Studies on the oxidative cross-linking of feruloylated arabinoxylans from wheat flour and wheat bran

Feruloylated arabinoxylans isolated from wheat flour and wheat bran were compared in their cross-linking behaviour with respect to viscosity properties and cross-linking products formed when various oxidative agents were applied to dilute solutions. Optimal conditions for each oxidative agent were investigated. In case of hydrogen peroxide and peroxidase, similar conditions were found for both types of arabinoxylans but wheat bran arabinoxylans gave a larger viscosity increase upon cross-linking than those of wheat flour.

When glucose, glucoseoxidase and peroxidase or ammonium persulphate were used as oxidative agents, differences in the concentration of reagent needed to induce cross-linking and in viscosity increase were observed. The distribution of coupling products for both types of arabinoxylans and the different oxidative treatments was approximately 5 : 3 : 1 : 1 for 8-5, 8-O-4, 8-8 and 5-5, respectively. The low ferulate recovery after oxidative treatment was assumed to be caused by formation of unknown compounds, such as higher oligomers and lignin-linked products.

A 1 : 1 mixture of flour arabinoxylan and feruloylated pectin showed a maximum synergistic effect on viscosity upon oxidative treatment using hydrogen peroxide and peroxidase. Both polysaccharides were shown to participate in cross-linking.     

Molecular characterization and solution properties of enzymatically tailored arabinoxylans

Two α-l-arabinofuranosidases with different substrate specificities were used to modify the arabinose-to-xylose ratio of cereal arabinoxylans: one enzyme (AXH-m) removed the l-arabinofuranosyl substituents from the monosubstituted xylopyranosyl residues and the other (AXH-d3) the (1 → 3)-linked l-arabinofuranosyl units from the disubstituted xylopyranosyl residue. In this study, we noticed that not only the arabinose-to-xylose ratio but also the position of the arabinofuranosyl substituents affects the water-solubility of arabinoxylans. The AXH-d3 treatment had no significant effect on the solution conformation of arabinoxylans, but the density of the arabinoxylan molecules decreased in DMSO solution after AXH-m modification. The possible heterogeneity of arabinoxylans complicated the interpretation of data describing the macromolecular properties of the enzymatically modified samples.     

Effects of cell wall components on the functionality of wheat gluten

Normal white wheat flours and especially whole meal flour contain solids from the inner endosperm cell walls, from germ, aleurone layer and the outer layers of cereal grains. These solids can prevent either gluten formation or gas cell structure. The addition of small amounts of pericarp layers (1–2%) to wheat flour had a marked detrimental effect on loaf volume. Microstructural studies indicated that in particular the epicarp hairs appeared to disturb the gas cell structure. The detrimental effects of insoluble cell walls can be prevented by using endoxylanases. It has been shown that some oxidative enzymes, naturally present in flour or added to the dough, will oxidise water-extractable arabinoxylans via ferulic acid bridges, and the resulting arabinoxylan gel will hinder gluten formation. The negative effects of water-unextractable arabinoxylans on gluten yield and rheological properties can be compensated by the addition of ferulic acid. Free ferulic acid can probably prevent arabinoxylan cross-linking via ferulic acid.     

Arabinoxylan gels: impact of the feruloylation degree on their structure and properties

Arabinoxylan (AX) samples of decreasing ferulic acid (FA) contents were chemically prepared from water-extractable wheat arabinoxylans without affecting their other structural properties. Gels were obtained from these partially feruloylated WEAX (PF-WEAX) by enzymatic covalent cross-linking of FA leading to the formation of diferulic (di-FA) and tri-ferulic acid (tri-FA). WEAX gelling ability was found related to the WEAX FA content whereas the gel structure and properties depended on the density of newly formed covalent cross-links. FA content of WEAX ranging from 1.4 to 2.3 microg/mg AX gave gels with di-FA cross-links contents from 0.20 to 0.43 microg/mg AX and G' values from 5 to 44 Pa. For WEAX gels with initial FA contents from 1.6 to 2.3 microg/mg AX, average mesh size ranging from 331 to 263 nm were calculated from swelling experiments. Cross-linking densities of gels, determined from swelling experiments, were higher than those that could be theoretically estimated from the di-FA and tri-FA content of WEAX gels. This result suggests that, in addition to di-FA and tri-FA, higher ferulate cross-linking and physical entanglements would contribute to the final WEAX gel structure.     

