Amino acid functionalized boron nitride nanosheet essential oil carrier as well as preparation method and application thereof

Abstract

The invention belongs to the field of nano pesticide preparations, and discloses an amino acid functionalized boron nitride nanosheet essential oil carrier as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) mixing hexagonal boron nitride and sulfur-containing amino acid, and carrying out ball milling to obtain a modified boron nitride nanosheet; (2) adsorbing essential oil by the modified boron nitride nanosheet to obtain an amino acid functionalized boron nitride nanosheet essential oil carrier; the nanosheet essential oil carrier disclosed by the invention has good adhesiveness, and the slow-release control capability on the essential oil is remarkably improved; through synergistic combination of the modified boron nitride nanosheets and the essential oil, the antibacterial activity of the essential oil is remarkably improved, and the boron nitride nanosheets can be used as an environment-friendly and stable essential oil carrier for preventing and treating plant diseases.

Description

Technical Field

[0001] The invention relates to the field of nano pesticide preparations, and in particular to an amino acid functionalized boron nitride nanosheet essential oil carrier and a preparation method and application thereof. Background Art

[0002] Bacterial wilt is a worldwide, devastating disease of Solanaceae crops (tomatoes, potatoes, peppers, ginger, eggplant, etc.) and tobacco crops caused by the soil-borne plant pathogen Ralstonia solanacearum. Ralstonia solanacearum infects the root system of crops, colonizes the vascular system and blocks the xylem tissue, causing the crops to wilt and die within 3-4 days. However, the chemical fungicides used in the prior art have problems such as causing crop resistance, cumulative toxicity and environmental pollution.

[0003] Pure natural plant essential oils extracted from plants, such as tea tree essential oil (TTO), cinnamon essential oil, oregano essential oil, thyme essential oil, lemon essential oil, citrus essential oil, garlic essential oil, etc., have broad-spectrum antibacterial activity and antioxidant effects. Although pure natural plant essential oils are safe, natural and effective agents, their use in the direct prevention and treatment of bacterial wilt is still limited by the following aspects: (1) Essential oils are easily affected by the environment, have poor stability, and are easily volatile under the influence of light, air and temperature conditions; (2) Essential oils have strong fluidity and insufficient fixation, and cannot stay at the roots of plants to form a barrier. When bacterial wilt infects the root system, it is difficult to accurately apply and control the release rate; (3) Essential oils are highly hydrophobic and insoluble in water, making them unsuitable for single application.

[0004] Hexagonal boron nitride (h-BN) is a two-dimensional material with a hexagonal honeycomb structure composed of alternating B and N atoms. Compared with graphene and other carbon-based two-dimensional materials, h-BN has more stable chemical properties and excellent biocompatibility. However, due to its two-dimensional hydrophobic surface, h-BN is easy to aggregate and has poor dispersibility. It only relies on π-π and hydrophobic effects, and its ability to precisely control and intelligently release essential oils is limited, which limits its application in the agricultural field. Therefore, it is urgent to further explore simple and scalable methods to achieve the modification of boron nitride two-dimensional materials in order to obtain ideal physical and chemical properties. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an amino acid functionalized boron nitride nanosheet essential oil carrier and a preparation method and application thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing an amino acid functionalized boron nitride nanosheet essential oil carrier, comprising the following steps:

[0008] (1) mixing hexagonal boron nitride and sulfur-containing amino acid and then ball milling to obtain modified boron nitride nanosheets;

[0009] (2) Adsorbing essential oil onto the modified boron nitride nanosheets to obtain amino acid functionalized boron nitride nanosheet essential oil carriers.

[0010] The present invention utilizes the advantages of diverse structures and biocompatibility of sulfur-containing amino acids (SSA) to conjugate hexagonal boron nitride nanosheets, wherein the sulfur-containing amino acids are acted on the boron nitride nanosheets through Lewis acid-base, and in addition to introducing hydrophilic groups amino and carboxyl groups, there are also methylthio (-S-CH3), sulfhydryl (-SH) and disulfide bonds (-SS-) unique to sulfur-containing amino acids. Sulfur is a nutrient element necessary for plant growth and development, and sulfur-containing amino acids play a role in improving plant defense mechanisms, including improving antioxidant defense, regulating plant hormones, participating in complex signal networks, etc., and the interaction between sulfur-containing amino acids and boron nitride realizes the stripping and modification of boron nitride nanosheets, forming a stable functionalized boron nitride carrier, enhancing its adhesion on the surface of plant tissues, and coordinating essential oils for preventing and controlling plant diseases, especially for preventing and controlling the invasion of Ralstonia solanacearum on crops. And the use of sulfur-containing amino acids as modifiers of boron nitride enhances its antibacterial properties and biocompatibility while improving the hydrophilicity of boron nitride, thereby promoting plant growth and development.

[0011] As a preferred embodiment of the preparation method of the amino acid functionalized boron nitride nanosheet essential oil carrier of the present invention, the sulfur-containing amino acid includes at least one of methionine, cysteine, and cystine; preferably, the sulfur-containing amino acid includes at least one of L-methionine, L-cysteine, and L-cystine; further preferably, the sulfur-containing amino acid is cystine; more preferably, the sulfur-containing amino acid is L-cystine.

