Muriel Subirade

Molecular differences in the formation and structure of fine‐stranded and particulate β‐lactoglobulin gels

In order to reveal at a molecular level differences between fine-stranded and particulate gels, we present an Fourier transform infrared spectroscopic study of the thermal behavior of beta-lactoglobulin (beta-lg) in salt-free D(2)O solutions and low ionic strength at different pDs. Differences are found in the denaturation mechanism, in the unfolded state of the protein, in the aggregate formation, and in the strength of the intermolecular interactions. For fine-stranded gels (pD 2.8 and 7.8), heating induces the dissociation of the dimers into monomers. The protein undergoes extensive structural modifications before aggregation begins. Aggregation is characterized by the appearance of a new band attributed to intermolecular beta-sheets which is located in the 1613-1619 cm(-1) range. For particulate gels (pD 4.4 and 5.4), the protein structure is almost preserved up to 75-80 degrees C with no splitting of the dimers. The band characteristic of aggregation originates from the component initially located at 1623 cm(-1), suggesting that at the beginning of aggregation, globular beta-lg in the dimeric form associate to constitute oligomers with higher molecular mass. Aggregation may result in the association of globular slightly denatured dimers, leading to the formation of spherical particles rather than linear strands. The aggregation band is always located in the 1620-1623 cm(-1) range for particulate gels showing that hydrogen bonds are weaker for these aggregates than for fine-stranded ones. This has been related to a more extensive protein unfolding for fine-stranded gels that allows a closer alignment of the polypeptide chains, and then to the formation of much stronger hydrogen bonds. Small differences are also found in protein organization and in intermolecular hydrogen bond strength vs pD within the same type of gel. Protein conformation and protein-protein interactions in the gel state may be responsible of the specific macroscopic properties of each gel network. A coarse representation of the different modes of gelation is described.

Kinetics of the breakdown of cross-linked soy protein films for drug delivery

The aim of the present work was to investigate the potential of soy protein isolate (SPI) films as controlled release systems for active compounds. Mechanical properties, dissolution and compound release kinetics of SPI films prepared with different concentrations of formaldehyde were measured over time in the absence or presence of digestive enzymes at gastric or intestinal pH. The effect of formaldehyde on tensile strength, elastic modulus, % elongation and swelling suggested that increasing its concentration increased film cross-linking density. Film bulk erosion in the presence of digestive enzymes followed first-order kinetics. Methylene blue or rifampicin release followed variable kinetics depending on compound solubility during a 1-2h initial phase, followed by zero-order release. Cross-linking density appears to provide effective means of regulating the erosion and release rate of SPI films. SPI film networks displayed excellent compound binding capacity, especially for hydrophobic molecules, and hence potential for use in controlled release systems based on matrix erosion.

Molecular basis of film formation from a soybean protein: comparison between the conformation of glycinin in aqueous solution and in films

Fourier transform infrared spectroscopy has been used to investigate the conformational changes of glycinin. a major storage protein of soybean seeds, upon film-forming. The results show that the secondary structure of glycinin is mainly composed of a beta-sheet (48%) and unordered (49%) structures. The amide I band of glycinin in film-forming conditions, i.e. in alkaline media and in the presence of plasticizing agent, reveals the conversion of 18% of the secondary structure of the protein from the beta-sheet (6%) and random coil (12%) to the alpha-helical conformation due to the helicogenic effect of the ethylene glycol used as the plasticizing agent. Conformational changes also occur upon the film-forming process leading to the formation of intermolecular hydrogen-bonded beta-sheet structures. Results obtained from other plant families indicate that, whatever the origin and conformation of protein, formation of films leads to the appearance of intermolecular hydrogen-bonded beta-sheet structures, suggesting that this type of structure might be essential for the network formation in films. Thus, it is hypothesized that, in the film state, intermolecular hydrogen bonding between segments of beta-sheet may act as junction zones in the film network. This study reveals for the first time that there is a close relationship between the conformation of proteins and the mechanical properties of films.

Elaboration and Characterization of Soy/Zein Protein Microspheres for Controlled Nutraceutical Delivery

Microspheres (15-25 microm) of soy protein isolate (SPI), zein, and SPI/zein blends were prepared using a cold gelation method as possible delivery systems for nutraceutical products. Microsphere matrix crystalline structure, swelling behavior, and nutrient load release kinetics in simulated gastrointestinal fluids were investigated. SPI microspheres showed early burst release of the model nutrient, whereas zein microspheres showed very slow release in both simulated gastric and intestinal fluids. Blending of SPI and zein provides a convenient method of adjusting the hydrophobicity and crystallinity of the protein matrix and hence its swelling behavior and in vivo nutrient release kinetics. Diffusion plays a major role in regulating nutrient release. SPI/zein microspheres blended at ratios of 5:5 and 3:7 showed near zero-order release kinetics over the test period in simulated intestinal buffer and thus have potential as delivery vehicles for nutraceutical products in functional foods.

Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy.

The interaction of a nonspecific wheat lipid transfer protein (LTP) with phospholipids has been studied using the monolayer technique as a simplified model of biological membranes. The molecular organization of the LTP-phospholipid monolayer has been determined by using polarized attenuated total internal reflectance infrared spectroscopy, and detailed information on the microstructure of the mixed films has been investigated by using epifluorescence microscopy. The results show that the incorporation of wheat LTP within the lipid monolayers is surface-pressure dependent. When LTP is injected into the subphase under a dipalmytoylphosphatidylglycerol monolayer at low surface pressure (< 20 mN/m), insertion of the protein within the lipid monolayer leads to an expansion of dipalmytoylphosphatidylglycerol surface area. This incorporation leads to a decrease in the conformational order of the lipid acyl chains and results in an increase in the size of the solid lipid domains, suggesting that LTP penetrates both expanded and solid domains. By contrast, when the protein is injected under the lipid at high surface pressure (> or = 20 mN/m) the presence of LTP leads neither to an increase of molecular area nor to a change of the lipid order, even though some protein molecules are bound to the surface of the monolayer, which leads to an increase of the exposure of the lipid ester groups to the aqueous environment. On the other hand, the conformation of LTP, as well as the orientation of alpha-helices, is surface-pressure dependent. At low surface pressure, the alpha-helices inserted into the monolayers are rather parallel to the monolayer plane. In contrast, at high surface pressure, the alpha-helices bound to the surface of the monolayers are neither parallel nor perpendicular to the interface but in an oblique orientation.

