Degreased rice bran carbon dot-gelatin-glucan three-layer film as well as preparation method and application thereof

Abstract

The invention relates to the technical field of packaging films, in particular to a defatted rice bran carbon dot-gelatin-glucan three-layer film as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing defatted rice bran powder with deionized water, and stirring and mixing at room temperature to obtain a mixed solution; transferring the mixed solution into a hydrothermal reaction kettle, carrying out hydrothermal reaction, naturally cooling, and carrying out centrifugal operation; collecting supernate, uniformly mixing the supernate with absolute ethyl alcohol, precipitating impurities, and collecting filtrate; concentrating the filtrate under reduced pressure, filtering the concentrated solution through a microfiltration membrane, dialyzing the filtrate, and freeze-drying to obtain defatted rice bran carbon dots; degreased rice bran carbon dots are added into a gelatin-dextran film forming solution to be stirred to form a gelatin-dextran film forming solution containing DRB-CDs, and then a layer-by-layer assembly method is adopted. The invention also relates to application of the three-layer film in preservation of aquatic products. The three-layer membrane disclosed by the invention can delay lipid oxidation and microbial proliferation, and shows good application prospects in the aspects of prolonging the shelf life of aquatic products and maintaining the quality of the aquatic products.

Description

Technical Field

[0001] The present invention relates to the technical field of packaging films, and particularly relates to a defatted rice bran carbon dots - gelatin - dextran three - layer film and its preparation method and application. Background Art

[0002] Aquatic products are extremely prone to spoilage and deterioration during processing, transportation, and storage due to factors such as the environment, endogenous enzymes, microorganisms, and lipid oxidation, resulting in food resource waste and economic losses, and even triggering food safety problems. Therefore, improving aquatic product preservation technology and maintaining the nutritional quality of aquatic products are key issues that need to be urgently solved. Currently, antibacterial and antioxidant degradable active packaging designed for two inducements of aquatic product deterioration, namely microbial reproduction and unsaturated lipid oxidation, has achieved remarkable results in the field of aquatic product preservation. The substrates of degradable active packaging mainly consist of biopolymers such as proteins and polysaccharides. Gelatin is a partial hydrolysis product of collagen, and dextran is an extracellular polysaccharide produced by lactic acid bacteria. Both have good safety, compatibility, degradability, and film - forming properties. Degradable active packaging films can be divided into single - layer, double - layer, and three - layer films. The outer layer of the three - layer film plays a role in blocking the external environment and preventing the leakage and loss of active substances in the middle layer. The middle layer is responsible for loading active substances, while the inner layer is responsible for improving the controlled - release effect of active substances. Therefore, three - layer films usually have better mechanical, barrier, and controlled - release properties than single - layer and double - layer films.

[0003] Current three - layer films include gelatin / gelatin - dextran / gelatin three - layer film loaded with citronellal and α - tocopherol, flaxseed gum / chitosan / flaxseed gum three - layer film loaded with eugenol and laurel essential oil, sodium alginate / chitosan - cinnamomum cassia essential oil / sodium alginate three - layer film, and gelatin / chitosan - nisin / corn starch three - layer film. However, large - sized active substances are hindered in the dense matrix of degradable active packaging films. Therefore, it is of great significance to develop a degradable active packaging film that can load nano - scale active ingredients. Summary of the Invention

[0004] The purpose of the present invention is to provide a defatted rice bran carbon dots - gelatin - dextran three - layer film and its preparation method and application, which can effectively solve the problem that large - sized active substances are hindered in the dense matrix of degradable active packaging films in the prior art.

[0005] To solve the above - mentioned technical problems, the present invention adopts the following technical solutions:

[0006] A defatted rice bran carbon dots - gelatin - dextran three - layer film, wherein the defatted rice bran carbon dots - gelatin - dextran three - layer film is obtained by loading defatted rice bran carbon dots in a gelatin / gelatin - dextran / gelatin three - layer film.

[0007] Meanwhile, the present invention also relates to a preparation method of a defatted rice bran carbon dots - gelatin - dextran three - layer film, comprising the following steps:

[0008] S1. Mix defatted rice bran powder with deionized water and stir at room temperature to obtain a mixed solution;

[0009] S2. Transfer the mixed solution to a hydrothermal reactor for hydrothermal reaction, react at 180 - 220 °C for 4 - 6 h, and perform centrifugation after natural cooling;

[0010] S3. Collect the supernatant and mix it with 3 - 5 times the volume of absolute ethanol to precipitate impurities, and collect the filtrate;

[0011] S4. Concentrate the filtrate under reduced pressure, then filter the concentrated solution through a microporous membrane, dialyze the filtrate, and obtain defatted rice bran carbon dots by freeze - drying;

[0012] S5. Add defatted rice bran carbon dots to the gelatin - dextran film - forming solution and stir to form a gelatin - dextran film - forming solution containing DRB - CDs. Then, cast in the order of one layer of gelatin film - forming solution, one layer of gelatin - dextran film - forming solution containing DRB - CDs, and one layer of gelatin film - forming solution to obtain a defatted rice bran carbon dot - gelatin - dextran three - layer film.

[0013] Further, in step S1, the mass - to - volume ratio of defatted rice bran powder to deionized water is 1∶4 - 6, and the stirring time is 4 - 6 hours.

[0014] Further, in step S2, the centrifugation conditions are centrifugation at 10000 rpm for 15 min.

[0015] Further, in step S4, the conditions for concentration under reduced pressure are: concentrate under reduced pressure at 50 °C until one - fifth of the original total volume; the dialysis conditions are: dialyze with a 500 Da dialysis bag for 48 h; the pore size of the microporous membrane is 0.22 μm.

[0016] Further, step S5 is specifically as follows:

[0017] S51. Mix gelatin with deionized water, and add glycerol as a plasticizer while stirring magnetically to prepare a gelatin film - forming solution;

[0018] S52. Mix gelatin, dextran with deionized water, and add glycerol as a plasticizer while stirring magnetically to prepare a gelatin - dextran film - forming solution;

[0019] S53. Add defatted rice bran carbon dots to the above - mentioned gelatin - dextran film - forming solution and stir magnetically to prepare a gelatin - dextran film - forming solution containing DRB - CDs;

[0020] S54. Cast the gelatin film-forming solution in a petri dish and dry it until a gelatin film is formed but the surface is still sticky; then continue to cast an equal volume of the gelatin-dextran film-forming solution containing DRB-CDs, and dry it until a gelatin-dextran film is formed but the surface is still sticky; then cast an equal volume of the gelatin film-forming solution again and dry it to obtain the defatted rice bran carbon dots-gelatin-dextran three-layer film.

[0021] In a further embodiment, in step S53, the weight percentages of the defatted rice bran carbon dots in the gelatin-dextran film-forming solution are 1-3% respectively, and this ratio is calculated based on the mass of the polymer in the gelatin-dextran film-forming solution.

[0022] In a further embodiment, the weight percentage of the defatted rice bran carbon dots in the gelatin-dextran film-forming solution is 3% respectively.

