A core-shell structure protein-nano zinc oxide antibacterial colloidal spray, a preparation method thereof and food preservation application

Abstract

This invention belongs to the field of nanomaterials technology and discloses a core-shell structured protein-nano zinc oxide antibacterial colloidal spray, its preparation method, and its application in food preservation. This invention discloses a wheat gliadin-nano zinc oxide colloid, which includes a nano-zinc oxide core and a wheat gliadin coating layer covering the surface of the nano-zinc oxide core. Unlike existing protein-zinc oxide composite powder materials, the wheat gliadin-nano zinc oxide colloid provided by this invention is a liquid-stable, dispersed nanocolloid system that can be directly used as a spray for food preservation. Its core-shell structure can effectively regulate Zn... 2+ The slow-release behavior of the nano zinc oxide is improved by reducing its potential cytotoxicity through a protein shell, thereby maintaining high antibacterial performance while improving biocompatibility and safety.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a core-shell structural protein-nano zinc oxide antibacterial colloidal spray, its preparation method, and its application in food preservation. Background Technology

[0002] With the increasing prominence of food safety issues, developing efficient, safe, and environmentally friendly antimicrobial preservation technologies has become an important research direction in the field of food science. Nano-zinc oxide, due to its broad-spectrum antimicrobial activity and extremely low risk of drug resistance, is widely used in food preservation and food contact materials. However, traditional nano-zinc oxide still suffers from problems such as easy aggregation, poor dispersibility, and uncontrollable activity release in practical applications, thus limiting its effectiveness in food systems.

[0003] In recent years, researchers have attempted to improve the performance of nano-zinc oxide by constructing composite systems. On the one hand, nano-zinc oxide composite materials based on biomolecular templates have gradually attracted attention, such as using proteins to regulate the formation of zinc oxide. However, such technical solutions mainly focus on solid powder material systems, which have poor dispersion stability in aqueous environments and are difficult to control in terms of active release behavior.

[0004] On the other hand, functional membrane materials containing nano-zinc oxide (such as antibacterial packaging films or edible coating films) have also been extensively studied. By introducing nano-zinc oxide into polysaccharide or protein matrices to form thin film structures, the antibacterial and barrier properties can be improved. However, such membrane materials usually require pre-forming or coating processes, which are relatively complex to prepare, difficult to scale up, and have low flexibility in use. Furthermore, the dispersion state and release behavior of nanoparticles in the membrane matrix are not easy to control precisely.

[0005] In addition, recent studies have reported on liquid-phase-based zinc oxide nanocomposites, such as nanocarrier systems for drug delivery or tumor therapy. These systems achieve stability and release behavior regulation of zinc oxide nanoparticles in specific physiological environments by constructing pH-responsive or biomacromolecule-encapsulated structures. These systems are typically targeted at biomedical applications, emphasizing responsiveness to specific environments and functional design. Their preparation processes often involve multiple reactions or functional modifications, resulting in relatively high raw material and process costs.

[0006] In contrast, gliadin derived from natural grains (such as wheat gliadin) is widely available, inexpensive, and biocompatible. Using it to construct nano-zinc oxide composite systems could potentially simplify the preparation process and reduce costs while maintaining antibacterial properties. However, current technologies lack methods for constructing nano-zinc oxide systems with controllable structure, stable dispersion, and suitability for direct food applications using these low-cost proteins.

[0007] Therefore, existing antimicrobial systems based on nano-zinc oxide, whether solid powders, membrane materials, or biomedical liquid systems, struggle to simultaneously meet requirements such as dispersion stability, potent antimicrobial activity, high biocompatibility, ease of use, and cost control. There is an urgent need to develop an antimicrobial system with controllable structure, stable dispersion, a mild preparation process, and suitability for direct application on food surfaces, in order to achieve efficient, safe, and environmentally friendly applications of nano-zinc oxide in food preservation. Summary of the Invention

[0008] This invention aims to solve at least one of the technical problems existing in the prior art. It provides a method for preparing a highly biocompatible nano-zinc oxide antibacterial colloid using a gliadin template in a green synthesis process. The resulting product exhibits good colloidal stability, pH adaptability, and broad-spectrum antibacterial activity, and can be directly sprayed for surface preservation treatment of fruits, vegetables, and baked goods.

[0009] The first objective of this invention is to provide a wheat gliadin-nano zinc oxide colloid.

[0010] The second objective of this invention is to provide a method for preparing wheat gliadin-nano zinc oxide colloids according to the first aspect of this invention.

