Yuhuang fungus stock culture medium, culture method and application of Yuhuang fungus stock culture medium

Abstract

The invention discloses a Yunhuang fungus stock culture medium, a culture method and application, and belongs to the technical field of fungus culture. According to the method, the stock culture medium and stock culture conditions are optimized, the stock culture medium and culture conditions are optimized, the stock culture effect of the poplar and yellow fungus is remarkably improved, the hypha growth speed, the hypha diameter, the hypha polysaccharide content, the flavone yield and the BMF dry weight of the poplar and yellow fungus are increased, meanwhile, the contamination rate of infectious microbes and the number of hypha tip branches are remarkably reduced, and the yield of the poplar and yellow fungus is increased. The extension speed of hyphae is promoted, and a technical support is provided for batch production of Yunhuang fungus.

Description

Technical Field

[0001] The present invention relates to the technical field of fungal culture, and more particularly to a culture medium, a culture method and an application of a fungus stock culture medium. Background Art

[0002] Yanghuang (Pomilloides yanghuang) is a medicinal fungus that grows on poplar trees. Its scientific name is Physcomitrella leucoderma, and it belongs to the phylum Basidiomycota, class Agaricaceae, order Pyrophorales, family Pyrophoraceae, and genus Pyrophora. Yanghuang (Pomilloides yanghuang) has extensive applications in Traditional Chinese Medicine. It contains a variety of active ingredients, such as polysaccharides and flavonoids, that exhibit anti-tumor, antioxidant, and anti-inflammatory effects. Furthermore, Yanghuang (Pomilloides yanghuang) has excellent immunomodulatory properties, enhancing immune function. Therefore, Yanghuang (Pomilloides yanghuang) holds great potential for medicinal applications.

[0003] The primary goal of the hierarchical propagation of fungal strains, from parent stock to stock, and then to cultivar, is to achieve efficient, stable, and scalable strain production. The parent stock generally ensures freedom from contamination and genetic stability. The stock (secondary stock) is propagated from the parent stock to further verify strain viability and eliminate contaminated or poorly growing strains. The cultivar (production stock) is a highly adaptable mycelium after stock propagation, allowing it to be directly used in cultivation, reducing the risk of large-scale contamination. Therefore, stock culture plays a crucial role in the entire hierarchical propagation process, not only improving strain adaptability but also increasing the number of strains, providing technical support for large-scale production. However, current research on Yanghuang fungus is largely limited to active ingredient extraction and pharmacological studies, as well as molecular biology and genetics. Few reports exist on optimizing stock culture formulations and acclimating Yanghuang fungus for large-scale cultivation. Furthermore, researchers have found that mycelium grows slowly and sparsely on conventional stock culture media, resulting in a limited supply of strains and hindering subsequent large-scale artificial cultivation trials.

[0004] Therefore, how to provide a culture medium, culture method and application of the original seed of Yanghuang mushroom to increase the reproduction speed and promote the large-scale production of the original seed is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a culture medium, culture method and application of the Yanghuang mushroom stock, which significantly improves the cultivation efficiency, accelerates the growth rate of mycelium, and improves the propagation coefficient through the optimization of the culture medium and the optimization of the culture method.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A culture medium for the stock of Euphorbia pulex comprises one of cottonseed hulls, straw or corn cobs; and further comprises wheat bran, rice hulls and oil residue.

[0008] As a preferred technical solution, the composition ratio of the boxwood fungus stock culture medium is as follows:

[0009] 85% cottonseed hulls, 5% wheat bran, 5% rice hulls, and 5% oilseed residue;

[0010] or 85% straw, 5% wheat bran, 5% rice husk and 5% oil residue;

[0011] Or 85% corn cobs, 5% wheat bran, 5% rice husks and 5% oil residue.

[0012] Another object of the present invention is to provide: application of the above-mentioned Yanghuangzi stock culture medium in Yanghuangzi stock culture.

