A compound preservative for food preservation and a preparation method thereof

Abstract

This invention relates to the field of food preservation technology, specifically to a composite preservative for food preservation and its preparation method. The invention prepares a composite preservative with broad-spectrum antibacterial activity, low sensory impact, and high stability by mixing trehalose, ε-polylysine, antioxidants, a preservative composition, xanthan gum, and nisin. The preservative composition is prepared from microsphere composition A and probiotic agent B. In microsphere composition A, oregano essential oil effectively disrupts the integrity of the cell membrane structure of pathogenic microorganisms, leading to bacterial death, but it is easily volatile and oxidized. Meanwhile, protamine sulfate effectively disrupts the bacterial cell membrane, thereby inhibiting bacterial growth and exhibiting strong antibacterial activity. Its three-dimensional gel network structure effectively encapsulates oregano essential oil, prolonging its antibacterial time. Probiotic agent B is *Lactobacillus reuteri* Fullarton-9-35 after heat inactivation treatment, which has more stable antibacterial properties and broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of food preservation technology, and in particular to a composite preservative for food preservation and its preparation method. Background Technology

[0002] With the development of the social economy and the continuous improvement of living standards, food safety has become a hot issue of common concern to consumers and the food industry. Baked goods are made from flour, yeast, salt, sugar, and water as basic raw materials, with the addition of appropriate amounts of oil, dairy products, eggs, and additives, through a series of complex baking processes. Due to their rich nutritional content, they are prone to microbial growth, leading to the risk of mold, rancidity, and excessive microbial levels within their shelf life. This not only reduces the hygienic quality and commercial value of the food, but also the toxins produced by microorganisms can harm consumers' health. Therefore, how to prevent food spoilage is increasingly attracting attention. Manufacturers need to adopt certain antibacterial measures to maintain the shelf life of baked goods, ensuring consumer safety within the shelf life. These technical measures include effectively reducing the initial microbial count of raw materials, controlling water activity, lowering pH, and strictly controlling on-site environmental hygiene. Another important chemical control method is the reasonable and legal addition of food preservatives to inhibit the growth and reproduction of microorganisms.

[0003] Currently, the preservatives commonly used in baked goods include chemical preservatives and preservatives derived from natural plant extracts. Although chemical preservatives have good antibacterial effects, their carcinogenic, teratogenic, and food poisoning-inducing properties pose a significant threat to human health. They can also impart unpleasant sensory qualities to food, such as astringency or bitterness. This often necessitates the addition of more additives, such as sugar, gums, and vegetable fats, to mask these unpleasant sensory effects. While some preservatives derived from natural plant extracts offer a degree of safety, their antibacterial effects do not match those of chemical preservatives. Furthermore, the odor residue of common plant-derived antibacterial agents can affect the flavor of raw materials, making them unsuitable for use in baked goods.

[0004] Therefore, based on the relevant technologies mentioned above, there is an urgent need to develop a composite preservative for food preservation and its preparation method. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a composite preservative for food preservation and its preparation method, so as to provide a composite preservative with broad-spectrum antibacterial properties, low sensory impact and high stability.

[0006] To achieve the above objectives, the present invention provides a composite preservative for food preservation and its preparation method.

[0007] A compound preservative for food preservation comprises the following raw materials in parts by weight: ε-Polylysine 0.5-1 part; Nisin 0.3-0.8 part; Preservative composition 2.8-3.6 part; Xanthan gum 0.1-0.2 part; Trehalose 1-2 part; Antioxidant 0.02-0.03 part; Deionized water 50-100 part; The preservative composition is obtained by mixing microsphere complex A and probiotic agent B at a mass ratio of 4.5-6:2-2.4; The probiotic agent B is Lactobacillus reuteri Fullarton-9-35 after heat inactivation treatment; The microsphere complex A was prepared from fish protamine nucleoprotein gel and oregano essential oil; The antioxidant is any one of vitamin E, glutathione, sodium erythritol, and vitamin C.

