Culture medium and method for inducing sclerotia of polyporus umbellatus

Abstract

The invention belongs to the technical field of microorganisms, and discloses a wood stick culture medium for inducing sclerotia of polyporus umbellatus, the wood stick culture medium contains hardwood small sticks, glucose, monopotassium phosphate, magnesium sulfate and water, and the mass ratio of the hardwood small sticks to the glucose to the monopotassium phosphate to the magnesium sulfate to the water is 2000: 2: 1: 1: 2000. The invention also discloses a method for inducing sclerotia of polyporus umbellatus. The method comprises the following steps: 1) preparing the stick culture medium according to claim 1; (2) disinfecting the polyporus umbellatus seed; and (3) inoculating an armillaria mellea strain, and carrying out co-culture on the armillaria mellea strain. According to the method, the growth cycle of the polyporus umbellatus sclerotia can be greatly shortened, and meanwhile the yield of the polyporus umbellatus sclerotia and the content of effective components can be increased.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a wooden stick culture medium for inducing sclerotia of *Polyporus umbellatus* and a method for inducing sclerotia of *Polyporus umbellatus* using the same culture medium. Background Technology

[0002] *Polyporus umbellatus* (Pers.) Fr., belonging to the Polyporaceae family of the Basidiomycetes phylum, is also known as wild boar umbellatus or ground pupa. It is a valuable medicinal fungus with a medicinal history in my country spanning over 2000 years. Its medicinal part is the dried sclerotium, which possesses both diuretic and detoxifying effects. Clinically, it is mainly used to treat symptoms such as systemic edema, jaundice, frequent and urgent urination, as well as diseases such as acute nephritis and acute hepatitis. The main chemical components of the *Polyporus umbellatus* sclerotium are polysaccharides and steroids. *Polyporus umbellatus* polysaccharides have the effects of regulating the body's immune function and anti-tumor activity; the steroids in *Polyporus umbellatus* (such as ergosterol and porphyrinone) are key components in exerting its diuretic and renal protective effects.

[0003] In the wild, the growth and development of *Polyporus umbellatus* sclerotia are inseparable from the infection of *Armillaria sp.*. A delicate and unique symbiotic relationship exists between *Armillaria sp.* and *Polyporus umbellatus* sclerotia. *Armillaria sp.* provides essential nutrients for the development of *Polyporus umbellatus* sclerotia, including amino acids, polysaccharides, and minerals; simultaneously, *Armillaria sp.* can also draw nutrients from *Polyporus umbellatus* sclerotia for its own growth and development. Currently, with the expansion of the medicinal scope and usage of *Polyporus umbellatus* sclerotia, coupled with the disorderly harvesting of wild *Polyporus umbellatus* resources and the lack of effective protection, the habitat of wild *Polyporus umbellatus* sclerotia has been severely damaged, and wild *Polyporus umbellatus* resources are becoming increasingly scarce. Although the semi-wild cultivation technology of *Polyporus umbellatus* has been successfully established, this technology suffers from unstable yields and inconsistent quality due to the degradation of *Armillaria sp.*, improper use, significant phenotypic heterogeneity and quality fluctuations in *Polyporus umbellatus* seed sclerotia, and the long growth cycle of *Polyporus umbellatus*. Therefore, the selection of high-quality seed sclerotia (standardized *Polyporus umbellatus* seed sources) is imperative.

[0004] The traditional cultivation method of *Polyporus umbellatus* (fungus substrate cultivation) requires a huge number of Fagaceae tree branches (at least 3 branches per hole for wild cultivation of *Polyporus umbellatus*). The Fagaceae tree branches used are 5-10 cm in diameter and 15-50 cm in length. The cost of purchasing and processing the branches into suitable lengths is high, and this method will inevitably cause serious damage to ecological resources. In addition, this cultivation method also requires a large amount of Fagaceae branches, short branches, humus soil and leaves, which further increases the planting cost of *Polyporus umbellatus*.

