ARTP mutagenic strain openable liquid fermentation, preparation method and application

Abstract

The present application relates to the technical field of microorganism, and more particularly to an ARTP mutagenized strain capable of open liquid fermentation, a preparation method and application, comprising: the strain is Ganoderma lucidum strain MKLGE251231, which has been preserved in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No.42590.The present application improves the anti-bacterial ability of open liquid fermentation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to an ARTP mutagenesis strain capable of open-air liquid fermentation, its preparation method, and its application. Background Technology

[0002] Currently, the preparation of mycelium through deep liquid fermentation followed by shallow static culture is one of the important methods for preparing mycelial materials. However, the reliance of traditional liquid fermentation on a strictly aseptic environment, while advantageous in ensuring pure culture, also brings several significant drawbacks. First, it substantially increases equipment investment and operating costs. From the sealed design of the fermenter and air filtration system to the batch sterilization of the culture medium, all require substantial financial and energy support. Second, these stringent conditions greatly increase the complexity of operations and the risk of contamination. Any minor oversight in any step, such as inoculation or pipeline sterilization, can lead to the scrapping of the entire batch, resulting in resource waste. Furthermore, the strict aseptic requirements limit the flexibility and scale of production, making continuous production difficult, and exhibiting poor adaptability to complex non-standard raw materials, indirectly restricting the application potential of fermentation technology in some low-cost, high-value-added product fields.

[0003] Atmospheric pressure room temperature plasma (ARTP) mutagenesis is a classic physical mutagenesis method with advantages such as high efficiency, safe operation, and broad mutation spectrum. It utilizes a plasma jet rich in active particles generated under atmospheric pressure to act on protoplasts or spores, causing DNA damage and inducing mutations, thereby screening for mutant strains with excellent traits. Studies have used ARTP mutagenesis technology to obtain excellent strains of edible fungi such as Ganoderma lucidum, which are used for liquid fermentation to produce mycelial raw materials.

[0004] However, existing mutagenic strains are mainly aimed at the food and health product fields, and there are no reports of strains specifically for the preparation of mycelial materials, especially strains that can achieve open liquid fermentation and form a continuous mycelial cortex.

[0005] Chinese Patent Publication No. CN119242454A discloses a *Ganoderma lucidum* strain and its mycelial culture method. This invention provides a *Ganoderma lucidum* strain ZHM1939 (accession number CGMCCNo: 41412). This invention is the first to provide a *Ganoderma lucidum* strain and its optimal culture method, providing new materials for the research and clinical application of the edible and medicinal value of *Ganoderma* fungi, and providing an excellent strain for the artificial cultivation and promotion of this fungus. It enriches the cultivable varieties of *Ganoderma*, effectively protects *Ganoderma lucidum* cultivation resources, and prevents the extinction threat to this fungus caused by human and natural factors. However, the aforementioned *Ganoderma lucidum* strain and its mycelial culture method have the following problems: The Ganoderma lucidum strains cannot inhibit the growth of contaminating bacteria and form a continuous mycelial cortex under open liquid fermentation conditions, which leads to the preparation of mycelial materials relying on a strict aseptic environment, resulting in high production costs and a high risk of contamination. After static culture of the fermentation broth, the mycelial skin integrity was poor and the molding quality was low. Summary of the Invention

[0006] Therefore, the present invention provides an ARTP mutagenesis strain capable of open-air liquid fermentation, its preparation method, and its application, in order to overcome the problem in the prior art of lacking a dedicated strain capable of achieving open-air liquid fermentation and forming a continuous mycelial cortex.

[0007] To achieve the above objectives, in one aspect, the present invention provides an ARTP mutagenesis strain capable of open-air liquid fermentation, comprising: The strain is Ganoderma lucidum strain MKLGE251231, which has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.42590.

[0008] Furthermore, this invention also provides the application of ARTP mutagenic strains capable of open-air liquid fermentation, wherein the Ganoderma lucidum strain is applied to mycelial materials, including... The activity coefficient of the Ganoderma lucidum strain was determined based on the pH value and reducing sugar concentration of the strain inoculated in the first culture medium, so as to determine the qualification of the seed liquid. The competitive inhibition coefficient of the Ganoderma lucidum strain was determined based on the biomass change rate and inhibition zone diameter of the qualified seed liquid inoculated in the unsterilized second culture medium, so as to obtain the fermentation broth in which the competitive inhibition of Ganoderma lucidum strain against miscellaneous bacteria met the standard. The mycelium formation coefficient was determined based on the mycelium coverage, mycelium thickness, and mycelium porosity during static fermentation, and the compliance of static fermentation was determined based on the total number of miscellaneous bacteria in the fermentation broth below the mycelium. The mycelial skin that has reached the standard after static culture is plasticized and hot-pressed to obtain mycelial material.

[0009] Furthermore, based on an activity coefficient greater than or equal to a preset activity coefficient, the activated seed culture is deemed qualified. The activity coefficient is determined based on the product of the pH change rate ratio and the reducing sugar consumption rate ratio, wherein the pH change rate ratio is the ratio of the pH change rate to the preset pH change rate, and the reducing sugar consumption rate ratio is the ratio of the reducing sugar consumption rate to the preset reducing sugar consumption rate.

[0010] Furthermore, based on the competitive inhibition coefficient being greater than or equal to a preset competitive inhibition coefficient, the competitive inhibition of the Ganoderma lucidum strain against contaminating bacteria was determined to be up to standard. The competitive inhibition coefficient is determined based on the product of the ratio of biomass change rate and the ratio of inhibition zone diameter. The ratio of biomass change rate is the ratio of biomass change rate to a preset biomass change rate, and the ratio of inhibition zone diameter is the ratio of inhibition zone diameter to a preset inhibition zone diameter.

[0011] Furthermore, based on the fact that the mycelial skin formation coefficient is greater than or equal to the preset mycelial skin formation coefficient, and the total number of contaminating bacteria is less than or equal to the preset total number of contaminating bacteria, the static culture is determined to have met the standard, wherein... The mycelium formation coefficient is determined based on the product of the mycelium coverage ratio, the mycelium thickness ratio, and the mycelium porosity ratio. The mycelium coverage ratio is the ratio of the mycelium coverage to the preset mycelium coverage ratio, the mycelium thickness ratio is the ratio of the mycelium thickness to the preset mycelium thickness, and the mycelium porosity is the ratio of the mycelium porosity to the preset mycelium porosity.

[0012] Further, the first culture medium consists of the following components: glucose 35 g / L, peptone 5.0 g / L, yeast extract 2.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium dihydrogen phosphate 1.0 g / L, vitamin B1 0.05 g / L, with the balance being water, pH 5.50, and sterilized at 121°C for 20 min; The second culture medium consists of the following components: 35 g / L glucose, 5 g / L peptone, 3 g / L beef extract, 3 g / L fine wheat bran, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, with the remainder being water. The pH value is 6.5–7.5 and is not adjusted. The second sterilized culture medium was prepared and then sterilized at 121°C for 20 minutes before inoculation. The unsterilized second culture medium is prepared and then directly inoculated.

[0013] Furthermore, the activation culture conditions are as follows: the slant culture of Ganoderma lucidum strain is made into 5 mm diameter blocks using a sterile punch, and 3 blocks are inoculated at a ratio of 100 mL of the first culture medium. The culture is then incubated at 25 °C and 150 rpm for 4 days to obtain the seed liquid.

[0014] Furthermore, the conditions for obtaining the fermentation broth are as follows: the activated and cultured seed liquid is inoculated into an unsterilized second culture medium at an inoculation rate of 5% by volume, and fermented on a shaker at 25°C and 150 rpm for 5 days.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention obtains regenerated strains through ARTP mutagenesis and regeneration, screens the mutagenic strains through antagonistic culture, quantifies the growth adaptability of the strains in an open environment using the fermentation coefficient, and solves the problem of high false screening rate caused by reliance on subjective morphology in mutagenesis screening. The activity coefficient is used to quantify the seed liquid activity, ensuring that the inoculated Ganoderma lucidum strains are in the logarithmic growth phase, thus improving the fermentation success rate. The shaking speed or fermentation time is dynamically adjusted by the competitive inhibition coefficient, which enhances the resistance of Ganoderma lucidum strains to contamination and reduces the batch scrap rate caused by contamination. Furthermore, the static culture is controlled based on the mycelial skin forming coefficient, and the plasticizing hot pressing process is optimized to obtain mycelial materials. The present invention synergistically realizes the control of open liquid fermentation and the preparation of mycelial materials.

