Preparation method and application of pea protein nanoparticles constructed based on dynamic phenol-polypeptide crosslinking and proton buffer shell

Abstract

The invention relates to the technical field of vegetable protein functional structuring, in particular to a preparation method and application of pea protein nanoparticles constructed based on dynamic phenol-polypeptide crosslinking and a proton buffer shell. The preparation method comprises the following steps: introducing an oxidized phenolic compound under an alkaline condition to generate dynamic charge bridge crosslinking with pea polypeptide, constructing an inner core network with reversible self-repairing capability, and forming a proton buffer type outer shell structure by utilizing chitosan oligosaccharide through enzymatic reaction, so as to realize stable regulation and control of interfacial potential. The prepared nano-particles are of a core-shell double-layer structure, the particle size of the particles is 80-160 nm, the particle size is uniform and controllable, and the Zeta potential is + 25-+ 35 mV, so that the particles are not prone to agglomeration due to electrostatic repulsion, long-term dispersion stability is kept within the pH range of 3.0-4.5, and excellent acid stability, thermal stability and oxidation resistance are shown. The prepared pea protein nanoparticles can be used as a stabilizer or a functional carrier of an acidic beverage system, can significantly improve the transparency and flavor retentivity of a vegetable protein beverage, and have a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant protein functional structuring and food ingredient stabilization technology, specifically to a method for preparing pea protein nanoparticles based on dynamic phenol-peptide crosslinking and supplemented with a proton buffer shell coated with oligochitosan, and its application. Background Technology

[0002] Pea protein, as an important source of high-quality plant protein, is rich in a balanced combination of essential amino acids, possessing both high nutritional value and low allergenicity, and has attracted attention from the beverage industry in recent years. However, pea protein is prone to flocculation, aggregation, and precipitation under acidic conditions (pH<4.5), and its stability near its isoelectric point is extremely poor, making it difficult to directly apply to acidic beverage systems such as fruit juices, tea drinks, and lactic acid bacteria drinks.

[0003] Currently, the application of plant proteins in acidic beverages still faces serious challenges in terms of stability and dispersibility. Several published patents have attempted to improve the acid stability and dispersibility of plant proteins through methods such as polysaccharide coating, enzymatic modification, chemical derivatization, or emulsion construction, but significant technical bottlenecks remain.

[0004] For example, patent CA2796643C uses polysaccharides such as pectin and gum arabic to coat whey protein to construct an acid-stable particle system. While this improves dispersibility to some extent, the particle size is relatively large (>250nm), affecting taste and transparency. Furthermore, it relies on animal-derived whey protein, which is inconsistent with the development trend of plant-based beverages. The polysaccharide coating is also mainly physical adsorption, resulting in poor structural stability and susceptibility to thermal disintegration. Patent CN202410216380.6 utilizes unhydrolyzed soybean globulin to construct an acidic emulsion system, obtaining high-purity globulin through gentle extraction and introducing vegetable oil to form an emulsion to improve storage stability. However, this approach is only suitable for cloudy beverages, not transparent acidic drinks; it also requires a high oil phase ratio, resulting in high energy density, which is not conducive to the development of low-fat beverages. Furthermore, the currently prepared systems have limitations such as structural stability mainly relying on single-point chemical modification, lacking spatial structural support, and having limited heat resistance, acid resistance, and shear resistance. In addition, the use of anhydride-based chemical modifiers results in low naturalness and consumer acceptance, or the preparation process can easily cause severe protein denaturation, uncontrollable structure, and wide particle size distribution, which is not conducive to the application of transparent beverages. Moreover, the process operation is complex and does not meet the requirements of green production and industrialization.

[0005] In summary, existing technologies mostly focus on improving solubility or acid stability at the protein molecule level, and generally suffer from problems such as large particle size, structural instability, lack of functionality, insufficient naturalness, and poor system adaptability. A systematic strategy for regulating acid stability at the structural network level is still lacking. Especially in the pH range of 3.0–4.5, how to balance particle structural integrity, proton buffering capacity, and interfacial potential stability remains a key technological gap in this field. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing technologies, such as instability, poor dispersibility, and insufficient functional expansion of pea protein in acidic beverage systems. It proposes a method for preparing nanoparticles based on the synergistic construction of a dynamic phenol-peptide crosslinking and a proton-buffered shell. This method achieves a synergistic bilayer structure of "core stability - outer buffering." Through reversible phenol-peptide covalent / charge bridge composites, the particles possess "self-rearrangement" and "charge migration" capabilities. Enzymatic regulation of the protonation degree of the oligochitosan shell forms a pH-self-regulating buffer interface, inhibiting acid aggregation. The resulting particles are small in size, highly positively charged, and highly acid-stable, maintaining transparent dispersion within a pH range of 3.0–4.5, making them suitable for clear and low-turbidity beverages.

