Preparation method of edible antibacterial packaging film of tilapia skin gelatin hydrolyzed peptides

Abstract

This invention relates to the field of food packaging materials technology, and discloses an edible antibacterial packaging film containing tilapia skin gelatin hydrolysate peptides, comprising the following components: tilapia skin gelatin: 55-65%; antibacterial peptides: 18-22%, wherein the molecular weight of the antibacterial peptides is less than 5 kDa, prepared by microwave-ultrasound synergistic directional enzymatic hydrolysis of gelatin; tea polyphenols: 0.8-1.2%; nanocellulose: 0.8-1.2%; citric acid: 0.8-1.2%; the balance being deionized water; in the preparation of the antibacterial peptides: the microwave parameters are 2.45 GHz, 300-500 W, pulse mode on for 25-35 seconds / off for 8-12 seconds; the ultrasonic parameters are 18-22 kHz, 180-220 W, and are synchronously turned on with the microwave pulse; the tea polyphenols and nanocellulose form a composite reinforcing agent through pre-adsorption and covalent cross-linking. By modifying the interface between tea polyphenols and nanocellulose, a uniformly dispersed composite reinforcing network is formed, which effectively enhances the mechanical strength and deformation resistance of the gelatin matrix, while reducing water vapor permeability, making the material more suitable for food packaging needs in high humidity environments.

Description

Technical Field

[0001] This invention relates to the field of food packaging materials technology, specifically a method for preparing an edible antibacterial packaging film made from hydrolyzed peptides of tilapia skin gelatin. Background Technology

[0002] Traditional gelatin-based food packaging materials face multiple technical bottlenecks in application. In existing technologies, to improve the mechanical properties of gelatin films, reinforcing agents are often added through physical blending. However, components such as nanocellulose are prone to agglomeration due to poor interfacial compatibility, leading to uneven stress distribution within the material and making it difficult to simultaneously achieve high toughness and low moisture permeability. Regarding antibacterial functionalization, while directly adding chemical preservatives or natural extracts can inhibit bacteria in the short term, problems such as easy migration of active ingredients and uncontrollable slow release exist. Furthermore, high-temperature processing can easily deactivate heat-sensitive substances, making it difficult to meet the requirements for long-term preservation.

[0003] Furthermore, enzymatic hydrolysis of collagen often relies on a single enzyme and static reaction conditions, resulting in a wide molecular weight distribution of the products and a low proportion of target functional peptides. This not only wastes raw materials but also affects the bioactivity and stability of the final product. More significantly, existing technologies often require the introduction of synthetic plasticizers or stabilizers to balance processing efficiency and activity retention, leading to decreased biocompatibility and contradicting the trend of green packaging development. These systemic defects restrict the application of gelatin-based packaging materials in the high-value-added food sector. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for preparing an edible antibacterial packaging film of hydrolyzed peptides from tilapia skin gelatin. This method solves the problems of uneven dispersion of reinforcing agents, short-lasting antibacterial activity, uncontrollable enzymatic hydrolysis, and poor mechanical properties and insufficient preservation caused by processing activity loss in traditional gelatin materials.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing an edible antibacterial packaging film of tilapia skin gelatin hydrolysate peptides, comprising the following components by weight percentage of total solids: Fish skin gelatin: 55-65%; Antimicrobial peptides: 18-22%, wherein the molecular weight of the antimicrobial peptides is less than 5 kDa, and they are prepared by microwave-ultrasound synergistic directional enzymatic hydrolysis of gelatin; Tea polyphenols: 0.8-1.2%; Nanocellulose: 0.8-1.2%; Citric acid: 0.8-1.2%; The remainder is deionized water.

[0006] Preferably, in the preparation of the antimicrobial peptide: The microwave parameters are 2.45GHz, 300-500W, and the pulse mode is on for 25-35 seconds and off for 8-12 seconds. The ultrasound parameters are 18-22kHz and 180-220W, and are activated synchronously with the microwave pulse.

[0007] Preferably, the tea polyphenols and nanocellulose form a composite reinforcing agent through pre-adsorption and covalent cross-linking, wherein: The mass ratio of tea polyphenols to nanocellulose is 0.8:1-1.2:1; Covalent crosslinking was performed using genipin crosslinking agent, with an addition amount of 0.5-1.5% of the mass of tea polyphenols. The crosslinking conditions were pH 3.8-4.2, temperature 45-55℃, and time 0.8-1.2 hours.

[0008] Preferably, the nanocellulose has a diameter of 20-50 nm and is dispersed in a citrate buffer solution with a pH of 3.5-4.5.

