Method for promoting stiffness of cordyceps sinensis susceptible insects through arginine and application

Abstract

The invention discloses a method for promoting stiffness of cordyceps sinensis susceptible insects through arginine and application, relates to the technical field of cordyceps sinensis cultivation, and solves the problems that hepialus armoricanus larva stress is caused by traditional mannitol injection, and the process is tedious and time-consuming. The breeding of cordyceps sinensis is improved, and the stiffness of infected insects is accelerated; arginine is loaded in a polyaspartic acid-chitosan oligosaccharide gel network to form arginine nano sustained-release particles, so that the arginine is slowly released in intestinal tracts of hepialus armoricanus larvae, the residence time of the arginine in the intestinal tracts is prolonged, and the utilization rate of the arginine is increased; the larvae of hepialus armoricanus are fed with the arginine nano sustained-release particles and the feed soaked with the arginine together, the problems that the concentration of the arginine in the early stage is high and the concentration of the arginine in the later stage is low are solved, and the effects that double nutrition supply is achieved for cordyceps sinensis, and the rigidifying speed is further increased are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cordyceps cultivation, more particularly, it relates to a method for promoting the mycosis of insects of cordyceps by arginine and application. BACKGROUND

[0002] Cordyceps is a complex of sclerotium and fungal stroma formed by the Cordyceps sinensis of the Clavicipitaceae family infecting the larvae of the bat moth in the alpine meadow soil of the Qinghai-Tibet Plateau, has the effects of tonifying kidney and lung, stopping bleeding and reducing phlegm, and is used for kidney deficiency and essence deficiency, impotence and spermatorrhea, soreness of waist and knees, chronic cough and shortness of breath, and labor cough and hemoptysis.

[0003] The formation of cordyceps depends on extremely special natural conditions, including: 3500-5000 meters of high altitude, thin oxygen, cold climate, suitable for the growth of cordyceps; low temperature and high humidity: the mycelium needs a humid environment, but it cannot be too humid, otherwise it will rot; soil contains organic matter: the larvae of the bat moth feed on plant roots and soil humus, so rich soil nutrients help the survival of the larvae; ecological balance: if the number of bat moth is too small, the cordyceps fungus has no host, and cannot reproduce, these harsh conditions make the wild cordyceps extremely scarce, and also one of the reasons for its high price.

[0004] The prior art records that the mycosis of insects is promoted by injecting mannitol into the body of the bat moth larvae, but this method is easy to cause the stress response of the bat moth larvae, and is tedious and time-consuming to inject one by one, and is not suitable for batch cultivation.

[0005] It is a problem to be solved to provide a method for improving the proliferation speed of cordyceps fungus in the body of mycosis of insects and accelerating the mycosis of insects without causing the stress response of the bat moth larvae and being simple. SUMMARY

[0006] To solve the problems in the prior art, the purpose of the present application is to provide a method for promoting the mycosis of insects of cordyceps by arginine and application, by soaking the feed in arginine and then feeding the bat moth larvae, the reproduction of cordyceps fungus is improved, and the mycosis of insects is accelerated; the arginine nano slow-release particles formed by loading arginine in the poly-aspartic acid-chitosan oligosaccharide gel network make the arginine slowly release in the intestinal tract of the bat moth larvae, prolong the residence time of arginine in the intestinal tract, and improve the utilization rate of arginine; Feeding the bat moth larvae with the arginine nano slow-release particles and the feed soaked in arginine avoids the problem of high arginine concentration in the early stage and low arginine concentration in the later stage, realizes the double nutrition supply of cordyceps fungus, and further accelerates the mycosis speed.

[0007] The above technical purpose of the present application is realized by the following technical scheme: In a first aspect, there is provided an application of arginine in promoting the mycosis-induced immobilization of O. sinensis.

[0008] In a second aspect, there is provided a method for promoting the mycosis-induced immobilization of O. sinensis by arginine, comprising the following steps: Pre-treating the feed with arginine, and feeding the mycosis-induced larvae with the pre-treated feed; Raising the larvae in a dark environment with a temperature of 15-18°C and a humidity of 70-80% until the larvae are immobilized.