[0016] Furthermore, this invention achieves efficient breeding of superior strains for open fermentation through an ARTP mutagenesis combined with a fermentation coefficient screening strategy. ARTP mutagenesis utilizes active particles generated by ambient pressure and room temperature plasma to act on Ganoderma lucidum protoplasts, causing DNA damage and inducing broad-spectrum mutations. Antagonistic culture is used to rapidly identify regenerated strains with genetic variations. The fermentation coefficient reflects the ability of strains to inhibit contaminating bacteria and compete for nutrients in an open environment through metabolites. This solves the problems of low screening efficiency and high false screening rate caused by traditional mutagenesis screening relying on subjective morphological observation and lacking quantitative evaluation indicators, thus improving the screening accuracy and providing strain guarantee for open fermentation.

[0017] Furthermore, this invention achieves quantitative control of seed culture viability by comprehensively reflecting the pH change rate and reducing sugar consumption rate during activation culture through the activity coefficient. The pH change rate characterizes the rate of acid production by mycelial metabolism, reflecting the intensity of mycelial respiration metabolism, while the reducing sugar consumption rate characterizes the efficiency of mycelial carbon source utilization, reflecting the mycelial growth rate. When both are at a high level simultaneously, it indicates that the mycelium is in the vigorous logarithmic growth phase. Compared with the traditional method of visually observing mycelial morphology and judging by experience, this invention solves the problems of lack of quantitative indicators for seed culture viability evaluation and high fermentation failure rate caused by large fluctuations in viability between different batches, thereby improving the seed culture qualification rate and ensuring the stability of open fermentation.

[0018] Furthermore, this invention establishes a competitive inhibition coefficient evaluation system to quantitatively assess the antimicrobial ability of Ganoderma lucidum strains in open fermentation. The competitive inhibition coefficient is calculated by combining the biomass change rate and the diameter of the inhibition zone in the fermentation broth supernatant. The biomass change rate reflects the growth rate of the strain and indirectly characterizes the metabolic activity intensity of the strain. The diameter of the inhibition zone directly characterizes the inhibitory effect of the antimicrobial substances produced by the strain's metabolism on other microorganisms. By increasing the shaking speed to increase dissolved oxygen supply to enhance the aerobic metabolic capacity of the strain, or by extending the fermentation time to allow the strain to fully establish population dominance, this invention solves the problem of inhibited growth and fermentation failure of Ganoderma lucidum strains due to competition from other microorganisms in open fermentation, reduces the contamination rate, and reduces batch scrap.

[0019] Furthermore, this invention achieves control over the quality of mycelial formation during static cultivation by constructing a mycelial formation coefficient. The mycelial formation coefficient comprehensively reflects the mycelial coverage, mycelial thickness, and mycelial porosity. Among them, the mycelial coverage directly affects the liquid surface sealing, determining whether contaminating bacteria can invade the liquid phase from the gas phase; the mycelial thickness determines the density of the mycelial network and the operability of hot pressing; and the mycelial porosity affects the penetration depth and uniformity of the plasticized liquid. These three factors jointly determine the integrity and forming quality of the mycelial. By extending the static time to promote the continued extension and coverage of mycelia, or by supplementing carbon and nitrogen sources to provide nutritional support for mycelial growth, the problems of incomplete mycelial, uneven thickness, and excessive porosity are solved, improving the integrity and structural uniformity of the mycelial and providing a raw material guarantee for the preparation of mycelial materials. Attached Figure Description

[0020] Figure 1 A logic diagram for determining whether a mutant strain is a Ganoderma lucidum strain in an embodiment of the present invention; Figure 2 This is a flowchart illustrating the steps involved in applying the ARTP mutagen strain capable of open-air liquid fermentation according to an embodiment of the present invention. Figure 3 A logic diagram for determining whether the seed culture solution for active culture is qualified in an embodiment of the present invention; Figure 4 This is a logic diagram showing whether the competitive inhibition of Ganoderma lucidum strains against miscellaneous bacteria meets the standard in an embodiment of the present invention. Figure 5 The image shows the actual mycelium obtained after static culture in an embodiment of the present invention. Figure 6 This is a scanning electron microscope image of the mycelial material in an embodiment of the present invention; Figure 7 This is a photograph of the mycelial material after hot pressing according to an embodiment of the present invention.

[0021] The Ganoderma lucidum strain MKLGE251231 involved in this invention is classified and named Ganoderma lucidum ( Ganoderma lingzhi The specimen was deposited on March 11, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42590. Detailed Implementation

[0022] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0023] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0024] It should be noted that in the description of this invention, the terms "upper", "lower", "left", "right", "inner", "outer", etc., which indicate directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and is not intended to indicate or imply that the device or element must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation of this invention.

[0025] In this embodiment of the invention, the starting strain is Ganoderma lingzhi, which was isolated in June 2025 from wild Ganoderma fruiting bodies collected from a broad-leaved forest in Chengdu, Sichuan Province, and pure cultures were obtained by tissue isolation method.

[0026] In this embodiment of the invention, an ITS sequence (SEQ ID NO:1) with a length of 654 bp was obtained through ITS sequence analysis (primers: ITS1F / ITS4). After comparison with NCBI BLAST, the ITS sequence of Ganoderma lingzhi showed a similarity of 99.8%, and the classification name was determined to be Ganoderma lingzhi.

[0027] In this embodiment of the invention, the starting strain was screened after ARTP mutagenesis to obtain Ganoderma lucidum strain MKLGE251231, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No.42590, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 11, 2026.

[0028] Specifically, the Ganoderma lucidum strain MKLGE251231 was prepared based on the following steps, including: The protoplasts prepared from the starting strain were diluted and mutagenized, and the resulting regenerated strain and the starting strain were antagonistically cultured to obtain the mutagenized strain. The fermentation coefficient of the mutant strain is determined based on the biomass of the mutant strain inoculated into the culture medium in order to identify the Ganoderma lucidum strain.

[0029] Specifically, the process of preparing protoplasts includes: Step S11: Inoculate the starting strain into PDB liquid medium and incubate it in a constant temperature incubator at 25℃ for 7 days to obtain mycelium; Step S12: Wash the mycelium three times with 0.6M mannitol solution. After each wash, centrifuge at 6000 r / min for 10 min, discard the supernatant, collect the mycelium precipitate, and add 2% (w / v) lysozyme solution at a ratio of 0.2 g wet mycelium to 1 mL enzyme solution. Place in a 31℃ constant temperature water bath for 3.5 h of enzymatic hydrolysis, shaking once every 0.5 h during the enzymatic hydrolysis process. Step S13: Filter the solution through a 0.45 μm microporous membrane to remove undigested mycelial fragments. Centrifuge the filtrate at 4000 r / min for 10 min, discard the supernatant, and obtain protoplast precipitate. Redissolve the protoplast precipitate in 0.6 M mannitol solution, centrifuge at 4000 r / min for 10 min, and repeat the washing process 3 times. Step S14: Add the washed protoplasts to a 0.6M mannitol solution to prepare a protoplast suspension.

[0030] In this embodiment of the invention, the composition of the PDB liquid culture medium includes 200 g / L potato, 20 g / L glucose, and natural pH.

[0031] In this embodiment of the invention, the protoplast suspension is counted and detected under an optical microscope using a hemocytometer to determine the protoplast concentration to be 2 × 10⁻⁶. 8 per mL.

[0032] Specifically, the process of obtaining regenerated strains includes: Step S21: Dilute the protoplast suspension with 0.6M mannitol solution to... cells / mL; Step S22: Take 90 μL of protoplast solution and 10 μL of glycerol and mix them evenly in a sterile EP tube. Take 20 μL of the mixed solution and spread it evenly on the surface of a sterile slide. Step S23: Place the slide in the ARTP mutagenesis breeding instrument, set the working gas to helium, power to 121W, distance of the worktable from the gas outlet to 2mm, and processing time to 55s for mutagenesis treatment. Step S24: After mutagenesis, transfer the sample on the slide to 650 μL of 0.6 M mannitol solution, mix gently, and take 200 μL of the suspension to spread on a regeneration culture medium plate. Step S25: Place the regeneration medium plate in a 25℃ constant temperature incubator and incubate in the dark for 5 days. Observe and count the number of regenerated colonies daily and calculate the regeneration rate. Step S26: Select regenerated colonies with a diameter greater than 2 mm and vigorous growth after regeneration culture, inoculate them into PDA plates for purification culture, and culture at 25℃ for 3 days to obtain pure culture of regenerated strain.

