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

Abstract

The invention provides composite gel based on enteromorpha prolifera insoluble dietary fiber (IDF), sodium alginate (SA) and euphausia superba protein (AKP), a preparation method of the composite gel and application of the composite gel in fruit and vegetable preservation. The composite gel is prepared from the following components in percentage by mass: 0 to 3 percent of enteromorpha prolifera IDF, 2.5 percent of SA, 4 percent of AKP, 1 to 2 percent of gluconic acid-delta-lactone (GDL) and the balance of water. Through synergistic crosslinking of enteromorpha IDF, SA and AKP, gel network compactness and film forming stability are improved, gentle acidification gel forming is realized by means of GDL, no exogenous calcium ions and chemical crosslinking agents are added in the whole process, and the enteromorpha IDF gel is green, safe and edible. The coating liquid is used for preservation of strawberries and can effectively lock water and block microorganisms and oxygen under the conditions that the temperature is 24-26 DEG C and the relative humidity is 75-85%, after the strawberries are stored for 8 days, the rot index of the strawberries is smaller than or equal to 65%, the weight loss rate is smaller than or equal to 13%, and quality deterioration is remarkably delayed. The method is simple and convenient in process and controllable in cost, can be adapted to berry fruits and vegetables such as blueberries, cherries and tomatoes besides strawberries, and has good industrial application prospects and environmental protection benefits.

Description

Technical Field

[0001] This invention relates to the field of food preservation and edible packaging materials technology, specifically to a preparation method and application of a composite gel based on seaweed insoluble dietary fiber-sodium alginate-Antarctic krill protein. Background Technology

[0002] Hydrogels are a class of three-dimensional hydrophilic polymer network materials that have been widely used in food packaging, biomedical carriers, and controlled release of active substances. As food-grade hydrogel materials, they typically need to meet requirements such as edibility, biocompatibility, structural stability, and a certain degree of functional responsiveness.

[0003] Antarctic krill protein (AKP) possesses good solubility, high amino acid activity, low allergenicity, and emulsifying, film-forming, and antioxidant potential, making it a suitable protein source for constructing composite hydrogels. Sodium alginate (SA) is a natural anionic polysaccharide that can form an ionic cross-linked network structure in the presence of Ca²⁺ and is often combined with proteins for hydrogel construction. However, the research and application of AKP–SA binary hydrogels face the following technical bottlenecks: 1. Gel construction strategies largely rely on traditional ionic cross-linking, raising concerns about environmental friendliness and safety. This is especially true for sodium alginate-based materials, which primarily use calcium ion (Ca²⁺) cross-linking, resulting in complex processes and the introduction of additional reagents or stringent conditions. Green and mild gelation methods that do not require chemical cross-linking agents still need to be developed; 2. The mechanical properties and long-term preservation effects of existing gel systems are still unsatisfactory. Existing SA-AKP binary composite gels often suffer from loose network structures, insufficient mechanical strength, and limited water-holding capacity. This directly results in poor adhesion of the coating to the surface of fruits and vegetables, easy cracking, and insufficient barrier properties against water vapor and oxygen, thus limiting its practical application effect.

[0004] To overcome the aforementioned shortcomings, this invention attempts to introduce insoluble dietary fiber (IDF) into a protein-polysaccharide binary system. IDF, due to its rigid structure and abundant functional groups, is considered an ideal candidate material for physical reinforcement. However, its mechanism of action as a major structural component or crosslinking / template component remains unclear, and its systematic integration within the gel network is still poorly studied, especially regarding the role of IDF derived from *Ulva prolifera* as a core component in gel construction. Therefore, there is an urgent need to develop a novel composite gel system based on all-natural components that can construct an edible coating with excellent mechanical properties and preservation functions without relying on chemical crosslinking agents, thereby meeting the market's urgent demand for high-performance, green preservation materials. Summary of the Invention

[0005] The first aspect of the present invention is to provide a composite gel based on insoluble dietary fiber (IDF) from seaweed, sodium alginate (SA), and Antarctic krill protein (AKP).

[0006] The second aspect of this invention aims to provide a method for preparing a composite gel based on insoluble dietary fiber (IDF) from seaweed, sodium alginate (SA), and Antarctic krill protein (AKP).

[0007] The third aspect of this invention aims to provide the application of a composite gel based on insoluble dietary fiber (IDF) from sea lettuce, sodium alginate (SA), and Antarctic krill protein (AKP) in the preservation of fruits and vegetables.

[0008] The fourth aspect of this invention aims to provide a method for preserving strawberries.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] In a first aspect, the present invention provides a composite gel based on insoluble dietary fiber (IDF) from seaweed, sodium alginate (SA), and Antarctic krill protein (AKP), characterized in that it comprises the following components by mass percentage: 0-3% insoluble dietary fiber (IDF) from seaweed; 2.5% sodium alginate (SA); 4% Antarctic krill protein isolate (AKP); 1-2% glucono-δ-lactone (GDL); and the balance being water.

