Method for improving content of adenosine in cordyceps sinensis based on metabolic regulation and synergistic induction

Abstract

The application provides a method for improving the adenosine content of Ophiocordyceps sinensis based on metabolic regulation and synergistic induction, wherein a metabolic intervention is performed on the larva and strain complex using a regulating agent during the infection period before low-temperature treatment to form the infected larva; first, deep low-temperature immobilization treatment is performed at 2-5 DEG C for 30-50 days, then culture is performed using a variable-temperature cycle with diurnal temperature difference during the stroma induction period, and mannitol is sprayed onto the surface of the culture medium at the beginning of the step; after harvesting, rapid temperature rising enzyme inactivation and drying treatment is performed on the harvested Ophiocordyceps sinensis, the rapid temperature rising enzyme inactivation and drying treatment comprises increasing the drying temperature from 40-45 DEG C to 60-70 DEG C within 30-45 minutes and maintaining the temperature until the moisture content is less than 10%. The application can solve the problem that the adenosine content is generally low in the existing full artificial cultivation technology, and can make the adenosine content of the full artificial cultivation Ophiocordyceps sinensis reach more than 0.020% (w / v), which is significantly better than the existing artificial cultivation level, and the product morphology is complete and the reproducibility is high.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular, it is a systematic method for significantly increasing the content of adenosine, the core active ingredient in the final cordyceps product, by intervening in and optimizing its internal biochemical metabolic pathways and environmental inducing factors during the artificial cultivation process that fully simulates the natural life cycle of cordyceps (i.e., feeding host insect larvae, inoculating with cordyceps fungus, and inducing the formation of stunted insects and stroma at low temperature). Background Technology

[0002] Cordyceps sinensis is a complex formed by the parasitic fungus *Ophiocordyceps sinensis* on the larvae of Hepialidae moths. Due to the near depletion of wild resources, developing fully artificial cultivation techniques (from larval rearing to obtaining complete Cordyceps with stroma) is the fundamental solution. Currently, this technology can achieve Cordyceps formation, but a common and prominent drawback is that the adenosine content of the obtained artificial Cordyceps sinensis is significantly lower than that of high-quality wild products, even below the pharmacopoeia standard, and inconsistent between batches, severely restricting its medicinal value and market competitiveness.

[0003] The main technological bottlenecks are as follows: 1. The "emphasis on morphology, neglect of metabolism" breeding philosophy: Current processes mainly focus on completing the morphological development process of "larva → molting → stroma," but lack active intervention in the dynamic metabolism of adenosine synthesis, accumulation, and degradation in this complex symbiotic system. Ignoring this intrinsic regulation leads to the failure to stimulate adenosine synthesis potential or its ineffective degradation during stroma development.

[0004] 2. Coarse environmental induction parameters: Low temperature treatment is the key to inducing rigor mortis and stroma germination, but the selection of temperature and duration in existing technologies is often based on experience and fails to accurately match the specific physiological state of the larva (rigor mortis) and the critical window period of adenosine metabolism.

[0005] 3. Post-processing causes component loss: Drying is the final step in cultivation and also the step where adenosine is most easily lost. Traditional low-temperature slow drying process keeps the insects in the activity temperature range of adenosine-degrading enzymes (such as adenosine deaminase) for a long time (such as around 40°C), resulting in a large loss of adenosine accumulated in the early stage after harvesting.

[0006] Therefore, there is an urgent need for an artificial breeding method that can systematically solve the problem of low adenosine content from three levels: metabolic origin, development induction, and finished product locking. Summary of the Invention

[0007] This invention provides a method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction. Addressing the problem of generally low adenosine content in existing fully artificially cultivated Cordyceps sinensis, this invention intervenes through three core technologies: First, before the formation of the stroma, exogenous precursor substances or metabolic regulators are added to target and regulate the metabolic pathways of CD73 hydrolase, nucleoside diphosphate kinase, and adenosine kinase related to adenosine synthesis and degradation. Second, the synergistic process of low-temperature treatment and stroma induction is optimized. Precise low-temperature treatment (e.g., 2-5℃) is applied at specific developmental stages to promote material transformation in the pre-stroma and stroma stages, and conditions such as light and temperature difference are matched to maximize adenosine accumulation during stroma development. Finally, a rapid heating and enzyme-inactivating drying method is used after harvesting to lock in adenosine content. Using this invention, the adenosine content of fully artificially cultivated Cordyceps sinensis (intact insect-fungus complex) can be stably maintained at over 0.020% (w / v), significantly superior to existing artificial cultivation levels, with intact product morphology and high reproducibility.

