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Carbohydrate Polymers 220 (2019) 247–255

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Carbohydrate Polymers 220 (2019) 247–255 Carbohydrate Polymers 220 (2019) 247–255 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Molecular, rheological and physicochemical characterisation of puka gum, an arabinogalactan-protein extracted from the Meryta sinclairii tree T ⁎ May S.M. Weea,b, Ian M. Simsc, Kelvin K.T. Goha, Lara Matia-Merinoa, a School of Food and Advanced Technology, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand b Clinical Nutrition Research Centre (CNRC), Singapore Institute of Clinical Sciences (SICS), Agency for Science, Technology and Research (A⁎STAR), Singapore c The Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Road, Lower Hutt 5040, New Zealand ARTICLE INFO ABSTRACT Keywords: A water-soluble polysaccharide (type II arabinogalactan-protein) extracted from the gum exudate of the native Arabinogalactan-protein New Zealand puka tree (Meryta sinclairii), was characterised for its molecular, rheological and physicochemical Polysaccharide 6 properties. In 0.1 M NaCl, the weight average molecular weight (Mw) of puka gum is 5.9 × 10 Da with an RMS Gum exudate, Meryta sinclarii radius of 56 nm and z-average hydrodynamic radius of 79 nm. The intrinsic viscosity of the polysaccharide is Puka 57 ml/g with a coil overlap concentration 15% w/w. Together, the shape factor, p, of 0.70 (exponent of RMS radius vs. hydrodynamic radius), Smidsrød-Haug’s stiffness parameter B of 0.031 and Mark-Houwink exponent α of 0.375 indicate that the polysaccharide adopts a spherical conformation in solution, similar to gum arabic. The pKa is 1.8. The polysaccharide exhibits a Newtonian to shear-thinning behaviour from 0.2 to 25% w/w. Viscosity − of the polysaccharide (1 s 1) decreases with decreasing concentration, increasing temperature, ionic strength, and at acidic pH. 1. Introduction Plant gum exudates usually consist of polysaccharide material in part or whole, many of which have been extracted and used in food Meryta sinclarii (family Araliaceae), also known as ‘puka’ in Māori, is applications such as gum arabic (from Acacia senegal; E414), gum tra- a small tree endemic to the Three Kings Island in New Zealand, com- gacanth (Astragalus gummifer; E413), gum karaya (Sterculia urens; E416) monly found in the Hen and Chicken islands and along coasts. It is and gum ghatti (Anogeissus latifolia; GRAS). They have been used in widely cultivated in the North Island of New Zealand as garden plant. dairy products, baked goods, beverages, dressings and sauces as stabi- Its distinctively large thick glossy leaves spanning up to 30–50 cm long lisers, emulsifiers and thickeners (Nussinovitch, 2010). Their uses are, and 20 cm wide, clustered fruits and flowers, and tall trunks up to 8 m however, not limited to only food applications, but the pharmaceutical high makes it easily distinguished from other New Zealand plants industry as well for drug delivery or as bioadhesives (Ololade, 2018; (Foster, 2008). When wounded, the trunk of the puka tree exudes a gum Salih, 2018). in defence to the external stress which dries up to a glassy resin (Fig. 1). The demand for gum arabic has been steadily increasing due to its Sims and Furneaux (2003) extracted and isolated the polysaccharide numerous applications in food products. It is one of the few poly- fraction from the gum and further studied its structure. The poly- saccharides with actual emulsifying activity, owing to the protein saccharide was found to be a type II arabinogalactan-protein (AGP), moiety on the polysaccharide (Randall, Phillips, & Williams, 1988). with > 95% w/w carbohydrate and 2% w/w protein. Constituent sugar Recently, it has also been extensively used in studies on protein-poly- and linkage analyses, together with 1H and 13C NMR spectroscopy, saccharide complexation and coacervate formation (Weinbreck, de revealed a highly branched backbone of 1,3-linked β-D-galactopyr- Vries, Schrooyen, & de Kruif, 2003). The weak polyelectrolyte nature of anosyl (Galp) residues, with side-chains made up of α-L-arabinofur- the polysaccharide makes it suitable for electrostatic interactions with anosyl- (Araf) containing oligosaccharides, terminated variously by α- proteins such as whey protein without resulting in precipitation. The L-rhamnopyranosyl (Rhap), α-L-Araf, β-L-arabinopyranosyl (Arap) and compositional and structural similarity of puka gum (PG) to gum arabic 4-O-methyl-β-D-glucuronopyranosyl (4-O-Me-GlcpA) residues. Its mo- may make it a potential substitute for gum arabic in certain applica- lecular weight was determined to be 4.45 × 106 Da with a low poly- tions, and it has been demonstrated to form highly viscous coacervates dispersity index of 1.03. with whey protein isolate (WPI) (Wee et al., 2014). To date, the ⁎ Corresponding author. E-mail address: [email protected] (L. Matia-Merino). https://doi.org/10.1016/j.carbpol.2019.05.076 