[0005] CN102037853B and CN1900267A describe methods for inducing sclerotia formation through *Polyporus umbellatus* mycelium. However, these methods are limited to forming *Polyporus umbellatus* sclerotia on solid culture media, petri dishes (CN1900267A), or test tube slant (CN102037853B), and can only form the white stage of *Polyporus umbellatus*, not the gray or black stages used for commercial purposes. Clearly, these methods lack medicinal value because, according to the Pharmacopoeia of the People's Republic of China (2025 edition), *Polyporus umbellatus* is characterized by a black, grayish-black, or brownish-black surface, wrinkled or with nodular protrusions. Furthermore, the pharmacodynamic components in sclerotia formed from *Polyporus umbellatus* mycelium are significantly lower than those in wild-cultivated *Polyporus umbellatus* sclerotia. Compared to the technical route of inducing sclerotia formation (white stage) through *Polyporus umbellatus* mycelium, the method of propagating sclerotia based on *Polyporus umbellatus* seed sclerotia (sclerotia) is more in line with the natural developmental biological characteristics of *Polyporus umbellatus* sclerotia and can effectively ensure the stability of its medicinal components.

[0006] Therefore, under the premise of ensuring that the content of pharmacodynamic components meets the content determination standards stipulated in the pharmacopoeia, it is necessary to establish a new method for culturing *Polyporus umbellatus* sclerotia that is green, environmentally friendly, inexpensive, and environmentally friendly. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a novel culture method for culturing *Polyporus umbellatus* sclerotia under aseptic conditions and using it for screening high-quality *Polyporus umbellatus* seed, in order to solve the problem of low yield and poor quality of cultivated *Polyporus umbellatus* due to significant phenotypic heterogeneity and quality fluctuations in *Polyporus umbellatus* seed.

[0008] The objectives of this invention and the solutions to its technical problems can be achieved by the following technical solutions.

[0009] On one hand, the present invention provides a wood stick culture medium for inducing *Polyporus umbellatus* sclerotia. This culture medium contains broadleaf wood sticks, glucose, potassium dihydrogen phosphate, magnesium sulfate, and water, wherein the mass ratio of broadleaf wood sticks, glucose, potassium dihydrogen phosphate, magnesium sulfate, and water is 2000:2:1:1:2000. In a specific embodiment, the wood stick culture medium may contain, for example, 200 g of broadleaf wood sticks, 0.2 g of glucose, 0.1 g of potassium dihydrogen phosphate, 0.1 g of magnesium sulfate, and 200 mL of water.

[0010] In embodiments of this aspect, the broadleaf wood can be from plants of the Fagaceae family. Those skilled in the art know that the Fagaceae family includes genera such as *Lithocarpus*, *Quercus*, *Castanea*, *Cyclobalanopsis*, *Formanodendron*, *Fagus*, and *Castanopsis*. Therefore, the broadleaf wood sticks can be from species within these genera, such as *Quercus glauca* Thunb. Broadleaf wood can also be from species of the Betulaceae family, such as *Betula platyphylla*.

[0011] On the other hand, the present invention provides a method for inducing *Polyporus umbellatus* sclerotia, comprising the following steps:

[0012] 1) Prepare the wooden stick culture medium described in the first aspect; 2) Sterilize the poria cocos seedlings; and 3) Inoculate with Armillaria mellea strain and co-culture.

[0013] In this embodiment, disinfection includes surface disinfection using a 75% ethanol solution and a 5% sodium hypochlorite solution.

[0014] In embodiments of this aspect, the Armillaria mellea strain may be Armillaria mellea, Armillaria gallica, or Armillaria ostoyae.

[0015] In the implementation scheme of this aspect, co-cultivation is carried out at 25±2℃ under indoor static conditions, in the dark.

[0016] In an embodiment of the present invention, the small wooden stick refers to a broad-leaved wood stick with a diameter of 2-3 cm and a length of 2-3 cm.

