An enzymatic hydrolysis of gleditsin-copper chelate and its preparation method and application in citrus canker prevention

Abstract

The application discloses an enzymatic Gleditsia japonica saponin-copper chelate, a preparation method thereof and application thereof in the prevention and treatment of citrus canker, and belongs to the technical field of crop prevention and treatment. The Gleditsia japonica saponin is extracted, purified and degraded by an enzyme method, CuSO4*5H2O is added to perform a chelation reaction, and the enzymatic Gleditsia japonica saponin-copper chelate is obtained. The initial contact angle of the aqueous solution of the chelate on citrus leaves is small, the aqueous solution can rapidly wet and spread on the leaves, the deposition effect is good, the chelate has excellent leaf adhesion and anti-washing capacity, the enzymatic Gleditsia japonica saponin-copper chelate has excellent inhibitory activity on Xanthomonas axonopodis, can be used as a green, environmentally-friendly, safe and efficient bactericide, and has excellent application potential in the prevention and treatment of citrus canker.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of crop prevention and control, and particularly relates to an enzymatic Gleditsia japonica Hance saponin-copper chelate, a preparation method thereof and application thereof in prevention and control of citrus canker. BACKGROUND

[0002] Citrus is the largest fruit crop in the world. The planting area and yield of citrus in China rank first in the world. In recent years, the planting scale of citrus in China is still expanding. However, diseases and pests have a great impact on the development of the citrus industry. Among them, citrus canker, due to its high infectivity, high pathogenicity, and difficult prevention and control, has become an important factor restricting the healthy development of the citrus industry.

[0003] Citrus canker is a bacterial disease mainly caused by Xanthomonas campestris pv. Citri. Xanthomonas campestris pv. Citri infects the fruit, leaves and shoots of plants through stomata, wounds and other channels during the young fruit stage or new shoot stage of citrus. After citrus plants are infected with Xanthomonas campestris pv. Citri, water-stained lesions are formed, and the lesions are gradually suberized and present as a volcanic crater in the later stage. When the disease is severe, it can cause a large number of leaf and fruit drop, affect fruit quality, and cause a large reduction in yield, and even cause plant death, which seriously affects economic benefits.

[0004] At present, there is no cure for citrus canker, and prevention and control methods are still the main method. Among them, the use of chemical reagents is the most common prevention and control method, especially the use of inorganic copper preparations such as copper sulfate, Bordeaux mixture and Kocide 3000 for prevention and control. However, these preparations have problems such as poor water solubility and weak plant conductivity, resulting in poor systemic treatment effect. Therefore, farmers often use multiple spraying to strengthen prevention and control. However, during the spraying process, 20-30% of the liquid will bounce, splash and slide off the leaf surface, making it difficult to adhere to the surface of the citrus leaf. In addition, the copper crystals attached to the leaf surface are also easily washed away by rain. This not only leads to waste of the liquid and low bioavailability of the liquid, but also requires farmers to increase the amount of liquid sprayed. At the same time, the copper that slides off can cause water, soil and metal pollution in the local fruit forest. Therefore, it is urgent to develop an active agent that can effectively deposit and adhere to the surface of the citrus leaf, resist rainwater washing, has stable performance and can efficiently inhibit Xanthomonas campestris pv. Citri.

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

[0006] Chinese patents CN115948299B and CN118978986A respectively disclose the application of Pseudomonas and Bacillus velezensis as fungicides in citrus canker, however, these microbial pesticides not only have slow effect, short product shelf life, but also poor wetting and adhesion on citrus leaves. SUMMARY

[0007] The first technical problem to be solved by the present application is to provide an enzymatic Gleditsia sinensis saponin-copper chelate complex which has excellent inhibitory effect on Xanthomonas campestris; the second technical problem to be solved by the present application is to provide a preparation method of the enzymatic Gleditsia sinensis saponin-copper chelate complex, which utilizes biological enzymes to degrade Gleditsia sinensis saponin and chelate with copper ions, thereby achieving stronger bacteriostatic activity and improving the wet deposition, adsorption and anti-washing performance of copper on the surface of citrus leaves; and the third technical problem to be solved by the present application is to provide the application of the enzymatic Gleditsia sinensis saponin-copper chelate complex in the prevention and treatment of citrus canker and the inhibition of Xanthomonas campestris.

