A cultivation method of Lepista sordida strain and its fruiting body

Abstract

The present invention provides a Lepista sordida strain, named G-0011, with a preservation number of CGMCC NO. 41166, preserved by the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 5, 2024. The content of total flavonoids in the fruiting body of the Lepista sordida strain is 5.530±0.004mg·g<supgt;−1< / supgt>. The cultivation method of the fruiting body of the above Lepista sordida strain is as follows: The Lepista sordida strain G-0011 is cultured successively through the stages of inoculation, spawn-running, and fruiting to obtain the fruiting body of Lepista sordida. The Lepista sordida strain G-0011 provided by the present invention has stable heredity. The flavonoid content in the fruiting body of Lepista sordida obtained by culturing this strain can reach 5.530±0.004mg·g<supgt;−1< / supgt>, and this strain has the potential for commercial production, with a yield of 4.62±0.17kg·m<supgt;‑2< / supgt> and a biological efficiency of 35.51±1.29%. It has a strong ability in synthesizing total flavonoids and can be used in the preparation of antioxidant health foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of edible mushroom breeding and cultivation, and particularly relates to a strain of Lepista sordida and a cultivation method for its fruiting bodies. Background Art

[0002] Edible mushrooms are a type of food with high nutritional value and delicious taste, capable of supplementing nutrients such as proteins, mineral elements, and vitamins required by the human body. As a cultivable edible mushroom, Lepista sordida is a rare edible mushroom variety that is expected to be successfully commercialized in recent years. Nevertheless, in production practice, there are still problems such as scarce germplasm resources, low yield, unstable traits, difficult cultivation, and lack of excellent varieties.

[0003] Lepista sordida has a rich fragrance, tender texture, and contains abundant nutrients such as amino acids, crude protein, crude fat, and crude fiber. It also has various bioactive substances such as polysaccharides, anthocyanins, and polyphenols. These bioactive components are often used in aspects such as analgesia and anti-inflammation, improving immunity, repairing damaged organs, and regulating body functions. Regarding the functional components of Lepista sordida, relevant research has been carried out on polysaccharides, anthocyanins, etc. Chinese Patent CN111386970 A discloses a strain of Lepista sordida mycelium rich in anthocyanins, its cultivation method and application. This patent mainly promotes the enrichment of anthocyanins in Lepista sordida mycelium by adding external substances such as copper, iron, zinc, and tea polyphenols that promote the generation of anthocyanin synthase and increase enzyme activity. However, this invention is limited to the mycelium stage and does not explore the anthocyanin content in the fruiting body stage. Although anthocyanins belong to flavonoids, their content in Lepista sordida is extremely low, and there is no relevant literature and data support. This invention discloses the anthocyanin content in Lepista sordida mycelium as 15.21 - 45.25 mg·g -1 which far exceeds the value of total flavonoids in the present invention, and the research parameters are very different from the content we disclosed.

[0004] Flavonoids, also known as bioflavonoids, are a class of compounds with a 2-phenyl-chromone structure that exist in free or glycoside forms. Currently, 4,000 flavonoids are known. Common flavonoids include flavones, bioflavonoids, flavanols, biflavonoids, anthocyanins, isoflavones, dihydroflavones, dihydroflavonols, and chalcones. Flavonoids are important secondary metabolites of edible fungi and play various roles in the growth and development of edible fungi. They cannot be directly synthesized in the human body and can only be obtained through food. Edible fungi have good antioxidant, anti-inflammatory, antibacterial, hypoglycemic, anti-cancer, and immune-enhancing activities due to their flavonoid content. In recent years, flavonoids have been regarded as a promising method for delaying aging and exerting a powerful protective effect on neuron damage involved in Parkinson's disease (PD) and Alzheimer's disease (AD). Therefore, flavonoids, as lead compounds for new drug research and development, are a resource worthy of attention and have important practical application significance.

