Review Atomic Force Microscopy Imaging of Food Polysaccharides

Food Sci. Technol. Res., 16 (1), 1–12, 2010 Review

Atomic Force Microscopy Imaging of Food Polysaccharides

* Takahiro Funami

Texture Design Division, San-Ei Gen F.F.I., Inc., 1-1-11, Sanwa-cho, Toyonaka, Osaka 561-8588, Japan

Received May 4, 2009; Accepted June 3, 2009

Atomic force microscopy (AFM) is a type of scanning probe microscopy that generates images (primarily topographical ones) by scanning the surface of samples with a sharp tip. AFM is applicable to samples with low electric conductivities, and its operating range spans that accessible to both light and electron microscopes, allowing for molecular resolution. These features of AFM enable soft materials to be visualized under natural conditions without harsh or damaging procedures. For food polysaccharides, AFM is capable of visualizing not only dispersed molecules but also molecular assemblies with an advantage over other physical techniques in quantifying the heterogeneity of samples. From these perspectives, AFM is one of the most versatile techniques for obtaining structural information on food polysaccharides, contributing to the progress of this research area. In this article, AFM images of various food polysaccharides are presented along with the usefulness and limitations of this microscopy technique.

Keywords: atomic force microscopy (AFM), food polysaccharides, dispersed molecule, molecular assembly, supermolecular structure

Introduction but also molecular assemblies or supermolecular structures; Atomic force microscopy (AFM), developed by Binning gel precursors, microgels (i.e., local networks and aggre- and Quate in 1986, is a type of scanning probe microscopy gates), and network structures of bulk gels. Here, food hy- that generates images, primarily topographical ones, by scan- drocolloids are defined as a state of edible colloid particles, ning the surface of samples with a sharp tip attached to a 10-1,000 nm in diameter, dispersing in water as a continuous cantilever. This means that AFM generates images by touch- phase, or, alternatively, polysaccharides and proteins them- ing samples rather than observing them. AFM is applicable selves that regulate such a dispersing state in food systems. to samples with low electric conductivities, differing from a Height and width on the AFM images provide information scanning tunneling microscope, and is free from a diffraction regarding the degree of molecular associations in a quantita- limit, allowing for a molecular resolution up to the sub-nano- tive or qualitative manner. Another advantage of AFM over meter level. These features of AFM enable the visualization other physical methodologies is the ability to directly observe of soft materials, including biomaterials and food materials and quantify the heterogeneity of samples. Food hydrocol- under natural conditions without harsh or damaging prepara- loids are from natural sources in most cases and have com- tive and imaging procedures used in conventional electron plex molecular structures with some heterogeneity, involving microscopy methods, including dyeing, drying, and metal various chain lengths and degrees of branching. From these deposition. AFM imaging works both in air and aqueous en- perspectives, AFM is one of the most versatile techniques for vironments, making it possible to visualize samples in a hy- obtaining structural information on food hydrocolloids, con- drated form such that the samples exhibit behaviors similar tributing to the progress of this research area. to those seen in real systems. Many food hydrocolloids, including carrageenan (Gun- As a tool for the study of food hydrocolloids, AFM is ca- ning et al., 1998; Ikeda et al., 2001), gellan gum (Gunning pable of visualizing not only dispersed or isolated molecules et al., 1996; Ikeda, Nitta, Temsiripong et al., 2004; Mor- ris et al., 1999a), xanthan gum (Camesano and Wilkinson, *To whom correspondence should be addressed. 2001), cellulose (Morris et al., 1999b), pectin (Kirby et al., E-mail: [email protected] 2006; Morris et al., 1999b), gum arabic (Cowman et al., T. Funami

2006; Ikeda et al., 2005), soy soluble polysaccharide (Ikeda contact as the scanning proceeds, realizing high resolution. et al., 2005), curdlan (Ikeda and Shishido, 2005), hyaluro- Contact mode is measurement within the repulsive regime of nan (Cowman et al., 1998), amylose (McIntire and Brant, the intermolecular force curve by keeping the deflection of 1999), starch (Ridout et al., 2002), xyloglucan (Ikeda, Nitta, the AFM cantilever constant. One of the drawbacks of this Kim et al., 2004), and gelatin (Mackie et al., 1998), have operation mode is that the sample is subjected to large lateral been imaged by AFM. Adsorption/desorption of milk-based forces as the tip is dragged over it, sometimes damaging and proteinaceous materials (i.e., β-casein, α-lactoalbumin, and displacing molecules, so called “molecular kicking”, of soft β-lactoglobulin) onto/from the interface of O/W type emul- materials even if the spring constant of the tip is extremely sions (Mackie et al., 2001) and also in-situ monitoring of low. Non-contact mode is measurement within the attractive polysaccharides decomposition with enzymes (Thomson et regime of the intermolecular force curve by keeping the non- al., 1994) are novel and interesting targets for the AFM im- contact distance between the tip and sample constant. In this aging recently investigated. operation mode, the cantilever must be oscillated above the In this article, some AFM images of food polysaccharides surface of the sample at a distance in which measurements are presented along with brief descriptions of this micros- are made outside the repulsive regime of the intermolecular copy technique, including the fundamentals, applications, force curve, resulting in relatively poor resolution. It is also usefulness, and limitations, for the best usage in this research difficult to operate this mode in ambient conditions due to a area. thin layer of water molecules on the surface, which may form a capillary bridge between the tip and sample, causing the AFM basics (fundamentals, applications, usefulness, and tip to “jump-to-contact”. Tapping mode, an intermediate be- limitations) tween the contact and the non-contact mode, detects a weak AFM uses a laser beam deflection system in most cases, repulsive force as a result of tapping the sample surface with where the laser is reflected from the back of the AFM canti- an oscillating AFM tip. The oscillation of the tip is gener- lever onto a position-sensitive detector (Fig. 1). The AFM ated by vibrating the AFM cantilever or the cantilever holder cantilever and tip are usually microfabricated from Si or at the resonant frequency, and utilizing a feedback function