[0008] According to a first aspect of the present invention, one or more embodiments of this application provide a method for increasing the adenosine content of Cordyceps sinensis based on metabolic regulation and synergistic induction. The method includes feeding host larvae, inoculating with Cordyceps sinensis fungus, low-temperature treatment to form stunted larvae and low-temperature induced culture of stromata, and drying after harvesting. The steps from low-temperature treatment to low-temperature induced culture of stromata to drying after harvesting include: a. During the infection period before the formation of stunted worms under low temperature treatment, metabolic intervention was performed on the larval and strain complex using regulators that target the adenosine synthesis pathway or inhibit adenosine degradation enzymes. b. The steps of forming stunted insects and inducing stroma formation by low temperature treatment include: firstly, deep low temperature stunting treatment is carried out at 2-5℃ for 30-50 days, and then a variable temperature cycle with day and night temperature difference is used for cultivation during the stroma induction period, and mannitol is sprayed on the surface of the cultivation substrate at the beginning of this step. c. During the drying process after harvesting, the harvested Cordyceps sinensis is subjected to rapid temperature-inactivation drying treatment. This rapid temperature-inactivation drying treatment involves raising the drying temperature from 40-45℃ to 60-70℃ within 30-45 minutes and maintaining it until the moisture content is below 10%.

[0009] According to the above-described technical solution of the present invention, the following improvements can also be made: Preferably, the regulators used in step a to metabolically intervene in the larval-strain complex include adenosine precursors.

[0010] Preferably, the regulator used in step a to intervene in the metabolism of the larval-strain complex is hypoxanthine.

[0011] Preferably, the regulator used in step a to intervene in the metabolism of the larval-strain complex includes an inhibitor of adenosine-degrading enzyme activity, wherein the inhibitor of adenosine-degrading enzyme activity is emodin methyl ether.

[0012] Preferably, the regulator is applied in solution form via microinjection or feeding, and the concentration of the regulator is 0.01%-0.05% (w / v).

[0013] Preferably, the concentration of mannitol sprayed onto the surface of the culture substrate during the initial 1-3 days of stroma induction is 0.05-0.1% (w / v).

[0014] Preferably, the induction period of the stroma in step b is carried out by a variable temperature cycle with a diurnal temperature difference, including: daytime temperature of 12-15℃ and light for 6-8 hours, nighttime temperature of 5-8℃, and a cycle of 25-40 days.

[0015] Preferably, the rapid temperature-inactivation and drying process described in step c includes: first drying the harvested material at 40-45°C for 1-2 hours, and then raising the temperature to 60-70°C within 30-45 minutes and maintaining it for 1-1.5 hours.

[0016] Preferably, the rapid temperature-inactivation and drying process described in step c includes: raising the temperature to 60-70°C within 30-45 minutes and maintaining it for 1-1.5 hours, then drying at 40-45°C until the moisture content is less than 10%.

[0017] The beneficial effects of this invention are as follows: This invention provides a method for increasing the adenosine content of Cordyceps sinensis based on metabolic regulation and synergistic induction. Compared with existing fully artificial cultivation techniques, this invention has the following significant advantages: 1. A qualitative leap in adenosine content: Through the triple guarantee of "metabolic initiation + synergistic induction + enzyme inactivation and locking", the adenosine content of the final artificial cordyceps product is steadily increased from the usual less than 0.010% to more than 0.02%, reaching or even exceeding the level of ordinary wild cordyceps.

