Preparation of H2O2 response type compound enzyme preparation and application of H2O2 response type compound enzyme preparation in prefabricating surimi product

Abstract

The invention provides preparation of an H2O2 response type compound enzyme preparation and application of the H2O2 response type compound enzyme preparation in pre-preparing minced fillet products. The compound enzyme preparation is prepared from maltodextrin, glucose, a rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase and moringa peroxidase through low-temperature mixing and spray drying. The preparation is added into minced fillet, and the minced fillet is subjected to ultrahigh pressure treatment for primary physical sterilization; h2O2 generated by high-pressure stress microorganisms activates glucose oxidase and catalyzes glucose to generate a large amount of H2O2 to realize signal amplification, so that moringa oleifera peroxidase is activated to catalyze polymerization of sulfonated moringa oleifera seed shell polyphenols, and a potent bactericide is generated in situ for secondary chemical sterilization; and the residual trace H2O2 can trigger the cascade reaction again to provide third long-acting protection, so that the texture, flavor and nutrition of the surimi are effectively maintained, and the shelf life is remarkably prolonged.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of prefabricated aquatic food processing, and particularly relates to preparation of an H2O2-responsive composite enzyme preparation and application thereof in prefabricated surimi products. BACKGROUND

[0002] With the economic transformation of the country, the rapid development of the food industry and the change of people's consumption mode, prefabricated dishes have become one of the fastest growing categories in the food industry. Aquatic prefabricated dishes have the characteristics of low fat, high protein and good nutritional balance, and have become a health food sweeping the world. Surimi products are a typical representative of aquatic prefabricated dishes, are rich in nutritional value, have a tender and elastic taste, are easy to digest, and are deeply loved by consumers, and are a modern aquatic prefabricated dish with good development prospects. However, surimi products are usually treated by high-temperature sterilization during production, and the sterilization temperature is usually above 120℃. High-temperature treated surimi products are beneficial to prolonging the shelf life, and are also beneficial to long-distance transportation and preservation. However, due to the high heating temperature, the protein is denatured excessively, the protein aggregates due to covalent bonding, the gel strength decreases, the meat fiber elasticity is poor, the meat is not solid, and there may be overcooked flavor, so that the inherent flavor and nutritional value are lost, thereby affecting the quality of surimi products. Therefore, it is an important content of surimi product research to study safe and efficient non-thermal surimi sterilization technology and comprehensive preservation methods, so that surimi products can maximize the original nutrition and good flavor during processing, and have a good shelf life.

[0003] Ultra-high pressure sterilization technology is one of the most promising physical sterilization technologies in the food industry in the 21st century. Ultra-high pressure non-thermal sterilization does not destroy the covalent bond in food, and the degree of influence on small molecules is also low, which can solve the problems of loss of food nutritional ingredients, change of color and flavor caused by traditional "thermal processing", and better meet the increasing demand of consumers for food safety, nutrition and flavor. Ultra-high pressure technology has been applied in aquatic prefabricated dishes, but the relatively high cost of the equipment has always been the biggest problem in its application in the food industry. The higher the pressure, the longer the action time, and the higher the cost. Research and development of synergistic sterilization measures to reduce ultra-high pressure treatment have become the key to solving this problem. Some scholars have studied synergistic measures to reduce ultra-high pressure treatment. The combination of preservatives and ultra-high pressure technology can play a barrier effect and ensure food quality. However, the state has clearly issued relevant regulations that preservatives cannot be added to prefabricated dishes. Natural plant extracts contain a variety of antibacterial components, which can be extracted and separated and added to aquatic prefabricated dishes to synergize with ultra-high pressure technology to achieve good sterilization effect, reduce processing cost, and improve food quality. It is an ideal synergistic sterilization treatment method.

[0004] Moringa oleifera is a tropical plant with unique economic value, known as the diamond of plants, and has attracted attention from the food, medical and nutritional fields. Studies have shown that the leaves and seed shells of Moringa oleifera are rich in bioactive substances such as polyphenols and flavonoids. These components not only have excellent antioxidant capacity, but also can effectively inhibit the growth of various food-borne pathogenic bacteria and spoilage bacteria, showing great potential for the development of natural preservatives. However, how to efficiently apply the natural active ingredients in Moringa oleifera to the food preservation system, especially in coordination with non-thermal processing technologies such as ultra-high pressure, to build an efficient sterilization "responsive" preservation system, rather than simple addition, is the key innovative direction to break through the current technical bottleneck. SUMMARY

[0005] The technical problem to be solved: In view of the above technical problems, the purpose of the present application is to provide a preparation method of H2O2-responsive composite enzyme preparation and its application in pre-prepared surimi products. The composite enzyme preparation is prepared by low-temperature mixing and spray drying of malt dextrin, glucose, rosemary extract, sulfonated Moringa oleifera seed shell polyphenol, glucose oxidase and Moringa oleifera peroxidase. The preparation is added to the surimi, which is first subjected to first heavy physical sterilization by ultra-high pressure treatment; H2O2 produced by high-pressure stressed microorganisms activates glucose oxidase, catalyzing glucose to produce a large amount of H2O2 to achieve signal amplification, and then activates Moringa oleifera peroxidase to catalyze sulfonated Moringa oleifera seed shell polyphenol polymerization to generate a strong bactericide in situ for second heavy chemical sterilization; residual trace amount of H2O2 can trigger the cascade reaction again to provide third heavy long-acting protection, effectively maintaining the texture, flavor and nutrition of surimi, and significantly prolonging the shelf life.

[0006] Technical scheme: A preparation method of H2O2-responsive composite enzyme preparation, comprising the following steps: S1. Dissolve malt dextrin in 0.1M pH 6.5-7.0 phosphate buffer at 4-10℃ to prepare a malt dextrin solution with a concentration of 20-30%; S2. Add glucose, rosemary extract, sulfonated Moringa oleifera seed shell polyphenol, glucose oxidase and Moringa oleifera peroxidase to the malt dextrin solution in sequence, stir uniformly, and spray dry to obtain the H2O2-responsive composite enzyme preparation.

[0007] Further, the addition amount of glucose in step S2 accounts for 15-25% of the composite enzyme preparation; the addition amount of rosemary extract accounts for 3-8% of the composite enzyme preparation; the addition amount of sulfonated Moringa oleifera seed shell polyphenol accounts for 5-10% of the composite enzyme preparation; the addition amount of glucose oxidase accounts for 1-3% of the composite enzyme preparation; and the addition amount of Moringa oleifera peroxidase accounts for 1-3% of the composite enzyme preparation.

[0008] Further, the preparation steps of sulfonated Moringa oleifera seed shell polyphenol are as follows: Step 1. Preparation of crude moringa seed shell polyphenol extract: Moringa seed shells are dried in an oven at 55-60°C for 30-48h, ground and sieved to obtain moringa seed shell powder; the moringa seed shell powder is mixed with 30-80% ethanol solution at a solid-liquid ratio of 1:(10-50), and ultrasonic extraction is performed at 25-65°C and 200-300W for 30-60min to obtain the crude moringa seed shell polyphenol extract; Step 2. Purification of moringa seed shell polyphenol: 20.00g of pre-processed D-101 macroporous resin is loaded into a glass chromatography column, and the crude moringa seed shell polyphenol extract with a concentration of 1-4mg / mL (previously adjusted to pH 3-7 with phosphate buffer) is loaded at a flow rate of 1-5mL / min for 30-180min. After adsorption saturation, distilled water is used for elution until the Molish reaction is negative. Then 25-75% ethanol solution is added for desorption at a flow rate of 1-4mL / min. The eluate is collected, concentrated and dried to constant weight to obtain the moringa seed shell polyphenol. Step 3. Preparation of sulfonated moringa seed shell polyphenol: The moringa seed shell polyphenol is dissolved in deionized water to obtain a moringa seed shell polyphenol solution, and sulfamic acid is added. The mixture is reacted at 60-70°C for 2-4h, cooled to room temperature, dialyzed and freeze-dried to obtain the sulfonated moringa seed shell polyphenol.

