Walnut protein isolate bacteriostatic biofilm and preparation method and application thereof

Abstract

The application discloses walnut protein isolate bacteriostatic biological membrane and a preparation method and application thereof, and the preparation method comprises the following steps: step (1), mixing a WNPI stock solution and a PGA stock solution, and stirring to prepare WNPI / PGA composite coacervates; step (2), slowly adding a CA stock solution to the WNPI-PGA composite coacervate solution, and stirring in the dark to prepare WNPI / PGA-CA composite coacervates; step (3), mixing and stirring a PVA solution and the WNPI / PGA-CA composite coacervates, and then being refrigerated overnight, then being dried and cooled, and then being balanced in a dryer to obtain walnut protein isolate bacteriostatic biological membrane. The WNPI / PGA composite is used as a wall material to fix CA, and then the WNPI / PGA-CA composite is combined with PVA to prepare a biological bacteriostatic membrane, and the walnut protein biological bacteriostatic membrane has strong antibacterial, antioxidant and oxygen barrier capabilities.

Description

Technical Field

[0001] This invention relates to the field of antibacterial biofilms, and more particularly to a walnut protein isolate antibacterial biofilm, its preparation method, and its application. Background Technology

[0002] Walnut protein isolate (WNPI) is the main protein component in walnut meal, but its poor solubility limits its application in food processing. Currently, numerous studies have employed physical, chemical, and biological methods to improve protein functionality. Among these, pH induction and thermal induction are two traditional and simple techniques widely used to improve protein function. For example, Jeong et al. reported that pH induction significantly improved the water-holding capacity and gelling properties of pea protein. However, these methods, especially heat treatment, still have many shortcomings. Protein-polysaccharide aggregation, as an emerging technology, has attracted widespread attention due to its high loading capacity, high encapsulation efficiency, high thermal stability, and complex structure, enabling the sustained release of core materials. However, research on WNPI-polysaccharide aggregation is currently limited.

[0003] Therefore, this invention proposes a WNPI / PGA composite to improve the functional properties of WNPI near its isoelectric point. PGA is a low-cost, widely used natural amphiphilic polysaccharide in beverages and jams; its electrostatic interaction with WNPI can improve the performance of the composite. The study will explore the electrostatic composite mechanism of PGA and WNPI under specific conditions and purposefully prepare composite aggregates with predetermined functions by adjusting internal (pH, ionic strength, and biopolymer ratio) and external (temperature) factors. Furthermore, given the antioxidant and antibacterial properties of chlorogenic acid, as well as its environmental sensitivity and easy degradation, this invention will also explore the use of WNPI / PGA composite aggregates as encapsulation carriers to enhance the stability of the encapsulated material. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a walnut protein isolate antibacterial biofilm, its preparation method, and its application. This invention uses a WNPI / PGA composite as a wall material to fix CA (chlorogenic acid), and then combines it with PVA (polyvinyl alcohol) to form a biofilm. The walnut protein biofilm provided by this invention has strong antibacterial, antioxidant, and oxygen barrier capabilities.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing a walnut protein isolate antibacterial biofilm, the method comprising the following steps:

[0007] Step (1) Mix WNPI stock solution and PGA stock solution and stir to prepare WNPI / PGA composite condensate;

[0008] Step (2) The CA stock solution was slowly added to the WNPI-PGA composite coagulant solution and stirred in the dark to obtain the WNPI / PGA-CA composite coagulant;

[0009] Step (3) PVA solution and WNPI / PGA-CA composite coagulant were mixed at room temperature and stirred, refrigerated overnight, then dried and cooled, and equilibrated in a desiccator to obtain walnut protein isolate antibacterial biofilm.

[0010] Preferably, the mass ratio of WNPI to PGA is 1.5-3:1, and the total concentration of both in the reaction system reaches 0.4-1.0% (w / v).

[0011] Preferably, the mass ratio of WNPI / PGA to CA is 2-8:1; more preferably, the mass ratio of WNPI / PGA to CA is 3-6:1.

[0012] The concentration of CA in the CA stock solution is 0.3-1.0% (w / v); the CA stock solution is slowly added to the WNPI-PGA composite coagulant solution to adjust the final concentration of CA to 0.05%-0.20% (w / v).

[0013] Preferably, in step (1), the pH of the reaction system is adjusted to 3.0-6.0 using 0.5-1.5 mol / L hydrochloric acid; the reaction temperature is 2-8℃.

[0014] Preferably, in step (2), the pH of the mixed solution is adjusted to 3.0-6.0 using 0.5-1.5 mol / L hydrochloric acid, then stirred in the dark for 20-60 minutes, and then centrifuged at a low speed of 2500-4000 rpm for 8-20 minutes to remove possible large aggregates and undissolved matter.

[0015] Preferably, glycerol and Tween-80 are also added to the reaction system in step (3); the concentration of glycerol in the reaction system is 0.5-1.0%; the concentration of Tween-80 in the reaction system is 0.05-0.2%; the concentration of the PVA solution is 3-10% (w / v); and the mass ratio of PVA, WNPI / PGA-CA, glycerol and Tween-80 is 3-10:0.3-1.0:0.5-1.0:0.05-0.2.

[0016] In a second aspect, the present invention also provides a walnut protein isolate antibacterial biofilm prepared by the method.

[0017] In a third aspect, the present invention also provides the application of the walnut protein isolate antibacterial biofilm in the preparation of antioxidants or antibacterial agents.

[0018] In a fourth aspect, the present invention also provides the application of walnut protein isolate antibacterial biofilm in the preparation of food preservatives or food packaging bags.

[0019] Preferably, the food is meat product, fruit, yogurt, or jelly.

[0020] The features of this invention are as follows: the WNPI-PGA complex retains a certain net negative charge during the complexation process, and the repulsive force in the system is sufficient to resist the aggregation of the complex. The mass ratio of protein to polysaccharide is another key factor affecting the complexation and aggregation process; different mass ratios affect the charge balance between the two, thereby affecting their interaction and complexation strength. In this invention, when the WNPI-PGA ratio is preferably 2:1, WNPI saturates and adsorbs PGA glycans, and the insoluble WNPI-PGA aggregate reaches its maximum value, showing that increasing the polysaccharide concentration can effectively inhibit further protein aggregation. After WNPI and PGA form an aggregate, the WNPI protein molecular structure undergoes changes, and the thermal stability of the aggregate is improved due to the addition of polysaccharide. This enhancement may stem from the formation of numerous hydrogen bonds during aggregation, making the crystal structure more stable and ordered, and forming a dense sheet-like structure. These findings indicate that WNPI-PGA aggregates are ideal for encapsulating and preserving bioactive compounds, suitable for controlled release and targeted delivery.

