A method for simultaneously improving the insect resistance and grain functional nutrients of sesame based on nano-chitosan

Abstract

The present invention discloses a method for simultaneously improving the insect resistance and the functional nutrients of sesame seeds based on nano-chitosan, belonging to the field of nano-agricultural regulation. The method for improving the control effect of Spodoptera litura larvae on sesame and the functional nutrients of sesame seeds specifically comprises formulating nano-chitosan into a suspension and spraying it on sesame plants; the average size of the nano-chitosan is 21±5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2±9.3 nm and 36.9±1.3 mV respectively. This method can not only efficiently activate the synthesis of sesame defense signals and insect-resistant substances, increase the resistance of sesame to Spodoptera litura, but also effectively improve the content of functional nutrients in sesame seeds; nano-chitosan is significantly superior to the traditional insecticide acetamiprid and traditional large-particle chitosan in the control effect of Spodoptera litura on sesame, and has broad application value in the field of controlling Spodoptera litura pests on crops.

Description

Technical Field

[0001] The present invention relates to a method for simultaneously improving the insect resistance and grain functional nutrients of sesame based on nano-chitosan, and belongs to the field of nano-agricultural regulation. Background Art

[0002] Phytophagous insects usually feed on crops, causing crop yield losses. Traditionally, the control of phytophagous insects mainly relies on the use of chemical pesticides. However, the overuse of pesticides not only exacerbates the resistance of pests, but also causes serious damage to the ecosystem and environmental health, especially posing a serious threat to non-target organisms and human health.

[0003] Sesame is an oil crop and has attracted much attention because it contains nutrients beneficial to human health. Spodoptera litura is a polyphagous pest and has gradually become the main phytophagous pest of different crops. Especially when it feeds on sesame plants, it will cause a large loss of sesame crop yield.

[0004] For the control of the sesame pest Spodoptera litura, the prior art usually uses targeted drugs to achieve the control effect; for example, the nano-pesticide preparation for controlling Spodoptera litura and its preparation method disclosed in Chinese Patent CN103109799A uses chitosan as a raw material to prepare chitosan nanoparticles as a drug delivery carrier, which loads the highly lethal target gene siRNA for Spodoptera litura, so as to achieve the treatment effect.

[0005] A slow-release nano-insecticide and its preparation method disclosed in Chinese Patent CN107821411A specifically disclose a slow-release nano-insecticide, in which nano-chitosan is used as a delivery carrier and the active ingredient of the drug is the core material of azadirachtin, so as to achieve the purpose of effectively controlling pests.

[0006] However, in the prior art, chitosan nanoparticles are usually used as carriers to prepare slow-release nano-pesticides with various pesticide reagents or biological reagents to control pests and diseases, which still brings the problems of overuse and abuse of active pesticides in the carrier, and does not involve directly using nano-chitosan as a plant immune inducer to activate the plant's own immune defense ability, promote the synthesis of anti-insect substances in the plant, enhance the insect resistance of the plant, especially enhance the resistance of the plant to Spodoptera litura, a kind of Lepidoptera pest that has a wide host and seriously endangers crop yield.

[0007] Therefore, there is an urgent need to develop new environmental protection strategies to control the sesame pest Spodoptera litura. Summary of the Invention

[0008] In view of the defects and deficiencies existing in the prior art, the present invention aims to synthesize a green nano-plant immune inducer with high activity and high biocompatibility for controlling phytophagous insects, which can not only effectively improve the control effect on Spodoptera litura larvae of sesame, but also increase the content of functional nutrients in sesame seeds at the same time; this method has the advantages of simple operation and strong versatility, and has broad application value in the field of Spodoptera litura control.

[0009] The object of the present invention is to provide a method for improving the control effect on Spodoptera litura larvae of sesame and the functional nutrients in sesame seeds, and the method includes: formulating nano-chitosan into a suspension and spraying it on sesame plants.

[0010] In one embodiment, the concentration of the suspension is 50-200 mg / L; preferably 100 mg / L.

[0011] In one embodiment, the average size of the nano-chitosan is 21±5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2±9.3 nm and 36.9±1.3 mV respectively.

[0012] In one embodiment, spraying on sesame plants specifically refers to spraying the nano-chitosan suspension on sesame leaves.

