Cordyceps sinensis bionic culture method

Abstract

The invention discloses a cordyceps sinensis bionic culture method which comprises the following steps: determining a target area in which cordyceps sinensis is naturally distributed, arranging a signal tower A for each area, and collecting climate data of the target area; a plurality of independent compartments are arranged in the culture base, each compartment corresponds to one target area, a signal tower B is arranged, and the signal tower B receives data transmitted by the signal tower A; the inoculated hepialus armoricanus larvae are evenly distributed in all the compartments; monitoring the growth information of the hepialus armoricanus larvae in different time periods in each compartment in real time, and recording the growth information of the hepialus armoricanus larvae in different time periods; growth information in different time periods is analyzed, climate data are reconstructed and stored in a signal tower B, and the climate data in different compartments are controlled to be unified by using the signal tower B; according to the method, the natural environment can be accurately simulated, it is ensured that the growth of cordyceps sinensis is closer to the natural state, the ecological requirements of cordyceps sinensis hyphae and hepialus armoricanus larvae are met, and quality improvement and accumulation of active ingredients are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of fungus cultivation, and in particular to a bionic cultivation method for cordyceps sinensis. Background Art

[0002] Although Cordyceps sinensis has the appearance of both insects and grass, it is neither an insect nor a grass, but belongs to the fungus and algae. Cordyceps sinensis and Cordyceps sinensis belong to the genus Cordyceps of the phylum Fungi, but they are different species. Their growth environment and medicinal effects are completely different. Cordyceps sinensis belongs to the family of ergot fungi. The fungus Cordyceps sinensis is a complex of the stroma and the corpse of the larvae of the insects of the family Bat Moth. It is a traditional precious tonic Chinese herbal medicine with multiple effects such as regulating immune system function, anti-tumor, and anti-fatigue. Cordyceps sinensis has the functions of protecting the liver and lungs, replenishing essence and filling marrow, stopping bleeding, resolving phlegm, and anti-tumor. It can also nourish the lungs and kidneys to resist various bacteria and lower blood pressure. One of its main ingredients, cordycepin, is a substance with antibiotic effects and the effect of inhibiting cell division. It is confirmed by medical and foreign medical isotope detection technology that the cordycepin and oxalic acid in Cordyceps sinensis are also confirmed by research that the cordycepic acid in the grass also has a high effect of nourishing the kidney, strengthening yang, benefiting essence, and relieving asthma, and can significantly fight cell aging. According to modern pharmacological research results, Qinghai Cordyceps contains about 7% cordycepic acid, 28.9% carbohydrates, about 8.4% fat, about 25% protein, and 82.2% of the fat is unsaturated fatty acids. In addition, it also contains vitamin B12, ergosterol, hexose alcohol, alkaloids, etc. Due to the narrow distribution area of ​​wild Cordyceps, low natural parasitism rate, and strict requirements for living environment conditions, the resources themselves are relatively limited. In recent years, due to the serious human damage to the ecological environment of the main production areas of Cordyceps, a large number of blind and unreasonable excavations have caused the resources to decrease day by day, and the output has decreased year by year. Since Cordyceps can be used as both medicine and food, and people continue to discover its new pharmacological effects, the demand for it in many countries has doubled, the price has risen year by year, and the international market is increasingly in short supply. In order to meet the needs of the global market, many regions such as Sichuan, Hubei, Henan, Shaanxi, Yunnan and Hunan have successively carried out research on Cordyceps. People's research on Cordyceps sinensis involves the confirmation of asexual forms, ecological environmental investigation, analysis of chemical composition and pharmacological effects, and artificial cultivation.