[0008] To solve the above technical problems, the technical solutions adopted by the present application are as follows:

[0009] A preparation method of an enzymatic Gleditsia sinensis saponin-copper chelate complex, comprising the following steps:

[0010] 1) Gleditsia sinensis saponin crude extract: Gleditsia sinensis powder is dissolved in an alcohol solution for extraction, centrifuged, and freeze-dried to obtain the Gleditsia sinensis saponin crude extract;

[0011] 2) Purified Gleditsia sinensis saponin: the Gleditsia sinensis saponin crude extract obtained in step 1) is redissolved in water, and then purified using a macroporous resin, eluted, and the eluate of the Gleditsia sinensis saponin is collected, rotary evaporated, dialyzed, and freeze-dried to obtain the purified Gleditsia sinensis saponin;

[0012] 3) Enzymatic degradation of Gleditsia sinensis saponin: the purified Gleditsia sinensis saponin obtained in step 2) is dissolved in water, and then enzyme is added for enzymatic degradation; after the enzymatic degradation is completed, the enzyme solution is centrifuged, and the supernatant is freeze-dried and then purified using a macroporous resin, eluted, and the eluate is collected, concentrated, and freeze-dried to obtain the purified enzymatic degradation saponin;

[0013] 4) Chelation reaction: the enzymatic degradation saponin obtained in step 3) is dissolved in water, and then CuSO4·5H2O is added for chelation reaction; after the reaction is completed, dialysis is performed, and the solution in the bag is collected and freeze-dried to obtain the enzymatic Gleditsia sinensis saponin-copper chelate complex.

[0014] Further, in step 1), the mass / volume ratio of Gleditsia sinensis powder to alcohol solution is 1:10; the concentration of the 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: first, using distilled water to elute 3 column volumes to remove impurities, and then using 90% ethanol solution to elute, and collecting the 90% ethanol eluent rich in saponin.

[0016] Further, in step 3), the enzyme is selected from one or a combination of cellulase, xylanase, hemicellulase, Viscozyme L and snailase; the mass ratio of saponin to enzyme is 1:50; the enzymolysis temperature is 30-50 DEG C, the enzymolysis time is 2 h, and the pH is 4.0-4.5.

[0017] Further, in step 3), the purification step is: first, using distilled water to elute to remove impurities, and then using an ethanol solution with a concentration of 10%-90% to elute.

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

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

[0020] Further, the enzyme-degraded Gleditsia japonica saponin-copper chelate prepared by the preparation method of the enzyme-degraded Gleditsia japonica saponin-copper chelate.

[0021] Further, the enzyme-degraded Gleditsia japonica saponin-copper chelate is applied in the prevention and treatment of citrus canker.

[0022] Further, the enzyme-degraded Gleditsia japonica saponin-copper chelate is applied in the inhibition of Xanthomonas axonopodis.

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

[0024] (1) The enzyme-degraded Gleditsia japonica saponin-copper chelate prepared by the present application has good solubility in water.

[0025] (2) The initial contact angle of the enzyme-degraded Gleditsia japonica saponin-copper chelate droplet on the surface of the citrus leaf is small, which is much lower than that of the copper sulfate droplet and the Bordeaux liquid droplet, and the wettability of the enzyme-degraded Gleditsia japonica saponin-copper chelate droplet on the surface of the citrus leaf is excellent.

[0026] (3) The deposition effect, adhesion capacity and anti-washing capacity of the enzyme-degraded Gleditsia japonica saponin-copper chelate on the citrus leaf are significantly improved compared with copper sulfate and Bordeaux liquid.