[0021] To enhance the stability of chlorogenic acid (CA), this invention selected WNPI / PGA composite aggregates as the carrier material for freeze-dried nanoparticles. The particle size, PDI (particulate density), and surface charge of the nanoparticles are key indicators for evaluating their long-term stability and controlled release behavior. Studies show that compared to WNPI / PGA:CA12:1, the particle sizes of WNPI / PGA:CA6:1, WNPI / PGA:CA4:1, and WNPI / PGA:CA3:1 are significantly increased. This change may be due to the increased CA content significantly expanding the nanoparticle size. The surface electrostatic charge of the composite nanoparticles decreases, possibly because of the reduced zeta potential of free protein and polysaccharide molecules in solution and the protein-polysaccharide complex. This is speculated to be because the increased chlorogenic acid content in the system may inhibit the potential response. The water contact angle of the composite nanoparticles is less than 90°, exhibiting good hydrophilicity, which may be a result of chlorogenic acid inhibiting the hydrophobicity of the protein surface. Therefore, WNPI / PGA:CA4:1, with its excellent hydrophilicity and stable structure, becomes the preferred choice for nanoparticles.

[0022] In terms of package efficiency (EE) and load capacity (LC), WNPI / PGA:CA4:1 The encapsulation contents reached 92.30% and 18.46% respectively, demonstrating superior encapsulation performance compared to other CA-loaded nanoparticles. This is likely due to the presence of more CA binding sites in the WNPI molecules. Furthermore, the addition of PGA increased the viscosity of the continuous phase, thereby inhibiting CA migration to some extent, resulting in a higher energy efficiency (EE). Therefore, WNPI / PGA:CA 4:1 Due to its high encapsulation capability, the delivery system is considered an ideal carrier for the further application of CA in food processing. Furthermore, WNPI / PGA:CA 4:1 The nanoparticles also showed significantly better performance than the other four groups of nanoparticles in terms of thermal stability, freeze-thaw stability, pH stability, temperature storage stability, and light stability.

[0023] To improve the stability of walnut protein (CA), a WNPI / PGA composite was used as the wall material to fix CA, which was then combined with PVA to form a film, aiming to develop packaging materials that can extend the shelf life of meat products and other foods. Compared with pure PVA film without nanoparticles, the film containing nanoparticles has a larger thickness due to the increased mass after solution casting. Generally, the barrier performance of a film is directly affected by its substrate composition. The results show that the water vapor permeability (WVP) of the walnut protein antibacterial film remains at a low level. The addition of CA slightly increases the WVP of the film. Simultaneously, the addition of glycerol, a hydrophilic plasticizer, improves certain physical properties of the composite film, such as flexibility, water solubility, and WVP. Considering all factors, the CA-containing composite film exhibits superior barrier performance, proving that the walnut protein antibacterial film is suitable for packaging materials. The light transmittance of the composite film after CA addition is significantly reduced, because CA absorbs ultraviolet light. Studies show that this composite film can effectively block ultraviolet rays and is suitable for food packaging. With increasing WNPI / PGA-CA content, the free radical scavenging and antioxidant capabilities of the membranes significantly improved, and all samples exhibited dose-dependent antioxidant properties. Furthermore, the PVA-based membranes loaded with WNPI / PGA-CA showed antibacterial effects against Staphylococcus aureus and Escherichia coli, indicating their potential as packaging materials for active foods.

[0024] Given that meat products are susceptible to microbial contamination and spoilage, and changes in the storage environment can also trigger fat oxidation, this invention mixes WNPI / PGA-CA with PVA to create a PVA-WNPI / PGA-CA packaging film, which is then applied to the anti-oxidation, antibacterial, and preservation of meat products. Studies have shown that the addition of PVA-WNPI / PGA-CA significantly extends the shelf life of pork and slows down changes in its appearance. Therefore, incorporating WNPI / PGA-CA nanoparticles into the packaging matrix can effectively extend the shelf life of food, and this invention provides a theoretical basis for the application of WNPI / PGA-CA nanoparticles in food packaging.

[0025] Compared with existing technologies, this invention has the following beneficial effects: This invention prepares a PVA-WNPI / PGA-CA composite membrane by mixing a WNPI / PGA-CA suspension with a PVA solution and then casting it. Studies have shown that as the WNPI / PGA-CA ratio increases, the surface roughness and thickness of the film increase, the tensile strength increases, and the elongation at break decreases. In particular, the tensile strength of the PVA-WNPI / PGA-CA membrane is higher than that of the pure PVA membrane, and the elongation at break is reduced. This is attributed to the hydrogen bonds and electrostatic interactions formed between WNPI / PGA-CA and the membrane matrix, which increase the viscosity of the film-forming solution, thereby improving the mechanical properties of the membrane. After adding WNPI / PGA-CA, the water vapor permeability (WVP) and oxygen permeability (OP) of the membrane both decrease. Simultaneously, the UV-Vis light blocking ability, antibacterial ability, and antioxidant ability of these membranes are also significantly enhanced. During food preservation, the PVA-WNPI / PGA-CA membrane exhibits strong antibacterial, antioxidant, and oxygen blocking capabilities. Attached Figure Description

[0026] Figure 1 These are scanning electron microscope (SEM) images (5000×) of the WNPI-PGA composite coagulants. Figures A, B, and C correspond to the composite coagulants formed at pH values ​​of 2.5, 3.5, and 6.0, respectively. Figures D, E, and F correspond to the composite coagulants formed after adding 5 mM, 10 mM, and 20 mM NaCl, respectively. Figures G, H, and I show the composite coagulants with WNPI to PGA ratios of 1:1, 2:1, and 1:2.

[0027] Figure 2 for WNPI, WNPI / PGA:CA 12:1 WNPI / PGA:CA 6:1 WNPI / PGA:CA 4:1 and WNPI / PGA:CA 3:1 Water contact angle;

[0028] Figure 3 WNPI / PGA:CA 12:1 WNPI / PGA:CA 6:1 WNPI / PGA:CA 4:1 and WNPI / PGA:CA 3:1 The impact of CA on EE and LC;

[0029] Figure 4 Cross-sectional SEM images of (A) PVA membrane, (B) PVA-CA membrane, (C) PVA-WNPI / PGA-CA I membrane, (D) PVA-WNPI / PGA-CAII membrane, and (E) PVA-WNPI / PGA-CA III membrane;

[0030] Figure 5 The tensile strength (A) and elongation at break (B) of the antibacterial film;

[0031] Figure 6 The viscosity of the film-forming solution;

[0032] Figure 7 This is a diagram showing the appearance of the antibacterial film;

[0033] Figure 8 The light transmittance of the antibacterial film;

[0034] Figure 9 The free radical scavenging rates of (A) DPPH and (B) ABTS of the antibacterial film;

[0035] Figure 10 Absorbance of bacterial suspensions treated with film-forming solution: (A) Escherichia coli, (B) Staphylococcus aureus;

[0036] Figure 11 The effect of different packaging films on the pH value of pork samples;

[0037] Figure 12 The effect of different packaging films on the yield of volatile basic nitrogen in pork samples during storage;

[0038] Figure 13 This represents the total bacterial count of each group of pork during storage. Detailed Implementation

[0039] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0040] Example 1

[0041] Preparation of walnut protein isolate

[0042] Walnut meal was pulverized and dried to constant weight at 45℃-48℃, then sieved through a 150-mesh sieve. The walnut meal was then mixed with n-hexane at a ratio of 1:5 (w / v) for defatting, and the mixture was continuously stirred with a constant magnetic force for 2 hours. Afterward, the mixture was filtered and the residue was collected. The defatting process was repeated until the filtrate became colorless and transparent. The defatted residue was placed in a fume hood to evaporate any remaining solvent. The dried residue was then pulverized in a grinder and sieved (200 mesh) to obtain defatted walnut meal powder. This powder was stored in a refrigerator at 4℃.

