0.1 M, pH 7.4) and to that, 0.2 ml H2O2 (10 mM) was
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added. The mixture was incubated at 37 °C in the dark for 1 h. After incubation, 1 ml of TCA
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(2.8%) and TBA (1%) were added to the mixture and then, were left to stand in a boiling
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water bath for 10 min. The absorbance was measured at 532 nm. If the mixture was turbid, the
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absorbance was measured after centrifugation. Scavenging activity (%) was calculated using
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the equation :
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Hydroxyl radical-scavenging activity (%) = ((Ablank-Asample)/Ablank) × 100
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where Ablank is the absorbance of the blank (containing all reagents except the sample) and
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Asample is the absorbance of the sample. The results were compared with L-mannitol and BHA
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as positive controls.
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2.9.5. DNA-nicking assay
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DNA nicking assay was performed using pGapZαA plasmid (Invitrogen) by the
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method of Lee et al. [23], with slight modifications. A mixture of 10 μl of CBSA at the
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concentrations of 1, 2 and 3 mg/ml and plasmid DNA (0.5 μg/well) were incubated for 10 min
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at room temperature followed by the addition of 10 μl of Fenton's reagent (30 mM H2O2, 50
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μM L-ascorbic acid and 80 μM FeCl3). The mixture was then incubated for 30 min at 37 °C.
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The DNA was analysed on 1% (w/v) agarose gel.
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2.10. Emulsifying activity and emulsion stability
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The emulsifying activity was assessed as described by Cooper and Goldenberg [24],
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with slight modifications. Briefly, 3 ml of vegetable oil were added to 3 ml of CBSA aqueous
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solution in a test tube (18 mm × 120 mm) and stirred in the vortex at 2400 rpm for 2 min.
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After 24 h, the emulsification index (E24) was determined as follows:
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E24=he/hT×100
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where he (mm) is the height of the emulsion layer and hT (mm) is the overall height of the
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mixture. Several vegetable oils including olive oil, corn oil, sunflower oil, soybean oil, ricin
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oil, almond oil and argan oil were used to study the emulsifying activity of CBSA. All
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experiments were carried out in triplicate. All tests were performed at room temperature (25
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°C±1 °C) and at pH 5.0.
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The emulsifying capacity and the emulsion stability were also evaluated at different
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concentrations of CBSA (0.25-3%), temperatures (25-100 °C) and pH (3.0-11.0). For that
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purpose, emulsion (CBSA-corn oil) was pretreated for 30 min at each temperature and each
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pH before stabilization during 24 h at normalized conditions (25 °C) in order to determine the
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emulsification index (E24).
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2.11. Statistical analysis
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Statistical analyses were performed with SPSS ver.17.0, professional edition using
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ANOVA analysis. Differences were considered significant at p-value < 0.05. All tests were
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carried out in triplicate.
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3. Results and discussion
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3.1. Extraction yield and chemical analysis of C. barbata sodium alginate
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Taking into account the results obtained in the literature, we have adopted to extract
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sodium alginate from C. barbata at 60 °C for 2 h as described in section 2.4. The extraction
262
yield and the chemical characteristics (sugar content, moisture and ash) of CBSA are shown
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in Table 1. The extraction yield of CBSA was 9.9% based on dry seaweed weight. The CBSA
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extraction yield was higher than that registered for Dictyota caribaea and Padina
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perindusiata (7.4 and 5.4%, respectively) [25].
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Table 1
Considering variations being dependent on the alginate extraction method used, Davis
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et al. [10] found values in the range of 21.1-24.5% for Sargassum fluitans and 16.3-20.5% for
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S. oligocystum. In another study, Davis et al. [26] obtained higher yields for the same species,
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of 45% and 37%, respectively. The extraction yields depend crucially on the algal species and
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the extraction method. Rahelivao et al. [27] reported that, in the absence of EDTA, the
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alginate extraction yield was lower (10-13%) in relation with the presence of calcium
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complexed in the cell wall. However, in the presence of EDTA, the yield of alginate isolated
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from three species of brown algae was around 30%.
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3.2. Structural characterization of C. barbata sodium alginate by cicular dichroism and
276
ATR-FTIR)
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CD spectrum of CBSA presented in Fig. 2A was characterized by a peak at 196 nm and a trough at 205 nm. Fig. 2A
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The M/G ratio was determined as described by Morris et al. [14], who have
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demonstrated an empirical correlation between the ratio of peak height (p) to trough depth (t)
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with overall composition (Fig. 2B). The M/G ratio of CBSA was 0.59. Hence, sodium alginate
283
extracted from C. barbata was composed of 37% mannuronic acid and 63% guluronic acid.
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M
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p/t = (1.82927/5.89431) = 0.31 ; M/G = 0.59
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Fig. 2B
IR spectroscopy has been used for identification of the type of polysaccharide derived
287
from different seaweeds. Two bands characterized the ATR-FTIR spectrum of CBSA (Fig.
288
3A) : a broad band centred at 3253.5 cm-1 assigned to hydrogen bonded O-H stretching
289
vibrations and a weak signal at 2937.1 cm-1 attributed to C-H stretching vibrations [28].
290
Fig. 3A
291
In addition, the ATR-FTIR spectrum of CBSA showed bands at 1597.3 and 1407.2
292
cm-1, which were attributed to the asymmetric and symmetric carboxylate group stretching
293
vibrations (COO-) on the polymeric backbone of alginate. ATR-FTIR spectrum of CBSA was
294
similar to that of commercial sodium alginate (Alfa Aesar) (Fig. 3B).
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Fig. 3B
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In fact, in the alginic acid, the stretching of protonated carboxylic group (C=O) occurs
297
at 1730 cm-1. When the proton is displaced by a monovalent ion (sodium), peaks appear at
298
approximately 1600 and 1400 cm-1, respectively, and are assigned to asymmetric and
299
symmetric stretching vibration of free carboxyl group of sodium alginate [28]. The anomeric
300
region of CBSA infrared spectrum (950-750 cm-1) showed characteristic absorption bands at
301
945.9, 903.4, 854.9, 806.3 and 778.0 cm-1 assigned to vibration of uronic acid residues
302
[29,30].
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FTIR spectroscopy has proven useful for quantitative estimation of the M/G ratio of
304
alginates. Pereira et al. [28] found that the ratio of absorption band intensities at
305
approximately 1100 and 1025 cm-1, which were attributed to mannuronic and guluronic units,
306
respectively, gave a fairly good estimation of the M/G ratio. The M/G ratio of CBSA
307
polymer, inferred from the relative intensity ratio of the 1083.5 and 1024.7 cm-1 bands, was
308
0.52. This ratio is comparable to that obtained by circular dichroism (0.59). Alginates with
309
low M/G ratio (1) is related to low values of
311
guluronic acid blocks producing soft and elastic gels [3,31]. This alginate heterogeneity
312
provides the versatility for many food and non food industrial applications.
314
M
d
te
Ac ce p
313
an
303
3.3. Intrinsic viscosity and molecular weight
315
The plot of reduced viscosity (ηsp/c) versus CBSA concentration is shown in Fig. 4A.
316
The intrinsic viscosity of CBSA in 0.1 M NaCl at 25 °C was 283 ml/g, which was comparable
317
to those of Fucus vesiculosis and A. nodosum alginates (250 and 280 ml/g, respectively) [32],
318
but lower than that of Sargassum species, which were in the range of 860-1520 ml/g [5].
14
Page 14 of 38
Fig. 4A
319
The viscometric-average molecular weight was determined from the intrinsic viscosity
321
in 0.1 M NaCl using the relation : [η] = 2 × 10-3 × M0.97. The average molar mass of CBSA
322
was 204 kDa, which was higher than that of F. vesiculosus alginate (125 kDa), but lower than
323
the molar mass of L. japonica alginate (750 kDa) [32].
ip t
320
The elution profile of CBSA on size exclusion chromatography shown in Fig. 4B
325
suggested that this polymer was homogeneous. Based on calibration with standard
326
polyethyleneoxide, Mn, Mw and polydispersity index (PI) were determined as 2.03 × 105
327
g/mole, 2.99 × 105 g/mole and 1.47±0.05, respectively. The PI obtained was < 2, indicating
328
that there is not much degradation during the extraction-purification steps adopted. From the
329
data obtained, the weight-average degree of polymerization and the number-average degree of
330
polymerization were determined (DPw = 1509 and DPn = 1025).
M
an
us
cr
324
Fig.4B
te
333
3.4. Rheolgical properties
The analysis of the viscosity of CBSA showed evidence of pseudoplastic fluid
Ac ce p
332
d
331
334
behaviour, as the viscosity was influenced by shear rate (Fig. 5A). This behaviour is expected
335
for solutions of polysaccharides, resulting from their polymeric structure and high molecular
336
weight [33]. The effect of temperature on the flow behaviour of CBSA aqueous solutions was
337
investigated by measuring the apparent viscosity at different temperatures (Fig. 5A). It is
338
evident that the CBSA polymer is sensitive to the temperature, as shown by the viscosity
339
reduction observed as the temperature was increased. Nevertheless, the pseudoplastic fluid
340
behaviour was retained even for the highest temperature tested.
