Application of magnesium aluminum silicate in promoting cordyceps sinensis to produce blastospores
By adding magnesium aluminum silicate to the Cordyceps sinensis culture medium, the generation of budding spores is promoted, which solves the problem of rapidly obtaining a large number of budding spores and achieves efficient artificial culture and cost savings.
Patent Information
- Application Number
- CN202211056583.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-08-31
- Publication Date
- 2025-12-16
- Estimated Expiration
- 2042-08-31
AI Technical Summary
How to quickly obtain large quantities of Cordyceps sinensis bud spores for infecting ghost moth larvae in order to shorten the artificial culture cycle and reduce costs.
Adding magnesium aluminum silicate to the Cordyceps sinensis culture medium, preferably at a concentration of 1.0–5.0 g/L, can promote an increase in the number of budding spores and enhance their tolerance and activity.
It can increase the number of budding spores by 4 to 14 times within a specified time, with an average increase of 8 times, extend the collection time by 45 to 60 days, and reduce the cost of artificial culture.
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Figure CN115505553B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of fungus culture, and particularly relates to application of magnesium aluminum silicate in promoting production of blastospores of Ophiocordyceps sinensis. BACKGROUND
[0002] Ophiocordyceps sinensis is a composite structure of larva cadaver and fungal stroma formed by Ophiocordyceps sinensis infecting host insects, i.e. larvae of Hepialidae. Due to good pharmacological and pharmacodynamic effects, Ophiocordyceps sinensis is called one of the three treasures of traditional Chinese medicine (ginseng, deer horn and Ophiocordyceps sinensis). Ophiocordyceps sinensis is mainly produced in the Qinghai-Tibet Plateau with an altitude of more than 3000 meters. The growth cycle is long, and the survival conditions are harsh. Therefore, the wild resources are extremely limited. With the expansion of the "sub-health" population, Ophiocordyceps sinensis is increasingly favored by people. Over-exploitation and climate warming lead to the wild Ophiocordyceps sinensis in danger. The only choice is to artificially cultivate Ophiocordyceps sinensis.
[0003] Since the late 1970s, China has begun to artificially cultivate Ophiocordyceps sinensis. Through fermentation culture of mycelium of the anamorphic form of Ophiocordyceps sinensis, various substitute products have been developed and produced, such as "Bailing capsules" made of fermented mycelium of Ophiocordyceps sinensis, which has been used as a national important new drug. In addition, through simulation of the ecological environment of the Qinghai-Tibet Plateau, Ophiocordyceps sinensis has also been successfully artificially cultivated by Ophiocordyceps sinensis infecting Hepialidae larvae in low-altitude environments. It is found that Ophiocordyceps sinensis exists in the form of blastospores in the hemocoel of Hepialidae larvae. Compared with surface infection, through injection of blastospores into the hemocoel of Hepialidae larvae, the cycle of artificially cultivating Ophiocordyceps sinensis is shortened due to the reduction of the steps of attaching to the surface of Hepialidae larvae and penetrating the body wall. Moreover, injection of high-concentration blastospores can shorten the proliferation time of blastospores in the hemocoel of Hepialidae larvae. Therefore, how to quickly obtain a large number of blastospores for infection is a problem to be solved in the process of artificially cultivating Ophiocordyceps sinensis, and solving the problem is conducive to saving the cost of artificially cultivating Ophiocordyceps sinensis. SUMMARY
[0004] Based on the above problems, the purpose of the present application is to provide application of magnesium aluminum silicate in promoting production of blastospores of Ophiocordyceps sinensis. The addition of magnesium aluminum silicate in the culture medium of Ophiocordyceps sinensis can increase the number of blastospores of Ophiocordyceps sinensis in the culture solution, and enhance the tolerance and activity of blastospores, thereby providing important technical support for the artificial cultivation industry of Ophiocordyceps sinensis.
[0005] The present application finds through experiments that the addition of magnesium aluminum silicate in the culture medium of Ophiocordyceps sinensis can increase the number of blastospores of Ophiocordyceps sinensis, and enhance the tolerance and activity of blastospores.
[0006] Therefore, the first purpose of the present application is to provide application of magnesium aluminum silicate in promoting production of blastospores of Ophiocordyceps sinensis.
[0007] Preferably, the application is adding magnesium aluminum silicate into the culture medium inoculated with O. sinensis, and then culturing.
[0008] Preferably, the concentration of the magnesium aluminum silicate in the culture medium is 1.0-5.0 g / L.
[0009] Preferably, the concentration of the magnesium aluminum silicate in the culture medium is 2.0-3.5 g / L.
[0010] The second object of the present application is to provide the application of magnesium aluminum silicate in improving the tolerance and activity of the blastospores of O. sinensis.
[0011] The third object of the present application is to provide the application of magnesium aluminum silicate in preparing a preparation for improving the quantity of the blastospores of O. sinensis, or the tolerance and activity of the blastospores of O. sinensis.
