Double-crosslinked animal and plant protein combined composite hydrogel as well as preparation and application thereof

Abstract

The invention relates to the technical field of hydrogel, in particular to double-crosslinked animal and plant protein combined composite hydrogel as well as preparation and application thereof. According to the invention, glutamine transaminase and skin-derived gelatin are adopted to carry out dual crosslinking on pea protein isolate, so as to prepare the animal and plant protein combined composite hydrogel. The method not only relieves adverse effects caused by excessive consumption of animal protein, but also enables the hydrogel to have high mechanical strength, excellent water-retaining property, good thermal stability and an adjustable porous structure by adjusting the proportion of the two phases, and effectively overcomes the problem of poor gel performance of single plant protein. Good application prospects are realized in the fields of food texture improvement and biological materials.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and more particularly to a composite hydrogel combining double-crosslinked animal and plant proteins, its preparation and application. Background Technology

[0002] Hydrogels, as polymeric materials with a three-dimensional network structure, have attracted considerable attention in food science, biomedicine, and tissue engineering due to their excellent physicochemical properties, biocompatibility, and controllable functions. Adding different matrices can produce gels with varying functional properties. Protein is considered one of the main sources of food hydrogels, and it is also an essential nutrient in the human diet, with the majority derived from animal products. However, the excessive consumption of animal protein products has had a significant negative impact on human health, environmental protection, and animal welfare. To mitigate these drawbacks and achieve healthy diets and sustainable food production systems, developing composite gels that combine animal and plant proteins to form synergistic properties has become a current research hotspot.

[0003] Pea protein isolate (PPI) is a relatively new plant protein that stands out from other plant proteins due to its high nutritional value, low allergenicity, and non-GMO properties. However, PPI has a low cysteine ​​content, resulting in weak gelling properties. Furthermore, the hydrophobic surface and low charge density of PPI lead to relatively low solubility in water, resulting in poor gel formation and limiting its application in food. Therefore, modifying PPI to improve its gelling properties is crucial.

[0004] To overcome these limitations, researchers have explored various modification strategies. One effective method is the addition of polysaccharides, such as sodium alginate, gellan gum, or pectin. These substances can non-covalently cross-link with proteins through hydrogen bonds, hydrophobic interactions, and electrostatic interactions, thereby enhancing gel strength and stability. Another strategy is enzymatic modification. By catalyzing intermolecular and intramolecular covalent cross-linking of proteins, the gelation behavior of pea proteins can be modulated.

[0005] Single covalent crosslinking systems offer limited improvement in the gel strength of PPIs, making it difficult to form high-strength, highly elastic network structures. While protein-polysaccharide complex systems can enhance gel performance, they often rely on non-covalent interactions, exhibiting poor stability and are prone to failure, especially under thermal or mechanical stress. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a composite hydrogel that combines double cross-linked animal and plant proteins.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a composite hydrogel combining animal and plant proteins, wherein the network structure of the composite hydrogel is obtained by cross-linking pea protein isolate with gelatin; the gelatin is skin-derived gelatin; the cross-linking includes covalent cross-linking and non-covalent cross-linking; the covalent cross-linking is covalent cross-linking catalyzed by transglutaminase; the non-covalent cross-linking is hydrogen bonding, hydrophobic interaction and electrostatic interaction formed between pea protein isolate and gelatin.

[0008] To address the problems of poor gel performance, low mechanical strength, insufficient water retention, and poor thermal stability of PPI in existing technologies, this invention constructs a composite gel system with high mechanical strength, good shape recovery, high water retention, and thermal stability by compounding pea protein isolate (PPI) with gelatin (GEL) and covalently cross-linking them under the catalysis of transglutaminase (TGase), combined with the non-covalent interaction between the two. This effectively overcomes the problem of poor gel performance of single plant proteins and has good application prospects in the fields of food texture improvement and biomaterials.

[0009] The pea protein isolate (PPI) of this invention is low in allergens, non-GMO, and has good emulsifying properties, but it contains few sulfur amino acids and has weak gelling properties on its own. The gelatin used in this invention is hide-derived gelatin (derived from bovine / pig hide), which has good thermal reversibility and can provide the main three-dimensional network framework. After the two are combined and cross-linked, a composite hydrogel with high mechanical strength, excellent water retention, good thermal stability, and a tunable porous structure can be obtained.