[0012] The disulfide bond (-SS-) introduced by the hexagonal boron nitride modified with cysteine ​​in the present invention can be broken in a reducing environment, thereby giving the boron nitride nanosheets stimulus responsiveness, further enhancing the adhesion, stability and antibacterial properties of the nanosheets on the surface of plant tissues. Compared with pure essential oils, the nanocarriers loaded with essential oils still maintain a relatively stable and lasting essential oil release rate under high temperature (60°C), and the antibacterial activity is increased by up to 80.06%, and the bacterial growth can be completely inhibited at a relatively low essential oil concentration (0.6 mg / mL).

[0013] As a preferred embodiment of the method for preparing the amino acid functionalized boron nitride nanosheet essential oil carrier of the present invention, the mass ratio of the hexagonal boron nitride to the sulfur-containing amino acid is 1:(1-4).

[0014] As a preferred embodiment of the method for preparing the amino acid functionalized sulfur-containing amino acid stripped and functionalized boron nitride nanosheet essential oil carrier of the present invention, the ball milling time is 8h-24h.

[0015] As a preferred embodiment of the preparation method of the amino acid functionalized boron nitride nanosheet essential oil carrier of the present invention, the essential oil includes at least one of cinnamon essential oil, oregano essential oil, thyme essential oil, lemon essential oil, citrus essential oil, tea tree essential oil, and garlic essential oil; preferably, the essential oil is tea tree essential oil.

[0016] As a preferred embodiment of the method for preparing the amino acid functionalized boron nitride nanosheet essential oil carrier of the present invention, the adsorption adopts a light-proof environment condition; the adsorption temperature is 20° C.-25° C., and the adsorption time is 18h-24h.

[0017] In a second aspect, the present invention provides an amino acid functionalized boron nitride nanosheet essential oil carrier prepared by the preparation method.

[0018] In a third aspect, the present invention provides the use of the amino acid functionalized boron nitride nanosheet essential oil carrier in plant disease prevention and control, such as being used to prepare plant antibacterial agents that are widely used in agricultural production to prevent and control a variety of plant diseases caused by Ralstonia solanacearum.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: first, the present invention improves the adhesion of the functionalized boron nitride nanosheet essential oil carrier on the surface of plant tissue by stripping and functionalizing boron nitride with sulfur-containing amino acids, limits the volatilization of essential oils, enables the essential oil molecules to be continuously and stably released, exerts a long-lasting antibacterial effect, significantly improves the utilization efficiency of essential oils, and realizes intelligent and precise regulation of essential oil molecules; secondly, the present invention modifies boron nitride so that it has different surface properties and structures, combines its physical destruction of bacteria with the chemical destruction of essential oils, synergistically enhances the overall antibacterial effect while significantly reducing the amount of essential oil used, and serves as an environmentally friendly and stable essential oil carrier for preventing and controlling plant diseases; finally, the preparation method described in the present invention has mild conditions, simple preparation steps, and is easy to repeat and quantitatively produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The schematic diagram of the preparation process and principle of the amino acid functionalized boron nitride nanosheet essential oil carrier of the present invention;

[0021] Figure 2 SEM images of BNNS and BNNS-SAAs of Example 1 of the present invention: (a) BNNS, (b) BNNS-MET, (c) BNNS-CYS, (d) BNNS-CYST;

[0022] Figure 3TEM images of BNNS and BNNS-SAAs for verification example 1 of the present invention: (a) h-BN, (b) BNNS, (c) BNNS-MET, (d) BNNS-CYS, (e) BNNS-CYST;

[0023] Figure 4 This is the AFM image of BNNS-SAAs of the verification example 1 of the present invention;

[0024] Figure 5 Cumulative release curves of essential oils from nanosheets loaded with essential oils for verification example 2-3 of the present invention: (a) a loading rate curve of tea oil from nanosheets loaded with essential oils, (b) a sustained release curve of tea oil from nanosheets loaded with essential oils at 40°C, (c) a sustained release curve of tea oil from nanosheets loaded with essential oils at 60°C;

[0025] Figure 6 This is a SEM image of amino acid functionalized boron nitride nanosheets on the surface of plant tissue in Experimental Example 1 of the present invention;

[0026] Figure 7 This is the S element distribution diagram of amino acid functionalized boron nitride nanosheets on the surface of plant seeds in Experimental Example 1 of the present invention: (a) BNNS-CYST, (b) BNNS-CYS;

[0027] Figure 8 The antibacterial effect diagram of the nanosheet loaded with essential oil in Experimental Example 2 of the present invention: (a) against Ralstonia solanacearum, (b) against Escherichia coli, (c) against Staphylococcus aureus, (d) a comparison diagram of the inhibition rate of the nanosheet loaded with essential oil against the above three bacteria;

[0028] Fig. 9 Minimum inhibitory concentration of the nanosheets loaded with essential oils against Ralstonia solanacearum in Experimental Example 3 of the present invention: (a) TTO, (b) BNNS-MET-TTO, (c) BNNS-CYS-TTO, (d) BNNS-CYST-TTO, (f) MIC bar graph;

[0029] Fig.10 This is a SEM image of Ralstonia solanacearum after being treated with nanosheets loaded with essential oils in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0030] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] Unless otherwise specified, the test methods used in the examples are conventional methods; the materials, reagents, equipment, etc. used, unless otherwise specified, can be obtained from commercial sources. The essential oils in the embodiments of the present invention are implemented by taking tea tree oil (TTO) and cinnamon essential oil (CIN) as examples. It should be understood by those skilled in the art that the tea tree oil or cinnamon essential oil of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solution of the present invention.