[0008] Preferably, the method for preparing the protamine nucleoprotein gel is as follows: Step A1. Place the testis tissue of *Pufferus thunbergii* in an ice-water bath at 0℃ and soak it in anhydrous ethanol for 12-14 hours. Then, centrifuge it at 2-4℃ and 10000-12000 rpm for 8-10 minutes to obtain precipitate 1. Wash it twice with ultrapure water. Add precipitate 1 to a buffer salt solution and homogenize it for 1-2 minutes. Then, centrifuge it at 2-4℃ and 10000-12000 rpm for 8-10 minutes to obtain precipitate 2. Add precipitate 2 to a buffer salt solution and homogenize it for 1-2 minutes. Then, centrifuge it at 2-4℃ and 10000-12000 rpm for 8-10 minutes to obtain precipitate 3. Freeze-dry the precipitate 3, grind it through a 100-mesh sieve, and obtain fish protamine nucleoprotein powder. Step A2. Add fish protamine nucleoprotein powder to a 10% sodium chloride solution and stir at 22-25℃ until it does not flow when inverted. Then place it in the water bath of an ultrasonic cleaner and sonicate for 4-5 hours to obtain fish protamine nucleoprotein gel.

[0009] Preferably, the ratio of the testis tissue of the dark-striped pufferfish in step A1 to anhydrous ethanol is 1-2g:4-8mL.

[0010] Preferably, the buffer salt solution in step A1 is an aqueous solution containing 0.14 mol / L sodium chloride and 0.01 mol / L sodium citrate dihydrate, with a pH value of 7.4-7.6.

[0011] Preferably, the ratio of fish protamine nucleoprotein powder to 10% sodium chloride solution in step A2 is 1-2g:10-20mL.

[0012] Preferably, the microsphere complex A is prepared by the following method: Deionized water was added to the fish protamine nucleoprotein gel and stirred evenly at room temperature. Then, oregano essential oil was added and sheared and emulsified at 10,000-12,000 rpm for 8-10 minutes. After freeze-drying and grinding through a 100-mesh sieve, microsphere composition A was obtained.

[0013] Preferably, the mass ratio of the fish protamine nucleoprotein gel, deionized water, and oregano essential oil is 3-5.8:6.3-10.5:0.6-0.9.

[0014] Preferably, the probiotic agent B is prepared as follows: Lactobacillus reuteri Fullarton-9-35 was inoculated into MRS culture medium and cultured statically at 37℃ for 22-24h. The fermentation broth was collected, and the concentration of probiotics in the fermentation broth was determined by measuring the OD600nm value with an enzyme-linked immunosorbent assay (ELISA) and by plate counting. The broth was then subjected to heat inactivation treatment at 90-100℃ for 10min to obtain probiotic agent B.

[0015] Preferably, the concentration of probiotics in the fermentation broth is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

[0016] A method for preparing a composite preservative for food preservation includes the following steps: Add trehalose, ε-polylysine succinate, and antioxidants to deionized water and mix and stir at 40-45℃ for 30-50 minutes. Then add the preservative composition, xanthan gum, and nisin and stir for 1-1.5 hours to obtain a compound preservative for food preservation.

[0017] The beneficial effects of this invention are: This invention provides a composite preservative for food preservation and its preparation method. The invention prepares a composite preservative with broad-spectrum antibacterial activity, low sensory impact, and high stability by mixing trehalose, ε-polylysine, antioxidants, a preservative composition, xanthan gum, and nisin. The preservative composition is prepared from microsphere composition A and probiotic agent B. In microsphere composition A, oregano essential oil is a natural plant extract with broad-spectrum antibacterial activity, effectively disrupting the cell membrane structure of pathogenic microorganisms, leading to cell death. However, it is easily volatile and oxidized, shortening the antibacterial time. Meanwhile, protamine sulfate effectively disrupts bacterial cell membranes, thereby inhibiting bacterial growth and exhibiting strong antibacterial activity. Its three-dimensional gel network structure effectively encapsulates oregano essential oil, prolonging its antibacterial time. Probiotic agent B is heat-inactivated Lactobacillus reuteri Fullarton-9-35, which has more stable antibacterial properties and works synergistically with microsphere composition A. Compared with existing technologies, this invention has broad application prospects. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0019] The sources and properties of some of the raw materials used in this invention are as follows: The Lactobacillus reuteri Fullarton-9-35 used in this invention is disclosed in the invention patent with authorization announcement number "CN121587310A" entitled "A space probiotic agent with antibacterial, antiseptic and preservation functions and its application", and accession number CGMCCNo.14939.

