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Rheological properties of a polysaccharide isolated from Adansonia digitata leaves
Accepted Manuscript Rheological properties of a polysaccharide isolated from Adansonia digitata leaves Louis M. Nwokocha, Peter A. Williams PII:
S0268-005X(16)30043-1
DOI:
10.1016/j.foodhyd.2016.02.013
Reference:
FOOHYD 3297
To appear in:
Food Hydrocolloids
Received Date: 21 December 2015 Revised Date:
10 February 2016
Accepted Date: 11 February 2016
Please cite this article as: Nwokocha, L.M., Williams, P.A., Rheological properties of a polysaccharide isolated from Adansonia digitata leaves, Food Hydrocolloids (2016), doi: 10.1016/j.foodhyd.2016.02.013. 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.
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Graphical Abstract Molar Mass vs. Volume
Adansonia 1st order
1.0x10 8
GPC elution profile of Adansonia digitata polysaccharide
Mw 1.0x10 7
Mw = 4.01 x 106 g/mol 1.0x10 6 16.0
18.0
20.0
22.0
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Volume (mL)
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Molar Mass (g/mol)
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Rheological properties of a polysaccharide isolated from Adansonia digitata leaves
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Louis M. Nwokocha1 and Peter A. Williams2,*
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Department of Chemistry, University of Ibadan, Ibadan, Nigeria
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Centre for Water Soluble Polymers, Glyndwr University, Wrexham, North Wales LL 11 2AW, UK
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*Corresponding author. Tel.: +44 1978293 083.
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E-mail address: [email protected] (P.A. Williams).
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Abstract
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The rheological properties of Adansonia digitata leaf polysaccharide were studied in dilute and
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semi-dilute solutions. The intrinsic viscosity of the polysaccharide obtained by Fedors equation
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and the combined Huggins and Kraemer extrapolations was ~3.27 dL/g. The polysaccharide
16
contained random coil macromolecules with mass average molecular mass of 4.01 x 106 g/mol.
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The polysaccharide in semi-dilute concentrations exhibited strong shear thinning property, and
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viscoelastic behaviour was observed with solutions within (3 - 5 % (w/w)) consistent with the
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formation of entangled random coil macromolecules in solution. The polysaccharide solutions
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were sensitive to temperature and the minimum energy to initiate flow in 4.0% polysaccharide
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solution calculated from Arrhenius plot of zero shear viscosity as a function of temperature was
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48.6 kJ/mol. The FTIR spectral studies of the polysaccharide confirmed the presence of uronic
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acid groups.
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Keywords: Adansonia digitata polysaccharide; molecular mass; rheological properties;
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activation energy of flow 1. Introduction
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Many of the hydrocolloids currently available as industrial gums were first used in an empirical
29
way in domestic cookery (Ndjouenkeu, Goycoolea, Morris, & Akingbala, 1996). There are many
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others, however, that are not yet exploited commercially but are extensively used in local
31
recipes, particularly in Nigeria and other tropical regions of the world. These materials are
32
obtained from plants that grow wild or are cultivated only on a limited scale, and their functional
33
properties as hydrocolloids remain largely unexplored. The performance of these plants in local
34
cookery indicates they have potential for exploitation; however, there is lack of information on
35
the properties of the hydrocolloids. Recently, some of them are receiving attention: Afzelia
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Africana (Ren, Ellis, Sutherland, & Ross-Murphy, 2003; Ren, Picout, Ellis, Ross-Murphy, &
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Grant Reid, 2005), Irvingia gabonensis (Ndjouenkeu, et al., 1996; Uzomah & Ahiligwo, 1999;
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Nwokocha & Williams, 2014a), Brachystegia eurycoma (Uzomah & Ahiligwo, 1999; Nwokocha
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& Williams, 2014b), Corchorus olitorius (Yamazaki, Kurita, & Matsumura, 2009), Detarium
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microcarpum (Picout, Ross-Murphy, Errington, & Harding, 2003), Mucuna flagellipes
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(Nwokocha & Williams, 2009) and Hibiscus esculentum (Ndjouenkeu et al., 1996).
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Adansonia digitata L.(Bombacaceae) is an African plant known as baobab tree. The baobab tree
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grows in most parts of West Africa as well as in parts of North and East Africa where the leaves
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are used as vegetable and as a soup thickener (Builders, Okeke, & Egieye, 2007; Woolfe,
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Chaplin, & Otchere, 1977). Woolfe et al., (1977) who analyzed the sugar composition reported it
46
contained mainly galacturonic and glucuronic acids with minor quantities of galactose,
47
rhamnose, glucose and arabinose; they also reported that the mucilage viscosity decreased with
48
increase in temperature. If the polysaccharide is to find expanded application in the food
49
industry, a detailed study is required to provide information on its application properties.
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Presently, we have not seen any studies on the molecular characteristics, intrinsic viscosity and
51
mechanical spectra of this important food thickener. In this work, we studied the molecular
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characteristics of the polysaccharide isolated from the leaves of Adansonia digitata using GPC-
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MALLS coupled to RI and UV detectors and the rheological properties using capillary
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viscometry in the dilute regime, and steady shear and small angle deformation oscillatory
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measurements in the semi dilute regime. We also studied the effect of temperature on the
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rheological properties of Adansonia digitata polysaccharide and confirmed the presence of
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uronic acid groups using FTIR. Some preliminary characteristics of this polysaccharide have
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been highlighted elsewhere (Nwokocha & Williams, 2012).
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2. Materials and Methods
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2.1 Sample preparation and isolation of polysaccharide
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Fresh leaves were collected from Adansonia digitata tree and air dried. The leaves were
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pulverized and defatted in a soxhlet extractor using hexane as solvent. The defatted flour was rid
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of the solvent by leaving to dry in a vacuum chamber. The flour, 10 g/L (w/v) was dispersed in
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deionised water by means of a mechanical stirrer for 3 h. The resulting dispersion was poured
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into centrifuge bottles and centrifuged at 2500 rpm for 2 h at 25oC. The supernatant was pooled
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together. The resulting supernatant was treated with excess isopropanol to precipitate the
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polysaccharide. The powdered polysaccharide was recovered by freeze drying.
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2.2 Fourier Transform Infra Red Spectroscopy
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FTIR spectra of Adansonia digitata polysaccharide and its H-form were obtained on a KBr disc
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using FTIR spectrophotometer (Perkin Elmer Spectrum Two, USA). The disc was prepared by
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grinding 2 mg of the native polysaccharide with 200 mg of FTIR grade KBr and pressing into a
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disc. The polysaccharide in H-form was prepared by acidifying the polysaccharide with
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hydrochloric acid solution during which all carboxylate groups (-COO-) were converted to free
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carboxylic acid groups (-COOH); this was precipitated with ethanol and washed with the same
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and dried. The polysaccharide (H-form) was prepared for FTIR as previously described for the
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native polysaccharide.
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2.3 Molecular mass determination
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The molecular mass was determined using gel permeation chromatography coupled to
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multiangle laser light scattering and refractive index and UV detectors (Optilab DSP, Wyatt
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Technology Corporation, Santa Barbara Ca93103). The polysaccharide solution (20 mL)
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containing 4.001 x 10-4 g/mL (w/w) was subjected to microwave bomb treatment for 40 s to
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ensure complete disaggregation (Ratcliffe, Williams, Viebke, & Meadows, 2005), filtered
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through a 0.45 µm syringe filter and injected through a rheodyne into a 200 µL loop connected to
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a combination of Suprema columns (100Å, 3000Å and 30000Å) packed with 10 µm beads of
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polyhydroxymethacrylate copolymer network through which the degassed (CSI 6150,
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Cambridge Scientific Instruments, England) eluent (0.1M NaNO3 + 10-6M NaN3 solution) was
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pumped (Waters: 515 HPLC Pump, Milford, MA 01757, USA) at a flow rate of 0.5 mL/min. The
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total injected mass was 8.002 x 10-5 g. The chromatogram was analyzed with Astra software with
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a predetermined dn/dc value of 0.140 mL/g ( Li & Xie, 2006) and the molecular mass,
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polydispersity and radius of gyration quantified using Berry first order polynomial.
