SAXS conformational tracking of amylose synthesized by amylosucrases.

Amylose, a linear polymer of α(1,4)-linked glucosyl units and a major constituent of starch granules, can also be enzymatically synthesized in vitro from sucrose by bacterial amylosucrases. Depending on the initial sucrose concentration and the enzyme used, amylose oligomers (or polymers) are formed and self-associate during synthesis into various semicrystalline morphologies. This work describes for the first time a synchrotron SAXS study of the structure in solution of two amylosucrases, namely, NpAS and the thermostable DgAS, under conditions of polymer synthesis and, simultaneously, the amylose conformation. The structure in solution of both amylosucrases during the reaction was shown to be similar to the known crystallographic structures. The conformation of amylose produced at an early stage consists of a mixture of wormlike chains and double helical cylindrical structures. In the case of NpAS, in a second stage, individual double helices pack into clusters before crystallizing and precipitating. Amylose produced by DgAS never self-associates in such clusters due to the higher temperature used for amylose synthesis. All the dimensions determined for wormlike chains and cylindrical conformations at different times of NpAS synthesis are in very good agreement with structural features usually observed on gels of amylose extracted from starch. This provides new insights in understanding the mechanisms of amylose gelation.

Foaming properties of protein/pectin electrostatic complexes and foam structure at nanoscale.

The foaming properties, foaming capacity and foam stability, of soluble complexes of pectin and a globular protein, napin, have been investigated with a "Foamscan" apparatus. Complementary, we also used SANS with a recent method consisting in an analogy between the SANS by foams and the neutron reflectivity of films to measure in situ film thickness of foams. The effect of ionic strength, of protein concentration and of charge density of the pectin has been analysed. Whereas the foam stability is improved for samples containing soluble complexes, no effect has been noticed on the foam film thickness, which is almost around 315Å whatever the samples. These results let us specify the role of each specie in the mixture: free proteins contribute to the foaming capacity, provided the initial free protein content in the bulk is sufficient to allow the foam formation, and soluble complexes slow down the drainage by their presence in the Plateau borders, which finally results in the stabilisation of foams.

Spatial structure and composition of polysaccharide-protein complexes from small angle neutron scattering.

We use small angle neutron scattering (SANS), with an original analysis method, to obtain both the characteristic sizes and the inner composition of lysozyme-pectin complexes depending on the charge density. Lysozyme is a globular protein and pectin a natural anionic semiflexible polysaccharide with a degree of methylation (DM) 0, 43, and 74. For our experimental conditions (buffer ionic strength I = 2.5 10(-2) mol/L and pH between 3 and 7), the electrostatic charge of lysozyme is always positive (from 8 to 17, depending on pH). The pectin charge per elementary chain segment is negative and can be varied from almost zero to one through the change of DM and pH. The weight molar ratio of lysozyme on pectin monomers is kept constant. The ratio of negative charge content per volume to positive charge content per volume, -/+, is varied between 10 and 0.007. On a local scale, for all charged pectins, a correlation peak appears at 0.2 A(-1) due to proteins clustering inside the complexes. On a large scale, the complexes appear as formed of spherical globules with a well-defined radius of 10 to 50 nm, containing a few thousands proteins. The volume fraction Phi of organic matter within the globules derived from SANS absolute cross sections is around 0.1. The protein stacking, which occurs inside the globules, is enhanced when pectin is more charged, due to pH or DM. The linear charge density of the pectin determines the size of the globules for pectin chains of comparable molecular weights whether it is controlled by the pH or the DM. The radius of the globules varies between 10 and 50 nm. In conclusion, the structure is driven by electrostatic interactions and not by hydrophobic interactions. The molecular weight also has a large influence on the structure of the complexes because long chains tend to form larger globules. This may be one reason why DM and pH are not completely equivalent in our system, because DM0 has a short mass, but this may not be the only one. For very low pectin charge (-/+ = 0.07), globules do not appear and the scattering signals a gel-like structure. We did not observe any beads-on-a-string structure.

Polysaccharide/Surfactant complexes at the air-water interface - effect of the charge density on interfacial and foaming behaviors.

