Water-saving surimi gel preparation method based on polyphenol-enzyme synergistic cross-linking and application thereof

Abstract

This application discloses a method for preparing water-saving surimi gel based on polyphenol-enzyme synergistic crosslinking and its application. The method includes: rinsing surimi raw materials no more than twice; sequentially adding EGCG and TGase to the rinsed surimi, utilizing their synergistic effect to induce protein crosslinking; and then filling and undergoing two-stage heating gelation to obtain the surimi gel. This application significantly improves the breaking strength, elasticity, water-holding capacity, and other textural properties of the surimi gel by introducing synergistic crosslinking of EGCG and TGase into the water-saving rinsing process, successfully overcoming the quality degradation problem caused by reduced rinsing. Simultaneously, the gel exhibits unique digestive-resistant and slow-release properties, delaying protein hydrolysis in the early stages of gastric digestion and ensuring the full release of essential amino acids, thus preserving its nutritional value. This application provides an effective solution for developing high-quality, high-nutrition, and environmentally friendly novel surimi products.

Description

Technical Field

[0001] This application relates to the field of food processing technology, specifically to a method for preparing water-saving surimi gel based on polyphenol-enzyme synergistic crosslinking and its application. Background Technology

[0002] Surimi products are important processed aquatic foods, and their core qualities (such as gel strength, elasticity, water retention and whiteness) are key to their market competitiveness.

[0003] In traditional surimi processing, multiple rinsing steps are essential for ensuring product quality, aiming to remove water-soluble impurities, fats, and colored substances that affect gel formation. However, this process consumes a large amount of water and leads to the loss of some soluble nutrients, resulting in significant production costs and environmental pressure. Therefore, developing and applying water-saving rinsing technologies (i.e., reducing the number of rinsing steps) has become an urgent need for the sustainable development of the industry.

[0004] However, reducing the number of rinsing cycles presents a direct technical challenge: impurities that are not adequately removed can severely interfere with and degrade the gel network of the surimi, resulting in insufficient strength, loose texture, poor water retention, and undesirable color, significantly reducing product quality and making it difficult to meet commercial standards. To address the quality defects caused by water-saving rinsing, existing technologies have introduced various physical, chemical, or enzymatic quality improvement methods. While these methods can strengthen the gel structure and improve texture or color to some extent, they generally have the following limitations when dealing with the specific application scenario of "water-saving rinsing":

[0005] 1. The contradiction between effectiveness and cost: The overall improvement effect of using a single improver is often limited. In order to achieve a gel quality close to that of traditional multi-rinsed fish paste, the amount of improver needs to be increased significantly, which leads to a significant increase in production costs and may introduce new flavor or safety issues.

[0006] 2. The contradiction between performance and digestibility: In order to achieve excellent gel performance, existing modification techniques (especially certain strong cross-linking methods) tend to construct highly dense protein networks. While this structure improves texture, it significantly reduces the sensitivity of the gel to digestive enzymes in the early stages of digestion, slows down the hydrolysis process of proteins, and may reduce the bioavailability of nutrients.

[0007] 3. One-sidedness in improvement objectives: Most technologies focus primarily on improving a single or a few gel indicators (such as hardness), lacking effective solutions for systematically and synergistically improving multiple dimensions of gels, such as strength, elasticity, water retention, and color, while simultaneously considering their subsequent digestibility and absorption characteristics. This makes it difficult to achieve a balance between quality and nutritional value.

[0008] Therefore, how to simultaneously optimize the textural quality of surimi gel and beneficially regulate its digestibility through innovative collaborative methods while achieving water-saving production goals has become a comprehensive problem that urgently needs to be solved in the existing technology. Summary of the Invention

[0009] The purpose of this application is to provide a water-saving method for preparing surimi gel based on polyphenol-enzyme synergistic crosslinking and its application. This method introduces a specific ratio of epigallocateching allate (EGCG) and transglutaminase (TGase) into a surimi system that has undergone water-saving rinsing (e.g., single rinsing). Utilizing the synergistic crosslinking effect of these two agents on surimi proteins, the formation of the gel network structure is efficiently induced and strengthened under the background of significantly reduced rinsing water consumption. This technique not only significantly improves the overall quality of the water-saving rinsed surimi gel, including breaking strength, elasticity, water retention, and whiteness, but also rationally influences its hydrolysis kinetics during in vitro digestion by controlling the density and crosslinking mode of the gel network, delaying rapid degradation in the early stages of digestion and promoting the full release of essential amino acids. This application ultimately achieves simultaneous optimization of the textural quality and nutritional digestibility of surimi products under the premise of water-saving and energy-saving green processing, providing an effective technical solution for its application in high-quality, high-value-added surimi products.

[0010] Therefore, this application provides the following technical solution:

[0011] In a first aspect, this application provides a method for preparing an EGCG-TGase crosslinked water-saving fish paste gel, comprising the following steps:

[0012] S1. Raw material preparation: Obtain fish meat, and after meat extraction and chopping, obtain fish paste raw material;

[0013] S2. Water-saving rinsing: The fish paste raw material is rinsed no more than twice to obtain rinsed fish paste;

[0014] S3. Synergistic cross-linking: EGCG and TGase are added sequentially to the rinsed fish paste and mixed by beating, and the synergistic effect of EGCG and TGase is used to induce cross-linking of fish paste proteins.

[0015] S4. Gel forming: The fish paste processed in step S3 is filled and formed. First, it is heated at 30-50 °C to gel the protein, and then heated at 80-100 °C to set and mature the gel, thus obtaining the fish paste gel.

[0016] Step S2 above defines the specific process scenario targeted by this application, namely water-saving rinsing, with rinsing times of 0, 1, or 2 times. Its direct effect is to achieve the processing goals of saving water, saving energy, and reducing nutrient loss.

[0017] Step S3 above is the core process of the technical solution. The process order of adding EGCG before TGase is beneficial for EGCG to bind to the protein first, creating a more favorable conformation or site for subsequent enzymatic cross-linking by TGase. The synergistic cross-linking effect of EGCG and TGase is key. EGCG can not only bind to the protein itself, but also activate TGase, jointly inducing the formation of a more efficient and denser protein cross-linking network.

[0018] In step S4 above, the first stage at a lower temperature (30-50 °C) is a crucial stage for the protein to fully unfold and initially form a three-dimensional network through the aforementioned cross-linking process (gelation). The second stage at a higher temperature (80-100 °C) aims to further stabilize the formed gel network, thoroughly denature it, and also serve to sterilize and set it (curing).

[0019] In some preferred embodiments, step S1 specifically involves the following steps:

[0020] After slaughtering the fresh fish, remove the scales, head, and tail. Then, manually remove the white meat from the belly. Wash the fish meat and drain it. Use a chopper to chop the fish meat for 5 minutes to obtain fish paste raw material. Control the preparation temperature to always be below 10 ℃.

[0021] In some preferred embodiments, step S2 specifically involves the following steps:

[0022] According to the material-to-liquid ratio (m / v) of 1:5, pre-cooled (4 ℃) deionized water is added to the fish paste raw material obtained by S1 and rinsed 1 to 2 times for 5 minutes each time. In the last rinse, 0.3% (w / w) NaCl is added to the ice water. After each rinse, the fish paste is dehydrated and filtered with gauze to control the moisture content to below 78% (w / w). This yields fish paste with different degrees of rinsing.

