Enzymatic gleditsia sinensis saponin-copper chelate, preparation method thereof and application of chelate in prevention and treatment of citrus canker

Abstract

The invention discloses an enzymolysis gleditsia sinensis saponin-copper chelate as well as a preparation method and application thereof in prevention and treatment of citrus canker, and belongs to the technical field of crop prevention and treatment. The preparation method comprises the following steps: extracting gleditsia sinensis saponin, purifying the gleditsia sinensis saponin, carrying out enzymatic degradation on the gleditsia sinensis saponin, and then adding CuSO4. 5H2O to carry out chelation reaction to obtain the enzymolysis gleditsia sinensis saponin-copper chelate. The initial contact angle of an aqueous solution of the chelate on citrus leaves is small, the chelate can be rapidly wetted and spread on the leaves, the deposition effect is good, the chelate has excellent leaf adhesion performance and anti-scouring capacity, meanwhile, the enzymolysis gleditsia sinensis saponin-copper chelate has excellent inhibitory activity on xanthomonas carpesium, and the enzymolysis gleditsia sinensis saponin-copper chelate can be used for preparing the citrus leaf-copper chelate. The compound can be used as a green, environment-friendly, safe and efficient bactericide, and has excellent application potential in the aspect of citrus canker prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of crop control technology, specifically relating to an enzymatic hydrolysis of saponin-copper chelate, its preparation method, and its application in the control of citrus canker. Background Technology

[0002] Citrus is the world's largest fruit crop. China ranks first in the world in both citrus planting area and output, and the scale of citrus cultivation in my country has continued to expand in recent years. However, pests and diseases have had a significant impact on the development of the citrus industry. Among them, citrus canker, due to its high infectivity, high pathogenicity, and difficulty in control, has become a major factor restricting the healthy development of the citrus industry.

[0003] Citrus canker is a bacterial disease primarily caused by *Xanthomonas campestris* pv. Citri. *Xanthomonas campestris* infects citrus fruits, leaves, and shoots through stomata and wounds during the young fruit stage or new shoot emergence period. Infected citrus plants develop water-soaked lesions, which later become corky and crater-like. Severe infection can cause significant leaf and fruit drop, affecting fruit quality, leading to substantial yield reduction, and even plant death, severely impacting economic benefits.

[0004] Currently, there is no cure for citrus canker, and prevention and control remain the primary methods. Chemical agents are the most common control method, particularly inorganic copper preparations such as copper sulfate, Bordeaux mixture, and Kocide 3000. However, these preparations suffer from poor water solubility and weak plant conductivity, resulting in ineffective systemic treatment. Growers often resort to multiple applications to strengthen control. However, during application, 20-30% of these inorganic copper preparations bounce, splash, and slide off the leaves, making it difficult to adhere. Furthermore, copper crystals adhering to the leaves are easily washed away by rain. This not only leads to waste and low bioavailability, forcing growers to increase application rates, but also poses a risk of water, soil, and metal pollution to local orchards from the sliding copper. Therefore, developing an active preparation that can effectively deposit and adhere to the surface of citrus leaves, resist rain erosion, and exhibit stable performance while efficiently inhibiting Xanthomonas carpetii is urgently needed.

[0005] Chinese patent CN115812734B discloses the application of a fungicide in the prevention and control of citrus canker. The fungicide solution contains one or more chemical reagents such as methanol, ethanol, glycerol, ethylene glycol, and acetonitrile, which poses potential hazards during application.

[0006] Chinese patents CN115948299B and CN118978986A respectively disclosed the application of Pseudomonas and Bacillus belye as fungicides in citrus canker. However, these microbial pesticides are not only slow to take effect and have a short shelf life, but also have poor wetting and adhesion on citrus leaves. Summary of the Invention

[0007] The first technical problem to be solved by this invention is to provide an enzymatic hydrolysis of saponin-copper chelate, which has an excellent inhibitory effect on Xanthomonas carpetii. The second technical problem to be solved by this invention is to provide a method for preparing the enzymatic hydrolysis of saponin-copper chelate, which utilizes the enzymatic degradation of saponin and the chelation reaction of copper ions to enhance the antibacterial activity and improve the properties of copper on the surface of citrus leaves, such as wetting deposition, adsorption, and anti-erosion. The third technical problem to be solved by this invention is to provide the application of the enzymatic hydrolysis of saponin-copper chelate in the prevention and control of citrus canker and the inhibition of Xanthomonas carpetii.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing an enzymatic hydrolysis saponin-copper chelate includes the following steps:

[0010] 1) Crude saponin extract: Soapberry powder was dissolved in an alcohol solution for extraction, centrifuged, and freeze-dried to obtain crude saponin extract;

[0011] 2) Purification of saponin: The crude saponin extract obtained in step 1) was redissolved in water and purified using macroporous resin. The eluent of saponin was collected, rotary evaporated, dialyzed, and freeze-dried to obtain purified saponin.

