Method for preparing mother culture by tissue isolation from wild bamboo fungus

Abstract

This invention discloses a method for preparing mother cultures from wild bamboo fungus tissue isolation, belonging to the field of agricultural technology. The method uses naturally grown wild bamboo fungus buds or open-capped bamboo fungus as raw materials, obtains the stipe-coated portion through tissue isolation, and finally cultivates and purifies it to obtain the mother culture. This invention effectively utilizes collected wild bamboo fungus resources, solving the problem of effectively isolating mature or open-capped bamboo fungus, and laying a technical foundation for the collection of high-quality bamboo fungus germplasm and the breeding of superior varieties.

Description

Technical Field

[0001] This invention relates to the field of agricultural science and technology, and in particular to a method for preparing mother cultures by isolating tissues from wild bamboo fungus. Background Technology

[0002] Bamboo fungus (Dictyophora indusiata (Vent. ex Pers) Fisch), also known as bamboo shoot or bamboo ginseng, has four common edible varieties: long-skirt bamboo fungus, short-skirt bamboo fungus, spiny-stemmed bamboo fungus, and red-stemmed bamboo fungus. It is a cryptogamic fungus that parasitizes the roots of dead bamboo. Its shape somewhat resembles a net-like dried white snakeskin. It has a dark green cap, a snow-white cylindrical stalk, and a pinkish egg-shaped volva. At the top of the stalk, a delicate, pure white net-like skirt spreads downwards from the cap, earning it the names "Snow-Skirt Fairy," "Flower of the Mountains," "Flower of Fungi," and "Queen of Fungi." Bamboo fungus is rich in nutrients, has a strong aroma, and a delicious flavor, and has been listed as one of the "Eight Treasures of Herbs" since ancient times.

[0003] The young basidiospores of *Dictyophora indica* are spherical, with three layers of inflorescence: a thin, smooth, grayish-white or light brownish-red outer layer; a gelatinous middle layer; and a tough, fleshy inner layer. When mature, the inflorescence splits open, and the stipe pushes out from under the cap. The stipe is hollow, 15–20 cm high, white, and composed of spongy pores. The inflorescence remains at the base of the stipe, forming a volva. The cap, borne at the apex of the stipe, is bell-shaped, with an uneven, net-like surface, the concave parts densely covered with basidiospores. Beneath the cap is a white, reticulated veil, hanging down like a skirt, reaching over 8 cm in length. The spores are smooth, transparent, elliptical, and 3–3.5 × 1.5–2 micrometers in size.

[0004] Currently, the main method for obtaining bamboo fungus strains is to collect immature fungal eggs and isolate them using agar medium. However, the success rate is not very high, and undesirable phenomena such as swelling may occur during the cultivation process. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to disclose a method for preparing mother cultures by isolating wild bamboo fungus tissue. This method effectively utilizes collected wild bamboo fungus resources, solves the problem of effectively isolating bamboo fungus with mature buds or already opened caps, and lays a technical foundation for the collection of high-quality bamboo fungus germplasm and the breeding of superior varieties.

[0006] Specifically, the present invention provides a method for preparing a mother culture from wild bamboo fungus tissue isolation. The method uses bamboo fungus buds or open-umbrella bamboo fungus that grow naturally in the wild as raw materials, obtains the stipe-coated fungus through tissue isolation, and finally obtains the mother culture after cultivation and purification.

[0007] Furthermore, for unopened bamboo fungus buds, the specific procedure for separating the stipe-covered tissue is as follows: on a sterile laminar flow hood, after surface disinfection by soaking in 75% alcohol, the surface moisture is dried with sterile absorbent paper, and then the bud is longitudinally cut open with a scalded scalpel. The stipe is removed with sterile forceps to expose the stipe-covered tissue. A 5mm square piece of the stipe-covered tissue is then transferred to the mother culture medium for further cultivation.