[0005] Currently, the research interest and research efforts in extracting flavonoids from edible fungi as raw materials are increasing continuously, becoming one of the most important and significant research topics in the field of nutrition recently. Existing research has shown that different species of edible fungi contain different flavonoid contents, and different strains of the same species of edible fungi also have different flavonoid contents. Moreover, the flavonoid contents of the same strain are also different under different cultivation substrates (Zhang Shaoyan. Development of a new cultivation substrate for Auricularia polytricha and its effect on flavonoid synthesis and metabolism. Shandong Agricultural University, Tai'an, 2021). The variety recognized in the industry as having a relatively high flavonoid content is Phellinus igniarius. Xie Chunqin et al. studied the flavonoid contents of 17 strains of Phellinus igniarius, and the results showed that the flavonoid contents were 1.16 - 7.17 mg·g -1 (Xie Chunqin, Yang Hetong, Wu Qinyan, et al. Screening test for excellent strains with high yields of flavonoids and polysaccharides in Phellinus igniarius. Jiangsu Agricultural Sciences, 2019, 47(14): 209 - 212). The total flavonoid contents of 9 common wild edible fungi in Pu'er City, Yunnan Province were 0.62 - 6.47 mg·kg -1 (Long Hua, Liu Bin, Li Zhengdong. Extraction, identification and analysis of total flavonoids from 9 common wild edible fungi. Edible Fungi of China, 2019, 38(8): 45 - 47). Fan Li et al. studied and showed that the crude flavonoid contents of 8 edible fungi (Lentinula edodes, Agaricus blazei, Auricularia auricula-judae, Tremella fuciformis, Russula vinosa, Russula puellaris, Boletus luridus, and Dictyophora indusiata) were 0.45 - 5.26 mg·g -1 (Fan Li, Jiang Xianbiao, Xu Zhengyi. Extraction of crude flavonoids from 8 edible fungi and their antioxidant activities. Food Research and Development, 2021, 42(1): 141 - 147). The flavonoid content of Hypsizygus marmoreus was 0.229 mg·g -1 , and the flavonoid content of Pleurotus nebrodensis was 0.375 mg·g -1(Lin Qunying, Ye Yunshou, Song Bin. Comparison of partial components of the fruiting bodies of Lepista sordida and Hypsizygus marmoreus. Guangdong Agricultural Sciences, 2012, (10): 53 - 55). The research by Miao Qianjiang et al. showed that the total flavonoid contents of 4 species of edible fungi (Pleurotus citrinopileatus, Pleurotus ostreatus, Pleurotus geesteranus, and Auricularia auricula) were 0.64 - 3.16 mg·g -1 (Miao Qianjiang, Liu Yu, Xu Feng, et al. Study on the biological functions of total flavonoids in 4 species of edible fungi. Food Science and Technology, 2014, 39(7): 206 - 209). However, there has been no report on the content of total flavonoid substances in Lepista sordida yet.

[0006] In view of the above, the present invention provides a strain of Lepista sordida suitable for commercial cultivation, which will lay a solid foundation for expanding the application fields of flavonoid compounds in Lepista sordida, etc., and at the same time provide a potentially available resource variety for commercial production. It is of great significance for the development and application of Lepista sordida germplasm resources. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a cultivation method for a strain of Lepista sordida and its fruiting body in view of the deficiencies of the above - mentioned existing technologies. The flavonoid content in the fruiting body of this strain of Lepista sordida can reach 5.530 ± 0.004 mg·g −1 and this strain has the potential for commercial production, with a yield that can reach 4.62 ± 0.17 kg·m -2 and the biological efficiency can reach 35.51 ± 1.29%; it has a strong ability in synthesizing total flavonoids and can be used in the preparation of antioxidant health foods.

[0008] To solve the above - mentioned technical problems, the technical solution adopted by the present invention is: A strain of Lepista sordida, named G - 0011, with a preservation number of CGMCC NO. 41166, preserved in the China General Microbiological Culture Collection Center, and the preservation date is February 5, 2024.

[0009] Preferably, the content of high - yield total flavonoids in the fruiting body of the strain of Lepista sordida is 5.530 ± 0.004 mg·g −1 .

[0010] The present invention also provides a cultivation method for the fruiting body of the above - mentioned strain of Lepista sordida, and the cultivation method is as follows:

[0011] S1. Preparation of cultivation materials:

[0012] S101. Mix soybean straw, cow dung, lime, gypsum, and superphosphate evenly to obtain the culture medium;

[0013] S102. Prepare the nutrient solution;

[0014] S103. Pre-fermentation: The culture medium obtained in S101 is pre-wetted with the nutrient solution obtained in S102 to obtain a pre-wetted culture medium. Then, the pre-wetted culture medium is built into a trapezoidal pile, and two rows of ventilation holes are vertically drilled on the trapezoidal pile. When the temperature of the trapezoidal pile reaches 65 - 70 °C, it is maintained for 24 - 48 h until white actinomycetes appear, and the first turning of the pile is started, and water is supplemented. A total of 4 turnings of the pile are carried out to obtain a pre-fermented pile of culture medium;

[0015] S104. Post-fermentation: First, the internal temperature of the pre-fermented pile of culture medium obtained in S103 is raised to 58 - 62 °C and maintained for 12 - 24 h, then the internal temperature of the pre-fermented pile of culture medium is lowered to 50 - 52 °C by ventilation and maintained for 4 - 6 d, and then it is lowered to normal temperature to obtain the cultivation raw material;

[0016] S105. Add water to the cultivation raw material obtained in S104 to make the water content reach 55 - 60%, and then add lime to adjust the pH to obtain the cultivation medium;

