Enteromorpha insoluble dietary fiber-sodium alginate-cod protein composite gel and preparation method thereof

Abstract

The invention provides complex gel based on enteromorpha prolifera insoluble dietary fiber (IDF), sodium alginate (SA) and cod protein (CP), a preparation method of the complex gel and application of the complex gel in aquatic products. The complex gel comprises the following components in percentage by mass: 0-3% of enteromorpha prolifera IDF, 2.5% of SA, 3% of CP, 2-3% of gluconic acid-delta-lactone (GDL) and the balance of water. By introducing IDF, the mechanical strength, the film forming integrity and the water retention capacity of the composite gel film are improved, so that water loss and quality deterioration of aquatic food in the refrigeration process are inhibited, and the shelf life is prolonged; all-natural degradable materials are adopted, safety and environmental protection are achieved, and the concept of green and sustainable development is met; an optimized gel formula and a preparation process enable the hydrogel to have good mechanical property, structural stability and barrier property, and meanwhile, the protective effect on aquatic food is improved; the preparation process is simplified, the process conditions are mild, the cost is controllable, and the method has large-scale application potential, can be expanded to be used for coating preservation of other aquatic products, fruits and vegetables and other foods besides the penaeus vannamei, and has good industrialization and market application prospects.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a composite gel of insoluble dietary fiber from seaweed, sodium alginate, and cod protein, and its preparation method. Background Technology

[0002] With the increasing emphasis on green and low-carbon strategies, natural hydrogels have attracted widespread attention due to their excellent biocompatibility, biodegradability, and tunable structural properties. These advantages are particularly important in scenarios such as cold chain food preservation, biological sample storage, and cryogenic medical applications. These applications typically require hydrogel materials to possess high structural stability, good freeze-thaw resistance, and food-grade safety. However, traditional hydrogels often rely on energy-intensive processes such as heat treatment, Ca²⁺ ion crosslinking, or chemical coupling for preparation, which may lead to the risk of crosslinking agent residue and make it difficult to precisely control structural properties, thus limiting their further promotion in the food and biomedical fields.

[0003] Cod myofibrillar protein (CP) is the main protein component extracted from the muscle of cod (Gadus morhua), rich in myosin and actin, and belongs to the high molecular weight protein category. The CP molecule contains various functional groups such as amino, carboxyl, and hydroxyl groups, giving it certain water-retention, gelling, and emulsifying properties. However, due to its low disulfide bond content, it often exhibits insufficient gel strength and poor structural integrity under freeze-thaw conditions. CP can form a gel network structure with insoluble dietary fiber (IDF) through physical entanglement and hydrogen bonding, but this binary system is still prone to structural collapse and water loss during low-temperature storage, resulting in limited long-term functional stability. Sodium alginate (SA) is a natural anionic polysaccharide that can form a gel by protonating the carboxylate groups through pH induction. Although the CP–SA binary system can form a gel, it is also prone to structural collapse and water loss under refrigeration conditions, indicating that its network strength is still insufficient. Therefore, it is necessary to construct a ternary reinforcement mechanism to improve the structural robustness and environmental adaptability of this type of hydrogel.

[0004] Existing research on protein-polysaccharide hydrogels largely focuses on protein systems requiring heat denaturation, such as whey protein or gelatin, and typically relies on thermal gelation or ionic gelation methods. These pathways are not suitable for low-temperature systems. Currently, in the field of cryopreservation, there is still a lack of ternary hydrogel systems that do not require external cross-linking agents and are formed based on pH triggering. In particular, research on introducing IDF as a natural activity-enhancing component into ternary systems to improve low-temperature performance remains relatively insufficient.

[0005] This invention proposes a green gelation strategy based on "pH-induced – IDF co-assembly" to construct a ternary hydrogel system composed of CP, SA, and IDF derived from Ulva prolifera. By triggering electrostatic assembly between proteins and polysaccharides through pH regulation, and utilizing the hydrogen bonding and space-filling effects provided by IDF to enhance the physical network structure, a green, safe, and high-performance composite gel is constructed. This gel can be used in cold chain food and biological preservation applications, and promotes the high-value utilization of cod by-products and Ulva prolifera resources. Summary of the Invention

[0006] The first aspect of the present invention is to provide a composite gel based on insoluble dietary fiber (IDF) from sea lettuce, sodium alginate (SA), and cod protein (CP).

[0007] The second aspect of this invention aims to provide a method for preparing a composite gel based on insoluble dietary fiber (IDF) from sea lettuce, sodium alginate (SA), and cod protein (CP).

[0008] The third aspect of this invention aims to provide the application of a composite gel based on insoluble dietary fiber (IDF) from seaweed (Ulva prolifera), sodium alginate (SA), and cod protein (CP) in the preservation of aquatic products.

[0009] The fourth aspect of this invention aims to provide a method for preserving Pacific white shrimp.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] In a first aspect, the present invention provides a composite gel based on insoluble dietary fiber (IDF) from sea lettuce, sodium alginate (SA), and cod protein (CP), characterized in that it comprises the following components by mass percentage: 0-3% insoluble dietary fiber (IDF) from sea lettuce; 2.5% sodium alginate (SA); 3% cod protein (CP); 2-3% glucono-δ-lactone (GDL); and the balance being water.

[0012] In some embodiments of the present invention, the mass percentage of the added insoluble dietary fiber from *Ulva prolifera* is 1-3%; in some embodiments of the present invention, the mass percentage of the added insoluble dietary fiber from *Ulva prolifera* is 1-2%; and in some embodiments of the present invention, the mass percentage of the added insoluble dietary fiber from *Ulva prolifera* is 2-3%.

[0013] In some embodiments of the present invention, the mass percentage of the added insoluble dietary fiber from *Ulva prolifera* is 0%; in some embodiments of the present invention, the mass percentage of the added insoluble dietary fiber from *Ulva prolifera* is 1%; in some embodiments of the present invention, the mass percentage of the added insoluble dietary fiber from *Ulva prolifera* is 2%; and in some embodiments of the present invention, the mass percentage of the added insoluble dietary fiber from *Ulva prolifera* is 3%.