[0007] The present invention also provides the application of the nanoparticles prepared by the above method as an additive in acidic beverages.

[0008] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a method for preparing pea protein nanoparticles based on dynamic phenol-peptide crosslinking and proton buffer shell construction, comprising the following steps: (1) Extraction of soluble protein: Add defatted pea flour to an ammonium acetate buffer solution containing calcium chloride, adjust the pH, stir and centrifuge to obtain the extract; (2) Limit enzymatic hydrolysis: Add protease to the extract to hydrolyze it, control the degree of hydrolysis, terminate the reaction, remove the insoluble matter by centrifugation, and take the supernatant as the pea polypeptide solution. (3) Dynamic phenol-peptide crosslinking: Phenolic compounds were added to the obtained pea peptide solution and reacted under alkaline conditions to oxidize the phenolic hydroxyl groups to form quinone groups and react with the peptide amino groups by Mike addition or Schiff base reaction to construct a dynamic crosslinking network with reversible charge bridges. After the reaction was completed, the phenol-peptide crosslinked nanocores were obtained by centrifugation. (4) Construction of proton buffer shell: The phenol-peptide cross-linked nanocore is dispersed in an oligochitosan solution and composited. The composite solution is then modified by enzymatic method to form an outer shell structure with adjustable proton binding sites.

[0009] Preferably, in step (1), the ratio of defatted pea flour to ammonium acetate buffer solution containing calcium chloride is 1g:10-30mL; the concentration of calcium chloride in the ammonium acetate buffer solution is 0.1–0.3mol / L; the pH value of the ammonium acetate buffer solution is 6; and the pH is adjusted to 8.0-9.0.

[0010] Preferably, in step (2), the protease is trypsin, and the amount of protease added is 1.0-2.0% (E / S); the hydrolysis is carried out at a temperature of 37-50°C for 40-50 min, and the degree of hydrolysis (DH) is controlled at 3-6%; then the temperature is raised to 80°C to terminate the reaction.

[0011] Preferably, in step (3), the amount of phenolic compound added accounts for 5% of the mass of pea polypeptide; the phenolic compound is ferulic acid, chlorogenic acid or gallic acid.

[0012] Preferably, in step (3), the ultrasonic treatment is performed for 10-15 minutes at pH 8.5; the ultrasonic conditions are 300-600W and 20kHz.

[0013] Preferably, in step (4), the mass ratio of the phenol-peptide crosslinked nanoparticles to the oligochitosan is 1:10; the enzymatic modification uses chitosanase or protease.

[0014] Preferably, in step (4), the molecular weight of the oligochitosan is 1–5 kDa and the degree of deacetylation is (70–95%); the concentration of the oligochitosan solution is 1–10 mg / mL; and the mass ratio of the phenol-peptide crosslinked nanoparticles to the oligochitosan is 1:5–1:15.

[0015] Preferably, in step (4), the specific process of enzymatic modification is as follows: Enzymatic modification reaction conditions: Adjust the pH of the composite solution to 5.0–6.0 and add chitosanase or protease at a constant temperature; reaction temperature is 30–45°C, reaction time is 15–60 min, and stirring speed is 300–800 rpm. Enzyme termination and post-treatment: Adjust the pH of the system to 8.0–9.0, heat the system to 75–90°C and hold for 3–10 min to inactivate chitosanase; after termination, dialyze to remove free small molecules, free enzymes and low molecular weight salts to obtain a stable dispersion or freeze-dry to obtain powder; The amount of chitosanase added is 0.1–1.0% (E / S, chitosanase based on the mass of added oligochitosan). The protease is papain, trypsin, or flavor protease, with an enzyme activity of 500–2000 U / mg; the amount of the protease added is 0.05–0.5% of the chitosan mass.

