Cultivation and bottle cultivation method of tremella sanguinea strain

Abstract

The invention belongs to the technical field of tremella sanguinea culture and cultivation, and particularly discloses a tremella sanguinea strain cultivation and bottle cultivation method which comprises the following steps: separating from a tremella sanguinea sporocarp based on a spore ejection method and carrying out purification culture to obtain tremella sanguinea basidiospore; separating from auricularia sanguinea wood tissues, purifying and culturing to obtain associated fungus hyphae; pairing the tremella sanguinea basidiospores in a culture medium based on a spore pairing method to obtain tremella sanguinea hyphae; respectively inoculating tremella sanguinea hyphae and associated fungus hyphae into a culture medium for light-proof activation culture to respectively obtain cultivation mother strains; the method comprises the following steps: putting a culture material into a wide-mouth bottle, compacting, punching an inoculation hole in the center, covering with a bottle cap with a breathable film, sterilizing under high pressure, inoculating a mixed cultivation mother strain under an aseptic condition, carrying out dark culture until hyphae grow full of the bottle, and carrying out fruiting management to obtain a tremella sanguinea fruiting body. The method provided by the invention has the advantages of early fruiting time, short growth cycle, capability of fruiting by substitute cultivation, stable strain, capability of obtaining pure tremella sanguinea hyphae and the like.

Description

Technical Field

[0001] The invention relates to the technical field of culturing and growing Sanguinea spp., and in particular to a method for cultivating Sanguinea spp. strains and bottling the strains. Background Art

[0002] In recent years, due to the value of edible fungi, people have gradually increased the collection of wild edible fungi, resulting in a significant reduction in edible fungi germplasm resources. In order to protect my country's edible fungi germplasm resources and species diversity, it is very necessary to carry out research on the isolation and identification of wild edible fungi.

[0003] Tremella sanguinea, also known as blood tremella, red ear, and blood wood ear, is a rare edible fungus in the genus Tremella. It possesses extremely high nutritional and medicinal value. However, due to its limited distribution and environmental conditions, wild resources of Tremella sanguinea are extremely scarce, and research on the subject is limited. Tremella sanguinea exhibits the parabiotic characteristics common to Tremella genus fungi, requiring the presence of these parabiotic fungi for growth. Current isolation techniques for Tremella sanguinea are immature, and only the yeast-like basidiospores and hyphae of the parabiotic fungi can be isolated. Consequently, using these as inoculum yields are inconsistent. Patent No. CN 105154335 B discloses a method for cultivating a mixed strain of blood ear fungus, specifically: separating the ear fungus and blood ear basidiospore species from wild blood ear fungus, purifying and preliminarily culturing them separately to obtain two strains; respectively, expanding the two strains in a liquid culture medium, mixing the two strains after the expansion culture is completed to obtain a mixed liquid strain; mixing the mixed liquid strain and inoculating it into a solid sawdust bran culture medium, cultivating to obtain a blood ear solid mixed strain for blood ear cultivation, and cultivating the mixed strain to maturity for log cultivation to obtain a blood ear finished product. The method has the disadvantages that the blood ear basidiospores do not germinate into mycelium, resulting in no ears or few ears, and there is also the disadvantage that blood ear can only be cultivated on logs, and substitute material cultivation has not yet been achieved. The present invention studies the separation of wild blood ear fungus species and substitute material cultivation in order to protect the blood ear germplasm resources and lay a certain foundation for subsequent development and application. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a method for cultivating and bottling a Hale fungus strain. The present invention solves the problems in the prior art of being unable to obtain pure Hale fungus mycelium and the instability of the strain, and solves the problems that Hale fungus can only be cultivated on logs, has a slow growth period, and cannot be grown on substitute materials. The present invention fills the gap in previous research on Hale fungus isolation and substitute material cultivation technology, and provides a theoretical basis for the research of Tremella fungi.

