Polylactic acid-based film with efficient active nitrogen-mediated photodynamic bacteriostatic activity and preparation method of polylactic acid-based film

Abstract

The invention discloses a polylactic acid-based film with efficient active nitrogen-mediated photodynamic bacteriostatic activity and a preparation method thereof, and relates to the technical field of food packaging films.The method comprises the following steps that PLA serves as a film matrix, DA serves as a surface modification raw material, and a PLA / PDA film is formed on the surface of a PLA film through polymerization and cross-linking reaction; bBRArgCMCDTAT is used as PS, carboxyl groups on the PS are utilized, coupling treatment with the PLA / PDA film subjected to surface modification is carried out through an amide reaction or an esterification reaction, covalent grafting is carried out, and the PLA / PDA / PS film with efficient photodynamic antibacterial activity is obtained. The PDI antibacterial film obtained from the polylactic acid-based film has excellent mechanical properties, and generates a large amount of RNS under the irradiation of white light to efficiently kill gt; the treatment time is short, the sterilization efficiency is high, and the operation is simple and convenient.

Description

Technical Field

[0001] This application relates to the field of food packaging film technology, and in particular to a polylactic acid-based film with highly efficient active nitrogen-mediated photodynamic antibacterial activity and its preparation method. Background Technology

[0002] Compared to traditional heat sterilization techniques, non-heat sterilization can preserve the natural color, aroma, and flavor of food to a greater extent, reducing negative impacts. Photodynamic inactivation (PDI), as a novel non-heat sterilization technology, is widely recognized as an effective method for killing microorganisms and ensuring food quality and safety. Due to its advantages such as being green and safe, non-toxic and residue-free, and not inducing antibiotic resistance in bacteria, it has gradually become a research hotspot in the food industry in recent years. The three essential elements of a PDI system are light, oxygen, and a photosensitizer (PS). Its principle is that under irradiation with a specific wavelength of light, the photosensitizer absorbs energy and undergoes a transition, transferring the energy to oxygen molecules, converting O2 into highly oxidizing reactive oxygen species (ROS). ROS damages cell microstructure, DNA, lipids, proteins, etc., leading to bacterial cell death.

[0003] To obtain polylactic acid (PLA) packaging materials with PDI antibacterial activity, some studies have involved plasma-modifying the surface of PLA films. This involves bombarding the material surface with high-energy electrons, ions, and free radicals via plasma to form active carboxyl sites on the PLA film surface. Furthermore, PS is covalently grafted onto the plasma-modified PLA film surface. The resulting PLA / ALA film exhibits high performance at 100.80 J / cm². 2 Blue light irradiation can kill ~3 Log CFU / mL Listeria monocytogenes. Furthermore, researchers have used electrospinning technology to prepare MZ-8 / PLA films by blending PLA with a photothermal titanium carbide / zeolite imidazole backbone-8 (MZ-8). This film can generate thermal and ROS effects based on photothermal and PDI effects, killing ~3 Log CFU / mL Escherichia coli and methicillin-resistant Staphylococcus aureus.

[0004] However, plasma etching can damage the surface structure of the film, significantly reducing its mechanical properties. More importantly, traditional PDI antibacterial films prepared using these methods primarily rely on generated ROS as the active substance to attack bacterial cells. The short half-life (3µs) and small effective radius (100nm) of ROS greatly limit the bactericidal efficiency of PDI films in practical applications. While photothermal effects combined with PDI effects can improve bactericidal efficiency, a single film exhibiting both photothermal and PDI effects often requires two independent excitation sources, which can complicate the bactericidal process. Summary of the Invention

[0005] This application provides a polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity and its preparation method, aiming to solve the technical problems of poor mechanical properties and low bactericidal efficiency of traditional PDI antibacterial films prepared by the prior art.

[0006] To address the aforementioned technical problems, this application provides a method for preparing a polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity, comprising the following steps: PLA / PDA film is formed by polymerizing PLA film on the surface of PLA film using PLA as film matrix and DA as surface modification material through polymerization and crosslinking reaction; Using BBR, Arg, CMCD, and TAT as PS, and utilizing the carboxyl groups on the PS, covalent grafting is performed by coupling them with surface-modified PLA / PDA films through amide or esterification reactions to obtain PLA / PDA / PS films with highly efficient photodynamic antibacterial activity.

[0007] As some optional embodiments of this application, the DA concentration is 2~2.5 mg / mL, and the dosage is 400~500 mg; the area of ​​the PLA film is 50 cm². 2 .

