Armillaria ostoyae for improving yield and quality of medicinal polyporus in qinba mountainous area

Abstract

This invention discloses a strain of *Armillaria ostoyae* that improves the yield and quality of medicinal *Polyporus umbellatus* in the Qinling-Bashan Mountains. The strain's preservation number is CGMCC No. 40374. The *Armillaria ostoyae* strain provided by this invention not only increases the yield of medicinal *Polyporus umbellatus*, but also increases the content of ergosterol, porphyrin B, and porphyrin polysaccharides in *Polyporus umbellatus*, thereby improving economic benefits.

Description

Technical Field

[0001] This invention relates to a strain of Armillaria mellea that improves the yield and quality of medicinal Poria cocos in the Qinling-Bashan Mountains and its application technology, belonging to the field of microbial technology. Background Technology

[0002] *Polyporus umbellatus* (Pers.) Fr. is a medicinal fungus belonging to the Polyporaceae family. Its sclerotium is used medicinally and has been included in successive editions of the *Chinese Pharmacopoeia*. The term *Polyporus umbellatus* generally refers to the sclerotium of *Polyporus umbellatus*. *Polyporus umbellatus* is sweet, bland, and neutral in nature. It enters the kidney and bladder meridians and has the effect of "promoting diuresis and eliminating dampness." Modern pharmacological studies have shown that the active substances responsible for the diuretic, kidney-protective, and anti-tumor effects of *Polyporus umbellatus* are mainly steroids and polysaccharides. The former, represented by ergosterol, has a significant diuretic effect; porphyrin A and B are characteristic chemical components of *Polyporus umbellatus* and have anti-tumor activity. Polysaccharides can enhance the body's immunity and also have certain anti-tumor effects, and are used clinically as adjuvant therapy for tumors.

[0003] The main producing area of ​​Poria cocos in my country is the Qinling-Bashan Mountains, mainly concentrated in Liuba County, Shaanxi Province. Liuba County has also been awarded the title of "National Poria cocos Seed Source Base County", which has a very good regional representativeness.

[0004] The formation of *Polyporus umbellatus* resources depends on *Armillaria mellea*, with the two establishing a symbiotic relationship. *Armillaria mellea* is not only the direct nutrient source for *Polyporus umbellatus*, but also affects its yield and quality. Co-cultivating *Armillaria mellea* with *Polyporus umbellatus* seeds to obtain *Polyporus umbellatus* sclerotia is currently the only method for cultivating *Polyporus umbellatus*. Therefore, the quality of *Armillaria mellea* is crucial to the development of the *Polyporus umbellatus* industry.

[0005] Currently, a common problem in the cultivation of *Polyporus umbellatus* is the degeneration of *Armillaria mellea*, which directly results in reduced yield and lower quality. Therefore, screening for superior *Armillaria mellea* strains for *Polyporus umbellatus* cultivation is an effective way to solve this problem and has significant practical implications. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of *Armillaria ostoyae* AP29 that can improve the yield and quality of *Polyporus umbellatus* (a medicinal herb) in the Qinling-Bashan Mountains. AP29 was isolated and purified from the sclerotia of wild *Polyporus umbellatus* in Ganzi Prefecture, Sichuan Province, and has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 6, 2023. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC No. 40374. The deposit acceptance notice and viability report for the above-mentioned strain are attached.

[0007] This invention analyzes and compares the rDNA-TEF sequence of AP29 to determine that AP29 belongs to Armillaria ostoyae in biological classification.

[0008] AP29 biological culture characteristics:

[0009] In PDA agar medium, under room temperature (20±5℃) and dark conditions, AP29 mycelia are white, gradually forming a plum blossom shape as the colonies grow. Mycelial cords originate at the inoculation point or can also be formed by hyphal differentiation, spreading outwards in the substrate and reaching the edge of the agar plate within 3 weeks. The tips of the mycelial cords are white, turning red, brownish-red, and then black as they mature. Lateral branching of the mycelial cords is vigorous, with apical branching rarely observed, and the mycelium is abundant. In the culture medium of the cultivated strain, under room temperature (20±5℃) and dark conditions, the AP29 mycelial cords of the cultivated strain grow vigorously, growing from the mouth of the bottle to the bottom. The mycelial cords are white, robust, flat, and highly branched, with some cords adhering to the branches in sheets. AP29 mainly attaches to branches as mycelia and invades the interior of branches as mycelial cords, which can be either mycelia or mycelial cords. During the culture process, the liquid gradually turns honey-yellow and paste-like.

[0010] AP29 can significantly increase the yield of medicinal Polyporus umbellatus in the Qinba Mountains and increase the content of ergosterol, polyporone B and polyporus polysaccharide in Polyporus umbellatus.