[0009] Further, the concentration of the moringa seed shell polyphenol solution in step 3 is 5-20mg / mL; the mass-volume ratio of sulfamic acid to moringa seed shell polyphenol solution is (2-5):1.

[0010] The H2O2-responsive complex enzyme preparation prepared by the above preparation method.

[0011] Application of the above H2O2-responsive complex enzyme preparation in pre-prepared surimi products.

[0012] Further, the specific steps of the pre-prepared surimi product are as follows: (1) Preparation of pre-prepared surimi: Fresh fish is cleaned, scaled, skinned and deboned, and the fish meat is washed with clean water for 3 times. The fish meat is chopped and mixed for 4-8min, and cornstarch, egg white, salt, sugar, onion and ginger, yeast extract, cooking wine are added and stirred for 10-20min. Then the H2O2-responsive complex enzyme preparation is added and stirred uniformly. The mixture is shaped, heat-treated, vacuum-packed and refrigerated to obtain the pre-prepared surimi. (2) Ultra-high pressure sterilization treatment: The pre-prepared surimi is subjected to ultra-high pressure sterilization at 100-400MPa and 25-55°C for 5-20min, and then depressurized to obtain the pre-prepared surimi product.

[0013] Further, the specific formula of the prefabricated surimi in step (1) is: 70-75% fish meat, 5-6% corn starch, 6-7% egg white, 2-2.5% salt, 1.5-2% sugar, 0.3-0.35% onion ginger, 0.2-0.25% yeast extract, 0.1-0.15% cooking wine, 0.1-0.5% H2O2 responsive complex enzyme preparation, and the rest is water.

[0014] Beneficial effects: The application prepares an H2O2 responsive complex enzyme preparation, and the super high pressure is applied together with the H2O2 responsive complex enzyme preparation for sterilization of the prefabricated surimi, which has the following advantages: (1) first, the moringa peroxidase (MPOD), sulfonated moringa seed shell polyphenol (SMSP), glucose oxidase (GOD), glucose, rosemary extract and malt dextrin are mixed to prepare a complex enzyme preparation powder by spray drying, the powder is added to the surimi as a food ingredient during the surimi grinding stage, and then the packaged prefabricated surimi is subjected to super high pressure treatment, so that the high pressure directly destroys the cell structure of microorganisms to achieve the first physical sterilization; (2) the super high pressure stress causes oxidative stress in the cells of microorganisms, and H2O2 is generated, which is then released by cell rupture, and trace H2O2 activates glucose oxidase, which starts to efficiently and continuously catalyze the substrate glucose, consume oxygen and produce a large amount of H2O2, achieving signal amplification; (3) sufficient H2O2 activates the moringa peroxidase, which can efficiently catalyze the substrate sulfonated moringa seed shell polyphenol to generate high-concentration sulfonated polyphenol oligomers / quinoxalines (strong bactericides) in situ in a short time, and the newly generated bactericides perform the second chemical sterilization on the microorganisms damaged by high pressure; (4) if there is pollution bacteria reproduction during storage, the trace H2O2 produced by the metabolism of the bacteria will be sensed again, and the system will be started again to achieve precise and efficient targeted sterilization and provide the third long-acting protection; (5) the surimi is rich in unsaturated fatty acids, which are easily oxidized and rancid under the action of oxygen, light and metal ions, producing an unpleasant smell, and the active ingredients (such as carnosic acid and carnosol) in the rosemary extract are strong natural antioxidants that can effectively scavenge free radicals, block the chain reaction of lipid oxidation and inhibit the lipid oxidation of the surimi; the rosemary extract can also protect the color and delay browning, thereby protecting the flavor and color of the surimi product.

[0015] The H2O2 response type composite enzyme preparation prepared by the application is prepared by modifying the moringa oleifera seed shell polyphenol into sulfonated moringa oleifera seed shell polyphenol, because the solubility of natural moringa polyphenol in water is limited, and the moringa polyphenol is easy to aggregate, cannot be quickly and uniformly dispersed in surimi, and is easy to combine with protein in surimi through hydrogen bond and hydrophobic interaction, so that the reaction activity and antibacterial activity are lost, therefore, the moringa oleifera seed shell polyphenol in the application is modified by sulfonation, the phenolic hydroxyl (-OH) in the molecular structure of the moringa oleifera seed shell polyphenol is reacted with aminosulfonic acid to generate sulfonated seed shell moringa polyphenol, after the introduction of the negatively charged sulfonic acid group, the sulfonated moringa oleifera seed shell polyphenol and the protein molecules with the same negative charge will produce electrostatic repulsion, thereby avoiding ineffective combination, and ensuring that most of the sulfonated moringa oleifera seed shell polyphenol can be free in the water phase to participate in effective reaction, and the sulfonation modification can guide the moringa peroxidase catalytic reaction to generate sulfonated polyphenol oligomers or sulfonated quinones, and these new products have stronger water solubility and antibacterial activity. DETAILED DESCRIPTION

[0016] The application will be further described in combination with examples, and the following examples are used to explain the application, and the application is not limited to the following examples. Example 1

[0017] A preparation method of an H2O2 response type composite enzyme preparation, comprising the following steps: S1. Preparation of moringa oleifera seed shell polyphenol crude extract: moringa oleifera seed shells are dried in a 60℃ oven for 48h, crushed and sieved to obtain moringa oleifera seed shell powder; the moringa oleifera seed shell powder is mixed with 50% ethanol solution at a material-liquid ratio of 1:20, ultrasonic extraction is carried out at 50℃ and 243 W for 30 min, and moringa oleifera seed shell polyphenol crude extract is obtained; S2. Purification of moringa oleifera seed shell polyphenol: 20.00 g of pre-processed D-101 macroporous resin is weighed and loaded into a glass chromatography column, and 1 mg / mL moringa oleifera seed shell polyphenol crude extract (previously adjusted to pH 5 with phosphate buffer) is selected for loading, the loading flow rate is 2 mL / min, the adsorption time is 180 min, after adsorption saturation, distilled water is used for elution until the Molish reaction is negative, then 50% ethanol solution is added for desorption, the elution speed is 2 mL / min, the eluate is collected, concentrated and dried to constant weight to obtain moringa oleifera seed shell polyphenol; S3. Preparation of sulfonated moringa oleifera seed shell polyphenol: moringa oleifera seed shell polyphenol is dissolved in deionized water to prepare a 10 mg / mL moringa oleifera seed shell polyphenol solution, aminosulfonic acid is added, the mass-volume ratio of aminosulfonic acid to moringa oleifera seed shell polyphenol solution is 3:1, 60℃ reaction is carried out for 4h, cooling to room temperature, dialysis and freeze-drying are carried out, and sulfonated moringa oleifera seed shell polyphenol is obtained; S4. Maltodextrin is dissolved in 0.1M pH7.0 phosphate buffer to prepare a 25% maltodextrin solution at 4℃ environment; S5. To the malt dextrin solution, glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase and moringa peroxidase were added in sequence (the addition amount of glucose was 15% of the complex enzyme preparation; the addition amount of the rosemary extract was 5% of the complex enzyme preparation; the addition amount of the sulfonated moringa seed shell polyphenol was 8% of the complex enzyme preparation; the addition amount of the glucose oxidase was 2% of the complex enzyme preparation; and the addition amount of the moringa peroxidase was 2% of the complex enzyme preparation), and stirred uniformly, and then spray dried to obtain the H2O2-responsive complex enzyme preparation.

