A method for cultivating a pupa of a mandarin moth and a cordyceps

Abstract

The present application relates to the field of biotechnology, and more particularly to a cultivation method of pupa of the Chinese oak silkworm and Cordyceps.The present application can effectively improve the germination rate of the fruiting body of pupa of the Chinese oak silkworm and Cordyceps, the germination rate can reach 96%, and the uniformity of the growth of the fruiting body is significantly improved, the generation of the waste of pupa without fruiting body is reduced, the utilization rate of the pupa of the Chinese oak silkworm is improved, the production cost is greatly reduced, the cultivation benefit is improved, the overexploitation of the wild Cordyceps resources is reduced, and the ecological environment is protected.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a method for cultivating Cordyceps militaris. Background Technology

[0002] Cordyceps militaris often refers to Cordyceps militaris, which is cultivated by inoculating Cordyceps militaris fungus onto a grain culture medium. It contains active substances such as cordycepin, cordycepic acid, adenosine, and cordyceps polysaccharides. It does not involve a live parasitic process and is mostly used in the food industry, with a relatively low market price.

[0003] Cordyceps militaris: This is a fungus-insect hybrid formed by inoculating Cordyceps militaris fungus onto tussah silkworm pupae, followed by the maturation of the pupae and the growth of the fruiting bodies. It contains active substances such as cordycepin, cordycepic acid, adenosine, and cordyceps polysaccharides. Because its growth process involves the living parasitism of Cordyceps militaris fungus, it can produce antimicrobial peptides. Its cultivation process is similar to that of Cordyceps sinensis. Cordyceps militaris combines the nutritional characteristics of both tussah silkworm pupae and Cordyceps, possessing various health benefits including enhanced immunity, anti-cancer properties, cardiovascular protection, liver protection, asthma relief, and lung and kidney tonification. It is commonly used in traditional Chinese medicine formulations and has a relatively high market price, exceeding 1,000 yuan per kilogram, making it economically viable.

[0004] In recent years, with the increasing demand for natural medicinal materials, the demand for Cordyceps militaris has gradually increased. However, under the traditional production model, the production of Cordyceps militaris often requires a large amount of manual labor and strict environmental control, as well as the collection of large quantities of wild Cordyceps resources for strain selection. This not only results in high production costs, but the over-collection of wild Cordyceps militaris may also damage ecological resources. Biomimetic cultivation of Cordyceps militaris can overcome the limitations of the natural environment, significantly increasing the yield of Cordyceps militaris for isolation and purification. At the same time, it can effectively reduce the over-harvesting of wild Cordyceps resources, protect the ecological environment and biodiversity, ensure the sustainable supply of Cordyceps resources, and maintain ecological balance. Biomimetic cultivation of Cordyceps militaris can achieve a yield per unit area far exceeding that of traditional field crops, making it a model for developing high-efficiency agriculture, specialty agriculture, and facility agriculture. Cultivating biomimetic Cordyceps militaris in areas with advantageous tussah silkworm farming can form a local specialty industry, drive the development of related industries, become a new way for farmers to increase their income and become wealthy, and contribute to rural revitalization.

[0005] The traditional methods for cultivating Cordyceps militaris pupae still have many problems, such as low fruiting body germination rate (70%-80%), a large amount of waste from sterile pupae without fruiting bodies, and high costs, which seriously affect the profitability of Cordyceps militaris cultivation. Therefore, there is an urgent need to provide a cultivation method that can significantly improve the germination rate and uniformity of fruiting bodies, minimize the generation of sterile pupae waste, thereby saving production costs, improving raw material utilization and production efficiency. Summary of the Invention

[0006] In view of this, the present invention provides a method for cultivating Cordyceps militaris pupae. The cultivation method provided by the present invention significantly improves the germination rate and uniformity of fruiting bodies, and minimizes the generation of waste from stagnant silkworm pupae, thereby saving production costs, improving raw material utilization, and increasing production efficiency.

[0007] The cultivation method of Cordyceps militaris provided by this invention includes: placing the stiffened pupae of the tussah silkworm on the soil surface, covering them with soil, and then cultivating them. Covering with soil refers to covering the stiffened pupae of the tussah silkworm with a thin layer of soil.

[0008] In some implementations, the soil moisture content is 65-75%, specifically 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%.

[0009] In some implementation schemes, the thickness of the soil layer is 0.5 to 1 cm, specifically 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, or 1 cm.

[0010] In some embodiments, the soil includes at least one of pine needle soil, coconut fiber soil, and garden soil; preferably, the garden soil is garden soil from Northeast China. In a specific embodiment of the present invention, the soil is garden soil from Kouqian Town, Yongji County, Jilin City, Jilin Province, and the soil composition analysis is shown in Table 2. Studies have found that, in addition to the soils in Table 2, garden soil from Northeast China with similar composition (e.g., the content of the main components differs by no more than 15%) is also suitable for the present invention. Furthermore, experiments show that using coconut fiber soil and pine needle soil to cultivate Cordyceps militaris pupae yields similar results to using garden soil from Northeast China, with a fruiting body germination rate of 90%-98%.

