A boletus planting culture medium and a preparation method thereof

Abstract

The present application relates to a boletus planting culture medium and a preparation method thereof, and belongs to the technical field of biology. The boletus planting culture medium is prepared by using bagasse fermented by Clostridium cellulolyticum, Halanaerobium sp. and Leuconostoc citreum as a culture medium raw material, and combining the bagasse with cottonseed hulls, bran, corn flour, gypsum powder, glucose, potassium dihydrogen phosphate and magnesium sulfate. The boletus cultivated by using the culture medium can effectively improve the mycelium growth of boletus, the time of boletus mushroom growth, the yield, the content of crude protein, polysaccharide and ergothioneine in the fruiting body, and the quality of boletus.

Description

Technical Field

[0001] This invention pertains to the field of biotechnology, specifically relating to a Boletus edulis culture medium and its preparation method. Background Technology

[0002] Boletus ( Boletus Boletus edulis (spp.) is a group of large fungi widely distributed in global forest ecosystems, highly favored by consumers for its delicious flavor, rich nutrition, and various bioactive components. my country possesses abundant wild Boletus edulis resources, with Yunnan province being particularly concentrated, earning it the title of "Kingdom of Wild Mushrooms." However, the artificial cultivation of Boletus edulis has long faced numerous technical bottlenecks. On the one hand, some Boletus edulis are mycorrhizal fungi, traditionally believed to require symbiosis with specific trees for growth, making artificial cultivation virtually impossible. On the other hand, even for Boletus edulis species that can be cultivated saprophytically, current cultivation techniques still suffer from slow mycelial growth, long fruiting cycles, and unstable yields. In recent years, with the accelerated industrialization of edible fungi, the development of novel edible fungi culture media using agricultural waste has become a research hotspot in the field of ecological agriculture. The preparation of solid culture media for edible fungi using byproducts such as bagasse, scum, and filter mud from sugar factories has attracted widespread attention. Against this backdrop, exploring new, efficient, low-cost culture media that can improve the yield and quality of Boletus edulis has significant economic and ecological value.

[0003] Currently, the culture media commonly used in the artificial cultivation of Boletus edulis mainly consist of sawdust, cottonseed hulls, corn cobs, and rice straw, supplemented with nutrients such as wheat bran and corn flour. However, existing culture media have the following shortcomings in practical applications: First, the mycelial growth rate is slow, and the cultivation cycle is long. Traditional cultivation media suffer from problems such as slow mycelial growth, inconsistent strain quality, and easy degeneration, which have become obstacles to the industrialization of Boletus edulis.

[0004] Secondly, the fruiting efficiency is low, and the biological conversion rate is not high. Existing culture medium formulations fail to adequately match the physiological needs and growth patterns of Boletus edulis, resulting in prolonged mycelial colonization time, difficulty in primordia formation, uneven fruiting, and low yield per unit area. The cultivation biological efficiency of some Boletus edulis varieties is far lower than that of commercially viable edible mushroom varieties, severely restricting the large-scale development of the industry.

[0005] Third, the nutritional quality of the fruiting bodies needs improvement. Boletus fruiting bodies are rich in crude protein, polysaccharides, and active ingredients such as ergothioneine. Among these, Boletus polysaccharides exhibit significant immunomodulatory and antioxidant activity; studies have shown that Boletus fruiting body polysaccharides can significantly increase the mass of the thymus and spleen, and promote the body's immune function. However, under current cultivation conditions, the content of these active ingredients in the fruiting bodies is often lower than in wild individuals, limiting the high-value-added development and utilization of Boletus.

[0006] In conclusion, developing a cultivation culture medium and its preparation method that can simultaneously improve the mycelial growth rate, fruiting rate, and nutritional quality of Boletus edulis is of great significance for promoting the quality improvement, efficiency enhancement, and sustainable development of the Boletus edulis industry. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies by providing [the following].

