Lentinan as well as preparation method and application thereof

Abstract

The invention provides lentinan as well as a preparation method and application thereof, and belongs to the technical field of plant protection. According to the preparation method, a ZTC 1 + 1 II type clarifying agent is used, and step-by-step flocculation purification is carried out at 50 DEG C according to a sequence of firstly adding a component B and then adding a component A and specific stirring parameters. According to the process, impurities such as protein are efficiently removed, the active conformation of lentinan is well reserved, and the antiviral activity of the product is remarkably improved. Meanwhile, the invention finds that when the lentinan obtained by the invention is mixed with oligosaccharin.chain protein in a mass ratio of 7: 3 or 9: 1, the lentinan shows a remarkable specific synergistic effect. The invention solves the problems of poor product stability and large activity loss in the prior art, and is especially suitable for prevention and treatment of plant virus diseases such as tomato mosaic virus diseases.

Description

Technical Field

[0001] This invention belongs to the field of plant protection technology, and in particular relates to a lentinan, its preparation method and application. Background Technology

[0002] Lentinan, a polysaccharide extracted from shiitake mushrooms (Lentinus edodes), possesses significant immunomodulatory, antiviral, and plant growth-promoting effects. It is widely used in plant protection (phytosanitary) applications, particularly in biocontrol pesticides such as sprays and fertigation products, effectively enhancing plant disease resistance, inhibiting viral infection, and improving crop quality. However, lentinan extract has a complex composition, containing impurities such as mucilage, gelatinized starch, tannins, proteins, pigments, resins, inorganic salts, pectin, and gums. These impurities can lead to poor product stability, precipitation, or flocculation, affecting the field efficacy and shelf life of phytosanitary products.

[0003] While existing technologies have addressed the purification issues of plant extracts to some extent, traditional methods such as alcohol precipitation often lead to decreased polysaccharide content and product instability due to the complex composition of lentinan extracts, making them particularly unsuitable for applications requiring plant protection spraying and fertigation. Existing flocculation technologies, though advantageous, lack systematic optimization for lentinan, resulting in imprecise flocculant selection and process parameter control, impacting impurity removal rates, polysaccharide retention rates, and floc settling efficiency. Therefore, an improved lentinan extraction process is needed to efficiently remove impurities while retaining active ingredients, thereby enhancing product stability and plant protection applicability to meet market demand for high-quality biocontrol pesticides. Summary of the Invention

[0004] The purpose of this invention is to provide a lentinan for the efficient prevention and control of plant viral diseases, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing enhanced lentinan to enhance disease resistance, the method comprising the following steps: (1) Obtain lentinan extract; (2) Adjust the temperature of the shiitake polysaccharide extract to 45-55℃, add ZTC 1+1 II type clarifying agent B component, and stir rapidly to obtain mixture A; (3) After standing, add ZTC 1+1 II type clarifying agent A component to the mixture A, first stir quickly, then stir slowly; (4) Allow to stand and settle, separate the supernatant to obtain crude shiitake polysaccharide solution; (5) Pass the crude shiitake polysaccharide solution through a 3kDa ultrafiltration membrane, collect the filtrate, and pass the filtrate through a 2kDa ultrafiltration membrane to collect the retentate; (6) The retentate is concentrated under reduced pressure to 1 / 10 of its original volume, and then freeze-dried to obtain lentinan. Preferably, the method for obtaining the lentinan extract in step (1) is as follows: (a) Grind the dried shiitake mushroom bodies into coarse powder using a grinder and pass them through a 10-mesh sieve; (b) Put the coarse shiitake mushroom powder into a container, add 15 times the amount of water, stir well, heat to 65°C, add 2.5g of papain, stir well, heat to 70°C and hydrolyze for 1-2 hours. (c) After enzymatic hydrolysis, rapidly raise the temperature to 100°C and maintain it for 8-15 minutes; (d) Filter to obtain filtrate 1, add 15 times the amount of water to filter residue 1, and heat at 100°C for 1-2 hours; (e) Filter to obtain filtrate 2, add 15 times the amount of water to filter residue 2, and heat at 100°C for 1-2 hours; (f) Filtrate 3 is obtained by filtration. Filtrate 1, Filtrate 2 and Filtrate 3 are combined to obtain lentinan filtrate; (g) The filtrate of shiitake polysaccharide was concentrated under reduced pressure to a shiitake polysaccharide concentration of 2% to obtain shiitake polysaccharide extract.

[0006] Preferably, in step (2), the amount of ZTC 1+1 II type clarifying agent component B added per 1L of the lentinan extract is 0.4g-0.6g; In step (3), the amount of ZTC 1+1 II type clarifying agent A component added per 1L of the shiitake polysaccharide extract is 0.2-0.3g.

[0007] Preferably, in step (2), the rapid stirring speed after adding component B is 1.31 m / s, and the stirring time is 2 min; In step (3), the settling time is 30 min; the rapid stirring speed after adding component A is 1.31 m / s and the stirring time is 2 min; the slow stirring speed is 0.26 m / s and the stirring time is 15 min.

