Enzymatically cross-linked tilapia gelatin hydrogels: physical, chemical, and hybrid networks.

This Article investigates different types of networks formed from tilapia fish gelatin (10% w/w) in the presence and absence of the enzymatic cross-linker microbial transglutaminase. The influence of the temperature protocol and cross-linker concentration (0-55 U mTGase/g gelatin) was examined in physical, chemical, and hybrid gels, where physical gels arise from the formation of triple helices that act as junction points when the gels are cooled below the gelation point. A combination of rheology and optical rotation was used to study the evolution of the storage modulus (G') over time and the number of triple helices formed for each type of gel. We attempted to separate the final storage modulus of the gels into its chemical and physical contributions to examine the existence or otherwise of synergism between the two types of networks. Our experiments show that the gel characteristics vary widely with the thermal protocol. The final storage modulus in chemical gels increased with enzyme concentration, possibly due to the preferential formation of closed loops at low cross-linker amount. In chemical-physical gels, where the physical network (helices) was formed consecutively to the covalent one, we found that below a critical enzyme concentration the more extensive the chemical network is (as measured by G'), the weaker the final gel is. The storage modulus attributed to the physical network decreased exponentially as a function of G' from the chemical network, but both networks were found to be purely additive. Helices were not thermally stabilized. The simultaneous formation of physical and chemical networks (physical-co-chemical) resulted in G' values higher than the individual networks formed under the same conditions. Two regimes were distinguished: at low enzyme concentration (10-20 U mTGase/g gelatin), the networks were formed in series, but the storage modulus from the chemical network was higher in the presence of helices (compared to pure chemical gels); at higher enzyme concentration (30-40 U mTGase/g gelatin), strong synergistic effects were found as a large part of the covalent network became ineffective upon melting of the helices.

Pressure cell assisted solution characterization of galactomannans. 3. Application of analytical ultracentrifugation techniques.

The pressure heating cell approach previously applied to galactomannans in two earlier studies is now used to prepare samples for characterization using the analytical ultracentrifuge. Sedimentation velocity data were obtained for both guar gum and locust bean gum samples. These were compared to our earlier light scattering and intrinsic viscosity measurements on samples prepared using identical temperature and pressure profiles. A number of methods were then employed to obtain chain persistence lengths, including the Hearst-Stockmayer and Bohdanecky wormlike chain approaches. These results were compared to earlier results obtained using methods appropriate for excluded volume coil and rodlike chains, respectively.

A novel xyloglucan from seeds of Afzelia africana Se. Pers.--extraction, characterization, and conformational properties.

This paper is the first multi-scale characterization of the xyloglucan extracted from seeds of the African tree Afzelia africana Se. Pers. It describes the extraction and characterization of this polysaccharide in terms of both primary monosaccharide and oligosaccharide composition. It also includes a study of the seed morphology. Morphological characterization includes optical, transmission, and scanning electron microscopy. The polysaccharide exists in thickened cell walls of the cotyledonary cells, and the extracted xyloglucan is structurally quite similar to those from tamarind seed and detarium. Nevertheless there are some subtle differences in the fine structure, particularly in the oligomeric xyloglucan composition. The chain flexibility of the polysaccharide is also discussed in the light of our recent measurements reported elsewhere [Biomacromolecules2004, 5, 2384-2391].

Fibrillar beta-lactoglobulin gels: Part 1. Fibril formation and structure.

As a prelude to experimental and theoretical work on the mechanical properties of fibrillar beta-lactoglobulin gels, this paper reports the structural characterization of beta-lactoglobulin fibrils by electron and atomic force microscopy (AFM), infrared and Raman spectroscopy, and powder X-ray diffraction. Aggregates formed by incubation of beta-lactoglobulin in various alcohol-water mixtures at pH 2, and in water-trifluoroethanol (TFE) at pH 7, were found to be wormlike (approximately 7 nm in width and <500 nm in length), with a "string-of-beads" appearance. Longer (approximately 7 nm in width, and >1 microm in length), smoother, and seemingly stiffer fibrils formed on heating aqueous beta-lactoglobulin solutions at pH 2 and low ionic strength, although there was little evidence for the higher-order structures common in most amyloid-forming systems. Time-lapse AFM also revealed differences in the formation of these two fibril types: thermally induced aggregation occurring more cooperatively, in keeping with a nucleation and growth process. Only short stiff-rods (<20 nm in length) formed on heating beta-lactoglobulin at pH 7, and only complex three-dimensional "amorphous"aggregates in alcohols other than TFE at this pH. Studies of all of the pH 2 fibrils from beta-lactoglobulin, by Raman and infrared spectroscopy confirmed beta-sheet as mediating the aggregation process. Interestingly, however, some evidence for de novo helix formation for the solvent-induced systems was obtained, although it remains to be seen whether this is actually incorporated into the fibril-structure. In contrast to other amyloid systems, X-ray powder diffraction provided no evidence for extensive repeating "crystalline" structures for any of the pH 2 beta-lactoglobulin fibrils. In relation to amyloid, the lactoglobulin fibrils bear more resemblance to protofilaments than to higher-order fibril structures, these latter appearing more convincingly for thermally induced insulin fibrils (pH 2) also included in the AFM study.

