Pulullan modified cerium oxide nano composite material as well as preparation method and application thereof

Abstract

The invention provides a Pulullan modified cerium oxide nano composite material, a preparation method and application. The preparation method of the Pulullan modified cerium oxide nano composite material comprises the following steps: mixing cerium salt and Pulullan in a solvent, and reacting to prepare the Pulullan modified cerium oxide nano composite material. The provided preparation method is simple in process and low in cost. Pulullan is used for modifying cerium oxide nanoparticles, and the obtained Pulullan modified cerium oxide nanoparticles are not prone to agglomeration, uniform in particle size and high in free radical scavenging capacity and antibacterial performance.

Description

Technical Field

[0001] The invention relates to the field of nanomaterials, and in particular to a pullulan-modified cerium oxide nanocomposite material, a preparation method and application thereof. Background Art

[0002] Cerium is a rare earth metal that belongs to the lanthanide series in the periodic table. 3+ ) and oxidation (Ce 4+ ) states, which enables CeO2 NPs to exhibit effective reactive oxygen species scavenging and cell protection against oxidative stress. CeO2 NPs have unique antibacterial properties and biocompatibility. The antibacterial activity of CeO2 NPs can be attributed to two main mechanisms: Ce 3+ and Ce 4+ Reversible redox transitions and generation of reactive oxygen species (ROS) between Ce 4+ It is a potent oxidant that can cause substantial damage to bacterial cell membranes, ultimately compromising their structural integrity and function. Due to its antioxidant, anti-inflammatory and antibacterial activities, cerium oxide nanoparticles are widely used in catalysts, sensors, solid oxide fuel cells, sunscreen cosmetics, bioimaging, biotransformation and antibacterial activity. However, due to the large number of oxygen vacancies on the surface of CeO2NPs, they have a strong tendency to agglomerate, resulting in low stability of their aqueous suspensions, which restricts the application of CeO2NPs. Summary of the invention

[0003] Aiming at the problem that the current cerium oxide nanoparticles are easy to agglomerate, a pullulan-modified cerium oxide nanocomposite material, a preparation method and application are provided.

[0004] In some embodiments, a method for preparing a pullulan-modified cerium oxide nanocomposite material is provided, comprising the following steps: mixing a cerium salt and pullulan in a solvent, and reacting the mixture to prepare the pullulan-modified cerium oxide nanocomposite material.

[0005] In some embodiments, the preparation method meets one or more of the following characteristics:

[0006] (1) In the step of mixing the cerium salt and pullulan in a solvent, the pH value of the obtained solution is 3.5-6;

[0007] (2) In the step of mixing the cerium salt and pullulan in a solvent, the final concentration of pullulan is 1% to 10% in g / mL;

[0008] (3) In the step of mixing the cerium salt and pullulan in a solvent, the mass ratio of pullulan to cerium salt is (6.8-68):(20-120) based on the amount of feed.

[0009] In some embodiments, the preparation method meets one or more of the following characteristics:

[0010] (1) In the step of mixing the cerium salt and pullulan in a solvent, the pH value of the obtained solution is 5 to 5.5;

[0011] (2) In the step of mixing the cerium salt and pullulan in a solvent, the final concentration of pullulan is 1% to 5% in g / mL;

[0012] (3) In the step of mixing the cerium salt and pullulan in a solvent, the mass ratio of pullulan to cerium salt is (6.8-34):(30-40) based on the amount of feed.

[0013] In some embodiments, the preparation method meets one or more of the following characteristics:

[0014] (1) The solvent comprises a first solvent and a second solvent, the first solvent is used to dissolve the cerium salt and the pH adjuster, the second solvent is used to dissolve pullulan, the first solvent is water, the second solvent is an alcohol solvent, and the pH adjuster is acetic acid; based on the amount of feed, the volume mass ratio of the first solvent to the cerium salt is (0.08-3) mL:1 g, and the volume ratio of the pH adjuster to the first solvent is (0.3-12):1;

[0015] (2) The cerium salt is cerium nitrate or cerium nitrate hydrate. Optionally, the cerium nitrate hydrate comprises cerium nitrate hexahydrate.

[0016] In some embodiments, the preparation method meets one or more of the following characteristics:

[0017] (1) In the step of mixing the cerium salt and pullulan in the solvent, the stirring speed is 350 rpm to 450 rpm;

[0018] (2) In the step of mixing the cerium salt and pullulan in a solvent to carry out a reaction, the reaction temperature is 150° C. to 170° C.;

[0019] (3) In the step of mixing the cerium salt and pullulan in a solvent to carry out a reaction, the reaction time is 140 min to 180 min;

[0020] (4) In the step of mixing the cerium salt and pullulan in a solvent to carry out a reaction, the reaction is carried out under a pressure of 1 to 3 MPa.

[0021] In some embodiments, in the preparation method, after the reaction is completed, the following steps are further included: solid-liquid separation, solid phase collection, water washing, drying, and grinding.

[0022] In some embodiments, a pullulan-modified cerium oxide nanocomposite material is provided, comprising cerium oxide and pullulan modified on the cerium oxide.

[0023] In some embodiments, the pullulan-modified cerium oxide nanocomposite material satisfies at least one of the following conditions:

[0024] (1) The particle size of the pullulan-modified cerium oxide nanocomposite material is 100 nm to 600 nm;

[0025] (2) The pullulan-modified cerium oxide nanocomposite material is prepared according to the preparation method of the pullulan-modified cerium oxide nanocomposite material.

[0026] In some embodiments, the particle size of the pullulan-modified cerium oxide nanocomposite material is 100 nm to 250 nm.

[0027] In some embodiments, the pullulan-modified cerium oxide nanocomposite material is used in one or more of the following:

[0028] (1) Application in at least one of eliminating free radicals, inhibiting microorganisms, bioimaging and biotransformation, wherein the free radicals include one or more of superoxide anion radicals, hydroxyl radicals, hydrogen peroxide and nitrogen radicals, and the microorganisms are at least one of Gram-negative bacteria and Gram-positive bacteria;

[0029] (2) Use in the preparation of at least one of skin products, medicines, catalysts, sensors and solid oxide fuel cells;

[0030] In some embodiments, the application is not a method for diagnosing or treating a disease.

[0031] In some embodiments, a skin product, a medicine, a catalyst, a sensor or a solid oxide fuel cell is provided, which contains the pullulan-modified cerium oxide nanocomposite material.