Formation of intermolecular β-sheet structures: a phenomenon relevant to protein film structure at oil–water interfaces of emulsions

Oil-in-water emulsions stabilized with beta-lactoglobulin (beta-lg) were made using a homogenizer or a high-speed blender. The protein was studied by Fourier transform infrared (FTIR) spectroscopy in the raw emulsion, in the bulk phase, and at the interface, as a function of pH, oil content, and homogenizing pressure. Results show that the amount of adsorbed protein varies with the available interfacial area. The protein that remains in the aqueous phase exhibit no spectral change, which suggests that homogenization causes no conformational modification or reversible ones. Strong and irreversible changes were observed in the adsorbed protein. Our findings reveal the formation of intermolecular antiparallel beta-sheets upon adsorption due to the protein self-aggregation. As deduced from transmission electronic microscopy, this surface aggregation leads to the formation of continuous and homogeneous membranes coating the globules. The structure of the adsorbed proteins is unaffected by the homogenizing pressures used in our study and slightly modified by the pH. FTIR spectroscopy allows to characterize the type of aggregates formed at the interface. An analysis of the spectra of beta-lg heat-induced gels shows that the aggregates at the interface are very close at a molecular scale to those that constitute particulate gels near the proteins isoelectric point. Since the type of aggregates is similar when the emulsion water phase is pure D(2)O and D(2)O at pD 4.4, the interface not only seems to induce aggregation, but seems to determine the type of aggregation as well. The mechanism that drives the formation of particulate aggregates (rather than fine-stranded ones) may reside in strong protein-protein interactions that are promoted by adverse oil-protein interactions.

Preparation and in vitro evaluation of calcium-induced soy protein isolate nanoparticles and their formation mechanism study

Soy protein isolate (SPI) nanoparticles (28-179 nm) were prepared by employing a cold gelation method with a slight modification. The obtained nanoparticles exhibited uniform size distribution and spherical shape with a unique honeycomb-like core structure. Nanoparticle characteristics including size, surface charge and hydrophobicity could be adjustable by changing calcium concentration and environmental pH. Generally, higher calcium concentration and lower pH led to formation of nanoparticles with larger size, lower surface charge and hydrophobicity. Both protein conformation and nanoparticle dissociation studies indicated that calcium likely shielded negative charges on the SPI polypeptide chains, and functioned as a salt-bridge to permit polypeptide chains to approach one another. In this process, calcium favoured the development of β-sheet structures to form SPI aggregates stabilised by hydrogen bonding. These aggregates were then associated to build SPI nano-networks through hydrophobic interactions. In vitro study indicated that the SPI nanoparticles were non-toxic and mainly located in the cytoplasm when uptaken into Caco-2 cells.

Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of beta-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of beta-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon beta-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding beta-sheet structure as evidenced by the appearance of a strong band at 1614 cm(-1) at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of beta-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular beta-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of beta-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular beta-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.

Replacement of Trifluoroacetic Acid with HCl in the Hydrophobic Purification Steps of Pediocin PA-1: a Structural Effect

ABSTRACT Trifluoroacetic acid (TFA) is a purification contaminant associated with pediocin PA-1 that interferes with Fourier transform infrared spectroscopy structural analysis. As revealed by circular dichroism, its presence affects the structural folding of pediocin. Consequently, we propose a new pediocin PA-1 purification procedure using HCl instead of TFA in all of the hydrophobic steps. This procedural change does not affect the purification yield or the amount of pediocin PA-1 purified. Furthermore, removing HCl, as opposed to TFA, after purification is an easier procedure to carry out. In fact, the removal of TFA requires more experimentation and results in protein loss. Thus, HCl is a good alternative to TFA in pediocin PA-1 purification and can be extended to the purification of other proteins. We also show that TFA-induced structural modifications do not significantly affect the antimicrobial activity of pediocin PA-1.

Coated whey protein/alginate microparticles as oral controlled delivery systems for probiotic yeast

Viable Saccharomyces boulardii, used as a biotherapeutic agent, was encapsulated in food-grade whey protein isolate (WP) and alginate (ALG) microparticles, in order to protect and vehicle them in gastrointestinal environment. Yeast-loaded microparticles with a WP/ALG ratio of 62/38 were produced with high encapsulation efficiency (95%) using an extrusion/cold gelation method and coated with ALG or WP by a simple immersion method. Swelling, yeast survival, WP loss and yeast release in simulated gastric and intestinal fluids (SGF and SIF, pH 1.2 and 7.5) with and without their respective digestive enzymes (pepsin and pancreatin) were investigated. In SGF, ALG network shrinkage limited enzyme diffusion into the WP/ALG matrix. Coated and uncoated WP/ALG microparticles were resistant in SGF even with pepsin. Survival of yeast cells in microparticles was 40% compared to 10% for free yeast cells and was improved to 60% by coating. In SIF, yeast cell release followed coated microparticle swelling with a desirable delay. Coated WP/ALG microparticles appear to have potential as oral delivery systems for Saccharomyces boulardii or as encapsulation means for probiotic cells in pharmaceutical or food processing applications.