[0011] The beneficial effects of this invention are that, compared with existing Armillaria mellea production strains, it can significantly improve the yield and quality of Poria cocos sclerotia, thereby increasing economic benefits. Specific implementation methods

[0012] Example 1: AP29 Armillaria mellea co-culture with Poria cocos experiment

[0013] Preparation of AP29 primary culture medium: Using sawdust as the basal culture medium, a primary culture medium was prepared by adding wheat bran to 4 parts sawdust. An appropriate amount of tap water was added, enough to allow water to flow out between the fingers when lightly squeezed, and the medium was mixed and soaked. The mixture was dispensed into wide-mouth plastic bottles (750ml) to 4 / 5 full volume and autoclaved at 122℃ for 3 hours, then allowed to cool to room temperature (20±5℃). In a clean bench, Armillaria mellea was transferred and cultured at room temperature (20±5℃) in the dark.

[0014] Preparation of AP29 cultivar: Take oak branches with a diameter of 2.0±1.0cm and cut them into short branches about 5cm in length. Fill plastic culture bottles to 9 / 10 full, and add tap water just enough to cover the branches. Autoclave at 122℃ for 3 hours, then allow to cool to room temperature (20±5℃). Inoculate with AP29 cultivar in a clean bench and incubate at room temperature (20±5℃) in the dark.

[0015] AP29 Armillaria mellea cultivation experiment with Poria co-cultivation: A cultivation experiment was conducted in Liuba County, Qinling-Bashan Mountains, in early April. A semi-shaded, semi-sunny forest slope was selected, and a continuous trench was dug along the slope, measuring 40cm x 25cm. Poria co-cultivation was carried out in holes, with three oak linden logs (cut into fish-scale shapes) approximately 10cm in diameter placed parallel and evenly spaced in each hole. Poria cocos seeds were placed at the fish-scale shapes and both ends of the logs, with a seed quantity of 250g per hole. One bottle of AP29 Armillaria mellea spawn was broken into pieces and placed at the location of the Poria cocos seeds. Approximately 500g of oak twigs were placed in the hole, followed by a 5cm layer of leaves, and then a 5cm layer of soil. The holes were separated by a 10cm layer of compacted soil. A commonly used Armillaria mellea strain in Liuba County was used as a control strain and cultivated using the same method. After 4 years, Poria cocos was harvested, and the yield was measured. The results are shown in Table 1.

[0016] At the same seeding rate (250g), the yield of *Polyporus umbellatus* cultivated with the local *Armillaria mellea* strain increased by 433.4%, while the yield of *Polyporus umbellatus* cultivated with the AP29 strain increased by 572.6%. Compared with the local *Armillaria mellea* strain in Liuba County, AP29 significantly increased the yield of *Polyporus umbellatus* (P<0.05), by 26.1%.

[0017] Table 1. Yield of *Polyporus umbellatus* in a cultivation experiment in Liuba County, Qinba Mountains (n=7)

[0018]

[0019] Example 2: Content of ergosterol, porcinione A and B in *Armillaria mellea* co-cultured with AP29

[0020] The contents of ergosterol, poria cocos A and B in the samples of *Polyporus umbellatus* were determined by the standard curve method.

[0021] Take appropriate amounts of ergosterol, poria cocos A, and B reference standards, accurately weighing 1.5 mg, 1.6 mg, and 1.6 mg respectively, and place them in separate 1 ml volumetric flasks. Dissolve them in an appropriate amount of methanol, and then dilute to the mark with methanol to obtain the reference standard stock solutions. Measure 20, 40, 60, 80, 120, and 140 μL of the ergosterol stock solution and 2, 6, 10, 14, 22, and 26 μL of the poria cocos A and B stock solutions respectively into 1 ml volumetric flasks, dilute to the mark with methanol, and mix thoroughly to obtain the mixed reference standard solutions. Accurately inject 20 μL into a high-performance liquid chromatograph (Agilent 1260 / VWD), and perform gradient elution at a rate of 1.0 ml / min in a Bridge RP18 column (250 mm × 4.6 mm column, 5 μm) (Table 2). Mobile phase A is ultrapure water, mobile phase B is acetonitrile, and mobile phase C is methanol. The column temperature was room temperature, and the detection wavelengths were 247 nm (porphyrin A and B) and 283 nm (ergosterol). The ergosterol standard curve equation was y = 24322x - 70.797 (R²). 2=0.999), the standard curve equation for poria cocos A is y = 28316x - 15.059 (R = 0.999). 2 =0.999), the standard curve equation for poria cocos B is y = 40549x - 12.368 (R = 0.999). 2 =0.999)(x is the concentration of the reference standard, and y is the peak area).

[0022] Take *Polyporus umbellatus* cultivated with AP29 and local *Armillaria mellea* strains, wash, dry, pulverize, and sieve (40 mesh). Weigh 3g of the test sample, add 60ml (20 times the volume) of 95% ethanol solution, and soak for 12 hours (overnight). Extract by ultrasonication for 15 minutes, allowing to stand for 5 minutes at intervals, repeating the operation 3 times. Filter using a Buchner funnel, concentrate the filtrate by rotary evaporation, and evaporate to dryness. Redissolve the residue with an appropriate amount of methanol, and dilute to 1ml in a volumetric flask to prepare the test solution. Test and calculate the contents of ergosterol, porcinione A, and B in the sample according to the above method.

