A composite coating solution for low-temperature cooked tuna and a preparation method and application thereof

Abstract

This invention pertains to food preservation materials and processing technologies, specifically relating to a composite coating liquid for low-temperature cooked tuna, its preparation method, and its application. The invention discloses a method for preparing the composite coating liquid for low-temperature cooked tuna, comprising the following steps: dissolving carboxymethyl chitosan in water, then adding a plasticizer and mixing to obtain a CMCS solution; dissolving sodium alginate in water, then adding a plasticizer and mixing to obtain an SA solution; first adding ergothioneine to the SA solution, then adding the CMCS solution and stirring continuously to obtain the composite coating liquid. This invention also provides the application of the composite coating liquid: for the preservation of vacuum-cooked tuna at low temperatures.

Description

Technical Field

[0001] This invention pertains to food preservation materials and processing technologies, specifically relating to a composite coating liquid for low-temperature cooked tuna, its preparation method, and its application. Background Technology

[0002] Vacuum sous-vide cooking technology can cook yellowfin tuna under mild conditions, preserving the original texture, nutrients, and natural flavor of the fish to the maximum extent, and has become an important development direction for high-end seafood processing. However, tuna products cooked at low temperatures are prone to quality deterioration during refrigerated distribution at 4°C due to low protein denaturation, high moisture content, and strong residual activity of endogenous enzymes. This leads to accelerated lipid oxidation, rapid microbial proliferation, protein degradation, texture deterioration, and loss of flavor substances, ultimately resulting in short shelf life and reduced commercial value, severely restricting the industrialization of vacuum sous-vide tuna products. Therefore, developing safe, efficient, and environmentally friendly preservation technologies to extend the refrigerated shelf life of cooked tuna is of great practical significance for improving the high-value processing level of tuna.

[0003] Edible composite coatings, as a green, mild, and efficient biopreservation technology, can form a semi-permeable barrier layer on the surface of food, effectively inhibiting moisture migration, oxygen penetration, and microbial contamination. Simultaneously, they can be loaded with natural active substances to achieve functional preservation such as anti-oxidation and antibacterial properties, demonstrating broad application prospects in the field of aquatic product preservation. Sodium alginate (SA) and carboxymethyl chitosan (CMCS) are both widely available, biodegradable, and highly safe natural polysaccharide substrates. Both exhibit good film-forming properties and compatibility, and carboxymethyl chitosan itself carries cationic groups, possessing certain antibacterial effects, making them ideal matrices for constructing edible composite coatings.

[0004] The invention CN119505379A, entitled "An edible antibacterial preservation film with ROS response and its preparation method", discloses that the edible antibacterial preservation film is composed of sodium alginate (SA), carboxymethyl chitosan (CS), ROS responsive groups (TK-NH2) and antibacterial small molecules (AB).

[0005] The invention CN121400496A, "Preparation Method of Liquid Preservative Film Loaded with Antibacterial and Antioxidant from Bamboo Nanocellulose", describes a method for preparing a functional spray by compounding sodium alginate, carboxymethyl chitosan, and bamboo fiber nanodispersion, and adding ε-polylysine and bamboo polyphenols. After spraying, calcium chloride solution is applied, and a stable, transparent, and strongly adhesive hydrogel film is rapidly formed through ionic cross-linking.

[0006] The invention CN107484814A, entitled "Composite Film-Forming Preservative," discloses the following components: 1.5-2 parts carboxymethyl chitosan, 1.5-2 parts sodium alginate, 2-3 parts tea polyphenols, 0.5-0.6 parts ε-polylysine, 0.2-0.3 parts vitamin C, 0.05-0.06 parts nano-TiO2, 2-2.6 parts bone polypeptide, 0.15-0.2 parts nisin, 0.3-0.5 parts sodium phytate, 0.3-0.5 parts sodium sorbate, 0.5-0.8 parts deodorizing agent, 0.3-0.6 parts plant extract, 1.2-1.5 parts film-forming agent, 0.5-0.6 parts emulsifier, and 90-100 parts distilled water. The beneficial effects are: simple to make, good film-forming effect and fast speed, with super deodorizing, antibacterial, antioxidant, enzyme activity inhibiting and delaying the fish meat from reaching the limit pH, etc. It is a compound film-forming preservative that can remove fishy smell, is highly efficient, safe and has no toxic residues.

[0007] Ergothioneine (EGT) is a stable, highly effective, and safe natural sulfur-containing antioxidant. It can effectively scavenge free radicals, inhibit lipid oxidation, and shield against ultraviolet radiation. It also has a certain inhibitory effect on some foodborne pathogens. It is commonly used for the preservation of fruits and vegetables, and can also be used for the low-temperature preservation of freshwater fish.

[0008] The invention CN117397721A, "A Compound Preservative Containing Ergothioneine and Its Application in Low-Temperature Preservation of Freshwater Fish," discloses that: the use of ergothioneine for low-temperature preservation of freshwater fish has proven that ergothioneine can be used as a low-temperature preservative for freshwater fish. The content of ergothioneine in the low-temperature preservative is 0.01%~5% (preferably 0.1%~2%, with 0.5% showing better results, 1.0% showing the best results, and 2.0% showing a slight decrease in effect); it also demonstrates that ergothioneine can be used in combination with Nisin as a low-temperature preservative for freshwater fish, and that the two have a good synergistic effect.

[0009] Currently, there is considerable research on the application of polysaccharide-based composite coatings in fresh aquatic products. However, research on the systematic exploration of the effects of combining vacuum low-temperature cooking technology with SA / CMCS / EGT active composite coatings on maintaining the quality, regulating microorganisms, and preserving the flavor of cooked yellowfin tuna remains lacking. Furthermore, the antibacterial mechanism, antioxidant pathways, microbial community succession patterns, and flavor compound changes of this composite coating on refrigerated tuna products are still unclear, limiting its application and promotion in actual production. Summary of the Invention

[0010] The technical problem to be solved by the present invention is a composite coating liquid for low-temperature cooked tuna, its preparation method and application.

[0011] To address the aforementioned technical problems, this invention provides a method for preparing a composite coating liquid for low-temperature cooked tuna, comprising the following steps: (1) Preparation of CMCS solution: Dissolve 1.5 ± 0.2 g of carboxymethyl chitosan (CMCS) in 100 mL of water (distilled water), then add plasticizer and mix well to obtain a CMCS solution; (2) Preparation of SA solution: Dissolve 1.5 ± 0.2 g sodium alginate (SA) in 100 mL of water (distilled water), then add plasticizer and mix well to obtain SA solution; (3) Preparation of composite coating liquid: Set the volume ratio of SA solution to CMCS solution to 1:1; first add ergothioneine to the SA solution, then add CMCS solution and stir continuously to obtain a composite coating solution (a composite coating solution for low-temperature cooked tuna), wherein the concentration of ergothioneine in the composite coating solution is 0.1~0.3 mg / mL.