[0043] Walnut meal defatted powder was mixed with water at a ratio of 1:20 (w / v). The pH of the solution was adjusted to 11 with 1M NaOH. The mixture was stirred with a constant magnetic force for 1.5 h, and then centrifuged (4℃, 3000 rpm, 10 min) to obtain a clear protein solution. The pH of the supernatant was adjusted to 4.5 with 1M HCl, and the mixture was allowed to stand for 1 h. It was then centrifuged (4℃, 3000 rpm, 15 min), and the precipitate was washed (with water). The precipitate was neutralized, and the pH was adjusted to neutral (7.0). Walnut protein isolate (WNPI) was obtained by freeze drying and stored in a brown reagent bottle at 4℃.

[0044] Preparation of WNPI-PGA composite solution

[0045] Accurately weigh 2.00 g of WNPI powder and dissolve it in 100 mL of deionized water to obtain a 2% (w / v) stock solution. Hydrate the solution at 4°C with a magnetic stirrer at 500 rpm for 12 h. Immediately afterwards, adjust the pH of the solution to 12 using 1.0 mol / L NaOH and maintain it at room temperature for 3 h. Then, slowly adjust the pH of the solution to neutral using 0.1 mol / L HCl. After centrifuging the solution at 4500 rpm for 30 min, remove insoluble components to obtain the WNPI solution. Dissolve 1.00 g of PGA powder in 100 mL of deionized water to prepare a 1% (w / v) PGA stock solution. Stir the solution at 25°C with a magnetic stirrer at 800 rpm until completely dissolved.

[0046] WNPI and PGA (propylene glycol alginate) stock solutions of different proportions were mixed and stirred for 2 h, then diluted to a total biological concentration of 0.6% to prepare WNPI / PGA composite solutions. The pH was then adjusted using NaOH and HCl solutions to induce coagulation reactions under different conditions. The interaction effects under different pH values ​​(2.0–10.0), sodium chloride concentrations (0–40 mM), proteoglycan mass ratios (1:2, 1:1, 2:1, 4:1, 10:1 w / w), and temperatures (4, 25, 35, 45 °C) were investigated by measuring turbidity, zeta potential, and coagulation yield.

[0047] This invention observed the appearance of WNPI-PGA aggregates at different pH values. The pH values ​​at which soluble complexes, aggregates, and maximum turbidity initially formed are typically expressed as pHc, respectively. and pHopt, and This corresponds to the pH value at which the charged groups of polysaccharides are completely protonated and lose their ability to electrostatically bind to proteins. The critical pH value. Defined as the intersection of the two tangents on the turbidity curve, pHopt is the pH value corresponding to the maximum turbidity. At pH 3.5 (pHopt), the turbidity reaches a peak of 0.946, forming obvious insoluble complex coagulants (suitable for film formation). The optimal reaction pH is 3.5.

[0048] Furthermore, to further understand the effect of the pH value of the mixture on the electrostatic interaction between WNPI and PGA, the present invention measured the Zeta-potential and particle size of the polymer. As the pH value changed (from 2 to 10.0), the Zeta-potential of the pure WNPI solution increased from -27.83 mV to 8.22 mV, indicating that pH affects the surface charge of WNPI. The charge magnitude of the ionizable groups (especially carboxyl and amino groups) on WNPI depends on the pH value. When the pH value was adjusted from much below the pKa value to much above the pKa value, the carboxyl group changed from neutral (-COOH) to completely negative (-COO-), while the amino group changed from positive (-NH3) to positive (-NH3). + The net charge on proteins changes from positive to negative as pH increases, meaning their solubility, structure, interactions, and function are all pH-dependent. At higher pH values, the WNPI-PGA polymer exhibits a higher Zeta potential, indicating that the formation of the WNPI-PGA polymer is stable. As the solution pH gradually decreases, the Zeta potential of the WNPI-PGA mixed solution continuously increases. Based on these findings, this application proposes that WNPI and PGA will electrostatically attract each other between pH 2 and 5, because within this pH range, WNPI carries a positive charge and PGA carries a negative charge.

[0049] Effect of Temperature on WNPI-PGA Composite Agglomeration Behavior: To investigate the effect of temperature on the interactions of WNPI-PGA composite aggregates, the phase behavior of the aggregates was analyzed within a temperature range of 4–55 °C, with room temperature as a control. When the temperature decreased to 4 °C, both the turbidity curve and the critical pH value of the composite aggregates shifted to lower pH values, indicating that low temperature enhanced hydrogen bonding and promoted aggregate formation. Conversely, when the temperature increased to 25–55 °C, the turbidity curve and critical pH value shifted to higher pH values ​​due to weakened hydrogen bonding, leading to unstable aggregate structures. Although hydrophobic interactions did not appear to have a direct effect on aggregate formation, they did contribute to stabilizing the structure of the WNPI-PGA complex. Furthermore, high temperature also appeared to promote electrostatic bonding between WNPI and PGA. Therefore, the optimal reaction temperature is approximately 4 °C.

[0050] Effect of ionic strength on the coagulation behavior of WNPI-PGA composites: This invention investigated the effect of different concentrations of NaCl (0, 5, 10, 20, and 40 mM) on the turbidity of WNPI-PGA mixtures with pH values ​​ranging from 1.0 to 10.0. With increasing salt ionic strength, pHc and... All shifted towards lower pH values, indicating that Na + With positively charged groups and Cl in proteins - It competitively adsorbs with the negatively charged groups in the polysaccharide side chain, interfering with the electrostatic binding between WNPI and PGA, thereby reducing the aggregation behavior of WNPI and PGA. Therefore, no additional salt ions are needed.

[0051] Effect of Protein / Polysaccharide Mass Ratio on WNPI-PGA Composite Coagulation Behavior: This invention investigated the pH-dependent behavior of the composite coagulated with WNPI to PGA ratios varying from 1:2 to 10:1. As the protein / polysaccharide ratio increased from 2:1 to 10:1, the turbidity curve shifted to lower pH values, and the maximum turbidity decreased, reaching its maximum at a 2:1 ratio. With increasing ratio, pH... c It dropped to about 6.5, while The initial pH of the aggregate formation remains at approximately 7.0. Similarly, as the proportion increases, the pH of the most significant interaction point in the mixture... opt The pH value at the initial stage of maximum turbidity also showed a similar change. Therefore, it can be concluded that at a 2:1 ratio, the sugar chains of PGA are saturated with WNPI adsorption, at which point the insoluble WNPI-PGA aggregate reaches its maximum. When the polysaccharide concentration is higher than the protein concentration (e.g., 1:2), the maximum turbidity value decreases significantly, the turbidity curve shifts to the left, and the pH... opt The concentration also decreased from 0.946 to 0.152. Under high polysaccharide concentrations, PGA molecules became supersaturated, leading to a reduction in the number of amino groups available for WNPI binding. Furthermore, the light scattering efficiency of polysaccharide molecules in the protein-polysaccharide mixture was lower than that of protein molecules. Therefore, the overall turbidity of the system decreased significantly. This large difference indicates that the presence of excess PGA affects the aggregation of the resulting aggregates, suggesting that increasing the overall polysaccharide concentration can inhibit further protein aggregation.