[0023] Finally, the present invention also relates to the application of the defatted rice bran carbon dots-gelatin-dextran three-layer film in the preservation of aquatic products.

[0024] In the defatted rice bran carbon dots-gelatin-dextran three-layer film provided by the above technical solution, DRB-CDs have small particle sizes, are rich in hydroxyl, carboxyl and amide groups on the surface, and have excellent ultraviolet absorption characteristics and biocompatibility (the survival rate is >95% after 72 hours of HaCaT cell incubation); the addition of DRB-CDs significantly improves the mechanical strength, water vapor barrier property, ultraviolet barrier property and thermal stability of the composite film, and optimizes the density of the composite film matrix through hydrogen bond interaction. The three-layer film exhibits the DRB-CD sustained release characteristic, and its release rate is negatively correlated with the ethanol concentration of the food simulant.

[0025] The composite film loaded with 3% DRB-CDs shows significant antioxidant activity (the ABTS and DPPH radical scavenging rates reach 72.56% and 50.24% respectively) and antibacterial ability (the inhibition rates against Staphylococcus aureus and Escherichia coli are 79.17% and 46.86% respectively).

[0026] The scallop cold storage experiment shows that the 3% composite film can effectively inhibit the increase of pH, TBARS, TVB-N and TVC, and delay lipid oxidation and microbial proliferation; the DRB-CDs enhanced gelatin-dextran three-layer film shows good application prospects in the extension of the shelf life and quality maintenance of aquatic products due to its multiple functional characteristics such as antioxidant, antibacterial and ultraviolet barrier. Description of the Drawings

[0027] Figure 1 It is the TEM image and particle size distribution diagram of DRB-CDs;

[0028] Figure 2UV-Vis absorption spectra of DRB-CDs (the insets in the figure show the colors of DRB-CDs under visible light and 365 nm);

[0029] Figure 3 Fluorescence emission spectra of DRB-CDs at different excitation wavelengths;

[0030] Figure 4 FTIR spectra of DRB-CDs;

[0031] Figure 5 XPS survey spectrum (A), C 1s fine spectrum (B), N 1s fine spectrum (C), and O 1s fine spectrum (D) of DRB-CDs;

[0032] Figure 6 XRD patterns of DRB-CDs;

[0033] Figure 7 Cell viability of HaCaT cells after incubation with different concentrations of DRB-CDs for 48 h and 72 h;

[0034] Figure 8 Scanning electron microscopy images of the surface (A) and cross-section (B) of composite films loaded with different contents of DRB-CDs;

[0035] Figure 9 FTIR spectra of composite films loaded with different contents of DRB-CDs;

[0036] Figure 10 TGA curves of composite films loaded with different contents of DRB-CDs;

[0037] Figure 11 XRD patterns of composite films loaded with different contents of DRB-CDs;

[0038] Figure 12 Appearance of composite films loaded with different contents of DRB-CDs;

[0039] Figure 13 UV / Vis transmittance of composite films loaded with different contents of DRB-CDs;

[0040] Figure 14 TS of composite films loaded with different contents of DRB-CDs;

[0041] Figure 15 EAB of composite films loaded with different contents of DRB-CDs;

[0042] Figure 16 Release rate of DRB-CDs from 3% composite films in food simulants;

[0043] Figure 17is the antioxidant capacity of composite films with different contents of DRB-CDs;

[0044] Figure 18 is the antibacterial ability of the composite film against Escherichia coli (A) and Staphylococcus aureus (B);

[0045] Figure 19 is the change of pH value during the cold storage of Patinopecten yessoensis under different packaging conditions;

[0046] Figure 20 is the change of TBARS value during the cold storage of Patinopecten yessoensis under different packaging conditions;

[0047] Figure 21 is the change of TVB-N during the cold storage of Patinopecten yessoensis under different packaging conditions;

[0048] Figure 22 is the change of TVC during the cold storage of Patinopecten yessoensis under different packaging conditions. Specific Embodiments

[0049] In order to make the purpose and advantages of the present invention more clear, the present invention will be specifically described below in conjunction with embodiments. It should be understood that the following text is only used to describe one or several specific implementation manners of the present invention, and does not strictly limit the specific protection scope claimed by the present invention.

[0050] 1 Materials and Methods

[0051] 1.1 Materials and Reagents

[0052] Prepare raw materials: defatted rice bran powder; HaCat cells, DMEM medium; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); gelatin (derived from fish), glycerol (98%), 2-thiobarbituric acid (98.5%); 1,1-diphenyl-2-picrylhydrazyl (DPPH, 98%), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, 98%), dextran (70000 Da): Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 6538); plate count agar, LB broth; fresh Patinopecten yessoensis, covered with crushed ice and transported to the laboratory within 1.5 h.

[0053] 1.2 Instruments and Equipment

[0054] JEM-2100F Transmission Electron Microscope; GeminiSEM 360 Scanning Electron Microscope; IRTracer 100 Fourier Transform Infrared Spectrometer; D8 Advance X-ray Diffractometer; Nexsa X-ray Photoelectron Spectrometer; STA2500 Thermogravimetric Analyzer; Zetasizer Nano ZS90 Zeta Potential Analyzer; U-3900 UV-Visible Spectrophotometer; F-7000 Fluorescence Spectrophotometer; SC-10 Color Difference Meter; TA.XTplus Texture Analyzer; K1100 Kjeldahl Nitrogen Analyzer.

[0055] 1.3 Methods

[0056] 1.3.1 Preparation of DRB-CDs

[0057] Mix 10 g of defatted rice bran powder (passed through a 40-mesh sieve) with 50 mL of deionized water and stir at room temperature for 5 h; transfer the mixture to a 100-mL stainless-steel hydrothermal reactor and react at 200 °C for 5 h. After natural cooling, centrifuge the reaction solution at 10000 rpm for 15 min; collect the supernatant and mix it with 4 times the volume of absolute ethanol to precipitate impurities, and collect the filtrate; concentrate the filtrate under reduced pressure at 50 °C until it is about one-fifth of the original total volume; filter the concentrated solution through a 0.22-μm microporous membrane, and dialyze the filtrate with a 500-Da dialysis bag for 48 h. The dialysate is obtained as DRB-CDs by freeze-drying.

[0058] 1.3.2 Preparation of Gelatin / Gelatin-Dextran / Gelatin Composite Films

[0059] Prepare a gelatin-dextran three-layer film by the layer-by-layer assembly method; mix 6 g of gelatin with 100 mL of deionized water and stir magnetically at 80 °C for 15 min, and simultaneously add 0.6 g of glycerol as a plasticizer to prepare a gelatin film-forming solution; mix 8 g of gelatin, 8 g of dextran with 100 mL of deionized water and stir magnetically at 80 °C for 15 min, and simultaneously add 4.0 g of glycerol as a plasticizer to prepare a gelatin-dextran film-forming solution; to prepare a three-layer film loaded with DRB-CDs, add different amounts of DRB-CDs (1%, 2%, and 3 wt%, based on the mass of the polymers in the gelatin-dextran film-forming solution) to the above gelatin-dextran film-forming solution and stir magnetically at 80 °C for 15 min.