[0013] In one embodiment, the spraying time can be any stage during the sesame seedling stage, growth stage, or flowering stage, or spraying treatments can be carried out at each stage.

[0014] In one embodiment, the spraying amount is 10-30 mL of nano-chitosan suspension with a concentration of 50-200 mg / L per sesame plant.

[0015] In one embodiment, the preparation of the nano-chitosan includes the following steps:

[0016] Dissolve chitosan in acetic acid, adjust the pH to 5-6, stir overnight at room temperature to form a chitosan solution; then add a tripolyphosphate solution to the chitosan solution, continuously stir to form a milky white chitosan nano-suspension, centrifuge, wash, and freeze-dry to obtain nano-chitosan.

[0017] In one embodiment, the acetic acid is an acetic acid aqueous solution with a volume fraction of 1%.

[0018] In one embodiment, the volume ratio of the tripolyphosphate solution to the chitosan solution is 1:3.

[0019] In one embodiment, the mass concentration of tripolyphosphate in the tripolyphosphate solution is 0.5-1%.

[0020] In one embodiment, the mass concentration of chitosan in the chitosan solution is 0.5-1%.

[0021] Another object of the present invention is to provide a method for increasing the contents of Ca 2+ flow rate, salicylic acid and jasmonic acid in sesame leaf cells, and the method includes: preparing nanosized chitosan into a suspension and spraying it on sesame plants; the average size of the nanosized chitosan is 21±5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2±9.3 nm and 36.9±1.3 mV respectively.

[0022] The third object of the present invention is to provide a method for increasing the contents of sesamin and gardenoside methyl ester, which are anti-insect substances, in sesame leaves, and the method includes: preparing nanosized chitosan into a suspension and spraying it on sesame plants; the average size of the nanosized chitosan is 21±5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2±9.3 nm and 36.9±1.3 mV respectively.

[0023] The fourth object of the present invention is to provide a method for controlling Spodoptera litura in sesame based on inducing an increase in the contents of sesamin and gardenoside methyl ester, which are anti-insect substances, in sesame leaves by nanosized chitosan, and the method includes: preparing nanosized chitosan into a suspension and spraying it on sesame plants; the average size of the nanosized chitosan is 21±5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2±9.3 nm and 36.9±1.3 mV respectively.

[0024] The fifth object of the present invention is a method for inducing an increase in sesamin and amino acids, which are functional nutrients of sesame, by nanosized chitosan, and the method includes: preparing nanosized chitosan into a suspension and spraying it on sesame plants; the average size of the nanosized chitosan is 21±5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2±9.3 nm and 36.9±1.3 mV respectively. Description of the Drawings

[0025] Figure 1 It is a transmission electron microscope characterization diagram of nanosized chitosan (CNPs) prepared in Example 1 of the present invention; (A) is the hydrodynamic diameter; (B) is the Zeta potential;

[0026] Figure 2 It is a Fourier transform infrared spectrum diagram and an X-ray diffraction diagram of nanosized chitosan (CNPs) prepared in Example 1 of the present invention; (A) is the Fourier transform infrared spectrum diagram; (B) is the X-ray diffraction diagram;

[0027] Figure 3 It is the control effect diagram of different concentrations of chitosan and nanosized chitosan on Spodoptera litura larvae in Example 2 of the present invention;

[0028] Figure 4 This is the control effect diagram of nano-chitosan and commercial insecticide acetamiprid on Spodoptera litura larvae in Example 3 of the present invention;

[0029] Figure 5 This is the data diagram of the influence of nano-chitosan and commercial insecticide acetamiprid on the content of signal substances in sesame leaves in Example 3 of the present invention;

[0030] Figure 6 This is the data diagram of the influence of nano-chitosan and commercial insecticide acetamiprid on the content of insect-resistant substances in sesame leaves in Example 3 of the present invention;

[0031] Figure 7 This is the data diagram of the regulation of nutrients in sesame seeds by nano-chitosan and commercial insecticide acetamiprid in Example 4 of the present invention. Detailed implementation manners

[0032] The following further describes the detailed implementation manners of the present invention in conjunction with the drawings and examples.

[0033] The chitosan CS (50 - 190KDa, degree of deacetylation 80%) involved in the present invention was purchased from Sigma-Aldrich Shanghai Trading Co., Ltd.

