Preparation method and application of walnut shell derived carbon dot antibacterial agent

Abstract

The present invention relates to a preparation method and application of a walnut shell-derived carbon dot antibacterial agent, and the preparation method of the carbon dot antibacterial agent comprises the following steps: 1) selecting walnut shell as a raw material; 2) crushing the walnut shell and dissolving it in water to form a solution A; 3) placing the solution A in a reactor for heating reaction; 4) filtering the product after the reaction in step 3), collecting the filtrate as a solution B, and the solution B is the walnut shell-derived carbon dot antibacterial agent. The beneficial effects of the present invention are as follows: 1) the present invention prepares a walnut shell-derived carbon dot antibacterial agent, which has low raw material cost, simple preparation method and excellent antibacterial effect. 2) the walnut shell-derived carbon dot antibacterial agent prepared by the method of the present invention has a broad-spectrum antibacterial effect, and has a very significant effect on Escherichia coli. 3) the walnut shell-derived carbon dot antibacterial agent prepared by the present invention has strong compatibility and can be used as a sewage treatment agent and an antibacterial additive to be added to various carriers that need antibacterial.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation method and application of antibacterial agents, and in particular to the technical field of preparation method of a carbon dot antibacterial agent. Background Art

[0002] The invention belongs to the technical field of antibacterial material preparation, and specifically relates to a method for preparing a walnut shell-derived carbon dot antibacterial agent.

[0003] Yunnan Province is the largest walnut producing province in China, but the development and utilization of walnut shells may need to be further improved. For local fruit farmers, walnut shells are usually backfilled into orchard soil to serve as natural fertilizers. For current development and research, it is mainly to use its physical properties to become an effective substitute for traditional abrasives (such as sand, glass beads or plastic particles) for cleaning and surface treatment, or to use walnut shell powder as a filling material for plastics, or to make natural anti-slip materials, or to make it into adsorbents, water purifiers, etc. There are also ways to extract the fragrance substances in the shells for the production of perfumes and cosmetics, and to use the shells to prepare biomass charcoal, ceramics, etc., or to use the shells to prepare some simple handicrafts, decorations, etc. But these are all in the minority, and there is no large-scale industrialization. In addition, there are very few reports on the preparation of carbon point antibacterial agents using walnut shells as the main raw material, and there are almost no related papers and patents. There are related patents that mention the use of walnut shells, but they only use the shells as a very small component and do not use them as the main body. For example, patents such as "A hard carbon material, its preparation method and application, sodium ion battery (Invention Publication 202311579178.1)"; "A Gastrodia elata cultivation matrix, its preparation method and application (Invention Publication 202311579512.3)"; "A fruit shell activated carbon, its preparation method and application (Invention Publication 2311266764.0)" show that there is still a large gap in the deep processing, development and utilization of walnut shells.

[0004] Therefore, in order to break through the deep processing technology of walnut shell byproducts, walnut shells are prepared into biomass-based antibacterial agents, which are green, safe, non-toxic and harmless. Based on this, through preliminary laboratory exploration and research into antibacterial materials, further industrial production into related antibacterial products. Turning waste into treasure, improving the comprehensive utilization rate of walnut resources, increasing the added value of walnuts, further broadening the walnut industry chain, and realizing the green and sustainable development of the walnut industry. In view of the low antibacterial activity of activated carbon, on the basis of the preliminary preparation of activated carbon inorganic composite antibacterial materials using walnut shells as raw materials, we have innovated new preparation technologies and obtained new antibacterial products. Moreover, the operation is simple and fast, and a new walnut shell-derived carbon point antibacterial agent with excellent antibacterial effect has been explored and prepared.

[0005] The simple activated carbon obtained by calcining walnut shells has a low antibacterial effect. It needs to add inorganic metal ions with good antibacterial effect to prepare activated carbon inorganic composite antibacterial materials to achieve excellent antibacterial effect. The preparation process is relatively complicated, and the preparation process conditions are relatively high. After adding chemical substances, it is not so pure, environmentally friendly, and green.

