Culture medium for promoting growth of sparassis crispa hyphae as well as preparation method and application of culture medium

Abstract

The invention discloses a culture medium for promoting growth of sparassis crispa hyphae as well as a preparation method and application of the culture medium, and belongs to the technical field of microorganism culture. The culture medium is prepared from the following raw materials: 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 invention provides a culture medium capable of rapidly growing sparassis crispa hyphae, so that the sparassis crispa hyphae can grow on a flat plate within 15-18 days, and the problem of slow growth of the sparassis crispa hyphae at present is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial culture, and particularly relates to a culture medium for growing hydrangea mycelium, a preparation method thereof and an application thereof. Background Art

[0002] Sparassia, a member of the Holobasidium subclass, Polyporales order, and Sparassidaceae family, is also known as Sparassia, flower fungus, dry fungus, cabbage fungus, honeycomb fungus, and white hydrangea. Sparassia is a rare and valuable medicinal and edible mushroom with anti-tumor, immune-boosting, blood sugar-lowering, lipid-regulating, anti-inflammatory, and antioxidant properties, earning it the nickname "the treasure of fungi." In 2005, my country achieved its first breakthrough in artificial cultivation of Sparassia, becoming the third country after Japan and South Korea to do so.

[0003] Currently, artificial cultivation of Hydrangea spp. is possible, but existing technologies commonly suffer from slow mycelial growth, low biomass, long production cycles, and low yields. Numerous researchers have continuously optimized Hydrangea spp. mother cultures, but Hydrangea spp. mycelium still suffers from slow germination (beginning after 7 days), slow growth (taking 25-35 days to fill a plate), and poor mycelial consistency on publicly available mother culture media. This results in inefficient basic biological research on Hydrangea spp. and long mother culture preparation cycles, severely hindering its industrial development. Therefore, developing an efficient culture medium is crucial to overcoming the bottlenecks in the industrialization of Hydrangea spp.

[0004] Chinese patents with publication numbers CN115226571A, CN111837811A, CN114303793A, CN106520561A, CN115322909A, CN104429604A, and CN110184200A disclose culture media and preparation methods for the synthesis of various hydrangea fungi and the cultivation of various strains. Each has its own advantages, but generally revolve around the optimization of liquid culture medium formula and the improvement of culture process of hydrangea fungi, mainly focusing on the optimization of carbon and nitrogen source ratios, aiming to increase mycelium yield, shorten the cycle or simplify the process flow, but still fail to solve the problems of slow mycelium growth, poor stability, and low mycelial biomass in solid mother culture medium.

[0005] References can be found in:

[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 nutritional value of protein in mycelium and fruiting body of Hydrangea latifolia[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 the mycelial growth of Hydrangea spp. [J]. Journal of Edible Fungi, 2011, 18(03): 22-26.

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

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

[0014] [9] A liquid culture medium and culture method for Hydrangea spp. CN111837811A.

[0015]

[10] A liquid culture medium for Hydrangea spp. and a culture method thereof CN115226571A.

[0016]

[11] A culture medium for hydrangea and its cultivation method CN114303793A.

[0017]

[12] Preparation method of culture medium for hydrangea cultivation CN106520561A.

[0018]

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

[0019]

[14] A liquid culture medium and culture method for Hydrangea spp. CN104429604A.

[0020]

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

[0021] The purpose of the present invention is to solve the problems existing in the prior art and provide a culture medium capable of shortening the growth period of hydrangea mycelium, as well as a preparation method and application thereof.

[0022] The technical solution adopted by the present invention is as follows:

[0023] A culture medium for promoting the growth of hydrangea mycelium. The raw materials and masses 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 the pH value is 5.5-5.8.

[0024] The preparation method of the culture medium for promoting the growth of hydrangea mycelium of the present invention is as follows:

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

[0026] (2) Heating the container, dissolving the solid raw materials and boiling, then diluting the volume to 1 L, bottling and sterilizing to obtain the culture medium;

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

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

[0029] The culture medium prepared by the method of the present invention is applied by inoculating hydrangea mushroom blocks into a culture dish containing the culture medium, and then placing the dish in a constant temperature and humidity incubator at 23±1°C for culturing in the dark for 15-18 days.

[0030] This study combines ferrous sulfate with Poria cocos sclerotium powder for the first time in the cultivation of Hydrangea mycelium. Iron ions are a key cofactor for lignin-degrading enzymes such as laccase, enhancing their activity. Poria cocos sclerotium powder is rich in β-glucan, which stimulates the growth of Hydrangea mycelium. The combination of ferrous sulfate and Poria cocos sclerotium powder promotes both mycelial growth and accumulation.