[0013] Another object of the present invention is to provide a method for culturing a stock of Populus flavescentis, comprising the following steps:

[0014] (1) Obtaining the mother strain of Yanghuang fungus: Select healthy and fresh Yanghuang fruiting bodies, cut small pieces of tissue after disinfection, and inoculate them into the mother strain culture medium for cultivation. After the mycelium grows, select the mycelium that grows rapidly and is healthy. After multiple transfers of the mother strain culture medium, the foreign bacteria are effectively removed and a healthy Yanghuang fungus mother strain is obtained;

[0015] (2) Cultivation of the mother culture of Buxus flavescentis: The mother culture of Buxus flavescentis obtained in step (1) is inoculated into the above-mentioned Buxus flavescentis stock culture medium for stock culture.

[0016] As a preferred technical solution, the disinfection in step (1) is carried out using 75% alcohol, and the number of disinfections is ≥5 times.

[0017] As a preferred technical solution, the mother culture medium in step (1) is based on potato and agar, and also includes a carbon source, a nitrogen source and water; the weight ratio of each component is as follows: 150-250g / L potato, 15-25g / L carbon source, 1-5g / L nitrogen source, 15-20g / L agar, and water is added to make up to 1000mL.

[0018] As a preferred technical solution, the carbon source is sucrose and / or maltose; and the nitrogen source is beef extract and / or peptone.

[0019] As a preferred technical solution, the mother culture temperature is 25-28°C, the lighting conditions are complete darkness or scattered light, and the culture pH is 5-9.

[0020] As a preferred technical solution, the mother culture temperature is 28°C, the lighting conditions are completely dark, and the culture pH is 8.

[0021] As a preferred technical solution, the temperature of the original seed culture is 25-26°C, the lighting condition is completely dark, and the culture pH is 7.0-7.5.

[0022] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a stock culture medium, a culture method and an application of a Pseudomonas aeruginosa. By optimizing the mother culture medium and the mother culture conditions as well as the stock culture medium and the culture conditions, the effect of the stock culture of the present invention is significantly improved, the mycelial growth rate, mycelial diameter, mycelial polysaccharide content, flavonoid yield and BMF dry weight of the Pseudomonas aeruginosa are improved, and the contamination rate of foreign bacteria and the number of mycelial tip branches are significantly reduced, the extension speed of the mycelium is promoted, and technical support is provided for the mass production of the Pseudomonas aeruginosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0025] Figure 1 Figure: Yanghuang (left) and the strain obtained after isolation (right).

[0026] Figure 2 This is the status diagram of the original seed after 30 days of cultivation.

[0027] Figure 3 For: Mushroom fruiting status diagram.

[0028] Figure 4 Figure 3: Status diagram of 30 days of growth on mother culture medium. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 making creative efforts are within the scope of protection of the present invention.

[0030] Example 1

[0031] Obtaining the mother strain of Yanghuang mushroom

[0032] Collect fresh Yanghuang, select healthy fresh Yanghuang fruiting bodies, make sure there is no obvious rot or lesion when selecting, use a clean brush to gently brush off impurities on the surface of Yanghuang, and be careful not to damage the base of the fruiting body. Disinfect the surface of Yanghuang with 75% alcohol and scrub 5 times to ensure that bacteria and other contaminants that may exist on the surface are completely eliminated. Place the sterilized Yanghuang fruiting body in a sterile beaker to avoid direct contact with air. On the clean bench, tear open the base of the Yanghuang fruiting body to expose the mycelium at the base. Use a sterilized scalpel to cut a small piece of tissue the size of a grain of rice from the inside of the base, making sure that the cut part is healthy and uncontaminated. Inoculate the removed tissue block into the mother culture medium, place it in a constant temperature box at 25°C for culture, and observe the growth. After the mycelium grows out, select the block with rapid growth and healthy mycelium, transplant it into another sterile mother culture medium, and continue to observe. After multiple transfers of the mother culture medium, the miscellaneous bacteria are effectively removed, and a healthy Yanghuang fungus mother culture ( Figure 1 ).

