Armillaria strains for increasing the biomass and active ingredient content of Polyporus umbellatus and their application

Abstract

The present invention belongs to the field of microbial technology, and discloses a strain of Armillaria mellea Ag001 that coexists with Polyporus umbellatus, and its deposit number is CGMCC No. 41458. The present invention also provides a biological preparation containing Armillaria mellea Ag001, and a method for increasing the biomass and active ingredient content of Polyporus umbellatus sclerotia. The Armillaria mellea provided by the present invention can significantly increase the biomass of Polyporus umbellatus sclerotia and the content of components such as polysaccharides and steroids, thereby improving economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a high-quality honey fungus capable of increasing the biomass of Polyporus umbellatus sclerotia and the content of active ingredients such as polysaccharides and steroids, and an application thereof. Background Art

[0002] Poria Polyporus umbellatus (Pers.) Fr.] is a precious medicinal fungus with a history of more than 2,000 years of medicinal use in China. Its dried sclerotium is the traditional Chinese medicine Polyporus, which is used as a diuretic and detoxifier to treat edema, acute nephritis, jaundice and other diseases. Polysaccharides and steroids are the main chemical components of Polyporus, among which Polyporus polysaccharide has the effects of inhibiting the proliferation of tumor cells, regulating immunity and anti-inflammatory. Sterols and sterones in steroid components have a strong diuretic effect. In clinical Chinese medicine, it is mainly used to treat acute nephritis, gonorrhea, diabetes, general edema and urinary problems.

[0003] Under natural conditions, the sclerotia of Polyporus must be separated from the sclerotia of Armillaria spp. Armillaria ) Fungi can only grow and develop through symbiosis. There is a special symbiotic relationship between Armillaria mellea and Polyporus umbellatus. Armillaria mellea infects Polyporus umbellatus to form an isolation cavity. Armillaria mellea maintains its own survival by digesting Polyporus umbellatus hyphae. Polyporus umbellatus absorbs the disintegrated Armillaria mellea, promoting its own sclerotium growth and accumulation of chemical components.

[0004] As a key biological factor in the growth and development of Polyporus, the species of Armillaria fungi will affect the biomass and content of pharmacological components of Polyporus sclerotia. At present, the degeneration of Armillaria species has led to a reduction in the yield or quality of Polyporus. Therefore, it is of great significance to screen and apply high-quality Armillaria for the cultivation and production of Polyporus. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a strain of Armillaria melleae ( Armillaria sp.) strain Ag001, which is used to solve the problem of reduced production or quality of Poria cocos caused by the degeneration of Armillaria species.

[0006] The purpose of the present invention and the solution to its technical problem can be achieved by adopting the following technical solutions.

[0007] In the first aspect, the present invention provides a honey fungus strain Ag001, which can increase the biomass of Polyporus sclerotia and the content of pharmacodynamically active ingredients. Honey fungus strain Ag001 was isolated from wild Polyporus sclerotia in Yanqing County, Beijing, and was deposited in the China General Microbiological Culture Collection Management Center on September 20, 2024. The address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101; the deposit number is: CGMCC No. 41458, and the taxonomic name of the strain Ag001 is: Honey fungus Armillariasp. .

[0008] In an embodiment of the first aspect of the present invention, the sequence of the internal transcribed spacer (IGS) of strain Ag001 is shown as SEQ ID NO: 1.

[0009] The present invention studies and analyzes the culture characteristics and rDNA-IGS sequence of Ag001 and determines that Ag001 is Armillaria sp.

[0010] Ag001 has the following morphological characteristics in PDA medium: when cultured in the dark at 25℃, Ag001 hyphae begin to germinate after 2 days, and the hyphae are white and dense; after 4 days, the mycelium begins to grow, and the new mycelium is milky white with bifurcated tips, and gradually becomes curved as the mycelium lengthens; after 6 days, the length of the new mycelium is about 5 mm, and then gradually elongates, circling inside and on the surface of the culture medium, and the color gradually changes from milky white to brown.

[0011] In an embodiment of the first aspect of the present invention, Ag001 can increase the biomass of Poria sclerotia.

[0012] In the embodiment of the first aspect of the present invention, Ag001 can increase the content of the pharmacodynamically active components of Polyporus. In the embodiment of the first aspect, the active components are polysaccharides and steroids. In a specific embodiment, the steroids are ergosterol, Polyporus ketone A and Polyporus ketone B, etc.

[0013] In a second aspect, the present invention provides a biological preparation for increasing the biomass and active ingredient content of Polyporus umbellatus sclerotia, comprising a culture of Armillaria mellea strain Ag001.

[0014] In an embodiment of the second aspect of the present invention, the active ingredients are polysaccharides and steroids. In a specific embodiment, the steroids are ergosterol, poliporin A and poliporin B.

