Carbohydrate Polymers 220 (2019) 247–255

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Carbohydrate Polymers

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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. water to reduce contamination by dust particles. Solutions of puka gum

(1% w/w) were prepared in the mobile phase (0.1 M NaCl with 0.02% 2.5. Determination of pKa of puka gum w/w sodium azide), filtered through a 0.2 μm syringe filter (Sartorius, Goettingen, Germany) and injected into the system at a flow rate of The pH of 1% w/w puka gum solution was first adjusted from ˜5to

248 M.S.M. Wee, et al. Carbohydrate Polymers 220 (2019) 247–255

1.7 with 0.1 or 1.0 M HCl before titration with 1 M NaOH. The titration radius is 3 times larger at 183 nm (Yuliarti, Goh, Matia-Merino, curve of pH vs. volume of titrant, Vt, was obtained in the pH range of Mawson, & Brennan, 2015). Lacebark mucilage with a smaller mole- 6 1.6 to 11.2. The first derivative of titration curve i.e. d(pH)/d(Vt) (or cular weight of 2.31 × 10 also has a larger RMS radius of 90.4 nm ΔpH/ΔVt) was also plotted. The volume of titrant at maximum d(pH)/d (Sims, Smith, Morris, Ghori, & Carnachan, 2018). This suggests that −pH (Vt) is the equivalence point volume, Ve. Next, the plot of Vt⋅10 vs puka gum has a compact spherical conformation (see Section 3.3). Vt, i.e. the Gran plot was constructed to determine the linear acidic Gum arabic is reported to have a molecular weight in the range of 5 region. The Ka values in the linear acidic region were calculated using 5–7×10 Da, depending on a range of factors including the species of Eq. (5) and averaged, giving pKa of puka gum using Eq. (6). Acacia, country of origin and degree of purity (Renard, Garnier, Lapp,

−pH Schmitt, & Sanchez, 2012; Sanchez, Renard, Robert, Schmitt, & −pH Vt ∙10 Vt ∙=10KVae ( −⇒= V t ) K a Lefebvre, 2002; Sims & Furneaux, 2003). Puka gum is therefore ap- VV− (5) et proximately 10 times larger and relatively more monodisperse than gum arabic. Molecular weights of some arabinogalactan-proteins are i) pKKa =−log10 a (6) 1.1 × 105 kDa from New Zealand Kanuka honey (Steinhorn, Sims, Carnachan, Carr, & Schlothauer, 2011), ii) 4 × 105 Da from mesquite 2.6. Zeta-potential gum (Lopez-Franco et al., 2012), iii) 1.268 × 105 Da from the tuberous cortex of the white-skinned sweet potato (Ozaki, Oki, Suzuki, & The zeta-potential of the samples was measured using the Malvern Kitamura, 2010), and iv) 0.76 × 105 Da from the fruits of Murr (Peng Zetasizer Nano ZS instrument (Malvern Instruments Ltd.) based on the et al., 2012). The molecular weight of puka gum is therefore con- combination of laser doppler velocimetry (LDV) and phase angle ana- siderably higher than that of many other arabinogalactan-proteins. lysis light scattering (M3-PALS) techniques. For zeta-potential mea- surements at different ionic strength, the samples were prepared by 3.2. Intrinsic viscosity diluting 1% to 0.1% w/w puka gum with the appropriate dialysate such that the same ionic strength is maintained. For zeta-potential mea- Based on Huggins and Kraemer plots of ηsp/c and ln ηrel/c vs. con- surements at different pH, 1% w/w puka gum solution was adjusted centration (Fig. 2a), the average intrinsic viscosity obtained for puka with 1 M or 0.1 M NaOH or HCl to the appropriate pH and diluted down gum in 0.1 M NaCl was 57.03 ± 1.55 ml/g. K′ was calculated to be 1.3 to a concentration of 0.1% w/w. The samples were filtered through and K″ was 1.1 (Table 1). The intrinsic viscosity ([η]w) determined 0.22 μm membrane filter (Sartorius AG, Germany) and 1 ml of the fil- tered sample was injected into a folded capillary cell (DTS1060C, Malvern Instruments Ltd.). The temperature was set and maintained at 20 ± 0.1°C by a Peltier system. All zeta-potential readings were re- ported as the mean and standard deviation of at least three readings.