[0012] As an improvement to the preparation method of the composite coating liquid of the present invention: In step (1): 1 ± 0.1 mL of glycerol is added as a plasticizer; In step (2): 1 ± 0.1 mL of glycerol is added as a plasticizer.

[0013] As a further improvement to the preparation method of the composite coating liquid of the present invention: The degree of substitution of the carboxymethyl chitosan is ≥80%; the ergothionein is L-ergothionein with a purity of ≥99%.

[0014] The present invention also provides a composite coating liquid prepared by any of the above methods.

[0015] The present invention also provides the use of the composite coating liquid for the preservation of tuna cooked at low temperature (vacuum low temperature).

[0016] An improvement to the use of the composite coating liquid of the present invention: low-temperature cooked tuna is immersed in the composite coating liquid (the low-temperature cooked tuna is completely submerged) for 2 ± 0.5 minutes; thereby forming a coating on the surface of the low-temperature cooked tuna with the composite coating liquid.

[0017] Note: After soaking, remove and drain. The composite coating solution forms a coating on the surface of the low-temperature cooked tuna.

[0018] An improvement to the use of the composite coating liquid of the present invention: Low-temperature cooked tuna refers to tuna meat that has been vacuum-cooked at 55±2°C (heated to a core temperature of 55±2°C and then maintained for 30±2 minutes).

[0019] Instructions: After coating, the yellowfin tuna is placed in a food-grade polyethylene-nylon vacuum bag, vacuum-sealed, and then refrigerated at 4°C.

[0020] This invention addresses the problem of short shelf life caused by lipid oxidation, microbial proliferation, and protein degradation in tuna products after low-temperature cooking during refrigerated distribution. The core technical approach is as follows: using sodium alginate (SA) and carboxymethyl chitosan (CMCS) as film-forming matrices (volume ratio 1:1), introducing ergothioneine (EGT) as an active substance, and glycerol as a plasticizer, a composite coating solution is constructed to immerse and coat vacuum-cooked tuna.

[0021] The beneficial effects of this invention are: It significantly slows down pH rise, browning, and textural deterioration; reduces TVB-N and TBARS values; inhibits the proliferation of Firmicutes and Bacillus; blocks the formation of oxidative odor substances such as hexanal; and extends the refrigerated shelf life by 4 days.

[0022] This invention employs a solution casting method to prepare an active composite membrane of sodium alginate / carboxymethyl chitosan / ergothioneine (SA / CMCS / EGT). The effects of different EGT addition amounts on the rheological properties, mechanical properties, thermal stability, microstructure, UV blocking ability, antioxidant properties, and antibacterial properties of the composite membrane were systematically studied. Furthermore, molecular docking technology was used to analyze the interaction patterns between EGT and key bacterial proteins at the molecular level, elucidating its potential antibacterial mechanism. Based on this, the SA / CMCS / EGT composite coating with the best overall performance was selected and applied to yellowfin tuna cooked at 55℃ under vacuum. The changes in pH, color, texture, lipid oxidation (TBARS), protein degradation (TVB-N), total bacterial count, microbial community structure, and volatile flavor compounds of the fish were dynamically monitored under 4℃ refrigeration conditions. The preservation effect and mechanism of action of this composite coating on vacuum-cooked tuna products were comprehensively evaluated. This can provide a theoretical basis and technical support for the application of ergothionein-based active composite coatings in the field of cooked aquatic products, and at the same time provide new ideas and methods for improving the quality stability and extending the shelf life of vacuum low-temperature cooked tuna products.

[0023] This invention introduces ergothionein into the sodium alginate / carboxymethyl chitosan composite system, which can give full play to the synergistic advantages of polysaccharide film-forming properties and EGT antioxidant and antibacterial properties, and prepare an edible active composite coating with excellent mechanical properties, moderate barrier properties and outstanding functional activity, providing efficient "secondary preservation" for vacuum low-temperature cooked tuna products.

[0024] The main technical advantages of this invention are: 1. This invention employs a process of separately preparing carboxymethyl chitosan (CMCS) solutions and sodium alginate (SA) solutions, followed by mixing and compounding. This avoids uneven dissolution, localized overconcentration, and phase separation caused by directly mixing two high-viscosity polysaccharides, ensuring a stable and uniform coating solution and effectively protecting the activity of ergothioneine (EGT). Existing technologies often directly mix and dissolve CMCS and SA powders. Due to the high viscosity and significant difference in solubility of the two polysaccharides, localized agglomeration, incomplete dissolution, or phase separation easily occur, leading to uneven distribution of ergothioneine and loss of activity. This invention, by separately dissolving and independently preparing the two polysaccharide solutions, and then gently mixing them in a specific ratio, effectively avoids agglomeration and phase separation, achieving a stable coating solution system, high EGT activity retention, and strong process controllability, making it suitable for large-scale production applications.

[0025] 2. This invention introduces EGT into the SA / CMCS composite membrane system for the first time, and clarifies that EGT and SA / CMCS mainly interact through hydrogen bonds without changing the chemical structure, thus constructing a multifunctional composite membrane liquid with strong free radical scavenging, ultraviolet shielding and certain antibacterial activity.

[0026] While SA and CMCS are commonly used in coating preservation, the stability, release behavior, and interaction between EGT and polysaccharides in this composite membrane matrix lack systematic research. Furthermore, the addition of EGT may affect the viscosity, uniformity, and antibacterial properties of the film-forming solution, leading to uncertainties in membrane performance. Therefore, despite the reduced cost of EGT, technological exploration remains limited, presenting technical obstacles. This invention considers combining these three components because SA is an anionic polysaccharide rich in hydroxyl groups, while CMCS, due to the introduction of carboxymethyl groups, has its amino groups mostly deprotonated under neutral conditions, and its carboxymethyl groups are negatively charged, resulting in a molecular chain rich in hydroxyl, amino, and acetylamino groups. EGT contains a strong hydrogen bond donor (NH) on its imidazole ring and a hydrogen bond acceptor (sulfur atom) on its thionyl group. Therefore, based on the molecular characteristics of the above three materials, a reasonable hypothesis is proposed: when EGT is introduced, the NH and C=S of EGT can form hydrogen bonds with the hydroxyl and amino groups of SA and CMCS, respectively. Since hydrogen bonding is milder than electrostatic interaction, the resulting film solution is less likely to precipitate and is more likely to obtain a uniform composite film casting solution.