[0023] In some embodiments, in step S3, the amount of EGCG added is 1.0 × 10⁻³ ~ 3.0 × 10⁻³ g / g. pro The amount of TGase added is 5~20 U / gpro .

[0024] In some embodiments, in step S3, before adding EGCG and TGase, salt is added to the rinsed fish paste, and the amount of salt added is 1.5%-3.5% of the fish paste mass.

[0025] In some embodiments, in step S4, the protein gelation time is 0.5-2 hours; the gel setting and ripening time is 20-40 minutes.

[0026] Secondly, this application provides a surimi gel prepared by the preparation method described in the first aspect.

[0027] Thirdly, this application provides the use of the surimi gel described in the second aspect in the preparation of digestible, slow-release protein foods.

[0028] Fourthly, this application provides the application of the surimi gel described in the second aspect in the preparation of highly water-holding gel foods, wherein the water-holding capacity of the surimi gel is not less than 90%.

[0029] Fifthly, this application provides the use of a composition in the preparation of a surimi gel product, the composition comprising EGCG and TGase.

[0030] Compared with the prior art, this application has at least the following advantages and beneficial effects:

[0031] 1. The preparation method of this application can be used to produce fish paste that has only been rinsed once or twice. Compared with untreated water-saving rinsed fish paste, the breaking force, breaking distance, gel strength, hardness, elasticity, chewiness and water retention of the gel are significantly and synergistically improved after EGCG and TGase synergistic crosslinking, successfully overcoming the quality deterioration problem caused by reducing the number of rinsing times.

[0032] 2. The surimi gel provided in this application has a synergistic cross-linked gel network that can effectively delay the initial digestion process. In vitro dynamic digestion experiments show that, compared with the control group, the digestibility of the synergistically cross-linked gel is reduced and the digestion half-life is significantly prolonged, demonstrating sustained-release properties.

[0033] 3. Although the surimi gel provided in this application has a slow digestion rate in the initial stage, the synergistic cross-linking does not reduce the final nutritional quality of the product. After complete gastrointestinal digestion, the proportion of essential amino acids in the total amino acids (EAA / TAA) in the gel digestion solution is consistently higher than 60%, indicating that it maintains high nutritional value and good amino acid release levels.

[0034] 4. This application solves the problems of improving gel quality and regulating digestibility of water-saving rinsing surimi through a specific process, providing an effective way to develop new surimi products that are high-quality, high-nutrition and environmentally friendly. Attached Figure Description

[0035] Figure 1The puncture characteristics of surimi gels with different rinsing times after cross-linking of EGCG and TGase are shown in Figure 1 (p<0.05), where A, B, and C are the breaking force, breaking distance, and gel strength of the surimi gel, respectively.

[0036] Figure 2 The water-holding capacity of surimi gels treated with EGCG and TGase after different rinsing times (p<0.05).

[0037] Figure 3 Digestibility results of water-saving rinsed surimi gel crosslinked with EGCG and TGase at different digestion stages (p < 0.05).

[0038] Figure 4 The particle size distribution of water-saving rinsed surimi gel crosslinked with EGCG and TGase at different digestion stages is shown in the diagram. A and B represent the particle size changes of the surimi gel during in vitro dynamic gastric and gastrointestinal digestion, respectively.

[0039] Figure 5 SDS-PAGE analysis of the digestion precipitate of surimi gel during in vitro dynamic gastrointestinal digestion, where (A): surimi gel; (B): 1.8 × 10⁻⁶. -3 g / g pro EGCG-crosslinked surimi gel; (C) 10 U / g pro TGase-crosslinked surimi gel; (D): 1.8 × 10⁻⁶ -3 g / g pro EGCG synergistic 10 U / g pro TGase-crosslinked surimi gels, 1-7 are samples digested in the stomach for 10, 30, 60, 90, 120, 150 and 180 min respectively. (a), (b), (c) and (d) are the gastrointestinal digestion samples corresponding to (A), (B), (C) and (D), and 1-8 are samples digested in the stomach for 30, 60, 90, 120, 150, 180, 210 and 240 min respectively.

[0040] Figure 6 The release of free amino acids and their fitting curves (Logistics model) during in vitro dynamic gastric and intestinal digestion of water-saving washed surimi gel crosslinked with EGCG and TGase.

[0041] Figure 7 Release of free amino acids and fitted curves (quasi-first-order kinetics) during in vitro gastric and intestinal digestion of water-saving washed surimi gel crosslinked with EGCG and TGase.

[0042] Figure 8The release of free amino acids and their fitted curves (pseudo-second-order kinetics) during in vitro gastric and intestinal digestion of water-saving washed surimi gel crosslinked with EGCG and TGase. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0044] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following definitions are provided to better understand it.

[0046] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0047] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0048] To enable those skilled in the art to more accurately understand this application, the following explanations are provided for some terms or definitions in the text of this application:

[0049] 1. Water-saving rinsing: This refers to a fish paste pretreatment process that involves rinsing no more than twice (including 0, 1, and 2 times). Its purpose is to reduce water consumption.

[0050] 2. Crushing: This involves chopping, grinding, and mixing the fish paste to break down the muscle fibers of the fish, dissolve salt-soluble proteins, and mix them evenly.

[0051] 3.g / g pro and U / gproAll of these refer to the calculation based on the mass of protein (myofibrillar) in the surimi raw material. The amounts of EGCG and TGase added in this application are used to calculate these amounts.

[0052] 4. Gel network: refers to the three-dimensional continuous network structure formed by proteins such as myosin in surimi through intermolecular forces under the action of heating and cross-linking agents.

[0053] 5. Synergistic cross-linking: This refers to the enhanced gel network structure produced when EGCG (through covalent / non-covalent binding) and TGase (through enzymatic catalysis to form heteropeptide bonds) work together on surimi proteins, which is superior to the effect produced by using either alone or simply superimposing them.

[0054] The technical solution of this application and the technical effects achieved will be described in detail below through more specific embodiments.

[0055] 1. Materials and Methods

[0056] 1.1 Experimental Materials

[0057] Silver carp: Each individual weighed approximately 1500 g and was purchased from the vegetable market of Huazhong Agricultural University. It was kept alive in a water tank and transported to the laboratory for slaughter. The head, skin, and internal organs were then removed, and the fish meat was used for later use. Transglutaminase (TGase, 3000 U / g): Purchased from Konatik (Switzerland) Ltd. Epigallocatechin gallate (EGCG, 98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0058] 1.2 Experimental Reagents

[0059] Sodium chloride, potassium chloride, sodium hydroxide, hydrochloric acid, sodium dihydrogen phosphate, calcium chloride, ammonium chloride, sodium bicarbonate, potassium dihydrogen phosphate, magnesium chloride, sodium dodecyl sulfate, sulfosalicylic acid, and other analytical and purification chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.; Tris(hydroxymethyl)aminomethane (Tris), β-mercaptoethanol (β-ME), Nile blue, Coomassie Brilliant Blue R-250, etc. were purchased from Shanghai Maclean Biotechnology Co., Ltd.; pepsin and trypsin were purchased from Sigma-Aldrich, Inc.; amino acid reagent kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.; SDS-PAGE pre-cast gels were purchased from Bio-Rad Laboratories, Inc.; high molecular weight markers were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0060] 1.3 Experimental Equipment

[0061] The experimental equipment is shown in Table 1 below.

