Curcumin / epsilon-polylysine antibacterial film, and preparation method and application thereof

Abstract

This invention belongs to the field of food preservation materials, and relates to a curcumin / ε-polylysine antibacterial film, its preparation method, and its application. A polyvinyl alcohol aqueous solution is added to an aqueous acetic acid solution containing carboxylated cellulose, and the mixture is heated and stirred to obtain a PVA / CC mixture. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, curcumin, and ε-polylysine are added to the PVA / CC mixture for a grafting reaction. After the reaction is complete, the reaction solution is dialyzed to obtain a reaction solution containing PVA / CC / CUR / PL. The obtained reaction solution is poured into a container and dried to obtain the curcumin / ε-polylysine antibacterial film. This invention successfully prepares a PVA / CC / CUR / PL film through solvent casting and chemical grafting. The PVA / CC / CUR / PL composite film exhibits good bactericidal effects.

Description

Technical Field

[0001] This invention belongs to the field of food preservation materials, and relates to a curcumin / ε-polylysine antibacterial film, its preparation method and application. Background Technology

[0002] Chilled chicken is not only unique in flavor but also high in moisture and rich in nutrients, resulting in consistently rising sales in the market. However, with the expansion of the market, a series of food safety issues have also emerged. For example, chilled chicken is susceptible to microbial contamination, with *P. lundensis* and *S. putrefaciens* being the most representative. Therefore, to ensure the food safety of chilled chicken and reduce food spoilage caused by microorganisms, many countries are committed to researching new antimicrobial technologies. [1] .

[0003] Curcumin (CUR) is one of the most common antibacterial agents. It is a lipophilic plant polyphenol extracted from turmeric and is widely used in the food industry as a non-toxic additive and colorant. Numerous studies have shown that CUR possesses antioxidant, antibacterial, and anti-inflammatory properties. However, its low water solubility and stability limit its applications. [2 -4]. ε-Polylysine (PL) is a naturally occurring cationic polypeptide, typically consisting of 25-35 polylysine residues. It is usually obtained from the metabolites of *Streptomyces albopictus*. PL is known to have broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria, is non-toxic, and exhibits good thermal stability. [5,6] However, due to the potential harm to humans, antimicrobial agents are rarely added directly to food. To address this issue, many researchers have incorporated antimicrobial agents directly into films to prepare antimicrobial films. Chen et al. found that polymer molecules containing CUR can improve photoinduced antimicrobial activity and stability in water. [7] According to Cheng et al.'s research, applying a chitosan-based coating containing an amino acid combination of PL and glutathione to the surface of beef can prevent bacterial growth and reproduction, thus extending shelf life. [8] However, this approach still has certain problems. For example, the antimicrobial agent has weak adhesion to the substrate, leading to its migration. This phenomenon often results in secondary contamination. Furthermore, the addition of antimicrobial agents may affect the taste of food; therefore, a stable covalent bond must be established between the matrix and the antimicrobial agent to fully utilize its antimicrobial function. [9,10] .

[0004] Grafting is reportedly a common method for establishing stable covalent bonds, which can firmly bind antimicrobial agents to the surface of various materials. Therefore, to fully utilize the antimicrobial advantages of CUR and PL and improve their stability, a suitable and reliable immobilization matrix is ​​needed for grafting. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a novel nano-antibacterial film that grafts curcumin and ε-polylysine onto carboxylated cellulose (CC) to inhibit the growth and reproduction of spoilage microorganisms in chilled fresh chicken, thereby extending the shelf life of the food.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned antibacterial film.

[0007] Finally, the technical problem to be solved by the present invention is to provide the application of the above-mentioned antibacterial film in the preparation of active packaging materials or food preservation materials.

[0008] Invention concept: First, a composite polymer material is prepared by mixing carboxylated cellulose and polyvinyl alcohol. Then, the carboxyl groups on the carboxylated cellulose are activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. Finally, the activated composite polymer material is grafted with curcumin and ε-polylysine to successfully prepare an antibacterial film.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0010] This invention discloses a method for preparing a curcumin / ε-polylysine antibacterial film, comprising the following steps:

[0011] (1) Mix the carboxylated cellulose dispersion with an aqueous acetic acid solution to obtain a carboxylated cellulose mixture; add polyvinyl alcohol to the carboxylated cellulose mixture, heat and stir to obtain a PVA / CC mixture;

[0012] (2) Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the PVA / CC mixture obtained in step (1) for activation; after activation, add curcumin dimethyl sulfoxide solution and ε-polylysine aqueous solution to the system for grafting reaction. After the reaction is completed, dialyze the reaction solution to obtain the reaction solution containing PVA / CC / CUR / PL.

[0013] (3) The reaction solution containing PVA / CC / CUR / PL obtained in step (2) is poured into a container, dried, and a PVA / CC / CUR / PL film is obtained, namely curcumin / ε-polylysine antibacterial film.

[0014] The carboxylated nanocellulose was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0015] The polyvinyl alcohol, with a purity of ≥90%, was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0016] The ε-polylysine in question was of analytical grade and purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0017] In some embodiments, in step (1), the solvent in the carboxylated cellulose dispersion is water; the concentration of carboxylated cellulose in the carboxylated cellulose dispersion is 5.5 to 6.5 wt%; the concentration of acetic acid in the acetic acid aqueous solution is 5 v / v% to 20 v / v%; the mass-volume ratio of the carboxylated cellulose dispersion to the acetic acid aqueous solution is 1 g: 1 to 10 mL; and the mass-volume ratio of polyvinyl alcohol to the acetic acid aqueous solution is 0.02 to 0.08 g: 1 mL.