[0017] The wooden stick culture medium of this invention can rapidly induce the formation of *Polyporus umbellatus* sclerotia, allowing for harvesting 2 to 2.5 years earlier compared to newly formed sclerotia from wild cultivation. It also increases the yield and effective component content of *Polyporus umbellatus* sclerotia, laying the foundation for establishing a new industrialized cultivation model for large-scale, standardized production of *Polyporus umbellatus* and for the breeding of superior varieties. Furthermore, the *Polyporus umbellatus* sclerotia induced by the method of this invention undergo the same transformation process as those from wild cultivation, from white to gray to black, while simultaneously forming unique isolation cavities and other structures resulting from *Armillaria mellea* infection of the sclerotia.

[0018] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0019] Figure 1 The growth status of newly formed gray and black sclerotia after induction of *Polyporus umbellatus* sclerotia and inoculation with *Polyporus umbellatus* seed and *Armillaria mellea* for 6 months is shown in tissue culture bottles with wooden sticks. Among them, 1.1 represents newly formed white sclerotia, 1.2 represents *Armillaria mellea*; 2.1 is newly formed black sclerotia; 2.2 is seed sclerotia; 2.3 and 2.4 are newly formed gray sclerotia.

[0020] Figure 2 This diagram shows the evolution of newly formed *Polyporus umbellatus* sclerotia from white to gray to black, where A, B, and D represent newly formed white sclerotia; C is the "isolation cavity" of newly formed *Polyporus umbellatus* sclerotia; E represents newly formed gray sclerotia; and F represents newly formed black sclerotia. Detailed Implementation

[0021] The preferred embodiments of the present invention will now be described in detail with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent, and all such modifications and substitutions fall within the scope of protection claimed in the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] Example 1: Method for inducing *Polyporus umbellatus* sclerotia on a wooden stick culture medium

[0024] Preparation of the wood stick culture medium: The total volume of the tissue culture flasks used is 740 mL (bottom diameter 9 cm, height 14.5 cm, mouth diameter 5.5 cm). Select small wood sticks of broadleaf oak (Q. glauca) with a diameter of 2-3 cm and a length of 2-3 cm, wash off the surface dust, soak in clean water for 1 day, and take 7-8 small wood sticks (about 200 g) and place them in the plastic culture flasks, with the volume of the wood sticks occupying about half of the total volume of the culture flasks. Weigh 0.2 g of glucose, 0.1 g of potassium dihydrogen phosphate, 0.1 g of magnesium sulfate, and 200 mL of water, stir thoroughly to completely dissolve the glucose and inorganic salts, then add them to the culture flasks, seal the flasks, autoclave at 122℃ for 3 hours, and then cool completely.

[0025] Disinfection of the sclerotium: Select 50 g of fresh, glossy black or gray, elastic *Polyporus umbellatus* sclerotia as the seed culture. Perform a series of disinfection procedures in a clean bench. First, depending on the size and texture of the sclerotium, surface disinfect with 75% ethanol solution for about 3-5 minutes, rinse three times with sterile deionized water, then blot dry the surface of the sclerotium with sterile filter paper, and then disinfect with 5% sodium hypochlorite solution for 3-5 minutes. Rinse the surface of the sclerotium three times with sterile water to remove excess disinfectant. Place the *Polyporus umbellatus* seed culture on the wooden stick at the top of the tissue culture bottle.

