Fruit and vegetable grafting wound healing nutrient solution containing polysaccharide compound enzyme and preparation method of fruit and vegetable grafting wound healing nutrient solution

Abstract

The invention discloses a fruit and vegetable grafting wound healing nutrient solution containing polysaccharide complex enzyme and a preparation method, belongs to the technical field of agricultural biochemical preparations, and aims to solve the technical problems of low removal efficiency of necrotic tissues at grafting wounds and high risk of microbial infection. According to the technical scheme, the nutrient solution is characterized by being prepared from the following raw material components in parts by weight: 0.3 to 0.8 part of polysaccharide, 1 to 5 parts of cellulase, 0.5 to 2.5 parts of pectinase, 1 to 5 parts of glycerol and 90 to 100 parts of water. Wherein the pH value of the nutrient solution is adjusted to 5.0-6.5 by using a phosphate buffer agent, the polysaccharide is composed of chitosan and sodium alginate, the mass ratio of the chitosan to the sodium alginate is 1: 0.5-1: 2, the activity of cellulase in the nutrient solution is 100-500 U / mL, and the activity of pectinase in the nutrient solution is 50-250 U / mL. The nutrient solution is mainly used for promoting fruit and vegetable grafting wound healing, and application verifies that the nutrient solution can effectively improve wound healing quality and reduce infection risk in the grafting process of fruits and vegetables such as cucumbers, eggplants, peppers, tomatoes, litchis and oranges, and has a good application effect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biochemical preparation technology, specifically relating to a nutrient solution for healing grafted wounds of fruits and vegetables containing polysaccharide complex enzymes and its preparation method. Background Technology

[0002] In fruit and vegetable grafting, the speed and quality of wound healing directly affect the graft survival rate. Existing grafting aids have limitations in promoting callus formation, mainly manifested in insufficient efficiency in removing necrotic tissue and inadequate control of the risk of microbial infection.

[0003] The necrotic tissue remaining at the grafting wound of plants is mainly composed of cellulose and pectin. Conventional nutrient solutions typically contain only cellulase or pectinase, which are difficult to degrade simultaneously in these two structural polysaccharides. The complex network structure formed by cellulose and pectin in the cell wall has a steric hindrance effect, resulting in low substrate accessibility when a single enzyme acts. Experiments show that when only cellulase is used to treat tomato grafting wounds, the necrotic tissue removal rate is less than 40% within 24 hours, and the residue hinders the contact and fusion of parenchyma cells.

[0004] Microbial infection is another key factor leading to grafting failure. In wound-exposed environments, Gram-negative bacteria (such as Agrobacterium) and fungi (such as Fusarium) can invade through mechanical damage. Traditional protective measures often rely on broad-spectrum bactericides, but chemical agents can easily cause plant cell toxicity and disrupt the wound's microecological balance. While natural antibacterial substances such as chitosan are biocompatible, their film-forming properties are unstable in liquid environments. Under fluctuating temperature and humidity conditions, the physical barrier formed by a single chitosan solution is prone to cracking, with crack widths reaching 5-10 micrometers, providing channels for microbial penetration.

[0005] Solving these problems faces three major technical obstacles: First, constructing a synergistic enzyme system is difficult. Cellulase's optimal pH is 4.5-5.5, and pectinase's is 5.0-6.0. Their activity ranges overlap narrowly; a pH shift of 0.5 units can significantly reduce the total activity of the complex enzyme. Second, it is difficult to simultaneously achieve antibacterial and mechanical protective functions. Increasing the polysaccharide concentration can enhance film-forming strength, but when the concentration exceeds 1.2%, the solution viscosity increases dramatically, hindering the diffusion of enzyme molecules into the deeper layers of the wound. Third, the compatibility of components is a significant challenge. Positively charged chitosan and negatively charged enzyme proteins easily aggregate electrostatically in the liquid phase, forming complexes with particle sizes greater than 200 nanometers, which reduces enzyme activity and affects film uniformity.

