The study of pH-dependent complexation between gelatin and gum arabic by morphology evolution and conformational transition
Graphical abstract
Introduction
The formation of self-assembled colloidal entities between protein and polysaccharide induced by electrostatic interaction refers to a physico-chemical phenomenon (Turgeon, Schmitt, & Sanchez, 2007). It is phase separation caused by the interaction of oppositely charged biopolymers in which a dilute colloidal phase coexists with a more concentrated colloidal phase (de Kruif, Weinbreck, & de Vries, 2004; Moschakis, Murray, & Biliaderis, 2010). The latter is called the coacervates phase. According to Bungenberg de Jong and Kruyt (1929), the word “complex coacervation” was created specifically for the interaction of two kinds of biopolymers, opposing to the “simple coacervation”, which is based on one biopolymer (Bungenberg de Jong & Kruyt, 1929).
Nowadays, considerable research efforts are being devoted to the reaction of complex coacervation, attributing to not only the biological significance of the protein–polysaccharide system but also its potential application in a variety of fields, including pharmaceutical and food industry (Huang, Cheng, Yu, Tsai, & Cham, 2007; Lee & Rosenberg, 2000; Weinbreck, Minor, & de Kruif, 2004; Xing, Cheng, Yang, & Ma, 2004). As shown in various studies, the reaction is highly sensitive to series of parameters, like pH (Schmitt et al., 2000), mixing ratio (MR), total concentration of biopolymers (Liu, Low, & Nickerson, 2009), ionic strength and type of ion (Wang, Wang, Ruengruglikit, & Huang, 2007). Generally, turbidity titration accompanied by some chemical analysis is the most common applied technology for disclosing the whole course of coacervates formation. However, in terms of mechanism elucidation for coacervates, the information collected already is far from satisfaction. Currently, few studies have been conducted to probe the relationship between two biopolymers at the molecular level.
Complex coacervation is a reaction driven mainly by electrostatic interaction, mostly occurring between protein (a kind of polyampholyte) and polysaccharide (anionically charged within a wide range of pH). In order to explore the conformational transition on protein or polysaccharide, versatile methodologies were chosen. For instance, circular dichroism (CD) was used to disclose the conformational switch of β-lactoglobulin upon complexation with gum arabic (GA) (Mekhloufi, Sanchez, Renard, Guillemin, & Hardy, 2005). It was concluded by a loss of α-helix during the coacervates formation. Similarly, Cousin et al. applied the Fourier transform infrared spectroscopy to test the conformational change of lysozyme in the presence of polystyrene sulfonate and acquired the same loss of helical structure (Cousin, Gummel, Ung, & BouÉ, 2005). However, due to the specific structure of protein, coacervates formation may also lead to an increase in α-helix. In a study on coacervates formation in poly (l)-lysine and pectin, a shift from coil to α-helix was demonstrated by CD technology (Paradossi, Chiessi, & Malovikova, 1999). Obviously, it seems difficult to generalize the protein's conformational change upon complexation from these variable results.
Regarding the other constituent during coacervates formation, polysaccharide, the investigation of its conformational change is challenging. Compared with the protein unit – amino acid, the structure of monosaccharide seems much more complicated. Due to dependence on the amount of carbon atom, existence of isomer, variable ways of inter-connection, the regularity of the monosaccharide is still less known. Besides, the conformational transition of polysaccharide could not be represented by simple terms like α-helix or β-sheet as protein. Thus, most researchers were only centered on protein's conformation, with little or even no reference to the polysaccharide. Burova et al. investigated the conformational changes in ι- and κ-carrageenan upon complexation with β-casein (Burova et al., 2007). The application of high-sensitivity differential scanning calorimetry revealed that β-casein reduced the helical structure of carrageenan, as the interaction with unordered parts of the polysaccharide chains was more favored.
Since it is difficult to derive a general rule for protein's conformational change, considering protein and polysaccharide simultaneously could be a better strategy. Therefore, the aim of this work is to elucidate the mechanism of coacervates formation by investigating the conformational aspects of both polyelectrolytes.
For our research, a classical system was chosen, where gelatin (G) acted as a positive polyelectrolyte while GA as a negative polymer. The pioneering research on the phenomenon of complex coacervation by Bungenberg de Jong and Kruyt was based on such system (Bungenberg de Jong & Kruyt, 1929). Although, G–GA system has the longest history in the scope of the complex coacervation, and it has been gradually fading out of the researchers' focus, the exploitation of this kind of system seems meaningful when applied as wall material in microencapsulation. Moreover, the mechanism on G–GA system at the molecular level has never been reported. In the present study, G–GA mixtures with three MRs were chosen for investigating the morphology evolution and size distribution with pH's transition. Furthermore, coacervates composition and their conformational transition were examined to disclose the formation mechanism during complexation.
Section snippets
Material
Gelatin (type B) and GA (type from Acacia senegal trees) were purchased from China Medicine (Group) Shanghai Chemical Reagent Corporation (Shanghai, China). From the analysis of the raw materials, the results are as follows: GA powder contained 8.10 ± 0.03% moisture, 2.04 ± 0.14% protein (using Kjeldahl analysis with N conversion factor of 6.60 (Blakeney, Harris, Henry, & Stone, 1983)) and 3.50 ± 0.05% ash (mineral content: 0.22% Mg2+, 0.98% Ca2+, 0.012% Na+, 0.73% K+); G granules contained
pH-dependent complexation evaluated by morphology evolution and size distribution
As previously mentioned, complex coacervation is triggered by electrostatic interaction. Thus, both parameters including the MR of biopolymers and pH should be considered simultaneously to obtain general rules of coacervates formation. In order to inspect the pH-dependent coacervates transition thoroughly, three MRs (2:1, 1:1 and 1:2) were chosen. The coacervates at specific MR were obtained over a wide range of pH to determine the suitable pH scope, corresponding to their specific drop-like
Conclusions
Complex coacervation between two polyelectrolytes, G and GA, has been investigated to disclose their coacervates transition with pH and MR. In the present research, with the aid of morphology observation and composition analysis, the individual transition pattern was presented and it seemed GA played a much significant role on the size variation. CD spectroscopy was also applied to explore the conformational transition of G and GA (MR: 2:1) simultaneously: G molecules experienced a
Acknowledgment
The authors would like to thank the National Key Technology R&D Program (2011BAD23B01) and Industry-Academia-Research Program of Guangdong (2010B090400139) for its financial support.
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