Gamma-cyclodextrin reduced Fe(III) nanomaterial with antibacterial function, and preparation method and application thereof

Abstract

The application discloses a kind of preparation methods of food-grade Fe-gamma-CD nano bacteriostatic material, belongs to food preservation technical field, and its specific steps include: FeCl3·6H2O and gamma-CD are dissolved in deionized water, ultrasonic treatment is carried out;The molar ratio of gamma-CD and FeCl3·6H2O is 1:2-1:3;Heating is carried out in 120-180 DEG C for 5-7 hours;After reaction, centrifugal, remove supernatant, collect precipitate, continue to wash precipitate with deionized water, repeat centrifugal, remove impurities completely, place the sample after centrifugal in blast drying oven, dry sample completely, complete the preparation of Fe-gamma-CD nano bacteriostatic material.Fe-gamma-CD nano bacteriostatic material has excellent peroxide catalytic activity and broad-spectrum antibacterial effect, and high tolerance to harsh environment, high stability and other advantages, Fe-gamma-CD is non-toxic to normal cells, and belongs to food-grade bacteriostatic material.

Description

Technical fields:

[0001] This invention belongs to the field of food preservation technology, specifically relating to a γ-cyclodextrin-reduced Fe(III) nanomaterial for antibacterial purposes, its preparation method, and its application. Fe-γ-CD nanomaterials are prepared via a one-pot hydrothermal method. The peroxide-like properties of Fe-γ-CD nanomaterials generate hydroxyl radicals to achieve antibacterial effects, demonstrating good antibacterial efficacy and being harmless to the human body. Background technology:

[0002] Foodborne illnesses are a growing global health concern, with the World Health Organization stating that approximately 70% of human diseases are caused by foodborne pathogens. Common pathogens causing food poisoning include Escherichia coli, Staphylococcus aureus, Salmonella, and Shigella. Currently, traditional methods for controlling foodborne bacteria involve physical sterilization techniques and the addition of food additives. For example, heat sterilization is an important method used in food processing. However, this technique often alters the color, aroma, and taste of food, while also reducing nutritional content. Compared to traditional heat sterilization, using microwaves, ultrasound, or pulsed electric fields during food processing can kill harmful bacteria while preserving nutrients, but their high cost limits their application potential. Sodium benzoate is a widely used food additive. However, its use alters protein structure, thereby inhibiting trypsin function and adversely affecting human digestion. Therefore, there is an urgent need for green, low-toxicity, and effective methods to control foodborne pathogens.

[0003] Currently, lysozyme is a natural biocatalyst that exhibits strong antibacterial activity against fungi, viruses, and bacteria by affecting cell membrane permeability, thus making it a potential preservative in food systems. However, natural enzymes like lysozyme have inherent drawbacks such as high production costs, low catalytic stability, and susceptibility to inactivation under harsh environments, which significantly limit their potential application as antibacterial agents. With the development of nanotechnology, various nanocomposite materials have been discovered. Among them, nanozymes are nanoparticles with enzyme catalytic properties that can be used to replace traditional antibacterial strategies.

[0004] Fe in synthetic antibacterial agents 2+ Iron ions are classic catalyst ions widely explored in antibacterial research due to their high POD-like catalytic efficiency and high biocompatibility. However, they are easily oxidized, thus necessitating the development of nanomaterials capable of maintaining their valence state. Gao Lizeng et al. discovered that Fe3O4 nanoparticles possess inherent peroxidase-like (POD-like) activity, which endows iron-based nanoparticles with more potential applications. Furthermore, the POD-like activity can be enhanced through Fenton (Fe2O3) reaction with H2O2. 2+ ) or class Fenton(Mn 2+ Co 3+The reaction generates reactive oxygen species (ROS) to achieve an antibacterial effect. Patent CN116764662A discloses a defective magnetic FeNi porous carbon nanozyme possessing multiple enzymatic activities. Using aquatic waste—waste shrimp shells—as raw material, it is prepared as a defective magnetic FeNi porous carbon nanozyme with high specific surface area, large pore capacity, and a hierarchical pore structure. It exhibits peroxidase-like activity, hydrogen peroxide-like mimicry activity, and oxidase-like activity, and can be used for highly selective colorimetric / photothermal / smartphone three-mode detection of total antioxidant levels and total polyphenol content in beverages and fruits. Patent CN102029163A discloses a catalase-like catalyst. The catalyst involves dissolving ferric nitrate and bismuth nitrate in ethylene glycol methyl ether, adding a coordinating agent and a dispersant to obtain a sol, which is then further gelled. High-temperature calcination and post-treatment yield a BiFeO3-based catalase catalyst, which can serve as a substitute for natural catalase. It can be used for the detection and analysis of H2O2 content in food, biological, and environmental samples, and also for the degradation and mineralization of organic pollutants. Summary of the Invention:

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a food-grade Fe-γ-CD nano-antibacterial material, its preparation method, and its application. In the prepared Fe-γ-CD nano-antibacterial material, the γ-CD ligand promotes the activity of Fe... 3+ Reduced to Fe 2+ This leads to the simultaneous presence of Fe in Fe-γ-CD. 2+ and Fe 3+ It has peroxidase-like activity, which enables it to effectively inhibit bacteria.

[0006] To achieve the above objectives, the present invention relates to a method for preparing food-grade Fe-γ-CD nano-antibacterial materials, the specific steps of which include:

[0007] (1) Dissolve FeCl3·6H2O and γ-CD in deionized water and perform ultrasonic treatment; the molar ratio of γ-CD and FeCl3·6H2O is 1:2-1:3, preferably 1:3;

[0008] (2) Heat at 120-180℃ for 5-7 hours;

[0009] (3) After the reaction, centrifuge to remove the supernatant, collect the precipitate, continue to wash the precipitate with deionized water, repeat centrifugation to remove impurities, place the centrifuged precipitate sample in a forced-air drying oven and dry until the sample is completely dry to complete the preparation of Fe-γ-CD nano antibacterial material.

[0010] Specifically, in step (2), the heating time is 5, 6 or 7 hours, preferably 6 hours; the reaction temperature is 120°C, 130°C, 140°C, 150°C, 160°C, 170°C or 180°C, preferably 120°C.

[0011] Step (3) is as follows: Take out the sample after the reaction, centrifuge at 8000 rpm / min for 10 minutes, remove the supernatant, collect the precipitate, continue to wash the precipitate with deionized water, repeat the centrifugation 3 times, put the centrifuged precipitate sample in a 60℃ forced-air drying oven, and dry until the sample is completely dry to complete the preparation of Fe-γ-CD nano antibacterial material.

[0012] The present invention relates to a method for preparing food-grade Fe-γ-CD nano-antibacterial materials. The Fe-γ-CD nanomaterials have a rod-like structure with a length ranging from 150 nm to 200 nm and a width of approximately 50 nm, and exhibit good biological activity.

[0013] This invention relates to γ-CD ligands in food-grade Fe-γ-CD nano-antibacterial materials, which promote the partial absorption of Fe. 3+ Reduced to Fe 2 + Fe-γ-CD contains 6 / 11 Fe 2+ Fe in a 5 / 11 ratio 3+ It exhibits excellent catalytic properties.

[0014] This invention relates to food-grade Fe-γ-CD nano-antibacterial materials with peroxidase activity, which enables them to catalyze the generation of hydroxyl radicals from hydrogen peroxide, thereby more effectively inhibiting bacteria.

[0015] This invention also provides the different antibacterial effects of the Fe-γ-CD nano-antibacterial material against drug-resistant Escherichia coli and drug-resistant Staphylococcus aureus, ultimately inhibiting the growth of drug-resistant Escherichia coli by 98.5% and drug-resistant Staphylococcus aureus by up to 61.8%.

[0016] The present invention also provides the application of the Fe-γ-CD nano-antibacterial material as an antibacterial material, specifically the application of the Fe-γ-CD nano-antibacterial material in the preservation of milk and orange juice.

[0017] This invention synthesizes a food-grade nano-antibacterial material, Fe-γ-CD, via a simple one-pot hydrothermal method. This material exhibits significant peroxidase-like activity, and γ-CD, acting as a reducing agent, can reduce the added Fe... 3+ Reduced to Fe 2+ Overcoming the limitations of directly using Fe 2+Its susceptibility to oxidation and negligible toxicity to normal mammalian cells make it an ideal candidate for use as an antibacterial agent in food. Compared to α-cyclodextrin (α-CD) and β-cyclodextrin (β-CD), nano-antibacterial materials prepared using γ-cyclodextrin as a reducing agent exhibit superior performance.