[0017] S2. Spreading the medium and sowing: Prepare the land and make beds, leaving operation paths. First, evenly sprinkle a layer of lime at the bottom of the bed. Sowing is carried out in the way of "3 layers of medium and 2 layers of bacteria" from bottom to top. The lower layer is paved with the cultivation medium obtained in S105 with a thickness of 5 - 6 cm. After paving it flat, sprinkle a layer of the Leucocoprinus birnbaumii strain with a thickness of 1 - 2 cm on the surface of the lower layer of medium. The middle layer is paved with the cultivation medium obtained in S105 with a thickness of 5 - 6 cm, and then sprinkle another layer of the Leucocoprinus birnbaumii strain with a thickness of 1 - 2 cm on the surface of the middle layer of medium. The upper layer is finally covered with a layer of the cultivation medium obtained in S105 with a thickness of 3 - 5 cm, and it is compacted and arranged into a turtle-back shape, and holes are drilled in the cultivation medium of the top layer;

[0018] S3. Spawn running: During the spawn running period, regulate the temperature, and keep the relative air humidity at 70 - 75%, and no light is required;

[0019] S4. Casing: 20 d after the spreading of the medium and sowing in S2, start casing, and spray water to make the humidity of the casing layer 60 - 65%;

[0020] S5. Mushroom fruiting management: 15 - 25 d after the casing in S4, the mycelium of Leucocoprinus birnbaumii fills the casing layer. After 4 - 5 d, knots begin to form on the surface. After budding, maintain the air humidity at 80 - 90% and control the temperature at 22 - 26 °C;

[0021] S6. Harvesting: 5 - 7 d after budding, when the edge of the fruit body of Leucocoprinus birnbaumii shows undulations, carry out harvesting.

[0022] Preferably, the culture medium described in S101 is prepared from the following components by mass fraction: soybean straw 62%, cow dung 35%, lime 1%, gypsum 1% and superphosphate 1%.

[0023] Preferably, the preparation method of the nutrient solution in S102 is as follows: add 20 g of corn flour, 2 g of yeast extract, 1.0 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, Mn 2+ 0.25 g, Zn 2+ 0.50 g and Ca 2+ in a ratio of 0.25 g to form a mixed solution, add xanthan gum to the mixed solution and stir evenly to obtain the nutrient solution; the addition amount of the xanthan gum is 0.2% of the dry weight of the culture material obtained in S101.

[0024] Preferably, the moisture content of the pre-wetted culture material in S103 is 55 - 60% (m / m); the height of the trapezoidal material pile is 1.2 - 1.5 m, the width is 2.5 - 3 m; the row spacing of the two rows of ventilation holes is 80 cm, and the hole spacing is 50 cm; the time interval between each turning of the pile is 4 - 6 d.

[0025] Preferably, in S105, lime is added to adjust the pH to 7.0 - 7.5.

[0026] Preferably, the thickness of a layer of lime in S2 is 5 mm, the width of the operation path is 40 - 50 cm, the bottom width of the ridge bed is 1.2 m, the surface width is 1 m, and the height of the ridge surface is 15 - 20 cm; the row spacing of the holes for punching is 10 cm, the hole spacing is 6 cm, and the hole diameter is 4 cm.

[0027] Preferably, in S3, the temperature is controlled at 22 - 26 °C.

[0028] Preferably, the thickness of the soil covering in S4 is 1 - 2 cm.

[0029] The present invention has the following advantages compared with the prior art:

[0030] The Lepista sordida strain G - 0011 cultivated by the present invention has stable heredity. The flavonoid content in the fruiting bodies of Lepista sordida obtained by culturing this strain can reach 5.530 ± 0.004 mg·g −1 -1, and this strain has the potential for commercial production, and the yield can reach 4.62 ± 0.17 kg·m -2 -2, and the biological efficiency can reach 35.51 ± 1.29%. It has strong ability in synthesizing total flavonoids and can be used in the preparation of antioxidant health foods.

[0031] The present invention will be further described in detail below with reference to the drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a photograph of the purified mycelium of Lepista sordida.

[0033] Figure 2It is a specific molecular marker of Lepista sordida strain G-0011. Detailed implementation mode

[0034] Example 1:

[0035] In this example, it is a Lepista sordida strain named G-0011, with the preservation number of CGMCC NO. 41166, the preservation unit being the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the preservation date being February 5, 2024. The content of total flavonoids with high yield in the fruiting body of the Lepista sordida strain is 5.530±0.004mg·g −1 (dry weight);

[0036] The specific primers of the Lepista sordida strain are: TCD1-1F as shown in SEQ ID NO: 1, and TCD1-1R as shown in SEQ ID NO: 2;

[0037] (1) Using the specific primers TCD1-1F / TCD1-1R as PCR primers, with the genomic DNA of the sample to be tested as the template DNA, perform PCR reaction. If there is a PCR product and it is all 193bp, it is identified that the sample to be tested is the Lepista sordida strain G-0011. This pair of specific primers can better identify and screen the Lepista sordida strain G-0011, thus protecting the strain resources.