[0032] The preparation process and principle diagram of amino acid functionalized boron nitride nanosheet essential oil carrier (BNNS-SAAs-TTO) are shown in Figure 1 shown.

[0033] Example 1: Preparation of essential oil-loaded methionine-boron nitride

[0034] (1) 2 g of hexagonal boron nitride powder (h-BN) and 6 g of methionine (MET) were placed in a beaker, 30 mL of deionized water was added, and ultrasonic cleaning was performed for 10 min. After the mixed solution was evenly dispersed, it was poured into a ball mill. The mass ratio of h-BN powder to ball milling beads was 1:50, the ball milling speed was set to 600 rpm, and the ball milling time was 12 h.

[0035] (2) The ball-milled mixture was centrifuged at 2000 rpm for 10 min, the lower layer was washed twice with water and then dried at 60° C. to obtain functionalized hexagonal boron nitride, abbreviated as BNNS-MET.

[0036] (3) 100 mg of BNNS-MET was placed in a filter paper funnel, 5 mL of TTO was added to immerse it, and it was placed in a dark and cool place for 24 h. After the essential oil on the sample surface evaporated completely, the boron nitride-based material sample loaded with essential oil was collected and labeled as BNNS-MET-TTO.

[0037] Example 2: Preparation of essential oil-loaded cysteine-boron nitride

[0038] (1) 2 g of hexagonal boron nitride powder (h-BN) and 2 g, 4 g, 6 g, and 8 g of cysteine ​​(CYS) were placed in a beaker, 30 mL of deionized water was added, and ultrasonic cleaning was performed for 10 min. After the mixed solution was evenly dispersed, it was poured into a ball mill. The mass ratio of h-BN powder to ball milling beads was 1:50, the ball milling speed was set to 600 rpm, and the ball milling time was 8 h-24 h.

[0039] (2) The ball-milled mixture was centrifuged at 2000 rpm for 10 min, the lower layer was washed twice with water and then dried at 60° C. to obtain functionalized hexagonal boron nitride, abbreviated as BNNS-CYS (BNNS-CYS-1, BNNS-CYS-2, BNNS-CYS-3, BNNS-CYS-4).

[0040] (3) Place 100 mg of BNNS-CYS in a filter paper funnel, add 5 mL of TTO to immerse it, and place it in a dark and cool place for 18 h to 24 h. After the essential oil on the sample surface evaporates completely, the boron nitride-based material samples loaded with essential oil are collected and labeled as BNNS-CYS-TTO (BNNS-CYS-TTO-1, BNNS-CYS-TTO-2, BNNS-CYS-TTO-3, BNNS-CYS-TTO-4).

[0041] Example 3: Preparation of cystine-boron nitride loaded with essential oil

[0042] (1) 2 g of hexagonal boron nitride powder (h-BN) and 6 g of cystine (CYST) were placed in a beaker, 30 mL of deionized water was added, and ultrasonic cleaning was performed for 10 min. After the mixed solution was evenly dispersed, it was poured into a ball mill. The mass ratio of h-BN powder to ball milling beads was 1:50, the ball milling speed was set to 600 rpm, and the ball milling time was 12 h.

[0043] (2) The ball-milled mixture was centrifuged at 2000 rpm for 10 min, the lower layer was washed twice with water and then dried at 60° C. to obtain functionalized hexagonal boron nitride, abbreviated as BNNS-CYST.

[0044] (3) 100 mg of BNNS-CYST was placed in a filter paper funnel, 5 mL of TTO was added to immerse it, and it was placed in a dark and cool place for 18 h. After the essential oil on the sample surface evaporated completely, the boron nitride-based material sample loaded with essential oil was collected and labeled as BNNS-CYST-TTO.

[0045] Example 4: Preparation of cystine-boron nitride loaded with different essential oils

[0046] (1) 2 g of hexagonal boron nitride powder (h-BN) and 6 g of cystine (CYST) were placed in a beaker, 30 mL of deionized water was added, and ultrasonic cleaning was performed for 10 min. After the mixed solution was evenly dispersed, it was poured into a ball mill. The mass ratio of h-BN powder to ball milling beads was 1:50, the ball milling speed was set to 600 rpm, and the ball milling time was 12 h.

[0047] (2) The ball-milled mixture was centrifuged at 2000 rpm for 10 min, the lower layer was washed twice with water and then dried at 60° C. to obtain functionalized hexagonal boron nitride, abbreviated as BNNS-CYST.

[0048] (3) 100 mg of BNNS-CYST was placed in a filter paper funnel, 5 mL of cinnamon essential oil (CIN) was added to immerse it, and the funnel was placed in a dark and cool place for 24 h. After the essential oil on the sample surface evaporated completely, the boron nitride-based material sample loaded with essential oil was collected and labeled as BNNS-CYST-CIN.

