A method for rapid indoor propagation of a species of the genus guepinia

Abstract

The application discloses an indoor rapid amplification method of Bresadola cristella, and belongs to the technical field of edible mushroom cultivation. The method comprises two stages of plate culture and liquid fermentation. The plate culture stage adopts a PDA culture medium with 2 mu m microporous special filter membrane, and the PDA culture medium is added with potassium dihydrogen phosphate, magnesium sulfate and proteose peptone. The liquid fermentation stage is cultured in a 300 mL triangular flask with a liquid loading amount of 120 mL, 4 glass beads with a diameter of 7 mm, and a shaking bed rotating speed of 160 r / min. The indoor rapid mass propagation of the Bresadola cristella is realized for the first time through the synergistic optimization of physical and nutritional conditions, the amplification efficiency and biomass of the Bresadola cristella mycelium are significantly improved, and a reliable strain propagation technology is provided for artificial domestication and cultivation of the Bresadola cristella.

Description

Technical Field

[0001] This invention relates to the field of edible fungi cultivation technology, specifically to a type of *Entoloma crystallineis* (Cryptospira). Entoloma clypeatum Indoor rapid amplification technology for bacterial strains. Background Technology

[0002] Crystal-capped pleurotus ( Entoloma clypeatum It belongs to the phylum Basidiomycota, class Agaricomycetes, order Agaricales, family Entolomataceae, and genus Entolomycetes. Entoloma *Entoloma crystallineis* is an edible macrofungus with a delicious taste, rich in nutrients, and possesses medicinal value, including anti-tumor properties. It is an important fungal resource for developing understory economy and enriching forest resources. Artificial domestication and cultivation of *Entoloma crystallineis* is very limited both domestically and internationally, especially regarding the early plate culture stage of the strain (Shishikura M, Sugawara R, Takemura Y, et al. First successful isolation of...). Entoloma clypeatum Species complex from basidiospores[J]. Mycoscience, 2019, 60(4): 221-227.). This article mainly studies the genus *Basidiospores*. Entoloma of" Entoloma clypeatum The challenge of this "species complex" study lies in the fact that there have been almost no previous reports of successfully obtaining pure culture strains of this fungus, and there is also a lack of live strain resources. As can be seen from the article, the overall growth of the strain is very slow; after one month of culture on MNC medium, the maximum colony diameter only reaches 12-13 mm.

[0003] Currently, *Entoloma crystallinum* exists only in the wild, with a short fruiting period, making wild collection insufficient to meet demand. Furthermore, the quality of wild mushrooms is difficult to guarantee, and they may pose safety risks due to the accumulation of heavy metals from the environment. Therefore, achieving the artificial cultivation and domestication of *Entoloma crystallinum* is crucial. *Entoloma crystallinum* is an ectomycorrhizal fungus, and its artificial domestication relies on mycorrhizal synthesis technology, which requires a large amount of high-quality mycelium or spores as starting material. However, research on *Entoloma* fungi in my country has mainly focused on taxonomy, with very little research on the cultivation characteristics and artificial cultivation of *Entoloma crystallinum*. In existing technologies, *Entoloma crystallinum* grows extremely slowly under indoor cultivation conditions, exhibits low mycelial amplification efficiency, and is difficult to induce spore production, posing a significant challenge to establishing a mycorrhizal synthesis pathway and constructing an artificial cultivation technology system.

[0004] Therefore, developing a technology that enables rapid indoor amplification of *Gynostemma pentaphyllum* is of significant theoretical and practical value for solving the bottleneck of strain sourcing and promoting its artificial domestication and cultivation.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a method for rapid indoor amplification of *Entoloma crystallineis*, so as to achieve efficient and rapid propagation of its mycelium.

[0007] To achieve the above objectives, the present invention provides a method for rapid indoor amplification of *Entoloma crystallineis*, comprising the following steps: Plate culture steps: Inoculate the *Entoloma crystallineis* strain onto a culture medium covered with a microporous filter membrane and culture it on a plate to obtain activated colonies; The microporous filter membrane has a pore size of 2 μm; the culture medium is a PDA-enriched medium, which is based on a conventional PDA formulation with the addition of potassium dihydrogen phosphate, magnesium sulfate and peptone.

