Culture medium for promoting growth of inonotus obliquus mycelium, and preparation method and application thereof

Abstract

The application discloses a culture medium for promoting the growth of Hygrophorus myxoides mycelium and a preparation method and application thereof, and belongs to the technical field of microbial culture. Raw materials for preparing the culture medium are as follows: 20g of glucose, 3g of fish meal peptone, 18g of agar, 1g of magnesium sulfate, 0.6-1g of ferrous sulfate, 10-12g of sclerotium powder and 1L of water, and the pH value is 5.5-5.8. The culture medium provided by the application can rapidly grow the mycelium of Hygrophorus myxoides, and the mycelium of Hygrophorus myxoides can grow on a flat plate within 15-18 days, so that the problem of slow growth of the mycelium of Hygrophorus myxoides is solved.

Description

Technical Field

[0001] This invention belongs to the field of microbial culture technology, specifically relating to a culture medium for the growth of Hydrangea macrophylla mycelium, its preparation method, and its application. Background Technology

[0002] *Sparassia*, belonging to the subclass Holobasidium-sidiomycetidae, order Polyporales, and family Sprassidaceae, is also known as hydrangea mushroom, variegated mushroom, cauliflower mushroom, honeycomb mushroom, and white hydrangea. It is a rare and valuable mushroom used for both medicinal and culinary purposes, possessing pharmacological effects such as anti-tumor activity, immune enhancement, blood sugar reduction, lipid regulation, anti-inflammation, and antioxidant properties, earning it the reputation of a "precious mushroom." In 2005, my country achieved a breakthrough in the artificial cultivation of *Sparassia*, becoming the third country after Japan and South Korea to do so.

[0003] Currently, *Hydrangea macrophylla* has been successfully cultivated artificially, but existing techniques generally suffer from problems such as slow mycelial growth, low biomass, long growth cycles, and low yields. Many researchers have continuously optimized the *Hydrangea macrophylla* mother culture, but the mycelium still exhibits slow germination (germination begins after 7 days), slow growth rate (25-35 days to fully colonize a plate), and poor mycelial uniformity on known publicly available mother culture media. This results in low efficiency in basic biological research on *Hydrangea macrophylla* and a long mother culture preparation cycle, severely hindering its industrial development. Therefore, developing a highly efficient culture medium is one of the keys to overcoming the bottlenecks in the industrialization of *Hydrangea macrophylla*.

[0004] Chinese patents with publication numbers CN115226571A, CN111837811A, CN114303793A, CN106520561A, CN115322909A, CN104429604A, and CN110184200A disclose some culture media and their preparation methods for the synthesis of various *Hydrangea hydrangea* strains and the cultivation of various strains. Each has its own advantages, but they generally focus on optimizing the formulation of liquid culture media for *Hydrangea hydrangea* and improving the cultivation process. They mainly focus on optimizing the carbon and nitrogen source ratio, aiming to increase mycelial yield, shorten the cycle, or simplify the process. However, they still do not solve the problems of slow mycelial growth, poor stability, and low mycelial biomass in solid mother culture media.

[0005] References are available at:

[0006] [1] Sou HD, Ryoo R, Ka KH et al. The mycelial growth and ligninolyticenzyme activity of cauliflower mushroom (sparassis latifolia) [J]. ForestScience and Technology, 2017,13(4), 158–163.

[0007] [2] Dai YC, Wang Z, Binder M, et al. Phylogeny and a new species ofsparassis (Polyporales, Basidiomycota): evidence from mitochondrial atp6,nuclear rDNA and rpb2 genes[J]. Mycologia, 2006, 98(4): 584-592.

[0008] [3] Zhu Douxi, He Ronghua. Economic value and development prospects of rare and precious hydrangea[J]. Edible Fungi, 2008, 3: 8.

[0009] [4]Kimura T. Natural products and biological activity of thepharmacologically active cauliflower mushroom Sparassis crispa[J]. BioMedresearch international, 2013:982317.