[0032] The preparation method provided in the present application is simple in process and low in cost. The cerium oxide nanoparticles are modified with pullulan, and the obtained pullulan-modified cerium oxide nanoparticles are not easy to agglomerate, have uniform particle size, and have strong free radical scavenging ability and antibacterial performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0034] Figure 1 This is a flow chart of the preparation process of pullulan-modified cerium oxide nanocomposite materials in one embodiment of the present application;

[0035] Figure 2 is a transmission electron microscope (TEM) image and a particle size distribution diagram of the pullulan-modified cerium oxide nanocomposite prepared in Example 1 and the cerium oxide nanospheres prepared in Comparative Example 1, wherein a is a transmission electron microscope image of the pullulan-modified cerium oxide nanocomposite prepared in Example 1, b is a particle size distribution diagram of the pullulan-modified cerium oxide nanocomposite prepared in Example 1, c is a transmission electron microscope image of the cerium oxide nanospheres prepared in Comparative Example 1, and d is a particle size distribution diagram of the cerium oxide nanospheres prepared in Comparative Example 1, wherein Count is the number and Particle Size is the particle size;

[0036] Figure 3 FTIR spectra of Pul-CeO2NPs prepared in Example 1, CeO2NPs prepared in Comparative Example 1, and Pul prepared in Comparative Example 2, wherein Wavenumber is the wave number;

[0037] FIG4 is a schematic diagram of a DPPH removal reaction of a cerium oxide dispersion in one embodiment of the present application, wherein Purple is purple and Yellow is yellow;

[0038] Figure 5 The DPPH free radical scavenging rates of Pul-CeO2NPs prepared in Example 1, CeO2NPs prepared in Comparative Example 1, and Pul prepared in Comparative Example 2;

[0039] Figure 6 The superoxide anion radical scavenging rates of Pul-CeO2NPs prepared in Example 1, CeO2NPs prepared in Comparative Example 1, and Pul prepared in Comparative Example 2;

[0040] Figure 7 The hydroxyl radical scavenging rates of Pul-CeO2NPs prepared in Example 1, CeO2NPs prepared in Comparative Example 1, and Pul prepared in Comparative Example 2;

[0041] Figure 8The inhibition zones of Pul-CeO2NPs prepared in Example 1, CeO2NPs prepared in Comparative Example 1, and Pul prepared in Comparative Example 2 against Escherichia coli and Staphylococcus aureus. DETAILED DESCRIPTION

[0042] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0044] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0045] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0046] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0047] In this application, the "suitable" mentioned in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0048] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0049] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0050] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0051] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0052] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0053] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0054] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0055] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0056] In this application, Pul and pullulan refer to pullulan polysaccharide. CeO2, CeO2 NPs, cerium oxide, and bare cerium oxide refer to unmodified cerium oxide nanoparticles. Pul-CeO2, Pul-CeO2 NPs, pullulan cerium oxide, and pullulan polysaccharide-modified cerium oxide refer to pullulan polysaccharide-modified cerium oxide nanocomposites.

[0057] Since there are a large number of oxygen vacancies on the surface of CeO2 NPs, they have a strong tendency to agglomerate, resulting in low stability of their aqueous suspensions, which restricts the application of CeO2 NPs.

[0058] The type and amount of polymer modified on cerium oxide nanoparticles affect the stability of the suspension. Pullulan is an extracellular water-soluble polysaccharide produced by microbial fermentation. It contains "maltose triacyl" as a repeating unit connected by α-(1,6) glycosidic bonds. Pullulan has no antibacterial effect because it is non-toxic, non-mutagenic, non-carcinogenic, biocompatible and biodegradable. Pul has excellent water solubility and a large number of hydroxyl groups, which form complexes with Ce. CeO2 is modified on the molecular structure of CeO2. The polyhydroxyl groups of pullulan form complexes with Ce to regulate its surface stability and functionalization to improve the solubility, stability and biocompatibility of CeO2 in water. Pullulan itself has no antibacterial effect, but it enhances the antibacterial properties of CeO2 by combining with CeO2. The surface functionalization of CeO2 NPs is controlled by the modification of pullulan to prevent the aggregation of cerium oxide nanoparticles. The prepared pullulan-modified cerium oxide nanocomposite material has high stability and biocompatibility, a smaller particle size than cerium oxide nanoparticles, better dispersibility, and lower toxicity than cerium oxide nanoparticles. It has better free radical scavenging ability and better microbial inhibition ability, and can effectively reduce the risk of bacterial infection.

[0059] In one embodiment, a method for preparing a pullulan-modified cerium oxide nanocomposite material is provided, comprising the following steps:

[0060] Cerium salt and pullulan are mixed in a solvent and reacted to prepare a pullulan-modified cerium oxide nanocomposite material. Figure 1 shown.

[0061] In one embodiment, in the step of mixing the cerium salt and pullulan in a solvent, the pH value of the obtained solution is 3.5-6, and the pH value of the obtained solution can also be 5-5.5, for example, 3.5, 4.0, 4.5, 5.0, 5.2, 5.5, 6 or a range consisting of any two of the foregoing values.

[0062] In one embodiment, in the step of mixing the cerium salt and pullulan in a solvent, the final concentration of pullulan is 1% to 10% in g / mL, and can also be 1% to 5%, and can further be 2% to 3%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a range consisting of any two of the foregoing values.

[0063] In one embodiment, in the step of mixing the cerium salt and pullulan in a solvent, the mass ratio of pullulan to the cerium salt, based on the amount of feed, is (6.8-68):(20-120), or (6.8-34):(30-40), or 6.8:20, 6.8:40, 6.8:80, 6.8:120, 34:20, 34:40, 34:80, 34:120, 68:20, 68:40, 68:80, 68:120 or a range selected from any two of the foregoing ratios.

[0064] In one embodiment, the solvent includes a first solvent and a second solvent.

[0065] In one embodiment, the first solvent is used to dissolve the cerium salt and the pH adjuster, and the second solvent is used to dissolve pullulan.

[0066] In one embodiment, the first solvent is water.

[0067] In one embodiment, the second solvent is an alcohol solvent.

[0068] In one embodiment, the pH adjuster is acetic acid.

[0069] In one embodiment, based on the feed amount, the volume mass ratio of the first solvent to the cerium salt is (0.08-3) mL: 1 g.

[0070] In one embodiment, based on the feed amount, the volume ratio of the pH adjuster to the first solvent is (0.3-12):1;

[0071] In one embodiment, the cerium salt is cerium nitrate or cerium nitrate hydrate.

[0072] In one embodiment, the cerium nitrate hydrate includes but is not limited to cerium nitrate hexahydrate.

[0073] In one embodiment, in the step of mixing the cerium salt and pullulan in the solvent, the stirring speed is 350 rpm to 450 rpm.

[0074] In one embodiment, in the step of mixing the cerium salt and pullulan in a solvent, in the step of reacting, the reaction temperature is 150° C. to 170° C.

[0075] In one embodiment, in the step of mixing the cerium salt and pullulan in a solvent, and in the step of reacting, the reaction time is 140 min to 180 min.

[0076] In one embodiment, in the step of mixing the cerium salt and pullulan in a solvent, in the step of reacting, the reaction is carried out under a pressure of 1 to 3 MPa.

