A method of liquid culture of armillaria fruit bodies, cortices and rhizomorphs
By using liquid culture medium preparation and variable temperature cultivation, the limitations of materials and long cycle in solid culture of Armillaria mellea have been solved, enabling efficient and low-cost production of Armillaria mellea fruiting bodies, mycelium, and mycelial cords, which is suitable for industrial applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANHUI UNIV
- Filing Date
- 2024-04-26
- Publication Date
- 2026-07-03
AI Technical Summary
Solid-state cultivation of Armillaria mellea is limited by raw materials, has a long cycle, is greatly affected by natural conditions, and is difficult to dispose of waste. Liquid fermentation equipment requires large investments, and there is a lack of economical, simple, and efficient cultivation techniques.
The method of liquid culture medium preparation, floating inoculation, variable temperature culture and oxygenation treatment is adopted. Food raw materials such as flour, corn flour and rice flour are used to carry out liquid culture of Armillaria mellea by wide-mouth glass bottles and double-layer newspaper wrapping. This promotes the growth of mycelial cords and the formation of mycelial colony. The culture conditions are controlled to realize the industrial production of Armillaria mellea fruiting bodies, mycelial colony and mycelial cords.
This method enables the efficient production of Armillaria mellea fruiting bodies, mycelium, and mycelial cords, shortening the cycle, reducing costs, avoiding the problem of waste substrate disposal, and improving biological efficiency and economic benefits.
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Figure CN118303269B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of edible and medicinal fungi cultivation technology, specifically relating to a method for liquid culture of Armillaria mellea fruiting bodies, mycelial vegetation, and mycelial cords. Background Technology
[0002] Armillaria mellea, belonging to the subphylum Basidiomycota, class Agaricomycetes, order Agaricales, family Tricholomataceae, and genus Armillaria, is a fungus that grows alongside the traditional Chinese medicines Gastrodia elata and Polyporus umbellatus. Rich in nutrients, Armillaria mellea is classified as a first-class food in some developed countries. It is also a valuable medicinal fungus, with similar therapeutic effects to Gastrodia elata in treating dizziness caused by hypertension, vertebrobasilar insufficiency, Meniere's disease, and autonomic nervous system dysfunction. It also has some therapeutic effects on limb numbness, insomnia, tinnitus, and post-stroke sequelae. Furthermore, it has protective and sedative effects on the brain.
[0003] Currently, the cultivation of Armillaria mellea primarily continues to rely on solid substrates such as wood and crop straw. This method presents numerous technical challenges, including limited raw materials, a long cultivation cycle, significant susceptibility to natural conditions, difficulty in fruiting, and the disposal of substrate waste. While liquid fermentation can produce Armillaria mellea mycelium, the equipment investment is substantial. Therefore, finding an economical, convenient, efficient, and feasible cultivation technique is an urgent problem to be solved. Summary of the Invention
[0004] To address the problems existing in current technologies, this invention, based on the research group's previous work on "Cultivation Methods of Armillaria mellea Fungi," focuses on operability, simplicity, controllable conditions, and the ability to achieve industrial-scale liquid cultivation of Armillaria mellea fruiting bodies, colonies, and mycelial cords. Through repeated research, a method for liquid cultivation of Armillaria mellea fruiting bodies, colonies, and mycelial cords has been provided. This method is simple to operate, has low production costs, controllable conditions, a short production cycle, and is easy to implement for industrial production, exhibiting significant application advantages.
[0005] The method for liquid culture of Armillaria mellea fruiting bodies, mycelial colonies, and mycelial cords of the present invention includes the following steps:
[0006] Step 1: Preparation of liquid culture medium
[0007] Using food ingredients, add water to 1L, boil for 8-12 minutes, and dispense while hot to obtain liquid culture medium; the mass concentration of food ingredients in the liquid culture medium is 5%;
[0008] Step 2: Dispensing and sterilization of liquid culture medium
[0009] Dispense the liquid culture medium prepared in step 1 into wide-mouth glass culture bottles, tighten the caps, ensure the pore diameter is 0.5cm, cover with newspaper and wrap tightly, sterilize at 105℃ for 4-5 hours, and set aside for later use.
