A method for preserving fresh fish fillets based on source reduction and endogenous inhibition of bacteria.

Abstract

This invention belongs to the field of aquatic product preservation technology and discloses a method for preserving fresh fish fillets based on source-based sterilization and endogenous antibacterial action. The method includes: non-thermal sterilization of fresh fish fillets, followed by ultrasonic-ultra-high pressure synergistic treatment in a nano-antibacterial emulsion, then draining, packaging, and refrigeration. The nano-antibacterial emulsion is prepared by dissolving natural antibacterial active substances in anhydrous ethanol as the oil phase and using an aqueous solution of a natural polymer as the aqueous phase. The oil and aqueous phases are mixed, homogenized under microfluidic high pressure, and then microfiltered. This invention utilizes a dual-core synergistic process of source-based non-thermal targeted inactivation of spoilage bacteria and enhanced penetration of a natural antibacterial system to significantly extend the shelf life of fresh fish fillets under 0-4℃ refrigeration conditions from the conventional 3-5 days to over 18 days, while preserving the texture, flavor, and nutrition of the fish fillets to the greatest extent possible.

Description

Technical Field

[0001] This invention relates to the field of aquatic product preservation technology, specifically to a method for preserving fresh fish fillets based on source reduction and endogenous inhibition of bacteria. Background Technology

[0002] Fresh fish fillets are popular with consumers due to their tender texture, rich nutrition, and convenient consumption, making them a core category in the seafood processing and catering industries. However, fresh fish fillets inherently possess characteristics such as high moisture content, fragile muscle fiber tissue, and high initial bacterial load after slaughter, making them highly susceptible to spoilage due to microbial growth during storage and transportation. Simultaneously, a series of quality deterioration issues, including protein denaturation, fat oxidation, and softening of the texture, result in a shelf life of only 3-5 days under standard 0-4℃ refrigeration conditions, severely restricting the product's distribution radius and the industry's large-scale development.

[0003] To address the above issues, existing technologies primarily focus on two directions: reducing bacteria at the source or inhibiting endogenous bacteria. In terms of source sterilization, the main methods used are irradiation, heat treatment, and immersion in chemical disinfectants to directly sterilize fresh fish fillets. However, these technologies all have significant shortcomings: irradiation technology requires large investments in equipment and has high operating costs, and consumers generally have low safety and acceptance of it; heat treatment (such as pasteurization) can effectively inactivate microorganisms, but it can cause thermal denaturation of fish proteins, resulting in a dry texture, nutrient loss, and completely destroying the unique freshness of fresh fish fillets; immersion in chemical disinfectants (such as chlorine-containing disinfectants) poses a risk of chemical residue, which contradicts the current market trend of consuming natural and healthy foods. Emerging non-thermal sterilization technologies (such as cold plasma and ultraviolet light) can avoid problems such as fish protein denaturation and chemical residue, but current applications are mostly single-treatment methods that can only kill some microorganisms on the surface of the fish fillets, failing to accurately inactivate dominant spoilage bacteria at the source, resulting in insufficient control of the initial bacterial load, extremely high pressure on subsequent preservation, and difficulty in achieving long-term freshness.

[0004] In terms of endogenous antibacterial properties, the mainstream research direction focuses on the application of natural antibacterial substances (such as plant polyphenols, plant essential oils, and antimicrobial peptides). However, existing technologies still have the following core pain points: First, most natural antibacterial substances (such as tea polyphenols and grape seed extract) have poor water solubility, low chemical stability, and are easily oxidized and deactivated, resulting in limited and unsustainable antibacterial effects when used alone. Second, conventional soaking processes can only allow antibacterial components to adhere to the surface of the fish fillets, making it difficult to effectively penetrate into the muscle fiber tissue and inhibit the reproduction of microorganisms inside the fish fillets, leading to a phenomenon of "intact surface, internal spoilage" and short shelf life. Third, single antibacterial technologies usually cannot simultaneously solve multiple quality deterioration problems such as microbial spoilage, fat oxidation, and protein denaturation, resulting in limited effects on extending shelf life.