[0009] Further, the pre-treatment process comprises the following steps: Preparing an arginine aqueous solution with a mass fraction of 1%; Soaking the feed in the arginine solution for 30 minutes; Draining the soaked feed to obtain the pre-treated feed.

[0010] In a third aspect, there is provided a method for promoting the mycosis-induced immobilization of O. sinensis by arginine, comprising the following steps: S1. Preparing a poly-aspartic acid-chitosan oligosaccharide composite gel; S2. Adding arginine to the poly-aspartic acid-chitosan oligosaccharide composite gel and performing probe sonication to obtain a nano-composite emulsion; S3. Freeze-drying the nano-composite emulsion to obtain poly-aspartic acid-chitosan oligosaccharide nano-coated arginine nano-sustained-release particles; S4. Mixing the nano-particles into the feed as described in the second aspect to feed the mycosis-induced larvae.

[0011] Further, in step S1, the poly-aspartic acid-chitosan oligosaccharide composite gel is prepared by the following steps: Placing a 5% w / v poly-aspartic acid solution on a magnetic stirrer and stirring at 500 rpm; According to a volume ratio of chitosan oligosaccharide solution: poly-aspartic acid solution of 1:3, slowly adding 2% w / v chitosan oligosaccharide solution to the poly-aspartic acid solution to obtain a poly-aspartic acid-chitosan oligosaccharide composite gel solution; In the above process, the pH is controlled at 5.0, the temperature is controlled at 23-27°C, and the drop rate is ≤1 mL / min.

[0012] Further, in step S2, the following steps are included: Adding an arginine solution to the poly-aspartic acid-chitosan oligosaccharide composite gel solution, and controlling the volume ratio of the poly-aspartic acid-chitosan oligosaccharide composite gel: arginine to be 4:1; Then emulsification is carried out by using a probe ultrasound, and the emulsification conditions are as follows: power 200 W, time 10 min, working mode pulse open 2s-close 1s, and ice bath temperature control ≤30 DEG C.

[0013] Further, in step S3, the following steps are included: The nanocomposite emulsion is transferred to a freeze-drying tray for pre-freezing, the thickness of the freeze-drying tray is ≤1 cm, and the pre-freezing process is quick freezing at-40 DEG C for 2 hours to prevent ice crystals from growing and damaging the structure; The pre-freezing product is subjected to freeze-drying, and is placed in an environment with a vacuum degree of ≤10 Pa and a temperature of-40 DEG C for 24 hours for primary drying; Then, the product is placed in an environment with a temperature of 25 DEG C for 2 hours for analytical drying, and combined water is completely removed to obtain the final product, i.e., polyaspartic acid-chitooligosaccharide nanocoated arginine nanoscale slow-release particles.

[0014] Further, the feed is ginseng fruit and pearlleaf smartweed.

[0015] Further, the feed pearlleaf smartweed is cut into small pieces with a volume of 1-2 cm. 3 .

[0016] Compared with the prior art, the present application has the following beneficial effects: 1. By soaking the feed in arginine and then feeding the bat moth larvae, the present application improves the propagation of Cordyceps sinensis and speeds up the pupation of the larvae; 2. By loading arginine into the arginine nanoscale slow-release particles formed in the polyaspartic acid-chitooligosaccharide gel network, the present application enables the slow release of arginine in the intestinal tract of the bat moth larvae, prolongs the residence time of arginine in the intestinal tract, and improves the utilization rate of arginine; 3. By feeding the bat moth larvae with the arginine nanoscale slow-release particles and the feed soaked in arginine, the present application forms a double-nutrition supply system, avoids the problem of high arginine concentration in the early stage and low arginine concentration in the later stage, and further speeds up the pupation of Cordyceps sinensis by realizing double-nutrition supply; 4. By using the swelling of polyaspartic acid in the alkaline environment of the intestinal tract of the bat moth larvae and the specific decomposition of the beta-1,4 glycosidic bond of chitooligosaccharide by chitinase, the present application releases arginine and forms an enzyme-controlled targeted delivery mechanism. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which are included to provide a further understanding of the embodiments of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings: Figure 1 The figures are tissue section images of the larvae infected with Cordyceps sinensis in Example 2 of the present application. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description of the present application is made below in combination with examples and drawings, the illustrative embodiments of the present application and the description thereof are only used to explain the present application and do not limit the present application.

