PPI-GA-CH-RRA composite emulsion capable of being used as natural substitute of nitrite as well as preparation method and application of PPI-GA-CH-RRA composite emulsion

Abstract

The invention discloses a PPI-GA-CH-RRA composite emulsion capable of being used as a natural substitute of nitrite as well as a preparation method and application of the PPI-GA-CH-RRA composite emulsion. The pea protein isolate, the Arabic gum, the chitosan and the carrot anthocyanin are used as raw materials to prepare a composite emulsion system, the composite emulsion system can replace nitrite to be applied to emulsified sausages, and the problems that pickled meat is insufficient in color stability, weak in antibacterial protection and poor in antioxidant stability, the quality degradation is accelerated during refrigeration storage, and the taste is poor are solved. And a single-component substitute cannot reproduce the multifunctional effect of sodium nitrite in the meat product.

Description

Technical Field

[0001] This invention belongs to the field of clean label processed meat technology, specifically involving a pea protein-gum arabic-chitosan-carrot anthocyanin complex system as a natural substitute for sodium nitrite. This system can stabilize the red color in emulsified sausages and inhibit microbial spoilage. Background Technology

[0002] Nitrites, especially sodium nitrite, are widely used in emulsified meat products such as sausages, serving functions including stabilizing curing color, delaying lipid oxidation, inhibiting microbial growth, and imparting unique flavor. However, with increasing public concern about the potential formation of carcinogenic N-nitrosamines during meat processing and storage, and the excessive intake of nitrites in processed meat products, relevant regulatory standards are becoming increasingly stringent. This has spurred a continued growth in the meat industry's demand for safer alternatives that better meet clean label standards.

[0003] The core challenge in reducing or replacing sodium nitrite lies in its multifunctionality. Nitrites form stable curing pigments through complex reactions with myoglobin, while also exerting antioxidant and antibacterial effects. The absence of nitrites often leads to adverse effects in cured meat products, such as dull color, accelerated lipid oxidation, reduced emulsion stability, and increased microbial activity during storage. Therefore, in emulsified meat product systems, simply replacing a single component often fails to replicate the comprehensive technical functions of nitrites.

[0004] To address these issues, various strategies have been explored, including the use of plant-derived pigments, natural antioxidants, organic acids, and antimicrobial agents. Anthocyanins and other natural colorants have been studied for their ability to improve meat color; however, their application is often limited by poor thermal stability, pH sensitivity, and weak interaction with meat proteins. Similarly, while natural antimicrobial agents such as chitosan or essential oils can inhibit microbial growth, their use alone may impair texture, sensory quality, or processing stability. Protein or polysaccharide additives, although enhancing emulsion stability, generally lack sufficient antioxidant or coloring capabilities.

[0005] In the field of nitrite substitution or reduction in meat products, existing patents have proposed several technical routes. However, some limitations still exist in practical applications, especially in emulsified sausage systems. For example, Chinese patent application 201911136425.4 discloses a color-developing agent to replace nitrite in processed meat products. Coagulase-negative staphylococci with nitric oxide synthase can produce nitric oxide in processed meat products, thereby forming red nitrosomyoglobin, giving the processed meat products a red color; the addition of L-arginine can further enhance the red color. However, it depends on microbial inoculation and strain activity, and the consistency of the product may be difficult to control under different processing and storage conditions. In addition, it does not provide a comprehensive solution that can simultaneously ensure the color stability, antioxidant protection, antibacterial properties, and emulsification stability of emulsified sausages. Furthermore, Chinese patent application 202410838608.5 discloses a method for preparing a food additive to replace or reduce nitrite in Cantonese sausage. This method uses sodium alginate and maltodextrin as wall materials to prepare polyphenol and astaxanthin composite microcapsules, which are then homogenized and freeze-dried before being applied to the sausage system. Therefore, this method can reduce nitrite residue under specific conditions. However, this preparation process is relatively complex, and the disclosed system is only applicable to specific types of sausages and processes. It does not establish a controllable aqueous-phase assembled composite material suitable for emulsifying sausage matrices, capable of simultaneously providing color stability, antibacterial protection, antioxidant stability, and emulsification stability during refrigeration.

[0006] Therefore, there is an urgent need to develop a stable, multifunctional, and food-safe composite system that can simultaneously achieve color stability, antioxidant activity, antibacterial protection, and emulsification stability in emulsified sausages. This system should be based on safe, natural raw materials, have controllable preparation conditions, and demonstrate high efficiency in actual meat processing and storage. This invention aims to overcome the aforementioned technical deficiencies and meet the growing market demand for clean-label nitrite alternatives. Summary of the Invention

[0007] To overcome the shortcomings of existing clean label nitrite alternatives in emulsified sausages, such as insufficient color stability in cured meat, weak antibacterial protection, poor antioxidant stability, accelerated quality deterioration during refrigeration, and the inability of single-component substitutes to reproduce the multifunctional effects of sodium nitrite in meat products, the primary objective of this invention is to provide a PPI-GA-CH-RRA composite emulsion that can serve as a natural substitute for nitrites. This emulsion can be conveniently applied to emulsified meat products to improve storage stability and product quality. To address the aforementioned technical challenges, this invention proposes a pea protein isolate (PPI), gum arabic (GA), chitosan (CH), and carrot anthocyanin (RRA) composite system (PGCR), using a specific ratio of PPI:GA:CH:RRA, and a controllable stepwise preparation method for application in emulsified sausages.

