Porous, highly dispersed Ag / Cu / Cu2O composite materials enhance the antibacterial properties of microbial extracellular polysaccharides (EPS).

Abstract

The application discloses a kind of porous high-dispersion Ag / Cu / Cu2O composite material to enhance the antibacterial characteristics of microbial extracellular polysaccharide (EPS) and be applied to food packaging, belongs to the field of antibacterial material.The application aims to solve the technical problem that the application of lactic acid bacterial extracellular polysaccharide in food preservation, anti-infection medical dressing and other fields is limited.The application uses PVP as carbon source and template agent, uses Cu (NO3) 2 as Cu source, and uses a small amount of AgNO3 as Ag source to synthesize porous high-dispersion Ag / Cu / Cu2O nanocomposite, which is simple in method and cheap in raw materials.After connecting EPS with Ag / Cu / Cu2O nanocomposite, the bactericidal characteristics of EPS-Ag / Cu / Cu2O composite are greatly improved compared with EPS-Ag and EPS-Cu / Cu2O.The Ag / Cu / Cu2O nanocomposite of the application can greatly reduce the use amount of noble metal Ag and exhibit very excellent antibacterial characteristics, so it has more extensive practical application value.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial materials, specifically relating to an EPS-Ag / Cu / Cu2O nanocomposite, its preparation method, and its application. Background Technology

[0002] Extracellular polysaccharides (EPS) of lactic acid bacteria are a class of natural high-molecular polymers secreted by lactic acid bacteria during their growth and metabolism. Their molecular structures are complex and diverse, composed of different types of monosaccharides linked by glycosidic bonds, forming linear or branched structures. These unique molecular architectures, like precision instruments in the microscopic world, endow them with excellent functional properties such as thickening, stabilizing, emulsifying, antioxidant, and immunomodulatory effects. In the pharmaceutical industry, they act as precise "deliverymen," leveraging their excellent biocompatibility and biodegradability to deliver active ingredients precisely to the lesion. Simultaneously, they can bind to receptors on the surface of immune cells, stimulating the human immune system and becoming a "booster" for boosting immunity. In cosmetics, their excellent moisturizing properties stem from the abundant hydrophilic groups on the polysaccharide molecules, forming a water-locking film on the skin surface to maintain skin hydration. In agriculture, as a biofilm material, they provide a physical barrier for plants, reducing water loss, and can also regulate soil microbial communities, providing strong support for plant growth and soil improvement.

[0003] Although lactic acid bacteria extracellular polysaccharides have a wide range of applications, their lack of antibacterial properties limits their use in areas such as anti-infective medical dressings. Furthermore, the difficulty in preventing infection when treating open wounds significantly restricts their application. Summary of the Invention

[0004] This invention aims to address the technical problem of limited application of lactic acid bacteria extracellular polysaccharides in anti-infective medical dressings and other fields, and provides an EPS-Ag / Cu / Cu2O nanocomposite, its preparation method, and its application.

[0005] To address the aforementioned technical problems, the present invention adopts the following technical solution: The purpose of this invention is to provide a method for preparing an EPS-Ag / Cu / Cu2O nanocomposite, characterized by comprising the following steps: Step 1: Dissolve Cu(NO3)2·3H2O and AgNO3 in deionized water, then add them dropwise to the PVP solution. Stir vigorously until homogeneous and stable, dry the water, grind until uniform, and carbonize under a nitrogen protective atmosphere to obtain Ag / Cu / Cu2O nanomaterials.

[0006] Step 2: Add Ag / Cu / Cu2O nanomaterials to ultrapure water, stir thoroughly, and sonicate until homogeneous. Then add lactic acid bacteria extracellular polysaccharide (EPS) solution, stir thoroughly, sonicate, and freeze-dry to obtain EPS-Ag / Cu / Cu2O nanocomposite material.

[0007] Further specifying, in step one, 1.5 g Cu(NO3)2·3H2O and 0.05 g AgNO3 are dissolved in 20 mL of deionized water.

[0008] Further specifying, in step one, the PVP solution is obtained by completely dissolving 1.0 g of polyvinylpyrrolidone (PVP) in 30 mL of deionized water.

[0009] Further specifying, in step one, the temperature is 5℃ min. -1 The temperature was increased to 600°C at a rising rate and held for 1 hour for carbonization.

[0010] Further specifying, in step two, 10 mg of Ag / Cu / Cu2O nanomaterials are added to 10 mL of ultrapure water, followed by 10 mL of EPS solution with a concentration of 5 mg / mL.

