Culture medium for armillaria mellea cultivation and preparation method thereof

Abstract

The invention provides a culture medium for armillaria mellea cultivation and a preparation method thereof, and relates to the technical field of armillaria mellea cultivation. The culture medium is prepared by adopting alkali-treated fermented dendrocalamus brandisii powder, wheat bran, dendrocalamus brandisii shoot shell extracting solution, peptone, gypsum, monopotassium phosphate and magnesium sulfate as raw materials. The bamboo-based armillaria mellea culture medium overcomes the defects in the prior art, a novel bamboo-based armillaria mellea culture medium is prepared by utilizing the current thought of replacing wood with bamboo, the growth efficiency and quality of armillaria mellea can be effectively ensured, and the effects of relieving the environmental pressure and reducing the gastrodia elata planting cost can be achieved on the basis of the bamboo-based armillaria mellea culture medium.

Description

Technical Field

[0001] The present invention relates to the technical field of Armillaria mellea cultivation, and particularly relates to a culture medium for cultivating Armillaria mellea and a preparation method thereof. Background Art

[0002] Armillaria mellea belongs to Basidiomycotina, Agaricales, Tricholomataceae, and is an important parasitic wood-rotting fungus in the genus Armillaria (Fr.) Staude. Fungi of this genus are widely distributed in regions such as Asia, Europe, North America, and Africa. Its life cycle includes two main stages: mycelial growth and fruiting body formation. In the mycelial stage, the fungus exhibits two forms: hyphae and rhizomorphs. Hyphae are tiny filamentous structures, usually hidden in the roots and trunks of decaying wood and almost invisible to the naked eye. Rhizomorphs, on the other hand, are bundle-like structures formed by the tight aggregation of hyphae, which represent the adaptive morphological changes of hyphae under adverse conditions and are responsible for transporting nutrients, water, and oxygen. In addition, rhizomorphs also have the functions of proliferation, expansion, and exploration of new nutrient sources, and are the key way for Armillaria mellea to infect hosts during the vegetative growth stage.

[0003] The cultivation process of Gastrodia elata is significantly different from that of conventional field crops. During the growth process of Gastrodia elata, since Gastrodia elata lacks roots and cannot directly absorb nutrients from other culture media, there is no need for additional fertilization. In addition, Gastrodia elata has no leaves and cannot perform photosynthesis, and it is a completely heterotrophic plant. In this unique ecological environment, a special symbiotic relationship has formed between Gastrodia elata and Armillaria mellea, and this relationship plays a crucial role in the growth and development of Gastrodia elata. More than 80% of its life is spent underground with mycorrhizal fungi. Armillaria mellea attaches to the epidermis of Gastrodia elata through its rhizomorphs and uses a variety of lignocellulose-degrading enzymes secreted by it, such as laccase, cellulase, xylanase, amylase, and pectinase, to decompose the plant cell wall. The activity of these enzymes enables Armillaria mellea to penetrate the cortex of Gastrodia elata and then spread along the cortical cells in the form of hyphae to obtain the required nutrients. When the hyphae of Armillaria mellea invade the cortical cells of Gastrodia elata, Gastrodia elata will activate its defense mechanism and produce antifungal proteins to degrade the invading hyphae. In addition, substances such as lysozyme, digestive enzymes, and chitinase released by Gastrodia elata cells can also digest the invading hyphae and degrade them into small molecule substances. In this way, Gastrodia elata can digest the invading hyphae of Armillaria mellea and convert them into organic nutrients and energy to meet its own growth and development needs. This process ultimately promotes Gastrodia elata to grow from the white tuber or rice tuber stage to the arrowhead tuber stage, completing an important transformation in its life cycle.