[0034] Example 1

[0035] A preparation method of nano-chitosan (CNPs), the method includes the following:

[0036] Dissolve CS (0.5% w / v) in 1% (v / v) acetic acid, adjust the pH to 5.2 with 1mol / L NaOH, and then stir overnight at room temperature to form a CS solution; dissolve tripolyphosphate (TPP: 0.5% w / v) in ultrapure water, and slowly add it to the CS solution (volume ratio 1:3) under magnetic stirring, and continue stirring for 8 hours to form a milky white chitosan nano-suspension; the suspension is centrifuged (4°C, 12000rpm, 15min) with ultrapure water and washed twice, and then freeze-dried to obtain nano-chitosan.

[0037] Characterization of nano-chitosan materials

[0038] The results of transmission electron microscopy (TEM) are as Figure 1 shown, the synthesized nano-chitosan is spherical, with an average size of 21 ± 5.8nm ( Figure 1 A); the hydrodynamic diameter and zeta potential of nano-chitosan are 262.2 ± 9.3nm and 36.9 ± 1.3mV respectively ( Figure 1 B). The results of Fourier transform infrared (FTIR) are as Figure 2 shown, nano-chitosan is at 3423.53cm -1, 1640.41 cm -1 , 1545.34 cm -1 and 1069.57 cm -1 have absorption bands at, among which, 1640.41 cm -1 and 1545.34 cm -1 The absorption bands at are due to the vibrations of amide I and amide II ( Figure 2 A), especially at 1545.34 cm -1 The absorption band indicates that the cations in chitosan crosslink with the anions in sodium tripolyphosphate. The XRD pattern of nanochitosan does not show peaks in the diffraction pattern ( Figure 2 B), indicating that the synthesized nanochitosan is an amorphous structure.

[0039] Example 2

[0040] Application of nanochitosan in controlling Spodoptera litura larvae in sesame, and the specific application includes:

[0041] Disperse the nanochitosan (CNPs) prepared in Example 1 in water to prepare 50, 100, 200 mg / L CNPs suspensions; disperse chitosan CS in water to prepare 50, 100, 200 mg / L CS suspensions.

[0042] Use greenhouse pot culture to grow multiple pots of sesame seedlings, 500 g of soil per pot (pH 6.8, total organic carbon = 11.3 g / kg, total nitrogen = 1.4 g / kg), 2 sesame seedlings per pot, growing in an artificial climate chamber (temperature is 25 °C, relative humidity is 60 ± 5%, light cycle is 14 h / 10 h (day / night), effective radiation of light is 15000 LX); the pots for growing sesame plants are randomly repositioned twice a week to minimize the position effect; after the sesame plants grow for six weeks, randomly select sesame seedlings with consistent growth vigor for the following treatments:

[0043] Control group: Use an equal amount of deionized water as the control;

[0044] CNPs group: 50, 100, 200 mg / L CNPs suspensions;

[0045] CS group: 50, 100, 200 mg / L CS suspensions

[0046] Use a handheld sprayer to spray the sesame leaves on the leaf surface, evenly spray 20 mL of the suspensions with different concentrations in each group above per pot, using deionized water as the control group; set 4 replicates for each concentration treatment; after spraying, inoculate 4 third-instar Spodoptera litura larvae per pot of sesame seedlings, and then cover with a 75 μm fine mesh bag to prevent Spodoptera litura from escaping, and observe the results 24 hours after inoculation.

[0047] The results showed that the treatments with different concentrations of nano-chitosan (CNPs) and chitosan (CS) generally inhibited the growth of Spodoptera litura larvae on sesame leaves. Figure 3 ) Compared with the control group, CS100 (CS concentration of 100 mg / L), CS200, CNPs100, and CNPs200 significantly reduced the growth of Spodoptera litura larvae, by 25.4%, 20.4%, 37.1%, and 30.0% respectively. Especially when the concentration of CNPs was 100 mg / L, the inhibitory effect was more prominent. This indicated that compared with other application doses of CNPs and CS, foliar application of 100 mg / L nano-chitosan was most effective in controlling Spodoptera litura and could prevent Spodoptera litura at an early stage.

