A soilless cultivation method for *Bacillus globosum*
By using soilless cultivation methods, with oak sawdust and wheat bran as the main culture medium, combined with liquid inoculum and automated operation, the biosafety and resource waste problems of industrialized production of *Gymnocypris edulis* have been solved, and efficient and stable production of *Gymnocypris edulis* has been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI MEDICAL COLLEGE
- Filing Date
- 2024-11-26
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies make it difficult to achieve the industrialized production of *Bacillus subtilis*, and field cultivation with soil covering poses biosafety risks and resource waste.
Soilless cultivation was adopted, using oak sawdust and wheat bran as the main culture medium raw materials, with the addition of appropriate amounts of corn flour, inorganic salts, etc. Through liquid inoculation and automated operation, the culture conditions were controlled to achieve efficient growth of the bright fungus and germination of fruiting bodies.
It has achieved stable and efficient production of luminescent fungi, avoided biological hazards caused by soil cultivation, reduced production costs, facilitated industrial-scale operation, and increased yield and profits.
Abstract
Description
Technical Field
[0001] This invention relates to the field of luminescent fungus cultivation technology, and more particularly to a soilless cultivation method for luminescent fungus. Background Technology
[0002] Armillaria mellea, also known as false honey fungus, belongs to the Tricholomataceae family and the Armillaria genus. It is a fungus used for both medicinal and culinary purposes. Market demand is high, but wild Armillaria mellea grows only in specific regions and seasons, and its yield is unstable, failing to meet market demand. Furthermore, due to the unknown conditions for fruiting body germination, it is difficult to cultivate artificially.
[0003] Glechomycosis fungus can parasitize tree roots, causing root rot and leading to tree death, which can be quite destructive to economic forests. Chinese patent "A Cultivation Method for Glechomycosis Fungus" (CN 110063201 A) discloses an outdoor raised bed cultivation method with soil covering, but this method requires arable land, complex culture medium conditions, and a growth promoter solution. Furthermore, field soil covering cultivation can lead to Glechomycosis infection of wild trees, posing a biosafety risk.
[0004] Technical problems to be solved Establish a soilless cultivation method for *Bleucanthus urinaria* to achieve industrialized production. Waste culture materials can be centrally composted for treatment, and preventative measures should be taken against *Bleucanthus urinaria* diseases.
[0005] Screening of raw materials and formulations for the cultivation medium of *Gymnocypris edulis* fruiting bodies was conducted to obtain suitable carbon and nitrogen sources, as well as inorganic salt combinations, for the growth of *Gymnocypris edulis* and the germination of fruiting bodies. The amount of growth promoters used or their components were reduced.
[0006] Reduce industrial production costs. This can be achieved through simplified operating procedures, the establishment of production methods suitable for automation, and a reduction in the types of materials used, thus enabling the easy production of *Gymnocypris edulis* fruiting bodies. Summary of the Invention
[0007] This invention proposes a soilless cultivation method for *Bacillus lucida* to solve the problems mentioned in the background section.
[0008] This invention proposes a soilless cultivation method for *Bleucobacterium brevicornu*, comprising: the composition and preparation of the cultivation medium, the preparation of liquid inoculum of *Bleucobacterium brevicornu*, the inoculation method of the cultivation medium, mycelial culture, and fruiting body culture.
[0009] Composition and preparation of cultivation medium The cultivation medium uses oak sawdust as the main carbon source and provides the necessary minerals and trace elements for the growth of *Gymnocypris edulis*, making it a key material for soilless cultivation. Wheat bran is the main nitrogen source and provides some carbon and minerals. Wheat bran is one of the important substances promoting the germination and growth of *Gymnocypris edulis* fruiting bodies. By weight, at least 50 parts wheat bran should be added to 100 parts oak sawdust to promote rapid and abundant germination of *Gymnocypris edulis*. The maximum amount of wheat bran added can be 100 parts. Corn flour, as a supplementary carbon source, provides basic nutrients for the growth of *Gymnocypris edulis* and can increase the yield of fruiting bodies, resulting in larger individual fruiting bodies. 10-50 parts corn flour can be added to 100 parts oak sawdust, generally 15-30 parts.