[0018] In some embodiments, preferably, in step (1), the solvent in the carboxylated cellulose dispersion is water; the concentration of carboxylated cellulose in the carboxylated cellulose dispersion is 6.0 wt%; the concentration of acetic acid in the acetic acid aqueous solution is 5 v / v%; the mass-volume ratio of the carboxylated cellulose dispersion to the acetic acid aqueous solution is 1 g: 6-10 mL; and the mass-volume ratio of polyvinyl alcohol to the acetic acid aqueous solution is 0.06 g: 1 mL.

[0019] In some embodiments, in step (1), the heating and stirring are performed at a temperature of 70-90°C, preferably 80°C, and for a stirring time of 90-120 min.

[0020] In some embodiments, in step (2), the mass ratio of carboxylated cellulose dispersion to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the PVA / CC mixture is 1:0.1-0.45:0.05-0.23; the activation temperature is room temperature and the activation time is 0.5-2h.

[0021] In some embodiments, preferably, in step (2), the mass ratio of carboxylated cellulose dispersion to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the PVA / CC mixture is 1:0.216:0.115; the activation temperature is room temperature and the activation time is 1 h.

[0022] In some embodiments, in step (2), the concentration of curcumin in the curcumin dimethyl sulfoxide solution is 50-200 mmol / L, preferably 200 mmol / L; the concentration of ε-polylysine in the ε-polylysine aqueous solution is 50-200 mg / mL, preferably 200 mg / mL.

[0023] In some embodiments, in step (2), the mass-to-volume ratio of the carboxylated cellulose dispersion to the curcumin dimethyl sulfoxide solution in the PVA / CC mixture is 1 g: 5-20 μL; the mass-to-volume ratio of the carboxylated cellulose dispersion to the ε-polylysine aqueous solution in the PVA / CC mixture is 1 g: 5-20 μL; the grafting reaction is carried out at room temperature for 24-48 h.

[0024] In some embodiments, preferably, in step (2), the mass-to-volume ratio of the carboxylated cellulose dispersion to the curcumin dimethyl sulfoxide solution in the PVA / CC mixture is 1 g: 10 μL; and the mass-to-volume ratio of the carboxylated cellulose dispersion to the ε-polylysine aqueous solution in the PVA / CC mixture is 1 g: 10 μL.

[0025] The grafting reaction needs to be carried out in the dark.

[0026] In some embodiments, in step (3), the drying temperature is 37-50°C.

[0027] The curcumin / ε-polylysine antibacterial film prepared by the above method is also within the scope of protection of this invention.

[0028] The application of the aforementioned curcumin / ε-polylysine antibacterial film in the preparation of active packaging materials or food preservation materials is also within the scope of protection of this invention.

[0029] The food in question is poultry, preferably chilled fresh chicken.

[0030] Beneficial effects:

[0031] (1) This invention successfully prepared PVA / CC / CUR / PL films through solvent casting and chemical grafting. The crystallinity of the grafted composite film decreased and its thermal stability improved. In addition, the PVA / CC / CUR / PL composite film showed good bactericidal effect.

[0032] (2) In actual packaging and preservation applications of chilled chicken, chilled chicken packaged with PVA / CC / CUR / PL film, when stored at 4℃ for 6 days, had a total bacterial count of 5.941g CFU / g, maintaining its freshness; and the TVB-N content was also significantly reduced, thus delaying spoilage. The PVA / CC / CUR / PL film prepared by this invention is a promising antibacterial material with the advantage of structural stability, and has broad application value in the field of chilled chicken preservation. Attached Figure Description

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0034] Figure 1 This is a schematic diagram of the design principle of the PVA / CC / CUR / PL thin film of the present invention.

[0035] Figure 2 These are cross-sectional electron micrographs of different antibacterial films; among them, Figure 2 'a' represents PVA. Figure 2 b is CC, Figure 2 c represents a PVA / CC film. Figure 2 d represents a PVA / CC / CUR film. Figure 2 e represents a PVA / CC / PL thin film. Figure 2 f represents a PVA / CC / CUR / PL film.

[0036] Figure 3 FT-IR spectra of different antibacterial film samples.

[0037] Figure 4 XRD patterns of different antibacterial film samples.

[0038] Figure 5 TGA and DTG curves for PVA, CC, PVA / CC, PVA / CC / CUR, PVA / CC / PL, and PVA / CC / CUR / PL films; among them, Figure 5 a is the TGA curve. Figure 5 b is the DTG curve.

[0039] Figure 6 The bactericidal effects of different films on *Pseudomonas lundii* and *Shewanella putrefactiveis* were studied; among them, Figure 6 a Lund Pseudomonas and Figure 6 b. The bactericidal effect of Shewanella putrefactive bacteria.

[0040] Figure 7a TVC for chilled chicken packaged in PVA / CC / CUR, PVA / CC / PL, and PVA / CC / CUR / PL films.

[0041] Figure 7b TVB-N for chilled chicken packaged in PVA / CC / CUR, PVA / CC / PL and PVA / CC / CUR / PL films.

[0042] Figure 7c TVC for chilled chicken packaged in PCCP and PVA / CC / CUR / PL films.

[0043] Figure 7d TVB-N for chilled chicken packaged in PCCP and PVA / CC / CUR / PL films. Detailed Implementation

[0044] To make the technical problems solved, technical solutions, and beneficial effects of this invention clearer, the following examples, in conjunction with embodiments, illustrate preferred embodiments of this invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent technologies discovered by the inventors that can be used to implement this invention, and therefore can be considered preferred solutions for implementing this invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this invention.

[0045] 1. Materials and Reagents

[0046] Chilled fresh chicken (brand: Xiangjia) was purchased from Suguo Supermarket in Tiejianying. Reagents are listed in the table below.

