1 INTRODUCTION
Soybean protein isolate (SPI) has been widely used due to its low cost and easy availability of materials. SPI is a complete protein of plant origin with the characteristics of high protein, low fat, calcium, and cholesterol (Liu etal., 2022). SPI can be equivalent to animal protein. It is one of the few plant proteins that can replace animal protein. In addition, edible quality and nutritional value are closely related to functional properties, and the structure of SPI determines its unique functional properties (Zhang, Du etal., 2023). The side chains of peptide bonds or amino acids in SPI interacted with water molecules, showing solubility. However, it contains hydrophilic groups and lipophilic groups. In the mixture of oil and water, the dispersed proteins tend to diffuse to the oil–water interface, thus showing emulsification. The interaction of the disulfide, hydrogen, and hydrophobic bonds in the SPl molecule makes the broken peptides cross-linked and aggregated into a network again, showing gel properties. Part of the SPI peptide chain is unfolded at the interface. Through the interaction between the peptide chains, a two-dimensional protection network is formed to strengthen the interface film to behave foaming. Because SPI has a variety of properties, such as solubility, emulsification, gelation, and foaming, it is widely used in the food industry. For example, SPI can be used as an emulsifier to make baked, frozen, and soup food so that the product state is stable, SPI can be used in flour foods to increase the strength of gluten. SPI can also be used as an antibacterial and fresh-preserving packaging material for food. These functional features significantly increase SPI in the market share trend. This trend will continue in the coming years (Zhang etal., 2024).
However, natural SPI has a compact spherical structure, the structure and functional properties during processing are easily affected by many factors such as pH and high temperature. For example, the pH near its isoelectric point, low heat treatment, and salt ions will induce protein denaturation and aggregation, leading to precipitation and limiting its industrial application. Therefore, functional modification is usually used to improve the structure and performance of plant proteins such as SPI in the food industry (Zhang, Li etal., 2023).
There are many strategies for protein modification to improve its performance, such as the enzyme, physical, and genetic engineering methods, a highly preferred method is chemically modifying, which has the advantages of short reaction time, low cost, no requirement for specialized equipment, and particular modification effects. Chemical modification methods include glycosylation, deacetylation, phosphorylation, covalent cross-linking, and so on. These methods target the functional groups of proteins, altering the spatial structure, the number of electric charges on the protein surface, and the hydrophilicity or hydrophobicity of the protein (Abedi & Pourmohammadi, 2021). Glycosylation has the advantages of environmental friendliness, efficient, and gentle, making it one of the most promising methods for altering the structure and function of natural proteins. More importantly, many toxicological and nutritional experiments showed glycosylated proteins were nontoxic; therefore, glycosylation is a reliable method for protein modification (Yu etal., 2020).
Since the 1990s, the study of protein glycosylation has been widely carried out, and it is still a research hotspot in protein modification. After glycosylation, SPI (Feng, Wu etal., 2024), whey protein isolate (Li etal., 2024), ovalbumin (Yang etal., 2022), and casein (Yang etal., 2023) (solubility, emulsification, foaming, thermal stability, gel properties, antioxidants, and antibacterial agents) were all improved. Notably, SPI, as one of the most common and promising proteins, has attracted much attention from glycosylation researchers; however, there is no new and comprehensive review on the effect of saccharides on the functional properties of SPI. Therefore, this review summarizes the impact of glycosylation on the structure, analytical methods, functional properties, and biological activities of SPI. The purpose is to provide a theoretical guidance and research ideas for applying SPI in the food industry.
2 MECHANISM AND CHARACTERISTICS OF FOOD PROTEIN GLYCOSYLATION
Protein glycosylation modification is a Maillard reaction mechanism in which the ε-amino group of a protein molecule is covalently bound to the carbon group of the reducing end of a sugar molecule, as shown in Figure1. It is usually considered the initial stage of the Maillard reaction. SPI does not change its molecular structure during the initial reaction and does not lose its original functional properties (Wu etal., 2022). The resulting protein-sugar covalent complex can generally be used as a high-quality multifunctional additive and is highly valued for its mild reaction conditions and high safety. Glycosylation involves many reactions such as condensation between carbon-based compounds and amino compounds, molecular rearrangement, degradation, aldol condensation, and polymerization, thereby affecting the functional properties, flavor, color, and nutrition of processed foods (Zhang, Li etal., 2019).
However, in the actual research process, the intermediate and end products of the Maillard reaction will also be produced. Such as black essence, which causes the reddish-brown color change in food during processing and can give attractive colors to foods such as coffee, bread, and cocoa (Starowicz & Zieliński, 2019). Moreover, Maillard reaction products (MRPs), such as reduced ketones, aldehydes, and heterocyclic compounds, have distinctive flavor profiles that confer aroma and flavor to baked goods, dairy products, and meat products (Fu etal., 2023). However, too much intake of MRPs may increase the risk of chronic diseases and even cancer, thus threatening human health (Peng etal., 2024). Appropriate reaction conditions can reduce the formation of harmful Maillard intermediate and end products (Liu etal., 2024). Alternatively, polysaccharides, which provide steric effect and reducibility, are used to modify the protein structure to avoid Maillard reaction entering an advanced stage, thereby reducing the harm to the human body (Shakoor etal., 2022).
3 PREPARATION AND QUANTIFICATION OF GLYCOSYLATION PRODUCTS
3.1 Methods for preparing the glycosylation products
As mentioned above, choosing the appropriate glycosylation method and reaction conditions is crucial. Glycosylation methods can be divided into dry-heating glycosylation, wet heating glycosylation, and composite modification.