[0027] 3. This invention further elucidates the interaction mode between EGT and key bacterial proteins that cause aquatic spoilage, and for the first time applies this composite membrane solution to the 4°C cold storage preservation of yellowfin tuna obtained by vacuum low-temperature cooking at 55°C. Unlike broad-spectrum sterilization, this composite membrane solution can target and inhibit the proliferation of dominant spoilage bacteria such as Firmicutes and Bacillus, and block the generation pathway of oxidative off-odor substances such as hexanal. This allows the composite membrane solution to successfully extend the shelf life of yellowfin tuna by 4 days, delay the increase in product pH, browning, and textural deterioration, and retain the characteristic flavor substances of fresh fish to the greatest extent, achieving precise control of product flavor and quality. Attached Figure Description

[0028] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] Figure 1 UV-Vis spectra of EGT coating solutions with different addition amounts.

[0030] Figure 2 The figure shows the ABTS radical scavenging ability of different amounts of EGT coating solution; different lowercase letters (a, b, c, d) in the figure indicate that the ABTS radical scavenging ability of different amounts of EGT coating solution is significantly different (p < 0.05).

[0031] Figure 3 The antibacterial effect of different amounts of EGT coating solution on bacteria: Figure 3 In the diagram: A represents the antibacterial effect against Staphylococcus aureus; B represents the antibacterial effect against Escherichia coli. Note: Different lowercase letters (a, b, c, d) indicate that the antibacterial effect of different amounts of EGT coating solution varies significantly (p < 0.05).

[0032] Figure 4 The effect of different treatments on the pH of chilled tuna meat was investigated. Different uppercase letters indicate significant differences between different time points within the same treatment group (P < 0.05); different lowercase letters indicate significant differences between different treatments within the same group at the same time point (P < 0.05). Intra-group comparisons were made between AC and AE, and between VC and VE.

[0033] Figure 5 The effect of different processing methods on the color difference of refrigerated tuna flesh: Figure 5 In the diagram: A represents the effect of different storage days on the L* (brightness) of refrigerated tuna meat; B represents the effect of different storage days on the a* (redness) of refrigerated tuna meat; C represents the effect of different storage days on the b* (yellowness) of refrigerated tuna meat. Note: Different uppercase letters indicate significant differences between different time points within the same treatment group (P < 0.05); different lowercase letters indicate significant differences between different treatments at the same time point within the group (P < 0.05). Within-group comparisons: AC vs. AE, VC vs. VE.

[0034] Figure 6 The effects of different processing methods on the texture of refrigerated tuna flesh: Figure 6 In the middle section: A represents the effect of different storage days on the hardness of chilled tuna flesh; B represents the effect of different storage days on the elasticity of chilled tuna flesh; C represents the effect of different storage days on the chewiness of chilled tuna flesh; D represents the effect of different storage days on the stickiness of chilled tuna flesh. Note: Different uppercase letters indicate significant differences between different time points within the same treatment group (P < 0.05); different lowercase letters indicate significant differences between different treatments at the same time point within the group (P < 0.05). Within-group comparisons: AC vs. AE, VC vs. VE.

[0035] Figure 7 To investigate the effects of different treatment methods on lipid oxidation in refrigerated tuna meat.

[0036] Figure 8 The effects of different processing methods on TVB-N in refrigerated tuna meat.

[0037] Figure 9 To investigate the effects of different treatment methods on the total bacterial count in refrigerated tuna.

[0038] Figure 10 Statistical analysis of the alpha diversity index of refrigerated tuna meat under different processing methods; Figure 10 In the middle: A: Observed features are used to characterize the number of species actually detected in the sample; B: Chao1 index is used to assess species richness; C, D: Shannon and Simpson indices together reflect species diversity.

[0039] Figure 11 To investigate the effects of different treatment methods on the microbial species diversity richness of refrigerated tuna.

[0040] Figure 12 This is a schematic diagram of the PCA analysis results.

[0041] Figure 13 This is a graph showing the changes in the relative abundance of microbial communities at the phylum level during the final stage of fish storage.

[0042] Figure 14 This is a graph showing the changes in the relative abundance of the microbial community at the genus level during the final stage of fish storage.

[0043] Figure 15 This is a scatter plot of ROAV relative odor activity values.

[0044] Figure 16 Heatmaps for cluster analysis of volatile substances treated by different methods. Detailed Implementation

[0045] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0046] Reagents used in the examples: Sodium alginate (SA), carboxymethyl chitosan (CMCS, degree of substitution ≥80%), and glycerol were purchased from Maclean's Biochemical Co., Ltd.; ergothioneine (EGT, L-ergothioneine purity ≥99%) was purchased from Bloomage Biotechnology Co., Ltd. TSA tryptone soybean agar was purchased from Qingdao Haibo Biotechnology Co., Ltd.; Staphylococcus aureus (ATCC6538) and Escherichia coli (ATCC11775) were purchased from Hunan Fenghui Biotechnology Co., Ltd. Plate count agar (PCA) was used (Qingdao Haibo Biotechnology Co., Ltd.). All other chemicals and reagents used were of analytical grade.

[0047] Example 1: Preparation method of EGT active composite coating liquids of different concentrations and their coatings, the following steps are performed in sequence: (1) Preparation of CMCS solution: Weigh 1.5 g of carboxymethyl chitosan (CMCS) and add it to 100 mL of distilled water. Place it on a magnetic stirrer at 50℃ and stir until the carboxymethyl chitosan dissolves. Then add 1 mL of glycerol as a plasticizer, mix well, and obtain CMCS solution.

[0048] (2) Preparation of SA solution: Weigh 1.5 g of sodium alginate (SA) and add it to 100 mL of distilled water. Stir continuously on a magnetic stirrer until the sodium alginate is completely dissolved. Add 1 mL of glycerol as a plasticizer, mix well, and obtain SA solution.

[0049] (3) Preparation of composite coating liquid: divided into four groups (control group and 3 experimental groups).

[0050] Control group (SA-CMCS, denoted as CK): The above SA solution and CMCS solution were directly mixed at a volume ratio of 1:1 and stirred continuously for 30 min.

[0051] Experimental Group 1 (EGT-0.1): Ergothioneine was added to the SA solution, followed by CMCS solution, and the mixture was stirred for 30 min to obtain a composite coating solution (SA-CMCS solution). The concentration of ergothioneine in the composite coating solution was 0.1 mg / mL. The volume ratio of SA solution to CMCS solution was 1:1. The resulting composite coating solution was named SA-CMCS-EGT-0.1 mg / mL.

[0052] Experimental Group 2 (EGT-0.2): Ergothioneine was introduced into the SA solution, and then CMCS solution was added and stirred continuously for 30 min to obtain a composite coating solution (SA-CMCS solution). The concentration of ergothioneine in the composite coating solution reached 0.2 mg / mL. The volume ratio of SA solution to CMCS solution was 1:1. The resulting composite coating solution was named SA-CMCS-EGT-0.2 mg / mL.

