Odosporium oudemansiella mutant strain M1 obtained through artificial mutagenesis, breeding method, special liquid fermentation culture medium and industrial cultivation method

Abstract

The invention discloses a hymenopellis raphanipes mutant strain M1 obtained through artificial mutagenesis, the hypha growth rate of the hymenopellis raphanipes mutant strain M1 is larger than or equal to 5.20 mm / d, the biological efficiency reaches 53.6%, the hymenopellis raphanipes mutant strain M1 has strong resistance to trichoderma harzianum, the proportion of first-grade mushrooms is larger than or equal to 40.6%, and the hymenopellis raphanipes mutant strain M1 is good in hereditary stability. The invention also provides a breeding method of the strain, a special liquid fermentation culture medium and an industrial cultivation process in an intelligent mushroom square cabin. The method solves the problems that traditional cultivation is long in period, high in pollution rate, difficult to factorize and the like, and a complete solution is provided for annual efficient production of oudemansiella oosporium.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi biotechnology and industrialized cultivation, specifically involving a high-yield and stable-yield mutant strain M1 of Hymenopellis raphanipes obtained through artificial mutagenesis, as well as the breeding method of this strain, a special liquid fermentation medium, and an industrialized cultivation method in an intelligent mushroom container. Background Technology

[0002] Hymenopellis raphanipes, formerly known as long-rooted mushroom and two-spore hymenopellis, and commercially known as black-skinned chicken mushroom, is a rare edible fungus with balanced nutrition and significant health benefits. Its fruiting body has a protein content exceeding 32.12%, rich in 18 amino acids (of which umami amino acids account for 76.73%), and its amino acid composition meets the FAO / WHO ideal protein standards. In terms of minerals, it is high in potassium (3230 mg / 100g), low in sodium, and contains abundant phosphorus, magnesium, calcium, and other trace elements. Furthermore, it contains various bioactive components, such as polysaccharides, flavonoids (rosinin, luteolin), and raphanipesin, which possess various physiological effects including antioxidant, anti-tumor, hypoglycemic, hypolipidemic, immune-enhancing, pathogen-inhibiting, and liver-protecting properties, showing great promise in the development of functional foods and pharmaceuticals.

[0003] Currently, *Millettia oleifera* has been cultivated on a certain scale, mainly using three methods: removing the bag and covering with soil, leaving the cultivation bag uncovered, or covering with soil. The greenhouse method of removing the bag and covering with soil is the most common. However, this method has significant drawbacks: the soil covering is susceptible to mold infection, leading to large-scale yield reduction; soil collection is difficult and labor costs are high; there is a risk of heavy metal contamination; the cultivation cycle is long (post-ripening period reaches 30-35 days); and it is difficult to adapt to fully enclosed, highly ventilated, and high-temperature environments. The demand for industrialized production in this environment. Existing commercial strains generally suffer from slow mycelial growth (9-12 days to fill a PDA plate, 150-180 hours for liquid fermentation), weak and easily aging mycelia, poor adaptability to intelligent modular environments (slow primordia formation, many deformed mushrooms, low and unstable biological efficiency), and insufficient genetic stability (severe degradation after 5-8 generations of continuous transfer), which seriously restricts their year-round, large-scale, and green production.

[0004] Although atmospheric and room temperature plasma (ARTP) mutagenesis technology has been successfully applied in the breeding of edible fungi such as enoki mushrooms, shiitake mushrooms, reishi mushrooms, and morels, and has advantages such as being non-toxic, safe, and having a high mutation rate, it has proven effective in these applications. , mutation spectrum, etc. However, there is no report on using it to breed high-yield and stable-yield strains of H. raphanipes suitable for cultivation in a fully closed intelligent shelter. <1500 ppm) and a long post-maturation period, the existing public technology is mostly limited to the "greenhouse + cold storage" stage, and lacks a complete process parameter system suitable for the full environmental precision control shelter.

[0005] Therefore, it is urgent to carry out full-chain innovation from the source of the strain to the end cultivation and break through the bottleneck of the industrial production of H. raphanipes. SUMMARY

[0006] The purpose of the present application is to provide a mutant strain M1 of H. raphanipes suitable for annual efficient cultivation in a fully closed intelligent mushroom shelter, and to solve the technical problems of slow growth, weak resistance, poor adaptability, and unstable yield of existing strains.

[0007] Another purpose of the present application is to provide an efficient breeding method of the strain, a special liquid fermentation medium, and a matching intelligent shelter precision cultivation process, to realize the full-process standardization, automation, and high yield and stability from strain preparation to fruiting body harvesting.

[0008] To achieve the above purpose, the first aspect of the present application provides:

[0009] The mutant strain M1 of H. raphanipes obtained by artificial mutagenesis is preserved in the Guangdong Microbial Culture Collection Center (GDMCC), with the preservation number GDMCC No: 67331 and the preservation date November 21, 2025.