[0008] Furthermore, for *Dictyophora indicum*, the specific procedure for obtaining the stipe-coated tissue through tissue isolation is as follows: first, cut off the stipe at the base of the volva; then, invert the fruiting body and rinse it thoroughly with 75% alcohol, followed by rinsing with sterile water; finally, wipe it with sterile paper; separate the volva with sterile forceps, remove the stipe, and then use sterile forceps to pick up a 5mm square piece of the stipe-coated tissue and transfer it into the mother culture medium for cultivation.

[0009] Furthermore, the method specifically includes the following steps:

[0010] S1: Collect bamboo fungus buds and open-umbrella bamboo fungus that grow naturally in the wild to obtain raw materials for separation;

[0011] S2: Obtain sterile isolated material stipe coating through aseptic processing, and transfer the stipe coating into the mother culture medium for cultivation;

[0012] S3: Cultivate until mycelial germination, then transfer to sawdust medium to obtain sawdust medium mother culture;

[0013] S4: Purified mother culture is obtained by purifying and rejuvenating the mycelial tips, then transferred to the original culture and the cultivated culture, and finally the fruiting bodies are obtained by cultivation through simulated wild planting.

[0014] S5: Through systematic selection and breeding by separating fruiting body tissues, and after 3-5 years of systematic cultivation and artificial domestication, a stable bamboo fungus mother seed that can be used for propagation and production is obtained.

[0015] Furthermore, each liter of the mother culture medium comprises the following raw materials: 40g of oak sawdust, 10g of arrow bamboo leaves, 200g of potato, 20g of glucose, 5g of peptone, 10g of agar powder, 1g of potassium dihydrogen phosphate, and 0.5g of magnesium sulfate.

[0016] Furthermore, the sawdust culture medium comprises the following raw materials in parts by weight: 78 parts by weight of oak sawdust, 20 parts by weight of wheat bran, 1 part by weight of potassium dihydrogen phosphate, 0.5 parts by weight of magnesium sulfate, and 0.5 parts by weight of gypsum.

[0017] Furthermore, in step S3, when the mycelium has germinated to 1-2 cm, it is transferred to a sawdust culture medium.

[0018] Furthermore, the raw materials of the mother culture medium also include nutrient slow-release granules, which are nutrient slow-release capsules with a core, the core being wrapped with a composite membrane layer, and camphor wood extract filling the space between the core and the composite membrane layer.

[0019] Furthermore, the nutrient sustained-release capsule uses a nutrient solution as the core and a sodium alginate / collagen composite material as the capsule shell.

[0020] Furthermore, the preparation method of the nutrient sustained-release granules specifically includes the following steps:

[0021] A1: Dissolve sodium alginate in water to obtain sodium alginate solution, add collagen solution to sodium alginate solution, adjust pH to 6-7, add ethanol, stir and mix well, heat under reflux for 30 minutes, cool to room temperature, add nutrient solution, stir and mix well, and then spray into a continuously stirred calcium chloride solution using high voltage electrostatic spraying method. Take out the microcapsules and wash them with deionized water to obtain nutrient sustained-release capsules.

[0022] A2: Weigh methylcellulose and dissolve it in deionized water to obtain a methylcellulose solution. After stirring and mixing, add chitosan solution and stir until well mixed. Homogenize at 10000 r / min for 10 min. During homogenization, add camphor wood extract dropwise, then add nutrient sustained-release microcapsules. After ultrasonic dispersion, add crosslinking agent and stir at 25℃ for 15 min. After the reaction is complete, filter and wash the filter cake with deionized water until neutral. Freeze-dry to obtain nutrient sustained-release granules.