Dry-heating glycosylation is the earliest method used for glycosylation reactions, and it is also the most common reaction happened in the spray drying of protein. The dry-heating method is to prepare a certain proportion of proteoglycan solution, that is freeze-dried and heated at a certain temperature (generally 60°C) and relative humidity (65% or 79%). The heating time depends on the nature of the protein and polysaccharide, ranging from a few hours to a few weeks. The reaction was terminated by rapidly cooling the cross-linked product. This method provided easily controlled reaction conditions and efficient grafting. However, the dry-heating method requires the raw material to be dry before the reaction, and the temperature and relative humidity need to be controlled during the reaction, and the reaction time is also long, which is not suitable for mass production (Ma etal., 2023).
Wet-heating glycosylation involves directly conducting glycosylation reactions in a liquid medium. It is suitable for the reaction between small sugar molecules and proteins. In the wet-heating glycosylation, proteins and sugar molecules come into more excellent contact, leading to a quicker reaction rate and shorter reaction time. Although wet-heating glycosylation significantly alters the functional properties of proteins, it also has some drawbacks. As a solvent of wet- heating method, water inhibits the glycosylation reaction from going forward in some extent because it is also the product of Maillard primary stage. Additionally, high temperatures under wet-heating condition and electrostatic charge interaction on molecular surface lead to aggregation and generation of complex Maillard end products (Lu etal., 2024). Therefore, the control of wet heating glycosylation reactions and the complexity of the products are issues that still need to be addressed.
Recent years, many scholars have incorporated other methods such as physical, chemical methods, and enzymatic methods on the basis of tradition or explored some new techniques to improve the efficiency of glycosylation or make proteins show better functional characteristics. For example, Chen etal. (2022) used ultrasonic treatment to make synergistic glycosylation modification of rice protein. The results showed that ultrasonic treatment significantly (p<0.05) accelerated the glycosylation reaction between rice protein and glucan and improved solubility, foaming, and emulsification. Wang etal. (2024) used double modification by succinylation and glycosylation improving the functional properties of walnut protein isolate, especially its potential for gelation and emulsification. Compound modification may provide a new approach to glycosylation modification in the future. Enzymes also used to perform glycosylation reactions between proteins and sugars. Transglutaminase is a commonly used enzyme that can promote and catalyze acyl transfer reactions between gamma-carboxyamide groups of protein/peptide glutamine residues (acyl donors) and various primary amino groups of sugars (acyl receptors) (Guo etal., 2023).
3.2 Methods for characterizing the glycosylation products
The main purpose of protein glycosylation is to obtain functionally enhanced glycosylation products. Therefore, whether protein glycosylation occurs or the depth to which the reaction occurs requires appropriate analytical methods to characterize the glycosylation products. Various methods have been used to characterize glycosylation products in recent years, such as spectrophotometry, gel electrophoresis analysis, chromatography, and microstructure analysis (Zhang, Li etal., 2019).
3.2.1 Microstructure analysis
Surface morphology can be recorded at the micrometer scale when the test sample is treated with a very finely focused high-power electron beam. Microscopic differences in surface morphology are useful information for identifying covalent cross-links between protein and polysaccharide molecules.
3.2.2 Spectrophotometry
Characteristic absorption peaks of the glycosylation reaction products in a specific absorption wavelength range. The degree of grafting was calculated by testing the content of free amino groups at 340nm using a modified O-phthalaldehyde method. Fluorescence spectrophotometry can be used to determine the surface hydrophobicity (H0) of glycosylated proteins. Infrared and Raman spectroscopy were used to determine the structure of glycosylated proteins.
3.2.3 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)
The protein standard was used as a marker to obtain the distribution of protein bands in the separation gel by producing different moving distances due to different molecular weights of proteins under a certain amount of electricity. The high sensitivity and resolution and the absence of absorption and electroosmotic effects are used to characterize protein glycosylation products based on the appearance or disappearance of protein bands and their color.
3.2.4 Chromatographic method
The difference in molecular weight of the macromolecules can be reflected by the size exclusion column in the liquid chromatography system. The size of the glycosylation product was significantly higher than that of the native protein or polysaccharide, and the amount of the glycosylation product was determined by high-performance size exclusion chromatography.
4 EFFECTS OF GLYCOSYLATION REACTIONS ON SPI STRUCTURE AND ANALYTICAL METHODS
4.1 Effect of the glycosylation reaction on SPI structure
4.1.1 Secondary structure of SPI
Secondary structure refers to a local protein structure consisting of α-helices, β-sheets, and random coils. These structures play a crucial role in the stability, function, and interactions of proteins (Chen etal., 2019). Studies have shown that in glycation reactions, various sugars can induce alterations in the secondary structure of SPI (Table1). In these secondary structures, α-helix is a compact helical structure in the protein's secondary structure, where the peptide chain is stabilized by hydrogen bonding. Hence, the α-helix structure is highly stable (Zhang, Zheng etal., 2019). An increase in α-helix content indicates a more ordered structure, whereas an increase in random coil content indicates that the protein tends to be loose and disordered. High addition of polysaccharides could make the secondary structure of the protein change from ordered α-helix to disordered β-sheet, β-turn, and random coil structure, and the complex conformation becomes more flexible and loose (Han, Zhao etal., 2022). This helps to improve the functional properties of proteins and the flexibility of protein molecules (Mi etal., 2021).