[0011] 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 2-3%.

[0012] 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%.

[0013] In some embodiments of the present invention, the glucono-δ-lactone has a mass percentage of 1%; in some embodiments of the present invention, the glucono-δ-lactone has a mass percentage of 2%.

[0014] In some embodiments of the present invention, the composite gel is formed by hydrolysis and acidification of the gluconate-δ-lactone (GDL), and no exogenous calcium ion crosslinking agent is added during the gelation process.

[0015] In some embodiments of the present invention, the composite gel is acidified by gradually hydrolyzing gluconic acid-δ-lactone (GDL) to produce gluconic acid, thereby inducing the formation of the composite gel.

[0016] 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 Antarctic krill protein (AKP), characterized by comprising the following steps:

[0017] (1) Prepare a sodium alginate aqueous solution with a mass fraction of 2.5% and an Antarctic krill protein isolate aqueous solution with a mass fraction of 4.0% respectively; (2) Mix the sodium alginate aqueous solution prepared in step (1) with the Antarctic krill protein isolate aqueous solution at a volume ratio of 3:4 to obtain a sodium alginate-protein complex precursor solution;

[0018] (3) Add insoluble dietary fiber from *Ulva prolifera* to the composite precursor solution and mix well;

[0019] (4) Add gluconate-δ-lactone to the mixture obtained in step (3) and mix well to obtain a coating solution;

[0020] (5) The coating solution is incubated at 20-30°C to complete gelation, and then placed at 2-8°C to stabilize the gel network to obtain the composite gel.

[0021] In some embodiments of the present invention, in step (2), the mass ratio of sodium alginate (SA) to Antarctic krill protein (AKP) is 5:8.

[0022] 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, washed 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, and 40 times (v / v) 50 mmol / L maleic acid buffer is added, along with heat-stable amylase, and extracted at above 95 ℃ for 35 min to obtain enzymatic hydrolysate 1; enzymatic hydrolysate 1 is taken, and amyloglucosidase is added for enzymatic hydrolysis at 37 ℃ for 16 h to obtain enzymatic hydrolysate 2; enzymatic hydrolysate 2 is taken, and protease is added, and enzymatic hydrolysis 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.

[0023] In some embodiments of the present invention, the sodium alginate-protein complex precursor solution is prepared as follows: sodium alginate (SA, 2.5% by mass) and Antarctic krill protein isolate (AKP, 4.0% 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 Antarctic krill protein solution, 16 g of AKP is dispersed in 400 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 Antarctic krill protein are mixed at a ratio of 3:4 (v / v) to obtain the sodium alginate-protein complex precursor solution.

[0024] In some embodiments of the present invention, the mass fraction of the added insoluble dietary fiber (IDF) extracted from Ulva prolifera is 1%, 2% or 3%, and the mixture is stirred for 5 min.

[0025] In some embodiments of the present invention, the coating solution is added with a mass fraction of 1% or 2% glucono-δ-lactone (GDL) and stirred for 10 min.

[0026] In some embodiments of the present invention, 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.

[0027] A third aspect of the present invention provides the application of a composite gel based on insoluble dietary fiber (IDF) from sea lettuce, sodium alginate (SA), and Antarctic krill protein (AKP) in the preservation of fruits and vegetables.

[0028] In some embodiments of the present invention, the fruits and vegetables are berries such as strawberries, blueberries, and cherry tomatoes.

[0029] A fourth aspect of the present invention provides a method for preserving strawberries, characterized by comprising the following steps:

[0030] (1) Immerse strawberries in the coating solution prepared in step (4) of the method; (2) Take out the immersed strawberries and air dry them to form an edible coating on their surface; (3) Store the strawberries treated in step (2).

[0031] In some embodiments of the present invention, the storage conditions are: temperature 24-26°C, relative humidity 75-85%.

[0032] In some embodiments of the present invention, the storage temperature is 24°C; in some embodiments of the present invention, the storage temperature is 25°C; in some embodiments of the present invention, the storage temperature is 26°C.

[0033] In some embodiments of the present invention, the relative humidity of the storage is 75%; in some embodiments of the present invention, the relative humidity of the storage is 80%; in some embodiments of the present invention, the relative humidity of the storage is 85%.

[0034] In some embodiments of the present invention, the food is stored in an environment with a temperature of 25°C and a relative humidity of 80±5%, and the preservation index is tested on days 0, 2, 4, 6, and 8.

