High-temperature-resistant modified embedded nisin and application thereof in high-temperature baked egg processing

Abstract

The invention relates to the technical field of roast egg preservation, in particular to high-temperature-resistant modified embedded nisin and application thereof in high-temperature roast egg processing. Comprising nisin, hydroxypropyl-beta-cyclodextrin wrapping the outer side of a non-bacteriostatic hydrophobic region of the nisin, and non-denatured type I collagen wrapping the outer sides of the nisin and the hydroxypropyl-beta-cyclodextrin, and the non-denatured type I collagen forms a continuous porous network structure. After the modified embedded nisin is applied to high-temperature baked egg processing, the total number of bacterial colonies of the baked eggs can be smaller than 103 CFU / g, the modified embedded nisin also has an excellent antibacterial effect on gram-negative bacteria of escherichia coli, the storage period reaches 3 months, and the shelf life is prolonged.

Description

Technical Field

[0001] This invention relates to the field of egg preservation technology, specifically to a high-temperature resistant modified encapsulated nisin and its application in high-temperature baked egg processing. Background Technology

[0002] Nisin is an approved natural antimicrobial peptide whose metabolites are amino acids. It has high safety and broad-spectrum inhibitory effects on Gram-positive pathogens such as Bacillus cereus and Staphylococcus aureus. It has been widely used in the field of food preservation.

[0003] High-temperature baked egg products, as a popular food category, have stringent requirements for the high-temperature and pH resistance of preservatives during processing, as well as specific needs for preservative safety and targeted application. However, nisin suffers from three core technical defects in the processing and application of high-temperature baked egg products, severely limiting its practical application:

[0004] 1. Poor high temperature resistance: The α-helical conformation of nisin is prone to unfolding in environments above 60°C. When egg products are baked at high temperatures, its activity is easily lost, and it cannot maintain effective antibacterial activity.

[0005] 2. Weak pH tolerance: The isoelectric point of nisin is about 8.5, while the pH range of the egg processing system is 6.5-7.0. In this environment, the solubility of nisin is low, and the amide bond is easily hydrolyzed, resulting in the loss of its antibacterial activity.

[0006] 3. Insufficient antibacterial targeting: Traditional nisin is indiscriminately dispersed in food matrix and is easily adsorbed and inactivated by proteins and lipids in the matrix. It cannot accurately target pathogens, which not only leads to low preservation efficiency, but also requires increased dosage to ensure effectiveness. In addition, it only has a broad-spectrum inhibitory effect on Gram-positive pathogens and cannot inhibit Gram-negative pathogens.

[0007] Although some nisin modification schemes have emerged in the existing technology, there are still obvious limitations: some schemes use chemical cross-linking modification to improve stability, but there is a safety risk of generating toxic byproducts; simple nisin inclusion technology can only slightly improve the stability of nisin, and cannot simultaneously solve the three core problems of high temperature resistance, pH resistance and targeted antibacterial action, and is prone to blocking the antibacterial sites of nisin due to the encapsulation effect, resulting in antibacterial failure.

[0008] Therefore, developing a modified encapsulated nisin that can be used in high-temperature baked egg products, possesses high-temperature resistance, polar pH resistance, targeted antibacterial function, and meets food-grade safety standards is an urgent problem to be solved. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the existing defects and provide a high-temperature resistant modified encapsulated nisin and its application in high-temperature baked egg processing, so as to solve the problems involved in the background art.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A heat-resistant modified encapsulated nisin comprises nisin, hydroxypropyl-β-cyclodextrin wrapped around the non-bacterial hydrophobic region of nisin, and non-denatured type I collagen wrapped around both nisin and hydroxypropyl-β-cyclodextrin, wherein the non-denatured type I collagen forms a continuous porous network structure.

[0012] Furthermore, the modified encapsulated nisin exhibits antibacterial effects against both Gram-negative and Gram-positive bacteria.

[0013] Furthermore, the hydroxypropyl-β-cyclodextrin does not encapsulate the antibacterial region of nisin, and the antibacterial region of nisin penetrates the pores of non-denatured type I collagen and binds to bacteria in a targeted manner.

[0014] The lactic acid nisin is obtained by fermentation with Lactococcus lactis, and hydroxypropyl-β-cyclodextrin is added in the later stage of fermentation to improve the activity of lactic acid nisin.

[0015] A method for preparing a high-temperature resistant modified encapsulated nisin.

[0016] (1) Preparation of the nisin-hydroxypropyl-β-cyclodextrin composition:

[0017] Under stirring conditions of 25~30℃ and 150~200rpm, nisin aqueous solution was slowly added dropwise to hydroxypropyl-β-cyclodextrin aqueous solution. After the addition was completed, the mixture was magnetically stirred for 2~3h. After stirring, the mixture was placed in a 4℃ refrigerator and allowed to stand for 12h to obtain nisin-hydroxypropyl-β-cyclodextrin composition.

[0018] (2) Preparation of modified encapsulated nisin:

[0019] Under stirring conditions of 30~35℃ and 100~150 rpm, non-denatured type I collagen aqueous solution was slowly added dropwise to the lactic acid nisin-hydroxypropyl-β-cyclodextrin composition obtained in (1), and then a composite buffer aqueous solution was added until the final concentration of the composite buffer aqueous solution was 0.02~0.05mol / L. The pH of the solution was adjusted to 6.5~7.0, and stirring was continued for 4~6h, so that the non-denatured type I collagen formed a porous network structure through self-assembly. After stirring, the solution was refrigerated at 4℃ for 8h to solidify the porous network structure and obtain the modified encapsulated lactic acid nisin solution.

[0020] (3) Freeze-drying preservation:

[0021] The modified encapsulated nisin solution was centrifuged at 4℃ and 20,000~30,000 rpm for 30~60 min. The precipitate obtained by centrifugation was added to a freeze-drying protectant and freeze-dried for 20~28 h to obtain modified encapsulated nisin powder.

[0022] Furthermore, the molar ratio of nisin to hydroxypropyl-β-cyclodextrin is 1:1 to 1:2.

[0023] Furthermore, the mass ratio of the lactic acid nisin to the non-denatured type I collagen is 1:3 to 1:5.

[0024] Furthermore,

[0025] The nisin aqueous solution is prepared using sterile water, with a concentration of 1.0~2.0 mg / mL and a pH of 6.0~6.5.

[0026] The aqueous solution of hydroxypropyl-β-cyclodextrin was prepared using sterile water and had a concentration of 2.0~4.0 mg / mL.