Si3N4. When the AFM tip approaches a sample, an attractive in the z-direction to keep the amplitude of vibration constant. “van der Waals” force occurs between the tip and sample, The tip taps the sample surface for a tiny fraction of its oscil- followed by a repulsive force based on the Pauli exclusion lation period, making this operation mode the most suitable principle upon closer approach (Fig. 2). AFM relies on the for soft materials, including food hydrocolloids, due to re- forces between the tip and sample, and these forces, either duced lateral force. The tapping mode is also advantageous attractive or repulsive, are calculated by both the deflection in imaging viscous samples, sometimes leading to poor reso- of the cantilever and its spring constant using the Hookean lution due to adhesion of the sample with the tip. law. AFM imaging modes are differentiated by the force For the visualization of food hydrocolloids, concentra- interaction involved, and three major modes are frequently tions of test solutions usually range from 0.1 to 10 µg/ml used, including contact mode, non-contact mode, and tap- prior to deposition. To observe molecular dispersion or ping mode (or intermediate contact mode); each having isolated individual molecules, very dilute solutions at 0.1 µg/ specific advantages and disadvantages. Contact mode is the ml, for example, should be deposited on a substrate. In most primary and most traditional method for AFM imaging. As the name suggests, the AFM tip and sample remain in close

Fig. 1. Principal of atomic force microscopy (schematic). Fig. 2. Measurement of forces in the AFM visualization (schematic). AFM Imaging of Food Polysaccharides cases, samples deposited are then dried in air for immobiliza- specified accessories, thus reducing capillary force. The type tion. During air drying, condensation and displacement of of liquid or solvent used is not an issue as long as the tip and the molecules may occur through so called “molecular comb- sample are completely immersed. Under-liquid imaging is ing”, resulting in associations and aggregations forming in also advantageous in avoiding excessive dehydration dur- situ on a substrate that are not originally present in test solu- ing scanning. In imaging food polysaccharides, n-butanol is tions; known as a “drying artifact”. A single molecule on an the most favorable since it works as a poor solvent to retain image does not necessarily represent a dispersed single mole- sample architectures on a substrate. For example, AFM vi- cule because it may, for example, involve end-to-end type as- sualization under n-butanol shows a different result from that sociations. Two types of substrates are used in most cases as in air though the nature of network formation is substantially a surface; mica and graphite. Mica is more frequently used the same for deacylated gellan gum (1 µg/ml) in the pres- to visualize food hydrocolloids than graphite due to its higher ence 0.01 mM NaCl (Fig. 4). This is attributable to a lower hydrophilicity. Combination of two substrates is helpful in degree of intermolecular associations under n-butanol as a imaging some food hydrocolloids with amphiphilic charac- result of preventing dehydration during scanning as repre- teristics or surface activity. Each substrate should be used af- sented by a decreased vertical height on the image. ter cleavage of the surface. The surface of mica is negatively Two types of intermolecular associations are presented charged immediately after the cleavage. When negatively- for food polysaccharides, end-to-end type and side-by-side charged samples are deposited onto freshly-cleaved mica, type intermolecular associations (Fig. 5). End-to-end type imaging resolution may sometimes be poor due to insuf- intermolecular associations contribute less to the vertical ficient immobilization, particularly for polyelectrolyte poly- height on the image than side-by-side type associations. The saccharides above their pKa values. Salts, chemical agents degree of molecular associations, particularly through the (e.g., 3-aminopropyltriethoxy silane APTES), and surfactants side-by-side type, is partially deducible from the height in- are used to promote immobilization of these samples onto formation. Care, however, should be taken so that resolution the mica surface by canceling electrostatic repulsions. For in the x-axis is inferior to that in the z-axis. This means that example, the number density of visible fibrils or molecular the width of features is comparable but is not very quantita- bundles increases on the APTES-treated mica in comparison tive as it is due to the curvature radius of the AFM tip, af- to those on freshly-cleaved mica for deacylated gellan gum fecting the contact between the tip and sample and resulting (1 µg/ml) along with increased vertical height observed on in overestimations in most cases (Fig. 6). The “measured the image (Fig. 3). Samples deposited and examined in air widths” are calibrated using the following equations to esti- may remain hydrated with a thin layer of water molecules on mate “real widths” by eliminating the tip broadening effects the surface. When the AFM tip is brought into contact with under an assumption that each molecular feature visualized sample in such a state, water molecules in the hydration layer is in a cylindrical form (Morris et al., 1997): coalesce. This may generate a strong adhesive force between 2 r = wm /16R the AFM tip and sample, damaging and/or displacing sample architectures during scanning. Thus, adhesive forces should wr = 2r be eliminated to obtain reproducible and reliable AFM im- where r and R represent the radii of the cylindrical feature ages. One of the solutions is to image under liquids using and the AFM tip, respectively. Also, wr and wm stand for