[0052] Microstructure of WNPI-PGA composite aggregates: Figure 1 The microstructures of WNPI-PGA composite aggregates prepared under various conditions are shown. It is well known that natural WNPI surfaces exhibit a complex structure composed of random flakes of various sizes and shapes. This flake-like structure is attributed to the high-density binding of protein molecules, which affects their dispersibility and solubility. PGA, a high-molecular-weight polysaccharide, exhibits a filamentous structure with a smooth surface. Figure 1 AC revealed differences in the microstructure of the aggregates under different pH conditions. At pH 6.0, the morphology exhibited a dense, sheet-like structure. As the pH decreased, a porous, sheet-like structure emerged. Notably, the surface was rougher at pH 3.5. This result further demonstrates that the strength of electrostatic interactions varies under different pH conditions; at a lower pH (2.5), electrostatic interactions are stronger, and the aggregated layer exhibits a denser network structure. Therefore, the macromolecules WNPI and PGA form an aggregated network due to electrostatic interactions, complicating the cohesive structure. With increasing salt ion concentration, the surface of the sheet-like structure gradually became rougher. Particularly when the salt ion concentration increased to 20 mM, the structure exhibited an irregular, rough sheet-like appearance. This phenomenon is attributed to the strong electrostatic shielding effect at high salt concentrations, which inhibits protein aggregation and disrupts the aggregate structure. When the mass ratio of protein to polysaccharide is 1:1 and 1:2, the surface roughness increases significantly, while the surface is relatively smooth when the ratio is 2:1. This indicates that when the ratio of WNPI to PGA is 2:1, the saturation of protein and polysaccharide is high and the interaction strength is the greatest, thus forming a tighter and denser network structure.

[0053] In summary, the preferred method for preparing WNPI-PGA composite coagulants is as follows: Stock solutions of WNPI and PGA are mixed at a 2:1 ratio and gently stirred on a magnetic stirrer for 120 minutes to adjust the total biopolymer concentration of the solution to 0.6% (w / v). Subsequently, the mixed solution is acidified with 1 mol / L hydrochloric acid to adjust the pH to 3.5. After acidification, the sample is allowed to stand at room temperature for two days to promote phase separation. The enriched phase at the bottom is then collected for later use. The enriched phase is lyophilized using a vacuum freeze-drying method to obtain the dried coagulant.

[0054] Preparation of CA-loaded WNPI / PGA composite nanoparticles

[0055] Mix WNPI and PGA stock solutions in a 2:1 ratio and gently stir on a magnetic stirrer for 30-120 minutes to ensure the total concentration of biopolymer reaches 0.6% (w / v) to prepare WNPI / PGA composite coagulants.

[0056] First, accurately weigh a certain amount of CA powder and add it to deionized water. Stir at 300 rpm in the dark for 1 hour at room temperature to prepare a CA stock solution (0.5%, w / v). Slowly add this CA stock solution to the WNPI-PGA composite coagulant solution, adjusting the final CA concentration to 0.05%, 0.10%, 0.15%, and 0.20% (w / v). Adjust the pH of the mixed solution to 3.5 using 1 mol / L hydrochloric acid. After stirring in the dark for 30 minutes, centrifuge the sample at low speed (3000 rpm, 10 minutes) to remove any large aggregates and undissolved matter, obtaining a CA-containing WNPI-PGA composite coagulant (WNPI / PGA-CA). Part of the composite coagulant sample was stored at 4℃, while the other part was freeze-dried to prepare solid freeze-dried nanoparticles. Therefore, the final nanoparticle sample had a WNPI / PGA:CA mass ratio of WNPI / PGA to CA. 12:1 WNPI / PGA:CA 6:1 WNPI / PGA:CA 4:1 and WNPI / PGA:CA 3:1 .

[0057] WNPI / PGA:CA 12:1 WNPI / PGA:CA 6:1 WNPI / PGA:CA 4:1 and WNPI / PGA:CA 3:1 The average diameter, polydispersity index (PDI), and zeta potential were determined using dynamic light scattering electrophoresis at 25 °C. Prior to measurement, all samples were diluted with deionized water to a total biopolymer concentration of 0.1 wt%.

[0058] Characterization of WNPI-PGA condensate self-assembly of embedded chlorogenic acid nanoparticles

[0059] Particle size and potential analysis of WNPI / PGA-CA nanoparticles

[0060] Table 1. Average particle size, Zeta potential, and polydispersity index of nanoparticles loaded with different concentrations of CA (P<0.05)

[0061]

[0062] Table 1 shows that the size of the nanoparticles is moderate, and tends to increase with the increasing content of chlorogenic acid. This may be related to the formation and dissociation of the WNPI / PGA electrostatic complex. A potential reason for the increased particle size is that peptides and amino acids released mainly through hydrophobic interactions during hydrolysis, accompanied by electrostatic attraction, electrostatic repulsion, and the exchange of thiol and disulfide bonds, promote protein aggregation. PDI is an indicator for evaluating the dispersibility of polymers. PDI is generally between 0 and 1, with PDI < 0.3 indicating monodispersity and PDI > 0.3 indicating high polydispersity. As shown in Table 1, the nanoparticles produced in this invention are all uniform. Especially the WNPI / PGA:CA... 4:1 The nanoparticles had a PDI value of 0.26 ± 0.02, lower than the other three groups. Zeta potential is one of the key parameters for evaluating the stability of nanoparticles. A higher absolute value of the Zeta potential indicates greater nanoparticle stability. As shown in Table 1, the Zeta potential of all nanoparticles was below -19.56 mV, with WNPI / PGA:CA... 6:1 and WNPI / PGA:CA 4:1 The zeta potential of this group of nanoparticles was lower than that of the other two groups, indicating higher stability. These results suggest that the addition of appropriate amounts of CA can transform large plant protein aggregates into stable nanoscale particles.

[0063] Water contact angle of WNPI / PGA-CA nanoparticles: In this embodiment, the water contact angle (WCA) of different nanoparticles was measured to determine their surface hydrophobicity / hydrophilicity. When the contact angle is close to 90°, the particles have the same affinity for both the oil and water phases and tend to adsorb strongly at the oil / water interface. When θ < 90°, the particles are mainly hydrophilic, and when θ > 90°, the particles are mainly hydrophobic. Before encapsulation, the contact angle of WNPI was 98.4°. After encapsulation, the contact angle of WNPI / PGA-CA nanoparticles first decreased and then increased. The smallest contact angle, 33.0°, was observed when the ratio of WNPI / PGA:CA was 4:1. Figure 2 The presence of chlorogenic acid inhibits the surface hydrophobicity of proteins, a result indicating that WNPI / PGA-CA nanoparticles possess good hydrophilicity.

