Method for preparing fish gelatin nanoparticle collagenous fiber slow-release antioxidant film loaded with ascorbic acid

Abstract

The invention discloses a preparation method of an ascorbic acid-loaded fish gelatin nanoparticle / collagenous fiber slow-release antioxidant film, which comprises the following steps: preparing ascorbic acid-loaded fish gelatin nanoparticles; extracting collagen fibers; preparing a film forming solution; and drying the film-forming solution to obtain the anti-oxidation film. The obtained anti-oxidation film is good in barrier property and mechanical property, has excellent anti-oxidation and slow-release effects, and is simple in preparation process, good in raw material biocompatibility, environment-friendly and capable of being widely applied to the field of food. The preparation method not only contributes to the preparation of the food packaging film with antioxidant activity, but also solves the problem of environmental pollution caused by the fishery waste, and provides theoretical guidance significance for improving the comprehensive utilization of the fishery waste.

Description

Technical Field

[0001] The present invention relates to a preparation method of a slow-release antioxidant film of mirror carp gelatin nanoparticles / collagen fibers loaded with ascorbic acid, belonging to the technical field of food packaging. Background Art

[0002] Fresh meat has seen a year-on-year increase in consumption recognition and demand due to its freshness and high nutritional value. However, fresh meat is perishable. Although cold-chain transportation has been adopted to maintain low temperature in the actual supply chain, its freshness is still prone to decline, losing its original nutritional value and even harming human health. In recent years, the preservation effect of active packaging films has received extensive attention: films / coatings containing antioxidants or antibacterial agents are used for meat food packaging in an attempt to preserve the meat. However, due to the direct combination of antioxidants or antibacterial agents with the film matrix, their rapid release in the food system results in a poor effect of extending the shelf life.

[0003] Collagen extracted from fish waste has a good effect of blocking oxygen and water vapor, which is beneficial to reducing the water loss of refrigerated foods and inhibiting food spoilage caused by air. In addition, improper disposal of fishery waste can cause environmental pollution. According to the statistics of the Food and Agriculture Organization of the United Nations, more than 20 million tons of fishery waste are generated every year. Therefore, using collagen in fish waste to prepare protein-based packaging materials can not only alleviate the environmental pollution problems brought by petroleum-based packaging materials, but also provide an opportunity to turn fishery waste into treasure. In biological tissues, collagen often exists in the form of collagen fibers, aggregating in a complex, hierarchical manner and finally forming observable macroscopic fibers and networks in tissues, bones and basement membranes. Due to its such complex multi-scale structure, collagen fibers exhibit strong tensile strength, excellent elasticity and good biodegradability, and are suitable for manufacturing various biomaterials, including sausage casings, biocompatible scaffolds, hydrogels and packaging films, etc. However, during its extraction process, the film-forming performance of collagen fibers becomes poor, and its antioxidant activity is low, and the preservation effect is not good, which limits its application in food packaging films.

[0004] Ascorbic acid (AA), commonly known as vitamin C, is widely used as a vitamin supplement and antioxidant in food. It can also combine with polymers to form highly active biopolymeric materials, such as packaging films. However, the controlled-release effect of the active film directly obtained by combining ascorbic acid with polymers is poor, which limits its application in extending the shelf life of food. As a hydrolyzed derivative of collagen, fish gelatin nanoparticles (FGNP) can not only improve the film-forming properties of collagen fibers but also encapsulate active substances. Therefore, the preparation of collagen fiber-based antioxidant films using ascorbic acid-loaded fish gelatin nanoparticles (AA-FGNP) has strong research potential. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of a slow-release antioxidant film of mirror carp fish gelatin nanoparticles / collagen fibers loaded with ascorbic acid. The antioxidant film prepared by this method can achieve the slow-release effect of antioxidants, extend the antioxidant effect of the antioxidant film, and has a simple preparation process and green and environmentally friendly raw materials.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] Step 1: Extraction of collagen fibers:

[0008] Fresh mirror carp fish skin is washed with ice water and soaked in 0.1 mol / L NaOH solution (1:10 w / v) for 36 h (the soaking solution is changed every 12 h) to remove non-collagen proteins. Then, the skin is rinsed with ice water until neutral and soaked in n-butanol solution (1:10 w / v) for 12 h to remove fat. The sample is extracted with 30 times the volume of 0.5 mol / L acetic acid solution for 48 h to expand the fish skin. The solution is centrifuged (10,000×g, 30 min), and the brown outer skin is removed by hand to obtain collagen fibers. Finally, the collagen fibers are homogenized at 4 °C for 30 s and freeze-dried for 24 h to obtain a dry sample.