[0005] In summary, existing fresh fish fillet preservation technologies struggle to simultaneously meet the four core requirements of "precise bacterial control at the source, natural safety, deep and long-lasting antibacterial effect, and no loss of quality." Therefore, there is an urgent need to develop a novel preservation method that effectively controls initial spoilage bacteria at the source, while simultaneously achieving a deep and even distribution of antibacterial components within the fish tissue, thus achieving the dual goals of extending shelf life and maintaining freshness.

[0006] In view of this, this invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preserving fresh fish fillets based on source reduction and endogenous inhibition of bacteria. Through a dual-core synergistic process of "source non-thermal targeted inactivation of spoilage bacteria" and "enhanced deep penetration of natural antibacterial system", the risk of spoilage is reduced from the source, and the problems of low penetration efficiency and short preservation time of natural antibacterial substances are solved. Under the premise of no chemical preservatives added and no heat damage, the refrigerated shelf life of fresh fish fillets is significantly extended, and the original texture, flavor quality and nutritional components of the fish fillets are preserved to the greatest extent.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preserving fresh fish fillets based on source-level bacterial reduction and endogenous bacterial inhibition includes: After non-thermal sterilization, the fresh fish fillets are placed in a nano-antibacterial emulsion for ultrasonic-ultra-high pressure synergistic treatment, then drained, packaged, and refrigerated. The preparation method of the nano antibacterial emulsion is as follows: natural antibacterial active substances are dissolved in anhydrous ethanol as the oil phase, and an aqueous solution of natural polymer materials is used as the water phase. The oil phase and water phase are mixed, and then microfiltration is performed after microfluidic high-pressure homogenization treatment.

[0009] Preferably, the fresh fish fillets are non-thermal sterilized to a total bacterial count ≤10² CFU / g.

[0010] Preferably, the non-thermal sterilization is atmospheric pressure cold plasma sterilization or pulsed intense light combined with weakly acidic electrolyzed water atomization sterilization.

[0011] Preferably, the ultrasonic-ultra-high pressure synergistic treatment involves first performing ultrasonic treatment to assist in impregnation, and then performing ultra-high pressure treatment to allow the nano-antibacterial emulsion to penetrate deeply.

[0012] Preferably, the ultrasonic treatment has a power of 100-300 W, a frequency of 20-40 kHz, a treatment time of 5-15 min, and an intermittent ratio of 1:1; the ultra-high pressure treatment has a pressure of 100-300 MPa and a pressure holding time of 5-20 min.

[0013] Preferably, the particle size of the nano-antibacterial milk is 100-300 nm.

[0014] Preferably, the natural antibacterial active substance is at least three of the following: rosmarinic acid, protamine sulfate, oregano oil, and mustard oil.

[0015] Preferably, the natural polymer material is at least one of chitosan, water-soluble soybean polysaccharide, and whey protein isolate.

[0016] Preferably, the pressure of the microfluidic high-pressure homogenization process is 800-1200 bar, and the cycle is 3-5 times.

[0017] Preferably, the packaging is vacuum packaging or modified atmosphere packaging.

[0018] The beneficial effects of this invention are as follows: This invention breaks through the limitations of existing technologies that focus on "suppressing bacteria but neglecting to control bacteria". It introduces cutting-edge non-thermal sterilization technology in the pretreatment stage to achieve precise inactivation of dominant spoilage bacteria in fresh fish fillets, controlling the initial total bacterial count to below 10² CFU / g, thereby significantly reducing the starting point of spoilage from the root and laying a solid foundation for subsequent long-term preservation.

[0019] This invention constructs a natural composite antibacterial nanosystem using microfluidic technology. It utilizes natural polymer wall materials to encapsulate and protect the active ingredients, effectively overcoming the technical bottlenecks of poor water solubility and easy oxidation and inactivation of natural antibacterial substances such as oregano oil and mustard oil. The nanoscale particle size further facilitates mass transfer and penetration into fish tissue. Furthermore, it employs a synergistic ultrasound-ultra-high pressure technology. First, ultrasonic cavitation opens the gaps between muscle fibers, then ultra-high pressure drives the nano-antibacterial emulsion to distribute evenly deep within the tissue. This overcomes the limitations of traditional soaking, which only provides surface antibacterial protection, achieving long-lasting, full-space antibacterial protection from the surface inwards, fundamentally preventing internal spoilage of the fish fillets.