[0077] In one embodiment, in the step of mixing the cerium salt and pullulan in a solvent, a reaction is carried out, and after the reaction is completed, the following steps are further included: solid-liquid separation, solid phase collection, water washing, drying, and grinding.

[0078] In one embodiment, a pullulan-modified cerium oxide nanocomposite material is provided, comprising cerium oxide and pullulan modified on the cerium oxide.

[0079] In one embodiment, the particle size of the pullulan modified cerium oxide nanocomposite material is 100nm~600nm, and can be 100nm~250nm, such as 100nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm or a range selected from any two of the foregoing values.

[0080] In one embodiment, the provided pullulan-modified cerium oxide nanocomposite material is prepared according to the aforementioned method for preparing the pullulan-modified cerium oxide nanocomposite material.

[0081] In one embodiment, a pullulan-modified cerium oxide nanocomposite material is provided for use in at least one of eliminating free radicals, inhibiting microorganisms, bioimaging, and biotransformation, wherein the free radicals include one or more of superoxide anion radicals, hydroxyl radicals, hydrogen peroxide, and nitrogen radicals, and the microorganisms are at least one of Gram-negative bacteria and Gram-positive bacteria.

[0082] In one embodiment, a use of a pullulan-modified cerium oxide nanocomposite material in the preparation of at least one of a skin product, a medicine, a catalyst, a sensor, and a solid oxide fuel cell is provided.

[0083] In one embodiment, the application provided is not a method for diagnosing or treating a disease.

[0084] In one embodiment, the application provided is a method for diagnosing and treating a disease.

[0085] In one embodiment, a skin product, a medicine, a catalyst, a sensor or a solid oxide fuel cell is provided, which contains a pullulan-modified cerium oxide nanocomposite material.

[0086] In one embodiment, the provided skin care product has at least one of antioxidant, anti-aging, microbial inhibition and inflammation elimination effects.

[0087] In one embodiment, the active ingredient of the provided medicine comprises a pullulan-modified cerium oxide nanocomposite material. Optionally, the provided medicine further comprises an acceptable carrier.

[0088] In this application, unless otherwise specified, "acceptable" means suitable for administration to subjects within the scope of reasonable judgment and commensurate with a reasonable benefit / risk ratio, and can be used to describe ligands, materials, compositions, dosage forms, etc. As an example, "pharmaceutically acceptable" means suitable for administration to subjects within the scope of reasonable medical judgment and commensurate with a reasonable benefit / risk ratio.

[0089] In some embodiments, "acceptable carrier" refers to an acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material.

[0090] The preparation method provided is simple in process and low in cost. The prepared pullulan polysaccharide-modified cerium oxide nanocomposite material is not easy to agglomerate, and has strong free radical scavenging ability and antibacterial performance, and has high biological activity. Through the design of single-factor experiments, it is found that a pullulan cerium oxide dispersion with good antioxidant properties can be prepared when the pullulan polysaccharide concentration is in the range of 1wt% to 10wt%.

[0091] The embodiments of the present application will be described in detail below in conjunction with some examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0092] 1. Preparation Example of Pullulan-modified Cerium Oxide Nanocomposite Material

[0093] Example 1

[0094] Weigh 13.6 g of pullulan, add 600 mL of ethylene glycol (HOCH2CH2OH), and stir at 400 rpm and 80°C until the solution is clear and transparent. Weigh 40 g of hexahydrated cerium nitrate crystals (Ce(NO3)3·6H2O) and add them to 40 mL of pure water for mixing. After the hexahydrated cerium nitrate crystals are completely dissolved, add 40 mL of acetic acid (CH3COOH). Pour the pullulan solution into the cerium nitrate solution and stir at room temperature at 400 rpm for 10 min to fully disperse the solution. The concentration of pullulan in the obtained mixed solution is 2% (g / mL), and the pH value of the mixed solution is 5.2. Transfer the above solution to a 1 L reactor, set the reaction pressure to 1.1 MPa, and place it in a preheated 170°C oven for reaction for 160 min. Centrifuge the reaction product at 10,000 rpm for 10 min, and wash the resulting precipitate twice with deionized water. The final precipitate after washing and centrifugation was placed in an oven at 60°C for 4 h and ground to obtain pullulan-modified cerium oxide nanocomposites (Pul-CeO2 NPs).

[0095] Embodiment 2 to Embodiment 7

[0096] The preparation method of the pullulan-modified cerium oxide nanocomposite material is similar to that of Example 1, except that the amount of pullulan added is different, that is, the pullulan concentration is different, and the pullulan concentration is shown in Table 1. The pullulan concentration refers to: pullulan mass ÷ (acetic acid + ethylene glycol + pure water) volume, and the calculation formula of the synthesis yield is as follows:

[0097] Synthesis yield = mass of cerium oxide / theoretical mass of cerium oxide, theoretical mass of cerium oxide = (mass of cerium nitrate / relative molecular mass of cerium nitrate) × relative molecular mass of cerium oxide.

[0098] Table 1

[0099]

[0100] Comparative Example 1

[0101] Weigh 40 g of hexahydrated cerium nitrate crystals (Ce(NO3)3·6H2O) and add them to 40 mL of pure water for mixing. After the hexahydrated cerium nitrate crystals are completely dissolved, add 40 mL of acetic acid (CH3COOH) for mixing, and then add 600 mL of ethylene glycol (HOCH2CH2OH) to the solution. Stir at room temperature for 10 min at 400 rpm to fully disperse the solution. Transfer the above solution to a 1 L reactor and place it in a preheated 170℃ oven for reaction for 160 min. The reaction product is centrifuged at 10,000 rpm for 10 min, and the resulting precipitate is washed twice with deionized water. The final precipitate after washing and centrifugation is placed in a 60℃ oven for drying for 4 h and ground to obtain cerium oxide nanosphere powder (CeO2 NPs).

[0102] Comparative Example 2

[0103] 1.5 g of pullulan was weighed, 10 ml of pure water was added, and the mixture was stirred at 400 rpm and 80°C until the pullulan was dissolved to obtain a pullulan aqueous solution (Pul).

[0104] Comparative Example 3

[0105] Weigh 13.6 g of levan, add 600 mL of ethylene glycol (HOCH2CH2OH), and stir at 400 rpm and 80°C until the solution is clear and transparent. Weigh 40 g of hexahydrated cerium nitrate crystals (Ce(NO3)3·6H2O) and add them to 40 mL of pure water for mixing. After the hexahydrated cerium nitrate crystals are completely dissolved, add 40 mL of acetic acid (CH3COOH). Pour the levan solution into the cerium nitrate solution and stir at room temperature at 400 rpm for 10 min to fully disperse the solution. Transfer the above solution to a 1 L reactor and place it in a preheated 170°C oven for reaction for 160 min. The reaction product was centrifuged at 10,000 rpm for 10 min, and the resulting precipitate was washed twice with deionized water. The final precipitate after washing and centrifugation was placed in a 60°C oven for 4 h and ground to obtain levan-CeO2 NPs.