[0033] In this embodiment of the invention, the regeneration culture medium consists of the following components: mannitol 109.3 g / L, glucose 4 g / L, maltose 10 g / L, yeast extract 4 g / L, agar 15 g / L, and natural pH.

[0034] Specifically, the process of obtaining mutant strains includes: In step S31, the pure culture of the regenerated strain and the original strain were inoculated onto PDA plates and cultured at 25°C for 3 days to obtain fresh bacterial blocks. Step S32: In the center of a PDA plate with a diameter of 90 mm, inoculate a starting strain block with a diameter of 5 mm, and inoculate 6 regenerated strain blocks at equal intervals around it, with each regenerated strain block 30 mm away from the central starting strain block. Step S33: Place the inoculated PDA plates in a 25°C constant temperature incubator and incubate in the dark for 7 days, observing the colony growth daily. Step S34: After the culture is completed, observe the boundary between the colonies of the starting strain and the regenerated strain, measure the width of the antagonistic line, measure it three times at different positions at the boundary with a vernier caliper, take the average value, and compare the width of the antagonistic line with the preset antagonistic line width. Step S35: If the antagonistic line width is greater than or equal to the preset antagonistic line width, then the regenerated strain is determined to be a mutagenic strain. Step S36: If the antagonistic line width is less than the preset antagonistic line width, the regenerated strain is determined to be a non-mutated strain and discarded. Step S37: Inoculate the mutant strain into a PDA slant, incubate at 25°C for 7 days, and store in a 4°C refrigerator for later use.

[0035] In this embodiment of the invention, the antagonistic line width is the width of the no-growth zone between the colony of the starting strain and the colony of the regenerated strain. The preset antagonistic line width is 2 mm, determined by: conducting open fermentation capacity tests on 30 regenerated strains with antagonistic line widths of 1 mm, 1.5 mm, 2 mm, 2.5 mm, and 3 mm respectively, with a fermentation coefficient greater than or equal to 0.95 as the standard for excellence. The results show that among the strains with an antagonistic line width greater than or equal to 2 mm, the proportion of excellent strains is 82%; while among the strains with an antagonistic line width less than 2 mm, the proportion of excellent strains is only 6%.

[0036] Please see Figure 1 As shown, it is a logic diagram for determining whether the mutant strain is a Ganoderma lucidum strain in an embodiment of the present invention.

[0037] Specifically, the slant culture of the mutant strain was made into 5mm diameter blocks using a sterile punch, and 3 blocks were inoculated at a ratio of 100mL of the first culture medium. The blocks were then cultured in a shaker at 25℃ and 150rpm for 4 days to obtain the seed culture.

[0038] Specifically, the seed culture was inoculated at a volume percentage of 5% into both unsterilized and sterilized secondary culture media, and cultured in a constant temperature shaker at 25℃ and 150 rpm for 5 days. After the culture was completed, the fermentation broth was centrifuged at 8000 r / min for 15 min, the supernatant was discarded, the mycelial precipitate was collected, washed three times with deionized water, dried at 60℃ to constant weight, and the biomass obtained from the culture in sterilized and unsterilized secondary culture media was recorded to calculate the fermentation coefficient and determine whether the mutant strain was a Ganoderma lucidum strain. Compare the fermentation coefficient with the preset fermentation coefficient; If the fermentation coefficient is greater than or equal to the preset fermentation coefficient, then the mutagenized strain is determined to be a Ganoderma lucidum strain. If the fermentation coefficient is less than the preset fermentation coefficient, then the mutant strain is determined not to be a Ganoderma lucidum strain.

[0039] In this embodiment of the invention, the fermentation coefficient is the ratio of the biomass obtained by the mutagenic strain in the unsterilized second culture medium to the biomass obtained by the mutagenic strain in the sterilized second culture medium. Biomass refers to the dry weight obtained by centrifuging and collecting mycelia after fermentation, drying them at 60°C to constant weight, and then weighing them. It can be understood that the ratio reflects the degree to which the growth ability of the mutagenic strain in the unsterilized culture medium is maintained relative to that in the sterilized culture medium. The higher the ratio, the stronger the growth adaptability of the mutagenic strain in the open environment.

[0040] In this embodiment of the invention, the preset fermentation coefficient ranges from 0.90 to 0.98, preferably set to 0.95. The value is determined based on the following experimental data: 82 mutant strains were subjected to open fermentation capacity tests, and the biomass ratio in unsterilized and sterilized culture media was measured. For strains with a fermentation coefficient greater than or equal to 0.95, open fermentation verification was performed with a success rate of over 95%. For strains with a fermentation coefficient less than 0.95, the verification success rate was less than 20%. Through receiver operating characteristic (ROC) curve analysis, 0.95 was determined to be the optimal cutoff value for distinguishing between good and bad open fermentation capacity, corresponding to a sensitivity of 92% and a specificity of 88%. However, the above value is not limited to this, and those skilled in the art can adjust the value according to actual needs.

[0041] Specifically, the first culture medium consists of the following components: 35 g / L glucose, 5.0 g / L peptone, 2.5 g / L yeast extract, 0.5 g / L magnesium sulfate heptahydrate, 1.0 g / L potassium dihydrogen phosphate, 0.05 g / L vitamin B1, with the balance being water, pH 5.50, and sterilized at 121°C for 20 min.

[0042] Specifically, the second culture medium consists of the following components: 35 g / L glucose, 5 g / L peptone, 3 g / L beef extract, 3 g / L fine wheat bran, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, with the remainder being water. The pH value is 6.5–7.5 and is not adjusted. The sterilized second culture medium is prepared by sterilizing at 121°C for 20 min before inoculation. The non-sterilized second culture medium is prepared by direct inoculation.

[0043] Specifically, if the mutant strain is not a Ganoderma lucidum strain, it is determined that the mutant strain does not have open fermentation capability and is discarded. Then, the remaining mutant strains are re-screened until a Ganoderma lucidum strain with a fermentation coefficient greater than or equal to the preset fermentation coefficient is obtained.

[0044] Specifically, this invention achieves efficient breeding of superior strains for open fermentation through an ARTP mutagenesis combined with a fermentation coefficient screening strategy. ARTP mutagenesis utilizes active particles generated by ambient pressure and room temperature plasma to act on Ganoderma lucidum protoplasts, causing DNA damage and inducing broad-spectrum mutations. Antagonistic culture is used to rapidly identify regenerated strains with genetic variations. The fermentation coefficient reflects the ability of strains to inhibit contaminating bacteria and compete for nutrients in an open environment through metabolites. This solves the problems of low screening efficiency and high false screening rate caused by traditional mutagenesis screening relying on subjective morphological observation and lacking quantitative evaluation indicators, thus improving the screening accuracy and providing strain guarantee for open fermentation.

[0045] Please see Figure 2 The diagram shown is a flowchart illustrating the steps involved in the application of the ARTP mutagen strain capable of open-air liquid fermentation according to an embodiment of the present invention.

[0046] The application of the ARTP mutagen strain capable of open-air liquid fermentation according to embodiments of the present invention includes: Step S1: In response to the mutagenized strain being a Ganoderma lucidum strain, the Ganoderma lucidum strain is inoculated into a first culture medium for activation culture to obtain a seed liquid. The pH value and reducing sugar concentration of the first culture medium during the activation culture process are obtained to calculate the mycelial activity coefficient and determine whether the activated culture seed liquid is qualified. Step S2: Inoculate the activated and qualified seed liquid into an unsterilized second culture medium for fermentation, and obtain the biomass of the Ganoderma lucidum strain and the diameter of the inhibition zone of the fermentation broth supernatant during the fermentation process to determine the competitive inhibition coefficient, and determine whether the competitive inhibition of the Ganoderma lucidum strain against miscellaneous bacteria meets the standard. If the competitive inhibition does not meet the standard, determine whether to increase the shaking speed or extend the fermentation time. Step S3: In response to the Ganoderma lucidum strain achieving competitive inhibition of miscellaneous bacteria, a fermentation broth is obtained, and the fermentation broth is statically cultured. The mycelial skin coverage, mycelial skin thickness, and mycelial skin porosity during the static culture process are obtained to determine the mycelial skin forming coefficient. The total number of miscellaneous bacteria in the fermentation broth below the mycelial skin is also obtained to determine whether the static culture has met the standard. Step S4: Plasticize the mycelial skin that has reached the standard after static culture and heat-press it into shape to obtain mycelial material.