[0020] Example 1: A method for preparing a composite preservative for food preservation, comprising the following steps: S1. Weigh 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g diammonium citrate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate and 0.25g manganese sulfate tetrahydrate, add 1000mL distilled water and 1mL Tween-80 and stir until completely dissolved, adjust pH to 6.8, filter and sterilize at 121℃ for 15min to obtain MRS culture medium; S2. *Lactobacillus reuteri* Fullarton-9-35 was inoculated into MRS culture medium and incubated statically at 37℃ for 22 h. The fermentation broth was collected, and the concentration of probiotics in the fermentation broth was determined to be 1 × 10⁻⁶ by measuring the OD600nm value using a microplate reader and plate counting. 8 CFU / mL, then heat-inactivated at 90℃ for 10 min to obtain probiotic agent B; S3. Place 1g of *Pufferus tumefaciens* testis tissue in an ice-water bath at 0℃, soak in 4mL of anhydrous ethanol for 12h, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 1, wash twice with ultrapure water, add precipitate 1 to an aqueous solution with pH 7.4 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 1min, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 2, add precipitate 2 to an aqueous solution with pH 7.4 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 1min, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 3, freeze-dry, grind through a 100-mesh sieve to obtain fish protamine nucleoprotein powder; S4. Add 1g of fish protamine nucleoprotein powder to 10mL of 10% sodium chloride solution, stir at 22℃ until it does not flow when inverted, and then place it in the water bath of an ultrasonic cleaner for ultrasonic treatment for 4h to obtain fish protamine nucleoprotein gel. S5. Add 6.3g of deionized water to 3g of fish protamine nucleoprotein gel, stir evenly at room temperature, then add 0.6g of oregano essential oil, shear emulsify at 10000rpm for 8min, freeze dry, grind through a 100-mesh sieve to obtain microsphere composition A; S6. Mix 4.5g of microsphere complex A and 2g of probiotic agent B to obtain a preservative composition; add 1g of trehalose, 0.5g of ε-polylysine and 0.02g of antioxidant to 50g of deionized water, mix and stir at 40℃ for 30min, then add 2.8g of the preservative composition, 0.1g of xanthan gum and 0.3g of nisin and stir for 1h to obtain a compound preservative for food preservation.

[0021] Example 2: A method for preparing a composite preservative for food preservation, comprising the following steps: S1. Weigh 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g diammonium citrate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate and 0.25g manganese sulfate tetrahydrate, add 1000mL distilled water and 1mL Tween-80 and stir until completely dissolved, adjust pH to 6.8, filter and sterilize at 121℃ for 15min to obtain MRS culture medium; S2. *Lactobacillus reuteri* Fullarton-9-35 was inoculated into MRS culture medium and incubated statically at 37℃ for 22 h. The fermentation broth was collected, and the concentration of probiotics in the fermentation broth was determined to be 1 × 10⁻⁶ by measuring the OD600nm value using a microplate reader and plate counting. 8 CFU / mL, then heat-inactivated at 93℃ for 10 min to obtain probiotic agent B; S3. Place 1.3g of testis tissue from *Pufferus tumefaciens* in an ice-water bath at 0℃, soak in 5.2mL of anhydrous ethanol for 12h, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 1, wash twice with ultrapure water, add precipitate 1 to an aqueous solution with pH 7.4 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 1-2min, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 2, add precipitate 2 to an aqueous solution with pH 7.4 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 1min, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 3, freeze-dry, grind through a 100-mesh sieve to obtain fish protamine nucleoprotein powder; S4. Add 1.2g of fish protamine nucleoprotein powder to 12mL of 10% sodium chloride solution, stir at 23℃ until it does not flow when inverted, and then place it in the water bath of an ultrasonic cleaner for ultrasonic treatment for 4.5h to obtain fish protamine nucleoprotein gel. S5. Add 7.2g of deionized water to 3.5g of fish protamine nucleoprotein gel, stir evenly at room temperature, then add 0.68g of oregano essential oil, shear emulsify at 10000rpm for 8min, freeze dry, grind through a 100-mesh sieve to obtain microsphere composition A; S6. Mix 4.8g of microsphere complex A and 2.1g of probiotic agent B to obtain a preservative composition; add 1.2g of trehalose, 0.6g of ε-polylysine and 0.02g of antioxidant to 60g of deionized water, mix and stir at 42℃ for 35min, then add 3g of the preservative composition, 0.1g of xanthan gum and 0.45g of nisin and stir for 1h to obtain a compound preservative for food preservation.