[0018] Example 2 A preparation method of an H2O2-responsive complex enzyme preparation, comprising the following steps: S1. Preparation of moringa seed shell polyphenol crude extract: moringa seed shells were dried in a 60℃ oven for 48h, crushed and sieved to obtain moringa seed shell powder; the moringa seed shell powder was mixed with 50% ethanol solution at a solid-liquid ratio of 1:20, ultrasonically extracted at 50℃ for 30min to obtain the moringa seed shell polyphenol crude extract; S2. Purification of moringa seed shell polyphenol: 20.00 g of pre-processed D-101 macroporous resin was loaded into a glass chromatography column, and 2mg / mL moringa seed shell polyphenol crude extract (previously adjusted to pH 5 with phosphate buffer) was selected for loading, with a loading flow rate of 2mL / min and an adsorption time of 180 min; after adsorption saturation, distilled water was used for elution until the Molish reaction was negative, and then 50% ethanol solution was added for desorption, with an elution speed of 2 mL / min; the eluate was collected, concentrated and dried to constant weight to obtain the moringa seed shell polyphenol; S3. Preparation of sulfonated moringa seed shell polyphenol: the moringa seed shell polyphenol was dissolved in deionized water to prepare a 10mg / mL moringa seed shell polyphenol solution, and sulfamic acid was added at a mass-volume ratio of 3:1, and reacted at 60℃ for 4h; after cooling to room temperature, dialysis and freeze-drying, the sulfonated moringa seed shell polyphenol was obtained; S4. Malt dextrin was dissolved in 0.1M pH 7.0 phosphate buffer at 4℃ to prepare a 25% malt dextrin solution; S5. To the malt dextrin solution, glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase and moringa peroxidase were added in sequence (the addition amount of glucose was 15% of the complex enzyme preparation; the addition amount of the rosemary extract was 5% of the complex enzyme preparation; the addition amount of the sulfonated moringa seed shell polyphenol was 8% of the complex enzyme preparation; the addition amount of the glucose oxidase was 2% of the complex enzyme preparation; and the addition amount of the moringa peroxidase was 2% of the complex enzyme preparation), and stirred uniformly, and then spray dried to obtain the H2O2-responsive complex enzyme preparation.

[0019] Example 3 A preparation method of an H2O2-responsive composite enzyme preparation, comprising the following steps: S1. Preparation of a crude moringa seed shell polyphenol extract: moringa seed shells were dried in an oven at 60°C for 48 h, crushed and sieved to obtain moringa seed shell powder; the moringa seed shell powder was mixed with 50% ethanol solution at a solid-liquid ratio of 1:20, and ultrasonic extraction was performed at 50°C for 30 min at 243 W to obtain the crude moringa seed shell polyphenol extract; S2. Purification of moringa seed shell polyphenol: 20.00 g of pre-processed D-101 macroporous resin was loaded into a glass chromatography column, and a crude moringa seed shell polyphenol extract with a concentration of 4 mg / mL (previously adjusted to pH 5 with a phosphate buffer) was selected for loading at a loading flow rate of 2 mL / min for 180 min of adsorption; after saturation, distilled water was used for elution until the Molish reaction was negative, and then 50% ethanol solution was added for desorption at an elution rate of 2 mL / min; the eluate was collected, concentrated and dried to constant weight to obtain the moringa seed shell polyphenol; S3. Preparation of sulfonated moringa seed shell polyphenol: the moringa seed shell polyphenol was dissolved in deionized water to prepare a moringa seed shell polyphenol solution with a concentration of 10 mg / mL; sulfamic acid was added at a mass-volume ratio of 3:1 to the moringa seed shell polyphenol solution, and the mixture was reacted at 60°C for 4 h; after cooling to room temperature, dialysis and freeze-drying, sulfonated moringa seed shell polyphenol was obtained; S4. Maltodextrin was dissolved in 0.1M pH 7.0 phosphate buffer at 4°C to prepare a maltodextrin solution with a concentration of 25%; S5. Glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase and moringa peroxidase were sequentially added to the maltodextrin solution (the amount of glucose added was 15% of the composite enzyme preparation; the amount of rosemary extract added was 5% of the composite enzyme preparation; the amount of sulfonated moringa seed shell polyphenol added was 8% of the composite enzyme preparation; the amount of glucose oxidase added was 2% of the composite enzyme preparation; and the amount of moringa peroxidase added was 2% of the composite enzyme preparation), and the mixture was stirred uniformly and spray dried to obtain the H2O2-responsive composite enzyme preparation.

[0020] Example 4 A preparation method of an H2O2-responsive composite enzyme preparation, comprising the following steps: S1. Preparation of a crude moringa seed shell polyphenol extract: moringa seed shells were dried in an oven at 60°C for 48 h, crushed and sieved to obtain moringa seed shell powder; the moringa seed shell powder was mixed with 50% ethanol solution at a solid-liquid ratio of 1:20, and ultrasonic extraction was performed at 50°C for 30 min at 243 W to obtain the crude moringa seed shell polyphenol extract; S2. Purification of moringa seed shell polyphenol: 20.00 g of pre-processed D-101 macroporous resin was weighed and loaded into a glass chromatographic column, and a crude moringa seed shell polyphenol solution with a concentration of 2 mg / mL (previously adjusted to pH 5 with a phosphate buffer) was selected for loading. The loading flow rate was 2 mL / min, and the adsorption time was 180 min. After adsorption saturation, distilled water was used for elution until the Molish reaction was negative. Then, 50% ethanol solution was added for desorption, and the elution rate was 2 mL / min. The eluate was collected, concentrated, and dried to constant weight to obtain the moringa seed shell polyphenol; S3. Preparation of sulfonated moringa seed shell polyphenol: Moringa seed shell polyphenol was dissolved in deionized water to prepare a 5 mg / mL moringa seed shell polyphenol solution. Amino sulfonic acid was added, and the mass-volume ratio of amino sulfonic acid to moringa seed shell polyphenol solution was 3:1. The solution was reacted at 60°C for 4 h, cooled to room temperature, dialyzed, and freeze-dried to obtain sulfonated moringa seed shell polyphenol; S4. Maltodextrin was dissolved in 0.1 M pH 7.0 phosphate buffer at 4°C to prepare a 25% maltodextrin solution; S5. Glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase, and moringa peroxidase were sequentially added to the maltodextrin solution (the amount of glucose added was 15% of the complex enzyme preparation; the amount of rosemary extract added was 5% of the complex enzyme preparation; the amount of sulfonated moringa seed shell polyphenol added was 8% of the complex enzyme preparation; the amount of glucose oxidase added was 2% of the complex enzyme preparation; and the amount of moringa peroxidase added was 2% of the complex enzyme preparation). The mixture was stirred uniformly and spray dried to obtain the H2O2-responsive complex enzyme preparation.