[0020] This invention utilizes a dual-core synergistic process—non-thermal targeted inactivation of spoilage-causing bacteria at the source combined with the enhanced penetration of a natural antibacterial system—to significantly extend the shelf life of fresh fish fillets under 0-4°C refrigeration conditions from the conventional 3-5 days to over 18 days. Even after 18 days of storage, the total bacterial count of the product remains controlled below 10%. 4 With CFU / g below the limit and volatile basic nitrogen (TVB-N) values ​​far below the national standard limit, the sensory quality remains excellent. This significant effect not only greatly expands the circulation radius and sales cycle of fresh fish fillets and effectively reduces storage and transportation losses, but also demonstrates extremely high industrial application value and market competitiveness.

[0021] The preservation method provided by this invention does not add any chemically synthesized preservatives throughout the entire process. All antibacterial ingredients used are food-grade raw materials with no harmful residues. It highly meets consumers' demand for natural, healthy, and additive-free foods, and improves the market acceptance of the product while ensuring food safety. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description of the invention is provided in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are merely exemplary and not intended to limit the scope of the invention. Furthermore, in the following description, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] Embodiments of the present invention provide a method for preserving fresh fish fillets based on source reduction of bacteria and endogenous inhibition of bacteria, comprising: After non-thermal sterilization, the fresh fish fillets are placed in a nano-antibacterial emulsion for ultrasonic-ultra-high pressure synergistic treatment, then drained, packaged, and refrigerated. The preparation method of the nano antibacterial emulsion is as follows: natural antibacterial active substances are dissolved in anhydrous ethanol as the oil phase, and an aqueous solution of natural polymer materials is used as the water phase. The oil phase and water phase are mixed, and then microfiltration is performed after microfluidic high-pressure homogenization treatment.

[0024] The spoilage of fresh fish fillets is mainly driven by the metabolic activity of specific dominant putrefactive bacteria. Studies have shown that *Pseudomonas*, *Shewanella*, *Aeromonas*, and *Thermus thermophilus* are the core spoilage bacteria in fresh fish fillets. These bacteria retain strong metabolic activity even at low temperatures, preferentially utilizing non-protein nitrogen in the fish meat to metabolize and produce amines (such as putrescine and cadaverine), sulfides, and volatile organic acids, thereby causing the fish fillets to develop a characteristic fishy odor, soften the tissue, and produce mucus.

[0025] During the processing of fresh fish fillets, steps such as cutting and deboning disrupt the natural structure of the fish meat, exposing the cut surface directly to environmental microorganisms. This leads to the accumulation of the aforementioned spoilage bacteria on the cut surface, with the initial total bacterial count typically reaching 10³-10⁻¹⁰. 5 CFU / g. A higher initial bacterial load significantly shortens the spoilage lag period, thus directly limiting the refrigerated shelf life of the product. This implementation method first inactivates 95% or more of the spoilage bacteria on the surface and shallow layer of fresh fish fillets through non-thermal sterilization, resulting in an initial bacterial count ≤10² CFU / g, fundamentally delaying the microbial growth cycle.

[0026] Non-thermal sterilization methods are selected that can efficiently kill microorganisms on the surface and shallow layers of fresh fish fillets at low temperatures with minimal impact on the quality of the fish. Atmospheric pressure cold plasma sterilization or pulsed intense light combined with weakly acidic electrolyzed water atomization sterilization are preferred. When using atmospheric pressure cold plasma sterilization, the process parameters are: discharge voltage 15-30 kV, discharge frequency 20-50 kHz, gas medium is an air / helium mixture (volume ratio 9:1), treatment time 30-120 s, single-sided treatment or alternating double-sided treatment. When using pulsed high-intensity light combined with weakly acidic electrolyzed water for atomization sterilization, the process parameters are as follows: pulse width of pulsed high-intensity light 2-10 μs, flash frequency 1-5 times / s, light intensity 3-8 J / cm², and treatment time 10-60 s; simultaneously, weakly acidic electrolyzed water is atomized and sprayed, wherein the effective chlorine concentration of the weakly acidic electrolyzed water is 10-30 mg / L, the pH value is 5.0-6.5, and the atomization rate is 0.5-2 mL / min. The photochemical effect of pulsed high-intensity light and the bactericidal effect of weakly acidic electrolyzed water work synergistically to significantly improve the inactivation efficiency of putrefactive bacteria. Preferably, before the non-thermal sterilization step, a washing and draining process is also included, specifically: the fresh fish fillets are rinsed in sterile ice water at 4℃ 1-2 times, 1-3 min each time, to fully remove blood and tissue debris, and then drained until there is no obvious water on the surface.