[0060] 6 mL of gelatin film-forming solution was cast in a plastic petri dish with a diameter of 90 mm and dried at 40 °C for about 3 h until a gelatin film was formed but the surface was still sticky; subsequently, another 6 mL of gelatin-dextran film-forming solution containing different contents of DRB-CDs was cast and dried at 40 °C for about 3 h until a gelatin-dextran film was formed but the surface was still sticky; finally, 6 mL of gelatin film-forming solution was cast again and dried at 40 °C for about 4 h to obtain a gelatin / gelatin-dextran / gelatin three-layer film; according to the content of DRB-CDs, the composite films were named blank composite film, 1% composite film, 2% composite film, and 3% composite film respectively. The composite films were stored at 25 °C and 50% relative humidity for 48 h for subsequent tests.

[0061] 1.3.3 Characterization of DRB-CDs and composite films

[0062] 1.3.3.1 TEM

[0063] DRB-CDs were dissolved in ultrapure water and ultrasonically dispersed, then dropped onto the surface of a ultra-thin carbon film copper grid. After drying, observations were made at an acceleration voltage of 200 kV to obtain their TEM images; about 100 carbon dot particles within the field of view were randomly selected, and their particle size distributions were statistically analyzed by Nano Measurer software and the average particle size was calculated.

[0064] 1.3.3.2 SEM

[0065] The surface micro-morphology of the composite film (magnified 5000 times) was obtained by SEM after sputtering with gold; the cross-sectional micro-morphology of the composite film (magnified 200 times) was obtained by SEM after brittle fracture in liquid nitrogen and sputtering with gold.

[0066] 1.3.3.3 FTIR

[0067] DRB-CDs and the composite film ground into powder with liquid nitrogen were respectively mixed with KBr and pressed into tablets. After drying, they were detected in the wavelength range of 4000 - 400 cm -1 with a resolution of 4 cm -1 .

[0068] 1.3.3.4 XRD

[0069] The diffraction angle range for DRB-CDs testing was 5 - 90°, the voltage was 40 kV, the current was 150 mA, and the scanning speed was 10° / min; the diffraction angle range for composite film testing was 5 - 90°, the voltage was 40 kV, the current was 40 mA, and the scanning speed was 4° / min.

[0070] 1.3.3.5 XPS

[0071] The excitation source type for DRB-CDs testing is a monochromatized Al target (E = 1486.68 eV), the full-spectrum scanning pass energy is 100 eV, the fine-spectrum scanning pass energy is 50 eV, and the Avantage software is used for peak-fitting processing.

[0072] 1.3.3.6 TGA

[0073] The composite film is cut into discs with a diameter of less than 3 mm and placed in an alumina crucible. Under nitrogen conditions, its weight loss curve is obtained by a thermogravimetric analyzer. The test temperature range is 50 - 600 °C, and the heating rate is 10 °C / min.

[0074] 1.3.3.7 Zeta potential

[0075] DRB-CDs are dissolved in ultrapure water to prepare a concentration of 0.5 mg / mL, and a Zeta potential tester is used to detect the Zeta potential of DRB-CDs.

[0076] 1.3.4 Determination of the optical properties of DRB-CDs and the composite film

[0077] DRB-CDs are dissolved in ultrapure water to prepare a concentration of 1.0 mg / mL; the composite film is cut into strips of 30 mm × 10 mm, and an ultraviolet-visible spectrophotometer is used to measure the absorption spectra of DRB-CDs and the composite film in the wavelength range of 200 - 800 nm. A fluorescence spectrometer is used to measure the emission spectra of DRB-CDs in the excitation wavelength range of 320 - 460 nm.

[0078] A color difference meter is used to measure the L*, a*, and b* values of the composite film, and ΔE is calculated according to formula (1):

[0079]

[0080] In the formula: ΔL*, Δa*, and Δb* respectively represent the differences in L*, a*, and b* between the composite film and the white standard color plate (L* = 98.55, a* = 0.29, b* = 0.91).

[0081] 1.3.5 Cytotoxicity determination of DRB-CDs

[0082] The MTT method is used to evaluate the toxicity of DRB-CDs to HaCat cells. The cell culture conditions are DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin; the cell suspension is at 5×10 3The cells were inoculated at a density of cells / well in a 96-well plate (100 μL of cell suspension was added to each well), and the group with only 100 μL of DMEM without cell suspension was used as the blank group. After 12 h of adherent culture, the original DMEM in each well was removed, and then the medium containing different concentrations of DRB-CDs was added. The medium group without DRB-CDs was used as the control group. After culturing for 48 h and 72 h, 20 μL of MTT solution (5 mg / mL) was added to each well, and after continuous incubation at 37 °C in the dark for 4 h, the absorbance value was measured at 490 nm using an enzyme-linked immunosorbent assay reader. The cell survival rate of the control group was 100%. The cell survival rate was calculated according to formula (2):

[0083]

[0084] 1.3.6 Determination of composite film thickness, water solubility, moisture content, and WVP

[0085] 1.3.6.1 Thickness

[0086] The thickness of the composite film was randomly measured at 6 different positions using a digital micrometer with a precision of 0.001 mm.

[0087] 1.3.6.2 Moisture content and water solubility

[0088] The composite film (2 cm × 2 cm) was dried at 105 °C for 24 h, and the percentage of the mass loss to the total mass of the composite film before drying was the moisture content of the composite film. Subsequently, the dried composite film was placed in 30 mL of deionized water and left at room temperature for 24 h. After taking it out, it was dried again at 105 °C for 24 h, and the percentage of the mass loss to the total mass of the dried composite film before rehydration was the water solubility of the composite film.

[0089] 1.3.6.3 WVP

[0090] The WVP of the composite film was determined according to the national standard GB / T 1037-2021. An appropriate amount of anhydrous calcium chloride was added to a weighing bottle (40 mm × 25 mm), and the bottle mouth was sealed with the composite film to ensure that the gap between the composite film and anhydrous calcium chloride was less than 3 mm; the weighing bottle was placed in a constant temperature and humidity chamber (38 °C, 90% relative humidity), and weighed at fixed intervals. The WVP (g / m·s·Pa) was calculated according to formula (3):

[0091]

[0092] where: Δw (g) is the increased mass of the weighing bottle; d (m) is the thickness of the film; A (m 2 ) is the effective area of the film; Δt (s) is the test time; ΔP (Pa) is the pressure difference across the film (6619.34 Pa).

[0093] 1.3.7 Determination of TS and EAB of the composite film

[0094] Cut the composite film into strips with a size of 15 mm × 60 mm. Use a texture analyzer to test the TS and EAB of the composite film, set the clamping distance to 40 mm, and the stretching rate to 1 mm / s.

[0095] 1.3.8 Determination of the release performance of the composite film

[0096] Determine the release amount of DRB-CDs in the 3% composite film in food simulants (deionized water, 10%, 50%, and 95% ethanol solutions, simulating aqueous foods and alcoholic foods respectively). Immerse a 2.5 cm × 2.5 cm composite film in 20 mL of food simulant, stir gently at 25 °C, and take 2 mL of samples every half hour to measure the absorbance value at a wavelength of 297 nm. Calculate its release amount based on the standard curve made of DRB-CDs.