On freshly-cleaved mica On APTES-treated mica

Av. height: 0.91 ± 0.19 nm (21.3 CV%) Av. height: 1.02 ± 0.25 nm (24.3 CV%)

Fig. 3. Topographical AFM images of deacylated gellan gum on freshly-cleaved mica or APTES-treated mica surface in air. Concentration of gellan gum was 1 µg/ml in test solutions prior to deposition. Scan size: 1 × 1 µm. T. Funami widths with and without calibration, respectively. and dairy desserts, as a water-holding agent in processed meat products, as well as a stabilizing and a thickening agent AFM images of food polysaccharides in sauces, dressings, and beverages (Piculell, 2006). Three There are two aspects of AFM in studying food polysac- types of carrageenan have been used frequently in the food charides. To correlate with the physicochemical parameters, industry, and kappa- and iota-carrageenan exhibit gelling including weight-average molecular weight Mw and root- properties. Carrageenan is basically composed of an al- mean-square radius of gyration Rg by light scattering, mo- ternating β-(1-3)-d-galactose and α-(1-4)-3, 6-anhydro-d- lecular dispersions should be imaged. To obtain molecular galactose repeating unit, in which the β-galactose is partially dispersions, some techniques are possible for preparing test sulfated at the C4 position for kappa or additionally at the C2 solutions for deposition; polysaccharide samples should be position of the α-anhydrogalactose for iota. dissolved in an aqueous medium in the dilute regime or in AFM imaging in air has elucidated the difference in the a hot state above the sol-gel transition temperature. Instead network structures between kappa- and iota-carrageenan of aqueous media, using organic solvents like dimethyl sulf- (Funami, Hiroe et al., 2007), and the molecular bundles of oxide (DMSO) is an option. On the other hand, to correlate iota-carrageenan seem to be more flexible with higher ho- with macroscopic behaviors, such as rheology (i.e., elasticity, mogeneity in the vertical height on the image (as presented viscosity), molecular assemblies should be imaged because by a smaller CV value) than those of kappa-carrageenan polysaccharides do not function as individual molecules in (Fig. 7). In this study, samples are both in sodium form, and real systems. macromolecular characteristics, including Mw and Rg, are Carrageenan Carrageenan recovered from marine red almost equivalent among the samples. Flexibility of iota- algae is one of the most important polysaccharides with a carrageenan molecules is attributable to the irregularities in wide range of food applications; as a gelling agent in jellies the structures; sulfated (1-4) galactose residue, resulting in

In air Under butanol

Av. height: 0.93 ± 0.22 nm (23.9 CV%) Av. height: 0.84 ± 0.17 nm (20.2 CV%)

Fig. 4. Topographical AFM images of deacylated gellan gum in the presence of NaCl on freshly-cleaved mica surface in air or under n-butanol. Concentrations of gellan gum and NaCl were 1 µg/ml and 0.01 mM, respectively in test solutions prior to deposition. Scan size: 1 × 1 µm.