[0027] The non-denatured type I collagen aqueous solution was prepared using sterile water and had a concentration of 3.0~5.0 mg / mL.

[0028] Furthermore,

[0029] The composite buffer is a sodium citrate-sodium dihydrogen phosphate buffer solution with a pH of 6.5-7.0.

[0030] Application of a heat-resistant modified encapsulated nisin in high-temperature baked egg processing.

[0031] (1) Egg shelling and filtering

[0032] Select fresh, undamaged, and odorless pre-washed sterile eggs, and check the integrity and cleanliness of the eggshells to ensure there are no cracks, stains, or signs of spoilage.

[0033] Put the eggs into a sheller to remove the shells;

[0034] Pour the shelled egg liquid into a filter with an 80-mesh screen to filter out residual eggshell fragments and ligature impurities, resulting in preliminarily purified egg liquid.

[0035] (2) Blending and Filtration

[0036] Pour the coarsely filtered egg liquid into a mixing container, add the auxiliary ingredients according to the formula, and stir with a mixer at a speed of 300-500 rpm for 5-8 minutes to fully mix the egg liquid and auxiliary ingredients to obtain the prepared egg liquid;

[0037] Meanwhile, take the freeze-dried modified nisin powder, reconstitute it with sterile water to 0.05-0.1 g / mL, stir at 30℃ and 100 rpm for 10 min until a uniform dispersion is formed, and add the reconstituted modified nisin solution to the pre-prepared modified egg liquid, adding 10 mL of modified nisin solution per kg of modified egg liquid;

[0038] The mixed egg liquid is filtered a second time through a 120-mesh filter to further remove fine impurities and ensure a smooth texture.

[0039] (3) Baking and cooling

[0040] Pour the finely filtered egg mixture into the mold, bake at a temperature greater than 100℃ for more than 6 minutes, and observe the solidification of the egg mixture during baking until the surface is evenly golden brown and the inside is fully cooked.

[0041] After baking, unmold the egg to obtain a shaped baked egg base; avoid contact with oil or impurities during the cooling process;

[0042] (4) Inspection and bagging

[0043] The appearance, smell, and taste of the baked egg embryos are tested, and unqualified products with cracked surfaces, uneven color, or off-odors are removed.

[0044] Pack qualified roasted eggs into food-grade composite packaging bags according to the set weight, vacuum pack them using a vacuum packaging machine, and seal them to ensure that the packaging bags are airtight and the seal is flat.

[0045] (5) Secondary sterilization and cooling

[0046] The sealed packaging bags are placed in a sterilization tank and pasteurized to kill bacteria, mold and other microorganisms inside the packaging bags.

[0047] After sterilization, the product is placed in a cooling tank and cooled to below 15°C.

[0048] (6) Inspection and warehousing

[0049] The X-ray foreign object detection machine is used to detect whether there are metal, glass, or stone foreign objects mixed in with the product. The product is then passed through a metal detector to further check for fine metal impurities to ensure product safety. Products that pass the inspection are packed into cartons, sealed, and labeled with the production date and shelf life. They are then sent to a constant temperature and humidity warehouse with a temperature of 0-8℃ and a humidity of ≤75% for storage, awaiting shipment.

[0050] Compared with the prior art, the beneficial effects of the present invention are:

[0051] The modified encapsulated nisin of this application, through the dual protection of hydroxypropyl-β-cyclodextrin and non-denatured type I collagen, increases the heat resistance and pH stability of nisin, significantly improving the targeted antibacterial efficiency and extending the shelf life of high-temperature baked egg products. In addition, the modified encapsulated nisin of this application also has a broad-spectrum antibacterial effect against both Gram-positive and Gram-negative pathogens, and can be widely used in various high-temperature baked egg products or other high-temperature processed foods. Attached Figure Description

[0052] Figure 1 This is a flowchart illustrating the preparation process of the baked eggs described in this application.

[0053] Figure 2 The results of the detection of the modified encapsulated lactic acid and the effect of lactic acid on the cell wall of Staphylococcus aureus are presented in this invention.

[0054] Figure 3 This invention relates to the modified encapsulated lactic acid nisin and the detection results of the effect of lactic acid nisin on the cell wall of Escherichia coli.

[0055] Figure 4 The graph shows the results of the modified encapsulated nisin and the thermal stability of nisin at different pH values.

[0056] Figure 5 The thermogravimetric analysis (TGA) results of the modified encapsulated nisin of this invention and nisin under pH 2 and pH 7 conditions are shown. Detailed Implementation

[0057] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] The raw materials used in this application are:

[0059] Nisin, food grade, purchased from Shandong Freda Biotechnology Co., Ltd.

[0060] Hydroxypropyl-β-cyclodextrin, food grade, purchased from Zhejiang Tianhe Food Biotechnology Co., Ltd.

[0061] Undenatured type I collagen, retaining the natural triple helix structure, food grade, purchased from Hebei Rencan Biotechnology Co., Ltd.

[0062] Sodium citrate, food grade, purchased from Henan Linbang Biotechnology Co., Ltd.

[0063] Sodium dihydrogen phosphate, food grade, purchased from Jiangsu Kelunduo Food Ingredients Co., Ltd.

[0064] Mannitol, food grade, purchased from Leshengyuan Biotechnology (Nanjing)

[0065] Hydroxypropyl distarch phosphate, prepared from corn starch or potato starch, has a viscosity range of 1600–2100 BU.

[0066] High-fructose corn syrup, model: SW-maltose syrup, purchased from Shandong Xinhongyan New Materials Co., Ltd.

[0067] Skipjack tuna powder, purchased from Qingdao Rishengchang Food Ingredients Co., Ltd.

[0068] Natural carotene, CAS No.: 7235-40-7, food grade, purchased from Jiangxi Huayuyuan Biotechnology Co., Ltd.

[0069] Hydroxypropyl starch, CAS: 9049-76-7, food grade, purchased from Zhengzhou Yuhe Food Additives Co., Ltd.

[0070] Soy lecithin, CAS: 8030-76-0, food grade, purchased from Hefei Shengrun Biological Products Co., Ltd.

[0071] Example 1:

[0072] A heat-resistant modified encapsulated nisin comprises nisin, hydroxypropyl-β-cyclodextrin encapsulating the non-bacterial hydrophobic region of nisin, and non-denatured type I collagen encapsulating both nisin and hydroxypropyl-β-cyclodextrin. The hydroxypropyl-β-cyclodextrin does not encapsulate the antibacterial region of nisin. The non-denatured type I collagen forms a continuous porous network structure. The antibacterial region of nisin penetrates the pores of the non-denatured type I collagen and binds to bacteria. The modified encapsulated nisin exhibits antibacterial effects against both Gram-negative and Gram-positive bacteria.