[0023] This invention adds slow-release nutrient granules to the mother culture medium. These granules are coated with a composite membrane. On one hand, the membrane protects the nutrient granules, facilitating preservation. On the other hand, the composite membrane uses methylcellulose and chitosan as raw materials. Chitosan, as a natural antibacterial substance, can prevent the mother culture medium from being contaminated by other microorganisms, reducing the infection rate of the tissue. Methylcellulose also serves as a nutrient for the subsequent growth of mycelial hyphae in isolated tissues. Furthermore, camphor wood extract is filled between the composite membrane and the slow-release capsules. Camphor wood extract has insect-repellent and antibacterial properties. During the preservation of the slow-release granules, it protects them. During the preparation of the mother culture medium, the outer composite membrane absorbs water, swells, and loosens, releasing the camphor wood extract, thereby further enhancing the antibacterial properties of the mother culture medium and further reducing the chance of contamination. Furthermore, this invention adds slow-release nutrient granules to the mother culture medium, which to a certain extent ensures a long-term and continuous supply of nutrients during the cultivation process, thereby maintaining the concentration of nutrients in the culture medium at a suitable level, thus promoting rapid mycelial growth and shortening the growth cycle.

[0024] Furthermore, the camphor wood extract is obtained by boiling camphor wood chips in water and then purifying them. Specifically, camphor wood chips are taken, deionized water is added, and the mixture is boiled for 2 hours. After filtration, a primary filtrate is obtained. The residue is then boiled again in deionized water for 1 hour and filtered to obtain a secondary filtrate. The filtrates are combined, concentrated under reduced pressure to 2 / 3 of their volume, and then chitosan solution is added. The mixture is stirred and mixed, then subjected to electrophoresis and filtered to obtain the camphor wood extract.

[0025] Camphor wood extract contains compounds such as alkaloids, flavonoids, proteins, and organic acids. By adding chitosan and performing electrophoresis, the content of alkaloid compounds in the camphor wood extract can be reduced, while retaining as many beneficial compounds as possible, such as flavonoids, proteins, and organic acids, as possible.

[0026] The beneficial effects of this invention are:

[0027] This invention discloses a method for preparing mother culture from wild bamboo fungus tissue isolation. Through tissue isolation studies on different parts of bamboo fungus buds at varying maturity levels, the inventors discovered that the stipe-encapsulated portion of the bamboo fungus yields excellent isolation results, with a higher success rate than using the stipe alone, and without any undesirable phenomena such as swelling. The discovery of the stipe-encapsulated portion as a material for spore isolation solves the problem of obtaining spores from mature or opened bamboo fungus buds through tissue isolation, and is also of great significance for the resource collection and preservation of bamboo fungus spores. Attached Figure Description

[0028] Figure 1 This is a schematic diagram showing the position and morphological structure of the stipe covering inside the bamboo fungus bud in Embodiment 1 of the present invention;

[0029] Figure 2 This is a schematic diagram of the anatomical structure of bamboo fungus buds in Embodiment 1 of the present invention;

[0030] Among them, the stipe is 1, the spore layer is 2, the skirt is 3, the cap is 4, the stipe sheath is 5, and the volva is 6. Detailed Implementation

[0031] The present invention will be described in detail below with reference to specific embodiments:

[0032] Example 1

[0033] S1: On a sunny day, collect three different types of mature (fully matured, slightly matured, and not matured) bamboo fungus buds, as well as a number of bamboo fungus fruiting bodies with open caps, which have not been washed by rain and whose bottom of the stipe has not been contaminated, to obtain the raw materials for separation.

[0034] The preparation of the mother culture medium and sawdust culture medium is as follows:

[0035] Each liter of mother culture agar medium is prepared using the following formula: 40g of oak sawdust, 10g of arrow bamboo leaves, 200g of potato, 20g of glucose, 5g of peptone, 10g of agar powder, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, with the remainder being water. The medium is prepared using common methods for preparing general edible fungi culture media. The oak sawdust, potato, and arrow bamboo leaves are boiled for 30 minutes, and the filtrate is used to prepare the medium. The medium is then dispensed into test tubes, sterilized at 121℃ for 30 minutes, cooled to 70℃, and placed on slant agar for mother culture isolation. The pH is set to natural.