[0106] 2. Test Analysis

[0107] (1) Transmission electron microscopy (TEM)

[0108] The size and morphology of the Pul-CeO2NPs prepared in Example 1 and the CeO2NPs prepared in Comparative Example 1 were analyzed by transmission electron microscopy (JEOL, JSM 2200 FS). For TEM observation, the sample was dispersed in an ethanol / water solution, and the diluted sample suspension was then dropped on a Cu grid and dried. The size of 50 particles was measured using Image J software (appropriately magnified images obtained from different microscope fields), the particle sizes of Pul-CeO2NPs and CeO2NPs were statistically calculated, and the particle size distribution histogram was drawn using Origin 2021 software. The results are shown in Figure 2, where a is a transmission electron micrograph of a pullulan-modified cerium oxide nanocomposite prepared in Example 1, b is a particle size distribution graph of a pullulan-modified cerium oxide nanocomposite prepared in Example 1, c is a transmission electron micrograph of cerium oxide nanospheres prepared in Comparative Example 1, and d is a particle size distribution graph of cerium oxide nanospheres prepared in Comparative Example 1. TEM images show that the particle sizes of the prepared Pul-CeO2 NPs and CeO2 NPs are basically uniform and the particles are spherical. The particle size of Pul-CeO2 NPs is 182nm, and the particle size of CeO2 NPs is 249nm. The particle size of Pul-CeO2 NPs is smaller than that of CeO2 NPs, which reduces the aggregation of CeO2 NPs. This may be due to the combination of the hydroxyl group of Pul with the Ce of CeO2, which reduces the particle size.

[0109] (2) Infrared

[0110] The functional groups were analyzed using ATR mode of Fourier transform infrared spectrometer (Spectrum 3, PerkinElmer, USA) to study the interaction between chitosan and cerium oxide. Each sample was scanned 6 times in the wavelength range of 4000–500 cm -1 , resolution 4cm -1 . Figure 3 FTIR spectra of Pul-CeO2NPs prepared in Example 1, CeO2NPs prepared in Comparative Example 1, and Pul prepared in Comparative Example 2. In the spectrum of Pul, the -1 There are two strong absorption bands at 1645 cm -1 is the stretching vibration of OCO. Pul at 1348 and 1155 cm -1 Other characteristic bands were observed nearby, corresponding to COH bending and COC stretching. In addition, at 925, 844, and 756 cm -1The absorption peaks at 844 cm-1 are α-(1,6) glycosidic bond, α-pyranose ring and α-(1,4) glycosidic bond, which are typical Pul bands and α-configuration of α-acyl-glucose units. By comparing with the FTIR spectrum of Pul-CeO2NPs, the characteristic bands of Pul in the sample can be observed. CeO2NPs have a peak at 844 cm-1. -1 There is no characteristic peak in Pul-CeO2NPs, but there is one in Pul-CeO2NPs, indicating that Pul is modified on CeO2NPs. 1000 cm -1 It is the CO bond of primary alcohol. There is no peak in CeO2NPs, but characteristic peaks appear in Pul-CeO2NPs, which may be due to the combination of Pul and Ce to produce Ce-O bond.

[0111] (3) DPPH free radical scavenging effect

[0112] DPPH (1,1-disphenyl-2-picryl-hydrazyl), chemical name 1,1-diphenyl-2-trinitrophenylhydrazine, molecular formula (C6H5)2N-NC6H2(NO2)3, this compound has an unpaired valence electron on one atom of the nitrogen bridge, and the orbital motion of this electron is almost offset by the molecular structure. Usually, the capture of DPPH free radicals is used as an indicator to assess antioxidant capacity. DPPH is a stable organic free radical with a maximum absorption peak of 517 nm in the visible light region. In ethanol solution, each DPPH molecule can generate a stable nitrogen-containing free radical in the solution, with a typical purple color, such as Figure 3 As shown in the figure, when it reacts with a free radical scavenger that provides 1 electron (for example, cerium oxide dispersion), a colorless product is generated, which makes the typical purple color of the solution lighter. The degree of fading is stoichiometrically related to the number of paired electrons. Therefore, spectrophotometry can be used for quantitative analysis to detect the free radical scavenging situation, thereby evaluating the free radical scavenging ability of the sample.

[0113] 1. Instruments and Reagents

[0114] Note: ① The brands and models marked in brackets for equipment are those used during laboratory method development, and are not exclusive. ② The brands, specifications, and other information commonly used in laboratories are marked in brackets for reagents and consumables, and are not exclusive.

[0115] (1) Analytical balance (d = 0.1 mg, Sartorius, BSA2202S-CW, Max 220 g);

[0116] (2) Vortex mixer (MX-F);

[0117] (3) Multifunctional microplate reader (Tecan), 96-well plate (jet);

[0118] (4) Pipette (5 mL, 200 μL, 1000 μL, Eppendorf) and matching tips;

[0119] (5) High-speed centrifuge (Thermo scientific);

[0120] (6) DPPH (sigma, analytical grade, C 18 H 12 N5O6, molecular weight 394.32);

[0121] (7) Deionized water;

[0122] (8) Anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., analytical grade).

[0123] 2. Experimental Preparation

[0124] 2.1 Reagent preparation

[0125] (1) 0.2 mmol / L DPPH solution

[0126] Weigh 0.0200 g DPPH, add anhydrous ethanol to dissolve and dilute to a 250 mL volumetric flask, and shake well.

[0127] (2) Sample dispersion

[0128] Take 10 mg of the pullulan-modified cerium oxide nanocomposites prepared in Examples 1 to 7 and the products obtained in Comparative Examples 1 to 3 as the test samples, respectively, disperse the test samples in 10 mL of deionized water, ultrasonically disperse for 10 minutes, and shake well to obtain 0.1 wt% test sample dispersions. Dilute them to 0.08 wt%, 0.06 wt%, 0.04 wt%, and 0.02 wt% test sample dispersions.

[0129] 2.2 Preparation of reagents and consumables

[0130] Before starting the test, prepare the prepared test solution, anhydrous ethanol, DPPH diluent, pipette and pipette tip in front of the EP tube rack.

[0131] 3. Experimental operation

[0132] Mix the same amount of the sample dispersion with the DPPH solution as group A (sample group), mix the same amount of anhydrous ethanol with the DPPH solution as group B (control group), and mix the same amount of anhydrous ethanol with the sample dispersion as group C (blank group). After reacting for 30 minutes at room temperature and in the dark, measure the absorbance of each group of solutions at 517 nm. Note: In order to avoid the influence of CeO2 particles in the solution on the absorbance measurement, all solutions in the experimental group (group A) and the blank group (group C) were centrifuged at 10,000 rpm for 10 minutes, and then the absorbance of the supernatant was measured.