[0073] The modified hydroxypropyl-β-cyclodextrin (HP-β-CD) of nisin in this application forms a hydrophobic inclusion link with the hydrophobic regions (non-antibacterial regions) of the N-terminal ring and C-terminal α-helix of nisin, thus fully exposing the core antibacterial sites in the hydrophilic region, physically blocking the destruction of the α-helix conformation of the peptide chain by high temperature, and stabilizing the structure of the peptide chain.

[0074] Undenatured type I collagen is linked to nisin and HP-β-CD via hydrogen bonds. Its own triple helix structure forms a porous network on the outermost layer. Furthermore, the hydrogen bond network between undenatured type I collagen and nisin can enhance the rigidity of nisin's peptide chain and reduce its unfolding probability.

[0075] A method for preparing a high-temperature resistant modified encapsulated nisin.

[0076] (1) Preparation of the nisin-hydroxypropyl-β-cyclodextrin composition:

[0077] Under stirring conditions of 25°C and 150 rpm, an aqueous solution of nisin was slowly added dropwise to an aqueous solution of hydroxypropyl-β-cyclodextrin. The aqueous solution of nisin was prepared using sterile water with a concentration of 1.5 mg / mL and a pH of 6.0. The aqueous solution of hydroxypropyl-β-cyclodextrin was prepared using sterile water with a concentration of 3 mg / mL. The molar ratio of nisin to hydroxypropyl-β-cyclodextrin was 1:1.5.

[0078] After the addition was completed, the mixture was magnetically stirred for 2 hours. After stirring, the mixture was placed in a 4°C refrigerator and allowed to stand for 12 hours to obtain the lactic acid nisin-hydroxypropyl-β-cyclodextrin composition.

[0079] (2) Preparation of modified encapsulated nisin:

[0080] Under stirring conditions of 30℃ and 100rpm, non-denatured type I collagen aqueous solution was slowly added dropwise to the lactic acid nisin-hydroxypropyl-β-cyclodextrin composition obtained in (1). The non-denatured type I collagen aqueous solution was prepared with sterile water and had a concentration of 4mg / mL. The lactic acid nisin was added at a mass ratio of 1:4 to non-denatured type I collagen.

[0081] Add a composite buffer solution until the final concentration of the composite buffer solution is 0.04 mol / L, and adjust the pH of the solution to 6.5. The composite buffer is sodium citrate-sodium dihydrogen phosphate buffer with a pH of 6.5. Continue stirring for 4 hours to allow the non-denatured type I collagen to form an outer porous network structure through self-assembly. After stirring, refrigerate at 4°C for 8 hours to solidify the porous network structure, obtaining the modified nisin-embedded solution.

[0082] (3) Freeze-drying preservation:

[0083] The modified encapsulated nisin solution was centrifuged at 4℃ and 20,000 rpm for 30 min. The precipitate obtained by centrifugation was added to food-grade mannitol as a freeze-drying protectant at a concentration of 5% (w / w), and then freeze-dried for 20 h to obtain modified encapsulated nisin powder.

[0084] The application of a heat-resistant modified encapsulated nisin in the high-temperature baking of eggs, with a flowchart of the preparation process of the baked egg product as shown below. Figure 1 As shown;

[0085] (1) Egg shelling and filtering

[0086] Select fresh, undamaged, and odorless pre-washed sterile eggs, and check the integrity and cleanliness of the eggshells to ensure there are no cracks, stains, or signs of spoilage.

[0087] Put the eggs into a sheller to remove the shells;

[0088] Pour the shelled egg liquid into a filter with an 80-mesh screen to filter out residual eggshell fragments and ligature impurities, resulting in preliminarily purified egg liquid.

[0089] (2) Blending and Filtration

[0090] 1 kg of coarsely filtered egg liquid was added to a mixing tank. 0.6 g of soy lecithin, 240 g of fructose syrup, 20 g of hydroxypropyl distarch phosphate, and 0.08 g of hydroxypropyl starch were added to 250 g of water and dissolved. The mixture was stirred at 300 rpm for 5 minutes. After dissolution, the mixture was added to the mixing tank and stirred at 30 rpm. 3 g of white vinegar, 10 g of benzoin, 30 g of bonito flakes, 20 g of sodium chloride, and 0.03 g of natural carotene were added in sequence. The mixture was stirred at 15°C for 3 hours and then passed through a 20-mesh sieve to obtain the prepared egg liquid.

[0091] Meanwhile, take the freeze-dried modified nisin powder, reconstitute it with sterile water to 0.08 g / mL (containing 13 mg / mL of nisin), stir at 30°C and 100 rpm for 10 min until a uniform dispersion is formed, add the reconstituted modified nisin solution to the prepared egg liquid, and add 10 mL of modified nisin solution per kg of prepared egg liquid;

[0092] The mixed egg liquid is filtered a second time through a 120-mesh filter to further remove fine impurities and ensure a smooth texture.

[0093] (3) Baking and cooling

[0094] Pour the finely filtered egg mixture into the mold, bake at a temperature greater than 100℃ for more than 6 minutes, and observe the solidification of the egg mixture during baking until the surface is evenly golden brown and the inside is fully cooked.

[0095] After baking, unmold the egg to obtain a shaped baked egg base; avoid contact with oil or impurities during the cooling process;

[0096] (4) Inspection and bagging

[0097] The appearance, smell, and taste of the baked egg embryos are tested, and unqualified products with cracked surfaces, uneven color, or off-odors are removed.

[0098] Pack qualified roasted eggs into food-grade composite packaging bags according to the set weight, vacuum pack them using a vacuum packaging machine, and seal them to ensure that the packaging bags are airtight and the seal is flat.

[0099] (5) Secondary sterilization and cooling

[0100] The sealed packaging bags are placed in a sterilization tank and pasteurized to kill bacteria, mold and other microorganisms inside the packaging bags.

[0101] After sterilization, the product is placed in a cooling tank and cooled to below 15°C.

[0102] (6) Inspection and warehousing

[0103] The X-ray foreign object detection machine is used to detect whether there are metal, glass, or stone foreign objects mixed in with the product. The product is then passed through a metal detector to further check for fine metal impurities to ensure product safety. Products that pass the inspection are packed into cartons, sealed, and labeled with the production date and shelf life. They are then sent to a constant temperature and humidity warehouse with a temperature of 0-8℃ and a humidity of ≤75% for storage, awaiting shipment.