[0010] Extensive research has revealed that not all combinations of animal and plant proteins can yield hydrogels with superior performance. For instance, when using gelatin as the animal protein, if soy protein isolate (SPI) is chosen as the plant protein for cross-linking, SPI typically exhibits higher gel strength than protein protein isolate (PPI), but it also has higher allergenicity. Furthermore, in the soy protein and gelatin composite system, the pore structure formed may be less uniform than that of PPI due to the different structures of soy globulins. Alternatively, using wheat gluten protein (WG) as the plant protein for cross-linking results in poor solubility, leading to a harder and less elastic hydrogel with significantly lower water-holding capacity (WHC) compared to the PPI system.

[0011] However, if bone-derived gelatin is chosen as the animal protein for cross-linking, given that pea protein isolate is the plant protein, its low isoelectric point and poor stability under neutral conditions (the phosphate buffer solution used in this invention is neutral to ensure maximum enzyme activity) make it unsuitable for this composite gel. Furthermore, bone-derived gelatin has relatively low gel strength. Alternatively, whey protein isolate (WPI) can be chosen as the animal protein for cross-linking. However, WPI is a thermosetting protein, unlike gelatin which is thermally reversible. Replacing gelatin with WPI significantly reduces the gel's elasticity and shape recovery ability, thus negating the "shape memory" property of this invention.

[0012] The choice of enzyme during the catalytic cross-linking process also affects the performance of the hydrogel. The transglutaminase (TGase) used in this invention is food-grade safe and specifically catalyzes the reaction between glutamine and lysine. If tyrosinase is used instead, the tyrosine content in gelatin is very low, resulting in extremely poor direct cross-linking effect on gelatin; its main action is on PPI. The overall network of the resulting composite gel will be weaker than that of the TGase system. Furthermore, enzymatic oxidation produces melanin-like substances, causing the hydrogel to turn brown or black, severely affecting the sensory properties of the food. While genipin is also a natural cross-linking agent, it causes the gel to turn blue, affecting its appearance; glutaraldehyde is cytotoxic and cannot be used in food. Therefore, TGase is the best choice balancing safety and appearance.

[0013] Preferably, the composite hydrogel has a porous honeycomb structure.

[0014] Secondly, the present invention provides a method for preparing the above-mentioned composite hydrogel combining animal and plant proteins, comprising the following steps: (1) Pea protein isolate and gelatin were dissolved in phosphate buffer to prepare pea protein isolate solution and gelatin solution, respectively; (2) Mix the pea protein isolate solution and the gelatin solution evenly to obtain mixture a; (3) Add transglutaminase to mixture a, stir evenly, and then react in a water bath to obtain mixture b; (4) The mixture b is subjected to enzyme inactivation treatment, cooled and refrigerated to obtain the composite hydrogel of the combination of animal and plant proteins.

[0015] This invention combines animal and plant proteins and prepares composite hydrogels through a dual covalent and non-covalent cross-linking mode (PPI and GEL can form non-covalent cross-links such as hydrogen bonds, hydrophobic interactions and electrostatic interactions, and the two undergo covalent cross-linking under TGase catalysis).

[0016] Preferably, in step (1), the pH of the phosphate buffer is 6.5-7.5.

[0017] Preferably, in step (1), the concentration of pea protein isolate in the pea protein isolate solution is 12%-18% (w / v); and the concentration of gelatin in the gelatin solution is 8%-12% (w / v).

[0018] More preferably, in step (1), the concentration of pea protein isolate in the pea protein isolate solution is 15% (w / v); and the concentration of gelatin in the gelatin solution is 10% (w / v).

[0019] Preferably, in step (1), the pea protein isolate is dissolved by stirring at room temperature for 1-3 hours; the gelatin is dissolved by heating at 45-60°C.

[0020] Preferably, in step (2), the volume ratio of the pea protein isolate solution to the gelatin solution is pea protein isolate solution: gelatin solution = (1-3): (1-2).

[0021] Through research, this invention has found that a compounding of gelatin and PPI within a specific ratio range can achieve better crosslinking and mechanical properties.

[0022] Preferably, in step (2), the amount of transglutaminase added is 10-30 U / g protein.