[0009] Step 2: Preparation of AA-FGNP:

[0010] Fish skin gelatin powder (1.20 g) was dissolved in 25 mL of water, and continuously heated and stirred (50 °C, 800 rpm, about 1 h) to obtain a uniform gelatin sol. After adding 25 mL of acetone, the mixture was stirred for 30 s and left standing for half an hour to precipitate the high molecular weight part of gelatin. The precipitate containing the high molecular weight part was washed three times with distilled water (5 mL), and then redissolved in water (25 mL) to form a gelatin sol again. After stirring for 2 h, the pH value of the gelatin sol was adjusted to 2.5, ascorbic acid (0.12 g) was added, and then the mixture was transferred to a round-bottom flask in an oil bath. Subsequently, acetone (70 mL) was added dropwise to the solution at a rate of 100 mL / h using an automatic injection pump until a white colloid was formed. Then, 360 μL of glutaraldehyde was added to synthesize AA-FGNP. After reacting for 12 h, glycine solution was added to stop the reaction, acetone was removed by evaporation, and the precipitate was collected by centrifugation.

[0011] Step 3: Preparation of the film-forming solution for the antioxidant film:

[0012] S1. Dissolve a certain mass of collagen fibers in an acetic acid solution with a certain concentration, and mix well to obtain a collagen fiber film-forming solution;

[0013] S2. Dissolve a certain mass of AA-FGNP in the collagen fiber film-forming solution prepared in S1, and mix well to obtain the film-forming solution for the antioxidant film;

[0014] S3. Add a certain amount of glycerol to the collagen fiber film-forming solution prepared in S2 to play a plasticizing role.

[0015] Step 4: Casting and forming of the antioxidant film:

[0016] Place the film-forming solution for the antioxidant film obtained in Step 3 in an ultrasonic cleaner and process it for a certain time to remove air bubbles; take a certain volume of the film-forming solution and pour it onto the film embryo, and dry and remove the film under certain conditions to obtain the antioxidant film;

[0017] Preferably, the mass concentration of collagen fibers in the collagen fiber film-forming solution in Step 3 S1 is 0.5 - 2 g / mL.

[0018] Preferably, the concentration of the acetic acid solution in the collagen fiber film-forming solution in Step 3 S1 is 0.2 - 1 mol / L.

[0019] Preferably, the addition amount of AA-FGNP in the collagen fiber film-forming solution in Step 3 S2 is 5 - 20% (based on the weight of collagen fibers).

[0020] Preferably, the addition amount of glycerol in the collagen fiber film-forming solution in Step 3 S3 is 10 - 30%.

[0021] Preferably, the ultrasonic cleaning time in step two is 20 - 40 min, the volume of the film-forming solution is 50 mL, and the drying conditions are a temperature of 20 - 30 °C, a relative humidity of 40 - 50%, and a time of 36 - 60 h.

[0022] The present invention uses hydrophilic ascorbic acid active substances and fish gelatin nanoparticles to prepare AA - FGNP by a two - step desolvation method, and mixes it with a collagen fiber solution to prepare an antioxidant film. The active substance ascorbic acid is embedded in the film by the nanoparticles and can be used for the preservation of meat products. When meat exudes juice under refrigeration conditions, it stimulates the release of hydrophilic ascorbic acid. The more juice exudes from the meat, the more ascorbic acid is released, thus achieving an efficient, antioxidant, and slow - release meat preservation effect. The raw materials of this composite film are green and environmentally friendly, and the preparation process is simple. It is an environmentally friendly and superior - performance antioxidant packaging material with extremely high application value.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) Extremely high environmental friendliness and by - product utilization rate:

[0025] The fish skin gelatin used in the present invention is of natural origin, and the collagen fiber is extracted from the skin of mirror carp, which is extremely safe and environmentally friendly. Both are derived from fishery by - products, which can improve the low comprehensive utilization rate of fishery by - products and the high - value processing degree.