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2.4 Determination of intrinsic viscosity
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0.2 % (dry basis, w/w) Adansonia digitata polysaccharide powder was dispersed in 0.1M NaCl
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solution at ambient temperature by placing on a roller mixer (SRT2, Staurt Scientific, UK)
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overnight. 7 mL of solution was transferred into a Cannon-Ubbelohde capillary viscometer (No
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75), which was immersed in a precision water bath to maintain the temperature at 25.0±0.1 oC.
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After equilibration for 10 min, the flow time was determined between the two etched marks.
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Serial isoionic dilution was performed in situ and three readings were taken for each dilution and
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averaged. The relative viscosity, ηr, was calculated as flow time of the solution divided by the
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flow time of the solvent. The intrinsic viscosity, [η], was determined by applying Fedors
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equation (Eq. 1) (Fedors, 1979).
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2(η r
1/ 2
− 1)
=
1 1 − [η ]C [η ]C max
(Eq. 1)
Where C is the concentration of polymer (g/dL), Cmax is a factor showing Fedors concentration
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limit.
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The specific viscosity, ηsp, is related to ηr by ηsp = ηr-1. The [η] was also evaluated by the
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combined Huggins (ηsp/C vs C) and Kraemer (ln (ηr)/C vs C) plots.
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2.5 Steady shear and small angle deformation oscillation studies
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Different concentrations of the Adansonia digitata polysaccharide solutions (0.5 - 6.0%, w/w)
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were prepared by dispersing the desired amount of the polysaccharide powder in distilled water
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while continuously solubilizing it overnight or more at ambient temperature by means of a roller
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mixer (SRT2, Stuart Scientific, UK). The rheological data were generated on a Controlled Stress
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Rheometer (AR 2000, TA Instruments, Newcastle, UK) at 25oC using cone and plate geometry
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(40 mm 2° steel cone, ser no 982525, truncation gap 53 µm) for sample concentrations 3 to 6%
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and standard-size recessed end geometry (coaxial cylinders with rotor outer radius 14 mm, gap
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4000 µm) for concentrations 0.5 and 2.0%. Solvent trap was used to prevent moisture loss. The
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sample was allowed to equilibrate for 2 min before each measurement. The flow properties were
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obtained by subjecting the polysaccharide solutions to a stepped-flow procedure at a shear rate of
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0.01 to 1000 s-1. In the oscillation procedure, strain sweep was performed on each gum solution
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from 0.003 to 100% at an angular frequency of 1 rad/s to locate the linear viscoelastic region. A
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frequency sweep was performed on the gum solutions in the region of 0.1 to 120 rad/s at an
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amplitude strain within the linear viscoelastic region. The resulting data was analyzed using TA
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Data Analysis Software.
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2.6 Effect of temperature
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The effect of temperature on the viscosity of Adansonia digitata polysaccharide was investigated
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in water by subjecting a 4% polysaccharide solution to a stepped flow procedure in the
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temperature range 5o to 55oC at 10o interval. The Cross model was fitted to the flow curves to
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determine the zero shear viscosities. The activation energy for viscous flow of 4%
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polysaccharide in water was determined from the Arrhenius plot of zero shear viscosities (ηo)
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versus the inverse of absolute temperature (T) (Eq. 2).
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ηo = ηo, T∞ exp(
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ηo = zero shear viscosity (Pa s), η o,T∞ zero shear viscosity (Pa s) at infinite temperature, Ea=
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activation energy for viscous flow (J/mol), R= gas constant (8.314 J/mol K), T= temperature (K).
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Since η o,T∞ represents the zero shear viscosity at infinite temperature, the equation can be written
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in the natural logarithmic form by choosing a reference temperature, in this case 308 K (Eq. 3).
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ln η o = ln η o, 308 +
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3. Results and Discussion
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3.1 FTIR of Adansonia digitata polysaccharide
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Figure 1 shows the FTIR spectra of Adansonia digitata polysaccharide and its H-form. The
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spectrum of the natural polysaccharide shows a broad absorption band in the region 1500 – 1800
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cm-1 centred at 1621 cm-1. In this region appear peaks associated with the presence of dissociated
140
carboxylate groups (-COO-).and the in-plane deformation of molecules of water hydrogen
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bonded to polysaccharide molecules (usually centred around 1640 cm-1). If carboxylate groups
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are present, upon acid treatment, a shift in position of the band to higher wave numbers or
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(Eq. 3)
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appearance of a new peak would indicate the carboxylate groups have been converted to the H-
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form (-COOH). The acid treated polysaccharide shows a new peak at 1720 cm-1 which is absent
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in the natural polysaccharide confirming that Adansonia digitata polysaccharide contains uronic
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acid groups (Garcia Vidal, 2013). Similar observations have been reported for other anionic
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polysaccharides (Marry, McCann, Kolpak, White, Stacey, & Roberts, 2000; Wang &
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Somasundaran, 2005; Garcia Vidal, 2013).
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3.2 Molecular mass
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The refractive index elution profile of Adansonia digitata polysaccharide and the molecular mass
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distribution are presented in Figure 2a. The result of the analysis (Table 1) shows that Adansonia
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digitata polysaccharide is characterized by a number-average molecular mass, Mn, of 3.647 x 106
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g/mol, mass-average molecular mass, Mw, of 4.01 x 106 g/mol and z-average molecular mass,
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Mz, of 4.33 x 106 g/mol. The polydispersity of the Adansonia polysaccharide (Mw/Mn =1.1)
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indicates a narrow range of mass distribution of the polysaccharide. The mass-average radius of
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gyration, Rg, of Adansonia digitata polysaccharide was 172.7 nm. The radius of gyration (Rg) is
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related to the molar mass (M) by Eq. 4.
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Rg ≈
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The log-log plot of Rg vs. M is linear from which the exponent ν is obtained as the slope. The
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value of the parameter ν gives information about the conformation of the polysaccharide
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macromolecules in solution. It has a value of 0.33 for spheres and 0.5-0.6 for random coils
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(Andersson, Wittgren, & Wahlund, 2003). Figure 2b shows the conformation plot for Adansonia
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digitata polysaccharide in aqueous solution. From the slope, ν had a value of 0.58; this is
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consistent with values reported for random coil polymers in aqueous solution (Picout et al., 2003;
(Eq. 4)
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Lim, Kim, Kim, Kim, Hwang, & Yun, 2005; Goh, Pinder, Hall, & Hemar, 2006). The presence
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of random coil polymers and the high molecular mass of Adansonia digitata polysaccharide will
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largely affect its solution viscosity and suggests the potential application of its polysaccharide as
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thickener in food. We have not seen any report on the molecular mass of Adansonia
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polysaccharide for comparison; however, it is higher than the molecular mass of 9.4 x 105 g/mol
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reported for the hydrocolloid from Corchorus olitorius leaves by Yamazaki et al. (2009).