Effects of soluble corn bran arabinoxylans on cecal digestion, lipid metabolism, and mineral balance (Ca, Mg) in rats

The effects of soluble corn bran arabinoxylans on cecal digestion, lipid metabolism, and mineral utilization [calcium (Ca) and magnesium (Mg)] were investigated in rats adapted to semipurified diets. The diets provided either 710 g/kg wheat starch alone (control) or 610 g/kg wheat starch plus 100 g/kg corn soluble fiber (arabinoxylans) and either 0 or 2 g/kg cholesterol (control + cholesterol and arabinoxylans + cholesterol, respectively). Compared with rats fed the control diets, rats fed the arabinoxylan diets had significant cecal hypertrophy (+50% after 3 days of the fiber adaptation) and an accumulation of short-chain fatty acids, especially propionic acid (up to 45% in molar percentage). Arabinoxylans enhanced the cecal absorption of Ca and Mg (from 0.07 to 0.19 micromol/min for Ca and from 0.05 to 0.23 micromol/min for Mg). Mg balance was enhanced by arabinoxylans (+25%). The arabinoxylan diet markedly reduced the cholesterol absorption from 50% of ingested cholesterol in controls up to approximately 15% in rats adapted to the arabinoxylans diet. Arabinoxylans were effective in lowering plasma cholesterol (approximately -20%). There was practically no effect of the diets on cholesterol in d > 1.040 lipoproteins (high density lipoproteins) whereas arabinoxylans were very effective in depressing cholesterol in d < 1.040 lipoproteins (especially in triglyceride-rich lipoproteins). Corn fermentable fiber decreased the accumulation of cholesterol in the liver. In parallel, the arabinoxylan diet counteracted the downregulation of 3-hydroxy-3-methylglutaryl-CoA by cholesterol. These data suggest that arabinoxylans may have a great impact on intestinal fermentation, mineral utilization, and cholesterol metabolism.     

Factors affecting xylanase functionality in the degradation of arabinoxylans

Endo-beta-1,4-xylanases are key enzymes in the degradation of arabinoxylans, the main non-starch polysaccharides from grain cell walls. Due to the heterogeneity of arabinoxylans, xylanases with different characteristics are required in industrial applications but the choice of the enzyme is still largely empirical. Although the classification into glycoside hydrolase families greatly helped to derive mechanistic information on the catalytic and substrate specificity of xylanases, other factors e.g. their sensitivity to endogenous inhibitors, the presence of carbohydrate-binding module(s) and their degree of selectivity towards soluble versus insoluble substrate may play a role in determining the functionality of these enzymes in the degradation of arabinoxylans.     

The Stiffness of Three-dimensional Ionic Self-assembling Peptide Gels Affects the Extent of Capillary-like Network Formation

Improving our ability to control capillary morphogenesis has implications for not only better understanding of basic biology, but also for applications in tissue engineering and in vitro testing. Numerous biomaterials have been investigated as cellular supports for these applications and the biophysical environment biomaterials provide to cells has been increasingly recognized as an important factor in directing cell function. Here, the ability of ionic self-assembling peptide gels to support capillary morphogenesis and the effect of their mechanical properties is investigated. When placed in a physiological salt solution, these oligopeptides spontaneously self-assemble into gels with an extracellular matrix (ECM)-like microarchitecture. To evaluate the ability of three-dimensional (3D) self-assembled peptide gels to support capillary-like network formation, human umbilical vein endothelial cells (HUVECs) were embedded within RAD16-I ((RADA)₄) or RAD16-II ((RARADADA)₂) peptide gels with various stiffness values. As peptide stiffness is decreased cells show increased elongation and are increasingly able to contract gels. The observation that capillary morphogenesis is favored in more malleable substrates is consistent with previous reports using natural biomaterials. The structural properties of peptide gels and their ability to support capillary morphogenesis in vitro make them promising biomaterials to investigate for numerous biomedical applications.     