[0008] Preferably, the formulation of the PDA-enriched medium for the amplification method is as follows: 200 g / L potato, 20 g / L glucose, 20 g / L agar, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, and 2 g / L peptone.

[0009] Preferably, the microporous filter membrane of the amplification method is made of polytetrafluoroethylene.

[0010] Preferably, the plate culture temperature of the amplification method is 25°C, and the culture method is dark culture.

[0011] Preferably, the amplification method includes a liquid fermentation step: inoculating the activated colonies into a liquid culture medium for fermentation culture; The fermentation conditions were as follows: 120 mL of liquid was added to a 300 mL Erlenmeyer flask, along with 4 glass beads with a diameter of 7 mm, and the flask was incubated in a shaker at 25°C and 160 r / min.

[0012] The liquid culture medium is preferably PDB liquid culture medium; the fermentation culture time is 14 days. Preferably, the amplification method further includes a microbial agent preparation step after the plate culture step and before the liquid fermentation step: by preparing the activated colonies into a mycelial cake and inoculating it in PDB liquid medium for pre-culture to obtain a seed liquid.

[0013] Preferably, the pre-culture conditions are: cultured at 25℃ and 160 r / min in the dark for 7 days.

[0014] The amplification method provided by this invention is applicable to *Entoloma crystallineis*, and especially to *Entoloma crystallineis* strain with accession number SICAUCC22-0090.

[0015] The present invention has the following advantages: This invention addresses the challenge of slow growth of *Entoloma crystallineis* in indoor culture by proposing a comprehensive and rapid amplification strategy that combines physical and nutritional optimization.

[0016] In terms of plate culture, through multiple experiments, this invention ultimately selected the use of a 2 μm pore size microporous filter membrane to cover the PDA-enriched medium, which significantly improved the microenvironment for mycelial growth, promoted the rapid expansion of colonies, and resulted in a colony diameter that was significantly better than that of ordinary PDA medium.

[0017] In terms of liquid fermentation, this invention optimizes the liquid volume (120 mL), the number of glass beads (4 beads), and the shaking speed (160 r / min). While providing sufficient dissolved oxygen, it utilizes the moderate shear force generated by the glass beads to break up the mycelial clumps, promoting the formation of more and more uniform mycelial balls, thus significantly increasing the mycelial biomass per unit volume. Attached Figure Description

[0018] Figure 1 This is a comparative diagram showing the effect of different types of culture media on the colony diameter of *Entoloma crystallineis* in the embodiments of the present invention. In this diagram, R represents the colony plate growth diagram of different treatments; S represents the quantitative diagram of colony diameter; A: control group; B: PDA medium; C: plum root medium; D: peach root medium; E: pear root medium; F: potato bran agar medium; different lowercase letters indicate significant differences (P<0.05), the same below.

[0019] Figure 2 This is a comparative diagram showing the effect of different filter membrane specifications on the colony diameter of *Gynostemma pentaphyllum* in the embodiments of the present invention. In the diagram, R represents the colony plate growth diagram of different treatments; S represents the quantitative diagram of colony diameter; A: control group; B: 0.1 μm; C: 0.22 μm; D: 0.45 μm; E: 1 μm; F: 2 μm; G: 5 μm.

[0020] Figure 3 This is a comparison chart of the effects of different liquid volumes on the dry weight of *Entoloma crystallineis* pellets in Example 2 of the present invention. In the chart, R is a picture of *Entoloma crystallineis* pellets under different treatments; S is a bar chart of the corresponding pellet dry weight; A: 80 mL; B: 100 mL; C: 120 mL; D: 140 mL; E: 160 mL.

[0021] Figure 4 This is a comparison chart of the effect of different numbers of glass beads on the dry weight of *Entoloma crystallineis* mycelial balls in Example 2 of the present invention. In the chart, R is a picture of *Entoloma crystallineis* mycelial balls under different treatments; S is a bar chart of the corresponding mycelial ball dry weight; A: 2 beads; B: 3 beads; C: 4 beads; D: 5 beads; E: 6 beads.

