A thesis submitted for M.Sc. degree of hygiene and food safety Evaluation of sodium hydrosulfite interaction with serum albumin protein using spectroscopic and molecular docking methods

Abstract

ract Aim: The structural and functional properties of the sodium caseinate complex and phenolic acids/ascorbic acid were studied, and their ability to form a solid particle- stabilized emulsion (Pickering emulsion), electrospinning fiber, and hydrogel was evaluated. Material and methods: Initially, a systematic optimization of key factors, including temperature, alkaline pH, reaction time, and the concentration of active substances (ascorbic acid or gallic acid), was conducted. Sodium caseinate was set at a weight concentration of 10 mg/mol, while the active ingredients were maintained at 1 mg/mol. To replicate the conditions of the Maillard reaction, the aqueous phase underwent a heat treatment at 80°C for 30 minutes. Subsequently, an in-depth analysis of the structural and physicochemical properties of the resulting colloidal particles was undertaken. This included the measurement of particle size, zeta potential, and free amine groups. Furthermore, a comprehensive examination was performed using analytical techniques such as Fourier transform infrared spectroscopy (FT-IR), polyacrylamide gel electrophoresis (SDS-PAGE), fluorescence spectroscopy, contact angle measurements, and droplet surface tension analysis. The capacity for forming Pickering emulsions was evaluated by combining the aqueous phase samples with corn oil in a 50:50 ratio, followed by homogenization using a homogenizer (10,000 revolutions for 5 minutes) and an ultrasound probe with a 13 mm diameter (operating at 50% power for 2 minutes). Subsequent analysis encompassed the investigation of structural characteristics, as well as the physical and oxidative stability of the emulsion droplets through droplet size testing, determination of the creaming index, confocal laser scanning microscopy (CLSM), and assessment of thiobarbituric acid reactive substances (TBARS). Furthermore, the potential of modified sodium caseinate in the formation of electrospun nanofibers was explored. A composite comprising equal amounts of sodium caseinate and gelatin (at a 50:50 ratio) was prepared with a weight concentration of 25%. Subsequently, varying concentrations (0.1%, 0.5%, 1%, 5%, and 10%) of gallic acid were incorporated. The resulting nanofibers were characterized using scanning electron microscopy, Fourier transform spectroscopy, and assays for antioxidant capacity. Results: Fluorescence analysis confirmed the occurrence of hydrophobic interactions in the sodium caseinate-ascorbic acid (SC-AA) system. The presence of Schiff base formations in colloidal particles

of hydrolyzed sodium caseinate-ascorbic acid (HSC-AA) was established through Fourier Transform Infrared (FTIR) spectroscopy, affirming the onset of the Maillard reaction between AA and SC. Notably, the HSC-AA colloidal particles, at a lower concentration (10 mg/mL SC and 1 mg/mL AA), exhibited effective stabilization of Pickering Emulsion (PE) within a 50:50 oil-to-water volume ratio, demonstrating robust physical and oxidative stability. Under the influence of heat treatment and alkaline conditions, the interaction between SC and AA was observed to increase the contact angle while significantly reducing the surface tension. Scanning Electron Microscopy (SEM) images illustrated that HSC-AA particles possessed a denser structure and the smallest particle size among the samples. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and the quantification of the percentage loss of free amino groups indicated the formation of covalent bonds in HSC-AA samples. Confocal Laser Scanning Microscopy (CLSM) imagery demonstrated effective encapsulation of oil droplets by HSC-AA particles in the oil-in-water Pickering emulsion. The analysis of Thiobarbituric Acid Reactive Substances (TBARS) strongly suggested a significant enhancement in the oxidative stability of HSC-AA-stabilized PE. A 50:50 ratio of sodium caseinate and gelatin emerged as the optimal polymer blend ratio for electrospinning. Among the different concentration ratios, 1% gallic acid exhibited the most favorable appearance properties and diameter size in the electrospun nanofibers. Additionally, the antioxidant capacity of the nanofibers was observed to increase proportionally with the concentration of gallic acid. Fourier Transform Infrared (FTIR) spectroscopy revealed predominant hydrophobic interactions between gallic acid and the protein complex in the nanofibers. Notably, the proposed modifications involving various thermal and alkaline treatments, as well as the introduction of ascorbic acid or gallic acid, exhibited the capacity to form a hydrogel structure when dissolved in sodium caseinate. Conclusion: The findings of the current study offer a promising approach for the utilization of ascorbic acid as a food-grade additive for protein modification, particularly in the development of emulsions predicated on solid particles. These results present an opportunity for the formulation of low-fat food products with natural constituents. Additionally, the utilization of gallic acid as a food additive is suggested for the formulation of products with enhanced appearance and heightened antioxidant attributes Key words: Ascorbic Acid, Phenolic Acid, Maillard Reaction, Reducing Agents, Cross-linkers