Crisp auricularia auricula strain and rejuvenation culture medium and cultivation method thereof

Abstract

The invention discloses an auricularia auricula strain and a rejuvenation culture medium and a cultivation method thereof, and belongs to the field of edible fungus cultivation. A new auricularia crispa strain is separated from miscellaneous trees at the top of Aoba in Nanpond village, Shixan town, eight-step area, Hexanzhou city, Guangxi Zhuang Autonomous Region, named 'Guiyun No.7', and the preservation number is GDMCC No.65145. The invention further discloses a preparation method of the auricularia crispa strain. The invention also discloses a rejuvenation culture medium which comprises the following components: 20 g / L of barks, 20 g / L of glucose, 2 g / L of yeast extract and 20 g / L of agar, and the barks are cyclobalanopsis barks or maple barks. Through the rejuvenation culture medium, the auricularia auricula strain can be rejuvenated, and strain degeneration is prevented. The crisp agaric strain can also be directly applied to cultivation and production, large-scale cultivation and production of crisp agaric are achieved, and the current situation that only a small-scale experimental research stage exists in crisp agaric cultivation at present is changed.

Description

Technical Field

[0001] The invention relates to the field of edible fungus cultivation, in particular to a friable fungus strain and a rejuvenation culture medium and a cultivation method thereof. Background Art

[0002] Auricularia fibrillifera is a species of the genus Auricularia in the family Auriculariales. It is an edible and medicinal fungus.

[0003] Auricularia auricula is mainly distributed in tropical and subtropical regions of my country. At present, it is mainly collected from the wild. At present, there is only one literature report on the artificial domestication of Auricularia auricula, and it has not yet been industrialized. Fungus strains and cultivation techniques are the key technical links for industrialization. The acquisition of Auricularia auricula strains is usually achieved by separating and purifying the fruiting bodies and directly applying them to cultivation production. However, due to the lack of Auricularia auricula varieties and cultivation techniques, there are currently few strains that can be cultivated and the cultivation conditions are in the stage of small-scale experimental research. Summary of the invention

[0004] The purpose of the present invention is to provide a brittle fungus strain and a rejuvenation culture medium and a cultivation method thereof, so as to solve the problems existing in the above-mentioned prior art. The brittle fungus strain can be rejuvenated by a culture medium containing cycad bark or maple bark to prevent strain degeneration, and the brittle fungus strain can be directly applied to cultivation production to realize large-scale cultivation and production of brittle fungus.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides an Auricularia fibrillifera strain, and the preservation number of the strain is GDMCC No.65145.

[0007] The invention also provides a mycelium or fruiting body of Auricularia auricula, which is obtained by culturing the Auricularia auricula strain.

[0008] The invention also provides a rejuvenation culture medium for the brittle fungus strain, which comprises components in the following concentrations: 20 g / L bark, 20 g / L glucose, 2 g / L yeast extract and 20 g / L agar, wherein the bark is Cyclobalanopsis glauca bark or maple bark.

[0009] The present invention also provides a method for preparing the rejuvenation medium, comprising the following steps:

[0010] Crush 20g of Cyclobalanopsis glauca bark or maple bark into blocks, then add 2-3 times the volume of water and boil for 20-30 minutes, filter and discard the residue, and collect the water extract;

[0011] According to the dosage of each component of the rejuvenation medium, glucose, yeast extract and agar are uniformly mixed with the water extract, and then distilled water is added to make up the total volume to 1L to obtain the rejuvenation medium.

[0012] The present invention also provides a rejuvenation method for the brittle fungus strain, comprising the step of culturing the brittle fungus strain in a rejuvenation medium; wherein the rejuvenation medium comprises components in the following concentrations: 20 g / L bark, 20 g / L glucose, 2 g / L yeast extract and 20 g / L agar, and the bark is cycad glauca bark or maple bark.

[0013] Preferably, the culture conditions are: 26.5°C for 7 days.