[0008] As some optional embodiments of this application, in the polymerization and crosslinking reaction, the concentration of the Tris-HCl buffer is 10~20 mM, the pH is 8.0~8.5, the shaking speed is 180~200 rpm, the reaction temperature is 25 ℃~50 ℃, and the reaction time is 6 h~30 h. The film preparation involves two steps: the first step is to immerse a PLA film in a DA solution, where DA self-aggregates under weakly alkaline conditions and simultaneously adheres to the PLA film to form a coating, thus obtaining a PLA / PDA film; the second step is to graft a photosensitizer onto the PLA / PDA film using an amide reaction or esterification reaction to obtain a PLA / PDA / PS film.

[0009] As some optional embodiments of this application, if it is an amide reaction, the concentration of PS is 100 μM~300 μM, and the solvent is sterile deionized water.

[0010] As some optional embodiments of this application, the PS is placed in an EDC / NHS conjugated solution for carboxyl group activation treatment. After the activation treatment is completed, it is coupled with the PLA / PDA film in the EDC / NHS conjugated solution. The activation treatment conditions are 180~200 rpm, 35~45 ℃, 2~2.5 h; The coupling treatment conditions are 180~200 rpm, 35~45 ℃, and 4~6 h.

[0011] As some optional embodiments of this application, the EDC / NHS conjugated solution system is prepared by 0.1~0.2 M of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.02~0.04 M of N-hydroxysuccinimide, and 0.1~0.2 M of 2-morpholinoethanesulfonic acid, and the pH value of the conjugated solution system is adjusted to 5.2~5.4.

[0012] As some optional embodiments of this application, if it is an esterification reaction, the concentration of PS is 100 μM~300 μM and the solvent is DMF.

[0013] As some optional embodiments of this application, the PS is placed in a DCC / DMAP conjugated solution for carboxyl group activation treatment. After the activation treatment is completed, it is coupled with the PLA / PDA film in a DCC / DMAP conjugated solution. The DCC solution has a concentration of 0.12~0.24 M and a volume of 2~4 mL; the DMAP has a concentration of 0.01~0.02 M and a volume of 0.5~0.1 mL. The activation treatment includes: a first activation treatment in a DCC solution, followed by a second activation treatment with the addition of DMAP; wherein the conditions for the first activation treatment are 180~200 rpm, 25~30 ℃, 30~40 min; and the conditions for the second activation treatment are 180~200 rpm, 25~30 ℃, 10~20 min. The coupling treatment conditions are 180~200 rpm, 35~45 ℃, and 4~6 h.

[0014] As some optional embodiments of this application, after obtaining a PLA / PDA / PS film with highly efficient active nitrogen-mediated photodynamic antibacterial activity, the following further methods are included: The PLA / PDA / PS film was rinsed and dried sequentially with DMF, ethanol and deionized water to remove unbound photosensitizers BBR / Arg / CMCD / TAT. The drying conditions are: temperature 25~35 ℃, time 12~24 h.

[0015] To address the aforementioned technical problems, this application also provides: a polylactic acid-based film with high photodynamic antibacterial activity, prepared by the method described above.

[0016] Compared to existing technologies, the advantages of this application lie in the fact that PDA-modified PLA films can introduce a large number of active groups onto their surface while ensuring the mechanical properties of the film. Furthermore, through an amidation reaction, PS with RNS-generating capabilities is covalently grafted onto the film surface, preparing PLA / PDA / PS films with high PDI activity. The PDI antibacterial film designed based on this application exhibits excellent mechanical properties and generates a large amount of RNS under white light irradiation, effectively killing >8 Log of foodborne pathogens with short processing time, high sterilization efficiency, and simple operation. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0018] Figure 1 The effect of different processing temperatures on the surface thickness of PLA / PDA films; Figure 2 The effect of different incubation times on the surface thickness of PLA / PDA films; Figure 3 PS content on PLA / PDA / PS films prepared by different covalent grafting methods; Figure 4 For PLA / PDA / PS films, tensile strength and elongation at break; Figure 5 ROS yield of PLA / PDA / PS films; Figure 6 NO yield of PLA / PDA / PS films; Figure 7 ONOO for PLA / PDA / PS films - Yield; Figure 8 PDI-mediated PLA / PDA / PS thin film pair L. monocytogenes The sterilization efficiency; Figure 9 PDI-mediated PLA / PDA / PS thin film pair V. parahaemolyticus The sterilization efficiency.