[0016] The present invention also provides nanoparticles prepared by the above preparation method, wherein the nanoparticles have a dynamic phenol-peptide cross-linked core and an oligochitosan proton buffer shell, and possess acid stability and reversible anti-aggregation ability, with a particle size of 80–160 nm and a Zeta potential of +25 to +35 mV.

[0017] Another object of the present invention is to provide the application of the above-mentioned nanoparticles as stabilizers, functional factor carriers or transparent conditioning agents in acidic beverage systems.

[0018] The nanoparticles provided by this invention are stable and do not precipitate within a pH range of 3.0–4.5, making them suitable for acidic beverage systems such as fruit juices and teas. The resulting acidic beverages are transparent or have low turbidity, exhibiting good dispersibility and flavor carrier capabilities.

[0019] In the preparation process of this invention, an enzymatic method is used, the purpose of which is to slightly cleave the chitosan chains to adjust the outer layer density / chain length, improve the uniformity of surface coverage and expose cationic sites, thereby optimizing the particle size distribution and zeta potential.

[0020] The beneficial effects of this invention are as follows: (1) By precisely controlling reaction conditions, this invention prepares functional nanoparticles with excellent performance. The particle size is 80–160 nm, the particle size is uniform and the size is controllable. The zeta potential is +25 to +35 mV, which makes it difficult for the particles to aggregate due to electrostatic repulsion. No precipitation occurs in the pH range of 3.0–4.5, which significantly improves the long-term stability of the system. The prepared nanoparticles have excellent acid stability, thermal stability and dispersibility, and are suitable for acidic beverage systems, with good application prospects.

[0021] (2) The particles prepared by this invention have small particle size, high positive charge and strong acid stability. They can maintain transparent dispersion in the pH range of 3.0–4.5, and are suitable for use in clear drinks and low turbidity beverages. This solves the problem that traditional functional materials are prone to agglomeration and instability in acidic environments. Detailed Implementation

[0022] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0023] In some embodiments, the enzyme activity of trypsin, papain or flavor protease is 2,000–10,000 U / g (measured using casein as a substrate at 37°C and pH 7.6).

[0024] Example 1: Ferulic acid dynamic crosslinking + 2kDa chitosan proton buffer shell (1) Add 1200mL of 0.2mol / L ammonium acetate buffer solution containing calcium chloride (pH of buffer solution is 6) to 100g defatted pea flour, adjust pH to 8.5, stir at 45℃ for 60min and then centrifuge (8000r / min, 15min) to obtain the extract; (2) Preparation of functional peptides: Add trypsin to the extract at an enzyme concentration of 1.5% (E / S), and carry out hydrolysis at 40°C (about 45 min). Control the degree of hydrolysis to 5%, raise the temperature to 80°C and keep it at 5 min to terminate the reaction. After centrifugation to remove insoluble matter, take the supernatant as the pea peptide solution. (3) Construction of phenol-peptide cross-linked nanoparticles: Add ferulic acid at 5% of the pea peptide solution to the obtained pea peptide solution, adjust the pH to 8.5, sonicate (500W, 20kHz) for 10min, and centrifuge to obtain phenol-peptide cross-linked nanoparticles. (4) Preparation of oligochitosan solution: Take 2 kDa oligochitosan and dissolve it in deionized water at a mass ratio of phenol-peptide crosslinked nanoparticles to oligochitosan of 1:10, so that the solution concentration is 10 mg / mL. Adjust the pH to 5.8 with dilute acetic acid and stir until clear; (5) The oligochitosan solution was slowly added dropwise to the phenol-peptide crosslinked nanoparticle dispersion under stirring (3500 rpm). The addition time was adjusted according to the volume to ensure the uniformity of the surface coating. The system temperature was maintained at 30°C during the addition process to obtain the composite solution. (6) Adjust the pH of the composite solution to 5.5 and add chitosanase at a constant temperature. The enzyme addition amount is 0.5% (E / S, based on the mass of the added oligochitosan); the reaction temperature is 40°C, the reaction time is 60 min, and the stirring speed is 500 rpm. Enzyme termination and post-treatment: The pH of the system was adjusted to 8.5, and the system was heated to 80°C and held for 5 minutes to inactivate chitosanase. After termination, the system was dialyzed to remove free small molecules, free enzymes and low molecular weight salts to obtain a stable dispersion. The dispersion was then freeze-dried to obtain powder (nanoparticles).