[0036] The culture medium for every 100 kg of dry sawdust is prepared by using 78 kg of oak sawdust, 20 kg of wheat bran, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 0.5 kg of gypsum. Specifically, the oak sawdust is soaked one day in advance, drained the next day, and then mixed according to the formula. After fermentation for 24 hours, it is bottled and sterilized at 123°C for 2 hours. After sterilization, it is cooled to room temperature and used for the transfer of agar mother culture. The pH should be natural.

[0037] S2: Obtain the aseptic separation material stipe coating through aseptic processing, specifically as follows:

[0038] For unopened bamboo fungus buds, first wash the surface of the buds, then soak them in 75% alcohol for 1 minute before placing them on a clean bench. Simultaneously, place the necessary test tubes, slant cultures, and separation tools on the worktable. Sterilize with UV light for 30 minutes, then open the worktable for tissue separation. For each bud, collect six tissue samples: spores, cap, upper, middle, and lower parts of the stipe, and the stipe sheath. Take a pea-sized tissue piece and place it on a slant culture. The separation method is the same for buds of different maturity levels. Label the samples and incubate them in the dark at 23°C. Observe daily and record the contamination and germination status of different tissue pieces in the culture medium.

[0039] For *Dictyophora indicum*, wash the volva thoroughly, being careful not to get water on the stipe or inside the eggs. Wipe the volva with 75% alcohol and place it on a clean bench. Simultaneously, place the necessary test tubes, slant cultures, and separation tools on the work surface. Sterilize with UV light for 30 minutes, then open the workbench for tissue separation. First, use a flamed and cooled inoculation needle to collect spores from the inner layer of the cap and streak them onto the culture medium. Then remove the volva and inoculate the base of the stipe, which has not been exposed to air, onto the culture medium.

[0040] S3: After the mother culture is isolated, it is cultured at 23℃ in the dark until the mycelium germinates to 1-2cm. The germination status is shown in Table 1 and Table 2. Then, it is transferred to sawdust medium to obtain sawdust medium mother culture.

[0041] Table 1 Germination status of bamboo fungus eggs.

[0042]

[0043] As shown in Table 1, after 2-7 days, the isolated tissues began to germinate, producing white mycelium. The germination rate of spores was zero in all tissues, and all were contaminated. The germination rate of the stipe did not vary significantly between different parts, but the germination rate varied considerably between stipes of different maturity levels. More mature stipes had lower germination rates, and secondary metabolites were produced after a certain period, causing some stipes to swell drastically to 2-3 times their original volume. The cap could grow white hairs, but could not continue to expand. The stipe-covered area had the highest germination rate, the shortest germination time, and the lowest contamination rate. Therefore, spores and caps are not suitable for tissue isolation. While the germination rate of different parts of the stipe is not significantly different, and they can still produce some white mycelium, the stipe-covered area has the highest germination rate and the lowest contamination rate, making it more suitable as a tissue for separating the mother culture.

[0044] Table 2. Germination status of fruiting body separation

[0045] Part Pollution rate Germination rate Germination time d stipe 75％ 0 0 Stipe 15％ 80％ 3 spore 100％ 0 0

[0046] Table 2 shows that the contamination rate of the fungal strain isolated from the fruiting bodies of *Dictyophora indica* after the cap has opened is relatively high. This is mainly because most of the tissue comes into contact with air after the cap opens, leading to severe contamination by other fungi. Even though only the bottom tissue was taken, some contamination was still unavoidable. Of the three parts, neither the spores nor the stipe germinated; all spore-separated tissues were contaminated. The stipe tissue began to swell after 3 days. The stipe sheath, located at the bottom of the stipe, had a contamination rate of only 10% and a germination rate of 80%. The mycelium after germination of the stipe is white, with a germination time of 3 days and dense hyphae, making it a relatively good material for isolation. Therefore, the stipe sheath remains the best material for tissue isolation from *Dictyophora indica* after the cap has opened.