[0011] In some implementation schemes, the temperature for cultivation after soil covering is 20-25℃, specifically 20℃, 21℃, 22℃, 23℃, 24℃ or 25℃.

[0012] In this invention, the tussah silkworm pupae are prepared by the following method:

[0013] S1. Inoculate the fruiting bodies of wild Cordyceps militaris into PDA solid medium and culture in the dark until the Cordyceps militaris fungus completely covers the medium, and then culture in the light.

[0014] S2. Take Cordyceps militaris mycelial blocks and inoculate them in PDA liquid medium and culture them in the dark to obtain Cordyceps militaris mycelial ball suspension;

[0015] S3. Inoculate the mycelial ball suspension into tussah silkworm pupae, culture, and obtain stiff tussah silkworm pupae.

[0016] In some implementations, in step S1, the temperature for the light-protected culture is 20~25℃, specifically 20℃, 21℃, 22℃, 23℃, 24℃ or 25℃; the temperature for the light-exposed culture is 20~25℃, specifically 20℃, 21℃, 22℃, 23℃, 24℃ or 25℃; and the light intensity for the light-exposed culture is 150-200 lx, specifically 150 lx, 160 lx, 170 lx, 180 lx, 190 lx or 200 lx.

[0017] In some implementations, in step S2, the light-protected culture is carried out at 20-25°C in the dark, on a shaker at 100-150 rpm, for 3-5 days. Specifically, the light-protected culture temperature is 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C; the shaker speed is 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, or 150 rpm; and the culture time is 3, 4, or 5 days.

[0018] In some implementation schemes, in step S3, the inoculation amount of the silkworm pupa is 0.2-0.3 mL of mycelial ball suspension per pupa, specifically 0.2 mL, 0.25 mL, or 0.3 mL; the culture temperature is 20-25℃, specifically 20℃, 21℃, 22℃, 23℃, 24℃, or 25℃; and the culture time is 10-15 days, specifically 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention, through a cultivation method of placing tussah silkworm pupae on the soil surface and mounding them in soil, effectively improves the germination rate of tussah silkworm pupae and Cordyceps fruiting bodies, achieving a germination rate of up to 96%. It also significantly improves the uniformity of fruiting body growth, reduces the generation of waste pupae without fruiting bodies, increases the utilization rate of tussah silkworm pupae raw materials, greatly reduces production costs, and increases cultivation profits. This method can be used for mass cultivation in agricultural greenhouses and under forest cover (see...). Figure 4 In addition, it can reduce the over-harvesting of wild cordyceps resources, which is beneficial to the protection of the ecological environment. Attached Figure Description

[0021] Figure 1 This invention illustrates the cultivation process of Cordyceps militaris from the tussah silkworm pupa, where a~h are schematic diagrams of the following steps: collection of wild Cordyceps militaris, preparation of liquid Cordyceps militaris inoculum, injection of Cordyceps militaris inoculum into tussah silkworm pupae, obtaining stiff tussah silkworm pupae, burying the stiff tussah silkworm pupae in the soil, germination of the fruiting bodies, and obtaining the final Cordyceps militaris from the tussah silkworm pupa.

[0022] Figure 2This refers to step (2) of the traditional method for cultivating silkworm pupae.

[0023] Figure 3 This invention describes the germination stage of the fruiting bodies of Cordyceps militaris cultivated using pine needle soil.

[0024] Figure 4 This invention relates to the cultivation of Cordyceps militaris pupae in agricultural greenhouses and under forest cover. Detailed Implementation

[0025] This invention provides a method for cultivating Cordyceps militaris pupae. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0026] In this document, the terms "including", "comprising", and "having" describe both closed-loop technical solutions consisting of the listed features and open-loop technical solutions that include the listed features.

[0027] In this document, the term “and / or” as used includes any and all combinations of one or more of the related listed items.

[0028] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0029] In this article, when referring to units for data ranges, if the unit is only followed by the right endpoint, it indicates that the units for the left and right endpoints are the same. For example, 65~75% means that the units for the left endpoint "65" and the right endpoint "75" are both %.

