International Journal of Biological Macromolecules 50 (2012) 925–931 Contents lists available at SciVerse ScienceDirect International Journal of Biological Macromolecules jo u r n al hom epa ge: ww w.elsevier.com/locate/ijbiomac Self-assembly of -lactoglobulin and the soluble fraction of gum tragacanth in aqueous medium Mahtab Hasandokht Firooz, Mohammad Amin Mohammadifar ∗ , Parivash Haratian Department of Food Science and Technology, Faculty of Nutrition Sciences, Food Science and Technology/National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran a r t i c l e i n f o Article history: Received 23 December 2011 Received in revised form 15 February 2012 Accepted 18 February 2012 Available online 25 February 2012 Keywords: Gum tragacanth -Lactoglobulin Self-assembly Complex coacervation Rheology a b s t r a c t Spectrophotometric and light scattering measurements, along with optical microscopy, were used to follow the complexation and coacervation process that occur when -lactoglobulin (BLG)/tragacanthin (T) mixed dispersions (0.3 wt.% total concentration; BLG:T ratio of 2:1) were brought from pH 6 to pH 2. In addition, the coupling of slow in situ acidification of the mixture and rheometry was utilised to gain deeper insights into pH-induced structural transitions during the assembly process. The results obtained by this multi-methodological approach allowed the associative phase separation process to be parameterised in terms of a set of characteristic pH values (∼5.3, ∼4.8, ∼4.5, ∼4.15, ∼4, ∼3.8, ∼2.5) at which critical structural changes took place. Investigation of the absorbance profiles of complexed/coacervated systems as a function of time revealed that several transitions could occur at different time scales. Morphological changes in the assemblies and the subsequent formation of some flocculant substances during the late stage of process were clearly visualised using microscopy. © 2012 Elsevier B.V. All rights reserved. 1. Introduction In the past decade, a great interest in the development and optimisation of multi-functional systems by interactions between macro-ions, especially proteins and polysaccharides, has appeared [1–3]. Because most of the novel self-assembled colloidal enti- ties are multi-component systems, it should be noted that for functionality of the multi-ingredient systems, the interactions between ingredients are more important than the composition of the ingredients [4]. In this respect, a particular associative phase separation (complex coacervation), mainly arising by electrostatic attractions between oppositely charged polymers, is an interest- ing phenomenon from both a fundamental and an applied point of view [5]. Protein–polysaccharide complexes and coacervates are relevant to many biological cognate pairs [1] and physiological processes such as DNA/histone collapse, gene replication, elastogenesis, channelling of enzymes, and cytoplasmic organisation [6]. In addi- tion to this fundamental biological significance, a huge potential application of non-cognate protein–polysaccharide complexes and coacervates has grown exponentially in the following domains: ∗ Corresponding author at: Department of Food Science and Technology, Faculty of Nutrition Sciences, Food Science and Technology/National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, P.O. Box 19395-4741, Tehran, Iran. Tel.: +98 2122648120; fax: +98 212237647. E-mail address: mohamdif@ut.ac.ir (M.A. Mohammadifar). nano-biotechnology, design of biomimetic systems, pharmaceuti- cal, cosmetic, and food industries [2,3,7]. The investigation of many protein–polysaccharide systems using a multi-methodological approach, covering a very wide range of structural levels, has provided good knowledge about pH-induced structural transitions during complexation and coacer- vation [8–10]. Roughly speaking, the self-assembly of an associative protein–polysaccharide system occurs as follows: (a) unlike macro- ions interact through electrostatic interactions to form primary soluble complexes; (b) after the completion of this process, inter- apolymeric complexes associate into interpolymer aggregates; and (c) finally, a liquid–liquid phase separation leads to the forma- tion of coacervate droplets [1,8]. It is worth mentioning that some protein–polysaccharide systems phase separate as co-precipitates instead of coacervates [3]. Phase-ordering kinetic studies revealed that electrostatic complexation/coacervation between BLG and pectin or acacia gum follows a nucleation and growth mecha- nism [11,12], which is characterised by initial short-range/high amplitude concentration fluctuations within the entire volume of the mixture [4]. However, a survey of the literature shows that the effects of many factors, such as pH, ionic strength, protein to polysaccharide ratio, total biopolymer concentration, biopolymer charge density, and molecular weight on the formation, struc- ture and yield of complexes and coacervates are well documented [1,3,7]; this unique phase separation phenomenon is still one of the most challenging issues of modern biopolymer science. BLG, the most prevalent protein in bovine milk whey, contains 162 amino acids, has a molecular mass of about 18,350 Da and an 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2012.02.020