[0022] Example 3: A method for preparing a composite preservative for food preservation, comprising the following steps: S1. Weigh 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g diammonium citrate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate and 0.25g manganese sulfate tetrahydrate, add 1000mL distilled water and 1mL Tween-80 and stir until completely dissolved, adjust pH to 6.8, filter and sterilize at 121℃ for 15min to obtain MRS culture medium; S2. *Lactobacillus reuteri* Fullarton-9-35 was inoculated into MRS culture medium and incubated statically at 37℃ for 23 h. The fermentation broth was collected, and the concentration of probiotics in the fermentation broth was determined to be 1 × 10⁻⁶ by measuring the OD600nm value using a microplate reader and plate counting. 9 CFU / mL, then heat-inactivated at 95℃ for 10 min to obtain probiotic agent B; S3. Place 1.5g of testis tissue from *Pufferus tumefaciens* in an ice-water bath at 0℃, soak in 6mL of anhydrous ethanol for 13h, centrifuge at 11000rpm for 9min at 3℃ to obtain precipitate 1, wash twice with ultrapure water, add precipitate 1 to an aqueous solution with pH 7.5 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 2min, centrifuge at 11000rpm for 9min at 3℃ to obtain precipitate 2, add precipitate 2 to an aqueous solution with pH 7.5 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 2min, centrifuge at 11000rpm for 9min at 3℃ to obtain precipitate 3, freeze-dry, grind through a 100-mesh sieve to obtain fish protamine nucleoprotein powder; S4. Add 1.4g of fish protamine nucleoprotein powder to 14mL of 10% sodium chloride solution, stir at 24℃ until it does not flow when inverted, and then place it in the water bath of an ultrasonic cleaner for ultrasonic treatment for 4.5h to obtain fish protamine nucleoprotein gel. S5. Add 8g of deionized water to 4.2g of fish protamine nucleoprotein gel, stir evenly at room temperature, then add 0.7g of oregano essential oil, shear emulsify at 11000rpm for 9min, freeze dry, grind through a 100-mesh sieve to obtain microsphere composition A; S6. Mix 5.3g of microsphere complex A and 2.2g of probiotic agent B to obtain a preservative composition; add 1.5g of trehalose, 0.7g of ε-polylysine and 0.025g of antioxidant to 75g of deionized water, mix and stir at 43℃ for 40min, then add 3.2g of the preservative composition, 0.15g of xanthan gum and 0.55g of nisin and stir for 1.5h to obtain a compound preservative for food preservation.

[0023] Example 4: A method for preparing a composite preservative for food preservation, comprising the following steps: S1. Weigh 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g diammonium citrate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate and 0.25g manganese sulfate tetrahydrate, add 1000mL distilled water and 1mL Tween-80 and stir until completely dissolved, adjust pH to 6.8, filter and sterilize at 121℃ for 15min to obtain MRS culture medium; S2. *Lactobacillus reuteri* Fullarton-9-35 was inoculated into MRS culture medium and incubated statically at 37℃ for 24 h. The fermentation broth was collected, and the concentration of probiotics in the fermentation broth was determined to be 1 × 10⁻⁶ by measuring the OD600nm value using a microplate reader and plate counting method. 9CFU / mL, then heat-inactivated at 98℃ for 10 min to obtain probiotic agent B; S3. 1.7g of testis tissue from *Pufferus tumefaciens* was placed in an ice-water bath at 0℃ and soaked in 6.8mL of anhydrous ethanol for 14h. After centrifugation at 12000rpm for 10min at 4℃, precipitate 1 was obtained. After washing twice with ultrapure water, precipitate 1 was added to an aqueous solution with pH 7.6 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate. After homogenization for 2min, it was centrifuged at 12000rpm for 10min at 4℃ to obtain precipitate 2. Precipitate 2 was then added to an aqueous solution with pH 7.6 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate. After homogenization for 2min, it was centrifuged at 12000rpm for 10min at 4℃ to obtain precipitate 3. After freeze-drying and grinding through a 100-mesh sieve, fish protamine nucleoprotein powder was obtained. S4. Add 1.8g of fish protamine nucleoprotein powder to 18mL of 10% sodium chloride solution, stir at 25℃ until it does not flow when inverted, and then place it in the water bath of an ultrasonic cleaner for ultrasonic treatment for 5h to obtain fish protamine nucleoprotein gel. S5. Add 9.4g of deionized water to 5.2g of protamine nucleoprotein gel, stir evenly at room temperature, then add 0.8g of oregano essential oil, shear emulsify at 12000rpm for 10min, freeze dry, grind through a 100-mesh sieve to obtain microsphere composition A; S6. Mix 5.5g of microsphere complex A and 2.3g of probiotic agent B to obtain a preservative composition; add 1.7g of trehalose, 0.83g of ε-polylysine and 0.03g of antioxidant to 88g of deionized water, mix and stir at 45℃ for 45min, then add 3.4g of the preservative composition, 0.2g of xanthan gum and 0.7g of nisin and stir for 1.5h to obtain a compound preservative for food preservation.