[0019] Example 1 A method for promoting the mycosis of O. sinensis by arginine is provided, comprising the following steps: The feed is pretreated with arginine, and the mycosis larvae are fed with the pretreated feed. The mycosis larvae are bred in a light-proof environment with a temperature of 15-18℃ and a humidity of 70-80% until the mycosis larvae are immobilized.

[0020] The pretreatment process comprises the following steps: preparing an arginine aqueous solution with a mass fraction of 1%; soaking the feed in the arginine solution for 30 minutes; and draining the soaked feed to obtain the pretreated feed.

[0021] The feed is ginseng fruit and Rubia cordifolia; the Rubia cordifolia is cut into small pieces with a volume of 1-2 cm before soaking 3 .

[0022] Compared with the normal cultured mycosis larvae, the concentration of arginine in the intestinal tract and hemolymph of the mycosis larvae fed with the feed treated with arginine is increased, which provides direct nutrition for O. sinensis and accelerates the proliferation of O. sinensis in the mycosis larvae, so that the number of immobilized mycosis larvae is significantly increased, and the immobilization of the mycosis larvae is accelerated.

[0023] The above method indirectly soaks the feed with arginine, avoids the risk of stress caused by directly treating the larvae, and is simple in operation and low in cost. The arginine solution can be recycled, and resources are not wasted.

[0024] Example 2 In existing research, it is recorded that the pH range of the intestinal fluid of the moth larvae is 9.69-10.99, and it is confirmed that O. sinensis invades the moth larvae through the mouthparts and digestive tract of the moth larvae, and arginine is the core precursor for synthesizing nucleic acids (such as adenine and guanine) and proteins. After the moth larvae are infected with O. sinensis, the mycelium growth and fruiting body formation of O. sinensis need a large amount of nitrogen source support.

[0025] In the feeding method in Example 1, arginine enters the intestinal tract of the moth larvae, most of the arginine is absorbed and utilized by the moth larvae, enters the hemolymph, and only a small part of the arginine can be absorbed by O. sinensis. The moth larvae intestinal tract and O. sinensis directly compete for the absorption of arginine, which leads to more arginine that cannot be directly absorbed by O. sinensis, which is not conducive to accelerating the immobilization and providing nutrition for O. sinensis.

[0026] To further improve the retention time of arginine in the intestine of the bat moth larvae and improve the utilization rate of arginine, and improve the proliferation of O. sinensis, and speed up the sclerotization of the infected larvae, another method for promoting the sclerotization of the infected larvae of O. sinensis by arginine is provided in the embodiment, which comprises the following steps: S1, preparing a poly-aspartic acid-chitosan oligosaccharide composite gel; S2, adding arginine to the poly-aspartic acid-chitosan oligosaccharide composite gel and emulsifying with a probe to obtain a nano composite emulsion; S3, freeze-drying the nano composite emulsion to obtain nano-sustained-release particles of arginine coated with poly-aspartic acid-chitosan oligosaccharide; S4, mixing the nano particles into the feed as described in Embodiment 1 to feed the infected larvae.

[0027] In the method of the embodiment, aspartic acid PASP is an inert skeleton and is not recognized by intestinal enzymes. In an alkaline environment (pH 9.69-10.99), the carboxyl group ionizes to form carboxylate ions, and the electrostatic repulsion makes the gel network swell but not disintegrate, and only relies on the slow dissolution in the alkaline environment to form a physical barrier to slow down the release of arginine and prolong the retention time of arginine in the intestine. The β-1,4 glycosidic bond of chitosan oligosaccharide COS needs to be hydrolyzed by specific enzymes (such as chitinase), and the intestine of the bat moth larvae lacks efficient COS-degrading enzyme systems (such as lysozyme), so the degradation rate is slow. The chitinase secreted by O. sinensis can slowly degrade COS to release arginine, which is directly absorbed and utilized by O. sinensis, forming an enzyme-controlled targeted delivery mechanism. Therefore, by feeding the feed soaked with arginine and preparing arginine nano-sustained-release particles coated with poly-aspartic acid (PASP)-chitosan oligosaccharide (COS) composite gel, the sclerotization speed can be further accelerated, and double nutritional supply can be realized for O. sinensis.