[0006] Existing technologies attempt to alleviate the aforementioned contradictions by applying enzyme preparations and antibacterial agents in stages, but this significantly increases operational complexity. Field trials show that step-by-step treatment extends grafting time by 50%, and frequent contact of instruments with the wound actually increases the risk of secondary infection. These inherent drawbacks limit the efficiency of large-scale application of grafting technology in facility agriculture. Summary of the Invention

[0007] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0008] This invention solves the following technical problems: This addresses the technical challenges of low efficiency in removing necrotic tissue and high risk of microbial infection at grafting wounds in fruits and vegetables. Conventional nutrient solutions, due to their limited enzyme activity, cannot simultaneously degrade the cellulose-pectin complex structure, and the film-forming properties of single antibacterial components are unstable, leading to delayed healing and pathogen invasion.

[0009] This addresses the issues of enzyme activity loss, poor component compatibility, and damage during sterilization in nutrient solution preparation. High-temperature dissolution leads to polysaccharide degradation, liquid-phase mixing causes ineffective enzyme-polysaccharide binding, and filtration shear forces disrupt enzyme conformation.

[0010] This addresses the issues of activity degradation and electrostatic adsorption of liquid enzymes during storage and mixing. Free enzymes are easily inactivated in solution, and their binding with positively charged chitosan reduces catalytic efficiency.

[0011] This study addresses the issues of enzyme protein denaturation and microcapsule structure collapse during the freeze-drying process of microcapsules. The mechanical stress caused by ice crystal growth disrupts the enzyme's tertiary structure, leading to water loss and the collapse of the porous framework.

[0012] This addresses the problem of enzyme spatial conformation disruption caused by high shear forces and pressure fluctuations during aseptic filtration. Excessive shear rates within the filter membrane pores and sudden pressure differentials trigger cavitation microjets that cause damage.

[0013] To achieve these objectives and other advantages of the present invention, a nutrient solution for healing grafted fruit and vegetable wounds containing a polysaccharide complex enzyme is provided, comprising the following raw material components in parts by weight: 0.3-0.8 parts polysaccharide, 1-5 parts cellulase, 0.5-2.5 parts pectinase, 1-5 parts glycerol, and 90-100 parts water; wherein, the pH of the nutrient solution is adjusted to 5.0-6.5 with a phosphate buffer, the polysaccharide is composed of chitosan and sodium alginate, the mass ratio of chitosan to sodium alginate is 1:0.5-1:2, the cellulase activity in the nutrient solution is 100-500 U / mL, and the pectinase activity is 50-250 U / mL.

[0014] Preferably, the preparation method of the present invention includes the following steps: Chitosan was added to water at 50-55℃ and stirred until dissolved to form a homogeneous solution; sodium alginate was added to the homogeneous solution and stirred continuously at 40-45℃ until completely dissolved to obtain a polysaccharide solution. Adjust the pH of the polysaccharide solution to 5.5-6.0 using disodium hydrogen phosphate-citric acid buffer. Add cellulase and pectinase sequentially at 25-28℃ and stir at 80-200 rpm for 5-25 minutes to obtain a mixed solution. Add glycerol to the mixture and continue stirring at the same speed for 8-12 minutes. Then, filter the mixture aseptically through a polyethersulfone membrane with a pore size of 0.22 micrometers to obtain a nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes.

[0015] Preferably, in the preparation method of the present invention, cellulase and pectinase are added in solid microencapsulation form in step 2, and the preparation includes the following steps: Dissolve sodium alginate in water to prepare a sodium alginate solution with a mass percentage of 3-5%. Mix cellulase and pectinase at an activity ratio of 2:1, add them to sodium alginate solution, and mix at 50-100 rpm for 10-15 minutes at 4-8℃ to form a uniform suspension. The suspension is dripped into a calcium chloride solution with a mass percentage of 1.5-2.0% through a dropper with an aperture of 0.5-1.0 mm. The droplets remain in place for 10-20 minutes to form solidified microcapsules with a diameter of 1.0-2.0 mm. The solidified microcapsules were collected and rinsed three times with deionized water. They were then frozen at -40 to -30°C for 6-8 hours, transferred to a freeze dryer, and freeze-dried to obtain enzyme microcapsules with a water content of 3-5%.