[0023] U / g protein indicates the number of enzyme activity units added per gram (of substrate or sample) of protein. U represents the enzyme activity unit, and g protein refers to per gram of substrate / sample. The transglutaminase (TGase, 200 U / g) in this invention contains 200 U of enzyme activity units per gram of transglutaminase.

[0024] Preferably, in step (3), the reaction conditions are: constant temperature reaction in a water bath at 45-55℃ for 1.5-3 hours.

[0025] Preferably, in step (4), the enzyme is inactivated by heating at 70-85°C for 5-15 minutes.

[0026] Preferably, in step (4), the refrigeration conditions are: refrigeration at 0-4℃ for at least 12 hours.

[0027] Thirdly, the present invention provides the application of the above-mentioned composite hydrogel combining animal and plant proteins in the preparation of food and biomaterials.

[0028] The beneficial effects of this invention are as follows: This invention utilizes transglutaminase and gelatin to perform dual cross-linking of pea protein isolate, preparing a composite hydrogel combining animal and plant proteins. This not only mitigates the adverse effects of excessive animal protein consumption but also, by adjusting the ratio of the two phases, achieves high mechanical strength, excellent water retention, good thermal stability, and a controllable porous structure. It effectively overcomes the poor performance of single-plant protein gels and shows promising application prospects in food texture improvement and biomaterials. Attached Figure Description

[0029] Figure 1 This is a microscopic image of a hydrogel sample.

[0030] Figure 2 This is a thermogravimetric analysis (TGA) graph of the hydrogel sample.

[0031] Figure 3 This is a differential scanning calorimetry (DSC) analysis graph of a hydrogel sample.

[0032] Figure 4 This is a graph showing the swelling of the hydrogel sample.

[0033] Figure 5 The figure shows the test results of water content and water holding capacity of the hydrogel sample.

[0034] Figure 6 The image shows the strength test results of the hydrogel sample.

[0035] Figure 7 The figure shows the test results of the fatigue resistance properties of the hydrogel sample.

[0036] Figure 8 This is a diagram showing the crystal structure of the hydrogel sample. Detailed Implementation

[0037] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0038] Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the materials and reagents used are commercially available products that can be obtained through commercial channels.

[0039] Materials used in this invention; Pea protein isolate (PPI): Green peas were purchased from a local supermarket, and pea protein isolate was extracted using the isoelectric point precipitation method, with a purity of ≥80%. It can also be purchased commercially.

[0040] Gelatin (GEL, Type A): Purchased from Aladdin Biochemical Technology Co., Ltd.

[0041] Type A gelatin is typically made from raw materials such as pigskin or cowhide, and is produced by short-term treatment under dilute acid (pH 1-3) conditions. It has a high isoelectric point (pH 7.0-9.0), high gel strength, good transparency, and low viscosity.

[0042] Transglutaminase (TGase, 200 U / g): purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China).

[0043] Solvent: Phosphate-buffered saline (PBS) with a pH of 6.5-7.5.

[0044] Example 1: Sample Preparation Pea protein isolate was dissolved in 50 mM phosphate buffer solution at pH 7.0 and stirred at 500 rpm for 2 hours at room temperature to prepare a 15% pea protein solution.

[0045] Gelatin was dissolved in a 50 mM phosphate buffer solution with pH=7.0 and swollen at room temperature for 20 min. Then it was heated in a water bath at 600 rpm and 50℃ for 30 min to prepare a 10% gelatin solution.

[0046] Prepare hydrogels in groups to achieve the final concentration (total volume of each sample is 12 mL): The mixtures were prepared as follows: 12 mL pure gelatin solution (1G0P), 8 mL gelatin solution and 4 mL pea protein solution (2G1P), 6 mL gelatin solution and 6 mL pea protein solution (1G1P), 4 mL gelatin solution and 8 mL pea protein solution (1G2P), 3 mL gelatin solution and 9 mL pea protein solution (1G3P), 12 mL pea protein solution (0G1P), 2 mL gelatin solution and 10 mL pea protein solution (1G5P), and 10 mL gelatin solution and 2 mL pea protein solution (5G1P).

