Method for regulating and controlling aggregation path and gel property of soybean protein isolate based on polymerization degree specific chitosan oligosaccharide

Abstract

The invention discloses a method for regulating and controlling an aggregation path and gel characteristics of soybean protein isolate based on polymerization degree specific chitosan oligosaccharide, which comprises the following steps: preheating an SPI (Serial Peripheral Interface) solution, and uniformly mixing the SPI solution with a COS (DPlt; the degree of aggregation is lt; on one hand, the COS of 1, 6 guides the LAs to perform ordered assembly, optimizes protein space orientation, and promotes efficient formation of disulfide bonds; on the other hand, the small aggregate SAs is maintained in a non-covalent interaction stable state which is compact in structure and rich in free sulfydryl. An ordered precursor composed of a covalent cross-linked LAs skeleton and a non-covalent stable SAs filling unit constructs a compact and continuous three-dimensional network structure with high storage modulus in the subsequent thermally induced gelation process, and the mechanical strength and the structural stability of the SPI gel are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method for regulating the aggregation behavior of soy protein isolate (SPI) by using chitosan oligosaccharides (COS) with a specific degree of polymerization (DP) in conjunction with heat treatment, thereby improving its gel properties. Background Technology

[0002] Spirulina protein (SPI), derived from soybeans, is a nutrient-rich plant protein widely used in various food products due to its excellent functional properties such as emulsification, foaming, and gelation abilities. Among these, SPI's gelation properties are particularly crucial, enabling its application in the production of tofu, soy yogurt, dried bean curd sticks, and plant-based meat substitutes. It also determines the texture, water retention, and sensory quality of these products. However, the heat-induced gelation of natural SPI typically suffers from low strength, poor water retention, and a non-dense structure, limiting its application in the high-end food sector.

[0003] To improve the gelation properties of SPI, physical modification (such as ultrasound and high pressure), chemical modification (such as glycosylation and phosphorylation), or compounding with polysaccharides are commonly used methods. Among these, the compounding of polysaccharides with proteins has attracted much attention due to its ease of operation and high safety. Chitosan oligosaccharides (COS), as a degradation product of chitosan, carry a positive charge and can theoretically bind to SPI through electrostatic interactions, thereby affecting the formation and properties of the gel. However, when COS and SPI are directly mixed, rapid and uncontrollable aggregation and phase separation often occur due to excessively strong electrostatic interactions, resulting in an uneven gel structure and degraded performance.

[0004] Existing research has attempted to improve the compatibility of SPI and COS through enzymatic crosslinking. For example, in Wang Dan et al.'s paper "Enzymatic Synthesis and Gel Properties of Soybean Protein-Chitosan Oligosaccharide Polymers," transglutaminase (TGase) was used to catalyze the graft copolymerization of SPI and COS, achieving a grafting rate of 57.49% under optimized conditions. This study focused on the effects of enzymatic reaction conditions (such as temperature, time, and mass ratio) on the grafting rate and gel properties, and found that the acidified gel of the polymer after enzymatic modification was significantly superior to the single SPI gel in terms of hardness, elasticity, and water retention. However, the study also pointed out that the thermoinduced gel of the polymer did not show significant differences in hardness and elasticity compared to the unmodified SPI gel, indicating that enzymatic grafting alone did not significantly improve the overall performance of the thermoinduced gel.

[0005] Furthermore, existing research largely focuses on the effects of process parameters such as reaction conditions, formulation ratios, and cross-linking methods on the grafting rate or apparent properties of the complex, without systematically investigating the regulatory role of polysaccharide structural characteristics on protein aggregation pathways and gel network construction from the underlying mechanisms of molecular interactions. Therefore, how to effectively improve the performance of SPI thermally induced gels by regulating key structural factors in the composite system remains a technical problem to be solved in this field. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for regulating the aggregation pathway and gel properties of soybean protein isolate (SPI) based on chitosan oligosaccharides with specific polymerization degree. By selecting chitosan oligosaccharides with a specific polymerization degree, the method utilizes the mixing of chitosan oligosaccharide solution with preheated SPI solution to form a gel under heat-induced conditions, thereby improving the properties of the composite gel and solving the problems mentioned above, such as the decrease in protein gel properties after adding chitosan oligosaccharides.