[0011] To further specify, in step two, the EPS is prepared according to the following steps: Step 1: Activate *Leuconostoc mesenteroides* DRP105 preserved in glycerol tubes by inoculating 1% (v / v) into 20 mL / 50 mL MRS medium. Incubate at 30°C and 140 rpm for 18 h on a shaker. Subculture 2-3 times on MRS agar solid medium. Transfer single colonies of strain DRP105 to 20 mL / 50 mL MRS medium and incubate at 30°C and 140 rpm for 18 h to obtain a seed culture (1×10⁻⁶). 8 (CFU / mL) Step 2: Inoculate the seed culture into MRS-S medium at a ratio of 2% and incubate at 30℃ and 140 rpm for 42 h; Step 3: Centrifuge the fermentation broth from Step 2 at 1000 rpm for 30 min at 4°C to remove the cells. Then, add three volumes of pre-cooled 95% (v / v) ethanol to the supernatant, let it stand overnight at 4°C for alcohol precipitation, centrifuge again, collect the polysaccharide precipitate, and completely dissolve it in 100 mL of double-distilled water. Add 100 mL of 10% (v / v) trichloroacetic acid solution, treat at 4°C for 4 h, centrifuge a third time to remove the protein precipitate from the polysaccharide solution, add three volumes of pre-cooled 95% (v / v) ethanol to the supernatant, let it precipitate overnight at 4°C, centrifuge the precipitated liquid, dissolve the polysaccharide precipitate in double-distilled water, use a dialysis bag with a molecular weight cutoff of 14 kDa, change the water every 8 h, dialyze for two days to obtain the crude EPS solution. Step 4: Then, elute with a Sephadex G-100 gel column using purified water as the eluent at a flow rate of 0.2 mL / min. Collect the eluent according to the peak time on the elution chromatogram. Freeze the collected eluent sequentially at -20℃ and -80℃ until it is close to the temperature of the freeze dryer, and then freeze-dry it under vacuum to obtain pure EPS dried product.

[0012] Another object of the present invention is to provide EPS-Ag / Cu / Cu2O nanocomposites prepared by any of the above methods.

[0013] The application of the EPS-Ag / Cu / Cu2O nanocomposite of the present invention in the preparation of anti-infective medical dressings.

[0014] The EPS-Ag / Cu / Cu2O nanocomposite of the present invention is used as an antibacterial agent.

[0015] This invention synthesizes porous, highly dispersed Ag / Cu / Cu2O nanocomposites using PVP as the carbon source and template agent, Cu(NO3)2 as the Cu source, and a small amount of AgNO3 as the Ag source. The method is simple and the raw materials are inexpensive. After linking EPS to the Ag / Cu / Cu2O nanocomposites, the bactericidal properties of the EPS-Ag / Cu / Cu2O composite are significantly improved compared to EPS-Ag and EPS-Cu / Cu2O. This is attributed to the unique electronic and interfacial effects of the Ag / Cu / Cu2O composite. The molar ratio of Cu to Ag in the Ag / Cu / Cu2O composite is 20.7:1; the mass ratio of Cu to Ag is 12.3:1. This means that by introducing inexpensive Cu, the amount of precious metal Ag used can be greatly reduced, and excellent antibacterial properties are exhibited, thus possessing broader practical application value, such as in anti-infective medical dressings.

[0016] To further understand the features and technical content of this invention, please refer to the following detailed description and accompanying drawings. However, the accompanying drawings are for reference and illustration only and are not intended to limit the invention. Attached Figure Description

[0017] Figure 1 This is the XRD pattern of Cu / Cu2O; Figure 2 It is the XRD pattern of Ag; Figure 3 The XRD pattern of Ag / Cu / Cu2O prepared by the method in Example 1 is shown. Figure 4 This is a SEM image of Cu / Cu2O; Figure 5 It is a SEM image of Ag; Figure 6 This is a SEM image of Ag / Cu / Cu2O prepared by the method in Example 1. Detailed Implementation

[0018] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, while not limiting the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0019] Example 1 Step 1: Preparation of Ag / Cu / Cu2O nanocomposite At room temperature (25°C), 1.0 g of polyvinylpyrrolidone (PVP) was dissolved in 30 mL of deionized water and completely dissolved under magnetic stirring. 1.5 g of copper nitrate trihydrate (Cu(NO3)2·3H2O) and 0.05 g of silver nitrate (AgNO3) were dissolved in 20 mL of deionized water to obtain a mixed metal ion solution. This mixed solution was added dropwise to the above PVP solution, and the mixture was stirred vigorously for 6 h to form a homogeneous and stable PVP-Ag solution. + -Cu 2+ Precursor solution. The precursor solution was dried in an oven at 100℃ for 24 hours. After drying, a gel-like PVP-Ag was obtained. + -Cu 2+ Solid precursors are prepared by grinding the dried product in a mortar until it becomes a uniform powder.