[0005] In order to achieve the above object, the present invention provides a method for cultivating and bottling Haemophilus fungi, comprising the following steps: S1. Separate the fruiting bodies of Sanguinea odorata by spore ejection and purify and culture them on PDA-modified + peanut solid medium to obtain Sanguinea odorata basidiospores; separate the woody tissue of Sanguinea odorata and purify and culture them on PDA-modified + peanut solid medium to obtain mycelium of the associated fungus; S2. Pairing the basidiospores of Sanguinea odorata in pairs in a PDA-modified + peanut solid culture medium based on a spore pairing method to obtain Sanguinea odorata hyphae; S3. Inoculating the mycelia of Sanguinea fuciformis and the mycelia of the associated fungus into a modified PDA+peanut solid culture medium for activation culture in the dark, respectively, to obtain a Sanguinea fuciformis mycelia cultivation mother culture and a associated fungus mycelia cultivation mother culture, respectively; wherein the Sanguinea fuciformis mycelia / associated fungus mycelia cultivation mother culture comprises the Sanguinea fuciformis mycelia / associated fungus mycelia and the remaining solid culture medium; S4. The culture medium is placed in a wide-mouth bottle, compacted, an inoculation hole is made in the center, the bottle cap with a breathable membrane is covered, and the bottle is sterilized under high pressure. Then, a mixed solid strain is inoculated under sterile conditions and dark culture is carried out until the mycelium fills the bottle, and then the mycelium is removed to obtain the blood ear fruiting body; wherein, the mixed solid strain includes the blood ear mycelium cultivation mother strain and the companion fungus mycelium cultivation mother strain.

[0006] According to one aspect of the present invention, the formula of the PDA improved + peanut solid culture medium is: 200 g / L potato, 20 g / L glucose, 5 g / L peptone, 3 g / L KH2PO4, 1.5 g / L MgSO4, 10 g / L peanut, 20 g / L agar, and the rest is distilled water.

[0007] According to one aspect of the present invention, in step S2, the spore pairing method is specifically as follows: placing different single yeast-like basidiospores in pairs on a PDA+peanut plate culture medium, about 0.5 cm apart, and culturing at 25°C until the two basidiospores grow to contact each other and hyphae germinate at the edges of the contact.

[0008] According to one aspect of the present invention, in step S3, the conditions for the light-proof activation culture are: light-proof culture at 25° C. for 7-14 days.

[0009] According to one aspect of the present invention, in step S4, the culture medium includes sawdust, wheat bran, corn flour, and gypsum; and the moisture content of the culture medium is 58-60%.

[0010] According to one aspect of the present invention, in step S4, the culture medium contains 80 parts of sawdust, 14 parts of wheat bran, 5 parts of corn flour, and 1 part of gypsum, calculated by weight.

[0011] According to one aspect of the present invention, in step S4, after the culture medium is loaded into the wide-mouth bottle, it is compacted to 1 / 3 to 2 / 3 of the bottle height; the temperature of the high-pressure sterilization is 121° C., and the time is 3 hours.

[0012] According to one aspect of the present invention, in step S4, the mass ratio of the mycelial cultivation mother culture of Sanguinea fuciformis to the mycelial cultivation mother culture of the associated fungi in the mixed solid spawn is 1:1.

[0013] According to one aspect of the present invention, in step S4, the dark culture temperature is 25° C., the humidity is 55-65%, and the cells are protected from light.

[0014] According to one aspect of the present invention, in step S4, the ear management is specifically: lowering the temperature to 20°C, increasing the humidity to 85-95°C, and ventilating twice a day.

[0015] Innovations of the present invention: (1) The present invention obtains basidiospores of Sanguinea fuciformis by using the spore ejection method, and obtains pure mycelium of Sanguinea fuciformis, a companion fungus of Sanguinea fuciformis, by the ear wood separation method. Then, the basidiospores of Sanguinea fuciformis are paired in pairs on PDA+peanut plate culture medium to find a basidiospore combination that can germinate Sanguinea fuciformis mycelium, and obtain pure Sanguinea fuciformis mycelium. Finally, the species status is determined by combining morphological identification with molecular biological identification, and a Sanguinea fuciformis strain that can be used for production is obtained.

[0016] (2) The present invention simultaneously inoculates the pure mycelium of Sanguinea odorata and its associated fungus Sanguinea odorata into bottled cultivation materials. After culturing for about 40 days, when red water droplets appear on the surface, the fungus is produced in an environment of 20°C (about 30 days), and the Sanguinea odorata fruiting body is obtained. This has preliminarily realized the artificial substitute domestication cultivation of Sanguinea odorata and found a method for bottle cultivation of Sanguinea odorata.