[0010] [5] Ma Lu, Lin Yanquan, Ying Zhenghe, et al. Evaluation of the protein nutritional value of mycelium and fruiting body of Hydrangea macrophylla [J]. Food Science, 2016, 37(5): 214-218.

[0011] [6] Lin Yanquan, Ma Lu, Ying Zhenghe, et al. Effects of carbon and nitrogen sources on mycelial growth of Hydrangea rotundifolia [J]. Journal of Edible Fungi, 2011, 18(03):22-26.

[0012] [7] Guo Ye, Chu Rungen, Wang Xiaojun, et al. Optimization of culture medium for Hydrangea macrophylla mother culture [J]. Agricultural Technology and Equipment, 2020, (09): 99-101.

[0013] [8] Chen Zhanghu, Yang Ruiheng, Wu Yingying, et al. Effects of sclerotium powder from two macrofungi on mycelial growth of 11 edible fungi [J]. Fungal Research, 1-9.

[0014] [9] A liquid culture medium for *Hydrangea hydrangea* and a culture method thereof CN111837811A.

[0015]

[10] A liquid culture medium for *Hydrangea hydrangea* and its culture method CN115226571A.

[0016]

[11] A culture medium for *Hydrangea fuciformis* and its cultivation method CN114303793A.

[0017]

[12] Preparation method of culture medium for Hydrangea spp. CN106520561A.

[0018]

[13] A method for preparing a culture medium for *Hydrangea fusiforme* CN115322909A.

[0019]

[14] A liquid culture medium for *Hydrangea hydrangea* and a culture method thereof CN104429604A.

[0020]

[15] A high-yield Hydrangea mycelium fermentation culture medium and its preparation method CN110184200A. Summary of the Invention

[0021] The purpose of this invention is to solve the problems existing in the prior art and to provide a culture medium that can shorten the mycelial growth cycle of Hydrangea fusiforme, its preparation method and application.

[0022] The technical solution adopted in this invention is as follows:

[0023] A culture medium for promoting the mycelial growth of *Hydrangea hydrangea*, wherein the raw materials and their quantities for preparing the culture medium are: 20g glucose, 3g fish meal peptone, 18g agar, 1g magnesium sulfate, 0.6-1g ferrous sulfate, 9-12g *Poria cocos* sclerotium powder, 1L water, and a pH of 5.5-5.8.

[0024] The method for preparing a culture medium to promote the mycelial growth of *Hydrangea hydrangea* according to the present invention is as follows:

[0025] (1) Weigh out glucose, fish meal peptone, agar, magnesium sulfate, ferrous sulfate, sclerotinia powder and water according to the raw material mass ratio, put them into a container and stir evenly;

[0026] (2) Heat the container, dissolve the solid raw material, boil it, and then bring the volume to 1 L. Bottle it for sterilization to obtain the culture medium.

[0027] (3) Dispense the culture medium into petri dishes.

[0028] Furthermore, in step (3) above, the culture medium is dispensed into 9 cm diameter petri dishes, with 15 mL of culture medium per dish.

[0029] The application of the culture medium prepared by the method of the present invention is to inoculate the *Hydrangea hydrangea* mycelium block into a petri dish containing the culture medium and then place it in a constant temperature and humidity incubator at 23±1℃ and culture in the dark for 15-18 days.

[0030] This invention marks the first application of a combination of ferrous sulfate and Poria cocos sclerotium powder to the culture of Hydrangea spp. mycelium. Iron ions are key cofactors for lignin-degrading enzymes such as laccase, enhancing the enzyme activity of Hydrangea spp. Poria cocos sclerotium powder is rich in β-glucan, which stimulates the growth of Hydrangea spp. mycelium; the two work synergistically to promote both mycelial growth and accumulation.