[0020] Finally, the powder was placed in a tube furnace and heated at 5°C for 1 minute under a nitrogen (N2) protective atmosphere. -1The temperature was increased to 600℃ at a rising rate and held for 1 h for carbonization, ultimately yielding carbon-supported porous and highly dispersed Ag / Cu / Cu2O nanocomposite materials. Individual carbon-supported Ag nanoparticles and Cu / Cu2O nanoparticles were also obtained using the same method, except that the addition of metal raw materials transformed them into individual AgNO3 and Cu(NO3)2·3H2O.

[0021] Step 2: Fermentation of the strain to produce EPS solution Leuconostoc mesenteroides DRP105, preserved in glycerol tubes, was activated by inoculation at 1% (v / v) into 20 mL / 50 mL MRS medium. The culture was carried out at 30°C and 140 rpm for 18 h on a shaker. The culture was then subcultured 2-3 times on MRS agar. Single colonies of strain DRP105 were picked and transferred to 20 mL / 50 mL MRS medium and cultured at 30°C and 140 rpm for 18 h to obtain a seed culture (1×10⁻⁶). 8 (CFU / mL).

[0022] The seed culture was inoculated at a ratio of 2% into MRS-S medium (120 g sucrose, 10 g beef extract, 10 g peptone, 5 g yeast extract, 0.1 g anhydrous sodium sulfite, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 2 g ammonium citrate, 2 g dipotassium hydrogen phosphate, 5 g anhydrous sodium acetate, 1 L distilled water; pH 5.5, autoclaved at 108℃ for 20 min) and cultured at 30℃ and 140 rpm for 42 h.

[0023] The fermentation broth after the above fermentation culture was centrifuged at 1000 rpm for 30 min at 4°C to remove bacterial cells. Then, three volumes of pre-cooled 95% (v / v) ethanol were added to the supernatant, and the mixture was allowed to stand overnight at 4°C for alcohol precipitation. The mixture was then centrifuged again (8000 rpm, 30 min) to collect the polysaccharide precipitate, which was completely dissolved in 100 mL of double-distilled water. 100 mL of 10% (v / v) trichloroacetic acid solution was added, and the mixture was treated at 4°C for 4 h. A third centrifugation (8000 rpm, 30 min) was performed to remove protein precipitate from the polysaccharide solution. Three volumes of pre-cooled 95% (v / v) ethanol were added to the supernatant, and the mixture was allowed to precipitate overnight at 4°C. Finally, the precipitated liquid was centrifuged, and the polysaccharide precipitate was dissolved in an appropriate amount of double-distilled water. Dialysis was performed using a dialysis bag with a molecular weight cutoff of 14 kDa, changing the water every 8 h for two days to obtain a crude EPS solution.

[0024] Step 3: Prepare a dry sample of pure EPS. (1) Pretreatment of gel column Soak Sephadex G-100 powder granules in excess purified water for 12 hours to allow the granules to fully swell. Gently stir to remove impurities and particles floating on the surface, then replace the water with fresh purified water. After vertically mounting the gel column, use a glass rod to guide the flow of the prepared gel solution. Alternate between allowing it to stand and filling the column until the gel height exceeds approximately half the column length, maintaining a 1-2 cm gap between the purified water and the gel surface. Ensure the system is airtight and allow the gel column to settle overnight, maintaining a stable liquid level. Before loading the sample, adjust the UV detector to ensure a stable absorbance value (A) at 220 nm.

[0025] (2) Purification of polysaccharides After the baseline stabilizes, sample loading begins, with a loading volume of 1-2 mL. Pure water is used as the eluent at a flow rate of 0.2 mL / min. The eluent is collected based on the peak times in the elution chromatogram. The collected eluent is then frozen sequentially at -20℃ and -80℃ until it reaches a temperature close to that of the freeze dryer, followed by vacuum freeze-drying to obtain pure EPS dried product.

[0026] Step 4: Prepare EPS-Ag / Cu / Cu2O, EPS-Ag, and EPS-Cu / Cu2O samples. 10 mg of Ag / Cu / Cu2O was dissolved in 10 mL of ultrapure water, stirred thoroughly for 1 h, and sonicated for 1 h. Then, 10 mL of 5 mg / mL EPS solution (prepared from the above-mentioned pure dried EPS) was added, stirred thoroughly for 12 h, and sonicated for 2 h. The EPS-Ag / Cu / Cu2O composite material was obtained by freeze-drying. The preparation methods of EPS-Ag and EPS-Cu / Cu2O samples were the same as above, except that Ag nanoparticles and Cu / Cu2O nanoparticles supported on carbon were used as the substrate.