[0019] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0020] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0021] As mentioned above, with the increasing awareness of environmental protection and the promotion of related policies, the use of natural biodegradable biopolymers to replace traditional non-degradable chemical materials has become a research hotspot and development trend in the food packaging field. Polylactic acid (PLA) is a biopolymer material synthesized from plant resources. It has good mechanical properties and is biodegradable. It has been approved by the U.S. Food and Drug Administration (FDA) as a raw material for developing food packaging films. PLA has no toxic side effects on the human body and does not cause environmental pollution after disposal, making it an ideal new green and sustainable packaging substrate. At present, PLA-based packaging materials have successfully entered the market and achieved commercial application. However, PLA is a bio-inert substance with poor natural hydrophilicity. Its molecular chain segments lack active structures and do not possess antibacterial properties, which prevents PLA films from further extending the shelf life of food and ensuring food safety. These shortcomings limit its application in the field of antibacterial active packaging for food.

[0022] Based on this, this application proposes a polylactic acid (PLA)-based food packaging film with highly efficient PDI antibacterial activity. The product uses PLA, a green and biodegradable material, as the film matrix and dopamine (DA) as the surface modification material. The two are polymerized on the PLA film surface through the polymerization and crosslinking of DA to form a PLA / PDA film, introducing a large number of active amino and hydroxyl groups onto the PLA film surface. The product further uses BBR / Arg / CMCD / TAT, which has RNS-generating ability, as a photosensitizer (PS). Utilizing the carboxyl groups on PS, it is covalently grafted onto the surface-modified PLA / PDA film through amide and esterification reactions to prepare a PLA / PDA / PS film with highly efficient active nitrogen-mediated photodynamic antibacterial activity. Under white light irradiation, this film can rapidly and efficiently generate RNS, effectively killing common pathogenic bacteria in food.

[0023] The concentration of DA is 2~2.5 mg / mL (preferably 2 mg / mL); in the PDA surface modification PLA reaction, the concentration of Tris-HCl buffer used is 10~20 mM (preferably 10 mM), and the pH is 8.0~8.5; the shaking speed is 180~200 rpm; the reaction temperature is 25 ℃~50 ℃, preferably 25 ℃, 30 ℃, 40 ℃ and 50 ℃, and optimally 30 ℃; the reaction time is 6 h~30 h, preferably 6 h, 12 h, 18 h, 24 h and 30 h, and optimally 12 h.

[0024] When the PLA / PDA film is covalently grafted using an amide reaction, the concentration of the PS is 100 μM to 300 μM, preferably 100 μM, 200 μM, and 300 μM; the solvent is sterile deionized water. The covalent grafting treatment then includes the following steps: The PS was placed in an EDC / NHS conjugated solution for carboxyl group activation treatment. After the activation treatment was completed, it was coupled with the PLA / PDA film in the EDC / NHS conjugated solution. The activation treatment conditions are 180~200 rpm, 35~45 ℃, 2~2.5 h; the coupling treatment conditions are 180~200 rpm, 35~45 ℃, 4~6 h; in a preferred embodiment, the activation treatment conditions are 200 rpm, 40 ℃, 2 h; the coupling treatment conditions are 200 rpm, 40 ℃, 4 h.

[0025] The EDC / NHS conjugated solution system is prepared by using 0.1-0.2 M of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.02-0.04 M of N-hydroxysuccinimide, and 0.1-0.2 M of 2-morpholinoethanesulfonic acid, with the pH of the conjugated solution system adjusted to 5.2-5.4. In a preferred embodiment, the EDC / NHS conjugated solution system is prepared by using 0.1 M of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.02 M of N-hydroxysuccinimide, and 0.1 M of 2-morpholinoethanesulfonic acid, with the pH of the conjugated solution system adjusted to 5.4.

[0026] When covalently grafting the PLA / PDA film using an esterification reaction, the concentration of PS is 100 μM to 300 μM, and the solvent is DMF. Specifically, the photosensitizers BBR / Arg / CMCD / TAT are dissolved in DMF at concentrations of 100 μM, 200 μM, and 300 μM, respectively, and then ultrasonically dissolved until clear. The ultrasonic power is 45 W, and the ultrasonic time is 1 min, with an on-off cycle of 5 s.

[0027] As some optional embodiments of this application, the PS is placed in a DCC / DMAP conjugated solution for carboxyl group activation treatment. After the activation treatment is completed, it is coupled with the PLA / PDA film in a DCC / DMAP conjugated solution. The DCC solution has a concentration of 0.12~0.24 M and a volume of 2~4 mL; the DMAP has a concentration of 0.01~0.02 M and a volume of 0.5~0.1 mL. The activation treatment includes: a first activation treatment in a DCC solution, followed by a second activation treatment with the addition of DMAP; wherein the conditions for the first activation treatment are 180~200 rpm, 25~30 ℃, 30~40 min; and the conditions for the second activation treatment are 180~200 rpm, 25~30 ℃, 10~20 min. The coupling treatment conditions are 180~200 rpm, 35~45 ℃, and 4~6 h.