[0014] The present invention also provides a method for cultivating the Auricularia auricula strain, comprising the following steps:

[0015] (1) collecting the brittle fungus separated from the tissue, inoculating the brittle fungus into a PDA slant culture medium aseptically, and culturing in a dark environment at a constant temperature of 26.5° C. until the mycelium covers the slant culture medium, thereby obtaining a production mother culture;

[0016] (2) The production mother seed is aseptically inoculated into a stock seed bag, and placed in a constant temperature dark culture at 26.5° C. until the mycelium is fully fed with the material, thereby obtaining the stock seed; the culture medium in the stock seed bag comprises the following raw materials in parts by weight: 100 parts of cottonseed hulls, 30 parts of wheat bran, 2.5 parts of superphosphate, 1 part of gypsum, and 60 parts of water;

[0017] (3) The original seed is aseptically inoculated into a production bag, and placed in a dark culture at a constant temperature of 26.5° C. until the mycelium is fully fed with the material, thereby obtaining the production seed; the culture medium in the production bag comprises the following raw materials in parts by weight: 50 parts of broad-leaved wood chips, 34 parts of bran, 50 parts of bagasse, 10 parts of cottonseed hulls, 1.2 parts of superphosphate, 2 parts of gypsum, and 57 parts of water; the broad-leaved wood chips contain at least 20% of Cyclobalanopsis glauca chips or maple wood chips;

[0018] (4) After the cultivation material is bagged and sterilized, the production species is inoculated, and the inoculated bacterial sticks are piled up in a "well" shape, the temperature is controlled at 26.5° C., and the cultivation is shaded; the culture medium loaded in the cultivation material includes the following raw materials in parts by weight: 85 parts of miscellaneous sawdust, 10 parts of wheat bran, 1 part of gypsum, 0.5 parts of lime, 15 parts of cottonseed hulls, and 70 parts of water; the miscellaneous sawdust contains at least 20% of Cyclobalanopsis glauca sawdust or maple sawdust;

[0019] (5) When the mycelium has grown all over the bag, the mycelium is punctured when it is physiologically mature. After puncturing, the mushroom sticks are stacked in a "well" shape for ventilation and cultivation. When primordia are formed at the puncture holes, the ears can be placed on the ground and managed;

[0020] (6) Harvest the fungus after it matures.

[0021] Preferably, in step (2), the inoculation amount of the production mother seed is 8 g per original seed bag.

[0022] Preferably, in step (3), the inoculation amount of the original seed is 8 g for each production bag.

[0023] Preferably, in step (4), the inoculation amount of the production seed is 10 g per cultivation bag.

[0024] The present invention discloses the following technical effects:

[0025] The present invention isolates a new brittle fungus strain from miscellaneous woods of Muaoding, Nantang Village, Hejie Town, Babu District, Hezhou City, Guangxi Zhuang Autonomous Region. The brittle fungus strain can be rejuvenated using a culture medium containing cycad bark or maple bark, which can ensure a large amount of hyphae growth and prevent strain degradation. The brittle fungus strain obtained by the present invention can also be directly applied to cultivation production to achieve large-scale planting, changing the current situation that the cultivation of brittle fungus is still in the small-scale experimental research stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 To purify the strain with better growth;

[0028] Figure 2 This is a diagram of the mycelial growth status of the culture dish of the Auricularia auricularia strains that were rejuvenated using different culture media;

[0029] Figure 3 To rejuvenate the Auricularia auriculariae strains on different culture media, and to count the mycelial growth rates of different groups;

[0030] Figure 4 The fungus growing out of the bag after being punctured (A) and the harvested fungus (B). DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1 Isolation and identification of Auricularia auricula strains

[0037] 1. Strain Isolation

[0038] (1) Fruiting body collection: The fruiting bodies were collected from miscellaneous trees in Muaoding, Nantang Village, Hejie Town, Babu District, Hezhou City, Guangxi Zhuang Autonomous Region in April 2018.

[0039] (2) Tissue separation: Rinse the debris on the fruiting body with running water and wipe dry. Place in 75% alcohol to sterilize the surface for 3-5 minutes. Wash the surface with sterilized water on a clean bench for 3-5 times, dry the water, cut the fruiting body with a blade, tear open the inner layer of the fruiting body, cut into 0.5-1 cm squares, and stick the inner layer on a culture dish containing PDA culture medium for separation and purification.

