A strain of Ganoderma lucidum and a mycelium culture method thereof

Abstract

The present invention belongs to the technical field of edible and medicinal fungi development, and specifically discloses a strain of Ganoderma lucidum and a mycelium culture method thereof. Specifically, the present invention provides a strain of Ganoderma lucidum ( Ganoderma changeable ) strain ZHM1939, with a preservation number of CGMCC No: 41412. The present invention provides a strain of Ganoderma lucidum and its optimal culture method for the first time, providing new materials for the research and clinical application of the edible and medicinal value of Ganoderma lucidum fungi, and providing excellent strains for the artificial cultivation and promotion and application of the fungus, enriching the cultivatable varieties of Ganoderma lucidum, effectively protecting the planting resources of Ganoderma lucidum, and preventing the extinction threat of the fungus caused by human and natural factors.

Description

Technical Field

[0001] The invention belongs to the technical field of development of edible and medicinal fungi, and particularly relates to a strain of Ganoderma lucidum and a mycelium culture method thereof. Background Art

[0002] Ganoderma mutabile is a rare new species of Ganoderma found in Yunnan in recent years. Wild Ganoderma mutabile has problems such as extremely low growth rate, long growth and development cycle, and high requirements for growth environment. It is also very difficult to collect the fruiting bodies and isolate the strains of Ganoderma mutabile. The research on it is only limited to the morphological observation and taxonomy of the fruiting bodies. At present, there is no artificially cultured strain of Ganoderma mutabile and no culture method.

[0003] The invention aims to provide a Ganoderma lucidum strain and a mycelium culture method thereof. Summary of the invention

[0004] The purpose of the present invention is to provide a Ganoderma mutabile ZHM1939 strain and a mycelium culture method of the strain. This is achieved by the following measures:

[0005] The invention provides a Ganoderma mutabile strain ZHM1939, which is classified and named as Ganoderma mutabile, and is deposited at the General Microbiological Center of China Microbiological Culture Collection Committee; the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit time is July 11, 2024; the deposit number is CGMCC No.41412, the biological material is received by the deposit center on July 11, 2024, and is registered. According to the request, the biological material is deposited for 30 years from that date. The viability of the biological material is tested by the deposit center on July 11, 2024, and the result is that the biological material is viable.

[0006] Furthermore, the present invention provides a culture medium for culturing the mycelium of the Ganoderma lucidum strain ZHM1939, wherein the culture medium comprises: 150-250 g / L potato, 15-30 g / L corn flour, 0.8-12.5 g / L wheat bran, 1.5-3 g / L KH2PO4, 0.01-0.04 g / L MnCl2 and 2.0-5.0 mg / L IBA.

[0007] Furthermore, the culture medium comprises: 200 g / L potato, 20 g / L corn flour, 12.5 g / L wheat bran, 2.5 g / L KH2PO4, 0.02 g / L MnCl2 and 2.0 mg / L IBA.

[0008] Furthermore, the culture medium also contains 2-30 g / L agar, and the pH is adjusted to 4.0-8.5.

[0009] Preferably, the culture medium is a solid culture medium containing 25 g / L agar, and the pH is adjusted to 5.0.

[0010] Furthermore, the present invention provides a method for culturing mycelium of the Ganoderma lucidum strain ZHM1939, comprising the following steps:

[0011] (1) preparing any of the culture media, sterilizing it, and cooling it for later use;

[0012] (2) Activation of Ganoderma lucidum strain ZHM1939;

[0013] (3) inoculating the activated strain of Ganoderma lucidum ZHM1939 into the culture medium for cultivation;

[0014] (3) The culture conditions are set as follows: the temperature is 20℃~28℃.

[0015] Furthermore, the culture method sets the culture conditions to a temperature of 25° C. and light-shielded culture.

[0016] The advantages and beneficial effects of the present invention are:

[0017] The invention provides a Ganoderma lucidum strain ZHM1939 which can be used for artificial cultivation, propagation and planting. Compared with other Ganoderma lucidum of the genus Ganoderma, the Ganoderma lucidum has a sessile cap, a slightly oval fresh fruiting body, lignified, and is difficult to separate and culture. The invention provides a pure strain of the Ganoderma lucidum strain and a cultivation method thereof for the first time, provides an excellent strain for the artificial cultivation and promotion and application of the Ganoderma lucidum, enriches the cultivatable varieties of the genus Ganoderma, effectively protects the germplasm resources of the Ganoderma lucidum, and prevents the extinction threat of the Ganoderma lucidum caused by human and natural factors.

