Gingko oligosaccharide chemical modification method, prepared gingko amino-oligosaccharide and application

Abstract

The invention relates to the field of preparation of amino-oligosaccharides, and particularly discloses a chemical modification method of gingko oligosaccharides, prepared gingko amino-oligosaccharides and application of the gingko amino-oligosaccharides, the method comprises the following steps: S1, adding a hydrogen peroxide solution into the gingko oligosaccharides, and stirring to prepare a primary mixed solution, the glycosyl of the gingko oligosaccharides at least comprising glucose; s2, adding ammonium carbonate into the primary mixed solution, stirring, and stopping stirring until no bubble is generated in the solution; and S3, after standing reaction, drying to obtain the gingko amino-oligosaccharide. The invention also discloses the gingko amino-oligosaccharide prepared by adopting the modification method and application of the prepared gingko amino-oligosaccharide in plant growth. The ginkgo oligosaccharide is chemically modified, the molecular structure of the ginkgo oligosaccharide is connected with an amino functional group, the ginkgo oligosaccharide is converted into the ginkgo amino oligosaccharide, the function is more comprehensive and the effect is better due to the introduction of the functional group, and the efficiency of the ginkgo oligosaccharide in plant disease resistance and quality improvement is improved.

Description

Technical Field

[0001] The present application relates to the field of amino oligosaccharide preparation, and more specifically, to a method for chemically modifying ginkgo oligosaccharides, the prepared ginkgo amino oligosaccharides, and their application. Background Art

[0002] In recent years, studies have found that oligosaccharides can be used for plant protection, enhance plant disease resistance, promote plant growth, and increase crop yields. However, most of the current research on oligosaccharides focuses on chitosan oligosaccharides and alginate oligosaccharides, and the types of oligosaccharides are limited.

[0003] Ginkgo oligosaccharides are biologically active oligosaccharides extracted from the outer seed coat of the Ginkgo biloba plant. Their glycosyl groups primarily consist of glucose, fructose, and mannose. Ginkgo oligosaccharides are used to induce stress resistance in plants and enhance their defenses against pests and diseases. Their application in plant growth has promising prospects for improving disease resistance and increasing productivity.

[0004] As a new type of plant growth regulator, it is of great significance to improve the efficacy of ginkgo oligosaccharides in plant disease resistance and quality improvement through chemical modification. Summary of the Invention

[0005] In order to chemically modify ginkgo oligosaccharides to enhance their efficacy in plant disease resistance and quality improvement, the present application provides a method for chemically modifying ginkgo oligosaccharides, the obtained ginkgo amino oligosaccharides, and their applications.

[0006] In the first aspect, the present application provides a method for chemically modifying ginkgo oligosaccharides, using the following technical solution: A method for chemically modifying ginkgo oligosaccharides comprises the following steps: S1. adding hydrogen peroxide solution to ginkgo oligosaccharide and stirring to prepare a primary mixed solution, wherein the glycosyl group of ginkgo oligosaccharide includes at least glucose; S2. Add ammonium carbonate to the initial mixed solution and stir until no bubbles are generated in the solution; then stop stirring; S3. After standing to react, drying is performed to obtain Ginkgo biloba oligosaccharide.

[0007] By adopting the above technical solution, the present application first uses hydrogen peroxide as an oxidant to oxidize ginkgo oligosaccharides, so that the functional group on the sixth carbon atom of glucose in the ginkgo oligosaccharides is oxidized to a carboxyl group, and then after adding ammonium carbonate, the ammonium carbonate is hydrolyzed under the action of hydrogen peroxide to generate ammonia and heat energy. Under the exothermic conditions of the reaction between ammonium carbonate and hydrogen peroxide, the ammonia reacts with the carboxyl group on the glucose to introduce an amino group, thereby achieving amino modification on the glucose molecule, and then achieving modification of ginkgo amino oligosaccharides to obtain ginkgo amino oligosaccharides.

[0008] Finally, through chemical reaction, amino functional groups are connected to the ginkgo oligosaccharide molecules to realize the conversion of ginkgo oligosaccharide into ginkgo amino oligosaccharide, which has more comprehensive functions and better efficacy, especially for the prevention and control of crop diseases such as tomato virus disease. The effective ingredient of ginkgo oligosaccharide with a concentration of 2% by mass is 45g / hm2. 2 The prevention and treatment effect of viral diseases is only 60%, while the effective ingredient of Ginkgo biloba oligosaccharide prepared by chemical modification in this application is 45g / hm2 at a concentration of 2%. 2 The effectiveness of preventing and controlling viral diseases can reach more than 80%, the prevention and control effect is significantly improved, and the resulting fruit has a better taste.