[0026] (2) It has dual effects of antioxidant and slow release:

[0027] The present invention uses fish gelatin nanoparticles to embed active ascorbic acid to form AA - FGNP, which has excellent antioxidant activity. The embedding rate of the nanoparticles can reach more than 65%, showing a spherical dense structure. After AA - FGNP and collagen fibers are mixed and cast into a film, ascorbic acid is embedded inside the nanoparticles, delaying the release of ascorbic acid. At the same time, AA - FGNP and collagen fibers have good compatibility and excellent biodegradability. Description of the Drawings

[0028] Figure 1 Particle size and morphology of the antioxidant film AA - FGNP prepared in Example 2;

[0029] Figure 2 Mechanical properties of the antioxidant film prepared in Example 2;

[0030] Figure 3 Antioxidant activity of the antioxidant film prepared in Example 2;

[0031] Figure 4 Slow - release performance of ascorbic acid in the antioxidant film prepared in Example 2 in food simulants (A and C: 50% ethanol, B and D: 95% ethanol);

[0032] Figure 5 Application effect of the antioxidant film prepared in Example 2 in fish meat. Detailed implementation manners

[0033] To make the objectives, technical solutions and beneficial effects of the present invention clearer, the following further describes the present invention in detail with reference to specific embodiments and the accompanying drawings. However, the protection scope of the present invention is not limited thereto;

[0034] Example 1:

[0035] Step 1: Extraction of fish skin collagen fibers:

[0036] The fresh mirror carp fish skin was washed with ice water and soaked in 0.1 mol / L NaOH solution (1:10 w / v) for 36 h (the soaking solution was changed every 12 h) to remove non-collagen proteins. Then, the skin was rinsed with ice water until neutral and soaked in n-butanol solution (1:10 w / v) for 12 h to remove fat. The sample was extracted with 30 volumes of 0.5 mol / L acetic acid solution for 48 h to swell the fish skin. The solution was centrifuged (10,000×g, 30 min), and the brown outer skin was removed to obtain collagen fibers. Finally, the collagen fibers were homogenized at 4°C for 30 s and freeze-dried for 24 h to obtain a dry sample.

[0037] Step 2: Preparation of AA-FGNP:

[0038] Fish skin gelatin powder (1.20 g) was dissolved in 25 mL of water, and continuously heated and stirred (50°C, 800 rpm, about 1 h) to obtain a uniform gelatin sol. After adding 25 mL of acetone, the mixture was stirred for 30 s and left to stand for half an hour to precipitate the high molecular weight part of gelatin. The precipitate containing the high molecular weight part was washed three times with distilled water (5 mL) and then redissolved in water (25 mL) to form a gelatin sol again. After stirring for 2 h, the pH value of the gelatin sol was adjusted to 2.5, ascorbic acid (0.12 g) was added, and then the mixture was transferred to a round-bottom flask in an oil bath. Then, acetone (70 mL) was added dropwise to the solution at a rate of 100 mL / h using an automatic injection pump until a white colloid was formed. Subsequently, 360 μL of glutaraldehyde was added to synthesize AA-FGNP. After reacting for 12 h, glycine solution was added to stop the reaction, acetone was removed by evaporation, and the precipitate was collected by centrifugation.

[0039] Step 3: Preparation of the film-forming solution for the antioxidant film:

[0040] S1. Take the collagen fibers and dissolve them in 0.2 mol / L acetic acid solution, and mix well to obtain a 0.5 g / mL collagen fiber film-forming solution;

[0041] S2. dissolving 5% (based on the weight of collagen fibers) of AA-FGNP in the collagen fiber film-forming solution prepared in S1, and mixing thoroughly to obtain an antioxidant film-forming solution;

[0042] S3. Add 10% (based on the weight of collagen fibers) of glycerol to the collagen fiber film-forming solution prepared in S2 to play a plasticizing role.