[0031] This invention provides a mother culture medium that promotes the growth of *Hydrangea macrophylla* mycelium. This medium accelerates the germination of *Hydrangea macrophylla* mycelium blocks within approximately 3 days after inoculation (compared to 5-7 days on traditional media), effectively shortening the mycelial growth cycle. This allows the mycelium to fully colonize the plate within 15-18 days, solving the problem of the long mycelial growth cycle currently observed in *Hydrangea macrophylla*. This invention plays a crucial role in subsequent basic biological research on *Hydrangea macrophylla* and in industrialized cultivation and seed production. Furthermore, the mother culture produced using this formula medium can produce fruiting bodies normally, with a shorter cycle and higher yield. Attached Figure Description

[0032] Figure 1 The germination of *Hydrangea macrophylla* on different inorganic salt culture media was shown;

[0033] Figure 2 The effects of different inorganic salts on Hydrangea syringae mycelium were shown (after 30 days of culture).

[0034] Figure 3 The effects of different concentrations of sclerotium powder on Hydrangea mycelium were shown;

[0035] Figure 4 The growth of Hydrangea syringae mycelium after 18 days of culture on different culture media;

[0036] Figure 5 ITS identification results of Hydrangea hydrangea mycelium in Example 1;

[0037] Figure 6 The fruiting of *Hydrangea macrophylla* mycelium cultured on the mother culture medium in Example 1 of this invention is shown. Detailed Implementation

[0038] The present invention will now be clearly and completely described in conjunction with specific embodiments.

[0039] The germination of *Hydrangea macrophylla* mycelium on different inorganic salt culture media was studied.

[0040] Using a basal culture medium of 20g glucose, 3g fishmeal peptone, and 18g agar, and supplemented with 1g each of ferrous sulfate, magnesium sulfate, zinc sulfate, potassium sulfate, sodium chloride, calcium chloride, and manganese sulfate, it was found that the addition of ferrous sulfate significantly promoted the mycelial germination of *Hydrangea rubra*. (See...) Figure 1 . Figure 1 In the table, A represents ferrous sulfate, B represents magnesium sulfate, C represents potassium dihydrogen phosphate, D represents zinc sulfate, E represents potassium sulfate, F represents manganese sulfate, G represents sodium chloride, and H represents calcium chloride. (Table 1 and...) Figure 2 It also showed that the addition of ferrous sulfate significantly promoted the growth rate of Hydrangea hydrangea mycelium (culture time 30 days). Figure 2 In the diagram, what are the meanings of A, B, C, D, E, F, G, and H? Figure 1 same.

[0041] Table 1. Average daily growth rate of *Hydrangea sylvestris* mycelium on different inorganic salt culture media.

[0042] deal with Mycelial growth rate (mm / d) Ferrous sulfate 1.7738 Magnesium sulfate 1.1363 Potassium dihydrogen phosphate 1.1120 Zinc sulfate 0.7871 potassium sulfate 0.9363 manganese sulfate 0.3651 Sodium chloride 0.9976 Calcium chloride 0.8948

[0043] However, in basal culture media with only ferrous sulfate added, the mycelium of *Hypericum hygroscopicum* was relatively sparse. But when 9-12g of *Poria cocos* sclerotium powder was added to the basal culture medium, the mycelium became dense and the growth rate was rapid. Figure 3 And Table 2.

[0044] Table 2. Average daily growth rate of *Hydrangea syringae* on culture media with different concentrations of sclerotium powder.

[0045] Sclerotium powder concentration (g / L) Average daily growth rate of mycelium (mm / d) 0 0.57 3 1.08 6 1.22 9 1.36 12 1.34 15 1.31 18 1.30 21 1.29

[0046] Figure 4 The growth of *Hydrangea hydrangea* mycelium was shown after 18 days of culture on different culture media. Figure 4 In the table, A represents the growth of *Hydrangea hydrangea* mycelium on PDA medium, B represents the growth of *Hydrangea hydrangea* mycelium on PDPA, C represents the growth of *Hydrangea hydrangea* mycelium on SM1 (from reference [5] the effect of carbon and nitrogen sources on *Hydrangea hydrangea* mycelium), D represents the growth of *Hydrangea hydrangea* mycelium on SM2 / pine sawdust juice composite medium (optimized *Hydrangea hydrangea* mother culture formula), E represents the growth of *Hydrangea hydrangea* mycelium on F1 / starch composite medium, F represents the growth of *Hydrangea hydrangea* mycelium on sclerotium powder medium, G represents the growth of *Hydrangea hydrangea* mycelium on ferrous sulfate medium, and H represents the growth of *Hydrangea hydrangea* mycelium on the medium of this invention.