[0012] The fourth object of the present application is to provide a method for improving the quantity of the blastospores of O. sinensis, or the tolerance and activity of the blastospores of O. sinensis, which is inoculating O. sinensis into a culture medium added with magnesium aluminum silicate for culturing.
[0013] Preferably, the concentration of the magnesium aluminum silicate in the liquid culture medium is 1.0-5.0 g / L.
[0014] Preferably, the liquid culture medium is PMG culture medium, and the formula of the PMG culture medium is: 200 g of peeled potato, 20 g of maltose, 10 g of peptone, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, 5 g of Galleria mellonella, and 0.02 g of vitamin B1 per liter of the culture medium, and the solvent is water.
[0015] Preferably, the culturing condition is: 14±1℃, 120 r / min, and dark condition.
[0016] The present application aims at how to quickly obtain a large amount of blastospores for infection in the process of artificial cultivation of O. sinensis, and adds magnesium aluminum silicate into the culture medium of O. sinensis to induce O. sinensis to produce blastospores efficiently. The culture medium added with 1.0-5.0 g / L of magnesium aluminum silicate is inoculated with the inoculum of O. sinensis, and the amount of blastospores produced by the culture medium added with magnesium aluminum silicate is increased by 4-14 times, and the average increase is about 8 times, compared with the control group without addition of magnesium aluminum silicate, and the most suitable addition amount is between 2.0-3.5 g / L, and the amount of blastospores produced is increased by about 10 times, compared with the control group without addition of magnesium aluminum silicate. The addition of magnesium aluminum silicate can also increase the tolerance and the preservation ability of the blastospores, and can prolong the collection time by 45-60 days. The present application can more efficiently obtain blastospores for injection and infection of artificial cultivation of O. sinensis, shorten the time of artificial cultivation of O. sinensis, and save the cost of artificial cultivation. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 Figure 1 is a graph showing the relationship between the addition amount of magnesium aluminum silicate and the spore yield.
[0018] Figure 2 Figure 4 is a graph showing the spore results of the culture of 2.0, 3.5 and 5.0 g / L magnesium aluminum silicate for 55 days.
[0019] Figure 3 Figure 5 is a graph showing the spore comparison of the culture of 2.0, 3.5 g / L magnesium aluminum silicate and the culture without magnesium aluminum silicate (CK) under electron microscope detection. DETAILED DESCRIPTION
[0020] The specific embodiments of the present application are described in detail below with specific examples, which are used to understand rather than limit the scope of the present application.
[0021] Example 1
[0022] 1. Method
[0023] (1) Preparation of culture medium: PMG culture medium (take peeled potatoes 200 g, maltose 20 g, peptone 10 g, potassium dihydrogen phosphate 3 g, magnesium sulfate 1.5 g, large wax moth 5 g, vitamin B1 0.02 g, dissolved in 1000 mL of pure water, stirred and dissolved, high temperature sterilization, cooling, and then obtained) is prepared. Different concentrations of magnesium aluminum silicate are added to the culture medium, and the culture medium containing different concentrations of magnesium aluminum silicate is prepared. The sterilized culture medium is placed in a sterile cooling room below room temperature (20°C) in a high-pressure sterilization pot at 121°C for 30 min. Then, the medium is divided into a triangular bottle, and the liquid volume of each bottle is 150 mL.
[0024] (2) Inoculation and culture: The collected cordyceps sinensis spore solution (10 6 mL, i.e. Chinese hirsutella blastospore solution) is operated in a clean bench, and a pipette gun is used to inject 500 μL of spore solution into the culture medium of step (1) according to the inoculation amount of 500 μL / bottle. The whole process is kept sterile. After inoculation, it is placed in a shaker for dark culture at a speed of 120 r / min and a temperature of 14±1°C.
[0025] (3) Sampling, counting and detection: After 20 days of culture, sampling is performed for observation, and sampling is performed every 5 days. The time gradient is set to 20d, 25d, 30d, 35d, 40d, 45d, 50d, 55d, 60d. 1.5 mL is taken from each bottle each time, and the culture bottle continues to be cultured after each sampling. The sample is counted for spores, and the counting is performed under a microscope. A blood cell counting plate is used for counting, and the liquid volume for counting each time is 10 μL. Each sample is counted twice.
[0026] (4) Collection and spore activity detection: at 30 days of culture, spore collection was performed, and then the collected spore liquid was counted, after counting, the spore liquid was diluted to 10 6 stratum, and finally, the diluted spore liquid was spotted on G5 medium (200 g of peeled potatoes, 20 g of glucose, 10 g of proteose peptone, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, 5 g of waxworms, 0.02 g of vitamin B1, 16 g of agar, dissolved in 1000 mL of pure water, stirred and dissolved, high-temperature sterilization, and cooling) for activity test. To avoid differences between spotting, 20 μL was selected for each plate, and after completion, the plates were placed in an environment at 14±1℃ for culture, and test observation was performed at regular time intervals.
[0027] (5) Detailed records were made for each observation (sampling count, collection count, and germination condition).