[0009] The chemical modification in this application not only fills the application gap of ginkgo oligosaccharides in the field of plant growth, but also has better effects and provides key technical support. In addition, in this application, ginkgo oligosaccharides are used as the base reactant. The glucose units in ginkgo oligosaccharides are connected by 1,4-glycosidic bonds. The C6 hydroxyl group is located at the end of the molecular chain, with a high degree of exposure and is more susceptible to oxidation. The C6 position of the internal glucose unit has a reduced oxidation efficiency due to steric hindrance. The control of the modification method and degree in this application is used to achieve selective modification of ginkgo oligosaccharides, realize the combination of the sixth carbon atom of the hexose sugar of ginkgo oligosaccharide with ammonia, and generate an amino modification of the six carbon atoms, which has a better anti-disease effect and quality improvement effect.

[0010] Optionally, the mass concentration of hydrogen peroxide in the hydrogen peroxide solution in step S1 is 3±0.5%, and the mass ratio of ginkgo oligosaccharides to hydrogen peroxide is 1:(4-5).

[0011] More preferably, the mass concentration of hydrogen peroxide in the hydrogen peroxide solution in step S1 is 3%, and the mass ratio of ginkgo oligosaccharide to hydrogen peroxide is 1:4.3.

[0012] By adopting the above technical solution, the concentration and addition amount of hydrogen peroxide are controlled to achieve selective oxidation of ginkgo oligosaccharides to achieve carboxylation, and then achieve subsequent amino modification. If the concentration of hydrogen peroxide is too high or the addition amount is too much, it will cause damage to the structure of ginkgo oligosaccharides. When the concentration is too low or the addition amount is too small, the degree of oxidation is insufficient, which affects the degree of subsequent amino modification and thus affects the efficacy of the final ginkgo amino oligosaccharides.

[0013] Optionally, in step S2, the mass ratio of ginkgo oligosaccharide to ammonium carbonate is 1:(0.1-0.25), more preferably 1:0.17.

[0014] By adopting the above technical solution, the amount of ammonium carbonate added is controlled for amino modification of ginkgo oligosaccharides, and the obtained ginkgo amino oligosaccharides have better efficacy.

[0015] Optionally, in step S1, the saccharide groups of the ginkgo oligosaccharide include glucose, fructose and mannose in a mass ratio of (3.5-4): (5.8-6.2): 1.

[0016] Optionally, the glycosyl groups of the ginkgo oligosaccharide in step S1 include: 226.22 μg / mg of glucose, 352.51 μg / mg of fructose, and 58.842 μg / mg of mannose.

[0017] Optionally, when adding ammonium carbonate in step S2, add it evenly in 2-4 times, and add it once every 5-10 minutes. After adding, stir until no bubbles are generated.

[0018] By adopting the above technical solution, ammonium carbonate is added in batches, a small amount is added each time and fully stirred after addition, so as to avoid adding too much ammonium carbonate at one time, which may cause the reaction to be too violent, generate a large number of bubbles, and affect the stability and controllability of the reaction.

[0019] Optionally, sodium pyrophosphate is added to the hydrogen peroxide solution in step S1, and the amount of sodium pyrophosphate added is 0.01-0.02 wt % of the amount of hydrogen peroxide added.

[0020] By adopting the above technical solution, hydrogen peroxide realizes the oxidation of ginkgo oligosaccharides, oxidizing the hydroxyl functional group on the sixth carbon atom of the hexose to a carboxyl group. The hydrogen peroxide also participates in the reaction with ammonium carbonate to generate NH3. Since the addition of ammonium carbonate in an alkaline environment will cause the decomposition of hydrogen peroxide, reducing the process of hydrogen peroxide participating in the reaction of ammonium carbonate to generate amines, the present application adds sodium pyrophosphate as a stabilizer for hydrogen peroxide to reduce the degradation of hydrogen peroxide, so that it can better react with ammonium carbonate to generate amines and thus achieve amino modification.

[0021] In a second aspect, the present application provides a ginkgo amino oligosaccharide, which adopts the following technical solution: A ginkgo amino oligosaccharide is prepared by a method for chemically modifying the ginkgo oligosaccharide.