End-to-end type Side-by-side type Cantilever

Fig. 6. Accuracy of vertical (z-axis) height and inaccuracy of Fig. 5. Intermolecular associations (schematic). horizontal (x-axis) width for AFM imaging. AFM Imaging of Food Polysaccharides kinks to reduce the space occupied by the molecules (Kirby geneity between gellan gum samples as presented by the et al., 1996). The fibrils on the image are composed mainly CV values of the vertical height. The fibrils on the image of nonaggregated double helices for each carrageenan sam- may involve side-by-side type intermolecular associations ple considering a corresponding value previously reported; between the helices locally because the vertical height of ca. 0.9 nm for iota-carrageenan (McIntire and Brant, 1999). a double helix for deacylated gellan gum is reportedly 0.5 For kappa-carrageenan, branching of the fibrils can be also nm by small-angle X-ray diffraction (Yuguchi et al., 1999). visualized with observable ends. For deacylated gellan gum, side-by-side type intermolecular Gellan gum Gellan gum produced by a microorganism associations are enhanced sporadically, causing a marked is a linear anionic heteropolysaccharide with tetrasaccharide increase in the vertical height sometimes up to ca. 2.0 nm. repeating unit of β-d-glucose, β-d-glucuronic acid, β-d- Moreover, a type of double strand is occasionally observable glucose, and α-l-rhamnose as the backbone. Gellan gum in for deacylated gellan gum with two molecular bundles ar- native form is esterified with l-glycerate and acetate at the ranging in parallel in the presence of added potassium (Fig. C2 and C6 (ca. 50%) positions of the (1-3)-linked d-glucose, 9), consistent with the proposed gelation model. Care should respectively (Kuo et al., 1986), and, in most cases, the be taken, however, as this phenomenon tends to happen when products commercially available are in the deacylated form double tipping occurs, where the shaper points on the end of through some alkaline treatments. Although each type of the AFM tip allow higher resolution images of polysaccha- gellan gum has been used in the food industry mainly as a ride chains. As for gellan gum, much more detailed studies gelling agent, physical and textural properties of gels differ have been carried out using well-characterized samples to from each other; the deacylated type forms hard and brittle discuss supermolecular structures in relation to the rheologi- gels in the presence of cations, whereas the acylated type cal behaviors exhibited during gelation (Noda et al., 2008; forms soft and elastic gels even in the absence of cations Funami, Noda et al., 2008; Funami et al., 2009). (Morris, 2006). AFM imaging is applicable not only to single polysac- AFM imaging in air elucidates the difference in the charide systems but also to binary systems. The mixture of network structures between acylated and deacylated gellan deacylated gellan gum and xyloglucan has been visualized gums, and the molecular bundles of the deacylated type seem by AFM (Ikeda, Nitta, Kim et al., 2004). Here, xyloglucan, to be less flexible on the image than those of the acylated recovered from the seed of the tamarind tree, has a molecular type (Fig. 8). In this study, samples are both in sodium form, backbone of (1-4)-linked β-d-glucose, about three-quarters but macromolecular characteristics are different between of which is substituted with α-d-xylose at the C6 position the samples; Mw and Rg for the acylated type is ca. 3.5 and 3 (Nishinari et al., 2000). About one-third of the xylose resi- times larger than those for the deacylated type, respectively. dues are further substituted with β-d-galactose at the C2 po- The results are similar to the case of carrageenan though no sition (Nishinari et al., 2000). Xyloglucan is soluble in water substantial differences are detected in the structural homo- without heating, forming gels only in the presence of alcohol

Iota carrageenan Acylated gellan gum -O-O SO H C 3 2 CCHH OOHH CH 22 O 3 O C O O O CH O 2 1/2 - + CH OH O COO M 2 OH O O O O O CH OSOOSO - O O OH 3 3 n OH OH OH OH OH OH OH O b-D--ggaallaaccttoossee--44--ssuullffaattee 3,6-anhydro-a-D--ggaallaaccttoossee C CH OH 2 n -2-sulfate O Av. height: Av. height: 0.76 ± 0.11 nm (14.5 CV%) 0.70 ± 0.05 nm (7.1 CV%)

-O SO H C 3 2 Deacylated gellan gum Kappa carrageenan CH OH 2 O O - + CH OH COO M CH OH OO 22 2 O O O O O O O O CH OH O OH OH 3 OH n OHOH OOHH OOHH OH OH OH n b-D--ggllaaccttoossee--44--ssuullffaattee 33,,66--aanhnhyyddrroo--a-D--ggaallaaccttoossee

→3)-β-D-Glcp-(1→4)-β-D-GlcpA-(1→4)-β-D-Glcp-(1→4)-α-L-Rhap-(1→

Av. height: 0.92 ± 0.15 nm (16.3 CV%) Av. height: 0.84 ± 0.24 nm (28.6 CV%)