[0014] In some embodiments of the present invention, the glucono-δ-lactone has a mass percentage of 2%; in some embodiments of the present invention, the glucono-δ-lactone has a mass percentage of 3%.

[0015] In some embodiments of the present invention, the mass percentage of the added insoluble dietary fiber from *Ulva prolifera* is 2%; the mass percentage of glucono-δ-lactone is 3%; the mass percentage of sodium alginate is 2.5%; and the mass percentage of cod protein isolate is 3%.

[0016] In some embodiments of the present invention, the mass percentage of the added insoluble dietary fiber from *Ulva prolifera* is 3%; the mass percentage of glucono-δ-lactone is 3%; the mass percentage of sodium alginate is 2.5%; and the mass percentage of cod protein isolate is 3%.

[0017] In some embodiments of the present invention, the gel is hydrolyzed and acidified by the gluconate-δ-lactone (GDL) to induce the formation of the composite gel, and no exogenous calcium ion crosslinking agent is added during the gelation process.

[0018] In some embodiments of the present invention, the composite gel is acidified by the stepwise hydrolysis of gluconic acid-δ-lactone (GDL) to produce gluconic acid, thereby inducing the formation of the composite gel.

[0019] A second aspect of the present invention provides a method for preparing a composite gel based on insoluble dietary fiber (IDF) from seaweed (Ulva prolifera), sodium alginate (SA), and cod protein (CP), characterized by comprising the following steps:

[0020] (1) Prepare a sodium alginate aqueous solution with a mass fraction of 2.5% and a cod protein isolate aqueous solution with a mass fraction of 3.0% respectively;

[0021] (2) The sodium alginate aqueous solution prepared in step (1) is mixed with the cod protein isolate aqueous solution at a volume ratio of 3:4 to obtain the sodium alginate-cod protein complex precursor solution.

[0022] (3) Add insoluble dietary fiber from *Ulva prolifera* to the composite precursor solution and mix well;

[0023] (4) Add gluconate-δ-lactone to the mixture obtained in step (3), mix well, and obtain a coating solution;

[0024] (5) The coating solution is incubated at 25 °C to complete gelation, and then placed at 4 °C to stabilize the gel network to obtain the composite gel.

[0025] In some embodiments of the present invention, the preparation of the insoluble dietary fiber of *Ulva prolifera* is as follows: *Ulva prolifera* is washed four times with 30 times (v / w) water, dried at 50 ℃ for 6 h, pulverized to 40 mesh, rinsed with 10 times (v / v) 85% ethanol, mixed for 2 min, repeated three times, and dried overnight at 40 ℃ to obtain desaccharified *Ulva prolifera*; the desaccharified *Ulva prolifera* is taken, 40 times (v / v) 50 mmol / L maleic acid buffer is added, heat-stable amylase is added, and extracted at above 95 ℃ for 35 min to obtain enzymatic hydrolysate 1; enzymatic hydrolysate 1 is taken, amyloglucosidase is added, and enzymatic hydrolysis is carried out at 37 ℃ for 16 h to obtain enzymatic hydrolysate 2; enzymatic hydrolysate 2 is taken, protease is added, and enzymatic hydrolysis is carried out at 60 ℃ for 30 min to obtain enzymatic hydrolysate 3; enzymatic hydrolysate 3 is filtered, the precipitate is washed twice with 70 ℃ hot water, dried at 105 ℃ to constant weight, and pulverized to obtain IDF.

[0026] In some embodiments of the present invention, the sodium alginate-cod protein complex precursor solution is prepared as follows: sodium alginate (SA, 2.5% by mass) and cod protein isolate (CP, 3.0% by mass) solutions are prepared separately with sterile water. Sodium alginate is stirred at 25 °C until completely dissolved, sonicated for 10 min, and then stored at 4 °C. In the cod protein solution, 3 g of CP is dispersed in 100 mL of sterile water and slowly dissolved in 100 mL of sterile water with magnetic stirring at 25 °C. After complete dissolution, the solution is sonicated at room temperature for 10 min to remove air bubbles. After the solution stabilizes, it is stored in a refrigerator at 4 °C. Sodium alginate and cod protein are mixed at a ratio of 3:4 (v / v) to form the sodium alginate-cod protein complex precursor solution.

[0027] In some embodiments of the present invention, the mass ratio of sodium alginate (SA) to cod protein isolate (CP) in step (2) is 5:8.

[0028] In some embodiments of the present invention, the composite gel is prepared as follows: Insoluble dietary fiber (IDF) extracted from *Ulva prolifera* is added at a mass fraction of 1%, 2%, or 3%, and the mixture is stirred for 5 min. Next, glucono-δ-lactone (GDL) is added at a mass fraction of 2% or 3%, and the mixture is stirred for 10 min. The mixture is then incubated at 25 °C for 2 h to gel, and stored at 4 °C overnight to stabilize the gel network, yielding the composite gel.

[0029] A third aspect of the invention provides the application of a composite gel based on insoluble dietary fiber (IDF) from sea lettuce, sodium alginate (SA), and cod protein isolate (CP) in the preservation of aquatic products.

[0030] In some embodiments of the present invention, the aquatic product is shrimp.

[0031] In some embodiments of the present invention, the aquatic product is Litopenaeus vannamei.

[0032] A fourth aspect of the present invention provides a method for preserving whiteleg shrimp, characterized by comprising the following steps:

[0033] (1) Immerse the whiteleg shrimp in the coating solution prepared in step (4) of the method as described in claim 5; (2) Remove the soaked whiteleg shrimp and air dry them to form an edible coating on their surface;

[0034] (3) Store the dried white shrimp in cold storage.

[0035] In some embodiments of the present invention, the storage temperature is 4 °C.