[0018] 2. Deep level of technological innovation: The key regulatory targets of adenosine metabolism discovered by transcriptomics are applied to the practice of artificial culture, intervening in the accumulation of components at the molecular level; at the same time, the low-temperature treatment is refined into a staged precision process to serve different physiological and metabolic goals.

[0019] 3. Strong controllability of the cultivation process: Each efficiency-enhancing module has clear timing, material, and parameter indicators, breaking the "black box" of traditional processes and greatly improving the controllability of the production process and the stability of product quality.

[0020] 4. Integration and universality: The steps provided by this invention can be flexibly embedded into the existing and relatively mature "larva-mute worm-stool" breeding framework without disrupting the original production line, making it easy to promote and apply. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the seven-septate bacteria inside the insect according to an embodiment of the present invention; Figure 2 This is a flowchart illustrating an embodiment of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings.

[0023] It should be noted that, unless otherwise defined, the technical or scientific terms used in one or more embodiments of this application should have the ordinary meaning understood by one of ordinary skill in the art to which this disclosure pertains. The terms "first," "second," and similar terms used in one or more embodiments of this application do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as "upper," "lower," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0024] like Figures 1-2 As shown, one or more embodiments of this application describe a method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction. This method incorporates the following three core enhancement modules into the standard process of larval rearing, inoculation with Cordyceps sinensis fungus, and low-temperature induction to form stunted worms and stroma: Module 1: Targeted Regulation of Metabolic Pathways Before the Formation of Stiff Worms Metabolic intervention was performed on the larval-fungus complex after the larvae were inoculated with Cordyceps sinensis and before they underwent deep low-temperature rigor mortis treatment.

[0025] Timing of intervention: After inoculation, wait until the larvae develop to the 6th-7th instar, when the bacteria in the larvae begin to show seven-septate bacteria (such as...). Figure 1 As shown in the image, when larvae exhibit symptoms such as sluggish activity and cessation of feeding.

[0026] Intervention method: Supplement the insect body with adenosine metabolism regulators through micro-injection or feeding.

[0027] The following regulators are selected: Predrug supplementation: Supplement with adenosine precursors such as hypoxanthine at a concentration of 0.01%-0.05% (w / v). Metabolic regulation: Add regulatory substances related to the adenosine metabolic pathway identified through transcriptomics analysis, aiming to relatively inhibit degradation pathways (e.g., affecting CD73 hydrolase activity) or promote synthetic pathways (e.g., enhancing nucleoside diphosphate kinase efficiency).

[0028] Function: This intervention aims to “initiate” a shift in the worm-fungus complex toward a metabolic pattern of high adenosine accumulation, laying the material and metabolic foundation for subsequent development.

[0029] Module 2: Synergistic Process Optimization of Low-Temperature Hardening and Stool Induction The single cryogenic treatment is broken down into a synergistic induction process that is precisely matched with developmental stages and metabolic targets.

[0030] Phase 1: Deep low-temperature rigorization and material transformation period.

[0031] Timing and target: For infected larvae treated with Module 1, the procedure should be carried out when they have completely ceased activity and their body color has begun to change.

[0032] Parameters: Place the larvae in a dark environment at 2-5℃ and 75-80% humidity for 30-50 days. This low temperature facilitates a slow maturation process, promoting the conversion of macromolecules such as proteins and nucleic acids in the larvae into small molecules that can be utilized by the mycelium, while avoiding abrupt metabolic cessation. The larvae gradually maturate to form a mushy larvae body.

[0033] The second stage: stroma germination and adenosine enrichment period.

[0034] Timing: Once the mycelial membrane on the surface of the worm has formed, the growth phase begins with the induction of the stroma.

[0035] Temperature strategy: A "low-temperature induction-diurnal temperature variation" cycle was adopted for 25-40 days. During the day, the temperature was raised to 12-15℃ and weak diffused light was provided (6-8 hours), while the nighttime temperature was lowered to 5-8℃. The large diurnal temperature variation is a key abiotic factor in simulating the high-altitude environment, stimulating the formation of fruiting primordia, and promoting the accumulation of secondary metabolites such as adenosine.