[0027] In this embodiment, the natural antibacterial active substances are made into nano-antibacterial emulsions with a particle size of 100-300 nm and then used in the processing of fresh fish fillets. This is to effectively solve the problems of poor water solubility and easy oxidation and inactivation of natural antibacterial active substances, while facilitating the active substances to penetrate the intercellular gaps and infiltrate the interior of the fish muscle tissue, thus maintaining the stability of the system.

[0028] Rosmarinic acid possesses strong antioxidant and broad-spectrum antibacterial activity, inhibiting both Gram-positive and Gram-negative bacteria; protamine is rich in arginine, which can disrupt bacterial cell membranes; oregano oil and mustard oil are both natural plant essential oils with strong antibacterial activity and mild flavor. Selecting at least three of these natural antibacterial active substances as core functional ingredients to construct the oil phase of the nano-antibacterial emulsion can cover different antibacterial mechanisms, broaden the antibacterial spectrum, and simultaneously provide antioxidant effects, inhibiting fat oxidation and flavor deterioration in fish fillets.

[0029] Regarding the dosage of natural antibacterial active substances, their concentration in the oil phase shall be the standard. For example, in the oil phase, the concentrations of rosmarinic acid, protamine, oregano oil, and mustard oil are 0.4-0.5 wt%, 0.3-0.5 wt%, 0.2-0.3 wt%, and 0.2-0.3 wt%, respectively, based on the mass of anhydrous ethanol.

[0030] Chitosan, water-soluble soybean polysaccharide, and whey protein isolate are all food-grade natural polymer materials that possess film-forming properties, biocompatibility, and certain antibacterial effects. Selecting at least one of these natural polymer materials as the wall material can achieve encapsulation and protection of the core antibacterial components. The amount of natural polymer material used is determined by its concentration in the aqueous phase. For example, the concentrations of chitosan, water-soluble soybean polysaccharide, and whey protein isolate in the aqueous phase are 0.5-1.2 wt%, 0.4-1.0 wt%, and 0.5-1.2 wt%, respectively. It should be noted that if the wall material includes chitosan, after dissolving it in water, glacial acetic acid must be used to adjust the pH of the aqueous phase to approximately 6 to ensure the stability and functionality of the nano-antibacterial emulsion.

[0031] After initially mixing the aqueous and oil phases at a volume ratio of 8-10:1, microfluidic high-pressure homogenization is performed. Under extremely high shear force, impact force, and cavitation effect, the oil phase is broken into nanoscale droplets, which are uniformly dispersed in the aqueous phase. Simultaneously, natural polymer materials are rapidly adsorbed at the newly formed oil-water interface, forming a stable interfacial film that prevents droplet aggregation and coalescence, thereby producing a uniform and stable nano-antibacterial emulsion. In a specific embodiment, the microfluidic high-pressure homogenization is performed at a pressure of 800-1200 bar for 3-5 cycles. Preferably, before microfluidic high-pressure homogenization, the mixture of oil and aqueous phases can be pre-emulsified by high-speed shearing to ensure initial uniform mixing, creating favorable conditions for subsequent high-pressure homogenization to prepare nano-sized droplets. After microfluidic high-pressure homogenization, the mixture is filtered through a 0.45 μm sterile filter membrane to obtain the nano-antibacterial emulsion.