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The first aspect of this invention provides a culture medium for cultivating Boletus edulis, comprising the following components in parts by weight: 65-75 parts fermented sugarcane bagasse; 10-18 parts cottonseed hulls; 8-12 parts wheat bran; 3-6 parts corn flour; 0.8-1.2 parts gypsum powder; 0.5-1.0 parts glucose; 0.1-0.3 parts potassium dihydrogen phosphate; and 0.05-0.1 parts magnesium sulfate.

[0009] Furthermore, the method for preparing the fermented sugarcane bagasse includes the following steps: S1, First Stage: Crush sugarcane bagasse to a particle size of 0.5-1.5 cm, add water to adjust the moisture content to 60%-65%, and add 0.5%-1.0% urea by dry weight of sugarcane bagasse to inoculate with Clostridium pyrolyticus (…). Caldicellulosiruptor besci i) Seed liquid, the inoculation amount is 5%-8% of the dry weight of sugarcane bagasse; put the material into a sealable fermentation container, remove the air to form a strict anaerobic environment, and let it ferment at 50-55℃ for 48-72 hours. S2, Second Stage: After S1, open the fermentation container, turn the pile to dissipate heat, and lower the material temperature to 35-40℃. Inoculate with halophilic Helicobacter pylori (HHP). Spirochaeta halophila Seed liquid, inoculated at 5%-8% of the dry weight of sugarcane bagasse, kept in an anaerobic state, fermented at 35-40℃ for 36-48 hours; S3, the third stage: After S2, the material is turned over to lower the temperature to 25-30℃, and then *Bacillus thuringiensis* is introduced. Myxococcus xanthus Seed liquid, inoculated at 2%-3% of the dry weight of sugarcane bagasse, spread the material in a clean environment, aerobic fermentation for 24-36 hours, then dry to a moisture content of 10-12% to obtain fermented sugarcane bagasse.

[0010] The second aspect of the present invention provides a method for preparing the culture medium of the first aspect, comprising the following steps: weighing each component according to the proportion and mixing them evenly, adding water and stirring until the mixture contains 62%-65% water, then packaging and sterilizing to obtain the culture medium.

[0011] The third aspect of the present invention provides a culture medium prepared by the method described in the first aspect.

[0012] The fourth aspect of the present invention provides the application of the culture medium described in the third aspect in the cultivation of Boletus edulis.

[0013] Beneficial effects: Using the culture medium provided by this invention to cultivate Boletus edulis effectively improves the mycelial growth of Boletus edulis, the fruiting time of garlic segments, increases the yield, and increases the content of crude protein, polysaccharides and ergothionein in the fruiting bodies, thereby improving the quality of Boletus edulis. Detailed Implementation

[0014] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0015] The pyrolytic cellulose bacterium (Caldicellulosiruptor bescii) used in this invention was purchased from Beijing Bio-Bio Biotechnology Co., Ltd., strain number: bio-138154; Spirochaeta halophila was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., strain number: B68160; Myxococcus xanthus was purchased from Beijing Bio-Bio Biotechnology Co., Ltd., strain number: bio-108869.

[0016] Example 1 A culture medium for cultivating Boletus edulis comprises the following components in parts by weight: 70 parts fermented sugarcane bagasse; 14 parts cottonseed hulls; 10 parts wheat bran; 4.5 parts corn flour; 1 part gypsum powder; 0.7 parts glucose; 0.2 parts potassium dihydrogen phosphate; and 0.08 parts magnesium sulfate.