[0022] By adopting the above technical solution and the method provided in the present application, the amino group modification on the sixth carbon atom of glucose in ginkgo oligosaccharides is achieved. The obtained ginkgo amino oligosaccharides have better disease resistance and quality improvement performance than ginkgo oligosaccharides used in plant growth. For example, for the prevention and treatment of viral diseases, 2% ginkgo oligosaccharides have a prevention and treatment effect of only 60%; while 2% ginkgo amino oligosaccharides can achieve a prevention and treatment effect of more than 80% for viral diseases.

[0023] Moreover, compared with conventional glucosamine, the ginkgo oligosaccharide in the present application also contains fructose and mannose in the sugar group. In the process of amino-modification of oligosaccharides, it also has a certain influence on fructose and mannose, and ultimately has a certain influence on the molecular structure of ginkgo oligosaccharides. Moreover, the complex three-dimensional conformation formed by the combination of multiple sugar groups containing the above three sugar groups is found to have better efficacy than single glucosamine after amino modification.

[0024] In a third aspect, the present application provides an application of Ginkgo biloba oligosaccharides, using the following technical solution: Application of ginkgo amino oligosaccharide in plant growth.

[0025] By adopting the above technical solution, the ginkgo amino oligosaccharide in the present application is prepared based on ginkgo oligosaccharide through chemical amino modification, which not only fills the gap in the application of ginkgo oligosaccharide in the plant growth process, but also has more comprehensive functions and better efficacy after amino modification, especially for plant growth, such as the prevention and treatment of tomato virus disease and the improvement of fruit quality.

[0026] In summary, this application has the following beneficial effects: 1. This application is based on ginkgo oligosaccharide. By chemical reaction, amino functional groups are connected to ginkgo oligosaccharide molecules to realize the conversion of ginkgo oligosaccharide into ginkgo amino oligosaccharide. It has more comprehensive functions and better efficacy, especially for the prevention and treatment of plant viral diseases such as tomato viral disease. The effective ingredient of ginkgo oligosaccharide with a concentration of 2% by mass is 45g / hm2. 2 The prevention and treatment effect of viral diseases is only 60%, while the effective ingredient of Ginkgo biloba oligosaccharide prepared by chemical modification in this application is 45g / hm2 at a concentration of 2%. 2 The effect of preventing and controlling viral diseases can reach more than 80%, the prevention and control effect is significantly improved, and the obtained fruit has a better taste; 2. In the present application, hydrogen peroxide is first used as an oxidant to oxidize ginkgo oligosaccharides, so that the functional group on the sixth carbon atom of glucose in the ginkgo oligosaccharides is oxidized to a carboxyl group. Then, after adding ammonium carbonate, the ammonium carbonate is hydrolyzed under the action of hydrogen peroxide to generate ammonia and heat energy. Under the exothermic conditions of the reaction between ammonium carbonate and hydrogen peroxide, the ammonia reacts with the carboxyl group on the glucose to introduce an amino group, thereby achieving amino modification on the glucose molecule, and then achieving modification of ginkgo amino oligosaccharides to obtain ginkgo amino oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the ion chromatogram of the mixed standard solution during the ion chromatography test in Example 1 of the present application; Figure 2This is a standard curve diagram of glucosamine in the ion chromatography test in Example 1 of the present application; Figure 3 This is a standard curve diagram of glucose in the ion chromatography test in Example 1 of the present application; Figure 4 This is a standard curve diagram of mannose in the ion chromatography detection test in Example 1 of the present application; Figure 5 This is a standard curve diagram of fructose in the ion chromatography detection test in Example 1 of the present application; Figure 6 This is the ion chromatogram of Ginkgo biloba oligosaccharide obtained in Example 1 of the present application; Figure 7 This is the ion chromatogram of the untreated ginkgo oligosaccharide in Example 1 of the present application. DETAILED DESCRIPTION

[0028] The present application is further described in detail below with reference to the examples. It is particularly noted that if no specific conditions are specified in the following examples, the reactions are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0029] Unless otherwise specified, the percentages in the following examples are by mass.