[0036] In some embodiments of the present invention, the freshness indicators of the whiteleg shrimp are tested on days 0, 3, 6, 9, 12, and 15.

[0037] The beneficial effects of the present invention: The composite gel based on insoluble dietary fiber (IDF) of sea lettuce (Ulva prolifera), sodium alginate (SA), and cod protein (CP) provided by the present invention: (1) By introducing insoluble dietary fiber (IDF) of sea lettuce as a structural reinforcing component into the SA–CP system, a more dense and stable three-dimensional network is constructed, which improves the mechanical strength, film integrity and water retention performance of the composite gel membrane, thereby helping to inhibit water loss and quality deterioration of aquatic food during refrigeration and extend shelf life. (2) The system uses food-grade natural raw materials such as IDF, sodium alginate and cod protein, and uses GDL to achieve mild acidification-induced gelation. No exogenous calcium ion crosslinking agent or any chemical crosslinking agent is added. The overall safety is high, it is degradable and has a low environmental burden, which meets the requirements of green, low-carbon and sustainable development. (3) The membrane layer can form a continuous and uniform protective barrier on the food surface, which has a certain structural stability and barrier capacity. It can reduce the impact of external oxygen, water migration and microbial action on product quality, thereby improving the comprehensive protection effect on aquatic food. (4) The preparation process of this invention is simplified, the process conditions are mild and the cost is controllable. It has the potential for large-scale application. In addition to whiteleg shrimp, it can also be used for coating preservation of other aquatic products and fruits and vegetables. It has good prospects for industrialization and market application. Attached Figure Description

[0038] Figure 1 The effect of different IDF addition amounts on the visual appearance of different gels (GDL=2% and GDL=3%).

[0039] Figure 2 The effect of different IDF addition amounts on the water-holding capacity of different gels (GDL=2% and GDL=3%).

[0040] Figure 3 A. Effect of different IDF addition amounts on the storage modulus (G′) of different gels (GDL=2% and GDL=3%); Figure 3 B. The effect of different IDF addition amounts on the loss modulus (G″) of different gels (GDL=2% and GDL=3%).

[0041] Figure 4 Fourier transform infrared spectra of cod protein, sodium alginate and insoluble dietary fiber from seaweed; Figure 4 Fourier transform infrared spectra of GDL=2% gel with different IDF addition amounts; Figure 4 C. Fourier transform infrared spectra of GDL=3% gel with different IDF addition amounts.

[0042] Figure 5 X-ray diffraction patterns of cod protein, sodium alginate and insoluble dietary fiber from seaweed; Figure 5 B. X-ray diffraction patterns of GDL=2% gel with different IDF addition amounts; Figure 5 C. X-ray diffraction patterns of GDL=3% gel with different IDF addition amounts.

[0043] Figure 6 A1 Thermogravimetric analysis (TGA) plot of cod protein, sodium alginate and insoluble dietary fiber from seaweed; Figure 6 B1 Thermogravimetric analysis (TGA) plots of GDL=2% gel with different IDF addition amounts; Figure 6 C1 Thermogravimetric analysis (TGA) plots of GDL=3% gel with different IDF addition amounts; Figure 6 A2 Microthermogravimetric analysis (DTG) plot of cod protein, sodium alginate and insoluble dietary fiber from seaweed; Figure 6 B2. Microthermogravimetric analysis (DTG) plots of GDL=2% gel with different IDF addition amounts; Figure 6 C2. Microthermogravimetric analysis (DTG) plots of GDL=3% gel with different IDF addition amounts.

[0044] Figure 7 The effects of different IDF additions on the morphology of different gels (GDL=2% and GDL=3%) were characterized by SEM.

[0045] Figure 8 Visual appearance of whiteleg shrimp preserved with different gels at 4℃.

[0046] Figure 9 A. The effect of different treatments on the pH value for preserving Litopenaeus vannamei; Figure 9 B. Effects of different treatments on the preservation of volatile amino nitrogen (TVB-N) in Litopenaeus vannamei; Figure 9 C. Effects of different treatments on total TVC in fresh Litopenaeus vannamei; Figure 9 D. Effects of different treatments on the malondialdehyde (TBA) content in fresh-keeping Litopenaeus vannamei; Figure 9 E. Effects of different treatments on the moisture content (MC) of Litopenaeus vannamei; Figure 9 F. Effects of different treatments on the water-holding capacity of Litopenaeus vannamei (whiteleg shrimp).

[0047] Detailed Implementation Methods (Examples)

[0048] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0049] It should be noted that the terms "comprising" and "having" and any variations thereof in the specification, claims and accompanying drawings of this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product or device.

[0050] Ulva prolifera (collected from the Yellow Sea coast of China, coordinates: [120°52', 35°37')) served as a source of insoluble dietary fiber (IDF). Sodium alginate (SA, AR, viscosity: 180-220 mPa·s (1% in water, 25 ℃), M / G ratio: 2:1), D-(+)-gluconic acid-δ-lactone (GDL, AR, purity 99%), and food-grade cod protein isolate (CP, purchased from Xi'an Xuhua Pharmaceutical Co., Ltd., China; this protein is derived from cold-water cod, with a protein content ≥90% (w / w), molecular weight approximately 40–220 kDa, and isoelectric point (pI) of 5.0). Thermoresistant α-amylase (BR, 5000 U / mg), amylase (BR, 300 U / mg), and protease (BR, 300 U / mg) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0051] The full-wavelength microplate reader, texture analyzer, rheometer, infrared spectrometer, scanning electron microscope, and XRD diffractometer were purchased from Thermo Fisher Scientific (China) Co., Ltd., and the thermogravimetric analyzer was purchased from PerkinElmer Instruments Co., Ltd.

[0052] The following are some definitions used in this invention:

[0053] In this invention, "composite gel" refers to a gel system formed by combining insoluble dietary fiber (IDF) from seaweed, sodium alginate (SA), and cod protein (CP) in a certain proportion. This gel is formed through acid coagulation induced by gluconate-δ-lactone (GDL) or calcium ion cross-linking, and has good mechanical strength, water retention, and film-forming properties, and can be used for food encapsulation, coating, and preservation.