[0017] The method for preparing fermented sugarcane bagasse includes the following steps: S1, First Stage: Crush sugarcane bagasse to a particle size of 0.5-1.5 cm, add water to adjust the moisture content to 60%-65%, add 0.8% urea (dissolved in water) of the dry weight of the sugarcane bagasse; inoculate with pyrolytic cellulose-degrading Clostridium (…). Caldicellulosiruptor besci i) Seed liquid, the inoculation amount is 6.5% (v / m) of the dry weight of sugarcane bagasse; the material is put into a sealable fermentation container, the air is discharged to form a strict anaerobic environment, and it is allowed to ferment at 50-55℃ for 60 hours. S2, Second Stage: After S1, open the fermentation container, turn the pile to dissipate heat, and lower the material temperature to 35-40℃. Inoculate with halophilic Helicobacter pylori (HHP). Spirochaeta halophila Seed liquid, inoculated at 6% of the dry weight of sugarcane bagasse, kept in an anaerobic state, fermented for 42 hours at 35-40℃; S3, the third stage: After S2, the material is turned over to lower the temperature to 25-30℃, and then *Bacillus thuringiensis* is introduced. Myxococcus xanthus Seed liquid, with an inoculation amount of 2.5% of the dry weight of sugarcane bagasse, is spread out in a clean environment and fermented aerobically for 30 hours. After drying, the water content is reduced to 10-12% to obtain fermented sugarcane bagasse.

[0018] The culture medium preparation method includes the following steps: weigh each component according to the proportion, mix them evenly, add water and stir until the mixture contains 62%-65% water, then pack it into bags and sterilize to obtain the culture medium.

[0019] Example 2 A culture medium for cultivating Boletus edulis comprises the following components in parts by weight: 65 parts fermented sugarcane bagasse; 10 parts cottonseed hulls; 8 parts wheat bran; 3 parts corn flour; 0.8 parts gypsum powder; 0.5 parts glucose; 0.1 parts potassium dihydrogen phosphate; and 0.05 parts magnesium sulfate.

[0020] The method for preparing fermented sugarcane bagasse includes the following steps: S1, First Stage: Crush sugarcane bagasse to a particle size of 0.5-1.5 cm, add water to adjust the moisture content to 60%-65%, add 0.5% urea (dissolved in water) based on the dry weight of the sugarcane bagasse; inoculate with pyrolytic cellulose-degrading Clostridium (…). Caldicellulosiruptor besci i) Seed liquid, the inoculation amount is 5% (v / m) of the dry weight of sugarcane bagasse; the material is put into a sealable fermentation container, the air is discharged to form a strict anaerobic environment, and it is allowed to ferment at 50-55℃ for 60 hours. S2, Second Stage: After S1, open the fermentation container, turn the pile to dissipate heat, and lower the material temperature to 35-40℃. Inoculate with halophilic Helicobacter pylori (HHP). Spirochaeta halophila Seed liquid, inoculated at 5% of the dry weight of sugarcane bagasse, kept in an anaerobic state, fermented for 36 hours at 35-40℃; S3, the third stage: After S2, the material is turned over to lower the temperature to 25-30℃, and then *Bacillus thuringiensis* is introduced. Myxococcus xanthus Seed liquid, inoculated at 2% of the dry weight of sugarcane bagasse, spread the material in a clean environment, aerobic fermentation for 24 hours, and then dry it to a moisture content of 10-12% to obtain fermented sugarcane bagasse.

[0021] The culture medium preparation method includes the following steps: weigh each component according to the proportion, mix them evenly, add water and stir until the mixture contains 62%-65% water, then pack it into bags and sterilize to obtain the culture medium.

[0022] Example 3 A culture medium for cultivating Boletus edulis comprises the following components in parts by weight: 75 parts fermented sugarcane bagasse; 18 parts cottonseed hulls; 12 parts wheat bran; 6 parts corn flour; 1.2 parts gypsum powder; 1.0 part glucose; 0.3 parts potassium dihydrogen phosphate; and 0.1 parts magnesium sulfate.