[0011] The third aspect of the present invention aims to provide the application of the preparation method of wheat gliadin-nano zinc oxide colloid of the first aspect of the present invention or the preparation method of the second aspect of the present invention.

[0012] The fourth aspect of this invention is to provide a product.

[0013] The fifth aspect of this invention aims to provide a method.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a wheat gliadin-nano zinc oxide colloid, the wheat gliadin-nano zinc oxide colloid comprising a nano zinc oxide core and a wheat gliadin coating layer covering the surface of the nano zinc oxide core.

[0015] In some embodiments of the present invention, the wheat gliadin-nano zinc oxide colloid is spherical with a particle size of 100-800 nm; further, 180-300 nm; and even further, 200-240 nm.

[0016] In some embodiments of the present invention, the wheat gliadin-nano zinc oxide colloid exists in a liquid dispersion form with a polydispersity index (PDI) of less than 0.20.

[0017] The wheat gliadin-nano zinc oxide colloid provided by this invention exhibits excellent broad-spectrum antibacterial activity, with minimum bactericidal concentrations (MBCs) as low as 37.50 μg / mL and 18.75 μg / mL against Escherichia coli and Staphylococcus aureus, respectively. Furthermore, it achieves a clearance rate of over 97% for mature biofilms at MBC concentrations. This wheat gliadin-nano zinc oxide colloid demonstrates high biocompatibility, with a hemolysis rate of less than 5%, and exhibits no significant toxicity to zebrafish embryos, demonstrating good application safety. This wheat gliadin-nano zinc oxide colloid can be used directly as a preservative spray, requiring no film formation or complex processing steps, offering ease of use and suitability for antibacterial preservation treatment of food surfaces. Using strawberries and bread as application examples, after uniformly spraying a solution containing wheat gliadin-nano zinc oxide colloid onto the surface of strawberries, the mold growth time was significantly delayed under room temperature storage conditions, with no significant mold growth observed in the 5×MBC group within 7 days. When sprayed onto the surface of bread, no significant spoilage or deterioration was observed within 15 days of room temperature storage. The above results indicate that the wheat gliadin-nano zinc oxide colloid provided by the present invention can effectively extend the shelf life of food and has broad application prospects in the field of fruit and vegetable and baked food preservation.

[0018] A second aspect of the present invention provides a method for preparing wheat gliadin-nano zinc oxide colloids according to the first aspect of the present invention, comprising the following steps: Wheat gliadin solution is mixed with zinc salt, pH value is adjusted, and antisolvent is added to obtain wheat gliadin-nano zinc oxide colloid.

[0019] Glycol protein, a natural wheat protein, boasts advantages such as wide availability, low cost, edibility, strong amphiphilicity, and richness in functional groups (carbonyl, amino, and hydroxyl groups). It can chelate metal ions and induce in-situ nucleation of ZnO under mild conditions. More importantly, glycol protein undergoes a conformational change (from α-helix to β-sheet) under antisolvent induction, driving the co-assembly of the protein shell and ZnO nanocore to form a stable core-shell colloidal structure. This protein shell can act as a biocompatibility barrier, regulating ZnO formation. 2+ Release kinetics, thereby balancing antibacterial efficacy and biosafety.

[0020] In some embodiments of the present invention, the solvent of the wheat gliadin solution is ethanol, preferably a 60%-80% aqueous ethanol solution.

[0021] In some embodiments of the present invention, the concentration of the wheat gliadin solution is 5-15 mg / mL, such as any value or a range formed by any combination of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg / mL.

[0022] In some embodiments of the present invention, the zinc salt includes a weak acid zinc salt.

[0023] In some embodiments of the present invention, the zinc salt is selected from zinc acetate or zinc carbonate.

[0024] In some embodiments of the present invention, the amount of zinc salt added is 30%-70% of the wheat alcohol-soluble protein content, such as any value or a range formed by any two of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%.

[0025] In some embodiments of the present invention, the pH value is adjusted to 10-13, such as any value of 10, 11, 12 or 13 or a range formed by both.

[0026] In some embodiments of the present invention, zinc ions can be induced to form zinc oxide nanocrystals in situ by adding sodium hydroxide solution to the system to adjust the pH value to 11-12.

[0027] In some embodiments of the present invention, the antisolvent includes a water or ethanol solution (such as a 10% ethanol solution or a 20% ethanol solution).

[0028] In some embodiments of the present invention, the amount of antisolvent added is 1-4 times the total volume of the reaction system after pH adjustment.

