Michael J. Gidley

Loss of crystalline and molecular order during starch gelatinisation: Origin of the enthalpic transition

The disruption of molecular oders which occur during the gelatinisation of starch granules has been studied by isolating dried samples from maize, waxy maize, wheat, potatoe, and tapioca starches after defined thermal pre-treatments. Residual molecular and crystalline order was quantified by 13C-c.p.-m.a.s.-n.m.r. spectroscopy and powder X-ray diffraction, respectively, and the results compared with residual gelatinisation enthalpy determined by d.s.c. For native starches, molecular (double-helical) order was significantly greater than crystalline order. Molecular and crystalline order were both found to correlated with the residual enthalpy of gelatinisation following thermal pre-treatment, indicating that both levels of structure are disrupted concurrently during gelatinisation. From the data obtained, predicted enthalpy values for the disruption of fully ordered crystalline analogues of the starches studied were calculated, and compared with values for essentially fully ordered and crystalline model material. This comparison suggests that the enthalpy of gelatinisation primarily reflects the loss of molecular (doube-helical) order.

Crystallisation of malto-oligosaccharides as models of the crystalline forms of starch: minimum chain-length requirement for the formation of double helices

The multigram preparation of malto-oligosaccharides of average d.p. ∼11, by the debranching of glycogen using Cytophaga isoamylase is described. Debranched glycogen and fractions derived therefrom readily crystallise from hot, concentrated aqueous solution to give 40-70% of crystalline materials having sharp X-ray diffraction patterns characteristic of A-, B-, and C-type (intermediate) starch polymorphs. The polymorphic form obtained is dependent on chain length, concentration, and temperature, the A-type being favoured by shorter chain-length, higher concentration, and higher crystallisation temperature. For pure oligomers, the minimum chain-length required for crystallisation (formation of double helices) is 10. In the presence of longer chains, oligomers as short as maltohexaose can co-crystallise. These results explain the known differences in aggregation properties of glycogens and amylopectins.

Conformations and interactions of pectins. I. Polymorphism between gel and solid states of calcium polygalacturonate

Ca2+ binding to poly-d-galacturonate (the main backbone sequence of the polysaccharide pectin from plant cell walls) has been investigated by equilibrium dialysis and circular dichroism to elucidate the nature of conformational ordering and chain association in the sol-gel transition. Of the total stoichiometric requirement of bound calcium, only (50 ± 5)% is resistant to displacement by swamping concentrations of univalent counterions. Closely similar behaviour has been reported for poly-l-guluronate (derived from the polysaccharide alginate) and was attributed to site-binding of Ca2+ within dimers of chains of 21 helical symmetry. The circular dichroism changes that accompany Ca2+ binding are also closely similar for the two polymers when allowance is made for their near mirror-image stereochemistry by inversion of sign. When polygalacturonate gels, with calcium as sole or principal counterion, are dried to solid films, very profound circular dichroism changes occur, suggesting that the chain conformation and/or packing undergoes some modification during interconversion between gel and solid states. No such circular dichroism changes are seen for polyguluronate, consistent with the persistence of similar (21) chain conformations and packing for this polysaccharide in both the gel and solid state. On the basis of all this evidence, we propose: 1. (1) The co-operative binding of Ca2+ in polyguluronate and polygalacturonate gels, is through “egg-box” complexes with the polysaccharide chains in analogous 21 conformations. 2. (2) Drying of calcium polygalacturonate gels, but not polyguluronate gels, is associated with a polymorphic phase transition, and it is for this reason that diffraction studies on dried films show the 31 helix.

Heterogeneity in the chemistry, structure and function of plant cell walls

Higher plants resist the forces of gravity and powerful lateral forces through the cumulative strength of the walls that surround individual cells. These walls consist mainly of cellulose, noncellulosic polysaccharides and lignin, in proportions that depend upon the specific functions of the cell and its stage of development. Spatially and temporally controlled heterogeneity in the physicochemical properties of wall polysaccharides is observed at the tissue and individual cell levels, and emerging in situ technologies are providing evidence that this heterogeneity also occurs across a single cell wall. We consider the origins of cell wall heterogeneity and identify contributing factors that are inherent in the molecular mechanisms of polysaccharide biosynthesis and are crucial for the changing biological functions of the wall during growth and development. We propose several key questions to be addressed in cell wall biology, together with an alternative two-phase model for the assembly of noncellulosic polysaccharides in plants.