[0054] In this invention, "Ulva prolifera insoluble dietary fiber" refers to a water-insoluble dietary fiber component extracted from the green seaweed Ulva prolifera, mainly including cellulose, hemicellulose and a small amount of lignin. It has excellent water absorption and swelling capacity and structural support capacity, and plays a skeletal reinforcement role in gel.

[0055] In this invention, "cod protein isolate" or "cod protein" refers to myofibrillar protein components derived from the muscle tissue of cold-water cod, mainly comprising structural proteins such as myosin and actin, with a protein content of not less than 90% (w / w), a molecular weight of approximately 40–220 kDa, and an isoelectric point (pI) of approximately 5.0. This protein molecule contains functional groups such as amino, carboxyl, and hydroxyl groups, and possesses certain water-retention and gel-forming abilities. In this invention, CP serves as the functional protein matrix of the composite gel, synergistically constructing a gel network with sodium alginate and insoluble dietary fiber from seaweed to improve the structural stability and mechanical properties of the gel membrane.

[0056] In this invention, "gluconic acid-δ-lactone" is a commonly used food-grade acid coagulant. It is used as a pH inducer for gel formation, gradually hydrolyzing to generate gluconic acid, thus lowering the pH of the system and promoting the cross-linking of SA and CP to form a gel.

[0057] In this invention, "coating liquid" refers to a liquid system formed by mixing and homogenizing composite components such as IDF, SA, and CP with water (or other food-grade solvents) in a certain proportion. It can be used to directly immerse food surfaces and form a dense, edible protective film after acidification with GDL.

[0058] In this invention, "mass percentage" refers to the percentage of the mass of a certain component to the total mass of the entire mixture, expressed as "% (w / w)". Unless otherwise stated, all component ratios described in the specification are mass percentages.

[0059] The invention will be further analyzed below with reference to specific examples.

[0060] Example 1: Effects of different IDF addition amounts and different GDL concentrations on the preparation of composite gels

[0061] Weigh 2.5 g of sodium alginate (SA) powder and slowly dissolve it in 100 mL of sterile water with magnetic stirring at 25°C. After complete dissolution, sonicate at room temperature for 10 min to remove air bubbles. After the solution is stable, store it in a refrigerator at 4°C.

[0062] Weigh 3 g of cod protein isolate and slowly dissolve it in 100 mL of sterile water with magnetic stirring at 25 °C. After complete dissolution, sonicate at room temperature for 10 min to remove air bubbles. After the solution is stable, store it in a refrigerator at 4 °C.

[0063] Sodium alginate and cod protein isolate were mixed at a ratio of 3:4 (v / v) to form a binary emulsion. Insoluble dietary fiber (IDF) extracted from *Ulva prolifera* was added at a mass fraction of 1%, 2%, or 3% (with a blank control of 0% IDF addition), and the mixture was stirred for 5 min. Next, glucono-δ-lactone (GDL) was added at a mass fraction of 2% or 3%, and the mixture was stirred for 10 min. The mixture was incubated at 25 °C for 2 h to gel, and then stored at 4 °C overnight to stabilize the gel network.

[0064] Example 2: Effects of different IDF addition amounts and different GDL concentrations on the visual appearance of the composite gel

[0065] like Figure 1 As shown, the morphology of the obtained gels showed a significant dependence on the amounts of both IDF and GDL added. In the system with 2% GDL, the hydrogel without IDF (0%) was opaque and shrunken, with a rough surface and obvious water separation. When 1% IDF was added, the gel formed a more regular shape and the surface became significantly smoother. When IDF was increased to 2%, the gel exhibited the clearest cylindrical appearance in this group, with the best surface smoothness. However, when IDF was further increased to 3%, the gel structure deteriorated, with phenomena such as collapse, surface cracking, and liquid exudation occurring.

[0066] Similar patterns of change were observed in the system with a GDL of 3% (see...). Figure 1However, at the same IDF addition level, its overall structural quality was superior to the system with 2% GDL. Specifically, the control gel (0% IDF) under 3% GDL conditions was more intact than the control gel under 2% GDL conditions; when IDF was 1% and 2%, the gel formation and surface smoothness were both good; notably, at 3% IDF, the gel in the 3% GDL group still maintained structural integrity well, significantly better than the collapsed state of the 2% GDL group under the same IDF conditions, but still had some appearance defects compared to the medium IDF group. The above results indicate that IDF can serve as an effective physical filler component, improving network density and enhancing water retention capacity; its improvement effect is significant in the range of 1%-3% IDF addition, with the most significant improvement in the 1%-2% range. When the IDF addition reaches 3%, the gel structural stability decreases, but by increasing the GDL addition to 3%, the structural deterioration caused by excessive IDF can be compensated to some extent. Therefore, by adjusting the addition levels of IDF (0-3%) and GDL (2-3%), the macroscopic morphology of the gel can be effectively controlled.

[0067] Example 3: Effects of different IDF addition amounts and different GDL concentrations on the colorimetric test of the composite gel.

[0068] Using a portable colorimeter (3nh, SC-10), the gel brightness (L*), red-green value (a*), and yellow-green value (b*) were measured against a white background. Each sample was repeated 9 times.

[0069] As shown in Table 1, when GDL was 2%, the L value first decreased and then slightly increased with the addition of IDF: 41.31±0.89 at 0% IDF, decreasing to 25.02±0.72 at 1% IDF, and then increasing to 28.71±0.46 at 3% IDF, indicating that an appropriate amount of IDF may improve surface uniformity and change light scattering characteristics. In contrast, under the condition of 3% GDL, the L variation range was narrower (21.19±0.41–26.41±0.34), indicating that the gel was denser and had better color stability at higher acidification levels. The a value was generally low: the 2% GDL sample was close to neutral (-0.16±0.10 to 0.63±0.10), while the 2% GDL sample shifted slightly towards the red direction, which is presumably related to the difference in light reflection caused by protein aggregation. The b-value increases with increasing IDF content, reaching a maximum of 10.07±0.78 under 3% GDL conditions, indicating that IDF can enhance the yellow tint of the gel, and this effect is more pronounced under relatively strong acidification conditions.