[0047] S4: Purified mother culture is obtained by purifying and rejuvenating the mycelial tips, then transferred to the original culture and the cultivated culture, and finally the fruiting bodies are obtained by cultivation through simulated wild planting.

[0048] S5: Through systematic selection and breeding by separating fruiting body tissues, and after 3-5 years of systematic cultivation and artificial domestication, a stable bamboo fungus mother seed that can be used for propagation and production is obtained.

[0049] Example 2

[0050] Compared with Example 1, the difference in this embodiment is that the raw materials in the mother culture medium also include nutrient slow-release particles. The nutrient slow-release particles have a nutrient slow-release capsule as the core, and the core is wrapped with a composite membrane layer. Camphor wood extract is filled between the core and the composite membrane layer. The nutrient slow-release capsule has a nutrient solution as the core and sodium alginate / collagen composite material as the capsule skin. The nutrient solution is the nutrient solution used in the existing bamboo fungus cultivation process.

[0051] Camphor wood extract is obtained by boiling camphor wood chips in water and then purifying them. Specifically, camphor wood chips are taken and added to 8 times the volume of camphor wood chips in deionized water. After boiling for 2 hours, the mixture is filtered to obtain a primary filtrate. The residue is then boiled again with 5 times the volume of the residue in deionized water for 1 hour and filtered to obtain a secondary filtrate. The filtrates are combined and concentrated under reduced pressure to 2 / 3 of their volume. Then, 0.1 times the mass of the filtrate and 2wt% chitosan solution are added. The mixture is stirred and mixed, and then subjected to electrophoresis and filtered to obtain the camphor wood extract.

[0052] The preparation of sustained-release nutrient granules is as follows:

[0053] A1: Dissolve sodium alginate in water to obtain a sodium alginate solution with a concentration of 60 g / L. Add saturated collagen solution to the sodium alginate solution, with a mass ratio of sodium alginate to collagen of 2:1. Adjust the pH to 6-7. Add anhydrous ethanol at a volume equal to that of the sodium alginate solution. Stir and mix well. Heat under reflux at 30°C for 30 min. Cool to room temperature. Add nutrient solution at a volume equal to that of the sodium alginate solution. Stir and mix well. Spray the mixture into a continuously stirred calcium chloride solution using a high-voltage electrostatic spraying method. Remove the microcapsules and wash them with deionized water to obtain nutrient sustained-release capsules.

[0054] A2: Weigh methylcellulose and dissolve it in deionized water to obtain a methylcellulose solution with a concentration of 4 g / L. After stirring and mixing, add an equal volume of chitosan solution (6 g / L) and stir until well mixed. Homogenize at 10000 r / min for 10 min. During homogenization, add 1 / 2 volume of camphor wood extract of chitosan solution dropwise. Then add nutrient sustained-release microcapsules at a solid-liquid ratio of 5 g / L. After ultrasonic dispersion, add crosslinking agent and stir at 300 r / min for 15 min at 25℃. After the reaction is complete, filter and wash the filter cake with deionized water until neutral. Freeze-dry to obtain nutrient sustained-release granules.

[0055] Each liter of the mother culture medium in this embodiment includes: 40g of oak sawdust, 10g of arrow bamboo leaves, 200g of potato, 20g of glucose, 5g of peptone, 10g of agar powder, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 10g of nutrient slow-release granules, and the remainder is water. It can be prepared using conventional methods.

[0056] Comparative Example 1

[0057] The difference between this comparative example and Example 2 is that this comparative example uses camphor wood extract directly in the preparation of nutrient slow-release granules without electrophoresis treatment.

[0058] Comparative Example 2

[0059] The difference between this comparative example and Example 2 is that nutrient slow-release capsules were directly added to the mother culture medium in this comparative example.