[0133] 4. Result processing

[0134] ,

[0135] Where:

[0136] A——Absorbance at 517 nm after the sample solution is mixed with DPPH solution;

[0137] B——Absorbance at 517 nm after mixing anhydrous ethanol and DPPH solution;

[0138] C——Absorbance at 517 nm after mixing anhydrous ethanol with the sample solution.

[0139] 5. Results Analysis

[0140] The results of DPPH free radical scavenging rate are shown in Tables 2, 3 and Figure 5 shown.

[0141] Table 2

[0142]

[0143] Table 3

[0144]

[0145] In order to intuitively reflect the relationship between Pul-CeO2 NPs and CeO2 NPs, the cerium oxide content in Levan-CeO2 NPs, Pul-CeO2NPs and CeO2 NPs was made the same. As shown in Table 2, the Pul-CeO2NPs prepared in Example 1 to Example 7 have a higher DPPH radical scavenging rate than the CeO2 NPs, Pul, and Levan-CeO2 NPs prepared in Comparative Example 1 to Comparative Example 3. The DPPH radical scavenging rate of Pul is low, and the DPPH radical scavenging effect is poor, which can be regarded as Pul having no scavenging rate for DPPH. The DPPH radical scavenging rate of Levan-CeO2 NPs is higher than that of CeO2 NPs, but lower than that of Pul-CeO2 NPs. The scavenging effect is stronger than that of CeO2 NPs, but weaker than that of Pul-CeO2 NPs. As shown in Tables 3 and Figure 5 As shown, it can be seen that the DPPH radical scavenging rates of Pul, CeO2 NPs, Pul-CeO2 NPs prepared in Example 1 and Levan-CeO2 NPs increase with increasing concentration, the purple color of the DPPH solution gradually turns lighter, and the scavenging rate is significantly improved.

[0146] When the CeO2 content in the Pul-CeO2 NPs and CeO2 NPs prepared in Example 1 reached 0.1%, the purple color of the DPPH solution turned into yellow (the clearance rates were 93.1 and 87.6). The DPPH solution treated with the Pul-CeO2 NPs prepared in Example 1 turned yellow faster and had a darker color than the DPPH solution treated with CeO2 NPs. At the same concentration, the Pul-CeO2 NPs and CeO2 NPs prepared in Example 1 have the same content of cerium oxide. The Pul-CeO2 NPs prepared in Example 1 have a higher DPPH radical scavenging rate than CeO2 NPs, while the DPPH radical scavenging rate of Pul is lower. The DPPH radical scavenging rate of Pul is also significantly lower than that of CeO2 NPs and Pul-CeO2. This shows that under the conditions of the example (Pul-CeO2 NPs), Pul modifies CeO2 without reducing its antioxidant capacity. Pul improves the DPPH radical scavenging effect of CeO2 NPs, improves the DPPH radical antioxidant capacity of CeO2 NPs, and improves the biological activity.

[0147] (4) Superoxide anion free radical scavenging effect

[0148] Superoxide anion (O2 -) is a kind of reactive oxygen, which is the product of single electron reduction of oxygen molecules in cells. It can diffuse through cells, participate in many physiological activities such as cell proliferation and apoptosis, and play an important role in the redox metabolism of cells. This method uses the self-oxidation of pyrogallol under alkaline conditions to produce superoxide anions. At the same time, a characteristic absorption peak will appear near 325 nm, and superoxide anions can further promote the oxidation of pyrogallol. As the oxidation reaction deepens, the absorption of the peak gradually becomes stronger. When the generated superoxide anions are removed, the reaction is inhibited, and its absorption near 325 nm will decrease. Therefore, the amount of superoxide anions in the solution can be judged according to the change of the characteristic absorption peak near 325 nm, thereby judging the scavenging effect of antioxidants on superoxide anions.

[0149] 1. Instruments and Reagents

[0150] Note: ① The brands and models marked in brackets for equipment are those used during laboratory method development, and are not exclusive. ② The brands, specifications, and other information commonly used in laboratories are marked in brackets for reagents and consumables, and are not exclusive.

[0151] (1) Analytical balance (d = 0.1 mg, Sartorius, BSA2202S-CW, Max 220 g);

[0152] (2) Vortex mixer (MX-F);

[0153] (3) Multifunctional microplate reader (Thermo scientific), 96-well plate (jet);

[0154] (4) Pipette (5 mL, 200 μL, 1000 μL, Eppendorf);

[0155] (5) High-speed centrifuge (Thermo scientific);

[0156] (6) Pyrogallol (Beijing Bio-Toda Technology Co., Ltd., analytical grade, C6H6O3, molecular weight 126.11);

[0157] (7) Tris (Beijing Bio-Toda Technology Co., Ltd., analytical grade, C4H 11 NO3, molecular weight 121.14);

[0158] (8) Concentrated hydrochloric acid (Modern Oriental (Beijing) Science and Technology Development Co., Ltd., analytical grade).

[0159] 2. Experimental Preparation

[0160] 2.1 Reagent preparation

[0161] (1) 0.1 mol / L HCl solution

[0162] Weigh 2.1 ml of concentrated hydrochloric acid, add deionized water to a 250 mL volumetric flask, and shake well.

[0163] (2) 50 mmol / L Tris-HCl solution

[0164] Weigh 0.6057 g Tris and dissolve it in deionized water. Add 22.9 ml 0.1 mol / L HCL solution and dilute to 100 mL volumetric flask with deionized water and shake well.

[0165] (3) 0.3 mmol / L pyrogallol solution

[0166] Take 3.7833 mg of pyrogallol solution, add 0.01 mol / L HCL solution to dissolve, make up to volume in a 100 mL volumetric flask, and shake well.

[0167] (4) Sample solution

[0168] Take 10 mg of the sample to be tested, disperse it in 10 mL of deionized water, ultrasonically disperse it for 10 minutes, and shake it to obtain a 0.1 wt% sample solution. Dilute it to 0.05 wt%, 0.01 wt%, 0.005 wt%, and 0.001 wt% concentrations.

[0169] 2.2 Preparation of reagents and consumables

[0170] Before starting the experiment, prepare the prepared test solution, 0.1 mol / L HCL solution, 50 mmol / L Tris-HCL solution, 0.3 mmol / L pyrogallol solution, pipette and pipette tip in front of the EP tube rack.

[0171] 3. Experimental operation

[0172] Add 500 μL Tris-HCl buffer and 100 μL pyrogallol solution to a 1.5 mL centrifuge tube, mix and shake, and place in a 37°C water bath for 10 min. Then add 100 μL sample solution, react for 5 min, and terminate the reaction with 100 μL 0.1 mol / L HCL solution. Measure the absorbance of each solution at 320 nm. Note: In order to prevent the CeO2 particles in the solution from affecting the absorbance measurement, A i and A j All solutions were centrifuged at 10,000 rpm for 10 min, and the absorbance of the supernatant was measured. The clearance rate of superoxide anions (R, %) was calculated according to the following formula.