[0008] Preferably, the enhancement of the disease resistance of lentinan is to enhance its resistance to tomato mosaic virus disease.

[0009] Secondly, the present invention provides a lentinan for the efficient prevention and control of tomato mosaic virus disease, wherein the lentinan is prepared by the above-described preparation method.

[0010] Thirdly, the present invention provides a pharmaceutical composition for preventing and controlling tomato mosaic virus disease, the pharmaceutical composition being composed of lentinan and oligosaccharide-chain protein; The lentinan was prepared by the above-described preparation method.

[0011] Preferably, the mass ratio of the lentinan to the oligosaccharide-chain protein is 7:3 or 9:1.

[0012] Fourthly, the present invention provides the application of the above-mentioned pharmaceutical composition in the preparation of drugs for the prevention and treatment of plant viral diseases.

[0013] Preferably, the plant viral disease is tomato mosaic virus disease; The mass ratio of the lentinan to the oligosaccharide-chain protein is 9:1.

[0014] The beneficial effects of this invention are as follows: This invention addresses the complex composition of shiitake polysaccharide extract by screening and optimizing a purification process centered on ZTC 1+1 II type clarifying agent. By precisely controlling the flocculation temperature (50℃), the order of sample addition (component B first, then component A), and specific stirring parameters, it effectively overcomes the problems of high polysaccharide loss, high energy consumption, and poor clarification effect of conventional flocculants (such as tannins and sodium alginate) in traditional alcohol precipitation methods. The resulting flocs have low liquid content and fast settling speed (significant interfacial settling), greatly improving production efficiency and facilitating large-scale industrial production.

[0015] Meanwhile, the inventors unexpectedly discovered during their research that the lentinan prepared using the specific ZTC 1+1Ⅱ type clarifying agent described in Examples 1 and 5 combined with a stepwise flocculation process exhibited significantly superior antiviral bioactivity compared to similar products prepared using chitosan flocculation, traditional alcohol precipitation, or conventional ultrafiltration processes. This is likely because the specific process of this invention efficiently removes impurities such as proteins and pigments while avoiding the destruction of the helical active conformation of lentinan by strong acids, strong alkalis, or violent chemical reagents. Simultaneously, this process effectively removes interfering macromolecules adsorbed on the polysaccharide surface, eliminating the shielding effect of impurities on the active sites of the polysaccharide, thereby significantly enhancing the ability of a unit mass of polysaccharide molecules to induce plant disease resistance responses.

[0016] Furthermore, this invention has discovered a highly specific synergistic combination. Studies have shown that when lentinan prepared solely by the process of this invention is mixed with oligosaccharide-chain protein in a mass ratio ranging from 7:3 to 9:1, it exhibits a significant synergistic effect, significantly superior to a simple additive effect. This synergistic effect was not observed when lentinan prepared by other processes was mixed with oligosaccharide-chain protein, or when lentinan of this invention was mixed with amino oligosaccharides or ningnanmycin. This is likely because the lentinan prepared by the special method of this invention has a specific physical affinity for oligosaccharide-chain protein, achieving a synergistic amplification of the inhibitory effect. This discovery provides a new technical pathway for developing highly efficient, low-dosage agents for the control of plant viral diseases. Attached Figure Description

[0017] Figure 1 The effect of chitosan dosage on the turbidity of the extract and the retention rate of effective components; Figure 2 The effect of chitosan dosage on protein removal rate; Figure 3 The effect of chitosan dosage on the liquid content of flocs; Figure 4 The effect of ZTC dosage on the turbidity of the extract and the retention rate of effective components; Figure 5 The effect of ZTC dosage on protein removal rate; Figure 6 The effect of ZTC dosage on the liquid content of flocs; Figure 7 The effect of the dosage of 101 fruit juice clarifying agent on the turbidity of the extract and the retention rate of effective components; Figure 8 The effect of 101 juice clarifying agent dosage on protein removal rate; Figure 9 The effect of 101 juice clarifying agent dosage on floc liquid content; Figure 10 The effect of flocculation temperature on the turbidity of the extract and the retention rate of effective components; Figure 11 The effect of flocculation temperature on protein removal rate; Figure 12 The effect of rapid stirring speed on the turbidity of the extract and the retention rate of effective components; Figure 13 The effect of rapid stirring time on the turbidity of the extract and the retention rate of effective components; Figure 14 The effect of high stirring speed on protein removal rate; Figure 15 The effect of rapid stirring time on protein removal rate; Figure 16The effect of slow stirring speed on the turbidity of the extract and the retention rate of effective components; Figure 17 The effect of slow stirring time on the turbidity and retention rate of active ingredients in the extract; Figure 18 The effect of slow stirring speed on protein removal rate; Figure 19 The effect of slow stirring time on protein removal rate. Detailed Implementation

[0018] The following details the specific implementation methods of the present invention. It should be noted that the implementation methods described are only used to explain the invention and are not intended to limit the scope of protection of the present invention. Any non-essential modifications and substitutions made in accordance with the present invention should be within the scope of protection of the present invention.

