Effect of Freezing on Soybean Protein Solution

Abstract

To investigate the impact of frozen storage conditions on the physicochemical properties of soybean protein and explore the underlying mechanisms, this study focused on soybean isolate (SPI), ß-soybean companion globulin (7S), and soybean globulin (11S). The protein solutions were prepared at a concentration of 2% and subjected to freezing for 1 and 5 days. Subsequently, the protein content, physicochemical properties, secondary structure, sulfhydryl content, and chemical interaction forces were assessed and analyzed using UV spectrophotometry, Zeta potential measurements, SDS-PAGE, Fourier infrared spectroscopy, and endogenous fluorescence photoemission spectroscopy. The obtained results revealed that the solubility and total sulfhydryl content of SPI, 7S, and 11S exhibited a decreasing trend with prolonged freezing time. Among them, 11S demonstrated the largest decrease in solubility and total sulfhydryl content, followed by SPI, and 7S the least. During freezing, the aromatic amino acids of SPI, 7S, and 11S molecules were exposed, leading to increased hydrophobicity, protein aggregation, and particle size enlargement, and the structure of the protein changed from disordered structure to ordered structure. After freezing, the polarity of the microenvironment of SPI, 7S, and 11S increased, and their maximum fluorescence emission wavelengths were red-shifted. Notably, the largest red shift of SPI was from 332 nm to 335 nm. As freezing time increased, the contribution of hydrogen bonding increased, while the contribution of hydrophobic interactions decreased. This indicates that freezing affects the hydrophobic interactions, hydrogen bonding, and other chemical forces of the protein. The growth of ice crystals leads to the unfolding of protein molecular chains, exposure of internal hydrophobic groups, enhancement of hydrophobicity, and alters the secondary structure of the protein.

Keywords: 11S; 7S; different freezing time; soybean isolate protein.

Figure 1

Molecular structure of 7S and 11S [8]. Note: (a) molecular structure characteristics of β-glycine; (b) molecular structure characteristics of glycine; (c) schematic diagram of 7S; (d) schematic diagram of 11S.

Figure 2

Distribution of soluble protein content in different protein frozen storage days. Note: F0 represents 0 days of freezing, F1 represents 1 day of freezing, and F5 represents 5 days of freezing. Different lowercase letters on the shoulders of the same indicator indicate significant differences (p < 0.05).

Figure 3

Relationship between viscosity and shear rate of different proteins at different tempera-tures. Note: (a) the relationship between viscosity and shear rate of SPI at different temperatures; (b) the relationship between viscosity and shear rate of 7S at different temperatures; (c) the relationship between viscosity and shear rate of 11S at different temperatures.

Figure 3

Relationship between viscosity and shear rate of different proteins at different tempera-tures. Note: (a) the relationship between viscosity and shear rate of SPI at different temperatures; (b) the relationship between viscosity and shear rate of 7S at different temperatures; (c) the relationship between viscosity and shear rate of 11S at different temperatures.

Figure 4

Particle size distribution and average particle size distribution of different protein solutions frozen for different days. Note: different lowercase letters on the shoulders of the same indicator indicate significant differences (p < 0.05).

Figure 5

Potential distribution of different protein solutions frozen for different days. Note: different lowercase letters on the shoulders of the same indicator indicate significant differences (p < 0.05).

Figure 6

UV absorption spectra and second-derivative spectra of different protein solutions frozen for different days. Note: a and b represent the difference between the two wave peaks and troughs.

Figure 7

Fluorescence emission spectra of protein solution frozen for different days.

Figure 8

Electrophoretic profiles of non-reduced (N) and reduced (R) protein solutions at different days of freezing. Note: (a) SPI non-reduction profile; (b) SPI reduction profile; (c) 7S non-reduction profile; (d) 7S reduction profile; (e) 11S non-reduction profile; (f) 11S reduction profile. Lane M: protein specimen. Lanes 1–8: protein powder, supernatant F0, supernatant F1, supernatant F5, precipitate F0, precipitate F1, precipitate F5, protein powder.

Figure 9

Fourier infrared spectra of protein solution frozen for different days.

Figure 10

Changes in total sulfhydryl content of proteins under different days of freezing. Different lowercase letters on the shoulders of the same indicator indicate significant differences (p < 0.05).

Figure 11

Effect of different days of freezing on the intermolecular forces of proteins. Different lowercase letters on the shoulders of the same indicator indicate significant differences (p < 0.05).