[0026] Inoculation and Culture Observation of Armillaria gallica: Under aseptic conditions in a laminar flow hood, one Armillaria gallica (A. gallica, purchased from Liuba County Qinzheng Zhuling Development Co., Ltd.) culture disc, which had been cultured in PDA medium for 30 days, was added to a plastic culture bottle (the disc was obtained using a 10 mm diameter punch). (See...) Figure 1 Add the solution (1.2) to the bottom of the culture flask near the wooden stick, seal the flask tightly, and incubate in the dark (25±2℃). Observe the growth of the sclerotia of *Polyporus umbellatus* regularly. After 60 days of incubation, new white *Polyporus umbellatus* can be observed germinating from the seed site. Figure 1 1.1 and Figure 2 As shown in A, B, and D, the sclerotia of *Polyporus umbellatus* gradually mature with increasing culture time. After 90 days of culture, the sclerotia turn gray, becoming newly formed gray *Polyporus umbellatus*. Figure 1 2.3 and 2.4 in the text; Figure 2 The E in the text, after 180 days of cultivation, gradually turns black, becoming newly formed black porridge with visible wrinkles and nodular protrusions on the surface. This morphological characteristic conforms to the characteristics of *Polyporus umbellatus* as defined in the Pharmacopoeia of the People's Republic of China. Figure 1 2.1 in the middle; Figure 2 (F in the text) Figure 2 This demonstrates the evolution of newly formed *Polyporus umbellatus* sclerotia from white to gray to black. During the growth of the new sclerotia, a characteristic "isolation cavity" structure formed due to *Armillaria mellea* infection can be observed, such as... Figure 2 As shown in C. The rDNA-ITS sequence of the newly formed sclerotia was identified using primers ITS1 and ITS4. The primer sequence for ITS1 was 5'-TCCGTAGGTGAACCTGCGG-3', and the primer sequence for ITS4 was 5'-TCCTCCGCTTATTGATATGC-3'. The obtained ITS sequence of the newly formed sclerotia was compared with the sequence of *Polyporus umbellatus* (GenBank: JX110719.1) submitted to the database on the NCBI website, and the similarity reached 100%. Therefore, the newly formed sclerotia induced by this invention are *Polyporus umbellatus* sclerotia. After culturing the newly formed *Polyporus umbellatus* for 180 days, they were taken out and weighed to calculate the biomass of the newly formed sclerotia.

[0027] Example 2: Wild Cultivation Experiment of Polyporus umbellatus

[0028] Preparation of Armillaria mellea solid culture: The basic components of the solid culture medium were wheat bran and sawdust, with a total mass of 100 g (mass ratio of wheat bran to sawdust of 1:3). The basic components were mixed with a prepared glucose solution (total volume 100 mL, of which 2 g of glucose was added) and poured into a plastic culture bottle (total volume 300 mL) until approximately 2 / 3 full. The bottle was then autoclaved (122℃, 3 h) and cooled for later use. Using a 10 mm diameter punch, one Armillaria mellea culture disc (A. gallica, purchased from Liuba County Qinzheng Zhuling Development Co., Ltd.) that had been cultured in PDA medium for 30 days was transferred to the cooled solid culture medium and cultured in the dark at (25±2) ℃ for 30 days to obtain the Armillaria mellea culture in the solid culture medium.

[0029] Preparation of Armillaria mellea spawn: Add 320 g ± 5 g of overnight-soaked oak branches (approximately 2 cm in diameter and 2-3 cm in length) to a plastic culture bottle (total volume 700 mL, empty bottle weight 25 g), filling the bottle to 2 / 3 full. Add 230 mL of tap water, autoclave at 122 ℃ for 3 h, and then cool. After inoculating with 10 g of Armillaria mellea solid spawn, weigh the plastic culture bottle; the weight is 585 g ± 5 g / bottle. After culturing at (25 ± 2 ℃) for 90 days (suitable for *Polyporus umbellatus* cultivation experiments), weigh again; the weight is 600 g ± 5 g. The weight of the Armillaria mellea spawn is estimated to be approximately 15 g / bottle using the weight reduction method.

[0030] Cultivation experiment of *Polyporus umbellatus*: The plants were cultivated in holes at the Beijing Medicinal Botanical Garden. The dimensions of each hole were 20cm × 20cm × 20cm. Three oak sticks, each about 15cm long and 5cm in diameter, with oblique fish-scale cuts, were placed parallel and equidistant from each hole. 50g of *Polyporus umbellatus* seed was evenly placed next to the fish-scale cuts and at both ends of the sticks. The *Armillaria mellea* spawn (total weight 120.0g ± 3g, of which the inoculation amount of *Armillaria mellea* strain was about 3g) was placed near the *Polyporus umbellatus* seed, and 50g of oak twigs were evenly placed on top. The top layer was covered with 40g of oak leaves.

[0031] In Examples 1 and 2, samples were taken after 1 year and 3 years, respectively, to analyze the yield of *Polyporus umbellatus* cultivated in the wild and cultivated in the laboratory. The results are shown in Table 1. The yield of *Polyporus umbellatus* in the wooden stick culture medium was significantly higher than that in the wild, with a statistically significant difference (P<0.05) (see Table 1).