[0025] The nanoparticle dispersion prepared in Example 1 was added to a citrate-sodium citrate buffer solution (0.01 mol / L, pH 3.5), and after standing for 24 h, the particle size and zeta potential were measured. Results: Particle size 132 nm, zeta potential +30.4 mV, no precipitation or flocculation under these conditions.

[0026] Example 2: Dynamic cross-linking of chlorogenic acid + 1kDa chitosan proton buffer shell Steps (1)-(2) are the same as in Example 1; (3) Construction of phenol-peptide cross-linked nanoparticles: 5% chlorogenic acid was added to the obtained pea peptide solution, the pH was adjusted to 8.5, and the solution was sonicated (500W, 20kHz) for 10min. After centrifugation, phenol-peptide cross-linked nanocores were obtained. (4) Preparation of oligochitosan solution: Take 1 kDa of oligochitosan and dissolve it in deionized water at a mass ratio of phenol-peptide crosslinked nanoparticles to oligochitosan of 1:10, so that the solution concentration is 10 mg / mL. Adjust the pH to 5.5 with dilute acetic acid and stir until clear; (5) The oligochitosan solution was slowly added dropwise to the phenol-peptide crosslinked nanoparticle dispersion under stirring (3500 rpm). The addition time was adjusted according to the volume to ensure the uniformity of the surface coating. The system temperature was maintained at 30°C during the addition process to obtain the composite solution. (6) Adjust the pH of the composite solution to 5.5 and add papain at a constant temperature. The amount of papain added is 0.5% of the mass of chitosan. The reaction temperature is 40°C, the reaction time is 40 min, and the stirring speed is 500 rpm. Enzyme termination and post-treatment: The pH of the system was adjusted to 8.5, and the system was heated to 80°C and held for 5 minutes to inactivate chitosanase. After termination, the system was dialyzed to remove free small molecules, free enzymes and low molecular weight salts to obtain a stable dispersion. The dispersion was then freeze-dried to obtain powder (nanoparticles).

[0027] The nanoparticle dispersion prepared in Example 2 was added to a citrate-sodium citrate buffer solution (0.01 mol / L, pH 3.2), and after standing for 24 h, the particle size and zeta potential were measured. Results: Particle size 120 nm, zeta potential +32 mV, no precipitation or flocculation under these conditions.

[0028] Comparative Example 1: No dynamic cross-linking (1) Add 1200 mL of 0.2 mol / L ammonium acetate buffer solution containing calcium chloride (pH 6) to 100 g defatted pea flour, adjust the pH to 8.5, stir at 45 °C for 60 min and then centrifuge (8,000 r / min, 15 min) to obtain the extract; (2) Preparation of functional peptides: Add trypsin to the extract at an enzyme concentration of 1.5% (E / S), and carry out hydrolysis at 40°C (about 45 min). Control the degree of hydrolysis to 5%, raise the temperature to 80°C and keep it at 5 min to terminate the reaction. After centrifugation to remove insoluble matter, take the supernatant as the pea peptide solution. (3) Preparation of oligochitosan solution: Take 2 kDa oligochitosan and dissolve it in deionized water at a mass ratio of phenol-peptide crosslinked nanoparticles to oligochitosan of 1:10, so that the solution concentration is 10 mg / mL. Adjust the pH to 5.8 with dilute acetic acid and stir until clear; (4) The oligochitosan solution was slowly added dropwise to the pea polypeptide solution (based on the mass ratio of pea polypeptide to oligochitosan in the cross-linked particles of Example 1, which was 1:10) under stirring (3500 rpm). The addition time was adjusted according to the volume to ensure the uniformity of the surface coating. The system temperature was maintained at 30°C during the addition process to obtain the composite solution. Step (6) is the same as in Example 1; freeze-drying yields powder (nanoparticles).

[0029] The nanoparticle dispersion prepared in Comparative Example 1 was added to a citrate-sodium citrate buffer solution (0.01 mol / L, pH 3.5), and after standing for 24 h, the particle size and Zeta potential were measured. Results: Particle size > 400 nm, Zeta potential +12 mV, precipitation occurred within 30 min at pH 3.5.

[0030] Comparative Example 2: Enzyme-free modified shell Steps (1)-(5) are the same as in Example 1; (6) The composite solution is dialyzed to remove free small molecules and low molecular weight salts to obtain a stable dispersion, which is then freeze-dried to obtain powder (nanoparticles).