[0003] The larvae are inoculated with mycelium and placed in a sterile greenhouse breeding room, where temperature, humidity and other conditions are strictly controlled to ensure the best growth environment; the bat moth larvae in the natural environment have adapted to the unique climatic conditions of high altitudes after a long period of natural selection. These conditions include a large temperature difference between day and night, low oxygen concentration and strong ultraviolet radiation. This adaptability gives the larvae stronger survival ability and stress resistance, and these characteristics may affect their symbiotic relationship with fungi; due to the complexity and diversity of the natural environment, bat moth larvae and fungi may produce more secondary metabolites, which are essential for the medicinal value of Cordyceps sinensis. For example, the active ingredients contained in Cordyceps sinensis, such as adenosine, cordycepin, and polysaccharides, are believed to be produced in the symbiotic process in the natural environment. The larvae cultured in the greenhouse lack the natural symbiotic process with the fungus, so they may not be able to produce the same types and quantities of active ingredients.

[0004] Therefore, in order to solve the above problems, it is necessary to design a bionic cultivation method for Cordyceps sinensis. Summary of the invention

[0005] The invention overcomes the shortcomings of the prior art and provides a bionic cultivation method for Cordyceps sinensis.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a bionic cultivation method of Cordyceps sinensis, comprising the following steps:

[0007] Step S1, determining the target area where Cordyceps sinensis is naturally distributed, arranging signal towers A for each area, and collecting climate data of the target area;

[0008] Step S2, arranging a plurality of independent compartments in the training base, each compartment corresponding to a target area, and arranging a signal tower B, the signal tower B receiving the climate data transmitted by the signal tower A of different target areas, and synchronizing in real time to each corresponding compartment;

[0009] Step S3, inoculating Chinese Cordyceps sinensis mycelium and healthy bat moth larvae of the same species, and evenly distributing the inoculated bat moth larvae in each compartment;

[0010] Step S4, real-time monitoring of the growth information of the bat moth larvae in each compartment at different time periods, and recording the growth information and climate data of the different time periods;

[0011] Step S5, integrating and analyzing the growth information of different time periods, recording the climate data under the high-quality growth information, reconstructing the climate data and storing it in the signal tower B, and using the signal tower B to control the climate data in different compartments to unify them;

[0012] Step S6: different types of bat moth larvae are selected and evenly inoculated with Chinese Cordyceps sinensis mycelium, and the inoculated bat moth larvae are evenly distributed in each compartment, and the signal tower B controls each compartment using the reconstructed climate data.

[0013] In a preferred embodiment of the present invention, in the step S2, the configurations in each compartment are the same, specifically, multiple layers of culture beds are arranged in the compartment, each layer is 16-18 cm high, 1.3-1.5 m wide, and the length corresponds to the length of the compartment. The top layer of the culture bed is 1-1.5 m high from the top of the compartment, and a 0.8-1 m wide passage is left between the beds for easy management.

[0014] Fill each layer of the bed frame with culture soil, requiring the soil layer thickness to be 8 to 10 cm. The culture soil is made by mixing 50 kg of river sand, 30 kg of garden soil, 10 kg of loose yellow mud, and 10 kg of artificial compound feed in proportion. Adjust the soil pH to 6 and the humidity to 50%, and then make the culture soil into ridges.

[0015] In a preferred embodiment of the present invention, in step S1, the target area is Yushu Prefecture and Guoluo Prefecture with an altitude between 4400 and 4600 m, as well as different valleys, hillsides or river areas in Yushu Prefecture and Guoluo Prefecture.

[0016] In a preferred embodiment of the present invention, in step S1, the climate data includes: temperature, humidity, light, air pressure, oxygen concentration, and soil moisture of the target area;

[0017] The data collection frequency of the signal tower A is set to 10 min / c.

[0018] In a preferred embodiment of the present invention, in step S3, the method of inoculating Chinese Cordyceps sinensis mycelium and healthy bat moth larvae comprises the following steps:

[0019] Step S31, inoculating the Cordyceps sinensis strain into a PDA culture medium, and culturing at 25° C. to 28° C. and a relative humidity of 70% to 80% for 7 to 14 days;

[0020] Step S32: Chop the cultured mycelium into small pieces and place them in sterile physiological saline to make a concentration of 10 6 CFU / ml bacterial suspension;

[0021] Step S33, placing the 2nd-instar bat larvae in a refrigerated environment at 4°C to 5°C for short-term anesthesia for 10 to 15 minutes, washing the surface of the bat larvae with sterile saline, and transferring them to a sterile environment to dry;

[0022] Step S34, use a sterile syringe to inject 5-10 μl of the bacterial solution into the abdomen of the bat larvae;

[0023] Step S35, evenly distribute the injected bat larvae to each compartment of the culture base.