[0017] Beneficial effects of the present invention: (1) Compared with the prior art, the present invention germinates mycelia by pairing the basidiospores of Haemophilus auriculariae in pairs, thereby obtaining separate pure mycelia of Haemophilus auriculariae, which has good vitality and stability; (2) The present invention adopts a method for cultivating the blood ear through a substitute material, grows the blood ear fruiting body through bottle cultivation, and shortens the time for the blood ear to grow (about 30 days). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a physical picture of Haemophilus influenzae collected by the spore ejection method in Example 1 of the present invention; Figure 2 This is a physical picture of the mycelium of the associated fungus obtained by the ear wood separation method in Example 1 of the present invention; Figure 3 This is a photo of Example 1 of the present invention showing that after two yeast-like basidiospores grow together and then germinate hyphae at the edges of the joints; Figure 4 This is a photo of pure mycelia of Sanguinea obtained by pure culture of mycelia germinated from two-by-two basidiospores of Sanguinea in Example 1 of the present invention; Figure 5 This is a photo of a cultivation bottle inoculated with mycelia of Sanguinea fuciformis and associated mycelia in Example 1 of the present invention and cultured in the dark at 25° C. until the mycelia fill the bottle; Figure 6 This is a photo of red water droplets appearing on the surface of the cultivation bag after the mycelium has fully covered the cultivation bag in Example 1 of the present invention; Figure 7 This is a physical picture of the ear management until the blood ear fruiting body is formed in Example 1 of the present invention; Figure 8 This is a food diagram of comparative example 3 of the present invention, in which the mycelia of blood fungus cannot be germinated by the method of obtaining golden fungus mycelia or white fungus mycelia. DETAILED DESCRIPTION

[0019] To make the present invention easier to understand, the present invention is further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in this field; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.

[0020] To address the existing problems of the inability to obtain pure mycelium and the instability of Tremella fuciformis strains, the present invention solves the problems of the strain being unable to be cultivated only on logs, resulting in delayed growth, a long growth cycle, and the inability to grow strains using substitute materials. This invention fills the gap in previous research on the isolation and substitute material cultivation techniques of Tremella fuciformis and provides a theoretical basis for the study of Tremella fuciformis. The present invention provides a method for cultivating and bottling Tremella fuciformis strains, comprising the following steps: S1. Separate the fruiting bodies of Sanguinea odorata by spore ejection and purify and culture them on PDA-modified + peanut solid medium to obtain Sanguinea odorata basidiospores; separate the woody tissue of Sanguinea odorata and purify and culture them on PDA-modified + peanut solid medium to obtain mycelium of the associated fungus; S2. Pairing the basidiospores of Sanguinea odorata in pairs in a PDA-modified + peanut solid culture medium based on a spore pairing method to obtain Sanguinea odorata hyphae; S3. Inoculating the mycelia of Sanguinea fuciformis and the mycelia of the associated fungus into a modified PDA+peanut solid culture medium for activation culture in the dark, respectively, to obtain a Sanguinea fuciformis mycelia cultivation mother culture and a associated fungus mycelia cultivation mother culture, respectively; wherein the Sanguinea fuciformis mycelia / associated fungus mycelia cultivation mother culture comprises the Sanguinea fuciformis mycelia / associated fungus mycelia and the remaining solid culture medium; S4. The culture medium is placed in a wide-mouth bottle, compacted, an inoculation hole is made in the center, the bottle cap with a breathable membrane is covered, and the bottle is sterilized under high pressure. Then, a mixed solid strain is inoculated under sterile conditions and dark culture is carried out until the mycelium fills the bottle, and then the mycelium is removed to obtain the blood ear fruiting body; wherein, the mixed solid strain includes the blood ear mycelium cultivation mother strain and the companion fungus mycelium cultivation mother strain.

[0021] It should be noted that the above-mentioned associated bacteria are Sanguis sanguineus.

[0022] In some specific embodiments of the present invention, the formula of the PDA improved + peanut solid culture medium is: 200 g / L potato, 20 g / L glucose, 5 g / L peptone, 3 g / L KH2PO4, 1.5 g / L MgSO4, 10 g / L peanut, 20 g / L agar, and the rest is distilled water.

[0023] In some specific embodiments of the present invention, in step S2, the spore pairing method is specifically as follows: placing different single yeast-like basidiospores in pairs on a PDA+peanut plate culture medium, about 0.5 cm apart, and culturing at 25°C until the two basidiospores grow to contact each other and hyphae germinate at the edges of the contact.

[0024] In some specific embodiments of the present invention, in step S3, the conditions for the light-proof activation culture are: light-proof culture at 25° C. for 7-14 days.