[0008] Another object of the present invention is to provide a PPI-GA-CH-RRA composite emulsion prepared by the above preparation method, which can be used as a natural substitute for nitrite.

[0009] Another object of the present invention is to provide the application of the above-mentioned PPI-GA-CH-RRA composite emulsion, which can be used as a natural substitute for nitrite, in emulsified sausage.

[0010] To achieve this objective, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a PPI-GA-CH-RRA composite emulsion that can be used as a natural substitute for nitrite, comprising the following steps:

[0012] (1) After adding pea protein isolate (PPI) to water for full hydration, adjust the pH, centrifuge, and take the supernatant to obtain pea protein isolate solution;

[0013] (2) Dissolve gum arabic (GA) in water to obtain a gum arabic solution;

[0014] (3) Dissolve chitosan (CH) in acetic acid solution, adjust pH to obtain chitosan solution;

[0015] (4) Mix the pea protein isolate solution and gum arabic solution and stir. During the stirring process, adjust the pH of the system to 3.8-4.0 and keep it stable to obtain the PPI-GA complex;

[0016] (5) Add the chitosan solution dropwise to the PPI-GA complex and stir to obtain the PPI-GA-CH complex;

[0017] (6) Dissolve concentrated carrot anthocyanin (RRA) extract in citrate buffer to obtain carrot anthocyanin solution;

[0018] (7) Add carrot anthocyanin solution to PPI-GA-CH complex, homogenize, and obtain PPI-GA-CH-RRA complex emulsion that can be used as a natural substitute for nitrite.

[0019] Preferably, the concentration of the pea protein isolate solution in step (1) is 2g:100mL-4g:100mL.

[0020] Preferably, in step (1), the pH is adjusted to 7.0-8.0; the reagent used to adjust the pH is a 0.1-1.0 mol / L sodium hydroxide solution.

[0021] Preferably, the time for full hydration in step (1) is 2-3 hours.

[0022] Preferably, the centrifugation speed in step (1) is 5000-8000 rpm and the time is 15-25 minutes.

[0023] Preferably, the concentration of the gum arabic solution in step (2) is 2g:100mL-4g:100mL.

[0024] Preferably, the volume concentration of the acetic acid solution in step (3) is 1-2%.

[0025] Preferably, the concentration of chitosan in the chitosan solution in step (3) is 1g:100mL-3g:100mL.

[0026] Preferably, in step (3), the pH is adjusted to 5.5-6.5; the reagent used to adjust the pH is a 0.1-1.0 mol / L sodium hydroxide solution.

[0027] Preferably, the volume ratio of the pea protein isolate solution, gum arabic solution, chitosan solution from step (3), and carrot anthocyanin solution from step (6) is 1:(0.25-0.5):(0.5-0.75):0.1.

[0028] Preferably, in step (4), the pH of the acid solution is adjusted to 3.8-4.0, and the acid solution is a hydrochloric acid solution with a concentration of 0.05-0.2 mol / L.

[0029] Preferably, the time to maintain stability in step (4) is 10-15 minutes.

[0030] Preferably, the anthocyanin equivalent in the carrot anthocyanin solution in step (6) is 10-20 mg / mL.

[0031] Preferably, the pH value of the citrate buffer solution in step (6) is 3-3.5.

[0032] Preferably, the homogenization temperature in step (7) is ≤35℃, the rotation speed is 8000-10000 rpm, and the time is 2-5 minutes.

[0033] Preferably, in step (7) of the PPI-GA-CH-RRA composite emulsion, the mass ratio of pea protein isolate (PPI), gum arabic (GA), chitosan (CH) and carrot anthocyanin (RRA) is 2:(0.5-1.0):(0.5-0.75):(0.1-0.15).

[0034] Secondly, the present invention provides a PPI-GA-CH-RRA composite emulsion prepared by the above preparation method, which can be used as a natural substitute for nitrite.

[0035] Thirdly, the present invention provides the application of the above-mentioned PPI-GA-CH-RRA composite emulsion, which can be used as a natural substitute for nitrite, in emulsified sausage.

[0036] At pH 3.8-4.0 (close to the isoelectric point pI of pea protein, pI 4.5), pea protein (PPI) exhibits a tendency to aggregate / precipitate due to its near-zero net surface charge, weakened intermolecular electrostatic repulsion, and dominant hydrophobic interactions. Therefore, traditional processes typically avoid this pH (choosing pH 7.0, far from the isoelectric point, or dissolving in strong acids with pH < 3.0). In the process of this invention: the PPI-gum arabic (GA) complex is actively adjusted to this pH range. At this point, PPI is in a thermodynamically unstable state, while GA (containing uronic acid groups, pKa≈3.5, with a dissociation degree of about 40-50% at this pH, carrying a partial negative charge) adsorbs onto the PPI surface through electrostatic-hydrophobic synergistic interactions, forming a spatially stable layer that prevents complete phase separation of PPI. In this state, the PPI-GA complex exhibits a kinetically hindered metastable state ("maintaining stability for 10-15 minutes" in S4 is experimental evidence). Subsequently, chitosan (CH, pH 5.5, positively charged) is introduced, preferentially binding with GA (negatively charged), thereby obtaining a stable composite emulsion.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] (1) The carrot anthocyanin used in this invention can provide a stable red pigment, which can simulate the color of nitrosomyoglobin in traditional cured meats and has the function of color fixation.

[0039] (2) The chitosan component used in this invention has a broad-spectrum antibacterial effect against common meat spoilage bacteria and pathogenic bacteria, and directly replaces the safety function of nitrite.