[0035] The beneficial effects of the present invention are as follows: (1) Enhanced structure and improved film stability: By introducing insoluble dietary fiber (IDF) of Ulva prolifera into the SA–AKP system as a physical reinforcement and cross-linking synergistic component, the composite gel network can be made denser and more continuous, improving the integrity and structural stability of the coating film, thereby improving the adhesion and coverage effect of the coating on the strawberry surface. (2) Significant water retention and water loss inhibition effect: The porous structure and hydrophilic functional groups of IDF, together with the synergistic effect of SA and AKP, can enhance the system's ability to retain water and reduce the water migration rate, so that strawberries can effectively slow down water loss and shrinkage under storage conditions of 25℃ and 80% relative humidity, and maintain appearance and texture quality. (3) Barrier and microenvironment regulation effect: The gel film formed can build a continuous barrier on the fruit surface, reduce oxygen and water vapor exchange and play a certain barrier role against the invasion of external microorganisms, thereby delaying the browning, rotting and other quality deterioration of strawberries during storage and improving shelf life stability. (4) Green, safe and mild process: This invention uses food-grade natural raw materials (IDF, SA, AKP) and glucono-δ-lactone (GDL) to achieve mild acidification and induce gelation. No Ca²⁺ crosslinking agent or chemical crosslinking agent needs to be added during the preparation process. The system is edible and biodegradable, and has higher safety and environmental friendliness. (5) Simple process, controllable cost and wide applicability: The preparation process is simple and the conditions are easy to achieve, and it has the potential for large-scale application. In addition to strawberries, it can also be promoted for surface coating preservation of berries such as blueberries and cherry tomatoes and other fruits and vegetables, and has good prospects for industrial application. Attached Figure Description

[0036] Figure 1 The effect of different IDF addition amounts on the visual appearance of different gels (GDL=1% and GDL=2%).

[0037] Figure 2 The effect of different IDF addition amounts on the water-holding capacity of different gels (GDL=1% and GDL=2%).

[0038] Figure 3 A. Effect of different IDF addition amounts on the storage modulus (G′) of different gels (GDL=1% and GDL=2%); Figure 3B. The effect of different IDF addition amounts on the loss modulus (G″) of different gels (GDL=1% and GDL=2%).

[0039] Figure 4 A Fourier transform infrared spectra of Antarctic krill protein, sodium alginate, and insoluble dietary fiber from seaweed. Figure 4 B. Fourier transform infrared spectra of GDL=1% gel with different IDF addition amounts; Figure 4 C. Fourier transform infrared spectra of GDL=1% gel with different IDF addition amounts.

[0040] Figure 5 A. X-ray diffraction pattern of Antarctic krill protein, sodium alginate and insoluble dietary fiber from seaweed; Figure 5 B. X-ray diffraction patterns of GDL=1% gel with different IDF addition amounts; Figure 5 C. X-ray diffraction patterns of GDL=1% gel with different IDF addition amounts.

[0041] Figure 6 A1 Thermogravimetric analysis (TGA) plot of Antarctic krill protein, sodium alginate and insoluble dietary fiber from seaweed; Figure 6 Thermogravimetric analysis (TGA) plot of GDL=1% gel with different IDF addition amounts; Figure 6 C1 Thermogravimetric analysis (TGA) plots of GDL=1% gel with different IDF addition amounts; Figure 6 A2 Microthermogravimetric analysis (DTG) plot of Antarctic krill protein, sodium alginate and insoluble dietary fiber from seaweed; Figure 6 B2. Microthermogravimetric analysis (DTG) plots of GDL=1% gel with different IDF addition amounts; Figure 6 Microthermogravimetric analysis (DTG) plots of C2 gels with different IDF addition amounts for GDL=1% gel.

[0042] Figure 7 The effects of different IDF additions on the morphology of different gels (GDL=1% and GDL=2%) were characterized by SEM.

[0043] Figure 8 Visual appearance of strawberries preserved with different gels at 4℃.

[0044] Figure 9 A. The effect of different treatments on the spoilage index of strawberries; Figure 9 B. Effects of different treatments on the weight loss rate of strawberries during preservation; Figure 9 C. Effects of different treatments on titratable acidity in strawberry preservation; Figure 9 D. Effects of different treatments on soluble solids content in strawberry preservation; Figure 9 E. Effects of different treatments on the firmness of strawberries during preservation; Figure 9 F. Effects of different treatments on ascorbic acid in strawberry preservation; Figure 9 G. Effects of different treatments on the total phenol content in strawberry preservation; Figure 9 H. Effects of different treatments on the total phenol content of strawberries during preservation; Figure 9 I. Effects of different treatments on malondialdehyde content in strawberry preservation.

[0045] Detailed Implementation Methods (Examples)

[0046] 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.