[0040] (3) Strain purification: Incubate in dark at 26.5°C for 4 days, remove contaminated culture dishes, and select uncontaminated culture dishes with good growth to obtain the strain (see Figure 1). Use an inoculation needle to pick up a 0.5 cm-sized bacterial mass and inoculate it onto the PDA culture medium for separation and purification to purify the strain. After 3 days, pick up a 0.5 cm-sized bacterial mass and inoculate it onto the PDA culture slope. Culture it in the dark at 26.5°C for 6 days to obtain the purified test tube strain of Auricularia auricula.

[0041] 2. Strain identification

[0042] The test fruiting bodies and corresponding mycelium were used to extract DNA according to the CTAB method, and PCR amplification and ITS sequence determination were performed. The ITS of the samples was compared by Blast and the Auricularia fibrillifera Identities = 558 / 562 (99%), Gaps = 4 / 562 (0%) in GENBANK. The analysis confirmed that the isolated pure culture was Auricularia fibrillifera mycelium.

[0043] Biological characteristics: The fruiting body is 6.33cm×7.46cm in size; the ear base is small and the ear blade is thin; the fruiting body is flaky, mostly clustered, and has obvious wrinkles. The fruiting body is elastic, soft, translucent, disc-shaped, light reddish brown, and leathery, thin, brown or reddish brown after drying. The sterile mask is sparsely pubescent; the cross section seems to have a medullary layer, which is not obvious.

[0044] The ITS sequence primers were: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO: 2) and ITS4 (5'-TCCTCCGCT TATTGATATGC-3', SEQ ID NO: 3).

[0045] Amplification reaction system: TaKaRa Ex Taq enzyme 0.25 μL, 10×Ex Taq Buffer 3 μL, MgCl 2 1.5μL, dNTP Mixture 2.5μL, ITS1 / ITS4 0.25μL each, DNA template 1μL, ddH 2 Add 25 μL of HO.

[0046] The reaction program was: 95°C for 3 min; 95°C for 30 s, 56°C for 45 s, 72°C for 1 min, 35 cycles; 72°C for 4 min.

[0047] The amplified ITS sequence (SEQ ID NO: 1) is as follows:

[0048] .

[0049] The strain isolated and identified above is Auricularia fibrillifera 2018031413, named "Guiyun No. 7", abbreviated as "413". The strain has been deposited in the Guangdong Microbiological Culture Collection Center on September 19, 2024, with the deposit number GDMCC No. 65145, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0050] Example 2 Bacterial strain rejuvenation

[0051] 1. Test culture medium

[0052] Basic medium (CK): 20 g glucose, 2 g yeast extract, 20 g agar, 1000 mL distilled water, natural pH.

[0053] Rejuvenation medium: Based on the basal medium, different groups were set up with the addition of Cyclobalanopsis glauca bark (20g / L), Castanopsis castanopsis bark 28g / L), Acer tung bark (20g / L), and Vc (0.01g / L), and the basal medium was used as a blank control. After the bark was crushed into blocks, 20g of bark was added to 3 times the water and boiled, then boiled for 30 minutes with a low fire, filtered and discarded the residue, and the bark water extract was obtained for the preparation of the medium. Specifically: the bark water extract was evenly mixed with the glucose, yeast extract, and agar in the basal medium, and then supplemented with distilled water to a total volume of 1000mL to obtain the rejuvenation medium.

[0054] 2. Biological characteristics test

[0055] 2.1 Strain activation

[0056] The strains used for the test were transferred to PDA medium for culture. When their colonies filled the culture dish, they were used for single-factor and multi-factor tests.

[0057] 2.2 Mycelial growth test of different strains

[0058] Use PDA culture medium to pour the plate, use a cork puncher (or ampoule tube) to punch a mycelium block with a diameter of 5 mm and inoculate it in the center of the plate, and culture it at a constant temperature of 28°C. Repeat each treatment 3 times, measure the colony diameter using the cross-hatch method, measure once every 24 hours after 3 days of inoculation, for a total of 3 times, and calculate the average value to determine the mycelium growth of different strains.

[0059] like Figure 2 and Figure 3 As shown, the results show that the medium with maple bark or cycad bark can rejuvenate Auricularia auricula-Guiyun No. 5. The above method can ensure the excellent properties of the strain and the purity of the strain, and prevent the strain from degenerating.