Effects of decorin proteoglycan on fibrillogenesis,ultrastructure, and mechanics of type I collagen gels

The proteoglycan decorin is known to affect both the fibrillogenesis and the resulting ultrastructure of in vitro polymerized collagen gels. However, little is known about its effects on mechanical properties. In this study, 3D collagen gels were polymerized into tensile test specimens in the presence of decorin proteoglycan, decorin core protein, or dermatan sulfate (DS). Collagen fibrillogenesis, ultrastructure, and mechanical properties were then quantified using a turbidity assay, 2 forms of microscopy (SEM and confocal), and tensile testing. The presence of decorin proteoglycan or core protein decreased the rate and ultimate turbidity during fibrillogenesis and decreased the number of fibril aggregates (fibers) compared to control gels. The addition of decorin and core protein increased the linear modulus by a factor of 2 compared to controls, while the addition of DS reduced the linear modulus by a factor of 3. Adding decorin after fibrillogenesis had no effect, suggesting that decorin must be present during fibrillogenesis to increase the mechanical properties of the resulting gels. These results show that the inclusion of decorin proteoglycan during fibrillogenesis of type I collagen increases the modulus and tensile strength of resulting collagen gels. The increase in mechanical properties when polymerization occurs in the presence of the decorin proteoglycan is due to a reduction in the aggregation of fibrils into larger order structures such as fibers and fiber bundles.     

Particle Trapping Mechanisms Are Different in Spatially Ordered and Disordered Interacting Gels

Using stochastic simulations, we study the influence of spatial disorder on the diffusion of a single particle through a gel that consists of rigid, straight fibers. The interaction between the particle and the gel fibers consists of an invariant short-range repulsion, the steric part, and an interaction part that can be attractive or repulsive and of varying range. The effect that spatial disorder of the gel structure has on the particle diffusivity depends crucially on the presence of nonsteric interactions. For attractive interactions, disorder slows down diffusion, because in disordered gels, the particle becomes strongly trapped in regions of locally increased fiber density. For repulsive interactions, the diffusivity is minimal for intermediate disorder strength, because highly disordered lattices exhibit abundant passageways of locally low fiber density. The comparison with experimental data on protein and fluorophore diffusion through various hydrogels is favorable. Our findings shed light on particle-diffusion mechanisms in biogels and thus on biological barrier properties, which can be helpful for the optimal design of synthetic diffusors as well as synthetic mucus constructs.     

Photo-cross-linking of type I collagen gels in the presence of smooth muscle cells: mechanical properties,cell viability,and function

The effectiveness of photomediated cross-linking of type I collagen gels in the presence of rat aortic smooth muscle cells (RASMC) as a method to enhance gel mechanical properties while retaining native collagen triple helical structure and maintaining high cell viability was investigated. Collagen was chemically modified to incorporate an acrylate moiety. Collagen methacrylamide was cast into gels in the presence of a photoinitiator along with RASMC. The gels were cross-linked using visible light irradiation. Neither acrylate modification nor the cross-linking reaction altered collagen triple helical content. The cross-linking reaction, however, moved the denaturation temperature beyond the physiologic range. A twelve-fold increase in shear modulus was observed after cross-linking. Cell viability in the range of 70% (n = 4, p > 0.05) was observed in the photo-cross-linked gels. Moreover the cells were able to contract the cross-linked gel in a manner commensurate with that observed for natural type I collagen. Methacrylate-mediated photo-cross-linking is a facile route to improve mechanical properties of collagen gels in the presence of cells while maintaining high cell viability. This enhances the potential for type I collagen gels to be used as scaffolds for tissue engineering.     