[0010] Step 3: Floating Inoculation
[0011] After the liquid culture medium is cooled to room temperature, it is inoculated with Armillaria mellea corn cob culture under aseptic conditions;
[0012] Step 4: Promote bacterial culture
[0013] After culturing at 25℃ for 30-40 days, once the mycelial cords have fully colonized the bottle, remove the wrapping paper to increase air permeability. Continue culturing at 27℃ for 5 days to promote the formation of a brown mycelial colony (thick mycelial skin). Then, cool the bottle to 20℃ and culture for 7 days to reach physiological maturity and promote the transition from vegetative growth to reproductive growth.
[0014] Step 5: Oxygenation and variable temperature incubation
[0015] Loosen the bottle cap to increase oxygen, and culture at varying temperatures under light conditions to promote the differentiation of Armillaria mellea fruiting body primordia, the formation of white dot-like primordia, and their gradual development.
[0016] Step 6: Management of Fruiting Body Development
[0017] When the fruiting bodies grow to about 1 cm, remove the bottle cap and, under good ventilation conditions, irradiate with diffused light at a temperature of 17℃-19℃ and a humidity of 80%-90%, cultivate for 14-16 days to harvest the fruiting bodies, mycelium, and mycelial cords of Armillaria mellea.
[0018] In step 1, the food ingredients are selected from one or more of wheat flour, corn flour, and rice flour, with a total mass of 50g. This invention uses wheat flour, corn flour, and rice flour to prepare a liquid culture medium for cultivating Armillaria mellea fruiting bodies, mycelial colonies, and mycelial cords, replacing the use of solid culture media made from resource-rich materials such as wood. This not only solves the problem of protecting natural resources but also eliminates the issue of post-processing waste culture media.
[0019] In step 2, 500 mL of liquid culture medium is dispensed into each 550 mL wide-mouth glass culture bottle. This invention uses wide-mouth glass bottles, which facilitates the growth of Armillaria mellea fruiting bodies from the bottle opening. The transparency of the glass also aids in observation. Filling each 550 mL wide-mouth glass bottle with 500 mL of liquid culture medium leaves sufficient space for oxygen exchange during the mycelial growth phase of Armillaria mellea. The bottle cap is wrapped with double layers of newspaper to enhance aseptic effect and increase permeability during temperature incubation, promoting the formation of the mycelial colony (thick mycelial skin).
[0020] In step 3, the *Armillaria mellea* corn cob spawn floats on the surface of the liquid culture medium. Inoculating the *Armillaria mellea* corn cob spawn onto the surface of the liquid culture medium and allowing it to float enables the *Armillaria mellea* mycelia to transport oxygen, promoting their growth into the liquid to obtain nutrients until the mycelia completely fill the bottle. Furthermore, the fact that the *Armillaria mellea* corn cob spawn floats on the surface of the liquid culture medium after inoculation solves the problem of mycelia that sink in the liquid and cannot grow under static conditions.
[0021] Furthermore, the Armillaria mellea corn cob cultivar is prepared by the following method:
[0022] Fresh corn cobs are sliced into thin slices of 0.5-1.0 cm, and water is added to a moisture content of 60%-65%. The slices are then placed in wide-mouthed bottles, plugged with cotton, and sealed tightly. The bottles are then sterilized at 121℃ for 2-3 hours. After cooling to room temperature, 10-20 mL of a conventional Armillaria mellea liquid inoculum is inoculated under aseptic conditions. The inoculum is then cultured at 25℃ for 14-16 days until it is fully colonized with mycelium, yielding the Armillaria mellea corn cob culture medium. The corn cobs used for the inoculum are sliced, with one slice used per bottle. Using corn cob slice culture medium solves both the nutritional and floating issues of the culture medium. Furthermore, corn cobs are non-toxic and edible, avoiding the introduction of harmful substances. The preparation of the corn cob culture medium is also simple and easy.
[0023] Not all edible fungi can be liquid cultured. Only edible fungi that have formed mycelial cords can be cultivated in liquid culture medium. The formation of colonies and thick mycelial coverings is the prerequisite and foundation for liquid suspension culture.