[0007] To achieve the above objectives, this invention first provides a method for regulating the aggregation pathway and gel properties of soybean protein isolate based on degree-of-polymerization specific chitosan oligosaccharides, comprising the following steps: (1) Protein solution preparation and preheating: Soy protein isolate was dispersed in water to prepare a soy protein isolate SPI solution and preheated to fully expand the protein structure and expose the internal active reaction sites, thus obtaining a preheated SPI solution; (2) Preparation of polysaccharide solution: Select chitosan oligosaccharide COS with DP < 6 and prepare COS solution; (3) Preparation of the composite: The COS solution from step (2) and the SPI solution after preheating in step (1) are mixed evenly under stirring conditions, and the pH is adjusted to neutral to obtain the SPI-COS composite dispersion solution. The SPI-COS composite powder is obtained by freeze drying. (4) Gel preparation: The SPI-COS composite powder from step (3) is dispersed in water and thermally induced to gel. After cooling, a high-strength SPI-COS composite gel is obtained.

[0008] In one embodiment of the present invention, the mass concentration of the soy protein isolate (SPI) solution in step (1) is 2%~8% (w / v), and the preheating treatment is performed by heating at 95-120 °C for 15-30 minutes.

[0009] In one embodiment of the present invention, the COS has a degree of polymerization DP < 6, a molecular weight ≤ 2000 Da, and a degree of polymerization DP ≥ 3. COS with DP < 3 is avoided because it easily induces the formation of disordered macroaggregates that are detrimental to improving gel performance.

[0010] In one embodiment of the present invention, the COS solution has a mass concentration of 0.04%–0.12%, w / v, preferably 0.08%.

[0011] In one embodiment of the present invention, the volume ratio of COS solution to preheated SPI solution in step (3) is 1:3 to 3:1, preferably 1:1.

[0012] In one embodiment of the present invention, the stirring speed in step (3) is 300~500 rpm, the stirring time is not less than 3h, preferably 3~5h, and the pH is adjusted to 6.5~7.5, preferably 7.0, using a 0.5~1mol / L sodium hydroxide solution.

[0013] In one embodiment of the present invention, in step (3), the concentration of SPI-COS composite powder after being dispersed in water is 9-12%.

[0014] In one embodiment of the present invention, in step (3), the temperature for thermally induced gelation is 100~120℃ and the time is 20~30min.

[0015] In one embodiment of the present invention, in step (3), after thermally induced gelation, the mixture is cooled at 0~4℃ for 10~12h.

[0016] The present invention also provides a high-strength SPI-COS composite gel prepared using the above method.

[0017] The present invention also provides an application of the above-mentioned high-strength SPI-COS composite gel in the food industry.

[0018] Beneficial effects: 1. Significantly improved gel strength: Compared with SPI gel alone or gel with COS containing low DP, the composite gel prepared by the present invention using COS with DP<6 has significantly increased energy storage modulus and hardness.

[0019] 2. Precise control of aggregation pathways: This invention reveals and utilizes the DP control mechanism of COS for the first time. Unlike the loose, disordered, and disulfide bond-resistant large particle aggregates induced by DP<3, COS with DP<6 can guide the preheated SPI to form compact and ordered LAs and SAs.

[0020] 3. Optimization of microstructure: DP<6-induced ordered LAs optimize protein spatial orientation and promote efficient disulfide bond formation (manifested as a significant reduction in total free thiol content); simultaneously, SAs maintain stability through non-covalent interactions. This "covalent backbone + non-covalent filler" model constructs a dense, continuous three-dimensional network structure, greatly improving gel quality.

[0021] 4. Simple process and high safety: This invention does not use any toxic chemical cross-linking agents. It achieves a leap in gel performance through physical heat treatment and natural polysaccharide compounding, making it suitable for large-scale production in the food industry.

[0022] 5. This invention effectively improves the gelation properties of proteins by mixing a chitosan oligosaccharide solution with a concentration of less than 0.12% with a preheated SPI solution. Further increasing the concentration of chitosan oligosaccharide not only fails to improve protein performance but also wastes resources or prevents gel formation. Attached Figure Description

[0023] Figure 1 The graph shows the protein content of LAs and SAs in the aggregate systems of Example 1 and Comparative Examples 1-3.

[0024] Figure 2 The particle size distribution diagrams of LAs and SAs in the aggregate systems of Example 1 and Comparative Examples 1-3 are shown.

[0025] Figure 3 The graph shows the total free thiol content of LAs and SAs in the aggregate systems of Example 1 and Comparative Examples 1-3.

[0026] Figure 4 The graph shows the surface hydrophobicity of LAs and SAs in the aggregate systems of Example 1 and Comparative Examples 1-3.