[0024] In a preferred embodiment of the present invention, in step S4, the different time periods include: larval stage, hyphae infection stage, and bacterial growth stage;

[0025] Monitoring of growth information includes: real-time detection of larval growth rate and survival rate, degree of mycelium infection, morphological characteristics of Cordyceps sinensis, and regular sampling to detect the content of key active ingredients of Cordyceps sinensis, such as cordycepin, polysaccharides and adenosine.

[0026] In a preferred embodiment of the present invention, in step S4, the growth information of the bat moth larvae in each compartment in different time periods is monitored in real time, and the climate data in each compartment in different time periods is recorded.

[0027] In a preferred embodiment of the present invention, in step S5, the high-quality growth information is specifically a data information set reflecting the optimal growth state of the inoculated bat moth larvae during the bionic cultivation process of Cordyceps sinensis.

[0028] In a preferred embodiment of the present invention, in step S6, the reconstructed climate data is used to culture the inoculated larvae of different species of bat moths, and the growth rate and survival rate of the larvae are monitored.

[0029] The present invention solves the defects existing in the background technology and has the following beneficial effects:

[0030] (1) The present invention provides a bionic cultivation method for Cordyceps sinensis, which utilizes a signal tower to collect and transmit climate data of a target area in real time, accurately restores the natural environment to a compartment, and avoids errors caused by human control. The high similarity between the compartment environment and the target area can accurately simulate the natural environment, ensure that the growth of Cordyceps sinensis is closer to the natural state, meet the ecological needs of Cordyceps sinensis mycelium and bat moth larvae, and help improve the quality and accumulation of active ingredients.

[0031] (2) The present invention dynamically adjusts the microclimate data of each compartment through real-time monitoring and data analysis to ensure that the growth needs of the inoculated bat moth larvae at each stage are met. Through dynamic linear regression and model prediction, the climate data affecting the growth of Cordyceps sinensis is extracted to generate the optimal climate data for the whole year, ensuring that each stage is in the optimal environmental conditions, thereby optimizing the environmental conditions in different time periods and greatly improving the growth rate and yield of Cordyceps sinensis.

[0032] (3) The present invention comprehensively controls the cultivation status by real-time monitoring of the larval growth rate, mycelial infection degree and active ingredient content in different time periods. By recording the correspondence between growth data and climate parameters, a scientific feedback mechanism is formed to provide a decision-making basis for subsequent cultivation. At the same time, growth disorders caused by sudden environmental changes or operational errors are avoided, ensuring the high stability of the cultivation process.

[0033] (4) The present invention can verify the actual effect and promotion potential of bionic cultivation technology by conducting simultaneous experiments on bat moth larvae from different locations under the same climate data conditions, thereby reducing excessive dependence on natural resources and effectively protecting the original ecological environment; at the same time, through standardized climate simulation and cultivation processes, it can also ensure the stability of the active ingredient content of Cordyceps sinensis and improve product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

[0035] Figure 1 The present invention is a flowchart of a bionic cultivation method of Cordyceps sinensis according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0038] In the description of the present application, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the scope of protection of the present application. In addition, the terms "first", "second", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the invention, unless otherwise specified, "multiple" means two or more.

[0039] In the description of this application, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood by specific circumstances.

[0040] like Figure 1 As shown, a bionic cultivation method of Cordyceps sinensis comprises the following steps:

[0041] Step S1, determining the target area where Cordyceps sinensis is naturally distributed, arranging signal towers A for each area, and collecting climate data of the target area;

[0042] Step S2, arranging a plurality of independent compartments in the training base, each compartment corresponding to a target area, and arranging a signal tower B, the signal tower B receiving the climate data transmitted by the signal tower A of different target areas, and synchronizing in real time to each corresponding compartment;

[0043] Step S3, inoculating Chinese Cordyceps sinensis mycelium and healthy bat moth larvae, and evenly distributing the inoculated bat moth larvae in each compartment;