[0062] Table 1 Equipment Information

[0063]

[0064] 1.4 Experimental Methods

[0065] 1.4.1 Preparation of fish paste with different rinsing degrees

[0066] Preparation of unwashed fish paste: After slaughtering fresh silver carp, remove the scales, head, and tail. Then, manually remove the white meat from the belly. Wash the fish meat and drain it. Chop the fish meat for 5 minutes using a chopper. Control the preparation temperature to be below 10 ℃ at all times.

[0067] Preparation of rinsed fish paste: Add pre-cooled (4 ℃) deionized water to unrinsed silver carp paste at a material-to-liquid mass-to-volume ratio (m / v) of 1:5, and rinse 1 to 2 times, each time for 5 minutes. During the final rinse, add 0.3% sodium chloride (NaCl) to the ice water. After each rinse, filter the paste using gauze to dehydrate it, controlling the moisture content to be below 78% (w / w based on the total mass of the fish paste). This yields fish paste with different degrees of rinsing.

[0068] 1.4.2 Preparation of EGCG-TGase cross-linked surimi gel

[0069] The moisture content of the fish paste with different rinsing degrees was uniformly adjusted to 78% (based on the total mass of the fish paste, w / w), and then pre-chopped in a food processor for 2 minutes; 2.5% (w / w) of sodium chloride and a concentration of 1.8×10⁻³g / g were added to the pre-chopped fish paste. pro The EGCG was then pounded for 5 minutes; the fish paste was left to stand at 4 °C for 30 minutes, and then a concentration of 10 U / g was added to the fish paste. gpro The TGase was crushed and processed for 2 minutes to obtain mixed fish paste. The mixed fish paste was degassed using a vacuum packaging machine, and then filled into 26 mm diameter casings using a sausage stuffer and sealed to obtain filled fish paste. The filled fish paste was placed in a 40 °C water bath for low-temperature gelation for 1 hour. Then it was transferred to a 90 °C water bath for high-temperature cooking (fish cake making) for 30 minutes. After cooking, the product was cooled immediately, and the resulting fish paste gel was stored at 4 °C for later use.

[0070] 1.4.3 Fish paste gel puncture test

[0071] A puncture test was performed using a texture analyzer. The specific steps are as follows: The fish paste gel sample was cut into cylinders with a height of 20 mm. A spherical puncture probe (model P / 0.25S) was used for measurement.

[0072] The test parameters were set as follows: compression distance 15 mm, pre-test speed 5 mm / s, test speed 1 mm / s, post-test speed 5 mm / s, trigger force 5 g. This test allows for the determination of the sample's breaking force and breaking distance, and the calculation of gel strength.

[0073] 1.4.4 Texture Analysis of Surimi Gel

[0074] The TPA test of myosin gel was performed on a texture analyzer equipped with a P / 36R probe. The specific steps are as follows:

[0075] The surimi gel sample was cut into cylinders 20 mm high and compressed at a compression rate of 40%. The compression speed was 5 mm / s before and after the test, and 1 mm / s during the test.

[0076] 1.4.5 Determination of whiteness of fish paste gel

[0077] The L* (lightness value), a* (red-green value), and b* (yellow-blue value) of the surimi gel were measured at random locations on its surface using a colorimeter. Whiteness (W) was calculated using the following formula:

[0078]

[0079] 1.4.6 Determination of water-holding capacity of surimi gel

[0080] The water holding capacity (WHC) of surimi gel was determined using a hardness tester. The test procedure is as follows:

[0081] Cut the surimi gel into 5 mm thin slices, weigh them and record the weight as W1. Then wrap each slice in filter paper and place it in the center of a hardness tester. Press down with a force of 5 g for 1 min, then remove the filter paper and weigh the gel after pressing, recording the weight as W2. Repeat the test 6 times. Calculate the water-holding capacity of the surimi gel using the following formula:

[0082]

[0083] 1.4.7 Effect of EGCG-TGase synergistic crosslinking on the in vitro dynamic digestion properties of water-saving rinsed fish paste gel

[0084] (1) Preparation of EGCG-TGase synergistic crosslinking single-rinse surimi gel

[0085] Based on the study of gel properties, four groups of surimi gels with minimal rinsing (one rinse) were prepared: uncrosslinked surimi gel, 1.8 × 10⁻⁶ mollusc gel, and 1.8 × 10⁻⁶ mollusc gel. -3 g / g pro EGCG cross-linked fish paste gel, 10 U / gproTGase cross-linked surimi gel and 1.8×10 -3 g / g pro EGCG synergistic 10 U / gpro The TGase cross-linked surimi gel was prepared using the same method as in 1.4.2.

[0086] (2) Determination of in vitro dynamic gastrointestinal digestive properties of fish paste gel

[0087] First, prepare the simulated digestion solution as follows:

[0088] The simulated gastric juice (pH 1.4) contained: 5.51 mg / mL NaCl, 1.65 mg / mL KCl, 0.53 mg / mL NaH2PO4, 0.60 mg / mL CaCl2, 0.61 mg / mL NH4Cl and 2000 U / mL pepsin, and the pH was adjusted with 1M hydrochloric acid.

[0089] The simulated intestinal fluid (pH 7.8) contained: 14.02 mg / mL NaCl, 1.13 mg / mL KCl, 6.78 mg / mL NaHCO3, 0.16 mg / mL KH2PO4, 0.10 mg / mL MgCl2, and 100 U / mL trypsin.

[0090] In vitro dynamic digestion is performed continuously in a human biomimetic stomach and intestine model (DIVHS-IV system). The specific steps are as follows:

[0091] First, 200 g of chopped sample was placed into the bionic stomach of the DIVHS-IV system for continuous gastrointestinal digestion for 4 hours (gastric digestion only takes 3 hours), with 30 mL of simulated gastric juice pre-added to the stomach model. Then, the DIVHS-IV system was started with the parameters shown in Table 2. Gastric digestion samples were then collected from the bionic stomach at different times after digestion began (10, 30, 60, 90, 120, 150, 180 min), and intestinal digestion samples were collected from the distal end of the bionic small intestine at different times after digestion began (30, 60, 90, 120, 150, 180, 210, 240 min). The collected digestion samples were immediately heated in a boiling water bath for 10 min to inactivate the digestive enzymes, then centrifuged at 4000×g for 15 min to separate the supernatant and precipitate, and stored at -80 ℃ for later use.

[0092] Table 2 Operating parameters of dynamic in vitro gastrointestinal digestive equipment

[0093]

[0094] (3) Determination of digestibility of digestion products

[0095] 5 g of chopped fish paste gel particles and the precipitate obtained in step (2) above were dried at 105 °C to constant weight. Digestibility was calculated using the following formula:

[0096]

[0097] Wherein, PD is the digestibility of surimi gel, and W0 and W1 represent the dry matter content (g) in surimi gel before and after digestion, respectively.