Production of very-high-amylose potato starch by inhibition of SBE A and B

High-amylose starch is in great demand by the starch industry for its unique functional properties. However, very few high-amylose crop varieties are commercially available. In this paper we describe the generation of very-high-amylose potato starch by genetic modification. We achieved this by simultaneously inhibiting two isoforms of starch branching enzyme to below 1% of the wild-type activities. Starch granule morphology and composition were noticeably altered. Normal, high-molecular-weight amylopectin was absent, whereas the amylose content was increased to levels comparable to the highest commercially available maize starches. In addition, the phosphorus content of the starch was increased more than fivefold. This unique starch, with its high amylose, low amylopectin, and high phosphorus levels, offers novel properties for food and industrial applications.

Conformations and interactions of pectins: II. Influence of residue sequence on chain association in calcium pectate gels

Abstract In the accompanying paper (Morris et al. , 1982) we present evidence of Ca 2+ -induced association of poly- d -galacturonate sequences from pectin into dimers of 2 1 chain symmetry, with co-operative (“egg-box”) binding of Ca 2+ on specific sites along the interior faces of each chain. We now investigate the role in calcium pectate gel networks of other structural features, in particular methyl esterification and 1,2-linked l -rhamnosyl residues in the polymer backbone. Acid hydrolysis of citrus, apple and sunflower pectins gave polygalacturonate blocks with a relatively narrow molecular weight distribution, and average chainlength of ~25 residues in each case. Since the known relative stabilities of glycosidic linkages would lead to chain cleavage predominantly at l -rhamnose, this result indicates that the length of polygalacturonate sequences between rhamnose interruptions is approximately constant within and between the pectins studied. Calcium pectate gel strength is reduced dramatically by the incorporation of these chain segments when they are de-esterified, but not when they are esterified. This interference with the development of a network structure that resists applied stress, provides further support for our model of junction zone formation from sequences of contiguous deesterified residues, with Ca 2+ -mediated chain dimers providing the primary associations that can offer resistance to deformation. Samples with different levels and patterns of esterification were prepared by enzymic (blockwise) and chemical (random) de-esterification of almost fully methyl esterified pectin. In the former series, the extent of Ca 2+ binding (as monitored by circular dichroism) increased almost linearly with the fraction of free carboxyl groups, whereas the latter showed a non-linear relationship of a form consistent with the requirement of this binding for blocks of contiguous non-esterified residues and, in the presence of excess univalent cations, binding was negligible when more than ~40% of the carboxyl groups were esterified. Statistical calculations of sequence length distribution at different degrees of random de-esterification show the best fit with experimental data when binding is assumed to require sequences with seven or more consecutive free carboxyl groups along the participating face of the chain. For 2 1 chain symmetry, this corresponds to a sequence length of 14 residues, in excellent agreement with previous independent studies of Ca 2+ binding to oligogalacturonates. In the absence of competing univalent counterions, circular dichroism changes are similar in form but so large in magnitude that site-binding of Ca 2+ must now go beyond the half-stoichiometry at which it is arrested in their presence. Ca 2+ binding monitored by circular dichroism, and gel strength (yield stress) measured mechanically, both show a similar dependence on the pattern as well as the level of esterification, as expected for network formation by co-operative binding of Ca 2+ within interchain junction zones. To fit this binding data quantitatively, it is necessary to postulate a two-stage process. (1) Initial dimerization, probably corresponding to the “strong associations” indicated by evidence from competitive inhibition (see above), for which a critical minimum sequence of seven residues is again required but esterified residues can now be accommodated within individual sites provided that they are paired with a free carboxylate on the complementary chain. (2) Subsequent Ca 2+ -induced aggregation of these preformed dimers, which can occur irrespective of the pattern of esterification on the external faces; the evidence from mechanical measurements shows that these contribute little to gel strength at high stress.