[0031] The nanoparticle dispersion prepared in Comparative Example 2 was added to a citrate-sodium citrate buffer solution (0.01 mol / L, pH 3.5), and after standing for 24 h, the particle size and zeta potential were measured. Results: Particle size 180 nm, zeta potential +18 mV, precipitation occurred within 2 h at pH 3.5.

[0032] Example 1 The antioxidant capacity of the pea protein nanoparticles prepared in Examples 1-2 and Comparative Examples 1-2 was detected by spectrophotometry to determine their DPPH free radical scavenging rate. The specific detection method was as follows: (1) Sample preparation: Pea protein nanoparticles prepared in the examples and comparative examples were added to ethanol to prepare a stock solution with a concentration of 1 mg / mL; the stock solution was diluted with ethanol to a series of concentration gradients of 0.2, 0.4, 0.6, 0.8 and 1.0 mg / mL; (2) Accurately weigh DPPH powder (purity ≥98%), dissolve it in anhydrous ethanol, and prepare a 0.1 mmol / L stock solution. After preparation, it should be kept in the dark for 30 min. The absorbance should be measured at 517 nm using a spectrophotometer to ensure that the absorbance value is between 0.7 ± 0.02. (3) Constructing the reaction system: Mix the sample and DPPH solution in proportion: Take 2 mL of sample solution of various concentrations, add 2 mL of 0.1 mmol / L DPPH solution, mix thoroughly; let stand at room temperature (25±2℃) in the dark for 30 min; Blank group: 2 mL solvent (such as ethanol) + 2 mL DPPH solution, used to calibrate the instrument background; Control group: 2 mL sample solution + 2 mL solvent, used to subtract the interference of the sample's own absorbance; Absorbance measurement: The absorbance of the sample group, blank group and control group was measured sequentially at a wavelength of 517 nm using a spectrophotometer. Each group was measured in parallel 3 times and the average value was taken. SR(%) = [1 - (Sample A - Control A) / Blank A] × 100; The specific results are shown in Table 1.

[0033] Table 1 Example 2 The thermal stability of the pea protein nanoparticles prepared in Examples 1-2 and Comparative Examples 1-2 was tested. The specific method was as follows: A dispersion of pea protein nanoparticles (concentration 2 mg / mL, neutral pH) was aliquoted into centrifuge tubes. Temperature gradients were set at 30℃, 50℃, and 70℃. At each temperature, the nanoparticles were heated in a constant-temperature water bath or oil bath for 30 min, and then rapidly cooled to room temperature in an ice-water bath. Dynamic light scattering (DLS) was used, with three parallel measurements performed at each temperature gradient. The average particle size was recorded, and the polydispersity index (PDI) was calculated. Specific results are shown in Table 2.

[0034] Table 2 Example 3 The pea protein nanoparticles prepared in Examples 1-2 and Comparative Examples 1-2 were subjected to acidification-alkalization cycle tests. The initial concentration was 2.0 mg / mL of nanoparticle dispersion.

[0035] Test instruments and methods: DLS (scattering angle 90°, temperature 25℃, diluted to a suitable concentration), Zeta measured on the same equipment; turbidity / absorbance at 600nm (A600).

[0036] Cyclic procedure (3 cycles): Initial (neutral): Adjust the sample to pH 7.0, let stand for 10 min, and measure DLS / PDI, Zeta, and A600; Acidification: Adjust the pH to 3.5 with citrate-sodium citrate buffer, let stand for 30 min (simulating short-term acidification of acidic beverages), and measure DLS / PDI, Zeta, and A600; Alkalinization and restoration: Adjust the pH of the system back to 8.0 with a small amount of 0.1M NaOH, let stand for 30 min, and measure DLS / PDI, Zeta, and A600.

[0037] Repeat steps 2–3 to complete a total of 3 acidification and alkalization cycles.

[0038] Judgment index: Particle size recovery rate = (particle size after each alkalization / initial neutral particle size) × 100%; Stability criterion example: Recovery rate ≥ 85% and no visible precipitation / A600 not significantly increased is considered "good reversibility / self-repair".