[0098] (4) Particle size determination of digestion products

[0099] The particle size of the fish paste gel digest was determined using a laser particle size analyzer. The supernatant obtained in step (2) was injected into the sample chamber of the laser particle size analyzer, ultrasonically dispersed, and then the measurement was started. The automatic measurement was performed 3 times, and the average value was taken as the measurement result. Five parallel measurements were performed for each sample. Measurement parameters: the dispersant was water, the refractive index of the dispersant was 1.330, and the refractive index of the sample was 1.540. The data was processed using Malvern Mastersizer software (version 5.12c, Malvern, Worcestershire, UK) to obtain the particle size information: Dx(10), Dx(50), Dx(90), Dx3,2, Dx4,3. Dx(10), Dx(50), and Dx(90) represent the particle size corresponding to the cumulative particle size distribution number of a sample reaching 10%, 50%, and 90%, respectively. Dx3,2 and Dx4,3 represent the average particle size of the specific surface area and the average particle size of the volume of a sample.

[0100] (5) Identification of digestion products by SDS-PAGE

[0101] The extraction method for total protein from the surimi gel digestion product was performed according to Morrissey's method. The process was carried out with slight modifications. The specific steps are as follows:

[0102] 1 g of the digested product precipitate was added to 9 mL of 5% SDS solution containing 0.1% β-mercaptoethanol, and then homogenized slowly for 3 min. The homogenate was then incubated in a water bath at 80 °C for 1 h. The resulting mixture was centrifuged at 10000 × g for 10 min, and the protein concentration in the supernatant was measured using the Lowry method. The protein concentration in the supernatant was adjusted to 1.0 mg / mL and mixed with sample buffer at a 1:1 (v / v) ratio. The mixture was placed in a tube and heated in a boiling water bath for 5 min. After cooling, the sample was added to a 10% pre-prepared SDS-PAGE gel, with 1 μL added to each well. Electrophoresis was performed at a constant voltage of 160 V. Finally, the gel was stained with Coomassie Brilliant Blue R250 for 30 min, and then destained with destaining solution until the bands were clear and the background was clean before photographing and observation.

[0103] (6) Determination of free amino acid composition of digestion products

[0104] The composition of free amino acids in surimi gel digest was determined using the sulfosalicylic acid method. The specific steps are as follows:

[0105] Add 100 µL of 10% sulfosalicylic acid to 400 µL of digestion solution, mix well, filter through a 0.45 µm filter membrane, and let stand at 4 °C for 60 min. Centrifuge the sample at 14500 rpm for 15 min, collect the supernatant, and centrifuge again at 14500 rpm for 5 min. Dilute the centrifuged sample twice with sample diluent, filter through a 0.22 µm filter membrane, and then analyze the filtered sample using a fully automated amino acid analyzer. The results of free amino acid analysis are expressed as the mass of amino acids contained in each mL of digestion solution.

[0106] (7) Fish paste gel digestion kinetic model

[0107] Following the method of Li et al (2017) with slight modifications, the specific steps are as follows:

[0108] The digestive kinetics of surimi gel were characterized by determining the concentration of free amino acids in the digestive fluid. The free amino acid content was determined using a trace amino acid assay kit. The digestive kinetics during the gastric digestion stage were fitted using the following logistic model:

[0109]

[0110] Where x(t) is the content of free amino acids produced at time t; X0 is the initial content of free amino acids during digestion (μmol / mL); M is the maximum value of free amino acids reached at the end of digestion (μmol / mL); t M / 2The time (in minutes) required to release half of the total digestible amino acid content; t is the digestion time (in minutes), and p is the fitting parameter.

[0111] The digestive kinetic equations for the intestinal digestion stage were fitted using pseudo-first-order kinetic equations (Formula 1) and pseudo-second-order kinetic equations (Formula 2).

[0112] (Formula 1)

[0113] (Formula 2)

[0114] Where x(t) is the content of free amino acids produced at time t, M is the maximum value of free amino acid content in the digestive fluid, and p is the fitting parameter.

[0115] 1.4.8 Data Processing and Analysis

[0116] Each experiment was repeated three times. Experimental data were processed using SPSS Statistics 26 and Excel software. Experimental data were plotted using Origin 9.0. The significance analysis was performed at a 95% confidence level (p<0.05).

[0117] 2. Results and Analysis

[0118] 2.1 Effect of EGCG-TGase crosslinking on the breaking force and breaking distance of surimi gel after different rinsing cycles

[0119] Breaking force represents the hardness and fracture resistance of surimi gel, while breaking distance is related to the elasticity of surimi gel. Gel strength is expressed as the product of breaking force and breaking distance. The combination of breaking force, breaking distance and gel strength can explain the quality of gel performance.

[0120] Figure 1 The puncture characteristics of surimi gels crosslinked with EGCG and TGase after different washing cycles (p<0.05) are shown, where A, B, and C represent the breaking force, breaking distance, and gel strength of the surimi gel, respectively. Figure 1 As shown, the unwashed surimi gel exhibited poor puncture properties, with a breaking force of 384.11 g and a breaking distance of 8.11 mm, the lowest among all groups. With increasing washing cycles, both the breaking force and breaking distance of the surimi gel improved. Compared to the uncrosslinked group, both EGCG and TGase crosslinking significantly improved the breaking force and breaking distance of the surimi gel, especially TGase, which had a very significant effect on improving the breaking force (10 U / g). gproThe addition of TGase increased the breaking strength of surimi gels after different rinsing cycles by 135.20%, 116.05%, and 200.01%, respectively. EGCG, however, primarily affected the breaking distance of the surimi gel, at 1.8 × 10⁻⁶. -3 g / g pro EGCG increased the breaking distance of surimi gels with different rinsing cycles by 0.79 mm, 0.11 mm, and 0.35 mm, respectively. Surimi gels co-crosslinked with EGCG and TGase exhibited the highest breaking force and breaking distance among groups with the same number of rinsing cycles. The breaking force and breaking distance of unrinsed, 1-rinsed, and 2-rinsed surimi gels were 918.55 g, 1015.46 g, and 1132.39 g, and 8.49 mm, 8.51 mm, and 9.25 mm, respectively. The synergistic crosslinking of EGCG and TGase further enhanced the fracture resistance and elasticity of the surimi gels, resulting in the highest breaking force and breaking distance among groups with the same number of rinsing cycles.

[0121] With increasing rinsing cycles, the gel strength of surimi gel gradually increases. During rinsing, salt-soluble proteins are retained to form a dense gel network, while water-soluble proteins such as tropomyosin and troponin, which do not participate in the formation of the surimi gel network, are washed away, improving the uniformity of the surimi gel structure and enhancing gel strength. Therefore, rinsing effectively improves the quality of surimi. The changes in gel strength and breaking distance of surimi gel with different rinsing cycles show a consistent trend. TGase can significantly improve the gel strength of surimi gel, and EGCG-TGase synergistic crosslinking can further enhance the gel strength of surimi gel. EGCG has an enhancing effect on the gel properties of surimi gel, and TGase modification also reduces the α-helix content and increases the β-sheet content in surimi gel. This makes it easier for surimi to fill into the surimi protein gel network through chemical bonds, forming a stronger gel complex. Surimi gel therefore has higher breaking force and breaking distance, showing a significant effect on improving the physical properties of surimi gel after a single rinse.