[0038] (2) The PCR reaction system is: the total system is 25μL, 1.0μL upstream primer (10μM), 1.0 μL downstream primer (10μM), 1.0μL DNA template, 2μL dNTP, 0.5μL Taq enzyme and 2.5μL 10 × PCR Buffer, 17μL ddH2O.

[0039] (3) The amplification program is: pre-denaturation at 95℃ for 4 minutes; denaturation at 94℃ for 30 seconds, annealing at 57℃ for 30 seconds, extension at 72℃ for 90 seconds, 30 cycles; extension at 72℃ for another 10 minutes. The amplification result is as Figure 2 shown. Cut the gel and recover the band in Figure 2 for cloning and sequencing, and obtain the following 193bp sequence result. The sequencing result is consistent with the size of the target band;

[0040] The DNA sequence amplified from the above template DNA is shown in SEQ ID NO: 3.

[0041] The inventor collected the fruiting bodies of wild fungi in 2018 from the Sandaoguan Nature Reserve in Mudanjiang City, Heilongjiang Province.

[0042] Pick the tissue at the junction of the mushroom cap and stipe, and then place it on a PDA medium. Incubate it in the dark at 25 °C for 2 weeks to obtain mycelia. Pick out the mycelia and purify them twice to obtain the fungus G-0011. The steps for each purification are as follows: Place the mycelia on a PDA medium and incubate them in the dark at 25 °C for 1 week.

[0043] 1. Morphological identification

[0044] (1) Morphological characteristics of the fruiting body. The fruiting body grows on the ground at the edge of the forest. The mushroom cap has a diameter of 6 cm, is purple, flat, and the edge shows a petal-like shape. The mushroom flesh is relatively thin, purple, with indistinct stripes. The gills are light purple, slightly sparse, adnate or sinuate, and of unequal length. The stipe is 4 cm long and 0.8 cm thick, the same color as the mushroom cap, curved near the base, with villi, and solid inside.

[0045] (2) Mycelial characteristics. Inoculate the mycelia of the fungus G-0011 on a PDA medium and incubate them at 25 °C for 8 days to observe the growth state of the mycelia.

[0046] The test results show that the colony on the PDA medium is white when young, turns light purple after culturing for 3 - 5 days, and fills the slant after incubating in the dark at 25 °C for 7 - 8 days. The aerial mycelia are vigorous, and the front of the colony is light purple and cottony ( Figure 1 )

[0047] 2. Molecular biological characteristics (ITS) identification

[0048] rDNA-ITS sequence analysis can substantially reflect the base pair differences among genera, species, and strains, and has specificity at the genus and species levels and a relatively fast evolutionary rate. In addition, its sequence fragment is small and easy to analyze. Currently, it has been widely used in genetic variation, phylogenetic relationship analysis, classification, and phylogenetic studies among different species within a genus or between closely related genera of fungi. The specific steps are as follows:

[0049] (1) Extract the genomic DNA of the fungus G-0011 and use it as a template. Perform PCR amplification using the primer pair composed of primer ITS1 (shown in SEQ ID NO: 4) and primer ITS4 (shown in SEQ ID NO: 5) to obtain a PCR amplification product.

[0050] (2) Take the PCR amplification product obtained in step (1) and sequence it.

[0051] The obtained sequence is shown in SEQ ID NO: 6. BLAST the nucleotide sequence obtained by sequencing online in the NCBI database, and then combine the morphological classification method to identify the fungus G-0011 strain as Lepista sordida Lepista sordida .

[0052] Example 2:

[0053] This example was carried out in the Mudanjiang area of Heilongjiang. Sowing was carried out from mid-to-late May to early June, and the bed cultivation method was adopted. The area of each cultivation plot was 15 m 2 , and the feeding amount of each plot was 200 kg (dry weight);

[0054] The cultivation method of the fruiting body of the Lepista sordida strain in Example 1 is as follows:

[0055] S1. Preparation of cultivation materials:

[0056] S101. Mix 62% soybean straw, 35% cow dung, 1% lime, 1% gypsum, and 1% superphosphate evenly to obtain the culture material;

[0057] S102. Prepare the nutrient solution: Add 20 g of corn flour, 2 g of yeast extract, 1.0 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.25 g of Mn 2+ , 0.50 g of Zn 2+ , and 0.25 g of Ca 2+ to 1 L of water and stir evenly to form a mixed solution. Add xanthan gum to the mixed solution and stir until the xanthan gum dissolves to obtain the nutrient solution; the addition amount of the xanthan gum is 0.2% of the dry weight of the culture material obtained in S101; (The nutrient solution is a Chinese invention patent previously applied for by the inventor, with the publication number: CN114946531A)