[0030] The ginkgo oligosaccharides in this application can be obtained from common commercial sources or prepared by the following methods. The ginkgo oligosaccharides in the following examples were prepared by the following methods: a. Crushing: crush the outer seed coat of ginkgo into a coarse powder with a diameter of 1-3mm; b. Wetting: put the coarse powder into an extraction tank equipped with an ultrasonic device, add 2 times the mass of water to moisten it until the moisture content is 50%; c. Ultrasonication to break up the cell walls: 400W ultrasound at 35°C for 15 minutes; d. Water extraction: Add purified water at a material-liquid ratio of 1:5 (i.e., the material-liquid ratio of the crushed coarse powder to water is 1:5), extract at 90°C for 5 minutes to obtain the extract; e. Filtration: Release the extract and filter with a 180-mesh filter to obtain a filtrate; f. Concentration: The filtrate was concentrated by evaporation at 95°C under normal pressure to a syrupy state to obtain a concentrate; g. Alcohol precipitation: add 95% ethanol by weight to the concentrate in a ratio of 3:1 and stir thoroughly; h. Precipitation: Let the mixture stand for 1 hour to allow the oligosaccharides to fully precipitate. Filter out the ethanol solution, then wash the precipitate with 95% ethanol and freeze-dry to obtain ginkgo oligosaccharides. The glycosyl groups in the obtained ginkgo oligosaccharides include 226.22 μg / mg of glucose, 352.51 μg / mg of fructose, and 58.842 μg / mg of mannose. The mass ratio of glucose, fructose, and mannose is 3.84:6:1.

[0031] Example 1

[0032] A method for chemically modifying ginkgo oligosaccharides comprises the following steps: S1. Add hydrogen peroxide solution to ginkgo oligosaccharide at room temperature and stir. Stir evenly and continue stirring for 10 minutes to prepare a primary mixed solution. The glycosyl group of ginkgo oligosaccharide includes: glucose 226.22 μg / mg, fructose 352.51 μg / mg, mannose 58.842 μg / mg, the mass concentration of hydrogen peroxide in the hydrogen peroxide solution is 3%, and the mass ratio of ginkgo oligosaccharide to hydrogen peroxide is 1:4.3. S2. Add ammonium carbonate to the initial mixture while stirring continuously. The mass ratio of ginkgo oligosaccharide to ammonium carbonate is 1:0.17. Add ammonium carbonate in three times, each time adding 1 / 3 of the total amount of ammonium carbonate, and add once every 10 minutes. After all ammonium carbonate is added, continue stirring until no bubbles are generated in the solution, then stop stirring. S3. After standing for 4 hours, the mixture was dried to obtain Ginkgo biloba oligosaccharide.

[0033] Example 2

[0034] A method for chemically modifying ginkgo oligosaccharides comprises the following steps: S1. Add hydrogen peroxide solution to ginkgo oligosaccharide at room temperature and stir. Stir evenly and continue stirring for 5 minutes to prepare a primary mixed solution. The glycosyl group of ginkgo oligosaccharide includes: glucose 226.22 μg / mg, fructose 352.51 μg / mg, mannose 58.842 μg / mg, the mass concentration of hydrogen peroxide in the hydrogen peroxide solution is 2.5%, and the mass ratio of ginkgo oligosaccharide to hydrogen peroxide is 1:5. S2. Add ammonium carbonate to the initial mixed solution while stirring continuously. The mass ratio of ginkgo oligosaccharide to ammonium carbonate is 1:0.1, more preferably 1:0.17. Add ammonium carbonate in three times, each time adding 1 / 3 of the total amount of ammonium carbonate, and add once every 5 minutes. After all the ammonium carbonate is added, continue stirring until no bubbles are generated in the solution, then stop stirring. S3. After standing for 3 hours, the mixture was dried to obtain ginkgo amino oligosaccharide.

[0035] Example 3

[0036] A method for chemically modifying ginkgo oligosaccharides comprises the following steps: S1. Add hydrogen peroxide solution to ginkgo oligosaccharide at room temperature and stir. Stir evenly and continue stirring for 15 minutes to prepare a primary mixed solution. The glycosyl group of ginkgo oligosaccharide includes: glucose 226.22 μg / mg, fructose 352.51 μg / mg, mannose 58.842 μg / mg, the mass concentration of hydrogen peroxide in the hydrogen peroxide solution is 3.5%, and the mass ratio of ginkgo oligosaccharide to hydrogen peroxide is 1:4. S2. Add ammonium carbonate to the initial mixed solution while stirring continuously. The mass ratio of ginkgo oligosaccharide to ammonium carbonate is 1:0.25, more preferably 1:0.17. Add ammonium carbonate in three times, each time adding 1 / 3 of the total amount of ammonium carbonate, and add once every 10 minutes. After all the ammonium carbonate is added, continue stirring until no bubbles are generated in the solution, then stop stirring. S3. After standing for 6 hours, the mixture was dried to obtain ginkgo amino oligosaccharide.