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3.3 Intrinsic viscosity
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The intrinsic viscosity was determined in 0.1M NaCl using isoionic dilution of the
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polysaccharide solution in the concentration range 0.19% to 0.065% (w/w) and relative viscosity,
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ηr, of 1.2 < ηr < 2.0. Sometimes, the intercept of the combined Huggins and Kraemer
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extrapolations do not meet at C = 0, which is the situation in this case, thus [η] was presented as
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the average of the Huggins and Kraemer intercepts. The combined Huggins and Kraemer plots
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(Figure 3a) gave an intrinsic viscosity of 3.27±0.07 dL/g which is in good agreement with the
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value of 3.28 dL/g obtained from the slope of Fedors plot (slope = 1/[η]) (Figure 3b). Fedors
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equation has been reported to give the same intrinsic viscosity values for both native and
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modified dextrans as the Huggins equation when applied in the dilute domain (Rotureau,
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Dellacherie, & Durand, 2006). The limiting concentration of a Fedors plot of Adansonia digitata
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polysaccharide calculated from the intercept (intercept = 1/Cm[η]) was 1.245 g/dL. The Huggins
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constant, hc, calculated from the slope (slope = hc[η]2) of the Huggins plot was 0.63. The value of
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the Kraemer constant from the slope (slope = - kc[η]2) was 0.02. Both hc and kc are constants
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related to polymer-solvent interactions. In a solvent where polymer-polymer interactions counter
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balances polymer-solvent interactions, both hc and kc are related through: hc = kc + 0.5. The
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intrinsic viscosity of Adansonia digitata polysaccharide is less than 4.4 dL/g and 7.6 dL/g
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reported for Irvingia gabonensis and okra, respectively (Ndjouenkeu et al., 1996) and 19.8 dL/g
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reported for the exopolysaccharide from Escherichia coli strain S61(Ren et al., 2003).
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3.4 Steady shear viscosity
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The polysaccharide concentrations (0.5 - 6.0% (w/w)) were subjected to steady shear in the shear
192
rate range 10-3 to 103 s-1 and the flow profiles are shown in Figure 4a. Two distinct flow regimes
193
were observed- a low shear Newtonian plateau in which the apparent viscosity remained constant
194
(ηo) and a shear thinning or power law regime in which apparent viscosity decreased at relatively
195
higher shear rates. According to Graessley (1974), a constant apparent viscosity is maintained in
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the low shear rate region because the rate of intermolecular disentanglements brought about by
197
shearing forces is nearly the same as that of entanglements newly formed. On the other hand, the
198
decrease in apparent viscosity in the power law region occurs because the rate of
199
disentanglements is higher than the rate of formation of new entanglements. The magnitude of
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the zero shear apparent viscosity is a macroscopic representation of the micro structural nature of
201
the biopolymer (Hwang & Shin, 2000).
202
Different types of models have been employed in describing the flow properties of biopolymer
203
solutions in the semi dilute concentrations. One of such models which we have found useful in
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this case is the Cross model (Eq. 5) which we have used to obtain the flow characteristics of
205
Adansonia digitata polysaccharide.
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Cross model:
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η = η∞ +
η° −η ∞ ⋅
1 + (τ ∗ γ )
(Eq.5) m
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where η, ηo, η∞ are the shear, zero shear and infinite shear viscosity (Pa s), respectively; τ is
208
Cross relaxation time (s),
γ& is the shear rate (1/s) and m is the Cross rate index (dimensionless). 9
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The apparent viscosity exhibited both concentration and shear rate dependence. The zero-shear
210
apparent viscosity, ηo, increased from 0.09 Pa s to 153.7 Pa s and τ from 1.657 to 19.97s as
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concentration increased from 0.5 % to 6.0%. The relaxation time, τ, is related to the critical shear
212
rate ( γ& crit) through 1/ γ& crit = τ. The γ& crit marks the onset of shear thinning of the polysaccharide
213
solution and the result indicates that the onset of shear thinning shifted to lower shear rates as
214
polysaccharide concentration increased. This has been explained in terms of the degree of chain
215
entanglements. At high polymer concentration there is restriction of movement of the individual
216
chains as a result of the corresponding increase in entanglements which results in an increase in
217
time to replace the entanglements disrupted by the imposed deformation (Graessley, 1974). The
218
Cross rate index, m, fell within the range 0.66 < m < 0.84 and indicates the degree of dependence
219
of apparent viscosity on shear rate in the shear thinning region for Adansonia digitata
220
polysaccharide. Both the magnitudes of the zero shear apparent viscosity and apparent viscosity
221
in the power law region are important in formulation of non-Newtonian fluids. The high zero
222
shear apparent viscosity is a positive application property of the polysaccharide as suspending
223
agent and stabilizer since high apparent viscosity of the continuous phase of polysaccharide
224
solution at low shear rate would prevent particle movements and therefore hinder flocculation,
225
coalescence and sedimentation while the high shear thinning as indicated by the high value of the
226
Cross rate index (0.66 < m < 0.84) makes engineering operations such as mixing, pumping,
227
packaging or bottling easier (Young, 2002; Rincón, Muñoz, Ramírez, Galán, & Alfaro, 2014).
228
In Figure 4b, η/ηo versus τ γ& was plotted at different concentrations of Adansonia
229
polysaccharide, it was observed the profiles superimposed to a single flow curve. At very low
230
shear rates, the flow curve approached unity. It was observed that profiles of low polysaccharide
231
concentrations (0.5% and 2%) which contained transition zones between the power law zone and
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second Newtonian viscosity deviated at high shear rates. Similar deviations were reported to
233
occur in xanthan gum (Launay, Cuvelier, & Martinez-Reyes, 1983).
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3.5 Small angle deformation oscillation studies
235
Figure 5 shows a plot of storage modulus G′ and loss modulus G″ versus angular frequency ω for
236
polysaccharide solutions of 3, 4, and 5%. G′ and G″ showed dependence on ω and concentration,
237
with the dependence decreasing with increase in concentration. At low angular frequency (i.e. ω
238
< 0.186 rad/s), G″ > G′ for the three polymer solutions indicating a predominantly liquid-like
239
response. As ω increased, G′ increased faster than G″ so that a crossover occurred to a
240
predominantly elastic response with G′ > G″. The crossover points occurred at lower angular
241
frequencies of 1.697, 0.2294 and 0.168 rad/s for 3, 4, and 5% polymer concentrations,
242
respectively. The angular frequency at crossover point decreased to lower values while the
243
storage modulus increased with increasing polymer concentration. Similar observation has been
244
reported for colanic acid (Ren, et al., 2003) and Detarium gum (Picout et al., 2003).
245
3.6 Effect of temperature
246
Figure 6a, b shows the effect of temperature on the viscosity of 4% Adansonia digitata
247
polysaccharide in water. The steady shear flow profiles had two distinct regions- zero shear and
248
shear thinning regions similar to those obtained for most polysaccharides in this concentration
249
regime. A progressive decrease in viscosity was observed as temperature increased. This is
250
because an increase in temperature increases the kinetic motion of the macromolecules and thus
251
promotes disentanglement of the chains leading to a decrease in viscosity. A similar loss of
252
viscosity with increase in temperature was reported by Woolfe et al (1977). The zero shear
253
viscosities were determined by fitting the flow curves to the Cross model. The Arrhenius plot of
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the zero shear viscosities versus inverse of absolute temperature (Figure 6b) was used to
255
determine the activation energy (Ea) of viscous flow in water. The Ea required to initiate flow in
256
4% polysaccharide in water was 48.6kJ/mol (R2 =0.974). The activation energy of flow is
257
influenced by polysaccharide concentration (Xu, Chen, & Zhang, 2007; Nwokocha & Williams,
258
2014b), nature of intra and inter-chain interactions and the molecular mass of the polysaccharide.
259
A cashew tree exudates polysaccharide with molecular mass of 1.6 × 104 g/mol had Ea of flow
260
~16 kJ/mol at 3% (de Paula, & Rodrigues, 1995).
261
4.