[0047] Table 1 Test Reagents

[0048]

[0049] 2. Instruments and Equipment

[0050] Table 2 Test Instruments and Equipment

[0051]

[0052] Example 1: Preparation of antibacterial film

[0053] Preparation of PVA / CC / CUR / PL thin films:

[0054] (1) At room temperature, 5 g of carboxylated nanocellulose aqueous dispersion (solvent: water, concentration of carboxylated nanocellulose: 6 wt%, carboxyl group content in carboxylated nanocellulose: 1.2–3.0 mmol / g) was mixed with 30 mL of 5 v / v% acetic acid aqueous solution to obtain a carboxylated cellulose mixture. The mixture was homogenized at 10,000 rpm for 3 min to ensure that CC was uniformly dispersed in the acetic acid aqueous solution. Then, the carboxylated cellulose mixture was continuously stirred at 80 °C and 1.8 g of PVA powder was added. The mixture was stirred for 24 h to ensure that the carboxylated nanocellulose was uniformly dispersed, thus preparing a PVA / CC mixture (the concentration of PVA in the mixture was 6% g / mL).

[0055] (2) After the PVA / CC mixture obtained in step (1) was allowed to stand for a period of time, 1.08 g EDC and 0.574 g NHS were gradually added to the system at room temperature and stirred for 1 h to activate the carboxyl group in CC; then 50 μL CUR dimethyl sulfoxide solution (200 mmol / L) and 50 μL PL aqueous solution (200 mg / mL) were added to the system and stirred at room temperature for 24 h to complete the grafting reaction; after the reaction was completed, the reaction solution was dialyzed to remove the ungrafted residue and obtain the reaction solution containing PVA / CC / CUR / PL.

[0056] (3) The reaction solution containing PVA / CC / CUR / PL obtained in step (2) is poured into a petri dish. Then, the petri dish containing the film solution is placed in an oven at 37°C and dried to obtain a smooth and uniform PVA / CC / CUR / PL film, which is then peeled off. The design principle diagram of the PVA / CC / CUR / PL film is shown below. Figure 1 .

[0057] Preparation of PVA / CC film: The preparation method is the same as that of PVA / CC / CUR / PL film, except that step (2) is not included. The PVA / CC mixture is poured and dried to obtain the PVA / CC film.

[0058] Preparation of PVA / CC / CUR thin films: The preparation method is the same as that for PVA / CC / CUR / PL thin films, except that PL is not added during the preparation process.

[0059] Preparation of PVA / CC / PL films: The preparation method of PVA / CC / CUR / PL films differs from that CUR is not added during the preparation process.

[0060] Preparation of PCCP thin films:

[0061] (1) At room temperature, 5 g of carboxylated nanocellulose aqueous dispersion (solvent: water, concentration of carboxylated nanocellulose: 6 wt%, carboxyl group content in carboxylated nanocellulose: 1.2–3.0 mmol / g) was mixed with 30 mL of 5 v / v% acetic acid aqueous solution to obtain a carboxylated cellulose mixture. The mixture was homogenized at 10,000 rpm for 3 min to ensure that CC was uniformly dispersed in the acetic acid aqueous solution. Then, the carboxylated cellulose mixture was continuously stirred at 80 °C and 1.8 g of PVA powder was added. The mixture was stirred for 24 h to ensure that the carboxylated nanocellulose was uniformly dispersed, thus preparing a PVA / CC mixture (the concentration of PVA in the mixture was 6% g / mL).

[0062] (2) After the PVA / CC mixture obtained in step (1) is left to stand for a period of time, 50 μL of CUR dimethyl sulfoxide solution (200 mmol / L) and 50 μL of PL aqueous solution (200 mg / mL) are added to the system at room temperature. The mixture is stirred for 24 h at room temperature to ensure thorough mixing and to obtain a reaction solution containing PCCP.

[0063] (3) Pour the reaction solution containing PCCP obtained in step (2) into a petri dish, and then place the petri dish containing the film solution in an oven at 37°C until dry to prepare a smooth and uniform PCCP film and peel it off.

[0064] Example 2: Measurement Methods for Thin Film Characterization

[0065] 1. Scanning electron microscope (SEM)

[0066] First, the fracture cross-section of the sample film was obtained by liquid nitrogen freezing treatment. Then, the cross-section was coated with gold sputtering and its cross-sectional morphology was observed by SEM at 1000x magnification under an accelerating voltage of 5kV.

[0067] 2. Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction spectroscopy (XRD)

[0068] At room temperature, the thin film sample is placed on a beam exposure stage, and an exposure is performed using a wavelength range of 4000–500 cm⁻¹. -1 Resolution is 4cm -1 The FTIR spectrometer was used to perform 32 scans to record the changes in functional groups.

[0069] The thin film sample was ground into powder, and the crystallinity of the thin film sample was determined using D8 Advance X-ray diffraction. The X-ray source was Cu Kα. The voltage is 20kV, the current is 5mA, the scanning rate is 4° / min, the step size is 0.028°, and the scanning range is 5~80°(2θ).

[0070] 3. Thermal weight loss

[0071] The sample film was cut into pieces of approximately 2–5 mg and placed in an aluminum dish. Under N2 at a flow rate of 50 mL / min, a heating rate of 10 °C / min was applied, starting at 30 °C and continuing until the temperature reached 600 °C. After this process, a thermal image of the film sample's TGA (DTG) was obtained.

[0072] 4. Mechanical properties

[0073] Before measuring mechanical properties, the film thickness was measured at five different locations using a digital micrometer, and the average value was used to represent the film thickness. The pre-cut film (4×1) was mounted in a specific area of ​​the equipment, with the measurement length set at 50 mm. Various mechanical data were measured using a universal tensile testing machine at a working speed of 5.0 mm / min. All samples were measured five times repeatedly, and the average of the five measurements was taken as the result.

