Chitosan for food packaging, preparation method of chitosan and preparation method of food packaging film

Abstract

The invention provides chitosan for food packaging, a preparation method of the chitosan and a preparation method of a food packaging film, and belongs to the field of food engineering.The preparation method of the chitosan comprises the steps that natural pigment is added into N, N-dimethylformamide, stirring is conducted till the natural pigment is completely dissolved, and a pigment solution is obtained; dissolving chitosan in a glacial acetic acid aqueous solution, and fully swelling to form a transparent solution; dropwise adding the pigment solution into the transparent solution at room temperature, and uniformly stirring and mixing to obtain a mixture; adding a sufficient amount of a formaldehyde solution into the mixture until the reaction is completed, dropwise adding a sodium hydroxide solution to enable a product to form a precipitate, collecting the precipitate, and washing and freeze-drying the precipitate to obtain chitosan powder; wherein the natural pigment comprises curcumin and alizarin. The chitosan prepared by the method can improve the dispersibility of natural pigments in a film, improve the fixation mode of the pigments, avoid migration and loss, improve the light and heat stability of the natural pigments, improve the structural stability and mechanical properties of the film, and realize the double functions of preservation and indication.

Description

Technical Field

[0001] This invention relates to the field of food engineering, and in particular to a chitosan for food packaging and its preparation method, as well as a method for preparing food packaging films. Background Technology

[0002] Natural pigments have a wide range of applications in the food packaging industry, such as in packaging films for preservation purposes.

[0003] Currently, natural pigments exhibit poor dispersibility in hydrophilic bio-based membranes, and their fixation within the membrane lacks effective control, leading to easy migration or loss. Furthermore, the photothermal stability of natural pigments is insufficient, causing color fading with prolonged use. In addition, the lack of molecular-level bonding between pigments and the matrix limits improvements in membrane structure and performance. Moreover, existing technologies primarily focus on antibacterial or functional release, lacking the synergistic effect of both preservation and indication functions. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a chitosan for food packaging and a method for preparing it, as well as a method for preparing food packaging film.

[0005] This invention provides a method for preparing chitosan for food packaging, comprising: adding a natural pigment to N,N-dimethylformamide (DMF) and stirring until completely dissolved to obtain a pigment solution; dissolving chitosan in an aqueous glacial acetic acid solution, allowing it to swell fully and form a transparent solution; adding the pigment solution dropwise to the transparent solution at room temperature and stirring until homogeneous to obtain a mixture; adding sufficient formaldehyde solution to the mixture until the reaction is complete, then adding sodium hydroxide solution dropwise to precipitate the product; collecting the precipitate and washing and freeze-drying it to obtain chitosan powder; wherein the natural pigment includes curcumin (CUR) and alizarin (AL).

[0006] According to the present invention, a method for preparing chitosan for food packaging is provided, wherein when the natural pigment is curcumin, the ratio of curcumin, chitosan and N,N-dimethylformamide is 934 mg: 1.0 g: 50 mL; and when the natural pigment is alizarin, the ratio of alizarin, chitosan and N,N-dimethylformamide is 634 mg: 1.0 g: 50 mL.

[0007] According to the present invention, a method for preparing chitosan for food packaging is provided, wherein the formaldehyde solution is 53 mL of a formaldehyde solution with a mass fraction of 37%.

[0008] According to a method for preparing chitosan for food packaging provided by the present invention, the step of adding sufficient formaldehyde solution to the mixture until the reaction is complete includes: after adding sufficient formaldehyde solution to the mixture, stirring the reaction system at 500 rpm for 24 hours at 60°C.

[0009] According to a method for preparing chitosan for food packaging provided by the present invention, the collection of precipitates includes: collecting the precipitates by centrifugation at 6000 rpm for 10 min.

[0010] The present invention also provides a chitosan for food packaging, which is prepared based on the chitosan preparation method for food packaging described in any of the above claims.

[0011] This invention also provides a method for preparing a food packaging film, comprising: adding gelatin to deionized water, heating and stirring until the gelatin is completely dissolved, and then cooling; adding a chitosan solution prepared from the above-mentioned chitosan for food packaging to the gelatin solution, and stirring evenly to mix; adding glycerol to the mixed solution and continuing to stir until fully dispersed; adding calcium chloride and stirring evenly to obtain a film-forming precursor solution; slowly pouring the film-forming precursor solution into a petri dish to spread the film-forming precursor solution evenly on the bottom of the dish, and drying to remove the solvent to form a solid film.

[0012] According to the food packaging film preparation method provided by the present invention, the ratio of gelatin to deionized water is 300 mg: 5 mL.

[0013] According to the food packaging film preparation method provided by the present invention, the chitosan solution concentration is 20 mg / mL.

[0014] The chitosan for food packaging and its preparation method, as well as the food packaging film preparation method provided by this invention, can improve the dispersibility of natural pigments in the film, improve the fixation mode of pigments and avoid migration and loss, improve the photothermal stability of natural pigments, improve the structural stability and mechanical properties of the film, and achieve the dual functions of preservation and indication. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 This is a schematic flowchart of the method for preparing chitosan for food packaging provided by the present invention; Figure 2 This is a schematic diagram of the synthetic route of the natural polymer pigment provided by the present invention; Figure 3 These are the ¹H NMR spectra of different chitosans in a D₂O / DCl mixed solution provided by this invention; Figure 4These are the relevant spectra of different chitosans and the comparison diagram of DPPH free radical scavenging activity provided by this invention; Figure 5 These are XPS full-spectrum images of different chitosans provided by this invention; Figure 6 These are SEM images of different chitosans provided by this invention; Figure 7 These are the FTIR spectra, XRD spectra, TGA curves, and DTG curves of different composite membranes provided by this invention; Figure 8 These are the visual color changes and color change rate curves of different composite membranes with different pH sensitivities provided by this invention; Figure 9 These are schematic diagrams of the composite membrane structure and molecular docking simulation results provided by this invention. Figure 10 These are actual images and color change diagrams of the composite film preservation experiment provided by this invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0018] The following is combined with Figures 1-10 This invention describes the chitosan for food packaging, its preparation method, and the preparation method of food packaging film. Figure 1 This is a schematic flowchart of the method for preparing chitosan for food packaging provided by the present invention, as shown below. Figure 1 As shown, the present invention provides a method for preparing chitosan for food packaging, comprising: 101. Add the natural pigments to N,N-dimethylformamide and stir until completely dissolved to obtain a pigment solution. The natural pigments include curcumin and alizarin.