[0036] Assisted induction: In the early stage of this phase, specifically within 1-3 days, a low concentration (0.05%-0.1% (w / v)) of mannitol or a specific polysaccharide solution can be sprayed onto the surface of the culture substrate to act as a signaling molecule to further promote stroma development and metabolic flow to adenosine synthesis.

[0037] Module 3: Post-harvest rapid heating and enzyme inactivation drying process To fix the adenosine accumulated throughout the cultivation cycle, a drying technique that differs from traditional methods is employed.

[0038] The specific process flow includes: 1. Pre-drying stage: Place the harvested fresh grass in an environment of 40-45℃ and dry it quickly for 1-2 hours to remove most of the free water.

[0039] 2. Enzyme Inactivation and Lock-in Stage: Within 30-45 minutes, the drying temperature is rapidly increased to 60-70℃ and maintained for 1-1.5 hours. This rapid heating process effectively inactivates enzyme systems (such as adenosine deaminase) within the cordyceps (especially at the base of the stroma) that may lead to adenosine degradation, thereby "locking" the synthesized adenosine in the product.

[0040] 3. Slow drying stage: Adjust the temperature back to 40-45℃ and continue drying until the moisture content is below 10%.

[0041] Function: The core of this process lies in "rapid temperature rise to inactivate enzymes," which is a crucial step to prevent all previous efforts from being wasted and to ensure that the high adenosine content is reflected in the final product.

[0042] The technical solution of the present invention will be illustrated below through specific embodiments and comparative examples: Example 1 (Applying the complete solution of this invention) 1. Preliminary Procedure: Healthy 5th instar larvae of the ghost moth are reared, and after their bodies are disinfected, they are inoculated with a suspension of *Hymenochrysis sinensis* spores. They are then reared in the dark at 12°C.

[0043] 2. Module 1 (Metabolic Regulation): After inoculation, when the larvae develop to the 6th-7th instar, 7-septate bacteria begin to appear in the larvae's body (e.g., Figure 1 (As shown). Inject 5 μL of 0.03% hypoxanthine solution into the hemocoel of each larva using a microsyringe.

[0044] 3. Module Two (Collaborative Induction): The injected larvae were transferred to a dark environment at 2°C and 78% humidity for 40 days (first stage), during which the larvae gradually stiffened and formed muscular bodies. They were then transferred to a stroma culture room with a daytime temperature of 14°C (8 hours of light), a nighttime temperature of 7°C (darkness), and a humidity of 80%. After approximately 40 days of culture, the stroma reached a length of 2-3 cm.

[0045] 4. Module 3 (Enzyme Inactivation and Drying): After harvesting, dry at 42℃ for 1.5 hours, then raise the temperature to 65℃ within 40 minutes and maintain it for 70 minutes, and finally dry at 45℃ until the moisture content is below 10%.

[0046] 5. Test results: Adenosine content was measured by random sampling, with an average value of 0.0223%.

[0047] Comparative Example 1 (Traditional low-temperature treatment, without metabolic regulation and enzyme inactivation drying) 1. Preliminary procedure (same as in Example 1): Healthy 5th instar larvae of the ghost moth were reared, and after their bodies were disinfected, they were inoculated with a suspension of *Hymenochrysis chinensis* spores. They were then reared at 12°C in the dark.

[0048] 2. After inoculation, place directly in a constant 10°C environment for cultivation until the stroma grows (approximately 90 days).

[0049] 3. After harvesting, dry at a constant temperature of 40℃ for 24 hours.

[0050] 4. Test results: Adenosine content was 0.0087%.

[0051] Comparative Example 2 (precursor only, without co-induction and enzyme inactivation drying) The preliminary process and modules are the same as in Example 1: Preliminary procedures: Healthy 5th instar larvae of the ghost moth are reared, and after their bodies are disinfected, they are inoculated with a suspension of *Hymenochrysis chinensis* spores. They are then reared at 12°C in the dark.