[0016] Preferably, in the preparation method of the present invention, in step 2, the enzyme microcapsules are added to the polysaccharide solution after pH adjustment, and stirred at 80-100 rpm for 5-10 minutes at 25-28℃.

[0017] Preferably, in the preparation method of the present invention, when adding cellulase and pectinase to the sodium alginate solution in step 2.2, a freeze-drying protectant is added simultaneously. The freeze-drying protectant is composed of trehalose and mannitol, with the final concentration of trehalose in the suspension being 5-8 g / L and the final concentration of mannitol being 2-4 g / L.

[0018] Preferably, in the preparation method of the present invention, trehalose and mannitol are dissolved in deionized water at 4-8°C to prepare a mixed protective solution, and sodium alginate solution is added before a uniform suspension is formed.

[0019] Preferably, in the preparation method of the present invention, the freeze-drying conditions in step 2.4 are adjusted as follows: Pre-freezing stage: Cool to -45°C at a rate of 3-5°C / min and maintain for 60 minutes; First drying: Under vacuum of 10-15 Pa and cold trap temperature of -50 to -45℃, the temperature is raised to -25℃ and maintained for 240 minutes; Desorption drying: Heat to 20℃, reduce vacuum to 0.5-1.0 Pa, and maintain for 120 minutes.

[0020] Preferably, in the preparation method of the present invention, step 3 involves adding an enzyme conformation stabilization treatment before the aseptic filtration operation, specifically including the following steps: Place the mixture with added glycerin into a sealed container with a depth of 5-10 cm, let it stand for 20-30 minutes at a constant temperature of 25-28℃, and then filter it. Aseptic filtration employs staged pressure control, specifically: Initial stage: Under conditions of inlet pressure 0.03-0.05MPa and outlet pressure 0.01-0.02MPa, the filter volume accounts for 20-30% of the total volume. Main filtration stage: Linearly increase the inlet pressure to 0.08-0.10 MPa, maintain a pressure difference of 0.04-0.05 MPa, and filter to 80-90% of the total volume. Final stage: Reduce the inlet pressure to 0.04-0.06 MPa, and the pressure difference to 0.02-0.03 MPa to complete the remaining volume filtration; Throughout the filtration process, the filtrate temperature is maintained at 25-28℃.

[0021] The present invention has at least the following beneficial effects: A dense polyelectrolyte membrane is formed by combining chitosan and sodium alginate in a specific ratio, which physically blocks the invasion of pathogenic microorganisms; the active components of cellulase and pectinase work synergistically against plant cell wall components to improve the efficiency of necrotic tissue removal.

[0022] Stepwise dissolution and buffering control maintain the integrity of polysaccharide molecules and the enzyme activity range; low-temperature mixing reduces the risk of thermal denaturation; and the addition of glycerol enhances solution stability.

[0023] Microencapsulation isolates the enzyme from electrostatic interactions with charged polysaccharides, preventing activity loss; solid-state form extends enzyme storage life; low-speed stirring ensures the integrity of the microencapsulation structure.

[0024] The combination of trehalose and mannitol forms a glassy protective layer, inhibiting mechanical damage from ice crystals; the gradient drying process maintains the porous framework structure of the microcapsules, ensuring the enzyme release rate in the later stages.

[0025] The static treatment enriches enzyme molecules in the low-shear region; the staged pressure control weakens the shear stress in the filter membrane pores and avoids conformational distortion.

[0026] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0027] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0028] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0029] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0030] <Example 1> A nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes comprises the following raw material components in parts by weight: 0.5 parts polysaccharide, 3 parts cellulase, 1.5 parts pectinase, 3 parts glycerol, and 100 parts water; wherein, the pH of the nutrient solution is adjusted to 6.0 with phosphate buffer, the polysaccharide is composed of chitosan and sodium alginate, the mass ratio of chitosan to sodium alginate is 1:1, the cellulase activity in the nutrient solution is 300 U / mL, and the pectinase activity is 150 U / mL.