[0104] Antibacterial process: Nisin crosses the network of pores in non-denatured type I collagen and binds to pathogens. The antibacterial zone (hydrophilic zone) of nisin binds to the phosphatidylglycerol of the pathogenic bacterial cell membrane. The binding strength of the protein is much higher than that of HP-β-CD and the non-inhibitory region (hydrophobic region) of nisin. It competitively replaces the connection between HP-β-CD and nisin through stronger electrostatic attraction, triggering targeted release. The subtilisin protease secreted by pathogenic bacteria degrades the irregular coiled region of non-denatured type I collagen, weakens its hydrogen bond connection with HP-β-CD and nisin, expands the porous channel, and increases the release rate of nisin.

[0105] Example 2:

[0106] A heat-resistant modified encapsulated nisin comprises nisin, hydroxypropyl-β-cyclodextrin encapsulating the non-bacterial hydrophobic region of nisin, and non-denatured type I collagen encapsulating both nisin and hydroxypropyl-β-cyclodextrin. The hydroxypropyl-β-cyclodextrin does not encapsulate the antibacterial region of nisin. The non-denatured type I collagen forms a continuous porous network structure. The antibacterial region of nisin penetrates the pores of the non-denatured type I collagen and binds to bacteria. The modified encapsulated nisin exhibits antibacterial effects against both Gram-negative and Gram-positive bacteria.

[0107] A method for preparing a high-temperature resistant modified encapsulated nisin.

[0108] (1) Preparation of the nisin-hydroxypropyl-β-cyclodextrin composition:

[0109] Under stirring conditions of 25°C and 150 rpm, an aqueous solution of nisin was slowly added dropwise to an aqueous solution of hydroxypropyl-β-cyclodextrin. The aqueous solution of nisin was prepared with sterile water, with a concentration of 1 mg / mL and a pH of 6.0. The aqueous solution of hydroxypropyl-β-cyclodextrin was prepared with sterile water, with a concentration of 2 mg / mL. The molar ratio of nisin to hydroxypropyl-β-cyclodextrin was 1:1.

[0110] After the addition was completed, the mixture was magnetically stirred for 2 hours. After stirring, the mixture was placed in a 4°C refrigerator and allowed to stand for 12 hours to obtain the lactic acid nisin-hydroxypropyl-β-cyclodextrin composition.

[0111] (2) Preparation of modified encapsulated nisin:

[0112] Under stirring conditions of 30℃ and 100rpm, non-denatured type I collagen aqueous solution was slowly added dropwise to the lactic acid nisin-hydroxypropyl-β-cyclodextrin composition obtained in (1). The non-denatured type I collagen aqueous solution was prepared with sterile water and had a concentration of 3mg / mL. The lactic acid nisin was added at a mass ratio of 1:3 to non-denatured type I collagen.

[0113] Add a composite buffer solution until the final concentration of the composite buffer solution is 0.02 mol / L, and adjust the pH of the solution to 6.5. The composite buffer is sodium citrate-sodium dihydrogen phosphate buffer with a pH of 6.5. Continue stirring for 4 hours to allow the non-denatured type I collagen to form an outer porous network structure through self-assembly. After stirring, refrigerate at 4°C for 8 hours to solidify the porous network structure, obtaining the modified nisin-embedded solution.

[0114] (3) Freeze-drying preservation:

[0115] The modified encapsulated nisin solution was centrifuged at 4℃ and 20,000 rpm for 30 min. The precipitate obtained by centrifugation was added to food-grade mannitol as a freeze-drying protectant at a concentration of 5% (w / w), and then freeze-dried for 20 h to obtain modified encapsulated nisin powder.

[0116] Application of a heat-resistant modified encapsulated nisin in high-temperature baked egg processing.

[0117] (1) Egg shelling and filtering

[0118] Select fresh, undamaged, and odorless pre-washed sterile eggs, and check the integrity and cleanliness of the eggshells to ensure there are no cracks, stains, or signs of spoilage.

[0119] Put the eggs into a sheller to remove the shells;

[0120] Pour the shelled egg liquid into a filter with an 80-mesh screen to filter out residual eggshell fragments and ligature impurities, resulting in preliminarily purified egg liquid.

[0121] (2) Blending and Filtration

[0122] 1 kg of coarsely filtered egg liquid was added to a mixing tank. 0.6 g of soy lecithin, 240 g of fructose syrup, 20 g of hydroxypropyl distarch phosphate, and 0.08 g of hydroxypropyl starch were added to 250 g of water and dissolved. The mixture was stirred at 300 rpm for 5 minutes. After dissolution, the mixture was added to the mixing tank and stirred at 30 rpm. 3 g of white vinegar, 10 g of benzoin, 30 g of bonito flakes, 20 g of sodium chloride, and 0.03 g of natural carotene were added in sequence. The mixture was stirred at 15°C for 3 hours and then passed through a 20-mesh sieve to obtain the prepared egg liquid.

[0123] Meanwhile, take the freeze-dried modified nisin powder, reconstitute it with sterile water to 0.05 g / mL (containing 11 mg / mL of nisin), stir at 30°C and 100 rpm for 10 min until a uniform dispersion is formed, add the reconstituted modified nisin solution to the pre-prepared modified egg liquid, and add 10 mL of modified nisin solution per kg of modified egg liquid;

[0124] The mixed egg liquid is filtered a second time through a 120-mesh filter to further remove fine impurities and ensure a smooth texture.

[0125] (3) Baking and cooling

[0126] Pour the finely filtered egg mixture into the mold, bake at a temperature greater than 100℃ for more than 6 minutes, and observe the solidification of the egg mixture during baking until the surface is evenly golden brown and the inside is fully cooked.

[0127] After baking, unmold the egg to obtain a shaped baked egg base; avoid contact with oil or impurities during the cooling process;

[0128] (4) Inspection and bagging

[0129] The appearance, smell, and taste of the baked egg embryos are tested, and unqualified products with cracked surfaces, uneven color, or off-odors are removed.

[0130] Pack qualified roasted eggs into food-grade composite packaging bags according to the set weight, vacuum pack them using a vacuum packaging machine, and seal them to ensure that the packaging bags are airtight and the seal is flat.

[0131] (5) Secondary sterilization and cooling

[0132] The sealed packaging bags are placed in a sterilization tank and pasteurized to kill bacteria, mold and other microorganisms inside the packaging bags.