[0019] In some embodiments, when the natural pigment is curcumin, the ratio of curcumin, chitosan and N,N-dimethylformamide includes 934 mg: 1.0 g: 50 mL.

[0020] Taking curcumin as an example, first add 934 mg of curcumin to 50 mL of N,N-dimethylformamide and dissolve it completely by magnetic stirring to obtain a homogeneous curcumin solution.

[0021] 102. Dissolve chitosan in an aqueous solution of glacial acetic acid, allowing it to swell fully and form a transparent solution.

[0022] Accordingly, 1.0 g of chitosan (CS) was dissolved in 100 mL of 1% (w / v) glacial acetic acid aqueous solution to allow it to swell fully and form a transparent solution.

[0023] 103. At room temperature, the pigment solution is added dropwise to the transparent solution and stirred until homogeneous to obtain a mixture.

[0024] The curcumin solution was added dropwise to the chitosan solution at room temperature and mixed evenly with magnetic stirring.

[0025] 104. Add sufficient formaldehyde solution to the mixture until the reaction is complete, then add sodium hydroxide solution dropwise to form a precipitate. Collect the precipitate and wash and freeze-dry it to obtain chitosan powder. In some embodiments, the formaldehyde solution is 53 mL of a formaldehyde solution with a mass fraction of 37%.

[0026] In some embodiments, adding sufficient formaldehyde solution to the mixture until the reaction is complete includes: after adding sufficient formaldehyde solution to the mixture, maintaining the reaction system at 60°C and stirring at 500 rpm for 24 hours.

[0027] In some embodiments, the concentration of the sodium hydroxide solution includes 0.5 mol / L.

[0028] Specifically, 53 mL of a 37% formaldehyde solution can be added to the above mixture to allow curcumin, chitosan and formaldehyde to undergo a Mannich reaction. The reaction system is kept at 60 °C and stirred at 500 rpm for 24 hours to ensure that the grafting reaction proceeds fully.

[0029] After the reaction was complete, the pH of the system was adjusted by adding 0.5M sodium hydroxide solution dropwise, causing the product to precipitate.

[0030] In some embodiments, collecting the precipitate includes collecting the precipitate by centrifugation at 6000 rpm for 10 minutes.

[0031] The resulting precipitate was washed four times with anhydrous ethanol and twice with distilled water to remove unreacted substances and solvent residues. The washed solid was freeze-dried to obtain curcumin-chitosan (CS-g-CUR) powder, while alizarin yielded alizarin-grafted chitosan (CS-g-AL) powder. This invention has the following advantages: Improving the dispersibility of natural pigments in membranes. In existing technologies, natural pigments (such as curcumin and alizarin) are highly hydrophobic and easily aggregate when directly added to aqueous solutions of bio-based polymers, leading to uneven film structures. This invention uses the Mannich reaction to covalently graft natural pigments onto chitosan molecular chains, allowing the natural pigments to exist stably within the chitosan backbone through chemical bonds, forming a molecular-level dispersion. The resulting CS-g-CUR and CS-g-AL nanoscale single-chain dispersion structures can be uniformly distributed in gelatin / chitosan mixtures, fundamentally improving the compatibility of pigments in aqueous systems and the structural uniformity of the membrane.

[0032] This invention improves the fixation method of pigments and prevents migration and loss. Natural pigments, when physically mixed, easily migrate from the membrane when humidity or temperature changes. This invention utilizes the Mannich reaction to covalently fix pigment molecules to the amino groups of chitosan via –CH2– bonds. Because the pigments are directly embedded in the chitosan chain structure, the membrane maintains stable pigment binding even under water absorption or heating conditions, preventing dissolution or precipitation. The presence of covalent bonds fundamentally solves the problems of weak pigment fixation and poor stability in existing technologies.

[0033] This invention improves the photothermal stability of natural pigments. Natural pigments are prone to degradation under light, oxygen exposure, and temperature increases, leading to color fading. The grafting method of this invention binds pigment molecules to chitosan segments by chemical bonds and protects them within a three-dimensional polymer network environment. After the composite film is dried and formed, a dense hydrogen-bonded network is created, placing the pigment molecules in a microenvironment relatively isolated from oxygen, light, and heat, significantly improving the structural stability and long-term color rendering ability of the pigment. Compared to physical mixing, covalently bonded pigments maintain more stable absorption peaks and color response performance under light and heat treatment conditions.

[0034] This invention improves the structural stability and mechanical properties of thin films. In existing technologies, natural pigments are physically embedded in the matrix, which disrupts the three-dimensional structure of the film and reduces its mechanical strength. The chitosan molecular chains grafted in this invention simultaneously carry pigment groups, giving them stronger intermolecular forces (such as π–π stacking and enhanced hydrogen bonding), thus forming a denser and more stable network structure when blended with gelatin. This structure enables the composite film to exhibit higher thermal stability, more suitable plasticity, and better mechanical integrity, solving the problem of poor mechanical properties in traditional pigmented films.