[0047] 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.

[0048] 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 Antarctic krill protein isolate (AKP, AR, protein content ≥90%) were also used. Thermoresistant α-amylase (BR, 5000 U / mg), amyloglucosidase (BR, 300 U / mg), and protease (BR, 300 U / mg) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0049] 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.

[0050] The following are some definitions used in this invention:

[0051] In this invention, "composite gel" refers to a gel system formed by combining insoluble dietary fiber (IDF) from seaweed, sodium alginate (SA), and Antarctic krill protein (AKP) in a certain proportion. This gel is formed through acid coagulation cross-linking induced by glucono-δ-lactone (GDL), and has good mechanical strength, water retention, and film-forming properties, and can be used for food encapsulation, coating, and preservation.

[0052] 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.

[0053] In this invention, Antarctic krill is used as the raw material. The protein components obtained through separation and extraction have good solubility and film-forming / gel-forming potential. The protein molecules contain functional groups such as amino, carboxyl, and hydroxyl groups, which can interact with polysaccharide systems. In this invention, AKP serves as the functional protein matrix of the composite gel. It forms a complex network with sodium alginate (SA) under acidification-induced conditions, and with the synergistic enhancement of insoluble dietary fiber (IDF) from seaweed, a more dense and stable three-dimensional structure is constructed. This improves the structural stability, mechanical properties, and water retention properties of the coating, and is beneficial for enhancing the preservation effect of the coating on fruit and vegetable surfaces.

[0054] 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 system pH and promoting the cross-linking of SA and AKP into a gel.

[0055] In this invention, "coating liquid" refers to a liquid system formed by mixing and homogenizing composite components such as IDF, SA, and AKP 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.

[0056] 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.

[0057] The invention will be further analyzed below with reference to specific examples.

[0058] Example 1: Effects of different IDF addition amounts and different GDL concentrations on the preparation of composite gels

[0059] 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.

[0060] Weigh 4 g of Antarctic krill protein AKP 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.

[0061] Sodium alginate and Antarctic krill protein 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 mass fractions of 1%, 2%, and 3% (with no IDF added as a blank control, i.e., IDF addition was 0%), and the mixture was stirred for 5 min. Next, glucono-δ-lactone (GDL) was added at mass fractions of 1% and 2%, 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.

[0062] Example 2: Effects of different IDF addition amounts and different GDL concentrations on the visual appearance of the composite gel

[0063] like Figure 1 As shown, under GDL = 1%, the hydrogel without added IDF exhibited a dull color, obvious edge water separation, and poor moldability. With the increase of IDF addition to 1%–2%, the gel surface became smoother, the color more uniform, and the edge contours clearer, indicating that IDF effectively promoted the stable construction of the gel network structure; however, at the 3% addition level, slight edge cracking began to appear in the samples upon macroscopic observation. Subsequent mechanical and water retention tests showed that the gel hardness and water holding capacity reached their peak at this addition level. In contrast, the overall macroscopic integrity of the gel formed under GDL = 2% was better than that at 1%. Although the surface of the group without IDF was relatively intact, slight gel flow was still visible; after adding an appropriate amount of IDF (1%–2%), the gel surface became fuller, the structure denser, and there was no obvious water separation; when the IDF addition increased to 3%, although the edge smoothness decreased slightly, the internal structure of the gel became more solid, indicating a good synergistic effect between higher acid induction levels and IDF filling.

[0064] Example 3: Effects of different IDF addition amounts and different GDL concentrations on the colorimetric test of the composite gel.

[0065] 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.

[0066] As shown in Table 1, the L value decreased significantly with increasing IDF ratio (P<0.05). Specifically, the L value decreased from 69.77±0.64 to 22.10±0.56 with GDL=1%, and from 57.93±0.73 to 10.03±0.63 with GDL=2%, indicating that the introduction of IDF significantly reduced the light transmittance and brightness of the gel system. This trend can be attributed to the enhanced multiple scattering effect of light due to the distribution of IDF particles in the gel matrix. Especially in the presence of natural pink pigments in krill protein (such as astaxanthin derivatives), the uneven particle size and refractive index differences in the system further amplified the interference of reflected light, leading to a decrease in sensory brightness. In addition, increased acidity (GDL=2%) also exacerbated the darkening of color, which is speculated to be related to the reduction of pigment embedding caused by changes in protein molecular conformation or changes in surface particle distribution. The a-value (red-green hue) showed a trend of first increasing and then decreasing, reaching its maximum at 1% IDF (6.48±0.32 at GDL=1%, 5.94±0.63 at GDL=2%), suggesting that an appropriate amount of IDF helps enhance the redness of the gel. This phenomenon may be related to the protein unfolding and exposure of aromatic amino acids (such as tyrosine and phenylalanine) promoted by IDF, which enhances the absorption of visible light and improves the red hue of the system. However, when the IDF addition increased to 3%, the a* value decreased significantly, possibly due to increased surface roughness and uneven light reflection caused by fiber aggregation, thus weakening the red expression. The b* value also showed a peak-shaped change, reaching its maximum at 1% IDF (18.32±0.50 at GDL=1%, 9.67±0.62 at GDL=2%), and then gradually decreasing. This indicates that high concentrations of IDF may reduce the system's reflectivity to yellow light through a particle masking effect, affecting its yellowness performance. Overall, the system with GDL=1% has higher L and b values ​​than that with GDL=2%, and the color is brighter and more natural.