[0047] Subsequently, TGase (20 U / g protein) was added to each sample, and the mixture was rapidly stirred for 1 min until dissolved. The sample was then heated in a 50°C water bath for 2 h, followed by heating in a 75°C water bath for 10 min to inactivate the TGase. The mixture was rapidly cooled and placed in a refrigerator overnight to obtain a hydrogel. These samples were named 1G0P, 2G1P, 1G1P, 1G2P, 1G3P, 0G1P, 1G5P, and 5G1P, respectively, for subsequent testing.

[0048] Example 2: Microstructure The network structure of the hydrogels prepared above was observed using a scanning electron microscope (SEM; Regulus 8230; Hitachi, Tokyo, Japan). Before measurement, all hydrogel samples were freeze-dried under vacuum for 48 hours. A small sample was fixed to a sample holder with conductive adhesive, and then the cross-section of the sample was sputtered with gold. Finally, the samples were observed and photographed at 100x magnification.

[0049] The results are as follows Figure 1As shown, all tested hydrogel samples exhibited a honeycomb-like porous network structure, but samples with different ratios showed significant differences in pore size, wall thickness, and network regularity. Pure gelatin hydrogel (1G0P) exhibited a relatively loose and open network structure with irregular pore distribution. Upon introduction of PPI, the network structure gradually became denser and more interconnected. Hydrogel 2G1P showed stronger cohesion, with smaller and more uniform pores. With increasing PPI ratios (1G1P, 1G2P, and 1G3P), the hydrogel matrix showed a trend towards further densification, increased wall thickness, and a significant decrease in average pore size. In contrast, pure PPI gel (0G1P) exhibited distinctly different morphological characteristics, displaying a coarse and aggregated granular network with less obvious pore structure, reflecting the inherent gelation behavior of plant proteins in gelatin-free conditions. This demonstrates that the introduction of gelatin can effectively regulate the porous structure of PPI hydrogels.

[0050] Hydrogel sample 1G5P contained excessive plant protein, resulting in significant phase separation and a discontinuous structure. Hydrogel sample 5G1P contained excessive gelatin, which caused excessive shrinkage leading to water separation and an overly hard, rubber-like texture; therefore, no further testing was conducted.

[0051] Example 3: Thermal Properties 1. Thermogravimetric analysis Thermogravimetric analysis (TGA) was performed on lyophilized hydrogel powders (5–10 mg) under a continuous nitrogen flow (40 mL / min) (TG209F1, Germany), heated from 25 °C to 600 °C at a rate of 10 °C / min. All hydrogel samples exhibited a similar two-stage thermal degradation pattern. Figure 2 ).

[0052] The first stage occurred between approximately 30°C and 150°C. The pure gelatin hydrogel showed a slightly larger initial weight loss than other samples, likely due to gelatin's high hygroscopicity, allowing it to retain more moisture even after freeze-drying. The second and most significant weight loss stage occurred in the temperature range of approximately 150°C to 450°C. This sharp weight decrease corresponds to the main thermal decomposition events involving the pyrolysis of the protein backbone, peptide bond breakage, and degradation of amino acid side chains. Furthermore, the thermal degradation curves of the composite hydrogels (2G1P, 1G1P, 1G2P, and 1G3P) fell between those of the pure 1G0P and 0G1P hydrogels. This indicates that the TGase-crosslinked pea protein isolate and gelatin did not negatively impact the overall thermal stability of the hydrogel system, and all hydrogels exhibited good thermal stability.

[0053] 2. Differential scanning calorimetry analysis The lyophilized hydrogel powder (6-10 mg) was placed in an aluminum pan, with an empty pan as a reference, and the measurement was performed using a differential scanning calorimeter (DSCQ2000, USA). The temperature was increased from 10 °C to 180 °C at a rate of 10 °C / min, while the heat flux was monitored as a function of temperature.

[0054] All hydrogels exhibited an endothermic peak around 120-140℃, corresponding to the mass loss in the first stage of the TG curve. The thermal denaturation temperature (Td), corresponding to the endothermic peak temperature in the DSC curve, is one of the key indicators for evaluating the thermal stability of hydrogels. A higher Td value indicates a more stable network structure, and its dissociation requires more energy. Figure 3 As shown, the Td values ​​of pure gelatin hydrogel (1G0P) and pure PPI hydrogel (0G1P) were 123.345℃ and 126.673℃, respectively. This indicates that under the experimental conditions, the initial thermal stability of the pea protein network cross-linked by transglutaminase (TGase) was slightly higher than that of the pure gelatin network. The Td values ​​of the composite hydrogel were 137.887℃, 121.743℃, 123.715℃, and 124.085℃, showing a trend of first decreasing and then increasing, indicating that the interaction between gelatin and PPI can significantly affect the thermal stability of the hydrogel.