[0012] 3) Enzymatic degradation of saponin from saponins: Dissolve the purified saponin from saponins obtained in step 2) in water, add enzyme for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, centrifuge the hydrolysate, freeze-dry the supernatant, purify it with macroporous resin, elute, collect the eluent, concentrate it, freeze-dry it, and obtain the purified enzymatically degraded saponin.

[0013] 4) Chelation reaction: Dissolve the enzyme-degraded saponin obtained in step 3) in water, then add CuSO4·5H2O to carry out the chelation reaction. After the reaction is completed, dialyze, collect the solution in the bag and freeze dry to obtain the enzyme-hydrolyzed saponin-copper chelate.

[0014] Further, in step 1), the mass-to-volume ratio of soapberry powder to alcohol solution is 1:10; the concentration of alcohol solution is 80%; the extraction temperature is 30-60 ℃; the extraction time is 1-4 h; and the stirring speed is 300-800 rpm.

[0015] Further, in step 2), the elution step is as follows: first, use distilled water to elute for 3 column volumes to remove impurities, then use 90% ethanol solution to elute, and collect the 90% ethanol eluent rich in saponins.

[0016] Further, in step 3), the enzyme is selected from one or more combinations of cellulase, xylanase, hemicellulase, Viscozyme L, and snail enzyme; the mass ratio of saponin to enzyme is 1:50; the enzymatic hydrolysis temperature is 30-50 ℃, the enzymatic hydrolysis time is 2 h, and the pH is 4.0-4.5.

[0017] Further, in step 3), the purification steps are as follows: first, use distilled water to wash away impurities, and then use a 10%~90% ethanol solution to wash away impurities.

[0018] Further, in step 4), the concentration of the saponin aqueous solution is 10 mg / mL; the pH of the system is adjusted to 4.0-6.0 using 0.01M hydrochloric acid solution.

[0019] Further, in step 4), the mass ratio of saponin aqueous solution to CuSO4·5H2O is 1:0.5~1, the reaction temperature is 25-50 ℃, and the reaction time is 0.5-2 h.

[0020] Furthermore, the enzymatic hydrolysis of saponin-copper chelate obtained by the aforementioned method is described.

[0021] Furthermore, the application of the enzymatic hydrolysis of saponins-copper chelates in the prevention and control of citrus canker.

[0022] Furthermore, the application of the described enzymatic hydrolysis of saponins-copper chelates in inhibiting Xanthomonas carpetii.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The enzymatic hydrolysed saponin-copper chelate prepared by the present invention has good solubility in water.

[0025] (2) The enzymatic hydrolysed saponin-copper chelate droplets prepared in this invention have a small initial contact angle on the surface of citrus leaves, which is much lower than that of copper sulfate droplets and Bordeaux mixture droplets, and have excellent wettability on the surface of citrus leaves.

[0026] (3) The enzymatic hydrolysed saponin-copper chelate prepared in this invention has significantly improved deposition effect, adhesion ability and erosion resistance on citrus leaves compared with copper sulfate and Bordeaux mixture.

[0027] (4) The copper in the enzymatic hydrolyzed saponin-copper chelate prepared by the present invention has good stability. When the pH is 10.0, the copper solubility in the chelate is greater than 80%, and the copper sulfate solubility is less than 1%. Therefore, the enzymatic hydrolyzed saponin-copper chelate can be applied together with other agents or foliar fertilizers in different pH ranges.

[0028] (5) In this invention, the inhibitory activity against Xanthomonas carpetii is significantly improved after copper sulfate is chelated with enzymatically hydrolyzed saponins. Attached Figure Description

[0029] Figure 1 The infrared spectra of the cellulosic hydrolysate and the hydrolysate-copper chelate prepared in Example 1 of this application are shown.