[0006] Therefore, the preparation of carbon dot antibacterial agents using walnut shells as the main raw material, that is, the antibacterial effect of simple carbon dot antibacterial agents can also achieve a very significant level, has become a technical problem that needs to be solved. Summary of the invention

[0007] In order to solve the above problems and defects, the present invention provides a preparation method and application of a walnut shell-derived carbon dot antibacterial agent. The present invention is a biomass-based derived carbon dot, which has the advantages of good biological safety, green, safe and efficient, simple preparation process, and simple and fast operation.

[0008] The present invention is implemented by the following technical solution.

[0009] A method for preparing a walnut shell-derived carbon dot antibacterial agent, the method comprising the following steps:

[0010] Step 1) Select walnut shell as raw material;

[0011] Step 2) crushing the walnut shells and dissolving them in water to form solution A;

[0012] Step 3) placing solution A in a reactor for heating reaction;

[0013] Step 4) filtering the product after the reaction in step 3) and collecting the filtrate as solution B, wherein the solution B is the walnut shell-derived carbon dot antibacterial agent.

[0014] Furthermore, step 2) in the preparation method of the present invention is specifically to grind and filter the walnut shells, the sieve used for filtering is 200-500 mesh, and the filtered brown powder is dispersed in a container filled with distilled water and stirred to form solution A; the material ratio of the powder after the walnut shells are crushed and filtered to distilled water is 0.1-5g: 5-100 ml.

[0015] Furthermore, in step 2 of the preparation method of the present invention, the sieve used for filtering the walnut shells after crushing is 200 mesh, and the ratio of the filtered powder to distilled water is 2g:60ml.

[0016] Furthermore, in step 3) of the preparation method of the present invention, the reaction in the reactor is: a hydrothermal reaction is carried out at 100-500° C. for 6-24 hours, and then naturally cooled to room temperature.

[0017] Furthermore, in step 3 of the preparation method of the present invention, the reaction in the reactor is: hydrothermal reaction is carried out at 180° C. for 12 hours, and then naturally cooled to room temperature.

[0018] Furthermore, in the preparation method of the present invention, a stirring step is provided between step 2) and step 3), and the stirring step is that solution A is placed on a magnetic stirrer and stirred at room temperature.

[0019] Furthermore, the filtration in step 4) of the preparation method of the present invention is filtering through a 0.1-1 μm water syringe filter.

[0020] Furthermore, the filtration in step 4) of the preparation method of the present invention is filtering through a 0.1 water system syringe filter.

[0021] As a product, the present invention protects the antibacterial agent obtained by the above preparation method.

[0022] As an application, the present invention protects the use of the antibacterial agent as a bactericidal additive or a sewage treatment agent.

[0023] Furthermore, the application of the present invention is to prepare an anti-Escherichia coli bacterial agent or a pharmaceutical agent.

[0024] The beneficial effects of the present invention are:

[0025] 1) The present invention adopts simple methods such as stirring method, hydrothermal reaction method and separation and impurity removal method to prepare walnut shell-derived carbon dot antibacterial agent, which has low raw material cost, simple preparation method and excellent antibacterial effect.

[0026] 2) The walnut shell-derived carbon dot antibacterial agent prepared by the method of the present invention has a broad-spectrum antibacterial effect, and is very effective against Escherichia coli.

[0027] 3) The walnut shell-derived carbon dot antibacterial agent prepared by the present invention has strong compatibility and can be used as a sewage treatment agent and an antibacterial additive and added to various carriers that require antibacterial properties.

[0028] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a real photo of the walnut shell-derived carbon dot antibacterial agent of the present invention.

[0030] Figure 2 This is a morphology diagram of the walnut shell-derived carbon dot antibacterial agent of the present invention.