[0004] At present, broad-leaved tree species such as traditional oak, cyclobalanopsis glauca, birch, and oak are widely used as the fungus materials for the culture medium of Armillaria mellea cultivated species. Existing research has found that cyclobalanopsis glauca is the most ideal fungus material for accompanying Gastrodia elata. When using cyclobalanopsis glauca as the fungus material for the Armillaria mellea cultivated species, it has excellent properties such as fast growth rate, prosperous hyphae and rhizomorphs, and large biomass (Zhou Yuan. Research on the Biological Characteristics of Gastrodia elata [D]. Northwest A&F University, 2005.). The inventor Zhang Shengming et al. proposed the preparation of a semi-solid Armillaria mellea strain, using broad-leaved tree sawdust as the fungus material for the cultivated species, which improved the germination rate and growth rate of Armillaria mellea (Patent Application No.: 202111503492.2). The inventor Wen Yuchao proposed using pine needles as the fungus material for the Armillaria mellea culture medium and applying it to accompany Gastrodia elata. The sufficient nutrient components of the culture medium enable Armillaria mellea to grow rapidly and improve the quality of Armillaria mellea (Patent Application No.: 201610447337.6). The inventor Yuan Yuan et al. used soybean straw as the Armillaria mellea fungus material to cultivate a culture medium for the Armillaria mellea cultivated species that can reduce the planting cost of Gastrodia elata (Patent Application No.: 202110337024.6). The inventor Ma Mingshan et al. prepared a culture medium using cut wood branches as the Armillaria mellea fungus material, resulting in high survival rate of Armillaria mellea, thick hyphae, and high yield (Patent Application No.: 201310681599.5).

[0005] In short, the current planting cost of Gastrodia elata is still very high. In recent years, the cultivation technology of Gastrodia elata has been popularized, which has also led to a large amount of felling of many tree species. In order to reduce the cost of Armillaria mellea cultivation and reduce environmental pressure, using other shrubs and agricultural waste to prepare the fungus material for the Armillaria mellea cultivated species is an important research direction at this stage. On this basis, how to reduce the acquisition cost of the fungus material and ensure the growth effect of Armillaria mellea is an important research direction at this stage. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the present invention provides a culture medium for cultivating Armillaria mellea and its preparation method, and prepares a new bamboo-based Armillaria mellea culture medium, which not only alleviates environmental pressure, but also reduces the cost problem of Gastrodia elata planting, and at the same time ensures the growth efficiency of Armillaria mellea.

[0007] To achieve the above objectives, the present invention is realized through the following technical solutions: A culture medium for cultivating Armillaria mellea, the formula of the culture medium for cultivating Armillaria mellea is 16-20 parts of alkali-treated fermented Dendrocalamus brandisii powder, 3-5 parts of wheat bran, 20-28 parts of Dendrocalamus brandisii bamboo shoot shell extract, and 0.28-0.32 parts of nutritional additive; the alkali-treated fermented Dendrocalamus brandisii powder is obtained by peeling fresh Dendrocalamus brandisii, cutting it into sections, soaking it in water, inoculating lactic acid bacteria for fermentation treatment, then adjusting the pH of the solution to alkaline, standing treatment, and then adjusting the pH to weakly acidic and drying and pulverizing; the Dendrocalamus brandisii bamboo shoot shell extract is obtained by boiling Dendrocalamus brandisii bamboo shoot shells and then pressing and separating.

[0008] Preferably, the preparation method of the alkali-treated fermented Dendrocalamus brandisii powder comprises the following steps: S1-1: After removing the surface green skin of fresh Dendrocalamus brandisii, cut it into small sections of Dendrocalamus brandisii with a length of 2-5 cm, and then soak it in 3-5 times the amount of clear water; S1-2: Inoculate lactic acid bacteria into the above-mentioned clear water, ferment for 24-30 h, then adjust the pH to 10-11, and let it stand for 10-12 h to obtain a fermented material for standby; S1-3: After adjusting the pH of the above-mentioned fermented material to 5-6, dry it at 60-80 °C until the water content ≤ 8%, then pulverize it, and sieve it through a 60-80 mesh sieve to obtain the alkali-treated fermented Dendrocalamus brandisii powder.

[0009] Preferably, the inoculation amount of lactic acid bacteria in the step S1-2 is 0.05%-0.1% of the total mass of the small sections of Dendrocalamus brandisii.