[0044] Step S4, real-time monitoring of the growth information of the bat moth larvae in each compartment at different time periods, and recording the growth information at different time periods;

[0045] Step S5, integrating and analyzing the growth information of different time periods, recording the climate data under the high-quality growth information, reconstructing the climate data and storing it in the signal tower B, and using the signal tower B to control the climate data in different compartments to unify them;

[0046] Step S6: different types of bat moth larvae are selected and evenly inoculated with Chinese Cordyceps sinensis mycelium, and the inoculated bat moth larvae are evenly distributed in each compartment, and the signal tower B controls each compartment using the reconstructed climate data.

[0047] In the present invention, in step S1, the target area screening basis is specifically that the altitude distribution is between 4400 and 4600m, which is the core altitude range of the natural distribution of Cordyceps sinensis, the average annual temperature is low, at 5-10°C, the temperature difference between day and night is large, the humidity is moderate, the sunlight is strong but evenly distributed, the soil layer is moderately thick, rich in organic matter, has strong water retention, and is slightly acidic, with a pH value between 5.5 and 6.5. The area should be covered with grassland, shrubs and moss vegetation suitable for the survival of bat moth larvae; therefore, Yushu Prefecture and Guoluo Prefecture, as well as different valleys, hillsides or river areas in Yushu Prefecture and Guoluo Prefecture were selected;

[0048] Multiple signal towers A are arranged in the target area to obtain representative climate data and monitor the climate data of temperature, humidity, light, air pressure, oxygen concentration, and soil moisture in the target area in real time; wherein the data collection frequency of the signal tower A is set to 10min / c.

[0049] In step S2, the configurations in each compartment are the same, specifically, multiple layers of culture beds are arranged in the compartment, each layer is 16-18 cm high, 1.3-1.5 m wide, and the length corresponds to the length of the compartment. The top layer of the culture bed is 1-1.5 m high from the top of the compartment, and a 0.8-1 m wide passage is left between the beds for easy management;

[0050] Fill each layer of the bed frame with culture soil, requiring the soil layer thickness to be 8 to 10 cm. The culture soil is made by mixing 50 kg of river sand, 30 kg of garden soil, 10 kg of loose yellow mud, and 10 kg of artificial compound feed in proportion. Adjust the soil pH to 6 and the humidity to 50%, and then make the culture soil into ridges.

[0051] Each compartment is equipped with an independent signal tower B, which is used to receive the target area climate data transmitted in real time by signal tower A. Signal tower B maintains real-time connection with signal tower A through a wireless network or local area network to ensure the continuity and accuracy of data transmission;

[0052] Signal towers are used to collect and transmit climate data of the target area in real time, and the natural environment can be accurately restored to the compartment to avoid errors caused by human control. The high similarity between the compartment environment and the target area can accurately simulate the natural environment, ensuring that the growth of Cordyceps sinensis is closer to its natural state, meeting the ecological needs of Cordyceps sinensis mycelium and bat moth larvae, and helping to improve quality and the accumulation of active ingredients.

[0053] In step S3, the method of inoculating Chinese Cordyceps sinensis mycelium and healthy bat moth larvae comprises the following steps:

[0054] Step S31, inoculating the Cordyceps sinensis strain into a PDA culture medium to provide a rich nutrient source for the strain, culturing for 7 to 14 days at 25° C. to 28° C. and a relative humidity of 70% to 80%, until the mycelium forms a dense and active bacterial body on the surface of the culture medium, wherein the Cordyceps sinensis strain is derived from the China General Microbiological Culture Collection Center;

[0055] Step S32: scrape the mycelium from the culture medium, chop it with sterile scissors to increase the suspension of mycelium, and place the chopped mycelium in sterile physiological saline to make a concentration of 10 6 CFU / ml of suspended bacterial solution ensures the activity and stability of the bacterial solution. All operations must be performed in a clean bench or sterile environment to prevent contamination by foreign bacteria. The mycelium of Chinese Cordyceps sinensis is obtained.