[0049] Comparative Example 1: Preparation of original hexagonal boron nitride loaded with essential oil

[0050] 100 mg of hexagonal boron nitride powder (h-BN) was placed in a filter paper funnel, 5 mL of TTO was added to immerse it, and it was placed in a dark and cool place for 24 hours. After the essential oil on the surface of the sample evaporated completely, the boron nitride material sample loaded with essential oil was collected and marked as h-BN-TTO.

[0051] Comparative Example 2: Preparation of exfoliated hexagonal boron nitride loaded with essential oil

[0052] (1) 2 g of hexagonal boron nitride powder (h-BN) was placed in a ball mill and ball milled for 12 h in a planetary ball mill under the same operating conditions as in Example 1 to obtain exfoliated boron nitride powder, which was labeled as BNNS.

[0053] (2) 100 mg of BNNS was placed in a filter paper funnel, 5 mL of TTO was added to immerse it, and it was placed in a dark and cool place for 24 h. After the essential oil on the sample surface evaporated completely, the boron nitride-based material sample loaded with essential oil was collected and labeled as BNNS-TTO.

[0054] Verification Example 1: Study on the morphology of functionalized boron nitride nanosheets

[0055] The morphology, microstructure and size of the BNNS-SAAs (BNNS-MET, BNNS-CYS is BNNS-CYS-3, BNNS-CYST) nanomaterials prepared in Examples 1-3 were studied using a scanning electron microscope (SEM), a transmission electron microscope (TEM) and an atomic force microscope (AFM).

[0056] like Figure 2 As shown in the figure, the lateral dimensions of the boron nitride nanosheets obtained by exfoliation and functionalization with MET and CYS are larger than those of BNNS. This is because during the ball milling process, the sulfur-containing amino acid acts as a lubricant and is evenly distributed on the surface of BNNS through Lewis acid-base action, causing the translational sliding of the sheet to increase the particle size.

[0057] The flakes obtained by exfoliation using CYST are stacked because the high CYST content causes the flakes to adhere and agglomerate, showing a more three-dimensional structure, indicating that the shear force and dissociation force provided by a simple ball milling method can effectively exfoliate boron nitride.

[0058] like Figure 3 As shown in the figure, after h-BN was exfoliated and functionalized with sulfur-containing amino acids, BNNS-MET and BNNS-CYS produced thinner sheets than BNNS, and the sheets became more transparent, which means that the sheets were thinner. Irregular dark areas formed where the sheets overlapped, which were attributed to the grafted sulfur-containing amino acids. BNNS-CYST, similar to h-BN, showed sheets with clear edges and similar sizes. Unlike the other two amino acids, the sheets it formed were more complete and the lateral dimensions of the single sheets were larger.

[0059] like Figure 4 As shown, the thickness of the nanosheets obtained after exfoliation with sulfur-containing amino acids is about 4-6 layers, the average lateral size of BNNS-MET is 425nm, the thickness of the sheet is 1.47nm, the average lateral size of BNNS-CYS is 123nm, the thickness is 2.15nm, and the average lateral size of BNNS-CYST is 820nm, the thickness is 1.78nm. The nanosheets obtained by exfoliation with MET are the thinnest, the nanosheets obtained by exfoliation with CYS have the smallest lateral size, and the nanosheets obtained by exfoliation with CYST have the largest lateral size.

[0060] Verification Example 2: Drug loading and encapsulation efficiency of essential oil carrier

[0061] The content of essential oil tea tree oil (pharmaceutical grade, Beijing Solebow) in the products obtained in Examples 1-3 and Comparative Examples 1-2 was determined using a synchronous thermal analyzer (TGA2, Mettler Toledo, Switzerland).

[0062] The test conditions were 40℃-600℃, and the heating rate was 10℃ / min. By comparing with the results of h-BN-TTO, the loading rate of tea tree essential oil in the boron nitride-based material (denoted as q) can be obtained. The calculation formula for the essential oil loading is as follows:

[0063]

[0064] Where m is the mass (mg) of the carrier after the essential oil has completely evaporated, and M is the mass (mg) of the initial carrier.

[0065] like Figure 5As shown in (a), h-BN-TTO and BNNS-TTO have only one weight loss step before 180℃, which is attributed to the weight loss of essential oils. The essential oil loading rates of h-BN and BNNS are 51.64% and 57.49%, and the essential oil loading rates of BNNS-MET, BNNS-CYS and BNNS-CYST are 36.40%, 36.06% and 37.11%, respectively. Compared with h-BN and BNNS, the loading rate of BNNS-SSAs for essential oils is lower, which is because after hydrophilic modification, the loading capacity of BNNS-SAAs for hydrophobic essential oils is reduced.

[0066] Verification Example 3: Cumulative sustained release rate of nanosheets loaded with essential oils

[0067] The sustained-release performance of the nanosheets loaded with essential oils of Examples 1-3 and Comparative Examples 1-2 was evaluated according to the following method:

[0068] The sustained release performance of pure essential oil and nanosheets loaded with essential oil at 40℃ and 60℃ for 10h was investigated using a synchronous thermal analyzer. The weight loss curve of essential oil was obtained for comparison.