[0009] Preferably, the citric acid is used to adjust the pH of the film-forming solution to 3.5-4.5.

[0010] A method for preparing an edible antibacterial packaging film using hydrolyzed peptides from tilapia skin gelatin includes the following steps: Step 1, Pre-treatment of tilapia skin: Degreasing and demineralization are performed sequentially; Step 2, Microwave-Ultrasound Synergistic Enzymatic Hydrolysis of Gelatin: Antimicrobial peptides are prepared by directional enzymatic hydrolysis using a dual-enzyme system under pulsed microwave (2.45GHz, 300-500W) and synchronous ultrasound (18-22kHz, 180-220W) conditions. Step 3: Preparation of composite reinforcing agent: Covalently crosslink tea polyphenols and nanocellulose at a mass ratio of 0.8:1-1.2:1; Step 4: Prepare the film-forming solution: Mix gelatin, antimicrobial peptides, composite enhancer and citric acid, and adjust the pH to 3.5-4.5; Step 5, Gradient Drying: Microwave drying and vacuum drying are performed sequentially to form the packaging film. Preferably, in step 1: The degreasing treatment uses 0.4-0.6M NaOH solution, with a material-to-liquid ratio of 1:8-1:12 (w / v), and is stirred at 25-30℃ for 1.5-2.5 hours. The demineralization treatment uses 0.2-0.4M HCl solution with a material-to-liquid ratio of 1:6-1:10 (w / v) and is carried out at 25-30℃ for 0.8-1.2 hours.

[0011] Preferably, in step 2: Enzymatic hydrolysis employs a dual-enzyme system of alkaline protease and flavor protease, with addition amounts of 0.8-1.2% and 0.4-0.6% of the gelatin mass, respectively. The enzymatic hydrolysis temperature was gradually reduced from 58-62℃ to 53-57℃, with a total time of 30-60 minutes.

[0012] Preferably, gradient drying in step 5 includes: First stage microwave drying: microwave power 280-320W, temperature gradually reduced from 38-42℃ to 33-37℃, time 4-6 minutes; The second stage is vacuum drying: the vacuum pressure is reduced from -0.07-0.09 MPa to -0.09-0.11 MPa, the temperature is 23-27℃, and the time is 8-12 minutes.

[0013] Preferably, the preparation of the composite reinforcing agent in step 3 includes: Disperse nanocellulose in a citrate buffer solution with a pH of 3.5-4.5 and sonicate for 8-12 minutes; After adding tea polyphenols and genipin crosslinking agent, stir at 55-65℃ for 1.5-2.5 hours, and collect the complex by centrifugation.

[0014] This invention provides a method for preparing an edible antibacterial packaging film of hydrolyzed peptides from tilapia skin gelatin. It has the following beneficial effects: 1. This invention modifies the interface between tea polyphenols and nanocellulose to form a uniformly dispersed composite reinforcing network, which effectively enhances the mechanical strength and deformation resistance of the gelatin matrix, while reducing water vapor permeability, making the material more suitable for food packaging needs in high humidity environments.

[0015] 2. This invention is based on dual-enzyme synergistic hydrolysis and physical field-assisted technology to release small molecule antimicrobial peptides in a targeted manner. Combined with the natural antibacterial properties of tea polyphenols, it endows the packaging film with a sustained inhibitory effect on a variety of foodborne pathogens. Moreover, the antimicrobial components are released slowly and stably, thus extending the food shelf life.

[0016] 3. This invention employs a dual-enzyme system combined with microwave-ultrasound synergy to overcome the bottleneck of traditional enzymatic hydrolysis efficiency, precisely cleaving the Gly-Pro-X sequence of collagen to achieve the directional preparation of high-value small molecule peptides and reduce the generation of ineffective byproducts.

[0017] 4. This invention uses a gradient drying process to avoid the damage of heat-sensitive antimicrobial peptides caused by high temperatures, while maintaining the compatibility between the composite reinforcing agent and the gelatin matrix, thus ensuring the functional integrity of the active ingredients after film formation. Attached Figure Description

[0018] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: Please see the appendix Figure 1 This invention provides a method for preparing an edible antibacterial packaging film made from hydrolyzed tilapia skin gelatin peptides, comprising: Step 1: Raw material pretreatment Degreasing: Soak 100g of tilapia skin in 0.5M NaOH solution (solid-to-liquid ratio 1:10 w / v) and stir at 28℃ for 2 hours.