[0032] In this embodiment, fresh fish fillets are placed in a nano-antibacterial emulsion for ultrasonic-ultra-high pressure synergistic treatment. Essentially, ultrasonic treatment assists in immersion, utilizing the cavitation effect to generate microjets and shock waves, forming micropores on the surface of the fish muscle fibers, opening the fiber gaps, reducing mass transfer resistance, and creating channels for the penetration of antibacterial components. Then, ultra-high pressure treatment propels the nano-sized antibacterial emulsion particles to rapidly penetrate deep into the muscle fibers, even into the fish cells, achieving full-space distribution from the surface to the interior. This addresses the limitation of traditional soaking, which only provides surface antibacterial protection, and significantly improves the antibacterial efficacy. In this treatment, the ratio of fresh fish fillets to nano-antibacterial emulsion is 1:5-10 (g / mL).

[0033] The parameter settings for ultrasonic and ultra-high pressure treatments must ensure that the nano-antibacterial emulsion fully penetrates the fish meat while maintaining its inherent tissue structure and textural properties. For example, the ultrasonic treatment power is 100-300W, the frequency is 20-40 kHz, the treatment time is 5-15 min, and the intermittent ratio is 1:1; the ultra-high pressure treatment pressure is 100-300 MPa, and the holding time is 5-20 min.

[0034] After the above-treated fresh fish fillets are dried with sterile air at 4℃ to remove surface moisture, they are packaged and refrigerated at 0-4℃ to achieve long-term freshness. Vacuum packaging or modified atmosphere packaging can be selected as the packaging method to better inhibit the growth of aerobic bacteria and fat oxidation. Specifically, vacuum packaging or modified atmosphere packaging is carried out in a Class 100 sterile environment, where the gas ratio for modified atmosphere packaging is CO2:N2 = 6:4-7:3.

[0035] It should be noted that all the above fresh fish fillet processing steps are completed at a low temperature of 4-10℃, without any heat treatment or irradiation. This effectively avoids heat damage that could lead to protein denaturation, softening of texture, and nutrient loss in the fresh fish fillets, thus preserving their tenderness, water retention, and original flavor to the greatest extent possible. Furthermore, all raw materials used (such as anhydrous ethanol and chitosan) are food-grade, ensuring the safety of this method during processing and the edible quality of the final product.

[0036] It should also be noted that the preservation method of the present invention has wide applicability and can be used for various freshwater and saltwater fish fillets, including grass carp, black carp, sea bass, sole, and cod. The processing parameters given in the above embodiments are preferred ranges determined based on fish fillets with a thickness of 2-4 mm, and are intended to exemplarily demonstrate the technical effects of the present invention. In actual operation or industrial applications, those skilled in the art can routinely adjust key process parameters such as ultrasound and ultra-high pressure according to factors such as the actual thickness and density of the fish fillets to obtain comparable preservation effects.

[0037] The following will disclose specific embodiments for implementing the present invention, as well as corresponding comparative examples to demonstrate the relevant technical effects of the present invention.

[0038] The raw materials used in the following examples were all purchased from the market and are food-grade materials.

[0039] Example 1: Method for Preserving Fresh Fish Fillets Fresh grass carp fillets, 3 mm thick, were rinsed twice with sterile ice water at 4°C for 2 minutes each time. After draining until no free moisture remained on the surface, they were subjected to atmospheric pressure cold plasma sterilization. The treatment parameters were: discharge voltage 15 kV, discharge frequency 20 kHz, gas medium of air / helium mixture (volume ratio 9:1), single-sided treatment time 15 s, and total double-sided treatment time 30 s. Post-treatment testing showed that the total bacterial count of the fresh grass carp fillets was 98 CFU / g, and the inactivation rate of dominant spoilage bacteria was 95.1%.

[0040] Chitosan was dissolved in sterile deionized water, and the pH was adjusted to 6 with glacial acetic acid to obtain an aqueous phase with a chitosan concentration of 0.5 wt%. Natural antibacterial active substances, including rosmarinic acid, protamine sulfate, and oregano oil, were dissolved in anhydrous ethanol to obtain an oil phase with concentrations of 0.4 wt%, 0.3 wt%, and 0.2 wt%, respectively. The aqueous and oil phases were mixed at a volume ratio of 9:1, pre-emulsified by high-speed shearing, and then subjected to microfluidic high-pressure homogenization with the following parameters: homogenization pressure of 800 bar, 3 cycles. The mixture was then filtered through a 0.45 μm sterile filter membrane to obtain nano-antibacterial emulsions with a particle size of 200-300 nm.