[0052] Metabolic regulation: After inoculation, when the larvae develop to the 6th-7th instar, 7-septate bacteria begin to appear in the larvae's body (e.g., Figure 1 (As shown). Inject 5 μL of 0.03% hypoxanthine solution into the hemocoel of each larva using a microsyringe.

[0053] They were then cultured at a constant temperature of 12°C under continuous low light until the stroma grew (approximately 70 days).

[0054] After harvesting, dry at a constant temperature of 40℃ for 24 hours.

[0055] Test results: Adenosine content was 0.0114%. This indicates that adding the precursor alone has a preliminary effect, but the lack of subsequent synergistic induction and enzyme inactivation limits the synergistic effect.

[0056] Comparative Example 3 (with synergistic induction, but improper drying process) The preliminary process and modules one and two are the same as in Example 1; Preliminary procedures: Healthy 5th instar larvae of the ghost moth are reared, and after their bodies are disinfected, they are inoculated with a suspension of *Hymenochrysis chinensis* spores. They are then reared at 12°C in the dark.

[0057] Metabolic regulation: After inoculation, when the larvae develop to the 6th-7th instar, 7-septate bacteria begin to appear in the larvae's body (e.g., Figure 1 (As shown). Inject 5 μL of 0.03% hypoxanthine solution into the hemocoel of each larva using a microsyringe.

[0058] Synergistic induction: After injection, the larvae were transferred to a dark environment at 2°C and 78% humidity for 40 days (first stage). During this stage, the larvae gradually stiffened and formed muscular bodies. Subsequently, they were transferred to a stroma culture room with a daytime temperature of 14°C (8 hours of light) and a nighttime temperature of 7°C (darkness) and humidity of 80%. After about 40 days of culture, the stroma length reached 2-3 cm.

[0059] After harvesting, the product is dried using a slow, stepwise temperature increase method (40℃ for 3 hours → 50℃ for 3 hours → 60℃ for 2 hours).

[0060] Test results: Adenosine content was 0.0145%. Although better than Comparative Examples 1 and 2, the adenosine content was partially degraded due to prolonged exposure to the enzyme activity temperature zone during the drying process, thus failing to achieve the optimal effect of Example 1.

[0061] Table 1. Processing technology and adenosine content The data in Table 1 above fully demonstrates that the three synergistic modules proposed in this invention have a significant synergistic effect, and none of them can be omitted. Module 1 enhances metabolic potential from the source, module 2 creates an optimal enrichment environment during development, and module 3 ensures that components are not lost at the endpoint. Only by combining the three can the adenosine content be maximized.

[0062] In addition, the following experimental scheme is designed to illustrate the drying process: Experimental materials and pretreatment Experimental materials: Fresh Cordyceps sinensis (stripe length 2-3cm, intact worm body, inoculated strain is Trichoderma sinensis, larvae are 6-7 instar infected organisms of Hepialus chinensis) were selected after treatment with "Module 1 (metabolic regulation) + Module 2 (synergistic induction)" of this invention. They were randomly divided into 4 groups, with 30 plants in each group and a fresh weight of (5.0±0.2)g. It was ensured that there was no significant difference in the initial adenosine content among the groups (preliminary detection, the average initial adenosine content was 0.0231%).

[0063] Uniform initial treatment: After harvesting, damaged individuals were removed from all experimental groups, surface impurities were quickly rinsed with sterile water, surface moisture was drained (time ≤ 5 minutes), and then immediately processed into different drying processes.

[0064] Experimental grouping and drying process parameters Experimental Group 1: The rapid temperature rise enzyme inactivation and drying process of this invention; Pre-drying stage: Place the fresh product in a drying oven at 42℃ and 50% humidity for 1.5 hours to remove free water.

[0065] Enzyme inactivation lock-in stage: Within 40 minutes, the temperature of the drying oven is rapidly increased from 42℃ to 65℃ and maintained for 70 minutes to inactivate adenosine deaminase.