[0010] Generally, the carbon and nitrogen source formulation for *Gymnocypris edulis* culture medium, by weight, consists of 100 parts of oak sawdust, 50-80 parts of wheat bran, and 15-30 parts of corn flour, which is sufficient to achieve the germination of *Gymnocypris edulis* fruiting bodies under hydroponics. In this invention, the weight ratio of oak sawdust, corn flour, and wheat bran to the total weight of carbon and nitrogen sources in the culture medium is greater than or equal to 80%, or greater than or equal to 90%. The proportion of oak sawdust to the total weight of carbon and nitrogen sources is greater than or equal to 50%. In a specific embodiment, the proportion of oak sawdust to the total weight of carbon and nitrogen sources is equal to 52%.
[0011] Other carbon or nitrogen sources can be added to the culture medium. Generally, adding appropriate amounts of monosaccharides or disaccharides can accelerate the growth rate of *Gymnocladus orientalis* mycelium. Glucose or sucrose is commonly used. The content of monosaccharides or disaccharides is less than 4%. 0-6 parts of glucose are added to 100 parts of *Quercus glauca* sawdust, generally 2-5 parts. In one specific example, 4 parts were added.
[0012] To further accelerate mycelial and fruiting body growth, inorganic salts, also known as minerals, are added. These are mainly calcium, magnesium, and potassium ions, typically selected from magnesium sulfate, potassium dihydrogen phosphate, and gypsum. In 100 parts of oak sawdust, 0.2-0.6 parts magnesium sulfate, 0.3-0.6 parts potassium dihydrogen phosphate, and 1.5-3 parts gypsum can be added.
[0013] In one specific embodiment, by weight, there are 100 parts of oak sawdust, 50-80 parts of wheat bran, 15-30 parts of corn flour, 2-5 parts of glucose, 0.2-0.6 parts of magnesium sulfate, 0.3-0.6 parts of potassium dihydrogen phosphate, and 1.5-3 parts of gypsum.
[0014] The cultivation medium is prepared by mixing the above formula evenly and adding water until the medium is fully moistened. The water content of the medium should be 50-70% by weight, with 55-65% being optimal. That is, add 150 parts water to every 100 parts of the medium.
[0015] Dispense the fully moistened culture medium into 500-1000g bags. Compact the medium, insert the inoculation punch, seal, and cap. Sterilize at 115-125℃ for 1-4 hours. To save energy, 121℃ for 1 hour is generally sufficient to effectively kill contaminating microorganisms, or the level of contaminating microorganisms will not affect the growth of fruiting bodies and mycelium.
[0016] To improve the yield and growth rate of *Gymnocladus orientalis* fruiting bodies, the *Quercus glauca* wood chips used in this invention are sawdust-like, with a particle size of 0.8 mm to 3 mm, preferably 1 mm to 2 mm. The preferred *Quercus glauca* wood chips contain both xylem and phloem. The *Quercus glauca* wood chips used in this invention are well-rotted. The rottenness method is well known in the art.
[0017] When inoculating the culture medium of this invention, the *Bleucobacterium* strain can be mycelium cultured in a solid medium or a liquid medium. Methods for culturing *Bleucobacterium* in solid or liquid media are well known in the art. For ease of automation, in one specific embodiment, the mycelium cultured in liquid media is inoculated into the cultivation medium, referred to as *Bleucobacterium* liquid strain. Liquid strains have shorter culture times and easier-to-control fermentation conditions.