[0047] Specifically, this invention obtains regenerated strains through ARTP mutagenesis and regeneration, screens the mutagenic strains through antagonistic culture, and quantifies the growth adaptability of the strains in an open environment using the fermentation coefficient, thus solving the problem of high false screening rate caused by reliance on subjective morphology in mutagenesis screening. The activity coefficient is used to quantify the seed liquid vigor, ensuring that the inoculated mycelium is in the logarithmic growth phase, thereby improving the fermentation success rate. The shaking speed or fermentation time is dynamically adjusted by the competitive inhibition coefficient, which enhances the strain's resistance to contaminants and reduces the batch rejection rate caused by contamination. Furthermore, the static culture is controlled based on the mycelial skin forming coefficient, and the plasticizing hot pressing process is optimized to obtain mycelial materials. This synergistically realizes the control of open liquid fermentation and the preparation of mycelial materials.

[0048] Please see Figure 3 As shown, it is a logic diagram for determining whether the seed culture solution for active culture is qualified in an embodiment of the present invention.

[0049] Specifically, in response to the mutagenized strain being a Ganoderma lucidum strain, the Ganoderma lucidum strain is inoculated into a first culture medium for activation culture to obtain a seed liquid, and the pH value and reducing sugar concentration of the first culture medium during the activation culture process are obtained to calculate the mycelial activity coefficient and determine whether the activated culture seed liquid is qualified. The activity coefficient was compared with the preset activity coefficient; If the activity coefficient is greater than or equal to the preset activity coefficient, the seed culture for activation culture is deemed qualified. If the activity coefficient is less than the preset activity coefficient, the seed culture is deemed unqualified.

[0050] In this embodiment of the invention, the activity coefficient is determined by the following formula:

[0051] in, The pH change rate from 1 to 4 days post-inoculation. To preset the pH change rate, This represents the rate of reducing sugar consumption from 1 to 4 days post-inoculation. To preset the reducing sugar consumption rate, the preset pH change rate ranges from 0.12 pH˙(8h). -1 ~0.18 pH˙(8h) -1The preferred setting is 0.15 pH (8h). -1 The preset reducing sugar consumption rate ranges from 1.0 g / (L·8h) to 1.4 g / (L·8h), and is preferably set to 1.2 g / (L·8h).

[0052] In this embodiment of the invention, the activation culture conditions are as follows: the slant culture of Ganoderma lucidum strain is made into 5mm diameter blocks using a sterile punch, and 3 blocks are inoculated at a ratio of 100mL of the first culture medium. The culture is then cultured at 25℃ and 150rpm for 4 days to obtain the seed liquid.

[0053] In this embodiment of the invention, the preset activity coefficient ranges from 0.7 to 0.9, preferably set to 0.8, but the above value is not limited to this, and those skilled in the art can adjust the value according to actual needs.

[0054] In this embodiment of the invention, the preset pH change rate and the preset reducing sugar consumption rate are determined based on the following: Several parallel culture experiments were conducted on the Ganoderma lucidum strain under the conditions of 25℃ and 150rpm shaker culture. The activated seed liquid was inoculated into the first culture medium at a rate of 5% (V / V). pH values ​​were measured at 1 day and 4 days after inoculation, and the reducing sugar concentration was determined using the 3,5-dinitrosalicylic acid method. Statistical results showed that when the mycelium was in a vigorous growth state, the pH change rate stabilized at 0.14 pH˙ (8h). -1 ~0.16 pH˙ (8h) -1 The reducing sugar consumption rate remained stable at 1.15 g / (L·8h) to 1.25 g / (L·8h). When the bacterial activity decreased or the culture conditions deviated from the optimal state, the pH change rate dropped to 0.1 pH˙ (8h). -1 Below, the reducing sugar consumption rate decreased to below 0.80 g / (L·8h). Based on receiver operating characteristic curve analysis of 50 batches of activated culture data, the optimal concentration was 1.2 g / (L·8h) and 0.15 pH˙ (8h). -1 As a threshold, the accuracy rate of judging whether the seed liquid is qualified or not is over 88%.

[0055] Specifically, if the seed culture activation culture fails and the seed culture activity is insufficient, it will not be used for subsequent fermentation. Instead, the bacterial blocks should be taken from the PDA slant culture and activated again at a ratio of 3 blocks per 100 mL of the first culture medium until the activity coefficient is greater than or equal to the preset activity coefficient, thus obtaining a qualified seed culture.

[0056] Specifically, this invention achieves quantitative control of seed culture viability by comprehensively reflecting the pH change rate and reducing sugar consumption rate during activation culture through an activity coefficient. The pH change rate characterizes the rate of acid production by mycelial metabolism, reflecting the intensity of mycelial respiration metabolism, while the reducing sugar consumption rate characterizes the efficiency of mycelial carbon source utilization, reflecting the mycelial growth rate. When both are at a high level simultaneously, it indicates that the mycelium is in the vigorous logarithmic growth phase. Compared with the traditional method of visually observing mycelial morphology and judging by experience, this invention solves the problems of lack of quantitative indicators for seed culture viability evaluation and high fermentation failure rate caused by large fluctuations in viability between different batches, thereby improving the seed culture qualification rate and ensuring the stability of open fermentation.

[0057] Please see Figure 4 As shown, it is a logic judgment diagram for whether the competitive inhibition of Ganoderma lucidum strain against miscellaneous bacteria in an embodiment of the present invention meets the standard.

[0058] Specifically, the activated and cultured seed liquid was inoculated into an unsterilized second culture medium for fermentation, and the biomass change rate of the Ganoderma lucidum strain and the diameter of the inhibition zone of the fermentation broth supernatant were obtained during the fermentation process to determine the competitive inhibition coefficient and whether the competitive inhibition of the Ganoderma lucidum strain against miscellaneous bacteria met the standard. Compare the competition inhibition coefficient with the preset competition inhibition coefficient; If the competitive inhibition coefficient is greater than or equal to the preset competitive inhibition coefficient, then the competitive inhibition of the Ganoderma lucidum strain against miscellaneous bacteria is determined to be up to standard. If the competitive inhibition coefficient is less than the preset competitive inhibition coefficient, it is determined that the Ganoderma lucidum strain does not meet the standard for competitive inhibition against miscellaneous bacteria.

[0059] In this embodiment of the invention, the activated and qualified seed culture was inoculated into an unsterilized second culture medium at an inoculation rate of 5% (V / V), at a temperature of 25°C, a shaking speed of 150 rpm, and a fermentation time of 5 days.

[0060] In this embodiment of the invention, the competition suppression coefficient is determined by the following formula:

[0061] Where I is the competition inhibition coefficient, The rate of biomass change from 0 to 5 days post-inoculation. To preset the rate of change in biomass, The diameter of the inhibition zone against Bacillus subtilis in the supernatant of the fermentation broth after 5 days of fermentation. The preset inhibition zone diameter is set to a value ranging from 0.08 g / (L·h) to 0.15 g / (L·h), preferably 0.11 g / (L·h), and the preset inhibition zone diameter is set to a value ranging from 14 mm to 20 mm, preferably 16 mm.

[0062] In this embodiment of the invention, the fermentation broth was cultured at 25℃ and 150 rpm for 5 days. After the culture, the fermentation broth was centrifuged at 8000 r / min for 15 min, the supernatant was discarded, and the mycelial precipitate was collected. The precipitate was washed three times with deionized water, dried at 60℃ to constant weight, and weighed to obtain the biomass. Using Bacillus subtilis as an indicator bacterium, the Oxford cup method was used to determine the antibacterial activity of the supernatant of the Ganoderma lucidum fermentation broth. Activated seed culture was inoculated at a rate of 5% (V / V) into an unsterilized second culture medium. The biomass was weighed and recorded at 0 days and 5 days after inoculation. On day 5 of fermentation, 200 μL of the supernatant was added to an Oxford cup and cultured at 37℃ for 24 hours. The diameter of the inhibition zone was then measured. Statistical results showed that when the Ganoderma lucidum strain had a strong competitive inhibition ability against contaminating bacteria, the biomass change rate was stable between 0.10 g / (L·h) and 0.13 g / (L·h). When the competitive inhibition ability was weak, the biomass increased slowly, and the biomass change rate was below 0.08 g / (L·h). g / (L·h), through correlation analysis of 40 batches of fermentation experimental data, the biomass change rate was significantly positively correlated with the diameter of the inhibition zone in the fermentation broth supernatant. R 2 =0.86. When the Ganoderma lucidum strain has a strong competitive inhibition ability against miscellaneous bacteria, the diameter of the inhibition zone is stable at 15mm to 18mm. When the competitive inhibition ability is weak, the diameter of the inhibition zone is less than 12mm. Through the analysis of the receiver operating characteristic curve of 40 batches of fermentation experimental data, with 16mm as the threshold, the accuracy rate of judging the competitive inhibition ability reaches more than 85%.