[0033] Example 2

[0034] Screening experiment of carbon and nitrogen sources in mother culture medium

[0035] Different sugar sources were used as nutrients, with 150–250 g / L potato and 15–20 g / L agar as the basic formula. Five carbon sources (15–25 g / L) and four nitrogen sources (1–5 g / L) were selected. The carbon source nutrients were sucrose, glucose, mannitol, maltose and lactose; the nitrogen sources were peptone, yeast extract, beef extract and ammonium chloride.

[0036] Different culture media were prepared, and the experiment was repeated three times for each nutrient to ensure data accuracy and reliability. A piece of Yanghuang fungus cake was removed with a sterile scalpel and inoculated into the center of each plate. The culture dish was covered and sealed with parafilm to ensure a sterile environment. The dish was then placed in a constant temperature incubator. Post-inoculation growth was recorded, and mycelial germination was observed. The results are shown in Tables 1 and 2.

[0037] Table 1 Growth results of mycelium under different carbon source conditions

[0038]

[0039] Table 2 Growth results of mycelium under different nitrogen source conditions

[0040]

[0041] Note: +++ indicates that the mycelium is growing very strong, thick and densely distributed; ++ indicates that the mycelium is growing relatively strong, thick and densely distributed; + indicates that the mycelium is growing relatively weak, not thick and sparsely distributed.

[0042] Result analysis: As shown in Table 1, the growth rate of mycelium showed significant differences in different carbon source culture media. The results showed that the mycelium grew fastest under the conditions of sucrose and maltose as carbon sources, and grew slowest under the condition of lactose.

[0043] As shown in Table 2, under the nitrogen source conditions of peptone and beef extract, the mycelium grew relatively fast, and under the condition of ammonium chloride, the mycelium grew the slowest.

[0044] Example 3

[0045] Screening of mother culture conditions

[0046] Screening conditions include: temperature experiment, light experiment, and pH experiment.

[0047] First, the above-mentioned mother culture medium is sterilized under high pressure at 121°C, cooled, and poured into a sterile flat plate culture dish, and allowed to solidify and dry. The culture medium required for the pH experiment needs to be pH-adjusted in advance with plant acid-base indicator solution.

[0048] Under a clean bench, use a sterile scalpel to remove a cake from the Yanghuang fungus strain and inoculate it in the center of the plate. Cover the petri dish and seal it with parafilm to ensure a sterile environment. Then, place it in the corresponding incubator. Each experiment was repeated three times to ensure the reliability of the results.

[0049] Temperature experiment: Five gradients of 22°C, 25°C, 28°C, 31°C and 34°C were set and cultured in incubators at different temperatures. The optimal temperature was determined by comparing the growth rate and quality of Yanghuang mycelium under different conditions (Table 3).

[0050] Lighting experiment: diffuse light, total darkness, and 12-hour light conditions were set respectively. By comparing the growth rates of Phellinus igniarius hyphae under different light conditions, the optimal light conditions were determined (Table 4).

[0051] pH experiment: Five different pH concentration gradients of 5.0, 6.0, 7.0, 8.0, and 9.0 were set, and the growth after inoculation was recorded and the germination of mycelium was observed (Table 5).

[0052] When the mycelium begins to germinate, use a marker to draw a starting line on the bottom of the plate. Regularly observe and record the progress of mycelium growth. When the mycelium grows rapidly and almost covers the entire culture dish, draw a termination line on the bottom of the plate. Measure the distance from the starting line to the termination line and record the growth rate of each repeated sample. Calculate the average value of mycelium growth rate under different temperatures, different light conditions, and different pH conditions, and compare the effects of the three different conditions on the growth of Yanghuang mycelium. The experimental results are shown in Tables 3, 4, and 5 (During the mycelium inoculation and observation process, try to minimize the time the culture dish is exposed to the air to reduce the risk of contamination by foreign bacteria. Each observation should be carried out in the same time period to ensure data consistency).