[0021] Example 5 A method for preparing an H2O2-responsive complex enzyme preparation, comprising the following steps: S1. Preparation of crude moringa seed shell polyphenol solution: Moringa seed shells were dried in a 60°C oven for 48 h, crushed, and sieved to obtain moringa seed shell powder. The moringa seed shell powder was mixed with 50% ethanol solution at a material-liquid ratio of 1:20, and ultrasonic extraction was performed at 50°C and 243 W for 30 min to obtain the crude moringa seed shell polyphenol solution; S2. Purification of moringa seed shell polyphenol: 20.00 g of pre-processed D-101 macroporous resin was weighed and loaded into a glass chromatographic column, and a crude moringa seed shell polyphenol solution with a concentration of 2 mg / mL (previously adjusted to pH 5 with a phosphate buffer) was selected for loading. The loading flow rate was 2 mL / min, and the adsorption time was 180 min. After adsorption saturation, distilled water was used for elution until the Molish reaction was negative. Then, 50% ethanol solution was added for desorption, and the elution rate was 2 mL / min. The eluate was collected, concentrated, and dried to constant weight to obtain the moringa seed shell polyphenol; S3. Preparation of sulfonated moringa seed shell polyphenol: dissolve the moringa seed shell polyphenol in deionized water to prepare a 20 mg / mL moringa seed shell polyphenol solution, add sulfamic acid, and the mass-volume ratio of sulfamic acid to the moringa seed shell polyphenol solution is 3:1, react at 60°C for 4h, cool to room temperature, dialysis, freeze-drying, and sulfonated moringa seed shell polyphenol is obtained; S4. Dissolve the malt dextrin in 0.1M pH7.0 phosphate buffer at 4°C to prepare a 25% malt dextrin solution; S5. Add glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase and moringa peroxidase (the addition amount of glucose is 15% of the complex enzyme preparation; the addition amount of the rosemary extract is 5% of the complex enzyme preparation; the addition amount of the sulfonated moringa seed shell polyphenol is 8% of the complex enzyme preparation; the addition amount of the glucose oxidase is 2% of the complex enzyme preparation; the addition amount of the moringa peroxidase is 2% of the complex enzyme preparation) to the malt dextrin solution in sequence, stir uniformly, and spray dry to obtain the H2O2-responsive complex enzyme preparation.

[0022] Example 6 A preparation method of an H2O2-responsive complex enzyme preparation, comprising the following steps: S1. Preparation of moringa seed shell polyphenol crude extract: dry the moringa seed shell in a 60°C oven for 48h, crush and sieve to obtain moringa seed shell powder; mix the moringa seed shell powder with 50% ethanol solution at a material-liquid ratio of 1:20, ultrasonically extract at 50°C for 30min, and moringa seed shell polyphenol crude extract is obtained; S2. Purification of moringa seed shell polyphenol: weigh 20.00 g of pre-processed D-101 macroporous resin into a glass chromatography column, select a 2 mg / mL moringa seed shell polyphenol crude extract (previously adjusted to pH 5 with phosphate buffer) for loading, the loading flow rate is 2 mL / min, the adsorption time is 180 min, after adsorption saturation, elute with distilled water until the Molish reaction is negative, then add 50% ethanol solution for desorption, the elution rate is 2 mL / min, collect the eluate, concentrate and dry to constant weight to obtain moringa seed shell polyphenol; S3. Preparation of sulfonated moringa seed shell polyphenol: dissolve the moringa seed shell polyphenol in deionized water to prepare a 10 mg / mL moringa seed shell polyphenol solution, add sulfamic acid, and the mass-volume ratio of sulfamic acid to the moringa seed shell polyphenol solution is 2:1, react at 60°C for 4h, cool to room temperature, dialysis, freeze-drying, and sulfonated moringa seed shell polyphenol is obtained; S4. Dissolve the malt dextrin in 0.1M pH7.0 phosphate buffer at 4°C to prepare a 25% malt dextrin solution; S5. To the malt dextrin solution, glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase and moringa peroxidase were added in sequence (the addition amount of glucose was 15% of the complex enzyme preparation; the addition amount of the rosemary extract was 5% of the complex enzyme preparation; the addition amount of the sulfonated moringa seed shell polyphenol was 8% of the complex enzyme preparation; the addition amount of the glucose oxidase was 2% of the complex enzyme preparation; and the addition amount of the moringa peroxidase was 2% of the complex enzyme preparation), and stirred uniformly, and then spray dried to obtain the H2O2-responsive complex enzyme preparation.

[0023] Example 7 A preparation method of an H2O2-responsive complex enzyme preparation, comprising the following steps: S1. Preparation of moringa seed shell polyphenol crude extract: moringa seed shells were dried in a 60℃ oven for 48h, crushed and sieved to obtain moringa seed shell powder; the moringa seed shell powder was mixed with 50% ethanol solution at a solid-liquid ratio of 1:20, ultrasonically extracted at 50℃ for 30min to obtain the moringa seed shell polyphenol crude extract; S2. Purification of moringa seed shell polyphenol: 20.00 g of pre-processed D-101 macroporous resin was loaded into a glass chromatography column, and 2mg / mL moringa seed shell polyphenol crude extract (previously adjusted to pH 5 with phosphate buffer) was selected for loading, with a loading flow rate of 2mL / min and an adsorption time of 180 min; after adsorption saturation, distilled water was used for elution until the Molish reaction was negative, and then 50% ethanol solution was added for desorption, with an elution speed of 2 mL / min; the eluate was collected, concentrated and dried to constant weight to obtain the moringa seed shell polyphenol; S3. Preparation of sulfonated moringa seed shell polyphenol: the moringa seed shell polyphenol was dissolved in deionized water to prepare a 10mg / mL moringa seed shell polyphenol solution, and sulfamic acid was added at a mass-volume ratio of 5:1, and reacted at 60℃ for 4h; after cooling to room temperature, dialysis and freeze-drying, the sulfonated moringa seed shell polyphenol was obtained; S4. Malt dextrin was dissolved in 0.1M pH 7.0 phosphate buffer at 4℃ to prepare a 25% malt dextrin solution; S5. To the malt dextrin solution, glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase and moringa peroxidase were added in sequence (the addition amount of glucose was 15% of the complex enzyme preparation; the addition amount of the rosemary extract was 5% of the complex enzyme preparation; the addition amount of the sulfonated moringa seed shell polyphenol was 8% of the complex enzyme preparation; the addition amount of the glucose oxidase was 2% of the complex enzyme preparation; and the addition amount of the moringa peroxidase was 2% of the complex enzyme preparation), and stirred uniformly, and then spray dried to obtain the H2O2-responsive complex enzyme preparation.