[0018] There are many formulations for liquid culture media: for example, PDB medium and corn flour and sweet potato flour mixed medium are well known in the field. For example, to prepare PDB liquid medium, peel and cut potatoes (200g / L) into pieces, boil for half an hour, filter with gauze, take the supernatant, add 2% glucose, and sterilize at 121℃ for 15-20 minutes.
[0019] The liquid culture is prepared by inoculating solid *Gymnocypris lucida* culture medium into a liquid culture medium. Specifically, activated solid *Gymnocypris lucida* blocks, approximately 0.5-2 cm in length, are aseptically inoculated into the liquid culture medium. The preferred inoculation ratio of solid blocks to liquid culture medium is 3-6 blocks, with a culture medium volume of 90-250 ml. The incubation temperature is 24-28℃, and the incubation time is 10-16 days, followed by static incubation. In one specific embodiment, 5 solid blocks are inoculated into 100 ml of liquid culture medium, incubated at 26℃ for 14 days, followed by static incubation. The *Gymnocypris lucida* mycelium in the liquid culture medium is then collected to obtain the liquid culture.
[0020] In one embodiment of the present invention, the inoculated culture medium is a liquid inoculum of *Gymnocypris edulis*. The collected *Gymnocypris edulis* mycelium is cut using a magnetic stirrer to form mycelial fragments approximately 0.5-2 cm in length. These mycelial fragments constitute the liquid inoculum. The liquid inoculum may contain liquid culture medium, the volume of which should be suitable for inoculation. For example, if the volume of liquid inoculum used for each inoculation is 5 ml, then that volume should contain at least one or more mycelial fragments.
[0021] Inoculation method of cultivation medium The inoculation of *Bleucobacterium brevicornu* liquid spawn into the culture medium of this invention is carried out in a sterile environment. The opening of the pre-sterilized culture medium bag is opened, and the aerated inoculation punch is removed. The *Bleucobacterium brevicornu* liquid spawn, along with the liquid culture medium, is inoculated into the culture medium. Mixing with the liquid culture medium facilitates the dispersion of the spawn in the culture medium and promotes mycelial growth. The preferred inoculation amount of the liquid spawn is 2%-10% of the culture medium, more preferably 5%-7%. The bag is then sealed, capped, and incubated.
[0022] Mycelial Culture In the mycelial culture stage described in this invention, the culture temperature is 20-30℃, preferably 24-28℃, more preferably 26℃; the culture humidity is preferably 40%-70%, more preferably 50%-60%; the culture is conducted in the dark; and the culture time is preferably 30-50 days. Mycelial culture is complete when the white mycelium covers more than 90% of the surface area of the mycelium substrate. Preferably, the mycelium covers more than 95% of the surface area of the mycelium substrate, and the culture time is 48-50 days.
[0023] Fruiting body cultivation stage The fruiting body cultivation stage can be divided into the fruiting body induction stage and the fruiting body growth stage.
[0024] During the fruiting body induction stage, after the white mycelium has fully colonized the substrate bag, remove the bag to obtain the bare substrate logs. Place the logs in a shaded, well-ventilated environment for cultivation at a temperature of 22-26℃, preferably 24℃, and a humidity of 80%-95%, more preferably 85%-90%. Oxygen is required during the fruiting body cultivation stage; ventilate twice daily for 10 minutes or longer each time. Alternatively, ventilation windows can be opened in the cultivation room. After 2-5 days of cultivation, the surface of the logs will be covered with white mycelium; continue cultivation for another 2-5 days, and the mycelium will turn from white to brown. During the fruiting body induction stage, spray with a mist of water every 3-8 hours at a water temperature of 10-20℃.
[0025] During the fruiting body growth stage, adjust the temperature to 26-28℃. The fruiting bodies will mature and be harvested in 5-15 days. Under normal circumstances, the fruiting bodies mature in 5-10 days.