[0031] Figure 3 This is an experimental diagram of the present invention on Gram-negative bacteria Escherichia coli. Figure 3Wherein A is blank, B is a walnut shell derived carbon dot antibacterial agent prepared at a ratio of 2g / 60ml (magnetic stirring for 5min, other conditions remain unchanged), C is a walnut shell derived carbon dot antibacterial agent prepared at a ratio of 2g / 60ml (reacted in a 100ml reactor for 20 hours, other reaction conditions remain unchanged), D is a walnut shell derived carbon dot antibacterial agent prepared at a ratio of 2g / 60ml (reacted in a 100ml reactor for 4 hours, other reaction conditions remain unchanged), and E is a walnut shell derived carbon dot antibacterial agent prepared at a ratio of 2g / 30ml (reacted in a 50ml reactor for 30 hours, other reaction conditions remain unchanged).

[0032] Figure 4 The graph shows the antibacterial test of E. coli with different amounts of carbon dot antibacterial agent (preparation ratio is 2g / 60ml). Figure 4 In the figure, A is blank, B is the antibacterial effect diagram of the antibacterial experiment with a dosage of 50 μL, C is the antibacterial effect diagram of the antibacterial experiment with a dosage of 100 μL, D is the antibacterial effect diagram of the antibacterial experiment with a dosage of 150 μL, E is the antibacterial effect diagram of the antibacterial experiment with a dosage of 200 μL, F is the antibacterial effect diagram of the antibacterial experiment with a dosage of 250 μL, G is the antibacterial effect diagram of the antibacterial experiment with a dosage of 300 μL, H is the antibacterial effect diagram of the antibacterial experiment with a dosage of 350 μL, I is the antibacterial effect diagram of the antibacterial experiment with a dosage of 400 μL, J is the antibacterial effect diagram of the antibacterial experiment with a dosage of 450 μL, and K is the antibacterial effect diagram of the antibacterial experiment with a dosage of 500 μL.

[0033] Figure 5 The bar graph shows the E. coli inhibition rate of different amounts of carbon dot antibacterial agent (preparation ratio is 2g / 60ml). Figure 5 The dosage of B for antibacterial experiment is 50μL, the dosage of C for antibacterial experiment is 100μL, the dosage of D for antibacterial experiment is 150μL, the dosage of E for antibacterial experiment is 200μL, the dosage of F for antibacterial experiment is 250μL, the dosage of G for antibacterial experiment is 300μL, the dosage of H for antibacterial experiment is 350μL, the dosage of I for antibacterial experiment is 400μL, the dosage of J for antibacterial experiment is 450μL, and the dosage of K for antibacterial experiment is 500μL.

[0034] Figure 6 Photos of the E. coli antibacterial experiment of carbon dot antibacterial agents prepared with different raw material ratios (the dosage for the antibacterial experiment is 500µL). Figure 6 A is blank, B is the antibacterial effect diagram with a preparation ratio of 2g / 60ml, C is the antibacterial effect diagram with a preparation ratio of 1g / 60ml, D is the antibacterial effect diagram with a preparation ratio of 0.5g / 60ml, E is the antibacterial effect diagram with a preparation ratio of 2g / 30ml, and F is the antibacterial effect diagram with a preparation ratio of 1g / 30ml.

[0035] Figure 7 This is a bar graph showing the E. coli inhibition rate of carbon dot antibacterial agents prepared with different raw material ratios (the dosage for the antibacterial experiment is 500µL). Figure 7 The preparation ratio of B is 2g / 60ml, the preparation ratio of C is 1g / 60ml, the preparation ratio of D is 0.5g / 60ml, the preparation ratio of E is 2g / 30ml, and the preparation ratio of F is 1g / 30ml.

[0036] Figure 8 Photos of the E. coli antibacterial experiment of preparing carbon dot antibacterial agents with different raw materials (the preparation ratio is 2g / 60ml, and the amount used in the antibacterial experiment is 500µL). Figure 8 A is blank, B is the antibacterial effect diagram of the carbon dot antibacterial agent prepared from melon seed shells, C is the antibacterial effect diagram of the carbon dot antibacterial agent prepared from peanut shells, D is the antibacterial effect diagram of the carbon dot antibacterial agent prepared from almond shells, and E is the antibacterial effect diagram of the carbon dot antibacterial agent prepared from walnut shells.