[0069] The calculation formula (2) for the essential oil release rate (R) in the system is as follows.

[0070]

[0071] Where M is the initial mass of the carrier (mg), m remain is the residual mass of the carrier at any time (mg), M TTO is the initial adsorbed essential oil content in the carrier (mg).

[0072] from Figure 5 As can be seen from (b), at 40 °C, the essential oil release rates of BNNS-MET-TTO, BNNS-CYS-TTO and BNNS-CYST-TTO were 39.33%, 28.64% and 21.83% at 100 min, while the essential oil release rate of pure TTO reached 93.28%; after 10 h of sustained release, the essential oil release rates of BNNS-MET-TTO, BNNS-CYS-TTO and BNNS-CYST- The cumulative release rates of essential oil from TTO were 87.42%, 72.64% and 63.34%, respectively, which were all lower than the 88.85% of the original h-BN, while the release rate of TTO had reached 97.86%, almost completely volatilized. Compared with pure TTO, the cumulative release rates of BNNS-MET-TTO, BNNS-CYS-TTO and BNNS-CYST-TTO were reduced by 10.44%, 25.22% and 34.52%, respectively.

[0073] from Figure 5As can be seen in (b), at 60°C, the cumulative release of pure TTO in the first 33 minutes has reached 98.40%, and the cumulative release has reached about 100% after 10 hours, indicating that TTO is extremely unstable in a high temperature environment. The cumulative release rates of TTO from BNNS-MET-TTO, BNNS-CYS-TTO and BNNS-CYST-TTO are 99.45%, 88.05% and 79.6%, respectively, which are the same as the release trend at 40°C, but the cumulative release rate of essential oil increases, which is due to the stimulation of high temperature, which increases the volatilization rate of essential oil.

[0074] The above-mentioned sustained-release results show that after the boron nitride loaded with sulfur-containing amino acids stripped and functionalized, it has a good sustained-release effect on TTO, which can effectively prevent the rapid volatilization of TTO. The use of BNNS-SAAs to load TTO not only changes its chemical properties by grafting sulfur-containing amino acids into the boron nitride structure, but also introduces additional steric hindrance, and the introduced functional groups (amino, carboxyl) can form hydrogen bonds or van der Waals forces with TTO molecules. This strong interaction significantly enhances the adsorption capacity of boron nitride for essential oils, allowing essential oil molecules to be more firmly bound to the surface of the material. For BNNS-CYST-TTO, due to the large amount of CYST grafted on boron nitride, a complex multilayer adsorption structure is constructed on the surface of boron nitride. This multilayer structure provides abundant adsorption sites for TTO molecules, allowing TTO molecules to be adsorbed on the surface of boron nitride in a multi-level, tightly arranged manner, achieving a long-distance transport and binding effect on TTO, making it more difficult for internal TTO molecules to be released, further limiting volatilization. Therefore, BNNS-CYST, as an essential oil carrier with the slowest release rate, can release TTO molecules from the carrier in a continuous and stable manner, thereby exerting a long-lasting antibacterial effect and significantly improving the utilization efficiency of essential oils.

[0075] Test Example 1: Adhesion of amino acid functionalized boron nitride materials on plant tissue surfaces

[0076] The adhesion properties of the functionalized boron nitride materials of Examples 1-3 and Comparative Examples 1-2 were analyzed according to the following method:

[0077] Scanning electron microscopy (SEM) was used to observe the adhesion and distribution of functionalized boron nitride materials on seeds and leaves.

[0078] (1) Tomato seeds were treated with a sample solution at a concentration of 1 mg / mL for 12 h, and then photographed after the moisture on the seed surface was air-dried.

[0079] (2) Soak the tomato leaves in a sample solution with a concentration of 1 mg / mL for 15 seconds, take them out to dry naturally, and then put them in a vacuum drying oven for low-temperature drying for 3 hours before photographing.

[0080] (3) Tomato seeds and leaves treated with deionized water were used as the control group (CK group).

[0081] The S content on the surface of the seeds treated with the functionalized boron nitride material prepared in Examples 1-3 was detected according to the following method.

[0082] The elemental analysis method was adopted, the surface of tomato seeds was observed with a scanning electron microscope, and the relative content of S element on the surface of tomato seeds was calculated using a photoelectron spectrometer (EDS).

[0083] like Figure 6 As shown in the figure, the surface of untreated tomato seeds is smooth, but after treatment, abundant boron nitride materials can be observed on the surface of the seeds. The original h-BN is accumulated in clusters on the surface of tomato seeds and is unevenly distributed; the distribution of the peeled BNNS on the surface of the seeds is too scattered and sparse due to its small particles; after modification with sulfur-containing amino acids, BNNS-SSAs have better adhesion ability and do not accumulate on the surface of seeds but are evenly distributed in a single layer, because the sulfur-containing amino acids improve the dispersibility of boron nitride.

[0084] For untreated tomato leaves, the surface is smooth with irregular textures. After treatment, BNNS-MET is able to form a continuous covering layer on the leaf surface, while the adhesion of BNNS-CYS and BNNS-CYST tends to fill in the grooves of the leaves.

