Spectrometric study of the interaction of phycocyanins with egg proteins

. Phycocyanins isolated from the biomass of Spirulina platensis are biologically active substances with antioxidant, anti-inflammatory and immunomodulatory properties. At the Federal State Budgetary Institution "Federal Research Center for Nutrition and Biotechnology" research is being conducted to create a food module - a carrier of these biologically active substances. In this work, the possible formation of contacts of phycocyanins with egg proteins was studied using molecular fluorescence spectroscopy. As a result of the studies, it was shown that a mixture of egg white and phycocyanin has a fluorescence maximum in the region characteristic of egg white (361 nm; 125 a.u.) and the fluorescence of phycocyanin has not been established. The results of the study indicate the formation of a bond between phycocyanin and the amino acids that make up the egg white, followed by shielding of phycocyanin molecules from the action of exciting radiation.


Introduction
The pandemic caused by the coronavirus infection has prompted specialists in various fields to search for effective means to increase the body's protective functions.It is known that nutritional factors can make a significant contribution to the formation of the body's resistance to various infections [1].For example, the antioxidant, anti-inflammatory and immunomodulatory properties of phycocyanins are described in the literature [2][3].On this basis, phycocyanins are introduced into specialized food products.The adequate daily intake of phycocyanins is 50 mg, and the upper tolerable daily intake should not exceed 150 mg [4].
A traditional source of phycocyanins is the blue-green prokaryotic microalgae Arthrospira platensis.Spirulina has a rich set of metabolites, including a large number of natural pigments [5][6].
However, phycocyanins are photo-and heat-sensitive compounds [7], which can lead to their degradation during the technological process or during storage.Phycocyanins can be protected from such external influences by forming protective complexes with various proteins.
Undoubtedly, egg whites are complete and even ideal in terms of amino acid composition.An egg intended for the development of a new organism contains all the components necessary for this.In Russia, industrial production and processing of chicken eggs is most common.This raw material has a relatively low cost and is available on the food market.Undoubtedly, egg whites are complete and even ideal in terms of amino acid composition.An egg intended for the development of a new organism contains all the components necessary for this.In Russia, industrial production and processing of chicken eggs is most common.This raw material has a relatively low cost and is available on the food market.
The amino acid composition of egg proteins does not depend on the level of protein in the chickens' diet, but is determined genetically.In terms of the amount of essential amino acids in 1 g of protein, a chicken egg has the most balanced ratio compared to most other food proteins (Table 1).*Amino acid composition of the reference protein according to the FAO/WHO scale (2007) Egg whites are heterogeneous in composition.The bulk -up to 75% of the total egg white -is ovalbumin.By chemical nature, it is a glycoprotein; its carbohydrate component is represented by mannose and glucose.Ovalbumin is divided into three fractions, differing in phosphorus content.It is known that of all egg protein fractions, ovalbumin is the most resistant to thermal denaturation.
Up to 13% of the total egg white is ovomucoid.It has a glycoprotein type and remains resistant to heat in an acidic environment [8].
About 7% of the total egg white is ovomucin.Like the previous two fractions, ovomucin is a glycoprotein, and in an aqueous solution it is very resistant to heat.
Approximately 3% of the total egg white is ovoconalbumin.It is also a glycoprotein, but is more sensitive to heat than ovalbumin.
Ovoglobulin has the smallest amount; its share in the total mass of egg white is only 2%.Ovoglobulin includes three fractions, one of which is considered lysozyme.
To identify potential contacts of phycocyanins with egg proteins, the instrumental method of molecular fluorescence spectroscopy (MFS) was used.This method allows you to measure the intensity of radiation resulting from the absorption of electromagnetic radiation of a different optical range by a molecular medium.The MFS method relies on the ability of substances exposed to UV radiation to fluoresce.Measuring the intensity of emitted radiation in the visible range allows us to obtain fluorescence spectra -optically excited electronic emission spectra of molecules.The advantages of fluorescence spectroscopy are high sensitivity, the ability to study biological objects in natural conditions without damaging them, high measurement speed and good spatial resolution [9] E3S Web of Conferences 463, 01021 (2023) EESTE2023 https://doi.org/10.1051/e3sconf/202346301021

Materials and methods
We used dry egg protein isolate with a mass fraction of egg white of 90% (manufacturer Ovopol Ltd., Poland).
To study complexation with egg protein, we used a phycocyanin concentrate from dry biomass of A. platensis, provided by the scientific and production association "Biosolar MSU", with a mass fraction of C-phycocyanin of 42.0% and allophycocyanin of 7.0%.
Fluorescence spectra were obtained using a Cary Eclipse fluorescence spectrometer (Agilent).The measurements were performed at an excitation wavelength of 200 nm and a monochromator slit width of 5 nm.
The objects of the study were 0.5% and 1% solutions of phycocyanin concentrate, egg white solutions and their mixture.
At the first stage, 0.5% and 1% aqueous solutions of phycocyanin and egg white extract were prepared.Next, to adsorb phycocyanins to the protein matrix, equal concentrations of aqueous solutions of phycocyanin concentrate were combined with aqueous solutions of egg white in a 1:1 ratio and thermostated at a temperature of (24.0 ± 0.5) o C for 60 minutes.

Results
Fluorescence spectroscopy, based on the measurement of emission intensity after excitation of molecules by higher energy photons, is one of the main physicochemical methods for characterizing both individual compounds and their complexes.The advantages of fluorescence spectroscopy include high sensitivity and speed of measurements, and the ability to study objects without damaging them.

Discussion
Egg white is a complex of compounds that have the ability to fluoresce.When using an excitation wavelength close to 200 nm, characteristic of egg white is the detection of emission peaks in the 360 nm region associated with the fluorescence of amino acids and nucleic acids [10], which was confirmed in our study.
Phycocyanins have 2 subunits: α and β, which are linked into an (αβ) protomer, which can exist as a trimer (αβ)3 or a hexamer (αβ)6.In this case, the maximum absorption of phycocyanins is observed at a wavelength of 620 nm [11].
As a result of the analysis of a mixture of egg white and phycocyanin, the maximum fluorescence was found in the region characteristic of egg white (361 nm; 125 a.u.) and the fluorescence of phycocyanin was not established.The mechanism of the noted effect of quenching the fluorescent signal of phycocyanin by egg white molecules may be the ability of phycocyanins to form bonds with proteins and other macromolecules through the hydroxyl groups of tyrosine and tryptophan, as well as the amino groups of lysine [12].

Conclusion
The interaction of phycocyanins with egg white was studied using fluorescence spectroscopy.The results of the study indicate the formation of a bond between phycocyanin and the amino acids that make up the egg white, followed by shielding of phycocyanin molecules from the action of exciting radiation.Based on the data obtained, it can be assumed that egg white molecules, isolating phycocyanin from the action of external factors, can increase its stability during storage and processing.

Table 1 .
Content of essential amino acids in chicken egg white, g per 100 g of protein.