[0019] Example 1 Obtaining Lentinan Extract (1) Take clean, mold-free dried shiitake mushroom bodies, crush them into coarse powder using a grinder, and pass them through a 10-mesh sieve; (2) Put 1000g of shiitake mushroom powder into a container, add 15 times the amount of water, stir well, heat to 65℃, add 2.5g of papain (enzyme activity ≥500,000 U / g), stir well, heat to 70℃, and continue enzymatic hydrolysis for 1 hour; (3) After the enzymatic hydrolysis is complete, quickly raise the temperature to 100°C and maintain it for 10 minutes; (4) Filter to obtain filtrate 1, add 15 times the amount of water to filter residue 1, and add at 100°C for 1 hour; (5) Filter to obtain filtrate 2, add 15 times the amount of water to filter residue 2, and add at 100°C for 1 hour; (6) Filtrate 3 was obtained by filtration. Filtrate 1, Filtrate 2 and Filtrate 3 were combined to obtain lentinan filtrate. (7) The filtrate of shiitake polysaccharide was concentrated under vacuum at -0.08 MPa and 60°C to a concentration of 2% (determined by phenol-sulfuric acid method) to obtain shiitake polysaccharide extract.

[0020] Example 2 Screening of flocculant types This embodiment compares the flocculation effects of different flocculants on lentinan extract to screen for suitable flocculants. Experimental conditions: extract volume 400 mL, flocculation temperature room temperature (25℃), rapid stirring speed 1.31 m / s (for 2 min), slow stirring speed 0.13 m / s (for 15 min). After flocculation, the morphology of the flocs, settling velocity, turbidity of the supernatant, protein removal rate, and lentinan retention rate were observed after standing.

[0021] The preparation method of the flocculant involved in this invention is as follows: Chitosan: Take 1g of chitosan, add 1% glacial acetic acid, place in a 100mL volumetric flask and make up to volume to prepare a 1% solution. Stir well and swell for 24 hours before use.

[0022] Tannin: Take 1g of tannin, add a certain amount of deionized water, place it in a 100mL volumetric flask and dilute to volume to prepare a 1% solution, stir well and set aside.

[0023] ZTC1+1Ⅱ type clarifying agent (Wuhan Zhengtiancheng Biotechnology Co., Ltd.): Take 1g of component A, add a certain amount of deionized water, swell for 24h, stir, filter through double-layer gauze, and prepare a 1% solution; take 2g of component B, dissolve in a certain amount of 1% acetic acid, swell for 24h, stir, filter through double-layer gauze, and prepare a 1% solution for later use.

[0024] Sodium alginate: Take 1g of sodium alginate, add a certain amount of deionized water, dilute to volume with a 100mL volumetric flask to prepare a 1% solution, stir well and let it swell for 24 hours, then set aside.

[0025] 101 Juice Clarifying Agent: Take 1g of 101 Juice Clarifying Agent, add a certain amount of deionized water, and dilute to volume in a 100mL volumetric flask to prepare a 1% solution for later use.

[0026] The specific experimental steps are as follows: (1) Measure 400 mL of shiitake mushroom extract into a beaker and place it on a stirrer.

[0027] (2) Set the operating parameters of the stirrer: fast stirring speed 1.31m / s, time 2min; slow stirring speed 0.13m / s, time 15min.

[0028] (3) Start the mixer and simultaneously add the corresponding amount of flocculant shown in Table 1 to each mixing cup, and observe the flocculation phenomenon. The addition method for ZTC1+1-Ⅱ type flocculant is as follows: first add component B, After stirring rapidly at 1.31 m / s for 2 minutes and letting stand for 30 minutes, add component A, stir rapidly at 1.31 m / s for 2 minutes, and then stir slowly at 0.13 m / s for 15 minutes; the remaining flocculants are added all at once.

[0029] (4) After flocculation, the mushroom liquid in the stirring cup is placed into a 250mL graduated cylinder for flocculation sedimentation experiment to observe sedimentation phenomenon and record the data of the clear and turbid interface position; the remaining liquid is poured into a beaker for later use.

[0030] (5) After sedimentation, draw out the supernatant from the graduated cylinder and place it in a bottle for measuring the effective concentration. The retention rate, turbidity, and impurity removal rate are measured. The flocs are placed in a small beaker to measure the liquid content of the flocs.

[0031] Table 1 Flocculant Dosage

[0032] The results are as follows: Comparison of the flocculation effects of different flocculants on lentinan extract: Table 2 Comparison of flocculation effects of different flocculants on lentinan extract

[0033] As can be seen from the results in Table 2, chitosan, ZTC1+1-II, and 101 fruit juice clarifying agent had a good flocculation and clarification effect on the extract of shiitake polysaccharide in this experiment. The flocs were obvious, the stratification phenomenon was significant during the sedimentation process, and the turbidity of the extract decreased significantly to below 400 NTU. However, tannin and sodium alginate did not play an obvious flocculation and clarification role. No obvious flocs appeared during the flocculation process, and the turbidity of the extract did not decrease significantly.