[0021] Demineralization: Degreased fish skin was treated with 0.3M HCl solution (solid-to-liquid ratio 1:8 w / v) for 1 hour and demineralized at 25℃.

[0022] Gelatin extraction: Fish skin was mixed with deionized water (solid-to-liquid ratio 1:15 w / v), extracted at 60℃ for 4 hours, centrifuged (8000 rpm, 20 minutes) and then freeze-dried.

[0023] Step 2: Microwave-ultrasound synergistic enzymatic hydrolysis Enzymatic hydrolysis system: 10g gelatin was dissolved in pH 8.0 phosphate buffer (4% w / v), and alkaline protease (1% w / w gelatin) and flavor protease (0.5% w / w gelatin) were added.

[0024] Physical field parameters: Microwave: 2.45GHz, 400W, pulse mode (30 seconds on / 10 seconds off), temperature gradually decreases from 60℃ to 55℃.

[0025] Ultrasound: 20kHz, 200W, synchronized with microwave pulse.

[0026] Enzymatic hydrolysis time: 20 minutes, followed by inactivation at 90°C for 2 minutes, centrifugation (10000 rpm, 15 minutes) to obtain antimicrobial peptide solution.

[0027] Step 3: Preparation of composite reinforcing agent Pre-adsorption: 1g of nanocellulose (30nm in diameter) was dispersed in pH 4.0 citrate buffer (1% w / v), sonicated (250W, 10 minutes), 1g of tea polyphenols was added, and stirred at 60℃ for 2 hours.

[0028] Covalent crosslinking: Add genipin (1% w / w tea polyphenols), react at 50℃ for 1 hour, and collect the complex by centrifugation (8000 rpm, 10 minutes).

[0029] Step 4: Preparation of film-forming solution Composition ratio (total solids): gelatin 60%, antimicrobial peptides 20%, composite reinforcing agent 1%, citric acid 1%, balance is water.

[0030] Mixing and casting: Stir magnetically at 50℃ for 1 hour, degas under vacuum (-0.08MPa, 10 minutes), and cast into a mold with a thickness of 0.5mm.

[0031] Step 5: Gradient drying Microwave drying: 300W, temperature 40℃→35℃, time 5 minutes.

[0032] Vacuum drying: Vacuum pressure -0.08→-0.1MPa, dry at 25℃ for 10 minutes.

[0033] Example 2: Example 2 (lower limit of parameters) Step 1: Raw material pretreatment Degreasing: 0.4M NaOH solution (solid-to-liquid ratio 1:8 w / v), stir at 30℃ for 2.5 hours.

[0034] Demineralization: 0.2M HCl solution (solid-to-material ratio 1:6 w / v), treated at 30℃ for 0.8 hours.

[0035] Gelatin extraction: Extract at 55℃ for 5 hours, then centrifuge (7000 rpm, 25 minutes).

[0036] Step 2: Microwave-ultrasound synergistic enzymatic hydrolysis Enzymatic hydrolysis system: gelatin concentration 3% w / v, alkaline protease 0.8% w / w, flavor protease 0.4% w / w.

[0037] Physical field parameters: Microwave: 300W, pulse mode (25 seconds on / 8 seconds off), temperature 58℃→53℃.

[0038] Ultrasound: 180W, synchronized with microwave.

[0039] Enzymatic hydrolysis time: 18 minutes, followed by inactivation at 88℃ for 2.5 minutes.

[0040] Step 3: Preparation of composite reinforcing agent Pre-adsorption: 0.8% w / v nanocellulose, tea polyphenols:nanocellulose mass ratio 0.8:1, stirred at 55℃ for 2.5 hours.

[0041] Covalent crosslinking: pH 3.8, genipin 0.5% w / w, reaction at 45℃ for 1.2 hours.

[0042] Step 4: Preparation of film-forming solution Composition ratio: gelatin 55%, antimicrobial peptides 18%, composite reinforcing agent 0.8%, citric acid 0.8%.

[0043] Step 5: Gradient drying Microwave drying: 280W, temperature 38℃→33℃, time 6 minutes.

[0044] Vacuum drying: Vacuum pressure -0.07→-0.09MPa, dry at 23℃ for 12 minutes.

[0045] Example 3: Step 1: Raw material pretreatment Degreasing: 0.6M NaOH solution (solid-to-material ratio 1:12 w / v), stir at 25℃ for 1.5 hours.

[0046] Demineralization: 0.4M HCl solution (solid-to-material ratio 1:10 w / v), treated at 25℃ for 1.2 hours.