[0067] Table 1. Effect of different IDF addition amounts on colorimetric tests of different gels (GDL=1% and GDL=2%)

[0068]

[0069] Example 4: Effects of different IDF addition amounts and different GDL concentrations on the water-holding capacity of the composite gel

[0070] 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:

[0071] WHC (%) = (Mt / M0) × 100

[0072] In the formula: M0 is the mass of the gel before centrifugation (g); Mt is the mass of the gel after centrifugation (g).

[0073] like Figure 2 As shown, the WHC of the AKP-SA-IDF composite gel increased significantly with the addition of IDF (P<0.05), from about 61% to about 83% under the condition of GDL=1%, and from 64% to nearly 90% under the condition of GDL=2%, indicating that the introduction of IDF can effectively enhance the water-holding capacity of the gel.

[0074] Example 5: Effects of different IDF addition amounts and different GDL concentrations on the determination of the texture of the composite gel

[0075] 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.

[0076] The results are shown in Table 2. Under the condition of GDL = 1%, as IDF increased from 0% to 3%, the gel hardness increased from 317.00 gf to 356.00 gf, the elasticity increased from 0.02 to 0.11, the adhesiveness increased from 14.11 to 26.68, and the chewiness increased from 13.77 to 23.69. This indicates that IDF promotes the transformation of the gel network from its initial loose state to a dense, ordered structure. IDF possesses a natural rigid framework and good water absorption and swelling properties, which can play a "filling-supporting" role in the proteoglycan network. Simultaneously, its hydroxyl groups form hydrogen bonds and van der Waals forces with AKP molecular chains, enhancing the physical stability of the network. Adhesion also showed a significant increasing trend, reaching a maximum value of 357.50 N, indicating an increase in cohesive force between the gel surface and the compression probe, reflecting improved interfacial continuity and structural integration. More importantly, the simultaneous improvement in elasticity and adhesiveness indicates that AKP, under the synergy of IDF, not only forms a dense network with compressive strength but also possesses good deformation recovery and structural plasticity, adapting to stress response under dynamic environments. Under the condition of GDL = 2%, the textural parameters are further enhanced, exhibiting a significant acid-induced strengthening effect. Especially in the 3% IDF group, the hardness reaches 421.00 gf, elasticity increases to 0.16, adhesiveness reaches 36.43, and chewiness reaches 31.14, all significantly better than the corresponding GDL = 1% group.

[0077] Table 2. Effects of different IDF addition amounts on gel texture determination of different gels (GDL=1% and GDL=2%).

[0078]

[0079] 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.

[0080] 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 .

[0081] like Figure 3 As shown, with the increase of IDF addition, the overall levels of G′ and G″ significantly increased (P < 0.05), especially in the mid-to-high frequency region, indicating that the introduction of IDF enhanced the structural rigidity and energy storage capacity of the gel. Specifically, the G′ value of the 0% IDF group increased slowly, and the network structure was relatively loose; while the 3% IDF group maintained the highest G′ value throughout the entire frequency range, suggesting that the gel structure of this group was the most dense and had a strong elastic energy storage capacity. Although the G″ value also increased with the increase of IDF concentration, reflecting some energy dissipation behavior, it never exceeded G′, further indicating that the viscoelastic response of the system was still dominated by elastic contribution.

[0082] Example 7: Fourier transform infrared spectra of composite gels with different IDF addition amounts and different GDL concentrations.

[0083] 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. -1 The scanning range is 4000 to 500 cm. -1 .

[0084] like Figure 4 As shown, SA is at 1605cm -1 and 1418cm -1 COO - Asymmetric and symmetric stretching peaks, and IDF in 1030–1070 cm⁻¹ -1 The C–O–C glycoside peak is a manifestation of its characteristic structure.

