Stephane Pezennec

Strong improvement of interfacial properties can result from slight structural modifications of proteins: the case of native and dry-heated lysozyme.

Identification of the key physicochemical parameters of proteins that determine their interfacial properties is still incomplete and represents a real stake challenge, especially for food proteins. Many studies have thus consisted in comparing the interfacial behavior of different proteins, but it is difficult to draw clear conclusions when the molecules are completely different on several levels. Here the adsorption process of a model protein, the hen egg-white lysozyme, and the same protein that underwent a thermal treatment in the dry state, was characterized. The consequences of this treatment have been previously studied: net charge and hydrophobicity increase and lesser protein stability, but no secondary and tertiary structure modification (Desfougères, Y.; Jardin, J.; Lechevalier, V.; Pezennec, S.; Nau, F. Biomacromolecules 2011, 12, 156-166). The present study shows that these slight modifications dramatically increase the interfacial properties of the protein, since the adsorption to the air-water interface is much faster and more efficient (higher surface pressure). Moreover, a thick and strongly viscoelastic multilayer film is created, while native lysozyme adsorbs in a fragile monolayer film. Another striking result is that completely different behaviors were observed between two molecular species, i.e., native and native-like lysozyme, even though these species could not be distinguished by usual spectroscopic methods. This suggests that the air-water interface could be considered as a useful tool to reveal very subtle differences between protein molecules.

Dry-heating makes hen egg white lysozyme an efficient foaming agent and enables its bulk aggregation.

Dry-heating is considered to be one of the most promising approaches to improving the functionality of food proteins. It has been shown that even if only minor structural modifications occur during dry-heating, the foaming properties of proteins are highly improved. With the recent results obtained in the field of foam stabilization by nanoparticles or protein aggregates in mind, a study was undertaken on the impact of dry-heating of lysozyme, used as a model protein, on its foaming properties. This work highlighted the fact that dry-heated hen egg white lysozyme simultaneously exhibited enhanced foaming properties and aggregation capacity. Although the conditions that favored bulk aggregation (high ionic strength, pH, treatment duration, and protein concentration) also favored foaming properties, the large bulk aggregates were not essential to obtain the best functionality. It is envisaged that heat-treated lysozyme may self-associate at the air/water interface, stabilizing air bubbles.

Surface properties and conformation of Nephila clavipes spider recombinant silk proteins at the air-water interface.

The dragline fiber of spiders is composed of two proteins, the major ampullate spidroins I and II (MaSpI and MaSpII). To better understand the assembly mechanism and the properties of these proteins, the adsorption behavior of the recombinant proteins of the spider Nephila clavipes produced by Nexia Biotechnologies Inc. has been studied at the air-water interface using ellipsometry, surface pressure, rheological, and infrared measurements. The results show that the adsorption is more rapid and more molecules are present at the interface for MaSpII than for MaSpI. MaSpII has thus a higher affinity for the interface than MaSpI, which is consistent with its higher aggregation propensity in water. The films formed at the interface consist of networks containing a high content of intermolecular beta-sheets as revealed by the in situ polarization modulation infrared absorption reflection spectra. The infrared results further demonstrate that, for MaSpI, the beta-sheets are formed as soon as the proteins adsorb to the interface while for MaSpII the beta-sheet formation occurs more slowly. The amount of beta-sheets is lower for MaSpII than for MaSpI, most likely due to the presence of proline residues in its sequence. Both proteins form elastic films, but they are heterogeneous for MaSpI and homogeneous for MaSpII most probably as a result of a more ordered and slower aggregation process for MaSpII. This difference in their mechanism of assembly and interfacial behaviors does not seem to arise from their overall hydrophobicity or from a specific pattern of hydrophobicity, but rather from the longer polyalanine motifs, lower glycine content, and higher proline content of MaSpII. The propensity of both spidroins to form beta-sheets, especially the polyalanine blocks, suggests the participation of both proteins in the silks beta-sheet crystallites.

Flexibility and Charge of Solutes as Factors That Determine Their Diffusion in Casein Suspensions and Gels

This work explores the influence of both the physicochemical characteristics of solutes and the solute-matrix interactions on diffusion in casein systems. Diffusion coefficients of three solute groups (dextrans, proteins, and peptides) presenting different physicochemical characteristics, such as molecular flexibility and charge, were measured using the technique of fluorescence recovery after photobleaching (FRAP). The casein systems had the same casein concentration, but different microstructures (suspension or gel), and/or a different pH (5.2 or 6.6). Flexible solutes diffused more rapidly through the casein systems than the rigid ones. Electrostatic interactions between charged solute molecules and the casein matrix were partly screened due to the high ionic strength of the systems. As a consequence, it was the flexibility of the solute molecule (rather than its charge) that most influenced its diffusion through casein systems.

Effects of citrate and NaCl on size, morphology, crystallinity and microstructure of calcium phosphates obtained from aqueous solutions at acidic or near-neutral pH.

Precipitation of calcium phosphates occurs in dairy products and depending on pH and ionic environment, several salts with different crystallinity can form. The present study aimed to investigate the effects of NaCl and citrate on the characteristics of precipitates obtained from model solutions of calcium phosphate at pH 6·70 maintained constant or left to drift. The ion speciation calculations showed that all the starting solutions were supersaturated with respect to dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP) and hydroxyapatite (HAP) in the order HAP>OCP>DCPD. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) analyses of the precipitates showed that DCPD was formed at drifting pH (acidic final pH) whereas poor crystallised calcium deficient apatite was mainly formed at constant pH (6·70). Laser light scattering measurements and electron microscopy observations showed that citrate had a pronounced inhibitory effect on the crystallisation of calcium phosphates both at drifting and constant pH. This resulted in the decrease of the particle sizes and the modification of the morphology and the microstructure of the precipitates. The inhibitory effect of citrate mainly acted by the adsorption of the citrate molecules onto the surfaces of newly formed nuclei of calcium phosphate, thereby changing the morphology of the growing particles. These findings are relevant for the understanding of calcium phosphate precipitation from dairy byproducts that contain large amounts of NaCl and citrate.

Antimicrobial activity of lysozyme isoforms: Key molecular features

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry‐heating increasing lysozyme activity. Dry‐heated lysozyme (DH‐L) is a mixture of isoforms (isoaspartyl, native‐like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH‐L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH‐L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.