[0030] Figure 2 The figure shows the copper element distribution of the cellulase hydrolysed saponin-copper chelate prepared in Example 1 of this application; in the figure, Figure (a) is the copper element distribution of cellulase hydrolysed saponin, and Figure (b) is the copper element distribution of cellulase hydrolysed saponin-copper chelate.

[0031] Figure 3 XRD pattern of the cellulosic hydrolysate saponin-copper chelate prepared in Example 1 of this application;

[0032] Figure 4 The diagram shows the contact angles of the enzymatic hydrolysed saponin-copper chelate, copper sulfate, and Bordeaux mixture droplets prepared in Examples 1-5 of this application on the surface of citrus leaves.

[0033] Figure 5 This image shows the deposition of cellulose enzymatic hydrolysate saponin-copper chelate, copper sulfate, and Bordeaux mixture droplets prepared in Example 1 of this application on the surface of citrus leaves. Detailed Implementation

[0034] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0035] The following examples illustrate the method for determining the purity of saponin, including the following steps: 0.5 mL of saponin solution was thoroughly mixed with an equal volume of 8% vanillin ethanol solution, followed by the addition of 4 mL of 77% sulfuric acid solution. The mixture was then incubated in a 60 °C water bath for 15 minutes. After the reaction, the mixture was rapidly cooled in an ice-water bath, and its absorbance was measured at 550 nm using a UV spectrophotometer (Specord 210 plus, Jena, Germany). Simultaneously, a standard curve was established using oleanolic acid as a standard to quantify the purity of saponin.

[0036] In the following examples, the yields of each enzymatically hydrolyzed saponin-copper chelate were determined by gravimetric method, and the calculation formulas are as follows:

[0037] Yield (%) = (mass of chelate / (mass of saponin + mass of copper sulfate)) × 100%

[0038] Example 1

[0039] (1) Crude saponin extract: The shelled soap pods were thoroughly crushed, passed through a 60-mesh sieve, and defatted with petroleum ether at a boiling range of 30-60 ℃ for 12 h. After drying to remove the solvent, the soap pod powder was fully dissolved in an 80% ethanol solution at a material-to-liquid ratio of 1:10 (g / mL). The extraction temperature was 50 ℃, the extraction time was 2 h, and the stirring speed was 500 rpm. After centrifugation of the extract, the supernatant was collected and freeze-dried to obtain the crude saponin extract.

[0040] (2) Purification of saponin: The crude saponin extract obtained in step (1) was redissolved in distilled water and purified using AB-8 macroporous resin. After eluting with distilled water for 3 column volumes, it was eluted with 90% ethanol solution for 3 column volumes. The ethanol eluent was collected, concentrated by rotary evaporation, and dialyzed with distilled water for 48 h using a dialysis bag with a molecular weight cutoff of 1000 Da. The solution in the bag was freeze-dried to obtain purified saponin with a purity of 75.6%.

[0041] (3) Enzymatic degradation of saponins: The purified saponins obtained in step (2) were fully dissolved in distilled water at a concentration of 10 mg / mL. Cellulase was added at an enzyme-to-substrate mass ratio of 1:50 for enzymatic degradation. The degradation temperature was 50 °C, the system pH was 4.2, and the degradation time was 2 h. After the enzymatic hydrolysis was completed, the hydrolysate was centrifuged, and the supernatant was purified using AB-8 macroporous resin. After eluting with distilled water for 3 column volumes, the supernatant was eluted with 10%, 30%, 50%, 70%, and 90% ethanol solutions in sequence. All ethanol eluents were collected, concentrated, and freeze-dried to obtain the enzymatically degraded saponins.

[0042] (4) Chelation reaction: The enzyme-degraded saponin obtained in step (3) was dissolved in distilled water at a concentration of 10 mg / mL. The pH of the system was adjusted to 6.0 with hydrochloric acid solution (0.01 M). The saponin aqueous solution was mixed with CuSO4·5H2O at a mass ratio of 1:1 and stirred at 40 °C for 1 h. After the reaction was completed, the solution was treated with a dialysis bag with a molecular weight cutoff of 100 Da for 24 h. The solution in the bag was freeze-dried to obtain cellulase-degraded saponin-copper chelate. The yield of the chelate was 44.6%. After wet digestion, the copper content in the chelate was determined by inductively coupled plasma atomic emission spectrometry (Optima 7000DV, PerkinElmer, USA). The copper content of the cellulase-degraded saponin-copper chelate was 24.7 mg / g.