[0010] Preferably, NaOH is used when adjusting the pH to 10-11, and any one or more of citric acid, tartaric acid, malic acid, and lactic acid are used when adjusting the pH to 5-6.

[0011] Preferably, the preparation method of the Dendrocalamus brandisii shell extract comprises the following steps: S2-1: After washing the Dendrocalamus brandisii shells, add 3-5 times the volume of clear water and grind them into a slurry to obtain a pretreated slurry; S2-2: Heat the pretreated slurry to 80-90 °C and cook it for 30-40 min, then press and filter it, and the filtrate is the Dendrocalamus brandisii shell extract.

[0012] Preferably, the nutritional additive is obtained by mixing peptone, gypsum, potassium dihydrogen phosphate, and magnesium sulfate in a mass ratio of 20∶7∶2∶1.

[0013] The preparation method of the above-mentioned culture medium comprises the following steps: (1) After pulverizing wheat bran, mix it with the alkali-treated fermented Dendrocalamus brandisii powder and bake it in an oven, then add the Dendrocalamus brandisii shell extract and stir evenly to obtain a premixed material for standby; (2) Add the nutritional additive to the above-mentioned premixed material, stir evenly and seal it, and then perform high-pressure steam sterilization to obtain the culture medium.

[0014] In the step (1), the wheat bran is pulverized and sieved through a 60-80 mesh sieve.

[0015] In the step (1), the baking method in the oven is to bake at 40-60 °C for 1-2 h.

[0016] In the step (2), the temperature of the high-pressure steam sterilization is 121 °C, the pressure is 100 kPa, and the sterilization time is 25-30 min.

[0017] The present invention provides a culture medium for cultivating Armillaria mellea and a preparation method thereof. Compared with the prior art, the advantages are as follows: The present invention uses easily obtainable and low-cost bamboo to replace wood, effectively saving the components for cultivating Armillaria mellea. And through alkali treatment after fermenting the bamboo, mixing the extract of bamboo shoots and other nutrient components can effectively improve the growth efficiency of Armillaria mellea mycelium, further promote the growth of thick and strong rhizomorphs, and comprehensively ensure the quality of the growth of Armillaria mellea, providing a basis for the subsequent industrial production of Armillaria mellea rods cultivated with bamboo-based fungus materials and the field companion cultivation of Gastrodia elata. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a broken line graph of the feeding depth of Armillaria mellea at different times in different culture media in the embodiment of the present invention; Figure 2 It is a bar graph of the average daily growth rate of Armillaria mellea within 14 days in the embodiment of the present invention; Figure 3 It is a bar graph of the mycelium germination time of Armillaria mellea in different culture media in the embodiment of the present invention; Figure 4 It is a broken line graph of the thickness of the rhizomorph of Armillaria mellea when cultured for 20 days in different culture media in the embodiment of the present invention; Figure 5 It is a schematic diagram of the growth of Armillaria mellea when cultured in different formula culture media for 4 days, 10 days, and 20 days in the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Embodiment:

[0020] Armillaria mellea culture experiment: 1. Preparation of culture medium raw materials: 1.1 Preparation of Dendrocalamus brandisii powder: (1) Preparation of alkali-treated and fermented Dendrocalamus brandisii powder: After removing the surface green skin of fresh Dendrocalamus brandisii, cut it into small sections of Dendrocalamus brandisii 2 - 5 cm long, and then soak it in 4 times the amount of clear water; inoculate lactic acid bacteria accounting for 0.08% of the total mass of the small sections of Dendrocalamus brandisii into the clear water (purchased from Henan Weilongka Biotechnology Co., Ltd., the viable bacteria count is 2×10 10Per gram, ferment for 26 h, then adjust the pH to 10 with NaOH, let stand for 12 h, and set aside the fermented material. After adjusting the pH of the fermented material to 6 with citric acid, dry it at 70 °C until the water content is ≤8%, then pulverize it and sieve it through a 60-80 mesh sieve to obtain alkali-treated fermented Dendrocalamus brandisii powder.