[0039] Table 3 Results of acidification-alkalization cycle test in Example 1 Table 4 Results of acidification-alkalization cycle test in Example 2 Table 5 Results of acidification-alkalization cycle test for Comparative Example 1 Table 6 Results of acidification-alkaliification cycle test for Comparative Example 2

Claims

1. A method for preparing pea protein nanoparticles based on dynamic phenol-peptide crosslinking and proton buffer shell construction, characterized in that, Includes the following steps: (1) Extraction of soluble protein: Add defatted pea flour to an ammonium acetate buffer solution containing calcium chloride, adjust the pH, stir and centrifuge to obtain the extract; (2) Limit enzymatic hydrolysis: Add protease to the extract to hydrolyze it, control the degree of hydrolysis, terminate the reaction, remove the insoluble matter by centrifugation, and take the supernatant as the pea polypeptide solution. (3) Dynamic phenol-peptide crosslinking: Phenolic compounds were added to the obtained pea peptide solution and reacted under alkaline conditions. After the reaction was completed, the phenol-peptide crosslinked nanocores were obtained by centrifugation. (4) Construction of proton buffer shell: The phenol-peptide cross-linked nanocore is dispersed in an oligochitosan solution and composited. The composite solution is then modified by enzymatic method to form an outer shell structure with adjustable proton binding sites.

2. The preparation method according to claim 1, characterized in that, In step (1), the ratio of defatted pea flour to ammonium acetate buffer solution containing calcium chloride is 1g:10-30mL; the concentration of calcium chloride in ammonium acetate buffer solution is 0.1–0.3mol / L; the pH of ammonium acetate buffer solution is 6; the pH of the extract is adjusted to 8.0-9.0 for subsequent steps.

3. The preparation method according to claim 1 or 2, characterized in that, In step (2), the protease is trypsin, and the amount of protease added is 1.0-2.0% (E / S); the hydrolysis is carried out at a temperature of 37-50°C for 40-50 min, and the degree of hydrolysis (DH) is controlled at 3-6%; then the temperature is raised to 80°C to terminate the reaction.

4. The preparation method according to claim 1, characterized in that, In step (3), the amount of phenolic compound added accounts for 5% of the mass of pea polypeptide; the phenolic compound is ferulic acid, chlorogenic acid or gallic acid.

5. The preparation method according to claim 1 or 4, characterized in that, In step (3), the ultrasonic treatment is performed for 10-15 minutes at pH 8.5; the ultrasonic conditions are 300-600W and 20kHz.

6. The preparation method according to any one of claims 1-5, characterized in that, In step (4), the mass ratio of the phenol-peptide cross-linked nanoparticles to the oligochitosan is 1:10; the enzymatic modification uses chitosanase or protease.

7. The preparation method according to claim 6, characterized in that, In step (4), the molecular weight of the oligochitosan is 1–5 kDa; the concentration of the oligochitosan solution is 1–10 mg / mL; and the mass ratio of the phenol-peptide crosslinked nanoparticles to the oligochitosan is 1:5–1:

15.

8. The preparation method according to claim 1 or 7, characterized in that, In step (4), The specific process of the enzymatic modification is as follows: Enzymatic modification reaction conditions: Adjust the pH of the composite solution to 5.0–6.0 and add chitosanase or protease at a constant temperature; reaction temperature is 30–45°C, reaction time is 15–60 min, and stirring speed is 300–800 rpm. Enzyme termination and post-treatment: Adjust the pH of the system to 8.0–9.0, heat the system to 75–90°C and hold for 3–10 min to inactivate chitosanase; after termination, dialyze to remove free small molecules, free enzymes and low molecular weight salts to obtain a stable dispersion or freeze-dry to obtain powder; The amount of chitosanase added is 0.1–1.0% (E / S, chitosanase based on the mass of added oligochitosan). The protease is papain, trypsin, or flavor protease, with an enzyme activity of 500–2000 U / g; the amount of the protease added is 0.05–0.5% of the chitosan mass.

9. Nanoparticles prepared by the preparation method according to any one of claims 1-8, characterized in that, The nanoparticles have a dynamic phenol-peptide cross-linked core and an oligomeric chitosan proton buffer shell, exhibiting acid stability and reversible anti-aggregation ability. Their particle size is 80–160 nm, and their zeta potential is +25 to +35 mV.

10. The application of the nanoparticles as described in claim 9 as a stabilizer, functional factor carrier, or transparent conditioning agent in acidic beverage systems.

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