[0097] 1.3.9 Determination of the antioxidant capacity of the composite film

[0098] Evaluate the antioxidant performance of the composite film through ABTS and DPPH free radical scavenging tests. First, dissolve 100 mg of the composite film in 10 mL of deionized water to prepare a composite film solution. The method for the ABTS free radical scavenging test is as follows: Mix 2.6 mM potassium persulfate with 7.4 mM ABTS (1:1, v / v), place it in the dark for 12 h and then measure the absorbance value at 732 nm, and then gradually dilute the solution until its absorbance value is about 1.1. Take 10 mL of the ABTS free radical solution and mix it with 0.5 mL of the composite film solution, incubate it in the dark at 25 °C for 0.5 h and then measure the absorbance value at 732 nm. At the same time, conduct a blank test with deionized water.

[0099] The method for the DPPH free radical scavenging test is as follows: Take 2 mL of 0.2 mM DPPH ethanol solution and mix it with 1 mL of the composite film solution, incubate it in the dark at 25 °C for 0.5 h and then measure the absorbance value at 517 nm. At the same time, conduct a blank test with deionized water. The ABTS and DPPH free radical scavenging rates k (%) are calculated according to formula (4):

[0100]

[0101] In the formula: A0 and A1 represent the absorbance values of the blank and the sample respectively.

[0102] 1.3.10 Determination of the antibacterial ability of the composite film

[0103] Testing the antibacterial activity of the composite film against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus: Streak-culture the two bacteria separately on plate count agar. After culturing at 37 °C for 12 - 18 h, pick 3 - 5 single colonies and inoculate them into 3 mL of sterile LB broth; culture at 37 °C for 8 - 11 h, at this time the bacterial liquid concentration is about 10 12 CFU / mL; then dilute the bacterial liquid with sterile normal saline to a bacterial liquid concentration of about 10 6 CFU / mL; take 5 mL of the diluted bacterial liquid and mix it with 0.5 g of the composite film sample, and culture at 37 °C for 12 h. Subsequently, dilute the culture solution 10-fold with sterile normal saline, take 100 μL and spread it on plate count agar, and record the number of colonies after culturing at 37 °C for 12 h. Calculate the antibacterial rate (%) according to formula (5):

[0104]

[0105] In the formula: N0 and N1 represent the TVC of the culture solutions of the blank composite film and the composite film loaded with DRB-CDs, respectively.

[0106] 1.3.11 Testing the freshness preservation performance of scallops

[0107] Shell the live Patinopecten yessoensis to obtain adductor muscles (average weight 14.8 ± 1.3 g) and randomly divide them into three treatment groups: the unpacked group, the blank composite film group, and the 3% composite film group. The unpacked group is placed naked in a 4 °C refrigerator, and the blank composite film group and the 3% composite film group are wrapped with the blank composite film and the 3% composite film respectively and then placed in a 4 °C refrigerator. The three groups of samples are refrigerated for 12 days, and samples are taken for testing every 3 days. The samples on the 0th day of storage of the three groups of samples are all unpacked fresh scallop adductor muscles.

[0108] 1.3.11.1 pH

[0109] Take 2.0 g of minced adductor muscle and mix it evenly with 20 mL of deionized water and homogenize. Let it stand at room temperature for half an hour, then centrifuge (4000 g, 5 min) and measure the pH of the supernatant.

[0110] 1.3.11.2 TBARS

[0111] Take 2.0 g of minced adductor muscle and mix it evenly with 20 mL of 7.5% trichloroacetic acid solution containing 0.1% ethylenediaminetetraacetic acid and homogenize. After centrifugation (4000 g, 5 min), take 1 mL of the supernatant and mix it with 1 mL of 2-thiobarbituric acid (20 mM) and 1 mL of 7.5% trichloroacetic acid, react in a boiling water bath for 0.5 h, cool, and measure its absorbance value (532 nm). The TBARS value (mg malondialdehyde (MDA) / kg) is calculated according to the standard curve made with 1,1,3,3-tetramethoxypropane.

[0112] 1.3.11.3 TVB-N

[0113] The TVB-N value of scallop adductor muscle was determined according to the national standard GB 5009.228-2016. 5.0 g of minced adductor muscle was mixed with 75 mL of deionized water and 1 g of magnesium oxide, and then determined by an automatic Kjeldahl nitrogen analyzer.

[0114] 1.3.11.4 TVC

[0115] The TVC of the sample was determined according to the national standard GB 4789.2-2022. 5.0 g of minced sample was put into a sterile homogenization bag, added with 45 mL of sterile normal saline, and homogenized by patting. The extract was continuously diluted 10-fold with sterile normal saline, and 1 mL of the diluted solution was inoculated into plate count agar. After incubation at 30 °C for 72 h, the total number of colonies (lg CFU / g) was calculated.

[0116] 1.4 Data analysis

[0117] The samples were determined in parallel 3 times, and the results were expressed as "mean ± standard deviation". Origin 2016 and SPSS 22.0 software were used for drawing and significance analysis. The significance of differences was determined by Duncan's multiple comparison (P<0.05).

[0118] 2 Results and discussion

[0119] 2.1 Characterization and cytotoxicity analysis of DRB-CDs

[0120] 2.1.1 TEM analysis

[0121] Figure 1 Figure and particle size distribution diagram of DRB-CDs are shown. As Figure 1 shown, DRB-CDs are monodispersed and have an approximately spherical morphological feature; the particle size of DRB-CDs is less than 10 nm, and its lattice spacing is 0.20 nm; particle size statistical analysis shows that the particle size distribution range of defatted rice bran carbon dots is 1.25 - 4.75 nm, and the average particle size is 3.19 ± 0.61 nm.

[0122] 2.1.2 Zeta potential analysis

[0123] Zeta potential is a key physicochemical parameter that characterizes the surface charge characteristics of particles. Its numerical value directly affects the electrostatic interaction between particles and the stability of the dispersed system. The surface charge of particles with a higher absolute value of Zeta potential tends to produce electrostatic repulsion, thereby maintaining the stable dispersion state of the system. In the carbon dot aqueous solution system, when the Zeta potential value is lower than -15mV or higher than +15mV, it can be determined that the carbon dot has good dispersion characteristics. In this embodiment, the Zeta potential of the DRB-CDs aqueous solution was measured to be -22.2±1.10mV, which fully confirmed that the carbon dot system has excellent stability and dispersibility.