[0040] (3) The anthocyanins and chitosan used in this invention have synergistic antioxidant effects, enabling the system to exhibit significant free radical (DPPH, ABTS) scavenging activity and effectively inhibit lipid oxidation and rancidity.

[0041] (4) The PPI-GA matrix used in this invention gives the system excellent emulsification stability and foam stability, which is crucial for the texture, water retention and cooking yield of emulsified sausage.

[0042] (5) The present invention uses pea protein isolate, gum arabic, chitosan and carrot anthocyanin as raw materials to prepare a composite emulsion system, which can replace nitrite in emulsified sausages and solves the problems of insufficient color stability of cured meat, weak antibacterial protection, poor antioxidant stability, accelerated quality deterioration during cold storage, and the inability of single-component substitutes to reproduce the multifunctional effects of sodium nitrite in meat products. Attached Figure Description

[0043] Figure 1 Optical images and optical microscope images of solutions prepared at different pH values ​​are shown.

[0044] Figure 2 The Fourier transform infrared spectra of each component and the PGCR composite system are presented.

[0045] Figure 3 The microstructure of the composite dispersion system and emulsion is illustrated using optical microscope images.

[0046] Figure 4 The sample demonstrates its DPPH radical scavenging activity.

[0047] Figure 5 The sample demonstrates its hydroxyl radical scavenging activity.

[0048] Figure 6 The antibacterial activity of the sample is demonstrated.

[0049] Figure 7 Demonstrates the emulsification stability of the PGCR composite system. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0051] Unless otherwise specified in the embodiments of this invention, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. All raw materials and reagents used, unless otherwise specified, are commercially available conventional products.

[0052] Comparative Example 1: Preparation of a PPI-GA Complex

[0053] S1: Pea Protein Isolate (PPI) Stock Solution (2% w / v): Place 2.0 g of pea protein isolate in a 250 mL beaker and add approximately 80 mL of deionized water. Stir with a magnetic stirrer at room temperature for 2 hours to ensure complete hydration. Adjust the pH to 7.5 with 0.1 mol / L NaOH solution while stirring. Transfer the dispersion to a 100 mL volumetric flask and bring the volume to 2% (w / v) with deionized water. Centrifuge at 5000 rpm for 15 minutes to remove insoluble matter and collect the supernatant for later use.

[0054] S2: Gum arabic (GA) stock solution (2% w / v): Dissolve 2.0 g of gum arabic in deionized water at room temperature with stirring, and react for 2 hours. Transfer the solution to a 100 mL volumetric flask and dilute to the marked volume to prepare a 2% (w / v) stock solution.

[0055] S3: Mix 100 mL of the PPI stock solution prepared in step S1 with 50 mL of the GA stock solution prepared in step S2 and stir (30 min) to promote protein-polysaccharide interaction. Adjust the pH of the mixture to 4.0 by adding 0.1 mol / L hydrochloric acid solution dropwise during stirring and keep it stable (e.g., for 15 min) to obtain the PPI-GA complex.

[0056] Comparative Example 2: Preparation of a PPI-GA-CH Complex

[0057] S1: Pea Protein Isolate (PPI) Stock Solution (2% w / v): Place 2.0 g of pea protein isolate in a 250 mL beaker and add approximately 80 mL of deionized water. Stir with a magnetic stirrer at room temperature for 2 hours to ensure complete hydration. Adjust the pH to 7.5 with 0.1 mol / L NaOH solution while stirring. Transfer the dispersion to a 100 mL volumetric flask and bring the volume to 2% (w / v) with deionized water. Centrifuge at 5000 rpm for 15 minutes to remove insoluble matter and collect the supernatant for later use.

[0058] S2: Gum arabic (GA) stock solution (2% w / v): Dissolve 2.0 g of gum arabic in deionized water at room temperature with stirring, and react for 2 hours. Transfer the solution to a 100 mL volumetric flask and dilute to the marked volume to prepare a 2% (w / v) stock solution.

[0059] S3: Chitosan (CH) Stock Solution (1% w / v): Prepare a 1% (v / v) glacial acetic acid solution. Add 1.0 g of chitosan to 80 mL of this solution and stir overnight at room temperature until completely dissolved. Then adjust the pH of the chitosan solution to 5.5 with 0.1 mol / L NaOH solution (added dropwise with stirring). Transfer the solution to a 100 mL volumetric flask and dilute to volume with deionized water to obtain a 1% (w / v) chitosan stock solution.

[0060] S4: Mix 100 mL of the PPI stock solution prepared in step S1 with 50 mL of the GA stock solution prepared in step S2 and stir (30 min) to promote protein-polysaccharide interaction. Adjust the pH of the mixture to 4.0 by adding 0.1 mol / L hydrochloric acid solution dropwise during stirring and keep it stable (e.g., for 15 min) to obtain the PPI-GA complex.

[0061] S5: Slowly (preferably dropwise) add 50 mL of the CH stock solution prepared in step S3 to the complex prepared in step S4 while stirring, and continue stirring (e.g., for 30 minutes) to form the intermediate PPI-GA-CH complex.

[0062] Example 1: Preparation of a PPI-GA-CH-RRA complex

[0063] S1: Pea Protein Isolate (PPI) Stock Solution (2% w / v): Place 2.0 g of pea protein isolate in a 250 mL beaker and add approximately 80 mL of deionized water. Stir with a magnetic stirrer at room temperature for 2 hours to ensure complete hydration. Adjust the pH to 7.5 with 0.1 mol / L NaOH solution while stirring. Transfer the dispersion to a 100 mL volumetric flask and bring the volume to 2% (w / v) with deionized water. Centrifuge at 5000 rpm for 15 minutes to remove insoluble matter and collect the supernatant for later use.