[0028] In the preferred embodiment, the DCC solution has a concentration of 0.12 M and a volume of 2 mL; the DMAP has a concentration of 0.01 M and a volume of 0.5 mL; the conditions for the first activation treatment are 200 rpm, 25 ℃, and 30 min; the conditions for the second activation treatment are 180 ℃ and 10 min; and the conditions for the coupling treatment are 200 rpm, 40 ℃, and 4 h.

[0029] It should be noted that after the PLA / PDA film is prepared, it is rinsed with a large amount of deionized water to remove unadsorbed PDA; after the PLA / PDA / PS film is prepared, it is rinsed sequentially with a large amount of DMF, ethanol, and deionized water to remove unbound photosensitizers BBR / Arg / CMCD / TAT. The PLA / PDA / PS film is dried at a temperature of 25~35 ℃ for 12~24 h.

[0030] The technical solution described in this application will be further explained in detail below with reference to specific implementation examples: Example 1: 1 Materials and Methods 1.1 Microbial strains Listeria monocytogenes Listeria monocytogenes (ATCC19115) is a representative Gram-positive pathogenic bacterium, with Vibrio parahaemolyticus as an example. Vibrio parahaemolyticus (ATCC17802, VPC17, VPC36, VPC49) are representative Gram-negative pathogens.

[0031] 1.2 Reagents PLA films were prepared in our laboratory by extrusion casting, with an preferred average thickness of 40 μm. DA Shanghai Maclean Biotechnology Co., Ltd.; The photosensitizers BBR / Arg / CMCD / TAT were prepared and synthesized in our laboratory. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-morpholine ethanesulfonic acid buffer (MES) Shanghai Mairui Chemical Technology Co., Ltd. N,N-Dimethylformamide (DMF), N,N'-Dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP) Shanghai Yuanye Biotechnology Co., Ltd. All chemical reagents were of analytical grade.

[0032] 1.3 Instruments IT-09A5 Heated Magnetic Stirrer, Shanghai Nenggong Industrial Co., Ltd. XZQ-X300C Thermostatic Oscillator, Shanghai Yiheng Scientific Instruments Co., Ltd.; 9272 Water-jacketed Constant Temperature Incubator, Shanghai Yiheng Technology Co., Ltd. DHG-9053A forced-air drying oven, Shanghai Yiheng Scientific Instruments Co., Ltd.

[0033] 1.4 Methods 1.4.1 Surface Modification of PLA / PDA Thin Films DA was dissolved in 10 mM Tris-HCl buffer (pH 8.0–8.5) to prepare a DA solution with a concentration of 2 mg / mL. PLA (5 × 5 cm) 2 The film was completely immersed in DA solution and continuously oscillated (180~200 rpm) at 25 ℃, 30 ℃, 40 ℃, and 50 ℃ for 6 h, 12 h, 18 h, 24 h, and 30 h, respectively. The PDA-modified PLA / PDA film was then rinsed with plenty of deionized water to remove unadsorbed PDA.

[0034] The surface coating thickness of PLA / PDA films prepared at different reaction temperatures and times was determined using atomic force microscopy. PLA films and PLA / PDA films were dried in an oven at 25 °C–35 °C in the dark for 12–24 h. Subsequently, they were scanned and imaged under an atomic force microscope at a rate of 1.0 Hz, and the film thickness was analyzed using NanoScope Analysis 1.7 (Bruker Co., Karlsruhe, Germany).

[0035] 1.4.2 Preparation of PLA / PDA / PS Thin Films by EDC / NHS amidation Reaction and Covalent Grafting PS (BBR / Arg / CMCD / TAT) was dissolved in sterile deionized water to prepare PS solutions with concentrations of 100 μM, 200 μM, and 300 μM. 0.1 M EDC, 0.02 M NHS, and 0.1 M MES were added to the PS solution to prepare a conjugated solution system for activating the carboxyl groups, and the pH of the conjugated solution was adjusted to 5.4. The PS activation solution system was activated at 40 °C for 2 h (200 rpm). Subsequently, a PLA / PDA film (5 × 5 cm) was applied. 2 The PLA / PDA / PS film was completely immersed in the PS conjugated solution and reacted with shaking at 40 °C and 200 rpm for 4 h. The prepared PLA / PDA / PS film was rinsed with plenty of deionized water to remove unbound PS. The film was then dried in an oven at 25–35 °C in the dark for 12–24 h. Films with different PS contents were named PLA / PDA / PS-1, PLA / PDA / PS-2, and PLA / PDA / PS-3, respectively.