[0133] After sterilization, the product is placed in a cooling tank and cooled to below 15°C.

[0134] (6) Inspection and warehousing

[0135] The X-ray foreign object detection machine is used to detect whether there are metal, glass, or stone foreign objects mixed in with the product. The product is then passed through a metal detector to further check for fine metal impurities to ensure product safety. Products that pass the inspection are packed into cartons, sealed, and labeled with the production date and shelf life. They are then sent to a constant temperature and humidity warehouse with a temperature of 0-8℃ and a humidity of ≤75% for storage, awaiting shipment.

[0136] Example 3:

[0137] A heat-resistant modified encapsulated nisin comprises nisin, hydroxypropyl-β-cyclodextrin encapsulating the non-bacterial hydrophobic region of nisin, and non-denatured type I collagen encapsulating both nisin and hydroxypropyl-β-cyclodextrin. The hydroxypropyl-β-cyclodextrin does not encapsulate the antibacterial region of nisin. The non-denatured type I collagen forms a continuous porous network structure. The antibacterial region of nisin penetrates the pores of the non-denatured type I collagen and binds to bacteria. The modified encapsulated nisin exhibits antibacterial effects against both Gram-negative and Gram-positive bacteria.

[0138] A method for preparing a high-temperature resistant modified encapsulated nisin.

[0139] (1) Preparation of the nisin-hydroxypropyl-β-cyclodextrin composition:

[0140] Under stirring conditions of 25°C and 150 rpm, an aqueous solution of nisin was slowly added dropwise to an aqueous solution of hydroxypropyl-β-cyclodextrin. The aqueous solution of nisin was prepared with sterile water, with a concentration of 2 mg / mL and a pH of 6.0. The aqueous solution of hydroxypropyl-β-cyclodextrin was prepared with sterile water, with a concentration of 4 mg / mL. The molar ratio of nisin to hydroxypropyl-β-cyclodextrin was 1:2.

[0141] After the addition was completed, the mixture was magnetically stirred for 2 hours. After stirring, the mixture was placed in a 4°C refrigerator and allowed to stand for 12 hours to obtain the lactic acid nisin-hydroxypropyl-β-cyclodextrin composition.

[0142] (2) Preparation of modified encapsulated nisin:

[0143] Under stirring conditions of 30℃ and 100rpm, non-denatured type I collagen aqueous solution was slowly added dropwise to the lactic acid nisin-hydroxypropyl-β-cyclodextrin composition obtained in (1). The non-denatured type I collagen aqueous solution was prepared with sterile water and had a concentration of 5mg / mL. The lactic acid nisin was added at a mass ratio of 1:5 to non-denatured type I collagen.

[0144] Add a composite buffer solution until the final concentration of the composite buffer solution is 0.05 mol / L, and adjust the pH of the solution to 6.5. The composite buffer is sodium citrate-sodium dihydrogen phosphate buffer with a pH of 6.5. Continue stirring for 4 hours to allow the non-denatured type I collagen to form an outer porous network structure through self-assembly. After stirring, refrigerate at 4°C for 8 hours to solidify the porous network structure, obtaining the modified nisin-embedded solution.

[0145] (3) Freeze-drying preservation:

[0146] The modified encapsulated nisin solution was centrifuged at 4℃ and 20,000 rpm for 30 min. The precipitate obtained by centrifugation was added to food-grade mannitol as a freeze-drying protectant at a concentration of 5% (w / w), and then freeze-dried for 20 h to obtain modified encapsulated nisin powder.

[0147] Application of a heat-resistant modified encapsulated nisin in high-temperature baked egg processing.

[0148] (1) Egg shelling and filtering

[0149] Select fresh, undamaged, and odorless pre-washed sterile eggs, and check the integrity and cleanliness of the eggshells to ensure there are no cracks, stains, or signs of spoilage.

[0150] Put the eggs into a sheller to remove the shells;

[0151] Pour the shelled egg liquid into a filter with an 80-mesh screen to filter out residual eggshell fragments and ligature impurities, resulting in preliminarily purified egg liquid.

[0152] (2) Blending and Filtration

[0153] 1 kg of coarsely filtered egg liquid was added to a mixing tank. 0.6 g of soy lecithin, 240 g of fructose syrup, 20 g of hydroxypropyl distarch phosphate, and 0.08 g of hydroxypropyl starch were added to 250 g of water and dissolved. The mixture was stirred at 300 rpm for 5 minutes. After dissolution, the mixture was added to the mixing tank and stirred at 30 rpm. 3 g of white vinegar, 10 g of benzoin, 30 g of bonito flakes, 20 g of sodium chloride, and 0.03 g of natural carotene were added in sequence. The mixture was stirred at 15°C for 3 hours and then passed through a 20-mesh sieve to obtain the prepared egg liquid.

[0154] Meanwhile, take the freeze-dried modified nisin powder, reconstitute it with sterile water to 0.1 g / mL (containing 14 mg / mL of nisin), stir at 30°C and 100 rpm for 10 min until a uniform dispersion is formed, and add the reconstituted modified nisin solution to the pre-prepared modified egg liquid, adding 10 mL of modified nisin solution per kg of modified egg liquid;

[0155] The mixed egg liquid is filtered a second time through a 120-mesh filter to further remove fine impurities and ensure a smooth texture.

[0156] (3) Baking and cooling

[0157] Pour the finely filtered egg mixture into the mold, bake at a temperature greater than 100℃ for more than 6 minutes, and observe the solidification of the egg mixture during baking until the surface is evenly golden brown and the inside is fully cooked.

[0158] After baking, unmold the egg to obtain a shaped baked egg base; avoid contact with oil or impurities during the cooling process;

[0159] (4) Inspection and bagging

[0160] The appearance, smell, and taste of the baked egg embryos are tested, and unqualified products with cracked surfaces, uneven color, or off-odors are removed.

[0161] Pack qualified roasted eggs into food-grade composite packaging bags according to the set weight, vacuum pack them using a vacuum packaging machine, and seal them to ensure that the packaging bags are airtight and the seal is flat.

[0162] (5) Secondary sterilization and cooling

[0163] The sealed packaging bags are placed in a sterilization tank and pasteurized to kill bacteria, mold and other microorganisms inside the packaging bags.

[0164] After sterilization, the product is placed in a cooling tank and cooled to below 15°C.