Simultaneously physically and chemically gelling polymer system utilizing a poly(NIPAAm-co-cysteamine)-based copolymer

The objective of this work was to create an in situ physically and chemically cross-linking hydrogel for in vivo applications. N-Isopropylacrylamide (NIPAAm) was copolymerized with N-acryloxysuccinimide (NASI) via free radical polymerization. Poly(NIPAAm-co-NASI) was further modified to obtain poly(NIPAAm-co-cysteamine) through a nucleophilic attack on the carbonyl group of the NASI by the amine group of the cysteamine. Modification was verified by nuclear magnetic resonance. In addition to thermoresponsive physical gelling due to the presence of NIPAAm, this system also chemically gels via a Michael-type addition reaction when mixed with poly(ethylene glycol) diacrylate. The presence of both physical and chemical gelation resulted in material properties that are much improved compared to purely physical gels. The chemical gelation time of the copolymers was not significantly affected by the amount of thiol present due to the increased pKa of the copolymer containing more thiols. In addition, the swelling of the copolymers was highly dependent on the temperature and thiol content. Last, the rate of nucleophilic attack in the Michael-type addition reaction was shown to be highly dependent on pH and on the mole ratio of thiol to acrylate. Due to the improved mechanical properties, this material may be better suited for long-term functional replacement applications than other thermosensitive physical gels. With further development and biocompatibility testing, this material could potentially be applied as a temperature-responsive injectable biomaterial for functional embolization.     

Of matrices and men

Matrices used in modern electrokinetic techniques are surveyed. They are essentially three: cellulose acetate, agarose and polyacrylamide gels. The use of cellulose acetate is confined mostly to analyses in clinical chemistry labs. The properties of agarose are discussed, in particular its capacity of forming large-pore structures via supercoiling, i.e. formation of suprafibers with average radii of approximately 20-25 nm. Several modified agaroses are reviewed, in particular the SeaPlaque, SeaPrep, NuSieve, NuFix, Seakem and Isogel brands and a composite agarose-polyacrylamide matrix, quite popular in the seventies for DNA and RNA separations. The field of polyacrylamide gels seems to be bursting, with the large number of crosslinkers described, imparting special properties to such matrices. The properties of new, modified acrylamide monomers, little known in the field of electrophoresis, are evaluated; in particular: trisacryl gels, hydroxyalkyl methacrylate gels and acryloylmorpholine-bisacrylylpiperazine gels, the latter formed by amphiphilic monomers, highly resistant to alkaline hydrolysis. The properties and formulas of a host of acidic and basic acrylamido derivatives (11 of them) used as buffers and titrants for isoelectric focusing in immobilized pH gradients are reviewed here for the first time. The review culminates with a glimpse at a new generation of amphiphatic matrices, such as HydroLink and 'shielded hydrophobic phase' gels, which appear to be the latest developments in the fields of electrophoresis and chromatography, respectively.     

Structure-function relationships in microbial exopolysaccharides

Sufficient well-characterized microbial exopolysaccharides are now available to permit extensive studies on the relationship between their chemical structure and their physical attributes. This is seen even in homopolysaccharides with relatively simple structures but is more marked when greater differences in structure exist, as are found in several heteropolysaccharides. The specific and sometimes unique properties have, in the case of several of these polymers, provided a range of commercial applications. The existence of "families" of structurally related polysaccharides also indicates the specific role played by certain structures and substituents; the characteristics of several of these microbial polysaccharide families will be discussed here. Thus, microbial exopolysaccharides frequently carry acyl groups which may profoundly affect their interactive properties although these groups often have relatively little effect on solution viscosity. Xanthan with or without acylation shows marked differences in synergistic gelling with plant gluco- and galacto-mannans, although the polysaccharides with different acylation patterns show similar viscosity. Similarly "gelrite" from the bacterium originally designated as Auromonas (Pseudomonas)elodea is of greater potential value after deacetylation, when it provides a valuable gelling agent, than it is as a viscosifier in the natural acylated form. The Klebsiella type 54 polysaccharide only forms gels when it, too, has been chemically deacetylated to give a structure equivalent to the Enterobacter XM6 polymer. Both these polysaccharides form gels due to the enhanced interaction with cations following deacylation and to the conformation adopted after removal of the acyl groups. Recent work in our laboratory suggests that deacetylation of certain bacterial alginates also significantly increases ion binding by these polysaccharides, making them more similar in their properties to algal alginates even although the alginates from some Pseudomonas species lack poly-L-guluronic acid sequences. The existence within families of polysaccharides of types in which monosaccharides are altered within a specific structure, or with varying side-chains, also gives an indication of the way in which such substituents affect the physical properties of the polymers in aqueous solution.     