[0053] Experimental Group 3 (EGT-0.3): Ergothioneine was introduced into the SA solution, and then CMCS solution was added and stirred continuously for 30 min to obtain a composite coating solution (SA-CMCS solution). The concentration of ergothioneine in the composite coating solution reached 0.3 mg / mL. The volume ratio of SA solution to CMCS solution was 1:1. The resulting composite coating solution was named SA-CMCS-EGT-0.3 mg / mL.

[0054] Experiment: The composite coating solutions obtained from the control group and experimental groups 1 to 3 were subjected to experiments to determine their UV resistance, ABTS free radical scavenging ability, and antibacterial properties.

[0055] Composite film preparation method: The composite coating solution was poured into a petri dish for routine degassing, and then placed in a 40℃ incubator for drying for 48 h. After the film was completely dry, it was removed from the plate, cooled to room temperature, and then peeled off. The resulting film was named the composite film. It was stored indoors at a temperature of 25±2℃ and a relative humidity of 50±2%.

[0056] Specifically as follows: 1. UV resistance Experimental methods: The UV shielding performance of the film was measured using a UV-Vis spectrophotometer in the wavelength range of 200–800 nm. The film was cut into 2 cm × 2 cm samples (thickness approximately 0.03 mm, with no significant difference between treatments), fixed inside the cuvette, and tested using an empty cuvette as a reference.

[0057] Experimental results: The UV and visible light transmittance of the SA / CMCS / EGT composite film are as follows: Figure 1As shown, the SA-CMCS composite film without ergothioneine exhibits high transmittance in the ultraviolet region (200–400 nm), indicating its limited ability to shield against ultraviolet radiation. After adding EGT, the transmittance of the film in the ultraviolet band decreased significantly, and the degree of decrease was positively correlated with the amount of EGT added. Adding 0.3 mg / ml EGT effectively shielded ultraviolet radiation, reducing it by approximately 30%–35% compared to the control group. In the 200–280 nm range, the composite film with 0.3 mg / ml EGT showed no peak, which is attributed to the synergistic effect between sodium alginate and carboxymethyl chitosan with ergothioneine, resulting in zero transmittance in this range.

[0058] II. ABTS free radical scavenging ability Experimental methods: ABTS was dissolved in water to prepare a 7 mM solution, which was then mixed with 2.45 mM potassium persulfate solution at a 1:1 ratio and reacted at room temperature in the dark for 12–16 h. The ABTS stock solution was diluted with ultrapure water to obtain an absorbance of 0.7 ± 0.02 at 734 nm, which was then used as the ABTS solution.

[0059] 25 mg of the composite film was added to 3 ml of ultrapure water and shaken for two hours to dissolve, resulting in a coating aqueous solution sample. 50 μL of ultrapure water and 150 μL of ABTS solution were transferred as the control group (A0), and 50 μL of coating aqueous solution sample and 150 μL of ABTS solution were transferred as the experimental group (A1).

[0060] The control group (A0) and the experimental group (A1) underwent the following procedures: Mix well in a 96-well microplate, incubate at room temperature in the dark for 6 minutes, and then measure the absorbance at 734 nm. The ABTS clearance rate is calculated using the formula: ABTS (%) = (A0 - A1) / A0 × 100%.

[0061] Experimental results: The antioxidant activity of sodium alginate, carboxymethyl chitosan, and ergothione was affected by the coating solution. Figure 2 As shown, the ABTS radical scavenging rate increased with the increase of ergothioneine addition, reaching 43%, 67%, and 81% respectively.

[0062] III. Antibacterial Performance Test Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria) were used as test strains. The activated bacterial solution was diluted to a specified absorbance of approximately 1.0. 2 mL of the bacterial solution was placed in sterile centrifuge tubes to form experimental and control groups. An equal volume of composite coating solution was added to the centrifuge tubes in the experimental group, while an equal volume of physiological saline was added to the centrifuge tubes in the control group. The final volume for both groups was 4 mL. After mixing, the mixture was incubated at 37°C with shaking for 1 h. 100 μL of the diluted solution was spread onto TSA agar plates and incubated at 37°C for 24 h. The total number of colonies was then counted. The inhibition rate of each coating solution was calculated using the following formula: Inhibition rate (%) = (Control group - Experimental group) / Control group × 100%.

[0063] Note: In the formula, the control group is physiological saline + bacterial solution, and the experimental group is film coating solution + bacterial solution.

[0064] Experimental results: Figure 3 Figures A and B show the inhibition rates of different amounts of EGT coating solution against Staphylococcus aureus (Gram-positive bacterium) and Escherichia coli (Gram-negative bacterium), respectively. With increasing EGT concentration (0.1 mg / mL~0.3 mg / mL), the inhibitory effect of the coating solution on both pathogens showed a dose-dependent trend. Figure 3 In group A, the inhibition rate against Staphylococcus aureus reached its highest level, approximately 45%, when the EGT concentration reached 0.3 mg / mL, significantly higher than that in the control group (approximately 10%). Similarly, in Figure 3 In group B, the group with added ergothioneine showed a better inhibition rate against Escherichia coli than the CK group.

[0065] Based on the above experimental results, and taking the UV blocking performance, ABTS free radical scavenging rate, and antibacterial performance of the composite membrane as the core evaluation indicators, the EGT-0.3 composite membrane was found to have the best performance in all aspects after comprehensive comparison.

[0066] Example 2: Application of composite coating liquid in the preservation of cooked yellowfin tuna The experimental group 3 (EGT-0.3) coating liquid with the best performance in Example 1 was used as the preservative for the experimental group, and distilled water was used as the control group.

[0067] (1) Sample processing: Yellowfin tuna meat that has been vacuum-cooked at 55℃ (heated to a core temperature of 55℃ and then kept at that temperature for 30 minutes) was selected and divided into two groups.

[0068] Experimental group (coating group): The cooked yellowfin tuna fillets were soaked in SA-CMCS-EGT-0.3 mg / mL composite coating solution for 2 min to ensure that the solution completely covered the surface of the fish fillets. After the soaking, the fillets were removed and drained to allow the coating to form a film on the surface naturally.

[0069] Control group (uncoated group): The cooked yellowfin tuna fillets were soaked in distilled water for 2 minutes, and then drained.

[0070] (2) Packaging and storage: The experimental group and the control group were each divided into two batches: The first batch (non-vacuum packaged): stored directly in a 4°C refrigerator. These were designated as the non-vacuum-controlled group (AC) and the non-vacuum-experimental group (AE).

[0071] Second batch (vacuum packaged): Samples were placed in sterile food-grade polyethylene-nylon vacuum bags (15 cm × 20 cm) in a clean bench, vacuumed, and then sealed. They were stored at 4°C. These were designated as the vacuum-control group (VC) and the vacuum-experiment group (VE), respectively.