[0034] 1. The effect of chitosan dosage on the turbidity of the extract and the retention rate of effective components is as follows: Figure 1 As shown. From Figure 1 It can be seen that when the amount of chitosan is less than 0.75 g / L, the turbidity of the extract decreases with the increase of its amount, while when the amount is greater than 0.75 g / L, the turbidity of the extract increases with the increase of its amount.

[0035] The retention rate of lentinan showed a trend of first increasing and then slightly decreasing with the increase of chitosan dosage, with little overall impact, reaching its maximum value at 0.5 g / L chitosan.

[0036] 2. Results of the effect of chitosan dosage on protein removal rate as follows: Figure 2 As shown. From Figure 2 It can be seen that when the amount of chitosan used does not exceed 0.75 g / L, the protein removal rate is relatively high, while the removal rate decreases when the amount exceeds 0.75 g / L.

[0037] Therefore, changes in the amount of chitosan used have a significant impact on the turbidity of the extract, the retention rate of shiitake polysaccharides, and the protein removal rate. The suitable dosage range for chitosan treatment of shiitake extract is 0.5 g / L to 0.75 g / L. At this concentration, the turbidity of the extract is low, the retention rate of effective components is above 90%, and the removal rate of impurities is high.

[0038] 3. Results of the effect of chitosan dosage on floc characteristics as follows: Figure 3 As shown. From Figure 3It can be seen that as the amount of chitosan flocculant gradually increases, the liquid content of the flocs first decreases and then increases, with the lowest liquid content of the flocs at 0.75 g / L.

[0039] 4. The results of the effect of chitosan dosage on the settling velocity of flocs are shown in Table 3.

[0040] Table 3 Effect of chitosan dosage on floc settling velocity

[0041] As can be seen from Table 3, the floc settling velocity is the highest at a dose of 1.0 g / L, and the floc settling velocity decreases at doses below or above this level.

[0042] A comprehensive analysis of the effects of different chitosan dosages on various indicators, including extract turbidity, retention rate of active ingredients, impurity removal rate, floc liquid content, and floc settling velocity, determined the optimal chitosan dosage to be 0.75 g / L–1.0 g / L, with all indicators showing good results. At lower chitosan dosages (below 0.25 g / L), the generated floc liquid content is relatively high, the flocs are relatively loose, the floc settling velocity is slow, the extract turbidity is high, the protein removal rate is low, and the retention rate of active ingredients is high. At higher chitosan dosages, the generated floc liquid content is relatively high, the floc density is increased, the floc settling velocity is increased, the flocculation efficiency is high, the extract turbidity is low, the protein removal rate is high, and the retention rate of active ingredients decreases somewhat. Excessive chitosan dosage (above 1.25 g / L) results in increased floc liquid content, decreased floc density, reduced floc settling speed, increased turbidity of the extract, lower protein removal rate, and a slight decrease in the content of effective components.

[0043] 5. Results of the effect of ZTC dosage on the turbidity of the extract and the retention rate of active ingredients are as follows: Figure 4 As shown. From Figure 4 It can be seen that when the dosage of ZTC1+1 is in the range of 0.6 g / L to 0.9 g / L, the turbidity of the flocculated extract is low and the retention rate of lentinan is high, remaining above 94%.

[0044] 6. Results of the effect of ZTC dosage on protein removal rate are as follows: Figure 5 As shown. From Figure 5 It can be seen that the protein removal rate in the flocculated extract was the highest when 0.75 g / L ZTC1+1 was added, and the turbidity of the extract also reached the lowest value at this time.

[0045] 7. Results of the effect of ZTC dosage on floc liquid content are as follows: Figure 6 As shown. From Figure 6 It can be seen that when the ZTC1+1 dosage is 0.75 g / L, the floc liquid content is low and the floc density is relatively high.

[0046] 8. The results of the effect of ZTC dosage on the settling velocity of flocs are shown in Table 4.

[0047] Table 4 Effect of ZTC dosage on floc settling velocity

[0048] As can be seen from the results in Table 4, the settling velocity of the flocs is relatively high when the ZTC1+1 dosage is 0.9 g / L.

[0049] A comprehensive analysis of the effects of different ZTC dosages on various indicators, including extract turbidity, retention rate of active ingredients, impurity removal rate, floc liquid content, and floc settling velocity, determined the optimal ZTC dosage to be 0.75 g / L–0.9 g / L, with all indicators showing good performance. At lower ZTC dosages (below 0.6 g / L), the generated floc liquid content is relatively high, the flocs are looser, the floc settling velocity is slower, the extract turbidity is higher, the protein removal rate is lower, and the retention rate of active ingredients is higher. At excessively high ZTC dosages (above 0.9 g / L), the generated floc liquid content increases, the floc density decreases, the floc settling velocity decreases, the extract turbidity increases, the protein removal rate is lower, and the content of active ingredients slightly decreases.