[0043] Depend on Figure 1 It can be seen that the position and intensity of the hydroxyl absorption peak of copper changed after enzymatic hydrolysis of saponins, indicating that hydroxyl groups are the main participants in the chelation of copper ions in enzymatic hydrolysis of saponins.

[0044] Depend on Figure 2 The presence of a uniformly distributed and strong copper signal in the chelate indicates that copper ions successfully chelated with enzymatically hydrolyzed saponins.

[0045] Depend on Figure 3 It can be seen that enzymatically hydrolyzed saponins are mainly composed of amorphous structures, but new diffraction peaks appear after chelation with copper ions, indicating that there is a crystalline structure in the enzymatically hydrolyzed saponin-copper chelate.

[0046] Example 2

[0047] The difference from Example 1 is that in step (3), the enzyme used is xylanase, the degradation temperature is 30 ℃, the system pH is 4.5, the degradation time is 2 h, and the copper content of the xylanase-degraded saponin-copper chelate is 26.9 mg / g.

[0048] Example 3

[0049] The difference from Example 1 is that in step (3), the enzyme used is hemicellulase, the degradation temperature is 30 ℃, the system pH is 4.5, the degradation time is 2 h, and the copper content of the saponin-copper chelate obtained by hemicellulase degradation is 23.1 mg / g.

[0050] Example 4

[0051] The difference from Example 1 is that in step (3), the enzyme used was Viscozyme L, the degradation temperature was 30 ℃, the system pH was 4.5, the degradation time was 2 h, and the copper content of the Viscozyme L enzyme-degraded saponin-copper chelate was 22.8 mg / g.

[0052] Example 5

[0053] The difference from Example 1 is that in step (3), the enzyme used is snail enzyme, the degradation temperature is 30 ℃, the system pH is 4.5, the degradation time is 2 h, and the copper content of the obtained snail enzyme-degraded saponin-copper chelate is 17.3 mg / g.

[0054] Example 6

[0055] The difference from Example 1 is that in step (4), the pH of the system was adjusted to 4.0, and the saponin aqueous solution and CuSO4·5H2O were mixed at a mass ratio of 1:1. The chelate yield was 41.2%, and the copper content of the saponin-copper chelate degraded by cellulase was 16.7 mg / g.

[0056] Example 7

[0057] The difference from Example 1 is that in step (4), the pH of the system was adjusted to 6.0, and the saponin aqueous solution and CuSO4·5H2O were mixed at a mass ratio of 1:0.5. The chelate yield was 56.0%, and the copper content of the saponin-copper chelate degraded by cellulase was 12.5 mg / g.

[0058] Example 8

[0059] The difference from Example 1 is that in step (4), the pH of the system was adjusted to 4.0, and the saponin aqueous solution and CuSO4·5H2O were mixed at a mass ratio of 1:0.5. The chelate yield was 51.3%, and the copper content of the saponin-copper chelate degraded by cellulase was 8.3 mg / g.

[0060] Comparative Example 1

[0061] The saponin from *Sapindus mukorossi* that had not undergone enzymatic degradation in Example 1 was fully dissolved in distilled water to a concentration of 10 mg / mL. The pH of the system was adjusted to 6.0 with hydrochloric acid. The saponin aqueous solution was mixed with CuSO4·5H2O at a mass ratio of 1:1, and the mixture was stirred at 40 °C for 1 h. After the reaction was completed, the mixture was treated with a dialysis bag with a molecular weight cutoff of 100 Da for 24 h. The solution in the bag was then freeze-dried to obtain a saponin-copper chelate. After wet digestion, the copper content in the chelate was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). The copper content of the saponin-copper chelate was 6.5 mg / g.

[0062] Example 9

[0063] The copper solubility of the saponin-copper chelates and copper sulfate prepared in Examples 1-5 and Comparative Example 1 was determined, including the following steps: The enzymatically hydrolyzed saponin-copper chelates and copper sulfate were dissolved separately in distilled water, the pH of the solution was adjusted to 5.0-10.0, and after thorough stirring, the solutions were centrifuged at 8000 rpm for 10 min, and the copper content in the supernatant was determined. The determination method was as follows: 1 mL of the supernatant was wet-digested, and the copper content was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). The results are shown in Table 1.