[0053] Table 3 Mycelial growth results under different temperature conditions

[0054]

[0055] Table 4 Mycelial growth results under different light conditions

[0056]

[0057] Note: +++ indicates that the mycelium is growing very strong, thick and densely distributed; ++ indicates that the mycelium is growing relatively strong, thick and densely distributed; + indicates that the mycelium is growing relatively weak, not thick and sparsely distributed.

[0058] Table 5 Mycelial growth results under different pH conditions

[0059]

[0060] Note: +++ indicates that the mycelium is growing very strong, thick and densely distributed; ++ indicates that the mycelium is growing relatively strong, thick and densely distributed; + indicates that the mycelium is growing relatively weak, not thick and sparsely distributed.

[0061] Result analysis: As shown in Table 3, the growth rate of mycelium showed significant differences in culture medium at different temperatures. The mycelium grew fastest when the temperature was between 25-28°C.

[0062] From the results in Table 4, we can see that the mycelium of Yanghuang can grow under three different light conditions, but the growth rate of the mycelium is relatively fast under the conditions of complete darkness and scattered light, and the growth rate is slow under the conditions of 12 hours of light.

[0063] From the results in Table 5, it can be seen that the mycelium can grow at a pH of 5.0-9.0. When the pH is 8.0, the mycelium grows relatively fast, and when the pH is 5.0, the mycelium grows the slowest.

[0064] Example 4

[0065] Stock culture medium screening experiment

[0066] Five different formulas were set up, and two repeated experiments were set up for each formula to ensure the reliability and accuracy of the experimental data. Rice husks, wheat grains, cottonseed hulls, straw, and corn cobs were used as the main ingredients. The five original formulas were:

[0067] Formula 1: 85% straw, 5% wheat bran, 5% rice husk, 5% oil residue;

[0068] Formula 2: 85% corn cobs, 5% wheat bran, 5% rice husks, 5% oil residue.

[0069] Formula 3: 85% rice husk, 5% wheat bran, 5% rice husk, 5% oil residue;

[0070] Formula 4: 85% cottonseed hulls, 5% wheat bran, 5% rice hulls, 5% oil residue;

[0071] Formula 5: 85% wheat grains, 5% wheat bran, 5% rice husks, 5% oil residue;

[0072] After the culture medium is prepared, it is stirred with water, the humidity is controlled within the range of 50-60%, and it is packaged in cans and placed in a high-pressure steam sterilizer for sterilization. Then, the mother strain of Yanghuang fungus prepared in Example 1 is inoculated on a clean bench. When the mycelium grows along the bottle wall, a line is drawn and the growth rate of each repeated sample is recorded. The effects of different formulas on the growth of Yanghuang fungus mycelium are compared. The experimental results are shown in Table 6. At the same time, the status of the original strain after 30 days of culture is shown in Table 6. Figure 2 shown.

[0073] Table 6 Growth results of mycelium under different stock formulation conditions

[0074]

[0075] Results analysis: As shown in Table 6, the mycelial growth rate of Yanghuang fungus in the cottonseed hull formula was the fastest, and the wheat grain and rice hull formula was the slowest;

[0076] Result analysis: Figure 2 The results showed that the yellow fuzzy primordia are in the early stages of mycelial growth transitioning to fruiting bodies. As can be seen from the figure, the rice husk primordia are relatively rare, with most still in the mycelial stage.

[0077] Furthermore, based on the mycelial growth rate and the primordium situation, the best formula cottonseed hull and the worst rice hull formula were selected for the next step of cultivation and fruiting experiment. The experimental results are as follows: Figure 3 shown.

[0078] Result analysis: 15 days after the cultivated species were inoculated, the mycelium of the cottonseed hull formula grew vigorously, densely, and rapidly; while the mycelium of the rice husk formula grew weakly, without vitality, and with a slow growth rate, and had a high contamination rate.

