A culture medium for mother culture of Chinese porcini mushroom and a preparation method and application thereof

Abstract

This invention discloses a Chinese saprophytic bolete ( Buchwaldoboletus xylophilus This invention relates to the mother culture medium, its preparation method, and its application, belonging to the field of microbial culture technology. The strain JSJ-Bx1 of the *Boletus spp.* mother culture, with accession number CGMCC No. 42007, is deposited at the China General Microbiological Culture Collection Center. The culture medium composition is: 10–30 g carbon source, 2–6 g nitrogen source, 1–3 g inorganic salts, 0.1–0.3 g suitable growth factors, 20 g agar, and 1 L water, with a pH of 4–6. Using the culture medium of this invention, the mycelium of the *Boletus spp.* mother culture exhibits rapid, vigorous, and dense growth, with neat colony edges, and does not produce pigments or exhibit mycelial degeneration such as exudation, significantly shortening the mother culture growth cycle.

Description

Technical Field

[0001] This invention belongs to the field of microbial culture technology, specifically relating to a formula for a mother culture medium of Boletus saprophyticus, its preparation method, and its specific applications. Background Technology

[0002] Chinese Boletus saprophyticus Buchwaldoboletus xylophilus (Petch) Both&B. Ortiz, belonging to the family Boletaceae, subfamily Chalciporoideae, genus *Boletus*. Buchwaldoboletus It is a typical tropical and subtropical rare bolete, a non-ectomycorrhizal fungus with saprophytic ability, and is edible. Its wild resources are very scarce and its yield is low.

[0003] Currently, there are approximately 2,300 species of edible and medicinal fungi worldwide, with over 1,000 described edible fungi in my country, including more than 500 mycorrhizal edible fungi. Nearly 200 of these macrofungi have been successfully domesticated and cultivated. *Boletus saprophyticus*, as the second newly cultivated bolete species, possesses significant scientific and commercial value. However, basic biological research on *Boletus saprophyticus* is currently weak, with no reports on its biological characteristics, domestication, or molecular biology. Existing authorized or published patents related to *Boletus saprophyticus* mainly concern strain selection, liquid spawn, solid spawn, and substrate formulation, while the screening and optimization of mother culture media remain unexplored. Therefore, a thorough understanding of the biological characteristics of *Boletus saprophyticus* and a systematic exploration of its culture environment and different nutrient sources are needed to screen for optimal culture conditions and the most suitable composition of the mother culture medium.

[0004] References are available at:

[0005] [1] Yunnan Tropical Crops Research Institute, Hainan Medical College. A strain of *Boletus sinensis*: CN113412763A[P].

[0006] [2] Yunnan Tropical Crops Research Institute. An artificial cultivation method for Boletus sinensis: CN113348963A[P].

[0007] [3] Yunnan Provincial Institute of Tropical Crops. A liquid culture medium and cultivation method for Boletus chinensis saprophyticus: CN113348966A[P].

[0008] [4] Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences. A method for culturing *Boletus chinensis* strain: CN118765716A[P].

[0009] [5] Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences. A cultivation method for *Boletus chinensis*: CN118923428A[P]. Summary of the Invention

[0010] The purpose of this invention is to provide a mother culture medium for *Boletus saprophyticus* with rapid mycelial growth, vigorous and dense mycelial growth, and no degeneration phenomena such as pigmentation and exudation, which can significantly shorten the cultivation cycle of *Boletus saprophyticus* and provide a good mother culture for artificial cultivation. This invention also provides the application of the aforementioned culture medium.

[0011] The technical solution adopted in this invention is as follows:

[0012] A type of Chinese saprophytic bolete ( Buchwaldoboletus xylophilus The mother culture medium, wherein the strain JSJ-Bx1 of *Boletus chinensis* mother culture is deposited at the China General Microbiological Culture Collection Center (CGMCC No. 42007) with the accession number CGMCC No. 42007; the raw material ratio of the culture medium is: 10–30 g carbon source, 2–6 g nitrogen source, 1–3 g inorganic salt, 0.1–0.3 g suitable growth factor, 20 g agar, 1 L water, and the pH of the culture medium is 4–6; the carbon source is glucose, maltose, or fructose; the nitrogen source is yeast extract, ammonium sulfate, or ammonium chloride; the inorganic salt is ferrous sulfate, manganese sulfate, or magnesium sulfate; and the suitable growth factor is 1 / 2 MS medium, L-tyrosine, or VB1.