[0165] (6) Inspection and warehousing

[0166] The X-ray foreign object detection machine is used to detect whether there are metal, glass, or stone foreign objects mixed in with the product. The product is then passed through a metal detector to further check for fine metal impurities to ensure product safety. Products that pass the inspection are packed into cartons, sealed, and labeled with the production date and shelf life. They are then sent to a constant temperature and humidity warehouse with a temperature of 0-8℃ and a humidity of ≤75% for storage, awaiting shipment.

[0167] Example 4:

[0168] The similarities between this embodiment and Embodiment 1 will not be repeated here. The differences are as follows:

[0169] In Example 1, a method for preparing a high-temperature resistant modified encapsulated nisin,

[0170] Replace step (1) of Example 1 with fermentation of Lactococcus lactis ATCC 11454 to obtain nisin, and add hydroxypropyl-β-cyclodextrin in the later stage of fermentation to obtain a nisin-hydroxypropyl-β-cyclodextrin composition. The specific steps are as follows:

[0171] 1) Seed culture: Lactococcus lactis ATCC 11454 was inoculated into 100 mL of food-grade microbial fermentation medium and cultured at 30 °C and 180 rpm for 12 h with shaking until OD600nm=0.8-1.0; then the above solution was inoculated into a fermenter containing 10 L of sterile medium and cultured at 32 °C and 150 rpm with aeration.

[0172] 2) Co-culture with hydroxypropyl-β-cyclodextrin: After 12 hours of fermentation, prepare a 50 mg / mL solution of 6.5 g of hydroxypropyl-β-cyclodextrin and slowly add it dropwise over 30 minutes; continue culturing for 4-8 hours, adding 100 g / L, 50 mL sterile glucose solution every 4 hours, and maintaining the pH at 6.0-6.2 with 1 mol / L citric acid; after fermentation, refrigerate at 4℃ for 12 hours to allow the hydroxypropyl-β-cyclodextrin to fully encapsulate with nisin.

[0173] 3) Purification and concentration adjustment: Centrifuge all fermentation broth at 4℃ and 8000rpm for 20min to remove bacterial precipitate. Pass the supernatant through a 5kDa ultrafiltration membrane to remove small molecule impurities. After vacuum concentration of the ultrafiltration retentate, desalinate by reverse osmosis and adjust the nisin concentration to 1.2mg / mL. Store at 4℃ for later use.

[0174] 4) Nisin potency test: The potency of nisin in 3) was tested. The potency determination method used the national standard: GB 1886.231-2023 "National Food Safety Standard for Food Additives: Nisin";

[0175] To verify whether adding hydroxypropyl-β-cyclodextrin in the later stage of fermentation would increase the activity of nisin, hydroxypropyl-β-cyclodextrin was removed from the preparation method described in this embodiment, and nisin was prepared as a comparative example according to steps 1)-3), and its potency was tested. The results are as follows:

[0176] The nisin potency in the nisin-hydroxypropyl-β-cyclodextrin composition obtained in this embodiment is 1700 IU / mg, while the nisin potency in the comparative example is 1200 IU / mg. The preparation method of this embodiment significantly improves the potency of nisin.

[0177] Comparative Example 1:

[0178] The modified encapsulated nisin powder in Example 1 was replaced with nisin, and the nisin was added to the prepared egg liquid according to the amount added in Example 1 to prepare the finished roasted egg product.

[0179] Comparative Example 2:

[0180] The modified encapsulated nisin powder in Example 2 was replaced with nisin, and the nisin was added to the prepared egg liquid according to the amount added in Example 2 to prepare the finished roasted egg product.

[0181] Comparative Example 3:

[0182] The modified encapsulated nisin powder in Example 3 was replaced with nisin, and the nisin was added to the prepared egg liquid according to the amount added in Example 3 to prepare the finished roasted egg product.

[0183] Result detection:

[0184] 1. Detection of antibacterial effect against Gram-negative bacteria

[0185] Detection of the effect of nisin on bacterial cell walls:

[0186] First, Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 were cultured to the logarithmic growth phase using LB liquid medium. Then, the final concentration of the two bacterial cultures was adjusted to OD=0.2 (OD630nm) using fresh LB liquid medium. The bacterial cells were collected by centrifugation at 2000rpm for 5min at room temperature. The bacterial cultures were then diluted with 1x PBS buffer to OD=0.2-0.3. The sample was added to a 96-well cell culture plate. 100 μL of bacterial culture was added to each well, and three experimental groups were set up: Group A: 100 μL of the modified encapsulated nisin powder reconstituted in Example 1 (after reconstitution, the actual concentration of nisin was 150 μg / mL); Group B: 100 μL of nisin solution (concentration 150 μg / mL); Group C: 100 μL of 1xPBS buffer as a negative control; Group D: 100 μL of Triton X-100 (300 μg / mL) as a positive control. The samples from each well were gently mixed and placed in a biochemical incubator at 37°C. The OD630nm absorbance was measured every 30 minutes using a microplate reader.

[0187] Experimental results are as follows Figure 2 As shown, after treatment with nisin (Group B), the OD630nm absorbance of Staphylococcus aureus decreased from 0.241 to 0.184; after treatment with modified nisin (Group A), the OD630nm absorbance also decreased from 0.237 to 0.181. The results suggest that both nisin and modified nisin significantly increase the permeability of the Staphylococcus aureus cell wall.

[0188] Depend on Figure 3 As shown, in the experiment targeting Escherichia coli, the OD630nm absorbance did not decrease significantly after treatment with nisin (Group B), decreasing from 0.252 to 0.235; however, after treatment with modified nisin, the OD630nm absorbance decreased significantly, decreasing from 0.263 to 0.151, indicating that modified nisin can significantly increase the permeability of Gram-negative bacteria—Escherichia coli.

[0189] Therefore, the modified encapsulated nisin prepared in this application has inhibitory effects on both Gram-positive and Gram-negative bacteria:

[0190] Nisin can only bind to the phosphatidylglycerol of bacterial cell membranes ( ) function, phosphatidylglycerol of Gram-negative bacteria ( Located on the inner membrane, and outside the inner membrane is a lipopolysaccharide (LPS) outer membrane. Lactococcus can not penetrate the LPS outer membrane of Gram-negative bacteria, so it is only effective against Gram-positive bacteria.

[0191] The modified encapsulated nisin of this application has a secondary hydroxyl group (-CH(OH)-) of HP-β-CD and a glycosidic oxygen (COC, ether oxygen) of the cyclodextrin parent compound that can form strong hydrogen bonds with the hydroxyl group of hydroxyproline (Hyp) in undenatured type I collagen, causing local weak hydrogen bond breakage in undenatured type I collagen, resulting in local loosening and unfolding of the triple helix of undenatured type I collagen, exposing the negatively charged Glu residues inside.