[0072] Samples were taken out on days 0, 3, 7, 10, 14, 17, and 21 of storage to determine their microbiological and physicochemical properties.

[0073] Specifically as follows: I. pH Measurement pH value is one of the core indicators for measuring the quality of meat products. This change mainly stems from the biochemical reactions following the death of muscle tissue: glycogen is fermented by lactic acid bacteria to produce lactic acid, causing a decrease in pH; while the decomposition of proteins by microorganisms produces alkaline nitrogenous substances such as amines, causing an increase in pH. pH stability is a key indicator for maintaining good edible quality in meat products and is regulated by multiple factors.

[0074] Test method: To measure the pH value of the fish samples, 2 g of each sample was mixed with 18 mL of distilled water and homogenized at 8000 rpm for 20 s. Subsequently, the pH value of the mixture was measured at room temperature using a calibrated pH meter (Mettler-Toledo International Ltd.).

[0075] Test results: by Figure 4 As shown, the initial pH of the fish meat was 6.69, and the overall pH of the samples gradually increased with prolonged storage. During storage, the edible active coating formed by the nanoemulsion maintained the pH stability of the fish meat. The two uncoated samples showed higher pH levels than the coated samples; the AC group reached a pH of 6.89 on day 21, while the AE and VE groups, after coating, maintained the pH of the fish meat better. Since the SA, CMCS, and EGT coating solutions themselves have certain antibacterial effects, CMCS and EGT may have produced a synergistic effect, inhibiting microbial growth and effectively suppressing the formation and accumulation of alkaline nitrogenous compounds such as amines, thus slowing down the rate of pH increase. In conclusion, fish meat with a surface coating solution exhibits better results.

[0076] II. Color difference Color is a core indicator for consumers' acceptance of meat products. Studies have shown that among various color indicators, a... * b * The value is more suitable for characterizing the redness of bluefin and yellowfin tuna flesh. Based on this method, this invention quantifies the redness of tuna flesh. The results show that a * b * The higher the value, the richer the bright red color, and the better the freshness of the fish.

[0077] Testing method: Samples were taken out after different storage days, and the color of the fish meat was evaluated using a colorimeter. The L... * (Brightness), a * (Redness), and b * (Yellow tint).

[0078] Test results: Figure 5 The study demonstrates the color changes of steamed fish meat with and without coating during storage. As storage time increases, the color of the AC and VC groups (with and without coating) shows a significant difference. * The overall trend of L values ​​is a gradual decrease, which may be related to the loss of moisture content in the samples during storage, or there may be an interaction among the three factors. The control group's L... * The decreasing trend was greater in the control group than in the experimental group, which may be related to protein and lipid oxidation. Increased lipid oxidation in fish meat during storage leads to a... * The decrease in the value indicates a certain degree of browning. Because the AE and VE groups contain EGT, which has strong antioxidant properties, the fish meat maintains its color better compared to the control group. The experimental group's a * The decreasing trend was less than that of the control group. The yellowness of the b-values ​​in all four groups of samples generally increased with storage time. * This increase in values ​​may be related to myoglobin accumulation and increased lipid oxidation, which are associated with both protein and lipid oxidation. As shown in the figure, the control group's AC and VC showed a greater increasing trend in the later stages than the experimental group's AE and VE. This is because the high EGT content reduces lipid oxidation, resulting in a slower rate of increase in values.

[0079] III. Changes in texture Texture characteristics are one of the core indicators for evaluating the quality of food storage. The dynamic changes in texture parameters during storage are presented for the control and experimental groups. Cohesion characterizes the ability of fish tissue to resist deformation and maintain structural integrity during chewing.

[0080] Testing method: The texture quality of the fish meat was determined using a TMS-PRO texture analyzer.

[0081] Test results: by Figure 6 It can be seen that with the extension of the storage period, the hardness and chewiness of the AC group samples showed a significant upward trend, while the elasticity of the AC and AE groups showed a continuous downward trend. The changes in VC and VE were relatively not very obvious. The cohesion of the AC control group first increased and then decreased, and the cohesion at 0 days compared to 21 days showed a decreasing trend. Among them, the increasing trend of the AC and VC control groups was greater than that of the VC and VE experimental groups. The increase in hardness may be due to the loss of juice, lipid oxidation, and microbial growth during storage. In contrast, the fish meat texture of the experimental groups AE and VE was better maintained because the SA, CMCS, and EGT coating solutions can inhibit lipid oxidation and have a certain degree of antibacterial properties in fish meat, with smaller changes. In summary, the decrease in each texture parameter was greater in the control group than in the experimental group, indicating that the composite coating solution of sodium alginate, carboxymethyl chitosan, and ergothioneine can better maintain the texture structure of fish meat during storage.

[0082] IV. Changes in lipid oxidation The TBARS assay is widely recognized as a reliable indicator of lipid oxidation and plays an important role in assessing food quality. It reflects the degree of fatty acid oxidation, which is considered one of the main factors contributing to food deterioration during storage. The TBARS value measures the MDA content.

[0083] Detection method: 2 g meat sample was homogenized with 20 mL of 5% (w / v) trichloroacetic acid (TCA), followed by centrifugation at 7500 r / min for 10 min. 2 mL of the filtrate was added to 2 mL of 0.02 mol / L TBA solution and vortexed. The mixture was incubated at 95℃ for 20 min and then rapidly cooled to room temperature. The absorbance of the reaction solution was measured spectrophotometrically at 532 nm. A standard curve was established using 1,1,3,3-tetraethoxypropane as a reference. The TBARS content was calculated by substituting the sample absorbance into the regression equation, and the unit was recorded as mgMDA / kg sample.

[0084] Results: During the 21-day storage period, TBARS increased in all samples. Figure 7 It can be seen that lipid oxidation in the AE and VE groups was significantly lower than in the uncoated samples. The lower TBARS values ​​in the coated samples are consistent with the antioxidant capacity of ergothioneine itself, indicating that the formulation with higher free radical scavenging activity provides stronger protection against lipid oxidation. This suggests that compared with the control group, the ergothioneine-coated group exhibits free radical scavenging activity through hydrogen donation and reducing capabilities, thereby terminating proliferation and reducing lipid oxidation.

[0085] V. TVB-N Measurement Volatile basic nitrogen (TVB-N) is a key physicochemical indicator for evaluating the freshness of meat. Its content reflects the accumulation of nitrogenous volatile components produced by the decomposition of proteins under the synergistic action of endogenous enzymes and microorganisms during storage. Generally speaking, the TVB-N value shows an increasing trend with the extension of the storage period, and its increase is positively correlated with the degree of deterioration in meat quality.