[0035] Achieving Dual Functions of Preservation and Indication. This invention combines pH-responsive grafted pigments (CS-g-CUR, CS-g-AL) with a chitosan / gelatin composite network, resulting in a film that simultaneously possesses food preservation and visual indication capabilities. Chitosan itself has antibacterial properties; after grafting, the bioactivity of the natural polysaccharide is retained, while the increased structural density of the composite film provides a certain barrier effect, delaying food quality degradation. The grafted pigments exhibit a significant color change when the pH rises due to the accumulation of volatile amines (TVB-N), enabling visual monitoring of the food spoilage process. Therefore, through the synergistic effect of covalently grafted natural pigments and the polysaccharide matrix, this invention successfully achieves the dual functions of preservation and indication, meeting the needs of smart packaging applications.

[0036] In some embodiments, when the natural pigment is alizarin, the ratio of alizarin, chitosan and N,N-dimethylformamide is 634 mg: 1.0 g: 50 mL.

[0037] The preparation process of alizarin chitosan (CS-g-AL) is similar to that of CS-g-CUR described above. First, 634 mg of alizarin (AL) was dissolved in 50 mL of DMF, and 1.0 g of chitosan was dissolved in 100 mL of 1% (w / v) acetic acid solution. Then, the alizarin solution was added dropwise to the chitosan solution and mixed, followed by the addition of 53 mL of 37% formaldehyde solution. The mixture was stirred at 500 rpm at 60°C for 24 h. After the reaction was complete, 0.5 M NaOH was added dropwise until a precipitate formed. The solid was collected by centrifugation at 6000 rpm for 10 min and washed with ethanol (four times) and distilled water (twice). Finally, alizarin chitosan (CS-g-AL) powder was obtained by lyophilization.

[0038] The present invention also provides a chitosan for food packaging, which is prepared based on the chitosan preparation method for food packaging described in any of the above claims.

[0039] This invention also provides a method for preparing a food packaging film, based on the above-described preparation of chitosan for food packaging. The method for preparing the food packaging film provided by this invention is described below, and the method described below can be referred to in correspondence with the method described above for preparing chitosan for food packaging. This method for preparing the food packaging film includes: 201. Add gelatin to deionized water, heat and stir until the gelatin is completely dissolved, then cool. 202. Add the chitosan solution prepared from the chitosan for food packaging in the above embodiment to the gelatin solution, and stir until evenly mixed. Then add glycerol to the mixed solution and continue stirring until fully dispersed. 203. Add calcium chloride and stir until homogeneous to obtain the film-forming precursor solution; 204. Slowly pour the film-forming precursor solution into a culture dish, allowing it to spread evenly on the bottom of the dish, and dry it to remove the solvent and form a solid film.

[0040] The food packaging film preparation method provided in this embodiment of the invention is based on the chitosan embodiment described above. For specific procedures and details, please refer to the chitosan preparation method for food packaging described above, which will not be repeated here.

[0041] In some embodiments, the ratio of gelatin to deionized water is 300 mg: 5 mL.

[0042] In some embodiments, the chitosan solution concentration is 20 mg / mL.

[0043] Specifically, 300 mg of gelatin was first added to 5 mL of deionized water and stirred at 300 rpm for 10 min under a 70°C water bath to completely dissolve the gelatin. After the gelatin solution became clear, it was allowed to cool naturally to 50°C. Then, 15 mL of the above-mentioned grafted chitosan solution with a concentration of 20 mg / mL was added to the solution, and the mixture was stirred continuously at 50°C and 300 rpm for 20 min to ensure uniform mixing of the two polymers. Next, 180 mg of glycerol (as a plasticizer) was added to the above mixture, and the mixture was stirred for another 10 min to ensure thorough dispersion of the plasticizer. Subsequently, 70 mg of calcium chloride was added as a crosslinking agent, and the mixture was stirred at 300 rpm for 5 min to obtain a uniform film-forming precursor solution.

[0044] After thorough mixing, the resulting film-forming solution was slowly poured into a 90 mm diameter polystyrene petri dish, allowing it to spread evenly on the bottom. The petri dish was then dried at 40°C for 36 hours to remove the solvent and form a solid film. After drying, the film was completely peeled off from the petri dish and stored in a desiccator in a sealed container.

[0045] The food packaging film preparation method provided in this embodiment of the invention has the same implementation principle and technical effect as the aforementioned chitosan preparation method for food packaging embodiment. For the sake of brevity, any parts not mentioned in the food packaging film preparation method embodiment can be referred to the corresponding content in the aforementioned chitosan preparation method for food packaging embodiment.

[0046] The products prepared by the above method were characterized and their performance tested. All the numbers and formulations are shown in Table 1. The composition and name of the pH-responsive gelatin-chitosan composite film containing CS-g-CUR and CS-g-AL are also listed.

[0047] Table 1

[0048] Proton nuclear magnetic resonance (¹H NMR) analysis: Proton nuclear magnetic resonance (¹H NMR) spectra were measured using an Avance NEONMR spectrometer (Bruker, Germany), with a proton resonance frequency of 600 MHz. Prior to testing, the natural polymer pigments (CS-g-CUR, CS-g-AL) and pure chitosan (CS) were dissolved in a mixed solvent of deuterated water (D₂O) and deuterated hydrochloric acid (DCl), respectively, to ensure complete dissolution and obtain clear spectra.

