Introduction Investigating anthocyanin-protein complexes is important for the food industry from several perspectives. 1) Utilizing the anthocyanin-protein complex formation method to stabilize anthocyanin pigments in food products against external factors. This is achieved through various physical interactions with different bond strengths established between their binding sites. Studies have shown that the higher the binding constant of this interaction and the stoichiometric ratio between them, the lower the concentration of free anthocyanin remains in the environment, and most anthocyanins are found bound to the protein. In this state, the stability of anthocyanin against external factors increases due to the physical bonds formed with the protein, and its structure is not degraded. 2) Changes in the physicochemical and physiological properties of anthocyanin. For example, it has been proven that the antioxidant properties of anthocyanins decrease in the presence of proteins. 3) The effect of this complex on protein digestibility, which plays a significant role in the nutritional value of the system under study. 4) Production of modified biopolymer for use in food formulation. As a results of these interactions, which are as covalent or non-covalent, the functional properties of proteins are altered. Consequently, suitable raw materials can be designed for various food products. Materials and Methods Fluorescence emission spectra were measured using a fluorimeter (Varian Cary Eclipse, Agilent, USA) equipped with a 10 mm cell and a temperature controller at temperatures of 298, 308, and 318 K. All samples were excited at a wavelength of 280 nm, and their emission spectra were recorded in the wavelength range of 280 to 500 nm. The slit width for both excitation and emission was set at 5 nm. To record the protein fluorescence quenching spectra, a 1 mg/mL solution of grass pea protein was first prepared and titrated against different concentrations of CYG pigment (0 to 4.5 × 10⁻ ⁶ M). The corresponding fluorescence emission spectrum for quenching was recorded at each step. For synchronous fluorescence spectra, simultaneous scanning was performed at the absorption and emission wavelengths of the tyrosine and tryptophan amino acid chromophores of grass pea protein, where their wavelength differences (Δλ) were set at 15 nm and 60 nm, respectively. Three-dimensional fluorescence spectra were recorded sequentially within the excitation wavelength range of 220 to 540 nm and the emission wavelength range of 220 to 600 nm, with a consecutive 10 nm increment in the excitation wavelength. To collect Resonance Light Scattering (RLS) data, the emission intensity of the protein monochromators was recorded simultaneously in the wavelength range of 220 to 700 nm at a zero wavelength difference (Δλ=0) between excitation and emission. In all experiments, the concentration of CYG pigment used was in the range of 0 to 4.5 × 10⁻ ⁶ M. Results and Discussion The results indicated a key role of hydrogen bonds in the formation of the grass pea protein-CYG complex through a combined static and dynamic quenching mechanism, with a binding constant of 1.73×103M⁻ ¹ and a binding site number of 0.78 at ambient temperature.Furthermore, the synchronous and three-dimensional fluorescence spectra of the protein revealed that the CYG pigment bound near tyrosine amino acid residues in the protein structure. This binding induced a local folding change in the protein\\\'s conformation. Changes in the RLS spectra of the protein indicated that the particle size of the complex decreased at low CYG concentrations. However, when the molar ratio of CYG to protein approached 2:1, the particle size of the complex increased. The results of the protein binding site saturation also demonstrated that grass pea protein is capable of binding CYG pigment at concentrations more than two times. Therefore, the use of this complex is recommended in systems that require natural and small quantities of protein to effectively trap the CYG pigment.

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