[0064] S2: Gum arabic (GA) stock solution (2% w / v): Dissolve 2.0 g of gum arabic in deionized water at room temperature with stirring, and react for 2 hours. Transfer the solution to a 100 mL volumetric flask and dilute to the marked volume to prepare a 2% (w / v) stock solution.

[0065] S3: Chitosan (CH) Stock Solution (1% w / v): Prepare a 1% (v / v) glacial acetic acid solution. Add 1.0 g of chitosan to 80 mL of this solution and stir overnight at room temperature until completely dissolved. Then adjust the pH of the chitosan solution to 5.5 with 0.1 mol / L NaOH solution (added dropwise with stirring). Transfer the solution to a 100 mL volumetric flask and dilute to volume with deionized water to obtain a 1% (w / v) chitosan stock solution.

[0066] S4: Mix 100 mL of the PPI stock solution prepared in step S1 with 50 mL of the GA stock solution prepared in step S2 and stir (30 min) to promote protein-polysaccharide interaction. Adjust the pH of the mixture to 4.0 by adding 0.1 mol / L hydrochloric acid solution dropwise during stirring and keep it stable (e.g., for 15 min) to obtain the PPI-GA complex.

[0067] S5: Slowly (preferably dropwise) add 50 mL of the CH stock solution prepared in step S3 to the complex prepared in step S4 while stirring, and continue stirring (e.g., for 30 minutes) to form the intermediate PPI-GA-CH complex.

[0068] S6: Carrot anthocyanin (RRA) extract: Dissolve the concentrated RRA extract in pH 3.5 citrate buffer to prepare a stock solution with a concentration of 10 mg / mL (anthocyanin equivalent).

[0069] S7: Add 10 mL of RRA stock solution (10 mg / mL) to the PPI-GA-CH complex intermediate from step S5. Place the final mixture in an ice bath (temperature below 35°C) and homogenize using a high-speed homogenizer at 10,000 rpm for 2 minutes. Store the resulting PGCR complex dispersion (final component mass ratio PPI:GA:CH:RRA = 2:1:0.5:0.1) in a light-protected container at 4°C.

[0070] Example 2: Preparation of a PPI-GA-CH-RRA complex

[0071] S1: Pea Protein Isolate (PPI) Stock Solution (2% w / v): Place 2.0 g of pea protein isolate in a 250 mL beaker and add approximately 80 mL of deionized water. Stir with a magnetic stirrer at room temperature for 2 hours to ensure complete hydration. Adjust the pH to 7.5 with 0.1 mol / L NaOH solution while stirring. Transfer the dispersion to a 100 mL volumetric flask and bring the volume to 2% (w / v) with deionized water. Centrifuge at 5000 rpm for 15 minutes to remove insoluble matter and collect the supernatant for later use.

[0072] S2: Gum arabic (GA) stock solution (2% w / v): Dissolve 2.0 g of gum arabic in deionized water at room temperature with stirring, and react for 2 hours. Transfer the solution to a 100 mL volumetric flask and dilute to the marked volume to prepare a 2% (w / v) stock solution.

[0073] S3: Chitosan (CH) Stock Solution (1% w / v): Prepare a 1% (v / v) glacial acetic acid solution. Add 1.0 g of chitosan to 80 mL of this solution and stir overnight at room temperature until completely dissolved. Then adjust the pH of the chitosan solution to 5.5 with 0.1 mol / L NaOH solution (added dropwise with stirring). Transfer the solution to a 100 mL volumetric flask and dilute to volume with deionized water to obtain a 1% (w / v) chitosan stock solution.

[0074] S4: Mix 100 mL of the PPI stock solution prepared in step S1 with 25 mL of the GA stock solution prepared in step S2 and stir (30 min) to promote protein-polysaccharide interaction. Adjust the pH of the mixture to 4.0 by adding 0.1 mol / L hydrochloric acid solution dropwise during stirring and keep it stable (e.g., for 15 min) to obtain the PPI-GA complex.

[0075] S5: Slowly (preferably dropwise) add 50 mL of the CH stock solution prepared in step S3 to the complex prepared in step S4 while stirring, and continue stirring (e.g., for 30 minutes) to form the intermediate PPI-GA-CH complex.

[0076] S6: Carrot anthocyanin (RRA) extract: Dissolve the concentrated RRA extract in pH 3.5 citrate buffer to prepare a stock solution with a concentration of 10 mg / mL (anthocyanin equivalent).

[0077] S7: Add 10 mL of RRA stock solution (10 mg / mL) to the ternary PPI-GA-CH complex from step S6. Place the final mixture in an ice bath (temperature below 35°C) and homogenize using a high-speed homogenizer at 10,000 rpm for 2 minutes. Store the resulting PGCR composite dispersion (final component mass ratio PPI:GA:CH:RRA = 2:0.5:0.5:0.1) in a light-protected container at 4°C.

[0078] Example 3: Preparation of a PPI-GA-CH-RRA complex

[0079] S1: Pea Protein Isolate (PPI) Stock Solution (2% w / v): Place 2.0 g of pea protein isolate in a 250 mL beaker and add approximately 80 mL of deionized water. Stir with a magnetic stirrer at room temperature for 2 hours to ensure complete hydration. Adjust the pH to 7.5 with 0.1 mol / L NaOH solution while stirring. Transfer the dispersion to a 100 mL volumetric flask and bring the volume to 2% (w / v) with deionized water. Centrifuge at 5000 rpm for 15 minutes to remove insoluble matter and collect the supernatant for later use.