341
Fig. 5A
15
Page 15 of 38
The viscosity of CBSA solutions at different concentrations (0.25-2%) was also
343
investigated (Fig. 5B). As expected, the apparent viscosity increased with increasing
344
concentration. As the polymer concentration becomes higher, the individual molecules start to
345
overlap, inducing the formation of intermolecular junctions and, hence, limiting polymer
346
chain arrangement and stretching. Consequently, there is an increase of the solution’s
347
viscosity [34]. Fig. 5B
us
348
3.5. Emulsifying properties
an
349
cr
ip t
342
The use of bioemulsifiers is advantageous comparing to chemical counterparts,
351
because they are biodegradable, less toxic and have activity under a wider variety of
352
conditions [35]. Due to their wide diversity in composition and structure, bioemulsifiers are
353
characterized by improved functionality and stability, which broadens the spectrum of
354
potential applications, including detergents, paints, cosmetics, pharmaceuticals, personal care
355
products and food processing [36]. In the present study, the emulsifying capacity of CBSA
356
polymer was studied using different vegetable oils including olive oil, corn oil, sunflower oil,
357
soybean oil, ricin oil, almond oil and argan oil. As reported in Table 2, the CBSA has proven
358
to possess significant (p < 0.05) emulsifying capacity for several oils (65.8-75.8%).
d
te
Ac ce p
359
M
350
Table 2
360
The highest emulsifying activity (75.8%) was obtained with the corn oil and the formed
361
emulsions were stable that did not break within several weeks after their preparation. The
362
high emulsification indexes observed reflect the stability of the emulsions thus formed.
363 364
The emulsifying capacity of CBSA at different concentrations (0.25-3% ; w/v), using corn oil, was also studied and shown in Table 3.
16
Page 16 of 38
Table 3
366
CBSA exhibited an appreciable emulsifying capacity, which increased with increasing
367
concentration. The emulsifying activity of CBSA is noteworthy (87.9% at a concentration of
368
3%), especially in comparison with that of Arabic gum (3%), that forms emulsion with corn
369
oil with E24 of 66.7%. Increasing the concentration of CBSA, the viscosity and accordingly
370
the emulsification indexes increased. Dickinson [37] reported that hydrocolloids are
371
commonly perceived to slow down or even prevent creaming by modifying the rheology of
372
the continuous phase. From these results, the differences of the emulsification capacity
373
observed among the various hydrocolloid polymers used as emulsion stabilizers are most
374
likely attributable to their diverse chemical composition and structure.
an
us
cr
ip t
365
In many industrial processes, emulsifiers are exposed to extreme temperatures and pH.
376
Emulsion prepared with corn oil and 2% CBSA aqueous solution was subjected to different
377
temperatures (25-100 °C) (Table 4).
d
Table 4
te
378
M
375
The emulsification capacity of corn oil in the presence of CBSA was average 80% at
380
25 °C. Thermal treatment of CBSA (40-100 °C) reduced 5% of the E24 at 25 °C. The decrease
381
of the emulsifying capacity caused by heating of the emulsion formed could be related to the
382
slight reduction of viscosity during heating. The slight reduction of emulsifying capacity at
383
temperature as high as 100 °C, indicates that the CBSA-oil emulsion is thermostable.
384 385 386
Ac ce p
379
The emulsion-forming capacity of CBSA with corn oil was noticeable for the pH
range tested (3.0-11.0) with a maximum at pH 3.0 (Table 4). Table 4
387
Acidic conditions increased the emulsifying activity of CBSA. In contrast, Guttierez et
388
al. [38] reported that acidic conditions decreased the emulsifying capacity of some
389
commercial polysaccharides such as xanthan gum and Arabic gum. The increase of the
17
Page 17 of 38
emulsifying capacity of CBSA at pH 3.0 could be related to the increase of the viscosity of
391
CBSA (formation of acidic gel stabilized by hydrogen bonds) due to protonation of carboxyl
392
acid groups and decrease of water solubility. As reported by Rinaudo [1], the viscosity of
393
alginate solution was nearly constant between pH 6.0 and 8.0, but it increased below pH 4.5
394
and reached a maximum around 3.0-3.5 and then decreased. At pH around 3 (~the intrinsic
395
pK of alginic acid), alginate formed gels resulting from H-bond attraction over dominating the
396
electrostatic repulsions [1].
us
cr
ip t
390
Dickinson [37] reported that the large molecular size and predominant hydrophilicity
398
of a polysaccharide emulsifier allows for the formation of a thicker stabilizing layer that is
399
capable of protecting droplets against aggregation over a wide range of unfavourable
400
conditions, such as thermal shock treatment and acidification. In contrast, protein-stabilized
401
emulsions were found highly sensitive to unfavourable environmental conditions due the low
402
surface coverage that makes the emulsions susceptible to destabilization. As the emulsion
403
formulated from CBSA was found less sensitive to temperature changes and stable at acidic
404
pH, CBSA would be useful as a potent emulsion stabilizer in food products.
405
3.6. Antioxidant activity
M
d
te
Ac ce p
406
an
397
Due to the diversity of oxidation processes, the use of a single method to evaluate the
407
antioxidant activity cannot provide a clear idea about their real antioxidant potential.
408
Therefore, CBSA was assayed for antioxidative activity using various antioxidant assays :
409
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing power, β-
410
carotene bleaching inhibition assay, hydroxyl radical-scavenging activity and DNA nicking
411
assay.
412
3.6.1. DPPH radical-scavenging activity
18
Page 18 of 38
The free radical scavenging activity of CBSA was tested through the DPPH method
414
and the results were compared with BHA used as positive control. As shown in Fig. 6A,
415
CBSA exhibited a concentration-dependent antiradical activity. BHA showed higher degree
416
of free radical scavenging activity than did CBSA at the same concentrations tested.
ip t
413
Fig. 6A
418
At 0.5 mg/ml, CBSA exerted great free radical scavenging activity (74%), but remain lower
419
than that of BHA (100%) at the same concentration. The antioxidant activity of CBSA
420
increases its importance as a potential new source of natural additives, primarily when
421
considering the inverse relationship between dietary intake of antioxidant-rich foods and
422
incidences of human diseases [39].
423
3.6.2. Reducing power
M
an
us
cr
417
The reducing capacity of a compound may serve as a significant indicator of its
425
potential antioxidant activity. Samples with higher reducing power have better abilities to
426
donate electrons. As reported in Fig. 6B, the reducing capacity of CBSA is concentration-
427
dependent.
te
Ac ce p
428
d
424
Fig. 6B
429
At 1.2 mg/ml, CBSA exerted significant reducing activity (OD at 700 nm = 2). However,
430
CBSA showed lower reducing power than did BHA at concentrations between 0.1 and 1
431
mg/ml. The obtained results demonstrated that CBSA can act as electron donors and can react
432
with free radicals to convert them to more stable products and thereby terminate radical chain
433
reactions.
434
3.6.3. β-carotene bleaching assay
19
Page 19 of 38
In this model system, the oxidation of linoleic acid generates free radicals (lipid
436
hydroperoxides, conjugated dienes and volatile byproducts) that attack the highly unsaturated
437
β-carotene molecules. When this reaction occurs, the β-carotene molecule looses its
438
conjugation and, as a consequence, the characteristic orange colour of β-carotene disappears
439
[40]. The presence of an antioxidant avoids the destruction of the β-carotene and the orange
440
colour is maintained. The antioxidant activity of CBSA measured by the inhibition of β-
441
carotene bleaching assay is reported in Fig. 6C.
us
cr
ip t
435
Fig. 6C
443
The effect of CBSA against the discoloration of β-carotene increased with increasing sample
444
concentration. CBSA displayed moderate ability to prevent bleaching of β-carotene. At 1.5
445
mg/ml, BHA was found to possess higher antioxidant activity (95%) than that of CBSA
446
(60%). Similarly, in the β-carotene-linoleic acid assay, the antioxidant activity of sulfated
447
fucans isolated from C. barbata was 62% [9], suggesting that C. barbata polysaccharides
448
have moderate antioxidant activity in the β-carotene-linoleate model system.
449
3.6.4. Hydroxyl radical-scavenging activity
M
d
te
Ac ce p
450
an
442
Among the oxygen radicals, the hydroxyl radical (OH) is the most reactive and can
451
induce oxidative damage to almost any biomolecule. Recently, many studies reported that
452
polysaccharides could stabilize radicals by providing electrons [41]. As shown in Fig .6D, the
453
hydroxyl radical scavenging activity increased by increasing the concentration of CBSA.
454
Fig .6D
455
At 4 and 5 mg/ml, CBSA exerted potent scavenging activities (80 and 82%, respectively),
456
which were higher than those of mannitol and BHA (71 and 47%, respectively). Therefore,
20
Page 20 of 38
457
CBSA might be a good candidate as an antioxidant that can help to prevent or postpone
458
oxidative damage of biomolecules.
459
3.6.5. DNA-nicking assay Antioxidative activities of CBSA using DNA nicking assay are reported in Fig. 6E.
461
Lane 1 represents the untreated plasmid (native DNA) with its three forms : the nicked form,
462
the linear and the supercoiled forms. Incubation of plasmid DNA with Fenton's reagent in the
463
absence of CBSA resulted in the complete degradation of the three DNA bands (lane 2). At 1
464
and 2 mg/ml, CBSA exerted a moderate DNA protection against hydroxyl radicals generated
465
from Fenton’s reaction (Lanes 3 and 4). However, at a concentration of 3 mg/ml, CBSA
466
exhibited a high protection against hydroxyl radical induced DNA breakage (Lane 5).
M
an
us
cr
ip t
460
Fig. 6E
467
Qi et al [42] reported that hydroxyl radical-scavenging activity was due to two different
469
antioxidant mechanisms. One suppresses the generation of hydroxyl radicals through Fe2+-
470
chelating activity and the other scavenges the hydroxyl radicals formed. As CBSA had proven
471
important hydroxyl radical-scavenging activity and as this biopolymer did not possess any
472
chelating ability of ferrous ions (data not shown), hence, the DNA protection recorded could
473
be entirely related to the scavenging effet of hydroxyl radicals. In this way, CBSA could be
474
effective against DNA damage.