[0010] Preferably, the strain has one or more of the following characteristics compared to the starting strain MC-OR-14:

[0011] (a) The protein content in the fruiting body of the strain is 25.6±0.5 g / 100g, which is significantly higher than that of the starting strain MC-OR-14 (24.4±0.7 g / 100g), and the cap diameter is ≤1.88 cm and the stem length is ≥6.21 cm;

[0012] (b) When cultured on PDA medium at 25°C, the average mycelial growth rate is ≥5.20 mm / d;

[0013] (c) The biological efficiency is ≥53.6%;

[0014] (d) The resistance to Trichoderma harzianum is significantly enhanced, and the plate infection rate is ≤46.4%;

[0015] (e) the proportion of primary mushrooms in fruiting bodies is greater than or equal to 40.6%;

[0016] (f) good genetic stability, and the mycelium growth rate fluctuation is not more than ±0.02 mm / d after continuous transfer for 5 generations.

[0017] The second aspect of the present application provides:

[0018] Use of the said strain M1 of Oudemansiella australis in industrialized cave cultivation.

[0019] The third aspect of the present application provides:

[0020] A breeding method of the said strain M1 of Oudemansiella australis, comprising the following steps:

[0021] (1) using commercially available strain MC-OR-14 of Oudemansiella australis as the starting strain to prepare a protoplast suspension;

[0022] (2) mixing the protoplast suspension with a glycerol protective agent, and then placing it in an ARTP system for mutagenesis treatment, wherein the radio frequency power is 120 W, the helium flow rate is 10 SLM, the working distance is 2 mm, and the mutagenesis time is 20-60 s;

[0023] (3) coating the mutagenized protoplasts on a regeneration medium and culturing at 25℃ until germination, and picking single colonies;

[0024] (4) screening mutagenized strains with a mycelium growth rate increased by more than or equal to 5% compared with MC-OR-14 and good genetic stability;

[0025] (5) further screening strains with high biological efficiency, strong resistance to Trichoderma, and good fruiting body commodity traits through fruiting test, to obtain the said strain M1.

[0026] The fourth aspect of the present application provides:

[0027] A special culture medium for liquid fermentation of the said strain M1 of Oudemansiella australis, comprising the following components per liter of culture medium: 20 g of glucose, 5 g of yeast extract powder, 1 g of potassium phosphate dibasic, 0.5 g of magnesium sulfate, 10 mg of vitamin B1, and the rest is water, and the pH is 6.0-7.0.

[0028] Preferably, the culture medium is used for preparing liquid inoculum, and the culture conditions are: 25℃, 150 rpm, and light-free culture for 8-9 days.

[0029] The fifth aspect of the present application provides:

[0030] A method for intelligent industrialized cave cultivation using the said strain M1 of Oudemansiella australis, comprising the following steps:

[0031] (1) inoculate M1 strain into a flask containing the liquid medium to prepare a liquid stock culture;

[0032] (2) after sterilizing the cultivation material in a bag, inoculate 30-40 mL of the liquid stock culture into each bag, and cultivate in the dark at 25°C and 70% humidity until the mycelium fills the bag, and then post-culture for 25 days;

[0033] (3) remove the collar and mechanically scrape off the old sclerotium by 0.5-1 mm;

[0034] (4) transfer into an intelligent mushroom shelter to induce primordia, with the control parameters being: temperature 18-20°C, relative humidity 96.5%-98.0%, CO2concentration 900-1100 ppm, light intensity 400-500 lx, photoperiod 12 h / d, and applying temperature difference stimulation: uniformly decreasing from 25°C to 20°C within 8 hours, and maintaining for 14-16 hours;

[0035] (5) after the primordia are formed, transfer into a fruiting body growth period, with the control parameters being: temperature 25-28°C, relative humidity 90%, CO2concentration 800-950 ppm, light intensity 600-800 lx, and photoperiod 12 h / d;

[0036] (6) harvest when the cap diameter of the fruiting body reaches 6-10 cm, the edge is involuted, and the cap is not opened.

[0037] Preferably, the intelligent mushroom shelter is provided with a full-automatic temperature, humidity, light and gas precise control system, with temperature control precision ±0.3°C, humidity control precision ±2%, and control precision ±50 ppm.

[0038] Preferably, the cultivation material comprises, by mass percentage, cottonseed hulls 45%, sawdust 32%, bran 16%, corn meal 4.2%, calcium carbonate 1.5%, and calcium oxide 1.3%, with a water content of 60%.

[0039] The sixth aspect of the present application provides:

[0040] A functional food comprising the egg-spore small oyster mushroom strain M1, the functional food containing one or more bioactive ingredients extracted from the fruiting body of the egg-spore small oyster mushroom strain M1, such as polysaccharides, flavonoids or long-root mushroom elements, and having one or more effects of antioxidant, blood glucose-lowering, blood lipid-lowering, immune system enhancement and liver protection.

