Immobilization of silver ions onto casein

Abstract

In this study, the physicochemical and biological properties of silver-casein complexes were investigated. The casein modification was carried out by a novel method, i.e., at basic conditions (pH 8) using silver precursor in a form of ammonia complex [Ag(NH3)2]+. The selected experimental conditions allowed us to obtain high colloidal stability of protein suspension and also minimized the risk of metal secondary reduction. Subsequently, the mechanism of silver immobilization was evaluated by using a batch sorption study (Freundlich, Langmuir isotherm modeling). Moreover, the influence of accompanying the protein ions on the silver binding process was studied (using ultrafiltration). Results revealed, that casein after the desalting step has much higher silver-binding capacity (258.2 mg/g) compared to untreated protein (157.1 mg/g). The nature of casein-silver interactions was comprehensively studied by multiple instrumental techniques, namely, spectrometric (ICP-MS), spectroscopic (DLS, ATR-FTIR, Fluorescence Spectroscopy), electron microscopy (STEM, SEM-EDX), and separation (SDS-PAGE). Results indicated, that silver sorption had a complex character. The heterogeneous character of metal binding processes was discovered. The DLS studies pointed out the great role of phosphoserine residues in silver-casein interactions and underlined their impact on the micelle size and structure. Moreover, STEM images confirmed the growth of silver nanoparticles upon metal binding to casein. According to FTIR analysis, carboxylic groups of aspartic and glutamic acid were identified as potential metal-binding sites which also could participate in silver reduction. Peptic digestion studies showed a relevant influence of silver incorporation on the structural stability of caseins. Finally, the biological activity of the 600Ag-CN complex was evaluated. Results indicated that the complex effectively inhibited the growth of both Gram-positive (S. aureus) and Gram-negative (K. pneumoniae) bacteria. Still, the treatment of mouse fibroblast cell line L929 with analyzed complex (0.32 mg/mL of 600Ag-CN corresponding to 7.5 µg/mL of silver) decrease cell viability up to 60 %. The toxic activity of the complex may be connected to the Ag+ release. The silver desorption study showed that 600Ag-CN complex released 21.5 %, 49.3 %, and 68.7 % in pH 6.8, 4.5, and 1.2 respectively.