[0029] The reaction system is rapidly added to an antisolvent for antisolvent treatment, which can induce protein conformational rearrangement and interfacial self-assembly, thereby forming a coating layer on the surface of zinc oxide nanocrystals, resulting in a core-shell structured wheat gliadin-nano zinc oxide colloid.

[0030] In some embodiments of the present invention, the preparation method further includes a concentration step, wherein the concentration includes rotary evaporation concentration.

[0031] In some embodiments of the present invention, the temperature of the rotary evaporation concentration is 50-60°C, such as any value or a range formed by any combination of 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60°C.

[0032] The preparation method provided by this invention uses alcohol-soluble proteins as structure-directing agents. In a water-alcohol system, zinc ion complexation and base-induced in-situ nucleation are achieved, enabling the controllable generation of zinc oxide nanocrystals under green and mild synthesis conditions. Subsequently, antisolvent-induced protein conformational rearrangement and interfacial self-assembly form dispersed colloidal particles with a stable core-shell structure. This preparation process does not require high temperature, high pressure, or complex equipment conditions; the reaction conditions are mild, and it has good operability and scalability.

[0033] A third aspect of the present invention provides the application of the preparation method of the wheat gliadin-nano zinc oxide colloid of the first aspect of the present invention or the preparation method of the second aspect of the present invention in any one of (1)-(6): (1) Antibacterial; (2) Preparation of antibacterial products; (3) Remove biofilm; (4) Prepare products for removing biofilms; (5) Food preservation; (6) Prepare food preservation products.

[0034] In some embodiments of the present invention, the bacteria described in (1)-(2) include Gram-positive bacteria and / or Gram-negative bacteria.

[0035] In some embodiments of the present invention, the bacteria described in (1)-(2) include Escherichia coli and / or Staphylococcus aureus.

[0036] In some embodiments of the present invention, the biofilms described in (3)-(4) include biofilms formed by Gram-positive bacteria (such as Staphylococcus aureus) and / or Gram-negative bacteria (such as Escherichia coli).

[0037] In some embodiments of the present invention, the food in (5)-(6) includes baked goods (such as bread, baguette, croissant, bagel, mooncake, shortbread, cake, pie, tart, cream puff, biscuit, etc.), fruits (such as grapes, kiwi, strawberry, blueberry, orange, tangerine, grapefruit, lemon, peach, plum, apricot, cherry, mango, apple, pear, hawthorn, banana, pineapple, papaya, durian, lychee, watermelon, cantaloupe, honeydew melon, walnut, almond, etc.), and vegetables (radish, carrot, sweet potato, bamboo shoot, lettuce, potato, lotus root, ginger, spinach, bok choy, celery, Chinese cabbage, cabbage, broccoli, cauliflower, daylily, tomato, chili, eggplant, cucumber, winter melon, pumpkin, green beans, peas, onion, garlic, shiitake mushroom, enoki mushroom, etc.).

[0038] In some embodiments of the present invention, the product includes antibacterial agents, disinfectants, and preservatives.

[0039] A fourth aspect of the present invention provides a product comprising wheat gliadin-nano zinc oxide colloid according to the first aspect of the present invention.

[0040] In some embodiments of the present invention, the product includes antibacterial agents, disinfectants, and preservatives.

[0041] In some embodiments of the present invention, the product has at least one of the following functions: antibacterial, biofilm removal, and food preservation (delaying quality deterioration).

[0042] A fifth aspect of the present invention provides a method comprising the step of treating a sample to be treated with the wheat gliadin-nano zinc oxide colloid of the first aspect of the present invention or the product of the fourth aspect of the present invention. The method is any one of (a1)-(a3): (a1) A method of antibacterial treatment; (a2) A method for removing biofilms; (a3) A method of food preservation.

[0043] In some embodiments of the present invention, the treatment includes applying wheat gliadin-nano zinc oxide colloid to the sample to be treated by means of smearing, spraying, soaking, etc.

[0044] In some embodiments of the present invention, the test sample includes solid surfaces contaminated with bacteria (such as tables, utensils, etc.) and food (baked goods, fruits, vegetables, etc.).