[0085] Under the condition of GDL=1%, IDF addition promoted 3285cm -1 The enhanced peak intensity and redshift in the region indicate its participation in hydrogen bond formation, strengthening the molecular association between the AKP and SA segments. Meanwhile, COO...- The asymmetric peak shifts slightly to 1590–1600 cm⁻¹ -1 This suggests that an electrostatic complex structure may form between the SA carboxylic acid group and AKP. The structural changes are more pronounced under stronger acid-induced conditions (GDL = 2%). (1730cm) -1 The newly appearing weak peak nearby may originate from the –COOH structure formed by carboxyl protonation, suggesting that the acidic environment promotes the activation of carboxylic acid functional groups and the co-entanglement of hydrogen bonds between proteoglycans. Furthermore, at 1040 cm⁻¹... -1 The intensity of the C–O–C glycoside peak increased, especially in the 3% IDF group, indicating that IDF plays a more stable and continuous spatial support role in the network. The carboxylate peak of AKP itself did not change significantly, suggesting that its gelation process mainly depends on non-covalent association conformations (such as hydrophobic interactions and hydrogen bonds), exhibiting a relatively flexible "soft crosslinking" behavior.

[0086] Example 8: Effects of different IDF addition amounts and different GDL concentrations on X-ray diffraction patterns of the composite gel.

[0087] 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°.

[0088] like Figure 5 As shown, the IDF single component exhibits a distinct diffraction peak at 2θ ≈ 22°, corresponding to the typical reflection signal of the semi-crystalline region in the cellulose structure; while the SA and AKP samples both show broad and gentle diffuse diffraction peaks in the range of 10°–30°, indicating that both are amorphous structures and lack regular lattice stacking.

[0089] Under the condition of GDL = 1% ( Figure 5 (B) The original crystalline peaks of IDF in the composite gel are still observable, but the peak intensity is significantly weakened, indicating that some crystalline regions of IDF are damaged or deconstructed during acid induction and network embedding. It is speculated that IDF undergoes physical entanglement and spatial cross-linking with protein-polysaccharide segments during this process, resulting in partial disturbance of its crystalline structure. Simultaneously, a new weak peak appears in the composite sample in the range of approximately 2θ = 19°–23°, possibly attributed to the conformational regularization of AKP segments under acid induction, leading to local segment recombination and ordered stacking under the synergistic effect of IDF. This suggests that although the overall system is disordered, there are small-scale quasi-ordered conformations. Under GDL = 2% induction conditions ( Figure 5(C) The further attenuation or even near disappearance of the characteristic diffraction peaks of IDF indicates that the increased acidity has a more severe destructive effect on the microcrystalline structure of IDF. Simultaneously, the enhanced and broadened diffuse peak region indicates a significant decrease in the overall crystallinity of the composite system. This change may be related to the rapid conformational unfolding of AKP induced by higher GDL concentrations and the formation of a denser electrostatic association network between its positively charged groups and the negatively charged carboxyl groups of SA. During this process, IDF is more deeply embedded in the amorphous structure, losing its original regular arrangement space. Furthermore, the increased densification of the gel network under high acid conditions also restricts the rearrangement and crystallinity recovery of IDF segments, further exacerbating the disordering trend.

[0090] Example 9: Thermogravimetric analysis of composite gels with different IDF addition amounts and different GDL concentrations.

[0091] 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 ℃.

[0092] like Figure 6 As shown, the three single components (SA, IDF, and AKP) exhibited different thermogravimetric characteristics during heat treatment: SA and AKP experienced major weight loss in the range of 250–340°C, with Tmax of approximately 286.4°C and 301.7°C, respectively, which were attributed to polysaccharide backbone breakage and protein peptide chain degradation; while IDF showed a significant decomposition peak at approximately 320°C, indicating strong thermal stability.

[0093] Under GDL = 1% conditions, the composite gel exhibited typical three-stage thermal decomposition behavior: the first stage (<150°C) was water evaporation, the second stage (220–340°C) was the thermal decomposition zone of the main organic matter, and the third stage (>400°C) was the slow decomposition period of residual carbon. With increasing IDF addition, both the initial degradation temperature (Tonset) and the maximum weight loss rate temperature (Tmax) of the samples increased significantly. The Tmax of the 3% IDF group increased to approximately 316.8°C, more than 15°C higher than the group without IDF, while the residual carbon content also increased, indicating that IDF effectively enhanced the thermal resistance to decomposition of the gel network. Under GDL = 2% conditions, this trend of improved thermal stability was even more pronounced. The Tonset of the 3% IDF group increased to approximately 260°C, the Tmax reached nearly 325°C, and the residual mass ratio increased to approximately 29%, significantly higher than the corresponding sample at 1% acidity. Further DTG curves show that with increased acid induction, the thermogravimetric peak shifts to the right in the high-temperature region, with increased peak width and decreased peak value, indicating that the thermal decomposition process of the system tends to slow down, decomposition is delayed, and the molecular structure is more stable. This change suggests that stronger acidity can promote the conformational unfolding of AKP segments and form more complete electrostatic association with SA, while synergistically forming a multiple hydrogen bond entanglement structure with IDF hydroxyl groups, constructing a dense, high-energy-consuming network, thereby significantly increasing the pyrolysis threshold.