[0060] According to the method of the present invention, tissue separation was performed to obtain the stipe-covered tissue of *Dictyophora indica*. Then, following the method of Example 1 to Comparative Example 2, the tissue was cultured at 23°C in the dark. The germination results are shown in Tables 3 and 4.

[0061] Table 3 Germination status of bamboo fungus eggs.

[0062] Pollution rate Germination rate Germination time d Example 1 10％ 80％ 3 Example 2 1％ 95％ 2 Comparative Example 1 5％ 87％ 2 Comparative Example 2 10％ 85％ 3

[0063] The data in Table 3, combined with the data in Tables 1 and 2, show that the mother culture medium prepared in this embodiment reduces the infection rate of isolated tissues and shortens the mycelial germination time. Furthermore, the addition of camphor wood extract can effectively reduce the infection rate of isolated tissues and improve the germination rate.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for preparing mother culture by tissue isolation from wild bamboo fungus, characterized in that, The method specifically includes the following steps: S1: Collect bamboo fungus buds and open-umbrella bamboo fungus that grow naturally in the wild, and obtain the stipe-coated material through tissue separation to obtain the isolated raw material; S2: Obtain sterile isolated material stipe coating through aseptic processing, and transfer the stipe coating into the mother culture medium for cultivation; S3: Cultivate until mycelial germination, then transfer to sawdust medium to obtain sawdust medium mother culture; S4: Purified mother culture is obtained by purifying and rejuvenating the mycelial tips, then transferred to the original culture and the cultivated culture, and finally the fruiting bodies are obtained by cultivation through simulated wild planting. S5: Through systematic selection and breeding by separating fruiting body tissues, and after 3-5 years of systematic cultivation and artificial domestication, a stable bamboo fungus mother seed that can be used for propagation and production is obtained; For unopened bamboo fungus buds, the specific procedure for separating the stipe-covered tissue is as follows: on a sterile laminar flow hood, after surface disinfection by soaking in 75% alcohol, the surface moisture is dried with sterile absorbent paper, the bud is longitudinally cut open with a scalded scalpel, the stipe is removed with sterile forceps to expose the stipe-covered tissue, and a 5mm square piece of the stipe-covered tissue is transferred into the mother culture medium for culture. For *Dictyophora indicum*, the specific procedure for separating the stipe-coated tissue is as follows: first, cut off the stipe at the base of the volva; then, rinse the fruiting body upside down with 75% alcohol, rinse with sterile water, wipe with sterile paper, separate the volva with sterile forceps, remove the stipe, and then use sterile forceps to pick up a 5mm square piece of tissue with the stipe-coated tissue and transfer it into the mother culture medium for culture. Each liter of the mother culture medium comprises the following ingredients: 40g of oak sawdust, 10g of arrow bamboo leaves, 200g of potato, 20g of glucose, 5g of peptone, 10g of agar powder, 1g of potassium dihydrogen phosphate, and 0.5g of magnesium sulfate. The raw materials of the mother culture medium also include nutrient slow-release granules, which are nutrient slow-release capsules with a core and a composite membrane layer wrapped around the core. Camphor wood extract is filled between the core and the composite membrane layer. The nutrient sustained-release capsule has a nutrient solution as the core and sodium alginate / collagen composite material as the capsule shell.

2. The method for preparing mother culture from wild bamboo fungus tissue according to claim 1, characterized in that, The sawdust culture medium comprises the following raw materials in parts by weight: 78 parts by weight of oak sawdust, 20 parts by weight of wheat bran, 1 part by weight of potassium dihydrogen phosphate, 0.5 parts by weight of magnesium sulfate, and 0.5 parts by weight of gypsum.

3. The method for preparing mother culture from wild bamboo fungus tissue according to claim 2, characterized in that, In step S3, when the mycelium has germinated to 1-2 cm, it is transferred to a sawdust culture medium.

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