[0025] In some specific embodiments of the present invention, in step S4, the culture medium includes sawdust, wheat bran, corn flour, and gypsum; and the moisture content of the culture medium is 58-60%.

[0026] In some specific embodiments of the present invention, in step S4, the culture medium contains 80 parts of sawdust, 14 parts of wheat bran, 5 parts of corn flour, and 1 part of gypsum, calculated by weight.

[0027] In some specific embodiments of the present invention, in step S4, after the culture medium is placed in the wide-mouth bottle, it is compacted to 1 / 3 to 2 / 3 of the bottle height; the high-pressure sterilization temperature is 121° C. and the time is 3 hours.

[0028] In some specific embodiments of the present invention, in step S4, the mass ratio of the mycelial cultivation mother culture of Sanguinea fuciformis to the mycelial cultivation mother culture of the associated fungi in the mixed solid spawn is 1:1.

[0029] In some specific embodiments of the present invention, in step S4, the temperature of the dark culture is 25° C., the humidity is 55-65%, and the cells are protected from light.

[0030] In some specific embodiments of the present invention, in step S4, the ear management is specifically: lowering the temperature to 20°C, increasing the humidity to 85-95°C, and ventilating twice a day.

[0031] The following is further described with reference to specific embodiments and comparative examples.

[0032] Example 1 A method for cultivating and bottling a Haemophilus fungus strain comprises the following steps: (1) Isolation and purification of strains: In a clean bench, the fresh fruiting bodies of the collected blood ear were wiped clean with 75% alcohol, the surface moisture was absorbed, and the fruiting bodies were hung with sterilized hooks in a conical flask with a thin layer of PDA culture medium (200 g potato, 20 g glucose, 15 g agar, 1 L water) at the bottom. The flask was covered with a breathable sealing film and cultured at 25 ° C. The fruiting bodies were taken out after one day. The spores began to germinate in about two days and appeared as white yeast. Single basidiospores were picked and purified in PDA modified + peanut solid culture medium (200 g / L potato, 20 g / L glucose, 5 g / L peptone, 3 g / L KH2PO4, 1.5 g / L MgSO4, 10 g / L peanut, 20 g / L agar, 1 L distilled water). The blood ear basidiospores ( Figure 1 Collect wood from the base of the blood ear, disinfect the surface with 75% alcohol, cut the wood into small pieces the size of rice grains in a clean bench, soak them in 75% alcohol for 30 seconds, then wash them three times with sterile water, and then dry the surface moisture with sterilized gauze or filter paper, and culture them on PDA modified + peanut solid medium (potato 200 g / L, glucose 20 g / L, peptone 5 g / L, KH2PO4 3 g / L, MgSO4 1.5 g / L, peanut 10 g / L, agar 20 g / L, distilled water 1L) at 25℃. Mycelia germinated after 3 days, and the terminal mycelia were picked and cultured on PDA modified + peanut solid medium (potato 200 g / L, glucose 20 g / L, peptone 5 g / L, KH2PO4 3 g / L, MgSO4 1.5 g / L, peanut 10 g / L, agar 20 g / L, distilled water 1L). g / L, distilled water 1L) for purification and culture, and the mycelium of the associated fungus was obtained ( Figure 2 ).

[0033] (2) Spore pairing: Pair the collected single Sanguinea basidiospores in pairs. Pick different Sanguinea basidiospores and use the spore pairing method to place different single yeast-like basidiospores in pairs on PDA modified + peanut solid culture medium (potato 200 g / L, glucose 20 g / L, peptone 5 g / L, KH2PO4 3 g / L, MgSO4 1.5 g / L, peanut 10 g / L, agar 20 g / L, distilled water 1L), about 0.5 cm apart, and culture at 25℃. When the two basidiospores grow to contact, hyphae will germinate at the edge of their contact ( Figure 3 ), observe the germination of mycelium, record the pairing combination of germinated mycelium, and then pick the germinated mycelium for pure culture to obtain pure mycelium of Haemophilus influenzae ( Figure 4 ), be careful not to touch the yeast-like spores when picking.

[0034] (3) Mother culture: The above-mentioned mycelia of the associated fungi and the pure mycelia of Sanguinea odorata were inoculated on PDA modified + peanut solid culture medium (potato 200 g / L, glucose 20 g / L, peptone 5 g / L, KH2PO4 3 g / L, MgSO4 1.5 g / L, peanut 10 g / L, agar 20 g / L, distilled water 1 L), and then placed at 25°C for dark culture for 10 days to obtain the cultivated mother culture.