[0043] Step 4: Tape casting of antioxidant film:

[0044] The film-forming solution for preparing the antioxidant film obtained in step 3 is placed in an ultrasonic cleaner for 20 minutes to remove bubbles; 50 mL of the film-forming solution is poured onto the film embryo, dried at a relative humidity of 40% and a temperature of 20°C for 36 hours, and the film is peeled off to obtain the antioxidant film.

[0045] Embodiment 2:

[0046] Step 1: Extraction of fish skin collagen fibers:

[0047] Fresh mirror carp skin was washed with ice water and soaked in 0.1mol / LNaOH solution (1:10w / v) for 36h (the soaking solution was changed every 12h) to remove non-collagen. Then, the skin was rinsed with ice water until neutral and soaked again in n-butanol solution (1:10w / v) for 12h to remove fat. The sample was extracted with 30 times the volume of 0.5mol / L acetic acid solution for 48h to swell the fish skin. The solution was centrifuged (10,000×g, 30min), the brown outer skin was removed, and collagen fibers were obtained. Finally, the collagen fibers were homogenized at 4°C for 30s and freeze-dried for 24h to obtain a dry sample.

[0048] Step 2: Preparation of AA-FGNPs:

[0049] Fish skin gelatin powder (1.20 g) was dissolved in 25 mL of water and heated and stirred continuously (50 ° C, 800 rpm, about 1 h) to obtain a uniform gelatin sol. After adding 25 mL of acetone, the mixture was stirred for 30 s and allowed to stand for half an hour to precipitate the high molecular weight portion of gelatin. The precipitate containing the high molecular weight portion was washed three times with distilled water (5 mL) and then redissolved in water (25 mL) to form a gelatin sol again. After stirring for 2 h, the pH value of the gelatin sol was adjusted to 2.5, ascorbic acid (0.12 g) was added, and the mixture was moved to a round-bottom flask in an oil bath. Next, acetone (70 mL) was added dropwise to the solution at a rate of 100 mL / h using an automatic syringe pump until a white colloid was formed. Subsequently, 360 μL of glutaraldehyde was added to synthesize AA-FGNPs. After reacting for 12 h, glycine solution was added to stop the reaction, acetone was evaporated to remove, and the precipitate was collected by centrifugation.

[0050] Step 3: Prepare the film-forming solution of the antioxidant film:

[0051] S1. Dissolve collagen fibers in 0.5 mol / L acetic acid solution and mix well to obtain a collagen fiber film-forming solution with a concentration of 1 g / mL.

[0052] S2. Dissolve 10% (based on the weight of collagen fibers) of AA-FGNP in the collagen fiber film-forming solution prepared in S1 and mix well to obtain a film-forming solution for the antioxidant film.

[0053] S3. Add 20% (based on the weight of collagen fibers) of glycerol to the collagen fiber film-forming solution prepared in S2 to play a plasticizing role.

[0054] Step Four: Casting and forming of the antioxidant film:

[0055] Place the film-forming solution for the antioxidant film obtained in Step Three in an ultrasonic cleaner and process for 30 min to remove air bubbles; take 50 mL of the film-forming solution and pour it onto the film embryo, and dry it for 48 h at a relative humidity of 45% and a temperature of 25 °C, then peel off the film to obtain the antioxidant film.

[0056] Example 3:

[0057] Step One: Extraction of fish skin collagen fibers:

[0058] Wash fresh mirror carp fish skin with ice water and soak it in 0.1 mol / L NaOH solution (1:10 w / v) for 36 h (change the soaking solution every 12 h) to remove non-collagen proteins. Then, rinse the skin with ice water until neutral and soak it in n-butanol solution (1:10 w / v) for 12 h to remove fat. Extract the sample with 30 times the volume of 0.5 mol / L acetic acid solution for 48 h to swell the fish skin. Centrifuge the solution (10,000×g, 30 min) to remove the brown outer skin and obtain collagen fibers. Finally, homogenize the collagen fibers at 4 °C for 30 s and freeze-dry for 24 h to obtain a dry sample.