[0050] 9.101 The effect of fruit juice clarifying agent dosage on extract turbidity and retention rate of active ingredients is as follows: Figure 7 As shown. From Figure 7 It can be seen that when the dosage of 101 fruit juice clarifying agent is in the range of 0.8g / L to 1.0g / L, the turbidity of the flocculated extract is low and the retention rate of lentinan is high, remaining above 90%.

[0051] The results of the effect of the clarification dosage on protein removal rate in juice 10.101 are as follows: Figure 8 As shown. From Figure 8 It can be seen that the protein removal rate in the flocculated extract was the highest when 1.0 g / L 101 juice clarifying agent was added, and the turbidity of the extract also reached the lowest value at this time.

[0052] 11. The results of the effect of 101 juice clarifying agent dosage on floc liquid content are as follows: Figure 9 As shown. From Figure 9 It can be seen that when the dosage of 101 juice clarifying agent is 1.0 g / L, the liquid content of the flocs is relatively low, and the density of the flocs is relatively high.

[0053] The results of the effect of the dosage of fruit juice clarifying agent on the settling velocity of flocs are shown in Table 5.

[0054] Table 5. Effect of 101 Juice Clarifying Agent Dosage on Floc Settling Velocity

[0055] As can be seen from Table 5, the settling velocity of flocs is relatively high when the dosage of 101 juice clarifying agent is 0.8 g / L.

[0056] A comprehensive analysis of the effects of different dosages of 101 fruit juice clarifying agent on various indicators such as extract turbidity, retention rate of effective components, impurity removal rate, floc liquid content, and floc settling velocity revealed that the optimal dosage of 101 fruit juice clarifying agent is 1.0 g / L, with all indicators showing good performance.

[0057] Example 3 The effect of flocculation temperature on flocculation effect Under the operating conditions of ZTC1+1Ⅱ clarifying agent dosage of 0.75 g / L, rapid stirring speed of 1.31 m / s for 120 s, slow stirring speed of 0.26 m / s for 15 min, the flocculation effect of the extract was studied at different temperature levels as designed in the experimental scheme.

[0058] Data on the changes in turbidity, retention rate of effective components, and removal rate of impurities in the extract after flocculation at different flocculation temperatures are as follows: Figure 10 and Figure 11 As shown. From Figure 10 and Figure 11 It can be seen that the flocculation effect is best when the flocculation temperature is 50℃.

[0059] Example 4 Effects of stirring speed and stirring time on flocculation effect Under the operating conditions of ZTC1+1Ⅱ clarifying agent dosage of 0.75 g / L, rapid stirring time of 2 min, slow stirring speed of 0.13 m / s, and slow stirring time of 15 min, the extract was subjected to flocculation treatment according to different rapid stirring speed levels designed in the experimental scheme.

[0060] Under the operating conditions of ZTC1+1Ⅱ clarifying agent dosage of 0.75 g / L, rapid stirring speed of 1.31 m / s, slow stirring speed of 0.13 m / s, and slow stirring time of 15 min, the extract was subjected to flocculation treatment according to different rapid stirring time levels designed in the experimental scheme.

[0061] Under the operating conditions of ZTC1+1Ⅱ clarifying agent dosage of 0.75 g / L, rapid stirring speed of 1.31 m / s, rapid stirring time of 120 s, and slow stirring time of 15 min, the extract was subjected to flocculation treatment according to different slow stirring speed levels designed in the experimental scheme.

[0062] Under the operating conditions of ZTC1+1Ⅱ clarifying agent dosage of 0.75 g / L, rapid stirring speed of 1.31 m / s, rapid stirring time of 120 s, and slow stirring speed of 0.26 m / s, the extract was subjected to flocculation experiments according to different slow stirring time levels designed in the experimental scheme.

[0063] The results obtained are as follows Figure 12-19 As shown, from Figure 12-19 It can be seen that when the fast stirring speed is 1.31 m / s and the fast stirring time is 120 s, and the slow stirring speed is 0.26 m / s and the slow stirring time is 15 min, the turbidity of the extract, the retention rate of effective components, and the protein removal rate are the best.

[0064] Based on the experimental results of various operational factors, this invention concludes that the suitable flocculation process conditions for shiitake mushroom extract using ZTC1+1Ⅱ clarifying agent and flocculant are as follows: ZTC1+1Ⅱ clarifying agent dosage is 0.75 g / L, flocculation temperature is 50℃, after both components are added, the rapid stirring speed is 1.31 m / s for 120 s, the slow stirring speed is 0.26 m / s for 15 min. Under these conditions, the liquid content of the formed flocs is low, the floc settling speed is high, the removal rate of protein in the flocculated extract is high, the retention rate of the effective component shiitake mushroom polysaccharide is high, and the clarity of the extract is high.