[0080] S2: Gum arabic (GA) stock solution (2% w / v): Dissolve 2.0 g of gum arabic in deionized water at room temperature with stirring, and react for 2 hours. Transfer the solution to a 100 mL volumetric flask and dilute to the marked volume to prepare a 2% (w / v) stock solution.

[0081] S3: Chitosan (CH) Stock Solution (1% w / v): Prepare a 1% (v / v) glacial acetic acid solution. Add 1.0 g of chitosan to 80 mL of this solution and stir overnight at room temperature until completely dissolved. Then adjust the pH of the chitosan solution to 5.5 with 0.1 mol / L NaOH solution (added dropwise with stirring). Transfer the solution to a 100 mL volumetric flask and dilute to volume with deionized water to obtain a 1% (w / v) chitosan stock solution.

[0082] S4: Mix 100 mL of the PPI stock solution prepared in step S1 with 50 mL of the GA stock solution prepared in step S2 and stir (30 min) to promote protein-polysaccharide interaction. Adjust the pH of the mixture to 4.0 by adding 0.1 mol / L hydrochloric acid solution dropwise during stirring and keep it stable (e.g., for 15 min) to obtain the PPI-GA complex.

[0083] S5: Slowly (preferably dropwise) add 75 mL of the CH stock solution prepared in step S3 to the complex prepared in step S4 while stirring, and continue stirring (e.g., for 30 minutes) to form the intermediate PPI-GA-CH complex.

[0084] S6: Carrot anthocyanin (RRA) extract: Dissolve the concentrated RRA extract in pH 3.5 citrate buffer to prepare a stock solution with a concentration of 15 mg / mL (anthocyanin equivalent).

[0085] S7: Add 10 mL of RRA stock solution (15 mg / mL) to the ternary PPI-GA-CH complex from step S5. Place the final mixture in an ice bath (temperature below 35°C) and homogenize using a high-speed homogenizer at 10,000 rpm for 2 minutes. Store the resulting PGCR composite dispersion (final component mass ratio PPI:GA:CH:RRA = 2:1:0.75:0.15) in a light-protected container at 4°C.

[0086] Comparative Example 3: Pea protein isolate (PPI).

[0087] Comparative Example 4: Gum Arabic (GA).

[0088] Comparative Example 5: Chitosan (CH).

[0089] Comparative Example 6: Preparation of a Radish Anthocyanin (RRA)

[0090] S1: Wash fresh carrots and cut them into small pieces of 3-4cm. First, dry them in a constant temperature oven at 60℃, then crush and sieve them to obtain carrot powder.

[0091] S2: Soak carrot powder in a 70% volume concentration ethanol solution at a material-to-liquid ratio of 1g:30mL. Extract anthocyanins with ultrasonic assistance for 21 minutes. Take the supernatant and evaporate it at 40℃ under light-protected conditions to obtain a pigment extract. Evaporate and concentrate the extract. Freeze-dry the concentrate for 48 hours to obtain carrot extract and store it at 4℃ in the dark.

[0092] The preparation method of concentrated RRA extract in the embodiments of this application and other comparative examples is the same as that in Comparative Example 6.

[0093] Comparative Example 7: Preparation of a PPI-GA complex (compared to Comparative Example 1, the pH value of S3 was adjusted to 7.5).

[0094] S1: Pea Protein Isolate (PPI) Stock Solution (2% w / v): Place 2.0 g of pea protein isolate in a 250 mL beaker and add approximately 80 mL of deionized water. Stir with a magnetic stirrer at room temperature for 2 hours to ensure complete hydration. Adjust the pH to 7.5 with 0.1 mol / L NaOH solution while stirring. Transfer the dispersion to a 100 mL volumetric flask and bring the volume to 2% (w / v) with deionized water. Centrifuge at 5000 rpm for 15 minutes to remove insoluble matter and collect the supernatant for later use.

[0095] S2: Gum arabic (GA) stock solution (2% w / v): Dissolve 2.0 g of gum arabic in deionized water at room temperature with stirring, and react for 2 hours. Transfer the solution to a 100 mL volumetric flask and dilute to the marked volume to prepare a 2% (w / v) stock solution.

[0096] S3: Mix 100 mL of the PPI stock solution prepared in step S1 with 50 mL of the GA stock solution prepared in step S2 and stir (for 30 minutes) to promote protein-polysaccharide interaction. During stirring, adjust the pH of the mixture to 7.5 by adding 0.1 mol / L NaOH solution dropwise and keep it stable (e.g., for 15 minutes) to obtain the PPI-GA complex.

[0097] Comparative Example 8: Preparation of a PPI-GA-CH-RRA complex (the pH of S4 was adjusted to 7.5 compared to Example 1).

[0098] S1: Pea Protein Isolate (PPI) Stock Solution (2% w / v): Place 2.0 g of pea protein isolate in a 250 mL beaker and add approximately 80 mL of deionized water. Stir with a magnetic stirrer at room temperature for 2 hours to ensure complete hydration. Adjust the pH to 7.5 with 0.1 mol / L NaOH solution while stirring. Transfer the dispersion to a 100 mL volumetric flask and bring the volume to 2% (w / v) with deionized water. Centrifuge at 5000 rpm for 15 minutes to remove insoluble matter and collect the supernatant for later use.