[0037] Example 4

[0038] A method for preparing ginkgo amino oligosaccharide is carried out according to the method in Example 1, except that the mass ratio of ginkgo oligosaccharide to hydrogen peroxide is 1:5.5.

[0039] Example 5

[0040] A method for preparing ginkgo amino oligosaccharide is carried out according to the method in Example 1, except that the mass ratio of ginkgo oligosaccharide to hydrogen peroxide is 1:3.

[0041] Example 6

[0042] A method for preparing ginkgo amino oligosaccharide is carried out according to the method in Example 1, except that the mass concentration of hydrogen peroxide in the hydrogen peroxide solution is 4%.

[0043] Example 7

[0044] A method for preparing ginkgo amino oligosaccharide is carried out according to the method in Example 1, except that sodium pyrophosphate is also added to the hydrogen peroxide solution in step S1, and the amount of sodium pyrophosphate added is 0.01wt% of the amount of hydrogen peroxide added.

[0045] Example 8

[0046] A method for preparing ginkgo amino oligosaccharide is carried out according to the method in Example 1, except that sodium pyrophosphate is also added to the hydrogen peroxide solution in step S1, and the amount of sodium pyrophosphate added is 0.02 wt% of the amount of hydrogen peroxide added.

[0047] Comparative Example 1 A method for preparing ginkgo amino oligosaccharides is carried out according to the method in Example 1, except that the hydrogen peroxide solution in step S1 is replaced by an equal amount of periodic acid solution, and the mass concentration of the periodic acid solution is 3%.

[0048] Comparative Example 2 A method for preparing ginkgo amino oligosaccharides is carried out according to the method in Example 1, except that the hydrogen peroxide solution in step S1 is replaced by an equal amount of nitric acid solution, and the mass concentration of the nitric acid solution is 10%.

[0049] Performance testing 1. The ginkgo amino oligosaccharide prepared in Example 1 was verified by an external standard method using an ion chromatograph (pulsed amperometric detector) to confirm the introduction of amino groups into the ginkgo oligosaccharide. The specific parameters are: Chromatographic conditions: chromatographic column PA100 (250 mm x 4.0 mm); flow rate 0.4 mL / min; column temperature 30°C; injection volume 25 µL; eluent: phase A: pure water; phase B: 100 mM NaOH solution + 50 mM NaOAc solution; elution gradient: 0-20 min: 95% phase A + 5% phase B (v / v); 35-60 min: 100% phase B; 60.1-75.0 min: 95% phase A + 5% phase B (v / v); Potential waveform: E1-E5: 0.10V / -2.00V / 0.60V / -0.10V / -0.10V; t1-t5: 0.40s / 0.02s / 0.01s / 0.01s / 0.06s; ts: 200ms; Detection method: pulsed amperometric mode; working electrode: Flexcell gold electrode.

[0050] The detection limit of glucosamine under the above chromatographic conditions was 0.008 mg / L.

[0051] Standard curve drawing: A commercially available mixed standard solution containing glucosamine, glucose, mannose, and fructose (glucosamine concentration 1000 mg / L, glucose concentration 1000 mg / L, mannose concentration 1000 mg / L, fructose concentration 1000 mg / L) was used for gradient dilution to prepare standard solutions of different concentrations as shown in Table 1. Table 1:

[0052] Under the above chromatographic conditions, the commercially available mixed standard solution was injected, and the chromatogram of the obtained mixed standard solution was as follows: Figure 1 As shown, the mass concentration of glucosamine (mg / L) is used as the horizontal axis to draw the standard curve as shown in Figure 2-5 As shown, Figure 2 is the standard curve of glucosamine, Figure 3 is the standard curve for glucose, Figure 4 is the standard curve of mannose, Figure 5 is the standard curve of fructose, and the corresponding linear equation is obtained.