262
The polysaccharide constituent of Adansonia digitata leaves, a popular food thickener in tropical
263
Africa was isolated and studied. The polysaccharide contained random coil macromolecules with
264
intrinsic viscosity of 3.27 dL/g and mass-average molecular mass of 4.01 x 106 g/mol. The
265
polysaccharide solutions exhibited shear thinning properties in the concentration range of 1.0 –
266
6.0 g/dL (w/w) and the polysaccharide solutions within 3 - 5 % (w/w) exhibit viscoelastic
267
behaviour consistent with the formation of entangled random coil macromolecules in solution.
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Acknowledgement: The authors are grateful to the Leverhulme Trust Foundation for funding.
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LMN was a Visiting Research Fellow to Glyndwr University, Wrexham, UK.
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conditions: Intrinsic viscosity and flow properties. In Gums and Stabilizers for the Food Industry
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2. Application of hydrocolloids. (Eds. G.O. Phillips, D.J. Wedlock and P.A. Williams) Pergamon
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Press, Oxford, England. p79-98
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Marry, M., McCann, M.C., Kolpak, F., White, A.R., Stacey, N.J., & Roberts, R. 2000. Extraction
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of pectic polysaccharides from sugar-beet cell walls. Journal of the Science of Food and
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Agriculture 80, 17-28.
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Ndjouenkeu, R., Goycoolea, F.M., Morris, E.R., & Akingbala, J.O. 1996. Rheology of Okra
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(Hibiscus esculentus L.) and dika nut (Irvingia gabonensis) polysaccharides. Carbohydrate
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Polymers 29, 263-269.
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Nwokocha, L.M. & Williams, P.A. 2009. Isolation and rheological characterization of Mucuna
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flagellipes seed gum. Food Hydrocolloids 23, 1394–1397.
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Nwokocha, L.M. & Williams, P.A. 2012. Evaluating the potential of Nigerian plants as a source
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of industrial hydrocolloids. In Gums and Stabilizers for the Food Industry 16. G.O. Phillips and
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P.A. Williams (Editors). Royal Society of Chemistry, Cambridge, United Kingdom. pp 27- 44.
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Nwokocha, L. M. & Williams, P. A. 2014a. Hydrodynamic and rheological properties of
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Irvingia gabonensis gum. Carbohydrate Polymers 114, 352–356.
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Nwokocha, L.M. & Williams, P.A. 2014b. Solution properties of Brachystegia eurycoma seed
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polysaccharide. In Gums and Stabilizers for the Food Industry 17: The Changing Face of Food
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Picout, D.R., Ross-Murphy,S.B., Errington, N., & Harding, S.E. 2003. Pressure cell assisted
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solubilization of xyloglucans: Tamarind seed polysaccharide and Detarium gum.
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Biomacromolecules 4, 799-807.
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Ratcliffe, I., Williams, P.A., Viebke, C., & Meadows, J. 2005. Physicochemical characterization
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of konjac glucomannan. Biomacromolecules 6, 1977-1986.
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Ren, Y., Ellis, P.R., Sutherland, I.W., & Ross-Murphy, S.B. 2003. Dilute and semi-dilute
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solution properties of an exopolysaccharide from Escherichia coli strain S61. Carbohydrate
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Polymers 52, 189-195.
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Ren, Y., Picout, D. R., Ellis, P. R., Ross-Murphy, S.B., & Grant Reid, J.S. 2005. A novel
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xyloglucan from seeds of Afzelia africana Se. Pers.—extraction, characterization and
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conformational properties. Carbohydrate Research 340, 997-1005.
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Rincón, F., Muñoz, J., Ramírez, P., Galán, H. & Alfaro, M. C. 2014. Physicochemical and 330
rheological characterization of Prosopis juliflora seed gum aqueous dispersions. Food
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Hydrocolloids 35, 348-357
Rotureau, E.; Dellacherie, E. & Durand, A. 2006. Viscosity of aqueous solutions of
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polysaccharides and hydrophobically modified polysaccharides: application of Fedors Equation.
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European Polymer Journal 42, 1086-1092.
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Uzomah A. & Ahiligwo R. N. 1999. Studies on the rheological properties and functional
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potentials of achi (Brachystegea eurycoma) and ogbono (Irvingia gabonesis) seed gums. Food
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Chemistry 67, 217-222.
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Wang, J & Somasundaran, P. 2005. Adsorption and conformation of carboxymethyl cellulose at
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solid–liquid interfaces using spectroscopic, AFM and allied techniques. Journal of Colloid and
340
Interface Science 291 75–83.
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Woolfe, M. L., Chaplin, M. F., & Otchere, G. 1977. Studies on the mucilages extracted from
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okra fruits (Hibiscus esculentus L.) and baobab leaves (Adansonia digitata L.). Journal of the
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Science of Food and Agriculture 28, 519–529.
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Xu, X., Chen, P. & Zhang, L. 2007. Viscoelastic properties of an exopolysaccharide: Aeromonas
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gum, produced by Aeromonas nichidenii 5797. Biorheology 44, 387-401.
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Yamazaki, E., Kurita, O. & Matsumura, Y. 2009. High viscosity of hydrocolloid from leaves of
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Corchorus olitorius L. Food Hydrocolloids 23, 65-660.
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Young, N.W.G. 2002. The yield stress phenomenon and related issues- an industrial view. In:
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Gums and Stabilizers for the Food Industry 11. G.O. Phillips and P.A. Williams (Editors). Royal
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Society of Chemistry, Cambridge, United Kingdom. Pp 226-234.
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Table 1. Molecular characteristics of Adansonia digitata polysaccharide
3.647± 0.01
Mw (x 10-6), g/mol
4.01± 0.08
Mz (x 10-6), g/mol
4.33± 0.15
Polydispersity 1.1 ±0.02
Mn/Mz
1.2 ± 0.04
R.M.S. radius moments Rn, nm
M AN U
Mw/Mn
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Mn (x 10-6), g/mol
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Molar mass moments
163.0± 4.2
Rg, nm
172.7± 5.1
Rz, nm
180.9± 5.9
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Data are mean of two determinations ± standard deviation
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Mn = number average molecular mass, Mw = mass average molecular mass, Mz = z-average molecular mass; Rn = number average radius of gyration, Rg = mass average radius of gyration, Rz = z-average radius of gyration.
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Natural
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H-form
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Figure 1. FTIR spectra of Adansonia digitata polysaccharide (natural and H-form)
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Molar Mass vs. Volume
Adansonia 1st order
1.0x108
Mw
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RI
1.0x107
1.0x106 16.0
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Molar Mass (g/mol)
a)
18.0
20.0
22.0
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Volume(mL) (mL) Volume
RMS Radius vs. Molar Mass
1000.0
10.0 1.0x10 6
TE D EP
100.0
AC C
R.M.S. Radius (nm)
b)
Adansonia 0.58 ± 0.00
1.0x10 7 Molar Mass (g/mol)
Mw (g/mol)
Figure 2. a). Refractive index and Mw GPC elution profile of Adansonia digitata polysaccharide.
b). Log-log plot of root mean square radius (Rg) versus molecular mass (Mw) of Adansonia digitata polysaccharide.
1.0x10 8
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5
y = 6.592x + 3.231
a)
ηsp/C
3.5 3
y = -0.244x + 3.305
ln(ηr)/C
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ln (ηr)/C, ηsp/C (dL/g)
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0.5 0 0
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b)
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y = 0.305x - 0.245 R² = 0.999
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1.5
1
0.5
0
0
5
10
15
20
1/C (dL/g)
Figure 3: Intrinsic viscosity of Adansonia digitata polysaccharide in 0.1M NaCl solution by (a) Combined Huggins and Kraemer extrapolations; (b) Fedors method , all determined at 25oC.