[0024] In step 4, the wrapping paper is removed to increase air permeability, and the temperature is raised to 27°C to promote the formation of a brown mycelial colony (thick mycelial skin) on the surface of the liquid culture medium, enhancing its resistance, especially its ability to resist other microorganisms. Then, the temperature is lowered to 20°C and cultured for 7 days to reach physiological maturity, promoting the transition from vegetative growth to reproductive growth, which is more conducive to the formation of fruiting body primordia on the mycelial colony and the development of fruiting bodies.
[0025] In step 5, the temperature variation refers to incubation at temperature ranges of 8℃-10℃ and 17℃-19℃, with 12-hour intervals. The light intensity is 300-400 LUX.
[0026] The cultivation method employed in this invention shortens the exposure time of the mycelium, reduces contamination by other microorganisms, increases aeration, and promotes the differentiation, formation, and development of *Armillaria mellea* fruiting body primordia. *Armillaria mellea* fruiting body development requires sufficient oxygen and exhibits a significant growth advantage. Removing the bottle cap and providing suitable temperature and humidity under good ventilation and diffused light conditions is beneficial to the growth and development of *Armillaria mellea* fruiting bodies. After the fruiting bodies mature, the three products of *Armillaria mellea*—the fruiting bodies, the mycelium, and the mycelial cords—can be harvested. A single cultivation can yield three or even four products simultaneously, making this cultivation method achieve unparalleled biological efficiency compared to other methods. It produces no waste and results in higher yields and economic benefits. Attached Figure Description
[0027] Figure 1 : A wide-mouthed glass bottle with a breathable cap.
[0028] Figure 2 Armillaria mellea corn cob culture.
[0029] Figure 3Floating inoculation.
[0030] Figure 4 The mycelial cords that cover the entire bottle and the mycelial covering (thick mycelial skin) formed on the surface.
[0031] Figure 5 The development process of the fruiting body.
[0032] Figure 6 : The fruiting body, mycelium, and mycelial cords of Armillaria mellea. Detailed Implementation
[0033] The present invention will be further described below with reference to specific embodiments. It should be understood that the examples are not intended to limit the scope of protection of the present invention.
[0034] Example 1:
[0035] 1. Preparation of Armillaria mellea corn cob culture
[0036] 1.1 Culture medium formula: Cut fresh corn cobs into thin slices of 0.5-1.0 cm and add water to a moisture content of 60%-65%.
[0037] 1.2 Culture medium preparation: Take corn cob slices, adjust the moisture content to 60%-65%, put them into wide-mouth bottles, plug them with cotton plugs, wrap them tightly, and sterilize at 121℃ for 2-3 hours.
[0038] 1.3 Inoculation: After cooling to room temperature, inoculate 15 ml of routinely produced Armillaria mellea liquid culture under aseptic conditions. The culture can be commercially available or isolated using conventional methods.
[0039] 1.4 Cultivation: Cultivate in strips at 25℃ for about 15 days until the mycelium is fully grown to obtain the Armillaria mellea corn cob spawn for later use.
[0040] 2. The process of liquid culture of Armillaria mellea trisomy
[0041] 2.1 Liquid culture medium
[0042] 2.1.1 Formula for liquid culture medium: 2.5 wt% corn flour, 2.5 wt% wheat flour, water to 1 L.
[0043] 2.1.2 Preparation of liquid culture medium: Weigh 25g of corn flour and 25g of wheat flour according to the ratio, and make up to 1L with tap water. Boil for about 10 minutes.
[0044] 2.1.3 Dispensing, wrapping, and sterilization of liquid culture medium: Dispense the liquid culture medium into 550ml wide-mouth glass culture bottles, tighten the caps (0.5cm diameter pores), cover with double layers of newspaper, wrap tightly, and sterilize at 105℃ for 4-5 hours before use.
[0045] 2.2 Floating inoculation: Cool the culture medium to room temperature and inoculate Armillaria mellea corn cob culture onto the surface of the liquid under aseptic conditions.
[0046] 2.3 Cultivation: Cultivate at 25℃ for about 30 to 40 days. After the mycelial cords have filled the bottle, remove the wrapping paper to increase air permeability. Continue cultivation at 27℃ for 5 days to promote the formation of brown mycelial hood (thick mycelial skin). Then, cool down to 20℃ and cultivate for 7 days to reach physiological maturity and promote the transition from vegetative growth to reproductive growth.