[0021] (2)Preparation of alkali-treated Dendrocalamus brandisii powder: Take fresh Dendrocalamus brandisii from Xishuangbanna, remove the surface green peel, cut it into small sections of Dendrocalamus brandisii 2-5 cm long, and then soak it in 4 times the volume of clear water. Adjust the pH to 10 with NaOH, let stand for 12 h, and set aside the alkali-treated material. After adjusting the pH of the alkali-treated material to 6 with citric acid, dry it at 70 °C until the water content is ≤8%, then pulverize it and sieve it through a 60-80 mesh sieve to obtain alkali-treated Dendrocalamus brandisii powder.

[0022] 1.2 Preparation of extraction solution: (1)Dendrocalamus brandisii bamboo shoot shell extraction solution: Take the Dendrocalamus brandisii bamboo shoot shells, wash them, add 4 times the volume of clear water, and grind them into a slurry to obtain the pretreated slurry. Heat the pretreated slurry to 85 °C and cook for 35 min, then press and filter. The filtrate is the Dendrocalamus brandisii bamboo shoot shell extraction solution.

[0023] (2)Potato extraction solution: Take the potatoes, peel and wash them, add 4 times the volume of clear water, and grind them into a slurry to obtain the pretreated slurry. Heat the pretreated slurry to 85 °C and cook for 35 min, then press and filter. The filtrate is the potato extraction solution.

[0024] 1.3 Compound preparation of nutritional additives: Compound peptone, gypsum, potassium dihydrogen phosphate, and magnesium sulfate in a mass ratio of 20∶7∶2∶1 to form a nutritional additive.

[0025] Preparation of culture medium: (1)Formula 1 (parts by mass): 18 parts of birch branch powder (pulverized and sieved through a 60-80 mesh sieve after drying) + 4 parts of wheat bran + 24 parts of clear water + 0.3 part of nutritional additive; Preparation: Pulverize the wheat bran and sieve it through a 60-80 mesh sieve, then mix it with the birch branch powder and bake it at 50 °C for 1.5 h. Then mix the clear water and the nutritional additive and stir evenly. Then place it in an autoclave and sterilize it at 121 °C and 100 kPa for 30 min.

[0026] (2)Formula 2 (parts by mass): 18 parts of alkali-treated Dendrocalamus brandisii powder + 4 parts of wheat bran + 24 parts of Dendrocalamus brandisii bamboo shoot shell extraction solution + 0.3 part of nutritional additive; Preparation: Crush wheat bran and sieve it through a 60 - 80 mesh sieve, then mix it with alkali - treated Dendrocalamus brandisii powder and bake at 50 °C for 1.5 h. Then mix it with Dendrocalamus brandisii bamboo shoot shell extract and nutritional additive, stir evenly, and then place it in an autoclave for sterilization at 121 °C and 100 kPa for 30 min.

[0027] (3) Formula III (parts by mass): Alkali - treated fermented Dendrocalamus brandisii powder 18 parts + wheat bran 4 parts + potato extract 24 parts + nutritional additive 0.3 parts; Preparation: Crush wheat bran and sieve it through a 60 - 80 mesh sieve, then mix it with alkali - treated fermented Dendrocalamus brandisii powder and bake at 50 °C for 1.5 h. Then mix it with potato extract and nutritional additive, stir evenly, and then place it in an autoclave for sterilization at 121 °C and 100 kPa for 30 min.

[0028] (4)Formula IV (parts by mass): Alkali - treated fermented Dendrocalamus brandisii powder 18 parts + wheat bran 4 parts + Dendrocalamus brandisii bamboo shoot shell extract 24 parts + nutritional additive 0.3 parts; Preparation: Crush wheat bran and sieve it through a 60 - 80 mesh sieve, then mix it with alkali - treated fermented Dendrocalamus brandisii powder and bake at 50 °C for 1.5 h. Then mix it with Dendrocalamus brandisii bamboo shoot shell extract and nutritional additive, stir evenly, and then place it in an autoclave for sterilization at 121 °C and 100 kPa for 30 min.