[0036] 1.4.3 Preparation of PLA / PDA / PS Thin Films by EDC / NHS amidation Reaction and Covalent Grafting PS (BBR / Arg / CMCD / TAT) was dissolved in DMF to prepare PS solutions with concentrations of 100 μM, 200 μM, and 300 μM. The PS was sonicated at 45 W for 1 min (5 s on, 5 s off) to promote dissolution. 2 mL of 0.12 M DCC was added dropwise to the PS solution to prepare a conjugated solution system for activating the carboxyl groups. The PS activation solution system was activated at 25 °C for 30 min (200 rpm). 0.5 mL of 0.01 M DMAP solution was added to the PS activation system, and the reaction was continued for 10 min to promote esterification. Subsequently, a PLA / PDA film (5 × 5 cm) was formed. 2 The PLA / PDA / PS film was completely immersed in a PS conjugated solution and reacted with shaking at 40 °C and 200 rpm for 4 h. The prepared PLA / PDA / PS film was then rinsed sequentially with large amounts of anhydrous DMF, ethanol, and deionized water to remove unbound PS. The film was then dried in an oven at 25 °C–35 °C in the dark for 12–24 h. Films with different PS contents were named PLA / PDA / PS-1, PLA / PDA / PS-2, and PLA / PDA / PS-3, respectively.

[0037] 1.4.4 Determination of PS content in PLA / PDA / PS films PS solution was dissolved in deionized water to prepare 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM PS solutions. The absorbance at 420 nm was measured to establish a PS concentration-absorbance standard curve.

[0038] PLA / PDA / PS film (2×2 cm) 2 The film was immersed in 1 mL of 2 M NaOH solution and reacted at 40 °C for 3 h to break the amide / ester bonds. The absorbance at 420 nm was then measured. The PS content in the film was calculated based on the established PS standard curve, in μM / cm². 2 .

[0039] 1.4.5 Determination of Reactive Oxygen Species (ROS) Yield of PLA / PDA / PS Thin Films PLA / PDA / PS (2×2 cm) 2 The thin film was placed at the bottom of a 12-well plate, and 990 μL of the film was added to the plate. V. parahaemolyticus bacterial solution (10) 6 Log CFU / mL) and 10 μL of ROS probe DCFH-DA were added. After incubation in the dark for 30 min, PDI treatment was performed for 20 min. The fluorescence intensity was then measured at an excitation wavelength of 488 nm and an emission wavelength of 535 nm.

[0040] 1.4.6 Determination of Active Nitrogen (RNS) Yield of PLA / PDA / PS Thin Films NO yield was determined using the nitric oxide (NO) probe DAF-FM. PLA / PDA / PS (2 × 2 cm⁻¹) 2 The thin film was placed at the bottom of a 12-well plate, and 995 μL of [unspecified material] was added to the plate. V. parahaemolyticus bacterial solution (10) 6 Log CFU / mL) and 5 μL DAF-FM were added. After incubation in the dark for 30 min, PDI was applied for 20 min. The fluorescence intensity was then measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

[0041] Using peroxynitroso anion (ONOO) - ) Rhodamine 123 probe (DHR123) determination of ONOO - Yield. PLA / PDA / PS (2×2 cm) 2 The thin film was placed at the bottom of a 12-well plate, and 900 μL of the film was added to the plate. V. parahaemolyticus bacterial solution (10) 6Log CFU / mL) and 100 μL DHR123 probe were added. After incubation in the dark for 30 min, PDI treatment was performed for 20 min. The absorbance was then scanned under UV light in the range of 350–800 nm.

[0042] 1.4.7 Photodynamic sterilization mediated by PLA / PDA / PS films The white LED (20 W) was purchased from Shenzhen, China (Getian Optoelectronics Co., Ltd., China). The LED tube was enclosed in a photography light box to prevent external light from entering. The distance between the LED light source and the bacterial suspension was adjusted to 10.0 cm, corresponding to an irradiation energy of 12.74 mW / cm². 2 The dose for each obtained sample is calculated using the following formula: E= Pt Where E = dose (energy density), the unit is J / cm³. 2 p = irradiance (power density), unit is W / cm² 2 t = time, in seconds.

[0043] by L. monocytogenes and V. parahaemolyticus These were used as representative Gram-positive and Gram-negative pathogens, respectively. After activation, the bacterial concentration was adjusted to ~8 Log CFU / mL with 0.85% sterile saline. 100 μL of the bacterial suspension was evenly dispersed onto a 2 × 2 cm membrane. 2 After incubating in the dark for 30 min, PDI treatment was performed for 5-20 min. The bacterial suspension on the membrane was diluted with 0.85% sterile saline. A suitable dilution was selected, and 100 μL of the diluted solution was plated. The plates were incubated at 37 °C for 24 h, and the colony count was calculated. Each treatment was performed in triplicate, and each dilution was replicated three times.