2.7. Rheology

Rotational rheological measurements were made using a Paar Physica rheometer (MCR 301,Anton-Paar, Graz, Austria) in controlled shear rate (CSR) mode at 20°C ± 0.1 °C (unless otherwise stated). The double gap (DG 26-7 geometry) and C-PTD 200-Peltier system were used. The viscosity curves were determined in the shear rate range of − 0.1-1000s 1 via a log-ramp increase with log time setting of 30 s (in- itial) and 5 s (final). Measurements were carried out in duplicate.

3. Results and discussion

3.1. Molecular weight

The molecular weight of puka gum was previously determined to be 6 4.45 × 10 Da in 0.1 M LiNO3 and a dn/dc of 0.145 ml/g by Sims and Furneaux (2003) using SEC-MALLS. The molecular weight determina- tion of puka gum was repeated in this research using SEC-MALLS coupled with a differential pressure unit (Viscotek), which allowed measurements of intrinsic viscosity and Mark-Houwink parameters at the same time. The mobile phase (solvent) was also changed to 0.1 M NaCl.

The new weight-average molecular weight (Mw) obtained was 5.91 × 106 Da, which is slightly higher than the previous value of 4.45 × 106 determined by Sims and Furneaux (2003). However, the 6 6 polydispersity index (Mw/Mn = 5.91 × 10 /4.79 × 10 ), 1.07, is in good agreement with the previous study i.e. 1.03. The difference in fi molecular weight may be due to the mobile phase used, t model or Fig. 2. a) Intrinsic viscosity of puka gum in 0.1 M NaCl obtained from Huggins ff other factors a ecting the entire sample population rather than het- and Kraemer plots of ηsp/c ( ) and lnηrel/c ( ) vs. concentration respectively; b) − erogeneity within the sample itself. The polydispersity index is close to Intrinsic viscosity of puka gum vs. I 0.5 where I is the total ionic strength ( ); unity, which indicates a high purity and monodispersity for the gum. intrinsic viscosity at infinite ionic strength [η]∞ is obtained by extrapolating the The z-average RMS radius of puka gum is 55.9 nm, which is relatively plot to the y-intercept i.e. 35.1 ± 2.5 ml/g; error bars represent standard de- small for its high molecular weight. For example, pectin extracted from viation (S.D.); Inset: intrinsic viscosity at 0, 0.01, 0.05, 0.1 & 0.5 M NaCl salt the kiwifruit has a similar molecular weight of 3.75 × 106, but the RMS concentrations ( ).

249 M.S.M. Wee, et al. Carbohydrate Polymers 220 (2019) 247–255

Table 1 viscosity, [η]∞ the intrinsic viscosity at infinite ionic strength, S a Intrinsic viscosity values of diluted puka gum solution in 0, 0.01, 0.05, 0.1, 0.25 parameter related to stiffness of polymer, and I the ionic strength of the & 0.5 M NaCl ( ± S.D.). system. The comparison of stiffness between polymers using S values Salt Concentration (M) Intrinsic Viscosity (ml/g) K′ K″ K′-K″ can be made, but only at similar molecular weights or counterion en- − vironment. Fig. 2b shows the plot of intrinsic viscosity vs. I 0.5 where 0 167.2 ± 25.8 3.7 1.8 1.9 [η]∞ of puka gum is 35.1 ± 2.5 ml/g based on extrapolation to the y- 0.01 95.2 ± 6.5 1.4 0.8 0.6 intercept and S is 6.25. In order to avoid the restriction of comparing 0.05 61.4 ± 6.2 1.7 0.9 0.8 0.1 57.0 ± 1.6 1.3 1.1 0.2 only polymers of the same molecular weight, Smidsrod and Haug 0.25 46.3 ± 1.9 1.6 1.0 0.6 (1971) introduced the Smidsrod-Haug stiffness parameter, B, which is 0.5 56.3 ± 0.2 0.4 0.4 0.2 related to the S parameter via Eq. (8) at a fixed ionic strength of 0.1 M NaCl. Using a value of v = 1.3, B is 0.031 for puka gum in 0.1 M NaCl. Polysaccharides with a stiff backbone e.g. xanthan gum have low values using the SEC-MALLS-Viscometer technique was 87.9 ml/g (in 0.1 M of B (0.005) (Tinland & Rinaudo, 1989) while semi-flexible to flexible NaCl), which is higher than the value obtained using Huggins and polysaccharides have higher values e.g. dextran (˜0.21) (Smidsrod, ff Kraemer equations. This discrepancy may be due to di erences in 1971). Therefore puka gum, with a B value of 0.031 is likely to be a measurement techniques and sample preparation, where one was dis- semi-flexible or flexible polymer, absent of a stiff backbone. solved in 0.1 M NaCl solution while the other was dialysed against it, −0.5 which might have resulted in different ionic strengths at equilibrium. [ηη][]=+∞ SI (7)