[0045] The beneficial effects of this invention are: Unlike existing protein-zinc oxide composite powder materials, the wheat gliadin-nano zinc oxide colloid provided by this invention is a liquid-stable, dispersed nanocolloid system that can be directly used as a spray (mist) agent for food preservation. Its core-shell structure can effectively regulate Zn... 2+ The slow-release behavior of the wheat gliadin-nano zinc oxide colloid, combined with the reduction of potential cytotoxicity of nano zinc oxide through a protein shell, improves biocompatibility and safety while maintaining high antibacterial performance. This invention's wheat gliadin-nano zinc oxide colloid exhibits good dispersion stability and sprayability, making it suitable for surface preservation treatment of fruits, vegetables, and baked goods. It inhibits microbial growth and delays quality deterioration, making it applicable to the field of green food preservation.

[0046] This invention utilizes wheat gliadin as a template to prepare a core-shell structured colloidal solution (i.e., wheat gliadin-nano zinc oxide colloid) in one step using a green and mild self-assembly method. This avoids the use of organic solvents or high-temperature and high-pressure methods, conforming to the principles of green chemistry. Wheat gliadin, as a byproduct of wheat starch processing, is a natural, widely available, and inexpensive ingredient with the potential for large-scale commercialization. This colloidal solution utilizes the in-situ regulatory effect of wheat gliadin on nano-zinc oxide, effectively inhibiting the aggregation behavior of nanoparticles and enabling them to form a stable and dispersed colloidal structure in an aqueous system. It exhibits good colloidal stability and pH adaptability, maintaining the integrity of the zinc oxide structure even under acidic conditions, making it suitable for complex food media environments. Attached Figure Description

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1Particle size distribution of wheat gliadin-nano zinc oxide (G-ZnO) colloids prepared in Examples 1-3 of this invention.

[0048] Figure 2 This is a SEM image of the G-ZnO colloid prepared in Example 2 of the present invention.

[0049] Figure 3 The image shows a TEM image of the G-ZnO colloid prepared in Example 2 of this invention, with a scale bar of 100 nm.

[0050] Figure 4 The G-ZnO colloids prepared in Examples 1-3 of this invention were used to treat Escherichia coli (E. coli) E. coli ) and Staphylococcus aureus ( S. aureus The result of bacterial survival rate is shown in the figure.

[0051] Figure 5 The zinc ion release rate of the G-ZnO colloid prepared in Example 2 of this invention within 120 min under different pH environments (pH 4.0, 7.4, 10.0).

[0052] Figure 6 The G-ZnO colloid prepared in Example 2 of this invention was used to study the effects of different zinc equivalent concentrations (9.38-300 μg / mL) on Escherichia coli (E. coli). E. coli The sterilization rate results are shown in the figure.

[0053] Figure 7 Images of inhibition zones for PBS, Gliadin, ZnO NPs, and G-ZnO.

[0054] Figure 8 The G-ZnO colloid prepared in Example 2 of this invention was used to study the effects of different zinc equivalent concentrations (9.38-300 μg / mL) on Staphylococcus aureus (Staphylococcus aureus). S. aureus The sterilization rate results are shown in the figure.

[0055] Figure 9 This is a graph showing the ratio of live to dead bacteria after the G-ZnO colloid prepared in Example 2 of this invention was applied to the biofilms of Escherichia coli and Staphylococcus aureus at the lowest bactericidal concentration (MBC) (i.e., at the zinc equivalent of MBC).

[0056] Figure 10 The graph shows the hemolysis rate of the G-ZnO colloid prepared in Example 2 of this invention at different concentrations (62.5-2000 mg / L).

[0057] Figure 11 The graph shows the fitting curve between the concentration of G-ZnO colloid prepared in Example 2 of this invention and the mortality rate of zebrafish embryos.

[0058] Figure 12 Comparison of the storage effects of bread treated with different preservatives.

[0059] Figure 13 Comparison of the storage effects of bread treated with different concentrations of G-ZnO colloid.

[0060] Figure 14 The image shows the preservation effect of the G-ZnO colloid prepared in Example 2 on strawberries. Detailed Implementation

[0061] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0062] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0063] The preparation method of wheat gliadin is as follows: Weigh 20 g of gluten powder (purchased from Henan Huafeng Flour Industry Co., Ltd.), add it to 200 mL of 70% ethanol solution at a material-to-liquid ratio of 1:10, stir and extract at room temperature for 5 h, then centrifuge the extract at 8000 r / min for 20 min, collect the supernatant and transfer it to a glass dish, and dry it in an oven at 60 ℃ for 12 h to obtain wheat gliadin.