[0018] The hetero-shelled Ganoderma lucidum provided by the present invention is a rare new species of the genus Ganoderma. Ganoderma lucidum is a large edible and medicinal fungus, rich in various active ingredients, such as polysaccharides, triterpenes, sterols, amino acids and other active ingredients, has high edible value and medicinal value, and provides new materials for the research and clinical application of the edible and medicinal value of Ganoderma fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The morphological characteristics of the wild fruiting bodies of the Ganoderma lucidum strain ZHM1939 of the present invention are shown; A: fresh fruiting bodies just collected from the wild; B&C: the front and back of the fruiting bodies after being shade-dried in the laboratory for 6 months; D: the fruiting bodies that have been cleaned and shade-dried and stored in the laboratory for 12 months.

[0020] Figure 2 The microscopic characteristic diagrams of the colony and body of the Ganoderma lucidum strain ZHM1939 of the present invention; A: colony cultured on PDA at 25°C for 9 days; B: colony cultured on PDA at 25°C for 20 days; C: skeleton hyphae from the fruiting body; D: basidiospore.

[0021] Figure 3 This is a phylogenetic tree diagram of the Ganoderma lucidum strain ZHM1939 of the present invention. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but any changes made according to the embodiments listed in the present invention fall within the protection scope of the present invention.

[0023] The invention provides a Ganoderma mutabile strain ZHM1939, which is classified and named as Ganoderma mutabile, and is deposited at the General Microbiological Center of China Microbiological Culture Collection Committee; the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit time is July 11, 2024; the deposit number is CGMCC No.41412, the biological material is received by the deposit center on July 11, 2024, and is registered. According to the request, the biological material is deposited for 30 years from that date. The viability of the biological material is tested by the deposit center on July 11, 2024, and the result is that the biological material is viable.

[0024] As an embodiment, a liquid culture medium for culturing mycelium of Ganoderma lucidum strain ZHM1939 comprises: 200 g / L potato, 30 g / L corn flour, 0.8 g / L wheat bran, 1.5 g / L KH2PO4, 0.01 g / L MnCl2 and 2.0 mg / L IBA, with a natural pH and is used after high temperature sterilization.

[0025] As an embodiment, a liquid culture medium for culturing the mycelium of the Ganoderma lucidum strain ZHM1939 includes: 150 g / L potato, 30 g / L corn flour, 4 g / L wheat bran, 1.5 g / L KH2PO4, 0.01 g / L MnCl2, 3.0 mg / L IBA, and 2 g / L agar, with a natural pH and is used after high-temperature sterilization.

[0026] As another embodiment, a solid culture medium for culturing mycelium of Ganoderma lucidum strain ZHM1939: 150 g / L potato, 25 g / L corn flour, 8 g / L wheat bran, 3 g / L KH2PO4, 0.04 g / L MnCl2, 5.0 mg / L IBA and 15 g / L agar, pH 8.0, used after high temperature sterilization.

[0027] As another embodiment, a solid culture medium for culturing mycelium of Ganoderma lucidum strain ZHM1939 comprises: 250 g / L potato, 15 g / L corn flour, 10 g / L wheat bran, 2 g / L KH2PO4, 0.02 g / L MnCl2, 2.0 mg / L IBA and 30 g / L agar, pH 4.0, and is used after high temperature sterilization.

[0028] As another embodiment, a solid culture medium for culturing mycelium of Ganoderma lucidum strain ZHM1939 comprises: 200 g / L potato, 20 g / L corn flour, 12.5 g / L wheat bran, 2.5 g / L KH2PO4, 0.02 g / L MnCl2, 2.0 mg / L IBA and 25 g / L agar, pH 5.0, and is used after high temperature sterilization.

[0029] Example 1 Morphological characteristics of wild fruiting bodies of Ganoderma lucidum strain ZHM1939

[0030] A giant wild Ganoderma lucidum fruiting body collected from Wulao Mountain, Linxiang District, Lincang City, Yunnan Province was observed for morphology. Its morphological characteristics are as follows: Figure 1 ): Fruiting body Fresh fruiting body is slightly oval, lignified, and clearly divided into 12 layers. It grows on the rotten wood substrate with a reduced base. The volume is quite large, and the fresh weight is 80.51kg. The mushroom body is measured to be 38.56cm high, and the long diameter × wide diameter × thickness of the mushroom cap is 63.86cm × 52.35cm × 21.63cm. The surface of the cap is brown-red, with obvious concentric grooves and annuli. The fresh mushroom flesh is dull and has blunt edges. The old mushroom flesh has a lacquer-like luster and smooth edges. The upper layer of the old mushroom flesh is full of white hyphae, and some mosses are attached in the grooves between the mushroom layers. After the fruiting body is placed indoors for 180 days, the surface turns gray-brown, the ventral surface is gray-white, and the mosses on the mushroom body are still alive; the mushroom body is cleaned and stored in the laboratory for 12 months. The fruiting body shrinks slightly and becomes smaller, and the surface color further deepens to dark brown.