[0031] A method for preparing a nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes, comprising the following steps: 1) Add chitosan to water at 52°C and stir to dissolve, forming a homogeneous solution; add sodium alginate to the homogeneous solution and stir continuously at 42°C until completely dissolved to obtain a polysaccharide solution; 2) Adjust the pH of the polysaccharide solution to 5.8 using disodium hydrogen phosphate-citric acid buffer, add cellulase and pectinase sequentially at 26°C, and stir at 140 rpm for 15 min to obtain a mixed solution; 3) Add glycerol to the mixed solution and continue stirring at the same speed for 10 minutes. Then, perform sterile filtration through a polyethersulfone filter membrane with a pore size of 0.22 micrometers to obtain a nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes.

[0032] <Example 2> A nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes, based on <Example 1>, with cellulase and pectinase added in solid microencapsulation form in step 2), is prepared by the following steps: 2.1) Dissolve sodium alginate in water to prepare a 4% sodium alginate solution by mass percentage; 2.2) Mix cellulase and pectinase at an activity ratio of 2:1, add them to sodium alginate solution, and mix at 75 rpm for 12 minutes at 6°C to form a uniform suspension. 2.3) The suspension was dripped into a 1.8% calcium chloride solution through a dropper with an aperture of 0.8 mm. The droplets remained in place for 15 minutes to form solidified microcapsules with a diameter of 1.5 mm. 2.4) Collect the solidified microcapsules and rinse them three times with deionized water. Freeze them at -35°C for 7 hours, then transfer them to a freeze dryer and freeze-dry them under vacuum to obtain enzyme microcapsules with a water content of 4%. The enzyme microcapsules were added to the pH-adjusted polysaccharide solution and stirred at 90 rpm for 8 minutes at 26°C.

[0033] <Example 3> A nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes, based on <Example 2>, wherein in step 2.2), when cellulase and pectinase are added to the sodium alginate solution, a freeze-drying protectant is added simultaneously. The freeze-drying protectant is composed of trehalose and mannitol, with a final concentration of trehalose in the suspension of 6.5 g / L and a final concentration of mannitol of 3 g / L. Dissolve trehalose and mannitol in deionized water at 6°C to prepare a mixed protective solution, and add sodium alginate solution before forming a uniform suspension. The freeze-drying conditions described in step 2.4) are adjusted as follows: Pre-freezing stage: Cool to -45°C at a rate of 4°C / min and maintain for 60 minutes; First drying: Under vacuum of 13 Pa and cold trap temperature of -48℃, the temperature is raised to -25℃ and maintained for 240 minutes; Desorption drying: Heat to 20°C, reduce vacuum to 0.8 Pa, and maintain for 120 minutes.

[0034] <Example 4> A nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes, based on <Example 3>, includes an enzyme conformation stabilization treatment in step 3) before aseptic filtration, specifically comprising the following steps: 3.1) Place the mixed solution after adding glycerin into a sealed container with a depth of 8 cm, let it stand at a constant temperature of 26℃ for 25 minutes, and then perform the filtration operation; 3.2) The aseptic filtration adopts staged pressure control, specifically: Initial stage: 25% of the total filtration volume under inlet pressure of 0.04 MPa and outlet pressure of 0.015 MPa; Main filtration stage: Linearly increase the inlet pressure to 0.09 MPa, maintain a pressure differential of 0.045 MPa, and filter to 85% of the total volume; Final stage: Reduce the inlet pressure to 0.05 MPa, and complete the remaining volume filtration with a pressure difference of 0.025 MPa; The filtrate temperature was maintained at 26℃ throughout the filtration process.

[0035] <Comparative Example 1> The difference from <Example 1> is that only chitosan is used, and sodium alginate is not included.

[0036] <Comparative Example 2> The difference from Example 1 is that only sodium alginate is used, and chitosan is not included.

[0037] <Comparative Example 3> The difference from <Example 1> is that the cellulase is 150 U / mL and does not contain pectinase.

[0038] <Comparative Example 4> The difference from <Example 1> is that the pectinase is 300 U / mL and does not contain cellulase.

[0039] <Comparative Example 5> Commercially available grafting nutrient solution is used, whose main components are chitosan oligosaccharide, bean sprout filtrate, and starch resin. Use according to the instructions.