[0035] (4) Preparation of cultivation bottles: Soak sawdust, wheat bran, corn flour, and gypsum in water in advance. After soaking thoroughly, place the materials in a cool place to drain the water for later use. Weigh each material according to the ratio in the formula (80 parts sawdust, 14 parts wheat bran, 5 parts corn flour, 1 part gypsum). After weighing, pile the materials together and stir them evenly. Add water to adjust the moisture content of the cultivation material to 60%. The moisture content is measured by grabbing the cultivation material with your hands and holding it tightly. After opening it, the cultivation material clumps together and falls to the ground and scatters. There are water stains on your hands but no clear water drips. Then the prepared cultivation material is divided into 1000ml glass bottles with a size of 9.2cm×18.9cm, with a bottling volume of 0.3-0.4kg per bottle (about half a bottle). The cultivation material is pressed tightly by hand, and an inoculation hole is made in the center. The lid with a breathable membrane is covered, and the surface stains of the filled bacteria bottle are wiped clean and placed in a high-pressure sterilizer for sterilization at 121°C for 3 hours. After sterilization, the bacteria bottle is taken out and placed in the inoculation room. After the bacteria bottle cools to room temperature, it is placed in a clean workbench and inoculated with bacteria. Each bottle is inoculated with the same proportion of blood ear mycelium and associated bacteria mycelium blocks (the mass ratio of blood ear mycelium cultivation mother strain and associated bacteria mycelium cultivation mother strain is 1:1; wherein, the blood ear mycelium / associated bacteria mycelium cultivation mother strain includes blood ear mycelium / associated bacteria mycelium and the remaining solid culture medium). After inoculation, the bacteria bottle is placed at 25°C and cultured in the dark until the mycelium fills the bottle ( Figure 5 ).

[0036] (5) Ear management: After the mycelium fills the cultivation bag, red water droplets appear on the surface ( Figure 6 ), lower the temperature to 20℃, increase the humidity to 85%-95%, ventilate twice a day, and cultivate for about 30 days until the blood ear fruiting body is formed ( Figure 7 ), harvest.

[0037] Comparative Example 1 A method for obtaining Tremella mycelium comprises the following steps: (1) Select healthy and mature Tremella fuciformis slices without diseases and insect pests, wash them thoroughly with sterile water in a clean bench, then dry them with sterile filter paper, and collect Tremella fuciformis single spores in a small triangular bottle using the hook hanging method; (2) The collected Tremella fuciformis spores were inoculated onto the germination-promoting medium plate, cultured at a constant temperature of 25°C for 7-10 days, and the germinated mycelium was selected; the formula of the germination-promoting medium (1L) was as follows: MgSO4·7H2O 1g, (NH4)2SO4 1g, KH2PO4 1.5g, glucose 10g, maltose 10g, agar powder 30g, ash fungus extract 400mL, and ddH2O to make up to 1L. The preparation method of the extract of the fragrant ash fungus is as follows: a sterile inoculation hook is used to pick up a fragrant ash fungus inoculum block from a test tube and place it in the center of a PDA enriched solid culture medium (200 g peeled potatoes, 20 g glucose, 20 g agar, 5 g yeast powder, 2 g peptone, and 1 L water), activate it at 24°C for 7 days, use a 6mm punch to punch a hole in the PDA enriched solid culture medium where the fragrant ash fungus has grown, then use a sterile inoculation hook to pick up a block and place it in the center of a sawdust culture medium (80 parts sawdust, 14 parts wheat bran, 5 parts corn flour, 1 part gypsum, and a water content of 60%), culture it at 24°C for 30 days, adjust the water content of the culture medium to 60%, accurately weigh 300 g of the sawdust culture medium containing the fragrant ash fungus, add 1500 mL of water, and boil it in a boiling water bath for 30 minutes, filter it twice through 4 layers of gauze, and add water to make the volume 1 L.

[0038] (3) Randomly select the hyphae germinated from the monospores of Tremella fuciformis for pairwise hybridization. Inoculate them 0.5 cm apart on the same plate and culture them at 25°C. When the two monokaryotic hyphae come into contact with the germinated hyphae, microscopic examination shows that there are lock-shaped united hyphae (indicating successful pairing).