[0064] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0065] Example 1 A method for preparing wheat gliadin-nano zinc oxide (G-ZnO) colloid includes the following steps: (1) Dissolve 0.4 g wheat gliadin in 50 mL of 70% ethanol aqueous solution and stir until completely dissolved; (2) Add 0.16 g of zinc acetate dihydrate, stir to dissolve, and obtain a mixed solution; (3) Add 4 M NaOH solution to adjust the pH to 12; (4) Under stirring conditions, the mixed solution is quickly added to 125 mL of deionized water for antisolvent treatment; (5) Then, the mixture was concentrated to 120 mL by rotary evaporation at 55 °C to obtain wheat gliadin-nano zinc oxide colloidal solution, which is G-ZnO colloid.

[0066] Example 2 A method for preparing wheat gliadin-nano zinc oxide (G-ZnO) colloid includes the following steps: (1) Dissolve 0.5 g of wheat gliadin in 50 mL of 70% ethanol aqueous solution and stir until completely dissolved; (2) Add 0.25 g of zinc acetate dihydrate, stir to dissolve, and obtain a mixed solution; (3) Add 4 M NaOH solution to adjust the pH to 12; (4) Under stirring conditions, the mixed solution is quickly added to 125 mL of deionized water for antisolvent treatment; (5) Then, the mixture was concentrated to 120 mL by rotary evaporation at 55 °C to obtain wheat gliadin-nano zinc oxide colloidal solution, which is G-ZnO colloid.

[0067] Example 3 A method for preparing wheat gliadin-nano zinc oxide (G-ZnO) colloid includes the following steps: (1) Dissolve 0.6 g of wheat gliadin in 50 mL of 70% ethanol aqueous solution and stir until completely dissolved; (2) Add 0.36 g of zinc acetate dihydrate, stir to dissolve, and obtain a mixed solution; (3) Add 4 M NaOH solution to adjust the pH to 12; (4) Under stirring conditions, the mixed solution is quickly added to 125 mL of deionized water for antisolvent treatment; (5) Then, the mixture was concentrated to 120 mL by rotary evaporation at 55 °C to obtain wheat gliadin-nano zinc oxide colloidal solution, which is G-ZnO colloid.

[0068] Effect Example 1. Structural characterization of G-ZnO colloids The particle size of the wheat gliadin-nano zinc oxide colloid (hereinafter referred to as G-ZnO colloid) prepared in Examples 1-3 was determined using a Malvern particle size analyzer. The results are as follows: Figure 1 As shown, the particle size of the G-ZnO colloid prepared in Example 1 is approximately 200 nm, and the PDI (Polymer dispersity index) is 0.154. The particle size of the G-ZnO colloid prepared in Example 2 is approximately 220 nm, and the PDI is 0.125. The particle size of the G-ZnO colloid prepared in Example 3 is approximately 232 nm, and the PD is 0.174.

[0069] The microstructure of the G-ZnO colloid prepared in Example 2 was characterized using scanning electron microscopy (SEM). The results are as follows: Figure 2As shown, the G-ZnO colloid is approximately spherical with a relatively uniform particle size distribution, consistent with the results measured by the particle size analyzer.

[0070] The internal structure of the G-ZnO colloid prepared in Example 2 was characterized using transmission electron microscopy (TEM), and the results are as follows: Figure 3 As shown in the image, the TEM image further reveals the internal structure of the particles, showing that spindle-shaped ZnO crystals exist inside the spherical particles, forming a stable core-shell composite particle structure.

[0071] The microstructure and internal structure of the G-ZnO colloids prepared in Examples 1 and 3 are similar to those in Example 2.

[0072] 2. Antibacterial activity test The broad-spectrum antibacterial activity of G-ZnO colloids was evaluated using a liquid diffusion method. *Escherichia coli* (GZnO colloids) were then subjected to the method. E. coli ATCC25922) and Staphylococcus aureus ( S. aureus After resuscitation, the bacterial culture (ATCC 6538) was inoculated into LB liquid medium and cultured at 37°C with shaking until the logarithmic growth phase. The culture was thoroughly washed and resuspended at 10⁻⁶ ppm. 7 CFU / mL available for use.

[0073] The G-ZnO colloids prepared in Examples 1-3 were diluted 50-fold with sterile PBS. 0.15 mL of each colloid was mixed with 0.15 mL of bacterial suspension and co-cultured at 37°C for 2 h. 100 μL of the mixture was then spread onto LB agar plates and incubated at 37°C for 24 h. Colony counting was performed, with three replicates for each group. Sterile water was used instead of G-ZnO colloid for the blank control. The bacterial survival rate was calculated as follows: Bacterial survival rate (%) = (Number of colonies on the plate in the sample treatment group / Number of colonies on the plate in the blank control group) × 100%.

