Protein-polysaccharide mixtures structure of the systems and the effect of shear studied by SANS

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Physica B 234-236 (1997) 289-291

Protein-polysaccharide mixtures structure of the systems and the effect of shear studied by SANS D. R e n a r d a'*, F. B o u 6 b, J. L e f e b v r e a aI.N.R.A., Centre de Recherches Agro-Alimentaires, Laboratoire de Physico-Chimie des Macromolkcules, BP 1627, 44316 Nantes Cedex 03, France bLaboratoire Lkon Brillouin (C.E.A.-C.N.R.S.), C.E.-Saclay, 91191 Gif-sur-Yvette Cedex, France

Abstract

We present results of a small-angle neutron scattering (SANS) study of a globular protein mixed with a neutral (or charged) polysaccharide, and discuss how the structure is affected by shear. According to SANS measurements, the structures of these mixtures located in the one-phase region of the phase diagram were heterogeneous, the level of flocculated particles being greatly dependent on the polysaccharide and the solvent conditions used. Shearing these types of solutions at low shear rates induced a disruption of the liquid-like short-range order (when it existed) between proteins and a slight anisotropy of the particles indicative of some preferential alignment of proteins in the direction of flow. Keywords: Polymers; Proteins; Small-angle neutron scattering

1. Introduction

Protein-polysaccharide mixtures show generally liquid-liquid phase separation above a critical polymer concentration [1]. The aim of this study was to investigate the structure of bovine serum albumin (BSA)/hydroxyethylcellulose (HEC) or BSA/carboxymethylcellulose (CMC) mixtures in the "one-phase" region of the phase diagram, and how it is affected by shear.

the signal from polysaccharide solutions was negligible and was further substracted from the intensity obtained for the protein/polysaccharide mixtures. Measurements under static and shear conditions were performed at ambient temperature using a quartz Couette shear cell (gap: 0.5 mm; shear rate range: 0.1-100 s-1). The intensities along the horizontal and vertical axes of the detector were determined by sectorial analysis as described previously [2] and were finally expressed in molecular mass (g mol- 1) as the density and the contrast variation of BSA in H 2 0 were known.

2. Experimental SANS measurements on polysaccharide/protein mixtures (1/10% w/w) in H 2 0 were carried out at the LLB, C.E.-Saclay using the PAXY instrument (d = 3.2 m; 2 = 10 ~). Using pure H 2 0 as the solvent, * Corresponding author.

3. Results 3.1. Scattering behaviour under static conditions.

We first observed BSA solutions alone. At pH 5.2, near the isoelectric point of the protein, a large

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D. Renard et al. / Physica B 234-236 (1997) 289-291

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Fig. 1. Scattering profiles for BSA solutions (10% w/w) and BSA/HEC or BSA/CMC mixtures (10%/1% w/w) in water. ( ) BSA pH 5.2; (...) BSA pH 7; (O) BSA/HEC pH 5.2; (0) BSA/CMC pH 7.

Fig. 2. Scattering profiles for BSA solutions (10% w/w) and BSA/HEC or CMC/BSA mixtures (10%/1% w/w) in water. ( ) BSA pH 5.2; (...) BSA pH 7; ((3) BSA/HEC pH 7; (0) BSA/CMC pH 5.2.

increase of intensity at low q values corresponding to an attractive interaction indicates incipient precipitation of the protein (Fig. 1). At pH 7, where BSA is negatively charged, a correlation peak is 1 observed (qm,x = 0.057A- ). This feature results from electrostatic repulsions between the particles (e.g. center-to-center distance of ca. 1.5 BSA diameters) [3]. In the presence of added polysaccharide, the mixtures show two different types of scattering behaviour. The first one (Fig. 1), corresponding to the cases of neutral protein-neutral polymer and charged protein-charged polymer with the same signs, is an increase of scattering intensity at low q values. It corresponds to a separation at large scale, may be micro-phase separation of BSA particles in the mixture. This flocculation process may come from the depletion forces exerted by the entangled polysaccharide chains on the BSA particles or, in the case where the two components are charged, may come from the great electrostatic repulsions. In the second type of scattering behaviour (Fig. 2), corresponding to the cases of neutral polymer-charged protein and charged polymer-neutral protein, a similar increase of intensity at large scale is observed but in addition a short-range order peak between particles at small scale appears; this

peak was not observed on BSA solution alone. We suggest that the liquid-like short-range order between BSA particles induced by the addition of the polymer could result from an adsorption of particles onto the polymer coil segments or an entrapping in the cross links of the entangled polymer coils network, hence a preferential distance between protein molecules in the mixture [4].

3.2. Scattering behaviour under shear conditions Whatever the shear rate (0.1-100s -1) applied, the peak intensity is reduced considerably as compared to the unsheared system, but the upturn of the scattering curves at low q values is unchanged. Besides, whereas the scattering of the unsheared system is isotropic, slight but definite anisotropy developed under moderate shear, the scattered intensity being larger in the direction normal to the shear direction [4]. It seems therefore that the initial short-range order of BSA molecules is disrupted under shear, and that some preferential alignment in the direction of flow develops giving a long-range anisotropic structure. This anisotropic character seems to be irreversible and disappears at high shear rate.

D. Renard et al. /Physica B 234-236 (1997) 289-291

References 1-13 V.B. Tolstoguzov, in: Functional Properties of Food Macromolecules, (Elsevier Applied Science Publishers, London, 1986) pp. 385-415. [2] J.D.F. Ramsay, S.W. Swanton and J. Bunce, J. Chem. Soc. Faraday Trans. 86 (1990) 3919.

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[3] D. Renard, M.A.V. Axelos, F. Bou~ and J. Lefebvre. Biopolymers, 39 (1996) 149. [4] D. Renard, F. Bou6 and J. Lefebvre. in: Food Colloids Proteins, Lipids and Polysaccharides, eds. E. Dickinson and B. Bergenstahl (The Royal Society of Chemistry, Cambridge, 1997), in press. -