[0023] The method for preparing fermented sugarcane bagasse includes the following steps: S1, First Stage: Crush sugarcane bagasse to a particle size of 0.5-1.5 cm, add water to adjust the moisture content to 60%-65%, add 1.0% urea (dissolved in water) of the dry weight of the sugarcane bagasse; inoculate with pyrolytic cellulose-degrading Clostridium (…). Caldicellulosiruptor besci i) Seed liquid, the inoculation amount is 8% (v / m) of the dry weight of sugarcane bagasse; the material is put into a sealable fermentation container, the air is discharged to form a strict anaerobic environment, and it is allowed to ferment at 50-55℃ for 72 hours. S2, Second Stage: After S1, open the fermentation container, turn the pile to dissipate heat, and lower the material temperature to 35-40℃. Inoculate with halophilic Helicobacter pylori (HHP). Spirochaeta halophila Seed liquid, inoculated at 8% of the dry weight of sugarcane bagasse, kept in an anaerobic state, fermented for 48 hours at 35-40℃; S3, the third stage: After S2, the material is turned over to lower the temperature to 25-30℃, and then *Bacillus thuringiensis* is introduced. Myxococcus xanthus The seed liquid is inoculated at 3% of the dry weight of sugarcane bagasse. The material is spread out in a clean environment and fermented aerobically for 36 hours. After drying, the water content is reduced to 10-12% to obtain fermented sugarcane bagasse.

[0024] The culture medium preparation method includes the following steps: weigh each component according to the proportion, mix them evenly, add water and stir until the mixture contains 62%-65% water, then pack it into bags and sterilize to obtain the culture medium.

[0025] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the fermented sugarcane bagasse is obtained by drying it after the first stage of fermentation is completed and the water content is 10-12%.

[0026] Comparative Example 2 The difference between Comparative Example 2 and Example 1 lies in the first stage of the preparation of fermented sugarcane bagasse: sugarcane bagasse is crushed to a particle size of 0.5-1.5 cm, water is added to adjust the moisture content to 60%-65%, and urea (dissolved in water) at 0.8% of the dry weight of the sugarcane bagasse is added before inoculating with only halophilic Helicobacter pylori. Spirochaeta halophila Seed liquid fermentation, and after fermentation, drying until the water content is 10-12% yields fermented sugarcane bagasse.

[0027] Comparative Example 3 The difference between Comparative Example 3 and Example 1 lies in the first stage of the preparation of fermented sugarcane bagasse: sugarcane bagasse is crushed to a particle size of 0.5-1.5 cm, water is added to adjust the moisture content to 60%-65%, and urea (dissolved in water) at 0.8% of the dry weight of the sugarcane bagasse is added before inoculating with only *Myxocytocium chrysogenum*. Myxococcus xanthus Seed liquid fermentation, and after fermentation, drying until the water content is 10-12% yields fermented sugarcane bagasse.

[0028] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that unfermented sugarcane bagasse was used as the culture medium material instead of fermented sugarcane bagasse.

[0029] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that cottonseed hulls were used as the culture medium raw material instead of fermented sugarcane bagasse.

[0030] Experimental Example The culture media from Examples 1 to 3 and Comparative Examples 1 to 5 were bagged (15cm*35cm polypropylene bags), sterilized, cooled, and inoculated with Boletus edulis spawn (10 bags per treatment, three replicates). They were then cultured indoors at 33-35℃, 80%-85% humidity, and 12h / 12h light-dark alternation. Mycelial growth was observed and the growth rate was measured. After the mycelium filled the bags, the bags were opened and covered with 3-4cm of paddy soil, maintaining a soil moisture content of 40-45%, relative humidity of 75-85%, and temperature of 26-28℃. Fruiting was then carried out in a fruiting house. Fruiting bodies were harvested before the caps fully expanded. Yield was tallied, and the crude protein, polysaccharide, and ergothioneine content in the fruiting bodies of each treatment were measured. The results are shown in Table 1 below.

[0031] The crude protein content in 100g of dried Boletus edulis was determined by the Kjeldahl method, the polysaccharide content in 100g of dried Boletus edulis was determined by the phenol-sulfuric acid method, and the ergothionein content in 100g of Boletus edulis was determined by high performance liquid chromatography.