[0044] 1.5 Data Analysis Experimental data were processed and analyzed using OriginPro 9.1 and SPSS 17.0 software packages (SPSS Inc., Chicago, USA). The least significant range (LSD) method was used to compare the significance of data points. p< 0.05).

[0045] 2 Results 2.1 PDA-modified PLA films This application utilizes the self-polymerization of DA in a weakly alkaline (8.0~8.5) aqueous solution to form a PDA coating strongly adhered to the surface of a PLA film. The optimal conditions for PDA-modified PLA were investigated by modifying PLA at temperatures of 25 ℃, 30 ℃, 40 ℃, and 50 ℃, and for reaction times of 6 h, 12 h, 18 h, 24 h, and 30 h. The surface coating thickness of the PLA / PDA films prepared at different reaction temperatures and times was determined using atomic force microscopy, and the results are shown below. Figure 1-2 As shown, the average surface thickness of the PLA film without PDA modification is 139.99 nm. In the reaction temperature parameter optimization experiment (reaction time fixed at 30 h), the surface thickness of the PLA / PDA film first increases and then decreases with increasing temperature, reaching a maximum of 202.11 nm at 30 ℃. Figure 1 Therefore, in this application, 30 °C is the optimal reaction temperature for PDA-modified PLA. Under the optimized temperature condition of 30 °C, the surface thickness of the PLA / PDA film increases with the extension of reaction time, reaching 212.33 nm at 24 h, and the thickness change is not significant after further extending to 30 h (216.19 nm). Figure 2 The above results indicate that the optimal process parameters for PDA-modified PLA films are 30 °C for 24 h, under which the benefits of PDA modification can be maximized. Therefore, all subsequent PDA-modified PLA film processes in this application will be carried out at 30 °C for 24 h.

[0046] 2.2 Covalent Grafting of PLA / PDA / PS Thin Films PDA modification introduces a large number of active amino and hydroxyl groups onto the surface of PLA / PDA films. These active groups can be further covalently coupled with carboxyl groups on PS (BBR / Arg / CMCD / TAT) to obtain antibacterial films with PDI activity. This application utilizes amidation and esterification reactions to covalently bond the amino and hydroxyl groups on PLA / PDA films to PS, respectively, and explores the optimal bonding scheme between PLA / PDA films and PS.

[0047] Strong bases can break the amide and ester bonds formed between the film and PS, releasing the PS grafted onto the PLA / PDA / PS film. Therefore, the PS content on films prepared by different covalent grafting methods was measured. The results are as follows: Figure 3 As shown, the PS content on PLA / PDA / PS-1, PLA / PDA / PS-2, and PLA / PDA / PS-3 films prepared by amidation reaction is 2.02 μM / cm², respectively. 2 2.99 μM / cm 2 and 3.66 μM / cm2 However, the PS content on PLA / PDA / PS-1, PLA / PDA / PS-2, and PLA / PDA / PS-3 films prepared by esterification was low, only 0.55 μM / cm. 2 0.86 μM / cm 2 and 1.04 μM / cm 2 The above results indicate that, compared to esterification, amidation can better promote the covalent grafting of amino groups on PLA / PDA films and carboxyl groups on PS via amide bonds. Through comparison and optimization, this application further determined the optimal grafting method for PS and PLA / PDA films, namely amidation. Based on this, all subsequent PLA / PDA / PS film preparation processes in this application will use amidation for covalent grafting.

[0048] 2.3 Mechanical properties of PLA / PDA / PS films The mechanical properties of a film are an important indicator for evaluating its suitability as a packaging material. This application utilizes PDA to modify the surface of PLA films, thereby imparting active groups to the PLA films and enhancing their mechanical properties. For example... Figure 4 As shown, compared to PLA film (tensile strength 52.64 MPa, elongation at break 5.43%), the tensile strength and elongation at break of PLA / PDA film significantly increased to 60.70 MPa and 9.04%, respectively. After grafting PS, the tensile strength of PLA / PDA / PS film did not show a significant change, but the elongation at break increased significantly. The elongation at break of PLA / PDA / PS-1, PLA / PDA / PS-2, and PLA / PDA / PS-3 films increased to 9.25%, 10.85%, and 11.93%, respectively, which is about twice that of PLA film. This is because PDA modification and PS covalent grafting restrict the migration of PLA molecular chains, thereby enhancing the mechanical properties of PLA / PDA / PS film. The PDA surface coating technology used in this application shows more significant application advantages in the field of packaging films compared to conventional surface modification methods such as plasma treatment.