[0047] Gelatin extraction: Extract at 65℃ for 3 hours, then centrifuge (9000 rpm, 15 minutes).

[0048] Step 2: Microwave-ultrasound synergistic enzymatic hydrolysis Enzymatic hydrolysis system: gelatin concentration 5% w / v, alkaline protease 1.2% w / w, flavor protease 0.6% w / w.

[0049] Physical field parameters: Microwave: 500W, pulse mode (35 seconds on / 12 seconds off), temperature 62℃→57℃.

[0050] Ultrasound: 220W, synchronized with microwave.

[0051] Enzymatic hydrolysis time: 22 minutes, followed by inactivation at 92℃ for 1.5 minutes.

[0052] Step 3: Preparation of composite reinforcing agent Pre-adsorption: 1.2% w / v nanocellulose, tea polyphenols:nanocellulose mass ratio 1.2:1, stirred at 65℃ for 1.5 hours.

[0053] Covalent crosslinking: pH 4.2, genipin 1.5% w / w, reaction at 55℃ for 0.8 hours.

[0054] Step 4: Preparation of film-forming solution Composition ratio: gelatin 65%, antimicrobial peptides 22%, composite reinforcing agent 1.2%, citric acid 1.2%.

[0055] Step 5: Gradient drying Microwave drying: 320W, temperature 42℃→37℃, time 4 minutes.

[0056] Vacuum drying: Vacuum pressure -0.09→-0.11MPa, dry at 27℃ for 8 minutes.

[0057] Comparative Example 1: Compared with Example 1, the difference is that no tea polyphenol-nanocellulose composite reinforcing agent was added, only an equal amount of nanocellulose (1%) was used, and the pre-adsorption and covalent cross-linking steps were not performed. The remaining steps are the same as in Example 1.

[0058] Comparative Example 2: The difference from Example 1 is that only alkaline protease (1% w / w gelatin) was used in the enzymatic hydrolysis process, and no flavor protease was added. The remaining steps are the same as in Example 1.

[0059] Comparative Example 3: Compared with Example 1, the difference is that the microwave-ultrasound synergistic enzymatic hydrolysis is replaced with traditional isothermal enzymatic hydrolysis (60°C constant temperature stirring, without physical field assistance), and the hydrolysis time is extended to 2 hours. The remaining steps are the same as in Example 1.

[0060] Comparative Example 4: Compared to Example 1, the difference is that the antimicrobial peptide is replaced with ordinary gelatin hydrolyzed peptide (molecular weight range 5-10 kDa, uncontrolled enzymatic hydrolysis sites). The remaining steps are the same as in Example 1.

[0061] Comparative Example 5: Compared to Example 1, the difference is that tea polyphenols and nanocellulose are only physically mixed (without pre-adsorption and covalent cross-linking), and then directly added to the film-forming solution after mixing. The remaining steps are the same as in Example 1.

[0062] Comparative Example 6: Compared with Example 1, the difference is that gradient drying is replaced with single hot air drying (constant temperature drying at 60°C for 30 minutes). The remaining steps are the same as in Example 1.

[0063] Test Example 1: Explanation of the Comparative Experiment on Antibacterial Activity Experimental materials and equipment: Tested bacterial strains: Escherichia coli (ATCC25922) and Staphylococcus aureus (ATCC6538).

[0064] Culture media: LB liquid medium, LB agar plates.

[0065] Test samples: Packaging film samples (6mm diameter discs) from Examples 1-3 and Comparative Examples 1, 3, 4, and 6.

[0066] Positive control: Nisin (0.1 mg / mL).

[0067] Negative control: sterile PBS buffer.

[0068] Equipment: constant temperature incubator, vernier calipers, enzyme-linked immunosorbent assay (ELISA) reader, sterile operating table.

[0069] Experimental steps: Preparation of bacterial culture: The test strain was inoculated into LB liquid medium and cultured at 37°C with shaking for 12 hours. The bacterial concentration was then adjusted to 1×10⁻⁶. 6 CFU / mL.

[0070] Antibacterial zone test: Take 100 μL of bacterial suspension and spread it evenly on the surface of an LB agar plate.

[0071] Press the test film sample (circular disc) lightly onto the agar surface, repeating 3 times for each group.

[0072] After incubating at 37℃ for 24 hours, the diameter of the inhibition zone (including the sample diameter) was measured.

[0073] Antimicrobial peptide sustained-release test: Immerse the membrane sample (1g) in 10mL PBS buffer (pH 6.8) and shake at 37°C (120rpm).