[0124] 2.1.3 UV and fluorescence spectroscopy analysis

[0125] Figure 2 is the UV-visible absorption spectrum of DRB-CDs. Figure 2 As shown, the DRB-CDs aqueous solution is yellow and clear and transparent in the visible light region, which confirms its excellent water-soluble properties. Under 365nm ultraviolet light excitation, the solution exhibits significant bright blue fluorescence. From the UV-visible absorption spectrum, it can be seen that DRB-CDs show characteristic absorption peaks at 289nm and 297nm, respectively, which can be attributed to the π-π* electronic transition of the CC bond and the n-π* electronic transition of the C=O / C-OH bond. Based on the significant ultraviolet absorption characteristics of DRB-CDs, the present invention confirms its feasibility as a functional filler in the development of ultraviolet barrier packaging materials, and provides a theoretical basis for the construction of a new food packaging system.

[0126] Figure 3 is the fluorescence spectrum of DRB-CDs. Figure 3 As shown in the figure, DRB-CDs exhibit excitation wavelength-dependent fluorescence characteristics. When the excitation wavelength is gradually increased from 320nm to 460nm in increments of 20nm, its fluorescence emission peak shows a red shift phenomenon. At the same time, the fluorescence intensity shows a trend of first increasing and then decreasing with the change of excitation wavelength, and reaches the maximum fluorescence intensity at an excitation wavelength of 440nm and an emission wavelength of 512nm.

[0127] 2.1.4 FTIR analysis

[0128] Figure 4 The FTIR spectrum of DRB-CDs is shown in Figure 2. -1 There is a broad absorption peak at 2933.57 and 1240.60 cm -1The characteristic peaks at [specific position] respectively correspond to the symmetric and asymmetric stretching vibrations of C-H and CH2 bonds in methyl and methylene groups; the absorption peaks at 1705.09, 1660.39, and 1599.74 cm -1 in the spectrum are caused by the presence of C=O, C=N, and C=C bonds respectively. In addition, the absorption peak at 1407.93 cm -1 is due to the symmetric and asymmetric stretching vibrations of C-N bonds, while the characteristic peak at 1093.17 cm -1 is related to the stretching vibration of C-O-C bonds.

[0129] The above spectral characteristics fully confirm that the surface of defatted rice bran carbon dots is rich in hydrophilic functional groups such as amide groups, carboxyl groups, and hydroxyl groups. The presence of these functional groups not only endows defatted rice bran carbon dots with excellent water solubility but also ensures their good biocompatibility.

[0130] 2.1.5 XPS Analysis

[0131] Figure 5 are the XPS full spectrum and fine spectrum of DRB-CDs. As shown in (A) of Figure 5 , the XPS full spectrum of DRB-CDs shows significant characteristic peaks at 285.26 eV (C 1s), 400.06 eV (N 1s), and 532.14 eV (O 1s), indicating that the sample contains three elements, C, N, and O, with their contents being 71.86%, 6.38%, and 21.76% respectively. Through the analysis of the fine spectrum, it can be seen that the C1s spectrum ( Figure 5 (B) in) shows three sub-peaks at 284.79 eV, 286.15 eV, and 288.16 eV, which are attributed to C-H / C-C, C-O / C-N, and C=O / O-C-O bonds respectively. The N 1s spectrum ( Figure 5 (C) in) shows three characteristic peaks at 398.62 eV, 399.91 eV, and 401.70 eV, corresponding to C-N-C, N-C3, and N-H bonds respectively. The O 1s spectrum ( Figure 5 (D) in) observes three sub-peaks at 531.13 eV, 532.03 eV, and 532.92 eV, corresponding to C-O / N-O, C-O-H, and C=O bonds respectively; the above analysis results show that the surface of DRB-CDs contains various functional groups such as amide groups, carboxyl groups, and hydroxyl groups, and this result is consistent with the infrared spectroscopy results.

[0132] 2.1.6 XRD Analysis

[0133] Figure 6 is the XRD spectrum of DRB-CDs. As shown in Figure 6As shown, the XRD pattern of DRB-CDs presents a relatively broad diffraction peak at 2θ = 20.08°, and no other obvious diffraction peaks are observed, indicating that it has a graphene-like structure. This broad diffraction peak further confirms that the defatted rice bran carbon dots belong to an amorphous carbon crystal structure.

[0134] 2.1.7 Cytotoxicity analysis of DRB-CDs

[0135] Figure 7 represents the survival rate of HaCaT cells after 48 h and 72 h of incubation with DRB-CDs. The biosafety of DRB-CDs is the core evaluation index for its use as a functional filler in active packaging films. In this invention, the in vitro toxic effect of these carbon dots on HaCaT cells was evaluated by the MTT method. As Figure 7 shown, in the concentration range of 10 - 2000 μg / mL, there was no significant difference in the survival rate of HaCaT cells between the DRB-CDs treatment group and the blank control group after 48 h and 72 h of exposure (P > 0.05), indicating that it did not produce cytotoxicity within the test concentration threshold (below 2 mg / mL) and had good biocompatibility. Based on the excellent biosafety characteristics of DRB-CDs, this invention confirms its good application value as a nano-functional filler in the field of active packaging materials.

[0136] 2.2 Characterization and property analysis of composite films

[0137] 2.2.1 SEM analysis

[0138] Figure 8 are the surface and cross-sectional SEM images of composite films loaded with different contents of DRB-CDs. As Figure 8 shown in A, the surfaces of all composite films exhibit a dense, uniform, and smooth microstructure. Since DRB-CDs are added to the middle layer of the composite film, the addition amount of DRB-CDs has no obvious effect on the surface microstructure of the composite film. As Figure 8As shown in Figure B, the cross-sections of all composite films have a clear three-layer structure of an outer gelatin layer, a gelatin-dextran intermediate layer, and an inner gelatin layer, indicating the successful preparation of the three-layer composite films. A relatively dense microstructure can also be seen in the cross-sectional images of all composite films, but there are a small number of cavities in the intermediate layer and at the junction of the intermediate layer and the inner and outer layers of the blank composite film and the 1% composite film; as the concentration of DRB-CDs increases, the cavities in the cross-section of the composite film decrease, and the cross-section of the 3% composite film is uniform and continuous without cavities. This is because the small nanoparticles DRB-CDs occupy the pores in the polymer film matrix, improving the density of the composite film. At the same time, due to the hydrogen bond interaction between DRB-CDs and the matrix polymer, it promotes the formation of a dense three-dimensional network structure in the intermediate layer, enhancing the interfacial adhesion and reducing the appearance of cavities; the barrier properties and mechanical properties enhanced by this dense network structure ensure good application prerequisites for the composite film.

[0139] 2.2.2 FTIR Analysis

[0140] Figure 9 The FTIR spectra of composite films loaded with different contents of DRB-CDs are shown as follows. As Figure 9 shown, the blank composite film, 1% composite film, 2% composite film, and 3% composite film all have broad absorption peaks at 3200 - 3500 cm -1 , corresponding to the stretching vibrations of -OH and N-H bonds. All composite films have characteristic peaks at 2941.46 cm -1 , 1653.60 cm -1 , 1545.63 cm -1 , and 1244.63 cm -1 , corresponding to the C-H stretching vibration, the C=O stretching vibration of the amide I band, the N-H bending vibration of the amide II band, and the C-N and N-H stretching vibrations of the amide III band, respectively. The absorption peaks of the blank composite film, 1% composite film, 2% composite film, and 3% composite film at 1027.52 and 918.10 cm -1 are related to the ether bonds in the dextran molecule. As the addition amount of DRB-CDs increases, the characteristic absorption peak of the blank composite film at 3375.43 cm -1 shows a red shift, shifting to 3353.58 cm -1 (1% composite film), 3351.28 cm -1 (2% composite film), and 3348.35 cm -1 (3% composite film) respectively, indicating that DRB-CDs form hydrogen bonds with the matrix polymer of the composite film.