The intrinsic viscosity of gum arabic in 0.1 M NaCl was reported to be v SB=×([ η ]I=0.1 ) (8) 18 ml/g (Gómez-Díaz, Navaza, & Quintáns-Riveiro, 2008), which is approximately three times lower than puka gum due to its lower mo- lecular weight. However, the intrinsic viscosity of puka gum is sig- 3.3. Molecular conformation nificantly lower when compared to other flexible coil polysaccharides such as the mamaku gum (848 ml/g;) (Goh, Matia-Merino, Pinder, α [ηkM] = v (9) Saavedra, & Singh, 2011) or xanthan gum (7650 ml/g) (Ndjouenkeu, Goycoolea, Morrisa, & Akingbala, 1996). The relationship between viscosity average molecular weight (Mv) The Huggins and Kraemer constants can be used to infer hydro- and intrinsic viscosity [η] was first defined by Mark and Houwink (as dynamic interactions between coils in solution and the quality of the cited in Harding, 1998) using Eq. 9. This equation is known as the solvent. Polymers in good solvents usually have K′-K″ values of 0.3-0.4 Mark-Houwink equation, α is the Mark-Houwink exponent, and k the and 0.5-0.8 for theta solvents (Lapasin & Pricl, 1999). Puka gum has a Mark-Houwink coefficient. Strictly speaking, this relationship is only K′-K″ of 0.2 in 0.1 M NaCl, indicating that the gum is soluble in the valid for monodisperse molecules (Guaita et al., 1991; Manaresi, presence of salts. However, it should be noted that the Kraemer plot Munari, Pilati, & Marianucci, 1988). For polydisperse molecules

(i.e. lnηrel/C vs. concentration in Fig. 2a) increased with increasing (usually polysaccharides), prior fractionation is likely required before polymer concentration, which is in contrary to other polysaccharides the Mark-Houwink rule can be applied although the puka gum is re- ffi reported such as the mamaku gum (Goh et al., 2011). This may be due latively monodisperse (M w/Mn = 1.07). The Mark-Houwink coe cient to concentration-dependent aggregation of the gum. Also, the Huggins and exponent are related to the molecular conformation of the poly- and Kraemer equations were derived for use with flexible coils and non- saccharide chain in a particular solvent, which can be broadly classified ionic polymers instead of branched, spherical polymers which would as compact spheres, random coils or linear chains. The α values are have contributed to inaccuracies. easier to define than K’ (Harding, 1998); expanded chains usually have The intrinsic viscosity of puka gum was further determined at var- high α values and compact spheres with small α values. According to ious ionic strengths (i.e salt concentrations: 0, 0.01, 0.05, 0.1, 0.25 and Brandrup and EImmergut (1975), spheres, random coils, stiff coils and 0.5 M NaCl as listed in Table 1) at the fixed pH of the native gum (pH rods have α values of 0, 0.5-0.8, 1.0 and 1.8 respectively. The Mark- ˜5.0). The intrinsic viscosity is highest at zero ionic strength and con- Houwink constant k determined by the HPLC-Viscotek unit was sistently decreases with salt concentration. Puka gum is therefore an 0.257 ± 0.012 ml/g, and α was 0.375 ± 0.000 i.e. η ⋅ 0.375 anionic polyelectrolyte, resultant of the glucuronic acid residues pre- [ ] = 0.257 M w = 89.0 ml/g in 0.1 M NaCl solution (Fig. 3). sent in the polysaccharide structure as found by Sims and Furneaux Therefore, it is likely to have a spherical conformation in 0.1 M NaCl, (2003). The cations from the salt (Na+) screen the negative charges in consistent with the small RMS radius of 55.9 nm found earlier. This is the polysaccharide, which reduces intramolecular repulsion between polysaccharide chains. The molecule is therefore able to adopt a more compact spherical configuration of lowest entropy. The slight increase in intrinsic viscosity at 0.5 M NaCl may be a result of subsequent ag- gregation between molecules since intermolecular electrostatic repul- sion would be reduced as well. Puka gum is an arabinogalactan-protein, which means that the protein core may also be subject to electroviscous effects (effects of particle surface charge on viscosity of fluid). At native pH (˜5.0, > pKa of most proteins), the carboxylate groups of the protein amino acids are − likely to be in the de-protonated form i.e. −COO which contributes to electrostatic repulsion. Previous amino acid analysis also shows that the protein core consists of 12.9 mol% glutamic acid/glutamine, an amino acid with an additional charged −COOH side group. The presence of all these negatively charged carboxylic groups from both the protein and carbohydrate fractions would contribute to its overall sensitivity to ionic strength.