A novel approach for calculating starch crystallinity and its correlation with double helix content: a combined XRD and NMR study.

A peak fitting procedure has been implemented for calculating crystallinity in granular starches. This methodology, widely used for synthetic polymers, is proposed to better reflect the crystalline content of starches than the method normally used, in which it is assumed that relatively perfect crystalline domains are interspersed with amorphous regions. The new approach takes into account irregularities in crystals that are expected to exist in semicrystalline materials. Therefore, instead of assuming that the amorphous background extends up to the base of diffraction peaks, the whole X‐ray diffraction (XRD) profile is fitted to an amorphous halo and several discrete crystalline diffraction peaks. The crystallinity values obtained from the XRD patterns of a wide range of native starches using this fitting technique are very similar to the double helix contents as measured by 13C solid state NMR, suggesting that double helices in granular starches are present within irregular crystals. This contrasts with previous descriptions of crystalline and noncrystalline double helices that were based on the analysis of XRD profiles as perfect crystals interspersed in a noncrystalline background. Furthermore, with this fitting methodology it is possible to calculate the contribution from the different crystal polymorphs of starch to the total crystallinity.

The phase transformations in starch during gelatinisation: a liquid crystalline approach.

The analogy between starch and a chiral side-chain polymeric liquid crystal is examined in relation to the processes involved during gelatinisation. There are three important parameters for characterisation of the molecular phase behaviour of the amylopectin: the lamellar order parameter (psi), the orientational order parameter of the amylopectin double helices (phi), and the helicity of the sample (h, the helix/coil ratio, a measure of the helix-coil transition of the double helices). The coupling between the double helices and the backbone through the flexible spacers is affected dramatically by the water content and it is this factor which dictates the particular phase adopted by the amylopectin inside the starch granule as a function of temperature. SAXS, WAXS and 13C CP/MAS NMR are used to examine these phenomena in excess water. Furthermore, previous experimental evidence pertaining to the limiting water case is reviewed with respect to this new theoretical framework.

Characterization of Starch by Size-Exclusion Chromatography: The Limitations Imposed by Shear Scission

Shear degradation is examined in size-exclusion chromatography (SEC, or GPC) of native starch in an eluent system (dimethylsulfoxide and LiBr) in which the starch is completely dissolved. Changes in apparent size distribution with flow rate suggested extensive shear scission of the amylopectin region. For smaller sizes, largely amylose, there was no significant scission for lower flow rates. Quantification by analogy to shear breakup of dispersed droplets gives a scaling law for conditions for shear scission of highly branched polymers. This shows both that it is impossible to obtain reliable size distributions for the amylopectin component of starch using current SEC technology and also that the amylose region is not significantly polluted by degraded amylopectin for lower flow rates. Hence, the complete size distribution of starch can only be obtained with SEC for smaller sizes (largely amylose), plus a size-separation technique with very low shear, such as field-flow fractionation, for the amylopectin region.

Hydrocolloid Gel Particles: Formation, Characterization, and Application

Hydrocolloid gel particles of micron and sub-micron size are particularly attractive for use in many applications in the food, agricultural, pharmaceutical, and chemical industries, due to their biocompatibility, perception as “natural” materials, and soft-solid texture. Industrial applications for such particles include uses as texturizers in confectionery and cosmetic products, slow-release encapsulation agents for flavors, nutrients, and pharmaceutical products, and thickeners in soups and sauces. Properties such as particle size, hardness, shape, texture, and molecular release rates can be important for individual applications. In addition, product formats will determine specific needs for physical form (e.g. dry or wet) and compatibility with other components. The diverse range of potential applications for hydrocolloid gel particles provide a driver for understanding-led tailoring of raw material and process conditions. This review introduces some of the materials that are used to form hydrocolloid gel particles and the corresponding gel formation mechanisms. One issue of importance in the production of hydrocolloid gel particles is the control of particle properties, such as release profiles, strength, and detectability within products. An alternative technique to traditional methods of hydrocolloid gel particle production is evaluated and a model for control of particle size, and subsequently other particle properties, is proposed. Key properties of hydrocolloid gel particles are identified and characterization methods for evaluating these properties are described.