The binding of a cationic surfactant (hexadecyltrimethylammonium bromide, CTAB) to a negatively charged natural polysaccharide (pectin) at air-solution interfaces was investigated on single interfaces and in foams, versus the linear charge densities of the polysaccharide. Besides classical methods to investigate polymer/surfactant systems, we applied, for the first time concerning these systems, the analogy between the small angle neutron scattering by foams and the neutron reflectivity of films to measure in situ film thicknesses of foams. CTAB/pectin foam films are much thicker than the pure surfactant foam film but similar for high- and low-charged pectin/CTAB systems despite the difference in structure of complexes at interfaces. The improvement of the foam properties of CTAB bound to pectin is shown to be directly related to the formation of pectin-CTAB complexes at the air-water interface. However, in opposition to surface activity, there is no specific behavior for the highly charged pectin: foam properties depend mainly upon the bulk charge concentration, while the interfacial behavior is mainly governed by the charge density of pectin. For the highly charged pectin, specific cooperative effects between neighboring charged sites along the chain are thought to be involved in the higher surface activity of pectin/CTAB complexes. A more general behavior can be obtained at lower charge density either by using a low-charged pectin or by neutralizing the highly charged pectin in decreasing pH.

Front-face fluorescence spectroscopy study of globular proteins in emulsions: displacement of BSA by a nonionic surfactant.

The displacement of a globular protein (bovine serum albumin, BSA) from the surface of oil droplets in concentrated oil-in-water emulsions by a nonionic surfactant (polyoxyethylene sorbitan monolauarate, Tween 20) was studied using front-face fluorescence spectroscopy (FFFS). This method relies on measurement of the change in intensity (I(MAX)) and wavelength (lambda(MAX)) of the maximum in the tryptophan emission spectrum. A series of oil-in-water emulsions (21 wt % n-hexadecane, 0.22 wt % BSA, pH 7.0) containing different molar ratios of Tween 20 to BSA (R = 0-131) were prepared. As the surfactant concentration was increased, the protein was progressively displaced from the droplet surfaces. At R > or = 66, the protein was completely displaced from the droplet surfaces. There was an increase in both I(MAX) and lambda(MAX) with increasing Tween 20 concentration up to R = 66, which correlated with the increase in the ratio of nonadsorbed to adsorbed protein. In contrast, there was a decrease in I(MAX) and lambda(MAX) with Tween 20 concentration in protein solutions and for R > or = 66 in the emulsions, which was attributed to binding of the surfactant to the protein. This study shows that FFFS is a powerful technique for nondestructively providing information about the interfacial composition of droplets in concentrated protein-stabilized emulsions in situ. Nevertheless, in general the suitability of the technique may also depend on protein type and the nature of the physicochemical matrix surrounding the proteins.

Front-face fluorescence spectroscopy study of globular proteins in emulsions: influence of droplet flocculation.

Measurement of the intensity (I(MAX)) and/or wavelength (lambda(MAX)) of the maximum in the tryptophan (TRP) emission spectrum using front-face fluorescence spectroscopy (FFFS) can be used to provide information about the molecular environment of proteins in nondiluted emulsions. Many protein-stabilized emulsions in the food industry are flocculated, and therefore, we examined the influence of droplet flocculation on FFFS. Stock oil-in-water emulsions stabilized by bovine serum albumin were prepared by high-pressure valve homogenization (30 wt % n-hexadecane, 0.35 wt % BSA, pH 7). These emulsions were used to create model systems with different degrees of droplet flocculation, either by changing the pH, adding surfactant, or adding xanthan. Emulsions (21 wt % n-hexadecane, 0.22 wt % BSA) with different pH (5 and 7) and molar ratios of Tween 20 to BSA (R = 0-131) were prepared by dilution of the stock emulsion. As the surfactant concentration was increased, the protein was displaced from the droplet surfaces, which caused an increase in both I(MAX) and lambda(MAX), because of the change in TRP environment. The dependence of I(MAX) and lambda(MAX) on surfactant concentration followed a similar pattern in emulsions that were initially flocculated (pH 5) and nonflocculated (pH 7). Relatively small changes in FFFS emission spectra were observed in emulsions (21 wt % n-hexadecane, 0.22 wt % BSA, pH 7) with different levels of depletion flocculation induced by adding xanthan. These results suggested that droplet flocculation did not have a major impact on FFFS. This study shows that FFFS is a powerful technique for nondestructively providing information about the molecular environment of proteins in concentrated and flocculated protein-stabilized emulsions. Nevertheless, in general the suitability of the technique may also depend on protein type and the nature of the physicochemical matrix surrounding the proteins.