[0031] Example 2 Microscopic characteristics of the colony and body of Ganoderma lucidum strain ZHM1939

[0032] By tissue separation method, soybean-sized fungus tissue was picked from the fruiting body of wild Ganoderma lucidum, and then inoculated in the center of a sterile PDA plate in a Φ9.0 cm culture dish and cultured in a constant temperature box at 25°C. The morphology of the colony was observed (photographed) in a timely manner and the diameter (mm) of each colony was measured. At the same time, the morphological characteristics (color, shape, separation, etc.) of hyphae and spores were observed under a microscope, and the size of 30 spores and the diameter (μm) of hyphae were measured. In addition, an appropriate amount of tissue was directly picked from the fruiting body to prepare a slice, and the morphological characteristics of the skeleton hyphae and basidiospores were observed under a microscope. The diameter of 30 hyphae and the size of 30 basidiospores were measured. The morphological characteristics are ( Figure 2): After 9 days of cultivation at 25℃ on PDA plates, the colonies were white, slightly round, with irregular edges and villi, Φ58.26±3.05mm (n=5), and the growth rate was 3.04mm / d; after 20 days of cultivation, a large number of spores formed on the surface of the colonies, making the surface powdery white. Under a microscope, the hyphae were transparent, separated, and branched at nearly right angles, Φ1.28~3.32μm (n=30); the lock-like joint structure formed on the hyphae was clear; the spores were oval or grain-shaped, colorless, single-celled, and small, 4.13~6.25μm×2.31~4.16μm (n=30). The skeleton hyphae of the fruiting body were light gray or reddish gray, separated, and some hyphae were spirally curved at the tips, Φ1.41~2.75μm (n=30). The basidiospores are black-gray, single-celled, oval, with rounded ends and one slightly pointed end, and the size is 6.52-10.26 μm × 4.68-7.17 μm (n=30).

[0033] Example 3 Molecular Biological Identification of the Ganoderma lucidum Strain ZHM1939

[0034] Genomic DNA was extracted from the mycelium of strain ZHM1939 using the cetyltrimethylammonium bromide method (CTAB). The ITS, TEF1-α and RPB2 gene sequence fragments were amplified from the DNA using primers ITS4 / ITS5, TEF1-983 / TEF1-1567 and RPB2-6f / fRPB2-7cR, respectively, and PCR amplified. The PCR amplification products were sequenced, and then the sequencing results were compared online by BLAST, and the phylogenetic tree was constructed and analyzed with the ITS sequences of other species of Ganoderma lucidum in the database ( Figure 3 ), the results showed that the strain was Ganoderma mutabile.

[0035] Example 4 Effect of nutritional conditions on mycelial growth of Ganoderma lucidum strain ZHM1939

[0036] 1) Experimental preparation

[0037] ① Preparation of culture medium: Weigh, heat, dissolve, and make up the volume of the drugs according to the formula, then dispense into 250 mL Erlenmeyer flasks, seal them, and place them in a steam sterilizer. Sterilize at 121°C for 30 min, pour the culture medium onto a plate while it is still hot, and set up 4 replicates for each treatment for later use.

[0038] ② Activation, inoculation and cultivation of strains: Take out the test tube strains of Ganoderma lucidum stored in a 4℃ refrigerator and place them in a 25℃ electric constant temperature incubator in a dark place for about 7 days. Then, using aseptic operation technology, take 1cm 3The edge hyphae of different sizes were re-inoculated in the center of the PDA medium plate and inverted to be cultured in the same environment for 7 days. The solid plate strains of Ganoderma lucidum with vigorous, dense and strong hyphae were selected, and a piece of Ganoderma lucidum with a diameter of 5 mm was taken and re-inoculated into the single factor screening solid plate and cultured in the same way.

[0039] ③ Determination parameters: Determine the mycelium germination time (d), mycelium culture time (d), use the cross method to determine the colony diameter of Ganoderma lucidum, calculate the mycelium growth rate (mm / d), observe the growth potential of mycelium ("+++" indicates dense and strong, "++" indicates relatively dense, "+" indicates sparse growth, and "-" indicates no growth), colony color, etc.