| Secondary structure | |||||||
|---|---|---|---|---|---|---|---|
| Protein | Polysaccharide | Method | α-Helix | β-Sheet | β-Turn | Random coils | References |
| Soybean protein isolate | Lentinan | Silt divergent ultrasonic (SDC) assisted Maillard reaction (wet-heating) | ↓ | ↑ | ↑ | ↑ | Wen etal. (2020) |
| Soybean protein isolate | Maltose | Radiation assisted Maillard reaction | ↓ | ↑ | ↑ | ↑ | Wang etal. (2020) |
| Soybean protein isolate | Flaxseed gum | High hydrostatic pressure (200MPa) assisted Maillard reaction | ↑ | ↑ | ↓ | ↓ | Liu etal. (2020) |
| Soybean protein isolate | Dextran | Maillard reaction (wet-heating) under molecular crowding (the concentrations of PEG were 0, 2wt%, respectively, the concentrations of PEG were 4, 6, 8, and 10wt%, respectively) SPI and dextran were dissolved separately in distilled water (2%, w/v) and stirred at 25°C for 3h to ensure complete dissolution (deacetylation, phosphorylation, and glycosylation) | ↓ | ↓ | ↓ | ↑ | Hu etal. (2020) |
| ↓ | ↓ | ↑ | ↑ | ||||
| ↑ | ↓ | ↑ | ↑ | Han etal. (2024) | |||
| ↓ | ↓ | ↑ | ↑ | ||||
| ↓ | ↑ | ↓ | ↓ | ||||
| Soybean protein isolate | Maltodextrin | Glycation conjugates with ultrasonic pretreatment | ↓ | ↑ | ↑ | ↓ | Zhao etal. (2021) |
| Soybean protein isolate | Chitosan | The polysaccharide stock solutions (0.2%, 0.4%, and 0.6%, w/v) were prepared by dispersing the CS, GUG, and GEG powder in deionized water, respectively, with continued high-speed shearing for at least 2h | ↑ | ↑ | ↑ | ↓ | Han etal. (2024) |
| Guar gum (GUG, a neutral polysaccharide) | Remained Almost Unchanged | ↑ | ↑ | ↓ | |||
| Gellan gum (GEG, an anionic polysaccharide) | ↑ | ↑ | ↑ | ↓ | |||
Xu and Zhao (2019) revealed the secondary structure characteristics of sugar-modified SPI through CD spectroscopy. The results showed that the sugar-modified SPI products had fewer β-sheets and β-turn compared to SPI. The secondary structure of sugar-modified SPI was more disordered than that of SPI, and maltose could interfere with the normal formation of β-sheets and β-turn. Zhao etal. (2021) showed ultrasonic treatment promoted glycosylation reaction between SPI and maltodextrin (MD) and proned to form β-type secondary structure and compact coupling compound, which controlled SPI–MD gelation properties.
4.1.2 Tertiary structure of SPI
The tertiary structure of SPI, also known as the three-dimensional conformation of the protein, is formed by the further coiling or folding of the polypeptide chain based on various secondary structures, the stabilization of the tertiary structure of SPI mainly depends on the secondary bonds, including hydrogen bonds, hydrophobic bonds, van der Waals forces, and so on (Wu etal., 2022).
Due to the spherical structure of SPI, the outer of sphere is composed of hydrophilic side chains, which appear hydrophilic, whereas the inner sphere is composed of hydrophobic groups, which appear hydrophobic (Liu etal., 2021). When SPI undergoes glycosylation, secondary bonds are broken, tertiary structure is unfolded, hydrophobic groups are exposed, and hydrophobicity is increased. However, as the protein structure unfolds, the surface area of the protein increases, making it easier for SPI to interact with sugars, thereby affecting the hydrophobicity of SPI. Therefore, the surface hydrophobicity (H0) of SPI can be used to determine the changes in the tertiary structure (Table2) (Ai etal., 2021).
| Protein | Polysaccharide | Method | Tertiary structure | Discussion and analysis | References |
|---|---|---|---|---|---|
| Soy protein isolate | Dextran | Maillard reaction (wet-heating) | The fluorescence intensity decreased | The strong steric hindrance effect of dextran shielded the fluorophore, reducing its exposure. This significantly quenched the intrinsic fluorescence of SPI and exposed the Trp residues to a more hydrophilic microenvironment | Han etal. (2024) |
| H0 decreased | The additional dextran can envelop SPI, shielding the hydrophobic groups of SPI and leading to an overall decrease in the H0 | ||||
| Soy protein isolate | Gum Arabic (GA) | Maillard reaction (wet-heating) | The fluorescence intensity decreased | After Maillard reaction, the chromophore groups (mainly tryptophan residues) were exposed to a More hydrophilic microenvironment | Feng etal. (2023) |
| H0 decreased | The binding of SPI with GA reduced the exposure of hydrophobic groups buried in protein | ||||
| Soy protein isolate | Maltodextrin (MD) | Maillard reaction (wet-heating) | The fluorescence intensity decreased | The shielding effect of the polysaccharide chain on the region around the Trp residues | Zhao etal. (2021) |
| H0 increased | Maillard reaction between SPI and MD inhibited thermal denaturation and unfolding of proteins, reducing exposure of hydrophobic residues | ||||
| Soy protein isolate | Lentinan | Maillard reaction (wet-heating) | The fluorescence intensity increased | The interaction between amino groups and carboxyl groups changed the protein structure | Wen etal. (2020) |
| H0 increased | Changes in protein structure exposed more hydrophobic residues from inside the molecule to the surface | ||||
| Soy protein isolate | Pectin | Maillard reaction (wet-heating) | The fluorescence intensity decreased | Trp residues reached a more exposed and polar microenvironment | Ma etal. (2020) |
| H0 decreased | Maillard reaction changed the SPI conformation and restrained the exposure of hydrophobic groups | ||||
| Soy protein isolate | l-arabinose | Maillard reaction (wet-heating) | The fluorescence intensity decreased | The fact that the shielding effect of polysaccharide chain bound to protein leads to the Trp residues in the conjugate being more easily surrounded by hydrophobic environment, and proteins in the conjugate may have a closer tertiary conformation | Zhang, Dou etal. (2023) |
| d-galactose | H0 increased | Maillard reaction enhanced the H0 of SPI, which may be because SPI bonded with hydrophilic saccharides, and its solubility increased, thereby exposing more hydrophobic groups |
- Abbreviation: Trp, tryptophan.