[0047] 2.4 Loosen the bottle cap, add oxygen, and alternate temperature and light: culture at 12-hour intervals at 8℃~10℃ and 17℃~19℃ with light (300-400LUX) to promote the differentiation of Armillaria mellea fruiting body primordia, the formation of white dot-like primordia, and their gradual development.
[0048] 2.5 Fruiting body development management: When the fruiting bodies grow to about 1 cm, remove the bottle cap and expose them to diffused light under good ventilation conditions, with a temperature of 17℃~19℃ and humidity of 80%~90%. Usually, the fruiting bodies, mycelium and mycelial cords of Armillaria mellea can be harvested in about 15 days.
[0049] Example 2:
[0050] 1. Preparation of Armillaria mellea corn cob culture
[0051] 1.1 Culture medium formulation: Fresh corn cobs are cut into 0.5–1.0 cm thin slices. Add water to a moisture content of 60%–65%.
[0052] 1.2 Culture medium preparation: Take corn cob slices, adjust the moisture content to 60%-65%, put them into wide-mouth bottles, plug them with cotton plugs, wrap them tightly, and sterilize at 121℃ for 2-3 hours.
[0053] 1.3 Inoculation: Cool to room temperature and inoculate with 15 ml of conventionally produced Armillaria mellea liquid culture under aseptic conditions.
[0054] 1.4 Cultivation: Cultivate at 25℃ for about 15 days until the mycelium is fully grown to obtain the Armillaria mellea corn cob culture medium for later use.
[0055] 2. The process of liquid culture of Armillaria mellea trisomy
[0056] 2.1 Liquid culture medium
[0057] 2.1.1 Formula for liquid culture medium: 2.5 wt% corn flour, 2.5 wt% rice flour, water added to 1 L.
[0058] 2.1.2 Preparation of liquid culture medium: Weigh 25g of corn flour and 25g of rice flour according to the ratio, and make up to 1L with tap water. Boil for about 10 minutes.
[0059] 2.1.3 Dispensing, wrapping, and sterilization of liquid culture medium: Dispense the liquid culture medium into 550ml wide-mouth glass culture bottles, tighten the caps (0.5cm diameter pores), cover with double layers of newspaper, wrap tightly, and sterilize at 105℃ for 4-5 hours before use.
[0060] 2.2 Floating inoculation: Cool the culture medium to room temperature and inoculate Armillaria mellea corn cob culture onto the surface of the liquid under aseptic conditions.
[0061] 2.3 Cultivation: Cultivate at 25℃ for about 30 to 40 days. After the mycelial cords have filled the bottle, remove the wrapping paper to increase air permeability. Continue cultivation at 27℃ for 5 days to promote the formation of brown mycelial hood (thick mycelial skin). Then, cool down to 20℃ and cultivate for 7 days to reach physiological maturity and promote the transition from vegetative growth to reproductive growth.
[0062] 2.4 Loosen the bottle cap, add oxygen, and alternate temperature and light: culture at 12-hour intervals at 8℃~10℃ and 17℃~19℃ with light (300-400LUX) to promote the differentiation of Armillaria mellea fruiting body primordia, the formation of white dot-like primordia, and their gradual development.
[0063] 2.5 Fruiting body development management: When the fruiting bodies grow to about 1 cm, remove the bottle cap and expose them to diffused light under good ventilation conditions, with a temperature of 17℃~19℃ and humidity of 80%~90%. Usually, the fruiting bodies, mycelium and mycelial cords of Armillaria mellea can be harvested in about 15 days.
[0064] Example 3:
[0065] 1. Preparation of Armillaria mellea corn cob culture
[0066] 1.1 Culture medium formulation: Fresh corn cobs are cut into 0.5–1.0 cm thin slices. Add water to a moisture content of 60%–65%.
[0067] 1.2 Culture medium preparation: Take corn cob slices, adjust the moisture content to 60%-65%, put them into wide-mouth bottles, plug them with cotton plugs, wrap them tightly, and sterilize at 121℃ for 2-3 hours.
[0068] 1.3 Inoculation: Cool to room temperature and inoculate with 15 ml of conventionally produced Armillaria mellea liquid culture under aseptic conditions.