Received 7 March 2019; Received in revised form 4 May 2019; Accepted 25 May 2019 Available online 26 May 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved. M.S.M. Wee, et al. Carbohydrate Polymers 220 (2019) 247–255 Fig. 1. Visual appearance of puka gum exudate resin (left) and freeze-dried puka gum (right). molecular and rheological properties of puka gum have yet to be 0.5 ml/min with an automatic syringe injector. The light scattering and characterised, which would be important to further understand the specific viscosity data was recorded and analysed using Astra software mechanism of PG-WPI coacervate formation, and its use in other ap- (version 5.3.4.20, Wyatt Technology) and the previously determined plications. Therefore, the aim of this present study was to characterise dn/dc value of 0.145 ml/g. these properties, namely molecular weight, molecular conformation, particle size, polydispersity, hydrodynamic radius, intrinsic viscosity, 2.3. Intrinsic viscosity charge density, acid dissociation constant (pKa), flow behaviour, and the effect of salt and pH on some of these properties. Samples for intrinsic viscosity determination were prepared by hy- drating freeze-dried puka gum in milliQ water (1% w/w) with 0.02% 2. Methodology w/w sodium azide to prevent microbial growth. The gum solution (30 g) was dialysed (6 –8000 MWCO; SpectraPor) against milliQ water 2.1. Isolation of puka gum or 0.01, 0.05, 0.1, 0.25, 0.5 M NaCl solutions (1 L) at 20°C for 48 h under continuous stirring. The samples were further diluted isotonically Puka gum was obtained from Meryta sinclairii trees in Gracefield, with the respective dialysate to final concentrations (c) of between Lower Hutt, Wellington (41.2353 °S, 174.9177 °E). The isolation of the 0.1–1.0% w/w. Efflux time of the sample (t) and the solvent (ts) were polysaccharide from the tree exudate was carried out by Sims and recorded using a size 75 Cannon-Ubbelohde calibrated capillary visc- Furneaux (2003) and described in the same paper. Briefly, the gum ometer (Cannon Instrument Co., PA, USA) at 20 ± 0.5°C. tears were obtained from the trunk of wounded Meryta sinclairii trees Measurements were taken a minimum of three times such that at least (25.0 g) and dispersed in hot water (250 ml, 80°C) for 1 h to dissolve three measurements were within ± 1.0 s of each other. Relative visc- fi completely. The hot solution was ltered under pressure (Whatman GF- osity (ηrel) and specific viscosity (ηsp) were determined empirically B glass fibre filter) and freeze-dried (crude gum). The crude gum was re- using Eqs. (1) and (2). Intrinsic viscosity [η] was subsequently de- dissolved in distilled water, dialysed against distilled water through a termined by constructing the Huggins and Kraemer plot using Eqs. (3) 12–14,000 MWCO membrane for 48 h and then freeze-dried again and (4) and extrapolating to zero concentration to obtain intrinsic (Fig. 1). The yield of filtered and freeze-dried puka gum is approxi- viscosity at the intercept. The Huggins (K’) and Kraemer (K”) constants mately 71% w/w of the original exudate (Sims & Furneaux, 2003). were obtained from the slope of the plots. η ==ηη// tts (1) 2.2. Size exclusion chromatography coupled with multi angle laser light rel s scattering and viscometry (SEC-MALLS-viscometry) ηsp =−()/ηηηs s = η rel −1 (2) The determination of the parameters for molecular weight, mole- 2 ηsp/[]'[]cηKη=+∙∙c (3) cular conformation, intrinsic viscosity, radius mean square (RMS), ffi 2 Mark-Houwink coe cient (K) and exponent (a) were done using a high lnηcrel /=+″∙∙ [] η K [] ηc (4) performance liquid chromatography (HPLC) system coupled to a multi- angle laser light scattering photometer (Dawn Heleos 8+, Wyatt Technology Corp., CA, USA), differential refractive index (DRI) detector 2.4. Particle size (Optilab rEX, Wyatt Technology), and viscometer (ViscoStar II, Wyatt Technology). A size-exclusion column (OHpak SB-804 HQ, Shodex, Particle size measurements of 0.1% w/w puka gum solution (with Tokyo, Japan) was used to separate the molecular species in puka gum. 0.02% w/w sodium azide) were made using dynamic light scattering The mobile phase was 0.1 M NaCl solution prepared using Milli-Q water (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). The and 0.02% w/w sodium azide, filtered through 0.22 μm membrane samples were diluted from the samples used for intrinsic viscosity filter (Millipore Corp., MA, USA) followed by a 0.025 μm membrane analysis using the dialysate (i.e. isotonic dilution). The samples were filter (Millipore Corp., MA, USA). All glassware for use in sample and measured in disposable polystyrene cuvettes at a temperature of mobile phase preparation were soaked overnight in Pyroneg solution, 20 ± 0.02 °C. The refractive index of the dispersant i.e. water used was acid-washed in 5% w/v nitric acid and rinsed thoroughly with Milli-Q 1.33.
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