[0018] Compared with existing technologies, the present invention has the following advantages: Fe-γ-CD nano-antibacterial materials have excellent peroxide catalytic activity and broad-spectrum antibacterial effects, and are highly tolerant to harsh environments and have high stability; Fe-γ-CD is non-toxic to normal cells and belongs to food-grade antibacterial materials; the preparation method is simple and pollution-free, the preparation method of Fe-γ-CD nano-antibacterial materials is simple, the preparation equipment is readily available, the preparation process is simple, and at the same time, the antibacterial effect is good and harmless to the human body. The whole process is green and pollution-free, and the application environment is friendly, providing a broad market for inhibiting the spread of foodborne pathogens and ensuring food safety. Attached image description:

[0019] Figure 1 This is a schematic diagram of the antibacterial mechanism of the Fe-γ-CD nano-antibacterial material involved in Example 1.

[0020] Figure 2 The images shown are transmission electron microscopy (TEM) image (a) and scanning electron microscopy (SEM) image (b) of the Fe-γ-CD nano-antibacterial material involved in Example 1.

[0021] Figure 3 The image shows the X-ray diffraction (XRD) pattern of the Fe-γ-CD nano-antibacterial material involved in Example 1.

[0022] Figure 4 The image shows an X-ray photoelectron spectroscopy (XPS) image of the Fe-γ-CD nano-antibacterial material involved in Example 1.

[0023] Figure 5 This is an X-ray energy dispersive spectroscopy (EDSMapping) diagram of the Fe-γ-CD nano-antibacterial material involved in Example 1.

[0024] Figure 6The figure shows the peroxidase-like activity characterization of the Fe-γ-CD nano-antibacterial materials involved in Examples 1-4. In the figure, (1) Fe-γ-CD+H2O2 is: the molar ratio of γ-CD and FeCl3·6H2O is 1:3, and the reaction temperature is 120℃; (2) Fe-γ-CD+H2O2 is: the molar ratio of γ-CD and FeCl3·6H2O is 1:3, and the reaction temperature is 180℃; (3) Fe-γ-CD+H2O2 is: the molar ratio of γ-CD and FeCl3·6H2O is 1:2, and the reaction temperature is 120℃; (4) Fe-α-CD+H2O2 is: the molar ratio of α-CD and FeCl3·6H2O is 1:3; (5) Fe-β-CD+H2O2 is: the molar ratio of β-CD and FeCl3·6H2O is 1:3.

[0025] Figure 7 The image shows the electron paramagnetic resonance (EPR) image of the Fe-γ-CD nano-antibacterial material involved in Example 1.

[0026] Figure 8 The image shows the antibacterial effect of Fe-γ-CD nano-antibacterial material in Example 1 against drug-resistant Escherichia coli and drug-resistant Staphylococcus aureus.

[0027] Figure 9 The graph shows the effect of Fe-γ-CD nano-antibacterial material involved in Example 1 on the bacterial activity of drug-resistant Escherichia coli (a) and drug-resistant Staphylococcus aureus (b).

[0028] Figure 10 The image shows the MTT cytotoxicity test results of the Fe-γ-CD nano-antibacterial material involved in Example 1, taking the activity of L929 cells treated with different concentrations of Fe-γ-CD as an example.

[0029] Figure 11 Example 1 describes the antibacterial effect of Fe-γ-CD nano-antibacterial material on drug-resistant Staphylococcus aureus in milk at 4℃ (a) and 25℃ (b), and on drug-resistant Escherichia coli in orange juice at 4℃ (c) and 25℃ (d). Detailed implementation method:

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] Example 1:

[0032] This embodiment relates to a method for preparing food-grade Fe-γ-CD nano-antibacterial materials. The specific preparation steps are as follows:

[0033] (1) Mix 0.336g FeCl3·6H2O and 0.972g γ-CD (the molar ratio of FeCl3·6H2O to γ-CD is 1:1).

[0034] 3) Dissolve in 30 mL of deionized water and sonicate to achieve uniform dispersion, then transfer to a 50 mL reaction vessel;

[0035] (2) Place the reactor in an oven and heat at 120°C for 6 hours;

[0036] (3) Take out the sample after the reaction, centrifuge at 8000 rpm / min for 10 minutes, remove the supernatant, collect the precipitate, continue to wash the precipitate with deionized water, repeat the centrifugation 3 times, put the centrifuged precipitate in a 60℃ forced-air drying oven and dry until the sample is completely dry, thus completing the preparation of Fe-γ-CD nano antibacterial material.