[0064] Table 1. Copper solubility of saponin-copper chelates prepared in Examples 1-5 and Comparative Example 1

[0065]

[0066] As shown in Table 1, the chelates prepared in all examples all exhibit excellent copper solubility. When the pH is neutral (pH=7), the copper solubility of the chelates in Examples 1-5 is greater than 90%. When the pH is 10, the copper solubility of the chelate prepared in Example 1 is 87.6%, which is 6% higher than that in Example 3.

[0067] Example 10

[0068] The contact angles of the saponin-copper chelates, copper sulfate, and Bordeaux mixture prepared in Examples 1-5 and Comparative Example 1 were measured. The steps included: preparing enzymatically hydrolyzed saponin-copper chelates, copper sulfate solutions, and Bordeaux mixtures with the same copper concentration (500 μg / mL); accurately transferring 5 μL of each solution onto a clean, flat citrus leaf surface; and characterizing the change in droplet contact angle at 25 °C using a dynamic contact angle meter (DSA 100, KRüSS, Germany), measuring every 30 s for 180 s. The results are shown in Table 2 and... Figure 4 .

[0069] Table 2. Contact angles of the saponin-copper chelates prepared in Examples 1-5 and Comparative Example 1

[0070]

[0071] Table 2 shows that the enzymatically hydrolyzed saponin-copper chelate droplets exhibit excellent wetting effects on the surface of citrus leaves. However, there are significant differences in the wetting performance of different enzymatically hydrolyzed saponin-copper chelate droplets on the surface of citrus leaves. Specifically, Example 2 showed the smallest initial contact angle at 39°, while Example 5 showed the smallest contact angle at 180 s at 37.4°. Figure 4 The contact angle diagrams of copper sulfate, Bordeaux mixture, and enzymatic hydrolysis of saponin-copper chelate droplets on the surface of citrus leaves show that the initial contact angle of the enzymatic hydrolysis of saponin-copper chelate droplets on the surface of citrus leaves is significantly smaller than that of copper sulfate and Bordeaux mixture droplets, indicating that the chelate droplets have better wetting properties on the surface of citrus leaves.

[0072] Example 11

[0073] The deposition diameter of the saponin-copper chelate, copper sulfate, and Bordeaux mixture prepared in Examples 1-5 and Comparative Example 1 was determined. The steps included: preparing enzymatically hydrolyzed saponin-copper chelate, copper sulfate, and Bordeaux mixture solutions with the same copper concentration (500 μg / mL); slowly and evenly extruding and releasing the solution through a needle with an inner diameter of 0.25 mm using a syringe, with the needle height controlled at 30 cm above the citrus leaf surface; capturing the dynamic process of droplet impact on the citrus leaf using a high-speed camera at a frame rate of 3000 frames / second; and analyzing the droplet deposition on the citrus leaf surface using Image-Pro 10 software. The results are shown in Table 3 and... Figure 5 .

[0074] Table 3. Deposition diameters of saponin-copper chelates prepared in Examples 1-5 and Comparative Example 1

[0075]

[0076] Table 3 shows that the deposition ability of different enzymatically hydrolyzed saponin-copper chelate droplets on the surface of citrus leaves varies significantly. The deposition diameter in Example 4 reached 8.23 ​​mm, a 7% increase compared to Example 5. Figure 5 The deposition patterns of copper sulfate, Bordeaux mixture, and cellulase-hydrolyzed saponin-copper chelate droplets on the surface of citrus leaves show that after impacting the citrus leaf surface, copper sulfate and Bordeaux mixture droplets undergo contact, spreading, shrinkage, rebound, and stabilization processes. In contrast, the cellulase-hydrolyzed saponin-copper chelate droplets spread and stabilize rapidly after impacting the leaf, exhibiting a significant "pinning" effect and a deposition diameter significantly larger than that of copper sulfate and Bordeaux mixture.

[0077] Example 12

[0078] The copper adsorption capacity of the saponin-copper chelate, copper sulfate, and Bordeaux mixture prepared in Examples 1-5 and Comparative Example 1 was determined, including the following steps: preparing enzymatically hydrolyzed saponin-copper chelate, copper sulfate solution, and Bordeaux mixture with the same copper concentration (500 μg / mL). Fresh citrus leaf discs with a diameter of 16 mm were prepared using a circular punch, and the discs were completely immersed in the above solution for 10 s. The discs were then removed and immersed in 2 mL of distilled water, and sonicated for 2 min. This process was repeated three times to obtain a leaf washing solution. After wet digestion, the copper content of the washing solution was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES), and the adsorption capacity of copper on the surface of the citrus leaves in each sample was calculated. The results are shown in Table 4.