The type of sugar in the glycosylation reaction can affect the H0 of SPI, and usually, the introduction of sugars will reduce the surface hydrophobicity of SPI. The introduction of neutral polysaccharide and anionic polysaccharide can reduce H0; however, introduction of the cationic polysaccharide can increase the H0, this could be due to the structure of polysaccharide, the effect of surface charge, and interaction (Han, Li etal., 2024). Du etal. (2018) observed that covalent cross-linking occurred between SPI and polysaccharide. This cross-linking increases the number of hydroxyl groups on the complex's surface, decreasing the surface hydrophobicity. Han, Li etal. (2024) found that the addition of highly hydrophilic glucan molecules increased the number of hydrophilic groups in the SPI peptide chain, which greatly enhanced the hydrophile surface of the molecule compared with other kinds of sugars.
The amount of sugar used in the glycosylation reaction can affect the surface hydrophobicity of SPI. Adding more polysaccharides leads to an increase in the number of hydroxyl groups on the surface of the complex, which decreases the surface hydrophobicity and showed some hydrophilicity. However, with a small amount of sugar, the hydrophilic group is insufficient to counteract the effect of the exposed hydrophobic group in SPI, thus showing hydrophobicity. Chang etal. (2021) found that the added excess polysaccharide molecules can encapsulate SPI, which has a specific shielding effect on the hydrophobic group of SPI and prevents the interaction between proteins, thereby inhibiting the aggregation of proteins and reducing the hydrophobicity of the surface, leading to an overall decrease of H0. Mu etal. (2010) found that the protein structure unfolded, exposing many hydrophobic groups. Although the polysaccharide contains a large number of hydrophilic hydroxyl groups, due to the small amount of polysaccharide added, there are not enough hydrophilic hydroxyl group in the system, which improves the surface hydrophobicity of the complex.
4.2 Analysis method of SPI structure in glycosylation modification
The investigation of SPI structure in glycosylation modification commonly uses techniques that include Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, circular dichroism spectroscopy, and fluorescence spectroscopy, and so on. Figure2 shows these instruments and the main functions applied in protein structure characterization.
4.2.1 Fourier transform infrared spectroscopy (FTIR)
Protein structure and function are frequently studied by using infrared spectroscopy. Glycosylation mainly alters the structure of the C–N bond and N–H bond in the primary protein reaction. These groups exhibit characteristic absorption peaks in the infrared spectrum (Zhan etal., 2021). By measuring the absorption peaks of proteins in the infrared spectrum range, information about protein secondary structure (such as α-helix and β-folding) can be obtained (Yang etal., 2020). Two essential sections utilized to describe the structure of the protein backbone are the amide I band (1600–1700cm−1, C–O stretch) and the amide III band (1220–130cm−1, N–H bond, and C–N stretch) (Sun etal., 2022). In the glycosylation reaction, the covalent interaction between the free amino group and the carbonyl group reduces the amino group content, affecting the intensity of the absorption peaks from 1030–1150 to 1600cm−1. Specifically, the peak at about 1600cm−1 is the bending vibration peak of N–H, which is the typical characteristic peak of glycosylation products (Musalli etal., 2020). Thus, the enhanced absorption peak in this region represents covalent cross-linking, increasing N–H vibrations. In addition, characteristic peaks generated by C–O and C–C stretching vibrations, representing the main typical absorption of saccharide, were demonstrated at 1030–1150cm−1. The absorption peak in this region tends to be enhanced, which may be due to the formation of new chemical bonds between carbon atoms and hydroxyl groups after glycosylation and cross-linking with hydroxyl groups (Zhang etal., 2023).
FTIR spectroscopy can be used to analyze SPI glycosylation products qualitatively. Namli etal. (2021) glycosylated SPI using a water bath method under pH 7 and pH 10 and compared with the traditional “water bath glycosylation” method. The structural changes of glycosylated SPI were studied using FTIR, which revealed C–O stretching vibrations as well as C–H and N–H bending vibrations and indicated that SPI had changed its structure. Han, Zhou etal. (2022) used FTIR spectroscopy to discover that the absorption peaks corresponding to the covalent complexes near the amide I band shifted relative to the corresponding peaks of natural SPI, indicating the involvement of protein amino groups in the reaction. In addition, the absorption intensity of glycosylation products at 1545cm−1 significantly decreases (p<0.05) or completely disappears. This indicates that the content of amino groups decreases due to the covalent binding of reducing sugars to SPI during the Maillard reaction. FTIR spectroscopy can be used for detecting the degree of glycosylation of SPI glycosylation products. In SPI-shiitake polysaccharide covalent complexes, the intensities of amide I (3200–3400cm−1) and amide II bands (1600–1700cm−1) show a decreasing trend, indicating a decrease in free amino groups and carbonyl groups, which can reflect an increase in the degree of grafting and, consequently, an increase in the number of glycosylation products (Ghaedi & Hosseini, 2021).
4.2.2 Raman spectra
Raman spectroscopy is a highly valuable spectroscopic technique for analyzing the structure and properties of SPI. Information about the molecular vibrations and chemical composition of the sample can be obtained by measuring the scattered light of the sample under laser irradiation. In the study of SPI, Raman spectroscopy can provide insights into protein secondary structure, such as α-helix, β-folding, and random crimp structure (Gómez-Mascaraque & Pinho, 2021). Different secondary structures correspond to distinct Raman spectral peaks and characteristic vibrations. Through the peak position and intensity of the Raman spectrum, the content and changes of various secondary structures in SPI can be quantitatively evaluated (Zhu etal., 2020).