[0041] Fresh grass carp fillets, after sterilization, were placed in nano-antibacterial emulsion at a material-to-liquid ratio of 1:5 (g / mL), and subjected to ultrasonic and ultra-high pressure treatments in sequence. The ultrasonic treatment parameters were: ultrasonic power 100 W, frequency 20 kHz, treatment time 5 min, and intermittent ratio 1:1. The ultra-high pressure treatment parameters were: treatment pressure 100 MPa, holding time 5 min, and sterile water as the pressure transmission medium.

[0042] All the above sterilization, ultrasonic and ultra-high pressure treatments were carried out at 4°C. After the treatment, the fresh grass carp fillets were dried with 4°C sterile cold air to remove surface moisture, then vacuum-packed in a Class 100 sterile environment, and then refrigerated at 4°C.

[0043] Example 2: Method for Preserving Fresh Fish Fillets Fresh sea bass fillets, 4 mm thick, were rinsed twice with sterile ice water at 4°C for 3 minutes each time. After draining until no free water remained on the surface, they were subjected to pulsed high-intensity light combined with weakly acidic electrolyzed water atomization sterilization treatment. The treatment parameters were: pulse width of 10 μs, flash frequency of 5 times / s, light intensity of 8 J / cm², and treatment time of 60 s. Simultaneously, weakly acidic electrolyzed water with an effective chlorine concentration of 30 mg / L, a pH of 6.5, and a misting rate of 2 mL / min was sprayed. After treatment, the total bacterial count of the fresh sea bass fillets was 68 CFU / g, and the inactivation rate of dominant spoilage bacteria was 97.8%.

[0044] A natural polymer material was dissolved in sterile deionized water to obtain an aqueous phase, wherein the natural polymer material included water-soluble soybean polysaccharide and whey protein isolate, with concentrations of 1.0 wt% and 1.2 wt%, respectively; a natural antibacterial active substance was dissolved in anhydrous ethanol to obtain an oil phase, wherein the natural antibacterial active substance included rosmarinic acid, protamine sulfate, and mustard oil, with concentrations of 0.5 wt%, 0.5 wt%, and 0.3 wt%, respectively; the aqueous phase and oil phase were mixed at a volume ratio of 10:1, pre-emulsified by high-speed shearing, and then subjected to microfluidic high-pressure homogenization with the following parameters: homogenization pressure 1200 bar, 5 cycles; then filtered through a 0.45 μm sterile filter membrane to obtain nano-antibacterial emulsion with a particle size of 100-200 nm.

[0045] Fresh sea bass fillets, after sterilization, were placed in nano-antibacterial emulsion at a material-to-liquid ratio of 1:10 (g / mL), and subjected to ultrasonic and ultra-high pressure treatments in sequence. The ultrasonic treatment parameters were: ultrasonic power 300 W, frequency 40 kHz, treatment time 15 min, and intermittent ratio 1:1. The ultra-high pressure treatment parameters were: treatment pressure 300 MPa, holding time 20 min, and sterile water as the pressure transmission medium.

[0046] All the above sterilization, ultrasonic and ultra-high pressure treatments were carried out at 10 ℃. After the treatment, the fresh sea bass fillets were dried with 4 ℃ sterile cold air to remove surface moisture, and then modified atmosphere packaging was carried out in a Class 100 sterile environment with a gas ratio of CO2:N2=6:4. After that, they were refrigerated at 2 ℃.

[0047] Example 3: Method for Preserving Fresh Fish Fillets Fresh sea bass fillets, 2 mm thick, were rinsed once with sterile ice water at 4°C for 3 minutes. After draining until no free water remained on the surface, they were subjected to pulsed high-intensity light combined with weakly acidic electrolyzed water atomization sterilization. The treatment parameters were: pulse width of 5 μs, flash frequency of 3 times / s, light intensity of 5 J / cm², and treatment time of 30 s. Simultaneously, weakly acidic electrolyzed water with an effective chlorine concentration of 20 mg / L, a pH of 5.5, and a misting rate of 1 mL / min was sprayed. After treatment, the total bacterial count of the fresh sea bass fillets was 72 CFU / g, and the inactivation rate of dominant spoilage bacteria was 97.5%.