[0037] Fig. 9 A bar graph showing the E. coli inhibition rate of carbon dot antibacterial agents prepared from different raw materials (the preparation ratio is 2g / 60ml, and the amount used in the antibacterial experiment is 500µL). Fig. 9 B is the carbon dots prepared from melon seed shells, C is the carbon dots prepared from peanut shells, D is the carbon dots prepared from almond shells, and E is the carbon dots prepared from walnut shells.

[0038] Fig.10 The antibacterial experiment diagram of sewage treatment with different amounts of carbon dot antibacterial agent (preparation ratio is 2g / 60ml). Fig.10 A is blank, B is the antibacterial effect diagram of the antibacterial experiment with a dosage of 500 μL, C is the antibacterial effect diagram of the antibacterial experiment with a dosage of 1000 μL, D is the antibacterial effect diagram of the antibacterial experiment with a dosage of 1500 μL, and E is the antibacterial effect diagram of the antibacterial experiment with a dosage of 2000 μL.

[0039] Fig.11 Actual photos of fabrics, sponges and foams with added carbon dot antimicrobial agents.

[0040] Fig.12 These are photos of antibacterial experiments on fabrics, sponges and foams with added carbon dot antibacterial agent (preparation ratio is 2g / 60ml). DETAILED DESCRIPTION

[0041] The following examples are only a part of the technical solution of the present invention, and are not intended to limit the entire technical solution of the present invention. The examples of the present invention are provided to further explain and illustrate the details of the technical solution of the present invention.

[0042] Example

[0043] The present invention uses transmission electron microscopy (TEM) to study the morphology of carbon dot antibacterial agents (see Figure 2 ). Figure 2Transmission electron microscopy image of carbon dot antibacterial agent (a), high-resolution transmission electron microscopy image of carbon dot antibacterial agent (b), and particle size distribution of carbon dot antibacterial agent (c). Figure 2 In a, the carbon dot antibacterial agent presents regular spherical particles. The high-resolution TEM (HRTEM) image shows that the carbon dot antibacterial agent has a good lattice spacing of 0.21 nm ( Figure 2 b), corresponding to the (100) plane of graphite carbon. Planar particle size distribution Figure 2 c shows that the particles are spherical with an average diameter of about 1.7 nm and a particle size range of 1-3 nm.

[0044] Example

[0045] A method for preparing a walnut shell carbon dot antibacterial agent comprises the following steps:

[0046] 1) Put the walnut shells into a grinder for crushing, and filter the crushed powder through a 200-mesh filter.

[0047] 2) Weigh 2.0 g of brown walnut shell powder and dissolve it in a small beaker containing 60 ml of distilled water (Solution A).

[0048] 3) Place solution A on a magnetic stirrer and stir at room temperature for 20 min.

[0049] 4) The stirred solution A was transferred into a 100 ml polytetrafluoroethylene-lined autoclave, subjected to a hydrothermal reaction at 180°C for 12 hours, and then naturally cooled to room temperature.

[0050] 5) Filter the large particles through a 0.1-m water syringe filter and collect the product sample solution B, which is the carbon dot antibacterial agent. Store solution B in a refrigerator at 4°C for later use.

[0051] 6) Antibacterial: Escherichia coli was used as the experimental bacteria, and the antibacterial properties of the material were examined by the coating plate method.

[0052] The experiment selected Gram-negative bacteria Escherichia coli (E. coli, CCTCC 204033) as the experimental strain for antibacterial detection.

[0053] First, prepare LB agar medium (5 g tryptone, 2.5 g yeast powder, 5 g sodium chloride, 500 mL distilled water, 7.5 g agar), pour it into a plate for later use; then prepare LB liquid medium (5 g tryptone, 2.5 g yeast powder, 5 g sodium chloride, 500 mL distilled water), add the strain to an appropriate amount of sterile liquid medium, cultivate it into a bacterial suspension, and dilute the bacterial suspension to 5×10 6CFU / mL. Mix sample solution B with diluted bacterial solution and place in a sterile test tube to prepare a suspension. Place all test tubes on a shaker at 37°C and 200rpm for 15 min to make them evenly distributed, then take them out, draw 100μL of the upper layer of liquid and drop it into the culture dish, and use the spread plate method to evenly spread the bacterial solution on the culture medium. Place the culture dish in a constant temperature incubator and culture for 18 to 24 hours.

