Screening and cultivating method of mutagenic grifola frondosa strain
The hiB3 strain was screened through carbon ion beam irradiation technology, and the culture medium and cultivation conditions were optimized, which solved the problem of poor adaptability of ash tree flower varieties in Heilongjiang area, and achieved the effect of early mushroom production, high yield and excellent commercial properties, which was suitable for large-scale promotion.
Patent Information
- Application Number
- CN202411765283.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-04
- Publication Date
- 2025-03-21
AI Technical Summary
The lack of Grifola frondosa varieties suitable for cultivation in Heilongjiang area in the prior art has led to late mushroom production, low yield, poor commodity performance, and inability to promote on a large scale.
Carbon ion beam irradiation technology was used to perform mutagenesis, and excellent hiB3 mutagenesis strains were screened out, and the proportion of raw materials of mycelium germination solid culture medium, mycelium rapid expansion liquid culture medium and tertiary strains were optimized, so as to improve the cultivation conditions to adapt to the environment in Heilongjiang area.
The obtained hiB3 strain has white mycelium, strong vitality, fast growth rate and short expansion cycle. It produces early mushrooms and has high yields. The fruiting bodies overlap and grows, looks like open flowers, has excellent commercial properties, and has high temperature resistance. It is suitable for mushroom production in mid-August to meet market demand.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and in particular to a kind of Grifola frondosa ( Grifola frondosa ) Screening and cultivation methods of mutagenic strains. Background Art
[0002] Grifola frondosa Grifola frondosa ), commonly known as "maitake mushroom", is a edible and medicinal mushroom that often grows around chestnut trees in summer and autumn. It is also called chestnut mushroom and belongs to the order Aphyllodes, the family Polyporaceae, the genus Grifola, and the species Grifola. Grifola frondosa ) has a beautiful appearance, shaped like a flower, overlapping into clusters, and looks like a blooming lotus from a distance. The flesh is tender and crisp, and the aroma is overflowing when eaten. It can be used for soup, frying, and stewing. In addition to being edible, Grifola frondosa also has unique therapeutic effects in medicine. According to records, the National Cancer Institute of the United States confirmed as early as 1992 that Grifola frondosa ( Grifola frondosa ) extract has the effect of resisting HIV. Dr. Hiroaki Namba of Japan found in his experiment that the extract of Grifola frondosa ( Grifola frondosa ) has anti-HIV effects, and has also been found to be effective against breast cancer, lung cancer, and liver cancer; at the same time, it can also improve various adverse reactions caused by tumor chemotherapy, such as lack of appetite, vomiting, nausea, hair loss, and leukopenia, and is known as the "king of fungi, anti-cancer wonder". In addition to its significant anti-cancer effects, Grifola frondosa ( Grifola frondosa ) also has the effects of controlling blood sugar, inhibiting fat cell accumulation, lowering blood pressure and enhancing immunity. In short, maitake mushroom ( Grifola frondosa ) As mushrooms with both edible and medicinal properties, they are loved by consumers, especially their medicinal value which gives them an unsurpassed status.
[0003] Grifola frondosa Grifola frondosa ) is mostly produced in Qingyuan and Tangshan, and has very high requirements for environmental conditions, strain activity and cultivation substrate. So far, no breeding and large-scale promotion of Grifola frondosa varieties in Heilongjiang Province have been retrieved. The present invention collects Grifola frondosa ( Grifola frondosa ) resources, separated and purified them, and then carried out fruiting experiments. During the cultivation process, it was found that the mushrooms were late to fruit, the yield was low, the cap was grayish white, and the commercial quality was poor, so it could not be promoted on a large scale in Heilongjiang Province. Grifola frondosa ) varieties, and also to speed up the breeding speed, the present invention adopts mutagenesis technology to quickly separate and screen mutant strains. Under normal circumstances, most edible and medicinal fungi use ultraviolet mutagenesis technology to induce mutations, but the efficiency is low, the number of positive mutagenesis is extremely small, and the screening work is time-consuming and laborious. The present invention uses carbon ion beam irradiation method for mutagenesis, which is a precedent in edible and medicinal fungi.