[0141] 2.2.3 TGA Analysis

[0142] Figure 10 The TGA curves of composite films loaded with different contents of DRB-CDs are shown as follows. AsFigure 10 As shown, all composite films have two main weight loss stages. In the first stage (50 - 150 °C), the thermal degradation is mainly due to the evaporation of physically adsorbed water in the composite film. The second stage of thermal degradation is the main mass loss stage of the composite film, which occurs at 180 - 420 °C. The thermal degradation in this stage is attributed to the decomposition of gelatin, dextran, and glycerol. From the thermogravimetric analysis curve, it can be seen that DRB-CDs have an impact on the thermal stability of the composite film. In the first stage of thermal degradation, the degradation rate of the composite film with added DRB-CDs is lower than that of the blank composite film. This is because the hydrophilic DRB-CDs encapsulate the physically adsorbed water molecules, resulting in a reduced evaporation rate. Compared with the blank composite film, the composite film with added DRB-CDs has a lower degradation degree in the second stage and shows higher thermal stability. This may be due to the enhanced intermolecular interaction through hydrogen bonding between DRB-CDs and the hydroxyl groups on the main chain of the composite film matrix polymer. In addition, the residual mass of the composite film with added DRB-CDs at 600 °C is higher than that of the blank composite film. The order of their residual mass from high to low is: 3% composite film > 2% composite film > 1% composite film > blank composite film. This is mainly attributed to the difference in the content of incombustible minerals and impurities in the composite film. Previous studies have shown that carbon dots from various natural sources can improve the thermal stability of biopolymer-based composite films.

[0143] 2.2.4 XRD analysis

[0144] Figure 11 Figure shows the XRD patterns of composite films loaded with different contents of DRB-CDs. As Figure 11 shown, all composite films have similar XRD patterns and show a relatively broad diffraction peak at 2θ = 19.30°, indicating a semi-crystalline structure. As the content of DRB-CDs increases, the intensity of the diffraction peak increases slightly. This may be due to the enhanced interaction between DRB-CDs and the molecules of the composite film matrix polymer, resulting in an increase in the crystallinity of the composite film, a more ordered polymer molecular structure, and ultimately an improvement in the physical properties of the composite film.

[0145] 2.2.5 Thickness, solubility, moisture content, and WVP analysis

[0146] Table 1 shows the thickness, water solubility, moisture content, and WVP of composite films loaded with different contents of DRB-CDs. As shown in Table 1, the thickness, moisture content, water solubility, and WVP of the blank composite film are 0.221 ± 0.010 mm, 17.68 ± 0.60%, 64.43 ± 0.55%, and 8.11 ± 0.34×10 11g / (m·s·Pa). With the increase in the addition amount of DRB-CDs, the thickness of the composite film increased significantly (P<0.05), while the moisture content, water solubility, and WVP decreased significantly (P<0.05). The increase in the thickness of the composite film was mainly attributed to the increase in the solid content of the middle active layer of the composite film by adding DRB-CDs. Since hydrogen bonds were formed between DRB-CDs and the polymer matrix of the middle active layer, the interaction between polymer molecules and water molecules was weakened, resulting in a decrease in the moisture content of the composite film with the increase in the addition amount of DRB-CDs. Since DRB-CDs were uniformly distributed in the composite film matrix and hydrogen bonds were formed between them and the matrix polymer, the number of free hydrophilic groups of the matrix polymer decreased, ultimately reducing the water solubility of the composite film. Since the nano-filler DRB-CDs filled the voids between the matrix polymer chains, it promoted the formation of a denser network structure in the composite film, increasing the tortuous path of water vapor diffusion, thus inhibiting the passage of water vapor and ultimately improving the water vapor barrier performance of the composite film. In addition, the hydrogen bonds formed between DRB-CDs and the polymer matrix further enhanced the water vapor barrier ability of the composite film.

[0147] Table 1 Thickness, water solubility, moisture content, and WVP of composite films loaded with different contents of DRB-CDs

[0148]

[0149] Note: Different letters in the same column indicate significant differences between samples (P<0.05).

[0150] 2.2.6 Optical property analysis

[0151] Figure 12 are the appearance pictures of composite films loaded with different contents of DRB-CDs. The color of food packaging films has a significant impact on the appearance of products and consumer preferences. As Figure 12 shown, the blank composite film was colorless and transparent, while the composite film containing DRB-CDs showed yellow, and with the increase in the content of DRB-CDs, the color of the composite film gradually deepened. In addition, since DRB-CDs themselves were yellow, the uniform color of the composite film further confirmed the uniform distribution of DRB-CDs in the polymer matrix.

[0152] Table 2 shows the color parameters of composite films loaded with different contents of DRB-CDs. With the increase in the addition amount of DRB-CDs, the L* value of the composite film decreased significantly (P<0.05), while the a*, b*, and ΔE values increased significantly (P<0.05). This result indicates that the introduction of DRB-CDs not only reduced the brightness of the composite film but also promoted the transformation of the composite film to yellow-red colors, resulting in a significant change in the overall color difference. The change in the color of the composite film was mainly due to the brown-yellow color of DRB-CDs themselves.

[0153] Table 2 L*, a*, b and ΔE values of composite films loaded with different contents of DRB-CDs

[0154]

[0155] Figure 13 The UV / visible light transmittance of composite films loaded with different contents of DRB-CDs is shown. The composite films exhibit high transmittance in the visible light region above 400 nm, but low transmittance in the UV light region below 400 nm, especially in the range of 200 - 320 nm, thus showing certain UV blocking performance. This characteristic is mainly attributed to the inherent UV blocking ability of dextran and gelatin. However, the addition of DRB-CDs significantly reduces the visible and UV light transmittance of the composite films, and the reduction rate is proportional to the concentration of DRB-CDs. The composite film loaded with 3% DRB-CDs almost completely blocks the transmission in the UV light band, which is mainly attributed to the light scattering and UV light absorption ability of DRB-CDs. The UV blocking property of this composite film is expected to protect packaged foods from photooxidation and photodegradation reactions caused by ultraviolet light.

[0156] 2.2.7 Mechanical property analysis

[0157] The mechanical properties of the composite films were evaluated by measuring TS and EAB. Figure 14 Shown is the TS of composite films loaded with different contents of DRB-CDs. As Figure 14 shown, the tensile strength of the blank composite film is 12.67 ± 0.79 MPa. With the increase in the addition concentration of DRB-CDs, the tensile strength of the composite film increases significantly (P < 0.05). DRB-CDs are uniformly dispersed in the composite film matrix, and the hydrogen bond interaction formed between its surface functional groups and the matrix polymer promotes the formation of a dense three-dimensional network structure in the composite film, resulting in an enhancement of the TS of the composite film. In addition, the SEM results show that the cross-section of the middle active layer of the composite film added with DRB-CDs is denser, which further confirms the formation of the dense network structure.