[0099] S2: Gum arabic (GA) stock solution (2% w / v): Dissolve 2.0 g of gum arabic in deionized water at room temperature with stirring, and react for 2 hours. Transfer the solution to a 100 mL volumetric flask and dilute to the marked volume to prepare a 2% (w / v) stock solution.

[0100] S3: Chitosan (CH) Stock Solution (1% w / v): Prepare a 1% (v / v) glacial acetic acid solution. Add 1.0 g of chitosan to 80 mL of this solution and stir overnight at room temperature until completely dissolved. Then adjust the pH of the chitosan solution to 5.5 with 0.1 mol / L NaOH solution (added dropwise with stirring). Transfer the solution to a 100 mL volumetric flask and dilute to volume with deionized water to obtain a 1% (w / v) chitosan stock solution.

[0101] S4: Mix 100 mL of the PPI stock solution prepared in step S1 with 50 mL of the GA stock solution prepared in step S2 and stir (30 min) to promote protein-polysaccharide interaction. During stirring, adjust the pH of the mixture to 7.5 by adding 0.1 mol / L NaOH solution dropwise and keep it stable (e.g., for 15 min) to obtain the PPI-GA complex.

[0102] S5: Slowly (preferably dropwise) add 50 mL of the CH stock solution prepared in step S3 to the complex prepared in step S4 while stirring, and continue stirring (e.g., for 30 minutes) to form the intermediate PPI-GA-CH complex.

[0103] S6: Carrot anthocyanin (RRA) extract: Dissolve the concentrated RRA extract in pH 3.5 citrate buffer to prepare a stock solution with a concentration of 10 mg / mL (anthocyanin equivalent).

[0104] S7: Add 10 mL of RRA stock solution (10 mg / mL) to the PPI-GA-CH complex intermediate from step S5. Place the final mixture in an ice bath (temperature below 35°C) and homogenize using a high-speed homogenizer at 10,000 rpm for 2 minutes. Store the resulting PGCR complex dispersion (final component mass ratio PPI:GA:CH:RRA = 2:1:0.5:0.1) in a light-protected container at 4°C.

[0105] Comparative Example 9: Preparation of a PPI-GA-CH-RRA complex (the pH of S4 was adjusted to 2.7 compared to Example 1).

[0106] S1: Pea Protein Isolate (PPI) Stock Solution (2% w / v): Place 2.0 g of pea protein isolate in a 250 mL beaker and add approximately 80 mL of deionized water. Stir with a magnetic stirrer at room temperature for 2 hours to ensure complete hydration. Adjust the pH to 7.5 with 0.1 mol / L NaOH solution while stirring. Transfer the dispersion to a 100 mL volumetric flask and bring the volume to 2% (w / v) with deionized water. Centrifuge at 5000 rpm for 15 minutes to remove insoluble matter and collect the supernatant for later use.

[0107] S2: Gum arabic (GA) stock solution (2% w / v): Dissolve 2.0 g of gum arabic in deionized water at room temperature with stirring, and react for 2 hours. Transfer the solution to a 100 mL volumetric flask and dilute to the marked volume to prepare a 2% (w / v) stock solution.

[0108] S3: Chitosan (CH) Stock Solution (1% w / v): Prepare a 1% (v / v) glacial acetic acid solution. Add 1.0 g of chitosan to 80 mL of this solution and stir overnight at room temperature until completely dissolved. Then adjust the pH of the chitosan solution to 5.5 with 0.1 mol / L NaOH solution (added dropwise with stirring). Transfer the solution to a 100 mL volumetric flask and dilute to volume with deionized water to obtain a 1% (w / v) chitosan stock solution.

[0109] S4: Mix 100 mL of the PPI stock solution prepared in step S1 with 50 mL of the GA stock solution prepared in step S2 and stir (30 min) to promote protein-polysaccharide interaction. Adjust the pH of the mixture to 2.7 by adding 0.1 mol / L hydrochloric acid solution dropwise during stirring and keep it stable (e.g., for 15 min) to obtain the PPI-GA complex.

[0110] S5: Slowly (preferably dropwise) add 50 mL of the CH stock solution prepared in step S3 to the complex prepared in step S4 while stirring, and continue stirring (e.g., for 30 minutes) to form the intermediate PPI-GA-CH complex.

[0111] S6: Carrot anthocyanin (RRA) extract: Dissolve the concentrated RRA extract in pH 3.5 citrate buffer to prepare a stock solution with a concentration of 10 mg / mL (anthocyanin equivalent).

[0112] S7: Add 10 mL of RRA stock solution (10 mg / mL) to the PPI-GA-CH complex intermediate from step S5. Place the final mixture in an ice bath (temperature below 35°C) and homogenize using a high-speed homogenizer at 10,000 rpm for 2 minutes. Store the resulting PGCR complex dispersion (final component mass ratio PPI:GA:CH:RRA = 2:1:0.5:0.1) in a light-protected container at 4°C.

[0113] The materials obtained in Comparative Examples 1-9 and Example 1 were characterized.