[0053] Sample testing: 1) Accurately weigh 0.5 g of the Ginkgo biloba oligosaccharide sample prepared in Example 1, dissolve it in pure water and dilute to 100 mL, dilute it appropriately and filter it through a 0.22 μm filter membrane to obtain a 003 sample mixed solution, and then inject the sample under the same chromatographic conditions as above to obtain the following: Figure 6 The chromatogram in the figure is the chromatogram of Ginkgo biloba amino oligosaccharide; 2) Accurately weigh 0.5g of the untreated ginkgo oligosaccharide sample from step S1 of Example 1 as a control group, dissolve, dilute and filter according to the method in 1) above to obtain 001 sample, inject the sample, and obtain the following: Figure 7 The chromatogram in Figure 2 is the chromatogram of ginkgo oligosaccharides.

[0054] In addition, the contents of glucosamine, glucose, mannose and fructose in the ginkgo oligosaccharide samples and ginkgo amino oligosaccharides were statistically analyzed, and the results are shown in Table 2 below.

[0055] Table 2:

[0056] As you can see, combined Figure 1 , Figure 7 There is no peak at the glucosamine peak, which does not contain glucosamine. Figure 6 The prepared ginkgo oligosaccharide showed a glucosamine peak. The present application provides a method for achieving amino modification of ginkgo oligosaccharide.

[0057] In addition, the above operation was performed on the ginkgo amino oligosaccharides prepared in the remaining examples and comparative examples to measure the glucosamine content therein. The results are shown in Table 3 below.

[0058] Table 3:

[0059] Combined with the test results in Table 3 above, the method provided in the embodiment of the present application realizes the amino modification of ginkgo oligosaccharides and generates glucosamine. Combined with the test results of Example 4 and Example 6, when the amount of hydrogen peroxide added is too much or the concentration is too high, it causes certain damage to the glucose structure, and the content of glucosamine generated is reduced. When the amount of hydrogen peroxide added in Example 5 is too little, the oxidation effect on glucose is limited, resulting in a decrease in the glucosamine generated. With reference to the test results of Example 7 and Example 8, when sodium pyrophosphate is added to hydrogen peroxide, it has a certain stabilizing protective effect on glucose, and the content of glucosamine obtained is improved. Combined with the test results of Comparative Example 1 and Comparative Example 2, when the oxidant is nitric acid or periodic acid solution, the content of glucosamine obtained is almost 0, and the type, concentration and amount of oxidant added will affect the final modification effect.

[0060] 2. Field trial of drug efficacy against tomato virus diseases Select areas with a history of tomato virus disease, with continuous cropping for at least three years, to ensure a high natural incidence rate. Before sowing, apply lime at a rate of 100 kg / mu for disinfection, plow the soil to a depth of 30 cm to reduce residual viruses, and maintain an average daily temperature of 25–30°C and a relative humidity of 60–70% in the greenhouse. Then, sow Provence cultivar tomatoes with a planting density of 60 cm in row and 40 cm in plant spacing, in 12 plots with 30 plants per plot.

[0061] The ginkgo oligosaccharide prepared in Example 1 was used as the test group. After being prepared into a 2% solution, the solution was diluted and sprayed at an effective ingredient rate of 45 g / hectare. In addition, the unmodified ginkgo oligosaccharide was used as the control group 1. After being prepared into a 2% solution, the solution was diluted and sprayed at an effective ingredient rate of 45 g / hectare. In addition, commercially available glucosamine was used as the control group 2. After being prepared into a 2% solution, the solution was diluted and sprayed at an effective ingredient rate of 45 g / hectare. Then, clean water was used as the blank group. The reagents of the test group, control group 1, control group 2, and blank group were sprayed on tomato plants in 12 planted areas as parallel tests, with each group spraying 3 areas. The spraying area of each group was separated from the spraying area of other groups. The spraying areas of the test group and the control group were treated areas, and the spraying area of the blank group was a blank area. In the experimental field, the first symptoms of tomatoes were seen by spraying.

[0062] 3. Viral disease monitoring Viral diseases are graded according to the following criteria.

[0063] Level 0: No symptoms (no visible lesions on leaves, stems and fruits).

[0064] Level 1: Heart leaves have clear veins and slight mosaic (only the top tender leaves have light green or yellow striped transparent veins (clear veins), or slight yellow and green mottled (mosaic)).

[0065] Level 3: Mosaic on the heart leaves and middle leaves (obvious yellow-green mosaic appears on the top 3-5 leaves and the middle mature leaves, and the mottled area accounts for 10-30% of the leaf area).