[0027] (4) The prepared enzymatic Gaojia saponin-copper chelate has good stability of copper, when pH is 10.0, the solubility of copper in the chelate is greater than 80%, and the solubility of copper sulfate is less than 1%; therefore, the enzymatic Gaojia saponin-copper chelate can be applied together with other agents or leaf fertilizers in different pH ranges.

[0028] (5) The enzymatic Gaojia saponin-copper chelate prepared in the application has significantly improved inhibition activity on Xanthomonas campestris. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The infrared spectrograms of cellulase enzymatic Gaojia saponin and cellulase enzymatic Gaojia saponin-copper chelate prepared in Example 1 of the application;

[0030] Figure 2 The copper element distribution diagrams of cellulase enzymatic Gaojia saponin-copper chelate prepared in Example 1 of the application; in the diagrams, diagram (a) is the copper element distribution diagram of cellulase enzymatic Gaojia saponin, and diagram (b) is the copper element distribution diagram of cellulase enzymatic Gaojia saponin-copper chelate;

[0031] Figure 3 The XRD diagram of cellulase enzymatic Gaojia saponin-copper chelate prepared in Example 1 of the application;

[0032] Figure 4 The contact angle diagrams of cellulase enzymatic Gaojia saponin-copper chelate, copper sulfate and Bordeaux liquid prepared in Examples 1-5 of the application on the surface of citrus leaves;

[0033] Figure 5 The deposition diagrams of cellulase enzymatic Gaojia saponin-copper chelate, copper sulfate and Bordeaux liquid prepared in Example 1 of the application on the surface of citrus leaves. DETAILED DESCRIPTION

[0034] The application will be further illustrated by specific examples, which are implemented on the premise of the technical scheme of the application, and it should be understood that the examples are only used to illustrate the application and not to limit the scope of the application.

[0035] In the following examples, the determination method of saponin purity includes the following steps: 0.5 mL of saponin solution is mixed with an equal volume of 8% vanillin ethanol solution, then 4 mL of 77% sulfuric acid solution is added, and the mixed solution is incubated in a 60 ℃ water bath for 15 minutes. After the reaction is completed, the mixed solution is quickly placed in an ice water bath for cooling, and the absorbance is measured at 550 nm wavelength by using a UV spectrophotometer (Specord210 plus, Jena, Germany). At the same time, using oleanolic acid as a standard to establish a standard curve, the saponin purity is quantified.

[0036] The yield of each enzymatic saponin-copper chelate was determined by gravimetric method in the following examples, and the calculation formula is as follows:

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

[0038] Example 1

[0039] (1) Saponin crude extract: The shelled Gleditsia sinensis L. was fully crushed and passed through a 60-mesh sieve, and then was degreased with 30-60 ℃ boiling petroleum ether for 12 h. After drying and removing the solvent, the Gleditsia sinensis L. powder was fully dissolved in an 80% ethanol solution, with a solid-liquid ratio of 1:10 (g / mL), an extraction temperature of 50 ℃, and an extraction time of 2 h, and the stirring speed was 500 rpm. After centrifugation of the extraction solution, the supernatant was collected and freeze-dried to obtain the saponin crude extract.

[0040] (2) Purified Gleditsia sinensis L. saponin: The saponin crude extract obtained in step (1) was redissolved in distilled water and then purified using AB-8 macroporous resin. After elution with three column volumes of distilled water, three column volumes of 90% ethanol solution were used for elution. The ethanol eluate was collected, concentrated by rotary evaporation, and then dialyzed against 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 Gleditsia sinensis L. saponin, and the saponin purity was 75.6%.