[0026] The fruiting bodies of the *Gymnocladus orientalis* fungus in this invention are initially white. After about 5 days of growth, the fruiting bodies (stalk and cap) turn pale yellow, and the yellow color gradually deepens. During the rapid growth period, the growth rate is approximately 0.5-1.5 cm per day. The period from bud germination to maturity is usually 5-10 days. A mature fruiting body is approximately 5-11 cm long, with a stipe diameter of approximately 0.3-0.7 cm and a weight of approximately 2-10 g.
[0027] Compared with existing technologies, the advantages of this invention are as follows: This invention employs soilless culture medium cultivation technology for *Gynostemma pentaphyllum*, allowing for sterilization of the bagged materials and controllable cultivation conditions. It also avoids the biological hazards caused by soil cultivation or soil-covered mushroom cultivation. After fruiting, the mycelium in the mushroom logs can be inactivated through composting. By using industrial materials such as oak sawdust as the main culture medium, the raw materials are readily available, waste is utilized, costs are low, energy is saved and emissions are reduced, and industrial production is easily achieved, resulting in stable fruiting and increased profits. This invention allows for the large-scale production of *Gynostemma pentaphyllum* fruiting bodies, which can be promoted and applied as a natural functional food, or the active substances can be extracted for use in health products or pharmaceuticals. Detailed Implementation
[0028] The present invention will be further described below with reference to specific embodiments. The detailed process of the present invention is described below through specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. Many reagents used in this invention do not specify the manufacturer and can be purchased through legitimate channels.
[0029] This invention provides a soilless cultivation method for *Bleucanthus urinaria*, comprising the following steps: preparing a cultivation medium, preparing a liquid inoculum of *Bleucanthus urinaria*, inoculating it into the cultivation medium, mycelial culture, and fruiting body culture.
[0030] In this invention, the *Bacillus lucida* strain was provided by the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, with strain number CGMCC 5.92.
[0031] Example 1:
[0032] The following formula is for preparing soilless culture medium for *Bacillus thuringiensis*: 1000g oak sawdust, 666g wheat bran, 222g corn flour, 33g glucose, 4.4g potassium dihydrogen phosphate, and 28g gypsum.
[0033] Culture medium preparation. Weigh out all the ingredients of the cultivation culture medium and mix thoroughly. Add 2.4L of water and stir until well mixed. Dispense into 6 mushroom bags, insert the aeration and inoculation punch, compact, and cover with a breathable cap. Autoclave at 121℃ for 60 minutes. The dimensions of the mushroom bags are 15cm wide and 25cm long.
[0034] Inoculum preparation. The mycelium in the vigorous growth stage in PDA solid medium is cut into pieces, each about 1 cubic centimeter, together with the medium, to form solid inoculum particles.
[0035] Preparation of mycelium substrate. In a sterile environment, open the cultivation bag, remove the venting and perforating rod, and inoculate the mycelium into the cultivation medium, with the inoculation volume being 8% of the medium. Cover with a venting cap. Place in the incubation room.
[0036] During the mycelial culture stage, the culture temperature is kept constant at 26℃, and the humidity is maintained at 60-70%. Culture is carried out in the dark. After 40 days, once the white mycelium has completely covered the substrate bag, the bag is removed to obtain the mycelial logs.
[0037] Fruiting body induction. Place the substrate logs naked in a shaded, well-ventilated environment at 26°C and 85%-90% humidity, changing the air twice daily with a 12-hour interval. After 4 days of cultivation, the logs will be covered with white mycelium. After another 3 days of cultivation, the mycelium will turn from white to brown. During the fruiting body induction stage, spray with a mist of water every 3-5 hours at a water temperature of 10-20°C. After 10 days of fruiting body induction, white mushroom buds will appear on the surface of all logs. These buds are generally clustered, with 5-10 buds per cluster; or scattered, with approximately 1-3 buds per square centimeter.
[0038] Fruiting body growth stage. After the fruiting bodies germinate, maintain a temperature of 26℃ and a humidity of 85-90%RH, and continue to cultivate for 12 days until the fruiting bodies mature.