[0049] 2.4 RNS generation capability of PLA / PDA / PS thin films After successful grafting of PS, the PLA / PDA / PS film exhibits PDI antibacterial activity under photoexcitation. Generally, natural PS, under irradiation with light of a specific wavelength, is excited to produce a large amount of ROS, which attack bacterial cells and cause bacterial death. However, the short half-life and small diffusion radius of ROS limit the antibacterial efficiency and application potential of PDI-mediated antibacterial films. In the PLA / PDA / PS film prepared in this application, the PS (BBR / Arg / CMCD / TAT) can further convert the generated ROS into RNS (NO, ONOO), which have stronger oxidizing power and longer lifespan. - (etc.), thereby significantly improving the antibacterial ability of the PDI antibacterial film. Therefore, the ROS yield of the PLA / PDA / PS film was first determined. From Figure 5 It can be seen that PLA and PLA / PDA films without PS grafting do not possess ROS generation capabilities. In the positive control group (BBR / CMCD / TAT), due to the lack of the RNS conversion factor Arg, ROS generation was significantly reduced under white light irradiation (15.29 J / cm²). 2 Under these conditions, a large amount of ROS is generated. The ROS yield of the PLA / PDA / PS film is significantly lower than that of the control group, which means that the PS (BBR / Arg / CMCD / TAT) grafted onto the film consumes the ROS generated during the PDI process.

[0050] Next, the NO yield of the PLA / PDA / PS film was further examined. From Figure 6 It is observed that ungrafted PS PLA and PLA / PDA films do not produce NO, while the NO yield of PLA / PDA / PS films increases significantly, and the NO yield gradually increases with increasing PS content. This means that PLA / PDA / PS films can effectively convert generated ROS into NO. Furthermore, the generated NO is further oxidized by ROS to peroxynitrite ions (ONOO). - ), Figure 7 ONOO for PLA / PDA / PS films - Yield. ONOO produced from PLA / PDA / PS thin films. - The trend is consistent with NO; as the PS content on the film increases, ONOO... - The yield increases accordingly, and the ONOO of PLA / PDA / PS-3 film... - The yield reached its maximum. In summary, under white light irradiation, PLA / PDA / PS films can efficiently generate RNS with stronger oxidizing power and longer lifespan, thereby improving the bactericidal efficiency of PDI-mediated antibacterial films.

[0051] 2.5 Sterilization effect of PDI-mediated PLA / PDA / PS films Figure 8 Demonstrates the effect of PDI-mediated PLA / PDA / PS films on Gram-positive pathogens after white light irradiation. L. monocytogenes The inactivation status was assessed. The negative control group consisted of an untreated bacterial suspension with an initial inoculum of 8.89 LogCFU / mL. After light exposure, the PLA film did not show a decrease in activity compared to the control group. L. monocytogenes Cell count (8.85 Log CFU / mL). The PLA / PDA membrane exhibited weak bactericidal activity, with a concentration of 15.29 J / cm³ after 20 min of PDI treatment. 2 ), L. monocytogenes The cell number decreased by only 0.58 Log CFU / mL. However, the PLA / PDA / PS film exhibited significant PDI activity, which increased with irradiation time from 5 min (3.82 J / cm²). 2 Extend to 20 min (15.29 J / cm) 2 PLA / PDA / PS-1 thin film treatment L. monocytogenes The cell count decreased from 6.69 Log CFU / mL (>99%) to 2.40 Log CFU / mL (~99.9999%). Notably, the PDI activity of the PLA / PDA / PS films increased with increasing PS content. PLA / PDA / PS-2 and PLA / PDA / PS-3 films were treated with PDI for 20 min (15.29 J / cm²). 2 ) after L. monocytogenes A reduction in cell count to an uncountable level indicates a bactericidal efficiency >8 Log CFU / mL (>99.999999%).

[0052] To further demonstrate the broad-spectrum antibacterial properties of PDI-mediated PLA / PDA / PS films, we selected... V. parahaemolyticus As a representative Gram-negative pathogen. See [link to data] for clearance effectiveness. Figure 9 PLA / PDA / PS film V. parahaemolyticus The cleaning efficiency and L. monocytogenes The trend was consistent. The initial cell count in the negative control group was 8.30 Log CFU / mL. With increasing PS content on the membrane and prolonged illumination time, V. parahaemolyticus The quantity gradually decreased. Finally, the PLA / PDA / PS-3 film was treated with PDI for 20 min (15.29 J / cm). 2 After that, make V. parahaemolyticusThe cell count decreased to an uncountable level (>99.999999%). These results indicate that the PDI-mediated PLA / PDA / PS film prepared in this application possesses broad-spectrum antibacterial activity, and that PS concentration and irradiation dose are important factors in the PDI-mediated antibacterial film. Under white light irradiation, the PLA / PDA / PS film can rapidly and efficiently generate RNS, which possesses strong oxidizing capabilities, acting on bacterial cells and leading to cell death.

