Effect of pea proteins extraction and vicilin/legumin fractionation on the phase behavior in admixture with alginate J.-L. Mession a , A. Assifaoui a, b , P. Cayot a , R. Saurel a, * a AgroSup Dijon, UMR PAM 02.102, Equipe PAPC (Procédés Alimentaires et Physico-Chimie),1 Esplanade Erasme, 21000 Dijon, France b School of Pharmacy, Université de Bourgogne, 7 bd Jeanne d’Arc, 21079 Dijon, France article info Article history: Received 23 May 2011 Accepted 12 March 2012 Keywords: Pea globulins Legumin Vicilin Heterogeneity Sodium alginate Segregative phase separation CLSM Microstructure abstract Soluble and natural mixed pea proteins (PP) were extracted from defatted pea seeds according to acidic precipitation (PPP) or ultrafiltration/diafiltration (PPDF) procedures. The isolates contained proteins with a low level of denaturation. Mixed pea globulins isolates presented quite similar solubility and thermal profiles, also a similar polypeptide composition. Vicilin/convicilin 7S (Vic) and legumin 11S (Leg) fractions were obtained by batch chromatography using a salt gradient for the elution. Several incompatible systems were built by mixing the pea proteins with an anionic polysaccharide (sodium alginate, SA), when biopolymers were both negatively charged. Most of mixtures exhibited a phase separation phenomenon. From phase diagrams, experimental binodal curves obtained with either mixed globulins or legumin fraction were apparently very close. However phase boundary was better-defined with the Leg fraction. No macroscopic phase separation was evidenced for mixtures with the vicilin fraction. Microstructure of the PP-SA mixtures was investigated by confocal microscopy (CLSM) according to PP composition and biopolymer initial composition. The Leg-SA and most of PPP-SA mixtures exhibited a droplet-like structure, while structure of PPDF-SA mixtures was aggregated-like. With mixed PP, an alginate entrapment within the PP-enriched phase would disturb phase separation. Also density and shape of the protein-enriched microdomains influenced kinetics of demixing. Polydispersity within the PP-SA mixtures, in terms of wide range molecular weights distribution and charge heterogeneity would explain such differences. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Proteins and polysaccharides are two major compounds encountered in almost all food systems. When mixed together in solution, interaction between those biopolymers governed the phase behavior of such blended systems, hence impacting their structural, textural properties and hence their stability. Biopoly- mers in admixture can be miscible or immiscible (unless limitedly co-soluble). Single-phase mixtures refer commonly to diluted biopolymers solutions and/or biopolymers with a low molecular weight so that they do not interact to each other. Increasing biopolymer initial concentrations led to unstable mixtures. Depending on the type of interaction, such phase separation phenomenon could be “associative” or “segregative” (De Kruif and Tuinier, 2001; Grinberg & Tolstoguzov, 1997; Syrbe, Fernandes, Dannemberg, Bauer, & Klostermeyer, 1995; Tolstoguzov, 1986, 1991). Associative phase separation occurs when protein and anionic polysaccharide are oppositely charged (below the protein’s isoelectric pH). Soluble or insoluble interbiopolymer electrostatic complexes are then formed, and concentrated in a lower phase while the upper phase is depleted with biopolymers (Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998; Tolstoguzov, 1991). For a mixture containing polymers (1, 2) with a random coil conformation, segregative phase separation is usually described by the FloryeHuggins theory; indeed, phase behavior of a mixture is a consequence of the balance between polymer1-polymer2 and polymer1(2)-solvent interactions (Grinberg et al., 1997). The appearance of an excluded volume for each polymer led to an enhancement of polymeresolvent interaction. Then mixture split into two liquid phases at the equilibrium, as a result of polymer segregation in separated phases. By thermodynamic incompati- bility, it is more favorable that each polymer is surrounded by like polymers than unlike polymers remain mixed together (Tolstoguzov, 1986, 1991). Nevertheless, the Flory theory could not be applied reliably for mixtures of dissimilar polymers, in terms of shape and structure; this is the case for globular proteins or colloidal protein particles mixed with polysaccharides of elongated shape (De Kruif & Tuinier, 2001). * Corresponding author. Tel.: þ33 380 774 051; fax: þ33 380 396 647. E-mail address: r.saurel@agrosupdijon.fr (R. Saurel). Contents lists available at SciVerse ScienceDirect Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2012.03.003 Food Hydrocolloids 29 (2012) 335e346