[0069] 1.4 Cultivation: Cultivate at 25℃ for about 15 days until the mycelium is fully grown to obtain the Armillaria mellea corn cob culture medium for later use.
[0070] 2. The process of liquid culture of Armillaria mellea trisomy
[0071] 2.1 Liquid culture medium
[0072] 2.1.1 Formula for liquid culture medium: 2.5 wt% rice flour, 2.5 wt% wheat flour, water added to 1 L.
[0073] 2.1.2 Preparation of liquid culture medium: Weigh 25g of rice flour and 25g of wheat flour according to the ratio, and make up to 1L with tap water. Boil for about 10 minutes.
[0074] 2.1.3 Dispensing, wrapping, and sterilization of liquid culture medium: Dispense the liquid culture medium into 550ml wide-mouth glass culture bottles, tighten the caps (0.5cm diameter pores), cover with double layers of newspaper, wrap tightly, and sterilize at 105℃ for 4-5 hours before use.
[0075] 2.2 Floating inoculation: Cool the culture medium to room temperature and inoculate Armillaria mellea corn cob culture onto the surface of the liquid under aseptic conditions.
[0076] 2.3 Cultivation: Cultivate at 25℃ for about 30 to 40 days. After the mycelial cords have filled the bottle, remove the wrapping paper to increase air permeability. Continue cultivation at 27℃ for 5 days to promote the formation of brown mycelial hood (thick mycelial skin). Then, cool down to 20℃ and cultivate for 7 days to reach physiological maturity and promote the transition from vegetative growth to reproductive growth.
[0077] 2.4 Loosen the bottle cap, add oxygen, and alternate temperature and light: culture at 12-hour intervals at 8℃~10℃ and 17℃~19℃ with light (300-400LUX) to promote the differentiation of Armillaria mellea fruiting body primordia, the formation of white dot-like primordia, and their gradual development.
[0078] 2.5 Fruiting body development management: When the fruiting bodies grow to about 1 cm, remove the bottle cap and expose them to diffused light under good ventilation conditions, with a temperature of 17℃~19℃ and humidity of 80%~90%. Usually, the fruiting bodies, mycelium and mycelial cords of Armillaria mellea can be harvested in about 15 days.
[0079] Example 4:
[0080] 1. Preparation of Armillaria mellea corn cob culture
[0081] 1.1 Culture medium formulation: Fresh corn cobs are cut into 0.5–1.0 cm thin slices. Add water to a moisture content of 60%–65%.
[0082] 1.2 Culture medium preparation: Take corn cob slices, adjust the moisture content to 60%-65%, put them into wide-mouth bottles, plug them with cotton plugs, wrap them tightly, and sterilize at 121℃ for 2-3 hours.
[0083] 1.3 Inoculation: Cool to room temperature and inoculate with 15 ml of conventionally produced Armillaria mellea liquid culture under aseptic conditions.
[0084] 1.4 Cultivation: Cultivate at 25℃ for about 15 days until the mycelium is fully grown to obtain the Armillaria mellea corn cob culture medium for later use.
[0085] 2. The process of liquid culture of Armillaria mellea trisomy
[0086] 2.1 Liquid culture medium
[0087] 2.1.1 Formula for liquid culture medium: Mix equal amounts of corn flour, wheat flour and rice flour, take 50g of the mixture and add water to 1L.
[0088] 2.1.2 Preparation of liquid culture medium: Weigh 50g of an equal mixture of corn flour, wheat flour, and rice flour, and dilute to 1L with tap water. Boil for about 10 minutes.
[0089] 2.1.3 Dispensing, wrapping, and sterilization of liquid culture medium: Dispense the liquid culture medium into 550ml wide-mouth glass culture bottles, tighten the cap (0.5 cm pore diameter), cover with double layers of newspaper, wrap tightly, and sterilize at 105℃ for 4-5 hours before use.
[0090] 2.2 Floating inoculation: Cool the culture medium to room temperature and inoculate Armillaria mellea corn cob culture onto the surface of the liquid under aseptic conditions.