[0047] Further, ferrous sulfate and sclerotium powder were combined and added to the basal culture medium to obtain the *Hydrangea hydrangea* mycelium cultured in the culture medium of this invention, see [link to article]. Figure 4 The sclerotium powder of *Poria cocos* contains various chemical components such as carbohydrates, polysaccharides, triterpenoids, sterols, and amino acids. These components can provide abundant carbon sources and necessary nutritional support for *Poria cocos* mycelia in the culture medium, thereby promoting its growth and development.

[0048] Example 1

[0049] A culture medium for promoting the mycelial growth of *Hydrangea hydrangea*, wherein the raw materials and their quantities for preparing the culture medium are: 20g glucose, 3g fish meal peptone, 18g agar, 1g magnesium sulfate, 0.8g ferrous sulfate, 12g *Poria cocos* sclerotium powder, 1L water, and pH 5.6.

[0050] The method for preparing the culture medium that promotes the growth of Hydrangea macrophylla mycelium is as follows:

[0051] (1) Weigh out glucose, fish meal peptone, agar, magnesium sulfate, ferrous sulfate, poria cocos sclerotium powder and water according to the raw material mass ratio, put them into a container and stir evenly;

[0052] (2) Heat the container, dissolve the solid raw material, boil it, and then bring the volume to 1 L. Bottle it for sterilization to obtain the culture medium.

[0053] (3) Dispense the culture medium into 9cm diameter petri dishes, 15 mL of culture medium per dish.

[0054] The *Hydrangea macrophylla* mycelium was inoculated into a petri dish containing the aforementioned culture medium and incubated in a constant temperature and humidity incubator at 23±1℃ in the dark for 18 days. The *Hydrangea macrophylla* mycelium then completely covered the plate. See [link / reference]. Figure 4 H in the middle.

[0055] ITS identification of germinating hyphae:

[0056] The mycelia cultured in this example were picked and transferred to centrifuge tubes, and DNA was extracted according to the kit (BaiTek Biotechnology Co., Ltd. Fungal Genomic DNA Extraction Kit). PCR amplification was performed using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') for the fungal ribosomal gene transcription spacer region, respectively. The total PCR amplification volume was 50 μL: Mix: 25 μL, ddH2O: 22 μL, ITS1 and ITS4: 1 μL each, DNA template: 1 μL. After mixing, the amplification reaction was performed in a PCR instrument. The PCR amplification program was: 94℃—5 min, 94℃—30 s, 56℃—45 s, 72℃—1 min, 35 cycles, 72℃—10 min, 4℃—∞ (unlimited time). The PCR amplification products were sent to Shanghai Sangon Biotech Co., Ltd. for bidirectional sequencing. The obtained sequence was submitted to NCBI for BLAST analysis. The sequence fragment showed 99.34%–99.5% homology with *Sparassis latifolia* in GenBank. (See attached image.) Figure 5 Therefore, the species was identified as *Hydrangea macrophylla*.

[0057] Further verification of the fruiting of *Hydrangea macrophylla* mycelium obtained from the culture medium of this invention was conducted. The results showed that the *Hydrangea macrophylla* mycelium obtained by this method could produce fruit normally, with a single fruit weight of 268.96 g and a biological conversion efficiency of 75.76%. Furthermore, the fruiting cycle was 7 days shorter than the control example (using a mother culture obtained by removing ferrous sulfate and sclerotium powder from the culture medium of this invention as a control). Figure 6 And Table 3. Figure 6 The left side of the image shows a comparative example, and the right side shows this embodiment.