[0031] The present invention provides a mother culture medium that promotes the growth of hydrangea mycelium. This medium can promote germination of hydrangea mycelium blocks within approximately three days of inoculation (compared to germination in traditional culture media, which takes 5-7 days). This effectively shortens the growth cycle of hydrangea mycelium, allowing mycelium to fully grow on a plate within 15-18 days of incubation. This solves the current problem of long mycelial growth cycles and is of great significance for subsequent basic biological research on hydrangea mycelium, as well as for industrial cultivation and seed production. Furthermore, mother cultures cultured with this formula can produce mushrooms normally, with a shorter growth cycle and higher yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The germination of Hydrangea on different inorganic salt media is shown;

[0033] Figure 2 The effects of different inorganic salts on the mycelium of Hydrangea oleracea (cultivated for 30 days) are shown;

[0034] Figure 3 The effects of different concentrations of sclerotium powder on the hyphae of Hydrangea spp. are shown;

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

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

[0037] Figure 6 This is the fruiting situation of Hydrangea mycelium cultured in the mother culture medium of Example 1 of the present invention. DETAILED DESCRIPTION

[0038] The present invention will be described clearly and completely below with reference to specific embodiments.

[0039] Study on the germination of Hydrangea mycelium on different inorganic salt culture media:

[0040] The basal medium was composed of 20 g glucose, 3 g fish meal peptone, and 18 g agar. 1 g of ferrous sulfate, magnesium sulfate, zinc sulfate, potassium sulfate, sodium chloride, calcium chloride, and manganese sulfate were added respectively. It was found that the addition of ferrous sulfate could significantly promote the germination of hydrangea mycelium. Figure 1 . Figure 1 In the table, A represents that the added inorganic salt is ferrous sulfate, B represents that the added inorganic salt is magnesium sulfate, C represents that the added inorganic salt is potassium dihydrogen phosphate, D represents that the added inorganic salt is zinc sulfate, E represents that the added inorganic salt is potassium sulfate, F represents that the added inorganic salt is manganese sulfate, G represents that the added inorganic salt is sodium chloride, and H represents that the added inorganic salt is calcium chloride. Figure 2 It also showed that the addition of ferrous sulfate significantly promoted the growth rate of Hydrangea mycelium (culture time 30 days). Figure 2 In the above, the meanings of A, B, C, D, E, F, G, and H are the same as those in Figure 1 same.

[0041] Table 1 Average daily growth rate of Hydrangea mycelium on different inorganic salt 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 the medium with only ferrous sulfate added to the basal medium, the hyphae of Hydrangea were relatively sparse, while when 9-12 g of Poria cocos sclerotium powder was added to the basal medium, the hyphae were dense and grew rapidly. Figure 3 and Table 2.

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

[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 Shown are the growth patterns of Hydrangea mycelium cultured on different media for 18 days. Figure 4 In the figure, A is the growth of Hydrangea mycelium on PDA medium, B is the growth of Hydrangea mycelium on PDPA, C is the growth of Hydrangea mycelium on SM1 (from reference [5] on the effects of carbon source and nitrogen source on Hydrangea mycelium), D is the growth of Hydrangea mycelium on SM2 / pine sawdust juice comprehensive medium (optimized Hydrangea mother culture formula), E is the growth of Hydrangea mycelium on F1 / starch comprehensive medium, F is the growth of Hydrangea mycelium on sclerotium powder medium, G is the growth of Hydrangea mycelium on ferrous sulfate medium, and H is the growth of Hydrangea mycelium on the culture medium of the present invention.

[0047] Further, ferrous sulfate and sclerotium powder are compounded into the basic culture medium to obtain the mycelium of Hydrangea cultured in the culture medium of the present invention. Figure 4 Poria sclerotium powder contains a variety of chemical components such as carbohydrates, polysaccharides, triterpenes, sterols, amino acids, etc. These components can provide a rich carbon source and necessary nutritional support for the mycelium of Hydrangea in the culture medium, thereby promoting its growth and development.

[0048] Example 1

[0049] A culture medium for promoting the growth of hydrangea mycelium. The raw materials and masses 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, and 1L water, with a pH of 5.6.

[0050] The preparation method of the culture medium for promoting the growth of hydrangea mycelium is as follows:

[0051] (1) Weigh 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) Heating the container, dissolving the solid raw materials and boiling, then diluting the volume to 1 L, bottling and sterilizing to obtain the culture medium;

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

[0054] The hydrangea block was inoculated into a culture dish containing the culture medium and placed in a constant temperature and humidity incubator at 23±1°C in the dark for 18 days until the hydrangea hyphae covered the plate. Figure 4 Middle H.