[0004] After a large number of screenings, multiple separations and purifications, the present invention finally obtains the hiB3 excellent mutagenic strain. The hiB3 mycelium is white, vigorous, fast-growing and has a short propagation cycle. At the same time, the field test performance is excellent. The mushrooms are early in fruiting, high in yield, and the fruiting bodies grow in an overlapping manner, resembling open flowers, with excellent commercial quality. In order to meet production needs, the present invention optimizes the mycelium germination solid culture medium and mycelium propagation liquid culture medium, as well as the three-level strain raw material ratio and cultivation method, laying a solid foundation for the large-scale promotion of hiB3 in Heilongjiang Province. In particular, hiB3 is a high-temperature resistant variety and can be put on the market in mid-August to meet the market demand for edible and medicinal fungi in the high temperature season, and also adds valuable rare edible and medicinal fungi germplasm resources to Heilongjiang Province. Summary of the invention
[0005] The technical problems to be solved by the present invention are: The existing technology lacks the maitake mushroom suitable for cultivation in Heilongjiang region. Grifola frondosa ) varieties often have problems such as late fruiting, low yield, and poor commercial quality. The present invention provides a mutant Grifola frondosa ( Grifola frondosa ) strain, this strain has white mycelium, strong vitality, fast growth rate, short propagation cycle, early fruiting and high yield, and is suitable for large-scale cultivation in Heilongjiang area.
[0006] Specifically, the present invention is realized through the following technical solutions: In a first aspect, the present invention provides a mutagenized Grifola frondosa ( Grifola frondosa ) Screening of hiB3 strains.
[0007] As an optional method, during the cultivation of the above-mentioned mutagenic strain, the mutagenic method is carbon ion beam irradiation, 80MeV / u.
[0008] As an optional method, during the cultivation of the above-mentioned mutagenized strain, the hiB3 strain has a preservation number of CCTCC NO: M 20242287, and was deposited in the China Center for Type Culture Collection on October 21, 2024, and the preservation address is Wuhan University, China.
[0009] In a second aspect, the present invention provides a method for cultivating hiB3 strain.
[0010] As an optional method, during the cultivation of the above-mentioned hiB3 strain, the formula of the solid culture medium for rapid mycelium germination is potato dextrose agar medium (PDA), PDA+peptone, PDA+beef extract+yeast powder, PDA+peptone+potassium dihydrogen phosphate, PDA+beef extract+yeast powder+potassium dihydrogen phosphate, PDA+peptone+beef extract+yeast powder+potassium dihydrogen phosphate, and the temperature is 25°C, 27°C and 29°C.
[0011] Preferably, in the above formula, the formula of the solid culture medium for rapid mycelium germination is PDA+peptone+beef extract+yeast powder+potassium dihydrogen phosphate, and the temperature is 27°C.
[0012] As an optional method, during the cultivation of the above-mentioned hiB3 strain, the formula of the mycelium rapid expansion liquid culture medium is glucose, glucose + corn flour, glucose + corn flour + soybean flour, glucose + corn flour + soybean flour + wheat bran, and the temperature is 25°C, 27°C and 29°C.
[0013] Preferably, in the above formula, the formula of the mycelium rapid propagation liquid culture medium is glucose + corn flour + soybean flour + wheat bran, and the temperature is 27°C.
[0014] As an optional method, during the cultivation process of the above-mentioned hiB3 strain, the raw material formula of the tertiary strain is sawdust + wheat bran + quicklime, sawdust + corn cob + quicklime, sawdust + wheat bran + corn cob + quicklime, homemade fermentation feed + wheat bran + quicklime, homemade fermentation feed + corn cob + quicklime, homemade fermentation feed + wheat bran + corn cob + quicklime.
[0015] Preferably, in the above formula, the tertiary strain raw material formula is homemade fermentation material + wheat bran + corn cob + quicklime.
[0016] As an optional method, the tertiary fungus cultivation method is: the tertiary fungus cultivation method is to build a bed in mid-July, with a bed width of 1.1m, a bed length of 5.0m, and a bed depth of 25cm. After the outer packaging of the tertiary fungus is removed, it is placed vertically in the bed, and the top layer of soil is kept no more than 2cm; mushroom stimulating treatment is carried out in mid-August to keep the mushrooms uniform.
[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention obtains Grifola frondosa ( Grifola frondosa ) strain hiB3. This mutagenesis technology is the first time it has been applied to the mutagenesis of edible and medicinal fungi, and has achieved excellent results.
[0018] After the culture medium of the hiB3 strain of the present invention is optimized, the hiB3 mycelium has stronger activity, faster growth rate and shorter propagation cycle.
[0019] The hiB3 of the present invention has excellent performance in field tests, has early fruiting, high yield, overlapping growth of fruiting bodies, and a shape resembling an open flower, and has excellent commercial quality.