[0086] Detection method: The TVB-N content in chilled tuna was determined using the automated Kjeldahl method as specified in GB 5009.228-2016, "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food". 10 g of tuna sample was accurately weighed and placed in a distillation tube. 75 mL of distilled water was added, and the mixture was shaken to ensure thorough dispersion. The sample was then mixed with 1.0 g of magnesium oxide and distilled at atmospheric pressure. The distillate was absorbed using a boric acid solution containing a methyl red-bromocresol green mixed indicator. After collection, the solution was titrated to the endpoint with 0.01 mol / L hydrochloric acid standard titrant. The TVBN content was calculated using the formula and expressed as milligram nitrogen equivalents (mg / 100 g) of volatile basic nitrogen per 100 g of tuna.

[0087]

[0088] In the formula, x is the TVB-N content in the sample (mg / 100 g), V1 is the volume of hydrochloric acid standard titration solution consumed by the sample (mL), V2 is the volume of hydrochloric acid standard titration solution consumed by the reagent blank (mL), c is the concentration of hydrochloric acid standard titration solution (mol / L), m is the sample mass (g), and 100 is the conversion factor.

[0089] Test results: The TVB-N values ​​for each group are as follows: Figure 8 As shown, the content of volatile basic nitrogen gradually increases with prolonged storage time, which is entirely consistent with the theory that TVB-N reflects protein degradation and indicates a decline in freshness. The initial TVB-N value of the fish after steaming was 10.85 mg / 100g. The AC control group reached its highest value of approximately 33 mg / 100g on day 17, showing a greater increasing trend than the AE coating group. Simultaneously, the trend in the VC control group was greater than that in the VE coating group, indicating that the coating solution can inhibit the growth of microorganisms and reduce protein degradation caused by microorganisms. The decomposition by spoilage microorganisms is the main factor contributing to the increase in TVB-N. This relationship is related to the favorable environment of high pH, ​​which promotes the growth of spoilage microorganisms. Increased pH helps microorganisms gradually transform from glycogen-dependent bacteria to protein-degrading bacteria. The coating solution contains carboxymethyl chitosan and ergothioneine, which can better inhibit bacteria and lipid oxidation, thus reducing the rate of spoilage of the fish. This conclusion is consistent with that of Fathihatunnisa et al.

[145] Similarly, coating tilapia can effectively reduce microbial growth and protein degradation, thus slowing down the rate at which TVB-N values ​​increase.

[0090] VI. Total bacterial count determination The total bacterial count reflects the degree of microbial contamination and can, to some extent, serve as a standard for testing food hygiene quality. For example... Figure 9 As storage time increased, the total bacterial count in all four groups of samples showed a continuous upward trend, but the rate of increase and the final colony count differed significantly, reflecting the positive effect of coating treatment on inhibiting microbial growth. According to the national standard "National Food Safety Standard for Cooked Meat Products" (GB 2726-2016), the limit for the total bacterial count in cooked meat products is that none of the results should exceed 10. 5 CFU / g.

[0091] Detection method: The total bacterial count was determined according to GB 4789.2-2022, "National Food Safety Standard - Microbiological Examination of Food: Determination of Total Colony Count". Under aseptic conditions, 10 g of fish sample was weighed into a homogenizing bag, and 90 mL of sterile physiological saline was added. Homogenize for 4 min, then take 1 mL of the homogenate and perform a 10-fold serial dilution. Spread the diluted solution onto nutrient agar plates and incubate at 30°C for 48 hours before counting.

[0092] Test results: The total colony count results for each group are as follows: Figure 9 As shown in the figure, a comparison between the uncoated and coated groups shows that, regardless of whether the environment is vacuum or non-vacuum, the total bacterial count in the coated group is lower than that in the corresponding uncoated control group. This confirms that the coating treatment can effectively inhibit the growth of microorganisms and has a certain antibacterial effect. This may be because the matrix formed by the carboxymethyl chitosan coating can stabilize ergothioneine through encapsulation, slowing down the release or degradation rate during storage. This slow-release effect allows ergothioneine to maintain an effective concentration for a long time, synergistically with the antibacterial activity of carboxymethyl chitosan, ultimately achieving sustained inhibition of microbial growth. A comparison under non-vacuum conditions (AC vs AE) shows that in the early stage of storage (1-7 days), the difference between the two groups gradually becomes apparent, with the total bacterial count in the AE group increasing slowly. By day 10 of storage, the total bacterial count in the AC group had reached 10... 6 CFU / g, while the AE group was 10. 4 CFU / g, in the AE group, only rose to 10 on day 14. 5CFU / g indicates that the coating has a more significant inhibitory effect on microorganisms under aerobic conditions, possibly related to oxygen blockage or the release of antibacterial components. Results under vacuum conditions showed that the total bacterial count in the VE group was lower than that in the VC group throughout the storage period, with the difference gradually widening from day 5. On day 17, the total bacterial count in the VC group reached 10. 5 CFU / g. The VE group ultimately exceeded the national microbiological testing standards at 21 days. The coating effect exhibited environmental dependence; the antibacterial effect was more pronounced under both vacuum and non-vacuum conditions, indicating that the antibacterial effect may be achieved through reducing initial microbial contamination and inhibiting the resurgence of residual bacteria. Therefore, it can be concluded that this coating material possesses excellent antibacterial properties and can be used as an effective preservation method to extend the shelf life of aquatic products.

[0093] VII. Microbial Diversity Analysis 1) Alpha diversity analysis Alpha diversity analysis relies on multiple statistical indices to quantify the abundance and diversity of microbial communities. By comparing the alpha diversity indices (such as Shannon and Chao1) of different treatment groups (e.g., chilled, modified atmosphere, coated), it can be determined whether the preservation method can significantly reduce the species richness and evenness of microorganisms. Ideal preservation should lead to a decrease in diversity indices, indicating that dominant spoilage bacteria are suppressed.

[0094] Detection method: DNA Extraction and PCR Amplification of 16S rDNA V3-V4 Region: Microbial diversity analysis was performed on samples stored for 21 days. Genomic DNA was extracted from the samples, and DNA quality was verified by 1% agarose gel electrophoresis. Following the methods of Chen et al., PCR amplification was performed on the V3-V4 region of the bacterial 16S rDNA gene using primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The amplified products were mixed, detected by 2% agarose gel electrophoresis, and purified by gel excision using an AxyPrep DNA gel extraction kit. The target fragment was obtained by elution with Tris-HCl. The purified product was quantified using a QuantiFluor™-ST blue fluorescence quantitative system, and an Illumina PE library was constructed. After PCR enrichment, the library was sequenced.

[0095] Bioinformatics analysis: Cluster analysis and splicing were performed on the sample data. Illumina PE sequencing data was spliced ​​into 51PE reads, and quality filtering and sample partitioning were performed. Various sequences were collected for further analysis. Colony structure at various levels was statistically analyzed. After processing, alpha diversity analysis was used, including species abundance statistics (observed_features, Chao) and species diversity statistics (Shannon index and Simpson index).