[0173] 4. Result processing

[0174] The calculation formula of superoxide anion free radical scavenging rate is: ,

[0175] Where:

[0176] A i ——Absorbance at 325 nm after the sample solution is mixed with Tris-HCl solution, pyrogallol solution and 0.1 mol / L HCl solution

[0177] A j ——Absorbance at 325 nm after the sample solution is mixed with Tris-HCl solution, deionized water and 0.1 mol / L HCl solution

[0178] A0——Absorbance at 325 nm after mixing deionized water with Tris-HCL solution, pyrogallol solution and 0.1 mol / L HCL solution.

[0179] 5. Results Analysis

[0180] The results of superoxide anion free radical scavenging rate are shown in Tables 4, 5 and Figure 6 shown.

[0181] Table 4

[0182]

[0183] Table 5

[0184]

[0185] In order to intuitively reflect the relationship between Pul-CeO2 NPs and CeO2 NPs, the cerium oxide content in Levan-CeO2 NPs, Pul-CeO2NPs and CeO2 NPs is made the same. As shown in Table 4, the Pul-CeO2NPs prepared in Example 1 to Example 7 have a higher superoxide anion radical scavenging rate than the CeO2 NPs, Pul, and Levan-CeO2 NPs prepared in Comparative Example 1 to Comparative Example 3. Pul has a poorer superoxide anion radical scavenging effect than CeO2 NPs and Pul-CeO2 NPs prepared in Example 1 to Example 7. The superoxide anion radical scavenging rate of Levan-CeO2 NPs is higher than that of CeO2 NPs, but lower than that of Pul-CeO2 NPs prepared in Example 1 to Example 7. The scavenging effect is stronger than that of CeO2 NPs, but weaker than that of Pul-CeO2 NPs. As shown in Tables 5 and Figure 6As shown, the superoxide anion radical scavenging rate of CeO2 NPs, Pul-CeO2 NPs and Levan-CeO2 NPs prepared in Example 1 increases with the increase of concentration, and the growth rate is stable. At the same concentration, the content of cerium oxide in Pul-CeO2 NPs and CeO2 NPs prepared in Example 1 is the same. The superoxide anion radical scavenging rate of Pul-CeO2 NPs prepared in Example 1 is higher than that of CeO2 NPs, while the superoxide anion radical scavenging rate of Pul is significantly lower than that of Pul-CeO2 NPs prepared in Example 1. It can be shown that under the conditions of the example (Pul-CeO2 NPs), Pul modified CeO2 without reducing its antioxidant capacity, Pul improved the superoxide anion radical scavenging effect of CeO2 NPs, improved the superoxide anion radical antioxidant capacity of CeO2 NPs, and improved the biological activity.

[0186] (5) Hydroxyl free radical scavenging effect

[0187] Hydroxyl radical (·OH) is an important reactive oxygen species that can react quickly with other molecules to become a more stable form. This method uses the reaction of FeSO4·7H2O and H2O2 to generate hydroxyl radical (·OH). The reaction of salicylic acid with the hydroxyl radical (·OH) generated by the Fenton reaction will generate 2.3-dihydroxybenzoic acid with a maximum absorption wavelength at 510nm. If a substance with the ability to scavenge hydroxyl radicals is added to the reaction system, the formation of 2,3-dihydroxybenzoic acid can be inhibited. The fixed reaction time method is used to detect the change in absorbance at 510nm using an enzyme marker to detect the antioxidant capacity of the added substance.

[0188] 1. Instruments and Reagents

[0189] Note: ① The brands and models marked in brackets for equipment are those used during laboratory method development, and are not exclusive. ② The brands, specifications, and other information commonly used in laboratories are marked in brackets for reagents and consumables, and are not exclusive.

[0190] (1) Analytical balance (d = 0.1 mg, Sartorius, BSA2202S-CW, Max 220 g);

[0191] (2) Vortex mixer (MX-F);

[0192] (3) Multifunctional microplate reader (Thermo scientific), 96-well plate (Corning);

[0193] (4) Pipette (5 mL, 200 μL, 1000 μL, Eppendorf);

[0194] (5) High-speed centrifuge (Thermo scientific);

[0195] (6) Salicylic acid (MACKLIN);

[0196] (7) Anhydrous ethanol (Tianjin Damao Chemical Reagent Factory);

[0197] (8) Ferrous sulfate (Sinopharm Chemical Reagent Co., Ltd.);

[0198] (9) Hydrogen peroxide (MREDA).

[0199] 2. Experimental Preparation

[0200] 2.1 Reagent preparation

[0201] (1) 9 mmol / L salicylic acid-ethanol solution

[0202] Take 0.124 g of salicylic acid, dissolve it in anhydrous ethanol and make up to 100 mL.

[0203] (2) 9 mmol / L ferrous sulfate solution

[0204] Take 0.2502 g of ferrous sulfate heptahydrate, dissolve it in distilled water and make up to 100 mL.

[0205] (3) 8.8 mmol / L hydrogen peroxide solution

[0206] Take 9.926g of 30% H2O2 solution and add distilled water to make up to 100mL; then take 1mL of the solution after the volume is adjusted to a 100mL volumetric flask and add water to make up to 100mL.

[0207] (4) Sample solution

[0208] Take 200 mg of the sample to be tested, disperse it in 10 mL of deionized water, ultrasonically disperse it for 10 minutes, and shake it well to obtain a 2 wt% sample solution. Dilute it to 1.5 wt%, 1 wt%, and 0.5 wt% concentrations.

[0209] 2.2 Preparation of reagents and consumables

[0210] Before starting the test, prepare the prepared test solution, 9 mmol / L ferrous sulfate solution, 9 mmol / L salicylic acid-ethanol solution, 8.8 mmol / L hydrogen peroxide solution, distilled water, pipette and pipette tip in front of the EP tube rack.

[0211] 3. Experimental operation

[0212] Add different solutions to a 1.5 mL centrifuge tube in the order from top to bottom listed in Table 6.

[0213] Table 6

[0214]

[0215] After adding in order, mix and shake well, heat in a 37℃ water bath for 15 minutes and then take out. Pipette 200μL of each solution and measure the absorbance at 510nm. Note: In order to prevent the CeO2 particles in the solution from affecting the absorbance measurement, A X and A X0 All solutions were centrifuged at 10,000 rpm for 10 min, and the absorbance of the supernatant was measured. The scavenging rate of hydroxyl free radicals (R, %) was calculated according to the following formula.