[0060] Example 3 Cultivation and application of crisp fungus

[0061] (1) Preparation of mother culture: Collect the strain of Auricularia auriculariae "Guiyun No. 7" separated by tissue, aseptically inoculate the strain into the mother culture medium (PDA slant culture medium), and place it in a 26.5°C incubator for constant temperature and dark culture until the mycelium grows all over the slant to obtain the production mother culture.

[0062] (2) Preparation of stock seed: The production mother seed is aseptically inoculated into a stock seed bag (the culture medium in the bag includes the following raw materials in parts by weight: 100 parts of cottonseed hulls, 30 parts of wheat bran, 2.5 parts of superphosphate, 1 part of gypsum, and 60 parts of water), with an inoculum amount of 8 g per bag, and placed in a 26.5° C. incubator for constant temperature dark culture until the mycelium is fully fed with the material, and the stock seed is obtained;

[0063] (3) Making production seeds: The original seeds are aseptically inoculated into a production seed bag (the culture medium in the bag includes the following raw materials by weight: 50 parts of broad-leaved wood chips (containing at least 20% of Cyclobalanopsis glauca chips or maple wood chips, and this embodiment uses 20% Cyclobalanopsis glauca chips), 34 parts of bran, 50 parts of bagasse, 10 parts of cottonseed hulls, 1.2 parts of superphosphate, 2 parts of gypsum, and 57 parts of water), with an inoculum size of 8 g per bag, and placed in a 26.5° C. incubator for constant temperature dark culture until the mycelium has eaten up all the materials, and the production seeds are obtained;

[0064] (4) Preparation of mushroom bags: A polyethylene bag with a size of 15 cm × 55 cm × 0.005 cm is selected, and a cultivation material (composed of the following components by weight: 85 parts of miscellaneous wood chips (containing at least 20% of Cyclobalanopsis glauca chips or maple wood chips, and this embodiment uses 20% of Cyclobalanopsis glauca chips), 10 parts of wheat bran, 1 part of gypsum, 0.5 parts of lime, 15 parts of cottonseed hulls, and 70 parts of water) is packed in a bag and sterilized in an autoclave. The temperature is raised to 100° C. within 4 hours after entering the pot, and the bag is sterilized at a constant temperature for 15 hours, and the heat cannot be turned off during the sterilization period. After sterilization, the temperature in the pot is naturally lowered to 50-60° C., and the material rod is taken out while hot and moved into a cooling room or an inoculation room. When the temperature of the material rod drops below 28° C., the material rod is inoculated according to the aseptic operation requirements.

[0065] (5) Inoculation: Make 4 holes in the bag for inoculation, with a hole diameter of 2.5 cm and a hole depth of 2.5 cm; the inoculation amount is 10 g;

[0066] (6) Cultivation: The inoculated mushroom sticks are piled up in a “well” shape, the temperature is controlled at 26.5°C, and the culture is shaded. The relative humidity in the pile is lower than 65%. The mycelium will grow fully in about 60 days. During this period, the pile is turned, spread, and ventilated according to the ambient temperature and mycelium growth.

[0067] (7) Puncture: Puncture when the mycelium fills the bag and reaches physiological maturity. Use the bag-free method to puncture holes. The number of holes is 250-300 per bag. The hole diameter is 0.4 cm and the hole depth is 0.5 cm. After puncturing, the mushroom sticks are stacked in a "well" shape and ventilated for 7 days. When primordia are formed at the hole mouth (see Figure 4 ) can be put to the ground and ears can be pulled out.

[0068] (8) Placement: Place the mushrooms in a greenhouse or outdoors on a sunny day with a temperature of about 25°C. Set up a simple rack with mushroom sticks leaning against it at an angle of about 60 degrees, with a distance of 15 cm between the mushroom sticks.

[0069] (9) Management of mushrooms: Use water mist spray belts or nozzles to maintain the humidity of mushroom sticks, spray 5 times a day, each time for 30 minutes; when the auricularia grows to more than 1 cm, the number of spraying can be reduced to 2 times, and the relative humidity can be maintained above 95%; harvest when the auricularia matures (see Figure 4 ).

[0070] Table 1 Determination of yield of different fungus

[0071]

[0072] It can be seen from the above data that the present invention successfully utilizes the separated brittle fungus for large-scale cultivation with considerable yield, which changes the current situation that brittle fungus cultivation is still in the small-scale experimental research stage, and provides a scientific method and basis for large-scale cultivation and promotion of brittle fungus.