[0013] Preferably, the raw material ratio of the culture medium is: 20g glucose, 2g yeast extract, 1.5g ferrous sulfate, 0.3g 1 / 2 MS medium, 20g agar, and 1L water.

[0014] Preferably, the pH of the culture medium is 4.

[0015] The present invention describes a Chinese saprophytic bolete (Boletus sinensis) Buchwaldoboletus xylophilus The method for preparing the mother culture medium is as follows:

[0016] (1) Weigh carbon source, nitrogen source, inorganic salt, suitable growth factor of culture medium, agar and water according to the raw material mass ratio, put them into a container and stir evenly;

[0017] (2) Heat the container and boil the liquid material in the container over a low flame to form a homogeneous mixed solution, which is the nutrient solution;

[0018] (3) After the above nutrient solution is bottled, it is sterilized to obtain liquid culture medium;

[0019] (4) Dispense the liquid culture medium into petri dishes to obtain the mother culture medium of the Chinese Boletus saprophyticus.

[0020] The present invention describes a Chinese saprophytic bolete (Boletus sinensis) Buchwaldoboletus xylophilus The application of the mother culture medium involves purifying the wild fruiting bodies of *Boletus spp.* using a modified PDA medium to obtain the mother strain JSJ-Bx1. This strain is then inoculated onto the *Boletus spp.* mother culture medium and cultured in a 25–35℃ constant temperature and humidity incubator under dark conditions to obtain *Boletus spp.* mycelium. The modified PDA medium consists of: 200g peeled potato, 20g glucose, 2g yeast extract, 1.5g MgSO4·7H2O, 3g KH2PO4, 20g agar, 1000mL distilled water, and a natural pH.

[0021] The culture medium of this invention, when used for dark cultivation of *Boletus spp.* mother culture at pH 4–6 and 25–35℃ for 3–4 days, allows *Boletus spp.* mycelium to completely cover a 9cm diameter culture dish. The mycelial growth is rapid, vigorous, and dense, without pigmentation or degenerative phenomena such as gibberish, significantly shortening the cultivation cycle and providing an excellent mother culture for artificial cultivation. Furthermore, by adding glucose (carbon source), yeast extract (nitrogen source), ferrous sulfate (inorganic salt), and 1 / 2MS (growth factor), the mycelial growth is excellent, effectively meeting the normal nutritional and physiological needs of the *Boletus spp.* mother culture. This is of great significance for further research on its basic biological characteristics, domestication cultivation, and industrial production. Attached Figure Description

[0022] Figure 1 The mycelial growth of the Chinese saprophytic bolete strain JSJ-Bx1 under different single-factor culture conditions;

[0023] Figure 2 The mycelial growth of the *Boletus saprophyticus* strain JSJ-Bx1 in an orthogonal experiment;

[0024] Figure 3 The mycelial growth in the culture medium and the modified PDA culture medium of Example 1 of this invention;

[0025] Figure 4 The results of ITS species identification of mycelia cultured in the culture medium described in this invention;

[0026] Figure 5 This is an example of mycelial growth in the culture medium of Example 2 of the present invention;

[0027] Figure 6 This shows the mycelial growth in the culture medium of Example 3 of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description of the invention is provided in conjunction with specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0029] The strain JSJ-Bx1 of the mother culture of *Boletus spp.* described in this invention has the accession number CGMCC No. 42007 and is deposited at the China General Microbiological Culture Collection Center, and is classified as *Boletus spp.* Buchwaldoboletus xylophilus The deposit date is May 15, 2025.

[0030] The parent strain of the *Boletus chinensis* strain of this invention was collected on June 5, 2024, from wild fruiting bodies in the forest under the trees of Jinghong City, Xishuangbanna Dai Autonomous Prefecture. Pure culture mycelia were obtained through tissue isolation, purification, and identification.