[0023] Table 2. Gradient elution conditions of mobile phase

[0024]

[0025]

[0026] The results are shown in Table 2. Compared with the local Armillaria mellea strain, AP29 increased the contents of ergosterol and porcini ketone B by 11.5% and 16.3%, respectively. There was no significant difference in the porcini ketone A content between AP29-cultured Porcini and the local strain.

[0027] Table 2. Contents of ergosterol, poria cocos A, and poria cocos B in cultivated *Polyporus umbellatus* in Liuba County, Qinba Mountain Area (n=5)

[0028]

[0029] Example 3: Detection of Polysaccharide Content in Poria Cocos Co-cultured with Armillaria mellea AP29

[0030] The content of polysaccharides in *Polyporus umbellatus* was determined by the phenol-sulfuric acid method (calculated as glucose).

[0031] Accurately weigh 10 mg of glucose reference standard and place it in a 10 ml volumetric flask. Dissolve in an appropriate amount of ultrapure water and dilute to the mark to obtain a 1 mg / ml glucose stock solution. Accurately measure 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, and 0.50 g of the standard solution into 10 ml stoppered test tubes, and add water to each to a final volume of 2.0 ml. Accurately add 1 ml of 5% phenol solution and shake well. Slowly add 5 ml of concentrated sulfuric acid along the wall and shake well. Let stand for 10 min, then incubate in a 40°C water bath for 15 min. Remove and quickly cool to room temperature in an ice-water mixture. Using an equal volume of distilled water as a blank, measure the absorbance at a wavelength of 490 nm. Set up three replicates for each concentration. Plot a standard curve y = 6.2507x - 0.5089 (R²). 2 =0.999).

[0032] Take *Polyporus umbellatus* cultivated with AP29 and local *Armillaria mellea* strains, wash, dry, pulverize, and sieve (40 mesh). Accurately weigh 2.0 g of the sample and place it in a 100 ml Erlenmeyer flask. Add 40 ml of ultrapure water and soak for 12 hours (overnight). Extract by sonication for 15 minutes, allowing to stand for 5 minutes at intervals, repeating this process 3 times. Filter using a Buchner funnel, concentrate the filtrate by rotary evaporation, add 3 times the volume of 85% ethanol solution, and precipitate overnight at -4℃. Centrifuge at 4500 r / min for 40 min, discard the supernatant, and obtain crude *Polyporus umbellatus* polysaccharide. Dissolve completely in 8 ml of deionized water, and record the volume of each sample after dissolution.

[0033] Accurately pipette 1 ml of each sample polysaccharide solution into a 10 ml stoppered test tube. Add 1 ml of water, then accurately add 1 ml of 5% phenol solution and shake well. Slowly add 5 ml of concentrated sulfuric acid along the wall and shake well. Let stand for 10 min, then incubate in a 40°C water bath for 15 min. Remove and rapidly cool to room temperature in an ice-water bath. Using an equal volume of distilled water as a blank, measure the absorbance at a wavelength of 490 nm and calculate the polysaccharide content of *Polyporus umbellatus* sclerotium.

[0034] Table 3 shows the polysaccharide content of *Polyporus umbellatus* cultivated in Liuba County, Qinba Mountains. Compared with the local *Armillaria mellea* strain, the polysaccharide content of *Polyporus umbellatus* cultivated with AP29 increased by 8.80%.

[0035] Table 6. Polysaccharide content of cultivated *Polyporus umbellatus* in Liuba County, Qinba Mountains (n=5)

[0036]

[0037] Compared with local Armillaria mellea strains, AP29 exhibits advantages in increasing the yield, polysaccharide content, and steroidal component content of *Polyporus umbellatus*. Therefore, AP29 is a high-quality Armillaria mellea strain capable of improving the yield and quality of the traditional Chinese medicine *Polyporus umbellatus*.

Claims

1. An application of Armillaria ostoyae in improving the yield of medicinal Polyporus umbellatus and the content of ergosterol, umbelatrine B and polysaccharide in Polyporus umbellatus in Qinba Mountainous Area, characterized in that, The Armillaria ostoyae has a preservation number of CGMCC No. 40374 and is classified and named as Armillaria ostoyae Armillaria ostoyae ; The transcription elongation factor sequence of Armillaria mellea is as follows: atggagtttt ggcattggaa ggaagtcttg cccttgacga caccggcctt ggtctccttg 60 gtccagccct tgtaccatgg catgctacag agaacgttaa gaggcagcac tggtcatact 120 taggtaagga cttacttggc ggactcctcc aacatgttat caccgtgcca tccagagatg 180 gggacgaaag caacggcctt ggggttgtag ccgaccttct tgatgaaggt ggaagtttcc 240 ttgacgattt cgttgaaccg gtcctcgctc cactgagata acagtcagat ttgcctaagg 300 aaaaaggtaa aacgatagat ctcgtacctt ggtggtgtcc atcttgttga cggcgacgat 360 gagctgcctg acaccgaggg tgaaggcaag gagggcgtgc tctcgggtct gcccgccttt 420 gggagataaa aaaatttgag c 441 The *Armillaria ostreatus* strain was isolated from the sclerotium of wild *Polyporus umbellatus* in Ganzi Prefecture, Sichuan Province.

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