[0027] Figure 5 The graph shows the dependence of the composite gel modulus of Example 1 and Comparative Examples 1-3 on frequency.

[0028] Figure 6 Comparison of the macroscopic appearance of the final gels obtained in Example 1 and Comparative Examples 1-3 (observed upside down).

[0029] Figure 7 Macroscopic appearance of the heterogeneous gels obtained in Comparative Example 4 and Comparative Example 5.

[0030] Figure 8 Comparison of the frequency dependence of the storage modulus of the homogeneous gels obtained in Example 1 and Comparative Example 5. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The COS with DP < 6 and molecular weight ≤ 2000 Da, and the COS with DP < 3 and molecular weight ≤ 1000 Da involved in the embodiments and comparative examples of this invention, were all sourced from Shanghai Aladdin Biochemical Technology Co., Ltd. The COS with DP < 9 and molecular weight < 3000 Da involved were sourced from Shanghai Yuanye Biotechnology Co., Ltd.

[0033] Example 1 A method for preparing a high-strength SPI-COS composite gel includes the following steps: (1) Preheating treatment SPI powder was dispersed in deionized water and magnetically stirred for 3 hours to fully hydrate it, preparing a 2% (w / v) SPI solution. The solution was then heated in a 100 °C water bath for 30 minutes, and immediately cooled to room temperature (25 °C) with running water after heating to obtain a preheated SPI solution with fully developed structure.

[0034] (2) Composite treatment Weigh COS with DP<6 and molecular weight≤2000 Da, and prepare a COS solution with a concentration of 0.08% (w / v) using deionized water. Under continuous stirring, mix the COS solution with the preheated SPI solution obtained in step (1) at a volume ratio of 1:1 to obtain a homogeneous mixed solution, and adjust the pH of the mixed solution to 7.0. Prepare SPI-COS composite powder by freeze drying. (3) Gelization of SPI-COS The SPI-COS composite powder obtained in step (2) was dissolved in deionized water at a protein concentration of 10% (w / v) and heated in a 100°C water bath for 30 minutes to induce gelation. After cooling, the SPI-COS composite gel was obtained.

[0035] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that in step (2), the same volume of deionized water is used instead of COS solution.

[0036] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that in step (2), COS with DP<3 and molecular weight ≤1000 Da is used instead of COS with DP<6 and molecular weight ≤2000 Da.

[0037] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the natural SPI powder was directly dissolved in deionized water at a protein concentration of 10% (w / v) and heated in a 100 °C water bath for 30 minutes to induce gelation. After cooling, the natural SPI gel was obtained.

[0038] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that in step (2), COS with DP<9 and molecular weight ≤3000 Da is used instead of COS with DP<6 and molecular weight ≤2000 Da.

[0039] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that the mass concentration of the COS solution was changed to 0.12% and 0.4% in step (2), respectively.

[0040] The composite powder obtained in step (2) of Example 1 was dissolved in deionized water at a protein concentration of 2% (w / v), heated at 100°C for 30 minutes to obtain an aggregate solution, and then separated by ultracentrifugation (400,000 g, 4°C, 60 minutes) to obtain LAs and SAs.

[0041] like Figure 1 As shown, in Example 1 (degree of polymerization < 6), the distribution ratio of aggregates in the LAs and SAs after centrifugation exhibits a specific distribution, demonstrating an altered aggregation pathway. The COS of a specific DP (DP < 6) acts as a regulatory factor, guiding the ordered assembly of protein molecules. Figure 2 As shown, the particle size distribution curve of the obtained aggregates exhibits relatively sharp, narrow, and symmetrical nanoscale peaks, indicating that the protein formed relatively uniform aggregate precursors. Further analysis revealed that the internal structure of the LAs formed in the examples was dense. Figure 3 As shown, its total free thiol content was significantly lower than that of Comparative Example 1 and Comparative Example 2, indicating that the ordered structure induced by DP<6 effectively shortened the distance between cysteine ​​residues, promoting the oxidation of a large number of free thiols to form disulfide bonds, thereby constructing a robust covalent backbone. Meanwhile, for SAs, such as Figure 4 As shown, the surface hydrophobicity of the SAs in this embodiment is significantly higher than that of Comparative Example 1 and other groups. This means that the SAs do not completely embed hydrophobic groups like the LAs, but retain a high level of surface hydrophobicity. These highly hydrophobic SAs, acting as "active fillers," can be adsorbed into the gel network through strong hydrophobic interactions, further enhancing the compactness of the system.