[0049] Result (corresponding) Figure 3 In the ¹H NMR spectrum of pure CS, a characteristic singlet of acetylmethyl (–NHCOCH3) appears at δ 1.9 ppm, and typical resonance signals of sugar ring protons are observed at δ 3.0, 3.2, 3.6, and 3.7 ppm, consistent with the skeletal structure of chitosan. Compared to pure CS, the spectra of CS-g-CUR and CS-g-AL show significant changes: the acetyl singlet at δ 1.9 ppm transforms into a distinct double signal, indicating a change in the chemical environment surrounding the acetyl group; simultaneously, new characteristic resonance peaks appear at δ 2.7 ppm and δ 2.9 ppm, attributed to the –CH2–N– protons introduced by the Mannich reaction; a new peak appears near δ 7.8 ppm, corresponding to the proton signal of the aromatic ring in the pigment molecule. These new characteristic peaks directly confirm that curcumin (CUR) and alizarin (AL) have been successfully grafted onto the chitosan backbone through a methylene bridge, forming a stable covalent bond structure.

[0050] Synthesis and Verification of Basic Properties of Natural Polymer Pigments: Natural polymer pigments are synthesized via the Mannich reaction, the specific route of which is as follows: Figure 2 As shown: using formaldehyde as a bridging agent, the amino group (–NH2) on the chitosan molecular chain undergoes a condensation reaction with the active hydrogen adjacent to the aromatic ring of the pigment to form a methylene linker (–CH2–), thereby achieving covalent grafting of the pigment and chitosan.

[0051] Result (corresponding) Figure 2 The synthesized products CS-g-CUR and CS-g-AL are orange-red powders and dark brown-gray powders, respectively. Both are insoluble in water but readily soluble in 1% (w / v) acetic acid aqueous solution. Compared with pure chitosan, the solubility of the grafted products did not deteriorate significantly, while effectively improving the water insolubility defects of curcumin and alizarin. Combined with the structural verification results of ¹H NMR spectrum, it is shown that the Mannich reaction successfully achieved covalent grafting of natural pigments and chitosan. The product structure is consistent with the designed route, providing stable functional raw materials for the subsequent preparation of composite membranes.

[0052] Fourier Transform Infrared (FTIR) Spectroscopy Analysis: Fourier transform infrared (FTIR) spectra were measured using a Nicolet iS50 FTIR spectrometer (Thermo Scientific, USA). Sample powders (CS, CUR, AL, CS-g-CUR, CS-g-AL) were thoroughly ground with dry potassium bromide (KBr) at a ratio of 2% (w / w) in an agate mortar, and then pressed into transparent KBr pellets under 10,000 psi in a hydraulic press. Each sample was analyzed at 400-4000 cm⁻¹. -1 The wavenumber range was scanned 16 times, with a spectral resolution of 4 cm⁻¹. -1 To obtain a clear infrared absorption spectrum.

[0053] Result (corresponding) Figure 4 Part A): In the FTIR spectrum of pure CS, 3362 cm⁻¹ -1 The peak at 2922 cm⁻¹ represents the stretching vibrations of O–H and N–H. -1 and 2864 cm -1 Peaks for asymmetric and symmetric C–H stretching vibrations of –CH2 and –CH3, 1590 cm⁻¹ -1 The peak is the N–H bending vibration peak of amino group, at 1321 cm⁻¹. -1 This is a C–N stretching or amine skeletal vibration peak, 1420 cm⁻¹. -1 and 1374 cm -1 These are the –CH2 bending vibration peak and the –CH3 symmetric deformation vibration peak, respectively. Compared with pure CS, CS-g-CUR and CS-g-AL have peaks at 1650 cm⁻¹. -1 and 1655 cm -1 A new strong absorption peak appeared at 1590 cm⁻¹, corresponding to the aromatic C=C stretching and ketone carbonyl C=O stretching vibrations in curcumin and alizarin, confirming the successful introduction of the aromatic structure into the chitosan backbone; meanwhile, the original 1590 cm⁻¹... -1 The N–H bending peaks at the location were redshifted to 1573 cm⁻¹. -1 and 1587 cm -1 This indicates that some amino groups participate in the grafting reaction, leading to a change in the chemical environment; 2922 cm -1 The C–H stretching peak intensity was slightly enhanced and shifted slightly, attributed to the increased –CH2– content due to the –CH2– bridging structure introduced by the Mannich reaction. These spectral changes collectively confirm that curcumin and alizarin were successfully grafted onto the chitosan backbone via the Mannich reaction.

[0054] X-ray diffraction (XRD) analysis: X-ray diffraction (XRD) patterns were determined using the EMPYREAN XRD system (Malvern PANalytical, Netherlands). Cu Kα radiation (K-Alpha1 = 1.54060 Å, K-Alpha2 = 1.54443 Å) was used, with a tube voltage of 45 kV, a tube current of 40 mA, a 2θ scan range of 5–40°, and a step size of 0.01°. Crystallinity analysis was performed on CS, CS-g-CUR, CS-g-AL, and pure pigment samples.

[0055] Result (corresponding) Figure 4 (B and C sections): The pure CS sample exhibits two broad diffraction peaks at 2θ = 11.5° and 20.1°, corresponding to the (020) and (200) crystal planes, respectively, indicating that CS mainly exists in an amorphous state. In the CS-g-CUR and CS-g-AL samples, the broad peak at 2θ = 20.1° is still retained, indicating that the chitosan backbone structure was not destroyed after grafting. However, the intensity of this peak is weakened and the peak width is increased, which is attributed to the participation of some free amino groups (–NH2) in the Mannich reaction to form C–N covalent bonds, reducing the hydrogen bonding between chitosan molecular chains and increasing the amorphous properties. In addition, the CS-g-AL sample shows new weak diffraction peaks at 2θ = 10.8°, 13.8°, 26.0°, and 26.9°, corresponding to the partial crystalline structure of alizarin molecules, further confirming that alizarin was successfully grafted to the chitosan backbone.