475
4. Conclusions
Ac ce p
te
d
468
476
Sodium alginate was isolated and purified from brown seaweeds (C. barbata)
477
collected from a Tunisian island (Kerkennah, Sfax). C. barbata sodium alginate (CBSA) was
478
characterized by chromatographic (HPSEC-MALLS) and spectroscopic methods (circular
21
Page 21 of 38
479
dichroism, ATR-FTIR). Its viscometric-average molar mass equals 204 kDa and the
480
composition M/G equal 0.59. CBSA had been demonstrated a potent emulsifier, having an original low sensitivity to
482
temperature. CBSA showed also interesting antioxidant properties using various antioxidant
483
assays. Taking into account these results, it can be concluded that purified C . barbata sodium
484
alginate could be used as a natural additive in food applications.
485
Acknowledgement
486
This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia.
492 493 494
te Ac ce p
491
d
488
490
cr
us
an M
487
489
ip t
481
495 496 497
22
Page 22 of 38
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499
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500
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Biochem. Biotechnol. 110 (2003) 75-90.
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polysaccharides from Microbacterium laevaniformans, Int. J. Biol. Macromol. 42 (2008), 10-
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surfactants, Appl. Microbiol. Biotechnol. 53 (2000) 495-508.
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[37] E. Dickinson, Hydrocolloids as emulsifiers and emulsion stabilizers, Food Hydrocoll. 23
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[38] T. Gutierrez, T. Shimmield, C. Haidon, K. Black, D.H. Green, Emulsifying and metal ion
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binding activity of a glycoprotein exopolymer produced by Pseudoalteromonas sp. Strain
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TG12, Appl. Environ. Microbiol. 74 (2008) 4867-4876.
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[39] Y. Lu, Y. Foo, Antioxidant radical scavenging activies of polyphenols from apple
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pomaceae, Food Chem. 68 (2000) 81-85.
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Page 26 of 38
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antioxidative activity of oregano essential oil, Food Chem. 85 (2004) 633-640.
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[41] W. Zhang, J. Wang, W. Jin, Q. Zhang, The antioxidant activities and neuroprotective
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effect of polysaccharides from the starfish Asterias rollestoni, Carbohydr Polym. 95 (2013) 9-
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15.
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[42] H.M. Qi, Q.B. Zhang, T.T. Zhao, R.G. Hu, K. Zhang, Z.E. Li, In vitro antioxidant
598
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pertusa (Chlorophyta), Bioorg. Med. Chem. Lett. 16 (2006) 2441-2445.
us
cr
ip t
592
an
600 601
Ac ce p
te
d
M
602
27
Page 27 of 38
602
Table 1. Yield and chemical analysis of C. barbata sodium alginate
603
Values (%, w/w)
604
Yielda
9.9±0.8
605
Moistureb
8.6±0.2
606
Neutral sugarc
9.3±0.6
Uronic acidc
58.1±1.3
Ashc
23.9±1.9
607
cr
608
ip t
Analytical data
us
609 610
All experiments were carried out in triplicate and expressed as mean values.
612
a
The yield was expressed on dry seaweed weight.
613
b
The moisture was expressed on purified sodium alginate weight.
614 615
c
M
an
611
Neutral sugars, uronic acid and ash contents were expressed on purified sodium alginate dry weight.
d
616
te
617
Table 2. Emulsifying capacity (E24) of C. barbata sodium alginate (1%) using different vegetable oils
Ac ce p
618
Oils
Olive oil
Sunflower oil
Corn oil
E24a
69.2±1.2b
69.2±1.2b
75.8±1.2a
Soybean oil 69.2±1.2b
Ricin oil 65.8±1.2c
619
a
620
Values are given as mean ± SD from triplicate determinations (n = 3).
621
Almond oil
Argan oil
69.2±1.2b
69.2±1.2b
Results are expressed as percentages of the total height occupied at 25°C.
Concentration (%, w/v)
0.25
0.5
1
2
3
CBSA
65.8±1.2e
70.8±1.2d
75.8±1.2c
80.8±1.2b
87.9±0.6a
Arabic gum
0±0.0b
0±0.0b
0±0.0b
66.7±0.0a
66.7±0.0a
Different letters indicate significant differences among the different oils (p < 0.05).
622
28
Page 28 of 38
623 624
Table 3. Emulsifying capacity (E24) of C. barbata sodium alginate at different concentrations
625 626
a
627
Values are given as mean ± SD from triplicate determinations (n = 3).
628 629
Different letters indicate significant differences among the different concentrations within the same line (p < 0.05).
cr
630
us
631 632
Table 4. Effect of pH (3.0-11.0) and heat (25-100°C) treatments on the emulsifying capacity (E24) of C. barbata sodium alginate (2%)
an
633 634
ip t
Emulsions were prepared with corn oil at 25°C.
Ac ce p
Temperature (°C)
te
d
Treatment
M
635
E24* (%)
25
80.8±1.2a
40
75.8±1.2b
60
75.8±1.2b
80
75.8±1.2b
100
75.8±1.2b
3
100±0.0a
5
80.8±1.2b
7
80.8±1.2b
9
75.8±1.2c
11
75.8±1.2c
pH
636
*
637
Values are given as mean ± SD from triplicate determinations (n = 3).
638 639
Different letters indicate significant differences among the different temperatures and different pH (p < 0.05).
Emulsions prepared with corn oil and CBSA (2%) were subjected to different temperatures and pH.
29
Page 29 of 38
643
Figure captions
644
Fig. 1. Molecular structure of sodium alginate.
646
Fig. 2. (A) Circular dichroism spectrum of C. barbata sodium alginate (800 ppm in distilled
647
water) at 25°C. (B) Determination of alginate composition from circular dichroism spectrum
648
according to Morris et al. (1980).
649 650
Fig. 3. ATR-FTIR spectra of (A) C. barbata sodium alginate and (B) commercial sodium alginate (Alfa Aesar).
651
Fig. 4. (A) Intrinsic viscosity of C. barbata sodium alginate at 25°C in 0.1 M NaCl solution.
652
(B) Determination of molecular weight of C. barbata sodium alginates by HPSEC-MALLS
653
eluted with 0.1 M NaCl at 25°C. Red signal is the light scattering intensity at the angle 90°
654
and the blue signal is the refractive index (related to polymer concentration).
655 656 657 658 659 660 661 662 663 664 665 666 667 668
Fig. 5. (A) Apparent viscosity of 2% CBSA aqueous solutions (in 0.1 M NaCl) during heating (25°C- 60°C) with increasing the shear rate from 1 s-1 to 50 s-1. (B) Apparent viscosity of CBSA aqueous solutions (in 0.1 M NaCl) as a function of polymer concentration at a shear rate of 5 s-1. The experiments were carried out at 25°C.
te
d
M
an
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cr
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645
Ac ce p
Fig. 6. (A) DPPH radical-scavenging activity of CBSA. (B) Fe2+-reducing power of CBSA. (C) β-carotene-bleaching inhibition activity of CBSA. (D) Hydroxyl radical-scavenging activity of CBSA at different concentrations. Values are means ± S.D. (n=3). (E) Protective effect of CBSA at different concentrations on hydroxyl radical induced DNA damage. Lane 1: untreated control: native pGapZαA plasmid DNA, Lane 2: plasmid DNA + Fenton’s reagent, Lane 3: plasmid DNA + Fenton’s reagent + 1 mg/ml of CBSA, Lane 4: plasmid DNA + Fenton’s reagent + 2 mg/ml of CBSA, Lane 5: plasmid DNA + Fenton’s reagent + 3 mg/ml of CBSA.
669 670 671 672 673
31
Page 30 of 38
674 675 676
Fig.1
us
cr
ip t
677
678
an
679 680
M
681
685 686 687 688 689 690 691
te
684
Ac ce p
683
d
682
692 693 694 695 696
32
Page 31 of 38
ip t
Fig. 2 (A)
an
us
cr
t
te
(B)
d
M
p
Ac ce p
697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740
33
Page 32 of 38
741
Fig. 3 (A)
742 743 744
cr us an M d
(B)
te
749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780
(a) (a)
Ac ce p
746 747 748
ip t
745
34
Page 33 of 38
Fig. 4 (A)
784 785 786
(B)
Ac ce p
te
d
M
an
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cr
ip t
781 782 783
788 790 792 794 796 798 800 802 804 806 808 810 812 814 816 818 820 822 824 826 828
829 830 831 832
35
Page 34 of 38
Fig. 5 (B)
te
d
M
an
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cr
ip t
(A)
Ac ce p
833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876
36
Page 35 of 38
M
an
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cr
ip t
Fig. 6 (A)
te
d
(B)
Ac ce p
877 878 880 882 884 886 888 890 892 894 896 898 900 902 904 906 907 908 909 910 911 913 915 917 919 921 923 925 927 929 931 933 935 937 939 941 942 943 944 945 946 947 948 949
37
Page 36 of 38
1018
an
us
cr
ip t
(C)
te
d
M
(D)
Ac ce p
950 951 953 955 957 959 961 963 965 967 969 971 973 975 977 979 980 981 982 984 986 988 990 992 994 996 998 1000 1002 1004 1006 1008 1009 1010 1011 1012 1013 1014 1016
(E)
1
2
3
4
5
Nicked circular form Linear form Circular supercoiled form
1020 1022
38
Page 37 of 38
1024 1025 1026 1027
Highlights Sodium alginate was isolated from the brown seaweed Cystoseira barbata harvested in Tunisia ; C. barbata sodium alginate (CBSA) was analysed by circular dichroism (CD) and
ip t
1023
ATR-FTIR spectroscopies ;
The emulsifying capacity of CBSA was studied at different concentrations (0.25-3%),
1029
temperatures (25-100 °C) and pH (3.0-11.0) and the emulsion formulated was found to
1030
be less sensitive to temperature changes and more stable at acidic pH ;
us
Isolated sodium alginate exhibited important antioxidant activity.
an
1031
cr
1028
1032
Ac ce p
te
d
M
1033
39
Page 38 of 38
S0141-8130(14)00692-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.10.016 BIOMAC 4664
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
11-8-2014 7-10-2014 8-10-2014
Please cite this article as: S. Sellimi, I. Younes, H.B. Ayed, H. Maalej, V. Montero, M. Rinaudo, M. Dahia, T. Mechichi, M. Hajji, M. Nasri, Structural, physicochemical and antioxidant properties of sodium alginate isolated from a Tunisian brown seaweed, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.10.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Structural, physicochemical and antioxidant properties of sodium alginate isolated from
2
a Tunisian brown seaweed
3
Sabrine Sellimi1*, Islem Younes1, Hanen Ben Ayed1, Hana Maalej1, Veronique Montero2,
4
Marguerite Rinaudo3, Mostefa Dahia4, Tahar Mechichi1, Mohamed Hajji1 and Moncef Nasri1
ip t
1
cr
5
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6 7 8
1
9
Nationale d’Ingénieurs de Sfax, B.P. 1173-3038 Sfax, Tunisie.
an
: Laboratoire de Génie Enzymatique et de Microbiologie, Université de Sfax, Ecole
2
11
Montpellier II, ENSCM, 8, rue de l’Ecole-Normale, 34296 Montpellier cedex, France.