[0056] Step S33, select healthy 2-year-old bat moth larvae. At this stage, the larvae are active and highly sensitive to mycelium infection. Place the 2-year-old bat larvae in a 4°C to 5°C refrigerated environment for short-term anesthesia for 10 to 15 minutes to reduce activity interference during the inoculation process. Rinse the surface of the bat larvae with sterile saline to remove attached bacteria and dirt. The rinsed larvae need to be placed in a sterile environment to dry to avoid water droplets remaining and causing subsequent operation failures;

[0057] Step S34, using a sterile syringe with a needle of 0.5 mm diameter to minimize the wound caused to the larvae, inject 5 to 10 μl of the bacterial solution into the abdomen of the bat larvae with a sterile syringe;

[0058] Step S35, evenly distribute the injected bat larvae to the compartments of the culture base, wherein the larvae are evenly placed in the ridged soil of the culture bed frame to ensure that the mycelium contact surface is maximized, and the distance between the larvae is about 5 to 10 cm to avoid mutual interference between the larvae.

[0059] Among them, the inoculated larvae need 12 to 24 hours to adapt to the environment. During this stage, the temperature and humidity in the compartment are kept stable to avoid drastic changes in the environment causing stress to the larvae. An ultraviolet sterilization lamp is installed in each compartment, and regular sterilization is carried out every day with a frequency of 2h / c. The culture soil is sterilized every week to prevent the growth of miscellaneous bacteria. Monitoring equipment is used to observe the activity, survival rate and mycelium infection status of the larvae in real time.

[0060] In step S4, different time periods include: larval stage, hyphae infection stage, and bacterial growth stage; the larval stage is generally 0 to 2 weeks, the hyphae infection stage is generally 2 to 6 weeks, the hyphae gradually invade the larvae and begin to reproduce, and the morphology of the larvae gradually changes; the bacterial growth stage is generally after 6 weeks, the hyphae completely occupy the larvae and form the typical morphological characteristics of Cordyceps sinensis, and the active ingredients gradually accumulate;

[0061] The monitoring of growth information includes: real-time detection of larval growth rate and survival rate, mycelium infection degree, morphological characteristics of Cordyceps sinensis, and regular sampling and detection of the content of key active ingredients of Cordyceps sinensis, such as cordycepin, polysaccharides and adenosine. Specifically:

[0062] Growth rate: Use high-definition video equipment to mark the larvae with fluorescent marker dyes, capture the growth dynamics of the larvae through fluorescent images under ultraviolet light, install high-precision electronic sensors on the culture substrate to record real-time changes in larval weight, and use image analysis software to convert changes in larval length into growth rate curves. Record data every 2 days to ensure the continuity of detection and monitor changes in growth rate over 14 days. Growth rate = (current weight or length - initial weight or length) / monitoring time.

[0063] Survival rate: Infrared activity sensors were used to monitor the behavior of the larvae, including movement frequency and crawling speed. The surviving larvae would show active behavior, while the non-surviving individuals would be inactive. The number of surviving larvae was visually inspected and recorded regularly, and the survival rate was calculated in each monitoring. Survival rate = (number of surviving larvae / total number of larvae) × 100%.

[0064] Mycelial infection degree: Every 3 days, randomly select inoculated bat moth larvae from different culture compartments for sample collection. The number of samples each time is 1% to 2% of the total number of larvae in each compartment to avoid affecting the overall growth;

[0065] The larvae were picked up with sterile forceps, placed in 75% alcohol for surface disinfection, and then rinsed with sterile PBS (phosphate buffered saline) to remove impurities. In a sterile environment, the larvae epidermis was cut open with a scalpel to expose the internal tissue to observe the distribution of hyphae;

[0066] Fluorescent dye (DAPI) was used to specifically stain the hyphae inside and on the surface of the larvae, and after staining, the samples were washed three times with PBS to remove unbound dye;

[0067] Use fluorescence microscopy or confocal microscopy to observe the infection of hyphae inside and outside the larvae, capture high-resolution images of hyphae distribution, and measure hyphae density and infection area. Hyphae coverage (%) = (hyphae coverage area / larvae surface area) × 100%;

[0068] The quantitative scores are as follows:

[0069] Grade 0: no mycelium infection;

[0070] Level 1: The surface is slightly covered with hyphae;

[0071] Level 2: hyphae cover more than 50% of the larvae;

[0072] Grade 3: Mycelium completely covers and invades internal tissues.