[0122] 2.2 Effect of EGCG-TGase crosslinking on TPA of surimi gel after different rinsing cycles

[0123] Texture properties are one of the sensory evaluation standards for surimi products and can be analyzed using the full texture mode of a texture analyzer. During sample testing, the sample is subjected to two compressions by the probe. Hardness refers to the maximum force applied after the first compression; elasticity refers to the height the sample recovers before the second compression; chewiness indicates the ease of chewing the surimi gel and is generally used as a supplementary parameter to hardness; cohesion is the ratio of the area of ​​force applied during the second compression to the area of ​​force applied during the first compression; resilience refers to the ability of the elastic gel to rebound after the first compression.

[0124] The TPA parameters of different surimi gel samples are shown in Table 3.

[0125] Table 3. TPA parameters of surimi gels treated with EGCG and TGase after different rinsing cycles.

[0126]

[0127] Note: 1: Fish paste gel; 2: 1.8×10 -3 g / g pro EGCG-crosslinked surimi gel; 3:10 U / g pro TGase-crosslinked surimi gel; 4: 1.8 × 10 -3 g / g pro EGCG synergistic 10 U / g pro TGase-crosslinked surimi gel; different lowercase letters in the same column indicate significant differences between samples (p < 0.05), the same below.

[0128] As shown in Table 3, the addition of TGase significantly improved the hardness, elasticity, chewiness, cohesiveness, and resilience of the surimi gel, showing significant differences compared to the blank group (1). The addition of EGCG mainly increased the elasticity of the surimi gel. The synergistic crosslinking of EGCG-TGase also improved the TPA parameter of the surimi gel to a certain extent, and it was higher than that of other experimental groups with the same number of rinsing cycles.

[0129] The number of rinsing cycles also affects the effects of EGCG and TGase. In unrinsed surimi gel, EGCG had limited effect on improving the texture properties, even decreasing the chewiness from 902.16 to 876.08. This is because other impurities and water-soluble proteins in the surimi reacted with it, producing substances that hindered gelation. TGase significantly improved the texture of surimi gels after different rinsing cycles, but the effect decreased with increasing rinsing cycles. Without rinsing, the hardness of the surimi gel increased by 111.82%; after one rinsing, it increased by 131.24%; and after two rinsings, it only increased by 69.36%. The effect of EGCG on the TPA parameters of the surimi gel was most pronounced in the one-rinsing group, with all indicators showing significant differences compared to the control group. This indicates that the effects of TGase and EGCG do not increase with increasing rinsing cycles. Furthermore, repeated rinsing significantly improves the gel properties of the surimi gel itself. The TPA parameter of the surimi gel in the rinsed group was significantly increased compared to the unrinsed group, which also reduced the effect of TGase.

[0130] 2.3 Effect of EGCG-TGase crosslinking on the color of surimi gel after different rinsing cycles

[0131] Color is an important factor in evaluating the appearance quality of surimi products, and the color of the product's appearance can affect consumers' choice and acceptance of the product.

[0132] The color of surimi gels treated with EGCG and TGase after different rinsing times is shown in Table 4 below.

[0133] Table 4. Color of surimi gel after different rinsing cycles treated with EGCG and TGase.

[0134]

[0135] Table 4 shows that the unwashed surimi gel had the lowest whiteness value (W), indicating that rinsing effectively removes pigments, especially myoglobin, from the surimi. Adding TGase to the unwashed surimi had no significant effect on the whiteness of the surimi gel. Adding EGCG increased the brightness of the surimi gel, thus increasing its whiteness. The synergistic effect of both substances also increased the whiteness of the surimi gel. After one rinse, the addition of both EGCG and TGase decreased the color of the surimi gel. The color of the surimi gel after two rinses was lower than that after one rinse, but both EGCG and TGase increased the color. The color of the surimi gel mainly depends on the properties of the surimi raw materials, and is also related to the refraction of light in the pores of the surimi gel, the scattering of free water on the gel surface, and the color of exogenous additives in the surimi gel. The more uniform and dense the network structure of the surimi gel, the more even the moisture distribution, the fewer the pores in the gel, and the smaller the light refraction and scattering effects. In addition, after EGCG itself oxidizes, the powder will turn light pink, and the products generated by self-degradation and oxidation during the gelation process will be yellowish-brown. Although this changes the color of the surimi gel, it is still within an acceptable range.

[0136] 2.4 Effect of EGCG-TGase crosslinking on the water-holding capacity of surimi gels after different rinsing cycles

[0137] The water-holding capacity of surimi gel is an important parameter for measuring its edibility. The higher the water-holding capacity, the stronger the ability of the surimi gel to retain its own moisture, indicating that more water is trapped in the network structure of the surimi gel.

[0138] Figure 2The figure shows the water-holding capacity of surimi gels treated with EGCG and TGase after different rinsing cycles (p<0.05). As can be seen from the figure, the unrinsed surimi gel had the lowest water-holding capacity at 82.29%. The TGase-treated surimi gel had lower water-holding capacity than the EGCG-treated surimi gel. This is because the unrinsed surimi contained a higher amount of actin, and TGase had little effect on actin. After rinsing, the surimi gels formed after gelation and fish cake formation showed significantly improved water-holding capacity. After two rinsing cycles, the improvement in water-holding capacity of the gels formed by adding TGase and EGCG decreased. Among all rinsing cycles, the EGCG-TGase synergistic cross-linking resulted in the highest water-holding capacity, at 93.73%, 95.43%, and 95.68%, respectively. This indicates that the EGCG-TGase cross-linked surimi gel structure is superior to the pure surimi gel, forming a more complex network structure.

[0139] 2.5 Effect of EGCG-TGase crosslinking on the in vitro dynamic digestion properties of water-saving rinsed fish paste gel

[0140] 2.5.1 Dry matter digestibility of digestion products

[0141] Dry matter digestibility is an important indicator for measuring the nutritional value of food, reflecting the efficiency of the absorption and utilization of dry matter in food by food.

[0142] Figure 3 Digestibility results for water-saving rinsed surimi gel crosslinked with EGCG and TGase at different digestion stages (p < 0.05). Figure 3 As shown, after a single rinse, the dry matter digestibility of the surimi gel obtained from different treatment groups ranged from 42.64% to 58.61% and from 50.80% to 69.75% after undergoing in vitro dynamic gastric and gastrointestinal digestion stages. During these two digestion stages, a consistent trend in the dry matter digestibility of the four different treatments of surimi gel was observed. The control group of surimi gel had the highest dry matter digestibility, with a pepsin digestion efficiency of approximately 60%. After further digestion by trypsin, the digestibility of the surimi gel reached approximately 70%. The digestibility of surimi gel with single or synergistic cross-linking of EGCG and TGase was significantly lower than that of the untreated group. EGCG and TGase promote myosin aggregation, and their cross-linking alters the conformation of proteins. This structural change affects the bioavailability of surimi proteins and, moreover, partially hides the protein recognition sites of digestive enzymes within the network structure, thus reducing the digestibility of the surimi gel. Therefore, the higher the degree of cross-linking, the lower the accessibility of pepsin to protein binding sites, resulting in a lower gastric degradation rate of the gel.