[0032] Observations show that the growth of *Polyporus umbellatus* in the wooden stick medium is similar to that in the wild environment, and it will go through the stages of white, gray and black. It can form unique isolation cavities and other structures formed by *Armillaria mellea* infection of *Polyporus umbellatus* sclerotia. Furthermore, when the yield of *Polyporus umbellatus* sclerotia increases, the wooden stick medium can shorten the growth cycle of *Polyporus umbellatus* sclerotia by at least 2 / 3, which greatly improves production efficiency.

[0033] Table 1. Yield of *Polyporus umbellatus* sclerotia (n=5)

[0034] Group Sclerotium yield of *Polyporus umbellatus* (g / hole or bottle) Field cultivation experimental group (Example 2) 185.4±3.19 Wooden stick culture medium test group (Example 1) 200.3±2.06

[0035] Example 3: Determination of ergosterol, porcinione A and polysaccharide content in *Polyporus umbellatus* sclerotium cultured under two different cultivation methods

[0036] Sclerotia of *Polyporus umbellatus* obtained in both wild cultivation and laboratory culture as described in Examples 1 and 2 were washed, dried, pulverized, and passed through a 40-mesh sieve as test samples. 1 mg of ergosterol and *Polyporus umbellatus* A (reference standards) were weighed separately, dissolved in methanol, and diluted to 1 mL in volumetric flasks, with a concentration of 1 mg / mL for each standard solution. The solutions were sonicated until completely dissolved to obtain the ergosterol standard solution. Different volumes of single *Polyporus umbellatus* A solution were accurately pipetted and diluted to 1 mL in volumetric flasks to obtain the mixed standard solution for quantitative analysis. 1000.00 mg of each test sample was weighed and soaked in 20 times its volume of 95% ethanol solution for 12 h, extracted by sonication for 1 h, centrifuged for 30 min, and the supernatant was collected. The supernatant was poured into an evaporating dish, concentrated in an 85 °C water bath, and the solvent was evaporated to dryness. The residue was diluted to 1 mL in a volumetric flask with methanol to prepare the sample solution. The high-performance liquid chromatography (HPLC) system was a 1260 InfinityLC evaporative light scattering HPLC system (Agilent Technologies, USA), and the chromatographic column was a Bridge RP18 column (250 mm × 4.6 mm, 5 μm). The flow rate was 1.0 mL / min, and the column temperature was 30 ℃. The detection wavelengths were 247 nm (porphyrin A) and 283 nm (ergosterol). Before injection, the reference standard and sample solutions were filtered through a 0.22 μm organic phase filter membrane. The mobile phases used for analysis were: Solution A: formic acid-water (formic acid: 0.1%, water: 99.9%), Solution B: acetonitrile, as detailed in Tables 2 and 3 for chromatographic conditions.

[0037] Table 2. Chromatographic conditions for poria cocos A

[0038] Serial Number time Flow rate [mL / min] Liquid A [%] Solution B [%] Maximum pressure limit [bar] 1 0 1.00 72.0 28.0 400 2 22.00 1.00 72.0 28.0 400 3 23.10 1.00 0.0 100.0 400 4 28.00 1.00 0.0 100.0 400 5 29.00 1.00 72.0 28.0 400 6 39.00 1.00 72.0 28.0 400

[0039] Table 3. Chromatographic conditions for ergosterol

[0040] Serial Number time Flow rate [mL / min] Liquid A [%] Solution B [%] Maximum pressure limit [bar] 1 0 1.00 0.0 100.0 400 2 20.00 1.00 0.0 100.0 400