[0074] Take 1 mL samples at 0, 6, 12, and 24 hours, centrifuge, and collect the supernatant.

[0075] Mix the supernatant with the bacterial culture (1×10) 6 The mixture (CFU / mL) was mixed in equal volumes and incubated at 37°C for 6 hours. The OD value at 600 nm was measured using a UV spectrophotometer to calculate the inhibition rate.

[0076] Data processing: Antibacterial rate formula: Antibacterial rate (%) = (1 - OD of experimental group) 600 / Blank Group OD 600 ) × 100%.

[0077] Table 1 Summary of antibacterial activity test data Diameter of the inhibition zone: Examples 1-3 showed significantly higher antibacterial activity than the comparative examples (e.g., the inhibition zone of conventional enzymatic hydrolysis in comparative example 3 was only 8.5 mm), demonstrating the synergistic antibacterial effect of microwave-ultrasound synergistic enzymatic hydrolysis and composite enhancer.

[0078] Comparative Example 4 (common hydrolyzed peptide) showed the lowest antibacterial activity, indicating the key role of small molecule antimicrobial peptides (<5kDa) in the antibacterial effect.

[0079] Sustained-release test: Examples 1-3 maintained an antibacterial rate of >80% after 24 hours, while Comparative Example 6 (single drying) only had a rate of 70.4%, demonstrating the protective effect of the gradient drying process on the activity of antimicrobial peptides.

[0080] Test Example 2: Explanation of Mechanical Performance Comparison Experiment Experimental materials and equipment: Test samples: Packaging film samples of Examples 1-3, Comparative Example 1 (without composite reinforcing agent), and Comparative Example 5 (physically mixed reinforcing agent) (thickness 0.5 mm, cut into dumbbell-shaped samples).

[0081] Test equipment: Universal testing machine (ASTM D882 standard, tensile rate 50 mm / min); Water vapor transmission rate tester (ASTM E96, temperature 25°C, humidity 75%). Digital thickness gauge (accuracy ±0.001mm).

[0082] Experimental steps: Tensile strength and elongation at break tests: Cut dumbbell-shaped specimens (total length 75mm, gauge length 25mm×5mm), and test 5 parallel specimens in each group.

[0083] Fix the specimen in the fixture and stretch it at a rate of 50 mm / min until it breaks. Record the maximum load and the elongation at break.

[0084] Calculation formula: Tensile strength (MPa) = Maximum load (N) / Specimen cross-sectional area (mm²) 2 ); Elongation at break (%) (gauge length elongation at break / original gauge length) × 100%.

[0085] Water vapor transmission rate (WVTR) test: Seal the membrane sample (6 cm in diameter) in a permeation cup (containing dry silica gel) and place it in a constant temperature and humidity chamber (25℃, 75%RH).

[0086] Weigh the permeation cup every hour, and continue testing for 24 hours to calculate the permeation rate per unit time.

[0087] Calculation formula: WVTR (g / (m) 2 ·24h))=(weight increment×24) / (test time×membrane area).

[0088] Data processing: Remove outliers (such as tearing at the edge of the sample) and take the average value ± standard deviation of the remaining valid data.

[0089] Table 2 Summary of Mechanical Performance Test Data Tensile strength: Examples 1-3 (45-50 MPa) were significantly higher than Comparative Example 1 (22.4 MPa) and Comparative Example 5 (29.8 MPa), indicating the key role of interfacial modification (pre-adsorption + covalent crosslinking) of the composite reinforcing agent in mechanical strength.

[0090] The strength of Comparative Example 5 (physical mixture) was higher than that of Comparative Example 1 (no tea polyphenols), indicating that even without cross-linking, tea polyphenols still have a certain enhancing effect.

[0091] Water vapor transmission rate: Examples 1-3 (108-118g / (m 2 The concentration of water vapor permeability was significantly lower than that of Comparative Example 1 (215.7) and Comparative Example 5 (189.3) after 24 hours, demonstrating that the composite reinforcing agent can effectively fill the pores of the gelatin network and reduce water vapor permeability.

[0092] Test Example 3: Experimental Description of Enzymatic Digestion Efficiency and Peptide Analysis Experimental materials and equipment: Test samples: Enzyme hydrolysates of Examples 1-3, Comparative Example 2 (alkaline protease only), and Comparative Example 3 (conventional enzymatic hydrolysis).

[0093] Reagents: BCA protein quantification kit, SDS-PAGE gel (4-20% gradient gel), molecular weight standards (5-100kDa), acetonitrile (HPLC grade), trifluoroacetic acid (TFA).