[0094] Example 10: SEM microstructure characterization of composite gels by different IDF addition amounts and different GDL concentrations.

[0095] After freeze-drying the gels under different conditions, they were sputter-coated with gold and observed by scanning electron microscopy at 300x.

[0096] like Figure 7 As shown, the surface structure of AKP-SA gel without added IDF is loose, with large and unevenly distributed pores, thin pore walls, obvious collapse, and even cracking in some areas, indicating that its three-dimensional network stability is limited and its spatial support is weak.

[0097] With increasing IDF addition, the internal microstructure of the gel gradually improved significantly. The 1% and 2% IDF addition groups exhibited a denser and more regular honeycomb porous structure, with significantly reduced pore size, thickened pore walls, and enhanced connectivity between pores. This indicates that IDF not only plays a physical filling role in the gel system but may also participate in network reconstruction through hydrogen bonding and electrostatic association. This mechanism helps improve the overall stability and structural integrity of the gel, constructing a synergistic and continuous three-dimensional network framework. This structural change trend is consistent with the observed –OH and COO groups in FTIR analysis. - The enhanced functional group association phenomenon is consistent with the improved elasticity and adhesion in the TPA results.

[0098] However, when the IDF content was further increased to 3%, the microstructure of the gel underwent further evolution: although it still maintained a porous structure, the regularity of the pores decreased, and the filling density of the IDF particles increased significantly, forming a denser composite network. This high-filling-content network structure may be directly related to the increased hardness, storage modulus, and water retention capacity observed in the aforementioned examples (Examples 5 and 6). The results indicate that the addition of IDF significantly alters the microstructure of the gel network, mainly forming a regular honeycomb structure at 1-2% addition, while a high-density, high-strength filled and reinforced network is formed at 3% addition.

[0099] Example 11: Appearance of strawberries preserved by different gel treatments

[0100] like Figure 8 The image shows a comparison of the appearance of strawberries treated with different coatings after storage. In the blank group, mold began to appear on day 4 (white mycelium on the fruit surface), the mold area expanded on day 6, and the fruit severely rotted and deteriorated rapidly on day 8. In the AKP-0 group, a small number of mold spots appeared on day 6, and the degree of mold was less than that of the blank group on day 8, but obvious white mycelium and fruit surface wrinkling were still visible, indicating limited delay in appearance deterioration. In the AKP-IDF group, after 8 days of storage, the fruit surface only slightly wrinkled, with no obvious mold or rot, and the color remained bright red and the fruit shape intact, with significantly better appearance quality than the blank group and the AKP-0 group.

[0101] Example 12: Effects of different IDF addition amounts and different GDL concentrations on the preservation index of composite gels for strawberries (Figure)

[0102] Figure 9 The A–F system demonstrates the impact of different treatments (blank, AKP-0, AKP-IDF) on nine key quality indicators of strawberries during 8 days of storage at 25°C, including spoilage index, weight loss, titratable acid (TA), soluble solids (TSS), firmness, ascorbic acid (Vc), total phenols, catalase activity (CAT), and malondialdehyde (MDA) content. This data is used to comprehensively evaluate the actual preservation effects of each membrane layer in delaying the mold growth process and maintaining the quality stability of strawberries.