[0041] The present application has the following beneficial effects:

[0042] ​​​1. The mycelium growth rate of M1 strain is increased by 6.3% compared with CK, the liquid fermentation period is shortened, the material consumption is fast, the pollution rate is low, the biological efficiency is 53.6%, which is significantly higher than that of the control (47.2%), the infection rate is only 5% (CK is 20%), the first grade mushroom accounts for 40.6%, and the commodity value is high.

[0043] 2. The precise cultivation parameter system suitable for the fully closed intelligent shelter is established for the first time, the original base occurrence rate is 99.2%, the present bud is neat (concentrated in 56-68 h after scraping the fungus), and the problems of dead mushroom, clump, and deformity in traditional cultivation are completely solved.

[0044] 3. The three core technologies of "ARTP mutagenesis-liquid fermentation optimization-shelter precise cultivation" are integrated, the efficient, stable and green production from strain to product is realized, and the complete solution for factory, annual and large-scale production of Oudemansiella australes is provided.

[0045] 4. The total cultivation period is shortened by 13 days compared with CK, the energy consumption and labor cost are reduced, the fruit body protein content is improved, the functional components are completely reserved, and the demand of the health industry is met. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 It is a schematic diagram of M1 mutagenic strain and control strain MC-OR-14 infected by Trichoderma harzianum on a plate (upper: M1; lower: CK).

[0047] Figure 2 It is a graph of the influence of different carbon sources on the dry weight of M1 strain mycelium.

[0048] Figure 3 It is a graph of the influence of different nitrogen sources on the dry weight of M1 strain mycelium.

[0049] Figure 4 It is a graph of the influence of different pH on the dry weight of M1 strain mycelium. DETAILED DESCRIPTION

[0050] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions will be described in detail below in combination with specific examples, and the described examples are only part of the examples of the present application, not all examples. The specific conditions are not specified in the following examples, which are usually implemented according to conventional conditions or according to the conditions recommended by the manufacturer.

[0051] Hymenopellis raphanipes, once named as Cymatoderma apios, Hymenochaete apios, and Hymenochaete bispora, and known as black-pored chicken nest fungus in the market, belongs to the kingdom Fungi, the phylum Basidiomycota, the class Agaricomycetes, the order Agaricales, the family Physalacriaceae, and the genus Hymenopellis. H. raphanipes is a rare edible fungus with balanced nutrition and potential health benefits. Its fruiting body is rich in protein (the content can reach more than 32.12%, which is significantly higher than that of common edible fungi such as Lentinula edodes and Agaricus bisporus) and 18 kinds of amino acids, among which the proportion of delicious amino acids is as high as 76.73%, which is closely related to its delicious taste. The amino acid composition meets the ideal protein standard of FAO / WHO, indicating that it is a high-quality protein source. In terms of minerals, H. raphanipes shows the characteristics of high potassium (3230 mg / 100 g) and low sodium, and the potassium content is much higher than that of common edible fungi such as Auricularia auricula and Pleurotus eryngii, making it a potential food beneficial to anti-hypertension. In addition, it also contains rich trace elements such as phosphorus, magnesium, and calcium. H. raphanipes is rich in polysaccharides, flavonoids (such as fisetin and luteolin), and long root fungus, as well as many other bioactive ingredients. Studies have shown that it has significant physiological functions such as antioxidant, anti-tumor, blood sugar and blood lipid reduction, immune enhancement, pathogenic bacteria inhibition, and liver protection, showing broad prospects in the development of functional foods and drugs. Based on the above-mentioned nutritional and functional components of H. raphanipes, it is determined that H. raphanipes has high edible and health value. At present, H. raphanipes has realized large-scale artificial cultivation, and the main cultivation methods include bag removal and soil covering, bag cultivation without soil covering, and bag cultivation with soil covering. At present, the main method is still the bag removal and soil covering in the greenhouse. Bag removal and soil covering in the greenhouse has the characteristics of high yield and good single fruiting body characteristics, but once the soil cultivation is infected by mold, it will easily lead to a large area of yield reduction. At the same time, there are also problems such as difficulty in taking soil, high labor cost of soil covering, high risk of heavy metal pollution, long cultivation period, backward technology, low efficiency of large-scale industrial cultivation, and production methods cannot meet the requirements of sustainable and green manufacturing, etc. which makes the cultivation scale of H. raphanipes unable to expand. Therefore, selecting and breeding excellent strains suitable for industrial cultivation is of great significance for year-round large-scale production. Long-term production practice shows that the existing commercial strains have the following outstanding defects:

[0052] (1) Slow mycelial growth: the full plate time on PDA plates at 25°C generally needs 9-12 days, and the liquid strain fermentation period is as long as 150-180 hours, which leads to limited inoculation period and difficult production scheduling of industrial production lines;