[0079] Example 5

[0080] Screening of stock culture conditions

[0081] After sterilizing the stock culture medium, the following temperature test, light test, and pH test were performed;

[0082] Temperature experiment: set four gradients of 22℃, 25℃, 26℃ and 28℃, and culture them in incubators at different temperatures. By comparing the growth rate and quality of Yanghuang mycelium under different conditions, the optimal temperature was determined. The experimental results showed that the most suitable temperature was 25-26℃.

[0083] Lighting experiment: set scattered light, total darkness, and 12-hour light conditions respectively. By comparing the growth rate of Phellinus igniarius mycelium under different lighting conditions, it was determined that the best lighting condition was total darkness.

[0084] pH experiment: Set the pH at five different concentration gradients of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, record the growth after inoculation, and observe the germination of mycelium. The experimental results show that the most suitable pH is 7.0-7.5.

[0085] Example 6

[0086] A method for cultivating the original species of Yanghuang mushroom comprises the following steps:

[0087] (1) Obtaining the mother culture of Yanghuang Fungus: Select healthy and fresh Yanghuang fruiting bodies, disinfect them with 75% alcohol for 5 times, cut small pieces of tissue, inoculate them into the mother culture medium (150g / L potato, 15g / L sucrose, 1g / L beef extract, 15g / L agar, add water to 1000mL), and culture them at a temperature of 25℃, in complete darkness, and under pH 5-9. After the mycelium grows, select the mycelium that grows rapidly and is healthy. After multiple transfers of the mother culture medium, the foreign bacteria are effectively removed to obtain a healthy Yanghuang Fungus mother culture;

[0088] (2) Cultivation of the stock culture of the Mycoplasma fusca: The Mycoplasma fusca mother culture obtained in step (1) was inoculated into the stock culture medium (85% straw, 5% wheat bran, 5% rice husk and 5% oil residue), and placed in a temperature of 25°C, completely dark, and pH 7.0 for stock culture.

[0089] Example 7

[0090] A method for cultivating the original species of Yanghuang mushroom comprises the following steps:

[0091] (1) Obtaining the mother culture of Yanghuang fungus: Select healthy and fresh Yanghuang fruiting bodies, disinfect them with 75% alcohol for 5 times, cut small pieces of tissue, inoculate them into the mother culture medium (250g / L potato, 25g / L carbon source maltose, 5g / L peptone, 20g / L agar, add water to 1000mL) and culture them at a temperature of 28℃, scattered light, and pH 9.0. After the mycelium grows, select the mycelium that grows rapidly and is healthy. After multiple transfers of the mother culture medium, the foreign bacteria are effectively removed to obtain a healthy Yanghuang fungus mother culture;

[0092] (2) Cultivation of the stock culture of the Mycoplasma fusca: The Mycoplasma fusca mother culture obtained in step (1) was inoculated into the stock culture medium (85% corn cobs, 5% wheat bran, 5% rice husks and 5% oil residue), and placed at a temperature of 26°C, completely dark, and pH 7.5 for stock culture.

[0093] Example 8

[0094] A method for cultivating the original species of Yanghuang mushroom comprises the following steps:

[0095] (1) Obtaining the mother culture of Yanghuang Fungus: Select healthy and fresh Yanghuang fruiting bodies, disinfect them with 75% alcohol for 5 times, cut small pieces of tissue, inoculate them into the mother culture medium (200g / L potato, 20g / L sucrose, 3g / L beef extract, 18g / L agar, add water to 1000mL) and culture them at a temperature of 28℃, in complete darkness, and with a pH of 8.0. After the mycelium grows out, select the mycelium that grows rapidly and is healthy. After multiple transfers of the mother culture medium, the foreign bacteria are effectively removed and a healthy Yanghuang Fungus mother culture is obtained. The mycelium grows on the mother culture medium for 30 days as shown in the figure. Figure 4 As shown, the yellow flocculent knot is the primordium of the yellow jasmine.

[0096] (2) Cultivation of the stock culture of the Mycoplasma fusca: The Mycoplasma fusca mother culture obtained in step (1) was inoculated into the stock culture medium (85% cottonseed hulls, 5% wheat bran, 5% rice hulls and 5% oil residue), and placed at a temperature of 26°C, completely dark, and pH 7.5 for stock culture.