[0074] The following expressions can be used to calculate the TS and EB of the thin film:

[0075]

[0076]

[0077] Where K is the cross-sectional area of ​​the thin film (m²) 2 L0 is the initial length of the film, EB is the elongation at break of the film, and L max F is the maximum elongation length of the film. max This represents the maximum tensile force at which the film breaks.

[0078] 5. Water vapor permeability and contact angle test

[0079] The water vapor permeability of the membrane was tested using the standard method ASTM E96-95. Anhydrous calcium chloride was first placed in a small beaker (40 mm in diameter, 25 mm deep) as a desiccant. The beaker was then sealed with a membrane, and its initial weight was recorded. The membrane-sealed beaker was placed in an environment with a relative humidity of 50%. During this time, the moisture absorbed by the desiccant through the membrane caused a change in the weight of the beaker. The weight of the beaker was recorded every 4 hours over 24 hours. Each sample was tested in triplicate. The water vapor transfer rate of the membrane was determined using the following equation.

[0080] WVTR(g / m 2 d)=ΔH / (D×Δt)

[0081] WVP=WVTR×L / Δp

[0082] Where WVP represents the water vapor permeability coefficient (g mm / hm)2 ΔP is the water vapor pressure difference between the two surfaces of the sample, L represents the average sample thickness (mm), Δt is the test time (h), and D is the sample size (m). 2 ), where ΔH is the weight change of the glass during the experiment.

[0083] The contact angle of the thin film was measured using the sitting drop method: The thin film sample was placed on a horizontal table, and 1 μL of deionized water was dropped at five random locations using a microsyringe to measure the contact angle of the thin film.

[0084] 6. Sterilization effect test

[0085] CUR and PL themselves have strong bactericidal effects, but whether grafting CUR and PL onto PVA / CC hydrogel films still maintains good antibacterial properties requires further investigation. Therefore, the inventors tested the bactericidal effect of the composite film on the dominant spoilage bacteria P. lundensis and S. putrefaciens in chilled fresh chicken.

[0086] First, the laboratory-preserved *P. lundensis* and *S. putrefaciens* were removed from the preservation tubes and placed in 5 mL of LB medium for primary culture at 30°C for 12 h. Then, 100 μL of the primary bacterial culture was cultured again for 5 h to achieve a total bacterial count of 91 g CFU / mL. Next, 100 μL of the bacterial suspension was spread onto a 1 × 1 cm antibacterial hydrogel. After 2 h, the membrane sections were placed into centrifuge tubes containing a certain amount of normal physiological saline, and the residual bacterial count of *P. lundensis* and *S. putrefaciens* was determined using a 10-fold serial dilution method.

[0087] 7. Determination of total bacterial count in chilled chicken meat

[0088] Fresh chicken breasts were purchased from Suguo Fresh Supermarket in Nanjing. The fresh chicken breasts were cut into 5g portions, and the weighed samples were packaged separately in PVA / CC / CUR, PVA / CC / PL, and PVA / CC / CUR / PL films. After irradiation with 465-475nm light for 30 minutes, the samples were stored in a freezer at 4℃. During the 8-day storage period, the total bacterial count was analyzed every 2 days. For each experiment, at least 3 samples from each of the PVA / CC / CUR, PVA / CC / PL, and PVA / CC / CUR / PL film-packaged samples should be analyzed.

[0089] 8. TVB-N content

[0090] The TVB-N content in the sample was determined according to GB 5009.228-2016 standard.

[0091] Example 3: Analysis of Characterization Results

[0092] 1. SEM analysis results

[0093] SEM image of the cross-section of the composite film as shown below Figure 2 As shown. From Figure 2 As can be seen from this, PVA ( Figure 2 a) and CC ( Figure 2 b) The cross-section of the film is uniform and smooth, without obvious cracks or voids. Even after PVA and CC are mixed, the entire cross-section remains relatively smooth, without obvious defects. Figure 2 c). This may be due to the relatively uniform distribution of CC in the PVA hydrogel, forming intermolecular hydrogen bonds. However, the PVA / CC / CUR film ( Figure 2 The morphology of d) has changed significantly, with the cross-section of the film becoming rough and uneven, which may be due to the covalent bonds formed between CUR and CC. PVA / CC / PL( Figure 2 e) The cross-sectional morphology exhibits a striped structure, possibly due to PL grafting. The cross-section of PVA / CC / CUR / PL is shown below. Figure 2 As shown in f, the cross-section is rough with irregular stripes, indicating that CUR and PL were successfully grafted onto the cross-section of PVA / CC.

[0094] 2. FTIR analysis results of the thin film

[0095] Figure 3 The FT-IR spectra of the thin films are shown for CC, PVA, PVA / CC, PVA / CC / CUR, PVA / CC / PL, and PVA / CC / CUR / PL. Figure 3 As can be seen from this, the FT-IR of all samples is at 3290 cm⁻¹. -1 A typical characteristic peak is observed at all locations, attributed to the OH tensile vibration. At 2900 cm⁻¹... -1 The peak shown is related to the symmetrical stretching vibration of aliphatic CH bonds.

[0096] For the FT-IR spectrum of PVA, at 1647 cm⁻¹ -1 The characteristic peak of the deformation vibration of the hydroxyl group appeared, and at 1456 cm⁻¹ -1 The characteristic peaks are caused by the deformation vibrations of CH2. The FT-IR spectrum of CC at 1610 cm⁻¹ -1 A sharp peak can be clearly observed, indicating the presence of oxidized carboxyl groups. PVA, CC, and PVA / CC hydrogels all exhibit remarkably similar FT-IR spectra, and no new chemical bonds are formed when mixing PVA and CC.