[0048] Whey protein isolate was dissolved in sterile deionized water to obtain an aqueous phase with a concentration of 1 wt%. Natural antibacterial active substances were dissolved in anhydrous ethanol to obtain an oil phase. The natural antibacterial active substances included protamine sulfate, oregano oil, and mustard oil, with concentrations of 0.3 wt%, 0.3 wt%, and 0.2 wt%, respectively. The aqueous and oil phases were mixed at a volume ratio of 9:1, pre-emulsified by high-speed shearing, and then subjected to microfluidic high-pressure homogenization. The processing parameters were: homogenization pressure 1000 bar, 4 cycles. After that, it was filtered through a 0.45 μm sterile filter membrane to obtain nano-antibacterial emulsion with a particle size of 150-250 nm.

[0049] Fresh sea bass fillets, after sterilization, were placed in nano-antibacterial emulsion at a material-to-liquid ratio of 1:8 (g / mL), and subjected to ultrasonic and ultra-high pressure treatments in sequence. The ultrasonic treatment parameters were: ultrasonic power 200 W, frequency 28 kHz, treatment time 10 min, and intermittent ratio 1:1. The ultra-high pressure treatment parameters were: treatment pressure 200 MPa, holding time 10 min, and sterile water as the pressure transmission medium.

[0050] The above sterilization, ultrasonic and ultra-high pressure treatments were all carried out at 6 ℃. After the treatment, the fresh sea bass fillets were dried with sterile cold air at 4 ℃ and then packaged in a Class 100 sterile environment with a gas ratio of CO2:N2=7:3. After that, they were refrigerated at 2 ℃.

[0051] Comparative Example 1 This comparative example is a conventional method for preserving fresh fish fillets. The specific steps are as follows: Fresh grass carp fillets with a thickness of 3 mm are rinsed three times with 4℃ ice water for 2 minutes each time, drained until there is no free water on the surface, vacuum-packed, and refrigerated at 4℃.

[0052] Comparative Example 2 The preservation method is the same as in Example 1, except that sterilization, ultrasonication and ultra-high pressure treatment are omitted, and the rinsed fresh grass carp fillets are directly soaked in nano antibacterial emulsion for 20 minutes.

[0053] Comparative Example 3 The preservation method is the same as in Example 1, except that the sterilization treatment is omitted.

[0054] Comparative Example 4 The preservation method is the same as in Example 1, except that the ultrasonic treatment is omitted.

[0055] Comparative Example 5 The preservation method is the same as in Example 1, except that the ultra-high pressure treatment is omitted.

[0056] Comparative Example 6 The preservation method is the same as in Example 1, except that only the oil phase is prepared, and the sterilized fresh grass carp fillets are placed directly into the oil phase for ultrasonic and ultra-high pressure treatment.

[0057] Experimental Example The quality changes of the fresh fish fillets in the above examples and comparative treatments during storage were detected. Specifically, samples were taken on days 0, 3, 7, 14, 18, and 21 of storage for total bacterial count and volatile basic nitrogen (TVB-N) testing, as well as sensory evaluation. The testing methods are as follows, and the results are shown in Tables 1-3: Total bacterial count: Refer to GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Bacterial Count"; TVB-N: Refer to GB 5009.228-2016 "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food"; Sensory evaluation: Ten professional judges scored the samples based on four dimensions: color, odor, texture, and morphology. The maximum score was 100 points, with 60 points being the acceptable lower limit.

[0058] Table 1. Results of total bacterial count (lg value) of fresh fish fillets during storage. .

[0059] Table 2. Results of TVB-N content detection during storage of fresh fish fillets (mg / 100g) .

[0060] Table 3 Sensory evaluation results of fresh fish fillets during storage (scores) .

