Physico-chemical characterisation and functionality of the polysaccharide extracted from the New Zealand black tree fern, Cyathea medullaris (Mamaku) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
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2015
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Massey University
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Abstract
The aim of this thesis was to characterise the polysaccharide extracted from the New Zealand black
tree fern, Cyathea medullaris, or mamaku in Maori using a combination of rheological, structural
and in vivo research techniques. Polysaccharides are biopolymers with diverse functionalities that
have found their way into many applications in the food, cosmetic or pharmaceutical industries.
Novel sources of polysaccharides may have promising functional properties for new or existing
applications, therefore it is essential to have fundamental knowledge of their properties. The
native and endemic New Zealand black tree fern produces mucilage (containing the polysaccharide)
which is extracted from the thick fleshy stem pith of the frond.
Rheological properties of the polysaccharide were characterised using rotational shear, oscillatory
shear, and extensional rheology. The combination of these techniques provided information on how
the polysaccharide deformed under shear, strain and extension. Rotational shear was further
classified into tests for shear- dependent viscosity/normal stresses, time-dependent viscosity, and
shear-history dependent viscosity. The polysaccharide (5% w/w) exhibited shear-thickening
(4-10s-1), positive first normal stress differences coinciding with shear-thickening,
anti-thixotropy (under constant shear with time at shear rates between 4-10s-1), and thixotropy (at
1s-1, pre-sheared at 10s-1) or rheopexy (at 10s-1, pre-sheared at 1000s-1) depending on shear-
history. Oscillatory shear was classified into linear and nonlinear rheology, i.e. small amplitude
(SAOS) and large amplitude oscillatory shear (LAOS) respectively. Under linear strain deformation,
the polysaccharide displayed viscoelasticity and a power-law dependence on concentration for
relaxation time (?s~c3.6). Complex
viscosity did not superimpose on shear viscosity at higher shear rates/angular frequency (nonlinear
region),
therefore not complying with the Cox-Merz rule. The LAOS response in the nonlinear region was
characterised by new large-strain and minimum-strain moduli parameters (G‘L and G‘M), as well
as the traditional first-harmonic storage modulus G‘. The polysaccharide (10% w/w mamaku) was
found to exhibit first a linear viscoelastic region (0.1-20% ?0), followed by strain-softening
(20-800% ?0), then strain hardening (800-2000% ?0) and finally a second strain-softening region due
to viscous flow (>2000% ?0) for all three elastic moduli measurements. Closer examination of
Lissajous plots in the intercycle strain-hardening region revealed deviation from ellipsoidality
i.e. sigmoidal shapes, which were representative of intracycle strain-stiffening. Finally, the
evolution of filament diameter with time and extensional relaxation time were characterised using a
capillary breakup extensional rheometer (CaBER). The polysaccharide exhibited long extensional
relaxation times (4.6s), high extensional viscosities (~104) and large Trouton ratios (~104).
Factors i.e. temperature, urea concentration, cations (ionic strength) and pH were tested to
investigate how changes in the environment would affect the rheological properties of the
polysaccharide. These factors are also intrinsically related to intermolecular interactions which
may be present in the polysaccharide e.g. hydrogen bonding, hydrophobic interaction and
electrostatic attractions. Thus the molecular origin of its rheological behaviour could also be
elucidated through these effects. Shear-thickening was lost at higher temperatures (?50?C) but
enhanced at low temperatures. The peak viscosity during shear-thickening exhibited an Arrhenius‘
Law dependency with an activation energy of flow of ~90 kJ/mol (5% w/w). Hydrogen bonds are
sensitive to temperature and inversely proportion to temperature in the order of kT, which
indicated that hydrogen bonds are likely to be involved in shear-thickening of the polysaccharide.
The addition of urea, a hydrogen-bond disruptor (chaotropic agent) suppressed shear-thickening
completely in 5% w/w mamaku solution at a concentration of 5M. Urea molecules compete for
hydrogen bonding sites with the polysaccharide and lower the lifetime of polymer-polymer
associations. Removal of salts from the native mamaku solution via dialysis resulted in loss of
shear-thickening as well. However, shear-thickening was reinstated upon addition of salts (NaCl,
KCl, N(CH3)4Cl, CaCl2, MgCl2, LaCl3?7H2O, AlCl3?6H2O) back. Mono-,
di- and trivalent cations screen the electrostatic charges on the polysaccharide thus lowering the
viscosity as
the polysaccharide adopts a more compact configuration. In addition, trivalent cations also cause
chain collapse (precipitation) and re-dissolution of the polysaccharide, a phenomenon known as
re-entrant condensation in polyelectrolytes. Lastly, shear-thickening was also recovered in the
dialysed extract at pH 2-4. Similarly, the protons (H+) screen the electrostatic charges which
lowered the viscosity of the polysaccharide. Screening of electrostatic repulsion appeared to
promote shear-thickening rather than ionic cross-linking, since monovalent cations and protons were
able to recover shear-thickening.
Chemical structure is an important identity for any polysaccharide. In addition, the chemical
structure can provide insight as to how the polysaccharide may have participated in
shear-thickening. The native mamaku extract was further purified prior to structural
characterisation via ultracentrifugation, starch hydrolysis, de- proteinisation and ethanol (80%
w/v) precipitation. This method of purification yielded approximately 15% of purified material,
removing most of the starch, minerals and simple sugars from the native extract. The purified
fraction retained its shear-thickening character and had a molecular weight of 1.94 x 106 Da.
Structural characterisation determining monosaccharide composition and glycosyl linkages were
carried out using methylation, HPLC/GC, GC-MS and NMR techniques. The structure of the mamaku
polysaccharide was
suggested to be a glucuronomannan backbone (methylesterified 4-GlcpA (27.9 mol%) with 2,3- (9.2
mol%) and
2,3,4-linked Manp (10.9 mol%)) with branched sugar side-chains of galactose, arabinose, xylose,
non- methylesterified glucuronic acid (8.2 mol%) and other simple sugars at the O-3 and O-4 of the
mannose residues.
Piecing the information obtained from the various characterisation techniques together helped to
elucidate the molecular origin of shear-thickening, anti-thixotropy, strain-hardening and
extensional-hardening. They were postulated to be of the same event, i.e. intra- to intermolecular
association between polysaccharide chains during shear, strain or extension via hydrogen bonding.
Stretching the polysaccharide exposed associative groups within the long chain, which interacted in
a cooperative zip-like manner. The hydrogen bonds were suggested to take place via the hydroxyl
(-OH) groups of mannose or carbonyl/carboxyl groups (-C=O/- COOH) of the glucuronic acids.
Finally, the satiety effects of the mamaku gum were tested in vivo in rats. The functional ability
of the polysaccharide to confer satiety was postulated to arise from its high viscosity as well as
its shear-thickening behaviour, which alters gastric antrocorporal contractions and delays gastric
emptying. Oral gavage of the rats with mamaku gum (15% w/w) showed a significant reduction in short
term food consumption (p<0.05), smaller weight gains (p<0.05), as well as prolonged gastric
emptying (p<0.05) as compared to rats gavaged with water. Therefore the polysaccharide could
potentially be used as a satiety aid in food products.
Biopolymers which exhibit such complex rheological properties that can be easily controlled by
manipulating environmental factors are rarely or never before encountered. Clearly, the mamaku
polysaccharide would find its way into novel applications, starting with satiety enhancers.
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Wee, May Sui Mei