[0097] Comparative Example 1

[0098] The operation is basically the same as that of Example 8, except that the culture pH in step (1) is 6.0.

[0099] Comparative Example 2

[0100] The operation is basically the same as that of Example 8, except that the main ingredient of the original seed culture medium in step (2) is cottonseed hulls instead of rice hulls.

[0101] Comparative Example 3

[0102] The operation is basically the same as that of Example 8, except that the original seed culture medium in step (2) does not contain oil residue, and the other components remain unchanged.

[0103] In order to verify the technical effects of Examples 6-8 and Comparative Examples 1-3, the following data measurement experiments were conducted, as shown in Table 7.

[0104] Table 7 Effects of different stock culture methods on mycelial growth

[0105]

[0106] Results analysis shows that, as shown in Table 7, the original seed culture methods of Examples 6-8 of the present invention have improved mycelial growth rate, mycelial diameter, mycelial polysaccharide content, flavonoid yield, and BMF dry weight to varying degrees compared with Comparative Examples 1-3. They also significantly reduced the contamination rate of foreign bacteria and the number of mycelial tip branches, thereby promoting the extension rate of mycelium.

[0107] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0108] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A culture medium for the stock of Yanghuang fungus, characterized in that: Includes one of cottonseed hulls, straw or corn cobs; also includes wheat bran, rice hulls and oilseed cakes.

2. The boxwood fungus stock culture medium according to claim 1, characterized in that: The proportions of the ingredients are as follows: 85% cottonseed hulls, 5% wheat bran, 5% rice hulls, and 5% oilseed residue; or 85% straw, 5% wheat bran, 5% rice husk and 5% oil residue; Or 85% corn cobs, 5% wheat bran, 5% rice husks and 5% oil residue.

3. A method for cultivating the original species of Yanghuang fungus, characterized in that: The steps include: (1) Obtaining the mother strain of Yanghuang fungus: Select healthy and fresh Yanghuang fruiting bodies, cut small pieces of tissue after disinfection, and inoculate them into the mother strain culture medium for cultivation. After the mycelium grows, select the mycelium that grows rapidly and is healthy. After multiple transfers of the mother strain culture medium, the foreign bacteria are effectively removed and a healthy Yanghuang fungus mother strain is obtained; (2) Cultivation of the mother culture of Boxwood fungus: The mother culture of Boxwood fungus obtained in step (1) is inoculated into the Boxwood fungus stock culture medium according to claim 1 or 2 for stock culture.

4. The method for cultivating the original species of Yanghuang fungus according to claim 3, characterized in that: The disinfection in step (1) is carried out using 75% alcohol, and the number of disinfections is ≥5 times.

5. The method for cultivating the original species of Yanghuang fungus according to claim 3, characterized in that: The mother culture medium in step (1) is based on potato and agar, and also includes a carbon source, a nitrogen source and water; the weight ratio of each component is as follows: 150-250g / L potato, 15-25g / L carbon source, 1-5g / L nitrogen source, 15-20g / L agar, and water is added to make up to 1000mL.

6. The method for cultivating the original species of Yanghuang fungus according to claim 5, characterized in that: The carbon source is sucrose and / or maltose; the nitrogen source is beef extract and / or peptone.

7. The method for cultivating the original species of Yanghuang fungus according to claim 3, characterized in that: The culture temperature of the mother culture medium is 25-28° C., the lighting condition is complete darkness or scattered light, and the culture pH is 5-9.

8. The method for cultivating the original species of Yanghuang fungus according to claim 7, characterized in that: The mother culture medium is cultured at a temperature of 28° C., a lighting condition of complete darkness, and a culture pH of 8.

9. The method for cultivating the original species of Yanghuang fungus according to claim 3, characterized in that: The temperature of the stock culture is 25-26° C., the lighting condition is completely dark, and the culture pH is 7.0-7.5.

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