[0031] Screening medium for mother cultures of *Boletus chinensis*:

[0032] (1) Single-factor experiments were conducted to test different culture temperatures, pH, light duration, carbon sources, nitrogen sources, inorganic salts and growth factors. The treatments are shown in Table 1.

[0033] Table 1 Different single-factor experimental treatments

[0034]

[0035] (1.1) A single-factor experimental method was used, setting seven temperature gradients: 5, 10, 15, 20, 25, 30, and 35℃. Using a 0.65cm diameter punch, JSJ-Bx1 *Boletus spp.* mycelial blocks were taken from the modified PDA medium after purification and isolation using a 0.65cm diameter punch. These blocks were inoculated onto the modified PDA medium, which consisted of: 200g peeled potato, 20g glucose, 2g yeast extract, 1.5g MgSO4·7H2O, 3g KH2PO4, 20g agar, 1000mL distilled water, and natural pH. Six replicates were performed per group. Incubation was stopped when the mycelium covered the entire surface of the medium at one of the temperature treatments. The colony diameter was measured using the cross-sectional method, and the mycelial growth potential was observed and recorded. See [link to relevant documentation]. Figure 1 Mycelial growth of *Boletus chinensis* JSJ-Bx1 under different temperature conditions.

[0036] The results showed that the mycelium of *Boletus sinensis* could not germinate and grow in the range of 5–15℃. In the range of 20–35℃, the mycelial growth rate was positively correlated with temperature, and the mycelial growth rate was the fastest at 35℃. The colonies were white, without pigment, and the mycelium was dense.

[0037] (1.2) A single-factor experimental method was used, setting up three light treatment gradients: 24h light, 12h / 12h light / dark alternation, and 24h darkness. Using a 0.65cm diameter punch, JSJ-Bx1 *Boletus spp.* mycelial blocks were taken from the same radius of the purified *Boletus spp.* medium obtained from the modified PDA medium and inoculated onto the modified PDA medium, with 6 replicates per group. When the mycelium covered the entire surface of the medium under one of the light treatments, the culture was stopped, and the colony diameter was measured using the cross-crossing method. The mycelial growth potential was observed and recorded. See [link to relevant documentation]. Figure 1 Mycelial growth of *Boletus chinensis* JSJ-Bx1 under different light conditions.

[0038] The results showed that the mycelium of *Boletus sinensis* could grow normally under all three light treatments, but the growth was significantly different under different light treatments.

[0039] There were differences in mycelial growth. Light inhibited the mycelial growth of Boletus sinensis to some extent. The mycelial growth rate was slower but the mycelium was denser when treated with 24h light, while the mycelial growth rate was faster but the mycelium was denser when treated with 24h darkness.

[0040] (1.3) A single-factor experimental method was used, setting up six pH gradients: 4, 5, 6, 7, 8, and 9. Using a 0.65 cm diameter punch, holes were punched at the same radius from the modified PDA medium containing *Boletus spp.* (a type of agar), and *Boletus spp.* (JSJ-Bx1) mycelial blocks were inoculated onto the modified PDA medium, with six replicates per group. When the mycelium covered the entire surface of the medium under one of the pH treatments, the culture was stopped. The colony diameter was measured using the cross-crossing method, and the mycelial growth potential was observed and recorded. See [link to relevant documentation]. Figure 1 Mycelial growth of JSJ-Bx1 Boletus sinensis under different pH conditions.

[0041] The results showed that the mycelium of *Boletus sinensis* exhibited significant differences in growth within the pH range of 4–9, with better growth under acidic conditions.

[0042] Furthermore, the mycelium grows vigorously at pH 4, and cannot germinate and grow in neutral or alkaline environments.