[0094] equipment: ELISA reader (BCA detection); High performance liquid chromatography (HPLC, C18 column, 0.1% TFA / acetonitrile gradient elution); SDS-PAGE electrophoresis system; Mass spectrometer (MALDI-TOF / TOF).

[0095] Experimental steps: Peptide yield determination: Take 1 mL of the enzyme hydrolysate and centrifuge (12000 rpm, 10 minutes) to remove unhydrolyzed residue.

[0096] The total peptide concentration in the supernatant was determined using a BCA kit, and the peptide yield was calculated. Yield (%) = (total peptide mass after hydrolysis / initial gelatin mass) × 100%.

[0097] Molecular weight distribution analysis: SDS-PAGE: Mix 20 μL of enzyme digest with loading buffer, boil for 5 minutes, load onto gel, and stain with Coomassie Brilliant Blue after electrophoresis.

[0098] HPLC: The enzymatic hydrolysate was filtered through a 0.22 μm filter membrane and injected at a flow rate of 1 mL / min. The detection wavelength was 214 nm. The peptide elution peaks were recorded and the molecular weight percentages were calculated.

[0099] Gly-Pro-X sequence identification: The enzymatic hydrolysate was lyophilized and then reconstituted in 0.1% TFA, followed by purification using a C18 desalting column.

[0100] Peptide sequences were analyzed using MALDI-TOF / TOF mass spectrometry, and the proportion of peptides containing Gly-Pro-X was identified by matching with the Mascot database.

[0101] Data processing: The molecular weight distribution range was calculated based on the HPLC elution time and the standard curve. Gly-Pro-X percentage = (number of peptides containing the target sequence / total number of identified peptides) × 100%.

[0102] Table 3: Summary of Enzymatic Hydrolysis Efficiency and Peptide Analysis Data Peptide yield: The yields of Examples 1-3 (79-84%) were significantly higher than those of Comparative Examples 2 (62.5%) and 3 (54.3%), demonstrating that the synergistic effect of the dual-enzyme system and physical field can improve hydrolysis efficiency.

[0103] Molecular weight distribution: In Examples 1-3, the proportion of <5kDa peptides was >73%, while in Comparative Example 3 (traditional enzymatic hydrolysis) it was only 38.9%, indicating that microwave-ultrasound synergy can effectively control the molecular weight of peptides.

[0104] Gly-Pro-X sequence percentage: In Examples 1-3, the target sequence accounted for >65%, while in Comparative Example 2 (single enzyme) it was only 43.6%, verifying the targeted cleavage of the collagen feature sequence (Gly-Pro-X) by the flavor protease.

[0105] Test Example 4: Explanation of the Experiment on Structural Stability and Activity Retention Experimental materials and equipment Test samples: Packaging film samples from Examples 1-3, Comparative Example 4 (ordinary hydrolyzed peptide), and Comparative Example 6 (single drying).

[0106] Reagents: Phosphate-buffered saline (PBS, pH 6.8); Escherichia coli bacterial suspension (1×10) 6 (CFU / mL) Circular dichroism (CD) buffer (10 mM phosphate, pH 7.0).

[0107] equipment: Constant temperature incubator (60℃ accelerated experiment); Scanning electron microscope (SEM, accelerating voltage 5kV); Circular dichroism chromatograph (wavelength 190-260nm); Ultraviolet spectrophotometer (OD600 detection).

[0108] Experimental steps Antimicrobial peptide thermal stability test: The membrane sample (0.5g) was sealed in an aluminum foil bag and placed in a 60℃ constant temperature oven for accelerated aging for 7 days.

[0109] After removal, soak in 10 mL of PBS buffer (37℃, 120 rpm for 2 hours), centrifuge and collect the supernatant.

[0110] The inhibition rate of the supernatant against Escherichia coli was determined (method as in Test Example 1), and the activity retention rate was calculated: Activity retention rate (%) = (inhibition rate after acceleration / initial inhibition rate) × 100%.

[0111] Membrane surface morphology analysis (SEM): The membrane sample was subjected to liquid nitrogen brittle fracture and then sputter-coated with gold (10 nm thickness).

[0112] SEM was used to observe the microstructure at the fracture surface (magnified 5000 times), and the porosity was statistically analyzed using ImageJ software.

[0113] Secondary structure analysis of antimicrobial peptides (circular dichroism chromatogram): Take a membrane sample (0.1g) and dissolve it in 10mL LCD buffer. Centrifuge (12000rpm, 10min) to remove insoluble matter.