[0143] 2.5.2 Particle size of digestion products

[0144] D x(10) D x(50) and D x(90) D represents the particle size corresponding to the cumulative particle size distribution number of the sample reaching 10%, 50%, and 90%, respectively. (3,2) and D (4,3) This refers to the average particle size based on surface area and the average particle size based on volume. Particle size and its distribution reflect the degree to which surimi gel has been digested and broken down. Through in vitro dynamic simulations of gastric and gastrointestinal digestion, the average particle size D in the digestive solutions of surimi gel treated with four different methods can be observed. (4,3) The significant reduction indicates that the fish paste gel has been digested and broken down.

[0145] Figure 4 Table 5 shows the particle size distribution and average particle size of the surimi gel digestive fluid. A and B represent the particle size changes of the surimi gel during in vitro dynamic gastric and gastrointestinal digestion, respectively.

[0146] Table 5. Particle size of digestive fluid at different digestion stages of water-saving rinsed surimi gel cross-linked with EGCG and TGase.

[0147]

[0148] from Figure 4 As shown in Table 5, during the gastric digestion stage, the particle size of the four types of surimi gels mainly exhibited a unimodal distribution, with the particle size generally less than 1000 μm. The blank group and the EGCG cross-linked group had the smallest particle size, followed by the TGase cross-linked group, with the EGCG-TGase synergistic cross-linked group having the largest. This indicates that different gel structures lead to differences in the enzymatic hydrolysis of surimi proteins by pepsin. During the simulated gastrointestinal digestion stage, the particle size of the four groups of surimi gels showed a bimodal or trimodal distribution, with almost all particles having a particle size less than 100 μm, indicating further hydrolysis of the protein particles by trypsin. The particle size order was consistent with the gastric digestion stage: the blank group had the smallest particle size, followed by the EGCG-treated group, with the TGase cross-linked and EGCG-TGase synergistic cross-linked groups having the largest. This is because the heteropeptide bonds formed by TGase-catalyzed cross-linking of surimi proteins are resistant to pepsin hydrolysis, and the higher the degree of cross-linking, the lower the accessibility of pepsin, resulting in a lower gel degradation rate, thus leading to larger protein particle sizes in the digestive fluid.

[0149] 2.5.3 SDS-PAGE of digestion products

[0150] Figure 5SDS-PAGE of digested precipitates of surimi gels during in vitro dynamic gastrointestinal digestion was presented, revealing differences in digestion patterns among four different surimi gels during the in vitro gastric digestion phase. Results showed that in the initial digestion phase (10 min, well 1), the myosin heavy chain (MHC, approximately 220 kDa) band was extremely prominent in the untreated surimi gel (A). As digestion time increased, the MHC band gradually faded, almost completely disappearing after 30 min of gastric digestion, indicating that MHC was essentially completely degraded within the first 30 min of gastric digestion. Similar to MHC, the actin (approximately 45 kDa) band in the untreated surimi gel also showed a similar trend. In addition, peptide bands with a molecular weight of less than 35 kDa in untreated surimi gel gradually darkened in the early stage of gastric digestion (0-120 min, wells 1-5), indicating that the peptides were broken down into smaller protein particles. However, after 120 min of gastric digestion, these bands began to lighten and gradually disappeared. This is because as digestion progresses, the small molecular weight proteins are further digested and broken down, eventually becoming undetectable on SDS-PAGE.

[0151] The MHC bands in the EGCG-treated surimi gel (B) were significantly lighter than those in the control group. This is because EGCG interacts with myofibrillar proteins in the surimi, altering the types and quantities of proteins produced after digestion. The myosin light chain (MLC, approximately 25 kDa) band also gradually disappeared after 90 minutes of gastric digestion, compared to the control group. This indicates that the highly cross-linked network structure in the surimi gel negatively impacts the protein digestion process, hindering the formation of protein breakdown products. In the EGCG-treated surimi gel, bands with molecular weights below 15 kDa initially darkened and then lightened.

[0152] For TGase-treated surimi gel (C), the MHC bands were significantly lighter before being broken down by pepsin, and some accumulation occurred near the sample wells. This indicates that TGase treatment promoted the aggregation of surimi proteins, forming some oversized aggregates. During digestion, the MHC band intensity of these TGase-treated surimi gels increased slightly, but the band intensity weakened to the point of complete disappearance after 60 minutes of gastric digestion. This suggests that the MHC degradation rate in TGase-treated surimi gels was significantly lower than that in the untreated group, indicating that TGase also hindered the digestion and degradation of proteins in surimi gels, thus prolonging the digestion time.

[0153] For EGCG-TGase co-treated surimi gel, there was significant accumulation at the loading wells during the initial digestion phase, indicating a high degree of cross-linking and the formation of numerous large molecular weight aggregates. Although the MHC bands remained diffuse under the continuous degradation of pepsin, the high molecular weight bands gradually faded while the low molecular weight bands gradually deepened in color, indicating that large peptides were gradually hydrolyzed into smaller protein particles by pepsin.

[0154] The surimi gel undergoes a period of digestion in an in vitro simulated stomach within a dynamic digestive system before entering the duodenum. Figure 5 (a)-(d) It can be observed that the MHC and actin bands in the untreated surimi gel are almost invisible to the naked eye, while the MLC band is relatively clear from the beginning of gastrointestinal digestion (30 min), gradually fading and disappearing after 120 min. The 15 kDa low molecular weight fragments are very dark in color initially, indicating that the digestion reaction of the untreated surimi gel occurs rapidly, and the color lightens after 210 min. In the early stage of intestinal digestion, the EGCG-treated surimi gel shows obvious accumulation in the sample wells, indicating that digestion is not thorough in the gastric stage, and the gel still contains a lot of large molecules. The MLC and smaller peptide bands gradually darken in color after 120 min, indicating that the EGCG-crosslinked surimi gel begins to be digested and broken down into small protein molecules at this time, and the digestion rate is slower compared with the blank group. The change pattern of the large peptide bands in the TGase-crosslinked surimi gel is similar to that of the EGCG group. After 90 min of intestinal digestion, the bands below 15 kDa gradually become clearer, and the color gradually darkens with the increase of digestion time. The EGCG-TGase co-crosslinked surimi gel exhibited some tailing near the sample wells, indicating that some large protein molecules were not completely digested. The protein bands were lighter in color and fewer in number compared to the other three groups. This may be because the digestible portion of the co-crosslinked surimi gel was broken down into smaller proteins during gastric digestion, leaving a highly crosslinked and densely packed gel structure that was difficult to digest, resulting in a reduced content of digestible proteins in the precipitate. However, SDS-PAGE results alone cannot fully reveal the specific composition of the digested products or the changes in the digestion rate during the digestion process; therefore, further research is needed.

[0155] 2.5.4 Analysis of the free amino acid composition of the digestion products

[0156] The release amount and composition of free amino acids in digestive fluids are important indicators for evaluating protein bioavailability. The types and contents of amino acids in water-saving rinsed fish paste gel cross-linked with EGCG and TGase after in vitro dynamic simulated digestion were determined using a fully automated amino acid analyzer. The results are shown in Table 6.