[0037] In this embodiment, the unique properties of Fe-γ-CD were investigated using transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and X-ray elemental mapping (EDS). Electron spin resonance (EPR) was used to characterize the prepared Fe-γ-CD nano-antibacterial materials. The characterization results are as follows: Figures 2-7 As shown.

[0038] from Figure 2 TEM and SEM images show that the Fe-γ-CD nano-antibacterial material has a rod-shaped structure with a length of 150nm-200nm and a width of about 50nm.

[0039] from Figure 3 , Figure 4 and Figure 5 The XRD, XPS, and EDS mapping images show that the material contains Fe, C, and O elements, and contains Fe in different valence states, proving that γ-CD acts as a reducing agent to reduce Fe. 3+ Reduced to Fe 2+ This facilitates the generation of free radicals through electron transfer.

[0040] The POD activity of Fe-γ-CD was evaluated by the oxidation of triborate ester (TMB). 50 μL of Fe-γ-CD (initial concentration: 1 mg / mL) was added to 1 mL of acetate-sodium acetate buffer (pH = 4), which contained hydrogen peroxide (H₂O₂) (final concentration: 5 mM) and TMB (final concentration: 1 mM). In the presence of H₂O₂, TMB was oxidized to form blue oxidized TMB (ox TMB), which showed an absorption peak at 652 nm as detected by UV-Vis spectroscopy. Figure 6 The enzyme activity diagram shows that the material has peroxidase-like activity and can catalyze the generation of hydroxyl radicals from hydrogen peroxide.

[0041] Electron spin resonance (ESR) was used to detect hydroxyl radicals. Fe-γ-CD (final concentration: 50 mg / mL), H₂O₂ (final concentration: 5 mM), and 5,5-dimethyl-1-oxypyrrolidone (DMPO, final concentration: 4 mM) were added to an acetate-sodium acetate buffer solution (0.1 mM, pH = 4). After a reaction time of 20 minutes, the hydroxyl radical signal was measured using an ESR spectrometer. Figure 7 The EPR spectrum shows that the ratio of each peak is 1:2:2:1, indicating that the material can generate free radicals, and the type of free radical is hydroxyl radical.

[0042] The Fe-γ-CD nano-antibacterial material prepared in Example 1 was applied to in vitro antibacterial experiments in milk and orange juice environments. Single colonies of drug-resistant bacteria on solid LB medium were inoculated into 50 mL of sterile liquid LB medium containing tryptone (0.5 g), yeast extract (0.25 g), and NaCl (0.5 g).

[0043] The suspension of drug-resistant bacteria was then incubated on a shaker at 37°C for 12 hours at 150 rpm / min. The bacteria were subsequently diluted to a concentration of 10⁻⁶ with sterile PBS. 7 CFU / mL was used to obtain bacterial culture. 50 μL of the bacterial culture (initial concentration 10) was added to a 500 μL system containing a final concentration of 300 μg / mL Fe-γ-CD and 250 μM H2O2. 7 The solution (CFU / mL) was incubated at 37°C for 30 minutes. Then, 100 μL of the solution was evenly spread onto a solid culture medium. The solid culture medium was then incubated at 37°C for 18 hours. Bacterial colony counts were performed using the CFU method. Sterile PBS was used as a blank control. Parallel control experiments were conducted with bacteria alone and with H2O2 or Fe-γ-CD nano-antibacterial materials. The measurement results are as follows: Figure 8 and Figure 9As shown, Fe-γ-CD nano-antibacterial materials exhibit different antibacterial effects against drug-resistant Escherichia coli and drug-resistant Staphylococcus aureus, with inhibition rates of 98.5% and 61.8%, respectively. This indicates that Fe-γ-CD nano-antibacterial materials possess good antibacterial properties in the presence of H2O2.

[0044] Example 1 describes the application of the Fe-γ-CD nano-antibacterial material prepared in biotoxicity testing. Mouse L929 cells were used in the experiment. Cells were placed in 96-well plates and treated with different concentrations (0 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL) of Fe-γ-CD nano-antibacterial material solution at 37°C for 24 hours. Subsequently, 100 μL of dimethyl sulfoxide (DMSO) was added to each well for another 4 hours of incubation. Finally, the absorbance values ​​were collected on a 490 nm microplate reader. Cell viability was set as 100% for cells not incubated with the Fe-γ-CD nano-antibacterial material solution. Figure 10 As shown, the results indicate that the survival rate of L929 cells gradually increases with the increase of Fe-γ-CD nano-antibacterial material concentration. In conclusion, Fe-γ-CD has no significant impact on the normal life activities of organisms, which is beneficial as a food antibacterial agent that will not cause harm to the human body, and it has great advantages in terms of biocompatibility.