[0097] In PVA / CC / CUR films, 1731 cm⁻¹-1 The characteristic peak indicates an esterification reaction between CC and CUR, and it corresponds to the 1727 cm⁻¹ peak of PVA. -1 Ester-stretched phases overlap. At 1650 and 1578 cm⁻¹ -1 The new peaks can be attributed to the stretching of the CUR benzene ring and the bending vibration of the CH group in the olefin.

[0098] For PVA / CC / PL films, 1640cm -1 The sharp peak at 1560 cm⁻¹ represents the C=O stretching of the amide, while 1560 cm⁻¹ represents the C=O stretching of the amide. -1 The peak shape change at 1262 cm⁻¹ is caused by the NH stretching of the primary amine, proving that PL was successfully grafted onto PVA / CC via a covalent coupling reaction between the primary amine group and the carboxyl group of CC. Furthermore, at 1262 cm⁻¹... -1 The small peak may be related to the protonation of some -NH2 in PL, and the protonation of -NH2 leads to NH3. + The formation of NH3 + It can form ionic bonds with -COO- in CC.

[0099] In the spectrum of PVA / CC / CUR / PL thin films, 3290 cm⁻¹ -1 and 2900cm -1 The peaks represent the stretching vibrations of the -OH and CH bonds, respectively. Because PL and CUR are grafted onto CC simultaneously, some characteristic peaks overlap. The FT-IR spectrum did not show significant changes, therefore no new peaks appeared.

[0100] 3. XRD Analysis Results

[0101] XRD patterns of various thin film samples, such as Figure 4 As shown. From Figure 4 As can be seen from the data, a sharp diffraction peak appears in the PVA film at 2θ = 19.5°, corresponding to the (101) plane of the PVA crystal. This peak is generated by the strong intermolecular hydrogen bonding of the PVA chains.

[0102] The CC film exhibits a broad peak at 2θ = 14.1° and a steep peak at 2θ = 22°, corresponding to the (-110) and (002) crystal planes of cellulose, respectively.

[0103] When PVA and CC are mixed, the peak intensity at 2θ = 19.5° decreases, leading to a decrease in crystallinity. This phenomenon may be a result of hydrogen bonding between PVA and CC, which reduces the crystallinity of the original material. Besides the strong peak near 2θ = 19.5°, the absorption intensity of the characteristic diffraction peak of the PVA / CC film at 2θ = 22° gradually weakens.

[0104] For PVA / CC / PL films, the diffraction peaks at 2θ = 19.5° and 2θ = 22° are broadened and weakened. This may be due to the change in the film's molecular structure caused by the reaction between the carboxyl groups of CC and the amino groups of PL.

[0105] The XRD pattern of the PVA / CC / CUR film also shows characteristic peaks with low intensity near 2θ = 19.5° and 2θ = 22°. The variation in peak intensity indicates a change in the morphology of the composite film's crystal structure. This decrease in crystallinity may be due to the interaction between the OH groups in CUR and the carboxyl groups in CC.

[0106] XRD images of the PVA / CC / CUR / PL thin film showed corresponding peaks at 2θ = 19.5° and 2θ = 22°, but the peak intensities decreased. This indicates that CC underwent esterification and amide reactions with CUR and PL, respectively, and the intermolecular hydrogen bond network was partially disrupted. Furthermore, during the grafting process, the intermolecular crosslinking between PVA and CUR or PL affected the crystallization of PVA. This is the main reason why the peak at 2θ = 19.5° of the grafted composite film is weaker and narrower than that of the PVA sample.

[0107] 4. Results of thermal stability analysis

[0108] Figure 5The thermal degradation processes of CC, PVA, PVA / CC, PVA / CC / CUR, PVA / CC / PL, and PVA / CC / CUR / PL films were shown. All samples could be divided into three degradation stages. The first stage of PVA film decomposition occurred in the temperature range of 30℃ to 245℃, likely due to mass loss caused by the evaporation of water and residual acetic acid. The second stage of thermal degradation occurred between 240℃ and 410℃, with a very rapid decrease in film weight. This stage was likely due to chain cleavage caused by the decomposition of hydroxyl groups in the PVA side chains. The third stage of PVA film degradation occurred approximately from 410℃ to 540℃, possibly related to the degradation of the PVA backbone. The composite films formed after grafting PVA / CC / CUR, PVA / CC / PL, and PVA / CC / CUR / PL also exhibited three distinct decomposition stages. The highest peak heat loss values ​​were 438℃, 446℃, and 450℃, respectively. Compared to PVA, CC, and PVA / CC films, the highest heat loss peaks of the grafted films PVA / CC / CUR, PVA / CC / PL, and PVA / CC / CUR / PL shift towards higher temperatures. For PVA / CC / CUR, this phenomenon may be due to the esterification reaction during grafting, which leads to a significant decrease in carboxyl and hydroxyl groups, thus strengthening intermolecular forces. For PVA / CC / PL, the improved thermal stability is likely due to the chemical reaction between CC and PL. The synthesis of PVA / CC / CUR / PL composite films involves both of these grafting processes, and the improvement in thermal stability is more pronounced.