[0048] Example 3

[0049] Application of Nano-chitosan (CNPs) and Commercial Insecticide Acetamiprid (Ace) in Controlling Spodoptera litura Larvae on Sesame

[0050] Sesame seedlings were cultivated according to the cultivation method of sesame seedlings in Example 2. After six weeks of plant growth, 20 mL of nano-chitosan (CNPs) with a concentration of 100 mg / L, 100 mg / L chitosan, and commercial insecticide acetamiprid were evenly sprayed on each pot, and an equal amount of ultrapure water was used as the control group (CK). Each treatment was repeated 4 times. Other experimental conditions and experimental operations were the same as those in Example 2.

[0051] Result determination

[0052] 1. Control effect of nano-chitosan (CNPs) and commercial insecticide acetamiprid (Ace) on Spodoptera litura larvae on sesame

[0053] The results were as Figure 4 shown. Foliar spraying of 100 mg / L nano-chitosan significantly inhibited the growth of Spodoptera litura larvae. The weight of Spodoptera litura larvae decreased by 40.5% compared with the control group, and this inhibitory effect was stronger than that of 100 mg / L commercial insecticide, by 17.1%. This indicated that the application of 100 mg / L nano-chitosan could be the best treatment for inhibiting the damage of Spodoptera litura on sesame.

[0054] 2. Effect of nano-chitosan (CNPs) on defense signaling substances in sesame leaves

[0055] The Ca 2+ flux in mesophyll cells of sesame leaves in different treatment groups was measured by a non-invasive micro-test system (NMT100S-SIM-XY). A sensor with a tip diameter of 4.5 ± 0.5 μm was used, and a filling solution (Ca 2+ : 100 mM CaCl2 ), about 50 μm of LIX reagent (Ca2+: XY-SJ-Ca-10) was aspirated from the LIX Holder by a sensor preparation device. After the silver wire was chlorinated, it was inserted into the sensor and calibrated with the corresponding calibration solution. The test could be started when the Nernst slope was within the range of 29 ± 3 mV. Before the test, sesame seedling leaves were fixed in the measurement buffer (0.1 mM CaCl 2 , pH 6.0) for 60 minutes for equilibration. During the measurement, ensure that the tip of the sensor is close to the position closest to the cell surface and keep a distance of about 1-2 μm from the cell surface. The step size for each measurement is 20 μm.

[0056] The contents of the phytohormones jasmonic acid (JA) and salicylic acid (SA) were determined using LC-MS / MS (Vanquish Flex, Germany). 100 mg of broad bean leaves were ground in liquid nitrogen and 1 mL of pre-cooled ethyl acetate (containing 10 μg·mL -1 butylated hydroxytoluene) was added. After vortexing for 15 minutes, the mixture was sonicated in an ice bath for 15 minutes, centrifuged at 4 °C and 12,000 rpm for 10 minutes, and the supernatant was transferred to a new centrifuge tube. After drying the supernatant with a nitrogen evaporator, it was re-dissolved with 200 μL of 70% methanol, vortexed for 5 minutes, then sonicated in an ice bath for 5 minutes again, and centrifuged at 4 °C and 12,000 rpm for 10 minutes to collect 100 μL of the supernatant. Finally, the supernatant was injected into the LC-MS / MS system. The concentrations of JA and SA were quantitatively calculated using the quantitative regression curve of the corresponding standard.

[0057] The results are as Figure 5 shown. Under the condition of no Spodoptera litura stress, compared with the control group, the treatment with nano-chitosan (CNPs) significantly increased the Ca 2+ flow rate by 102.9%, and the effect was higher than that of the treatment with conventional large-particle chitosan (CS) (39.6%) ( Figure 5 A). Especially when sesame was damaged by Spodoptera litura, the treatment with CNPs significantly increased the Ca 2+ flow rate by 193.7%, and the effect was significantly higher than that of the CS treatment (63.3%) ( Figure 5 A).

[0058] In addition, the contents of JA and SA also showed the same trend. When there was no Spodoptera litura, the treatment with CNPs significantly increased the contents of JA and SA by 66.7% and 183.7% respectively, and the treatment with CS significantly increased the contents of JA and SA by 23.7% and 91.5% respectively ( Figure 5 B, C). Similarly, when sesame was damaged by Spodoptera litura, the treatment with CNPs significantly increased the contents of JA and SA by 56.3% and 103.6% respectively, and the treatment with CS significantly increased the contents of JA and SA by 19.6% and 66.0% respectively (Figure 5 B, C), indicating that compared with conventional large - particle chitosan, nano - sized chitosan has a more obvious effect on activating plant defense signals.