[0064] EE and LC of WNPI / PGA-CA nanoparticles for chlorogenic acid: EE (encapsulation efficiency) and LC (loading capacity) are important indicators for evaluating the bioactivity loading performance of a carrier. The EE and LC of WNPI-PGA encapsulating different concentrations of CA are shown below. Figure 3As shown, a high energy efficiency (EE) was observed when the ratio of CA (chlorogenic acid) to WNPI / PGA was 4:1. With increasing concentration, the EE of the nanoparticles initially increased and then decreased. This is because excessive CA causes protein aggregation, preventing successful entry into the hydrophobic region of WNPI and thus preventing encapsulation by WNPI-PGA. CA is primarily loaded into WNPI-PGA through hydrophobic interactions. The loading capacity (LC) of CA increases with increasing concentration, indicating that the water solubility of the natural pigment is effectively improved.

[0065] Thermal stability of WNPI / PGA-CA nanoparticles: The average particle size and PDI value of four types of nanoparticles treated at different temperatures, as well as the retention rate of chlorogenic acid, showed specific changes. With increasing temperature, the particle size of all nanoparticles first increased and then decreased, with WNPI / PGA-CA showing the most significant changes. 12:1 The particle size of WNPI was consistently smaller than that of the other three groups. At 95℃, the particle size of all groups decreased significantly. This is because WNPI denatures at high temperatures and its tertiary structure dissociates, causing internal nonpolar amino acids to migrate to the protein surface. This promotes hydrophobic interactions and disulfide bond formation between WNPI molecules, ultimately leading to aggregation. The PDI values ​​of the four nanoparticles change with temperature, WNPI / PGA-CA 12:1 The PDI value increases significantly with temperature, while WNPI-PGA-CA 4:1 The PDI value of WNPI-PGA-CA showed little change, consistently remaining below 0.3. With increasing temperature, the chlorogenic acid retention rate decreased for all nanoparticles, but not for WNPI-PGA-CA. 4:1 More chlorogenic acid was retained at the same temperature, likely due to their smaller particle size and more uniform distribution, which helps maintain the chlorogenic acid content. These results suggest that the optimal storage method for nanoparticles to maintain system stability is under refrigeration or after high-temperature treatment.

[0066] Freeze-thaw stability of WNPI / PGA-CA nanoparticles: Effects of freeze-thaw cycles on the average particle size, PDI, and chlorogenic acid retention of four nanoparticle types. After freezing, the average particle size and PDI of all four nanoparticle types increased slightly. In particular, WNPI / PGA:CA... 6:1 The average particle size changed significantly before and after freeze-thaw, increasing from 550.22±8.774 nm to 951.82±6.1962 nm. Although the PDI value increased, the change was not significant, indicating that freeze-thaw had little effect on the dispersibility of nanoparticles. WNPI / PGA:CA 12:1 The PDI value remained below 0.3, indicating that it maintained good dispersibility after freeze-thaw cycles. Freeze-thaw cycles significantly affected the retention rate of chlorogenic acid, particularly the WNPI / PGA:CA ratio. 3:1The retention rate of WNPI (79.6 ± 1.1%) was significantly higher than that of the other three groups. This may be because the relatively loose structure of WNPI makes it easier for ice crystals formed during freezing to disrupt its structure, increasing the instability of the nanoparticles. However, the increase in CA enhances the interaction strength between CA and WNPI, forming a denser shell structure and reducing the adverse effects of ice crystals. This indicates that WNPI / PGA:CA 4:1 Its good stability under freeze-thaw conditions enhances its potential for use in frozen foods.

[0067] pH Stability of WNPI / PGA-CA Nanoparticles: The properties of nanoparticles are highly pH-dependent. At pH 4, the widespread aggregation of WNPI nanoparticles indicates their instability, with a significantly increased average size. Previous studies have shown that WNPI is unstable at pH 4, leading to precipitation, but the composite nanoparticles remain stable despite increased particle size at pH 4. This phenomenon suggests that the presence of WNPI / PGA contributes to a more compact structure in the composite nanoparticles, resisting particle aggregation at pH 4. Different pH values ​​alter the magnitude of electrostatic interactions, affecting the physical stability of the composite nanoparticles. At pH 2, the PDI of the composite nanoparticles is close to 0.25, indicating excellent anti-aggregation stability in a pH range far from the isoelectric point, likely due to enhanced electrostatic repulsion between particles. Above pH 4, the PDI increases significantly, indicating poorer dispersibility of nanoparticles in alkaline environments. The chlorogenic acid retention rate of the three nanoparticles reaches its highest at pH 6, but decreases in more alkaline environments. This is because chlorogenic acid is more stable under acidic conditions. When the pH value drops below 4, approaching the isoelectric point of walnut protein isolate, the stability of the nanoparticles is compromised, leading to the exposure of chlorogenic acid and a significant reduction in its content. Analytical data indicates that WNPI / PGA:CA 4:1 Its stability is better maintained when used in a weakly acidic environment.

[0068] Temperature storage stability of WNPI / PGA-CA nanoparticles: After 30 days of refrigeration (4℃), WNPI / PGA:CA 12:1 The average particle size increased from 213.52±6.61 nm to 775.73±4.68 nm, while WNPI / PGA:CA 4:1 The particle size increased from 469.13±7.81 nm to 783.41±8.81 nm. Furthermore, WNPI / PGA:CA 4:1 The PDI value (0.348) is lower than that of WNPI / PGA:CA. 12:1 The smaller value (0.394) indicates that the former has a more uniform and stable particle distribution. Under the same refrigeration conditions, after 30 days, WNPI / PGA:CA 4:1The chlorogenic acid retention rate reached 71.21±0.2%, which was significantly higher than that of WNPI / PGA:CA. 12:1 The percentage was 61.17 ± 1.82%. After being stored at room temperature (25℃) for 30 days, the WNPI / PGA:CA ratio was... 12:1 The particle size increased from 213.52±6.61 nm to 827.32±1.15 nm, while WNPI / PGA:CA 4:1 The particle size increased from 284.1±7.3 nm to 823.58±110.37 nm. At this point, WNPI / PGA:CA 12:1 The PDI value rose to 0.384, while WNPI / PGA:CA 4:1 Maintaining a lower value of 0.134 indicates that the latter has better stability at room temperature. Furthermore, after 30 days, WNPI / PGA:CA 4:1 It retained 67.98±0.50% chlorogenic acid, which is higher than WNPI / PGA:CA. 12:1 54.87±1.7%. These results indicate that, regardless of refrigeration or ambient temperature conditions, WNPI / PGA:CA 4:1 It exhibits superior stability and higher chlorogenic acid retention, demonstrating that low-temperature storage may help extend the service life of nanoparticles.

[0069] Photostress stability of WNPI / PGA-CA nanoparticles: WNPI / PGA:CA after illumination 12:1 WNPI / PGA:CA 6:1 WNPI / PGA:CA 4:1 and WNPI / PGA:CA 3:1 The chlorogenic acid retention rates of the nanoparticles were 60.4%, 65.21%, 81.53%, and 72.43%, respectively. The results indicate that WNPI / PGA:CA... 4:1 It exhibits better photostability because the protein shell encapsulating CA acts as a physical barrier to light. Secondly, the aromatic amino acid residues and double bond functional groups of walnut isolate can absorb light.