[0073] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Auricularia fibrillifera, characterized in that: The deposit number of this strain is GDMCC No.65145.

2. A mycelium or fruiting body of Auricularia auricula, characterized in that: The method is obtained by culturing the Auricularia auricularia auricula strain according to claim 1.

3. A rejuvenation medium for the Auricularia auricula strain according to claim 1, characterized in that: The rejuvenation medium comprises the following components in concentrations: 20 g / L bark, 20 g / L glucose, 2 g / L yeast extract and 20 g / L agar, wherein the bark is Cyclobalanopsis glauca bark or maple bark.

4. The method for preparing the rejuvenation medium according to claim 3, characterized in that: The following steps are involved: Crush 20g of Cyclobalanopsis glauca bark or maple bark into blocks, then add 2-3 times the volume of water and boil for 20-30 minutes, filter and discard the residue, and collect the water extract; According to the dosage of each component of the rejuvenation medium, glucose, yeast extract and agar are uniformly mixed with the water extract, and then distilled water is added to make up the total volume to 1L to obtain the rejuvenation medium.

5. A method for rejuvenating the Auricularia auricula strain according to claim 1, characterized in that: The method comprises the step of culturing the brittle fungus strain in a rejuvenation medium; wherein the rejuvenation medium comprises components of the following concentrations: 20 g / L bark, 20 g / L glucose, 2 g / L yeast extract and 20 g / L agar, and the bark is Cyclobalanopsis glauca bark or maple bark.

6. The rejuvenation method according to claim 5, characterized in that: The culture conditions are: 26.5° C. for 7 days.

7. A method for cultivating the Auricularia auricula strain according to claim 1, characterized in that: The following steps are involved: (1) collecting the brittle fungus separated from the tissue, inoculating the brittle fungus into a PDA slant culture medium aseptically, and culturing in a dark environment at a constant temperature of 26.5° C. until the mycelium covers the slant culture medium, thereby obtaining a production mother culture; (2) The production mother seed is aseptically inoculated into a stock seed bag, and placed in a constant temperature dark culture at 26.5° C. until the mycelium is fully fed with the material, thereby obtaining the stock seed; the culture medium in the stock seed bag comprises the following raw materials in parts by weight: 100 parts of cottonseed hulls, 30 parts of wheat bran, 2.5 parts of superphosphate, 1 part of gypsum, and 60 parts of water; (3) The original seed is aseptically inoculated into a production bag, and placed in a dark culture at a constant temperature of 26.5° C. until the mycelium is fully fed with the material, thereby obtaining the production seed; the culture medium in the production bag comprises the following raw materials in parts by weight: 50 parts of broad-leaved wood chips, 34 parts of bran, 50 parts of bagasse, 10 parts of cottonseed hulls, 1.2 parts of superphosphate, 2 parts of gypsum, and 57 parts of water; the broad-leaved wood chips contain at least 20% of Cyclobalanopsis glauca chips or maple wood chips; (4) After the cultivation material is bagged and sterilized, the production species is inoculated, and the inoculated bacterial sticks are piled up in a "well" shape, the temperature is controlled at 26.5° C., and the cultivation is shaded; the culture medium loaded in the cultivation material includes the following raw materials in parts by weight: 85 parts of miscellaneous sawdust, 10 parts of wheat bran, 1 part of gypsum, 0.5 parts of lime, 15 parts of cottonseed hulls, and 70 parts of water; the miscellaneous sawdust contains at least 20% of Cyclobalanopsis glauca sawdust or maple sawdust; (5) When the mycelium has grown all over the bag and the mycelium is physiologically mature, the mycelium is punctured and the mycelium sticks are stacked in a "well" shape for ventilation and cultivation; When the primordium is formed at the puncture hole, the ears can be placed on the ground and managed; (6) Harvest the fungus after it matures.

8. The cultivation method according to claim 7, characterized in that In step (2), the inoculation amount of the production mother seed is 8 g per original seed bag.

9. The cultivation method according to claim 7, characterized in that In step (3), the inoculation amount of the original seed is 8 g for each production bag.

10. The cultivation method according to claim 7, characterized in that In step (4), the inoculation amount of the production seed is 10 g per cultivation bag.

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