[0055] Example 4: Swelling Behavior Different lyophilized hydrogel samples were weighed and immersed in beakers containing phosphate buffered saline (PBS, 50 mM, pH 7) until swelling equilibrium was reached. Samples were recovered at 1, 2, 4, 8, 12, and 24 h, and residual PBS solution on the sample surface was gently blotted away with filter paper. All experiments were performed at room temperature and repeated three times.

[0056] The swelling ratio of a hydrogel sample is calculated using the following formula: Where M0 is the initial weight of the sample, M t The values ​​represent the weights of the samples after swelling for 1, 2, 4, 8, 12, and 24 hours, respectively.

[0057] The results are as follows Figure 4 As shown, all hydrogels exhibited rapid initial swelling within the first 2 hours, which gradually slowed down over 2 to 8 hours. By 8 hours, the swelling of all hydrogels tended to reach equilibrium. The pure gelatin hydrogel (1G0P) showed the highest equilibrium swelling ratio (703.15 ± 25.67%). In contrast, the pure PPI hydrogel (0G1P) had the lowest swelling capacity (355.52 ± 14.53%).

[0058] For composite hydrogels, the swelling ratio gradually decreases with increasing PPI content, specifically: 2G1P (632.85±37.74%) > 1G1P (450.10±19.40%) > 1G2P (446.56±33.85%) > 1G3P (405.90±17.18%). This trend indicates that introducing gelatin into the PPI matrix can enhance the crosslinking density and reduce the free volume in the hydrogel network, thereby limiting water absorption capacity.

[0059] To comprehensively evaluate the swelling process of hydrogels, this invention employs a second-order kinetic model proposed by Schott to analyze their swelling behavior parameters. The results show that the dynamic swelling behavior of all hydrogels in PBS buffer conforms to Schott's second-order kinetic model.

[0060] Example 5: Moisture content and water holding capacity 1. Moisture content Water content is defined as the percentage of water in a hydrogel, and is measured by the weight change of the gel before and after freeze-drying. Water content is calculated using the following formula: Wherein, W0 and W correspond to the initial weight and freeze-dried weight of the hydrogel, respectively.

[0061] 2. Water holding capacity The water-holding capacity (WHC) of the hydrogel was determined using a gravimetric method. Approximately 3g of sample was weighed and placed in a 50mL centrifuge tube. The tube was centrifuged at 10,000×g for 15 minutes at 25°C, and the separated water was removed using filter paper. The water-holding capacity can be calculated using the following formula: Where W0 and W represent the weights of the hydrogel before and after centrifugation, respectively.

[0062] Water content is a crucial characteristic of hydrogels, directly affecting their performance and applications. For example... Figure 5 As shown, the water content of different hydrogels all reached over 80%, indicating that almost all water molecules participated in the formation of the network.

[0063] The water-holding capacity (WHC) of a hydrogel reflects the interactions between the polymer and the solvent, as well as between the polymer itself. These interactions are closely related to the mechanical behavior of the gel and influence its texture and sensory properties. Figure 5 The pure PPI hydrogel (0G1P) exhibited the lowest water content (WHC) of all samples. In contrast, all samples with added gelatin achieved relatively high WHC. The introduction of gelatin enhanced the overall network stability, allowing the gel to retain more water.

[0064] Example 6: Mechanical Properties 1. Gel strength The hydrogel used for gel strength measurement was prepared in a 10 mL beaker. Gel strength was measured at 25°C using a TA-XT Plus texture analyzer (TA.XT Plus990000, UK) equipped with a cylindrical probe (P / 0.5 probe with a diameter of 12.7 mm). The probe speed was 2.0 mm / s before testing, 5.0 mm / s during testing, and 5.0 mm / s after testing. The trigger force was 5 g. The maximum force (g) was recorded when the probe penetrated 40% of the hydrogel sample, and each sample was tested three times.