[0070] Preparation of PVA-WNPI / PGA-CA composite membrane

[0071] Polyvinyl alcohol (PVA) powder was dissolved in deionized water and heated and stirred at 50°C for 240 min to obtain a 5% (w / v) PVA solution. Based on previous research, WNPI / PGA:CA was selected. 4:1 Nanoparticles are used as the membrane matrix. The formulation of the film-forming solution is shown in Table 2.

[0072] Table 2 Formulation of PVA-WNPI / PGA-CA nanoparticle film-forming solution

[0073]

[0074] Note: After mixing PVA, WNPI / PGA-CA, and CA, add ultrapure water to bring the volume to 100mL, then add Tween and glycerin.

[0075] Glycerin was added as a plasticizer and Tween-80 as an emulsifier. All film-forming solutions were stirred at 500 rpm at room temperature (10-25℃) for 60 min. The prepared film-forming solution was then placed in a 4℃ refrigerator overnight to defoam and remove any residual air. 15 mL of the film solution was spread evenly on a 9 cm diameter plastic petri dish. To maintain film uniformity, the surface area, volume, and drying environment remained constant. All films were dried in an incubator at 37℃ and 50±2% relative humidity for 10 h. After cooling, the films were peeled off and placed in a desiccator (relative humidity 53%) at room temperature for at least equilibration for at least 12 h to obtain the walnut protein antibacterial biofilm.

[0076] Example 2: Structural Characterization of PVA-WNPI / PGA-CA Thin Films

[0077] Microstructure of thin films: SEM images of the cross-sectional areas of pure PVA film, PVA-CA film, and different types of PVA-WNPI / PGA-CA film, as shown in the figure. Figure 4 As shown, all films except PVA-CA exhibit discontinuities in the form of micropores or cavities. The presence of these micropores may be related to the evaporation of glycerol during film formation (drying). Furthermore, with increasing concentrations of bioactive compounds, the addition of certain amounts of glycerol and Tween to the films leads to the formation of pores or voids, resulting in an amorphous structure with vacuoles and voids distributed along the fracture surfaces. This may correspond to oil droplets and, to some extent, disrupts the continuous structure of the membrane matrix.

[0078] Mechanical strength of the thin film: The mechanical properties of the thin film are shown in [link to relevant documentation]. Figure 5 Pure PVA films exhibit high elongation at break (257.78%) and low tensile strength (37.25 MPa), demonstrating typical ductile behavior. With the addition of CA, the tensile strength of the PVA-CA film increased to 89.42 MPa, while the elongation at break decreased to 103.96%. This is likely because the addition of CA improved the rigidity and brittleness of the coaxial nanofiber film. The interaction between phenolic resin and protein reduced the effect of plasticizers on the film, thereby increasing its rigidity. However, after adding WNPI / PGA-CA nanoparticles, the tensile strength of the film gradually decreased, while the elongation at break significantly increased, remaining at around 225.39%. This is because hydrogen bonds are formed between the carboxyl groups of WNPI and the hydroxyl groups of CA, enhancing the interaction between walnut protein isolate molecules. These interactions contribute to the formation of a denser structure, thereby improving the tensile strength of the nanofiber film.

[0079] FTIR analysis of thin films: Fourier transform infrared spectroscopy was used to understand intermolecular interactions and structural changes in the films at the molecular level. FTIR spectra of PVA films with added CA and those with added WNPI / PGA-CA are presented. The characteristic peak of the PVA film is approximately 3281 cm⁻¹. -1 (OH stretching), 2928cm -1 (CH stretching), 1029cm -1 (CO stretching). All developing films showed similar peaks with different amplitudes. No additional peaks were observed in the bioactive blend film, confirming that no new covalent bonds were formed between the control film and the blend film. The first peak of all films fell within the range of 3282–3298 cm⁻¹. -1 Within this range, it exhibits OH tensile vibration. The pure PVA film has the highest wavenumber, at 3298 cm⁻¹. -1 The fewest hydrogen bonds. PVA-WNPI / PGA-CA II and PVA-CA have the lowest wavenumbers (3282 cm⁻¹). -1 The intermembranous hydrogen bonding interaction is strongest, likely due to the increase in free OH groups, which cannot interact with the polymer matrix. The second peak is located at 2914–2926 cm⁻¹. -1 The interval between 1730 and 1720 cm represents the carbon-hydrogen vibration. -1 The peak indicates C=O stretching. There is no significant difference in C=O vibrations between different films. The characteristic peak is at 1085 cm⁻¹. -1 , representing the tensile vibration of CO. PVA characteristic peaks were observed in all sample films, indicating that the addition of WNPI / PGA-CA nanoparticles to the PVA matrix did not alter the molecular structure of PVA.

[0080] XRD analysis is commonly used to identify the crystal structure of thin films. The PVA film shows a strong peak at 2θ = 19.3° (indicating a crystalline region), while two weaker diffraction peaks (representing amorphous regions) are observed at 2θ = 11.3° and 40.6°, indicating that PVA exhibits semi-crystalline characteristics. After adding CA, the intensity of the diffraction peak at 2θ = 19.7° further increases, due to the crystalline nature of CA, indicating that the blend material has stronger amorphous properties. When PVA is combined with WNPI / PGA-CA nanoparticles, the intensity of the diffraction peak at 2θ = 19.3° of the composite film weakens. The intensity of the diffraction peak can reflect the morphology of the crystal grains in the crystalline region; generally, the larger the grains, the higher the intensity of the diffraction peak. Therefore, the addition of nanoparticles reduces the crystallinity of the film matrix, which is attributed to the hydrogen bonds formed between PVA and WNPI / PGA-CA nanoparticles disrupting the original arrangement of PVA. Strong intermolecular and intramolecular hydrogen bonds exist between PVA and CA.

[0081] Rheological analysis of membranes: Viscosity changes can reflect the effect of shearing on the intermolecular entanglement structure; medium-viscosity film-forming solutions are considered ideal because lower or higher viscosities may lead to the formation of inhomogeneous films. The PGA molecule exhibits a certain viscosity due to the network structure formed by numerous hydroxyl and amino groups; the shear-thinning characteristic is mainly due to the disruption of this network structure by rapid shearing processes. Figure 6 As shown, the addition of WNPI / PGA-CA to the solution resulted in an increase in solution viscosity, indicating that the polymer network in the solution was strengthened. This strengthening is mainly due to the electrostatic interactions and hydrogen bonding between WNPI, PGA, and CA. With increasing WNPI / PGA-CA concentration, its interaction with PVA became more significant, leading to a further increase in the viscosity of the film-forming solution. Viscosity analysis results indicate that the electrostatic interactions and hydrogen bonding between the PVA film matrix and WNPI / PGA-CA enhanced the polymer network structure in the film-forming solution.