[0020] The hiB3 of the present invention is a high temperature resistant variety, which produces mushrooms in mid-August, meeting the market demand for edible and medicinal fungi in the high temperature season, and also adding valuable rare edible and medicinal fungi germplasm resources to Heilongjiang. BRIEF DESCRIPTION OF THE DRAWINGS The drawings described herein are used to provide a further understanding of the application and constitute a part of the application. The illustrative embodiments of the application and their descriptions are used to explain the application and do not constitute an improper limitation of the application. In the drawings: Figure 1 This is an antagonistic test between hiB3 of the present invention and market varieties; Figure 2 is the ITS sequencing result of hiB3 strain; Figure 3 It is a commercial comparison between the hiB3 strain and market products. DETAILED DESCRIPTION
[0021] In order to enable those skilled in the art to better understand the scheme of the present invention, exemplary implementations or embodiments of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described implementations or embodiments are only implementations or embodiments of a part of the present invention, not all of them. Based on the implementations or embodiments of the present invention, all other implementations or embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present invention.
[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the drawings. Example
[0023] The collected resources were separated and purified from the mother strains, and the fruiting test was carried out in the ordinary greenhouse of the No. 4 courtyard of Harbin Academy of Agricultural Sciences. The indoor temperature was maintained at 27±2℃ and the humidity was 80%. No primordium appeared after 20 days. The fruiting method was changed and soil covering cultivation was adopted. After the fruiting, it was found that the fruiting bodies were grayish white in color, the overlap of the caps was poor, the single caps were large and thin, the commercial quality was poor, and the weight of a single plant was only 150g-250g. The fruiting rate was less than 25%, and it could not be cultivated on a large scale in Heilongjiang Province. The fruiting bodies with good growth and no pollution were selected for mother strain separation again to obtain the starting strain for mutagenesis.
[0024] 1.1 Mutagenesis of starting strain (1) The mother strain was propagated using a potato glucose liquid medium with a small amount of soybean powder added. The propagation conditions were 27±2℃ and 150rpm. When the number of bacterial balls reached a certain level, centrifuged at 12000rpm, the supernatant was removed, and the bacterial solution was added again and centrifuged at 12000rpm. This process was repeated 3-4 times. Finally, the mother strain was suspended in a potato liquid medium and diluted to 10 -5 The bacterial suspension of the concentration was sent to Harbin Institute of Technology for mutagenesis as the starting strain.
[0025] (2) This experiment uses carbon ion beam irradiation. As a type of heavy ion beam, carbon ion beam is widely used in the breeding of crops such as Chinese herbal medicine, sorghum, wheat and flowers, and has produced good mutagenic effects, but its application in edible and medicinal fungi has not been reported. The present invention uses 80Mev / u 12 C 6+ Carry out irradiation.
[0026] 1.2 Screening of mutagenic strains The induced strains were numbered hiB1, hiB2, hiB3, and hiB10, and the culture conditions were: dark culture at 27°C, and the culture medium was PDA. The control was a market product strain, cultured under the same conditions. After 30 days, it was found that the hiB3 strain performed best. The growth rate was measured using a caliper, and the results showed that hiB3 germinated first after 4 days, and the hyphae length reached 10mm after 7 days, with an average growth rate of 1.4mm / d; after 15 days, the hyphae length reached 32mm, with an average growth rate of 2.1mm / d, which was 220% faster than the control. The results are shown in Table 1.
[0027] Table 1 Determination of mycelial growth rate Strain name Mycelium length after 7 days (mm) Mycelium length after 10 days (mm) Mycelium length after 15 days (mm) Control strain 3 7 10 hiB1 5 15 21 hiB2 4 9 12 hiB3 10 20 32 hiB4 0 0 0 hiB5 0 0 2 hiB6 0 3 5 hiB7 3 6 6 hiB8 2 2 6 hiB9 0 3 7 hiB10 5 7 11 1.3 Antagonism test The hiB3 strain was propagated three times to observe its stability. PDA culture medium was prepared, and the hiB3 strain and the market product (with a little original culture medium) were picked up with an inoculation loop and inoculated into the same culture dish, leaving a spacer in the middle. The hiB3 strain was cultured in the dark at 27°C for 15 days, and the antagonistic effect was observed. The test showed that the growth of the induced hiB3 strain was significantly stronger than that of the control, and a clear antagonistic line was formed with the market product. The results are as follows Figure 1 shown.