[0039] Comparative Example 2 A method for obtaining Tremella fuciformis mycelium comprises the following steps: (1) Select healthy and mature golden ear pieces without diseases and insect pests, wash them thoroughly with sterile water in a clean bench, then dry them with sterile filter paper, and collect golden ear single spores in a small triangular bottle using the hook hanging method; (2) The collected T. aurantii spores were inoculated onto the germination-promoting medium plate, cultured at a constant temperature of 25°C for 7-10 days, and the germinated mycelium was selected; wherein, the formula of the germination-promoting medium (1 L) is (1 L): 200 g potato, 20 g glucose, 5 g yeast extract, 20 g agar powder, 700 mL of the associated bacteria extract, and ddH2O to a constant volume of 1 L. The preparation method of the associated bacteria extract is as follows: a sterile inoculation hook is used to pick up the associated bacteria inoculum block from the test tube and place it in the center of a PDA enriched solid culture medium (200 g peeled potatoes, 20 g glucose, 20 g agar, 5 g yeast powder, 2 g peptone, and 1 L water) plate, and activated at 24°C for 7 days. A 6mm hole punch is used to punch holes in the PDA enriched solid culture medium with the associated bacteria grown. Then, under sterile conditions, the inoculation hook is used to pick up a piece of PDA enriched solid culture medium containing the associated bacteria and place it in the center of a sawdust culture medium (80 parts sawdust, 14 parts wheat bran, 5 parts corn flour, and 1 part gypsum, with a water content of 60%). The culture is cultured at 24°C for 30 days, and the water content of the culture medium is adjusted to 60%. 300 g of the sawdust culture medium containing the associated bacteria is accurately weighed, 1500 mL of water is added, and a boiling water bath is placed for 30 minutes. The culture is filtered through 4 layers of gauze twice, and the volume is fixed to 1 L with water.

[0040] (3) Randomly select the hyphae germinated from the monospores of Golden Ear and perform pairwise hybridization. Inoculate them 0.5 cm apart on the same plate and culture them at 25°C. When the two monokaryotic hyphae come into contact with the germinated hyphae, microscopic examination shows that there are lock-shaped united hyphae (indicating successful pairing).

[0041] Comparative Example 3 An experiment on obtaining blood ear using the method of obtaining Tremella fuciformis hyphae (Comparative Example 1) or Auricularia auriculariae hyphae (Comparative Example 2): (1) Select healthy and mature pieces of Sanguinea odorata without pests and diseases, wash them thoroughly with sterile water in a clean bench, then dry them with sterile filter paper, and collect Sanguinea odorata monospores in a small triangular bottle using the hook hanging method; (2) The collected monospores of Haemophilus influenzae were inoculated onto germination-promoting culture medium plates, cultured at a constant temperature of 25°C for 7-10 days, and the germinated mycelium was selected; the formula of the germination-promoting culture medium (1L) was as follows: 200 g / L potato, 20 g / L glucose, 5 g / L peptone, 3 g / L KH2PO4, 1.5 g / L MgSO4, 10 g / L peanut, 300 ml of Haemophilus influenzae extract, 20 g / L agar, and distilled water was added to make up the volume to 1L. The preparation method of the blood-stain leather fungus extract is as follows: a sterile inoculation hook is used to pick up a blood-stain leather fungus inoculum block from a test tube and place it in the center of a PDA enriched solid culture medium (200 g peeled potatoes, 20 g glucose, 20 g agar, 5 g yeast powder, 2 g peptone, and 1 L water), activate it at 24°C for 7 days, use a 6mm punch to punch a hole in the PDA enriched solid culture medium where the blood-stain leather fungus has grown, then use a sterile inoculation hook to pick up a block and place it in the center of a sawdust culture medium (80 parts sawdust, 14 parts wheat bran, 5 parts corn flour, 1 part gypsum, and a water content of 60%), culture it at 24°C for 30 days, adjust the water content of the culture medium to 60%, accurately weigh 300 g of the sawdust culture medium containing the blood-stain leather fungus, add 1500 mL of water, and boil it in a boiling water bath for 30 minutes, filter it twice through 4 layers of gauze, and add water to make the volume 1 L.