[0054] After cultivation, the antibacterial rate of each sample was calculated using the colony counting method. The calculation formula is as follows:

[0055]

[0056] Within the above range, if Figure 3 As shown in the figure, for the Gram-negative bacteria Escherichia coli (E.coli, CCTCC204033), B: the preparation ratio is 2g / 60ml, but in step S3, the magnetic stirring is performed at room temperature for 30min, and other conditions remain unchanged; C: the preparation ratio is 2g / 60ml, but in step S4, the reaction is performed in a 100ml reactor for 20 hours, and other reaction conditions remain unchanged. The antibacterial effects of B and C are very significant.

[0057] D outside the above range: the preparation ratio is 2g / 60ml, but in step S4, the reaction is carried out in a 150ml reactor for 4 hours, and other reaction conditions remain unchanged; E: the preparation ratio is 2g / 30ml, but in step S4, the reaction is carried out in a 50ml reactor for 30 hours, and other reaction conditions remain unchanged. The antibacterial effects of C and D are very unsatisfactory.

[0058] It can be seen that the antibacterial effect of the carbon dot antibacterial agent prepared within this range is relatively excellent.

[0059] Figure 3 Middle A: blank;

[0060] B: Preparation ratio 2g / 60ml (magnetic stirring for 5min, other conditions remain unchanged);

[0061] C: Preparation ratio 2g / 60ml (100ml reactor for 20 hours, other reaction conditions remain unchanged);

[0062] D: Preparation ratio 2g / 60ml (150ml reactor for 4 hours, other reaction conditions remain unchanged);

[0063] E: Preparation ratio 2g / 30ml (50ml reactor for 30 hours, other reaction conditions remain unchanged); E. coli antibacterial photos and antibacterial rate bar graph.

[0064] Example

[0065] See Figure 4 Photos of the E. coli antibacterial experiment with different amounts of carbon dot antibacterial agent (preparation ratio is 2g / 60ml). Figure 5 Photographs of the antibacterial rate bar graph of the E. coli antibacterial experiment with different amounts of carbon dot antibacterial agent (preparation ratio is 2g / 60ml).

[0066] from Figure 4 , 5 It can be seen that the carbon dot antibacterial agent has an excellent antibacterial effect on Escherichia coli. As its dosage continues to increase, when the dosage reaches 450µL, its antibacterial rate reaches more than 90%.

[0067] Figure 4 , 5 A: Blank; B: 50 μL; C: 100 μL; D: 150 μL; E: 200 μL; F: 250 μL; G: 300 μL; H: 350 μL; I: 400 μL; J: 450 μL; K: 500 μL.

[0068] Example

[0069] See Figure 6 Photos of the E. coli antibacterial experiment of carbon dot antibacterial agents prepared with different raw material ratios (the dosage for the antibacterial experiment is 500µL). Figure 7 Bar graph of E. coli inhibition rate of carbon dot antibacterial agents prepared with different raw material ratios (the dosage for antibacterial experiment is 500µL).

[0070] At the same time, compared with the carbon dot antibacterial agents prepared with different raw material ratios, Figure 6 , 7 The antibacterial effect of carbon dot antibacterial agents prepared with different raw material ratios on Escherichia coli is relatively excellent. When the antibacterial experiment dosage is 500µL, the antibacterial effect of carbon dot antibacterial agents prepared with a raw material ratio of 2g / 60ml is the best.

[0071] Figure 6 , 7 In the table, A: blank; B: 2g / 60ml; C: 1g / 60ml; D: 0.5g / 60ml; E: 2g / 30ml; F: 1g / 30ml.

[0072] Example

[0073] See Figure 8 Photos of the E. coli antibacterial experiment of carbon dot antibacterial agents prepared from different raw materials (preparation ratio is 2g / 60ml, and the amount used in the antibacterial experiment is 500µL). Fig. 9 Bar graph of E. coli inhibition rate of carbon dot antibacterial agents prepared from different raw materials (preparation ratio is 2g / 60ml, and the dosage for antibacterial experiment is 500µL).