[0053] (2) Weak mycelial growth and easy aging: sparse mycelium, yellow autolysate in the later stage, yellow and fragile liquid strain pellets, slow eating after inoculation, and high contamination rate;

[0054] (3) Poor adaptability to factoryization habitat: most of the existing strains are selected for traditional soil or simple greenhouse, and in the fully closed, strong ventilation, high intelligent mushroom habitat, the slow formation of primordium, irregular differentiation of fruiting body, high proportion of deformed mushroom, low and unstable biological efficiency, and it is difficult to achieve high and stable yield all year round;

[0055] (4) Insufficient genetic stability: some introduced strains appear serious degradation after continuous transfer for 5-8 generations in China, and the yield decreases year by year, so the mushroom farmers change the strain frequently.

[0056] In order to break through the above bottleneck, the research team uses atmospheric and room temperature plasma (ARTP) mutagenesis technology to transform market strains. ARTP technology has the advantages of non-toxicity, safety, high mutation rate , wide mutation spectrum, and has been successful in the breeding of many edible fungi such as shiitake, shiitake, ganoderma, and morel. In the field of factory cultivation, the egg-spore small oyster mushroom has strict requirements for temperature (25-28℃), high humidity (≥85%), low (<1500 ppm), long post-maturation period (30-35 days), and traditional experience parameters are easy to lose control in a fully closed environment, resulting in dead mushrooms or clumped deformed mushrooms. The current public cultivation technology is mostly limited to the "greenhouse + cold storage" stage, and lacks a complete parameter system for precise control of the habitat of the full environment, which seriously restricts the large-scale development of this rare species.

[0057] After long-term research by the inventors of the present application, the commercially available main strain (MC-OR-14) was subjected to multiple rounds of ARTP mutagenesis, which significantly improved the mycelial growth rate and environmental tolerance; combined with precise screening of liquid fermentation carbon and nitrogen sources, and finally with the self-built intelligent mushroom habitat for multiple batches of continuous cultivation verification, a superior mutant strain M1 of egg-spore small oyster mushroom was successfully obtained, which can realize rapid mycelial consumption, uniform primordium formation, good commodity nature of fruiting body, and stable biological efficiency in a fully closed habitat, thereby realizing a full-chain breakthrough from the strain source to the end of cultivation, and providing a complete technical solution for the factoryization, year-round, and high-efficiency production of this rare mushroom.

[0058] Example 1

[0059] I. Mutagenesis method

[0060] A small piece of tissue was picked from the cultivated fruiting body of the market strain under sterile operation, and transferred to a comprehensive PDA slant medium. It was cultured at about 25°C to obtain the original strain MC-OR-14. After the test tube was full of mycelium, the MC-OR-14 strain was inoculated into a 250 mL conical flask containing 100 mL of comprehensive PD liquid medium, and cultured at 25°C and 140 rpm for 4 days. 2 mL of bacterial liquid with bacterial balls was sucked with a syringe, ground with a homogenizer, and then transferred to a new comprehensive PD liquid medium. After standing for 1 day, it was cultured in a 25°C shaking bed at 140 rpm for 7 days.

[0061] The mycelium was collected in a sterile centrifuge tube on an ultraclean bench, 8000 rpm, 10 minutes, and the supernatant was removed. It was washed with 0.6M mannitol three times at room temperature, 8000 rpm, 10 minutes, and the supernatant was removed. A 2% enzyme solution was prepared (freshly prepared, filtered with a disposable needle, and a part of the enzyme solution was lost during filtration, so more was prepared), 1 mL of enzyme solution was added per 150 mg of mycelium, and it was placed in a 30°C water bath for 3 hours (during which it was inverted and mixed 2-3 times). The enzyme solution was filtered with a 200-mesh cell sieve, and the filtrate was transferred to a new centrifuge tube. It was centrifuged at 4000 rpm and 4°C for 10 minutes, the supernatant was removed, and it was washed twice with 1 mL of mannitol, and finally resuspended to 500 μL of protoplast suspension. The concentration was determined with a cell counter, and finally diluted to 1×10 6 -1×10 7 / mL of suspension with 0.6M mannitol.

[0062] Turn on the power switch of the ARTP device, the instrument is powered on, and the cooling water circulation system is turned on. After confirming that the cooling water circulation is normal, open the gas cylinder valve, adjust the pressure, and ensure that the input pressure is between 0.15-0.2 MPa. Ultraviolet sterilization for 20 minutes. Set the experimental parameters: radio frequency power 120W, working distance 2mm, helium gas flow 10SLM, mutagenesis irradiation time is set as 0s, 20s, 30s, 40s, 45s, 50s, 60s, 0s treatment is the control group. Add 10 μL of protoplast suspension and 10 μL of 10% glycerol (final concentration 5%) as a protective agent to the slide after sterilization, place the slide in the corresponding groove of the ARTP operation room, and start ARTP mutagenesis. After all the samples are treated, close the gas path. Wipe the groove with 75% alcohol cotton, and turn on the ultraviolet sterilization for 20 minutes. Turn off the cooling water circulation machine and the ARTP system. The treated samples are placed in a centrifuge tube containing 600 μL of regeneration medium, vortexed for 2 minutes, and then 150 μL of the suspension is spread on the regeneration medium. Place it in a 25°C incubator, wait for germination, and pick out the mutagenized strains in time.