[0070] Table 1. Effect of different IDF addition amounts on colorimetric tests of different gels (GDL=2% and GDL=3%)

[0071]

[0072] Example 4: Effects of different IDF addition amounts and different GDL concentrations on the water-holding capacity of the composite gel

[0073] Accurately weigh 2 g of each gel stored at 4℃ for 12 h, denoted as M0. Centrifuge at 5000 rpm at 4℃ for 30 min, blot off the released water with filter paper, and weigh again, denoted as M1. Repeat this process 4 times for each sample. The water holding capacity (WHC) is expressed as the percentage of gel mass after centrifugation relative to the initial mass. The formula is as follows:

[0074] WHC (%) = (Mt / M0) × 100

[0075] In the formula: M0 is the mass of the gel before centrifugation (g); Mt is the mass of the gel after centrifugation (g).

[0076] like Figure 2 As shown, the WHC of the CP–SA–IDF hydrogel was significantly increased after the introduction of IDF (P<0.05). Under the condition of 2% GDL, the WHC increased from about 58±0.6% to 80±1.2%; under the condition of 3% GDL, the WHC increased from about 60±0.4% to nearly 84.3±2.1%.

[0077] These results indicate that under the GDL (2-3%) induction conditions, the introduction of IDF generally and significantly enhances the water-holding capacity of the CP-SA gel system, and the water-holding capacity increases with the increase of IDF addition (0-3%). Even at the lowest addition (1%), the water-holding capacity is significantly improved, confirming that IDF plays a positive water-retention role throughout the experimental range.

[0078] Example 5: Effects of different IDF addition amounts and different GDL concentrations on the determination of the texture of the composite gel

[0079] The hardness, adhesiveness, elasticity, cohesiveness, and chewiness of the gel under different conditions were determined using a texture analyzer. The conditions and parameters were as follows: speed 1 mm / s, rotation speed 2 mm / s, compression distance 7 mm, and trigger force 100 N.

[0080] As shown in Table 2, under acid-induced conditions with GDL concentrations of 2% and 3%, the hardness, adhesion, elasticity, cohesiveness, and chewiness of the CP-SA hydrogel significantly improved (P < 0.05) as the IDF addition increased from 0% to 3%, indicating that the introduction of IDF effectively improved the structural strength and stress response of the gel network. Specifically, the 3% GDL group generally showed higher performance in all indicators than the 2% GDL group, suggesting that stronger acid induction is more conducive to protein molecule conformation unfolding, exposure of charged groups, and enhanced interaction with SA / IDF, resulting in a more fully cross-linked and denser gel structure.

[0081] Specifically, the control group without added IDF had generally low texture parameters, and its gel compressive strength and structural recovery ability were limited. When 1% IDF was added, all indicators showed significant improvement, and the gel changed from a weak network state of "easy to collapse / easy to exude water" to a structure with better moldability and more obvious rebound. When IDF was increased to 2%, most indicators continued to rise, showing further enhancement of hardness and chewiness, and simultaneous improvement of elasticity and cohesion. This suggests that at this stage, IDF, as a "skeleton filler + physical entanglement" component, can significantly improve the continuity and load-bearing capacity of the network, enabling the gel to have better rebound and structural retention after compression. Under 3% GDL conditions, the hardness reaches 472.25±2.99 g when IDF is 3%, and the elasticity and chewiness are 1.02±0.06 and 23.93±0.19, respectively, showing stronger compressive strength and chewiness support. At the same time, the adhesion and cohesion also increase with the increase of IDF. For example, under 2% IDF conditions, the adhesion can reach 321.75±7.41 N, and the cohesion reaches 0.89±0.05 when IDF is 3%, indicating that the internal binding of the gel is tighter, the interfacial interaction is enhanced, and the energy dissipation capacity is improved.

[0082] It should be noted that, under the conditions of this implementation, when the IDF increases to 3%, although hardness, elasticity, and chewiness can still maintain a high level, adhesion declines (see Table 2), which is consistent with the localized collapse / cracking observed in the macroscopic appearance (see Table 2). Figure 1 This indicates that excessive fiber may lead to particle aggregation and increased interfacial friction, disrupting the continuous phase structure of the gel matrix and introducing defect points. Under stress, local network failure is more likely to occur, demonstrating the "non-monotonicity" of the IDF enhancement effect. The above data shows that within the IDF (0-3%) and GDL (2-3%) addition range provided by this invention, the hardness, elasticity, cohesiveness, and other textural properties of the composite gel can be effectively improved, showing an overall trend of increasing enhancement with increasing IDF and GDL dosage. This indicates that by adjusting two variables, this invention can achieve gradient design and control of gel mechanical properties over a wide range.

[0083] Table 2. Effects of different IDF addition amounts on gel texture determination of different gels (GDL=2% and GDL=3%).

[0084]

[0085] Example 6: Effects of different IDF addition amounts and different GDL concentrations on the storage modulus (G′) and loss modulus (G″) of the composite gel.

[0086] The rheological properties of the samples were examined using a rheometer. Before testing, a 100 s... -1 Pre-shear at the shear rate for 1 min to remove air bubbles and other factors causing sample inhomogeneity. After pre-shear, the sample was allowed to stand still for 2 min to eliminate residual stress. Frequency scanning conditions: frequency 1 Hz, strain 1%, program-set frequency range 0.1 rad / s - 100 rad / s; viscosity testing conditions: shear rate scan range 1-100 s. -1 .

