# A Study on the Stoichiometry of Casein/Chitosan Gel Complexes as a Delivery System for Quercetin
## 3 Results and Discussion  
### Formation and Characterization of Casein/Chitosan Gel Polyelectrolyte Complexes  
The pH level plays a crucial role in the complexation process because it regulates the degree of ionization of ionic groups and influences the intensity of the electrostatic interactions between charged components. In the present research, the pH of the synthesis medium was specifically chosen to ensure the difference in charge of the chitosan and casein molecules during the synthesis process. Due to the attractive electrostatic interaction, casein/chitosan polyelectrolyte complexes were successfully created. Based on [PERSON]'s theory about weak polyelectrolytes [26], the degree of ionization of chitosan at pH 6 was calculated to be 72% and that of casein was calculated to be 94%. Therefore, both polyelectrolytes were characterized with high charge density, which causes the formulation of insoluble precipitates. Similar results were already presented by other authors [27]. The complexation process between casein and chitosan was confirmed by FT-IR analysis. The FT-IR spectra of casein, chitosan, and their complex are presented in Figure 1.  
The spectrum of casein shows characteristic peaks at 3292 cm\({}^{-1}\), around 1550 cm\({}^{-1}\), and 1241 cm\({}^{-1}\) corresponding to N-H stretching, Amide II (N-H bending and C-H stretching), and Amide III (\(\beta\)-sheet structure) [26]. Detected characteristic peaks for chitosan  
Figure 1: FT-IR spectra of casein, chitosan, and their complex at casein/chitosan charge ratio 1:1.  
are at 1652 cm\({}^{-1}\) N-H deformation in Amide I, 1318 cm\({}^{-1}\) Amide III, and 1091 cm\({}^{-1}\) C-O vibration of tensional bonds [28]. Evidence for the complex formation can be found in the complexes' spectrum, showing characteristic bands of chitosan around 3500 cm\({}^{-1}\) and 2359 cm\({}^{-1}\) and a shift in the band of the primary amide of chitosan and the tertiary amide of casein along with a change in the peak shape and intensity around 1073 cm\({}^{-1}\)[29]. The composition and some main characteristics of the gel particles are presented in Table 1.  
The casein/chitosan complexes S1, S2, and S4 were characterized as submicron structures with mean diameters of 460, 460, and 377 nm, respectively. These results demonstrate a relatively narrow monomodal size distribution in the size range between 250 and 1000 nm with a Span around 1. While samples S1, S2, and S4 do not vary greatly in size, the size of sample S6 increases sharply and shifts into the micron range (5.720 \(\upmu\)m). For this sample, the size distribution changes to bimodal, and the Span increases to 7.068, indicating an aggregation tendency not observed in the other samples. A possible explanation for this could be related to the stabilizing role of chitosan. Casein is an amphiphilic protein with distinct hydrophobic and hydrophilic domains, which would result in the self-assembly of the casein molecules into stable micellar structures in aqueous solutions [30]. Chitosan is a hydrophilic polysaccharide and at a pH below its pKa, its primary amine groups become protonated, which would lead to the dissolution of the polysaccharide. Because of its high charge density (usually in the abovementioned conditions), the chitosan macromolecule is in a stretched conformation [31]. This way, it is located around the casein micelle and flocculates casein at low polysaccharide concentrations, while stabilizing it at moderate to high concentrations [32]. Casein/chitosan complex morphology was examined by SEM and AFM--Figures 2 and 3. The SEM microphotograph shows gel particles with three different patterns: rough spherical particles, wrinkled spherical particles, and wrinkled irregularly shaped particles. A pronounced tendency to aggregation is noticed. Similar aggregation behaviour was observed for casein phosphopeptides-chitosan composite nanoparticles in the research of [PERSON] et al.