[0091] 2.3 Cultivation: Cultivate at 25℃ for about 30 to 40 days. After the mycelial cords have filled the bottle, remove the wrapping paper to increase air permeability. Continue cultivation at 27℃ for 5 days to promote the formation of brown mycelial hood (thick mycelial skin). Then, cool down to 20℃ and cultivate for 7 days to reach physiological maturity and promote the transition from vegetative growth to reproductive growth.
[0092] 2.4 Loosen the bottle cap, add oxygen, and alternate temperature and light: culture at 12-hour intervals at 8℃~10℃ and 17℃~19℃ with light (300-400LUX) to promote the differentiation of Armillaria mellea fruiting body primordia, the formation of white dot-like primordia, and their gradual development.
[0093] 2.5 Fruiting body development management: When the fruiting bodies grow to about 1 cm, remove the bottle cap and expose them to diffused light under good ventilation conditions, with a temperature of 17℃~19℃ and humidity of 80%~90%. Usually, the fruiting bodies, mycelium and mycelial cords of Armillaria mellea can be harvested in about 15 days.
Claims
1. A method for liquid culture of Armillaria mellea fruiting bodies, mycelial colonies, and mycelial cords, characterized in that... Includes the following steps: Step 1: Preparation of liquid culture medium Using food-grade ingredients, add water to 1L and boil for 8-12 minutes to obtain a liquid culture medium; Step 2: Dispensing and sterilization of liquid culture medium Dispense the liquid culture medium prepared in step 1 into wide-mouth glass culture bottles, tighten the caps, ensure the pore diameter is 0.5cm, cover with newspaper and wrap tightly, sterilize at 105℃ for 4-5 hours, and set aside for later use. Step 3: Floating Inoculation After the liquid culture medium is cooled to room temperature, the Armillaria mellea corn cob cultivar is inoculated onto the surface of the liquid culture medium under aseptic conditions and allowed to float on the surface. The Armillaria mellea corn cob culture was prepared by the following method: Cut fresh corn cobs into 0.5-1.0cm thin slices, add water to a moisture content of 60%-65%, pack into wide-mouth bottles, plug with cotton, seal tightly, and sterilize at 121℃ for 2-3 hours; cool to room temperature, inoculate with 10-20mL of conventional Armillaria mellea liquid inoculum under aseptic conditions, and culture at 25℃ for 14-16 days until mycelium is fully grown, thus obtaining Armillaria mellea corn cob cultivar; Step 4: Promote bacterial culture After culturing at 25℃ for 30-40 days, once the mycelial cords have fully colonized the bottle, remove the wrapping paper to increase air permeability. Continue culturing at 27℃ for 5 days to promote the formation of brown mycelial colony. Then, cool the bottle to 20℃ and culture for 7 days to reach physiological maturity and promote the transition from vegetative growth to reproductive growth. Step 5: Oxygenation and variable temperature incubation Loosen the bottle cap to increase oxygen, and culture under light conditions at varying temperatures to promote the differentiation of Armillaria mellea fruiting body primordia, the formation of white dot-like primordia, and their gradual development; the varying temperatures are 8℃-10℃ and 17℃-19℃, with 12-hour intervals; the light intensity is 300-400 LUX. Step 6: Management of Fruiting Body Development When the fruiting bodies grow to about 1 cm, remove the bottle cap and, under good ventilation conditions, irradiate with diffused light, at a temperature of 17℃-19℃ and a humidity of 80%-90%, cultivate for 14-16 days to harvest the fruiting bodies, mycelium, and mycelial cords of Armillaria mellea. In step 1, the food ingredients are selected from one or more of flour, corn flour, and rice flour; the mass concentration of the food ingredients in the liquid culture medium is 5%.
2. The method according to claim 1, characterized in that: In step 2, 500 mL of liquid culture medium is dispensed into each 550 mL wide-mouth glass culture bottle.
3. The method according to claim 1, characterized in that: In step 4, the wrapping paper is removed to increase air permeability, and the temperature is raised to 27°C to promote the formation of brown mycelial colonies on the surface of the liquid culture medium, thereby enhancing stress resistance. Then, the temperature is lowered to 20°C and cultured for 7 days to reach physiological maturity, promoting the transition from vegetative growth to reproductive growth, which is more conducive to the formation of fruiting body primordia on the mycelial colony and the development of fruiting bodies.