[0024] Example 8 A preparation method of an H2O2-responsive composite enzyme preparation, comprising the following steps: S1. Preparation of a crude moringa seed shell polyphenol extract: moringa seed shells were dried in an oven at 60°C for 48 h, crushed and sieved to obtain moringa seed shell powder; the moringa seed shell powder was mixed with 50% ethanol solution at a solid-liquid ratio of 1:20, and ultrasonic extraction was performed at 50°C for 30 min at 243 W to obtain the crude moringa seed shell polyphenol extract; S2. Purification of moringa seed shell polyphenol: 20.00 g of pre-processed D-101 macroporous resin was loaded into a glass chromatography column, and a crude moringa seed shell polyphenol extract with a concentration of 2 mg / mL (previously adjusted to pH 5 with a phosphate buffer) was selected for loading, with a loading flow rate of 2 mL / min and an adsorption time of 180 min; after adsorption saturation, distilled water was used for elution until the Molish reaction was negative, and then 50% ethanol solution was added for desorption, with an elution speed of 2 mL / min; the eluate was collected, concentrated and dried to constant weight to obtain the moringa seed shell polyphenol; S3. Preparation of sulfonated moringa seed shell polyphenol: the moringa seed shell polyphenol was dissolved in deionized water to prepare a moringa seed shell polyphenol solution with a concentration of 10 mg / mL, and sulfamic acid was added at a mass-volume ratio of 3:1 with respect to the moringa seed shell polyphenol solution; the mixture was reacted at 60°C for 4 h, cooled to room temperature, dialyzed and freeze-dried to obtain the sulfonated moringa seed shell polyphenol; S4. Maltodextrin was dissolved in 0.1M pH 7.0 phosphate buffer at 4°C to prepare a maltodextrin solution with a concentration of 20%; S5. The maltodextrin solution was sequentially added with glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase and moringa peroxidase (the amount of glucose added was 15% of the composite enzyme preparation; the amount of rosemary extract added was 5% of the composite enzyme preparation; the amount of sulfonated moringa seed shell polyphenol added was 8% of the composite enzyme preparation; the amount of glucose oxidase added was 2% of the composite enzyme preparation; and the amount of moringa peroxidase added was 2% of the composite enzyme preparation), and stirred uniformly, followed by spray drying to obtain the H2O2-responsive composite enzyme preparation.

[0025] Example 9 A preparation method of an H2O2-responsive composite enzyme preparation, comprising the following steps: S1. Preparation of a crude moringa seed shell polyphenol extract: moringa seed shells were dried in an oven at 60°C for 48 h, crushed and sieved to obtain moringa seed shell powder; the moringa seed shell powder was mixed with 50% ethanol solution at a solid-liquid ratio of 1:20, and ultrasonic extraction was performed at 50°C for 30 min at 243 W to obtain the crude moringa seed shell polyphenol extract; S2. Purification of moringa seed shell polyphenol: 20.00 g of pre-processed D-101 macroporous resin was weighed and loaded into a glass chromatographic column, and a crude moringa seed shell polyphenol solution with a concentration of 2 mg / mL (previously adjusted to pH 5 with a phosphate buffer) was selected for loading. The loading flow rate was 2 mL / min, and the adsorption time was 180 min. After adsorption saturation, distilled water was used for elution until the Molish reaction was negative. Then, 50% ethanol solution was added for desorption, and the elution rate was 2 mL / min. The eluate was collected, concentrated, and dried to constant weight to obtain the moringa seed shell polyphenol; S3. Preparation of sulfonated moringa seed shell polyphenol: Moringa seed shell polyphenol was dissolved in deionized water to prepare a 10 mg / mL moringa seed shell polyphenol solution. Amino sulfonic acid was added, and the mass-volume ratio of amino sulfonic acid to moringa seed shell polyphenol solution was 3:1. The solution was reacted at 60°C for 4 h, cooled to room temperature, dialyzed, and freeze-dried to obtain sulfonated moringa seed shell polyphenol; S4. Maltodextrin was dissolved in 0.1 M pH 7.0 phosphate buffer at 4°C to prepare a 30% maltodextrin solution; S5. Glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase, and moringa peroxidase were sequentially added to the maltodextrin solution (the amount of glucose added was 15% of the complex enzyme preparation; the amount of rosemary extract added was 5% of the complex enzyme preparation; the amount of sulfonated moringa seed shell polyphenol added was 8% of the complex enzyme preparation; the amount of glucose oxidase added was 2% of the complex enzyme preparation; and the amount of moringa peroxidase added was 2% of the complex enzyme preparation). The mixture was stirred uniformly and spray dried to obtain the H2O2-responsive complex enzyme preparation.

[0026] Example 10 A method for preparing an H2O2-responsive complex enzyme preparation, comprising the following steps: S1. Preparation of crude moringa seed shell polyphenol solution: Moringa seed shells were dried in a 60°C oven for 48 h, crushed, and sieved to obtain moringa seed shell powder. The moringa seed shell powder was mixed with 50% ethanol solution at a material-liquid ratio of 1:20, and ultrasonic extraction was performed at 50°C and 243 W for 30 min to obtain the crude moringa seed shell polyphenol solution; S2. Purification of moringa seed shell polyphenol: 20.00 g of pre-processed D-101 macroporous resin was weighed and loaded into a glass chromatographic column, and a crude moringa seed shell polyphenol solution with a concentration of 2 mg / mL (previously adjusted to pH 5 with a phosphate buffer) was selected for loading. The loading flow rate was 2 mL / min, and the adsorption time was 180 min. After adsorption saturation, distilled water was used for elution until the Molish reaction was negative. Then, 50% ethanol solution was added for desorption, and the elution rate was 2 mL / min. The eluate was collected, concentrated, and dried to constant weight to obtain the moringa seed shell polyphenol; S3. Preparation of sulfonated moringa seed shell polyphenol: Moringa seed shell polyphenol was dissolved in deionized water to prepare a 10 mg / mL moringa seed shell polyphenol solution, and sulfamic acid was added. The mass-volume ratio of sulfamic acid to moringa seed shell polyphenol solution was 3:1. The solution was reacted at 60°C for 4 hours, cooled to room temperature, dialyzed, and freeze-dried to obtain sulfonated moringa seed shell polyphenol. S4. Maltodextrin was dissolved in 0.1M pH 7.0 phosphate buffer at 4°C to prepare a 25% maltodextrin solution. S5. Glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase, and moringa peroxidase were sequentially added to the maltodextrin solution (the amount of glucose added was 15% of the complex enzyme preparation; the amount of rosemary extract added was 5% of the complex enzyme preparation; the amount of sulfonated moringa seed shell polyphenol added was 10% of the complex enzyme preparation; the amount of glucose oxidase added was 2% of the complex enzyme preparation; and the amount of moringa peroxidase added was 2% of the complex enzyme preparation). The mixture was stirred uniformly and spray dried to obtain the H2O2-responsive complex enzyme preparation.

[0027] Comparative Example 1 The difference between this comparative example and Example 10 is that the moringa seed shell polyphenol was not modified. The preparation method is as follows: A preparation method of an H2O2-responsive complex enzyme preparation includes the following steps: S1. Preparation of moringa seed shell polyphenol crude extract: Moringa seed shells were dried in a 60°C oven for 48 hours, crushed, and sieved to obtain moringa seed shell powder. The moringa seed shell powder was mixed with 50% ethanol solution at a solid-liquid ratio of 1:20, and ultrasonically extracted at 50°C for 30 minutes at 243 W to obtain the moringa seed shell polyphenol crude extract. S2. Purification of moringa seed shell polyphenol: 20.00 g of pre-treated D-101 macroporous resin was loaded into a glass chromatography column, and 2 mg / mL moringa seed shell polyphenol crude extract (previously adjusted to pH 5 with phosphate buffer) was selected for loading. The loading flow rate was 2 mL / min, and the adsorption time was 180 min. After adsorption saturation, distilled water was used for elution until the Molish reaction was negative. Then, 50% ethanol solution was added for desorption at an elution rate of 2 mL / min. The eluate was collected, concentrated, and dried to constant weight to obtain the moringa seed shell polyphenol. S3. Maltodextrin was dissolved in 0.1M pH 7.0 phosphate buffer at 4°C to prepare a 25% maltodextrin solution. S4. Adding glucose, rosemary extract, moringa seed shell polyphenol, glucose oxidase and moringa peroxidase into the maltodextrin solution in sequence (the addition amount of glucose is 15% of the complex enzyme preparation; the addition amount of the rosemary extract is 5% of the complex enzyme preparation; the addition amount of the moringa seed shell polyphenol is 10% of the complex enzyme preparation; the addition amount of the glucose oxidase is 2% of the complex enzyme preparation; and the addition amount of the moringa peroxidase is 2% of the complex enzyme preparation), stirring uniformly, and spray drying to obtain the H2O2-responsive complex enzyme preparation.