[0042] The rheological tests were performed on the gel obtained in Example 1, and the results are as follows: Figure 5 As shown. The composite gel prepared in Example 1 exhibited the highest storage modulus. Notably, its strength was not only significantly higher than that of the preheated control group (Comparative Example 1) and the DP<3 group (Comparative Example 2), but even surpassed that of natural soy protein isolate (Comparative Example 3). This confirms that the synergistic effect of the "disulfide-rich ordered LAs backbone" and "hydrophobic SAs filling" constructed in step (2) is not simply to repair the denaturation damage caused by preheating, but rather to construct a novel high-strength three-dimensional network structure superior to the natural SPI heat-induced gel through the ordered assembly of long-chain COS. Figure 6As shown, the sample prepared in Example 1 exhibited excellent self-supporting ability after being inverted. The gel surface was smooth, the texture was fine and uniform, and no obvious water layering or graininess was observed. This intuitively confirms that the "ordered dense network" constructed by COS with DP < 6 exhibits a high-strength solid gel morphology on a macroscopic scale.

[0043] In the Comparative Example 1 (preheated SPI) system, protein aggregation behavior was mainly driven by thermal denaturation, lacking orderly regulation. For example... Figure 3 As shown, the total free thiol content in its LAs is significantly higher than that in Example 1, indicating a lower disulfide bond formation efficiency. Meanwhile, as... Figure 4 As shown, the surface hydrophobicity of its LAs is the highest, indicating that the protein underwent disordered denaturation and unfolding during aggregation, resulting in a large number of hydrophobic groups being exposed on the surface without being embedded. This loose structure is not conducive to the formation of a strong gel backbone. The surface hydrophobicity of its SAs is significantly lower than that of Example 1, indicating that its small aggregates lack sufficient surface active sites and are difficult to participate in subsequent gel network enhancement as an efficient filler.

[0044] The rheological properties of the gel obtained in Comparative Example 1 are as follows: Figure 5 As shown, due to the fragile framework and weak infill bonding, its energy storage modulus is significantly lower than that of Example 1. Figure 6 As shown, the sample failed to form a robust gel structure, exhibiting significant fluidity or a loose, semi-fluid state when inverted, with a rough surface and possible dehydration shrinkage. This further demonstrates that simply preheating the protein cannot construct an effective gel network.

[0045] For the sample in Comparative Example 2 (degree of polymerization < 3), the short-chain COS did not play a good regulatory role. Figure 2 As shown, compared to Example 1, its particle size distribution curve has a wider peak, indicating that the aggregate size is not uniform. Figure 3 As shown, the total free thiol content in its LAs is at a high level, failing to convert to disulfide bonds as efficiently as in Example 1. Figure 4 As shown, the surface hydrophobicity of its SAs is also at a low level, with insufficient skeletal strength and a lack of highly active filling units.

[0046] The rheological properties of the gel obtained in Comparative Example 2 are as follows: Figure 5 As shown, its storage modulus, while slightly higher than Comparative Example 1, is significantly lower than that of Example 1 (DP<6) and the natural SPI group. This indicates that although DP<3 induces a certain degree of aggregation, this aggregation is disordered and fails to form a "dense framework + active filler" structure similar to DP<6, thus failing to achieve a qualitative leap in gel strength. Figure 6As shown, the sample is in a weak gel state. Although it may not flow completely, the gel structure appears fragile and soft. This indicates that short-chain COS cannot induce disordered aggregates to form a strong gel network.

[0047] The relevant performance of the natural SPI in Comparative Example 3 is as follows: Figure 5 As shown, natural SPI, retaining its original globulin structure, can form a self-supporting gel under thermal induction, exhibiting acceptable gel strength, significantly higher than Comparative Example 1 and Comparative Example 2. However, compared to Example 1 of this invention (DP<6), the storage modulus of natural SPI remains lower. This strongly demonstrates the inventiveness of the method described in this invention: by utilizing COS synergistic preheating treatment with DP<6, a more dense and robust network structure than the natural SPI's own thermal gel was successfully constructed, achieving a substantial improvement in the gel properties of SPI. Figure 6 As shown, natural SPI can form a self-supporting gel, but compared to Example 1 (DP<6), its gel is slightly less firm and finer (for example, Example 1 appears denser and harder when pressed or visually observed). This direct comparison of macroscopic states strongly supports the significant advantages of the present invention in improving gel quality.