[0056] Elemental analysis (EA) and degree of substitution (DS) calculation: Elemental analysis was performed using an Elementar Unicube elemental analyzer (Langenselbold, Germany). The mass fractions of carbon (C), hydrogen (H), and nitrogen (N) in the CS, CS-g-CUR, and CS-g-AL samples were determined, as shown in Table 2. Each sample was measured in triplicate, and the average value was taken. Based on the elemental composition, the degree of substitution (DS) was calculated using the following formula:

[0057] Table 2

[0058] UV-Vis Spectroscopy Analysis: UV-Vis absorption spectra were measured using a UH5300 UV-Vis spectrophotometer (Hitachi, Japan), with a wavelength range of 200–800 nm. Sample Preparation: 1 mg of CS, CS-g-CUR, or CS-g-AL powder was dissolved in 10 mL of 1% (w / v) acetic acid solution. Control groups were prepared simultaneously: a physical mixture of 1 mg CS and 40 μM CUR / AL was dissolved in 10 mL of 1% (w / v) acetic acid solution, and a pure 40 μM CUR / AL solution was dissolved in 10 mL of ethanol. All samples were ensured to be completely dissolved before testing.

[0059] Result (corresponding) Figure 4 (D and E portions): Pure CS showed no obvious characteristic absorption peaks in the visible light region; CS-g-CUR showed a new characteristic absorption peak at 370 nm, and CS-g-AL showed a characteristic absorption peak at 448 nm, corresponding to the chromophores of curcumin and alizarin after grafting, respectively, confirming that the pigments were successfully chemically bound to the chitosan backbone. In the control group, the absorption peaks of the physical mixture of CS and CUR / AL were located at 428 nm and 430 nm, respectively, which were significantly different from the absorption peak wavelengths of the grafted samples. This was attributed to the grafting reaction altering the electronic structure and molecular environment of the pigment molecules, further proving that the pigments and chitosan were chemically grafted rather than physically mixed.

[0060] X-ray photoelectron spectroscopy (XPS) analysis: X-ray photoelectron spectroscopy (XPS) measurements were performed using a K-ALPHA spectrometer (Thermo Fisher Scientific, USA). First, a low-resolution full spectrum in the binding energy range of 0–1200 eV was acquired (through energy 80 eV, step size 1.0 eV). Then, high-resolution spectra for C 1s, O 1s, and N 1s were acquired (through energy 20 eV, step size 0.1 eV). A Gaussian function was used to perform peak fitting and deconvolution analysis on the high-resolution spectra.

[0061] Result (corresponding) Figure 5A, B, and C are the XPS full spectra of CS, CS-g-CUR, and CS-g-AL, respectively; D, E, and F are the high-resolution C 1s XPS spectra of CS, CS-g-CUR, and CS-g-AL, respectively. In the high-resolution C 1s spectrum of pure CS, the characteristic peaks at 284.8 eV, 286.7 eV, and 288.0 eV correspond to C–C / C–H, C–O / C–N, and C=O functional groups, respectively. In the C 1s spectra of CS-g-CUR and CS-g-AL samples, new characteristic peaks at 283.0 eV and 282.6 eV, respectively, are added, attributed to the introduction of C=C bonds in the aromatic ring of the pigment; at the same time, the C–O / C–N and C=O related peaks shift and increase in intensity, indicating that the oxygen- / nitrogen-containing functional groups in the pigment interact chemically with chitosan, changing the electronic environment of the corresponding carbon atoms. The results corroborate the shift trends of the amino and carbonyl absorption peaks in the FTIR spectrum and the increase in the C / N ratio in the elemental analysis, further confirming that CUR and AL were successfully grafted onto the chitosan backbone through chemical means.

[0062] In vitro antioxidant activity (DPPH free radical scavenging rate) analysis: The in vitro antioxidant activity of the samples was evaluated using the DPPH free radical scavenging method. Sample preparation: CS, CS-g-CUR, and CS-g-AL powders were dissolved in 1% (v / v) acetic acid solution to prepare 1 mg / mL sample solutions; a fresh 0.1 mM DPPH methanol solution was prepared. Test procedure: 1 mL of sample solution was mixed with 4 mL of DPPH solution and incubated at room temperature in the dark for 30 min, followed by centrifugation at 8000 rpm for 10 min at 4℃. The supernatant was collected, and the absorbance was measured at 517 nm. The control group consisted of a mixture of 1 mL of 1% acetic acid solution and 4 mL of DPPH solution. Each sample was measured in triplicate. The DPPH free radical scavenging rate was calculated using the following formula:

[0063] in, The absorbance of the sample-DPPH mixture is given. The absorbance is for the control group.

[0064] Result (corresponding) Figure 4(F portion): The DPPH free radical scavenging rate of pure CS was only 5.95±0.12%, indicating its inherently weak antioxidant activity. After grafting modification, the scavenging rate of CS-g-CUR significantly increased to 72.13±0.17%, and the scavenging rate of CS-g-AL increased to 64.42±0.16% (p<0.05). This result confirms that curcumin and alizarin, after being grafted onto the chitosan backbone, still retain their excellent free radical scavenging ability, significantly enhancing the antioxidant performance of the material and providing support for the preservation function of the composite film.

[0065] Morphological analysis of the composite film (SEM): The surface and cross-sectional microstructure of the composite film were observed using a scanning electron microscope (SEM, SU8010, Hitachi, Japan). Surface samples were dried and then sputter-coated with gold to improve conductivity; cross-sectional samples were prepared by liquid nitrogen embrittlement and subsequently subjected to the same gold sputtering process. All samples were imaged at 5000× (surface) and 1000× (cross-section) magnifications at an accelerating voltage of 10 kV.