[0059] Step Two: Preparation of AA-FGNP:

[0060] Fish skin gelatin powder (1.20 g) was dissolved in 25 mL of water, and continuously heated and stirred (50 °C, 800 rpm, about 1 h) to obtain a uniform gelatin sol. After adding 25 mL of acetone, the mixture was stirred for 30 s and left standing for half an hour to precipitate the high molecular weight part of the gelatin. The precipitate containing the high molecular weight part was washed three times with distilled water (5 mL), and then redissolved in water (25 mL) to form a gelatin sol again. After stirring for 2 h, the pH value of the gelatin sol was adjusted to 2.5, ascorbic acid (0.12 g) was added, and then the mixture was transferred to a round-bottom flask in an oil bath. Subsequently, acetone (70 mL) was added dropwise to the solution at a rate of 100 mL / h using an automatic injection pump until a white colloid was formed. Then, 360 μL of glutaraldehyde was added to synthesize AA-FGNP. After reacting for 12 h, a glycine solution was added to stop the reaction, acetone was removed by evaporation, and the precipitate was collected by centrifugation.

[0061] Step 3: Preparation of the film-forming solution for the antioxidant film:

[0062] S1. Collagen fibers were dissolved in 1 mol / L acetic acid solution and fully mixed to obtain a collagen fiber film-forming solution of 2 g / mL;

[0063] S2. 20% (based on the weight of collagen fibers) of AA-FGNP was dissolved in the collagen fiber film-forming solution prepared in S1 and fully mixed to obtain the film-forming solution for the antioxidant film;

[0064] S3. 30% (based on the weight of collagen fibers) of glycerol was added to the collagen fiber film-forming solution prepared in S2 to play a plasticizing role.

[0065] Step 4: Casting and forming of the antioxidant film:

[0066] The film-forming solution for the antioxidant film obtained in Step 3 was placed in an ultrasonic cleaner and treated for 40 min to remove air bubbles; 50 mL of the film-forming solution was poured onto the film embryo and dried at a relative humidity of 50% and a temperature of 30 °C for 60 h, and then the film was peeled off to obtain the antioxidant film.

[0067] Performance test of a slow-release antioxidant film of mirror carp gelatin nanoparticles / collagen fibers loaded with ascorbic acid prepared in Example 2;

[0068] (1) Determination of the particle size and morphology of AA-FGNP:

[0069] The FGNP sample (0.1 mg / mL) was dissolved in distilled water, and the particle size of the nanoparticles was measured using a nanoparticle size and zeta potential analyzer (Malvern Instruments Ltd., Malvern, UK). The morphology of the nanoparticles was imaged using a transmission electron microscope (TEM) (Hitachi HT-7800, Hitachi, Tokyo, Japan).

[0070] (2) Analysis of the particle size and morphology results of AA-FGNP:

[0071] As Figure 1 shown, AA-FGNP are spherical nanoparticles with a particle size of 169.26 nm.

[0072] (3) Mechanical property testing:

[0073] The film samples were cut into rectangles (5 cm × 1 cm), and a tensile test was performed using a texture analyzer (TA-XTplusC, Stable Micro System, UK). The calculation formulas for the tensile strength and elongation at break are as follows:

[0074]

[0075] (4) Analysis of the mechanical property results:

[0076] As Figure 2 shown, the effect of the addition amount of AA-FGNP on the mechanical properties of the composite film. Compared with the pure collagen fiber film, after adding AA-FGNP, the tensile strength and elongation at break of the antioxidant film increased by 39.31 and 63.19%, respectively.

[0077] (5) Antioxidant property testing:

[0078] ABTS radical scavenging rate: The film samples were chopped and completely dissolved in 7 mL of distilled water (60 °C), then 3 mL of ethanol was added, and the supernatant was obtained by centrifugation (10000×g, 10 min). An ABTS cation solution was prepared by reacting a 7 mmol / L ABTS solution with 2.45 mM potassium persulfate (1:1, v / v) in the dark for 12 h. The ABTS cation solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. 0.5 mL of the supernatant of different film samples was added to 4.5 mL of the ABTS solution, and the reaction was carried out at room temperature for 6 min. The absorbance value (As) was measured at 734 nm using a 720G visible spectrophotometer. At this time, 0.5 mL of methanol was used as a blank to measure the absorbance value (A0). The scavenging rate calculation formula is:

[0079]