[0039] In this embodiment, after about 5 days of cultivation during the fruiting body growth stage, the mushroom body (stipe and cap) turns pale yellow and gradually deepens in color. During the rapid growth period, the daily growth length is about 0.5-1.2 cm. It takes 12 days from mushroom bud germination to maturity. The mature fruiting bodies are clustered, with the longest single fruiting body reaching 10 cm in length, a stipe diameter of about 0.4 cm, a cap diameter of 3.3 cm, and a weight of 6 g.
[0040] The bioconversion rate in this embodiment is 15.6%.
[0041] Example 2:
[0042] The following formula is for preparing soilless culture medium for *Bacillus thuringiensis*: 1000g oak sawdust, 666g wheat bran, 222g corn flour, 33g glucose, 3.3g magnesium sulfate, 4.4g potassium dihydrogen phosphate, and 28g gypsum.
[0043] Culture medium preparation. Weigh out all the ingredients of the cultivation culture medium and mix thoroughly. Add 3L of water and stir until well mixed. Dispense into 6 mushroom bags, insert the aeration and inoculation punch, compact, and cover with a breathable cap. Autoclave at 121℃ for 60 minutes.
[0044] Preparation of mycelium logs. The process of inoculating the liquid spawn of *Brightia* into the cultivation medium is carried out in a sterile environment. The cultivation bag is opened, the venting and perforating rod is removed, and the liquid fermentation spawn, along with the fermentation liquid, is inoculated into the cultivation medium at a volume of 5% of the medium. A ventilated cover is then placed in the incubation room. During the mycelial growth stage, the temperature is 20-28℃ (room temperature), and the humidity is 70-80%. The mixture is incubated in the dark. After 35 days, when the white mycelium has completely covered the bag, the bag is removed to obtain the mycelium logs.
[0045] Fruiting body induction. Place the substrate logs naked in a shaded, well-ventilated environment at a temperature of 22-28℃ and humidity of 80%-95%, changing the air twice daily, with a 12-hour interval between each ventilation. After 2-3 days of cultivation, the logs will be covered with white mycelium. After another 2-3 days of cultivation, the mycelium will turn from white to brown. During the fruiting body induction stage, spray with a mist of water every 3-7 hours at a water temperature of 10-20℃. On day 5 of fruiting body induction, mushroom primordia will appear on the surface of the logs. By day 7, white mushroom primordia will appear on all logs, mainly in clusters of 10-30, with others scattered about 2-3 per square centimeter.
[0046] Fruiting body growth stage. Adjust the temperature to 26-28℃ and humidity to 80-100%RH, and continue culturing for 10 days until the fruiting bodies mature.
[0047] In this embodiment, during the fruiting body growth stage, the mushroom body (stipe and cap) turns pale yellow after about 3 days of cultivation, and then the yellow color gradually deepens. During the rapid growth period, it grows 0.9-1.5 cm per day. It takes 10 days from mushroom bud germination to maturity. The mature fruiting bodies are clustered, with the longest fruiting body being 11 cm in length, the stipe diameter being about 0.7 cm, the cap diameter being 4.1 cm, and the weight being about 8 g.
[0048] The bioconversion rate in this embodiment is 37.5%.
[0049] Implementation: 3:
[0050] The following is the formula for preparing soilless culture medium for *Bacillus lucida*: 100kg of oak sawdust, 66kg of wheat bran, 20kg of corn flour, 4kg of sucrose, 0.4kg of magnesium sulfate, 0.45kg of potassium dihydrogen phosphate, and 3kg of gypsum.
[0051] Culture medium preparation. (1) Decompose oak sawdust. Sprinkle gypsum into wheat bran and mix well, then pour it into oak sawdust and mix well. Add 1.5 times its weight of hot water at a temperature of about 60°C and mix well. Maintain the culture medium temperature at 55-65°C for 10 days.