[0028] Comparative Example 2 The difference between the present comparative example and Example 10 is that glucose oxidase is absent, and the preparation is as follows: A preparation method of an H2O2-responsive complex enzyme preparation includes the following steps: S1. Preparation of moringa seed shell polyphenol crude extract: drying moringa seed shells in a 60°C oven for 48h, crushing and sieving to obtain moringa seed shell powder; mixing the moringa seed shell powder with 50% ethanol solution at a solid-liquid ratio of 1:20, ultrasonic extraction at 50°C and 243 W for 30min to obtain the moringa seed shell polyphenol crude extract; S2. Purification of moringa seed shell polyphenol: weighing 20.00 g of pre-processed D-101 macroporous resin into a glass chromatography column, selecting the moringa seed shell polyphenol crude extract with a concentration of 2 mg / mL (previously adjusted to pH 5 with phosphate buffer) for loading, loading flow rate is 2 mL / min, adsorption time is 180 min, after adsorption saturation, eluting with distilled water until the Molish reaction is negative, then adding 50% ethanol solution for desorption, elution speed is 2 mL / min, collecting the eluate, concentrating and drying to constant weight to obtain the moringa seed shell polyphenol; S3. Preparation of sulfonated moringa seed shell polyphenol: dissolving the moringa seed shell polyphenol in deionized water to prepare a moringa seed shell polyphenol solution with a concentration of 10 mg / mL, adding sulfamic acid, the mass-volume ratio of sulfamic acid to moringa seed shell polyphenol solution is 3:1, reacting at 60°C for 4h, cooling to room temperature, dialysis and freeze-drying to obtain the sulfonated moringa seed shell polyphenol; S4. Dissolving maltodextrin in 0.1M pH 7.0 phosphate buffer at 4°C to prepare a maltodextrin solution with a concentration of 25%; S5. Adding glucose, rosemary extract, sulfonated moringa seed shell polyphenol and moringa peroxidase into the maltodextrin solution in sequence (the addition amount of glucose is 15% of the complex enzyme preparation; the addition amount of the rosemary extract is 5% of the complex enzyme preparation; the addition amount of the sulfonated moringa seed shell polyphenol is 10% of the complex enzyme preparation; and the addition amount of the moringa peroxidase is 2% of the complex enzyme preparation), stirring uniformly, and spray drying to obtain the H2O2-responsive complex enzyme preparation.

[0029] Comparative Example 3 The difference between this comparative example and Example 10 is the absence of moringa peroxidase, as follows: A preparation method of an H2O2-responsive composite enzyme preparation, comprising the following steps: S1. Preparation of a crude extract of moringa seed shell polyphenols: moringa seed shells were dried in an oven at 60°C for 48 h, crushed and sieved to obtain moringa seed shell powder; the moringa seed shell powder was mixed with 50% ethanol solution at a solid-liquid ratio of 1:20, and ultrasonic extraction was performed at 50°C for 30 min at 243 W to obtain the crude extract of moringa seed shell polyphenols; S2. Purification of moringa seed shell polyphenols: 20.00 g of pre-processed D-101 macroporous resin was loaded into a glass chromatography column, and a crude extract of moringa seed shell polyphenols with a concentration of 2 mg / mL (previously adjusted to pH 5 with a phosphate buffer) was selected for loading, with a loading flow rate of 2 mL / min and an adsorption time of 180 min; after adsorption saturation, distilled water was used for elution until the Molish reaction was negative, and then 50% ethanol solution was added for desorption, with an elution speed of 2 mL / min; the eluate was collected, concentrated and dried to constant weight to obtain the moringa seed shell polyphenols; S3. Preparation of sulfonated moringa seed shell polyphenols: the moringa seed shell polyphenols were dissolved in deionized water to prepare a moringa seed shell polyphenol solution with a concentration of 10 mg / mL, and sulfamic acid was added at a mass-volume ratio of 3:1 with respect to the moringa seed shell polyphenol solution; the mixture was reacted at 60°C for 4 h, cooled to room temperature, dialyzed and freeze-dried to obtain the sulfonated moringa seed shell polyphenols; S4. Maltodextrin was dissolved in 0.1M pH 7.0 phosphate buffer at 4°C to prepare a maltodextrin solution with a concentration of 25%; S5. The maltodextrin solution was sequentially added with glucose, rosemary extract, sulfonated moringa seed shell polyphenols and glucose oxidase (the amount of glucose added was 15% of the composite enzyme preparation; the amount of rosemary extract added was 5% of the composite enzyme preparation; the amount of sulfonated moringa seed shell polyphenols added was 10% of the composite enzyme preparation; and the amount of glucose oxidase added was 2% of the composite enzyme preparation), and stirred uniformly, followed by spray drying to obtain the H2O2-responsive composite enzyme preparation.

[0030] Performance test: (1) Inhibition zone diameter Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) were selected as test strains, which were activated and inoculated in LB broth medium, and cultured at 37°C for 24 h, and then the bacterial solution was diluted with physiological saline to a concentration of 1×10 6CFU / mL of bacterial suspension, 100 μL of the above bacterial suspension was removed and added to LB agar medium, shaken well, and used as needed; 50 mg of H2O2-responsive composite enzyme preparation prepared in Examples 1-10 and Comparative Examples 1-3 was weighed, dissolved with phosphate buffer, and glucose (simulated reaction substrate) and trace H2O2 (e.g., 50 μM, simulating the initial signal of ultra-high pressure stress) were added to a final concentration of 10 mM to trigger the bactericidal cascade reaction. After 30 min of reaction at 37°C, the sample solution after triggering the reaction was obtained; Oxford cups were placed on the surface of the coagulated plate, and 200 μL of the sample solution after triggering the reaction was added to the Oxford cups, and a negative control (phosphate buffer containing only glucose and H2O2) was set. The plate was placed in a 4°C refrigerator for 3 h, then transferred to a 37°C constant temperature incubator, and cultured for 24 h. The diameter of each inhibition zone was measured with a vernier caliper, and the average value of three parallel measurements was taken.

[0031] Table 1 Inhibition zone diameter

[0032] As can be seen from Table 1, the H2O2-responsive composite enzyme preparations prepared in the examples all showed significant and strong antibacterial activity, among which Example 10 had the best effect. In Comparative Example 1, unmodified moringa seed shell polyphenol was added, and the phenolic hydroxyl group of natural polyphenol is easily combined with protein in surimi through hydrogen bonding and hydrophobic interaction. In the culture medium for measuring the inhibition zone, it will also combine with the components in the agar or culture medium. This "ineffective combination" consumes the reaction substrate, making it impossible to participate in the oxidative polymerization reaction catalyzed by MPOD, so as to generate sufficient amounts of highly effective bactericides. In Comparative Example 2, glucose oxidase was lacking, and the antibacterial activity was weak. The catalytic activity of moringa peroxidase was highly dependent on the concentration of H2O2. Without sufficient "fuel" (H2O2) provided by GOD, the MPOD enzyme activity was extremely low, and it could not effectively catalyze the generation of bactericides from sulfonated polyphenol. In Comparative Example 3, moringa peroxidase was lacking, and the antibacterial activity was the lowest. Without MPOD, the system only had H2O2 generated by GOD as a bactericidal factor. Although H2O2 has certain bactericidal effect, its efficacy and speed of action are far inferior to the polyphenol derivative bactericides catalyzed by MPOD.