[0040] I. Passion Fruit Grafting Experiment Test materials: Rootstock: Passiflora edulis f. flavicarpa, stem diameter 6-8 mm, seedling age 90 days.

[0041] Scion: Passiflora edulis, semi-lignified branch with 1 bud.

[0042] Grafting method: Cleft grafting (the rootstock is horizontally cut 10cm above the ground, and the longitudinal cut is 1.5cm deep; the scion is cut into a double-sided wedge shape).

[0043] The processing group settings are shown in Table 1 below: Operating steps: Wound treatment: Immediately after grafting, use a sterile cotton swab to apply nutrient solution / distilled water evenly to the joint surface of the rootstock and scion; Spraying volume control: 0.5 mL / plant (atomized particle size 50-100 μm).

[0044] Bandaging and cultivation: The wound was bandaged with a moisture-permeable grafting film (0.03 mm thick); Greenhouse conditions: daytime temperature 35±2℃ / nighttime temperature 28±2℃, relative humidity 80±5%, light intensity 12000 Lux (12h / d).

[0045] (a) Survival rate results Survival rate criteria: 21 days after grafting, the scion buds sprout and the leaves unfold; there is no browning or gumming at the scion-rootstock junction. Or symptoms of pathogen infection.

[0046] The survival rate test results are shown in Table 2.

[0047] Table 2 As shown in Table 2, the survival rate of passion fruit grafts in Examples 1-4 of this invention was significantly higher than that in the comparative group and the blank control group (p<0.05). The survival rates of the example groups ranged from 83.3% to 96.7%, with Example 4 having the highest survival rate at 96.7%, followed by Example 3 at 93.3%, and Examples 2 and 1 at 90.0% and 83.3%, respectively. In contrast, the survival rates of Comparative Examples 1-5 and the blank control group were generally lower, ranging from 56.7% to 73.3%, with the blank control group having the lowest survival rate at only 56.7%. This indicates that the nutrient solution for healing grafting wounds in fruits and vegetables containing polysaccharide complex enzymes can effectively improve the grafting survival rate, and with the optimization of the preparation process (such as the microencapsulation of enzymes in Example 2, the addition of freeze-drying protectants in Example 3, and the optimization of the aseptic filtration process in Example 4), the survival rate showed a gradual increasing trend.

[0048] (II) Tensile strength testing methods and results 1. Sample preparation Source of grafted seedlings: Passion fruit grafted 14 days after grafting (complete healing period); Sampling specifications: Retain a 20mm stem segment above and below the rootstock-scion junction (total length 40±2mm), and remove lateral roots and leaves; Sample size: per group of plants; Pretreatment: Soak in distilled water at 25℃ for 1 hour, then blot dry the surface moisture with absorbent paper (to avoid the influence of material moisture content differences).

[0049] 2. Test equipment and parameters Equipment type: Universal testing machine; Fixture: Pneumatic flat-push fixture (clamping surface with anti-slip texture); Clamping distance: 20mm (ensure the interface is centered between the two clamps); Force sensor range: 0-500 N (accuracy ±0.1 N); Data acquisition frequency: 50 Hz (complete recording of the fracture process).

[0050] 3. Testing Process Calibration: Zero the load and correct the clamp spacing to (20.0±0.1) mm; Sample preparation: The stem segment is held vertically with the rootstock end in the lower clamp and the scion end in the upper clamp; Loading: Stretch at a speed of 5 mm / min until complete separation; Records: Peak tensile force (N): the highest point of the force-displacement curve; Fracture location: at the joint / rootstock / scion (to judge the quality of healing); Energy absorption (J): area under the force-displacement curve (reflects toughness).

[0051] 4. Data Processing Remove outliers (fracture locations not in the interface area and force values ​​<2N); Calculate the mean ± standard deviation, and perform ANOVA analysis to analyze differences between groups (p < 0.05).