[0082] Water vapor and oxygen barrier properties of the membrane: With the addition of WNPI / PGA-CA, the water vapor permeability (WVP) increased from 0.46 × 10⁻⁶ for the PVA membrane. –6 The g / m·s·Pa value decreased to 0.38 × 10⁻⁶ for the PVA-WNPI / PGA-CA II membrane. –6 The oxygen permeability (OP) of the PVA membrane is 1.11 × 10⁻⁶ g / m·s·Pa. –2 The g / m·s decreased to 0.79 × 10⁻⁶ for the PVA-WNPI / PGA-CA I membrane. –2 g / m·s. First, the reduction in WVP and OP is related to the spatial structure of the membrane. WNPI / PGA-CA dispersed in the membrane matrix and the hydrogen bonds formed with PVA reduce the availability of hydrophilic groups, thus limiting the membrane's water absorption and oxygen uptake capacity. Simultaneously, water molecules pass through the membrane in three stages: adsorption, diffusion, and decomposition. This means that the membrane's hydrophilicity also has a significant impact on WVP.

[0083] The apparent morphology of the thin film: such as Figure 7As shown, all films possess a certain degree of transparency and a smooth surface, free of dents, pores, wrinkles, bubbles, or cracks. The PVA film itself is nearly colorless and transparent. With the gradual addition of WNPI / PGA-CA, the film exhibits a yellowish-brown hue, primarily due to the inherent color of WNPI. The depth of the yellowish-brown gradually increases with the increase in the WNPI / PGA-CA ratio. However, when the WNPI / PGA-CA addition ratio is high, the film partially obscures the leaf patterns, slightly affecting the visual observation of the food inside the packaging. Table 3 lists the chromaticity values, total color difference (ΔE), and thickness data for each group of films. Compared to pure PVA film, the PVA film with added WNPI / PGA-CA exhibits higher L and b values, indicating a gradual decrease in film transparency and a more yellowish trend, consistent with the appearance changes of the composite film.

[0084] Table 3. Thickness and color parameters of the film

[0085]

[0086] Film thickness is an important parameter of a film, as it is related to its mechanical properties, air permeability, and light transmittance. Table 3 shows that the thickness range of all films is 0.100-0.167 mm. Compared to PVA films, the film thickness significantly increases with increasing amounts of CA and WNPI / PGA-CA (P<0.05).

[0087] like Figure 8 As shown, all films exhibited a high transmittance pattern, but significant differences existed in their transparency values. Among the films, the PVA-WNPI / PGA-CA II hybrid film had the lowest transparency, while the pure PVA film exhibited the highest UV transmittance. The transmittance of the film gradually decreased as the WNPI / PGA-CA content increased. The PVA-WNPI / PGA-CA II film showed almost zero UV transmittance in the 200nm-390nm range. The results indicate that the PVA-WNPI / PGA-CA II film exhibits strong UV and visible light blocking properties, which helps prevent oxidation and spoilage of packaged food caused by light exposure.

[0088] Membrane morphology: Pure PVA membranes exhibit low antioxidant activity; at a membrane concentration of 12.5 mg / mL, their scavenging efficiencies for DPPH and ABTS free radicals are only 3.59% and 2.72%, respectively (see [link to article]). Figure 9With increasing WNPI / PGA-CA content, the free radical scavenging capacity and antioxidant properties of the membrane were significantly enhanced. This is attributed to the increased antioxidant content and the expanded interaction area between the reagent and the membrane, demonstrating that the antioxidant capacity of all membrane samples exhibited a dose-related enhancement effect. The results indicate that in PVA-WNPI / PGA-CA films, the excellent antioxidant capacity mainly originates from the antioxidant activity of CA, which can scavenge free radicals through its aromatic structure and single-electron donor.

[0089] Antimicrobial activity analysis of the film: Figure 10 The antibacterial properties of five groups of films against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria were demonstrated. The absorbance of bacterial suspensions of Staphylococcus aureus and Escherichia coli in PVA films without CA loading increased, indicating that PVA had no antibacterial activity. When CA and WNPI / PGA-CA nanoparticles were added to the PVA films, the absorbance of the bacterial suspensions decreased to varying degrees, indicating that CA loading had an inhibitory effect on Staphylococcus aureus and Escherichia coli. However, the antibacterial activity of all CA-loaded films against Staphylococcus aureus and Escherichia coli showed little difference. The PVA-WNPI / PGA-CA II film exhibited slightly greater antibacterial activity than the other groups. The antibacterial activity of the films was related to the CA content and the distance of CA from the fiber interior to the surface. The results indicate that loading WNPI / PGA-CA can give PVA-based films a certain inhibitory effect against Staphylococcus aureus and Escherichia coli, making it a potential active food packaging material.

[0090] Example 3: Application of PVA-WNPI / PGA-CA composite film in refrigerating fresh pork

[0091] Preparation of pork samples: Fresh pork was purchased from the local market (Kunming, Yunnan, China). The hind leg meat was removed, tendons were removed, and the meat was cut into equal-sized pieces (3×3×1.0cm). The pieces were pre-cooled, rinsed with deionized water, and dried. The pork pieces were randomly divided into 5 groups and placed in sterile petri dishes (90mm×90mm). The dishes were sealed with PVA film, PVA-CA film, PVA-WNPI / PGA-CA I film, PVA-WNPI / PGA-CA II film, and PVA-WNPI / PGA-CA III film, respectively. The sealed samples were then placed on trays and stored in a 4℃ constant temperature refrigerator. Samples were randomly selected from each group on days 0, 2, 4, 6, and 8 of the storage period for quality evaluation.

[0092] The Influence of Films on Pork Color: Color is one of the most important quality parameters determining the quality of fresh meat. Therefore, measuring color changes during storage is a crucial step. In this invention, changes in brightness, chromaticity, and hue are monitored using L*, a*, and b* values. Chromaticity is an important parameter for measuring the color of fresh meat. It measures the color saturation of pork samples; a higher a* value indicates fresher pork, while a higher b* value indicates spoilage. During storage, the surface color of pork changed. The brightness of all samples decreased, but the pure PVA film showed the largest decrease, indicating a decline in the gloss of the pork after storage. Furthermore, the oxidation of myoglobin to oxymyoglobin is the main factor leading to the decrease in a*. In the mid-to-late storage period (4-8 days), the a* of PVA-WNPI / PGA-CA II was significantly higher than that of other groups (P<0.05), indicating that WNPI / PGA-CA inhibited myoglobin oxidation, which may be related to the antioxidant properties of WNPI / PGA-CA. Changes in b* values ​​are related to lipid oxidation. The b* value of PVA-WNPI / PGA-CA II was lower than that of the other groups, possibly because the WNPI / PGA-CA nanoparticles inhibited lipid oxidation in muscle. This indicates that the addition of WNPI / PGA-CA has a certain effect on delaying the color change of pork. Based on all the results, the spoilage rate of pork in the PVA-WNPI / PGA-CA II group was significantly lower than that of the other two groups; therefore, PVA-WNPI / PGA-CA II has the strongest effect on delaying the preservation of pork.