[0028] 1.4 Sequencing and preservation The hiB3 strain was sent to Hangzhou Lianchuan Company for ITS sequencing. The results are as follows Figure 2 As shown. After confirmation, the strain was deposited. The deposit number is CCTCC NO: M 20242287, and it was deposited in the China Center for Type Culture Collection on October 21, 2024. The deposit address is Wuhan University, China. Example
[0029] 2.1 Optimization of culture medium and culture temperature for rapid germination of strains Using market products and hiB3 mother strain as test materials, and PDA as the basic culture medium for strain germination, the effects of various factors on the growth of Grifola frondosa ( Grifola frondosa) The effect of the culture temperature on the germination of the strain was studied. Each combination was repeated 3 times, and the average number of days for mycelium germination was used as the detection index. (1) Carbon source: Potatoes and glucose in PDA medium were used as carbon sources for strain germination, and the optimal addition amount was adjusted to 45g / L. (2) Nitrogen source: Peptone, beef extract, and yeast powder were added as nitrogen sources, and the optimal addition amount was adjusted to 2.5g / L. (3) Inorganic salts: Potassium dihydrogen phosphate was added as inorganic salts, and the optimal addition amount was adjusted to 1g / L. (4) Temperature: The culture temperature was set to 25℃, 27℃, and 29℃, with 3 gradients. The optimal culture medium for rapid germination of the strain obtained through the experiment was: PDA+peptone+beef extract+yeast powder+potassium dihydrogen phosphate, and the optimal culture temperature was 27℃. The results are shown in Table 2.
[0030] Table 2 Effects of different culture factors on mycelial germination
[0031] 2.2 Optimization of liquid culture medium and culture temperature for rapid mycelial growth The hiB3 strain was propagated three times on PDA+peptone+beef extract+yeast powder+potassium dihydrogen phosphate medium and used as the test material after propagation. Potatoes and glucose were used as controls. By adding different auxiliary materials to the control medium and changing the culture temperature, the effects of various factors on the propagation of the hiB3 strain were explored. Each combination was repeated three times, and the average number of bacterial balls formed at 7 days and 10 days was used as the detection index. (1) Carbon source: Potatoes and glucose were used as carbon sources for mycelial growth, and the optimal addition amount was adjusted to 10%. (2) Nitrogen source: Corn flour, soybean flour, and wheat bran were added as nitrogen sources, and the optimal addition amount was adjusted to 10%. (3) Temperature: The culture temperature was set at 25℃, 27℃, and 29℃, with three gradients. The optimal liquid culture medium for rapid mycelial growth obtained through the experiment was: glucose+corn flour+soybean flour+wheat bran, and the optimal culture temperature was 27℃. The results are shown in Table 3.
[0032] Table 3 Effects of different culture factors on mycelial propagation Culture medium and culture conditions Number of bacterial balls formed in 7 days (pieces / mL) Number of bacterial balls formed in 10 days (pieces / mL) Control 25℃ 0 1 Control 27℃ 0 1 Control 29℃ 0 2 Glucose + corn flour 25℃ 0 1 Glucose + corn flour 27℃ 0 2 Glucose + corn flour 29℃ 1 3 Glucose + corn flour + soybean flour 25℃ 2 9 Glucose + corn flour + soybean flour 27℃ 3 10 Glucose + corn flour + soybean flour 29℃ 2 6 Glucose + corn flour + soybean flour + wheat bran 25℃ 4 20 Glucose + corn flour + soybean flour + wheat bran 27℃ 8 25 Glucose + corn flour + soybean flour + wheat bran 29℃ 5 21 2.3 Optimization of the formula of tertiary strain raw materials Prepare the tertiary strain raw materials, formulate them according to the carbon-nitrogen ratio of 40:1, and sterilize them by high pressure after bagging. At the same time, prepare the cultured liquid strains and inject 20mL into the raw materials per bag. Repeat each combination 3 times, and use the average time for the mycelium to fill the bag as the detection index to screen the optimal tertiary strain raw material formula. The raw materials are: sawdust, homemade fermentation feed, wheat bran, corn cobs and quicklime. The best tertiary strain raw material formula obtained through the experiment is: homemade fermentation feed + wheat bran + corn cobs + quicklime, and the results are shown in Table 4.