12
3
: Biomaterials Applications, 6, rue Lesdiguières 38000 Grenoble, France
13
4
: Département de Biologie, Faculté des sciences de la nature et de la vie, Université de
14
Djelfa, Algérie.
M
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Ac ce p
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: Laboratoire de Glycochimie et Reconnaissance Moléculaire, UMR 5032, Université
15 16 17 18 19 20
*
Corresponding author. E-mail address: [email protected]. Tel. : +216 20 096 945
1
Page 1 of 38
21
Abstract An original sodium alginate from Tunisian seaweed (Cystoseira barbata) was purified
23
and characterized by circular dichroism (CD) and ATR-FTIR spectroscopies. ATR-FTIR
24
spectrum of C. barbata sodium alginate (CBSA) showed the characteristic bands of
25
mannuronic (M) and guluronic acids (G). The M/G ratio was estimated by CD (M/G=0.59)
26
indicating that CBSA was composed of 37% mannuronic acid and 63% guluronic acid. The
27
analysis of viscosity of CBSA showed evidence of pseudoplastic fluid behaviour. The
28
emulsifying capacity of CBSA was evaluated at different concentrations (0.25-3%),
29
temperatures (25-100 °C) and pH (3.0-11.0). Compared to most commercial emulsifiers, the
30
emulsion formulated by CBSA was found to be less sensitive to temperature changes and
31
more stable at acidic pH. CBSA was examined for antioxidant properties using various
32
antioxidant assays. CBSA exhibited important DPPH radical-scavenging activity (74%
33
inhibition at a concentration of 0.5 mg/ml) and considerable ferric reducing potential.
34
Effective hydroxyl-radical scavenging activity (82% at a concentration of 5 mg/ml) and
35
potent protection activity against DNA breakage were also recorded for CBSA. However, in
36
the linoleate-β-carotene system, CBSA exerted moderate antioxidant activity (60% at a
37
concentration of 1.5 mg/ml). Therefore, CBSA can be used as a natural ingredient in food
38
industry or in the pharmaceutical field.
cr
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Ac ce p
39
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22
40
Keywords : Cystoseira barbata ; sodium alginate ; cicular dichroism ; ATR-FTIR ; viscosity ;
41
emulsifying capacity ; antioxidant activity
42 43
2
Page 2 of 38
44
1. Introduction Phycocolloids are polysaccharides associated with the cell wall and intercellular
46
spaces of some seaweed species [1]. The major structural polysaccharide of brown seaweeds
47
is alginate, which usually exists in the cell wall and in the matrix as a mixture of all the
48
cationic salt forms found in seawater. In its native state, alginate exists as calcium,
49
magnesium and sodium salts of alginic acid, providing both strength and flexibility to the
50
algal tissue [2]. The most important algal sources of alginate are Macrocystis pyrifera,
51
Ascophyllum nodosum, Laminaria spp, Ecklonia maxima, Eisenia bicyclis, Lessonia
52
nigrecans and Sargassum spp [3]. Alginate has been widely used in many fields such as cell
53
immobilization, tissue engineering, microencapsulation of nutraceuticals and drugs, as well as
54
in food applications as thickening, stabilizing and gelling agents and as alginate-based edible
55
films and coatings for food products [3,4].
M
an
us
cr
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45
Alginate is a linear anionic copolymer of β-D-mannuronic acid (M) and α-L-guluronic
57
acid (G) (1-4)-linked residues, arranged either in heteropolymeric (MG) and/or
58
homopolymeric (M or G) blocks (Fig. 1) [4,5].
te
Ac ce p
59
d
56
Fig. 1
60
The mannuronic acid forms β (1-4) linkages, then, M-block segments show linear and flexible
61
conformation. The guluronic acid, differently, gives rise to α (1-4) linkages, introducing a
62
steric hindrance around the carboxyl groups, then, the G-block segments provides folded and
63
rigid structural conformations, responsible of a pronounced stiffness of the molecular chains
64
[4]. In the presence of divalent cations such as calcium, a strong interaction with the COO-
65
groups of guluronic acid from different chains is formed, giving rise to a water insoluble and
66
thermo-irreversible three-dimensional network (gel), whose conformation is often
67
named « egg box » [6].
3
Page 3 of 38
Alginates are typically described by their M/G ratio and average molecular weight,
69
since these parameters are closely related to the functionality of the alginates [1]. Generally,
70
the extraction and purification processes of alginates are based on the conversion from the
71
insoluble form in the seaweed cell walls to the soluble one, normally the sodium salt,
72
followed by successive dissolutions and precipitations to eliminate impurities [5,7,8].
ip t
68
Cystoseira barbata is a brown seaweed abundant along the Tunisian seacoast used in a
74
few number of studies. Recently, structural features and antioxidant activity of fucans
75
(sulfated polysaccharides) isolated from C. barbata have been reported by Sellimi et al. [9].
76
However, alginate extracted from the C. barbata seaweeds was not yet studied. Then, the
77
aims of the present study were to extract and purify sodium alginate from C. barbata. The
78
extracted sodium alginate (CBSA) was characterized by circular dichroism, ATR-FTIR and
79
size exclusion chromatography. Finally, viscosity, emulsifying capacity and antioxidant
80
properties of CBSA were evaluated.
81
2. Materials and methods
82
2.1. Reagents
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an
M
d te
Ac ce p
83
cr
73
1,1-Diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxyanisole (BHA), linoleic
84
acid, β-carotene, polyethylene oxide, ferrous sulfate heptahydrate (FeSO4. 7H2O),
85
galacturonic acid and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma
86
Chemical Co. (St. Louis, MO, USA). Arabic gum and alginate under sodium salt form were
87
purchased, respectively, from Merck (Germany) and Alfa Aesar (Tianjin, China). All other
88
chemicals, namely sodium carbonate (Na2CO3), potassium ferricyanide, sodium chloride,
89
trichloroacetic acid (TCA), ferric chloride (FeCl3), D-deoxyribose, hydrogen peroxide (H2O2),
90
thiobarbituric acid (TBA), L-mannitol, L-ascorbic acid, 3,5 dimethylphenol, anthrone reagent,
91
Tween 80 and other solvents, were of analytical grade. The vegetable oils used (olive oil,
4
Page 4 of 38
sunflower oil, corn oil, soybean oil, ricin oil, almond oil and argan oil) were purchased from
93
the local supermarket.
94
2.2. Biological material
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92
A brown seaweed C. barbata collected from Kerkennah island (Sfax, Tunisia), in
96
November 2012, was studied. The freshly collected seaweed fronds were washed thoroughly
97
with tap water to remove all sand particles and epiphytes. Then, they were dried at room
98
temperature for about 20 days away from sunlight or heat such as to preserve as much as
99
possible the quality of the initial material including alginates. The dried samples were ground
100
using a mixer grinder (Moulinex) and were sieved in a 0.2 mm mesh size. The powder was
101
kept in a clean, dried, and well-sealed amber glass container to protect it from sunlight.
102
2.3. Preliminary treatments
M
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cr
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The C. barbata powder (50 g) was depigmented and deffated sequentially with
104
acetone-methanol (7:3, 500 ml) (twice) and chloroform (300 ml) (twice) for 24 h at 30 °C
105
under constant stirring (250 rpm) [9]. The algal material was air-dried to yield depigmented
106
and deffated algal powder (45 g).
107
2.4. Extraction and purification of sodium alginate
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Ac ce p
108
d
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The extraction of alginate from C. barbata seaweed was carried out using high
109
temperature alkaline extraction according to Davis et al. [10] with slight modifications.
110
Depigmented, defatted and dried seaweed powder (25 g) was treated twice at pH around 2.0
111
with 500 ml of 0.1 M HCl for 2 h at 60 °C under constant stirring (250 rpm) to ensure the
112
powder demineralization. The supernatant was eliminated by centrifugation (5000 rpm, 15
113
min, 4 °C). The residue was washed with distilled water and then treated with 3% Na2CO3
114
(pH = 11.0) at 60 °C for 2 h under constant stirring to solubilize the alginate in sodium salt
5
Page 5 of 38
form. Then, the supernatants were collected and precipitated with absolute ethanol (2v). This
116
precipitate, recovered by centrifugation, is suspended in distilled water and acidified with 6 N
117
HCl to pH < 3.0 to precipitate alginic acid. The precipitate formed was resuspended in
118
distilled water and neutralized by an aqueous solution of 1 M NaOH to pH 7.5. At end, the
119
sodium alginate was purified by a second precipitation with absolute ethanol. The precipitate
120
obtained was resuspended in distilled water and freeze dried to yield C. barbata sodium
121
alginate powder (CBSA).