Fig. 7. Topographical AFM images of carrageenan on freshly- Fig. 8. Topographical AFM images of gellan gum on freshly- cleaved mica surface in air. Concentration of carrageenan was 10 cleaved mica surface in air. Concentration of gellan gum was 10 µg/ml in test solutions prior to deposition. Scan size: 1 × 1 µm. µg/ml in test solutions prior to deposition. Scan size: 1 × 1 µm. T. Funami or a substantial amount (> 40 w/w%) of sugar (Nishinari et raphy (SEC) is now used most frequently for determination al., 2000). AFM imaging in air shows that gellan gum forms the molar mass but may be unreliable for some polysaccha- network structures in the mixed system at a concentration rides due to insufficient separation through column(s) with much lower than the gelation threshold of the polysaccharide limited exclusion size, giving overestimation in most cases. alone and indicates that xyloglucan residues swell within Instead of SEC, field flow fractionation (FFF), which sepa- the network formed by gellan gum without participating in rates samples on the basis of the molecular size utilizing both it. This function of xyloglucan leads to synergistic elasticity cross and channel flows, minimizes the problems associated enhancement as a result of increased effective concentration with SEC. Using xanthan gum, FFF is demonstrated to bet- of gellan gum in the system. ter correlate with AFM than SEC in terms of determining Xanthan gum Xanthan gum produced by a microor- molar mass (Figs. 10 & 11). ganism has a linear structure of (1-4)-linked β-d-glucose as Curdlan Curdlan produced by a microorganism is a the backbone as found in cellulose with a trisaccharide side linear neutral homopolymer of d-glucose with β-1, 3-link- chain, comprising mannose, glucuronic acid, and mannose in ages (Harada et al., 1966). Curdlan is insoluble in water at this sequence, on every other glucose. Xanthan gum is solu- neutral pH conditions, but its aqueous dispersion is capable ble in water without heating, and the solutions exhibit pseu- of forming gels by heating alone. Two types of gels have doplastic flow behavior. Viscosity of the solutions is stable been presented, termed low-set gel and high-set gel (Harada over a wide range of temperatures and pH and is resistant to et al., 1987). The low-set gel, forming as a result of heating enzymatic degradation. Moreover, xanthan gum shows syn- the dispersion to 55-60℃ and then cooling to below 40℃, ergistic interactions with locust bean gum or glucomannan is thermo-reversible, whereas the high-set gel, forming as a to form elastic gels and with guar gum to enhance viscosity, result of heating the dispersion to above 80℃, is higher in extending the application of the gum in the food industry thermo-irreversibility against reheating (Funami et al., 1999; (Morris, 2006). Harada et al., 1987; Konno and Harada, 1991). The mecha- AFM imaging enables us to directly determine the con- nism of gel formation is different between the low-set and tour length of certain polymers, particularly those with great- high-set gel (Konno et al., 1994). For the high-set gel, cross- ly extended and stiff fibrous structures, such as xanthan gum, linking between curdlan micelles (Fulton and Atkins, 1980), providing the information on the molecular weight, size, and which are occupied by molecules of multiple-chain helix polydispersity. If the conformation of a polymer is known, (Marchessault and Deslandes, 1979) or triple-stranded helix then its molar mass per unit chain length is known, and the (Kasai and Harada, 1980), is accomplished with hydropho- contour length can be converted into the molar mass, though bic interactions, whereas for the low-set gel, cross-linking several hundred measurements may be required for fairly ac- between curdlan micelles, which are occupied by molecules curate data on the contour length. Size exclusion chromatog- of single helix (Saito et al., 1989), is accomplished with hydrogen bonds. Curdlan also forms gels when its alkaline solutions are neutralized with acids under a static condition (Harada et al., 1987), in which inter- or intramolecular hydrogen bonds are broken and the preparation swells by dispersing curdlan in an alkaline condition, followed by the formation of new hydrogen bonds through neutralization. This type of gel is thermo-reversible, similar to the low-set gel, transforming to the high-set gel upon heating at higher temperatures (Harada et al., 1993). Therefore, dispersing curdlan in an alkaline medium has the same effect on its swelling as heating the polysaccharide dispersions to 55-60℃. That is, multiple modes of molecular conformation and intermolecular associations are involved in the gelation of curdlan. AFM imaging has elucidated the structural heterogeneity of curdlan in an alkaline medium (0.01 M NaOH), con- Fig. 9. Topographical AFM images of deacylated gellan gum in sisting of microfibrils and single molecular chains, partially the presence of added potassium on freshly-cleaved mica surface in air. Concentrations of gellan gum and KCl were 10 µg/ml and dissociated from the microfibrils (Ikeda and Shishido, 2005). 0.01 mM, respectively in test solutions prior to deposition. Scan Heating such a sol forms a densely cross-linked microgel size: 1 × 1 µm. network, in which the partially dissociated single chains AFM Imaging of Food Polysaccharides

(a) 6 6 Mw = 6.6×10 g/m ol (b) Mw = 4.0× 10 g/mol ) ) l V o ( m e / s g ( n o s p s s a e r m I r R a l o M

Contour length on the image: ca. 5.6 mm D. P. = 10,000～12,000 Volume (m l) Volume (ml) 6 ∴MW = 3.2～4.0×10 g/mol (Assuming double helix conformation) Fig. 11. Elusion profiles of xanthan gum by SEC-MALS (a) and FFF-MALS (b). Concentration of xanthan gum was 0.2 mg/ml for SEC and 0.5 mg/ml for FFF. Dot: molar mass; Line: refractive in- dex.