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1000 6% 5% 4%
viscosity (Pa.s)
10.00
3%
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100.0
a)
1.000 2% 0.1000
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0.5%
1.000E-3 1.000E-3
0.01000
0.1000
100
0.1
0.01 0.001
1.00E+00
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1.000 10.00 shear rate (1/s)
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η/ηo
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b)
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10
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0.01000
1.00E+02
100.0
1000
10000
6% 5% 4% 3% 2% 0.50%
1.00E+04
τأل
Figure 4. a). Viscosity dependence of shear rate and concentration; b). Generalized flow curves of dependence of viscosity on shear rate for different concentrations of Adansonia digitata polysaccharide in water at 25oC
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100
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10
SC
0.1
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G', G" (Pa)
1
0.01
0.001
0.01
5% G'
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0.0001 0.1
5% G"
1 Angular frequency (rad/s) 4% G'
4% G"
10
3% G'
100
3% G"
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Figure 5. Frequency sweep showing G′, G″ versus angular frequency, ω for Adansonia digitata polysaccharide at 3%, 4% and 5%, (w/w) solutions in water at 25oC.
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1000
a) 5 deg C 15 deg C 25 deg C
10.00
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35 deg C 45 deg C 55 deg C
1.000
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viscosity (Pa.s)
100.0
0.01000 1.000E-3
0.01000
0.1000
6
3 2 1
100.0
1000
10000
y = 5840x + 3.4394 R² = 0.9746
-0.0002
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1.000 10.00 shear rate (1/s)
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ln ηo, Pas
4
b)
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5
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0.1000
0
(1/T-1/308),
0.0002
0.0004
K-1
Figure 6. a) Effect of temperature on viscosity-shear rate profiles of 4% Adansonia digitata polysaccharide in water; b) Arrhenius plot of zero shear viscosity versus inverse of absolute temperature
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The manuscript highlights
•
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The molecular mass of Adansonia digitata polysaccharide was determined as 4.01 x 106 g/mol. The polysaccharide macromolecules exhibited random coil conformation in aqueous solution. The intrinsic viscosity was 3.27 dL/g. The polysaccharide contained uronic acid groups which were confirmed by FTIR
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S0268-005X(16)30043-1
DOI:
10.1016/j.foodhyd.2016.02.013
Reference:
FOOHYD 3297
To appear in:
Food Hydrocolloids
Received Date: 21 December 2015 Revised Date:
10 February 2016
Accepted Date: 11 February 2016
Please cite this article as: Nwokocha, L.M., Williams, P.A., Rheological properties of a polysaccharide isolated from Adansonia digitata leaves, Food Hydrocolloids (2016), doi: 10.1016/j.foodhyd.2016.02.013. 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.
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Graphical Abstract Molar Mass vs. Volume
Adansonia 1st order
1.0x10 8
GPC elution profile of Adansonia digitata polysaccharide
Mw 1.0x10 7
Mw = 4.01 x 106 g/mol 1.0x10 6 16.0
18.0
20.0
22.0
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Volume (mL)
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Molar Mass (g/mol)
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Rheological properties of a polysaccharide isolated from Adansonia digitata leaves
2
Louis M. Nwokocha1 and Peter A. Williams2,*
2
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Department of Chemistry, University of Ibadan, Ibadan, Nigeria
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Centre for Water Soluble Polymers, Glyndwr University, Wrexham, North Wales LL 11 2AW, UK
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*Corresponding author. Tel.: +44 1978293 083.
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E-mail address: [email protected] (P.A. Williams).
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Abstract
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The rheological properties of Adansonia digitata leaf polysaccharide were studied in dilute and
14
semi-dilute solutions. The intrinsic viscosity of the polysaccharide obtained by Fedors equation
15
and the combined Huggins and Kraemer extrapolations was ~3.27 dL/g. The polysaccharide
16
contained random coil macromolecules with mass average molecular mass of 4.01 x 106 g/mol.
17
The polysaccharide in semi-dilute concentrations exhibited strong shear thinning property, and
18
viscoelastic behaviour was observed with solutions within (3 - 5 % (w/w)) consistent with the
19
formation of entangled random coil macromolecules in solution. The polysaccharide solutions
20
were sensitive to temperature and the minimum energy to initiate flow in 4.0% polysaccharide
21
solution calculated from Arrhenius plot of zero shear viscosity as a function of temperature was
22
48.6 kJ/mol. The FTIR spectral studies of the polysaccharide confirmed the presence of uronic
23
acid groups.
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Keywords: Adansonia digitata polysaccharide; molecular mass; rheological properties;
26
activation energy of flow 1. Introduction
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Many of the hydrocolloids currently available as industrial gums were first used in an empirical
29
way in domestic cookery (Ndjouenkeu, Goycoolea, Morris, & Akingbala, 1996). There are many
30
others, however, that are not yet exploited commercially but are extensively used in local
31
recipes, particularly in Nigeria and other tropical regions of the world. These materials are
32
obtained from plants that grow wild or are cultivated only on a limited scale, and their functional
33
properties as hydrocolloids remain largely unexplored. The performance of these plants in local
34
cookery indicates they have potential for exploitation; however, there is lack of information on
35
the properties of the hydrocolloids. Recently, some of them are receiving attention: Afzelia
36
Africana (Ren, Ellis, Sutherland, & Ross-Murphy, 2003; Ren, Picout, Ellis, Ross-Murphy, &
37
Grant Reid, 2005), Irvingia gabonensis (Ndjouenkeu, et al., 1996; Uzomah & Ahiligwo, 1999;
38
Nwokocha & Williams, 2014a), Brachystegia eurycoma (Uzomah & Ahiligwo, 1999; Nwokocha
39
& Williams, 2014b), Corchorus olitorius (Yamazaki, Kurita, & Matsumura, 2009), Detarium
40
microcarpum (Picout, Ross-Murphy, Errington, & Harding, 2003), Mucuna flagellipes
41
(Nwokocha & Williams, 2009) and Hibiscus esculentum (Ndjouenkeu et al., 1996).
42
Adansonia digitata L.(Bombacaceae) is an African plant known as baobab tree. The baobab tree
43
grows in most parts of West Africa as well as in parts of North and East Africa where the leaves
44
are used as vegetable and as a soup thickener (Builders, Okeke, & Egieye, 2007; Woolfe,
45
Chaplin, & Otchere, 1977). Woolfe et al., (1977) who analyzed the sugar composition reported it
46
contained mainly galacturonic and glucuronic acids with minor quantities of galactose,
47
rhamnose, glucose and arabinose; they also reported that the mucilage viscosity decreased with
48
increase in temperature. If the polysaccharide is to find expanded application in the food
49
industry, a detailed study is required to provide information on its application properties.
50
Presently, we have not seen any studies on the molecular characteristics, intrinsic viscosity and
51
mechanical spectra of this important food thickener. In this work, we studied the molecular
52
characteristics of the polysaccharide isolated from the leaves of Adansonia digitata using GPC-
53
MALLS coupled to RI and UV detectors and the rheological properties using capillary
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viscometry in the dilute regime, and steady shear and small angle deformation oscillatory
55
measurements in the semi dilute regime. We also studied the effect of temperature on the
56
rheological properties of Adansonia digitata polysaccharide and confirmed the presence of
57
uronic acid groups using FTIR. Some preliminary characteristics of this polysaccharide have
58
been highlighted elsewhere (Nwokocha & Williams, 2012).