122
2.5. Chemical analysis
cr us
Moisture and ash contents were determined according to the AOAC methods (927.05
an
123
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115
and 942.05, respectively) [11].
Uronic acids (UA) were determined following the
125
colorimetric method using galacturonic acid as standard and 3,5 dimethylphenol as a reagent
126
[12] and neutral sugars (NS) were measured by anthrone colorimetric method [13].
127
2.6. Circular dichroism and ATR-FTIR spectroscopies
te
d
M
124
Circular dichroism (CD) spectrum was recorded on Jasco model J-815 CD
129
Spectrometer, using measurement range at 190-250 nm and sample concentration of 0.8
130
mg/ml (800 ppm in distilled water). The CD spectrum is often reported in degrees of
131
ellipticity (θ) expressed as millidegrees (mdeg). The ratio of peak height to trough depth was
132
calculated using the equation described by Morris et al. [14].
133
Ac ce p
128
Peak/trough ratio = (θ trough - θ peak)/ θ trough
134
Infrared (IR) spectra of sodium alginates were determined on a Fourier transform (FT)
135
spectrophotometer (Perkin Elmer®, Spectrum™ 100, Singapore) equipped with attenuated
136
total reflection (ATR) accessory containing a diamond/ZnSe crystal. An extra accessory plate
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Page 6 of 38
for powdered samples with a conic awl was used without need for previous sample
138
preparation. ATR-FTIR spectra were obtained in the 4000-600 cm-1 range at room
139
temperature, using 10 scans and 4 cm-1 resolution.
140
2.7. Capillary viscometry
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137
The dynamic viscosity measurements were carried out on a capillary viscosimeter
142
(Ubbelohde) immersed in a thermostated bath with a precision of ± 0.1 °C. Solutions at
143
different concentrations were prepared in 0.1 M NaCl. The measurements were carried out at
144
25 °C according to the method of Haug and Larsen [15]. The intrinsic viscosity [η] was
145
determined using Huggins equation (1) by extrapolating ηsp/c against concentration curve to
146
zero, and averaging the value of the intercept.
an
us
cr
141
η sp/c = [η] + k1 × [η]² × c (1)
148
ηsp = (η – η0)/ η0
149
where ηsp, k1 and c were the specific visocsity, the Huggins constant and the concentration of
150
the alginate solution expressed as g/ml. η and η0 were the absolute viscosities of the alginate
151
solution and that of the solvent (0.1 M NaCl), respectively. The viscometric-average
152
molecular weight (M) (Da) was determined using the corresponding Mark-Houwink equation
153
(2) with K = 2 × 10-3 (ml/g) and a = 0.97 in 0.1 M NaCl solvent at 25 °C [16].
155
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154
M
147
[η] = K × Ma (2)
2.8. Molecular weight distribution
156
The weight-average molecular mass (Mw), the number-average molecular mass (Mn)
157
and polydispersity (Mw/Mn) of 0.3% of CBSA, dissolved in HPLC grade water, were
158
determined after elution with 0.1 M NaCl at 25°C in a High Performance Size Exclusion
159
Chromatography (HPSEC) Waters Alliance GPCV2000 (USA) equipped with a Multi-Angle
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Page 7 of 38
Laser Light Scattering (MALLS) detector from Wyatt (USA). The weight-average degree of
161
polymerization DPw (DPw = Mw/m0 ; m0 = 198 represents the repeat unit in the sodium salt
162
form expressed as g/mole) and the number-average degree of polymerization DPn (DPn =
163
Mn/m0) were obtained [17].
ip t
160
Prior to measurements, the apparatus was calibrated using HPLC grade toluene and
165
normalized using polyethylene oxide (72 kDa) in 0.1 M NaCl. Before injection, the sample
166
was filtered on a 0.45 µm pore membrane to eliminate dust particules. The concentration
167
injected was 3 mg/ml, with an injection volume of 100 µl using a column TSK-G2000 SWXL
168
(7.8 mm × 300 mm). The eluent used was 0.1 M NaCl at 25 °C elution temperature and a flow
169
rate of 0.5 ml/min. The specific refractive index increment (dn/dc) adopted is equal to 0.155
170
[18]. Data were collected from the refractive index detector (DRI) and MALLS and evaluated
171
with the ASTRA software version 4.72.03.
172
2.9. Antioxidant activity
173
2.9.1. DPPH assay
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M
d te
Ac ce p
174
cr
164
The DPPH radical-scavenging activity of CBSA was determined by the method of
175
Kirby and Schmidt [19], with some modifications. Briefly, a volume of 500 µl of CBSA at
176
different concentrations (0.01-1 mg/ml) was added to 375 µl of 99% ethanol and 125 µl of
177
DPPH solution (0.02% (w/v) in ethanol). The mixture was incubated for 60 min in the dark at
178
room temperature. Scavenging capacity was measured spectrophotometrically (T70 UV-
179
visible spectrometer PG Instruments Ltd, Japan) by monitoring the decrease of absorbance at
180
517 nm. In its radical form, DPPH has an absorption band at 517 nm which disappears upon
181
reduction by an antiradical compound. Lower absorbance of the reaction mixture indicated
182
higher DPPH free radical-scavenging activity (expressed as percentage). BHA was used as
183
positive standard. DPPH radical-scavenging activity was calculated as follows:
8
Page 8 of 38
184 185
DPPH radical-scavenging activity (%) =
A control + A blank - A sample
× 100
A control
186
where A
187
sample), A
188
solution) and A sample is the absorbance of the CBSA with the DPPH solution. The experiment
189
was carried out in triplicate and the results are mean values.
190
2.9.2. Ferric-reducing activity
is the absorbance of the control reaction (containing all reagents except the
ip t
is the absorbance of the CBSA (containing all reagents except the DPPH
cr
blank
us
control
The reducing power of CBSA was determined by the method of Yildirim et al. [20].
192
A 0.5 ml of CBSA solution at different concentrations (0.1-1.2 mg/ml) was mixed with 1.25
193
ml of 0.2 M phosphate buffer (pH 6.6) and 1.25 ml of 1% (w/v) potassium ferricyanide. The
194
mixture was incubated for 30 min at 50 °C. After incubation, 1.25 ml of trichloroacetic acid
195
(10%, w/v) was added and the reaction mixture was centrifuged for 10 min at 3000 g. A 1.25
196
ml aliquot of the supernatant from each sample mixture was mixed with 1.25 ml of distilled
197
water and 0.25 ml of 0.1% (w/v) ferric chloride solution in a test tube. After 10 min, the
198
absorbances of the resulting solutions were measured at 700 nm. BHA was used as standard.
199
Values presented are the mean of triplicate analyses.
201
M
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Ac ce p
200
an
191
2.9.3. ß-carotene-linoleic acid assay The ability of CBSA to prevent bleaching of ß-carotene was assessed as described by
202
Koleva et al. [21]. A stock solution of β-carotene/linoleic acid mixture was prepared by
203
dissolving 0.5 mg of β-carotene, 25 µl of linoleic acid and 200 µl of Tween 80 in 1 ml of
204
chloroform. The chloroform was completely evaporated under vacuum in a rotary evaporator
205
at 40 °C, then, 100 ml of distilled water were added and the resulting mixture was vigorously
206
stirred. The emulsion obtained was freshly prepared before each experiment. Aliquots (2.5
9
Page 9 of 38
ml) of the β-carotene/linoleic acid emulsion were transferred to test tubes containing 0.2 ml of
208
CBSA solution at different concentrations (0.05-1.5 mg/ml). Following incubation for 2 h at
209
50 °C, the absorbance of each sample was measured at 470 nm. BHA was used as positive
210
standard. The control tube contained no sample. Antioxidant activity in β-carotene bleaching
211
model expressed as percentage was calculated with the following equation:
ip t
207
ß-carotene-bleaching inhibition (%) = [1 – ((A0 – At) / (A’0 – A’t))] × 100
213
where A0 and A’0 are the absorbances of the sample and the control, respectively, measured at
214
time zero, and At and A’t are the absorbances of the sample and the control, respectively,
215
measured after 2 h. Tests were carried out in triplicate.
216
2.9.4. Hydroxyl radical-scavenging activity
M
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cr
212
Hydroxyl radical-scavenging activity of CBSA was determined as described by
218
Chung et al. [22] with slight modifications. Briefly, 0.2 ml of CBSA solution at different
219
concentrations (0.25-5 mg/ml) was added to the reaction mixture containing 0.2 ml FeSO4 .7
220
H2O (10 mM), 0.2 ml EDTA (10 mM) and 0.2 ml D-deoxyribose (10 mM). The volume was
221
made up to 2 ml with phosphate buffer (0.1 M, pH 7.4) and to that, 0.2 ml H2O2 (10 mM) was
222
added. The mixture was incubated at 37 °C in the dark for 1 h. After incubation, 1 ml of TCA
223
(2.8%) and TBA (1%) were added to the mixture and then, were left to stand in a boiling
224
water bath for 10 min. The absorbance was measured at 532 nm. If the mixture was turbid, the
225
absorbance was measured after centrifugation. Scavenging activity (%) was calculated using
226
the equation :
227
Ac ce p
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d
217
Hydroxyl radical-scavenging activity (%) = ((Ablank-Asample)/Ablank) × 100
10
Page 10 of 38
where Ablank is the absorbance of the blank (containing all reagents except the sample) and
229
Asample is the absorbance of the sample. The results were compared with L-mannitol and BHA
230
as positive controls.