[0015] In a third aspect, the present invention provides a method for increasing the biomass and active ingredient content of Polyporus umbellatus sclerotia, comprising inoculating the cultivated species of Armillaria mellea strain Ag001 at a Polyporus umbellatus seedling planting site.

[0016] In an embodiment of the third aspect of the present invention, the active ingredients are polysaccharides and steroids. In a specific embodiment, the steroids are ergosterol, poliporin A and poliporin B.

[0017] In an embodiment of the third aspect of the present invention, the inoculum amount of Armillaria strain Ag001 cultivar can be 12-18 g / hole. In a specific embodiment, the inoculum amount of Armillaria strain Ag001 cultivar can be 12 g / hole, 12.5 g / hole, 13 g / hole, 13.5 g / hole, 14 g / hole, 14.5 g / hole, 15 g / hole, 15.5 g / hole, 16 g / hole, 16.5 g / hole, 17 g / hole, 17.5 g / hole, 18 g / hole, and values ​​between any two of the above values, preferably 15 g / hole.

[0018] In a fourth aspect, the present invention provides the use of Armillaria mellea strain Ag001 in the preparation of a biological preparation for increasing the biomass and active ingredient content of Polyporus umbellatus sclerotia.

[0019] Compared with the existing Armillaria strains widely used in the cultivation and production of Polyporus umbellatus, the Ag001 strain of the present invention can significantly increase the biomass and the content of polysaccharides and steroid components of Polyporus umbellatus sclerotia. Therefore, using the Ag001 strain to cultivate Polyporus umbellatus can save production costs and improve economic benefits.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following specifically cites a preferred embodiment and describes it in detail with the accompanying drawings as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The colony growth status of Armillaria mellea Ag001 cultured in PDA medium for 30 days is shown. The Ag001 mycelium is soft, winding and curled.

[0022] Figure 2 The colony growth status of Armillaria mellea Am100 (CGMCC No. 21080) cultured in PDA medium for 30 days is shown. The new mycelial cords of Am100 are short, dense and straight. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention, and all of these modifications and substitutions fall within the scope of the claims of the present invention.

[0024] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0025] Example 1: Isolation, culture characteristics and identification of Armillaria mellea strain Ag001

[0026] 1.1 Isolation and culture characteristics of Ag001

[0027] Preparation of PDA solid culture medium: 200 g potato, 20 g glucose, 12 g agar, 1000 mL distilled water, autoclave at 121°C for 30 min.

[0028] Isolation of strains: Take tissue blocks (1 mm × 1 mm) containing Armillaria cordi from the isolation chamber of wild Polyporus umbellatus, rinse with sterile water three times, then disinfect with 75% alcohol solution for 15 s, then disinfect with 5% NaClO solution for 0.5 min, and finally rinse with sterile water three times. Use sterile filter paper to dry the surface water of cordi, and inoculate them into the above-mentioned PDA solid medium with double antibiotics (penicillin and streptomycin).

[0029] Cultivation and morphological observation of the strain: Cultivated in the dark at 25℃, Ag001 hyphae began to germinate after 2 days, and the hyphae were white and dense; after 4 days, the mycelium began to grow, and the new mycelium was milky white with bifurcated tips, and gradually became curved as the mycelium lengthened; after 6 days, the length of the new mycelium was about 5 mm, and then gradually elongated, circling inside and on the surface of the culture medium, and the color gradually changed from milky white to brown.

[0030] After culturing Ag001 and Am100 (CGMCC No. 21080) in PDA medium for 30 days, the colony morphology of the two strains was significantly different: Ag001 had soft, winding and curly roots. Figure 1 ; Am100 new mycelial cords are numerous, short and straight, Figure 2 .

[0031] 1.2 Identification of Ag001 by intraribosome intergenic spacer (IGS) sequence

[0032] The DNA of Armillaria mellea was extracted by CTAB method. Using DNA as template, primers LR12R (5'-GAACGCCTCTAAGTCAGAATCC-3') / 5SRNA (5'-ATCAGACGGGATGCGGT-3') were designed according to the IGS sequence for PCR amplification. The amplification system was 25 μL, containing 12.5 μL of 2×Taq PCR MasterMix (Beijing Aidelai Biotechnology Co., Ltd.), 1 μL of LR12R / 5SRNA primers (10 μmol / L), 2 mL of template DNA (50 ng / mL), and ddH2O to a total volume of 25 μL. The amplification conditions were pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 1 min; annealing at 60 ℃ for 50 s; extension at 72 ℃ for 1.5 min; a total of 38 cycles; and finally, complete extension at 72 ℃ for 10 min, with a termination temperature of 4 ℃. The amplified sequence was sent to Genewise Biotechnology Co., Ltd. for sequencing. After sequencing, the nucleotide sequence of the IGS sequence of the Ag001 strain is shown in SEQ ID NO: 1.