[0033] Table 4 Effects of different culture materials on mycelial growth status Combination formula Time for mycelium to fill the bag (days) Sawdust+wheat bran+quicklime 60 Sawdust + corn cob + quicklime Not fully grown Sawdust + wheat bran + corn cobs + quicklime 48 Homemade fermented material + wheat bran + quicklime 50 Homemade fermented material + corn cob + quicklime Not fully grown Homemade fermented material + wheat bran + corn cob + quicklime 40 Example
[0034] 3.1 Fruiting test Data show that under normal circumstances, Grifola frondosa ( Grifola frondosa ) directly produce mushrooms in mushroom bags. In order to increase the yield and to improve the Grifola frondosa )'s high temperature resistance, this study adopted the soil covering cultivation method. In the ordinary greenhouse of No. 4 Courtyard of Harbin Academy of Agricultural Sciences, the bed was trimmed according to the size of the tertiary fungus. The bed width is 1.1m, the bed length is 5.0m, and the bed depth is 25cm. After the outer packaging of the tertiary fungus is removed, it is placed vertically in the bed, and the top layer of soil is kept no more than 2cm. The soil temperature is constant and contains a large amount of nutrients, which can supplement the nutritional deficiencies caused by the tertiary fungus after fruiting. The induced gray fungus ( Grifola frondosa Compared with the market product strains, the hiB3 strain has early fruiting, strong high temperature resistance and high yield. Grifola frondosa ) The yield of strain hiB3 is 250g-350g per plant, while the yield of the control market product is only 150g-250g per plant, with a difference of 40%-66% in weight per plant. Grifola frondosa ) The caps of strain hiB3 grow in overlapping patterns, resembling blooming flowers, with a gray-brown color and excellent commercial quality. Figure 3 10 plants were taken for weight measurement, and the results are shown in Table 5.
[0035] Table 5 Market products after Grifola frondosa ( Grifola frondosa )Single plant weight determination name Market product weight (g) hiB3 strain (g) 1 157 350 2 166 326 3 250 275 4 150 300 5 198 250 6 152 321 7 169 299 8 173 287 9 204 254 10 211 309 3.2 High temperature resistance test Under normal circumstances, the optimum growth temperature for edible and medicinal mushrooms is 20℃-35℃, and the best fruiting temperature is 18℃-32℃. According to the adaptability of varieties to temperature, they are divided into high-temperature varieties, medium-high-temperature varieties, medium-temperature varieties and low-temperature varieties. Grifola frondosa ) is a medium-temperature or medium-high-temperature variety, and the fruiting body can occur at 16℃-24℃, with the optimum temperature being 18℃-21℃. Grifola frondosa ) The high temperature resistance of hiB3 strain is significantly improved. In August, the outdoor temperature in Heilongjiang is the highest in the year, reaching above 32°C. In the absence of cooling conditions in ordinary greenhouses, hiB3 strains can produce mushrooms normally, and their commercial quality is better than that of market products. During the growth of fruiting bodies, the present invention observes and records the formation of primordia under different temperature conditions. It was found that the hiB3 strain has a large number of primordia at 30°C and above. However, under the condition of 30°C, the primordia of market products degenerate or die in large numbers. The results are shown in Table 6.
[0036] Table 6 High temperature resistance test Strain name 20℃ 25℃ 30℃ 32℃ Comparison No primordium formation 25% primordium formation 10% primordium formation No primordium formation hiB3 No primordium formation No primordium formation 80% primordium formation 80% primordium formation Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. A mutagenic Grifola frondosa ( Grifola frondosa ) hiB3 strain, characterized in that: The strain has strong vitality, fast growth rate, early fruiting, high yield, and high temperature resistance, and is suitable for cultivation in Heilongjiang. Its preservation number is CCTCC NO: M20242287, and it was preserved in the China Center for Type Culture Collection on October 21, 2024, and the preservation address is Wuhan University, China.
2. The hiB3 strain according to claim 1, characterized in that: The mutagenesis method was carbon ion beam irradiation, 80MeV / u.
3. The hiB3 strain according to claim 1 and claim 2, characterized in that: The mycelium rapid germination solid culture medium is PDA+peptone+beef extract+yeast powder+potassium dihydrogen phosphate, and the culture temperature is 27°C.
4. The hiB3 strain according to claim 1 and claim 2, characterized in that: The mycelium rapid propagation liquid culture medium is glucose+corn flour+soy flour+wheat bran, and the temperature is 27°C.
5. The hiB3 strain according to claim 1 and claim 2, characterized in that: The raw material formula of the third-level bacteria is homemade fermentation material + corn cob + wheat bran + quicklime.
6. The tertiary bacterial strain according to claim 5, characterized in that: The cultivation method of the tertiary fungus strains is to build a bed in mid-July with a bed width of 1.1m, a bed length of 5.0m and a bed depth of 25cm. After the outer packaging of the tertiary fungus strains is removed, they are placed vertically in the bed with the top layer of soil covering no more than 2cm. In mid-August, mushroom stimulating treatment is carried out to ensure consistent mushroom production.