[0063] In this embodiment of the invention, the preset competitive inhibition coefficient ranges from 0.8 to 0.95, preferably set to 0.88. The value is determined by a correlation analysis of the competitive inhibition coefficients of 40 batches of fermentation experiments with the total number of contaminating microorganisms after static culture. Statistical results show that when the competitive inhibition coefficient is greater than or equal to 0.88, the total number of contaminating microorganisms after static culture is less than or equal to 1 × 10⁻⁶. 3 CFU / mL, meeting sterility requirements; when the competitive inhibition coefficient is less than 0.80, the total number of contaminating bacteria is greater than 1×10⁻⁶. 5 CFU / mL, indicating severe contamination by other microorganisms. Analyzing the receiver operating characteristic (ROC) curve, 0.88 was determined to be the optimal cutoff value for distinguishing whether competitive inhibition was achieved. However, this value is not limited to this, and those skilled in the art can adjust it according to actual needs.

[0064] Specifically, if the competitive inhibition is not up to standard, the shaking speed can be increased from 150 rpm to 180 rpm, or the fermentation time can be extended from 24 hours to 36 hours.

[0065] In this embodiment of the invention, since the Ganoderma lucidum strain has insufficient competitive inhibition ability against miscellaneous bacteria, the shaking speed is increased to increase dissolved oxygen supply, promote the metabolic activity of the Ganoderma lucidum strain, and extend the fermentation time to allow the Ganoderma lucidum strain to fully establish competitive inhibition advantage. The competitive inhibition coefficient of the fermentation broth after increasing the shaking speed or extending the fermentation time is measured again. If it still does not meet the standard, the strain batch is replaced or the activation culture is repeated.

[0066] Specifically, this invention establishes a competitive inhibition coefficient evaluation system to quantitatively assess the antimicrobial ability of Ganoderma lucidum strains in open fermentation. The competitive inhibition coefficient is calculated by combining the biomass change rate and the diameter of the inhibition zone in the fermentation broth supernatant. The biomass change rate reflects the growth rate of the strain and indirectly characterizes the metabolic activity intensity of the strain. The diameter of the inhibition zone directly characterizes the inhibitory effect of the antimicrobial substances produced by the strain's metabolism on other microorganisms. Furthermore, by increasing the shaking speed to increase dissolved oxygen supply to enhance the aerobic metabolic capacity of the strain, or by extending the fermentation time to allow the strain to fully establish its population dominance, this invention solves the problem of inhibited growth and fermentation failure of Ganoderma lucidum strains due to competition from other microorganisms in open fermentation, thereby reducing the contamination rate and minimizing batch scrap.

[0067] Specifically, in response to the Ganoderma lucidum strain achieving competitive inhibition against miscellaneous bacteria, a fermentation broth is obtained, and the fermentation broth is statically cultured. During the static culture process, the mycelial skin coverage, mycelial skin thickness, and mycelial skin porosity are obtained to determine the mycelial skin formation coefficient. The total number of miscellaneous bacteria in the fermentation broth below the mycelial skin is also obtained to determine whether the static culture has met the standard. The mycelium formation coefficient and the total number of miscellaneous bacteria were compared with the preset mycelium formation coefficient and the preset total number of miscellaneous bacteria, respectively. If the mycelium formation coefficient is greater than or equal to the preset mycelium formation coefficient, and the total number of miscellaneous bacteria is less than or equal to the preset total number of miscellaneous bacteria, then the static culture is determined to have met the standard. If the mycelium formation coefficient is less than the preset mycelium formation coefficient, or the total number of miscellaneous bacteria is greater than the preset total number of miscellaneous bacteria, then the static culture is determined to be substandard.

[0068] In this embodiment of the invention, the mycelial skin forming coefficient is determined by the following formula:

[0069] Where B is the mycelial skin formation coefficient. This represents the mycelial coverage rate on the 10th day of static incubation. To preset the mycelial coverage rate, The thickness of the mycelium skin on the 10th day of static incubation. To preset the mycelial skin thickness, The porosity of the mycelium skin on the 10th day of static incubation. The preset mycelium porosity is set to a value ranging from 85% to 100%, preferably 95%. The preset mycelium thickness is set from 1.0 mm to 1.8 mm, preferably 1.5 mm. The preset mycelium porosity is set from 25% to 40%, preferably 32%.

[0070] In this embodiment of the invention, the preset mycelial coverage rate is determined by transferring the fermentation broth, after achieving the competitive inhibition standard, to sterile petri dishes, 50 mL per dish, and incubating them statically at 25°C for 10 days. The formation of mycelial skin on the liquid surface is observed daily, and the percentage of mycelial skin coverage area is statistically analyzed using a grid method. The statistical results show that when the Ganoderma lucidum strain has good film-forming ability, the mycelial skin coverage rate is stable at 92%–98% on the 10th day of static culture; when the film-forming ability is poor, the coverage rate is less than 80%. Through the analysis of 60 batches of static culture experimental data, using 95% as the threshold, the accuracy rate of judging the quality of mycelial skin formation reaches more than 90%.

[0071] In this embodiment of the invention, the preset mycelial skin thickness is determined by taking the mycelial skin from the liquid surface after 10 days of static culture, measuring it three times at the center and edge of the mycelial skin with calipers, and taking the average value as the mycelial skin thickness. Statistical results show that when the Ganoderma lucidum strain has good film-forming ability, the mycelial skin thickness is stable between 1.0 mm and 1.8 mm; when the mycelial skin thickness is less than 0.5 mm, it is easy to break during hot pressing. Through verification of 60 batches of static culture experimental data and molding process, 1.5 mm was determined to be the optimal mycelial skin thickness.

[0072] In this embodiment of the invention, the preset porosity of the mycelium peel is determined by freeze-drying the mycelium peel after 10 days of static culture and then analyzing the porosity using scanning electron microscopy. The electron microscopy images are magnified 500 times using ImageJ software for binarization, and the pore area ratio is calculated. Statistical results show that when the Ganoderma lucidum strain has good film-forming ability, the porosity of the mycelium peel is stable at 28% to 36%. When the porosity is lower than 25%, the plasticizing solution penetration effect is poor, and the material is brittle after hot pressing. When the porosity is higher than 40%, the material density is insufficient and the mechanical strength decreases. Through verification of 40 batches of static culture experimental data and molding process, 32% was determined to be the optimal porosity.

[0073] In this embodiment of the invention, the process of obtaining the total number of miscellaneous bacteria is as follows: on the 10th day of static culture, 1.0 mL of fermentation broth is taken from 1 cm below the mycelial skin using a sterile pipette, and plate count is performed. The broth is spread on LB medium and incubated at 37°C for 24 h for bacterial counting, and on PDA medium and incubated at 28°C for 48 h for fungal counting. The total number of colonies is then counted.

[0074] In this embodiment of the invention, the preset mycotoxin formation coefficient ranges from 0.80 to 0.95, preferably set to 0.88, and the preset total number of miscellaneous bacteria is less than or equal to 1 × 10⁻⁶. 3 CFU / mL, preferably less than or equal to 5 × 10⁻⁶ 2 The value is CFU / mL, but the above value is not limited to this. Those skilled in the art can adjust the value according to actual needs.

[0075] In this embodiment of the invention, the preset total number of miscellaneous bacteria is determined as follows: on the 10th day of static culture, 1.0 mL of fermentation broth is aspirated from 1 cm below the mycelial skin using a sterile pipette, and the total number of miscellaneous bacteria is determined using the plate count method. Statistical results show that when the mycelial skin formation quality meets the standards, with a coverage rate greater than or equal to 95%, a thickness of 1.2–1.5 mm, and a porosity of 28%–36%, the total number of miscellaneous bacteria in the fermentation broth below the mycelial skin stabilizes at 1 × 10⁻⁶. 2 ~5×10 2 CFU / mL; when the mycelial coating quality is poor, the total number of contaminating bacteria can reach 1×10⁻⁶. 5 CFU / mL or higher.