231
2.9.5. DNA-nicking assay
ip t
228
DNA nicking assay was performed using pGapZαA plasmid (Invitrogen) by the
233
method of Lee et al. [23], with slight modifications. A mixture of 10 μl of CBSA at the
234
concentrations of 1, 2 and 3 mg/ml and plasmid DNA (0.5 μg/well) were incubated for 10 min
235
at room temperature followed by the addition of 10 μl of Fenton's reagent (30 mM H2O2, 50
236
μM L-ascorbic acid and 80 μM FeCl3). The mixture was then incubated for 30 min at 37 °C.
237
The DNA was analysed on 1% (w/v) agarose gel.
238
2.10. Emulsifying activity and emulsion stability
M
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232
The emulsifying activity was assessed as described by Cooper and Goldenberg [24],
240
with slight modifications. Briefly, 3 ml of vegetable oil were added to 3 ml of CBSA aqueous
241
solution in a test tube (18 mm × 120 mm) and stirred in the vortex at 2400 rpm for 2 min.
242
After 24 h, the emulsification index (E24) was determined as follows:
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Ac ce p
243
d
239
E24=he/hT×100
244
where he (mm) is the height of the emulsion layer and hT (mm) is the overall height of the
245
mixture. Several vegetable oils including olive oil, corn oil, sunflower oil, soybean oil, ricin
246
oil, almond oil and argan oil were used to study the emulsifying activity of CBSA. All
247
experiments were carried out in triplicate. All tests were performed at room temperature (25
248
°C±1 °C) and at pH 5.0.
249
The emulsifying capacity and the emulsion stability were also evaluated at different
250
concentrations of CBSA (0.25-3%), temperatures (25-100 °C) and pH (3.0-11.0). For that
11
Page 11 of 38
purpose, emulsion (CBSA-corn oil) was pretreated for 30 min at each temperature and each
252
pH before stabilization during 24 h at normalized conditions (25 °C) in order to determine the
253
emulsification index (E24).
254
2.11. Statistical analysis
ip t
251
Statistical analyses were performed with SPSS ver.17.0, professional edition using
256
ANOVA analysis. Differences were considered significant at p-value < 0.05. All tests were
257
carried out in triplicate.
258
3. Results and discussion
259
3.1. Extraction yield and chemical analysis of C. barbata sodium alginate
M
an
us
cr
255
Taking into account the results obtained in the literature, we have adopted to extract
261
sodium alginate from C. barbata at 60 °C for 2 h as described in section 2.4. The extraction
262
yield and the chemical characteristics (sugar content, moisture and ash) of CBSA are shown
263
in Table 1. The extraction yield of CBSA was 9.9% based on dry seaweed weight. The CBSA
264
extraction yield was higher than that registered for Dictyota caribaea and Padina
265
perindusiata (7.4 and 5.4%, respectively) [25].
267
te
Ac ce p
266
d
260
Table 1
Considering variations being dependent on the alginate extraction method used, Davis
268
et al. [10] found values in the range of 21.1-24.5% for Sargassum fluitans and 16.3-20.5% for
269
S. oligocystum. In another study, Davis et al. [26] obtained higher yields for the same species,
270
of 45% and 37%, respectively. The extraction yields depend crucially on the algal species and
271
the extraction method. Rahelivao et al. [27] reported that, in the absence of EDTA, the
272
alginate extraction yield was lower (10-13%) in relation with the presence of calcium
12
Page 12 of 38
complexed in the cell wall. However, in the presence of EDTA, the yield of alginate isolated
274
from three species of brown algae was around 30%.
275
3.2. Structural characterization of C. barbata sodium alginate by cicular dichroism and
276
ATR-FTIR)
cr
278
CD spectrum of CBSA presented in Fig. 2A was characterized by a peak at 196 nm and a trough at 205 nm. Fig. 2A
279
us
277
ip t
273
The M/G ratio was determined as described by Morris et al. [14], who have
281
demonstrated an empirical correlation between the ratio of peak height (p) to trough depth (t)
282
with overall composition (Fig. 2B). The M/G ratio of CBSA was 0.59. Hence, sodium alginate
283
extracted from C. barbata was composed of 37% mannuronic acid and 63% guluronic acid.
286
M
d
te
285
p/t = (1.82927/5.89431) = 0.31 ; M/G = 0.59
Ac ce p
284
an
280
Fig. 2B
IR spectroscopy has been used for identification of the type of polysaccharide derived
287
from different seaweeds. Two bands characterized the ATR-FTIR spectrum of CBSA (Fig.
288
3A) : a broad band centred at 3253.5 cm-1 assigned to hydrogen bonded O-H stretching
289
vibrations and a weak signal at 2937.1 cm-1 attributed to C-H stretching vibrations [28].
290
Fig. 3A
291
In addition, the ATR-FTIR spectrum of CBSA showed bands at 1597.3 and 1407.2
292
cm-1, which were attributed to the asymmetric and symmetric carboxylate group stretching
293
vibrations (COO-) on the polymeric backbone of alginate. ATR-FTIR spectrum of CBSA was
294
similar to that of commercial sodium alginate (Alfa Aesar) (Fig. 3B).
13
Page 13 of 38
Fig. 3B
295
In fact, in the alginic acid, the stretching of protonated carboxylic group (C=O) occurs
297
at 1730 cm-1. When the proton is displaced by a monovalent ion (sodium), peaks appear at
298
approximately 1600 and 1400 cm-1, respectively, and are assigned to asymmetric and
299
symmetric stretching vibration of free carboxyl group of sodium alginate [28]. The anomeric
300
region of CBSA infrared spectrum (950-750 cm-1) showed characteristic absorption bands at
301
945.9, 903.4, 854.9, 806.3 and 778.0 cm-1 assigned to vibration of uronic acid residues
302
[29,30].
us
cr
ip t
296
FTIR spectroscopy has proven useful for quantitative estimation of the M/G ratio of
304
alginates. Pereira et al. [28] found that the ratio of absorption band intensities at
305
approximately 1100 and 1025 cm-1, which were attributed to mannuronic and guluronic units,
306
respectively, gave a fairly good estimation of the M/G ratio. The M/G ratio of CBSA
307
polymer, inferred from the relative intensity ratio of the 1083.5 and 1024.7 cm-1 bands, was
308
0.52. This ratio is comparable to that obtained by circular dichroism (0.59). Alginates with
309
low M/G ratio (1) is related to low values of
311
guluronic acid blocks producing soft and elastic gels [3,31]. This alginate heterogeneity
312
provides the versatility for many food and non food industrial applications.
314
M
d
te
Ac ce p
313
an
303
3.3. Intrinsic viscosity and molecular weight
315
The plot of reduced viscosity (ηsp/c) versus CBSA concentration is shown in Fig. 4A.
316
The intrinsic viscosity of CBSA in 0.1 M NaCl at 25 °C was 283 ml/g, which was comparable
317
to those of Fucus vesiculosis and A. nodosum alginates (250 and 280 ml/g, respectively) [32],
318
but lower than that of Sargassum species, which were in the range of 860-1520 ml/g [5].
14
Page 14 of 38
Fig. 4A
319
The viscometric-average molecular weight was determined from the intrinsic viscosity
321
in 0.1 M NaCl using the relation : [η] = 2 × 10-3 × M0.97. The average molar mass of CBSA
322
was 204 kDa, which was higher than that of F. vesiculosus alginate (125 kDa), but lower than
323
the molar mass of L. japonica alginate (750 kDa) [32].
ip t
320
The elution profile of CBSA on size exclusion chromatography shown in Fig. 4B
325
suggested that this polymer was homogeneous. Based on calibration with standard
326
polyethyleneoxide, Mn, Mw and polydispersity index (PI) were determined as 2.03 × 105
327
g/mole, 2.99 × 105 g/mole and 1.47±0.05, respectively. The PI obtained was < 2, indicating
328
that there is not much degradation during the extraction-purification steps adopted. From the
329
data obtained, the weight-average degree of polymerization and the number-average degree of
330
polymerization were determined (DPw = 1509 and DPn = 1025).
M
an
us
cr
324
Fig.4B
te
333
3.4. Rheolgical properties
The analysis of the viscosity of CBSA showed evidence of pseudoplastic fluid
Ac ce p
332
d
331
334
behaviour, as the viscosity was influenced by shear rate (Fig. 5A). This behaviour is expected
335
for solutions of polysaccharides, resulting from their polymeric structure and high molecular
336
weight [33]. The effect of temperature on the flow behaviour of CBSA aqueous solutions was
337
investigated by measuring the apparent viscosity at different temperatures (Fig. 5A). It is
338
evident that the CBSA polymer is sensitive to the temperature, as shown by the viscosity
339
reduction observed as the temperature was increased. Nevertheless, the pseudoplastic fluid
340
behaviour was retained even for the highest temperature tested.
341
Fig. 5A
15
Page 15 of 38
The viscosity of CBSA solutions at different concentrations (0.25-2%) was also
343
investigated (Fig. 5B). As expected, the apparent viscosity increased with increasing
344
concentration. As the polymer concentration becomes higher, the individual molecules start to
345
overlap, inducing the formation of intermolecular junctions and, hence, limiting polymer
346
chain arrangement and stretching. Consequently, there is an increase of the solution’s
347
viscosity [34]. Fig. 5B
us
348
3.5. Emulsifying properties
an
349
cr
ip t
342
The use of bioemulsifiers is advantageous comparing to chemical counterparts,
351
because they are biodegradable, less toxic and have activity under a wider variety of
352
conditions [35]. Due to their wide diversity in composition and structure, bioemulsifiers are
353
characterized by improved functionality and stability, which broadens the spectrum of
354
potential applications, including detergents, paints, cosmetics, pharmaceuticals, personal care
355
products and food processing [36]. In the present study, the emulsifying capacity of CBSA
356
polymer was studied using different vegetable oils including olive oil, corn oil, sunflower oil,
357
soybean oil, ricin oil, almond oil and argan oil. As reported in Table 2, the CBSA has proven
358
to possess significant (p < 0.05) emulsifying capacity for several oils (65.8-75.8%).
d
te
Ac ce p
359
M
350
Table 2
360
The highest emulsifying activity (75.8%) was obtained with the corn oil and the formed
361
emulsions were stable that did not break within several weeks after their preparation. The
362
high emulsification indexes observed reflect the stability of the emulsions thus formed.