[0043] (1.4) A single-factor experimental method was used, setting up 8 carbon source treatments: blank, glucose, maltose, soluble starch, lactose, sucrose, fructose, and mannitol. Using a 0.65 cm diameter punch, JSJ-Bx1 *Boletus spp.* mycelial blocks were taken from the same radius of the purified *Boletus spp.* culture medium obtained from modified PDA medium. These blocks were inoculated onto a carbon source basal medium (2 g yeast extract, 1.5 g MgSO4·7H2O, 3 g KH2PO4, 20 g agar, 1000 mL distilled water, natural pH), with 6 replicates per group. When the mycelium covered the entire surface of the medium under one carbon source treatment, the culture was stopped, and the colony diameter was measured using the cross-crossing method. The mycelial growth potential was observed and recorded. See [link to relevant documentation]. Figure 1 Mycelial growth of *Boletus chinensis* JSJ-Bx1 under different carbon sources.

[0044] The results showed that *Boletus spp.* mycelium could grow with all seven carbon sources. Compared with the carbon source control group, all tested carbon sources promoted the mycelial growth of *Boletus spp.*. Among them, glucose showed the fastest mycelial growth rate, with vigorous and dense mycelium; maltose, fructose, and soluble starch showed the next fastest growth rates; sucrose and mannitol showed slower growth rates and sparser mycelium; lactose showed the slowest growth rate but densest mycelium. In summary, *Boletus spp.* has a wide range of carbon source adaptability, with glucose, maltose, and fructose being suitable carbon sources, and glucose being the most suitable.

[0045] (1.5) A single-factor experimental method was used, with eight nitrogen source treatments: blank, yeast extract, peptone, beef extract, urea, ammonium tartrate, ammonium sulfate, and ammonium chloride. Using a 0.65 cm diameter punch, JSJ-Bx1 *Boletus saprophyticus* mycelial blocks were extracted from the modified PDA medium and purified using a punch at the same radius. These blocks were inoculated onto a nitrogen-based basal medium (20 g glucose, 1.5 g MgSO4·7H2O, 3 g KH2PO4, 20 g agar, 1000 mL distilled water, natural pH), with six replicates per group. Cultivation was stopped when the mycelium covered the entire surface of the medium under one nitrogen source treatment. The colony diameter was measured using the cross-crossing method, and the mycelial growth potential was observed and recorded. See [link to relevant documentation]. Figure 1 Mycelial growth of *Boletus chinensis* JSJ-Bx1 under different nitrogen sources.

[0046] The results showed that the mycelium of *Boletus sinensis* exhibited significant differences in growth among the seven nitrogen sources. Compared with the nitrogen source control group, the growth was best with urine.

[0047] When nitrogen is used as a nitrogen source, mycelia do not grow; when yeast extract is used as a nitrogen source, the mycelial growth rate is faster, the mycelia are denser, and the growth is better; when ammonium salts such as ammonium tartrate, ammonium sulfate, and ammonium chloride are used as nitrogen sources, the mycelial growth rate is fast, but the mycelia are weak and sparse, and the growth is poor; when peptone is used as a nitrogen source, the effect on mycelial growth is not obvious; when beef extract is used as a nitrogen source, the mycelial growth is the worst, and the colony edges are irregular. In summary, yeast extract, ammonium sulfate, and ammonium chloride are suitable nitrogen sources for *Boletus sinensis*, with yeast extract being the most suitable nitrogen source.

[0048] (1.6) A single-factor experimental method was used, setting up 8 inorganic salt treatments: blank, magnesium sulfate, manganese sulfate, ferrous sulfate, potassium dihydrogen phosphate, calcium sulfate, zinc sulfate, and potassium chloride. Using a 0.65 cm diameter punch, JSJ-Bx1 *Boletus sinensis* mycelial blocks were taken from the same radius of the purified *Boletus sinensis* culture medium obtained from modified PDA medium and inoculated onto an inorganic salt basal medium (20 g glucose, 2 g yeast extract, 20 g agar, 1000 mL distilled water, natural pH), with 6 replicates per group. When the mycelium covered the entire surface of the culture medium under one of the inorganic salt treatments, the culture was stopped, and the colony diameter was measured using the cross-crossing method. The mycelial growth potential was observed and recorded. See [link to relevant documentation]. Figure 1 Mycelial growth of *Boletus chinensis* JSJ-Bx1 under different inorganic salt conditions.

[0049] The results showed that the mycelia of *Boletus sinensis* exhibited significant differences in growth among the seven inorganic salts, with ZnSO4 being the most suitable inorganic salt.