[0066] Level 5: The heart and middle leaves are mosaic, a few leaves are deformed, wrinkled or the plant is slightly dwarfed (the mosaic area is >30%, some leaf edges are curled and wrinkled, and the plant height is 10-20% lower than that of healthy plants).

[0067] Level 7: Severe mosaic, most leaves are deformed, wrinkled or the plant is dwarfed (more than 70% of the leaves are severely mosaic, the veins are wrinkled into "chicken claws", and the plant height is reduced by 30-50%).

[0068] Level 9: Severe mosaic, leaves are obviously deformed and linear, the plant is severely dwarfed or even dead (the leaves are as thin as silk threads, only the main veins are retained, the plant height is less than 50% of the normal plant, and some plants are dead).

[0069] 4. Calculation of drug efficacy The efficacy calculation method and formula used are as follows: calculate the disease index and control effect of each area, and calculate the average control effect of each treatment.

[0070] Disease index (%) = [∑ (number of diseased plants × disease level) / (total number of plants × highest disease level)] × 100%; Control effect (%) = [(disease index of blank area - disease index of treated area) / disease index of blank area] × 100%.

[0071] Ten days after application, observe whether the pesticide causes any damage to the tomato plants.

[0072] When conducting efficacy investigations, it is also necessary to examine the effects of the pesticide on non-target organisms (such as beneficial insects) and surrounding crops.

[0073] The statistical results of the prevention effect are shown in Table 4.

[0074] Table 4:

[0075] Referring to the test results in Table 4 above, the ginkgo amino oligosaccharide prepared in the present application has a better preventive and therapeutic effect on tomato virus disease than ginkgo oligosaccharide, and compared with single glucosamine, the ginkgo amino oligosaccharide in the present application, as a three-dimensional polysaccharide structure, has a better preventive and therapeutic effect on tomato virus disease.

[0076] In addition, the taste of the tomatoes obtained during the field test was evaluated. The tomatoes obtained after the treatment in Example 1 had an excellent taste of sandy but not floury, solid but not hollow, and sweet but not sticky. Ginkgo amino oligosaccharide has a certain effect on improving the taste of tomatoes.

[0077] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for chemical modification of ginkgo oligosaccharides, characterized in that: The following steps are involved: S1. adding hydrogen peroxide solution to ginkgo oligosaccharide and stirring to prepare a primary mixed solution, wherein the glycosyl group of ginkgo oligosaccharide includes at least glucose; S2. Add ammonium carbonate to the initial mixed solution and stir until no bubbles are generated in the solution; then stop stirring; S3, after standing for reaction, drying to obtain ginkgo amino oligosaccharide; The mass concentration of hydrogen peroxide in the hydrogen peroxide solution in step S1 is 3±0.5%, and the mass ratio of ginkgo oligosaccharide to hydrogen peroxide is 1:(4-5); In step S2, the added mass ratio of ginkgo oligosaccharide to ammonium carbonate is 1:(0.1-0.25).

2. The method for chemical modification of ginkgo oligosaccharides according to claim 1, characterized in that: In step S2, the added mass ratio of ginkgo oligosaccharide to ammonium carbonate is 1:0.

17.

3. The method for chemical modification of ginkgo oligosaccharides according to claim 1, characterized in that: In step S1, the glycosyl groups of ginkgo oligosaccharides include glucose, fructose and mannose in a mass ratio of (3.5-4): (5.8-6.2):

1.

4. The method for chemical modification of ginkgo oligosaccharides according to claim 1, characterized in that: The glycosyl groups of the ginkgo oligosaccharide in step S1 include: 226.22 μg / mg of glucose, 352.51 μg / mg of fructose, and 58.842 μg / mg of mannose.

5. The method for chemical modification of ginkgo oligosaccharides according to claim 1, characterized in that: When adding ammonium carbonate in step S2, add it evenly in 2-4 times, and add it once every 5-10 minutes. After adding, stir until no bubbles are generated.

6. The method for chemical modification of ginkgo oligosaccharides according to claim 1, characterized in that: In step S1, sodium pyrophosphate is added to the hydrogen peroxide solution, and the amount of sodium pyrophosphate added is 0.01-0.02 wt % of the amount of hydrogen peroxide added.

7. Ginkgo amino oligosaccharide prepared by the method for chemical modification of ginkgo oligosaccharide according to any one of claims 1 to 6.

8. Use of the ginkgo amino oligosaccharide according to claim 7 in plant growth.

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