[0073] Randomly sample some Cordyceps sinensis from different compartments every 2 to 4 weeks, dry the samples to constant weight, crush them, sieve them (80 mesh), and set aside;

[0074] Cordycepin: weigh 1.0 g of powder, add 10 ml of 70% methanol solution, extract with ultrasonic assistance for 30 minutes (frequency 40 kHz, power 200 W), let stand at 4°C, take the supernatant, filter the extract with a 0.22 μm microporous membrane, and set aside;

[0075] Analyzed by HPLC

[0076] Chromatographic conditions:

[0077] Column: C18 reverse phase column (4.6 mm × 250 mm, 5 μm);

[0078] Mobile phase: A (0.1% formic acid in water) / B (methanol), gradient elution;

[0079] Flow rate: 1.0ml / min;

[0080] Detection wavelength: 260nm;

[0081] Column temperature: 30℃.

[0082] Inject 20 μL of sample extract into HPLC, prepare a standard curve based on cordycepin standards of known concentration, substitute the sample peak area into the standard curve to calculate the cordycepin content, and the result is expressed in mg / g (dry weight).

[0083] Polysaccharide: Weigh 2.0g of dry Cordyceps sinensis powder, add 50ml of deionized water, extract in a water bath at 85℃ for 2 hours, cool, centrifuge (4000rpm, 10min), take the supernatant, add 4 times the volume of 95% ethanol to the supernatant, precipitate at 4℃ overnight, centrifuge again (4000rpm, 10min), collect the precipitate, wash with anhydrous ethanol, acetone and ether in sequence, and dry in vacuum;

[0084] Analysis by phenol-sulfuric acid method combined with UV spectrophotometry

[0085] Reagent preparation:

[0086] Prepare 5% phenol solution and 98% concentrated sulfuric acid, add 1 ml of 5% phenol solution to 1 ml of sample solution, quickly add 5 ml of concentrated sulfuric acid, mix well, let stand at room temperature for 10 minutes, then heat in a 90°C water bath for 15 minutes, and measure the absorbance after cooling.

[0087] Measurement wavelength: 490nm;

[0088] A standard curve was prepared using a glucose standard solution, and the polysaccharide content was calculated based on the sample absorbance value. The result was expressed in mg / g (dry weight).

[0089] Adenosine: weigh 1.0 g of Cordyceps sinensis powder, add 10 ml of 80% methanol solution, and extract with ultrasonic wave for 20 minutes (frequency 40 kHz, power 200 W). Take the supernatant, filter the extract with a 0.22 μm filter membrane, and set aside;

[0090] Analysis using high performance liquid chromatography (HPLC)

[0091] Chromatographic conditions:

[0092] Column: C18 reverse phase column (4.6 mm × 250 mm, 5 μm);

[0093] Mobile phase: A (0.1% formic acid in water) / B (methanol), isocratic elution;

[0094] Flow rate: 1.0ml / min;

[0095] Detection wavelength: 260nm;

[0096] Column temperature: 30℃.

[0097] Inject 20 μl of sample extract into HPLC, prepare a standard curve based on adenosine standard, substitute the sample peak area into the standard curve to calculate the adenosine content, and express the result in μg / g (dry weight).

[0098] In step S4, the growth information of the bat moth larvae in each compartment at different time periods is monitored in real time, and the climate data in each compartment at different time periods needs to be recorded, so as to compare the time period with the best growth information of the bat moth larvae;

[0099] Through real-time monitoring and data analysis, the microclimate data of each compartment is dynamically adjusted to ensure that the growth needs of the inoculated bat moth larvae at each stage are met. Through dynamic linear regression and model prediction, the key climate data affecting the growth of Cordyceps sinensis are extracted, and the optimal climate data for the whole year is generated to ensure that each stage is in the optimal environmental conditions, so that the environmental conditions are optimized in different time periods, greatly improving the growth rate and yield of Cordyceps sinensis.