[0033] The IGS sequence was subjected to Blast similarity analysis in the GenBank nucleic acid database. After Blast sequence alignment and phylogenetic tree analysis, it was determined that Ag001 was a fungus of the genus Armillaria.

[0034] Example 2: Cultivation experiment in Beijing medicinal botanical garden

[0035] Preparation of Ag001 solid culture: The basic components of the solid culture medium are wheat bran and sawdust, with a mass ratio of 1:3. Add 1% sucrose solution to adjust the water content of the culture medium to 70%. After mixing, put it into a plastic culture bottle (total volume of 300mL) to about 2 / 3 of the volume, sterilize it by high pressure (122℃, 3h), and then cool it for use. Use a 10 mm diameter puncher to take a piece of Ag001 cultured in PDA culture medium for 30 days, transfer it to the cooled solid culture medium, and culture it in the dark at (25±2)℃ for 30 days to obtain Ag001 solid culture.

[0036] Preparation of Ag001 cultivar: Add 2 cm diameter and 3-5 cm length thick branches (320 g ± 5 g) soaked overnight to 2 / 3 of the plastic culture bottle (total volume 700 mL, empty bottle mass 25 g), add 230 mL of tap water to the plastic bottle, autoclave at 122 ℃ for 3 h, and cool. After inoculating 10 g of Ag001 solid bacteria, weigh the plastic culture bottle to 585 g ± 5 g / bottle, and after culturing at (25 ± 2) ℃ for 90 days (can be used for Polyporus cultivation test), weigh again to 600 g ± 5 g. The mass of the Armillaria strain Ag001 cultivar is estimated to be about 15 g by weight loss method.

[0037] Poria cultivation experiment: Cultivation was carried out in holes, and the specifications of each hole were 30 cm × 30 cm × 30 cm in length × width × height. Three wooden sticks with beveled fish scales were placed in parallel and equidistantly in each hole. 250 g of Poria seedlings were evenly placed beside the fish scales and at both ends of the wooden sticks. One bottle of Ag001 cultivar (total weight of 600.0 g ± 5 g, of which the inoculation amount of each bottle of Armillaria strain Ag001 was about 15 g) was placed near the Poria seedlings, and 250 g of twigs were evenly placed on it, and 200 g of leaves were covered on the top layer. At the same time, cultivation experiments were carried out with Armillaria production strain A9 (CK) (purchased from Qinzheng Poria Development Co., Ltd., Liuba County), Jing-234 (CK) (purchased from Qinzheng Poria Development Co., Ltd., Liuba County) and Am100 cultivar. After 3 years, samples were taken and analyzed for the yield of Poria, and the results are shown in Table 1. Compared with A9(CK) and Jing-234(CK), Ag001 could significantly increase the sclerotium yield of Polyporus umbellatus ( P <0.05); Ag001 could also significantly increase the sclerotium yield of Polyporus umbellatus compared with Am100 ( P <0.05) (Table 1).

[0038] Table 1. The yield of Polyporus umbellatus sclerotia (n=10)

[0039]

[0040] Example 3: Contents of ergosterol, policosanone A and policosanone B in sclerotia of Polyporus umbellatus cultivated in Beijing Medicinal Botanical Garden

[0041] Take the sclerotia of Polyporus umbellatus cultivated with Ag001, Am100 and A9 (CK) and Jing-234 (CK) obtained in Example 2, wash the mud and sand, dry, crush, and pass through a 40-mesh sieve as test samples. Weigh 1 mg of each of the reference substances ergosterol, polyporus ketone A and polyporus ketone B, dissolve them with methanol, and dilute them in 1 mL volumetric flasks, and the concentration of the reference substance solution is 1 mg / mL; ultrasonic treatment is performed until it is completely dissolved to obtain an ergosterol reference substance solution; accurately pipette different volumes of single reference substance polyporus ketone A and polyporus ketone B solutions and mix them, dilute them in a 1 mL volumetric flask, and obtain a mixed reference substance solution for quantitative analysis. Weigh 1000.00 mg of each test sample, soak it in 20 times the volume of 95% ethanol solution for 12 h, ultrasonically extract it for 1 h, centrifuge it for 30 min, take the supernatant, pour the supernatant into an evaporating dish, concentrate it in a water bath at 85 °C, evaporate the solvent, and dilute the residue to a 1 mL volumetric flask with methanol to prepare the sample solution. The HPLC system is a 1260 InfinityLC evaporative light scattering HPLC (Agilent, USA), and the chromatographic column is a Bridge RP18 column (250 mm×4.6 mm, 5 μm). Flow rate: 1.0 mL / min, column temperature 30 °C. Detection wavelengths are 247 nm (polyporin A and polyporin B) and 283 nm (ergosterol). Before injection, the reference and sample solutions were filtered through a 0.22 μm organic phase filter membrane. Mobile phases used for analysis: Phase A: formic acid-water (formic acid: 0.1%, water: 99.9%), Phase B: acetonitrile, see Tables 2 and 3 for chromatographic conditions.