[0080] DPPH radical scavenging rate: Mix 0.5 mL of the supernatant of different film samples with 4.5 mL of a 0.1 mM DPPH ethanol solution thoroughly, and place it in the dark at room temperature for 30 min, then centrifuge (8000×g, 5 min) to obtain the supernatant. At a wavelength of 517 nm, read the absorbance (As) of the supernatant. At this time, use 0.5 mL of methanol as a blank to measure the absorbance value (A0). The calculation formula for the DPPH radical scavenging rate of the sample is as follows:

[0081]

[0082] (6) Analysis of antioxidant performance results:

[0083] Figure 4 Show the antioxidant activity of the AA-FGNP / collagen composite film. Compared with the pure collagen fiber film, after adding AA-FGNP, the DPPH radical scavenging rate and ABTS radical scavenging rate of the composite film increased from 15.99 and 24.81% to 81.6 and 87.4% respectively.

[0084] (7) Sustained-release performance test:

[0085] Place the film sample (2×2 cm) in a brown bottle containing 60 mL of food simulants (simulant A: 50% ethanol and simulant B: 95% ethanol), and store it in the dark at 25 and 4 °C for 132 h respectively. Collect the food simulant (0.5 mL) and measure the AA content. The calculation formula for the cumulative release amount of AA is as follows:

[0086]

[0087] Among them, Mt is the content of AA in the food simulant, and M0 is the initial content of AA in the film.

[0088] (8) Analysis of sustained-release performance results:

[0089] Figure 5Show the sustained-release performance of the AA-FGNP / collagen composite film. Ethanol with concentrations of 50% (representing semi-fat foods) and 95% (representing fatty foods) was used as food simulants to evaluate the release behavior of ascorbic acid in the film at 25 °C and 4 °C. All samples showed rapid release within the first 24 h, followed by continuous slow release during the remaining time. Meanwhile, the release rate of the composite film containing AA-FGNP was significantly lower than that of the film containing only AA (P<0.05). This phenomenon indicates that the unencapsulated AA in the nanoparticles was released rapidly first, while the AA encapsulated in FGNP required a longer release time. In addition, a high ambient temperature was beneficial to the release of active substances, and the release rate and amount of AA at 25 °C were higher than those of the samples at 4 °C. However, the film containing AA-FGNP still showed a sustained-release effect. Interestingly, the release rate of AA in 50% ethanol was higher than that in 95% ethanol. This is because the surface wettability and swelling property of the film were relatively good, and ascorbic acid is hydrophilic, thus improving the release of ascorbic acid in a relatively hydrophilic environment.

[0090] Application effect of a sustained-release antioxidant film of mirror carp gelatin nanoparticles / collagen fibers loaded with ascorbic acid prepared in Example 2 in fish meat;

[0091] Cut the mirror carp muscle into fish slices of 4 cm×3 cm×1.5 cm, wash them clean with deionized water, and divide them into two treatment groups: an unpacked control group and a treatment group wrapped with an AA-FGNP / collagen antioxidant film. All samples were stored in a 4 °C refrigerator for 0 d, 2 d, 4 d, 6 d, 8 d, and 10 d, and various indicators were measured.

[0092] (1) Determination of fish meat freshness:

[0093] The total volatile salt-based nitrogen (TVB-N) of fish meat was measured using a Kjeldahl nitrogen analyzer by the semi-micro Kjeldahl method referring to the national standard GB 5009.228-2016, and the results were expressed as mg / 100 g.

[0094] (2) Analysis of fish meat freshness results:

[0095] As Figure 5 shown, with the extension of refrigeration time, the TVB-N value of fish meat showed a significant increasing trend (P<0.05). Especially after the storage time of the control group exceeded 6 d, it exceeded the acceptable TVB-N value (15 mg / 100 g) of freshwater fish, indicating that the fish meat in the control group was spoiled at this time. However, when the refrigeration time of the AA-FGNP / collagen antioxidant film treatment group reached 10 d, the TVB-N value of the fish meat was 18.09 mg / 100 g, which was 28.07% less than that of the control group.