59
2. Materials and Methods
60
2.1 Sample preparation and isolation of polysaccharide
61
Fresh leaves were collected from Adansonia digitata tree and air dried. The leaves were
62
pulverized and defatted in a soxhlet extractor using hexane as solvent. The defatted flour was rid
63
of the solvent by leaving to dry in a vacuum chamber. The flour, 10 g/L (w/v) was dispersed in
64
deionised water by means of a mechanical stirrer for 3 h. The resulting dispersion was poured
65
into centrifuge bottles and centrifuged at 2500 rpm for 2 h at 25oC. The supernatant was pooled
66
together. The resulting supernatant was treated with excess isopropanol to precipitate the
67
polysaccharide. The powdered polysaccharide was recovered by freeze drying.
68
2.2 Fourier Transform Infra Red Spectroscopy
69
FTIR spectra of Adansonia digitata polysaccharide and its H-form were obtained on a KBr disc
70
using FTIR spectrophotometer (Perkin Elmer Spectrum Two, USA). The disc was prepared by
71
grinding 2 mg of the native polysaccharide with 200 mg of FTIR grade KBr and pressing into a
72
disc. The polysaccharide in H-form was prepared by acidifying the polysaccharide with
73
hydrochloric acid solution during which all carboxylate groups (-COO-) were converted to free
74
carboxylic acid groups (-COOH); this was precipitated with ethanol and washed with the same
75
and dried. The polysaccharide (H-form) was prepared for FTIR as previously described for the
76
native polysaccharide.
77
2.3 Molecular mass determination
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The molecular mass was determined using gel permeation chromatography coupled to
79
multiangle laser light scattering and refractive index and UV detectors (Optilab DSP, Wyatt
80
Technology Corporation, Santa Barbara Ca93103). The polysaccharide solution (20 mL)
81
containing 4.001 x 10-4 g/mL (w/w) was subjected to microwave bomb treatment for 40 s to
82
ensure complete disaggregation (Ratcliffe, Williams, Viebke, & Meadows, 2005), filtered
83
through a 0.45 µm syringe filter and injected through a rheodyne into a 200 µL loop connected to
84
a combination of Suprema columns (100Å, 3000Å and 30000Å) packed with 10 µm beads of
85
polyhydroxymethacrylate copolymer network through which the degassed (CSI 6150,
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Cambridge Scientific Instruments, England) eluent (0.1M NaNO3 + 10-6M NaN3 solution) was
87
pumped (Waters: 515 HPLC Pump, Milford, MA 01757, USA) at a flow rate of 0.5 mL/min. The
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total injected mass was 8.002 x 10-5 g. The chromatogram was analyzed with Astra software with
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a predetermined dn/dc value of 0.140 mL/g ( Li & Xie, 2006) and the molecular mass,
90
polydispersity and radius of gyration quantified using Berry first order polynomial.
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2.4 Determination of intrinsic viscosity
92
0.2 % (dry basis, w/w) Adansonia digitata polysaccharide powder was dispersed in 0.1M NaCl
93
solution at ambient temperature by placing on a roller mixer (SRT2, Staurt Scientific, UK)
94
overnight. 7 mL of solution was transferred into a Cannon-Ubbelohde capillary viscometer (No
95
75), which was immersed in a precision water bath to maintain the temperature at 25.0±0.1 oC.
96
After equilibration for 10 min, the flow time was determined between the two etched marks.
97
Serial isoionic dilution was performed in situ and three readings were taken for each dilution and
98
averaged. The relative viscosity, ηr, was calculated as flow time of the solution divided by the
99
flow time of the solvent. The intrinsic viscosity, [η], was determined by applying Fedors
100
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equation (Eq. 1) (Fedors, 1979).
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101
2(η r
1/ 2
− 1)
=
1 1 − [η ]C [η ]C max
(Eq. 1)
Where C is the concentration of polymer (g/dL), Cmax is a factor showing Fedors concentration
103
limit.
104
The specific viscosity, ηsp, is related to ηr by ηsp = ηr-1. The [η] was also evaluated by the
105
combined Huggins (ηsp/C vs C) and Kraemer (ln (ηr)/C vs C) plots.
106
2.5 Steady shear and small angle deformation oscillation studies
107
Different concentrations of the Adansonia digitata polysaccharide solutions (0.5 - 6.0%, w/w)
108
were prepared by dispersing the desired amount of the polysaccharide powder in distilled water
109
while continuously solubilizing it overnight or more at ambient temperature by means of a roller
110
mixer (SRT2, Stuart Scientific, UK). The rheological data were generated on a Controlled Stress
111
Rheometer (AR 2000, TA Instruments, Newcastle, UK) at 25oC using cone and plate geometry
112
(40 mm 2° steel cone, ser no 982525, truncation gap 53 µm) for sample concentrations 3 to 6%
113
and standard-size recessed end geometry (coaxial cylinders with rotor outer radius 14 mm, gap
114
4000 µm) for concentrations 0.5 and 2.0%. Solvent trap was used to prevent moisture loss. The
115
sample was allowed to equilibrate for 2 min before each measurement. The flow properties were
116
obtained by subjecting the polysaccharide solutions to a stepped-flow procedure at a shear rate of
117
0.01 to 1000 s-1. In the oscillation procedure, strain sweep was performed on each gum solution
118
from 0.003 to 100% at an angular frequency of 1 rad/s to locate the linear viscoelastic region. A
119
frequency sweep was performed on the gum solutions in the region of 0.1 to 120 rad/s at an
120
amplitude strain within the linear viscoelastic region. The resulting data was analyzed using TA
121
Data Analysis Software.
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2.6 Effect of temperature
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The effect of temperature on the viscosity of Adansonia digitata polysaccharide was investigated
124
in water by subjecting a 4% polysaccharide solution to a stepped flow procedure in the
125
temperature range 5o to 55oC at 10o interval. The Cross model was fitted to the flow curves to
126
determine the zero shear viscosities. The activation energy for viscous flow of 4%
127
polysaccharide in water was determined from the Arrhenius plot of zero shear viscosities (ηo)
128
versus the inverse of absolute temperature (T) (Eq. 2).
129
ηo = ηo, T∞ exp(
130
ηo = zero shear viscosity (Pa s), η o,T∞ zero shear viscosity (Pa s) at infinite temperature, Ea=
131
activation energy for viscous flow (J/mol), R= gas constant (8.314 J/mol K), T= temperature (K).
132
Since η o,T∞ represents the zero shear viscosity at infinite temperature, the equation can be written
133
in the natural logarithmic form by choosing a reference temperature, in this case 308 K (Eq. 3).
134
ln η o = ln η o, 308 +
135
3. Results and Discussion
136
3.1 FTIR of Adansonia digitata polysaccharide
137
Figure 1 shows the FTIR spectra of Adansonia digitata polysaccharide and its H-form. The
138
spectrum of the natural polysaccharide shows a broad absorption band in the region 1500 – 1800
139
cm-1 centred at 1621 cm-1. In this region appear peaks associated with the presence of dissociated
140
carboxylate groups (-COO-).and the in-plane deformation of molecules of water hydrogen
141
bonded to polysaccharide molecules (usually centred around 1640 cm-1). If carboxylate groups
142
are present, upon acid treatment, a shift in position of the band to higher wave numbers or
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(Eq. 3)
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appearance of a new peak would indicate the carboxylate groups have been converted to the H-
144
form (-COOH). The acid treated polysaccharide shows a new peak at 1720 cm-1 which is absent
145
in the natural polysaccharide confirming that Adansonia digitata polysaccharide contains uronic
146
acid groups (Garcia Vidal, 2013). Similar observations have been reported for other anionic
147
polysaccharides (Marry, McCann, Kolpak, White, Stacey, & Roberts, 2000; Wang &
148
Somasundaran, 2005; Garcia Vidal, 2013).