363 364
The emulsifying capacity of CBSA at different concentrations (0.25-3% ; w/v), using corn oil, was also studied and shown in Table 3.
16
Page 16 of 38
Table 3
366
CBSA exhibited an appreciable emulsifying capacity, which increased with increasing
367
concentration. The emulsifying activity of CBSA is noteworthy (87.9% at a concentration of
368
3%), especially in comparison with that of Arabic gum (3%), that forms emulsion with corn
369
oil with E24 of 66.7%. Increasing the concentration of CBSA, the viscosity and accordingly
370
the emulsification indexes increased. Dickinson [37] reported that hydrocolloids are
371
commonly perceived to slow down or even prevent creaming by modifying the rheology of
372
the continuous phase. From these results, the differences of the emulsification capacity
373
observed among the various hydrocolloid polymers used as emulsion stabilizers are most
374
likely attributable to their diverse chemical composition and structure.
an
us
cr
ip t
365
In many industrial processes, emulsifiers are exposed to extreme temperatures and pH.
376
Emulsion prepared with corn oil and 2% CBSA aqueous solution was subjected to different
377
temperatures (25-100 °C) (Table 4).
d
Table 4
te
378
M
375
The emulsification capacity of corn oil in the presence of CBSA was average 80% at
380
25 °C. Thermal treatment of CBSA (40-100 °C) reduced 5% of the E24 at 25 °C. The decrease
381
of the emulsifying capacity caused by heating of the emulsion formed could be related to the
382
slight reduction of viscosity during heating. The slight reduction of emulsifying capacity at
383
temperature as high as 100 °C, indicates that the CBSA-oil emulsion is thermostable.
384 385 386
Ac ce p
379
The emulsion-forming capacity of CBSA with corn oil was noticeable for the pH
range tested (3.0-11.0) with a maximum at pH 3.0 (Table 4). Table 4
387
Acidic conditions increased the emulsifying activity of CBSA. In contrast, Guttierez et
388
al. [38] reported that acidic conditions decreased the emulsifying capacity of some
389
commercial polysaccharides such as xanthan gum and Arabic gum. The increase of the
17
Page 17 of 38
emulsifying capacity of CBSA at pH 3.0 could be related to the increase of the viscosity of
391
CBSA (formation of acidic gel stabilized by hydrogen bonds) due to protonation of carboxyl
392
acid groups and decrease of water solubility. As reported by Rinaudo [1], the viscosity of
393
alginate solution was nearly constant between pH 6.0 and 8.0, but it increased below pH 4.5
394
and reached a maximum around 3.0-3.5 and then decreased. At pH around 3 (~the intrinsic
395
pK of alginic acid), alginate formed gels resulting from H-bond attraction over dominating the
396
electrostatic repulsions [1].
us
cr
ip t
390
Dickinson [37] reported that the large molecular size and predominant hydrophilicity
398
of a polysaccharide emulsifier allows for the formation of a thicker stabilizing layer that is
399
capable of protecting droplets against aggregation over a wide range of unfavourable
400
conditions, such as thermal shock treatment and acidification. In contrast, protein-stabilized
401
emulsions were found highly sensitive to unfavourable environmental conditions due the low
402
surface coverage that makes the emulsions susceptible to destabilization. As the emulsion
403
formulated from CBSA was found less sensitive to temperature changes and stable at acidic
404
pH, CBSA would be useful as a potent emulsion stabilizer in food products.
405
3.6. Antioxidant activity
M
d
te
Ac ce p
406
an
397
Due to the diversity of oxidation processes, the use of a single method to evaluate the
407
antioxidant activity cannot provide a clear idea about their real antioxidant potential.
408
Therefore, CBSA was assayed for antioxidative activity using various antioxidant assays :
409
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing power, β-
410
carotene bleaching inhibition assay, hydroxyl radical-scavenging activity and DNA nicking
411
assay.
412
3.6.1. DPPH radical-scavenging activity
18
Page 18 of 38
The free radical scavenging activity of CBSA was tested through the DPPH method
414
and the results were compared with BHA used as positive control. As shown in Fig. 6A,
415
CBSA exhibited a concentration-dependent antiradical activity. BHA showed higher degree
416
of free radical scavenging activity than did CBSA at the same concentrations tested.
ip t
413
Fig. 6A
418
At 0.5 mg/ml, CBSA exerted great free radical scavenging activity (74%), but remain lower
419
than that of BHA (100%) at the same concentration. The antioxidant activity of CBSA
420
increases its importance as a potential new source of natural additives, primarily when
421
considering the inverse relationship between dietary intake of antioxidant-rich foods and
422
incidences of human diseases [39].
423
3.6.2. Reducing power
M
an
us
cr
417
The reducing capacity of a compound may serve as a significant indicator of its
425
potential antioxidant activity. Samples with higher reducing power have better abilities to
426
donate electrons. As reported in Fig. 6B, the reducing capacity of CBSA is concentration-
427
dependent.
te
Ac ce p
428
d
424
Fig. 6B
429
At 1.2 mg/ml, CBSA exerted significant reducing activity (OD at 700 nm = 2). However,
430
CBSA showed lower reducing power than did BHA at concentrations between 0.1 and 1
431
mg/ml. The obtained results demonstrated that CBSA can act as electron donors and can react
432
with free radicals to convert them to more stable products and thereby terminate radical chain
433
reactions.
434
3.6.3. β-carotene bleaching assay
19
Page 19 of 38
In this model system, the oxidation of linoleic acid generates free radicals (lipid
436
hydroperoxides, conjugated dienes and volatile byproducts) that attack the highly unsaturated
437
β-carotene molecules. When this reaction occurs, the β-carotene molecule looses its
438
conjugation and, as a consequence, the characteristic orange colour of β-carotene disappears
439
[40]. The presence of an antioxidant avoids the destruction of the β-carotene and the orange
440
colour is maintained. The antioxidant activity of CBSA measured by the inhibition of β-
441
carotene bleaching assay is reported in Fig. 6C.
us
cr
ip t
435
Fig. 6C
443
The effect of CBSA against the discoloration of β-carotene increased with increasing sample
444
concentration. CBSA displayed moderate ability to prevent bleaching of β-carotene. At 1.5
445
mg/ml, BHA was found to possess higher antioxidant activity (95%) than that of CBSA
446
(60%). Similarly, in the β-carotene-linoleic acid assay, the antioxidant activity of sulfated
447
fucans isolated from C. barbata was 62% [9], suggesting that C. barbata polysaccharides
448
have moderate antioxidant activity in the β-carotene-linoleate model system.
449
3.6.4. Hydroxyl radical-scavenging activity
M
d
te
Ac ce p
450
an
442
Among the oxygen radicals, the hydroxyl radical (OH) is the most reactive and can
451
induce oxidative damage to almost any biomolecule. Recently, many studies reported that
452
polysaccharides could stabilize radicals by providing electrons [41]. As shown in Fig .6D, the
453
hydroxyl radical scavenging activity increased by increasing the concentration of CBSA.
454
Fig .6D
455
At 4 and 5 mg/ml, CBSA exerted potent scavenging activities (80 and 82%, respectively),
456
which were higher than those of mannitol and BHA (71 and 47%, respectively). Therefore,
20
Page 20 of 38
457
CBSA might be a good candidate as an antioxidant that can help to prevent or postpone
458
oxidative damage of biomolecules.
459
3.6.5. DNA-nicking assay Antioxidative activities of CBSA using DNA nicking assay are reported in Fig. 6E.
461
Lane 1 represents the untreated plasmid (native DNA) with its three forms : the nicked form,
462
the linear and the supercoiled forms. Incubation of plasmid DNA with Fenton's reagent in the
463
absence of CBSA resulted in the complete degradation of the three DNA bands (lane 2). At 1
464
and 2 mg/ml, CBSA exerted a moderate DNA protection against hydroxyl radicals generated
465
from Fenton’s reaction (Lanes 3 and 4). However, at a concentration of 3 mg/ml, CBSA
466
exhibited a high protection against hydroxyl radical induced DNA breakage (Lane 5).
M
an
us
cr
ip t
460
Fig. 6E
467
Qi et al [42] reported that hydroxyl radical-scavenging activity was due to two different
469
antioxidant mechanisms. One suppresses the generation of hydroxyl radicals through Fe2+-
470
chelating activity and the other scavenges the hydroxyl radicals formed. As CBSA had proven
471
important hydroxyl radical-scavenging activity and as this biopolymer did not possess any
472
chelating ability of ferrous ions (data not shown), hence, the DNA protection recorded could
473
be entirely related to the scavenging effet of hydroxyl radicals. In this way, CBSA could be
474
effective against DNA damage.
475
4. Conclusions
Ac ce p
te
d
468
476
Sodium alginate was isolated and purified from brown seaweeds (C. barbata)
477
collected from a Tunisian island (Kerkennah, Sfax). C. barbata sodium alginate (CBSA) was
478
characterized by chromatographic (HPSEC-MALLS) and spectroscopic methods (circular
21
Page 21 of 38
479
dichroism, ATR-FTIR). Its viscometric-average molar mass equals 204 kDa and the
480
composition M/G equal 0.59. CBSA had been demonstrated a potent emulsifier, having an original low sensitivity to
482
temperature. CBSA showed also interesting antioxidant properties using various antioxidant
483
assays. Taking into account these results, it can be concluded that purified C . barbata sodium
484
alginate could be used as a natural additive in food applications.