Raman spectroscopy can also offer information about the vibration of amino acid residues and side chains of proteins. Raman spectra of different amino acids exhibit specific vibrations. They can be utilized to determine the presence and relative content of amino acids and the conformation and interaction of side chains in SPI (Wang, Yang etal., 2020). Raman spectroscopy analysis can be used to provide information about glycosylation modifications. By observing specific glycosyl vibrations and peak shifts, the presence and extent of glycosylation modifications in proteins can be detected and quantitatively evaluated (Gibbons etal., 2022). Raman spectra can be used to identify protein–polysaccharide complexes by comparing and analyzing the position and strength of absorption peaks. Ni etal. (2023) used Raman shift information of lactose as the extraction index for processing spectral data. The Maillard reaction between lactose and protein was confirmed by high-resolution mass spectrometry.
4.2.3 Circular dichroism spectrum
Protein structure can be examined by using a scientific technique called circular dichroism spectroscopy. It measures the difference in the absorption of left-handed and right-handed circularly polarized light to provide information about the secondary structure of the protein. By comparing the circular dichroism spectra before and after glycosylation modification, the influence of glycosylation modification on the structure of SPI can be evaluated (Meng etal., 2019). The amino acid residues and disulfide bonds of proteins absorb far-ultraviolet light with a wavelength range of 190–250nm. The conformation of the main chain can be described using this method, especially in protein solutions that are soluble in water. An α-helix often displays a negative peak at 208–222nm and a noticeable positive band at 191–193nm (Liu etal., 2020). The existence of β-folding is shown by the broad negative band at 216–218nm and the strong positive band at 195–200nm. At 195–200nm, there is a noticeable negative band that shows irregular curling (Wang etal., 2021).
Structurally, Tirgarian etal. (2022) used circular dichroism to monitor changes in protein secondary structure due to the Maillard reaction, the results showed that the binding of SPI to anionic polysaccharide increased the content of α-helix and β-sheet but decreased the content of β-turn and random coil. Fu etal. (2021) analyzed MRPs of SPI and α-lactose monohydrate by circular dichroism spectrum and scanning electron microscopy and confirmed significant (p<0.05) changes in the protein structure that the α-helix and β-sheet content decreased, whereas the β-turn and random coil content increased, which indicated that after Maillard reaction, the protein structure transitions from ordered to disordered. Boonlao etal. (2023) confirmed that the secondary structure of the SPI-MD conjugate prepared by both dry and wet-thermal methods changed conformationally through circular dichroism spectra.
4.2.4 Fluorescence spectrum
Measurement of emission spectra by exciting fluorophore or tryptophan (Trp) residues in the sample can provide information about the fluorescence characteristics and conformation of the sample. The principle of fluorescence spectroscopy in the study of SPI glycosylation is illustrated in Figure3.
Intrinsic fluorescence spectroscopy is an analytical method that relies on the internal fluorescence emission of molecules. It is utilized to investigate the structural changes and functional characteristics of proteins. In the process of SPI glycosylation, the degree of grafting and browning can be determined through intrinsic spectroscopy (Ai etal., 2021). The Trp fluorescence-emitting group is more exposed to the solvent, as indicated by the redshift of the fluorescence peak. This could be attributed to a number of factors, including increased polarity of the microenvironment surrounding the fluorescence-emitting group, peptide chain elongation, and improved spatial conformation. The blueshift indicates that the emitting group Trp is in a more hydrophobic microenvironment (Jia etal., 2022). Wang etal. (2020) prepared maltose-modified SPI through gamma-ray treatment to enhance functional properties and assess structural changes. Fluorescence spectra showed that a Maillard reaction occurred between SPI and maltose. As the degree of grafting increased, the fluorescence intensity significantly (p<0.05) decreased. This was attributed to the unfolding of protein molecules, which exposed the fluorescent chromophores to the solvent, leading to fluorescence quenching and a subsequent reduction in fluorescence intensity.
5 EFFECT OF GLYCOSYLATION ON FUNCTIONAL PROPERTIES OF SPI
Researchers have found that glycosylation can enhance the functional properties of proteins, thereby expanding the range of applications and commercial potential of proteins in the food industry (Table3). These functions are influenced by the molecular properties (surface charge, surface hydrophobicity, surface topological molecular weight, isoelectric point, structure, etc.) of proteins in their natural, intermediate, or denatured states (Li etal., 2021).