[0065] like Figure 6 As shown, the gel strength also varies significantly with the ratio of gelatin to PPI. The pure gelatin sample (1G0P) exhibits the second highest gel strength after 2G1P. However, the gel strength gradually decreases as the proportion of PPI in the composite system increases. In contrast, PPI may have fewer accessible cross-linking sites or form less ordered structures, thus hindering the formation of a coherent gel matrix when used alone or added in high proportions. Furthermore, the competitive reaction of the two proteins to the enzyme may further limit the cross-linking efficiency, resulting in a weaker gel structure with increasing PPI content. Notably, the pure PPI sample (0G1P) shows the lowest gel strength, indicating that PPI alone cannot form a strong hydrogel under the same cross-linking conditions, while the introduction of gelatin can significantly improve the mechanical strength of the hydrogel.

[0066] 2. Texture properties The textural properties of the samples were investigated using a TA-XT Plus texture analyzer (TA.XT Plus990000, UK). The analyzer was equipped with a 50 kg force sensor, and a p / 36R probe was used for measurements. The hydrogel was removed from the beaker and allowed to stand at 25°C for 0.5 h. Two compressions were performed at a pre-compression speed of 2 mm / s, a test speed of 2 mm / s, and a post-compression speed of 2 mm / s, with a 5-second interval between measurements. The trigger force for each compression was set to 5.0 g, and the compressive strain was 40%. Each sample was tested three times. Gel hardness was defined as the maximum force generated by the gel during the initial compression.

[0067] Table 1 As shown in Table 1, the 1G0P hydrogel exhibited the highest hardness value, while the hardness gradually decreased with the increase of PPI proportion in the composite system, indicating that the gel structure was disrupted. Theoretically, gelatin forms a thermally reversible triple helix network upon cooling due to hydrogen bonding. In contrast, PPI gels typically exhibit a more disordered and brittle structure due to thermally induced aggregation and disulfide bond formation. However, steric hindrance caused by ε-(γ-Glu)-Lys isopeptide bond formation may hinder the rearrangement and renaturation of irregular gel chains, ultimately affecting the order and stability of the three-dimensional network structure. Furthermore, differences in intramolecular or intermolecular covalent bond activity are considered another important reason for the differences in gel structure properties. Elasticity is a measurement of compression, indicating the speed of structural recovery, while chewiness and resilience reflect the uniformity of the gel network, and cohesion represents the strength of internal bonds. Notably, elasticity, cohesion, chewiness, and resilience all decrease with increasing PPI content. This indicates that the degree of recovery of the gel after yielding to external force gradually decreases, while hydrogels 1G0P and 2G1P exhibit better chewiness due to the combined effect of hardness and elasticity, which means improved gel performance.

[0068] 3. Anti-fatigue properties A rectangular hydrogel sample measuring 25 mm in length, width, and height (15 mm) was placed on the lower plate and compressed by the upper plate using a cylindrical probe (P / 36R) at speeds of 2.0 mm / s before testing, 1.0 mm / s during testing, and 2.0 mm / s after testing. The trigger force was 5 g. The compression distance of the sample was 6 mm, and the compressive strain was 40%. The sample was cyclically compressed 8 times. The strain of the hydrogel was estimated as the length change related to the initial length of the sample, and the stress was calculated by dividing the force by the initial cross-sectional area of ​​the hydrogel sample. Gel strength measurements and compression tests were performed at 25 °C.

[0069] like Figure 7 As shown, all hydrogels could easily withstand eight compression cycles and exhibited high resilience and rapid recovery behavior, indicating that the hydrogels had good fatigue resistance and shape recovery properties. Figure 7 As can be seen from a-7b, hydrogels 1G0P and 2G1P exhibit the highest compressive stress compared to other hydrogels, and recover the most completely after each cycle, indicating that they have excellent elastic recovery properties and minimal permanent deformation.

[0070] It is worth noting that, such as Figure 7 As shown in d-7f, hydrogels 1G2P, 1G3P, and 0G1P exhibited significant plastic deformation during compression testing, which delayed the increase in stress during subsequent cycles, accompanied by a gradual decrease in compressive stress. From... Figure 7As can be seen from bc, 2G1P and 1G1P did not experience significant stress reduction during compression, indicating their excellent shape recovery properties. Furthermore, the pure PPI hydrogel (0G1P) exhibited the lowest compressive stress, suggesting that its network structure was more brittle and less tough.