[0085] The improvement in adhesion is due to the following reasons: first, BNNS-SSAs have a larger specific surface area and a "softer" lamellar structure than h-BN, which can provide more contact points, which can increase the affinity between the carrier and different interfaces, and can better adapt to irregular surfaces, fill microscopic protrusions and depressions, and contact with irregular surfaces more effectively, thereby enhancing the adhesion effect; secondly, the functional groups (amino and carboxyl groups) provided by sulfur-containing amino acids enable them to produce more non-covalent interactions with different surfaces, such as the formation of hydrogen bonds, van der Waals forces, etc., thereby enhancing their adhesion ability; and as Figure 7 As shown, the distribution of S elements on the surface of seeds treated with BNNS-SAAs also supports this view. The atomic contents of S elements on the surface of seeds treated with BNNS-CYST and BNNS-CYS were 1.15±0.187% and 0.37±0.015%, respectively, while the content of S elements on the surface of seeds treated with BNNS-MET was lower, below the detection limit, and no S element could be detected.

[0086] Test Example 2: Antibacterial activity of nanosheets loaded with essential oils

[0087] The antibacterial activity of the essential oil-loaded nanosheets prepared in Examples 1-3 and Comparative Examples 1-2 was analyzed according to the following method:

[0088] The inhibition zone method was used to investigate the inhibitory effects of essential oil-loaded boron nitride nanosheets on Ralstonia solanacearum, Escherichia coli and Staphylococcus aureus.

[0089] (1) Determination of antibacterial activity of Escherichia coli and Staphylococcus aureus: Place the prepared agar solution and deionized water in an autoclave and sterilize at 121°C for 20 min. Dilute the corresponding bacterial solution (Escherichia coli or Staphylococcus aureus) with sterile water in a clean bench to a concentration of about 10 5 CFU / mL, use a pipette to transfer 100 μL of the corresponding bacterial solution to the solidified agar medium, spread it evenly with a spreading rod, and then use a hole puncher to punch a hole with a diameter of 5 mm in the middle of the plate, and put the corresponding sample in the hole (each boron nitride nanosheet loaded with the same amount of TTO (20 mg), according to the given essential oil loading rate, calculate the exact mass required for each sample, loading rate h-BN: 51.64%, BNNS: 57.49%, BNNS-MET: 36.40%, BNNS-CYS: 36.06%, BNNS-CYST: 37.11%), after the operation is completed, culture in a 37°C constant temperature biochemical incubator for 24 hours, observe the results, and measure the diameter of the inhibition zone with a vernier caliper.

[0090] (2) Determination of antibacterial activity of Ralstonia solanacearum: CPG culture medium was used, and the preparation steps were the same as step (1). The required items were placed in an autoclave and sterilized at 115°C for 20 min. In the clean bench, 100 μL of 2% TTC dye solution was first transferred to the solidified culture medium and evenly spread with a coating rod. Then 100 μL of Ralstonia solanacearum bacterial solution (concentration of about 10 9 CFU / mL) was evenly spread on the culture medium with the staining solution, and the samples were placed in the holes after punching (each boron nitride nanosheet was loaded with the same amount of TTO (20 mg), and the exact mass required for each sample was calculated according to the given essential oil loading rate, with loading rates of h-BN: 51.64%, BNNS: 57.49%, BNNS-MET: 36.40%, BNNS-CYS: 36.06%, and BNNS-CYST: 37.11%). After the operation was completed, the samples were cultured in a constant temperature biochemical incubator at 30°C for 48 hours, and the results were observed, and the diameter of the inhibition zone was measured with a vernier caliper.

[0091] like Figure 8As shown in (a), h-BN, BNNS and BNNS-SSAs had no inhibitory effect on Ralstonia solanacearum, indicating that the main antibacterial active ingredient was the essential oil TTO. For BNNS-SAAs-TTO, the diameter of the inhibition zone of BNNS-CYST-TTO was 31.76±0.78nm, which was the best and better than TTO (25.03±0.64mm) and h-BN-TTO (30.97±1.39mm). BNNS-CYS-TTO had a moderate antibacterial effect (22.40±1.15mm), and BNNS-MET-TTO had the smallest inhibition zone (22.14±0.39mm).

[0092] like Figure 8 As shown in (b), the diameter of the inhibition zone of BNNS-CYST-TTO is 41.63±0.29mm, which is the best and better than TTO (23.12±0.37mm) and h-BN-TTO (29.36±0.13mm). The antibacterial effect of BNNS-CYS-TTO is moderate (26.07±0.40mm), and the inhibition zone of BNNS-MET-TTO is the smallest (18.68±0.10mm).

[0093] like Figure 8 As shown in (c), the diameter of the inhibition zone of BNNS-CYST-TTO is 47.96±0.51nm, which is the best and better than TTO (30.46±0.69mm) and h-BN-TTO (41.62±0.83mm). The antibacterial effect of BNNS-CYS-TTO is moderate (28.77±0.20mm), and the inhibition zone of BNNS-MET-TTO is the smallest (23.51±0.70mm).