[0157] Table 6. Amino acid composition of the digestive fluid of water-saving rinsed surimi gel crosslinked with EGCG and TGase.

[0158]

[0159] As shown in Table 6, the total amino acid content in the surimi gel digestion solution cross-linked with EGCG alone was the highest at all digestion stages. The total amino acid content was 287.61 μg / mL in the gastric digestion stage and 193.8 μg / mL in the intestinal digestion stage. The theoretical total amino acid content should be higher after simulated intestinal digestion, but the actual result was lower because the injection volume of intestinal digestion solution was about twice that of gastric digestion solution during in vitro simulated digestion, thus diluting the amino acid concentration in the intestinal digestion solution. The total amino acid content in the surimi gel digestion solution of the blank group (1) and the TGase cross-linked group ranked second and third at different digestion stages. The EGCG-TGase synergistic cross-linked group was always the lowest, but after gastrointestinal digestion, there was no significant difference between it and the third-ranked TGase group. The total free amino acid content of the surimi gel cross-linked with EGCG was the highest, indicating that although EGCG reduced protein digestibility, it promoted amino acid release. TGase catalyzes the reaction between the glutamine (Glu)-γ-amide group and the ε-amino group of lysine (Lys) to form an isopeptide bond. The ε-(γ-Glu)-lys bond is resistant to pepsin and trypsin. TGase cross-links the surimi gel to form a dense gel network structure, thereby inhibiting the action of pepsin. However, after further intestinal digestion, the network structure of the surimi gel gradually disintegrates, and the digestibility increases in the later stages of gastrointestinal digestion. This indicates that although TGase reduces the digestibility in the early gastric digestion stage, the total amount of free amino acids released in the digestive fluid of the EGCG-TGase cross-linked surimi gel is not reduced, and the EGCG-TGase synergistic cross-linking does not reduce the nutritional value of the surimi gel.

[0160] During gastric digestion, digestive juices are rich in phenylalanine, histidine, and arginine. During intestinal digestion, digestive juices are rich in phenylalanine, histidine, and arginine, while the content of lysine and serine increases significantly compared to the gastric digestion stage. Throughout the digestion process, basic amino acids (histidine, arginine, and lysine) are more easily broken down and released than acidic amino acids. During digestion, pepsin mainly acts on hydrophobic amino acid residues, such as phenylalanine and leucine. EGCG-TGase cross-linking reduces the hydrophobic interactions of myosin gel; therefore, the content of phenylalanine, which was the most abundant amino acid in the control group during surimi gel digestion, decreases after cross-linking. Trypsin mainly acts on lysine and arginine. After gastrointestinal digestion, the lysine and arginine content in the EGCG and TGase cross-linked groups alone and the EGCG-TGase synergistic cross-linked groups were significantly higher than in the uncross-linked group, indicating that the dense network structure formed by cross-linking has been destroyed, exposing the trypsin action sites inside the protein and increasing the release of amino acids.

[0161] The nutritional value of dietary protein is usually evaluated using EAA / TAA (essential amino acids / total amino acids) and NAA / TAA (non-essential amino acids / total amino acids). During gastric digestion, the EAA / TAA ratios of the four groups of surimi gel were 71.36%, 67.64%, 70.38%, and 65.79%, respectively. During intestinal digestion, the EAA / TAA ratios were 73.07%, 67.43%, 79.67%, and 70.94%, respectively. The free amino acids detected in the digestive fluids of all four groups of surimi gel showed EAA / TAA ratios greater than the FAO / WHO standard for evaluating amino acids in proteins (EAA / TAA = 40%), indicating that surimi gel is a high-quality protein source rich in essential amino acids.

[0162] 2.5.5 Digestion kinetics model of fish paste gel

[0163] Figure 6 This displays the release of free amino acids and their fitted curves (Logistics model) during in vitro dynamic gastric and intestinal digestion of water-saving washed surimi gel crosslinked with EGCG and TGase. Where A(a): surimi gel; B(b): 1.8 × 10⁻⁶. -3 g / g pro EGCG cross-linked surimi gel; C(c): 10 U / g pro TGase-crosslinked surimi gel; D(d): 1.8 × 10⁻⁶ -3 g / g pro EGCG synergistic 10 U / g pro TGase-crosslinked surimi gel; uppercase letters represent gastric digestion processes, and lowercase letters represent intestinal digestion processes. For example... Figure 6As shown, the release of free amino acids followed an S-shaped curve with the digestion time of the surimi gel. This S-shaped curve represented three stages: from stable to increasing and then stabilizing. Compared to gastric digestion, the initial digestibility of the surimi gel increased faster during the initial stage of intestinal digestion. During the dynamic gastric digestion stage, the initial amino acid content of the blank group surimi gel was the highest at 0.98 μmol / mL. The release rate was relatively fast in the first 60 minutes of digestion, and the curve flattened out in the later stages of gastric digestion, with a smaller increase in amino acid concentration. The EGCG and TGase cross-linked groups and the EGCG-TGase synergistic cross-linked group showed a slower increase in amino acid concentration in the early stages of gastric digestion, but the release rate increased after 60 minutes. After gastric digestion, the amino acid concentrations were all higher than those in the blank group. During the intestinal digestion stage, the amino acid release curves of the four groups of surimi gels were quite similar, with the EGCG-TGase synergistic cross-linked group showing the slowest increase in release rate.

[0164] Logistic regression is a statistical method widely used in classification problems, often to predict the probability of a certain situation occurring under different independent variables. Table 7 shows the in vitro dynamic gastric and intestinal digestion kinetic parameters (Logistic model) of water-saving washed surimi gels crosslinked with EGCG and TGase. As can be seen from Table 7, the R² values ​​of the kinetic parameters for different surimi gels are all greater than 0.98, indicating a high degree of fit. This suggests that the surimi gels undergo gastric and intestinal digestion, and the release of free amino acids is consistent with the digestion kinetic model of the gastric digestion stage. The Logistic model can describe the gastric and intestinal digestion processes of surimi gels. X0 represents the initial free amino acid content during digestion. At t=0 min, although pepsin is inactive, gastric juice is acidic. Some amino acids are hydrolyzed. The X0 values ​​of surimi gels from different treatment groups showed significant differences during the gastric digestion stage, with the TGase-treated surimi gel having a maximum value of 1.00 μmol / mL, while the values ​​were close to 0 during the intestinal digestion stage. The M value (i.e., the free amino acid content at the end of digestion) of the EGCG-synergistic TGase cross-linked surimi gel was the highest value of 14.97 μmol / mL, which was also the highest value among all treatment groups. When x (t M / 2 When )=M / 2, the slope of the model fitting curve will change, and the t for each group of surimi gels will change. M / 2 All values ​​were positive, indicating that the digestibility of surimi gel first increased and then decreased over time during digestion, and the surimi gel co-crosslinked with EGCG-TGase was significantly longer than that of the control group.

[0165] Table 7. In vitro dynamic gastric and intestinal digestion kinetics of water-saving rinsed surimi gel crosslinked with EGCG and TGase.