[0028] In step S1, the poly-aspartic acid-chitosan oligosaccharide composite gel is prepared, comprising the following steps: Place a 5% w / v poly-aspartic acid solution on a magnetic stirrer and stir at 500 rpm; According to the volume ratio of chitosan oligosaccharide solution: poly-aspartic acid solution of 1:3, slowly add 2% w / v chitosan oligosaccharide solution to the poly-aspartic acid solution to obtain a poly-aspartic acid-chitosan oligosaccharide composite gel solution; The above process is controlled at PH=5.0 and temperature of 23-27 degrees, and the drop rate is ≤1 mL / min.

[0029] In step S2, the following steps are included: Add arginine solution to the poly-aspartic acid-chitosan oligosaccharide composite gel solution, and control the volume ratio of poly-aspartic acid-chitosan oligosaccharide composite gel: arginine to be 4:1; Then emulsification is performed by using a probe ultrasound, and the emulsification conditions are as follows: power 200 W, time 10 min, working mode pulse open 2 s-close 1 s, and ice bath temperature control ≤30℃.

[0030] In step S3, the following steps are included: The nano-composite emulsion is transferred to a freeze-drying tray for pre-freezing, the thickness of the freeze-drying tray is ≤1 cm, and the pre-freezing process is quick freezing at-40℃ for 2 h to prevent ice crystals from growing and damaging the structure. The pre-frozen product is subjected to freeze-drying, and is placed in an environment of a vacuum degree ≤10 Pa and-40℃ for 24 h for primary drying. Then, the product is placed in an environment of 25℃ for 2 h for analytical drying to completely remove the bound water, and the final product, the nano-sustained-release particle of polyaspartic acid-chitooligosaccharide nano-loaded arginine, is obtained.

[0031] The loading principle is as follows: in the preparation stage, pH=5.0, the ionization of carboxyl and amino groups is the most sufficient, and the electrostatic attraction is maximized. In the acidic environment of PASP, -COO- is negatively charged, and COS contains amino groups (-NH2, pKa≈6.5) which are protonated and positively charged (-NH3 + ) in the acidic environment. Electrostatic cross-linking occurs, -NH3 + of COS forms an ionic bond with -COO - of PASP to generate a PASP-COS gel network. The cross-linking time is 30 min to form a stable three-dimensional network structure. The guanidino group (+) of arginine electrostatically combines with the free carboxyl group (-) of PASP, and the PASP-COS gel network forms a microporous structure with a pore size of 50 nm. Arginine molecules (diameter≈0.7 nm) enter the pore channel by diffusion and are physically trapped inside the network. Ultrasonic emulsification uniformly disperses arginine into the gel core region.

[0032] After entering the intestinal alkaline environment (pH>9.5), the carboxyl group of PASP is ionized to -COO - , and the electrostatic repulsion causes the gel to swell, the pore size shrinks to <10 nm, and arginine is locked in a narrower space, so arginine cannot freely diffuse to the external environment.

[0033] The chitinase secreted by Cordyceps sinensis specifically hydrolyzes the β-1,4 glycosidic bond of COS, causing the gel network to disintegrate. The guanidino group of arginine is deprotonated (charge zero) at pH>10, the electrostatic anchoring is released, and arginine is released to the infection site, achieving enzyme-controlled targeting. At the same time, the encapsulated arginine also avoids being utilized by other symbiotic bacteria.

[0034] The particle size of the nano-sustained-release particle is 220-280 nm, and the PDI is ≤0.25.

[0035] The arginine nano-sustained-release particles with too large particle size are easily discharged by intestinal peristalsis, and the gel cross-linking density is too high when the particle size is too small, so chitinase cannot penetrate. The particle size between 220-280 nm is easy to adhere to the mucosa, which can prolong the retention time of arginine nano-sustained-release particles in the intestine and improve the utilization efficiency of arginine.

[0036] PASP is hydrolyzed to aspartic acid in the alkaline environment of the intestine; COS is hydrolyzed to N-acetylglucosamine by chitinase, which promotes mycelium branching, and the carrier degradation products are directly converted into mycelium growth promoters, without biological accumulation risk.