[0041] (3) Enzymatic degradation of Gleditsia sinensis L. saponin: The purified Gleditsia sinensis L. saponin obtained in step (2) was fully dissolved in distilled water to a concentration of 10 mg / mL, and cellulase was added according to an enzyme-to-substrate mass ratio of 1:50 for enzymatic degradation. The degradation temperature was 50 ℃, the system pH was 4.2, and the degradation time was 2 h. After the enzymatic degradation was completed, the enzyme solution was centrifuged, and the supernatant was purified using AB-8 macroporous resin. After elution with three column volumes of distilled water, 10%, 30%, 50%, 70%, and 90% ethanol solutions were used for elution in sequence. All ethanol eluates were collected, concentrated, and freeze-dried to obtain the enzyme-degraded saponin.

[0042] (4) Chelation reaction: The enzyme-degraded saponin obtained in step (3) was dissolved in distilled water to a concentration of 10 mg / mL, and the system pH was adjusted to 6.0 using a hydrochloric acid solution (0.01 M). The saponin aqueous solution was mixed with CuSO4·5H2O at a mass ratio of 1:1, and the mixture was stirred at 40 ℃ for 1 h. After the reaction was completed, the solution was treated using a dialysis bag with a molecular weight cutoff of 100 Da for 24 h. The solution in the bag was freeze-dried to obtain the cellulase-degraded saponin-copper chelate. The yield of the chelate was 44.6%. After wet digestion, the copper content in the chelate was determined using an inductively coupled plasma emission spectrometer (Optima7000DV, PerkinElmer, USA), and the copper content in the cellulase-degraded saponin-copper chelate was 24.7 mg / g.

[0043] FromFigure 1 It can be seen that the position and intensity of the hydroxyl absorption peak of copper after the chelation of enzymatic soapnut saponin changed, indicating that the main hydroxyl group in the enzymatic soapnut saponin participated in the chelation of copper ions.

[0044] From the above results, it can be seen that the copper signal observed in the chelate is evenly distributed and strong, indicating that the copper ions are successfully chelated with the enzymatic soapnut saponin. Figure 2 From the above results, it can be seen that the copper signal observed in the chelate is evenly distributed and strong, indicating that the copper ions are successfully chelated with the enzymatic soapnut saponin.

[0045] Figure 3 From the above results, it can be seen that the copper signal observed in the chelate is evenly distributed and strong, indicating that the copper ions are successfully chelated with the enzymatic soapnut saponin.

[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, and the degradation time is 2 h. 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, and the degradation time is 2 h. The copper content of the hemicellulase-degraded saponin-copper chelate is 23.1 mg / g.

[0050] Example 4

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

[0052] Example 5

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

[0054] Example 6

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

[0056] Example 7​

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

[0058] Example 8

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

[0060] Comparative Example 1

[0061] The saponin from Gleditsia sinensis L. that is not subjected to enzymatic degradation in Example 1 is dissolved in distilled water at a concentration of 10 mg / mL, and the pH of the system is adjusted to 6.0 with hydrochloric acid. The saponin aqueous solution is mixed with CuSO4·5H2O at a mass ratio of 1:1, and the reaction is stirred at 40°C for 1 h. After the reaction is completed, the solution in the dialysis bag with a molecular weight cut-off of 100 Da is treated for 24 h, and the saponin-copper chelate is obtained by lyophilizing the solution in the bag. After wet digestion, the copper content in the chelate is determined by inductively coupled plasma emission spectrometry, and the copper content of the saponin-copper chelate is 6.5 mg / g.

[0062] Example 9

[0063] The copper solubility of the saponin-copper chelates prepared in Examples 1-5 and Comparative Example 1 and copper sulfate is determined, including the steps of: dissolving the enzymatically degraded saponin-copper chelate and copper sulfate in distilled water, adjusting the pH of the solution to 5.0-10.0, and then centrifuging at 8000 rpm for 10 min. The copper content in the supernatant is determined. The determination method is as follows: 1 mL of the supernatant is subjected to wet digestion, and the copper content is determined by inductively coupled plasma emission spectrometry. The results are shown in Table 1.