[0058] Table 3 Fruiting data of *Hydrangea macrophylla*

[0059] sample Cycle (days) Weight of a single flower (g / bag) Biological conversion efficiency Example 72 268.96 75.76% Comparison Example 77 236.78 66.70%

[0060] Example 2

[0061] A culture medium for promoting the mycelial growth of *Hydrangea hydrangea*, wherein the raw materials and their quantities for preparing the culture medium are: 20g glucose, 3g fish meal peptone, 18g agar, 1g magnesium sulfate, 1g ferrous sulfate, 9g *Poria cocos* sclerotium powder, 1L water, and pH 5.5.

[0062] The method for preparing the culture medium that promotes the growth of Hydrangea macrophylla mycelium is as follows:

[0063] (1) Weigh out glucose, fish meal peptone, agar, magnesium sulfate, ferrous sulfate, poria cocos sclerotium powder and water according to the raw material mass ratio, put them into a container and stir evenly;

[0064] (2) Heat the container, dissolve the solid raw material, boil it, and then bring the volume to 1 L. Bottle it for sterilization to obtain the culture medium.

[0065] (3) Dispense the culture medium into 9cm diameter petri dishes, 15 mL of culture medium per dish.

[0066] The *Hydrangea macrophylla* mycelium blocks were inoculated into a petri dish containing the culture medium and placed in a constant temperature and humidity incubator at 23±1℃ for 16 days in the dark, until the *Hydrangea macrophylla* mycelium covered the plate.

[0067] Example 3

[0068] A culture medium for promoting the mycelial growth of *Hydrangea hydrangea*, wherein the raw materials and their quantities for preparing the culture medium are: 20g glucose, 3g fish meal peptone, 18g agar, 1g magnesium sulfate, 0.6g ferrous sulfate, 11g *Poria cocos* sclerotium powder, 1L water, and pH 5.8.

[0069] The method for preparing the culture medium that promotes the growth of Hydrangea macrophylla mycelium is as follows:

[0070] (1) Weigh out glucose, fish meal peptone, agar, magnesium sulfate, ferrous sulfate, poria cocos sclerotium powder and water according to the raw material mass ratio, put them into a container and stir evenly;

[0071] (2) Heat the container, dissolve the solid raw material, boil it, and then bring the volume to 1 L. Bottle it for sterilization to obtain the culture medium.

[0072] (3) Dispense the culture medium into 9cm diameter petri dishes, 15 mL of culture medium per dish.

[0073] The *Hydrangea macrophylla* mycelium blocks were inoculated into a petri dish containing the culture medium and placed in a constant temperature and humidity incubator at 23±1℃ for 15 days in the dark. The *Hydrangea macrophylla* mycelium then covered the plate.

Claims

1. A method to promote the growth of *Hydrangea macrophylla* (… Sparassis latifolia The culture medium for mycelial growth is characterized by, The raw materials and quality for preparing the culture medium are as follows: glucose 20 g, fish meal peptone 3 g, agar 18 g, magnesium sulfate 1 g, ferrous sulfate 0.6-1 g, poria sclerotium powder 9-12 g, water 1 L, and pH is 5.5-5.

8.

2. The method of claim 1, wherein the medium for promoting hyphal growth of G. virens is prepared by the steps of: Sparassis latifolia The method is as follows: ​ (1) glucose, fish meal peptone, agar, magnesium sulfate, ferrous sulfate, poria sclerotium powder and water are weighed according to the quality ratio of raw materials, put into a container and stirred uniformly; (2) heat the container, dissolve the solid raw materials, boil and then make up to 1 L, sterilize the bottle, and obtain the culture medium; (3) the culture medium is divided into culture dishes.

3. A method of producing a medium for promoting hyphal growth of a Ganoderma applanatum according to claim 2, characterized by, Sparassis latifolia ) adding a water-soluble extract of a Ganoderma applanatum to a medium. In step (3), the culture medium is divided into culture dishes with a diameter of 9 cm, and each dish contains 15 mL of culture medium.

4. The culture medium prepared by the method of claim 2 or 3 for *Hydrangea macrophylla* (… Sparassis latifolia In mycelial culture, the *Hydrangea hydrangea* mycelial blocks are inoculated into a petri dish containing the culture medium and then placed in a constant temperature and humidity incubator at 23±1℃ and cultured in the dark for 15-18 days.

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