[0052] 5. Test Results Table 3. Results of tensile strength test at the grafting interface on day 14 post-grafting. Table 3 shows the tensile strength test results, further illustrating the impact of different treatments on graft healing quality. Regarding peak tensile strength, the example groups were significantly higher than the comparative and blank control groups. Example 4 had the highest peak tensile strength at 15.3±1.2N, followed by Example 3 at 14.5±1.3N, and Examples 2 and 1 at 13.8±1.1N and 12.6±1.0N, respectively. In contrast, the comparative group's peak tensile strength ranged from 8.9±0.9N to 11.2±1.1N, while the blank control group only reached 7.5±0.7N. The proportion of fracture sites at the graft union was significantly lower in the example groups, with Example 4 at only 8% and Example 3 at 15%, indicating stronger graft union healing. In contrast, the proportion of fracture sites at the graft union in the comparative and blank control groups generally exceeded 65%, reaching as high as 95% in the blank control group. Energy absorption data also showed a significant advantage for the example groups, with Example 4 achieving the highest at 0.78±0.07J, far exceeding the comparative group (0.25±0.03J-0.45±0.07J), indicating stronger graft union toughness.

[0053] (III) Methods and Results of Scion Growth Rate Detection 1. Operating Procedures Starting mark: On the 7th day after grafting (when the buds of the scion swell and turn green), use a marker to make a reference point at the base of the new tender stem of the scion.

[0054] Measurement cycle: Measure once every 3 days until the survival rate is statistically analyzed 21 days after grafting.

[0055] Measure the length of the tender stem of the same scion each time (from the reference point to the highest point of the stem tip).

[0056] Calculate the average daily increment: Formula: (Current length - Previous length) / Interval days; Final daily average increase: The average daily increase over the entire 7-21 days after grafting.

[0057] The results are shown in Table 4.

[0058] Table 4 As shown in Table 4, the daily average growth rate of the scions in Examples 1-4 of this invention exhibited significantly better growth performance than the comparative and blank control groups. The daily average growth rate of the example groups showed a gradual increasing trend, with Example 4 showing the highest rate of 2.5-2.7 mm / day, Example 3 at 2.2-2.4 mm / day, Example 2 at 2.0-2.2 mm / day, and Example 1 at 1.8-2.0 mm / day. This trend is consistent with the optimization direction of the preparation process—with the improvement of enzyme microencapsulation, the addition of freeze-drying protectant, and the aseptic filtration process, the scion growth rate continued to increase, indicating that the nutrient solution, while promoting wound healing, can provide a more suitable growth environment for the scions and accelerate the development of new tissue.

[0059] In comparison, the growth rates of the scions in the comparative and blank control groups were generally low. The average daily growth rate of Comparative Example 5 (commercially available grafting nutrient solution) was 1.5–1.6 mm / day, higher than other comparative examples and the blank control, but still lower than Example 1. The growth rate of Comparative Example 3 was 1.2–1.4 mm / day, Comparative Example 2 was 1.1–1.3 mm / day, and Comparative Example 4 and the blank control were both 1.0–1.2 mm / day, which are at a low level. Furthermore, the data from Comparative Example 1, "9.1±0.8," differed significantly from the data format and numerical range of other groups, suggesting a recording error. Therefore, it was not included in the valid comparison for the time being, but this does not affect the overall trend assessment.

[0060] Overall, the nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes can not only improve the graft survival rate and wound healing quality, but also effectively promote scion growth. Furthermore, with the optimization of the preparation process (such as the process improvement in Examples 2 to 4), the growth-promoting effect is further enhanced, demonstrating the significant advantage of this nutrient solution in ensuring the continuous development of scions after successful grafting.

[0061] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A nutrient solution for healing grafted wounds in fruits and vegetables containing polysaccharide complex enzymes, characterized in that, The raw material components include the following parts by weight: 0.3-0.8 parts polysaccharide, 1-5 parts cellulase, 0.5-2.5 parts pectinase, 1-5 parts glycerol, and 90-100 parts water; wherein, the pH of the nutrient solution is adjusted to 5.0-6.5 with phosphate buffer, the polysaccharide is composed of chitosan and sodium alginate, the mass ratio of chitosan to sodium alginate is 1:0.5-1:2, the cellulase activity in the nutrient solution is 100-500 U / mL, and the pectinase activity is 50-250 U / mL.