149
3.2 Molecular mass
150
The refractive index elution profile of Adansonia digitata polysaccharide and the molecular mass
151
distribution are presented in Figure 2a. The result of the analysis (Table 1) shows that Adansonia
152
digitata polysaccharide is characterized by a number-average molecular mass, Mn, of 3.647 x 106
153
g/mol, mass-average molecular mass, Mw, of 4.01 x 106 g/mol and z-average molecular mass,
154
Mz, of 4.33 x 106 g/mol. The polydispersity of the Adansonia polysaccharide (Mw/Mn =1.1)
155
indicates a narrow range of mass distribution of the polysaccharide. The mass-average radius of
156
gyration, Rg, of Adansonia digitata polysaccharide was 172.7 nm. The radius of gyration (Rg) is
157
related to the molar mass (M) by Eq. 4.
158
Rg ≈
159
The log-log plot of Rg vs. M is linear from which the exponent ν is obtained as the slope. The
160
value of the parameter ν gives information about the conformation of the polysaccharide
161
macromolecules in solution. It has a value of 0.33 for spheres and 0.5-0.6 for random coils
162
(Andersson, Wittgren, & Wahlund, 2003). Figure 2b shows the conformation plot for Adansonia
163
digitata polysaccharide in aqueous solution. From the slope, ν had a value of 0.58; this is
164
consistent with values reported for random coil polymers in aqueous solution (Picout et al., 2003;
(Eq. 4)
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Lim, Kim, Kim, Kim, Hwang, & Yun, 2005; Goh, Pinder, Hall, & Hemar, 2006). The presence
166
of random coil polymers and the high molecular mass of Adansonia digitata polysaccharide will
167
largely affect its solution viscosity and suggests the potential application of its polysaccharide as
168
thickener in food. We have not seen any report on the molecular mass of Adansonia
169
polysaccharide for comparison; however, it is higher than the molecular mass of 9.4 x 105 g/mol
170
reported for the hydrocolloid from Corchorus olitorius leaves by Yamazaki et al. (2009).
171
3.3 Intrinsic viscosity
172
The intrinsic viscosity was determined in 0.1M NaCl using isoionic dilution of the
173
polysaccharide solution in the concentration range 0.19% to 0.065% (w/w) and relative viscosity,
174
ηr, of 1.2 < ηr < 2.0. Sometimes, the intercept of the combined Huggins and Kraemer
175
extrapolations do not meet at C = 0, which is the situation in this case, thus [η] was presented as
176
the average of the Huggins and Kraemer intercepts. The combined Huggins and Kraemer plots
177
(Figure 3a) gave an intrinsic viscosity of 3.27±0.07 dL/g which is in good agreement with the
178
value of 3.28 dL/g obtained from the slope of Fedors plot (slope = 1/[η]) (Figure 3b). Fedors
179
equation has been reported to give the same intrinsic viscosity values for both native and
180
modified dextrans as the Huggins equation when applied in the dilute domain (Rotureau,
181
Dellacherie, & Durand, 2006). The limiting concentration of a Fedors plot of Adansonia digitata
182
polysaccharide calculated from the intercept (intercept = 1/Cm[η]) was 1.245 g/dL. The Huggins
183
constant, hc, calculated from the slope (slope = hc[η]2) of the Huggins plot was 0.63. The value of
184
the Kraemer constant from the slope (slope = - kc[η]2) was 0.02. Both hc and kc are constants
185
related to polymer-solvent interactions. In a solvent where polymer-polymer interactions counter
186
balances polymer-solvent interactions, both hc and kc are related through: hc = kc + 0.5. The
187
intrinsic viscosity of Adansonia digitata polysaccharide is less than 4.4 dL/g and 7.6 dL/g
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reported for Irvingia gabonensis and okra, respectively (Ndjouenkeu et al., 1996) and 19.8 dL/g
189
reported for the exopolysaccharide from Escherichia coli strain S61(Ren et al., 2003).
190
3.4 Steady shear viscosity
191
The polysaccharide concentrations (0.5 - 6.0% (w/w)) were subjected to steady shear in the shear
192
rate range 10-3 to 103 s-1 and the flow profiles are shown in Figure 4a. Two distinct flow regimes
193
were observed- a low shear Newtonian plateau in which the apparent viscosity remained constant
194
(ηo) and a shear thinning or power law regime in which apparent viscosity decreased at relatively
195
higher shear rates. According to Graessley (1974), a constant apparent viscosity is maintained in
196
the low shear rate region because the rate of intermolecular disentanglements brought about by
197
shearing forces is nearly the same as that of entanglements newly formed. On the other hand, the
198
decrease in apparent viscosity in the power law region occurs because the rate of
199
disentanglements is higher than the rate of formation of new entanglements. The magnitude of
200
the zero shear apparent viscosity is a macroscopic representation of the micro structural nature of
201
the biopolymer (Hwang & Shin, 2000).
202
Different types of models have been employed in describing the flow properties of biopolymer
203
solutions in the semi dilute concentrations. One of such models which we have found useful in
204
this case is the Cross model (Eq. 5) which we have used to obtain the flow characteristics of
205
Adansonia digitata polysaccharide.
206
Cross model:
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η = η∞ +
η° −η ∞ ⋅
1 + (τ ∗ γ )
(Eq.5) m
207
where η, ηo, η∞ are the shear, zero shear and infinite shear viscosity (Pa s), respectively; τ is
208
Cross relaxation time (s),
γ& is the shear rate (1/s) and m is the Cross rate index (dimensionless). 9
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The apparent viscosity exhibited both concentration and shear rate dependence. The zero-shear
210
apparent viscosity, ηo, increased from 0.09 Pa s to 153.7 Pa s and τ from 1.657 to 19.97s as
211
concentration increased from 0.5 % to 6.0%. The relaxation time, τ, is related to the critical shear
212
rate ( γ& crit) through 1/ γ& crit = τ. The γ& crit marks the onset of shear thinning of the polysaccharide
213
solution and the result indicates that the onset of shear thinning shifted to lower shear rates as
214
polysaccharide concentration increased. This has been explained in terms of the degree of chain
215
entanglements. At high polymer concentration there is restriction of movement of the individual
216
chains as a result of the corresponding increase in entanglements which results in an increase in
217
time to replace the entanglements disrupted by the imposed deformation (Graessley, 1974). The
218
Cross rate index, m, fell within the range 0.66 < m < 0.84 and indicates the degree of dependence
219
of apparent viscosity on shear rate in the shear thinning region for Adansonia digitata
220
polysaccharide. Both the magnitudes of the zero shear apparent viscosity and apparent viscosity
221
in the power law region are important in formulation of non-Newtonian fluids. The high zero
222
shear apparent viscosity is a positive application property of the polysaccharide as suspending
223
agent and stabilizer since high apparent viscosity of the continuous phase of polysaccharide
224
solution at low shear rate would prevent particle movements and therefore hinder flocculation,
225
coalescence and sedimentation while the high shear thinning as indicated by the high value of the
226
Cross rate index (0.66 < m < 0.84) makes engineering operations such as mixing, pumping,
227
packaging or bottling easier (Young, 2002; Rincón, Muñoz, Ramírez, Galán, & Alfaro, 2014).
228
In Figure 4b, η/ηo versus τ γ& was plotted at different concentrations of Adansonia
229
polysaccharide, it was observed the profiles superimposed to a single flow curve. At very low
230
shear rates, the flow curve approached unity. It was observed that profiles of low polysaccharide
231
concentrations (0.5% and 2%) which contained transition zones between the power law zone and
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second Newtonian viscosity deviated at high shear rates. Similar deviations were reported to
233
occur in xanthan gum (Launay, Cuvelier, & Martinez-Reyes, 1983).