[0109] 5. Analysis results of mechanical properties, water vapor permeability and contact angle

[0110] Mechanical properties are a crucial factor in evaluating food packaging films. Table 3 shows that CC film exhibits a high total strength (TS) (24.17 ± 0.48 mPa) and a low elasticity (EB) (10.99% ± 2.24%), indicating that CC is relatively hard and has poor elasticity. PVA film has a TS of 14.97 mPa and an EB of 161.59% ± 5.17%, indicating that it is elastic and relatively soft. For PVA / CC, the TS and EB are 18.00 ± 0.29 mPa and 104.88% ± 5.12%, respectively, indicating that adding CC can increase the TS and reduce the EB of PVA film; other related studies have yielded similar conclusions. The increase in TS of the composite material is due to the formation of hydrogen bonds between CC and PVA. After grafting PL into the film, the TS of the PVA / CC / PL film is 20.30 ± 0.29 mPa. The interaction between NH2 in PL and COOH in CC produces amide bonds, promoting a tight bond between the two polymers, which is likely the main reason for the increased TS. Furthermore, the cohesion of the PVA / CC polymer chains is disrupted by the addition of PL, and the hydrogen bonds generated during the grafting process of PVA reduce the flexibility of the composite film. For PVA / CC / CUR, the tensile strength (TS) of the film is improved due to the chemical bond interaction between CC and CUR, and the number of hydrogen bonds in the film grafted with CUR also increases, leading to a decrease in the tensile strain at break (EB) of the composite film containing CUR. When CUR and PL are grafted into the PVA / CC film, the PVA / CC / CUR / PL film exhibits a higher TS (24.53 ± 2.01 mPa) and a lower tensile strain at break (59.46% ± 2.93%). This phenomenon may indicate that the grafting reaction of the two antibacterial substances forms more strongly bonded chemical bonds, increasing the mechanical strength of the composite film, but restricting intermolecular movement, resulting in a decrease in EB.

[0111] Table 3 shows the WVP values ​​and contact angles of different thin film samples. The contact angle of CC was 96.8°±0.6°, indicating it is a hydrophobic material. The WVP of the PVA film was 6.98±0.81×10⁻⁶. -9 g Pa -1 s -1 cm -1 This is a highly hydrophilic polymer, which is related to the abundance of hydrophilic hydroxyl groups in the side chains of PVA. When PVA is combined with CC to form a thin film, its WVP is approximately 1.97 ± 0.15 × 10⁻⁶. -8 g Pa -1 s -1 cm -1The contact angle was 45.0°±0.6°. This indicates that adding CC to the PVA film can improve the film's hydrophilicity. For the PVA / CC / PL film, the contact angle decreased significantly, while the WVP increased significantly, reaching 3.39±0.40×10⁻⁶. -8 g Pa -1 s -1 cm -1 The enhanced hydrophilicity is mainly due to the presence of numerous hydrophilic groups in PL, such as hydroxyl and amino groups. The WVP of the PVA / CC / CUR film is approximately 9.64 ± 0.83 × 10⁻⁶. -9 g Pa -1 s -1 cm -1 This is due to the reduced hydrophilicity of the membrane caused by the ester bonds formed after CUR grafting. Compared to PVA / CC / PL, the contact angle of the PVA / CC / PL / CUR composite membrane is slightly increased, which may be due to the reduction in the distance between polymer chains caused by CUR in the composite membrane. This finding is consistent with the report by Roy et al., who revealed that adding CUR to CMC composite membranes increases surface hydrophobicity. In addition, the hydrophobic CUR is uniformly distributed throughout the polymer matrix, acting as a physical barrier in the water vapor channels, which may also be one of the reasons. The WVP of the PVA / CC / CUR / PL film is 3.26 ± 0.24 × 10⁻⁶. -8 g Pa -1 s -1 cm -1 This may also be a result of the interaction between CUR and PL during grafting.

[0112] Table 3. Thickness, tensile strength (TS), elongation at break (EB), water vapor transmission rate (WVP), and contact angle (CA) of six types of films.

[0113]

[0114] 6. Results of bactericidal performance analysis

[0115] Figure 6The bactericidal effects of different films were demonstrated. *P. lundensis* and *S. putrefaciens* strains were used to represent dominant spoilage bacteria to demonstrate the bactericidal activity of PVA / CC composite films grafted with CUR and PL. The PVA / CC film did not show significant antibacterial activity against the two dominant spoilage bacteria present in chilled chicken. Conversely, the composite hydrogel grafted with CUR showed a significant bactericidal effect. After 2 hours of treatment with the PVA / CC / CUR film, the total colony count of *P. lundensis* was approximately 1.04 lg CFU / mL, and the viable count of *S. putrefaciens* decreased by 2.23 lg CFU / mL, with bactericidal rates of 90.6% and 99.5%, respectively. After 2 hours of treatment with the PVA / CC / PL film, approximately 1.13 lg CFU / mL of *P. lundensis* and 2.23 lg CFU / mL of *S. putrefaciens* were killed, with bactericidal rates of 90.9% and 99.5%, respectively. For the PVA / CC / CUR / PL film, the synergistic effect of CUR and PL significantly improved the bactericidal effect. Compared with the PVA / CC film, the viable bacterial counts of *P. lundensis* and *S. putrefaciens* decreased by 1.97 lg CFU / mL and 2.67 lg CFU / mL, respectively, with bactericidal rates reaching 99.0% and 99.8%, respectively. This result indicates that under aerobic conditions, irradiation with light of a specific wavelength can activate CUR in the film. During this process, reactive oxygen species (ROS) are formed, causing oxidative damage to bacterial lipid membranes, proteins, and nucleic acids, leading to cell necrosis or apoptosis. CUR itself can also inhibit the polymerization of ftsz, a protein essential for cell division and bacterial survival. It interferes with the formation of Z-loops by preventing the assembly of ftsz protists, inhibiting cell migration, and inactivating bacteria. In addition, PL is a potent antimicrobial peptide that can bind to the bacterial cell membrane, causing outer membrane separation, leading to cytoplasmic leakage and irregular distribution, and then killing the bacteria. Therefore, these results collectively demonstrate the antimicrobial properties of the composite PVA / CC / CUR / PL film.