[0066] Slow drying stage: Reduce the temperature to 45°C and continue drying until the moisture content is below 10% (approximately 2 hours).

[0067] Key controls: The heating rate is kept stable at 0.575℃ / minute (65℃-42℃=23℃, taking 40 minutes), and the temperature of the insect's core is monitored in real time throughout the process to ensure consistency with the temperature of the drying chamber.

[0068] Control group 1: Traditional low-temperature slow drying process Referring to existing drying methods that emphasize morphology and downplay metabolism, the parameters are as follows: Drying temperature: 40℃ throughout the process, humidity 50%.

[0069] Drying time: Continue drying for 24 hours until the moisture content is below 10%.

[0070] There is no rapid heating step; the temperature remains constant throughout the process.

[0071] Control group 2: Step-by-step temperature drying process The simulation part uses a "gradient temperature increase" scheme in existing technologies but without enzyme inactivation targeting design, with the following parameters: First stage: Dry at 40℃ for 3 hours.

[0072] Second stage: Dry at 50℃ for 3 hours.

[0073] Third stage: Dry at 60℃ for 2 hours.

[0074] Endpoint: Moisture content below 10%, overall heating rate ≤ 0.055℃ / min, no concentrated enzyme inactivation stage.

[0075] Control group 3: Direct high-temperature drying process Drying method: After harvesting, place directly in a drying oven at 65℃ and 50% humidity for 4 hours until the moisture content is below 10%.

[0076] There is no pre-drying or slow drying stage; it is directly treated at high temperature.

[0077] Adenosine content determination: High performance liquid chromatography (HPLC) was used, referring to the adenosine content determination method. The chromatographic column was a C18 column (4.6 mm × 250 mm, 5 μm), the mobile phase was methanol-water (15:85), the detection wavelength was 260 nm, the flow rate was 1.0 mL / min, and the column temperature was 30 ℃. The mean adenosine content of each group and the retention rate relative to the initial content were calculated.

[0078] Adenosine deaminase activity assay: The residual activity of adenosine deaminase in the dried insect tissue was detected by colorimetric method, expressed in "U / mg protein". The lower the activity, the better the enzyme inactivation effect.

[0079] Product form integrity rating: A 10-point rating standard is adopted, which is scored from four dimensions: fullness of the worm body (3 points), no breakage of the stroma (3 points), no shrinkage of the worm body (2 points), and color uniformity (2 points). A total score of ≥8 points is excellent, 6-7 points is good, and ≤5 points is unqualified.

[0080] The experimental results and data records are shown in Table 2: Table 2 Experimental Results and Data Records As shown in Table 2, the technical solution of this embodiment significantly improves the adenosine retention rate: the adenosine retention rate of experimental group 1 reached 98.27%, which is much higher than that of control group 1 (62.77%) and control group 2 (70.56%). This proves that the drying process of the present invention can effectively avoid the degradation of adenosine during the drying process and solves the core pain point of "early accumulation and later loss" in the prior art.

[0081] Although the control group 3 had a higher adenosine retention rate (81.82%) than the traditional process due to the direct high-temperature inactivation of some enzymes, it was still lower than the experimental group 1, and the morphological integrity score was only 5.3 points (unqualified). This shows that high temperature alone cannot achieve both "adenosine retention" and "morphological integrity". The "three-stage temperature gradient" design of this invention achieves the dual goals.

[0082] The highly efficient inactivation of degrading enzymes demonstrates the "highlighting of substantial features": the residual activity of adenosine deaminase in experimental group 1 was only 0.03 U / mg protein, significantly lower than that in control group 1 (0.89 U / mg protein) and control group 2 (0.45 U / mg protein). This proves that the design of "rapidly heating to 60-70℃ and maintaining it for 1-1.5 hours" can accurately target and inactivate degrading enzymes, while the constant temperature or slow heating of existing technologies cannot achieve efficient enzyme inactivation.