[0030] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0031] The present invention will be further illustrated below with reference to the embodiments:

[0032] Example 1

[0033] The cultivation method of this invention includes the following steps:

[0034] (1) Wild Cordyceps militaris with well-developed fruiting bodies were collected from Jingyue Park, Changchun City, Jilin Province, and surface-sterilized with 75% ethanol. The parts of the fruiting bodies with good color change were inoculated into PDA solid medium and cultured at 23°C in the dark for 12 days until the Cordyceps militaris mycelium completely covered the medium before being exposed to light. Vigorous growth and strong color change of Cordyceps militaris mycelium blocks (approximately the size of a soybean) from the solid medium were inoculated into 100 mL of PDA liquid medium and cultured at 23°C in the dark on a shaker at 120 r / min for 4 days to obtain a suspension of Cordyceps militaris mycelium balls. The inoculation amount for each silkworm pupa was 0.2-0.3 mL. The silkworm pupae inoculated with Cordyceps militaris were placed at 23°C in the dark for 12 days to obtain stiff silkworm pupae. Note that the solid medium culture was for mycelial isolation and purification and was not included in the total culture days of Cordyceps militaris.

[0035] (2) Bury the stiff tussah silkworm pupae in soil with a moisture content of 70% (garden soil from Kouqian Town, Yongji County, Jilin City, Jilin Province), with a soil depth of about 0.5cm, natural light, pH of about 5.5-6.5, and cultivate for 20 days to obtain soil-grown tussah silkworm pupae and cordyceps.

[0036] The total cultivation period for the cultivation method of this invention is 36 days.

[0037] Example 2

[0038] The difference from Example 1 is that the soil used in step (2) is pine needle soil; all other steps and conditions are the same, see [link to example]. Figure 3 .

[0039] Example 3

[0040] The difference from Example 1 is that the soil used in step (2) is coconut soil, while the other steps and conditions are the same.

[0041] Test case

[0042] This test case cultivates Cordyceps militaris using both the traditional method and the method described in Example 1.

[0043] The traditional method is the traditional glass bottle cultivation method. The steps (1) of this method are the same as those in Example 1. The difference between this method and Example 1 is that step (2) is different.

[0044] Traditional glass bottle culturing methods include the following steps:

[0045] (1) Same as step (1) in Example 1;

[0046] (2) Place the stiffened silkworm pupae in a glass bottle with a humidity of about 70%, a light intensity of 200 lx, and a natural pH. Cultivate for about 34 days to obtain silkworm pupa cordyceps. See [link to article]. Figure 2 .

[0047] The traditional glass bottle cultivation method takes about 50 days in total.

[0048] The testing steps included: obtaining 1000 tussah silkworm pupae according to step (1) of Example 1, dividing them into two groups of 500 each, and cultivating them according to step (2) of Example 1 and step (2) of the traditional method, respectively. The germination rate of the fruiting bodies was counted, and the content of active substances in the cultivated tussah silkworm pupae Cordyceps was determined (Table 3). The results of the garden soil composition analysis of Example 1 are shown in Table 2.

[0049] Table 1. Fruiting body germination rate

[0050]

[0051] Table 2 Soil composition analysis

[0052]

[0053] Table 3 Content of active substances

[0054]

[0055] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for cultivating Cordyceps militaris, characterized in that, Place the stiffened pupae of the tussah silkworm on the soil surface, cover them with soil, and then cultivate them. The soil moisture content is 65-75%; The thickness of the soil layer is 0.5-1cm.

2. The cultivation method according to claim 1, characterized in that, The soil moisture content is 70%.

3. The cultivation method according to claim 1, characterized in that, The thickness of the soil layer is 0.5 cm.

4. The cultivation method according to claim 1, characterized in that, The soil includes at least one of pine needle soil, coconut soil, and garden soil.

5. The cultivation method according to claim 4, characterized in that, The garden soil in question is from the Northeast region.

6. The cultivation method according to claim 1, characterized in that, The culture temperature is 20-25℃.

7. The cultivation method according to any one of claims 1 to 6, characterized in that, The tussah silkworm pupae were obtained by the following method: S1. Inoculate the fruiting bodies of wild Cordyceps militaris into PDA solid medium and culture in the dark until the Cordyceps militaris fungus completely covers the medium, and then culture in the light; the temperature of the light culture is 20-25℃ and the light intensity is 150-200 lx. S2. Take the Cordyceps militaris mycelium blocks obtained in step S1 and inoculate them in PDA liquid medium for culture in the dark to obtain Cordyceps militaris mycelium ball suspension. S3. Inoculate the mycelial ball suspension into tussah silkworm pupae, culture, and obtain stiff tussah silkworm pupae.

8. The cultivation method according to claim 7, characterized in that, In step S1, the temperature for the light-protected culture is 23°C; the temperature for the light-exposed culture is 20~25°C, and the light intensity is 150~200 lx.

9. The cultivation method according to claim 7, characterized in that, In step S2, the light-protected culture includes: culture at 20~25℃ in the dark, on a shaker at 100~150r / min; the light-protected culture time is 3~5 days.

10. The cultivation method according to claim 7, characterized in that, In step S3, the inoculation amount of the silkworm pupa is 0.2-0.3 mL of mycelial ball suspension per pupa; the culture time is 10-15 days.

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