[0074] from Figure 8 , 9 It can be seen that for the carbon dot antibacterial agents prepared from different raw materials, when using the same antibacterial test volume of 500µL, the carbon dot antibacterial agents prepared from melon seed shells, peanut shells, and almond shells have poor antibacterial effects on Escherichia coli. The carbon dot antibacterial agent prepared from walnut shells with a raw material preparation ratio of 2g / 60ml has the best antibacterial effect.

[0075] Figure 8 , 9 In the figure, A: blank; B: melon seed shell-derived carbon dots; C: peanut shell-derived carbon dots; D: almond shell-derived carbon dots; E: walnut shell-derived carbon dots.

[0076] Example

[0077] See Fig.10 Antibacterial experimental photos of sewage treatment in Dianchi Lake, Yunnan with different amounts of carbon dot antibacterial agents (preparation ratio is 2g / 60ml).

[0078] from Fig.10 It can be seen that the walnut shell-derived carbon dot antibacterial agent exhibits excellent broad-spectrum antibacterial effect in sewage treatment. It has excellent antibacterial effect when the dosage is 1000μL.

[0079] Fig.10 In the figure, A: blank; B: 500 μL; C: 1000 μL; D: 1500 μL; E: 2000 μL.

[0080] Example

[0081] See Fig.12 , photos of antibacterial experiments with carbon dot antibacterial agents added to fabrics, sponges and foams (preparation ratio is 2g / 60ml).

[0082] In such Fig.11 The fabrics, sponges and foams shown were immersed in the carbon dot antibacterial agent for 24 h and used in the antibacterial experiment. Fig.12 It can be seen that the fabrics and sponges with added carbon dots show excellent antibacterial properties, and the foams with added carbon dots can also achieve an antibacterial effect of 70%.

[0083] The above are only some specific embodiments of the present invention (since the present invention includes a numerical range, the embodiments cannot be exhaustive, and the protection scope recorded in the present invention includes the numerical range of the present invention and other technical key points). The specific content or common sense known in the scheme is not described in detail here (including but not limited to abbreviations, abbreviations, and units commonly used in the art). It should be pointed out that the above embodiments do not limit the present invention in any way. For those skilled in the art, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention. The scope of protection claimed in this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a walnut shell-derived carbon dot antibacterial agent, characterized in that: The preparation method of the carbon dot antibacterial agent consists of the following steps: Step 1) Select walnut shell as raw material; Step 2) After the walnut shells are crushed and filtered, the filtered brown powder is dispersed in a container filled with distilled water and stirred to dissolve in the water to form a solution A; Step 3) placing solution A in a reactor for heating reaction, specifically placing solution A in a reactor for heating reaction; the reaction in the reactor is: performing a hydrothermal reaction at 100-500° C. for 6-24 hours, and then naturally cooling to room temperature; Step 4) filtering the product after the reaction in step 3) and collecting the filtrate as solution B, wherein the solution B is the walnut shell-derived carbon dot antibacterial agent; The filtration in step 4) of the preparation method is filtering through a 0.1 μm water system syringe filter; In step 2 of the preparation method, the sieve used for filtering the walnut shells after crushing is 200 mesh, and the ratio of the filtered powder to distilled water is 2g:60ml.

2. The method for preparing a walnut shell-derived carbon dot antibacterial agent according to claim 1, characterized in that: In step 3 of the preparation method, the reaction in the reactor is: hydrothermal reaction is carried out at 180° C. for 12 hours, and then naturally cooled to room temperature.

3. The method for preparing a walnut shell-derived carbon dot antibacterial agent according to claim 1, characterized in that: In the preparation method, a stirring step is provided between step 2) and step 3), wherein the stirring step is that solution A is placed on a magnetic stirrer and stirred at room temperature.

4. An antibacterial agent obtained by the preparation method of a walnut shell-derived carbon dot antibacterial agent as described in any one of claims 1 to 3.

5. Use of the antibacterial agent as claimed in claim 4 as a bactericidal additive or a sewage treatment agent.

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