[0100] In step S5, the high-quality growth information is specifically a data information set reflecting the optimal growth state of the inoculated bat moth larvae during the bionic cultivation process of Cordyceps sinensis.

[0101] In step S6, the reconstructed climate data is used to cultivate the inoculated larvae of different species of bat moths, and the growth rate and survival rate of the larvae are monitored;

[0102] The different types of bat moth larvae are specifically the bat moth larvae in Qinghai and the bat moth larvae in Yunnan and Guizhou.

[0103] In order to verify whether Cordyceps sinensis from other places can achieve similar active ingredient content under the same climatic conditions, the following experimental steps can be designed; this experiment is based on the present invention, and focuses on verifying the comparison of the cultivation effects of Cordyceps sinensis in Qinghai, Yunnan-Guizhou, Taihang Mountains, Nanling Mountains, and Wuyi Mountains under target climate data.

[0104] Experimental example

[0105] (I) Experimental purpose

[0106] To verify whether the optimal climate data of Yushu Prefecture and Golog Prefecture can be used to cultivate Cordyceps sinensis in Qinghai, Yunnan-Guizhou, Taihang Mountains, Nanling Mountains, and Wuyi Mountains, respectively, to achieve similar active ingredient contents.

[0107] (II) Test materials

[0108] The bat moth larvae were selected from Qinghai (plateau area), Yunnan-Guizhou (mountainous forest area), Taihang Mountains (temperate mountainous area), Nanling Mountains (tropical rainforest area), and Wuyi Mountains (southern humid mountainous area).

[0109] Cordyceps sinensis mycelium: prepared by the present invention.

[0110] (III) Experimental steps

[0111] The Cordyceps sinensis strain was inoculated into PDA medium and cultured at 25°C and 75% relative humidity for 8 days. The cultured mycelium was chopped and placed in sterile physiological saline to prepare a concentration of 10 6CFU / ml of suspended bacterial liquid was used to select several 2-year-old bat larvae, of which half were from Qinghai and half from Yunnan and Guizhou. They were placed in a 5°C cold storage environment for short anesthesia for 15 minutes, the surface of the bat larvae was rinsed with sterile saline, and the larvae were transferred to a sterile environment to dry. A sterile syringe was used to inject 610 μl of the bacterial liquid into the abdomen of the bat larvae. The bat larvae after injection were evenly distributed to various compartments of the culture base, and the reconstructed climate data was used to control each compartment, and Cordyceps sinensis was cultivated. The content of active ingredients in Cordyceps sinensis cultivated by bat moth larvae from other places in the same environment was detected, as shown in Table 1.

[0112] Table 1

[0113]

[0114]

[0115] Compared with the above experiments, by simulating the optimal climate data of the target area and using bat moth larvae from other places to achieve efficient cultivation of Cordyceps sinensis under the same environmental conditions, the actual effect and promotion potential of bionic cultivation technology can be verified, which can reduce excessive dependence on natural resources and effectively protect the original ecological environment; at the same time, through standardized climate simulation and cultivation processes, the content of active ingredients in Cordyceps sinensis can be ensured to be stable and product quality can be improved.

[0116] The above is based on the ideal embodiment of the present invention. Through the above description, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the content in the specification, and the technical scope must be determined according to the scope of the claims.

Claims

1. A bionic cultivation method for Cordyceps sinensis, characterized in that: The following steps are involved: Step S1, determining the target area where Cordyceps sinensis is naturally distributed, arranging signal towers A for each area, and collecting climate data of the target area; Step S2, arranging a plurality of independent compartments in the training base, each compartment corresponding to a target area, and arranging a signal tower B, the signal tower B receiving the climate data transmitted by the signal tower A of different target areas, and synchronizing in real time to each corresponding compartment; Step S3, inoculating Chinese Cordyceps sinensis mycelium and healthy bat moth larvae of the same species, and evenly distributing the inoculated bat moth larvae in each compartment; Step S4, real-time monitoring of the growth information of the bat moth larvae in each compartment at different time periods, and recording the growth information and climate data of the different time periods; Step S5, integrating and analyzing the growth information of different time periods, recording the climate data under the high-quality growth information, reconstructing the climate data and storing it in the signal tower B, and using the signal tower B to control the climate data in different compartments to unify them; Step S6: different types of bat moth larvae are selected and evenly inoculated with Chinese Cordyceps sinensis mycelium, and the inoculated bat moth larvae are evenly distributed in each compartment, and the signal tower B controls each compartment using the reconstructed climate data.