[0032] Table 1. Statistical table of mycelia, yield and fruiting body indicators of *Boletus edulis* cultured on culture media prepared by each treatment. deal with mycelial growth Mycelial growth rate (mm / d) Mushroom emergence time (d) Production (g / bag) Crude protein content (%) Polysaccharide content (g) Ergothioneine content (mg) Example 1 Growing well, with dense mycelium 6.28 37 146.87 29.71 19.50 1.88 Example 2 Growing well, with dense mycelium 6.21 38 145.42 28.92 19.35 1.82 Example 3 Growing well, with dense mycelium 6.32 35 147.09 31.05 21.43 1.91 Comparative Example 1 Good growth, dense mycelium 4.21 51 128.23 26.65 17.83 1.68 Comparative Example 2 Average growth, relatively dense mycelium 3.75 58 123.14 25.62 16.56 1.62 Comparative Example 3 Good growth, dense mycelium 4.08 55 125.67 26.21 17.20 1.59 Comparative Example 4 Average growth, sparse mycelium 3.21 61 105.33 25.77 15.31 1.44 Comparative Example 5 Average growth, sparse mycelium 3.35 58 110.29 26.02 15.68 1.48 As can be seen from Table 1, using the culture medium of the present invention to cultivate Boletus can effectively improve the mycelial growth of Boletus, the fruiting time of garlic segments, increase the yield, and increase the content of crude protein, polysaccharides and ergothionein in the fruiting bodies, thereby improving the quality of Boletus.

[0033] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technical or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A culture medium for cultivating Boletus edulis, characterized in that: It contains the following components in parts by weight: 65-75 parts fermented sugarcane bagasse; 10-18 parts cottonseed hulls; 8-12 parts wheat bran; 3-6 parts corn flour; 0.8-1.2 parts gypsum powder; 0.5-1.0 parts glucose; 0.1-0.3 parts potassium dihydrogen phosphate; and 0.05-0.1 parts magnesium sulfate.

2. The culture medium according to claim 1, characterized in that: The method for preparing fermented sugarcane bagasse includes the following steps: S1, First Stage: Crush sugarcane bagasse to a particle size of 0.5-1.5 cm, add water to adjust the moisture content to 60%-65%, and add 0.5%-1.0% urea by dry weight of sugarcane bagasse to inoculate with Clostridium pyrolyticus (…). Caldicellulosiruptor besci i) Seed liquid, the inoculation amount is 5%-8% of the dry weight of sugarcane bagasse; put the material into a sealable fermentation container, remove the air to form a strict anaerobic environment, and let it ferment at 50-55℃ for 48-72 hours. S2, Second Stage: After S1, open the fermentation container, turn the pile to dissipate heat, and lower the material temperature to 35-40℃. Inoculate with halophilic Helicobacter pylori (HHP). Spirochaeta halophila Seed liquid, inoculated at 5%-8% of the dry weight of sugarcane bagasse, kept in an anaerobic state, fermented at 35-40℃ for 36-48 hours; S3, the third stage: After S2, the material is turned over to lower the temperature to 25-30℃, and then *Bacillus thuringiensis* is introduced. Myxococcus xanthus Seed liquid, inoculated at 2%-3% of the dry weight of sugarcane bagasse, spread the material in a clean environment, aerobic fermentation for 24-36 hours, then dry to a moisture content of 10-12% to obtain fermented sugarcane bagasse.

3. The method for preparing the culture medium according to any one of claims 1 or 2, characterized in that: The process includes the following steps: weigh each component according to the proportion, mix them thoroughly, add water and stir until the mixture contains 62%-65% water, then pack it into bags and sterilize to obtain the culture medium.

4. The culture medium prepared by the method according to any one of claims 1-2.

5. The culture medium as described in claim 3 is used in the cultivation of Boletus.