[0024] Example 5: A method for preparing a composite preservative for food preservation, comprising the following steps: S1. Weigh 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g diammonium citrate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate and 0.25g manganese sulfate tetrahydrate, add 1000mL distilled water and 1mL Tween-80 and stir until completely dissolved, adjust pH to 6.8, filter and sterilize at 121℃ for 15min to obtain MRS culture medium; S2. *Lactobacillus reuteri* Fullarton-9-35 was inoculated into MRS culture medium and incubated statically at 37℃ for 24 h. The fermentation broth was collected, and the concentration of probiotics in the fermentation broth was determined to be 1 × 10⁻⁶ by measuring the OD600nm value using a microplate reader and plate counting method. 9 CFU / mL, then heat-inactivated at 100℃ for 10 min to obtain probiotic agent B; S3. Place 2g of testis tissue from *Pufferus thunbergii* at 0℃ in an ice-water bath, soak in 8mL of anhydrous ethanol for 14h, centrifuge at 12000rpm at 4℃ for 10min to obtain precipitate 1, wash twice with ultrapure water, add precipitate 1 to an aqueous solution with pH 7.6 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 2min, centrifuge at 12000rpm at 4℃ for 10min to obtain precipitate 2, add precipitate 2 to an aqueous solution with pH 7.6 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 2min, centrifuge at 12000rpm at 4℃ for 10min to obtain precipitate 3, freeze-dry, grind through a 100-mesh sieve to obtain fish protamine nucleoprotein powder; S4. Add 2g of fish protamine nucleoprotein powder to 20mL of 10% sodium chloride solution, stir at 25℃ until it does not flow when inverted, and then place it in the water bath of an ultrasonic cleaner for ultrasonic treatment for 5h to obtain fish protamine nucleoprotein gel. S5. Add 10.5g of deionized water to 5.8g of fish protamine nucleoprotein gel, stir evenly at room temperature, then add 0.9g of oregano essential oil, shear emulsify at 12000rpm for 10min, freeze dry, grind through a 100-mesh sieve to obtain microsphere composition A; S6. Mix 6g of microsphere complex A and 2.4g of probiotic agent B to obtain a preservative composition; add 2g of trehalose, 1g of ε-polylysine and 0.03g of antioxidant to 100g of deionized water, mix and stir at 45℃ for 50min, then add 3.6g of the preservative composition, 0.2g of xanthan gum and 0.8g of nisin and stir for 1.5h to obtain a compound preservative for food preservation.

[0025] Comparative Example 1: Compared with Example 1, this comparative example did not undergo heat inactivation treatment during the preparation of probiotic agent B. All other steps and parameters were the same, and will not be repeated here. The final product was a composite preservative.

[0026] Comparative Example 2: This comparative example differs from Example 1 only in that the "preservative composition" is replaced with "microsphere composition A". All other steps and parameters are the same, and will not be repeated here. The final composite preservative is obtained.

[0027] Comparative Example 3: Compared with Example 1, this comparative example only replaces the "preservative composition" with "probiotic agent B". All other steps and parameters are the same, and will not be repeated here. The final composite preservative is obtained.

[0028] Comparative Example 4: This comparative example differs from Example 1 only in that "microsphere composition A" is replaced with "oregano essential oil". All other steps and parameters are the same, and will not be repeated here. The final product is a composite preservative.