[0058] S103. Pre-fermentation: Pre-wet the culture material obtained in S101 with the nutrient solution obtained in S102 to obtain a pre-wetted culture material with a moisture content of 57% (m / m). Then, build the pre-wetted culture material into a trapezoidal material pile with a height of 1.2 - 1.5 m, a width of 2.5 - 3 m, and an unlimited length. Make two rows of ventilation holes on the trapezoidal material pile with a row spacing of 80 cm and a hole spacing of 50 cm. When the temperature of the trapezoidal material pile reaches 65 - 70 °C, maintain the temperature for 24 - 48 h until white actinomycetes appear, and start the first turning of the pile and supplement water. A total of 4 turnings of the pile are carried out during the entire pre-fermentation period. The second turning of the pile is carried out 6 days after the first turning, the third turning of the pile is carried out 5 days after the second turning, and the fourth turning of the pile is carried out 4 days after the third turning. Pay attention to turning evenly each time, and turn through the upper, lower, inner, and outer parts. The pre-fermentation process takes 20 - 25 days to obtain the pre-fermented material pile;

[0059] S104. Post-fermentation: First, raise the internal temperature of the pre-fermented material pile obtained in S103 to 58 - 62 °C and maintain it for 12 - 24 h, then ventilate to lower the internal temperature of the pre-fermented material pile to 50 - 52 °C and maintain it for 4 - 6 d, and then lower it to normal temperature (25 - 28 °C) to obtain the cultivation raw material; the cultivation raw material: When grabbed by hand, the material is soft, elastic, odorless of ammonia, without lumps, and without the smell of manure, and a distinct white actinomycete layer can be seen on the surface of the material;

[0060] S105. Add water to the cultivation raw material obtained in S104 to make the water content reach 65% (m / m), and then add lime to adjust the pH to 7.2 to obtain the cultivation material.

[0061] S2. Spreading the material and sowing: Prepare the land and make ridges. The bottom width of the ridge bed is 1.2 m, the top width is 1 m, and the height of the ridge surface is 15 - 20 cm. Reserve an operation path (ridge spacing) with a width of 40 - 50 cm. First, evenly sprinkle a layer of lime with a thickness of 5 mm on the bottom of the ridge bed, and then sow in the way of "3 layers of material and 2 layers of bacteria" from bottom to top. Lay a layer of the cultivation material obtained in S105 with a thickness of 6 cm at the lower layer. After laying it flat, sprinkle a layer of Leucocoprinus birnbaumii strain with a thickness of 2 cm on the surface of the material. Lay a layer of the cultivation material obtained in S105 with a thickness of 6 cm at the middle layer, and then sprinkle another layer of Leucocoprinus birnbaumii strain with a thickness of 2 cm on the surface of the material. Finally, cover the upper layer with a layer of the cultivation material obtained in S105 with a thickness of 5 cm, and compact and arrange it into a turtle-back shape. Punch holes on the cultivation material of the top layer, with the hole row spacing of 10 cm, the hole spacing of 6 cm, and the hole diameter of 4 cm.

[0062] S3. Spawn running: During the spawn running period, control the temperature at 24 °C. When the temperature of the material is too high, cool it down through ventilation. The relative air humidity is preferably maintained at 72%, and no light is required.

[0063] S4. Covering with soil: 20 days after the inoculation described in S2, when the mycelium starts to consume the material and covers 80% of the bed surface, start covering with soil. It is preferred to use garden soil with fertile soil quality and good air permeability for covering with soil. The thickness of the covered soil is 2 cm, and spray water to make the humidity of the covered soil layer 63%.

[0064] S5. Mushroom fruiting management: 15 - 25 days after the covering with soil described in S4, 4 - 5 days after the Leucocoprinus birnbaumii mycelium covers the covered soil layer, knots start to appear on the surface. After budding, adjust the air humidity to 85% and control the temperature at 24 °C.

[0065] S6. Harvesting: 5 - 7 days after budding in S5, when the edge of the Leucocoprinus birnbaumii fruit body shows undulation, start harvesting. When harvesting, hold the mushroom stalk and gently rotate it. After harvesting, cover the exposed mycelium with soil again.