[0037] After arginine enters the intestine of the bat moth larva, the bat moth larva intestine absorbs arginine through amino acid transporters. The intestinal epithelial cells of the bat moth larva have the ability to efficiently absorb free amino acids. About 60%-70% of the arginine ingested by the bat moth larva is first absorbed by the midgut epithelial cells, and then transported to tissues such as fat bodies through hemolymph. Most of the arginine in the intestine of the bat moth larva is directly absorbed into the hemolymph, and the arginine that is not absorbed by the host (about 30%-40%) remains in the intestinal lumen, becoming a potential nutrient source for symbiotic or parasitic microorganisms. The arginine can be directly absorbed and utilized by Cordyceps sinensis. In addition, Cordyceps sinensis can absorb and utilize the arginine absorbed by the host through two mechanisms. One is hemolymph penetration: after the mycelium invades the host hemolymph, it directly absorbs the free arginine in the hemolymph. The other is metabolic hijacking: by inhibiting the host's arginine metabolic pathway (such as the urea cycle), the host's cells are forced to accumulate arginine and excrete it in reverse. Although Cordyceps sinensis can absorb arginine through reverse mechanisms, it also causes time waste, and most of the arginine cannot be directly utilized by Cordyceps sinensis in the first place.

[0038] In this embodiment, the problem of competition for nutrients between the host and the mycelium can be solved because: First, the bat moth larva intestine efficiently absorbs arginine (through amino acid transporters), resulting in most of the arginine being utilized by the host (60%-70% into the hemolymph) in the direct soaking method, and only a small amount being available for Cordyceps sinensis. The nano-particles ensure that arginine is preferentially supplied to Cordyceps sinensis through sustained and enzyme-controlled release.

[0039] Second, prolonging the retention time in the intestine and improving the sustained-release efficiency: the particle size design of the nano-particles (220-280 nm) optimizes the adhesion to the intestine. The particles swell in the alkaline environment (PASP ionization), the pore size shrinks to <10 nm, and the arginine is locked; chitinase triggers the release, which prolongs the action time to several hours compared to the direct soaking method, which solves the shortcoming of rapid metabolism of arginine in the direct soaking method.

[0040] Moreover, to achieve dual nutrition supply and optimize the rigid process: the direct soaking method provides "instant arginine", but the concentration decreases with feeding, and the nanoparticles act as a "supplementary library" to release arginine at a later stage. Experiments show that dual supply increases the rigid rate by 6.4% (96.5% vs. 90.1%), proving a synergistic effect.

[0041] Finally, the necessity of enzyme-controlled targeted delivery: the chitinase-rich site of O. sinensis infection (intestinal tract) can specifically degrade the chitosan component of the nanoparticles, which achieves "release where needed", while the direct soaking method cannot be targeted. The higher proportion of pseudohyphae (38.2% vs. 30.1%) in experimental analysis confirms this mechanism.

[0042] Experimental analysis and conclusion The present application first contacts and infects the small golden bat moth with O. sinensis, and selects healthy and aged infected bat moth larvae, and ensures that the initial body weight and the state of the fungus are consistent, a total of 200, randomly allocated to 4 groups, each group of 50, respectively, control group 1, control group 2, experimental group 1, experimental group 2, as follows: Control group 1: mannitol injection group, each larva in this group is injected with 20% mannitol 20 μL into the stoma; Control group 2: normal feeding, the feed is not treated, and is directly fed; Experimental group 1: arginine nanoparticle group, the feed is first soaked in 1% arginine solution, and then mixed with nanoparticle slow-release particles for feeding; Experimental group 2: arginine-soaked feed group, the feed is soaked in 1% arginine solution for 30 minutes, and then fed after draining.

[0043] The feed uses ginseng fruit and pearl barberry, with a mass ratio of 1:1, and the pearl barberry is cut into 1-2 cm 3 small pieces.

[0044] Each group of infected insects is placed in a constant temperature and humidity box, with a temperature of 13±1℃ and a humidity of 75±5%, and is cultured in the dark. The single bottle is fed; all groups are fed with equal amounts of feed, with a ginseng fruit: pearl barberry ratio of 5g:5g per insect.