[0028] The test was repeated 3 times, 11 bottles were inoculated at each concentration each time, of which 3 bottles were used as sampling bottles, and the other 8 bottles were used as collection bottles at each time point. A medium without magnesium aluminum silicate was selected as CK. Finally, data analysis was performed to determine the optimal addition amount.
[0029] 2. Result analysis
[0030] The spore yield data counted by the present application are all sampling spore count data (not collected spore count data), and the data of sampling count and collection count have a multiple relationship (generally 10 times).
[0031] The influence relationship of different addition amounts of magnesium aluminum silicate on spore yield is shown in Figure 1 The spore results of 2.0, 3.5 and 5.0 g / L magnesium aluminum silicate addition and 55 d culture sampling detection are shown in Figure 2 The spore comparison of 2.0, 3.5 g / L magnesium aluminum silicate addition culture and no magnesium aluminum silicate addition (CK) culture under electron microscope detection is shown in Figure 3
[0032] From Figure 1 It can be seen from the amount of spore production that the medium added with magnesium aluminum silicate has a significant difference in the number of spore growth compared with CK; within the specified spore collection time (25-45d), the spore production of the medium added with 1.0-5.0 g / L magnesium aluminum silicate is 4-14 times higher than that of the control group, and the addition amount between 2.0-3.5 g / L shows the best performance, and the spore production is about 10 times higher than that of the control group. In addition, it can be seen from the figure that the spore production of 5.0 g / L addition is lower than that of 2.0 g / L and 3.5 g / L addition, which indicates that the activity of 5.0 g / L addition has exceeded the upper limit value, and more addition has no effect on increasing spore production. In terms of spore production persistence, the medium added with active agent shows an upward trend in spore production after 55d of culture, and remains at a high level, like a secondary self-proliferation process. Sampling observation shows that the spores are full and there is no empty spore (see Figure 2 ), which indicates that the spores are still in an active state, while the CK shows almost no spores after 60d, which indicates that the spores cultured with the addition of active agent greatly enhance the tolerance and activity maintenance of the spores. Comprehensive analysis shows that: magnesium aluminum silicate added at 1.0-5.0 g / L can increase the spore production, and within the specified collection time, the spore production can be increased by 4 times at the lowest and 14 times at the highest, and the optimal addition amount is between 2.0-3.5 g / L, and the spore production is increased by about 10 times. In addition, the addition of magnesium aluminum silicate can also greatly improve the tolerance and activity maintenance of the spores, and can prolong the collection time by 45-60d.
[0033] Result analysis summary: ①1.0-5.0 g / L magnesium aluminum silicate addition can improve the spore production capacity, and the spore production can be increased by 4-14 times, with an average increase of about 8 times; ② the optimal addition amount is between 2.0-3.5 g / L, and the average can be increased by about 10 times; ③ magnesium aluminum silicate addition can also increase the tolerance and activity of the spores, and can prolong the collection time by 45-60d.
[0034] The above is only a preferred embodiment of the present application, and it should be noted that the above preferred embodiment should not be regarded as a limitation of the present application, and the protection scope of the present application should be limited by the scope defined in the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.
Claims
1. Application of magnesium aluminosilicate in promoting the production of conidium of Ophiocordyceps sinensis; the application is adding magnesium aluminosilicate into the culture medium inoculated with Ophiocordyceps sinensis, and then culturing; the concentration of magnesium aluminosilicate in the culture medium is 1.0-5.0 g / L.
2. Use according to claim 1, characterized in that, The concentration of magnesium aluminosilicate in the culture medium is 2.0-3.5 g / L.
3. Application of magnesium aluminosilicate in improving the tolerance and activity of conidium of Ophiocordyceps sinensis; the application is adding magnesium aluminosilicate into the culture medium inoculated with Ophiocordyceps sinensis, and then culturing; the concentration of magnesium aluminosilicate in the culture medium is 1.0-5.0 g / L.
4. Application of magnesium aluminosilicate in preparing a preparation for increasing the number of conidium of Ophiocordyceps sinensis; the application is adding magnesium aluminosilicate into the culture medium inoculated with Ophiocordyceps sinensis, and then culturing; the concentration of magnesium aluminosilicate in the culture medium is 1.0-5.0 g / L.
5. A method for increasing the number of blastospores and / or the tolerance and activity of blastospores of Ophiocordyceps sinensis, characterized in that, Ophiocordyceps sinensis is inoculated into the culture medium added with magnesium aluminosilicate for culturing; the concentration of magnesium aluminosilicate in the culture medium is 1.0-5.0 g / L.
6. The method of claim 5, wherein, The culture medium is PMG culture medium, and the formula is: 200 g of peeled potato, 20 g of maltose, 10 g of peptone, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, 5 g of Galleria mellonella, and 0.02 g of vitamin B1 per liter of culture medium, and the solvent is water.
7. The method of claim 5, wherein, The culture condition is: 14±1℃, 120 r / min, and dark condition.
Citation Information
Patent Citations
Method for stimulating cordyceps sinensis to generate conidia
CN103782794A
Solid fermentation method of cordyceps sinensis
CN104130947A