[0042] Table 2. Chromatographic conditions of policosanone A and policosanone B

[0043]

[0044] Table 3. Ergosterol chromatographic conditions

[0045]

[0046] The test results are shown in Table 4: Compared with the Armillaria production strains A9 (CK) and Jing-234 (CK), Ag001 can significantly increase the contents of ergosterol, policogenone A and policogenone B in Polyporus sclerotia ( P <0.05); Compared with Am100, Ag001 can also significantly increase the contents of ergosterol, policogenone A and policogenone B in Polyporus sclerotia ( P <0.05) (Table 4).

[0047] Table 4. Contents of ergosterol and policogenone A / B in Polyporus umbellatus (n=3)

[0048]

[0049] Example 4: Detection of Polyporus polysaccharide content in cultivated Polyporus umbellatus in Beijing Medicinal Botanical Garden

[0050] The phenol-sulfuric acid method (Lu Wenjing, Liang Zongsuo, Wu Yuanting, et al. Effects of different drying methods on the content of polysaccharides and ergosterol in Polyporus umbellatus [J]. Journal of Northwest Forestry University, 2013, 28(4): 5) was used to determine the content of polysaccharides in Polyporus umbellatus. The polyporus sclerotia cultivated with Ag001, Armillaria mellea production strain A9(CK), A9(CK) and Am100 (i.e., the polyporus sclerotia obtained in Example 2) were dried at 60°C to constant weight, 0.05 g was accurately weighed, 1 mL of water was added, the mixture was shaken thoroughly, and the mixture was extracted in a water bath at 100°C for 2 h. After cooling, 10,000 × g Centrifuge for 10 min and take the supernatant. Pipette 0.2 mL of the supernatant into a 1.5 mL centrifuge tube, slowly add 0.8 mL of anhydrous ethanol, mix well and let stand at 4 °C overnight. g Centrifuge for 10 min, discard the supernatant, wash the precipitate with 80% ethanol twice, 1 mL each time, dissolve the precipitate with deionized water, and adjust the volume to 1 mL in a volumetric flask as the sample for analysis. Accurately pipette 200 μL of the sample to be tested, add 100 μL of 5% phenol solution and 0.5 mL of concentrated sulfuric acid, mix well, and place in a 90 ℃ water bath for 20 min. After taking it out, immediately place it in an ice water bath for 30 min to terminate the reaction, and then measure its absorbance at a wavelength of 490 nm on a spectrophotometer, and calculate the content of Polyporus sclerotiorum polysaccharide.

[0051] Compared with Armillaria mellea production strains A9 (CK) and Jing-234 (CK), Ag001 can significantly increase the content of Polyporus polysaccharide ( P <0.05); compared with Am100, Ag001 can also significantly increase the content of Polyporus polysaccharide ( P <0.05), as shown in Table 5.

[0052] Table 5. Content of Polyporus polysaccharide (n=3)

[0053]

Claims

1. Armillaria mellea strain Ag001, whose deposit number is CGMCC No. 41458.

2. The Armillaria mellea strain Ag001 according to claim 1, characterized in that The intraribosome gene spacer (IGS) sequence of the strain Ag001 is shown in SEQ ID NO:

1.

3. A biological preparation for increasing the biomass and active ingredient content of Polyporus umbellatus, characterized in that: The biological preparation comprises the Armillaria strain Ag001 described in claim 1.

4. The biological agent according to claim 3, characterized in that The active ingredients are polyporus polysaccharide, ergosterol, polyporus ketone A and polyporus ketone B.

5. A method for increasing the biomass and active ingredient content of Polyporus umbellatus, characterized in that: The method comprises inoculating the Armillaria strain Ag001 described in claim 1 into a polyporus seedling planting site, wherein the active ingredients are polyporus polysaccharide, ergosterol, polyporus ketone A and polyporus ketone B.

6. The method according to claim 5, characterized in that The inoculation amount of the cultivar of the Armillaria strain Ag001 is 12-18 g / hole.

7. Use of the Armillaria strain Ag001 according to claim 1 in the preparation of a biological preparation for increasing the biomass and active ingredient content of Polyporus umbellatus sclerotia, wherein the active ingredients are Polyporus umbellatus polysaccharide, ergosterol, Polyporus ketone A and Polyporus ketone B.

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