[0216] 4. Result processing

[0217] The calculation formula of hydroxyl radical scavenging rate is: ,

[0218] Where:

[0219] A0——absorbance value after mixing ferrous sulfate solution, salicylic acid-ethanol solution and H2O2 solution;

[0220] A X ——Absorbance value after the sample solution is mixed with ferrous sulfate solution, salicylic acid-ethanol solution, H2O2 solution and distilled water;

[0221] A X0 ——Absorbance value after the sample solution is mixed with ferrous sulfate solution, salicylic acid-ethanol solution and distilled water.

[0222] 5. Results Analysis

[0223] Table 7 shows the test results of the hydroxyl radical scavenging efficacy.

[0224] Table 7

[0225]

[0226] Table 8

[0227]

[0228] In order to intuitively reflect the relationship between Pul-CeO2 NPs and CeO2 NPs, the cerium oxide content in Levan-CeO2 NPs, Pul-CeO2NPs and CeO2 NPs was made the same. As shown in Table 7, the Pul-CeO2NPs prepared in Example 1 to Example 7 have a higher hydroxyl radical scavenging rate than the CeO2 NPs, Pul, and Levan-CeO2 NPs prepared in Comparative Example 1 to Comparative Example 3. The hydroxyl radical scavenging rate of Pul is low, and the hydroxyl radical scavenging effect is poor, which can be regarded as Pul has no scavenging rate for hydroxyl radicals. The hydroxyl radical scavenging rate of Levan-CeO2 NPs is higher than that of CeO2 NPs, but lower than that of Pul-CeO2 NPs. The scavenging effect is stronger than that of CeO2 NPs, but weaker than that of Pul-CeO2 NPs. As shown in Tables 8 and Figure 7 As shown, the hydroxyl radical scavenging rates of CeO2 NPs, Pul-CeO2 NPs prepared in Example 1 and Levan-CeO2 NPs increase with increasing concentration, and the growth rate is stable. At the same concentration, the content of cerium oxide in Pul-CeO2 NPs and CeO2 NPs prepared in Example 1 is the same. The hydroxyl radical scavenging rate of Pul-CeO2 NPs prepared in Example 1 is higher than that of CeO2 NPs, while the hydroxyl radical scavenging rate of Pul is significantly lower than that of CeO2 NPs. It can be shown that under the conditions of Example (Pul-CeO2 NPs), Pul modifies CeO2 without reducing its antioxidant capacity. Pul improves the hydroxyl radical scavenging effect of CeO2 NPs, improves the hydroxyl radical antioxidant capacity of CeO2 NPs, and improves biocompatibility.

[0229] (6) Antibacterial activity test

[0230] Adsorption, redox, and toxicity are considered as three possible interactions between CeO2 NPs and bacteria. The first mechanism involves CeO2 NPs, which carry a positive charge, attaching to the negatively charged bacterial cell wall through electrostatic interactions. This attachment may block the membrane and become trapped, compromising the viscosity of the cell wall and disrupting transport exchanges between the solution and the bacterial cell. Redox is the second interaction and occurs close to the bacterial wall. High cytotoxicity is caused by redox, which is related to oxidative stress on the bacteria during nanoparticle adsorption. The key to understanding how cerium oxide nanoparticles affect bacterial cell survival is oxidative stress. The fact that toxicity persists after 1 or 5 hours of contact suggests that there is a rapid mechanism for transmembrane adsorption of nanoparticles that alters the bacterial membrane and disrupts specific ion pumps, which significantly changes the cell-solution interaction and reduces cell viability. The third mechanism involves the generation of reactive oxygen species (ROS), which are produced due to the Ce 3+ and Ce4+ ROS are known to attack nucleic acids, proteins, and polysaccharides, leading to loss of function and ultimately to the destruction and decomposition of bacteria.

[0231] 1. Instruments and Reagents

[0232] Note: The test site, test instruments, bacterial strains and culture media used in this experiment were all provided by the Beijing Beinong Testing Center of Beijing Beinong Technology Co., Ltd.

[0233] (1) Bacterial strains: Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538) were purchased from China Industrial Microbiological Culture Collection Center;

[0234] (2) Culture medium: Broth agar was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0235] (3) Instruments: clean bench, analytical balance, 50 μL pipette and matching tips, coating rod, centrifuge tube, test tube (with matching rubber stopper), culture dish, alcohol lamp, 1 L conical flask, biochemical incubator, shaker, tweezers, medicine spoon, hole puncher (8 mm in diameter);

[0236] (4) Sterile water.

[0237] 2. Experimental Preparation

[0238] 2.1 Sterilization

[0239] Place a 1 L conical flask, sterile water, matching gun tip, coating rod, and centrifuge tube in a high pressure sterilizer for sterilization at 120°C for 25 min. Wrap the culture dish with kraft paper and place it in a blast drying oven with the test tube for sterilization at 170°C for 2h. Before the experiment, the clean bench and operating room must be sterilized with ultraviolet light for more than 30 min before use.

[0240] 2.2 Preparation of culture medium

[0241] Solid culture medium: Weigh 30 g of broth agar and dissolve it in 750 mL of distilled water. Pour the solid culture medium into the culture dish while it is still hot. Shake the culture medium well before use. Pour about 20 mL into each culture dish. After pouring, let it stand horizontally until it is completely solidified. Thus, 12 culture dishes containing broth agar solid culture medium are prepared.

[0242] Liquid culture medium: Weigh 0.45 g of broth and dissolve it in 25 mL of sterile water to prepare two bottles of bacterial culture medium.

[0243] 2.3 Sample solution

[0244] Take 1 g of the sample to be tested (Example 1, Comparative Examples 1 to 3), disperse it in 10 mL of deionized water, ultrasonically disperse it for 10 minutes, shake it well, and get a 100 mg / mL sample solution. Dilute it to 80 mg / mL, 60 mg / mL, and 40 mg / mL concentrations.

[0245] 3. Experimental operation

[0246] Notes: ① The bacteria used in this test are Escherichia coli and Staphylococcus aureus, and broth agar is used for culture. ② Three parallel control groups are set up for each sample test. ③ Sterile gloves should be worn during the test, and the experimental instruments should be sterilized on an alcohol lamp before and after use.

[0247] 3.1 Inoculation of test bacteria

[0248] Take the strain out of the -80 ℃ freezer and wait for it to thaw at room temperature. Use a pipette to transfer 25 μL of the strain into the corresponding liquid culture medium, and place it in a shaker at 37 ℃ and 120 rpm for 12 h. Take 50 μL of the corresponding bacterial solution and evenly apply it to the corresponding solid culture medium.

[0249] 3.2 Adding cerium oxide

[0250] Each inoculated culture dish was divided into four areas of equal area, a hole was punched in the center of each area with a hole puncher, and 30 μL of sample was taken into the agar well with a pipette.