[0114] Figure 1 Optical images show that the PPI-GA system in Comparative Example 1 (pH 4.0) is a stable milky white emulsion, while Comparative Example 7 (pH 7.5) is a clear solution, and Comparative Example 9 (pH 2.7) is an unstable turbid or precipitated state. Further observation with a 10x optical microscope confirmed that the PPI-GA system in Comparative Example 1 (pH 4.0) is a uniformly dispersed solution, and the solution becomes even finer and more uniform after the addition of chitosan (Comparative Example 2), indicating the formation of a stable metastable complex and structure. In contrast, Comparative Example 9 (pH 2.7) shows a large number of irregular flocculent clumps, and the PPI-GA system in Comparative Example 7 (pH 7.5) has almost no visible particles (due to electrostatic repulsion and non-composite). However, after the addition of chitosan (Comparative Example 8), a large number of flocculent precipitates appear (self-aggregation of chitosan). Thus, from both macroscopic and microscopic perspectives, it is confirmed that pH 4.0 is a necessary condition for the formation of a stable metastable complex and ordered structure. Deviating from this condition will result in the inactivation of gum arabic or electrostatic repulsion, preventing the construction of the expected structure.

[0115] Figure 2 The FTIR spectra of Comparative Examples 3-6 and Example 1 are shown. Characteristic spectral bands include: approximately 3690 cm⁻¹. -1 (OH / NH stretching vibration) Approximately 2940 cm -1 (CH stretching vibration) Approximately 1680 cm -1 (C=O stretching vibration) Approximately 1478 cm -1 (CH bending vibration and / or COO) - Vibration), approximately 1200 cm -1 (COC / CO stretching vibration), and approximately 880 cm -1(Glycoside / ring vibration). The spectrum of Example 1 contains the above bands, indicating the formation of the complex system.

[0116] Figure 3 Optical microscope images of Comparative Examples 1-6 and Example 1 are shown. Figure 3 As shown, the droplets in the single-component system are large and unevenly distributed. The dispersibility of Comparative Examples 1-2 is improved, but aggregation still exists. The droplets in Example 1 are smaller and more uniform, indicating that the microstructure and stability of its emulsion are improved.

[0117] Figure 4 The DPPH radical scavenging rates of different samples are shown. The DPPH radical scavenging rate test method is as follows: A 0.2 mmol / L solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was prepared with anhydrous ethanol and stored in the dark for later use. 0.5 mL of different sample solutions were added to each test tube, followed by 4.5 mL of DPPH solution to obtain the sample group solutions; 4.5 mL of ethanol solution and 0.5 mL of different sample solutions were added to each test tube to obtain the control group solutions; 0.5 mL of ethanol solution and 4.5 mL of DPPH solution were added to each test tube to obtain the blank group solutions; each experiment was repeated three times, and the test tubes were shaken in the dark for 30 minutes, and the absorbance was measured at 517 nm. In the formula: A 样品 =Sample + DPPH free radical scavenger; A 对照 =Sample + Ethanol; A 空白 =DPPH free radical scavenger + ethanol.

[0118] like Figure 4 As shown, the single systems of Comparative Examples 3-6 exhibited limited scavenging activity, while the composite systems demonstrated higher scavenging activity. In particular, Example 1 showed the highest scavenging rate, indicating that the introduction of Comparative Example 6 significantly enhanced the antioxidant capacity of the composite system. Different letters indicate significant differences between groups.

[0119] Figure 5 The hydroxyl radical scavenging rate of different samples is shown. The test method for hydroxyl radical scavenging rate is as follows: Accurately measure 1.0 mL of each different sample solution into a test tube, add 1.0 mL of 9 mmol / L FeSO4, then add 1.0 mL of 8.8 mmol / L H2O2 solution, shake well, and let stand for 10 min. Finally, add 1.0 mL of 9 mmol / L salicylic acid solution, shake well, and let stand for 30 min. This is sample A. 样品 This is the sample group; salicylic acid is replaced with distilled water, and all other conditions are the same as the sample group, designated as A. 对照The control group (A) was formed by replacing the sample solution with distilled water and keeping all other conditions the same as the sample group. 空白 The blank group was used for detection at a wavelength of 510 nm. The scavenging capacity of different samples for hydroxyl radicals was calculated using the following formula: In the formula: A 样品 The absorbance value of the sample solution after adding salicylic acid; A 对照 Replace the absorbance value of salicylic acid with distilled water; A 空白 Replace the absorbance value of the sample solution with distilled water.

[0120] like Figure 5 As shown, Comparative Examples 3 and 4 exhibited relatively low scavenging activity, while Comparative Examples 5 and 6 showed stronger free radical scavenging capabilities. The composite system demonstrated a further enhancing effect. In particular, Example 1 showed the highest hydroxyl radical scavenging rate, indicating that the incorporation of Comparative Examples 5 and 6 significantly improved the antioxidant capacity of the composite system. Different letters indicate significant differences between groups.

[0121] The antibacterial test was conducted as follows: First, the *S. aureus* and *E. coli* strains were activated and bacterial suspensions were prepared. After thoroughly shaking the frozen *S. aureus* and *E. coli* bacterial suspensions, 100 μL was inoculated into 10 mL of freshly sterilized liquid culture medium and incubated at 37°C for 24 h. Using a sterilized inoculation loop, the cultured bacterial suspension was spread onto petri dishes and incubated again at 37°C for 18–24 h. Single colonies were picked and cultured in 10 mL of liquid culture medium for 24 h (37°C). This process was repeated 2–3 times for subculturing to achieve normal activity. *E. coli* and *S. aureus* bacterial suspensions with a concentration of 1108–1109 CFU / mL were obtained and stored at 4°C for later use. The bacterial suspensions were then mixed with different sample solutions and incubated for 1 h, with 100 μL of bacterial suspension and 100 μL of sample solution. Finally, the treated suspension was serially diluted with physiological saline, and 50 μL of each concentration was spread onto an agar plate and incubated overnight in a 37 ℃ biochemical incubator. The colony count was then calculated. Figure 6 The results of antibacterial plate counts for different samples showed strong inhibition of Escherichia coli and Staphylococcus aureus compared to the control group. Comparative Examples 5 and 6 exhibited strong inhibitory effects, as did the complex (Example 1, which significantly reduced colony formation). Therefore, this complex system showed superior antibacterial properties.