[0045] The rigidity is monitored, and the activity state, food intake, and rigidity rate are recorded daily. Abnormal behaviors such as convulsions or death during injection or treatment are recorded. The observation period is 0-10 days, and the key time points are recorded at 1d, 3d, 5d, 7d, and 10d.

[0046] In this example, the longitudinal section of the rigid insect tissue sample of each group is as follows: Figure 1As shown in the figure, A is a tissue section diagram of the control group 1, C is a tissue section diagram of the control group 2, B is a tissue section diagram of the experimental group 1, and D is a tissue section diagram of the experimental group 2, and it can be known from the figure that, compared with the control group 1, the number of O. anthropophaga in the experimental group 1 and the experimental group 2 is obviously increased; compared with the control group 2, the number of O. anthropophaga in the experimental group 1 and the experimental group 2 is obviously increased; compared with the experimental group 2, the number of O. anthropophaga in the experimental group 1 is more.

[0047] Table 1 Rigidity rate comparison (%)

[0048] In Table 1, in terms of rigidity effect and speed, the experimental group 1 (arginine + nanoparticles) is optimal: the rigidity rate is 92.1% (10d), the peak time is 7d, which is 6d faster than that of the control group 1 mannitol, which benefits from double nutrition supply (soaking feed provides instant arginine, and nanoparticles slow down and prolong retention), arginine as a nucleic acid precursor directly promotes the proliferation of O. anthropophaga; the experimental group 2 (only arginine) is suboptimal: the rigidity rate is 84.2%, but slower than the experimental group 1, verifying the value-added effect of nanoparticles (enzyme-controlled delivery mechanism improves utilization rate); the control group 1 (mannitol) is effective but limited: the rigidity rate is 66.3%, although mannitol accelerates rigidity by about 5 days, but the speed and quality are lower than those of the experimental group 1; the control group 2 (normal) is the worst: the rigidity rate is 46.2%, and the peak is 20d.

[0049] Table 2 Rigidity insect quality index (7d data)

[0050] In Table 2, hardness and fungal development: the hardness (68.5HC) and the proportion of pseudohyphae (38.2%) of the experimental group 1 are the highest, as shown in the figure, showing that the mycelium is dense, proving that the enzyme-controlled targeted delivery of nanoparticles (alkaline environment PASP swelling, chitinase releasing arginine) effectively improves the development of mycelium; the control group 1 has a stress rate of 15%, which is caused by convulsions caused by injection, and the hardness (56.8HC) is low, and the hardness increases slowly after injection. Figure 1 Pseudohyphae are the transitional form in the process of O. anthropophaga infecting Thitarussa larva, and are also the key marker of the rigidity process, and the proportion of pseudohyphae directly reflects the infection activity of O. anthropophaga and the rigidity process, and the formation process is: blastospore→ swollen blastospore→ pseudohyphae→ mature mycelium. Pseudohyphae is a necessary stage for O. anthropophaga to transform from spore to invasive mycelium, and the increase of the proportion of pseudohyphae indicates that the fungus body is converted from the dormant state to the active proliferation state, accelerating the death of the host and the rigidity of the pseudohyphae.

[0051] Based on the experimental analysis, the method for promoting the rigidity of O. anthropophaga by arginine in the application is superior to the injection method of mannitol in rigidity effect, speed and quality, and the main technical effects are as follows: High efficiency and low rigidity: Compared with mannitol injection, the rigidity rate increased by 25.8%, and the peak time was shortened to 7 days.

[0052] High-quality caterpillar: hardness increased by 20.6% (68.5 HC vs 56.8 HC), mycelial development rate increased by 15.7% (38.2% vs 22.5%), high Ophiocordyceps sinensis density, ensuring the quality of subsequent Cordyceps, such as Figure 1 The mycelial density shown in the figure is conducive to the formation of fruiting bodies.

[0053] Low-risk operation: Compared with mannitol 15% convulsion rate, there is no stress reaction during the treatment process, and the soaking feeding is simpler than the injection operation, which is suitable for large-scale cultivation.

[0054] Innovative mechanism: dual nutrition supply (soaking + nano slow release) and enzyme-controlled targeted delivery (PASP-chitosan oligosaccharide gel network) significantly improve the utilization rate of arginine, and its degradation products have no biological accumulation risk.