Pals and Hermans (1952) proposed a relationship between intrinsic Fig. 3. Mark-Houwink plot of Log[η] vs. Log(Mw) for puka gum in 0.1 M NaCl viscosity and ionic strength using Eq. (7), where [η] is the intrinsic ( ).

250 M.S.M. Wee, et al. Carbohydrate Polymers 220 (2019) 247–255 also in agreement with the Smidsrød-Haug stiffness parameter found earlier, which suggested that puka gum is not a stiff polymer but rather one with a flexible conformation. The conformation of puka gum can also be inferred from another 21/2 term, known as the shape factor, p (Eq. 10). The term (Rgz) is known as the root mean square (RMS) radius (of gyration), obtained from static light scattering measurements (see Section 3.1) and Rh is the hydro- dynamic radius obtained from dynamic light scattering (see Section 3.5). The shape factor of puka gum in 0.1 M NaCl is therefore 55.9/ 79.3 = 0.70. According to Lapasin and Pricl (1999), compact mono- dispersed spheres, linear flexible chains and extended coils have shape factors, p, of 0.775, 1.86, and > 2. Therefore, based on the shape factor, puka gum has a conformation that of a monodispersed sphere.

21/2 ()Rgz p = Rh (10) Both the shape factor p and Mark-Houwink exponent α suggest that the conformation of puka gum (in 0.1 M NaCl) is a compact sphere. Gum arabic, is also known to have a spherical conformation due to its highly-branched structure, has values of k = 0.013 and α = 0.54 (Anderson & Rahman, 1967; Renard et al., 2012). It is not likely for hydrocolloids to have the conformation of a perfect sphere i.e. α =0 like the case of globular proteins (Harding, 1998). Instead, the con- formation of puka gum and gum arabic may be a pseudo-spherical one, where the highly branched arabinogalactan (carbohydrate) blocks covalently linked to the flexible polypeptide backbone forms a spherical conformation (Connolly, Fenyo, & Vandevelde, 1987; Dror, Cohen, & Yerushalmi-Rozen, 2006).

3.4. Acid dissociation constant, ka

Using pH titration (Fig. 4), the acid dissociation constant of puka −1.8 gum (1% w/w), Ka was determined to be 10 , and thus a pKa = 1.8. This is similar to the pKa of gum arabic (2.2), as determined based on

Fig. 5. – a) Particle size distributions (z-average diameter; r.nm) of puka gum ( ) and gum arabic ( ) in 0.1 M NaCl; arrows indicate deviation from a monomodal size distribution b) particle size of 0.1% w/w puka gum in 0, 0.05, 0.1, 0.25 & 0.5 M salt (NaCl) concentration ( ); c) particle size of 0.1% w/w puka gum in pH 1, 2, 3, 4, 5.5 (native), 7.5, 9, 10, 11.5 ( ); error bars represent standard deviation (S.D.).

turbidity changes during complexation with whey protein (Weinbreck et al., 2003).The glucuronic acid groups in puka gum are most likely to

be responsible for its acidic properties. Understanding the pKa of puka gum allows better prediction of its properties and functionality at dif-

ferent pH values. At pH > pKa (1.8), the carboxyl groups are dis- sociated and exist as COO-. At pH < pKa (1.8), the carboxyl groups are largely protonated and exists as COOH. Properties such as solubility, cation-binding or protein-polysaccharide complexation will be affected

by pH, especially if the thresholds of pKa are crossed. For example, Fig. 4. a) pH titration curve ( ) and slope of titration curve ( ) of 1% w/w coacervate formation is based on the electrostatic interaction between puka gum with 1 M NaOH at 20°C; b) Gran plot with linear fit at the linear the oppositely charged puka gum and WPI. At pH below pKa, (< 1.8), acidic region ( ). both puka gum and WPI would be positively charged and complex