[0116] 7. Practical application of composite film in the storage of chilled chicken

[0117] 7.1 Total bacterial count

[0118] Total bacterial count (TVC) is closely related to the safety of chilled chicken. From... Figure 7aThe initial TVC value of all samples was 4.69 lg CFU / g, and the TVC value of all samples gradually increased with the extension of storage time. PVA / CC / CUR, PVA / CC / PL, and PVA / CC / CUR / PL all successfully inhibited bacterial growth and reproduction in chilled chicken. After 6 days of storage, the total colony count of the blank control group was 7.69 lg CFU / g; for the chilled chicken samples packaged and stored with PVA / CC / CUR, the TVC value was lower, approximately 6.84 lg CFU / g. This may be because light irradiation activated the CUR in the composite membrane, generating reactive oxygen species (ROS) through a series of complex reactions. ROS can kill bacteria in chilled chicken to achieve the preservation effect. In addition, the PVA / CC / PL group also showed good antibacterial activity, with a TVC value of 6.63 lg CFU / g on the 6th day of storage. PL is a cationic surfactant that has electrostatic adsorption on the surface of microbial cells, which can lead to the destruction of their external structure and irregular cytoplasmic distribution, ultimately causing damage to bacterial cells. In contrast, the TVC value of the PVA / CC / CUR / PL group was only 5.94 lg CFU / g, possibly because CUR and PL worked together to strongly inhibit bacterial growth in chilled chicken. After 8 days of storage, the TVC of the sample treated with PVA / CC / CUR / PL was 6.69 lg CFU / g, which was much lower than that of the blank control group.

[0119] like Figure 7cAs shown, the TVC content of the control group, PCCP group, and PVA / CC / CUR / PL group all gradually increased with time, while the TVC content of the PCCP and PVA / CC / CUR / PL groups was consistently lower than that of the control group (p < 0.05). At 4 days of storage, the TVC content of the control group was 6.401g CFU / g, while the TVC content of the PCCP group was 5.911g CFU / g, and the TVC content of the PVA / CC / CUR / PL group was approximately 5.401g CFU / g. By day 6 of storage, the TVC content of the control group was approximately 7.691g CFU / g, while the TVC content of the PCCP group was 6.671g CFU / g, all significantly exceeding the national standard. The TVC value of the PVA / CC / CUR / PL group was 5.941g CFU / g. This indicates that during the first 4 days of storage, both the PCCP and PVA / CC / CUR / PL groups exhibited good antibacterial effects, maintaining the freshness of chilled chicken. As storage time increased, the difference in antibacterial effects between the PCCP and PVA / CC / CUR / PL groups became increasingly apparent, with the PVA / CC / CUR / PL grafted film exhibiting a more sustained antibacterial effect. This may be because CUR and PL form stable covalent bonds when grafted into the PVA / CC substrate, allowing CUR and PL to better exert their antibacterial effects. In contrast, the PCCP film is prepared through blending, and the antibacterial substances CUR and PL may migrate, leading to a reduction in the content of antibacterial substances and thus affecting the antibacterial effect of the blended film.

[0120] 7.2 TVB-N Analysis

[0121] The increase in TVB-N content in chilled chicken during storage is often closely related to bacterial growth and increased endogenous enzyme activity. Therefore, TVB-N can be used to determine whether chilled chicken meets food standards in order to ensure its freshness. Figure 7bIt can be seen that the TVB-N content in chilled chicken increases with the extension of storage time. On day 0 of storage, the TVB-N content is low, approximately 11.9 mg / 100g. After day 4 of storage, the TVB-N content in the blank control group reaches 32.43 mg / 100g, indicating that the chilled chicken has spoiled and is inedible (National Standard GB 5009.228-2016 stipulates that chilled chicken with a TVB-N content exceeding 25 mg / 100g is considered spoiled meat). The TVB-N contents in the PVA / CC / CUR, PVA / CC / PL, and PVA / CC / CUR / PL groups are 24.27 mg / 100g, 23.8 mg / 100g, and 20.3 mg / 100g, respectively, all below 25 mg / 100g. Chilled chicken packaged with PVA / CC / CUR / PL showed the best preservation effect, with its TVB-N content increasing slowly, reaching 22.4 mg / 100g on day 6. After 6 days of storage, the chilled chicken meat still remained fresh, indicating that the composite film containing CUR and PL can effectively reduce the rate of TVB-N formation and delay meat spoilage.

[0122] from Figure 7d It can be seen that the TVB-N content in the blank group, PCCP group, and PVA / CC / CUR / PL group all gradually increased during storage. According to the Chinese national standard GB 5009.228-2016, the TVB-N content of fresh meat is ≤25mg / 100g, and that of spoiled meat is >25mg / 100g. Therefore, the TVB-N content of the control blank group after 4 days of storage was 32.43mg / 100g, which exceeded the range for fresh meat, indicating that the chicken breast had spoiled. However, the PCCP-treated sample only spoiled after 6 days of storage. The TVB-N value of the PVA / CC / CUR / PL group only exceeded the safety limit after 8 days of storage. This indicates that the PCCP group and the PVA / CC / CUR / PL group can effectively reduce TVB-N production and prevent protein oxidation to a certain extent, but the PVA / CC / CUR / PL group is more effective. This may be because CUR and PL in the PCCP film separated from the film, leading to unstable antimicrobial properties of the film. The antibacterial substances CUR and PL on the PVA / CC / CUR / PL film are covalently bonded, making them less prone to migration and effectively delaying the deterioration of chicken quality.

[0123] This invention successfully prepared a PVA / CC / CUR / PL film through solvent casting and chemical grafting. SEM results showed that the PVA / CC / CUR / PL composite film grafted with CUR and PL exhibited rough, irregular striations in cross-section. FT-IR results showed that the grafting of CC with CUR and PL was successful. The crystallinity of the grafted composite film decreased, and its thermal stability improved. Furthermore, the PVA / CC / CUR / PL composite film showed good bactericidal effects against *P. lundensis* and *S. putrefaciens*. In actual packaging and preservation applications of chilled chicken, chilled chicken packaged with PVA / CC / CUR / PL film, stored at 4°C for 6 days, had a total bacterial count of 5.94 lg CFU / g, and the chicken remained fresh. The TVB-N content was also significantly reduced, thus delaying spoilage. Therefore, the PVA / CC / CUR / PL film is a promising antibacterial material with the advantage of structural stability and can be widely used in the field of chilled chicken preservation in the future.