[0050] During growth, when KCl was used as the inorganic salt, the inoculum only germinated without significant growth; both KCl and CaSO4 inhibited mycelial growth, with ZnSO4 showing the most significant inhibitory effect. When KH2PO4 and CaSO4 were used as inorganic salts, their effects on promoting mycelial growth were not significant, resulting in poor mycelial growth and sparse mycelia. When MgSO4, MnSO4, and FeSO4 were used as inorganic salts, they significantly promoted mycelial growth, resulting in vigorous and dense mycelia, with FeSO4 showing the most significant promoting effect. In summary, MgSO4, MnSO4, and FeSO4 are suitable inorganic salts for *Boletus sinensis*, with FeSO4 being the most suitable inorganic salt.

[0051] (1.7) A single-factor experimental method was used, with eight growth factor treatments: blank, vitamin B1, 1 / 2 MS, adenine, L-arginine, L-tyrosine, 6-benzylaminopurine, and indole-3-butyric acid. Using a 0.65 cm diameter punch, JSJ-Bx1 *Boletus spp.* mycelial blocks were taken from the same radius of the purified *Boletus spp.* culture medium obtained from modified PDA medium. These blocks were inoculated onto a growth factor-based basal medium (20 g glucose, 2 g yeast extract, 1.5 g MgSO4·7H2O, 3 g KH2PO4, 20 g agar, 1000 mL distilled water, pH natural), with six replicates per group. Culture was stopped when the mycelium covered the entire surface of the medium under one of the growth factor treatments. The colony diameter was measured using the cross-cross method, and the mycelial growth potential was observed and recorded. See [link to relevant documentation]. Figure 1 Mycelial growth of *Boletus chinensis* JSJ-Bx1 on different growth factor basal media.

[0052] The results showed that the mycelial growth of *Boletus spp.* differed significantly among the seven tested growth factors. Compared with the control group, mycelial growth was not observed when 6-BA was used as a growth factor; IBA significantly inhibited mycelial growth, resulting in the slowest growth rate, but the mycelia were dense; adenine and arginine inhibited mycelial growth, but the mycelial vigor was good and the mycelia were dense; VB1, 1 / 2MS, and tyrosine had no significant effect on promoting mycelial growth, but 1 / 2MS resulted in better and denser mycelial growth. In conclusion, 1 / 2MS is the suitable growth factor for *Boletus spp.*

[0053] (2) Based on the results of the single-factor experiments, the optimal carbon source (glucose), optimal nitrogen source (yeast extract), optimal inorganic salt (FeSO4), and optimal growth factor (1 / 2MS) were selected as direct factors for L9(3) experiments. 4 The orthogonal experiment was conducted, with treatments shown in Table 2. Each treatment was repeated six times. Using a 0.65 cm diameter punch, JSJ-Bx1 *Boletus spp.* mycelial blocks were punched at the same radius from the purified *Boletus spp.* culture medium obtained from the modified PDA medium. These blocks were inoculated onto the nine combined culture media and cultured under optimized conditions. The orthogonal experiment procedure and data recording were the same as for the single-factor experiment. The modified PDA medium was selected as the control group.

[0054] Table 2 L9(3) 4 Orthogonal experimental design factor level table

[0055]

[0056] The results showed that the mycelia germinated and grew rapidly in all nine experimental treatments. Compared with the control group, the mycelia on the modified PDA medium were just beginning to germinate and showed poor growth. In contrast, all nine treatments in the orthogonal experiment using the medium described in this invention showed good growth-promoting effects. Experiment 4 showed the fastest growth rate and best growth, followed by Experiment 8, which also showed good growth. Experiment 4 can be considered the most suitable treatment for the mycelial growth of *Boletus spp.* mother culture. In other words, the mycelial growth of the mother culture medium described in this invention is significantly better than that of the modified PDA medium, and it is well-suited for the growth of *Boletus spp.* mother culture.

[0057] Based on the results of single-factor and orthogonal experiments, the formula for the mother culture medium of Boletus sinensis was determined, with the following mass ratio: glucose 10–30g, yeast extract 2–6g, ferrous sulfate 1–3g, 1 / 2 MS medium 0.1–0.3g, agar 20g, and water 1L.