485
Acknowledgement
486
This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia.
492 493 494
te Ac ce p
491
d
488
490
cr
us
an M
487
489
ip t
481
495 496 497
22
Page 22 of 38
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[35] I.M. Banat, R.S. Makkar, S.S. Cameotra, Potential commercial applications of microbial
582
surfactants, Appl. Microbiol. Biotechnol. 53 (2000) 495-508.
583
[36] E. Rosenberg, E.Z. Ron, High and low-molecular-mass microbial surfactants, Appl.
584
Microbiol. Biotechnol. 52 (1999) 154-162.
585
[37] E. Dickinson, Hydrocolloids as emulsifiers and emulsion stabilizers, Food Hydrocoll. 23
586
(2009) 1473-1482.
587
[38] T. Gutierrez, T. Shimmield, C. Haidon, K. Black, D.H. Green, Emulsifying and metal ion
588
binding activity of a glycoprotein exopolymer produced by Pseudoalteromonas sp. Strain
589
TG12, Appl. Environ. Microbiol. 74 (2008) 4867-4876.
590
[39] Y. Lu, Y. Foo, Antioxidant radical scavenging activies of polyphenols from apple
591
pomaceae, Food Chem. 68 (2000) 81-85.
Ac ce p
te
d
M
an
us
cr
ip t
567
26
Page 26 of 38
[40] T. Kulisic, A. Radonic, V. Katalinic, M. Milos, Use of different methods for testing
593
antioxidative activity of oregano essential oil, Food Chem. 85 (2004) 633-640.
594
[41] W. Zhang, J. Wang, W. Jin, Q. Zhang, The antioxidant activities and neuroprotective
595
effect of polysaccharides from the starfish Asterias rollestoni, Carbohydr Polym. 95 (2013) 9-
596
15.
597
[42] H.M. Qi, Q.B. Zhang, T.T. Zhao, R.G. Hu, K. Zhang, Z.E. Li, In vitro antioxidant
598
activity of acetylated and benzoylated derivatives of polysaccharide extracted from Ulva
599
pertusa (Chlorophyta), Bioorg. Med. Chem. Lett. 16 (2006) 2441-2445.
us
cr
ip t
592
an
600 601
Ac ce p
te
d
M
602
27
Page 27 of 38
602
Table 1. Yield and chemical analysis of C. barbata sodium alginate
603
Values (%, w/w)
604
Yielda
9.9±0.8
605
Moistureb
8.6±0.2
606
Neutral sugarc
9.3±0.6
Uronic acidc
58.1±1.3
Ashc
23.9±1.9
607
cr
608
ip t
Analytical data
us
609 610
All experiments were carried out in triplicate and expressed as mean values.
612
a
The yield was expressed on dry seaweed weight.
613
b
The moisture was expressed on purified sodium alginate weight.
614 615
c
M
an
611
Neutral sugars, uronic acid and ash contents were expressed on purified sodium alginate dry weight.
d
616
te
617
Table 2. Emulsifying capacity (E24) of C. barbata sodium alginate (1%) using different vegetable oils
Ac ce p
618
Oils
Olive oil
Sunflower oil
Corn oil
E24a
69.2±1.2b
69.2±1.2b
75.8±1.2a
Soybean oil 69.2±1.2b
Ricin oil 65.8±1.2c
619
a
620
Values are given as mean ± SD from triplicate determinations (n = 3).
621
Almond oil
Argan oil
69.2±1.2b
69.2±1.2b
Results are expressed as percentages of the total height occupied at 25°C.
Concentration (%, w/v)
0.25
0.5
1
2
3
CBSA
65.8±1.2e
70.8±1.2d
75.8±1.2c
80.8±1.2b
87.9±0.6a
Arabic gum
0±0.0b
0±0.0b
0±0.0b
66.7±0.0a
66.7±0.0a
Different letters indicate significant differences among the different oils (p < 0.05).
622
28
Page 28 of 38
623 624
Table 3. Emulsifying capacity (E24) of C. barbata sodium alginate at different concentrations
625 626
a
627
Values are given as mean ± SD from triplicate determinations (n = 3).
628 629
Different letters indicate significant differences among the different concentrations within the same line (p < 0.05).
cr
630
us
631 632
Table 4. Effect of pH (3.0-11.0) and heat (25-100°C) treatments on the emulsifying capacity (E24) of C. barbata sodium alginate (2%)
an
633 634
ip t
Emulsions were prepared with corn oil at 25°C.
Ac ce p
Temperature (°C)
te
d
Treatment
M
635
E24* (%)
25
80.8±1.2a
40
75.8±1.2b
60
75.8±1.2b
80
75.8±1.2b
100
75.8±1.2b
3
100±0.0a
5
80.8±1.2b
7
80.8±1.2b
9
75.8±1.2c
11
75.8±1.2c
pH
636
*
637
Values are given as mean ± SD from triplicate determinations (n = 3).
638 639
Different letters indicate significant differences among the different temperatures and different pH (p < 0.05).
Emulsions prepared with corn oil and CBSA (2%) were subjected to different temperatures and pH.
29
Page 29 of 38
643
Figure captions
644
Fig. 1. Molecular structure of sodium alginate.
646
Fig. 2. (A) Circular dichroism spectrum of C. barbata sodium alginate (800 ppm in distilled
647
water) at 25°C. (B) Determination of alginate composition from circular dichroism spectrum
648
according to Morris et al. (1980).
649 650
Fig. 3. ATR-FTIR spectra of (A) C. barbata sodium alginate and (B) commercial sodium alginate (Alfa Aesar).
651
Fig. 4. (A) Intrinsic viscosity of C. barbata sodium alginate at 25°C in 0.1 M NaCl solution.
652
(B) Determination of molecular weight of C. barbata sodium alginates by HPSEC-MALLS
653
eluted with 0.1 M NaCl at 25°C. Red signal is the light scattering intensity at the angle 90°
654
and the blue signal is the refractive index (related to polymer concentration).
655 656 657 658 659 660 661 662 663 664 665 666 667 668
Fig. 5. (A) Apparent viscosity of 2% CBSA aqueous solutions (in 0.1 M NaCl) during heating (25°C- 60°C) with increasing the shear rate from 1 s-1 to 50 s-1. (B) Apparent viscosity of CBSA aqueous solutions (in 0.1 M NaCl) as a function of polymer concentration at a shear rate of 5 s-1. The experiments were carried out at 25°C.
te
d
M
an
us
cr
ip t
645
Ac ce p
Fig. 6. (A) DPPH radical-scavenging activity of CBSA. (B) Fe2+-reducing power of CBSA. (C) β-carotene-bleaching inhibition activity of CBSA. (D) Hydroxyl radical-scavenging activity of CBSA at different concentrations. Values are means ± S.D. (n=3). (E) Protective effect of CBSA at different concentrations on hydroxyl radical induced DNA damage. Lane 1: untreated control: native pGapZαA plasmid DNA, Lane 2: plasmid DNA + Fenton’s reagent, Lane 3: plasmid DNA + Fenton’s reagent + 1 mg/ml of CBSA, Lane 4: plasmid DNA + Fenton’s reagent + 2 mg/ml of CBSA, Lane 5: plasmid DNA + Fenton’s reagent + 3 mg/ml of CBSA.
669 670 671 672 673
31
Page 30 of 38
674 675 676
Fig.1
us
cr
ip t
677
678
an
679 680
M
681
685 686 687 688 689 690 691
te
684
Ac ce p
683
d
682
692 693 694 695 696
32
Page 31 of 38
ip t
Fig. 2 (A)
an
us
cr
t
te
(B)
d
M
p
Ac ce p
697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740
33
Page 32 of 38
741
Fig. 3 (A)
742 743 744
cr us an M d
(B)
te
749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780
(a) (a)
Ac ce p
746 747 748
ip t
745
34
Page 33 of 38
Fig. 4 (A)
784 785 786
(B)
Ac ce p
te
d
M
an
us
cr
ip t
781 782 783
788 790 792 794 796 798 800 802 804 806 808 810 812 814 816 818 820 822 824 826 828
829 830 831 832
35
Page 34 of 38
Fig. 5 (B)
te
d
M
an
us
cr
ip t
(A)
Ac ce p
833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876
36
Page 35 of 38
M
an
us
cr
ip t
Fig. 6 (A)
te
d
(B)
Ac ce p
877 878 880 882 884 886 888 890 892 894 896 898 900 902 904 906 907 908 909 910 911 913 915 917 919 921 923 925 927 929 931 933 935 937 939 941 942 943 944 945 946 947 948 949
37
Page 36 of 38
1018
an
us
cr
ip t
(C)
te
d
M
(D)
Ac ce p
950 951 953 955 957 959 961 963 965 967 969 971 973 975 977 979 980 981 982 984 986 988 990 992 994 996 998 1000 1002 1004 1006 1008 1009 1010 1011 1012 1013 1014 1016
(E)
1
2
3
4
5
Nicked circular form Linear form Circular supercoiled form
1020 1022
38
Page 37 of 38
1024 1025 1026 1027
Highlights Sodium alginate was isolated from the brown seaweed Cystoseira barbata harvested in Tunisia ; C. barbata sodium alginate (CBSA) was analysed by circular dichroism (CD) and
ip t
1023
ATR-FTIR spectroscopies ;
The emulsifying capacity of CBSA was studied at different concentrations (0.25-3%),
1029
temperatures (25-100 °C) and pH (3.0-11.0) and the emulsion formulated was found to
1030
be less sensitive to temperature changes and more stable at acidic pH ;
us
Isolated sodium alginate exhibited important antioxidant activity.
an
1031
cr
1028
1032
Ac ce p
te
d
M
1033
39
Page 38 of 38