[0096] In summary, the present invention designs a preparation method of a sustained-release antioxidant film of mirror carp gelatin nanoparticles / collagen fibers loaded with ascorbic acid. All raw materials are of natural origin, and it is a green and environmentally friendly antioxidant film. By improving the properties of the collagen fiber film and encapsulating the active substances with fish gelatin nanoparticles, an antioxidant film with good performance and dual effects of antioxidant and sustained release can be achieved, thereby ensuring the wide application of the packaging film in foods (especially meat foods).

[0097] Note: The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention shall be covered by the scope of the claims of the present invention.

Claims

1. A method for preparing a mirror carp gelatin nanoparticle / collagen fiber sustained-release antioxidant film loaded with ascorbic acid, characterized in that: The following steps are involved: Step 1: Extraction of collagen fibers: Fresh mirror carp skin was washed with ice water and soaked in 0.1mol / LNaOH solution (1:10w / v) for 36h (the soaking solution was changed every 12h) to remove non-collagen. Then, the skin was rinsed with ice water until neutral and soaked again in n-butanol solution (1:10w / v) for 12h to remove fat. The sample was extracted with 30 times the volume of 0.5mol / L acetic acid solution for 48h to swell the fish skin. The solution was centrifuged (10,000×g, 30min), and the brown outer skin was removed by hand to obtain collagen fibers. Finally, the collagen fibers were homogenized at 4°C for 30s and freeze-dried for 24h to obtain a dry sample. Step 2: Preparation of AA-FGNPs: Fish skin gelatin powder (1.20 g) was dissolved in 25 mL of water and heated and stirred continuously (50 ° C, 800 rpm, about 1 h) to obtain a uniform gelatin sol. After adding 25 mL of acetone, the mixture was stirred for 30 s and allowed to stand for half an hour to precipitate the high molecular weight portion of gelatin. The precipitate containing the high molecular weight portion was washed three times with distilled water (5 mL) and then redissolved in water (25 mL) to form a gelatin sol again. After stirring for 2 h, the pH value of the gelatin sol was adjusted to 2.5, ascorbic acid (0.12 g) was added, and the mixture was moved to a round-bottom flask in an oil bath. Next, acetone (70 mL) was added dropwise to the solution at a rate of 100 mL / h using an automatic syringe pump until a white colloid was formed. Subsequently, 360 μL of glutaraldehyde was added to synthesize AA-FGNPs. After reacting for 12 h, glycine solution was added to stop the reaction, acetone was evaporated to remove, and the precipitate was collected by centrifugation. Step 3: Prepare the film-forming solution of the antioxidant film: S1. dissolving a certain mass of collagen fibers in an acetic acid solution of a certain concentration and mixing them thoroughly to obtain a collagen fiber film-forming solution; S2. dissolving a certain amount of AA-FGNP in the collagen fiber film-forming solution prepared in S1, and mixing thoroughly to obtain an antioxidant film-forming solution; S3. Add a certain amount of glycerol to the collagen fiber film-forming solution prepared in S2 to play a plasticizing role. Step 4: Tape casting of antioxidant film: The film-forming solution for preparing the antioxidant film obtained in step three is placed in an ultrasonic cleaner for a certain period of time to remove bubbles; a certain volume of the film-forming solution is poured onto the film embryo, and the antioxidant film is obtained by drying and peeling off the film under certain conditions.

2. The preparation method according to claim 1, characterized in that: Step 3: The mass concentration of collagen fibers in the S1 collagen fiber membrane-forming solution is 0.5-2 g / mL.

3. The preparation method according to claim 1, characterized in that: Step 3: The concentration of the acetic acid solution in the S1 collagen fiber membrane-forming solution is 0.2-1 mol / L.

4. The preparation method according to claim 1, characterized in that: Step 3: The amount of AA-FGNP added to the S2 collagen fiber film-forming solution is 5-20% (based on the weight of the collagen fibers).

5. The preparation method according to claim 1, characterized in that: Step 3: The amount of glycerol added to the S3 collagen fiber membrane-forming solution is 10-30%.

6. The preparation method according to claim 1, characterized in that: The ultrasonic cleaning time in step 2 is 20 to 40 minutes, the volume of the film-forming solution is 50 mL, and the drying conditions are temperature 20 to 30° C., relative humidity 40 to 50%, and time 36 to 60 hours.

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