251 M.S.M. Wee, et al. Carbohydrate Polymers 220 (2019) 247–255 coacervation would be hindered (Wee et al., 2014).

3.5. Particle size and distribution

The particle size and distributions of 0.1% w/w puka gum (and gum arabic) were measured using dynamic light scattering (Fig. 5a). The z- average particle size puka gum and gum arabic in 0.1 M NaCl are 79.3 ± 1.6 nm and 28.6 ± 0.3 nm respectively. The polydispersity index are 0.446 ± 0.019 and 0.370 ± 0.010 respectively. The size distribution curves are very similar for both puka gum and gum arabic, with a distinctive mean peak but the small kinks in the distribution curve as indicated by the arrows in Fig. 5a indicate the samples are not strictly monomodal. This is supported by the polydispersity indices of the gums, both similarly at around 0.4 (PDI of 1 represents a highly polydispersed sample). Therefore there may be another species with a slightly larger size, possibly the protein-rich fraction as found in gum arabic (Connolly, Fenyo, & Vandevelde, 1988). The particle size of puka gum is approximately three times as large as gum arabic, due to its larger molecular weight. The PDI of both gums are, however, relatively similar as demonstrated by the size distribution curves. On increasing salt concentration from 0 to 0.1 M NaCl, the particle size of 0.1% w/w puka gum decreases approximately by half from ˜165 to ˜85 d.nm. The presence of salt ions (Na+) screens the negatively charged groups on the polysaccharide, which reduces intramolecular electrostatic repulsion therefore decreasing the particle size. However, on further increasing salt concentration to 0.5 M NaCl, the particle size increases again to ˜130 nm. This is probably due to intermolecular ag- gregation between the neutralised puka gum particles, which results in the size increment. This agrees with the decreasing intrinsic viscosity of the gum with increasing ionic strength (Table 1) as discussed pre- viously, as well as the higher intrinsic viscosity observed at 0.5 M NaCl. With the exception of pH 1, puka gum shows the largest particle size Fig. 6. Zeta-potential of 1% w/w puka gum with a) NaCl and b) pH (inset: slow at its native pH (˜5.5) and becomes smaller upon either increasing or acidification with glucono-delta-lactone instead of HCl); error bars represent decreasing its pH. Similar to increasing salt concentration, decreasing − standard deviation (S.D.). the pH protonates the carboxyl groups from COO to COOH which screens the charges, resulting in less intramolecular electrostatic re- was also measured using slow acidification with glucono-delta-lactone pulsion and a smaller particle size. At pH 1 (< pKa), the polysaccharide is largely protonated which can lead to intermolecular aggregation and (GDL). For formation of complex coacervates with whey protein isolate, fi hence the large particle size. However, the particle size decreases as slow acidi cation is required (Wee et al., 2014) and therefore also in- well with increasing pH from native pH, even though an increasing cluded in this study. An unusual phenomenon was observed, where the particle size was expected with further intramolecular electrostatic re- zeta-potential abruptly approached neutrality near native pH (Fig. 6b fi pulsion at higher pH. Zeta-potential measurements with pH (Fig. 6b) inset). This was not detected when the gum was acidi ed with HCl. The show that the polysaccharide is still negatively charged at high pH, pH of the solution is unstable when adjusted in this region, and the although the large particle size at native pH in particular could also be neutral charge at this pH could possibly explain the large particle size attributed to sudden reduction of zeta-potential (Fig. 6b inset), resulting detected (Fig. 5c). in molecular aggregation. The reduction in particle size is therefore not likely to have been related to charge-screening effects. In this case, size 3.7. Rheological properties reduction could be related to structural changes to the protein moiety of the polysaccharide or an increase in protein solubility in sodium hy- The viscosity profiles of puka gum at concentrations from 0.2 to droxide. 25% w/w are shown in Fig. 7a. The gum exhibits a Newtonian flow (i.e. viscosity independent of shear rate) at low concentrations (0.2–2% w/ 3.6. Zeta-potential w) and progressively becomes shear-thinning as concentration in- creases (> 2% w/w). However, compared to other random coil poly- Zeta-potential measurements show that 1% w/w puka gum is ne- saccharides, the shear-thinning behaviour is considerably less distinct gatively charged at native conditions i.e. 0 mM salt and pH ˜5. The zeta- due to its spherical conformation (Tirtaatmadja, Dunstan, & Boger, potential indicates the charge density of puka gum. Compared to 2001). The (zero-shear) viscosity of puka gum is also very low, at < − strongly acidic polysaccharides e.g. alginate or pectin with zeta-po- 0.01 Pa.s (at shear rate of 1s 1) for 1% w/w concentration in com- tentials in the range of −50 mV (Bengoechea, Jones, Guerrero, & parison to other random coil polysaccharides such as carrageenan. At McClements, 2011), the zeta-potential of puka gum (˜−35 mV) shows 1.5% w/w, the zero-shear viscosity of lambda carrageenan is ˜3 Pa.s that it is a relatively weaker polyelectrolyte. However, it is fairly similar (Morris, Cutler, Ross-Murphy, Rees, & Price, 1981), making it at least a to 0.1% w/w gum arabic, with a zeta-potential of −30 mV (results not hundred times more viscous than puka gum. Its extremely low viscosity shown). The charge-screening effect in the presence of salt is also allows a high concentration puka gum solution to be achieved as few shown by the reduction in zeta-potential with salt concentration polysaccharides are able to be solubilised beyond 25% w/w in solution. (Fig. 6a). The zeta-potential plateaus after around 0.25 M NaCl, in- Dextran, a highly branched compact polysaccharide with molecular dicating that the salt ions were in excess of the charged groups at this weight of ˜2×106 Da is one such example, exhibiting low viscosity, concentration or more. The zeta-potential of puka gum at different pH Newtonian flow behaviour even at 30% w/w concentration