Fig. 10. Topographical AFM images of xanthan gum on freshly- ecules. The AGP fraction adsorbs preferentially onto the sur- cleaved mica surface in air for determination of the contour length of the chains. Concentration of xanthan gum was 0.1 µg/ml in test face of the oil droplets with hydrophobic polypeptide chains solutions prior to deposition. Scan size: 3 × 3 µm. acting as an anchor, while hydrophilic carbohydrate blocks inhibit flocculation and coalescence between the droplets work as cross-linkers between parent microfibrils presum- through steric repulsions. ably through the formation of triple-stranded helices. Results A commercial product of gum arabic from Acasia senegal from AFM are consistent with the macroscopic gelation be- is visualized by AFM in air using mica or graphite as a sur- haviors described above, suggesting multiple steps of heat- face. Spherical lumps are visualized when freshly-cleaved induced gelation for this polysaccharide and also supporting mica is used as a surface with the very occasional occurrence the gelation mechanism known as the fibrous model that has of linear fibrils (Fig. 12). Extended linear arrays of glob- been developed for cold-set gels of carrageenan and gellan ules are visualized, on the other hand, when freshly-cleaved gum through the coil-helix transition (Gunning et al., 1996; graphite is used as a surface with connecting strands (Fig. Gunning and Morris, 1990; Ikeda et al., 2001; Ikeda, Nitta, 12). These developed molecular assemblies are consistent Temsiripong et al., 2004; Morris et al., 1999a). with the wattle blossom type structure model, presumably Gum arabic Gum arabic or gum acacia recovered from from the AGP fraction. Molecular features of gum arabic tree exudate is of commercial importance mainly as a food have been described alternatively by the twisted hairy rope emulsifier. Gum arabic is a complex polysaccharide and model, in which a rod-like molecule with numerous small contains a small amount of proteinaceous materials bound polysaccharide substituents, regularly arranged along a covalently to the carbohydrate moiety, detected as a high highly periodic polypeptide backbone based hypothetically molecular mass (Dickinson et al., 1989; Dickinson et al., on a 10 to 12 residue repetitive peptide motif and forming a 1991; Randall et al., 1988). Three types of macromolecular twisted hairy rope through intermolecular hydrogen bond- fractions have been identified, including arabinogalactan, ing between the substituents (Qi et al., 1991). Molecular arabinogalactan-protein conjugate (AGP), and glycoprotein assemblies consistent with the twisted hairy rope model are (Al-Assaf et al., 2003; Islam et al., 1997; Randall et al., visualized when a different commercial sample of A. senegal 1988, 1989; Williams et al., 1990). The carbohydrate frac- is used (Fig. 13). tion of gum arabic consists mainly of 1, 3-linked β-galactan Polysaccharides that exhibit surface activity and high wa- as the backbone with the galactan branches through the 1, ter-solubility sometimes cause molecular aggregation upon 6 positions and with arabinose, rhamnose, and glucuronic dehydration during sample preparation for AFM, and this is acid in ramified side chains as terminating groups (Islam et the case with gum arabic. A non-ionic surfactant Tween 20 al., 1997; Randall et al., 1989). The AGP fraction, which has been tried in test solutions prior to deposition, preventing amounts to ca. 10% of the whole gum and is explained by aggregation among polymer chains upon air drying and suc- wattle blossom type molecular structures (Connolly et al., cessfully visualizing isolated polymer chains with a reason- 1987) (Fig. 12), plays a major role in emulsifying (Al-Assaf able resolution (Ikeda et al., 2005). In the presence of the et al., 2003; Islam et al., 1997; Randall et al., 1988). This surfactant, gum arabic has predominantly linear structures molecular model predicts a few large polysaccharide blocks with a contour length on the order of 100 nm, presumably along the polypeptide backbone of spheroidal macromol- from the arabinogalactan, the major fraction of this polysac- T. Funami