Biocompatible wound dressings based on chemically degradable triblock copolymer hydrogels

The synthesis of a series of thermo-responsive ABA triblock copolymers in which the outer A blocks comprise poly(2-hydroxypropyl methacrylate) and the central B block is poly(2-(methacryloyloxy)ethyl phosphorylcholine) is achieved using atom transfer radical polymerization. These novel triblock copolymers form thermo-reversible physical gels with critical gelation temperatures and mechanical properties that are highly dependent on the copolymer composition and concentration. TEM studies on dried dilute copolymer solutions indicate the presence of colloidal aggregates, which is consistent with micellar gel structures. This hypothesis is consistent with the observation that incorporating a central disulfide bond within the B block leads to thermo-responsive gels that can be efficiently degraded using mild reductants such as dithiothreitol (DTT) over time scales of minutes at 37 degrees C. Moreover, the rate of gel dissolution increases at higher DTT/disulfide molar ratios. Finally, these copolymer gels are shown to be highly biocompatible. Only a modest reduction in proliferation was observed for monolayers of primary human dermal fibroblasts, with no evidence for cytotoxicity. Moreover, when placed directly on 3D tissue-engineered skin, these gels had no significant effect on cell viability. Thus, we suggest that these thermo-responsive biodegradable copolymer gels may have potential applications as wound dressings.     

Advances in biomedical applications of pectin gels

Pectin, due to its simple and cytocompatible gelling mechanism, has been recently exploited for different biomedical applications including drug delivery, gene delivery, wound healing and tissue engineering. Recent studies involving pectin for the biomedical field are reviewed, with the aim to capture the state of art on current research about pectin gels for biomedical applications, moving outside the traditional fields of application such as the food industry or pharmaceutics. Pectin structure, sources and extraction procedures have been discussed focussing on the properties of the polysaccharide that can be tuned to optimize the gels for a desired application and possess a fundamental role in application of pectin in the biomedical field.     

Stereocontrolled Synthesis of an Octasaccharide Part of I-Active Glycolipids

The effects of glycerin and ethylene glycol on the elastic modulus and DSC thermograms of agarose and kappa-carrageenan gels were examined to clarify the relation between structure and properties. The elastic modulus of these gels as a function of the concentration of polyols increased up to a certain concentration and then decreased with increasing concentration of polyols. These polyols shifted the melting temperature of the gel to higher temperatures in kappa-carrageenan gels but to lower temperatures in agarose gels. The temperature dependence of elastic modulus was changed in opposite directions in agarose and kappa-carrageenan gels by the addition of polyols, and this is discussed on the basis of model consisting of junction zones which are connected by Langevin chains. It was suggested that the mean distance between junction zones became shorter in the presence of a small amount of polyols.     

Chitin and carbon aerogels from chitin alcogels

Supercritical point drying of gels is a common technique for the production of a specific category of nano-porous materials called aerogels. We have successfully prepared chitin aerogels by extracting the solvent from the alcogels (gels with an alcohol as the solvent) with carbon dioxide under supercritical conditions. The produced nano-porous materials exhibit the typical properties of aerogels such as high porosity, high surface area, and low density, which make them quite attractive for many applications. The use of chitin, however, is of particular interest for the production of aerogels not only for being abundant and cheap but also because it has important inherent properties such as biocompatibility, non toxicity, thermal and chemical stability. In this work we examine the influence of different parameters on the porosity characteristics of the aerogels, such as the drying conditions (temperature and pressure), the nature of the solvent, and the gel concentration. Since these aerogels collapse in liquid medium, we also investigated the possibility of their utilization as carbon aerogel precursors.     