[0061] Note: GB 2733-2015, the National Food Safety Standard for Fresh and Frozen Aquatic Animal Products, stipulates that the TVB-N content of marine fish and shrimp must be ≤30mg / 100g. If the total bacterial count of fresh aquatic products is >10... 5 If the CFU / g (lg value > 5), the product is obviously spoiled and deteriorated.

[0062] According to the test results in Table 1-3, the fish fillets treated in Examples 1-3 of this invention still had a total bacterial count below 10 when refrigerated at 0-4℃ for 18 days. 4The CFU / g and TVB-N content were far below the national standard limit, and the sensory scores were all above 75 points, indicating that the products were still edible and of good quality, with a shelf life of over 18 days. Comparative Example 1, with conventionally treated fish fillets, was nearly spoiled after 7 days of refrigeration, and the TVB-N content completely exceeded the standard after 14 days, with a shelf life of only 3-5 days, significantly different from the present invention. Comparative Example 2, lacking source control and enhanced penetration steps, only underwent conventional soaking for antibacterial effect. After 14 days of refrigeration, the TVB-N content was close to the acceptable limit, and after 18 days, the TVB-N content also completely exceeded the standard, with a preservation effect far inferior to the present invention. Comparative Examples 3-6, after refrigeration for 18 days, all products spoiled and deteriorated, and were deemed unacceptable, especially Comparative Examples 3 and 6, where the TVB-N content exceeded the standard, and the preservation effect was inferior to the present invention.

[0063] In summary, this invention achieves long-term preservation of fresh fish fillets by combining targeted bacterial control at the source with the synergistic effect of a natural antibacterial system that enhances deep penetration, while perfectly preserving the quality of the fish fillets. It possesses extremely high innovation and industrial application value.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preserving fresh fish fillets based on source-level bacterial reduction and endogenous bacterial inhibition, characterized in that, include: After non-thermal sterilization, the fresh fish fillets are placed in a nano-antibacterial emulsion for ultrasonic-ultra-high pressure synergistic treatment, then drained, packaged, and refrigerated. The preparation method of the nano antibacterial emulsion is as follows: natural antibacterial active substances are dissolved in anhydrous ethanol as the oil phase, and an aqueous solution of natural polymer materials is used as the water phase. The oil phase and water phase are mixed, and then microfiltration is performed after microfluidic high-pressure homogenization treatment.

2. The method for preserving fresh fish fillets as described in claim 1, characterized in that, The fresh fish fillets are non-thermal sterilized to a total bacterial count ≤10² CFU / g.

3. The method for preserving fresh fish fillets as described in claim 1 or 2, characterized in that, The non-thermal sterilization is atmospheric pressure cold plasma sterilization or pulsed intense light combined with weak acidic water electrolysis atomization sterilization.

4. The method for preserving fresh fish fillets as described in claim 1, characterized in that, The ultrasonic-ultra-high pressure synergistic treatment involves first performing ultrasonic treatment to assist in impregnation, followed by ultra-high pressure treatment to allow the nano-antibacterial emulsion to penetrate deeply.

5. The method for preserving fresh fish fillets as described in claim 4, characterized in that, The ultrasonic treatment has a power of 100-300 W, a frequency of 20-40 kHz, a treatment time of 5-15 min, and an intermittent ratio of 1:1; the ultra-high pressure treatment has a pressure of 100-300 MPa and a holding time of 5-20 min.

6. The method for preserving fresh fish fillets as described in claim 1, characterized in that, The particle size of the nano-antibacterial milk is 100-300 nm.

7. The method for preserving fresh fish fillets as described in claim 1, characterized in that, The natural antibacterial active substance is at least three of the following: rosmarinic acid, protamine, oregano oil, and mustard oil.

8. The method for preserving fresh fish fillets as described in claim 1, characterized in that, The natural polymer material is at least one of chitosan, water-soluble soybean polysaccharide, and whey protein isolate.

9. The method for preserving fresh fish fillets as described in claim 1, characterized in that, The microfluidic high-pressure homogenization process is carried out at a pressure of 800-1200 bar for 3-5 cycles.

10. The method for preserving fresh fish fillets as described in claim 1, characterized in that, The packaging is either vacuum packaging or modified atmosphere packaging.