[0065] Example 5 Preparation of lentinan (1) Take 500 mL of the lentinan extract prepared in Example 1 and adjust the temperature to 50°C; (2) Add 25 mL of ZTC 1+1 II type clarifying agent component B with a concentration of 1%, and stir rapidly at a speed of 1.31 m / s for 2 minutes; (3) Stop stirring and let stand for 30 minutes. Then add 12.5 mL of ZTC 1+1 II type clarifying agent component A with a concentration of 1%. Stir rapidly at a speed of 1.31 m / s for 2 minutes, and then stir slowly at a speed of 0.26 m / s for 15 minutes. (4) Stop stirring, let stand at 50°C for 4 hours to settle, filter and collect the supernatant to obtain crude shiitake polysaccharide solution; (5) Pass the crude shiitake polysaccharide solution through a 3 kDa ultrafiltration membrane, collect the filtrate, pass the filtrate through a 2 kDa ultrafiltration membrane, and collect the retentate; (6) The retentate was concentrated under reduced pressure to 1 / 10 of its original volume and then freeze-dried to obtain lentinan 1.

[0066] Comparative Example 1 (1) Take 500 mL of the lentinan extract prepared in Example 1 and adjust the temperature to 50°C; (2) Add 37.5 mL of chitosan solution, stir rapidly at 1.31 m / s for 2 minutes, and then stir slowly at 0.26 m / s for 15 minutes; (3) Stop stirring, let stand at 50°C for 4 hours to settle, filter and collect the supernatant, concentrate under reduced pressure to 1 / 10 of the original volume, and freeze dry to obtain lentinan 2.

[0067] Comparative Example 2 (1) Take 500 mL of the lentinan extract prepared in Example 1 and adjust the temperature to 50°C; (2) Add 50 mL of 101 fruit juice flocculant, stir rapidly at a speed of 1.31 m / s for 2 minutes, and then stir slowly at a speed of 0.26 m / s for 15 minutes; (3) Stop stirring, let stand at 50°C for 4 hours to settle, filter and collect the supernatant, concentrate under reduced pressure to 1 / 10 of the original volume, and freeze dry to obtain lentinan 3.

[0068] Comparative Example 3 (1) Take 500mL of shiitake mushroom liquid, add 1.2 times the volume of 95% ethanol to the alcohol content of 60%, precipitate for 8 hours, discard the supernatant after the alcohol precipitation is completed, turn on the stir, add 3 times the amount of precipitate of 95% ethanol, stir for 20 minutes, let stand for 4 hours, and discard the supernatant.

[0069] (2) Centrifuge to collect the precipitate, freeze dry to obtain lentinan 4.

[0070] Comparative Example 4 (1) Take 500 mL of the lentinan extract prepared in Example 1 and pass it through a 3 kDa ultrafiltration membrane, and collect the permeate; (2) Concentrate under reduced pressure to 1 / 10 of the permeate volume, and freeze dry to obtain lentinan 5.

[0071] Example 6 Investigating the effects of different shiitake polysaccharides against tomato mosaic virus (1) Preparation of Tomato Mosaic Virus (ToMV) Sap Fresh, obviously diseased leaves were cut from diseased tomatoes in the laboratory. Each time, 10g of diseased leaves were weighed, and a small amount of quartz sand and distilled water were added to a mortar and pestle and ground together. The juice was then filtered through gauze for later use.

[0072] (2) Indoor single-dose toxicity assay For every 20 tomato plants treated, one fully expanded true leaf was selected from each plant, and the treatment was repeated three times. ToMV sap was inoculated using the friction method, and the leaves were rinsed with water after inoculation. Spraying began on the 3rd day after inoculation, with a second application 7 days after the first, for a total of two applications. Five days after the last application, the disease incidence was assessed on the entire plant, graded according to a 0-4 scale, and the disease index and relative efficacy were calculated. The virulence regression equation and EC50 were obtained using DPS software. 50 .

[0073] The concentrations of lentinan were prepared at 600, 500, 400, 300, and 200 mg / L.

[0074] Viral disease classification standards: Level 0: Asymptomatic; Grade 1: Slight mosaic pattern, with lesion area <10%; Grade 2: Obvious mosaic or wrinkled leaves, with lesions covering 10%–30% of the leaf area; Grade 3: Severe deformity, lesion area > 30%; Grade 4: Severe deformity, necrosis or yellowing, with lesion area >50%.

[0075] Disease index = [∑(number of diseased plants × disease level value) / (total number of plants × highest disease level)] × 100 Prevention and control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100.

[0076] The results of indoor toxicity tests of different lentinan to ToMV are shown in Table 6.

[0077] Table 6. Results of indoor toxicity assays of different lentinan to ToMV

[0078] The results show that, compared to lentinan 2, lentinan 3, lentinan 4, and lentinan 5, lentinan 1 exhibited the strongest anti-ToMV effect among all tested samples. It not only had the highest relative efficacy but also required the lowest effective concentration, indicating its superior virus inhibition potential. This result also suggests that the preparation method used in this invention may endow lentinan 1 with unique advantages in terms of molecular structure, purity, or structure-activity relationship, thus giving it better application prospects in plant virus control.