[0071] Example 7: X-ray diffraction The crystal structure of the lyophilized hydrogel powder was analyzed using an X-ray diffractometer (D8 Advance, Bruker GmbH, Germany) with Cu-Kα radiation (λ = 1.54 Å). The measurement conditions were 40 kV, 40 mA, scan range 5–60° (2θ), and scan rate 2 / min.

[0072] To better understand the formation mechanism of hydrogels, the crystal structure of the hydrogels was analyzed using X-ray diffraction. The results are as follows: Figure 8 As shown, pure gelatin hydrogel (1G0P) exhibits a characteristic broad diffraction peak centered at approximately 2θ = 20°, with a weak peak near 2θ = 7°–8°. The weak peak at the lower angle is related to the diameter of the triple helix structure, and its intensity reflects the number of this structure; while the broad diffraction halo at 20° corresponds to the spacing between polypeptide chains in the amorphous region. This confirms the semi-crystalline nature of gelatin—the triple helix structure is disrupted during gelation and drying. Similarly, the pure PPI sample (0G1P) shows two broad diffraction peaks at 2θ = 9° and 19.5°, respectively, which is typical of globular proteins that are mainly amorphous. For the composite hydrogels (2G1P, 1G1P, 1G2P, and 1G3P), the XRD patterns show a smooth transition between the two pure components. All composite samples exhibited broad amorphous halos similar to 1G0P and 0G1P, without the appearance of new sharp peaks. Therefore, the amorphous structure was maintained regardless of the mixing ratio. With increasing PPI ratio, the intensity and position of the main diffraction peak slightly shifted towards the characteristic spectral lines of 0G1P. However, the retention of the overall amorphous state indicates that the incorporation of gelatin did not significantly alter the disordered structural arrangement of the PPI matrix, which plays a positive role in maintaining the swelling capacity and structural uniformity of the hydrogel.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A composite hydrogel combining animal and plant proteins, characterized in that, The network structure of the composite hydrogel is obtained by cross-linking pea protein isolate with gelatin; the gelatin is skin-derived gelatin; the cross-linking includes covalent cross-linking and non-covalent cross-linking; the covalent cross-linking is covalent cross-linking catalyzed by transglutaminase; the non-covalent cross-linking is hydrogen bonding, hydrophobic interaction and electrostatic interaction formed between pea protein isolate and gelatin.

2. The composite hydrogel combining animal and plant proteins as described in claim 1, characterized in that, The composite hydrogel has a porous honeycomb structure.

3. A method for preparing a composite hydrogel combining animal and plant proteins as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) Pea protein isolate and gelatin were dissolved in phosphate buffer to prepare pea protein isolate solution and gelatin solution, respectively; (2) Mix the pea protein isolate solution and the gelatin solution evenly to obtain mixture a; (3) Add transglutaminase to mixture a, stir evenly, and then react in a water bath to obtain mixture b; (4) The mixture b is subjected to enzyme inactivation treatment, cooled and refrigerated to obtain the composite hydrogel of the combination of animal and plant proteins.

4. The preparation method according to claim 3, characterized in that, In step (1), the concentration of pea protein isolate in the pea protein isolate solution is 12%-18% (w / v); the concentration of gelatin in the gelatin solution is 8%-12% (w / v).

5. The preparation method according to claim 3, characterized in that, In step (1), the pea protein isolate is dissolved by stirring at room temperature for 1-3 hours; the gelatin is dissolved by heating at 45-60℃.

6. The preparation method according to claim 4, characterized in that, In step (2), the volume ratio of the pea protein isolate solution to the gelatin solution is pea protein isolate solution: gelatin solution = (1-3): (1-2).

7. The preparation method according to claim 3, characterized in that, In step (2), the amount of transglutaminase added is 10-30 U / g protein.

8. The preparation method according to claim 3, characterized in that, In step (3), the reaction conditions are: constant temperature reaction in a water bath at 45-55℃ for 1.5-3 hours.

9. The preparation method according to claim 3, characterized in that, In step (4), the refrigeration conditions are: refrigerate at 0-4℃ for at least 12 hours.

10. The application of the composite hydrogel combining animal and plant proteins as described in any one of claims 1-2 in the preparation of food and biomaterials.

Images