[0096] Test results: After denoising and species annotation of the ASV sequences, the resulting community structure information is shown in Figure 10. Among the evaluation indices, Observed features characterize the actual number of species detected in the samples; the Chao1 index assesses species richness, with higher values ​​indicating a greater number of species; the Shannon and Simpson indices comprehensively reflect species diversity, and these values ​​are positively correlated with community diversity levels. Under refrigerated conditions, microbial diversity was measured on day 21 of storage. The Chao1 index plot shows that on day 21, the richness of the four sample groups was higher in the coated sample group than in the uncoated group, regardless of whether the samples were in vacuum or non-vacuum conditions. The Shannon and Simpson index plots also indicate that species diversity was higher in the coated group than in the uncoated group. This may be because, in the later stages of storage, the dominant spoilage bacteria in the uncoated group grew rapidly, inhibiting the growth of other bacteria and leading to a decrease in species richness and diversity. The α-diversity analysis for each group is shown below. Figure 11 As shown, with increasing sequencing depth, the curves for each sample gradually flattened, indicating that the current sequencing data volume has reached saturation and can reliably reflect the species composition of the microbial community in the samples. Testing was conducted on day 21 of storage; the figure shows that the AE group samples exhibited the highest bacterial diversity, while the samples at the end of storage showed the lowest bacterial diversity. The dilution curves for all four groups of samples gradually flattened with increasing sequencing depth, essentially ceasing at 61,000 sequences. This indicates that the current sequencing depth can adequately cover the vast majority of microbial species in the samples, providing sufficient sequencing data to accurately reflect the changes in microbial richness and diversity in each sample.

[0097] 2) β-diversity analysis β-diversity analysis is an indicator of the degree of difference in microbial community composition between different preservation treatment groups. By calculating the β-diversity distance between different groups (such as Bray-Curtis, UniFrac, etc.), it can be determined whether different preservation methods (such as modified atmosphere packaging, addition of preservatives, different temperatures, etc.) have led to significant differentiation of the microbial community. If the β-diversity difference between the treatment group and the control group (such as fresh samples) is large, it indicates that the preservation method has significantly altered the microbial community structure.

[0098] Detection method: DNA extraction and PCR amplification of 16S rDNA V3-V4 regions: (same as α diversity analysis); Bioinformatics analysis: Cluster analysis and splicing were performed on the sample data. Illumina PE sequencing data were spliced ​​into 51 PE reads, and quality filtering and sample partitioning were performed. Various sequences were collected for further analysis. Colony structures at various levels were statistically analyzed. After processing, β-diversity analysis was used to analyze the fish flesh microbial community structure.

[0099] Test results: β-diversity analysis such as Figure 12 As shown, the variance contribution rates of the first principal component (PC1) and the second principal component (PC2) are 82.59% and 14.48%, respectively, with a cumulative contribution rate of 97.07%, effectively representing the main differences in the original data. The AC and AE groups are widely dispersed, and within the same group, the horizontal axis overlaps well during the same storage period, indicating stable microbial community structures and good parallel aggregation within the same group. The dispersion distance between the two groups also confirms that the coating preservation in the AE group alters the microbial community structure. Similarly, the VC and VE groups also show a clear trend of separation between groups. From the overall distribution, the four groups of samples in the figure exhibit clear dispersion, indicating certain differences among the samples.

[0100] 3) Microbial community structure analysis By employing high-throughput sequencing and other methods, the species, abundance, and succession patterns of microbial communities under different preservation conditions (such as temperature, modified atmosphere packaging, and preservative treatment) were tracked to identify the dominant bacterial groups (such as specific Pseudomonas and Shewanella) leading to yellowfin tuna spoilage and their key succession nodes. By comparing the differences in microbial community structure between different preservation treatment groups and the control group, it was determined whether the preservation measures significantly inhibited spoilage bacteria or caused abnormal changes in the microbial community structure, thereby scientifically evaluating the targeting and effectiveness of preservation technologies.

[0101] Detection method: Same as above for β-diversity analysis; Test results: Analysis of the microbial community structure of each group as follows Figure 13 The figure shows the changes in the microbial community structure at the phylum level in four groups of fish samples (coated and uncoated) on day 21 of storage. As can be seen from the figure, Firmicutes and Proteobacteria are the absolutely dominant phyla in the four groups (AC, AE, VC, and VE), with their combined relative abundance exceeding 90% in each group. This indicates that these two phyla are the core groups determining the microbial community characteristics at the end of storage. Other phyla include Bacteroidota, Actinobacteriota, and Chloroflexi. Towards the end of storage, Firmicutes were the dominant phylum in groups AC and VC, while in groups AE and VE, the abundance of Firmicutes decreased and the abundance of Proteobacteria increased after coating preservation, indicating that the growth and reproduction of Firmicutes were inhibited to some extent, while the abundance of other communities increased. The vast majority of Firmicutes are Gram-positive bacteria, including some capable of forming spores. Spores typically possess strong tolerance, resisting extreme environments and dry conditions. Changes in Firmicute abundance are related to the quality of fish meat during refrigeration, increasing in abundance towards the end of storage. Compared to the control groups AC and VC, the coating group improved the diversity of community structure and promoted the relatively stable growth of other microorganisms. This conclusion is related to the α-diversity mentioned above; the dominant community in the control group inhibited the growth of other communities to some extent, resulting in lower α-diversity than the experimental group. At the genus level, such as... Figure 14As shown, the four groups of samples mainly included Bacillus, Staphylococcus, Stenotrophomona, and Ralstonia. In the control group (AC and VC), Bacillus was the most prevalent bacterial genus, with a relative abundance of around 80%. Bacillus was one of the main causes of spoilage in the control group, becoming the dominant bacteria. Coating preservation reduced the Bacillus community, thus inhibiting its growth and reproduction. In the AE and VE groups, community diversity increased, Bacillus decreased, and other genera maintained a certain abundance. The figure shows that after coating preservation, Bacillus content in the AE and VE groups decreased to below 30% and 10%, respectively. This may be due to the antibacterial effect of substances in the coating solution, which inhibited microbial reproduction. The coating solution contains carboxymethyl chitosan and ergothioneine, which have certain antibacterial effects. Studies have shown that Bacillus species produce some putrid odors during metabolism, which is related to the off-odor content measured in the following section.

[0102] VIII. Effects of different processing methods on volatile flavor compounds in refrigerated tuna meat Odor Activity Value (ROAV) is a core indicator for quantifying the contribution of flavor compounds to the overall flavor of a sample. It can quantitatively evaluate the preservation effect of plastic wrap on refrigerated tuna, dynamically monitor the deterioration process of yellowfin tuna freshness, and distinguish key flavor compounds.