[0029] Comparative Example 5: S1. Weigh 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g diammonium citrate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate and 0.25g manganese sulfate tetrahydrate, add 1000mL distilled water and 1mL Tween-80 and stir until completely dissolved, adjust pH to 6.8, filter and sterilize at 121℃ for 15min to obtain MRS culture medium; S2. *Lactobacillus reuteri* Fullarton-9-35 was inoculated into MRS culture medium and incubated statically at 37℃ for 22 h. The fermentation broth was collected, and the concentration of probiotics in the fermentation broth was determined to be 1 × 10⁻⁶ by measuring the OD600nm value using a microplate reader and plate counting. 8 CFU / mL, then heat-inactivated at 90℃ for 10 min to obtain probiotic agent B; S3. Place 1g of *Pufferus tumefaciens* testis tissue in an ice-water bath at 0℃, soak in 4mL of anhydrous ethanol for 12h, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 1, wash twice with ultrapure water, add precipitate 1 to an aqueous solution with pH 7.4 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 1min, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 2, add precipitate 2 to an aqueous solution with pH 7.4 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 1min, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 3, freeze-dry, grind through a 100-mesh sieve to obtain fish protamine nucleoprotein powder; S4. Add 1g of fish protamine nucleoprotein powder to 10mL of 10% sodium chloride solution and stir at 22℃ until it does not flow when inverted to obtain fish protamine nucleoprotein gel; S5. Add 6.3g of deionized water to 3g of fish protamine nucleoprotein gel, stir evenly at room temperature, then add 0.6g of oregano essential oil, shear emulsify at 10000rpm for 8min, freeze dry, grind through a 100-mesh sieve to obtain microsphere composition A; S6. Mix 4.5g of microsphere complex A and 2g of probiotic agent B to obtain a preservative composition; add 1g of trehalose, 0.5g of ε-polylysine and 0.02g of antioxidant to 50g of deionized water, mix and stir at 40℃ for 30min, then add 2.8g of the preservative composition, 0.1g of xanthan gum and 0.3g of nisin and stir for 1h to obtain a compound preservative for food preservation.

[0030] Comparative Example 6: S1. Weigh 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g diammonium citrate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate and 0.25g manganese sulfate tetrahydrate, add 1000mL distilled water and 1mL Tween-80 and stir until completely dissolved, adjust pH to 6.8, filter and sterilize at 121℃ for 15min to obtain MRS culture medium; S2. *Lactobacillus reuteri* Fullarton-9-35 was inoculated into MRS culture medium and incubated statically at 37℃ for 22 h. The fermentation broth was collected, and the concentration of probiotics in the fermentation broth was determined to be 1 × 10⁻⁶ by measuring the OD600nm value using a microplate reader and plate counting. 8 CFU / mL, then heat-inactivated at 90℃ for 10 min to obtain probiotic agent B; S3. Place 1g of *Pufferus tumefaciens* testis tissue in an ice-water bath at 0℃, soak in 4mL of anhydrous ethanol for 12h, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 1, wash twice with ultrapure water, add precipitate 1 to an aqueous solution with pH 7.4 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 1min, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 2, add precipitate 2 to an aqueous solution with pH 7.4 containing 0.14mol / L sodium chloride and 0.01mol / L sodium citrate dihydrate, homogenize for 1min, centrifuge at 10000rpm for 8min at 2℃ to obtain precipitate 3, freeze-dry, grind through a 100-mesh sieve to obtain fish protamine nucleoprotein powder; S4. Add 1g of fish protamine nucleoprotein powder to 10mL of 10% sodium chloride solution, stir at 22℃ until it does not flow when inverted, and then place it in the water bath of an ultrasonic cleaner for ultrasonic treatment for 8h to obtain fish protamine nucleoprotein gel. S5. Add 6.3g of deionized water to 3g of fish protamine nucleoprotein gel, stir evenly at room temperature, then add 0.6g of oregano essential oil, shear emulsify at 10000rpm for 8min, freeze dry, grind through a 100-mesh sieve to obtain microsphere composition A; S6. Mix 4.5g of microsphere complex A and 2g of probiotic agent B to obtain a preservative composition; add 1g of trehalose, 0.5g of ε-polylysine and 0.02g of antioxidant to 50g of deionized water, mix and stir at 40℃ for 30min, then add 2.8g of the preservative composition, 0.1g of xanthan gum and 0.3g of nisin and stir for 1h to obtain a compound preservative for food preservation.

[0031] Comparative Example 7: Compared with Example 1, this comparative example only replaces "heat inactivation treatment at 90°C for 10 min" with "heat inactivation treatment at 121°C for 10 min". All other steps and parameters are the same, and will not be repeated in this comparative example. The final composite preservative is obtained.