[0048] Comparative Example 4 used COS with a DP greater than 6 and less than 9 to react with SPI protein. Due to the excessively long polysaccharide chain and its strong bridging ability, a violent bridging flocculation reaction occurred instantly upon mixing with the preheated SPI in step (2). A large amount of white flocculent precipitate was visible to the naked eye in the mixture, and severe phase separation occurred in the system, making it impossible to form a homogeneous mixed solution similar to that in Example 1.

[0049] Heat treatment did not improve the homogeneity of the system; on the contrary, it exacerbated phase separation. Figure 7 (Left). Due to the presence of large particle sediments, the three-dimensional cross-linking of the protein network was hindered, and a self-supporting gel was ultimately not formed. Instead, it exhibited a solid-liquid separation state (i.e., a state in which rough, hard clumps were completely separated from the water), thus losing its application value as a food gel.

[0050] Comparative Example 5 compared the effects of different COS addition amounts. For the gel system with a COS concentration of 0.12%, mixing the COS solution with the protein resulted in a homogeneous mixture similar to that in Example 1. Although the 0.12% (w / v) COS group formed a homogeneous gel, the rheological data ( Figure 8The results show that the increase in storage modulus compared to Example 1 is minimal. This indicates that the COS gel strengthening mechanism with DP < 6 described in this invention has reached functional saturation at a concentration of 0.08% (w / v). Conventional methods of increasing COS concentration to improve gel strength are ineffective in this system and instead result in a waste of raw materials. This demonstrates the unexpectedly high efficiency of the 0.08% (w / v) concentration selected in this invention.

[0051] If the concentration of COS is further increased to 0.4%, in step (2), the 0.4% (w / v) COS carries an excess of positive charge, which completely neutralizes the protein surface charge. The solution becomes turbid immediately upon mixing, producing a large amount of white electrostatic precipitate. After gelation, the 0.4% (w / v) COS group, after thermal induction, exhibits a tofu-like consistency with solid-liquid separation. Figure 7 (Right), completely unable to form a self-supporting gel.

[0052] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for regulating the preheating SPI aggregation path based on DP-specific COS, characterized in that, The method includes the following steps: (1) Protein solution preparation and preheating: Soy protein isolate was dispersed in water to prepare a soy protein isolate SPI solution and preheated to fully expand the protein structure and expose the internal active reaction sites, thus obtaining a preheated SPI solution; (2) Preparation of polysaccharide solution: Select chitosan oligosaccharide COS with DP < 6 and prepare COS solution; (3) Preparation of the complex: The COS solution from step (2) and the SPI solution from step (1) were mixed evenly under stirring, and the pH was adjusted to neutral to obtain the SPI-COS complex dispersion solution.

2. The method according to claim 1, characterized in that, In step (1), the mass concentration of the soy protein isolate (SPI) solution is 2% to 8%, and the preheating treatment is performed at 95-120 °C for 15–30 minutes.

3. The method according to claim 1, characterized in that, The COS has a degree of polymerization (DP) < 6, a molecular weight ≤ 2000 Da, and a degree of polymerization (DP) ≥ 3. In one embodiment of the present invention, the mass concentration of the COS solution is 0.04%–0.12%, w / v, preferably 0.08%.

4. The method according to claim 1, characterized in that, In step (3), the volume ratio of COS solution to preheated SPI solution is 1:3~3:

1.

5. The method according to claim 1, characterized in that, In step (3), the stirring speed is 300~500 rpm and the stirring time is not less than 3 hours. The pH is adjusted to 6.5~7.5 using a 0.5~1 mol / L sodium hydroxide solution.

6. A method for regulating the properties of preheated SPI gel based on DP-specific COS, characterized in that, The SPI-COS composite dispersion obtained according to any one of claims 1 to 5 is freeze-dried, then dispersed in water and subjected to thermally induced gelation. After cooling, a high-strength SPI-COS composite gel is obtained.

7. The method according to claim 6, characterized in that, The concentration of SPI-COS composite powder after dispersion in water is 9-12%.

8. The method according to claim 1, characterized in that, In step (3), the temperature for thermally induced gelation is 100~120℃ and the time is 20~30min. After thermally induced gelation, the gel is cooled at 0~4℃ for 10~12h.

9. The high-strength SPI-COS composite gel prepared by the method according to any one of claims 6 to 8.

10. The application of the high-strength SPI-COS composite gel according to claim 9 in the food industry.

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