[0059] 3. Effects of nano - chitosan (CNPs) on anti - insect substances in sesame leaves

[0060] The relative contents of anti - insect substances in sesame leaves were determined using LC - MS / MS (Vanquish Flex, Germany). 100 mg of sesame leaves were ground in liquid nitrogen and transferred to a 2 - mL centrifuge tube. 1.5 mL of 80% methanol aqueous solution (containing 0.1% formic acid and 0.2 mg / L 2 - chloro - L - phenylalanine as an internal standard) was added. Subsequently, the mixture was vortexed for 1 minute, sonicated in an ice - water bath for 30 minutes, and then centrifuged at 4 °C and 12,000 rpm for 15 minutes to collect the supernatant. After freeze - drying the supernatant, it was re - dissolved in 200 μL of methanol - acetonitrile - water (4:4:2, v / v / v) solution, and then centrifuged at 4 °C and 12,000 rpm for 15 minutes to collect 150 μL of the supernatant. Finally, the supernatant was injected into the LC - MS / MS system for metabolome analysis and quantification. Quality control (QC) samples were prepared with a mixture of all samples in the same amount. For metabolic data, the online tool MetaboAnalyst 5.0 was used. Metabolites with a VIP value > 1 and p < 0.05 (inter - group difference) based on PLS - DA analysis were considered differential biomarker metabolites.

[0061] The results are as Figure 6 shown. Under the stress of Spodoptera litura, the nano - chitosan treatment (CNPs) significantly increased the content of sesamin, an anti - insect substance in sesame leaves, by 204.7%, which is more than 5 times that of the large - particle chitosan treatment CS (39.9%) ( Figure 6 A). In addition, the CNPs treatment significantly increased the content of gardenoside methyl ester, an anti - insect substance in sesame leaves, by 178.1%, which was also significantly higher than that of the CS treatment (23.6%) ( Figure 6 B).

[0062] Example 4

[0063] A method for improving the functional nutrients of sesame seeds based on nano - chitosan, the method comprising the following:

[0064] The field experiment adopted a randomized complete block design, with each treatment group replicated 4 times. After 35 days (before flowering) and 45 days (after flowering) of sesame growth, foliar spraying material treatments were carried out: deionized water (CK), 100 mg / L CS (CS100), and 100 mg / L CNPs (CNPs100) were sprayed respectively, with 10 mL sprayed per plant; in addition to pest management, weeding and fertilization were carried out according to the needs of the crops; finally, after 100 days of sesame plant growth, the nutritional quality of sesame seeds was analyzed by combining metabolomics.

[0065] The results are as Figure 7 shown. Foliar application of 100 mg / L CNPs significantly increased the contents of sesamin ( Figure 7 A), proline ( Figure 7 B), and valine ( Figure 7 C) in sesame seeds by 86.2%, 70.5%, and 247.9% respectively, which were significantly higher than those of sesamin (40.4%) ( Figure 7 A), proline (44.6%) ( Figure 7 B), and valine (198.4%) ( Figure 7 C) after CS treatment.

[0066] The embodiments provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit their execution order. Obvious improvements made by those skilled in the art in combination with the existing well-known common sense also fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving the control effect of Spodoptera litura larvae on sesame and the functional nutrients in sesame seeds, characterized in that, the method comprises: preparing a suspension of nano-chitosan and spraying it on sesame plants; the average size of the nano-chitosan is 21±5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2±9.3 nm and 36.9±1.3 mV respectively; the preparation of the nano-chitosan comprises the following steps: dissolving chitosan in acetic acid, adjusting the pH to 5-6, stirring overnight at room temperature to form a chitosan solution; then adding a tripolyphosphate solution to the chitosan solution, continuously stirring to form a milky white chitosan nano-suspension, centrifuging, washing, and freeze-drying to obtain nano-chitosan; the volume ratio of the tripolyphosphate solution to the chitosan solution is 1:3; the mass concentration of tripolyphosphate in the tripolyphosphate solution is 0.5-1%; the mass concentration of chitosan in the chitosan solution is 0.5-1%.