[0074] The results are as follows Figure 4 As shown, the G-ZnO colloid obtained in Example 2 is effective against Escherichia coli (E. coli). E. coli ATCC 25922) and Staphylococcus aureus ( S. aureus (ATCC 6538) has the strongest antibacterial activity and the lowest bacterial survival rate.

[0075] The G-ZnO colloid obtained in Example 2 was further diluted with sterile PBS to different concentrations and mixed with bacterial suspensions at a 1:1 volume ratio to achieve final zinc equivalent concentrations of 300, 150, 75, 37.5, 18.75, and 9.38 μg / mL. The sterilization rate was calculated using sterile PBS as a control, with three replicates for each group. After co-culturing at 37°C for 2 h, 100 μL was plated onto LB agar plates and incubated at 37°C for 24 h, followed by colony counting. The minimum bactericidal concentration (MBC) was defined as the lowest concentration required to kill 99.99% of bacteria.

[0076] The results are as follows Figure 5 and Figure 6 As shown, the G-ZnO colloid prepared in Example 2 has an MBC of 37.50 μg / mL against Escherichia coli and an MBC of 18.75 μg / mL against Staphylococcus aureus, exhibiting excellent antibacterial activity.

[0077] Further inhibition zone testing was used to compare the antibacterial effects of G-ZnO colloid and zinc oxide obtained in Example 2, as follows: 15 mL of sterilized, unsolidified LB broth solid culture medium was pipetted into a petri dish, maintaining a uniform culture medium height in the plate. The initial bacterial suspension (10 mL) was diluted with sterile water. 7 The bacterial concentration (CFU / mL) was diluted to 1×10⁻⁶. 6 CFU / mL E. coli ) and 5×10 5 CFU / mL S. aureus 100 μL of working bacterial culture was spread onto LB solid medium and air-dried. After air-drying, 7.5 mm diameter sterile punches were used to create wells. 200 μL of PBS, Gliadin, ZnONPs (purchased from Aladdin, catalog number Z141332), and G-ZnO colloidal solution were then added to the wells. The cultures were incubated at 37°C for 12 hours. Colonies were photographed and recorded, with three replicates per sample.

[0078] The results are as follows Figure 7 The results showed that no inhibition zones were generated in the PBS, Gliadin, and ZnO NPs groups. In contrast, the G-ZnO group exhibited a significant and larger inhibition zone, indicating that the G-ZnO colloid has good dispersibility and antibacterial activity.

[0079] 3. Tolerance testing The wheat gliadin-zinc oxide nano-colloid obtained in Example 2, which exhibited the best uniformity, moderate size, and high zinc oxide content (the zinc content in this wheat gliadin-zinc oxide nano-colloid was 600 ppm as determined by ICP-OES), was used for subsequent tests. The zinc ion release kinetics of this G-ZnO colloid under different pH conditions were determined using inductively coupled plasma atomic emission spectrometry (ICP-OES). Phosphate-buffered saline (PBS) was used to simulate different pH values, which also allowed for testing the salt tolerance of the colloid. The specific steps were as follows: the G-ZnO colloid was placed in PBS at pH 4.0, 7.4, and 10.0 for 120 min, respectively, and then measured using inductively coupled plasma atomic emission spectrometry.

[0080] The results are as follows Figure 8 As shown, the overall zinc ion release rate was less than 1.6%, and the G-ZnO colloidal solution did not exhibit significant aggregation or sedimentation throughout the process, demonstrating good colloidal stability and tolerance.