[0079] Table 4. Copper adsorption capacity of saponin-copper chelates prepared in Examples 1-5 and Comparative Example 1

[0080]

[0081] Table 4 shows that copper in each enzymatic saponin-copper chelate exhibits high adsorption capacity on citrus leaves, exceeding 20 μg / cm³. 2 However, the copper adsorption capacity of the chelates prepared in different embodiments varied significantly. The adsorption capacity of Example 1 was the lowest, at 20.40 μg / cm³. 2 The adsorption capacity was increased by 88.4% compared to copper sulfate and by 17.6% compared to Bordeaux mixture. Example 4 showed the highest adsorption capacity at 22.51 μg / cm³. 2 It is 107.8% higher than copper sulfate and 29.8% higher than Bordeaux mixture.

[0082] Example 13

[0083] The erosion resistance of the saponin-copper chelate, copper sulfate, and Bordeaux mixture prepared in Examples 1-5 and Comparative Example 1 was determined, including the following steps: preparing enzymatically hydrolyzed saponin-copper chelate, copper sulfate solution, and Bordeaux mixture with the same copper concentration (500 μg / mL). Fresh citrus leaf discs with a diameter of 16 mm were prepared using a circular punch, and the discs were completely immersed in the above solution for 10 s. After the leaves were removed and allowed to dry naturally, they were fixed (at an angle of 45° to the horizontal plane). A syringe with an inner diameter of 0.58 mm was connected to a constant flow pump, and the water flow rate was controlled at 9 mL / min to simulate raindrops continuously washing the leaves for 30 s. After the simulated rain washing, the leaves were removed and immersed in 2 mL of distilled water, and sonicated for 2 min to obtain a leaf washing solution. After wet digestion of the washing solution, the copper content was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). The residual amount of copper on the surface of the citrus leaves in each sample was determined, and the results are shown in Table 5. The erosion resistance was calculated using the following formula:

[0084] Erosion resistance (%) = Residual copper content on blades / Copper adsorption capacity on blades

[0085] Table 5. Erosion resistance of the saponin-copper chelates prepared in Examples 1-5 and Comparative Example 1

[0086]

[0087] As shown in Table 5, the chelation of copper ions with enzymatically hydrolyzed saponins significantly enhances the erosion resistance. Example 2 exhibits the strongest erosion resistance at 49.67%, a 141% improvement over copper sulfate and a 55% improvement over Bordeaux mixture. Example 4 shows the weakest erosion resistance at 43.28%, a 110% improvement over copper sulfate and a 35% improvement over Bordeaux mixture.

[0088] Example 14

[0089] The saponin-copper chelate, copper sulfate, and Bordeaux mixture prepared in Examples 1-5 and Comparative Example 1 were used to test the inhibitory activity of Xanthomonas carpetii, including the following steps: Xanthomonas carpetii was purchased from the China Pharmaceutical Culture Collection Center (ID: CPCC S230694). The inhibitory effect of copper in copper sulfate, Bordeaux mixture, and copper chelate on Xanthomonas carpetii was determined by the two-fold isocratic dilution method (copper concentration: 500-3.91 μg / mL). The strain was inoculated into liquid culture medium and cultured at 37 ℃ with shaking until the logarithmic growth phase. The bacterial culture in the logarithmic growth phase was then diluted to 1×10⁻⁶ with liquid culture medium. 6CFU / mL was prepared for use. 100 μL of the chelate solution and copper sulfate solution were added to each well of a 96-well plate, followed by an equal volume of the above bacterial suspension. The mixture was thoroughly mixed, with culture medium without added active ingredients serving as a control. The 96-well plates were incubated at 30 °C for 36 h using a microplate reader, and the EC50 value was calculated. The results are shown in Table 6.

[0090] Table 6. Antibacterial effects of saponin-copper chelates prepared in Examples 1-5 and Comparative Example 1

[0091]

[0092] As shown in Table 6, the antibacterial activity was greatly enhanced after copper ions were chelated with enzymatically hydrolyzed saponins, but the inhibitory activity of different enzymatically hydrolyzed saponin-copper chelates against Xanthomonas carpetii varied. Among them, the chelate prepared in Example 1 had the strongest antibacterial performance, with an EC50 value of only 15.4 μg / mL against Xanthomonas carpetii, while the EC50 value of the copper chelate prepared in Example 2 was 37.9 μg / mL.