2. The bionic culture method of Cordyceps sinensis according to claim 1, characterized in that: In step S2, the configurations in each compartment are the same, specifically, multiple layers of culture beds are arranged in the compartment, each layer is 16-18 cm high, 1.3-1.5 m wide, and the length corresponds to the length of the compartment. The top layer of the culture bed is 1-1.5 m high from the top of the compartment, and a 0.8-1 m wide passage is left between the beds for easy management; Fill each layer of the bed frame with culture soil, requiring the soil layer thickness to be 8 to 10 cm. The culture soil is made by mixing 50 kg of river sand, 30 kg of garden soil, 10 kg of loose yellow mud, and 10 kg of artificial compound feed in proportion. Adjust the soil pH to 6 and the humidity to 50%, and then make the culture soil into ridges.

3. The bionic culture method of Cordyceps sinensis according to claim 1, characterized in that: In step S1, the target area is Yushu Prefecture and Guoluo Prefecture with an altitude between 4400 and 4600 m, as well as different valleys, hillsides or river areas in Yushu Prefecture and Guoluo Prefecture.

4. The bionic culture method of Cordyceps sinensis according to claim 1, characterized in that: In step S1, the climate data includes: temperature, humidity, light, air pressure, oxygen concentration, and soil moisture of the target area; The data collection frequency of the signal tower A is set to 10 min / c.

5. The bionic culture method of Cordyceps sinensis according to claim 1, characterized in that: In step S3, the method of inoculating Chinese Cordyceps sinensis mycelium and healthy bat moth larvae comprises the following steps: Step S31, inoculating the Cordyceps sinensis strain into a PDA culture medium, and culturing at 25° C. to 28° C. and a relative humidity of 70% to 80% for 7 to 14 days; Step S32: Chop the cultured mycelium into small pieces and place them in sterile physiological saline to make a concentration of 10 6 CFU / ml bacterial suspension; Step S33, placing the 2nd-instar bat larvae in a refrigerated environment at 4°C to 5°C for short-term anesthesia for 10 to 15 minutes, washing the surface of the bat larvae with sterile saline, and transferring them to a sterile environment to dry; Step S34, use a sterile syringe to inject 5-10 μl of the bacterial solution into the abdomen of the bat larvae; Step S35, evenly distribute the injected bat larvae to each compartment of the culture base.

6. The bionic culture method of Cordyceps sinensis according to claim 1, characterized in that: In step S4, the different time periods include: larval stage, mycelium infection stage, and bacterial growth stage; Monitoring of growth information includes: real-time detection of larval growth rate and survival rate, degree of mycelium infection, morphological characteristics of Cordyceps sinensis, and regular sampling to detect the content of key active ingredients of Cordyceps sinensis, such as cordycepin, polysaccharides and adenosine.

7. The bionic culture method of Cordyceps sinensis according to claim 1, characterized in that: In step S4, the growth information of the bat moth larvae in each compartment in different time periods is monitored in real time, and the climate data in each compartment in different time periods is recorded.

8. The bionic culture method of Cordyceps sinensis according to claim 6, characterized in that: In step S5, the high-quality growth information is specifically a data information set reflecting the optimal growth state of the inoculated bat moth larvae during the bionic cultivation process of Cordyceps sinensis.

9. The bionic culture method of Cordyceps sinensis according to claim 1, characterized in that: In step S6, the reconstructed climate data is used to culture the inoculated larvae of different species of bat moths, and the growth rate and survival rate of the larvae are monitored.

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