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

[0065]

[0066] As can be seen from Table 1, all the chelates prepared in the examples have 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 increased by 6% compared with Example 3.

[0067] Example 10

[0068] The gleditsinidin-copper chelate prepared in Examples 1-5 and Comparative Example 1, copper sulfate and Bordeaux solution were subjected to contact angle determination, including the steps of: preparing gleditsinidin-copper chelate, copper sulfate solution and Bordeaux solution with the same copper concentration (500 μg / mL), accurately pipetting 5 μL of the solution onto the surface of a clean and flat citrus leaf, using a dynamic contact angle measuring instrument (DSA 100, KRüSS, Germany) to characterize the contact angle change of the droplet at 25 ℃, measuring once every 30 s for 180 s. The results are shown in Table 2 and Figure 4 .

[0069] Table 2 Contact angle of gleditsinidin-copper chelate prepared in Examples 1-5 and Comparative Example 1

[0070]

[0071] As can be seen from Table 2, the gleditsinidin-copper chelate droplet has excellent wetting effect on the surface of the citrus leaf. However, the wetting performance of different gleditsinidin-copper chelate droplets on the surface of the citrus leaf is significantly different. Among them, the initial contact angle of Example 2 is the smallest, which is 39°, and the contact angle of Example 5 at 180 s is the smallest, which is 37.4°. From the contact angle-time curves of copper sulfate, Bordeaux solution and gleditsinidin-copper chelate droplets on the surface of the citrus leaf, it can be seen that the initial contact angle of the gleditsinidin-copper chelate droplet on the surface of the citrus leaf is significantly smaller than that of copper sulfate and Bordeaux solution, indicating that the wetting performance of the chelate droplet on the surface of the citrus leaf is better. Figure 4

[0072] Example 11

[0073] The gleditsinidin-copper chelate prepared in Examples 1-5 and Comparative Example 1, copper sulfate and Bordeaux solution were subjected to deposition diameter determination, including the steps of: preparing gleditsinidin-copper chelate, copper sulfate solution and Bordeaux solution with the same copper concentration (500 μg / mL), using a syringe to slowly and uniformly extrude and release the solution from a needle with an inner diameter of 0.25 mm, controlling the height of the needle from the surface of the citrus leaf to be 30 cm, using a high-speed camera to capture the dynamic process of the droplet impacting the citrus leaf at a frame rate of 3000 frames / s, and analyzing the deposition of the droplet on the surface of the citrus leaf by image-Pro 10 software. The results are shown in Table 3 and Figure 5 .

[0074] Table 3 Deposition diameter of gleditsinidin-copper chelate prepared in Examples 1-5 and Comparative Example 1

[0075]

[0076] ​From Table 3, it can be seen that the deposition abilities of different enzyme-degraded gossypiol-copper chelate droplets on the surface of citrus leaves are obviously different. The deposition diameter of Example 4 is 8.23 mm, which is increased by 7% compared with Example 5. From the deposition diagrams of copper sulfate, Bordeaux mixture and cellulase-degraded gossypiol-copper chelate droplets on the surface of citrus leaves, it can be seen that after the copper sulfate and Bordeaux mixture droplets impact the surface of citrus leaves, they undergo the processes of contact, spreading, shrinkage, rebound and stability. However, after the cellulase-degraded gossypiol-copper chelate droplets impact the leaves, they spread and then quickly stabilize, and the "pinned" effect is obvious, and the deposition diameter is obviously larger than that of copper sulfate and Bordeaux mixture. Figure 5 From Table 3, it can be seen that the deposition abilities of different enzyme-degraded gossypiol-copper chelate droplets on the surface of citrus leaves are obviously different. The deposition diameter of Example 4 is 8.23 mm, which is increased by 7% compared with Example 5. From the deposition diagrams of copper sulfate, Bordeaux mixture and cellulase-degraded gossypiol-copper chelate droplets on the surface of citrus leaves, it can be seen that after the copper sulfate and Bordeaux mixture droplets impact the surface of citrus leaves, they undergo the processes of contact, spreading, shrinkage, rebound and stability. However, after the cellulase-degraded gossypiol-copper chelate droplets impact the leaves, they spread and then quickly stabilize, and the "pinned" effect is obvious, and the deposition diameter is obviously larger than that of copper sulfate and Bordeaux mixture.