[0158] Figure 15 Shown is the EAB of composite films loaded with different contents of DRB-CDs. As Figure 15 shown, with the increase in the addition concentration of DRB-CDs, the EAB of the composite film shows a trend of first increasing and then decreasing; when the concentration of DRB-CDs is 2%, the elongation at break reaches the maximum value of 74.64 ± 0.86%. It should be noted that the elongation at break of all composite films added with DRB-CDs is significantly higher than that of the blank composite film (P < 0.05). When the addition amount of DRB-CDs exceeds a certain threshold, with the improvement of mechanical strength, the rigidity of the composite film increases, which may be the main reason for the decrease in the elongation at break of the composite film.

[0159] 2.2.8 Release characteristic analysis

[0160] The application of active packaging technology mainly depends on the release ability of active substances from the packaging film. Research shows that the release behavior of nanoparticles is affected by various factors, including the swelling index of the composite film, the solubility of the matrix polymer, and the dispersibility and solubility of nanoparticles in aqueous solution. Figure 16 Figure 3% shows the release rate of DRB-CDs in four food simulants: water, 10% ethanol solution, 50% ethanol solution, and 95% ethanol solution. The results show that the release patterns of DRB-CDs in the four food simulants are similar, all showing rapid release within 1 hour, followed by a slowdown in the release rate within 1 - 1.5 hours, and finally reaching a steady state after 1.5 hours, indicating that the composite film has a significant sustained-release effect on DRB-CDs. In addition, the release rate of DRB-CDs is the highest in water and 10% ethanol solution, followed by 50% ethanol solution, and the slowest in 95% ethanol solution, indicating that an increase in the ethanol content in the food simulant significantly reduces the release rate of DRB-CDs. This phenomenon is mainly attributed to the hydrophilicity of the composite film matrix polymers (dextran and gelatin), which allows water molecules to rapidly penetrate the matrix, promoting full hydration of the composite film and a more open matrix network structure, thus facilitating the rapid release of DRB-CDs. However, ethanol cannot effectively interact with gelatin and dextran, resulting in DRB-CDs being trapped in the matrix, with significantly reduced diffusibility and mobility. Therefore, compared with high-concentration ethanol solutions, the release rate and release amount of DRB-CDs in water are faster and larger.

[0161] 2.2.9 Antioxidant capacity analysis

[0162] The antioxidant capacity of the composite film was evaluated by ABTS and DPPH free radical scavenging methods. Figure 17Antioxidant capacity of composite films loaded with different contents of DRB-CDs. The results showed that the blank composite film exhibited an ABTS radical scavenging capacity of 2.88 ± 0.56% and a DPPH radical scavenging capacity of 1.56 ± 0.11%, which might be due to some antioxidant peptides contained in gelatin. With the increase in the addition concentration of DRB-CDs, the antioxidant capacity of the composite film increased significantly (P<0.05). Adding 3% DRB-CDs increased the ABTS and DPPH radical scavenging capacities of the composite film to 72.56 ± 1.28% and 50.24 ± 0.94% respectively. The strong antioxidant capacity of the composite film was attributed to the excellent radical scavenging ability of the loaded DRB-CDs. Defatted rice bran carbon dots might achieve the scavenging of DPPH and ABTS radicals through the following three mechanisms: 1) In the hydrogen atom transfer mechanism, functional groups such as hydroxyl and carboxyl groups on the surface of defatted rice bran carbon dots act as hydrogen donors, transferring hydrogen atoms to DPPH and ABTS radicals, reducing them to stable DPPH-H and ABTS-H; 2) In the electron transfer mechanism, defatted rice bran carbon dots transfer electrons to DPPH and ABTS radicals through the electron transfer process, generating DPPH - anions and ABTS - anions, and at the same time, defatted rice bran carbon dots themselves are transformed into cation radicals. Subsequently, the cation radicals transfer protons to DPPH - anions and ABTS - anions, further transforming them into DPPH-H and ABTS-H; 3) In the sp 2 carbon site radical adduct formation mechanism, the sp 2 carbon-rich domain of defatted rice bran carbon dots combines with radicals to form radical adducts, and then secondary adducts are generated through the hydrogen atom transfer or electron transfer process of surface functional groups, thus achieving the scavenging of radicals. It should be noted that the scavenging activity of defatted rice bran carbon dots against ABTS radicals was significantly higher than that against DPPH radicals, which might be due to the hydrophilic properties of defatted rice bran carbon dots, making their solubility in ABTS aqueous solution better than that in DPPH ethanol solution, thus interacting more effectively with ABTS radicals.

[0163] 2.2.10 Analysis of antibacterial ability

[0164] Figure 18 Antibacterial ability of the composite film against Escherichia coli and Staphylococcus aureus. As Figure 18As shown, with the increase in the concentration of DRB-CDs, the antibacterial performance of the composite film was significantly enhanced (P<0.05). The antibacterial rates of the 3% composite film against Escherichia coli and Staphylococcus aureus reached 46.86±2.62% and 79.17±2.71% respectively. Functional groups such as amide groups and amino groups rich on the surface of DRB-CDs made the surface of DRB-CDs carry a positive charge. These positive charges could combine with the negative potential on the surface of bacteria, and then the reactive oxygen species derived from DRB-CDs damaged the bacterial cell wall, resulting in the leakage of cell contents and ultimately causing bacterial death. In addition, at the same concentration, the antibacterial rate of DRB-CDs against the Gram-positive bacterium Staphylococcus aureus was significantly higher than that against the Gram-negative bacterium Escherichia coli, which was mainly attributed to the differences in the cell wall structures of the two types of bacteria. The single-layer peptidoglycan cell wall structure of Gram-positive bacteria was conducive to the strong interaction between DRB-CDs and peptide sequences, thus promoting the penetration of carbon dots into the cell interior; while the double-layer cell wall structure of Gram-negative bacteria hindered their effective interaction with DRB-CDs, resulting in a relatively weak antibacterial effect.

[0165] 2.2.11 Analysis of the freshness preservation performance of scallops

[0166] Figures 19 - 22 They were the changes in the pH, TBARS, TVB-N and TVC values of scallop adductor muscles during cold storage under different packaging conditions respectively. During the storage period, the pH, TBARS, TVB-N and TVC values of scallop adductor muscles in the unpacked group, the blank composite film group and the 3% composite film group all showed a trend of gradually increasing with the extension of storage time. Among them, the increase in pH value was mainly attributed to the degradation of proteins and nitrogen-containing non-protein substances, as well as the accumulation of alkaline substances such as ammonia and trimethylamine caused by microbial action. The increase in TBARS value was mainly due to the oxidation of unsaturated fatty acids rich in scallop adductor muscles to generate aldehyde substances. The increase in TVB-N value was due to the combined action of exogenous enzymes secreted by microorganisms and endogenous enzymes of scallop adductor muscles themselves, promoting the accumulation of trimethylamine, ammonia and other volatile basic nitrogen-containing substances. The increase in TVC value directly reflected the growth and reproduction of microorganisms in scallop adductor muscles.