[0022] Figure 5 This is a comparison chart of the effects of different shaking speeds on the dry weight of *Entoloma crystallineis* pellets in Example 2 of the present invention. R represents actual images of *Entoloma crystallineis* pellets under different treatments; S represents a bar chart of the corresponding pellet dry weight; A: 140 r / min; B: 150 r / min; C: 160 r / min; D: 170 r / min; E: 180 r / min.

[0023] Figure 6 This is a trend graph showing the influence of various factors on the dry weight of bacterial pellets during liquid fermentation in Example 2 of the present invention. R, S, and T represent the dry weight of bacterial pellets under different liquid volumes, different numbers of pellets, and different shaking speeds, respectively.

[0024] Figure 7 This is a response surface plot of the response surface optimization results for liquid fermentation conditions in Example 2 of the present invention, wherein... Figure 7 a, c, and e in the figure are contour plots, corresponding to the interaction of rotation speed × number of beads, rotation speed × liquid volume, and number of beads × liquid volume, respectively; b, d, and f are three-dimensional response surface plots, corresponding to the interaction of rotation speed × number of beads, rotation speed × liquid volume, and number of beads × liquid volume, respectively, with the vertical axis representing the dry weight of the bacterial pellets. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0027] The strain used in this invention is *Entoloma crystallineis*, deposited in the Herbarium of Sichuan Agricultural University Culture Collection (SICAUCC), strain number SICAUCC 22-0090. The source can be found in: Xu Ruolan, Zhou Yijie, Hu Saixue, et al. Identification, nutritional evaluation and mycelial biological characteristics of wild *Entoloma crystallineis* [J / OL]. Fungal Research, 1-26 [2026-07-30]. This biological material is available to the public from the applicant.

[0028] Example 1: Rapid Amplification via Plate Culture 1. Preparation of different test culture media: The plate culture in this experiment involved six different culture media for comparative experiments, namely PDA medium, PDA enriched medium, plum root medium, peach root medium, pear root medium, and potato bran agar medium. The preparation methods of each culture medium are as follows. After all the culture media were prepared, they were autoclaved at 121℃ for 30 min and then cooled for later use.

[0029] (1) PDA culture medium Formula: 200 g / L peeled potatoes, 20 g / L anhydrous glucose, 20 g / L agar, with distilled water as the solvent.

[0030] Preparation method: Weigh 200 g of peeled potatoes, cut them into small pieces, add an appropriate amount of distilled water and simmer for 20-30 minutes. Filter the solution through four layers of gauze and collect the filtrate. Add 20 g of anhydrous glucose and 20 g of agar to the filtrate, heat and stir until the solutes are completely dissolved, and add distilled water to make up to 1000 mL.

[0031] (2) PDA-enriched medium Formula: 200 g / L peeled potatoes, 20 g / L anhydrous glucose, 20 g / L agar, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, 2 g / L peptone, and distilled water as solvent.

[0032] Preparation method: The preparation process of the basic filtrate is exactly the same as that of PDA culture medium; after the glucose and agar are completely dissolved, add potassium dihydrogen phosphate, magnesium sulfate and peptone, and stir continuously until all solutes are dissolved, and then bring the volume to 1000 mL with distilled water.

[0033] (3) Plum root culture medium Formula: 200 g / L plum root extract, 20 g / L anhydrous glucose, 20 g / L agar, with distilled water as the solvent.

[0034] Preparation method: Take 200 g of fresh plum tree roots, chop them, add water and boil to extract. Filter through four layers of gauze to obtain the root extract. Add anhydrous glucose and agar to the extract, heat to dissolve, and then distill water to a final volume of 1000 mL.

[0035] (4) Peach root culture medium Formula: 200 g / L peach root extract, 20 g / L anhydrous glucose, 20 g / L agar, with distilled water as the solvent.

[0036] Preparation method: The extraction and preparation process is the same as that of plum root culture medium, except that the raw materials are replaced with fresh peach roots.

[0037] (5) Pear root culture medium Formula: 200 g / L pear root extract, 20 g / L anhydrous glucose, 20 g / L agar, with distilled water as the solvent.