[0041] The sclerotia of *Polyporus umbellatus* obtained by the two culture methods were dried to constant weight at 60°C, and 0.05 g of each were accurately weighed. 1 mL of water was added, and the mixture was shaken thoroughly and extracted in a 100°C water bath for 2 h. After cooling, the mixture was centrifuged at 10,000×g for 10 min, and the supernatant was collected. 0.2 mL of the supernatant was transferred to a 1.5 mL centrifuge tube, and 0.8 mL of anhydrous ethanol was slowly added. After mixing, the mixture was allowed to stand overnight at 4°C, centrifuged at 10,000×g for 10 min, and the supernatant was discarded. The precipitate was washed twice with 1 mL of 80% ethanol each time. The precipitate was dissolved in deionized water and diluted to a 1 mL volumetric flask as the analytical sample. 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, incubate in a 90 ℃ water bath for 20 min, remove and immediately place in an ice-water bath to cool for 30 min to terminate the reaction, then measure its absorbance at a wavelength of 490 nm using a spectrophotometer and calculate the content of polysaccharide from the sclerotium of *Polyporus umbellatus*.

[0042] The results are shown in Table 4. As can be seen from Table 4, compared with the field cultivation experiment, the sclerotia of *Polyporus umbellatus* cultured on the stick medium significantly increased the contents of ergosterol, poria cocos A and polysaccharides (P<0.05).

[0043] Table 4. Content of ergosterol and porphyrin A in Polyporus umbellatus (n=3)

[0044] Group Ergosterol content (μg / g) Content of poria cocos A (μg / g) Polysaccharide content (%) Field cultivation experimental group (Example 2) 1980.8±4.81 116.73±4.95 1.91%±0.03% Wood stick culture medium test group (Implementation 1) 2000.5±5.48 138.03±5.60 2.29%±0.04%

[0045] In summary, the culture medium of this invention does not contain soil, but instead adds a certain proportion of carbon source (glucose), inorganic salts (potassium dihydrogen phosphate and magnesium sulfate) and water, which can meet the nutrient absorption and utilization requirements of *Polyporus umbellatus* seeds and *Armillaria mellea*. This invention uses small broadleaf wood sticks with a diameter of 2-3 cm and a length of 2-3 cm to meet the culture medium requirements, thus saving resources and avoiding the forest resource damage caused by felling broadleaf wood sticks with a diameter of 5 cm or more and a length of 15-50 cm in current *Polyporus umbellatus* cultivation. Furthermore, compared to the fresh, large wood sticks used in existing technologies, the small wood sticks of this invention, after soaking in clean water for 1 day and then autoclaving for 3 hours, release some of the nutrients from the wood sticks and mix them with the water added to the culture medium, which is beneficial for the absorption and utilization of nutrients by *Polyporus umbellatus* seeds and *Armillaria mellea*. Within a limited volume, the small wooden stick is closer to the poria cocos seed and Armillaria mellea, which accelerates the establishment of the symbiotic relationship between poria cocos and Armillaria mellea, promotes the growth of poria cocos sclerotia, and enables poria cocos to accumulate more pharmacodynamic substances in a shorter period of time.

Claims

1. A wood stick culture medium for inducing sclerotia of *Polyporus umbellatus*, containing broadleaf wood sticks, glucose, potassium dihydrogen phosphate, magnesium sulfate and water, wherein the mass ratio of broadleaf wood sticks, glucose, potassium dihydrogen phosphate, magnesium sulfate and water is 2000:2:1:1:2000.

2. The wooden stick culture medium according to claim 1, characterized in that, The broad-leaved trees mentioned can be species from the Fagaceae or Betulaceae families.

3. The wooden stick culture medium according to claim 2, characterized in that, The broad-leaved tree may be Quercus glauca Thunb. or Betula platyphylla.

4. A method for inducing sclerotia of *Polyporus umbellatus*, comprising the following steps: 1) Prepare the wood stick culture medium as described in claim 1; 2) Disinfect the poria cocos seeds; and 3) Inoculate with Armillaria strain and co-culture.

5. The method according to claim 4, characterized in that, The surface is disinfected using a 75% ethanol solution and a 5% sodium hypochlorite solution.

6. The method according to claim 4, characterized in that, The Armillaria mellea can be Armillaria mellea, Armillaria gallica, or Armillaria ostoyae.

7. The method according to claim 4, characterized in that, The co-culture was carried out under static conditions in a light-proof room at 25±2℃.

Images