[0122] The emulsion stability test was conducted as follows: The emulsion formulation had an oil-to-water ratio of 40 / 60 (v / v), where the aqueous phase was 4.8 mL of the pH-adjusted composite dispersion prepared in Example 1, and the oil phase was 3.2 mL of edible oil. The specific procedure was as follows: 4.8 mL of the pH-adjusted composite dispersion was transferred to a small beaker, and 3.2 mL of the oil phase was added. The mixture was premixed at 700 rpm for 2 min on a magnetic stirrer; subsequently, it was homogenized at 6000 rpm for 3 min using a high-speed homogenizer. The resulting emulsion was immediately transferred to a 10 mL graduated cylinder or polypropylene tube, sealed, and allowed to stand at room temperature for 3 days. The emulsion stability index was calculated by measuring the volume of the bottom clear liquid (aqueous phase). V B V represents the initial aqueous phase volume (4.8 mL). A This represents the volume of the precipitated aqueous phase after standing for 3 days. A higher ES value indicates better emulsion stability. Figure 7 The stability of the emulsion from Example 1 after 6 days of treatment at different pH values ​​was demonstrated. The stability initially increased at pH 2.5, peaked at pH 4.0 (approximately 81%), and then gradually decreased in the pH range of 4.5–5.0. Therefore, a pH of approximately 4.0 is most favorable for maintaining emulsion stability.

[0123] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A PPI-GA-CH-RRA composite emulsion that can serve as a natural substitute for nitrite, characterized in that, Includes the following steps: (1) After adding the pea protein isolate to water for full hydration, adjust the pH, centrifuge, and take the supernatant to obtain the pea protein isolate solution; (2) Dissolve gum arabic in water to obtain gum arabic solution; (3) Dissolve chitosan in acetic acid solution, adjust pH to obtain chitosan solution; (4) Mix the pea protein isolate solution and gum arabic solution and stir. During the stirring process, adjust the pH of the system to 3.8-4.0 and keep it stable to obtain the PPI-GA complex; (5) Add the chitosan solution dropwise to the PPI-GA complex and stir to obtain the PPI-GA-CH complex; (6) Dissolve the concentrated carrot anthocyanin extract in citrate buffer to obtain carrot anthocyanin solution; (7) Add carrot anthocyanin solution to PPI-GA-CH complex, homogenize, and obtain PPI-GA-CH-RRA complex emulsion that can be used as a natural substitute for nitrite.

2. The composite emulsion according to claim 1, characterized in that, In step (7), the mass ratio of pea protein isolate, gum arabic, chitosan and carrot anthocyanin in the PPI-GA-CH-RRA composite emulsion is 2:(0.5-1.0):(0.5-0.75):(0.1-0.15).

3. The composite emulsion according to claim 1 or 2, characterized in that, The concentration of the pea protein isolate solution in step (1) is 2g:100mL-4g:100mL; The concentration of the gum arabic solution in step (2) is 2g:100mL-4g:100mL; In step (3), the concentration of chitosan in the chitosan solution is 1g:100mL-3g:100mL; In step (6), the anthocyanin equivalent in the carrot anthocyanin solution is 10-20 mg / mL; The volume ratio of the pea protein isolate solution in step (4), gum arabic solution, chitosan solution in step (3), and carrot anthocyanin solution in step (6) is 1:(0.25-0.5):(0.5-0.75):0.

1.

4. The composite emulsion according to claim 1 or 2, characterized in that, Step (4) uses an acid solution to adjust the pH of the system to 3.8-4.0, wherein the acid solution is a hydrochloric acid solution with a concentration of 0.05-0.2 mol / L; The time to maintain stability in step (4) is 10-15 minutes.

5. The composite emulsion according to claim 1 or 2, characterized in that, The homogenization process in step (7) involves a temperature ≤35℃, a rotation speed of 8000-10000 rpm, and a time of 2-5 minutes.

6. The composite emulsion according to claim 1 or 2, characterized in that, Step (1) Adjust the pH to 7.0-8.0; the reagent used to adjust the pH is a 0.1-1.0 mol / L sodium hydroxide solution; The sufficient hydration time in step (1) is 2-3 hours; In step (1), the centrifugation speed is 5000-8000 rpm and the time is 15-25 minutes.

7. The composite emulsion according to claim 1 or 2, characterized in that, The volume concentration of the acetic acid solution in step (3) is 1-2%; Step (3) Adjust the pH to 5.5-6.5; the reagent used to adjust the pH is a 0.1-1.0 mol / L sodium hydroxide solution.

8. The composite emulsion according to claim 1 or 2, characterized in that, The pH value of the citrate buffer in step (6) is 3-3.

5.

9. A PPI-GA-CH-RRA composite emulsion that can be used as a natural substitute for nitrite, obtained by the preparation method according to any one of claims 1-8.

10. The application of the PPI-GA-CH-RRA composite emulsion as a natural substitute for nitrite as described in claim 9 in emulsified sausage.

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