[0029] Inoculation and culture experiment: The Armillaria mellea used in the following experiments was purchased from Shaanxi Chunsen Fungi Industry Co., Ltd., and the tissue culture bottles were purchased from Bickman Biotechnology Co., Ltd. (size: 30 * 150 mm) (1)After taking the Armillaria mellea out of the 4 °C refrigerator, disinfect the bottle body with 75% alcohol and then place it in the ultra - clean workbench overnight to break the strain in low - temperature dormancy; (2)Place the raw materials of each group of culture media in the disinfected tissue culture bottles respectively, and the amount of the culture medium raw materials in the tissue culture product accounts for 1 / 2 of the volume of the tissue culture bottle; (3)Use an inoculation knife to cut small pieces of Armillaria mellea about 0.5 * 0.5 * 0.5, and about 3 - 4 pieces in one tissue culture bottle. For each group of culture media, three parallel experiments are conducted; (4)Place the inoculated tissue culture bottles of each group in an incubator for culture, adjust the temperature to 25 °C, no light, and humidity to 80 - 85% for culture; 4. Experimental results: 4.1. Further observe the culture growth process of each group of Armillaria mellea, record the feeding depth of each group of Armillaria mellea at different growth days, and at the same time calculate the average daily growth rate of Armillaria mellea within 14 days. The specific results are as Figure 1 and Figure 2 shown; From Figure 1It can be seen that the mycelia of Formulation 3 and Formulation 4 had grown to the bottom on the 8th day, the mycelia of Formulation 2 grew to the bottom on the 11th day, while the growth of Formulation 1 was relatively slow and grew to the bottom on the 14th day.

[0030] It can be seen from Figure 2 that the fastest average daily growth rate is Formulation 4, with an average of 0.825 cm per day. The slowest is Formulation 1, with an average of 0.467 cm per day. For the other Formulation 2 and Formulation 3, the average daily growth rates are 0.599 cm and 0.799 cm respectively.

[0031] 4.2. Detect and record the mycelial germination time of Armillaria mellea in each group of culture media and the thickness of the rhizomorphs at 20 days of culture, as specifically shown in Figure 3 and Figure 4 shown; The sensory observations of different Armillaria mellea culture media are as shown in Figure 3 shown. The mycelia of Formulation 3 and Formulation 4 germinated on the 2nd day, while the mycelia of Formulation 1 and Formulation 2 began to germinate on the 3rd day. It can be seen from Figure 4 that after 20 days of culture, the rhizomorphs of Formulation 1 grew the thinnest, only 0.349 cm. The rhizomorphs of Formulation 4 grew the thickest, 0.626 cm; those of Formulation 2 and Formulation 3 were 0.469 cm and 0.559 cm respectively; indicating that the rhizomorphs of Armillaria mellea grew the thickest during the culture process in the culture medium of Formulation 4.

[0032] 4.3. Observe and record once every four days. Use a digital vernier caliper to measure the depth of material consumption from outside the bottle, and observe and record the growth conditions of each group of Armillaria mellea at different times; It can be seen from Figure 5 that among the four culture medium formulations, on the 4th day, the growth was relatively lush. Formulation 3 and Formulation 4 were particularly prominent. On the 10th day, Formulation 2, Formulation 3 and Formulation 4 had all reached the bottom, while the growth of Formulation 1 was relatively slow and reached the bottom on the 14th day. Between 10 and 20 days in the middle period, all four formulations showed normal growth. After the 10th day, Formulation 2, Formulation 3 and Formulation 4 began to grow towards the bottom of the tissue culture bottle. Since the culture medium at the bottom of the bottle was relatively dense, the growth of the mycelia and rhizomorphs was relatively slow. But by the 20th day, almost all had filled the bottom of the bottle, and only Formulation 1 had grown a little. After 20 days, the growth changes of the four formulations of the culture medium were relatively microscopic and all grew normally. Brown rhizomorphs began to appear in the culture medium, which also indicated that Armillaria mellea was absorbing nutrients for its own growth and development. At this time, the fermented broth would also turn light yellow, and a brown bacterial ring would appear on the upper layer. This was because after the top layer contacted the external air, it was a self-condition reaction of Armillaria mellea to the external environment and was a normal phenomenon. The specific results are shown in Table 1 below: Table 1