[0066] In the present embodiment S103, the moisture content of the pre-wetted culture material may also be 55, 56, 58, 59 or 60% (m / m); the height of the trapezoidal material pile may be 1.2, 1.3, 1.4 or 1.5 m, and the width may be 2.5, 2.6, 2.7, 2.8, 2.9 or 3 m; when the temperature of the trapezoidal material pile reaches 65, 66, 67, 68, 69 or 70 °C, it is maintained for 24, 25, 30, 33, 35, 40, 45, 47 or 48 h; in S104, first raise the internal temperature of the pre-fermented material pile body to 58, 59, 60, 61 or 62 °C and maintain it for 12, 15, 17, 20, 22 or 24 h, and then ventilate to lower the internal temperature of the pre-fermented material pile body to 50, 51 or 52 °C and maintain it for 4, 5 or 6 d; in S105, add lime to adjust the pH to 7.0, 7.1, 7.3, 7.4 or 7.5; in S2, the thickness of the cultivation material laid in the lower layer may also be 5 cm. After laying it flat, sprinkle a layer of 1 cm thick Lepista sordida strain on the lower layer material surface. The middle layer is laid with the cultivation material obtained in S105 with a thickness of 5 cm, and then a layer of 1 cm thick Lepista sordida strain is sprinkled on the middle layer material surface. The upper layer is finally covered with a layer of the cultivation material obtained in S105 with a thickness of 3 cm or 4 cm; in S3, the regulated temperature may also be 22, 23, 25 or 26 °C, and the relative air humidity may also be maintained at 70, 71, 73, 74 or 75%; in S4, the thickness of the soil covering may also be 1 cm, and water is sprayed to make the humidity of the soil covering layer 60, 61, 62, 64 or 65%; in S5, adjust the air humidity to 80, 82, 84, 87, 89 or 90%, and control the temperature to 22, 23, 25 or 26 °C.

[0067] Experiment 1: The total flavonoid contents of the fruiting bodies of Lepista sordida cultivated with different cultivation materials are different;

[0068] Cultivation material formula:

[0069] T1: 62% soybean straw, 35% cow dung, 1% lime, 1% gypsum, 1% superphosphate.

[0070] T2: 62% rice straw, 35% cow dung, 1% lime, 1% gypsum, 1% superphosphate.

[0071] T3: 62% corn cob, 35% cow dung, 1% lime, 1% gypsum, 1% superphosphate.

[0072] (1) Preparation of total flavonoid and total polyphenol samples

[0073] Precisely weigh 0.5 g of the fruiting body powder, place it in a 50 mL stoppered centrifuge tube, add 25 mL of methanol, weigh it, perform ultrasonic treatment (250 W, 40 kHz) for 30 min, let it stand until room temperature, then precisely weigh it, make up the weight loss with methanol, shake well, 10 000 r·min－1 Centrifuge for 20 min, take the supernatant for determination of total flavonoid content and total phenol content. All measurement indexes are repeated 3 times.

[0074] (2)Determination of total flavonoid and total polyphenol contents

[0075] The determination of total flavonoid content adopts the nitrite - aluminum nitrate method. Respectively take 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mL of 0.307 mg·mL -1 rutin standard solution into a 10.0 mL volumetric flask, add 0.3 mL of 5% NaNO2, shake well and let stand for 5 min; add 0.3 mL of 5% Al(NO3)3, shake well and let stand for 5 min; add 4.0 mL of 4% NaOH, make up the volume to 10.0 mL with water, shake well and let stand for 10 min, measure the absorbance A at 504 nm. Take the mass concentration of rutin (c, mg·mL -1 ) as the abscissa and the absorbance (A) as the ordinate, measure in parallel 3 times to make a standard curve. Measure the absorbance of the sample solution at 504 nm, and calculate the mass concentration of rutin (c, mg·mL -1 ) in the reaction system from the regression equation of the rutin standard curve. Flavonoid content = flavonoid concentration in the sample / flavonoid concentration in the crude extract of the sample × 100%;

[0076] The determination method of total phenol content adopts the Folin - Ciocalteu colorimetric method with slight modification. Take 4 mL of the extract of Lepista sordida, add 1 mL of 0.5 mol·L －1 Folin - Ciocalteu reagent into a 25 mL stoppered test tube, shake well and let stand for 3 min, add 4 mL of 16% sodium carbonate, shake well and let stand in the dark at room temperature for 2 h, measure the absorbance at 760 nm. Take gallic acid as the reference substance to draw a standard curve (0.015 - 0.06 mg·L －1 ), and obtain the standard equation. The measurement result is expressed as mg of gallic acid per g of dry weight. －1

[0077] (3)Preparation and determination of anthocyanin samples

[0078] Sample preparation: Place the fruiting bodies of Lepista sordida in an oven at 60 °C for drying for 2 - 3 d. After complete drying, weigh and grind them into powder. Then add 70% ethanol containing acetic acid according to the mass - to - volume ratio of material to liquid of 1:20, perform microwave extraction at 70 °C with 600 W for 0.5 h and then filter. Collect the filtrate, repeat the extraction operation on the filter cake 2 times, combine the filtrates collected by each filtration, and concentrate by vacuum distillation to a density of 1.0 g·ml −1The obtained extract was added with ethyl acetate in a volume twice that of the extract and extracted twice. The lower layers after each extraction were combined and collected as the retention solution. The obtained retention solution was filtered through a microporous filter membrane with a membrane pressure of 0.25 MPa and a filtration temperature of 55 °C. After rotary evaporation of the filtered filtrate, an anthocyanin extract was obtained.