[0087] like Figure 3 As shown, the addition of IDF significantly enhances the viscoelasticity of the gel system, particularly in the mid-to-high frequency range (>10 rad / s). Taking the 3% IDF group as an example, its storage modulus G′ increases significantly, indicating enhanced gel network rigidity and improved energy storage capacity; simultaneously, the loss modulus G″ also increases, indicating increased intermolecular friction and viscous dissipation, thus strengthening the damping properties. Compared to the 0% IDF control group, which exhibits a slow increase in frequency response and a relatively loose network structure, the hydrogel with added IDF shows higher frequency sensitivity, reflecting a denser internal network and stronger molecular entanglement.

[0088] The rheological results are consistent with the texture and water retention properties. Correlation analysis showed a significant positive correlation between the storage modulus G′ and gel hardness (r = 0.92, p < 0.01) and the water-holding capacity (WHC) (r = 0.88, p < 0.01). These results indicate that as the three-dimensional network becomes more dense and continuous, the elasticity and rigidity of the gel increase (G′ increases), macroscopically manifested as improved resistance to deformation, i.e., increased hardness. Simultaneously, the strengthened network structure, with its stronger capillary binding effect and higher hydrogen bonding capacity, can more effectively trap and fix water molecules, thus leading to an increase in WHC.

[0089] Example 7: Fourier transform infrared spectra of composite gels with different IDF addition amounts and different GDL concentrations.

[0090] Take 5 mg of dried sample and mix it with 100 mg of potassium bromide powder to prepare the sample. Measure the sample using an infrared spectrometer with a resolution of 4 cm⁻¹. -1The scanning range is 4000 to 500 cm. -1 .

[0091] like Figure 4 As shown, all three raw materials—CP, SA, and IDF—can be used at 3280–3300 cm⁻¹. -1 A broad peak was observed at 1605 cm⁻¹, corresponding to the stretching vibration of –OH / –NH, indicating abundant hydrogen bonding sites in the system. SA peaks at 1605 cm⁻¹. -1 With 1418 cm -1 The COO is presented separately at each location. - Characteristic peaks of asymmetric and symmetric stretching vibrations; a typical protein absorption band is visible in the CP spectrum, approximately 1650 cm⁻¹. -1 The region is located at the amide I band (dominated by C=O stretching vibrations), approximately 1540 cm. -1 The region is an amide II band (N–H bending / C–N stretching vibration); IDF is between 1030 and 1070 cm⁻¹. -1 The presence of C–O–C glycosidic bond vibration peaks reflects the structural characteristics of its cellulose skeleton.

[0092] Under the condition of 2% GDL, IDF incorporation into CP-SA gel resulted in multiple band changes: 3280–3300 cm⁻¹. -1 The –OH / –NH broad peak at SA shows a slight red shift and increased intensity, indicating enhanced hydrogen bonding; - The slight shift in characteristic peaks indicates a change in the electrostatic environment. Simultaneously, changes in the shape and intensity of the C–O–C characteristic peaks of IDF suggest that the cellulose microfibrils are not simply physically mixed, but rather participate in the gel network through interfacial interactions. These results demonstrate that IDF can participate in the construction of a three-dimensional network within the system through hydrogen bonding and electrostatic interactions, thereby improving the network crosslinking density and structural stability.

[0093] The spectral changes were more significant at a GDL of 3%: approximately 1730 cm⁻¹ -1 The appearance of a new absorption peak nearby indicates that part of the –COO in SA - Protonation occurs to form –COOH, thereby increasing the number of proton donors available for hydrogen bonding; simultaneously, the amide I and amide II bands of CP undergo more pronounced shifts, indicating a conformational change in the protein induced by strong acid, exposing more polar groups and enhancing its interaction with SA and IDF; furthermore, IDF in the composite gel is at approximately 1040 cm⁻¹ -1 The C–O–C peaks at the point tend to be sharper and / or shift, especially under the condition of 3% IDF, which reflects the enhanced interaction of cellulose components in the network and the acquisition of more stable spatial fixation.

[0094] Example 8: Effects of different IDF addition amounts and different GDL concentrations on X-ray diffraction patterns of the composite gel.

[0095] The effects of pH level and IDF concentration on the binding gel formation and crystallinity were evaluated using X-ray diffraction analysis. The measurement conditions were as follows: a copper target was used in the diffractometer, the scanning speed was 2° / min, and the scanning range was 2θ = 10~80°.

[0096] like Figure 5 As shown, the IDF raw material exhibits a distinct diffraction peak at 2θ≈22°, corresponding to the crystalline region stacking characteristics of cellulose microfibrils; while CP and SA mainly show broad diffuse peaks in the range of 2θ=10°–30°, indicating that their overall structure is mainly amorphous.

[0097] Under the condition of 2% GDL, the characteristic peak of IDF at 2θ≈22° can still be observed in the composite hydrogel, and it is more obvious in the groups with 2% and 3% IDF. However, its peak intensity is significantly lower than that of pure IDF, indicating that the IDF crystalline regions are partially embedded in the gel matrix or the crystal order is disturbed. At the same time, a new weak diffraction signal appears in the composite system, which is speculated to be related to the local short-range ordered arrangement of CP and SA molecular chains induced by mild acidic conditions.

[0098] When GDL increased to 3%, the IDF characteristic peak weakened further, almost disappearing in the 3% IDF group, indicating that the microcrystalline ordered structure was more severely disrupted. Under stronger acid-induced conditions, the conformational changes of the CP molecular chain were more complete, which facilitated the formation of a tighter electrostatic association with SA and promoted the transformation of IDF from its original microcrystalline state to a more amorphous, embedded state. The crystallinity index calculated under the condition of 2% GDL decreased from 15.2% at 0% IDF to 8.7% at 3% IDF, further verifying that the composite network tends to become amorphous.

[0099] Example 9: Thermogravimetric analysis of composite gels with different IDF addition amounts and different GDL concentrations.