[0066] Result (corresponding) Figure 6 Parts B and C, as shown in Table 1): Figure 6 Part A describes the macroscopic appearance of the composite membranes. The CS / GL composite membrane is colorless and transparent with a smooth surface. SEM images show that chitosan and gelatin form a dense and continuous network structure, with both the surface and cross-section exhibiting a uniform and smooth morphology. The 0.3CgC / CS / GL and 0.3CgA / CS / GL membranes change color to orange-yellow and dark brownish-red, respectively, with wavy and uneven micro / nano structures appearing on the surface. The cross-section still maintains a dense layered structure without breakage or delamination, indicating that pigment grafting only affects surface assembly and does not destroy the overall structural integrity. The 0.3CgCA / CS / GL membrane (co-grafted with curcumin and alizarin) deepens in color to dark reddish-brown, with a finer and more uniform surface texture, significantly reduced unevenness, and a tendency towards smoothness and density. The cross-section still maintains a continuous and dense structure without pores or delamination defects, attributed to the synergistic effect of the two pigments optimizing intermolecular interactions and suppressing microscale phase separation.

[0067] Optical performance analysis of the composite film: color parameters, thickness, and opacity testing. Color parameters (L, a, b) and whiteness index (WI) were measured using a colorimeter (CR400, Minolta, Japan). The instrument was calibrated with a white standard plate (L=94.48, a=0.35, b=0.55), and WI was calculated according to a predetermined method. Film thickness was measured at 5 random points using a digital micrometer (C / N 293-240-30, Mitutoyo, Japan), and the average value was taken. Light blocking performance was measured using a UV-Vis spectrophotometer (UH5300, Hitachi, Japan). Transmittance in the 200-800 nm wavelength range was measured using an empty quartz cuvette as a reference. Opacity was calculated using the following formula (A600 is the absorbance at 600 nm, and d is the film thickness):

[0068] Result (corresponding) Figure 6 (Part E, Table 3): Compared with the CS / GL film (L=86.38±0.31, WI=84.33±0.38), the L and WI values ​​of all modified films were significantly reduced, with the 0.3CgCA / CS / GL film having the lowest (L=26.39±0.20, WI=22.66±0.27), indicating the deepest color. The a value was significantly increased, with the 0.3CgC / CS / GL film having the highest (27.51±0.25), showing the most pronounced red hue. Regarding the b value, the 0.3CgC / CS / GL film had the highest (17.21±0.31) (consistent with the yellow characteristics of curcumin), while the 0.3CgCA / CS / GL film had the lowest (5.17±0.18). In terms of thickness, the CS / GL film was 0.122±0.006 mm, while the modified film thickness decreased slightly to 0.112-0.114 mm, attributed to the pigment grafting enhancing the internal density of the film structure. Regarding opacity, the CS / GL film is 2.06 ± 0.23 Å·mm. -1 The modified membranes successively increased to 12.65 (0.3CgC / CS / GL), 14.46 (0.3CgA / CS / GL), and 15.87 (0.3CgCA / CS / GL) A·mm. -1 The 0.3CgCA / CS / GL film exhibits the best light-blocking performance, which is attributed to the superposition effect of light absorption by the two pigments.

[0069] Table 3

[0071] Water contact angle (WCA) analysis of the composite membrane: The water contact angle was measured using an OCA15EC contact angle meter (Dataphysics, Germany). 2.0 μL of distilled water was dropped onto the center of the membrane surface, and the contact angle values ​​were analyzed using SCA20 software. Each sample was measured in triplicate, and the average value was taken.

[0072] Result (corresponding) Figure 6 (Part D, Table 3): The WCA of the CS / GL film was 101.77±2.29°, exhibiting moderate hydrophobicity. After the incorporation of pigment grafts, the WCA significantly increased (p < 0.05): 111.96±0.93° for the 0.3CgC / CS / GL film, 122.27±1.44° for the 0.3CgA / CS / GL film, and 123.22±0.41° for the 0.3CgCA / CS / GL film. This change is attributed to the grafting of hydrophobic aromatic groups from curcumin and alizarin molecules onto the chitosan backbone, which reduced the exposure of hydrophilic groups (hydroxyl and amino groups) on the film surface, thereby enhancing hydrophobicity.

[0073] Intermolecular interaction (FTIR) analysis of the composite membrane: The infrared spectrum of the composite membrane was measured using a Nicolet iS50 FTIR spectrometer (Thermo Scientific, USA), with a scanning range of 400-4000 cm⁻¹. -1 4cm resolution -1 Each sample was scanned a total of 16 times.

[0074] Result (corresponding) Figure 7 Part A): CS / GL membrane at 3254 cm⁻¹ -1 (Amide A, NH stretching), 2878 cm -1 (Amide B, CH stretching), 1637 cm -1 (Amide I, C=O stretching), 1544 cm -1 (Amide II, NH4+ bend) and 1245 cm -1 A characteristic peak appears at (amide III, CN stretching and NH bending), and a peak is also present at 2930 cm⁻¹. -1 (CH2 / CH3 asymmetric bending), 1151 cm -1 Absorption peaks such as (CO stretching). No new characteristic peaks appeared after the addition of CS-g-CUR and CS-g-AL, but the amide A peak showed a red shift: the 0.3CgC / CS / GL film shifted to 3231 cm⁻¹. -1 The 0.3CgA / CS / GL membrane migrated to 3247 cm⁻¹. -1 The 0.3CgCA / CS / GL membrane migrated to 3224 cm⁻¹. -1This indicates that the phenolic hydroxyl and carbonyl groups of the pigment molecules form stronger hydrogen bonds with the hydroxyl and amino groups of chitosan / gelatin, with the 0.3CgCA / CS / GL membrane exhibiting the strongest hydrogen bonding and the most significant peak shift.