Fibrillar beta-lactoglobulin gels: Part 3. Dynamic mechanical characterization of solvent-induced systems.

Oscillatory shear rheometry has been used to study the gelation of beta-lactoglobulin at ambient in 50% v/v trifluoroethanol (TFE)/pH 7 aqueous buffer and in 50% v/v ethanol (EtOH)/water at pH 2. In contrast to what was found on heating aqueous solutions at pH 2 (Part 2 of this series), a more expected "chemical gelation"-like profile was found with modulus components G' and G' ' crossing over as the gels formed and then with G' ' passing through a maximum. In addition, for the EtOH system, there was a significant modulus increase at long time, suggestive of a more complex two-step aggregation scheme. Modulus-concentration relationships were obtained for both systems by extrapolating cure data to infinite time. For the TFE gels, this data was accurately described by classical branching theory, although it could also be approximated by a constant power--law relationship. Only the latter described the modulus--concentration data for the gels in ethanol, but there were problems here of greater frequency dependence of the modulus values and much less certain extrapolation. Gel times for the TFE systems showed higher power laws in the concentration than could be explained by the branching theory in its simplest form being similar, in this respect, to the heat-set systems at pH 2. Such power laws were harder to establish for the EtOH gels as for these there was evidence of gel time divergence close to a critical concentration. Reduced G'/G'inf versus t/tgel data were difficult to interpret for the gels in ethanol, but for the TFE system they were consistent with previous results for the heat-set gels and approximated master curve superposition. The frequency and temperature dependences of the final gel moduli were also studied. In general, the networks induced by alcohols appeared more flexible than those obtained by heating.

Solution properties of the xyloglucan polymer from Afzelia africana.

In this paper we describe the solution properties of a new xyloglucan polysaccharide extracted from the African legume Afzelia africana Se. Pers. The polysaccharide is of high weight-average molecular weight (Mw), but application of the "pressure cell" method enabled a range of Mw fractions to be prepared. Results from the light scattering/intrinsic viscosity measurements on these fractions suggest that like other xyloglucans from tamarind and detarium it occurs in solution as a polymeric coil, with a small amount of excluded volume. Measurement of dilute and semidilute solution rheology suggests that, like these polymers, and the related galactomannan series, it forms viscous solutions at higher concentrations via entanglements.

Fibrillar beta-lactoglobulin gels: Part 2. Dynamic mechanical characterization of heat-set systems.

Oscillatory shear rheometry (mechanical spectroscopy) has been used to study the heat-set gelation of beta-lactoglobulin at pH 2. Modulus-concentration relationships were obtained by extrapolating cure data to infinite time. In terms of theory, these fail to provide a clear distinction between the fractal description of biopolymer gels and the classical random f-functional polycondensation branching theory (cascade) approach, though the latter is preferred. Critical exponents for the sol-gel transition, derived from these data, are also discussed. Where gel time-concentration results are concerned the fractal model makes no predictions, and the cascade approach in its simplest form must be rejected in favor of a more sophisticated version involving delivery of fibrils by nucleation and growth into the random aggregation process. Over the limited concentration range accessed experimentally, cure data for the different beta-lactoglobulin solutions, reduced to the universal form G'/G'inf versus t/tgel, superimposed well for samples heated both at 80 and 75 degrees C and for different batches of protein. Studies of the frequency responses of the fully cured gels confirm the validity of the gel description given to these materials, and a study of the temperature dependence of the frequency spectrum suggests a fall in the elastic component of the modulus as temperature decreases. This contrasts with what has been found for other heat-set globular protein gels such as those from serum albumin where the gel modulus increases at lower temperatures. The present results are in good agreement with more limited amounts of pH 2 beta-lactoglobulin data published earlier, though some differences arise through a previous neglect of measurement "dead time".