[0077] Example 12

[0078] The copper adsorption amounts of the gossypiol-copper chelates prepared in Examples 1-5 and Comparative Example 1, copper sulfate and Bordeaux mixture were determined, including the steps of: preparing enzyme-degraded gossypiol-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 by a circular puncher, and the circular leaf discs were completely immersed in the above-mentioned solutions for 10 s. Then the leaf discs were taken out and immersed in 2 mL of distilled water, and ultrasonic treatment was performed for 2 min. This process was repeated for 3 times to obtain the leaf washing solution. After wet digestion of the washing solution, the copper content was determined by inductively coupled plasma emission spectrometry, and the adsorption amount of copper on the surface of the citrus leaf discs was calculated. The results are shown in Table 4.

[0079] Table 4 Copper adsorption amount of gossypiol-copper chelate prepared in Examples 1-5 and Comparative Example 1

[0080]

[0081] From Table 4, it can be seen that the copper in each enzyme-degraded gossypiol-copper chelate has a high adsorption amount on the citrus leaf, which is more than 20 μg / cm 2 . However, the copper adsorption amounts of the chelates prepared in different examples are obviously different. Among them, the adsorption amount of Example 1 is the lowest, which is 20.40 μg / cm 2 , which is increased by 88.4% compared with copper sulfate and by 17.6% compared with Bordeaux mixture. The adsorption amount of Example 4 is the highest, which is 22.51 μg / cm 2 , which is increased by 107.8% compared with copper sulfate and by 29.8% compared with Bordeaux mixture.

[0082] Example 13

[0083] The gleditsin-copper chelate prepared in Examples 1-5 and Comparative Example 1, copper sulfate and Bordeaux solution were subjected to the anti-erosion capacity determination, including the steps of: preparing the enzymatic saponin-copper chelate, copper sulfate solution and Bordeaux solution with the same copper concentration (500 μg / mL). Fresh citrus leaf discs with a diameter of 16 mm were prepared by a circular punch, and the circular leaf discs were completely immersed in the above-mentioned solution for 10 s. After the leaf discs were taken out and naturally dried, the dried leaf discs were fixed (45° angle with the horizontal plane). A needle tube with an inner diameter of 0.58 mm was connected with a constant flow pump, and the water flow rate was controlled at 9 mL / min to simulate raindrops to continuously erode the leaf discs for 30 s. After the simulated rainwater erosion, the leaf discs were taken out and immersed in 2 mL of distilled water, and ultrasonic treatment was performed for 2 min to obtain the leaf washing solution. After the washing solution was subjected to wet digestion, the copper content was determined by inductively coupled plasma emission spectrometry, and the residual amount of copper on the surface of the citrus leaf discs in each sample was determined, and the results are shown in Table 5. The anti-erosion capacity calculation formula is:

[0084] Anti-erosion capacity (%) = residual amount of copper on leaf discs / adsorbed amount of copper on leaf discs

[0085] Table 5 Anti-erosion capacity of gleditsin-copper chelate prepared in Examples 1-5 and Comparative Example 1

[0086]

[0087] As can be seen from Table 5, after the copper ions were chelated with the enzymatic saponin, the anti-erosion capacity was greatly improved. Among them, the anti-erosion capacity of Example 2 was the strongest, which was 49.67%, increased by 141% compared with copper sulfate and by 55% compared with Bordeaux solution; the anti-erosion capacity of Example 4 was the weakest, which was 43.28%, increased by 110% compared with copper sulfate and by 35% compared with Bordeaux solution.