[0032] Performance testing: Freshness test: The composite preservatives prepared in Examples 1-5 and Comparative Examples 1-7 were uniformly sprayed onto the surface of the cooled baked goods (the concentration of the composite preservatives was 1%). A group without any preservatives was set up as a blank control group. The average shelf life under refrigeration (8-12℃, RH35-75%) was tested. Antibacterial performance test: The antibacterial effect of the compound preservative of this invention was determined using the Oxford cup method. *Escherichia coli*, *Salmonella*, and *Bacillus subtilis* were selected for the experiment, and each was activated and cultured in a suitable culture medium until the viable count reached 10⁻⁶. 7 -10 8 CFU / mL, the specific operation is as follows: After activation, the test strain was mixed with sterilized nutrient agar medium (the medium temperature was controlled at 45℃), poured into plates, and after the plates solidified, placed in Oxford cups. Then, the mouth of the Oxford cups was opened, nutrient agar was poured in, and after solidification, the Oxford cups were removed. Sterile water (as a blank control group) and the composite preservatives prepared in Examples 1-5 and Comparative Examples 1-7 were inoculated into the wells respectively. The wells were placed in an incubator for cultivation, and the diameter of the inhibition zone was tested (in mm). Sensory evaluation: A double-blind evaluation panel of 30 people was randomly selected to score the taste, color and flavor of baked goods (bread) after 15 days of refrigeration (out of 10). The sensory evaluation criteria are shown in Table 2.

[0033] Table 1. Summary of experimental data from Examples 1-5 and Comparative Examples 1-7

[0034] Table 2 Sensory Evaluation Criteria

[0035] Table 3 Summary of experimental data from Examples 1-5 and Comparative Examples 1-7

[0036] Data Analysis: As can be seen from Tables 1-3, the composite preservative prepared by this invention has better preservation and anti-corrosion properties and better flavor synergy. This may be because the preservative composition is prepared by microsphere composition A and probiotic agent B. In microsphere composition A, oregano essential oil is a natural plant extract with broad-spectrum antibacterial activity. It can effectively destroy the integrity of the cell membrane structure of pathogenic microorganisms, leading to cell death. However, it is volatile and easily oxidized, resulting in a shortened antibacterial time. Meanwhile, protamine can effectively destroy the cell membrane of bacteria, thereby inhibiting bacterial growth. It has strong antibacterial properties, and its three-dimensional gel network structure can effectively encapsulate oregano essential oil, prolonging its antibacterial time. Probiotic agent B is Lactobacillus reuteri Fullarton-9-35 after heat inactivation treatment. It appears in the form of undamaged inactive cells, cell walls and membranes, and intracellular parts. It can also exert antibacterial properties well and work synergistically with microsphere composition A to achieve better preservation and anti-corrosion effects. Comparative Example 1, which did not undergo heat inactivation treatment during the preparation of probiotic agent B, and Comparative Example 7, which replaced "heat inactivation treatment at 90℃ for 10 min" with "heat inactivation treatment at 121℃ for 10 min," exhibited poorer preservation, antibacterial properties, and sensory evaluation. This may be because probiotic agents without heat inactivation treatment are easily inactivated by external environmental influences, resulting in significantly weakened antibacterial activity. Furthermore, heat inactivation treatment at 121℃ significantly alters the bioactive properties of the probiotic agents; excessive heating leads to the degradation of heat-sensitive active ingredients and a reduction in their bioavailability, thus hindering their antibacterial effect. Comparative Example 2, which replaced "preservative composition" with "microsphere composition A," and Comparative Example 3, which replaced "preservative composition" with "probiotic agent B," also showed poorer preservation, antibacterial properties, and sensory evaluation. This may be because the synergistic effect of microsphere composition A and probiotic agent B in this invention significantly enhances antibacterial and preservation properties. Significant improvement was observed, but the antibacterial effect was limited when using a single component. Comparative Example 4, due to replacing the "microsphere composition" with "oregano oil," exhibited poor preservation, antibacterial properties, and sensory evaluation. This may be due to the volatility and easy oxidation of oregano oil, leading to a shortened antibacterial time and poor preservation of baked goods. Comparative Example 5, without ultrasonic treatment of the protamine nucleoprotein gel, also showed poor preservation, antibacterial properties, and sensory evaluation. This may be because ultrasonic treatment significantly enhances the mechanical properties of the protein gel. Its mechanism mainly involves regulating disulfide bond recombination and hydrophobic interactions to induce conformational changes in the secondary structure of proteins, thereby promoting the formation of molecular cross-linking networks. This improves the stability of microsphere composition A and increases the utilization rate of oregano oil. Comparative Example 6, due to excessively long ultrasonic treatment time of the protamine nucleoprotein gel, exhibited poor preservation, antibacterial properties, and sensory evaluation. This may be because prolonged ultrasonic treatment easily leads to the degradation of the cross-linking network, causing the main structure to collapse and reducing antibacterial properties.