[0192] Exposed Glu residues With the N-terminal loop Lys / Arg of nisin Electrostatic crosslinking is formed, generating a tensile force on the N-terminal ring. This tensile force is transmitted through the main chain to the C-terminal α-helix of nisin, causing the C-terminal α-helix to undergo gentle twisting or unwinding. After the C-terminal α-helix unwinds, the Lys / Arg positively charged residues in the antibacterial region of nisin form a high-density cluster of cationic sites.

[0193] The cationic site clusters formed by nisin can match the local negatively charged clusters of the lipopolysaccharide outer membrane of Gram-negative bacteria, forming a strong electrostatic bond and disrupting the integrity of the outer membrane. At the same time, the hydrophobic fragments exposed by the unfolding of non-denatured type I collagen bind to the hydrophobic regions of the phospholipid bilayer of the outer membrane, causing the phospholipid bilayer structure to become disordered. Nisin can then penetrate the outer membrane and act on the inner membrane of Gram-negative bacteria, thereby inhibiting Gram-negative bacteria.

[0194] 2. Minimum inhibitory concentration (MIC) test:

[0195] The minimum inhibitory concentration (MIC) was determined using the microdilution method, and the specific steps are as follows:

[0196] The test bacteria were cultured in TSB medium to an appropriate concentration. The concentration was first adjusted to 0.5 McFarland units (approximately 1 × 10⁻⁶ CFU / mL) using a McFarland turbidimeter, and then diluted to 2 × 10⁻⁶ CFU / mL. 5 CFU / mL.

[0197] The modified nisin-embedded lactococcal powder from Example 1 was serially diluted to different concentrations using fresh, autoclaved liquid culture medium. 50 μL of the diluted bacterial suspension and 50 μL of the diluted liquid culture medium containing the modified nisin-embedded lactococcal powder were added to a 96-well plate and mixed thoroughly. The liquid culture medium served as a negative control. After incubation in a biochemical incubator for 16–18 h, the absorbance was measured at OD600 nm. The state of each well was visually inspected; the absence of a misty suspension in the well was considered growth inhibition, and the lowest concentration showing significant inhibition was taken as the MIC value. The experimental results are shown in Table 1.

[0198] Table 1. Results of minimum inhibitory concentration (MIC) of modified encapsulated nisin against different bacteria.

[0199]

[0200] 3. Thermal stability

[0201] Experimental group: Using PBS as a buffer system, a solution was prepared with the modified encapsulated nisin powder from Example 1, with a final concentration of nisin of 1 mg / ml. The solution was thoroughly mixed and shaken at 250 r / min for 30 min at room temperature (25°C) to ensure thorough mixing.

[0202] Control group: A solution of nisin with a final concentration of 1 mg / ml was prepared using PBS as a buffer system, thoroughly mixed, and shaken at 250 r / min for 30 min at room temperature (25°C) to mix thoroughly.

[0203] After adjusting the pH of each group to 2 and 7 with NaOH, the samples were thoroughly mixed using a magnetic stirrer at low temperature (4°C) for 30 minutes. After treatment at 25°C and 121°C for 20 minutes, the samples were diluted with PBS at the corresponding pH values ​​for potency determination.

[0204] The potency determination method uses the national standard: GB 1886.231-2023 "National Food Safety Standard for Food Additives - Nisin".

[0205] The results are as follows Figure 4 As shown, the potency loss rate of the nisin group after high-temperature heat treatment at 121°C increased with increasing pH at pH 2 and 7. The potency loss rate reached 96.23% at pH 7. The modified encapsulated nisin experimental group showed a lower potency loss rate at pH 7 after high-temperature heat treatment at 121°C.

[0206] The cations dissociated from the modified nisin-encapsulated composite buffer of this application are adsorbed onto the negatively charged carboxyl groups of the hydrophilic region on the surface of the triple helix structure of non-denatured type I collagen via ionic bonds, that is, distributed around nisin, maintaining the pH of the environment around nisin and preventing the hydrolysis of the amide bonds of nisin.

[0207] 4. Thermogravimetric analysis

[0208] The modified encapsulated nisin and nisin prepared in Example 1 were analyzed by thermogravimetric analysis. Approximately 1 mg of sample was placed in an Al2O3 crucible and heated from 20°C to 750°C at a rate of 10°C / min under nitrogen atmosphere.

[0209] The starting temperature of the thermal decomposition stage is determined by the percentage of the sample's decomposed and vaporized portion reaching 5% of its total mass. Figure 5 Thermogravimetric analysis (TGA) plots show the thermal changes of nisin and modified encapsulated nisin at pH 2 and pH 7 as the temperature increases. The dehydration stage of the samples occurs before 120°C. The initial decomposition temperatures of nisin at pH 2 and pH 7 are 132°C and 126°C, respectively, indicating that nisin at pH 2 has higher thermal stability than that at pH 7.

[0210] The curves of modified encapsulated nisin showed a significant rightward shift under both pH 2 and pH 7 conditions, indicating that its main thermal decomposition stage had a higher temperature and higher thermal stability. Specifically, the main decomposition peak temperature was 148°C under pH 2 conditions and 141°C under pH 7 conditions.

[0211] 5. Test on the preservative effect of roasted eggs:

[0212] The total bacterial count of the roasted egg products prepared in Examples 1-3 and Comparative Examples 1-3 was tested according to the method provided in GB4789.2-2016 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count". The percentage reduction in total bacterial count was calculated using the total bacterial count of the roasted egg product without preservatives (control group) as the baseline. The spoilage rate after 3 months of storage at room temperature and away from light was calculated by the ratio of the mass of spoiled roasted egg products in the same batch to the total mass of roasted egg products within 3 months. The data obtained are shown in Table 2.

[0213] Table 2. Spoilage rate of roasted egg products within 3 months

[0214]

[0215] Examples 1-3 using modified encapsulated nisin showed excellent preservative effects, reducing the total bacterial count to the 10² CFU / g level, a reduction of over 99.9% in total bacterial count, and a spoilage rate of only 0%-2% after 3 months. In contrast, the comparative examples 1-3 using free nisin showed significantly poorer preservative effects, with total bacterial counts ranging from 8.2 × 10³ to 1 × 10³. 5 With a CFU / g and a reduction of only about 98% in total bacterial count, and a spoilage rate of 20%-32% after 3 months, the modified encapsulated nisin has a far superior preservative effect on baked eggs compared to nisin alone, and can effectively achieve stable storage of baked eggs for 3 months.