[0058] The optimal mass ratio of each component is as follows: 20g glucose, 2g yeast extract, 1.5g ferrous sulfate, 0.3g 1 / 2 MS medium, 20g agar, and 1L water. Figure 2 The mycelial growth of JSJ-Bx1 *Boletus sinensis* in an orthogonal experiment is shown. Example 1

[0059] A mother culture medium for *Boletus chinensis*, wherein the raw materials of the medium are in the following proportions: 20g glucose, 2g yeast extract, 1.5g ferrous sulfate, 0.1g 1 / 2 MS medium, 20g agar, and 1L water. The pH of the medium is 4.

[0060] The method for preparing the mother culture medium of *Boletus chinensis* is as follows:

[0061] (1) Weigh glucose, yeast extract, ferrous sulfate, 1 / 2MS medium, agar and water according to the raw material mass ratio, put them into a container and stir evenly;

[0062] (2) Heat the container and boil the liquid material in the container over a low flame to form a homogeneous mixed solution, which is the nutrient solution;

[0063] (3) After the above nutrient solution is bottled, it is sterilized to obtain liquid culture medium;

[0064] (4) Dispense the liquid culture medium into petri dishes, 20 ml per dish, to obtain the mother culture medium of the Chinese saprophytic bolete.

[0065] Using a 0.65cm diameter punch, holes were punched at the same radius to obtain JSJ-Bx1 *Boletus saprophyticus* mycelial blocks from the modified PDA medium after purification. These blocks were then inoculated onto the *Boletus saprophyticus* mother culture medium described in Example 1 of this invention. After inoculation, the medium was placed in a 35℃ constant temperature and humidity incubator and cultured in the dark for 3 days to obtain *Boletus saprophyticus* mycelia.

[0066] The mycelial growth in the mother culture medium of this invention and the modified PDA medium was compared, see... Figure 3 As can be seen, using the mother culture medium described in Example 1 of this invention, the mycelium of *Boletus spp.* germinates rapidly, covering a 9cm diameter culture dish in 3-4 days. The mycelium grows vigorously and densely, with neat colony edges, resulting in high-quality mother culture. In contrast, the existing modified PDA culture medium exhibits slow mycelial germination, requiring 12-15 days to cover the culture dish, with poor mycelial growth and irregular colony edges. The mycelial growth of the culture medium described in this invention is significantly superior to that of the modified PDA culture medium.

[0067] Mycelia cultured using the mother culture medium of the present invention were molecularly identified, and their ITS sequence (NCBI accession number PV335977) showed a similarity of over 98% with the ITS sequences of *Boletus saprophyticus* (accession numbers ON707263.1 and PQ498146.1) reported in the GenBank database. Phylogenetic analysis showed that it clustered with the previously reported *Boletus saprophyticus* species, with a support rate of 99%, indicating that strain JSJ-Bx1 is *Boletus saprophyticus*. Figure 4 The results of ITS species identification of mycelia cultured on the mother culture medium described in this invention. Example 2

[0068] A mother culture medium for *Boletus chinensis*, wherein the raw materials of the medium are in the following mass ratio: 20g maltose, 2g ammonium sulfate, 1.5g magnesium sulfate, 0.1g L-tyrosine, 20g agar, and 1L water. The pH of the medium is 5.

[0069] The method for preparing the mother culture medium of *Boletus chinensis* is as follows:

[0070] (1) Weigh maltose, ammonium sulfate, magnesium sulfate, L-tyrosine, agar and water according to the raw material mass ratio, put them into a container and stir evenly;

[0071] (2) Heat the container and boil the liquid material in the container over a low flame to form a homogeneous mixed solution, which is the nutrient solution;

[0072] (3) After the above nutrient solution is bottled, it is sterilized to obtain liquid culture medium;

[0073] (4) Dispense the liquid culture medium into petri dishes, 20 ml per dish, to obtain the mother culture medium of the Chinese saprophytic bolete.