[0037] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

[0038] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention are not permitted.

Claims

1. A compound preservative for food preservation, characterized in that, Includes the following quantities of raw materials: ε-Polylysine 0.5-1 part; Nisin 0.3-0.8 part; Preservative composition 2.8-3.6 part; Xanthan gum 0.1-0.2 part; Trehalose 1-2 part; Antioxidant 0.02-0.03 part; Deionized water 50-100 part; The preservative composition is obtained by mixing microsphere complex A and probiotic agent B at a mass ratio of 4.5-6:2-2.4; The probiotic agent B is Lactobacillus reuteri Fullarton-9-35 after heat inactivation treatment; The microsphere complex A was prepared from fish protamine nucleoprotein gel and oregano essential oil; The antioxidant is any one of vitamin E, glutathione, sodium erythritol, and vitamin C.

2. The composite preservative for food preservation according to claim 1, characterized in that, The method for preparing the protamine nucleoprotein gel is as follows: Step A1. Place the testis tissue of *Pufferus thunbergii* in an ice-water bath at 0℃ and soak it in anhydrous ethanol for 12-14 hours. Then, centrifuge it at 2-4℃ and 10000-12000 rpm for 8-10 minutes to obtain precipitate 1. Wash it twice with ultrapure water. Add precipitate 1 to a buffer salt solution and homogenize it for 1-2 minutes. Then, centrifuge it at 2-4℃ and 10000-12000 rpm for 8-10 minutes to obtain precipitate 2. Add precipitate 2 to a buffer salt solution and homogenize it for 1-2 minutes. Then, centrifuge it at 2-4℃ and 10000-12000 rpm for 8-10 minutes to obtain precipitate 3. Freeze-dry the precipitate 3, grind it through a 100-mesh sieve, and obtain fish protamine nucleoprotein powder. Step A2. Add fish protamine nucleoprotein powder to a 10% sodium chloride solution and stir at 22-25℃ until it does not flow when inverted. Then place it in the water bath of an ultrasonic cleaner and sonicate for 4-5 hours to obtain fish protamine nucleoprotein gel.

3. The composite preservative for food preservation according to claim 2, characterized in that, The ratio of the testis tissue of the dark-spotted pufferfish described in step A1 to anhydrous ethanol is 1-2g:4-8mL.

4. The composite preservative for food preservation according to claim 2, characterized in that, The buffer salt solution mentioned in step A1 is an aqueous solution containing 0.14 mol / L sodium chloride and 0.01 mol / L sodium citrate dihydrate, with a pH value of 7.4-7.

6.

5. The composite preservative for food preservation according to claim 2, characterized in that, The ratio of fish protein nucleoprotein powder to 10% sodium chloride solution in step A2 is 1-2g:10-20mL.

6. The composite preservative for food preservation according to claim 1, characterized in that, The microsphere complex A is prepared as follows: Deionized water was added to the fish protamine nucleoprotein gel and stirred evenly at room temperature. Then, oregano essential oil was added and sheared and emulsified at 10,000-12,000 rpm for 8-10 minutes. After freeze-drying and grinding through a 100-mesh sieve, microsphere composition A was obtained.

7. The composite preservative for food preservation according to claim 6, characterized in that, The mass ratio of the fish protamine nucleoprotein gel, deionized water, and oregano essential oil is 3-5.8:6.3-10.5:0.6-0.

9.

8. The composite preservative for food preservation according to claim 1, characterized in that, The preparation method of the probiotic agent B is as follows: Lactobacillus reuteri Fullarton-9-35 was inoculated into MRS culture medium and cultured statically at 37℃ for 22-24h. The fermentation broth was collected, and the concentration of probiotics in the fermentation broth was determined by measuring the OD600nm value with an enzyme-linked immunosorbent assay (ELISA) and by plate counting. The broth was then subjected to heat inactivation treatment at 90-100℃ for 10min to obtain probiotic agent B.

9. The composite preservative for food preservation according to claim 8, characterized in that, The probiotic concentration in the fermentation broth is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

10. The method for preparing the composite preservative for food preservation according to any one of claims 1-9, characterized in that, Includes the following steps: Add trehalose, ε-polylysine succinate, and antioxidants to deionized water and mix and stir at 40-45℃ for 30-50 minutes. Then add the preservative composition, xanthan gum, and nisin and stir for 1-1.5 hours to obtain a compound preservative for food preservation.

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