Collagen fiber alignment does not explain mechanical anisotropy in fibroblast populated collagen gels

Many load-bearing soft tissues exhibit mechanical anisotropy. In order to understand the behavior of natural tissues and to create tissue engineered replacements, quantitative relationships must be developed between the tissue structures and their mechanical behavior. We used a novel collagen gel system to test the hypothesis that collagen fiber alignment is the primary mechanism for the mechanical anisotropy we have reported in structurally anisotropic gels. Loading constraints applied during culture were used to control the structural organization of the collagen fibers of fibroblast populated collagen gels. Gels constrained uniaxially during culture developed fiber alignment and a high degree of mechanical anisotropy, while gels constrained biaxially remained isotropic with randomly distributed collagen fibers. We hypothesized that the mechanical anisotropy that developed in these gels was due primarily to collagen fiber orientation. We tested this hypothesis using two mathematical models that incorporated measured collagen fiber orientations: a structural continuum model that assumes affine fiber kinematics and a network model that allows for nonaffine fiber kinematics. Collagen fiber mechanical properties were determined by fitting biaxial mechanical test data from isotropic collagen gels. The fiber properties of each isotropic gel were then used to predict the biaxial mechanical behavior of paired anisotropic gels. Both models accurately described the isotropic collagen gel behavior. However, the structural continuum model dramatically underestimated the level of mechanical anisotropy in aligned collagen gels despite incorporation of measured fiber orientations; when estimated remodeling-induced changes in collagen fiber length were included, the continuum model slightly overestimated mechanical anisotropy. The network model provided the closest match to experimental data from aligned collagen gels, but still did not fully explain the observed mechanics. Two different modeling approaches showed that the level of collagen fiber alignment in our uniaxially constrained gels cannot explain the high degree of mechanical anisotropy observed in these gels. Our modeling results suggest that remodeling-induced redistribution of collagen fiber lengths, nonaffine fiber kinematics, or some combination of these effects must also be considered in order to explain the dramatic mechanical anisotropy observed in this collagen gel model system.     

Accessibility of tobacco lipid transfer protein cavity revealed by 15N NMR relaxation studies and molecular dynamics simulations

Plant LTP1 are small helical proteins stabilized by four disulfide bridges and are characterized by the presence of an internal cavity, in which various hydrophobic ligands can be inserted. Recently, we have determined the solution structure of the recombinant tobacco LTP1_1. Unexpectedly, despite a global fold very similar to the structures already known for cereal seed LTP1, its binding properties are different: Tobacco LTP1_1 is able to bind only one monoacylated lipid, whereas cereal LTP1 can bind either one or two. The 3D structure of tobacco LTP1_1 revealed the presence of a hydrophobic cluster, not observed on cereal LTP1 structures, which may hinder one of the two entrances of the cavity defined for wheat LTP1. To better understand the mechanism of lipid entrance for tobacco LTP1_1 and to define the regions of the protein monitoring the accessibility of the cavity, we have complemented our structural data by the study of the internal dynamics of tobacco LTP1_1, using (15)N magnetic relaxation rate data and MD simulations at room and high temperatures. This work allowed us to define two regions of the protein experiencing the largest motions. These two regions delineate a portal that opens up during the simulation constituting a unique entrance of the hydrophobic cavity, in contrast with wheat LTP1 where two routes were detected. The hydrophobic interactions resulting from a few point mutations are strong enough to completely block the second portal so that the accessibility of the cavity is restricted to one entrance, explaining why this particular LTP1 binds only one lipid molecule.     

Microstructure and rheological behavior of pure and mixed pectin gels

The microstructure and the rheological properties of pure HM (high methoxyl) and LM (low methoxyl) pectin gels and of mixed HM/LM pectin gels have been investigated. Gel formation of either the HM or LM pectin, or both, was initiated in the mixed gels by varying the sucrose and Ca(2+) content. The microstructure was characterized by transmission electron microscopy, light microscopy, and confocal laser scanning microscopy. HM and LM pectin gels showed aggregated networks with large pores around 500 nm and network strands of similar character. Small differences could be found, such as a more inhomogeneous LM pectin network with shorter and more branched strands of flexible appearance. LM pectin also formed a weak gel in 60% sucrose in the absence of calcium. A highly inhomogeneous mixed gel structure was formed in the presence of 60% sucrose and Ca(2+) ions, which showed large synergistic effects in rheological properties. Its formation was explained by the behavior of the corresponding pure gels. In the presence of 60% sucrose alone, a homogeneous, fine-stranded mixed network was formed, which showed weak synergistic effects. It is suggested that LM pectin interacts with HM pectin during gel formation, thereby hindering secondary aggregation leading to the aggregated networks observed for the pure gels.