[0103] Corruption Index (CPI) Figure 9 A) is a direct indicator for evaluating the degree of fruit spoilage. In the early stages of storage, the three groups of strawberries showed slow spoilage development. From day 4 onwards, the spoilage degree in the control group rapidly increased, reaching nearly 90% on Day 8. In contrast, the AKP-0 and AKP-IDF groups significantly delayed the spoilage process, with the latter maintaining a spoilage rate of around 65%, a significant difference (p<0.05). This indicates that the membrane coating can effectively slow down the infection of pathogenic microorganisms, especially the IDF composite membrane, which enhances the membrane's airtightness and structural stability. Changes in weight loss rate ( Figure 9 (B) Shows the water loss process in strawberries. The control group suffered the most severe water loss, with a cumulative weight loss rate of approximately 22% over 8 days; the weight loss rates of the AKP-0 and AKP-IDF groups were controlled at approximately 17% and 13%, respectively, indicating that the composite membrane has better water retention capacity. It is speculated that the introduction of IDF improved the hydrophilicity and pore structure distribution of the membrane, effectively inhibiting fruit transpiration. Titratable acid content ( Figure 9 C) is an important indicator for judging the respiration intensity and acidity changes of fruit. The initial acid content of the three groups was close to 1.10 g / 100 g, with the control group showing the fastest degradation, reaching only 0.40 g / 100 g on Day 8; while the AKP-IDF group maintained above 0.85 g / 100 g. This indicates that the membrane structure effectively delayed the metabolic consumption of organic acids, possibly related to its inhibitory effect on gas exchange and microbial metabolism. Soluble solids ( Figure 9 D) The TSS content initially increased slightly, then gradually decreased. The control group's TSS content decreased from an initial 7.2°Brix to approximately 5.9°Brix on Day 8, while the AKP-IDF group maintained above 6.6°Brix. Membrane treatment slowed sugar breakdown and stabilized the changes in fruit flavor compounds. (Firmness changes...) Figure 9 E) Shows the integrity of strawberry tissue structure. During storage, the firmness of the control group decreased significantly, with a retention rate of less than 50% after 8 days; the AKP-IDF group maintained approximately 80% of its initial firmness, significantly better than other groups. The addition of IDF may help form a dense membrane structure, inhibit cell wall enzyme activity, and improve the membrane's mechanical protective efficacy against the pulp. Ascorbic acid (ASA) Figure 9 F) is an important parameter for measuring the fruit's ability to retain nutrients. The initial vitamin C content of all three groups was around 80 mg / 100g, while the control group only had 32 mg / 100g at the end of storage, and the AKP-IDF group still maintained a retention rate of over 70%. This indicates that its membrane structure has a significant advantage in delaying oxidative degradation and protecting nutrients. Total phenolic content ( Figure 9 G) The overall trend was "increase first, then stabilize". The AKP-IDF group reached its peak on day 4 (approximately 0.80 g / kg), then slowly decreased, but remained significantly higher than the control group (p<0.05). It is speculated that the membrane structure indirectly regulates the balance between the synthesis and degradation of phenolic substances through the slow release of oxygen and free radicals. Catalase (CAT) activity ( Figure 9 H) reflects the degree of oxidative stress. In the blank group, CAT activity increased rapidly over time, reaching 140 U / g on Day 8; the activity increase in the AKP-IDF group was significantly slower, only about 97 U / g, indicating that it possesses good antioxidant regulation capabilities and helps to delay the expansion of the oxidation reaction chain. Malondialdehyde (MDA) Figure 9I) MDA is the end product of lipid oxidation, and its level reflects the degree of oxidative damage to the cell membrane. In the control group, MDA levels significantly increased in the later stages of storage, while they were slightly lower in the AKP-0 group. The AKP-IDF group accumulated the least MDA, reaching only 0.72 nmol / g on Day 8, showing a significant difference. This indicates that the synergistic effect of IDF can reduce free radical levels and block the lipid oxidation chain reaction.

[0104] Comprehensive analysis shows that the AKP-IDF composite film outperformed the control group and the single film group in all nine indicators, exhibiting multiple effects such as delaying spoilage, locking in moisture, anti-oxidation, and preserving quality and flavor. Its mechanism of action may include: ① increased gel film density, limiting the migration of external oxygen and moisture; ② functional groups provided by IDF enhancing antioxidant / antibacterial capabilities; ③ synergistic inhibition of respiration and enzyme activity fluctuations, maintaining metabolic stability within the fruit. Therefore, this film system has promising applications in preservation, particularly suitable for short-term room-temperature storage of high-moisture, perishable fruits.

Claims

1. A composite gel based on insoluble dietary fiber (IDF) from seaweed (Ulva prolifera), sodium alginate (SA), and Antarctic krill protein (AKP), 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); 4% Antarctic krill protein isolate (AKP); 1-2% 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 insoluble dietary fiber in the *Ulva prolifera* is 1-3%.

4. The composite gel according to claim 3, characterized in that, The mass percentage of insoluble dietary fiber in the *Ulva prolifera* 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 an Antarctic krill protein isolate aqueous solution with a mass fraction of 4.0% respectively; (2) Mix the sodium alginate aqueous solution prepared in step (1) with the Antarctic krill protein isolate aqueous solution at a volume ratio of 3:4 to obtain a sodium alginate-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 Antarctic krill protein (AKP) is 5:

8.

7. The application of the composite gel as described in any one of claims 1-4 in the preservation of fruits and vegetables.

8. A method for preserving strawberries, characterized in that, The method includes the following steps: (1) immersing strawberries in the coating solution prepared in step (4) of the method as described in claim 5; (2) removing the immersed strawberries and air-drying them to form an edible coating on their surface; and (3) storing the strawberries treated in step (2).

9. The preservation method according to claim 8, characterized in that, The storage conditions are: temperature 24-26℃, relative humidity 75-85%.

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