[0100] Thermogravimetric analysis was performed using a thermogravimetric analyzer. At room temperature, a precise weight of 10 mg of sample was placed in the thermogravimetric analyzer for measurement. The measurement conditions were as follows: nitrogen flow rate was set to 40 mL / min, heating rate was 20 ℃ / min, and temperature range was 30–600 ℃.

[0101] like Figure 6 As shown, among the single components, IDF has the highest DTG maximum weight loss rate temperature (Tmax), indicating that its thermal stability is better than that of SA and CP. SA and CP exhibit the main weight loss process in the range of about 260–320 °C, which is mainly attributed to the breakage and degradation of the polysaccharide backbone and protein peptide chain.

[0102] Under the condition of 2% GDL, the composite hydrogel exhibits a three-stage thermal degradation characteristic: the first stage is water evaporation (<150 °C), the second stage is the main degradation zone of organic components (approximately 220–340 °C), and the third stage is the slow degradation of carbonized residues (>400 °C). With the increase of IDF content, the onset temperature (Tonset) and Tmax of the samples both shift towards higher temperatures, and the residual mass at 600 °C increases significantly. Among them, the Tmax of the 3% IDF group can reach 317.2 °C, indicating that IDF can improve the heat resistance of the system through its rigid framework and enhanced hydrogen bond network.

[0103] When the GDL content was increased to 3%, the thermal stability of the composite hydrogel was further enhanced. Taking the 3% IDF group as an example, its Tmax increased to 328.6 °C, and the residual mass at 600 °C was close to 30%. At the same time, the DTG peak shifted to the right and the peak intensity decreased, indicating that the thermal decomposition process was delayed and the thermal reactivity was reduced. These changes indicate that under stronger acid-induced conditions, the CP conformational change was more complete and formed stronger electrostatic association with SA, while IDF further strengthened the network structure through hydroxyl-mediated entanglement / action, resulting in a denser three-dimensional cross-linked network.

[0104] In summary, the introduction of IDF and the increase in GDL dosage both improve the thermal stability of the composite gel. Throughout the entire study range, with IDF additions of 0-3% and GDL concentrations of 2-3%, the thermal decomposition temperature of the gel was higher than that of the control group without IDF, demonstrating that this ternary system exhibits superior structural robustness across a wide formulation range.

[0105] Example 10: Effects of different IDF addition amounts and different GDL concentrations on the SEM microstructure of the composite gel.

[0106] After freeze-drying the gels under different conditions, they were sputter-coated with gold and then observed using a scanning electron microscope at 300x magnification.

[0107] like Figure 7As shown, without the addition of IDF, the hydrogel exhibits a porous structure with large, irregular, and unevenly distributed pores. The pore walls are thin, and local collapse or breakage is observed, indicating that the gel network structure is relatively loose and lacks stability. With increasing IDF addition, the gel microstructure gradually transforms into a more dense and continuous honeycomb porous structure, with reduced pore size, thickened pore walls, and enhanced network connectivity. These results indicate that, in addition to its physical filling effect, IDF's surface hydroxyl and carboxyl groups and other active groups participate in network construction through hydrogen bonding and electrostatic interactions, promoting the entanglement and co-assembly of protein and polysaccharide molecular chains, thereby improving the regularity, spatial closure, and overall stability of the network structure. When the IDF addition is 2–3%, the pore wall strength and overall connectivity of the gel are further improved, which can, to some extent, inhibit network collapse and water separation.

[0108] Example 11: Effects of different IDF addition amounts and different GDL concentrations on the appearance of composite gel for preserving Litopenaeus vannamei.

[0109] like Figure 8 As shown, the blank group exhibited surface dehydration and browning from day 6; by day 12, obvious reddish-brown putrefaction spots appeared on the sample surface; by day 15, the entire sample was dark brown, softened, and showed signs of severe spoilage. The CP-0 group (protein coating without IDF) showed some improvement in appearance during the early storage period (days 6–9), but the color darkened and elasticity decreased significantly after day 9; by day 15, significant browning and surface collapse were observed, indicating that the structural protection of the single protein coating was limited. In contrast, the CP–IDF group maintained the natural color, surface morphology, and overall structural integrity of the samples better throughout the storage period; even on day 15, only slight fading and edge darkening were observed, with no obvious putrefaction spots, and the sensory appearance quality was significantly better than the previous two groups. These results indicate that the introduction of IDF can significantly improve the structural stability and preservation effect of the CP coating.

[0110] Example 12: Effects of different IDF addition amounts and different GDL concentrations on the preservation index of composite gel for Litopenaeus vannamei.

[0111] Figure 9The pH values ​​of all samples in the A–F series showed a slight decrease in the first 3 days (initially approximately 6.92±0.02), presumably related to the production of acidic substances from glycogen degradation. Subsequently, the pH gradually increased, with the blank group showing the most significant increase, reaching 7.84±0.04 by day 15; the CP-0 group followed (7.62±0.02); and the CP–IDF group showed the smallest increase (7.44±0.03). These results indicate that coating treatment can delay the accumulation of alkaline metabolites, and the introduction of IDF further reduces membrane permeability and slows down internal gas diffusion, thus having a stronger inhibitory effect on pH rise.

[0112] like Figure 9 As shown in Figure B, the total volatile basic nitrogen (TVB-N) increased overall with prolonged storage time, indicating a gradual increase in protein degradation. On day 15, the TVB-N in the control group reached 78.65 ± 4.5 mg / 100 g, significantly exceeding the spoilage threshold; the TVB-N in the CP-0 group was 33.94 ± 4.0 mg / 100 g; and the TVB-N in the CP–IDF group was 41.62 ± 3.7 mg / 100 g. Notably, while the TVB-N value in the CP-0 group was lower than that in the CP–IDF group, the CP–IDF group showed less fluctuation and higher stability. This suggests that the adsorption and buffering effects of IDF may have a certain "retention / fixation" effect on nitrogen-containing volatile substances, reducing their release rate and making the measured values ​​more stable. In the relatively loosely structured CP-0 membrane, TVB-N may be more easily volatilized or migrated, leading to an underestimation at the measurement level. Therefore, a single indicator may not be sufficient to comprehensively reflect the overall preservation effect.