2. The method according to claim 1, characterized in that, the concentration of the suspension is 50-200 mg / L.

3. The method according to claim 1, characterized in that, the spraying time can be any stage of the sesame seedling stage, growth stage, or flowering stage, or spraying treatment can be carried out in each stage.

4. The method according to claim 1, characterized in that, the spraying amount is 10-30 mL of a nano-chitosan suspension with a concentration of 50-200 mg / L per sesame plant.

5. A method for increasing the Ca 2+ flow rate, salicylic acid and jasmonic acid contents in sesame leaf cells, characterized in that, the method comprises: preparing a suspension of nano-chitosan and spraying it on sesame plants; the average size of the nano-chitosan is 21±5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2±9.3 nm and 36.9±1.3 mV respectively; the preparation of the nano-chitosan comprises the following steps: dissolving chitosan in acetic acid, adjusting the pH to 5-6, stirring overnight at room temperature to form a chitosan solution; then adding a tripolyphosphate solution to the chitosan solution, continuously stirring to form a milky white chitosan nano-suspension, centrifuging, washing, and freeze-drying to obtain nano-chitosan; the volume ratio of the tripolyphosphate solution to the chitosan solution is 1:3; the mass concentration of tripolyphosphate in the tripolyphosphate solution is 0.5-1%; the mass concentration of chitosan in the chitosan solution is 0.5-1%.

6. A method for increasing the contents of sesamin and gardenoside methyl ester, the anti-insect substances in sesame leaves, characterized in that, the method comprises: preparing a suspension of nano-chitosan and spraying it on sesame plants; the average size of the nano-chitosan is 21±5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2±9.3 nm and 36.9±1.3 mV respectively; the preparation of the nano-chitosan comprises the following steps: dissolving chitosan in acetic acid, adjusting the pH to 5-6, stirring overnight at room temperature to form a chitosan solution; then adding a tripolyphosphate solution to the chitosan solution, continuously stirring to form a milky white chitosan nano-suspension, centrifuging, washing, and freeze-drying to obtain nano-chitosan; The volume ratio of the tripolyphosphate solution to the chitosan solution is 1:3; The mass concentration of tripolyphosphate in the tripolyphosphate solution is 0.5 - 1%; The mass concentration of chitosan in the chitosan solution is 0.5 - 1%.

7. A method for controlling Spodoptera litura in sesame by inducing anti-insect substances in sesame leaves based on nano-chitosan, characterized in that, the method includes: preparing nano-chitosan into a suspension and spraying it on sesame plants; the average size of the nano-chitosan is 21 ± 5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2 ± 9.3 nm and 36.9 ± 1.3 mV respectively; The preparation of the nano-chitosan includes the following steps: dissolving chitosan in acetic acid, adjusting the pH to 5 - 6, stirring overnight at room temperature to form a chitosan solution; then adding a tripolyphosphate solution to the chitosan solution, continuously stirring to form a milky white chitosan nano-suspension, centrifuging, washing, and freeze-drying to obtain nano-chitosan; The volume ratio of the tripolyphosphate solution to the chitosan solution is 1:3; The mass concentration of tripolyphosphate in the tripolyphosphate solution is 0.5 - 1%; The mass concentration of chitosan in the chitosan solution is 0.5 - 1%.

8. A method for inducing the improvement of sesamin and amino acids, the functional nutrients in sesame, based on nano-chitosan, characterized in that, the method includes: preparing nano-chitosan into a suspension and spraying it on sesame plants; the average size of the nano-chitosan is 21 ± 5.8 nm, and the hydrodynamic diameter and zeta potential are 262.2 ± 9.3 nm and 36.9 ± 1.3 mV respectively; The preparation of the nano-chitosan includes the following steps: dissolving chitosan in acetic acid, adjusting the pH to 5 - 6, stirring overnight at room temperature to form a chitosan solution; then adding a tripolyphosphate solution to the chitosan solution, continuously stirring to form a milky white chitosan nano-suspension, centrifuging, washing, and freeze-drying to obtain nano-chitosan; The volume ratio of the tripolyphosphate solution to the chitosan solution is 1:3; The mass concentration of tripolyphosphate in the tripolyphosphate solution is 0.5 - 1%; The mass concentration of chitosan in the chitosan solution is 0.5 - 1%.

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