[0088] Example 14

[0089] The gleditsin-copper chelate prepared in Examples 1-5 and Comparative Example 1, copper sulfate and Bordeaux solution were subjected to the anti-erosion capacity determination, including the steps of: preparing the enzymatic saponin-copper chelate, copper sulfate solution and Bordeaux solution with the same copper concentration (500 μg / mL). Fresh citrus leaf discs with a diameter of 16 mm were prepared by a circular punch, and the circular leaf discs were completely immersed in the above-mentioned solution for 10 s. After the leaf discs were taken out and naturally dried, the dried leaf discs were fixed (45° angle with the horizontal plane). A needle tube with an inner diameter of 0.58 mm was connected with a constant flow pump, and the water flow rate was controlled at 9 mL / min to simulate raindrops to continuously erode the leaf discs for 30 s. After the simulated rainwater erosion, the leaf discs were taken out and immersed in 2 mL of distilled water, and ultrasonic treatment was performed for 2 min to obtain the leaf washing solution. After the washing solution was subjected to wet digestion, the copper content was determined by inductively coupled plasma emission spectrometry, and the residual amount of copper on the surface of the citrus leaf discs in each sample was determined, and the results are shown in Table 5. The anti-erosion capacity calculation formula is: 6CFU / mL. After adding 100 μL of the chelate solution and copper sulfate solution into each well of the 96-well plate respectively, an equal volume of the above bacterial suspension was added and mixed uniformly, and a culture medium without the active ingredient was used as a control. The 96-well plate was placed in an enzyme marker at 30 °C for 36 h, and the EC50value was calculated. The results are shown in Table 6.

[0090] Table 6 Bacteriostatic effect of the Gleditsia sinensis - copper chelate prepared in Examples 1-5 and Comparative Example 1

[0091]

[0092] As shown in Table 6, the bacteriostatic activity of the copper ion chelated with the enzymatic saponin was greatly improved, but the inhibition activity of different enzymatic saponin-copper chelates on Xanthomonas axonopodis was different. Among them, the chelate prepared in Example 1 had the strongest bacteriostatic performance, and the EC50value of Xanthomonas axonopodis was only 15.4 μg / mL, while the EC50value of the copper chelate prepared in Example 2 was 37.9 μg / mL.

[0093] In summary, the preparation method of the enzymatic Gleditsia sinensis saponin-copper chelate of the present application is simple and easy to operate, the enzymatic Gleditsia sinensis saponin has strong ability to chelate copper ions, the obtained chelate has stable properties, high solubility in alkaline conditions, greatly improved inhibition activity on Xanthomonas axonopodis than copper sulfate and Bordeaux liquid, small initial contact angle on citrus leaves, can quickly wet and spread on the leaves, large effective deposition area, strong leaf adhesion performance and anti-washing ability, and is a green, environmentally friendly, safe and efficient bacteriostatic active substance. The enzymatic Gleditsia sinensis saponin-copper ion chelate prepared by the present application has great potential in preventing and treating citrus canker.

[0094] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A process for the preparation of an enzymatic saponin-copper chelate complex, 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: The purified saponin from saponins obtained in step 2) is dissolved in water, and then an enzyme is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the hydrolysate is centrifuged, the supernatant is freeze-dried, purified using macroporous resin, eluted, the eluent is collected, concentrated, and freeze-dried to obtain purified enzymatically degraded saponins; the enzyme is selected from one of cellulase, hemicellulase, Viscozyme L, and snail enzyme. 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 process for the preparation of enzymatic 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 process for the preparation of enzymatic 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 process for the preparation of enzymatic saponin-copper chelate of claim 1, wherein: In step 3), 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 of claim 1, wherein the enzymatic preparation of saponin-copper chelate is characterized by: 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 process for the preparation of enzymatic saponin-copper chelate of claim 1, wherein: 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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