[0079] Example 7 Investigating the field efficacy of different lentinan Plot arrangement: A greenhouse with an area of ​​1.5 acres was selected at the early stage of tomato mosaic virus infection and divided into 6 treatments, totaling 24 plots. A randomized block design was used, with a protection row set up between each plot. Experimental Groups: Treatment 1: Prepare 5% concentration of lentinan 1, apply at a rate of 60ml / acre, diluted 500 times for spraying.

[0080] Treatment 2: Lentinan 2, with a concentration of 5%, is applied at a rate of 60 ml / acre, diluted 500 times for spraying.

[0081] Treatment 3: Lentinan 3, with a concentration of 5%, is applied at a rate of 60 ml / acre, diluted 500 times for spraying.

[0082] Treatment 4: Lentinan 4, with a concentration of 5%, is applied at a rate of 60 ml / acre, diluted 500 times for spraying.

[0083] Treatment 5: Lentinan 5, with a concentration of 5%, was applied at a rate of 60 ml / acre, diluted 500 times for spraying.

[0084] Treatment 6: Water control Application period and method: Apply the pesticide twice via foliar spray, with a 7-day interval between applications. Seven days after the second application, investigate and collect data on the incidence of tomato mosaic virus. Appendix: Classification Standards for Viral Diseases Grade 0: Asymptomatic, healthy leaves.

[0085] Grade 1: Slight mosaic or chlorotic spots, with the affected area covering less than 10% of the leaf area.

[0086] Grade 2: Obvious mosaic patterns, wrinkled or necrotic spots, with the lesions covering 10%-30% of the leaf area.

[0087] Grade 3: Severe deformity, with lesions covering more than 30% of the leaf area.

[0088] Grade 4: Severe deformity, necrosis, or yellowing of the entire leaf, with lesions covering more than 50% of the leaf area.

[0089] Disease index = [∑(number of diseased plants × disease level value) / (total number of plants × highest disease level)] × 100 Prevention and control efficacy (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100 The results are shown in Table 7.

[0090] Table 7 Field efficacy of different lentinan

[0091] As can be seen from the results in Table 7, similar to the results of indoor testing, the actual field efficacy of Lentinan 1 of this invention is also significantly higher than that of other processed products.

[0092] Example 8 Detecting the effects of combining different lentinan with oligosaccharides and chain proteins Five stock solutions were prepared using lentinan 1-5 as agent A (concentration 200 mg / L) and oligosaccharide-chain protein as agent B (6% oligosaccharide-chain protein wettable powder, purchased from Hebei Zhongbaolv Agricultural Technology Co., Ltd., concentration 1300 mg / L). These stock solutions were prepared at mass ratios of 1:9, 3:7, 5:5, 7:3, and 9:1, and then diluted to create five concentration gradients. Inoculation and application methods were the same as above. The EC50 values ​​for each ratio were calculated. 50 The co-toxicity coefficient (CTC) was calculated using oligosaccharide-chain protein single agent as the standard agent.

[0093] Relative toxicity index (TI) = EC50 of standard reagent / EC50 of test reagent 50 ×100 Actual Toxicity Index (ATI) of Mixture = EC of Standard Formulation 50 EC of the test mixture 50 ×100 Theoretical Toxicity Index (TTI) of a mixture = ATI of agent A × Percentage of agent A in the mixture + ATI of agent B × Percentage of agent B in the mixture

[0094] CTC = Actual Toxicity Index (ATI) of the mixture / Theoretical Toxicity Index (TTI) of the mixture × 100% If the CTC of the mixture is ≥120, it indicates a synergistic effect; if the CTC is between 80 and 120, it indicates an additive effect; if the CTC is ≤80, it indicates an antagonistic effect.

[0095] Viral Disease Classification Standards Grade 0: Asymptomatic, healthy leaves.

[0096] Grade 1: Slight mosaic or chlorotic spots, with the affected area covering less than 10% of the leaf area.

[0097] Grade 2: Obvious mosaic patterns, wrinkled or necrotic spots, with the lesions covering 10%-30% of the leaf area.

[0098] Grade 3: Severe deformity, with lesions covering more than 30% of the leaf area.

[0099] Grade 4: Severe deformity, necrosis, or yellowing of the entire leaf, with lesions covering more than 50% of the leaf area.

[0100] Disease index = [∑(number of diseased plants × disease level value) / (total number of plants × highest disease level)] × 100 Prevention and control efficacy (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100 The results are shown in Table 8.

[0101] Table 8. Virulence of mixed agents of lentinan 1, lentinan 2, lentinan 3, lentinan 4, lentinan 5 and oligosaccharide-chain protein against combined virulence of tomato viral diseases.