[0052] (2) Dissolve sucrose, magnesium sulfate and potassium dihydrogen phosphate in 40 liters of water, then add corn flour and mix well.
[0053] (3) Mix the decomposed sawdust and corn flour thoroughly. Add water until the content is about 60%. At this point, when you squeeze the culture medium tightly, 1-2 drops of water can be dripped out.
[0054] The subsequent conditions for mycelium preparation, fruiting body induction, and fruiting body growth are the same as in Example 2.
[0055] In this embodiment, during the fruiting body growth stage, the mushroom body (stipe and cap) turned pale yellow after about 3 days of cultivation, and the yellow color gradually deepened. During the rapid growth period, it grew 0.5-1.5 cm per day. From bud germination to maturity, the fastest time was 9 days. All fruiting bodies were harvested by the 12th day. The bioconversion rate was 58.1%.
[0056] Comparative Example 1: Compared with Example 1, the only difference is that poplar wood chips were used instead of oak wood chips. Mycelium could grow normally in the mushroom sticks, but no mushroom buds appeared and fruiting bodies could not be formed.
[0057] Comparative Example 2: Compared with Example 1, the only difference is that willow bark was used instead of oak sawdust, the mycelium in the mushroom sticks grew slowly, and no mushroom buds appeared on the sticks, and no fruiting bodies were formed.
[0058] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A cultivation medium for H. lucidum, which is composed of a carbon source, a nitrogen source, and inorganic salts, characterized in that The carbon source includes cypress sawdust and corn flour, and the nitrogen source includes bran; the proportion of the cypress sawdust in the total weight of the carbon source and the nitrogen source is greater than or equal to 50%.
2. The cultivated medium of claim 1, wherein, The raw materials are as follows in terms of weight ratio: cypress sawdust 100 parts, bran 50-80 parts, corn flour 15-30 parts, glucose 0-6 parts, magnesium sulfate 0.2-0.6 parts, potassium dihydrogen phosphate 0.3-0.6 parts, and gypsum 1.5-3 parts.
3. The cultivation medium for H. lucidum according to claim 1, characterized in that, The cypress sawdust is in the form of sawdust, and the particle size is 0.8 mm to 3 mm.
4. The cultivated medium of claim 2, wherein, The cypress sawdust is decomposed cypress sawdust.
5. A soilless culture method of H. lucidum, characterized by, The method comprises the following steps: The cultivation medium for the Armillaria mellea is as claimed in any one of claims 1-4. The Armillaria mellea spores are inoculated into the cultivation medium for the Armillaria mellea. In the mycelium cultivation stage, the cultivation time is 30-50 days, the cultivation temperature is 20-30 DEG C, and the humidity is 40-70% RH. In the fruiting body cultivation stage, the cultivation time is 10-20 days, the cultivation temperature is 22-30 DEG C, and the humidity is 70-100% RH.
6. The soilless culture method of H. lucidum according to claim 5, characterized in that, The spores are specifically liquid spores.
7. The soilless culture method of H. lucidum according to claim 6, characterized in that, The liquid spores of the Armillaria mellea are obtained by inoculating the Armillaria mellea solid medium cultivation mother spores into PDB liquid medium, and then culturing at a temperature of 24-26 DEG C for 12-16 days.
8. The soilless culture method of H. lucidum according to claim 5, characterized in that, The fruiting body cultivation stage can be divided into a fruiting body induction stage and a fruiting body growth stage.
9. The soilless culture method of Armillaria mellea according to any one of claims 5 to 8, characterized in that, In the fruiting body induction stage, the mycelium rods are placed in a light-shielded and ventilated environment, and the temperature is 22-26 DEG C and the humidity is 80%-95%. In the fruiting body growth stage, the temperature is adjusted to 26-28 DEG C, and the fruiting bodies are harvested after 5-10 days. In the fruiting body induction stage, the water temperature is 10-20 DEG C.