[0079] Content determination: Using the conventional liquid chromatography method under the same conditions (the volume ratio of water, acetonitrile, tetrahydrofuran and phosphoric acid is 30:35:30:5, the flow rate is 1.2 ml·min −1 , the column temperature is 40 °C, the injection volume is 20 μL, the detector is an ultraviolet detector, the detection wavelength is 525 nm, and the chromatographic column is an ODS C18 reverse-phase column, 4.6*250 mm, 5 μm), the prepared anthocyanin extract was quantified, and its content was expressed as a mass fraction with the unit of mg·g −1 .

[0080] Result analysis:

[0081] Yield distribution and biological efficiency of Lepista sordida under different cultivation substrates.

[0082] Table 1 Yield differences of Lepista sordida under different cultivation substrates

[0083]

[0084] The total yields of three flushes of mushrooms were counted. The data in Table 1 showed that the total yields of T1, T2, and T3 were 4.62±0.17 kg·m -2 , 4.13±0.12 kg·m -2 , 4.31±0.06 kg·m -2 , and the total yield of treatment T1 was the highest at 4.62±0.17 kg·m -2 , significantly higher than the other two treatments. The first flush of mushrooms accounted for about 50% of the total yield, and the second flush of mushrooms accounted for about 30% of the total yield. The fresh weight of the first flush of mushrooms in different treatments ranged from 2.08±0.08 - 2.40±0.10 kg·m -2 , and the yield of the T1 group was significantly higher than the other two groups; the fresh weight of the second flush of mushrooms ranged from 1.15±0.10 kg·m -2 - 1.23±0.12 kg·m -2 , and there was no significant difference among the treatments. The yield range of the third flush was 0.87±0.11 kg·m -2 - 0.99±0.03 kg·m -2 . The biological efficiencies of T1, T2, and T3 were 35.51±1.29%, 31.77±0.94%, and 33.15±0.48% respectively. The biological efficiency of treatment T1 was the highest at 35.51±1.29%, significantly higher than the other treatments.

[0085] Table 2 Effects of Different Cultivation Substrates on Active Substances of Lepista sordida

[0086]

[0087] As shown in Table 2, there were significant differences in the contents of total flavonoids and total polyphenols of Lepista sordida under each cultivation substrate, and there was no significant difference in the anthocyanin content. Moreover, there was no obvious connection between the anthocyanin content and the contents of total flavonoids and total polyphenols. The T1 treatment had higher contents of total flavonoids and total polyphenols, and the T2 treatment had the lowest contents of total flavonoids and total polyphenols. The contents of total flavonoids and total polyphenols in T3 were between those of T1 and T2.

[0088] Experiment 2: Flavonoid Yields of Different Strains on Soybean Straw Substrate (T1 Culture Substrate in Experiment 1)

[0089] Using soybean straw as the cultivation substrate, the ability of different strains to synthesize flavonoids in the fruiting bodies in the same cultivation substrate was investigated. The preparation of the soybean straw culture medium and the cultivation management method were carried out according to Example 2, and the preparation and determination of the flavonoid samples were carried out according to Experiment 1. The selected control Lepista sordida strain was the patented strain DCH618 (authorization number: CN111742778B), and the biological characteristics and flavonoid synthesis ability of different strains were evaluated. The results were as follows:

[0090] (1) Growth of Mycelia and Formation of Primordia

[0091] On the soybean straw culture medium (shown in Table 3), taking the patented strain DCH618 as the control, it was found that there was no obvious difference in the mycelium growth time of the two strains, and the primordium appearance time of the strain of the present invention was 4 days earlier than that of the patented strain DCH618.

[0092] Table 3 Mycelium Growth Time and Primordium Appearance Time of Different Strains

[0093]

[0094] (2) Yield and Biological Efficiency

[0095] The yields of different strains on soybean straw were different. The yields of each flush, the total yield and the biological efficiency of different strains on the soybean straw substrate were statistically analyzed. The results are shown in Table 4. From the statistical data in the table, it can be seen that although the data were different for the yields of each flush, the total yield and the biological efficiency of the two strains, there was no significant difference statistically.

[0096] Table 4 Yields of Different Strains

[0097]

[0098] (3) Synthesis Ability of Total Flavonoids and Total Polyphenols

[0099] The determination results of the total flavonoid content and total polyphenol content of different strains are shown in the following table. It is found that there are significant differences in the abilities of different strains to produce flavonoids and polyphenols. In this invention, the total flavonoid content of the strain reaches 5.555 ± 0.042 mg·g −1 , which is 70.50% higher than that of the control strain DCH618 (3.258 ± 0.007 mg·g −1 ). The corresponding total polyphenol content is 3.744 ± 0.044 mg·g −1 , which is 1.26 times higher than that of the control strain (1.656 ± 0.006 mg·g −1 ).

[0100] Table 5 Total flavonoid content and total polyphenol content of different strains

[0101]

[0102] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent variations made to the above embodiments based on the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A Lepista sordida strain, characterized in that, The strain of Lepista sordida is named G-0011, with the preservation number of CGMCC NO. 41166. The preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the preservation date is February 5, 2024.