[0027] Antibacterial performance analysis The cultured indicator bacteria Staphylococcus aureus ATCC 6538, Micrococcus luteus ATCC 4698, Bacillus subtilis KLM HLJU, and Escherichia coli ATCC 68004 were inoculated into undiluted LB medium to prepare indicator agar plates. 6 mm diameter wells were punched in the plates using Oxford cups. 200 μL of sample was added to each well, using EPS, EPS-Ag, EPS-Cu / Cu2O, and EPS-Ag / Cu / Cu2O as samples (EPS concentration 5 mg / mL for all samples). The plates were incubated at 30°C for 24 h. The size of the inhibition zone was measured using calipers to determine the antibacterial properties of the composite material.

[0028] 6. Minimum inhibitory concentration test The minimum inhibitory concentration (MIC) of the composite material against different indicator bacteria was determined using a two-fold dilution method. The specific steps were as follows: 10 μL of the composite material, after a two-fold serial dilution, was added to a suspension containing 90 μL of indicator bacteria (10...). 6 96-well plates (CFU / mL) were prepared and sealed with the plate caps to prevent contamination from environmental microorganisms. The plates were then incubated at 30°C for 16 h. Sterile culture medium without indicator bacteria served as a negative control, and bacterial suspension with added Nisin served as a positive control. The OD values ​​of the samples in the 96-well plates were measured using a microplate reader. 600 nm MIC is defined as the minimum composite concentration required to inhibit the growth of indicator bacteria.

[0029] This invention employs X-ray diffraction (XRD) to systematically characterize the crystal structure of the samples. For example... Figure 1 As shown in (Cu / Cu2O), characteristic diffraction peaks corresponding to the (111), (200), and (220) crystal planes of metallic Cu (JCPDS No. 04#0836) were observed at 2θ = 43.297°, 50.433°, and 74.130°, respectively. Meanwhile, characteristic peaks of the (110), (111), (200), (220), and (311) crystal planes of Cu2O (JCPDS No. 78#2076) were detected at 29.582°, 36.441°, 42.328°, 61.406°, and 77.414°, confirming the coexistence of Cu and Cu2O phases. Figure 2 (Ag) showed clear diffraction peaks at 2θ = 38.114°, 44.298°, 64.441°, and 77.395°, indicating the successful preparation of metallic silver nanoparticles. It is worth noting that the ternary composite material (JCPDS No. 87-0597) exhibited clear diffraction peaks of the (111), (200), (220), and (311) crystal planes. Figure 3 The XRD pattern of Ag / Cu / Cu2O included characteristic peaks of Cu (43.297°, 50.433°, 74.130°), Cu2O (36.441°, 61.406°, 77.414°) and Ag (38.114°, 64.441°), indicating that the Ag / Cu / Cu2O complex was successfully synthesized.

[0030] Scanning electron microscopy (SEM) revealed that all three samples possessed a honeycomb-like porous structure. Figure 4-6This three-dimensional honeycomb structure consists of interconnected carbon walls, forming uniform microporous channels with diameters in the micrometer range. SEM images clearly show that a large number of nanoparticles are uniformly distributed in the carbon matrix without obvious aggregation. This hierarchical structure, which combines a microporous carbon framework with highly dispersed active nanoparticles, provides an extremely high specific surface area and abundant active sites, which is crucial for improving antibacterial properties by enhancing interfacial interactions.

[0031] The inhibitory effects of different materials on indicator bacteria are shown in Table 1. EPS itself has no inhibitory effect on indicator bacteria, while the other three composite materials all exhibit significant antibacterial activity. Among them, the EPS-Ag / Cu / Cu2O composite material has the strongest antibacterial ability, followed by the EPS-Ag and EPS-Cu / Cu2O composite materials. It can be seen that the addition of Cu and Ag gives EPS antibacterial ability, and Ag has a stronger antibacterial ability than Cu. When EPS is compounded with Cu and Ag, the antibacterial ability of the material is further enhanced. The diameters of the inhibition zones of the EPS-Ag / Cu / Cu2O composite material against S. aureus, M. luteus, B. subtilis, and E. coli were 14.89±0.32 mm, 14.93±0.58 mm, 14.55±0.51 mm, 14.03±0.66 mm, 10.83±0.34 mm, and 10.77±0.23 mm, respectively, which were significantly higher than the antibacterial abilities of EPS-Ag and EPS-Cu / Cu2O. This indicates that the introduction of Ag / Cu / Cu2O enhanced the antibacterial properties of EPS.