[0081] 4. Anti-biofilm activity test To evaluate the ability of the G-ZnO colloid prepared in Example 2 to remove biofilms from food contact surfaces, food-grade 304 stainless steel was used as a carrier to culture mature biofilms of *Escherichia coli* and *Staphylococcus aureus*, respectively. (Taking *Staphylococcus aureus* as an example, the preparation process of the mature biofilm is as follows: First, frozen *Staphylococcus aureus* liquid was inoculated onto nutrient-rich agar plates and cultured at 37°C for 16 to 20 hours to isolate single colonies. Selected colonies were transferred to liquid culture medium (3% tryptone soybean broth, TSB + 1% glucose) and cultured overnight at 37°C on a shaker at 200 rpm. One portion (4 mL) of the bacterial suspension was taken and centrifuged (3000 rpm, 5 minutes), and then the precipitate was washed three times with 4 mL of sterile water under the same centrifugation conditions. The washed cells were resuspended in fresh culture medium (3% TSB + 1% glucose) and analyzed using a multi-functional microplate reader (Synergy H1, BioTek).) Optical density (OD) at nanometers 600Adjust the concentration to 0.005. Dispense 400 mL of the bacterial suspension into the inner wells of a 48-well plate, and fill the outer wells with 0.5 mL of sterile water to maintain humidity. After incubating at 37°C for 24 hours, replace half of the culture medium with fresh medium and continue incubation for another 24 hours. Confirm successful biofilm formation by observing a dense film layer. Then, add G-ZnO colloid at MBC concentration and co-culture for 2 hours. After incubation, determine the ratio of live to dead bacteria in the biofilm using a live / dead bacteria viability assay kit (purchased from Aladdin Biochemical Technology Co., Ltd. The LIVE / DEAD™ kit).

[0082] After the live / dead bacterial activity test, the results are as follows: Figure 9 As shown, the bacterial mortality rate in the biofilm after treatment with G-ZnO colloid was as high as 97%, indicating that the G-ZnO colloid prepared in Example 2 has a good ability to remove biofilms on food contact surfaces.

[0083] 5. Biocompatibility evaluation (1) In vitro hemolysis assessment Fresh goat blood was collected, and red blood cells were separated by centrifugation. The cells were thoroughly washed with physiological saline to prepare a 5% red blood cell suspension. Subsequently, the G-ZnO colloid prepared in Example 2 was co-incubated with the red blood cell suspension at different concentrations (62.50-2000 mg / L, calculated from the solid content of the G-ZnO colloid after freeze-drying, and then diluted) (37°C for 3 hours). Physiological saline and deionized water were used as negative and positive controls, respectively. After centrifugation, the absorbance (OD) at 540 nm was measured in the supernatant. 540 ), calculate the hemolysis rate = (OD sample - OD saline) / (OD deionized water - OD saline).

[0084] The results are as follows Figure 10 As shown, at a concentration of 2000 mg / L, the hemolysis rate of G-ZnO colloid is still less than 5%, which meets the safety standards for biomedical materials.

[0085] (2) Acute toxicity test of zebrafish embryos in vivo Following the OECD 236 guideline (i.e., Fish Embryo Acute Toxicity (FET) Test, TG 236), zebrafish fertilized eggs were exposed to different concentrations of G-ZnO colloid (125-4000 mg / L based on colloid particle concentration) for 96 h, embryo mortality was recorded, and mortality curves were fitted.

[0086] The mortality rate fitting curve is as follows Figure 11 As shown, the results indicate that the median lethal concentration (LC50) of G-ZnO colloid is... 50 The concentration was 681.02 mg / L, which is much higher than 100 mg / L, indicating that its acute toxicity is at the lowest level.

[0087] 6. Bread preservation test (1) Comparison of preservation effects of different preservatives The G-ZnO colloid prepared in Example 2 was diluted to 5 times the MBC concentration (93.75 μg / mL), then placed in a spray bottle and sprayed evenly onto the surface of 5 g fresh bread. After drying at room temperature, it was stored at room temperature (25±2℃). Unsprayed blank bread, bread sprayed with Gliadin solution (the preparation method of Gliadin in the Gliadin solution differed from that of G-ZnO colloid in Example 2 only in that weak acid zinc salt was not added. That is, if the G-ZnO colloid was 5 times the MBC concentration (93.75 μg / mL), then the concentration of Gliadin in the Gliadin solution was approximately 0.63 mg / mL), and bread sprayed with ZnO nanoparticle dispersion (ZnO nanoparticles (<100 nm, 99.8%) purchased from Aladdin, catalog number Z141332. The Zn concentration of zinc oxide was the same as that of G-ZnO colloid) served as the control groups (referred to as blank control group, Gliadin group, and ZnO nanoparticle group, respectively). The mold growth on the bread surface was observed on days 1, 3, 5, 7, 9, 12, and 15.

[0088] The results are as follows Figure 12 As shown, bread from the blank control group, Gliadin group, and ZnO nanoparticle group developed mold after 5 days of storage, while bread coated with G-ZnO colloid showed no obvious mold growth on the surface after 15 days of storage, fully demonstrating that the colloid has excellent antibacterial and preservation effects.