[0076] In this embodiment of the invention, the preset mycelium formation coefficient is determined by performing a correlation analysis between the mycelium formation coefficient of 60 batches of static culture experiments and the tensile strength of the material after hot pressing. The statistical results show that when the mycelium formation coefficient is greater than or equal to 0.88, the tensile strength of the mycelium material after hot pressing is greater than or equal to 2.2 MPa, which meets the application requirements; when the mycelium formation coefficient is less than 0.80, the tensile strength of the material is less than 1 MPa. Through receiver operating characteristic (ROC) curve analysis, 0.88 is the optimal cutoff value for distinguishing whether static culture has met the standards.

[0077] Specifically, if the static culture is not up to standard, the static culture time is extended from 10 days to 12-14 days to observe whether the mycelium continues to grow and cover, or sterile glucose solution and sterile peptone solution are added to the fermentation broth to a final concentration of 35 g / L and 5 g / L, respectively.

[0078] In this embodiment of the invention, if the standard is still not met after adjustment, the fermentation broth is determined to be discarded and fermentation culture is restarted from step S2.

[0079] Specifically, this invention achieves control over the quality of mycelial formation during static cultivation by constructing a mycelial formation coefficient. The mycelial formation coefficient comprehensively reflects the mycelial coverage, mycelial thickness, and mycelial porosity. Among them, the mycelial coverage directly affects the liquid surface sealing, determining whether contaminating bacteria can invade the liquid phase from the gas phase; the mycelial thickness determines the density of the mycelial network and the operability of hot pressing; and the mycelial porosity affects the penetration depth and uniformity of the plasticized liquid. These three factors jointly determine the integrity and forming quality of the mycelial. By extending the static time to promote the continued extension and coverage of mycelia, or by supplementing carbon and nitrogen sources to provide nutritional support for mycelial growth, the problems of incomplete mycelial, uneven thickness, and excessive porosity are solved, improving the integrity and structural uniformity of the mycelial and providing a raw material guarantee for the preparation of mycelial materials.

[0080] Specifically, the mycelial skin that has reached the standard after static culture is removed from the liquid surface and rinsed three times with clean water to remove residual culture medium; the washed mycelial skin is placed in a 20% glycerol solution and soaked at room temperature for 12 hours to plasticize; the plasticized mycelial skin is taken out and air-dried naturally, and then hot-pressed at 100℃ and 10MPa for 1 minute to obtain mycelial material.

[0081] In this embodiment of the invention, the starting strain is Ganoderma lucidum, which was isolated from the fruiting bodies of Ganoderma lucidum in broad-leaved forests in Chengdu, Sichuan Province, and the strain was identified by gene sequencing. Example 1

[0082] Protoplasts were prepared at a concentration of 2 × 10⁻⁶. 8 Cells / mL, diluted to 1×10⁻⁶ 8 After the number of cells / mL was reduced, ARTP mutagenesis was performed. The conditions for ARTP mutagenesis were: working gas: helium; power: 121W; treatment time: 55s. After mutagenesis, the cells were spread on regeneration medium plates and incubated at 25℃ in the dark for 5 days. Regenerated colonies were picked, and a total of 205 regenerated strains were obtained. Through antagonistic culture, mutant strains with an antagonistic line width greater than or equal to 2 mm were screened, and a total of 82 mutant strains were obtained. The slant cultures of the 82 mutant strains were made into 5 mm diameter blocks using a sterile punch. Three blocks were inoculated at a ratio of 100 mL of the first culture medium and cultured in a shaker at 25℃ and 150 rpm for 4 days to obtain seed liquid. The seed liquid was inoculated into unsterilized and sterilized second culture medium at a volume percentage of 5% and cultured at 25℃ and 150 rpm for 5 days. The biomass was measured and referred to Table 1. The biomass of mutant strain 5 in the unsterilized culture medium was 12.5 g / L and the biomass in the sterilized culture medium was 12.6 g / L. The fermentation coefficient was 0.99, which was greater than the preset fermentation coefficient of 0.95. It was identified as a Ganoderma lucidum strain. The mutant strain 5 was inoculated into the first culture medium and activated for 4 days. The pH change rate was measured to be 0.15 pH˙ (8h).-1 The reducing sugar consumption rate was 1.2 g / (L·8h), and the activity coefficient was 1.0, which is greater than the preset activity coefficient of 0.8, indicating that the seed liquid is qualified. The seed culture was inoculated into an unsterilized second culture medium and fermented for 5 days. The biomass change rate was measured to be 0.12 g / (L·h), the inhibition zone diameter was 17 mm, and the competitive inhibition coefficient was 1.16, which was greater than the preset competitive inhibition coefficient of 0.88. This confirmed that the Ganoderma lucidum strain met the standard for competitive inhibition against miscellaneous bacteria. The fermentation broth was allowed to stand for 10 days. The mycelial cover coverage was 96%, the mycelial cover thickness was 1.5 mm, the mycelial cover porosity was 31%, and the mycelial cover formation coefficient was 0.98, greater than the preset mycelial cover formation coefficient of 0.88. The total number of miscellaneous bacteria in the fermentation broth beneath the mycelial cover was 2.5 × 10⁻⁶. 2 CFU / mL, less than the preset total bacterial count of 1×10⁻⁶. 3 CFU / mL, static incubation meets the standard; Remove the mycelium from the liquid surface, rinse with water to remove residual culture medium, soak in 20% glycerol solution at 40°C for 12 hours to plasticize, air dry naturally, and then hot press at 100°C and 10MPa for 1 minute to obtain the mycelial material 1 prepared in Example 1. Example 2

[0083] Please refer to Table 1. Mutagenic strain 18 was selected from 82 mutagenic strains. Its biomass in unsterilized medium was 9.8 g / L, and its biomass in sterilized medium was 11.7 g / L. The fermentation coefficient was 0.84, which was less than the preset fermentation coefficient of 0.95. This indicates that mutagenic strain 18 does not have the ability to directly ferment in an open environment. The mutant strain 18 was inoculated into the first culture medium and activated for 4 days. The pH change rate was measured to be 0.14 pH˙ (8h). -1 The reducing sugar consumption rate was 1.15 g / (L·8h), and the activity coefficient was 0.89, which is greater than the preset activity coefficient of 0.8, indicating that the seed liquid is qualified. The seed culture was inoculated into an unsterilized secondary culture medium and fermented for 5 days. The biomass change rate was measured to be 0.09 g / (L·h), the inhibition zone diameter was 15 mm, and the competitive inhibition coefficient was 0.77, which was less than the preset competitive inhibition coefficient of 0.88. This indicated that the Ganoderma lucidum strain did not meet the standard for competitive inhibition against contaminating bacteria. The shaking speed was increased from 150 rpm to 180 rpm, and fermentation was repeated for 24 hours. The biomass change rate was then measured to be 0.11 g / (L·h). 1 The inhibition zone diameter was 18 mm, and the competitive inhibition coefficient was 1.13, indicating that the Ganoderma lucidum strain met the standard for competitive inhibition against miscellaneous bacteria. After the fermentation broth met the standards, it was allowed to stand for 10 days. The mycelial coverage rate was measured to be 92%, the mycelial thickness to be 1.2 mm, the mycelial porosity to be 34%, and the mycelial formation coefficient to be 0.82, which was less than the preset mycelial formation coefficient of 0.88. Therefore, the standing culture was not up to standard. The standing time was then extended to 12 days. The mycelial coverage rate was measured to be 95%, the mycelial thickness to be 1.5 mm, the mycelial porosity to be 32%, and the mycelial formation coefficient to be 1.0. The total number of miscellaneous bacteria in the fermentation broth below the mycelial layer was 3.8 × 10⁻⁶. 2 CFU / mL, less than the preset total bacterial count of 1×10⁻⁶. 3 CFU / mL, static culture reaches the standard. By increasing the shaking speed and static culture time, it is not only applicable to the mutant strain 5 of the excellent strain in Example 1 with the fermentation coefficient reaching the standard, but also applicable to the mutant strain 18 with the fermentation coefficient not reaching the standard but which can be compensated by subsequent processes. Remove the mycelium from the liquid surface, rinse with water to remove residual culture medium, soak in 20% glycerol solution at 40°C for 12 hours to plasticize, air dry naturally, and then hot press at 100°C and 10MPa for 1 minute to obtain the mycelial material 2 prepared in Example 2.