[0063] The mutagenized strains germinated on the plate were selected and cultured, and the original strain (CK) was synchronously cultured. The strains that grew faster than CK were selected and transferred to PDA medium, and CK was used as a blank control. When the colony diameter reached 20 mm, D1 was drawn, and then the culture was continued until the fastest growing strain covered the plate. At this time, D2 was drawn at the edge of the colony. The distance between D1 and D2 was measured, and the average mycelial growth rate was calculated as (D2-D1) / d. The mutagenized strains with a growth rate increased by more than 5% were selected, and the mutagenized strains and CK were inoculated on the same plate to observe the phenomenon. At the same time, the selected mutagenized strains were subcultured for 5 generations to observe their stability.

[0064] The results are as follows: the concentration of protoplast prepared by the MC-OR-14 strain is 1.04×10 7 / mL, and the concentration of active protoplast is 9.87×10 6 / mL. The control strain and the mutagenized strain transferred after 20s can germinate. The control group has more than 200 germinated colonies per plate, and the mutagenized strain after 20s has only 5-7 germinated colonies per plate, which is lower than the control group. This indicates that the mutagenesis lethality of the strain MC-OR-14 reaches more than 97% under the conditions of helium gas flow 10 MSL, radio frequency power 120 W, working distance 2 mm, and mutagenesis time 20s. The germinated colonies are selected to new medium to observe their growth. A total of 23 germinated strains are selected from the four plates.

[0065] The 23 mutagenized strains and the control strain are transferred to PDA plates for culture, and the time taken for each plate to be fully covered is recorded. The results show that there are a total of 8 mutagenized strains that grow faster than the control strain.

[0066] Table 1 Growth rate of mutagenized strains and control strains

[0067] Note: The data is represented as mean ± standard error, and different letters indicate significant differences between different strains (P<0.05).

[0068] Each mutagenized strain is used as a control with MC-OR-14, and each group has five replicates. Five rounds of iterative growth are performed, and the average growth rate of each round is shown in Table 2. This indicates that the 8 strains are stable mutagenized strains in terms of growth rate.

[0069] Table 2 Genetic stability determination of mutagenized strains

[0070]

[0071] The 8 mutagenized strains have a growth rate increased by more than 5% compared to CK, and are stably inherited within 5 generations. Therefore, these 8 mutagenized strains are selected for subsequent mushroom production experiments.

[0072] The medium formula used in the fruiting experiment is as follows:

[0073] The stock culture medium (liquid): 5 g / L proteose peptone, 2 g / L yeast extract powder, 20 g / L glucose, 1 g / L potassium phosphate dibasic, 0.5 g / L magnesium sulfate, 10 mg / L vitamin B1, pH 6.4±0.4;

[0074] The cultivation bag medium: 45% cotton seed hulls, 32% wood chips, 1.5% GaCO3, 16% bran, 4.2% corn flour, 1.3% GaO.

[0075] Preparation of mother culture and stock culture

[0076] The strain in the plate was inoculated into the slant comprehensive PDA test tube as the mother culture, and cultured at 25°C in the dark for 14 days. The stock culture was prepared by liquid fermentation. The culture was cultured at 150 rpm, 25°C in the dark for 9 days.

[0077] The cultivation material included wood chips, cotton seed hulls, bran, corn flour, calcium oxide and calcium carbonate. The cotton seed hulls and wood chips were pre-wetted one day before sterilization, and the bran, calcium oxide and the like were added the next day. After being fully stirred and mixed in the blender, the water content was adjusted to about 60%, and the material was loaded into high-temperature-resistant polypropylene bags with a size of 17 cm x 35 cm. The dry weight of each bag was 410 g before sterilization. The bags were sterilized at 128°C for 1.5 hours. After sterilization and when the temperature decreased to below 50°C, the bags were transferred to a sterile room, and when the temperature decreased to about 25°C, 30-40 mL of the stock culture was inoculated into each bag. Twenty bags were inoculated for each type. After inoculation, the bags were cultured in the dark in a room with an air humidity of 70% and a temperature of 25°C. The bags were observed every 3-4 days, and the contaminated bags were discarded in time. After the mycelium grew fully in the bags, the bags were post-cultured for 10-20 days.