On freshly-cleaved mica

On freshly-cleaved graphite Wattle blossom model

Fig. 12. Topographical AFM images of gum arabic in air and a Fig. 13. Topographical AFM images of gum arabic on freshly- schematic drawing of the “wattle-blossom” model. Imaging was cleaved mica surface in air. Concentration of gum arabic was 1 µg/ carried out on freshly-cleaved mica and freshly-cleaved graphite ml in test solutions prior to deposition. Scan size: 1 × 1 µm. surface. Concentration of gum arabic was 1 µg/ml in test solutions prior to deposition. Scan size: 2 × 2 µm for mica and 3 × 3 µm for graphite. er than 100 nm, and that the length of long branches ranges from ca. 20 to 80 nm (Ikeda et al., 2005). The imaging has charide. been carried out in the presence of Tween 20 to prevent Soybean soluble polysaccharide Soybean soluble poly- molecular aggregation upon dehydration and to visualize saccharide recovered from defatted soybean after protein isolated polymer chains, confirming that molecular features extraction has been used in the food industry as a stabilizer visualized do not represent overlapping multiple chains but a in acidified milk beverages (Nakamura et al., 2003) and also single branched polymer. as an emulsifier in oil-in-water emulsions. Similar to gum Sugar beet pectin Pectin has been widely used in the arabic, emulsifying properties of this polysaccharide are as- food industry utilizing its gelling, thickening, and stabilizing cribed to the proteinaceous moiety that is covalently bound properties. Pectin products commercially available are ex- to a high molecular weight fraction of the carbohydrate back- tracted from citrus peel and apple pomace in most cases, but bone, adsorbing onto the oil-water interface as an anchor, pectin is also obtained from sugar beet pulp as a byproduct whereas the carbohydrate fraction contributes to emulsion during the extraction of sugar. A common feature of pec- stability through steric repulsions, preventing aggregation or tin molecules is that the backbone consists of α-1, 4-linked coalescence of the oil droplets by forming a hydrated layer d-galacturonic acid units interrupted by the insertion of 1, (Nakamura, Takahashi et al., 2004; Nakamura, Yoshida et 2-linked l-rhamnose in adjacent or alternative positions. The al., 2004; Nakamura et al., 2006). Soybean soluble poly- side chains consist mainly of d-galactose and l-arabinose saccharide is abundant in galactose, galacturonic acid, and as found in galactan, arabinogalactan, and arabinan with a arabinose, in this order, consisting predominantly of galac- considerable chain length, linked glycosidically to the O4 turonan and rhamnogalacturonan as the backbone with long and O3 positions of the rhamnose residue. Physicochemi- lateral polymer chains of 1, 4-linked β-galactan and 1, 3- cal differences between pectin from sugar beet and that from or 1, 5-linked α-arabinan, linked to the rhamnose residues conventional sources include that sugar beet pectin has a within the rhamnogalacturonan backbone (Nakamura et al., higher proportion of neutral-sugar side chains (Williams et 2001, 2002). The degree of polymerization for the galactan al., 2005), a higher content of acetyl groups within the ga- side-chain is reportedly ca. 43-47 on average (Nakamura lacturonic backbone (Rombouts and Thibault, 1986), a high- et al., 2002). An intriguing, highly branched and spherical er content of phenolic esters in the side chains, especially structural feature is found in this polysaccharide, occupying arabinose and galactose, (Colquhoun et al., 1994; Guillon et a relatively compact space in an aqueous system in compari- al., 1989; Ralet et al., 1994; Rombouts and Thibault, 1986), son to an ordinary linear polysaccharide with an equivalent and a higher content of the proteinaceous materials bound to molecular weight. the side chains through covalent linkages (Williams et al., AFM imaging in air has visualized that soybean soluble 2005). Sugar beet pectin does not form gels thermally even polysaccharide has highly-branched, star- or comb-shaped in the presence of a high concentration of soluble solids (e.g., structures, the overall dimension of which is typically small- sugar) at low pH (< 3-4) conditions or in the presence of AFM Imaging of Food Polysaccharides 9

(a) (b)

Fig. 14. AFM images of sugar beet pectin on freshly-cleaved graphite in air. Concentration of sugar beet pectin in test solutions prior to deposition was 10 µg/ml. (a): control sample; (b): processed sample. Scan size: 2 × 2 µm. calcium ions. The main application envisaged for sugar beet is primarily designed to image very fl at surfaces at high reso- pectin in the food industry is thus as an emulsifi er rather than lution, and starch usually generates an effective roughness as a gelling or stabilizing agent. Emulsifying properties of larger than the z-limitation when deposited onto a substrate sugar beet pectin have been attributed predominantly to the as is. One practical solution to this problem is to embed the proteinaceous moiety (Funami, Zhang et al., 2007). starch granules, decreasing the effective roughness of the AFM imaging under n-butanol has visualized liner carbo- surface (Thomson et al., 1994; Baldwin et al., 1997) (Fig. hydrate chains, branched carbohydrate chains, and carbohy- 15). Differences in the surface morphology have been re- drate-protein complexes for sugar beet pectin (Kirby et al., ported for the starch granules between wheat and potato, and 2006). This is the fi rst study demonstrating that the protein the surface protrusions are attributable to the ends of ordered moiety is attached to one end of the carbohydrate chains and clusters of amylopectin side-chains (Baldwin et al., 1997). not randomly. AFM imaging in air has visualized the super- AFM imaging in air elucidates the differences in the molecular structures of sugar beet pectin using two different surface morphology of the starch granules between corn and samples for comparison (Fig. 14); one is the starting material rice (Fig. 16). Hexagonal crystalline structures arranging and the other is a processed one to enhance the emulsifying like fish scales are visualized for corn starch, and streaky properties (Funami, Nakauma et al., 2008). Extended and crystalline structures elongating in a longitudinal direction well-aligned chain confi gurations are visualized for the pro- are visualized for rice starch. These morphological differ- cessed sample, in which the carbohydrate chains seem to in- ences presumably originate from the clusters or blocklets of teract with each other via the proteinaceous moiety detected each starch source, particularly of the amylopectin fraction. as blob features (circled on the image) for polymerization. Results indicate that corn starch comprises predominantly Starch Starch is one of the most abundant food hydro- B-type crystalline structures, whereas rice starch comprises colloids and has been used not only as a main ingredient of A-type crystalline structures. staple foods such as bread and noodles but also as a thick- ener, gelling agent, stabilizer, and fat replacer in processed Conclusion food products. Starch is insoluble in water but swells upon It is diffi cult to understand the macroscopic properties of heating to enhance viscosity in an excess amount of water polysaccharide gels or sols without information regarding (i.e., gelatinization). Although its ultrastructure depends the microscopic molecular assemblies or network structures on the botanical source, particle diameter of starch granules (a) (b) ranges from 1 to 200 µm on average. The size along with Cantilever the morphology of native and swollen granules may deter- Embedding medium mine the suitability of starch in food applications (Virtanen Mica and Autio, 1993). From this perspective, there is increasing interest in imaging intact starch granules rather than constitu- Fig. 15. Schematic drawings representing the diffi culties encoun- tered in imaging large objects. Large starch granules will contact tional polysaccharides; amylose and amylopectin. The rela- the cantilever forcing the AFM tip off the surface as shown in (a). tively large size of the starch granules can be a problem in Embedding starch granules reduces the effective roughness of the AFM imaging due to the z-limitation of the cantilever. AFM surface, making it possible to image the surface as shown in (b). 10 T. Funami