[0084] Comparative Example 1 Use starting strains that have not undergone ARTP mutagenesis, without screening for fermentation coefficients; The biomass of the starting strain was 3.2 g / L in unsterilized medium and 12.6 g / L in sterilized medium, with a fermentation coefficient of 0.25. The starting strain was inoculated into the first culture medium and activated for 4 days. The pH change rate was measured to be 0.08 pH˙ (8h). -1 The reducing sugar consumption rate was 0.65 g / (L·8h), and the activity coefficient was 0.29, which was less than the preset activity coefficient of 0.8. The seed liquid was unqualified, but it was used directly for fermentation. The seed culture was inoculated into the second culture medium and fermented for 5 days. The biomass change rate was 0.05 g / (L·h), the inhibition zone diameter was 8 mm, and the competitive inhibition coefficient was 0.23. The competitive inhibition of the Ganoderma lucidum strain against miscellaneous bacteria did not meet the standard, and no adjustment was made. The fermentation broth was allowed to stand for 10 days. The mycelial cover rate was 35%, the mycelial cover thickness was 0.8 mm, the mycelial cover porosity was 52%, the mycelial cover formation coefficient was 0.32, and the total number of miscellaneous bacteria was 2.5 × 10⁻⁶. 5 CFU / mL, static incubation did not meet the standard; After hot pressing, the mycelial material becomes brittle and cannot be formed into a complete sheet, making it impossible to measure its tensile strength.

[0085] Comparative Example 2 Mutagenic strain 5 obtained by screening in Example 1 was a Ganoderma lucidum strain; The activity coefficient determination step was omitted, and the pH change rate and reducing sugar consumption rate were not measured. Seed liquid that had not been evaluated for activity was used directly. The steps for determining the competitive inhibition coefficient are omitted, the biomass change rate and inhibition zone diameter are not measured, and the shaking speed or fermentation time is not adjusted; the seed culture is directly placed in a static environment. After 10 days of static incubation, the mycelial skin coverage was 78%, the skin thickness was 1.8 mm, the skin porosity was 43%, the skin formation coefficient was 1.32, and the total number of contaminating microorganisms was 8.5 × 10⁻⁶. 3 The CFU / mL concentration was insufficient for static culture. After hot pressing, the surface of the mycelial material was uneven with local cracks, and the tensile strength was 1.52 MPa.

[0086] Comparative Example 3 The mutant strain 5 obtained by screening in Example 1 was a Ganoderma lucidum strain, and its activity coefficient was evaluated according to the steps in Example 1. The pH change rate and reducing sugar consumption rate were measured. The activity coefficient was 1.0, which is qualified for seed liquid activity. In competitive inhibition regulation, the biomass change rate and inhibition zone diameter were measured. The competitive inhibition coefficient was 1.0, which is qualified for competitive inhibition of miscellaneous bacteria by the Ganoderma lucidum strain. After 10 days of static culture, the mycelium formation coefficient reached 0.93, which met the standard. However, when the formation coefficient of a certain batch of mycelium was 0.75, no adjustment treatment was made by extending the static time or adding carbon and nitrogen sources. The mycelium was directly used for hot pressing. After hot pressing, the thickness of the mycelium material was uneven, and the tensile strength of the local area was 1.85 MPa.

[0087] Comparative Example 4 The mutant strain 5 obtained by screening in Example 1 was a Ganoderma lucidum strain, and the activity coefficient was evaluated in sequence according to the steps of Example 1. The activity coefficient was 1.0, which was qualified for seed liquid activity. The competitive inhibition regulation was 1.0, which was qualified for competitive inhibition of Ganoderma lucidum strain against miscellaneous bacteria. After static culture, the mycelial skin formation coefficient was 0.93, which was qualified for static culture. Before hot pressing, the plasticizing step is omitted, and the mycelium is not soaked in glycerol solution. Instead, the mycelium is directly air-dried and then hot-pressed. The tensile strength of the mycelium material after hot pressing is 1.21 MPa.

[0088] Table 1. Biomass of 82 mutant strains strain Biomass (g / L) in unsterilized secondary culture medium Biomass (g / L) in sterilized secondary culture medium Original strain 3.2 12.6 Mutagenic strain 1 2.3 12.4 Mutagenized strain 2 1.9 11.9 Mutagenic strain 3 2.6 12.0 Mutagenic strain 4 2.5 12.4 Mutagenic strain 5 12.5 12.6 Mutagenic strain 6 1.5 11.6 Mutagenic strain 7 3.6 10.3 Mutagenic strain 8 3.3 11.5 Mutagenic strain 9 2.8 9.5 Mutagenic strain 10 2.5 12.1 Mutagenic strain 11 2.3 10.9 Mutagenic strain 12 2.1 10.4 Mutagenic strain 13 2.2 12.0 Mutagenized strain 14 2.6 8.4 Mutagenic strain 15 3.0 12.4 Mutagenized strain 16 3.1 7.9 Mutagenic strain 17 2.7 8.9 Mutagenized strain 18 9.8 11.7 Mutagenic strain 19 3.5 10.6 Mutagenic strain 20 3.2 11.6 Mutagenic strain 21 4.0 12.4 Mutagenic strain 22 2.9 11.8 Mutagenic strain 23 3.1 10.7 Mutagenic strain 24 3.7 8.9 Mutagenic strain 25 3.5 10.1 Mutagenic strain 26 3.2 11.9 Mutagenic strain 27 2.6 12.1 Mutagenic strain 28 4.0 10.7 Mutagenic strain 29 3.6 10.4 Mutagenic strain 30 3.9 9.9 Mutagenic strain 31 2.0 11.2 Mutagenic strain 32 3.1 12.6 Mutagenic strain 33 2.1 10.6 Mutagenic strain 34 2.8 9.4 Mutagenic strain 35 3.7 12.5 Mutagenic strain 36 2.7 11.6 Mutagenic strain 37 2.5 10.5 Mutagenic strain 38 3.5 11.6 Mutagenic strain 39 2.6 12.1 Mutagenic strain 40 3.4 9.0 Mutagenic strain 41 3.6 11.6 Mutagenic strain 42 3.5 11.6 Mutagenic strain 43 3.5 12.1 Mutagenic strain 44 3.2 11.8 Mutagenic strain 45 3.8 12.4 Mutagenic strain 46 4.2 11.8 Mutagenic strain 47 1.2 9.5 Mutagenic strain 48 1.9 10.2 Mutagenic strain 49 1.5 12.3 Mutagenic strain 50 3.6 8.9 Mutagenic strain 51 2.5 9.9 Mutagenic strain 52 2.0 11.2 Mutagenic strain 53 2.9 10.5 Mutagenic strain 54 3.9 12.5 Mutagenic strain 55 4.1 9.3 Mutagenic strain 56 4.9 10.5 Mutagenic strain 57 4.2 11.2 Mutagenic strain 58 4.1 12.1 Mutagenic strain 59 3.6 11.8 Mutagenic strain 60 3.2 10.5 Mutagenic strain 61 2.9 9.8 Mutagenic strain 62 2.5 10.4 Mutagenic strain 63 2.0 11.2 Mutagenic strain 64 1.5 12.5 Mutagenic strain 65 3.7 9.5 Mutagenic strain 66 3.2 10.2 Mutagenic strain 67 3.8 10.6 Mutagenic strain 68 3.6 10.3 Mutagenic strain 69 4.2 12.4 Mutagenic strain 70 4.0 12.9 Mutagenic strain 71 1.9 12.4 Mutagenic strain 72 2.9 11.6 Mutagenic strain 73 2.5 9.5 Mutagenic strain 74 1.7 9.9 Mutagenic strain 75 3.7 8.7 Mutagenic strain 76 3.5 10.6 Mutagenic strain 77 4.2 10.5 Mutagenic strain 78 4.5 11.6 Mutagenic strain 79 2.3 11.9 Mutagenic strain 80 2.9 11.4 Mutagenic strain 81 3.1 12.5 Mutagenic strain 82 3.3 12.2 Table 2 Properties of Mycelial Materials

[0089] Note: The mycelium in Comparative Example 1 could not form a complete sheet, so its tensile strength could not be measured.