[0040] 2) Single factor test

[0041] Based on the single factor screening basal medium (Table 1), 9 carbon sources (glucose, maltose, fructose, sucrose, sawdust, mannitol, glycerol, soluble starch, corn flour), 16 nitrogen sources (peptone, glycine, (NH4)2SO4, soybean powder, wheat bran, phenylalanine, tyrosine, NH4Cl, urea, yeast extract, beef extract, proline, tryptophan, methionine, lysine, arginine), 5 macroelements (KH2PO4, MgSO4 with mass concentrations of 0.5, 1.0, 1.5, and 2.0 g / L, respectively) were prepared. 4, NaCl, CaCl2, KCl), 5 trace elements (ZnSO4, FeSO4, CoCl2, CuSO4, MnCl with mass concentrations of 0.01, 0.02, 0.03, and 0.04 g / L), and 11 growth factors (vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, vitamin C, naphthylacetic acid, indolebutyric acid, indoleacetic acid, and gibberellin with mass concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / L).

[0042] Table 1 Experimental basal culture medium and formula

[0043]

[0044] The results of the single-factor experiment showed that the optimal carbon source, nitrogen source, macroelements, trace elements and growth factors for the growth of Ganoderma lucidum mycelium were 20g / L corn flour, 2g / L wheat bran, 2.0g / L KH2PO4, 0.02g / L MnCl2 and 4.0mg / L indolebutyric acid, respectively.

[0045] 3) Orthogonal optimization test

[0046] Taking mycelium growth rate as the evaluation index, based on the above single factor experiment, L 16 (45 ) was used to conduct an orthogonal test on the mycelium of Ganoderma lucidum (Table 2).

[0047] Table 2 Orthogonal test factors and levels based on mycelium growth rate

[0048]

[0049] The results of orthogonal optimization test showed (Table 3): According to the verification test, the mycelium of Ganoderma lucidum grew fastest on the combination of A2B4C3D2E1 medium. That is, the optimal combination was 20g / L corn flour, 12.5g / L wheat bran, 2.5g / L KH2PO4, 0.02g / L MnCl2, and 2.0mg / L growth factor IBA.

[0050] Table 3 Orthogonal combination effect

[0051]

[0052]

[0053] Note: Capital letters represent extremely significant differences at the 0.01 level; lowercase letters represent significant differences at the 0.05 level. The same below.

[0054] Example 5 Effect of potato-enriched medium on mycelial growth of Ganoderma lucidum strain ZHM1939

[0055] 1) Experimental preparation

[0056] The preparation of culture medium, activation of bacterial strains, inoculation, culture and measurement parameters are the same as in Example 4.

[0057] 2) Culture medium plate preparation and culture method

[0058] The test formula is as follows (Table 4). Solid plates with a culture dish diameter of 60 mm were prepared and placed in a 25°C electric constant temperature incubator for 7 days in a dark environment.

[0059] Table 4 Experimental culture medium and formula

[0060]

[0061] The experimental results showed that the mycelium of Ganoderma lucidum had the best overall performance on the enriched culture medium, and its growth rate was the fastest, reaching 7.11±0.25mm / d (Table 5). That is, the optimal culture medium for the fungus was 200g / L potato, 20g / L corn flour, 12.5g / L wheat bran, 2.5g / L KH2PO4, 0.02g / L MnCl2, 2.0mg / L indolebutyric acid (IBA), 25g / L agar, 1L deionized water, and pH 5.0.

[0062] Table 5 Effects of different culture media on the growth of mycelium of Ganoderma lucidum

[0063]

Claims

1. A kind of Ganoderma lucidum with different shells ( Ganoderma mutabile ) strain ZHM1939, characterized in that The deposit number is CGMCC No: 41412.

2. A Ganoderma lucidum with a different shell as claimed in claim 1 ( Ganoderma mutabile ) A method for culturing mycelium of strain ZHM1939, characterized in that: The following steps are involved: (1) Prepare culture medium, sterilize it, and cool it for later use; (2) Ganoderma lucidum Ganoderma mutabile ) strain ZHM1939 strain activation; (3) Inoculate the activated Ganoderma lucidum into the culture medium ( Ganoderma mutabile ) strain ZHM1939 was cultured; (4) Set the culture conditions as follows: culture at a temperature of 20°C to 28°C; The culture medium comprises 200 g / L potato, 20 g / L corn flour, 12.5 g / L wheat bran, 2.5 g / L KH2PO4, 0.02 g / L MnCl2 and 2.0 mg / L IBA.

3. The culture method according to claim 2, characterized in that The culture conditions were set at 25°C and light-shielded.

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