[0093] In summary, the enzymatic hydrolysis method for preparing saponin-copper chelates in this application is simple and easy to implement. The enzymatic hydrolysis of saponin exhibits a strong ability to chelate copper ions, resulting in a stable chelate with high solubility under alkaline conditions. Its inhibitory activity against *Xanthomonas carpetii* is significantly enhanced compared to copper sulfate and Bordeaux mixture. It has a small initial contact angle on citrus leaves, allowing for rapid wetting and spreading on the leaves, resulting in a large effective deposition area. It also demonstrates strong leaf adhesion and resistance to erosion, making it a green, environmentally friendly, safe, and highly effective bactericidal agent. The enzymatic hydrolysis method for preparing saponin-copper ion chelates using this application shows great potential in the prevention and control of citrus canker.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an enzymatic hydrolysis saponin-copper chelate, characterized in that, Includes the following steps: 1) Crude saponin extract: Soapberry powder was dissolved in an alcohol solution for extraction, centrifuged, and freeze-dried to obtain crude saponin extract; 2) Purification of saponin: The crude saponin extract obtained in step 1) was redissolved in water and purified using macroporous resin. The eluent of saponin was collected, rotary evaporated, dialyzed, and freeze-dried to obtain purified saponin. 3) Enzymatic degradation of saponin from saponins: Dissolve the purified saponin from saponins obtained in step 2) in water, add enzyme for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, centrifuge the hydrolysate, freeze-dry the supernatant, purify it with macroporous resin, elute, collect the eluent, concentrate it, freeze-dry it, and obtain the purified enzymatically degraded saponin. 4) Chelation reaction: Dissolve the enzyme-degraded saponin obtained in step 3) in water, then add CuSO4·5H2O to carry out the chelation reaction. After the reaction is completed, dialyze, collect the solution in the bag and freeze dry to obtain the enzyme-hydrolyzed saponin-copper chelate.

2. The method for preparing the enzymatic hydrolysis saponin-copper chelate according to claim 1, characterized in that: In step 1), the mass-to-volume ratio of soapberry powder to alcohol solution is 1:10; the concentration of alcohol solution is 80%; the extraction temperature is 30-60 ℃; the extraction time is 1-4 h; and the stirring speed is 300-800 rpm.

3. The method for preparing the enzymatic hydrolysis saponin-copper chelate according to claim 1, characterized in that: In step 2), the elution steps are as follows: first, use distilled water to elute for 3 column volumes to remove impurities, then use 90% ethanol solution to elute, and collect the 90% ethanol eluent rich in saponins.

4. The method for preparing the enzymatic hydrolysis saponin-copper chelate according to claim 1, characterized in that: In step 3), the enzyme is selected from one or more combinations of cellulase, xylanase, hemicellulase, Viscozyme L, and snail enzyme; the mass ratio of saponin to enzyme is 1:50; the enzymatic hydrolysis temperature is 30-50 ℃, the enzymatic hydrolysis time is 2 h, and the pH is 4.0-4.

5.

5. The method for preparing the enzymatic hydrolysis saponin-copper chelate according to claim 1, characterized in that: In step 3), the purification steps are as follows: first, use distilled water to wash away impurities, and then use a 10%~90% ethanol solution to wash away impurities.

6. The method for preparing the enzymatic hydrolysis saponin-copper chelate according to claim 1, characterized in that: In step 4), the concentration of the saponin aqueous solution is 10 mg / mL; the pH of the system is adjusted to 4.0-6.0 using 0.01M hydrochloric acid solution.

7. The method for preparing the enzymatic hydrolysis saponin-copper chelate according to claim 1, characterized in that: In step 4), the mass ratio of saponin aqueous solution to CuSO4·5H2O is 1:0.5~1, the reaction temperature is 25-50 ℃, and the reaction time is 0.5-2 h.

8. The enzymatically hydrolyzed saponin-copper chelate prepared by the method according to any one of claims 1 to 7.

9. The application of the enzymatic hydrolysis of saponin-copper chelate according to claim 8 in the prevention and control of citrus canker.

10. The application of the enzymatic hydrolysis of saponins-copper chelates according to claim 8 in the inhibition of Xanthomonas carpetii.

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