[0114] The proportions of α-helices, β-sheets, and random coils were calculated using a circular dichroism spectroscopy system (1 mm optical path, 50 nm / min scanning rate) (fitted using CDPro software).

[0115] Table 4: Summary of Structural Stability and Activity Retention Test Data Thermal stability: Examples 1-3 showed an activity retention rate of >85%, while Comparative Example 4 (ordinary hydrolyzed peptide) showed only 43.2%, indicating that small molecule antimicrobial peptides (<5kDa) are more resistant to thermal degradation.

[0116] Comparative Example 6 (single drying) showed a retention rate of 65.4%, demonstrating that the gradient drying process can reduce the damage to heat-sensitive components.

[0117] Surface morphology: The surface porosity of the example (11-15%) was significantly lower than that of Comparative Example 4 (28.7%), indicating that the antimicrobial peptide-composite reinforcing agent system can maintain the compactness of the membrane structure.

[0118] Secondary structure: The α-helix content of the examples (29-32%) was higher than that of the comparative examples (<25%), indicating that the targeted enzymatically hydrolyzed peptides are more likely to form a stable conformation.

[0119] Test Example 5: Experimental Description for Verifying the Dispersibility of Composite Reinforcing Agents Experimental materials and equipment: Test samples: film-forming solutions and dried film samples of Examples 1-3, Comparative Example 1 (without composite reinforcing agent), and Comparative Example 5 (physically mixed reinforcing agent).

[0120] Reagents: 2% phosphotungstic acid (negative staining agent); Anhydrous ethanol (for cleaning TEM samples).

[0121] equipment: Transmission electron microscope (TEM, accelerating voltage 120kV); Atomic force microscopy (AFM, tapping mode, silicon probe). High-speed centrifuge (maximum 12000 rpm); UV-Vis spectrophotometer (OD) 600 (Detection).

[0122] Experimental steps TEM observation of dispersion uniformity: Add 10 μL of film-forming solution to a copper grid, let it stand for 5 minutes, and then use filter paper to absorb the excess liquid.

[0123] Add 2% phosphotungstic acid to stain for 1 minute, then rinse and air dry.

[0124] TEM observation of the distribution of nanocellulose (at least 5 fields of view), and statistical analysis of the proportion of aggregates (area > 1 μm). 2 (The percentage of aggregates).

[0125] AFM interface bonding strength test: The dried film sample was cut into 1×1cm² cubes and fixed on the sample stage.

[0126] The adhesion force (nN level) between nanocellulose and gelatin matrix was measured by scanning the surface (5×5μm² area) with an AFM probe at a constant rate (0.5Hz).

[0127] The average adhesion force was calculated by randomly selecting 10 points.

[0128] Centrifugal sedimentation rate test: Take 10 mL of film-forming solution (not dried) into a centrifuge tube and centrifuge at 6000 rpm for 15 minutes.

[0129] Take the supernatant and measure the OD value at 600 nm to calculate the precipitation rate: Sedimentation rate (%) = (initial OD) 600 -Supernatant OD 600 ) / Initial OD 600 ×100%.

[0130] Table 5: Summary of Dispersibility Test Data for Composite Reinforcing Agents TEM aggregate ratio: In Examples 1-3, the proportion of aggregates was <10%, while significant aggregation was observed in Comparative Examples 1 (36.5%) and 5 (27.4%), demonstrating that the pre-adsorption-covalent crosslinking process can inhibit the aggregation of nanocellulose.

[0131] AFM adhesion: The adhesion strength of the example (48-55 nN) was much higher than that of the comparative example (<30 nN), indicating that covalent cross-linking significantly enhances the interfacial bonding strength between nanocellulose and gelatin.

[0132] Centrifugal sedimentation rate: The sedimentation rate of the examples was <7%, while that of comparative examples 1 and 5 was as high as 32.7% and 24.9%, respectively, indicating that physically mixed or unmodified nanocellulose is prone to sedimentation.

[0133] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An edible antibacterial packaging film made from tilapia skin gelatin hydrolyzed peptides, characterized in that, Based on the percentage of total solids by mass, it includes the following components: Fish skin gelatin: 55-65%; Antimicrobial peptides: 18-22%, wherein the molecular weight of the antimicrobial peptides is less than 5 kDa, and they are prepared by microwave-ultrasound synergistic directional enzymatic hydrolysis of gelatin; Tea polyphenols: 0.8-1.2%; Nanocellulose: 0.8-1.2%; Citric acid: 0.8-1.2%; The remainder is deionized water.