[0089] (2) Comparison of the preservation effects of different concentrations of G-ZnO colloid The G-ZnO colloid prepared in Example 2 was diluted with sterile PBS to different concentrations (1×MBC, 2×MBC, 5×MBC, calculated using the MBC of E. coli). Approximately 300 μL of each colloid was evenly sprayed onto the surface of 5 g fresh bread, air-dried at room temperature, and then stored at room temperature (25±2℃). Unsprayed blank bread was used as a control group, and mold growth on the bread surface was observed on days 1, 3, 5, 7, 9, 12, and 15.

[0090] The results are as follows Figure 13As shown, the blank control group bread developed obvious mold spots after 5 days of storage; the 1×MBC group bread began to show mold on the 9th day of storage; the 2×MBC group bread showed mold on the 12th day of storage; while the 5×MBC group bread showed no obvious mold after 15 days of storage. The results indicate that the preservation effect of G-ZnO colloid is concentration-dependent, and an effective preservation period of 15 days can be achieved at a concentration of 5×MBC.

[0091] 7. Strawberry Preservation Test The G-ZnO colloid prepared in Example 2 was diluted with sterile PBS to different concentrations (1×MBC, 2×MBC, 5×MBC, calculated using the MBC of E. coli). Strawberries without spraying, those sprayed with Gliadin solution (same as bread preservation test), and those sprayed with ZnO nanoparticle dispersion (same as bread preservation test) served as control groups (referred to as unsprayed group, Gliadin solution group, and ZnO nanoparticle dispersion group, respectively). The strawberries were then subjected to preservation treatment. Approximately 300 μL of the corresponding solution was uniformly sprayed onto the surface of each group of strawberries. After air-drying at room temperature, the strawberries were stored at room temperature (25±2℃). The mold growth on the strawberry surface was observed and recorded on days 1, 3, 5, and 7.

[0092] The results are as follows Figure 14 As shown, on the 3rd day of storage, mold began to appear on the surface of strawberries in the unsprayed group, the Gliadin solution group, and the ZnO nanoparticle dispersion group. Mold appeared in the 1×MBC group on the 5th day, and in the 2×MBC group on the 7th day, while the 5×MBC group showed no obvious mold after 7 days of storage. This indicates that the preservation effect of G-ZnO colloid is concentration-dependent, and high concentration (5×MBC) treatment can significantly delay the mold growth of strawberries.

[0093] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A wheat gliadin-nano zinc oxide colloid, characterized in that, The wheat gliadin-nano zinc oxide colloid comprises a nano zinc oxide core and a wheat gliadin coating layer covering the surface of the nano zinc oxide core.

2. The wheat gliadin-nano zinc oxide colloid according to claim 2, characterized in that, The wheat gliadin-nano zinc oxide colloid is spherical with a particle size of 100-800 nm.

3. The preparation method of wheat gliadin-nano zinc oxide colloid according to claim 1 or 2, comprising the following steps: Wheat gliadin solution is mixed with zinc salt, pH value is adjusted, and antisolvent is added to obtain wheat gliadin-nano zinc oxide colloid.

4. The preparation method according to claim 3, characterized in that, The solvent of the wheat gliadin solution is ethanol; and / or, the concentration of the wheat gliadin solution is 5-15 mg / mL.

5. The preparation method according to claim 3, characterized in that, The zinc salts include weakly acidic zinc salts; Preferably, the zinc salt is selected from zinc acetate or zinc carbonate.

6. The preparation method according to claim 5, characterized in that, The amount of zinc salt added is 30%-70% of the wheat alcohol-soluble protein content.

7. The preparation method according to any one of claims 3-6, characterized in that, The pH value is adjusted to 10-13; and / or the antisolvent includes a water or ethanol solution.

8. The application of the wheat gliadin-nano zinc oxide colloid according to claim 1 or 2 or the preparation method according to any one of claims 3-7 in any one of (1)-(6): (1) Antibacterial; (2) Preparation of antibacterial products; (3) Remove biofilm; (4) Prepare products for removing biofilms; (5) Food preservation; (6) Prepare food preservation products; Preferably, the biofilms described in (3)-(4) include biofilms formed by Gram-positive bacteria and / or Gram-negative bacteria.

9. A product comprising the wheat gliadin-nano zinc oxide colloid as described in claim 1 or 2.

10. A method comprising the step of treating a sample to be treated with the wheat gliadin-nano zinc oxide colloid of claim 1 or 2 or the product of claim 9; The method is any one of (a1)-(a3): (a1) A method of antibacterial treatment; (a2) A method for removing biofilms; (a3) A method of food preservation.

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