[0090] Please refer to Table 2. The mycelial materials prepared in Examples 1 and 2 both achieved a skin integrity of over 95%, and their tensile strengths after hot pressing were 2.37 MPa and 2.29 MPa, respectively, significantly better than all comparative examples. Comparative example 1, due to its fermentation coefficient of only 0.25, could not form a complete skin after static culture, and its tensile strength could not be measured, indicating that the starting strain did not have open fermentation capability. Although comparative example 2 used the same mutant strain, its skin integrity decreased to 78%, and its tensile strength was only 1.52 MPa, proving that seed culture activity and fermentation process control are key factors in ensuring material performance. The tensile strength of comparative example 3 was 1.85 MPa, lower than that of example 1, indicating that the control measures during static culture were crucial to material performance. The tensile strength of comparative example 4 was only 1.21 MPa, far lower than that of example 1, proving that glycerol plasticization treatment can significantly enhance the binding force between mycelia and the flexibility of the material.

[0091] The mutant strain 5 obtained in Example 1 was continuously passaged on PDA slant for 10 times. Open liquid fermentation and static culture experiments were carried out every 2 generations to determine the skin formation time and the material properties after hot pressing.

[0092] Please refer to Table 3. The mycelial skin formation time for generations 1 to 10 was 10 days. After hot pressing, the thickness was stable at 0.3–0.4 mm, and the tensile strength was stable at 2.29–2.40 MPa. The results show that the Ganoderma lucidum strain MKLGE251231 still maintains good resistance to contaminants and the ability to form mycelial skin after 10 generations. It has good genetic stability and is suitable for industrial production.

[0093] Table 3. Effects of different passage numbers on the properties of the prepared mycelial biomaterials

[0094] In Example 1, the fermentation coefficient of mutant strain 5 was 0.99, significantly higher than the 0.25 of the original strain in Comparative Example 1. This demonstrates that ARTP mutagenesis combined with fermentation coefficient screening can yield superior strains for open fermentation. Please refer to [link to relevant documentation]. Figure 5 and Figure 7 As shown, in Example 1, the mycelial crust formed after 10 days of static cultivation completely, continuously, and uniformly covered the liquid surface. Please refer to [link / reference]. Figure 6 As shown, the microstructure of the mycelium is a three-dimensional network with densely interwoven hyphae, proving that the mutant strain 5 can form a high-quality mycelium under non-sterile conditions.

[0095] Comparative Example 2 omitted the evaluation of activity coefficient and competitive inhibition regulation. Although the same strain was used, the mycelial skin formation coefficient was only 0.58 and the tensile strength was only 1.52 MPa, which was significantly lower than 2.37 MPa in Example 1. This proves that seed culture activity and fermentation process regulation are key quality control links.

[0096] Comparative Example 3, without any adjustment when the mycelial skin forming coefficient did not meet the standard, had a tensile strength of only 1.85 MPa, which was lower than that of Example 1, demonstrating that the control measures during the static culture process are crucial to the material performance.

[0097] Comparative Example 4, which omitted the plasticizing step, had a tensile strength of only 1.21 MPa, far lower than that of Example 1. This demonstrates that glycerol plasticizing treatment significantly enhances the bonding force between hyphae and the flexibility of the material by penetrating into the gaps between hyphal fibers.

[0098] The same batch of seed liquid that had passed activation in Example 1 was inoculated into sterilized and unsterilized secondary culture media at an inoculation rate of 5% by volume. Fermentation was carried out at 25°C and 150 rpm for 5 days. After fermentation, the mycelial skin was collected and soaked in 20% glycerol at room temperature for 12 hours, followed by hot pressing at 100°C and 10 MPa for 1 minute to prepare mycelial materials. Measurements showed that the mycelial skin thickness in the sterilized group was 0.4 mm and the tensile strength was 2.33 MPa; the mycelial skin thickness in the unsterilized group was 0.4 mm and the tensile strength was 2.37 MPa. There was no significant difference in mycelial skin thickness and tensile strength, indicating that the mycelial material prepared by the Ganoderma lucidum strain MKLGE251231 under open (unsterilized) conditions had performance comparable to that under strictly aseptic conditions.

[0099] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. An ARTP mutagenizable strain capable of open-air liquid fermentation, characterized in that, The strain is Ganoderma lucidum strain MKLGE251231, which has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.42590.

2. The application of the ARTP mutagen strain capable of open-air liquid fermentation as described in claim 1, characterized in that, The Ganoderma lucidum strain is applied to mycelial material based on the following steps, including: The activity coefficient of the Ganoderma lucidum strain was determined based on the pH value and reducing sugar concentration of the strain inoculated in the first culture medium, so as to determine the qualification of the seed liquid. The competitive inhibition coefficient of the Ganoderma lucidum strain was determined based on the biomass change rate and inhibition zone diameter of the qualified seed liquid inoculated in the unsterilized second culture medium, so as to obtain the fermentation broth in which the competitive inhibition of Ganoderma lucidum strain against miscellaneous bacteria met the standard. The mycelium formation coefficient was determined based on the mycelium coverage, mycelium thickness, and mycelium porosity during static fermentation, and the compliance of static fermentation was determined based on the total number of miscellaneous bacteria in the fermentation broth below the mycelium. The mycelial skin that has reached the standard after static culture is plasticized and hot-pressed to obtain mycelial material.

3. The application of the ARTP mutagen strain capable of open-air liquid fermentation according to claim 2, characterized in that, Based on an activity coefficient greater than or equal to a preset activity coefficient, the seed culture for activation culture is deemed qualified. The activity coefficient is determined based on the product of the pH change rate ratio and the reducing sugar consumption rate ratio, wherein the pH change rate ratio is the ratio of the pH change rate to the preset pH change rate, and the reducing sugar consumption rate ratio is the ratio of the reducing sugar consumption rate to the preset reducing sugar consumption rate.

4. The application of the ARTP mutagen strain capable of open-air liquid fermentation according to claim 2, characterized in that, Based on a competitive inhibition coefficient greater than or equal to a preset competitive inhibition coefficient, the Ganoderma lucidum strain was determined to have achieved the target of competitive inhibition against contaminating bacteria. The competitive inhibition coefficient is determined based on the product of the ratio of biomass change rate and the ratio of inhibition zone diameter. The ratio of biomass change rate is the ratio of biomass change rate to a preset biomass change rate, and the ratio of inhibition zone diameter is the ratio of inhibition zone diameter to a preset inhibition zone diameter.

5. The application of the ARTP mutagen strain capable of open-air liquid fermentation according to claim 2, characterized in that, Based on the fact that the mycelial skin formation coefficient is greater than or equal to the preset mycelial skin formation coefficient, and the total number of contaminating bacteria is less than or equal to the preset total number of contaminating bacteria, the static culture is determined to have met the standard. The mycelium formation coefficient is determined based on the product of the mycelium coverage ratio, the mycelium thickness ratio, and the mycelium porosity ratio. The mycelium coverage ratio is the ratio of the mycelium coverage to the preset mycelium coverage ratio, the mycelium thickness ratio is the ratio of the mycelium thickness to the preset mycelium thickness, and the mycelium porosity is the ratio of the mycelium porosity to the preset mycelium porosity.

6. The application of the ARTP mutagen strain capable of open-air liquid fermentation according to claim 2, characterized in that, The first culture medium consists of the following components: glucose 35 g / L, peptone 5.0 g / L, yeast extract 2.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium dihydrogen phosphate 1.0 g / L, vitamin B1 0.05 g / L, with the balance being water, pH 5.50, and sterilized at 121°C for 20 min. The second culture medium consists of the following components: 35 g / L glucose, 5 g / L peptone, 3 g / L beef extract, 3 g / L fine wheat bran, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, with the remainder being water. The pH value is 6.5–7.5 and is not adjusted. The second sterilized culture medium was prepared and then sterilized at 121°C for 20 minutes before inoculation. The unsterilized second culture medium is prepared and then directly inoculated.

7. The application of the ARTP mutagen strain capable of open-air liquid fermentation according to claim 2, characterized in that, The activation culture conditions are as follows: the slant culture of Ganoderma lucidum strain is made into 5 mm diameter blocks using a sterile punch, and 3 blocks are inoculated at a ratio of 100 mL of the first culture medium. The culture is then incubated at 25 °C and 150 rpm for 4 days to obtain the seed liquid.

8. The application of the ARTP mutagen strain capable of open-air liquid fermentation according to claim 2, characterized in that, The conditions for obtaining the fermentation broth are as follows: the activated and cultured seed liquid is inoculated into the unsterilized second culture medium at an inoculation rate of 5% by volume, and fermented on a shaker at 25°C and 150 rpm for 5 days.

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