Rheology of biopolymer solutions and gels.

Rheological techniques and methods have been employed for many decades in the characterization of polymers. Originally developed and used on synthetic polymers, rheology has then found much interest in the field of natural (bio) polymers. This review concentrates on introducing the fundamentals of rheology and on discussing the rheological aspects and properties of the two major classes of biopolymers: polysaccharides and proteins. An overview of both their solution properties (dilute to semi-dilute) and gel properties is described.

Pressure cell assisted solubilization of xyloglucans: tamarind seed polysaccharide and detarium gum.

To improve the solubilization of two water-soluble xyloglucans, tamarind seed polysaccharide and detarium gum, by reducing substantially molecular aggregation, a "pressure cell" heating method was used. Conditions allowing solubilization and chain depolymerization were produced by varying appropriately the pressure, time, and temperature applied. The various MW fractions of solubilized xyloglucans were characterized by capillary viscometry and light scattering techniques in order to extract, with reliability, fundamental macromolecular parameters. Mark-Houwink and Flory exponents were found to be 0.67 +/- 0.04 and 0.51 +/- 0.06, respectively for both xyloglucan data combined, consistent with linear random coil behavior. A detailed analysis of the data seems to suggest that tamarind gum solutions are slightly perturbed by the effect of excluded volume, whereas detarium gum samples are close to the theta state. Chain flexibility parameters such characteristic ratio, C( proportional, variant ), and persistence length, L(p), were calculated for tamarind and detarium using the Burchard-Stockmayer-Fixman (BSF) geometric method. L(p) values of 6-8 nm were estimated for xyloglucans. The seemingly linear structure of tamarind and detarium, as suggested by the value of the Mark-Houwink and Flory exponents obtained, follows from analysis of the data by the classical Zimm method but not when employing the square root or Berry method which suggests a more branched chain profile. This was the approach adopted in our previous work on the characterization of detarium samples.

On the chain flexibility of arabinoxylans and other beta-(1-->4) polysaccharides.

The recent paper by Dervilly-Pinel and co-workers (Carbohydr. Res. 2001, 330, 365-372) presents a complete macromolecular characterisation of a series of de-esterified arabinoxylans extracted and fractionated from wheat flour. From their measurements, they were able to extract parameters related to chain flexibility, such as the Mark-Houwink exponent a and the chain persistence length. However, the estimate they obtain for the latter parameter is rather larger than would be expected, since the arabinoxylan backbone is beta-(1-->4) like cellulose and the galactomannans. By treating their data in an alternative, but well accepted manner, we are able to obtain a lower value of persistence length, which agrees well with estimates for this parameter for cellulose, in the literature, and our own recent measurements for a series of differently substituted galactomannans. These results suggest that the parameters obtained may be applicable to other beta-(1-->4) polysaccharides.

Pressure cell assisted solution characterization of polysaccharides. 2. Locust bean gum and tara gum.

Following the work carried out on guar gum in our first paper of a series, the "pressure cell" solubilization method was applied to two other less highly substituted galactomannans: locust bean gum (LBG) and tara gum. True molecular solution of the polymers was achieved using appropriate temperature, time, and pressure regimes. The technique of capillary viscometry was used to determine the intrinsic viscosity [eta] of the "pressure cell" treated and untreated samples. Molecular weight (M(w)) and radius of gyration (R(g)) were determined by light scattering. The data obtained for LBG and tara gum were compared statistically with reliable data found for guar gum in the literature. The variation in [eta] with M(w) followed the Mark-Houwink-Sakurada relationship, giving the exponent alpha = 0.74 +/- 0.01 for galactomannans consistent with random coil behavior. The characteristic ratio, C(infinity), and the chain persistence length, L(p), were both calculated for LBG and tara gum using the Burchard-Stockmayer-Fixman (BSF) method which is appropriate for flexible to semiflexible chains. A general value of 9 < C(infinity) < 16 and 3 < L(p) < 5 nm can now be estimated with statistical confidence for all galactomannans. According to our statistical analysis, the chain persistence length was found to be insensitive to the degree of galactose substitution.