2. The method for preparing the nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes as described in claim 1, characterized in that, Includes the following steps: 1) Add chitosan to water at 50-55℃ and stir to dissolve, forming a homogeneous solution; add sodium alginate to the homogeneous solution and stir continuously at 40-45℃ until completely dissolved to obtain a polysaccharide solution; 2) Adjust the pH of the polysaccharide solution to 5.5-6.0 with disodium hydrogen phosphate-citric acid buffer, add cellulase and pectinase sequentially at 25-28℃, and stir at 80-200 rpm for 5-25 minutes to obtain a mixed solution; 3) Add glycerol to the mixed solution and continue stirring at the same speed for 8-12 minutes. Then, filter the solution aseptically through a polyethersulfone membrane with a pore size of 0.22 micrometers to obtain a nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes.

3. The method for preparing the nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes according to claim 2, characterized in that, In step 2), cellulase and pectinase are added in solid microencapsulation form, and their preparation includes the following steps: 2.1) Dissolve sodium alginate in water to prepare a sodium alginate solution with a mass percentage of 3-5%; 2.2) Mix cellulase and pectinase at an activity ratio of 2:1, add them to sodium alginate solution, and mix at 50-100 rpm for 10-15 minutes at 4-8℃ to form a uniform suspension. 2.3) The suspension is dripped into a calcium chloride solution with a mass percentage of 1.5-2.0% through a dropper with an aperture of 0.5-1.0 mm. The droplets remain in place for 10-20 minutes to form solidified microcapsules with a diameter of 1.0-2.0 mm. 2.4) Collect the solidified microcapsules and rinse them three times with deionized water. Freeze them at -40 to -30°C for 6-8 hours and transfer them to a freeze dryer for vacuum drying to obtain enzyme microcapsules with a water content of 3-5%.

4. The method for preparing the nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes according to claim 3, characterized in that, In step 2), the enzyme microcapsules are added to the polysaccharide solution after pH adjustment, and stirred at 80-100 rpm for 5-10 minutes at 25-28℃.

5. The method for preparing the nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes according to claim 3, characterized in that, In step 2.2), when adding cellulase and pectinase to the sodium alginate solution, a freeze-drying protectant is added simultaneously. The freeze-drying protectant is composed of trehalose and mannitol, with the final concentration of trehalose in the suspension being 5-8 g / L and the final concentration of mannitol being 2-4 g / L.

6. The method for preparing the nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes according to claim 5, characterized in that, Dissolve trehalose and mannitol in deionized water at 4-8℃ to prepare a mixed protective solution, and add sodium alginate solution before forming a uniform suspension.

7. The method for preparing the nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes according to claim 6, characterized in that, The freeze-drying conditions described in step 2.4) are adjusted as follows: Pre-freezing stage: Cool to -45°C at a rate of 3-5°C / min and maintain for 60 minutes; First drying: Under vacuum of 10-15 Pa and cold trap temperature of -50 to -45 °C, heat to -25 °C and maintain for 4 hours; Desorption drying: Heat to 20 °C, reduce vacuum to 0.5-1.0 Pa and maintain for 2 hours.

8. The method for preparing the nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes according to claim 2, characterized in that, In step 3), an enzyme conformation stabilization treatment is added before the aseptic filtration operation, specifically including the following steps: 3.1) Place the mixed solution after adding glycerin into a sealed container with a depth of 5-10 cm, let it stand for 20-30 minutes in a constant temperature environment of 25-28℃, and then perform the filtration operation; 3.2) The aseptic filtration adopts staged pressure control, specifically: Initial stage: Under conditions of inlet pressure 0.03-0.05MPa and outlet pressure 0.01-0.02MPa, the filter volume accounts for 20-30% of the total volume. Main filtration stage: Linearly increase the inlet pressure to 0.08-0.10 MPa, maintain a pressure difference of 0.04-0.05 MPa, and filter to 80-90% of the total volume. Final stage: Reduce the inlet pressure to 0.04-0.06 MPa, and the pressure difference to 0.02-0.03 MPa to complete the remaining volume filtration; Throughout the filtration process, the filtrate temperature is maintained at 25-28℃.

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