[0166]

[0167] Pseudo-first-order kinetics is often used to describe the rate equations of certain chemical reactions, where the rate, although dependent on the concentrations of two or more reactants, can be simplified to appear to depend only on the concentration of one reactant. This situation typically occurs when the concentration of one reactant is much higher than that of the others, making its concentration change very little during the reaction and thus considered constant. Pseudo-second-order kinetics is a theoretical model describing adsorption processes and is commonly used in fields such as chemical kinetics and biophysics. In this study, the initial amino acid content is treated as constant during digestion. Pseudo-first-order and pseudo-second-order kinetic equations are used to fit the kinetic curves of free amino acid release, aiming to explore the dynamic digestion kinetics of EGCG and TGase-treated surimi gels during digestion.

[0168] Figure 7 Table 8 shows the in vitro dynamic gastric and intestinal digestion kinetics (pseudo-first-order kinetics) of the water-saving rinsed surimi gel crosslinked with EGCG and TGase during gastric and intestinal digestion. The table also includes the in vitro dynamic gastric and intestinal digestion kinetic parameters (pseudo-first-order kinetics) of the water-saving rinsed surimi gel crosslinked with EGCG and TGase. Figure 7 As shown in Table 8, the digestive kinetics of surimi gel after dynamic intestinal digestion showed a higher degree of agreement with the pseudo-first-order kinetic equation, with its R... 2 The values ​​all exceeded 0.98. Furthermore, the reaction rate constant p of the TGase-treated surimi gel was the highest at 0.0112, indicating that the cross-linking of TGase with proteins in the surimi resulted in a faster digestion rate. After fitting the amino acid content released during gastric digestion of the surimi gel, the R value of the blank group was significantly higher. 2 Less than 0.95, R in other treatment groups 2 Although the value is greater than 0.95, the fitted curve does not converge, therefore the gastric digestion kinetics of surimi gel does not conform to the pseudo-first-order equation.

[0169] Table 8. In vitro dynamic gastric and intestinal digestion kinetics of water-saving rinsed surimi gel crosslinked with EGCG and TGase.

[0170]

[0171] Figure 8 The table shows the release of free amino acids and fitted curves (pseudo-second-order kinetics) during the in vitro dynamic gastric and intestinal digestion of the water-saving surimi gel crosslinked with EGCG and TGase. Table 9 shows the in vitro dynamic gastric and intestinal digestion kinetic parameters (pseudo-second-order kinetics) of the water-saving surimi gel crosslinked with EGCG and TGase. Figure 8 As shown in Table 9, only the surimi gel treated with TGase and EGCG-TGase exhibited a release pattern of free amino acids following pseudo-second-order kinetics after intestinal digestion, but its R...2 The values ​​are all less than the pseudo-first-order kinetic equation, indicating that the pseudo-second-order kinetics have a low degree of fit with the amino acid release during the in vitro dynamic simulation of surimi gel digestion, and the actual digestion process of surimi gel cannot be predicted based on this equation.

[0172] Table 9. In vitro dynamic gastric and intestinal digestion kinetics of water-saving rinsed surimi gel crosslinked with EGCG and TGase.

[0173]

[0174] Based on the above-mentioned effects of the water-saving rinsing fish paste gel crosslinked with EGCG and TGase, the embodiments of this application also provide the application of the fish paste gel in the following aspects:

[0175] 1. Application in the preparation of digestible, sustained-release protein foods. As described, the surimi gel exhibits unique sustained-release properties during simulated digestion: the release kinetics of free amino acids during the gastrointestinal digestion phase conform to the Logistic model, and the key parameter—the digestive half-life—is significantly longer than that of uncrosslinked ordinary surimi gel. This demonstrates its ability to effectively slow down the excessively rapid hydrolysis rate of proteins in the early stages of digestion, making it suitable for nutritional foods or special medical purpose formulations that require a slow and continuous supply of amino acids.

[0176] 2. Application in the preparation of high water-holding capacity gel foods. Experimental results show that, especially for surimi raw materials that have undergone one rinse, the water-holding capacity (WHC) of the gel prepared by the method of this invention can be increased to over 95%. Products targeted by this application exhibit excellent juiciness, tenderness, and yield, making them suitable for surimi products with high requirements for texture and water retention.

[0177] Based on this, this application also provides an application of the composition in the preparation of surimi gel products, the composition comprising EGCG and TGase. The above embodiments demonstrate that it is the synergistic use of this composition (rather than any single component therein) that simultaneously achieves the aforementioned dual effects of "improving the overall quality of the gel" and "regulating digestive properties".

[0178] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for preparing an EGCG-TGase crosslinked water-saving fish paste gel, characterized in that, Includes the following steps: S1. Raw material preparation: Obtain fish meat, and after meat extraction and chopping, obtain fish paste raw material; S2. Water-saving rinsing: The fish paste raw material is rinsed no more than twice to obtain rinsed fish paste; S3. Synergistic cross-linking: EGCG and TGase are added sequentially to the rinsed fish paste and mixed by beating, and the synergistic effect of EGCG and TGase is used to induce cross-linking of fish paste proteins. S4. Gel forming: The fish paste processed in step S3 is filled and formed. First, it is heated at 30-50 °C to gel the protein, and then heated at 80-100 °C to set and mature the gel, thus obtaining the fish paste gel.

2. The preparation method according to claim 1, characterized in that, The specific steps of step S1 are as follows: After the fresh fish is slaughtered, the scales, head and tail are removed. Then the white meat from the belly is manually removed. The fish meat is washed and drained. The fish meat is then chopped for 5 minutes using a chopper to obtain fish paste. The temperature of the entire preparation process is below 10 ℃.

3. The preparation method according to claim 1, characterized in that, The specific steps of step S2 are as follows: According to the mass-volume ratio of 1:5, add 4 ℃ pre-cooled deionized water to the fish paste raw material obtained by S1 and rinse 1 to 2 times, each time for 5 minutes. In the last rinse, add 0.3% NaCl of the fish paste raw material mass to the ice water. After each rinse, use gauze to dehydrate and filter, and control the moisture content to be below 78%. In this way, rinsed fish paste with different degrees of rinsing is obtained.

4. The preparation method according to claim 1, characterized in that, In step S3, the amount of EGCG added is 1.0×10⁻³~3.0×10⁻³ g / g pro The amount of TGase added is 5~20 U / gpro .

5. The preparation method according to claim 1, characterized in that, In step S3, before adding EGCG and TGase, salt is added to the rinsed fish paste, and the amount of salt added is 1.5%-3.5% of the fish paste mass.

6. The preparation method according to claim 1, characterized in that, In step S4, the protein gelation time is 0.5-2 hours; the gel setting and ripening time is 20-40 minutes.

7. A fish paste gel, characterized in that, Prepared by the preparation method according to any one of claims 1-6.

8. The use of the surimi gel according to claim 7 in the preparation of digestible, slow-release protein foods.

9. The use of the surimi gel of claim 7 in the preparation of highly water-holding gel foods, wherein the surimi gel has a water-holding capacity of not less than 90%.

10. The use of a composition in the preparation of a fish paste gel product, said composition comprising EGCG and TGase.

Images