[0053] Verification has shown that this application can effectively covalently graft carboxyl-containing PS (BBR / Arg / CMCD / TAT) onto the surface of PLA films through PDA surface modification and amidation reactions, resulting in PLA / PDA / PS films with excellent mechanical properties. Under white light irradiation, the PLA / PDA / PS films generate highly reactive oxidative stress (RNS), achieving rapid and efficient killing of common Gram-positive and Gram-negative pathogenic bacteria in food. This novel PDI-mediated antibacterial packaging film effectively ensures food safety and has significant application potential.

[0054] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A method for preparing a polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity, characterized in that, Includes the following steps: PLA / PDA film is formed by polymerizing PLA film on the surface of PLA film using PLA as film matrix and DA as surface modification material through polymerization and crosslinking reaction; Using BBR, Arg, CMCD, and TAT as PS, and utilizing the carboxyl groups on the PS, covalent grafting is performed by coupling them with surface-modified PLA / PDA films through amide or esterification reactions to obtain PLA / PDA / PS films with highly efficient photodynamic antibacterial activity.

2. The method for preparing the polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity according to claim 1, characterized in that, The concentration of DA is 2~2.5 mg / mL, and the dosage is 400~500 mg; the area of ​​the PLA film is 50 cm². 2 .

3. The method for preparing the polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity according to claim 1, characterized in that, In the polymerization and crosslinking reaction, the concentration of Tris-HCl buffer used is 10~20 mM, the pH is 8.0~8.5, the shaking speed is 180~200 rpm, the reaction temperature is 25 ℃~50 ℃, and the reaction time is 6 h~30 h.

4. The method for preparing the polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity according to claim 1, characterized in that, If the reaction is an amide reaction, the concentration of PS is 100 μM to 300 μM, and the solvent is sterile deionized water.

5. The method for preparing the polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity according to claim 4, characterized in that, The PS was placed in an EDC / NHS conjugated solution for carboxyl group activation treatment. After the activation treatment was completed, it was coupled with the PLA / PDA film in the EDC / NHS conjugated solution. The activation treatment conditions are 180~200 rpm, 35~45 ℃, 2~2.5 h; The coupling treatment conditions are 180~200 rpm, 35~45 ℃, and 4~6 h.

6. The method for preparing the polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity according to claim 5, characterized in that, The EDC / NHS conjugated solution system was prepared by using 0.1-0.2 M of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.02-0.04 M of N-hydroxysuccinimide, and 0.1-0.2 M of 2-morpholinoethanesulfonic acid, with the pH of the conjugated solution system adjusted to 5.2-5.

4.

7. The method for preparing the polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity according to claim 1, characterized in that, If it is an esterification reaction, the concentration of PS is 100 μM to 300 μM, and the solvent is DMF.

8. The method for preparing the polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity according to claim 7, characterized in that, The PS was placed in a DCC / DMAP conjugated solution for carboxyl group activation treatment. After the activation treatment was completed, it was coupled with the PLA / PDA film in a DCC / DMAP conjugated solution. The DCC solution has a concentration of 0.12~0.24 M and a volume of 2~4 mL; the DMAP has a concentration of 0.01~0.02 M and a volume of 0.5~0.1 mL. The activation treatment includes: a first activation treatment in a DCC solution, followed by a second activation treatment with the addition of DMAP; wherein the conditions for the first activation treatment are 180~200 rpm, 25~30 ℃, 30~40 min; and the conditions for the second activation treatment are 180~200 rpm, 25~30 ℃, 10~20 min. The coupling treatment conditions are 180~200 rpm, 35~45 ℃, and 4~6 h.

9. The method for preparing a polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity according to claim 1, characterized in that, After obtaining PLA / PDA / PS films with highly efficient photodynamic antibacterial activity, the following steps are also included: The PLA / PDA / PS film was rinsed and dried sequentially with DMF, ethanol and deionized water to remove unbound photosensitizers BBR / Arg / CMCD / TAT. The drying conditions are: temperature 25~35 ℃, time 12~24 h.

10. A polylactic acid-based thin film with highly efficient active nitrogen-mediated photodynamic antibacterial activity, characterized in that, Prepared by the method described in any one of claims 1-9.

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