[0251] 4. Result processing

[0252] Place the treated culture dish in a biochemical incubator for culturing at 37 °C for 24 h. Finally, observe the inhibition and measure the average diameter of the inhibition zone with a vernier caliper and record it. Note: When measuring the inhibition zone, a uniform and completely sterile inhibition zone should be selected. The diameter should be measured with the outer edge of the inhibition zone as the boundary.

[0253] 5. Results Analysis

[0254] Table 9, Table 10 and Figure 8 These are the results of Pul, CeO2 NPs, Pul-CeO2 NPs of Example 1, and Levan-CeO2 NPs inhibiting Escherichia coli and Staphylococcus aureus.

[0255] Table 9 Diameters of inhibition zones of Pul, CeO2 NPs, Pul-CeO2 NPs of Example 1, and Levan-CeO2 NPs against Escherichia coli

[0256]

[0257] Table 10 Diameters of inhibition zones of Pul, CeO2 NPs, Pul-CeO2 NPs of Example 1, and Levan-CeO2 NPs against Staphylococcus aureus

[0258]

[0259] Table 9, Table 10 and Figure 8 All of them can intuitively reflect the antibacterial ability of Pul (Comparative Example 2), CeO2 NPs (Comparative Example 1), Levan-CeO2 NPs (Comparative Example 3) and Pul-CeO2 NPs (Example 1). Pure Pul has no effective inhibitory ability against Escherichia coli (E.coli) and Staphylococcus aureus (S. aureus). When the concentration of CeO2 added is 40 mg / mL, Escherichia coli and Staphylococcus aureus have the ability to inhibit these bacteria, that is, an inhibition zone similar to the inhibition zone can be observed, but due to the influence of insufficient antibacterial ability, there are still sporadic bacterial colonies in the inhibition zone. When the concentration of CeO2 added reaches 60 mg / mL or above, CeO2, Levan-CeO2 and Pul-CeO2 of Example 1 have a relatively strong inhibitory effect on these bacteria, that is, a very obvious inhibition zone can be observed, and the antibacterial ability gradually increases with the increase of CeO2 content, and the inhibition zone also increases and becomes more obvious. This experiment shows that when the CeO2 content reaches a certain level, it has strong antibacterial properties against both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), and the inhibition ability against Escherichia coli is stronger than that against Staphylococcus aureus. It is also obvious that the inhibition zone of Pul-CeO2 NPs is larger than that of Levan-CeO2 and CeO2 NPs, and the antibacterial effect is stronger.

[0260] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0261] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A method for preparing a pullulan-modified cerium oxide nanocomposite material, characterized in that: The method comprises the following steps: mixing cerium salt and pullulan in a solvent, reacting and preparing a pullulan-modified cerium oxide nanocomposite material.

2. The preparation method according to claim 1, characterized in that: Meet one or more of the following characteristics: (1) In the step of mixing the cerium salt and pullulan in a solvent, the pH value of the obtained solution is 3.5-6; (2) In the step of mixing the cerium salt and pullulan in a solvent, the final concentration of pullulan is 1% to 10% in g / mL; (3) In the step of mixing the cerium salt and pullulan in a solvent, the mass ratio of pullulan to cerium salt is (6.8-68):(20-120) based on the amount of feed.

3. The preparation method according to claim 1 or 2, characterized in that: Meet one or more of the following characteristics: (1) In the step of mixing the cerium salt and pullulan in a solvent, the pH value of the obtained solution is 5 to 5.5; (2) In the step of mixing the cerium salt and pullulan in a solvent, the final concentration of pullulan is 1% to 5% in g / mL; (3) In the step of mixing the cerium salt and pullulan in a solvent, the mass ratio of pullulan to cerium salt is (6.8-34):(30-40) based on the amount of feed.

4. The preparation method according to any one of claims 1 to 3, characterized in that Meet one or more of the following characteristics: (1) The solvent comprises a first solvent and a second solvent, the first solvent is used to dissolve the cerium salt and the pH adjuster, the second solvent is used to dissolve pullulan, the first solvent is water, the second solvent is an alcohol solvent, and the pH adjuster is acetic acid; based on the amount of feed, the volume mass ratio of the first solvent to the cerium salt is (0.08-3) mL:1 g, and the volume ratio of the pH adjuster to the first solvent is (0.3-12):1; (2) The cerium salt is cerium nitrate or cerium nitrate hydrate. Optionally, the cerium nitrate hydrate comprises cerium nitrate hexahydrate.

5. The preparation method according to any one of claims 1 to 4, characterized in that Meet one or more of the following characteristics: (1) In the step of mixing the cerium salt and pullulan in the solvent, the stirring speed is 350 rpm to 450 rpm; (2) In the step of mixing the cerium salt and pullulan in a solvent to carry out a reaction, the reaction temperature is 150° C. to 170° C.; (3) In the step of mixing the cerium salt and pullulan in a solvent to carry out a reaction, the reaction time is 140 min to 180 min; (4) In the step of mixing the cerium salt and pullulan in a solvent to carry out a reaction, the reaction is carried out under a pressure of 1 to 3 MPa.

6. The preparation method according to any one of claims 1 to 5, characterized in that After the reaction is finished, the following steps are further included: solid-liquid separation, solid phase collection, water washing, drying, and grinding.

7. A pullulan-modified cerium oxide nanocomposite material, characterized in that: The invention comprises cerium oxide and pullulan modified on the cerium oxide.

8. The pullulan-modified cerium oxide nanocomposite material according to claim 7, characterized in that: The pullulan-modified cerium oxide nanocomposite material satisfies at least one of the following conditions: (1) The particle size of the pullulan-modified cerium oxide nanocomposite material is 100 nm to 600 nm; (2) The pullulan modified cerium oxide nanocomposite material is prepared according to the method for preparing the pullulan modified cerium oxide nanocomposite material according to any one of claims 1 to 6.

9. The pullulan-modified cerium oxide nanocomposite material according to claim 7 or 8, characterized in that: The particle size of the pullulan-modified cerium oxide nanocomposite material is 100 nm to 250 nm.

10. Use of the pullulan-modified cerium oxide nanocomposite material according to any one of claims 7 to 9 in one or more of the following: (1) Application in at least one of free radical elimination, microbial inhibition, bioimaging and biotransformation, wherein: The free radicals include one or more of superoxide anion free radicals, hydroxyl free radicals, hydrogen peroxide, and nitrogen free radicals, and the microorganism is at least one of Gram-negative bacteria and Gram-positive bacteria; (2) Use in the preparation of at least one of skin products, medicines, catalysts, sensors and solid oxide fuel cells; Optionally, the application is not a method for diagnosing or treating a disease.

11. A skin product, medicine, catalyst, sensor or solid oxide fuel cell, characterized in that: A pullulan-modified cerium oxide nanocomposite material comprising the pullulan-modified cerium oxide nanocomposite material according to any one of claims 7 to 9.

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