[0078] The fully cultured bags were transferred to the fruiting room, and the covers were removed and placed vertically. After opening the bags, the room temperature was 26±1°C, the air relative humidity was 75-85%, the light intensity was 800 lux, and the light was on for 12 hours. The room was ventilated 3 times a day for 10 minutes each time, so that the carbon dioxide content in the space was similar to that outside, and was maintained at 350-1000 ppm. After 3 days, the air relative humidity was increased to 90-95% while the other conditions remained unchanged. After the primordia appeared, the air humidity was maintained at 95%, and the ventilation was selected according to the carbon dioxide concentration, so that the indoor carbon dioxide content was maintained at about 2000 ppm. Before the fruiting bodies opened, the harvesting was completed, and the agronomic traits and yield were counted. The biological efficiency, which was the fresh fruiting body weight produced per unit mass of cultivation material, was calculated and expressed as a percentage. The results are as follows:

[0079] Table 3 Time used in the total growth cycle

[0080]

[0081] The results showed that M9 and M12 mutagenic strains significantly shortened the cultivation cycle of O. oregana spores, 28 days and 25 days respectively, compared with the control strain.

[0082] Table 4 Average yield and biological efficiency of strains

[0083]

[0084] Except for M1 mutagenic species, the yield of the rest of the mutagenic species and CK is similar, even lower than CK. In order to determine whether the yield of M1 mutagenic species is significantly improved, the yield of CK and M1 mutagenic species is selected for independent t test. First, the two groups of data are subjected to Levene variance homogeneity test, p = 0.008 < 0.05, indicating that the variances of the two groups of data are different; without assuming equal variance, independent sample t test is performed, at this time p = 0.012 < 0.05, indicating that the yield of M1 mutagenic species is significantly improved compared with CK. Therefore, M1 mutagenic species is a high-yield excellent strain.

[0085] In the middle and late stage of fruiting, the cultivation material of multiple strains of mushroom bags is infected by Trichoderma (no Trichoderma is seen on O. oregana spores), and the infected part of the cultivation material is removed in time, and the subsequent fruiting can still be normal, but the yield will decline to a certain extent. Therefore, the infection rate of each strain is counted. It can be seen that the infection rate of the control strain is high, while the infection rate of most mutagenic strains is lower than that of CK. Among them, the infection rate of M1 mutagenic strain is the lowest, and the yield of the affected mushroom bag is the least. It is shown that compared with CK, the resistance of M1 mutagenic strain to the Trichoderma has been improved. The specific infection rate of each variety is shown in the following table.

[0086] Table 5 Infection rate statistics

[0087]

[0088] The cultivated fruiting bodies were measured for agronomic traits, among which the O. oregana spores without opening the cap had the advantages of transportation and market sales. Among all the mutagenic strains, the cap diameter of M1 was the smallest, which was beneficial to market transportation. The agronomic traits of the remaining O. oregana spores are shown in the following table.

[0089] Table 6 Statistics of agronomic traits of O. oregana spores of each strain

[0090]

[0091] According to the grade standard of NY / T 4377-2023 Black Poria Hypocrea Grade Specification, the picked egg spore small oyster mushroom fruiting bodies are graded. In the same fruiting environment, the CK grade I and grade II mushrooms account for 20.8% and 30.4% respectively; while the M1 mutant strain grade I and grade II mushrooms account for 40.6% and 33.4% respectively, indicating that it has more advantages in producing high-quality mushrooms compared to CK. The proportion of fruiting body grades of each strain is shown in the following table.

[0092] Table 7 Quality grade distribution of fruiting bodies

[0093]

[0094] The M1 strain selected in the above experiment was subjected to plate resistance analysis. The common pathogenic fungus Trichoderma harzianum was used to infect the M1 mutant strain and CK during the cultivation of egg spore small oyster mushrooms. CK and M1 mutant strains were inoculated at the quarter of the plate. After the diameter of the strains reached 20 mm, T. harzianum was inoculated at the other quarter of the plate. After the plate was completely covered with the fungus, the infection rate was measured. The infection of M1 mutant strain and CK is shown in Figure 1 The results showed that the infection rate of CK strain was 75.6±10.5a%, while the infection rate of M1 mutant strain was 46.4±5.8%. Meanwhile, independent sample t test was performed on the two groups of data, p<0.05, indicating that compared with CK, the ability of M1 mutant strain to resist T. harzianum was significantly improved.

[0095] The protein content of fruiting bodies of M1 mutant strain and CK strain was detected. The results showed that the protein content (g / 100g) of CK was 24.4±0.7, and the protein content (g / 100g) of M1 mutant strain was 25.6±0.5.

[0096] Based on the above experiment screening, the high-quality egg spore small oyster mushroom strain M1 was obtained.