[0094] from Figure 8 As can be seen in (d), the inhibitory effect of BNNS-SAAs-TTO materials on Escherichia coli and Staphylococcus aureus maintained the same trend as that on Ralstonia solanacearum. Among them, BNNS-CYST showed the best antibacterial effect on all three bacteria. Compared with pure TTO, the antibacterial activity of BNNS-CYST-TTO on Ralstonia solanacearum, Escherichia coli and Staphylococcus aureus increased by 26.89%, 80.06% and 57.45%, respectively. The antibacterial effects of TTO loaded on carriers obtained by different amino acid modifications were different, and BNNS-CYST-TTO had more obvious advantages as an antibacterial material. This is because BNNS-CYST has a better restraining effect on TTO, effectively preventing the premature volatilization and loss of tea tree oil, and enhancing its antibacterial activity.

[0095] Test Example 3: Determination of the minimum inhibitory concentration of essential oil-loaded nanosheets against Ralstonia solanacearum

[0096] The minimum inhibitory concentration (MIC) of the nanosheets loaded with essential oils prepared in Examples 1-3 and Comparative Examples 1-2 against Ralstonia solanacearum was analyzed according to the following method:

[0097] (1) Pre-culture: A series of BNNS-SSAs-TTO materials (BNNS-SSAs nanosheets loaded with TTO in a concentration gradient of 0.2 mg / mL to 2.0 mg / mL) were added to a liquid culture medium (CPG culture medium without agar), and after being evenly dispersed by ultrasonication, a bacterial solution was added to form a bacterial solution (Ralstonia solanacearum) with a concentration of 10 6 CFU / mL of bacterial culture medium, and then placed in a shaking incubator at 30°C for 24 hours.

[0098] (2) Inhibitory culture: On the second day, 100 μL of 2% TTC staining solution was spread on the solid culture medium (CPG medium), and then 100 μL of the bacterial culture solution in step (1) was evenly spread on the solid culture medium. After the operation was completed, the culture was placed in a 30°C incubator for 48 h and the results were observed. The concentration corresponding to the plate without bacterial growth was the minimum inhibitory concentration (MIC concentration).

[0099] like Fig. 9 As shown in the figure, the MIC concentration of pure TTO is 1.8 mg / mL, the MIC concentration of BNNS-MET-TTO and BNNS-CYS-TTO is 1 mg / mL, and the MIC concentration of BNNS-CYST-TTO is 0.6 mg / mL. Compared with pure TTO, the three materials of BNNS-MET-TTO, BNNS-CYS-TTO and BNNS-CYST-TTO, which are boron nitride nanosheets loaded with tea tree oil and stripped and functionalized by sulfur-containing amino acids, significantly reduce the minimum inhibitory concentration of Ralstonia solanacearum, and the antibacterial efficiency is increased by 44.44% and 66.67%. In particular, BNNS-CYST-TTO can completely inhibit bacterial growth at a lower TTO concentration (0.6 mg / mL). This shows that the introduction of sulfur-containing amino acids not only improves the performance of BNNS and enhances its interaction with TTO, but also significantly reduces the dosage of tea tree oil and improves the antibacterial performance of the composite material.

[0100] Experimental Example 4: Effect of the morphology of Ralstonia solanacearum after treatment with nanosheets loaded with essential oils

[0101] In order to further illustrate the interaction between BNNS-SAAs-TTO and bacteria, the effect of the treatment of the nanosheets loaded with essential oils prepared in Examples 1-3 and Comparative Examples 1-2 on the morphology of Ralstonia solanacearum was analyzed according to the following method:

[0102] (1) Each sample was added to a liquid culture medium, dispersed evenly by ultrasonication, and an appropriate amount of bacterial solution (Ralstonia solanacearum) was added. The culture was carried out at 30°C and 180 rpm for 24 h.

[0103] (2) The culture medium was centrifuged at 6000 rpm for 5 min, and the culture medium was removed by repeated washing three times with 2 mL of 0.1 M PBS buffer. The bacterial cells were collected by centrifugation and fixed with 2 mL of 2.5% glutaraldehyde solution at 4°C overnight.

[0104] (3) Collect the cells by centrifugation and wash them twice with 0.1 M PBS buffer. Dehydrate them with ethanol in a gradient manner (concentrations are 30%, 50%, 70%, 80%, 90% and 100%, 100% treatment twice), with an interval of 3 min between each treatment. After centrifugation, freeze-dry and take a small amount of cells for photography to observe their morphology.

[0105] like Fig.10 As shown in the figure, the R. solanacearum in the CK group is cylindrical and has a smooth surface and grows evenly. After being treated with TTO, the surface of the bacteria becomes wrinkled and damaged and concave, indicating that TTO has a destructive effect on R. solanacearum and affects bacterial growth; after being treated with BNNS-SAAs-TTO, it can be seen that more nanosheets are attached to the outside of the bacteria, but there are differences in the adhesion mode, which is the reason why they exert different antibacterial activities.