(a) (b)

Fig. 16. AFM amplitude images of the starch granule on freshly-cleaved mica surface in air. (a) normal corn; (b) normal rice. Scan size: 0.5 × 0.5 µm. because polysaccharides do not function as individual mol- atomic force microscopy of hyaluronan: Extended and intramo- ecules in real systems. Food polysaccharides have been uti- lecularly interesting chains. Biophysical Journal, 75, 2030-2037. lized for decades as texture modifiers and quality improving Dickinson, E., Elverson, D.J., and Murray, B.S. (1989). On the film- agents in various food items. However, for the development forming and emulsion-stabilizing properties of gum arabic: dilu- of new products with higher functionality and palatability tion and flocculation aspects.Food Hydrocolloids, 3, 101-114. based on polysaccharide technology, it is necessary to better Dickinson, E., Galazka, V.B., and Anderson, D.M.W. (1991). Emul- understand behaviors of polysaccharide molecules in food sifying behaviour of gum arabic. Carbohydrate Polymers, 14, systems. AFM should be more widely utilized to provide 373-392. strategies for the best usage of food polysaccharides and sub- Fulton, W.S. and Atkins, E.D.T. (1980). The gelling mechanism and sequently advance and innovate areas of the food industry. relationship to molecular structure of microbial polysaccharide curdlan. American Chemical Society Symposium Series, 141, References 385-410. Al-Assaf, S., Katayama, T., Phillips, G.O., Sasaki, Y., and Williams, Funami, T., Funami, M., Yada, H., and Nakao, Y. (1999). Rheologi- P.A. (2003). Quality control of gum arabic. Foods & Food Ingre- cal and thermal studies on gelling characteristics of curdlan. Food dients Journal of Japan, 208, 771-780. Hydrocolloids, 13, 317-324. Baldwin, P.M., Davies, M.C., and Melia, C.D. (1997). Starch Funami, T., Hiroe, M., Noda, S., Asai, I., Ikeda, S., and Nishinari, granule surface imaging using low-voltage scanning electron mi- K. (2007). Influence of molecular structure imaged with atomic croscopy and atomic force microscopy. International Journal of force microscopy on the rheological behavior of carrageenan Biological Macromolecules, 21, 103-107. aqueous system in the presence or absence of cations. Food Hy- Camesano, T.A. and Wilkinson, K.J. (2001). Single molecule study drocolloids, 21, 617-629. of xanthan conformation using atomic force microscopy. Biomac- Funami, T., Nakauma, M., Noda, S., Ishihara, S., Al-Assaf, S., and romolecules, 2, 1184-1191. Phillips, G.O. (2008). Enhancement of the performance of sugar Colquhoun, I.J., Ralet, M.C., Thibault, J.F., Faulds, C.B., and Wil- beet pectin as an emulsifier. Foods & Food Ingredients Journal liamson, G. (1994). Feruloylated oligosaccharides from cell-wall of Japan, 213, 347-356. polysaccharides, Part II. Structure identification of feruloyrated Funami, T., Noda, S., Nakauma, M., Ishihara, S., Takahashi, R., oligosaccharides from sugar-beet pulp by NMR spectroscopy. Al-Assaf, S., Ikeda, S., Nishinari, K., and Phillips, G.O. (2008). Carbohydrate Research, 263, 243-256. Molecular structures of gellan gum imaged with atomic force Connolly, S., Fenyo, J.C., and Vandevelde, M.C. (1987). Hetero- microscopy in relation to the rheological behavior in aqueous geneity and homogeneity of an arabinogalactan-protein: Acacia systems in the presence or absence of various cations. Journal of senegal gum. Food Hydrocolloids, 1, 477-480. Agricultural and Food Chemistry, 56, 8609-8618. Cowman, M.K., Funami, T., Al-Assaf, S., Kudasheve, D.S., Mohan, Funami, T., Noda, S., Nakauma, M., Ishihara, S., Takahashi, R., D., and Phillips, G.O. (2006). Use of atomic force microscopy Al-Assaf, S., Ikeda, S., Nishinari, K., and Phillips, G.O. (2009). to investigate the structure of arabinogalactan proteins. Foods & Molecular structures of gellan gum imaged with atomic force mi- Food Ingredients Journal of Japan, 211, 3, 207-215. croscopy (AFM) in relation to the rheological behavior in aque- Cowman, M.K., Li, M., and Balazs, E.A. (1998). Tapping mode ous systems in the presence of sodium chloride. Food Hydrocol- AFM Imaging of Food Polysaccharides 11

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