252 M.S.M. Wee, et al. Carbohydrate Polymers 220 (2019) 247–255

()ηη− η =+η 0 ∞ ∞ [1+ (λγ˙ )]1−n (11)

3.7.1. Effect of temperature, salt and pH The effects of salt, temperature and pH on the flow behaviour of puka gum were tested. Food systems are commonly subjected to changes in ionic strength, temperature and/or pH as a result of pro- cessing conditions and presence of other ingredients. Therefore, the effect on flow behaviour (bulk property) at various conditions would be a good indicator of its performance and stability in practical applica- tions. A clear trend on the effect of temperature on flow behaviour is − observed (Fig. 7a). As expected, the viscosity e.g. at shear rate of 1s 1

(η1.0) decreases with increasing temperature, due to the increased mobility of the polysaccharide chains in solution. By applying Ar-

rhenius’ law to η1.0 with temperature, the activation energy of viscous flow, EA, obtained is 19.0 kJ/mol. This is similar to other poly- saccharides such as pectin (6.7–13.5 kJ/mol) (Arslan & Kar, 1998) and gum arabic (17–20 kJ/mol) (as cited in Peter, Glyn, Alistair, & Shirley, 2010). The overall shear-thinning behaviour is preserved from 5 to

80°C, as indicated by only a slight change to EA at η1000.0 of 13.8 kJ/ mol. The viscosity of puka gum decreases in the presence of salt (Fig. 8b). This is attributed to the charge-screening effect of the Na+ ions as previously demonstrated by the decrease in negative zeta-potential in the presence of salt (Fig. 6a). Despite larger particle sizes at 0.25 and 0.5 M salt concentrations, these were, as discussed previously, due to intermolecular aggregation which would be easily broken up under − shear. In 0.1 M NaCl, the viscosity of puka gum at shear rate of 1s 1 reduces from approximately 0.28 to 0.24 Pa.s. Effects of ionic strength on viscosity plateaued at 0.25 M NaCl, in line with zeta-potential results Fig. 7. a) Viscosity curves of puka gum solutions at 0.2–25% w/w at 20°C; (—) (Fig. 6a). viscosity curves fitted with Cross’ equation; b) Concentration dependence of Fig. 8c shows the viscosity profiles of puka gum in various pH zero shear specific viscosity on coil overlap parameter, c[η]. ranging from pH 1 to 12. The viscosity is relatively stable in alkaline pH, except for a slight decrease in viscosity at pH 12, possibly due to (Tirtaatmadja et al., 2001). The rheological properties of puka gum are polysaccharide degradation. Since viscosity is not affected at alkaline – also similar to gum arabic low viscosities at high concentrations pH, the decrease in particle size (Fig. 5c) is likely due to disaggregation (soluble up to 50% w/w), and mostly Newtonian to mildly shear- or solubility effects rather than changes in conformation and hydro- thinning behaviours (Sanchez et al., 2002, 2018). Nonetheless, gum dynamic radius. However, puka gum is not as stable in acidic pH. A fi arabic is still signi cantly less viscous than puka gum due to its lower small reduction in pH from 4.6 (native) to 3.0 resulted in significant molecular weight and intrinsic viscosities. At a concentration 10% w/w, viscosity changes and drops further at lower pH. In this case, the de- the viscosity of puka gum is approximately 0.3 Pa.s (at shear rate of crease in viscosity could be attributed to charge-screening effects and −1 1s ) as compared to 0.09 Pa.s of gum arabic (Mothe & Rao, 1999). therefore reducing hydrodynamic radius, agreeing with particle size Although it may not be practical to use puka gum as a thickener, its low (Fig. 5c) and zeta-potential (Fig. 6b) data, or additional acid hydrolysis viscosity at high concentrations may be useful where low system visc- of the acid-labile arabinofuranose residues (Aspinall & Whitehead, osities are required. 