[0097] II. Liquid fermentation formula screening

[0098] Further screening of the liquid fermentation medium formula of M1 strain, taking the mycelial biomass per unit time (8 Day) as the index

[0099] Selection of each factor:

[0100] 1. Carbon source: glucose, maltose, fructose, sucrose, lactose

[0101] 2. Nitrogen source: tryptone, yeast extract, beef extract, acid hydrolyzed casein peptone, malt extract powder, urea, ammonium sulfate

[0102] 3. pH value: 5, 6, 7, 8, 9

[0103] Carbon source, nitrogen source, pH value as a variable, respectively, do single factor variable control experiment, each group of culture medium set 4 groups of repeats.

[0104] Basic medium configuration method

[0105] 1. Carbon source: glucose 20 g / L

[0106] 2. Nitrogen source: peptone 5 g / L

[0107] 3. pH: natural

[0108] 4. Liquid volume: 150 mL medium per bottle (250 ml flask)

[0109] Strain: 9d old oospore small oyster mushroom-M1 plate strain.

[0110] Inoculation: sterile operation, inoculate 5 pieces of equal amount of mycelium block (diameter about 0.7 cm) of vigorous growth (growth cycle for 9 days) in each bottle, ensure the consistency of inoculum.

[0111] Culture conditions: constant temperature shaker, temperature 25℃, placed in incubator for light-free culture. The speed is 150 rpm, all samples are taken out after 8 days of culture to observe and record the growth, measure the fresh weight and dry weight.

[0112] Mycelium fresh weight determination:

[0113] Collect the fermentation broth in the conical flask, filter through the weighed quantitative filter paper. Wash the mycelium with distilled water for 2-3 times to remove the residual medium, and dry to the surface of the filter paper without water accumulation. Then use clean filter paper to dry the surface moisture, immediately weigh the fresh weight of mycelium (W) with electronic balance.

[0114] Mycelium dry weight determination

[0115] ① Put the filter paper and mycelium measured for fresh weight together into the oven, dry at 60℃, measure the constant weight (W2).

[0116] ② Calculation: mycelium dry weight (W3) (g / L) = (constant weight after drying (W2) / fermentation broth volume 150 mL) × 1000

[0117] Through shake flask fermentation of oospore small oyster mushroom, the mycelium biomass was measured, and through the significant difference analysis of dry weight, the optimal carbon source of oospore small oyster mushroom M1 strain was glucose, the optimal nitrogen source was yeast extract powder, the optimal pH was 6~7 weak acid and neutral, and the optimal liquid volume was 150 mL per bottle. Figures 2-4 .

[0118] Three, self-built square warehouse cultivation parameter research

[0119] In the self-built 60 m² intelligent mushroom shelter (full-automatic temperature, humidity, light, and gas precision control, i.e. equipped with temperature, humidity, light, , and full-automatic precision control system of wind speed, precision: temperature ±0.3℃, humidity ±2%, ±50 ppm), five batches were continuously cultivated, and the most suitable high-yield and stable-yield parameters for M1 strain were found out.

[0120] The preparation of the bag, inoculation, and cultivation of the fungus were the same as before. After the mycelium filled the bag, it was continued to mature for 25 days. Then the old periderm was mechanically scraped off 0.5-1 mm after removing the collar, and the primordium formation was stimulated. The induction parameters were as follows: temperature: 18-20℃

[0121] relative humidity: 96.5%-98.0% (needing atomization for humidification), concentration: 900-1100 ppm, light intensity: 400-500 lx (cold white light LED, 12 h / d), temperature difference stimulation: the temperature was uniformly reduced from 25℃ to 20℃ within 8 hours, and the constant stimulation window was maintained for 14-16 hours. Under the above parameters, the primordium formation rate of M1 reached 99.2%, and the present bud time was concentrated in 56-68 hours after scraping the fungus, and the present bud was very neat.

[0122] The management of the fruiting body growth period was as follows: temperature was set to 25-28℃, humidity was 90%, 800-950 ppm, light intensity was 600-800 lx, and the illumination time was 12 h / d. When the fruiting body cap diameter was 6-10 cm, the edge was involuted, and the spores had not yet been ejected, it could be harvested.

[0123] Through the whole-chain technology integration of ARTP multi-round mutagenesis-liquid fermentation optimization-shelter precision cultivation, the special strain M1 for Oudemansiella austral was successfully cultivated, which could realize high yield, stable yield, and high quality in a fully enclosed intelligent environment. The industry pain point that the rare mushroom was difficult to produce in a factory for a long time was completely solved, and it had very high economic value and promotion prospect.

[0124] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can make equivalent replacement or change according to the technical solution and concept of the present application within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.

Claims

1. Mutant strain M1 of Hymenopellis raphanipes obtained by artificial mutagenesis, characterized in that, Preserved in GDMCC No: 67331.