[0038] Preparation method: The extraction and preparation process is the same as that of plum root culture medium, except that the raw materials are replaced with fresh pear roots.

[0039] (6) Potato bran agar medium Formula: 200 g / L peeled potatoes, 30 g / L wheat bran, 20 g / L anhydrous glucose, 20 g / L agar, with distilled water as the solvent.

[0040] Preparation method: After boiling and filtering potatoes to obtain filtrate, add wheat bran and heat and stir for 10 minutes. Then add anhydrous glucose and agar, and after completely dissolving, distilled water to bring the volume to 1000 mL.

[0041] 2. Gradient setting and cultivation of microporous membranes with different pore sizes: Under aseptic conditions, sterilized PDA-enriched medium was poured into petri dishes. After the medium solidified, sterile microporous membranes (PTFE) with pore sizes of 0.1 μm, 0.22 μm, 0.45 μm, 1 μm, 2 μm, and 5 μm were smoothly attached to the surface of the medium, grouped separately. PDA-enriched medium without membranes served as a control.

[0042] Using a sterile punch, cut 0.5 cm diameter mycelial discs from the edge of activated *Entoloma crystallineis* colonies and inoculate them into the center of a culture medium plate. Incubate in the dark at 25°C. Measure colony diameter every 7 days using the cross-sectional method until colony growth ceases. Five replicates were performed.

[0043] 3. Effects of different culture media on the growth of *Entoloma crystallineis*: Colony diameters under different types of culture media are shown below. Figure 1As shown in the figure. The results indicated that not all strains grew normally under different types of culture media. The strains did not grow on plum root, peach root, and pear root media. Significant differences in colony diameter were observed on other media (P<0.05). Growth was inhibited on potato bran medium, with an average diameter of only 16.8 mm. Colonies grew normally on PDA medium, but the average diameter was 29.3 mm, slightly smaller than the 31.4 mm on enriched medium. The colony diameter, from largest to smallest, was as follows: enriched PDA medium > control group (PDA medium) > potato bran medium > plum root = peach root = pear root. In summary, enriched PDA medium is the optimal growth medium for *Entoloma crystallineis*.

[0044] 4. Effects of different filter membrane specifications on the growth of *Entoloma crystallineis*: Colony diameters under different filter membrane specifications are shown in the figure. Figure 2 The results showed that the bacterial strains could grow normally under different filter membrane pore sizes. The average colony diameter was 39.7 mm when the filter membrane pore size was 2 μm, slightly higher than the 37.3 mm of the 0.22 μm filter membrane, and significantly higher than the 33.3 mm of the control group and other filter membranes with different pore sizes. The colony diameter of the 2 μm filter membrane was significantly larger than that of other filter membranes (P<0.05). The colony diameters under different filter membrane pore sizes, from largest to smallest, were: 2 μm > 0.22 μm > control group > 1 μm > 5 μm > 0.45 μm > 0.1 μm. Based on comprehensive analysis, the optimal filter membrane pore size for *Entoloma crystallineis* growth was 2 μm.

[0045] Example 2: Rapid Expansion via Liquid Fermentation 1. Preparation of microbial agents: Using a sterile punch, cut a 0.5 cm mycelial disc from the edge of an activated *Gyromitra crystallineis* colony and inoculate it into PDB liquid medium. Pre-culture at 25°C and 160 r / min in the dark for 7 days as a pre-inoculum.

[0046] 2. Fermentation conditions optimization: Different liquid volumes (80, 100, 120, 140, 160 mL), numbers of glass beads (0, 2, 3, 4, 5, 6 beads, 7 mm in diameter), and shaking speeds (140, 150, 160, 170, 180 r / min) were set in 300 mL Erlenmeyer flasks. The pre-cultured seed culture bacterial cake was transferred into the Erlenmeyer flasks of each treatment group. Each treatment was set up with 5 replicates and cultured in a shaker at 25℃ for 14 days before biomass determination.

[0047] After cultivation, collect the mycelial balls, rinse them with purified water, dry them in a 50℃ oven until constant weight, and weigh the dry weight of the mycelium.