[0033] 4.4. After 20 days of cultivation, the polysaccharide content of Armillaria mellea in each group of culture media was measured, and the specific results are shown in Table 2 below: Table 2

[0034] It can be seen from the above detection that culturing Armillaria mellea with the culture medium of Formula 4 can effectively increase the polysaccharide content in Armillaria mellea and comprehensively improve the quality of Armillaria mellea cultivation.

[0035] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for cultivating Armillaria mellea, characterized in that: The formula of the culture medium for honey fungus cultivation is 16-20 parts of alkali-treated fermented sweet bamboo powder, 3-5 parts of wheat bran, 20-28 parts of sweet bamboo shoot shell extract, and 0.28-0.32 parts of nutritional additives; The alkaline treated fermented sweet bamboo powder is prepared by peeling fresh sweet bamboo, cutting it into sections, soaking it in water, inoculating it with lactic acid bacteria and fermenting it, then adjusting the pH of the solution to alkaline, allowing it to stand for treatment, adjusting the pH to weakly acidic, and then drying and crushing it; The sweet bamboo shoot shell extract is obtained by boiling the sweet bamboo shoot shell and then squeezing and separating it.

2. The culture medium according to claim 1, characterized in that The preparation method of alkali-treated fermented sweet bamboo powder comprises the following steps: S1-1. Take fresh sweet bamboo, remove the green skin on the surface, cut into 2-5cm sweet bamboo segments, and soak in 3-5 times the amount of clean water; S1-2, inoculate lactic acid bacteria into the above clean water, ferment for 24-30 hours, then adjust the pH to 10-11, let stand for 10-12 hours, and obtain the fermented material for use; S1-3, adjusting the pH of the fermented material to 5-6, drying it at 60-80°C to a moisture content of ≤8%, and then crushing it, passing it through a 60-80 mesh sieve to obtain alkali-treated fermented sweet bamboo powder.

3. The culture medium according to claim 2, characterized in that: The inoculation amount of lactic acid bacteria in step S1-2 is 0.05%-0.1% of the total mass of the small segment of sweet dragon bamboo.

4. The culture medium according to claim 2, characterized in that: When adjusting the pH to 10-11, NaOH is used; when adjusting the pH to 5-6, any one or more of citric acid, tartaric acid, malic acid, and lactic acid are used.

5. The culture medium according to claim 1, characterized in that The preparation method of the sweet dragon bamboo shoot shell extract comprises the following steps: S2-1, wash the shells of sweet dragon bamboo shoots, add 3-5 times the volume of clean water, grind into pulp, and obtain pre-treated pulp; S2-2, heating the pretreated slurry to 80-90°C and boiling for 30-40 minutes, and then squeezing and filtering, and the filtrate is the sweet dragon bamboo shoot shell extract.

6. The culture medium according to claim 1, characterized in that: The nutritional additive is obtained by mixing peptone, gypsum, potassium dihydrogen phosphate and magnesium sulfate in a mass ratio of 20:7:2:

1.

7. A method for preparing a culture medium as claimed in any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: (1) crushing wheat bran, mixing with alkali-treated fermented sweet bamboo powder, baking in an oven, adding sweet bamboo shoot shell extract, and stirring evenly to obtain a premix for later use; (2) Add the nutrient additives to the above premix, stir evenly and seal, then sterilize with high-pressure steam to obtain the culture medium.

8. The preparation method according to claim 7, characterized in that: The wheat bran in (1) is crushed and passed through a 60-80 mesh sieve.

9. The preparation method according to claim 7, characterized in that: The method of oven baking in (1) is baking at 40-60° C. for 1-2 hours.

10. The preparation method according to claim 7, characterized in that: The temperature of the high-pressure steam sterilization in (2) is 121° C., the pressure is 100 kPa, and the sterilization time is 25-30 min.

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