[0045] Application experiments of the Fe-γ-CD nano-antibacterial material prepared in Example 1 in the environmental antibacterial effect of milk and orange juice: The Fe-γ-CD nano-antibacterial material prepared in Example 1 was applied to the preservation of milk and orange juice. Infection models of drug-resistant Staphylococcus aureus and Escherichia coli in milk and orange juice were established, respectively. The samples were divided into four treatment groups at 4℃ and 25℃: a blank group with only phosphate-buffered saline (PBS) and no other treatment; a control group with H2O2; a control group with Fe-γ-CD; and an experimental group with Fe-γ-CD + H2O2. The bacterial content of different treatment groups was measured at regular intervals. Figure 11 It can be seen that the Fe-γ-CD+H2O2 treatment group had the lowest bacterial content, indicating that the Fe-γ-CD nano-antibacterial material has significant antibacterial properties against a variety of drug-resistant bacteria and can be used as a food antibacterial agent.

[0046] Example 2:

[0047] The difference between this embodiment and embodiment 1 is that the reaction temperature in step (2) is 180°C, while all other aspects are the same.

[0048] Example 3:

[0049] The difference between this embodiment and embodiment 1 is that the molar ratio of γ-CD and FeCl3·6H2O in step (1) is 1:2, while all other aspects are the same.

[0050] Example 4:

[0051] The difference between this embodiment and Embodiment 1 is that γ-CD is replaced with α-CD, and the molar ratio of α-CD and FeCl3·6H2O in step (1) is 1:3, while all other aspects are the same.

[0052] Example 5:

[0053] The difference between this embodiment and Embodiment 1 is that γ-CD is replaced with β-CD, and the molar ratio of β-CD and FeCl3·6H2O in step (1) is 1:3, while all other aspects are the same.

Claims

1. A method for preparing food-grade Fe-γ-CD nano-antibacterial material, characterized in that, The specific steps include: (1) Dissolve FeCl3•6H2O and γ-CD in deionized water and sonicate; the molar ratio of γ-CD and FeCl3•6H2O is 1:2-1:3; (2) Heat at 120℃ for 5-7 hours; (3) After the reaction, centrifuge to remove the supernatant, collect the precipitate, continue to wash the precipitate with deionized water, repeat centrifugation to remove impurities, place the centrifuged precipitate sample in a forced-air drying oven to dry, and complete the preparation of Fe-γ-CD nano antibacterial material.

2. The method for preparing food-grade Fe-γ-CD nano-antibacterial material according to claim 1, characterized in that, The heating time in step (2) is 6 hours.

3. The method for preparing food-grade Fe-γ-CD nano-antibacterial material according to claim 1, characterized in that, Step (3) is as follows: Take out the sample after the reaction, centrifuge at 8000 rpm for 10 minutes, remove the supernatant, collect the precipitate, continue to wash the precipitate with deionized water, repeat the centrifugation 3 times, put the centrifuged precipitate sample in a 60 ℃ forced-air drying oven, and dry until the sample is completely dry to complete the preparation of Fe-γ-CD nano antibacterial material.

4. The preparation method of the food-grade Fe-γ-CD nano-antibacterial material according to claim 1, characterized in that, Fe-γ-CD nanomaterials have a rod-like structure with a length of 150 nm-200 nm and a width of 50 nm.

5. The method for preparing food-grade Fe-γ-CD nano-antibacterial material according to claim 1, characterized in that, When the molar ratio of γ-CD to FeCl3•6H2O is 1:3, the heating time is 6 hours, and the heating temperature is 120℃, the prepared Fe-γ-CD nano-antibacterial material contains a 6 / 11 ratio of Fe. 2+ Fe in a 5 / 11 ratio 3+ .

6. Fe-γ-CD nano-antibacterial material prepared by the method for preparing food-grade Fe-γ-CD nano-antibacterial material according to any one of claims 1-5.

7. The application of the Fe-γ-CD nano-antibacterial material according to claim 6 as an antibacterial material for non-therapeutic purposes.

8. The application of the Fe-γ-CD nano-antibacterial material according to claim 6 in the preservation of milk and orange juice.

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