[0103] Detection method: Solid-phase microextraction (SPME) was performed automatically on a PAL orbital system with the following parameters: incubation temperature 60℃, preheating for 15 min, extraction and adsorption for 30 min, and desorption for 4 min. Separation and detection of volatile components were performed using an Agilent 7890 gas chromatograph-5977B mass spectrometer (GC-MS). Raw mass spectrometry data were processed using Chroma TOF software (V4.3x, LECO), sequentially performing peak extraction, baseline correction, deconvolution, peak integration, peak alignment, and NIST mass spectrometry database retrieval to achieve compound identification.

[0104] Test results: Scatter plot of relative odor activity (ROAV) values ​​of volatile flavor compounds in samples with different treatments, as shown in the figure. Figure 15As shown in the figure, the overall distribution pattern of the four sample groups conforms to the typical distribution characteristics of flavor substances during the cold storage of aquatic products: the overall flavor of the samples is dominated by a very small number of core substances with high ROAV. In addition to the baseline flavor substance with ROAV=100, the AC group also has a secondary core high ROAV substance with ROAV of about 75. This type of substance is a characteristic marker of fishy and rancid fatty acid flavors from lipid oxidation. In summary, the ROAV values ​​of the core undesirable flavor substances in the coated groups (AE, VE) are significantly lower than those in the corresponding uncoated groups (AC, VC), and there is no significant difference in the distribution of low-contribution modifying substances with ROAV<1 among the groups. The composite coating treatment only targets and regulates the core undesirable flavor substances from lipid oxidation, achieving a precise flavor improvement effect.

[0105] Cluster analysis thermograms of volatile substances under different treatment methods are shown below. Figure 16 As shown. The contribution of volatile flavor compounds to the product is determined by both content and threshold. This invention evaluates the contribution of each component using ROAV. Components with ROAV ≥ 1 are core flavor compounds, components with 0.1 ≤ ROAV < 1 are modifying flavor compounds, and components with ROAV < 0.01 are potential flavor compounds. The test results show that aldehydes and alcohols dominate the flavor composition.

[0106] In summary, in the tuna preservation experiment, the SA / CMCS / EGT composite coating effectively delayed the increase in pH, browning, textural deterioration, lipid oxidation, and protein degradation of the tuna during refrigeration, significantly reduced TVB-N and TBARS values, and inhibited the growth of total bacterial count. Combined with 16S rDNA microbial community analysis, the coating significantly inhibited the proliferation of dominant spoilage bacteria—Firmicobacteria and Bacillus—in the later stages of storage, improving microbial diversity and delaying the spoilage process. Simultaneously, the coating effectively inhibited the formation of typical oxidative off-odor substances such as hexanal and 1-octen-3-ol, improving the flavor and quality of the tuna. Ultimately, the composite coating extended the shelf life of non-vacuum-packed and vacuum-packed, low-temperature cooked tuna by 4 days, demonstrating a significant preservation effect. In summary, the SA / CMCS / EGT active composite membrane possesses excellent film-forming properties, antioxidant properties, antibacterial properties, and UV blocking properties. It can effectively maintain the physicochemical, microbiological, and flavor qualities of vacuum-cooked yellowfin tuna, extend the product's shelf life, and provide a reliable technical solution and theoretical support for the green and efficient preservation of cooked aquatic products.

[0107] Comparative Example 1, refer to Chitosan edible coating: a potential control of toxicbiogenic amines and enhancing the quality and shelf life of chilled tunafilets In Comparative Example 1, Elsabagh et al. used chitosan combined with curcumin, garlic, or beet extract to preserve tuna fillets under cold storage. Their optimal group had a TBA value of 4.24 ± 0.12 mg MDA / kg and a TVB value of 27.96 ± 0.44 mg / 100 g on day 14, maintaining sensory quality for up to 14 days. In contrast, the VE group of this invention maintained TBARS at approximately 2.4 mg MDA / kg and TVB-N at approximately 21 mg / 100 g on day 21, with a shelf life extended to 21 days. This indicates that the combination of the SA / CMCS / EGT coating and vacuum cryogenic cooking provides more sustained inhibition of lipid oxidation and protein spoilage.

[0108] Comparative Example 2, refer to Shelf life extension for Bluefin tuna slices ( Thunnus thynnus ) wrapped with myofibrillar protein film incorporated with catechin-Kradon extract In Comparative Example 2, Kaewprachu et al. used a fish myofibrillar protein active membrane to coat bluefin tuna slices. Although this delayed the growth of PV, TBARS, TVB-N, and microorganisms, its longest shelf life was only 8 days, and all samples showed weight loss and increased hardness during storage. In contrast, the SA / CMCS / EGT composite coating of this invention can extend the shelf life of the non-vacuum group to 14 days and the vacuum group to 21 days, and can delay color and texture deterioration, indicating that the system studied in this study has a greater advantage in terms of actual shelf life extension and quality maintenance.

[0109] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a composite coating liquid for low-temperature cooked tuna, characterized in that... Includes the following steps: (1) Preparation of CMCS solution: Dissolve 1.5 ± 0.2 g of carboxymethyl chitosan in 100 mL of water, then add plasticizer and mix well to obtain CMCS solution; (2) Preparation of SA solution: Dissolve 1.5 ± 0.2 g sodium alginate in 100 mL of water, then add plasticizer and mix well to obtain SA solution; (3) Preparation of composite coating liquid: Set the volume ratio of SA solution to CMCS solution to 1:1; first add ergothioneine to the SA solution, then add CMCS solution and stir continuously to obtain a composite coating solution, wherein the concentration of ergothioneine in the composite coating solution is 0.1~0.3 mg / mL.

2. The method for preparing the composite coating liquid according to claim 1, characterized in that: In step (1): 1 ± 0.1 mL of glycerol is added as a plasticizer; In step (2): 1 ± 0.1 mL of glycerol is added as a plasticizer.

3. The method for preparing the composite coating liquid according to claim 2, characterized in that: The degree of substitution of the carboxymethyl chitosan is ≥80%; the ergothionein is L-ergothionein with a purity of ≥99%.

4. The composite coating liquid prepared by any one of claims 1 to 3.

5. The use of the composite coating liquid prepared by any one of claims 1 to 3, characterized in that: Used for preserving the freshness of tuna cooked at low temperatures.

6. The use of the composite coating liquid according to claim 5, characterized in that: Low-temperature cooked tuna is immersed in a composite coating solution for 2 ± 0.5 minutes, thereby forming a coating on the surface of the low-temperature cooked tuna.

7. The use of the composite coating liquid according to claim 5 or 6, characterized in that: Low-temperature cooked tuna refers to tuna meat that has been vacuum-cooked at 55±2℃.

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