[0074] Using a 0.65cm diameter punch, holes were punched at the same radius from the modified PDA medium to obtain *Boletus spp.* culture medium containing *Boletus spp.* JSJ-Bx1 mycelial blocks were taken and inoculated onto the *Boletus spp.* mother culture medium described in Example 2 of this invention. After inoculation, the medium was placed in a 30℃ constant temperature and humidity incubator and cultured in the dark for 4 days to obtain *Boletus spp.* mycelia. See [link to relevant documentation]. Figure 5 As can be seen, using the mother culture medium described in Example 2 of this invention, the mycelium of *Boletus chinensis* mother culture germinates relatively quickly, covering a culture dish with a diameter of 9 cm in 4 days. However, its mycelium is relatively sparse, and the quality of the mother culture is generally average. Example 3

[0075] A mother culture medium for *Boletus chinensis*, wherein the raw materials of the medium are in the following mass ratio: fructose 20g, ammonium chloride 2g, manganese sulfate 1.5g, vitamin B1 0.1g, agar 20g, and water 1L. The pH of the medium is 6.

[0076] The method for preparing the mother culture medium of *Boletus chinensis* is as follows:

[0077] (1) Weigh fructose, ammonium chloride, manganese sulfate, VB1, agar and water according to the raw material mass ratio, put them into a container and stir evenly;

[0078] (2) Heat the container and boil the liquid material in the container over a low flame to form a homogeneous mixed solution, which is the nutrient solution;

[0079] (3) After the above nutrient solution is bottled, it is sterilized to obtain liquid culture medium;

[0080] (4) Dispense the liquid culture medium into petri dishes, 20 ml per dish, to obtain the *Boletus saprophyticus* mother culture medium. Using a 0.65 cm diameter punch, punch holes at the same radius to obtain *Boletus saprophyticus* mycelial blocks (JSJ-Bx1). Inoculate these blocks into the *Boletus saprophyticus* mother culture medium described in Example 3 of this invention. After inoculation, place the medium in a 25°C constant temperature and humidity incubator and culture in the dark for 3-4 days to obtain *Boletus saprophyticus* mycelia. See [link to relevant documentation]. Figure 6 As can be seen, using the mother culture medium described in Example 3 of this invention, the mycelial germination rate of the Boletus sinensis mother culture is generally slow, and it can fill a culture dish with a diameter of 9 cm in 6 days, but its mycelium is sparse and the quality of the mother culture is poor.

Claims

1. A type of Chinese saprophytic bolete ( Buchwaldoboletus xylophilus Mother culture medium, characterized in that, The strain JSJ-Bx1 of the *Boletus chinensis* mother culture is deposited at the China General Microbiological Culture Collection Center (CGMCC No. 42007) with the accession number CGMCC No. 42007. The raw material ratio of the culture medium is as follows: 20g glucose, 2g yeast extract, 1.5g ferrous sulfate, 0.3g 1 / 2 MS medium, 20g agar and 1L water, with a pH of 4–6.

2. The *Boletus sinensis* species as described in claim 1 (… Buchwaldoboletus xylophilus Mother culture medium, characterized in that, The pH of the culture medium is 4.

3. A type of Chinese saprophytic bolete as described in claim 1 or 2 ( Buchwaldoboletus xylophilus The method for preparing the mother culture medium is characterized by, The method is as follows: (1) Weigh glucose, yeast extract, ferrous sulfate, 1 / 2MS medium, agar and water according to the raw material mass ratio, put them into a container and stir evenly; (2) Heat the container and boil the liquid material in the container over a low flame to form a homogeneous mixed solution, which is the nutrient solution; (3) After the above nutrient solution is bottled, it is sterilized to obtain liquid culture medium; (4) Dispense the liquid culture medium into petri dishes to obtain the mother culture medium of the Chinese Boletus saprophyticus.

4. A type of Chinese saprophytic bolete as described in claim 1 or 2 ( Buchwaldoboletus xylophilus The application of the mother culture medium involves inoculating the strain JSJ-Bx1 of the Boletus saprophyticus mother culture as described in claim 1 into the Boletus saprophyticus mother culture medium, and then culturing it in a constant temperature and humidity incubator at 25–35℃ under dark conditions to obtain Boletus saprophyticus mycelium.

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