[0093] The effect of the membrane on the pH of pork: pH changes in all samples are as follows Figure 11 As shown in the figure, the pH value of fresh pork before storage was 7.25. During storage, the pH values ​​of all treatment groups showed the same trend, initially decreasing and then increasing. The initial decrease in pH was related to lactic acid and phosphoric acid produced by anaerobic glycolysis and ATP degradation, while the later increase in pH was due to the accumulation of alkaline compounds. In the early stage of storage (0-2 days), the decrease in pH value may be due to the decomposition of glycogen in fresh pork under anaerobic conditions, producing acids such as lactic acid. During the storage period of 4-8 days, the proteins in the pork may be metabolized and degraded into alkaline nitrogenous molecules by microorganisms and proteases, leading to an increase in pH value. In addition, during 4-8 days, the pH value of the group loaded with CA was lower than that of the other two groups, indicating that the CA-containing membrane prevented the deterioration of fresh pork quality to some extent. However, since pH value is affected by microbial metabolites (such as lactic acid), pH value is a reliable indicator of changes in pork quality.

[0094] The effect of film on TVB-N (volatile basic nitrogen) in pork: TVB-N refers to ammonia and amino nitrogen substances produced by protein decomposition in animal meat during storage, under the combined action of endogenous enzymes and bacteria. It is one of the important indicators for measuring the degree of spoilage of meat products. The change of TVB-N value in pork with prolonged storage time is shown below. Figure 12 As shown, the initial TVB-N value of fresh pork was 5.27 ± 0.067 mg / 100g. On day 8, the TVB-N value of the PVA group reached its maximum at 23.64 ± 0.643 mg / 100g, while the TVB-N value of the PVA-WNP / PGA-CA II film group on day 8 was the lowest compared to the other five groups, at 14.58 ± 0.087 mg / 100g. With prolonged storage, the TVB-N value increased to varying degrees, indicating a decrease in pork muscle quality. On day 8 of storage, the TVB-N of pork stored with the pure PVA film exceeded the limit of 20 mg / 100g specified in GB 2733-2015. This indicates that WNPI / PGA-CA nanoparticles effectively extended the acceptable freshness period of fresh pork and inhibited the accumulation of TVB-N, which was reflected in the increase in pH value during the later stages of storage. These results may be due to CA limiting protein breakdown and microbial growth, similar to the effects of nanoemulsion-based edible coatings containing fennel oil used to preserve pork patties.

[0095] The Influence of Membranes on Pork Texture: The texture of pork products significantly impacts consumer acceptance. Maintaining tenderness is crucial during refrigerated pork. Furthermore, textural characteristics are important indicators of pork quality. This invention measured the hardness, elasticity, resilience, chewiness, cohesiveness, and adhesiveness of various groups of pork during storage. With prolonged storage, the texture of all groups of pork showed a decreasing trend. This is due to the oxidation of pork tenderloin proteins, leading to intramolecular / intermolecular protein cross-linking via disulfide and non-disulfide bonds, resulting in protein aggregation and the formation of complexes. Microbial activity promotes this process. Pure PVA membranes showed the largest variation and severe textural degradation, while PVA-WNP / PGA-CA II membranes may have mitigated this degradation by inhibiting microbial activity during storage. This is mainly because PVA-WNP / PGA-CA II membranes exhibit superior moisture barrier properties and stronger antioxidant activity compared to pure PVA membranes, thus more effectively reducing pork weight loss and preventing protein oxidation.

[0096] Effects of membranes on pork microorganisms: All sample groups, including PVA, PVA-CA, PVA-WNPI / PGA-CA I, PVA-WNPI / PGA-CA II, and PVA-WNPI / PGA-CA I membranes, were stored at 4℃. Figure 13Bacterial colonies were collected from pork samples from each group and observed using the plate counting method. Although the types and quantities of bacterial colonies were not studied, the density of bacterial colonies on agar plates can indicate the ability of the membrane sample to inhibit microbial growth. On day 8, both the PVA and PVA-WNPI / PGA-CA membrane groups had a large number of colonies. The number of colonies in pork packaged with PVA membrane was higher than that in the control group. The PVA-WNPI / PGA-CA II film showed a significantly greater inhibitory effect on microbial growth in chilled pork than the pure PVA membrane group, which is closely related to the antibacterial ability of CA. After 6 days of storage, the TVC of PVA reached 7.61 log 10 CFU / g, exceeding the 7 log [restriction] recommended by the International Committee for Microbiological Standards in Foods. 10 CFU / g. This indicates that the addition of WNPI / PGA-CA nanoparticles effectively inhibits the growth of microorganisms in pork, while the PVA-WNPI / PGA-CA II film can prolong the time for microbial growth in pork (at least 8 days).

[0097] The PVA-WNPI / PGA-CA II membrane exhibits broad-spectrum antibacterial activity, which is mainly due to the antibacterial activity of WNPI / PGA-CA.

[0098] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any modifications, equivalent substitutions, or improvements made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a walnut protein isolate antibacterial biofilm, characterized in that, The preparation method includes the following steps: Step (1) Mix WNPI stock solution and PGA stock solution and stir to prepare WNPI / PGA composite condensate; the mass ratio of WNPI and PGA is 2:1, and the total concentration of the two in the reaction system reaches 0.4-1.0% (w / v); Step (2) Slowly add the chlorogenic acid (CA) stock solution to the WNPI-PGA composite coagulant solution obtained in step (1), wherein the mass ratio of WNPI / PGA composite coagulant to CA is 4:1; adjust the pH of the mixed solution to 3.0-3.5 using hydrochloric acid, stir in the dark for 20-60 min, and then centrifuge at 2500-4000 rpm for 8-20 min to remove large aggregates and undissolved matter, thereby obtaining the WNPI / PGA-CA composite coagulant; The concentration of CA in the CA stock solution is 0.3-1.0% (w / v); the CA stock solution is slowly added to the WNPI-PGA composite coagulant solution to adjust the final concentration of CA to 0.05%-0.20% (w / v); Step (3) PVA solution and WNPI / PGA-CA composite coagulant are mixed and stirred, refrigerated overnight, then dried and cooled, and then equilibrated in a desiccator to obtain walnut protein isolate antibacterial biofilm; In step (3), glycerol and Tween-80 are also added to the reaction system; the concentration of glycerol in the reaction system is 0.5-1.0%; the concentration of Tween-80 in the reaction system is 0.05-0.2%; the concentration of the PVA solution is 3-10% (w / v); the mass ratio of PVA, WNPI / PGA-CA, glycerol and Tween-80 is 3-10:0.3-1.0:0.5-1.0:0.05-0.

2.

2. The preparation method according to claim 1, characterized in that, In steps (1) and (2), the pH of the reaction system is adjusted to 3.

5.

3. The preparation method according to claim 1, characterized in that, The concentration of PVA in step (3) is 5% (w / v).

4. The preparation method according to claim 1, characterized in that, The mass ratio of PVA, WNPI / PGA-CA, glycerol and Tween-80 in step (3) is 5:0.5:0.75:0.

1.

5. The application of a walnut protein isolate antibacterial biofilm prepared by the method of any one of claims 1-4 in the cold storage and preservation packaging of fresh meat products.

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