[0048] Single-factor experiments are shown Figure 3-5 The results compare the effects of different liquid volumes, different numbers of glass beads, and different shaking speeds on the dry weight of *Gynostemma pentaphyllum* mycelial pellets. The trends in dry weight variation under different factors are shown in [Figure 1]. Figure 6 As shown in the figure. The results indicate that the bacterial pellets had the highest dry weight when the liquid volume was 120 mL; the bacterial pellets had the highest dry weight when the number of glass beads was 4; and the bacterial pellets had the highest dry weight when the shaking speed was 160 r / min.

[0049] Based on the results of single-factor experiments, response surface methodology was used to optimize the study of the effect of two-factor interaction on the dry weight of bacterial pellets. The factors and levels of the response surface experiment are shown in Table 1, and the resulting response surface plot is shown in [Table 1]. Figure 7 The results of the response surface methodology are shown in Table 2. The optimal fermentation combination was determined to be: 120 mL liquid volume, 4 glass beads, and a shaking speed of 160 r / min. A verification experiment was conducted under these optimal conditions, yielding a bacterial pellet dry weight of 0.17 ± 0.005 g, which was largely consistent with the predicted value. This indicates that the liquid fermentation conditions can stably and efficiently achieve rapid propagation of *Entoloma crystallineis* mycelium.

[0050] Table 1. Factors and levels in response surface methodology ; Table 2 Response Surface Experimental Design and Results ; Example 3: Combined application of plate culture and liquid fermentation The *Gynostemma pentaphyllum* colonies obtained through rapid plate amplification in Example 1 were used to cut mycelial cakes using a sterile punch and used as inoculum for liquid fermentation. The culture was then expanded according to the optimized liquid fermentation conditions of Example 2. The results confirmed that the combined application of the two methods could obtain sufficient high-quality mycelium in a shorter time, meeting the needs of subsequent research such as mycorrhizal synthesis.

[0051] In summary, this invention provides a method for rapid indoor amplification of *Entoloma crystallineis*. By optimizing the physical and nutritional conditions of plate culture and the key process parameters of liquid fermentation, the problem of slow growth is solved in a coordinated manner, laying the foundation for the large-scale cultivation and development of *Entoloma crystallineis*.

[0052] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for rapid indoor amplification of *Entoloma crystallineis*, characterized in that, Includes the following steps: Plate culture steps: Inoculate the *Entoloma crystallineis* strain onto a culture medium covered with a microporous filter membrane and culture it on a plate to obtain activated colonies; The microporous filter membrane has a pore size of 2 μm; the culture medium is a PDA-enriched medium, which is based on a conventional PDA formulation with the addition of potassium dihydrogen phosphate, magnesium sulfate and peptone.

2. The method according to claim 1, characterized in that, The formula for the PDA-enriched medium is as follows: 200 g / L potato, 20 g / L glucose, 20 g / L agar, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, and 2 g / L peptone.

3. The method according to claim 1, characterized in that, The microporous filter membrane is made of polytetrafluoroethylene.

4. The method according to claim 1, characterized in that, The plate culture was conducted at a temperature of 25°C in the dark.

5. The method according to any one of claims 1-4, characterized in that, It also includes a liquid fermentation step: inoculating the activated colonies into a liquid culture medium for fermentation culture; The fermentation conditions were as follows: 120 mL of liquid was added to a 300 mL Erlenmeyer flask, along with 4 glass beads with a diameter of 7 mm, and the flask was incubated in a shaker at 25°C and 160 r / min.

6. The method according to claim 5, characterized in that, The liquid culture medium is PDB liquid culture medium.

7. The method according to claim 5, characterized in that, The fermentation culture time is 14 days.

8. The method according to claim 5, characterized in that, After the plate culture step and before the liquid fermentation step, there is also a microbial agent preparation step: the activated colonies are made into mycelial cakes, inoculated into PDB liquid culture medium for pre-culture, and seed liquid is obtained.

9. The method according to claim 8, characterized in that, The pre-culture conditions were: cultured at 25℃ and 160 r / min in the dark for 7 days.

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