[0167] During the storage period of 3 to 12 days, the pH value, TBARS value, TVB-N value, and TVC value of the scallop adductor muscle in the 3% composite film group were significantly lower than those in the blank composite film group and the unpackaged group. This phenomenon is mainly attributed to the strong antibacterial activity of DRB-CDs released from the composite film, which effectively inhibits microbial activities, reduces the accumulation of alkaline substances, and simultaneously decreases the generation of trimethylamine, ammonia, and other volatile basic nitrogen-containing substances, thus slowing down the rising rate of the above indicators. In addition, the excellent free radical scavenging ability of DRB-CDs blocks the chain reaction of the auto-oxidation of unsaturated lipids in the scallop adductor muscle, reduces the degree of lipid oxidation, and further inhibits the increase in the thiobarbituric acid value. The experimental results show that the composite film loaded with 3% DRB-CDs can effectively improve the quality stability of scallop adductor muscle during cold storage.

[0168] In this study, a gelatin / gelatin-dextran / gelatin three-layer composite film loaded with DRB-CDs was successfully constructed, and its application potential in scallop preservation was systematically evaluated. The results showed that DRB-CDs prepared with defatted rice bran as the carbon source had small particle size (3.19 ± 0.61 nm), high dispersion stability (Zeta potential -22.2 mV), were rich in active functional groups such as hydroxyl and carboxyl groups on the surface, exhibited significant ultraviolet shielding characteristics, and had no cytotoxicity (HaCaT cell survival rate > 95%). The addition of DRB-CDs optimized the density of the composite film matrix through hydrogen bonding, significantly improving its mechanical strength (tensile strength increased by 37.5%), water vapor barrier property (WVP decreased by 35.0%), ultraviolet barrier property, and thermal stability. The three-layer film structure endows the composite film with excellent slow-release performance. In the scallop cold storage experiment, the 3% composite film effectively improved the quality stability of the scallop adductor muscle by inhibiting lipid oxidation (TBARS value decreased by 53.8%) and microbial proliferation (TVC value decreased by 1.2 lg CFU / g). This invention not only confirms the feasibility of the application of food-derived carbon dots in the field of active packaging but also provides a solution for the development of green preservation technologies for aquatic products. Future research can further explore the applicability of the composite film in complex food systems to promote its practical application process.

[0169] The above has described the embodiments of the present invention in detail in combination with the examples. However, the present invention is not limited to the above embodiments. For those of ordinary skill in the art in this technical field, after learning the content recorded in the present invention, without departing from the principle of the present invention, several equivalent transformations and substitutions can still be made, and these equivalent transformations and substitutions should also be regarded as belonging to the protection scope of the present invention.

Claims

1. A defatted rice bran carbon dot - gelatin - dextran three - layer film, characterized in that: The degreased rice bran carbon dot - gelatin - dextran three - layer film is prepared by loading degreased rice bran carbon dots into a gelatin / gelatin - dextran / gelatin three - layer film.

2. A preparation method of a defatted rice bran carbon dot - gelatin - dextran three - layer film, characterized in that, It includes the following steps: S1. Mix degreased rice bran powder with deionized water and stir at room temperature to obtain a mixed solution. S2. Transfer the mixed solution to a hydrothermal reaction kettle for hydrothermal reaction, react at 180 - 220 °C for 4 - 6 h, and perform centrifugation after natural cooling. S3. Collect the supernatant and mix it with 3 - 5 times the volume of absolute ethanol to precipitate impurities, and collect the filtrate. S4. Concentrate the filtrate under reduced pressure, then filter the concentrated solution through a microporous membrane, dialyze the filtrate, and obtain degreased rice bran carbon dots by freeze - drying. S5. Add degreased rice bran carbon dots to the gelatin - dextran film - forming solution and stir to form a gelatin - dextran film - forming solution containing DRB - CDs. Then, cast in the order of one layer of gelatin film - forming solution, one layer of gelatin - dextran film - forming solution containing DRB - CDs, and one layer of gelatin film - forming solution to obtain the degreased rice bran carbon dot - gelatin - dextran three - layer film.

3. The preparation method of the defatted rice bran carbon dot - gelatin - dextran three - layer film according to claim 2, wherein: In step S1, the mass - to - volume ratio of degreased rice bran powder to deionized water is 1∶4 - 6, and the stirring time is 4 - 6 hours.

4. The preparation method of the defatted rice bran carbon dots - gelatin - dextran three - layer film according to claim 2, characterized in that: In step S2, the centrifugation conditions are centrifugation at 10000 rpm for 15 min.

5. The preparation method of the defatted rice bran carbon dots - gelatin - dextran three - layer film according to claim 2, characterized in that, In step S4, the conditions for concentration under reduced pressure are: concentrate under reduced pressure at 50 °C until one - fifth of the original total volume. The dialysis conditions are: dialyze with a 500 Da dialysis bag for 48 h. The pore size of the microporous membrane is 0.22 μm.

6. The preparation method of the defatted rice bran carbon dot - gelatin - dextran three - layer film according to claim 2, characterized in that, Step S5 is specifically as follows: S51. Mix gelatin with deionized water, and add glycerol as a plasticizer while stirring magnetically to prepare a gelatin film - forming solution. S52. Mix gelatin, dextran with deionized water, and add glycerol as a plasticizer while stirring magnetically to prepare a gelatin - dextran film - forming solution. S53. Add degreased rice bran carbon dots to the above - mentioned gelatin - dextran film - forming solution and stir magnetically to prepare a gelatin - dextran film - forming solution containing DRB - CDs. S54. Cast the gelatin film - forming solution in a petri dish and dry it until the gelatin forms a film but the surface is still sticky; continue to cast an equal volume of the gelatin - dextran film - forming solution containing DRB - CDs and dry it until the gelatin - dextran forms a film but the surface is still sticky; then cast an equal volume of the gelatin film - forming solution again and dry it to obtain the degreased rice bran carbon dot - gelatin - dextran three - layer film.

7. The preparation method of the degreased rice bran carbon dot - gelatin - dextran three - layer film according to claim 6, wherein: In step S53, the weight percentage of degreased rice bran carbon dots in the gelatin - dextran film - forming solution is 1 - 3%, and this ratio is calculated based on the mass of the polymer in the gelatin - dextran film - forming solution.

8. The preparation method of the defatted rice bran carbon dots - gelatin - dextran three - layer film according to claim 7, characterized in that: The weight percentage of degreased rice bran carbon dots in the gelatin - dextran film - forming solution is 3%.

9. Application of the degreased rice bran carbon dot - gelatin - dextran three - layer film prepared by the preparation method according to any one of claims 2 - 8 in the preservation of aquatic products.

10. The application according to claim 9, characterized in that: The aquatic product is scallop.

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