[0055] Economy: The cost is lower than that of mannitol injection, and the arginine solution can be recycled.

[0056] Recommended application: arginine nano slow-release granules method is preferred in artificial cultivation of Cordyceps sinensis, especially suitable for batch production; only arginine soaking feed can be used as a low-cost alternative.

[0057] The above specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. Application of arginine in promoting the rigor mortis of Cordyceps sinensis fungal insects.

2. A method for promoting the mutagenesis of Cordyceps sinensis fungal insects using arginine, characterized in that, Includes the following steps: Arginine was used to pretreat the feed, and the pretreated feed was used to feed the infected larvae. They are raised in a dark environment with a temperature of 15–18℃ and a humidity of 70–80% until the infected larvae become mummified.

3. The method for promoting the mutagenesis of Cordyceps sinensis fungal insects using arginine according to claim 2, characterized in that, The preprocessing process includes the following steps: Prepare a 1% (w / w) arginine aqueous solution; Soak the feed in the arginine solution for 30 minutes; After soaking, the feed is drained to obtain pre-treated feed.

4. A method for promoting the mutagenesis of Cordyceps sinensis fungal insects using arginine, characterized in that, Includes the following steps: S1. Preparation of polyaspartic acid-chitooligosaccharide composite gel; S2. Arginine is added to the polyaspartic acid-chitooligosaccharide composite gel and ultrasonically emulsified with a probe to obtain a nanocomposite emulsion. S3. The nanocomposite emulsion is freeze-dried to obtain polyaspartic acid-chitosan oligosaccharide nano-coated arginine sustained-release particles. S4. The nanoparticles are mixed into the feed as described in claim 2 and fed to the susceptible larvae.

5. The method for promoting the mutagenesis of Cordyceps sinensis fungal insects using arginine according to claim 4, characterized in that, The preparation of the polyaspartic acid-chitosan oligosaccharide composite gel in step S1 includes the following steps: Place a 5% w / v polyaspartic acid solution on a magnetic stirrer and stir at 500 rpm. According to the volume ratio of chitosan oligosaccharide solution to polyaspartic acid solution of 1:3, 2% w / v chitosan oligosaccharide solution was slowly added dropwise to the polyaspartic acid solution to obtain polyaspartic acid-chitosan oligosaccharide composite gel solution. During the above process, the pH should be controlled at 5.0, the temperature at 23-27 degrees Celsius, and the dropping rate at ≤1 mL / min.

6. The method for promoting the mutagenesis of Cordyceps sinensis fungal insects using arginine according to claim 4, characterized in that, Step S2 includes the following steps: Arginine solution was added to the polyaspartic acid-chitooligosaccharide composite gel solution to control the volume ratio of polyaspartic acid-chitooligosaccharide composite gel to arginine to be 4:

1. Then, emulsification was performed using ultrasound with a probe. The emulsification conditions were: power 200W, time 10min, working mode: pulse on 2s-off 1s, and ice bath temperature control ≤30℃.

7. The method for promoting the mutagenesis of Cordyceps sinensis fungal insects using arginine according to claim 4, characterized in that, Step S3 includes the following steps: The nanocomposite emulsion was transferred to a freeze-drying tray for pre-freezing. The thickness of the freeze-drying tray was ≤1cm. The pre-freezing process was to place it at -40℃ for 2 hours to prevent ice crystals from growing and damaging the structure. The pre-frozen product was freeze-dried and placed in an environment with a vacuum degree ≤10Pa and a temperature of -40℃ for 24 hours for primary drying. Then, the mixture was placed at 25°C for 2 hours for desorption and drying to completely remove the bound water, yielding the final product, polyaspartic acid-chitosan oligosaccharide nano-coated arginine sustained-release particles.

8. A method for promoting the mutagenesis of Cordyceps sinensis fungal insects using arginine according to any one of claims 7, characterized in that, The nano-sized sustained-release particles have a particle size of 220-280 nm and a PDI ≤ 0.

25.

9. The product prepared by the method of promoting the mutagenesis of Cordyceps sinensis fungal insects by arginine as described in claim 2 or 3.

10. The product prepared by any one of claims 4-7 using arginine to promote the mutagenesis of Cordyceps sinensis fungal insects.

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