2. The edible antibacterial packaging film of tilapia skin gelatin hydrolyzed peptides according to claim 1, characterized in that, In the preparation of the antimicrobial peptide: The microwave parameters are 2.45GHz, 300-500W, and the pulse mode is on for 25-35 seconds and off for 8-12 seconds. The ultrasound parameters are 18-22kHz and 180-220W, and are activated synchronously with the microwave pulse.

3. The edible antibacterial packaging film of tilapia skin gelatin hydrolyzed peptides according to claim 1, characterized in that, The tea polyphenols and nanocellulose form a composite reinforcing agent through pre-adsorption and covalent cross-linking, wherein: The mass ratio of tea polyphenols to nanocellulose is 0.8:1-1.2:1; Covalent crosslinking was performed using genipin crosslinking agent, with an addition amount of 0.5-1.5% of the mass of tea polyphenols. The crosslinking conditions were pH 3.8-4.2, temperature 45-55℃, and time 0.8-1.2 hours.

4. The edible antibacterial packaging film of tilapia skin gelatin hydrolyzed peptides according to claim 1, characterized in that, The nanocellulose has a diameter of 20-50 nm and is dispersed in a citrate buffer solution with a pH of 3.5-4.

5.

5. The edible antibacterial packaging film of tilapia skin gelatin hydrolyzed peptides according to claim 1, characterized in that, The citric acid is used to adjust the pH of the film-forming solution to 3.5-4.

5.

6. A method for preparing an edible antibacterial packaging film of hydrolyzed tilapia skin gelatin peptides, according to any one of claims 1-5, characterized in that, Includes the following steps: Step 1, Pre-treatment of tilapia skin: Degreasing and demineralization are performed sequentially; Step 2, Microwave-Ultrasound Synergistic Enzymatic Hydrolysis of Gelatin: Antimicrobial peptides are prepared by directional enzymatic hydrolysis using a dual-enzyme system under pulsed microwave (2.45 GHz, 300-500 W) and synchronous ultrasound (18-22 kHz, 180-220 W) conditions. Step 3: Preparation of composite reinforcing agent: Covalently crosslink tea polyphenols and nanocellulose at a mass ratio of 0.8:1-1.2:1; Step 4: Prepare the film-forming solution: Mix gelatin, antimicrobial peptides, composite enhancers and citric acid, and adjust the pH to 3.5-4.5; Step 5, Gradient drying: Microwave drying and vacuum drying are performed sequentially to form a packaging film.

7. The method for preparing an edible antibacterial packaging film of tilapia skin gelatin hydrolysate peptides according to claim 6, characterized in that, In step 1: The degreasing treatment uses 0.4-0.6M NaOH solution, with a material-to-liquid ratio of 1:8-1:12 (w / v), and is stirred at 25-30℃ for 1.5-2.5 hours. The demineralization treatment uses 0.2-0.4M HCl solution with a material-to-liquid ratio of 1:6-1:10 (w / v) and is carried out at 25-30℃ for 0.8-1.2 hours.

8. The method for preparing an edible antibacterial packaging film of tilapia skin gelatin hydrolysate peptides according to claim 6, characterized in that, In step 2: Enzymatic hydrolysis employs a dual-enzyme system of alkaline protease and flavor protease, with addition amounts of 0.8-1.2% and 0.4-0.6% of the gelatin mass, respectively. The enzymatic hydrolysis temperature was gradually reduced from 58-62℃ to 53-57℃, with a total time of 30-60 minutes.

9. The method for preparing an edible antibacterial packaging film of tilapia skin gelatin hydrolysate peptides according to claim 6, characterized in that, Step 5, gradient drying, includes: First stage microwave drying: microwave power 280-320W, temperature gradually reduced from 38-42℃ to 33-37℃, time 4-6 minutes; The second stage is vacuum drying: the vacuum pressure is reduced from -0.07-0.09 MPa to -0.09-0.11 MPa, the temperature is 23-27℃, and the time is 8-12 minutes.

10. The method for preparing an edible antibacterial packaging film of tilapia skin gelatin hydrolysate peptides according to claim 6, characterized in that, Step 3 involves the preparation of the composite reinforcing agent, including: Disperse nanocellulose in a citrate buffer solution with a pH of 3.5-4.5 and sonicate for 8-12 minutes; After adding tea polyphenols and genipin crosslinking agent, stir at 55-65℃ for 1.5-2.5 hours, and collect the complex by centrifugation.

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