234
3.5 Small angle deformation oscillation studies
235
Figure 5 shows a plot of storage modulus G′ and loss modulus G″ versus angular frequency ω for
236
polysaccharide solutions of 3, 4, and 5%. G′ and G″ showed dependence on ω and concentration,
237
with the dependence decreasing with increase in concentration. At low angular frequency (i.e. ω
238
< 0.186 rad/s), G″ > G′ for the three polymer solutions indicating a predominantly liquid-like
239
response. As ω increased, G′ increased faster than G″ so that a crossover occurred to a
240
predominantly elastic response with G′ > G″. The crossover points occurred at lower angular
241
frequencies of 1.697, 0.2294 and 0.168 rad/s for 3, 4, and 5% polymer concentrations,
242
respectively. The angular frequency at crossover point decreased to lower values while the
243
storage modulus increased with increasing polymer concentration. Similar observation has been
244
reported for colanic acid (Ren, et al., 2003) and Detarium gum (Picout et al., 2003).
245
3.6 Effect of temperature
246
Figure 6a, b shows the effect of temperature on the viscosity of 4% Adansonia digitata
247
polysaccharide in water. The steady shear flow profiles had two distinct regions- zero shear and
248
shear thinning regions similar to those obtained for most polysaccharides in this concentration
249
regime. A progressive decrease in viscosity was observed as temperature increased. This is
250
because an increase in temperature increases the kinetic motion of the macromolecules and thus
251
promotes disentanglement of the chains leading to a decrease in viscosity. A similar loss of
252
viscosity with increase in temperature was reported by Woolfe et al (1977). The zero shear
253
viscosities were determined by fitting the flow curves to the Cross model. The Arrhenius plot of
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the zero shear viscosities versus inverse of absolute temperature (Figure 6b) was used to
255
determine the activation energy (Ea) of viscous flow in water. The Ea required to initiate flow in
256
4% polysaccharide in water was 48.6kJ/mol (R2 =0.974). The activation energy of flow is
257
influenced by polysaccharide concentration (Xu, Chen, & Zhang, 2007; Nwokocha & Williams,
258
2014b), nature of intra and inter-chain interactions and the molecular mass of the polysaccharide.
259
A cashew tree exudates polysaccharide with molecular mass of 1.6 × 104 g/mol had Ea of flow
260
~16 kJ/mol at 3% (de Paula, & Rodrigues, 1995).
261
4.
262
The polysaccharide constituent of Adansonia digitata leaves, a popular food thickener in tropical
263
Africa was isolated and studied. The polysaccharide contained random coil macromolecules with
264
intrinsic viscosity of 3.27 dL/g and mass-average molecular mass of 4.01 x 106 g/mol. The
265
polysaccharide solutions exhibited shear thinning properties in the concentration range of 1.0 –
266
6.0 g/dL (w/w) and the polysaccharide solutions within 3 - 5 % (w/w) exhibit viscoelastic
267
behaviour consistent with the formation of entangled random coil macromolecules in solution.
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Acknowledgement: The authors are grateful to the Leverhulme Trust Foundation for funding.
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LMN was a Visiting Research Fellow to Glyndwr University, Wrexham, UK.
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Table 1. Molecular characteristics of Adansonia digitata polysaccharide
3.647± 0.01
Mw (x 10-6), g/mol
4.01± 0.08
Mz (x 10-6), g/mol
4.33± 0.15
Polydispersity 1.1 ±0.02
Mn/Mz
1.2 ± 0.04
R.M.S. radius moments Rn, nm
M AN U
Mw/Mn
SC
Mn (x 10-6), g/mol
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Molar mass moments
163.0± 4.2
Rg, nm
172.7± 5.1
Rz, nm
180.9± 5.9
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Data are mean of two determinations ± standard deviation
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Mn = number average molecular mass, Mw = mass average molecular mass, Mz = z-average molecular mass; Rn = number average radius of gyration, Rg = mass average radius of gyration, Rz = z-average radius of gyration.
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Natural
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H-form
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Figure 1. FTIR spectra of Adansonia digitata polysaccharide (natural and H-form)
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Molar Mass vs. Volume
Adansonia 1st order
1.0x108
Mw
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RI
1.0x107
1.0x106 16.0
SC
Molar Mass (g/mol)
a)
18.0
20.0
22.0
M AN U
Volume(mL) (mL) Volume
RMS Radius vs. Molar Mass
1000.0
10.0 1.0x10 6
TE D EP
100.0
AC C
R.M.S. Radius (nm)
b)
Adansonia 0.58 ± 0.00
1.0x10 7 Molar Mass (g/mol)
Mw (g/mol)
Figure 2. a). Refractive index and Mw GPC elution profile of Adansonia digitata polysaccharide.
b). Log-log plot of root mean square radius (Rg) versus molecular mass (Mw) of Adansonia digitata polysaccharide.
1.0x10 8
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5
y = 6.592x + 3.231
a)
ηsp/C
3.5 3
y = -0.244x + 3.305
ln(ηr)/C
2.5 2
SC
ln (ηr)/C, ηsp/C (dL/g)
4
1.5
0.5 0 0
0.05
0.1
5 4.5
b)
4
EP
3.5
0.15
TE D
C (g/dL)
M AN U
1
3 2.5 2
0.2
0.25
y = 0.305x - 0.245 R² = 0.999
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4.5
1.5
1
0.5
0
0
5
10
15
20
1/C (dL/g)
Figure 3: Intrinsic viscosity of Adansonia digitata polysaccharide in 0.1M NaCl solution by (a) Combined Huggins and Kraemer extrapolations; (b) Fedors method , all determined at 25oC.
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1000 6% 5% 4%
viscosity (Pa.s)
10.00
3%
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100.0
a)
1.000 2% 0.1000
SC
0.5%
1.000E-3 1.000E-3
0.01000
0.1000
100
0.1
0.01 0.001
1.00E+00
AC C
0.0001 1.00E-02
1.000 10.00 shear rate (1/s)
TE D
η/ηo
1
b)
EP
10
M AN U
0.01000
1.00E+02
100.0
1000
10000
6% 5% 4% 3% 2% 0.50%
1.00E+04
τأل
Figure 4. a). Viscosity dependence of shear rate and concentration; b). Generalized flow curves of dependence of viscosity on shear rate for different concentrations of Adansonia digitata polysaccharide in water at 25oC
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100
RI PT
10
SC
0.1
M AN U
G', G" (Pa)
1
0.01
0.001
0.01
5% G'
TE D
0.0001 0.1
5% G"
1 Angular frequency (rad/s) 4% G'
4% G"
10
3% G'
100
3% G"
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Figure 5. Frequency sweep showing G′, G″ versus angular frequency, ω for Adansonia digitata polysaccharide at 3%, 4% and 5%, (w/w) solutions in water at 25oC.
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1000
a) 5 deg C 15 deg C 25 deg C
10.00
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35 deg C 45 deg C 55 deg C
1.000
SC
viscosity (Pa.s)
100.0
0.01000 1.000E-3
0.01000
0.1000
6
3 2 1
100.0
1000
10000
y = 5840x + 3.4394 R² = 0.9746
-0.0002
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0 -0.0004
1.000 10.00 shear rate (1/s)
TE D
ln ηo, Pas
4
b)
EP
5
M AN U
0.1000
0
(1/T-1/308),
0.0002
0.0004
K-1
Figure 6. a) Effect of temperature on viscosity-shear rate profiles of 4% Adansonia digitata polysaccharide in water; b) Arrhenius plot of zero shear viscosity versus inverse of absolute temperature
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The manuscript highlights
•
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The molecular mass of Adansonia digitata polysaccharide was determined as 4.01 x 106 g/mol. The polysaccharide macromolecules exhibited random coil conformation in aqueous solution. The intrinsic viscosity was 3.27 dL/g. The polysaccharide contained uronic acid groups which were confirmed by FTIR
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•