| Protein | Carbohydrate | Method | Functional and bioactive properties | Application or research implications in food processing | Reference | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| S | EP | GP | F | TS | FS | AO | AM | |||||
| Soy protein isolate (SPI) | d-galactose | Wet-heating | ↑ | ↑ | - | ↑ | - | - | ↑ | ↑ | It has better hypoglycemic effect, fat binding ability, and bile acid binding ability. Improve the biological activity of food | Zhang, Dou etal. (2023) |
| Arabinose conjugate | ↑ | ↑ | - | ↑ | - | - | ↑ | ↑ | ||||
| Soy protein isolate (SPI) | β-Glucan | Wet-heating | ↑ | - | - | - | - | - | - | - | The reaction of SPI with β -Glucan glycosylation provided theoretical support for the modification of glycans with SPI | Chu etal. (2023) |
| Soy protein isolate | xylose | Wet-heating | ↑ | ↓ | - | - | ↑ | - | - | - | Further demonstrated that the humid heat method is a promising method for glycosylation of SPI | Wang etal. (2018) |
| xylose | ↑ | ↓ | - | - | ↑ | - | - | - | ||||
| Soy protein isolate | Orange pectin | Dry-heating | ↑ | ↑ | - | - | - | - | - | - | Provide theoretical support for SPI and pectin glycosylation modifications. It is used as a food additive in food | Ma etal. (2020) |
| Orange pectin | ↑ | ↑ | - | - | - | - | - | - | ||||
| Soy protein isolate | Glucose | Wet-heating | - | ↑ | - | - | ↑ | - | - | - | It provides essential guidance on how pH affects the structure and emulsification properties of glycosylation products | Sun etal. (2022) |
| Soy protein isolate | Soy hull hemicelluloses | Dry-heating | - | ↑ | - | - | ↑ | - | - | - | Demonstration the effect of glycosylation modification on the emulsification of SPI. It is used as a food additive in food | Wang etal. (2017) |
| Soy protein isolate | Xanthan gum | Wet-heating | - | ↑ | - | ↑ | ↑ | - | - | - | Glycation products as food additives to improve the functional characteristics of food | Li etal. (2015) |
| d-glucose | - | ↑ | - | ↑ | ↑ | - | - | - | ||||
| Soy protein isolate | Okara dietary fiber | Dry-heating | ↑ | ↑ | - | - | ↑ | - | - | - | The glycosylation-modified SPI was used for the preparation of the Pickering emulsion and investigated for changes in hydrophobicity | Ashaolu and Zhao (2020) |
| Soy protein isolate | Maltodextrin | Ultrasonic treatment+wet heating | - | - | ↑ | - | - | - | - | - | To explore the effect of ultrasound-assisted application on the gel performance of SPI glycosylation products | Zhao etal. (2021) |
| Soy protein isolate | Soy oligosaccharide | Wet-heating | - | - | ↑ | - | ↑ | - | - | - | Altering the structure of SPI by controlling the degree of glycosylation, thereby improving the physicochemical properties and stability of the emulsion gel | Zhao etal. (2023) |
| Soy protein isolate | Konjac starch | Wet-heating | - | - | ↑ | - | ↑ | - | - | - | SPI with glycosylation modification 1 has excellent gel properties and enhanced function in crushed meat products | Hu etal. (2023) |
| Soy protein isolate | Dextran conjugates | Dry-heating | - | - | - | - | ↑ | - | - | - | To provide theoretical support for the SPI glycosylation modifications | Zhuo etal. (2013) |
| Soy protein isolate | Maltose | Irradiation technology | - | ↑ | - | - | - | ↑ | - | - | Enrich and develop the protein modification system to provide a theoretical basis for the application of comprehensive modification technology in food processing | Wang etal. (2020) |
| Soy protein isolate | Maltodextrin | Wet-heating | - | - | - | - | - | - | ↑ | - | The relationship between structural modifications and antioxidant capacity is discussed, and the validity of the approach for the functional properties of SPI glycosylation products is confirmed | Zhang etal. (2018) |
| Soy protein isolate | Chitosan | Dry-heating | - | ↑ | - | - | - | - | - | ↑ | Character described the effect of chitosan attachment on reducing soybean protein sensitization for further development of functional protein-polysaccharide conjugates | Usui etal. (2004) |
| Soy protein isolate | d-galactose | Wet-heating | ↑ | ↑ | - | ↑ | - | - | ↑ | ↑ | Provide ideas for the application of SPI in the food industry and as a potential functional food | Zhang etal. (2022) |
| Mung bean protein | Dextran | Wet-heating | ↑ | ↑ | - | - | - | - | - | - | Expanded the knowledge of the functional changes caused by glycosylation reactions and provides an efficient way to improve the functional properties of proteins for better application in the food industry | Zhou etal. (2017) |
| Casein | Pectin | Wet-heating | ↑ | ↑ | - | ↑ | ↑ | - | ↑ | - | To provide theoretical support for protein-polysaccharide interactions and structural changes as well as functional changes | Yang etal. (2023) |
| Arabinogalactan | ↑ | ↑ | - | ↑ | ↑ | - | ↑ | - | ||||
| Pea protein isolate | Glucose | High-intensity ultrasound+wet-heating | ↑ | ↑ | - | ↑ | - | - | - | - | To provide valuable insights into the critical role of plant protein solubility on the extent of the glycosylation reaction and the function of the conjugates | Gao etal. (2023) |
| Rice protein | Dextran+macromolecular crowding conditions | Wet-heating | ↑ | ↑ | - | ↑ | - | - | - | - | The application of crowding conditions can be used as a new method to enhance the reaction and improve its solubility while maintaining the protein structure, thus contributing to the industrial application of rice protein | Cheng etal. (2022) |
| Sunflower meal | Xylose | Dry-heating | - | ↑ | - | - | - | - | ↑ | ↑ | May be used as food ingredients in food products because of their relatively high antimicrobial and antioxidant activities. On the other hand, it may have significant potential for the food industry as a functional food | Habinshuti etal. (2019) |
- Note: ↓: Decreased; ↑: Enhanced; -: Not reported.
- Abbreviations: AM, antimicrobial activity; AO, antioxidant activity; EP, emulsifying property; F, foaming; FS, freeze-thaw stability; GP, gelation property; S, solubility; TS, thermal stability.
5.1 Solubility
The solubility of SPI falls under the category of hydration properties and is one of the most fundamental functional characteristics. SPI contains numerous polar groups along its peptide chain skeleton, enabling it to absorb and retain water (OHYPERL etal., 2021). It is related to the protein properties (amino acid composition and sequence) and external conditions, such as pH (Yan etal., 2021), ionic strength (Feng, Zhao etal., 2024), and temperature (Yan etal., 2024). During the glycosylation modification, SPI reacts with sugar, depleting the amino groups of SPI and incorporating sugar chains with numerous hydroxyl groups. This alteration affects the number of hydrophilic groups, resulting in a change in solubility (Zhang, Li etal., 2019).