[0033] (2) Lipid antioxidant activity determination (DPPH free radical scavenging method) DPPH stock solution preparation: accurately weigh DPPH powder, prepare a 0.1 mM stock solution with absolute ethanol, and store in a 4°C refrigerator in the dark; Sample solution preparation: accurately weigh an equal amount (e.g., 10 mg) of composite enzyme preparation powder of Examples 1-10 and Comparative Examples 1-3, prepare a 1 mg / mL solution with phosphate buffer, and vortex to fully dissolve or disperse.

[0034] Sample group: take 2 mL sample solution, add 2 mL DPPH ethanol solution, vortex mix well; Sample blank group: take 2 mL sample solution, add 2 mL anhydrous ethanol, vortex mix well; Blank control group: take 2 mL phosphate buffer, add 2 mL DPPH ethanol solution, vortex mix well; Put all test tubes in the dark, react at room temperature for 30 min, use 1 cm cuvette, measure the absorbance value of each tube at 517 nm wavelength with anhydrous ethanol as reference, measure in parallel for 3 times, calculate the DPPH free radical scavenging rate (%) according to the following formula: DPPH free radical scavenging rate (%) = [1- (A 样品组 -A 样品空白组 ) / A 空白对照组 ] × 100% Table 2 DPPH free radical scavenging rate

[0035] As can be seen from Table 2, the DPPH free radical scavenging rates of all examples and comparative examples are relatively high, and the scavenging rate of Comparative Example 1 slightly decreases after sulfonation modification; the scavenging rates of Comparative Example 2 lacking glucose oxidase and Comparative Example 3 lacking moringa peroxidase do not change much, and the main function of these two enzymes in the system is to catalyze biochemical reactions, and together constitute a H2O2-responsive bactericidal system, and the antioxidant capacity mainly comes from rosemary extract and sulfonated moringa seed shell polyphenol itself.

[0036] Example 11 The application of the H2O2-responsive composite enzyme preparation in the prepared surimi product is as follows: (1) Preparation of prepared surimi: after cleaning, scaling, peeling and removing the spine of fresh fish, the fish meat is scraped and washed with clean water for 3 times, 70% of the fish meat is put into a chopper, 14.6% of ice water is added, and choppering is carried out for 6 min, then 5% of corn starch, 6% of egg white, 2% of salt, 1.5% of sugar, 0.3% of onion and ginger, 0.2% of yeast extract and 0.1% of cooking wine are added in sequence and stirred for 15 min, finally, 0.3% of the H2O2-responsive composite enzyme preparation prepared in Example 10 is added and stirred uniformly, and then the prepared surimi is obtained after shaping treatment, 90℃ heat treatment for 30 min, vacuum packaging and low-temperature refrigeration; (2) Ultra-high pressure sterilization treatment: the prepared surimi is subjected to ultra-high pressure sterilization at 200 MPa and 25℃ for 10 min, and then the pressure is released, and the prepared surimi product is obtained.

[0037] Comparative Example 4 The difference between this comparative example and Example 11 is that the H2O2-responsive composite enzyme preparation is not added, which is as follows: Application of H2O2-responsive complex enzyme preparation in pre-prepared surimi products, the steps are as follows: (1) Preparation of pre-prepared surimi: after cleaning, scaling, peeling and removing the spine of fresh fish, the fish meat was scraped and rinsed with clean water for 3 times, 70% of the fish meat was put into a chopper, 14.6% of ice water was added at the same time, and the chopper was operated for 6 min, then 5% of corn starch, 6% of egg white, 2% of salt, 1.5% of sugar, 0.3% of onion and ginger, 0.2% of yeast extract and 0.1% of cooking wine were added in sequence and stirred for 15 min, then the pre-prepared surimi was obtained after molding treatment, 90℃ heat treatment for 30 min, vacuum packaging and low-temperature refrigeration; (2) Ultra-high pressure sterilization treatment: the pre-prepared surimi was subjected to ultra-high pressure sterilization at 200 MPa and 25℃ for 10 min, and then the pressure was released to obtain the pre-prepared surimi product.

[0038] Comparative Example 5 The difference between this comparative example and Example 11 is that no ultra-high pressure treatment is used, and the specific steps are as follows: Application of H2O2-responsive complex enzyme preparation in pre-prepared surimi products, the steps are as follows: Preparation of pre-prepared surimi: after cleaning, scaling, peeling and removing the spine of fresh fish, the fish meat was scraped and rinsed with clean water for 3 times, 70% of the fish meat was put into a chopper, 14.6% of ice water was added at the same time, and the chopper was operated for 6 min, then 5% of corn starch, 6% of egg white, 2% of salt, 1.5% of sugar, 0.3% of onion and ginger, 0.2% of yeast extract and 0.1% of cooking wine were added in sequence and stirred for 15 min, then the pre-prepared surimi was obtained after molding treatment, 90℃ heat treatment for 30 min, vacuum packaging and low-temperature refrigeration;

[0039] Comparative Example 6 The difference between this comparative example and Example 11 is that no H2O2-responsive complex enzyme preparation is prepared, but sulfonated Khaya senegal seed shell polyphenol is directly added, and the specific steps are as follows: Application of H2O2-responsive complex enzyme preparation in pre-prepared surimi products, the steps are as follows: (1) Preparation of pre-prepared surimi: after cleaning, scaling, peeling and removing the spine of fresh fish, the fish meat was scraped and rinsed with clean water for 3 times, 70% of the fish meat was put into a chopper, 14.6% of ice water was added at the same time, and the chopper was operated for 6 min, then 5% of corn starch, 6% of egg white, 2% of salt, 1.5% of sugar, 0.3% of onion and ginger, 0.2% of yeast extract and 0.1% of cooking wine were added in sequence and stirred for 15 min, then the pre-prepared surimi was obtained after molding treatment, 90℃ heat treatment for 30 min, vacuum packaging and low-temperature refrigeration; (2) Ultra-high pressure sterilization treatment: the pre-prepared surimi is subjected to ultra-high pressure sterilization at 200 MPa and 25°C for 10 min, and then depressurized to obtain the pre-prepared surimi product.

[0040] Comparative Example 7 The difference between this comparative example and Example 11 is that the H2O2-responsive complex enzyme preparation is not prepared, but the moringa seed shell polyphenol is directly added, as follows: The application of the H2O2-responsive complex enzyme preparation in the pre-prepared surimi product is as follows: (1) Preparation of pre-prepared surimi: after the fresh fish is cleaned, scaled, skinned and debarbed, the fish meat is scraped and rinsed with clean water for 3 times. 70% of the fish meat is put into a chopper, and 14.6% of ice water is added. The chopper is operated for 6 min. Then 5% of corn starch, 6% of egg white, 2% of salt, 1.5% of sugar, 0.3% of onion and ginger, 0.2% of yeast extract, and 0.1% of cooking wine are added and stirred for 15 min. Finally, 0.3% of moringa seed shell polyphenol is added and stirred uniformly. The pre-prepared surimi is obtained after shaping, 90°C heat treatment for 30 min, vacuum packaging, and low-temperature refrigeration. (2) Ultra-high pressure sterilization treatment: the pre-prepared surimi is subjected to ultra-high pressure sterilization at 200 MPa and 25°C for 10 min, and then depressurized to obtain the pre-prepared surimi product.

