Complex coacervation process occurs due to electrostatic attraction of oppositely charged biopolymers (Devi, Sarmah, Khatun, & Maji, 2016; Lv, Zhang, Zhang, Abbas, & Karangwa, 2013) that induce the formation of biopolymer soluble or insol-uble complexes. The formation of insoluble complexes results in macroscopic phase separation into the coacervate phase and sol-vent rich phase (Anema & de Kruif, 2013; Kaushik, Dowling,
Barrow, & Adhikari, 2015; Wee et al., 2014; Wu & McClements, 2015). Beside, other weak energy interactions especially hydrogen bonding, hydrophobic interactions can also contribute to the for-mation of complexes and coacervates (Turgeon, Schmitt, & Sanchez, 2007). The complexation process and the resultant coac-ervate types formed are driven and influenced by various param-eters; pH, protein to polysaccharide ratio (Prs), total biopolymer concentration, molecular conformation and flexibility among others (Schmitt & Turgeon, 2011; Timilsena, Wang, Adhikari, & Adhikari, 2016; Wee et al., 2014; Wu et al., 2011). Generally, co-
acervates are formed when biopolymers with a low charge density and/or very flexible backbone are utilized, implying that charge density is critical in the formation of liquid coacervates (Lv, Zhang, Abbas, & Karangwa, 2012; Turgeon & Laneuville, 2009). The com-plex coacervation method has been used to develop effective de-livery systems to encapsulate, protect, and release bioactives and flavors and has several benefits, such as high pay load, encapsula-tion efficiency (Prata & Grosso, 2015; Santos, Bozza, Thomazini, &Favaro-Trindade, 2015; Aziz, Gill, Dutilleul, Neufeld, & Kermasha,2014) and controlled release of encapsulated materials (Dong et al.,2011).
Proteins and polysaccharides have been extensively utilized in recent years as functional ingredients to improve texture, structure and shelf-life of most food products and for bioactives coating and delivery (Anvari, Pan, Yoon, & Chung, 2015; Niu et al., 2015; Qiu, Zhao, & McClements, 2015; Wu & McClements, 2015). The in-teractions between proteins and polysaccharides are either attractive or repulsive. They can be manipulated to form avariety of biopolymer complexes, such as soluble complexes, coacervates or precipitates (Anvari et al., 2015; Jones, Lesmes, Dubin, & McClements, 2010; Souza, Garcia Rojas, Melo, Gaspar, & Lins, 2013). Protein and polysaccharide complex coacervates exhibit
new functional properties by combining advantages of both bio-polymers (Yan & Zhang, 2014).
Gelatin is a natural non-toxic water-soluble protein derived from collagen. Polypeptide structure of gelatin molecule facilitates its interactions with other oppositely charged ingredients (Dai, Wu,Li, Zhou, Li, & Chen., 2010; Milanovi ? c, Petrovi ? c, Sovilj, & Katona,2014), which makes it a very important wall material used for microcapsules production by complex coacervation (Dai et al.,2010; Wu & McClements, 2015; Zhang, Zhang, Hu, Bao, & Huang,2012).
Sodium carboxymethyl cellulose (CMC) is one of the most important anionic nontoxic water-soluble polysaccharides widely used in food, pharmaceutical and medical industry (Koupantsis, Pavlidou, & Paraskevopoulou, 2016; Nur Hazirah, Isa, & Sarbon, 2016). Structurally, CMC (degree of substitution, DS, viscosity and molecular weight, Mw) influences the stability of protein in acid environment (Du et al., 2009). The DS, usually in the range of 0.4e1.5 is an important characteristic of CMC owning to its charge distribution, which defines its electrostatic interactions. CMCs with higher DS have stronger negative charges that induce more elec-trostatic attraction with protein (Iwasaki, Shioi, & Kouno, 1977; Li et al., 2017). For those with DS above 1.5, it is hard to produceunclustered coacervates and microcapsules, whereas that of DS
below 0.4 exhibits a very low solubility (Du et al., 2009; Iwasaki, Shioi, & Kouno, 1977).
The combination of gelatin and gum Arabic is the most common system used in studies of complex coacervation (Anvari et al., 2015; Lv et al., 2013). For food systems, CMC is the most widely used material compared to GA due to its biodegradability, biocompati-bility and relatively low cost (Carpineti, Martinez, Pilosof, & P ? erez, 2014; Devi et al., 2016). However, the few studies, which reported the use of gelatin and CMC to prepare microcapsules (Devi & Maji,2011; Wu et al., 2011), surfactants were added to achieve protein/CMC microcapsules by complex coacervation method. This stems from the fact that CMC has a weak surface-activity and difficult to form protein/CMC microcapsules when used alone in complex coacervation technique (Dai et al., 2010; Wu et al., 2011). Addi-tionally, the use of CMC is limited by the narrow pH range of pro-tein/CMC coacervation compared to GA. To the best of our knowledge, gelatin and CMC complex coacervation and its forma-tion mechanism still have not been well documented.
The aim of this work was therefore to study the G-CMC complex coacervate formation process and its mechanism. We systemati-cally studied the complex coacervate formation process between G and different CMC types and investigated their conformational transition and the nature of interaction at the molecular level. The complex coacervates formation process as a function of pH and protein topolysaccharide mixing ratio (G/CMC MR) was carried out. Optical microscopy was used to evaluate the coacervates morphology during complex formation. ATR-FTIR and Far-UV CD were used to evaluate the nature of interaction between G and CMC and theirconformational transitions during the complex formation. The thermal stability of the resultant product was investigated based on TGA and DSC measurements.