[0075] Crystallinity (XRD) analysis of the composite membrane: The XRD pattern of the composite membrane was determined by the EMPYREAN XRD system (Malvern PANalytical, Netherlands) using Cu Kα radiation (K-Alpha1=1.54060 Å, K-Alpha2=1.54443 Å), tube voltage 45 kV, tube current 40 mA, 2θ scan range 5-40°, step size 0.01°.

[0076] Result (corresponding) Figure 7 Part B): All composite membranes exhibited characteristic diffraction signals near 2θ=8.4° (collagen triple helix crystal region), 11.4° (chitosan type II crystal), and 22° (amorphous phase). After incorporating pigment grafts, the intensity of the amorphous diffraction peak at 22° was significantly enhanced, with the 0.3CgCA / CS / GL membrane showing the most significant enhancement. This indicates that the hydrogen bond network between pigment molecules and chitosan / gelatin segments strengthened the intermolecular interactions and improved the segment flexibility. Furthermore, no new crystallization peaks appeared, suggesting that pigment grafting did not alter the crystal phase structure of the composite membrane, which remained dominated by the amorphous phase of the natural polymer.

[0077] Thermal stability (TGA / DTG) analysis of the composite membrane: Thermal stability was assessed using a thermogravimetric analyzer (TGA, DSC200PC / TG209C, Netzsch, Germany). Approximately 10 mg of film fragments were placed in an alumina sample dish and incubated at 10 °C·min under a dynamic nitrogen atmosphere (nitrogen flow rate 50 mL / min). -1 The heating rate was increased from room temperature to 600 °C, and the mass change and thermal decomposition rate were recorded.

[0078] Result (corresponding) Figure 7Parts C and D): The thermal decomposition process of all composite membranes is divided into three stages: Stage I (95-160 ℃, evaporation of physically adsorbed water, mass loss 5-8%), Stage II (160-260 ℃, release of bound water and glycerol and degradation of molecular chain side groups, mainly the weight loss stage), and Stage III (260-360 ℃, cracking and carbonization of the macromolecular skeleton). The residual weights at 600 ℃ are: CS / GL membrane 33.58%, 0.3CgC / CS / GL membrane 34.33%, 0.3CgA / CS / GL membrane 34.99%, and 0.3CgCA / CS / GL membrane 36.49%, indicating that pigment grafting improves the thermal resistance of the membrane. The maximum decomposition temperature (Tmax) of Stage II is 250-260℃. The Tmax of the modified film is slightly lower than that of the CS / GL film. However, the 0.3CgCA / CS / GL film exhibits the lowest weight loss rate and the highest residual weight throughout the pyrolysis process. This is attributed to the fact that the multi-hydrogen bond network formed by the synergistic grafting inhibits chain segment cleavage and carbonization, thereby enhancing structural stability.

[0079] pH sensitivity (color response) analysis of composite membranes: 2×2 cm membrane samples were immersed in buffer solutions with pH 2-12 for 10 min each. After removal, the color changes were photographed and recorded under consistent illumination (corresponding to...). Figure 8 Part A of the calculation involves measuring color parameters (L, a, b values) using a colorimeter (CR400, Minolta, Japan) and calculating the total color difference ΔE to quantify the response intensity (corresponding to...). Figure 8 (Part B). The total color difference ΔE value is calculated based on the CIE Lab color space, reflecting the overall color difference before and after sample immersion. The larger the ΔE value, the more significant the color response.

[0080] Result (corresponding) Figure 8(Parts A and B): The 0.3CgC / CS / GL membrane remained yellow to orange-yellow throughout the pH range of 2-12, with no obvious visual color change, indicating low visual sensitivity to pH changes; the 0.3CgA / CS / GL membrane was reddish-brown at pH 2, gradually turning dark brown as the pH increased to 10, and blackish-brown at pH 12, with significant color variations; the 0.3CgCA / CS / GL membrane (co-grafted with curcumin and alizarin) showed more layered color changes, being dark reddish-brown at pH 2, brownish-brown at pH 4-6, transitioning to dark brownish-black at pH 7-9, and remaining blackish-brown at pH 10-12. The overall color changes were more vivid and the transitions smoother than those of the 0.3CgA / CS / GL membrane, resulting in higher visual recognition. The ΔE value of the 0.3CgC / CS / GL membrane remained consistently below 35%, exhibiting gentle fluctuations, confirming its low pH response sensitivity. The ΔE values ​​of both the 0.3CgA / CS / GL and 0.3CgCA / CS / GL membranes showed significant fluctuations, exhibiting two distinct peaks under acidic (pH 2-4) and alkaline (pH 8-10) conditions, with maximum values ​​exceeding 140%, indicating a strong response to extreme pH changes. The ΔE peak value of the 0.3CgCA / CS / GL membrane in the pH 7-9 range (close to the slightly alkaline environment of early-stage freshwater fish spoilage) (145.3% ± 2.1%) was slightly higher than that of the 0.3CgA / CS / GL membrane (138.7% ± 1.8%). This is attributed to the synergistic effect of curcumin and alizarin enhancing the dynamic response of intermolecular hydrogen bonds and π-π stacking, resulting in richer color gradations and higher response intensity, making it more suitable for monitoring slight pH fluctuations during food spoilage.

[0081] Molecular docking simulation validation: Rigid molecular docking was performed using the LibDock module of Discovery Studio 2019 software to explore the interaction mechanism between chitosan, pigment grafts, and gelatin. The three-dimensional structure of gelatin was obtained from the Protein DataBank (PDB), and the two-dimensional structures of chitosan and pigments were retrieved from the PubChem database. After being drawn using ChemDraw 20.0, the three-dimensional structures were constructed and energy minimization was performed using Discovery Studio 2019 for docking simulation.