[0102] As can be seen from the results in Table 8, only lentinan 1 (prepared by ZTC flocculation process) showed a significant synergistic effect with oligosaccharide-chain protein at mass ratios of 7:3 and 9:1. Among them, the 9:1 ratio had the highest CTC of 150.69, showing the most significant synergistic effect. All ratios of lentinan and oligosaccharide-chain protein prepared by the other four processes showed only an additive effect.

[0103] Example 9 To determine whether the combined use of lentinan 1 with amino oligosaccharides or ningnanmycin has a synergistic inhibitory effect on tomato mosaic virus disease. Following the same method as in Example 8, the effect of combining lentinan 1 with amino oligosaccharide or ningnanmycin was tested.

[0104] The results are shown in Table 9.

[0105] Table 9. Virulence of Lentinan 1, Amino Oligosaccharide, and Ningnanmycin Mixtures against Combined Virus Diseases in Tomato

[0106] As shown in Table 9, the mixtures of lentinan 1 with amino oligosaccharides and ningnanmycin did not exhibit a synergistic effect. This indicates that lentinan 1 prepared in this invention has a highly specific synergistic effect with oligosaccharide-chain proteins, while similar synergistic phenomena were not found in conventional plant virus disease control agents such as amino oligosaccharides and ningnanmycin.

Claims

1. A method for preparing lentinan with enhanced disease resistance, characterized in that, The preparation method comprises the following steps: (1) obtaining a lentinan extraction solution; (2) adjusting the temperature of the lentinan extraction solution to 45-55℃, adding ZTC 1+1 II type clarifier B component, and stirring rapidly to obtain a mixed solution A; (3) after standing, adding ZTC 1+1 II type clarifier A component to the mixed solution A, stirring rapidly first, and then stirring slowly; (4) standing and settling, separating the supernatant to obtain a crude lentinan solution; (5) passing the crude lentinan solution through a 3kDa ultrafiltration membrane, collecting the filtrate, passing the filtrate through a 2kDa ultrafiltration membrane, and collecting the retentate; (6) concentrating the retentate under reduced pressure to 1 / 10 of the original volume, and freeze-drying to obtain lentinan.

2. The production method according to claim 1, characterized by, The method for obtaining the lentinan extraction solution in step (1) is as follows: (a) crushing dried lentinan entities into coarse powder using a crusher, and passing the coarse powder through a 10-mesh sieve; (b) placing the lentinan coarse powder into a container, adding 15 times the amount of water, stirring uniformly, heating to 65℃, adding 2.5g of papain, stirring uniformly, and heating to 70℃ for enzymatic hydrolysis for 1-2 hours; (c) after the enzymatic hydrolysis is completed, rapidly heating to 100℃ and maintaining for 8-15 minutes; (d) filtering to obtain filtrate 1, adding 15 times the amount of water to the residue 1, and heating at 100℃ for 1-2 hours; (e) filtering to obtain filtrate 2, adding 15 times the amount of water to the residue 2, and heating at 100℃ for 1-2 hours; (f) filtering to obtain filtrate 3, and combining the filtrate 1, the filtrate 2 and the filtrate 3 to obtain a lentinan filtrate; (g) concentrating the lentinan filtrate under reduced pressure to a lentinan concentration of 2% to obtain a lentinan extraction solution.

3. The production method according to claim 2, characterized by, In step (2), the addition amount of the ZTC 1+1 II type clarifier B component corresponding to 1L of the lentinan extraction solution is 0.4g-0.6g; In step (3), the addition amount of the ZTC 1+1 II type clarifier A component corresponding to 1L of the lentinan extraction solution is 0.2-0.3g.

4. The production method according to claim 3, characterized by, In step (2), the rapid stirring speed after adding the B component is 1.31 m / s, and the stirring time is 2 min; In step (3), the standing time is 30 min; the rapid stirring speed after adding the A component is 1.31 m / s, the stirring time is 2 min; the slow stirring speed is 0.26 m / s, and the stirring time is 15 min.

5. The preparation method according to claim 4, characterized in that, The enhanced lentinan disease resistance is the ability to enhance the resistance of lentinan to tomato mosaic virus disease.

6. A lentinan for efficiently preventing and treating tomato spotted wilt virus, characterized by, The lentinan is prepared by the preparation method in any one of claims 1-5.

7. A pharmaceutical composition for controlling tomato leaf mould, characterized by comprising the compound of claim 1. The pharmaceutical composition is composed of lentinan and oligosaccharide·chain protein; The lentinan is prepared by the preparation method in any one of claims 1-5.

8. The pharmaceutical composition of claim 7, wherein, The mass ratio of the lentinan to the oligosaccharide·chain protein is 7:3 or 9:

1.

9. The use of the pharmaceutical composition of claim 7 in the preparation of a medicine for preventing and treating plant viral diseases.

10. Use according to claim 9, characterized in that, The plant viral disease is tomato mosaic virus disease; The mass ratio of the lentinan to the oligosaccharide·chain protein is 9:1.

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