[0124] This invention provides a curcumin / ε-polylysine antibacterial film, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

[0125] References:

[0126] [1]Abdou ES,Galhoum GF,Mohamed E N.Curcumin loaded nanoemulsions / pectincoatings for refrigerated chicken fillets[J].Food Hydrocolloids,2018,83:445-53.

[0127] [2]Madusanka N,De Silva KMN,Amaratunga GA curcuminactivatedcarboxymethyl cellulose-montmorillonite clay nanocomposite havingenhancedcurcumin release in aqueous media[J].Carbohydrate Polymers,2015,134:695-9.

[0128] [3]Roy S,Rhim J-W.Preparation of antimicrobial and antioxidantgelatin / curcumincomposite films for active food packaging application[J].Colloids and SurfacesB-Biointerfaces,2020,188.

[0129] [4]Roy S,Rhim J-W.Preparation of bioactive functional poly(lacticacid) / curcumincomposite film for food packaging application[J].InternationalJournal of BiologicalMacromolecules,2020,162:1780-9.

[0130] [5]Li H,Cui R,Peng L,et al.Preparation of Antibacterial CellulosePaper UsingLayer-by-Layer Assembly for Cooked Beef Preservation at AmbientTemperature[J].

[0131] Polymers,2018,10(1).

[0132] [6]Zhou C,Li P,Qi X,et al.A photopolymerized antimicrobial hydrogelcoatingderived from epsilon-poly-L-lysine[J].Biomaterials,2011,32(11):2704-12.

[0133] [7]Chen L,Dong Q,Shi Q,et al.Novel 2,3-Dialdehyde Cellulose-BasedFilms withPhotodynamic Inactivation Potency by Incorporating thebeta-Cyclodextrin / Curcumin Inclusion Complex[J].Biomacromolecules,2021,

[0134] 22(7):2790-801.

[0135] [8]Cheng Y,Hu J,Wu S.Chitosan based coatings extend the shelf-life ofbeef slicesduring refrigerated storage[J].Lwt-Food Science and Technology,2021,138.

[0136] [9]Nie C,Shen T,Hu W,et al.Characterization and antibacterialproperties ofepsilon-poly-L-lysine grafted multi-functional cellulose beads[J].CarbohydratePolymers,2021,262.

[0137]

[10] Sun H,Shao X,Zhang M,et al.Mechanical,barrier and antimicrobialproperties ofcorn distarch phosphate / nanocrystalline cellulose filmsincorporated with Nisin andepsilon-polylysine[J].International Journal ofBiological Macromolecules,2019,136:839-46.

Claims

1. A method for preparing a curcumin / ε-polylysine antibacterial film, characterized in that, Includes the following steps: (1) Mix the carboxylated cellulose dispersion with an aqueous acetic acid solution to obtain a carboxylated cellulose mixture; add polyvinyl alcohol to the carboxylated cellulose mixture, heat and stir to obtain a PVA / CC mixture; (2) Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the PVA / CC mixture obtained in step (1) for activation; after activation, add curcumin dimethyl sulfoxide solution and ε-polylysine aqueous solution to the system for grafting reaction. After the reaction is completed, dialyze the reaction solution to obtain the reaction solution containing PVA / CC / CUR / PL. (3) The reaction solution containing PVA / CC / CUR / PL obtained in step (2) is poured into a container, dried, and a PVA / CC / CUR / PL film is obtained, namely curcumin / ε-polylysine antibacterial film.

2. The preparation method according to claim 1, characterized in that, In step (1), the solvent in the carboxylated cellulose dispersion is water; the concentration of carboxylated cellulose in the carboxylated cellulose dispersion is 5.5~6.5wt%; the concentration of acetic acid in the acetic acid aqueous solution is 5v / v%~20v / v%; the mass-volume ratio of the carboxylated cellulose dispersion to the acetic acid aqueous solution is 1 g: 1~10 mL; and the mass-volume ratio of polyvinyl alcohol to the acetic acid aqueous solution is 0.02~0.08 g: 1 mL.

3. The preparation method according to claim 1, characterized in that, In step (1), the heating and stirring are carried out at a temperature of 70~90℃.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of carboxylated cellulose dispersion to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the PVA / CC mixture is 1:0.1~0.45:0.05~0.23; the activation temperature is room temperature and the activation time is 0.5~2 h.

5. The preparation method according to claim 1, characterized in that, In step (2), the concentration of curcumin in the curcumin dimethyl sulfoxide solution is 50~200 mmol / L; the concentration of ε-polylysine in the ε-polylysine aqueous solution is 50~200 mg / mL.

6. The preparation method according to claim 1, characterized in that, In step (2), the mass-to-volume ratio of carboxylated cellulose dispersion to curcumin dimethyl sulfoxide solution in the PVA / CC mixture is 1 g: 5~20 μL; the mass-to-volume ratio of carboxylated cellulose dispersion to ε-polylysine aqueous solution in the PVA / CC mixture is 1 g: 5~20 μL; the grafting reaction is carried out at room temperature for 24~48 h.

7. The preparation method according to claim 1, characterized in that, In step (3), the drying temperature is 37~50℃.

8. The curcumin / ε-polylysine antibacterial film prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the curcumin / ε-polylysine antibacterial film according to claim 8 in the preparation of active packaging materials or food preservation materials.

10. The application according to claim 9, characterized in that, The food in question is poultry food.

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