[0055] ITS identification of germinated hyphae:

[0056] Mycelia cultured in this example were collected into a centrifuge tube and DNA was extracted using a fungal genomic DNA extraction kit (Biotech Biotechnology Co., Ltd.). PCR amplification was performed using the universal primers for the fungal ribosomal gene transcribed spacer region, ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), as forward and reverse primers, respectively. The total PCR amplification volume was 50 μL: Mix: 25 μL, ddH2O: 22 μL, 1 μL each of ITS1 and ITS4, and 1 μL DNA template. After mixing, amplification was performed in a PCR instrument using the following PCR program: 94°C for 5 minutes, 94°C for 30 seconds, 56°C for 45 seconds, 72°C for 1 minute, 35 cycles, 72°C for 10 minutes, and 4°C for ∞ (in this case, an unlimited time). The PCR products were sent to Shanghai Sangon Biotechnology Co., Ltd. for bidirectional sequencing. The obtained sequence was submitted to NCBI for BLAST, and the sequence fragments showed 99.34% to 99.5% homology with Sparassis latifolia in GenBank. Figure 5 Therefore, the species was identified as Hydrangea latifolia.

[0057] The mycelium of the hydrangea obtained by culturing with the medium of the present invention was further verified to produce mushrooms. The results showed that the mycelium of the hydrangea obtained by the method could produce mushrooms normally, with a single flower weight of 268.96 g and a biological conversion efficiency of 75.76%. The cycle was shortened by 7 days compared with the control example (the mother culture obtained by removing ferrous sulfate and sclerotium powder from the medium of the present invention was used as the control). Figure 6 and Table 3. Figure 6 The left side is a control example, and the right side is this embodiment.

[0058] Table 3 Data on fruiting of Hydrangea chinensis

[0059] sample Cycle (days) Single flower weight (g / bag) Biological conversion efficiency Example 72 268.96 75.76% Control Example 77 236.78 66.70%

[0060] Example 2

[0061] A culture medium for promoting the growth of hydrangea mycelium. The raw materials and masses 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, and 1L water, with a pH of 5.5.

[0062] The preparation method of the culture medium for promoting the growth of hydrangea mycelium is as follows:

[0063] (1) Weigh 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) Heating the container, dissolving the solid raw materials and boiling, then diluting the volume to 1 L, bottling and sterilizing to obtain the culture medium;

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

[0066] The hydrangea spp. block was inoculated into a culture dish containing the culture medium, and the dish was placed in a constant temperature and humidity incubator at 23±1° C. in the dark for culturing for 16 days until the hydrangea spp. hyphae covered the plate.

[0067] Example 3

[0068] A culture medium for promoting the growth of hydrangea mycelium. The raw materials and masses 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 the pH value is 5.8.

[0069] The preparation method of the culture medium for promoting the growth of hydrangea mycelium is as follows:

[0070] (1) Weigh 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) Heating the container, dissolving the solid raw materials and boiling, then diluting the volume to 1 L, bottling and sterilizing to obtain the culture medium;

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

[0073] The hydrangea spp. block was inoculated into a culture dish containing the culture medium, and the dish was placed in a constant temperature and humidity incubator at 23±1° C. in the dark for culturing for 15 days until the hydrangea spp. hyphae covered the plate.

Claims

1. A culture medium for promoting the growth of hydrangea mycelium, characterized in that The raw materials and mass for preparing the culture medium are: 20 g glucose, 3 g fish meal peptone, 18 g agar, 1 g magnesium sulfate, 0.6-1 g ferrous sulfate, 9-12 g Poria cocos sclerotium powder, 1 L water, and the pH is 5.5-5.

8.

2. The method for preparing a culture medium for promoting the growth of hydrangea mycelium according to claim 1, characterized in that: Here’s how: (1) Weigh glucose, fish meal peptone, agar, magnesium sulfate, ferrous sulfate, sclerotium powder, and water according to the raw material mass ratio, put them into a container, and stir evenly; (2) Heating the container, dissolving the solid raw materials and boiling, then diluting the volume to 1 L, bottling and sterilizing to obtain the culture medium; (3) Dispense the culture medium into culture dishes.

3. The method for preparing a culture medium for promoting the growth of hydrangea mycelium according to claim 2, wherein: In step (3), the culture medium was dispensed into 9-cm diameter culture dishes, with 15 mL of culture medium per dish.

4. Use of the culture medium prepared by the method of claim 2 or 3, inoculating a hydrangea colony into a culture dish containing the culture medium, and culturing in a constant temperature and humidity incubator at 23±1°C in the dark for 15-18 days.

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