[0082] Result (corresponding) Figure 9 (BD part): where, Figure 9Part A is a schematic diagram of the composite membrane structure. Docking results show that serine (Ser), threonine (Thr), arginine (Arg), and leucine (Leu) in gelatin are the main binding sites, forming hydrogen bonds with chitosan segments through hydroxyl and amino groups. In the unmodified CS-GL composite, the lysine (Lys) residues of gelatin exhibit unfavorable electrostatic repulsion with the free -NH2 of chitosan; however, after pigment grafting, some of the free -NH2 is consumed, reducing electrostatic repulsion, while the carbonyl groups of the pigment molecules form new hydrogen bond crosslinks with the hydroxyl and amino groups of gelatin. Furthermore, the synergistic effect of π-π stacking and van der Waals forces between the pigment and gelatin further enhances the barrier properties and environmental responsiveness of the composite membrane, consistent with the FTIR analysis results.

[0083] Application of composite membranes in freshwater fish preservation monitoring: Fresh mandarin fish was selected as the model food. CS / GL, 0.3CgC / CS / GL, 0.3CgA / CS / GL, and 0.3CgCA / CS / GL membranes were cut into 2×2 cm squares and attached to the inside of the petri dish lid. Fish pieces (15±2 g) were washed, dried, and placed in petri dishes, stored at room temperature (25 ℃) for 36 h. The color changes of the membranes were recorded by photographing at 0, 12, 24, and 36 h, and the total volatile basic nitrogen (TVB-N) content of the fish meat was determined. TVB-N determination: 5 g of fish meat was homogenized with 100 mL of distilled water, centrifuged at 10000 rpm for 15 min, and the supernatant was analyzed using an automated Kjeldahl nitrogen analyzer (K1160, Hanon, China).

[0084] Result (corresponding) Figure 10 During storage, the TVB-N content in the fish meat gradually increased from an initial 8.8 mg N / 100 g to 12.7 (12 h), 20.7 (24 h), and 25.2 (36 h) mg N / 100 g, indicating gradual spoilage. The CS / GL membrane showed no significant color change and was insufficiently responsive to pH fluctuations; the 0.3CgC / CS / GL membrane remained stable in color within 12 h, showed significant discoloration at 24 h, and deepened further at 36 h; the 0.3CgA / CS / GL and 0.3CgCA / CS / GL membranes showed significant color change trends within 36 h, with the 0.3CgCA / CS / GL membrane being the most sensitive to TVB-N changes, exhibiting a significant color change when TVB-N approached 20 mg N / 100 g (24 h, the freshness threshold of fish meat). The color change was well correlated with the TVB-N content, allowing for precise and visual monitoring of fish meat freshness.

[0085] The embodiments described above are merely illustrative, and some or all of the modules can be selected to achieve the purpose of this embodiment according to actual needs. Those skilled in the art can understand and implement these embodiments without any creative effort.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing chitosan for food packaging, characterized in that, include: The natural pigment was added to N,N-dimethylformamide and stirred until completely dissolved to obtain a pigment solution. Chitosan was dissolved in an aqueous solution of glacial acetic acid, allowing it to swell fully and form a transparent solution. The pigment solution was added dropwise to the transparent solution at room temperature and stirred until homogeneous to obtain a mixture. Add sufficient formaldehyde solution to the mixture until the reaction is complete, then add sodium hydroxide solution dropwise to form a precipitate. Collect the precipitate and wash and freeze-dry it to obtain chitosan powder. The natural pigments mentioned include curcumin and alizarin.

2. The method for preparing chitosan for food packaging according to claim 1, characterized in that, When the natural pigment is curcumin, the ratio of curcumin, chitosan and N,N-dimethylformamide is 934 mg: 1.0 g: 50 mL. When the natural pigment is alizarin, the ratio of alizarin, chitosan and N,N-dimethylformamide is 634 mg: 1.0 g: 50 mL.

3. The method for preparing chitosan for food packaging according to claim 1, characterized in that, The formaldehyde solution is 53 mL of a formaldehyde solution with a mass fraction of 37%.

4. The method for preparing chitosan for food packaging according to claim 1, characterized in that, The step of adding sufficient formaldehyde solution to the mixture until the reaction is complete includes: After adding sufficient formaldehyde solution to the mixture, the reaction system was stirred at 500 rpm at 60°C for 24 hours.

5. The method for preparing chitosan for food packaging according to claim 1, characterized in that, The collection of precipitates includes centrifugation at 6000 rpm for 10 minutes.

6. The method for preparing chitosan for food packaging according to claim 1, characterized in that, The concentration of the sodium hydroxide solution includes 0.5 mol / L.

7. A chitosan for food packaging, characterized in that, Chitosan for food packaging was prepared according to any one of claims 1-6.

8. A method for preparing a food packaging film, characterized in that, include: Add gelatin to deionized water, heat and stir until the gelatin is completely dissolved, then cool. Add the chitosan solution prepared from the chitosan for food packaging as described in claim 7 to the gelatin solution, and stir until evenly mixed. Then add glycerol to the mixed solution and continue stirring until fully dispersed. Add calcium chloride and stir until homogeneous to obtain the film-forming precursor solution; The film-forming precursor solution is slowly poured into a culture dish to spread evenly on the bottom of the dish. After drying to remove the solvent, a solid film is formed.

9. The method for preparing food packaging film according to claim 8, characterized in that, The ratio of gelatin to deionized water is 300 mg: 5 mL.

10. The method for preparing food packaging film according to claim 8, characterized in that, The chitosan solution concentration includes 20 mg / mL.

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