[0083] This design is not a conventional choice for those skilled in the art: the core objective of existing drying processes is "removing moisture and maintaining form", without recognizing the key issue that "adenosine deaminase has the highest activity at around 40°C". This invention is the first to take "enzyme inactivation" as the core objective of the drying process, and achieves the synergistic effect of enzyme activity inhibition and form protection through precise temperature gradient design.

[0084] The morphological integrity score of experimental group 1 was 8.6 (excellent), which was comparable to that of control group 1 (8.2) and control group 2 (8.4), and much higher than that of control group 3 (5.3). This proves that the three-stage process of "pre-drying + enzyme inactivation + slow drying" of the present invention not only solves the problem of adenosine loss in traditional processes, but also avoids morphological defects such as insect shrinkage and stroma breakage caused by direct high temperature. It achieves dual optimization of "component content" and "product form", which is a structural innovation of existing drying processes.

[0085] This embodiment, through multiple comparative experiments, increases the adenosine retention rate by more than 30% while maintaining the integrity of the product form, thus resolving the contradiction that existing technologies cannot achieve simultaneously.

[0086] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0087] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction, the method comprising feeding host larvae, inoculating with Cordyceps sinensis fungus, low-temperature treatment to form stunted larvae and low-temperature induction culture of stromata, and drying after harvesting, characterized in that, The process from low-temperature treatment to form stunted worms and low-temperature induction culture of stromata to post-harvest drying includes: a. During the infection period before the formation of stunted worms under low temperature treatment, metabolic intervention was performed on the larval and strain complex using regulators that target the adenosine synthesis pathway or inhibit adenosine degradation enzymes. b. The steps of forming stunted insects and inducing stroma formation by low temperature treatment include: firstly, deep low temperature stunting treatment is carried out at 2-5℃ for 30-50 days, then the stroma induction period is carried out by temperature cycling with day and night temperature difference, and mannitol is sprayed on the surface of the culture substrate in the early stage of stroma induction. c. During the drying process after harvesting, the harvested Cordyceps sinensis is subjected to rapid temperature-inactivation drying treatment. This rapid temperature-inactivation drying treatment involves raising the drying temperature from 40-45℃ to 60-70℃ within 30-45 minutes and maintaining it until the moisture content is below 10%.

2. The method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction according to claim 1, characterized in that, The regulators used in step a to intervene in the metabolism of the larval-strain complex include adenosine precursors.

3. The method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction according to claim 2, characterized in that, The regulator used in step a to intervene in the metabolism of the larval-strain complex is hypoxanthine.

4. The method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction according to claim 2, characterized in that, The regulators used in step a to intervene in the metabolism of the larval and strain complex include an inhibitor of adenosine-degrading enzyme activity, wherein the inhibitor of adenosine-degrading enzyme activity is emodin methyl ether.

5. The method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction according to claim 4, characterized in that, The regulator is administered in solution via microinjection or feeding at a concentration of 0.01%–0.05% (w / v).

6. The method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction according to claim 1, characterized in that, During the initial 1-3 days of stroma induction, mannitol at a concentration of 0.05-0.1% (w / v) should be sprayed onto the surface of the culture substrate.

7. The method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction according to claim 1, characterized in that, The induction period of the stroma described in step b is carried out by a variable temperature cycle with a diurnal temperature difference, including: daytime temperature of 12-15℃ and light for 6-8 hours, nighttime temperature of 5-8℃, and a cycle of 25-40 days.

8. The method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction according to claim 1, characterized in that, The rapid temperature-inactivation and drying process described in step c includes: first drying the harvested material at 40-45℃ for 1-2 hours, and then raising the temperature to 60-70℃ within 30-45 minutes and maintaining it for 1-1.5 hours.

9. The method for increasing adenosine content in Cordyceps sinensis based on metabolic regulation and synergistic induction according to claim 8, characterized in that, The rapid temperature-inactivation and drying process described in step c includes: raising the temperature to 60-70°C within 30-45 minutes and maintaining it for 1-1.5 hours, then drying at 40-45°C until the moisture content is below 10%.

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