Chu etal. (2023) prepared an SPI glycosylation product modified by β-glucan from oats and studied the structures and properties. The results showed that after the glycosylation of β-glucan, the secondary structure of SPI becomes looser. This was because of the unfolding of the peptide chain and the introduction of hydrophilic groups in the polysaccharide; this significantly (p<0.05) increased the solubility of SPI. Wang etal. (2018) prepared xylose-SPI and fructose-SPI MRPs. The solubility of SPI increased by 43% and 59%, respectively, compared to the original state. This is due to significant changes in the structure of SPI; glycosylation disrupts the hydrogen bonds and hydrophobic interactions within the protein molecules, making them more prone to interact with water molecules, thereby increasing their solubility. Ma etal. (2020) used orange pectin and apple pectin as grafted polysaccharides to prepare soybean protein-pectin conjugate. The grafting degree of orange pectin and apple pectin was 25.00% and 21.85%, respectively.
5.2 Emulsifying property
SPI is a common surfactant with practical emulsifying properties because protein molecules are amphiphilic. Generally, the glycosylation modification adds hydrophilic group to the SPI molecule, allowing it to arrange more orderly and tightly at the interface of oil and water. This results in the formation of a denser protein film and significantly (p<0.05) improving its emulsifying properties (Tapal & Tiku, 2012). The type of sugar, reaction process, and other factors can affect the structure of the protein, thereby influencing its emulsifying properties.
Gharibzahedi and Smith (2021) found that through glycosylation reactions, the charge density on the protein's surface can be altered, resulting in improved hydrophilicity and lipophilicity, thus enhancing the emulsifying characteristics of SPI. Sun etal. (2022) explored the preparation of glycosylated conjugate of SPI and glucose (SPI-G) after subjecting them to different pH treatments. The results showed the SPI-G exhibited excellent emulsifying activity at pH 9.0 and emulsion stability at pH 8.0. There was a significant (p<0.05) correlation between the molecular flexibility and emulsifying properties of SPI-G conjugate. This indicates that pH treatment can significantly enhance the structure and emulsifying properties of natural SPI and SPI-G conjugates (p<0.05). Ashaolu and Zhao (2020) refined and decorated tofu dietary fiber (ODF) with SPI by glycosylation modification under dry heat conditions at 60°C. The ODF-SPI conjugate demonstrates excellent potential for emulsion stability, thermal stability, and hydrophilicity.
5.3 Gelation property
Gelatinicity is a crucial functional property of proteins in the food industry. Gelation refers to the property of proteins forming a branched structure, which gives proteins high viscosity, plasticity, and elasticity. The glycosylation modification can result in protein covalent cross-linking. When a small amount of reducing sugar react with SPI by glycosylation, the insufficient hydrophilic hydroxyl groups relatively increases the hydrophobic groups in the long chain of SPI. This enhances hydrophobic interactions between conjugate molecules, increases gelation properties, and gives it high viscoelasticity and breakage resistance.
Zhao etal. (2021) utilized SPI and MD to create SPI/MD glycosylation complexes via the Maillard reaction, and their rheological properties and microstructure were examined. The results show that the gel storage modulus of the SPI/MD mixture significantly (p<0.05) increases due to the phase separation between SPI and MD. The microstructure of the gel indicates that SPI/MD glycosylation complexes can form a high-strength, uniform, and dense gel network, thereby enhancing the gelling properties of SPI. Zhao etal. (2023) prepared heat-denatured SPI and soy oligosaccharide conjugates (HSPI-SOS) by heating themixed dispersion at 90°C and investigated the structural characteristics and the gel properties of acid-induced emulsion. By controlling the degree of glycosylation, the structure of HSPI-SOS conjugate can be altered, thereby enhancing the physical and chemical properties and stability of the gel. Hu etal. (2023) evaluated the effects of different industrial modification methods (heat (H), alkali (A), glycosylation (G), and oxidation (O)) on the structure and gel properties of SPI. The study found that the four industrial modification methods did not affect the subunit composition of SPI. G-SPI had the highest (p<0.05) disulfide bond content and optimal (p<0.05) gel properties.
5.4 Other functional features
SPI possesses other functional characteristics, particularly in processing, such as foaming. The foam is created by a soluble protein film that acts as a surface-active liquid, and the bubbles are separated by an elastic liquid film and a semisolid film to prevent them from coalescing. This process relies on the inner surface of protein molecules, allowing for rapid spreading. This property can give processed foods a loose structure and achieve a crisp taste. The lower the polymerization degree, the stronger the foaming property. When the mass ratio of SPI to xanthan gum is 10:1, the foaming stability of SPI increases from 2.6% to 8.2%, and its maximum foaming activity increases from 155 to 205mL, This may be due to the high molecular weight of xanthan gum, and glycosylation may increase the flexibility of natural protein structures, conformational changes, and protein surface rearrangements, which may lead to enhanced protein adsorption at the air–water interface during bubbling (Li etal., 2015).
Thermal stability refers to a protein's ability to maintain its properties when heated. Researchers prepared SPI-dextran conjugate via Maillard reaction, the conjugate showed higher denaturation temperature and enthalpy than SPI. This is due to the covalent bond between dextran and SPI, which increases the electronegativity, steric hindrance, and electrostatic repulsion of the molecules, reducing aggregation during heating and improving thermal stability (Zhuo etal., 2013).