[0041] Comparative Example 8 The difference between this comparative example and Example 11 is that the H2O2-responsive complex enzyme preparation prepared in Comparative Example 1 is used.

[0042] Comparative Example 9 The difference between this comparative example and Example 11 is that the H2O2-responsive complex enzyme preparation prepared in Comparative Example 2 is used.

[0043] Comparative Example 10 The difference between this comparative example and Example 11 is that the H2O2-responsive complex enzyme preparation prepared in Comparative Example 3 is used.

[0044] Performance test: (1) Total number of colonies, TVB-N (volatile salt-based nitrogen) content Refer to GB 4789.2-2022 "National Food Safety Standard Food Microbiological Examination Determination of Total Number of Colonies"; Refer to GB 5009.228-2016 "National Food Safety Standard Determination of Volatile Salt-based Nitrogen in Food".

[0045] Table 3 Total number of colonies and TVB-N content in pre-prepared surimi product

[0046] As shown in Table 3, the super-high pressure physical destruction, H2O2 response type composite enzyme preparation of Example 11 has high sterilization, rosemary antioxidant, and triple synergistic sterilization, and microorganisms are inhibited efficiently, and freshness is perfectly maintained; Comparative Example 4 has only super-high pressure single physical sterilization, lacks chemical long-acting sterilization, and the residual bacteria content increases; Comparative Example 5 does not use super-high pressure treatment, lacks initial damage of physical sterilization, and cannot generate a large amount of H2O2 to activate subsequent reactions, and only relies on heat treatment and weak natural antibacterial, and the effect is poor; Comparative Example 6 only adds sulfonated polyphenol, and Comparative Example 7 only adds natural polyphenol, and the antibacterial and fresh-keeping effects are also relatively poor; Comparative Example 8 uses an unmodified enzyme preparation, and even if the enzyme is complete, it cannot catalyze sufficient sterilizing agents, and the effect is similar to that of Comparative Example 6 / 7; Comparative Example 9 uses an enzyme preparation lacking GOD, and Comparative Example 10 uses an enzyme preparation lacking MPOD, and the entire H2O2 response system is not complete, and the sterilization effect is reduced.

[0047] (2) Texture measurement The TPA was used for texture measurement, and the prepared surimi product was cut into a cylinder with a diameter of 20 mm and a height of 20 mm, a TA-XTPlus texture analyzer was selected, and the measurement parameters were set as follows: pre-measurement rate 1.00 mm·s -1 , test rate 1.00 mm·s -1 , post-measurement rate 1.00 mm·s -1 , compression ratio 40%, trigger force 5 g, and then a P50 cylindrical probe was used for a second compression test on the surimi cylinder.

[0048] Table 4: Texture properties of prepared surimi products

[0049] As shown in Table 4, the prepared surimi product of Example 11 has low hardness, high elasticity and chewiness, and it can be seen that the surimi product prepared by the H2O2 response type composite enzyme preparation and super-high pressure synergistic treatment has advantages in texture properties.

[0050] The above description is only a preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification of the above embodiments according to the technical essence of the present application, which does not deviate from the technical solutions of the present application, still belongs to the protection scope of the technical solutions of the present application.

Claims

1. A method for preparing an H202-responsive complex enzyme preparation, characterized by, Comprising the following steps: S1. Dissolve malt dextrin in 0.1M pH 6.5-7.0 phosphate buffer at 4-10℃ to prepare a malt dextrin solution with a concentration of 20-30%; S2. Add glucose, rosemary extract, sulfonated moringa seed shell polyphenol, glucose oxidase and moringa peroxidase to the malt dextrin solution in sequence, stir evenly, and spray dry to obtain the H2O2-responsive composite enzyme preparation.

2. The method for preparing an H2O2-responsive complex enzyme preparation according to claim 1, characterized in that: The amount of glucose added in step S2 accounts for 15-25% of the composite enzyme preparation; the amount of rosemary extract added accounts for 3-8% of the composite enzyme preparation; the amount of sulfonated moringa seed shell polyphenol added accounts for 5-10% of the composite enzyme preparation; the amount of glucose oxidase added accounts for 1-3% of the composite enzyme preparation; and the amount of moringa peroxidase added accounts for 1-3% of the composite enzyme preparation.

3. The method for preparing an H2O2-responsive complex enzyme preparation according to claim 2, characterized in that, The preparation steps of the sulfonated moringa seed shell polyphenol are as follows: Step 1. Preparation of moringa seed shell polyphenol crude extract: Dry the moringa seed shell in a 55-60℃ oven for 30-48h, crush and sieve to obtain moringa seed shell powder; mix the moringa seed shell powder with 30-80% ethanol solution at a solid-liquid ratio of 1:(10-50), ultrasonically extract at 25-65℃ for 30-60min to obtain the moringa seed shell polyphenol crude extract; Step 2. Purification of moringa seed shell polyphenol: Weigh D-101 macroporous resin and load it into a glass chromatography column, select the moringa seed shell polyphenol crude extract with a concentration of 1-4mg / mL for loading, the loading flow rate is 1-5mL / min, the adsorption time is 30-180min, after adsorption saturation, elute with distilled water until the Molish reaction is negative, then add 25-75% ethanol solution for desorption, the elution rate is 1-4mL / min, collect the eluate, concentrate and dry to constant weight to obtain the moringa seed shell polyphenol; Step 3. Preparation of sulfonated moringa seed shell polyphenol: Dissolve the moringa seed shell polyphenol in deionized water to prepare a moringa seed shell polyphenol solution, add sulfamic acid, react at 60-70℃ for 2-4h, cool to room temperature, dialyze and freeze-dry to obtain the sulfonated moringa seed shell polyphenol.

4. The method for preparing an H2O2-responsive complex enzyme preparation according to claim 3, characterized in that: The concentration of the moringa seed shell polyphenol solution in step 3 is 5-20mg / mL; the mass-volume ratio of sulfamic acid to moringa seed shell polyphenol solution is (2-5):

1.

5. The H2O2-responsive composite enzyme preparation prepared by the preparation method of any one of claims 1-4.

6. The use of the H2O2-responsive composite enzyme preparation of claim 5 in the preparation of a pre-prepared surimi product.

7. Use according to claim 6, characterized in that, The specific steps of the pre-prepared surimi product are as follows: (1) Preparation of pre-prepared surimi: Chop and mix the fish meat for 4-8min, add corn starch, egg white, salt, sugar, onion and ginger, yeast extract, cooking wine and stir for 10-20min, then add the H2O2-responsive composite enzyme preparation and stir evenly, shape, heat treatment, vacuum packaging, low-temperature refrigeration to obtain the pre-prepared surimi; (2) Ultra-high pressure sterilization treatment: the prefabricated surimi is subjected to ultra-high pressure sterilization at 100-400 MPa and 25-55 ℃ for 5-20 min, and then depressurized to obtain the prefabricated surimi product.

8. Use according to claim 7, characterized in that, The specific formula of the prefabricated surimi in step (1) is as follows: 70-75% fish meat, 5-6% corn starch, 6-7% egg white, 2-2.5% salt, 1.5-2% sugar, 0.3-0.35% onion and ginger, 0.2-0.25% yeast extract, 0.1-0.15% cooking wine, 0.1-0.5% H2O2 responsive compound enzyme preparation, and the rest is water.

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