[0032] Table 1. Inhibitory effects of different materials on indicator bacteria

[0033] The MICs of the EPS-Ag / Cu / Cu2O composite material against indicator bacteria are shown in Table 2. The EPS-Ag / Cu / Cu2O composite material can effectively inhibit... S. aureus, M. luteus, B. subtilis and E. coli The MICs were 4.93 μg / mL, 4.03 μg / mL, 3.74 μg / mL and 2.82 μg / mL, respectively.

[0034] Table 2 MIC of EPS-Ag / Cu / Cu2O composite material against indicator bacteria

[0035] The specific embodiments of the present invention have been described above. It should be noted that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing EPS-Ag / Cu / Cu2O nanocomposite, characterized in that, Includes the following steps: Step 1: Dissolve Cu(NO3)2·3H2O and AgNO3 in deionized water, then add them dropwise to the PVP solution, stir vigorously until homogeneous and stable, dry the water, grind until uniform, and carbonize under a nitrogen protective atmosphere to obtain Ag / Cu / Cu2O nanomaterials. Step 2: Add Ag / Cu / Cu2O nanomaterials to ultrapure water, stir thoroughly, and sonicate until homogeneous. Then add lactic acid bacteria extracellular polysaccharide (EPS) solution, stir thoroughly, sonicate, and freeze-dry to obtain EPS-Ag / Cu / Cu2O nanocomposite material.

2. The method according to claim 1, characterized in that, 1.5 g Cu(NO3)2·3H2O and 0.05 g AgNO3 were dissolved in 20 mL of deionized water; the PVP solution was obtained by completely dissolving 1.0 g polyvinylpyrrolidone (PVP) in 30 mL of deionized water.

3. The method according to claim 1, characterized in that, At 5℃min -1 The temperature was increased to 600°C at a rising rate and held for 1 hour for carbonization.

4. The method according to claim 1, characterized in that, Add 10 mg of Ag / Cu / Cu2O nanomaterials to 10 mL of ultrapure water, and then add 10 mL of EPS solution with a concentration of 5 mg / mL.

5. The method according to claim 1, characterized in that, EPS is prepared according to the following steps: Step 1: Activate *Leuconostoc mesenteroides* DRP105, preserved in glycerol tubes, by inoculating at 1% (v / v) on MRS medium. Incubate at 30°C and 140 rpm for 18 h on a shaker. Subculture 2-3 times on MRS agar solid medium. Select single colonies of strain DRP105 and transfer them to MRS medium. Incubate at 30°C and 140 rpm for 18 h to obtain a seed culture (1×10⁻⁶). 8 (CFU / mL) Step 2: Inoculate the seed culture into MRS-S medium at a ratio of 2% and incubate at 30℃ and 140 rpm for 42 h; Step 3: Centrifuge the fermentation broth from Step 2 at 1000 rpm for 30 min at 4°C to remove cells. Then, add three volumes of pre-cooled 95% (v / v) ethanol to the supernatant, let it stand overnight at 4°C for alcohol precipitation, centrifuge again, collect the polysaccharide precipitate, and completely dissolve it in 100 mL of double-distilled water. Add 100 mL of 10% (v / v) trichloroacetic acid solution, treat at 4°C for 4 h, centrifuge a third time to remove protein precipitate from the polysaccharide solution, add three volumes of pre-cooled 95% (v / v) ethanol to the supernatant, let it precipitate overnight at 4°C, centrifuge the precipitated liquid, dissolve the polysaccharide precipitate in double-distilled water, use a dialysis bag with a molecular weight cutoff of 14 kDa, change the water every 8 h, dialyze for two days to obtain crude EPS solution; Step 4: Then, elute with a Sephadex G-100 gel column using purified water as the eluent at a flow rate of 0.2 mL / min. Collect the eluent according to the peak time on the elution chromatogram. Freeze the collected eluent sequentially at -20℃ and -80℃ until it is close to the temperature of the freeze dryer, and then freeze-dry it under vacuum to obtain pure EPS dried product.

6. An EPS-Ag / Cu / Cu2O nanocomposite prepared according to any one of claims 1-5.

7. The application of the EPS-Ag / Cu / Cu2O nanocomposite prepared according to any one of claims 1-5 in the preparation of anti-infective medical dressings.

8. An EPS-Ag / Cu / Cu2O nanocomposite prepared according to any one of claims 1-5 is used as an antibacterial agent.

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