[0216] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A high-temperature resistant modified encapsulated nisin, characterized in that: It includes nisin, hydroxypropyl-β-cyclodextrin wrapped around the non-bacterial hydrophobic region of nisin, and non-denatured type I collagen wrapped around nisin and hydroxypropyl-β-cyclodextrin, wherein the non-denatured type I collagen forms a continuous porous network structure.

2. The high-temperature resistant modified encapsulated nisin according to claim 1, characterized in that: The modified encapsulated nisin exhibits antibacterial effects against both Gram-negative and Gram-positive bacteria.

3. The high-temperature resistant modified encapsulated nisin according to claim 1, characterized in that: The antibacterial zone of the lactic acid nisin penetrates the pores of the non-denatured type I collagen network structure and binds to bacteria in a targeted manner.

4. The high-temperature resistant modified encapsulated nisin according to claim 1, characterized in that: The lactic acid nisin is obtained by fermentation with Lactococcus lactis, and hydroxypropyl-β-cyclodextrin is added in the later stage of fermentation to improve the activity of lactic acid nisin.

5. A method for preparing a high-temperature resistant modified encapsulated nisin according to any one of claims 1-4, characterized in that: (1) Preparation of the nisin-hydroxypropyl-β-cyclodextrin composition: Under stirring conditions of 25~30℃ and 150~200rpm, nisin aqueous solution was slowly added dropwise to hydroxypropyl-β-cyclodextrin aqueous solution. After the addition was completed, the mixture was magnetically stirred for 2~3h. After stirring, the mixture was placed in a 4℃ refrigerator and allowed to stand for 12h to obtain nisin-hydroxypropyl-β-cyclodextrin composition. (2) Preparation of modified encapsulated nisin: Under stirring conditions of 30~35℃ and 100~150 rpm, non-denatured type I collagen aqueous solution was slowly added dropwise to the lactic acid nisin-hydroxypropyl-β-cyclodextrin composition obtained in (1), and then a composite buffer aqueous solution was added until the final concentration of the composite buffer aqueous solution was 0.02~0.05mol / L. The pH of the solution was adjusted to 6.5~7.0, and stirring was continued for 4~6h. The non-denatured type I collagen formed a porous network structure through self-assembly. After stirring, the solution was refrigerated at 4℃ for 8h to solidify the porous network structure and obtain the modified encapsulated lactic acid nisin solution. (3) Freeze-drying preservation: The modified encapsulated nisin solution was centrifuged at 4℃ and 20,000~30,000 rpm for 30~60 min. The precipitate obtained by centrifugation was added to a freeze-drying protectant and freeze-dried for 20~28 h to obtain modified encapsulated nisin powder.

6. The method for preparing a high-temperature resistant modified encapsulated nisin according to claim 5, characterized in that: The molar ratio of nisin to hydroxypropyl-β-cyclodextrin is 1:1 to 1:2; the mass ratio of nisin to undenatured type I collagen is 1:3 to 1:

5.

7. The method for preparing a high-temperature resistant modified encapsulated nisin according to claim 5, characterized in that: The nisin aqueous solution is prepared using sterile water, with a concentration of 1.0~2.0 mg / mL and a pH of 6.0~6.

5.

8. The method for preparing a high-temperature resistant modified encapsulated nisin according to claim 5, characterized in that: The hydroxypropyl-β-cyclodextrin aqueous solution was prepared using sterile water and had a concentration of 2.0~4.0 mg / mL. The non-denatured type I collagen aqueous solution was prepared using sterile water and had a concentration of 3.0~5.0 mg / mL.

9. The method for preparing a high-temperature resistant modified encapsulated nisin according to claim 5, characterized in that: The composite buffer is a sodium citrate-sodium dihydrogen phosphate buffer solution with a pH of 6.5-7.

0.

10. The application of a high-temperature resistant modified encapsulated nisin according to any one of claims 1-4 in the high-temperature baking of eggs, characterized in that: (1) Egg shelling and filtering Select fresh, undamaged, and odorless pre-washed sterile eggs, and check the integrity and cleanliness of the eggshells to ensure there are no cracks, stains, or signs of spoilage. Put the eggs into a sheller to remove the shells; Pour the shelled egg liquid into a filter with an 80-mesh screen to filter out residual eggshell fragments and ligature impurities, resulting in preliminarily purified egg liquid. (2) Blending and Filtration Pour the coarsely filtered egg liquid into a mixing container, add the auxiliary ingredients according to the formula, and stir with a mixer at a speed of 300-500 rpm for 5-8 minutes to fully mix the egg liquid and auxiliary ingredients to obtain the prepared egg liquid; Meanwhile, take the freeze-dried modified nisin powder, reconstitute it with sterile water to 0.05-0.1 g / mL, stir at 30℃ and 100 rpm for 10 min until a uniform dispersion is formed, and add the reconstituted modified nisin solution to the pre-prepared modified egg liquid, adding 10 mL of modified nisin solution per kg of modified egg liquid; The mixed egg liquid is filtered a second time through a 120-mesh filter to further remove fine impurities and ensure a smooth texture. (3) Baking and cooling Pour the filtered egg mixture into the mold, bake at a temperature greater than 100℃ for more than 6 minutes, and observe the solidification of the egg mixture during baking until the surface is evenly golden brown and the inside is fully cooked. After baking, unmold the egg to obtain a shaped baked egg base; avoid contact with oil or impurities during the cooling process; (4) Inspection and bagging The appearance, smell, and taste of the baked egg embryos are tested, and unqualified products with cracked surfaces, uneven color, or off-odors are removed. Pack qualified roasted eggs into food-grade composite packaging bags according to the set weight, vacuum pack them using a vacuum packaging machine, and seal them to ensure that the packaging bags are airtight and the seal is flat. (5) Secondary sterilization and cooling The sealed packaging bags are placed in a sterilization tank and pasteurized to kill bacteria, mold and other microorganisms inside the packaging bags. After sterilization, the product is placed in a cooling tank and cooled to below 15°C. (6) Inspection and warehousing The X-ray foreign object detection machine is used to detect whether there are metal, glass, or stone foreign objects mixed in with the product. The product is then passed through a metal detector to further check for fine metal impurities to ensure product safety. Products that pass the inspection are packed into cartons, sealed, and labeled with the production date and shelf life. They are then sent to a constant temperature and humidity warehouse with a temperature of 0-8℃ and a humidity of ≤75% for storage, awaiting shipment.

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