1970; McGarvie & Parolis, 1981). Fig. 7b shows the concentration dependence of zero-shear viscosity of puka gum as characterised by plotting the log of zero-shear viscosity 4. Conclusion against the coil overlap parameter i.e. logηsp0 vs. log c[η]. The zero- shear viscosity was estimated by fitting Cross’ Eq. (11) to the viscosity The molecular and physicochemical properties of puka gum char- curves, whereby η and η∞ are the plateau viscosities at zero- and high- 0 acterised by various viscometry, light scattering, velocimetry and po- shear rates, λ is the relaxation time and (1-n) is the value of the slope tentiometric titration techniques are summarised in Table 2. Based on between zero- and high-shear viscosities. Two concentration regions i.e. molecular parameters and flow behaviour, puka gum is likely to have a dilute and semi-dilute was observed for puka gum (Fig. 7b), and a spherical conformation in solution. The spherical conformation resulted power law equation (y = a⋅xb) was fitted to the data for each con- in low solution viscosity, Newtonian or slightly shear-thinning flows, centration region. The exponent of concentration dependence (b) in the and high solubility up to 25% w/w concentration. Most poly- dilute and semi-dilute region is 1.4 and 2.3 respectively. The b value of saccharides, such as pectin or guar gum, have a flexible or semi-flexible random coil polysaccharides are typically 1.4 and 3.3 in the dilute and coil type conformation, and therefore the functional properties of puka semi-dilute regions, independent of polysaccharide type (Morris et al., gum are likely to be distinctly different from these types of poly- 1981). The exponent of concentration dependence in the semi-dilute saccharides. On the other hand, puka gum is similar to gum arabic in region for puka gum is smaller, likely due to its spherical conformation terms of molecular structure, whereby gum arabic also has a spherical which occupies a smaller hydrodynamic volume. The coil overlap conformation. Compared to gum arabic, puka gum has a larger mole- concentration of puka gum (the intersection between the two fit lines) cular weight, hydrodynamic radius and particle size, which makes it was estimated as 15% w/w. more viscous at the same concentrations. Its charge density (or zeta- potential) is similar (˜−30 mV), making both puka gum and gum arabic

253 M.S.M. Wee, et al. Carbohydrate Polymers 220 (2019) 247–255

Table 2 Summary of molecular and physicochemical properties of puka gum.

SEC-MALLS

6 Weight-average molecular weight, Mw 5.911 × 10 Da

Polydispersity, Mw/Mn 1.074 RMS radius 55.9 nm Viscometry (in 0.1 M NaCl) Intrinsic viscosity, [η] 57.0 ± 1.6 ml/g Huggins’ constant, K’ 1.3 Kraemer’s constant, K” 1.1 Parameter B 0.031 Mark-Houwink coefficient, k 0.257 ± 0.012 ml/g Mark-Houwink exponent, α 0.375 Coil overlap concentration 15% w/w Shape factor, p 0.70 Dynamic light scattering Particle size 79.2 ± 1.6 d.nm Polydispersity index 0.446 Physicochemical properties

pKa 1.8 Zeta-potential (0 mM salt) ˜−35 mV

Acknowledgements

This research was supported in part by the Massey University Doctoral Scholarship for May Wee. The authors would also like to thank Kevin Tan for help with determining the acid dissociation constant

(pKa) of puka gum.

References

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