2. A cultivation method for the fruiting body of the Lepista sordida strain as described in claim 1, characterized in that, The cultivation method is as follows: S1. Preparation of cultivation materials: S101. Mix 62% of soybean straw, 35% of cow dung, 1% of lime, 1% of gypsum and 1% of superphosphate evenly to obtain the culture medium; S102. Prepare the nutrient solution; S103. Pre-fermentation: Pre-wet the culture medium obtained in S101 with the nutrient solution obtained in S102 to obtain the pre-wetted culture medium. Then, build the pre-wetted culture medium into a trapezoidal pile, and vertically drill two rows of ventilation holes on the trapezoidal pile. When the temperature of the trapezoidal pile reaches 65-70 °C, maintain it for 24-48 h until white actinomycetes appear, and start the first turning of the pile and supplement water. A total of 4 turnings of the pile are carried out to obtain the pre-fermented pile; S104. Post-fermentation: First, raise the internal temperature of the pre-fermented pile obtained in S103 to 58-62 °C and maintain it for 12-24 h, then ventilate to lower the internal temperature of the pre-fermented pile to 50-52 °C and maintain it for 4-6 d, and then lower it to normal temperature to obtain the cultivation raw materials; S105. Add water to the cultivation raw materials obtained in S104 to make the water content reach 55-60%, and then add lime to adjust the pH to obtain the cultivation materials; S2. Spreading materials and sowing: Prepare the land and make beds, reserve operation roads, first evenly sprinkle a layer of lime at the bottom of the bed, and sow in the way of "3 layers of materials and 2 layers of bacteria" from bottom to top. Lay a layer of the cultivation materials obtained in S105 with a thickness of 5-6 cm at the lower layer. After spreading it flat, sprinkle a layer of the Lepista sordida strain with a thickness of 1-2 cm on the surface of the lower layer of materials. Lay a layer of the cultivation materials obtained in S105 with a thickness of 5-6 cm at the middle layer, and then sprinkle another layer of the Lepista sordida strain with a thickness of 1-2 cm on the surface of the middle layer of materials. Finally, cover the upper layer with a layer of the cultivation materials obtained in S105 with a thickness of 3-5 cm, and compact and arrange it into a turtle-back shape, and punch holes on the cultivation materials at the top layer; S3. Spawn running: During the spawn running period, regulate the temperature, and keep the relative air humidity at 70-75%, and no light is required; S4. Covering soil: 20 d after the spreading materials and sowing in S2, start to cover the soil with a thickness of 1-2 cm, and spray water to make the humidity of the covered soil layer 60-65%; S5. Mushroom fruiting management: 15-25 d after the covering soil in S4, the mycelium of Lepista sordida fills the covered soil layer. After 4-5 d, knots begin to appear on the surface. After budding, maintain the air humidity at 80-90%, and control the temperature at 22-26 °C; S6. Harvesting: 5-7 d after budding, harvest before the edge of the fruiting body of Lepista sordida shows undulations.

3. The cultivation method of the fruiting body of the Lepista sordida strain according to claim 2, characterized in that, The preparation method of the nutrient solution in S102 is as follows: add 20 g corn flour, 2 g yeast extract, 1.0 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, and 0.5 g Mn into 1 L of water. 2+ 0.25g, Zn 2+ 0.50g and Ca 2+ A mixed solution is prepared in a ratio of 0.01 to 0.25g, xanthan gum is added to the mixed solution and stirred evenly to obtain a nutrient solution; the amount of xanthan gum added is 0.2% of the dry weight of the culture medium obtained in S101.

4. The cultivation method of the fruiting body of a Lepista sordida strain according to claim 2, characterized in that, The water content of the pre-wetted culture medium described in S103 is 55-60% (m / m); the height of the trapezoidal pile is 1.2-1.5 m, and the width is 2.5-3 m; the row spacing of the two rows of ventilation holes is 80 cm, and the hole spacing is 50 cm; the time interval between each turning of the pile is 4-6 d.

5. The cultivation method of the fruiting body of the Lepista sordida strain according to claim 2, characterized in that, In S105, add lime to adjust the pH to 7.0-7.

5.

6. The cultivation method of the fruiting body of a Lepista sordida strain according to claim 2, characterized in that, The thickness of one layer of lime in S2 is 5 mm, the width of the operation path is 40 - 50 cm, the bottom width of the border bed is 1.2 m, the top width is 1 m, and the height of the border surface is 15 - 20 cm; the row spacing of the holes for drilling is 10 cm, the hole spacing is 6 cm, and the hole diameter is 4 cm.

7. The cultivation method of the fruiting body of a Lepista sordida strain according to claim 2, characterized in that, In S3, the temperature is regulated to 22 - 26 °C.

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