Historical PerspectiveHeteroprotein complex coacervation: Focus on experimental strategies to investigate structure formation as a function of intrinsic and external physicochemical parameters for food applications
Graphical Abstract
Introduction
Protein is the main component of human tissues and organs and is essential for their growth, maintenance, and metabolism, so a sufficient intake of high-quality protein is essential for human health. In food systems, apart from providing nutrition, proteins can also modify food texture, color, and flavor as a result of their many physicochemical functional properties, and the enormous diversity of their intrinsic structures [1]. Many amino acids in proteins have hydrophobic hydrocarbon, or aromatic side-chains, and the hydrophobic attraction between them cause protein chains to fold into “molten globules”, which become stable globular proteins after “polishing” by van der Waals forces, hydrogen bonds, and ionic bonds [2]. In contrast, non-globular proteins exist mainly as expanded disordered chains and intermediate structural states [3]. The physicochemical properties of amino acid side-chains also impart amphipathic and polyampholytic properties to proteins, which facilitates their self-assembly, into complex multi-component and multi-phase structures, with specific surface anisotropy [4].
The earliest research in the field of biopolymer complex coacervates was reported by Bungenberg et al. [5] a century ago. Since then, complex coacervation, especially in protein-polysaccharide systems, has developed rapidly in food research, with a wide range of applications. For example, coacervates play an important structural role in controlling the macroscopic characteristics of foods (such as texture, stability, and taste), producing edible films, and designing controlled-delivery systems for encapsulated nutraceuticals [6]. Heteroprotein complex coacervation (HPCC) is a specific form of biopolymer complex coacervation and usually refers to the associative phase separation between two or more proteins, driven by the entropy gain from the release of protein-associated counter-ions and water molecules. This process results in a protein-enriched colloidal coacervate phase, stabilized by electrostatic interactions, which co-exists with a protein-poor dilute bulk phase. The resulting assembly structure has novel functional properties, compared with the original proteins and provides a new source of soft, condensed material for the partial replacement of animal proteins by sustainable plant, or microbial proteins. It should be noted that the term “coacervation” is restricted to the colloidal systems with two liquid phases, whereas associative phase separation, caused by electrostatic complexation between different proteins can also generate a solid complex precipitate [7,8]. Therefore, the terminology “complex coacervation” should be used with caution and it is necessary to determine the physical state of any new concentrated phase, using microscopy to distinguish between liquid-liquid and solid-liquid colloids. The metastable nature of liquid assemblies means that both liquid-liquid and solid-liquid phase separation can be triggered by environmental, or intrinsic factors. This review therefore covers both complex coacervates (liquid-liquid phase separation) and amorphous complex precipitates (solid-liquid phase separation) to aid a complete understanding of these assembly architectures.
HPCC is not a new concept and it appears in some early reports [[9], [10], [11]], although not referred to as “heteroprotein complex coacervation”. Yan et al. [12] studied the interaction and phase separation behavior of β-Lactoglobulin (β-LG), combined with lactoferrin (LF) and used the terminology “heteroprotein complex coacervation” for the first time. Since then, there have been many reports on the interactions and supramolecular assemblies between oppositely charged proteins (Table 1), and the rapid development in the field of HPCC has led to considerable interest in practical applications. Croguennec [4] reviewed the studies related to HPCC, in relation to the types of proteins involved, describing LF based coacervates, lysozyme (LYS) based coacervates, and other binary systems. Similarly, Boire et al. [13,14] briefly described the progress of HPCC and suggested that it is important to study protein-based multi-component systems because pure protein systems rarely exist in real food matrices.
In this review, we first introduce the research methods and techniques used in HPCC in detail, which may help to remedy deficiencies in past research and provide guidelines and considerations to facilitate further progress in this field. Many studies have focused on the effect of the predominant parameters on the organizational structure and phase behavior of HPCC; these variables are summarized in terms of environmental conditions and intrinsic factors in the second part. This is highly relevant because real food matrices are often affected by a wide variety of factors. Finally, the potential applications of HPCC and heteroprotein assemblies in the food field are described, based on current reports on the functional properties of these architectures.
Section snippets
Research methods and techniques
Several primary and innovative experimental methods and simulation tools have been developed to understand the formation conditions, driving forces, multi-scale structure, phase behavior, and rheology, as well as the interactions between the proteins and the sorrounding medium (water and counter-ions) of HPCC materials (Fig. 1). Although most methods and tools have limitations, the application of complementary approaches facilitates improved understanding of the structure and properties of
Effect of environmental and intrinsic factors on HPCC
Naturally occurring liquid-liquid phase separation is usually subject to regulation by multiple factors (changes in protein and/or RNA concentration, ionic strength, post-translational modification, and ligand binding). The food matrix is often affected by similar factors during processing, storage, and transportation. HPCC arises from a combination of electrostatic interaction, short-range forces, structural characteristics, and thermodynamic factors. Electrostatic interaction is strongly
Application
As multi-functional building blocks, proteins can form a variety of self-assembled structures that have been widely applied in the food field [105]. Although there have been far fewer reports on HPCC than on polyelectrolyte-based complex coacervation systems, the current studies have highlighted the application of HPCC and related complexes in areas such as nutraceutical encapsulation, nanogels, emulsions stabilization, and protein separation.
Conclusions and future prospects
As novel materials, with high protein concentration and formation driven mainly by electrostatic interaction, HPCC materials have wide application potential in the food and other fields. At present, there has been some consensus on the HPCC, especially its response to environmental factors (Fig. 7). As mentioned above, many types of proteins can form HPCCs, such as globulins, expanded disordered proteins, intermediate structural proteins, and even polyelectrolyte polypeptides. Hence, it is
Declaration of Competing Interest
We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with above work submitted to Advances in Colloid and Interface Science.
Acknowledgements
This work was supported by the National Key R&D Program of China (No. 2016YFD0401504); the National Natural Science Foundation of China (31671870); Pearl River S&T Nova Program of Guangzhou (201610010105); the Science and Technology Program of Guangzhou (201807010102); the Special Support Project of Guangdong Province for Science and Technology Innovative Young Talents (2014TQ01N538) and the 111 Project (B17018).
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