[0042] (3) Randomly pick the hyphae of the single spore germinated from the blood ear for pairwise hybridization, inoculate them 0.5 cm apart on the same plate, and culture them at 25℃. It was found that the blood ear basidiospores could not germinate single spore hyphae on the culture medium containing the extract of their respective associated bacteria like the golden ear or the white ear. Figure 8 ). Instead, it is necessary to pair the collected single Sanguinea basidiospores in pairs as in Example 1 of the present application. Different Sanguinea basidiospores are picked and, using the spore pairing method, different single yeast-like basidiospores are placed in pairs on a PDA+peanut plate culture medium, about 0.5 cm apart, and cultured at 25°C. When the two basidiospores grow to contact each other, hyphae will germinate at the edge of the contact to obtain pure Sanguinea mycelium.

Claims

1. A method for cultivating and bottling Haemophilus influenzae strains, characterized in that: The following steps are involved: S1. Separate the fruiting bodies of Sanguinea odorata by spore ejection and purify and culture them on PDA-modified + peanut solid medium to obtain Sanguinea odorata basidiospores; separate the woody tissue of Sanguinea odorata and purify and culture them on PDA-modified + peanut solid medium to obtain mycelium of the associated fungus; S2. Pairing the basidiospores of Sanguinea odorata in pairs in a PDA-modified + peanut solid culture medium based on a spore pairing method to obtain Sanguinea odorata hyphae; S3. Inoculating the mycelia of Sanguinea fuciformis and the mycelia of the associated fungus into a modified PDA+peanut solid culture medium for activation culture in the dark, respectively, to obtain a Sanguinea fuciformis mycelia cultivation mother culture and a associated fungus mycelia cultivation mother culture, respectively; wherein the Sanguinea fuciformis mycelia / associated fungus mycelia cultivation mother culture comprises the Sanguinea fuciformis mycelia / associated fungus mycelia and the remaining solid culture medium; S4. The culture medium is placed in a wide-mouth bottle, compacted, an inoculation hole is made in the center, the bottle cap with a breathable membrane is covered, and the bottle is sterilized under high pressure. Then, a mixed solid strain is inoculated under sterile conditions and dark culture is carried out until the mycelium fills the bottle, and then the mycelium is removed to obtain the blood ear fruiting body; wherein, the mixed solid strain includes the blood ear mycelium cultivation mother strain and the companion fungus mycelium cultivation mother strain.

2. The method for cultivating and bottling Haemophilus fungi according to claim 1, characterized in that: The formula of the PDA improved + peanut solid culture medium is: 200 g / L potato, 20 g / L glucose, 5 g / L peptone, 3 g / L KH2PO4, 1.5 g / L MgSO4, 10 g / L peanut, 20 g / L agar, and the rest is distilled water.

3. The method for cultivating and bottling Haemophilus fungi according to claim 1, characterized in that: In step S2, the spore pairing method is specifically as follows: placing different single yeast-like basidiospores in pairs on a PDA+peanut plate culture medium, about 0.5 cm apart, and culturing at 25°C until the two basidiospores grow to contact each other and hyphae germinate at the edges of the contact.

4. The method for cultivating and bottling Haemophilus fungi according to claim 1, characterized in that: In step S3, the conditions for the light-proof activation culture are: light-proof culture at 25° C. for 7-14 days.

5. The method for cultivating and bottling Haemophilus fungi according to claim 1, characterized in that: In step S4, the culture medium includes sawdust, wheat bran, corn flour, and gypsum; and the water content of the culture medium is 58-60%.

6. The method for cultivating and bottling Haemophilus fungi according to claim 5, characterized in that: In step S4, based on parts by weight, the culture material contains 80 parts of sawdust, 14 parts of wheat bran, 5 parts of corn flour, and 1 part of gypsum.

7. The method for cultivating and bottling Haemophilus fungi according to claim 1, characterized in that: In step S4, after the culture medium is put into the wide-mouth bottle, it is compacted to 1 / 3 to 2 / 3 of the bottle height; the temperature of the high-pressure sterilization is 121° C. and the time is 3 hours.

8. The method for cultivating and bottling Haemophilus fungi according to claim 1, characterized in that: In step S4, the mass ratio of the mycelial cultivation mother culture of Sanguinea fuciformis to the mycelial cultivation mother culture of the associated fungi in the mixed solid spawn is 1:

1.

9. The method for cultivating and bottling Haemophilus fungi according to claim 1, characterized in that: In step S4, the dark culture temperature is 25° C., the humidity is 55-65%, and the cells are protected from light.

10. The method for cultivating and bottling Haemophilus fungi according to claim 1, characterized in that: In step S4, the ear management is specifically as follows: lowering the temperature to 20°C, increasing the humidity to 85-95°C, and ventilating twice a day.

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