The freeze-thaw stability of SPI is an important functional property crucial for its application in frozen foods. Freeze-thaw stability refers to the ability of a gel system to remain stable when exposed to repeated cycles of freezing and thawing. During the low-temperature freezing process, water in frozen foods freezes into ice, leading to phase separation. Upon thawing, ice crystals melt into water, which can be easily released from the food matrix, the process known as the dehydration shrinkage phenomenon (Liu etal., 2019). After the protein undergoes the glycosylation, the molecular structure extends, exposing the lysine residues inside. This increases the intramolecular and intermolecular forces of the protein, thus cross-linking effects increases. Consequently, the gel network structure becomes tight and orderly, making it more resistant to external environmental damage. The enhancement in freeze-thaw stability can effectively extend the shelf life of food and improve product quality (Yu, Wang etal., 2018). Wang etal. (2020) combined microwave technology with Maillard reaction and found that glycosylated products treated by high frequency microwave have a looser structure and stronger freeze-thaw stability.
6 EFFECT OF GLYCOSYLATION MODIFICATION ON BIOLOGICAL ACTIVITY OF SPI
6.1 Antioxidant activity
Modifying glycosylation alters protein structure and function, impacting antioxidant activity. It can cause conformational changes, shielding certain amino acid residues from oxidation. It also affects the interaction between proteins and antioxidants, altering antioxidant effects. Hence, glycosylation modification may improve SPI of antioxidant properties (Vhangani & Van Wyk, 2013).
The antioxidant capacity of MRPs is enhanced by prolonging the reaction time and increasing the reaction temperature. This generates new molecules with antioxidant activity, such as melanoidins and volatile compounds, which scavenge free radicals, thereby increasing the proteins’ antioxidant capacity (Yu, He etal., 2018). Zhang etal. (2018) explored the conformation and antioxidant properties of the SPI–MD complex. The results showed that glycosylated SPI products exhibited good reducing power and resistance to lipid oxidation, as well as higher free radical scavenging ability and iron ion chelating activity, which were attributed to the spherical structure of the SPI–MD complex, which positively influences its antioxidant properties.
6.2 Antibacterial activity
Glycosylation modification may also affect the antibacterial activity of SPI. Some studies have shown that glycosylation modification can change the charge distribution and spatial conformation of proteins, thus affecting their interaction with bacterial cells. Specifically, glycosylation modification may cause protein change the surface charge and its adsorption capacity with the bacterial surface, thus enhancing its antibacterial activity (Sun etal., 2017). In addition, glycosylation modification may also affect the interaction between proteins and bacterial cell membranes and interfere with the physiological functions of bacteria, thus exhibiting antibacterial properties (Zhang etal., 2015). However, different glycosylation modifications and degrees may lead to other effects, so the specific antibacterial effect needs to be evaluated by experimental study.
The inhibitory effect of SPI-chitosan conjugates on Escherichia coli was significantly (p<0.05) better than that of SPI. This is because the SPI-chitosan conjugates have surface activity, which can more effectively disrupt the stability of E. coli cell membranes, thereby enhancing their antibacterial activity. Therefore, SPI-chitosan conjugates can be utilized as a novel functional soy protein with antibacterial properties in food applications (Usui etal., 2004). Zhang etal. (2022) studied the glycosylation of SPI with d-lactose. The results showed introducing hydroxyl groups through glycosylation improved the solubility of SPI conjugates and enhanced the antibacterial activity of SPI. In addition, the antibacterial activity of SPI increased with the increase of d-lactose in glycosylation.
7 CONCLUSIONS AND OUTLOOK
Glycosylation is a green and effective method of protein modification and is an area of research with potential use in the food industry. In this review, we focused on the effects of modification of the glycosylation regulatory mechanism on the structure and function of SPI. The quantitative analysis method and structure-detection analysis of SPI glycosylation products were further discussed. Furthermore, the function of SPI was demonstrated in detail. In addition to the common functional properties such as solubility, emulsification, and gelation, the antioxidant and antibacterial properties of the glycosylation-modified products are also worthy of further study, which provides a theoretical basis for their application as functional ingredients in food development. In addition, the future development of SPI glycosylation products warrants further analysis.
The following is a specific perspective on the development of SPI glycosylation:
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Flavor: Glycosylation enhances food flavor and can be used as a new additive in the food industry. It can also create food products with unique sensory characteristics, making it a valuable tool.
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Methods: A single glycosylation reaction seems unable to meet the development of protein in the food field. In the process of glycosylation modification, physical methods can be introduced to assist the glycosylation reaction in increasing the degree of modification. In addition, enzymatic glycosylation may also be a better direction for development. Therefore, using two or more approaches to modify SPI may be a good option in future studies.
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Reaction environment: Both dry glycosylation and wet glycosylation will be affected by the reaction environment, such as temperature, time, and pH value. Therefore, it is necessary to optimize the reaction conditions.
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Application: The glycosylation function of SPIs has been improved and is widely used in the food field. However, glycosylated SPI is rarely used in research on food packaging, 3D printing, medical dressings, and other fields, which can be further investigated in future research.
AUTHOR CONTRIBUTIONS
Jinjing Chen: Writing—original draft; writing—review and editing; methodology. Wanting Zhang: Validation; visualization. Yiming Chen: Investigation; software. Meng Li: Supervision; data curation. Chang Liu: Conceptualization; writing—review and editing; funding acquisition. Xiuli Wu: Formal analysis; resources; project administration.
ACKNOWLEDGMENTS
We thank scientific research project of Education Department of Jilin Province [JJKH20240752KJ], Scholar Climbing Program of Changchun University [zpk202122].
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
REFERENCES