2. The mutant strain Ml of Hymenopellis raphanipes according to claim 1, characterized in that, The strain has one or more of the following characteristics compared with the starting strain MC-OR-14: (a) The protein content in the fruiting body of the strain is 25.6±0.5 g / 100g, which is significantly higher than that of the starting strain MC-OR-14, 24.4±0.7 g / 100g, and the cap diameter is ≤1.88 cm and the stem length is ≥6.21 cm; (b) When cultured on PDA medium at 25℃, the average mycelial growth rate is ≥5.20 mm / d; (c) The biological efficiency is ≥53.6%; (d) The resistance to Trichoderma harzianum is significantly enhanced, and the plate infection rate is ≤46.4%; (e) The proportion of primary mushrooms in the fruiting body is ≥40.6%; (f) The genetic stability is good, and the mycelial growth rate fluctuates by no more than ±0.02 mm / d after 5 generations of continuous transfer.

3. Use of the strain M1 of Oudemansiella australis in factory cabin cultivation according to claim 1 or 2.

4. The method for breeding the strain Ml of Orseroia allomycis oospore according to claim 1 or 2, wherein the method is characterized by, Comprising the following steps: (1) Using commercially available Oudemansiella australis strain MC-OR-14 as the starting strain, prepare a protoplast suspension; (2) Mix the protoplast suspension with a glycerol protective agent, and then place it in an ARTP system for mutagenesis treatment, wherein the radio frequency power is 120 W, the helium flow rate is 10 SLM, the working distance is 2 mm, and the mutagenesis time is 20-60 s; (3) After mutagenesis, the protoplasts are coated on a regeneration medium and cultured at 25℃ until germination, and single colonies are picked; (4) Screen for mutagenized strains with a mycelial growth rate increased by ≥5% compared with MC-OR-14 and good genetic stability; (5) Further screen for strains with high biological efficiency, strong resistance to Trichoderma, and good fruiting body commodity traits through fruiting tests, and obtain the strain M1.

5. A special medium for liquid fermentation of the Oudemansiella juglandi strain Ml according to claim 1 or 2, characterized in that, Each liter of culture medium comprises the following components: 20g of glucose, 5g of yeast extract powder, 1g of potassium phosphate dibasic, 0.5g of magnesium sulfate, 10mg of vitamin B1, and the balance is water, with a pH of 6.0-7.

0.

6. The specialized medium of claim 5, wherein, The medium is used to prepare liquid inoculum, and the culture conditions are: 25℃, 150 rpm, and dark culture for 8-9 days.

7. A method for intelligent factory cultivation of the shelter using the strain M1 of Orseroia emersa according to claim 1 or 2, characterized by, Comprising the following steps: (1) Inoculate the M1 strain into a flask containing the liquid medium of claim 5 or 6 to prepare liquid inoculum; (2) After sterilizing the cultivation material, inoculate 30-40 mL of the liquid inoculum into each bag, and culture in the dark at 25℃ and 70% humidity until the mycelium fills the bag, and then post-culture for 25 days; (3) Remove the collar and mechanically remove the old skin 0.5-1mm; (4) Transfer into the intelligent mushroom shelter, and carry out primordium induction. The control parameters are as follows: temperature 18-20℃, relative humidity 96.5%-98.0%, concentration 900-1100 ppm, light intensity 400-500 lx, photoperiod 12 h / d, and temperature difference stimulation: uniformly reduced from 25℃ to 20℃ within 8 hours, and maintained for 14-16 hours; temperature 18-20℃, relative humidity 96.5%-98.0%, concentration 900-1100 ppm, light intensity 400-500 lx, photoperiod 12 h / d, and temperature difference stimulation: uniformly reduced from 25℃ to 20℃ within 8 hours, and maintained for 14-16 hours; (5) After the primordium formation, the fruiting body growth stage is entered, and the control parameters are: temperature 25-28℃, relative humidity 90%, concentration 800-950 ppm, light intensity 600-800 lx, photoperiod 12 h / d; (6) Harvest when the cap diameter of the fruiting body reaches 6-10 cm, the edge is involuted, and the umbrella is not opened.

8. The method of claim 7, wherein, The intelligent mushroom-shaped cabin is equipped with a fully automatic temperature, humidity, light, and gas precision control system, with a temperature control accuracy of ±0.3℃ and a humidity control accuracy of ±2%. Control accuracy ±50ppm.

9. The method of claim 7, wherein, The cultivation material comprises, by mass percentage: 45% of cottonseed hulls, 32% of sawdust, 16% of bran, 4.2% of corn meal, 1.5% of calcium carbonate, and 1.3% of calcium oxide, with a water content of 60%.

10. A functional food comprising the *Millettia oleifera* strain M1 as described in claim 1 or 2, characterized in that, The functional food contains one or more bioactive components of polysaccharides, flavonoids or xylariopsis longipes strain M fruiting body extract, and has one or more effects of antioxidant, hypoglycemic, hypolipidemic, immune enhancement and liver protection.

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