[0106] Specifically, BNNS-MET-TTO tends to adhere to both sides of the outer wall of Ralstonia solanacearum, inhibiting the growth of bacteria through compression and contact. At the same time, the TTO component in the composite material can also be continuously released and act on the bacteria, exacerbating the roughness and collapse of its surface. BNNS-CYS-TTO nanosheets are inserted into the head and tail of Ralstonia solanacearum. This direct penetration of bacteria has a stronger inhibitory effect on the bacteria, and some gaps and partial detachment appear on the surface of the bacteria. The BNNS-CYST-TTO sheet is thicker, and the bacteria are twisted and entangled by squeezing and wrapping. This strong physical effect plus the antibacterial effect of TTO leads to severe wrinkles on the surface of the bacteria. Under the same magnified field of view, the number of bacteria is greatly reduced, which is due to the more excellent antibacterial effect of BNNS-CYST-TTO.

[0107] However, h-BN-TTO and BNNS-TTO have less adhesion on the surface of R. solanacearum, which is mainly due to their higher surface energy, which makes it easy for the nanosheets to agglomerate to form larger particles, reducing the contact opportunity with the bacterial surface, reducing the adhesion and antibacterial effect.

[0108] In contrast, the BNNS-SAAs-TTO composites stripped and functionalized with sulfur-containing amino acids can more effectively disperse and adhere to the bacterial surface, exerting their excellent antibacterial properties.

[0109] Since bacterial extracellular polysaccharides have strong suspension, stability and viscosity, and have high affinity with BNNS-SAAs-TTO, BNNS-SAAs-TTO tends to bind tightly to bacteria. The amino, carboxyl and other polar groups introduced by sulfur-containing amino acid molecules can form hydrogen bonds with proteins and lipids on the bacterial surface, which helps to stabilize the adhesion of boron nitride nanosheets. Among them, BNNS-CYST-TTO has a more excellent antibacterial effect, especially at low concentrations of bioactive ingredients. Its mechanism of action is as follows: (i) BNNS-CYST-TTO aggregates and adheres to the outer membrane of Ralstonia solanacearum. Thicker nanosheets can wrap and squeeze bacteria, affecting their normal growth; nanosheets can extract phospholipids from the lipid bilayer through strong van der Waals forces, thereby destroying the integrity of the cell membrane. The continuous release of TTO in the nanochannels within BNNS-CYST can increase the concentration of local active ingredients. (ii) The released TTO can directly act on the bacterial cell membrane, destroying the integrity of the cell membrane, causing leakage of cell contents, and ultimately damaging the bacterial metabolic function and inhibiting its growth and proliferation. (iii) The nanosheets can target key bacterial surface proteins involved in cell division, such as FtsP, EnvC, and TolB proteins, thereby hindering bacterial reproduction and division. The disulfide bond (-SS-) in the introduced cystine has high reactivity and can undergo thiol-disulfide exchange reactions with sulfhydryl groups or disulfide bonds in bacterial membrane proteins, leading to conformational changes or depolymerization of proteins. The presence of disulfide bonds can also trigger local redox reactions, further affecting the structure and function of proteins. The unique surface properties of BNNS-CYST enhance the interaction between bacteria and nanosheets, and together with the loaded TTO, they form an efficient and long-lasting antibacterial system.

[0110] In conclusion, sulfur-containing amino acid-modified boron nitride has different adhesion and antibacterial effects on bacteria due to their different surface properties and structures, and the overall antibacterial effect can be enhanced by combining the physical destruction of bacteria by functionalized boron nitride nanosheets with the chemical destruction by essential oils.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing an amino acid functionalized boron nitride nanosheet essential oil carrier, characterized in that: The following steps are involved: (1) mixing hexagonal boron nitride and sulfur-containing amino acid and then ball milling to obtain modified boron nitride nanosheets; (2) Adsorbing essential oil onto the modified boron nitride nanosheets to obtain amino acid functionalized boron nitride nanosheet essential oil carriers.

2. The method for preparing the amino acid functionalized boron nitride nanosheet essential oil carrier according to claim 1, characterized in that: The sulfur-containing amino acid includes at least one of methionine, cysteine ​​and cystine.

3. The method for preparing the amino acid functionalized boron nitride nanosheet essential oil carrier according to claim 2, characterized in that: The sulfur-containing amino acid is cystine.

4. The method for preparing the amino acid functionalized boron nitride nanosheet essential oil carrier according to claim 1, characterized in that: The mass ratio of the hexagonal boron nitride to the sulfur-containing amino acid is 1:(1-4).

5. The method for preparing the amino acid functionalized boron nitride nanosheet essential oil carrier according to claim 1, characterized in that: The ball milling time is 8h-24h.

6. The method for preparing the amino acid functionalized boron nitride nanosheet essential oil carrier according to claim 1, characterized in that: The essential oil comprises at least one of cinnamon essential oil, oregano essential oil, thyme essential oil, lemon essential oil, citrus essential oil, tea tree essential oil and garlic essential oil.

7. The method for preparing the amino acid functionalized boron nitride nanosheet essential oil carrier according to claim 6, characterized in that: The essential oil is tea tree essential oil.

8. The method for preparing the amino acid functionalized boron nitride nanosheet essential oil carrier according to claim 1, characterized in that: The adsorption is carried out in a light-proof environment; the adsorption temperature is 20° C.-25° C., and the adsorption time is 18 h-24 h.

9. An amino acid functionalized boron nitride nanosheet essential oil carrier obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the amino acid functionalized boron nitride nanosheet essential oil carrier according to claim 9 in plant disease prevention and control.

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