[0113] To avoid bias caused by a single indicator and to comprehensively evaluate preservation performance, this study used the Combined Freshness Index (CFI) to normalize and average the six key indicators (pH, TVB-N, TVC, MDA, total moisture content, and WHC) on day 15, obtaining a comprehensive score. The results showed that the CFI of the CP-IDF group was 0.82±0.01, significantly higher than that of the CP-0 group (0.71±0.02) and the control group (0.49±0.04) (p<0.05), indicating that the CP-IDF coating has a more balanced and overall superior preservation effect in maintaining multidimensional quality. Even though CP-0 performed lower in TVB-N, this advantage was insufficient to offset its overall disadvantages in microbial control, oxidative stability, and water retention.

[0114] like Figure 9As shown in Figure C, the total colony count (TVC) reflects the microbial proliferation trend. The initial colony counts in all groups were below 3 ± 0.3 log CFU / g; the control group exceeded 7 ± 0.5 log CFU / g on day 6 and reached approximately 9 ± 0.3 log CFU / g on day 15; the CP-0 group had 7.6 ± 0.4 log CFU / g on day 15; and the CP–IDF group had a significantly lower count of 6.9 ± 0.4 log CFU / g (p < 0.05). These results indicate that the protein-based coating can form a certain physical barrier, and the introduction of IDF may further inhibit rapid microbial proliferation by reducing water migration and local water activity, adsorbing metabolites, and constructing a denser and more stable membrane microenvironment.

[0115] like Figure 9 As shown in Figure D, malondialdehyde (MDA) content was used to characterize lipid oxidation levels. In the control group, MDA increased from an initial 0.59 to 2.10 ± 0.04 nmol / mg on day 15; in the CP-0 group, it was 1.68 ± 0.04 nmol / mg; and in the CP–IDF group, the increase was the smallest, only 1.43 ± 0.03 nmol / mg. These results indicate that, in addition to enhancing structure, the addition of IDF may also synergistically reduce lipid oxidation by blocking oxygen diffusion and providing a certain degree of free radical scavenging / adsorption, thereby improving flavor and quality stability.

[0116] like Figure 9 E and Figure 9 As shown in Figure F, the total water content and water-holding capacity (WHC) of the blank group decreased most significantly, dropping to 73.4±0.4% and 72.6±0.3%, respectively, on day 15; while the CP-IDF group maintained higher levels, at 75.1±0.4% and 74.3±0.4%, respectively. These results indicate that the CP-IDF coating can effectively slow down water loss and improve bound water retention. This can be attributed to the fact that the porous structure of IDF adsorbs, traps, and fixes free water; simultaneously, the improved integrity and density of the composite membrane help reduce cell collapse and water leakage, thus achieving better water retention stability.

[0117] In summary, compared with the uncoated control group, the CP-SA binary gel coating (IDF=0%) already provided a significant preservation effect. Furthermore, the ternary composite gel coating formed by introducing IDF further enhances the overall preservation performance. The CP-IDF composite coating exhibits stronger comprehensive preservation capabilities in pH control, nitrogen metabolism regulation, antibacterial activity, antioxidant properties, and water retention. Its mechanism of action can be summarized as follows: the CP base film layer provides a basic physical barrier; the introduction of IDF enhances network density and microenvironment stability; and non-covalent interactions effectively regulate the migration of moisture and small molecules, thereby achieving green, safe, and efficient preservation of high-moisture-content aquatic products under refrigeration conditions.

Claims

1. A composite gel based on insoluble dietary fiber (IDF) from seaweed (Ulva prolifera), sodium alginate (SA), and cod protein (CP), characterized in that, The product comprises the following components by weight percentage: 0-3% insoluble dietary fiber (IDF) from seaweed; 2.5% sodium alginate (SA); 3% cod protein isolate (CP); 2-3% glucono-δ-lactone (GDL); and the balance being water.

2. The composite gel according to claim 1, characterized in that, The composite gel is formed by hydrolysis and acidification of gluconate-δ-lactone (GDL), and no exogenous calcium ion crosslinking agent is added during the gelation process.

3. The composite gel according to claim 1 or 2, characterized in that, The mass percentage of the IDF is 1-3%.

4. The composite gel according to claim 3, characterized in that, The mass percentage of the IDF is 2%.

5. A method for preparing the composite gel according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Prepare a sodium alginate aqueous solution with a mass fraction of 2.5% and a cod protein isolate aqueous solution with a mass fraction of 3.0% respectively; (2) The sodium alginate aqueous solution prepared in step (1) is mixed with the cod protein isolate aqueous solution at a volume ratio of 3:4 to obtain the sodium alginate-cod protein complex precursor solution. (3) Add insoluble dietary fiber from *Ulva prolifera* to the composite precursor solution and mix well; (4) Add gluconate-δ-lactone to the mixture obtained in step (3), mix well, and obtain a coating solution; (5) The coating solution is incubated at 25 °C to complete gelation, and then placed at 4 °C to stabilize the gel network to obtain the composite gel.

6. The method according to claim 5, characterized in that, In step (2), the mass ratio of sodium alginate (SA) to cod protein (CP) is 5:

8.

7. The application of the composite gel as described in any one of claims 1-4 in the preservation of aquatic products.

8. The application according to claim 7, characterized in that, The aquatic product in question is Litopenaeus vannamei.

9. A method for preserving fresh Litopenaeus vannamei, characterized in that, Includes the following steps: (1) Immerse the whiteleg shrimp in the coating solution prepared in step (4) of the method as described in claim 5; (2) Take out the immersed whiteleg shrimp and air dry them to form an edible coating on their surface; (3) Store the dried white shrimp in cold storage.

10. The preservation method according to claim 9, characterized in that, The storage temperature is 4°C.

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